Listeriosis

Appl Environ Microbiol, 2004 Apr, 70(4), 2289 - 95
Pulsed electric field alters molecular chaperone expression and sensitizes Listeria monocytogenes to heat; Lado BH et al.; Pulsed electric field (PEF)-resistant and PEF-sensitive Listeria monocytogenes strains were sublethally treated with electric pulses at 15 kV/cm for 29 micro s and held at 25 degrees C for 5 to 30 min prior to protein extraction . The levels of the molecular chaperones GroEL, GroES, and DnaJ were determined by immunoblotting . After 10 to 20 min after sublethal PEF treatment, a transient decrease in molecular chaperone expression was observed in the PEF-sensitive strain (Scott A) . The levels of GroEL and DnaJ increased back to the basal expression level within 30 min . A substantial decrease in GroES expression persisted for at least 30 min after PEF treatment . Chaperone expression was suppressed after PEF treatment to a smaller extent in the PEF-resistant (OSY-8578) than in the PEF-sensitive strain, and no clear expression pattern was identified in OSY-8578 . Inactivation of Scott A and OSY-8578 in phosphate buffer was compared when lethal PEF (27.5 kV/cm, 144 micro s) and heat (55 degrees C, 10 min) were applied in sequence . When PEF and heat treatments were applied separately, the populations of L . monocytogenes Scott A and OSY-8578 decreased 0.5 to 0.6 log CFU/ml . Cells treated first with PEF and incubated at 25 degrees C for 10 min showed substantial sensitivity to subsequent heat treatment; the decrease in counts for Scott A and OSY-8578 was 6.1 and 2.8 log CFU/ml, respectively . The sequence and time lapse between the two treatments were crucial for achieving high inactivation rates . It is concluded that PEF sensitized L . monocytogenes to heat and that maximum heat sensitization occurred when chaperone expression was at a minimum level.

Appl Environ Microbiol, 2004 Apr, 70(4), 2193 - 203
Multilocus sequence typing of Listeria monocytogenes by use of hypervariable genes reveals clonal and recombination histories of three lineages; Meinersmann RJ et al.; In an attempt to develop a method to discriminate among isolates of Listeria monocytogenes, the sequences of all of the annotated genes from the fully sequenced strain L . monocytogenes EGD-e (serotype 1/2a) were compared by BLASTn to a file of the unfinished genomic sequence of L . monocytogenes ATCC 19115 (serotype 4b) . Approximately 7% of the matching genes demonstrated 90% or lower identity between the two strains, and the lowest observed identity was 80% . Nine genes (hisJ, cbiE, truB, ribC, comEA, purM, aroE, hisC, and addB) in the 80 to 90% identity group and two genes (gyrB and rnhB) with approximately 97% identity were selected for multilocus sequence analysis in two sets of L . monocytogenes isolates (a 15-strain diversity set and a set of 19 isolates from a single food-processing plant) . Based on concatenated sequences, a total of 33 allotypes were differentiated among the 34 isolates tested . Population genetics analyses revealed three lineages of L . monocytogenes that differed in their history of apparent recombination . Lineage I appeared to be completely clonal, whereas representatives of the other lineages demonstrated evidence of horizontal gene transfer and recombination . Although most of the gene sequences for lineage II strains were distinct from those of lineage I, a few strains with the majority of genes characteristic of lineage II had some that were characteristic of lineage I . Genes from lineage III organisms were mostly similar to lineage I genes, with instances of genes appearing to be mosaics with lineage II genes . Even though lineage I and lineage II generally demonstrated very distinct sequences, the sequences for the 11 selected genes demonstrated little discriminatory power within each lineage . In the L . monocytogenes isolate set obtained from one food-processing plant, lineage I and lineage II were found to be almost equally prevalent . While it appears that different lineages of L . monocytogenes can share habitats, they appear to differ in their histories of horizontal gene transfer.

Appl Environ Microbiol, 2004 Apr, 70(4), 2180 - 5
Use of PCR-restriction fragment length polymorphism of inlA for rapid screening of Listeria monocytogenes strains deficient in the ability to invade Caco-2 cells; Rousseaux S et al.; A PCR-restriction fragment length polymorphism (RFLP) method was developed in order to screen a large number of strains for impaired adhesion to epithelial cells due to expression of truncated InlA . inlA polymorphism was analyzed by PCR-RFLP in order to correlate inlA PCR-RFLP profiles and production of truncated InlA . Thirty-seven Listeria monocytogenes strains isolated from various sources, including five noninvasive and two invasive reference strains, were screened . Two endonucleases (AluI and Tsp509I) were used, and they generated five composite profiles . Thirteen L . monocytogenes isolates were characterized by two specific PCR-RFLP profiles similar to PCR-RFLP profiles of noninvasive reference strains previously described as strains that produce truncated InlA . Ten of the 13 isolates showed low abilities to invade human epithelial Caco-2 cells . However, 4 of the 13 isolates were able to invade Caco-2 cells like reference strains containing complete InlA . Sequencing of inlA and Western blot analysis confirmed that truncated InlA was expressed in the 10 L . monocytogenes strains which were isolated from food . This PCR-RFLP method allowed us to identify 10 new strains expressing a truncated internalin . Based on the results obtained in this study, the PCR-RFLP method seems to be an interesting method for rapidly screening L . monocytogenes strains deficient in the ability to invade Caco-2 cells when a sizeable number of strains are studied.

Appl Environ Microbiol, 2004 Apr, 70(4), 1935 - 43
The htrA (degP) gene of Listeria monocytogenes 10403S is essential for optimal growth under stress conditions; Wonderling LD et al.; This report describes a mutant of Listeria monocytogenes strain 10403S (serotype 1/2a) with a defective response to conditions of high osmolarity, an environment that L . monocytogenes encounters in some ready-to-eat foods . A library of L . monocytogenes clones mutagenized with Tn917 was generated and scored for sensitivity to 4% NaCl in order to identify genes responsible for growth or survival in elevated-NaCl environments . One of the L . monocytogenes Tn917 mutants, designated strain OSM1, was selected, and the gene interrupted by the transposon was sequenced . A BLAST search with the putative translated amino acid sequence indicated that the interrupted gene product was a homolog of htrA (degP), a gene coding for a serine protease identified as a stress response protein in several gram-positive and gram-negative bacteria . An htrA deletion strain, strain LDW1, was constructed, and the salt-sensitive phenotype of this strain was complemented by introduction of a plasmid carrying the wild-type htrA gene, demonstrating that htrA is necessary for optimal growth under conditions of osmotic stress . Additionally, strain LDW1 was tested for its response to temperature and H(2)O(2) stresses . The results of these growth assays indicated that strain LDW1 grew at a lower rate than the wild-type strain at 44 degrees C but at a rate similar to that of the wild-type strain when incubated at 4 degrees C . In addition, strain LDW1 was significantly more sensitive to a 52 degrees C heat shock than the wild-type strain . Strain LDW1 was also defective in its response to H(2)O(2) challenge at 37 degrees C, since 100 or 150 micro g of H(2)O(2) was more inhibitory for the growth of strain LDW1 than for that of the parent strain . The stress response phenotype observed for strain LDW1 is similar to that observed for other HtrA(-) organisms, which suggests that L . monocytogenes HtrA may play a role in degrading misfolded proteins that accumulate under stress conditions.

Mol Microbiol, 2004 Apr, 52(2), 601 - 11
Negative control of Listeria monocytogenes virulence genes by a diffusible autorepressor; Ermolaeva S et al.; Virulence genes from the facultative intracellular pathogen Listeria monocytogenes are controlled by the transcriptional regulator PrfA . Although PrfA synthesis is activated at 37 degrees C, PrfA-dependent expression remains low in rich medium . However, a strong induction of the PrfA regulon is observed when L . monocytogenes is cultured in the presence of activated charcoal . Here, we show that the 'charcoal effect' results from the adsorption of a diffusible autorepressor substance released by L . monocytogenes during exponential growth . Analyses using an L . monocytogenes strain in which the prfA gene is expressed constitutively at 37 degrees C from a plasmid indicate that the autoregulatory substance represses PrfA-dependent expression by inhibiting PrfA activity . PrfA presumably functions via an allosteric activation mechanism . The inhibitory effect is bypassed by a PrfA* mutation that locks PrfA in fully active conformation, suggesting that the autorepressor interferes with the allosteric shift of PrfA . Our data indicate that the listerial autorepressor is a low-molecular-weight hydrophobic substance . We suggest that this diffusible substance mediates a quorum-sensing mechanism by which L . monocytogenes restricts the expression of its PrfA virulence regulon . This autoregulatory pathway could serve L . monocytogenes to ensure the silencing of virulence genes during extracellular growth at 37 degrees C . It may also play a role during intracellular infection, by limiting the damage to the host cell caused by an excess production of cytotoxic PrfA-dependent virulence factors in the PrfA-activating cytosolic compartment.

J Biotechnol, 2004 Apr 8, 109(1-2), 13 - 20
Purification and characterization of a recombinant listeriolysin O expressed in Escherichia coli and possible diagnostic applications; Giammarini C et al.; The secreted pore-forming toxin listeriolysin O (LLO) is an essential virulence factor that allows the food-borne bacterial pathogen Listeria monocytogenes to escape from the phagocytic vacuole and reach the host cytosol . This protein belongs to the group of cholesterol-binding sulfhydryl-activated toxins, expressed by a large number of Gram-positive bacteria . A protocol for large-scale expression and purification of recombinant LLO was previously optimized . By a simple two-step purification method, we achieved a high-level LLO synthesis (4.5 mg l(-1) of cell culture) in a hemolytically active form (1.2 x 10(6) HU mg(-1) of protein) . This procedure can solve the problem of LLO isolation from L . monocytogenes cultures which is a difficult task, mainly owing to the low levels of toxin released in the culture media . Here we report the characterization of toxin properties and its preliminary application in an ELISA diagnostic test for listeriosis.

FEMS Microbiol Lett, 2004 Apr 15, 233(2), 257 - 67
Simultaneous quantitative detection of Listeria spp . and Listeria monocytogenes using a duplex real-time PCR-based assay; Rodriguez-Lazaro D et al.; We report a duplex real-time PCR-based assay for the simultaneous quantitative detection of Listeria spp . and the food-borne pathogen Listeria monocytogenes . The targets of this single tube reaction were the 23S rDNA and hly genes of Listeria spp . and L . monocytogenes, respectively . Our assay was efficient, 100% selective (i.e., it allowed accurate simultaneous identification of 52 L . monocytogenes and 120 Listeria spp . strains through the FAM-labelled hly and the VIC-labelled 23S rDNA probes, respectively); and had a detection limit of one target molecule in 100% (23S rDNA) and 56% (hly) of the reactions . Simultaneous quantification was possible along a 5-log dynamic range, with an upper limit of 30 target molecules and R2 values > 0.995 in both cases . Our results indicate that this assay based on the amplification of the 23S rDNA gene can accurately quantify any mixture of Listeria species and simultaneously unambiguously quantify L . monocytogenes.

J Microbiol Methods, 2004 May, 57(2), 251 - 8
Atypical colonial morphology and low recoveries of Listeria monocytogenes strains on Oxford, PALCAM, Rapid'L.mono and ALOA solid media; Leclercq A; The performance of four commercial media, polymyxin-acriflavine-LiCl-ceftazidime-aesculin-mannitol (PALCAM), Oxford, Rapid'L.mono (Bio-Rad, Marne la Coquette, France) and Agar Listeria according to Ottaviani and Agosti (ALOA: AES Laboratoire, Combourg, France; Biolife, Milan, Italy), used to detect and enumerate 176 Belgian strains of Listeria monocytogenes of human and food origin, was evaluated . Four strains showed a low recovery and/or atypical colonies on one or more media . These results showed that a combination of these media, especially alternative media (Rapid'L.mono and/or ALOA) with esculin-containing media (PALCAM and/or Oxford), should therefore be recommended to detect or enumerate atypical strains of L . monocytogenes . In outbreak case investigation for example, incubation of plates should be extended to at least 96 h if no colonies are typical or growth does not appear after 48 h . This is a cost/benefit calculation that should be done in the context of recent listeriosis risk assessments.

C R Biol, 2004 Feb, 327(2), 115 - 23
Exploitation of host cell cytoskeleton and signalling during Listeria monocytogenes entry into mammalian cells; Pizarro-Cerda J et al.; Deciphering how Listeria monocytogenes exploits the host cell machinery to invade mammalian cells during infection is a key issue for the understanding how this food-borne pathogen causes a pleiotropic disease ranging from gastro-enteritis to meningitis and abortions . Using multidisciplinary approaches, essentially combining bacterial genetics and cell biology, we have identified two bacterial proteins critical for entry into target cells, InlA and InlB . Their cellular ligands have been also identified: InlA interacts with the adhesion molecule E-cadherin, while InlB interacts with the receptor for the globular head of the complement factor C1q (gC1q-R), with the hepatocyte growth factor receptor (c-Met) and with glycosaminoglycans (including heparan sulphate) . The dynamic interaction between these cellular receptors and the actin cytoskeleton is currently under investigation . Several intracellular molecules have been recognized as key effectors for Listeria entry into target cells, including catenins (implicated in the connection of E-cadherin to actin) and the actin depolymerising factor/cofilin (involved in the rearrangement of the cytoskeleton in the InlB-dependent internalisation pathway) . At the organism level, species specificity has been discovered concerning the interaction between InlA and E-cadherin, leading to the generation of transgenic mice expressing the human E-cadherin, in which the critical role of InlA in the crossing of the intestinal barrier has been clearly determined . Listeria appears as an instrumental model for addressing critical questions concerning both the complex process of bacterial pathogenesis and also fundamental molecular processes, such as phagocytosis.

Curr Microbiol, 2004 May, 48(5), 373 - 8
Lack of apoptosis in Listeria monocytogenes-infected thymocytes from mice fed with dietary lipids; Puertollano MA et al.; The potential action of certain fatty acids has been studied since the early 1970s . Numerous effects on immune system functions have been related to dietary lipid administration; therefore, several of them have been applied in the treatment of inflammatory disorders . Nevertheless, n-3 polyunsaturated fatty acids may affect host resistance to infectious diseases . In addition, several studies have demonstrated that certain fatty acids are involved in apoptosis induction . Here, we have examined the action of different dietary lipids on the promotion of apoptosis in thymocytes from mice fed with dietary lipids and infected with Listeria monocytogenes . Thus, L . monocytogenes promoted an important cytotoxic effect in all of the groups, but it did not increase the percentage of DNA fragmentation . Similarly, an important increase of caspase-3 activity was demonstrated in OO and FO groups, but infection with L . monocytogenes exerted an inhibitory effect . Finally, L . monocytogenes did not modify proteasome activity among groups fed with dietary lipids . On the basis of this preliminary study, we can state that the infection of thymocytes from mice fed with dietary lipids does not promote a synergistic effect in the induction of apoptosis . Hence, these results may partially serve to elucidate the immune mechanisms involved in cells from mice fed with dietary lipids in an infectious process.

Pediatr Emerg Care, 2004 Apr, 20(4), 233 - 7
Fulminant Listeria monocytogenes meningitis complicated with acute hydrocephalus in healthy children beyond the newborn period; Ulloa-Gutierrez R et al.; We describe 3 previously healthy Costa Rican children who had Listeria monocytogenes meningitis, an uncommon cause of bacterial meningitis beyond the newborn period in normal subjects . Two of them had initial normal brain computed tomography, but all 3 developed acute hydrocephalus at days 7, 3, and 5, respectively . All required immediate ventriculostomy placement and only 1 of 3 survived . L . monocytogenes should be considered among the etiologies of bacterial meningitis in children who do not respond initially to conventional antimicrobial treatment or who deteriorate rapidly.

J Antimicrob Chemother, 2004 May, 53(5), 863 - 6 Epub 2004 Mar 31.
Effect of listeriolysin O-loaded erythrocytes on Mycobacterium avium replication within macrophages; Rossi L et al.; OBJECTIVE: To evaluate the efficacy of erythrocytes loaded with the haemolytic toxin listeriolysin O against Mycobacterium avium replication within human macrophages . METHODS: Recombinant listeriolysin O was loaded in human erythrocytes by a procedure of hypotonic dialysis and isotonic resealing . Loaded erythrocytes were modified to allow them to be recognized and taken up by human macrophages infected with M . avium . The antimycobacterial activity of the erythrocytes loaded with listeriolysin O was evaluated by supernatant and intracellular cfu counts on days 4 and 7 post-erythrocyte administration . RESULTS: Recombinant listeriolysin O was encapsulated in human erythrocytes to reach final concentrations ranging from 1 to 4 ng/mL of erythrocytes . Erythrocytes loaded with increasing quantities of recombinant protein were able to reduce (at most by 50%) M . avium replication in a dose-dependent fashion when administered to infected macrophages . CONCLUSIONS: Erythrocytes loaded with listeriolysin O are effective against M . avium replication within macrophages . We are confident that the strategy presented could be useful against mycobacteria other than M . avium (such as Mycobacterium tuberculosis and Mycobacterium leprae) by itself or as part of an antimycobacterial treatment.

Roum Arch Microbiol Immunol, 2002 Oct-Dec, 61(4), 275 - 83
Listeria monocytogenes--host interaction; Zlei M et al.; Both innate and adaptive immunity against Listeria monocytogenes are essential in circumventing or fighting the disease, although there is no clear delineation of the precise role of either in listeriosis . A consistent amount of in vivo experiments have generated a complex picture over the impact of listerial infection on the hosts, and they are shortly reviewed in this paper.

Mol Microbiol, 2004 Apr, 52(1), 257 - 71
GW domains of the Listeria monocytogenes invasion protein InlB are required for potentiation of Met activation; Banerjee M et al.; The Listeria monocytogenes protein InlB promotes intracellular invasion by activating the receptor tyrosine kinase Met . Earlier studies have indicated that the LRR fragment of InlB is sufficient for Met activation, but we show that this is not the case unless the LRR fragment is artificially dimerized through a disulphide bond . In contrast, activation of Met proceeds through monomers of intact InlB and, at physiologically relevant concentrations, requires coordinated action in cis of both InlB N-terminal LRR region and C-terminal GW domains . The GW domains are shown to be crucial for potentiating Met activation and inducing intracellular invasion, with these effects depending on association between GW domains and glycosaminoglycans . Glycosaminoglycans do not alter the monomeric state of InlB, and are likely to enhance Met activation through a receptor-mediated mode, as opposed to the ligand-mediated mode observed for the LRR fragment . Surprisingly, we find that gC1q-R, a host protein implicated in InlB-mediated invasion, specifically antagonizes rather than enhances InlB signalling, and that interaction between InlB and gC1q-R is unnecessary for bacterial invasion . Lastly, we demonstrate that HGF, the endogenous ligand of Met, substitutes for InlB in promoting intracellular invasion, suggesting that no special properties are required of InlB in invasion besides its hormone-like mimicry of HGF.

Mol Microbiol, 2004 Apr, 52(1), 39 - 52
In vitro transcription of the Listeria monocytogenes virulence genes inlC and mpl reveals overlapping PrfA-dependent and -independent promoters that are differentially activated by GTP; Luo Q et al.; Most known virulence genes of Listeria monocytogenes are regulated by the transcriptional factor PrfA . Using our recently established in vitro transcription system, we have studied the PrfA-dependent promoter (PinlC) regulating the expression of the small, secreted internalin C . PrfA-dependent and PrfA-independent transcription is observed starting from PinlC in vitro and in vivo, suggesting the presence of two apparently overlapping promoters both of which use the same -10 box . Although the PrfA-dependent transcription requires, as expected, the PrfA-box, PrfA-independent transcription depends on a -35 box located directly downstream of the PrfA-box . PrfA-independent transcription starts at A, 7 bp downstream of the common -10 box (A7), and is strongly inhibited by PrfA because of the close proximity of the PrfA binding site to the -35 box . PrfA-dependent transcription starts preferentially at G5 but, in the absence of this start nucleotide, alternative start sites at A positions 7 or 8 bp downstream of the -10 box can also be used . The -35 box of the PrfA-independent promoter can be functionally inactivated without affecting PrfA-dependent transcription as long as the distance between the PrfA-box and the -10 box remains fixed to 22 (or 23) bp . Vice versa, the PrfA-box can be deleted without affecting PrfA-independent transcription from PinlC, which is no longer inhibited by PrfA . The PrfA-dependent transcription initiation needs, in contrast to the PrfA-independent one, the presence of a high concentration of GTP (and ATP) but not of CTP and UTP . Overlapping PrfA-dependent and PrfA-independent promoter activity was also demonstrated for the mpl promoter (Pmpl) . Again, PrfA-dependent transcription starting at Pmpl is dominant at high GTP concentration and PrfA-independent transcription at low GTP . Here too, the PrfA-dependent and the PrfA-independent promoters share the same -10 box characteristic of SigA-loaded RNA polymerase . High GTP concentration also appears to be necessary for transcription initiation at other PrfA-dependent promoters (Phly, PactA) but not at the PrfA-independent promoter PinlC-m8.

Eur J Immunol, 2004 Apr, 34(4), 913 - 20
Mini-review: Presentation of pathogen-derived antigens in vivo; Lauvau G et al.; Most intracellular pathogens induce robust T cell responses upon infection of mammalian hosts . In most cases, these T cell responses are protective and result in pathogen clearance . It is therefore important to determine how T cells are primed and how they differentiate into cytokine-secreting and/or cytotoxic effector cells . In contrast to B cells, which recognize soluble Ag, CD8(+) and CD4(+) T cells react to Ag-derived peptides bound to MHC I or MHC II molecules, respectively . Therefore, elucidating the mechanisms by which pathogen-derived Ag become available for presentation is necessary to understand how pathogens trigger T cell responses in vivo . Although many excellent reviews have focused on the mechanisms involved in Ag processing, very few have pointed to the specificity of host-pathogen interactions . In this respect, it should be noticed that these interactions are very different from one pathogen to another, and may result in the involvement of different cells and molecules . Because of space limitations, we have decided to focus this review on two intracellular pathogens--vaccinia virus and Listeria monocytogenes . We have chosen these two pathogens because they both induce a strong CD8(+) T cell response and because they have been extensively studied by both microbiologists and immunologists.

Biosens Bioelectron, 2004 May 15, 19(10), 1331 - 5
A generic approach for the detection of whole Listeria monocytogenes cells in contaminated samples using surface plasmon resonance; Leonard P et al.; The opportunistic food pathogen Listeria monocytogenes is of great concern to the food industry and its rapid detection is of major importance . This paper describes the detection of L . monocytogenes with a polyclonal antibody by means of a new subtractive inhibition assay using a BIAcore 3000 biosensor . Incubating L . monocytogenes cells and antibody for a short period of time, followed by subsequent separation of free unbound antibody with a stepwise centrifugation process, allowed the detection of 1 x 10(5) L . monocytogenes cells/ml in less than 30 min . Free antibody was passed over an anti-Fab ligand-coated sensor chip surface with the generated response being inversely proportional to the inhibiting cell concentration . The method was simple, rapid and needed minimum sample preparation . This assay format has the potential for the quick and sensitive detection of pathogens with limited sample handling and preparation.

FEMS Microbiol Lett, 2004 Apr 1, 233(1), 159 - 64
Listeria monocytogenes: comparative interpretation of mouse virulence assay; Liu D; Being an opportunistic bacterial pathogen, Listeria monocytogenes demonstrates significant strain variations in virulence and pathogenicity . The availability of laboratory procedures to ascertain the pathogenic potential of L . monocytogenes bacteria would greatly enhance the control and prevention of listerial infections . As a method that measures all virulent determinants, mouse virulence assay has been frequently used for assessing L . monocytogenes virulence . The pathogenic potential of a given L . monocytogenes strain as determined by mouse virulence assay is often calculated from mouse mortality data in combination with colony forming units (CFUs) derived from plate counts, and expressed by medium lethal dose (LD(50)) . In this report, we describe an alternative method {i.e., relative virulence (%)} that does not involve CFU estimation, and is comparable to LD(50) for interpretation of mouse virulence assay for L . monocytogenes . The relative virulence (%) is obtained by dividing the number of dead mice with the total number of mice tested for a particular strain using a known virulent strain (e.g., L . monocytogenes EGD) as reference . Besides providing a more direct interpretation in comparison with LD(50) values for mouse virulence assay, this method requires fewer dosage groups per L . monocytogenes strain, and eliminates CFU estimation that is step subject to variations between runs and also between laboratories.

Nat Rev Immunol, 2004 Feb, 4(2), 100 - 9
p47 GTPases: regulators of immunity to intracellular pathogens; Taylor GA et al.; Activation of the innate immune system by interferon-gamma (IFN-gamma is crucial for host resistance to infection . IFN-gamma induces the expression of a wide range of mediators that undermine the ability of pathogens to survive in host cells, including a newly discovered family of 47-kDa GTPases . Elimination of different p47 GTPases in mice by gene targeting severely cripples IFN-gamma-regulated defence against Toxoplasma gondii, Listeria monocytogenes, Mycobacterium spp . and other pathogens . In this article, we review our understanding of the role of p47 GTPases in resistance to intracellular infection and discuss the present evidence concerning their mode of action.

Int Immunol, 2004 Apr, 16(4), 597 - 605
DNA vaccination with gp96-peptide fusion proteins induces protection against an intracellular bacterial pathogen; Rapp UK et al.; Effective vaccination using in vitro peptide-loaded heat-shock proteins (HSP), tumor-derived HSP and HSP fusion proteins has been shown in viral, parasite and tumor model systems . We demonstrate protective DNA vaccination using gp96-peptide fusion proteins against the intracellular bacterial pathogen Listeria monocytogenes in a mouse model . In contrast to previous studies using pathogen-derived HSP as vaccine vehicles, we used recombinant endogenous (Mus musculus) gp96 (GRP94) as a carrier for immunodominant listerial peptides . Analyses of the cellular immune response revealed profound epitope-specific IFN-gamma and cytotoxic T cell responses . Our findings suggest a predominantly MHC I-restricted T cell response to DNA vaccination with gp96 fusion proteins in the model employed . Most importantly, DNA vaccination induced protection against an otherwise lethal dose of L . monocytogenes.

Infect Immun, 2004 Apr, 72(4), 2131 - 9
Toll-like receptor 2 is required for optimal control of Listeria monocytogenes infection; Torres D et al.; The control of Listeria monocytogenes infection depends on the rapid activation of the innate immune system, likely through Toll-like receptors (TLR), since mice deficient for the common adapter protein of TLR signaling, myeloid differentiation factor 88 (MyD88), succumb to Listeria infection . In order to test whether TLR2 is involved in the control of infections, we compared the host response in TLR2-deficient mice with that in wild-type mice . Here we show that TLR2-deficient mice are more susceptible to systemic infection by Listeria than are wild-type mice, with a reduced survival rate, increased bacterial burden in the liver, and abundant and larger hepatic microabscesses containing increased numbers of neutrophils . The production of tumor necrosis factor, interleukin-12, and nitric oxide and the expression of the costimulatory molecules CD40 and CD86, which are necessary for the control of infection, were reduced in TLR2-deficient macrophages and dendritic cells stimulated by Listeria and were almost abolished in the absence of MyD88, coincident with the high susceptibility of MyD88-deficient mice to in vivo infection . Therefore, the present data demonstrate a role for TLR2 in the control of Listeria infection, but other MyD88-dependent signals may contribute to host resistance.

Infect Immun, 2004 Apr, 72(4), 2014 - 21
Induction of protective cellular immunity against Mycobacterium tuberculosis by recombinant attenuated self-destructing Listeria monocytogenes strains harboring eukaryotic expression plasmids for antigen 85 complex and MPB/MPT51; Miki K et al.; We report here the induction of specific protective cellular immunity against Mycobacterium tuberculosis by the employment of vaccination with recombinant attenuated Listeria monocytogenes strains . We constructed self-destructing attenuated L . monocytogenes Delta 2 strains carrying eukaryotic expression plasmids for the antigen 85 complex (Ag85A and Ag85B) and for MPB/MPT51 (mycobacterial protein secreted by M . bovis BCG/mycobacterial protein secreted by M . tuberculosis) molecules . Infection of these recombinant bacteria allowed expression of the genes in the J774A.1 murine macrophage cell line . Intraperitoneal vaccination of C57BL/6 mice with these recombinant bacteria was capable of inducing purified protein derivative-specific cellular immune responses, such as foot pad reactions, proliferative responses of splenocytes, and gamma interferon production from splenocytes, suggesting the efficacy of vaccination against mycobacterial infection by use of these recombinant L . monocytogenes strains . Furthermore, intravenous vaccination with recombinant bacteria carrying expression plasmids for Ag85A, Ag85B, or MPB/MPT51 in BALB/c mice elicited significant protective responses, comparable to those evoked by a live Mycobacterium bovis BCG vaccine . Notably, this is the first report to show that MPB/MPT51 is a major protective antigen in addition to Ag85A and Ag85B, which have been reported to be major mycobacterial protective antigens.

Curr Opin Microbiol, 2004 Feb, 7(1), 45 - 50
T cell responses to Listeria monocytogenes; Lara-Tejero M et al.; Owing to its unique intracellular biology that allows it to gain access to the host cell cytosol, Listeria monocytogenes induces potent, protective CD8 responses . The study of these responses has served as a paradigm to understand cell-mediated immunity to microbial pathogens . The availability of mutants specifically defective in unique aspects of the intracellular biology of this pathogen has greatly aided these studies . During the past few years, progress has been made to understand the contribution of the innate immune system and CD4 T cells in the generation of robust, long lasting CD8 responses to L . monocytogenes.

Biochemistry, 2004 Mar 30, 43(12), 3688 - 95
Membrane fusion induced by the catalytic activity of a phospholipase C/sphingomyelinase from Listeria monocytogenes; Montes LR et al.; Listeria monocytogenes is a bacterium responsible for localized and generalized infections in humans and animals . It has the ability to spread from the cytoplasm of an infected cell to neighboring cells without becoming exposed to the extracellular space . The bacterium secretes a phospholipase C (PLC(LM)) that is active on glycerophospholipids, e.g., phosphatidylcholine, and on sphingomyelin; thus, PLC(LM) should be described more appropriately as a phospholipase C/sphingomyelinase . We have obtained PLC(LM) free from a frequent contaminant, listeriolysin O, using an improved purification procedure . PLC(LM) has been assayed on large unilamellar liposomes of defined lipid composition . The enzyme is activated by K(+) and Mg(2+), and readily degrades phospholipids in bilayer form, in the absence of detergents . Enzyme activity is accompanied by important changes in the structure of the phospholipid vesicles, namely, vesicle aggregation, intervesicular mixing of lipids, and mixing of aqueous contents, with very low leakage of vesicular contents . The data are interpreted as indicative of PLC(LM)-induced vesicle fusion . This is confirmed by the demonstration of intervesicular mixing of inner monolayer lipids, using a novel procedure . The observation of PLC(LM)-induced membrane fusion suggests a mechanism for the cell-to-cell propagation of the bacterium, which requires disruption of a double-membrane vacuole.

J Food Prot, 2004 Mar, 67(3), 480 - 5
Inhibition of Listeria monocytogenes on the surface of individually packaged hot dogs with a packaging film coating containing nisin; Franklin NB et al.; The objective of this study was to determine the effectiveness of packaging films coated with a methylcellulose/hydroxypropyl methylcellulose-based solution containing 10,000, 7,500, 2,500, or 156.3 IU/ml nisin for controlling Listeria monocytogenes on the surfaces of vacuum-packaged hot dogs . Barrier film coated with a methylcellulose/hydroxypropyl methylcellulose-based solution containing nisin or no nisin (control) was heat sealed to form individual pouches . Hot dogs were placed in control and nisin-containing pouches and inoculated with a five-strain L . monocytogenes cocktail (approximately 5 log CFU per package), vacuum sealed, and stored for intervals of 2 h and 7, 15, 21, 28, and 60 d at 4 degrees C . After storage, hot dogs and packages were rinsed with 0.1% peptone water . Diluent was spiral plated on modified oxford agar and tryptic soy agar and incubated to obtain counts (CFU per package) . L . monocytogenes counts on hot dogs packaged in films coated with 156.3 IU/ml nisin decreased slightly (approximately 0.5-log reduction) through day 15 of refrigerated storage but was statistically the same (P > 0.05) as hot dogs packaged in films without nisin after 60 d of storage . Packaging films coated with a cellulose-based solution containing 10,000 and 7,500 IU/ml nisin significantly decreased (P < 0.05) L . monocytogenes populations on the surface of hot dogs by greater than 2 log CFU per package throughout the 60-d study . Similar results were observed for hot dogs packaged in films coated with 2,500 IU/ml nisin; however, L . monocytogenes populations were observed to be approximately 4 log CFU per package after 60 d of refrigerated storage from plate counts on tryptic soy and modified oxford agars.

J Food Prot, 2004 Mar, 67(3), 475 - 9
Development and characterization of an antimicrobial packaging film coating containing nisin for inhibition of Listeria monocytogenes; Grower JL et al.; The purpose of this study was to develop and characterize a packaging film coating containing nisin . A spot-on-lawn assay was used to determine the effect of acid type (ascorbic, acetic, hydrochloric, lactic) and nisin level (equal increments from 10,000 IU to 9 IU) to be used in the formulation of the film coating . Zones of inhibition were measured after incubation on tryptic soy agar (37 degrees C, 48 h) . Low-density polyethylene films coated with differing levels of nisin were characterized by field emission scanning electron microscopy, tensile strength, elongation, and water vapor transmission rate . The MIC of nisin in solution was 157 mg/ml . All acids were equally inhibitory (P > 0.05), but acetic acid produced the largest zone of inhibition (21 mm) . Field emission scanning electron microscopy confirmed that the cloudy appearance of the films was due to sodium chloride found in the commercially prepared nisin . Tensile strength increased as nisin concentration increased, which also corresponded to increasing film thickness . The nisin coatings (10,000 and 2,500 IU/ml) did not have a significant effect (P > 0.05) on the water vapor transmission rate of the low-density polyethylene film.

J Food Prot, 2004 Mar, 67(3), 470 - 4
Radiation resistance and virulence of Listeria monocytogenes Scott A following starvation in physiological saline; Mendonca AF et al.; The influence of starvation on the resistance of Listeria monocytogenes Scott A to electron beam irradiation in 0.85% (wt/vol) NaCl (saline) and in ground pork was investigated . Exponential- or stationary-phase cells (control) were grown at 35 degrees C in tryptic soy broth supplemented with 0.6% yeast extract . Washed cells were starved for 12 days in saline, and virulence of the pathogen was evaluated at 0, 8, and 12 days during starvation . Samples of saline and irradiation-sterilized ground pork, inoculated with control or starved cells, were irradiated at doses ranging from 0.0 to 2.5 kGy . L . monocytogenes survivors were determined by plating diluted samples of saline or pork on tryptic soy agar supplemented with 0.6% yeast extract and counting bacterial colonies following incubation (35 degrees C, 48 h) . Virulence of starved cells and control was not significantly different (P > 0.05) . Cells exhibited the highest radiation resistance at 8 days of starvation . Irradiation (0.5 kGy) in saline resulted in approximately 7.14, 5.55, and 2.38 log reduction in exponential, stationary, and starved cells, respectively . Irradiation of ground pork at 2.5 kGy reduced controls by approximately 6.0 log, whereas starved cells were reduced by only 3.8 log . Starved cells consistently exhibited higher irradiation D10-values than controls (P < 0.05) . D10-values for exponential, stationary, and starved cells were 0.07, 0.09, and 0.21 kGy and 0.35, 0.42, and 0.66 kGy in saline and ground pork, respectively . These results indicate that starvation cross-protects L . monocytogenes Scott A against radiation inactivation and should be considered when determining this pathogen's irradiation D-value.

J Food Prot, 2004 Mar, 67(3), 463 - 9
Temperature effect on Listeria monocytogenes growth in the event of contamination of cooked pork products; Membre JM et al.; The aim of this study was to describe the effect of temperature on the growth of Listeria monocytogenes in the event of postprocess contamination of packaged pork meats . This study was carried out in two steps . In the first step, the effect of temperature on L . monocytogenes growth rates was determined in duplicates at 13 temperatures between 2 and 43 degrees C by turbidimetric methods and adjusted by a quantitative secondary model . Then, seven sets of growth kinetics were collected by challenge testing in white pudding and roulade, both cooked pork products prepared according to an industrial process and stored at suboptimal temperatures ranging from 2 to 20 degrees C . In the second step, objectives were to (i) collect direct information on the temperature effect of L . monocytogenes on the two pork products, (ii) compare the two products regarding L . monocytogenes exposure, and (iii) compare results given by modeling (step i) with results obtained independently and then evaluate the model application domain . Each kinetic was built with at least 10 experimental data and two replicates . Comparison between L . monocytogenes behavior at 4 degrees C on white pudding and roulade indicated that both meat products were affected by food safety problems . Indeed, after contamination and storage for 10 days at 4 degrees C, the bacterial population increased by 2 log CFU/g in both products . Comparison between growth kinetic simulations and experimental data obtained separately gave satisfactory conclusions; the difference between observed and predicted bacterial population values was always less than 1 log CFU/g and a bias factor of 1.18 when growth rates were compared . These results applied to L . monocytogenes contamination of white pudding or roulade can now be used either in the management of optimal process and distribution networks or in risk assessment (exposure assessment).

J Food Prot, 2004 Mar, 67(3), 456 - 62
Attachment of Listeria monocytogenes on ready-to-eat meats; Foong SC et al.; Five individual strains of Listeria monocytogenes and a mixed cocktail of all five were studied for attachment on frankfurters, ham, bologna, and roast beef relative to their cell surface characteristics . The ratio of strongly attached (sessile) L . monocytogenes cells compared with total (sessile and planktonic) attached cells on ready-to-eat meats was also determined . Because bacterial cell surfaces were characterized by net negative charge and hydrophobicity, electrostatic interaction chromatography and cationized ferritin methods were chosen to study net negative charge distribution on the bacterial cell surface, whereas hydrophobic interaction chromatography and contact angle measurement were used to examine the cell surface hydrophobicity . No differences (P > 0.05) were observed in cell surface charge or cell surface hydrophobicity among strains . Approximately 84 to 87% L . monocytogenes were found to attach strongly to ready-to-eat meats within 5 min . No differences (P > 0.05) were found among strains or among meats . Micrographs observed from scanning electron microscopy showed no differences among the strains but showed a difference in age of cells (mixed culture) in terms of surface negative charge distribution . More surface negatively charged sites were observed at 0 and 7 days and much fewer at 3 days during storage of washed, harvested cells in buffer at 4 degrees C (aged cells under cold and nutrient deprivation), indicating a possible change in cell surface properties . Because no difference in strains was observed, the contact angle measurement study was carried out with the five-strain mixed culture . The surface hydrophobicity increased in frankfurters, decreased in roast beef, and was unchanged in ham and bologna as a result of inoculation.

J Immunol, 2004 Apr 1, 172(7), 4418 - 24
The Ly-6Chigh monocyte subpopulation transports Listeria monocytogenes into the brain during systemic infection of mice; Drevets DA et al.; Mononuclear phagocytes can be used by intracellular pathogens to disseminate throughout the host . In the bloodstream these cells are generically referred to as monocytes . However, blood monocytes are a heterogeneous population, and the exact identity of the leukocyte(s) relevant for microbial spreading is not known . Experiments reported in this study used Listeria monocytogenes-infected mice to establish the phenotype of parasitized blood leukocytes and to test their role in systemic dissemination of intracellular bacteria . More than 90% of the blood leukocytes that were associated with bacteria were CD11b(+) mononuclear cells . Analysis of newly described monocyte subsets showed that most infected cells belonged to the Ly-6C(high) monocyte subset and that Ly-6C(high) and Ly-6C(neg-low) monocytes harbored similar numbers of bacteria per cell . Interestingly, systemic infection with wild-type or DeltaactA mutants of L . monocytogenes, both of which escape from phagosomes and replicate intracellularly, caused expansion of the Ly-6C(high) subset . In contrast, this was not evident after infection with Deltahly mutants, which neither escape phagosomes nor replicate intracellularly . Importantly, when CD11b(+) leukocytes were isolated from the brains of lethally infected mice, 88% of these cells were identified as Ly-6C(high) monocytes . Kinetic analysis showed a significant influx of Ly-6C(high) monocytes into the brain 2 days after systemic infection . This coincided with both bacterial invasion and up-regulation of brain macrophage chemoattractant protein-1 gene expression . These data indicate that the Ly-6C(high) monocyte subset transports L . monocytogenes into the brain and establish their role as Trojan horses in vivo.

J Immunol, 2004 Apr 1, 172(7), 4410 - 7
Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response; Sunderkotter C et al.; Blood monocytes are well-characterized precursors for macrophages and dendritic cells . Subsets of human monocytes with differential representation in various disease states are well known . In contrast, mouse monocyte subsets have been characterized minimally . In this study we identify three subpopulations of mouse monocytes that can be distinguished by differential expression of Ly-6C, CD43, CD11c, MBR, and CD62L . The subsets share the characteristics of extensive phagocytosis, similar expression of M-CSF receptor (CD115), and development into macrophages upon M-CSF stimulation . By eliminating blood monocytes with dichloromethylene-bisphosphonate-loaded liposomes and monitoring their repopulation, we showed a developmental relationship between the subsets . Monocytes were maximally depleted 18 h after liposome application and subsequently reappeared in the circulation . These cells were exclusively of the Ly-6C(high) subset, resembling bone marrow monocytes . Serial flow cytometric analyses of newly released Ly-6C(high) monocytes showed that Ly-6C expression on these cells was down-regulated while in circulation . Under inflammatory conditions elicited either by acute infection with Listeria monocytogenes or chronic infection with Leishmania major, there was a significant increase in immature Ly-6C(high) monocytes, resembling the inflammatory left shift of granulocytes . In addition, acute peritoneal inflammation recruited preferentially Ly-6C(med-high) monocytes . Taken together, these data identify distinct subpopulations of mouse blood monocytes that differ in maturation stage and capacity to become recruited to inflammatory sites.

Int J Food Microbiol, 2004 Apr 1, 92(1), 95 - 103
Effects of several environmental factors on the anti-Listeria monocytogenes activity of an essential oil of Picea excelsa; Canillac N et al.; The effects of several environmental factors on the anti-Listeria monocytogenes activity of an essential oil of Picea excelsa were explored by determination of active concentrations using two methods and with survival curves . In trial conditions, the serovars 1/2c and 4b behaved similarly . A dose of 0.2-0.3% (v/v) of essential oil was bactericidal for 10(5)-10(7) cells contained in 1 ml of Tryptone Soy Broth Yeast Extract at pH 6 and 7 incubated at 13 and 37 degrees C and of medium supplemented with levan . Introduction of sodium caseinate, agar or fat into the test medium and use of a cheese medium decreased the bactericidal effects of the essential oil . Basic pH, addition of NaCl or use of Tryptone Soy Broth and saline solution increased its antilisterial activity . Serovar 1/2c survival curves exhibited an exponential death rate followed by a tailing effect in the presence of the minimal bactericidal concentration of the essential oil . A three log10 reduction of cell viability was obtained within 100 min in Tryptone Soy Broth Yeast Extract, within longer exposure in media supplemented with NaCl or at basic pH.

Int J Food Microbiol, 2004 Apr 1, 92(1), 89 - 94
Growth of Listeria monocytogenes in melon, watermelon and papaya pulps; Penteado AL et al.; Growth of Listeria monocytogenes in low-acid fruits (melon, watermelon and papaya) at different times of incubation and at temperatures of 10, 20 and 30 degrees C was studied . Fruit pulp portions with an average pH of 5.87, 5.50 and 4.87 for melon, watermelon and papaya, respectively, were obtained aseptically, homogenized, weighed and inoculated with suspensions (approximately 10(2) CFU/g) of L . monocytogenes . Generation times of 7.12, 13.03 and 15.05 h at 10 degrees C, 1.74, 2.17 and 6.42 h at 20 degrees C and 0.84, 1.00 and 1.16 h at 30 degrees C were obtained, respectively, for melon, watermelon and papaya . The results showed that L . monocytogenes grew in low-acid fruits at all tested temperatures, although growth was diminished, but not inhibited at 10 degrees C.

Int J Food Microbiol, 2004 Apr 1, 92(1), 15 - 33
Listeria monocytogenes and listeriosis: a review of hazard characterisation for use in microbiological risk assessment of foods; McLauchlin J et al.; Considerable effort has been put into the application of quantitative microbiological risk assessment for Listeria monocytogenes, and data are available for England and Wales (probably more so than most other countries) on the adverse health effects, together with incidence data on different age and risk groups for human L . monocytogenes infections . This paper reviews aspects of Listeria and human listeriosis, especially from a public health perspective and provide hazard characterisation data, i.e . the qualitative and/or quantitative evaluation of the adverse health effect associated with the hazard, which is the relationship between exposure levels (dose) and frequency of illness . The majority of cases of human listeriosis are food-borne; however, the disease process is complex with multiple routes of infection . The dose-response relationship is poorly understood, and data from human volunteer studies are not available and would be unethical to produce . Data are available from a range of different animal and in vitro models, although these poorly mimic the natural disease process in route of infection, end point, host and history of prior exposure to the bacterium . Epidemiological data provide some information on infective doses and dose responses, but because of the characteristics of the disease (the hugely variable and potentially very long incubation periods, the low attack rates and the rarity of identification of specific food vehicles), this also provides limited data for calculation of dose responses . There is some, albeit limited, evidence for strain variation, but this is an area of considerable uncertainty despite great advances in the genetic basis of the virulence of this bacterium, and almost all strains seem capable of causing serious disease . A variety of mathematical approaches have been used to model dose responses . The review is written to provide a clinical and epidemiological background to the mathematically oriented, as well as to outline the mathematical approaches to those interested in food-borne infection.

Curr Drug Targets Inflamm Allergy, 2004 Mar, 3(1), 97 - 104
Estrogen, a double-edged sword: modulation of TH1- and TH2-mediated inflammations by differential regulation of TH1/TH2 cytokine production; Salem ML; Estrogen appears to play a central role in the immune response and immune-mediated diseases . Estrogen receptors are expressed in a variety of immunocompetent cells, including CD4(+) and CD8(+) T cells and macrophages . Clinical observations indicate that some autoimmune diseases, such as rheumatoid arthritis and multiple sclerosis, frequently remit during pregnancy but exacerbate, or have their onset during the postpartum period . Pharmacological levels of estrogen also appear to ameliorate certain autoimmune diseases . In addition, estrogen is known to suppress certain infectious diseases, as well as T cell-mediated responses toward oxazolone, keyhol lympet hemocyanin, Listeria soluble protein and purified protein derivatives . The immune basis for these phenomena is poorly understood . Based on a distinctive profile of cytokine production, data accumulated thus far have revealed modulatory effects for estrogen on the TH1-type and TH2-type cells, which represent two polarized forms of the effector specific immune response . Recent evidence indicates that estrogens inhibit the production of TH1 proinflammatory cytokines, such as IL-12, TNF-alpha and IFN-gamma, whereas they stimulate the production of TH2 anti-inflammatory cytokines, such as IL-10, IL-4, and TGF-beta . This can explain why estrogen suppresses and potentiates TH1- and TH2-mediated diseases, respectively . We hypothesize that exacerbation or suppression of inflammatory diseases by estrogen is mediated by skewing TH1-type to TH2-type response . This view represents a novel mechanism for the modulatory effect of estrogen on certain inflammatory diseases that can lead to beneficial or detrimental impacts depending on the type of immune involved . Such a concept is valuable when considering the application of combination therapies that include estrogen.

FDA Consum, 2004 Jan-Feb, 38(1), 10 - 1
Preventing Listeria contamination in foods; Rados C; Keeping ready-to-eat foods cold is key to reducing listeriosis, a serious infection in humans . That's one of the conclusions of a recent Food and Drug Administration risk assessment on the relationships between foodborne listeriosis and human health.

Biochem Biophys Res Commun, 2004 Apr 2, 316(2), 379 - 86
Characterization of the calcium-binding sites of Listeria monocytogenes InlB; Marino M et al.; The Listeria monocytogenes protein InlB promotes invasion of mammalian cells through activation of the receptor tyrosine kinase Met . The InlB N-cap, a approximately 40 residue part of the domain that binds Met, was previously observed to bind two calcium ions in a novel and unusually exposed manner . Because subsequent work raised questions about the existence of these calcium-binding sites, we assayed calcium binding in solution to the InlB N-cap . We show that calcium ions are bound with dissociation constants in the low micromolar range at the two identified sites, and that the sites interact with one another . We demonstrate that the calcium ions are not required for structure, and also find that they have no appreciable effect on Met activation or intracellular invasion . Therefore, our results indicate that the sites are fortuitous in InlB, but also suggest that the simple architecture of the sites may be adaptable for protein engineering purposes.

Am J Ophthalmol, 2004 Mar, 137(3), 579 - 81
Listeria monocytogenes-induced endogenous endophthalmitis: bioultrasonic findings; Mendez-Hernandez C et al.; PURPOSE: To report bioultrasonic findings in Listeria monocytogenes-induced endophthalmitis (LMIE) that have not been described previously . DESIGN: Interventional case report . METHODS: To rule out intraocular tumor, ultrasound biomicroscopy was performed in a patient referred for a 2-day history of uveitis with elevated intraocular pressure, dark hypopyon, and pigment dispersion in the anterior chamber . RESULTS: Ultrasound biomicroscopy examination showed increased iris thickness with rarefaction of tissue and irregular echogenicity as well as iris pigment epithelial detachment . A small choroidal detachment was also detected . The anterior chamber and vitreous sample confirmed the LMIE diagnosis . CONCLUSIONS: The detection of both pigment epithelial detachment and changes in the iris tissue could explain why black hypopyon frequently develops in LMIE with significant pigment dispersion in some cases.

FEBS Lett, 2004 Mar 12, 561(1-3), 99 - 104
The interplay between classical and alternative isoprenoid biosynthesis controls gammadelta T cell bioactivity of Listeria monocytogenes; Begley M et al.; Isoprenoids are synthesised either through the classical, mevalonate pathway, or the alternative, non-mevalonate, 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway . The latter is found in many microbial pathogens and proceeds via (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), a potent activator of human Vgamma9/Vdelta2 T cells . Listeria monocytogenes is the only pathogenic bacterium known to contain both pathways concurrently . Strategic gene knockouts demonstrate that either pathway is functional but dispensable for viability . Yet, disrupting the mevalonate pathway results in a complementary upregulation of the MEP pathway . Vgamma9/Vdelta2 T cell bioactivity is increased in DeltalytB mutants where HMB-PP accumulation is expected, and lost in DeltagcpE mutants which fail to produce HMB-PP.

Mol Microbiol, 2004 Mar, 51(6), 1601 - 14
Auto, a surface associated autolysin of Listeria monocytogenes required for entry into eukaryotic cells and virulence; Cabanes D et al.; Listeria monocytogenes is an opportunistic food-borne human and animal pathogen . Several surface proteins expressed by this intracellular pathogen are critical for the infectious process . By in silico analysis we compared the surface protein repertories of L . monocytogenes and of the non-pathogenic species Listeria innocua and identified a gene encoding a surface protein of L . monocytogenes absent in L . innocua . This gene that we named aut encodes a protein (Auto) of 572 amino acids containing a signal sequence, a N-terminal autolysin domain and a C-terminal cell wall-anchoring domain made up of four GW modules . We show here that the aut gene is expressed independently of the virulence gene regulator PrfA and encodes a surface protein with an autolytic activity . We provide evidence that Auto is required for entry of L . monocytogenes into cultured non-phagocytic eukaryotic cells . The low invasiveness of an aut deletion mutant correlates with its reduced virulence following intravenous inoculation of mice and oral infection of guinea pigs . During infection, the autolytic activity of Auto may also be critical . Auto appears thus as a novel type of L . monocytogenes virulence factor.

J Obstet Gynaecol Res, 2004 Apr, 30(2), 117 - 9
Maternal listeriosis in pregnancy associated with measles virus infection; Naruse K et al.; A case of maternal listeriosis associated with measles infection is described . Maternal listeriosis without fetal or neonatal involvement is relatively rare . This case was diagnosed early and treated appropriately, and the baby showed no symptoms or signs of feto-maternal infection.

Appl Environ Microbiol, 2004 Mar, 70(3), 1669 - 79
pbp2229-mediated nisin resistance mechanism in Listeria monocytogenes confers cross-protection to class IIa bacteriocins and affects virulence gene expression; Gravesen A et al.; It was previously shown that enhanced nisin resistance in some mutants was associated with increased expression of three genes, pbp2229, hpk1021, and lmo2487, encoding a penicillin-binding protein, a histidine kinase, and a protein of unknown function, respectively . In the present work, we determined the direct role of the three genes in nisin resistance . Interruption of pbp2229 and hpk1021 eliminated the nisin resistance phenotype . Interruption of hpk1021 additionally abolished the increase in pbp2229 expression . The results indicate that this nisin resistance mechanism is caused directly by the increase in pbp2229 expression, which in turn is brought about by the increase in hpk1021 expression . We also found a degree of cross-protection between nisin and class IIa bacteriocins and investigated possible mechanisms . The expression of virulence genes in one nisin-resistant mutant and two class IIa bacteriocin-resistant mutants of the same wild-type strain was analyzed, and each mutant consistently showed either an increase or a decrease in the expression of virulence genes (prfA-regulated as well as prfA-independent genes) . Although the changes mostly were moderate, the consistency indicates that a mutant-specific change in virulence may occur concomitantly with bacteriocin resistance development.

Appl Environ Microbiol, 2004 Mar, 70(3), 1366 - 77
Quantitative detection of Listeria monocytogenes and Listeria innocua by real-time PCR: assessment of hly, iap, and lin02483 targets and AmpliFluor technology; Rodriguez-Lazaro D et al.; We developed and assessed real-time PCR (RTi-PCR) assays for the detection and quantification of the food-borne pathogen Listeria monocytogenes and the closely related nonpathogenic species L . innocua . The target genes were hly and iap for L . monocytogenes and lin02483 for L . innocua . The assays were 100% specific, as determined with 100 Listeria strains and 45 non-Listeria strains, and highly sensitive, with detection limits of one target molecule in 11 to 56% of the reactions with purified DNA and 3 CFU in 56 to 89% of the reactions with bacterial suspensions . Quantification was possible over a 5-log dynamic range, with a limit of 15 target molecules and R(2) values of >0.996 . There was an excellent correspondence between the predicted and the actual numbers of CFU in the samples (deviations of <23%) . The hly-based assay accurately quantified L . monocytogenes in all of the samples tested . The iap-based assay, in contrast, was unsuitable for quantification purposes, underestimating the bacterial counts by 3 to 4 log units in a significant proportion of the samples due to serovar-related target sequence variability . The combination of the two assays enabled us to classify L . monocytogenes isolates into one of the two major phylogenetic divisions of the species, I and II . We also assessed the new AmpliFluor technology for the quantitative detection of L . monocytogenes by RTi-PCR . The performance of this system was similar to that of the TaqMan system, although the former system was slightly less sensitive (detection limit of 15 molecules in 45% of the reactions) and had a higher quantification limit (60 molecules).

Mol Biol Cell, 2004 May, 15(5), 2164 - 75 Epub 2004 Mar 05.
Listeria monocytogenes actin-based motility varies depending on subcellular location: a kinematic probe for cytoarchitecture; Lacayo CI et al.; Intracellular Listeria monocytogenes actin-based motility is characterized by significant individual variability, which can be influenced by cytoarchitecture . L . monocytogenes was used as a probe to transmit information about structural variation among subcellular domains defined by mitochondrial density . By analyzing the movement of a large population of L . monocytogenes in PtK2 cells, we found that mean speed and trajectory curvature were significantly larger for bacteria moving in mitochondria-containing domains (generally perinuclear) than for bacteria moving in mitochondria-free domains (generally peripheral) . Analysis of bacteria that traversed both mitochondria-containing and mitochondria-free domains revealed that these motile differences were not intrinsic to bacteria themselves . Disruption of mitochondrial respiration did not affect bacterial mean speed, speed persistence, or trajectory curvature . In contrast, microtubule depolymerization lead to decreased mean speed per bacterium and increased mean speed persistence of L . monocytogenes moving in mitochondria-free domains compared with untreated cells . L . monocytogenes were also observed to physically collide with mitochondria and push them away from the bacterial path of motion, causing bacteria to slow down before rapidly resuming their speed . Our results show that subcellular domains along with microtubule depolymerization may influence the actin cytoskeleton to affect L . monocytogenes speed, speed persistence, and trajectory curvature.

Mol Biol Cell, 2004 May, 15(5), 2312 - 23 Epub 2004 Mar 05.
Biophysical parameters influence actin-based movement, trajectory, and initiation in a cell-free system; Cameron LA et al.; Using a biochemically complex cytoplasmic extract to reconstitute actin-based motility of Listeria monocytogenes and polystyrene beads coated with the bacterial protein ActA, we have systematically varied a series of biophysical parameters and examined their effects on initiation of motility, particle speed, speed variability, and path trajectory . Bead size had a profound effect on all aspects of motility, with increasing size causing slower, straighter movement and inhibiting symmetry-breaking . Speed also was reduced by extract dilution, by addition of methylcellulose, and paradoxically by addition of excess skeletal muscle actin, but it was enhanced by addition of nonmuscle (platelet) actin . Large, persistent individual variations in speed were observed for all conditions and their relative magnitude increased with extract dilution, indicating that persistent alterations in particle surface properties may be responsible for intrinsic speed variations . Trajectory curvature was increased for smaller beads and also for particles moving in the presence of methylcellulose or excess skeletal muscle actin . Symmetry breaking and movement initiation occurred by two distinct modes: either stochastic amplification of local variation for small beads in concentrated extracts, or gradual accumulation of strain in the actin gel for large beads in dilute extracts . Neither mode was sufficient to enable spherical particles to break symmetry in the cytoplasm of living cells.

J Immunol, 2004 Mar 15, 172(6), 3725 - 35
Increased dendritic cell numbers impair protective immunity to intracellular bacteria despite augmenting antigen-specific CD8+ T lymphocyte responses; Alaniz RC et al.; Dendritic cells (DCs) reside in tissues, where they function as sentinels, providing an essential link between innate and adaptive immunity . Increasing the numbers of DCs in vivo augments T cell responses, and can cause dramatic CTL-dependent tumor regression . To determine whether greater DC numbers promoted T cell-mediated protection in the context of host defense against intracellular bacteria, we treated mice with Flt3 ligand (Flt3-L) to increase DCs in vivo and challenged them with Listeria monocytogenes . Unexpectedly, after primary challenge with Listeria, the overall control of Listeria infection was impaired in Flt3-L-treated mice, which had greater bacterial burden and mortality than controls . Similar results were obtained when DC numbers were increased by treatment with polyethylene glycol-conjugated GM-CSF rather than Flt3-L and in mice infected with Mycobacterium tuberculosis . Impaired protection was not due to dysfunctional T cell responses, as Flt3-L-treated mice had a greater frequency and absolute number of Ag-specific CD8+ T cells, which produced IFN-gamma, exhibited cytolytic activity, and transferred protection . The increased Listeria burden in Flt3-L-treated mice was preferentially associated with DCs, which were unable to kill Listeria and more resistant to CTL lysis compared with macrophages in vitro . Although we cannot exclude the possibility that other potential effects, in addition to increased numbers of DCs, are shared by Flt3-L and polyethylene glycol-conjugated GM-CSF and contributed to the increase in susceptibility observed in treated mice, these results support the notion that DC numbers must be properly controlled within physiological limits to optimize host defense to intracellular bacterial pathogens.

J Immunol, 2004 Mar 15, 172(6), 3491 - 500
Prolonged antigen presentation, APC-, and CD8+ T cell turnover during mycobacterial infection: comparison with Listeria monocytogenes; van Faassen H et al.; We expressed the CTL epitope of OVA (OVA(257-264)) in an acute (Listeria monocytogenes (LM)-OVA) and a chronic intracellular pathogen (Mycobacterium bovis (BCG)-OVA), to evaluate the kinetics of Ag presentation . LM-OVA proliferated rapidly in vivo, resulting in profound LM-OVA expansion within the first 24 h of infection, culminating in the generation of a potent CD8+ T cell response, which peaked on day 7 but underwent a rapid attrition subsequently . In contrast, BCG-OVA exhibited reduced growth in vivo, resulting in a delayed CD8+ T cell response that increased progressively with time . Relative to LM-OVA, BCG-OVA induced persistently increased numbers of apoptotic (annexin V+) CD8+ T cells . Ag presentation in vivo was evaluated by transferring Thy1.2+ carboxyfluorescein-labeled OT1 transgenic CD8+ T cells into infected Thy1.1+ congeneic recipient mice . LM-OVA induced rapid Ag presentation that was profound in magnitude, with most of the transferred cells getting activated within 4 days and resulting in a massive accumulation of activated donor CD8+ T cells . In contrast, Ag presentation induced by BCG-OVA was delayed, weaker in magnitude, which peaked around the second week of infection and declined to a low level subsequently . Increasing the dose of BCG-OVA while enhancing the magnitude of Ag presentation did not change the kinetics . Furthermore, a higher dose of BCG-OVA also accelerated the attrition of OVA(257-264)-specific CD8+ T cells . Relative to LM-OVA, the dendritic cells in BCG-OVA-infected mice were apoptotic for prolonged periods, suggesting that the rapid death of APCs may limit the magnitude of Ag presentation during chronic stages of mycobacterial infection.

J Immunol, 2004 Mar 15, 172(6), 3385 - 9
Cutting edge: long-lived CD8 memory and protective immunity in the absence of CD40 expression on CD8 T cells; Sun JC et al.; CD8 T cells need CD4 T cells to develop into long-lived, functional memory cells that provide protection against pathogen rechallenge . We investigated whether signaling via CD40 expressed on the CD8 cells themselves is involved in this cooperation . In murine responses to Listeria monocytogenes and lymphocytic choriomeningitis virus, we found no evidence of any requirement for CD40-CD40 ligand interaction at this level . No differences were observed between CD40(-/-) and CD40(+/+) CD8 T cells that had matured in the same environment when comparing their expansion in a primary or secondary response, their contribution to memory, and their ability to enter nonlymphoid tissues such as the liver . Thus, we find no evidence that CD40 ligand-expressing CD4 T cells are required to activate CD40 on CD8 T cells directly for the full differentiation of the cytotoxic T cell response.

J Comp Pathol, 2004 Feb-Apr, 130(2-3), 130 - 6
Suppurative gastritis in BALB/c mice infected with Listeria monocytogenes via the intragastric route; Park JH et al.; Suppurative gastritis was demonstrated in BALB/c mice 3 days after intragastric inoculation with 10(9) organisms of Listeria monocytogenes strain ATCC19113 (serotype 3) . Also tested were four other strains of mice (C3H, C57BL/6, FVB and ICR) and three other strains of L . monocytogenes (HPB 3 {serotype 4b}, HPB 410 {serotype 1/2a} and HPB 503 {serotype 1/2b}) . After inoculation with ATCC19113 the numbers of bacteria found in the stomach wall were greater in C57BL/6 and ICR mice than in C3H and FVB mice; moreover, the gastritis produced in BALB/c and C57BL/6 mice was more severe than that produced in the other mouse strains . The gastritis produced in BALB/c mice with L . monocytogenes HPB 3, HPB 410 and HPB 503 was much more severe than that produced by ATCC19113 . The inflammatory response occurred in the lamina muscularis and mucosa of the fundus . Massive necrosis of the gastric epithelium was observed, and there was oedema in a large part of the mucosal layer of the fundus . In addition, the submucosal layer was apparently expanded due to oedema, and in the cardia, the mucosal layer had become thin and flattened . Immunohistochemically, a polyclonal antibody against Listeria spp . produced labelling in areas of the gastric mucosa in which there was an inflammatory response and gastric epithelial necrosis.

J Mol Biol, 2004 Mar 19, 337(2), 453 - 61
Folding and stability of the leucine-rich repeat domain of internalin B from Listeri monocytogenes; Freiberg A et al.; Internalin B (InlB), a surface protein of the human pathogen Listeria monocytogenes, promotes invasion into various host cell types by inducing phagocytosis of the entire bacterium . The N-terminal half of InlB (residues 36-321, InlB321), which is sufficient for this process, contains a central leucine-rich repeat (LRR) domain that is flanked by a small alpha-helical cap and an immunoglobulin (Ig)-like domain . Here we investigated the spectroscopic properties, stability and folding of InlB321 and of a shorter variant lacking the Ig-like domain (InlB248) . The circular dichroism spectra of both protein variants in the far ultraviolet region are very similar, with a characteristic minimum found at approximately 200 nm, possibly resulting from the high 3(10)-helical content in the LRR domain . Upon addition of chemical denaturants, both variants unfold in single transitions with unusually high cooperativity that are fully reversible and best described by two-state equilibria . The free energies of GdmCl-induced unfolding determined from transitions at 20 degrees C are 9.9(+/-0.8)kcal/mol for InlB321 and 5.4(+/-0.4)kcal/mol for InlB248 . InlB321 is also more stable against thermal denaturation, as observed by scanning calorimetry . This suggests, that the Ig-like domain, which presumably does not directly interact with the host cell receptor during bacterial invasion, plays a critical role for the in vivo stability of InlB.

DNA Cell Biol, 2004 Feb, 23(2), 93 - 106
Cytotoxic T-lymphocyte-, and helper T-lymphocyte-oriented DNA vaccination; Nagata T et al.; DNA vaccines have advantages over other types of vaccines in that they can induce strong cellular immune responses, namely cytotoxic T lymphocytes (CTL) and helper T lymphocytes (Th) . DNA vaccines are therefore considered a promising alternative to attenuated live vaccines in the field of infectious diseases . So far, various DNA vaccines have been generated and tried to induce a particular cellular immune response by virtue of recombinant DNA technology . DNA vaccines have been designed for efficient transcription and translation of target genes by a variety of strategies . Also, various DNA vaccine strategies for induction of specific CTL and Th have been reported by taking into consideration antigen presentation pathways and the strategies have been shown to be effective to elicit particular T-cell responses . In this paper, we have reviewed these strategies, including our study on epitope-specific T-cell induction by DNA vaccination against Listeria monocytogenes infection . From this review, it has been surmised that, to induce strong immune responses by DNA vaccines, the immunization route and the immunization regimen, such as heterologous "prime-boost" regimen, should also be considered.

Int J Food Microbiol, 2004 Mar 1, 91(2), 167 - 74
Incidence of Listeria monocytogenes in two milk processing environments, and assessment of Listeria monocytogenes blood agar for isolation; Kells J et al.; A year-long survey of two Northern Ireland milk processing plants for Listeria monocytogenes was carried out . Sample sites included the milk processing environment (walls, floors, drains, and steps), processing equipment, raw and pasteurised milk . The FDA listeria-selective enrichment procedure was used to process samples and an additional agar medium, L . monocytogenes Blood Agar (LMBA), was utilized as part of the isolation procedure in order to compare its performance to that of the recommended Oxford and Palcam agars . LMBA proved to be a very useful tool and was able to detect L . monocytogenes from 94.1% of sites compared to the 76.5% and 79.4% detection rate displayed by Oxford and Palcam agars, respectively . The overall incidence of listeria on equipment was 18.8% (6.3% L . monocytogenes), in the environment was 54.7% (40.6% L . monocytogenes) and in raw milk 44.4% (22.2% L . monocytogenes) . On one occasion, L . welshimeri was isolated from pasteurised milk, probably demonstrating post-pasteurisation contamination of product . The main environmental sources of L . monocytogenes were considered to be a floor drain and stainless steel steps.

Int J Food Microbiol, 2004 Mar 1, 91(2), 119 - 27
A contribution to the improvement of Listeria monocytogenes enumeration in cold-smoked salmon; Besse NG et al.; For the enumeration of Listeria monocytogenes in food, a sensitive enumeration method based on membrane filtration followed by transfer of the filter to a selective medium has been developed . This study was carried out with cold-smoked salmon, a product likely to be contaminated with L . monocytogenes . The operating protocol utilizes three filtration runs in parallel (5, 15 and 30 ml) of a 1 in 10 dilution of the salmon suspension through 0.45-microm pore-size cellulose ester membranes, and then culture of the filters on Aloa agar (AES Laboratoires, Combourg, France) . The results obtained with the technique were compared with those from the reference EN ISO 11290-2 method and found to provide more precise results in the enumeration of L . monocytogenes from both artificially and naturally contaminated cold-smoked salmon . Moreover, for several samples contaminated at low levels, L . monocytogenes could be recovered only by the filtration method . The examination of increasing volumes of salmon suspension enabled readable results to be obtained for all levels of L . monocytogenes and competitive microflora investigated . In most cases, the optimised operating protocol enabled 5.1 g of salmon to be examined, instead of 0.01-0.1 g with the reference EN ISO 11290-2 method, thus improving the sensitivity of the method.

Int J Food Microbiol, 2004 Mar 15, 91(3), 297 - 304
Use of PCR primers derived from a putative transcriptional regulator gene for species-specific determination of Listeria monocytogenes; Liu D et al.; Listeria monocytogenes is an opportunistic bacterial pathogen that has accounted for an important portion of human foodborne diseases worldwide . In this study, through comparative analysis of L . innocua and L . monocytogenes genomic sequences, we selected a L . monocytogenes specific gene (lmo0733) that has the potential for specific detection of L . monocytogenes . Using PCR primers (lmo0733F and lmo0733R) derived from this gene, a specific fragment of 453 bp was amplified only from genomic DNA of L . monocytogenes strains . PCR products from other Listeria species as well as other Gram-positive and -negative species were not detectable, confirming the specificity of this assay . Thus, the PCR test employing primers lmo0733F and lmo0733R represents an additional tool in the diagnostic arsenal for rapid, sensitive and specific detection and identification of human infections due to L . monocytogenes.

Cell Motil Cytoskeleton, 2004 May, 58(1), 17 - 29
Palladin is a novel binding partner for Ena/VASP family members; Boukhelifa M et al.; Palladin is an actin-associated protein that contains proline-rich motifs within its amino-terminal sequence that are similar to motifs found in zyxin, vinculin, and the Listeria protein ActA . These motifs are known to be potential binding sites for the Vasodilator-Stimulated Phosphoprotein (VASP) . Here, we demonstrate that palladin is an additional direct binding partner for VASP, by using co-immunoprecipitation and blot overlay techniques with both endogenous palladin and recombinant myc-tagged palladin . These results show that VASP binds to full-length palladin and also to the amino-terminal half of palladin, where the polyproline motifs are located . Using a synthetic peptide array, two discrete binding sites for VASP were identified within palladin's proline-rich amino-terminal domain . Using double-label immunofluorescence staining of fully-spread and actively-spreading fibroblasts, the extent of co-localization of palladin and VASP was explored . These proteins were found to strongly co-localize along stress fibers, and partially co-localize in focal adhesions, lamellipodia, and focal complexes . These results suggest that the recently described actin-associated protein palladin may play an important role in recruiting VASP to sites of actin filament growth, anchorage, and crosslinking .

Heart Lung, 2004 Jan-Feb, 33(1), 61 - 4
Listeria monocytogenes encephalitis mimicking West Nile encephalitis; Cunha BA et al.; We present a case of a 50-year-old man who presented to Winthrop-University Hospital in the midst of the 2002 West Nile encephalitis (WNE) outbreak with the cardinal clinical findings of WNE, ie, fever, encephalopathy, weakness, and muscle tremors . During the summer of 2002, several cases of aseptic meningitis/viral encephalitis were admitted to our emergency room weekly . In addition, cases of WNE were being admitted at the same time . During this period we had 3 cases of WNE . Our patient presented with the clinical findings of WNE . However, laboratory and radiologic findings suggested the possibility of Listeria monocytogenes encephalitis . The cerebrospinal fluid findings included red blood cells, which, in the absence of a traumatic tap or HSV encephalitis, argue against the diagnosis of WNE but are consistent with L . monocytogenes encephalitis . Computed tomography scan showed communicating hydrocephalus, which also suggests the possibility of L . monocytogenes and argued against the diagnosis of WNE . Clinicians should be vigilant for the mimics of WNE in geographical areas where WNE outbreaks are occurring.

Mol Microbiol, 2004 Mar, 51(5), 1483 - 92
Listeria monocytogenes virulence proteins induce surface expression of Fas ligand on T lymphocytes; Zenewicz LA et al.; Virulence factors secreted by Listeria monocytogenes are known to interfere with host cellular signalling pathways . We investigated whether L . monocytogenes modulates T-cell receptor signalling by examining surface expression of proteins known to be upregulated on activated T cells . In vitro culture of murine splenocytes with L . monocytogenes resulted in a specific and dose-dependent upregulation of Fas ligand (FasL) . Induction of FasL expression was also observed for pathogenic Listeria ivanovii but not for non-pathogenic Listeria innocua, indicating involvement of Listeria virulence protein(s) . Examination of L . monocytogenes strains deficient in different virulence genes demonstrated that FasL upregulation was dependent on the expression of two secreted proteins: listeriolysin O (LLO) and phosphatidylcholine-preferring phospholipase C (PC-PLC) . Treatment of cells with purified proteins demonstrated that LLO was sufficient for inducing FasL, while PC-PLC synergized with LLO for the induction of FasL expression . FasL-expressing cells induced by L . monocytogenes were capable of killing Fas-expressing target cells . Furthermore, L . monocytogenes infection results in upregulation of FasL on T cells in mice . These results describe a novel function for LLO and PC-PLC and suggest that L . monocytogenes may use these virulence factors to modulate the host immune response.

J Immunol, 2004 Mar 1, 172(5), 3167 - 72
Depletion of CD4+ T cells during immunization with nonviable Listeria monocytogenes causes enhanced CD8+ T cell-mediated protection against listeriosis; Kursar M et al.; Immunization of mice with nonviable Listeria monocytogenes generates an insufficient CD8(+) T cell response and consequently only limited protection against subsequent L . monocytogenes infection . We have recently demonstrated that depletion of regulatory CD4(+) T cells during immunization significantly enhances CD8(+) T cell responses . In the present study, we determined the impact of CD4(+) T cell depletion on the CD8(+) T cell response against heat-killed LISTERIA: Treatment of mice with anti-CD4 mAb during boost immunization with heat-killed Listeria significantly increased numbers of Listeria-specific CD8(+) T cells and improved protection against subsequent infection with L . monocytogenes . During challenge infection, numbers of Listeria-specific CD8(+) T cells were enhanced, and these cells expressed effector functions in terms of IFN-gamma production . In summary, we demonstrate that combining nonviable L . monocytogenes vaccination and CD4(+) T cell depletion improves generation of long-lasting and functional Listeria-specific CD8(+) memory T cells.

Int Immunol, 2004 Mar, 16(3), 415 - 21
Protective T cell response against intracellular pathogens in the absence of Toll-like receptor signaling via myeloid differentiation factor 88; Kursar M et al.; Toll-like receptors (TLR) have been indicated as germline-encoded receptors for sensing a variety of pathogens . Although the role of TLR in innate immunity is beyond question, their function in acquired immunity, in particular in T cell immunity, is less clear . Here, we used experimental Listeria monocytogenes infection of mice to analyze requirements for TLR2, TLR4 and the central TLR adaptor protein myeloid differentiation factor 88 (MyD88) in the generation of specific T cell responses . We demonstrate that following L . monocytogenes infection, mice deficient in TLR2, TLR4 and MyD88 can generate Listeria-specific CD8+ and CD4+ Th1 responses . These T cell responses are sufficient to control secondary infection with a high dose of L . monocytogenes even in the absence of TLR signaling via MyD88 . Thus, TLR2-, TLR4- and MyD88-dependent signals are not essential for the generation of CD4+ Th1 and CD8+ T cells, and T cells can protect mice against infection in the absence of these signals.

Front Biosci, 2004 May 01, 9, 1294 - 310
ENA/VASP proteins: multifunctional regulators of actin cytoskeleton dynamics; Sechi AS et al.; The spatial and temporal regulation of the actin cytoskeleton is fundamental to several cellular processes as diverse as cell motility and immune responses . At the molecular level, the remodelling of the actin cytoskeleton depends on two key events: actin filament nucleation and elongation . Seminal studies on the actin-based intracellular motility of the bacterial pathogen Listeria monocytogenes have been instrumental for the characterisation of a class of actin filament elongating factors, the proteins of the Ena/VASP family . Ena/VASP proteins enhance actin filament elongation via the recruitment of profilin:actin complexes to sites of active actin remodelling such as the tips of spreading lamellipodia and the surface of intracellular Listeria . Moreover, Ena/VASP proteins not only enhance actin filament elongation but also influence the activity of the Arp2/3 complex and counteract the inhibition of actin polymerisation by capping proteins . These findings, taken together with the observation that Ena/VASP proteins can influence actin filament architecture by affecting the actin filament branching activity of the Arp2/3 complex, define Ena/VASP proteins as multifunctional organisers of the actin cytoskeleton.

Rev Argent Microbiol, 2003 Oct-Dec, 35(4), 224 - 7
{Monitoring of a HACCP (Hazard Analysis Critical Control Point) plan for Listeria monocytogenes control}; Mengoni GB et al.; The monitoring of a HACCP (Hazard Analysis Critical Control Point) plan for the Listeria monocytogenes control in the cooked and frozen meat section of a thermo-processing meat plant was evaluated . Seventy "non-product-contact" surface samples and fourteen finished product samples were examined . Thirty eight positive sites for the presence of Listeria sp . were obtained . Twenty-two isolates were identified as L . monocytogenes, two as L . seeligeri and fourteen as L . innocua . Non isolates were obtained from finished product samples . The detection of L . monocytogenes in cooked and frozen meat section environment showed the need for the HACCP plan to eliminate or prevent product contamination in the post-thermal step.

An Med Interna, 2004 Feb, 21(2), 75 - 8
{Listeriosis in the adult . Revision of 10 cases}; Arias Miranda IM et al.; Listeria monocytogenes is still a very rare opportunist infection in immunosuppressive patients . The clinical-epidemiological and therapeutic characteristics in 10 patients with infection produced by LM are reported--four of them had primary bacteriemia, three patients had a meningeal involvement, there were two patients with spontaneous bacterial peritonitis and one suffered from abdominal access . All of the patients had underlying disorders favouring the infection . Sepsis and meningeal syndrome were the most common presenting forms . Ampicillin was the most used antibiotic . The overall mortality was 40%.

Mol Cell Biochem, 2004 Jan, 255(1-2), 257 - 65
Metal composition and solubility determine lung toxicity induced by residual oil fly ash collected from different sites within a power plant; Antonini JM et al.; Residual oil fly ash (ROFA) is a particulate pollutant comprised of soluble and insoluble metals and is produced by the combustion of fossil fuels . The objective was to examine the pulmonary responses to chemically distinct ROFA samples collected from either a precipitator or air heater within the same power plant . The collected ROFA samples were suspended in saline (total sample), incubated for 24 h at 37 degrees C, centrifuged, separated into soluble and insoluble fractions, and the metal composition was determined . In addition, electron spin resonance (ESR) was used to detect short-lived free radical intermediates produced by the ROFA samples and the different fractions . On day 0, Male Sprague-Dawley rats were intratracheally instilled with saline (vehicle control) or the ROFA samples (1 mg/100 g body wt) . At day 1, bronchoalveolar lavage was performed, and lung inflammation was assessed . On day 3, additional rats that had been treated with ROFA were intratracheally inoculated with 5 x 10(5) Listeria monocytogenes, and pulmonary bacterial clearance was measured at days 6, 8, and 10 . The precipitator ROFA was found to be more soluble and acidic with a significantly greater mass of each metal compared with the air heater ROFA . A prominent hydroxyl radical signal was measured for the total and soluble precipitator ROFA after the addition of H2O2, whereas the air heater ROFA and its fractions did not produce a signal . Precipitator ROFA induced a greater inflammatory response than air heater ROFA illustrated by a significant elevation in lung neutrophils . In addition, pulmonary clearance of L . monocytogenes was greatly diminished in the rats treated with the soluble and total precipitator ROFA samples . None of the air heater ROFA samples had an effect on lung bacterial clearance . In conclusion, precipitator ROFA, particularly the soluble fraction, generated a metal-dependent hydroxyl radical as measured by ESR and was shown to cause more inflammation and result in reduced lung defense against infection compared with air heater ROFA . These results are most likely due to differences in metal composition and solubility of the ROFA samples.

Ann Ig, 2003 Sep-Oct, 15(5), 575 - 81
{Lysteria monocytogenes in samples of swines sent to the human alimentary chain}; Adorisio E et al.; Animal food-stuffs are known to be potential vehicles of Listeria monocytogenes . The contamination can be caused from processing or enviromental sources and from infected animals . This hypothesis has been checked in the present work . The authors found that 13.2% of 189 swines were carriers of Listeria monocytogenes, the microrganism was isolated from salivary glands, mesenterial gangles and tonsils . The authors suggest some preventive intervention to reduce both the environmental circulation of Listeria monocytogenes and the human risk of infection.

J Food Prot, 2004 Feb, 67(2), 383 - 6
Thermal inactivation of Listeria innocua in salmon (Oncorhynchus keta) caviar using conventional glass and novel aluminum thermal-death-time tubes; Al-Holy M et al.; Differences in the come-up times and thermal inactivation parameters of Listeria innocua in salmon (Oncorhynchus keta) caviar containing 2.5% salt using conventional thermal-death-time (TDT) glass tubes and a novel aluminum tube were tested and compared . Generally, the come-up times and decimal reduction times (D-values) were shorter and the change in temperature required to change the D-value (z-value) was longer in the aluminum than in the glass tubes . The D-values at 60, 63, and 65 degrees C for the aluminum TDT tubes were 2.97, 0.77, and 0.40 min, respectively, and for the glass TDT tubes, these values were 3.55, 0.84, and 0.41 min . The z-values were 5.7 degrees C in the aluminum and 5.3 degrees C in the glass . Because of the shorter come-up time, the aluminum TDT tubes may provide a more precise measurement of microbial thermal inactivation than the glass TDT tubes, particularly for viscous materials, solid foods, and foods containing particulate matter.

J Food Prot, 2004 Feb, 67(2), 378 - 82
Survival of Listeria monocytogenes in vanilla-flavored soy and dairy products stored at 8 degrees C; Tipparaju S et al.; The survival of Listeria monocytogenes V37 in vanilla-flavored yogurt (low-fat and nonfat) and soy milk (low-fat and Plus) stored at 8 degrees C for 31 days was investigated . Commercial samples of yogurt and soy milk were used . These samples were inoculated with either 10(4) or 10(7) CFU of L . monocytogenes per ml . Sampling was carried out every 3 to 4 days initially and was then carried out weekly, for a total storage time of 31 days . Each time a sample was collected, the pH of the sample was measured . After 31 days, low-fat plain, low-fat vanilla, and nonfat plain yogurt samples inoculated with 10(4) CFU/ml showed 2.5-log reductions in viable cell populations, and nonfat vanilla yogurt showed a 3.5-log reduction . For yogurt inoculated with 10(7) CFU/ml, reductions of 2.5 log CFU/ml were observed for plain low-fat and nonfat yogurts, and reductions of 5 log CFU/ml were observed for vanilla-flavored low-fat and nonfat yogurts . In vanilla-flavored and plain low-fat and Plus soy milk samples, cell counts increased from 10(4) and 10(7) CFU/ml to 10(9) CFU/ml at 7 and 3 days of storage, respectively, at 8 degrees C . Coagulation in soy milk samples was observed when the cell population reached 10(9) CFU/ml . In soy milk, the L . monocytogenes population did not change for up to 31 days . Vanillin had an inhibitory effect on L . monocytogenes in yogurt but not in soy milk.

J Food Prot, 2004 Feb, 67(2), 328 - 41
Tracking of Listeria monocytogenes in smoked fish processing plants; Thimothe J et al.; Four smoked fish processing plants were used as a model system to characterize Listeria monocytogenes contamination patterns in ready-to-eat food production environments . Each of the four plants was sampled monthly for approximately 1 year . At each sampling, four to six raw fish and four to six finished product samples were collected from corresponding lots . In addition, 12 to 14 environmental sponge samples were collected several hours after the start of production at sites selected as being likely contamination sources . A total of 234 raw fish, 233 finished products, and 553 environmental samples were tested . Presumptive Listeria spp . were isolated from 16.7% of the raw fish samples, 9.0% of the finished product samples, and 27.3% of the environmental samples . L . monocytogenes was isolated from 3.8% of the raw fish samples (0 to 10%, depending on the plant), 1.3% of the finished product samples (0 to 3.3%), and 12.8% of the environmental samples (0 to 29.8%) . Among the environmental samples, L . monocytogenes was found in 23.7% of the samples taken from drains, 4.8% of the samples taken from food contact surfaces, 10.4% of the samples taken from employee contact surfaces (aprons, hands, and door handles), and 12.3% of the samples taken from other nonfood contact surfaces . Listeria spp . were isolated from environmental samples in each of the four plants, whereas L . monocytogenes was not found in any of the environmental samples from one plant . Overall, the L . monocytogenes prevalence in the plant environment showed a statistically significant (P < 0.0001) positive relationship with the prevalence of this organism in finished product samples . Automated EcoRI ribotyping differentiated 15 ribotypes among the 83 L . monocytogenes isolates . For each of the three plants with L . monocytogenes-positive environmental samples, one or two ribotypes seemed to persist in the plant environment during the study period . In one plant, a specific L . monocytogenes ribotype represented 44% of the L . monocytogenes-positive environmental samples and was also responsible for one of the two finished product positives found in this plant . In another plant, a specific L . monocytogenes ribotype persisted in the raw fish handling area . However, this ribotype was never isolated from the finished product area in this plant, indicating that this operation has achieved effective separation of raw and finished product areas . Molecular subtyping methods can help identify plant-specific L . monocytogenes contamination routes and thus provide the knowledge needed to implement improved L . monocytogenes control strategies.

J Food Prot, 2004 Feb, 67(2), 316 - 21
Changes in heat resistance resulting from pH and nutritional shifts of acid-adapted and non-acid-adapted Listeria monocytogenes Scott A; Bayles DO; Stationary-phase Listeria monocytogenes cells that were either pH dependent acid adapted or not acid adapted were heat challenged at 60 degrees C in a two-level full factorial design for three variables . The three variables and the levels consisted of tryptic soy broth (TSB) and sterile cell-free culture supernatant (sterile TSB), the presence and absence of 1% added glucose, and pH 4.8 and pH 7 . Non-acid-adapted cells were most heat resistant when challenged in TSB (mean decimal reduction times at 60 degrees C: D60 = 1.16 min) . In the absence of added glucose, non-acid-adapted cells had similar D60-values for inactivations at pH 4.8 and pH 7; however, the presence of glucose caused non-acid-adapted cells challenged at pH 4.8 to be more heat sensitive (D60 = 0.65 min) than those inactivated at pH 7 (D60 = 1.03 min), indicating an interaction between glucose and pH . Overall, the significantly decreased heat resistance of the acid-adapted cells was due to the presence of glucose (D60 = 0.78 min without glucose, D60 = 0.59 min with glucose) . Acid-adapted cells heat challenged in TSB had similar D60-values for inactivations at pH 4.8 and pH 7; however, acid-adapted cells in sterile TSB challenged at pH 4.8 (D60 = 0.52 min) had significantly lower heat resistance than did cells challenged at pH 7 (D60 = 0.76 min), indicating an interaction between the medium and pH . The L . monocytogenes survivor data were modeled to extract information on the frequency distribution of heat resistance within heat-challenged populations, and the frequency distribution characteristics of mean, mode, and variance were compared among treatment conditions . Significant differences in the frequency distribution data were compared with the D60-values . These data indicated that the presence and level of cross-protection is highly dependent on the physiological state of the cells and nutrient availability at the time of heat challenge . Such conditions should be considered to ensure that stressed pathogens in foods are destroyed or inactivated.

Int J Food Microbiol, 2004 Feb 15, 91(1), 63 - 72
Influence of the sigB gene on the cold stress survival and subsequent recovery of two Listeria monocytogenes serotypes; Moorhead SM et al.; The influence of serotype and the role of the sigB gene of Listeria monocytogenes during the survival and recovery on different substrates were determined . Wild-type and sigB mutants of two serotypes of L . monocytogenes were inoculated into buffer and onto beef steaks, and incubated at 4 degrees C for 8 weeks during which samples were removed and Listeria numbers determined . Growth kinetics of stationary phase wild-type and sigB mutant cells were compared, without prechilling and after prechilling at 4 degrees C . The two serotypes had similar survival capabilities under the conditions examined, and the sigB gene was influential in survival of chill stress, but was dependent upon additional nutritional factors . Prechilling cells prior to growth extended the lag phase of both strains, and this lag phase extension was compounded by the absence of a functional sigB gene . In conclusion, the sigB gene is involved in the survival and recovery from chill stress by the two serotypes tested . Additional factors such as previous growth conditions, nutritional requirements and serotype susceptibility are also contributory . This study adds relevant information regarding the influence of the sigB gene, in conjunction with the historical growth conditions and serotype differences . Understanding the significance of these factors may be useful in creating improved recovery systems for the detection of L . monocytogenes from at-risk foods.

Int J Food Microbiol, 2004 Feb 15, 91(1), 13 - 8
Effects of chlorine and pH on efficacy of electrolyzed water for inactivating Escherichia coli O157:H7 and Listeria monocytogenes; Park H et al.; The effects of chlorine and pH on the bactericidal activity of electrolyzed (EO) water were examined against Escherichia coli O157:H7 and Listeria monocytogenes . The residual chlorine concentration of EO water ranged from 0.1 to 5.0 mg/l, and the pH effect was examined at pH 3.0, 5.0, and 7.0 . The bactericidal activity of EO water increased with residual chlorine concentration for both pathogens, and complete inactivation was achieved at residual chlorine levels equal to or higher than 1.0 mg/l . The results showed that both pathogens are very sensitive to chlorine, and residual chlorine level of EO water should be maintained at 1.0 mg/l or higher for practical applications . For each residual chlorine level, bactericidal activity of EO water increased with decreasing pH for both pathogens . However, with sufficient residual chlorine (greater than 2 mg/l), EO water can be applied in a pH range between 2.6 (original pH of EO water) and 7.0 while still achieving complete inactivation of E . coli O157:H7 and L . monocytogenes.

Biochemistry, 2004 Feb 24, 43(7), 1939 - 49
A ratiometric expressible FRET sensor for phosphoinositides displays a signal change in highly dynamic membrane structures in fibroblasts; Cicchetti G et al.; Phosphoinositides are important signal transduction intermediates in cell growth, survival, and motility . We have invented a fluorescence sensor for polyphosphorylated phosphoinositides based on a peptide derived from the Listeria protein ActA that undergoes a random coil to helix transition upon lipid binding . The sensor, termed CAY, is a fusion protein of cyan and yellow fluorescent proteins flanking the peptide at its N- and C-termini, respectively . CAY displays fluorescence resonance energy transfer in vitro in the absence of phosphorylated phosphoinositides, and this energy transfer is lost upon interaction with these phospholipids . These results demonstrate that a short peptide undergoing a coil to helix transition can be sufficient for the engineering of a FRET-based biosensor . CAY is predominantly localized to the cytoplasm in fibroblasts expressing the sensor but shows loss of fluorescence resonance energy transfer in regions of active actin dynamics such as ruffles that have previously been demonstrated to contain high levels of phosphoinositides.

Infez Med, 1997, 5(2), 118 - 24
{A case report of Listeria monocytogenes infection in a patient with AIDS . Efficacy of treatment with cotrimoxazole associated with rHuG-CSF (filgrastim)}; Manfredi R et al.; Listeriosis is an emerging opportunistic infection in the immunocompromised host . A case of sepsis due to Listeria monocytogenes in a patient with advanced HIV infection and severe neutropenia, treated for an underlying non-Hodgkin's lymphoma, is described . Therapy with cotrimoxazole associated with rHuG-CSF (filgrastim) led to a rapidly favourable clinical and microbiological outcome, and to the correction of concurrent neutropenia . The case report is discussed according to a literature review of all cases of listeriosis reported until now in the setting of HIV infection and AIDS . In particular, the role of both cotrimoxazole and rHuG-CSF adjunct in the treatment of listeriosis in the immunocompromised patient is focused.

Lett Appl Microbiol, 2004, 38(3), 181 - 4
A comparison of the traditional method of counting viable cells and a quick microplate method for monitoring the growth characteristics of Listeria monocytogenes; Horakova K et al.; AIMS: To determine: (i) the growth parameters (specific growth rate, lag time, asymptotic amount of growth, generation time and time for maximum growth rate) of Listeria monocytogenes in different broths by standard cultivation methods and (ii) whether a microplate method in conjunction with a standard nondedicated plate reader could be adapted to routine assay . METHODS AND RESULTS: Growth curves were determined from cell numbers in a standard tube method at 2 h intervals by serial dilution and plating, and in a microplate method by absorbance measurements . Growth curves were fitted with a modified Gompertz function . CONCLUSIONS: The microplate method was similar to the standard cultivation methods in accuracy, required less chemical reagents, and considerably reduced the time required for analyses . This work also illustrates that growth characteristics of bacteria are not necessarily constant, and depend on the methodology used . SIGNIFICANCE AND IMPACT OF THE STUDY: It is not the intended purpose of this paper to present all the data for the media tested but instead to illustrate the success of the microplate method for studying growth kinetics compared to a standard cultivation method and system precision . The method will be of considerable benefit to laboratories unable to afford dedicated workstations.

Microbiology, 2004 Feb, 150(Pt 2), 335 - 40
Physiological implications of class IIa bacteriocin resistance in Listeria monocytogenes strains; Vadyvaloo V et al.; High-level resistance to class IIa bacteriocins has been directly associated with the absent EIIAB(Man) (MptA) subunit of the mannose-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS) (EIIt(MAN)) in Listeria monocytogenes strains . Class IIa bacteriocin-resistant strains used in this study were a spontaneous resistant, L . monocytogenes B73-MR1, and a defined mutant, L . monocytogenes EGDe-mptA . Both strains were previously reported to have the EIIAB(Man) PTS component missing . This study shows that these class IIa bacteriocin-resistant strains have significantly decreased specific growth and glucose consumption rates, but they also have a significantly higher growth yield than their corresponding wild-type strains, L . monocytogenes B73 and L . monocytogenes EGDe, respectively . In the presence of glucose, the strains showed a shift from a predominantly lactic-acid to a mixed-acid fermentation . It is here proposed that elimination of the EIIAB(Man) in the resistant strains has caused a reduced glucose consumption rate and a reduced specific growth rate . The lower glucose consumption rate can be correlated to a shift in metabolism to a more efficient pathway with respect to ATP production per glucose, leading to a higher biomass yield . Thus, the cost involved in obtaining bacteriocin resistance, i.e . losing substrate transport capacity leading to a lower growth rate, is compensated for by a higher biomass yield.

Microbiology, 2004 Feb, 150(Pt 2), 321 - 33
Deregulation of Listeria monocytogenes virulence gene expression by two distinct and semi-independent pathways; Milenbachs Lukowiak A et al.; Expression of the major virulence cluster in Listeria monocytogenes is positively regulated by the transcription factor PrfA and is influenced by several environmental factors, including the presence of readily metabolized carbohydrates such as cellobiose and glucose . Although little is understood about the mechanisms through which environmental factors influence expression of the PrfA regulon, evidence for structural and functional similarities of PrfA to the CRP-FNR family of regulatory proteins suggests the possibility that PrfA activity could be modulated by a small molecule ligand . The identity of components of the PrfA-associated regulatory pathway was sought through the isolation of mutants that exhibit high levels of PrfA-controlled gene expression in the presence of cellobiose or glucose . Here are described the properties and preliminary genetic analysis in two different genetic loci, gcr and csr, both unlinked by general transduction to the major virulence cluster . A mutation in gcr deregulates the expression of PrfA-controlled genes in the presence of several repressing sugars and other environmental conditions, a phenotype similar to that of a G145S substitution in PrfA itself . A mutation in the csr locus, within csrA, results in a cellobiose-specific defect in virulence gene regulation . Gene products encoded by the csr locus share homology with proteins involved in the sensing and transport of beta-glucosides in other bacteria . Mutations in both gcr and csr are required for full relief of cellobiose-mediated repression of the PrfA regulon . These results suggest the existence of two semi-independent pathways for cellobiose-mediated repression and further reconcile conflicting reports in previous literature concerning the repressive effects of carbohydrates on virulence gene expression in L . monocytogenes.

Appl Environ Microbiol, 2004 Feb, 70(2), 913 - 20
Multi-virulence-locus sequence typing of Listeria monocytogenes; Zhang W et al.; A multi-virulence-locus sequence typing (MVLST) scheme was developed for subtyping Listeria monocytogenes, and the results obtained using this scheme were compared to those of pulsed-field gel electrophoresis (PFGE) and the published results of other typing methods, including ribotyping (RT) and multilocus sequence typing (MLST) . A set of 28 strains (eight different serotypes and three known genetic lineages) of L . monocytogenes was selected from a strain collection (n > 1,000 strains) to represent the genetic diversity of this species . Internal fragments (ca . 418 to 469 bp) of three virulence genes (prfA, inlB, and inlC) and three virulence-associated genes (dal, lisR, and clpP) were sequenced and analyzed . Multiple DNA sequence alignment identified 10 (prfA), 19 (inlB), 13 (dal), 10 (lisR), 17 (inlC), and 16 (clpP) allelic types and a total of 28 unique sequence types . Comparison of MVLST with automated EcoRI-RT and PFGE with ApaI enzymatic digestion showed that MVLST was able to differentiate strains that were indistinguishable by RT (13 ribotypes; discrimination index = 0.921) or PFGE (22 profiles; discrimination index = 0.970) . Comparison of MVLST with housekeeping-gene-based MLST analysis showed that MVLST provided higher discriminatory power for serotype 1/2a and 4b strains than MLST . Cluster analysis based on the intragenic sequences of the selected virulence genes indicated a strain phylogeny closely related to serotypes and genetic lineages . In conclusion, MVLST may improve the discriminatory power of MLST and provide a convenient tool for studying the local epidemiology of L . monocytogenes.

Science, 2004 Feb 6, 303(5659), 851 - 3
Extracellular replication of Listeria monocytogenes in the murine gall bladder; Hardy J et al.; The bacterium Listeria monocytogenes can cause a life-threatening systemic illness in humans . Despite decades of progress in animal models of listeriosis, much remains unknown about the processes of infection and colonization . Here, we report that L . monocytogenes can replicate in the murine gall bladder and provide evidence that its replication there is extracellular and intraluminal . In vivo bioluminescence imaging was employed to determine the location of the infection over time in live animals, revealing strong signals from the gall bladder over a period of several days, in diseased as well as asymptomatic animals . The data suggest that L . monocytogenes may be carried in the human gall bladder.

Cell Microbiol, 2004 Mar, 6(3), 235 - 42
Characterization of flagellin expression and its role in Listeria monocytogenes infection and immunity; Way SS et al.; Flagellin is the structural component of flagella produced by many pathogenic bacteria and is a potent proinflammatory molecule that mediates these effects through Toll-like receptor (TLR) 5 . In Listeria monocytogenes (LM), flagellin expression is regulated by temperature and has been described as being shut off at 37 degrees C . In this study, we demonstrate that TLR5-mediated cell activation and flagellin expression is maintained at 37 degrees C in some laboratory-adapted strains and in approximately 20% of LM clinical isolates . To determine the role of flagellin in LM infection, a targeted mutation in the structural gene for flagellin (flaA) was generated in a parental LM strain that expressed flagellin under all conditions examined . In vitro studies demonstrated that this deltaflaA mutant was (i) . non-motile; (ii) . not able to activate TLR5-transfected HeLa cells; and (iii) . induced tumour necrosis factor (TNF)-alpha production in approximately 50% fewer CD11b+ cells in splenocytes from normal mice compared with the parental strain . However, there was no significant alteration in virulence of the deltaflaA mutant after either intravenous or oral murine infection . Similarly, there was no difference in the generation of LM-specific CD8 or CD4 T cells after intravenous or oral infection . These data indicate that flagellin is not essential for LM pathogenesis or for the induction of LM-specific adaptive immune responses in normal mice.

Diagn Microbiol Infect Dis, 2004 Jan, 48(1), 63 - 7
Recurrent Listeria monocytogenes aortic graft infection: confirmation of relapse by molecular subtyping; Rohde H et al.; Based on molecular typing methods, we identified a rare case of a recurrent L . monocytogenes infection resulting from an infected aortic prosthesis as detected by 18-F-Fluoro-d-deoxyglucose positron emission tomography (FDG PET) . Our case highlights the usefulness of molecular typing and nuclear imaging methods for understanding L . monocytogenes pathogenesis and epidemiology.

Enferm Infecc Microbiol Clin, 2004 Jan, 22(1), 18 - 21
{Listeriosis in patients with human immunodeficiency virus infection in Spain . Three new cases and literature review}; Guerra J et al.; INTRODUCTION: Listeriosis has been considered infrequent among patients infected with the human immunodeficiency virus (HIV) . Only a limited number of listeriosis cases among HIV-infected patients have been published in Spain, where the prevalence of HIV infection is relatively high . We present our experience in this field and provide a review of the reported cases in our country . METHODS: We report three cases of listeriosis among the 903 HIV-infected patients attended in our service . Age, sex, stage of HIV infection, clinical form of listeriosis, treatment, and prognosis are analyzed in these three patients and in the published cases . RESULTS: Sixteen (76%) of the 21 cases analyzed were males . Ten had central nervous system infection, ten had bacteremia alone or bacteremia associated with another condition, and one had pneumonia . Two patients died (9.5%) and none of them were treated with the antibiotics of choice for listeriosis . CONCLUSION: Although Listeria monocytogenes infection is infrequent in HIV-infected patients (0.33% of patients attended in our service), clinicians should be alert to this possibility, since even the severe clinical forms have a favorable prognosis with proper treatment.

Rev Neurol, 2004 Jan 16-31, 38(2), 143 - 4
{Meningitis by Listeria in children}; del Rio-Camacho G et al.; INTRODUCTION: Listeria monocytogenes is a rare cause of bacterial meningoencephalitis in the non-immunocompromised pediatric population . On occasions, the absence of differential characteristics with other bacteria that cause meningitis delays diagnosis and hence treatment, worsening the prognosis . CASE REPORT: We present a case of a previously healthy, non-immunocompromised teenager who was admitted to hospital with meningitis caused by Listeria . DISCUSSION: We review the literature related to this case, noting the increasing incidence of this microorganism in the etiopathogenesis of meningoencephalitis, reason for which it has to be kept in mind in the differential diagnosis at the time of admission.

Int J Food Microbiol, 2004 Feb 1, 90(3), 349 - 56
Occurrence of Listeria monocytogenes in fresh and processed foods in Navarra (Spain); Vitas AI et al.; The presence of Listeria spp . was investigated in a total of 3685 food samples obtained from different industries and markets of Northern Spain in the last 4 years . The samples analyzed include fresh raw products (meat, milk and poultry) and treated products (cooked and cured meats, frozen vegetables and smoked salmon) . Occurrence of Listeria spp . varied from 8.1% in soft cheese to 76.3% in raw poultry samples . The highest incidence of L . monocytogenes also occurred in raw poultry (36.1% positive samples) . Despite this high incidence of contamination, these kinds of products carry a low risk of listeriosis transmission because of the heat treatment prior to consumption . On the other hand, the ready-to-eat products (RTE) tested in this study showed incidences that could pose serious health problems, taking into account that the storage conditions may allow for rapid growth of the pathogen . It was also found that up to 75.5% of the L . monocytogenes strains isolated in this study belonged to serogroup 1, mainly serotype 1/2a, while the clinical cases observed in Navarra in the same period of time belonged mainly to serotype 4b/4bx.

Int J Food Microbiol, 2004 Feb 1, 90(3), 341 - 7
Characterization of Listeria monocytogenes and Listeria innocua from a vegetable processing plant by RAPD and REA; Aguado V et al.; The incidence of Listeria monocytogenes in a vegetable processing plant was investigated over a 23-month period . Frozen ready-to-eat vegetable samples, well as the plant environment, were sampled . The molecular subtyping techniques, Random Amplified Polymorphic DNA (RAPD) and Restriction Endonuclease Analyses (REA), were performed to help investigate the origin and routes of Listeria dissemination.The low and sporadic incidence of L . monocytogenes made it impossible to establish an epidemiological sequence in the processing plant, though a case of cross-contamination between tomato and ratatouille was detected . Listeria innocua subtyping, however, allowed us to determine the prevalence of several strains in vegetables, and their presence on machinery samples suggested the possibility of cross-contamination during processing.The low incidence of L . monocytogenes indicated that the risk of listeriosis transmission by vegetable consumption is low . On the other hand, the isolation of the same strain of L . innocua in several surveys pointed out the risk of colonisation on surfaces and machinery . The persistence of Listeria spp . is a cause for concern as can lead to future contamination of vegetables processed in the plant and to a possible increased risk for health . Therefore, periodic controls for the presence of Listeria spp . and a further review of the cleaning and disinfection procedures used in frozen vegetable plants are recommended.

Acta Microbiol Immunol Hung, 2003, 50(4), 331 - 7
Increased salt- and nisin-sensitivity of pressure-injured bioluminescent Listeria monocytogenes; Farkas J et al.; Suspensions of a bioluminescent (luxAB) transformant of Listeria monocytogenes in pH 7.0 phosphate buffer were pressurised and the effect of the pressure treatment was monitored by plate counting . When the bacteria were suspended in NaCl- and nisin-free buffer the number of colony forming units (CFU) decreased by 3 and 6 log cycles after 300 MPA for 10 and 30 min, respectively . Supplementing the plating medium with 5% NaCl did not influence the colony forming capacity of non-pressurised cells, however, CFU of residual populations after respective treatments of 300 MPa for 10 and 30 min were reduced by a further 2 and 3.5 log cycles in case of salt containing plates . Nisin-addition to the plating medium caused less than one log unit decrease in the CFU of the non-pressurised population . However, the CFU of 10 min-pressurised sample was 4 log cycles less in the nisin-containing plates than in the nisin-free ones, whereas no colonies were formed in the nisin-containing plates even when 1 ml was inoculated from the originally 10(10) CFU/ml population after 300 MPa for 30 min . The luciferase activities (bioluminescence intensities) decreased concomitant with the reduction of the viable cell counts, however, they were approx . 0.6-0.8 log units less in the presence of 5% NaCl in the pressurised suspension than those expected from the previously established linear correlation between the logarithmic light outputs and the logarithmic viable cell counts.

Eur Radiol . 2004 Jan 29; {Epub ahead of print}
Cranial bacterial infection; Anslow P; Early diagnosis of cranial sepsis is mandatory if morbidity is to be avoided . In the case of structural integrity of the skull, haematogenous spread or extension from adjacent structures, especially the sinuses, are the most common sources of infection . Infections may be limited to compartments by the meninges or spread diffusely . Focal disease includes brain abscess as well as subdural and extradural empyaema . A history or signs of sinus disease should always be sought . Tuberculosis, lyme disease and listeriosis may present specific pathological findings . A series of cases is presented to illustrate the role of imaging in infective disease and to draw attention to diagnostic and management points of which radiologists should be aware.

Lett Appl Microbiol, 2004, 38(2), 151 - 7
Identification of a gene encoding a putative phosphotransferase system enzyme IIBC in Listeria welshimeri and its application for diagnostic PCR; Liu D et al.; AIMS: To identify a Listeria welshimeri-specific gene that can be used for identification of this species by PCR . METHODS AND RESULTS: Through comparative analysis of genomic DNA from Listeria species using dot blot hybridization, an L . welshimeri-specific clone was isolated that contained a gene segment whose translated protein sequence is similar to enzyme IIBC from phosphotransferase systems in other bacteria . Using oligonucleotide primers derived from this L . welshimeri-specific clone, a 608-bp fragment was amplified from L . welshimeri genomic DNA and not from other Listeria species or other Gram-negative and Gram-positive species . CONCLUSION AND SIGNIFICANCE: The PCR employing L . welshimeri-specific primers shows promise as a useful method for differentiating L . welshimeri from other Listeria species and related bacteria.

J Infect Dis, 2004 Feb 1, 189(3), 393 - 401 Epub 2004 Jan 27.
Inefficient replication of Listeria innocua in the cytosol of mammalian cells; Slaghuis J et al.; The efficiency of adherence to, internalization by, and replication in the cytosol of J774 macrophages and HEp-2 epithelial cells was compared between a nonspreading Listeria monocytogenes actA mutant and L . innocua . The studied L . innocua strains were equipped either with listeriolysin alone or with listeriolysin O (LLO) and the recently identified hexose-phosphate transporter of L . monocytogenes . All listerial strains expressed green fluorescent protein (GFP) under the control of the PrfA-dependent actA promoter . GFP expression was observed exclusively in the cytosol of host cells . Escape from the phagosome of LLO-expressing L . innocua strains was as efficient as that from L . monocytogenes . hpt-positive L . innocua showed significantly enhanced adherence to HEp-2 cells, but internalization was only slightly increased, compared with hpt-negative L . innocua . Subsequent replication of L . monocytogenes in the cytosol of the host cells proceeded within the next 6 h in most infected host cells, with a generation time <40 min . L . innocua prfA hly replicated more slowly (with a generation time of 60-90 min), and, in most host cells, bacterial replication stopped after 2-3 rounds of replication . In some cells, bacterial replication did not occur . Twenty-four hours after infection, the majority of J774 cells (>90%) infected with L . monocytogenes actA were dead, whereas most host cells infected with L . innocua were still alive . L . innocua equipped with the prfA, hly, and hpt genes of L . monocytogenes did not show significantly increased cytosolic replication, which indicates that expression of this sugar phosphate uptake system is not sufficient for extensive listerial replication in the cytosol of host cells.

Nat Immunol, 2004 Feb, 5(2), 159 - 68 Epub 2004 Jan 25.
MHC class Ia-restricted memory T cells inhibit expansion of a nonprotective MHC class Ib (H2-M3)-restricted memory response; Hamilton SE et al.; Listeria monocytogenes infection generates major histocompatibility complex (MHC) class Ia-restricted and MHC class Ib-(H2-M3)-restricted effector and memory CD8+ T cells . However, only MHC class Ia-restricted memory cells expand after rechallenge, and it is unknown if MHC class Ib-restricted memory CD8+ T cells generated by vaccination are protective . We show here that H2-M3-restricted memory CD8+ T cells were capable of secondary expansion but, in contrast to primary H2-M3-restricted effector cells, failed to provide protective immunity . In lm-immune mice, MHC class Ia-restricted memory CD8+ T cells prevented the expansion of H2-M3-restricted memory T cell populations by limiting dendritic cell antigen presentation . Thus, protective immunity by H2-M3-restricted T cells is limited to primary infection, indicating that memory MHC class Ia-restricted T cells prevent nonessential immune responses during secondary infection.

Ann Pharmacother, 2004 Jan, 38(1), 58 - 61
Listeria meningitis associated with infliximab; Bowie VL et al.; OBJECTIVE: To report a case of Listeria monocytogenes meningitis in a 73-year-old man receiving infliximab for rheumatoid arthritis . CASE SUMMARY: A 73-year-old white man taking infliximab for rheumatoid arthritis developed listeria meningitis following his second dose . He was receiving other immunosuppressants; however, these remained constant immediately prior to the infection . Diagnosis was confirmed with L . monocytogenes isolated in the cerebrospinal fluid . The patient received 21 days of antibiotic therapy and recovered without any complications . DISCUSSION: L . monocytogenes is a gram-positive, non-spore-forming rod that has been associated with the ingestion of undercooked foods . This organism can cause sepsis or meningitis; however, immunocompromised patients, elderly patients, pregnant women, and neonates appear to be at greater risk for this type of infection . Tumor-necrosis factor-alpha (TNF-alpha) plays an important role in resistance to this type of infection, and listeria infections have been reported in 26 patients receiving TNF-alpha inhibitors . In our patient, the listeria infection occurred following his second course of infliximab, which provides a temporal relationship between the listeria infection and infliximab . However, his underlying rheumatoid arthritis and chronic steroid therapy would also increase his risk for a listeria infection . CONCLUSIONS: The listeria infection in our patient was a possible adverse event of infliximab according to the Naranjo probability scale . Because the majority of listeria infections occur in patients who are immunosuppressed, it would be reasonable to provide education for healthcare professionals on preventing these infections in all patients receiving immunosuppressants, including anti-TNF-alpha therapy . Those at risk due to their underlying health conditions should also be monitored closely.

Infect Immun, 2004 Feb, 72(2), 1072 - 83
New aspects regarding evolution and virulence of Listeria monocytogenes revealed by comparative genomics and DNA arrays; Doumith M et al.; Listeria monocytogenes is a food-borne bacterial pathogen that causes a wide spectrum of diseases, such as meningitis, septicemia, abortion, and gastroenteritis, in humans and animals . Among the 13 L . monocytogenes serovars described, invasive disease is mostly associated with serovar 4b strains . To investigate the genetic diversity of L . monocytogenes strains with different virulence potentials, we partially sequenced an epidemic serovar 4b strain and compared it with the complete sequence of the nonepidemic L . monocytogenes EGDe serovar 1/2a strain . We identified an unexpected genetic divergence between the two strains, as about 8% of the sequences were serovar 4b specific . These sequences included seven genes coding for surface proteins, two of which belong to the internalin family, and three genes coding for transcriptional regulators, all of which might be important in different steps of the infectious process . Based on the sequence information, we then characterized the gene content of 113 Listeria strains by using a newly designed Listeria array containing the "flexible" part of the sequenced Listeria genomes . Hybridization results showed that all of the previously identified virulence factors of L . monocytogenes were present in the 93 L . monocytogenes strains tested . However, distinct patterns of the presence or absence of other genes were identified among the different L . monocytogenes serovars and Listeria species . These results allow new insights into the evolution of L . monocytogenes, suggesting that early divergence of the ancestral L . monocytogenes serovar 1/2c strains from the serovar 1/2b strains led to two major phylogenetic lineages, one of them including the serogroup 4 strains, which branched off the serovar 1/2b ancestral lineage, leading (mostly by gene loss) to the species Listeria innocua . The identification of 30 L . monocytogenes-specific and several serovar-specific marker genes, such as three L . monocytogenes serovar 4b-specific surface protein-coding genes, should prove powerful for the rapid tracing of listeriosis outbreaks, but it also represents a fundamental basis for the functional study of virulence differences between L . monocytogenes strains.

Infect Immun, 2004 Feb, 72(2), 1057 - 64
Chemokine receptor 5 is dispensable for innate and adaptive immune responses to Listeria monocytogenes infection; Zhong MX et al.; Chemokine receptor 5 (CCR5) binds macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, RANTES, and members of the monocyte chemotactic protein family and is also a receptor for human immunodeficiency virus (HIV) . CCR5 ligands can suppress HIV-1 entry into cells . In humans, homozygous mutations of the ccr5 gene confer resistance to HIV-1 infection . The role of CCR5 in defense against microbial infection is unclear . In this study we examined the innate and adaptive immune responses of CCR5-deficient mice to the intracellular bacterial pathogen Listeria monocytogenes . We found that migration of monocytic cells, formation of L . monocytogenes-containing lesions, and bacterial clearance occurred normally in the spleens and livers of CCR5-deficient animals . Activation of macrophages and dendritic cells during the first 3 days postinfection was normal in the absence of CCR5, as demonstrated by intact expression of inducible nitric oxide synthase (iNOS) and production of the cytokines tumor necrosis factor alpha, gamma interferon, and interleukin-12 . Priming of L . monocytogenes-specific CD8 T cells also occured independently of CCR5 expression . Previously immunized, CCR5-deficient animals mounted normal secondary CD8 T-cell responses and cleared bacteria from infected organs similarly to wild-type controls, suggesting that CCR5 is dispensable for migration and activation of memory CD8 T cells . Our data indicate that CCR5-mediated chemotaxis is not required for defense against infection with L . monocytogenes.

Infect Immun, 2004 Feb, 72(2), 931 - 6
Heat shock protein 60 acts as a receptor for the Listeria adhesion protein in Caco-2 cells; Wampler JL et al.; The 104-kDa Listeria adhesion protein (LAP) in Listeria monocytogenes is involved in binding to various mammalian cell lines . However, the receptor that interacts with LAP in eukaryotic cells is unknown . In this study, scanning immunoelectron microscopy qualitatively demonstrated greater binding capacity of wild-type (WT) L . monocytogenes strain (F4244) than a LAP-deficient mutant strain (KB208) to Caco-2 cells . The goal of this study was identification of the host cell receptor for LAP . Using a Western blot ligand overlay assay, we identified a protein of 58 kDa to be the putative receptor for LAP from Caco-2 cells . N-terminal sequencing and subsequent database search identified this protein as heat shock protein 60 (Hsp60) . Modified immunoseparation with protein A-Sepharose beads bound to the LAP-specific monoclonal antibody H7 (MAb-H7) and a sequential incubation with LAP preparation and Caco-2 lysate confirmed the receptor to be the same 58-kDa protein . Western blot analysis with anti-Hsp60 MAb of whole-cell adhesion between Caco-2 and WT also revealed the receptor protein to be a 58-kDa protein, thus corroborating the identification of Hsp60 as a host cell receptor for LAP . Furthermore, the anti-Hsp60 antibody also caused approximately 74% reduction in binding of L . monocytogenes WT to Caco-2 cells, whereas a control antibody, C11E9, had no effect on binding . The adhesion mechanism of L . monocytogenes to eukaryotic cells is a complex process, and identification of Hsp60 as a receptor for LAP adds to the list of previously discovered ligand-receptor modules that are essential to achieve successful adhesion.

Infect Immun, 2004 Feb, 72(2), 717 - 27
Disruption of putative regulatory loci in Listeria monocytogenes demonstrates a significant role for Fur and PerR in virulence; Rea RB et al.; The ability to adapt to adverse environmental conditions encountered in food and during host infection is a sine qua non for a successful Listeria monocytogenes infection . This ability is likely to depend on complex regulatory pathways controlled by a number of key regulators . We utilized the pORI19 plasmid integration system to analyze the role of six putative regulatory loci in growth under suboptimal environmental conditions and during murine infection . Disruption of loci encoding a topoisomerase III (lmo2756), a putative methyltransferase (lmo0581), and a regulator of the MarR family (lmo1618) revealed roles for the methyltransferase and the MarR regulator in growth under environmental stress conditions . However, plasmid integration into these loci had no impact on virulence potential in the murine model of infection . Disruption of the alternative sigma factor Sigma-H resulted in a mutant that demonstrated reduced growth potential in minimal medium . Murine studies indicated a minor role for this sigma factor in the infectious process . Strikingly, disruption of both perR and fur loci resulted in mutants that are significantly affected in virulence for mice, with the fur mutant demonstrating the greatest reduction in virulence potential . Both perR and fur mutants demonstrated increased resistance to hydrogen peroxide and the fur mutant was sensitive to low-iron conditions . The virulence defect of both fur and perR mutants could be rescued by iron-overload after esculetin treatment of mice, suggesting that the in vivo role of these gene products is to procure iron for bacterial growth.

FEBS Lett, 2004 Jan 16, 557(1-3), 88 - 92
CRP2 is an autonomous actin-binding protein; Grubinger M et al.; Cysteine-rich proteins (CRPs) have been shown to be involved in cell differentiation, transcriptional regulation and the organisation of the actin cytoskeleton . Thus far, the latter function has been inferred solely from the in vitro interaction of CRP1, CRP2, and CRP3 with alpha-actinin and zyxin . We show here that purified, recombinant CRP2 binds directly to F-actin in vitro in co-sedimentation assays . Using a green fluorescent protein (GFP)-tagged construct of CRP2 we analysed its localisation and dynamics in A7r5 rat smooth muscle cells . CRP2 was associated with the actin cytoskeleton and decorated actin stress fibres in a continuous fashion, unlike the periodic labelling pattern observed for alpha-actinin and zyxin, which also accumulate in focal adhesions . Using live video fluorescence microscopy we observed the behaviour of GFP-CRP2 during the dynamic rearrangement of the actin cytoskeleton in phorbol 12,13-dibutyrate-treated A7r5 cells . In contrast to the actin-binding proteins SM22alpha and alpha-actinin, GFP-CRP2 did not translocate into the podosomes induced by this treatment, but remained preferentially bound to the stress fibres, suggesting an actin filament-stabilising role for CRP2 . When fused to the mitochondrial targeting sequence from the Listeria protein ActA, GFP-CRP2 was almost completely localised to mitochondria, but no significant recruitment of either alpha-actinin or zyxin could be observed . Taken together, our results demonstrate that CRP2 can bind to F-actin directly and that the association with the actin cytoskeleton is independent of alpha-actinin or zyxin localisation in the cell, thus questioning the role of CRP2 as a regulator of alpha-actinin function in vivo.

J Parasitol, 2003 Dec, 89(6), 1237 - 9
Absence of interferon-gamma-inducible gene IGTP does not significantly alter the development of chagasic cardiomyopathy in mice infected with Trypanosoma cruzi (Brazil strain); de Souza AP et al.; Interferon-gamma (IFN-gamma) contributes to host resistance during acute infection with Trypanosoma cruzi, the causative agent of Chagas' disease . Inducibly expressed guanosine triphosphatase (IGTP), a 48-kDa guanosine triphosphatase (GTPase), is a member of a family of GTPase proteins inducibly expressed by IFN-gamma . The expression pattern of IGTP suggests that it may mediate IFN-gamma-induced responses in a variety of cell types . IGTP has been demonstrated to be important for control of Toxoplasma gondii infection but not for resistance against Listeria monocytogenes . We evaluated the role of IGTP in development of chronic chagasic cardiomyopathy in IGTP null mice and C57X129sv (wild type {WT}) mice infected with the Brazil strain for 6 mo . There was no significant difference in parasitemia or cardiac histopathology between null and WT mice . Right ventricular remodeling was observed in infected IGTP null mice, suggesting that IGTP does not significantly alter the course of T . cruzi infection.

Microbes Infect, 2004 Jan, 6(1), 8 - 16
Antigen-specific CD8+ T cell responses in intestinal tissues during murine listeriosis; Kursar M et al.; Infection of mice with Listeria monocytogenes induces a strong CD8+ T cell response, which is critical for the control of bacteria and for protection against re-infection . We analyzed the CD8+ T cell response in different intestinal tissues following oral and intravenous (i.v.) L . monocytogenes infection . After oral infection, bacterial titers in small intestine and large intestine, and the listeria-specific CD8+ T cell response in the mucosa of both parts of the intestine, were highly correlated . Oral infection of CD28-deficient mice revealed that this response was strictly dependent on CD28 costimulation . Significant listeria-specific CD8+ T cell responses also occurred in all intestinal tissues analyzed after i.v . infection or after DNA vaccination, indicating that the accumulation of listeria-specific CD8+ T cells in these tissues only partially depends on local antigen presentation and inflammation.

J Immunol, 2004 Feb 1, 172(3), 1595 - 601
Escherichia coli expressing recombinant antigen and listeriolysin O stimulate class I-restricted CD8+ T cells following uptake by human APC; Hu PQ et al.; Vaccination against cancer or intracellular pathogens requires stimulation of class I-restricted CD8(+) T cells . It is therefore important to develop Ag delivery vectors that will promote cross-presentation by APCs and stimulate appropriate inflammatory responses . Toward this goal, we tested the potential of Escherichia coli as an Ag delivery vector in in vitro human culture . Bacteria expressing enhanced green fluorescent protein were internalized efficiently by dendritic cells, as shown by flow cytometry and fluorescence microscopy . Phenotypic changes in DC were observed, including up-regulation of costimulatory molecules and IL-12p40 production . We tested whether bacteria expressing recombinant Ags could stimulate human T cells using the influenza matrix protein as a model Ag . Specific responses against an immunodominant epitope were seen using IFN-gamma ELISPOT assays when the matrix protein was coexpressed with listeriolysin O, but not when expressed alone . THP-1 macrophages were also capable of stimulating T cells after uptake of bacteria, but showed slower kinetics and lower overall levels of T cell stimulation than dendritic cells . Increased phagocytosis of bacteria induced by differentiation of THP-1 increased their ability to stimulate T cells, as did opsonization . Presentation was blocked by proteasome inhibitors, but not by lysosomal protease inhibitors leupeptin and E64 . These results demonstrate that recombinant E . coli can be engineered to direct Ags to the cytosol of human phagocytic APCs, and suggest possible vaccine strategies for generating CD8(+) T cell responses against pathogens or tumors.

Int Immunol, 2004 Feb, 16(2), 335 - 43
Roles of caspase-1 in Listeria infection in mice; Tsuji NM et al.; Caspase-1 {IL-1beta-converting enzyme (ICE)} processes substrate precursor molecules to yield the biologically active form of IL-1beta and IL-18, both of which are considered to play important roles in the host defense by activation of both innate and adaptive immunity . We evaluated the immune response of caspase-1(-/-) mice to Listeria monocytogenes (LM) infection . LM eradication in the early phase of infection was impaired in the mutant mice with a prominent decrease in IL-18 and IFN-gamma production, but not in IL-12 . Caspase-1(-/-) spleen cells including dendritic cells and NK cells produced less IFN-gamma in response to heat-killed LM than wild-type cells in vitro . IFN-gamma production and bactericidal activity in LM-infected caspase-1(-/-) mice was reconstituted to normal levels by adding back IL-18 at the initial phase of infection, suggesting that the lack of this cytokine is primarily responsible for the susceptibility of caspase-1(-/-) mice against LM infection . Moreover, IFN-gamma injection of caspase-1(-/-) mice corrected the deficiency in pathogen clearance . In contrast, LM-specific acquired immunity in caspase-1(-/-) mice was normal and they successfully cleared the pathogen following secondary infection, in spite of a moderate skewing of cytokine profile to T(h)2 when compared to wild-type mice . These data shed light on the importance of caspase-1-mediated IL-18 processing in innate immunity against facultative intracellular pathogens.

Arch Otolaryngol Head Neck Surg, 2004 Jan, 130(1), 92 - 7
Regression of HPV-positive tumors treated with a new Listeria monocytogenes vaccine; Sewell DA et al.; BACKGROUND: Human papillomavirus (HPV) has been implicated in the pathogenesis of 15% to 23% of head and neck squamous cell carcinomas as well as most oropharyngeal carcinomas . The viral oncoproteins E6 and E7 are expressed in HPV-positive tumor cells and therefore provide ideal targets for tumor immunotherapy . Because of its unique ability to induce a cellular immune response, the intracellular bacteria Listeria monocytogenes has been studied as a potential HPV-positive tumor vaccine . OBJECTIVE: To present a new recombinant strain of L monocytogenes that is effective in treating HPV-positive tumors in a murine model . DESIGN: A new recombinant L monocytogenes vaccine, Lm-ActA-E7, was designed by transforming an attenuated Listeria strain with an E7 expression cassette . The cassette consists of the HPV-16 E7 sequence fused to the Listeria protein ActA . The resultant strain of bacteria secretes E7 antigen as a fusion protein with ActA . METHODS: Tumors were established in C57BL/6 mice with a syngeneic HPV-positive cell line prior to treatment with vaccine . INTERVENTION: The Lm-ActA-E7 vaccine was administered intraperitoneally to the mice 5 days after tumors were established . A booster dose was administered 7 days after the first dose . Tumor progression was measured in 2 dimensions periodically after the vaccination . RESULTS: In C57BL/6 mice, the administration of Lm-ActA-E7 caused the complete regression of HPV-positive tumors in 6 of 8 mice tested . A cytotoxic T-lymphocyte assay revealed that administration of the vaccine caused the generation of cytotoxic T cells specific for E7 . CONCLUSION: Our results demonstrate the ability of a new Listeria-based vaccine to generate a specific antitumor T-cell response and cause the regression of HPV-positive tumors in a murine model.

Int J Infect Dis, 2004 Mar, 8(2), 97 - 102
Prosthetic valve endocarditis due to Listeria monocytogenes . Report of two cases and reviews; Fernandez Guerrero ML et al.; INTRODUCTION: Endocarditis due to Listeria monocytogenes is a rare but serious disease often leading to valve dysfunction and heart failure . Two cases of listerial prosthetic valve endocarditis are reviewed along with 66 cases previously reported . RESULTS: The mean age of patients with listerial endocarditis increased from 47.1 years in the decades from 1955-1984 to 65.5 years from 1985-2000 . Chronic debilitating diseases, solid tumours and immunosuppression associated with organ transplantation, hematologic neoplasia or AIDS were found in 41.1% of cases . Listerial endocarditis was a vegetative and destructive process, with dehiscense of the prosthesis and occasionally, abscess formation, fistulization and pericarditis . Treatment with penicillin or ampicillin alone or combined with gentamicin was adequate therapy in most cases . Vancomycin together with gentamicin may be a reasonable alternative therapy . CONCLUSIONS: Despite problems associated with microbial persistence and relapses in other forms of human listeriosis, antimicrobial therapy alone may be a successful treatment for listerial endocarditis, including cases occurring on prosthetic valves . Valve replacement may be reserved for complicated cases with valve dehiscense, cardiac failure or myocardial abscess . Overall mortality was 35.3%, although most patients who died did so before 1985 and since then mortality has been significantly reduced to 12%.

Proteomics, 2004 Jan, 4(1), 123 - 35
Global changes in gene expression observed at the transition from growth to stationary phase in Listeria monocytogenes ScottA batch culture; Weeks ME et al.; Listeria monocytogenes is a food-borne Gram-positive bacterium that is responsible for a variety of infections (worldwide) annually . The organism is able to survive a variety of environmental conditions and stresses, however, the mechanisms by which L . monocytogenes adapts to environmental change are yet to be fully elucidated . An understanding of the mechanism(s) by which L . monocytogenes survives unfavourable environmental conditions will aid in developing new food processing methods to control the organism in foodstuffs . We have utilized a proteomic approach to investigate the response of L . monocytogenes batch cultures to the transition from exponential to stationary growth phase . Proteomic analysis showed that batch cultures of L . monocytogenes perceived stress and began preparations for stationary phase much earlier (approximately A(600) = 0.75, mid-exponential) than predicted by growth characteristics alone . Global analysis of the proteome revealed that the expression levels of more than 50% of all proteins observed changed significantly over a 7-9 h period during this transition phase . We have highlighted ten proteins in particular whose expression levels appear to be important in the early onset of the stationary phase . The significance of these findings in terms of functionality and the mechanistic picture are discussed.

J Med Microbiol, 2004 Feb, 53(Pt 2), 87 - 91
Lactoferricin influences early events of Listeria monocytogenes infection in THP-1 human macrophages; Longhi C et al.; Bovine lactoferrin (BLf) and its derivative peptide lactoferricin B (LfcinB) are known for their antimicrobial activity towards several pathogens, including Listeria monocytogenes, a food-borne Gram-positive invasive bacterium that infects a wide variety of host cells, including professional phagocytes . To add further information on the antibacterial effects of these compounds, the influence of BLf, LfcinB and the antimicrobial centre of LfcinB, the hexapeptide LfcinB(4-9), on the invasive behaviour of L . monocytogenes was analysed in IFN-gamma-activated human macrophagic cells (THP-1) . Significant inhibition of bacterial entry in THP-1 cells was observed at LfcinB concentrations that were unable to produce any bacteriostatic or bactericidal effect, compared with BLf and LfcinB(4-9) peptide . This inhibition occurred when LfcinB was incubated during the bacterial infection step and was not due only to competition for common glycosaminoglycan receptors . Assays performed through a temperature shift from 4 to 37 degrees C showed that inhibition of invasion took place at an early post-adsorption step, although an effect on a different step of intracellular infection could not be ruled out.

J Antimicrob Chemother, 2004 Feb, 53(2), 240 - 6 Epub 2004 Jan 16.
Enhancement of antibiotic activity by sub-lethal concentrations of enterocin CRL35; Minahk CJ et al.; OBJECTIVE: The aim of this study was to evaluate the interaction of several conventional antibiotics with sub-lethal concentrations of enterocin CRL35, a cationic peptide, on Listeria innocua 7 . METHODS: Susceptibility of L . innocua 7 cells to the combination of enterocin CRL35 and non-peptide antibiotics (cefalexin, ampicillin, ciprofloxacin, nalidixic acid, erythromycin, chloramphenicol, vancomycin and tetracycline) was assayed using the broth dilution method and killing curves . Fractional inhibitory concentration (FIC) index was calculated to assess synergy . The transmembrane electrical potential and pH gradient were determined by specific fluorescent probes . RESULTS: We found positive interactions between the cationic peptide and three conventional antibiotics (tetracycline, erythromycin and chloramphenicol) which are excluded from the cells by efflux pumps dependent on the membrane proton gradient . Furthermore, enterocin CRL35 even at sub-lethal concentrations induced the dissipation of both components of the proton motive force (Deltap), i.e . transmembrane electrical potential and pH gradient and hence the alteration of processes dependent on it . CONCLUSION: We hypothesize that enterocin CRL35 increases the effectiveness of these antibiotics by impairment of the bacterial active efflux systems and the consequent accumulation of these toxic compounds in the cytoplasm.

J Bacteriol, 2004 Feb, 186(3), 794 - 802
Regulation of transcription of compatible solute transporters by the general stress sigma factor, sigmaB, in Listeria monocytogenes; Cetin MS et al.; Listeria monocytogenes is well known for its durable physiological characteristics, which allow the organism to grow at low temperature and pH and high osmolarity . Growth under high osmolarity depends on the accumulation of compatible solutes, among which glycine betaine and carnitine are the preferred solutes for this organism . Three different transport systems, Gbu, BetL, and OpuC, have been identified in L . monocytogenes which serve to scavenge the preferred compatible solutes . The general stress response regulator sigma(B) has been shown to play an important role in osmotic adaptation in L . monocytogenes, presumably by directing transcription from one or more of the solute transport genes . In the studies presented here, we have used primer extension analyses to identify the promoter elements responsible for transcription of the opuC, gbuA, and betL genes . All three genes are osmotically inducible to some degree . betL is transcribed from a sigma(B)-independent promoter, while gbuA is transcribed from dual promoters, one of which is sigma(B) dependent . opuC is transcribed exclusively from a sigma(B)-dependent promoter . The betL promoter is similar in sequence to the sigma(B)-independent gbuAP1 promoter . Kinetic analysis of transcript accumulation after osmotic upshift demonstrated that sigma(B)-dependent transcripts from gbuAP2 and sigB accumulate for an extended period after upshift, suggesting that sigma(B) activity may provide a mechanism for sustained high-level expression during osmotic challenge . In contrast to osmotic upshift, expression from the sigma(B)-dependent opuC and gbuAP2 promoters after temperature upshift and ethanol stress was minimal, suggesting that additional mechanisms may also participate in regulating transcription from these sigma(B)-dependent promoters.

J Appl Microbiol, 2004, 96(2), 398 - 408
Effects of high-pressure processing on Listeria monocytogenes, spoilage microflora and multiple compound quality indices in chilled cold-smoked salmon; Lakshmanan R et al.; AIMS: To evaluate the effect of high-pressure processing (HPP) on Listeria monocytogenes, microbial and chemical changes and shelf-life in chilled cold-smoked salmon (CSS) . METHODS AND RESULTS: First, challenge tests with L . monocytogenes were carried out using HPP of the product at 0.1 (control), 150, 200 and 250 MPa . Secondly, storage trials with the naturally contaminated product and HPP at 0.1 (control) and 200 MPa were realized . Shelf-life, microbial changes and chemical changes were determined and existing predictive models and multiple compound quality indices evaluated . HPP with 250 MPa did not inactivate L . monocytogenes but significant lag phases of 17 and 10 days were observed at ca 5 and 10 degrees C, respectively . HPP with 200 MPa had a marked effect on both colour and texture of CSS . CONCLUSIONS: High-pressure processing was unable to prevent growth of L . monocytogenes or spoilage of chilled CSS . Existing mathematical models allowed growth rates of L . monocytogenes and shelf-life of samples without high-pressure treatments to be predicted . SIGNIFICANCE AND IMPACT OF THE STUDY: High-pressure processing seems more appropriate for new types of salmon products than for a classical product like CSS where consumers expect specific quality attributes.

Ned Tijdschr Geneeskd, 2003 Dec 27, 147(52), 2565 - 9
{The importance of a complete diagnostic workup in patients with nontraumatic (partial) paraplegia}; Schinagl DA et al.; In two women, aged 86 and 56 years, respectively, who suffered from back pain and loss of strength, and in a 55-year-old man who lost sensation and strength in his left leg, spinal-cord compression in connection with vertebral destruction was seen on radiological examination . When spinal-cord compression is the result of a local malignant tumour, the therapy often entails emergency radiotherapy . In the first two patients, histological examination revealed a solitary plasmocytoma and curative high-dose radiotherapy was applied . The third patient also had a lung tumour and received low-dose palliative radiotherapy to the vertebrae, as a metastasis was suspected . Later, however, histopathologic examination of the vertebral lesion revealed osteomyelitis due to Listeria monocytogenes and the lung tumour was diagnosed as a pT2N0M0 broncho-alveolar carcinoma which was surgically removed . When a patient is referred with a nontraumatic spinal-cord injury, it is important to complete the radiological and histological examinations before starting emergency radiotherapy in order to prevent an inadequate or even incorrect treatment.

J Food Prot, 2004 Jan, 67(1), 77 - 82
Reduction and survival of Listeria monocytogenes in ready-to-eat meats after irradiation; Foong SC et al.; A five-strain Listeria monocytogenes culture was inoculated onto six different types of ready-to-eat (RTE) meats (frankfurters, ham, roast beef, bologna, smoked turkey with lactate, and smoked turkey without lactate) . The meats were vacuum packed and stored at 4 degrees C for 24 h prior to irradiation . Populations of L . monocytogenes were recovered by surface plating on nonselective and selective media . The margins of safety studied include 3-log (3D) and 5-log (5D) reduction of pathogenic bacteria to achieve an optimal level of reduction while retaining organoleptic qualities of the meats . A 3-log reduction of L . monocytogenes was obtained at 1.5 kGy when nonselective plating medium was used . The dosages for 3-log reduction were 1.5 kGy for bologna, roast beef, and both types of turkey and 2.0 kGy for frankfurters and ham on the basis of use of selective medium . The D10-values ranged from 0.42 to 0.44 kGy . A 5-log reduction of L . monocytogenes was obtained at 2.5 kGy with nonselective medium . With selective medium, the dosages were 2.5 kGy for bologna, roast beef, and both types of turkey and 3.0 kGy for frankfurters and ham . Survival of L . monocytogenes in the same RTE meat types after irradiation was also studied . Meats were inoculated with 5 log L . monocytogenes per g and irradiated at doses of 2.0 and 4.0 kGy . Recovery of the surviving organisms was observed during storage at temperatures of 4 and 10 degrees C for 12 weeks . Preliminary results showed no growth in meats irradiated at 4.0 kGy . Survivors were observed for irradiated meats at 2.0 kGy stored at 10 degrees C after the second week . No growth was observed in samples irradiated at 2.0 kGy stored at 4 degrees C until the fifth week.

J Food Prot, 2004 Jan, 67(1), 71 - 6
Effect of reheating on viability of a five-strain mixture of Listeria monocytogenes in vacuum-sealed packages of frankfurters following refrigerated or frozen storage; Porto AC et al.; The purpose of this study was to assess consumer preferences for storing and reheating frankfurters and to use this information to assess the effect of product formulation and storage times and temperatures on the viability of Listeria monocytogenes after reheating of frankfurters . Individual links were inoculated with about 8.0 log CFU per package of a five-strain mixture of the pathogen, vacuum sealed, and stored at 4 degrees C for 3 and 15 days and at -18 degrees C for 30 days . Frankfurters formulated with and without 2% added potassium lactate were heated to a surface temperature of 60, 70, 80, or 90 degrees C for up to 8 min by submersing the packages in a thermostatically controlled circulating water bath . Surviving bacteria were recovered and counted by rinsing the contents of each package with sterile peptone water and plating this solution directly onto modified Oxford selective agar plates . In general, the results revealed that about a 5-log unit reduction was achieved by reheating to a surface temperature of 70 degrees C for about 2 min or 80 or 90 degrees C for about 0.6 min regardless of storage conditions or formulation . Product formulation did not appreciably affect the viability of the pathogen after heating; there was no appreciable difference in the number of cells surviving the heat treatment in product prepared with or without potassium lactate . These findings can be used to establish reheating guidelines for consumers to ensure that frankfurters, which may become contaminated with low levels of L . monocytogenes prior to packaging and after unpackaging, are adequately reheated prior to consumption.

J Clin Microbiol, 2004 Jan, 42(1), 276 - 85
Comparative analysis of multilocus sequence typing and pulsed-field gel electrophoresis for characterizing Listeria monocytogenes strains isolated from environmental and clinical sources; Revazishvili T et al.; One hundred seventy-five Listeria monocytogenes strains were characterized by serotyping, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) based on loci in actA, betL, hlyA, gyrB, pgm, and recA . One hundred twenty-two sequence types (STs) were identified by MLST based on allelic profiles of the four housekeeping genes (betL, gyrB, pgm, and recA), and 34 and 38 alleles were identified for hlyA and actA, respectively . Several actA and hlyA alleles appeared to be predominantly associated with clinical isolates . MLST differentiated most of the L . monocytogenes strains better than did PFGE, and the discriminating ability of PFGE was better than that of serotyping . Several strains with different serotypes were found, by MLST and PFGE, to have very closely related genetic backgrounds, which suggested possible "antigen switching" among them . MLST can be a useful typing tool for differentiating L . monocytogenes strains (including strains undistinguishable by PFGE typing and serotyping), and it may be of value during investigations of food-borne outbreaks of listeriosis.

J Clin Microbiol, 2004 Jan, 42(1), 172 - 8
Decision support tools for clinical diagnosis of disease in cows with suspected bovine spongiform encephalopathy; Saegerman C et al.; Reporting of clinically suspected cattle is currently the most common method for detecting cases of bovine spongiform encephalopathy (BSE) . Improvement of clinical diagnosis and decision-making remains crucial . A comparison of clinical patterns, consisting of 25 signs, was made between all 30 BSE cases, confirmed in Belgium before October 2002, and 272 suspected cases that were subsequently determined to be histologically, immunohistochemically, and scrapie-associated-fiber negative . Seasonality in reporting suspected cases was observed, with more cases being reported during wintertime when animals were kept indoors . The median duration of illness was 30 days . The 10 most relevant signs of BSE were kicking in the milking parlor, hypersensitivity to touch and/or sound, head shyness, panic-stricken response, reluctance to enter in the milking parlor, abnormal ear movement or carriage, increased alertness behavior, reduced milk yield, teeth grinding, and temperament change . Ataxia did not appear to be a specific sign of BSE . A classification and regression tree was constructed by using the following four features: age of the animal, year of birth, number of relevant BSE signs noted, and number of clinical signs, typical for listeriosis, noted . The model had a sensitivity of 100% and a specificity of 85% . This approach allows the use of an interactive decision-support tool, based entirely on odds ratios, a statistic independent of disease prevalence.

Avian Dis, 2003 Oct-Dec, 47(4), 1496 - 502
Pathology of listerial encephalitis in chickens in Japan; Kurazono M et al.; Neural signs (torticollis, drowsiness) and mortality were observed in five chickens of a native chicken flock (reared for meat) that included 450 male birds on a farm that had 2300 native chickens and 1120 layers . Histologic lesions were observed in the medulla oblongata, optic lobe, cerebellum, and spinal cord of the affected birds . The lesions, which were most severe in the medulla oblongata, were massive abscesses with rarefaction (demyelination and malacia) of the parenchyma with gram-positive bacteria . The degenerative and necrotic areas were characterized by fibrin thrombosis, hemorrhages, and congestion in the blood vessels . Immunohistochemically, the bacteria positive for L . monocytogenes antigen were observed in the medulla oblongata, cerebellum, and spinal cord . Ultrastructurally, the small rod-shaped and thin-cell-walled bacteria were observed in the parenchyma of the medulla oblongata . Listeria monocytogenes (serotype 4b) was isolated from the medulla oblongata and spinal cord . The pathogenesis of listerial encephalitis in chickens was discussed.

J Immunol, 2004 Jan 15, 172(2), 1163 - 8
Mice deficient in LRG-47 display increased susceptibility to mycobacterial infection associated with the induction of lymphopenia; Feng CG et al.; Although IFN-gamma is essential for host control of mycobacterial infection, the mechanisms by which the cytokine restricts pathogen growth are only partially understood . LRG-47 is an IFN-inducible GTP-binding protein previously shown to be required for IFN-gamma-dependent host resistance to acute Listeria monocytogenes and Toxoplasma gondii infections . To examine the role of LRG-47 in control of mycobacterial infection, LRG-47(-/-) and wild-type mice were infected with Mycobacterium avium, and host responses were analyzed . LRG-47 protein was strongly induced in livers of infected wild-type animals in an IFN-gamma-dependent manner . LRG-47(-/-) mice were unable to control bacterial replication, but survived the acute phase, succumbing 11-16 wk postinfection . IFN-gamma-primed, bone marrow-derived macrophages from LRG-47(-/-) and wild-type animals produced equivalent levels of TNF and NO upon M . avium infection in vitro and developed similar intracellular bacterial loads . In addition, priming for IFN-gamma production was observed in T cells isolated from infected LRG-47(-/-) mice . Importantly, however, mycobacterial granulomas in LRG-47(-/-) mice showed a marked lymphocyte deficiency . Further examination of these animals revealed a profound systemic lymphopenia and anemia triggered by infection . As LRG47(-/-) T lymphocytes were found to both survive and confer resistance to M . avium in recipient recombinase-activating gene-2(-/-) mice, the defect in cellular response and bacterial control in LRG-47(-/-) mice may also depend on a factor(s) expressed in a nonlymphocyte compartment . These findings establish a role for LRG-47 in host control of mycobacteria and demonstrate that in the context of the IFN-gamma response to persistent infection, LRG-47 can have downstream regulatory effects on lymphocyte survival.

Cell Microbiol, 2004 Feb, 6(2), 155 - 66
Evidence implicating the 5' untranslated region of Listeria monocytogenes actA in the regulation of bacterial actin-based motility; Wong KK et al.; The ActA protein of Listeria monocytogenes is a major virulence factor, essential for the recruitment and polymerization of host actin filaments that lead to intracellular motility and cell-to-cell spread of bacteria within the infected host . The expression of actA is tightly regulated and is strongly induced only when L . monocytogenes is within the host cytosol . Intracellular induction of actA expression is mediated through a single promoter element that directs the expression of a messenger RNA with a long (150 bp) 5' untranslated region (UTR) . Deletion of the actA+3 to +130 upstream region was found to result in bacterial mutants that were no longer capable of intracellular actin recruitment or cell-to-cell spread, thus indicating that this region is important for actA expression . L . monocytogenes strains that contained smaller deletions (21-23 bp) within the actA upstream region demonstrated a range of actA expression levels that coincided with the amount of bacterial cell-to-cell spread observed within infected monolayers . A correlation appeared to exist between levels of actA expression and the ability of L . monocytogenes to transition from uniform actin accumulation surrounding individual bacteria (actin clouds) to directional assembly and the formation of actin tails . Bacterial mutants containing deletions that most significantly altered the predicted secondary structure of the actA mRNA 5' UTR had the largest reductions in actA expression . These results suggest that the actA 5' UTR is required for maximal ActA synthesis and that a threshold level of ActA synthesis must be achieved to promote the transition from bacteria-associated actin clouds to directional actin assembly and movement.

J Biol Chem, 2004 Mar 19, 279(12), 11672 - 9 Epub 2003 Dec 30.
Identification and characterization of a phosphoinositide phosphate kinase homolog; Chang JD et al.; Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) plays a central role in regulating the actin cytoskeleton as a substrate for phosphoinositide 3-kinase and phospholipase C as well as by binding directly to proteins that control the processes of actin monomer sequestration, filament severing, capping, nucleation, cross-linking, and bundling (Ma, L., Cantley, L . C., Janmey, P . A., and Kirschner, M . W . (1998) J . Cell Biol . 140, 1125-1136; Hinchliffe, K . (2000) Curr . Biol . 10, R104-R1051) . Three related phosphatidylinositol 4-phosphate 5-kinases (PI(4)P 5-kinases) have been identified in mammalian cells (types Ialpha, Ibeta, and Igamma) and appear to play distinct roles in actin remodeling . Here we have identified a fourth member of this family by searching the human genome and EST data bases . This new protein, which we have designated phosphatidylinositol phosphate kinase homolog (PIPKH), is expressed at relatively high levels in brain and testis . Immunoprecipitates of PIPKH expressed in mammalian cells contain PI(4)P 5-kinase activity, but this activity is not affected by mutations in residues that inactivate other type I PI(4)P 5-kinases . We show that the PI(4)P 5-kinase activity in PIPKH immunoprecipitates can be explained by the ability of PIPKH to heterodimerize with other type I PI(4)P 5-kinases . Transfection of 293t cells with PIPKH resulted in >8-fold increase in total phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) without a significant net increase in total PI(4,5)P(2) . When coexpressed with PIPKH, green fluorescent protein (GFP) fusion construct of the pleckstrin homology domain from Bruton's tyrosine kinase (GFP-BTK-PH) localized in intracellular vesicular structures, suggesting an unusual intracellular site of PI(3,4,5)P(3) production . Finally, expression of PIPKH induced the reorganization of actin from predominantly stress fibers to predominantly foci and comets similar to those observed previously in cells infected with the intracellular pathogen Listeria or transfected with recombinant PIPKIalpha . These results suggest that PIPKH acts as a scaffold to localize and regulate type I PI(4)P 5-kinases and the synthesis of PI(3,4,5)P(3).

Int J Food Microbiol, 2004 Jan 15, 90(2), 219 - 36
Evaluation of a challenge testing protocol to assess the stability of ready-to-eat cooked meat products against growth of Listeria monocytogenes; Uyttendaele M et al.; Challenge testing of ready-to-eat (RTE) foods with Listeria monocytogenes is recommended to assess the potential for growth . The present study was undertaken to evaluate a protocol for challenge testing applied to RTE cooked meat products . In order to choose L . monocytogenes strains with a representative behaviour, initially, the variability of the response of multiple L . monocytogenes strains of human and food origin to different stress and growth conditions was established . The strains were not inhibited in their growth at moderate acid pH (5.25) and the four strains tested in particular showed a similar acid-adaptive response . Growth of the various strains under four different combined stress conditions indicated that no L . monocytogenes strain had consistently significant longer or shorter lag phase or higher or lower maximum specific growth rates . The effect of choice of strain and history (pre-incubation temperature 7 or 30 degrees C) on growth of L . monocytogenes under optimum conditions (Brain Heart Infusion, BHI) and modified BHI simulating conditions of cooked ham and pate was studied . In general, all four L . monocytogenes strains behaved similarly . In BHI, no difference in lag phase was observed for the cold-adapted and standard inoculum, whereas in BHI adjusted to ham and pate conditions, a ca . 40-h reduction of the lag phase was noted for the cold-adapted inoculum . Subsequently, microbial challenge testing of L . monocytogenes in modified atmosphere packaged sliced cooked ham and pate was performed . A mixed inoculum of four L . monocytogenes strains and an inoculum level of ca . 1-10 cfu/g was used . On vacuum packed sliced cooked ham, the concentration of 100 cfu/g, the safety limit considered as low risk for causing listeriosis, was exceeded after 5 days whereas ca . 10(5) cfu/g were obtained after 14 days when also LAB spoilers reached unacceptable numbers (ca . 10(7) cfu/g) whether standard or cold-adapted inoculum was used . The concentration of sodium lactate determined the opportunities for growth of L . monocytogenes in pate . If growth of L . monocytogenes in pate was noticed, the threshold of 100 cfu/ml was crossed earlier for the cold-adapted inoculum compared to the standard inoculum.

J Leukoc Biol, 2004 Mar, 75(3), 541 - 52 Epub 2003 Dec 23.
Macrophage activation and Fcgamma receptor-mediated signaling do not require expression of the SLP-76 and SLP-65 adaptors; Nichols KE et al.; The Src-homology 2 domain-containing, leukocyte-specific phosphoprotein of 76 kDa (SLP-76) is a hematopoietic adaptor that plays a central role during immunoreceptor-mediated activation of T lymphocytes and mast cells and collagen receptor-induced activation of platelets . Despite similar levels of expression in macrophages, SLP-76 is not required for Fc receptor for immunoglobulin G (IgG; FcgammaR)-mediated activation . We hypothesized that the related adaptor SLP-65, which is also expressed in macrophages, may compensate for the loss of SLP-76 during FcgammaR-mediated signaling and functional events . To address this hypothesis, we examined bone marrow-derived macrophages (BMM) from wild-type (WT) mice or mice lacking both of these adaptors . Contrary to our expectations, SLP-76(-/-) SLP-65(-/-) BMM demonstrated normal FcgammaR-mediated activation, including internalization of Ig-coated sheep red blood cells and production of reactive oxygen intermediates . FcgammaR-induced biochemical events were normal in SLP-76(-/-) SLP-65(-/-) BMM, including phosphorylation of phospholipase C and the extracellular signaling-regulated kinases 1 and 2 . To determine whether macrophages functioned normally in vivo, we infected WT and SLP-76(-/-) SLP-65(-/-) mice with sublethal doses of Listeria monocytogenes (LM), a bacterium against which the initial host defense is provided by activated macrophages . WT and SLP-76(-/-) SLP-65(-/-) mice survived acute, low-dose infection and showed no difference in the number of liver or spleen LM colony-forming units, a measure of the total body burden of this organism . Taken together, these data suggest that neither SLP-76 nor SLP-65 is required during FcgammaR-dependent signaling and functional events in macrophages.

Nat Immunol, 2004 Jan, 5(1), 38 - 44 Epub 2003 Dec 14.
Gadd45beta is important for perpetuating cognate and inflammatory signals in T cells; Lu B et al.; Gadd45beta (growth arrest and DNA damage-inducible, beta) is involved in cell cycle arrest, apoptosis, signal transduction and cell survival . In T cells, Gadd45b was rapidly induced by T cell receptor (TCR) and inflammatory signals . Deficiency of Gadd45beta in CD4+ T cells impaired their responses to TCR stimulation or inflammatory cytokines . ERK, p38 and JNK activation were all substantially suppressed in Gadd45beta-deficient CD4+ T cells . Cytokine production by Gadd45beta-deficient CD4+ T cells was also impaired . Furthermore, Gadd45beta mediated inflammatory cytokine production by dendritic cells, and Gadd45beta-deficient mice showed an impaired T helper type 1 response during Listeria monocytogenes infection . Gadd45beta is therefore a critical feedback regulator that perpetuates both cognate and inflammatory signals.

Comp Immunol Microbiol Infect Dis, 2004 Mar, 27(2), 141 - 8
Isolation of pathogenic Listeria monocytogenes and detection of antibodies against phosphatidylinositol-specific phospholipase C in buffaloes; Chaudhari SP et al.; The isolation of pathogenic Listeria spp . in bacteriological samples, and anti-phosphatidylinositol-specific phospholipase C (anti-PIPLC) antibodies in sera of buffaloes were studied . Isolation of the pathogen was attempted from the samples by selective enrichment in University of Vermont Medium and plating onto Dominguez-Rodriguez isolation agar . Pathogenicity of the isolates was tested by Christie, Atkins, Munch Petersen test and mice incoulation test . Listeria spp . and L . monocytogenes were isolated from 8.8 and 2.4%, and 4.8 and 1.6% of 125 each meat and blood samples, respectively . Out of the 125 samples each of feacal, nasal and vaginal swabs from buffaloes 8 and 4%, 13.6 and 2.4%, and 6.4 and 2.4% were positive for Listeria spp . and L . monocytogenes, respectively . L . ivanovii was confirmed from 0.8% vaginal sample . A total of 125 serum samples were tested by phosphatidylinositol-specific phospholipase C (PIPLC) based indirect ELISA of which 4.0% turned out to be seropositive.

Cancer Biol Ther, 2003 Nov-Dec, 2(6), 687 - 93
An essential role of Th1 responses and interferon gamma in infection-mediated suppression of neoplastic growth; Rankin EB et al.; We had previously demonstrated that in mice acute toxoplasmosis leads to systemic inhibition of angiogenesis and, consequently, strong suppression of neoplastic growth . Here we investigated the role of Th1 cytokines, in particular interferon gamma (IFN-gamma), in this phenomenon . Besides toxoplasma, neoplastic growth was readily blocked during acute infection with other Th1 response-inducing pathogens such as Listeria monocytogenes and lymphocytic choriomeningitis virus (LCMV) . In contrast, chronic infection with LCMV (when Th1 responses were strongly suppressed) and acute infection with Schistosoma mansoni (when Th2 responses predominated) afforded no anti-tumor protection . To corroborate the involvement of Th1 cytokines in infection-mediated suppression of neoplastic growth, we utilized mice deficient in interleukin-10 (IL10), a suppressor of Th1 responses . When challenged with B16 cells concomitantly with toxoplasma infection, both IL10-null and wild type mice exhibited resistance to neoplastic growth . However, tumors borne by IL10-null animals were even smaller than those borne by their wild type counterparts . This enhanced resistance correlated with dramatically elevated levels of circulating IFN-gamma, a principal Th1 cytokine . Furthermore, while interleukin-12 and tumor necrosis factor a were dispensable for tumor suppression, in animals deficient in IFN-gamma production or signaling, tumor growth and neovascularization were markedly enhanced . Interestingly, the enhancement was also apparent in uninfected animals suggesting that IFN-gamma and its anti-angiogenic effects underlie both infection-dependent and -independent tumor surveillance.

Proc Natl Acad Sci U S A, 2004 Jan 6, 101(1), 129 - 34 Epub 2003 Dec 19.
Detection of bacteria in suspension by using a superconducting quantum interference device; Grossman HL et al.; We demonstrate a technique for detecting magnetically labeled Listeria monocytogenes and for measuring the binding rate between antibody-linked magnetic particles and bacteria . This sensitive assay quantifies specific bacteria in a sample without the need to immobilize them or wash away unbound magnetic particles . In the measurement, we add 50-nm-diameter superparamagnetic magnetite particles, coated with antibodies, to an aqueous sample containing L . monocytogenes . We apply a pulsed magnetic field to align the magnetic dipole moments and use a high-transition temperature superconducting quantum interference device, an extremely sensitive detector of magnetic flux, to measure the magnetic relaxation signal when the field is turned off . Unbound particles randomize direction by Brownian rotation too quickly to be detected . In contrast, particles bound to L . monocytogenes are effectively immobilized and relax in about 1 s by rotation of the internal dipole moment . This Neel relaxation process is detected by the superconducting quantum interference device . The measurements indicate a detection limit of (5.6 +/- 1.1) x 10(6) L . monocytogenes in our sample volume of 20 microl . If the sample volume were reduced to 1 nl, we estimate that the detection limit could be improved to 230 +/- 40 L . monocytogenes cells . Time-resolved measurements yield the binding rate between the particles and bacteria.

Infect Immun, 2004 Jan, 72(1), 489 - 97
Listeriosis in the pregnant guinea pig: a model of vertical transmission; Bakardjiev AI et al.; Feto-placental infections represent a major cause of pregnancy complications, and yet the underlying molecular and cellular mechanisms of vertical transmission are poorly understood . Listeria monocytogenes, a facultative intracellular pathogen, is one of a group of pathogens that are known to cause feto-placental infections in humans and other mammals . The purpose of this study was to evaluate possible mechanisms of vertical transmission of L . monocytogenes . Humans and guinea pigs have a hemochorial placenta, where a single layer of fetally derived trophoblasts separates maternal from fetal circulation . We characterized L . monocytogenes infection of the feto-placental unit in a pregnant guinea pig model and in primary human trophoblasts and trophoblast-derived cell lines . The clinical manifestations of listeriosis in the pregnant guinea pigs and the tropism of L . monocytogenes to the guinea pig placenta resembled those in humans . Trophoblast cell culture systems were permissive for listerial growth and cell-to-cell spread and revealed that L . monocytogenes deficient in internalin A, a virulence factor that mediates invasion of nonphagocytic cells, was 100-fold defective in invasion . However, crossing of the feto-placental barrier in the guinea pig model was independent of internalin A, suggesting a negligible role for internalin-mediated direct invasion of trophoblasts in vivo . Further understanding of vertical transmission of L . monocytogenes will help in designing more effective means of treatment and disease prevention.

Przegl Epidemiol, 2003, 57(3), 439 - 47
{Assessment of etiological diagnostics in adults with aseptic encephalomeningitis--own material}; Piekarska A et al.; OBJECTIVE: Epidemiologic, etiologic and clinical assessment of patients with aseptic encephalomeningitis (AE); evaluation of efficacy of diagnostic process . METHODS: From January 1996 to August 2002, in seventy seven patients AE was diagnosed on the basis of lymphocytic predominance in cerebrospinal fluid (CSF) and negative culture of CSF . Analysis comprised: etiology, course of the disease and spectrum of used diagnostic tests . RESULTS: In 48/77 patients (62.3%) etiology was not identified . In 8/77 patients (10.3%) herpes simplex encephalitis was diagnosed, in 7/77 (9.1%) neuroborreliosis, in 6/77 (7.8%) tuberculosis encephalitis, in 3/77 (3.9%) listerial meningitis, in 3/77 (3.9%) mumps meningitis, and in remaining 2/77 (2.6%) tick-borne encephalitis . The incidence of AE was higher in summer (42.9%) than in any other season . In 14/77 patients (18.2%) the episode of unconsciousness occurred in the course of the disease . In 6 of these 14 patient etiology was unknown, in 4 tuberculosis AE, in 2 herpes simplex encephalitis and in 2 neuroborreliosis was diagnosed . In 7 of 77 patients (9.0%) et least one episode of convulsions occurred . CONCLUSIONS: In 62.3% of patients etiology remained unknown due to clinical and economic reasons . Aseptic encephalomeningitis in adults not always mean the viral etiology and mild course of the disease.

J Toxicol Environ Health A, 2004 Feb 13, 67(3), 251 - 63
Soluble metals associated with residual oil fly ash increase morbidity and lung injury after bacterial infection in rats; Roberts JR et al.; Inhalation of residual oil fly ash (ROFA) has been shown to impair lung defense mechanisms in laboratory animals and susceptible populations . Bioavailability of soluble transition metals has been shown to play a key role in lung injury caused by ROFA exposure . The goal of this study was to evaluate the effect of soluble metals on lung defense and injury in animals preexposed to ROFA followed by pulmonary challenge with a bacterial pathogen . ROFA was suspended in saline (ROFA-TOTAL), incubated overnight at 37 degrees C, and separated by centrifugation into soluble (ROFA-SOL) and insoluble (ROFA-INSOL) fractions . A portion of the soluble sample was treated with the metal-binding resin Chelex for 24 h at 37 degrees C . Sprague-Dawley rats were intratracheally dosed at d 0 with ROFA-TOTAL (1.0 mg/100 g body weight), ROFA-INSOL, ROFA-SOL, saline, saline + Chelex, or ROFA-SOL + Chelex . At d 3, 5 x 10(5) Listeria monocytogenes were intratracheally instilled into rats from each treatment group . At d 6, 8, and 10, left lungs were removed, homogenized, and cultured to assess bacterial clearance . Histopathological analysis was performed on the right lungs . Pulmonary exposure of ROFA-TOTAL or ROFA-SOL before infection led to a marked increase in lung injury and inflammation at all three time points after inoculation, and an increase in morbidity in comparison to saline control rats . Treatment with ROFA-INSOL, saline + Chelex, or ROFA-SOL + Chelex caused no significant increases in lung damage and morbidity when compared to control . By d 10, the ROFA-SOL and ROFA-TOTAL groups had approximately 200-fold more bacteria in the lung than saline control, indicating the inability of these groups to effectively respond to the infection . None of the other treatment groups had significant impairments in bacterial clearance when compared to saline . In conclusion, exposure to ROFA-TOTAL and ROFA-SOL significantly suppressed the lung response to infection . These results suggest that soluble metals present in ROFA may play a key role in increased susceptibility to pulmonary infection in exposed populations.

Cad Saude Publica, 1994 Dec, 10(4), 440 - 5 Epub 2003 Dec 15.
In vitro activity of naturally occurring peptides (defensins) against Listeria monocytogenes; Nascimento Mda G et al.; Autoclaved distilled water samples were inoculated with L . monocytogenes strain V7 and strain VPH-1, and incubated aerobically, at 30 C for 48 hours . Each strain was tested individually, and growth curves were determined at 1, 2, 3, 4, 5, 21, 24, and 48 hours . The growth or survival of L . monocytogenes was similar for both strains, with survivors at 24 hour-incubation . The microbicidal activity of one synthetic cationic peptide (NP-2) was examined against L . monocytogenes strain V7, in a water system . Antibacterial activity of NP-2 (1, 5, and 10 g/ml) was best expressed at 60 minute-incubation, with 10 g/ml of peptide, at 30 C.

Int J Food Microbiol, 2004 Jan 1, 90(1), 15 - 22
Effective control of Listeria monocytogenes by combination of nisin formulated and slowly released into a broth system; Chi-Zhang Y et al.; In order to identify conditions for efficient food preservation by nisin, the sensitivity of Listeria monocytogenes to this preservative was studied under the following three model conditions: (1) the instantaneous addition of nisin into broth medium to simulate the formation of nisin in foods, (2) the slow delivery of nisin solution into broth medium using a pump to simulate the slow release of nisin from packaging materials to foods, (3) a combination of the two delivery methods . Based on the following results, we conclude that the antimicrobial effectiveness of nisin strongly depends on its mode of delivery . The instantaneous and slow methods for adding nisin inhibited L . monocytogenes, but over time of exposure, L . monocytogenes developed tolerance to nisin . Our data indicate that cells treated with instantaneously added nisin developed resistance to higher concentrations of nisin (200 IU/ml), compared to cells treated with slowly added nisin at the same total amount of the antimicrobial . Further studies indicated that nisin-tolerant cells recovered from treatments in which 200 IU/ml nisin was added instantaneously were likely to be mutants, which became resistant to the bacteriocin . In contrast, when 200 IU/ml of the antimicrobial was added slowly to the cells, only a temporary tolerance was developed; these cells became nisin-sensitive after passage through nisin-free medium . Due to the development of nisin-resistant cells, excessive amounts of nisin in the model system did not further inhibit L . monocytogenes . These results signify that excess nisin in foods does not necessarily improve the efficiency of controlling L . monocytogenes . Our data suggest that the combination of packaging material containing nisin used in conjunction with nisin-containing foods will provide the most effective means of preventing L . monocytogenes growth.

J Food Prot, 2003 Dec, 66(12), 2362 - 6
Pathogenicity of food and clinical Listeria monocytogenes isolates in a mouse bioassay; Takeuchi K et al.; Serotype distributions of Listeria monocytogenes in clinical samples and foods often differ . It is unknown whether such differences reflect a variation in the virulence of strains or are due to other factors that are not directly related to the strains' ability to cause illnesses . Fifty-two food and eight clinical isolates of L . monocytogenes were obtained from France, Japan, and the United States . Their pathogenicity in nonimmunocompromised female ICR mice was determined by intraperitoneal (i.p.) injection of the mice with test strains at 10(8) to 10(9) CFU per mouse . Five mice were injected with each Listeria strain and observed for 5 days . Listeria isolates that caused at least one death in 5 days were considered pathogenic . Isolates that caused no deaths in 5 days were considered nonpathogenic . All strains except Listeria innocua and one L . monocytogenes serotype 4b strain (RM3-1) isolated from bovine raw milk were pathogenic to nonimmunocompromised mice . Three food isolates of L . monocytogenes serotype 1/2c were weakly pathogenic to nonimmunocompromised mice, killing a maximum of 50% of mice at 10(8) CFU . Strains with no pathogenicity or reduced pathogenicity were further tested for their pathogenicity to immunocompromised mice . Each strain was inoculated i.p . into five mice at 10(3) to 10(10) CFU per mouse . No deaths of immunocompromised mice inoculated with 10(8) CFU were observed, but 20 to 40% of the mice died when inoculated with 10(9) CFU of L . monocytogenes RM3-1 . The three L . monocytogenes serotype 1/2c isolates were also weakly pathogenic to immunocompromised mice, with two of the three isolates killing < or = 60% of mice at doses of < or = 10(8) CFU . The hemolytic activity of the three weakly pathogenic serotype 1/2c isolates was similar to that of pathogenic strains . However, the nonpathogenic strain RM3-1 was not found to be hemolytic on horse blood agar . We have identified several L . monocytogenes strains with reduced virulence levels . Further characterization of such isolates may aid in understanding factors affecting the variation in virulence among strains.

J Food Prot, 2003 Dec, 66(12), 2252 - 7
Nitrite-induced injury of Listeria monocytogenes and the effect of selective versus nonselective recovery procedures on its isolation from frankfurters; Ngutter C et al.; Sodium nitrite (NaNO2) is used as a curing agent in frankfurters . Although previous studies have documented the bacteriostatic abilities of NaNO2 toward Listeria monocytogenes, few if any studies have been conducted that consider the possibility of sublethal injury to L . monocytogenes by exposure to NaNO2 . The goals of this study were to determine whether NaNO2 has the ability to injure L . monocytogenes, to determine whether nitrite injury is reversible, and to compare the recovery of L . monocytogenes from frankfurters containing nitrite with Listeria repair broth (LRB) and University of Vermont modified Listeria enrichment broth (UVM) . NaNO2, when used at concentrations of 100 and 200 ppm, was found to injure L . monocytogenes . The injury was completely reversible, or growth of uninjured Listeria occurred in LRB when injury was between 98.5 and 98.7% . However, total recovery was not observed in LRB when injury exceeded 99% . UVM was unable to reverse the effects of nitrite-injured L . monocytogenes . With respect to time, inoculum, and meat type, LRB was found to be consistently superior to UVM at recovering L . monocytogenes from frankfurters . Nitrite injury might be a factor influencing detection and recovery of L . monocytogenes from frankfurters.

Immunity, 2003 Dec, 19(6), 891 - 901
Sequential MyD88-independent and -dependent activation of innate immune responses to intracellular bacterial infection; Serbina NV et al.; Microbial infections induce chemokine and cytokine cascades that coordinate innate immune defenses . Infection with the intracellular bacterial pathogen Listeria monocytogenes induces CCR2-dependent monocyte recruitment and activation, an essential response for host survival . Herein we show that invasive L . monocytogenes, but not killed or noninvasive bacteria, induce secretion of MCP-1, the requisite chemokine for monocyte recruitment . Induction of MCP-1, but not TNF or IL-12, following L . monocytogenes infection is MyD88 independent . Consistent with these results, MyD88 deficiency does not impair monocyte recruitment to L . monocytogenes infected spleens, but prevents monocyte activation . Our results indicate that distinct microbial signals activate innate immune responses in an ordered, step-wise fashion, providing a mechanism to specify and modulate antimicrobial effector functions.

Immunity, 2003 Dec, 19(6), 793 - 802
Phosphorylation of the Stat1 transactivation domain is required for full-fledged IFN-gamma-dependent innate immunity; Varinou L et al.; Stat1 is phosphorylated on serine 727 within its transactivating domain (TAD) in response to interferons or other immunological signals . We generated gene-targeted mutant mice expressing a serine727-alanine mutant of Stat1 . These animals showed increased mortality upon infection with Listeria monocytogenes and impaired clearance of the bacteria from spleen and liver . The Stat1S727A mice were more resistant to the LPS-induced septic shock syndrome, suggesting that Stat1 serine phosphorylation promotes inflammatory responses . Expression of IFN-gamma-induced genes was strongly reduced in macrophages expressing Stat1(S727A) . While mutation of Stat1 at S727 did not reduce its binding to chromatin, association with the coactivator CBP and histone acetylation at the interferon-responsive GBP promoter was strongly reduced, suggesting defective recruitment of histone acetylases as the mechanism underlying IFN-gamma hyporesponsiveness . Our data demonstrate that the increase in transcription factor activity caused by Stat serine phosphorylation contributes to macrophage activation and to IFN-gamma-dependent immune responses in vivo.

Syst Appl Microbiol, 2003 Nov, 26(4), 539 - 45
Characterisation of persistent and sporadic Listeria monocytogenes strains by pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP); Autio T et al.; This study was set up to evaluate the genetic similarity or dissimilarity of persistent and sporadic Listeria monocytogenes strains existing in eleven food processing facilities, including fish, dairy, meat and poultry processing plants . In each plant persistent and sporadic strains were selected on the basis of PFGE typing results . A total of 17 strains representing persistent strains and 38 sporadic strains originating from eleven food processing plants were included in the study . PFGE macrorestriction patterns of persistent and sporadic strains from different processing plants were compared and the strains were further studied by amplified fragment length polymorphism (AFLP), being a characterisation method giving more whole genome based information . The 17 persistent and 38 sporadic strains showed 14 and 35 pulsotypes, 14 and 28 AFLP types, respectively . The combination of PFGE and AFLP typing results yielded a total of 48 genotypes . Thirteen of 15 genotypes presented by persistent strains were only associated with persistent strains and similarly 94% (33/35) of genotypes showed by sporadic strains were recovered among sporadic strains only . Our results showed that L . monocytogenes strains causing persistent contamination differ from sporadic strains . In AFLP analysis persistent strains did not, however, form any specific clusters and neither was there any difference between the known two genomic groups . These results indicate that even though persistent strains differ from sporadic strains there seems not to be any specific evolutional lineage of persistent strains.

Eur Biophys J, 2004 Jul, 33(4), 310 - 20 Epub 2003 Dec 09.
The effect of diffusion, depolymerization and nucleation promoting factors on actin gel growth; Plastino J et al.; In eukaryotic cells, localized actin polymerization is able to deform the plasma membrane and push the cell forward . Depolymerization of actin filaments and diffusion of actin monomers ensure the availability of monomers at sites of polymerization, and therefore these processes must play an active role in cellular actin dynamics . Here we reveal experimental evidence that actin gel growth can be limited by monomer diffusion, consistent with theoretical predictions . We study actin gels formed on beads coated with ActA (and ActA fragments), the bacterial factor responsible for actin-based movement of Listeria monocytogenes . We observe a saturation of gel thickness with increasing bead radius, the signature of diffusion control . Data analysis using an elastic model of actin gel growth gives an estimate of 2x10(-8) cm(-2) s(-1) for the diffusion coefficient of actin monomers through the gel, ten times less than in buffer, and in agreement with literature values in bulk cytoskeleton, providing corroboration of our model . The depolymerization rate of actin filaments and the elastic modulus of the gel are also evaluated . Furthermore, we qualitatively examine the different actin gels produced when ActA fragments interact with either VASP or the Arp2/3 complex.

J Clin Microbiol, 2003 Dec, 41(12), 5537 - 40
Listeria monocytogenes serotype identification by PCR; Borucki MK et al.; Serotyping is a universally accepted subtyping method for Listeria monocytogenes . Identification of the strain serotype permits differentiation between important food-borne strains (1/2a, 1/2b, and 4b) and provides a "gold standard" for comparing isolates analyzed in different labs and with different techniques . Although an efficient enzyme-linked immunosorbent assay serotyping protocol was described recently, identification of PCR serotyping primers would further increase the ease and accessibility of this classification system . Serotyping PCR primers were designed from variable regions of the L . monocytogenes genome . Three primer sets were used in conjunction with a previously described Division III primer set in order to classify 122 L . monocytogenes strains into five serotype groups {1/2a(3a), 1/2b, 1/2c(3c), 4b(d,e), and 4a/c} . Results of the PCR method agreed with those of the conventional slide agglutination method for 97, 100, 94, and 91% of strains belonging to serotypes 1/2a, 1/2b, 1/2c, and 4b, respectively.

J Immunol, 2003 Dec 15, 171(12), 6488 - 94
The interaction of gamma delta T cells with activated macrophages is a property of the V gamma 1 subset; Dalton JE et al.; Immunoregulation is an emerging paradigm of gammadelta T cell function . The mechanisms by which gammadelta T cells mediate this function, however, are not clear . Studies have identified a direct role for gammadelta T cells in resolving the host immune response to infection, by eliminating populations of activated macrophages . The aim of this study was to identify macrophage-reactive gammadelta T cells and establish the requirements/outcomes of macrophage-gammadelta T cell interactions during the immune response to the intracellular bacterium, Listeria monocytogenes (Lm) . Using a macrophage-T cell coculture system in which peritoneal macrophages from naive or Lm-infected TCRdelta(-/-) mice were incubated with splenocytes from naive and Lm-infected alphabeta/gammadelta T cell-deficient and wild-type mice, the ability to bind macrophages was shown to be restricted to gammadelta T cells and the GV5S1 (Vgamma1) subset of gammadelta T cells . Macrophage adherence resulted in a 4- to 10-fold enrichment of Vgamma1(+) T cells . Enrichment of Vgamma1 T cells was dependent upon the activation status of macrophages, but independent of the activation status of gammadelta T cells . Vgamma1 T cells were cytotoxic for activated macrophages with both the binding to and killing of macrophages being TCR dependent because anti-TCRgammadelta Abs inhibited both Vgamma1 binding and killing activities . These studies establish the identity of macrophage cytotoxic gammadelta T cells, the conditions under which this interaction occurs, and the outcome of this interaction . These findings are concordant with the involvement of Vgamma1 T cells in macrophage homeostasis during the resolution of pathogen-mediated immune responses.

Appl Environ Microbiol, 2003 Dec, 69(12), 7514 - 6
Effect of food processing-related stresses on acid tolerance of Listeria monocytogenes; Koutsoumanis KP et al.; Stationary-phase cells of Listeria monocytogenes grown in glucose-free or glucose-containing media were exposed for 90 min to various stresses, including acid stress (pH 4.0 to 7.0), osmotic stress (10.5 to 20.5% NaCl), and various temperatures (-5 to 50 degrees C), and were further exposed to pH 3.5 . Exposure to a mildly acidic (pH 5.0 to 6.0) environment provided protection of the pathogen against acid upon subsequent exposure . This adaptive response, however, was found to be strongly dependent on other environmental conditions during the shock, such as temperature or the simultaneous presence of a second stress factor (NaCl) . Growth of L . monocytogenes in the presence of glucose resulted in enhanced survival of the pathogen at pH 3.5 . Sublethal stresses other than acidic stresses, i.e., osmotic, heat, and low-temperature stresses, did not affect the acid resistance of L . monocytogenes (P > 0.5) . More-severe levels of these stresses, however, resulted in sensitization of the pathogen to acid.

Appl Environ Microbiol, 2003 Dec, 69(12), 7492 - 8
Role of the glycine betaine and carnitine transporters in adaptation of Listeria monocytogenes to chill stress in defined medium; Angelidis AS et al.; The food-borne pathogen Listeria monocytogenes proliferates at refrigeration temperatures, rendering refrigeration ineffective in the preservation of Listeria-contaminated foods . The uptake and intracellular accumulation of the potent compatible solutes glycine betaine and carnitine has been shown to be a key mediator of the pathogen's cold-tolerant phenotype . To date, three compatible solute systems are known to operate in L . monocytogenes: glycine betaine porter I (BetL), glycine betaine porter II (Gbu), and the carnitine transporter OpuC . We investigated the specificity of each transporter towards each compatible solute at 4 degrees C by examining mutant derivatives of L . monocytogenes 10403S that possess each of the transporters in isolation . Kinetic and steady-state compatible solute accumulation data together with growth rate experiments demonstrated that under cold stress glycine betaine transport is primarily mediated by Gbu and that Gbu-mediated betaine uptake results in significant growth stimulation of chill-stressed cells . BetL and OpuC can serve as minor porters for the uptake of betaine, and their action is capable of providing a small degree of cryotolerance . Under cold stress, carnitine transport occurs primarily through OpuC and results in a high level of cryoprotection . Weak carnitine transport occurs via Gbu and BetL, conferring correspondingly weak cryoprotection . No other transporter in L . monocytogenes 10403S appears to be involved in transport of either compatible solute at 4 degrees C, since a triple mutant strain yielded neither transport nor accumulation of glycine betaine or carnitine and could not be rescued by either osmolyte when grown at that temperature.

Rev Epidemiol Sante Publique, 2003 Oct, 51(5), 493 - 503
{Quantification of the probability of milk contamination by Listeria monocytogenes during manufacture of hard cheese}; Schaffner E et al.; BACKGROUND: The present work is concerned with the probability of contamination by Listeria monocytogenes in the artisanal manufacture of Swiss Emmental hard cheese made from raw milk . The simulation model follows the evolution of the contaminant flora from raw milk at the farm to milk mixing, storage at the cheese factory and to the cheese manufacturing process . METHODS: The simulations are based on models of predictive microbiology, namely the exponential growth model of bacteria including the lag-time, a cardinal growth model and a Log-linear model of thermal deactivation of bacteria . RESULTS: The results of the actual simulation indicate that the contamination of milk at the farm is a rare event (P=0.0036), but the mixing of milk at the cheese factory leads to a higher probability of contamination of cheese milk (P=0.07) . Elevated bacterial concentrations are mainly due to cases of mastitis involving Listeria monocytogenes . The decline in bacterial counts during cheese manufacture depends on the curing temperature (52-54 degrees C) and varies between 1.5 and 3.2 Log cfu/ml . Freshly manufactured Emmental-cheese made from contaminated raw milk is expected to have only 4.6 cfu of heat injured Listeria monocytogenes /kg cheese mass in the press . CONCLUSION: Depending on listeria evolution, from the press to the product consumption, consumer exposure has been evaluated and might result in 1 to 10 Listeria monocytogenes per portion of cheese . The bacterial presence could be due to recontamination during packaging, distribution and cheese preparation by the consumer . Based on the presented data and estimations, it is concluded that the consumption of traditionally/artisanal manufactured Swiss Emmental hard cheese presents an extremely low, but existent risk, especially for people with a deficient or diminished immune system.

Toxicol Sci, 2004 Feb, 77(2), 263 - 71 Epub 2003 Dec 02.
Suppression of cell-mediated immune responses to listeria infection by repeated exposure to diesel exhaust particles in brown Norway rats; Yin XJ et al.; Diesel exhaust particles (DEP) have been shown to alter pulmonary immune responses to bacterial infection . Exposure of rats to 100 mg/m(3) DEP for 4 h was found to aggravate Listeria monocytogenes(Listeria) infection at 3 days postinfection, but the bacteria were largely cleared at 7 days postinfection due to the development of a strong T cell-mediated immunity . In the present study, we examined the effects of repeated DEP exposure at lower doses on pulmonary responses to bacterial infection . Brown Norway rats were exposed to DEP by inhalation at 20.62 +/- 1.31 mg/m 3 for 4 h/day for 5 days, followed by intratracheal inoculation with 100,000 Listeria at 2 h after the last DEP exposure . DEP-exposed rats showed a significant increase in lung bacterial load at both 3 and 7 days postinfection . The repeated DEP exposure was shown to suppress both the innate, orchestrated by alveolar macrophages (AM), and T cell-mediated responses to Listeria . DEP inhibited AM production of interleukin- (IL-) 1beta, tumor necrosis factor- (TNF-) alpha, and IL-12 but enhanced Listeria-induced AM production of IL-10, which has been shown to prolong the survival of intracellular pathogens such as Listeria . DEP exposure also suppressed the development of bacteria-specific lymphocytes from lung-draining lymph nodes, as indicated by the decreased numbers of T lymphocytes and their CD4(+) and CD8(+) subsets . Furthermore, the DEP exposure markedly inhibited the Listeria-induced lymphocyte secretion of IL-2 at day 7, IL-10 at days 3 and 7, and interferon- (IFN-) gamma at days 3 to 10 postinfection when compared to air-exposed controls . These results show a sustained pattern of downregulation of T cell-mediated immune responses by repeated low-dose DEP exposure, which is different from the results of a single high-dose exposure where the acute effect of DEP aggravated bacteria infection but triggered a strong T cell-mediated immunity.

Oper Dent, 2003 Nov-Dec, 28(6), 740 - 6
Effect of mouthrinses on microhardness and wear of composite and compomer restoratives; Yap AU et al.; This study investigated the effect of commercially available mouthrinses on the microhardness and wear of composite (Esthet-X, Dentsply) and compomer (Dyract Posterior, Dentsply) restoratives . Fifty-four hardness and 36 wear specimens of each material were fabricated and stored in distilled water at 37 degrees C for two weeks . The specimens were then randomly divided into six equal groups and exposed to the following solutions for 24 hours at 37 degrees C: distilled water {WC} (control); Listerine Original {AP} (alcohol-containing essential oil/phenolic compound mouthrinse); Colgate Chloropharm {AC} (alcohol-containing chlorhexidine mouthrinse); Oral B Tooth & Gum Care {AF} (alcohol-containing fluoride mouthrinse); Oral B Tooth & Gum Care Alcohol Free {OF} (alcohol free fluoride mouthrinse) and Oral B Sensitive {PF} (phosphoric acid containing fluoride mouthrinse) . After conditioning, the specimens were subjected to hardness testing using a digital microhardness tester (load = 500 gf; dwell time = 15 seconds) and wear testing with a reciprocal compression-sliding system (contact stress = 20 MPa) . Wear depth was measured every 1,000 cycles up to 10,000 cycles using profilometry . Data was analyzed using ANOVA/Scheffe's test at significance level 0.05 . Dyract was significantly softened by AP, while Esthet-X was significantly softened by AC and AP . The wear resistance of Dyract was significantly reduced after exposure to PF, while the wear resistance of Esthet-X was significantly reduced by AC . The effect of mouthrinses on hardness and wear was material dependent.

J Bacteriol, 2003 Dec, 185(24), 7140 - 4
Transcriptional regulation and posttranslational activity of the betaine transporter BetL in Listeria monocytogenes are controlled by environmental salinity; Sleator RD et al.; While the genetic elements contributing to the salinity tolerance of Listeria monocytogenes have been well characterized, the regulatory signals and responses (genetic and/or biochemical) that govern these mechanisms have yet to be elucidated . Encoded by betL, the first genetic element to be linked to listerial osmotolerance, the secondary betaine uptake system BetL is a member of the betaine-carnitine-choline transporter family . Preceded by consensus sigma(A)- and sigma(B)-dependent promoter sites, betL is constitutively expressed and transcriptionally up-regulated in response to salt stress . The nisin-controlled expression system was used to achieve salinity-independent, controlled betL expression in LISTERIA: In the absence of NaCl-activated transcriptional control, BetL activity was found to be a function of environmental salinity, showing optimal activity in buffer supplemented with 1 to 2% NaCl (osmolality, 417 to 719 mosmol/kg) . In addition, BetL was activated rapidly (half-life, 2 min) in response to an osmotic upshift imposed by adding 2% NaCl to 50 mM potassium phosphate buffer.

Cell Microbiol, 2003 Dec, 5(12), 901 - 11
Exploration of host-pathogen interactions using Listeria monocytogenes and Drosophila melanogaster; Mansfield BE et al.; The facultative intracellular bacterial pathogen Listeria monocytogenes is capable of replicating within a broad range of host cell types and host species . We report here the establishment of the fruit fly Drosophila melanogaster as a new model host for the exploration of L . monocytogenes pathogenesis and host response to infection . Listeria monocytogenes was capable of establishing lethal infections in adult fruit flies and larvae with extensive bacterial replication occurring before host death . Bacteria were found in the cytosol of insect phagocytic cells, and were capable of directing host cell actin polymerization . Bacterial gene products necessary for intracellular replication and cell-to-cell spread within mammalian cells were similarly found to be required within insect cells, and although previous work has suggested that L . monocytogenes virulence gene expression requires temperatures above 30 degrees C, bacteria within insect cells were found to express virulence determinants at 25 degrees C . Mutant strains of Drosophila that were compromised for innate immune responses demonstrated increased susceptibility to L . monocytogenes infection . These data indicate L . monocytogenes infection of fruit flies shares numerous features of mammalian infection, and thus that Drosophila has the potential to serve as a genetically tractable host system that will facilitate the analysis of host cellular responses to L . monocytogenes infection.

Cell Microbiol, 2003 Dec, 5(12), 875 - 85
Drosophila S2 cells: an alternative infection model for Listeria monocytogenes; Cheng LW et al.; Listeria monocytogenes is a Gram-positive facultative intracellular bacterial pathogen that infects humans and animals . Its pathogenic strategy involves the expression of virulence proteins that mediate intracytosolic growth and cell-to-cell spread . A key virulence protein is the cholesterol-dependent cytolysin, listeriolysin O (LLO), which is largely responsible for mediating escape from the phagosome into the host cytosol . To study further the host processes exploited during L . monocytogenes infection, we sought to develop Drosophila S2 cells as a model for infection . Here, we show that S2 cells share a number of properties with mammalian cell culture models of infection . As with mouse macrophages, LLO was required for phagosomal escape from S2 cells . Furthermore, vacuolar escape was dependent on their acidification via the ATPase proton pumps, as bafilomycin A1 treatment sharply decreased escape . However, unlike in mouse macrophages, LLO mutants replicated in the phagosome of S2 cells . Drosophila cells are cholesterol auxotrophs, and exogenous cholesterol increased the infection rate of L . monocytogenes (LLO independent) and also augmented the efficiency of vacuolar escape (LLO dependent) . With available genetic tools such as RNA interference, S2 cells could become an important model in the study of host-pathogen interactions.

J Pediatr (Rio J), 2000 Mar, 76(2), 115 - 118
{Tuberculin test in the diagnosis of childhood tuberculosis: Analysis of quantitative and qualitative features}; Santacute;Anna CC et al.; OBJECTIVE: To evaluate the role of the tuberculin test in the diagnosis of tuberculosis in children . METHODS: Test diagnosis study; Tuberculin test with PPD Rt 23 (2 UT) was performed in 158 children, distributed in 2 groups: 101 no tuberculous, BCG vaccinated children and 57 tuberculous ones (diagnosis by clinical-radiological and epidemiological features) . The interpretation of the tuberculin test was made by quantitative analysis (Mantoux test) and qualitative analysis (Koch and Listeria phenomena) . RESULTS: Using cutoff = 10mm in Mantoux test, we found sensitivity of 85.9% and specificity of 86.1% . The qualitative analysis (Koch phenomenon), showed sensitivity of 77.2% and specificity of 98% . CONCLUSION: The qualitative analysis of the tuberculin test was useful in the diagnosis of tuberculosis in children, associated to the Mantoux test interpretation.

Blood, 2004 Mar 15, 103(6), 2214 - 20 Epub 2003 Nov 26.
Mast cell-mediated inflammatory responses require the alpha 2 beta 1 integrin; Edelson BT et al.; Although the alpha 2 beta 1 integrin is widely expressed and has been extensively studied, it has not been previously implicated in mast cell biology . We observed that alpha 2 integrin subunit-deficient mice exhibited markedly diminished neutrophil and interleukin-6 responses during Listeria monocytogenes- and zymosan-induced peritonitis . Since exudative neutrophils of wild-type mice expressed little alpha 2 beta 1 integrin, it seemed unlikely that this integrin mediated neutrophil migration directly . Here, we demonstrate constitutive alpha 2 beta 1 integrin expression on peritoneal mast cells . Although alpha 2-null mice contain normal numbers of peritoneal mast cells, these alpha 2-null cells do not support in vivo mast cell-dependent inflammatory responses . We conclude that alpha 2 beta 1 integrin provides a costimulatory function required for mast cell activation and cytokine production in response to infection.

Infect Immun, 2003 Dec, 71(12), 6754 - 65
Listeria monocytogenes mutants that fail to compartmentalize listerolysin O activity are cytotoxic, avirulent, and unable to evade host extracellular defenses; Glomski IJ et al.; Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a phagosome and grows in the host cell cytosol . Escape of the bacterium from the phagosome to the cytosol is mediated by the bacterial pore-forming protein listeriolysin O (LLO) . LLO has multiple mechanisms that optimize activity in the phagosome and minimize activity in the host cytosol . Mutants that fail to compartmentalize LLO activity are cytotoxic and have reduced virulence . We sought to determine why cytotoxic bacteria have attenuated virulence in the mouse model of listeriosis . In this study, we constructed a series of strains with mutations in LLO and with various degrees of cytotoxicity . We found that the more cytotoxic the strain in cell culture, the less virulent it was in mice . Induction of neutropenia increased the relative virulence of the cytotoxic strains 100-fold in the spleen and 10-fold in the liver . The virulence defect was partially restored in neutropenic mice by adding gentamicin, an antibiotic that kills extracellular bacteria . Additionally, L . monocytogenes grew more slowly in extracellular fluid (mouse serum) than within tissue culture cells . We concluded that L . monocytogenes controls the cytolytic activity of LLO to maintain its nutritionally rich intracellular niche and avoid extracellular defenses of the host.

Infect Immun, 2003 Dec, 71(12), 6721 - 7
The Listeria monocytogenes lemA gene product is not required for intracellular infection or to activate fMIGWII-specific T cells; D'Orazio SE et al.; Clearance of the intracellular bacterial pathogen Listeria monocytogenes requires antigen-specific CD8(+) T cells . Recently it was shown that activation of class Ib major histocompatibility complex (MHC)-restricted CD8(+) T cells alone is sufficient for immune protection against listeriae . A major component of the class Ib MHC-restricted T-cell response is T cells that recognize formylated peptide antigens presented by M3 molecules . Although three N-formylated peptides derived from L . monocytogenes are known to bind to M3 molecules, fMIGWII is the immunodominant epitope presented by M3 during infection of mice . The source of fMIGWII peptide is the L . monocytogenes lemA gene, which encodes a 30-kDa protein of unknown function . In this report, we describe the generation of two L . monocytogenes lemA deletion mutants . We show that lemA is not required for growth of listeriae in tissue culture cells or for virulence during infection of mice . Surprisingly, we found that fMIGWII-specific T cells were still primed following infection with lemA mutant listeriae, suggesting that L . monocytogenes contains at least one additional antigen that is cross-reactive with the fMIGWII epitope . This cross-reactive antigen appears to be a small protease-resistant molecule that is secreted by L . monocytogenes.

Immunol Cell Biol, 2003 Dec, 81(6), 431 - 439
Listeria species escape from the phagosomes of interleukin-4-deactivated human macrophages independent of listeriolysin; Neumann K et al.; Listeria monocytogenes is the causative agent of infections like sepsis and meningitis, especially in immunocompromised hosts . Human macrophages are able to phagocytose and digest L . monocytogenes but IL-4 prevents human macrophages from killing the bacteria, the mechanisms of which are unknown . In the present study, we examined various listeria species and strains including wild-type and deletion mutants in human macrophages pretreated with IL-4 . To analyse the IL-4-mediated deactivation process, we combined quantitative infection assays with various morphologic methods . IL-4 facilitates survival and escape of the pathogenic L . monocytogenes wild-type strain 10403S from the macrophage phagosomes . In untreated macrophages, the isogenic listeriolysin deletion mutant strain DP-L2161 was killed and did not escape from the phagolysosomes . However, after macrophage deactivation with IL-4 DP-L2161 survived and escaped from the phagosomes . This was also the case, but to a lesser extent, even for the naturally avirulent L . innocua . As detected by confocal laser-scanning fluorescence microscopy and electron microscopy, IL-4 permitted the escape of all listeria species tested, including DP-L2161 and L . innocua from the phagosomal compartment of the macrophages . We conclude that escape from the phagosome and survival of the listeria species tested in IL-4-deactivated human macrophages is independent of the virulence factor listeriolysin.

J Evol Biol, 2003 Mar, 16(2), 289 - 301
Frequency dependence in matings with water-borne sperm; Pemberton AJ et al.; Negative frequency-dependent mating success--the rare male effect--is a potentially powerful evolutionary force, but disagreement exists as to whether previous work, focusing on copulating species, has robustly demonstrated this phenomenon . Noncopulating sessile organisms that release male gametes into the environment but retain their eggs for fertilization may routinely receive unequal mixtures of sperm . Although promiscuity seems unavoidable it does not follow that the resulting paternity obeys 'fair raffle' expectations . This study investigates frequency dependence in the mating of one such species, the colonial ascidian Diplosoma listerianum . In competition with an alternative sperm source males fathered more progeny if previously mated to a particular female than if no mating history existed . This suggests positive frequency-dependent selection, but may simply result from a mate order effect involving sperm storage . With fewer acclimation matings, separated by longer intervals, this pattern was not found . When, in a different experimental design, virgin females were given simultaneous mixtures of gametes at widely divergent concentrations, sperm at the lower frequency consistently achieved a greater than expected share of paternity--a rare male effect . A convincing argument as to why D . listerianum should favour rare sperm has not been identified, as sperm rarity is expected to correlate very poorly with ecological or genetic male characteristics in this pattern of mating . The existence of nongenetic female preferences at the level of colony modules, analogous in effect to fixed female preferences, is proposed . If visible to selection, indirect benefits from increasing the genetic diversity of a sibship appear the only likely explanation of the rare male effect in this system as the life history presents virtually no costs to multiple mating, and a near absence of direct (resource) benefits, whereas less controversial hypotheses of female promiscuity (e.g . trade up, genetic incompatibility) do not seem appropriate.

J Immunol, 2003 Dec 1, 171(11), 6032 - 8
Activation of antigen-specific CD8 T cells results in minimal killing of bystander bacteria; Jiang J et al.; Memory CD8 T cells play a critical role in protective immunity against intracellular pathogens . In addition to their ability to specifically recognize and lyse infected targets, activated CD8 T cells secrete cytokines that induce phagocytic cells to engulf and kill bacterial pathogens . In this study, we asked whether activation of Ag-specific CD8 T cells results in nonspecific killing of bystander bacteria during a mixed infection . Mice with epitope-specific memory CD8 T cells were coinfected with two isogenic strains of recombinant Listeria monocytogenes that differ in the cognate epitope . Recall responses by epitope-specific CD8 T cells rapidly inhibited the growth of epitope-bearing bacteria, impeding the course of infection within 6 h after challenge . This rapid inhibition was highly specific and did not affect the growth of coinfecting bacteria without the epitope . CTL recall did not enhance activation of innate immune cells, as evidenced by the absence of inducible NO synthase production in infectious foci . Our observations demonstrate the remarkable specificity of the bactericidal mechanisms of CTL and reveal the possibility for escape mutants to prevail in the hostile environment of a specific immune response . This implication has a bearing on subunit vaccine design strategies and understanding failure of immunization against bacterial infection.

J Immunol, 2003 Dec 1, 171(11), 5948 - 55
Promiscuity of MHC class Ib-restricted T cell responses; Ploss A et al.; Murine infection with the Gram-positive intracellular bacterium Listeria monocytogenes activates CD8(+) T cells that recognize bacterially derived N-formyl methionine peptides in the context of H2-M3 MHC class Ib molecules . Three peptides, fMIGWII, fMIVIL, and fMIVTLF, are targets of L . monocytogenes-specific CD8(+) T cells . To investigate epitope cross-recognition by H2-M3-restricted CD8(+) T cells, we deleted the sequence encoding fMIGWII from a virulent strain of L . monocytogenes . Infection with fMIGWII-deficient L . monocytogenes unexpectedly primed CD8(+) T cells that stain with fMIGWII/H2-M3 tetramers and lyse fMIGWII-coated target cells in vivo . Because the fMIGWII sequence is nonredundant, we speculated that other bacterially derived Ags are priming these responses . HPLC peptide fractionation of bacterial culture supernatants revealed several distinct L . monocytogenes-derived peptides that are recognized by fMIGWII-specific T cells . Our results demonstrate that the dominant H2-M3-restricted CD8(+) T cell population, although reactive with fMIGWII, is primed by other, non-fMIGWII peptides derived from L . monocytogenes . Although this degree of Ag receptor promiscuity is unusual for the adaptive immune system, it may be a more common feature of T cell responses restricted by nonpolymorphic MHC class Ib molecules.

J Appl Microbiol, 2003, 95(5), 958 - 66
PCR detection of Listeria monocytogenes: a study of multiple factors affecting sensitivity; Aznar R et al.; AIMS: To test, under comparable conditions, several parameters affecting sensitivity of PCR detection in order to establish a PCR procedure suitable for the routine detection of Listeria monocytogenes in food . METHODS AND RESULTS: Beef samples artificially inoculated were used to determine sensitivity of PCR detection under different parameters . As few as 1 CFU g(-1) were detected by DNA extraction using a DNeasy Tissue Kit (Qiagen GmhH, Hilden, Germany) of 1 ml aliquot and PCR amplification with primers directed to the hlyA gene . This PCR protocol was applied in 60 naturally contaminated foods, comparing two enrichment procedures with the traditional culture method . The highest number of positives was recorded by PCR following a 24-h pre-enrichment step at 30 degrees C and a 24-h enrichment step at 37 degrees C . Afterwards, it was applied in 217 naturally contaminated foods and 56 of them tested positive for L . monocytogenes in which only 17 tested positive using the culture method . CONCLUSIONS: The PCR procedure described has proved to be a rapid and sensitive method suitable for the routine analysis of different types of food . SIGNIFICANCE AND IMPACT OF THE STUDY: The method proposed for the detection of L . monocytogenes, has been validated in naturally contaminated food and is suitable to implement in the food industry.

J Food Prot, 2003 Nov, 66(11), 2062 - 9
Persistent and nonpersistent Listeria monocytogenes contamination in meat and poultry processing plants; Lunden JM et al.; Contamination analysis of persistent and nonpersistent Listeria monocytogenes strains in three meat processing plants and one poultry processing plant were performed in order to identify factors predisposing to or sustaining persistent plant contamination . A total of 596 L . monocytogenes isolates were divided into 47 pulsed-field gel electrophoresis (PFGE) types by combining the restriction enzyme patterns of AscI (42 patterns) and ApaI (38 patterns) . Persistent and nonpersistent strains were found in all plants . Nonpersistent PFGE types were found mostly at one sampling site, with the processing environment being the most common location, whereas the persistent strains were found at several sampling sites in most cases . The processing machines were frequently contaminated with persistent L . monocytogenes PFGE types, and it was of concern that surfaces having direct contact with the products were contaminated . The role of the processing machines in sustaining contamination and in contaminating the products appeared to be important because the final product of several processing lines was contaminated with the same L . monocytogenes PFGE type as that found in the processing machine . The proportion of persistent PFGE types in heat-treated products was eight times higher than in the raw products, showing the importance of the persistent PFGE types as contaminants of the final heat-treated products . The contamination status of the processing lines and machines appeared to be influenced by the compartmentalization of the processing line, with poor compartmentalization increasing L . monocytogenes contamination . The separation of raw and post-heat treatment areas seemed especially important in the contamination status of post-heat treatment lines.

J Food Prot, 2003 Nov, 66(11), 2057 - 61
Pressure death and tailing behavior of Listeria monocytogenes strains having different barotolerances; Tay A et al.; The objectives of this study were to investigate the variability among Listeria monocytogenes strains in response to high-pressure processing, identify the most resistant strain as a potential target of pressure processing, and compare the inactivation kinetics of pressure-resistant and pressure-sensitive strains under a wide range (350 to 800 MPa) of pressure treatments . The pressure resistance of Listeria innocua and nine strains of L . monocytogenes was compared at 400 or 500 MPa and 30 degrees C . Significant variability among strains was observed . The decrease in log CFU/ml during the pressure treatment was from 1.4 to 4.3 at 400 MPa and from 3.9 to >8 at 500 MPa . L . monocytogenes OSY-8578 exhibited the greatest pressure resistance, Scott A showed the greatest pressure sensitivity, and L . innocua had intermediate resistance . On the basis of these findings, L . monocytogenes OSY-8578 is a potential target strain for high-pressure processing efficacy studies . The death kinetics of L . monocytogenes Scott A and OSY-8578 were investigated at 350 and 800 MPa . Survivors at 350 MPa were enumerated by direct plating, and survivors at 800 MPa were enumerated by the most-probable-number technique . Both pressure-resistant and pressure-sensitive strains exhibited non-first-order death behavior, and excessive pressure treatment did not eliminate the tailing phenomenon.

J Food Prot, 2003 Nov, 66(11), 2051 - 6
Radiation (gamma) resistance and postirradiation growth of Listeria monocytogenes suspended in beef bologna containing sodium diacetate and potassium lactate; Sommers C et al.; Listeria monocytogenes, a psychrotrophic foodborne pathogen, is a frequent postprocessing contaminant of ready-to-eat (RTE) meat products, including frankfurters and bologna . Ionizing radiation can eliminate L . monocytogenes from RTE meats . When they are incorporated into fine-emulsion sausages, sodium diacetate (SDA) and potassium lactate (PL) mixtures inhibit the growth of L . monocytogenes . The radiation resistance of L . monocytogenes, and its ability to proliferate during long-term refrigerated storage (9 degrees C), when inoculated into beef bologna that contained 0% SDA-0% PL, 0.07% SDA-1% PL, and 0.15% SDA-2% PL, were determined . The radiation doses required to eliminate 90% of the viable L . monocytogenes cells were 0.56 kGy for bologna containing 0% SDA-0% PL, 0.53 kGy for bologna containing 0.07% SDA-1% PL, and 0.46 kGy for bologna containing 0.15% SDA-2% PL . L . monocytogenes was able to proliferate on bologna containing 0% SDA-0% PL during refrigerated storage, but the onset of proliferation was delayed by the addition of the SDA-PL mixtures . An ionizing radiation dose of 3.0 kGy prevented the proliferation of L . monocytogenes and background microflora in bologna containing 0.07% SDA-1% PL and in bologna containing 0.15% SDA-2% PL over 8 weeks of storage at 9 degrees C . Little effect on lipid oxidation and color of the control bologna, or bologna containing SDA-PL mixtures, was observed upon irradiation at either 1.5 or 3.0 kGy.

Int J Hyg Environ Health, 2003 Oct, 206(6), 583 - 90
The possible effect of a sanitization program on intraspecies differentiation of Listeria monocytogenes strains isolated from a fish processing plant; Medrala D et al.; Forty-seven Listeria monocytogenes strains isolated during a year in a selected Polish fish-processing plant as well as 7 L . monocytogenes strains of different origins (including a reference strain) were analyzed in our studies . Strains were isolated from raw fish fillets (flounder), frozen coated flounder fillets, coating ingredients, and the processing environment . Isolation of strains covered the period of a sanitization program introduced in the plant . L . monocytogenes was identified using conventional microbiological methods and the PCR technique . RAPD (random amplified polymorphic DNA) technique for fingerprinting was applied to analyze the intraspecies diversity . Six RAPD types (A-F) and seven unique strains were revealed as a result of fingerprinting with one persistent type isolated from July 1999 to February 2000 . It was detected for the first time after one month of sanitization . Its occurrence could have been promoted by clone selection either due to ineffective disinfection or to resistance against the disinfectant . As L . monocytogenes mostly occurred on frozen products, this indicates that contamination could start during product freezing, cold storage, or handling . The results revealed that there is a crucial need for preparing sanitization schemes precisely targeted at L . monocytogenes to avoid its recurrence as persistent 'in-house' strains . The possibility of incorrect interpretations of classical microbiological test results as well as the necessity to introduce assays based on nucleic acid analysis into epidemiological investigations were emphasized.

J Exp Med, 2003 Nov 17, 198(10), 1583 - 93
Memory CD8+ T cells provide innate immune protection against Listeria monocytogenes in the absence of cognate antigen; Berg RE et al.; Interferon (IFN)-gamma plays an important role in the innate immune response against intracellular bacterial pathogens . It is commonly thought that natural killer cells are the primary source of this cytokine that is involved in activating antibacterial effects in infected cells and polarizing CD4+ T cells toward the Th1 subset . However, here we show that both effector and memory CD8+ T cells have the potential to secrete IFN-gamma in response to interleukin (IL)-12 and IL-18 in the absence of cognate antigen . We demonstrate that memory CD8+ T cells specific for the ovalbumin protein secrete IFN-gamma rapidly after infection with wild-type Listeria monocytogenes (LM) . Furthermore, small numbers of ovalbumin-specific, memory CD8+ T cells can reduce spleen and liver bacterial counts in IFN-gamma-deficient mice 3 d after LM infection . Up-regulation of the receptors for IL-12 and IL-18 provides a mechanism for the ability of memory CD8+ T cells to respond in this antigen nonspecific manner . Thus, CD8+ T cells play an important role in the innate immune response against intracellular pathogens by rapidly secreting IFN-gamma in response to IL-12 and IL-18.

Int J Food Microbiol, 2003 Dec 31, 89(2-3), 287 - 90
The VIT technology for rapid detection of Listeria monocytogenes and other Listeria spp; Stephan R et al.; Detection of Listeria monocytogenes is generally performed in a two-step cultural enrichment process and takes on average 1 week until the biochemical identification of a L . monocytogenes suspicious colony is completed . However, food processing companies depend increasingly on test methods, which attempt to generate results comparable to standard methods but in reduced time-frame and which allow to release produced batches dependent on such results . In the present study, the vermicon identification technology (VIT), a rapid commercial test system using fluorescently labelled gene probes, was compared to a cultural standard method . In total, 298 naturally contaminated samples were analysed . The sensitivity and the specificity of the VIT system were 100% for the detection of L . monocytogenes and 97.1% and 100%, respectively, for the detection of the genus Listeria.

J Bacteriol, 2003 Dec, 185(23), 6801 - 8
Identification and characterization of a peptidoglycan hydrolase, MurA, of Listeria monocytogenes, a muramidase needed for cell separation; Carroll SA et al.; A novel cell wall hydrolase encoded by the murA gene of Listeria monocytogenes is reported here . Mature MurA is a 66-kDa cell surface protein that is recognized by the well-characterized L . monocytogenes-specific monoclonal antibody EM-7G1 . MurA displays two characteristic features: (i) an N-terminal domain with homology to muramidases from several gram-positive bacterial species and (ii) four copies of a cell wall-anchoring LysM repeat motif present within its C-terminal domain . Purified recombinant MurA produced in Escherichia coli was confirmed to be an authentic cell wall hydrolase with lytic properties toward cell wall preparations of Micrococcus lysodeikticus . An isogenic mutant with a deletion of murA that lacked the 66-kDa cell wall hydrolase grew as long chains during exponential growth . Complementation of the mutant strain by chromosomal reintegration of the wild-type gene restored expression of this murein hydrolase activity and cell separation levels to those of the wild-type strain . Studies reported herein suggest that the MurA protein is involved in generalized autolysis of L . monocytogenes.

Immunity, 2003 Nov, 19(5), 701 - 11
GATA-3 promotes maturation, IFN-gamma production, and liver-specific homing of NK cells; Samson SI et al.; The GATA-3 transcription factor has a determinant role in T cell specification and is an essential mediator of T helper 2-type polarized immune responses . While both committed NK precursors and mature NK cells express GATA-3, a role of this transcription factor in murine NK cell differentiation is not known . We found that NK cells, in contrast to T cells, can be generated in the absence of GATA-3 . However, while GATA-3 antagonizes IFN-gamma production in differentiating T cells, GATA-3-deficient NK cells paradoxically produced less IFN-gamma compared to control NK cells and failed to provide early protection in vivo against infection with Listeria monocytogenes . Surprisingly, GATA-3 was essential for NK cell homing to the liver . Our results suggest that GATA-3 promotes NK cell maturation and acts in this lineage to specify distinct effector phenotypes.

J Vet Sci, 2000 Dec, 1(2), 77 - 80
Immunohistochemical study of constitutive neuronal and inducible nitric oxide synthase in the central nervous system of goat with natural listeriosis; Shin T et al.; The expression of both constitutive and inducible forms of nitric oxide synthase (NOS) was investigated by immunohistochemical staining of formalin-fixed paraffin-embedded sections in normal and Listeria monocytogenes-infected brains of goats . In normal control goats, a small number of neurons showed immunoreactivity of both iNOS and nNOS, and the number of iNOS-positive neurons was higher than the number of nNOS-positive neurons . In natural listeriosis, listeria antigens were easily immunostained in the inflammatory cells of microabscesses . In this lesion, the immunoreactivity of iNOS in neurons was more intense than the control, but nNOS was not . In microabscesses, nNOS was weakly visualized in macrophages and neutrophils, while iNOS was expressed in macrophages, but not in neutrophils . These findings suggest that normal caprine brain cells, including neurons, constitutively express iNOS and nNOS, and the expressions of these molecules is increased in Listeria monocytogenes infections . Furthermore, inflammatory cells, including macrophages, expressing both nNOS and iNOS may play important roles in the pathogenesis of bacterial meningoencephalitis in goat.

J Med Microbiol, 2003 Dec, 52(Pt 12), 1065 - 70
Characterization of virulent and avirulent Listeria monocytogenes strains by PCR amplification of putative transcriptional regulator and internalin genes; Liu D et al.; Listeria monocytogenes is an opportunistic bacterial pathogen that is an important cause of human food-borne illness worldwide . However, L . monocytogenes strains demonstrate considerable variation in pathogenic potential . In this report, virulent and avirulent L . monocytogenes isolates were compared by using a comparative screening strategy . Two clones were identified that contained DNA that was only present in virulent L . monocytogenes strains . PCR primers were designed for three genes from these clones and for five other selected L . monocytogenes genes . All eight primer sets predominantly detected virulent L . monocytogenes isolates, as determined by a mouse virulence assay; one of the putative internalin genes, lmo2821, was detected in all strains that were considered to be virulent . Primers from these eight genes were then tested by PCR against a larger panel of bacterial strains; each of the genes was detected predominantly in clinical or food L . monocytogenes isolates, rather than environmental isolates . The findings from this study suggest that virulent L . monocytogenes strains may possess genes that are not present in avirulent isolates, which could serve as markers for PCR assessment of L . monocytogenes virulence.

EMBO J, 2003 Nov 17, 22(22), 6161 - 73
Beta-casein-derived peptides, produced by bacteria, stimulate cancer cell invasion and motility; Oliveira MJ et al.; In colon cancer, enteric bacteria and dietary factors are major determinants of the microenvironment but their effect on cellular invasion is not known . We therefore incubated human HCT-8/E11 colon cancer cells with bacteria or bacterial conditioned medium on top of collagen type I gels . Listeria monocytogenes stimulate cellular invasion through the formation of a soluble motility-promoting factor, identified as a 13mer beta-casein-derived peptide (HKEMPFPKYPVEP) . The peptide is formed through the combined action of Mpl, a Listeria thermolysin-like metalloprotease, and a collagen-associated trypsin-like serine protease . The 13mer peptide was also formed by tumour biopsies isolated from colon cancer patients and incubated with a beta-casein source . The pro- invasive 13mer peptide-signalling pathway implicates activation of Cdc42 and inactivation of RhoA, linked to each other through the serine/threonine p21- activated kinase 1 . Since both changes are necessary but not sufficient, another pathway might branch upstream of Cdc42 at phosphatidylinositol 3-kinase . Delta opioid receptor (deltaOR) is a candidate receptor for the 13mer peptide since naloxone, an deltaOR antagonist, blocks both deltaOR serine phosphorylation and 13mer peptide-mediated invasion.

J Immunol, 2003 Nov 15, 171(10), 5454 - 60
Increased CD8+ T cell memory to concurrent infection at the expense of increased erosion of pre-existing memory: the paradoxical role of IL-15; Chapdelaine Y et al.; The use of cytokines during vaccination, particularly IL-15, is being considered due to the unique ability of IL-15 to enhance the proliferation of memory CD8(+) T cells . However, as homeostatic mechanisms limit excessive lymphocyte expansion, we addressed the consequences of this enhancement of T cell memory by IL-15 . Infection of mice with either recombinant Mycobacterium bovis (BCG) expressing IL-15 (BCG-IL-15) or BCG and purified IL-15 resulted in an increased CD44, IL-2Rbeta expression and increased frequency of IFN-gamma-secreting CD8(+) T cells . Surprisingly, the enhancement of memory to concurrent infection by IL-15 exacerbated the attrition of pre-existing memory . Infection of mice with Listeria monocytogenes expressing OVA resulted in potent OVA(257-264)-specific CD8(+) T cell memory, and a challenge of these mice with either BCG-IL-15 or BCG and purified IL-15 resulted in an increased erosion of OVA(257-264)-specific CD8(+) T cell memory, relative to BCG . Enhancement in the erosion of OVA-specific CD8(+) T cell memory by BCG-IL-15 resulted in a consequently greater impairment in protection against a challenge with OVA-expressing tumor cells . We thus raise important questions regarding vaccinations that are aimed at maximizing T cell memory without considering the impact on pre-existing T cell memory.

J Immunol, 2003 Nov 15, 171(10), 5447 - 53
Localized reactive oxygen and nitrogen intermediates inhibit escape of Listeria monocytogenes from vacuoles in activated macrophages; Myers JT et al.; Listeria monocytogenes (Lm) evades being killed after phagocytosis by macrophages by escaping from vacuoles into cytoplasm . Activated macrophages are listericidal, in part because they can retain Lm in vacuoles . This study examined the contribution of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) to the inhibition of Lm escape from vacuoles . Lm escaped from vacuoles of nonactivated macrophages within 30 min of infection . Macrophages activated with IFN-gamma, LPS, IL-6, and a neutralizing Ab against IL-10 retained Lm within the vacuoles, and inhibitors of ROI and RNI blocked inhibition of vacuolar escape to varying degrees . Measurements of Lm escape in macrophages from gp91(phox-/-) and NO synthase 2(-/-) mice showed that vacuolar retention required ROI and was augmented by RNI . Live cell imaging with the fluorogenic probe dihydro-2',4,5,6,7,7'-hexafluorofluorescein coupled to BSA (DHFF-BSA) indicated that oxidative chemistries were generated rapidly and were localized to Lm vacuoles . Chemistries that oxidized DHFF-BSA were similar to those that retained Lm in phagosomes . Fluorescent conversion of DHFF-BSA occurred more efficiently in smaller vacuoles, indicating that higher concentrations of ROI or RNI were generated in more confining volumes . Thus, activated macrophages retained Lm within phagosomes by the localization of ROI and RNI to vacuoles, and by their combined actions in a small space

J Microbiol Methods, 2003 Dec, 55(3), 821 - 7
Distribution of turbidity detection times produced by single cell-generated bacterial populations; Metris A et al.; The distributions of the times to turbidity for wells inoculated with single cells of Listeria innocua were determined in different environmental conditions (pH 4.5 to 7 and with 0.5% to 8% of NaCl at 30 degrees C) . It was established by statistical analysis that the main source of the variability of the detection times, T, is the variability of individual lag times . A linear relation dev(T) approximately T was observed between the detection times and their standard deviation . At slow growth, other sources of variability became increasingly significant.

J Microbiol Methods, 2003 Dec, 55(3), 763 - 73
Comparison of a cultural method with ListerScreen plus Rapid'L.mono or PCR-ELISA methods for the enumeration of L . monocytogenes in naturally contaminated sewage sludge; Garrec N et al.; Cultural methods used to count Listeria monocytogenes in sewage sludge are laborious and time consuming, and alternative methods are needed to reduce analysis time and improve detection limits . In this study, a survey of L . monocytogenes in sewage sludge is presented with a comparative study between a cultural method and immunomagnetic separation using a ListerScreen test followed by identification of L . monocytogenes with Rapid'L.mono agar or PCR-ELISA . These two alternative methods improved the detection of L . monocytogenes in different types of sludge, irrespective of their physical and chemical characteristics . The ListerScreen method coupled with detection of L . monocytogenes on Rapid'L.mono offers the advantage of being less sophisticated than the molecular method and allows isolation of the organism, which may be useful in epidemiological studies . However, ListerScreen coupled with PCR-ELISA proved best for high-sensitivity detection of L . monocytogenes in sewage samples.

J Clin Microbiol, 2003 Nov, 41(11), 5308 - 9
Fatal case of Listeria innocua bacteremia; Perrin M et al.; Listeria innocua is widespread in the environment and in food . This species has to date never been described in association with human disease . We report a case of fatal bacteremia caused by L . innocua in a 62-year-old patient.

Appl Environ Microbiol, 2003 Nov, 69(11), 6943 - 5
Development of a synthetic minimal medium for Listeria monocytogenes; Tsai HN et al.; A defined solid and liquid minimal medium, HTM, which contained methionine and cysteine as the sole amino acids, was developed for Listeria monocytogenes . Complex broth-grown L . monocytogenes had to adapt to HTM by inducing amino acid biosyntheis . HTM is the simplest minimal medium available for growth of L . monocytogenes.

Appl Environ Microbiol, 2003 Nov, 69(11), 6393 - 8
Assessment of photodynamic destruction of Escherichia coli O157:H7 and Listeria monocytogenes by using ATP bioluminescence; Romanova NA et al.; Antimicrobial photodynamic therapy was shown to be effective against a wide range of bacterial cells, as well as for fungi, yeasts, and viruses . It was shown previously that photodestruction of yeast cells treated with photosensitizers resulted in cell destruction and leakage of ATP . Three photosensitizers were used in this study: tetra(N-methyl-4-pyridyl)porphine tetratosylate salt (TMPyP), toluidine blue O (TBO), and methylene blue trihydrate (MB) . A microdilution method was used to determine MICs of the photosensitizers against both Escherichia coli O157:H7 and Listeria monocytogenes . To evaluate the effects of photodestruction on E . coli and L . monocytogenes cells, a bioluminescence method for detection of ATP leakage and a colony-forming assay were used . All tested photosensitizers were effective for photodynamic destruction of both bacteria . The effectiveness of photosensitizers (in microgram-per-milliliter equivalents) decreased in the order TBO > MB > TMPyP for both organisms . The MICs were two- to fourfold higher for E . coli O157:H7 than for L . monocytogenes . The primary effects of all of the photosensitizers tested on live bacterial cells were a decrease in intracellular ATP and an increase in extracellular ATP, accompanied by elimination of viable cells from the sample . The time courses of photodestruction and intracellular ATP leakage were different for E . coli and L . monocytogenes . These results show that bioluminescent ATP-metry can be used for investigation of the first stages of bacterial photodestruction.

Appl Environ Microbiol, 2003 Nov, 69(11), 6386 - 92
Rapid identification of Listeria species by using restriction fragment length polymorphism of PCR-amplified 23S rRNA gene fragments; Paillard D et al.; A molecular method based on restriction fragment length polymorphism (RFLP) of PCR-amplified fragments of the 23S rRNA gene was designed to rapidly identify Listeria strains to the species level . Two fragments (S1, 460 bp, and S2, 890 bp) were amplified from boiled DNA . S2 was cut with the restriction enzymes XmnI or CfoI and, if needed, S1 was digested by either AluI or ClaI . This method was first optimized with six reference strains and then applied to 182 isolates collected from effluents of treatment plants . All isolates were also identified by the API Listeria kit, hemolysis, and phosphatidylinositol-specific phospholipase C production (PI-PLC) on ALOA medium . The PCR-RFLP method unambiguously identified 160 environmental strains, including 131 in concordance with the API system, and revealed that 22 isolates were mixed cultures of Listeria monocytogenes and Listeria innocua . Discrepant results were resolved by a multiplex PCR on the iap gene, which confirmed the PCR-RFLP data for 49 of the 51 discordances, including the 22 mixed cultures . Sequencing of the 16S rRNA gene for 12 selected strains and reconstruction of a phylogenetic tree validated the molecular methods, except for two unclassifiable strains . The 158 single identifiable isolates were 92 L . monocytogenes (including seven nonhemolytic and PI-PLC-negative strains), 61 L . innocua, 4 Listeria seeligeri, and 1 Listeria welshimeri strain . The PCR-RFLP method proposed here provides rapid, easy-to-use, inexpensive, and reliable identification of the six Listeria species . Moreover, it can detect mixtures of Listeria species and thus is particularly adapted to environmental and food microbiology.

Poult Sci, 2003 Oct, 82(10), 1559 - 64
The role of dietary vitamin E in experimental Listeria monocytogenes infections in turkeys; Zhu M et al.; The current study was designed to determine if dietary vitamin E influenced either the gut clearance or levels of peripheral blood CD4+ and CD8+ T lymphocytes in adult turkeys experimentally infected with Listeria monocytogenes . Turkeys were fed vitamin E (0, 100, or 200 IU) from day of hatch to time of necropsy . After 6 wk on the experimental diet, turkeys were orally inoculated with L . monocytogenes (approximately 10(9) cfu) . To monitor infection status, cloacal swabs were taken on selected days post-inoculation (DPI) . At necropsy, samples of viscera, including liver, spleen, cecum, duodenum, ileum, and colon were collected and cultured for L . monocytogenes . In experiments 1 and 2, recovery of L . monocytogenes from cloacal swabs, tissues, and intestines from turkeys fed vitamin E was generally lower than that from turkeys fed the control diet, although these differences were not statistically significant . When data from both trials were combined, L . monocytogenes was cultured less frequently from cloacal swabs of the vitamin E-treated group (200 IU) on 2 and 3 DPI, when compared to controls (0 IU, P < 0.01) . There were no changes in virulence characteristics of L . monocytogenes cells, as measured by in vitro killing of Ped-2E9 cells, recovered from cloacal swabs or tissues of experimentally infected turkeys fed the control or a vitamin E treatment diet . Flow cytometric analysis indicated that CD4+ and CD8+ peripheral blood T lymphocytes were elevated at 6 and 8 DPI in infected turkeys given 200 IU vitamin E.

Microbiology, 2003 Nov, 149(Pt 11), 3247 - 56
Sigma(B)-dependent expression patterns of compatible solute transporter genes opuCA and lmo1421 and the conjugated bile salt hydrolase gene bsh in Listeria monocytogenes; Sue D et al.; Listeria monocytogenes is a food-borne pathogen that can persist and grow under a wide variety of environmental conditions including low pH and high osmolarity . The alternative sigma factor sigma(B) contributes to L . monocytogenes survival under extreme conditions . The purpose of this study was to identify and confirm specific sigma(B)-dependent genes in L . monocytogenes and to characterize their expression patterns under various stress conditions . opuCA, lmo1421 and bsh were identified as putative sigma(B)-dependent genes based on the presence of a predicted sigma(B)-dependent promoter sequence upstream of each gene . opuCA and lmo1421 encode known and putative compatible solute transporter proteins, respectively, and bsh encodes a conjugated bile salt hydrolase (BSH) . Reporter fusions and semi-quantitative RT-PCR techniques were used to confirm sigma(B)-dependent regulation of these stress-response genes and to determine their expression patterns in response to environmental stresses . RT-PCR demonstrated that opuCA, lmo1421 and bsh transcript levels are reduced in stationary-phase L . monocytogenes deltasigB cells relative to levels present in wild-type cells . Furthermore, BSH activity is abolished in a L . monocytogenes deltasigB strain . RT-PCR confirmed growth-phase-dependent expression of opuCA, with highest levels of expression in stationary-phase cells . The L . monocytogenes wild-type strain exhibited two- and threefold induction of opuCA expression and seven- and fivefold induction of lmo1421 expression following 10 and 15 min exposure to 0.5 M KCl, respectively, as determined by RT-PCR, suggesting rapid induction of sigma(B) activity in exponential-phase L . monocytogenes upon exposure to salt stress . Single-copy chromosomal opuCA-gus reporter fusions also showed significant induction of opuCA expression following exposure of exponential-phase cells to increased salt concentrations (0.5 M NaCl or 0.5 M KCl) . In conjunction with recent findings that indicate a role for opuCA and bsh in L . monocytogenes virulence, the data presented here provide further evidence of specific sigma(B)-mediated contributions to both environmental stress resistance and intra-host survival in L . monocytogenes.

Eur J Epidemiol, 2003, 18(10), 1001 - 6
Incidence of Listeria monocytogenes in food and environmental samples in Italy between 1990 and 1999: serotype distribution in food, environmental and clinical samples; Gianfranceschi M et al.; We report the findings of the study of 4185 food samples and 958 environmental samples collected in Italy in the period 1990-1999 and tested for the presence of Listeria monocytogenes . The strains isolated were biochemically and serologically characterised . We found a fairly high percentage of L . monocytogenes contamination in food (12.8%), whereas the level of contamination was lower in the environment (environment and work surfaces in food processing plants) (6.1%) . Serotyping showed a prevalence of a few serotypes (i.e., 1/2a, 1/2b, 1/2c and 4b), which were the same as those found in clinical samples collected during outbreaks and from sporadic cases of listeriosis reported in Italy in the period considered . The geographical distribution of the strains of L . monocytogenes isolated from food samples is very similar to that of the clinical strains.

Int J Food Microbiol, 2003 Dec 1, 88(2-3), 241 - 5
Homemade traditional cheeses for the isolation of probiotic Enterococcus faecium strains; Saavedra L et al.; One hundred twenty-two strains of Enterococcus faecium isolated from Tafi Cheese, a homemade traditional cheese of the highlands in the province of Tucuman, Argentina, were evaluated for their potential application as starter cultures in the manufacture of this traditional cheese . Eleven of the 122 strains showing limited delays in growth in oxgall were selected for the study of bile salts hydrolase activity (BSH), cholesterol reduction, antimicrobial activity, and virulence determinants . Nine strains were able to remove cholesterol in in vitro assays, a property that was closely related to the bile salt hydrolase activity . Only two strains produced active bacteriocins against Listeria strains although genetic evidence for the bacteriocin structural gene was found in six other enterococci strains . No virulence factors were detected in any of the 11 selected strains of enterococci.

Acta Microbiol Pol, 2003, 52(2), 131 - 42
Ampicillin resistance in Listeria monocytogenes acquired as a result of transposon mutagenesis; Poros-Gluchowska J et al.; We have used plasmid pLTV3, which carries transposon Tn917, to obtain a series of mutants of Listeria monocytogenes EGD showing varied degrees of resistance to ampicillin and other beta-lactam antibiotics, including imipenem . In this paper we focus on the characteristics of two strains in which decreased susceptibility to ampicillin is accompanied by changes in the structure of the cell wall murein and cell-wall related changes of phenotype.

Acta Microbiol Pol, 2003, 52(2), 113 - 29
Antimicrobial resistance of Listeria monocytogenes; Poros-Gluchowska J et al.; Listeria monocytogenes is an opportunistic pathogen that causes rare but frequently fatal infections, termed listerioses . In general, strains of L . monocytogenes are susceptible to a wide range of antibiotics, except for the cephalosporins, fluorochinolones and fosfomycin (Hof, 1991) . The current therapy of choice is a combination of ampicillin and aminoglycoside, usually gentamicin (Lorber, 1997) . In cases when it is not possible to use a beta-lactam antibiotic, second-choice therapy involves the use of an association of trimethoprim with a sulfonamide, such as in co-trimoxazole, in which the more active in the combination seems trimethoprim, synergized by the sulfa compound . Other second line agents for listeriosis include erythromycin and vancomycin (Temple and Nahata, 2000) . The first strains of L . monocytogenes resistant to antibiotics were reported in 1988 (Poyart-Salmeron et al . 1990) The present paper reviews the current state of affairs with regard to the resistance of L . monocytogenes isolated from food products and clinical material to different antibiotics, with particular emphasis on those used in the therapy of listeriosis.

J Immunol Methods, 2003 Oct 1, 281(1-2), 119 - 28
Characterization and application of a Listeria monocytogenes reactive monoclonal antibody C11E9 in a resonant mirror biosensor; Lathrop AA et al.; Typical detection of Listeria monocytogenes involves selective enrichment, isolation and biochemical testing . Development of antibodies to Listeria species has improved detection; however, most antibodies detect all species of Listeria . A previously developed monoclonal antibody (MAb)-C11E9 was examined for its reaction to 13 L . innocua and 40 L . monocytogenes strains representing all 13 serotypes by ELISA . Absorbance values for L . monocytogenes strains were 0.44-3.58 and for L . innocua 0.22-1.44 . ELISA reactions were divided into three arbitrary groups of high (Abs 1.0 or higher), intermediate (0.6-0.99) and low (0.18-0.59) . Most L . monocytogenes strains (32/41, 78%) were in the high group while only 23% (3/13) of L . innocua were in the same group . In the Western blot assay, antibody reacted with phosphate-buffered saline (PBS) extracted protein preparations of 52, 66 and 97 kDa . Ribopattern of all strains was analyzed and no clear relationship was observed for antibody reaction and ribotype of a given strain . MAb C11E9 was used in a resonant mirror biosensor (IAsys sensor), but failed to detect any viable intact L . monocytogenes cells at levels as high as 10(8) cells/ml; however, it showed binding (85-150 arc/s) with the surface protein preparations containing the 97-, 66- and 52-kDa proteins at 208 mug/ml . Binding kinetics of L . monocytogenes and L . innocua surface protein extracts showed significantly (p<0.05) higher responses than the three other Listeria species (L . ivanovii, L . welshimeri and L . grayi), which could be detected in 10-20 min . These data corroborate with ELISA results . In summary, this study suggest that MAb-C11E9 is suitable for detection of all serotypes of L . monocytogenes despite cross-reaction with L . innocua and could be used for detection of soluble protein extracts in the resonant mirror (IAsys) biosensor.

Antimicrob Agents Chemother, 2003 Nov, 47(11), 3421 - 9
CesRK, a two-component signal transduction system in Listeria monocytogenes, responds to the presence of cell wall-acting antibiotics and affects beta-lactam resistance; Kallipolitis BH et al.; Listeria monocytogenes is a food-borne pathogen that can cause a variety of illnesses ranging from gastroenteritis to life-threatening septicemia . The beta-lactam antibiotic ampicillin remains the drug of choice for the treatment of listeriosis . We have previously identified a response regulator of a putative two-component signal transduction system that plays a role in the virulence and ethanol tolerance of L . monocytogenes . Here we present evidence that the response regulator, CesR, and a histidine protein kinase, CesK, which is encoded by the gene downstream from cesR, are involved in the ability of L . monocytogenes to tolerate ethanol and cell wall-acting antibiotics of the beta-lactam family . Furthermore, CesRK controls the expression of a putative extracellular peptide encoded by the orf2420 gene, located immediately downstream from cesRK . Inactivation of orf2420 revealed that it contributes to ethanol tolerance and pathogenesis in mice . Interestingly, we found that transcription of orf2420 was strongly induced by subinhibitory concentrations of various cell wall-acting antibiotics, ethanol, and lysozyme . The induction of orf2420 expression was abolished in the absence of CesRK . Our data suggest that CesRK is involved in regulating aspects of the cell envelope architecture and that changes in cell wall integrity provide a potent stimulus for CesRK-mediated regulation . These results further our understanding of how L . monocytogenes senses and responds to antibiotics that are used therapeutically in the treatment of infectious diseases.

Infect Immun, 2003 Nov, 71(11), 6372 - 80
DNA vaccination protects mice against challenge with Listeria monocytogenes expressing the hepatitis C virus NS3 protein; Simon BE et al.; The goal of this study was to develop a new surrogate challenge model for use in evaluating protective cell-mediated immune responses against hepatitis C virus (HCV) antigens . The use of recombinant Listeria monocytogenes organisms which express HCV antigens provides novel tools with which to assay such in vivo protection, as expression of immunity against this hepatotropic bacterial pathogen is dependent on antigen-specific CD8(+) T lymphocytes . A plasmid DNA vaccine encoding a ubiquitin-NS3 fusion protein was generated, and its efficacy was confirmed by in vivo induction of NS3-specific, gamma interferon-secreting T cells following vaccination of BALB/c mice . These immunized mice also exhibited specific in vivo protection against subsequent challenge with a recombinant L . monocytogenes strain (TC-LNS3) expressing the NS3 protein . Notably, sublethal infection of naive mice with strain TC-LNS3 induced similar NS3-specific T-cell responses . These findings suggest that recombinant strains of L . monocytogenes expressing HCV antigens should prove useful for evaluating, or even inducing, protective immune responses against HCV antigens.

J Food Prot, 2003 Oct, 66(10), 1920 - 3
Validation of the USDA/ARS package rinse method for recovery of Listeria monocytogenes from naturally contaminated, commercially prepared frankfurters; Wallace FM et al.; The utility of the U.S . Department of Agriculture/Agricultural Research Service (USDA/ARS) package rinse method for recovering Listeria monocytogenes from the surface of contaminated foods was validated in comparison to the standard USDA/Food Safety and Inspection Service (FSIS) product composite enrichment method and two other methods using frankfurters from a lot with a known package prevalence rate of approximately 16% for this pathogen . One hundred packages from this batch of naturally contaminated, commercially prepared frankfurters were examined as follows: (i) the package exudative fluid was removed and tested using the standard USDA/FSIS product composite enrichment method; (ii) approximately 5 to 7 portions of frankfurters were removed to obtain a 25-g composite of meat that was then processed using the standard USDA/FSIS product composite enrichment method: (iii) 50 ml of 0.1% peptone water was added to each package, and the USDA/ARS package rinse method was performed on the remaining contents; and (iv) after removing the rinse fluid, the solid contents remaining in each package were directly enriched using the USDA/FSIS product composite enrichment method . These four methods identified that 7, 6, 15, and 9 of the 100 packages tested positive for the pathogen, respectively . Although no single approach yielded a positive result for every package that tested positive for L . monocytogenes by any one of the four sampling strategies, the USDA/ARS package rinse method was appreciably (P < 0.05) better than either the package exudate enrichment method or the standard USDA/FSIS product composite enrichment method at recovering the bacterium . These findings validate the sensitivity and ease of use of the USDA/ARS package rinse method using naturally contaminated frankfurters and argue strongly for its adoption for routine screening of ready-to-eat products that are prone to surface contamination with undesirable microbes such as L . monocytogenes.

J Food Prot, 2003 Oct, 66(10), 1840 - 4
Survival of Listeria monocytogenes in a simulated recirculating brine chiller system; Gailey JK et al.; Contamination by Listeria monocytogenes of processed meats after cooking presents a significant food safety risk . The purpose of this study was to determine the survival of L . monocytogenes in a simulated recirculating brine chiller system using pH values of 5, 6, and 7 with free chlorine concentrations of 0, 3, 5, and 10 ppm in 20% salt brine at -12 degrees C . At pH values of 5, 6, and 7 with chlorine concentrations of 2 and 3 ppm, using 10(8) CFU in a test tube system, an immediate drop of 0.28 log CFU/ml with no significance between treatments (P > 0.05), followed by a steady survival phase with a slope close to 0, was observed . In brine at a pH of 5 with 5 and 10 ppm of chlorine, an initial drop of 0.8 log CFU/ml was observed, which was followed by a steady survival phase with a destruction slope close to zero . At an inoculation concentration of 10(2) CFU in a test tube system (pH values of 5 and 7 with 0 and 10 ppm of chlorine), the average initial drop for all treatments was 0.1 log CFU/ml, which was followed by a steady survival phase . In a recirculating system, very few cells were destroyed during the brine chilling process, but only low numbers of L . monocytogenes were recovered from the brine and uninoculated hot dogs . Although little destruction of L . monocytogenes was noted, the dilution effect observed during the study indicates that environmental contamination of a brine chiller system poses little danger of postcooking contamination for processed meats if the system is regularly cleaned and sanitized.

J Food Prot, 2003 Oct, 66(10), 1832 - 9
Prevalence of Listeria monocytogenes in, and microbiological and sensory quality of, rainbow trout, whitefish, and vendace roes from Finnish retail markets; Miettinen H et al.; The prevalence of Listeria monocytogenes in retail roe, as well as the microbiological and sensory qualities of the roe, were studied for three fish species under three different storage conditions . A total of 147 Finnish rainbow trout (Oncorhynchus mykiss), whitefish (Coregonus lavaretus), vendace (Coregonus albula), and burbot (Lota lota) roe samples were bought fresh, frozen, or frozen-thawed from Finnish retail markets . The overall prevalence of L . monocytogenes was 5%; however, the prevalence of the pathogen in fresh roe was 18% . Fresh-bought roe tested positive for Listeria spp . and for L . monocytogenes, respectively, 5 and 20 times as often as did frozen and frozen-thawed roe products combined . The microbiological quality (analyzed as total aerobic heterotrophic bacteria and coliform bacteria) of 78% of the roe samples was unacceptable . Frozen roe samples were found to have the best microbiological quality . According to the results of a sensory evaluation, at least one sensory attribute (appearance, odor freshness, texture, and freshness of taste) was unacceptable for 29% of the roe samples studied . The sensory quality of roe samples bought fresh was better than that of roe samples bought frozen or frozen-thawed . From the results of this study, it is concluded that both the microbiological and the sensory qualities of roe at the retail level need to be improved.

Annu Rev Cell Dev Biol, 2003, 19, 541 - 64
Ena/VASP proteins: regulators of the actin cytoskeleton and cell migration; Krause M et al.; Ena/VASP proteins are a conserved family of actin regulatory proteins made up of EVH1, EVH2 domains, and a proline-rich central region . They have been implicated in actin-based processes such as fibroblast migration, axon guidance, and T cell polarization and are important for the actin-based motility of the intracellular pathogen Listeria monocytogenes . Mechanistically, these proteins associate with barbed ends of actin filaments and antagonize filament capping by capping protein (CapZ) . In addition, they reduce the density of Arp2/3-dependent actin filament branches and bind Profilin at sites of actin polymerization . Vertebrate Ena/VASP proteins are substrates for PKA/PKG serine/threonine kinases . Phosphorylation by these kinases appears to modulate Ena/VASP function within cells, although the mechanism underlying this regulation remains to be determined.

Inhal Toxicol, 2003 Nov, 15(13), 1347 - 68
Effect of asphalt fume inhalation exposure at simulated road paving conditions prior to bacterial infection on lung defense responses in rats; Antonini JM et al.; Asphalt fume inhalation has been suspected of affecting immune function in exposed workers . The objective of this study was to evaluate the effect of asphalt exposure on lung immune responses in rats using a bacterial infectivity model . Pathogen-free male Sprague-Dawley rats were exposed by inhalation to asphalt fumes (72.6 +/- 4.95 mg/m3) or filtered air for 6 h/day for 5 days . One day after the final asphalt exposure, rats were intratracheally inoculated with 5 x 10(5) Listeria monocytogenes . At 0 (prior to bacterial inoculation), 3, and 7 days after L . monocytogenes instillation, the lungs of each animal were divided . Bronchoalveolar lavage (BAL) was performed on right lungs . The recovered BAL cells were then differentiated and counted, and alveolar macrophage (AM) function was determined . Albumin and lactate dehydrogenase (LDH), two indices of lung injury, were measured in the acellular BAL fluid . To assess bacterial clearance, the left lungs were removed, homogenized, and bacterial colony-forming units (CFUs) were counted . In addition, lung-draining lymph nodes were removed, and lymphocyte phenotype and lymphocyte-induced cytokine production were examined . Asphalt fume exposure did not cause lung injury or inflammation in rats in the absence of infection . Infection induced elevations in AMs, neutrophils (PMNs), albumin, and LDH . Importantly, no significant differences were seen when comparing the asphalt group with the air and nonexposed naive groups at any time before or after infection . Also, asphalt fume inhalation exposure did not affect the rate of pulmonary clearance of L . monocytogenes or AM production of reactive oxygen and nitrogen species . However, asphalt-related increases in lymphocyte secretion of interferon (IFN)-gamma, interleukin (IL)-6, and IL-10 were observed at different times after bacterial infection, whereas the total number of lymph-node cells and the percentage of CD4+ and CD8+ cells were not significantly different among the treatment groups . Despite the asphalt-induced changes observed in lymphokine secretion, adaptive immune function seemed to function properly in lung defense against bacterial infection . Because innate nonspecific lung responses and pulmonary clearance of L . monocytogenes were unaffected by asphalt fume exposure, lung defenses were sufficient to control the infection . It was concluded that acute inhalation of asphalt fumes at a high concentration had a minimal effect on lung immune responses to infection in rats.

Water Res, 2003 Nov, 37(19), 4810 - 4
Development of a protocol for the isolation of Listeria monocytogenes from sludge; Garrec N et al.; Given the high level of background flora in sludge, methods for detecting Listeria monocytogenes are not well established . In this study, two critical parameters for the detection of L . monocytogenes were evaluated: the concentration of Listeria sp . in a modified Fraser broth (first stage of the method) and the proportion of L . monocytogenes on Palcam agar (second stage of the method) . Concentrations of Listeria sp . estimated in 118 modified Fraser enrichment broths inoculated with four types of sludge, reached 10(4) bacteria per mL for 83% of the positive enrichment broths . Proportion of L . monocytogenes on Palcam agar, which was estimated by transferring all characteristic colonies of Listeria sp . onto Rapid'L Mono agar, was highly variable regardless of the type of sludge . According to these results, we proposed a protocol that consisted of an enrichment in modified Fraser broth for 48 h at 37 degrees C, followed by plating 0.1 mL of appropriate dilutions of broth onto Palcam agar . After an incubation of 48 h at 37 degrees C, a systematic identification of characteristic colonies of Listeria sp . on Rapid'L Mono agar allowed to enhance the detection of Listeria monocytogenes.

Gene Ther, 2003 Nov, 10(24), 2036 - 45
Bactofection of mammalian cells by Listeria monocytogenes: improvement and mechanism of DNA delivery; Pilgrim S et al.; Bacteria-mediated transfer of plasmid DNA into mammalian cells (bactofection) is a potent approach to express plasmid-encoded heterologous proteins (protein antigens, toxins or enzymes) in a large set of different cell types including phagocytic and nonphagocytic mammalian cells . Previously, we have described a Listeria monocytogenes-mediated DNA delivery system, which releases plasmid DNA directly into the cytosol of mammalian cells by partial self-destruction of the carrier bacteria . Here we report on a second generation of this phage lysin supported bactofection system, which is greatly improved with respect to plasmid stability, transfer efficacy and biosafety . In this case, DNA release is initiated by spontaneous bacterial lysis in the infected cells cytosol which is subsequently enhanced by the simultaneously released phage lysin produced by the intracellular carrier bacteria . Bacteria that are capable of cell-to-cell spread are found to be much more efficient in bactofection than their non spreading counterparts.

Science, 2003 Oct 17, 302(5644), 462 - 4
Listeria intracellular growth and virulence require host-derived lipoic acid; O'Riordan M et al.; Listeria monocytogenes is a Gram-positive intracytosolic pathogen that causes severe disease in pregnant and immunocompromised individuals . We found that L . monocytogenes lacking the lipoate protein ligase LplA1 was defective for growth specifically in the host cytosol and was less virulent in animals by a factor of 300 . A major target for LplA1, the E2 subunit of pyruvate dehydrogenase (PDH), lacked a critical lipoyl modification when the DeltalplA1 strain was grown intracellularly, which suggests that abortive growth was due to loss of PDH function . Thus, the use of host-derived lipoic acid may be a critical process for in vivo replication of bacterial pathogens.

J Bacteriol, 2003 Nov, 185(21), 6295 - 307
Requirement of the Listeria monocytogenes broad-range phospholipase PC-PLC during infection of human epithelial cells; Grundling A et al.; In this study, we investigated the requirement of the Listeria monocytogenes broad-range phospholipase C (PC-PLC) during infection of human epithelial cells . L . monocytogenes is a facultative intracellular bacterial pathogen of humans and a variety of animal species . After entering a host cell, L . monocytogenes is initially surrounded by a membrane-bound vacuole . Bacteria promote their escape from this vacuole, grow within the host cell cytosol, and spread from cell to cell via actin-based motility . Most infection studies with L . monocytogenes have been performed with mouse cells or an in vivo mouse model of infection . In all mouse-derived cells tested, the pore-forming cytolysin listeriolysin O (LLO) is absolutely required for lysis of primary vacuoles formed during host cell entry . However, L . monocytogenes can escape from primary vacuoles in the absence of LLO during infection of human epithelial cell lines Henle 407, HEp-2, and HeLa . Previous studies have shown that the broad-range phospholipase C, PC-PLC, promotes lysis of Henle 407 cell primary vacuoles in the absence of LLO . Here, we have shown that PC-PLC is also required for lysis of HEp-2 and HeLa cell primary vacuoles in the absence of LLO expression . Furthermore, our results indicated that the amount of PC-PLC activity is critical for the efficiency of vacuolar lysis . In an LLO-negative derivative of L . monocytogenes strain 10403S, expression of PC-PLC has to increase before or upon entry into human epithelial cells, compared to expression in broth culture, to allow bacterial escape from primary vacuoles . Using a system for inducible PC-PLC expression in L . monocytogenes, we provide evidence that phospholipase activity can be increased by elevated expression of PC-PLC or Mpl, the enzyme required for proteolytic activation of PC-PLC . Lastly, by using the inducible PC-PLC expression system, we demonstrate that, in the absence of LLO, PC-PLC activity is not only required for lysis of primary vacuoles in human epithelial cells but is also necessary for efficient cell-to-cell spread . We speculate that the additional requirement for PC-PLC activity is for lysis of secondary double-membrane vacuoles formed during cell-to-cell spread.

Rev Med Chil, 2003 Aug, 131(8), 921 - 8
{Rhomboencephalitis caused by listeria monocytogenes infection in the central nervous system}; Illanes S et al.; Listeria monocytogenes infections can involve the central nervous system in the form of a rhomboencephalitis . Three possible cases of rhomboencephalitis by Listeria monocytogenes are reported (2 females, aged 44 and 49 and a man of 36 years old) . The three cases were preceded by an unspecific prodrome of headache, vertigo and fever in absence of a meningeal syndrome . The neurological stage was defined by the unilateral involvement of cranial nerves and the cerebellum and a clear inflammatory cerebrospinal fluid (CSF) with the presence of polymorphonuclear leukocytes, and normal glucose and protein levels . A magnetic nuclear resonance (MRI) showed the appearance of characteristic images, present in the bulboprotuberancial region . These images are one of the most constant features of this disease, reported in the literature . The early diagnosis of rhomboencephalitis was based on the clinical picture, the study of the CSF and the MRI, allowing the use of antimicrobials, prior to microbiological identification . Therefore, the risk of brain stem and cardiac complications of the disease is reduced.

J Cell Biol, 2003 Oct 13, 163(1), 131 - 42
How VASP enhances actin-based motility; Samarin S et al.; The function of vasodilator-stimulated phosphoprotein (VASP) in motility is analyzed using a biomimetic motility assay in which ActA-coated microspheres propel themselves in a medium containing actin, the Arp2/3 complex, and three regulatory proteins in the absence or presence of VASP . Propulsion is linked to cycles of filament barbed end attachment-branching-detachment-growth in which the ActA-activated Arp2/3 complex incorporates at the junctions of branched filaments . VASP increases the velocity of beads . VASP increases branch spacing of filaments in the actin tail, as it does in lamellipodia in living cells . The effect of VASP on branch spacing of Arp2/3-induced branched actin arrays is opposed to the effect of capping proteins . However, VASP does not compete with capping proteins for binding barbed ends of actin filaments . VASP enhances branched actin polymerization only when ActA is immobilized on beads or on Listeria . VASP increases the rate of dissociation of the branch junction from immobilized ActA, which is the rate-limiting step in the catalytic cycle of site-directed filament branching.

Account Res, 2003 Jan-Mar, 10(1), 1 - 16
Children as guinea pigs: historical perspective; Lederer SE; Experimentation involving children is not a new phenomenon . Children have been used as research subjects in a diverse set of experiments, including the trials of new vaccines and sera, in efforts to understand normal pediatric anatomy and physiology and in the development of new drugs and procedures . Concern about child participants in research is also not a new development . For more than a century, critics of medical research have called attention to the fact that children and other vulnerable populations--pregnant women, prisoners, the mentally ill--have too often served as the unwitting and unwilling subjects of medical experiments . This paper looks at several early cases in which children participated, including the first trial of cowpox vaccine, the first human trial of rabies vaccine, and the first treatment of Listerian wound antisepsis . The history of concern for children, especially institutionalized children, in medical research is considered along with the development of regulations or guidelines, including the Declaration of Helsinki (1964).

Virus Res, 2003 Nov, 97(1), 7 - 16
Enhanced immunogenicity of SIV Gag DNA vaccines encoding chimeric proteins containing a C-terminal segment of Listeriolysin O; Ye L et al.; We investigated the potential of the C-terminal 59-amino acid segment of Listeriolysin O (LLO) in enhancing immune responses against the SIV Gag antigen in the context of DNA immunization . Genes with codons optimized for mammalian expression were synthesized for the SIVmac239 Gag, a secreted SIV Gag protein with the tissue plasminogen antigen (tPA) signal fused to its N-terminus (tPA/Gag), as well as their corresponding chimeric proteins Gag/LLO and tPA/Gag/LLO containing the C-terminal 59 amino acids of LLO . Analysis of immune responses to these DNA constructs in a Balb/c mouse model showed that the Gag/LLO construct induced higher levels of both CD4 and CD8 T cell responses against SIV Gag, whereas the tPA/Gag construct induced higher levels of CD4 T cell responses . Moreover, immunization with the tPA/Gag/LLO construct further enhanced both CD4 and CD8 T cell responses . DNA constructs encoding secreted Gag proteins (tPA/Gag and tPA/Gag/LLO) were also more effective in eliciting antibody responses against SIV Gag . Our results demonstrate that the C-terminal segment of LLO can be effectively employed to enhance both cellular and humoral immune responses in the context of a DNA vaccine.

J Immunol, 2003 Oct 15, 171(8), 4352 - 8
Selective depletion of nonspecific T cells during the early stage of immune responses to infection; Jiang J et al.; Transient T cell depletion occurs before the development of an effective immune response to infection . In this study we show that most T cells, regardless of specificity, are induced to express early activation markers soon after infection with Listeria monocytogenes or lymphocytic choriomeningitis virus . Ag-specific T cells are further activated to display late activation markers and undergo extensive proliferation . As Ag-specific T cells begin to expand, nonspecific T cells are depleted en masse and exhibit no sign of further activation or proliferation before their depletion . This selective depletion of nonspecific T cells is due to in situ death via apoptosis, as visualized by confocal microscopy . Thus, early activation and subsequent depletion of nonspecific T cells are integral parts of the immune response to proinflammatory infections . These results have important implications for our understanding of early events in the development of a robust T cell response.

J Immunol, 2003 Oct 15, 171(8), 4254 - 62
Deficient anti-listerial immunity in the absence of perforin can be restored by increasing memory CD8+ T cell numbers; Messingham KA et al.; Compared with wild-type (WT) mice, Listeria monocytogenes (LM)-vaccinated perforin-deficient (PKO) mice have elevated levels of CD8(+) T cell memory, but exhibit reduced levels of protection against virulent LM . In this study, Ag-specific CD8(+) T cells from LM-vaccinated WT and PKO mice were used in adoptive transfer assays to determine the contribution of perforin-dependent cytolysis in protective immunity to LM . Perforin deficiency resulted in an approximately 5-fold reduction in the per-cell protective capacity of Ag-specific memory CD8(+) T cells that was not caused by differences in memory cell quality as measured by CD62L/CD27 expression, TCR repertoire use, functional avidity, differences in expansion of Ag-specific cells upon infection, or maintenance of memory levels over time . However, perforin-deficient CD8(+) T cells exhibited reduced in vivo cytotoxic function compared to WT CD8(+) T cells . Consistent with the existence of perforin-independent effector pathways, double-vaccinated PKO mice were as resistant to challenge with LM as single-vaccinated WT mice . Thus, increasing the number of memory CD8(+) T cells can overcome diminished per-cell protective immunity in the absence of perforin.

J Immunol, 2003 Oct 15, 171(8), 3970 - 6
Highly biased type 1 immune responses in mice deficient in LFA-1 in Listeria monocytogenes infection are caused by elevated IL-12 production by granulocytes; Emoto M et al.; LFA-1 (CD11a/CD18) plays a key role in various inflammatory responses . Here we show that the acquired immune response to Listeria monocytogenes is highly biased toward type 1 in the absence of LFA-1 . At the early stage of listeriosis, numbers of IFN-gamma producers in the liver and spleen of LFA-1(-/-) mice were markedly increased compared with heterozygous littermates and Valpha14(+)NKT cell-deficient mice, and NK cells were major IFN-gamma producers . Numbers of IL-12 producers were also markedly elevated in LFA-1(-/-) mice compared with heterozygous littermates, and endogenous IL-12 neutralization impaired IFN-gamma production by NK cells . Granulocyte depletion diminished numbers of IL-12 producers and IFN-gamma-secreting NK cells in the liver of LFA-1(-/-) mice . Granulocytes from the liver of L . monocytogenes-infected LFA-1(-/-) mice were potent IL-12 producers . Thus, in the absence of LFA-1, granulocytes are a major source of IL-12 at the early stage of listeriosis . We assume that highly biased type 1 immune responses in LFA-1(-/-) mice are caused by increased levels of IL-12 from granulocytes and that granulocytes play a major role in IFN-gamma secretion by NK cells . In conclusion, LFA-1 regulates type 1 immune responses by controlling prompt infiltration of IL-12-producing granulocytes into sites of inflammation.

J Microbiol Methods, 2003 Nov, 55(2), 425 - 32
An SNP-based PCR assay to differentiate between Listeria monocytogenes lineages derived from phylogenetic analysis of the sigB gene; Moorhead SM et al.; The alternative sigma factor sigB gene is involved in the stress response regulation of Listeria monocytogenes, and contributes towards growth and survival in adverse conditions . This gene was examined to determine if it could be a useful indicator of lineage differentiation, similar to the established method based on ribotyping . The sigB sequence was resolved in four local L . monocytogenes strains and the phylogenetic relationship among these, and a further 21 sigB gene sequences from strains of different serotype and lineage including two Listeria innocua strains, obtained from the GenBank database were determined . The sigB nucleotide sequences of these 25 Listeria strains were then examined for single nucleotide polymorphic (SNP) sites that could differentiate between the three lineages . Based on nucleotide sequences L . monocytogenes lineage I/serotype 1/2b and 4b clustered together, lineage II/serotype 1/2a and 1/2c strains clustered together, lineage III/serotypes 4a and 4c strains clustered together and L . innocua strains clustered together as an outgroup . SNPs differentiating the three lineages were identified . Individual allele-specific PCR reactions based on these polymorphisms were successful in grouping known and a further 37 local L . monocytogenes isolates into the three lineages.

Arch Latinoam Nutr, 2003 Jun, 53(2), 184 - 7
{Occurrence of Listeria monocytogenes in pre-sliced vacuum-packaged}; Sakate RI et al.; There is scarce information in Brazil and other South American countries about the occurrence of Listeria monocytogenes in food, mainly refrigerated ready-to-eat products . The consumption of sliced vacuum-packaged meat products has increased in the last few years . Nevertheless, a complete assessment of the risk associated with L . monocytogenes in these products is still necessary . Because of the production and storage characteristics of these products, they can be considered potential vehicles for L . monocytogenes to humans, mainly immunocompromised, elderly, and pregnant women . The objectives of this study was to evaluate the population of L . monocytogenes in salami, a ready-to-eat meat product with extended shelf life, acquired in retail stores in Sao Paulo-Brazil . The three-tube most probable number technique was used and the methodology was that from Health Canada . Strains were biochemically identified and serotyped . Among the 45 samples, 3 (6.7%) harboured 9.2 MPN/g of L . monocytogenes and the others < 0.3 MPN/g . All the strains belonged to serotypes 1/2a and 1/2b, the most frequent serotypes found in food everywhere . Even being low, the population of L . monocytogenes found in this product could be a cause of concern to public health authorities as it can pose a threat to population at risk . This contamination highlights the importance of implementing systems like HACCP to assure safe products to consumers.

Proc Natl Acad Sci U S A, 2003 Oct 14, 100(21), 12432 - 7 Epub 2003 Oct 03.
SecA2-dependent secretion of autolytic enzymes promotes Listeria monocytogenes pathogenesis; Lenz LL et al.; Pathogenic bacteria secrete proteins that promote invasion of host tissues and resistance to immune responses . However, secretion mechanisms that contribute to the enormous morbidity and mortality of Gram-positive bacteria are largely undefined . An auxiliary protein secretion system (SecA2) has recently emerged in Listeria monocytogenes and eight other Gram-positive pathogens . Here, a proteomics approach identified seventeen SecA2-dependent secreted and surface proteins of L . monocytogenes, the two most abundant of which {the p60 and N-acetylmuramidase (NamA) autolysins} hydrolyze bacterial peptidoglycan (PGN) and contribute to host colonization . SecA2-deficient (DeltaSecA2) bacteria were rapidly cleared after systemic infection of murine hosts, and in cultured cells showed reduced cell-cell spread . p60 or NamA deficiencies (Deltap60 and DeltaNamA) caused intermediate reductions in bacterial virulence in vivo, yet showed no defect for infection of cultured cells . Restoration of virulence in Deltap60 bacteria required full-length p60 with an intact catalytic domain, suggesting that PGN hydrolysis by p60 is crucial for L . monocytogenes virulence . Coordinated PGN hydrolysis by p60 and NamA activities is predicted to generate a muramyl glycopeptide, glucosaminylmuramyl dipeptide (GMDP), which is known to modify host inflammatory responses . Thus, SecA2-dependent secretion may promote release of muramyl peptides that subvert host pattern recognition.

J Bacteriol, 2003 Oct, 185(20), 5953 - 8
Restricted translocation across the cell wall regulates secretion of the broad-range phospholipase C of Listeria monocytogenes; Snyder A et al.; The virulence of Listeria monocytogenes is directly related to its ability to spread from cell to cell without leaving the intracellular milieu . During cell-to-cell spread, bacteria become temporarily confined to secondary vacuoles . Among the bacterial factors involved in escape from these vacuoles is a secreted broad-range phospholipase C (PC-PLC), the activation of which requires processing of an N-terminal prodomain . Mpl, a secreted metalloprotease of Listeria, is involved in the proteolytic activation of PC-PLC . We previously showed that, during intracellular growth, bacteria maintain a pool of PC-PLC that is not accessible to antibodies and that is rapidly released in its active form in response to a decrease in pH . pH-regulated release of active PC-PLC is Mpl dependent . To further characterize the mechanism regulating secretion of PC-PLC, the bacterial localization of PC-PLC and Mpl was investigated . Both proteins were detected in the bacterial supernatant and lysate with no apparent changes in molecular weight . Extraction of bacteria-associated PC-PLC and Mpl required cell wall hydrolysis, but there was no indication that either protein was covalently bound to the bacterial cell wall . Results from pulse-chase experiments performed with infected macrophages indicated that the rate of synthesis of PC-PLC exceeded the rate of translocation across the bacterial cell wall and confirmed that the pool of PC-PLC associated with bacteria was efficiently activated and secreted upon acidification of the host cell cytosol . These data suggest that bacterially associated PC-PLC and Mpl localize at the cell wall-membrane interface and that translocation of PC-PLC across the bacterial cell wall is rate limiting, resulting in the formation of a bacterially associated pool of PC-PLC that would readily be accessible for activation and release into nascent secondary vacuoles.

Diagn Microbiol Infect Dis, 2003 Oct, 47(2), 393 - 8
Listeria monocytogenes infection in patients with cancer; Rivero GA et al.; Listeriosis (LT) is an important infection in immunocompromised patients, but no large series of LT in cancer patients have been recently described . We reviewed the records of 34 cancer patients with LT at our institution (1990-2001) . Twenty patients (59%) had an underlying hematologic malignancy . In 11 patients, LT complicated bone marrow transplantation . Lymphocytopenia was observed in 62% of the patients . Twenty-six patients (76%) received prior corticosteroids . Bacteremia was the most common presentation of LT (74%) followed by meningoencephalitis (21%) . The most common treatment of LT was ampicillin with or without gentamicin (68%) . The median duration of treatment was 26 days (range, 8-74 days) . The rate of response to antimicrobial therapy was 79% . No relapses were identified . LT contributed to death in 9 (75%) of the 12 patients who died . Meningoencephalitis had the worst prognosis (3 of 6 cases were fatal) . Treatment of central nervous system LT continues to have a high failure rate.

Zhonghua Liu Xing Bing Xue Za Zhi, 2003 Aug, 24(8), 657 - 60
{Active surveillance on Listeria monocytogenes in seven kinds of food in 11 provinces of China in 2001}; Wu SY et al.; OBJECTIVE: To carry out national active surveillance on Listeria monocytogenes in foods in China . METHODS: Four thousand and thirty-four random samples from raw meat, meat product, raw milk, vegetable, yoghurt, icecream and aquatic product were collected in 11 provinces (cities), and examined for Listeria monocytogenes according to the national standard method and confirmed by BAX system (DuPont Qualicon, Wilmington, DE) . RESULTS: Seventy isolates four kinds of foods in seven provinces were found to have LM according to the national standard method with a total isolate rate of 1.74% . In Fujian, the rate was higher than in the other provinces . Raw meat was found to be most heavily contaminated in seven kinds of foods . Comparing to national standard method, BAX system showed good sensitivity (> 98%) and specificity (> 97%) . CONCLUSION: In each province seven kinds of food were all contaminated by Listeria monocytogenes to some degrees, suggesting that local sanitary surveillance should be strengthened . BAX system can be used to correctly and quickly screen Listeria monocytogenes.

Eur J Immunol, 2003 Oct, 33(10), 2875 - 85
Differences in maintenance of CD8+ and CD4+ bacteria-specific effector-memory T cell populations; Schiemann M et al.; Our knowledge about the kinetics and dynamics of complex pathogen-specific CD8(+) T cell responses and the in vivo development of CD8(+) memory T cells has increased substantially over the past years; in comparison, relatively little is known about the CD4(+) T cell compartment . We monitored and directly compared the phenotypical changes of pathogen (Listeria monocytogenes)-specific CD8(+) and CD4(+) T cell responses under conditions leading to effective and long-lasting protective immunity . We found that the general kinetics of bacteria-specific CD8(+) and CD4(+) T cells during the effector and post-effector phases are synchronized . However, later during the memory phase, CD8(+) and CD4(+) T cell populations differ substantially . Whereas CD8(+) memory T cell populations with immediate effector function are readily detectable in lymphoid and non-lymphoid tissues and remain remarkably stable in size, antigen-specific CD4(+) effector-memory T cells decline continuously in frequency over time . These findings have important implications for the better understanding of the in vivo development of protective immunity towards intracellular pathogens.

Eur J Immunol, 2003 Oct, 33(10), 2666 - 75
In vivo role of IFN-gamma produced by antigen-presenting cells in early host defense against intracellular pathogens; Suzue K et al.; Antigen-presenting cells (APC), including dendritic cells and macrophages, produce a large amount of interferon (IFN)-gamma, a crucial cytokine for the control of infectious diseases . To elucidate the role of IFN-gamma from APC in vivo, we employed cytokine receptor common gamma subunit (gamma c) and recombination-activating gene (Rag)-2 double-knockout (gamma c(-/-(y))-Rag-2(-/-)) mice, which are severely impaired in IFN-gamma production and are extremely susceptible to infection of intracellular pathogens including Listeria monocytogenes and Toxoplasma gondii . Adoptive transfer of IFN-gamma-producing APC increased levels of serum IFN-gamma and the resistance to Listeria . Although depletion of NK cells from Rag-2(-/-) mice slightly increased the susceptibility to bacterial infection, they are substantially more resistant than gamma c(-/-(y))-Rag-2(-/-) mice, which are also devoid of all lymphoid cells . These results demonstrate that the APC-derived IFN-gamma contributes to the control of infectious agents in vivo.

Toxicol Sci, 2003 Dec, 76(2), 347 - 56 Epub 2003 Sep 26.
Immunological function in mice exposed to JP-8 jet fuel in utero; Keil DE et al.; Immunological parameters, host resistance, and thyroid hormones were evaluated in F1 mice exposed in utero to jet propulsion fuel-8 (JP-8) . C57BL/6 pregnant dams (mated with C3H/HeJ males) were gavaged daily on gestation days 6-15 with JP-8 in a vehicle of olive oil at 0, 1000, or 2000 mg/kg . At weaning (3 weeks of age), no significant differences were observed in body, liver, spleen, or thymus weight, splenic and thymic cellularity, splenic CD4/CD8 lymphocyte subpopulations, or T-cell proliferation . Yet, lymphocytic proliferative responses to B-cell mitogens were suppressed in the 2000 mg/kg treatment group . In addition, thymic CD4-/CD8+ cells were significantly increased . By adulthood (8 weeks of age), lymphocyte proliferative responses and the alteration in thymic CD4-/CD8+ cells had returned to normal . However, splenic weight and thymic cellularity were altered, and the IgM plaque forming cell response was suppressed by 46% and 81% in the 1000 and 2000 mg/kg treatment groups, respectively . Furthermore, a 38% decrease was detected in the total T4 serum hormone level at 2000 mg/kg . In F1 adults, no significant alterations were observed in natural killer cell activity, T-cell lymphocyte proliferation, bone marrow cellularity and proliferative responses, complete blood counts, peritoneal and splenic cellularity, liver, kidney, or thymus weight, macrophage phagocytosis or nitric oxide production, splenic CD4/CD8 lymphocyte subpopulations, or total T3 serum hormone levels . Host resistance models in treated F1 adults demonstrated that immunological responses were normal after challenge with Listeria monocytogenes, but heightened susceptibility to B16F10 tumor challenge was seen at both treatment levels . This study demonstrates that prenatal exposure to JP-8 can target the developing murine fetus and result in impaired immune function and altered T4 levels in adulthood.

Inorg Chem, 2003 Oct 6, 42(20), 6300 - 5
Constrained synthesis of cobalt oxide nanomaterials in the 12-subunit protein cage from Listeria innocua; Allen M et al.; The protein cage of the 12-subunit ferritin-like protein from Listeria innocua has been utilized as a size and shape constrained reaction environment for the synthesis of two cobalt oxide minerals, Co(3)O(4) and Co(O)OH . Reaction of Co(II) with H(2)O(2) at pH 8.5 under either elevated temperature (65 degrees C) or ambient temperature (23 degrees C) resulted in the formation of cobalt oxide nanoparticles encapsulated within the protein cage . At elevated temperatures, Co(3)O(4) was formed while at lower temperature the oxyhydroxide Co(O)OH was found . Mineral particles, commensurate in size with the internal dimensions of the protein (5 nm), were imaged by transmission electron microscopy and shown to be surrounded by the intact protein cage . The minerals were investigated by electron diffraction and revealed a crystalline Co(3)O(4) phase and an amorphous Co(O)OH phase . Further investigation of these composite materials using size exclusion chromatography, gel electrophoresis, dynamic light scattering, and zeta potential measurements indicated that the mineral was encapsulated within the protein cage giving rise to properties of both the mineral and protein components.

Biochimie, 2003 Jul, 85(7), 633 - 8
Characterization of a unique sigma54-dependent PTS operon of the lactose family in Listeria monocytogenes; Dalet K et al.; The sigma(54) subunit of the RNA polymerase directs the expression of specific operons in association with cognate activators . Three different activators have been detected in the Listeria monocytogenes genome on the basis of the high conservation of a specific domain . Among them, the LacR activator, of the LevR family, was found just upstream from a newly described sigma(54)-dependent operon, lpo, which presents a classical -24/-12 consensus promoter . The lpo operon encodes proteins similar to subunits of a PTS permease (EII) of the lactose family, namely LpoA (IIA) and LpoB (IIB) . It also encodes a third putative protein, LpoO, with an unknown function but sharing high similarity with proteins also encoded within PTS operons from other bacteria and bearing a RGD motif . The expression of lpo was clearly dependent on LacR and sigma(54), and was induced by cellobiose, chitobiose and lactose . It underlies that the lpo operon likely encodes proteins involved in the utilization of these sugars by L . monocytogenes.

J Food Prot, 2003 Sep, 66(9), 1658 - 65
A PCR method based on 16S rRNA sequence for simultaneous detection of the genus Listeria and the species Listeria monocytogenes in food products; Somer L et al.; The genus Listeria comprises six closely related species, of which only Listeria monocytogenes is a human pathogen . The rapid and sensitive detection of L . monocytogenes is important in the food industry as well as in medical diagnosis . In this study, a PCR-based method for the rapid, specific, and sensitive detection of L . monocytogenes in food products was developed . The PCR is based on DNA sequences and primer pairs that are found within the 16S subunit of the rRNA gene and are specific to the Listeria genus and to L . monocytogenes within the Listeria genus . The primers for the Listeria genus and for L . monocytogenes were used in the same reaction mix for their simultaneous detection . In addition, a pair of bacterial primers universal to any bacterial DNA at the 16S subunit of the rRNA gene were developed as a positive control . For the detection of Listeria and L . monocytogenes in food products, the method includes selective enrichment for Listeria followed by DNA extraction and a specific PCR reaction . The method detects 1 to 5 CFU in a 25-g sample in < or = 24 h . It can be easily incorporated into the routine screening of diverse food products and readily adapted for clinical use.

J Food Prot, 2003 Sep, 66(9), 1623 - 30
Prepackage surface pasteurization of ready-to-eat meats with a radiant heat oven for reduction of Listeria monocytogenes; Gande N et al.; In this paper, a thermal process for the surface pasteurization of ready-to-eat (RTE) meat products for the reduction of Listeria monocytogenes on such products (turkey bologna, roast beef, corned beef, and ham) is described . The process involves the passage of products through a "tunnel" of heated coils on a stainless steel conveyor belt at various treatment times relevant to the manufacture of processed meat for the surface pasteurization of RTE meat products . Two inoculation procedures, dip and contact inoculation, were examined with the use of a four-strain cocktail of L . monocytogenes prior to heat processing . With the use of radiant heat prepackage surface pasteurization, 1.25 to 3.5-log reductions of L . monocytogenes were achieved with treatment times of 60 to 120 s and air temperatures of 475 to 750 degrees F (246 to 399 degrees C) for these various RTE meats . Reduction levels differed depending on the type of inoculation method used, the type of product used, the treatment temperature, and the treatment time . Prepackage pasteurization (60 s) was also combined with postpackage submerged water pasteurization for formed ham (60 or 90 s), turkey bologna (45 or 60 s), and roast beef (60 or 90 s), resulting in reductions of 3.2 to 3.9 . 2.7 to 4.3, and 2.0 to 3.75 log cycles, respectively . These findings demonstrate that prepackage pasteurization, either alone or in combination with postpackage pasteurization, is an effective tool for controlling L . monocytogenes surface contamination that may result from in-house handling.

J Food Prot, 2003 Sep, 66(9), 1618 - 22
Thermal inactivation of Listeria monocytogenes on ready-to-eat turkey breast meat products during postcook in-package pasteurization with hot water; Murphy RY et al.; The inactivation of Listeria monocytogenes during postcook in-package pasteurization was evaluated for fully cooked turkey breast meat products (4-kg packages) . The products were surface-inoculated to contain 10(7) CFU of L . monocytogenes per cm2 of product surface . The inoculated products were vacuum-packaged in different thicknesses (0.08 to 0.33 mm) of packaging films and treated with hot water at 96 degrees C . After heat treatment, the products were immediately cooled in an ice water bath at 0 degrees C . The relationship between heating time and product surface temperature was determined for different thicknesses of packaging films . The effectiveness of heat treatment for inactivating the pathogen was affected by product surface roughness . About 50 min of heating time was needed to achieve a thermal kill of 7 log10 CFU/cm2 on products with surface roughness up to 15 mm in depth . The cooling time needed after a heat treatment increased with an increasing endpoint temperature of the heated product and the heat penetration depth reached in the product . The cooling time needed to cool the product from 71 degrees C to 4 degrees C was about 2.5-fold the heating time.

J Food Prot, 2003 Sep, 66(9), 1611 - 7
Thermal inactivation, growth, and survival studies of Listeria monocytogenes strains belonging to three distinct genotypic lineages; De Jesus AJ et al.; Twenty-one Listeria monocytogenes strains belonging to three different genotypic lineages were evaluated for differences between lineages and between individual strains with respect to thermal inactivation, growth, and survival . Three sets of heat inactivation conditions (60 degrees C, pH 6.0, and 0.5 M lactate; 55 degrees C, pH 6.0, and 0.5 M lactate; and 50 degrees C, pH 4.0, and 0.5 M lactate) were used on strains grown in modified brain heart infusion (BHI) broth with and without glucose . Two sets of growth conditions (35 degrees C, pH 6.5, and 0.1 M lactate and 5 degrees C, pH 6.5, and 0.1 M lactate) were used with modified BHI broths to determine lag phases and exponential growth rates . Two sets of conditions (28 degrees C, pH 4.0, and 1 M lactate and 28 degrees C, pH 4.5, and 0.5 M lactate) were used with modified BHI broth to determine survival times (D-values) . Thermal inactivation D-values were consistently lowest for lineage III, but differences were not significant for any set of conditions tested . Some significant differences were observed between lineages with respect to some of the growth and survival conditions tested . Extensive strain-to-strain variation was observed for all parameters tested . Average coefficients of variation for the thermal inactivation, growth, and survival studies were 0.31, 0.18, and 0.26, respectively . Strain-to-strain variations were approximately equal to the uncertainties associated with the analytical procedures . The results obtained indicate a diversity among strains encountered in food processing that must be accounted for in process calculations and risk assessments.

Nucleic Acids Res . 2003 Oct 1;31(19):e117.
Real-time closed tube single nucleotide polymorphism (SNP) quantification in pooled samples by quencher extension (QEXT); Rudi K et al.; Quencher extension (QEXT) is a novel single step closed tube real-time method to quantify SNPs using reporters and quenchers in combination with primer extension . A probe with a 5'-reporter dye is single base extended with a dideoxy nucleotide containing a quencher dye if the target SNP allele is present . The extension is recorded from the quenching (reduced fluorescence) of the reporter dye . This avoids the influence of the unincorporated dye-labeled nucleotides, resulting in high accuracy and a high signal-to-noise ratio . The relative amount of a specific SNP allele is determined from the nucleotide incorporation rate in a thermo-cycling reaction . We tested the QEXT assay using five SNPs in the Listeria monocytogenes inlA gene as a model system . The presence of the target SNP alleles was determined with high statistical confidence (P < 0.0005) . The quantitative detection limits were between 0 and 5% for the targeted SNP alleles on a background of other SNP alleles (P < 0.05) . The QEXT method is directly adaptable to current real-time PCR equipment and is thus suited for high throughput and a wide application range.

Curr Biol, 2003 Sep 16, 13(18), R734 - 44
Biomimetic systems for studying actin-based motility; Upadhyaya A et al.; Actin polymerization provides a major driving force for eukaryotic cell motility . Successive intercalation of monomeric actin subunits between the plasma membrane and the filamentous actin network results in protrusions of the membrane enabling the cell to move or to change shape . One of the challenges in understanding eukaryotic cell motility is to dissect the elementary biochemical and biophysical steps that link actin polymerization to mechanical force generation . Recently, significant progress was made using biomimetic, in vitro systems that are inspired by the actin-based motility of bacterial pathogens such as Listeria monocytogenes . Polystyrene microspheres and synthetic phospholipid vesicles coated with proteins that initiate actin polymerization display motile behavior similar to Listeria, mimicking the leading edge of lamellipodia and filopodia . A major advantage of these biomimetic systems is that both biochemical and physical parameters can be controlled precisely . These systems provide a test bed for validating theoretical models on force generation and polarity establishment resulting from actin polymerization . In this review, we discuss recent experimental progress using biomimetic systems propelled by actin polymerization and discuss these results in the light of recent theoretical models on actin-based motility.

FEMS Microbiol Lett, 2003 Sep 12, 226(1), 101 - 5
Susceptibility of Listeria monocytogenes to antimicrobial peptides; Lopez-Solanilla E et al.; We assessed the susceptibility of several pathogenic and non-pathogenic Listeria species to antimicrobial peptides of animal and plant origin . Human defensins and thionins were highly inhibitory, whereas protamine, snakin and magainin showed an intermediate effect . A temperature dependence in the activity of potato defensin was observed for Listeria monocytogenes and Listeria ivanovii . PrfA* L . monocytogenes mutants, that overexpress constitutively PrfA-dependent genes, were sensitive to the peptide independently of the temperature whereas isogenic PrfA(-) derivatives were constitutively resistant . These data indicate that the thermoregulated transcription factor PrfA controls the expression of bacterial products that influence the susceptibility or resistance to some antimicrobial peptides.

J Biol Chem, 2003 Dec 5, 278(49), 49469 - 77 Epub 2003 Sep 15.
Maturation of lipoproteins by type II signal peptidase is required for phagosomal escape of Listeria monocytogenes; Reglier-Poupet H et al.; Lipoproteins of Gram-positive bacteria are involved in a broad range of functions such as substrate binding and transport, antibiotic resistance, cell signaling, or protein export and folding . Lipoproteins are also known to initiate both innate and adaptative immune responses . However, their role in the pathogenicity of intracellular microorganisms is yet poorly understood . In Listeria monocytogenes, a Gram-positive facultative intracellular human pathogen, surface proteins have important roles in the interactions of the microorganism with the host cells . Among the putative surface proteins of L . monocytogenes, lipoproteins constitute the largest family . Here, we addressed the role of the signal peptidase (SPase II), responsible for the maturation of lipoproteins in listerial pathogenesis . We identified a gene, lsp, encoding a SPase II in the genome of L . monocytogenes and constructed a deltalsp chromosomal deletion mutant . The mutant strain fails to process several lipoproteins demonstrating that lsp encodes a genuine SPase II . This defect is accompanied by a reduced efficiency of phagosomal escape during infection of eucaryotic cells, and leads to an attenuated virulence . We show that lsp gene expression is strongly induced when bacteria are still entrapped inside phagosomes of infected macrophages . The data presented establish, thus, that maturation of lipoproteins is critical for efficient phagosomal escape of L . monocytogenes, a process temporally controlled by the regulation of Lsp production in infected cells.

J Cell Biol, 2003 Sep 15, 162(6), 1079 - 88
Molecular requirements for actin-based lamella formation in Drosophila S2 cells; Rogers SL et al.; Cell migration occurs through the protrusion of the actin-enriched lamella . Here, we investigated the effects of RNAi depletion of approximately 90 proteins implicated in actin function on lamella formation in Drosophila S2 cells . Similar to in vitro reconstitution studies of actin-based Listeria movement, we find that lamellae formation requires a relatively small set of proteins that participate in actin nucleation (Arp2/3 and SCAR), barbed end capping (capping protein), filament depolymerization (cofilin and Aip1), and actin monomer binding (profilin and cyclase-associated protein) . Lamellae are initiated by parallel and partially redundant signaling pathways involving Rac GTPases and the adaptor protein Nck, which stimulate SCAR, an Arp2/3 activator . We also show that RNAi of three proteins (kette, Abi, and Sra-1) known to copurify with and inhibit SCAR in vitro leads to SCAR degradation, revealing a novel function of this protein complex in SCAR stability . Our results have identified an essential set of proteins involved in actin dynamics during lamella formation in Drosophila S2 cells.

J Antimicrob Chemother, 2003 Oct, 52(4), 610 - 5 Epub 2003 Sep 12.
Intracellular accumulation and activity of ampicillin used as free drug and as its phthalimidomethyl or pivaloyloxymethyl ester (pivampicillin) against Listeria monocytogenes in J774 macrophages; Chanteux H et al.; AIMS: To determine the intracellular accumulation in a macrophage cell line of ampicillin and ampicillin esters, and to measure their activity against intracellular Listeria monocytogenes . METHODS: Quantitative evaluation of the activity of ampicillin, phthalimidomethylampicillin (PIMA) or pivaloyloxymethylampicillin (PIVA) against intracellular L . monocytogenes, and direct measurement of cellular ampicillin concentration in J774 macrophages . RESULTS: Ampicillin, PIMA and PIVA caused a 0.5 log decrease in cell-associated cfu within 5 h when used at an extracellular concentration of 3.6 microM {10 x MIC of ampicillin (1.25 mg/L); 1.83 mg/L for PIMA and 1.67 mg/L for PIVA} . Addition of beta-lactamase in the extracellular milieu abolished the activity of ampicillin and of PIMA but not that of PIVA . At low extracellular concentrations {0.5 x MIC ampicillin (62.5 microg/L); equimolar concentrations for PIMA (91.5 microg/L) and PIVA (83.5 microg/L)}, ampicillin and PIMA lost all activity (compared with controls), but PIVA remained as active as at the higher concentration . Incubation of cells with PIVA at the low concentration (83.5 microg/L) for 20 h caused a 2 log reduction of cfu if the medium was changed every 5 h (to compensate for the degradation of extracellular PIVA) . Incubation of cells with PIVA allowed for a marked (four- to 25-fold) cell accumulation of ampicillin, whereas no ampicillin accumulation was seen for cells incubated with ampicillin or with PIMA . CONCLUSIONS: This is the first demonstration that PIVA (a prodrug of ampicillin) can be used to promote ampicillin cellular accumulation and, thereby to increase ampicillin intracellular activity . PIVA could be useful for control of the intracellular multiplication of L . monocytogenes.

Lett Appl Microbiol, 2003, 37(4), 309 - 13
Quantification of Listeria spp . contamination on shell and flesh of cooked black tiger prawns (Penaeus monodon); Dykes GA et al.; AIMS: To quantify Listeria levels on the shell and flesh of artificially contaminated cooked prawns after peeling, and determine the efficacy of Listeria innocua as a model for L . monocytogenes in this system . METHODS AND RESULTS: A L . monocytogenes and L . innocua strain were inoculated separately onto cooked black tiger prawns using two protocols (immersion or swabbing with incubation) . Prawns were peeled by two methods (gloved hand or scalpel and forceps) and numbers of Listeria on shells, flesh and whole prawn controls were determined . Prawns were exposed to crystal violet dye to assess the penetration of liquids . Regardless of preparation method or bacterial strain there were ca 1log10 CFU more Listeria per shell than per peeled prawn . Dye was able to penetrate to the flesh in all cases . CONCLUSIONS: Shell-on prawns may be only slightly safer than shell-off prawns . Listeria innocua is an acceptable model for L . monocytogenes in this system . SIGNIFICANCE AND IMPACT OF THE STUDY: Reduced risk from L . monocytogenes on prawns can only be assured by adequate hygiene or heating.

J Appl Microbiol, 2003, 95(4), 839 - 46
Betaine and carnitine uptake systems in Listeria monocytogenes affect growth and survival in foods and during infection; Sleator RD et al.; AIMS: To establish the relative importance of the osmo- and cryoprotective compounds glycine betaine and carnitine, and their transporters, for listerial growth and survival, in foods and during infection . METHODS AND RESULTS: A set of Listeria monocytogenes mutants with single, double and triple mutations in the genes encoding the principal betaine and carnitine uptake systems (gbu, betL and opuC, respectively) was used to determine the specific contribution of each transporter to listerial growth and survival . Food models were chosen to represent high-risk foods of plant and animal origin i.e . coleslaw and frankfurters, which have previously been linked to major human outbreaks of listeriosis . BALB/c mice were used as an in vivo model of infection . Interestingly, while betaine appeared to confer most protection in foods, the hierarchy of transporter importance differs depending on the food type: Gbu>BetL>OpuC for coleslaw, as opposed to Gbu>OpuC>BetL in frankfurters . By contrast in the animal model, OpuC and thus carnitine, appears to play the dominant role, with the remaining systems contributing little to the infection process . CONCLUSIONS: This study demonstrates that the individual contribution of each system appears dependent on the immediate environment . In foods Gbu appears to play the dominant role, while during infection OpuC is most important . SIGNIFICANCE AND IMPACT OF THE STUDY: It is envisaged that this information may ultimately facilitate the design of effective control measures specifically targeting this pathogen in foods and during infection.

J Appl Microbiol, 2003, 95(4), 762 - 72
Expression of cellular antigens of Listeria monocytogenes that react with monoclonal antibodies C11E9 and EM-7G1 under acid-, salt- or temperature-induced stress environments; Geng T et al.; AIMS: To study the expression of cellular antigens of Listeria monocytogenes that react with monoclonal antibodies (MAbs) C11E9 and EM-7G1 under acid-, salt- or temperature-induced stress environments . METHODS AND RESULTS: The reaction patterns of antibodies to L . monocytogenes held in stressful environments for a short duration (3 h) or grown for extended periods (16-72 h) were investigated . During both short or prolonged exposure to stress environments of high temperature (45 degrees C) and NaCl (>1.5%, w/v), reactions of whole cells of L . monocytogenes to antibodies were severely affected as determined by ELISA and by the reduced expression of the antibody-reactive 66 kDa antigen in the Western blot assay . Conversely, cold (4-15 degrees C) or acid (pH 2-3) stress environments had very little effect on antigen expression or antibody reaction . Additionally, heat-killed cells showed reduced reactions to these antibodies when compared with unheated cells . Artificially created stress environments in hotdog slurry also affected the antigen expression in L . monocytogenes . Immunoelectron microscopy revealed that the antibody-reactive antigens were uniformly present on the surface of the cells . Morphological characteristics following growth in stressed environments revealed that heat stress at 45 degrees C caused L . monocytogenes cells to be elongated and to form clumps; whereas, osmotic stress (5.5% NaCl, w/v) caused filamentous appearance with multiple septa along the length of the cell . CONCLUSIONS: These results indicated that MAb C11E9 or EM-7G1 could detect L . monocytogenes from cold or acid-stress environments; however, they may show weaker reactions with heat or osmotically stressed cells or cells grown at 4 degrees C . SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteria in food are routinely subjected to various stresses, induced by cold, heat, salt or acid during processing and storage . Whether stresses would modify the expression of cellular antigens of L . monocytogenes is of a great concern for immunodetections in food products.

Am J Dent, 2003 Jun, 16(3), 155 - 60
Dental calculus composition following use of essential-oil/ZnCl2 mouthrinse; LeGeros RZ et al.; PURPOSE: To test the hypothesis that anticalculus agents cannot completely inhibit calculus formation but can influence the types of calcium phosphate which form, i.e., they can influence the composition of the inorganic component of human dental calculus (HDC) . MATERIALS AND METHODS: The composition of HDC specimens obtained from a 16-week multi-center clinical study using three regimens were analyzed, investigators blinded . The treatment regimens were: (a) standard dentifrice (SD), (b) pyrophosphate antitartar dentifrice, and (c) SD with Tartar Control Listerine Antiseptic mouthrinse (containing essential oils and 0.09% zinc chloride) . 25 individual samples and eight pooled samples from each group were analyzed using X-ray diffraction, infrared spectroscopy, and scanning electron microscopy . RESULTS: (1) relative frequency of occurrence for: (a) bacteria: Group A = 100%, Group B = 60%, and Group C = 25%; (b) Carbonate hydroxyapatite (CHA): Groups A, B, and C = 100%; (c) dicalcium phosphate dihydrate (DCPD): Group A = 55%; Group B = 45%; Group C = 80%; (2) The relative amount of DCPD is inversely proportional to that of CHA in HDC: the higher the amount of DCPD, the lower the amount of CHA . Group C regimen with essential oil/ZnCl2 mouthrinse and standard dentifrice showed a significant anti-microbial effect and favored the formation of DCPD, the most soluble Ca-P.

Zh Mikrobiol Epidemiol Immunobiol, 2003 Jul-Aug, (4), 31 - 6
{Listeria pathogenicity factors and their role in pathogenesis and laboratory diagnosis of listeriosis}; Tartakovskii IS et al.; The mechanisms of relationships between Listeria and eukaryotic cells on the level of the main biomolecules are analyzed . These mechanisms determine the penetration and multiplication of Listeria and their importance for the pathogenesis of listeriosis . The theoretical and practical aspects of the regulation of the expression of pathogenicity factors in Listeria, connected with the activation of the regulatory protein PrfA and autoregulatory mechanisms, are considered . The elimination of the autorepressor from the cultivation medium results in the activation of the regulatory protein and the induction of the pathogenicity factors . The effective identification method for L . monocytogenes, permitting the differentiation of pathogenic and non-pathogenic Listeria, has been developed on the basis of the study of lecithinase expression in the presence of activated charcoal.

Medicina (Kaunas), 2003, 39(8), 767 - 72
Influence of cadmium and zinc on the mice resistance to Listeria monocytogenes infection; Simonyte S et al.; The objective of this study was to examine the effect of chronic exposure to cadmium and zinc on the mice resistance to experimental Listeria monocytogenes infection . MATERIALS AND METHODS: At the day beginning of experiment outbred mice were injected with suspension of bacteria and 8 weeks were given the following oral intake treatment: control group (n=28) deionized drinking water, Cd- group (n=37) water containing CdCl2 10 mg/l and Cd+Zn- group (n=33) water containing CdCl2 10 mg/l and ZnSO4 100 mg/l . The delayed type hypersensitivity was evaluated by the inflammatory response during so-called "foot" test . Listerial proteins solution was injected under plantare of lower aponeurosis of rear foot of experimental animals . Survival of L . monocytogenes in organs of experimental animals was evaluated by the presence of bacteria colonies after 30 days incubation of livers homogenates in broth medium at +4 degrees C and inoculation on CASO-agar . Kidneys, liver and spleen were used for metals analysis . Differences were significant if the P value was below 0.05 . RESULTS: Chronic exposure to Cd or to Cd with Zn for 8 weeks caused influence on survival of mice: Cd- and Cd+Zn groups mice died more rapidly than control group ones . Bacterial growth in organs was observed for all groups until fourth week . From sixth-week, control and Cd+Zn- group's mice more rapidly eliminated bacteria from organs, demonstrating that Zn- treated mice were more resistant to listerial infection than Cd- intoxicated ones . On the other hand, mice from Cd+Zn- group had significantly decreased spleen index (up to 74%, p<0.01) as compared to control group . Chronic poisoning of mice with low doses cadmium and zinc during infection significantly affected (p<0.05) their growth rate from fourth week in both experimental groups . Cadmium and zinc insignificantly decreased the delayed type hypersensitivity response to L . monocytogenes allergens in Cd+Zn- group of mice, and no differences were observed in Cd- group, as compared with control group . CONCLUSIONS: 1 . Cadmium-exposed mice are more susceptible to Listeria monocytogenes and to other opportunistic infections than not intoxicated mice . 2 . Zinc significantly reduces the negative effect of cadmium on the antimicrobial defense of mice . 3 . Cadmium and zinc no significantly decrease the delayed type hypersensitivity response to L . monocytogenes allergens as compared with control.

J Pharm Sci, 2003 Sep, 92(9), 1860 - 8
Modulation of hepatic cytochrome P450 during Listeria monocytogenes infection of the brain; Garcia Del Busto Cano E et al.; Hepatic cytochrome P450 enzymes can be modulated during systemic infections . Inflammatory responses in the brain have also been shown to cause a significant decrease in the levels and activities of important cytochrome P450 isoforms in the liver . We determined some of the effects of central nervous system (CNS) Listeria monocytogenes infection on hepatic cytochrome P450 systems in rats . Intracerebroventricular injection of L . monocytogenes resulted in a time-dependent modulation of CYP1A, CYP2B, and CYP3A activities in the liver . Total hepatic cytochrome P450 content was significantly lowered 48 h after administration of the bacterium, and hepatic CYP1A and CYP2B activities were significantly altered 48 and 72 h after infection, respectively, whereas CYP3A activity and protein content were depressed 72 h after the insult . Bacterial load in the brain increased dramatically over a 72-h period, but the number of bacteria cultured from liver over this time period was relatively small . Therefore, an infection largely confined to the CNS in the rat results in abnormal activity levels of certain hepatic cytochrome P450 enzymes crucial in drug metabolism . If such a response also occurs in humans, this has the potential to produce serious complications with drug and endogenous substrate metabolism in patients with an infectious disease involving the CNS .

J Bacteriol, 2003 Sep, 185(18), 5573 - 84
Genome diversification in phylogenetic lineages I and II of Listeria monocytogenes: identification of segments unique to lineage II populations; Zhang C et al.; Thirteen different serotypes of Listeria monocytogenes can be distinguished on the basis of variation in somatic and flagellar antigens . Although the known virulence genes are present in all serotypes, greater than 90% of human cases of listeriosis are caused by serotypes 1/2a, 1/2b, and 4b and nearly all outbreaks of food-borne listeriosis have been caused by serotype 4b strains . Phylogenetic analysis of these three common clinical serotypes places them into two different lineages, with serotypes 1/2b and 4b belonging to lineage I and 1/2a belonging to lineage II . To begin examining evolution of the genome in these serotypes, DNA microarray analysis was used to identify lineage-specific and serotype-specific differences in genome content . A set of 44 strains representing serotypes 1/2a, 1/2b, and 4b was probed with a shotgun DNA microarray constructed from the serotype 1/2a strain 10403s . Clones spanning 47 different genes in 16 different contiguous segments relative to the lineage II 1/2a genome were found to be absent in all lineage I strains tested (serotype 4b and 1/2b) and an additional nine were altered exclusively in 4b strains . Southern hybridization confirmed that conserved alterations were, in all but two loci, due to absence of the segments from the genome . Genes within these contiguous segments comprise five functional categories, including genes involved in synthesis of cell surface molecules and regulation of virulence gene expression . Phylogenetic reconstruction and examination of compositional bias in the regions of difference are consistent with a model in which the ancestor of the two lineages had the 1/2 somatic serotype and the regions absent in the lineage I genome arose by loss of ancestral sequences.

Epidemiol Infect, 2003 Aug, 131(1), 637 - 45
Real-time application of automated ribotyping and DNA macrorestriction analysis in the setting of a listeriosis outbreak; Inglis TJ et al.; A cluster of three cases of listeriosis cases occurred against a background of endemic listeriosis in Western Australia . Human and environmental isolates of Listeria monocytogenes obtained during the outbreak investigation were rapidly subtyped by automated ribotyping using an EcoRI protocol and a RiboPrinter . DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) was used to confirm the relatedness of isolates . Serogroup 1/2 predominated among the food samples and the four clinical isolates from the outbreak cluster were also of this serogroup . All isolates from chicken material were serogroup 1/2 and indistinguishable by ribotype pattern . PFGE subdivided strains of this ribotype into four subtypes . The preliminary analysis had an immediate impact on hypothesis generation, environmental health investigations, environmental specimen collection and initial control measures . Sufficient typing data to guide environmental health and disease control initiatives was generated in less than one week by combining automated ribotyping with PCR-based detection of L . monocytogenes in suspect foodstuffs and an L . monocytogenes DNA probe . There were no further cases of bacteriologically confirmed listeriosis in Western Australia for six months after completion of the investigation.

Mol Cell Biol, 2003 Sep, 23(18), 6609 - 17
Apparently normal tumor necrosis factor receptor 1 signaling in the absence of the silencer of death domains; Endres R et al.; The silencer of death domains (SODD) has been proposed to prevent constitutive signaling of tumor necrosis factor receptor 1 (TNFR1) in the absence of ligand . Besides TNFR1, death receptor 3 (DR3), Hsp70/Hsc70, and Bcl-2 have been characterized as binding partners of SODD . In order to investigate the in vivo role of SODD, we generated mice congenitally deficient in expression of the sodd gene . No spontaneous inflammatory infiltrations were observed in any organ of these mice . Consistent with this finding, in the absence of SODD no alteration in the activation patterns of nuclear factor kappaB (NF-kappaB), stress kinases, or ERK1 or -2 was observed after stimulation with tumor necrosis factor (TNF) . Activation of NF-kappaB by DR3 was also unchanged . The extents of DR3- and TNF-induced apoptosis were comparable in gene-deficient and wild-type cells . Protection of cells against heat shock as mediated by the Hsp70 system and against staurosporine-induced apoptosis was independent of SODD . Furthermore, resistance to high-dose lipopolysaccharide (LPS) injections, LPS-D-GalN injections, and infection with listeriae was similar in wild-type and gene-deficient mice . In conclusion, our data do not support the concept of a unique, nonredundant role of SODD for the functions of TNFR1, Hsp70, and DR3.

Mol Microbiol, 2003 Sep, 49(5), 1361 - 75
Ena/VASP proteins contribute to Listeria monocytogenes pathogenesis by controlling temporal and spatial persistence of bacterial actin-based motility; Auerbuch V et al.; The Listeria monocytogenes surface protein ActA mediates actin-based motility by interacting with a number of host cytoskeletal components, including Ena/VASP family proteins, which in turn interact with actin and the actin-binding protein profilin . We employed a bidirectional genetic approach to study Ena/VASP's contribution to L . monocytogenes movement and pathogenesis . We generated an ActA allelic series within the defined Ena/VASP-binding sites and introduced the resulting mutant L . monocytogenes into cell lines expressing different Ena/VASP derivatives . Our findings indicate that Ena/VASP proteins contribute to the persistence of both speed and directionality of L . monocytogenes movement . In the absence of the Ena/VASP proline-rich central domain, speed consistency decreased by sixfold . In addition, the Ena/VASP F-actin-binding region increased directionality of bacterial movement by fourfold . We further show that both regions of Ena/VASP enhanced L . monocytogenes cell-to-cell spread to a similar degree, although the Ena/VASP F-actin-binding region did so in an ActA-independent manner . Surprisingly, our ActA allelic series enabled us to uncouple L . monocytogenes speed from directionality although both were controlled by Ena/VASP proteins . Lastly, we showed the pathogenic relevance of these findings by the observation that L . monocytogenes lacking ActA Ena/VASP-binding sites were up to 400-fold less virulent during an adaptive immune response.

Mol Microbiol, 2003 Sep, 49(5), 1227 - 38
The CtsR regulator of Listeria monocytogenes contains a variant glycine repeat region that affects piezotolerance, stress resistance, motility and virulence; Karatzas KA et al.; A spontaneous high hydrostatic pressure (HHP)-tolerant mutant of Listeria monocytogenes ScottA, named AK01, was isolated previously . This mutant was immotile and showed increased resistance to heat, acid and H2O2 compared with the wild type (wt) (Karatzas, K.A.G . and Bennik, M.H.J . 2002 Appl Environ Microbiol 68: 3183-3189) . In this study, we conclusively linked the increased HHP and stress tolerance of strain AK01 to a single codon deletion in ctsR (class three stress gene repressor) in a region encoding a highly conserved glycine repeat . CtsR negatively regulates the expression of the clp genes, including clpP, clpE and the clpC operon (encompassing ctsR itself), which belong to the class III heat shock genes . Allelic replacement of the ctsR gene in the wt background with the mutant ctsR gene, designated ctsRDeltaGly, rendered mutants with phenotypes and protein expression profiles identical to those of strain AK01 . The expression levels of CtsR, ClpC and ClpP proteins were significantly higher in ctsRDeltaGly mutants than in the wt strain, indicative of the CtsRDeltaGly protein being inactive . Further evidence that the CtsRDeltaGly protein lacks its repressor function came from the finding that the Clp proteins in the mutant were not further induced upon heat shock, and that HHP tolerance of a ctsR deletion strain was as high as that of a ctsRDeltaGly mutant . The high HHP tolerance possibly results from the increased expression of the clp genes in the absence of (active) CtsR repressor . Importantly, the strains expressing CtsRDeltaGly show significantly attenuated virulence compared with the wt strain; however, no indication of disregulation of PrfA in the mutant strains was found . Our data highlight an important regulatory role of the glycine-rich region of CtsR in stress resistance and virulence.

Infect Immun, 2003 Sep, 71(9), 5202 - 9
Influence of pregnancy on the pathogenesis of listeriosis in mice inoculated intragastrically; Hamrick TS et al.; Pregnancy increases the risk of listeriosis, a systemic disease caused by Listeria monocytogenes . However, there is incomplete agreement on the reasons for this increased risk . We examined two features of listeriosis in gravid and nongravid female mice following intragastric (gavage) inoculation, namely, (i) disease severity (measured by lethality) and (ii) listerial infectivity (measured by liver and spleen colonization levels up to 120 h postinoculation) . Two listerial strains of differing serotype (1/2a and 4nonb) were initially employed . Neither strain produced a lethal infection in nonpregnant female mice (dose range, 10(6) to 10(9) CFU/mouse), and only the 4nonb strain produced lethalities in pregnant mice (dose range, 10(6) to 10(8) CFU/mouse) . The 4nonb strain also produced a higher level of liver and spleen colonization than the 1/2a strain following gavage administration . (The two strains showed similar levels of colonization if parenterally administered.) Both strains were equally capable of binding to and forming plaques upon cultured mouse enterocytes . The ability of the 4nonb strain to produce a lethal infection in pregnant animals did not correlate with an increased incidence or level of liver and spleen colonization over that in nonpregnant females . However, the lethality rate did correlate well with the rate at which embryos and their surrounding decidual covering became infected, suggesting that intrauterine infection could be responsible for the increased disease severity in the gravid females.

J Food Prot, 2003 Aug, 66(8), 1469 - 73
Effects of pH and agitation on the growth of Listeria monocytogenes Scott A in brain heart infusion broth containing combined potassium lactate and sodium diacetate during storage at 4 or 10 degrees C; Yoon KS et al.; The objective of this study was to compare the effects of pH on the growth kinetics of Listeria monocytogenes Scott A in static and agitated broths stored at 4 and 10 degrees C with and without a combination of 1.85% potassium lactate (PL) and 0.13% sodium diacetate (SDA) (3.3% of a 60% commercial solution, PURASAL P Opti.Form 4) . The pH of brain heart infusion broth without (control) or with 1.85% PL + 0.13% SDA was adjusted to 5.5, 6.0, 6.5, and 7.5 . L . monocytogenes Scott A was inoculated (at 10(2) CFU/ml) into pH-adjusted broth, which was stored at 4 or 10 degrees C with or without agitation . At pH 5.5, a listeriostatic effect was observed for the broth containing 1.85% PL + 0.13% SDA at 4 and 10 degrees C both with and without agitation . At pH 6.0, 1.85% PL + 0.13% SDA fully controlled the growth of L . monocytogenes Scott A in static broth at 4 degrees C for up to 20 days and significantly slowed the growth of the pathogen in agitated broth . At 10 degrees C, the growth of L . monocytogenes Scott A was significantly reduced by 1.85% PL + 0.13% SDA in agitated and unagitated broths . At pH 6.5, 1.85% PL + 0.13% SDA significantly suppressed the growth of L . monocytogenes Scott A at both 4 degrees C (P < 0.001) and 10 degrees C (P < 0.01) . At pH 7.5, 1.85% PL + 0.13% SDA had a limited effect on the growth of L . monocytogenes Scott A in broth stored at 4 and 10 degrees C . At 4 degrees C, agitation decreased the lag time and increased the growth rate of L . monocytogenes Scott A at all tested pHs . A similar but less obvious trend was observed for broths stored at 10 degrees C . These results indicate that lactate-diacetate combinations effectively acted with low pH and temperature to inhibit the growth of L . monocytogenes Scott A.

J Food Prot, 2003 Aug, 66(8), 1465 - 8
Use of pulsed-field gel electrophoresis to monitor a five-strain mixture of Listeria monocytogenes in frankfurter packages; Porto AC et al.; In a previous study, the viability of a five-strain mixture of Listeria monocytogenes (including Scott A {serotype 4b, clinical isolate}, 101M {serotype 4b, beef-pork sausage isolate}, F6854 {serotype 1/2a, turkey frankfurter isolate}, H7776 {serotype 4b, frankfurter isolate}, and MFS-2 {serotype 1/2a, pork plant isolate}) was monitored during refrigerated storage of frankfurters prepared with and without 3.0% added potassium lactate . Throughout a 90-day period of storage at 4 degrees C, the initial inoculum level of 20 CFU per package remained relatively constant in packages containing frankfurters prepared with potassium lactate, but pathogen counts increased to 4.6 log10 CFU in packages containing frankfurters prepared without added potassium lactate . To determine which of the five strains persisted under these conditions, randomly selected colonies obtained after 28 and 90 days of refrigerated storage of frankfurters were analyzed by pulsed-field gel electrophoresis (PFGE) with the restriction enzyme SmaI to generate distinct banding patterns for each of the five strains . Then, with the use of PFGE as a tool for identification, the percentages of the strains on days 28 and 90 of the growth study were compared . In the absence of any added potassium lactate in the product, 43% of the 58 isolates recovered on day 28 were identified as strain Scott A, 12% were identified as strain 101M, 22% were identified as strain F6854, 10% were identified as strain H7776, and 12% were identified as strain MFS-2 . However, by day 90, an appreciable number (83%) of the 60 isolates analyzed were identified as strain MFS-2 . In packages containing frankfurters formulated with 3.0% potassium lactate, all five strains were present at frequencies of 5 to 36% among the 19 isolates tested on day 28; however, by day 90, strain MFS-2 made up the statistical majority (63%) of the 27 isolates tested . The results of this study indicate that strain MFS-2, a serotype 1/2a isolate recovered from a pork processing plant, was more persistent than strains Scott A, 101M, F6854, or H7776 during the extended refrigerated storage of frankfurters.

J Food Prot, 2003 Aug, 66(8), 1420 - 5
Inhibition of Listeria monocytogenes in cold-smoked salmon by Carnobacterium piscicola CS526 isolated from frozen surimi; Yamazaki K et al.; Strain CS526 was isolated from frozen surimi and identified as a bacteriocin producer that had strong inhibitory activity against Listeria monocytogenes . Strain CS526 was identified as Carnobacterium piscicola by partial 16S rDNA sequence similarity . The ability of this bacteriocinogenic strain and nonbacteriocinogenic C . piscicola JCM5348 to inhibit the growth of L . monocytogenes was examined in culture broth incubated at 12 degrees C and cold-smoked salmon stored at 4, 12, and 20 degrees C . L . monocytogenes viable counts in the culture broth rapidly declined from 10(6) colony-forming units per ml to less than 10 colony-forming units per ml within 1 day at 12 degrees C in the presence of C . piscicola CS526 . At 4 and 12 degrees C, inhibition of L . monocytogenes on salmon depended on the initial inoculum level of C . piscicola CS526 . However, C . piscicola CS526 was bactericidal to L . monocytogenes within 21 and 12 days at 4 and 12 degrees C in cold-smoked salmon, respectively, even when the initial inoculum levels were low . C . piscicola CS526 suppressed the maximum cell number of L . monocytogenes by two and three log cycles, even at 20 degrees C . However, C . piscicola JCM5348 did not prevent the growth of the pathogen, except at 4 degrees C . Bacteriocin was detected in the samples coinoculated with C . piscicola CS526 . The study shows that C . piscicola CS526 might have potential for biopreservation of refrigerated foods against L . monocytogenes.

J Food Prot, 2003 Aug, 66(8), 1413 - 9
Prevalence of Listeria monocytogenes subtypes in bulk milk of the Pacific Northwest; Muraoka W et al.; The prevalence of Listeria monocytogenes in bulk milk from three Pacific Northwest states was assessed for 474 herds at three time points . For samples collected in November 2000 and June 2001, the L . monocytogenes prevalence levels were 4.9 and 7.0%, respectively . All isolates were subtyped by serotyping and by pulsed-field gel electrophoresis (PFGE) . Forty-nine of the 55 isolates belonged to serogroup 1/2a, while 6 belonged to serogroup 4 . Subtyping by PFGE revealed that isolates from 31 herds shared 10 patterns; there was a weak but significant association between PFGE subtype and geographical distance . Six herds were positive for L . monocytogenes at both time points . Of these six herds, four had indistinguishable PFGE patterns at both time points . Twenty-five of the 33 herds that were positive in June 2001 were sampled again in June 2002 . L . monocytogenes was recovered from 17 of these 25 herds (68%), with the ApaI restriction enzyme digestion profiles (REDP) for 8 herds being identical to those of isolates recovered from these herds the previous year . The ApaI REDP for the bulk milk isolates were compared with those for isolates recovered from environmental and human samples that were collected by the Washington Department of Health (n = 23) . Analysis of ApaI digestion profiles revealed that only two of the Washington Department of Health isolates had digestion profiles similar to those for isolates from bulk milk; however, further analysis with the use of a second enzyme (AscI) was capable of discriminating between isolates from the two sources . Thus, we found no direct REDP matches between bulk milk and clinical isolates.

J Food Prot, 2003 Aug, 66(8), 1393 - 401
Selection of fluorescent probes for flow cytometric viability assessment of Listeria monocytogenes exposed to membrane-active and oxidizing disinfectants; Luppens SB et al.; The aim of this study was to select fluorescence methods for use as alternatives to plate counting to assess the viability of Listeria monocytogenes cells exposed to benzalkonium chloride (BAC) and hydrogen peroxide, two disinfectants with different mechanisms of action . A further aim of this study was to determine whether growth phase influences fluorescence labeling and whether it is possible to predict whether a probe will be a good viability indicator for cells exposed to a certain disinfectant on the basis of the mechanism of action of the disinfectant and the target of the fluorescent probe . The fluorescence methods used were labeling with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC; dehydrogenase activity), labeling with TOTO-1 iodide (TOTO; membrane-impermeant probe), and assessment of pH gradient maintenance in a low-pH buffer after labeling with the pH-sensitive probe 5-(and 6)-carboxyfluorescein succinimidyl ester (CFSE) (the pH(in) method) . Growth phase influenced fluorescent labeling . However, the cutoff value for distinction between viable and nonviable cells was the same for both growth phases . The viability (determined by plate counts) of BAC-exposed cells correlated well with CTC labeling and TOTO exclusion . For both BAC-exposed and hydrogen peroxide-exposed cells, the pH(in) method gave a good qualitative indication of viability, sublethal damage, and cell death . CTC labeling and TOTO exclusion did not correlate with the viability of hydrogen peroxide-exposed cells . Our results demonstrate that even if the mechanism of action of a disinfectant is known, in some cases it is still difficult to predict whether a certain fluorescent probe is suitable for viability assessment . Thus, the proper selection of fluorescent probes for the assessment of the efficacy of antimicrobial agents is essential.

Int J Food Microbiol, 2003 Oct 15, 87(1-2), 173 - 9
Bacteriocin-producing Enterococcus casseliflavus IM 416K1, a natural antagonist for control of Listeria monocytogenes in Italian sausages ("cacciatore"); Sabia C et al.; The bacteriocinogenic Enterococcus casseliflavus IM 416K1 (Bac+) isolated from Italian sausages or its bacteriocin Enterocin 416K1, with strong anti-listerial activity, were used in trials to evaluate the effect on Listeria monocytogenes NCTC 10888 in artificially inoculated Italian sausages ("cacciatore") . In trials with Enterocin 416K1 added, L . monocytogenes showed a significant reduction as compared to the control inoculated with L . monocytogenes alone . The elimination of L . monocytogenes was only obtained in sausages added with E . casseliflavus IM 416K1 Bac+.

Int J Food Microbiol, 2003 Oct 15, 87(1-2), 87 - 95
The influence of process parameters for the inactivation of Listeria monocytogenes by pulsed electric fields; Alvarez I et al.; The influence of the electric field strength, the treatment time, the total specific energy and the conductivity of the treatment medium on the Listeria monocytogenes inactivation by pulsed electric fields (PEF) has been investigated . L . monocytogenes inactivation increased with the field strength, treatment time and specific energy . A maximum inactivation of 4.77 log(10) cycles was observed after a treatment of 28 kV/cm, 2000 micros and 3490 kJ/kg . The lethal effect of PEF treatments on L . monocytogenes was not influenced by the conductivity of the treatment medium in a range of 2, 3 and 4 mS/cm when the total specific energy was used as a PEF control parameter . A mathematical model based on the Weibull distribution was fitted to the experimental data when the field strength (15-28 kV/cm), treatment time (0-2000 micros) and specific energy (0-3490 kJ/kg) were used as PEF control parameters . A linear relationship was obtained between the log(10) of the scale factor (b) and the electric field strength when the treatment time and the total specific energy were used to control the process . The total specific energy, in addition to the electric field strength and the treatment time, should be reported in order to evaluate the microbial inactivation by PEF.

Int J Food Microbiol, 2003 Oct 15, 87(1-2), 35 - 43
Interactions of the bacteriocins sakacin P and nisin with food constituents; Aasen IM et al.; Bacteriocins are amphiphilic peptides susceptible to adsorption to food macromolecules and proteolytic degradation . These properties may limit their use as preservation agents . The aim of the present work has been to elucidate the fate of the bacteriocin sakacin P in food . Nisin was used in a few experiments for comparison . Recovery of bacteriocins was studied in homogenates of cold-smoked salmon, chicken cold cuts and raw chicken, with verification of the results in the corresponding food products . More than 80% of the added sakacin P and nisin were quickly adsorbed to proteins in the food matrix . In foods that had not been heat-treated, proteolytic activity caused a rapid degradation of the bacteriocins, with less than 1% of the total activity left after 1 week in cold-smoked salmon, and even less in raw chicken . In heat-treated foods, the bacteriocin activity was stable for more than 4 weeks . The high fat content in salmon compared to chicken had no adverse effect on bacteriocin recovery or activity . However, mixing of triglyceride oils and bacteriocin solutions caused a considerable loss of activity . No principal differences were observed between sakacin P and nisin, but less nisin was adsorbed to muscle proteins at low pH, and the negative effect of oils was less pronounced for nisin . Growth of Listeria monocytogenes was completely inhibited for at least 3 weeks in both chicken cold cuts and cold-smoked salmon by addition of sakacin P (3.5 microg/g), despite the proteolytic degradation in the salmon.

J Vet Med B Infect Dis Vet Public Health, 2003 May, 50(4), 155 - 60
Listeria spp . contamination in piggeries: comparison of three sites of environmental swabbing for detection and risk factor hypothesis; Belceil PA et al.; Listeria monocytogenes is a foodborne pathogen of major concern for public health in industrialized countries . Listeria carriage by pigs at the herd level could be a primary source for carcass contamination . Forty-seven finishing pig facilities were involved in the present study designed to compare three environmental swabbing sites in order to detect Listeria spp . in piggeries . Swabs were taken from the pen walls, the perianal regions of the pigs and the trough/feeder of the piggery premises . Listeria contamination of wet or dry feed given to the pigs was also investigated . The capacity of the various sampling sites for Listeria spp . detection were compared with a maximum likelihood estimation method . Listeria spp . were recovered in 74% of the pens studied and L . monocytogenes was detected in 15% of pens . With a specificity of 99%, sensitivity estimates (and 95% CI) of the Listeria spp . detection method were 93.4% (72.7-98.7) for pen walls, 73.1% (54.9-85.9) for pigs and 66.6% (48.6-80.7) for the trough/feeder . Listeria spp . were isolated from 84% of wet feed samples and 5% of dry feed samples . Listeria monocytogenes was found in 13% of wet feed samples . The type of feeding (wet versus dry) was associated (P < 0.001) with Listeria spp . contamination of both the pen and the feed . The results of this study confirm that Listeria spp., including L . monocytogenes, are present in pig facilities . Pen wall swabbing appears to be an effective way to assess Listeria spp . status of finishing pigs . The type of feeding (wet versus dry) could play a role in pig contamination.

Int J Food Microbiol, 2003 Sep 15, 86(3), 283 - 92
Influence of marinades on survival during storage of acid-adapted and nonadapted Listeria monocytogenes inoculated post-drying on beef jerky; Calicioglu M et al.; The objective of the present study was to investigate the survival of acid-adapted and nonadapted Listeria monocytogenes inoculated post-drying on dried beef slices (beef jerky), which were treated (24 h, 4 degrees C) with the following marinades before drying at 60 degrees C for 10 h: (1) control (C), (2) traditional marinade (TM), (3) modified marinade; double the amount of TM with added 1.2% sodium lactate, 9% acetic acid, and 68% soy sauce with 5% ethanol (MM), (4) dipping into 5% acetic acid and then TM (AATM), and (5) dipping into 1% Tween 20 and then into 5% acetic acid followed by the TM (TWTM) . Dried meat slices were inoculated with acid-adapted or nonadapted L . monocytogenes (ca . 5.7 log CFU/cm(2)) prior to aerobic storage at 25 degrees C for 60 days . Survivors were determined using tryptic soy agar with 0.1% pyruvate (TSAP) and PALCAM agar . Results showed that surviving bacterial populations on TWTM, AATM, and MM treatments were significantly (P<0.05) lower than those surviving on C and TM until 42 days of storage . By the end of 60 days of storage, bacterial populations in all treatments were not different regardless of acid adaptation or recovery media, except for treatment C inoculated with nonadapted cultures, which had significantly higher TSAP counts than other treatments . There was no significant (P> or =0.05) difference in survival of previously acid-adapted and nonadapted bacterial populations in samples of TWTM, AATM, and MM treatments . However, bacterial populations that were nonadapted were significantly higher than those that were acid-adapted on products of C and TM treatments on days 60 and 24, respectively . The earliest complete elimination (enrichment negative) of the pathogen occurred by day 28 (AATM) in products inoculated with acid-adapted cultures and by day 42 (TWTM and AATM) in products inoculated with nonadapted cultures . These results indicate that use of modified marinades in jerky processing and low water activity provided antimicrobial effects against possible post-processing contamination with L . monocytogenes.

Ned Tijdschr Geneeskd, 2003 Jul 26, 147(30), 1470 - 2
{Meningitis due to Listeria monocytogenes as a complication of infliximab therapy}; Joosten AA et al.; A 41-year-old woman was admitted to the hospital with meningitis caused by Listeria monocytogenes . Because of her Crohn's disease she used prednisolone and azathioprine . Two weeks before presenting with meningitis, infliximab had been given as the other immunosuppressant drugs had no effect . This tumour necrosis factor alpha (TNF alpha) blocking agent is known to increase the risk of opportunistic infections . This is the first Dutch patient described with meningitis caused by L . monocytogenes after treatment with infliximab . She recovered after antibiotic therapy . When antibiotic treatment is chosen, the possibility of opportunistic infections in patients who use infliximab concurrently with other immunosuppressant drugs should be taken into account.

Lett Appl Microbiol, 2003, 37(3), 234 - 8
Listeria monocytogenes occurrence and characterization in meat-producing plants; Peccio A et al.; AIMS: The prevalence, level of contamination and epidemiological profile of Listeria monocytogenes were investigated in two meat-producing plants during a 20-month period . METHODS AND RESULTS: Sampling for L . monocytogenes was carried out in a cattle slaughterhouse (n = 72) and a swine meat-processing plant (n = 68) during a 20-month period . Swabs and food samples were analysed with the most probable number (MPN) technique for L . monocytogenes and the isolated strains were characterized by AscI-restriction analysis pulsed-field gel electrophoresis (REA-PFGE) . Contamination of meat and meat products was always at low level (below 50 MPN per gram) . The seven L . monocytogenes positive samples isolated in the bovine slaughterhouse yielded strains with the same REA-PFGE profile . However, the seven strains isolated in the swine meat processing plant showed six different profiles . Two of them showed indistinguishable profiles with L . monocytogenes strains collected from other meat processing facilities located in the same area . SIGNIFICANCE AND IMPACT OF THE STUDY: The genotyping method is a valuable tool to investigate contamination sources . The study of REA-PFGE profiles indicated that environmental contamination was probably responsible for the persistence of over 16 months of one strain of L . monocytogenes in the cattle slaughterhouse . Several meat suppliers could be responsible for the contamination in the pig meat processing facility, and this is confirmed by the finding of some identical strain in other meat processing facilities located in the same area.

Appl Environ Microbiol, 2003 Aug, 69(8), 4519 - 26
Biocontrol of Listeria monocytogenes on fresh-cut produce by treatment with lytic bacteriophages and a bacteriocin; Leverentz B et al.; The fresh-cut produce industry has been the fastest-growing portion of the food retail market during the past 10 years, providing consumers with convenient and nutritious food . However, fresh-cut fruits and vegetables raise food safety concerns, because exposed tissue may be colonized more easily by pathogenic bacteria than intact produce . This is due to the higher availability of nutrients on cut surfaces and the greater potential for contamination because of the increased amount of handling . We found that applied Listeria monocytogenes populations survived and increased only slightly on fresh-cut Red Delicious apples stored at 10 degrees C but increased significantly on fresh-cut honeydew melons stored at 10 degrees C over 7 days . In addition, we examined the effect of lytic, L . monocytogenes-specific phages via two phage application methods, spraying and pipetting, on L . monocytogenes populations in artificially contaminated fresh-cut melons and apples . The phage mixture reduced L . monocytogenes populations by 2.0 to 4.6 log units over the control on honeydew melons . On apples, the reduction was below 0.4 log units . In combination with nisin (a bacteriocin), the phage mixture reduced L . monocytogenes populations by up to 5.7 log units on honeydew melon slices and by up to 2.3 log units on apple slices compared to the control . Nisin alone reduced L . monocytogenes populations by up to 3.2 log units on honeydew melon slices and by up to 2.0 log units on apple slices compared to the control . The phage titer was stable on melon slices, but declined rapidly on apple slices . The spray application of the phage and phage plus nisin reduced the bacterial numbers at least as much as the pipette application . The effectiveness of the phage treatment also depended on the initial concentration of L . monocytogenes.

Appl Environ Microbiol, 2003 Aug, 69(8), 4431 - 7
Differences in susceptibility of Listeria monocytogenes strains to sakacin P, sakacin A, pediocin PA-1, and nisin; Katla T et al.; Two hundred strains of Listeria monocytogenes collected from food and the food industry were analyzed for susceptibility to the class IIa bacteriocins sakacin P, sakacin A, and pediocin PA-1 and the class I bacteriocin nisin . The individual 50% inhibitory concentrations (IC(50)) were determined in a microtiter assay and expressed in nanograms per milliliter . The IC(50) of sakacin P ranged from 0.01 to 0.61 ng ml(-1) . The corresponding values for pediocin PA-1, sakacin A, and nisin were 0.10 to 7.34, 0.16 to 44.2, and 2.2 to 781 ng ml(-1), respectively . The use of a large number of strains and the accuracy of the IC(50) determination revealed patterns not previously described, and for the first time it was shown that the IC(50) of sakacin P divided the L . monocytogenes strains into two distinct groups . Ten strains from each group were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins and amplified fragment length polymorphism . The results from these studies essentially confirmed the grouping based on the IC(50) of sakacin P . A high correlation was found between the IC(50) of sakacin P and that of pediocin PA-1 for the 200 strains . Surprisingly, the correlation between the IC(50) of the two class IIa bacteriocins sakacin A and sakacin P was lower than the correlation between the IC(50) of sakacin A and the class I bacteriocin nisin.

Microb Pathog, 2003 Aug, 35(2), 81 - 6
Sodium pentobarbital anesthesia transiently enhances the severity of infection following intragastric, but not intravenous, inoculation of Listeria monocytogenes in mice; Czuprynski CJ et al.; In the present study we observed that mice anesthetized with sodium pentobarbital were far more susceptible to gastrointestinal challenge with Listeria monocytogenes than were unanaesthetized mice . The effect of pentobarbital anesthesia was transient (gone within 2 h) and did not alter the severity of infection following i.v . challenge with L . monocytogenes . Treatment with pharmacological inhibitors of gastric acidity (i.e . cimetidine and omeprazole), or intestinal motility (loperamide), did not duplicate the effect of pentobarbital on gastrointestinal listeriosis . These findings suggest that sodium pentobarbital anesthesia causes a short-lived but striking diminution in resistance to gastrointestinal listeriosis in mice, via an undefined mechanism.

Microb Pathog, 2003 Aug, 35(2), 57 - 62
Altering tissue tropism of Listeria monocytogenes by ectopically expressing human E-cadherin in transgenic mice; Luo Y et al.; The Gram-positive bacterium Listeria monocytogenes invades a variety of cells in vitro and in vivo . Here, we show that L . monocytogenes is able to enter muscle cells if provided with the appropriate host receptor . Using a cardiac-specific promoter, human E-cadherin was misexpressed in the heart of transgenic mice . First, we demonstrate that L . monocytogenes is capable of infecting the E-cadherin-expressing, but not the N-cadherin-expressing cardiomyocytes in vitro . Second, we show that L . monocytogenes infects the heart of transgenic animals after administering the bacteria intravenously . This unique system may prove useful for understanding basic aspects of L . monocytogenes cell biology by comparing infection of muscle versus epithelial cells.

New Microbiol, 2003 Jul, 26(3), 269 - 74
Detection of listeria monocytogenes in foodstuffs using chromogenic isolation media; Sacchetti R et al.; A study was carried out to compare two selective plating media commonly used to detect Listeria monocytogenes in food samples (Palcam and Oxford agar) with two more recently formulated chromogenic media (ALOA and Rapid'L.mono) . A comparison was also made between the identification systems API-Listeria and Monocytogenes ID Discs . The results obtained from the analysis of 132 food samples showed that the chromogenic media allowed Listeria monocytogenes to be detected more rapidly and with higher levels of specificity and sensitivity . The efficiency of the Monocytogenes ID Discs test was confirmed, giving results comparable to those obtained with API-Listeria, but in a simpler and quicker manner.

Ned Tijdschr Geneeskd, 2003 Jul 19, 147(29), 1404 - 8
{Clinical reasoning and decision-making in practice . A young woman with fever, shortness of breath, and reduced consciousness}; Taal W et al.; A 23-year-old woman with mild psychomotor retardation presented with fever, coughing, reduced consciousness and a stiff neck . A chest X-ray revealed an infiltrate in the left lower lobe; the cerebrospinal fluid was cloudy with a mild pleocytosis . Ceftriaxone was prescribed and the fever subsided . On the second day of admission she had a seizure, and a paraparesis emerged . Despite changes in the antibiotic regimen, her clinical condition hardly improved . On the fifth day, antibodies against Mycoplasma pneumoniae were found to be strongly positive and the diagnosis was M . pneumoniae infection . This accounted for the pneumonia together with meningoencephalitis and a transverse myelitis . The antibiotics were switched to doxycycline and the clinical condition improved dramatically . Six weeks after discharge, the patient had made a complete recovery . In patients suffering from meningitis with an atypical presentation, uncommon causes of infection should be considered . Together with a pneumonia, M . pneumoniae, Chlamydia pneumoniae, Legionella pneumophila and Listeria monocytogenes should be high on the list of potential causes for bacterial meningitis.

Am J Infect Control, 2003 Aug, 31(5), 322 - 4
Listeria monocytogenes cross-contamination in a nursery {corrected}; Colodner R et al.; Molecular evidence of Listeria monocytogenes cross-contamination in a nursery is presented . Listeria monocytogenes serotype 4b was isolated from the blood and the conjunctiva of a baby with neonatal sepsis who was born after septic amnionitis and premature rupture of membrane . Nine days later, the same bacterium was isolated from the cerebrospinal fluid of a second baby presenting with meningitis . Cervical cultures from the second baby's healthy mother were negative for Listeria sp . An in-depth epidemiologic investigation revealed that the same nurse administered routine treatments to both babies in the nursery during a 1-hour interval of time {corrected} . Pulse-field gel electrophoresis analysis of both strains with 2 different restriction enzymes demonstrated that they were identical and differ from other wild strains of L monocytogenes serotype 4b isolated in Israel . This fact strongly suggests that the second baby was infected during admittance to the nursery as a result of a hospital cross-contamination.

Clin Infect Dis, 2003 Aug 1, 37(3), 359 - 64 Epub 2003 Jul 22.
Listeriosis in patients at a comprehensive cancer center, 1955-1997; Safdar A et al.; Listeria monocytogenes infection occurred in 94 patients during 1955-1997 at Memorial Sloan-Kettering Cancer Center . The incidence was 0.5 (1955-1966), 0.96 (1970-1979), and 0.14 (1985-1997) cases per 1000 new admissions . Eighty-five patients (90%) were bacteremic, and 34 (36%) had evidence of intracranial infection . In 91 patients with cancer, 70 (77%) received chemotherapy for advanced or relapsed malignancy (n=51; 56%); 64 (68%) received corticosteroids . Breast cancer was the most common solid-organ cancer (n=14; 45%), and 34 (36%) had preexisting advanced liver disease . In 14 (39%) of 37 patients who died of listeriosis, death occurred within 48 h of L . monocytogenes isolation . Four (80%) of 5 patients with extracranial foci of infection died of their infection, compared with 33 (37%) of 89 patients with isolated bacteremia and/or intracranial infection (odds ratio, 2.34; P=.05) . Most infections (60%) were due to L . monocytogenes serotype 1/2, and the remainder (40%) were due to serovar 4b . Listeriosis in these patients with cancer occurred most often in individuals receiving antineoplastic therapy for advanced or relapsed malignancy and systemic corticosteroids . The presence of advanced liver disease may have increased the risk of systemic listeriosis in susceptible patients with underlying cancer.

Transpl Int, 2003 Nov, 16(11), 820 - 7 Epub 2003 Jul 22.
Calcineurin inhibitor-free immunosuppression based on antithymocyte globulin and mycophenolate mofetil in cadaveric kidney transplantation: results after 5 years; Grinyo JM et al.; Kidney grafts from suboptimal donors are more likely to suffer the nephrotoxic side-effects of cyclosporine than kidneys from standard donors . In an attempt to avoid the use of cyclosporine, we carried out a prospective study in low-immunological risk recipients of suboptimal kidneys, using an immunosuppressive protocol combining Thymoglobuline in induction with a bi-therapy of mycophenolate mofetil (MMF) and steroids . Patients with panel reactive antibodies (PRA) <50% receiving a first renal transplant from a suboptimal donor (age >or=50, non heart beating, arterial hypertension, or acute renal failure) or a kidney at risk of delayed graft function (DGF) because of a prolonged cold ischaemia time (CIT) of 24 h or more, were eligible for this trial . Between September 1996 and December 1999, 30 patients were enrolled for the trial and treated with MMF 2 g orally, pre-operatively, and 3 g daily, post-operatively; Thymoglobuline 2 mg/kg IV pre-operatively, 1.5 mg/kg IV the next day, and for doses of 1 mg/kg IV given on alternate days; and prednisolone 0.25 mg/kg per day, reduced progressively from the end of the first month to 0.1 mg/kg per day by 3 months post-transplant . Cyclosporine was added only if rejection grade II or higher, or a reduction in MMF below 1 g daily, occurred . Ten patients (30%) suffered from DGF, and one kidney suffered primary non function . Seven patients (24%) suffered acute rejection (six were biopsy proven, 3 grade I and 3 grade II) . MMF dosage was reduced in 28 patients because of adverse events, and calcineurin inhibitors were introduced in 16 patients . There were 14 episodes of opportunistic infection (cytomegalovirus (CMV 10), Herpes zoster 2, Listeria monocytogenes 1, Pseudomonas aeuruginosa 1), and 7 malignancies (skin 2, thyroid 1, lung 1, Kaposi's sarcoma 2, post-transplantation lymphoproliferative disorder 1) . Mean serum creatinine was 178, 199, 213, and 218 micromol/l at 1, 2, 3 and 5 years after transplantation, respectively . Actuarial patient and graft (after censoring for death) survival was 94% and 83% after 1 year and 79% and 65% after 5 years, respectively . These results show that with the combination of MMF, Thymoglobuline and steroids the use of cyclosporine can be delayed, and in a few cases completely avoided, with good efficacy in terms of prevention of rejection and recovery of renal function . Regardless of acceptable patient and graft survival, side-effects of MMF at the doses used in this protocol were common and led to overimmunosuppression in the long-term . Starting MMF at low dose, MPA monitoring and probably CMV prophylaxis may improve the results of this regimen.

Int J Food Microbiol, 2003 Aug 25, 85(3), 301 - 6
Influence of the adaptation of Listeria monocytogenes populations to structured or homogeneous habitats on subsequent growth on chilled processed meat; Dykes GA; The influence of adaptation to habitat structure on the growth of Listeria monocytogenes on processed meats was investigated . Triplicate populations of each of two L . monocytogenes strains were adapted to growth in homogeneous (Tryptic Soy Broth, TSB) or structured (Tryptic Soy Agar) environments by serial transfer in these media incubated at 25 degrees C for 48 h . Adapted (serial transfer for 56 days) and unadapted control (single 48 h growth period) populations were inoculated onto the surface of bologna or farmers' sausage disks, vacuum packaged and stored at 4 degrees C for 4 weeks . Initially and after 4 weeks, L . monocytogenes numbers were determined on Modified Oxford Agar incubated at 25 degrees C for 48 h . Control populations of both strains grown in the structured habitat reached significantly higher levels (1-1.5 log cfu g(-1), P<0.05) on both products than their counterparts grown in homogeneous habitat . Populations of both strains adapted to homogeneous habitats grew to significantly higher levels (0.4-1 log cfu g(-1), P<0.05), while those adapted to growth in structured habitats grew to significantly lower levels (0.3-1 log cfu g(-1), P<0.05), on both products as compared to their unadapted control populations . Populations of both strains adapted to homogeneous habitats grew to significantly higher levels (0.2 to 0.5 log cfu g(-1), P<0.05) on farmers' sausage, and significantly lower levels (0.2 to 0.4 log cfu g(-1), P<0.05) on bologna, than those adapted to structured habitats . Habitat adaptation affected L . monocytogenes growth on processed meats, but the practical significance and reasons for these findings were not established.

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