Listeriosis

Infect Immun, 2003 Aug, 71(8), 4398 - 404
Oral pretreatment of mice with CpG DNA reduces susceptibility to oral or intraperitoneal challenge with virulent Listeria monocytogenes; Ray NB et al.; Listeria monocytogenes is an enteroinvasive intracellular bacterial pathogen that infects humans and other animals, including mice, sometimes resulting in severe systemic infections . Previous studies showed that intraperitoneal (i.p.) pretreatment of susceptible BALB/c mice with immune-stimulatory CpG DNA 48 to 96 h prior to i.p . challenge with virulent L . monocytogenes reduces bacterial numbers in livers by greater than 100-fold, correlating with recovery from infection . Here we show that oral pretreatment of BALB/c mice with CpG DNA results in decreased susceptibility to either oral or i.p . challenge with L . monocytogenes . A single dose of 200 microg of CpG DNA administered to BALB/c mice orally by gavage 48 h or 7 days before oral challenge with virulent L . monocytogenes reduces bacterial numbers approximately 10- to 100-fold in livers and spleens . Lymphotoxin alpha knockout mice lacking Peyer's patches (PPs) and pretreated orally with CpG DNA 48 h prior to oral challenge with L . monocytogenes also have reduced susceptibility to infection, suggesting that PPs are required neither for oral infection nor for CpG-induced resistance against oral infection with L . monocytogenes . Surprisingly, 48-h oral pretreatment of BALB/c mice with 100 to 200 microg of CpG DNA results in approximately 100-fold-decreased bacterial numbers in livers following i.p . challenge with L . monocytogenes, suggesting, along with other data in this report, that orally delivered CpG DNA induces systemic resistance to infection . These results indicate that oral administration of CpG DNA induces systemic innate immune defenses against either oral or systemic infection with virulent L . monocytogenes.

J Immunol, 2003 Aug 1, 171(3), 1148 - 55
Macrophages of the splenic marginal zone are essential for trapping of blood-borne particulate antigen but dispensable for induction of specific T cell responses; Aichele P et al.; Rapid removal of pathogens from the circulation by secondary lymphoid organs is prerequisite for successful control of infection . Blood-borne Ags are trapped mainly in the splenic marginal zone . To identify the cell populations responsible for Ag trapping in the marginal zone, mice were selectively depleted of marginal zone macrophages and marginal metallophilic macrophages . In the absence of these cells, trapping of microspheres and Listeria monocytogenes organisms was lost, and early control of infection was impaired . Depletion of marginal zone macrophages and marginal metallophilic macrophages, however, did not limit Ag presentation because Listeria-specific protective T cell immunity was induced . Therefore, marginal zone macrophages and marginal metallophilic macrophages are crucial for trapping of particulate Ag but dispensable for Ag presentation.

Immunity, 2003 Jul, 19(1), 59 - 70
TNF/iNOS-producing dendritic cells mediate innate immune defense against bacterial infection; Serbina NV et al.; Dendritic cells (DCs) present microbial antigens to T cells and provide inflammatory signals that modulate T cell differentiation . While the role of DCs in adaptive immunity is well established, their involvement in innate immune defenses is less well defined . We have identified a TNF/iNOS-producing (Tip)-DC subset in spleens of Listeria monocytogenes-infected mice that is absent from CCR2-deficient mice . The absence of Tip-DCs results in profound TNF and iNOS deficiencies and an inability to clear primary bacterial infection . CD8 and CD4 T cell responses to L . monocytogenes antigens are preserved in CCR2-deficient mice, indicating that Tip-DCs are not essential for T cell priming . Tip-DCs, as the predominant source of TNF and iNOS during L . monocytogenes infection, orchestrate and mediate innate immune defense against this intracellular bacterial pathogen.

Immunity, 2003 Jul, 19(1), 2 - 4
Monocyte heterogeneity and innate immunity; Taylor PR et al.; Peripheral monocyte heterogeneity is widely acknowledged in humans but until now comparable heterogeneity has not been characterized in mice . In this issue, Geissmann et al . use chemokine receptors to define two monocyte subsets and Serbina et al . highlight the importance of selective monocyte recruitment in the innate immune response to Listeria.

J Food Prot, 2003 Jul, 66(7), 1283 - 7
Evaluation of antibodies for immunomagnetic separation combined with flow cytometry detection of Listeria monocytogenes; Jung YS et al.; Four polyclonal anti-Listeria antibodies were evaluated for the detection of Listeria monocytogenes in direct and indirect assays using immunomagnetic separation with flow cytometry . The efficiency of immunocapturing using magnetic beads was also determined . None of the tested antibodies exhibited sufficient specificity or avidity to allow sufficient separation and detection of L . monocytogenes for a useful test that differentiated between negative (without cell) and positive (with cell) samples . Plating results confirmed that cells were captured with Dynabeads anti-Listeria and magnetic beads coated with goat anti-Listeria antibody with recovery ranging from 7 to 23% . Fluorescent-labeled polyclonal antibodies used in this study were not sufficiently specific to allow the detection of L . monocytogenes cells captured by the beads.

J Food Prot, 2003 Jul, 66(7), 1216 - 21
A predictive model to determine the effects of temperature, sodium pyrophosphate, and sodium chloride on thermal inactivation of starved Listeria monocytogenes in pork slurry; Lihono MA et al.; The effects and interactions of 27 combinations of heating temperature (57.5 to 62.5 degrees C), sodium pyrophosphate (SPP) level (0 to 0.5%, wt/vol), and salt (NaCl) level (0 to 6%, wt/vol) on the thermal inactivation of starved Listeria monocytogenes ATCC 19116 in pork slurry were investigated . A split-split plot experimental design was used to compare all 27 combinations . L . monocytogenes survivors were enumerated on tryptic soy agar supplemented with 0.6% yeast extract . The natural logarithm (loge) of the means of decimal reduction times (D-values) were modeled as a function of temperature, SPP level, and NaCl level . Increasing concentrations of SPP or NaCl protected starved L . monocytogenes from the destructive effect of heat . For example, D-values for the pathogen at 57.5 degrees C in pork slurry with 0, 3, and 6% NaCl were 2.79, 7.75, and 14.59 min, respectively . All three variables interacted to affect the thermal inactivation of L . monocytogenes . A mathematical model describing the combined effect of temperature, SPP level, and NaCl level on the thermal inactivation of starved L . monocytogenes was developed . There was strong correlation (R2 = 0.97) between loge D-values predicted by the model and those observed experimentally . The model can predict D-values for any combination of variables that falls within the range of those tested . This predictive model can be used to assist food processors in designing thermal processes that include an adequate margin of safety for the control of L . monocytogenes in processed meats.

J Food Prot, 2003 Jul, 66(7), 1208 - 15
Growth kinetics and cell morphology of Listeria monocytogenes Scott A as affected by temperature, NaCl, and EDTA; Zaika LL et al.; Growth kinetics and morphological characteristics of Listeria monocytogenes Scott A grown under stress conditions induced by increasing levels of NaCl and EDTA were studied as a function of temperature . L . monocytogenes Scott A was inoculated into brain heart infusion broth (pH 6) at 19, 28, 37, and 42 degrees C . Test cultures contained NaCl (at concentrations of 4.5, 6.0, and 7.5%) or EDTA (at concentrations of 0.1, 0.2, and 0.3 mM); control cultures contained 0.5% NaCl . Growth curves were fitted from plate count data by the Gompertz equation, and growth kinetics parameters were derived . Stationary-phase cells were examined by scanning and transmission electron microscopy . Generation times (GTs) and lag phase duration times (LPDs) increased as additive levels were increased . The bacterium grew at all NaCl levels . At 37 and 42 degrees C, growth was slow in media containing 7.5% NaCl, and no growth occurred in media containing 0.3 mM EDTA . Temperature was a major factor in certain stress conditions that led to cell elongation and loss of flagella . Cells in control media at 28 degrees C grew as short rods (0.5 by 1.0 to 2.0 microm), while at 42 degrees C most cells were 4 to 10 times as long . Higher levels of NaCl at higher temperatures resulted in longer and thicker cells . At 28 degrees C, 0.1 mM EDTA had little effect on growth kinetics and morphology; however, 0.3 mM EDTA caused a sixfold increase in GT and LPD and loss of flagellae, with most cells being two to six times as long as normal . Cell length did not correlate with growth kinetics . The results of this study suggest that the effect of altered morphological characteristics of L . monocytogenes cells grown under stress on the virulence and subsequent survival of these cells should be investigated.

Syst Appl Microbiol, 2003 Jun, 26(2), 236 - 44
An improved amplified fragment length polymorphism (AFLP) protocol for discrimination of Listeria isolates; Keto-Timonen RO et al.; Nine restriction enzyme combinations and 108 different primer combinations were initially tested for suitability for amplified fragment length polymorphism (AFLP) analysis of Listeria monocytogenes; the combination of HindIII and HpyCH4IV showed consistently strong signals on gels, amplified an adequate number of DNA fragments and detected polymorphism among closely related strains based on AscI macrorestriction profiles . AFLP also distinguished between L . monocytogenes, L . innocua, L . ivanovii, L . seeligeri, L . welshimeri and L . grayi species . All Listeria species showed species-specific clusters, with less than 33% similarity between different species . A total of 34 L . monocytogenes strains were characterised by using both AFLP and pulsed-field gel electrophoresis (PFGE) . The results of AFLP analysis of L . monocytogenes strains were in concordance with those obtained by PFGE . Both methods identified 29 different genotypes of L . monocytogenes and had a high discrimination index (> 0.999) . By combining the results of AFLP and PFGE, subtype discrimination was further improved . Numerical analysis of both AFLP and PFGE profiles yielded three genomic groups of L . monocytogenes strains . AFLP was found to be faster and less labour-intensive than PFGE . We conclude that the AFLP protocol is a highly discriminatory, reproducible and valuable tool in characterisation of Listeria strains and may also be suitable for Listeria species identification.

Indian J Med Res, 2003 Jan, 117, 19 - 24
Typing of Listeria monocytogenes isolates by random amplification of polymorphic DNA; Dhanashree B et al.; BACKGROUND & OBJECTIVES: Listeria monocytogenes is an important food-borne pathogen causing meningitis and septicaemia in newborns and immunocompromised persons, abortion and preterm labour in pregnant women . Though various methods are available for typing L . monocytogenes, RAPD analysis has been used for epidemiological purposes in developed countries due to its greater discriminating ability . However, as there are no published reports from India on the typing of L . monocytogenes by RAPD technique the present study was undertaken to type isolates of L . monocytogenes from clinical, food and veterinary samples . METHODS: Isolates of L . monocytogenes were subjected to RAPD using four decamer random primers R1, R2, R3 and R4 . Amplified products were analysed by agarose gel electrophoresis . RESULTS: Eight strains of L . monocytogenes on RAPD analysis generated 4 distinct profiles each with R1 and R4 primers and 3 different profiles with R2 and R3 primers . The isolates from fish, clinical and veterinary samples showed different profiles with respect to each other . Isolate from flat fish (serovar 4) showed a different profile from that of clams (serovar 1) . Two isolates from placenta (serovar 1) showed similar profiles and all the isolates from veterinary samples generated similar profiles . INTERPRETATION & CONCLUSION: RAPD analysis in the present study allowed discrimination of isolates among the same serotype but from different sources . Since RAPD is a rapid technique and offers greater discrimination of strains, this method may be used for typing L . monocytogenes in India.

Immunol Res, 2003, 27(2-3), 451 - 62
Rational approaches to immune regulation; Paterson Y; Our laboratory is interested in the properties of proteins that render them immunogenic, and how such immunogenicity may be modulated in vivo . We are attempting to enhance the immune response in the design of more effective vaccines against viral diseases, such as HIV, and against tumor antigens expressed on breast, ovarian, and cervical cancer and B cell lymphomas . Our main approach is to use a facultative intracellular bacterium, Listeria monocytogenes, which has the unusual ability to live and grow in the cytoplasm of the cell and is thus an excellent vector for targeting passenger antigens to the major histocompatibility complex (MHC) class I pathway of antigen processing with the generation of authentic cytotoxic T lymphocytes (CTL) epitopes . In the field of tumor immunotherapy, we are also developing nonliving vaccine vectors for tumor antigens.

J Biol Chem, 2003 Sep 19, 278(38), 36810 - 8 Epub 2003 Jul 10.
Design of N-substituted peptomer ligands for EVH1 domains; Zimmermann J et al.; Ena/VASP proteins are implicated in cytoskeletal reorganization during actin-dependent motility processes . Recruitment to subcellular sites of actin polymerization is mediated by the highly conserved N-terminal EVH1 domain, which interacts with target proteins containing proline-rich motifs . The VASP EVH1 domain specifically binds peptides with the consensus motif FPPPP present in all its binding partners, including the Listerial ActA protein . Previous studies have shown that the Phe and first and final Pro residues are highly conserved and cannot be substituted with any other natural amino acid without significant loss of binding affinity . We have incorporated peptoid building blocks (sarcosine derived, non-natural amino acids) into the peptide SFEFPPPPTEDEL from the Listerial ActA protein and were able to substitute the most highly conserved residues of this motif while maintaining binding to the VASP EVH1 domain with affinities in the range of 45-180 microm . We then used NMR chemical shift perturbations to locate specific domain residues involved in particular interactions . These studies may open up the way for designing selective modulators of VASP function for biological studies and for the development of novel therapeutics for diseases involving pathologically altered cell adhesion or cell motility.

J Immunol, 2003 Jul 15, 171(2), 533 - 7
Cutting edge: protective cell-mediated immunity to Listeria monocytogenes in the absence of myeloid differentiation factor 88; Way SS et al.; In addition to their role in triggering innate immune responses, Toll-like receptors are proposed to play a key role in linking the innate and adaptive arms of the immune response . The majority of cellular responses downstream of Toll-like receptors are mediated through the adapter molecule myeloid differentiation factor 88 (MyD88), and mice with a targeted deletion of MyD88 are highly susceptible to bacterial infections, including primary infection with Listeria monocytogenes (LM) . In contrast, herein we demonstrate that MyD88-deficient mice have only a modest impairment in their LM-specific CD4 T cell response, and no impairment in their CD8 T cell response following infection with ActA-deficient LM . Furthermore, CD8 T cells from immunized MyD88-deficient mice protected naive recipient mice following adoptive splenocyte transfer, and immunized MyD88-deficient mice were protected from infection with wild-type LM . These results indicate that adaptive immune responses can be generated and provide protective immunity in the absence of MyD88.

Acta Paediatr Taiwan, 2003 Mar-Apr, 44(2), 106 - 8
Early-onset listeriosis in prematurity; Chen JM et al.; Listeria monocytogenes has been recognized as a human pathogen for more than 70 years . It causes illness mainly in pregnant women, newborns, elderly, and immunocompromised persons . Although L . Monocytogenes is a relatively uncommon pathogen in neonates, it can cause considerable morbidity and mortality in this age group, especially in the early-onset form of the disease . In Taiwan, neonatal listeriosis is rarely reported . We report one case of a premature newborn with early-onset listeria sepsis and meningitis.

J Dairy Sci, 2003 Jun, 86(6), 1865 - 75
ADSA Foundation Scholar Award--An integrated science-based approach to dairy food safety: Listeria monocytogenes as a model system; Wiedmann M; ADSA Foundation; Transmission of food- and milkborne pathogens often involves complex interactions among the pathogen, the environment, and one or multiple host species . A complete understanding of these interactions is critical to allow the development of science-based, effective intervention strategies for foodborne infectious diseases . This article summarizes our studies on the transmission, ecology, pathogenesis and population genetics of Listeria monocytogenes, which we have used as model for a food- and milkborne pathogen that infects multiple hosts and also has considerable ability to survive and multiply in nonhost environments . Application of molecular subtyping tools in conjunction with phenotypic characterization of selected strains has allowed us to define distinct L . monocytogenes subtypes and clonal groups that appear to differ in relevant phenotypic characteristics that may affect their abilities to be transmitted through food systems . For example, a genetic group designated as lineage I has been shown to be not only more common among human listeriosis cases than among animal cases, but lineage I strains also appear to show an increased in vitro ability to spread intracellularly from host cell to host cell . These findings are consistent with the fact that while genetically diverse strains may be classified to one bacterial species, these strains often differ from one another in important genetic and phenotypic characteristics . I thus propose that evolutionary- and molecular subtyping-based definitions of bacterial subtypes and clonal groups will provide critical insight into the microbial ecology of dairy food systems, including not only foodborne pathogens, but also organisms important for dairy fermentation and spoilage.

J Biol Chem, 2003 Sep 12, 278(37), 35102 - 8 Epub 2003 Jun 27.
Tumor cell killing enabled by listeriolysin O-liposome-mediated delivery of the protein toxin gelonin; Provoda CJ et al.; Gelonin is a type I plant toxin that has potential as an effective anti-tumor agent by virtue of its enzymatic capacity to inactivate ribosomes and arrest protein synthesis, thereby effectively limiting the growth of cancer cells . Being a hydrophilic macromolecule, however, gelonin has limited access to its target subcellular compartment, the cytosol; it is effectively plasma membrane-impermeant and subject to rapid degradation within endosomes and lysosomes upon cellular uptake as it lacks the membrane-translocating capability that is typically provided by a disulfide-linked B polypeptide found in the type II toxins (e.g . ricin) . These inherent characteristics generate the need for the development of a specialized cytosolic delivery strategy for gelonin as an effective anti-tumor therapeutic agent . Here we describe an efficient means of delivering gelonin to the cytosol of B16 melanoma cells . Gelonin was co-encapsulated inside pH-sensitive liposomes with listeriolysin O, the pore-forming protein that mediates escape of the intracellular pathogen Listeria monocytogenes from the endosome into the cytosol . In in vitro experiments, co-encapsulated listeriolysin O enabled liposomal gelonin-mediated B16 cell killing with a gelonin IC50 of approximately 0.1 nM with an extreme efficiency requiring an incubation time of only 1 h . By contrast, cells treated with equivalent concentrations of unencapsulated gelonin or gelonin encapsulated alone in pH-sensitive liposomes exhibited no detectable cytotoxicity . Moreover, treatment by direct intratumor injection into subcutaneous solid tumors of B16 melanoma in a mouse model showed that pH-sensitive liposomes containing both listeriolysin O and gelonin were more effective than control formulations in curtailing tumor growth rates.

FEMS Microbiol Lett, 2003 Jun 27, 223(2), 205 - 10
Identification of Listeria innocua by PCR targeting a putative transcriptional regulator gene; Liu D et al.; Listeria innocua is a common, non-pathogenic bacterial species that shares morphological, biochemical and molecular characteristics with the pathogenic species L . monocytogenes . The presence of L . innocua may cause difficulty or confusion in the laboratory identification of L . monocytogenes or other Listeria spp . In this report, through examining the recently published genome sequence of L . innocua strain CLIP 11262 (serovar 6a), we identified a L . innocua-specific gene (lin0464) encoding a putative transcriptional regulator and evaluated its efficacy for species-specific detection by polymerase chain reaction (PCR) . The specificity of the oligonucleotide primers (lin0464F and lin0464R) derived from this gene was confirmed with the formation of a 749-bp fragment in PCR from genomic DNA of L . innocua strains only . We expect that this assay will be useful in confirming identification of L . innocua or in studies where rapid detection of L . innocua is necessary.

Cell Mol Life Sci, 2003 May, 60(5), 904 - 18
Pathogen, host and environmental factors contributing to the pathogenesis of listeriosis; Roberts AJ et al.; Listeriosis is a severe human and animal disease caused by two species of pathogenic bacteria from the genus Listeria, L . monocytogenes and L . ivanovii . In humans, listeriosis is overwhelmingly a foodborne disease, yet much remains to be learned regarding the transmission dynamics of pathogenic Listeria from the environment, through food, to humans . Similarly, our understanding of the various host, pathogen and environmental factors that impact the pathogenesis of listeriosis at the cellular and molecular level is incomplete . This review will summarize what is currently known about animal and human listeriosis, detail the pathogen, host and environmental factors that contribute to pathogenesis and, finally, examine the interactions among those factors that influence the occurrence of human infection.

Transplant Proc, 2003 Jun, 35(4), 1485 - 7
Infection with Listeria monocytogenes following orthotopic liver transplantation: case report and review of the literature; Rettally CA et al.; Infection with Listeria monocytogenes is rare with a reported annual incidence of 4.4 cases/million individuals . Epidemiological data have identified certain groups to be higher risk of developing listeriosis, including neonates, pregnant women, adults older than 60 years of age, individuals afflicted with hematologic malignancies, acquired immunodeficicency syndrome, cirrhosis, and those receiving corticosteroid therapy and organ transplants . Within this last group, multiple cases have been described following bone marrow and renal transplantation, but only a few following liver transplantation . We report a case of a 66-year-old woman presenting with Listeria monocytogenes bacteremia at 32 months following orthotopic liver transplantation.

Jpn J Infect Dis, 2003 Apr, 56(2), 60 - 1
Listeriosis in second trimester of pregnancy: case report from India; Gupta V et al.; Although Listeria monocytogenes infection occurs in sporadic and epidemic forms throughout the world, there are certain countries (especially Asian countries) that have reported only a few cases or failed to report even a single case . During her third visit at 17(+5) weeks of gestation, a 22-year-old primigravida presented with the complaint of an acute painful abdomen, leaking per vaginum and low-grade fever for the 2 preceding days . On ultrasonography, a single live fetus with no amniotic fluid was seen and the pregnancy was therefore terminated . L . monocytogenes was isolated from a high vaginal swab.

J Immunol, 2003 Jul 1, 171(1), 291 - 8
Class Ia MHC-deficient BALB/c mice generate CD8+ T cell-mediated protective immunity against Listeria monocytogenes infection; D'Orazio SE et al.; CD8(+) T cells are required for protective immunity against intracellular pathogens such as Listeria monocytogenes . In this study, we used class Ia MHC-deficient mice, which have a severe reduction in circulating CD8(+) T cells, to determine the protective capacity of class Ib MHC-restricted T cells during L . monocytogenes infection . The K(b-/-)D(b-/-) mutation was backcrossed onto a C.B10 (BALB/c congenic at H-2 locus with C57BL/10) background, because BALB/c mice are more susceptible to Listeria infection than other commonly studied mouse strains such as C57BL/6 . C.B10 K(b-/-)D(b-/-) mice immunized with a sublethal dose of L . monocytogenes were fully protected against a subsequent lethal infection . Adoptive transfer of Listeria-immune splenocyte subsets into naive K(b-/-)D(b-/-) mice indicated that CD8(+) T cells were the major component of this protective immune response . A CD8(+) T cell line isolated from the spleen of a Listeria-infected class Ia MHC-deficient mouse was shown to specifically recognize Listeria-infected cells in vitro, as determined by IFN-gamma secretion and cytotoxicity assays . Adoptive transfer of this T cell line alone resulted in significant protection against L . monocytogenes challenge . These results suggest that even a limited number of class Ib MHC-restricted T cells are sufficient to generate the rapid recall response required for protection against secondary infection with L . monocytogenes.

J Immunol, 2003 Jul 1, 171(1), 37 - 46
The uterine NK cell population requires IL-15 but these cells are not required for pregnancy nor the resolution of a Listeria monocytogenes infection; Barber EM et al.; During pregnancy in mice, uterine natural killer (uNK) cells abundantly accumulate on the mesometrial side of the placenta . In this study, we show that the presence of both mature and immature uNK cells requires IL-15 . Bone marrow transplantation of NK cell-negative mice due to null mutations in the recombination-activating gene (Rag) 2/common cytokine receptor gamma-chain (Rag2(-/-)gamma(c)(-/-)) genes indicated that uNK cells originate from the bone marrow and require IL-15 to develop . NK cells are thought to be central players in the immune response to intracellular pathogens such as Listeria monocytogenes, a bacterium that also has a predilection for replication in the placenta . However, IL-15(-/-), NK cell-deficient mice were relatively protected from this infection compared with wild-type mice, and during pregnancy the absence of NK cells did not compromise the immune response at this site . The loss of uNK cells results in decidual abnormalities, including thickening of the arterial walls with luminal narrowing and a hypocellular decidua basalis . These defects were rescued by bone marrow transplantation of the Rag2(-/-)gamma(c)(-/-) mice that restored the uNK cell population . The decidual abnormalities in the IL-15(-/-) mice however did not result in infertility as gestation times and litter sizes were comparable to those of wild-type mice . Fetal weights were mildly compromised, consistent with the arterial pathologies . These results show that uNK cells are not required for successful pregnancy and that NK cells are not essential for an adequate immune response to L . monocytogenes in either pregnant or nonpregnant mice.

Cell Microbiol, 2003 Jul, 5(7), 455 - 68
Cytoplasmic bacteria can be targets for autophagy; Rich KA et al.; Autophagy is an important constitutive cellular process involved in size regulation, protein turnover and the removal of malformed or superfluous subcellular components . The process involves the sequestration of cytoplasm and organelles into double-membrane autophagic vacuoles for subsequent breakdown within lysosomes . In this work, we demonstrate that the intracellular pathogen Listeria monocytogenes can also be a target for autophagy . If infected macrophages are treated with chloramphenicol after phagosome lysis, the bacteria are internalized from the cell cytoplasm into autophagic vacuoles . The autophagic vacuoles appear to form by fusion of small cytoplasmic vesicles around the bacteria . These vesicular structures immunolabel with antibodies to protein disulphide isomerase, a marker for the rough ER . Internalization of metabolically arrested cytoplasmic L . monocytogenes represents an autophagic process as the vacuoles have double membranes and the process can be inhibited by the autophagy inhibitors 3-methyladenine and wortmannin . Additionally, the rate of internalization can be accelerated under starvation conditions and the vacuoles fuse with the endocytic pathway . Metabolic inhibition of cytoplasmic bacteria prevents them from adapting to the intracellular niche and reveals a host mechanism utilizing the autophagic pathway as a defence against invading pathogens by providing a route for their removal from the cytoplasm and subsequent delivery to the endocytic pathway for degradation.

Int J Food Microbiol, 2003 Aug 1, 84(3), 285 - 97
Genetic variability among isolates of Listeria monocytogenes from food products, clinical samples and processing environments, estimated by RAPD typing; Martinez I et al.; RAPD analysis with four primers was used to examine the genetic relationship among 432 strains of Listeria monocytogenes isolated from clinical and veterinarian cases of listeriosis, dairy, vegetable, meat- and fish-based food items, environmental samples and samples collected from one transport terminal, one poultry-processing company and four Atlantic salmon-processing plants . The purpose of the study was to determine whether clinical isolates belonged to a specific genetic group, whether links could be made between food groups and clinical cases and whether specific genetic groups were associated with specific food products or processing units . There was great genetic variability among the isolates, which produced a total of 141 RAPD composites based on the RAPD analysis with four primers . The RAPD composites divided in two major clusters and clinical isolates were evenly distributed in both of them . None of the isolates from food products had the same RAPD composite as isolates from human patients, thus, no particular food commodity could be linked to clinical cases . Each food-processing environment was contaminated with more than one RAPD composite and the genetic variability found within each company was, in most cases, of approximately the same magnitude as the variability found when considering all the samples . In each plant, one or a few types persisted over time, indicating the presence of an established in-house flora . Our results indicate that most of the analysed cases of listeriosis were sporadic and, further, that these cases cannot be traced to a few specific food sources . We also found that no particular RAPD composite was better suited for survival in specific food types or food-processing environments, indicating that although differences may be found in virulence properties of individual strains, all L . monocytogenes must be treated as potentially harmful.

Gene Ther, 2003 Jul, 10(13), 1105 - 15
Cytosolic delivery of antisense oligonucleotides by listeriolysin O-containing liposomes; Mathew E et al.; Antisense oligodeoxynucleotides (ODNs) possess great potential as sequence-specific therapeutic agents . Sufficient concentrations of intact ODN must bypass membrane barriers and access the cytosol and nucleus, for ODNs to be therapeutically effective . A cytosolic delivery strategy was designed to improve the efficiency of ODN delivery in bone-marrow-derived macrophages . This liposome-based formulation utilizes listeriolysin O (LLO), the endosomolytic hemolysin from Listeria monocytogenes, to mediate the escape of ODN from endocytic compartments into the cytosol . To monitor the cytosolic delivery of ODN, subcellular trafficking of fluorescently labeled ODNs was visualized using epifluorescence microscopy . The expression of target protein and mRNA after delivery was measured using flow cytometry and Northern blot analysis, respectively . ODN specific for murine intercellular adhesion molecule-1 (ICAM-1) encapsulated in LLO-liposomes was released to the cytosol and trafficked to the nucleus, efficiently and specifically suppressing activation-induced expression of ICAM-1 at both protein and mRNA levels . Delivery without LLO resulted in sequestration of ODN in vesicular compartments leading to little inhibition of ICAM-1 expression, which supports the requirement of LLO for efficient cytosolic delivery using this system . The data clearly demonstrate that LLO-mediated escape of ODN from intracellular vesicles is an effective approach to achieve full therapeutic antisense activity in cultured macrophages.

Immunology, 2003 Jul, 109(3), 450 - 60
Anti-human immunodeficiency virus-gag CD8+ memory T cells generated in vitro from Listeria-immunized mice; Rayevskaya M et al.; The goal of vaccination is the generation of immune memory, an immune state that permits rapid and intense recall responses to a pathogen . Considerable effort is being made to understand the nature of memory T cells . We report here that by extending the length of in vitro culture following a single restimulation with specific peptide, preparations of highly enriched, highly active antigen-specific CD8+ memory T cells could be obtained . These cultures were begun with splenocytes from mice primed by infection either with an attenuated strain of Listeria monocytogenes or vaccinia virus, both expressing the human immunodeficiency virus-1-gag gene . In the cultures, antigen-specific cytotoxic T lymphocyte (CTL) activity reached a maximum at about 9 days and thereafter fell to negligible values . Concomitant with the fall of CTL activity, however, we observed enrichment for a subset of CD11ahigh antigen-specific gag-tetramerpos CD8+ T cells . The cells showed little or no 4-hr CTL activity, but had high delayed (18-hr) CTL activity, and very high cytolytic activity after restimulation . They rapidly expressed interferon-gamma production . Their growth and survival after sorting was completely dependent on interleukin-2 or -15 . As few as 5000 of the fluorescence-activated cell sorting-purified cells protected recipients against challenge 3 months after transfer . In response to the challenge, the cells repopulated lymphoid and non-lymphoid organs and showed a sizeable increase in number . The cells therefore demonstrate high protective activity for long periods of time . These cultured cells are thus a potential source of enriched natural memory T cells for reperfusion studies and in which the mechanisms that underlie the generation, differentiation and persistence of memory can be examined.

Immunology, 2003 Jul, 109(3), 407 - 14
Induction of interleukin-12 production in mouse macrophages by berberine, a benzodioxoloquinolizine alkaloid, deviates CD4+ T cells from a Th2 to a Th1 response; Kim TS et al.; In this study we investigated whether berberine-mediated induction of interleukin-12 (IL-12) production in antigen-presenting cells could regulate a cytokine profile of antigen-primed CD4+ T helper (Th) cells . Pretreatment with berberine induced IL-12 production in both macrophages and dendritic cells, and significantly increased the levels of IL-12 production in lipopolysaccharide-stimulated macrophages and in CD40 ligand-stimulated dendritic cells . Importantly, berberine pretreatment of macrophages increased their ability to induce interferon-gamma (IFN-gamma) and reduced their ability to induce IL-4 in antigen-primed CD4+ T cells . Berberine did not influence the macrophage cell surface expression of the class II major histocompatibility complex molecule, the co-stimulatory molecules CD80 and CD86, and intracellular adhesion molecule-1 . Addition of neutralizing anti-IL-12p40 monoclonal antibody to cultures of berberine-pretreated macrophages and CD4+ T cells restored IL-4 production in antigen-primed CD4+ T cells . The in vivo administration of berberine resulted in the enhanced induction of IL-12 production by macrophages when stimulated in vitro with lipopolysaccharide or heat-killed Listeria monocytogenes, leading to the inhibition of the Th type 2 cytokine profile (decreased IL-4 and increased IFN-gamma production) in antigen-primed CD4+ T cells . These findings may point to a possible therapeutic use of berberine or medicinal plants containing berberine in the Th type 2 cell-mediated immune diseases such as allergic diseases.

AIDS Res Hum Retroviruses, 2003 May, 19(5), 409 - 20
Enhancement of immune responses to an HIV env DNA vaccine by a C-terminal segment of listeriolysin O; Bu Z et al.; An effective vaccine against AIDS should induce both cellular and humoral immune responses . Here we report that immunization of mice with a DNA plasmid encoding a chimeric protein consisting of HIV89.6 Env gp140 and the listeriolysin O (LLO) C-terminal segment (59 amino acids) significantly enhanced both humoral and cellular immune responses against the HIV89.6 Env protein . Plasmid DNA expression vectors with genes codon-optimized for mammalian expression were synthesized for HIV89.6 gp140 as well as for chimeric protein gp140-LLO, in which the coding sequence for the C-terminal 59 amino acids of LLO were fused in frame to the 3' end of the codon-optimized gene for gp140 . All plasmid vectors produced high levels of protein expression, and the gp140-LLO chimeric protein was cleaved and secreted as efficiently as gp140 . Analysis of humoral immune responses by ELISA showed that the chimeric gp140-LLO construct induced higher antibody responses than the gp140 construct in immunized mice, more notably in the IgG2a antibody subtype . Intracellular cytokine staining and flow cytometry analysis showed that the gp140-LLO construct induced significantly higher levels of cytotoxic T lymphocyte immune responses against the HIV 89.6 Env protein than those observed with the gp140 construct . Our results thus demonstrate that the C-terminal segment of LLO can be effectively employed to enhance both cellular and humoral immune responses against the HIV89.6 Env antigen in the context of a DNA vaccine.

J Food Prot, 2003 Jun, 66(6), 993 - 8
Ionizing radiation sensitivity of Listeria monocytogenes ATCC 49594 and Listeria innocua ATCC 51742 inoculated on endive (Cichorium endiva); Niemira BA et al.; Ionizing radiation inactivates the pathogenic bacteria that can contaminate leafy green vegetables . Leaf pieces and leaf homogenate of endive (Cichorium endiva) were inoculated with the pathogen Listeria monocytogenes (ATCC 49594) or Listeria innocua (ATCC 51742), a nonpathogenic surrogate bacterium . The radiation sensitivity of the two strains was similar, although L . innocua was more sensitive to the type of suspending leaf preparation . During refrigerated storage after irradiation, the population of L . monocytogenes on inoculated endive was briefly suppressed by 0.42 kilogray (kGy), a dose calibrated to achieve a 99% reduction . However, the pathogen regrew after 5 days until it exceeded the bacterial levels on the control after 19 days in storage . Treatment with 0.84 kGy, equivalent to a 99.99% reduction, suppressed L . monocytogenes throughout refrigerated storage . Doses up to 1.0 kGy had no significant effect on the color of endive leaf material, regardless of whether taken from the leaf edge or the leaf midrib . The texture of leaf edge material was unaffected by doses up to 1.0 kGy, whereas the maximum dose tolerated by leaf midrib material was 0.8 kGy . These results show that endive leaves may be treated with doses sufficient to achieve at least a 99.99% reduction of L . monocytogenes with little or no impact on the product's texture or color.

J Food Prot, 2003 Jun, 66(6), 985 - 92
Acid adaptation does not promote survival or growth of Listeria monocytogenes on fresh beef following acid and nonacid decontamination treatments; Ikeda JS et al.; The objective of this study was to evaluate the survival and growth of acid-adapted and nonadapted Listeria monocytogenes inoculated onto fresh beef subsequently treated with acid or nonacid solutions . Beef slices (2.5 by 5 by 1 cm) from top rounds were inoculated with acid-adapted or nonadapted L . monocytogenes (4.6 to 5.0 log CFU/cm2) and either left untreated (control) or dipped for 30 s in water at 55 degrees C, water at 75 degrees C, 2% lactic acid at 55 degrees C, or 2% acetic acid at 55 degrees C . The beef slices were vacuum packaged and stored at 4 or 10 degrees C and were analyzed after 0, 7, 14, 21, and 28 days of storage . Dipping in 75 degrees C water, lactic acid, and acetic acid resulted in immediate pathogen reductions of 1.4 to 2.0, 1.8 to 2.6, and 1.4 to 2.4 log CFU/cm2, respectively . After storage at 10 degrees C for 28 days, populations of L . monocytogenes on meat treated with 55 degrees C water increased by ca . 1.6 to 1.8 log CFU/cm2 . The pathogen remained at low population levels (1.6 to 2.8 log CFU/cm2) on acid-treated meat, whereas populations on meat treated with 75 degrees C water increased rapidly, reaching levels of 3.6 to 4.6 log CFU/cm2 by day 14 . During storage at 4 degrees C, there was no growth of the pathogen for at least 21 days in samples treated with 55 and 75 degrees C water, and periods of no growth were longer for acid-treated samples . There were no differences between acid-adapted and nonadapted organisms across treatments with respect to survival or growth . In conclusion, the dipping of meat inoculated with L . monocytogenes into acid solutions reduced and then inhibited the growth of the pathogen during storage at 4 and 10 degrees C, while dipping in hot water allowed growth despite initial reductions in pathogen contamination . The results of this study indicate a residual activity of acid-based decontamination treatments compared with water-based treatments for refrigerated (4 degrees C) or temperature-abused (10 degrees C) lean beef tissue in vacuum packages, and these results also indicate that this activity may not be counteracted by prior acid adaptation of L . monocytogenes.

J Food Prot, 2003 Jun, 66(6), 962 - 9
Survival and recovery of Listeria monocytogenes on ready-to-eat meats inoculated with a desiccated and nutritionally depleted dustlike vector; De Roin MA et al.; Dust from construction was theorized to serve as a vector for L . monocytogenes transmission to ready-to-eat (RTE) meats after heat processing but before packaging . A five-strain Listeria monocytogenes culture including serotype 4b was continually stressed on a sand vector under four sets of nutritionally depleted and dry conditions to simulate postprocessing contamination by dustlike particulates . The stresses included that associated with sand stored at different temperatures (10 and 22 degrees C) and levels of humidity (40% relative humidity {RH}, 88% RH, or complete desiccation) . Irradiated RTE meats, including frankfurters, bologna, chopped ham, and deli-style roast beef, were inoculated with the L . monocytogenes-contaminated sand every 2 to 3 days over a period of 1 1/2 months . After inoculation, the RTE meats were vacuum packed and stored at 4 degrees C for 24 h . Populations of L . monocytogenes were enumerated by surface plating on nonselective and selective media to recover cells on the basis of the different stresses presented (osmotic or antibiotic) . L . monocytogenes was demonstrated to be capable of surviving on the sand vector for > 151 days at 10 degrees C and 88% RH, 136 days at 10 degrees C and 0% RH, 73 days at 22 degrees C and 40% RH, and 82 days at 22 degrees C and 0% RH . These results show that under the most conservative scenario, the 73-day-old L . monocytogenes-contaminated sand was able to attach to and be recovered from the RTE meats . This study illustrated that dust contaminated with L . monocytogenes, once in contact with meat surfaces, can survive and grow, posing a health hazard to consumers.

Biotechnol Bioeng, 2003 Aug 20, 83(4), 416 - 27
Micro-assembly of functionalized particulate monolayer on C18-derivatized SiO2 surfaces; Huang TT et al.; This work describes a simple approach to immobilize functionalized colloidal microstructures onto a C(18)-coated SiO(2) substrate via specific or non-specific bio-mediated interactions . Biotinylated bovine serum albumin pre-adsorbed onto a C(18) surface was used to mediate the surface assembly of streptavidin-coated microbeads (2.8 microm), while a bare C(18) surface was used to immobilize anti-Listeria antibody-coated microbeads (2.8 microm) through hydrophobic interactions . For a C(18) surface pre-adsorbed with bovine serum albumin, hydrophobic polystyrene microbeads (0.8 microm) and positively charged dimethylamino microbeads (0.8 microm) were allowed to self-assemble onto the surface . A monolayer with high surface coverage was observed for both polystyrene and dimethylamino microbeads . The adsorption characteristics of Escherichia coli and Listeria monocytogenes on these microbead-based surfaces were studied using fluorescence microscopy . Both streptavidin microbeads pre-adsorbed with biotinylated anti-Listeria antibody and anti-Listeria antibody-coated microbeads showed specific capture of L . monocytogenes, while polystyrene and dimethylamino microbeads captured both E . coli and L . monocytogenes non-specifically . The preparation of microbead-based surfaces for the construction of microfluidic devices for separation, detection, or analysis of specific biological species is discussed .

J Dent, 2003 Jul, 31(5), 313 - 9
The erosive potential of commercially available mouthrinses on enamel as measured by Quantitative Light-induced Fluorescence (QLF); Pretty IA et al.; DESIGN: Longitudinal in vitro . METHODS: Previously extracted, caries free, human premolars were selected and prepared by gentle pumicing and coating in an acid-resistant nail-varnish save for an exposed enamel window on the buccal surface . Each was assigned to one of eight groups (six per group, 10 in positive control); positive control (citric acid, pH 2.7, F(-) 0 ppm), negative control (pH 7.0, F(-) 0 ppm) Listerine (pH 3.87, F(-) 0.021 ppm), Tesco Value (pH 6.05, F(-) 289.00 ppm), Tesco Total Care (pH 6.20, F(-) 313.84 ppm), Sainsbury's (pH 6.15, F(-) 365.75 ppm), Sensodyne (pH 6.12, F(-) 285.30 ppm) and Corsodyl (pH 5.65, F(-) 0 ppm) . The titratable acid values (TAV) for each rinse were established using volume (ml) of 0.1 M NaOH to achieve pH 7 . Fluoride values were obtained by ion selective electrode . The solutions were kept at 37 degrees C and gently agitated . Teeth were removed at hourly intervals for 15 h, air-dried and subjected to Quantitative Light-induced Fluorescence (QLF) examination by a blinded examiner and DeltaQ values recorded . At the conclusion of the study each of the positive control teeth and one from each other group were sectioned through the eroded lesion, ground and polished to 100 micrometers and subjected to transverse microradiography and DeltaZ recorded for validation . RESULTS: TAVs were: Listerine 2.45 L > Sainsbury's 0.35 ml >Tesco Total Care 0.14 ml > Tesco Value 0.08 ml > Corsodyl 0.10 ml >Sensodyne 0.9 ml . DeltaQ increased over time for the positive control, (0 h 0.2, 10 h 95.2, 15 h 152.3) . Negative controls remained stable . The increase in DeltaQ for each rinse after 15 h was Listerine (9.3(+/-7.2)), Corsodyl (1.5(+/-1.2)), Tesco Value (1.8(+/-1.2)), Tesco Total Care (1.4(+/-1.1)), Sainsbury's (3.4(+/-2.2)), Sensodyne (0.9(+/-1.6)) . TMR confirmed the presence/absence of erosive lesions . CONCLUSIONS: QLF effectively monitored erosion in the positive controls and lack of erosion in the NC . Only one mouthrinse (Listerine) caused any erosion compared to the negative control, but this was only significant after 14 h of continuous use.

Cell Immunol, 2003 Mar, 222(1), 1 - 14
IL-12-assisted immunization generates CD4+ T cell-mediated immunity to Listeria monocytogenes; Miller MA et al.; Mice infected with virulent Listeria monocytogenes develop long-lived acquired immunity . We previously reported that acquired immunity to Listeria could also be elicited by immunizing mice with non-viable Listeria or listerial proteins/peptides in combination with IL-12 . Here we show that this IL-12-assisted immunization strategy was effective in class I but not in class II MHC-deficient mice, suggesting that antigen-specific CD4(+) T cells are selectively generated using this adjuvant system . We have also evaluated the importance of endogenous production of IFN-gamma and IL-12 for the efficacy of IL-12-assisted immunization . IFN-gamma-deficient mice immunized with HKLM and IL-12 failed to produce effective Listeria-specific responses . In contrast, IL-12-deficient mice were able to generate protective antigen-specific T cell responses in response to immunization with HKLM and IL-12, indicating that exogenous IL-12 is sufficient to initiate a cytokine cascade that results in a potent T(H)1 response . IL-12-assisted immunization provides a model in which both the generation and effector mechanisms of anti-bacterial antigen-specific CD4(+) effector cells can be analyzed.

Int J Immunopathol Pharmacol, 2003 May-Aug, 16(2), 119 - 27
Heterogeneity of virulence-related properties in Listeria monocytogenes strains isolated from patients with haematological malignancies; Longhi C et al.; Listeria monocytogenes is an intracellular foodborne pathogen of humans and animals for which there are indications of virulence differences among strains . Various virulence properties related to different phases of infection process were investigated in L . monocytogenes strains isolated from patients affected by haematological malignancies . In these isolates, besides to the clinical history, we analysed the haemolysin production, the survival to acidic pH, the ability to enter and proliferate in human intestinal-like and human macrophagic-like cells, as well as the allelic polymorphism of the actA gene involved intracellular movement . A general heterogeneity in the virulence properties was detected which did not appear correlated with the clinical outcome of listeriosis but more probably was influenced by the status of the immune defence of the host.

Mol Microbiol, 2003 Jun, 48(6), 1537 - 51
Isolation of Listeria monocytogenes mutants with high-level in vitro expression of host cytosol-induced gene products; Shetron-Rama LM et al.; The facultative intracellular bacterial pathogen Listeria monocytogenes dramatically increases the expression of several key virulence factors upon entry into the host cell cytosol . actA, the protein product of which is required for cell-to-cell spread of the bacterium, is expressed at low to undetectable levels in vitro and increases in expression more than 200-fold after L . monocytogenes escape from the phagosome . To identify bacterial factors that participate in the intracellular induction of actA expression, L . monocytogenes mutants expressing high levels of actA during in vitro growth were selected after chemical mutagenesis . The resulting mutant isolates displayed a wide range of actA expression levels, and many were less sensitive to environmental signals that normally mediate repression of virulence gene expression . Several isolates contained mutations affecting actA gene expression that mapped at least 40 kb outside the PrfA regulon, supporting the existence of additional regulatory factors that contribute to virulence gene expression . Two actA in vitro expression mutants contained novel mutations within PrfA, a key regulator of L . monocytogenes virulence gene expression . PrfA E77K and PrfA G155S mutations resulted in high-level expression of PrfA-dependent genes, increased bacterial invasion of epithelial cells and increased virulence in mice . Both prfA mutant strains were significantly less motile than wild-type L . monocytogenes . These results suggest that, although constitutive activation of PrfA and PrfA-dependent gene expression may enhance L . monocytogenes virulence, it may conversely hamper the bacterium's ability to compete in environments outside host cells.

Mol Microbiol, 2003 Jun, 48(6), 1525 - 36
Aromatic amino acids at the surface of InlB are essential for host cell invasion by Listeria monocytogenes; Machner MP et al.; The surface protein InlB of the pathogen Listeria monocytogenes promotes invasion of this bacterium into host cells by binding to and activating the receptor tyrosine kinase Met . The curved leucine-rich repeat (LRR) domain of InlB, which is essential for this process, contains a string of five surface-exposed aromatic amino acid residues positioned along its concave face . Here, we show that the replacement of four of these residues (F104, W124, Y170 or Y214) by serine leads to a complete loss of uptake of latex beads coated with InlB', a truncated functional variant of InlB . The mutants correspondingly display severely reduced binding to Met . To abrogate fully invasion of bacteria expressing full-length InlB, exchange of at least four aromatic amino acids is required . We conclude that InlB binds to Met through its concave surface of the LRR domain, and that aromatic amino acids are critical for binding and signalling before invasion.

Appl Environ Microbiol, 2003 Jun, 69(6), 3640 - 5
Adhesion, invasion, and translocation characteristics of Listeria monocytogenes serotypes in Caco-2 cell and mouse models; Jaradat ZW et al.; Adhesion is a crucial first step in Listeria monocytogenes pathogenesis . In this study, we examined how the adhesion properties of serotypes correlate with their invasion efficiencies in a cell culture model (Caco-2) and in a mouse model . Adhesion characteristics of all 13 serotypes of L . monocytogenes (25 strains) were analyzed, which yielded three distinct groups (P < 0.05) with high-, medium-, and low-level-adhesion profiles . The efficiency of these strains in invading the Caco-2 cell line was analyzed, which produced two groups; however, the overall correlation (R(2)) was only 0.1236 . In the mouse bioassay, all selected strains, irrespective of their adhesion profiles, translocated to the liver and the spleen with almost equal frequencies that did not show any clear relationship with adhesion profiles . However, the serotypes with increased adhesion showed a slightly increased translocation to the brain (R(2) = 0.3371) . Collectively, these results indicate that an in vitro adhesion profile might not be an accurate assessment of a strain's ability to invade a cultured cell line or organs or tissues in a mouse model.

Appl Environ Microbiol, 2003 Jun, 69(6), 3368 - 76
Development of a Listeria monocytogenes EGDe partial proteome reference map and comparison with the protein profiles of food isolates; Ramnath M et al.; A partially annotated proteome reference map of the food pathogen Listeria monocytogenes was developed for exponentially growing cells under standardized, optimal conditions by using the sequenced strain EGDe (serotype 1/2a) as a model organism . The map was developed by using a reproducible total protein extraction and two-dimensional (2-D) polyacrylamide gel electrophoresis analysis procedure, and it contained 33 identified proteins representing the four main protein functional classes . In order to facilitate analysis of membrane proteins, a protein compartmentalization procedure was assessed . The method used provided partial fractionation of membrane and cytosolic proteins . The total protein 2-D profiles of three serotype 1/2a strains and one serotype 1/2b strain isolated from food were compared to the L . monocytogenes EGDe proteome . An average of 13% of the major protein spots in the food strain proteomes were not matched in the strain EGDe proteome . The variation was greater for the less intense spots, and on average 28% of these spots were not matched . Two of the proteins identified in L . monocytogenes EGDe were missing in one or more of the food isolates . These two proteins were proteins involved in the main glycolytic pathway and in metabolism of coenzymes and prosthetic groups . The two corresponding genes were found by PCR amplification to be present in the four food isolates . Our results show that the L . monocytogenes EGDe reference map is a valuable starting point for analyses of strains having various origins and could be useful for analyzing the proteomes of different isolates of this pathogen.

Appl Environ Microbiol, 2003 Jun, 69(6), 3137 - 43
Identification of Listeria monocytogenes genes involved in salt and alkaline-pH tolerance; Gardan R et al.; The capacity of Listeria monocytogenes to tolerate salt and alkaline stresses is of particular importance, as this pathogen is often exposed to such environments during food processing and food preservation . We screened a library of Tn917-lacZ insertional mutants in order to identify genes involved in salt and/or alkaline tolerance . We isolated six mutants sensitive to salt stress and 12 mutants sensitive to salt and alkaline stresses . The position of the insertion of the transposon was located in 15 of these mutants . In six mutants the transposon was inserted in intergenic regions, and in nine mutants it was inserted in genes . Most of the genes have unknown functions, but sequence comparisons indicated that they encode putative transporters.

J Clin Periodontol, 2003, 30 Suppl 5, 13 - 6
Evidence-based control of plaque and gingivitis; Santos A; Most adults brush and floss inadequately, and constant education and/or reinforcement is often required . Bacteria are usually left behind with mechanical oral health routines, and chemotherapeutic agents may have a key role as adjuncts to daily home-care . To date, two antiseptic mouthwashes have received the ADA Seal of Acceptance: Peridex (Zila Pharmaceuticals, Phoenix, AZ, USA; CHX, chlorhexidine) and Listerine (Pfizer Consumer Healthcare, Morris Plains, NJ, USA; essential oil (EO) mouthwash) . CHX has a strong affinity for tooth and tissue surfaces, but can cause brown staining on the teeth and tongue . Patients must also wait until all traces of toothpaste are removed before rinsing with CHX . Long-term use of an EO mouthwash is microbiologically safe, with no changes observed in the bacterial composition of supragingival plaque, and no evidence of antimicrobial resistance . A number of trials have demonstrated the long-term plaque- and gingivitis-reducing properties of both CHX and EO mouthwashes . These studies clearly demonstrate that these agents have lasting efficacy, and can access hard-to-reach areas.

Int J Immunopathol Pharmacol, 1999 Sep, 12(3), 149 - 155
The anti-invasive effect of bovine lactoferrin requires an interaction with surface proteins of Listeria Monocytogenes; Conte MP et al.; The anti-invasive effect of bovine lactoferrin (BLf) and of bovine transferrin (BTf) towards L . monocytogenes, an intracellular facultative food-borne pathogen, was assayed in the enterocyte-like cell line Caco-2 . When 0.5 mg/ml BLf were added during the infection time or preincubated with bacteria the number of internalized bacteria was noticeably decreased whereas BLf was ineffective when preincubated with the enterocytes or added post infection . BTf was deprived of any effect . Results from direct binding and Western blotting assays provided evidence that two L . monocytogenes surface proteins, of approximately 80 and 60 kDa, specifically reacted with BLf . These findings strongly support the hypothesis that the antiinvasive mechanism of BLf is due to its interaction with bacterial surfaces, but not to its binding with eukaryotic cells.

Int J Parasitol, 2003 May, 33(5-6), 495 - 505
Haemolysin A and listeriolysin--two vaccine delivery tools for the induction of cell-mediated immunity; Dietrich G et al.; Haemolysin A of Escherichia coli and listeriolysin of Listeria monocytogenes represent important bacterial virulence factors . While such cytolysins are usually the reason for morbidity and even mortality, vaccine researchers have turned haemolysin A and listeriolysin into tools for vaccine delivery . Both cytolysins have found widespread application in vaccine research and are highly suitable for the elicitation of cell-mediated immunity . In this paper, we will review vaccine delivery mediated by the haemolysin A secretion system and listeriolysin and will highlight their use in vaccination approaches against protozoan parasites.

Int J Food Microbiol, 2003 Jul 15, 84(1), 79 - 85
Rapid enumeration of Listeria monocytogenes in milk using competitive PCR; Choi WS et al.; Competitive polymerase chain reaction (cPCR) was used to develop a direct enumeration method of Listeria monocytogenes in milk . Sterile milk was artificially inoculated with L . monocytogenes and DNA was extracted using guanidine thiocyanate/phenol/chloroform, followed by PCR . Several primers for L . monocytogenes hlyA gene were tested for specific detection and DG69/DG74 primer set was selected . The primer set produced a 636-bp band from L . monocytogenes, but no band appeared from the other six Listeria spp . tested . A detection limit was as few as 10(3) colony-forming unit (cfu) per 0.5 ml of milk with this primer set . When the samples were cultured at 25 degrees C for 15 h in a TSBY medium, even a single bacterium could be detected with this primer set by PCR . For the cPCR, hlyA gene segment was cloned in pGem-4Z vector and was modified to produce competitor DNA . The competitor DNA has the same primer binding sites and sequences as the target DNA except EcoRI site . Known amount of competitor DNA was coamplified with L . monocytogenes total DNA isolated from artificially inoculated milk . The target DNA and competitor DNA were distinguished by EcoRI digestion after cPCR . The cell number determined by cPCR was approximately equal to the colony-forming unit from conventional plate counting method . For the whole procedure, it took only 5 h.

Int J Food Microbiol, 2003 Jul 25, 84(2), 237 - 44
Effects of a bacteriocin-like inhibitory substance from Carnobacterium piscicola against human and salmon isolates of Listeria monocytogenes; Schobitz R et al.; The aim of this study was to characterize the antagonism of a bacteriocin-like inhibitory substance (BLIS) produced by Carnobacterium piscicola L103 against Listeria monocytogenes strains isolated from salmon and human samples . The inhibitory effect of the BLIS was evaluated in Tryptic soy agar (TSA) during different growth phases of L . monocytogenes at 5 degrees C, using the well diffusion method . Also, the type of inhibition, either bacteriostatic or bactericidal of the BLIS in Tryptic soy broth (TSB), was studied and the development of resistant cells investigated.Results showed an antagonistic effect of the BLIS on all the strains of L . monocytogenes . Four selected strains presented a higher sensitivity to the BLIS in the exponential growth phase and were more resistant in the stationary phase . In TSB, the inhibitory substance showed a partially bactericidal effect on L . monocytogenes . After inactivation of the BLIS with a protease, however, a regrowth of L . monocytogenes was found . The isolate most affected by the action of the BLIS was one of salmon origin . From the 86 isolated colonies that grew in the presence of the BLIS, 93% showed total resistance and 7% partial resistance, which was maintained through five consecutive culture cycles in the absence of the BLIS.

Int J Food Microbiol, 2003 Jul 25, 84(2), 207 - 16
Stress response of Listeria monocytogenes isolated from cheese and other foods; Faleiro ML et al.; The responses to pH and sodium chloride of four strains of Listeria monocytogenes isolated from Portuguese cheese, with a sodium chloride concentration of about 2% (w/v) and a pH value from 5.1 to 6.2, were studied . Two isolates from meat and two clinical isolates related to food-borne listeriosis, in which the implicated food product had about 2-3.5% (w/v) sodium chloride, also were studied . The effect of temperature on pH and sodium chloride sensitivity was also determined . The results show that natural isolates vary in response to these stresses and the data were often at variance with previously published data . Strains varied in sensitivity to low pH and to high sodium chloride concentration but the cheese isolates tended to be more resistant . A lower temperature was associated with a decrease in resistance to low pH and to sodium chloride . All strains showed an acid tolerance response induction when grown at pH 5.5 and although the time required for maximum induction of the response varied between strains, 2 h of acid adaptation, at least, was necessary which is longer than previously reported . Some strains showed an osmotolerance response after incubation in 3.5% (w/v) sodium chloride . Osmoadaptation, in addition to inducing an osmotolerance response, also induced cross-protection against acid shock conditions (pH 3.5) . The acid tolerance response also induced a cross-protection against osmotic shock conditions (20% (w/v) sodium chloride) . In some cases there was a relationship between the degree of resistance and adaptation, but usually the behaviour of a particular strain was independent of the conditions from which it was isolated.

Int J Food Microbiol, 2003 Jul 25, 84(2), 133 - 43
The effect of growth atmosphere on the ability of Listeria monocytogenes to survive exposure to acid, proteolytic enzymes and bile salts; King T et al.; Four isolates of Listeria monocytogenes from food, human and environmental sources were grown separately in broth (pH 6.0 at 8 degrees C) under atmospheres of air, 100% N(2), 40% CO(2):60% N(2) or 100% CO(2) . Exponential and stationary phase cells were harvested to determine if growth atmosphere and growth phase influenced this pathogen's ability to survive exposure to an acid environment coupled with proteolytic enzymes, and the activity of bile salts . In general, isolates were more resistant to the acid environment than the bile salts environment and stationary phase cells were significantly more resistant to both environments than exponential phase cells . Irrespective of prior growth atmosphere, none of the isolates when in exponential phase remained detectable following full exposure to the acid environment (110 min at 37 degrees C) or the bile environment (3 h at 37 degrees C) . With the exception of one isolate grown under the atmosphere of 40% CO(2):60% N(2), all isolates when in stationary phase were detectable following full exposure to the acid environment but death rates varied significantly . Stationary phase cells of all isolates grown under 40% CO(2):60% N(2) and 100% CO(2) were highly susceptible to the bile salts environment: cells were not detectable after a 2-min exposure whereas stationary phase cells grown under air or 100% N(2) were recovered following full exposure to the bile environment . Survival curves were characterised by a population decline of at least 3 log(10)/ml (from an initial level of 7 log(10) CFU/ml) in the first 15 min; thereafter a constant population number of approximately 4 log(10)/ml was maintained over the remaining exposure period . No survival was observed when stationary phase cells of L . monocytogenes FRRB 2538 grown in air and 100% N(2) were subjected to the acid environment followed by immediate exposure to the bile salts environment . The results showed that growth atmosphere and growth phase could influence survival of this pathogen against conditions that imitate the extremes of the most important nonspecific defence mechanisms against microbial infection: the acid environment of the stomach coupled with the activity of proteolytic enzymes, and the activity of bile salts in the small intestine.

Int Immunopharmacol, 2003 Jun, 3(6), 889 - 900
Protective effects of Chlorella vulgaris in lead-exposed mice infected with Listeria monocytogenes; Queiroz ML et al.; Chlorella vulgaris extract (CVE) was examined for its chelating effects on the myelosuppression induced by lead in Listeria monocytogenes-infected mice . The reduction in the number of bone marrow granulocyte-macrophage progenitors (CFU-GM) observed after the infection was more severe in the groups previously exposed to lead . Extramedullar hematopoiesis, which was drastically increased after the infection, was not altered by the presence of lead . Treatment with CVE, given simultaneously or following lead exposure, restored to control values the myelosuppression observed in infected/lead-exposed mice and produced a significant increase in serum colony-stimulating activity . The benefits of the CVE treatment were also evident in the recovery of thymus weight, since the reduction produced by the infection was further potentiated by lead exposure . The efficacy of CVE was evident when infected and infected/lead-exposed mice were challenged with a lethal dose of L . monocytogenes after a 10-day treatment with 50 mg/kg CVE/day, given simultaneously to the exposure to 1300 ppm lead acetate in drinking water . Survival rates of 30% for the infected group and of 20% for the infected/lead-exposed groups were observed . Evidence that these protective effects of CVE are partly due to its chelating effect was given by the changes observed in blood lead levels . We have observed in the group receiving the CVE/lead simultaneous exposure a dramatic reduction of 66.03% in blood lead levels, when compared to lead-exposed nontreated control . On the other hand, CVE treatment following lead exposure produced a much less effective chelating effect . CVE treatments for 3 or 10 days, starting 24 h following lead exposure, produced a reduction in blood lead levels of 13.5% and 17%, respectively, compared to lead-exposed nontreated controls . The significantly better response observed with the simultaneous CVE/lead administration indicates that the immunomodulation effect of CVE plays an important role in the ability of this algae to reduce blood lead levels . In this regard, additional experiments with gene knockout C57BL/6 mice lacking a functional IFN-gamma gene demonstrated that this cytokine is of paramount importance in the protection afforded by CVE . The antibacterial evaluation measured by the rate of survival demonstrated that, in face of a 100% survival in the control group composed of normal C57BL/6 mice, which are resistant to L . monocytogenes, we observed no protection whatsoever in the IFN-gamma knockout C57BL/6 mice treated with CVE and inoculated with L . monocytogenes.

Emerg Infect Dis, 2003 Jun, 9(6), 672 - 80
Molecular subtyping to detect human listeriosis clusters; Sauders BD et al.; We analyzed the diversity (Simpson's Index, D) and distribution of Listeria monocytogenes in human listeriosis cases in New York State (excluding New York City) from November 1996 to June 2000 by using automated ribotyping and pulsed-field gel electrophoresis (PFGE) . We applied a scan statistic (p<or=0.05) to detect listeriosis clusters caused by a specific Listeria monocytogenes subtype . Among 131 human isolates, 34 (D=0.923) ribotypes and 74 (D=0.975) PFGE types were found . Nine (31% of cases) clusters were identified by ribotype or PFGE; five (18% of cases) clusters were identified by using both methods . Two of the nine clusters (13% of cases) corresponded with investigated multistate listeriosis outbreaks . While most human listeriosis cases are considered sporadic, highly discriminatory molecular subtyping approaches thus indicated that 13% to 31% of cases reported in New York State may represent single-source clusters . Listeriosis control and reduction efforts should include broad-based subtyping of human isolates and consider that a large number of cases may represent outbreaks.

EMBO Rep, 2003 May, 4(5), 523 - 9
A crucial role for profilin-actin in the intracellular motility of Listeria monocytogenes; Grenklo S et al.; We have examined the effect of covalently crosslinked profilin-actin (PxA), which closely matches the biochemical properties of ordinary profilin-actin and interferes with actin polymerization in vitro and in vivo, on Listeria monocytogenes motility . PxA caused a marked reduction in bacterial motility, which was accompanied by the detachment of bacterial tails . The effect of PxA was dependent on its binding to proline-rich sequences, as shown by the inability of PH133SxA, which cannot interact with such sequences, to impair Listeria motility . PxA did not alter the motility of a Listeria mutant that is unable to recruit Ena (Enabled)/VASP (vasodilator-stimulated phosphoprotein) proteins and profilin to its surface . Finally, PxA did not block the initiation of actin-tail formation, indicating that profilin-actin is only required for the elongation of actin filaments at the bacterial surface . Our findings provide further evidence that profilin-actin is important for actin-based processes, and show that it has a key function in Listeria motility.

Toxicol Sci, 2003 Aug, 74(2), 325 - 34 Epub 2003 May 28.
Immune changes during acute cold/restraint stress-induced inhibition of host resistance to Listeria; Cao L et al.; Experiments were conducted to delineate the cellular changes modulated by acute cold/restraint stress (ACRS), a physical and psychological stressor, in response to a Listeria monocytogenes(LM) infection . In addition to wild type (WT) BALB/c mice, CD4-deficient (CD4-/-) BALB/c mice, which have no effective adaptive immunity, were used to determine the involvement of adaptive versus innate immunity . ACRS-induced suppression of host resistance to LM was not observed in CD4-/- mice, suggesting the involvement of CD4+T cells in the acute cold/restraint stress (ACRS)-induced inhibition . The in vivo splenic leukocyte phenotypes and activities of WT BALB/c mice after infection and in vitro lymphocyte responses to heat-killed LM (HKLM) also were examined . There were no significant differences in the numbers of splenic T and B lymphocytes, natural killer cells, macrophages, or neutrophils between nonstressed and ACRS-treated WT mice . However, higher levels of activated T cells and non-T lymphocytes were observed in the ACRS-treated mice; beta-adrenergic receptor (beta-ADR) antagonists (propranolol and atenolol) eliminated these elevated levels of activation, as well as the ACRS-induced suppression of host resistance . ACRS and control mice also had equivalent activation of macrophages . With in vitro HKLM stimulation, splenocytes from ACRS-treated mice produced significantly higher levels of IFNgamma and slightly higher levels of IL-6 in comparison with the nonstressed mice, although equivalent levels of lymphocyte proliferation were obtained . Additionally, ACRS-treated mice showed comparable elevation of serum nitric oxide after infection, indicating macrophage bactericidal activity similar to nonstressed mice . Thus, it appears that ACRS inhibits host resistance through regulatory CD4+ T cells and/or effector cell functions downstream of CD4+ T cell activation, as well as through beta-ADR signaling, in that blockage of these receptors appears to aid host defenses by means other than elevation of helper T cell activity . Because CD4 T cell deficiency and beta-ADR blockage produced equivalent effects, beta-ADR+ CD4+ T cells may have a negative role on host defenses after ACRS.

Int J Cancer, 2003 Jul 20, 105(6), 811 - 9
Recombinant E . coli efficiently delivers antigen and maturation signals to human dendritic cells: presentation of MART1 to CD8+ T cells; Radford KJ et al.; The generation of tumour-specific cytotoxic T-lymphocyte (CTL) responses is the primary focus in the design of immunotherapeutic cancer vaccines . We have recently demonstrated generation of ovalbumin (OVA)-specific CTLs and tumour-protection in a murine tumour model using vaccination with dendritic cells (DCs) pulsed with E . coli expressing listeriolysin O (LLO) and OVA as a model antigen . In this system paraformaldehyde fixation of E . coli/LLO provided an additional safety feature without compromising vaccine efficacy . We therefore reasoned that paraformaldehyde-fixed recombinant E . coli expressing LLO would be an efficient vehicle for the delivery of human tumour antigens to human DCs . In the present study, we demonstrate that fixed E . coli expressing LLO are taken up efficiently by human monocyte-derived DCs (MoDCs) with minimal toxicity . As a consequence of the interaction with bacteria, human DCs undergo marked phenotypic and functional maturation . Furthermore, we show that fixed E . coli/LLO expressing the well-characterised human melanoma antigen, MART1, efficiently deliver the HLA-A2-restricted MART1(27-35) epitope for processing and presentation on human MoDCs, suggesting the potential of this system as a novel strategy for human tumour immunotherapy .

Apoptosis, 2003 Mar, 8(2), 179 - 90
Potential role of the EPEC translocated intimin receptor (Tir) in host apoptotic events; Malish HR et al.; Apoptosis, or programmed cell death, is a well-ordered process that allows damaged or diseased cells to be removed from an organism without severe inflammatory reactions . Multiple factors, including microbial infection, can induce programmed death and trigger reactions in both host and microbial cellular pathways . Whereas an ultimate outcome is host cell death, these apoptotic triggering mechanisms may also facilitate microbial spread and prolong infection . To gain a better understanding of the complex events of host cell response to microbial infection, we investigated the molecular role of the microorganism Enteropathogenic Escherichia coli (EPEC) in programmed cell death . We report that wild type strain of EPEC, E2348/69, induced apoptosis in cultured PtK2 and Caco-2 cells, and in contrast, infections by the intracellularly localized Listeria monocytogenes did not . Fractionation and concentration of EPEC-secreted proteins demonstrated that soluble protein factors expressed by the bacteria were capable of inducing the apoptotic events in the absence of organism attachment, suggesting adherence is not required to induce host cell death . Among the known EPEC proteins secreted via the Type III secretion (TTS) system, we identified the translocated intimin receptor (Tir) in the apoptosis-inducing protein sample . In addition, host cell ectopic expression of an EPEC GFP-Tir showed mitochondrial localization of the protein and produced apoptotic effects in transfected cells . Taken together, these results suggest a potential EPEC Tir-mediated role in the apoptotic signaling cascade of infected host cells.

Clin Nutr, 2003 Jun, 22(3), 313 - 9
Anti-oxidant properties of N-acetyl-L-cysteine do not improve the immune resistance of mice fed dietary lipids to Listeria monocytogenes infection; Puertollano MA et al.; BACKGROUND & AIMS: Current knowledge of the potential effects that several dietary lipids exert on immune functions indicates that these substances participate actively in the modulation of immune system by which they contribute to the improvement of the conditions of patients suffering from inflammatory disorders . However, long-chain n-3 polyunsaturated fatty acids induce an immunosuppressive status that leads to a reduction of the host natural resistance to infectious agents as well as to an enhancement of oxidative damage . Hence, the present study has been designed to evaluate the effects on the immune system of the antioxidant N-acetyl-L-cysteine (NAC) in mice fed dietary lipids and infected with Listeria monocytogenes . METHODS: Balb/c mice were fed for 4 weeks with diets containing either olive oil (OO, 20% by weight), fish oil (FO, 20% by weight) or hydrogenated coconut oil (HCO, 20% by weight) . After dietary lipid administration mice were experimentally infected with L . monocytogenes or treated with NAC (25mg/ml intraperitoneally) . RESULTS: NAC at a concentration of 1mM promoted a loss of cell viability, although no differences were observed among the four groups . After injection of NAC in combination with L . monocytogenes, 25% of mice fed a low-fat (LF) diet survived . However, in the groups fed dietary lipids no effect on survival of mice was found . NAC participated in the reduction of superoxide anion generation measured with nitroblue tetrazolium (NBT) in the group fed a FO diet . Finally, NAC reduced the recovery of L . monocytogenes from spleen of mice fed diets containing LF or HCO . CONCLUSIONS: On the basis of these results, we can confirm that the administration of NAC improves survival in mice fed LF diet, whereas a reduction in the generation of superoxide radicals was measured in mice fed a FO diet and infected with L . monocytogenes . Similarly, bacterial recovery was diminished in mice fed diets containing LF or HCO . Hence, these data reveal a beneficial effect of NAC in mice fed LF or HCO and a detrimental action of this antioxidant in mice fed diets containing FO or OO.

Vaccine, 2003 Jun 1, 21 Suppl 2, S102 - 9
Induction of immune responses by attenuated isogenic mutant strains of Listeria monocytogenes; Darji A et al.; We have generated isogenic Listeria monocytogenes mutant strains to study the induction of protective immunity in mice . These strains harbored either a specific deletion within the actin nucleator (actA) and/or have multiple deletions within the actA and phospholipase B (plcB) genes . In comparison to the wild type parental L . monocytogenes EGDe strains, the mutant strains were extremely low in virulence and were rapidly eliminated by the host during the first days of infection . Nevertheless, a single immunization with both mutant strains (EGDe DeltaactA2 and DeltaactADeltaplcB) efficiently induced and maintained effector memory (CD8(+)) T cells and has provided animals with a state of long-lasting protective immunity against wild type L . monocytogenes . These mutant strains can be used as live vaccines against the corresponding virulent pathogen and as carriers for introducing heterologous protective antigens into animals and humans.

Infect Immun, 2003 Jun, 71(6), 3614 - 8
Listeriolysin O-mediated calcium influx potentiates entry of Listeria monocytogenes into the human Hep-2 epithelial cell line; Dramsi S et al.; To investigate factors which modulate the entry of Listeria monocytogenes into mammalian cells, we have analyzed the role of Ca(2+) . We show that L . monocytogenes induced Ca(2+) transients into the human Hep-2 epithelial cell line . The nonpathogenic species L . innocua or a L . monocytogenes mutant strain defective in listeriolysin O (LLO) production was unable to induce these calcium fluxes . Addition of plasma membrane calcium channel antagonists or chelation of extracellular calcium markedly reduced L . monocytogenes entry . In contrast, chelation of host cytosolic Ca(2+) or blockade of Ca(2+) release from intracellular stores did not affect invasion . These results indicate that L . monocytogenes-induced mobilization of extracellular Ca(2+) by LLO and activation of downstream Ca(2+)-dependent signaling are required for efficient cell invasion.

Infect Immun, 2003 Jun, 71(6), 3473 - 84
Deletion of the gene encoding p60 in Listeria monocytogenes leads to abnormal cell division and loss of actin-based motility; Pilgrim S et al.; Protein p60 encoded by the iap gene is regarded as an essential gene product of Listeria monocytogenes . Here we report, however, the successful construction of a viable iap deletion mutant of L . monocytogenes EGD . The mutant, which produces no p60, shows abnormal septum formation and tends to form short filaments and hooked forms during logarithmic growth . These abnormal bacterial cells break into almost normal sized single bacteria in the late-stationary-growth phase . The iap mutant is strongly attenuated in a mouse model after intravenous injection, demonstrating the importance of p60 during infection, and the invasiveness of the Deltaiap mutant for 3T6 fibroblasts and Caco-2 epithelial cells is slightly reduced . Upon uptake by epithelial cells and macrophages, the iap mutant escapes from the phagosome into the cytosol with the same efficiency as the wild-type strain, and the mutant bacteria also grow intracellularly at a rate similar to that of the wild-type strain . Intracellular movement and cell-to-cell spread are drastically reduced in various cell lines, since the iap-negative bacteria fail to induce the formation of actin tails . However, the bacteria are covered with actin filaments . Most intracellular bacteria show a nonpolar and uneven distribution of ActA around the cell, in contrast to that for the wild-type strain, where ActA is concentrated at the old pole . In an iap(+) revertant strain that produces wild-type levels of p60, intracellular movement, cell-to-cell spread, and polar distribution of ActA are fully restored . In vitro analysis of ActA distribution on the filaments of the Deltaiap strain shows that the loss of bacterial septum formation leads to ActA accumulation at the presumed division sites . In the light of data presented here and elswhere, we propose to rename iap (invasion-associated protein) cwhA (cell wall hydrolase A).

Infect Immun, 2003 Jun, 71(6), 3429 - 36
Experimental validation of low virulence in field strains of Listeria monocytogenes; Roche SM et al.; Several reports have described Listeria monocytogenes strains which were nonpathogenic or weakly pathogenic, but little is known about these low-virulence strains . We found that 9 field L . monocytogenes strains were hypovirulent and 17 were avirulent, based on the number of mice contaminated and the colonization of their spleens after subcutaneous inoculation . All these strains possessed the known virulence genes . We have now assessed the low virulence of these strains in other assays before determining how they differ from virulent strains . We have shown that the low-virulence strains exhibited a phenotypic stability and were not a mixture of virulent and avirulent bacteria . They did not recover virulence after many passages in mice and colonized the spleens of mice more poorly than virulent strains after i.v . inoculation . Their lethal capacities, determined by 50% lethal dose (LD(50)), were lower than those of virulent strains . Like Listeria innocua, 14 of 17 avirulent strains had no LD(50) and were eliminated by the lymph nodes after subcutaneous inoculation . The virulent, hypovirulent, and avirulent strains were always significantly different, whatever the tests of virulence used, confirming the importance of these low-virulence field strains in identifying the proteins involved in virulence.

Curr Infect Dis Rep, 2003 Jun, 5(3), 220 - 226
Microbiology and Treatment of Halitosis; Loesche WJ; The many thousands of individuals who experience oral malodor that stems from the overgrowth of proteolytic, anaerobic bacteria on their tongue surfaces can be successfully treated by a regimen that includes tongue brushing and tooth brushing, often in combination with a mouthrinse containing an antibacterial agent . Several candidate mouthrinses containing essential oils (Listerine; Warner-Lambert, Morris Plains, NJ), ZnCl(2), chlorine dioxide, or an oil:water-cetylpyridium chloride mouthrinse have reduced the organoleptic scores of individuals with moderate levels of oral malodor in the absence of tongue brushing . Very little long-term data beyond 6 weeks of use are available.

C R Biol, 2003 Feb, 326(2), 161 - 70
Actin-based motility as a self-organized system: mechanism and reconstitution in vitro; Carlier MF et al.; Site-directed actin polymerisation in response to signalling is responsible for the formation of cell protrusions . These elementary 'actin-based motility processes' are involved in cell locomotion, cell metastasis, organ morphogenesis and microbial pathogenesis . We have reconstituted actin-based propulsive movement of particles of various sizes and geometries (rods, microspheres) in a minimum motility medium containing five pure proteins . The ATP-supported treadmilling of actin filaments, regulated by Actin Depolymerizing Factor (ADF/cofilin), profilin and capping proteins provides the thermodynamic basis for sustained actin-based movement . Local activation of Arp2/3 complex at the surface of the particle promotes autocatalytic barbed end branching of filaments, generating a polarized arborescent array . Barbed end growth of branched filaments against the surface generates a propulsive force and is eventually arrested by capping proteins . Understanding the mechanism of actin-based movement requires elucidation of the biochemical properties and mode of action of Arp2/3 complex in filament branching, in particular the role of ATP binding and hydrolysis in Arp2/3, and a physical analysis of the movement of functionalised particles . Because the functionalisation of the particle by an activator of Arp2/3 complex (N-WASP or the Listeria protein ActA) and the concentrations of effectors in the medium are controlled, the reconstituted motility assay allows an analysis of the mechanism of force production at the mesoscopic and molecular levels.

Neurol Sci, 2003 Apr, 24(1), 40 - 3
Syringomyelia following Listeria meningoencephalitis: report of a case; Nardone R et al.; A case of symptomatic syringomyelia which appeared six years after Listeria meningoencephalitis is described . Chronic spinal arachnoiditis, as shown by standard MRI and dynamic phase contrast (PC) cine-MRI, may occur after spinal infection and is likely the cause of syringomyelia . To our knowledge, there are no previous reports of delayed spinal complications following Listeria monocytogenes infection . The possibility of developing syringomyelia should be always considered in any patient with a history of central nervous system infection.

Surg Neurol, 2003 Apr, 59(4), 320 - 8
Multiple cerebral abscesses because of Listeria monocytogenes: three case reports and a literature review of supratentorial listerial brain abscess(es); Cone LA et al.; BACKGROUND: Central nervous system involvement often follows bacteremia because of Listeria monocytogenes . Meningitis is clinically the most common manifestation, while brain abscess occurs in about 1% of patients . Brain abscess is usually solitary but in recent years, probably in part because of the availability of computerized tomography and magnetic resonance imaging, several reports have described two or more separate supratentorial abscesses . METHODS: We have described three patients with listerial brain abscesses and reviewed the North American and European literature of brain abscess(es) because of L . monocytogenes through December 2001 . We have evaluated the role of underlying diseases and therapeutic immunosuppression on the development of solitary or greater than one brain abscess . RESULTS: In contrast to meningitis, where immunosuppression does not predispose either to disease incidence or to higher mortality, patients with solitary and particularly those with more than one supratentorial abscess usually are immunosuppressed either by disease or by therapy . Corticosteroids in particular are significant predisposing factors, especially in those patients with two or more brain abscesses . Mortality resulting from listerial brain abscess, whether solitary or multiple, is nearly three times higher than nonlisterial brain abscess, probably in part because of both underlying diseases and immunosuppressive therapy . CONCLUSIONS: Therapy with high-dose ampicillin in combination with gentamicin appear to be the drugs of choice, followed by trimethoprim/sufamethoxazole and vancomycin . In general, antimicrobial therapy appears to be satisfactory treatment without surgical intervention.

J Food Prot, 2003 May, 66(5), 819 - 24
Gamma irradiation of fine-emulsion sausage containing sodium diacetate; Sommers C et al.; Listeria monocytogenes, a psychrotrophic foodborne pathogen, is a frequent postprocess contaminant of ready-to-eat (RTE) meat products, including frankfurters and bologna . Ionizing radiation can eliminate L . monocytogenes from RTE meats . Sodium diacetate (SDA) incorporated into fine-emulsion sausages inhibits the growth of L . monocytogenes . Irradiation of L . monocytogenes suspended in SDA solutions resulted in synergistic reductions of the microorganism . L . monocytogenes populations were reduced by > 9 log10 units at a radiation dose of 1.5 kGy when suspended in 0.125% SDA solution . In contrast, the D10-values (the ionizing radiation doses required to reduce the population by 90%) were 0.58, 0.59, 0.57, and 0.53 kGy for L . monocytogenes populations suspended in emulsions containing 0, 0.125, 0.25, and 0.5% SDA, respectively . The D10-values for L . monocytogenes surface inoculated onto frankfurters dipped in 0, 0.125, 0.25, and 0.5% SDA solutions were 0.58, 0.53, 0.54, and 0.52 kGy, respectively . Postirradiation growth of L . monocytogenes suspended in beef bologna emulsion at 9 degrees C was dependent on SDA concentration and ionizing radiation dose . Very small, but statistically significant, changes in bologna redness, lipid oxidation, and shear force were observed for the beef bologna emulsion with the highest SDA concentration (0.5%) and irradiation dose (3.0 kGy) . SDA can inhibit the proliferation of L . monocytogenes surviving the irradiation process with minimal impact on fine-emulsion sausage color, lipid oxidation, and firmness when used within regulatory limits.

J Food Prot, 2003 May, 66(5), 812 - 8
Reducing levels of Listeria monocytogenes contamination on raw salmon with acidified sodium chlorite; Su YC et al.; The antimicrobial activity of acidified sodium chlorite (ASC) against Listeria monocytogenes in salmon was studied . Raw salmon (whole fish and fillets) inoculated with L . monocytogenes (10(3) CFU/cm2 or 10(4) CFU/g) were washed with ASC solution (50 ppm) for 1 min and stored at -18 degrees C for 1 month (whole salmon) or in ice for 7 days (fillets) . L . monocytogenes populations were determined for whole salmon after frozen storage and for fillets on days 1, 3, 5, and 7 of storage . A wash with ASC solution followed by ASC glazing did not reduce L . monocytogenes on the skin of whole salmon during frozen storage . However, the wash resulted in an L . monocytogenes reduction of 0.5 log CFU/g for salmon fillets . The populations of L . monocytogenes in fillets increased slowly during ice storage, but the growth of these populations was retarded by ASC ice . By day 7, the populations were 0.25 log units smaller in fillets stored in ASC ice and 0.62 log units smaller in fillets that had been washed with ASC solution and stored in ASC ice than in control fillets . Treatment with ASC also reduced total plate counts (TPCs) by 0.43 log CFU/cm2 on the skin of whole salmon and by 0.31 log CFU/g in fillets . The TPCs for skin decreased during frozen storage but increased gradually for fillets stored at 5 degrees C or in ice . However, TPCs of ASC-treated samples were lower than those for controls at any point during the study . Washing with ASC solution significantly (P < 0.05) reduced TPCs on the skin of whole salmon and in fillets, as well as L . monocytogenes in fillets . The antimicrobial activity of ASC was enhanced when salmon was washed with ASC solution and stored in ASC ice.

J Food Prot, 2003 May, 66(5), 804 - 11
Predictive model for the combined effect of temperature, sodium lactate, and sodium diacetate on the heat resistance of Listeria monocytogenes in beef; Juneja VK; The effects of heating temperature (60 to 73.9 degrees C), sodium lactate (NaL; 0.0 to 4.8% {wt/wt}), and/or sodium diacetate (SDA; 0.0 to 0.25% {wt/wt}) and of the interactions of these factors on the heat resistance of a five-strain mixture of Listeria monocytogenes in 75% lean ground beef were examined . Thermal death times for L . monocytogenes in filtered stomacher bags in a circulating water bath were determined . The recovery medium was tryptic soy agar supplemented with 0.6% yeast extract and 1% sodium pyruvate . Decimal reduction times (D-values) were calculated by fitting a survival model to the data with a curve-fitting program . The D-values were analyzed by second-order response surface regression for temperature, NaL level, and SDA level . The D-values observed for beef with no NaL or SDA at 60, 65, 71.1, and 73.9 degrees C were 4.67, 0.72, 0.17, and 0.04 min, respectively . The addition of 4.8% NaL to beef increased heat resistance at all temperatures, with D-values ranging from 14.3 min at 60 degrees C to 0.13 min at 73.9 degrees C . Sodium diacetate interacted with NaL, thereby reducing the protective effect of NaL and rendering L . monocytogenes in beef less resistant to heat . A mathematical model describing the combined effect of temperature, NaL level, and SDA level on the thermal inactivation of L . monocytogenes was developed . This model can predict D-values for any combination of temperature, NaL level, and SDA level that is within the range of those tested . This predictive model will have substantial practical importance to processors of cooked meat, allowing them to vary their thermal treatments of ready-to-eat meat products in a safe manner.

Int J Food Microbiol, 2003 Jun 25, 83(3), 325 - 30
Susceptibility of Listeria monocytogenes isolated from food in Italy to antibiotics; Aureli P et al.; The susceptibility of 148 strains of Listeria monocytogenes isolated from food to antibiotics currently used in veterinary and human therapy was determined by standard agar dilution and disk diffusion methods . The antibiotics included amikacin, amoxicillin, cefazolin, chloramphenicol, erythromycin, flumequine, fosfomycin, gentamicin, kanamycin, lincomycin, oxytetracycline, rifampicin, spiramycin, streptomycin, tetracycline, tobramycin and vancomycin . Soussy's breakpoints and MIC(50)-MIC(90) values were used to classify the strains into sensitive, moderately sensitive and resistant groups.This work is part of a wider surveillance program on listeriosis started in Italy in 1995.

Proc Natl Acad Sci U S A, 2003 May 27, 100(11), 6493 - 8 Epub 2003 May 08.
Compression forces generated by actin comet tails on lipid vesicles; Giardini PA et al.; Polymerizing networks of actin filaments generate force for a variety of movements in living cells, including protrusion of filopodia and lamellipodia, intra- and intercellular motility of certain bacterial and viral pathogens, and motility of endocytic vesicles and other membrane-bound organelles . During actin-based motility, coexisting populations of actin filaments exert both pushing and retarding forces on the moving cargo . To examine the distribution and magnitude of forces generated by actin, we have developed a model system where large artificial lipid vesicles coated with the protein ActA from the bacterial pathogen Listeria monocytogenes are propelled by actin polymerization in cytoplasmic extract . We find that motile vesicles associated with actin comet tails are significantly deformed due to an inward compression force exerted by actin polymerization orthogonal to the direction of motion, which is >10-fold greater in magnitude than the component of the force exerted in the direction of motion . Furthermore, there is a spatial segregation of the pushing and retarding forces, such that pushing predominates along the sides of the vesicle, although retarding forces predominate at the rear . We estimate that the total net (pushing minus retarding) force generated by the actin comet tail is approximately 0.4-4 nN . In addition, actin comet tail formation is associated with polarization of the ActA protein on the fluid vesicle surface, which may reinforce the persistence of unidirectional motion by helping to maintain a persistent asymmetry of actin filament density.

Med Microbiol Immunol (Berl), 2003 May, 192(2), 85 - 91 Epub 2002 Oct 19.
A Listeria adhesion protein-deficient Listeria monocytogenes strain shows reduced adhesion primarily to intestinal cell lines; Jaradat ZW et al.; Listeria monocytogenes adheres and penetrates intestinal cell linings for systemic infection . A 104-kDa Listeria adhesion protein (LAP) from L . monocytogenes was previously demonstrated to be responsible for adhesion to intestinal enterocyte-like Caco-2 cells . We investigated the adhesion and invasion characteristics of a LAP-deficient mutant L . monocytogenes strain (A572) to various human intestinal and non-intestinal cell lines to assess the possible target host cells . Among the intestinal cell lines, A572 showed significantly reduced adhesion than the wild type (WT) strain to the cells of ileum-cecum (HCT-8) and colon (Caco-2 and HT-29), whereas A572 and WT did not show any significant differences in adhesion to other intestinal cell lines from duodenum (HuTu-80) or jejunum (Int-407) . Differences in adhesion between A572 and WT were little or none in non-intestinal cell lines from liver, kidney, bladder, ovary, cervix, breast, larynx, or skin . Invasion data showed that A572 was invasive but the invasion efficiency was proportional to its adhesion characteristics to respective cell lines . In mouse bioassay, A572 was not found in liver following oral administration, suggesting that LAP mutant was possibly unable to pass through intestinal cell linings . Immuno-electron microscopy revealed that the LAP is localized in the bacterial surface as well as the cytoplasm . In summary, this study indicated that the LAP-mediated adhesion is associated with the intestinal cells originating from the lower part of small intestine and from the upper part of large intestine, and possibly plays an important role during the intestinal phase of infection.

J Immunol, 2003 May 15, 170(10), 5228 - 34
Neutrophilia in LFA-1-deficient mice confers resistance to listeriosis: possible contribution of granulocyte-colony-stimulating factor and IL-17; Miyamoto M et al.; LFA-1 (CD11a/CD18) plays a crucial role in various inflammatory responses . In this study, we show that LFA-1(-/-) mice are far more resistant to Listeria monocytogenes infection than LFA-1(+/-) mice . Consistent with this, we found the following: 1) the numbers of granulocytes infiltrating the liver were markedly higher in LFA-1(-/-) mice than in LFA-1(+/-) mice, 2) increased antilisterial resistance in LFA-1(-/-) mice was abrogated by depletion of granulocytes, and 3) the numbers of granulocytes in peripheral blood, and the serum levels of both G-CSF and IL-17 were higher in LFA-1(-/-) mice than in LFA-1(+/-) mice . Neither spontaneous apoptosis nor survival of granulocytes from LFA-1(-/-) mice were affected by physiological concentrations of G-CSF . Our data suggest regulatory effects of LFA-1 on G-CSF and IL-17 secretion, and as a corollary on neutrophilia . Consequently, we conclude that increased resistance of LFA-1(-/-) mice to listeriosis is due to neutrophilia facilitating liver infiltration by granulocytes promptly after L . monocytogenes infection, although it is LFA-1 independent.

J Immunol, 2003 May 15, 170(10), 5210 - 8
The lymphotoxin beta receptor is critically involved in controlling infections with the intracellular pathogens Mycobacterium tuberculosis and Listeria monocytogenes; Ehlers S et al.; Containment of intracellularly viable microorganisms requires an intricate cooperation between macrophages and T cells, the most potent mediators known to date being IFN-gamma and TNF . To identify novel mechanisms involved in combating intracellular infections, experiments were performed in mice with selective defects in the lymphotoxin (LT)/LT beta R pathway . When mice deficient in LT alpha or LT beta were challenged intranasally with Mycobacterium tuberculosis, they showed a significant increase in bacterial loads in lungs and livers compared with wild-type mice, suggesting a role for LT alpha beta heterotrimers in resistance to infection . Indeed, mice deficient in the receptor for LT alpha(1)beta(2) heterotrimers (LT beta R-knockout (KO) mice) also had significantly higher numbers of M . tuberculosis in infected lungs and exhibited widespread pulmonary necrosis already by day 35 after intranasal infection . Furthermore, LT beta R-KO mice were dramatically more susceptible than wild-type mice to i.p . infection with Listeria monocytogenes . Compared with wild-type mice, LT beta R-KO mice had similar transcript levels of TNF and IFN-gamma and recruited similar numbers of CD3(+) T cells inside granulomatous lesions in M . tuberculosis-infected lungs . Flow cytometry revealed that the LT beta R is expressed on pulmonary macrophages obtained after digestion of M . tuberculosis-infected lungs . LT beta R-KO mice showed delayed expression of inducible NO synthase protein in granuloma macrophages, implicating deficient macrophage activation as the most likely cause for enhanced susceptibility of these mice to intracellular infections . Since LIGHT-KO mice proved to be equally resistant to M . tuberculosis infection as wild-type mice, these data demonstrate that signaling of LT alpha(1)beta(2) heterotrimers via the LT beta R is an essential prerequisite for containment of intracellular pathogens.

J Immunol, 2003 May 15, 170(10), 5176 - 87
The induction of HIV Gag-specific CD8+ T cells in the spleen and gut-associated lymphoid tissue by parenteral or mucosal immunization with recombinant Listeria monocytogenes HIV Gag; Peters C et al.; The induction of mucosal immunity is crucial in controlling viral replication during HIV infection . In this study we compare the ability of a recombinant Listeria monocytogenes that expresses and secretes the HIV Ag Gag to induce CD8(+) T cells against this Ag in the spleen, mesenteric lymph nodes, and Peyer's patches and the ability to provide effector Gag-specific CD8(+) T cells to the lamina propria after i.v., oral, or rectal administration of the vaccine . The levels of Ag-specific CD8(+)-activated T cells were measured ex vivo using intracellular cytokine staining for IFN-gamma and H-2K(d) Gag peptide tetramer staining . We found that all routes of immunization induced Gag-specific CD8(+) T cells in the spleen . After secondary infection, we observed substantial increases in splenic levels of CD8(+) T cells, and levels of Gag-specific cells were similar to those against listeriolysin O, the immunodominant Ag of L . monocytogenes . Both primary and secondary oral immunization resulted in abundant Gag-specific CD8(+)-activated T cells in the lamina propria that constituted approximately 35% of the CD8 compartment . However, significant levels of Gag and listeriolysin O-specific CD8(+) T cells were observed in mucosal lymphoid tissue only after two immunizations, perhaps because they had already entered the lamina propria compartment after a single immunization . In the context of HIV, a mucosally administered vaccine seems best calculated to prompt an immune response that is capable of preventing infection . The data presented in this report demonstrate that mucosally administered Listeria can prompt such a response and that booster doses can maintain this response.

J Immunol, 2003 May 15, 170(10), 4933 - 42
Regulation of CD8+ T cells undergoing primary and secondary responses to infection in the same host; Badovinac VP et al.; Naive Ag-specific CD8(+) T cells expand, contract, and become memory cells after infection and/or vaccination . Memory CD8(+) T cells provide faster, more effective secondary responses against repeated exposure to the same pathogen . Using an adoptive transfer system with low numbers of trackable nontransgenic memory CD8(+) T cells, we showed that secondary responses can be comprised of both primary (naive) and secondary (memory) CD8(+) T cells after bacterial (Listeria monocytogenes) and/or viral (lymphocytic choriomeningitis virus) infections . The level of memory CD8(+) T cells present at the time of infection inversely correlated with the magnitude of primary CD8(+) T cell responses against the same epitope but directly correlated with the level of protection against infection . However, similar numbers of Ag-specific CD8(+) T cells were found 8 days postinfection no matter how many memory cells were present at the time of infection . Rapid contraction of primary CD8(+) T cell responses was not influenced by the presence of memory CD8(+) T cells . However, contraction of secondary CD8(+) T cell responses was markedly prolonged compared with primary responses in the same host mice . This situation occurred in response to lymphocytic choriomeningitis virus or L . monocytogenes infection and for CD8(+) T cell responses against multiple epitopes . The delayed contraction of secondary CD8(+) T cells was also observed after immunization with peptide-coated dendritic cells . Together, the results show that the level of memory CD8(+) T cells influences protective immunity and activation of naive precursors specific for the same epitope but has little impact on the magnitude or program of the CD8(+) T cell response.

Curr Microbiol, 2003 Jun, 46(6), 461 - 6
The role of the sigB gene in the general stress response of Listeria monocytogenes varies between a strain of serotype 1/2a and a strain of serotype 4c; Moorhead SM et al.; The ability of Listeria monocytogenes to resist many adverse environmental conditions has been attributed in part to activation of the alternative sigma factor sigma(B), encoded by the sigB gene . The ability of this pathogen to survive and grow under stress conditions varies between strains within the species . The current study was undertaken to determine whether the role played by the sigB gene in the stress response varies among strains of different serotypes . Null mutations were generated in the sigB genes of L . monocytogenes L61 (serotype 1/2a) and L99 (serotype 4c), and the survival of the resulting mutants was compared with that of the wild-type strains under osmotic, oxidative, and carbon starvation stress conditions and on exposure to bacteriocins, ethanol, acid, and heat . Except in a few cases, strain L61 displayed greater dependence on the sigB products for survival of adverse conditions than did strain L99 . The results of this study indicated that the relative importance of the sigB gene in the stress response is not the same in all strains of L . monocytogenes, and this difference may be specific to serotype groupings within the species.

Appl Environ Microbiol, 2003 May, 69(5), 3020 - 3
An improved cloning vector for construction of gene replacements in Listeria monocytogenes; Li G et al.; Listeria monocytogenes is a gram-positive, facultative intracellular bacterium implicated in severe food-borne illness (listeriosis) in humans . The construction of well-defined gene replacements in the genome of L . monocytogenes has been instrumental to several genetic studies of the virulence and other attributes of the organism . Construction of such mutations by currently available procedures, however, tends to be labor intensive, and gene replacement mutants are sometimes difficult to recover due to lack of direct selection for the construct . In this study we describe the construction and use of plasmid vector pGF-EM, which can be conjugatively transferred from Escherichia coli S17-1 to L . monocytogenes and which provides the genetic means for direct selection of gene replacements.

Appl Environ Microbiol, 2003 May, 69(5), 2692 - 8
Role of Listeria monocytogenes sigma(B) in survival of lethal acidic conditions and in the acquired acid tolerance response; Ferreira A et al.; The food-borne pathogen Listeria monocytogenes can acquire enhanced resistance to lethal acid conditions through multiple mechanisms . We investigated contributions of the stress-responsive alternative sigma factor, sigma(B), which is encoded by sigB, to growth phase-dependent acid resistance (AR) and to the adaptive acid tolerance response in L . monocytogenes . At various points throughout growth, we compared the relative survival of L . monocytogenes wild-type and DeltasigB strains that had been exposed to either brain heart infusion (pH 2.5) or synthetic gastric fluid (pH 2.5) with and without prior acid adaptation . Under these conditions, survival of the DeltasigB strain was consistently lower than that of the wild-type strain throughout all phases of growth, ranging from 4 orders of magnitude less in mid-log phase to 2 orders of magnitude less in stationary phase . Survival of both DeltasigB and wild-type L . monocytogenes strains increased by 6 orders of magnitude upon entry into stationary phase, demonstrating that the L . monocytogenes growth phase-dependent AR mechanism is sigma(B) independent . sigma(B)-mediated contributions to acquired acid tolerance appear to be greatest in early logarithmic growth . Loss of a functional sigma(B) reduced the survival of L . monocytogenes at pH 2.5 to a greater extent in the presence of organic acid (100 mM acetic acid) than in the presence of inorganic acid alone (HCl), suggesting that L . monocytogenes protection against organic and inorganic acid may be mediated through different mechanisms . sigma(B) does not appear to contribute to pH(i) homeostasis through regulation of net proton movement across the cell membrane or by regulation of pH(i) buffering by the GAD system under the conditions examined in this study . In summary, a functional sigma(B) protein is necessary for full resistance of L . monocytogenes to lethal acid treatments.

Biochem Biophys Res Commun, 2003 May 16, 304(4), 807 - 11
Effects of iron and oxygen species scavengers on Listeria spp . chemiluminescence; Andre P et al.; Listeria monocytogenes and Listeria innocua are able, under certain conditions, to produce chemiluminescence (CL), which is amplified by luminol . Kinetic studies of CL by L . monocytogenes and L . innocua show a close parallelism between CL and growth curves during the exponential phase, with a maximum of CL reached just before entrance of bacteria into the stationary phase . CL is tightly correlated with the release of oxygen compounds . The reactive oxygen species scavengers tryptophan, mannitol, and tiron, as well as cellobiose and high temperature, were assessed with regard to CL in the two Listeria species . Only tiron strongly reduced the CL emitted by L . monocytogenes and L . innocua . On the other hand, charcoal pretreatment of the growth medium inhibited the CL, whereas ferric citrate strongly increased the CL of L . monocytogenes and L . innocua . These data suggest that iron and superoxide radical are implicated in the CL produced by these bacteria, but this phenomenon is not correlated to virulence.

Microbiology, 2003 May, 149(Pt 5), 1249 - 55
Modification of the signal sequence cleavage site of listeriolysin O does not affect protein secretion but impairs the virulence of Listeria monocytogenes; Lety MA et al.; Listeriolysin O (LLO, hly-encoded), a major virulence factor secreted by the bacterial pathogen Listeria monocytogenes, is synthesized as a precursor of 529 residues . To impair LLO secretion, the four residues of the predicted signal sequence cleavage site (EA-KD) were deleted and the mutant LLO protein was expressed in a hly-negative derivative of L . monocytogenes . Unexpectedly, the mutant protein was secreted in normal amounts in the culture supernatant and was fully haemolytic . N-terminal sequencing of the secreted LLO molecule revealed that N-terminal processing of the preprotein occurred three residues downstream of the natural cleavage site . L . monocytogenes expressing this truncated LLO showed a reduced capacity to disrupt the phagosomal membranes of bone marrow macrophages and of hepatocytes; and the mutant strain showed a 100-fold decrease in virulence in the mouse model . These results suggest that the first N-terminal residues of mature LLO participate directly in phagosomal escape and bacterial infection.

J AOAC Int, 2003 Mar-Apr, 86(2), 340 - 54
Modification of enrichment protocols for TECRA Listeria Visual Immunoassay method 995.22: collaborative study; Hughes D et al.; A collaborative study was conducted to validate new enrichment methods for the TECRA Listeria Visual Immunoassay (TLVIA) . These new methods incorporate a newly formulated medium, TECRA Listeria Enrichrment Broth, which does not contain the highly toxic antifungal agent, cycloheximide . The new procedures will provide an alternative to the enrichment procedures described in AOAC Method 995.22 . Three food types (raw ground beef, lettuce, and ice cream) were analyzed in the United States, and 2 food types (cooked turkey and cooked fish fillets) were analyzed in Australasia . Thirty collaborators participated in the study, 16 in Australasia and 14 in the United States . With the exception of one batch of ground beef, comparison of the proportion of positive test portions (p > or = 0.05) showed no significant difference between the TLVIA and the reference method for the 5 foods at 3 inoculation levels . For the one batch of naturally contaminated raw ground beef, the TLVIA gave significantly more confirmed positive results than the reference method.

Can J Microbiol, 2003 Feb, 49(2), 78 - 84
Low-level iron-dependent mutants of Listeria monocytogenes and their virulence in macrophages; Andre P et al.; Listeria monocytogenes is an opportunistic intracellular pathogen capable of growth that requires iron for growth within phagocytic cells and virulence expression . In the presence of an appropriate concentration tropolone, an iron-chelating agent, growth of L . monocytogenes is completely inhibited . However, this inhibition can be relieved by addition of dopamine, norepinephrine, or ferric citrate . By selection on streptonigrin medium supplemented with tropolone and norepinephrine, we have obtained two spontaneous mutants, Lm-8 and Lm-15, with the same iron dependence but lower iron dependence than the wild-type Lm-B38 . The association between iron requirement and virulence of the two mutants and the wild type was studied in the J774 macrophage cell line . One hour after phagocytosis by the J774 macrophage cell line, the two mutants and the parental strain displayed no difference in the number of phagocytosed bacteria . Twenty-four hours after phagocytosis, the number of bacteria within the surviving macrophages was identical for the wild strain and the two clones . However, only 40% of macrophage cells infected with Lm-8 and 90% of those infected with Lm-15 were alive after 24 h in comparison with macrophage cells infected with the parental strain Lm-B38 . These data demonstrate that there is no direct correlation between iron requirement and virulence of L . monocytogenes in the J774 macrophage cell line.

J Antimicrob Chemother, 2003 Jun, 51(6), 1365 - 71 Epub 2003 Apr 25.
The bacteriocin piscicolin 126 retains antilisterial activity in vivo; Ingham A et al.; OBJECTIVE: We have conducted a series of experiments to show that the bacteriocin piscicolin 126 (P126) retains antilisterial activity after injection into a mouse . METHODS: Groups of mice were challenged intravenously with Listeria monocytogenes and treated with purified P126 at varying times before and after challenge to determine whether administration of this peptide reduced numbers of colonizing L . monocytogenes and the symptoms of listeriosis . RESULTS: The bacteriocin P126 retained antilisterial activity after injection into the mouse . During the early time-points of listerial infection, the purified P126 was found to significantly reduce the listerial load in the liver and spleen and, further, that this reduction translated to reduced clinical signs of disease . CONCLUSIONS: This is the first report of a Class IIA bacteriocin displaying in vivo antimicrobial activity . Such a result provides preliminary evidence that this class of molecules may be useful in controlling systemic bacterial infections.

Neurol Neurochir Pol, 2002 Nov-Dec, 36(6), 1221 - 6
{Neural listeriosis presenting as leptomeningitis: a case report}; Lysiak Z et al.; The Listeria monocytogenes infection is rare and difficult to diagnosis . However, it is associated with a high mortality in adults . A case is presented of a 78-year-old woman with an immunological deficit following viral infection (herpes zoster).

Vaccine, 2003 May 16, 21(17-18), 2122 - 32
Protection of interferon-gamma knockout mice against Listeria monocytogenes challenge following intramuscular immunization with DNA vaccines encoding listeriolysin O; Barry RA et al.; In this study we evaluated the efficacy of DNA vaccination of IFN-gamma knockout (GKO) mice against Listeria monocytogenes, as these immunodeficient mice are highly susceptible to infection with low numbers of this intracellular bacterial pathogen . Following intramuscular immunization of BALB/c GKO mice with plasmid DNA constructs encoding recombinant forms of the L . monocytogenes hemolysin, listeriolysin O (LLO), we detected the in vivo induction of a LLO(91-99) peptide-specific, protective immune CTL response equivalent to that observed following similar DNA vaccination of normal BALB/c mice . The observed protection represented greatly enhanced immunity for the GKO host, suggesting that DNA vaccination may provide a useful vaccine alternative for certain immunocompromised host populations.

Int J Food Microbiol, 2003 Jun 15, 83(2), 133 - 45
Hypovirulent Listeria monocytogenes strains are less frequently recovered than virulent strains on PALCAM and Rapid' L . mono media; Gracieux P et al.; Several selective media have been developed to detect Listeria monocytogenes contaminated foodstuffs . Polymyxin-acriflavine-LiCl-ceftazidime-aesculin-mannitol (PALCAM) and Oxford media, required for the EN ISO method 11 290-1, are used for the detection of Listeria spp . in 2 days based on the expression of esculinase activity . Selective agar media such as Rapid' L . mono and Agar Listeria according to Ottaviani and Agosti (ALOA), based on the activity of phosphatidylinositol phospholipase C (PI-PLC) that allows the specific detection of L . monocytogenes in 2 days, are also used . However, no medium can assess the level of virulence of L . monocytogenes strains . Using a plaque-forming assay followed by subcutaneous footpad inoculation in mice, 15 virulent, 8 hypovirulent and 17 avirulent strains were discriminated among L . monocytogenes strains mainly originating from food (36/40) . Their growth was tested on the four selective media . After 2 days, the number of colony forming units (cfu) of all the virulent strains was significantly superior to the number obtained with avirulent strains on all the four media tested, and superior to the number obtained with hypovirulent strains on PALCAM and Oxford media . These results showed a relationship between the level of virulence of L . monocytogenes strains and their growth on the selective agar media tested . Moreover, 1 out of 8 hypovirulent and 5 out of 17 avirulent strains did not grow on Rapid' L . mono medium, and 1 hypovirulent and 8 avirulent strains grew but did not express PI-PLC activity during the 7 days of incubation . The lack of detection of PI-PLC activity on Rapid' L . mono was not related to a gene mutation since these strains expressed enzymatic activity on ALOA medium, which detected up to 92% of the hypo- and avirulent strains . In contrast, some of these strains without growth or enzymatic activity expression would not be detected with PALCAM and Rapid' L . mono in foodstuffs on the second day.

Immunity, 2003 Apr, 18(4), 463 - 74
Viral infection results in massive CD8+ T cell expansion and mortality in vaccinated perforin-deficient mice; Badovinac VP et al.; Perforin-mediated cytotoxicity is essential for clearance of primary LCMV infection . BALB/c-perforin-deficient (PKO) mice survived LCMV infection by deleting NP(118)-specific CD8(+) T cells whereas vaccination of PKO mice with Listeria expressing NP(118) generated a stable memory CD8(+) T cell population . However, >85% of vaccinated BALB/c-PKO mice died after LCMV infection . Mortality was associated with enormous expansion of NP(118)-specific CD8(+) T cells in both lymphoid and nonlymphoid tissues and aberrant CD8(+) T cell cytokine production . Depletion of CD8(+) T cells or treatment with anti-IFNgamma antibody rescued vaccinated mice from mortality . Thus, perforin was essential for resistance to secondary LCMV infection, and, in the absence of perforin, vaccination resulted in lethal disease mediated by dysregulated CD8(+) T cell expansion and cytokine production.

Infect Immun, 2003 May, 71(5), 2447 - 54
Differences in gamma interferon production induced by listeriolysin O and ivanolysin O result in different levels of protective immunity in mice infected with Listeria monocytogenes and Listeria ivanovii; Kimoto T et al.; Two pathogenic species in the genus Listeria, Listeria monocytogenes and Listeria ivanovii, are characterized by the production of hemolysins belonging to cholesterol-dependent cytolysins, listeriolysin O (LLO) and ivanolysin O (ILO), respectively . LLO, produced by L . monocytogenes, is able to induce gamma interferon (IFN-gamma) production and contributes to the generation of Th1-dependent protective immunity . On the other hand, nothing is known about the role of ILO, produced by L . ivanovii, in this regard . In this study, we immunized mice with 0.1 50% lethal dose (LD(50)) of L . monocytogenes and L . ivanovii . Protective immunity against a challenge with 10 LD(50) was generated in mice infected with L . monocytogenes, whereas L . ivanovii infection did not induce protection . After immunization, the level of IFN-gamma in serum samples was increased in mice given L . monocytogenes but not in those given L . ivanovii . To determine the IFN-gamma-inducing activity of cytolysins, recombinant protein was constructed . Recombinant ILO exhibited significantly lower IFN-gamma-inducing activity than LLO . By comparing the IFN-gamma-inducing activity of a chimera incorporating LLO and ILO, it was found that domains 1 to 3 of LLO were critical for IFN-gamma-inducing activity while the counterpart in ILO was unable to induce cytokine production . These results suggested that the weak ability of ILO to induce IFN-gamma production is responsible for the failure of L . ivanovii to generate effective protective immunity.

J Antimicrob Chemother, 2003 May, 51 Suppl 1, 37 - 42
Maximizing efficacy and reducing the emergence of resistance; Wise R; An understanding of the pharmacokinetic and pharmacodynamic properties of antimicrobial agents enables better choices to be made in the clinical situation . The fluoroquinolones share several useful pharmacokinetic properties, such as good bioavailability (in most cases >85%) and the ability to penetrate and concentrate intracellularly, giving them activity against pathogens such as Legionella pneumophila and Listeria monocytogenes . Nevertheless, there are some important differences between the fluoroquinolones, and even the newer fluoroquinolones demonstrate a range of pharmacodynamic properties . When considering the area under the inhibition curve (AUIC) and the Cmax/MIC, the comparative figures are: ciprofloxacin and ofloxacin (5-25, 1-5); levofloxacin, grepafloxacin and gatifloxacin (25-75, 5-10); trovafloxacin (75-250, 10-20) and moxifloxacin, clinafloxacin and gemifloxacin (>250, >20) . The development of resistance is also a concern, and selecting an agent that reaches an adequate concentration above the MIC will reduce the opportunity for resistance to develop . These properties should be considered when selecting a fluoroquinolone either for inclusion in a formulary, or for use in an individual patient.

Int J Food Sci Nutr, 2003 Mar, 54(2), 127 - 33
Antimicrobial effects of garlic, clove and red hot chilli on Listeria monocytogenes in broth model systems and soft cheese; Leuschner RG et al.; Antimicrobial activity of 1% (w/v) fresh garlic, ground clove and red dried chilli on Listeria monocytogenes was tested in broth systems at 37 degrees C and at 4 degrees C for 7 h . The initial cell concentration in the broth systems was between 2 x 10(6) and 4 x 10(6) CFU/ml . At 37 degrees C, growth to viable numbers of 3 x 10(8) CFU/ml in 7 h was measured . Clove had bacteriocidal activity and reduced the count to 1 CFU/ml . Garlic displayed bacteriostatic properties, and a count of 4 x 10(6) CFU/ml was maintained . Red chilli displayed an inhibitory effect and resulted in 50% lower counts than the control . L . monocytogenes had a slow growth rate at 4 degrees C and increased from an initial value of 3 x 10(6) to 5 x 10(6) CFU/ml during 7 h . The addition of garlic resulted in 3 x 10(6) CFU/ml, and clove reduced the viable cell concentration to 1 x 10(3) CFU/ml after 7 h . Two batches of soft cheese were produced in the laboratory using milk that was supplemented with L . monocytogenes . The final cheese containing L . monocytogenes with about 1 x 10(5) CFU/g . Half of each cheese batch was supplemented with either 1% garlic or 1% clove, whereby the other half served as a control . After 7 or 11 days incubation at 4 degrees C, the cheese was incubated at abuse temperature of 25 degrees C for 7 or 3 days, respectively . No antimicrobial effects of 1% (w/w) fresh garlic or clove powder on L . monocytogenes were observed in cheese after 1 or 2 weeks at the lower or higher temperature.

Curr Biol, 2003 Apr 15, 13(8), R302 - 4
Listeria motility: biophysics pushes things forward; Merz AJ et al.; Recent studies have provided important new insights into the forces exerted by actin polymerization during Listeria motility . The results also expose deficiencies in our understanding of this process, and suggest future directions for complete understanding of the molecular mechanisms involved.

J Endotoxin Res, 2002, 8(6), 453 - 8
Immunostimulatory activity of aminoalkyl glucosaminide 4-phosphates (AGPs): induction of protective innate immune responses by RC-524 and RC-529; Baldridge JR et al.; Earlier we showed that the structural requirements for adjuvanticity among the aminoalkyl glucosaminide 4-phosphate (AGP) class of synthetic immunostimulants may be less strict than those for other endotoxic activities, including the induction of nitric oxide synthase in murine macrophages and cytokine production in human whole blood . The known role of nitric oxide and pro-inflammatory cytokines in the activation of host defenses against infection prompted us to examine the ability of certain AGPs to enhance non-specific resistance in mice to Listeria monocytogenes and influenza infections as well as to stimulate the production of pro-inflammatory cytokines in mouse splenocytes, human PBMCs, and human U937 histiocytic lymphoma cells . Intranasal administration of RC-524 or RC-529 to mice 2 days prior to a lethal influenza challenge provided significant protection in each case . Similarly, the intravenous administration of these AGPs induced resistance to L . monocytogenes infection as measured by survival or reduction of bacteria in the spleen . Activation of the innate immune response by AGPs appears to involve activation of Toll-like receptor 4 (TLR4) because RC-524 failed to elicit a protective effect in C3H/HeJ mice which have a defect in TLR4 signaling or induce significant cytokine levels in C3H/HeJ splenocytes . Both AGPs also stimulated pro-inflammatory cytokine release in human cell cultures in a dose-dependent manner.

J Food Prot, 2003 Apr, 66(4), 584 - 91
Recovery rate of Listeria monocytogenes from commercially prepared frankfurters during extended refrigerated storage; Wallace FM et al.; To assess the prevalence of Listeria monocytogenes in vacuum-sealed packages of frankfurters, about 33,000 packages (1 lb each) were obtained by a third-party contractor from 12 volunteer commercial manufacturers over a 2-year period . The 12 producers, each of which contributed about 2,700 packages of frankfurters from one production run, comprised 9 large and 3 small plants located in eight U.S . Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS) districts in 10 states . Five days after manufacture, 500 packages were sampled at the USDA/Agricultural Research Service (ARS) Eastern Regional Research Center (ERRC) in Wyndmoor, Pa., by the USDA/ARS package rinse method . At regular intervals during subsequent storage at 4 and 10 degrees C, an additional 200 packages were tested for the pathogen at each sampling point . From a statistical perspective, L . monocytogenes was not recovered from any of the products of nine of the producers, whereas the pathogen was recovered at rates of 1.5% (plant 367), 2.2% (plant 439), and 16% (plant 133) from the products of the remaining three plants . In total, 532 of 32,800 (1.6%) packages of frankfurters tested positive for the pathogen . The recovery rates did not change appreciably over time, there was no appreciable difference in L . monocytogenes recovery rates with respect to frankfurter storage temperature (4 or 10 degrees C), and the seasonality of manufacture had no influence on recovery rate . Molecular subtyping of multiple L . monocytogenes-positive isolates from each plant revealed that profile A (serotype 1/2a) was displayed by about 90% of the 1,105 isolates tested . However, in some cases it was also possible to recover more than one profile from a given plant . This study provides estimates of the prevalence, types, and viability of L . monocytogenes associated with commercially prepared frankfurters during extended refrigerated storage.

J Food Prot, 2003 Apr, 66(4), 578 - 83
Determination of thermal lethality of Listeria monocytogenes in fully cooked chicken breast fillets and strips during postcook in-package pasteurization; Murphy RY et al.; Fully cooked chicken breast fillets and strips were surface inoculated with a cocktail of Listeria monocytogenes culture . The inoculation level was 10(7) to 10(8) CFU/g meat . The inoculated products were vacuum packaged and pasteurized at 90 degrees C with a pilot-scale steam or hot water cooker . After heat treatment, the survivors of L . monocytogenes were enumerated . No significant difference was found on survivors of L . monocytogenes between steam- and hot water-treated products . To achieve a 7-log10 (CFU/g) reduction, approximately 5, 25, and 35 min were needed for single-packaged fillets, 227-g package strips, and 454-g strips, respectively . The results from this study were subsequently verified by a computer model that could predict the thermal lethality of pathogens in fully cooked meat and poultry products during postcook in-package pasteurization.

J Food Prot, 2003 Apr, 66(4), 570 - 7
Listeria monocytogenes: low levels equal low risk; Chen Y et al.; Because of the public health significance of L . monocytogenes, U.S . regulatory agencies established a policy whereby ready-to-eat foods contaminated with the organism at a detectable level are deemed adulterated . This "zero tolerance" policy, however, makes no distinction between foods contaminated at high and low levels . We have reported elsewhere that a survey of over 31,000 ready-to-eat retail food samples, representing eight product categories, showed an overall prevalence rate of 1.82% for these foods . In this study, we used the food survey data in combination with concurrent data regarding illness in the population consuming the foods, together with other variable factors, to derive a dose-response model . The confidence interval for prevalence was 1.68 to 1.97% . L . monocytogenes levels, which ranged from -2 to 6 log CFU/g, were adequately described by the distribution beta (0.29, 2.68, -1.69, 6.1) . An exponential dose-response model was obtained, with an R value (essentially the probability of a single cell causing illness) of 1.76 x 10(-10) for the population at the highest risk . A microbial risk assessment based on the model shows that an alternative to the zero tolerance strategy has a greater risk reduction potential and suggests that a management strategy focusing on the concentration of L . monocytogenes rather than its presence alone may have a greater impact on the improvement of public health by facilitating the development of control measures to limit the maximum levels of L . monocytogenes in foods.

J Food Prot, 2003 Apr, 66(4), 559 - 69
Survey of Listeria monocytogenes in ready-to-eat foods; Gombas DE et al.; The purpose of this study was to develop data on the risk of listeriosis to support a science-based strategy for addressing Listeria monocytogenes in foods in the United States . Eight categories of ready-to-eat foods were collected over 14 to 23 months from retail markets at Maryland and northern California FoodNet sites . The product categories included luncheon meats, deli salads, fresh soft "Hispanic-style" cheeses, bagged salads, blue-veined and soft mold-ripened cheeses, smoked seafood, and seafood salads . The presence and levels of L . monocytogenes in the samples were determined by rapid DNA-based assays in combination with culture methods . Of 31,705 samples tested, 577 were positive . The overall prevalence was 1.82% . with prevalences ranging from 0.17 to 4.7% among the product categories . L . monocytogenes levels in the positive samples varied from <0.3 MPN (most probable number) per g to 1.5 x 10(5) CFU/g, with 402 samples having levels of <0.3 MPN/g, 21 samples having levels of >10(2) CFU/g, and the rest of the samples having intermediate levels . No obvious trends with respect to seasonality were observed . Significant differences (P < 0.05) between the sampling sites were found, with higher prevalences for threes categories in northern California and for two categories in Maryland . Significantly (P < 0.001) higher prevalences were found for in-store-packaged samples than for manufacturer-packaged samples of luncheon meats, deli salads, and seafood salads, while 16 of the 21 samples with higher counts were manufacturer packaged . The data collected in this study help to fill gaps in the knowledge about the occurrence of L . monocytogenes in foods, and this new information should be useful in the assessment of the risk posed by L . monocytogenes to consumers.

J Appl Microbiol, 2003, 94(5), 879 - 85
Detection of Listeria monocytogenes in Italian-style soft cheeses; Longhi C et al.; AIMS: A rapid detection system specific for Listeria monocytogenes and based on the polymerase chain reaction (PCR) was developed . METHODS AND RESULTS: Primers annealing to the coding region of the actA gene, critically involved in virulence and capable of discrimination between two different alleles naturally occurring in L . monocytogenes, have been utilized . The procedure was applied to recover L . monocytogenes cells in artificially contaminated fresh Italian soft cheeses (mozzarella, crescenza and ricotta) . Low levels of L . monocytogenes were detected in mozzarella and crescenza homogenates (0.04-0.4 and 4 CFU g(-1), respectively) whereas in ricotta the detection limit was higher (40 CFU g(-1)) . CONCLUSIONS: This PCR-based assay is highly specific as primers used recognize the DNA from different L . monocytogenes strains of clinical and food origin, while no amplification products result with any other Listeria spp . strains . SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted a low-cost and rapid procedure that can be appropriated for the detection in real time of low L . monocytogenes levels in soft cheese.

Am J Dent, 2002 Dec, 15(6), 351 - 5
Comparative effectiveness of an essential oil mouthrinse and dental floss in controlling interproximal gingivitis and plaque; Sharma NC et al.; PURPOSE: To compare the effectiveness of rinsing with an essential oil-containing antimicrobial mouthrinse with that of dental floss in reducing interproximal gingivitis and plaque in an unsupervised 6-month clinical trial designed in accordance with ADA Acceptance Program Guidelines . MATERIALS AND METHODS: 319 qualifying subjects, aged 18-63, were randomized into one of three groups: essential oil mouthrinse (Listerine Antiseptic); dental floss (Reach Dental Floss); or a negative control rinse . At baseline, subjects received a complete oral soft tissue examination and scoring of the Modified Gingival Index (MGI), modified Quigley-Hein Plaque Index (PI), and bleeding index (BI) . Following a complete dental prophylaxis and receiving flossing or rinsing instructions, subjects started on their respective regimen . They continued on their assigned regimen unsupervised at home, in addition to toothbrushing, and were reexamined at 3 and 6 months . The treatment groups were compared with respect to baseline demographic and clinical variables . The primary efficacy variables were mean interproximal MGI and PI at 6 months . Intergroup differences at 3 and 6 months were tested using a one-way analysis of covariance model with treatment as a factor and the respective baseline value as the covariate . In addition, the essential oil mouthrinse was compared to floss for interproximal gingivitis reduction using "at least as good as" statistical criteria . RESULTS: 301 subjects were considered evaluable . There were no statistically significant differences among the 3 groups at baseline, with the exception of the essential oil mouthrinse group having significantly fewer AfroAmerican subjects than the other two groups . For the interproximal MGI, the essential oil mouthrinse and flossing were both significantly more effective than the negative control (P < 0.001) at 3 and 6 months . The essential oil mouthrinse was shown to be "at least as good as" dental floss for the control of interproximal gingivitis . For the interproximal PI, the essential oil mouthrinse was significantly more effective than the negative control at 3 and 6 months (P < 0.001) while flossing was significantly more effective than the negative control at 3 months (P < 0.05) but not at 6 months . The essential oil mouthrinse was significantly more effective than floss (P < 0.001) at both these time periods.

Science, 2003 Apr 11, 300(5617), 339 - 42
Defective CD8 T cell memory following acute infection without CD4 T cell help; Sun JC et al.; The CD8+ cytotoxic T cell response to pathogens is thought to be CD4+ helper T cell independent because infectious agents provide their own inflammatory signals . Mice that lack CD4+ T cells mount a primary CD8 response to Listeria monocytogenes equal to that of wild-type mice and rapidly clear the infection . However, protective memory to a challenge is gradually lost in the former animals . Memory CD8+ T cells from normal mice can respond rapidly, but memory CD8+ T cells that are generated without CD4 help are defective in their ability to respond to secondary encounters with antigen . The results highlight a previously undescribed role for CD4 help in promoting protective CD8 memory development.

J Clin Microbiol, 2003 Apr, 41(4), 1694 - 700
Listeria monocytogenes isolates from invasive infections: variation of sero- and genotypes during an 11-year period in Finland; Lukinmaa S et al.; Listeria monocytogenes strains that were isolated from 314 human listeriosis cases in Finland during an 11-year period were analyzed by O:H serotyping and pulsed-field gel electrophoresis (PFGE) . Serotyping divided the isolates into five serotypes, the most common being 1/2a (53%) and 4b (27%) . During the study period, the number of cases caused by serotype 1/2a increased from 22% in 1990 to 67% in 2001, and those caused by serotype 4b decreased from 61 to 27%, respectively . PFGE with restriction enzyme AscI divided the strains into 81 PFGE genotypes; among strains of serotypes 1/2a and 4b, 49 and 18 PFGE types were seen, respectively . PFGE type 1 (serotype 1/2a) was the most prevalent single type (37 strains) . Together with six other, closely related PFGE types, PFGE type 1 formed a group of 71 strains, representing 23% of all 314 strains . Strains of PFGE type 1 have also been isolated from cold smoked fish, suggesting a source of human infections caused by this type . Moreover, PFGE type 24 (serotype 1/2c) was significantly associated with gender: 5% of 180 male subjects but none of 132 female subjects (P = 0.012) . An electronic database library was created from the PFGE profiles to make possible the prompt detection of new emerging profiles and the tracing of potential infection clusters in the future.

J Wildl Dis, 2003 Jan, 39(1), 136 - 44
Characterization and clinical manifestations of Arcanobacterium phocae infections in marine mammals stranded along the central California coast; Johnson SP et al.; Between 1994 and 2000, 141 Arcanobacterium phocae isolates were recovered from marine mammals that stranded along the central California coast (USA) . Arcanobacterium phocae was cultured from tissue sites with abnormal discharge or evidence of inflammation in 66 California sea lions (Zalophus californianus), 50 Pacific harbor seals (Phoca vitulina richardii), 19 northern elephant seals (Mirounga angustirostris), five southern sea otters (Enhydra lutris nereis), and one common dolphin (Delphinus delphis) . The overall prevalence of A . phocae among cultured stranded marine mammals was 8% . This is the first report of A . phocae in animals from the Pacific Ocean . Sequence analysis of a portion of the 16S ribosomal RNA gene confirmed recent isolates as A . phocae . Prior to phylogenetic testing and the routine use of the esculin hydrolysis and motility tests, A . phocae isolates may have been misidentified as Listeria ivanovii . Arcanobacterium phocae was commonly isolated from superficial abscesses, was often present in mixed infections, and was susceptible to all antimicrobial agents tested.

Med Microbiol Immunol (Berl), 2003 May, 192(2), 107 - 15 Epub 2003 Mar 05.
Role of heparan sulfate in interactions of Listeria monocytogenes with enterocytes; Henry-Stanley MJ et al.; Heparan sulfate is known to participate in binding a wide variety of microbes to mammalian cells, but few studies have focused on the enterocyte . Normal human colonic and small intestinal enterocytes, and cultured HT-29 (but not Caco-2) enterocytes, reacted prominently with antibodies specific for heparan sulfate and for the core protein of syndecan-1 (a heparan sulfate proteoglycan) . The heparan sulfate analog, heparin, inhibited interactions of Listeria monocytogenes (adherence and internalization) with HT-29, but not Caco-2, enterocytes . Internalization of L . monocytogenes by HT-29 enterocytes was inhibited by heparan sulfate and to a lesser extent by chondroitin sulfate, but not by the non-sulfated glycosaminoglycan hyaluronic acid . Compared to plasmid control ARH-77 cells, adherence of L . monocytogenes, was increased using ARH-77 cells transfected with syndecan-1 cDNA . Heparin binding protein(s) on L . monocytogenes were confirmed using biotinylated heparin . To determine if these in vitro observations might have in vivo relevance, L . monocytogenes was preincubated with heparin and then orally inoculated into mice . Compared to L . monocytogenes not pretreated with heparin, L . monocytogenes pretreated with heparin was associated with decreased extraintestinal dissemination to the mesenteric lymph nodes and liver of orally inoculated mice . Thus, heparan sulfate (possibly as the heparan sulfate proteoglycan syndecan-1) appears to participate in interactions of L . monocytogenes with enterocytes.

Appl Environ Microbiol, 2003 Apr, 69(4), 2223 - 9
Selection and identification of a Listeria monocytogenes target strain for pulsed electric field process optimization; Lado BH et al.; Nine Listeria monocytogenes strains were treated individually with a continuous pulsed electric field (PEF) apparatus, and their sensitivities to the treatment were compared at 25 kV/cm . When cell suspensions of these strains in 0.1% NaCl (pH 7.0) were treated at 23 degrees C for 144 micro s, inactivation ranged from 0.7 to 3.7 log(10) CFU/ml . Inactivation by 72- micro s PEF treatments at 37 degrees C ranged from 0.3 to 2.5 log(10) CFU/ml . L . monocytogenes OSY-8578 was substantially more resistant than other strains when cells were PEF treated in 0.1% NaCl, whereas Scott A was one of the most sensitive strains . The superiority of OSY-8578's resistance to that of Scott A was confirmed in 50% diluted acid whey (pH 4.2) . Changes in sensitivity to PEF during phases of growth were minimal in OSY-8578 and substantial in Scott A . Use of L . monocytogenes OSY-8578, therefore, is recommended in studies to optimize PEF processes that target L . monocytogenes . The nine L . monocytogenes strains were genotyped with pulsed-field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR) techniques . These strains were better differentiated with PFGE than with AP-PCR . The target strain (OSY-8578) was characterized by both molecular typing techniques, but resistance to PEF, in general, was not associated with a particular genotype group.

Appl Environ Microbiol, 2003 Apr, 69(4), 2015 - 22
Role of sigmaB in regulating the compatible solute uptake systems of Listeria monocytogenes: osmotic induction of opuC is sigmaB dependent; Fraser KR et al.; The regulation of the compatible solute transport systems in Listeria monocytogenes by the stress-inducible sigma factor sigma(B) was investigated . Using wild-type strain 10403S and an otherwise isogenic strain carrying an in-frame deletion in sigB, we have examined the role of sigma(B) in regulating the ability of cells to utilize betaine and carnitine during growth under conditions of hyperosmotic stress . Cells lacking sigma(B) were defective for the utilization of carnitine but retained the ability to utilize betaine as an osmoprotectant . When compatible solute transport studies were performed, the initial rates of uptake of both betaine and carnitine were found to be reduced in the sigB mutant; carnitine transport was almost abolished, whereas betaine transport was reduced to approximately 50% of that of the parent strain . Analysis of the cytoplasmic pools of compatible solutes during balanced growth revealed that both carnitine and betaine steady-state pools were reduced in the sigB mutant . Transcriptional reporter fusions to the opuC (which encodes an ABC carnitine transporter) and betL (which encodes an a secondary betaine transporter) operons were generated by using a promoterless copy of the gus gene from Escherichia coli . Measurement of beta-glucuronidase activities directed by opuC-gus and betL-gus revealed that transcription of opuC is largely sigma(B) dependent, consistent with the existence of a potential sigma(B) consensus promoter motif upstream from opuCA . The transcription of betL was found to be sigB independent . Reverse transcriptase PCR experiments confirmed these data and indicated that the transcription of all three known compatible solute uptake systems (opuC, betL, and gbu), as well as a gene that is predicted to encode a compatible solute transporter subunit (lmo1421) is induced in response to elevated osmolarity . The osmotic induction of opuCA and lmo1421 was found to be strongly sigma(B) dependent . Together these observations suggest that sigma(B) plays a major role in the regulation of carnitine utilization by L . monocytogenes but is not essential for betaine utilization by this pathogen.

Environ Health Perspect, 2003 Apr, 111(4), 524 - 30
Alteration of pulmonary immunity to Listeria monocytogenes by diesel exhaust particles (DEPs) . II . Effects of DEPs on T-cell-mediated immune responses in rats; Yin XJ et al.; Previously, we showed that diesel exhaust particles (DEPs) suppressed pulmonary clearance of Listeria monocytogenes (Listeria) and inhibited the phagocytosis of alveolar macrophages and their response to Listeria in the secretion of interleukin (IL)-1 beta, tumor necrosis factor alpha, and IL-12 . In this report we examined the effects of DEPs and/or Listeria on T-cell development and secretion of IL-2, IL-6, and interferon (IFN)-gamma . We exposed Brown Norway rats to clean air or DEPs at 50 or 100 mg/m3 for 4 hr by nose-only inhalation and inoculated with 100,000 Listeria . Lymphocytes in the lung-draining lymph nodes were isolated at 3 and 7 days postexposure, analyzed for CD4+ and CD8+ cells, and measured for cytokine production in response to concanavalin A or heat-killed L . monocytogenes . Listeria infection induced lymphocyte production of IL-6 . At 7 days postinfection, lymphocytes from Listeria-infected rats showed significant increases in CD4+ and CD8+ cell counts and the CD8+/CD4+ ratio and exhibited increased production of IFN-gamma and IL-2 receptor expression compared with the noninfected control . These results suggest an immune response that involves the action of IL-6 on T-cell activation, yielding Listeria-specific CD8+ cells . DEP exposure alone enhanced lymphocyte production of both IL-2 and IL-6 but inhibited lymphocyte secretion of IFN-gamma . In rats exposed to 100 mg/m3 DEPs and Listeria, a 10-fold increase occurred in pulmonary bacterial count at 3 days postinfection when compared with the Listeria-only exposure group . The isolated lymphocytes showed a significant increase in the CD4+ and CD8+ cell counts and the CD8+/CD4+ ratio and exhibited increased IL-2 responsiveness and increased capacity in the secretion of IL-2, IL-6, and IFN-gamma . This T-cell immune response was sufficient to allow the Brown Norway rats to clear the bacteria at 7 days postinfection and overcome the down-regulation of the innate immunity by the acute DEP exposure.

Brain Behav Immun, 2003 Apr, 17(2), 121 - 33
Acute cold/restraint stress inhibits host resistance to Listeria monocytogenes via beta1-adrenergic receptors; Cao L et al.; We previously reported that acute cold/restraint stress (ACRS) significantly inhibits host resistance to Listeria monocytogenes (LM) in BALB/c mice and that the sympathetic nervous system plays a major role in this inhibition . Here, we have further investigated the involvement of adrenergic receptor (ADR) subtypes . beta-ADR antagonist propranolol, but not alpha-ADR antagonist phentolamine significantly enhanced host resistance of ACRS mice . Pro-inflammatory cytokine (IL-6, IL-1beta, and TNFalpha) and IFNgamma levels positively correlated with the LM levels in all groups of mice . Furthermore, beta1-ADR antagonist atenolol but not beta2-ADR antagonist ICI118,551 significantly decreased LM burden in ACRS mice . In addition, SCID mice on the same genetic background (BALB/c), which have no adaptive immune potential, were used to assess the immune responses targeted by ACRS . ACRS-induced suppression of host resistance was not observed in SCID mice, and propranolol pretreatment provided no further improvement of host resistance, indicating that ACRS mainly affects adaptive immunity, which is less critical in mice with greater innate than adaptive immunity . In summary, the data suggest that ACRS inhibition of host resistance to LM is mediated through beta1-ADR stimulation, which appears to directly or indirectly modify activation of T cells or subsequent T cell functions involved in adaptive immunity, thus inhibiting overall host resistance . Interestingly, with heightened innate immunity and the absence of adaptive immunity, as observed in the SCID mice, ACRS does not affect host resistance, which emphasizes the importance of innate immunity in defense against bacterial infection.

Bull Math Biol, 2003 Mar, 65(2), 219 - 34
Estimating microbial inactivation parameters from survival curves obtained under varying conditions--the linear case; Peleg M et al.; When the isothermal semi-logarithmic survival curves of heat inactivated microbial cells or spores are known to be linear it is possible to calculate their survival parameters from curves obtained under nonisothermal conditions, provided that the temperature history ('profile') satisfies certain simple mathematical requirements . These requirements have been identified . The concept was tested by retrieving the survival parameters of a Listeria-like organism from generated survival curves for linear and nonlinear heating profiles on which noise had been superimposed . The availability of such a procedure eliminates the need to determine the survival parameters under perfect isothermal conditions, which are difficult to create for technical reasons . It will also enable determination of the survival parameters in the actual medium of interest, which may contain particles or may be too viscous to be treated in a capillary or narrow tube as is currently done . The method can also be used to assess survival parameters in nonthermal inactivation . A treatment with a dissipating chemical agent or anti-microbial is an example . In principle, the concept can be extended to the more general situation where the isothermal or iso-concentration semi-logarithmic survival curves are clearly nonlinear, but this will require a modification of the model and a different numerical calculation procedure.

J Nutr, 2003 Apr, 133(4), 1163 - 9
Dietary fish oil impairs primary host resistance against Listeria monocytogenes more than the immunological memory response; Irons R et al.; The primary objective of this study was to determine whether dietary (n-3) polyunsaturated fatty acids (PUFA) impair the ability of mice to generate an immunological memory response against the bacterial pathogen, Listeria monocytogenes . Weanling BALB/c female mice were fed for 28 d one of two semipurified high fat diets containing either lard or refined menhaden fish oil, rich in long-chain (n-3) PUFA . Mice were immunized with 10(4) or 10(3) colony forming units (cfu) bacteria . Thirty-five days later, these immune mice and age-matched naive (i.e., unimmunized) mice were challenged with 10(5) cfu bacteria . Three days postchallenge, bacterial clearance was determined . Compared with lard-fed mice, naive mice in the fish oil treatment group had higher bacterial loads in their liver and spleen (P < 0.001) . When mice were immunized with 10(4) cfu bacteria before rechallenge with 10-fold more bacteria, both lard- and fish oil-fed mice had significantly lower bacterial loads in their liver and spleen (e.g., approximately 2 log(10); P < 0.001) compared with their naive counterparts . However, when the immunization dose was reduced to 10(3) bacteria, a modest diet treatment effect was observed, such that compared with immune lard-fed mice, immune fish oil-fed mice had significantly greater bacterial loads in their liver and spleen (i.e., approximately 0.5 log(10); P < 0.01) . These data demonstrate for the first time that although dietary (n-3) PUFA can significantly impair host resistance to a primary as well as a secondary L . monocytogenes infection, the impairment of the immunological memory response is much less severe.

Best Pract Res Clin Haematol, 2003 Mar, 16(1), 33 - 40
Clinical manifestations and infectious complications of hairy-cell leukaemia; Kraut EH; Hairy-cell leukaemia is an indolent lymphoproliferative malignancy characterized by infiltration of the bone marrow, liver, spleen, and occasionally lymph nodes with a malignant B cell with hair-like cytoplasmic projections . This involvement leads to splenomegaly with secondary consumption of red cells, platelets and neutrophils as well as other complications of an enlarged spleen, including infarction-or-rarely rupture . The common haematological complications of anaemia, neutropenia and thrombocytopenia are due not only to the enlarged spleen but probably also to hairy cells in the bone marrow inducing cytokine-mediated suppression of haematopoiesis . Hepatic involvement, although frequent, only occasionally leads to liver dysfunction . Infections are a major cause of morbidity and mortality in patients with hairy-cell leukaemia, presumably owing to neutropenia and monocytopenia in these patients . The infections seen may be due to unusual pathogens, including Mycobacterium and Listeria.Autoimmune disease, including polyarthitis and vasculitis, occurs frequently and does not correlate with the severity of the disease . Other rare complications include bone involvement, meningitis and ascites . A wide range of secondary malignancies have been reported in patients with hairy-cell leukaemia, but it is still unclear whether the incidence is increased and whether they are related to the disease or treatment.

Vasc Endovascular Surg, 2003 Mar-Apr, 37(2), 145 - 9
Rapidly enlarging iliac aneurysm secondary to listeria monocytogenes infection: a case report; Clouse WD et al.; Infected aneurysms caused by Listeria monocytogenes are rare . Worldwide, 16 cases have been reported, none in the iliac system . The authors report the case of an 80-year-old man being followed for small aortic and right common iliac artery (RCIA) aneurysms who presented with progressive gastrointestinal symptoms . Serial computed tomography demonstrated a 200% increase in RCIA diameter with development of infection over 1 month . Right axillobifemoral bypass and aneurysm resection were performed . The authors believe this case represents the first description of bacteremic seeding of an iliac degenerative aneurysm by Listeria monocytogenes . The natural history and aggressive course of vascular infection with this organism are documented.

Genetics, 2003 Mar, 163(3), 1169 - 75
Mapping quantitative trait loci in the case of a spike in the phenotype distribution; Broman KW; A common departure from the usual normality assumption in QTL mapping concerns a spike in the phenotype distribution . For example, in measurements of tumor mass, some individuals may exhibit no tumors; in measurements of time to death after a bacterial infection, some individuals may recover from the infection and fail to die . If an appreciable portion of individuals share a common phenotype value (generally either the minimum or the maximum observed phenotype), the standard approach to QTL mapping can behave poorly . We describe several alternative approaches for QTL mapping in the case of such a spike in the phenotype distribution, including the use of a two-part parametric model and a nonparametric approach based on the Kruskal-Wallis test . The performance of the proposed procedures is assessed via computer simulation . The procedures are further illustrated with data from an intercross experiment to identify QTL contributing to variation in survival of mice following infection with Listeria monocytogenes.

Proc Natl Acad Sci U S A, 2003 Apr 15, 100(8), 4521 - 6 Epub 2003 Mar 25.
Probing polymerization forces by using actin-propelled lipid vesicles; Upadhyaya A et al.; Actin polymerization provides a powerful propulsion force for numerous types of cell motility . Although tremendous progress has been made in identifying the biochemical components necessary for actin-based motility, the precise biophysical mechanisms of force generation remain unclear . To probe the polymerization forces quantitatively, we introduce an experimental system in which lipid vesicles coated with the Listeria monocytogenes virulence factor ActA are propelled by actin polymerization . The polymerization forces cause significant deformations of the vesicle . We have used these deformations to obtain a spatially resolved measure of the forces exerted on the membrane using a model based on the competition between osmotic pressure and membrane stretching . Our results indicate that actin exerts retractile or propulsive forces depending on the local membrane curvature and that the membrane is strongly bound to the actin gel . These results are consistent with the observed dynamics . After a slow elongation of the vesicle from a spherical shape, the strong bonds between the actin gel and the membrane rupture if the retractile forces exceed a critical value, leading to a rapid release of the vesicle's trailing edge.

Mol Microbiol, 2003 Apr, 48(1), 173 - 86
Functional regulation of the Listeria monocytogenes bacteriophage A118 holin by an intragenic inhibitor lacking the first transmembrane domain; Vukov N et al.; We have dissected the functional properties of the holin encoded by Listeria monocytogenes bacteriophage A118 . Native hol118 was cloned into lambdaDeltaSthf, devoid of the S holin, and tested in an E . coli background . Surprisingly, it caused very late cell lysis, beginning at 80 min after induction . Immunological analyses demonstrated that Hol118 appears in the cytoplasmic membrane shortly after infection . The hol118 gene features a dual start motif similar to lambda S . Therefore, different N-terminally modified Hol118 variants were tested . However, in contrast to lambda S, inactivation of AUG-1 or AUG-2 showed no significant influence on lysis timing . In addition, Hol118-mediated lysis could not be triggered by energy poisons, indicating a functional regulation different from that of S . Toeprinting assays on hol118 mRNA revealed an unexpected translational start codon (AUG-3) at nucleotide position 40 . We demonstrated by in vitro and in vivo approaches that the predicted Hol118(83) product is actually produced together with the full-length polypeptide . However, although the truncated holin lacking its first transmembrane domain appeared in the cytoplasmic membrane, it was shown to be functionally deficient and unable to support lambda R-mediated lysis . In contrast, specific mutations introduced to abolish translation initiation at AUG-3 drastically accelerated lysis, pointing to an inhibitor function of Hol118(83) . This hypothesis was supported by the observation that hol118(83) inhibited holin function when expressed in trans . A deviation from the lambda S paradigm is proposed, which represents a new model of holin functional regulation: the intragenic, in frame translated Hol118(83) product, which is devoid of its first transmembrane domain, acts as a functional inhibitor and constitutes a key part of the lysis clock of A118 . Presence of the dominant inhibitor function also explains the long latent period of A118, where the onset of lysis takes about 70 min, more than twice the time needed by lambda.

Infect Immun, 2003 Apr, 71(4), 1748 - 54
Induction of protective immunity to Listeria monocytogenes with dendritic cells retrovirally transduced with a cytotoxic T lymphocyte epitope minigene; Nakamura Y et al.; In the present study, we developed a cytotoxic T lymphocyte (CTL) epitope minigene-transduced dendritic cell (DC)-based vaccine against Listeria monocytogenes . Murine bone marrow-derived DCs were retrovirally transduced with a minigene for listeriolysin O (LLO) 91-99, a dominant CTL epitope of L . monocytogenes, and were injected into BALB/c mice intravenously . We found that the DC vaccine was capable of generating peptide-specific CD8+ T cells exhibiting LLO 91-99-specific cytotoxic activity and gamma interferon production, leading to induction of protective immunity to the bacterium . Furthermore, we demonstrated that the retrovirally transduced DC vaccine was more effective than a CTL epitope peptide-pulsed DC vaccine and a minigene DNA vaccine for eliciting antilisterial immunity . These results provide an alternative strategy in which retrovirally transduced DCs are used to design vaccines against intracellular pathogens.

J Microbiol Methods, 2003 May, 53(2), 235 - 43
Detection of bacterial pathogens in environmental samples using DNA microarrays; Call DR et al.; Polymerase chain reaction (PCR) is an important tool for pathogen detection, but historically, it has not been possible to accurately identify PCR products without sequencing, Southern blots, or dot-blots . Microarrays can be coupled with PCR where they serve as a set of parallel dot-blots to enhance product detection and identification . Microarrays are composed of many discretely located probes on a solid substrate such as glass . Each probe is composed of a sequence that is complimentary to a pathogen-specific gene sequence . PCR is used to amplify one or more genes and the products are then hybridized to the array to identify species-specific polymorphism within one or more genes . We illustrate this type of array using 16S rDNA probes suitable for distinguishing between several salmonid pathogens . We also describe the use of microarrays for direct detection of either RNA or DNA without the aid of PCR, although the sensitivity of these systems currently limits their application for pathogen detection . Finally, microarrays can also be used to "fingerprint" bacterial isolates and they can be used to identify diagnostic markers suitable for developing new PCR-based detection assays . We illustrate this type of array for subtyping an important food-borne pathogen, Listeria monocytogenes.

Protein Expr Purif, 2003 Mar, 28(1), 78 - 85
High-level expression of the Listeria monocytogenes listeriolysin O in Escherichia coli and preliminary characterization of the purified protein; Giammarini C et al.; Listeriolysin O (LLO) is a cholesterol-binding sulfhydryl-activated hemolysin encoded by Listeria monocytogenes hlyA gene . After analyzing the nucleotide coding sequence of this gene from the ATCC 9525 L . monocytogenes strain, we cloned it in a pET vector for expression in Escherichia coli . Thanks to the optimization of the induction protocol, we achieved a high-level LLO synthesis (about 10% of total cell proteins) in hemolytically active form . The expressed hemolysin was then purified to homogeneity, as revealed by SDS-PAGE and Western blot analysis, by a hydroxyapatite adsorption chromatography, followed by an SP Sepharose ion-exchange chromatography . The recombinant protein showed the same properties determined for LLO purified from L . monocytogenes cultures and the characteristics of the sulfhydryl-activated toxins such as inactivation by oxidation and by reaction with cholesterol . By a combination of the pET expression system and the simple purification method, we obtained a significant amount of toxin (4.5 mg/litre cell culture) in a hemolytically active form (1.25 x 10(6)HU/mg 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 . The recombinant hemolysin, purified in sufficient quantities, could be very useful for structural studies and for diagnostic and pharmaceutical applications.

FEMS Immunol Med Microbiol, 2003 Apr 1, 35(3), 269 - 73
Efficiency of high pressure treatment for destruction of Listeria monocytogenes in fruit juices; Alpas H et al.; The objective of this study was to compare high pressure resistance of Listeria monocytogenes strains at 25 degrees C and 50 degrees C at 350 MPa and to use high pressure (250 MPa and 350 MPa) at 30 degrees C and 40 degrees C for the inactivation of the relatively most pressure resistant strain inoculated in pasteurized apple, apricot, cherry and orange juices . L . monocytogenes CA was found to be the relatively most pressure resistant strain and increasing pressurization from 250 MPa to 350 MPa at 30 degrees C had an additional three to four log cycle reduction in viability, still leaving viable cells after 5 min . When 350 MPa at 40 degrees C for 5 min was applied more than eight log cycle reduction in cell population of all fruit juices was achieved . This study demonstrated that low temperature (40 degrees C) high pressure (350 MPa) treatment has the potential to inactivate relatively pressure resistant L . monocytogenes strains inoculated in different fruit juices within 5 min.

FEMS Immunol Med Microbiol, 2003 Apr 1, 35(3), 255 - 62
Production of IL-12 and IL-18 in human dendritic cells upon infection by Listeria monocytogenes; Kolb-Maurer A et al.; Dendritic cells (DCs) are major antigen-presenting cells of the immune system, which need to be activated in order to initiate an immune response . Here, we describe the immunostimulatory effects on human monocyte-derived DCs observed upon infection with Listeria monocytogenes or after treatment with listerial lipoteichoic acid (LTA) and lipopolysaccharide (LPS), respectively . All stimuli caused upregulation of costimulatory molecules, induced T-cell proliferative responses and secretion of cytokines in vitro . Infection of DCs with L . monocytogenes induced release of interleukin (IL)-12 and IL-18 . In contrast treatment with purified listerial LTA yielded high levels of IL-18 release, but only minimal IL-12 production . Treatment of DCs with LPS conversely induced significant amounts of IL-12 production, but no IL-18 . The release of both stimulating cytokines IL-12 and IL-18 upon infection with entire bacteria suggests that attenuated strains of L . monocytogenes may be a valuable tool for subunit vaccine delivery.

FEMS Immunol Med Microbiol, 2003 Apr 1, 35(3), 243 - 53
Tailoring host immune responses to Listeria by manipulation of virulence genes -- the interface between innate and acquired immunity; Peters C et al.; Although attenuated strains of microbial pathogens have triggered vaccine development from its origin, the role of virulence factors in determining host immunity has remained largely unexplored . Using the murine listeriosis model, we investigated whether the induction and expansion of protective and inflammatory T cell responses may be modified by selective manipulation of virulence genes . We intentionally deleted specific genes of Listeria monocytogenes, including those encoding the positive regulatory factor (prfA), hemolysin (hly), the actin nucleator (actA), and phospholipase B (plcB) . The resulting strains showed decisive differences in their immunogenic properties . In particular, we identified a double-deletion mutant that retained Listeria's profound ability to induce protective CD8(+) T cells, but that is strongly attenuated and exhibits a significantly reduced ability to induce CD4(+) T cell-mediated inflammation . We conclude that this mutant, L . monocytogenes DeltaactADeltaplcB, is at present the most promising mutant for a bacterial vaccine vector and is able to safely induce potent CD8(+) T cell-mediated immunity.

FEMS Immunol Med Microbiol, 2003 Apr 1, 35(3), 235 - 42
CD8 T cell immunome analysis of Listeria monocytogenes; Kamm C et al.; The identification of T cell epitopes is crucial for the understanding of the host response during infections with pathogenic microorganisms . Generally, the identification of relevant T cell responses is based on the analysis of T cell lines propagated in vitro . We used an ex vivo approach for the analysis of the CD8 T cell response against Listeria monocytogenes that is based upon the fractionation of naturally processed antigenic peptides and subsequent analysis with T cells in an enzyme-linked immunospot (ELISPOT) assay . Our data indicate that the direct ex vivo ELISPOT analysis of peptides extracted from infected tissues represents a versatile and potent test system for the analysis of the CD8 T cell immunome of microorganisms that furthermore requires neither the knowledge of the microbial genome nor of the specificity of responding T cells.

FEMS Immunol Med Microbiol, 2003 Apr 1, 35(3), 215 - 25
Nucleic acid-based, cultivation-independent detection of Listeria spp and genotypes of L monocytogenes; Schmid M et al.; Based on comparative analysis of 16S rRNA gene sequences, two oligonucleotide probes for in situ detection of all members of the genus Listeria were designed . These probes allowed fast and reliable in situ detection of Listeria spp . even in complex samples like raw milk . Almost full-length iap (invasion-associated protein) gene sequences were determined for 69 Listeria monocytogenes strains of all 13 known serotypes . A comparison of these sequences revealed that the L . monocytogenes strains can be grouped into three distinct genotypes . These clusters correlate well with distinct serotypes . Thus, strains of serotypes b and d belong to genotype I, a and c to genotype II, and 4a and 4c, which are rarely isolated from humans, group together within genotype III . These results could be corroborated by further comparative sequence analysis of genes encoding two phospholipases - plcA and plcB . Based on the iap gene sequences, a highly specific and reproducible competitive PCR detection method was developed . Primer pairs targeting genotype-specific regions of the iap gene were designed . The amplification of non-specific PCR products from DNA of non-target strains was prevented by adding competitive primers . By applying this method, the rapid and reliable distinction of the three L . monocytogenes genotypes was possible.

FEMS Immunol Med Microbiol, 2003 Apr 1, 35(3), 203 - 5
Listeriosis: therapeutic options; Hof H; Since overt listeriosis occurs mainly in immunocompromised persons it is quite consistent to try to restore the hampered defence system by supportive measurements . For direct antimicrobial treatment a series of different antibiotics is available, since Listeria strains isolated from patients are in general susceptible to a wide range of antibiotics, except fosfomycin, quinolones and cephalosporins of the third generation, although a few exceptional strains exist . Unfortunately, most antibiotics are not bactericidal for Listeria . Drug combinations may exert a synergistic effect . Furthermore, the efficacy of therapy is limited by the fact of intracellular habitat of pathogenic Listeria . Few agents, such as macrolides and quinolones, are accumulated within host cells and may attack the intracellular Listeriae . The clinical experience shows that the combination of amoxicillin and gentamicin is the best option.

FEMS Immunol Med Microbiol, 2003 Apr 1, 35(3), 199 - 202
History and epidemiology of listeriosis; Hof H; Listeriae are used as a tool by different specialities in biomedical research . There are now at least four major fields of interest in LISTERIA: (1) . the role in medical microbiology: Listeria monocytogenes causes severe diseases of men and animals and is difficult to treat; (2) . the role in food microbiology: Listeria is a food-borne pathogen and is found in various food items; (3) . the role in cell biology: L . monocytogenes is a facultative intracellular parasite having an intense cross-talk and interactions with the host cell; (4) . the role in immunology: basic knowledge on cell-mediated immunity has been acquired through the model of listeriosis . This paper presents information on the past and the actual situation in research on Listeria and listeriosis.

FEMS Immunol Med Microbiol, 2003 Apr 1, 35(3), 191 - 7
Listeria monocytogenes: diagnostic problems; Beumer RR et al.; The first isolation methods for the detection of Listeria spp . were generally based on the direct culture of samples on simple agar media, but isolation of the pathogenic Listeria monocytogenes was difficult . In time, new techniques were developed, based on a variety of selective and elective agents in isolation and enrichment media, which gained better and quicker results . Current reference methods allow the recovery of L . monocytogenes from a variety of foods with relative ease . However, more comparative studies are needed to select one horizontal method . It is suggested that the procedure of the International Organization for Standardization is a good base for such comparisons.

FEMS Immunol Med Microbiol, 2003 Apr 1, 35(3), 183 - 9
Listeria: growth, phenotypic differentiation and molecular microbiology; Allerberger F; The identification of Listeria species is based on a limited number of biochemical markers, among which absence or presence of hemolysis and arylamidase are used to differentiate between L . monocytogenes and L . innocua . The CAMP (Christie, Atkins, Munch-Petersen) test must be interpreted with caution . Chromogenic media are based on both the specific chromogenic detection of phosphatidylinositol phospholipase C and the xylose fermentation and give specific and direct identification of L . monocytogenes and L . ivanovii . Isolates of L . monocytogenes with atypical properties require tools of molecular biology for final identification . Serotyping, although not allowing speciation, serves a useful purpose for confirming the genus diagnosis Listeria . Polymerase chain reaction is particularly useful when prior administration of antimicrobial agents compromises culture . For clinical specimens the importance of trying to isolate the pathogen as a prerequisite for an epidemiological work-up and finally for prevention of further cases cannot be overstressed.

FEMS Immunol Med Microbiol, 2003 Apr 1, 35(3), 177 - 82
Murine model of pregnancy-associated Listeria monocytogenes infection; Abram M et al.; Listeria monocytogenes has been recognized as a significant pathogen, occurring worldwide, capable of causing animal and human infections . In its most severe form, listeriosis is an invasive disease that affects immunocompromised patients . Additionally, pregnant women represent a high-risk group for L . monocytogenes infection . Abortion, stillbirth or severe neonatal infection can be the serious outcome of such an infection . In an experimental murine model of pregnancy-associated listeriosis we studied the impact of L . monocytogenes on the maternal immune response and pregnancy outcome . In comparison to virgin animals, pregnant mice mounted lower levels of protective cytokines and were unable to eliminate the pathogen . The impaired maternal immune response that has been found both on the systemic and local level, facilitated bacterial multiplication in the liver, placenta and ultimately in the fetal tissues . This resulted in severe necrotizing hemorrhagic hepatitis and Listeria-induced placental necrosis, increasing the incidence of postimplantation loss and poor pregnancy outcome.

FEMS Immunol Med Microbiol, 2003 Apr 1, 35(3), 173 - 5
Listeriosis: clinical presentation; Doganay M; Listeria monocytogenes is an uncommon cause of illness in the general population . However, this bacterium is an important cause of severe infections in neonates, pregnant women, the elderly, transplant recipients and other patients with impaired cell-mediated immunity . Various clinical syndromes due to L . monocytogenes have been described such as sepsis, central nervous system infections, endocarditis, gastroenteritis and localized infections . A review of the clinical presentation of listeriosis is given in this paper.

FEMS Microbiol Lett, 2003 Mar 14, 220(1), 9 - 14
Multi locus fingerprinting of Listeria monocytogenes by sequence-specific labeling of DNA probes combined with array hybridization; Rudi K et al.; We have developed an alternative multi locus sequence typing (MLST) approach that targets the variable genetic changes directly in a DNA array format . Our approach is based on DNA array hybridization in combination with sequence-specific labeling of oligonucleotide probes . Listeria monocytogenes was chosen for the development and evaluation of the assay . The genes hlyA, iap, flaA, inlA and actA were targeted . Twenty-nine suitable probe regions were identified within these genes . The DNA array results from 32 different strains were compared to serotype and amplified fragment length polymorphism data . This comparison showed that our DNA array method gave good discrimination between the strains analyzed . In conclusion, the DNA array-based MLST method is a promising tool for fingerprint bacteria.

Lett Appl Microbiol, 2003, 36(4), 230 - 3
An improved selective isolation medium for the recovery of Listeria monocytogenes from smoked fish; Neamatallah AA et al.; AIMS: The aim of this study was to improve the selective isolation of Listeria monocytogenes from smoked haddock fillets . METHODS AND RESULTS: Listeria selective agar (LSA)--Oxford formulation was supplemented with 25 microg x ml(-1) of colistin sulphate and 30 microg x ml(-1) of nalidixic acid . Inocula from four smoked haddock fillets produced colonies (approx . 2-13 bacteria x g(-1)), identified as L . monocytogenes, on LSA supplemented with antimicrobial compounds (MLSA) . Moreover, there was only negligible evidence of bacteria which were not L . monocytogenes on MLSA . In contrast, LSA supported dense bacterial growth, which was not equated with L . monocytogenes . SIGNIFICANCE AND IMPACT OF THE STUDY: The modified medium permitted the recovery of L . monocytogenes from smoked haddock fillets and reduced the growth of contaminating bacteria.

J Clin Invest, 2003 Mar, 111(6), 805 - 10
Measles virus infection results in suppression of both innate and adaptive immune responses to secondary bacterial infection; Slifka MK et al.; Among infectious agents, measles virus (MV) remains a scourge responsible for 1 million deaths per year and is a leading cause of childhood deaths in developing countries . Although MV infection itself is not commonly lethal, MV-induced suppression of the immune system results in a greatly increased susceptibility to opportunistic bacterial infections that are largely responsible for the morbidity and mortality associated with this disease . Despite its clinical importance, the underlying mechanisms of MV-induced immunosuppression remain unresolved . To begin to understand the basis of increased susceptibility to bacterial infections during MV infection, we inoculated transgenic mice expressing the MV receptor, CD46, with MV and Listeria monocytogenes . We found that MV-infected mice were more susceptible to infection with Listeria and that this corresponded with significantly decreased numbers of macrophages and neutrophils in the spleen and substantial defects in IFN-gamma production by CD4(+) T cells . The reduction in CD11b(+) macrophages and IFN-gamma-producing T cells was due to reduced proliferative expansion and not to enhanced apoptosis or to altered distribution of these cells between spleen, blood, and the lymphatic system . These results document that MV infection can suppress both innate and adaptive immune responses and lead to increased susceptibility to bacterial infection.

J Food Prot, 2003 Mar, 66(3), 436 - 40
Detection of Listeria monocytogenes in salmon using the Probelia polymerase chain reaction system; Wan J et al.; A validation was conducted on the performance of a commercially available polymerase chain reaction (PCR) kit (Probelia) in comparison with International Organization for Standardization (ISO) method 11290-1 (adopted as an Australian New Zealand Standard Method, AS/NZS 1766.2.16.1:1998) for the detection of Listeria monocytogenes in salmon samples . The validation was conducted following the guidelines of an Australian New Zealand Standard (Guide to Determining the Equivalence of Food Microbiology Test Methods, Part 1, Qualitative Tests, AS/NZS 4659.1:1999), which adopts an approach similar to that recommended by the Association of Analytical Communities Microbiology Method Validation Program for Performance Tested and Peer Verified Methods . The validation study involved the use of five cultures of L . monocytogenes, each challenged at a single level of inoculation into five different types of salmon samples . A total of 60 salmon samples (30 unchallenged and 30 challenged) were tested using both the PCR method and the ISO method . Results from this study indicated that the Probelia PCR method is equivalent to the ISO method . In addition, the detection sensitivity of the Probelia PCR system was determined as approximately 0.5 CFU per PCR assay (equivalent to 20 CFU/ml broth culture) for a pure culture of L . monocytogenes . The Probelia PCR method offers the advantage of detecting L . monocytogenes to genetic specificity within 48 to 50 h, whereas the ISO method requires 5 days for negative results with additional days for confirmed positive results by the use of other biochemical and cultural tests.

J Food Prot, 2003 Mar, 66(3), 390 - 5
A novel method for the reduction of numbers of Listeria monocytogenes cells by freezing in combination with an essential oil in bacteriological media; Cressy HK et al.; The use of multiple freeze (-20 degrees C)-thaw cycles in combination with isoeugenol and polysorbate 80 was investigated as a method for the reduction of numbers of Listeria monocytogenes cells in a bacteriological medium . Three freeze (1 h, -20 degrees C)-thaw cycles in the presence of isoeugenol at concentrations of 0, 100, and 300 ppm resulted in average L . monocytogenes reductions of 0.69, 2.65, and 3.3 log10 MPN (most probable number) per ml, respectively . Increasing the number of freeze-thaw cycles further decreased cell numbers, with reductions of nearly 5 log10 MPN/ml being obtained with six freeze-thaw cycles . Freeze-thaw cycles were effective in reducing cell numbers at isoeugenol concentrations down to 25 ppm . Rapid freezing rates with liquid nitrogen were found to be less effective in reducing numbers of L . monocytogenes cells . Two rapid freeze-thaw cycles in the presence of 100 ppm isoeugenol and polysorbate 80 resulted in a reduction of 1.45 log10 MPN/ml . Two freezing (-20 degrees C) cycles involving slow freezing and thawing rates with samples being held frozen for 6 h for each cycle resulted in reductions larger than those obtained with faster freezing rates . It was found that complete thawing in freeze-thaw cycles was not necessary to achieve bactericidal action . The application of multiple freeze-thaw cycles in combination with low concentrations of isoeugenol could effectively reduce numbers of L . monocytogenes cells in bacteriological media.

Microbiology, 2003 Mar, 149(Pt 3), 611 - 20
Capacity of ivanolysin O to replace listeriolysin O in phagosomal escape and in vivo survival of Listeria monocytogenes; Frehel C et al.; Listeriolysin O (LLO, hly-encoded) is a major virulence factor secreted by the pathogen Listeria monocytogenes . The amino acid sequence of LLO shows a high degree of similarity with that of ivanolysin O (ILO), the cytolysin secreted by the ruminant pathogen Listeria ivanovii . Here, it was tested whether ILO could functionally replace LLO by expressing the gene encoding ILO under the control of the hly promoter, in an hly-deleted strain of L . monocytogenes . It is shown that ILO allows efficient phagosomal escape of L . monocytogenes in both macrophages and hepatocytes . Moreover, expression of ILO is not cytotoxic and promotes normal intracellular multiplication . In vivo, the ILO-expressing strain can multiply and persist for several days in the liver of infected mice but is unable to survive in the spleen . This work underscores the key role played by the cytolysin in the virulence of pathogenic Listeria.

J Appl Microbiol, 2003, 94(4), 720 - 32
Subtyping Listeria monocytogenes through the combined analyses of genotype and expression of the hlyA virulence determinant; Rudi K et al.; AIMS: A major challenge for Listeria monocytogenes diagnostics is that this bacterium is ubiquitous in the environment, and that only a small fraction of the lineages are potential human pathogens . The aim of this work was to obtain a better subtyping of L . monocytogenes through utilization of combined analyses of genotype and the expression of the virulence determinant hlyA . METHODS AND RESULTS: We investigated the effect of growth temperature and medium on the hlyA expression . The gene expression levels were determined by real-time quantitative reverse transcription PCR . The expression pattern of hlyA was highly diverse among the different strains tested . The expression ranged from repression to a 1000-fold induction for growth at 42 degrees C, as compared with 0 degrees C . The expression patterns were compared with the corresponding genotypes . There were surprisingly low correlations between the expression patterns and the genotype clusterings.This is exemplified for the virulent type strain NTNC 7973 and non-virulent type strain DSMZ 20600 . These strains are genetically nearly identical, while the hlyA gene expression patterns are very different . CONCLUSIONS: The hlyA gene expression was highly diverse even within genetically clustered subgroups of L . monocytogenes . Consequently, the gene expression patterns can be used to further differentiate the strains within these genetic subgroups . SIGNIFICANCE AND IMPACT OF THE STUDY: A major limitation in the control of L . monocytogenes is that the current tools for subtyping are not accurate enough in determining the potential virulent strains . The impact of this study is that we have developed a subtyping approach that actually targets a virulence property.

J Appl Microbiol, 2003, 94(4), 633 - 40
Molecular epidemiological survey of Listeria monocytogenes in broilers and poultry products; Rorvik LM et al.; AIMS: To investigate the prevalence of Listeria monocytogenes in poultry products, and to elucidate whether poultry products may be linked to listeriosis cases . A further goal was to identify contamination routes for L . monocytogenes to broiler carcasses . METHODS AND RESULTS: Poultry products (385 samples) were screened for L . monocytogenes . The recovered isolates and 19 patient isolates were characterized by multilocus enzyme electrophoresis and restriction enzyme analysis . The poultry isolates showed great genetic diversity, but no identical subclones were identified from poultry sources and patients . One slaughterhouse was examined in detail during a 16-month period . The contamination rates increased along the processing line, and one subclone was found during the whole period . Only low prevalence of the bacteria was revealed from broiler faeces . CONCLUSIONS: The prevalence of L . monocytogenes in poultry products was high, but no listeriosis cases was linked to poultry products . Broilers seem to be contaminated during the slaughter process, and specific strains may persist in the processing environment . Broiler faeces does not seem to be an important source of L . monocytogenes in poultry products . SIGNIFICANCE AND IMPACT OF THE STUDY: Preventive measures to avoid contamination of poultry products by L . monocytogenes must be taken in the processing plants.

Folia Microbiol (Praha), 2002, 47(6), 659 - 62
Effects of nitrogen sources on bacteriocin production by Enterococcus faecium A 2000; Pantev A et al.; The production of a novel broad-spectrum antimicrobial peptide enterococcin A 2000, active against Gram-positive and Gram-negative microorganisms including Listeria subsp . and Escherichia coli, by Enterococcus faecium strain A 2000 isolated from the surface of traditional Bulgarian yellow cheese "kash-kaval" is considerably influenced by complex nitrogen sources in the production medium . Medium components, especially peptone and yeast extract, and their concentration contributed to the increase in bacteriocin production during the stationary phase (16-46 h) of cultivation even in the absence of one of the components present in the basal cultivation MRS medium.

J Immunol, 2003 Mar 15, 170(6), 3289 - 95
Engineered recombinant peanut protein and heat-killed Listeria monocytogenes coadministration protects against peanut-induced anaphylaxis in a murine model; Li XM et al.; Peanut allergy (PNA) is the major cause of fatal and near-fatal anaphylactic reactions to foods . Traditional immunotherapy using peanut (PN) protein is not an option for PNA therapy because of the high incidence of adverse reactions . We investigated the effects of s.c . injections of engineered (modified) recombinant PN proteins and heat-killed Listeria monocytogenes (HKLM) as an adjuvant on anaphylactic reactions in a mouse model of PN allergy . PN-allergic C3H/HeJ mice were treated s.c . with a mixture of the three major PN allergens and HKLM (modified (m)Ara h 1-3 plus HKLM) . The effects on anaphylactic reactions following PN challenge and the association with Ab levels and cytokine profiles were determined . Although all mice in the sham-treated groups exhibited anaphylactic symptoms with a median symptom score of 3, only 31% of mice in the mAra h 1-3 plus HKLM group developed mild anaphylaxis, with a low median symptom score of 0.5 . Alterations in core body temperature, bronchial constriction, plasma histamine, and PN-specific IgE levels were all significantly reduced . This protective effect was markedly more potent than in the mAra h 1-3 protein alone-treated group . HKLM alone did not have any protective effect . Reduced IL-5 and IL-13, and increased IFN-gamma levels were observed only in splenocytes cultures from mAra h 1-3 plus HKLM-treated mice . These results show that immunotherapy with modified PN proteins and HKLM is effective for treating PN allergy in this model, and may be a potential approach for treating PNA.

J Clin Microbiol, 2003 Mar, 41(3), 1055 - 61
Use of listeriolysin O and internalin A in a seroepidemiological study of listeriosis in Swiss dairy cows; Boerlin P et al.; Recombinant listeriolysin O and internalin A were used as antigens in an enzyme-linked immunosorbent assay (ELISA) for the specific detection of anti-Listeria monocytogenes antibodies in cattle . The results showed sensitivities and specificities of 82 and 92%, respectively, for the listeriolysin O ELISA, and 100 and 90%, respectively, for the internalin A ELISA, respectively . The test may be useful for the confirmation of listeria-related abortions and mastitis but does not seem to be indicated for use in the diagnosis of listeria-related encephalitis in cattle . A representative sample of 1,652 serum samples from the healthy dairy cattle population in Switzerland was tested by both ELISAs . The results showed that 11% of the healthy dairy cows in Switzerland simultaneously presented antibodies toward listeriolysin O and internalin A, and 48% of the farms had one or several animals simultaneously positive by assays with both antigens . Multivariable analysis at the farm level confirmed that feeding of silage represents a significant risk factor for a positive listeria serology . Detailed analysis identified corn silage but not grass silage as the major factor in this association . Cattle breed and hygiene on the farm were also identified as significant factors associated with the serological status of farms . In conclusion, the results of the study show that internalin A is a promising new antigen for use in listeria serology and that specific anti-L . monocytogenes antibodies are found in a significant proportion of healthy dairy cows in Switzerland.

Mol Microbiol, 2003 Mar, 47(6), 1613 - 25
Transcriptome analysis of Listeria monocytogenes identifies three groups of genes differently regulated by PrfA; Milohanic E et al.; PrfA is the major regulator of Listeria virulence gene expression . This protein is a member of the Crp/Fnr family of transcription regulators . To gain a deeper understanding of the PrfA regulon, we constructed a whole-genome array based on the complete genome sequence of Listeria monocytogenes strain EGDe and evaluated the expression profiles of the wild-type EGDe and a prfA-deleted mutant (EGDe Delta prfA) . Both strains were grown at 37 degrees C in brain-heart infusion broth (BHI) and BHI supplemented with either activated charcoal, a compound known to enhance virulence gene expression, or cellobiose, a sugar reported to downregulate virulence gene expression in spite of full expression of PrfA . We identified three groups of genes that are regulated differently . Group I comprises, in addition to the 10 already known genes, two new genes, lmo2219 and lmo0788, both positively regulated and preceded by a putative PrfA box . Group II comprises eight negatively regulated genes: lmo0278 is preceded by a putative PrfA box, and the remaining seven genes (lmo0178-lmo0184) are organized in an operon . Group III comprises 53 genes, of which only two (lmo0596 and lmo2067) are preceded by a putative PrfA box . Charcoal addition induced upregulation of group I genes but abolished regulation by PrfA of most group III genes . In the presence of cellobiose, all the group I genes were downregulated, whereas group III genes remained fully activated . Group II genes were repressed in all conditions tested . A comparison of the expression profiles between a second L . monocytogenes strain (P14), its spontaneous mutant expressing a constitutively active PrfA variant (P14prfA*) and its corresponding prfA-deleted mutant (P14 Delta prfA) and the EGDe strain revealed interesting strain-specific differences . Sequences strongly similar to a sigma B-dependent promoter were identified upstream of 22 group III genes . These results suggest that PrfA positively regulates a core set of 12 genes preceded by a PrfA box and probably expressed from a sigma A-dependent promoter . In contrast, a second set of PrfA-regulated genes lack a PrfA box and are expressed from a sigma B-dependent promoter . This study reveals that PrfA can act as an activator or a repressor and suggests that PrfA may directly or indirectly activate different sets of genes in association with different sigma factors.

Biosci Biotechnol Biochem, 2003 Jan, 67(1), 89 - 93
Effect of lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin on the susceptibility to Listeria infection; Sugita-Konishi Y et al.; We studied the effect of lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the susceptibility to Listeria infection of offspring in C57BL /6NCji mice . The offspring were nursed by TCDD-treated dams and exposed to TCDD from birth to weaning via milk . The exposure had little effect on the weights of immune organs and the spleen or the thymus cell population in the dams and offspring, but it enhanced the production of tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) in the serum after Listeria infection . The clearance of Listeria monocytogenes from the spleen was impaired in the off-spring . These results suggest that the exposure to TCDD of the offspring via milk disrupted the host resistance of the offspring, even though the main immune parameters were unchanged.

J Exp Med, 2003 Mar 3, 197(5), 575 - 84
Flt3 ligand-treated neonatal mice have increased innate immunity against intracellular pathogens and efficiently control virus infections; Vollstedt S et al.; Flt-3 ligand (FL), a hematopoetic growth factor, increases the number of dendritic cells (DCs), B cells, and natural killer cells in adult mice but the effect in neonates was unknown . We show that FL treatment of newborn mice induced a >100-fold increase in the innate resistance against infection with herpes simplex virus type 1 and Listeria monocytogenes . This resistance required interferon (IFN)-alpha/beta for viral and interleukin (IL)-12 for bacterial infections . Long-term survival after viral but not bacterial infection was increased approximately 100-fold by FL treatment . After treatment, CD11c(+)/major histocompatibility complex type II(+) and CD11c(+)/B220(+) DC lineage cells were the only cell populations increased in the spleen, liver, peritoneum, and skin . DC induction was independent of IFNs, IL-2, -4, -7, -9, -15, and mature T and B cells . The data suggest that FL increases the number of DCs in neonates and possibly in other immune-compromised individuals, which in turn improves IFN-alpha/beta- and IL-12-associated immune responses.

Annu Rev Immunol, 2003, 21, 425 - 56 Epub 2001 Dec 19.
IL-13 effector functions; Wynn TA; IL-13 was first recognized for its effects on B cells and monocytes, where it upregulated class II expression, promoted IgE class switching and inhibited inflammatory cytokine production . It was also thought to be functionally redundant with IL-4 . However, studies conducted with knockout mice, neutralizing antibodies, and novel antagonists demonstrate that IL-13 possesses several unique effector functions that distinguish it from IL-4 . Resistance to most gastrointestinal nematodes is mediated by type-2 cytokine responses, in which IL-13 plays a dominant role . By regulating cell-mediated immunity, IL-13 modulates resistance to intracellular organisms including Leishmania major, Leishmania mexicana, and Listeria monocytogenes . In the lung, IL-13 is the central mediator of allergic asthma, where it regulates eosinophilic inflammation, mucus secretion, and airway hyperresponsiveness . Manipulation of IL-13 effector function may also prove useful in the treatment of some cancers like B-cell chronic lymphocytic leukemia and Hodgkin's disease, where IL-13 modulates apoptosis or tumor cell growth . IL-13 can also inhibit tumor immunosurveillance . As such, inhibitors of IL-13 might be effective as cancer immunotherapeutics by boosting type-1-associated anti-tumor defenses . Finally, IL-13 was revealed as a potent mediator of tissue fibrosis in both schistosomiasis and asthma, which indicates that it is a key regulator of the extracellular matrix . The mechanisms that regulate IL-13 production and/or function have also been investigated, and IL-4, IL-12, IL-18, IFN-gamma, IL-10, TGF-beta, TNF-alpha, and the IL-4/IL-13 receptor complex play important roles . This review highlights the effector functions of IL-13 and describes multiple pathways for modulating its activity in vivo.

Biophys J, 2003 Mar, 84(3), 1591 - 605
Force generation by actin polymerization II: the elastic ratchet and tethered filaments; Mogilner A et al.; The motion of many intracellular pathogens is driven by the polymerization of actin filaments . The propulsive force developed by the polymerization process is thought to arise from the thermal motions of the polymerizing filament tips . Recent experiments suggest that the nucleation of actin filaments involves a phase when the filaments are attached to the pathogen surface by a protein complex . Here we extend the "elastic ratchet model" of Mogilner and Oster to incorporate these new findings . We apply this "tethered ratchet" model to derive the force-velocity relation for Listeria and discuss relations of our theoretical predictions to experimental measurements . We also discuss "symmetry breaking" dynamics observed in ActA-coated bead experiments, and the implications of the model for lamellipodial protrusion in migrating cells.

Kansenshogaku Zasshi, 2002 Dec, 76(12), 1025 - 9
{An elderly case with Listeria monocytogenes sepsis and pulmonary non-tuberculous mycobacterial infection}; Hontsu S et al.; A 88-year-old woman, who had lived in a nursing home, was admitted to our hospital because of the suspicion of pulmonary tuberculosis . She had a cough, fever and diarrhea on admission . She suffered from sepsis because Listeria monocytogenes was isolated from only the blood culture twice . We immediately administered imipenem/cilastatin to her on admission . She simultaneously had pulmonary non-tuberculous mycobacterial infection because the chest roentgenogram showed a cavity in the right upper lung field and Mycobacterium intracellulare was isolated from the sputum many times . She was treated with isoniazid, rifampicin and clarithromycin for the pulmonary non-tuberculous mycobacterial infection . Her condition improved soon after the administration of IPM/CS but a low grade fever and cough persisted . L . monocytogenes and M . intracellulare are important pathogens in the elderly because cell-mediated immunity mainly works as host defenses against both organisms.

Wei Sheng Yan Jiu, 2002 Aug, 31(4), 301 - 3
{Studies on the effect of combined treatment of irradiation with vacuum packaging on ready-to-eat meat products contaminated with Listeria monocytogenes and spoilage bacteria}; Fu P et al.; Altogether one hundred and fifty samples of Beijing roast duck, roast chicken and cooked meat products inoculated with approximately 1.5 x 10(3)-2.3 x 10(3) cfu/g L . monocytogenes are packed under vacuum and are irradiated with 60Co radiation at doses of 1.0, 2.0, 2.5 and 3 kGy . The results showed that L . monocytogenes in samples can be eliminated by a dose of 2.5 kGy . The resistance of the model strain 54004 to irradiation is stranger than three isolates (X20, G4 and g2) . In addition, the spoilage bacteria in 150 samples of roast duck, roast chicken and cooked meat products can be killed at doses of 10.15 and 20 kGy, respectively.

Rev Argent Microbiol, 2002 Oct-Dec, 34(4), 219 - 21
{Isolation and identification of Listeria monocytogenes and Listeria spp . in dry sausages obtained in markets in the city of La Plata, Argentina}; Pellicer K et al.; A total of 60 samples of dry sausages were analyzed (50 of "salami" and 10 of "chorizo" "candelario" type) obtained at random in markets authorized for their commercialization, for the purpose of evidencing the presence of bacteria of the genus Listeria (Listeria monocytogenes and Listeria spp.) . The results obtained in salami were the following: 10 (20%) isolates of Listeria spp., were characterized: 1 (2%) strain as L . monocytogenes type 1, 7 (14%) strains as L . innocua, 2 (4%) strains as L . welshimeri . In chorizo candelario type 6 (60%) isolates of Listeria spp., were characterized: 2 (33%) strains as L . monocytogenes type 1 and 4 (66%) strains as L . innocua . The total percentages of isolations were: 26.6% of Listeria spp., 5% of L . monocytogenes type 1, 18.3% of L . innocua and 3.3% of L . welshimeri . In conclusion, we consider that methodologies of control must be developed and implemented in order to guarantee the inocuity of these products.

J Endod, 2003 Feb, 29(2), 95 - 9
In vitro evaluation of the cytotoxicity of two root canal sealers on macrophage activity; de Oliveira Mendes ST et al.; Although some studies have been concerned with the cytotoxicity of endodontic sealers and their components, few have approached the effects of endodontic sealers on macrophage viability and activity . In this study the effect of two zinc oxide-eugenol-based sealers, freshly prepared or after setting for 24 h, was determined on macrophage activity in vitro . Sealers were placed inside a glass capillary tube and added to mouse-elicited macrophage cultures . Sealers did not affect macrophage viability; however, adherence to glass and phagocytosis were impaired . Moreover, nitric oxide production in response to activation with interferon-gamma was diminished, but interleukin-12 production in response to Listeria monocytogenes was not altered . Interestingly, freshly mixed and solid test samples had similar inhibitory activities . In conclusion, the tested sealers did not affect a pro-inflammatory response (interleukin-12 production) but had an inhibitory effect on the effector responses measured (phagocytosis and nitric oxide production).

J Food Prot, 2003 Feb, 66(2), 265 - 71
Impact of preheating on the behavior of Listeria monocytogenes in a broth that mimics Camembert cheese composition; Helloin E et al.; The effect of preheating on the survival of L . monocytogenes in Richard's broth, which mimics the composition of Camembert cheese composition, was examined . Experiments were carried out to reproduce contamination of cheese with environmental heat-stressed cells of L . monocytogenes surviving hot-cleaning procedures . Cells in mid-log phase were heated for 30 min at 56 degrees C before being inoculated into Richard's broth . The pHs and temperatures of Richard's broth were chosen to recreate the conditions of curd dripping (pH 5, 25 degrees C), of the beginning of cheese ripening (pH 5, 12 degrees C), and of the beginning (pH 5, 4 degrees C) and the end (pH 7, 4 degrees C) of cheese storage . Immediately after heat treatment, the viability loss was especially high for strain 306715, which exhibited only 0.6% +/- 0.2% survival, compared with 22% +/- 8.7% for strain EGD . The percentages of the surviving heated cells that were injured were 93% +/- 8% for strain 306715 and 98% +/- 3% for strain EGD . The destruction of the surviving L . monocytogenes cells was accelerated when they encountered the pH and temperature conditions of Camembert cheese during manufacturing, ripening, and cold storage (pH 5 at 25, 12, and 4 degrees C, respectively) . The multiplication of the surviving heated cells was retarded under favorable growth conditions similar to those of storage by the distributor and the consumer (pH 7 at 4 and 12 degrees C, respectively).

J Food Prot, 2003 Feb, 66(2), 256 - 64
Assessment of control measures to achieve a food safety objective of less than 100 CFU of Listeria monocytogenes per gram at the point of consumption for fresh precut iceberg lettuce; Szabo EA et al.; The important new concept of the food safety objective (FSO) offers a strategy to translate public health risk into a definable goal such as a specified maximum frequency or concentration of a hazardous agent in a food at the time of consumption that is deemed to provide an appropriate level of health protection . For the foodborne pathogen Listeria monocytogenes, there is a proposed FSO of < 100 CFU/g in ready-to-eat (RTE) products at the time of consumption . Fresh precut iceberg lettuce is one of these RTE products . In this study, we worked with a commercial manufacturer to evaluate the effectiveness of two antimicrobial washing agents (sodium hypochlorite and a mixture of hydrogen peroxide and peroxyacetic acid) against L . monocytogenes under simulated fresh precut washing conditions and evaluated the growth potential of this pathogen on lettuce packaged in a gas-permeable film and stored at 4 or 8 degrees C for 14 days . We used the results of this experiment to demonstrate how the commercial manufacturer could meet the FSO for L . monocytogenes in fresh precut lettuce through the application of performance, process, and microbiological criteria.

J Food Prot, 2003 Feb, 66(2), 249 - 55
Typing of Listeria monocytogenes isolates originating from the food processing industry with automated ribotyping and pulsed-field gel electrophoresis; Aarnisalo K et al.; A total of 486 Listeria monocytogenes isolates originating from 17 Finnish food processing plants (representing meat, poultry, fish, and dairy production) were collected and typed by automated ribotyping using EcoRI as the restriction enzyme . The isolates were divided into 16 different ribotypes (RTs) . Some of these isolates (121), representing all EcoRI types and 16 food plants, were subjected to ribotyping with the PvuII enzyme, to pulsed-field gel electrophoresis (PFGE) typing with AscI and SmaI restriction enzymes, and to serotyping with O-antigen antisera . Nineteen ribotypes were generated with PvuII, 42 macrorestriction patterns were generated with AscI and 24 with SmaI, and three serotypes were generated with antisera . When the results were combined, the overall number of RTs was 23, and that of the PFGE types was 46 . Thus, the overall discrimination power of PFGE was higher (discrimination index {DI} 0.966) than that of ribotyping (DI 0.906) . The most common serotype (90.1% of the isolates) was 1/2, and isolates of serotype 4 (3.3%) were rare . There was no connection between food sectors and RTs or PFGE types, but PFGE indicated the single plants (78.3% of the types) better than ribotyping (56.5%) . On the basis of its automation and on the availability of identification databases, automated ribotyping had some advantages over PFGE . Overall, automated ribotyping can be considered a practical and rapid tool when Listeria contamination is suspected and when screening a large number of isolates is necessary, e.g., when tracing contamination sources . However, in cases of outbreaks, the identical patterns must be confirmed by PFGE, which is a more discriminatory method.

J Food Prot, 2003 Feb, 66(2), 237 - 41
Polymerase chain reaction detection of Listeria monocytogenes on frankfurters using oligonucleotide primers targeting the genes encoding internalin AB; Jung YS et al.; A polymerase chain reaction (PCR) assay targeting the genes encoding internalin AB (inlAB) was developed for detecting Listeria monocytogenes in pure cell cultures and on artificially contaminated frankfurters . Four sets of oligonucleotide primers were evaluated . The set targeting a 902-bp region of the inlAB gene was the most specific . This PCR product was detected in 51 L . monocytogenes strains belonging to four different serogroups (1/2a, 1/2b, 1/2c, and 4b) . In contrast, the PCR product was not detected in other Listeria spp . (Listeria innocua, Listeria ivanovii, Listeria seeligeri, Listeria welshimeri, or Listeria grayi) or in gram-positive, non-Listeria bacteria, indicating that the primer set was highly specific for L monocytogenes . The detection limit of the PCR assay was 10(5) CFU per ml of pure cell culture . However, the assay could detect as few as 10(1) CFU of L . monocytogenes in 25 g of frankfurter with 16 h of enrichment in modified Listeria enrichment broth at 30 degrees C . The total assay time including enrichment was approximately 24 h . These results suggest that the PCR assay can be used to rapidly detect L . monocytogenes on frankfurters and possibly other types of ready-to-eat meat products.

Infect Immun, 2003 Mar, 71(3), 1580 - 3
N-terminal E-cadherin peptides act as decoy receptors for Listeria monocytogenes; da Silva Tatley F et al.; The observation that E-cadherin is the principal epithelial receptor for the bacterial pathogen Listeria monocytogenes led us to investigate whether N-terminal fragments of E-cadherin containing the L . monocytogenes binding domain could inhibit entry of the bacteria into cultured epithelial cells . Here we demonstrate that a conditioned medium from a gastric cancer cell line (Kato III) that carries a truncating CDH-1 mutation 3' of the L . monocytogenes binding domain can inhibit the uptake of the bacteria into Caco-2 cells . The inhibitory activity of the Kato III conditioned medium could be mimicked by incubation of the bacteria with a recombinant 26-kDa N-terminal E-cadherin peptide prior to infection . Furthermore, these data suggest that cleavage of the 80-kDa extracellular domain of E-cadherin from the cell surface may provide an innate form of pathogen defense by acting as a decoy receptor for L . monocytogenes.

Infect Immun, 2003 Mar, 71(3), 1574 - 9
Nonhuman primate model for Listeria monocytogenes-induced stillbirths; Smith MA et al.; Listeria monocytogenes, isolated from outbreaks in either human or nonhuman primate populations, was administered orally at doses ranging from 10(6) to 10(10) CFU . Four of 10 treated animals delivered stillborn infants . L . monocytogenes was isolated from fetal tissue, and the pathology was consistent with L . monocytogenes infection as the cause of pregnancy loss . For all pregnancies resulting in stillbirths, L . monocytogenes was isolated from maternal feces, indicating that L . monocytogenes had survived and had probably colonized the gastrointestinal tract . Antibodies and antigen-specific lymphocyte proliferation against Listeria increased in animals that had stillbirths.

Infect Immun, 2003 Mar, 71(3), 1217 - 24
Expression of truncated Internalin A is involved in impaired internalization of some Listeria monocytogenes isolates carried asymptomatically by humans; Olier M et al.; Fourteen human carriage Listeria monocytogenes isolates were compared to sporadic and epidemic-associated human strains in order to ascertain the pathogenic behavior of these unrecognized asymptomatic strains . Experimental infection of 14-day-old chick embryos revealed that the majority of the carriage strains were attenuated for virulence . Of the 10 attenuated carriage strains, 5 were affected in their invasion capacities in vitro . Western blot analysis with antibody directed against InlA, the surface protein implicated in the internalization in host cells, allowed correlation between the ability of the carriage strains to enter Caco-2 cells and InlA expression . Indeed, these five carriage strains produced truncated forms of InlA . Four of the five truncated forms of InlA had an apparent molecular mass of 47 kDa . In order to assess the existence of a genetic lineage, partial sequences of inlA gene of these four strains were compared and revealed that they had a high degree of sequence conservation at the gene (99.86%) and amino acid (100%) levels . Comparison of their nucleotide sequences with that of the corresponding segment of inlA from EGD-e and Scott A strains, taken as epidemic references, showed more divergence . Taken together, these observations suggest the presence of specific traits that characterize L . monocytogenes strains isolated during asymptomatic carriage . Some of these traits could provide some explanations about the determinants that make them unable to cause systemic human infection.

Infect Immun, 2003 Mar, 71(3), 1083 - 90
Enteral immunization with attenuated recombinant Listeria monocytogenes as a live vaccine vector: organ-dependent dynamics of CD4 T lymphocytes reactive to a Leishmania major tracer epitope; Saklani-Jusforgues H et al.; Listeria monocytogenes is considered as a potential live bacterial vector, particularly for the induction of CD8 T cells . The CD4 T-cell immune response triggered after enteral immunization of mice has not yet been thoroughly characterized . The dynamics of gamma interferon (IFN-gamma)- and interleukin-4 (IL-4)-secreting CD4 T cells were analyzed after priming through intragastric delivery of an attenuated delta actA recombinant L . monocytogenes strain expressing the Leishmania major LACK protein; a peptide of this protein, LACK(158-173) peptide (pLACK), is a well-characterized CD4 T-cell target in BALB/c mice . Five compartments were monitored: Peyer's patches, mesenteric lymph nodes (MLN), spleen, liver, and blood . A single intragastric inoculation of delta actA-LACK-LM in BALB/c mice led to colonization of the MLN and spleen at a significant level for at least 3 days . Efficient priming of IFN-gamma-secreting pLACK-reactive CD4 T cells was observed in all tested compartments . Interestingly, IL-4-secreting pLACK-reactive CD4 T cells were detectable at day 6 or 7 only in blood and liver . The absence of translocation of viable bacteria through the intestinal epithelium after further delta actA-LACK-LM inoculations was concomitant with the absence of an increase in the level of IFN-gamma secreted by the MLN, blood, and splenic pLACK-reactive Th1 T cells, although the levels remained significantly above the basal level . No change in this population size was detected in the spleen . However, an increase in the number of intragastric inoculations had a clinical beneficial effect in L . major-infected BALB/c mice . L . monocytogenes thus presents the potential of an efficient vector for induction of CD4 T cells when administered by the enteral route.

J Immunol, 2003 Mar 1, 170(5), 2621 - 8
Severe impairment in early host defense against Listeria monocytogenes in mice deficient in acid sphingomyelinase; Utermohlen O et al.; The phagolysosomal compartment is crucial for the defense against infection with intracellular pathogens . Within this compartment, the TNF- and IFN-gamma-responsive acid sphingomyelinase (ASMase) generates the signaling molecule ceramide, resulting in the activation of proteases like cathepsin D . To investigate the possible role of ASMase as a mediator of the antibacterial effects of TNF and IFN-gamma, ASMase(-/-) mice were infected with Listeria monocytogenes . ASMase(-/-) mice showed a dramatically increased susceptibility to L . monocytogenes (LD(50) approximately 100 CFU) when compared with syngeneic wild-type mice (LD(50) approximately 10,000 CFU) . In L . monocytogenes-challenged ASMase(-/-) mice, IFN-gamma serum levels as well as IL-1 beta and IL-6 secretion by macrophages were similar to those observed in wild-type C57BL/6 mice . Although macrophages and granulocytes from ASMase(-/-) mice showed intact production of reactive nitrogen intermediates and oxidative burst, ASMase(-/-) macrophages proved completely incapable of restricting the growth of L . monocytogenes in vitro . The results of this study suggest that ASMase is crucially required for the intracellular control of L . monocytogenes in macrophages and granulocytes by nonoxidative mechanisms.

Int J Food Microbiol, 2003 May 15, 82(3), 265 - 72
Adaptive and cross-adaptive responses of persistent and non-persistent Listeria monocytogenes strains to disinfectants; Lunden J et al.; Persistent and non-persistent Listeria monocytogenes strains were tested for initial resistance and adaptive and cross-adaptive responses towards two quaternary ammonium compounds, alkyl-benzyl-dimethyl ammonium chloride and n-alkyldimethyl ethylbenzyl ammonium chloride, one tertiary alkylamine, 1,3-propanediamine-N-(3-aminopropyl)N-dodecyl, sodium hypochlorite and potassium persulphate . The initial resistance of two persistent and two non-persistent L . monocytogenes strains was observed to differ . Both types of strains adapted after a 2-h sublethal exposure to the quaternary ammonium compounds and the tertiary alkylamine, the highest increase in the minimum inhibitory concentration (MIC) being 3-fold . Progressively increasing disinfecting concentrations at 10 and 37 degrees C resulted in adaptation of L . monocytogenes to all disinfectants except potassium sulphate . The highest observed increase in MIC was over 15-fold, from 0.63 to 10 microg/ml of n-alkyldimethyl ethylbenzyl ammonium chloride . All strains reached approximately similar MICs . Stability of the increased resistance was tested by measuring MICs every seventh day for 28 days . The increased resistance to sodium hypochlorite disappeared in 1 week, but the quaternary ammonium compounds and the tertiary alkylamine showed increased resistance for 28 days . These results suggest that cellular changes due to adaptive responses continue to have an effect on the resistance some time after the exposure . All disinfectants were shown to cause cross-adaptation of L . monocytogenes, the highest increase in MIC being almost 8-fold . The only agent that L . monocytogenes could not be shown to cross-adapt to was potassium persulphate which did, however, cause cross-adaptation to the other disinfectants . The mechanism behind these adaptive responses seemed to be non-specific as cross-adaptation was observed not only between related but also unrelated disinfectants . These findings suggest that sustaining high disinfectant effectiveness may be unsuccessful by rotation, even when using agents with different mechanisms of action.

Curr Biol, 2003 Feb 18, 13(4), 329 - 32
The force-velocity relationship for the actin-based motility of Listeria monocytogenes; McGrath JL et al.; The intracellular movement of the bacterial pathogen Listeria monocytogenes has helped identify key molecular constituents of actin-based motility (recent reviews ) . However, biophysical as well as biochemical data are required to understand how these molecules generate the forces that extrude eukaryotic membranes . For molecular motors and for muscle, force-velocity curves have provided key biophysical data to distinguish between mechanistic theories . Here we manipulate and measure the viscoelastic properties of tissue extracts to provide the first force-velocity curve for Listeria monocytogenes . We find that the force-velocity relationship is highly curved, almost biphasic, suggesting a high cooperativity between biochemical catalysis and force generation . Using high-resolution motion tracking in low-noise extracts, we find long trajectories composed exclusively of molecular-sized steps . Robust statistics from these trajectories show a correlation between the duration of steps and macroscopic Listeria speed, but not between average step size and speed . Collectively, our data indicate how the molecular properties of the Listeria polymerization engine regulate speed, and that regulation occurs during molecular-scale pauses.

J Gynecol Obstet Biol Reprod (Paris), 2003 Feb, 32(1), 39 - 42
{The role of amniocentesis in the management of chorioamnionitis with Listeria monocytogene}; Benhaim Y et al.; Listeriosis is prevalent in pregnant women . Associated morbidity includes miscarriage, chorioamnionitis, intrauterine and neonatal death . Maternal symptoms are not specific and the diagnosis is difficult with a high rate of false-negative microbiology results . We report here the case a patient who developed a chorioamnionitis at 31 weeks gestation . Diagnosis was established by examination of the amniotic fluid . We report a case of Literiosis in pregnancy diagnosed by direct examination of amniotic fluid obtained by amniocentesis.

Neurologia, 2003 Jan-Feb, 18(1), 34 - 7
{Listeria rhombencephalitis . Neuroradiological findings}; Pericot I et al.; Rhombencephalitis due to listeria monocytogenes is an uncommon and serious form of brainstem infection . The disease has a characteristic biphasic course: a nonspecific prodrome of headache, nausea or vomiting, and fever lasting for a several days is followed by progressive asymmetrical cranial-nerve palsies . We report two cases of Listeria monocytogenes rhombencephalitis . The first case is a previously healthy 20 year-old-man who developed fever, headache, nausea and vomiting, followed by numbness in left trigeminal nerve . The second case is an immunosuppressed 77 year-old-man, who developed sudden left hemiparesis, followed by fever and severe brainstem dysfunction with ophthalmoplegia and dysphagia . In both cases, a brain magnetic resonance imaging (MRI) scan, showed increased intensity on T2-weighted lesions in the brainstem that enhanced after contrast on T1-weighted sequences . Both patients had a favorable outcome with full clinical recovery . We conclude that MRI aids in the early detection of parenchymal infections, therefore, MRI is crucial for early diagnosis and is very useful for follow-up examinations.

Mol Genet Genomics, 2003 Feb, 268(5), 607 - 17 Epub 2002 Nov 22.
Identification and characterisation of regions in the cellular protein LaXp180 and the Listeria monocytogenes surface protein ActA necessary for the interaction of the two proteins; Bauer S et al.; The Listeria monocytogenes surface protein ActA is an important virulence factor that plays an essential role in intracellular movement of Listeria cells by inducing actin polymerisation . The ActA protein is known to interact with several mammalian proteins including the phosphoprotein VASP, actin and the Arp2/3 complex . In a search for additional ActA-binding proteins we recently employed the yeast two-hybrid system to search for proteins that interact with ActA, and identified, among others, the mammalian protein LaXp180 as a binding partner . In the present study the interaction of the two proteins was investigated in more detail . A number of variants were tested in the yeast two-hybrid system for their ability to interact . On the basis of these assays, the 14 C-terminal amino acids of LaXp180 were identified as being necessary for the interaction with ActA . The proline-rich repeat (PRR) region of ActA was found to be necessary for the interaction with LaXp180, but upstream or downstream sequences are also required to enhance the specificity of the interaction . The second and third repeats in ActA are especially important, and the minimal sequence of ActA capable of interacting with LaXp180 was a proline- and glutamate-rich stretch of PRR3 fused to part of the N-terminal sequence of ActA . Further analysis using site-specific mutations located in either the C-terminal region of LaXp180 or the proline-rich motif of PRR3 of ActA showed that three positively charged amino acids in LaXp180 and two negatively charged amino acids in ActA are critical for the interaction of the two proteins.

J Appl Microbiol, 2003, 94(3), 483 - 94
Image analysis method for evaluation of specific and non-specific hand contamination; Hansen TB et al.; AIMS: To evaluate a quantifying image analysis method for assessing the degree of hand contamination and efficacy of hand washing procedures . METHODS AND RESULTS: Two types of experimental design were used . In one, different concentrations of pure cultures of Escherichia coli, Listeria innocua and Pseudomonas flourescens were applied to hands . In the other, hands were contaminated by handling various raw foods . Imprints of the contaminated palms were made on 24.5 x 24.5 cm agar plates using appropriate agars . After incubation, digital photographs of the plates were analysed using image analysis . In pure culture studies with selective agars, levels from 1 to 10(6) CFU cm(-2) palm could be monitored . For aerobic, mesophilic organisms from raw chicken, levels from 10(3) to 10(6) CFU cm(-2) palm were correlated linearly to image analysis data . CONCLUSIONS: The image analysis of palm imprints made on agar plates was suitable for assessing the degree of contamination from foods on the palms . Sensitivity and specificity depended on the agar used and the type of contamination encountered . SIGNIFICANCE AND IMPACT OF THE STUDY: Data capture by the image analysis method is simple and can be partly automated . Sampling time is short for the person to be tested, which makes it an attractive method for assessing hand hygiene status in larger field trials.

FEMS Microbiol Lett, 2003 Jan 21, 218(1), 155 - 60
Different cellular fatty acid pattern behaviours of two strains of Listeria monocytogenes Scott A and CNL 895807 under different temperature and salinity conditions; Chihib NE et al.; Cells of two strains of Listeria monocytogenes CNL 895807 and Scott A were grown to late exponential phase at different growth temperatures (37, 20 and 4 degrees C) with or without NaCl (7%), and their fatty acid compositions were analysed . The results showed that low thermal adaptation response of L . monocytogenes CNL was different than that of the Scott A strain, and it was based on both an increase of anteiso-branched-chain fatty acids and a significant decrease of straight-chain fatty acids . However, the main modifications observed in the Scott A strain when grown at a low temperature were a decrease of the proportion of ai17:0 and an increase of ai15:0 . In hyperosmotic medium and over the entire temperature range (4 degrees C, 20 degrees C and 37 degrees C) the two L . monocytogenes strains showed a cellular fatty acid profile dominated by ai15:0 . In addition, a decrease of the two major straight-chain fatty acids (14:0 and 16:0) was observed in the CNL strain . These results demonstrated that the CNL strain showed different behaviours of low thermal and salt adaptation to maintain membrane fluidity, which are based both on an increase of anteiso-branched-chain fatty acids, and a significant decrease of straight-chain fatty acids.

Comp Immunol Microbiol Infect Dis, 2003 May, 26(3), 157 - 74
In vitro study of Listeria monocytogenes infection to murine primary and human transformed B cells; Menon A et al.; Immunity to Listeria monocytogenes is largely mediated by T lymphocytes . Recently, B lymphocytes or their secreted products are implicated to provide immunity against L . monocytogenes infection . To understand whether L . monocytogenes can infect and kill B cells as a possible strategy to initiate an infection, we examined the effects of L . monocytogenes on a human B lymphoma (Ramos RA-1) and mouse primary B cells in vitro . L . monocytogenes infection resulted in significantly (p<or=0.05) high cytotoxicity (58-79%) for Ramos and 39-68% cytotoxicity for mouse primary B cells . In contrast, non-pathogenic L . innocua caused only 1.2% cytotoxicity for Ramos and 19% for primary B cells . Bacterial cells were found frequently adhered to the B cell surfaces; however, active invasion was not a prerequisite for infection . L . monocytogenes caused loss of B cell surface molecules, pore formations, cell swelling, membrane damages and apoptosis . This study demonstrates that L . monocytogenes can infect and kill B cells as a possible strategy to initiate a successful infection.

Microbiology, 2003 Jan, 149(Pt 1), 111 - 20
Negative regulation of PrfA, the key activator of Listeria monocytogenes virulence gene expression, is dispensable for bacterial pathogenesis; Greene SL et al.; Listeria monocytogenes is a facultative intracellular bacterial pathogen that regulates the expression of virulence-associated gene products in response to specific host cell compartment environments . The PrfA protein of L . monocytogenes functions as a key regulatory factor required for the differential expression of bacterial virulence gene products within infected host cells . PrfA both positively and negatively regulates its own expression, and while PrfA positive regulation is required for cell-to-cell spread of L . monocytogenes and for full virulence in infected mice, a role for negative regulation has been of presumed importance but has yet to be established . To address the role of negative regulation of prfA expression in L . monocytogenes pathogenesis, prfA promoter mutations designed to reduce or eliminate negative regulation were introduced into L . monocytogenes and analysed for their effects on patterns of PrfA-dependent gene expression and virulence in murine models of infection . High level PrfA production resulting from the prfA promoter mutations produced significantly increased levels of PrfA-regulated gene expression in broth-grown cultures; however the apparent loss of negative prfA regulation had no deleterious effects on growth and spread of the bacteria within infected tissue culture cells or on virulence in mice . The results indicate that while negative regulation of prfA expression exists and provides a feedback system for the control of PrfA synthesis, this feedback system is dispensable for virulence.

J Immunol, 2003 Feb 15, 170(4), 2053 - 63
Role of CD4 T cell help and costimulation in CD8 T cell responses during Listeria monocytogenes infection; Shedlock DJ et al.; CD4 T cells are known to assist the CD8 T cell response by activating APC via CD40-CD40 ligand (L) interactions . However, recent data have shown that bacterial products can directly activate APC through Toll-like receptors, resulting in up-regulation of costimulatory molecules necessary for the efficient priming of naive T cells . It remains unclear what role CD4 T cell help and various costimulation pathways play in the development of CD8 T cell responses during bacterial infection . In this study, we examined these questions using an intracellular bacterium, Listeria monocytogenes, as a model of infection . In CD4 T cell-depleted, CD4(-/-), and MHC class II(-/-) mice, L . monocytogenes infection induced CD8 T cell activation and primed epitope-specific CD8 T cells to levels commensurate with those in normal C57BL/6 mice . Furthermore, these epitope-specific CD8 T cells established long-term memory in CD4(-/-) mice that was capable of mounting a protective recall response . In vitro analysis showed that L . monocytogenes directly stimulated the activation and maturation of murine dendritic cells . The CD8 T cell response to L . monocytogenes was normal in CD40L(-/-) mice but defective in CD28(-/-) and CD137L(-/-) mice . These data show that in situations where infectious agents or immunogens can directly activate APC, CD8 T cell responses are less dependent on CD4 T cell help via the CD40-CD40L pathway but involve costimulation through CD137-CD137L and B7-CD28 interactions.

J Immunol, 2003 Feb 15, 170(4), 1862 - 9
H2-M3-restricted memory T cells: persistence and activation without expansion; Kerksiek KM et al.; H2-M3-restricted T cells respond more rapidly to primary Listeria monocytogenes infection than conventional MHC class Ia-restricted T cells . Reinfection with L . monocytogenes, while inducing explosive proliferation of H2-K(d)-restricted T cells, does not stimulate significant expansion of H2-M3-restricted CTL . These disparate responses to reinfection are apparent within 5 days of primary L . monocytogenes infection . However, H2-M3-restricted memory T cells are generated, and are indistinguishable from classically restricted T cells in terms of cell surface memory markers and longevity . Early responses of H2-M3- and H2-K(d)-restricted memory T cells to reinfection are similar, with increases in size and expression of activation markers . Interestingly, priming of H2-M3-restricted T cells with an L . monocytogenes-derived N-formyl peptide plus anti-CD40 generates memory T cells that expand upon re-exposure to Ag during L . monocytogenes infection . Our data indicate that disparate H2-M3- and MHC class Ia-restricted memory T cell responses reflect intrinsic differences between these T cell populations . Although distinct proliferative programs appear to be hardwired in these populations during primary L . monocytogenes infection, under different inflammatory circumstances M3-restricted T cell populations can maintain the ability to expand upon re-exposure to Ag.

J Immunol, 2003 Feb 15, 170(4), 1737 - 45
Preferential survival of CD8 T and NK cells expressing high levels of CD94; Gunturi A et al.; The Qa-1(b)/Qdm tetramer binds to CD94/NKG2 receptors expressed at high levels on approximately 50% of murine NK cells . Although very few CD8 T cells from naive mice express CD94/NKG2 receptors, approximately 50% of CD8 T cells taken from mice undergoing a secondary response against Listeria monocytogenes (LM) are CD94(high) and bind the tetramer . Although CD94(int) NK cells do not bind the tetramer, CD94(int) CD8 T cells do, and this binding is dependent on the CD8 coreceptor . We found that the extent of apoptosis in CD8 T and NK cells was inversely related to the expression of CD94, with lower levels of apoptosis seen in CD94(high) cells after 1-3 days of culture . The difference in CD8 T cell survival was evident as early as 6 h after culture and persisted until nearly all the CD94(neg/int) cells were apoptotic by 48 h . In contrast, expression of inhibitory Ly-49A,G2,C/I molecules was associated with higher levels of apoptosis . Cross-linking CD94/NKG2 receptors on CD8 T cells from a mouse undergoing an LM infection further reduced the percentage of apoptotic cells on the CD94-expressing populations, while cross-linking Ly-49I had no effect on CD8 T cells expressing Ly-49I . Cross-linking CD3 on CD8 T cells from a mouse undergoing a secondary LM infection increases the extent of apoptosis, but this is prevented by cross-linking CD94/NKG2 receptors at the same time . Similar results were observed with NK cells in that the CD94(high) population displayed less apoptosis than CD94(int) cells after 1-3 days in culture . Therefore, the expression of CD94/NKG2 is correlated with a lower level of apoptosis and may play an important role in the maintenance of CD8 T and NK cells.

J Clin Microbiol, 2003 Feb, 41(2), 757 - 62
Development of a multilocus sequence typing method for analysis of Listeria monocytogenes clones; Salcedo C et al.; This study is a first step in the development of multilocus sequence typing (MLST) method for Listeria monocytogenes . Nine housekeeping genes were analyzed in a set of 62 strains isolated from different sources and geographic locations in Spain . These strains were previously characterized by pulsed-field gel electrophoresis (PFGE) . Because of low diversity, two loci were discarded from the study . The sequence analysis of the seven remaining genes showed 29 different allelic combinations, with 22 of them represented by only one strain . The results of this sequence analysis were generally consistent with those of PFGE . Because MLST allows the easy comparison and exchange of results obtained in different laboratories, the future application of this new molecular method could be a useful tool for the listeriosis surveillance systems that will allow the identification and distribution of analysis of L . monocytogenes clones in the environment.

J Clin Microbiol, 2003 Feb, 41(2), 564 - 71
Serotyping of Listeria monocytogenes by enzyme-linked immunosorbent assay and identification of mixed-serotype cultures by colony immunoblotting; Palumbo JD et al.; Routine analysis of Listeria monocytogenes by serotyping using traditional agglutination methods is limited in use because of the expense and limited availability of commercially prepared antisera and intra- and interlaboratory discrepancies arising from differences in antiserum preparation and visual determination of agglutination . We have adapted a commercially available set of L . monocytogenes antisera to an enzyme-linked immunosorbent assay (ELISA) format for high-throughput, low-cost serotype determination . Rather than subjective visualization of agglutination, positive antigen and antiserum reactions were scored by a quantitative, colorimetric reaction . ELISA serotyping of 89 of 101 L . monocytogenes isolates agreed with slide agglutination serotyping data, and 100 previously uncharacterized isolates were serotyped unambiguously by the ELISA method . In addition, mixed-serotype cultures of L . monocytogenes were identified by a colony immunoblot procedure, in which serogroup 1/2 and serogroup 4 colonies were discriminated by differential staining.

Arthritis Rheum, 2003 Feb, 48(2), 319 - 24
Listeria monocytogenes infection as a complication of treatment with tumor necrosis factor alpha-neutralizing agents; Slifman NR et al.; OBJECTIVE: Tumor necrosis factor alpha (TNFalpha) has been implicated in the pathogenesis of certain inflammatory diseases . Two TNFalpha-neutralizing agents are licensed in the US . Infliximab is licensed for the treatment of Crohn's disease (CD) and, when used with methotrexate, for the treatment of rheumatoid arthritis (RA) . Etanercept is licensed for the treatment of RA, including juvenile RA, and, more recently, was licensed for the treatment of psoriatic arthritis . Because of the potential for decreased host resistance to infectious agents due to treatment with anti-TNFalpha agents, we sought to evaluate postlicensure cases of opportunistic infection, including Listeria monocytogenes, in patients treated with these products . METHODS: The FDA Adverse Event Reporting System, a passive monitoring system, was reviewed to identify all reports of adverse events (through December 2001) associated with L monocytogenes infection in patients treated with infliximab or etanercept . RESULTS: Fifteen cases of L monocytogenes infection associated with infliximab or etanercept treatment were identified . In 14 of these cases, patients had received infliximab . The median age of all patients was 69.5 years (range 17-80 years); 53% were female . Six deaths were reported . Among patients for whom an indication for use was reported, there were 9 patients (64%) with RA and 5 patients (36%) with CD (information was not reported for 1 patient) . All patients for whom information was reported were receiving concurrent immunosuppressant drugs . CONCLUSION: Postlicensure surveillance suggests that L monocytogenes infection may be a serious complication of treatment with TNFalpha-neutralizing agents, particularly infliximab.

Appl Environ Microbiol, 2003 Feb, 69(2), 1082 - 8
Detection of Listeria monocytogenes from a model food by fluorescence resonance energy transfer-based PCR with an asymmetric fluorogenic probe set; Koo K et al.; It has been shown that fluorescence resonance energy transfer (FRET)-based PCR, including the TaqMan assay and molecular beacons, has potential for rapid detection of pathogens . In these promising real-time detection assays a single internal oligonucleotide probe labeled on both the 5' (reporter) and 3' (quencher) ends is used for selective generation of fluorescence . In this paper, we describe the use of a previously reported novel probe design for FRET-based PCR detection of Listeria monocytogenes in pure culture and in a model food commodity . In the assay described here an asymmetric probe set is used; this probe set consists of a long 5' fluorescein-labeled reporter probe and a short, complementary 3' DABCYL-labeled quencher oligonucleotide, which are used in a 5' nuclease amplification and detection assay . By using the listeriolysin O (hly) and p60 (iap) genes as amplification targets, the performance of two primer-probe sets in amplification and subsequent detection of target DNA was evaluated . In studies performed with pure cultures of L . monocytogenes, the PCR profiles indicated that the relative change in fluorescence intensity was correlated with both the initial number of cells and the accumulation of specific amplicons for both hly and iap gene fragments . Experiments were also done to determine the applicability of the method to the detection of L . monocytogenes by targeting hly DNA and its short-lived mRNA product in a model food commodity . Twenty-five-milliliter samples of reconstituted nonfat dry milk (NFDM) were seeded with L . monocytogenes and processed to concentrate the bacteria by centrifugation, and this was followed by nucleic acid extraction and amplification with hly-specific primers . Endpoint detection of PCR and reverse transcription-PCR amplicons could be achieved at inoculum levels of 10(3) and 10(4) CFU of L . monocytogenes/25 ml of NFDM, respectively . This study demonstrated that this asymmetric FRET-based amplification and detection protocol provides an alternative approach for endpoint detection of nucleic acid amplification products as applied to detection of pathogens in a model food.

Appl Environ Microbiol, 2003 Feb, 69(2), 1013 - 22
Three transporters mediate uptake of glycine betaine and carnitine by Listeria monocytogenes in response to hyperosmotic stress; Angelidis AS et al.; The uptake and accumulation of the potent osmolytes glycine betaine and carnitine enable the food-borne pathogen Listeria monocytogenes to proliferate in environments of elevated osmotic stress, often rendering salt-based food preservation inadequate . To date, three osmolyte transport systems are known to operate in L . monocytogenes: glycine betaine porter I (BetL), glycine betaine porter II (Gbu), and a carnitine transporter OpuC . We investigated the specificity of each transporter towards each osmolyte by creating mutant derivatives of L . monocytogenes 10403S that possess each of the transporters in isolation . Kinetic and steady-state osmolyte accumulation data together with growth rate experiments demonstrated that osmotically activated glycine betaine transport is readily and effectively mediated by Gbu and BetL and to a lesser extent by OpuC . Osmotically stimulated carnitine transport was demonstrated for OpuC and Gbu regardless of the nature of stressing salt . BetL can mediate weak carnitine uptake in response to NaCl stress but not KCl stress . No other transporter in L . monocytogenes 10403S appears to be involved in osmotically stimulated transport of either osmolyte, 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 under elevated osmotic stress.

Int J Food Microbiol, 2003 Apr 25, 82(2), 121 - 31
Growths kinetics comparison of clinical and seafood Listeria monocytogenes isolates in acid and osmotic environment; Vialette M et al.; Comparison of pathogenic bacterial strains of clinical origin with strains of the same species isolated from the environment may be a valuable tool for microbial risk assessment, especially for foodborne pathogens . Thus, a number of Listeria monocytogenes strains responsible for human cases of listeriosis, in relation to the consumption of contaminated seafood, have been compared with "natural" L . monocytogenes strains isolated from similar seafood products . Complete factorial designs were used to assess quantitatively the growth abilities of four clinical and four seafood isolates of L . monocytogenes placed in various environmental conditions . The cells were submitted to acid and osmotic stress as they were in stationary phase (constant condition) or in exponential phase (dynamic condition) . The effects and interactions of pH (5-7) and NaCl concentration (0.5-8% v/v) were studied at two growth temperatures (10 and 20 degrees C) . Growth parameters (lag and generation times calculated with Gompertz equation) were used to compare the behavior of the strains with respect to the conditions of culture . The results indicated an overall weak effect of acid stress alone, whereas osmotic stress clearly affected bacterial growth and a synergic effect between these two factors was observed . Clinical strains displayed better adaptation than seafood strains in stationary phase, however, this difference was not verified in exponential phase . Low temperature (10 degrees C) usually confirmed the observations at 20 degrees C, and the differences between clinical and food strains were more pronounced . Finally, a classification of the eight strains, based on the collected data, showed three groups: (i) seafood strains, (ii) three clinical strains and (iii) the last clinical strain, alone due to its high resistance to adverse conditions.

Drug Discov Today, 2003 Jan 15, 8(2), 54 - 5
Bad bugs: good for cancer therapy?
Burnham CM.
Components of Listeria and Escherichia coli have been used to prime the immune system in a novel approach to fighting cancer.

Roum Arch Microbiol Immunol, 2001 Oct-Dec, 60(4), 329 - 35
Involvement of Listeria monocytogenes in the abortive disease; Caplan DM; Listeria monocytogenes, an intracellular facultative germ that causes the invasion, sometimes fatal, in susceptible hosts is a food borne pathogen with ubiquitary spread that has generated a public health problem for such risk groups as: pregnant women, foetuses, new borns . 504 women with abortive disease were serologically investigated in 1999 for serotype 1a circulating in Romania . The most affected age group proved to be that in the range of 20-30 yrs: 378 (75%) cases . 107 (21.23%) female patients had the diagnostic titer (> or = 1/320): among these, 38 (7.53%) had miscarriages in the IVth-VIIIth month and 18 (3.57%) gave birth to dead foetuses; during pregnancy, 10 (1.98%) female patients received treatment with Ampicillin and 2 (0.39%) treatment with Erythromycin . In the age group > 31 yrs, the 1/320 titer was noticed in 21 (4.16%) female patients but among these only 4 (0.79%) had a history of miscarriage in the final pregnancy months; they were administered Ampicillin during pregnancy . Although there is no clear-cut evidence, our results point to the conclusion that these female patients were contaminated with Listeria monocytogenes.

FEBS Lett, 2003 Jan 30, 535(1-3), 195 - 9
Antibacterial activity of peptides derived from envelope glycoproteins of HIV-1; Cole AM et al.; Recent reports have highlighted the anti-HIV-1 activities of defensins, whose structure and charge resemble portions of the HIV-1 transmembrane envelope glycoprotein gp41 . The current report explores the obverse, whether peptides derived from HIV-1 envelope glycoproteins can exert antimicrobial activity . Fifteen-residue peptides spanning the entire sequence of HIV-1(MN) gp120 and gp41 were subjected to radial diffusion assays against laboratory strains of Escherichia coli and Listeria monocytogenes . Twenty-four active peptides corresponded predominantly to membrane-active domains of gp120 and gp41 . Several peptides retained significant activity in higher ionic conditions and may serve as templates for the development of novel peptide antibiotics . The strategies employed herein could uncover additional antimicrobial peptides from envelope proteins of other lytic viruses.

Vaccine, 2003 Mar 7, 21(11-12), 1187 - 94
Enhancing the immunogenicity of bioengineered Listeria monocytogenes by passaging through live animal hosts; Peters C et al.; Bioengineered Listeria monocytogenes can be used as a recombinant bacterial vaccine vector for the induction of strong cell-mediated immunity to passenger antigens . Listeria loses virulence after undergoing bioengineering techniques, thus decreasing its efficacy as a vaccine vector . We addressed this problem by examining the virulence, and the ability to induce CD8(+) T-cells, of Listeria monocytogenes vaccine strains before and after passaging through mice . We found that two in vivo passages are required to restore the induction of cell-mediated immunity to passenger antigens and maximum virulence to these strains . In addition, we found that after each passage, harvested bacteria must be cloned and checked for expression of the bioengineered gene to counter selection in favor of antigen loss mutants .

Vet Microbiol, 2003 Apr 29, 92(4), 351 - 62
Discrimination among Listeria monocytogenes isolates using a mixed genome DNA microarray; Borucki MK et al.; Listeria monocytogenes can cause serious illness in humans, usually following the ingestion of contaminated food . Epidemiologic investigation requires identification of specific isolates, usually done by a combination of serotyping and subtyping using pulsed-field gel electrophoresis (PFGE) . DNA microarrays provide a new format to resolve genetic differences among isolates and, unlike PFGE, to identify specific genes associated with the infecting pathogen . A 585 probe, mixed genome microarray was constructed and 24 strains of L . monocytogenes were hybridized to the array . Microarray analysis allowed discrimination among L . monocytogenes isolates within a serotype and obtained from similar geographic and epidemiologic sources . Importantly, the microarray results preserved previously described phylogenetic relationships between major serogroups and, in a limited comparison, agreed with PFGE subtypes . The association of individual probes with isolates allowed identification of specific genes . Sequencing of 10 polymorphic probes identified nine matches with previously described bacterial genes including several suspected virulence factors . These results demonstrate that mixed genomic microarrays are useful for differentiating among closely related L . monocytogenes isolates and identifying genetic markers that can be used in epidemiologic and possibly pathogenesis studies.

Ugeskr Laeger, 2002 Dec 9, 164(50), 5947 - 50
{Inflammatory bowel disease--do microorganisms play a role?}; Nielsen SE et al.; This review focuses on the potential pathogenic role of microorganisms in relation to inflammatory bowel diseases, i.e . Crohn's disease and ulcerative colitis . Pathogenic microorganism such as Mycobacterium paratuberculosis, measles and mumps viruses, Epstein-Barr virus, and Listeria monocytogenes are discussed, as well as involvement of the normal intestinal flora . Furthermore, the influence of microorganisms in experimental animal colitis models is discussed . The available results are inconclusive, but there seems to be basis for proposing the hypothesis that the inflammation in inflammatory bowel disease reflects an immune imbalance with loss of tolerance for normally harmless antigens in the mucosal microflora.

Infect Immun, 2003 Feb, 71(2), 682 - 9
A/J mice are susceptible and C57BL/6 mice are resistant to Listeria monocytogenes infection by intragastric inoculation; Czuprynski CJ et al.; Previous studies demonstrated that the innate resistance of mice to Listeria monocytogenes infection by intravenous or intraperitoneal inoculation is regulated principally by the Hc locus on mouse chromosome 2 . The A/J and C57BL/6 mouse strains were identified as prototype L . monocytogenes-susceptible and -resistant strains, respectively . In the present study, we compared the relative susceptibilities of A/J and C57BL/6 mice to intragastric (i.g.) inoculation with L . monocytogenes . The results of our study indicate that A/J mice are significantly more susceptible than C57BL/6 mice to an i.g . challenge with L . monocytogenes . This was reflected in the estimated 50% lethal doses for the two strains (10(6) and 10(8) CFU for A/J and C57BL/6 mice, respectively) and a more rapid and severe dissemination of the infection to the spleen and liver in A/J mice than in C57BL/6 mice . Histopathological examination of tissues from the infected mice confirmed the greater severity of disease in A/J mice . Clearance of a primary infection enhanced the resistance of both A/J and C57BL/6 mice to reinfection with L . monocytogenes via the gastrointestinal tract . However, the relative difference in susceptibility between the two strains was evident even after immunization . The A/J mouse holds promise as a model for investigating the pathogenesis of gastrointestinal listeriosis because of its ability to develop systemic infection following challenge with numbers of organisms similar to those recovered from some L . monocytogenes-contaminated food products.

J Food Prot, 2003 Jan, 66(1), 65 - 71
Comparison of different most-probable-number methods for enumeration of Listeria in poultry; Capita R et al.; To estimate levels of Listeria spp . in poultry and to select the most appropriate enumeration method for routine analysis, 40 naturally contaminated retail chicken carcasses were tested in Ponferrada (Leon, N.W . Spain) using the direct plate count technique and various most-probable-number (MPN) designs (UVM I {University of Vermont modified Listeria enrichment broth}, Fraser enrichment broth, or both were used in 3-, 5-, and 10-tube MPN techniques) . MPN estimation was obtained from the number of tubes with Listeria confirmed (after streaking on PALCAM and modified Oxford agars: "true" MPN) and from the number of dark Fraser broth tubes ("predictive" MPN) . Samples were analyzed in duplicate . Low levels of Listeria were found (< 110 CFU/g) . The direct plate count technique was totally ineffective for enumerating Listeria in poultry . The single-step (UVM I) and the two-step (UVM I-Fraser) MPN methods gave comparable estimations and a low number of significantly discrepant predictions . Using a single-step method with Fraser broth, lower true MPNs were obtained . The number of tubes used (3, 5, or 10) did not have a substantial influence on the results . Similar estimations, highly correlated (r = 0.538 to 0.968; P < 0.001), were found with (true MPN) and without (predictive MPN) plating confirmation when using the two-step MPN method . The statistical evaluation of the differential character of Fraser broth as part of the two-step MPN method showed high sensitivity (87.5 to 92.5%), specificity (95.2 to 98.6%), efficiency (94.2 to 97.6%), and predictive values (73.6 to 89.9% for a positive test and 98.0 to 98.9% for a negative test) . Taking into account these results, we suggest the convenience of using a 3- or 5-tube two-step (UVM I-Fraser) MPN method with estimations obtained from the number of tubes with darkening, without confirmation, in order to achieve great savings in time and money.

J Food Prot, 2003 Jan, 66(1), 61 - 4
Effectiveness of trisodium phosphate against Listeria monocytogenes on excised and nonexcised chicken skin; Capita R et al.; The influence of sample type (i.e., excised versus nonexcised chicken skin) on the efficiency of trisodium phosphate (TSP) solutions in reducing Listeria monocytogenes populations and inhibiting their growth during refrigerated storage was studied . Whole chicken legs and excised chicken leg skin fragments inoculated with 10(8) CFU of L . monocytogenes per ml were dipped for 15 min in sterile tap water (control) or in a solution containing 8, 10, or 12% TSP . L . monocytogenes counts were determined after 0, 1, 3, and 5 days of refrigerated storage (2 degrees C) . The decontamination effect of TSP was greater for excised skin than for whole legs . Microbial differences between control and TSP-treated samples were significantly larger for excised skin than for whole legs for 9 (75%) of 12 tested combinations of TSP concentrations and storage times . These differences varied from 1.05 +/- 0.26 log10 cycles (day 1) to 3.30 +/- 0.14 log10 cycles (day 5) for nonexcised-skin samples (whole legs) and from 1.54 +/- 0.48 log10 cycles (day 1) to 4.28 +/- 0.86 log10 cycles (day 5) for excised-skin samples . Significantly larger reductions were observed from the third day of refrigerated storage onward . The TSP concentration was a significant factor in the reduction of L . monocytogenes populations . These results suggest that bacteria are more readily accessible to TSP in excised than in nonexcised chicken skin and that the type of sample used to ascertain the efficacy of antimicrobial surface treatments may influence the findings of this type of study.

J Food Prot, 2003 Jan, 66(1), 52 - 60
Listeria monocytogenes contamination patterns for the smoked fish processing environment and for raw fish; Hoffman AD et al.; Reliable data on the sources of Listeria monocytogenes contamination in cold-smoked fish processing are crucial in designing effective intervention strategies . Environmental samples (n = 512) and raw fish samples (n = 315) from two smoked fish processing facilities were screened for L . monocytogenes, and all isolates were subtyped by automated ribotyping to examine the relationship between L . monocytogenes contamination from raw materials and that from environmental sites . Samples were collected over two 8-week periods in early spring and summer . The five types of raw fish tested included lake whitefish, sablefish, farm-raised Norwegian salmon, farm-raised Chilean salmon, and feral (wild-caught) salmon from the U.S . West Coast . One hundred fifteen environmental samples and 46 raw fish samples tested positive for L . monocytogenes . Prevalence values for environmental samples varied significantly (P < 0.0001) between the two plants; plant A had a prevalence value of 43.8% (112 of 256 samples), and plant B had a value of 1.2% (3 of 256 samples) . For plant A, 62.5% of drain samples tested positive for L . monocytogenes, compared with 32.3% of samples collected from other environmental sites and 3.1% of samples collected from food contact surfaces . Ribotyping identified 11 subtypes present in the plant environments . Multiple subtypes, including four subtypes not found on any raw fish, were found to persist in plant A throughout the study . Contamination prevalence values for raw fish varied from 3.6% (sablefish) to 29.5% (U.S . West Coast salmon), with an average overall prevalence of 14.6% . Sixteen separate L . monocytogenes subtypes were present on raw fish, including nine that were not found in the plant environment . Our results indicate a disparity between the subtypes found on raw fish and those found in the processing environment . We thus conclude that environmental contamination is largely separate from that of incoming raw materials and includes strains persisting, possibly for years, within the plant . Operational and sanitation procedures appear to have a significant impact on environmental contamination, with both plants having similar prevalence values for raw materials but disparate contamination prevalence values for the environmental sites . We also conclude that regular L . monocyrogenes testing of drains, combined with molecular subtyping of the isolates obtained, allows for efficient monitoring of persistent L . monocytogenes contamination in a processing plant.

South Med J, 2002 Nov, 95(11), 1350 - 2
Intra-abdominal abscess caused by Listeria monocytogenes in a patient with acquired hemolytic anemia and thrombocytopenia; Sile H et al.; We report a case of an intra-abdominal abscess caused by Listeria monocytogenes in a postoperative patient with Evans syndrome (acquired hemolytic anemia and thrombocytopenia) . Focal infections with L . monocytogenes are uncommon but have been reported in immunocompromised patients . A few cases of liver abscess in diabetic patients have also been reported . The present case is significant because of the paucity of previously described focal intra-abdominal infections caused by L . monocytogenes, particularly as a postsplenectomy pathogen.

Toxicol Ind Health, 2001 Jun, 17(5-10), 192 - 209
Evaluation of immunotoxicity induced by single or concurrent exposure to N,N-diethyl-m-toluamide (DEET), pyridostigmine bromide (PYR), and JP-8 jet fuel; Peden-Adam MM et al.; Approximately 5,000 to 80,000 of the US service personnel involved in the Persian Gulf War have complained of a variety of nonspecific symptoms since their return in 1991 . These symptoms have been collectively labeled Gulf War Illness and include muscle fatigue, general malaise, myalgia, impaired cognition, ataxia, headaches, fever, joint pain, skin rash, gastrointestinal disturbances, sleep disturbances, and respiratory difficulties . Exposures of military and service personnel were diverse and included the prescribed anti-nerve gas agent pyridostigmine bromide (PYR), N.N-diethyl-m-toluamide (DEET) insect repellent, and environmental exposures to jet fuel . Thus, studies in our laboratory were undertaken to determine if concurrent exposure to these agents, singly or in combination, would contribute to significant alterations in immunological function and disease susceptibility . To assess immune status, eight-week old B6C3F1 female mice were exposed for 14 days to single compounds or tertiary mixtures of 15.5 mg/kg DEET, 2 mg/kg PYR, and 500 mg/kg JP-8 (termed low dose), or 31 mg/kg DEET, 5 mg/kg PYR, and 1,000 mg/kg JP-8 (termed high dose) . Immunosuppression was assessed 24 h after the last exposure . No remarkable alterations were evident in hematological parameters, spleen and thymus organ weight and total cellularity, natural killer (NK) cell activity, cytotoxic T-cell activity, or mitogen-induced lymphocyte proliferation after exposure to either single or tertiary mixtures at low or high doses . A few changes in CD4/CD8 flow cytometric lymphocyte subpopulations were detected after exposure to the tertiary mixture at the high dose . Delayed type hypersensitivity (DTH) was decreased by 88% after exposure to the high-dose mixture, and suppression of antibody-specific IgM immune responses (plaque-forming cell, PFC) occurred after exposure to all single and tertiary mixtures at both dose levels . In the PFC response, antagonism was apparent in the mixture, while coexposure to these agents resulted in a synergistic effect in the DTH response . Susceptibility to B16F10 tumor or Listeria monocytogenes challenge was not affected after single or tertiary exposures . These data suggest that combined exposure to DEET, PYR, and JP-8 does not profoundly alter many immunological endpoints, but does selectively target functional endpoints such as the PFC and DTH response . This should be considered when assessing human health risks in the military environment.

J Immunol, 2003 Feb 1, 170(3), 1443 - 51
A specific role for B cells in the generation of CD8 T cell memory by recombinant Listeria monocytogenes; Shen H et al.; In this study, we investigated whether B cells play a role in the induction and maintenance of CD8 T cell memory after immunization with an intracellular bacterium, Listeria monocytogenes . Our results show that B cells play a minimal role in the initial activation and Ag-driven expansion of CD8 T lymphocytes . However, absence of B cells results in increased death of activated CD8 T cells during the contraction phase, leading to a lower level of Ag-specific CD8 T cell memory . Once memory is established, B cells are no longer required for the long-term maintenance and rapid recall response of memory CD8 T cells . Increased contraction of Ag-specific CD8 T cells in B cell-deficient mice is not due to impaired CD4 T cell responses since priming of epitope-specific CD4 T cell responses is normal in B cell-deficient mice following L . monocytogenes infection . Furthermore, no exaggerated contraction of Ag-specific CD8 T cells is evident in CD4 knockout mice . Thus, B cells play a specific role in modulating the contraction of CD8 T cell responses following immunization . Elucidation of factors that regulate the death phase may allow us to manipulate this process to increase the level of immunological memory and thus, vaccine efficacy.

Genome Biol . 2003;4(1):R2 . Epub 2002 Dec 23.
A gene-expression program reflecting the innate immune response of cultured intestinal epithelial cells to infection by Listeria monocytogenes; Baldwin DN et al.; BACKGROUND: Listeria monocytogenes is a Gram-positive, facultative, intracellular bacterial pathogen found in soil, which occasionally causes serious food-borne disease in humans . The outcome of an infection is dependent on the state of the infected individual's immune system, neutrophils being key players in clearing the microorganism from the body . The first line of host defense, however, is the intestinal epithelium . RESULTS: We have examined the transcriptional response of cultured human intestinal epithelial cells to infection by L . monocytogenes, which replicates in the host cell cytoplasm and spreads from cell to cell using a form of actin-based motility . We found that the predominant host response to infection was mediated by NFkappaB . To determine whether any host responses were due to recognition of specific virulence factors during infection, we also examined the transcriptional response to two bacterial mutants; actA which is defective in actin-based motility, and prfA, which is defective in the expression of all L . monocytogenes virulence genes . Remarkably, we found no detectable difference in the host transcriptional response to the wild-type and mutant bacteria . CONCLUSIONS: These results suggest that cultured intestinal epithelial cells are capable of mounting and recruiting a powerful innate immune response to L . monocytogenes infection . Our results imply that L . monocytogenes is not specifically detected in the host cytoplasm of Caco-2 cells by intracellular signals . This suggests that entry of bacteria is mediated in the host cell post-translationally, and that these bacteria seek the cytosol not only for the nutrient-rich environment, but also for protection from detection by the immune system.

Hunan Yi Ke Da Xue Xue Bao, 2001 Aug 28, 26(4), 305 - 8
{Listeria monocytogenes induces thymocyte apoptosis in mice}; Chen LY et al.; The murine thymocyte apoptosis induced by Listeria monocytogenes(LM) was detected with morphology, FCM, and DNA electrophoresis . The results were that LM elicited typical morphological changes of thymocyte apoptosis; the typical apoptosis peak was displayed with FCM, and typical "ladder pattern" with agarose gel electrophoresis . The apoptotic cells were found at 8 h after the mice had infected LM and reached climax at 48 h . The thymus weight significantly reduced at 16 h, and reached the lowest at 48 h after the mice had infected LM . The percentage of apoptotic cells was raised with the increasing of LM . These results suggest that LM induces thymocyte apoptosis in dose- and time-dependent manner.

Am J Gastroenterol, 2003 Jan, 98(1), 104 - 11
Safety and steroid-sparing experience using infliximab for Crohn's disease at a pediatric inflammatory bowel disease center; Stephens MC et al.; OBJECTIVES: The published experience using infliximab (Remicade, Centocor, Malvern, PA) for the treatment of pediatric Crohn's disease is limited but suggests utility in the treatment of refractory disease . Experience using infliximab at a large pediatric center is reviewed . METHODS: A retrospective review of all infliximab infusions administered to patients with Crohn's disease (CD) was undertaken . Data were obtained from database and pharmacy records . Chart review and interviews with physicians, patients, and families were used to obtain missing data . RESULTS: A total of 432 infusions were administered to 82 patients (34 female and 48 male) with CD . The number of infusions each patient received ranged from one to 18, with a mean of 5.3 (SD 4.6) and median of 3 . Of 33 patients, 19 (57.6%) became independent and remained free of corticosteroids . There was a statistically significant difference in the steroid dose between 0 and 4 wk and 0 and 8 wk . In all, 23 infusion reactions occurred (5.3%) . Three patients developed herpes zoster, and one developed Listeria monocytogenes meningitis . No patients were documented to have delayed hypersensitivity reactions or malignancies . CONCLUSIONS: Infliximab is safe and effective for treating pediatric patients with CD . A steroid-sparing effect was demonstrated . The most common adverse reaction to infliximab was infusion reaction . These reactions did not preclude further use of the agent . Serious infections were seen in a small number of patients.

Cell, 2002 Dec 13, 111(6), 825 - 36
Structure of internalin, a major invasion protein of Listeria monocytogenes, in complex with its human receptor E-cadherin; Schubert WD et al.; Listeria monocytogenes, a food-borne bacterial pathogen, enters mammalian cells by inducing its own phagocytosis . The listerial protein internalin (InlA) mediates bacterial adhesion and invasion of epithelial cells in the human intestine through specific interaction with its host cell receptor E-cadherin . We present the crystal structures of the functional domain of InlA alone and in a complex with the extracellular, N-terminal domain of human E-cadherin (hEC1) . The leucine rich repeat (LRR) domain of InlA surrounds and specifically recognizes hEC1 . Individual interactions were probed by mutagenesis and analytical ultracentrifugation . These include Pro16 of hEC1, a major determinant for human susceptibility to L . monocytogenes infection that is essential for intermolecular recognition . Our studies reveal the structural basis for host tro-pism of this bacterium and the molecular deception L . monocytogenes employs to exploit the E-cadherin system.

Gene Ther, 2003 Jan, 10(1), 72 - 83
Enhanced cytosolic delivery of plasmid DNA by a sulfhydryl-activatable listeriolysin O/protamine conjugate utilizing cellular reducing potential; Saito G et al.; Listeriolysin O (LLO), a sulfhydryl-activated pore-forming protein from Listeria monocytogenes, was tested and utilized for promoting plasmid DNA (pDNA) delivery into the cytosol of cells in culture . To render pDNA-complexing capability to LLO, the unique cysteine 484 of LLO was conjugated to polycationic peptide protamine (PN) at a 1:1 molar ratio through a reversible, endosome-labile disulfide bond . The sulfhydryl-oxidized LLO construct, LLO-s-s-PN, completely lacked its pore-forming activity, yet regained its original activity upon reduction . The enhanced cytosolic delivery using this construct therefore relies on the requisite reduction of the disulfide bond in LLO-s-s-PN by endogenous cellular reducing capacity . Condensed PN/pDNA complexes incorporating LLO-s-s-PN were tested for their enhanced gene delivery capability monitoring reporter gene expression in HEK293, RAW264.7, P388D1 cell lines and bone-marrow-derived macrophages in the presence of serum . Dramatic enhancement was observed for all tested complexes with varying weight ratios . The effect was most prominent at 0.64-0.80 (w/w) of PN/pDNA upon replacing 1-4% of PN with LLO-s-s-PN, resulting in approximately three orders of magnitude higher luciferase expression compared to PN/pDNA without apparent toxicity . These results demonstrate that incorporation of endosomolytic LLO into pDNA delivery systems in a controlled fashion is a promising approach of enhancing delivery into the cytosol of target cells in gene delivery strategies.

Wei Sheng Yan Jiu, 2000 Jul, 29(4), 246 - 7
{Identification of Listeria species in 167 kinds of foods}; Chen T et al.; Through preliminary identification of isolation of Listeria species in six types foods, which consisted of 167 kinds of foods, we found both S-form and R-form colonies were suspected colonies of Listeria species, and some of them were catalase-negative, urease-positive, methyl red(MR) negative and Voges-Proskauer(VP) negative . The results showed that S-form, catalase-positive, urease-negative, MR-positive and VP-positive colony should not be used as the strict criteria for the identification of Listeria.

J Immunol, 2003 Jan 15, 170(2), 823 - 30
Indoleamine 2,3-dioxygenase is regulated by IFN-gamma in the mouse placenta during Listeria monocytogenes infection; Mackler AM et al.; The tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) is expressed in macrophages that have been differentiated in the presence of CSF-1 and is important in the containment of intracellular pathogens . IDO also appears to play a role in suppression of T cell responses in a variety of contexts . In the placenta, its enzymatic activity is believed to establish a chemical barrier that protects the fetal allograft from T cell-mediated immune aggression . We have studied the regulation of IDO in the utero-placental unit of mice following infection with the Gram-positive, intracellular bacterium Listeria monocytogenes that has a predilection for replication in the decidua basalis . IDO mRNA and protein expression is enhanced in the utero-placental unit following infection with L . monocytogenes . However, in contrast to the human where IDO is expressed by the CSF-1R-positive syncytial trophoblast, IDO is not expressed in murine trophoblastic tissue but instead is found in stromal cells of the decidua basalis and metrial gland and following infection, in endothelial cells . Using mice carrying null mutations in cytokine/growth factor genes, we explored the regulation of IDO in the placenta . Consistent with the absence of CSF-1R expression in the IDO-expressing cells of mice, neither the basal levels of IDO nor its induction following infection is affected by the absence of CSF-1 . However, although the basal level of IDO is normal, the enhanced expression during Listeriosis is completely abrogated in the absence of IFN-gamma, a cytokine required for the resolution of this infection . These data suggest that IDO plays a role in resolving bacterial infection in the placenta while at the same time maintaining a barrier to T cells whose presence might result in fetal rejection.

J Clin Microbiol, 2003 Jan, 41(1), 483 - 5
Antimicrobial activities against 84 Listeria monocytogenes isolates from patients with systemic listeriosis at a comprehensive cancer center (1955-1997); Safdar A et al.; Listeriosis is a serious complication in patients undergoing treatment for cancer . We present antimicrobial susceptibility profiles of 84 clinical Listeria monocytogenes isolates . During 1955 to 1997, in vitro susceptibility for penicillin (97.6%), ampicillin (90.7%), erythromycin (98.8%), tetracycline (96.9%), and gentamicin (98.0%) remained unchanged . All isolates were susceptible to amikacin, ciprofloxacin, imipenem, rifampin, trimethoprim-sulfamethoxazole (TMP-SMX), and vancomycin . High prevalence of clindamycin resistance (96.2%) was unexpected . Ampicillin plus gentamicin is standard therapy for systemic listerosis, and TMP-SMX may be used for patients with beta-lactam intolerance . In vitro susceptibility profiles for carbapenem and fluoronated quinolone are promising, although clinical validation is critically needed before routine use is advocated, especially for listeric patients with severe cellular immune defects.

Biotechnol Bioeng, 2003 Mar 5, 81(5), 618 - 24
Composite surface for blocking bacterial adsorption on protein biochips; Huang TT et al.; The design and fabrication of protein biochips requires characterization of blocking agents that minimize nonspecific binding of proteins or organisms . Nonspecific adsorption of Escherichia coli, Listeria innocua, and Listeria monocytogenes is prevented by bovine serum albumin (BSA) or biotinylated BSA adsorbed on SiO(2) surfaces of a biochip that had been modified with a C(18) coating . Biotinylated BSA forms a protein-based surface that in turn binds streptavidin . Because streptavidin has multiple binding sites for biotin, it in turn anchors other biotinylated proteins, including antibodies . Hence, biotinylated BSA simultaneously serves as a blocking agent and a foundation for binding an interfacing protein, avidin or streptavidin, which in turns anchors biotinylated antibody . In our case, the antibody is C11E9, an IgG-type antibody that binds Listeria spp . Nonspecific adsorption of another bacterium, Escherichia coli, is also minimized due to the blocking action of the BSA . The blocking characteristics of BSA adsorbed on C(18)-derivatized SiO(2) surfaces for construction of a protein biochip for electronic detection of pathogenic organisms is investigated .

Appl Environ Microbiol, 2003 Jan, 69(1), 258 - 66
Attachment of Listeria monocytogenes to radish tissue is dependent upon temperature and flagellar motility; Gorski L et al.; Outbreaks of listeriosis and febrile gastroenteritis have been linked to produce contamination by Listeria monocytogenes . In order to begin to understand the physiology of the organism in a produce habitat, the ability of L . monocytogenes to attach to freshly cut radish tissue was examined . All strains tested had the capacity to attach sufficiently well such that they could not be removed during washing of the radish slices . A screen was developed to identify Tn917-LTV3 mutants that were defective in attachment to radish tissue, and three were characterized . Two of the three mutations were in genes with unknown functions . Both of the unknown genes mapped to a region predicted to contain genes necessary for flagellar export; however, only one of the two insertions caused a motility defect . The third insertion was found to be in an operon encoding a phosphoenolpyruvate-sugar phosphotransferase system . All three mutants were defective in attachment when tested at 30 degrees C; the motility mutant had the most severe phenotype . However, not all of the mutants were defective when tested at other temperatures . These results indicate that L . monocytogenes may use different attachment factors at different temperatures and that temperature should be considered an important variable in studies of the molecular mechanisms of Listeria fitness in complex environments.

Int J Food Microbiol, 2003 Jan 26, 82(1), 87 - 94
A simple method for the differentiation of Listeria monocytogenes based on induction of lecithinase activity by charcoal; Ermolaeva S et al.; The PlcB phospholipase C, or lecithinase, is an important listerial virulence factor of potential use as a pathogenicity marker for Listeria spp . food isolates . However, wild-type strains of Listeria monocytogenes express virulence factors very poorly in vitro and their lecithinase activity is normally difficult to detect on agar plates . We recently reported that the production of listerial virulence factors is strongly induced if L . monocytogenes is grown in the presence of activated charcoal . We report here a simple method for the rapid differentiation of L . monocytogenes from other Listeria spp . based on a comparison of lecithinase reactions in egg yolk agar plates with and without charcoal supplementation . All L . monocytogenes wild-type isolates tested showed a clear induction of lecithinase activity in charcoal-supplemented medium (CEYM), while nonpathogenic Listeria spp . remained negative in CEYM . The animal pathogen L . ivanovii was easily differentiated from L . monocytogenes because it showed a strong lecithinase reaction independently of the presence or absence of charcoal in the medium.

J Infect, 2003 Jan, 46(1), 70 - 1
Listeria monocytogenes meningitis in a patient with chronic hepatitis C infection, treated by interferon alfa and ribavirin; Vander T et al.; We present a patient with chronic hepatitis C infection without cirrhosis, treated by interferon alfa and ribavirin, who developed listeria monocytogenes meningitis . The possible pathophysiological association is discussed, along with therapeutic implications.

J Gen Appl Microbiol, 1998 Jun, 44(3), 183 - 191
Physiological and biochemical aspects of the acid survival of Listeria monocytogenes; Phan-Thanh L; The physiological aspects of the response to acidic conditions and the correlated protein synthesis were studied by using Listeria monocytogenes grown in a chemically defined synthetic medium . This growth was greatly affected by pH of the medium . It decreased when pH declined and was arrested at pH 4 . When pH went under 4, the bacteria began to die . If the bacteria had been adapted to an intermediary sublethal pH before imposition of lethal pH stress, they would have resisted better lethal pH . A prolonged treatment at intermediary pH, however, rendered the bacteria more sensitive to subsequent lethal pH . Organic volatile acids exerted a more deleterious effect on L . monocytogenes than inorganic acids at the same stressing pH . The acquired acid tolerance was conserved after several weeks of storage of the adapted bacteria at 4 degrees C . Acid stress and acid adaptation (tolerance) affected the synthesis patterns of bacterial proteins: Many proteins were repressed and several others increased in expression level . These acid-induced proteins were separated by two-dimensional (2D-) electrophoresis and analyzed by a computer-aided 2D-gel analysis system . The results obtained suggested that acid tolerance and acid stress responses require the synthesis of a certain number of shared proteins and that additional acid-induced proteins are needed when the bacteria must face more severe acidic pH.

Infect Immun, 2003 Jan, 71(1), 234 - 41
Seeligeriolysin O, a cholesterol-dependent cytolysin of Listeria seeligeri, induces gamma interferon from spleen cells of mice; Ito Y et al.; Seeligeriolysin O (LSO), one of the cholesterol-dependent cytolysins produced by Listeria seeligeri, shows 80% homology to listeriolysin O (LLO) produced by Listeria monocytogenes at the amino acid sequence level . In addition to cytolytic activity, LLO has been shown to exhibit cytokine-inducing activity . In order to determine whether LSO is also capable of exhibiting these two different activities, we constructed a recombinant full-length LSO (rLSO530) and a noncytolytic truncated derivative with a C-terminal deletion (rLSO483) and compared these molecules with recombinant LLO . The cytolytic rLSO530 molecule could induce gamma interferon (IFN-gamma) production in spleen cells when the cytolytic activity was blocked by treatment with cholesterol . The noncytolytic truncated rLSO483 molecule also induced IFN-gamma production . Anti-LLO polyclonal antibody inhibited not only LLO-induced IFN-gamma production but also LSO-induced IFN-gamma production . Both NK cells and CD11b(+) cells were required for LSO-induced IFN-gamma production . Among the various cytokines expressed in CD11b(+) cells, interleukin-12 (IL-12) and IL-18 appeared to be essential . We concluded that LSO exhibits the same biological activity as LLO.

Infect Immun, 2003 Jan, 71(1), 95 - 100
Antibodies present in normal human serum inhibit invasion of human brain microvascular endothelial cells by Listeria monocytogenes; Hertzig T et al.; Listeria monocytogenes causes meningitis and encephalitis in humans and crosses the blood-brain barrier by yet unknown mechanisms . The interaction of the bacteria with different types of endothelial cells was recently analyzed, and it was shown that invasion into, but not adhesion to, human brain microvascular endothelial cells (HBMEC) depends on the product of the inlB gene, the surface molecule InlB, which is a member of the internalin multigene family . In the present study we analyzed the role of the medium composition in the interaction of L . monocytogenes with HBMEC, and we show that invasion of HBMEC is strongly inhibited in the presence of adult human serum . The strong inhibitory activity, which is not present in fetal calf serum, does not inhibit uptake by macrophage-like J774 cells but does also inhibit invasion of Caco-2 epithelial cells . The inhibitory component of human serum was identified as being associated with L . monocytogenes-specific antibodies present in the human serum . Human newborn serum (cord serum) shows only a weak inhibitory activity on the invasion of HBMEC by L . monocytogenes.

Avian Dis, 2002 Oct-Dec, 46(4), 1051 - 4
Unidentified coryneform bacterial strain from cases of polyarthritis in chickens: phenotype and fatty acid profile; Mohan K et al.; We report isolation of a strain of fermentative coryneform bacteria from an outbreak of polyarthritis in chickens . This strain could not be assigned to any recognized bacterial taxon because its peculiar phenotype is not yet reported . The strain possessed phenotypic characteristics and fatty acid profile similar to Erysipelothrix but, on the other hand, exhibited temperature-dependent motility like Listeria . We found no evidence of either Mycoplasma synoviae or Chlamydia infection . Details of the phenotype and fatty acid profile of the isolate and measures undertaken to contain the outbreak have been described.

J Food Prot, 2002 Dec, 65(12), 1981 - 3
Use of vacuum-steam-vacuum and ionizing radiation to eliminate Listeria innocua from ham; Sommers C et al.; Listeria spp . are a frequent postprocess contaminant of ready-to-eat (RTE) meat products, including ham . Vacuum-steam-vacuum (VSV) technology has been used successfully to eliminate Listeria innocua from hot dogs . Ionizing radiation can eliminate Listeria spp . from RTE meats . However, the excessive application of either technology can cause changes in product quality, including structural changes, changes in cure color (redness), and lipid oxidation . In this study, two cycles of VSV were combined with 2.0 kGy of ionizing radiation to obtain 4.40- and 4.85-log10 reductions of L . innocua on ham meat and skin, respectively . The use of both treatments resulted in an additive, as opposed to synergistic, reduction of L . innocua on ham . The combination treatment did not cause statistically significant changes in product structure, color (redness), or lipid oxidation.

J Food Prot, 2002 Dec, 65(12), 1888 - 93
Incidence and susceptibility to antimicrobial agents of Listeria spp . and Listeria monocytogenes isolated from poultry carcasses in Porto, Portugal; Antunes P et al.; The occurrence of Listeria spp . and Listeria monocytogenes in 63 samples of Portuguese poultry carcasses obtained from two local butcher shops and one canteen in the city of Porto, Portugal, and the susceptibility of these bacteria to antimicrobial agents allowed for use in human or animal therapeutics were evaluated . All poultry samples were contaminated with Listeria spp., and L . monocytogenes was isolated from 41% (26 of 63) of the samples . Other Listeria species, including L . innocua, L . welshimeri, and L . seeligeri, were also isolated from poultry samples . A multiplex polymerase chain reaction method was used for the identification of all of the Listeria isolates; this method showed total conformity with the conventional method of biochemical identification and proved to be more reliable, faster, and less arduous . In addition, high percentages of Listeria spp . (84%) and L . monocytogenes (73%) isolates were found to be resistant to one or more antimicrobial agents of different groups, and 12 different resistance profiles were recorded . The frequency of the resistance of L . monocytogenes isolates to enrofloxacin and clindamycin is notable . The results of this study suggest a high incidence of L . monocytogenes on Portuguese poultry products available for consumers and indicate that poultry could be a potential vehicle of foodborne infections due to strains of L . monocytogenes that are resistant to antimicrobial agents.

J Appl Microbiol, 2003, 94(1), 48 - 53
Cold and carbon dioxide used as multi-hurdle preservation do not induce appearance of viable but non-culturable Listeria monocytogenes; Li J et al.; AIMS: To study whether the exposure to cold (4 degrees C) and carbon dioxide which results in the elongation of Listeria cells, induces a viable but nonculturable (VBNC) state . METHODS AND RESULTS: When cold and CO2 stressed L . monocytogenes were observed under a fluorescence microscope, using the LIVE/DEAD BacLight bacteria viability kit (Molecular Probes, Eugene, OR, USA), the healthy, mildly injured, and the putative VBNC cells accounted for 31.0% of the stressed cell population . By using the selective plate count, 31.4% of the same stressed cell population was found to be healthy and mildly injured (putative VBNC cells not included) . If there were VBNC state cells present, we should have observed a significant difference between the above two numbers . In fact, there was no significant difference between the results obtained from those two methods . CONCLUSIONS: There were no VBNC state cells observed in the stressed cell population . We conclude that cold and CO2 do not induce L . monocytogenes to enter a VBNC state . SIGNIFICANCE AND IMPACT OF THE STUDY: Cold and modified atmospheres are widely used in fresh muscle food and fruit preservation . Whether they would induce L . monocytogenes into a VBNC state is of a great concern for microbial food safety.

J Biol Chem, 2003 Mar 7, 278(10), 7783 - 9 Epub 2002 Dec 17.
Multiple regions of internalin B contribute to its ability to turn on the Ras-mitogen-activated protein kinase pathway; Copp J et al.; Internalin B (InlB) is a protein present on the surface of Listeria monocytogenes that mediates bacterial entry into mammalian cells . It is thought that InlB acts by binding directly to the hepatocyte growth factor (HGF) receptor, present on the surface of host cells . Binding of InlB to the HGF receptor results in mitogen-activated protein (MAP) kinase and phosphoinositide 3-kinase activation, followed by changes in the organization of the actin cytoskeleton . Here we have compared signaling by HGF and InlB . Whereas stimulation with equivalent concentrations of HGF and InlB elicits similar activation of the HGF receptor, we observed striking differences in downstream activation of MAP kinase . InlB leads to a greater activation of the Ras-MAP kinase pathway than does HGF . The leucine-rich repeat region, which was previously shown to be sufficient for binding and activation of the HGF receptor, lacks the ability to super-activate the Ras-MAP kinase pathway . Analysis of a series of deletion mutants suggests that it is the B repeat region between the leucine-rich repeat and GW domains that endows InlB with an increased ability to turn on the Ras-MAP kinase pathway . These unexpected observations suggest that HGF and InlB use alternative mechanisms to turn on cellular signaling pathways.

Biophys Chem, 2002 Dec 10, 101-102, 347 - 58
Listeria monocytogenes phosphatidylinositol-specific phospholipase C: activation and allostery; Ryan M et al.; The animal and human pathogen Listeria monocytogenes secretes several virulence factors, including a phosphatidylinositol-specific phospholipase C (PI-PLC) . Sufficient quantities of L . monocytogenes PI-PLC for biophysical studies were obtained by overexpression of the enzyme in Escherichia coli . The purified PI-PLC was examined in enzyme kinetics experiments using a new fluorogenic substrate, methyl-FLIP . Methyl-FLIP is a water-soluble monomeric substrate cleaved in a manner similar to the natural aggregate substrate, phosphatidylinositol (PI) . Michaelis-Menten kinetics were observed with K(M) = 61 +/- 7 microM and V(max) = 120 +/- 5 micromol min(-1) mg(-1), corresponding to k(cat) = 66+/-3 s(-1) . The catalysis is activated by the addition of a short-chain phospholipid, dihexanoyl phosphatidylcholine (diC(6)PC) . The kinetics were fitted to a two-site model in which the substrate binds to the active site and diC(6)PC binds to a second site, with an interaction between the two sites . The result is a decrease in K(M) and an increase in V(max), producing an overall four to five-fold increase in catalytic efficiency (k(cat)/K(M)) . The interaction is not a regulatory mechanism, as is the case for multimeric enzymes; rather, it suggests interfacial cooperativity between the active site and a lipid-binding subsite, presumably adjacent to the active site .

Intern Med, 2002 Nov, 41(11), 1073 - 8
Successful treatment of listerial brain abscess: a case report and literature review; Maezawa Y et al.; Listerial brain abscess is extremely rare; only two cases have been reported in Japan . We encountered a female patient with immunoblastic lymphadenopathy, who developed listerial brain abscess after 8 years of treatment with antineoplastic agents and corticosteroids . Brain MRI revealed multiple space occupying lesions, suggesting abscesses which were possibly caused by hematogenous spread of the bacteria . Immediate blood culture enabled early diagnosis, and she entered into complete remission with high-dose ampicillin . Blood culture and brain imaging seem to play a crucial role in making an early diagnosis, and the administration of high dose of antibiotics is recommended for improvement of this disease.

J Exp Med, 2002 Dec 16, 196(12), 1585 - 92
Regulatory CD4+CD25+ T cells restrict memory CD8+ T cell responses; Kursar M et al.; CD4+ T cell help is important for the generation of CD8+ T cell responses . We used depleting anti-CD4 mAb to analyze the role of CD4+ T cells for memory CD8+ T cell responses after secondary infection of mice with the intracellular bacterium Listeria monocytogenes, or after boost immunization by specific peptide or DNA vaccination . Surprisingly, anti-CD4 mAb treatment during secondary CD8+ T cell responses markedly enlarged the population size of antigen-specific CD8+ T cells . After boost immunization with peptide or DNA, this effect was particularly profound, and antigen-specific CD8+ T cell populations were enlarged at least 10-fold . In terms of cytokine production and cytotoxicity, the enlarged CD8+ T cell population consisted of functional effector T cells . In depletion and transfer experiments, the suppressive function could be ascribed to CD4+CD25+ T cells . Our results demonstrate that CD4+ T cells control the CD8+ T cell response in two directions . Initially, they promote the generation of a CD8+ T cell responses and later they restrain the strength of the CD8+ T cell memory response . Down-modulation of CD8+ T cell responses during infection could prevent harmful consequences after eradication of the pathogen.

Int J Food Microbiol, 2003 Mar 25, 81(3), 241 - 8
Occurrence of Listeria spp . in critical control points and the environment of Minas Frescal cheese processing; Silva IM et al.; Critical control points (CCPs) associated with Minas Frescal cheese (a Brazilian soft white cheese, eaten fresh) processing in two dairy factories were determined using flow diagrams and microbiological tests for detection of Listeria monocytogenes and other species of Listeria . A total of 218 samples were collected along the production line and environment . The CCPs identified were reception of raw milk, pasteurization, coagulation and storage . Thirteen samples were positive for Listeria; 9 samples were Listeria innocua, 2 were Listeria grayi and 2 were L . monocytogenes . In factory A, Listeria was found in 50% of raw milk samples, 33.3% of curd samples, 16.7% of pasteurized milk samples, 16.7% of cheese samples and 25% of rubber pipes used to transport the whey . The microorganism was not obtained from environmental samples in this plant . In factory B, Listeria was found in one sample of raw milk (16.7%) and in three samples of environment (17.6%) and L . monocytogenes was obtained from raw milk (16.7%) and the floor of the cheese refrigeration room (14.3%) . Two serotypes, 4b and 1/2a, were observed among the strains of L . monocytogenes isolated, both which are frequently involved in outbreaks of food-borne listeriosis and sporadic cases of the disease all over the world.

Symp Ser Soc Appl Microbiol, 2002, (31), 111S - 120S
Biocide use in the food industry and the disinfectant resistance of persistent strains of Listeria monocytogenes and Escherichia coli; Holah JT et al.; AIMS: The aims of the project were threefold: to survey the use of disinfectants in the UK food industry; to assess the product and environmental microflora of selected food factories for the persistence of Listeria monocytogenes and Escherichia coli; and to determine the disinfectant resistance of any persistent strains . METHODS AND RESULTS: A survey of the use of disinfectants in the UK food industry was undertaken in which a total of 40 sites were visited and a further 77 postal questionnaires were returned from farms, food manufacture, food transport and food retail sites . Quaternary ammonium compounds (QACs) were predominantly used, applied in small volumes as a mist . Approximately 30,000 samples from the product and environment of five chilled food factories were examined for L . monocytogenes and E . coli over a 3 year period . A total of 181 L . monocytogenes and 176 E . coli isolates were ribotyped to yield 19 and 34 ribogroups, respectively . Some strains were isolated only from the product, a number only from the environment and others from both niches . Some strains were seen to be persistent for the duration of the sampling exercise (2-3 years) . The most common L . monocytogenes and E . coli strains, together with two environmental L . monocytogenes strains, were assessed for any resistance to commercial disinfectants as compared with a laboratory L . monocytogenes disinfectant testing strain . The resistance of the L . monocytogenes and E . coli strains isolated from the factory were not significantly different from the laboratory control strain . CONCLUSIONS: Persistent strains of L . monocytogenes and E . coli are found in the UK food industry, though this persistence is not related to their increased susceptibility to the most commonly used disinfectants . SIGNIFICANCE AND IMPACT OF THE STUDY: The concept of a persistent microflora in food factories will have an impact on the future selection of suitable control options, including the use of biocides.

Brain Behav Immun, 2002 Dec, 16(6), 654 - 62
Chemical sympathectomy increases numbers of inflammatory cells in the peritoneum early in murine listeriosis; Rice PA et al.; Here, we investigated the effects of sympathectomy on systemic bacterial loads following infection with Listeria monocytogenes, and on innate and specific immune responses in the peritoneum . Sympathectomy decreased systemic bacterial loads, and increased the number of peritoneal leukocytes and the percentage of peritoneal macrophages three days postinfection . This suggests that sympathectomy-induced decreases systemic bacterial loads are associated with increased recruitment of inflammatory cells into tissues during the innate immune response.

Trends Cell Biol, 2003 Jan, 13(1), 23 - 31
Invasion of mammalian cells by Listeria monocytogenes: functional mimicry to subvert cellular functions; Cossart P et al.; The bacterium Listeria monocytogenes has the unusual capacity to enter and to multiply in nonphagocytic cells . Bacterially induced phagocytosis is triggered mainly by the two surface proteins internalin (also called InlA) and InlB, which interact with host cell receptors and either mimic or act in place of the normal cellular ligands . Internalin interacts specifically with human E-cadherin, whereas InlB activates the tyrosine kinase receptor Met and also interacts with the ubiquitous receptor gC1qR and proteoglycans . Signals induced by crosstalk between the bacterium and the host cell allow internalization, which is a prelude to intracellular multiplication, actin-based movement and spread of the bacterium from cell to cell . Manipulating the bacterial invasion proteins offers us an unprecedented tool with which to understand the complex phenomenon of phagocytosis.

Am J Clin Oncol, 2002 Dec, 25(6), 576 - 9
CNS listeriosis confused with leptomeningeal carcinomatosis in a patient with a malignant insulinoma; Mileshkin L et al.; We describe a case of presumed listeria monocytogenes rhomboencephalitis, which was initially confused with leptomeningeal carcinomatosis in a patient with a malignant carcinoid tumor . Long-term corticosteroid treatment and immunosuppression caused by malignancy predisposed the patient to developing listeriosis . The clinical and radiologic features of this illustrative case are described . Listeriosis is an important treatable differential diagnosis in patients with malignancy presenting with neurologic signs.

Scand J Infect Dis, 2002, 34(10), 735 - 41
Listeriosis in Iceland, 1978-2000: a description of cases and molecular epidemiology; Hjaltested EK et al.; The purpose of our study was to review all cases of listeriosis in Iceland during the period 1978-2000 and to analyse the genetic relatedness of their isolates . Case records of all patients in Iceland with listeriosis during the period were reviewed and the isolates compared using serotyping and pulsed-field gel electrophoresis (PFGE) using SmaI, AseI and ApaI restriction enzymes . Forty cases of listeriosis were diagnosed during the period, resulting in a mean annual incidence of 6.9 cases per million and a case fatality rate of 33% . In the first 5 y of the study only serotype 4b was observed; subsequently serotypes 1/2a and 1/2b appeared and serotype 4b declined in prevalence . PFGE yielded 24 different genotypes with 7 clusters of indistinguishable genotypes, each comprising 2-6 cases . During 1992-95 the annual incidence of listeriosis in Iceland rose to 15 cases per million . This was largely due to 2 clusters, 1 of 3 cases and the other of 6 . No cases of listeriosis were diagnosed during 1998-2000 . Our data show an increased number of cases within clusters in the latter half of the period . At the same time, food processing and distribution has become increasingly centralized in Iceland, suggesting an increased risk of listeriosis outbreaks.

Medicina (Kaunas), 2002, 38(9), 910 - 5
{Effects of aluminum ions on the resistance of mice to experimental Listeria monocytogenes infection}; Cerkasinas G et al.; This study presents evaluation of effects of aluminium ions on the development of experimental infection induced by the injection of Listeria monocytogenes bacteria into growing mice . We show that single exposure of mice to 0.05 LD50 or 0.5 LD50 of aluminium ions does not affect the accumulation of bacteria in liver and spleen of experimental animals . Long-term exposure of mice to aluminium ions (injection of 0.05 LD50 Al3+ every 3 days for totally 6 weeks) did not slow down the increase in weight of animals while in the infected animals aluminium significantly reduced their growth . Animals subjected to chronic aluminium exposure have shown lower accumulation of bacteria in liver 24 h after the initiation of experimental infection as compared to the mice after single injection of 0.05 LD50 Al3+ . Also, long-term exposure to aluminium causes more complicated development of experimental infection, which was evidenced by the decreased survival of aluminium-treated animals (77% compared to 83% in control group) as well as by the increase in the fraction of animals carrying infection after 6 weeks (36% versus 5% in control group) . In addition, aluminium-exposed animals had significantly lower blood serum agglutination titer to the listeria, and decrease in the delayed type of hypersensitivity to the listeria antigens . Results, presented here, indicate that the long-term exposure to low aluminium doses activates the antibacterial defence in experimental animals while the specific immunity becomes suppressed.

Medicina (Kaunas), 2002, 38(6), 650 - 4
{Effects of mercury on the course of Listeria monocytogenes infection in mice}; Cerkasinas G et al.; Effects of mercury ions on the colonization of mice organs by Listeria monocytogenes were investigated . It was found that single injection of 0.05 LD50 mercury ions has little effect on listeria spreading in mice internal organs . Dissemination of bacteria in the liver of mice, which received 0.5 LD50, was similar to that one in control animals, which received physiologic solution, while higher numbers of bacteria were found in spleen of experimental animals at later stages of infection (after 24 and 48 h), when inflammation processes begin to take place . Chronic exposure to 0.05 LD50 mercury ions for 6 weeks caused decrease in body weight and survival of mice as well as increase in the infection degree of their internal organs . In addition, we demonstrate the mercury ions results in decreased synthesis of specific antibodies against listeria proteins.

Cell Immunol, 2002 Jul-Aug, 218(1-2), 59 - 73
Adjuvanticity of an IL-12 fusion protein expressed by recombinant deltaG-vesicular stomatitis virus; Klas SD et al.; The remarkable immunomodulatory and adjuvant properties of rIL-12 have been well described . Many early studies documenting the adjuvanticity of IL-12 were performed using the murine model of Listeria monocytogenes infection . In this report, we describe the construction of an attenuated recombinant vesicular stomatitis virus (VSV-deltaG) that encodes a single-chain IL-12 fusion protein (IL-12F), and the use of this virus as an expression vector to produce large quantities of IL-12F . VSV-expressed IL-12F (vIL-12F) was then co-administered to mice along with a poorly immunogenic listerial antigen preparation as a vaccine regimen and the resulting immune responses were monitored . The vIL-12F was found to have adjuvant properties similar to those observed for rIL-12 . Co-administration of vIL-12F and listerial antigen elicited powerful cell-mediated immune responses that conferred long-lived protective listerial immunity . These studies demonstrated that VSVdeltaG-IL12F-infected cells secrete bioactive single-chain IL-12, and laid the foundation for studies using VSVdeltaG-IL12F as a vector for delivery of IL-12F in vivo.

Curr Drug Targets Infect Disord, 2002 Sep, 2(3), 247 - 64
The role of the cytoskeleton in the life cycle of viruses and intracellular bacteria: tracks, motors, and polymerization machines; Bearer EL et al.; Recent advances in microbiology implicate the cytoskeleton in the life cycle of some pathogens, such as intracellular bacteria, Rickettsia and viruses . The cellular cytoskeleton provides the basis for intracellular movements such as those that transport the pathogen to and from the cell surface to the nuclear region, or those that produce cortical protrusions that project the pathogen outwards from the cell surface towards an adjacent cell . Transport in both directions within the neuron is required for pathogens such as the herpesviruses to travel to and from the nucleus and perinuclear region where replication takes place . This trafficking is likely to depend on cellular motors moving on a combination of microtubule and actin filament tracks . Recently, Bearer et al . reconstituted retrograde transport of herpes simplex virus (HSV) in the giant axon of the squid . These studies identified the tegument proteins as the viral proteins most likely to recruit retrograde motors for the transport of HSV to the neuronal nucleus . Similar microtubule-based intracellular movements are part of the biological behavior of vaccinia, a poxvirus, and of adenovirus . Pathogen-induced surface projections and motility within the cortical cytoplasm also play a role in the life cycle of intracellular pathogens . Such motility is driven by pathogen-mediated actin polymerization . Virulence depends on this actin-based motility, since virulence is reduced in Listeria ActA mutants that lack the ability to recruit Arp2/3 and polymerize actin and in vaccinia virus mutants that cannot stimulate actin polymerization . Inhibition of intracellular movements provides a potential strategy to limit pathogenicity . The host cell motors and tracks, as well as the pathogen factors that interact with them, are potential targets for novel antimicrobial therapy.

Lett Appl Microbiol, 2002, 35(6), 538 - 42
Influence of interactions between temperature, ferric ammonium citrate and glycine betaine on the growth of Listeria monocytogenes in a defined medium; Dykes GA et al.; AIMS: To investigate interactions, if any, between temperature, ferric ammonium citrate and glycine betaine on the growth of Listeria monocytogenes in modified Pine's medium (Pine et al . 1989) . METHODS AND RESULTS: Modified Pine's medium containing 0, 0.044, 0.088 or 0.176 g l(-1) ferric ammonium citrate, and 0 or 1 mM glycine betaine, was inoculated with each of two L . monocytogenes strains and incubated at 4, 25 or 37 degrees C . The optical density at 600 nm, and cell numbers, were determined at appropriate time intervals . At 4 degrees C, but not other temperatures, increasing ferric ammonium citrate resulted in improved growth in the absence, but not the presence, of glycine betaine . The presence of glycine betaine was inhibitory at 25 and 37 degrees C, but not at 4 degrees C . CONCLUSIONS: Interactions affecting the growth kinetics of L . monocytogenes were apparent between the parameters investigated . SIGNIFICANCE AND IMPACT OF THE STUDY: Limitations on the use of modified Pine's medium, and the significance of iron metabolism at lower temperatures, were revealed.

Lett Appl Microbiol, 2002, 35(6), 513 - 7
Detection of single nucleotide polymorphisms within the Listeria genus using an 'asymmetric' fluorogenic probe set and fluorescence resonance energy transfer based-PCR; Koo K et al.; AIMS: We describe a novel and inexpensive fluorescence energy transfer (FRET)-based PCR protocol to distinguish single nucleotide polymorphisms (SNPs) within the genus Listeria . METHODS AND RESULTS: Sequence information for the 16S rRNA gene of representative Listeria species was used to design genus-specific primers and two species-specific probes that differed in sequence by one single nucleotide . The probes were 5' labelled with either fluorescein or Texas Red, quenched with a shorter yet complementary 3' dimethyl-amino-phenyloazo benzoic acid (DABCYL) labelled oligonucleotide, and then incorporated into a previously reported 'asymmetric' FRET-based PCR detection protocol . CONCLUSIONS: Listeria monocytogenes could be readily distinguished from other members of the Listeria genus after PCR amplification and measurement of endpoint fluorescence at two different wavelengths . SIGNIFICANCE AND IMPACT OF THE STUDY: The relatively low cost and high flexibility of this system will benefit laboratories in their efforts to develop rapid and specific methods to detect minor sequence differences between related microorganisms.

Lett Appl Microbiol, 2002, 35(6), 489 - 93
Environmental factors influencing the inactivation of Listeria monocytogenes by pulsed electric fields; Alvarez I et al.; AIMS: To investigate the influence of the growth phase, growth temperature, storage time, pH and aw of the treatment medium on the resistance of Listeria monocytogenes to pulsed electric fields (PEF) . METHODS AND RESULTS: Square wave pulses of 2 micros at a frequency of 1 Hz and 25 and 28 kV cm(-1) were used . Cells were more PEF resistant in the stationary than in the exponential phase at both incubation temperatures investigated (4 and 35 degrees C) . Cells grown at 4 degrees C were more PEF sensitive than cells grown at 35 degrees C independent of the growth phase . After a treatment of 25 kV cm(-1) and 800 micros, 1.48, 3.86 and 5.09 log10 cycles of inactivation were obtained at pH 7.0, 5.4 and 3.8, respectively . A reduction in the aw of the treatment medium protected cells against PEF treatments . CONCLUSIONS: The PEF resistance of L . monocytogenes depended on different environmental factors . The influence of growth conditions and treatment medium characteristics should be known and controlled to obtain reproducible and reliable PEF inactivation data . SIGNIFICANCE AND IMPACT OF THE STUDY: Erroneous conclusions and misinterpretation of results are possible if factors affecting the PEF resistance of L . monocytogenes are not considered during PEF inactivation studies.

Rev Clin Esp, 2002 Dec, 202(12), 638 - 43
{Listeria monocytogenes meningitis in the adult in Spain . Report of 10 cases and review of the literature}; Alcoba Leza M et al.; INTRODUCTION: Ten new cases of Listeria monocytogenes meningitis (LMM) in the adult are here reported . Also, a review is made of reported cases by Spanish authors in the last 30 years, with an analysis of the predisposing conditions and mortality rate from this type of bacterial meningitis (BM) throughout the study period . METHODS: The reported cases met two criteria: CSF biochemistry consistent with BM and positive CSF and/or blood culture for Listeria monocytogenes . The bibliographic search of previously reported cases was made through Medline . Cases were divided into two periods: from 1974 to 1988, and from 1989 onwards . Predisposing conditions, therapy, and mortality were analyzed and compared between the two study periods . RESULTS: The features of the cases reported here did not differ from those in the cases reported so far, with the single feature of two HIV-positive patients . Of the analyzed cases, 73% had some predisposing condition . Thirty-eight and 39% of the total of patients and of patients with some predisposing conditions, respectively, died, with a statistically significant difference (p < 0.001) between those with and without predisposing conditions . Among the treated patients, 87% received either ampicillin or penicillin and the mortality rate among these patients was 30% . No differences regarding mortality was rate observed between the two study periods, the total of cases of between those treated with the aforementioned antibiotics . CONCLUSIONS: Patients with LMM commonly have a predisposing condition . The mortality rate in this type of BM is still high among those treated with ampicillin or penicillin, and a decrease in the mortality rate was not observed when the reported cases in Spain in the last 30 years were analyzed . The optimal therapy for this condition is still to be defined.

Int J Food Microbiol, 2003 Mar 15, 81(2), 123 - 9
The development of an efficient and rapid enzyme linked fluorescent assay method for the detection of Listeria spp . from foods; Sewell AM et al.; Conventional isolation methods, including the Health Products and Food Branch (HPFB), Health Canada method used for the isolation and identification of Listeria species and Listeria monocytogenes from foods and environmental samples, can take a week or more to complete and are usually labor-intensive . This has led to the development of various rapid methods which attempt to generate results comparable to standard methods but in a reduced time-frame with less hands-on operation . Our previous work with rapid detection systems indicated that the recommended enrichment protocols failed to grow the Listeria to detectable levels in a reliable and consistent manner.In the present study, a novel enrichment protocol is described and consists of samples being pre-enriched in Palcam broth (incubated at 35 degrees C for 26 h), enriched in UVM 2 (30 degrees C for 26 h) and then plated and analysed by a rapid detection kit, with results being generated after 52 h of incubation.In total, 200 naturally contaminated samples were analysed by both the HPFB standard method and the Palcam method . The results showed that the Palcam method is comparable to the HPFB method . Further analysis involved a rapid detection system, which applies ELISA techniques and automation in an enzyme-linked fluorescent assay (ELFA) system . This system, referred to as the Vitek Immuno Diagnostic Assay System or VIDAS, can identify Listeria to the genus or species (L . monocytogenes) level.In this comparison, an additional 324 naturally contaminated samples were analysed by both the Palcam and ELFA methods . The sensitivity and specificity of the ELFA method were 98.1% and 97.0%, respectively, while the efficiency was 97.5% . False-negative and false-positive rates were 1.9% and 3.0%, respectively . These results show that the ELFA method (when using the Palcam method for pre- and secondary enrichment) was efficient and gave reliable results after 52 h of incubation, and met Health Canada's criteria for approval as a rapid method.

Int J Food Microbiol, 2003 Mar 15, 81(2), 87 - 104
Estimation of uncertainty and variability in bacterial growth using Bayesian inference . Application to Listeria monocytogenes; Pouillot R et al.; The usefulness of risk assessment is limited by its ability or inability to model and evaluate risk uncertainty and variability separately . A key factor of variability and uncertainty in microbial risk assessment could be growth variability between strains and growth model parameter uncertainty . In this paper, we propose a Bayesian procedure for growth parameter estimation which makes it possible to separate these two components by means of hyperparameters . This model incorporates in a single step the logistic equation with delay as a primary growth model and the cardinal temperature equation as a secondary growth model . The estimation of Listeria monocytogenes growth parameters in milk using literature data is proposed as a detailed application . While this model should be applied on genuine data, it is highlighted that the proposed approach may be convenient for estimating the variability and uncertainty of growth parameters separately, using a complete predictive microbiology model.

J Appl Microbiol, 1997 Mar, 82(3), 345 - 50
Modelling the growth, survival and death of Listeria monocytogenes; Membre JM et al.; In this paper, the predictive microbiology approach has been generalized to the study of growth, survival and death of Listeria monocytogenes . As this micro-organism is involved in food poisoning, its growth, survival and death were studied as functions of low temperatures, NaCl and phenol compounds, in a synthetic medium, by a factorially designed experiment . A significant inactivation of L . monocytogenes was obtained with 20 ppm of phenol and 4% (w/v) NaCl at temperatures from 4 to 12 degrees C . An empirical model is proposed to describe, in a single step, the biomass profile vs studied factors . Thereby, the influence of temperature, NaCl and phenol concentration on L . monocytogenes biomass quantity (0.5-8 log cfu ml(-1)) are presented as a function of storage duration . The comparisons of the proposed model with existing models (Gompertz for growth, vitalistic for survival and death) were performed . The use of a single equation allows the prediction of contamination levels in all experimental conditions without knowledge a priori . The model offers considerable prospects for its use in food microbiology.

Poult Sci, 2002 Nov, 81(11), 1751 - 7
Quality improvement of kosher chilled poultry; Zuckerman H et al.; Pathogens like Listeria monocytogenes are of great concern in the poultry industry . Poultry, which are subjected to the kosher slaughtering and koshering process, may introduce even higher risks than conventially slaughtered poultry due to the potential for microbial cross-contamination at several critical points in the koshering line, particularly in the chillers . The effect of Microgard (MIC) and Nisin (NIS) on reducing total microbial counts, inhibiting L . monocytogenes, and prolonging shelf life was evaluated . In this work we applied dips of poultry into solutions of NIS, active against Gram-positive bacteria, and MIC, which is a bacteriocin mixture that retards growth of Gram-negative bacteria, and a mixture of organic acids . These treatments inhibited both inoculated and naturally occurring L . monocytogenes on poultry, and increased shelf life, at 6 C, from 2 to 4 d (end of shelf life was considered when total aerobic counts reached 7 log cfu/g).

J Clin Microbiol, 2002 Dec, 40(12), 4720 - 8
Identification of Listeria species by microarray-based assay; Volokhov D et al.; We have developed a rapid microarray-based assay for the reliable detection and discrimination of six species of the Listeria genus: L . monocytogenes, L . ivanovii, L . innocua, L . welshimeri, L . seeligeri, and L . grayi . The approach used in this study involves one-tube multiplex PCR amplification of six target bacterial virulence factor genes (iap, hly, inlB, plcA, plcB, and clpE), synthesis of fluorescently labeled single-stranded DNA, and hybridization to the multiple individual oligonucleotide probes specific for each Listeria species and immobilized on a glass surface . Results of the microarray analysis of 53 reference and clinical isolates of Listeria spp . demonstrated that this method allowed unambiguous identification of all six Listeria species based on sequence differences in the iap gene . Another virulence factor gene, hly, was used for detection and genotyping all L . monocytogenes, all L . ivanovii, and 8 of 11 L . seeligeri isolates . Other members of the genus Listeria and three L . seeligeri isolates did not contain the hly gene . There was complete agreement between the results of genotyping based on the hly and iap gene sequences . All L . monocytogenes isolates were found to be positive for the inlB, plcA, plcB, and clpE virulence genes specific only to this species . Our data on Listeria species analysis demonstrated that this microarray technique is a simple, rapid, and robust genotyping method that is also a potentially valuable tool for identification and characterization of bacterial pathogens in general.

J Appl Microbiol, 1997 Feb, 82(2), 225 - 32
Development of a surface adhesion immunofluorescent technique for the rapid isolation of Listeria monocytogenes and Listeria innocua from meat; Sheridan JJ et al.; The use of a novel surface adhesion technique to isolate Listeria monocytogenes and Listeria innocua from an enrichment meat system was developed . Minced beef samples inoculated with L . monocytogenes (10 cfu g(-1)) were incubated at 30 degrees C for 14-18 h in a suitable enrichment broth . Listeria monocytogenes cells were isolated from the enriched meat sample by surface adhesion onto a polycarbonate membrane which was attached to a glass microscope slide . The Listeria cells on the membrane were subsequently visualized using an immunofluorescent microscopy procedure . The antibody used in this technique reacts with L . monocytogenes and L . innocua . The technique was demonstrated to have a detection level of log10 3.11 cfu ml(-1) . There was excellent correlation (r2 = 0.98) between the counts obtained by this surface adhesion immunofluorescent (SAIF) technique and counts obtained using traditional methods, i.e . plate counts on PALCAM . When the regression equation relating the rapid and standard methods was validated using the data from 50 retail beef mince samples, an rsd value of +/- 0.25 was obtained . No false-negative or false-positive results were recorded for L . monocytogenes or L . innocua species using the SAIF technique.

J Appl Microbiol, 1997 Feb, 82(2), 168 - 76
A comparison of quantitative structure-activity relationships for the effect of benzoic and cinnamic acids on Listeria monocytogenes using multiple linear regression, artificial neural network and fuzzy systems; Ramos-Nino ME et al.; The ability of artificial neural networks (ANN), fuzzy systems (FS) and multiple linear regression (MLR) to fit the biological activity surface describing the inhibition of Listeria monocytogenes by benzoic and cinnamic acid derivatives was compared . MLR and ANN were also compared for their ability to select the properties that best describe the biological activity of the compounds . The criteria used for comparing surface fits of all models were the coefficient of determination r2 and the standard deviation of the error, s(e) . The ANN method gave a better correlation, r2 = 0.96, compared with either MLR, r2 = 0.81, or FS, r2 = 0.92, and also a lower standard error, possibly indicating non-linearity in the data . The ANN was shown to generalize better than MLR using the leave-one-out method . The ANN selection algorithm for the selection of the parameters that contributed most to the biological activity of the phenols (log K and pKa) agreed with the selected parameters of the MLR system.

Appl Environ Microbiol, 2002 Dec, 68(12), 6405 - 9
Sensitivity of Listeria monocytogenes to sanitizers used in the meat processing industry; Romanova N et al.; Nineteen Listeria monocytogenes strains were characterized by automated ribotyping, pulsed-field gel electrophoresis, and plasmid profiling to determine the relationship between genotype and sanitizer resistance . Isolates within a ribogroup had a consistent sensitivity or resistance phenotype except for ribogroup C isolates . All isolates with resistance phenotypes harbored two plasmids . The sensitivity of L . monocytogenes strains to quaternary ammonium compounds (QACs) was correlated with sensitivity to sanitizers and antibiotics with other modes of action . All isolates tested contained the mdrL gene, which encodes an efflux pump that confers resistance to QACs and is both chromosome and plasmid borne.

Appl Environ Microbiol, 2002 Dec, 68(12), 6273 - 82
Direct identification in food samples of Listeria spp . and Listeria monocytogenes by molecular methods; Cocolin L et al.; A new molecular approach for the detection and identification of Listeria spp . and Listeria monocytogenes in food is presented here . The method is based on the PCR amplification of a fragment of the iap gene from the five species belonging to the genus and on the analysis of the PCR products obtained by denaturing gradient gel electrophoresis (DGGE) . The protocol was first optimized by using strains from international collections . Based on the differences present in the sequences amplified, it was possible to obtain species-specific DGGE migration that allowed fast and easy identification of L . monocytogenes, L . innocua, L . welshimeri, L . seeligeri, and L . ivanovii . Moreover, for L . monocytogenes serotypes, partial differentiation was possible . The optimized protocol was used for identification of Listeria strains traditionally isolated from food and for direct detection and identification of Listeria members in food after an overnight enrichment . Identification of 48 food isolates and direct detection of Listeria spp . in 73 food samples show the potential of the method that can be used as a fast screening test to investigate the presence of Listeria spp . and L . monocytogenes in food.

Appl Environ Microbiol, 2002 Dec, 68(12), 5904 - 10
Temperature- and surfactant-induced membrane modifications that alter Listeria monocytogenes nisin sensitivity by different mechanisms; Li J et al.; Nisin interacts with target membranes in four sequential steps: binding, insertion, aggregation, and pore formation . Alterations in membrane composition might influence any of these steps . We hypothesized that cold temperatures (10 degrees C) and surfactant (0.1% Tween 20) in the growth medium would influence Listeria monocytogenes membrane lipid composition, membrane fluidity, and, as a result, sensitivity to nisin . Compared to the membranes of cells grown at 30 degrees C, those of L . monocytogenes grown at 10 degrees C had increased amounts of shorter, branched-chain fatty acids, increased fluidity (as measured by fluorescence anisotropy), and increased nisin sensitivity . When 0.1% Tween 20 was included in the medium and the cells were cultured at 30 degrees C, there were complex changes in lipid composition . They did not influence membrane fluidity but nonetheless increased nisin sensitivity . Further investigation found that these cells had an increased ability to bind radioactively labeled nisin . This suggests that the modification of the surfactant-adapted cell membrane increased nisin sensitivity at the binding step and demonstrates that each of the four steps can contribute to nisin sensitivity.

J Vet Med B Infect Dis Vet Public Health, 2002 Oct, 49(8), 379 - 83
Demonstration of Listeria monocytogenes by immunohistochemistry in formalin-fixed brain tissues from natural cases of ovine and bovine encephalitis; Campero CM et al.; In the present work, evidence of Listeria monocytogenes antigens based on the avidin-biotin complex (ABC) immunoperoxidase technique was performed on formalin-fixed central nervous system tissues (CNS) from a total of 23 natural cases of encephalitis (four ovine and 19 bovine) . Listeria monocytogenes serotype 4 was isolated from 10 of 17 cultured specimens . Meningoencephalitis characterized by focal necrosis, microabscesses, perivascular cuffing, and gliosis with presence of macrophages and/or neutrophils was observed at histological examination . Positive L . monocytogenes antigens were successfully identified by immunohistochemistry (IHC) in the CNS of all 23 cases . Paraffin-embedded tissues assayed were stored up for 17 years . Morbidity of the outbreaks was between 0.3-3% and 0.1-1% for ovine and bovine cases, respectively . In all the ovine cases, flocks involved were under extensive grazing conditions . In nine of the 19 bovine cases (47.3%), supplementation with corn silage was used . The ABC test can help as a practical tool for the diagnosis of natural cases of L . monocytogenes encephalitis on formalin-fixed specimens from ovine and bovine.

Int J Cancer, 2002 Dec 20, 102(6), 629 - 37
Oral vaccination with recombinant Listeria monocytogenes expressing human papillomavirus type 16 E7 can cause tumor growth in mice to regress; Lin CW et al.; Listeria monocytogenes is a Gram-positive, facultative intracellular bacterium with the ability to present secreted proteins to the major histocompatibility complex class I pathway to stimulate cell-mediated immune response . In our study, we constructed the recombinant L . monocytogenes encoding human papillomavirus type 16 E7 gene (rLM-E7) . When orally administered to syngeneic mice, rLM-E7 could induce a cytotoxic T-lymphocyte (CTL) response . Furthermore, in vitro flow cytometric assay and in vivo immune deficiency assays showed that rLM-E7 could prevent and eradicate tumor growth via CD8+-dependent CTLs . Hence, the potency of rLM-E7 as a therapeutic vaccine for cervical cancer is the result of the induction E7-specific cell-mediated immunity by L . monocytogenes . In addition to potency, this vaccine also offers ease of administration and reduced cost of production compared with other vaccines formulated for injection . Thus, L . monocytogenes encoding HPV-16 E7 may be a useful oral vaccine for cervical cancer treatment .

J Neuroimmunol, 2002 Dec, 133(1-2), 132 - 43
Suppression of host resistance to Listeria monocytogenes by acute cold/restraint stress: lack of direct IL-6 involvement; Cao L et al.; We conducted kinetic studies to evaluate the effects of acute cold/restraint stress (ACRS) on both primary and secondary host resistance to Listeria monocytogenes (LM) . The involvement of IL-6 also was investigated using IL-6 knockout (KO) mice on the BALB/c background . ACRS dramatically increased the serum corticosterone levels, indicating that ACRS activated the hypothalamic-pituitary-adrenal (HPA) axis . ACRS significantly inhibited host resistance to LM during a primary but not a secondary LM infection . During the primary infection, ACRS caused a significant delay in clearance of LM, loss of body weight, reduced food/water intake, and elevated levels of pro-inflammatory cytokines (IL-6, IL-1beta, and TNFalpha) and IFNgamma . ACRS IL-6 KO mice showed higher LM burdens than did IL-6 KO controls, suggesting that IL-6 is not required for the ACRS-impairment of host resistance . Elevated levels of IL-1beta and TNFalpha may compensate for the absence of IL-6 and maintain the ACRS-induced impairment, in that the serum and splenic IL-1beta and TNFalpha levels were significantly higher in infected ACRS IL-6 KO mice, but not in control IL-6 KO mice, as compared to respective wild type controls . ACRS appears to inhibit IL-6 independent mechanisms associated with innate immunity and/or the development of adaptive immunity, but these reactions are unable to modulate the more efficient secondary immune responses.

J Immunol, 2002 Dec 1, 169(11), 6522 - 9
Production of type I IFN sensitizes macrophages to cell death induced by Listeria monocytogenes; Stockinger S et al.; Type I IFNs (IFN-alpha/beta) modulate innate immune responses . Here we show activation of transcription factor IFN regulatory factor 3, the synthesis of large amounts of IFN-beta mRNA, and type I IFN signal transduction in macrophages infected with Listeria monocytogenes . Expression of the bacterial virulence protein listeriolysin O was necessary, but not sufficient, for efficient IFN-beta production . Signaling through a pathway involving the type I IFN receptor and Stat1 sensitized macrophages to L . monocytogenes-induced cell death in a manner not requiring inducible NO synthase (nitric oxide synthase 2) or protein kinase R, potential effectors of type I IFN action during microbial infections . The data stress the importance of type I IFN for the course of infections with intracellular bacteria and suggest that factors other than listeriolysin O contribute to macrophage death during Listeria infection.

Microbes Infect, 2002 Nov, 4(13), 1335 - 43
Macrophage intracellular signaling induced by Listeria monocytogenes; Goldfine H et al.; Macrophages are critical for control of Listeria monocytogenes infections; accordingly, the interactions of L . monocytogenes with these cells have been intensively studied . It has become apparent that this facultative intracellular pathogen interacts with macrophages both prior to entry and during the intracellular phase . This review covers recent work on signaling induced in macrophages by L . monocytogenes, especially intracellular signals induced by secreted proteins including listeriolysin O and two distinct phospholipases C.

J Gene Med, 2002 Nov-Dec, 4(6), 655 - 67
Listeria monocytogenes mediated CFTR transgene transfer to mammalian cells; Krusch S et al.; BACKGROUND: Several approaches for gene therapy of cystic fibrosis using viral and non-viral vectors are currently being undertaken . Nevertheless, the present data suggest that vectors currently being used will either have to be further modified or, alternatively, novel vector systems need to be developed . Recently, bacteria have been proven as suitable vehicles for DNA transfer to a wide variety of eukaryotic cells . In this study, we assessed the ability of the facultative intracellular pathogen Listeria monocytogenes to deliver a cDNA encoding the human cystic fibrosis transmembrane conductance regulator (CFTR) to CHO-K1 cells, since these cells have been extensively used for heterologous CFTR expression . METHODS: An established in vitro gene transfer system based on antibiotic-mediated lysis of intracellular L . monocytogenes was exploited to transfer eukaryotic expression plasmids . Transient as well as stable CFTR transgene expression was analyzed by microscopical and biochemical methods; functionality was tested by whole-cell patch-clamp recordings . RESULTS: L . monocytogenes mediated gene transfer to CHO-K1 cells was facilitated by an improved transfection protocol . In addition, the use of the isogenic mutant L . monocytogenes hlyW491A, engineered to produce a hemolysin variant with low toxigenic activity, greatly enhanced the efficiency of gene transfer . This strain allowed the transfer of functional CFTR to CHO-K1 cells . CONCLUSIONS: This is the first demonstration of L . monoyctogenes mediated CFTR transgene transfer . The successful in vitro transfer suggests that L . monocytogenes might be a potential vector for cystic fibrosis gene therapy or alternative applications and deserves further investigation in vitro as well as in vivo .

J Clin Invest, 2002 Nov, 110(10), 1493 - 501
Rescue of CD8 T cell-mediated antimicrobial immunity with a nonspecific inflammatory stimulus; Tuma RA et al.; Reconstitution of protective immunity by adoptive transfer of pathogen-specific T cells has been successful in patients with compromised cellular immunity . The in vivo effectiveness of in vitro-expanded CD8 CTLs is variable, however . For example, adoptively transferred Listeria monocytogenes-specific CD8 CTLs only confer protective immunity if challenge infection occurs within 48 hours of T cell infusion . Herein we show that transferred CTLs persist in lymphoid compartments for many weeks, but that their response to bacterial challenge decreases during the first week following transfer . While T cells transferred less than 48 hours before infection proliferate, those transferred 7 days before infection die . Remarkably, treatment of mice with anti-CD40 at the time of T cell infusion reprograms transferred T cells, allowing them to proliferate and confer protective immunity upon bacterial challenge 7 days later . Our study demonstrates, for the first time to our knowledge that CD40-mediated stimuli can influence CD8 T cell activation independent of concurrent antigen exposure . The ability to modulate long-term responsiveness of CD8 T cells with a transient, nonspecific inflammatory stimulus has importation implications for adoptive immunotherapy.

Infect Immun, 2002 Dec, 70(12), 7179 - 81
Expression of NADPH oxidase-dependent resistance to listeriosis in mice occurs during the first 6 to 12 hours of liver infection; LaCourse R et al.; Wild-type mice inoculated with Listeria monocytogenes intravenously were capable of reducing the bacterial load in their livers by 90% within 6 h . In contrast, mice with deletions of the gene for NADPH oxidase were incapable of expressing this early oxygen-dependent anti-Listeria defense and consequently showed higher levels of liver infection at later times.

Infect Immun, 2002 Dec, 70(12), 6638 - 45
Differences in expression of toll-like receptors and their reactivities in dendritic cells in BALB/c and C57BL/6 mice; Liu T et al.; We have previously reported that differences in early production of interleukin 12 (IL-12) by dendritic cells (DC) underlies the difference between the susceptibilities to Listeria monocytogenes of C57BL/6 and BALB/c mice . To elucidate mechanisms for the different abilities of DC to produce cytokine in C57BL/6 and BALB/c mice, we examined Toll-like receptor (TLR) expression by DC and their responses in vitro to known microbial ligands for TLRs . We found that DC isolated from the spleens of naive C57BL/6 mice preferentially expressed TLR9 mRNA, whereas DC from naive BALB/c mice strongly expressed TLR2, -4, -5, and -6 mRNAs . C57BL/6 DC produced a higher level of IL-12p40 in response to the ligands for TLR4 (lipopolysaccharide), TLR2 (lipoprotein), and TLR9 (CpG), whereas BALB/c DC responded to these ligands by producing a larger amount of monocyte chemoattractant protein 1 . C57BL/6 DC expressed higher levels of CD40 and Stat4 than BALB/c DC did, suggesting that naive C57BL/6 mice contained more-mature subsets of DC than naive BALB/c mice . Differences in reactivities of DC to microbial molecules through TLRs may be associated with susceptibility and resistance to Listeria infection in BALB/c and C57BL/6 mice.

New Microbiol, 2002 Oct, 25(4), 449 - 54
Typing of food-borne Listeria monocytogenes by the optimized repetitive extragenic palindrome-based polymerase chain reaction; Pangallo D et al.; The repetitive extragenic palindrome-based polymerase chain reaction was optimized for typing Listeria monocytogenes by 1) using the QlAamp method to increase the reproducibility of DNA isolation, 2) running PCR with three different DNA concentrations in parallel, 3) using antibody-protected therrnostable DNA polymerase to reduce non-specific priming, and 4) using an improved temperature programme to increase the amplification yield . When applied to 42 L . monocytogenes strains isolated from food in the Czech and Slovak republics during 1999-2000, profiles of 7-15 DNA fragments of 330-3,310 bp were amplified . Based on REP-profiles, strains (serotypes 1/2 and 4) could be divided into 12 groups.

MMWR Morb Mortal Wkly Rep, 2002 Oct 25, 51(42), 950 - 1
Outbreak of listeriosis--northeastern United States, 2002.
{Genome analysis and primer sets determination for listeria species detection and gene typing}
Lymans'kyi OP, Shapoval VF, Volians'ka NP, Al'saban A, Kuchma IIu.

Institute of Microbiology and Immunology, Academy of Medical Sciences of Ukraine, 14 Pushkinskaya St., Kharkov, 61057, Ukraine . lim@vet.Kharkov.ua

Computer analysis of listeria genome isolates of sequences from EMBL, GenBank, DDBJ data bases has been made . Variable and highly conservative (homology degree is 90-100% for all known isolates) genes loci iap and listeriolysin (cytolysin) gene locuses have been determined . Primer sets for detection and differentiation of Listeria species by polymerase chain reaction (PCR) were designed by computer and following thermodynamic analysis . Primer sets can provide detection of Listeria monocytogenes, L . ivanovii, L . seeligeri, L . grayi, L . innocua, L . welshimeri and detection of only pathogenic L . monocytogenes and Listeria species with listeriolysin (cytolysin) gene . Differentiation of 6 Listeria species can be done by means of two primer sets for iap and listeriolysin genes . Amplified fragments of listeria species DNA have different amplicon size . Primers melting temperatures selection allows one to carry out listeria species typing by multiplex PCR in a single tube.

Int J Hematol, 2002 Aug, 76 Suppl 1, 277 - 9
Role of NKT cells and alpha-galactosyl ceramide; Shimosaka A; Alfa-Galactosyl Ceramide was isolated from Ocean sponge which has antitumor effect against several tumors in in vivo animal model with no cytotoxicity . KRN7000(KRN) is the most potent alpha-Galactosyl Ceramide modified from the one isolated from Ocean sponge . KRN is also active against metastatic tumors through the activation ofanimal immune system . Research efforts in learning the mechanism of action, we found the important role of dendritic cells(DC) and NKT cells . NKT cells was first characterized in 1988 which is overlap some part with NK cells and T-Cells and majority is different from NK and T . KRN is active through the activation of DC and NKT in giving antigen specific immune stimulation in animal . This antigen specific stimulation is memorized by immune system and can reject second tumor challenge . KRN is not active in nude mice and NKT deficient animal . NKT cells level in blood is lower in patients with autoimmune disease, cancer, HIV positive or aplastic anemia . NKT rapidly releases IL-4 and IFN-gamma at high level when activated . NKT is CD1d and TCR restricted . NKT plays important role in autoimmune disease such as Type 1 Diabetes, Scleroderma and Systemic Lupus Erythematosus, infections such as Mycobacteria, Listeria and Malaria, GVHD control and tumor rejection . NKT acts as double edge sword, aggressive and suppressive ways . KRN can prevent the onset of Type 1 Diabetes, inhibit replication of hepatitis virus B in liver and suppress malaria replication in activating NKT cells . KRN can activate NKT through DC and activated NKT activates NK, T and macrophage . KRN also expands NKT cells and expanded NKT has full function . Although the exact role of DC and NKT is not clear, KRN clinical study results in conjunction with DC and NKT cell activation are expected.

Int J Food Microbiol, 2003 Jan 15, 80(1), 1 - 15
Estimation of low bacterial concentration: Listeria monocytogenes in raw milk; Meyer-Broseta S et al.; A time-series bacteriological analysis has been carried out on milk collected on farms from 1997 to 2001 by a plant producing raw milk soft cheese, with the purpose of assessing the time course of the presence/absence of Listeria monocytogenes . A standard data collection procedure was used, in which farms were tested on a monthly or biweekly basis and 2-3 days after the detection of milk tanker contamination . This procedure yielded low figures for contamination frequencies . The average value and the median of the monthly prevalence of farms detected positive for L . monocytogenes were 2.4 and 0%, respectively . A seasonal effect (with peaks in winter) was observed . Between 1997 and 2001, there was no significant decrease of contamination rates, in spite of the efforts on the contaminated farms . Over the last year of the study (from March 2000 to February 2001), a new data collection procedure was implemented that allowed much better detection of sporadic occurrences . Milk samples were collected from the bulk tank of each participating farm just before pick-up, then stored and subsequently analysed whenever the milk tanker was found contaminated . The average value and the median of the monthly prevalence of positive farms were found equal to 7.7 and 0%, respectively (for a mean prevalence of L . monocytogenes in the milk tanker of 3.2%) . These results confirm that farm milk contamination is, most often, a sporadic event In addition to this prevalence study, contamination levels were quantified by enumerating L . monocytogenes using direct plating of small volumes of farm milk previously tested positive . Most often, these levels were extremely low . A simple simulation model shows that, when milk tankers were found positive, contamination levels in the corresponding bulk-tank milk are themselves very low (typically, below 3 L . monocytogenes per millilitre with most probable concentration 0.1 Colony Forming Unit (CFU)/ml and median ranging from 5.10(-2) to 0.1 CFU/ml) . Such low levels are very likely to be due to environmental contamination.

J Food Prot, 2002 Nov, 65(11), 1811 - 29
Listeria monocytogenes virulence and pathogenicity, a food safety perspective; Kathariou S; Several virulence factors of Listeria monocytogenes have been identified and extensively characterized at the molecular and cell biologic levels, including the hemolysin (listeriolysin O), two distinct phospholipases, a protein (ActA), several internalins, and others . Their study has yielded an impressive amount of information on the mechanisms employed by this facultative intracellular pathogen to interact with mammalian host cells, escape the host cell's killing mechanisms, and spread from one infected cell to others . In addition, several molecular subtyping tools have been developed to facilitate the detection of different strain types and lineages of the pathogen, including those implicated in common-source outbreaks of the disease . Despite these spectacular gains in knowledge, the virulence of L . monocytogenes as a foodborne pathogen remains poorly understood . The available pathogenesis and subtyping data generally fail to provide adequate insight about the virulence of field isolates and the likelihood that a given strain will cause illness . Possible mechanisms for the apparent prevalence of three serotypes (1/2a, 1/2b, and 4b) in human foodborne illness remain unidentified . The propensity of certain strain lineages (epidemic clones) to be implicated in common-source outbreaks and the prevalence of serotype 4b among epidemic-associated stains also remain poorly understood . This review first discusses current progress in understanding the general features of virulence and pathogenesis of L . monocytogenes . Emphasis is then placed on areas of special relevance to the organism's involvement in human foodborne illness, including (i) the relative prevalence of different serotypes and serotype-specific features and genetic markers; (ii) the ability of the organism to respond to environmental stresses of relevance to the food industry (cold, salt, iron depletion, and acid); (iii) the specific features of the major known epidemic-associated lineages; and (iv) the possible reservoirs of the organism in animals and the environment and the pronounced impact of environmental contamination in the food processing facilities . Finally, a discussion is provided on the perceived areas of special need for future research of relevance to food safety, including (i) theoretical modeling studies of niche complexity and contamination in the food processing facilities; (ii) strain databases for comprehensive molecular typing; and (iii) contributions from genomic and proteomic tools, including DNA microarrays for genotyping and expression signatures . Virulence-related genomic and proteomic signatures are expected to emerge from analysis of the genomes at the global level, with the support of adequate epidemiologic data and access to relevant strains.

J Food Prot, 2002 Nov, 65(11), 1796 - 9
Antimicrobial resistance of Listeria monocytogenes isolated from various cabbage farms and packing sheds in Texas; Prazak MA et al.; Twenty-one isolates of Listeria monocytogenes from cabbage, environmental, and water samples were evaluated for antimicrobial resistance by the disk diffusion method . Ninety-five percent (20 of 21) of the isolates tested were resistant to two or more antimicrobial agents . This finding is significant, since multiresistant strains of Listeria spp . are not commonly found in nature . Eighty-five percent (17 of 20) of the multiresistant strains were resistant to penicillin, and the remaining multiresistant isolates were somewhat sensitive to penicillin . A multiresistant strain showing intermediate sensitivity to penicillin was resistant to gentamicin . One isolate was susceptible to all antimicrobial agents except penicillin . Penicillin- and gentamicin-resistant L . monocytogenes have not previously been reported from human, food, or environmental samples . This study provides evidence of the emergence of multiresistant L . monocytogenes strains, pointing to an increase in the potential threat to human health posed by this pathogen.

J Food Prot, 2002 Nov, 65(11), 1750 - 5
Antioxidant power, lipid oxidation, color, and viability of Listeria monocytogenes in beef bologna treated with gamma radiation and containing various levels of glucose; Sommers CH et al.; Ionizing radiation can be used to pasteurize ready-to-eat (RTE) meat products . Thermal processing of RTE meats that contain dextrose results in the production of antioxidants that may interfere with ionizing radiation pasteurization of RTE meat products . Beef bologna was manufactured with dextrose concentrations of 0, 2, 4, 6, and 8% . Antioxidant activity, as measured by the Ferric Reducing Antioxidant Power assay, increased with dextrose concentration but was unaffected by ionizing radiation . Lipid oxidation increased significantly in irradiated bologna (4 kGy) that contained dextrose . Hunter color analysis indicated that the addition of dextrose reduced the ionizing radiation-induced loss of redness (a-value) but promoted the loss of brightness (L-value) . The radiation resistance, D10-value, of Listeria monocytogenes that was surface-inoculated onto bologna slices was not affected by dextrose concentration . L . monocytogenes strains isolated from RTE meats after listeriosis outbreaks were utilized . Increased antioxidant activity generated by thermal processing of dextrose in fine emulsion sausages does not present a barrier to radiation pasteurization of RTE meats . However, a high dextrose concentration in combination with gamma irradiation increases lipid oxidation significantly.

J Food Prot, 2002 Nov, 65(11), 1745 - 9
Behavior of Listeria monocytogenes in avocado pulp and processed guacamole; Iturriaga MH et al.; The potential ability of Listeria monocytogenes to grow or survive in avocado pulp (AP) and processed guacamole (PG) stored at 22, 4 to 7, and -18 degrees C was studied . Both products were obtained from a factory in Michoacan, Mexico . PG consisted of AP mixed with dehydrated vegetables, antioxidants, and preservatives . Populations of L monocytogenes in AP stored at 22 degrees C increased from 2 to 6 and 9 log CFU/g after 24 and 48 h, respectively . At 4 to 7 degrees C, the growth rate of L monocytogenes in AP was greatly decreased; generation time was 8.2 h, in contrast with 1.35 h observed at 22 degrees C . L . monocytogenes populations did not increase in PG either at 22 degrees C for 48 h or at 4 to 7 degrees C for 15 days . The bacteriostatic effect in PG may have resulted from the presence of added substances, especially citric acid and disodium dihydrogen pyrophosphate . Aerobic plate counts and coliforms increased in AP and PG stored at ambient temperature and under refrigeration . However, these increments did not affect the growth of the pathogen . L . monocytogenes (50,000 most probable number {MPN}/g) survived at least 58 weeks in both products stored frozen at -18 degrees C; the final population was 335 MPN/g in AP and 23 MPN/g in PG . Although the composition of avocado fruit differs significantly (high content of lipids and scarcity of simple carbohydrates) from that typical of most fruits, these results underline AP as a potential vehicle of human listeriosis and indicate that freezing should not be used as the sole mechanism to control this pathogen.

J Food Prot, 2002 Nov, 65(11), 1740 - 4
Effect of gamma irradiation on Listeria monocytogenes in frozen, artificially contaminated sandwiches; Clardy S et al.; Gamma irradiation has been shown to effectively control L monocytogenes in uncooked meats but has not been extensively studied in ready-to-eat foods . The presence of Listeria in ready-to-eat foods is often due to postprocess contamination by organisms in the food-manufacturing environment . Because gamma irradiation is applied after products are packaged, the treated foods are protected from environmental recontamination . Currently, a petition to allow gamma irradiation of ready-to-eat foods is under review by the Food and Drug Administration . This study was conducted to determine if gamma irradiation could be used to control L . monocytogenes in ready-to-eat sandwiches . Ham and cheese sandwiches were contaminated with L . monocytogenes, frozen at -40 degrees C, and exposed to gamma irradiation . Following irradiation, sandwiches were assayed for L . monocytogenes . A triangle test was performed to determine if irradiated and nonirradiated sandwiches differed in sensory quality . We found that the D10-values ranged from 0.71 to 0.81 kGy and that a 5-log reduction would require irradiation with 3.5 to 4.0 kGy . The results of a 39-day storage study of sandwiches inoculated with 10(7) CFU of L monocytogenes per g indicated that counts for nonirradiated sandwiches remained fairly constant . Counts for sandwiches treated with 3.9 kGy decreased by 5 log units initially and then decreased further during storage at 4 degrees C . Sensory panelists could distinguish between irradiated and nonirradiated sandwiches but were divided on whether irradiation adversely affected sandwich quality . Our results suggest that manufacturers of ready-to-eat foods could use gamma irradiation to control L . monocytogenes and improve the safety of their products.

J Food Prot, 2002 Nov, 65(11), 1735 - 9
Detection of Listeria in crawfish processing plants and in raw, whole crawfish and processed crawfish (Procambarus spp.); Thimothe J et al.; The foodborne pathogen Listeria monocytogenes represents a major concern to the food industry and particularly to producers of ready-to-eat (RTE) foods because of the severity of human listeriosis infections and because of the ubiquitous nature of this organism . Although several studies on the prevalence and sources of L monocytogenes in various RTE seafoods have been conducted, limited information is available on the presence and potential sources of this organism in RTE crawfish products . We thus monitored the presence of L monocytogenes and other Listeria spp . in the processing environment, in raw, whole crawfish, and in cooked crawfish meat from two processing plants . Samples were collected from the two plants throughout one crawfish season (April to June 2001) at 5 and 8 separate visits, respectively . At each visit, 6 raw, whole crawfish, 6 finished product samples (crawfish meat), and 14 mid- or end-of-processing environmental sponge samples were collected and tested for L . monocytogenes and Listeria spp . Of the 337 samples tested, 31 contained Listeria spp . Although Listeria innocua was the predominant Listeria spp . found (20 samples), four samples were positive for L monocytogenes . L . monocytogenes was detected in three raw material samples and in one environmental sample . Listeria spp . were found in 29.5% of raw, whole crawfish (n = 78) and in 4.4% of environmental samples (n = 181) but in none of the finished product samples . Among the environmental samples, Listeria spp . were found in 15.4% of the drains (n = 39) and in 5.1% of the employee contact surfaces (gloves and aprons) (n = 39) but in none of the samples from food contact surfaces . Even though a high prevalence of Listeria spp . was detected on raw materials, it appears that the heat treatment during the processing of crawfish and the practices preventing postprocessing recontamination can significantly reduce Listeria contamination of RTE crawfish meat.

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