J Food Prot, 2002 Nov, 65(11), 1728 - 34 Prevalence of Listeria monocytogenes during production and postharvest processing of cabbage; Prazak AM et al.; From November 1999 to May 2000, analyses of 425 cabbage, 205 water, and 225 environmental sponge samples from four cabbage farms with packing sheds and from two packing sheds in the Rio Grande Valley and Uvalde, Tex., were conducted to determine whether Listeria monocytogenes was present . Samples were tested by the Food and Drug Administration method for the isolation of Listeria spp., and confirmed isolates were DNA fingerprinted by repetitive-element sequence-based polymerase chain reaction (rep-PCR) . L monocytogenes was isolated from 3% (26 of 855) of the samples . Twenty of these isolates were obtained from cabbage (7 isolates from farms and 13 from packing sheds) . Three isolates were from water samples(two from farms and one from a packing shed), and three were from environmental sponge samples of packing shed surfaces . Rep-PCR-generated fingerprints of 21 of the isolates revealed 18 distinctive banding patterns . Four isolates from environmental sponge samples of conveyor belts and from cabbage samples shared identical banding patterns, suggesting common sources of contamination . These identical environmental isolates suggest that contact with packing shed surfaces may be a source of contamination of cabbage . However, the cabbage samples could have arrived contaminated, since they were not washed. Am J Crit Care, 2002 Nov, 11(6), 567 - 70 Effectiveness of 0.12% chlorhexidine gluconate oral rinse in reducing prevalence of nosocomial pneumonia in patients undergoing heart surgery; Houston S et al.; BACKGROUND: Decreasing the levels of bacteria in the oropharynx should reduce the prevalence of nosocomial pneumonia . OBJECTIVES: To test the effectiveness of 0.12% chlorhexidine gluconate oral rinse in decreasing microbial colonization of the respiratory tract and nosocomial pneumonia in patients undergoing open heart surgery . METHODS: A prospective, randomized, case-controlled clinical trial design was used . Peridex (0.12% chlorhexidine gluconate) was the experimental drug, and Listerine (phenolic mixture) was the control drug . A total of 561 patients undergoing aortocoronary bypass or valve surgery requiring cardiopulmonary bypass were randomized to an experimental (n = 270) or a control (n = 291) group . Nosocomial pneumonia was diagnosed by using the criteria established by the Centers for Disease Control and Prevention . RESULTS: The overall rate of nosocomial pneumonia was reduced by 52% (4/270 vs 9/291; P = .21) in the Peridex-treated patients . Among patients intubated for more than 24 hours who had cultures that showed microbial growth (all pneumonias occurred in this group), the pneumonia rate was reduced by 58% (4/19 vs 9/18; P = .06) in patients treated with Peridex . In patients at highest risk for pneumonia (intubated > 24 hours, with cultures showing the most growth), the rate was 71% lower in the Peridex group than in the Listerine group (2/10 vs 7/10; P = .02) . CONCLUSIONS: Although rates of nosocomial pneumonia were lower in patients treated with Peridex than in patients treated with Listerine, the difference was significant only in those patients intubated more than 24 hours who had the highest degree of bacterial colonization. Int J Food Microbiol, 2003 Feb 25, 81(1), 73 - 84 Applicability of a bacteriocin-producing Enterococcus faecium as a co-culture in Cheddar cheese manufacture; Foulquie Moreno MR et al.; Two strains, Enterococcus faecium RZS C5 and E . faecium DPC 1146, produce listericidal bacteriocins, so-called enterocins . E . faecium RZS C5 was studied during batch fermentation in both a complex medium (MRS) and in milk to understand the influence of environmental factors, characteristic for milk and cheese, on both growth and bacteriocin production . Fermentation conditions were chosen in view of the applicability of in situ enterocin production during Cheddar cheese production . Enterocin production by E . faecium RZS C5 in MRS started in the early logarithmic growth phase, and enterocin activity decreased during the stationary phase . The effect of pH on enterocin production and decrease of activity was as intense as the effect on bacterial growth . Higher enterocin production took place at pH 5.5 compared with pH 6.5 . The use of lactose instead of glucose increased the production of enterocin, and at higher lactose concentration, production increased more and loss of activity decreased . The production in skimmed milk compared to MRS was lower and was detected mainly in the stationary phase . When casein hydrolysate was added to the milk, enterocin production was higher and started earlier, indicating the importance of an additional nitrogen source for growth of E . faecium in milk . For co-cultures of E . faecium RZS C5 with the starters used during Cheddar cheese manufacture, no enterocin activity was detected during the milk fermentation . Furthermore, the applicability of E . faecium RZS C5 and E . faecium DPC 1146 strains was tested in Cheddar cheese manufacture on pilot scale . Enterocin production took place from the beginning of the cheese manufacturing and was stable during the whole ripening phase of the cheese . This indicates that both an early and late contamination of the milk or cheese can be combated with a stable, in situ enterocin production . The use of such a co-culture is an additional safety provision beyond good manufacturing practices. FEMS Microbiol Lett, 2002 Oct 29, 216(1), 91 - 7 Endosomal/lysosomal targeting of a single helper T-cell epitope of an intracellular bacterium by DNA immunisation induces a specific T-cell subset and partial protective immunity in vivo; Uchiyama H et al.; We evaluated here the effect of the intracellular targeting of a helper T-cell (Th) epitope, literiolysin O 215-226 derived from Listeria monocytogenes, on induction of a specific Th by gene gun immunisation . Immunisation of C3H/He mice with pE215LAMP plasmid encoding the Th epitope fused with the endosomal/lysosomal targeting signal of lysosome-associated membrane protein (LAMP)-1 gave the epitope-specific proliferative responses of CD4(+) T lymphocytes . In addition, specific interferon-gamma production from the splenocytes was observed . Concomitantly, pE215LAMP-immunised mice showed moderate, but significant protective immunity against listerial challenge . These results suggest that the intracellular targeting of a Th epitope to endosomal/lysosomal compartments by DNA immunisation is useful for eliciting a specific Th subset in vivo. J Immunol, 2002 Nov 15, 169(10), 5813 - 7 Inducible costimulator protein controls the protective T cell response against Listeria monocytogenes; Mittrucker HW et al.; The inducible costimulator protein (ICOS) was recently identified as a costimulatory molecule for T cells . Here we analyze the role of ICOS for the acquired immune response of mice against the intracellular bacterium Listeria monocytogenes . During oral L . monocytogenes infection, low levels of ICOS expression were detected by extracellular and intracellular Ab staining of Listeria-specific CD4(+) and CD8(+) T cells . Blocking of ICOS signaling with a soluble ICOS-Ig fusion protein markedly impaired the Listeria-specific T cell responses . Compared with control mice, the ICOS-Ig treated mice generated significantly reduced numbers of Listeria-specific CD8(+) T cells in spleen and liver, as determined by tetramer and intracellular cytokine staining . In contrast, the specific CD8(+) T cell response in the intestinal mucosa did not appear to be impaired by the ICOS-Ig treatment . Analysis of the CD4(+) T cell response revealed that ICOS-Ig treatment also affected the specific CD4(+) T cell response . When restimulated with listerial Ag in vitro, reduced numbers of CD4(+) T cells from infected and ICOS-Ig-treated mice responded with IFN-gamma production . The impaired acquired immune response in ICOS-Ig treated mice was accompanied by their increased susceptibility to L . monocytogenes infection . ICOS-Ig treatment drastically enhanced bacterial titers, and a large fraction of mice succumbed to the otherwise sublethal dose of infection . Thus, ICOS costimulation is crucial for protective immunity against the intracellular bacterium L . monocytogenes. J Immunol, 2002 Nov 15, 169(10), 5805 - 12 Nonsecreted bacterial proteins induce recall CD8 T cell responses but do not serve as protective antigens; Zenewicz LA et al.; Secreted or nonsecreted Ag expressed by recombinant Listeria monocytogenes can prime CD8 T cells . However, Ag-specific memory CD8 T cells confer protection against bacteria secreting Ag, but not against bacteria expressing the nonsecreted form of the same Ag . This dichotomy may be explained by a long-standing hypothesis that nonsecreted Ags are less effective than secreted Ags at inducing a protective immune response at the onset of infection . We tested this hypothesis by examining whether these two different forms of Ag induce different primary and secondary CD8 T cell responses . The primary responses to secreted and nonsecreted Ags expanded and contracted almost synchronously, although the responses to nonsecreted Ags were of lower magnitude . These results demonstrate that the kinetics of the CD8 T cell response are similar regardless of whether Ag is accessible to the endogenous MHC class I pathway or can only be presented through cross-presentation . No differences were detected in the CD8 T cell recall response to L . monocytogenes expressing secreted or nonsecreted Ags . Nonsecreted Ags are as effective as secreted Ags at the induction of a rapid recall response by memory CD8 T cells . Thus, the inability of nonsecreted bacterial proteins to serve as protective Ags cannot be attributed to a defective CD8 T cell response. Trends Mol Med, 2002 Nov, 8(11), 537 - 42 Genetically-modified-animal models for human infections: the Listeria paradigm; Lecuit M et al.; Several human pathogens exhibit a restricted host-tropism, relying on the species-specific interaction of microbial ligand(s) with host receptor(s) . This specificity accounts for some of the difficulties in modeling human infections in animals . The discovery of L . monocytogenes host-specificity and elucidation of the underlying mechanism has led to the generation of transgenic mice expressing one of its human receptors, E-cadherin . This model is presented here as a paradigm of a genetically-modified-animal model for studying a human infectious disease. Syst Appl Microbiol, 2002 Oct, 25(3), 456 - 61 Amplified fragment length polymorphism (AFLP) analysis of Listeria monocytogenes; Guerra MM et al.; An agarose gel based single enzyme AFLP method using EcoR1 digestion of Listeria monocytogenes DNA was developed for epidemiological typing . The method was evaluated with 84 L . monocytogenes cultures, and results were compared with those obtained with serotyping, phage-typing and cadmium and arsenic resistance typing . The AFLP method was reproducible and 14 different banding patterns comprising between five and eight DNA fragments were produced . All except two of the AFLP patterns were serorype specific . Different AFLP patterns were recognised within serovar 4b (four patterns), 1/2a (two patterns), 1/2b (six patterns): single patterns were obtained from cultures of serovars 1/2c, 3a, 3b and 3c . There were associations with AFLP results and those from phage-typing and cadmium and arsenic resistance typing, although each method showed some independence . This preliminary evaluation suggests that this AFLP method will be useful for epidemiological typing of L . monocytogenes. Environ Health Perspect, 2002 Nov, 110(11), 1105 - 11 Alteration of pulmonary immunity to Listeria monocytogenes by diesel exhaust particles (DEPs) . I . Effects of DEPs on early pulmonary responses; Yin XJ et al.; It has been hypothesized that diesel exhaust particles (DEPs) aggravate pulmonary bacterial infection by both innate and cell-mediated immune mechanisms . To test this hypothesis, we investigated the effects of DEP exposure on the functions of alveolar macrophages (AMs) and lymphocytes from lung-draining lymph nodes using a rat Listeria monocytogenes infection model . In the present study, we focused on the effects of DEP exposure on AM functions, including phagocytic activity and secretion of proinflammatory cytokines . The Listeria infection model was characterized by an increase in neutrophil count, albumin content, and acellular lactate dehydrogenase activity in the bronchoalveolar lavage (BAL) fluid at 3 and 7 days postinfection . Short-term DEP inhalation (50 and 100 mg/m(3), 4 hr) resulted in a dose-dependent suppression of lung clearance of Listeria, with the highest bacteria count occurring at day 3 . This aggravated bacterial infection was consistent with the inhibitory effect of DEPs on macrophage functions . DEPs suppressed phagocytosis and Listeria-induced basal secretion of interleukin-1ss (IL-1ss) and IL-12 by AMs in a dose-dependent manner . The amount of IL-1ss and IL-12 in the BAL fluid was also reduced by DEP exposure . In addition, DEPs decreased Listeria-induced lipopolysaccharide-stimulated secretion of tumor necrosis factor-alpha (TNF-alpha), IL-1ss, and IL-12 from AMs . These results suggest that DEPs retard bacterial clearance by inhibiting AM phagocytosis and weaken the innate immunity by inhibiting AM secretion of IL-1ss and TNF-alpha . DEPs may also suppress cell-mediated immunity by inhibiting AM secretion of IL-12, a key cytokine for the initiation of T helper type 1 cell development in Listeria infection. J Neuroimmunol, 2002 Nov, 132(1-2), 129 - 39 Compromised peripheral immunity of mice injected intrastriatally with six-hydroxydopamine; Filipov NM et al.; Intracisternal or intracerebroventricular administration of six-hydroxydopamine (6-OHDA), which results in decreased norepinephrine (NE) and dopamine (DA) levels throughout the brain, causes impaired peripheral immunity . However, in vivo immunocompetence following selective striatal depletion of DA by 6-OHDA has not been investigated . Thus, we sought to determine whether striatal DA depletion compromises host resistance to Listeria monocytogenes (LM) and impairs the immune response to keyhole limpet hemocyanin (KLH) . Mice treated with 6-OHDA (90% decrease in striatal DA) had (i) increased LM colonization in liver and spleen, (ii) lower primary IgM and IgG(1) antibody titers, as well as secondary IgM titers, and (iii) compromised DTH response compared to controls . Co-administration of a DA uptake inhibitor partially (40%) spared striatal DA depletion and completely prevented the increase in LM burden, but was ineffective in preventing any of the 6-OHDA-induced suppressions of the immune responses to KLH . Thus, striatal DA is suggested to play a response-specific role in peripheral immunological functions. Vet Microbiol, 2002 Dec 20, 90(1-4), 299 - 309 The intramacrophagic environment of Brucella suis and bacterial response; Kohler S et al.; Phagocytes have developed various antimicrobial defense mechanisms to eliminate pathogens . They comprise the oxidative burst, acidification of phagosomes, or fusion of phagosomes with lysosomes . Facultative intracellular bacteria, in return, have developed strategies counteracting the host cell defense, resulting in intramacrophagic survival . Until lately, only very little was known about the phagosomal compartment containing Brucella spp., the environmental conditions the bacteria encounter, and the pathogen's stress response . Recently, we have determined that the phagosomes acidify rapidly to a pH of 4.0-4.5 following infection, but this early acidification is crucial for intracellular replication as neutralization results in bacterial elimination . A vacuolar proton-ATPase is responsible for this phenomenon that is not linked to phagosome-lysosome fusion . On the contrary, in vitro reconstitution assays revealed association only between phagosomes containing killed B . suis and lysosomes, describing the absence of phagolysosome fusion due to specific recognition inhibition for live bacteria . Further evidence for the necessity of an intact, acidic phagosome as a predominant niche of brucellae in macrophages was obtained with a strain of B . suis secreting listeriolysin . It partially disrupts the phagosomal membranes and fails to multiply intracellularly . How does B . suis adapt to this environment? We have identified and studied a series of genes that are involved in this process of adaptation . The bacterial heat shock protein and chaperone DnaK is induced in phagocytes and it is essential for intracellular multiplication . A low-level, constitutive expression of dnaK following promoter exchange does not restore intramacrophagic survival . Another chaperone and heat shock protein, ClpB, belonging to the family of ClpATPases, is important for the resistance of B . suis to several in vitro stresses, but does not contribute to intramacrophagic survival of the pathogen . Additional bacterial genes specifically induced within the phagocyte were identified by an intramacrophagic screen of random promoter fusions to the reporter gene gfp . A large majority of these genes are encoding proteins involved in transport of nutrients (sugars, amino acids), or cofactors, such as nickel . Analysis of the intracellular gene activation reveals that low oxygen tension is encountered by B . suis . Altogether, these results suggest three major stress conditions encountered by brucellae in the phagosome: acid stress, starvation and low oxygen tension . EMBO J, 2002 Nov 1, 21(21), 5623 - 34 GW domains of the Listeria monocytogenes invasion protein InlB are SH3-like and mediate binding to host ligands; Marino M et al.; InlB, a surface-localized protein of Listeria monocytogenes, induces phagocytosis in non-phagocytic mammalian cells by activating Met, a receptor tyrosine kinase . InlB also binds glycosaminoglycans and the protein gC1q-R, two additional host ligands implicated in invasion . We present the structure of InlB, revealing a highly elongated molecule with leucine-rich repeats that bind Met at one end, and GW domains that dissociably bind the bacterial surface at the other . Surprisingly, the GW domains are seen to resemble SH3 domains . Despite this, GW domains are unlikely to act as functional mimics of SH3 domains since their potential proline-binding sites are blocked or destroyed . However, we do show that the GW domains, in addition to binding glycosaminoglycans, bind gC1q-R specifically, and that this binding requires release of InlB from the bacterial surface . Dissociable attachment to the bacterial surface via the GW domains may be responsible for restricting Met activation to a small, localized area of the host cell and for coupling InlB-induced host membrane dynamics with bacterial proximity during invasion. Mol Cell, 2002 Sep, 10(3), 437 - 9 Measuring the immeasurable; Newman JC et al.; Many bacterial pathogens turn on virulence genes at host body temperature . In the September 6, 2002, issue of Cell, Johansson et al . show that the Listeria monocytogenes thermosensor is an RNA structure in the 5' untranslated region of the mRNA for the virulence-activating transcription factor PrfA . The stem-loop structure blocks translation initiation at 30 degrees C but melts away at 37 degrees C. Dig Liver Dis, 2002 Sep, 34 Suppl 2, S34 - 6 Cytoskeletal proteins and resident flora; Biancone L et al.; Recent observations demonstrate that enteropathogenetic and enterohaemorrhagic bacteria, as well as other non enteropathogenetic bacteria (Listeria, Coxiella Burnetii), may subvert the host cell cytoskeleton . Models from enteropathogenic bacteria demonstrate that cytoskeletal proteins are required for bacteria binding to the enterocytes and that they play a role in the immune response of the host to intestinal bacteria . The cytoskeletal protein family Tropomyosins is present in all eukaryotic cells, with multiple isoforms regulated by multiple genes . Of the different Tropomyosin isoforms, TM5 has been shown to be expressed in colonic and jejunal epithelial cells, while TM1 in colonic and jejunal smooth muscle . In vitro studies have shown the presence of serum and mucosal IgG against TM5 in almost two thirds of patients with ulcerative colitis, suggesting: a . a possible autoimmune response to Tropomyosin in these patients; b . the hypothesis that the development of pouchitis may be related to the expression of TM5 in the ileal pouch; c . the use of probiotics in the treatment of pouchitis . Overall, the new expression of cytoskeletal proteins on the cell surface appears to be possibly induced by several mechanisms, including intestinal bacteria and apoptosis . The expression of cytoskeletal proteins on the cell surface may induce tolerance or autoimmune response on target cells . Further investigations are, however needed on the possible role of cytoskeletal proteins in human diseases. J Ind Microbiol Biotechnol, 2002 Nov, 29(5), 228 - 32 Multimethod assessment of commercial nisin preparations; Cleveland J et al.; Nisin is a GRAS preservative effective against several Gram-positive organisms including Listeria monocytogenes . Commercial preparations are usually fermentation products containing 2.5% pure nisin along with insoluble material which, in this study, was found to influence the quantification and activity of nisin under different conditions . Commercially available samples of nisin were tested for efficacy using various methods, such as well diffusion, time to turbidity, and GUS (where a reporter compound is induced in response to nisin) . SDS-PAGE detected a single peptide band, corresponding with the molecular weight of nisin . Protein quantified using the Bradford method indicated that the carrier of some samples was proteinaceous . Though the activity of commercially available nisin preparations is indicated on the label, end users should determine the effect of changing their source of nisin. Int Immunol, 2002 Nov, 14(11), 1343 - 50 A regulatory role for suppressor of cytokine signaling-1 in T(h) polarization in vivo; Fujimoto M et al.; Suppressor of cytokine signaling (SOCS)-1 is an inhibitory molecule for JAK, and its deficiency in mice leads to lymphocyte-dependent multi-organ disease and perinatal death . Crossing of SOCS-1(-/-) mice on an IFN-gamma(-/-), STAT1(-/-) and STAT6(-/-) background revealed that the fatal disease of SOCS-1(-/-) mice is also dependent on IFN-gamma/STAT1 and IL-4/STAT6 signaling pathways . Since IFN-gamma and IL-4 are representative T(h)1 and T(h)2 cytokines respectively, here we investigated the role of SOCS-1 in T(h) differentiation . Freshly isolated SOCS-1(-/-) CD4(+) T cells stimulated with anti-CD3 rapidly produced larger amounts of IFN-gamma and IL-4 than control cells, suggesting that these mutant T cells had already differentiated into T(h)1 and T(h)2 cells in vivo . In addition, SOCS-1(+/-) CD4(+) T cells cultured in vitro produced significantly larger amounts of IFN-gamma and IL-4 than SOCS-1(+/+) cells . Similarly, SOCS-1(+/-) CD4(+) T cells produced more IFN-gamma and IL-4 than SOCS-1(+/+) cells after infection with Listeria monocytogenes and Nippostrongyrus braziliensis respectively . Since IL-12-induced STAT4 and IL-4-induced STAT6 activation is sustained in SOCS-1(-/-) T cells, the enhanced T(h) functions in SOCS-1(-/-) and SOCS-1(+/-) mice appear to be due to the enhanced effects of these cytokines . These results suggest that SOCS-1 plays a regulatory role in both T(h)1 and T(h)2 polarizations. Appl Environ Microbiol, 2002 Nov, 68(11), 5698 - 703 In vitro and in vivo invasiveness of different pulsed-field gel electrophoresis types of Listeria monocytogenes; Larsen CN et al.; The virulence of different pulsed-field gel electrophoresis (PFGE) types of Listeria monocytogenes was examined by monitoring their ability to invade Caco-2 cells . Strains belonging to seven different PFGE types originating from both foods and humans were included . No significant differences in invasiveness were detected between strains isolated from humans and those isolated from food . Strains belonging to PFGE type 1 expressed a significantly lower ability to invade cells compared to strains belonging to other PFGE types . Although strains of PFGE type 2 also seemed to invade at a low level, this was not significant in the present study . PFGE types 1 and 2 as well as type 14 are more frequently found in food than the four other PFGE types examined and moreover have a relatively low prevalence in humans compared to their prevalence in food . Thus, the hypothesis that some PFGE types are less virulent than others is supported by this study showing that certain PFGE types of L . monocytogenes commonly found in food are less invasive than others to Caco-2 cells . In contrast to the differences in invasion, identical intracellular growth rates between the different PFGE types were observed . In vivo studies of the actual ability of the strains to invade the liver and spleen of cimetidine-treated rats following an oral dose of 10(9) L . monocytogenes cells were performed for isolates of PFGE types 1, 2, 5, and 15 . After 2 days, equal amounts of bacteria were observed in the liver and spleen of the rats for any of the PFGE types tested. Appl Environ Microbiol, 2002 Nov, 68(11), 5647 - 55 Gbu glycine betaine porter and carnitine uptake in osmotically stressed Listeria monocytogenes cells; Mendum ML et al.; The food-borne pathogen Listeria monocytogenes grows actively under high-salt conditions by accumulating compatible solutes such as glycine betaine and carnitine from the medium . We report here that the dominant transport system for glycine betaine uptake, the Gbu porter, may act as a secondary uptake system for carnitine, with a K(m) of 4 mM for carnitine uptake and measurable uptake at carnitine concentrations as low as 10 microM . This porter has a K(m) for glycine betaine uptake of about 6 micro M . The dedicated carnitine porter, OpuC, has a K(m) for carnitine uptake of 1 to 3 microM and a V(max) of approximately 15 nmol/min/mg of protein . Mutants lacking either opuC or gbu were used to study the effects of four carnitine analogs on growth and uptake of osmolytes . In strain DP-L1044, which had OpuC and the two glycine betaine porters Gbu and BetL, triethylglycine was most effective in inhibiting growth in the presence of glycine betaine, but trigonelline was best at inhibiting growth in the presence of carnitine . Carnitine uptake through OpuC was inhibited by gamma-butyrobetaine . Dimethylglycine inhibited both glycine betaine and carnitine uptake through the Gbu porter . Carnitine uptake through the Gbu porter was inhibited by triethylglycine . Glycine betaine uptake through the BetL porter was strongly inhibited by trigonelline and triethylglycine . These results suggest that it is possible to reduce the growth of L . monocytogenes under osmotically stressful conditions by inhibiting glycine betaine and carnitine uptake but that to do so, multiple uptake systems must be affected. Appl Environ Microbiol, 2002 Nov, 68(11), 5258 - 64 Postadaptational resistance to benzalkonium chloride and subsequent physicochemical modifications of Listeria monocytogenes; To MS et al.; Many studies have demonstrated that bacteria, including Listeria monocytogenes, are capable of adapting to disinfectants used in industrial settings after prolonged exposure to sublethal concentrations . However, the consequent alterations of the cell surface due to sanitizer adaptation of this pathogen are not fully understood . Two resistant and four sensitive L . monocytogenes strains from different sources were progressively subcultured with increasing sublethal concentrations of a surfactant, benzalkonium chloride (BC) . To evaluate the effects of acquired tolerance to BC, parent and adapted strains were compared by using several morphological and physiological tests . Sensitive strains showed at least a fivefold increase in the MIC, while the MIC doubled for resistant strains after the adaptation period . The hydrophobicities of cells of parent and adapted strains were similar . Serological testing indicated that antigen types 1 and 4 were both present on the cell surface of adapted cells . The data suggest that efflux pumps are the major mechanism of adaptation in sensitive strains and are less important in originally resistant isolates . A different, unknown mechanism was responsible for the original tolerance of resistant isolates . In an originally resistant strain, there was a slight shift in the fatty acid profile after adaptation, whereas sensitive strains had similar profiles . Electron micrographs revealed morphological differences after adaptation . The changes in cell surface antigens, efflux pump utilization, and fatty acid profiles suggest that different mechanisms are used by resistant and sensitive strains for adaptation to BC. Appl Environ Microbiol, 2002 Nov, 68(11), 5223 - 30 Membranes of class IIa bacteriocin-resistant Listeria monocytogenes cells contain increased levels of desaturated and short-acyl-chain phosphatidylglycerols; Vadyvaloo V et al.; A major concern in the use of class IIa bacteriocins as food preservatives is the well-documented resistance development in target Listeria strains . We studied the relationship between leucocin A, a class IIa bacteriocin, and the composition of the major phospholipid, phosphatidylglycerol (PG), in membranes of both sensitive and resistant L . monocytogenes strains . Two wild-type strains, L . monocytogenes B73 and 412, two spontaneous mutants of L . monocytogenes B73 with intermediate resistance to leucocin A (+/-2.4 and +/-4 times the 50% inhibitory concentrations {IC50} for sensitive strains), and two highly resistant mutants of each of the wild-type strains (>500 times the IC50 for sensitive strains) were analyzed . Electrospray mass spectrometry analysis showed an increase in the ratios of unsaturated to saturated and short- to long-acyl-chain species of PG in all the resistant L . monocytogenes strains in our study, although their sensitivities to leucocin A were significantly different . This alteration in membrane phospholipids toward PGs containing shorter, unsaturated acyl chains suggests that resistant strains have cells with a more fluid membrane . The presence of this phenomenon in a strain (L . monocytogenes 412P) which is resistant to both leucocin A and pediocin PA-1 may indicate a link between membrane composition and class IIa bacteriocin resistance in some L . monocytogenes strains . Treatment of strains with sterculic acid methyl ester (SME), a desaturase inhibitor, resulted in significant changes in the leucocin A sensitivity of the intermediate-resistance strains but no changes in the sensitivity of highly resistant strains . There was, however, a decrease in the amount of unsaturated and short-acyl-chain PGs after treatment with SME in one of the intermediate and both of the highly resistant strains, but the opposite effect was observed for the sensitive strains . It appears, therefore, that membrane adaptation may be part of a resistance mechanism but that several resistance mechanisms may contribute to a resistance phenotype and that levels of resistance vary according to the type of mechanisms present. Mol Microbiol, 2002 Oct, 46(2), 367 - 79 Critical role of the N-terminal residues of listeriolysin O in phagosomal escape and virulence of Listeria monocytogenes; Lety MA et al.; A putative PEST sequence was recently identified close to the N-terminus of listeriolysin O (LLO), a major virulence factor secreted by the pathogenic Listeria monocytogenes . The deletion of this motif did not affect the secretion and haemolytic activity of LLO, but abolished bacterial virulence . Here, we first tested whether the replacement of the PEST motif of LLO by two different sequences, with either a very high or no PEST score, would affect phagosomal escape, protein stability and, ultimately, the virulence of L . monocytogenes . Then, we constructed LLO mutants with an intact PEST sequence but carrying mutations on either side, or on both sides, of the PEST motif . The properties of these mutants prompted us to construct three LLO mutants carrying single amino acid substitutions in the distal portion of the PEST region (P49A, K50A and P52A; preprotein numbering) . Our data demonstrate that the susceptibility of LLO to intracellular proteolytic degradation is not related to the presence of a high PEST score sequence and that the insertion of two residues immediately downstream of the intact PEST sequence is sufficient to impair phagosomal escape and abolish bacterial virulence . Furthermore, we show that single amino acid substitutions in the distal portion of the PEST motif are sufficient to attenuate bacterial -virulence significantly, unravelling the critical role of this region of LLO in the pathogenesis of L . -monocytogenes. Virchows Arch, 2002 Oct, 441(4), 368 - 79 Epub 2002 Jul 27. Effects of pregnancy-associated Listeria monocytogenes infection: necrotizing hepatitis due to impaired maternal immune response and significantly increased abortion rate; Abram M et al.; The impact of L . monocytogenes infection on maternal immune responses as well as on the outcome of pregnancy was studied in a murine model of pregnancy-associated listeriosis . Mice infected i.v . with L . monocytogenes at day 15 of pregnancy showed a significantly impaired bacterial elimination, which resulted in a severe necrotizing hemorrhagic hepatitis . The aggravated course of the infection could be attributed to a suppressed transcription and production of anti-listerial, pro-inflammatory cytokines and chemokines, namely interferon-gamma, tumor necrosis factor, interleukin-12p40, inducible nitric oxide synthase, murine monokine induced by interferon-gamma, and interferon-gamma-inducible protein-10 . In addition, listeriosis significantly increased the abortion rate . Infection of the placenta and fetuses was characterized by placental and fetal necrosis with unrestricted bacterial multiplication . A weak transcription of anti-listerial cytokines in the placenta in the absence of a cellular immune response could not prevent the fatal outcome of pregnancy-associated listeriosis. FEMS Microbiol Lett, 2002 Oct 8, 215(2), 183 - 8 A proteomic analysis of the salt stress response of Listeria monocytogenes; Duche O et al.; Protein variations in Listeria monocytogenes were analyzed by 2-D electrophoresis . Bacteria were grown either in a rich medium or in a chemically defined medium . Three proteins, which are more expressed in the chemically defined medium than in the rich medium, were identified by mass spectrometry . They are closely related to AppA, Ctc and YvyD . After an osmotic shock, according to the medium and the NaCl concentration, the synthesis rate (P<0.05) of 59 proteins is altered by salinity . Half of them were more expressed, some of these proteins were closely related to Ctc, GbuA and the 30S ribosomal protein S6 . Among the proteins which were down-expressed in the presence of salt, two were similar to AckA and PdhD. J Immunol, 2002 Nov 1, 169(9), 5202 - 8 Perforin-mediated CTL cytolysis counteracts direct cell-cell spread of Listeria monocytogenes; San Mateo LR et al.; The immune system has evolved various effector cells and functions to combat diverse infectious agents equipped with different virulence strategies . CD8 T cells play a critical role in protective immunity to Listeria monocytogenes (Lm), a bacterium that grows within the host cell cytosol and spreads directly into neighboring cells . The importance of CD8 T cells during Lm infection is currently attributed to the cytosolic niche of this organism, which allows it to evade many aspects of immune surveillance . CTL lysis of infected cells is believed to be an essential protective mechanism, presumably functioning to release intracellular bacteria, although its precise role remains to be fully defined . In this study, we examined the contribution of perforin-mediated CTL cytolysis to protective immunity against recombinant Lm capable of or defective in cell-cell spread . We found that CTL cytolysis is critical for protective immunity to Lm capable of cell-cell spread while protective immunity against spread-defective Lm is largely independent of CTL cytolysis . These results demonstrate that an important function of CTL cytolysis is to counter the microbial virulence strategy of direct cell-cell spread . We propose a model that advances the current view of the role of CTL cytolysis in immunity to intracellular pathogens. J Immunol, 2002 Nov 1, 169(9), 4936 - 44 Quantitation of CD8+ T cell expansion, memory, and protective immunity after immunization with peptide-coated dendritic cells; Hamilton SE et al.; Dendritic cells (DCs) are potent APCs for naive CD8(+) T cells and are being investigated as vaccine delivery vehicles . In this study, we examine the CD8(+) T cell response to defined peptides from Listeria monocytogenes (LM), lymphocytic choriomeningitis virus, and murine CMV coated singly and in combination onto mature bone marrow-derived DCs (BMDCs) . We show that immunization of mice with 2 x 10(5) mature BMDCs coated with multiple MHC class I peptides generates a significant Ag-specific CD8(+) T cell response in both the spleen and nonlymphoid organs . This immunization resulted in a peptide-specific hierarchy in the magnitude of CD8(+) T cell priming and noncoordinate kinetics in response to different peptide epitopes . Kinetics were not exclusively due to specific characteristics of the MHC class I molecule, and were not altered in an Ag-independent manner by concurrent LM infection . Mice immunized with listeriolysin O 91-99-coated BMDCs are protected against high dose challenge with virulent LM . This protection was enhanced by diversifying the memory CD8(+) T cell compartment, even in the absence of a large increase in Ag-specific CD8(+) memory T cells. Mol Cell Biol, 2002 Nov, 22(22), 8035 - 43 Inactivation of the F4/80 glycoprotein in the mouse germ line; Schaller E et al.; Macrophages play a crucial role in the defense against pathogens . Distinct macrophage populations can be defined by the expression of restricted cell surface proteins . Resident tissue macrophages, encompassing Kupffer cells of the liver and red pulp macrophages of the spleen, characteristically express the F4/80 molecule, a cell surface glycoprotein related to the seven transmembrane-spanning family of hormone receptors . In this study, gene targeting was used to simultaneously inactivate the F4/80 molecule in the germ line of the mouse and to produce a mouse line that expresses the Cre recombinase under the direct control of the F4/80 promoter (F4/80-Cre knock-in) . F4/80-deficient mice are healthy and fertile . Macrophage populations in tissues can develop in the absence of F4/80 expression . Functional analysis revealed that the generation of T-cell-independent B-cell responses and macrophage antimicrobial defense after infection with Listeria monocytogenes are not impaired in the absence of F4/80 . Interestingly, tissues of F4/80-deficient mice could not be labeled with anti-BM8, another macrophage subset-specific marker with hitherto undefined molecular antigenic structure . Recombinant expression of a F4/80 cDNA in heterologous cells confirmed this observation, indicating that the targets recognized by the F4/80 and BM8 monoclonal antibodies are identical. Isr Med Assoc J, 2002 Oct, 4(10), 776 - 80 Listeria infection during pregnancy: a 10 year experience; Benshushan A et al.; BACKGROUND: Although Listeria monocytogenes is widely distributed in nature, it rarely causes clinical infection in previously healthy people . This microorganism, however, may cause severe invasive disease in pregnant women and newborns . OBJECTIVES: To investigate--in our pregnant population--the impact, severity and outcome of listeriosis on both mother and fetus . METHOD: The study was carried out at a level III, university two-hospital complex . In a retrospective chart review of 65,022 parturients during a 10 year period (1990-1999), we identified and evaluated 11 pregnant patients and their offspring with Listeria infection . RESULTS: Chorioamnionitis with multiple placental abscesses were observed in all five placentae examined . Clinically, 4 of 11 parturients had a cesarean section for fetal distress (36.3%), as compared to the 14% mean CS rate in our general population . Two of 11 had a late abortion (18.1%), as compared with the 4% rate in our hospital . Four of 11 had premature labor (36%), which was about four times the rate in our population . Finally, although no intrauterine fetal death was recorded in our series, there was one neonatal death of a term infant (1/11, 9%), which is about 10 times higher than our corrected perinatal mortality rate . CONCLUSIONS: If not promptly and adequately treated, listeriosis in pregnancy may present serious hazards to the fetus and newborn through direct infection of the placenta and chorioamnionitis. Rev Neurol, 2002 Sep 16-30, 35(6), 508 - 12 {A study of the incidence and a descriptive analysis of adult non-tuberculous primary bacterial meningitis in a population in Argentina}; Barboza AG et al.; INTRODUCTION: Adult non tuberculous primary bacterial meningitis (PBM) represents an important cause of morbidity and mortality in hospitals . The shortage of studies based on the population in Latin America provided the motivation for this work . AIMS: To determine the incidence of PBM in the captive population of our hospital and carry out a descriptive analysis of the cases detected . PATIENTS AND METHODS: We performed an epidemiological study of the captive population (CP) of the hospital (an average of 85,200 patients in 11 years) and a retrospective descriptive examination of patients who had been admitted . The clinical histories of all patients over the age of 18 who had been admitted with PBM between 1 January 1988 and 31 December 1998 were studied . RESULTS and CONCLUSIONS: A total number of 87 cases of primary bacterial meningitis were registered, of which 70 belonged to the CP . The overall gross rate of PBM incidence in the CP was 8.6/100,000 per year . The annual incidence rate, adjusted to the 1991 National Census on the Argentinean Population, was 5.4/100,000 per year, with a greater frequency between the ages of 70 and 79: 21/100,000 per year . Median age: 73 (lower quartile, 66; upper quartile, 78) . Clinical manifestations included high temperatures (90%), consciousness disorders (87%), and a stiff neck (81%) . The frequency with which it appeared remained constant over the 11 year period, without showing any seasonal variations . The most frequent etiological agent was pneumococcus (50%) . No cases of PBM by Listeria were reported . Overall fatality during the stay in hospital was 23%, without any type of modification over the period we studied. Toxicol Sci, 2002 Nov, 70(1), 110 - 9 Residual oil fly ash increases the susceptibility to infection and severely damages the lungs after pulmonary challenge with a bacterial pathogen; Antonini JM et al.; Inhalation of residual oil fly ash (ROFA), a component of ambient particulate matter, has been shown to increase pulmonary morbidity and impair lung defense mechanisms in exposed workers . Our objective was to evaluate the effect of ROFA preexposure on lung defense and injury after pulmonary challenge with a bacterial pathogen . Male Sprague-Dawley rats were dosed intratracheally at day 0 with saline (control) or ROFA (0.2 or 1 mg/100 g body weight) . Three days later, a low (5 x 10(3)) or high (5 x 10(5)) dose of Listeria monocytogenes was instilled intratracheally into the ROFA- and saline-treated rats . Bronchoalveolar lavage was performed on the right lungs at days 6, 8, and 10 . The recovered cells were differentiated, and chemiluminescence (CL) and nitric oxide (NO) production, two indices of alveolar macrophage (AM) function, were measured . At the same time points, the left lung and spleen were removed, homogenized, and cultured, and colony-forming units were counted after an overnight incubation . Exposure to ROFA and the high dose of L . monocytogenes led to marked lung injury and inflammation as well as to an increase in mortality, compared with rats treated with saline and the high dose of L . monocytogenes . Preexposure to ROFA significantly enhanced injury and delayed the pulmonary clearance of L . monocytogenes at both bacterial doses when compared to the saline-treated control rats . ROFA had no effect on AM CL but caused a significant suppression of AM NO production, as compared to the saline control rats . We have demonstrated that acute exposure to ROFA slowed the pulmonary clearance of L . monocytogenes . The suppression in AM NO production by ROFA pretreatment likely plays an important role . These results suggest that pulmonary exposure to ROFA may alter AM function and lead to increased susceptibility to lung infection in exposed populations. Blood, 2002 Nov 1, 100(9), 3175 - 82 NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse model for engraftment of human cells; Ito M et al.; To establish a more appropriate animal recipient for xenotransplantation, NOD/SCID/gamma(c)(null) mice double homozygous for the severe combined immunodeficiency (SCID) mutation and interleukin-2Rgamma (IL-2Rgamma) allelic mutation (gamma(c)(null)) were generated by 8 backcross matings of C57BL/6J-gamma(c)(null) mice and NOD/Shi-scid mice . When human CD34+ cells from umbilical cord blood were transplanted into this strain, the engraftment rate in the peripheral circulation, spleen, and bone marrow were significantly higher than that in NOD/Shi-scid mice treated with anti-asialo GM1 antibody or in the beta2-microglobulin-deficient NOD/LtSz-scid (NOD/SCID/beta2m(null)) mice, which were as completely defective in NK cell activity as NOD/SCID/gamma(c)(null) mice . The same high engraftment rate of human mature cells was observed in ascites when peripheral blood mononuclear cells were intraperitoneally transferred . In addition to the high engraftment rate, multilineage cell differentiation was also observed . Further, even 1 x 10(2) CD34+ cells could grow and differentiate in this strain . These results suggest that NOD/SCID/gamma(c)(null) mice were superior animal recipients for xenotransplantation and were especially valuable for human stem cell assay . To elucidate the mechanisms involved in the superior engraftment rate in NOD/SCID/gamma(c)(null) mice, cytokine production of spleen cells stimulated with Listeria monocytogenes antigens was compared among these 3 strains of mice . The interferon-gamma production from dendritic cells from the NOD/SCID/gamma(c)(null) mouse spleen was significantly suppressed in comparison with findings in 2 other strains of mice . It is suggested that multiple immunological dysfunctions, including cytokine production capability, in addition to functional incompetence of T, B, and NK cells, may lead to the high engraftment levels of xenograft in NOD/SCID/gamma(c)(null) mice. Gene, 2002 Aug 21, 296(1-2), 121 - 8 The expression of the dodecameric ferritin in Listeria spp . is induced by iron limitation and stationary growth phase; Polidoro M et al.; The Gram-positive bacterium Listeria innocua possesses an authentic ferritin with an unusual dodecameric assemblage that resembles the quaternary structure of the DNA-binding proteins designated Dps (DNA-binding proteins from starved cells) . The L . innocua gene encoding the above protein, termed ferritin from Listeria innocua (fri), has been localized on a 3-kb HindIII chromosomal fragment cloned in the Escherichia coli strain DH5alphaF' . DNA sequence analysis reveals an open reading frame of 468 nucleotides matching perfectly the amino acid sequence of the protein . Primer extension analysis indicates the presence of two transcriptional startpoints located 36 (proximal) and 85 nt (distal) upstream the fri start codon, respectively . Each transcriptional startpoint is preceded by suitably located -10 and -35 elements, which match the sigma(A) (proximal) and sigma(B) (distal) consensus sequences.In L . innocua and Liseria monocytogenes, fri expression increases both upon entry into stationary phase and, more markedly, under low-iron growth conditions . The effect of iron is apparent in the exponential and stationary phases of growth . An up-regulation by iron limitation has never been observed in other proven ferritins and bacterioferritins, but has been reported for several members of the Dps family . The unusual regulation by iron of the Listeria ferritin gene provides further support to the evolutionary link with the Dps family and suggests that the iron storage function may not be the unique role of ferritin in the physiology of this bacterium. Biochem J, 2003 Feb 1, 369(Pt 3), 681 - 5 Cd2+ and the N-terminal metal-binding domain protect the putative membranous CPC motif of the Cd2+-ATPase of Listeria monocytogenes; Bal N et al.; CadA, the Cd(2+)-ATPase of Listeria monocytogenes, contains four cysteine residues: two in the CTNC (Cys-Thr-Asn-Cys) sequence in the cytoplasmic metal-binding domain (MBD), and two in the CPC (Cys-Pro-Cys) sequence in the membrane domain . Taking advantage of DeltaMBD, a truncated version of CadA that lacks the MBD but which still acts as a functional Cd(2+)-ATPase {Bal, Mintz, Guillain and Catty (2001) FEBS Lett . 506, 249-252}, we analysed the role of the membrane cysteine residues (studied using DeltaMBD) separately from that of the cysteine residues of the MBD, which were studied using full-length CadA . The role of the cysteines was assessed by reacting DeltaMBD and CadA with N -ethylmaleimide (NEM), an SH-specific reagent, in the presence or absence of Cd(2+) . We show here that (i) in both DeltaMBD and CadA, the cysteine residues in the CPC motif are essential for phosphorylation; (ii) in both proteins, Cd(2+) protects against alkylation by NEM; and (iii) in the absence of Cd(2+), the MBD of CadA also protects against alkylation by NEM . Our results suggest that the CPC motif is present in the membrane Cd(2+) transport site(s) and that the MBD protects these site(s). Int J Food Microbiol, 2002 Nov 15, 79(1-2), 47 - 53 Physiological damages of Listeria monocytogenes treated by high hydrostatic pressure; Ritz M et al.; High hydrostatic pressure is a new food preservation technology known for its capacity to inactivate spoilage and pathogenic microorganisms . This study investigated the damages inflicted on Listeria monocytogenes cells treated by high pressure for 10 min at 400 MPa in pH 5.6 citrate buffer . Under these conditions, no cell growth occurred after 48 h on plate count agar . Scanning electron microscopy (SEM) revealed that cellular morphology was not really affected . Measuring propidium iodide (PI) staining followed by flow cytometry demonstrated that membrane integrity was damaged in a small part of the population, although the membrane potential evaluated by oxonol fluorescence or measured by analytical methods was reduced from - 86 to - 5 mV . These results for the first time showed that such combined methods as fluorescent dyes monitored by flow cytometry and physiological activity measurements provide valuable indications on cellular viability. Crit Care Nurse, 2002 Oct, 22(5), 38 - 43 Caring for a patient with Listeria endocarditis: use of antibiotic desensitization; Candela L; Occurrence of Listeria endocarditis is rare, and the mortality rate is high, 100% in untreated cases . The use of antibiotics, specifically ampicillin, is considered a first-line treatment . Coadministration of ampicillin and gentamicin provides a synergistic effect in killing the bacteria . Antibiotics are among the most common causes of hypersensitivity reactions . Of all antibiotics, penicillin is the one that most often causes a reaction . Skin testing adds time until treatment, and all patients with sensitivity to penicillin may not be detected . In the case presented, the patient had antibiotic desensitization with ampicillin . He did not have any allergic reactions to the drug . However, his history of allergy to penicillin was uncertain, so perhaps he did not have a true, serious penicillin allergy . Also, most likely he was anergic and could not mount an immune response to ampicillin, even if truly allergic . Therefore, his response may not be a typical response to antibiotic desensitization . Understanding possible hypersensitivity reactions can help guide the medical and nursing management of patients having antibiotic desensitization. J Food Prot, 2002 Oct, 65(10), 1663 - 6 Inactivation of Listeria monocytogenes Scott A 49594 in apple juice supplemented with cinnamon; Yuste J et al.; Normal (pH 3.7) and adjusted (pH 5.0) pasteurized apple juice containing cinnamon (0, 0.1, 0.2, and 0.3%) was inoculated with Listeria monocytogenes Scott A 49594 at 10(4) CFU/ml and stored at 5 and 20 degrees C for 7 days . Counts on tryptic soy agar (TSA), modified Oxford (MOX) medium, and thin agar layer (TAL) were determined at 1 h and 1, 3, and 7 days . The TAL method (MOX medium overlaid with TSA) was used for the recovery of injured cells . In apple juice, both at normal and adjusted pH, with any doses of cinnamon, no L . monocytogenes (a 4.6-log CFU/ml reduction) was detected after 1 h of storage at both temperatures, and no growth occurred at any points of storage . Therefore, cinnamon by itself (regardless of pH) had a pronounced killing effect . A further enrichment step with brain heart infusion agar showed that L monocytogenes was completely inactivated in apple juice stored at 20 degrees C, except in pH 5.0 samples with 0.1% of cinnamon . The TAL method was as effective as TSA in recovering injured cells of L . monocytogenes . Cinnamon considerably inactivates L . monocytogenes in apple juice and thus enhances the safety of this product. J Food Prot, 2002 Oct, 65(10), 1574 - 9 Molecular characterization of Listeria monocytogenes isolated from a poultry further processing facility and from fully cooked product; Berrang ME et al.; This study was undertaken to explore environmental sources of Listeria monocytogenes in a commercial chicken further processing facility and to compare the isolates obtained from this facility with others obtained from fully cooked product . In a survey conducted at the processing facility, 40 environmental sites (encompassing two production lines and representing areas in which raw and cooked products are processed) were cultured for L . monocytogenes . The resulting isolates were subjected to molecular subtyping by ribotyping, and these isolates were compared with 25 isolates collected by plant personnel from product contact surfaces and from fully cooked product . Eighty-nine environmental and product isolates were divided into 15 distinct ribogroups . Two ribogroups included isolates from fully cooked product; the members of these two ribogroups were subjected to further analysis by pulsed-field gel electrophoresis, resulting in four clusters . L . monocytogenes isolates from fully cooked product produced on line 1 were found to be indistinguishable from isolates collected from (i) drains on the raw-product side of line 1 and (ii) the floor surface in the cooked-product area of line 1 . L . monocytogenes isolates from fully cooked product from line 2 were found to be indistinguishable from isolates collected from (i) the spiral freezer exit conveyor on line 2, (ii) raw product contact surfaces on line 1, and (iii) drains in the cooked-product area of line 1 . These data suggest that L . monocytogenes can colonize a poultry further processing facility and eventually be transferred to fully cooked product. Br J Oral Maxillofac Surg, 2002 Oct, 40(5), 442 - 3 Oral cancer, fever of unknown origin, and listeriosis; Morritt AN et al.; Listeriosis is a rare cause of fever of unknown origin in patients with oral cancer . We report two patients who, because of pain and discomfort, ate large quantities of soft cheeses; this caused listeriosis and fever . Both cases responded to high doses of amoxycillin. Trop Anim Health Prod, 2002 Sep, 34(5), 359 - 81 Listeric infections in humans and animals in the Indian subcontinent: a review; Malik SV et al.; Listeriosis is an important bacterial zoonosis caused by the intracellular pathogen Listeria monocytogenes . The disease has been reported in animals from the Indian subcontinent, usually in the form of sporadic cases but occasionally as outbreaks . Cases of listeriosis arise mainly from the ingestion of contaminated food . Listeriosis has been reported to cause encephalitis, abortion, mastitis, repeat breeding and endometriosis in animals . Listeric infections occur in children and women with a poor obstetric history . The epidemiological aspects and pathogenesis of listeriosis in animals and humans are not yet fully understood . This review offers comprehensive information on experimental studies and field cases in animals and on cases of human listeriosis . There are also sections on isolation from foods, diagnosis and treatment in humans and animals. Gene Ther, 2002 Nov, 9(21), 1455 - 63 A recombinant E . coli vaccine to promote MHC class I-dependent antigen presentation: application to cancer immunotherapy; Radford KJ et al.; We have examined the potential of recombinant Escherichia coli expressing listeriolysin O (LLO) to deliver tumour antigens to dendritic cells (DCs) for cancer immunotherapy . Using OVA as a model tumour antigen, we have shown in murine DCs that E . coli expressing cytoplasmic LLO and OVA proteins can deliver the OVA K(b)-restricted epitope SIINFEKL for MHC class I presentation . In contrast, when E . coli expressing OVA alone were used, MHC class II presentation of the OVA 323-339 I-A(b)-restricted peptide was predominant . When injected in vivo, DCs pulsed with E . coli expressing LLO and OVA induced production of cytotoxic T-lymphocytes capable of lysing an OVA-expressing melanoma cell line (B16-OVA) and resulted in suppression of tumour growth following challenge with B16-OVA . Immunisation of mice by direct injection of E . coli LLO/OVA provided a more potent anti-tumour response, resulting in complete protection in 75% of mice . Injection of live bacteria was not necessary as immunisation with paraformaldehyde-fixed E . coli LLO/OVA provided an even stronger anti-tumour response against B16-OVA . Altogether, our data highlight the potential of this system as a novel and efficient strategy for tumour immunotherapy. Immunopharmacol Immunotoxicol, 2002 Aug, 24(3), 483 - 96 Effects of Chlorella vulgaris extract on cytokines production in Listeria monocytogenes infected mice; Queiroz ML et al.; In this study, we have investigated the effects of the unicellular-green-algae Chlorella vulgaris on the production of INF-gamma, IL-2, IL-4 and IL-10 in normal and Listeria monocytogenes infected mice . Our results demonstrated that in normal/non infected mice, CVE administration produced no effects in the levels of all cytokines studied . However, Listeria monocytogenes infection enhanced the production of INF-gamma and IL-2 at 48 and 72 h after the bacteria inoculation . Interestingly, the treatment with five consecutive doses of 50 mg/Kg/day of Chlorella vulgaris given previously to infection, led to further increases in INF-gamma and IL-2 levels at 48 and 72 h in relation to the presence of infection alone . No changes in IL-4 and IL-10 production were observed in Listeria monocytogenes and CVE treated/infected mice . These results are in accordance with the literature, which shows that CVE is a biological response modifier that enhances resistance to Listeria monocytogenes through augmentation of IL-2 and IFN-gamma. J Bacteriol, 2002 Nov, 184(21), 5935 - 45 Inducible control of virulence gene expression in Listeria monocytogenes: temporal requirement of listeriolysin O during intracellular infection; Dancz CE et al.; We have constructed a lac repressor/operator-based system to tightly regulate expression of bacterial genes during intracellular infection by Listeria monocytogenes . An L . monocytogenes strain was constructed in which expression of listeriolysin O was placed under the inducible control of an isopropyl-beta-D-thiogalactopyranoside (IPTG)-dependent promoter . Listeriolysin O (LLO) is a pore-forming cytolysin that mediates lysis of L . monocytogenes-containing phagosomes . Using hemolytic-activity assays and Western blot analysis, we demonstrated dose-dependent IPTG induction of LLO during growth in broth culture . Moreover, intracellular growth of the inducible-LLO (iLLO) strain in the macrophage-like cell line J774 was strictly dependent upon IPTG . We have further shown that iLLO bacteria trapped within primary phagocytic vacuoles can be induced to escape into the cytosol following addition of IPTG to the cell culture medium, thus yielding the ability to control bacterial escape from the phagosome and the initiation of intracellular growth . Using the iLLO strain in plaque-forming assays, we demonstrated an additional requirement for LLO in facilitating cell-to-cell spread in L2 fibroblasts, a nonprofessional phagocytic cell line . Furthermore, the efficiency of cell-to-cell spread of iLLO bacteria in L2 cells was IPTG dose dependent . The potential use of this system for determining the temporal requirements of additional virulence determinants of intracellular pathogenesis is discussed. J AOAC Int, 2002 Sep-Oct, 85(5), 1201 - 3 Comparison of visual immunoassay and chromogenic culture medium for the presence of Listeria spp . in foods; Istafanos P et al.; Two rapid screening methods {the TECRA Listeria Visual Immunoassay (LIS-VIS) kit, an AOAC-approved 48 h visual test, which detects Listeria through colorimetry, and BCM Listeria isolation and differentiation plating agar} were used to screen U.S . Food and Drug Administration-regulated commodities for the presence of Listeria spp . Seventy-four different food samples were screened for the presence of Listeria spp . by using both protocols . Test results for the TECRA LIS-VIA showed 66 negative samples and 1 false positive, with 4 confirmed as L . monocytogenes and 3 as L . innocua . With the BCM agar, 67 samples were negative, 4 were confirmed as L . monocytogenes, and 3 were confirmed as L . innocua . Both methods showed similar results and were effective screening tools for Listeria spp . in foods . The BCM agar method proved to be a rapid, sensitive, and excellent tool for early screening and differentiation of Listeria spp . present in foods. Eur J Immunol, 2002 Oct, 32(10), 2807 - 16 Contribution of CD8+ T cells to innate immunity: IFN-gamma secretion induced by IL-12 and IL-18; Berg RE et al.; The role of CD8+ T cells in adaptive immunity is well documented and involves numerous effector mechanisms including direct cytolysis of targets and secretion of cytokines . The role of CD8+ T cells in innate immunity has not been previously appreciated . Using J774 macrophages infected in vitro with the intracellular bacterium, Listeria monocytogenes (LM), we show that CD8+ T cells isolated from naive C57BL/6 (B6) mice respond rapidly by secreting IFN-gamma . CD8+ T cells secreting IFN-gamma can also be found in naive B6 mice 16 h after infection with LM . This rapid IFN-gamma response is TCR-independent and mediated through the actions of IL-12 and IL-18 . Cell surface staining and cell sorting experiments indicate that these novel CD8+ T cells express memory markers . In vitro CFSE-labeling experiments show that IFN-gamma-secreting CD8+ T cells proliferate rapidly after 2 days in culture and after 4 days constitute the majority of the CD8+ T cell population . Together, these data suggest an important role for IFN-gamma-secreting CD8+ T cells in the innate response to bacterial pathogens. Clin Infect Dis, 2002 Oct 15, 35(8), 943 - 9 Epub 2002 Sep 23. An outbreak of febrile gastroenteritis associated with delicatessen meat contaminated with Listeria monocytogenes; Frye DM et al.; In June 2001, the Los Angeles County Department of Health Services/Public Health conducted a cohort study of an outbreak of acute febrile gastroenteritis among 16 of 44 healthy attendees of a catered party . The median age of the attendees who became ill was 15.5 years . Symptoms included body aches (in 88% of attendees), fever (81%), headache (81%), diarrhea (63%), and vomiting (56%) . Illness was associated with ingestion of precooked, sliced turkey (P=.000004) . Six stool specimens yielded Listeria monocytogenes . Leftover turkey yielded L . monocytogenes, 1.6x10(9) cfu/g . All isolates were serotype 1/2a and had matching molecular fingerprints . Clusters of suspect cases were identified among attendees at 2 other catered events, but no additional cases were confirmed . This is only the third reported outbreak of L . monocytogenes-associated gastroenteritis in the United States . In cases of febrile gastroenteritis for which routine cultures for enteric pathogens are negative, clinicians should suspect listeriosis and should consider asking laboratories to retain stool specimens to expedite testing for Listeria organisms. J Comp Pathol, 2002 Aug-Oct, 127(2-3), 178 - 85 Metastatic Listeria monocytogenes infection of the peritoneum in mice with cyclosporine a-induced peritonitis; Prats N et al.; Inoculation of mice with Listeria monocytogenes intragastrically or by parenteral routes has not been reported to cause peritonitis . In this study, however, severe listerial peritonitis was induced in mice infected subcutaneously and treated intraperitoneally with cyclosporin A (Cs A) in an oil carrier . In both uninfected and listeria-infected mice, intraperitoneal administration of Cs A consistently produced overexpression of P-selectin in the peritoneal microvasculature and pyogranulomatous inflammation of the peritoneum, suggesting that Cs A causes endothelial damage . We suggest that in listeria-infected mice the non-specific irritant peritonitis induced by the intraperitoneal administration of Cs A results in transfer of listeria-infected phagocytes from the liver and spleen to the peritoneal microvasculature, producing metastatic infection. Appl Environ Microbiol, 2002 Oct, 68(10), 4876 - 83 Glucose and nutrient concentrations affect the expression of a 104-kilodalton Listeria adhesion protein in Listeria monocytogenes; Jaradat ZW et al.; Growth media and environmental conditions influence the expression of adhesion and invasion proteins in Listeria monocytogenes . Here, the expression of the 104-kDa Listeria adhesion protein (LAP) was studied in nutrient-rich media (Trypticase soy broth {TSB} and brain heart infusion {BHI}), minimal medium (Luria-Bertani {LB}), or nutrient-deficient medium (peptone water {PW}) by immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy . Also, the effect of incorporating different concentrations of glucose on LAP expression was studied . Immunoblotting showed that LAP expression was at least twofold higher in LB medium than in TSB or BHI, while PW supported very poor cell growth and LAP expression . ELISA and immunoblotting results showed that higher concentrations of glucose (>1.6 g/liter) lowered the culture pH and suppressed LAP expression by more than 75%; however, the addition of K(2)HPO(4) reduced this effect . L . monocytogenes cells grown in LB media with lower concentrations of glucose showed higher adhesion to Caco-2 cells (3,716 and 4,186 cpm of attached bacteria for 0 and 0.2 g of glucose/liter, respectively), while L . monocytogenes cells grown in LB with higher glucose concentrations exhibited lower adhesion (2,126 and 2,221 cpm for 1.6 and 3.2 g of glucose/liter, respectively) . A LAP-negative L . monocytogenes strain (A572) showed low adhesion profiles regardless of the amount of glucose added . Transmission electron microscopy revealed that LAP is localized mainly in the cytoplasm, with only a few molecules located on the cell surface . Growth in LB with high glucose (3.2 g/liter) showed the presence of only a few molecules in the cells, corroborating the results observed with ELISA or immunoblotting . In summary, nutrient-rich media and high concentrations of glucose suppressed LAP expression, which possibly is due to the changes in the pH of the media during growth from the accumulation of sugar fermentation by-products. Appl Environ Microbiol, 2002 Oct, 68(10), 4710 - 6 Multiple deletions of the osmolyte transporters BetL, Gbu, and OpuC of Listeria monocytogenes affect virulence and growth at high osmolarity; Wemekamp-Kamphuis HH et al.; The success of Listeria monocytogenes as a food-borne pathogen owes much to its ability to survive a variety of stresses, both in the food environment and, after ingestion, within the animal host . Growth at high salt concentrations is attributed mainly to the accumulation of organic solutes such as glycine betaine and carnitine . We characterized L . monocytogenes LO28 strains with single, double, and triple deletions in the osmolyte transport systems BetL, Gbu, and OpuC . When single deletion mutants were tested, Gbu was found to have the most drastic effect on the rate of growth in brain heart infusion (BHI) broth with 6% added NaCl . The highest reduction in growth rate was found for the triple mutant LO28BCG (DeltabetL DeltaopuC Deltagbu), although the mutant was still capable of growth under these adverse conditions . In addition, we analyzed the growth and survival of this triple mutant in an animal (murine) model . LO28BCG showed a significant reduction in its ability to cause systemic infection following peroral coinoculation with the wild-type parent . Altering OpuC alone resulted in similar effects (R . D . Sleator, J . Wouters, C . G . M . Gahan, T . Abee, and C . Hill, Appl . Environ . Microbiol . 67:2692-2698, 2001), leading to the assumption that OpuC may play an important role in listerial pathogenesis . Analysis of the accumulation of osmolytes revealed that betaine is accumulated up to 300 micro mol/g (dry weight) when grown in BHI broth plus 6% NaCl whereas no carnitine accumulation could be detected . Radiolabeled-betaine uptake studies revealed an inability of BGSOE (DeltabetL Deltagbu) and LO28BCG to transport betaine . Indeed, for LO28BCG, no accumulated betaine was found, but carnitine was accumulated in this strain up to 600 micro mol/g (dry weight) of cells, indicating the presence of a possible fourth osmolyte transporter. J Immunol, 2002 Oct 1, 169(7), 3869 - 75 MyD88-dependent but Toll-like receptor 2-independent innate immunity to Listeria: no role for either in macrophage listericidal activity; Edelson BT et al.; We have assessed the requirements for Toll-like receptor (TLR) signaling in vivo during early infection with Listeria monocytogenes . Mice deficient for TLR2, a receptor required for the recognition of Gram-positive peptidoglycan, showed equivalent Listeria resistance to wild-type mice . However, mice deficient for MyD88, an adaptor molecule used by all TLRs, showed profound susceptibility with 3-4 logs greater Listeria burden and severe spleen and liver pathology at day 3 postinfection . Listeria-infected MyD88-deficient mice also showed markedly diminished IFN-gamma, TNF-alpha, and NO responses, despite evidence of macrophage activation and up-regulation of MHC class II molecules . We demonstrate that although minor MyD88-independent responses to live Listeria do occur, these are insufficient for normal host defense . Lastly, we performed experiments in vitro in which macrophages deficient in TLR2 or MyD88 were directly infected with Listeria: Although TLR signaling was required for macrophage NO and cytokine production in response to Listeria, handling and direct killing of Listeria by activated macrophages occurred by TLR2- and MyD88-independent mechanisms. J Immunol, 2002 Oct 1, 169(7), 3863 - 8 Critical roles of myeloid differentiation factor 88-dependent proinflammatory cytokine release in early phase clearance of Listeria monocytogenes in mice; Seki E et al.; Listeria monocytogenes (LM), a facultative intracellular Gram-positive bacterium, often causes lethal infection of the host . In this study we investigated the molecular mechanism underlying LM eradication in the early phase of infection . Upon infection with LM, both IL-12 and IL-18 were produced, and then they synergistically induced IFN-gamma production, leading to normal LM clearance in the host . IFN-gamma knockout (KO) mice were highly susceptible to LM infection . IL-12/IL-18 double knockout mice were also highly susceptible . Their susceptibility was less than that of IFN-gamma KO mice, but more than that of single IL-12 or IL-18 KO mice . Mice deficient in myeloid differentiation factor 88 (MyD88), an essential adaptor molecule used by signal transduction pathways of all members of the Toll-like receptor (TLR) family, showed an inability to produce IL-12 and IFN-gamma following LM infection and were most susceptible to LM . Furthermore, MyD88-deficient, but not IFN-gamma-deficient, Kupffer cells could not produce TNF-alpha in response to LM in vitro, indicating the importance of MyD88-dependent TNF-alpha production for host defense . As TLR2 KO, but not TLR4 KO, mice showed partial impairment in their capacity to produce IL-12, IFN-gamma, and TNF-alpha, TLR2 activation partly contributed to the induction of IL-12-mediated IFN-gamma production . These results indicated a critical role for TLRs/MyD88-dependent IL-12/TNF-alpha production and for IL-12- and IL-18-mediated IFN-gamma production in early phase clearance of LM. J Rheumatol Suppl, 2002 Sep, 65, 33 - 8 What are the risks of biologic therapy in rheumatoid arthritis? An update on safety; Weisman MH; The tumor necrosis factor-alpha (TNF-alpha) blockers infliximab and etanercept and the recombinant interleukin 1 (IL-1) receptor antagonist anakinra are effective in patients with active rheumatoid arthritis (RA) . Here, information in the medical literature and public domain is used to consider the safety of these biologic agents . TNF-alpha inhibition with infliximab has been associated with reactivation of tuberculosis and possibly development of other opportunistic infections (histoplasmosis, listeriosis . and pneumocystis) . Exacerbations of multiple sclerosis and other central nervous system events have been reported with etanercept and infliximab . Recently, a review of preliminary data from an ongoing phase II study suggests that infliximab may worsen congestive heart failure . On the basis of clinical trials, there appears to be a higher incidence of serious infections seen in anakinra patients compared with controls; the particular combination of anakinra and etanercept may be associated with a higher incidence of serious infections and clinically significant leukopenia . Additional data are needed to understand whether all these safety issues are unique to an individual biologic agent or representative of a class effect . At this time, treating physicians must carefully weigh the benefits of these new biologics against their risks, particularly in patients at risk of infection. J Food Prot, 2002 Sep, 65(9), 1470 - 4 Detection of Listeria monocytogenes in pigs and pork; Kanuganti SR et al.; In this study, we surveyed hogs (n = 300) as well as pork products (ground pork and raw chitterlings) for Listeria monocytogenes . Pig specimens collected before (tonsil swabs) and after slaughter (tonsils, lymph nodes, carcass swabs, and rectal contents) were examined for L . monocytogenes by enrichment with conventional enrichment broths followed by subculturing to selective agar . A multiplex polymerase chain reaction assay targeting the highly conserved 16S rRNA gene of the Listeria species as well as the hlyA gene unique to L . monocytogenes was used to screen aliquots of the enrichment (method I) as well as to confirm presumptive Listeria colonies from Columbia agar with 0.05% glucose supplemented with polymyxin B-acriflavine-lithium chloride-ceftazidime-aesculin-mannitol (PALCAM; method II) . Subculturing to PALCAM agar was the more sensitive of the two methods on the basis of the overall detection of Listeria . For hog tissues, method I detected L . monocytogenes (0.87% positive) and no other Listeria spp . in all samples (n = 1,849) . In contrast, method II detected significantly more (P < 0.05) L . monocytogenes (2.38%) and Listeria spp . (0.38%) in these tissues . For small intestines (n = 300 raw chitterlings), L . monocytogenes was identified in 8.3% of enrichments with University of Vermont modified Listeria enrichment broth; plating to PALCAM slightly improved recovery (9%) . Overall, ground pork samples (n = 340) harbored L . monocytogenes (45% positive) and other Listeria species (1.5% positive), as determined by method I . Subculturing to PALCAM significantly (P < 0.05) improved the detection of L . monocytogenes (50.2%) but not that of other Listeria species (1.7%) . L . monocytogenes isolates (n = 243) were assigned to serotype 1 (53.5%), serotype 4 (25%), and serotypes other than 1 and 4 (21.4%). J Food Prot, 2002 Sep, 65(9), 1411 - 6 Control of Listeria monocytogenes on turkey frankfurters by generally-recognized-as-safe preservatives; Islam M et al.; Generally-recognized-as-safe chemicals applied to the surfaces of turkey frankfurters were evaluated for their ability to reduce populations of or inhibit the growth of Listeria monocytogenes . Frankfurters were treated prior to inoculation by dipping for 1 min in a solution of one of four preservatives (sodium benzoate, sodium propionate, potassium sorbate, and sodium diacetate) at three different concentrations (15, 20, and 25% {wt/vol}), with < 0.3% of the preservative being present for each frankfurter . Subsequently, 0.1 ml of a five-strain mixture of L . monocytogenes (10(6) CFU/ml) was used to surface inoculate each frankfurter separately in a sterile stomacher bag . Inoculated frankfurter bags were held at 4, 13, and 22 degrees C, and L . monocytogenes cells were enumerated at 0, 3, 7, 10, and 14 days of storage . The results of this study revealed that at all three concentrations of all four preservatives, the initial populations of L . monocytogenes decreased immediately by 1 to 2 log10 CFU/g . After 14 days of storage at 4 degrees C, L . monocytogenes counts for all treated frankfurters were 3 to 4 log10 CFU/g less than those for the untreated frankfurters . After 14 days of storage at 13 degrees C, L . monocytogenes counts for frankfurters treated with 25% sodium benzoate or 25% sodium diacetate were 3.5 to 4.5 log10 CFU/g less than those for untreated frankfurters, and those for frankfurters treated with 25% sodium propionate or 25% potassium sorbate were 2.5 log10 CFU/g less than those for untreated frankfurters . In all instances, the degree of growth inhibition was directly proportional to the concentration of the preservative . Only frankfurters treated with 25% sodium diacetate or sodium benzoate were significantly inhibitory to L . monocytogenes when held at 22 degrees C for 7 days or longer . Interestingly, the untreated frankfurters held at 22 degrees C were spoiled within 7 days, with copious slime formation, whereas there was no evidence of slime on any treated frankfurters after 14 days of storage. J Food Prot, 2002 Sep, 65(9), 1406 - 10 Irradiation temperature influences product quality factors of frozen vegetables and radiation sensitivity of inoculated Listeria monocytogenes; Niemira BA et al.; Four frozen vegetables (broccoli, corn, lima beans, and peas) were gamma irradiated at subfreezing temperatures ranging from -5 to -20 degrees C to determine (i) the radiation sensitivity of an inoculated outbreak strain of Listeria monocytogenes (ATCC 49594), (ii) the effect of changing irradiation conditions (i.e., temperature) on that sensitivity, and (iii) the effect of the recommended radiation dose on the texture and color of irradiated frozen vegetables . The amounts of radiation necessary to reduce the bacterial population by 90% (D10-values) for L . monocytogenes differed significantly among vegetables at each irradiation temperature . D10 increased significantly with decreasing temperature for all vegetables, with each vegetable showing a different response pattern . At an irradiation temperature of -5 degrees C, D10 ranged from 0.505 kGy for broccoli to 0.613 kGy for corn . At -20 degrees C, D10 ranged from 0.767 kGy for lima beans to 0.916 kGy for peas . At -20 degrees C, radiation doses sufficient to achieve a 5-log10 kill (3.9 to 4.6 kGy) caused significant softening of peas and broccoli stems but not of corn or lima beans . Lower doses of comparable antimicrobial efficacy delivered at -5 degrees C (2.5 to 3.1 kGy) did not cause significant changes in texture in any vegetable . Color varied significantly among the dose-temperature combinations only for broccoli florets; this variation did not demonstrate a clear pattern of quality changes in response to irradiation. Cell, 2002 Sep 6, 110(5), 551 - 61 An RNA thermosensor controls expression of virulence genes in Listeria monocytogenes; Johansson J et al.; In Listeria monocytogenes, virulence genes are maximally expressed at 37 degrees C, almost silent at 30 degrees C and controlled by PrfA, a transcriptional activator whose expression is thermoregulated . Here, we show that the untranslated mRNA (UTR) preceding prfA, forms a secondary structure, which masks the ribosome binding region . Mutations predicted to destabilize this structure led to virulence gene expression and invasion of mammalian cells at 30 degrees C . Chemical probing, native gel electrophoresis, in vitro translation, and "compensatory" and "increased stability" mutations demonstrated that the UTR switches between a structure active at high temperatures, and another inactive at low temperatures . Strikingly, when the DNA corresponding to the UTR was fused to gfp in E . coli, bacteria became fluorescent at 37 degrees C, but not at 30 degrees C . This mechanism of posttranscriptional thermoregulation may have important applications. Infect Immun, 2002 Oct, 70(10), 5800 - 7 The Mycobacterium tuberculosis phagosome in human macrophages is isolated from the host cell cytoplasm; Clemens DL et al.; Knowledge of whether Mycobacterium tuberculosis resides within a relatively impermeable membrane-bound vacuole or is free within the cytoplasm within its host cell is central to an understanding of the immunobiology of this intracellular parasite but is a matter of controversy . To explore this issue, we assessed the accessibility of medium-size protein molecules (Fab fragments of 50,000 Da) to M . tuberculosis within human macrophages . We infected the macrophages with wild-type or green fluorescent protein-expressing M . tuberculosis, microinjected Fab fragments directed against a major surface antigen of M . tuberculosis into the host cell, and assayed the accessibility of the bacteria to the Fab fragments by both immunofluorescence microscopy and immunogold electron microscopy . Whereas microinjected intact immunoglobulin G molecules against cytoplasmic early endosomal antigen 1 readily stained this antigen, microinjected Fab fragments against M . tuberculosis did not stain the bacterium within its phagosome . In contrast, microinjected Fab fragments against Listeria monocytogenes, an intracellular bacterium known to permeabilize its phagosomal membrane, strongly stained this bacterium . Our study shows that M . tuberculosis resides in an isolated phagosome that is relatively impermeable to cytoplasmic constituents. Int J Food Microbiol, 2002 Oct 25, 78(3), 235 - 43 A predictive model that evaluates the effect of growth conditions on the thermal resistance of Listeria monocytogenes; Chhabra AT et al.; A predictive model for Listeria monocytogenes was developed using cells grown in different pH and milkfat levels before subsequent thermal inactivation in identical pH and milkfat conditions . Inactivation of the cells used combinations of temperature (55, 60, 65 degrees C), pH (5.0, 6.0, 7.0), and milkfat (0%, 2.5%, 5.0%) in a complete 3 x 3 x 3 factorial design with each test done in triplicate . A modified Gompertz equation was used to model nonlinear survival curves with the following three parameter estimates: A for the shouldering region, B for the maximum death rate, and C for the tailing region . All treatment sets were analyzed together in a regression model using the modified Gompertz equation . There was good confidence in the overall model when it was used to predict values for the entire data set . The correlation of determination, R2, between the observed log surviving fraction (LSF) of cells from each of the conditions studied in the experiment, for the overall model was 0.811 . For the A and B parameter estimates, temperature or milkfat alone, and the interaction of temperature and milkfat significantly (p < 0.05) affected the shouldering region and maximum death rate of a survival curve, respectively . These results were compared to a previously published predictive model, generated for cells grown under optimum conditions (pH 7.0, 0% milkfat), where pH was the only significant (p < 0.05) factor affecting the shoulder region . These results suggested that the conditions of the growth environment had an important impact on survival curve shape and the estimates of the predictive model . Specifically, there were more factor interactions involving temperature and milkfat level . These growth factors affected the shoulder region and maximum rate of death of the survival curve when cells were grown in identical medium conditions to which they were heated . Differences related to shouldering and inactivation rates for cells grown in different conditions may have important and practical importance for estimating inactivation of L . monocytogenes . This study provides some evidence on the importance of growing conditions when evaluating microbial heat resistance. J Microbiol Methods, 2002 Nov, 51(3), 421 - 3 Development of a microslide agglutination assay with the aid of an inexpensive projection microscope; Abolmaaty A et al.; A microslide agglutination assay was developed involving the mixing of 2.5 microl each of antiserum and a cell suspension of Listeria monocytogenes . Cell agglutination in the final volume of 5.0 microl was visually observed at a direct magnification of 22 x on the projection screen of an inexpensive 20 US dollar projection microscope . The procedure has the advantage of increasing by a factor of 20 the number of agglutination assays that can be performed with a given volume of antiserum with the use of an inexpensive optical projection system . J Microbiol Methods, 2002 Nov, 51(3), 361 - 8 Factors affecting the performance of 5' nuclease PCR assays for Listeria monocytogenes detection; Lunge VR et al.; The design and operating parameters affecting the performance of 5' nuclease PCR (TaqMan) assays for the detection of Listeria monocytogenes was investigated . A system previously developed and based on the hlyA gene was used as a model {Appl . Environ . Microbiol . 61 (1995) 3724} . A series of fluorogenic probes labeled with a reporter and a quencher dye was synthesized to explore the effect of probe position and sequence content on the efficiency of probe hydrolysis . In addition, a series of PCR primer pairs that altered the distance between the upstream primer and the interceding probe was examined . The effects of various assay parameters were evaluated by measuring the ratio of the fluorescence intensity of the reporter dye over the quencher dye (deltaRQ) . For a given probe sequence, the deltaRQ was typically lower if the 5' terminus was a G residue . Decreasing the probe concentration increased the deltaRQ, although this was at the expense of reproducibility in the assay readout . The distance between the upstream primer and the interceding probe has a significant effect on probe hydrolysis . Reducing the primer-probe distance from, for example, 127 to 4 nt increased the deltaRQ from 2.87 to 5.00 . These general rules were used to develop a 5' nuclease PCR (TaqMan) assay with enhanced signal output, providing higher and more reproducible deltaRQ values for L . monocytogenes detection . Exp Toxicol Pathol, 2002 Aug, 54(2), 127 - 33 Pulmonary microbial infection in mice: comparison of different application methods and correlation of bacterial numbers and histopathology; Munder A et al.; Many investigations have been performed in characterising experimental bacterial infections in the lung of mice using several pathogens . Robust experimental pulmonary infection models require a reproducible method of application with defined numbers of pathogens to the respiratory tract without contaminating extrapulmonary tissues . At the same time trauma due to the experimental procedure should be kept to a minimum . So far several routes of administration have been used but a systematic comparison of these methods is still missing . Here we provide a comprehensive evaluation of view controlled i.t . instillation, tracheotomy, intranasal application, blind instillation and aerosol infection . An infection dose of up to 5 x 10(4) bacteria (L . monocytogenes) was applied to a group of ten mice by each technique and the animals were killed after 1 h or 24h . The number of viable bacteria was estimated by plating homogenates of the lungs and spleens . In addition, pathological effects on lung tissue were examined by histology 24h after infection . The highest reproducibility was achieved after applying Listeria directly in the trachea under view or by tracheotomy . However, mice were severely affected in their vitality after tracheotomy . Thus, for topical application of bacterial suspension into the lung the view controlled i.t . instillation is most appropriate. FEMS Microbiol Lett, 2002 Aug 27, 214(1), 69 - 75 Participation of DnaK in expression of genes involved in virulence of Listeria monocytogenes; Hanawa T et al.; DnaK is required for adaptation to environmental stress and is also involved in bacterial growth under normal conditions . To examine whether DnaK plays a role in the expression of genes related to pathogenicity of Listeria monocytogenes, the transcription of flaA, iap and lmaA in a dnaK mutant was analyzed . Northern blot analysis showed that expression of flaA and lmaA mRNAs was reduced in the dnaK mutant, THY-LK1 . A reporter assay revealed that transcription of lmaB in the dnaK mutant, LKS01, constructed in this study was lower than that in the parental strain . In contrast, the dnaK mutation had no effect on the transcription of iap . These results suggest that DnaK is involved in the transcription of flaA and lmaB. J Clin Microbiol, 2002 Sep, 40(9), 3319 - 25 Rational design of DNA sequence-based strategies for subtyping Listeria monocytogenes; Cai S et al.; The ability to differentiate bacteria beyond the species level is essential for identifying and tracking infectious disease outbreaks and to improve our knowledge of the population genetics, epidemiology, and ecology of bacterial pathogens . Commonly used subtyping methods, such as serotyping, phage typing, ribotyping, and pulsed-field gel electrophoresis, can yield ambiguous results that are difficult to standardize and share among laboratories . DNA sequence-based subtyping strategies can reduce interpretation ambiguity . We report the development of a rational approach for designing sequence-based subtyping methods . Listeria monocytogenes was selected as the model organism for testing the efficacy of this approach . Two housekeeping genes (recA and prs), one stress response gene (sigB), two virulence genes (actA and inlA), and two intergenic regions (hly-mpl and plcA-hly) were sequenced for 15 L . monocytogenes isolates . Isolates were chosen from a representative collection of more than 1,000 L . monocytogenes isolates to reflect the genetic diversity of this species . DNA sequences were aligned, and sliding window analyses were performed for each gene to define 600-bp-long regions that were (i) most polymorphic (using ProSeq) or (ii) most discriminatory (using a new algorithm implemented in WINDOWMIN) . Complete gene sequences for actA (1,929 bp) and inlA (2,235 bp) provided the highest discrimination (identifying 15 and 14 allelic types, respectively) . WINDOWMIN allowed identification of 600-bp regions within these genes that provided similar discriminatory power (yielding 15 and 13 allelic types, respectively) . The most discriminatory 600-bp fragments identified in the housekeeping and stress response genes differentiated the isolates into 8 to 10 subtypes; intergenic region sequences yielded 8 and 12 allelic types based on 335- and 242-bp sequences for hly-mpl and plcA-hly, respectively . Regions identified as most polymorphic were not necessarily most discriminatory; therefore, application of the WINDOWMIN algorithm provided a powerful tool for determining the best target regions for DNA sequence-based subtyping . Our specific results also show that inclusion of virulence gene target sequences in a DNA sequence-based subtyping scheme for L . monocytogenes is necessary to achieve maximum subtype differentiation. Immunity, 2002 Aug, 17(2), 211 - 20 In vivo depletion of CD11c(+) dendritic cells abrogates priming of CD8(+) T cells by exogenous cell-associated antigens; Jung S et al.; Cytotoxic T lymphocytes (CTL) respond to antigenic peptides presented on MHC class I molecules . On most cells, these peptides are exclusively of endogenous, cytosolic origin . Bone marrow-derived antigen-presenting cells, however, harbor a unique pathway for MHC I presentation of exogenous antigens . This mechanism permits cross-presentation of pathogen-infected cells and the priming of CTL responses against intracellular microbial infections . Here, we report a novel diphtheria toxin-based system that allows the inducible, short-term ablation of dendritic cells (DC) in vivo . We show that in vivo DC are required to cross-prime CTL precursors . Our results thus define a unique in vivo role of DC, i.e., the sensitization of the immune system for cell-associated antigens . DC-depleted mice fail to mount CTL responses to infection with the intracellular bacterium Listeria monocytogenes and the rodent malaria parasite Plasmodium yoelii. Scand J Immunol, 2002 Sep, 56(3), 233 - 47 Extensive and preferential Fas/Fas ligand-dependent death of gammadelta T cells following infection with Listeria monocytogenes; Mukasa A et al.; In the spleens of mice infected intraperitoneally with the bacterium Listeria monocytogenes, both alphabeta and gammadelta T cells became rapidly activated, followed by a massive apoptotic death response predominantly within the gammadelta population . The death response involved two major splenic gammadelta T-cell subsets and was Fas/Fas ligand (Fas-L)-dependent . Among T cells isolated from the Listeria-infected spleen, Fas-L was almost exclusively expressed in gammadelta T cells . gammadelta T cells coexpressed Fas and Fas-L, suggesting activation-induced suicide as a mechanism of their death . In vivo treatment with an antibody specific for CD3epsilon induced activation, preferential Fas-L expression and apoptosis of gammadelta T cells, resembling the response pattern in listeriosis, whereas antibodies specific for T-cell receptor-beta (TCR-beta) or TCR-delta did not, suggesting that the complete response seen in listeriosis requires both gammadelta TCR engagement and additional stimuli . L . monocytogenes causes early nonspecific, Fas-independent lymphocyte death in heavily infected tissues . In contrast, the death response described here is selective, Fas-dependent and triggered at low local levels of bacteria, suggesting that it is controlled by interactions with other infection-activated host cells, and perhaps part of a regulatory circuit specifically curtailing gammadelta T cells. ANZ J Surg, 2002 Aug, 72(8), 583 - 8 David Murray Morton: father figure of surgery at St Vincent's Hospital, bush lawyer and thwarted reformer of the medico-legal system; Vellar ID; When the first Golden Age of surgery at St Vincent's Hospital Melbourne (a period covering the first 25 years of the twentieth century) is discussed, the names that spring to mind are usually those of Sir Thomas Dunhill, Sir Hugh Devine, Sir Douglas Shields and Julian Smith . A name which is often overlooked, and by now almost forgotten, is that of David Murray Morton . Murray -Morton's career both as a medical student, general practitioner, anaesthetist and surgeon coincided with a revolution in the practice of medicine . He witnessed the progression from Listerian antisepsis to asepsis in surgical practice, the improvements in anaesthesia, the introduction of antibiotics and, before he died in 1959, the beginnings of organ transplants and open heart surgery . Morton was fond of saying that he grew up with the hospital . From the time of his first appointment as an anaesthetist in l896 to the time of his retirement from St Vincent's as senior surgeon in 1931, Morton, admired by all as a highly competent surgeon and a man of incorruptible probity, can rightly be described as the father figure of surgery during the formative years of St Vincent's Hospital . His innate sense of justice and fair play formed the basis of his distinguished tenure of the Presidency of the Medical Defence Association of Victoria, where his constructive attempts at reform of the medico-legal system were only thwarted by the predictable opposition of the legal profession and politicians. Acta Microbiol Pol, 2002, 51(1), 5 - 12 Penicillin-binding proteins of listeria monocytogenes--a re-evaluation; Korsak D et al.; Intact Listeria monocytogenes cells or membranes isolated from them were treated with {3H}penicillin to allow identification of the penicillin binding proteins (PBPs) located in the cytoplasmic membrane . In the former case the PBPs were released from the cells following disruption of the cell wall murein with Listeria monocytogenes bacteriophage lysin . The procedure described by Dougherty et al . (1996) for Escherichia coli, with some modifications, was used to evaluate the M(r)s of the individual PBPs and allowed direct quantitation of their copy number. Infect Immun, 2002 Sep, 70(9), 4805 - 11 Comparison of host resistance to primary and secondary Listeria monocytogenes infections in mice by intranasal and intravenous routes; Mizuki M et al.; There have been no studies on the susceptibility and host immune responses to an intranasal infection with Listeria monocytogenes . In this study, we compared the susceptibilities and cytokine responses between intranasal and intravenous infections with L . monocytogenes in mice . Moreover, we compared efficiency of acquisition of host resistance to L . monocytogenes infection between intranasally and intravenously immunized mice because an intranasal immunization of vaccines is reportedly available for induction of adaptive immunity against various infectious pathogens . The susceptibility to an intranasal infection with L . monocytogenes was markedly lower than that to the intravenous infection . The bacterial growth in the lungs, spleens, and livers was substantially similar between intranasally and intravenously infected mice . Titers of endogenous gamma interferon (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in the spleens, livers, and lungs were parallel to bacterial numbers in each organ of mice during intranasal infection and intravenous infection . IFN-gamma-deficient mice and TNF-alpha-deficient mice were highly susceptible to intranasal infection as well as intravenous infection . Susceptibilities to intranasal and intravenous L . monocytogenes infection were the same in these cytokine-deficient mice . These results suggest that both IFN-gamma and TNF-alpha play critical roles in host resistance to intranasal L . monocytogenes infection as well as the intravenous infection . Acquisition of host resistance to intravenous and intranasal L . monocytogenes infection was induced in intranasally immunized mice as well as intravenously immunized mice, suggesting that intranasal immunization is effective for prevention of a systemic infection with L . monocytogenes. Antimicrob Agents Chemother, 2002 Sep, 46(9), 2784 - 90 The LisRK signal transduction system determines the sensitivity of Listeria monocytogenes to nisin and cephalosporins; Cotter PD et al.; The Listeria monocytogenes two-component signal transduction system, LisRK, initially identified in strain LO28, plays a significant role in the virulence potential of this important food-borne pathogen . Here, it is shown that, in addition to its major contribution in responding to ethanol, pH, and hydrogen peroxide stresses, LisRK is involved in the ability of the cell to tolerate important antimicrobials used in food and in medicine, e.g., the lantibiotic nisin and the cephalosporin family of antibiotics . A (Delta)lisK mutant (lacking the LisK histidine kinase sensor component) displays significantly enhanced resistance to the lantibiotic nisin, a greatly enhanced sensitivity to the cephalosporins, and a large reduction in the expression of three genes thought to encode a penicillin-binding protein, another histidine kinase (other than LisK), and a protein of unknown function . Confirmation of the role of LisRK was obtained when the response regulator, LisR, was overexpressed using both constitutive and inducible (nisin-controlled expression) systems . Under these conditions we observed a reversion of the (Delta)lisK mutant to wild-type growth kinetics in the presence of nisin . It was also found that overexpression of LisR complemented the reduced expression of two of the aforementioned genes . These results demonstrate the important role of LisRK in the response of L . monocytogenes to a number of antimicrobial agents. J Food Prot, 2002 Aug, 65(8), 1333 - 7 Comparison of growth kinetics for healthy and heat-injured Listeria monocytogenes in eight enrichment broths; Silk TM et al.; Detection of Listeria in food products is often limited by performance of enrichment media used to support growth of Listeria to detectable levels . In this study, growth curves were generated using healthy and heat-injured Listeria monocytogenes strain F5069 in three nonselective and five selective enrichment broths . Nonselective enrichment media included the current Food and Drug Administration Bacteriological Analytical Manual Listeria enrichment broth base (BAM), Listeria repair broth (LRB), and Trypticase soy broth . Selective enrichment media included BAM with selective agents and LRB with selective agents, BCM L . monocytogenes preenrichment broth, Fraser broth, and UVM-modified Listeria enrichment broth . The Gompertz equation was used to model the growth of L . monocytogenes . Gompertz parameters were used to calculate exponential growth rate, lag-phase duration (LPD), generation time, maximum population density (MPD), and time required for repair of injured cells . Statistical differences (P < 0.05) in broth performance were noted for LPD and MPD when healthy and injured cells were inoculated into the broths . With the exception of Fraser broth, there were no significant differences in the time required for the repair of injured cells . Results indicate that the distinction between selective and nonselective broths in their ability to grow healthy Listeria and to repair sublethally injured cells is not solely an elementary issue of presence or absence of selective agents. J Food Prot, 2002 Aug, 65(8), 1329 - 32 Rapid enumeration of Listeria monocytogenes in artificially contaminated cabbage using real-time polymerase chain reaction; Hough AJ et al.; A quantitative real-time polymerase chain reaction (PCR) detection method specific for Listeria monocytogenes was developed, and studies involving pure culture showed that the response of the assay was linear over 7 log10 (log) cycles . The method was then applied to the detection of L . monocytogenes artificially inoculated onto cabbage, a vegetable chosen because it is a major component of coleslaw, which has been associated with an outbreak of listeriosis . After being allowed to attach to the food, cells were washed from the cabbage leaf surface and recovered by centrifugation . The DNA was purified by an organic solvent extraction technique and analyzed by real-time PCR . In this matrix, the method again produced a linear response over 7 log cycles from 1.4 x 10(2) to 1.4 x 10(9) CFU of L . monocytogenes in 25 g of cabbage, and analysis of the reproducibility of the system showed that log differences in L . monocytogenes numbers added to cabbage could be reliably distinguished . The system allowed quantitative results to be obtained within 8 h and was relatively inexpensive, showing good potential for routine analytical use. J Food Prot, 2002 Aug, 65(8), 1259 - 66 Assessment of the potential for Listeria monocytogenes survival and growth during alfalfa sprout production and use of ionizing radiation as a potential intervention treatment; Schoeller NP et al.; Alfalfa seeds (Australian, nondormant, nonscarified) were treated with 20,000 ppm active chlorine, sprouted in canning jars for 5 days, and packaged and stored at 5 degrees C for up to 9 days . Seeds or sprouts were inoculated with a three-strain cocktail of Listeria monocytogenes at one of three points during the process-day 0 (before 24-h aqueous seed soak), day 1 (after 24-h aqueous seed soak), or day 5 (after sprouting, before prepackaging 10 ppm chlorine rinse)--or control (no inoculum), and the ability of the inoculum to survive and grow was evaluated . Total bacterial numbers on uninoculated seeds increased dramatically during the first 24-h the seeds were soaked, from 3.5 to ca . 8.0 log CFU/g, and remained at this level during refrigerated storage . When the seeds were inoculated with a cocktail of L . monocytogenes (log 5 CFU/10 ml) on day 0 or 1, the population of the pathogen increased dramatically, to within 1 to 2 logs of the total, and remained high during refrigerated storage . When sprouted seeds were inoculated with L . monocytogenes later in the process (day 5), the inoculum survived but did not grow more than ca . 1 log CFU/g, regardless of whether the inoculation level in each jar was low (10(3)) or high (10(5)) . Irradiation of sprouts with beta radiation at 3.3 or 5.3 kGy, but not 1.5 kGy, was effective at eliminating L . monocytogenes from inoculated sprouts (6 log CFU/g) without causing noticeable changes in appearance or odor . In summary, L . monocytogenes can grow on sprouts during production, can survive on refrigerated sprouts, and may be eliminated on sprouts with beta radiation. Mol Microbiol, 2002 Aug, 45(4), 1043 - 56 Identification of a second Listeria secA gene associated with protein secretion and the rough phenotype; Lenz LL et al.; We describe the identification and characterization of a second secA gene in Listeria monocytogenes . This gene, termed secA2, is involved in smooth-rough phenotypic variation and secA2 expression contributes to bacterial virulence . Spontaneous rough (R-) variants of L . monocytogenes grow in chains and form rough colonies on solid media . A subset of R-variants, classified here as type I, also shows reduced secretion of an autolysin, p60 . We find that disruptions and in frame deletions in secA2 confer phenotypes identical to those of spontaneous type I R-variants . Additionally, the secA2 genes from two spontaneous type I R-variants encoded truncated SecA2 proteins . Mutations were not found in the secA2 genes from the remaining five independent R-variants, four of which showed a distinct (type II) rough morphology and secreted wild-type levels of p60 . Expression of an epitope-tagged SecA2 in the DeltasecA2 strain and a spontaneous R-variant restored normal cell septation and smooth colony morphology . These data suggest that mutations in both secA2 and other genes contribute to smooth-rough phase variation in L . monocytogenes . Expression of the full-length SecA2 also promotes secretion of p60 and a set of additional L . monocytogenes proteins . We hypothesize that SecA2-dependent protein secretion plays a role in the colonization of environmental and host surfaces. Berl Munch Tierarztl Wochenschr, 2002 Jul-Aug, 115(7-8), 259 - 66 {The role of nitric oxide in Listeria encephalitis of ruminants and in rats intracisternally infected with Listeria monocytogenes}; Remer KA et al.; Listeria monocytogenes is a Gram-positive facultative intracellular bacteria which infects a wide range of hosts . In ruminants, infection with L . monocytogenes frequently causes encephalitis, which is usually fatal in sheep and goat, while cattle often recover with antibiotic therapy . Since the role of NO in the control of Listeria is controversial, we have studied the expression of iNOS in the brains of cattle, sheep and goats which had succumbed to listeria encephalitis . iNOS was demonstrated in decreasing intensity in the M phi of microabscesses from cattle, sheep and goat . iNOS expression was accompanied by NT in the microabscesses of cattle, but was only present to a low degree in sheep and was absent in goats . This is indirect evidence for differences in the ability to produce NO in the three species . Presence of iNOS and NT were inversely correlated with the numbers of bacteria . While microabscesses of goats contained high amounts of L . monocytogenes they occurred only rarely in cattle . To corroborate our hypothesis that NO is involved in the control of listeria encephalitis a new animal model was developed . Eleven day old infant rats were infected intracisternally with a low dose of L . monocytogenes . This resulted in a transient meningoencephalitis with moderate clinical signs and low mortality .