Transfer of Microorganisms, Including Listeria monocytogenes, from Various Materials to Beef

The quantity of microorganisms that may be transferred to a food that comes into contact with a contaminated surface depends on the density of microorganisms on the surface and on the attachment strengths of the microorganisms on the materials. We made repeated contacts between pieces of meat and various surfaces (stainless steel and conveyor belt materials [polyvinyl chloride and polyurethane]), which were conditioned with meat exudate and then were contaminated with Listeria monocytogenes, Staphylococcus sciuri, Pseudomonas putida, or Comamonas sp. Attachment strengths were assessed by the slopes of the two-phase curves obtained by plotting the logarithm of the number of microorganisms transferred against the order number of the contact. These curves were also used to estimate the microbial population on the surface by using the equation of A. Veulemans, E. Jacqmain, and D. Jacqmain (Rev. Ferment. Ind. Aliment. 25:58-65, 1970). The biofilms were characterized according to their physicochemical surface properties and structures. Their exopolysaccharide-producing capacities were assessed from biofilms grown on polystyrene. The L. monocytogenes biofilms attached more strongly to polymers than did the other strains, and attachment strength proved to be weaker on stainless steel than on the two polymers. However, in most cases, it was the population of the biofilms that had the strongest influence on the total number of CFU detached. Although attachment strengths were weaker on stainless steel, this material, carrying a smaller population of bacteria, had a weaker contaminating capacity. In most cases the equation of Veulemans et al. revealed more bacteria than did swabbing the biofilms, and it provided a better assessment of the contaminating potential of the polymeric materials studied here.     

Antibiotic resistance among Listeria, including Listeria monocytogenes, in retail foods

AIMS: In the past eight to 10 years, reports of antibiotic resistance in food-borne isolates in many countries have increased, and this work examined the susceptibility of 1001 food isolates of Listeria species. METHODS AND RESULTS: Susceptibility/resistance to eight antibiotics was determined using the Bauer-Kirby disc diffusion assay, and 10.9% of the isolates examined displayed resistance to one or more antibiotics. Resistance to one or more antibiotics was exhibited in 0.6% of Listeria monocytogenes isolates compared with 19.5% of Listeria innocua isolates. Resistance was not observed in Listeria seeligeri or Listeria welshimeri. Resistance to tetracycline (6.7%) and penicillin (3.7%) was the most frequently observed, and while resistance to one antibiotic was most common (9.1%), isolates resistant to two or more antibiotics (1.8%) were also observed. CONCLUSION: While resistance to the antibiotics most commonly used to treat human listeriosis was not observed in L. monocytogenes, the presence of such resistance in other Listeria species raises the possibility of future acquisition of resistance by L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The higher level of resistance in L. innocua compared with L. monocytogenes suggests that a species-related ability to acquire resistance to antibiotics exists.     

The risk of Listeria monocytogenes infection in beef cattle operations

Aim:  To determine the prevalence of Listeria monocytogenes and associated risk factors among beef operations (cow-calf and feedlot) in central and southern California.
Methods and Results:  A repeated cross-sectional study where faecal and environmental samples were collected from 50 operations three times a year at different seasons was carried out. Samples were tested for presence of L. monocytogenes using a combination of enrichment and polymerase chain reaction tests. Data on putative risk factors were also collected. Listeria monocytogenes was detected in faecal samples from cows, calves and other animals on calf-cow operations at proportions of 3·1%, 3·75% and 2·5%, respectively. The organism was detected in 5·3% of cut-grass, 5·3% of soil, 14·3% of irrigation ditches, 3·1% of the ponds and 6·5% of water troughs samples. Listeria monocytogenes was less common in faecal (0·3%) and soil (0·75%) samples collected from feedlots.
Conclusions:  Listeria monocytogenes was present at a higher proportion among cow-calf operations than feedlots. There was no significant seasonal variation in the occurrence of this pathogen within the two types of operations.
Significance and Impact of the Study:  If risk mitigation strategies were implemented to reduce the public health risk these should focus in cow-calf operations.     

Listeria monocytogenes adheres to many materials found in food-processing environments

AIMS: To investigate the adhesion of Listeria monocytogenes 10403S to 17 different, food-use approved materials representing metals, rubbers and polymers. METHODS AND RESULTS: Adhesion assays were conducted by placing 'coupons' of the materials in planktonic cultures at 30 degrees C, and then immediately withdrawing them ('short contact') or leaving them submerged in the cultures for 2 h. Adherent cells were recovered by sonication. In the short contact experiments, the logarithm of the mean viable counts ranged from 3.67 +/- 0.43 to 4.78 +/- 0.38. After 2 h contact time, the numbers of adherent cells had increased significantly for all materials with the exception of polypropylene. The highest count (6.33 +/- 0.31) recorded was for stainless steel 405. CONCLUSION: Adhesion to a wide range of materials was time-dependent and characterized by reversible and irreversible stages. SIGNIFICANCE AND IMPACT OF THE STUDY: Adhesion test protocols must account for cell carry-over and cells which are only weakly bound. Material selection may only have a limited role in reducing food contamination by listeria.     

Control of Listeria monocytogenes in a biofilm by competitive-exclusion microorganisms

Biofilms from drains in food processing facilities with a recent history of no detectable Listeria monocytogenes in floor drains were cultured for microorganisms producing antilisterial metabolites. A total of 413 microbial isolates were obtained from 12 drain biofilm samples and were assayed at 15 and 37 degrees C for activities that were bactericidal or inhibitory to L. monocytogenes, by two agar plate assays. Twenty-one of 257 bacterial isolates and 3 of 156 yeast isolates had antilisterial activity. All 24 isolates which produced metabolites inhibitory to L. monocytogenes were assayed for antilisterial activity in coinoculated broth cultures containing tryptic soy broth with yeast extract (TSB-YE). A five-strain mixture of 10(3) CFU of L. monocytogenes/ml and 10(5) CFU of the candidate competitive-exclusion microorganism/ml was combined in TSB-YE and incubated at 37 degrees C for 24 h, 15 degrees C for 14 days, 8 degrees C for 21 days, and 4 degrees C for 28 days. Substantial inhibition of L. monocytogenes growth (4 to 5 log CFU/ml) was observed for nine bacterial isolates at 37 degrees C, two at 15 and 8 degrees C, and three at 4 degrees C. The inhibitory isolates were identified as Enterococcus durans (six isolates), Lactococcus lactis subsp. lactis (two isolates), and Lactobacillus plantarum (one isolate). The anti-L. monocytogenes activity of these isolates was evaluated in biofilms of L. monocytogenes on stainless steel coupons at 37, 15, 8, and 4 degrees C. Results revealed that two isolates (E. durans strain 152 and L. lactis subsp. lactis strain C-1-92) were highly inhibitory to L. monocytogenes (growth inhibition of >5 log(10) CFU of L. monocytogenes/cm(2)). These two bacterial isolates appear to be excellent competitive-exclusion candidates to control L. monocytogenes in biofilms at environmental temperatures of 4 to 37 degrees C.     

Chitin hydrolysis by Listeria spp., including L. monocytogenes

Listeria spp., including the food-borne pathogen Listeria monocytogenes, are ubiquitous microorganisms in the environment and thus are difficult to exclude from food processing plants. The factors that contribute to their multiplication and survival in nature are not well understood, but the ability to catabolize various carbohydrates is likely to be very important. One major source of carbon and nitrogen in nature is chitin, an insoluble linear beta-1,4-linked polymer of N-acetylglucosamine (GlcNAc). Chitin is found in cell walls of fungi and certain algae, in the cuticles of arthropods, and in shells and radulae of molluscs. In the present study, we demonstrated that L. monocytogenes and other Listeria spp. are able to hydrolyze alpha-chitin. The chitinolytic activity is repressed by the presence of glucose in the medium, suggesting that chitinolytic activity is subjected to catabolite repression. Activity is also regulated by temperature and is higher at 30 degrees C than at 37 degrees C. In L. monocytogenes EGD, chitin hydrolysis depends on genes encoding two chitinases, lmo0105 (chiB) and lmo1883 (chiA), but not on a gene encoding a putative chitin binding protein (lmo2467). The chiB and chiA genes are phylogenetically related to various well-characterized chitinases. The potential biological implications of chitinolytic activity of Listeria are discussed.     

Immune responses to Listeria monocytogenes

Listeria monocytogenes is a Gram-positive bacterium that is often used to study the mammalian immune response to infection because it is easy to culture, is relatively safe to work with and causes a highly predictable infection in laboratory mice. The broad application of this mouse model has resulted in a torrent of studies characterizing the contributions of different cytokines, receptors, adaptors and effector molecules to resistance against infection with Listeria monocytogenes. These studies, which are yielding one of the most comprehensive pictures of the 'battle' between host and microorganism, are reviewed here.     

Neuroinfections due to Listeria monocytogenes

Listeria monocytogenes is not a rare pathogen causing meningitis, mainly in small children and in close contacts to livestock. The pathogen is naturally resistant to cephalosporins and some glycopeptides as well, therefore despite of syndromologic diagnosis of meningitis and initial therapy with 3rd generation cephalosporins according to the guidelines therapeutic failures with clinical consequences may occur.     

Lipoteichoic acid from Listeria monocytogenes.

A lipoteichoic acid (LTA) was extracted from Listeria monocytogenes (serotype 1) by phenol-water partition and isolated by gel-filtration chromatography. The LTA exhibited amphiphilic properties by changes in gel-filtration mobility in the presence of detergent buffers and after mild base hydrolysis. In a hemagglutination assay, Listeria LTA bound antibody prepared against a known LTA from Streptococcus spp. Listeria LTA inhibited the binding of anti-LTA antibody to a Lactobacillus LTA in a hemagglutination inhibition assay. The Listeria LTA contained glucose, galactose, fatty acids, glycerol, and phosphate with molar ratios of 0.05, 0.07, 0.21, 0.94, and 1.0 to phosphate, respectively. Adjacent glycerols were linked between the C-1 and C-3 positions by phosphodiesters (structural type 1). The average chain length was 19 +/- 2 (standard deviation) glycerol-phosphate repeating units. Approximately one glycosyl side chain was present per LTA molecule. The side chain was a galactose-containing disaccharide. The lipid portion of the LTA was a galactose- and glucose-containing glycolipid which may have been a phosphoglycolipid, but the structure was not confirmed. Major fatty acids of LTA and the glycolipid were 17:anteiso, 15:anteiso, 16:iso, 16:n, and 18:n. L. monocytogenes contained cell wall products typical of gram-positive bacteria which is in contrast to the reports by others of the presence of lipopolysaccharides from L. monocytogenes.     

Isolation of Listeria monocytogenes from raw milk

During a recent outbreak of listeriosis, we examined 121 raw milk samples and 14 milk socks (filters). Listeria monocytogenes was recovered from 15 (12%) of 121 milk specimens and 2 (14%) of 14 milk socks. The optimal processing method consisted of cold enriching diluted milk for 1 month with culture to selective broth, followed by plating.     

Monoclonal antibody specific for Listeria monocytogenes, Listeria innocua, and Listeria welshimeri

Eight hundred fifty-nine murine hybridomas were produced from eight fusions, and 27 were characterized for secretion of antibodies reactive to Listeria monocytogenes. One monoclonal antibody (MAb), P5C9, reacted with all test strains of L. monocytogenes (31 of 31), L. innocua (3 of 3), and L. welshimeri (1 of 1) but not with any strains of the other four Listeria species or with any of 22 gram-positive or 11 gram-negative species of bacteria when tested in microtiter and dot blot enzyme immunoassays. Of the other 26 antibodies, 20 reacted with either L. monocytogenes Scott A or V7 and with some or all of the other six Listeria species but also cross-reacted with some or all of the non-Listeria bacteria tested. MAb P5C9 is of the immunoglobulin G1 murine subclass. In Western blot (immunoblot) analyses, this MAb reacted with a single antigen with a molecular weight of 18,500, and it is shared in common with all three reactive species, L. monocytogenes, L. innocua, and L. welshimeri. This antigen was extracted with detergent and appeared to be cell bound.     

Recurrent and sporadic Listeria monocytogenes contamination in alheiras represents considerable diversity, including virulence-attenuated isolates

Microbiological characterization of alheiras, traditional smoked meat sausages produced in northern Portugal, had previously shown that more than 60% of the lots analyzed were contaminated with Listeria monocytogenes at levels higher than 100 CFU/g. In order to better understand L. monocytogenes contamination patterns in alheiras, we characterized 128 L. monocytogenes isolates from alheiras using a variety of subtyping techniques (i.e., molecular serotyping; arsenic, cadmium, and tetracycline resistance typing; and pulsed-field gel electrophoresis [PFGE]). Subtyping of isolates from products collected on two separate dates provided evidence for the persistence of specific L. monocytogenes PFGE types in the production and distribution chains of alheiras from four different processors. A subset of 21 isolates was further characterized using ribotyping and Caco-2 cell invasion assays to evaluate the pathogenic potential of L. monocytogenes present in alheiras. Caco-2 invasion assays revealed seven isolates with invasion efficiencies that were less than 20% of that of the control strain 10403S. All seven isolates had premature stop codons in inlA that represented three distinct mutations, which had previously been observed in isolates from the United States or France. Our findings indicate the need for a comprehensive approach to control L. monocytogenes in alheiras, including strategies to reduce persistence. The presence of considerable diversity in invasion phenotypes among L. monocytogenes strains present in alheiras, including the presence of subtypes likely to be virulence attenuated, may provide an opportunity to initially focus control strategies on the subtypes most likely to cause human disease.     

Listeria monocytogenes, a Food-Borne Pathogen

[This corrects the article on p. 485 in vol. 55.].     

Listeria monocytogenes Surface Proteins: from Genome Predictions to Function

Summary: The genome of the human food-borne pathogen Listeria monocytogenes is predicted to encode a high number of surface proteins. This abundance likely reflects the ability of this bacterium to survive in diverse environments, including soil, food, and the human host. This review focuses on the various mechanisms by which listerial proteins are attached at the bacterial surface and their many functions, including peptidoglycan metabolism, protein processing, adhesion to host cells, and invasion of host tissues. Extensive in silico analysis of the domains or motifs present in these mosaic proteins reveals that diverse structural features allow the surface proteome to interact with diverse bacterial or host components. This diversity offers new clues about the molecular bases of Listeria pathogenesis.     

Response of Listeria monocytogenes to liquid smoke

Aims:  To investigate the effect of liquid smoke on growth, survival, proteomic pattern and haemolytic potential of Listeria monocytogenes.
Methods and Results:  Growth and survival curves were recorded in brain–heart infusion broth supplemented with three concentrations of liquid smoke. L. monocytogenes growth was inhibited in the presence of 15 μg ml⁻¹ phenol while a rapid decrease in cell viability occurred in the presence of 30 μg ml⁻¹ phenol. The proteome of L. monocytogenes cytosoluble proteins was slightly modified after 2-h incubation with 30 μg ml⁻¹ phenol but no protein already characterized in response to other known stresses was induced, except the protease ClpP. Liquid smoke inhibited the haemolytic potential without affecting hly gene expression, showing a potential inhibition of protein activity or stability.
Conclusions:  The presence of liquid smoke in a rich medium strongly affected growth and survival of L. monocytogenes . Brief smoke stress affected the metabolic pathways and inhibited the haemolytic activity of L. monocytogenes .
Significance and Impact of Study:  This study is a first step in the investigation of the influence of a smoked product on L. monocytogenes strains.     

Contribution of Chitinases to Listeria monocytogenes Pathogenesis

Listeria monocytogenes secretes two chitinases and one chitin binding protein. Mutants lacking chiA, chiB, or lmo2467 exhibited normal growth in cultured cells but were defective for growth in the livers and spleens of mice. Mammals lack chitin; thus, L. monocytogenes may have adapted chitinases to recognize alternative substrates to enhance pathogenesis.Listeria monocytogenes is a Gram-positive bacterium that is found as an inhabitant of soil, water, and decaying vegetation, where the organism is believed to live as a saprophyte (12, 13, 16). Upon consumption of the bacterium by a susceptible host, L. monocytogenes adopts a pathogenic lifestyle that can lead to serious and sometimes fatal infections (1, 3, 15, 22, 32). L. monocytogenes is considered a major food-borne pathogen and has been responsible for some of the largest and most expensive food recalls in U.S. history (6-10, 27, 31). While significant attention has focused on the identification of L. monocytogenes gene products that specifically contribute to bacterial life within host cells, relatively less is known regarding the function of gene products that may be adapted to contribute to bacterial life both inside and outside the infected host.Chitinases catalyze the hydrolysis of chitin, a linear polymer of N-acetylglucosamine residues linked in β-1,4 glycosidic bonds (5). Microbial chitinases have been associated with nutrient acquisition, as chitin is abundant in the environment and can serve as a source of carbon and nitrogen (17). Recently, a chitinase and a chitin binding protein have been linked to bacterial virulence for two environmental pathogens, Legionella pneumophila (14) and Vibrio cholerae (19, 21). Although chitin is not synthesized by mammals, L. pneumophila chitinase has been implicated in enhancing bacterial colonization of the lungs of infected mice (14), while a chitin binding protein produced by V. cholerae has been implicated in intestinal colonization (19, 21).L. monocytogenes produces two chitinases encoded by lmo1883 (chiA) and lmo0105 (chiB) and one chitin binding protein encoded by lmo2467 (Fig. ​(Fig.11 A) (26). chiA and chiB encode proteins containing a glycosyl hydrolase family 18 chitinase domain, and both have been demonstrated to possess chitinolytic activity (25, 26). Interestingly, the expression of chiA has recently been reported to be induced within the cytosol of infected macrophages (11). lmo2467 encodes a hypothetical chitin binding protein that lacks a chitinase domain (26). To investigate whether chiA, chiB, or lmo2467 contributes to host infection, in-frame deletion mutants were constructed in each gene in the L. monocytogenes chromosome using allelic exchange (18). Each of the resulting in-frame deletion mutants was found to resemble wild-type L. monocytogenes with respect to bacterial growth in brain heart infusion (BHI) broth culture (S. Chaudhuri and N. Freitag, data not shown), and the deletion of either chiA or chiB significantly reduced secreted chitinase activity as detected in the culture supernatants of mutant strains following 48 h of growth in LB at 30°C (Fig. ​(Fig.1B1B).Open in a separate windowFIG. 1.L. monocytogenes genes encoding chitinases and chitin binding proteins. (A) chiA (lmo1883) encodes a chitinase of 352 amino acids, chiB (lmo0105) encodes a chitinase of 756 amino acids, and lmo2467 encodes a predicted chitin binding protein of 478 amino acids (26). Chromosomal deletion mutants were constructed for each gene indicated in black. Surrounding coding regions are represented by white arrows. (B) Chitinase activity detected in supernatants derived from wild-type, ΔchiA, ΔchiB, and Δlmo2467 strains. Bacterial cultures were grown for 48 h at 30°C in LB broth. Bacterial supernatants were assayed for secreted chitinase activity using the substrate 4-nitrophenyl N,N′-diacetyl-β-d-chitobioside (14, 25). Chitinase units are expressed as the absorbance at 405 nm times 100 divided by the culture optical density measured at 600 nm (OD₆₀₀). Data shown are representative of two independent experiments.The ability of L. monocytogenes chitinase and chitin binding protein mutants to invade host cells, escape from the phagosome, and replicate in the cytosol was investigated via bacterial infection of the human intestinal Caco-2 cell line (Fig. ​(Fig.22 A). Monolayers of Caco-2 cells were grown on glass coverslips and infected with L. monocytogenes at a multiplicity of infection (MOI) of 10 bacteria per cell. At 1 h postinfection, monolayers were washed and medium containing 10 μg/ml gentamicin was added to kill extracellular bacteria. Intracellular bacterial replication was monitored by removing the coverslips at the indicated time points postinfection, lysing the infected host cells by vortexing the coverslips in water, and plating for viable CFU on LB agar media (29). Bacterial invasion and intracellular growth of each mutant strain in Caco-2 cells were found to be similar to those of wild-type L. monocytogenes (Fig. ​(Fig.2A).2A). Identical patterns of bacterial replication were also observed in the murine-derived J774 macrophage-like cell line using a similar approach (S. Chaudhuri and N. Freitag, data not shown).Open in a separate windowFIG. 2.Chitinase and chitin binding protein deletion mutants exhibit normal patterns of intracellular growth and cell-to-cell spread in tissue culture cells. (A) Monolayers of Caco-2 intestinal epithelial cells grown on glass coverslips were infected with L. monocytogenes wild-type (•), ΔchiA (▪), ΔchiB (▴), and Δlmo2467 (▾) strains at an MOI of 10:1. After 1 h, monolayers were washed with phosphate-buffered saline (PBS) and gentamicin was added to kill any remaining extracellular bacteria. Coverslips were removed at the indicated time points, followed by host cell lysis and enumeration of bacterial CFU on agar media. Data are representative of three independent experiments. (B) Monolayers of L2 fibroblast cells were infected with wild-type, ΔchiA, ΔchiB, and Δlmo2467 bacteria as previously described (30). Bacterial intracellular growth and cell-to-cell spread were monitored by measuring the diameter of the zones of clearance (plaques) in the infected monolayers. Relative plaque diameters are indicated as a percentage of those of the wild type (WT), which was set to 100%. Data shown are the average plaque diameter ± standard error from results from three independent experiments.The ability of L. monocytogenes to spread to adjacent cells during tissue culture infection can be rapidly assessed by measuring the ability of bacteria to form small zones of cell clearing or plaques during the infection of mouse fibroblast cell monolayers (30). Mouse L2 fibroblast cells were grown in 3.5-cm wells and infected with L. monocytogenes as previously described (30). One hour postinfection, the monolayers were washed and gentamicin was added along with a Dulbecco modified Eagle medium (DMEM)-0.7% agar overlay. Plaque formation was assessed at 3 days postinfection by staining viable L2 cells with neutral red. The chitinase and chitin binding protein mutants formed plaques in L2 cell monolayers with the same efficiency and of the same diameter as those formed by wild-type L. monocytogenes (Fig. ​(Fig.2B),2B), indicating that each of the mutant strains was fully capable of intracellular bacterial replication and cell-to-cell spread.The chitinase and chitin binding protein mutants were then tested for virulence in a mouse model of infection. ND4 Swiss Webster mice were infected via tail vein injection (2) with the ΔchiA and ΔchiB chitinase deletion mutants as well as with the Δlmo2467 mutant and wild-type L. monocytogenes (Fig. ​(Fig.3).3). Each mouse was inoculated with 2 × 10⁴ CFU, and at 3 days postinfection, the animals were sacrificed and the numbers of bacterial CFU were determined from homogenates of isolated livers and spleens. Each of the mutants exhibited a significant defect in bacterial replication within infected mice (Fig. ​(Fig.3).3). The Δlmo2467 mutant exhibited the most modest level of attenuation, with approximately 4-fold fewer bacteria recovered from the livers and 6-fold fewer from the spleens of infected animals than from those infected with wild-type L. monocytogenes (Fig. ​(Fig.3).3). The ΔchiB mutant exhibited more significant levels of attenuation, with approximately 8-fold and 14-fold fewer bacteria recovered from the livers and spleens, respectively (Fig. ​(Fig.3).3). Attempts to complement the defect of the ΔchiB mutant by providing a wild-type copy of the gene in trans using plasmid vector pPL2 (24) were not successful, as it was not possible to recover stable L. monocytogenes plasmid integrants for reasons that are not apparent (S. Chaudhuri and N. Freitag, data not shown). The most dramatic virulence defect was observed for strains lacking chiA (Fig. ​(Fig.3).3). The absence of chiA reduced bacterial colonization of the liver and spleen more than 19-fold and 45-fold, respectively, and the defect could be fully complemented in the liver and almost fully complemented in the spleen by providing a PCR-amplified wild-type copy of chiA in trans on the integrative plasmid vector pPL2 (24). These data indicate a significant role for the chitinase ChiA in L. monocytogenes virulence and suggest that additional contributions are made by chiB and lmo2467 during mouse infection.Open in a separate windowFIG. 3.L. monocytogenes chitinase mutants are defective for growth in the livers and spleens of infected mice. ND4 Swiss Webster mice were infected via tail injection (2) with 2 × 10⁴ CFU of wild-type, ΔchiA, ΔchiB, Δlmo2467, and ΔchiA bacteria complemented with plasmid-borne chiA (ΔchiA+pPL2-chiA). After 72 h of infection, the animals were euthanized and the bacterial burdens were measured in the livers (A) and spleens (B). A minimum of 5 mice were inoculated per strain tested, and the mean and standard deviation are shown. Statistical significance was determined using one-way analysis of variance with Tukey''s multiple-comparison test (*, P < 0.01; **, P < 0.001; ***, P < 0.0001).Given the absence of chitin in mammals, what are the potential roles for chitinases and chitin binding proteins in L. monocytogenes virulence? For the secreted V. cholerae chitin binding protein GbpA, it has been reported that the protein serves to enhance bacterial colonization of the intestine through the binding of mucin (4, 21), which consists of glycoproteins rich in carbohydrates that include N-acetylglucosamine (28). Kawada et al. have recently reported that a chitin binding protein expressed by Serratia marcescens increased bacterial adhesion to colonic epithelial cells via binding to the chitinase 3-like 1 (CHI3L1) protein expressed on the host cell surface (20). The L. pneumophila chitinase has been reported to contribute to bacterial colonization of mouse lung; however, the mechanism by which the enzyme enhances bacterial colonization of lung epithelium has not yet been established (14). For L. monocytogenes, neither ChiA, ChiB, nor Lmo2467 appeared to have any influence on bacterial invasion or replication within tissue culture cell lines (Fig. ​(Fig.2),2), although the proteins clearly contributed to virulence via bloodstream infection of mice (Fig. ​(Fig.3).3). It remains possible that one or more of the secreted proteins recognize glycoproteins or carbohydrate moieties present on host cells (but not expressed by the tissue culture cell lines examined in this study) and that this recognition somehow serves to enhance bacterial uptake. Alternatively, the expression of chiA has been reported to be induced within infected macrophages (11) and also by the L. monocytogenes central virulence regulator PrfA (23), raising the possibility that ChiA and/or the other chitin binding proteins may serve to modify host immune responses by binding to glycoproteins or other carbohydrate moieties that contribute to immune signaling. Elucidation of the role of chitinases and chitin binding proteins in L. monocytogenes pathogenesis should provide insight into how bacterial factors that contribute to survival in the outside environment can be exploited for additional roles within an infected mammalian host.