Determination of virulence of different strains of Listeria monocytogenes and Listeria innocua by oral inoculation of pregnant mice.

A pregnant mouse model was developed to follow the course of infection after peroral inoculation with six different strains of Listeria monocytogenes and one strain of Listeria innocua. Tissues were sampled and analyzed by microbiologic and histologic methods for 5 days postinoculation. In gnotobiotic pregnant BALB/c mice, L. monocytogenes Scott A (SA), serotype 4b, colonized the gastrointestinal tract, translocated to the livers and spleens of mice by day 1 postinoculation, and multiplied in these tissues until day 4. Infection of the placental tissues occurred by days 3 and 4 and was followed by infection of the fetuses. Little damage of colonic and cecal tissues was evident by histologic examination. Livers and spleens showed a cellular immune response; a similar immune response was not detected in the placentas or fetuses. A rough variant of L. monocytogenes SA which was as virulent as the parent strain in mice when injected intraperitoneally was less virulent perorally and did not consistently infect the fetuses. L. monocytogenes ATCC 19113, serotype 3a, did not colonize the gastrointestinal tract, nor was it isolated from any internal tissue. L. monocytogenes strains of serotypes 1/2a and 1/2b behaved like the SA strain in this mouse model. L. innocua colonized the gastrointestinal tract and translocated to the livers and spleens but did not survive in these organs and rapidly disappeared without infecting placental and fetal tissues. In comparison with gnotobiotic mice, conventional pregnant mice inoculated with L. monocytogenes strains showed less consistent infection. These results suggest that the gnotobiotic pregnant mouse is a useful model for detecting differences in virulence relating to colonization, invasiveness, and uteroplacental infection which cannot be detected by intraperitoneal inoculation of mice.     

Determination of virulence of different strains of Listeria monocytogenes and Listeria innocua by oral inoculation of pregnant mice.

A pregnant mouse model was developed to follow the course of infection after peroral inoculation with six different strains of Listeria monocytogenes and one strain of Listeria innocua. Tissues were sampled and analyzed by microbiologic and histologic methods for 5 days postinoculation. In gnotobiotic pregnant BALB/c mice, L. monocytogenes Scott A (SA), serotype 4b, colonized the gastrointestinal tract, translocated to the livers and spleens of mice by day 1 postinoculation, and multiplied in these tissues until day 4. Infection of the placental tissues occurred by days 3 and 4 and was followed by infection of the fetuses. Little damage of colonic and cecal tissues was evident by histologic examination. Livers and spleens showed a cellular immune response; a similar immune response was not detected in the placentas or fetuses. A rough variant of L. monocytogenes SA which was as virulent as the parent strain in mice when injected intraperitoneally was less virulent perorally and did not consistently infect the fetuses. L. monocytogenes ATCC 19113, serotype 3a, did not colonize the gastrointestinal tract, nor was it isolated from any internal tissue. L. monocytogenes strains of serotypes 1/2a and 1/2b behaved like the SA strain in this mouse model. L. innocua colonized the gastrointestinal tract and translocated to the livers and spleens but did not survive in these organs and rapidly disappeared without infecting placental and fetal tissues. In comparison with gnotobiotic mice, conventional pregnant mice inoculated with L. monocytogenes strains showed less consistent infection. These results suggest that the gnotobiotic pregnant mouse is a useful model for detecting differences in virulence relating to colonization, invasiveness, and uteroplacental infection which cannot be detected by intraperitoneal inoculation of mice.     

Specific gene probe for detection of biotyped and serotyped Listeria strains

A total of 284 strains of Listeria, including all known serovars and biovars together with Listeria grayi and Listeria murrayi, were biotyped and serotyped. Biotyping and serotyping could be done in 2 days. A gene probe encoding a delayed hypersensitivity factor (DTH) was used in the detection of pathogenic biotypes and serotypes of the tested strains. The gene was found in all 117 tested Listeria monocytogenes strains of serogroups 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4c, 4d, 4e, 4ab, and 7. It was also present in Listeria ivanovii. Of 78 L. monocytogenes strains of serogroup 4b, 77 strains contained the gene, whereas it was absent in all 10 tested L. monocytogenes strains of serogroup 4a. Furthermore, the gene was absent in Listeria seeligeri, L. grayi, L. murrayi, and L. innocua of serogroups 3c, 4b, and 6a and in L. welshimeri of serogroups 1/2b, 3b, 6a, and 6b. Since L. monocytogenes and L. ivanovii are the only two biotypes of the genus Listeria considered pathogens, the data obtained indicate that the DNA probe tested may be a useful tool in the detection of virulent Listeria isolates in clinical, environmental, and food samples.     

Specific gene probe for detection of biotyped and serotyped Listeria strains.

A total of 284 strains of Listeria, including all known serovars and biovars together with Listeria grayi and Listeria murrayi, were biotyped and serotyped. Biotyping and serotyping could be done in 2 days. A gene probe encoding a delayed hypersensitivity factor (DTH) was used in the detection of pathogenic biotypes and serotypes of the tested strains. The gene was found in all 117 tested Listeria monocytogenes strains of serogroups 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4c, 4d, 4e, 4ab, and 7. It was also present in Listeria ivanovii. Of 78 L. monocytogenes strains of serogroup 4b, 77 strains contained the gene, whereas it was absent in all 10 tested L. monocytogenes strains of serogroup 4a. Furthermore, the gene was absent in Listeria seeligeri, L. grayi, L. murrayi, and L. innocua of serogroups 3c, 4b, and 6a and in L. welshimeri of serogroups 1/2b, 3b, 6a, and 6b. Since L. monocytogenes and L. ivanovii are the only two biotypes of the genus Listeria considered pathogens, the data obtained indicate that the DNA probe tested may be a useful tool in the detection of virulent Listeria isolates in clinical, environmental, and food samples.     

Genetic characterization of Listeria monocytogenes food isolates and pathogenic potential within serovars 1/2a and 1/2b

A total of 39 Listeria monocytogenes strains isolated from raw milk, smoked meat, chicken carcass and reference strains, belonging to serovars 1/2a, 4a, 1/2b, 3b and 4b, were analysed by RAPD and by polymorphisms of the virulent genes inlAB and iap. Ten isolates, belonging to serovars 1/2a and 1/2b and, collected from raw milk and smoked meat, were further tested for pathogenicity by IP injection into mice. The clustering of the 39 L. monocytogenes strains in 3 groups at 0.45 similarity level, based on molecular typing, was observed. Distribution of serovars in these clusters was in agreement with the proposed three Listeria monocytogenes lineages. Within serovar 1/2b, the 50% lethal dose (LD50) ranged from 8.4 x 10(4) to 1.7 x 10(6) cfu.ml(-1). One of the serovar 1/2b strains, isolated from smoked meat, exhibited the lowest virulence potential evaluated by LD50 and by mean time to death (MTD) and, from this point of view, was completely different from the other strains. Our results suggest the existence of heterogeneity in virulence levels within serovars 1/2a and 1/2b. However, when comparing the isolates based on genotyping, virulence indicators and food origin, no relation could be assessed.     

Bias in the Listeria monocytogenes enrichment procedure: lineage 2 strains outcompete lineage 1 strains in University of Vermont selective enrichments

Listeria monocytogenes can be isolated from a range of food products and may cause food-borne outbreaks or sporadic cases of listeriosis. L. monocytogenes is divided into three genetic lineages and 13 serotypes. Strains of three serotypes (1/2a, 1/2b, and 4b) are associated with most human cases of listeriosis. Of these, strains of serotypes 1/2b and 4b belong to lineage 1, whereas strains of serotype 1/2a and many other strains isolated from foods belong to lineage 2. L. monocytogenes is isolated from foods by selective enrichment procedures and from patients by nonselective methods. The aim of the present study was to investigate if the selective enrichment procedure results in a true representation of the subtypes of L. monocytogenes present in a sample. Eight L. monocytogenes strains (four lineage 1 strains and four lineage 2 strains) and one Listeria innocua strain grew with identical growth rates in the nonselective medium brain heart infusion (BHI), but differed in their growth rate in the selective medium University of Vermont medium I (UVM I). When coinoculated in UVM I, some strains completely outgrew other strains. This outcome was dependent on the lineage of L. monocytogenes rather than the individual growth rate of the strains. When inoculated at identical cell densities in UVM I, L. innocua outcompeted L. monocytogenes lineage 1 strains but not lineage 2 strains. In addition, lineage 2 L. monocytogenes strains outcompeted lineage 1 L. monocytogenes strains in all combinations tested, indicating a bias in strains selected by the enrichment procedures. Bias also occurred when coinoculating two lineage 2 or lineage 1 strains; however, it did not appear to correlate with origin (clinical versus food). Identical coinoculation experiments in BHI suggested that the selective compounds in UVM I and II influenced this bias. The results of the present study demonstrate that the selective procedures used for isolation of L. monocytogenes may not allow a true representation of the types present in foods. Our results could have a significant impact on epidemiological studies, as lineage 1 strains, which are often isolated from clinical cases of listeriosis, may be suppressed during enrichment by other L. monocytogenes lineages present in a food sample.     

Combined sigB allelic typing and multiplex PCR provide improved discriminatory power and reliability for Listeria monocytogenes molecular serotyping

Conventional serotyping has traditionally been used to subtype Listeria monocytogenes, but has several limitations, including low discriminatory power and poor reproducibility. Molecular serotyping methods have been developed for L. monocytogenes, but generally show limited discriminatory power and high misclassification rates. We selected 157 Listeria isolates to evaluate a combination of a previously described multiplex PCR assay and sigB allelic typing as an alternative molecular serotyping and subtyping strategy for L. monocytogenes. While the multiplex PCR assay differentiated five L. monocytogenes subtypes (Simpson's Index of Discrimination [SID]=0.78), including classification of the most common disease-associated serotypes (1/2a, 1/2b, 1/2c, and lineage I 4b) into four distinct groups, it misclassified 3.8% of the isolates studied here. sigB allelic typing differentiated 29 subtypes (SID=0.87) and also allowed identification of lineage III L. monocytogenes, which could not be differentiated from the other Listeria spp. by the multiplex PCR assay. sigB allelic typing failed to differentiate serotype 1/2c and 1/2a isolates and one sigB allelic type included serotype 4b and 1/2b isolates. A molecular serotyping approach that combines multiplex PCR and sigB sequence data showed increased discriminatory power (SID=0.91) over either method alone as well as conventional serotyping (SID=0.87) and classifies the four major serotypes (i.e., 1/2a, 1/2b, 1/2c, and 4b) into unique subgroups with a lower misclassification rate as compared to the multiplex PCR assay. This combined approach also differentiates lineage I serotype 4b isolates from the genetically distinct serotype 4b isolates classified into lineage III.     

Typing of human, animal, food, and environmental isolates of Listeria monocytogenes by multilocus enzyme electrophoresis.

In order to elucidate some aspects of the epidemiology of listeriosis in Switzerland, 181 strains of Listeria monocytogenes isolated from humans, animals, food, and the environment have been analyzed by multilocus enzyme electrophoresis at 21 enzyme loci. The clone responsible for several recent food-borne outbreaks in Switzerland and in North America (marked by electrophoretic type 1 and serovar 4b) has been found frequently among strains isolated from animals. Thus, animals may represent a major source of diffusion of this clone in the environment and in food, in which it has been found only sporadically, however. Two other unrelated clones (including strains belonging to serovars 1/2b and 1/2c) have often been isolated from meat but not from animals. These findings indicate that contamination of meat with L. monocytogenes might originate mainly from the environment in which it is processed rather than from animals themselves. This could explain the differences in the distribution of L. monocytogenes serovars isolated from meat and from animals.     

Typing of human, animal, food, and environmental isolates of Listeria monocytogenes by multilocus enzyme electrophoresis

In order to elucidate some aspects of the epidemiology of listeriosis in Switzerland, 181 strains of Listeria monocytogenes isolated from humans, animals, food, and the environment have been analyzed by multilocus enzyme electrophoresis at 21 enzyme loci. The clone responsible for several recent food-borne outbreaks in Switzerland and in North America (marked by electrophoretic type 1 and serovar 4b) has been found frequently among strains isolated from animals. Thus, animals may represent a major source of diffusion of this clone in the environment and in food, in which it has been found only sporadically, however. Two other unrelated clones (including strains belonging to serovars 1/2b and 1/2c) have often been isolated from meat but not from animals. These findings indicate that contamination of meat with L. monocytogenes might originate mainly from the environment in which it is processed rather than from animals themselves. This could explain the differences in the distribution of L. monocytogenes serovars isolated from meat and from animals.     

Physical and antigenic heterogeneity in the flagellins of Listeria monocytogenes and L. ivanovii

Listeria monocytogenes serotypes 4a, 4b and 7, and L. ivanovii, all grown at 20 degrees C, were negatively stained and examined by electron microscopy. Crude extracts of the cell surface of L. monocytogenes serotypes 1/2b, 3b, 3c, 4a, 4b, 4d and 7 and of L. ivanovii (all grown at 20 degrees C) were examined by SDS-PAGE and Western blotting using (i) affinity-purified polyclonal monospecific antibody, and (ii) monoclonal antibody, each raised against 29 kDa flagellin of serotype 4b. No flagella were seen on serotype 7 by electron microscopy and no flagellin was detected in crude cell surface extracts of serotype 7 either in silver-stained gels or in Western blots. The monospecific polyclonal antibody detected flagellins of approximate molecular mass 29 kDa in each of the seven flagellate strains including L. ivanovii. The monoclonal antibody detected 29 kDa flagellin in serotypes 1/2b, 3b, 4a, 4b and 4d, but not the flagellins of serotype 3c or L. ivanovii, which had a slightly lower molecular mass. Following prolonged electrophoresis of crude flagellar extracts the 29 kDa complex was resolved into three closely migrating bands. In a heterologous system using serotype 1/2b crude flagellar extract, all three bands were detected using the polyclonal antibody whereas only two bands were detected by the monoclonal antibody. It is concluded that polyclonal anti-flagellin antibodies are not useful tools with which to distinguish serotypes of L. monocytogenes sensu lato in immunoblotting, but that differences can be determined using a monoclonal antibody directed against particular components of the flagellar complex. These differences did not fully correspond to those anticipated from results of agglutination tests.     

Host ranges of Listeria-specific bacteriophages from the turkey processing plant environment in the United States

Even though at least 400 Listeria phages have been isolated from various sources, limited information is available on phages from the food processing plant environment. Phages in the processing plant environment may play critical roles in determining the Listeria population that becomes established in the plant. In this study, we pursued the isolation of Listeria-specific phages from environmental samples from four turkey processing plants in the United States. These environmental samples were also utilized to isolate Listeria spp. Twelve phages were isolated and classified into three groups in terms of their host range. Of these, nine (group 1) showed a wide host range, including multiple serotypes of Listeria monocytogenes, as well as other Listeria spp. (L. innocua, L. welshimeri, L. seeligeri, and L. ivanovii). The remaining phages mostly infected L. monocytogenes serotype 4b as well as L. innocua, L. ivanovii, and/or L. welshimeri. All but one of the strains of the serotype 4b complex (4b, 4d, 4e) from the processing plant environment could be readily infected by the wide-host-range phages isolated from the environment of the processing plants. However, many strains of other serotypes (1/2a [or 3a] and 1/2b [or 3b]), which represented the majority of L. monocytogenes strains isolated from the environmental samples, were resistant to infection by these phages. Experiments with two phage-resistant strains showed reduced phage adsorption onto the host cells. These findings suggest that phage resistance may be an important component of the ecology of L. monocytogenes in the turkey processing plants.     

Heteroduplex mobility assay for the identification of Listeria sp and Listeria monocytogenes strains: application to characterisation of strains from sludge and food samples

One hundred and ten Listeria sp. isolates from sewage sludge were identified according to phenotypic and genotypic methods. The Listeria sp. strains isolated from five types of sludge from three sewage treatment plants in Angers (France) and the surrounding area included L. monocytogenes (55.5%), L. innocua (29.1%), L. seeligeri (13.6%) and L. welshimeri (1.8%). The majority of L. monocytogenes strains belonged to serotypes 4b, 1/2b and 1/2a. Moreover, a heteroduplex mobility assay based on the 16S rRNA sequences was tested for its ability to identify the six species of the genus Listeria. This study, performed on 283 Listeria sp. strains from human, food and sewage sludge samples, showed that all the species were distinguishable from one another. L. innocua and L. seeligeri showed respectively three and two distinct banding patterns. Within L. monocytogenes, four groups (I-IV) were defined. The majority of food and environmental isolates were clustered in group I and it is noteworthy that group IV clustered epidemiologic isolates and strains belonging to serotypes 4b, 1/2a and 1/2b.     

Differentiation of epidemic-associated strains of Listeria monocytogenes by restriction fragment length polymorphism in a gene region essential for growth at low temperatures (4 degrees C).

The growth of Listeria monocytogenes in food stored in the cold has often been implicated in outbreaks of listeriosis. Many subtyping schemes have suggested that epidemic-associated strains belong to a unique genetic group. It has not yet been possible, however, to identify molecular or bacteriologic markers unique to epidemic-associated strains. Recently we cloned three genes of L. monocytogenes, ltrA, ltrB, and ltrC, which are essential for growth at low temperatures (4 degrees C). The use of a 1.2-kb PstI fragment derived from ltrB as a probe in Southern blots of HindIII-digested DNA revealed three hybridization patterns: the first (a 5.0-kb band) was observed in strains of serotypes 4b, 1/2b, and 3b; the second (a 3.1-kb band) was seen in strains of serotypes 1/2a, 3a, 1/2c, and 3c; and the third (a 9.5-kb band) was characteristic of epidemic-associated serotype 4b strains. These and other data suggest that probes derived from this gene region that is essential for growth at low temperatures can be useful molecular tools for the subtyping of strains implicated in food-borne listeriosis.     

进口水产品中单增李斯特菌的分子流行病学特点

摘要：[目的] 探明进口水产品中单增李斯特菌的污染状况、致病性和分子特征。[方法] 针对2007年7月至2008年11月间从29个国家进口的1275批水产品,进行单增李斯特菌鉴定、谱系与血清型分析、小鼠毒力试验与多位点序列分析。[结果] 检出单增李斯特菌33批次（2.6%）,其中以4b型为主（65.2%）,而1/2a型、1/2b型与1/2c型仅分别占13.0%、17.4%与4.4%。这些分离株对小鼠均具有与强毒参考株相当的毒力。基于actA-hisJ-ribC-sigB的多位点序列分析可将32个菌株分为23个序列型,分辨力达0.97。其中3个序列型包含3个以上分离株,其中序列型9属于流行性克隆I。[结论] 进口水产品中单增李斯特菌污染率与国内水产品相近,但血清型分布以4b型为主,且有流行性克隆I检出,因此要加强对进口水产品中单增李斯特菌的监测。     

Combined ribotyping and random multiprimer DNA analysis to probe the population structure of Listeria monocytogenes

To improve our understanding of the genetic links between strains originating from food and strains responsible for human diseases, we studied the genetic diversity and population structure of 130 epidemiologically unrelated Listeria monocytogenes strains. Strains were isolated from different sources and ecosystems in which the bacterium is commonly found. We used rRNA gene restriction fragment length polymorphism analysis with two endonucleases and random multiprimer DNA analysis with seven oligonucleotide primers to study multiple genetic features of each strain. We used three clustering methods to identify genetic links between individual strains and to determine the precise genetic structure of the population. The combined results confirmed that L. monocytogenes strains can be divided into two major phylogenetic divisions. The method used allowed us to demonstrate that the genetic structure and diversity of the two phylogenetic divisions differ. Division I is the most homogeneous and can easily be divided into subgroups with dissimilarity distances of less than 0.30. Each of these subgroups mainly, or exclusively, contains a single serotype (1/2b, 4b, 3b, or 4a). The serotype 4a lineage appears to form a branch that is highly divergent from the phylogenetic group containing serotypes 1/2b, 4b, and 3b. Division II contains strains of serotypes 1/2a, 1/2c, and 3a. It exhibits more genetic diversity with no peculiar clustering. The fact that division II is more heterogeneous than division I suggests that division II evolved from a common ancestor earlier than division I. A significant association was found between division I and human strains, suggesting that strains from division I are better adapted to human hosts.     

An SNP-based PCR assay to differentiate between Listeria monocytogenes lineages derived from phylogenetic analysis of the sigB gene

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

Adherence of Listeria monocytogenes strains to stainless steel coupons

An assay was developed to measure the number of Listeria monocytogenes cells adhering to stainless steel, and was used to investigate the adherence of 111 strains of the organism, which included representatives with respect to serotype, carriage of plasmids, source and persistence in the food processing environment. Growth and adherence curves of four L. monocytogenes strains over 48 h were obtained. While the growth curves of all four micro-organisms were seen to reach similar levels at stationary phase, there was still substantial variation among the adherence curves. In addition, a scatter-graph of growth vs adherence counts at 24 h showed poor correlation. These factors indicated that interstrain variation in adherence at stationary phase is due to factor(s) intrinsic to each strain of L. monocytogenes. Persistent strains were found to adhere in significantly greater numbers than sporadic strains, and variation was also found among serotypes, with serotype 1/2c showing significantly greater adherence than serotypes 1/2a and 4b; 4b strains were significantly higher than those of 1/2a strains. No significant difference was found between strains according to source or plasmid carriage.     

Flagellin from Listeria monocytogenes is glycosylated with beta-O-linked N-acetylglucosamine

Glycan staining of purified flagellin from Listeria monocytogenes serotypes 1/2a, 1/2b, 1/2c, and 4b suggested that the flagellin protein from this organism is glycosylated. Mass spectrometry analysis demonstrated that the flagellin protein of L. monocytogenes is posttranslationally modified with O-linked N-acetylglucosamine (GlcNAc) at up to six sites/monomer. The sites of glycosylation are all located in the central, surface-exposed region of the protein monomer. Immunoblotting with a monoclonal antibody specific for beta-O-linked GlcNAc confirmed that the linkage was in the beta configuration, this residue being a posttranslational modification commonly observed in eukaryote nuclear and cytoplasmic proteins.     

Characteristics and frequency of detection of fecal Listeria monocytogenes shed by livestock, wildlife, and humans

Listeria monocytogenes is a facultative intracellular pathogen that can be carried asymptomatically in various animals and can be shed in feces. We investigated the prevalence and characteristics of L. monocytogenes isolated from livestock, wildlife, and human potential sources of contamination in 2 areas in Ontario, Canada. From February 2003 to November 2005, a total of 268 fecal samples were collected from different animals. Listeria monocytogenes was isolated using selective enrichment, isolation, and confirmation procedures, and 15 samples (6%) yielded to the isolation of 84 confirmed strains. Listeria monocytogenes was isolated from livestock (beef and dairy), wildlife (deer, moose, otter, and raccoon), and human (biosolids and septic) fecal sources. Thirty-two isolates were from serovar 1/2a, 34 from serovar 1/2b, 1 from serovar 3a, and 17 from serovar 4b. Listeria monocytogenes populations were resolved into 13 EcoRI ribotypes, and 18 ApaI and 18 AscI pulsotypes, with Simpson indexes of discrimination of 0.878 and 0.907, respectively. A majority (59%) of L. monocytogenes isolates exhibited potential virulence linked to the production of a functional internalin A, which was supported by higher entry into Caco-2 cells (9.3%) than isolates producing truncated and secreted internalin A (1.3% of entry). Listeria monocytogenes fecal isolates were on average resistant to 6.4 +/- 2.5 antibiotics out of 17 tested, and potentially virulent isolates exhibited an enhanced resistance to kanamycin, gentamicin, streptomycin, and rifampicin. Livestock, wildlife, and human L. monocytogenes fecal communities exhibited overlapping but distinct populations, and some genotypes and phenotypes were similar to those previously described for surface water isolates in the same area.     

A novel serotype-specific gene cassette (gltA-gltB) is required for expression of teichoic acid-associated surface antigens in Listeria monocytogenes of serotype 4b

Listeria monocytogenes serotype 4b strains account for about 40% of sporadic cases and many epidemics of listeriosis. Mutations in a chromosomal locus resulted in loss of reactivity with all three monoclonal antibodies (MAbs) which were specific to serotype 4b and the closely related serotypes 4d and 4e. Here we show that this locus contains a serotype 4b-4d-4e-specific gene cassette (3,071 bp) which consists of two genes, gltA and gltB, and is flanked by palindromic sequences (51 and 44 nucleotides). Complete loss of reactivity with the three serotype-specific MAbs resulted from insertional inactivation of either gltA or gltB. The gltA and gltB mutants were characterized by loss and severe reduction, respectively, of glucose in the teichoic acid, whereas galactose, the other serotype-specific sugar substituent in the teichoic acid, was not affected. Within L. monocytogenes, only strains of serotypes 4b, 4d, and 4e harbored the gltA-gltB cassette, whereas coding sequences on either side of the cassette were conserved among all serotypes. Comparative genomic analysis of a serotype 1/2b strain showed that the 3,071-bp gltA-gltB cassette was replaced by a much shorter (528-bp) and unrelated region, flanked by inverted repeats similar to their counterparts in serotype 4b. These findings indicate that in the evolution of different serotypes of L. monocytogenes, this site in the genome has become occupied by serotype-specific sequences which, in the case of serotype 4b, are essential for expression of serotype-specific surface antigens and presence of glucose substituents in the teichoic acids in the cell wall.