A sheep in wolf's clothing: Listeria innocua strains with teichoic acid-associated surface antigens and genes characteristic of Listeria monocytogenes serogroup 4

Listeria monocytogenes serotype 4b has been implicated in numerous food-borne epidemics and in a substantial fraction of sporadic listeriosis. A unique lineage of the nonpathogenic species Listeria innocua was found to express teichoic acid-associated surface antigens that were otherwise expressed only by L. monocytogenes of serotype 4b and the rare serotypes 4d and 4e. These L. innocua strains were also found to harbor sequences homologous to the gene gtcA, which has been shown to be essential for teichoic acid glycosylation in L. monocytogenes serotype 4b. Transposon mutagenesis and genetic studies revealed that the gtcA gene identified in this lineage of L. innocua was functional in serotype 4b-like glycosylation of the teichoic acids of these organisms. The genomic organization of the gtcA region was conserved between this lineage of L. innocua and L. monocytogenes serotype 4b. Our data are in agreement with the hypothesis that, in this lineage of L. innocua, gtcA was acquired by lateral transfer from L. monocytogenes serogroup 4. The high degree of nucleotide sequence conservation in the gtcA sequences suggests that such transfer was relatively recent. Transfer events of this type may alter the surface antigenic properties of L. innocua and may eventually lead to evolution of novel pathogenic lineages through additional acquisition of genes from virulent listeriae.     

Screening and characterization of monoclonal antibodies to the surface antigens of Listeria monocytogenes serotype 4b

Aims:  This study aims to develop and characterize monoclonal antibodies (Mabs) with high specificity and affinity for surface antigens of an epidemiologically important serotype 4b of Listeria monocytogenes .
Methods and Results:  Hybridoma clones were derived from B lymphocytes of mice immunized with L. monocytogenes serotype 4b and screened against this strain by an enzyme-linked immunosorbent assay. Twenty-nine clones secreting Mabs reactive with formalin-killed bacteria were obtained; 15, 8, 5 and 1 Mabs were immunoglobulin subclasses IgG2a, IgG2b, IgM and IgG1, respectively. Immuofluoresence or immunogold labelling demonstrated all except five IgM and one IgG2a Mabs bound to the surface of a live L. monocytogenes serotype 4b. The majority of the 23 surface-binding Mabs recognized linear epitopes on a 77-kDa protein. These surface-binding Mabs exhibited little or no cross-reactivity with non-4b serotypes (1/2a, 1/2b, 3a, etc.) of L. monocytogenes , five other Listeria species, Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium.
Conclusions:  The Mabs recognizing a 77-kDa surface protein are novel antibodies with specificity and affinity for L. monocytogenes serotype 4b.
Significance and Impact of the Study:  These anti-77 kDa surface protein Mabs may be explored as reagents for the development of Mabs-based diagnostic immunoassays for L. monocytogenes serotype 4b strains.     

Growth of Listeria monocytogenes in meat juice under a modified atmosphere at 4°C with or without members of a microbial association from chilled lamb under a modified atmosphere

Listeria monocytogenes together with Gram-positive and Gram-negative bacteria was grown in axenic cultures in meat juice under an atmosphere enriched with carbon dioxide at 4°C. The size of the final population of listeriae was determined by that of the initial inoculum. Growth of L. monocytogenes ceased with glucose depletion. Glucose-6-phosphate was not catabolized by listeriae. In aerobic axenic cultures generation times of L. monocytogenes were shorter and glucose depletion was faster than with a carbon dioxide-enriched atmosphere. Oxidation of L- and D-lactic to acetic acid was probably associated with the subsequent die-off of L. monocytogenes.     

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.     

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.     

Restriction fragment length polymorphisms detected with novel DNA probes differentiate among diverse lineages of serogroup 4 Listeria monocytogenes and identify four distinct lineages in serotype 4b.

Listeria monocytogenes of serotype 4b has been implicated in numerous outbreaks of food-borne listeriosis and in ca. 40% of sporadic cases. Strains of this serotype appear to be relatively homogeneous genetically, and molecular markers specific for distinct serotype 4b lineages have not been frequently identified. Here we show that DNA fragments derived from the putative mannitol permease locus of Listeria monocytogenes had an unexpectedly high potential to differentiate among different strains of serotype 4b when used as probes in Southern blotting of EcoRI-digested genomic DNA, yielding four distinct restriction fragment length polymorphism (RFLP) patterns. Strains of two epidemic-associated lineages, including the major epidemic clone implicated in several outbreaks in Europe and North America, had distinct RFLPs which differed from those of all other serotype 4b strains that we screened but which were encountered among strains of serotypes 1/2b and 3b. In addition, three serogroup 4 lineages were found to have unique RFLPs that were not encountered among any other L. monocytogenes strains. One was an unusual lineage of serotype 4b, and the other two were members of the serotype 4a and 4c group. The observed polymorphisms may reflect evolutionary relationships among lineages of L. monocytogenes and may facilitate detection and population genetic analysis of specific lineages.     

Factors controlling acid tolerance of Listeria monocytogenes: effects of nisin and other ionophores.

The acid tolerance of a Listeria monocytogenes serotype 4b strain was studied by measuring its ability to survive at an acidic pH at 37 degrees C. The acid tolerance of L. monocytogenes was much lower than those of Escherichia coli O157:H7 and Shigella flexneri strains. This observation suggested a higher infective dose for L. monocytogenes than E. coli O157:H7 and Shigella. The susceptibility of L. monocytogenes to acidic pH was dependent upon growth medium pH and growth phase of the culture. Nisin and some other ionophores reduced the acid tolerance of both stationary-phase and log-phase cultures of L. monocytogenes. These studies indicated that nisin might be a useful candidate for controlling acid tolerance of L. monocytogenes.     

Atypical Listeria monocytogenes serotype 4b strains harboring a lineage II-specific gene cassette

Listeria monocytogenes is the etiological agent of listeriosis, a severe food-borne illness. The population of L. monocytogenes is divided into four lineages (I to IV), and serotype 4b in lineage I has been involved in numerous outbreaks. Several serotype 4b epidemic-associated clonal groups (ECI, -II, and -Ia) have been identified. In this study, we characterized a panel of strains of serotype 4b that produced atypical results with a serotype-specific multiplex PCR and possessed the lmo0734 to lmo0739 gene cassette that had been thought to be specific to lineage II. The cassette was harbored in a genomically syntenic locus in these isolates and in lineage II strains. Three distinct clonal groups (groups 1 to 3) were identified among these isolates based on single-nucleotide polymorphism-based multilocus genotyping (MLGT) and DNA hybridization data. Groups 1 and 2 had MLGT haplotypes previously encountered among clinical isolates and were composed of clinical isolates from multiple states in the United States. In contrast, group 3 consisted of clinical and environmental isolates solely from North Carolina and exhibited a novel haplotype. In addition, all group 3 isolates had DNA that was resistant to MboI, suggesting methylation of adenines at GATC sites. Sequence analysis of the lmo0734 to lmo0739 gene cassette from two strains (group 1 and group 3) revealed that the genes were highly conserved (>99% identity). The data suggest relatively recent horizontal gene transfer from lineage II L. monocytogenes into L. monocytogenes serotype 4b and subsequent dissemination among at least three distinct clonal groups of L. monocytogenes serotype 4b, one of which exhibits restrictions in regional distribution.     

Generalized transduction of serotype 1/2 and serotype 4b strains of Listeria monocytogenes

This is the first report of generalized transduction in the gram-positive, food-borne pathogen Listeria monocytogenes. Bacteriophages were isolated from the environment and from lysogens, or were obtained from other laboratories. Of the 59 bacteriophages tested, 34 proved to be capable of transduction. We exploited the ability of L. monocytogenes to grow at room temperature and isolated bacteriophages that were incapable of growth at 37 degrees C. Transductions at this temperature therefore eliminated transductant killing and lysogeny, as did inclusion of citrate and the use of a low multiplicity of infection. Transducing bacteriophages were found for each of the well-characterized L. monocytogenes strains: EGD, 10403, Mack (serotype1/2a), L028 (serotype 1/2c), Scott A (serotype 4b) and strains from the Jalisco and Halifax, Nova Scotia outbreaks (serotype 4b). P35 (phiLMUP35) is a particularly useful generalized transducing bacteriophage with a wide host range (75% of all serotype 1/2 strains tested). Its disadvantages are that it is small and transduction is relatively infrequent. U153(phiCU-SI153/95) is larger than P35 and transduction frequency increased 100-fold, but it has a very narrow host range. We demonstrated interstrain transduction and used transduction to test linkage between transposon insertions and mutant phenotypes in a variety of strains.     

Estimating size and diversity of nitrifying communities in deodorizing filters using PCR and immunofluorescence

Thirty isolates of Listeria monocytogenes and 18 of L. innocua obtained from different short-ripened cheeses manufactured in Asturias (northern Spain), were compared with each other and with reference strains using serotype, phage type and pulsed-field restriction endonuclease digestion profiles analysis of the total DNA. Restriction enzymes Apa I and Sma I defined five clusters in L. monocytogenes ( m1 to m5 ) and two main clusters in L. innocua ( i1 and i2 ). Cluster i2 was further arranged into three subclusters ( i2a , i2b and i2c ) based on the different Eco 52I ( Xma III) and Crf 42I ( Sac II) patterns of its isolates. Clusters of L. innocua were clearly different whereas those of L. monocytogenes were more closely related to each other. In this latter species, serotype 4b isolates ( m4 and m5 ) constituted a more homogeneous group than serogroup 1 isolates ( m1 , m2 and m3 ). Cluster m3 contained two strains of serotype 1/2a whereas m1 and m2 harboured strains of both serotypes, 1/2a and 1/2b. Therefore, the combined use of restriction patterns and serotype may be useful to differentiate L. monocytogenes strains showing identical restriction profiles but differing in serotype. The cheese source of Listeria strains proved that isolates from cluster m1 were repeatedly detected as a contaminant in the same type of cheese. Comparison of L. monocytogenes Apa I profiles showed a genetic proximity of m4 and m5 to the recognized pathogenic strains ATCC 13932 and NCTC 11994, responsible for meningitis cases in other countries. Finally, bacteriophage typing data indicated that m4 , the sole phage typable group, had a phage type resembling that of strains causing the Auckland (New Zealand) outbreak of listeriosis in 1969. These data suggest a wide distribution of closely related types which might cause, under several circumstances, sporadic cases of listeriosis.     

Ability of the Listeria monocytogenes strain Scott A to cause systemic infection in mice infected by the intragastric route

Listeriosis is an important food-borne disease that causes high rates of morbidity and mortality. For reasons that are not clear, most large outbreaks of human listeriosis involve Listeria monocytogenes serotype 4b. Relatively little is known about the pathogenesis of listeriosis following gastrointestinal exposure to food-borne disease isolates of L. monocytogenes. In the present study, we investigated the pathogenesis of systemic infection by the food-borne isolate Scott A in an intragastric (i.g.) mouse challenge model. We found that the severity of infection with L. monocytogenes Scott A was increased in mice made neutropenic by administration of monoclonal antibody RB6-8C5. This observation was similar to a previous report on a study with the laboratory strain L. monocytogenes EGD. Prior administration of sodium bicarbonate did not enhance the virulence of L. monocytogenes strain Scott A for i.g. inoculated mice. Following i.g. inoculation of mice, two serotype 4b strains of L. monocytogenes (Scott A and 101M) achieved a greater bacterial burden in the spleen and liver and elicited more severe histopathological damage to those organs than did a serotype 1/2a strain (EGD) and a serotype 1/2b stain (CM). Of the four strains tested, only strain CM exhibited poor survival in synthetic gastric fluid in vitro. The other three strains exhibited similar patterns of survival at pHs of greater than 5 and relatively rapid (<30 min) loss of viability at pHs of less than 5.0. Growth of L. monocytogenes Scott A at temperatures of 12.5 to 37 degrees C did not affect its ability to cause systemic infection in i.g. inoculated mice. These observations suggest that the serotype 4b L. monocytogenes strains Scott A and 101M possess one or more virulence determinants that make them better able to cause systemic infection following inoculation via the g.i. tract than do the serotype 1/2 strains EGD and CM.     

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.     

Competitive fitness of Listeria monocytogenes serotype 1/2a and 4b strains in mixed cultures with and without food in the U.S. Food and Drug Administration enrichment protocol

Thirteen different serotypes of the food-borne pathogen Listeria monocytogenes have been described. Serotype 4b strains are most often associated with illness, and serotype 1/2a strains are most often isolated from foods and processing plants. Different abilities to respond to stresses have been described for serotype 4b and 1/2a strains. One of the common enrichment protocols used to test foods for the presence L. monocytogenes is described in the U.S. Food and Drug Administration (FDA) Bacterial Analytical Manual (BAM). We compared three strains of L. monocytogenes serotype 4b and five strains of serotype 1/2a in direct competition with each other in two-strain mixed cultures by using the FDA BAM enrichment protocol, which includes both enrichment broth and selective agar, with and without added food to mimic the conditions that occur during attempts to isolate Listeria species from contaminated foods. Using a colony immunoblot procedure and analyzing over 112,000 colonies, we observed differences in strain fitness, but these differences were not attributable to serotype or genetic lineage.     

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.     

DNA fragments from regions involved in surface antigen expression specifically identify Listeria monocytogenes serovar 4 and a subset thereof: cluster IIB (serotypes 4b, 4d, and 4e).

Listeria monocytogenes serotype 4b has frequently been implicated in sporadic as well as epidemic listeriosis. On the basis of pulsed-field fingerprinting, serotype 4b strains, along with strains of serotypes 4d and 4e, constitute one genomic cluster (IIB). We have identified two genomic regions essential for the expression of surface antigens which previously were shown to be specific to cluster IIB strains. A DNA probe of 1.1 kb derived from one of the regions (probe 1) hybridized only with strains of serotypes 4b, 4d, and 4e in Southern blots and dot blots. A different DNA probe of 0.3 kb (probe 2), derived from the other region, hybridized with all serovar 4 strains (serotypes 4b, 4a, 4c, 4d, and 4e). All other L. monocytogenes serotypes were negative with probe 1 or 2. Use of probe 1 in Southern blots of EcoRI-digested genomic DNA revealed a restriction fragment length polymorphism in serotype 4b strains, with the hybridizing EcoRI fragments being 4.5 kb (strains of the epidemic clone) and either 4.5 or 5.0 kb (all other serotype 4b strains). Although the probes hybridized with a special group of Listeria innocua strains which also expressed the surface antigens, the latter could be readily distinguished by the size of the hybridizing EcoRI fragment with probe 1 (ca. 2.2 kb). These data suggest that the combined use of these probes with L. monocytogenes can readily and specifically identify cluster IIB strains as well as the entire serovar 4 complex.     

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.     

Purification of a delayed hypersensitivity-inducing protein from Listeria monocytogenes

Abstract Cell-envelope fragments were prepared from Listeria monocytogenes L242, serotype 4b. Delayed hypersensitivity (DH)-inducing proteins were extracted with deoxycholate and separated into two fractions by filtration through a Sephacryl S-200 column equilibrated with deoxycholate buffer. The second peak eluting from the Sephacryl column was fractionated using ion exchange chromatography on a DEAE Sepharose CL-6B column in the presence of 6 M urea. A purified 20 400-Da protein which induced DH against L. monocytogenes was obtained by isocratic elution. Three other DH-inducing fractions containing several protein bands were eluted by a gradient of potassium thiocyanate (KSCN) in urea buffer. Our results indicate that denaturing conditions can be employed for the fractionation and purification of DH inducing proteins from L. monocytogenes . In addition, it is suggested that the procedure described might also be useful for the purification of other antigens involved in cellular immune reactions.     

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.     

Monoclonal antibodies with a high degree of specificity for Listeria monocytogenes serotype 4b.

Strains of Listeria monocytogenes serotype 4b account for a large fraction of sporadic listeriosis cases, as well as all major food-borne epidemics attributed to this pathogen. We have identified a set of three monoclonal antibodies which showed a high degree of specificity for strains of L. monocytogenes serotype 4b. Two of these antibodies (c74.33 and c74.180, isotypes immunoglobulin M [IgM] and IgG3, respectively) recognized all serotype 4b strains, whereas antibody c74.22 (isotype IgG1) failed to recognize certain epidemic-associated strains. The corresponding antigens were located on the surface of the bacteria and were expressed following bacterial growth in different media and over a wide range of temperatures (4, 22, and 37 degrees C). Heating L. monocytogenes cells at 80,90, or 100 degrees C abolished reactivity for c74.22 but not for c74.33 MAb. These MAbs were negative for all of the non-Listeria strains tested, including representatives of several gram-negative and gram-positive species. The surface antigen recognized by c74.22 appeared to be associated with the ability of the bacteria to enter (invade) mammalian cells in culture.     

Absence of serotype-specific surface antigen in laboratory variants of epidemic-associated Listeria monocytogenes strains

Variants that lacked reactivity with the serotype 4b-specific monoclonal antibody c74.22 and that lost susceptibility to certain Listeria- or serotype 4b-specific phages were identified in the course of genetic studies with serotype 4b Listeria monocytogenes strains H7550 and F2381L (epidemic clones I and II, respectively). Our findings suggest that such variants can become inadvertently established under laboratory conditions and suggest caution in work involving serotype 4b strains and genetic constructs thereof.