Pyrosequencing as a Method for Grouping of Listeria monocytogenes Strains on the Basis of Single-Nucleotide Polymorphisms in the inlB Gene

By using pyrosequencing (i.e., sequencing by synthesis) 106 strains of different serovars of Listeria monocytogenes were rapidly grouped into four categories based on nucleotide variations at positions 1575 and 1578 of the inlB gene. Strains of serovars 1/2a and 1/2c constituted one group, and strains of serovars 1/2b and 3b constituted another group, whereas serovar 4b strains were separated into two groups.     

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°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.     

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.     

Analysis of surface proteins of Listeria in relation to species, serovar and pathogenicity.

SDS extracts of whole bacteria, representing five species and 15 serovars of Listeria, were analysed by SDS-PAGE and by immunoblotting with serum directed against whole formalin-treated L. monocytogenes. Profiles of L. monocytogenes were very different from those of other species of Listeria (i.e. L. innocua,L.welshimeri, L. seeligeri and L. ivanovii). This low degree of similarity between species was found even in the case of common serovars. Within the species L. monocytogenes, protein patterns were characterized, on the one hand, by a high degree of homogeneity between all strains of the same serovar and, on the other hand, by large differences between serovars, especially between sv. 1/2 and 4b. Thus we have identified major, surface-located protein antigens, specific for L. monocytogenes, either common to all serovars (64 and 68 kDa) or characteristic of certain serovars: 98 kDa for sv. 1/2 and 3; 76 and 78 kDa for sv. 4b, 4d and 4e; and 80 and 100 kDa for sv. 4a and 4c. Moreover, some of these bands (68 and 98 kDa) might be related to virulence, since differences were noticed between the profiles of haemolytic L. monocytogenes vs. 1/2a differing only in their virulence for immunocompromised mice. All these results confirmed, for the first time, the classification of Listeria obtained previously by genomic studies. They should help in the identification of new virulence factors and the development of easier and more specific methods of detection and identification.     

Comparative Proteomic Analysis of Listeria monocytogenes Strains F2365 and EGD

Listeria monocytogenes is a gram-positive, food-borne pathogen that causes disease in both humans and animals. There are three major genetic lineages of L. monocytogenes and 13 serovars. To further our understanding of the differences that exist between different genetic lineages/serovars of L. monocytogenes, we analyzed the global protein expression of the serotype 1/2a strain EGD and the serotype 4b strain F2365 during early-stationary-phase growth at 37°C. Using multidimensional protein identification technology with electrospray ionization tandem mass spectrometry, we identified 1,754 proteins from EGD and 1,427 proteins from F2365, of which 1,077 were common to both. Analysis of proteins that had significantly altered expression between strains revealed potential biological differences between these two L. monocytogenes strains. In particular, the strains differed in expression of proteins involved in cell wall physiology and flagellar biosynthesis, as well as DNA repair proteins and stress response proteins.     

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.     

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.     

Trends in the Epidemiology of Pandemic and Non-pandemic Strains of Vibrio parahaemolyticus Isolated from Diarrheal Patients in Kolkata,India

A total of 178 strains of V. parahaemolyticus isolated from 13,607 acute diarrheal patients admitted in the Infectious Diseases Hospital, Kolkata has been examined for serovar prevalence, antimicrobial susceptibility and genetic traits with reference to virulence, and clonal lineages. Clinical symptoms and stool characteristics of V. parahaemolyticus infected patients were analyzed for their specific traits. The frequency of pandemic strains was 68%, as confirmed by group-specific PCR (GS-PCR). However, the prevalence of non-pandemic strains was comparatively low (32%). Serovars O3:K6 (19.7%), O1:K25 (18.5%), O1:KUT (11.2%) were more commonly found and other serovars such as O3:KUT (6.7%), O4:K8 (6.7%), and O2:K3 (4.5%) were newly detected in this region. The virulence gene tdh was most frequently detected in GS-PCR positive strains. There was no association between strain features and stool characteristics or clinical outcomes with reference to serovar, pandemic/non-pandemic or virulence profiles. Ampicillin and streptomycin resistance was constant throughout the study period and the MIC of ampicillin among selected strains ranged from 24 to >256 µg/ml. Susceptibility of these strains to ampicillin increased several fold in the presence of carbonyl cyanide-m-chlorophenyldrazone. The newly reported ESBL encoding gene from VPA0477 was found in all the strains, including the susceptible ones for ampicillin. However, none of the strains exhibited the β-lactamase as a phenotypic marker. In the analysis of pulsed-field gel electrophoresis (PFGE), the pandemic strains formed two different clades, with one containing the newly emerged pandemic strains in this region.     

Comparative transcriptome analysis of Listeria monocytogenes strains of the two major lineages reveals differences in virulence, cell wall, and stress response

Listeria monocytogenes is a food-borne, opportunistic, bacterial pathogen causing a wide spectrum of diseases, including meningitis, septicemia, abortion, and gastroenteritis, in humans and animals. Among the 13 L. monocytogenes serovars described, human listeriosis is mostly associated with strains of serovars 4b, 1/2b, and 1/2a. Within the species L. monocytogenes, three phylogenetic lineages are described. Serovar 1/2a belongs to phylogenetic lineage I, while serovars 4b and 1/2b group in phylogenetic lineage II. To explore the role of gene expression in the adaptation of L. monocytogenes strains of these two major lineages to different environments, as well as in virulence, we performed whole-genome expression profiling of six L. monocytogenes isolates of serovars 4b, 1/2b, and 1/2a of distinct origins, using a newly constructed Listeria multigenome DNA array. Comparison of the global gene expression profiles revealed differences among strains. The expression profiles of two strains having distinct 50% lethal doses, as assessed in the mouse model, were further analyzed. Gene ontology term enrichment analysis of the differentially expressed genes identified differences in protein-, nucleic acid-, carbon metabolism-, and virulence-related gene expression. Comparison of the expression profiles of the core genomes of all strains revealed differences between the two lineages with respect to cell wall synthesis, the stress-related sigma B regulon and virulence-related genes. These findings suggest different patterns of interaction with host cells and the environment, key factors for host colonization and survival in the environment.     

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.     

RAPD- and actA gene-typing of Listeria monocytogenes isolates of human listeriosis, the intestinal contents of cows and beef

Seventy-five L. monocytogenes isolates of human listeriosis, the intestinal contents of cows and beef were divided into 5 major clusters, 17 sub-clusters and 28 minor clusters by typing using random amplification of polymorphic DNA (RAPD). According to their major RAPD category, L. monocytogenes isolates serotyped as 1/2b and 4b were distinguished from L. monocytogenes isolates of serovars 1/2a and 1/2c. Moreover serovar 4b was distinguished from serovar 1/2b by a difference in the RAPD sub-cluster category. All L. monocytogenes were found to possess either actA gene Type I or II, and only one actA gene type was detected in each RAPD minor cluster. actA gene Type II was observed in 32.0%, 38.5% and 18.9% of isolates from humans, cows and beef, respectively, and was detected more frequently in serovar 4b (46.9%) than in serovars 1/2a (22.2%), 1/2b (7.7%) and 1/2c (0.0%). Twenty (80%) of 25 human isolates fell within three minor RAPD types (II-d (16%), V-p-1 (36%), V-p-2 (28%)). Two isolates from humans and beef were found to have the same RAPD type (Type IV-k-1), actA gene type (Type I) and serovar (1/2b). Our results suggest that only a few genotypes of L. monocytogenes are predominant in human listeriosis in Japan, although the human isolates were collected over a broad span of time and a wide geographical range. Our results also suggest that RAPD-, actA gene- and sero-typing can be useful for epidemiological analysis.     

Epidemic Clone I-Specific Genetic Markers in Strains of Listeria monocytogenes Serotype 4b from Foods

Listeria monocytogenes contamination of ready-to-eat foods has been implicated in numerous outbreaks of food-borne listeriosis. However, the health hazards posed by L. monocytogenes detected in foods may vary, and speculations exist that strains actually implicated in illness may constitute only a fraction of those that contaminate foods. In this study, examination of 34 serogroup 4 (putative or confirmed serotype 4b) isolates of L. monocytogenes obtained from various foods and food-processing environments, without known implication in illness, revealed that many of these strains had methylation of cytosines at GATC sites in the genome, rendering their DNA resistant to digestion by the restriction endonuclease Sau3AI. These strains also harbored a gene cassette with putative restriction-modification system genes as well as other, genomically unlinked genetic markers characteristic of the major epidemic-associated lineage of L. monocytogenes (epidemic clone I), implicated in numerous outbreaks in Europe and North America. This may reflect a relatively high fitness of strains with these genetic markers in foods and food-related environments relative to other serotype 4b strains and may partially account for the repeated involvement of such strains in human food-borne listeriosis.     

Incidence of Listeria monocytogenes in Nature

During a research project on the occurrence of Listeria monocytogenes 194 strains were isolated in southern West Germany during the years 1972 to 1974: 154 from soil and plant samples (20.3%), 16 from feces of deer and stag (15.7%), 9 from old moldy fodder and wildlife feeding grounds (27.2%), and 8 from birds (17.3%). The highest number of isolates was obtained from uncultivated fields. The beta-hemolytic serovars 1/2b and 4b were predominant; other serovars (some of them identified for the first time), including nonhemolyzing strains, have been encountered frequently. It is suggested that Listeria monocytogenes is a saprophytic organism which lives in a plant-soil environment and therefore can be contracted by humans and animals via many possible routes from many sources.     

Pyrosequencing as a method for grouping of Listeria monocytogenes strains on the basis of single-nucleotide polymorphisms in the inlB gene.

By using pyrosequencing (i.e., sequencing by synthesis) 106 strains of different serovars of Listeria monocytogenes were rapidly grouped into four categories based on nucleotide variations at positions 1575 and 1578 of the inlB gene. Strains of serovars 1/2a and 1/2c constituted one group, and strains of serovars 1/2b and 3b constituted another group, whereas serovar 4b strains were separated into two groups.     

The Prevalence of Listeria monocytogenes in the Environment of Dairy Farms

The prevalence of Listeria monocytogenes in the environment of dairy farms was surveyed from December 1993 to June 1994 in one city of Hokkaido. L. monocytogenes was isolated from 3 out of 5 farms investigated. Serovar 4b organism was isolated from the brain stem of a cow from one farm which was clinically diagnosed as having listeriosis. The same serovar of L. monocytogenes was also isolated from the rectal contents of a healthy cow, straw on the floor, straw in the barn, and silage scattered around the silo from the same farm. At another farm, with no reported cases of bovine listeriosis, serovar 1/2 organism was isolated from the same types of samples as the above mentioned farm except from straw on the floor. The difference in the isolation rates of the organism from straw on the floor between the two farms (22%: 5/23 vs 0%: 0/24) is considered to be caused by the different feeding methods of silage between the two farms.     

Identification and Characterization of Nucleotide Sequence Differences in Three Virulence-Associated Genes of Listeria monocytogenes Strains Representing Clinically Important Serotypes

Listeria monocytogenes is a Gram-positive, facultative intracellular bacterium that causes invasive, often fatal, disease in susceptible hosts. As a foodborne pathogen, the bacterium has emerged as a significant public health problem and has caused several epidemics in the United States and Europe. Three serotypes (1/2a, 1/2b, 4b) of L. monocytogenes are responsible for nearly 95% of all reported cases of human listeriosis. L. monocytogenes serotype 4b has caused all well-characterized foodborne epidemic outbreaks in North America and Europe between 1981 and 1993. However, most of the genetic studies to characterize virulence factors of L. monocytogenes have been done by using serotypes 1/2a and 1/2c. In this investigation, we examined three virulence-associated genes (hly encoding listeriolysin, plcA encoding phosphotidylinositol-specific phospholipase C, and inlA encoding internalin) of two serotype 4b and two serotype 1/2b strains. We chose these virulence-associated genes on the basis of published sequence differences among strains from Listeria subgroups containing serotypes 1/2a and 1/2c versus 4b, respectively. They correspond to sequence homologies that include very highly conserved (hlyA), highly conserved (plcA) and mostly conserved (inlA). We found by using nucleotide sequence analysis of the hly, plcA, and inlA genes, the two L. monocytogenes strains (including a strain associated with a foodborne disease outbreak in California in 1985) in this study, two serotype 1/2b strains from a study that we recently reported, and other similar published data for serotypes 1/2a, 1/2c, and 4b, had a high degree of sequence conservation at the gene and protein levels for all three genes. However, the sequences for the hly gene of L. monocytogenes strains of serotypes 1/2b and 4b were more closely related to each other and showed significant divergence from serotypes 1/2a and 1/2c. A unique nonsynonymous mutation was found in the hly gene of L. monocytogenes isolates that were associated with the 1985 California outbreak and were the epidemic phage type. When 158 L. monocytogenes isolates from the collection at the Centers for Disease Control and Prevention were screened, the mutation was found only in one other strain that had been isolated in California 3 years before the epidemic. Although the California epidemic clone was lactose negative, other L. monocytogenes serotype 4b isolates that were lactose negative did not possess the unique mutation observed in that epidemic clone. Received: 18 June 1997 / Accepted: 4 December 1997     

The Presence of the Internalin Gene in Natural Atypically Hemolytic Listeria innocua Strains Suggests Descent from L. monocytogenes

The atypical hemolytic Listeria innocua strains PRL/NW 15B95 and J1-023 were previously shown to contain gene clusters analogous to the pathogenicity island (LIPI-1) present in the related foodborne gram-positive facultative intracellular pathogen Listeria monocytogenes, which causes listeriosis. LIPI-1 includes the hemolysin gene, thus explaining the hemolytic activity of the atypical L. innocua strains. No other L. monocytogenes-specific virulence genes were found to be present. In order to investigate whether any other specific L. monocytogenes genes could be identified, a global approach using a Listeria biodiversity DNA array was applied. According to the hybridization results, the isolates were defined as L. innocua strains containing LIPI-1. Surprisingly, evidence for the presence of the L. monocytogenes-specific inlA gene, previously thought to be absent, was obtained. The inlA gene codes for the InlA protein which enables bacterial entry into some nonprofessional phagocytic cells. PCR and sequence analysis of this region revealed that the flanking genes of the inlA gene at the upstream, 5′-end region were similar to genes found in L. monocytogenes serotype 4b isolates, whereas the organization of the downstream, 3′-end region was similar to that typical of L. innocua. Sequencing of the inlA region identified a small stretch reminiscent of the inlB gene of L. monocytogenes. The presence of two clusters of L. monocytogenes-specific genes makes it unlikely that PRL/NW 15B95 and J1-023 are L. innocua strains altered by horizontal transfer. It is more likely that they are distinct relics of the evolution of L. innocua from an ancestral L. monocytogenes, as postulated by others.     

Examination of Food Chain-Derived Listeria monocytogenes Strains of Different Serotypes Reveals Considerable Diversity in inlA Genotypes,Mutability, and Adaptation to Cold Temperatures

Listeria monocytogenes strains belonging to serotypes 1/2a and 4b are frequently linked to listeriosis. While inlA mutations leading to premature stop codons (PMSCs) and attenuated virulence are common in 1/2a, they are rare in serotype 4b. We observed PMSCs in 35% of L. monocytogenes isolates (n = 54) recovered from the British Columbia food supply, including serotypes 1/2a (30%), 1/2c (100%), and 3a (100%), and a 3-codon deletion (amino acid positions 738 to 740) seen in 57% of 4b isolates from fish-processing facilities. Caco-2 invasion assays showed that two isolates with the deletion were significantly more invasive than EGD-SmR (P < 0.0001) and were either as (FF19-1) or more (FE13-1) invasive than a clinical control strain (08-5578) (P = 0.006). To examine whether serotype 1/2a was more likely to acquire mutations than other serotypes, strains were plated on agar with rifampin, revealing 4b isolates to be significantly more mutable than 1/2a, 1/2c, and 3a serotypes (P = 0.0002). We also examined the ability of 33 strains to adapt to cold temperature following a downshift from 37°C to 4°C. Overall, three distinct cold-adapting groups (CAG) were observed: 46% were fast (<70 h), 39% were intermediate (70 to 200 h), and 15% were slow (>200 h) adaptors. Intermediate CAG strains (70%) more frequently possessed inlA PMSCs than did fast (20%) and slow (10%) CAGs; in contrast, 87% of fast adaptors lacked inlA PMSCs. In conclusion, we report food chain-derived 1/2a and 4b serotypes with a 3-codon deletion possessing invasive behavior and the novel association of inlA genotypes encoding a full-length InlA with fast cold-adaptation phenotypes.     

Molecular and experimental virulence of Listeria monocytogenes strains isolated from cases with invasive listeriosis and febrile gastroenteritis

We analyzed 27 Listeria monocytogenes strains of serotypes 1/2b and 4b, from invasive and gastroenteric listeriosis, for molecular and experimental virulence. Molecular virulence was tested by PCR for the presence of 8 major virulence-associated genes and genetic polymorphisms through restriction enzyme analysis; genomic DNA typing using pulsed-field gel electrophoresis was also performed. Experimental virulence was evaluated through intra-peritoneal and intra-gastric mouse virulence assays. Our results showed no significant differences in the virulence-related molecular properties of the strains analyzed. All strains were equally pathogenic following intra-peritoneal inoculation of mice. In mice inoculated intra-gastric with 4 representative strains of the 2 types of listeriosis, there were no significant differences in the bacterial count when comparing invasive and gastroenteric strains, suggesting that the strains were comparable in terms of mean oral infectivity.