Molecular Typing by Pulsed-Field Gel Electrophoresis of Spanish Animal and Human Listeria monocytogenes Isolates

A total of 153 strains of Listeria monocytogenes isolated from different sources (72 from sheep, 12 from cattle, 18 from feedstuffs, and 51 from humans) in Spain from 1989 to 2000 were characterized by pulsed-field gel electrophoresis. The strains of L. monocytogenes displayed 55 pulsotypes. The 84 animal, 51 human, and 18 feedstuff strains displayed 31, 29, and 7 different pulsotypes, respectively, indicating a great genetic diversity among the Spanish L. monocytogenes isolates studied. L. monocytogenes isolates from clinical samples and feedstuffs consumed by the diseased animals were analyzed in 21 flocks. In most cases, clinical strains from different animals of the same flock had identical pulsotypes, confirming the existence of a listeriosis outbreak. L. monocytogenes strains with pulsotypes identical to those of clinical strains were isolated from silage, potatoes, and maize stalks. This is the first study wherein potatoes and maize stalks are epidemiologically linked with clinical listeriosis.     

Characterization of Listeria monocytogenes recovered from 41 cases of sporadic listeriosis in Austria by serotyping and pulsed-field gel electrophoresis

41 clinical Listeria monocytogenes strains recovered from seven feto-maternal and 34 non-pregnancy associated cases of human listeriosis documented between 1997 and 2000 underwent serotyping and typing by pulsed-field gel electrophoresis (PFGE) applying the enzymes AscI, ApaI and SmaI. The pulsotypes of the clinical strains were compared to the pulsotypes of three L. monocytogenes strains isolated from healthy fecal carriers and nine reference strains isolated from seven outbreaks in Europe and the USA. The 41 clinical strains of Austrian provenance showed 37 pulsotypes. Five sets of two Austrian strains each were indistinguishable by PFGE typing. Epidemiological links were absent between these indistinguishable isolates. One unique pulsotype (AB) was found in three fecal isolates. Five pulsotypes (A, Q, R, AC and AD) were distinguished among the strains associated with outbreaks. Clusters consisting of two, five and six Austrian strains each were indistinguishable from the outbreak-associated pulsotypes A, Q and R, respectively, after PFGE analysis with AscI. Three strains of AscI pulsotype Q and five strains of AscI pulsotype R could be further differentiated by restriction with ApaI and SmaI. One strain each from sporadic cases shared a combined pulsotype with the outbreak strains of pulsotypes A and R, respectively. These PFGE data suggest that a similar genetic background can be found in strains which have been contributing to outbreaks world-wide and in isolates associated with sporadic listeriosis in Austria.     

Isolation of Listeria monocytogenes from Vegetation

A rural area in Virginia, in which clinical listeriosis of man and animals has been rare, was selected for this study. Vegetatino, which had died and remained in the fields over the winter, was collected and examined for Listeria monocytogenes by Gray's cold-holding procedure. Twelve fields were sampled and cultured, and eight strains of L. monocytogenes were isolated. Only two of the strains were pathogenic for the mouse. Compared with strains of L. monocytogenes isolated from listeric humans and animals in Virginia and the United States as a whole, these represented serotypes of low frequency.     

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.     

Molecular epidemiological survey of Listeria monocytogenes in seafoods and seafood-processing plants

To evaluate the role of seafoods in the epidemiology of human listeriosis and the role of the processing environment as a source of Listeria monocytogenes in seafood products, 305 L. monocytogenes isolates were characterized by multilocus enzyme electrophoresis using 21 genetic loci and restriction enzyme analysis of total DNA. Forty-four isolates were recovered from patients in Norway; 93 were isolated from seafoods, seafood-processing environments, and seawater from 55 different producers; and the remaining 168 isolates originated from six seafood-processing plants and one transport terminal examined in detail for L. monocytogenes. The patient isolates fell into 11 electrophoretic types, with four of them being responsible for 77% of the listeriosis cases in 1992 to 1996. Isolates from Norwegian seafoods and processing environments showed great genetic diversity, indicating that seafoods and seafood-processing environments do not offer a niche for specific L. monocytogenes strains. On the other hand, isolates from individual processing plants were genetically more homogenous, showing that plants are likely to be colonized with specific subclones of L. monocytogenes. The isolation of identical subclones of L. monocytogenes from both human patients and seafoods, including ready-to-eat products, suggests that such products may have been possible sources for listeriosis cases in Norway.     

Relatedness of Listeria monocytogenes Isolates recovered from selected ready-to-eat foods and listeriosis patients in the United States

Pulsed-field gel electrophoresis and serotyping were performed for 544 isolates of Listeria monocytogenes, including 502 isolates recovered from contaminated samples from 31,705 retail ready-to-eat (RTE) food products and 42 isolates recovered from human cases of listeriosis. The isolates were from Maryland (294 isolates) and California (250 isolates) and were collected in 2000 and 2001. The isolates were placed into 16 AscI pulsogroups (level of relatedness within each group, > or =66%), 139 AscI pulsotypes (levels of relatedness, > or =25% to 100%), and eight serotypes (serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, 4b, 4c, and 4d). The most frequently found pulsotypes belonged to either pulsogroup A (150 food isolates plus 4 clinical isolates) or pulsogroup B (104 food isolates plus 5 clinical isolates). The majority of the 502 food isolates were either serotype 1/2a (298 isolates) or serotype 1/2b (133 isolates), whereas the majority of the 42 clinical isolates were either serotype 1/2a (19 isolates) or serotype 4b (15 isolates). Additionally, 13 clinical isolates displayed pulsotypes also found in food isolates, whereas the remaining 29 clinical isolates displayed 24 unique pulsotypes. These data indicate that most (86%) of the L. monocytogenes subtypes found in the RTE foods sampled belonged to only two serotypes and that 90% of the isolates displayed 73 pulsotypes, with 107 isolates displaying pulsotype 1. These data should help define the distribution and relatedness of isolates found in RTE foods in comparison with isolates that cause listeriosis.     

Genotyping by REA-PFGE of Listeria monocytogenes isolated from clinical, environmental and food samples

Listeria monocytogenes strains isolated from clinical food and environmental samples were genotyped by Restriction Enzyme Analysis with Pulsed Field Gel Electrophoresis (REA-PFGE) using ApaI and AscI enzymes according to PulseNet Europe procedure. Analysis of DNA fragments profiles obtained by AscI digestion demonstrated presence of 62 REA-PFGE profiles grouped in 2 lineages (FI, FII). Diversity of strains source among both lineages was observed. Statistical analysis showed, that strains isolated from clinical samples more frequently are included to lineage FI, then lineage FII. Non-clinical strains were more frequently included to lineage FII. Combined analysis of REA-PFGE profiles for ApaI and AscI enzymes showed 8 unique pulsotypes characteristic for two or more L. monocytogenes isolates. Moreover researched L. monocytogenes strains were analyzed by multiplex-PCR according Doumith et al methodology. PCR-group 4B was most frequent among strains isolated from clinical samples. Correlation between PCR-group and pulsotype was observed only in few cases.     

Ecology and transmission of Listeria monocytogenes infecting ruminants and in the farm environment

A case-control study involving 24 case farms with at least one recent case of listeriosis and 28 matched control farms with no listeriosis cases was conducted to probe the transmission and ecology of Listeria monocytogenes on farms. A total of 528 fecal, 516 feed, and 1,012 environmental soil and water samples were cultured for L. monocytogenes. While the overall prevalence of L. monocytogenes in cattle case farms (24.4%) was similar to that in control farms (20.2%), small-ruminant (goat and sheep) farms showed a significantly (P < 0.0001) higher prevalence in case farms (32.9%) than in control farms (5.9%). EcoRI ribotyping of clinical (n = 17) and farm (n = 414) isolates differentiated 51 ribotypes. L. monocytogenes ribotypes isolated from clinical cases and fecal samples were more frequent in environmental than in feed samples, indicating that infected animals may contribute to L. monocytogenes dispersal into the farm environment. Ribotype DUP-1038B was significantly (P < 0.05) associated with fecal samples compared with farm environment and animal feedstuff samples. Ribotype DUP-1045A was significantly (P < 0.05) associated with soil compared to feces and with control farms compared to case farms. Our data indicate that (i) the epidemiology and transmission of L. monocytogenes differ between small-ruminant and cattle farms; (ii) cattle contribute to amplification and dispersal of L. monocytogenes into the farm environment, (iii) the bovine farm ecosystem maintains a high prevalence of L. monocytogenes, including subtypes linked to human listeriosis cases and outbreaks, and (iv) L. monocytogenes subtypes may differ in their abilities to infect animals and to survive in farm environments.     

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.     

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.     

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.     

Survival and biofilm formation of Listeria monocytogenes in simulated vaginal fluid: influence of pH and strain origin

Listeria monocytogenes, the agent responsible for listeriosis, can be transmitted from mother to fetus/neonates by vertical transmission, transplacentally or during passage through the birth canal. The purpose of this study was to investigate the survival and biofilm formation of L. monocytogenes (isolated from clinical cases or from food) in simulated vaginal fluid at different pH values (4.2, 5.5 and 6.5). The results demonstrated that this pathogen is inhibited by the normal vaginal pH, but may proliferate when it increases. Clinical strains were significantly more resistant to pH 4.2 than food isolates. Listeria monocytogenes survived and even grew at the higher pHs investigated, suggesting that fetus/neonates from women having increased vaginal pH values during pregnancy may be at a higher risk of listeriosis. All isolates tested were producers of biofilm at different pH values; however, L. monocytogenes produced higher quantities of biofilm in a nutrient-rich medium. No significant differences in biofilm production were detected between food and clinical isolates. As L. monocytogenes are biofilm producers, this increases the probability of occurrence of neonatal infection.     

Characterization of Listeria monocytogenes strains involved in invasive and noninvasive listeriosis outbreaks by PCR-based fingerprinting techniques

A total of 32 Listeria monocytogenes strains (16 from a recent outbreak of invasive listeriosis and 16 from two outbreaks of noninvasive listeriosis, all three occurring in Italy) were characterized by PCR-ribotyping, arbitrarily primed PCR (AP-PCR), and the recently developed infrequent-restriction-site PCR (IRS-PCR). The discriminatory ability of the techniques, first evaluated on 29 unrelated L. monocytogenes food isolates using Simpson's index of diversity, was 0.714 for PCR-ribotyping, 0.690 for AP-PCR, and 0.919 for IRS-PCR. IRS-PCR was also more capable of distinguishing among strains from the invasive listeriosis outbreak: three different clusters were identified by IRS-PCR compared to two clusters identified by both PCR-ribotyping and AP-PCR. Within each of the two outbreaks of noninvasive listeriosis, the patterns were practically identical, as demonstrated by all three techniques. Only IRS-PCR succeeded in clearly discriminating the strains related to noninvasive listeriosis from all of the other strains included in this study, including those from the outbreak of invasive listeriosis. This finding may suggest the presence of unique differences in their DNA sequences.     

Antimicrobial resistance of Listeria monocytogenes

Listeria monocytogenes is an opportunistic pathogen that causes rare but frequently fatal infections, termed listerioses. In general, strains of L. monocytogenes are susceptible to a wide range of antibiotics, except for the cephalosporins, fluorochinolones and fosfomycin (Hof, 1991). The current therapy of choice is a combination of ampicillin and aminoglycoside, usually gentamicin (Lorber, 1997). In cases when it is not possible to use a beta-lactam antibiotic, second-choice therapy involves the use of an association of trimethoprim with a sulfonamide, such as in co-trimoxazole, in which the more active in the combination seems trimethoprim, synergized by the sulfa compound. Other second line agents for listeriosis include erythromycin and vancomycin (Temple and Nahata, 2000). The first strains of L. monocytogenes resistant to antibiotics were reported in 1988 (Poyart-Salmeron et al. 1990) The present paper reviews the current state of affairs with regard to the resistance of L. monocytogenes isolated from food products and clinical material to different antibiotics, with particular emphasis on those used in the therapy of listeriosis.     

Characterization of Listeria monocytogenes isolates from food animal clinical cases: PFGE pattern similarity to strains from human listeriosis cases

Twenty-one isolates of Listeria monocytogenes from food animal clinical cases that involved meningitis or meningoencephalitis, encephalitis, mastitis and abortion were characterized by serotyping and pulsed-field gel electrophoresis (PFGE) in order to improve our understanding of the genetic links between individual strains and strains recovered from human listeriosis cases. Results showed that five of the isolates were serotype 1/2a, six were 1/2b, nine were 4b, and one was untypeable. A caprine, two bovine and an ovine brain isolate shared identical PFGE patterns indicating that strains of L. monocytogenes are not host specific. Other isolates exhibited distinct patterns that were not shared, indicating a genetic diversity. Dendrogram analysis revealed that PFGE patterns of the isolates clustered primarily according to serotype. We compared the PFGE types obtained for these isolates with PFGE types for human clinical isolates present in the CDC national PulseNet database. Six (29%) of the twenty-one strains had patterns that were indistinguishable from pathogenic human isolates in the database. Our observations offer preliminary evidence that food animals could be significant reservoirs of L. monocytogenes that lead to human infections and support the inclusion of PFGE patterns of veterinary clinical isolates in the national PulseNet database for increased surveillance.     

The diversity of Listeria monocytogenes strains from 10 Icelandic sheep farms

AIMS: The purpose of this study was to examine the diversity of Listeria monocytogenes strains from healthy sheep, winter feed and environment of sheep farms in Iceland. METHODS AND RESULTS: A total of 104 L. monocytogenes isolates from animals, winter feed and environment on 10 Icelandic sheep farms were compared by serotyping, ribotyping, and pulsed-field gel electrophoresis with ApaI and AscI. The isolates were divided into 24 genotypes, all identified as serovars 1/2a, 1/2b, or 4b. Nine genotypes were detected on more than one farm. On three of the farms there seemed to be a dominant strain of L. monocytogenes. Isolates from incidents of listeriosis in animals occurring on two of the farms belonged to the genotype most commonly found on the particular farm. Nine of the 24 genotypes found on the sheep farms have been associated with disease in animals and/or humans elsewhere in Iceland. CONCLUSIONS: Certain strains of L. monocytogenes seem to be widely distributed on Icelandic sheep farms. On some farms there appears to be a dominant strain of L. monocytogenes. Incidents of listeriosis in animals may tend to be associated with strains commonly found on the farm. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the diversity of L. monocytogenes present in healthy sheep and their environment.     

Comparison of methods for discrimination between strains of Listeria monocytogenes from epidemiological surveys.

Total cellular DNA from 28 strains of Listeria monocytogenes isolated from food implicated in food-borne illness and from patients with listeriosis was digested with the restriction endonucleases HindIII, HaeIII, and EcoRI. Following agarose gel electrophoresis, the fragments were subjected to Southern blot hybridization with a digoxigenin-labeled cDNA probe transcribed from Escherichia coli 16S and 23S rRNA. The patterns of bands from genomic (DNA fingerprints) and rDNA fingerprints (ribotypes) were used for classifying L. monocytogenes strains, and the resulting subtypes were compared with serotyping and multilocus enzyme electrophoresis classification schemes. A total of 15 distinct and identical groups were obtained when genomic DNA was digested with either HindIII or HaeIII. The most discriminating enzyme for ribotyping of strains was EcoRI, which divided the 28 strains of L. monocytogenes into 6 ribotype groups. DNA fingerprinting and ribotyping differentiated L. monocytogenes from other Listeria spp., including L. ivanovii, L. welshimeri, and L. innocua as well as the lactic acid bacteria Lactococcus lactis subsp. lactis and subsp. cremoris. L. monocytogenes strains isolated from four independent food-borne illness incidents were analyzed by all typing methods. Patient and product isolates were not distinguishable by serotyping, ribotyping, or multilocus enzyme electrophoresis. DNA fingerprinting was the only method capable of differentiating these strains, or conversely, of proving relatedness of patient-product pairs of isolates. This method was a relatively simple, sensitive, reproducible, and highly discriminating method for epidemiological tracking of L. monocytogenes implicated in food-borne illness.     

Select Listeria monocytogenes subtypes commonly found in foods carry distinct nonsense mutations in inlA, leading to expression of truncated and secreted internalin A, and are associated with a reduced invasion phenotype for human intestinal epithelial cells

The surface protein internalin A (InlA) contributes to the invasion of human intestinal epithelial cells by Listeria monocytogenes. Screening of L. monocytogenes strains isolated from human clinical cases (n=46), foods (n=118), and healthy animals (n=58) in the United States revealed mutations in inlA leading to premature stop codons (PMSCs) in L. monocytogenes ribotypes DUP-1052A and DUP-16635A (PMSC mutation type 1), DUP-1025A and DUP-1031A (PMSC mutation type 2), and DUP-1046B and DUP-1062A (PMSC mutation type 3). While all DUP-1046B, DUP-1062A, DUP-16635A, and DUP-1031A isolates (n=76) contained inlA PMSCs, ribotypes DUP-1052A and DUP-1025A (n=72) contained isolates with and without inlA PMSCs. Western immunoblotting showed that all three inlA PMSCs result in the production of truncated and secreted InlA. Searches of the Pathogen Tracker database, which contains subtype and source information for more than 5,000 L. monocytogenes isolates, revealed that the six ribotypes shown to contain isolates with inlA PMSCs were overall more commonly isolated from foods than from human listeriosis cases. L. monocytogenes strains carrying inlA PMSCs also showed significantly (P=0.0004) reduced invasion of Caco-2 cells compared to isolates with homologous 3' inlA sequences without PMSCs. Invasion assays with an isogenic PMSC mutant further supported the observation that inlA PMSCs lead to reduced invasion of Caco-2 cells. Our data show that specific L. monocytogenes subtypes which are common among U.S. food isolates but rare among human listeriosis isolates carry inlA mutations that are associated with, and possibly at least partially responsible for, an attenuated invasion phenotype.     

In vivo and in vitro assessment of the virulence of Listeria monocytogenes strains

To evaluate whether the in vitro model (invasion and intracellular growth in Caco-2 cells) for determining virulence is a suitable alternative to the in vivo model (50% lethal dose), we compared the levels of virulence obtained with the two models. We tested L. monocytogenes strains isolated from food and clinical samples during three episodes of listeriosis occurring in Italy in the period 1993-1995. We also tested L. monocytogenes strains isolated from food during official control activities. The results obtained from the tested strains varied according to the experimental method adopted: the L. monocytogenes strains featuring the same genetic pattern showed a greater uniformity of response in vivo than in vitro. We can conclude that the in vitro model may be used as an alternative to the animal model to determine Listeria spp pathogenicity, though it cannot distinguish levels of virulence within the L. monocytogenes species.