Plasmids in Listeria monocytogenes in relation to cadmium resistance.

One hundred and seventy-three unrelated Listeria monocytogenes strains isolated from humans, animals, the environment, and food were analyzed for the presence of plasmids. Extrachromosomal DNA was found in 28% of the strains. Plasmid DNA was extracted more frequently from L. monocytogenes serogroup 1 strains (35%) than from serogroup 4 strains (15%). Among strains from food and the environment, 40% and 29%, respectively, harbored plasmids, whereas only 13% of the strains from humans and animals with listeriosis bore plasmids. We also investigated the susceptibility of 90 strains to seven antibiotics and four heavy-metal salts. No antibiotic resistance could be detected, but 95.3% of the plasmid-positive strains and only 12.7% of the plasmid-negative strains were resistant to cadmium. The plasmid-determined genetic basis of cadmium resistance was proven by conjugation between strains of L. monocytogenes and by cure of the plasmid. This is the first time that plasmids of L. monocytogenes have been shown to be associated with cadmium resistance.     

Subtyping of Listeria monocytogenes on the basis of plasmid profiles and arsenic and cadmium susceptibility

The susceptibilities to arsenic and cadmium together with the detection of plasmid DNA were evaluated for use as epidemiological markers for the subtyping of Listeria monocytogenes. Plasmid DNA was detected in 34% of 322 apparently unrelated isolates of L. monocytogenes. The resistance to cadmium and arsenic differentiated 565 apparently unrelated cultures into four groups, the smallest being 5% of cultures resistant to both agents, and the largest (53%) being sensitive to cadmium and resistant to arsenic. The resistance patterns to these agents and the presence of plasmid DNA varied markedly between the serotypes of the cultures. The detection of plasmid DNA was strongly associated with cadmium resistance in serogroup 1/2 cultures, but not within those of serogroup 4. Arsenic resistance was not associated with plasmid DNA. All methods were sufficiently stable to be useful for epidemiological investigations. The techniques described here offer simple methods which can be easily utilized in laboratories without a specialized expertise for this bacterium.     

Application of multilocus enzyme electrophoresis and restriction fragment length polymorphism analysis to the typing of Listeria monocytogenes strains isolated from raw milk, nondairy foods, and clinical and veterinary sources.

The powerful discriminatory typing capabilities of multilocus enzyme electrophoresis and restriction fragment length polymorphism analysis were applied to Listeria monocytogenes strains from raw milk, nondairy foods, and clinical and veterinary sources. The raw milk and nondairy food strains were sequential isolates obtained over a year-long period from a number of different producers and manufacturers. Results obtained by the two typing methods were in substantial agreement and showed that both raw milk and nondairy foods frequently contain recurrent L. monocytogenes strains, thus suggesting that the presence of these organisms in such commodities often arises because of contamination from within their respective processing environments. Most recurrent strains were serogroup 1/2, with only one instance of recurrent serogroup 4 strains. Some recurrent L. monocytogenes strains, including the serogroup 4 strains, were found by analysis of multilocus enzyme electrophoresis results to be closely related to clinical and veterinary strains, thus suggesting that strains adapted for survival in the food-processing environment retain their potential for pathogenicity.     

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.     

Plasmid-borne cadmium resistance genes in Listeria monocytogenes are present on Tn5422, a novel transposon closely related to Tn917.

The complete (6,449-bp) nucleotide sequence of the first-described natural transposon of Listeria monocytogenes, designated Tn5422, was determined. Tn5422 is a transposon of the Tn3 family delineated by imperfect inverted repeats (IRs) of 40 bp. It contains two genes which confer cadmium resistance (M. Lebrun, A. Audurier, and P. Cossart, J. Bacteriol. 176:3040-3048, 1994) and two open reading frames that encode a transposase (TnpA) and a resolvase (TnpR) of 971 and 184 amino acids, respectively. The cadmium resistance genes and the transposition genes are transcribed in opposite directions and are separated by a putative recombination site (res). The structural elements presumed to be involved in transposition of Tn5422 (IRs, transposase, resolvase, and res) are very similar to those of Tn917, suggesting a common origin. The transposition genes were not induced by cadmium. Analysis of sequences surrounding Tn5422 in nine different plasmids of L. monocytogenes indicated that Tn5422 is a functional transposon, capable of intramolecular replicative transposition, generating deletions. This transposition process is probably the reason for the size diversity of the L. monocytogenes plasmids. Restriction analysis and Southern hybridization revealed the presence of Tn5422 in all the plasmid-mediated cadmium-resistant L. monocytogenes strains tested but not in strains encoding cadmium resistance on the chromosome.     

Characterization of recurrent and sporadic Listeria monocytogenes isolates from raw milk and nondairy foods by pulsed-field gel electrophoresis, monocin typing, plasmid profiling, and cadmium and antibiotic resistance determination

Following previous surveys to assess the incidence of Listeria monocytogenes in raw milk and nondairy foods processed in Northern Ireland, isolates were characterized as recurrent or sporadic on the basis of multilocus enzyme electrophoresis (MEE) analysis and restriction fragment length polymorphism typing. In the present study, 45 representative recurrent and sporadic electrophoretic types (ETs) previously identified by MEE were subjected to pulsed-field gel electrophoresis (PFGE) of genomic DNA macrorestriction fragments, monocin typing, plasmid profiling, and an examination of resistance to cadmium and nine different antibiotics. Although PFGE proved to be capable of subdividing a number of recurrent and sporadic ETs, the grouping of strains arrived at by PFGE and MEE were in broad agreement, and previous conclusions regarding the designation of L. monocytogenes strains as recurrent or sporadic remained unaltered. It is considered that PFGE was able to detect minor genetic changes in recurrent ETs which occurred during the time period in which food surveys were carried out. Production of type E monocin (Types A to E were found among the 45 strains), plasmid carriage, and resistance to cadmium occurred more frequently in recurrent than in sporadic strains and may be important with regard to the ability of L. monocytogenes to persist in food and food-processing environments. Only 2 of 45 strains showed resistance to any of the nine antibiotics tested: two sporadic strains were resistant to tetracycline (MIC, 64 microg x ml(-1)).     

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.     

Antibiotic resistance in strains of Listeria spp. from meat products

The susceptibility or resistance to 26 antimicrobial agents was determined for 64 strains of Listeria monocytogenes and 102 strains of L. innocua isolated from Italian meat products. Some strains of L. monocytogenes were found to be resistant to tetracycline, erythromycin, co-trimoxazole and clindamycin. No plasmids were found in any L. monocytogenes strain. Five strains of L. innocua contained a 7.9 kbp plasmid, but these isolates were not resistant to any antibiotic in common and treatment with curing agents could not eliminate resistance to antibiotics. These results suggest that antibiotic resistance was not likely to be plasmid mediated in our strains.     

Diverse geno- and phenotypes of persistent Listeria monocytogenes isolates from fermented meat sausage production facilities in Portugal

The persistence of Listeria monocytogenes in food-associated environments represents a key factor in transmission of this pathogen. To identify persistent and transient strains associated with production of fermented meat sausages in northern Portugal, 1,723 L. monocytogenes isolates from raw material and finished products from 11 processors were initially characterized by random amplification of polymorphic DNA (RAPD), PCR-based molecular serotyping, and epidemic clone characterization, as well as cadmium, arsenic, and tetracycline resistance typing. Pulsed-field gel electrophoresis (PFGE) typing of 240 representative isolates provided evidence for persistence of L. monocytogenes for periods of time ranging from 10 to 32 months for all seven processors for which isolates from different production dates were available. Among 50 L. monocytogenes isolates that included one representative for each PFGE pattern obtained from a given sample, 12 isolates showed reduced invasion efficiency in Caco-2 cells, including 8 isolates with premature stop codons in inlA. Among 41 isolates representing sporadic and persistent PFGE types, 22 isolates represented lysogens. Neither strains with reduced invasion nor lysogens were overrepresented among persistent isolates. While the susceptibility of isolates to lysogenic phages also did not correlate with persistence, it appeared to be associated with molecular serotype. Our data show the following. (i) RAPD may not be suitable for analysis of large sets of L. monocytogenes isolates. (ii) While a large diversity of L. monocytogenes subtypes is found in Portuguese fermented meat sausages, persistence of L. monocytogenes in this food chain is common. (iii) Persistent L. monocytogenes strains are diverse and do not appear to be characterized by unique genetic or phenotypic characteristics.     

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.     

Plasmids in Listeria monocytogenes and other Listeria species.

One hundred and twenty-two food, clinical, and veterinary strains of Listeria monocytogenes were examined for the presence of plasmids. Twenty-five (20%) contained plasmids, which varied from 1.3 to 66 MDa in size. Of 10 strains of other Listeria species (L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, L. grayi, and L. murrayi) examined, seven (70%) contained plasmids, varying from 38 to 53 MDa. No strains with multiple plasmids were found. Plasmids of identical size were isolated from related strains in some, although not all, cases. The presence of a plasmid in a strain was not related to phenotypic characters of known extrachromosomal inheritance.     

Evaluation of the international phage typing set and some experimental phages for typing of Listeria monocytogenes from poultry in Spain

AIMS: The validity of the international phage set and 13 experimental phages for subtyping Listeria monocytogenes strains isolated from poultry in Spain was investigated. METHODS AND RESULTS: Ninety-six L. monocytogenes strains (52 from serogroup 1/2 and 44 from serogroup 4) were phage-typed using the international phage set, 10 experimental phages for typing serogroup 1/2 strains (seven isolated in France: 1313, 9425, 1807, 351, 881, 717 and 586-, and three from Denmark: 5775, 12682 and 6223-) and three experimental phages isolated in France for typing serogroup 4 strains (2425 A, 4286 and 197). Percentages of serogroup 1/2, serogroup 4 and total phage-typeable strains were 57.7%, 52.3% and 55.2%, respectively. Important differences in the behaviour of the phages tested were found. The typeability rate, the specificity index and the percentage of strong reactions were greater in the phages of international set than in the experimental phages. The number of phage typeable strains and the number of phage types (42) were not modified by the use of experimental phages. CONCLUSIONS: The phage set used was not effective for typing L. monocytogenes strains from poultry in Spain, because a low typeability rate was found. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest the importance of the availability of new phages specific to a geographical area in order to improve the typeability of the system.     

Phenotypic and genotypic evaluation of Listeria monocytogenes strains isolated from fish and fish processing plants

The aim of this study was to perform the phenotypic and genotypic evaluation of Listeria monocytogenes strains isolated from fish and equipment used in fish processing plants. The prevalence of selected gene-encoding virulence factors among L. monocytogenes strains was assessed by multiplex PCR. The genetic (PFGE method) and protein similarities (MALDI-TOF MS technique) of isolates were determined. Their drug resistance (disk-diffusion method and MIC values), serogroup classification (multiplex-PCR), and the ability to co-aggregate with Salmonella enteritidis were also evaluated. Among 37 L. monocytogenes isolates, 36 strains were found, one of which included two genetically identical isolates (PFGE method). In all examined strains, the following genes were found: hlyA, plcB, plcA, inlA, inlB, prfA, iap, and actA. The presence of virulence genes, mpl, and fbpA was confirmed in 32 (88.9%) strains. It was reported that 30 (83.3%) of the strains belonged to serogroup 1/2a-3a. It was also found that the rate of coaggregation with S. enteritidis bacilli was 16.5–36.3%. Among the investigated L. monocytogenes strains, 25 (69.4%) were sensitive to all antibiotics used. Resistance to penicillin was reported most often among strains (n = 6, 16.7%). The assessment of L. monocytogenes virulence level is an important aspect for the protection of public health. It was reported that strains isolated from fish contain genes coding for virulence factors and some of them are antibiotic-resistant. In our study, it was found that strains with a high degree of genetic similarity also showed a high degree of similarity at the level of protein profiles.     

Intraspecific phylogeny and lineage group identification based on the prfA virulence gene cluster of Listeria monocytogenes

Listeria monocytogenes is a serious food-borne pathogen that can cause invasive disease in humans and other animals and has been the leading cause of food recalls due to microbiological concerns in recent years. In order to test hypotheses regarding L. monocytogenes lineage composition, evolution, ecology, and taxonomy, a robust intraspecific phylogeny was developed based on prfA virulence gene cluster sequences from 113 L. monocytogenes isolates. The results of the multigene phylogenetic analyses confirm that L. monocytogenes comprises at least three evolutionary lineages, demonstrate that lineages most frequently (lineage 1) and least frequently (lineage 3) associated with human listeriosis are sister-groups, and reveal for the first time that the human epidemic associated serotype 4b is prevalent among strains from lineage 1 and lineage 3. In addition, a PCR-based test for lineage identification was developed and used in a survey of food products demonstrating that the low frequency of association between lineage 3 isolates and human listeriosis cases likely reflects rarity of exposure and not reduced virulence for humans as has been previously suggested. However, prevalence data do suggest lineage 3 isolates may be better adapted to the animal production environment than the food-processing environment. Finally, analyses of haplotype diversity indicate that lineage 1 has experienced a purge of genetic variation that was not observed in the other lineages, suggesting that the three L. monocytogenes lineages may represent distinct species within the framework of the cohesion species concept.     

Plasmid-borne cadmium resistance genes in Listeria monocytogenes are similar to cadA and cadC of Staphylococcus aureus and are induced by cadmium.

pLm74 is the smallest known plasmid in Listeria monocytogenes. It confers resistance to the toxic divalent cation cadmium. It contains a 3.1-kb EcoRI fragment which hybridizes with the cadAC genes of plasmid pI258 of Staphylococcus aureus. When introduced into cadmium-sensitive L. monocytogenes or Bacillus subtilis strains, this fragment conferred cadmium resistance. The DNA sequence of the 3.1-kb EcoRI fragment contains two open reading frames, cadA and cadC. The deduced amino acid sequences are similar to those of the cad operon of plasmid pI258 of S. aureus, known to prevent accumulation of Cd2+ in the bacteria by an ATPase efflux mechanism. The cadmium resistance determinant of L. monocytogenes does not confer zinc resistance, in contrast to the cadAC determinant of S. aureus, suggesting that the two resistance mechanisms are slightly different. Slot blot DNA-RNA hybridization analysis showed cadmium-inducible synthesis of L. monocytogenes cadAC RNA.     

Heavy Metal and Disinfectant Resistance of Listeria monocytogenes from Foods and Food Processing Plants

The persistence of Listeria monocytogenes in food processing plants and other ecosystems reflects its ability to adapt to numerous stresses. In this study, we investigated 138 isolates from foods and food processing plants for resistance to the quaternary ammonium disinfectant benzalkonium chloride (BC) and to heavy metals (cadmium and arsenic). We also determined the prevalence of distinct cadmium resistance determinants (cadA1, cadA2, and cadA3) among cadmium-resistant isolates. Most BC-resistant isolates were resistant to cadmium as well. Arsenic resistance was encountered primarily in serotype 4b and was an attribute of most isolates of the serotype 4b epidemic clonal group ECIa. Prevalence of the known cadmium resistance determinants was serotype associated: cadA1 was more common in isolates of serotypes 1/2a and 1/2b than 4b, while cadA2 was more common in those of serotype 4b. A subset (15/77 [19%]) of the cadmium-resistant isolates lacked the known cadmium resistance determinants. Most of these isolates were of serotype 4b and were also resistant to arsenic, suggesting novel determinants that may confer resistance to both cadmium and arsenic in these serotype 4b strains. The findings may reflect previously unrecognized components of the ecological history of different serotypes and clonal groups of L. monocytogenes, including exposures to heavy metals and disinfectants.     

Use of molecular typing methods to trace the dissemination of Listeria monocytogenes in a shrimp processing plant.

Molecular typing of bacteria has been widely used in epidemiological studies but not as extensively for tracing the transmission of pathogenic bacteria in food plants. This study was conducted to examine the potential use of two molecular typing methods, random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE), to trace Listeria monocytogenes contamination in a shrimp processing plant. Ribotyping and phase typing were also performed on a select number of strains. One hundred fifteen strains of L. monocytogenes collected in different areas of a shrimp processing plant were first serotyped and then subtyped by molecular typing. RAPD and PFGE showed great promise for typing L. monocytogenes isolates since distinguishable and reproducible DNA polymorphisms were obtained. When the composite profile from both (RAPD and PFGE) methods was generated, there was an increase in the discriminatory power to discern differences between strains of L. monocytogenes. The results indicated that environmental strains all fell into composite profile groupings unique to the environment, while strains from both water and utensils shared another composite profile group. L. monocytogenes fresh shrimp isolates belonging to one profile group were found in different areas of the processing line. This same profile group was also present in food handlers from the processing and packaging areas of the plant.