Distribution and Characteristics of Listeria monocytogenes Isolates from Surface Waters of the South Nation River Watershed, Ontario, Canada

Listeria monocytogenes is a facultative intracellular pathogen thought to be widely distributed in the environment. We investigated the prevalence and characteristics of L. monocytogenes isolates from surface waters derived from catchments within the South Nation River watershed (Ontario, Canada). This watershed is dominated by urban and rural development, livestock and crop production, and wildlife habitats. From June to November 2005, a total of 314 surface water samples were collected biweekly from 22 discrete sampling sites characterized by various upstream land uses. Presumptive Listeria spp. were isolated using a selective enrichment and isolation procedure, and 75 L. monocytogenes isolates were identified based on colony morphology, hemolytic activity, and amplification of three pathogenicity genes: iap, inlA, and hlyA. Thirty-two of 314 (10%) surface water samples were positive for the presence of L. monocytogenes, but detection ranged between 0 and 27% depending on the sampling date. Isolates belonging to serovar group 1/2a, 3a (50%) and group 4b, 4d, 4e (32%) were dominant. L. monocytogenes populations were resolved into 13 EcoRI ribotypes and 21 ApaI and 21 AscI pulsotypes. These had Simpson indexes of discrimination of up to 0.885. Lineage I-related isolates were dominant (61%) during the summer, whereas lineage II isolates were dominant (77%) in the fall. Isolates were, on average, resistant to 6.1 ± 2.1 antibiotics out of 17 tested. Half of the L. monocytogenes isolates exhibited potential virulence linked to the production of a functional internalin A, and some isolates were found to be moderately to highly virulent by in vitro Caco-2 plaque formation assay (up to 28% of entry). There was a statistically significant link between the occurrence of L. monocytogenes and proximity to an upstream dairy farm and degree of cropped land. Our data indicate that L. monocytogenes is widespread in the studied catchments, where it could represent a public health issue related to agricultural land use.     

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.     

Restriction enzyme analysis of Listeria monocytogenes strains associated with food-borne epidemics

Listeria monocytogenes (serotype 4b) has caused four major food-borne epidemics in North America. In this study, L. monocytogenes isolates from the Nova Scotia (Canada), Boston (Mass.), and Los Angeles (Calif.) outbreaks were examined by restriction enzyme analysis with the endonuclease HhaI. Human isolates (n = 32) from the 1981 Canadian outbreak were compared with a strain recovered from coleslaw, which was epidemiologically incriminated as the vehicle of infection. After HhaI digestion, 29 of 32 isolates exhibited the restriction enzyme pattern of the reference coleslaw isolate. The restriction enzyme patterns of the nine clinical isolates from the 1983 Massachusetts outbreak were identical to each other but differed from those of raw milk isolates recovered from sources supplying the pasteurizer. Isolates (n = 48) from the 1985 California outbreak were evaluated. The restriction enzyme patterns of the L. monocytogenes isolates from humans and from the suspect cheese samples were identical to those of four of five cheese factory environmental isolates. Isolates from each of these outbreaks exhibited a restriction enzyme pattern that was characteristic of that outbreak. The case with which restriction enzyme analysis can be applied to all serotypes of L. monocytogenes argues for its use in the epidemiology of L. monocytogenes.     

Restriction enzyme analysis of Listeria monocytogenes strains associated with food-borne epidemics.

Listeria monocytogenes (serotype 4b) has caused four major food-borne epidemics in North America. In this study, L. monocytogenes isolates from the Nova Scotia (Canada), Boston (Mass.), and Los Angeles (Calif.) outbreaks were examined by restriction enzyme analysis with the endonuclease HhaI. Human isolates (n = 32) from the 1981 Canadian outbreak were compared with a strain recovered from coleslaw, which was epidemiologically incriminated as the vehicle of infection. After HhaI digestion, 29 of 32 isolates exhibited the restriction enzyme pattern of the reference coleslaw isolate. The restriction enzyme patterns of the nine clinical isolates from the 1983 Massachusetts outbreak were identical to each other but differed from those of raw milk isolates recovered from sources supplying the pasteurizer. Isolates (n = 48) from the 1985 California outbreak were evaluated. The restriction enzyme patterns of the L. monocytogenes isolates from humans and from the suspect cheese samples were identical to those of four of five cheese factory environmental isolates. Isolates from each of these outbreaks exhibited a restriction enzyme pattern that was characteristic of that outbreak. The case with which restriction enzyme analysis can be applied to all serotypes of L. monocytogenes argues for its use in the epidemiology of L. monocytogenes.     

Effects of pH on distribution of Listeria ribotypes in corn, hay, and grass silage.

Listeria app, isolated from 13 of 129 (10%) corn silage samples, 21 of 76 (28%) hay silage samples, and 3 of 5 (60%) grass silage samples during a previous Vermont survey were subjected to automated ribotype (RT) analysis. The 13 positive corn silage samples contained 3 Listeria monocytogenes isolated (three RTs, including one known clinical RT) and 10 L. innocua isolates (four RTs). Similarly, 2 L. monocytogenes isolates (two RTs) and 19 L. innocua isolates (three RTs) were identified in the 21 positive hay silage samples. The three positive grass silage samples contained two L. innocua isolates (two RTs) and one isolate of L. welshimeri. One hundred seven of 129 (83%) high-quality (pH < 4.0) corn silage samples accounted for 8 of 13 Listeria isolates from corn silage, including isolates belonging to one L. monocytogenes clinical RT. In contrast, low-quality hay silage (70 of 76 [92%] samples having a pH of > or = 4.0) harbored 20 of 21 isolates, including isolates belonging to two nonclinical L. monocytogenes RTs. Poor-quality silage is readily discernible by appearance; however, these findings raise new concerns regarding the safety of high-quality (pH < 4.0) corn silage, which can contain Listeria spp., including L. monocytogenes strains belonging to RTs of clinical importance in cases of food-borne listeriosis.     

Antibiotic resistance among Listeria, including Listeria monocytogenes, in retail foods

AIMS: In the past eight to 10 years, reports of antibiotic resistance in food-borne isolates in many countries have increased, and this work examined the susceptibility of 1001 food isolates of Listeria species. METHODS AND RESULTS: Susceptibility/resistance to eight antibiotics was determined using the Bauer-Kirby disc diffusion assay, and 10.9% of the isolates examined displayed resistance to one or more antibiotics. Resistance to one or more antibiotics was exhibited in 0.6% of Listeria monocytogenes isolates compared with 19.5% of Listeria innocua isolates. Resistance was not observed in Listeria seeligeri or Listeria welshimeri. Resistance to tetracycline (6.7%) and penicillin (3.7%) was the most frequently observed, and while resistance to one antibiotic was most common (9.1%), isolates resistant to two or more antibiotics (1.8%) were also observed. CONCLUSION: While resistance to the antibiotics most commonly used to treat human listeriosis was not observed in L. monocytogenes, the presence of such resistance in other Listeria species raises the possibility of future acquisition of resistance by L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The higher level of resistance in L. innocua compared with L. monocytogenes suggests that a species-related ability to acquire resistance to antibiotics exists.     

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.     

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

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

Recovery of different Listeria ribotypes from naturally contaminated, raw refrigerated meat and poultry products with two primary enrichment media.

Isolation rates for Listeria monocytogenes and the other Listeria spp. typically improve when samples are enriched in more than one primary enrichment medium. This study evaluated the abilities of two primary enrichment media, University of Vermont-modified Listeria enrichment broth (UVM) and Listeria repair broth (LRB), to recover different ribotypes of Listeria spp. from raw meat and poultry samples. Forty-five paired 25-g retail samples of ground beef, pork sausage, ground turkey, and chicken (160 samples) underwent primary enrichment in UVM and LRB (30 degrees C for 24 h) followed by secondary enrichment in Fraser broth (35 degrees C for 24 and 40 h) and plating on modified Oxford agar. After 24 h of incubation of 35 degrees C, 608 Listeria colonies from selected positive samples were biochemically confirmed as L. monocytogenes (245 isolates), L innocua (276 isolates), and L. welshimeri (89 isolates) and then ribotyped with the automated Riboprinter microbial characterization system (E. I. du Pont de Nemours & Co., Inc.). Thirty-six different Listeria strains comprising 16 L. monocytogenes (including four known clinical ribotypes), 12 L. innocua, and 8 L. welshimeri ribotypes were identified from selected positive samples (15 samples of each product type; two UVM and two LRB isolates per sample). Twenty-six of 36(13 L. monocytogenes) ribotypes were detected with both UVM and LRB, whereas 3 of 36 (1 L. monocytogenes) and 7 of 36 (3 L. monocytogenes) Listeria ribotypes were observed with only UVM or LRB, respectively. Ground beef, pork sausage, ground turkey, and chicken yielded 22 (8 L. monocytogenes), 21 (12 L. monocytogenes), 20 (9 L. monocytogenes), and 19 (11 L. monocytogenes) different Listeria ribotypes, respectively, with some Listeria ribotypes confined to a particular product. More importantly, major differences in both the number and distribution of Listeria ribotypes, including previously recognized clinical and nonclinical ribotypes of L. monocytogenes, were observed when 10 UVM and 10 LRB isolates from five samples of each product were ribotyped. When a third set of six samples per product type was examined from which two Listeria isolates were obtained by using only one of the two primary enrichment media, UVM and LRB failed to detect L. monocytogenes (both clinical and nonclinical ribotypes) in two and four samples, respectively. These findings stress the importance of using more than one primary enrichment medium and picking a sufficient number of colonies per sample when attempting to isolate specific L. monocytogenes strains during investigations of food-borne listeriosis.     

Incidence and characterization of Listeria monocytogenes in foods available in Taiwan.

A variety of foods were examined for the incidence of Listeria monocytogenes, and the bacterial isolates were further characterized. L. monocytogenes was selected on LiCl-phenylethanol-moxalactam agar after enrichments and identified by several biochemical, mobility, and CAMP tests. L. monocytogenes was isolated from 58.8% of pork samples, 50% of chicken carcasses, 38% of turkey parts, 34% of frozen semiready foods, 24% of beef steaks, 12.2% of vegetables, 10.5% of seafoods, and 4.4% of frozen dim sum but was not found in the Chinese pickles and fermented milks. Isolates from seafoods, turkey parts, and beef samples had higher hemolytic activity than those from other samples. The isolates were highly susceptible to ampicillin, cephalothin, chloramphenicol, erythromycin, gentamicin, kanamycin, neomycin, novobiocin, penicillin, and streptomycin. About 14.5% of the isolates were resistant to methicillin, and 14.5% were resistant to tetracycline. The majority of the isolates from turkey parts and beef steaks were serotype 1, and those from chicken and pork samples were serotype 4 and others. Hemolytic activity, methicillin susceptibility, and serotype distribution of the isolates from domestic and imported food samples were significantly different. The results suggest the presence of food- or geography-specific L. monocytogenes strains.     

Sensitivity of Listeria monocytogenes to sanitizers used in the meat processing industry

Nineteen Listeria monocytogenes strains were characterized by automated ribotyping, pulsed-field gel electrophoresis, and plasmid profiling to determine the relationship between genotype and sanitizer resistance. Isolates within a ribogroup had a consistent sensitivity or resistance phenotype except for ribogroup C isolates. All isolates with resistance phenotypes harbored two plasmids. The sensitivity of L. monocytogenes strains to quaternary ammonium compounds (QACs) was correlated with sensitivity to sanitizers and antibiotics with other modes of action. All isolates tested contained the mdrL gene, which encodes an efflux pump that confers resistance to QACs and is both chromosome and plasmid borne.     

Listeria monocytogenes in poultry and poultry products: epidemiological investigations in seven Danish abattoirs

Listeria monocytogenes was isolated from 11/236 (4·7%) caecal samples from parent flocks, providing broilers to the abattoirs investigated. Caecal samples from 2078 broilers representing 90 randomly selected broiler flocks were negative for L. monocytogenes. A total of 3080 samples from seven abattoirs including poultry processing line samples, and final products were also examined for L. monocytogenes. Listeria monocytogenes was isolated in 0·3% to 18·7% of the samples collected in the different abattoirs. Epidemiological typing of 247 L. monocytogenes isolates, including serotyping, phage typing, pulsed-field gel electrophoresis and ribotyping revealed 62 different clones. Based upon typability and discriminatory power, DNA typing methods used were found equally suitable as epidemiological markers. Serotyping and phage typing were not found useful as epidemiological markers for poultry isolates of L. monocytogenes since only 120/247 (48·6%) isolates were typable by phage typing and 230/247 (93·1%) L. monocytogenes belonged to serotype 01 while 6/247 (2·4%) belonged to 04. The discovery of a few dominating clones in each abattoir might indicate an endemic occurrence of L. monocytogenes. It is concluded that L. monocytogenes in the broiler production is primarily localized to the abattoirs. The incidence of L. monocytogenes may be reduced by improving the hygiene.     

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.     

API Listeria, a new and promising one-day system to identify Listeria isolates.

API Listeria is a new 10-test strip for 24-h biochemical identification of Listeria isolates. With this commercial system, 85% of 646 Listeria strains, including atypical isolates selected for this study, were recognized at the species and subspecies level without a complementary test. A new test differentiates Listeria monocytogenes from L. innocua on the basis of the absence of arylamidase from the former. With this system, 97.7% (252 of 258) of the L. monocytogenes strains tested were correctly identified and differentiated from 99.4% (175 of 176) of the L. innocua strains also tested. Gram-positive bacteria other than Listeria spp. gave quite different biochemical patterns. This system considerably reduced the time needed for conventional identification, since results were available within 18 to 24 h.     

API Listeria, a new and promising one-day system to identify Listeria isolates.

API Listeria is a new 10-test strip for 24-h biochemical identification of Listeria isolates. With this commercial system, 85% of 646 Listeria strains, including atypical isolates selected for this study, were recognized at the species and subspecies level without a complementary test. A new test differentiates Listeria monocytogenes from L. innocua on the basis of the absence of arylamidase from the former. With this system, 97.7% (252 of 258) of the L. monocytogenes strains tested were correctly identified and differentiated from 99.4% (175 of 176) of the L. innocua strains also tested. Gram-positive bacteria other than Listeria spp. gave quite different biochemical patterns. This system considerably reduced the time needed for conventional identification, since results were available within 18 to 24 h.     

Prevalence, characterization, and antimicrobial resistance of Listeria monocytogenes isolates from bovine hides and carcasses

Listeria monocytogenes isolates from bovine hides and carcasses (n = 812) were mainly of serogroup 1/2a. All strains were positive for internalin genes. Several isolates were resistant to oxacillin (72.2%) or clindamycin (37.0%). These findings indicate that L. monocytogenes of beef origin can be considered a public health concern.     

进口水产品中单增李斯特菌的分子流行病学特点

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

Diversity of Listeria species in urban and natural environments

A total of 442 Listeria isolates, including 234 Listeria seeligeri, 80 L. monocytogenes, 74 L. welshimeri, 50 L. innocua, and 4 L. marthii isolates, were obtained from 1,805 soil, water, and other environmental samples collected over 2 years from four urban areas and four areas representing natural environments. Listeria spp. showed similar prevalences in samples from natural (23.4%) and urban (22.3%) environments. While L. seeligeri and L. welshimeri were significantly associated with natural environments (P ≤ 0.0001), L. innocua and L. monocytogenes were significantly associated with urban environments (P ≤ 0.0001). Sequencing of sigB for all isolates revealed 67 allelic types with a higher level of allelic diversity among isolates from urban environments. Some Listeria spp. and sigB allelic types showed significant associations with specific urban and natural areas. Nearest-neighbor analyses also showed that certain Listeria spp. and sigB allelic types were spatially clustered within both natural and urban environments, and there was evidence that these species and allelic types persisted over time in specific areas. Our data show that members of the genus Listeria not only are common in urban and natural environments but also show species- and subtype-specific associations with different environments and areas. This indicates that Listeria species and subtypes within these species may show distinct ecological preferences, which suggests (i) that molecular source-tracking approaches can be developed for Listeria and (ii) that detection of some Listeria species may not be a good indicator for L. monocytogenes.     

Physicochemical surface properties of five Listeria monocytogenes strains from a pork-processing environment in relation to serotypes, genotypes and growth temperature

Physicochemical surface properties, related to electrostatic, van der Waals and Lewis acid–base interactions, of five Listeria monocytogenes strains isolated from pork-processing environments were determined after two subcultures at 37 °C and a final culture at three temperatures: 37, 10 and 4 °C. Three strains (Lm1, Lm114 and Lm191) were genetically related while two were unrelated (Lm25 and Lm74) according to Apa I-macrorestriction and pulsed-field gel electrophoresis (PFGE) typing.
Listeria monocytogenes cell surfaces were generally negatively charged regardless of pH and tended to be hydrophilic due to a basic character. However, variable physicochemical surface properties of the five Listeria monocytogenes isolates were observed after growth at 37 °C. After growth at 10 °C, the three genetically related isolates exhibited similar surface properties and were slightly more hydrophilic and basic than the others. After growth at 4 °C, the five isolates displayed the same weak affinity for all kinds of solvents and low electrophoretic mobility values.
A sharp decrease of temperature and subsequent growth of various Listeria monocytogenes strains resulted in loss of the physicochemical surface property variability, which may suggest the role of common chill adaptation mechanisms affecting surface properties.     

Occurrence of Listeria sp and L monocytogenes in sewage sludge used for land application: effect of dewatering,liming and storage in tank on survival of Listeria species

The application of sewage sludge to agricultural land is widely used in France. To determine the impact of sludge treatments, concentrations of Listeria sp., Listeria monocytogenes and faecal indicators were monitored in five types of sludge from three sewage treatment plants in Angers (France) and its suburbs over a 1-year period. On the whole, bacteria were reduced in numbers through sludge treatments. Apart from liming, which leads to reduced levels of bacteria below detection limits, other sludge treatments did not eliminate Listeria sp. and faecal indicators. Listeria sp. and L. monocytogenes were found respectively in 87% and 73% of dewatered sludges and in 96% and 80% of sludges stored in tanks. Concentrations of L. monocytogenes, ranging from 0.15 to 20 MPN g(-1) dry matter in dewatered sludge and from 1 to 240 MPN g(-1) dry matter in sludge stored in tanks, did not show seasonal variations. Spreading of sanitised sludge onto agricultural land results in the addition of 10(6)-10(8) L. monocytogenes per hectare per year, which may contribute to the increase in the dissemination of this pathogenic species in the environment.