Inactivation of Escherichia coli O157:H7 using ultraviolet light-treated bacteriophages.

Escherichia coli O157:H7 causes serious foodborne infections warranting the development of effective control measures. One control option is to use bacteriophages (phages), which are regarded as safe to humans and an environmentally friendly alternative to chemical antimicrobials. One of the few remaining safety concerns is the potential for phages to facilitate genetic exchange between bacteria so resulting in undesirable mobilisation of genes. UV treatment of phages causes a rapid loss in their ability to replicate, while maintaining their antibacterial activity, and so the use of UV-treated phages could be an alternative to the use of viable phages. Data presented here show the inactivation of E. coli O157:H7 by UV-treated phages in milk and on the surface of raw and cooked meat. A minimum concentration of approximately 10(5) PFU cm(-2) (pre-UV treatment titre) of UV-treated phages was required before inactivation of E. coli O157:H7 on the surface of meat was measurable, and 1-2 log10 CFU cm(-2) reductions were typically obtained at concentrations of around 10(7) UV-treated phages cm(-2) (pre-UV treatment titre). Inactivation of E. coli O157:H7 by UV-treated phages was less than that for untreated phages. The production of UV-treated phages was not optimised and it is possible that better reductions in pathogen concentration could be achieved for the same input UV-treated phages concentrations.

Effect of phage and host concentration on the inactivation of Escherichia coli O157:H7 on cooked and raw beef.

A previously described phage infecting Escherichia coli O157:H7 was added to raw and cooked beef pieces at concentrations ranging from 10(1)-10(8) plaque forming units/cm(2) to either low (<100 CFU/cm(2)) or high (10(4) CFU/cm(2)) concentrations of host bacterial cells. Incubation for up to 24 h was performed at 5 ℃ and 24 ℃ to simulate refrigerated and room temperature storage/temperature abuse. Surviving bacteria were enumerated during the incubation period, with phages being counted at the first and last sampling times. Significant reductions of E. coli O157:H7 of the order of >4 log10 CFU/cm(2) at both temperatures could be achieved compared to phage-free controls. There was a trend for greater inactivation to occur with increasing phage concentration. While re-growth of surviving cells occurred in nearly all samples incubated for 24 h at 24 ℃, these conditions are not typical of those experienced by perishable foods. It was concluded that phages can be used to reduce the concentration of a bacterial pathogen on meat, but the concentration of phages needs to be high (>4-5 log10 plaque forming units/cm(2)) for reductions to occur. A concentration of the order 8 log10 plaque forming units/cm(2) was needed to achieve a 4 log10 CFU/cm(2) reduction.

Use of a bacteriophage to inactivate Escherichia coli O157:H7 on beef.

A number of outbreaks of Escherichia coli O157:H7 infections involving beef have been reported. Options for controlling bacterial pathogens in raw foods are limited, but one is to use bacteriophages (phages). We describe the isolation and characterisation of phage FAHEc1, which infects E. coli O157, and its ability to kill its host in vitro and on beef. The phage belonged to the family Myoviridae and lysed 28 of 30 E. coli O157 (:H7, :HNM and :H not specified) isolates, only one other non-O157 E. coli serotype (O162:H7), and none of the other 13 bacterial species tested. The phage did not contain stx1, stx2, eae or ehxA virulence genes as assessed by PCR. An approximate 4 log10 inactivation of E. coli O157:H7 occurred at 5 °C in the presence of phage FAHEc1 at >107 PFU/ml in broth in vitro. On thinly sliced beef pieces incubated at 37 °C, a > 2.7 log10 reduction occurred with 3.2 × 107 PFU/4 cm² meat piece. At lower phage concentrations (10³-104 PFU/4 cm² piece) phage replication occurred on beef at 37 °C. When the phage was applied to beef pieces under conditions simulating hot boning and conventional carcass cooling, inactivation of E. coli O157:H7 of approximately 2 log10 was measured under optimal conditions with phages applied at 3.2 × 107 PFU/4 cm² meat piece.

Application of molecular epidemiology to understanding campylobacteriosis in the Canterbury region of New Zealand.

Pulsed-field gel electrophoresis genotypes of Campylobacter isolates from 603 human patients were compared with 485 isolates from retail offal (primarily chicken and lamb) to identify temporal clusters and possible sources of campylobacteriosis. Detailed epidemiological information was collected from 364 of the patients, and when combined with genotyping data allowed a putative transmission pathway of campylobacteriosis to be assigned for 88% of patients. The sources of infection were 47% food, 28% direct animal contact, 7% overseas travel, 4% person-to-person transmission and 3% water-related. A significant summer increase in campylobacteriosis cases was primarily attributed to an increase in food-related cases. Genotyping of isolates was essential for identifying the likely cause of infection for individuals. However, a more rapid and cheaper typing tool for Campylobacter is needed, which if applied to human and animal isolates on a routine basis could advance greatly our understanding of the ongoing problem of Campylobacter infection in New Zealand.

Epidemiology of Shiga toxin-producing Escherichia coli O157 in very young calves in the North Island of New Zealand.

AIMS: To study the occurrence and spatial distribution of Shiga toxin-producing Escherichia coli (STEC) O157 in calves less than 1-week-old (bobby calves) born on dairy farms in the North Island of New Zealand, and to determine the association of concentration of IgG in serum, carcass weight, gender and breed with occurrence of E. coli O157 in these calves. METHODS: In total, 309 recto-anal mucosal swabs and blood samples were collected from bobby calves at two slaughter plants in the North Island of New Zealand. The address of the farm, tag number, carcass weight, gender and breed of the sampled animals were recorded. Swabs were tested for the presence of E. coli O157 using real time PCR (RT-PCR). All the farms were mapped geographically to determine the spatial distribution of farms positive for E. coli O157. K function analysis was used to test for clustering of these farms. Multiplex PCR was used for the detection of Shiga toxin 1 (stx1), Shiga toxin 2 (stx2), E. coli attaching and effacing (eae) and Enterohaemolysin (ehxA) genes in E. coli O157 isolates. Genotypes of isolates from this study (n = 10) along with human (n = 18) and bovine isolates (n = 4) obtained elsewhere were determined using bacteriophage insertion typing for stx encoding. RESULTS: Of the 309 samples, 55 (17.7%) were positive for E. coli O157 by RT-PCR and originated from 47/197 (23.8%) farms. E. coli O157 was isolated from 10 samples of which seven isolates were positive for stx2, eae and ehxA genes and the other three isolates were positive for stx1, stx2, eae and ehxA. Bacteriophage insertion typing for stx encoding revealed that 12/18 (67%) human and 13/14 (93%) bovine isolates belonged to genotypes 1 and 3. K function analysis showed some clustering of farms positive for E. coli O157. There was no association between concentration of IgG in serum, carcass weight and gender of the calves, and samples positive for E. coli O157, assessed using linear mixed-effects models. However, Jersey calves were less likely to be positive for E. coli O157 by RT-PCR than Friesian calves (p = 0.055). CONCLUSIONS: Healthy bobby calves are an asymptomatic reservoir of E. coli O157 in New Zealand and may represent an important source of infection for humans. Carriage was not associated with concentration of IgG in serum, carcass weight or gender.

A PCR-DGGE method for detection and identification of Campylobacter, Helicobacter, Arcobacter and related Epsilobacteria and its application to saliva samples from humans and domestic pets.

AIMS: To develop a PCR-denaturing gradient gel electrophoresis (PCR-DGGE) method for the detection and identification of Campylobacter, Helicobacter and Arcobacter species (Epsilobacteria) in clinical samples and evaluate its efficacy on saliva samples from humans and domestic pets. METHODS AND RESULTS: A semi-nested PCR was developed to allow sensitive detection of all Epsilobacteria, with species separation undertaken by DGGE. A database was constructed in BioNumerics using 145 strains covering 51 Campylobacter, Arcobacter and Helicobacter taxa; Nineteen distinct DGGE profile-groups were distinguished. This approach detected Epsilobacteria in all saliva samples collected from humans, cats and dogs, and identified Campylobacter concisus and/or Campylobacter gracilis in the human samples. The pet animal samples were taken from individuals with oral/dental diseases; PCR-DGGE identified up to four different species in each sample. The most common species detected included Wolinella succinogenes, Arcobacter butzleri and two hitherto uncultured campylobacters. The enteropathogen Campylobacter lari was also found. CONCLUSIONS: PCR combined with DGGE is a useful tool for direct detection and preliminary identification of Epsilobacteria in the oral cavity of humans and small animals. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR-DGGE method should allow determination of the true prevalence and diversity of Epsilobacteria in clinical and other samples. Contact with the oral cavity of domestic pets may represent a route of transmission for epsilobacterial enteric diseases.

Comparison of virulence-associated in vitro properties of typed strains of Campylobacter jejuni from different sources.

Campylobacter jejuni is a major cause of human diarrhoeal disease, but specific virulence mechanisms have not been well defined. This blinded study was undertaken with 40 C. jejuni isolates from different sources to determine their haemolytic, cytotoxic and adhesion and invasion activities towards mammalian cells. The results were correlated with source of isolation and genetic makeup by amplified fragment length polymorphism (AFLP) typing. The isolates had variable degrees of haemolytic activity against rabbit erythrocytes and cytotoxicity towards CaCo-2, HeLa and Vero cells. The data indicated that the haemolytic and cytotoxic activities were due to separate factors. A range of cytotoxicity was exhibited, whereby some strains had no activity against the target cells and others had activity against all three cell lines. Certain strains had activity against CaCo-2 cells but little or no activity against the other cells, while others exhibited the opposite phenotype. The data suggested that the cytotoxicity assay with the different cell lines may have detected more than one cytotoxin. A wide variation between isolates was observed for both adherence and invasion with all three cell lines, yet, overall, the strains showed a significantly greater invasion capacity for CaCo-2. There was no clear relationship between source of isolation or disease manifestation and possession of statistically significantly higher levels of particular virulence-associated factors although, in some cases, a correlation between cytotoxicity and cell invasion was evident. Five AFLP clusters, each representing two to eleven isolates with similar profiles, were observed at the 90 % similarity level. Some AFLP groups contained isolates with a common serotype, but each group had C. jejuni isolates from more than one source with the exception of group IV, which contained only human isolates. Isolates with high cytotoxic activity against CaCo-2 cells were confined to groups I, III and IV and a group of unrelated strains (U). Group II isolates had uniformly low cytotoxicity. Isolates in groups I, V and U were more invasive for CaCo-2 cells than isolates in groups II, III and IV. The strain differences in cytotoxicity or invasion did not correlate with source of isolation.

Species diversity of campylobacteria in a wild bird community in Sweden.

AIMS: To analyse the occurrence and host species distribution of campylobacteria species in shorebirds, geese and cattle on grazed coastal meadows in Sweden. METHODS AND RESULTS: Species identification was performed through a polyphasic approach, incorporating Amplified Fragment Length Polymorphism (AFLP) profiling, 16S RNA gene sequence analysis together with extensive phenotypic characterization. From 247 sampled birds and 71 cattle, we retrieved 113 urease positive thermophilic Campylobacter (UPTC) and 16 Campylobacter jejuni ssp. jejuni isolates. Furthermore, 18 isolates of Helicobacter canadensis, and five isolates that potentially represent a new genus of micro-aerophilic, spiral and Gram-negative bacteria were isolated. The distribution of bacterial species on hosts was uneven: all H. canadensis isolates were retrieved from geese, while all but one of the Campylobacter lari UPTC isolates were found in shorebirds. AFLP type distribution of Camp. lari UPTC isolates among individual, resampled and breeding-paired Redshank birds generally indicated a constant shift in strain populations over time and absence of geographical clustering. CONCLUSIONS: The large number of isolated campylobacteria, including species that are zoonotic enteropathogens, indicates that these wild birds potentially may serve as reservoirs of human infections. However, despite a common environment, the different host species largely carried their own campylobacteria populations, indicating that cross-species transmission is rare. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study is one of few that provide data on the occurrence of campylobacteria in wild animals, adding information on the ecology and epidemiology of micro-organisms that are of public health concern.

Identification and molecular epidemiology of Campylobacter coli isolates from human gastroenteritis, food, and animal sources by amplified fragment length polymorphism analysis and Penner serotyping.

Campylobacter coli is an infrequently studied but important food-borne pathogen with a wide natural distribution. We investigated its molecular epidemiology by use of amplified fragment length polymorphism (AFLP)-based genotyping and Penner serotyping. Serotype reference strains and 177 Danish isolates of diverse origin identified by routine phenotyping as C. coli were examined. Molecular tools identified some 12% of field isolates as Campylobacter jejuni, emphasizing the need for improved identification methods in routine laboratories. Cluster analysis of AFLP profiles of 174 confirmed C. coli isolates revealed a difference in the distribution of isolates from pig and poultry (chicken, duck, turkey, and ostrich) species and indicated the various poultry species, but not pigs, to be likely sources of human C. coli infection. A poor correlation was observed between serotyping and AFLP profiling, suggesting that the former method has limited value in epidemiological studies of this species.

Genotyping and genetic diversity of Arcobacter butzleri by amplified fragment length polymorphism (AFLP) analysis.

AIMS: To investigate the potential of amplified fragment length polymorphism (AFLP) profiling for genotyping Arcobacter butzleri and to obtain further data on the genetic diversity of this organism. METHODS AND RESULTS: Seventy-three isolates of Danish, British, Turkish, Swedish, Nigerian and North American origin from human infections, chickens, turkeys, ducks, sheep and poultry abbatoir effluent were studied by use of a protocol that involved stringent PCR amplification of fragments derived from digestion of genomic DNA with restriction enzymes BglII and Csp6I. The mean similarity value of duplicate profiles of 10 isolates was 91.15%, indicating the method to be reproducible. Numerical analysis of all 73 isolates distinguished 51 subtypes at the 91% similarity level, of which 39 comprised single strains. The remaining 34 isolates were distributed among 12 subtypes, each of which contained strains homogeneous with respect to their respective source of isolation. However, contemporaneous strains from the same source could also be distinguished. CONCLUSIONS: AFLP profiling is an effective method for typing the genetically diverse organism A. butzleri. SIGNIFICANCE AND IMPACT OF THE STUDY: The study represents a comprehensive analysis of the genetic diversity of A. butzleri by use of isolates from six countries spanning three continents and also shows that several distinct A. butzleri genotypes may be found in a given environment. AFLP profiling appears to have considerable potential for molecular epidemiological studies of this ubiquitous emerging pathogen that is implicated as a causative agent of both human and animal disease.

Genetic relatedness among Campylobacter jejuni serotyped isolates of diverse origin as determined by numerical analysis of amplified fragment length polymorphism (AFLP) profiles.

AIMS: To use amplified fragment length polymorphism (AFLP) analysis to evaluate the genetic relatedness among 254 Campylobacter jejuni reference and field strains of diverse origin representing all defined 'Penner' serotypes for this species. METHODS AND RESULTS: Field strains (n = 207) from human diarrhoea and diverse animal and environmental sources were collected mainly through a National surveillance programme in Denmark and serotyped by use of the established 'Penner' scheme. Genetic relationships among these isolates, and the archetypal serotype reference strains, were assessed by numerical analysis of AFLP profiles derived from genomic DNA. Extensive genetic diversity was seen among the strains examined; however, 43 groups of isolates were identified at the 92% similarity (S-) level. Thirteen groups contained isolates from a single host, possibly representing genotypes of 'low risk' to human health. The remaining 30 groups contained isolates from humans, chickens and associated food products, cattle, sheep, turkeys, ostriches and/or dogs. Strains assigned to serotypes 2, 6/7, 11 and 12 formed major clusters at the 77.6% S-level. Most other serotypes did not form homogeneous clusters. CONCLUSIONS: High-resolution genotyping applied to strains from a comprehensive range of sources provides evidence for multiple sources of sporadic C. jejuni infection. The results suggest that public health protection measures should be directed at all foods of animal origin. SIGNIFICANCE AND IMPACT OF THE STUDY: The genetic relatedness among all 'Penner' serotypes of C. jejuni is assessed by AFLP analysis. In addition, further evidence of epidemic and host-specific clones of C. jejuni is provided.

Differentiation of Arcobacter species by numerical analysis of AFLP profiles and description of a novel Arcobacter from pig abortions and turkey faeces.

AIMS: To evaluate the efficacy of amplified fragment length polymorphism (AFLP)-based genetic profiling for taxonomic and epidemiological analyses of diverse Arcobacter species. METHODS AND RESULTS: Seventy-two isolates of A. butzleri, A. cryaerophilus, A. skirrowii and A. nitrofigilis, and a previously unclassified porcine abortion strain were studied. AFLP profiling was performed using a BglII-Csp6I-based protocol previously used to characterize Campylobacter species. Duplicate profiles of 20 isolates were 93.25% similar, indicating high reproducibility. Numerical analysis of all 72 strains revealed five phenons at the 29% similarity level, four of which represented each of the known species studied. The remaining phenon was further characterized by phenotypic and 16S rDNA sequence analyses, the results of which indicated it to be a novel Arcobacter species. The genetically distinct subgroups of A. cryaerophilus were differentiated at the 39.5% similarity level. For strain typing, 62 distinct types were defined, with evidence of clonal lineages within A. butzleri, A. cryaerophilus and A. skirrowii. CONCLUSIONS: AFLP profiling is an effective means of determining taxonomic and strain relationships for arcobacters. SIGNIFICANCE AND IMPACT OF THE STUDY: First use of AFLP profiling for diverse Arcobacter species; indication of clonality in A. butzleri, A. cryaerophilus and A. skirrowii; potentially novel Arcobacter taxon identified.

Toward an international standard for PCR-based detection of food-borne thermotolerant Campylobacters: assay development and analytical validation.

As part of a European research project (FOOD-PCR), we developed a standardized and robust PCR detection assay specific for the three most frequently reported food-borne pathogenic Campylobacter species, C. jejuni, C. coli, and C. lari. Fifteen published and unpublished PCR primers targeting the 16S rRNA gene were tested in all possible pairwise combinations, as well as two published primers targeting the 23S rRNA gene. A panel of 150 strains including target and nontarget strains was used in an in-house validation. Only one primer pair, OT1559 plus 18-1, was found to be selective. The inclusivity and exclusivity were 100 and 97%, respectively. In an attempt to find a thermostable DNA polymerase more resistant than Taq to PCR inhibitors present in chicken samples, three DNA polymerases were evaluated. The DNA polymerase Tth was not inhibited at a concentration of 2% (vol/vol) chicken carcass rinse, unlike both Taq DNA polymerase and DyNAzyme. Based on these results, Tth was selected as the most suitable enzyme for the assay. The standardized PCR test described shows potential for use in large-scale screening programs for food-borne Campylobacter species under the assay conditions specified.

Arcobacter butzleri strains from poultry abattoir effluent in Nigeria [corrected].

OBJECTIVE: To investigate the prevalence, species distribution and genetic diversity of zoonotic Arcobacter species. DESIGN: Prospective study. SETTING: Drainage system of a cosmopolitan chicken abattoir in Lagos, Nigeria. METHODS: One hundred and fifty drainage water samples were enriched in a minimal antibiotics-containing medium at room temperature and bacteria then isolated by use of a membrane filtration method. RESULTS: Twenty six (14%) of samples were positive for Arcobacter spp. Of these, 20 were examined by a comprehensive probabilistic identification scheme for Epsilobacteria and all strains identified as A. butzleri. AFLP analysis of these strains revealed considerable genetic diversity among the strains, with 12 genotypes defined at the 90% similarity level. CONCLUSION: The prevalence of A. butzleri in Nigerian poultry abattoir effluent indicates this species may constitute a public health problem in this country. AFLP profiling could be a useful tool for molecular epidemiological and population genetic studies of this organism. This is the first known report of A. butzleri in Nigeria, and first application of AFLP analysis for genotyping the species.

Genetic heterogeneity of Campylobacter concisus determined by pulsed field gel electrophoresis-based macrorestriction profiling.

In order to investigate the genetic diversity of Campylobacter concisus to assist molecular typing studies, the use of macrorestriction profiling was examined. A suitable protocol was developed that included the use of formaldehyde pretreatment to prevent DNA degradation, and restriction enzyme NotI for pulsed field gel electrophoresis-based genotyping. Subsequently, 53 strains of C. concisus, principally from cases of diarrhoea in children, were examined. Fifty-one distinct patterns were obtained, indicating the high discriminatory potential of the method. Patterns comprised between one and 14 restriction fragments, with type and reference strains of two well-defined genomospecies of oral and faecal origin containing six and 12 fragments respectively. Our results show that C. concisus is genetically diverse and suggest the species as currently defined to be a taxonomic continuum comprised of several genomospecies. The pulsed field gel electrophoresis typing method described here has considerable potential for molecular epidemiological studies of C. concisus and may be a useful adjunctive method for helping to resolve key taxonomic issues for this species.

Campylobacter hyointestinalis subsp. hyointestinalis, a common Campylobacter species in reindeer.

AIMS: To study the prevalence of Campylobacter spp. in the faecal material of reindeer, and to identify the isolates by means of a polyphasic approach. In addition, to study the genetic diversity of Camp. hyointestinalis subsp. hyointestinalis reindeer isolates by pulsed-field gel electrophoresis (PFGE). METHODS AND RESULTS: The material, collected during the slaughter period in autumn 1998, comprised 399 faecal contents from the reindeer (Rangifer tarandus), a semi-domesticated, meat-producing ruminant of northern Finland. These samples came from 16 herds in the areas of eight reindeer slaughterhouses. Samples were cultured by methods suitable for isolation of fastidious Campylobacter species. Of all samples, 6% (24/399) were Campylobacter-positive. Phenotypic characteristics, SDS-PAGE protein patterns, dot blot DNA-DNA hybridization, 23S rDNA restriction fragment polymorphism analysis and PFGE identified the isolates as Camp. hyointestinalis subsp. hyointestinalis. CONCLUSIONS: Campylobacter hyointestinalis subsp. hyointestinalis was the only Campylobacter species isolated from reindeer in this study. The isolates showed high genomic diversity in PFGE with the restriction enzymes SmaI and KpnI. SIGNIFICANCE AND IMPACT OF THE STUDY: PFGE analysis is a useful subtyping method for epidemiological studies. Contaminated reindeer meat can be a source for human infections.

A cultured strain of "Helicobacter heilmannii," a human gastric pathogen, identified as H. bizzozeronii: evidence for zoonotic potential of Helicobacter.

We compared the characteristics of a cultured human "Helicobacter heilmannii" isolate with those of other helicobacters found in animals. Phenotypic, protein profile, 16S rDNA sequence, and DNA-DNA hybridization analyses identified the human strain as H. bizzozeronii, a species frequently found in dogs. Thus, H. bizzozeronii may have zoonotic potential.

Helicobacter ganmani sp. nov., a urease-negative anaerobe isolated from the intestines of laboratory mice.

Spiral bacteria were isolated from the intestines of laboratory mice during a study examining the presence of Helicobacter species and other spiral organisms naturally infecting mice maintained at four different animal facilities in Sydney, Australia. One group of 17 isolates, cultured from mice from three of the four facilities, were found to be helicobacters but did not fall within any of the 18 currently recognized species. These isolates were unusual in that they only grew anaerobically at 37 degrees C and were incapable of growth under microaerobic conditions. Like Helicobacter rodentium, isolates possessed single, bipolar, unsheathed flagella and were urease-negative. They were positive for oxidase and reduced nitrate to nitrite but did not hydrolyse hippurate or indoxyl acetate, grew on charcoal agar and were resistant to cephalothin. 16S rDNA sequences from four strains were determined and found to be identical to one another. H. rodentium was the most closely related species in terms of 16S rDNA sequence similarity (98.2%). Numerical analysis of whole-cell proteins by SDS-PAGE for nine isolates was carried out with a comparison to all known Helicobacter species, including newly determined profiles from three H. rodentium strains. The new isolates were clearly differentiated from H. rodentium and other Helicobacter spp. On the basis of this data, including genetic, biochemical and protein analysis, it is proposed that these isolates belong to Helicobacter ganmani sp. nov. (type strain CMRI H02T = CCUG 43526T = CIP 106846T).

Comparison of genotypes and serotypes of Campylobacter jejuni isolated from Danish wild mammals and birds and from broiler flocks and humans.

The incidence of human infection with Campylobacter jejuni is increasing in most developed countries and the reason for this is largely unknown. Although poultry meat is considered to be a major source, it is evident that other reservoirs exist, possibly common to humans and poultry. Environmental sources are believed to be important reservoirs of Campylobacter infection in broiler chicken flocks. We investigated the potential importance of wildlife as a source of infection in commercial poultry flocks and in humans by comparing the serotype distributions, fla types, and macrorestriction profiles (MRPs) of C. jejuni isolates from different sources. The serotype distribution in wildlife was significantly different from the known distributions in broilers and humans. Considerable sero- and genotype diversity was found within the wildlife collection, although two major groups of isolates within serotype O:12 and the O:4 complex were found. Common clonal lines among wildlife, chicken, and/or human isolates were identified within serotype O:2 and the O:4 complex. However, MRPs of O:12 and O:38 strains isolated from wildlife and other sources indicated that some clonal lines propagated in a wide selection of animal species but were not detected in humans or broilers in this study. The applied typing methods successfully identified different clonal groups within a strain collection showing large genomic diversity. However, the relatively low number of wildlife strains with an inferred clonal relationship to human and chicken strains suggests that the importance of wildlife as a reservoir of infection is limited.