Towards a typing strategy for Arcobacter species isolated from humans and animals and assessment of the in vitro genomic stability

Arcobacter species have a widespread distribution with a broad range of animal hosts and environmental reservoirs, and are increasingly associated with human illness. To elucidate the routes of infection, several characterization methods such as pulsed-field gel electrophoresis (PFGE), amplified fragment-length polymorphism, and enterobacterial repetitive intergenic consensus (ERIC)-PCR have already been applied, but without proper validation or comparison. At present, no criterion standard typing method or strategy has been proposed. Therefore, after the validation of PFGE, those commonly applied typing methods were compared for the characterization of six human- and animal-associated Arcobacter species. With a limited number of isolates to be characterized, PFGE with restriction by KpnI is proposed as the first method of choice. However, ERIC-PCR represents a more convenient genomic fingerprinting technique when a large number of isolates is involved. Therefore, a first clustering of similar patterns obtained after ERIC-PCR, with a subsequent typing of some representatives per ERIC cluster by PFGE, is recommended. As multiple genotypes are commonly isolated from the same host and food, genomic plasticity has been suggested. The in vitro genomic stability of Arcobacter butzleri and A. cryaerophilus was assessed under two temperatures and two oxygen concentrations. Variability in the genomic profile of A. cryaerophilus was observed after different passages for different strains at 37 degrees C under microaerobic conditions. The bias due to these genomic changes must be taken into account in the evaluation of the relationship of strains

Relation between serology of meat juice and bacteriology of tonsils and feces for the detection of enteropathogenic Yersinia spp. in pigs at slaughter

The association between positive serology and culture detection of Yersinia spp. in individual pigs was determined. Pieces of diaphragm from 370 pig carcasses were collected for serological analysis, and tonsils and feces of the same carcass were collected for bacteriological analysis. Detection of anti-Yersinia antibodies in meat juice samples was done using an indirect enzyme-linked immunosorbent assay (ELISA) based on Yops (Yersinia outer proteins). Tonsils and feces were tested for the presence of enteropathogenic Yersinia spp. by direct plating on cefsulodin–irgasan–novobiocin agar plates. Of the 370 meat juice samples, 241 (65.1%) gave a positive serological reaction using a cutoff value of 20%. Enteropathogenic Yersinia spp. (Yersinia enterocolitica serotype O:3 and Yersinia pseudotuberculosis) were found in tonsils of 161 pigs and feces of 30 pigs. Recovery of enteropathogenic Yersinia from the tonsils was highly correlated with positive serotiters, whereas no correlation was found between serology and fecal excretion. Results demonstrated that serology has an acceptable sensitivity, but a relatively low specificity for the rapid detection of enteropathogenic Yersinia spp. in tonsils of pigs at slaughter

Enterohemorrhagic Escherichia coli with particular attention to the German outbreak strain O104:H4

This review deals with the epidemiology and ecology of enterohemorrhagic Escherichia coli (EHEC), a subset of the verocytotoxigenic Escherichia coli (VTEC), and subsequently discusses its public health concern. Attention is also given to the outbreak strain O104:H4, which has been isolated as causative agent of the second largest outbreak of the hemolytic uremic syndrome worldwide, which started in Germany in May 2011. This outbreak strain is not an EHEC as such but possesses an unusual combination of EHEC and enteroaggregative E. coli (EAggEC) virulence properties

Genotyping and antimicrobial resistance patterns of Escherichia coli O157 originating from cattle farms

During a Escherichia coli O157 prevalence study on cattle farms, 324 E. coli O157 isolates were collected from 68 out of 180 cattle farms. All isolates harbored the eaeA gene and the enterohemolysin (ehxA) gene. The majority of the strains only contained vtx2 (245 isolates), the combination of vtx1 and vtx2 was detected in 50 isolates, and in 29 isolates none of the vtx genes was present. Pulsed-field gel electrophoresis (PFGE) revealed that at a similarity level of 98% the isolates grouped into 83 different genotypes, 76 of which were only detected on one farm. Twenty-two out of the 68 positive farms harbored isolates belonging to more than one PFGE type, with a maximum of four different PFGE types. Minimal inhibitory concentrations of 10 antimicrobial agents were determined on a subset of 116 isolates, that is, one isolate per positive age category per farm. Acquired resistance to at least one antimicrobial agent was detected in 18 isolates and within a farm, only one resistance pattern was observed. All these 18 isolates were resistant toward streptomycin, and 16 of them also showed resistance toward sulfisoxazole. Six isolates were resistant to three or more antimicrobial agents

Effect of the enrichment medium on the detection and diversity of Salmonella from porcine duodenal content

This study assesses the effect of the enrichment medium used on the isolation of Salmonella from the duodenal content of naturally infected slaughter pigs. At six slaughterhouses, the duodenum was collected from 458 randomly chosen pigs and examined in the laboratory. Three semi-solid enrichment media (modified semisolid Rappaport-Vassiliadis medium [MSRV], diagnostic semi-solid Salmonella medium [DIASALM], and Simple Method Salmonella [SMS] agar) and three enrichment broths (Rappaport-Vassiliadis, Rappaport Vassiliadis broth with Soya [RVS], and Muller Kauffmann Tetrathionate novobiocin broth [MKTTn]) were evaluated. If a migration zone was present on the semi-solid media, a loopful was taken both near the inoculation drop and at the edge of the migration zone and streaked on a Xylose Lysine Desoxycholate (XLD) agar plate. Each enrichment broth was streaked on XLD, and three presumptive colonies were further examined. Detection rate was calculated, and isolates were, after serotyping, genotyped by performing pulsed-field gel electrophoresis (PFGE). The overall frequency of Salmonella isolated in at least one of the six different media was 15.5% (71/458). No significant differences in relative sensitivity were obtained within semi-solid media and within liquid media. Semi-solid media showed a significant higher relative sensitivity than the one obtained with liquid media. A relative sensitivity higher than 83.1%, namely of 94.4%, could only be obtained by combining three different enrichment media (MSRV or DIASALM+RVS+MKTTn). In 13.4% of the positive pigs, more than one serotype was found within the duodenum of one pig. In 12.9% of the duodenal contents, different genotypes were found within the same serotype. Differences in serotypes and genotypes were found predominantly within the same enrichment medium. In conclusion, to obtain the highest Salmonella detection rate in naturally contaminated pig samples, MSRV should be used as enrichment medium. However, to obtain a realistic picture of the sero-and genotypes present, different samples per enrichment medium and different enrichment media should be tested

Spatial distribution of the emerging foodborne pathogen Arcobacter in the gastrointestinal tract of pigs

Pigs are important reservoirs for Arcobacter. Since 1978, Arcobacter species have been associated with reproduction disorders, but excretion by clinically healthy pigs has been frequently reported as well. Information on Arcobacter colonization of the porcine gastrointestinal tract is lacking. In the present study, gastrointestinal tracts of 12 pigs were collected, and the content and mucus of eight sections were examined. Arcobacters were enumerated and isolated by a selective quantitative and qualitative method, respectively, and identified by multiplex-polymerase chain reaction (PCR). Their genetic diversity was examined by enterobacterial repetitive intergenic consensus PCR and pulsed-field gel electrophoresis. Arcobacter species were isolated from at least two gastrointestinal sections of all pigs in levels up to 10(5) colony-forming units (CFU) g(-1) in content and 10(4) CFU g(-1) in mucus. Characterization of the isolates revealed a high degree of genotypic diversity. In general, the highest counts, and greatest species and strain diversity was obtained from the large intestine, and especially from the rectum. Though Arcobacter strains were mostly detected in one gastrointestinal section, several unique strains were also recovered from the content and/or mucus of various gastrointestinal sections of individual pigs. In the gastrointestinal tract, Arcobacter is present with species distributions, numbers, and strain heterogeneity comparable to those reported on porcine carcasses post slaughter, thus confirming the potential route of transmission to carcasses by fecal contamination during processing