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

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

Analyses of the bacterial contamination on Belgian broiler carcasses at retail level

Broilers are not equally exposed to bacterial contamination during rearing and processing, resulting in areas with different bacterial communities on carcasses at retail. The determination of these communities is also affected by the examination methods applied. The present study aimed to assess the bacterial communities on neck, breast, and back skin on broiler carcasses at retail through classical International Organization of Standardization based isolation methods combined with identification by matrix-assisted laser desorption ionization time-of-flight mass spectrum (MALDI-TOF MS) and 16S amplicon sequencing. Twelve commercially and eight organically reared broilers slaughtered in four slaughterhouses were examined. Significantly higher anaerobic bacterial counts were observed on the neck skin than on the breast and back skin. By the combination of cultivation and amplicon sequencing, remarkable shifts in bacterial communities were determined on the breast and back skin, but not on the neck skin. Although the aerobic bacteria contamination levels were not different between the areas, different bacterial communities were observed. The impact of the slaughterhouse to the overall microbial composition was rather small. Organically reared broilers had unique bacterial communities. In conclusion, compared to the breast skin, the neck, and back skin had a larger potential for bacterial spoilage, in particular when anaerobic storage conditions are applied. The distribution of bacteria on the different areas could be related to the contamination during slaughter as well as the bird-rearing methods

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

Variation in the prevalence of enteropathogenic Yersinia in slaughter pigs from Belgium, Italy, and Spain

Tonsils of 829 fattening pigs originating from Belgium (n = 201), Italy (n = 428), and Spain (n 200) were collected between 2005 and 2007 to study the prevalence of enteropathogenic Yersinia in slaughter pigs. Isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis was done by selective enrichment and by cold enrichment for 7 and 14 days. Pathogenic Y. enterocolitica and Y. pseudotuberculosis isolates were identified by polymerase chain reaction targeting the chromosomal genes ail and inv, respectively, as well as the plasmid-encoded virF of both species. A significantly higher (p < 0.001) prevalence of ail-positive Y. enterocolitica in Spain (93%) than in Belgium (44%) or Italy (32%) was observed. virF-positive Y. enterocolitica was present in 77% of ail-positive samples. Bioserotype 4/O:3 was the most common type in all three countries. Bioserotypes 2/O:5 and 3/O:9 were found in Italy (1%) and Belgium (9%), respectively. The prevalence of inv- and virF-positive Y. pseudotuberculosis was 2% and 1% in Belgium and Italy, respectively. Y. pseudotuberculosis was not detected in pigs from Spain. Bioserotypes 1/O:1 (20%), 1/O:2 (20%), and 2/O:3 (60%) were found in Belgium, and 1/O:1 (60%) and 2/O:3 (20%) in Italy. The most efficient method for isolation of Y. enterocolitica was combined cold enrichment for 7 and 14 days; however, the isolation method for Y. pseudotuberculosis was cold enrichment for 14 days. Fattening pigs seemto be an important reservoir of pathogenic Y. enterocolitica in Belgium, Italy, and Spain. Bioserotype 4/O:3 of Y. enterocolitica and bioserotypes 2/O:3 and 1/O:1 of Y. pseudotuberculosis have been shown to predominate

Microbiology and Epidemiology of Escherichia albertii—An Emerging Elusive Foodborne Pathogen

Escherichia albertii, a close relative of E. coli, is an emerging zoonotic foodborne pathogen associated with watery diarrhea mainly in children and immunocompromised individuals. E. albertii was initially classified as eae-positive Hafnia alvei, however, as more genetic and biochemical information became available it was reassigned to its current novel taxonomy. Its infections are common under conditions of poor hygiene with confirmed transmission via contaminated water and food, mainly poultry-based products. This pathogen has been isolated from various domestic and wild animals, with most isolates being derived from birds, implying that birds among other wild animals might act as its reservoir. Due to the absence of standardized isolation and identification protocols, E. albertii can be misidentified as other Enterobacteriaceae. Exploiting phenotypes such as its inability to ferment rhamnose and xylose and PCR assays targeting E. albertii-specific genes such as the cytolethal distending toxin and the DNA-binding transcriptional activator of cysteine biosynthesis encoding genes can be used to accurately identify this pathogen. Several gaps exist in our knowledge of E. albertii and need to be bridged. A deeper understanding of E. albertii epidemiology and physiology is required to allow the development of effective measures to control its transmission and infections. Overall, current data suggest that E. albertii might play a more significant role in global infectious diarrhea cases than previously assumed and is often overlooked or misidentified. Therefore, simple, and efficient diagnostic tools that cover E. albertii biodiversity are required for effective isolation and identification of this elusive agent of diarrhea

Faecal shedding of arcobacter species in Belgian pigs

The prevalence of Arcobacter was determined in porcine faecal samples collected at slaughterhouse and two unrelated finishing barns (A and B) using the previously developed Arcobacter isolation procedure. In 43.9% of the slaughterhouse samples tested (n=82) arcobacters were detected, and identified as A. butzleri and A. cryaerophilus. Two pigs shedded both species simultaneously. On farm A (n=98), arcobacters were isolated from 16.3% of the samples and identified as A. cryaerophilus and A. skirrowii. In samples (n=118) collected at farm B, arcobacters were detected in 45.1% of the samples. A. butzleri was the most frequently occurring species. Co-infections were found in 11 animals. Arocobacters were detected in clinically healthy pigs at contamination levels up to 103 cfu/g faeces

Boviene sarcosporidiosis of eosinofiele myositis?

Sarcosporidiosis is a misused term in meat inspection to name multifocal grey-green lesions observed in muscles of cattle. Instead the correct morphological diagnosis is bovine eosinophilic myositis. The confusion in terminology can not only lead to problems in insurance cases, it also results in incorrect European data reports. This article summarizes the current knowledge of Sarcocystis and bovine eosinophilic myositis in cattle, as a plea for the correct us of terminology

Prevalence of Arcobacter species among humans, Belgium, 2008-2013

We examined fecal samples from 6,774 patients with enteritis in Belgium, 2008–2013. Members of the genus Arcobacter were the fourth most common pathogen group isolated, and the isolation rate was higher than previously reported. Culturing Arcobacter in a microbiology laboratory is feasible and should thus be tested for in cases of diarrheal disease