Prevalence of Type VI Secretion System in Spanish Campylobacter jejuni Isolates.

Infections from Campylobacter jejuni pose a serious public health problem and are now considered the leading cause of foodborne bacterial gastroenteritis throughout the world. Sequencing of C. jejuni genomes has previously allowed a number of loci to be identified, which encode virulence factors that aid survival and pathogenicity. Recently, a Type VI secretion system (T6SS) consisting of 13 conserved genes was described in C. jejuni strains and recognised to promote pathogenicity and adaptation to the environment. In this study, we determined the presence of this T6SS in 63 Spanish C. jejuni isolates from the food chain and urban effluents using whole-genome sequencing. Our findings demonstrated that nine (14%) strains harboured the 13 ORFs found in prototype strain C. jejuni 108. Further studies will be necessary to determine the prevalence and importance of T6SS-positive C. jejuni strains.

Campylobacter shared between free-ranging cattle and sympatric wild ungulates in a natural environment (NE Spain).

Campylobacter infections are a public health concern and an increasingly common cause of food-borne zoonoses in the European Union. However, little is known about their spill-over from free-ranging livestock to sympatric wild ungulates, especially in regards to uncommon Campylobacter species. In this study, we aim to determine the prevalence of C. coli, C. jejuni and other C. spp. in game ungulates (wild boar Sus scrofa and Iberian ibex Capra pyrenaica) and free-ranging sympatric cattle in a National Game Reserve in NE Spain. Furthermore, we explore the extent to which Campylobacter species are shared among these co-habiting hosts. Faecal samples from Iberian ibex (n = 181) were negative for C. spp. By direct plating, two wild boars out of 150 were positive for C. coli (1.3%, 95% CI 0.16-4.73), and one was positive for C. jejuni (0.67%, 95% CI 0.02-3.66). The latter was predominant in cattle: 5.45% (n = 55, 95% CI 1.14-5.12), while C. coli was not isolated from this host. C. lanienae was the most frequent species in wild boar at 10% (95% CI 5.7-15.96), and one cow cohabiting with positive wild boars in the same canyon also carried C. lanienae. Four enrichment protocols (using Bolton or Preston broth combined with either mCCDA or CFA) were added for 172 samples (57 from wild boars, 55 cattle and 60 Iberian ibexes) to increase the number of isolates obtained allowing the detection of statistically significant differences. The prevalence of C. lanienae was statistically significantly higher in wild boar than in cattle (P < 0.01), but the prevalence of C. jejuni was higher in the latter (P = 0.045). These results suggest that wild boar and cattle carry their own predominant Campylobacter species, while Iberian ibex do not seem to play an important role in the epidemiology of Campylobacter. However, there is a potential spill-over of C. spp., and thus, further research is needed to elucidate the factors determining inter-species transmission.

The effect of different isolation protocols on detection and molecular characterization of Campylobacter from poultry.

We determined whether different methods to isolate Campylobacter (including the ISO standard 10272:2006-1) affected the genotypes detectable from poultry, at three points during slaughter: caecal content, neck skin and meat. Carcasses from 28 independent flocks were thus sampled (subset A). In addition, ten neck skin samples from four flocks, ten caecal samples from ten different flocks and ten unrelated meat samples obtained from local supermarkets were collected (subset B). Campylobacter was isolated using eight different protocols: with and without enrichment using Bolton broth, Preston broth or Campyfood broth (CFB), followed by culture on either modified Charcoal Cefoperazone Deoxycholate Agar (mCCDA) or Campyfood agar (CFA). All obtained isolates were genotyped for flaA-SVR, and over half of the isolates were also typed by MLST. The strain richness, as a measure of number of detected fla-genotypes, obtained from subset A neck skin and caecal samples was higher than that of meat samples. In half of the cases, within a flock, at least one identical fla-genotype was obtained at all three slaughter stages, suggestive of autologous contamination of carcasses. Enrichment reduced the observed richness of isolates, while CFA plates increased richness compared to mCCDA plates, irrespective of inclusion of an enrichment step. Because the isolation protocol used influences both the yield and the fla-genotype richness obtained from poultry, this variable should be taken into account when different studies are being compared.

Evaluation of four protocols for the detection and isolation of thermophilic Campylobacter from different matrices.

AIMS: To identify the optimal method for detection of thermophilic Campylobacter at various stages in the food chain, three culture-dependent (direct plating, Bolton and Preston enrichment) and one molecular method (qPCR) were compared for three matrices: poultry faeces (n = 38), neck skin (n = 38) and packed fresh meat (n = 38). METHODS AND RESULTS: Direct plating was compared to enrichment with either Bolton broth (ISO 10272:2006-1) or Preston broth, followed by culture on two selective agars: modified charcoal cefoperazone desoxycholate agar (mCCDA) and Campyfood agar (CFA). Direct plating on CFA provided the highest number of positive samples for faeces and neck skin samples. Enrichment of meat samples in Preston followed by plating on mCCDA gave significantly higher number of positives than the recommended ISO method. Real-time qPCR yielded the highest number of positive samples. CONCLUSION: Direct plating on CFA is optimal for Campylobacter isolation from highly contaminated samples such as faeces or neck skin. When enrichment is required for less-contaminated samples such as poultry meat, Preston broth is the best choice. The maximum of detectable cells predicted by qPCR is a sensitive and powerful evaluation tool. SIGNIFICANCE AND IMPACT OF THE STUDY: The recommended ISO protocol had the least sensitivity, and application of this method could result in underreporting. We detected a high prevalence of Campylobacter on packed meat to be distributed, which suggests this is still a significant risk for consumers.