Quantitative assessment of Campylobacter spp. levels with real-time PCR methods at different stages of the broiler food chain.

The importance of thermotolerant Campylobacter spp. as a food-borne pathogen continues to increase and there is a great need for rapid quantitative results in routine diagnostics. However, currently, only the culture-based ISO method is authorized for use in the context of official food control. The present study therefore aimed to assess the suitability of a qPCR method for a rapid quantitative determination of Campylobacter spp. at different stages in the poultry production chain and its equivalence with the culture-based method. Samples from two processors were collected and evaluated both separately and together. Censored regression (Tobit) models have been used to establish a relationship between Campylobacter qPCR counts on the carcasses and explanatory variables of processor and meat counts. Further, correlations of qPCR Campylobacter spp. counts at the different stages of production were calculated. In addition, the comparative data between microbiological enumeration and qPCR results were statistically analyzed. In the correlation calculation of the qPCR results, a highly significant relationship between the Campylobacter spp. counts of the neck skin samples to breast fillet and leg samples could be calculated, indicating a good prediction of Campylobacter spp. loads in these samples. The intercalating dye ethidium monoazide (EMA) was used to see whether the correlations between microbiological counts and qPCR results were improved by pretreating fecal and cecal samples before qPCR analysis. It was shown that the observed values of scatter plots between the qPCR-based and the culture-based methods were strongly correlated. However, on average, the qPCR results were two log10 CFU/mL levels higher than the microbiological counts. However, the classical culture-based method for food hygiene risk assessment cannot be replaced one-to-one by the qPCR or EMA-qPCR. The qPCR method can rather be used for the rapid identification of particularly highly contaminated flocks.

Adaptation of Campylobacter field isolates to propionic acid and sorbic acid is associated with fitness costs.

AIMS: To reduce the burden of Campylobacter at different stages of the food chain, recent studies have shown the effectiveness of organic acids as a risk mitigation strategy. However, very little is known about possible adaptation responses of Campylobacter that lead to reduced susceptibility to organic acids. Here we investigated the adaptive responses of Campylobacter field isolates to organic acids and estimated the fitness costs. METHODS AND RESULTS: Exposure of two Campylobacter jejuni and one Campylobacter coli isolate to subinhibitory concentrations of propionic acid or sorbic acid resulted in twofold to fourfold increased minimal inhibitory concentration values for the adapted variants. With one exception, the decreased susceptibility was stable in at least 10 successive subcultures without selection pressure. Growth competition experiments revealed a reduced fitness of adapted variants compared to the wild-type isolates. A linear regression model allowed an estimation of the fitness cost. Growth kinetics experiments showed significantly prolonged lag phases in five of six adapted isolates while there was not a direct correlation in the maximum growth rates compared to the wild-type isolates. CONCLUSIONS: The results of the study showed that a stepwise adaptation of Campylobacter to organic acids is possible, but at the detriment of changes in growth behaviour and reduced fitness. SIGNIFICANCE AND IMPACT OF THE STUDY: The study contributes to the understanding of adaptive responses of Campylobacter to organic acids treatments, for example, as part of risk mitigation strategies.

Genetic diversity and antibiotic susceptibility of Staphylococcus aureus isolates from wild boars.

We here report the occurrence of S. aureus in wild boars and characterize isolates genotypically and phenotypically in order to get knowledge about the occurrence of clonal lineages and genotypes in free-living wild animals. Forty-one S. aureus isolates obtained from 111 wild boars hunted in Lower Saxony, Germany, were investigated and compared to human and livestock isolates. The S. aureus belonged to multilocus sequence types ST1, ST7, ST30, ST133, ST425, ST804, ST890 and to the new ST3237, ST3238, ST3255 and ST3369. The livestock associated CC398-MRSA lineage, however, was not found. In addition to well-known spa types, the new types t14999, t15000, t15001 and t15002 were detected. Macrorestriction analysis revealed a variety of different SmaI fragment patterns. Most isolates were susceptible to all antimicrobials tested, including methicillin, and resistance was detected only to ampicillin, penicillin and erythromycin. PCR analysis confirmed the presence of staphylococcal enterotoxin genes (seh) in all t127-ST1 isolates. A high degree of genetic diversity was detected with many spa types and clonal lineages previously reported in humans and livestock animals.