Association of Antimicrobial Resistance in Campylobacter spp. in Broilers and Turkeys with Antimicrobial Use.

We investigated trends in antimicrobial resistance (AMR) in Campylobacter jejuni and Campylobacter coli in poultry between 2010 and 2016 in Germany and their association with antimicrobial use. Campylobacter had been isolated from the caeca of broilers and turkeys at slaughter by regional laboratories according to current ISO methods in the framework of a national monitoring program. Isolates were submitted to the National Reference Laboratory for Campylobacter and tested for AMR using broth microdilution methods. Minimum inhibitory concentrations were evaluated according to epidemiological cut-off values. Antimicrobial use (AMU) data from 2014 to 2016 were taken from a government report. AMR was higher in C. coli than in C. jejuni and higher in turkeys than in broilers. AMR was highest to tetracycline and the tested (fluoro)quinolones while it was rare to gentamicin in both bacterial species, infrequent to erythromycin in C. jejuni, and moderate in C. coli. AMR to tetracycline and erythromycin decreased over time while it increased to (fluoro)quinolones. An association of AMU and AMR was observed for resistance to tetracycline and erythromycin, while it was not observed for the aminoglycosides. Resistance to nalidixic acid and ciprofloxacin increased despite a decrease of fluoroquinolone use between 2014 and 2016, indicating that other factors have a strong influence on resistance to (fluoro)quinolones in Campylobacter.

Internal sample process control improves cultivation-independent quantification of thermotolerant Campylobacter.

Quantification of Campylobacter is challenging and one major reason is the fact that bacteria lose cultivability due to cold or oxygen stress during storage at retail. Alternative live/dead discriminatory qPCR currently lacks standardization and might overestimate live cells in the presence of dead cells. In this study an internal sample process control (ISPC) was developed. The ISPC consists of a specified number of peroxide-killed C. sputorum cells to be added to each sample in order to monitor (i) the level of reduction of the signal from dead cells and (ii) DNA losses during sample processing. A species-specific fragment of the 16S rRNA gene of C. sputorum was selected as real-time PCR target, based on its similar size and gene copy number compared to the C. jejuni/coli/lari target and confirmed in an exclusivity study. Extension of the amplification oligonucleotides for the target of thermotolerant Campylobacter improved real-time PCR efficiency, rendering the method suitable for quantification according to international standards. Concordant PCR signal variation of both C. jejuni and C. sputorum targets in co-inoculated chicken rinses verified the suitability of the ISPC. This provides a crucial step towards implementation of cultivation-independent quantification for improved food safety of fastidious bacteria.

Campylobacter detection along the food chain--towards improved quantitative risk analysis by live/dead discriminatory culture-independent methods.

Death, although absolute in its consequence, is not measurable by an absolute parameter in bacteria. Viability assays address different aspects of life, e. g. the capability to form a colony on an agar plate (CFU), metabolic properties or mem- brane integrity. For food safety, presence of infectious potential is the relevant criterion for risk assessment, currently only partly reflected by the quantification of CFU. It will be necessary for future improved risk assessment, in particular when fastidious bacterial pathogens are implicated, to enhance the informative value of CFU. This might be feasible by quantification of the number of intact and potentially infectious Campylobacter, impermeable to the DNA intercalating dye propidium monoazide (PMA). The latter are quantifiable by the combination of PMA with real-time PCR, although thorough controls have to be developed for standardization and the circumvention of pitfalls. Under consideration of differ- ent physiological states of the food-borne pathogen, we provide an overview of current and future suitable detection/quantification targets along the food chain, including putative limitations of detection.

"Limits of control"--crucial parameters for a reliable quantification of viable campylobacter by real-time PCR.

The unsuitability of the "CFU" parameter and the usefulness of cultivation-independent quantification of Campylobacter on chicken products, reflecting the actual risk for infection, is increasingly becoming obvious. Recently, real-time PCR methods in combination with the use of DNA intercalators, which block DNA amplification from dead bacteria, have seen wide application. However, much confusion exists in the correct interpretation of such assays. Campylobacter is confronted by oxidative and cold stress outside the intestine. Hence, damage caused by oxidative stress probably represents the most frequent natural death of Campylobacter on food products. Treatment of Campylobacter with peroxide led to complete loss of CFU and to significant entry of any tested DNA intercalator, indicating disruption of membrane integrity. When we transiently altered the metabolic state of Campylobacter by abolishing the proton-motive force or by inhibiting active efflux, CFU was constant but enhanced entry of ethidium bromide (EtBr) was observed. Consistently, ethidium monoazide (EMA) also entered viable Campylobacter, in particular when nutrients for bacterial energization were lacking (in PBS) or when the cells were less metabolically active (in stationary phase). In contrast, propidium iodide (PI) and propidium monoazide (PMA) were excluded from viable bacterial cells, irrespective of their metabolic state. As expected for a diffusion-limited process, the extent of signal reduction from dead cells depended on the temperature, incubation time and concentration of the dyes during staining, prior to crosslinking. Consistently, free protein and/or DNA present in varying amounts in the heterogeneous matrix lowered the concentration of the DNA dyes at the bacterial membrane and led to considerable variation of the residual signal from dead cells. In conclusion, we propose an improved approach, taking into account principles of method variability and recommend the implementation of process sample controls for reliable quantification of intact and potentially infectious units (IPIU) of Campylobacter by real-time PCR.