Identification of knowledge gaps in whole-genome sequence analysis of multi-resistant thermotolerant Campylobacter spp.

BACKGROUND: Campylobacter spp. is the most frequent cause of bacterial food-borne gastroenteritis and a high priority antibiotic resistant bacterium according to the World Health Organization (WHO). European monitoring of thermotolerant Campylobacter spp. does not reflect the global burden of resistances already circulating within the bacterial population worldwide. METHODS: We systematically compared whole genome sequencing with comprehensive phenotypic antimicrobial susceptibility, analyzing 494 thermotolerant Campylobacter poultry isolates from Vietnam and Germany. Any discrepancy was checked by repeating the wet lab and improving the dry lab part. Selected isolates were additionally analyzed via long-read Oxford Nanopore technology, leading to closed chromosomes and plasmids. RESULTS: Overall, 22 different resistance genes and gene variants (e. g. erm(B), aph(3')-IIIa, aph(2'')-If, catA, lnu(C), blaOXA, sat4) and point mutations in three distinct genes (gyrA, 23S rRNA, rpsL) associated with AMR were present in the Campylobacter isolates. Two AMR genes were missing in the database and one falsely associated with resistance. Bioinformatic analysis based on short-read data partly failed to identify tet(O) and aadE, when the genes were present as duplicate or homologous gene variants. Intriguingly, isolates also contained different determinants, redundantly conferring resistance to chloramphenicol, gentamicin, kanamycin, lincomycin and streptomycin. We found a novel tet(W) in tetracycline sensitive strains, harboring point mutations. Furthermore, analysis based on assemblies from short-read data was impaired to identify full length phase variable aad9, due to variations of the poly-C tract within the gene. The genetic determinant responsible for gentamicin resistance of one isolate from Germany could not be identified. GyrT86I, presenting the main determinant for (fluoro-)quinolone resistance led to a rare atypical phenotype of ciprofloxacin resistance but nalidixic acid sensitivity. Long-read sequencing predicted AMR genes were mainly located on the chromosome, and rarely on plasmids. Predictions from long- and short-read sequencing, respectively, often differed. AMR genes were often organized in multidrug resistance islands (MDRI) and partially located in proximity to transposase genes, suggesting main mobilization of resistance determinants is via natural transformation and transposition in Campylobacter. CONCLUSIONS: The results of this study suggest that there is frequent resistance gene duplication, mosaicism, and mutation leading to gene variation and truncation in Campylobacter strains that have not been reported in previous studies and are missing from databases. Furthermore, there is a need for deciphering yet unknown resistance mechanisms and resistance spread in thermotolerant Campylobacter spp. that may pose a challenge to global food safety.

Comparison of Antimicrobial Resistance of Thermophilic Campylobacter Isolates from Conventional and Organic Turkey Meat in Germany.

The objective of this study was to compare the prevalence and antimicrobial resistance (AMR) rates of Campylobacter spp. isolated from conventional and organic turkey meat sold at retail in Germany. Samples of conventional (N = 527) and organic (N = 245) fresh turkey meat without skin were collected at retail markets throughout Germany and tested for Campylobacter spp.. Campylobacter isolates were tested for resistance to six antimicrobials (gentamicin, streptomycin, ciprofloxacin, nalidixic acid, erythromycin, and tetracycline) using broth microdilution. Prevalence of Campylobacter spp. was higher in organic (32.7%) than in conventional (19.4%) turkey meat. The proportion of fully susceptible isolates was lower in Campylobacter coli (6.8%) than in Campylobacter jejuni (33.9%) and higher in isolates from organic (38.4%) than from conventional production (17.4%). Overall, resistance rates were the highest to ciprofloxacin, nalidixic acid, and tetracycline. Resistance to erythromycin was only observed in C. coli and resistance to gentamicin was absent. Overall, resistance rates to tetracycline and fluoroquinolones were higher in isolates from conventional (60.9% and 78.9%) than from organic meat (32.9% and 58.9%, respectively). However, this significant difference was only observed for C. jejuni, but not for C. coli. Further studies are needed to identify the reasons for the differences in the association of production type of turkeys with AMR in the different Campylobacter spp. and the critical parameters for the reduction of AMR in Campylobacter from turkey meat.

Chicken Skin Decontamination of Thermotolerant Campylobacter spp. and Hygiene Indicator Escherichia coli Assessed by Viability Real-Time PCR.

Thermotolerant Campylobacter spp. are fecal contaminants of chicken meat with serious implications for human health. E. coli is considered as hygiene indicator since, in contrast to Campylobacter. spp., the bacterium is generally present in the avian gut. Stress exposure may transiently cease bacterial division. Therefore, colony forming units (CFU) may underestimate the infection risk of pathogens. We developed a viability real-time PCR (v-qPCR) for the quantification of viable E. coli targeting the uidA gene, encoding ß-glucuronidase, which is usually detected for phenotypic species identification. The short- and long-term effects of decontaminating chicken skin on the survival of both C. jejuni and an ESBL-producing E. coli were evaluated by CFU and v-qPCR. The results showed that freezing and storage in cool conditions are potentially underestimated by CFU but not by v-qPCR. The effect of treatment with peroxyacetic acid on survival was consistently detected by CFU and v-qPCR. v-qPCR analysis detected bacterial survival upon the application of lactic acid, which awaits further analysis. Interestingly, both bacteria showed similar kinetics of inactivation upon the application of reduction strategies, suggesting that E. coli might be a complementary hygiene indicator. We conclude that v-qPCR can improve food safety under the consideration of some limitations.

Challenging the "gold standard" of colony-forming units - Validation of a multiplex real-time PCR for quantification of viable Campylobacter spp. in meat rinses.

Campylobacter jejuni is the leading bacterial food-borne pathogen in Europe. Despite the accepted limits of cultural detection of the fastidious bacterium, the "gold standard" in food microbiology is still the determination of colony-forming units (CFU). As an alternative, a live/dead differentiating qPCR has been established, using propidium monoazide (PMA) as DNA-intercalating crosslink agent for inactivating DNA from dead, membrane-compromised cells. The PMA treatment was combined with the addition of an internal sample process control (ISPC), i.e. a known number of dead C. sputorum cells to the samples. The ISPC enables i), monitoring the effective reduction of dead cell signal by the light-activated DNA-intercalating dye PMA, and ii), compensation for potential DNA losses during processing. Here, we optimized the method for routine application and performed a full validation of the method according to ISO 16140-2:2016(E) for the quantification of live thermophilic Campylobacter spp. in meat rinses against the classical enumeration method ISO 10272-2:2017. In order to render the method applicable and cost-effective for practical application, the ISPC was lyophilized to be distributable to routine laboratories. In addition, a triplex qPCR was established to simultaneously quantify thermophilic Campylobacter, the ISPC and an internal amplification control (IAC). Its performance was statistically similar to the two duplex qPCRs up to a contamination level of 4.7 log10Campylobacter per ml of meat rinse. The limit of quantification (LOQ) of the alternative method was around 20 genomic equivalents per PCR reaction, i.e. 2.3 log10 live Campylobacter per ml of sample. The alternative method passed a relative trueness study, confirming the robustness against different meat rinses, and displayed sufficient accuracy within the limits set in ISO 16140-2:2016(E). Finally, the method was validated in an interlaboratory ring trial, confirming that the alternative method was fit for purpose with a tendency of improved repeatability and reproducibility compared to the reference method for CFU determination. Campylobacter served as a model organism, challenging CFU as "gold standard" and could help in guidance to the general acceptance of live/dead differentiating qPCR methods for the detection of food-borne pathogens.