Journal Article

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

Knüver, Marie-Theres, Bretschneider, Nancy, Deneke, Carlus, Hoang, Ha Thi Thu, Huber, Ingrid, Stingl, Kerstin, Thieck, Maja, Pham, Ngoc Thi, Luu, Huong Quynh, Zarske, Michael

2024

Overview

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), bla OXA , 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.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Acid resistance / AMR / Analysis / Animal Genetics and Genomics / Antibiotic resistance / Antibiotics / Antimicrobial agents / Bacteria / Biomedical and Life Sciences / Campylobacter / Chloramphenicol / Chloromycetin / Chromosomes / Ciprofloxacin / DNA sequencing / Drug resistance in microorganisms / Ethylenediaminetetraacetic acid / Food / Food contamination & poisoning / Food safety / Gastroenteritis / Gene duplication / Gene mutations / Gene sequencing / Genes / Genetic aspects / Genetic variation / Genomes / Genomics / Gentamicin / Guillain-Barre syndrome / Infections / Kanamycin / Laboratories / Life Sciences / Lincomycin / Long-read sequencing / Methods / Microarrays / Microbial Genetics and Genomics / Microbiological research / Mosaicism / Multidrug resistance / Multidrug resistance islands / Multidrug resistant organisms / Mutation / Nalidixic acid / Neomycin / NGS / Nucleotide sequence / Nucleotide sequencing / Oxytetracycline / Pathogens / Phenotypes / Plant Genetics and Genomics / Plasmids / Poultry / Proteomics / Public health / Quinolones / RNA / rRNA / rRNA 23S / Safety and security measures / Scientific equipment and supplies industry / Strains (organisms) / Streptomycin / Susceptibility testing / Transposase / Transposition / Whole genome sequencing / Zoonoses