A Pasteurella multocida genetikai változatossága, a gazdaadaptáció jelei és az antibiotikum rezisztencia terjedése

Abstract

Pasteurella multocida is a globally distributed, Gram-negative bacterial species with zoonotic potential, that has significant economic and public health impacts. Strains of this species show a high genotypic and phenotypic heterogeneity, which contributes to the development of diverse diseases based on different pathomechanism affecting different host species. These infections are typically treated with antibiotics, however, the increasing emergence and spread of multidrug-resistant (MDR) strains is a challenge of increasing importance to both veterinary and human healthcare. Detailed molecular characterization of these strains, along with a deeper understanding of their antibiotic resistance is vital to elucidate the pathogenesis and epidemiological dynamics of infections, to develop effective therapeutic strategies and to mitigate zoonotic risks. Collectively, these insights will facilitate the development of targeted prevention and treatment protocols. The aim of this study was the detailed pheno- and genotypic characterization of P. multocida strains. We aimed to explore and statistically analyse the associations between virulence gene profiles (VGPs), clusters established on the basis of ompA gene sequences, and other characteristics of the strains – including serotype, Multi-host (MH) and the Rural Industries Research and Development Corporation (RIRDC) sequence type (ST), and host origin. Another objective was to assess the antibiotic susceptibility profiles of Hungarian and French P. multocida strains using both phenotypic (disk diffusion, broth microdilution) and genotypic (whole-genome sequencing) approaches. In this study, the antibiotic susceptibility of 80 P. multocida isolates of Hungarian and French origin, collected from various host species between 2004 and 2023, was examined. Particular attention was given to the occurrence of MDR strains, and the heterogeneity of the sample set enabled the identification of resistance-related trends. The strains were tested against antibiotics from ten antibiotic classes: penicillins, cephalosporins, aminoglycosides, tetracyclines, macrolides, fluoroquinolones, lincosamides, phenicols, polymixins and sulfonamides. Antibiotic resistance genes (ARGs) and single-nucleotide polymorphisms (SNPs) were identified using Bacterial and Viral Bioinformatics Resource Center (BV-BRC) and the Comprehensive Antibiotic Resistance Database (CARD) Resistance Gene Identifier (RGI) tool. Plasmid carriage in MDR strains was examined using the Solu server. To increase the reliability of the statistical analyses, detailed genotypic characterization was also performed for an additional 50 P. multocida genomes available in the National Center for Biotechnology Information (NCBI) database. Strain sequences were aligned to different reference genomes, allowing characterization of virulence gene carriage, and further VGP groups were separated. Capsule type and lipopolysaccharide (LPS) genotype were identified using a Danish bioinformatics server, while MH- and 8 RIRDC-STs were determined via the Public databases for molecular typing and microbial genome diversity (PubMLST) database. A phylogenetic tree based on ompA gene sequences revealed twenty-five wellseparated clusters. Based on the analysed virulence gene patterns, fourteen new VGP profiles were identified, thus currently twenty-seven VGP groups are known. The most common capsule-LPS genotype combination (serotype) was A:L3 (n=46), followed by A:L1 (n=23) and F:L3 (n=18). Some strains carried multiple point mutations in the tadD primerbinding region, resulting false-negative polymerase chain reactions (PCR). To overcome this, new primers were design based on whole-genome sequences, enabling reliable diagnostic identification of the gene. Phenotypic antibiotic susceptibility testing indicated that cephalosporins and phenicols were the most effective agents for treating P. multocida infections. In addition, it was observed that the majority of strains showed high susceptibility to fluoroquinolones and tetracyclines. In contrast, high resistance rates were observed against sulfamethoxazole and clindamycin. The most frequently identified resistance genes were strA (n=7), sul2, and tetH (n=6), while none of the strains carried the bla-TEM or erm(42) genes. Based on whole-genome sequencing data and plasmid carriage analysis, none of the isolates harboured integrative-conjugative elements (ICE). Significant associations were observed between the VGP groups of strains, the ompA clusters, and some other characteristics of the isolate, primarily host species, serotype, and sequence type. These relationships contribute to a deeper understanding of the pathogenesis and epidemiological dynamics of the diseases caused by P. multocida. Comparison of the results of antibiotic susceptibility tests revealed consistency between phenotypic susceptibility and the presence of resistance genes in most cases. However, in the case of certain antibiotic classes – sulfonamides, macrolides, and β- lactams – there were discrepancies between the genotypic and phenotypic results. In these cases, the genetic background of the phenotypically observed resistance could not be clearly identified, suggesting that, in addition to the currently known mechanisms, other, as yet undiscovered factors may also play a role in the development and maintenance of resistance