Bacteriophage-Mediated Risk Pathways Underlying the Emergence of Antimicrobial Resistance via Intrageneric and Intergeneric Recombination of Antibiotic Efflux Genes Across Natural populations of Human Pathogenic Bacteria.

Antimicrobial resistance continues to be a significant and growing threat to global public health, being driven by the emerging drug-resistant and multidrug-resistant strains of human and animal bacterial pathogens. While bacteriophages are generally known to be one of the vehicles of antibiotic resistance genes (ARGs), it remains largely unclear how these organisms contribute to the dissemination of the genetic loci encoding for antibiotic efflux pumps, especially those that confer multidrug resistance, in bacteria. In this study, the in-silico recombination analyses provided strong statistical evidence for bacteriophage-mediated intra-species recombination of ARGs, encoding mainly for the antibiotic efflux proteins from the MF superfamily, as well as from the ABC and RND families, in Salmonella enterica, Staphylococcus aureus, Staphylococcus suis, Pseudomonas aeruginosa, and Burkholderia pseudomallei. Events of bacteriophage-driven intrageneric recombination of some of these genes could be also elucidated among Bacillus thuringiensis, Bacillus cereus and Bacillus tropicus natural populations. Moreover, we could also reveal the patterns of intergeneric recombination, involving the MF superfamily transporter-encoding genetic loci, induced by a Mycobacterium smegmatis phage, in natural populations of Streptomyces harbinensis and Streptomyces chartreusis. The SplitsTree- (fit: 100; bootstrap values: 92.7-100; Phi p ≤ 0.2414), RDP4- (p ≤ 0.0361), and GARD-generated data strongly supported the above genetic recombination inferences in these in-silico analyses. Thus, based on this pilot study, it can be suggested that the above mode of bacteriophage-mediated recombination plays at least some role in the emergence and transmission of multidrug resistance across a fairly broad spectrum of bacterial species and genera including human pathogens.

Metagenomic and Recombination Analyses of Antimicrobial Resistance Genes from Recreational Waters of Black Sea Coastal Areas and Other Marine Environments Unveil Extensive Evidence for Their both Intrageneric and Intergeneric Transmission across Genetically Very Diverse Microbial Communities.

Microbial communities of marine coastal recreation waters have become large reservoirs of AMR genes (ARGs), contributing to the emergence and transmission of various zoonotic, foodborne and other infections that exhibit resistance to various antibiotics. Thus, it is highly imperative to determine ARGs assemblages as well as mechanisms and trajectories of their transmission across these microbial communities for our better understanding of the evolutionary trends of AMR (AMR). In this study, using metagenomics approaches, we screened for ARGs in recreation waters of the Black Sea coastal areas of the Batumi City (Georgia). Also, a large array of the recombination detection algorithms of the SplitsTree, RDP4, and GARD was applied to elucidate genetic recombination of ARGs and trajectories of their transmission across various marine microbial communities. The metagenomics analyses of sea water samples, obtained from across the above marine sites, could identify putative ARGs encoding for multidrug resistance efflux transporters mainly from the Major Facilitator and Resistance Nodulation Division superfamilies. The data, generated by SplitsTree (fit ≥95.619; bootstrap values ≥ 95; Phi p ≤ 0.0494), RDP4 (p ≤ 0.0490), and GARD, provided strong statistical evidence not only for intrageneric recombination of these ARGs, but also for their intergeneric recombination across fairly large and diverse microbial communities of marine environment. These bacteria included both human pathogenic and nonpathogenic species, exhibiting collectively the genera of Vibrio, Aeromonas, Synechococcus, Citromicrobium, Rhodobacteraceae, Pseudoalteromonas, Altererythrobacter, Erythrobacter, Altererythrobacter, Marivivens, Xuhuaishuia, and Loktanella. The above nonpathogenic bacteria are strongly suggested to contribute to ARGs transmission in marine ecosystems.

In-silico analyses provide strong statistical evidence for intra-species recombination events of the gyrA and CmeABC operon loci contributing to the continued emergence of resistance to fluoroquinolones in natural populations of Campylobacter jejuni.

OBJECTIVES: The continued emergence of Campylobacter jejuni strains resistant to fluoroquinolones (FQs) has posed a significant threat to global public health, leading frequently to undesirable outcomes of human campylobacteriosis treatment. The molecular genetic mechanisms contributing to the increased retention of resistance to FQs in natural populations of this species, especially in antibiotic-free environments, are not clearly understood. This study aimed to determine whether genetic recombination could be such a mechanism. METHODS: We applied a large array of algorithms, imbedded in the SplitsTree and RDP4 software packages, to analyse the DNA sequences of the chromosomal loci, including the gyrA gene and the CmeABC operon, to identify events of their genetic recombination between C. jejuni strains. RESULTS: The SplitsTree analyses of the above genetic loci resulted in several parallelograms with the bootstrap values being in a range of 94.7 to 100, with the high fit estimates being 99.3 to 100. These analyses were further strongly supported by the Phi test results (P ≤ 0.02715) and the RDP4-generated statistics (P ≤ 0.04005). The recombined chromosomal regions, along with the gyrA gene and CmeABC operon loci, were also found to contain the genetic loci that included, but were not limited to, the genes encoding for phosphoribosyltransferase, lipoprotein, outer membrane motility protein, and radical SAM domain protein. CONCLUSION: These findings strongly suggest that the genetic recombination of the chromosomal regions involving gyrA, CmeABC, and their adjacent loci may be an additional mechanism underlying the constant emergence of epidemiologically successful FQ-resistant strains in natural populations of C. jejuni.