Genetic recombination-mediated evolutionary interactions between phages of potential industrial importance and prophages of their hosts within or across the domains of Escherichia, Listeria, Salmonella, Campylobacter, and Staphylococcus.

BACKGROUND: The in-depth understanding of the role of lateral genetic transfer (LGT) in phage-prophage interactions is essential to rationalizing phage applications for human and animal therapy, as well as for food and environmental safety. This in silico study aimed to detect LGT between phages of potential industrial importance and their hosts. METHODS: A large array of genetic recombination detection algorithms, implemented in SplitsTree and RDP4, was applied to detect LGT between various Escherichia, Listeria, Salmonella, Campylobacter, Staphylococcus, Pseudomonas, and Vibrio phages and their hosts. PHASTER and RAST were employed respectively to identify prophages across the host genome and to annotate LGT-affected genes with unknown functions. PhageAI was used to gain deeper insights into the life cycle history of recombined phages. RESULTS: The split decomposition inferences (bootstrap values: 91.3-100; fit: 91.433-100), coupled with the Phi (0.0-2.836E-12) and RDP4 (P being well below 0.05) statistics, provided strong evidence for LGT between certain Escherichia, Listeria, Salmonella, and Campylobacter virulent phages and prophages of their hosts. The LGT events entailed mainly the phage genes encoding for hypothetical proteins, while some of these genetic loci appeared to have been affected even by intergeneric recombination in specific E. coli and S. enterica virulent phages when interacting with their host prophages. Moreover, it is shown that certain L. monocytogenes virulent phages could serve at least as the donors of the gene loci, involved in encoding for the basal promoter specificity factor, for L. monocytogenes. In contrast, the large genetic clusters were determined to have been simultaneously exchanged by many S. aureus prophages and some Staphylococcus temperate phages proposed earlier as potential therapeutic candidates (in their native or modified state). The above genetic clusters were found to encompass multiple genes encoding for various proteins, such as e.g., phage tail proteins, the capsid and scaffold proteins, holins, and transcriptional terminator proteins. CONCLUSIONS: It is suggested that phage-prophage interactions, mediated by LGT (including intergeneric recombination), can have a far-reaching impact on the co-evolutionary trajectories of industrial phages and their hosts especially when excessively present across microbially rich environments.

Comparison of Antimicrobial Susceptibility Profiles of Thermotolerant Campylobacter spp. Isolated from Human and Poultry Samples in Georgia (Caucasus).

Antimicrobial resistance remains a public health concern globally. This study presents antimicrobial resistance by microdilution and genetic diversity by the whole-genome sequencing of Campylobacter spp. from human and poultry samples isolated in Georgia in 2020/2021. The major species in poultry samples was C. coli, while C. jejuni was preferentially isolated from human samples. Resistance against tetracycline was highest (100%) in C. coli from industrial chicken and lowest in C. jejuni from clinical isolates (36%), while resistance against ciprofloxacin varied from 80% in C. jejuni from backyard chicken to 100% in C. jejuni and C. coli from industrial chicken. The point mutations in gyrA (T86I) and tet (O) genes were detected as resistance determinants for (fluoro-)quinolone or tetracycline resistance, respectively. Ertapenem resistance is still enigmatic. All isolates displayed sensitivity towards erythromycin, gentamicin and chloramphenicol. Multi-resistance was more frequently observed in C. coli than in C. jejuni, irrespective of the isolation matrix, and in chicken isolates compared to human isolates, independent of the Campylobacter species. The Georgian strains showed high variability of multi-locus sequence types (ST), including novel STs. This study provides the first antibiotic resistance data from Campylobacter spp. in Georgia and addresses the need for follow-up monitoring programs.

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.

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.

Risk-benefit in food safety and nutrition - Outcome of the 2019 Parma Summer School.

Risk-benefit assessment is the comparison of the risk of a situation to its related benefits, i.e. a comparison of scenarios estimating the overall health impact. The risk-benefit analysis paradigm mirrors the classical risk analysis one: risk-benefit assessment goes hand-in-hand with risk-benefit management and risk-benefit communication. The various health effects associated with food consumption, together with the increasing demand for advice on healthy and safe diets, have led to the development of different research disciplines in food safety and nutrition. In this sense, there is a clear need for a holistic approach, including and comparing all of the relevant health risks and benefits. The risk-benefit assessment of foods is a valuable approach to estimate the overall impact of food on health. It aims to assess together the negative and positive health effects associated with food intake by integrating chemical and microbiological risk assessment with risk and benefit assessment in food safety and nutrition. The 2019 Parma Summer School on risk-benefit in food safety and nutrition had the objective was to provide an opportunity to learn from experts in the field of risk-benefit approach in food safety and nutrition, including theory, case studies, and communication of risk-benefit assessments plus identify challenges for the future. It was evident that whereas tools and approaches have been developed, more and more case studies have been performed which can form an inherent validation of the risk-benefit approach. Executed risk-benefit assessment case studies apply the steps and characteristics developed: a problem formulation (with at least 2 scenarios), a tiered approach until a decision can be made, one common currency to describe both beneficial and adverse effects (DALYs in most instances). It was concluded that risk-benefit assessment in food safety and nutrition is gaining more and more momentum, while also many challenges remain for the future. Risk-benefit is on the verge of really enrolling into the risk assessment and risk analysis paradigm. The interaction between risk-benefit assessors and risk-benefit managers is pivotal in this, as is the interaction with risk-benefit communicators.

Bi- and Multi-directional Gene Transfer in the Natural Populations of Polyvalent Bacteriophages, and Their Host Species Spectrum Representing Foodborne Versus Other Human and/or Animal Pathogens.

Unraveling the trends of phage-host versus phage-phage coevolution is critical for avoiding possible undesirable outcomes from the use of phage preparations intended for therapeutic, food safety or environmental safety purposes. We aimed to investigate a phenomenon of intergeneric recombination and its trajectories across the natural populations of phages predominantly linked to foodborne pathogens. The results from the recombination analyses, using a large array of the recombination detection algorithms imbedded in SplitsTree, RDP4, and Simplot software packages, provided strong evidence (fit: 100; P ≤ 0.014) for both bi- and multi-directional intergeneric recombination of the genetic loci involved collectively in phage morphogenesis, host specificity, virulence, replication, and persistence. Intergeneric recombination was determined to occur not only among conspecifics of the virulent versus temperate phages but also between the phages with these different lifestyles. The recombining polyvalent phages were suggested to interact with fairly large host species networks, including sometimes genetically very distinct species, such as e.g., Salmonella enterica and/or Escherichia coli versus Staphylococcus aureus or Yersinia pestis. Further studies are needed to understand whether phage-driven intergeneric recombination can lead to undesirable changes of intestinal and other microbiota in humans and animals.

Phage Transduction is Involved in the Intergeneric Spread of Antibiotic Resistance-Associated blaCTX-M, mel, and tetM Loci in Natural Populations of Some Human and Animal Bacterial Pathogens.

The horizontal genetic transfer (HGT) of antibiotic resistance genes (ARGs) mediated by species-specific bacteriophages contributes to the emergence of antibiotic-resistant strains in natural populations of human and animal bacterial pathogens posing a significant threat to global public health. However, it is unclear and needs to be determined whether polyvalent bacteriophages play any role in the intergeneric transmission of ARGs. In this study, we examined the genome sequences of 2239 bacteriophages from different sources for the presence of ARGs. The identified ARG-carrying bacteriophages were then analyzed by PHACTS, PHAST, and HostPhinder programs to determine their lifestyles, genes coding for bacterial cell lysis, recombinases, and a spectrum of their potential host species, respectively. We employed the SplitsTree, RDP4 and SimPlot software packages in recombination tests to identify HGT events of ARGs between these bacteriophages and bacteria. In our analyses, some ARG-carrying bacteriophages exhibited temperate and/or polyvalent patterns. The bootstrap values (97-100) for the SplitsTree-generated parallelograms, fit values (97-100) for splits networks, Phi P values (< 10-17 to 3.9 × 10-16), RDP4 P values (≤ 7.8 × 10-03), and the SimPlot results, provided strong statistical evidence for the phage transduction events of blaCTX-M, mel, and tetM loci on inter-species level. These events involved several host species such as Escherichia coli, Salmonella enterica, Shigella sonnei, Streptococcus pneumoniae and Bacillus coagulans. HGT of mel loci between Erysipelothrix and Streptococcus phages were also detected. These results firmly suggest that certain bacteriophages possibly with temperate properties induce the intergeneric dissemination of blaCTX-M, mel and tetM in the above species.