Microbial ecology of antibiotic resistance genes: a one health perspective

This chapter starts with a brief history of the growing public interest in the ecology of resistance genes and then moves on to a survey of some of the conceptual problems that have emerged. It focuses on a few groups of bacteria that are major players in the oral and intestinal ecosystems of humans and animals, the obligate anaerobes. Perhaps the first modern example of the sudden importance of understanding the ecology of antibiotic resistance genes arose in connection with the debate over the safety of GM plants. Studies have shown that many soil bacteria are naturally transformable, although it is still unclear what significance this fact has in the overall ecology of antibiotic resistance genes. Bacteria in and on the human body have participated in the distribution of resistance genes. An interesting case in point is the population of bacteria that makes its home in the periodontal pocket, the region between the gums and the roots of the teeth. Prominent among these are the Porphyromonas and Prevotella species. These bacteria have been of particular interest in dentistry because they are thought to be instrumental in the development of periodontal disease, the main cause of tooth loss in adults. Currently, the treatment for periodontal disease is surgery that cuts into the gums, exposing the buried surface of the teeth to allow scraping of the plaque that has accumulated there and is causing inflammation.

Genital tract pathogens resistant to antibiotics are now commonly encountered in many parts of the world. The emergence of such strains may clearly be accounted for, at least in part, by the selection of mutant strains which would have a selective advantage in the face of widespread antibiotic usage. Molecular and genetic analysis of antibioticresistant organisms, however, indicates that such an explanation is an over simplification. In many instances genes coding for antibiotic resistance appear highly mobile, being capable of transfer, not only between strains of the same species, but between organisms of differing species or genus. Mechanisms of gene transfer which have been demonstrated in vitro include transformation and conjugal transfer of genes located on plasmids. In addition, antibiotic resistance genes located on transposons may be transferred between DNA molecules (e.g. between plasmid and chromosomal DNA). Genetic analysis indicates, moreover, that some antibiotic resistance genes found in genital tract pathogens may have been acquired from enteric bacteria, or bacteria commonly found in the respiratory tract. These observations clearly demonstrate that the pathogenic flora of the human genital tract comprises a highly complex molecular ecosystem. Keywords: Genital tract; Pathogens; Antibiotic resistance

Antibiotic resistance represents a global health problem, requiring better understanding of the ecology of antibiotic resistance genes (ARGs), their selection and their spread in the environment. Antibiotics are constantly released to the environment through wastewater treatment plant (WWTP) effluents. We investigated, therefore, the effect of these discharges on the prevalence of ARGs and bacterial community composition in biofilm and sediment samples of a receiving river. We used culture-independent approaches such as quantitative PCR to determine the prevalence of eleven ARGs and 16S rRNA gene-based pyrosequencing to examine the composition of bacterial communities. Concentration of antibiotics in WWTP influent and effluent were also determined. ARGs such as qnrS, bla TEM, bla CTX-M, bla SHV, erm(B), sul(I), sul(II), tet(O) and tet(W) were detected in all biofilm and sediment samples analyzed. Moreover, we observed a significant increase in the relative abundance of ARGs in biofilm samples collected downstream of the WWTP discharge. We also found significant differences with respect to community structure and composition between upstream and downstream samples. Therefore, our results indicate that WWTP discharges may contribute to the spread of ARGs into the environment and may also impact on the bacterial communities of the receiving river.

Antibiotic-resistant pathogenic bacteria pose a high threat to human health, but the environmental reservoirs of resistance genes are poorly understood. The origins of antibiotic resistance in the environment are relevant to human health because of the increasing importance of zoonotic diseases as well as the requirement for predicting emerging resistant pathogens. Only little is known about the antibiotic resistomes of the great majority of environmental bacteria, although there have been calls for a greater understanding of the environmental reservoirs of antibiotic resistance. The data on antibiotic resistance before the antibiotic era and in soil show how far away we are from a complete picture about the ecology of antibiotic resistance genes (ARGs). Most of the natural antibiotic producers reside in soil, but soil is a particularly challenging habitat due to its chemical and physical heterogeneity. The prevalence and diversity of ARGs in the environment led to hypotheses about the native roles of resistance genes in natural microbial communities.

A new perspective on the topic of antibiotic resistance is beginning to emerge based on a broader evolutionary and ecological understanding rather than from the traditional boundaries of clinical research of antibiotic-resistant bacterial pathogens. Phylogenetic insights into the evolution and diversity of several antibiotic resistance genes suggest that at least some of these genes have a long evolutionary history of diversification that began well before the 'antibiotic era'. Besides, there is no indication that lateral gene transfer from antibiotic-producing bacteria has played any significant role in shaping the pool of antibiotic resistance genes in clinically relevant and commensal bacteria. Most likely, the primary antibiotic resistance gene pool originated and diversified within the environmental bacterial communities, from which the genes were mobilized and penetrated into taxonomically and ecologically distant bacterial populations, including pathogens. Dissemination and penetration of antibiotic resistance genes from antibiotic producers were less significant and essentially limited to other high G+C bacteria. Besides direct selection by antibiotics, there is a number of other factors that may contribute to dissemination and maintenance of antibiotic resistance genes in bacterial populations.

Faculty Opinions recommendation of Ecology of antibiotic resistance genes: characterization of enterococci from houseflies collected in food settings.

Genital tract pathogens resistant to antibiotics are now commonly encountered in many parts of the world. The emergence of such strains may clearly be accounted for, at least in part, by the selection of mutant strains which would have a selective advantage in the face of widespread antibiotic usage. Molecular and genetic analysis of antibiotic-resistant organisms, however, indicates that such an explanation is an over simplification. In many instances genes coding for antibiotic resistance appear highly mobile, being capable of transfer, not only between strains of the same species, but between organisms of differing species or genus. Mechanisms of gene transfer which have been demonstrated in vitro include transformation and conjugal transfer of genes located on plasmids. In addition, antibiotic resistance genes located on transposons may be transferred between DNA molecules (e.g. between plasmid and chromosomal DNA). Genetic analysis indicates, moreover, that some antibiotic resistance genes found in genital tract pathogens may have been acquired from enteric bacteria, or bacteria commonly found in the respiratory tract. These observations clearly demonstrate that the pathogenic flora of the human genital tract comprises a highly complex molecular ecosystem.

Faculty Opinions recommendation of Ecology of antibiotic resistance genes: characterization of enterococci from houseflies collected in food settings.

In this project, enterococci from the digestive tracts of 260 houseflies (Musca domestica L.) collected from five restaurants were characterized. Houseflies frequently (97% of the flies were positive) carried enterococci (mean, 3.1 x 10(3) CFU/fly). Using multiplex PCR, 205 of 355 randomly selected enterococcal isolates were identified and characterized. The majority of these isolates were Enterococcus faecalis (88.2%); in addition, 6.8% were E. faecium, and 4.9% were E. casseliflavus. E. faecalis isolates were phenotypically resistant to tetracycline (66.3%), erythromycin (23.8%), streptomycin (11.6%), ciprofloxacin (9.9%), and kanamycin (8.3%). Tetracycline resistance in E. faecalis was encoded by tet(M) (65.8%), tet(O) (1.7%), and tet(W) (0.8%). The majority (78.3%) of the erythromycin-resistant E. faecalis isolates carried erm(B). The conjugative transposon Tn916 and members of the Tn916/Tn1545 family were detected in 30.2% and 34.6% of the identified isolates, respectively. E. faecalis carried virulence genes, including a gelatinase gene (gelE; 70.7%), an aggregation substance gene (asa1; 33.2%), an enterococcus surface protein gene (esp; 8.8%), and a cytolysin gene (cylA; 8.8%). Phenotypic assays showed that 91.4% of the isolates with the gelE gene were gelatinolytic and that 46.7% of the isolates with the asa1 gene aggregated. All isolates with the cylA gene were hemolytic on human blood. This study showed that houseflies in food-handling and -serving facilities carry antibiotic-resistant and potentially virulent enterococci that have the capacity for horizontal transfer of antibiotic resistance genes to other bacteria.

Ecology of Antibiotic Resistance Genes

Antibiotic resistance in the soil ecosystem: A One Health perspective

Streptomycetes are special: arcane applications

Cyanobacteria are ubiquitous in freshwater environments, but their role in aquatic resistome remains unclear. In this work, we performed whole genome sequencing on 43 cyanobacterial strains isolated from Portuguese fresh/wastewaters. From 43 available non-axenic unicyanoabacterial cultures (containing only one cyanobacterial strain and their co-occurring bacteria), it was possible to recover 41 cyanobacterial genomes from the genomic assemblies using a genome binning software, 26 of which were classified as high-quality based on completeness, contamination, N50 and contig number thresholds. By using the comprehensive antibiotic resistance database (CARD) on the assembled samples, we detected four antibiotic resistance gene (ARG) variants, conferring resistance in pathogenic bacteria to tetracyclines, fluoroquinolones (adeF-type) and macrolides (ermF-type, mefC-type and mphG-type). Among these, adeF-type was the most prevalent gene, found across 11 cyanobacterial genomes from the Nostocales order. Planktothrix presented the highest variety of close ARG matches, with hits for the macrolide resistance genes ermF-type, mefC-type and mphG-type. An analysis of the genomic assemblies also revealed an additional 12 ARGs in bacteria from the phyla Firmicutes, Proteobacteria and Bacteroidetes, present in the cyanobacterial cultures, foreseeing the horizontal gene transfer of ARGs with cyanobacteria. Additionally, more than 200 partial ARGs were detected on each recovered cyanobacterial genome, allowing for future studies of antibiotic resistance genotype/phenotype in cyanobacteria. These findings highlight the importance of further efforts to understand the role of cyanobacteria on the aquatic resistome from a One Health perspective.

Antimicrobial resistance is a global health concern, which is partly driven by rising meat consumption, which has led to the intensive farming of livestock that relies on antibiotics. ready-to-eat animal products can carry antibiotic-resistant bacteria, posing risks to humans since they are often consumed without further cooking. While countries such as Switzerland limit antibiotic use in agriculture, contamination of meat with antibiotic-resistant bacteria can still occur during meat processing, and non-antibiotic agents such as heavy metals may contribute to the co-selection of resistance. This study aimed to characterize antibiotic-resistant bacteria in ready-to-eat meat products from various Swiss butcheries. Presumptive resistant bacteria were isolated using selective plating and analyzed phenotypically and genotypically. A total of 53 bacteria-antibiotic resistance combinations were identified, including Enterobacterales resistant to third-generation cephalosporins, vancomycin-resistant Enterococci, and one strain of methicillin-resistant Staphylococcus aureus. Of the 804 products sampled, 177 antibiotic-resistant bacteria were isolated, 148 of which showed multidrug resistance. Notably, these strains remained susceptible to last-resort antibiotics such as carbapenems and colistin. Whole-genome sequencing of 31 selected isolates revealed 164 antibiotic resistance genes spanning 25 classes, confirming resistance to beta-lactams, cephalosporins, and tetracyclines. We also detected genes conferring resistance to metals, suggesting co-selection pressures. Long-read sequencing revealed that the majority of the antibiotic resistance genes were chromosomal, while others were plasmid-encoded, indicating the potential for horizontal gene transfer. This study demonstrates that ready-to-eat meat products are reservoirs of antibiotic and metal resistance genes, as well as antibiotic-resistant bacteria, even at low levels. From a One Health perspective, our results highlight the importance of extending AMR surveillance across the food chain and underscore the need to include non-traditional bacterial indicators.

Spatiotemporal dynamics of the resistome and virulome of riverine microbiomes disturbed by a mining mud tsunami