Antibiotic Resistance Genes in the Bacteriophage DNA Fraction of Human Fecal Samples

Abstract

A group of antibiotic resistance genes (ARGs) (bla TEM , bla CTX-M-1 , mecA, armA, qnrA, and qnrS) were analyzed by real-time quantitative PCR (qPCR) in bacteriophage DNA isolated from feces from 80 healthy humans. Seventy-seven percent of the samples were positive in phage DNA for one or more ARGs. bla TEM , qnrA, and, bla CTX-M-1 were the most abundant, and armA, qnrS, and mecA were less prevalent. Free bacteriophages carrying ARGs may contribute to the mobilization of ARGs in intra-and extraintestinal environments.A ntibiotic resistance may be obtained by spontaneous mutations or acquired by the incorporation of antibiotic resistance genes (ARGs) (1). ARGs spread between cells by using genetic platforms known as mobile genetic elements (MGEs). The most commonly studied MGEs are plasmids, transposons, integrons, and, more recently, bacteriophages (2).Bacteriophages or phage-related elements carry ARGs in Gram-positive (3-6) and Gram-negative (7-10) bacteria. Recently, some studies have suggested that the role of phages carrying ARGs in the environment is much more important than previously thought (2, 11-13). Abundant ARGs have been reported in the bacteriophage DNA fraction of fecally contaminated water (14-16), and metagenomic analyses indicate that there are abundant ARGs in viral DNA (17). As a result of their higher incidence in clinical settings, much effort has been devoted to the study of plasmids, integrons, and transposons. However, there is little information on phages carrying ARGs in clinical settings.This study analyzes a group of ARGs in phage DNA isolated from stool samples. The ARGs studied include two groups of beta-lactamase genes from Gram-negative bacteria (bla TEM and bla CTX-M-1 group ); mecA, responsible for resistance to methicillin in Staphylococcus spp.; armA, a gene which confers high-level resistance to aminoglycosides in Gram-negative bacteria; and qnrA and qnrS, plasmid-mediated genes that provide some degree of reduced quinolone susceptibility.The study was performed using 80 human fecal samples from 46 females and 34 males from 6 months to 102 years of age who visited the Sant Pau Hospital (Barcelona, Spain) during a 6-month period. Stool samples were processed according to conventional protocols for the isolation of enteropathogenic bacteria, rotavirus, and adenovirus and were microscopically examined for protozoa. Only samples that were negative for these pathogens were included in the study. None of the patients selected was involved in a food-borne outbreak or showed any severe gastrointestinal pathology. To our knowledge, none of the patients were receiving antibiotic treatment during the time of the study, although previous antibiotic treatments could not be excluded.Fecal samples were homogenized to a 1:5 (wt/vol) dilution in phosphate-buffered saline (PBS) by magnetic stirring for 15 min. Fifty milliliters of the homogenate was centrifuged at 3,000 ϫg, and the phage lysate was purified and concentrated as described previously (15, 16). Phage suspensions were treated wit...

Phages rarely encode antibiotic resistance genes: a cautionary tale for virome analyses

“…β-Lactamases were closely inspected, as several reports have suggested their presence on phages (Quiros et al, 2014;Asare et al, 2015a), sometimes with atypical length. Indeed, one such protein annotated as a β-lactamase is, in fact, a tail fiber protein (Cresawn et al, 2015).…”

Bacteriophage-mediated spread of bacterial virulence genes

“…In the same vein, a longitudinal metagenomic analysis investigating the composition and dynamics of the human gut virome (including eukaryotic viruses and phages) revealed that in addition to complete or partial bacteriophage genomes, most of the DNA contigs encoded gene products of unexpected functions, such as those of viral CRISPR arrays and antibiotic resistances [26]. Finally, analysis of the packaged DNA from the feces of healthy humans revealed the presence of antibiotic resistance genes, bla TEM , bla CTX-M-1 , mecA, armA, qnrA, and qnrS in 77 out 80 samples analysed [27 ]. These findings suggest that phages are involved in the spread of antibiotic resistance and highlight the importance of transduction in the emergence of novel virulent clones.…”

Improving Detection of Shiga Toxin-Producing Escherichia coli by Molecular Methods by Reducing the Interference of Free Shiga Toxin-Encoding Bacteriophages

“…Regarding the implementation of these techniques for routine diagnosis of pathogens, the interference that phages could cause in the assembly of a genome sequence should be considered when DNA is obtained directly from the samples and not from isolates, particularly from the less abundant members of a microbial community (40). For example, virulence genes (stx or others) or antibiotic resistance genes, both reported in phages or phage-like elements (41)(42)(43), will certainly be detected, but the strain or species to which they belong, if any, will be unknown. The reduction of possible interferences would improve the efficiency and reliability of genomics when routinely applied.…”