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...

Bacteriophages can package part of their host’s genetic material, including antibiotic resistance genes (ARGs), contributing to a rapid dissemination of resistances among bacteria. Phage particles containing ARGs were evaluated in meat, pork, beef and chicken minced meat, and ham and mortadella, purchased in local retailer. Ten ARGs (blaTEM, blaCTX-M-1, blaCTX-M-9, blaOXA-48, blaVIM, qnrA, qnrS, mecA, armA and sul1) were analyzed by qPCR in the phage DNA fraction. The genes were quantified, before and after propagation experiments in Escherichia coli, to evaluate the ability of ARG-carrying phage particles to infect and propagate in a bacterial host. According to microbiological parameters, all samples were acceptable for consumption. ARGs were detected in most of the samples after particle propagation indicating that at least part of the isolated phage particles were infectious, being sul1the most abundant ARG in all the matrices followed by β-lactamase genes. ARGs were also found in the phage DNA fraction of thirty-seven archive chicken cecal samples, confirming chicken fecal microbiota as an important ARG reservoir and the plausible origin of the particles found in meat. Phages are vehicles for gene transmission in meat that should not be underestimated as a risk factor in the global crisis of antibiotic resistance.

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b Shiga toxin-converting bacteriophages (Stx phages) carry the stx gene and convert nonpathogenic bacterial strains into Shiga toxin-producing bacteria. Previous studies have shown that high densities of free and infectious Stx phages are found in environments polluted with feces and also in food samples. Taken together, these two findings suggest that Stx phages could be excreted through feces, but this has not been tested to date. In this study, we purified Stx phages from 100 fecal samples from 100 healthy individuals showing no enteric symptoms. The phages retrieved from each sample were then quantified by quantitative PCR (qPCR). In total, 62% of the samples carried Stx phages, with an average value of 2.6 ؋ 10 4 Stx phages/g. This result confirms the excretion of free Stx phages by healthy humans. Moreover, the Stx phages from feces were able to propagate in enrichment cultures of stx-negative Escherichia coli (strains C600 and O157:H7) and in Shigella sonnei, indicating that at least a fraction of the Stx phages present were infective. Plaque blot hybridization revealed lysis by Stx phages from feces. Our results confirm the presence of infectious free Stx phages in feces from healthy persons, possibly explaining the environmental prevalence observed in previous studies. It cannot be ruled out, therefore, that some positive stx results obtained during the molecular diagnosis of Shiga toxin-producing Escherichia coli (STEC)-related diseases using stool samples are due to the presence of Stx phages.

Detection of Shiga toxin-producing Escherichia coli (STEC) by culture methods is advisable to identify the pathogen, but recovery of the strain responsible for the disease is not always possible. The use of DNA-based methods (PCR, quantitative PCR [qPCR], or genomics) targeting virulence genes offers fast and robust alternatives. However, detection of stx is not always indicative of STEC because stx can be located in the genome of temperate phages found in the samples as free particles; this could explain the numerous reports of positive stx detection without successful STEC isolation. An approach based on filtration through low-protein-binding membranes and additional washing steps was applied to reduce free Stx phages without reducing detection of STEC bacteria. River water, food, and stool samples were spiked with suspensions of phage 933W and, as a STEC surrogate, a lysogen harboring a recombinant Stx phage in which stx was replaced by gfp. Bacteria were tested either by culture or by qPCR for gfp while phages were tested using qPCR targeting stx in phage DNA. The procedure reduces phage particles by 3.3 log 10 units without affecting the recovery of the STEC population (culturable or assessed by qPCR). The method is applicable regardless of phage and bacteria densities and is useful in different matrices (liquid or solid). This approach eliminates or considerably reduces the interference of Stx phages in the detection of STEC by molecular methods. The reduction of possible interference would increase the efficiency and reliability of genomics for STEC detection when the method is applied routinely in diagnosis and food analysis. Shiga toxin-producing Escherichia coli (STEC) bacteria are a class of enteric pathogens capable of causing severe gastrointestinal disease (hemorrhagic colitis) that can develop undesirable complications, such as acute kidney failure (hemolytic-uremic syndrome [HUS]) that could require lifelong treatment (1, 2). STEC strains belonging to different serotypes, notably strains of serotype O157:H7, have been the causative agents of large outbreaks of food-borne disease. However, the emergence of non-O157 STEC strains with various combinations of virulence genes also represents a serious challenge for the protection of consumers from food-borne disease (3, 4).The identification of STEC strains, which requires culture enrichment on selective medium, is advisable to confirm and characterize the pathogen. However, recovery of the strain responsible for the disease is not always possible because it could be present in low concentrations, the cells might not be in a culturable state, or there could be interference from commensal E. coli within the microbiota in the sample. The need for early identification of STEC demands the use of faster and more robust methods.STEC strains present genomic plasticity that complicates discrimination of the pathogenic strains among other E. coli strains present in a sample. For this reason, DNA and protein detection methods have been developed to target the g...