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...

In this review we highlight recent work that has increased our understanding of the distribution of Shiga toxin-converting phages that can be detected as free phage particles, independently of Shiga toxin-producing bacteria (STEC). Stx phages are a quite diverse group of temperate phages that can be found in their prophage state inserted within the STEC chromosome, but can also be found as phages released from the cell after activation of their lytic cycle. They have been detected in extraintestinal environments such as water polluted with feces from humans or animals, food samples or even in stool samples of healthy individuals. The high persistence of phages to several inactivation conditions makes them suitable candidates for the successful mobilization of stx genes, possibly resulting in the genes reaching a new bacterial genomic background by means of transduction, where ultimately they may be expressed, leading to Stx production. Besides the obvious fact that Stx phages circulating between bacteria can be, and probably are, involved in the emergence of new STEC strains, we review here other possible ways in which free Stx phages could interfere with the detection of STEC in a given sample by current laboratory methods and how to avoid such interference.

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...

In Shiga toxin-producing Escherichia coli (STEC), induction of Shiga toxin-encoding bacteriophages (Stx phages) causes the release of free phages that can later be found in the environment. The ability of Stx phages to survive different inactivation conditions determines their prevalence in the environment, the risk of stx transduction, and the generation of new STEC strains. We evaluated the infectivity and genomes of two Stx phages (⌽534 and ⌽557) under different conditions. Infectious Stx phages were stable at 4, 22, and 37°C and at pH 7 and 9 after 1 month of storage but were completely inactivated at pH 3. Infective Stx phages decreased moderately when treated with UV (2.2-log 10 reduction for an estimated UV dose of 178.2 mJ/cm 2 ) or after treatment at 60 and 68°C for 60 min (2.2-and 2.5-log 10 reductions, respectively) and were highly inactivated (3 log 10 ) by 10 ppm of chlorine in 1 min. Assays in a mesocosm showed lower inactivation of all microorganisms in winter than in summer. The number of Stx phage genomes did not decrease significantly in most cases, and STEC inactivation was higher than phage inactivation under all conditions. Moreover, Stx phages retained the ability to lysogenize E. coli after some of the treatments. Shiga toxin-producing Escherichia coli (STEC) strains are pathogenic and cause a wide range of diseases, with symptoms varying from noncomplicated diarrhea to the life-threatening hemolytic-uremic syndrome (HUS) (1, 2). STEC produces two immunologically distinct toxins known as Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2), and both toxins present diverse variants (1).The genes encoding Stx in E. coli are located in the genomes of inducible temperate bacteriophages (Stx phages) (3). Induction of the lytic cycle of Stx phages causes an increase in production of Shiga toxin, which is the virulence factor responsible for severe complications associated with the infection, such as HUS (2, 4). In addition to the increase in Stx expression, the lysis of the cell caused by Stx phages allows the release of Stx outside the cell and also the dissemination of Stx phages. Free Stx phages spread within the gut and are excreted with the feces (5). In terms of occurrence in the environment, Stx phages have been found in water bodies containing fecal contamination of human or animal origin (6-12). Infectious Stx phages have also been detected in food samples, and despite the abundance of Stx phages in these samples, they showed levels of bacterial indicators (aerobic colony counts and E. coli) that make them acceptable for consumption according to European regulations (13).The widespread distribution of Stx phages in different environments indicates that they must be able to persist under diverse conditions. Previous studies suggested the persistence of environmental Stx phages (7). Newly developed molecular methods for the quantification of Stx phages (14), the development of new approaches for the optimal detection of plaques formed by infectious Stx phages (15), and optimized protocols for th...