Pablo Quirós

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

ABSTRACT A group of antibiotic resistance genes (ARGs) (blaTEM, blaCTX-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. blaTEM, qnrA, and, blaCTX-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.

Shiga toxin 2-encoding bacteriophages in human fecal samples from healthy individuals.

ABSTRACT 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 × 104 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.

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

ABSTRACT 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 log10 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.

Free Shiga toxin 1-encoding bacteriophages are less prevalent than Shiga toxin 2 phages in extraintestinal environments.

Stx bacteriophages are involved in the pathogenicity of Stx-producing Escherichia coli. Induction of the Stx phage lytic cycle increases Stx expression and releases Stx phages that reach extracellular environments. Stx phage family comprises different phages that harbour any stx subtype. Stx2 is closely related with severe disease and therefore previous studies focused on free Stx2 phages in extraintestinal environments. To provide similar information regarding Stx1 phages, we evaluate free Stx1 phages in 357 samples of human and animal wastewater, faeces, river water, soil, sludge and food. Our method, based on quantification of stx1 in the DNA from the viral fraction, was validated using electron microscopy counting of phages and infectivity. The overall prevalence of Stx1 phages was very low: 7.6% of positive samples and values below 3 × 10(3) GC (gene copies) ml(-1) . These results contrast starkly with the abundance of Stx2 phages in the samples (68.4%). This environmental scarcity of free Stx1 phages is attributed to their lower rates of induction and the fact that Stx1 does not require phage induction to be expressed because it possesses an independent promoter. The implications of the low prevalence of free Stx1 phages for the emergence of new pathogenic strains in the environment are discussed.

Spread of bacterial genomes in packaged particles

Department of Microbiology, University of Barcelona, Diagonal 645, Annex, Floor 0, 08028 Barcelona, Spain *Author for correspondence: Tel.: +34 93 403 9386; Fax: +34 93 403 9047; mmuniesa@ub.edu

Contribution of cropland to the spread of Shiga toxin phages and the emergence of new Shiga toxin-producing strains

A growing interest in healthy eating has lead to an increase in the consumption of vegetables, associated with a rising number of bacterial outbreaks related to fresh produce. This is the case of the outbreak in Germany, caused by a O104:H4 enteroaggregative E. coli strain lysogenic for a Stx phage. Temperate Stx phages released from their hosts occur as free particles in various environments. This study reports the occurrence of Stx phages in vegetables (lettuce, cucumber, and spinach) and cropland soil samples. Infectious Stx2 phages were found in all samples and many carried also Stx1 phages. Their persistence in vegetables, including germinated sprouts, of Stx phage 933 W and an E. coli C600 (933 W∆stx::gfp-cat) lysogen used as surrogate, showed reductions below 2 log10 units of both microorganisms at 23 °C and 4 °C over 10 days. Higher reductions (up to 3.9 log10) units were observed in cropland soils at both temperatures. Transduction of a recombinant 933 W∆stx::kan phage was observed in all matrices. Protecting against microbial contamination of vegetables is imperative to ensure a safe food chain. Since the emergence of new Stx strains by Stx phage transduction is possible in vegetable matrices, methods aimed at reducing microbial risks in vegetables should not neglect phages.

Closed Genome and Comparative Phylogenetic Analysis of the Clinical Multidrug Resistant Shigella sonnei Strain 866

Abstract Shigella sonnei is responsible for the majority of shigellosis infections in the US with over 500,000 cases reported annually. Here, we present the complete genome of the clinical multidrug resistant (MDR) strain 866, which is highly susceptible to bacteriophage infections. The strain has a circular chromosome of 4.85 Mb and carries a 113 kb MDR plasmid. This IncB/O/K/Z-type plasmid, termed p866, confers resistance to five different classes of antibiotics including ß-lactamase, sulfonamide, tetracycline, aminoglycoside, and trimethoprim. Comparative analysis of the plasmid architecture and gene inventory revealed that p866 shares its plasmid backbone with previously described IncB/O/K/Z-type Shigella spp. and Escherichia coli plasmids, but is differentiated by the insertion of antibiotic resistance cassettes, which we found associated with mobile genetic elements such as Tn3, Tn7, and Tn10. A whole genome-derived phylogenetic reconstruction showed the evolutionary relationships of S. sonnei strain 866 and the four established Shigella species, highlighting the clonal nature of S. sonnei.

Phage particles harboring antibiotic resistance genes in fresh-cut vegetables and agricultural soil

Bacteriophages are ubiquitously distributed prokaryotic viruses that are more abundant than bacteria. As a consequence of their life cycle, phages can kidnap part of their hosts genetic material, including antibiotic resistance genes (ARGs), which released phage particles transfer in a process called transduction. The spread of ARGs among pathogenic bacteria currently constitutes a serious global health problem. In this study, fresh vegetables (lettuce, spinach and cucumber), and cropland soil were screened by qPCR for ten ARGs (blaTEM, blaCTX-M-1 group, blaCTX-M-9 group, blaOXA-48, blaVIM, mecA, sul1, qnrA, qnrS and armA) in their viral DNA fraction. The presence of ARGs in the phage DNA was analyzed before and after propagation experiments in an Escherichia coli host strain to evaluate the ability of the phage particles to infect a host. ARGs were found in the phage DNA fraction of all matrices, although with heterogeneous values. ARG prevalence was significantly higher in lettuce and soil, and the most common overall were β-lactamases. After propagation experiments, an increase in ARG densities in phage particles was observed in samples of all four matrices, confirming that part of the isolated phage particles were infectious. This study reveals the abundance of free, replicative ARG-containing phage particles in vegetable matrices and cropland soil. The particles are proposed as vehicles for resistance transfer in these environments, where they can persist for a long time, with the possibility of generating new resistant bacterial strains. Ingestion of these mobile genetic elements may also favor the emergence of new resistances, a risk not previously considered.