Heterogeneity in phage induction enables the survival of the lysogenic population.

Lysogeny by temperate phages provides novel functions for bacteria and shelter for phages. However, under conditions that activate the phage lytic cycle, the benefit of lysogeny becomes a paradox that poses a threat for bacterial population survival. Using Escherichia coli lysogens for Shiga toxin (Stx) phages as model, we demonstrate how lysogenic bacterial populations circumvent extinction after phage induction. A fraction of cells maintains lysogeny, allowing population survival, whereas the other fraction of cells lyse, increasing Stx production and spreading Stx phages. The uninduced cells were still lysogenic for the Stx phage and equally able to induce phages as the original cells, suggesting heterogeneity of the E. coli lysogenic population. The bacterial population can modulate phage induction under stress conditions by the stress regulator RpoS. Cells overexpressing RpoS reduce Stx phage induction and compete with and survive better than cells with baseline RpoS levels. Our observations suggest that population heterogeneity in phage induction could be widespread among other bacterial genera and we propose this is a mechanism positively selected to prevent the extinction of the lysogenic population that can be modulated by environmental conditions.

BaeSR, involved in envelope stress response, protects against lysogenic conversion by Shiga toxin 2-encoding phages.

Infection and lysogenic conversion with Shiga toxin-encoding bacteriophages (Stx phages) drive the emergence of new Shiga toxin-producing Escherichia coli strains. Phage attachment to the bacterial surface is the first stage of phage infection. Envelope perturbation causes activation of envelope stress responses in bacterial cells. Although many external factors are known to activate envelope stress responses, the role of these responses in the phage-bacterium interaction remains unexplored. Here, we investigate the link between three envelope signaling systems in E. coli (RcsBC, CpxAR, and BaeSR) and Stx2 phage infection by determining the success of bacterial lysogenic conversion. For this purpose, E. coli DH5α wild-type (WT) and mutant strains lacking RcsBC, CpxAR, or BaeSR signaling systems were incubated with a recombinant Stx2 phage (933W). Notably, the number of lysogens obtained with the BaeSR mutant was 5 log10 units higher than with the WT, and the same differences were observed when using 7 different Stx2 phages. To assess whether the membrane receptor used by Stx phages, BamA, was involved in the differences observed, bamA gene expression was monitored by reverse transcription-quantitative PCR (RT-qPCR) in all host strains. A 4-fold-higher bamA expression level was observed in the BaeSR mutant than in the WT strain, suggesting that differential expression of the receptor used by Stx phages accounted for the increase in the number of lysogenization events. Establishing the link between the role of stress responses and phage infection has important implications for understanding the factors affecting lysogenic conversion, which drives the emergence of new pathogenic clones.

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.

Improving detection of Shiga toxin-producing Escherichia coli by molecular methods by reducing the interference of free Shiga toxin-encoding bacteriophages.

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.

Antibiotic resistance genes in the bacteriophage DNA fraction of human fecal samples.

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.

Persistence of infectious Shiga toxin-encoding bacteriophages after disinfection treatments.

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 (Φ534 and Φ557) 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-log10 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-log10 reductions, respectively) and were highly inactivated (3 log10) 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 2-encoding bacteriophages in human fecal samples from healthy individuals.

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.

Implications of free Shiga toxin-converting bacteriophages occurring outside bacteria for the evolution and the detection of Shiga toxin-producing Escherichia coli.

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.

Type III effector genes and other virulence factors of Shiga toxin-encoding Escherichia coli isolated from wastewater.

Pathogenic Shiga toxin-producing Escherichia coli (STEC) strains share the genes encoding Shiga toxins (stx) and many other virulence factors. The classification and evolutionary studies of pathogenic E. coli based on their virulence genes have been conducted with E. coli isolated from human and animal infections or outbreaks. In this study, we used 103 STEC strains isolated from faecally polluted water environments to analyse 23 virulence genes (stx1 , cdt, hlyA, saa, eae, three type III effector genes encoded within the locus of enterocyte effacement (LEE) and 15 non-LEE-encoded type III effector genes). Despite the presence of several stx2 variants, our isolates demonstrated low prevalence of the virulence genes (only 46.6% of the strains were positive for virulence determinants). Among these, the largest repertoire was found in a few O157:H7 isolates (most from cattle wastewater and one from sewage), while other serotypes showed fewer virulence determinants. The occurrence of most virulence genes seemed to be independent from one another. This was clear for hlyA (the most prevalent), cdt and cif (the least prevalent). Other effector genes, could be found or not in combination with others, suggesting that they can be mobilized independently. Our data suggest that E. coli strains can evolve separately by independently acquiring mobile genetic elements.