Complex effects of the exo-xis region of the Shiga toxin-converting bacteriophage Φ24B genome on the phage development and the Escherichia coli host physiology.

Lambdoid bacteriophages are excellent models in studies on molecular aspects of virus-host interactions. However, some of them carry genes encoding toxins which are responsible for virulence of pathogenic strains of bacteria. Shiga toxin-converting bacteriophages (Stx phages) encode Shiga toxins that cause virulence of enterohemorrhagic Escherichia coli (EHEC), and their effective production depends on Stx prophage induction. The exo-xis region of the lambdoid phage genome consists of genes which are dispensable for the phage multiplication under laboratory conditions; however, they might modulate the virus development. Nevertheless, their exact effects on the phage and host physiology remained unclear. Here, we present results of complex studies on the role of the exo-xis region of bacteriophage Φ24B, one of Stx2b phages. Transcriptomic analyses, together with proteomic and metabolomic studies, provided the basis for understanding the functions of the exo-xis region. Genes from this region promoted lytic development of the phage over lysogenization. Moreover, expression of the host genes coding for DnaK, DnaJ, GrpE, and GroELS chaperones was impaired in the cells infected with the Δexo-xis phage mutant, relative to the wild-type virus, corroborating the conclusion about lytic development promotion by the exo-xis region. Proteomic and metabolomic analyses indicated also modulation of gad and nrf operons, and levels of amino acids and acylcarnitines, respectively. In conclusion, the exo-xis region controls phage propagation and host metabolism by influencing expression of different phage and bacterial genes, directing the virus to the lytic rather than lysogenic developmental mode.

Influence of cheese making process on STEC bacteriophage release.

Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens implicated in diseases including hemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC). The main virulence factor are Shiga toxins; their production and secretion are by-products of the expression of late genes of prophages upon sub-lethal environmental stimuli exposure. Hence, the lysogenic prophage after a stress switch to lytic cycle spreading the Stx phages. In the present study, 35 STEC were screened for the presence and the ability to release Shiga toxin-encoding bacteriophages. Three bacterial strains showed signals of prophage presence both in plate and in PCR. Subsequently, these bacterial strains were subjected to stressors that simulate cheese manufacturing conditions: NaCl (1, 1.5 and 2% w/v), lactic acid (0.5, 1.5 and 3% v/v), anaerobic growth, pasteurization (72°C for 15 s), UV irradiation. The ability to release prophage was evaluated by Real Time qPCR. Induction of the prophages showed that the addition of NaCl at 1.5 and 2% significantly increased viral release compared to control. Conversely, the addition of lactic acid had a significant repressive effect. The other applied stressors had no significant effect in phage release according to the experimental conditions adopted.

Bacteriophages of Shiga Toxin-Producing Escherichia coli and Their Contribution to Pathogenicity.

Shiga toxins (Stx) of Shiga toxin-producing Escherichia coli (STEC) are generally encoded in the genome of lambdoid bacteriophages, which spend the most time of their life cycle integrated as prophages in specific sites of the bacterial chromosome. Upon spontaneous induction or induction by chemical or physical stimuli, the stx genes are co-transcribed together with the late phase genes of the prophages. After being assembled in the cytoplasm, and after host cell lysis, mature bacteriophage particles are released into the environment, together with Stx. As members of the group of lambdoid phages, Stx phages share many genetic features with the archetypical temperate phage Lambda, but are heterogeneous in their DNA sequences due to frequent recombination events. In addition to Stx phages, the genome of pathogenic STEC bacteria may contain numerous prophages, which are either cryptic or functional. These prophages may carry foreign genes, some of them related to virulence, besides those necessary for the phage life cycle. Since the production of one or more Stx is considered the major pathogenicity factor of STEC, we aim to highlight the new insights on the contribution of Stx phages and other STEC phages to pathogenicity.

Persistence of a Stx-Encoding Bacteriophage in Minced Meat Investigated by Application of an Improved DNA Extraction Method and Digital Droplet PCR.

Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens with Shiga toxins as the main virulence factor. Shiga toxins are encoded on Shiga toxin-encoding bacteriophages (Stx phages). Stx phages may exist as free bacteriophages in the environment or in foods or as prophages integrated into the host genome. From a food safety perspective, it is important to have knowledge on the survival and persistence of Stx phages in food products since these may integrate into the bacterial hosts through transduction if conditions are right. Here, we present the results from a study investigating the survival of a Stx phage in minced meat from beef stored at a suboptimal temperature (8°C) for food storage along with modifications and optimizations of the methods applied. Minced meat from beef was inoculated with known levels of a labeled Stx phage prior to storage. Phage filtrates were used for plaque assays and DNA extraction, followed by real-time PCR and digital droplet PCR (ddPCR). The results from the pilot study suggested that the initial DNA extraction protocol was not optimal, and several modifications were tested before a final protocol was defined. The final DNA extraction protocol comprised ultra-centrifugation of the entire phage filtrate for concentrating phages and two times phenol-chloroform extraction. The protocol was used for two spiking experiments. The DNA extraction protocol resulted in flexibility in the amount of DNA available for use in PCR analyses, ultimately increasing the sensitivity of the method used for quantification of phages in a sample. All three quantification methods employed (i.e., plaque assays, real-time PCR, and ddPCR) showed similar trends in the development of the phages during storage, where ddPCR has the benefit of giving absolute quantification of DNA copies in a simple experimental setup. The results indicate that the Stx phages persist and remain infective for at least 20 days under the storage conditions used in the present study. Stx phages in foods might represent a potential risk for humans. Although it can be speculated that transduction may take place at 8°C with subsequent forming of STEC, it can be expected to be a rare event. However, such an event may possibly take place under more optimal conditions, such as an increase in storage temperature of foods or in the gastrointestinal tract of humans.

Investigation of the Causes of Shigatoxigenic Escherichia coli PCR Positive and Culture Negative Samples.

Molecular methods may reveal the presence of pathogens in samples through the detection of specific target gene(s) associated with microorganisms, but often, the subsequent cultural isolation of the pathogen is not possible. This discrepancy may be related to low concentration of the cells, presence of dead cells, competitive microflora, injured cells and cells in a viable but non-culturable state, free DNA and the presence of free bacteriophages which can carry the target gene causing the PCR-positive/culture-negative results. Shiga-toxigenic Escherichia coli (STEC) was used as a model for studying this phenomenon, based on the phage-encoded cytotoxins genes (Stx family) as the detection target in samples through real-time qPCR. Stx phages can be integrated in the STEC chromosome or can be isolated as free particles in the environment. In this study, a combination of PCR with culturing was used for investigating the presence of the stx1 and stx2 genes in 155 ovine recto-anal junction swab samples (method (a)-PCR). Samples which were PCR-positive and culture-negative were subjected to additional analyses including detection of dead STEC cells (method (b)-PCR-PMA dye assay), presence of Stx phages (method (c)-plaque assays) and inducible integrated phages (method (d)-phage induction). Method (a) showed that even though 121 samples gave a PCR-positive result (78%), only 68 samples yielded a culturable isolate (43.9%). Among the 53 (34.2%) PCR-positive/culture-negative samples, 21 (39.6%) samples were shown to have STEC dead cells only, eight (15.1%) had a combination of dead cells and inducible stx phage, while two samples (3.8%) had a combination of dead cells, inducible phage and free stx phage, and a further two samples had Stx1 free phages only (3.8%). It was thus possible to reduce the samples with no explanation to 20 (37.7% of 53 samples), representing a further step towards an improved understanding of the STEC PCR-positive/culture-negative phenomenon.

The Probiotic Escherichia coli Strain Nissle 1917 Combats Lambdoid Bacteriophages stx and λ.

Shiga toxin (Stx) producing E. coli (STEC) such as Enterohemorrhagic E. coli (EHEC) are the major cause of foodborne illness in humans. In vitro studies showed the probiotic Escherichia coli strain Nissle 1917 (EcN) to efficiently inhibit the production of Stx. Life threatening EHEC strains as for example the serotype O104:H4, responsible for the great outbreak in 2011 in Germany, evolutionary developed from certain E. coli strains which got infected by stx2-encoding lambdoid phages turning the E. coli into lysogenic and subsequently Stx producing strains. Since antibiotics induce stx genes and Stx production, EHEC infected persons are not recommended to be treated with antibiotics. Therefore, EcN might be an alternative medication. However, because even commensal E. coli strains might be converted into Stx-producers after becoming host to a stx encoding prophage, we tested EcN for stx-phage genome integration. Our experiments revealed the resistance of EcN toward not only stx-phages but also against lambda-phages. This resistance was not based on the lack of or by mutated phage receptors. Rather it involved the expression of a phage repressor (pr) gene of a defective prophage in EcN which was able to partially protect E. coli K-12 strain MG1655 against stx and lambda phage infection. Furthermore, we observed EcN to inactivate phages and thereby to protect E. coli K-12 strains against infection by stx- as well as lambda-phages. Inactivation of lambda-phages was due to binding of lambda-phages to LamB of EcN whereas inactivation of stx-phages was caused by a thermostable protein of EcN. These properties together with its ability to inhibit Stx production make EcN a good candidate for the prevention of illness caused by EHEC and probably for the treatment of already infected people.

Influence of Stress Factors Related to Cheese-Making Process and to STEC Detection Procedure on the Induction of Stx Phages from STEC O26:H11.

Shiga toxin-producing Escherichia coli (STEC) are responsible for human infections, ranging from mild watery diarrhea to hemorrhagic colitis (CH) that may be complicated by hemolytic uremic syndrome (HUS). The main STEC virulence factor is Shiga toxin encoded by the stx gene, located in the genome of a bacteriophage integrated into the bacterial chromosome. The serotype O26:H11 is the second HUS-causing serotype worldwide (after O157:H7), and the first found in dairy products such as raw-milk cheeses. A small number of HUS cases identified each year in France are caused by serotype O26:H11. Stx phage induction is known to result in STEC lysis and release of new Stx phages particles. This phenomenon could negatively impact STEC screening in foods based on stx gene detection by PCR. Here, we evaluated the influence of physicochemical parameters related to cheese-making process on the induction rate of Stx phages from STEC O26:H11, including H2O2, NaCl, lactic acid and temperature. In addition, selective agents from the analytical STEC enrichment and detection procedure (XP CEN ISO/TS 13136) were tested, including novobiocin, acrifavin, cefixim-tellurite, and bile salts. An impact of H2O2 and NaCl on Stx phage induction was observed. Production of Stx phages was also observed during a real cheese-making process. By contrast, no significant effect could be demonstrated for the chemical agents of the STEC detection procedure when tested separately, except for acriflavin and novobiocin which reduced Stx1 phage production in some cases. In conclusion, these results suggest that the cheese-making process might trigger the production of Stx phages, potentially interfering with the analysis of STEC in food.

A novel transducible chimeric phage from Escherichia coli O157:H7 Sakai strain encoding Stx1 production.

Shiga toxin-producing Escherichia coli (STEC), and especially enterohaemorrhagic E. coli (EHEC) are important, highly virulent zoonotic and food-borne pathogens. The genes encoding their key virulence factors, the Shiga toxins, are distributed by converting bacteriophages, the Stx phages. In this study we isolated a new type of inducible Stx phage carrying the stx1 gene cluster from the prototypic EHEC O157:H7 Sakai strain. The phage showed Podoviridae morphology, and was capable of converting the E. coli K-12 MG1655 strain to Shiga toxin-producing phenotype. The majority of the phage genes originate from the stx2-encoding Sakai prophage Sp5, with major rearrangements in its genome. Beside certain minor recombinations, the genomic region originally containing the stx2 genes in Sp5 was replaced by a region containing six open reading frames from prophage Sp15 including stx1 genes. The rearranged genome, together with the carriage of stx1 genes, the morphology and the capability of lysogenic conversion represent a new type of recombinant Stx1 converting phage from the Sakai strain.

Shiga toxin-converting phages and the emergence of new pathogenic Escherichia coli: a world in motion.

Shiga toxin (Stx)-producing Escherichia coli (STEC) are pathogenic E. coli causing diarrhea, hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). STEC are characterized by a constellation of virulence factors additional to Stx and have long been regarded as capable to cause HC and HUS when possessing the ability of inducing the attaching and effacing (A/E) lesion to the enterocyte, although strains isolated from such severe infections sometimes lack this virulence feature. Interestingly, the capability to cause the A/E lesion is shared with another E. coli pathogroup, the Enteropathogenic E. coli (EPEC). In the very recent times, a different type of STEC broke the scene causing a shift in the paradigm for HUS-associated STEC. In 2011, a STEC O104:H4 caused a large outbreak with more than 800 HUS and 50 deaths. Such a strain presented the adhesion determinants of Enteroaggregative E. coli (EAggEC). We investigated the possibility that, besides STEC and EAggEC, other pathogenic E. coli could be susceptible to infection with stx-phages. A panel of stx2-phages obtained from STEC isolated from human disease was used to infect experimentally E. coli strains representing all the known pathogenic types, including both diarrheagenic E. coli (DEC) and extra-intestinal pathogenic E. coli (ExPEC). We observed that all the E. coli pathogroups used in the infection experiments were susceptible to the infection. Our results suggest that the stx2-phages used may not have specificity for E. coli adapted to the intestinal environment, at least in the conditions used. Additionally, we could only observe transient lysogens suggesting that the event of stable stx2-phage acquisition occurs rarely.