Analysis of a phase-variable restriction modification system of the human gut symbiont Bacteroides fragilis.

The genomes of gut Bacteroidales contain numerous invertible regions, many of which contain promoters that dictate phase-variable synthesis of surface molecules such as polysaccharides, fimbriae, and outer surface proteins. Here, we characterize a different type of phase-variable system of Bacteroides fragilis, a Type I restriction modification system (R-M). We show that reversible DNA inversions within this R-M locus leads to the generation of eight specificity proteins with distinct recognition sites. In vitro grown bacteria have a different proportion of specificity gene combinations at the expression locus than bacteria isolated from the mammalian gut. By creating mutants, each able to produce only one specificity protein from this region, we identified the R-M recognition sites of four of these S-proteins using SMRT sequencing. Transcriptome analysis revealed that the locked specificity mutants, whether grown in vitro or isolated from the mammalian gut, have distinct transcriptional profiles, likely creating different phenotypes, one of which was confirmed. Genomic analyses of diverse strains of Bacteroidetes from both host-associated and environmental sources reveal the ubiquity of phase-variable R-M systems in this phylum.

Review: Indicator bacteriophages in sludge, biosolids, sediments and soils.

Solid or semisolid matrices polluted with fecal remnants can be highly loaded with pathogens, especially viruses, and play a substantial role in the persistence and dispersion of pathogens in the water cycle. Water quality regulations and guidelines are increasingly including bacteriophages infecting enteric bacteria as indicators of fecal and/or viral pollution. However, more data are needed about viral indicators in contaminated solids to develop effective sanitation strategies for the management of raw and treated sludge, fecal sludge, manures and slurries. Also, the exact role of sediments and soil in the transmission cycle of viral pathogens still needs to be determined. This review aims to provide an update on available data for concentrations of indicator bacteriophages in different solid matrices as well as their resistance to treatments and persistence in solids. The conclusion reached is that there is a need for improved and standardized methodologies for bacteriophage extraction, detection and enumeration in solids. Reports indicate that these contain higher levels of somatic coliphages in comparison with traditional bacterial indicators and F-specific RNA coliphages. Water body sediments and soil have been found to be notable reservoirs of somatic coliphages, which are more persistent in nature and resistant to sludge treatments than Escherichia coli and fecal coliforms and F-specific RNA coliphages. Thus, somatic coliphages show up as excellent complementary indicators for the prediction of pathogenic viruses in solids.

Use of non-linear mixed-effects modelling and regression analysis to predict the number of somatic coliphages by plaque enumeration after 3 hours of incubation.

The present study aimed to establish the kinetics of the appearance of coliphage plaques using the double agar layer titration technique to evaluate the feasibility of using traditional coliphage plaque forming unit (PFU) enumeration as a rapid quantification method. Repeated measurements of the appearance of plaques of coliphages titrated according to ISO 10705-2 at different times were analysed using non-linear mixed-effects regression to determine the most suitable model of their appearance kinetics. Although this model is adequate, to simplify its applicability two linear models were developed to predict the numbers of coliphages reliably, using the PFU counts as determined by the ISO after only 3 hours of incubation. One linear model, when the number of plaques detected was between 4 and 26 PFU after 3 hours, had a linear fit of: (1.48 × Counts3 h + 1.97); and the other, values >26 PFU, had a fit of (1.18 × Counts3 h + 2.95). If the number of plaques detected was <4 PFU after 3 hours, we recommend incubation for (18 ± 3) hours. The study indicates that the traditional coliphage plating technique has a reasonable potential to provide results in a single working day without the need to invest in additional laboratory equipment.

Microbial source markers assessment in the Bogotá River basin (Colombia).

The microbiological indicators traditionally used to assess fecal contamination are insufficient to identify the source. The aim of this study was to detect microbial markers to identify the source of fecal pollution in the Bogotá River (Colombia). For this, we determined non-discriminating indicators such as Escherichia coli, somatic coliphages and phages infecting strain RYC2056 of Bacteroides, and potential source tracking markers as phages infecting strains GA17, HB13, and CA8 of Bacteroides, sorbitol-fermenting bifidobacteria, and molecular markers of Bifidobacterium adolescentis, Bifiodobacterium dentium, and Bacteroidetes in raw municipal wastewaters, slaughterhouse wastewaters, and the Bogotá River. Bacteriophages infecting Bacteroides strain GA17 and the molecular markers identified the wastewater sources. In contrast, sorbitol-fermenting bifidobacteria failed regarding specificity. In the Bogotá River, phages infecting strain GA17 were detected in all samples downstream of Bogotá, whereas they should be concentrated from 1 l samples in upstream samples containing less than 10(3) E. coli/100 ml to be detected. In the river water, the fraction of positive detections of molecular markers was lower than that of phages infecting strain GA17. The ratio SOMCPH/GA17PH was shown also to be a good marker. These results provide information that will allow focusing measures for sanitation of the Bogotá River.

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.

Quinolone resistance genes (qnrA and qnrS) in bacteriophage particles from wastewater samples and the effect of inducing agents on packaged antibiotic resistance genes.

OBJECTIVES: This study quantifies quinolone antibiotic resistance genes (qnrA and qnrS) in DNA of phage particles isolated from faecally polluted waters and evaluates the influence of phage inducers on the abundance of antibiotic resistance genes in packaged DNA. METHODS: qnrA and qnrS were quantified by qPCR in DNA of phage particles isolated from 18 raw urban wastewater samples, 18 river samples and 28 archived samples of animal wastewater. The bacterial fraction of the samples was treated with mitomycin C, ciprofloxacin, EDTA or sodium citrate under different conditions, and the number of resistance genes in DNA of phage particles was compared with the non-induced samples. RESULTS: qnrA was more prevalent than qnrS, with 100% of positive samples in urban wastewater and river and 71.4% of positive samples in animal wastewater. Densities of qnrA ranged from 2.3 × 10(2) gene copies (GC)/mL in urban wastewater to 7.4 × 10(1) GC/mL in animal wastewater. qnrS was detected in 38.9% of urban wastewater samples, in 22.2% of river samples and only in one animal wastewater sample (3.6%). Despite the lower prevalence, qnrS densities reached values of 10(3) GC/mL. Both qnr genes and other resistance genes assayed (blaTEM and blaCTX-M) showed a significant increase in DNA of phage particles when treated with EDTA or sodium citrate, while mitomycin C and ciprofloxacin showed no effect under the different conditions assayed. CONCLUSIONS: This study confirms the contribution of phages to the mobilization of resistance genes and the role of the environment and certain inducers in the spread of antibiotic resistance genes by means of phages.

Sludge as a potential important source of antibiotic resistance genes in both the bacterial and bacteriophage fractions.

The emergence and prevalence of antibiotic resistance genes (ARGs) in the environment is a serious global health concern. ARGs found in bacteria can become mobilized in bacteriophage particles in the environment. Sludge derived from secondary treatment in wastewater treatment plants (WWTPs) constitutes a concentrated pool of bacteria and phages that are removed during the treatment process. This study evaluates the prevalence of ARGs in the bacterial and phage fractions of anaerobic digested sludge; five ARGs (blaTEM, blaCTX-M, qnrA, qnrS, and sul1) are quantified by qPCR. Comparison between the wastewater and sludge revealed a shift in the prevalence of ARGs (blaTEM and sul1 became more prevalent in sludge), suggesting there is a change in the bacterial and phage populations from wastewater to those selected during the secondary treatment and the later anaerobic mesophilic digestion of the sludge. ARGs densities were higher in the bacterial than in the phage fraction, with high densities in both fractions; particularly for blaTEM and sul1 (5 and 8 log10 gene copies (GC)/g, respectively, in bacterial DNA; 5.5 and 4.4 log10 GC/g, respectively, in phage DNA). These results question the potential agricultural uses of treated sludge, as it could contribute to the spread of ARGs in the environment and have an impact on the bacterial communities of the receiving ecosystem.

Inflammation and bacteriophages affect DNA inversion states and functionality of the gut microbiota.

Reversible genomic DNA inversions control the expression of numerous gut bacterial molecules, but how this impacts disease remains uncertain. By analyzing metagenomic samples from inflammatory bowel disease (IBD) cohorts, we identified multiple invertible regions where a particular orientation correlated with disease. These include the promoter of polysaccharide A (PSA) of Bacteroides fragilis, which induces regulatory T cells (Tregs) and ameliorates experimental colitis. The PSA promoter was mostly oriented "OFF" in IBD patients, which correlated with increased B. fragilis-associated bacteriophages. Similarly, in mice colonized with a healthy human microbiota and B. fragilis, induction of colitis caused a decline of PSA in the "ON" orientation that reversed as inflammation resolved. Monocolonization of mice with B. fragilis revealed that bacteriophage infection increased the frequency of PSA in the "OFF" orientation, causing reduced PSA expression and decreased Treg cells. Altogether, we reveal dynamic bacterial phase variations driven by bacteriophages and host inflammation, signifying bacterial functional plasticity during disease.

Stability and infectivity of cytolethal distending toxin type V gene-carrying bacteriophages in a water mesocosm and under different inactivation conditions.

Two cytolethal distending toxin (Cdt) type V-encoding bacteriophages (Φ62 and Φ125) were induced spontaneously from their wild-type Escherichia coli strains and from the lysogens generated in Shigella sonnei. The stability of Cdt phages was determined at various temperatures and pH values after 1 month of storage by means of infectivity tests using a plaque blot assay and analysis of phage genomes using real-time quantitative PCR (qPCR): both were highly stable. We assessed the inactivation of Cdt phages by thermal treatment, chlorination, UV radiation, and in a mesocosm in both summer and winter. The results for the two Cdt phages showed similar trends and were also similar to the phage SOM23 used for reference, but they showed a much higher persistence than Cdt-producing E. coli. Cdt phages showed maximal inactivation after 1 h at 70°C, 30 min of UV radiation, and 30 min of contact with a 10-ppm chlorine treatment. Inactivation in a mesocosm was higher in summer than in winter, probably because of solar radiation. The treatments reduced the number of infectious phages but did not have a significant effect on the Cdt phage particles detected by qPCR. Cdt phages were quantified by qPCR in 73% of river samples, and these results suggest that Cdt phages are a genetic vehicle and the natural reservoir for cdt in the environment.

Bacteriophages carrying antibiotic resistance genes in fecal waste from cattle, pigs, and poultry.

This study evaluates the occurrence of bacteriophages carrying antibiotic resistance genes in animal environments. bla(TEM), bla(CTX-M) (clusters 1 and 9), and mecA were quantified by quantitative PCR in 71 phage DNA samples from pigs, poultry, and cattle fecal wastes. Densities of 3 to 4 log(10) gene copies (GC) of bla(TEM), 2 to 3 log(10) GC of bla(CTX-M), and 1 to 3 log(10) GC of mecA per milliliter or gram of sample were detected, suggesting that bacteriophages can be environmental vectors for the horizontal transfer of antibiotic resistance genes.

Isolation of bacteriophage host strains of Bacteroides species suitable for tracking sources of animal faecal pollution in water.

Microbial source tracking (MST) methods allow the identification of specific faecal sources. The aim is to detect the sources of faecal pollution in a water body to allow targeted, efficient and cost-effective remediation efforts in the catchment. Bacteriophages infecting selected host strains of Bacteroides species are used as markers to track faecal contaminants in water. By using a suitable Bacteroides host from a given faecal origin, it is possible to specifically detect bacteriophages of this faecal origin. It can thus be used to detect specific phages of Bacteroides for MST. With this objective, we isolated several Bacteroides strains from pig, cow and poultry faeces by applying a previously optimized methodology used to isolate the host strains from humans. The isolated strains belonged to Bacteroides fragilis and Bacteroides thetaiotaomicron. These strains, like most Bacteroides species, detected phages of the Siphoviridae morphology. Using the newly isolated host strains for phage enumeration in a range of samples, we showed that these detect phages in faecal sources that coincide with their own origin (70-100% of the samples), and show no detection or very low percentages of detection of phages from other animal origins (from 0 to 20% of the samples). Only strains isolated from pig wastewater detected phages in 50% of human sewage samples. Nevertheless, those strains detecting phages from faecal origins other than their own detected fewer phages (2-3 log10 pfu·100 ml⁻¹) than the phages detected by the specific strain of the same origin. On the basis of our results, we propose that faecal source tracking with phages infecting specific Bacteroides host strains is a useful method for MST. In addition, the method presented here is feasible in laboratories equipped with only basic microbiological equipment, it is more rapid and cost-effective than other procedures and it does not require highly qualified staff.

Quantification of Shiga toxin-converting bacteriophages in wastewater and in fecal samples by real-time quantitative PCR.

Shiga toxin-converting bacteriophages (Stx phages) are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7. Stx phages are released from their bacterial hosts after lytic induction and remain free in the environment. Samples were analyzed for the presence of free Stx phages by an experimental approach based on the use of real-time quantitative PCR (qPCR), which enables stx to be detected in the DNA from the viral fraction of each sample. A total of 150 samples, including urban raw sewage samples, wastewater samples with fecal contamination from cattle, pigs, and poultry, and fecal samples from humans and diverse animals, were used in this study. Stx phages were detected in 70.0% of urban sewage samples (10 to 10(3) gene copies [GC] per ml) and in 94.0% of animal wastewater samples of several origins (10 to 10(10) GC per ml). Eighty-nine percent of cattle fecal samples were positive for Stx phages (10 to 10(5) GC per g of sample), as were 31.8% of other fecal samples of various origins (10 to 10(4) GC per g of sample). The stx(2) genes and stx(2) variants were detected in the viral fraction of some of the samples after sequencing of stx(2) fragments amplified by conventional PCR. The occurrence and abundance of Stx phages in the extraintestinal environment confirm the role of Stx phages as a reservoir of stx in the environment.

Bacteriophage Isolation and Characterization: Phages of Escherichia coli.

Here we introduce methods for the detection, enumeration, and isolation of bacteriophages from Escherichia coli. In bacteria, horizontal gene transfer may be mediated by virulent and temperate phages. Strict virulent phages, able to propagate in a suitable strain following the lytic pathway, can be isolated directly from different natural environments. In temperate phages, the lytic cycle must be activated, and phages are detected after their induction. In both cases, detection is based on the production of visible plaques in a confluent lawn of the host strain using a double agar layer method. Further purification and characterization are achieved by density gradients, electron microscopy studies, and genomic analysis. This straightforward methodology can be applied to the detection, enumeration, and isolation of bacteriophages from any bacterial species, using the appropriate host strain, media, and culture conditions.

Fast and easy methods for the detection of coliphages.

Somatic and F-specific coliphages are gaining ground as indicators of fecal/viral pollution. Guidelines and regulations worldwide for monitoring water, biosolids and food are including them as parameters to assess quality and treatment efficiency. Robust methods to detect and quantify both groups of phages in water samples have been launched by agencies such as the International Standardization Organization (ISO) and the USA Environmental Protection Agency (USEPA). Although these methods have proved readily implementable in routine microbiology laboratories, faster and more user-friendly protocols will be highly welcome if coliphage detection becomes routine in water quality analysis. We here provide an overview of new approaches seeking to facilitate the detection of infectious coliphages included in guidelines and regulations. The improvements achieved suggest that streamlined kits able to provide results in a few hours at very reasonable costs will become available in the near future. The potential of molecular procedures and methods based on microelectronic sensors is also briefly discussed.

Genotypic and phenotypic diversity among induced, stx2-carrying bacteriophages from environmental Escherichia coli strains.

Shiga toxin 2 (stx(2)) gene-carrying bacteriophages have been shown to convert Escherichia coli strains to Shiga toxin-producing Escherichia coli (STEC). In this study, 79 E. coli strains belonging to 35 serotypes isolated from wastewaters of both human and animal origin were examined for the presence of stx(2)-carrying bacteriophages in their genomes. The lytic cycle of the bacteriophages was induced by mitomycin, and the bacteriophage fraction was isolated and used for morphological and genetic characterization. The induced bacteriophages showed morphological diversity, as well as restriction fragment length polymorphism variation, in the different strains belonging to different serotypes. The ability to infect new hosts was highly variable, although most of the induced phages infected Shigella sonnei host strain 866. In summary, in spite of carrying either the same or different stx(2) variants and in spite of the fact that they were isolated from strains belonging to the same or different serotypes, the induced bacteriophages were highly variable. The high level of diversity and the great infectious capacity of these phages could enhance the spread of the stx(2) gene and variants of this gene among different bacterial populations in environments to which humans may be exposed.

Phage-mediated Shiga toxin 2 gene transfer in food and water.

Shiga toxin (stx) transduction in various food matrices has been evaluated with lysogens of Stx phages. stx transduction events were observed for many phages under appropriate conditions. Transduction did not occur at low pH and low temperatures. A total of 10(3) to 10(4) CFU ml(-1) was the minimal amount of donor and recipient strains necessary to generate transductants.

Differential persistence of F-specific RNA phage subgroups hinders their use as single tracers for faecal source tracking in surface water.

The four subgroups of F-specific RNA bacteriophages (I-IV) have been proposed as potential tracers for faecal source tracking. Groups II and III predominate in human sources while groups I and IV are most abundant in animal sources. The four subgroups of naturally occurring F-specific RNA bacteriophages were identified in different samples by plaque hybridization with genotype-specific probes and the persistence of each subgroup was evaluated. The proportions of the F-specific RNA bacteriophage subgroups were measured in wastewaters, after inactivation in surface waters or after wastewater treatment and in mixtures of wastewater of human and animal origin. Our results indicate that phage groups differ in their persistence in the environment and to different disinfecting treatments. The greater survival of subgroups I and II in treated samples hinders the interpretation of results obtained with F-specific RNA bacteriophages. The phages of subgroups III and IV were the least resistant to all treatments. These results should be considered when using genotypes of F-specific RNA as sole tracers for faecal source tracking.

New approach for the simultaneous detection of somatic coliphages and F-specific RNA coliphages as indicators of fecal pollution.

Two groups of coliphages have been recently included in different water management policies as indicators of viral fecal pollution in water and food: somatic coliphages, which infect E. coli through cell wall receptors, and F-specific RNA coliphages, which infect through the F-pili. Somatic coliphages are more abundant in fecally contaminated waters, except reclaimed waters, those disinfected by UV irradiation, and some groundwater samples that show a higher level of F-specific coliphages. Somatic coliphages are morphologically similar to DNA enteric viruses while F-specific coliphages are similar to RNA viruses such as norovirus and hepatitis A viruses, which are the viral pathogens of concern in sewage. The use of strains sensitive to both types of phages has been proposed for total coliphage enumeration, thereby avoiding double analysis. The standardized methods available for coliphage detection are robust and cost-effective, but the introduction of ready-to-use methods would facilitate routine implementation in laboratories. The fastest available tool for somatic coliphage enumeration is the recently developed Bluephage, which uses a modified ß-glucuronide-overexpressing E. coli strain unable to take up the glucuronide substrate. The overexpressed enzyme accumulates inside the bacterial cells until released by phage-induced cell lysis, whereupon it encounters its substrate and the medium changes from yellow to blue. The present method uses E. coli strain CB12, sensitive to somatic coliphages and F-specific coliphages due to the expression of the F-pili. The Bluephage approach incorporating CB12 detects both types of coliphages in a time range of 1:30 to 4:00 h, as assayed with coliphages from raw sewage, river water, sludge and mussels. This strategy can be applied to obtain qualitative and quantitative results and is applicable to microplates as well as to large sample volumes (100 ml). Moreover it can provide monitoring of water bodies at real time, as for example for ambient recreational beach monitoring.

Evaluation of Escherichia coli host strain CB390 for simultaneous detection of somatic and F-specific coliphages.

Escherichia coli WG5, the strain recommended by the International Organization for Standardization (ISO) to detect somatic coliphages, was transformed to F(+) by introducing the plasmid Famp, which rendered it capable of simultaneously detecting both somatic and F-specific coliphages. Indeed, this strain, CB390, proved as effective in detecting similar numbers of phages as the sum of somatic and F-specific bacteriophages detected by the host strains recommended by both the ISO and the U.S. Environmental Protection Agency standardized methods.

Isolation of Bacteriophages of the Anaerobic Bacteria Bacteroides.

Here we describe the detection, enumeration, and isolation of bacteriophages infecting Bacteroides. The method is based on the infection of Bacteroides host strains and the production of visible plaques in a confluent lawn of the host strain using the double-layer agar method. This is a straightforward methodology that can be applied for the detection, enumeration and isolation of bacteriophages for other anaerobic bacteria, using an appropriate host strain and culture conditions. In the case of bacteriophages of Bacteroides the results can be obtained in less than 24 h, although the time could vary depending on the growth rate of the host strain.