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.

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.

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.

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.

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.

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.

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.

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.

Determination of crAssphage in water samples and applicability for tracking human faecal pollution.

In recent decades, considerable effort has been devoted to finding microbial source-tracking (MST) markers that are suitable to assess the health risks of faecally polluted waters, with no universal marker reported so far. In this study, the abundance and prevalence of a crAssphage-derived DNA marker in wastewaters of human and animal origins were studied by a new qPCR assay with the ultimate aim of assessing its potential as an MST marker. crAssphage showed up to 106 GC/ml in the sewage samples of human origin, in both the total DNA and the viral DNA fraction. In wastewaters containing animal faecal remains, 39% of the samples were negative for the presence of the crAssphage sequence, while those showing positive results (41% of the samples) were at least 1 log10 unit lower than the samples of human origin. Noteworthy, the log10 values of the ratio (R) crAssphage (GC/ml)/Escherichia coli (CFU/ml) varied significantly depending on the human or animal origin (R > 1.5 for human samples and R < -1.5 for animal wastewater samples. This study opens the way for further research to explore if different specific animal variants of crAssphage exist and whether other zones of the crAssphage genome are better suited to source discrimination.

Simultaneous detection of somatic and F-specific coliphages in different settings by Escherichia coli strain CB390.

Bacteriophages are increasingly being used as water quality indicators. Two groups of phages infecting Escherichia coli, somatic and F-specific coliphages, are being considered as indicators of fecal and viral contamination for several types of water around the world. However, some uncertainties remain regarding which coliphages to assess. Recently, E. coli strain CB390 has been reported to be suitable for simultaneous detection of both groups, which seems to be more informative than determining only one of the groups. Here, a significant number of samples from different settings, mostly those where F-specific phages have been reported to outnumber somatic coliphages, are analyzed for somatic coliphages, F-specific RNA phages by standardized methods and coliphages detected by host strain CB390. The results presented here confirm that the numbers of phages counted using CB390 are equivalent to the sum of the somatic and F-specific coliphages counted independently in all settings. Hence the usefulness of this strain for simultaneous detection of somatic and F-specific coliphages is confirmed. Also, sets of data on the presence of coliphages in reclaimed and groundwater are reported.

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 bacterial and bacteriophage fractions of Tunisian and Spanish wastewaters as markers to compare the antibiotic resistance patterns in each population.

The emergence and increased prevalence of antibiotic resistance genes (ARGs) in the environment may pose a serious global health concern. This study evaluates the abundance of several ARGs in bacterial and bacteriophage DNA via real-time qPCR in samples from five different sampling points in Tunisia; three wastewater treatment plants (WWTP 1, 2 and 3) and wastewater from two abattoirs slaughtering different animals. Results are compared with those obtained in the Barcelona area, in northeast Spain. Eight ARGs were quantified by qPCR from total and phage DNA fraction from the samples. Three ß-lactamases (bla(TEM), bla(CTX-M) cluster 1 and bla(CTX-M) cluster 9), two quinolone resistance genes (qnrA and qnrS), the mecA gene that confers resistance to methicillin in Staphylococcus aureus, the emerging armA gene, conferring resistance to aminoglycosides and sul1, the most extended gene conferring resistance to sulfonamides, were evaluated. Sul1 and bla(TEM) were the most prevalent ARGs detected at all five Tunisian sampling points, similarly with the observations in Barcelona. bla(CTX-M-9) was more prevalent than bla(CTX-M-1) both in bacterial and DNA within phage particles in all samples analysed. mecA and armA were almost absent in Tunisian waters from human or animal origin in contrast with Barcelona that showed a medium prevalence. qnrA was more prevalent than qnrS in bacterial and phage DNA from all sampling points. In conclusion, our study shows that ARGs are found in the bacterial and is reflected in the phage DNA fraction of human and animal wastewaters. The densities of each ARGs vary depending on the ARGs shed by each population and is determined by the characteristics of each area. Thus, the evaluation of ARGs in wastewaters seems to be suitable as marker reflecting the antibiotic resistance patterns of a population.

Identifying and analyzing bacteriophages in human fecal samples: what could we discover?

The human gut is a complex ecosystem, densely populated with microbes including enormous amounts of phages. Metagenomic studies indicate a great diversity of bacteriophages, and because of the variety of gut bacterial species, the human or animal gut is probably a perfect ecological niche for phages that can infect and propagate in their bacterial communities. In addition, some phages have the capacity to mobilize genes, as demonstrated by the enormous fraction of phage particles in feces that contain bacterial DNA. All these facts indicate that, through predation and horizontal gene transfer, bacteriophages play a key role in shaping the size, structure and function of intestinal microbiomes, although our understanding of their effects on gut bacterial populations is only just beginning.

Characterization of ciprofloxacin-resistant isolates from a wastewater treatment plant and its receiving river.

In this study we characterised the ciprofloxacin-resistant strains isolated in biofilm and sediments from a wastewater treatment plant (WWTP) discharge point and its receiving river. We also examined the prevalence of qnrA, qnrB, qnrS and aac(6')-Ib-cr genes in these isolates and determined whether they harbour plasmid-encoded ß-lactamases such as TEM, SHV and CTX-M. Moreover, antibiotic concentrations were also measured to evaluate the level of contamination of these pharmaceuticals in the sampling area. Antibiotics were found in the range of ng L(-1) in WWTP effluents, but most of them were no longer found in downstream river. However, some fluoroquinolones were detected in sediment downstream demonstrating their high persistence and their capacity to be retained in the river sediments. Most of the ciprofloxacin-resistant isolates belonged to the Gammaproteobacteria class and 17 of them, 8 (7.6%) from the first sampling and 9 (6.1%) from the second sampling, carried a qnr gene. In particular, 15 isolates carried the qnrS gene and 2 carried the qnrB gene. Among the qnr-positive isolates, 12 harboured the aac(6')-lb-cr gene and 2 of them also carried a ß-lactamase on the same plasmid, indicating that they may be transferred simultaneously. It is also noteworthy that all qnr-positive isolates identified as Aeromonas species harboured the same qnrS allele, namely the qnrS2. This study reinforces the importance of environmental bacteria as vehicles for dissemination of antibiotic resistance genes.

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.

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.

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.

Prevalence of antibiotic resistance genes and bacterial community composition in a river influenced by a wastewater treatment plant.

Antibiotic resistance represents a global health problem, requiring better understanding of the ecology of antibiotic resistance genes (ARGs), their selection and their spread in the environment. Antibiotics are constantly released to the environment through wastewater treatment plant (WWTP) effluents. We investigated, therefore, the effect of these discharges on the prevalence of ARGs and bacterial community composition in biofilm and sediment samples of a receiving river. We used culture-independent approaches such as quantitative PCR to determine the prevalence of eleven ARGs and 16S rRNA gene-based pyrosequencing to examine the composition of bacterial communities. Concentration of antibiotics in WWTP influent and effluent were also determined. ARGs such as qnrS, bla TEM, bla CTX-M, bla SHV, erm(B), sul(I), sul(II), tet(O) and tet(W) were detected in all biofilm and sediment samples analyzed. Moreover, we observed a significant increase in the relative abundance of ARGs in biofilm samples collected downstream of the WWTP discharge. We also found significant differences with respect to community structure and composition between upstream and downstream samples. Therefore, our results indicate that WWTP discharges may contribute to the spread of ARGs into the environment and may also impact on the bacterial communities of the receiving river.

Could bacteriophages transfer antibiotic resistance genes from environmental bacteria to human-body associated bacterial populations?

Environments without any contact with anthropogenic antibiotics show a great abundance of antibiotic resistance genes that use to be chromosomal and are part of the core genes of the species that harbor them. Some of these genes are shared with human pathogens where they appear in mobile genetic elements. Diversity of antibiotic resistance genes in non-contaminated environments is much greater than in human and animal pathogens, and in environments contaminated with antibiotic from anthropogenic activities. This suggests the existence of some bottleneck effect for the mobilization of antibiotic resistance genes among different biomes. Bacteriophages have characteristics that make them suitable vectors between different biomes, and as well for transferring genes from biome to biome. Recent metagenomic studies and detection of bacterial genes by genomic techniques in the bacteriophage fraction of different microbiota provide indirect evidences that the mobilization of genes mediated by phages, including antibiotic resistance genes, is far more relevant than previously thought. Our hypothesis is that bacteriophages might be of critical importance for evading one of the bottlenecks, the lack of ecological connectivity that modulates the pass of antibiotic resistance genes from natural environments such as waters and soils, to animal and human microbiomes. This commentary concentrates on the potential importance of bacteriophages in transferring resistance genes from the environment to human and animal body microbiomes, but there is no doubt that transduction occurs also in body microbiomes.

Effect of gamma irradiation on bacteriophages used as viral indicators.

This study aimed to examine the susceptibility of indicator bacteriophages towards γ-radiation to evaluate their appropriateness as viral indicators for water quality control. The effects of γ-radiation on naturally occurring somatic coliphages, F-specific coliphages and Escherichia coli were examined in raw sewage and sewage sludge. As well, the effects of radiation on bacteriophages ΦX174 and MS2, and E. coli all grown in the laboratory and seeded in distilled water, autoclaved raw sewage and a 1% peptone solution were evaluated. The inactivation of E. coli was fairly similar in all matrices. In contrast, inactivation of bacteriophages was significantly greater in distilled water than in the other matrices. These results showed the great influence of the matrix characteristics on virus inactivation. Somatic coliphages in raw sewage and sewage sludge and ΦX174 in autoclaved sewage were inactivated similarly and were far more resistant than F-specific coliphages, MS2 and E. coli. As well, F-specific RNA bacteriophages in raw sewage and sewage sludge and MS2 in autoclaved sewage were inactivated similarly and were more resistant than E. coli. In contrast, MS2 was more susceptible to γ-radiation than E. coli in distilled water. Our results showed that ΦX174 is a suitable indicator for estimating virus inactivation by γ-irradiation and corroborate the use of somatic coliphages to survey the viral quality of treated water and sludges.