Bacteriophages Are Good Estimators of Human Viruses Present in Water.

The detection of fecal viral pathogens in water is hampered by their great variety and complex analysis. As traditional bacterial indicators are poor viral indicators, there is a need for alternative methods, such as the use of somatic coliphages, which have been included in water safety regulations in recent years. Some researchers have also recommended the use of reference viral pathogens such as noroviruses or other enteric viruses to improve the prediction of fecal viral pollution of human origin. In this work, phages previously tested in microbial source tracking studies were compared with norovirus and adenovirus for their suitability as indicators of human fecal viruses. The phages, namely those infecting human-associated Bacteroides thetaiotaomicron strain GA17 (GA17PH) and porcine-associated Bacteroides strain PG76 (PGPH), and the human-associated crAssphage marker (crAssPH), were evaluated in sewage samples and fecal mixtures obtained from different animals in five European countries, along with norovirus GI + GII (NoV) and human adenovirus (HAdV). GA17PH had an overall sensitivity of ≥83% and the highest specificity (>88%) for human pollution source detection. crAssPH showed the highest sensitivity (100%) and specificity (100%) in northern European countries but a much lower specificity in Spain and Portugal (10 and 30%, respectively), being detected in animal wastewater samples with a high concentration of fecal indicators. The correlations between GA17PH, crAssPH, or the sum of both (BACPH) and HAdV or NoV were higher than between the two human viruses, indicating that bacteriophages are feasible indicators of human viral pathogens of fecal origin and constitute a promising, easy to use and affordable alternative to human viruses for routine water safety monitoring.

Microbial indicators and molecular markers used to differentiate the source of faecal pollution in the Bogotá River (Colombia).

Intestinal pathogenic microorganisms are introduced into the water by means of faecal contamination, thus creating a threat to public health and to the environment. Detecting these contaminants has been difficult due to such an analysis being costly and time-intensive; as an alternative, microbiological indicators have been used for this purpose, although they cannot differentiate between human or animal sources of contamination because these indicators are part of the digestive tracts of both. To identify the sources of faecal pollution, the use of chemical, microbiological and molecular markers has been proposed. Currently available markers present some geographical specificity. The aim of this study was to select microbial and molecular markers that could be used to differentiate the sources of faecal pollution in the Bogotá River and to use them as tools for the evaluation and identification of the origin of discharges and for quality control of the water. In addition to existing microbial source markers, a phage host strain (PZ8) that differentiates porcine contamination was isolated from porcine intestinal content. The strain was identified biochemically and genotypically as Bacteroides. The use of this strain as a microbial source tracking indicator was evaluated in bovine and porcine slaughterhouse wastewaters, raw municipal wastewaters and the Bogotá River. The results obtained indicate that the selected microbial and molecular markers enable the determination of the source of faecal contamination in the Bogotá River by using different algorithms to develop prediction models.

Detection of Bacteriophage Particles Containing Antibiotic Resistance Genes in the Sputum of Cystic Fibrosis Patients.

Cystic fibrosis (CF) is a chronic disease in which the bacterial colonization of the lung is linked to an excessive inflammatory response that leads to respiratory failure. The microbiology of CF is complex. Staphylococcus aureus is the first bacterium to colonize the lungs in 30% of pediatric CF patients, and 80% of adult patients develop a chronic Pseudomonas aeruginosa infection, but other microorganisms can also be found. The use of antibiotics is essential to treat the disease, but antibiotic performance is compromised by resistance mechanisms. Among various mechanisms of transfer of antibiotic resistance genes (ARGs), the recently been reported bacteriophages are the least explored in clinical settings. To determine the role of phages in CF as mobile genetic elements (MGEs) carrying ARGs, we evaluated their presence in 71 CF patients. 71 sputum samples taken from these patients were screened for eight ARGs (blaTEM, blaCTX-M-1-group, blaCTX-M-9-group, blaOXA-48, blaVIM, mecA, qnrA, and qnrS) in the bacteriophage DNA fraction. The phages found were also purified and observed by electron microscopy. 32.4% of CF patients harbored ARGs in phage DNA. ß-lactamase genes, particularly blaVIM and blaTEM, were the most prevalent and abundant, whereas mecA, qnrA, and qnrS were very rare. Siphoviridae phage particles capable of infecting P. aeruginosa and Klebsiella pneumoniae were detected in CF sputum. Phage particles harboring ARGs were found to be abundant in the lungs of both CF patients and healthy individuals and could contribute to the colonization of multiresistant strains.

Is Genetic Mobilization Considered When Using Bacteriophages in Antimicrobial Therapy?

The emergence of multi-drug resistant bacteria has undermined our capacity to control bacterial infectious diseases. Measures needed to tackle this problem include controlling the spread of antibiotic resistance, designing new antibiotics, and encouraging the use of alternative therapies. Phage therapy seems to be a feasible alternative to antibiotics, although there are still some concerns and legal issues to overcome before it can be implemented on a large scale. Here we highlight some of those concerns, especially those related to the ability of bacteriophages to transport bacterial DNA and, in particular, antibiotic resistance genes.

Bacteriophages infecting Bacteroides as a marker for microbial source tracking.

Bacteriophages infecting certain strains of Bacteroides are amid the numerous procedures proposed for tracking the source of faecal pollution. These bacteriophages fulfil reasonably well most of the requirements identified as appropriate for a suitable marker of faecal sources. Thus, different host strains are available that detect bacteriophages preferably in water contaminated with faecal wastes corresponding to different animal species. For phages found preferably in human faecal wastes, which are the ones that have been more extensively studied, the amounts of phages found in waters contaminated with human fecal samples is reasonably high; these amounts are invariable through the time; their resistance to natural and anthropogenic stressors is comparable to that of other relatively resistant indicator of faecal pollution such us coliphages; the abundance ratios of somatic coliphages and bacteriophages infecting Bacteroides thetaiotaomicron GA17 are unvarying in recent and aged contamination; and standardised detection methods exist. These methods are easy, cost effective and provide data susceptible of numerical analysis. In contrast, there are some uncertainties regarding their geographical stability, and consequently suitable hosts need to be isolated for different geographical areas. However, a feasible method has been described to isolate suitable hosts in a given geographical area. In summary, phages infecting Bacteroides are a marker of faecal sources that in our opinion merits being included in the "toolbox" for microbial source tracking. However, further research is still needed in order to make clear some uncertainties regarding some of their characteristics and behaviour, to compare their suitability to the one of emerging methods such us targeting Bacteroidetes by qPCR assays; or settling molecular methods for their determination.

Detection of quinolone-resistant Escherichia coli isolates belonging to clonal groups O25b:H4-B2-ST131 and O25b:H4-D-ST69 in raw sewage and river water in Barcelona, Spain.

OBJECTIVES: The present study was carried out to evaluate the prevalence of clonal group O25b:H4-B2-ST131 in water environments with faecal pollution (urban sewage and river water) in the north-east of Spain and to study the virulence gene content of environmental isolates and to compare them with isolates causing human extraintestinal infections in Spain. METHODS: This study was performed with 10 sewage samples (collected in Catalonia, north-eastern Spain, in autumn 2009 from the influent raw urban sewage of a wastewater treatment plant that serves a large urban area) and 6 river water samples (collected monthly from February to April 2010 in the Llobregat river catchment area, near Barcelona, a watercourse subjected to heavy anthropogenic pressure). Escherichia coli colonies were screened by PCR for the rfbO25b gene associated with the clonal group O25b:H4-B2-ST131. Sequence types (STs), serotypes, virulence genes, PFGE profiles, antimicrobial resistance and extended-spectrum ß-lactamase (ESBL) enzymes were determined in 75 E. coli isolates positive for the O25b molecular subtype. RESULTS: Of the 75 O25b-positive isolates, 51 belonged to the O25b:H4-B2-ST131 clonal group and the remaining 24 belonged to clonal group O25b:H4-D-ST69. The majority of ST69 isolates (23 of 24) were isolated from urban sewage, whereas ST131 isolates were isolated from urban sewage (25 isolates) as well as from river water (26 isolates). ST131 and ST69 isolates carried 4-13 virulence genes, the majority (82%) being quinolone resistant. CONCLUSIONS: We showed the presence of E. coli isolates belonging to clonal groups O25b:H4-B2-ST131 and O25b:H4-D-ST69 in raw sewage and river water in Barcelona. Furthermore, we observed that the environmental O25b:H4-B2-ST131 isolates showed similar virulence and macrorestriction profiles to clinical human isolates. To our knowledge, this is the first study describing the O25b:H4-D-ST69 clonal group.

Detection of somatic coliphages through a bioluminescence assay measuring phage mediated release of adenylate kinase and adenosine 5'-triphosphate.

The feasibility of detecting somatic coliphages by phage infection of Escherichia coli WG5 and measurement of phage propagation by the lysis mediated release of the bacterial host adenylate kinase (AK) and adenosine 5'-triphosphate (ATP) detected by a bioluminescent signal was evaluated. After 2h of incubation, all cultures infected with reference bacteriophage phiX174 showed a significant increase in the bioluminescent signal, even with number of phages as low as less of 10 plaque forming units (PFU). Naturally occurring somatic coliphages ensured a significant bioluminescent signal after 3h of infection when >10 PFU were inoculated. These results indicate that an easy and reliable method to detect low numbers of coliphages in less than 3h is feasible.

Microbial indicators and pathogens: removal, relationships and predictive capabilities in water reclamation facilities.

Four water reclamation facilities in north-eastern Spain were monitored over 2 years to determine the occurrence and concentrations of a set of microbial indicators (total coliforms, Escherichia coli, enterococci, spores of sulphite reducing clostridia, somatic coliphages, F-specific RNA phages, phages infecting Bacteroides fragilis strain RYC2056 and phages infecting Bacteroides tethaiotaomicron strain GA-17), and two selected pathogens (cytopathogenic enteroviruses and viable Cryptosporidium oocysts). The indicator (survival) and index (presence) functions of the various indicators tested were evaluated through the wastewater treatments. The inactivation pattern of all groups of bacteriophages tested was closer to the inactivation of enteroviruses than to the inactivation of the conventional bacterial indicators tested. The inactivation of sulfite reducing clostridia spores and bacteriophages more closely approximates the reduction of viable Cryptosporidium than do the conventional bacterial indicators. We observed neither index functions nor a predictive relationship between any of microbial indicators and viable Cryptosporidium oocysts. In contrast, several regression models (r>0.6) and discriminant functions (67-88% well classified samples) based mostly on numbers of bacteriophages were able to predict both the presence and concentrations of enteroviruses. A combination of both bacterial and bacteriophage indicators seem to be the best choice for ensuring the microbial quality of reclaimed water.

Integrated analysis of established and novel microbial and chemical methods for microbial source tracking.

Several microbes and chemicals have been considered as potential tracers to identify fecal sources in the environment. However, to date, no one approach has been shown to accurately identify the origins of fecal pollution in aquatic environments. In this multilaboratory study, different microbial and chemical indicators were analyzed in order to distinguish human fecal sources from nonhuman fecal sources using wastewaters and slurries from diverse geographical areas within Europe. Twenty-six parameters, which were later combined to form derived variables for statistical analyses, were obtained by performing methods that were achievable in all the participant laboratories: enumeration of fecal coliform bacteria, enterococci, clostridia, somatic coliphages, F-specific RNA phages, bacteriophages infecting Bacteroides fragilis RYC2056 and Bacteroides thetaiotaomicron GA17, and total and sorbitol-fermenting bifidobacteria; genotyping of F-specific RNA phages; biochemical phenotyping of fecal coliform bacteria and enterococci using miniaturized tests; specific detection of Bifidobacterium adolescentis and Bifidobacterium dentium; and measurement of four fecal sterols. A number of potentially useful source indicators were detected (bacteriophages infecting B. thetaiotaomicron, certain genotypes of F-specific bacteriophages, sorbitol-fermenting bifidobacteria, 24-ethylcoprostanol, and epycoprostanol), although no one source identifier alone provided 100% correct classification of the fecal source. Subsequently, 38 variables (both single and derived) were defined from the measured microbial and chemical parameters in order to find the best subset of variables to develop predictive models using the lowest possible number of measured parameters. To this end, several statistical or machine learning methods were evaluated and provided two successful predictive models based on just two variables, giving 100% correct classification: the ratio of the densities of somatic coliphages and phages infecting Bacteroides thetaiotaomicron to the density of somatic coliphages and the ratio of the densities of fecal coliform bacteria and phages infecting Bacteroides thetaiotaomicron to the density of fecal coliform bacteria. Other models with high rates of correct classification were developed, but in these cases, higher numbers of variables were required.

Tracking the origin of faecal pollution in surface water: an ongoing project within the European Union research programme.

The objectives of this study are to generate knowledge about methods to track the sources of faecal pollution in surface waters, with the aim of having one or a few easy procedures applicable to different geographic areas in Europe. For this, a first field study using already proposed methods (genotypes of F-specific RNA bacteriophages, bacteriophages infecting Bacteroides fragilis, phenotypes of faecal coliforms and enterococci, and sterols) has been done in five areas representing a wide array of conditions in Europe. The present faecal indicators (faecal coliforms, enterococci, sulfite reducing clostridia and somatic coliphages) have also been included in this first field study. At the same time some emerging methods have been settled or adapted to water samples and assayed in a limited number of samples. The results of this first field study indicate that no single parameter alone is able to discriminate the sources, human or non-human, of faecal pollution, but that a 'basket' of 4 or 5 parameters, which includes one of the present faecal indicators, will do so. In addition, numerical analysis of the data shows that this 'basket' will allow the successful building of predictive models. Both the statistical analyses and the studied predictive models indicate that genotype II of F-specific RNA bacteriophages, the coprostanol and the ratio coprostanol: coprostanol+epicoprostanol are, out of the studied parameters, those with a greater discriminating power. Either because unsuccessful adaptation of the methods to water samples or because the preliminary assays in water samples indicated low discriminating capability, only three (sorbitol-fermenting bifidobacteria, some species of bifidobacteria detected by PCR with specific primers and phages infecting Bacteroides tethaiotaomicron) of the newly assayed methods have been considered for a second field study, which is currently underway. Expectations are that these new tools will minimize the number of parameters in the 'basket', or at least minimize the difficulty in assaying them.

Homogeneity of the morphological groups of bacteriophages infecting Bacteroides fragilis strain HSP40 and strain RYC2056.

Bacteriophages infecting Bacteroides fragilis strains RYC2056 and HSP40 have been proposed as indicators of water quality. To accomplish this function, homogeneity of the group of phages detected by these strains is necessary to ensure that the final results are not due to the different kinetics of inactivation of the phages. To evaluate homogeneity, we observed by electron microscopy bacteriophages isolated from sewage with two Bacteroides fragilis strains (HSP40 and RYC2056). A predominant group of phages was observed, Siphoviridae with slightly curved tails. Detection of other minority groups, which could be present in the sample, was done with neutralization experiments by using antiserum against the majority group and with host mutants resistant to infection with the predominant phage. Although two other minority groups were observed, results showed that bacteriophages infecting B. fragilis strain HSP40 and strain RYC2056 form a homogeneous group, Siphoviridae with slightly curved tails being the most predominant in sewage.

The effect of reducing agents on the recovery of injured Bifidobacterium cells.

Bifidobacterium spp. have been proposed as a microbial indicator of faecal pollution in water and as possible probiotic in fermented dairy products. These anaerobic bacteria can be subject to metabolic stress in environmental samples because of the presence of oxygen or its derivatives. In this case, their recovery is compromised, especially in selective media. Three reducing agents were tested to improve the recovery of oxygen-stressed bifidobacteria: L-cysteine, sodium pyruvate and sodium thioglycolate. These agents were evaluated individually at various concentrations, and in combination by measuring recovery on rich and selective media. The addition of several mixtures of reducing agents to the water samples and pre-incubation for 4 h at 37 degrees C increased the recovery of Bifidobacterium spp. on culture media.

Genetic analysis of hepatitis A virus strains recovered from the environment and from patients with acute hepatitis.

The molecular epidemiology of hepatitis A virus (HAV) was studied by analysing HAV strains recovered from environmental water samples over a 7 year period and strains recovered from patients with acute hepatitis over a 5 year period. A total of 54 samples of raw domestic sewage and 66 samples of river water were collected. HAV particles were concentrated and detected by nested RT-PCR. HAV infection in patients with acute hepatitis was serologically diagnosed in 26 of 74 serum samples, which were also analysed by nested RT-PCR. HAV RNA was detected in 57.4% of sewage samples, 39.2% of Llobregat river water samples, 20% of Ter river water samples and 61.6% of serum samples. The HAV genomes detected were characterized further by directly sequencing a region of the 5' non-translated region, the VP1/2A junction region and, in some samples, the 2B region. Results showed a 95% prevalence of genotype I, with nearly 50% being either subgenotype IA or subgenotype IB. Various strains were found simultaneously in both environmental and clinical samples. These strains were closely related to those described in distant geographical areas. Genotype IIIA was also found in 5% of sewage samples and in 12.5% of serum samples. Strains belonging to a common endemic genotype were not identified. The abundance of HAV in the environment produces a situation of sanitary risk, especially considering the low prevalence of antibodies in the young population.

Identification of cell wall proteins of Bacteroides fragilis to which bacteriophage B40-8 binds specifically.

Bacteriophage infecting Bacteroides fragilis, one of the most abundant bacteria in the human colon, have been proposed as indicators of virological faecal pollution. The first identification of a receptor for a bacteriophage in B. fragilis is reported here. First, resistant mutants were characterized following phage inactivation, and it was shown that cell wall proteins are involved in phage binding. Then the proteins involved were identified by various approaches: (i) comparison of the protein profiles of wild-type B. fragilis HSP40 and phage-resistant mutants; (ii) application of a modification of the virus overlay protein blot assay (VOPBA). At least two proteins of B. fragilis, with apparent molecular masses of 35 +/- 5 kDa and 65 +/- 5 kDa, bind to B40-8. This result was later confirmed by running a complex consisting of this phage bound to radiolabelled proteins of B. fragilis on an immunoaffinity column loaded with a specific antibody against the phage. Cell proteins retained in the column also coincided with the proteins that differed in the profiles of resistant mutants. Finally, to identify the potential function of these two proteins, their N-terminal sequences were determined and compared to published sequences, but no homologies were found.