Effect of high hydrostatic pressure on four genotypes of F-specific RNA bacteriophages

AIMS: The pressure responses of four genotypes of F-specific RNA bacteriophages, f2, GA, Qbeta and SP, were evaluated with respect to pressure magnitude, treatment temperature and suspending medium. METHOD AND RESULTS: The pressure responses were studied with respect to pressure magnitude (350 to 600 MPa), treatment temperature (-10 to 50 degrees C) and suspending media. Phages f2 and GA had much higher pressure resistances than Qbeta and SP. Pressure resistances of Qbeta and SP were enhanced with increase in salt concentrations in the range of 350 to 600 MPa from -10 to 50 degrees C in PBS. Qbeta and SP had greater pressure resistances when suspended in phosphate-buffered saline (PBS) with added glucose (5%, w/w), UHT whole milk and Dulbecco's Modified Eagle's Medium plus 10% fetal bovine sera than they did in PBS. Two surfactants, sucrose laurate and monolaurin, and one chelating agent, ethylenediamine tetraacetic acid (EDTA), increased the pressure resistance of Qbeta and SP, but had modest effect on either f2 or GA. CONCLUSIONS: Four representative F-specific RNA bacteriophages, f2 (serotype I), GA (serotype II), Qbeta (serotype III) and SP (serotype IV) showed different resistances to hydrostatic pressure in the range of 350-600 MPa. Significance and Impact of the Study: This study screened for practical surrogates of HAV for validation of commercial high hydrostatic pressure processing.     

Distribution of F-specific bacteriophages and coliphages in wastewater

A new method for quantifying F-specific bacteriophages in wastewater is described. Somatic coliphages were also determined. Host-strainSalmonella typhimurium WG 49 was sensitive to only a few bacteriophages and this could have arisen from infection by F-RNA phages. Host-strainEscherichia coli ATCC 9723 C, however, supported multiplication of a wide range of bacteriophages present in sewage, giving plaque counts one to three orders of magnitude greater than those on F⁺ and F^- salmonellas.L. Bonadonna, R. Liberti and L. Volterra are with the Istituto Superiore di Sanita, Lab. Igiene Ambientale, Viale Regina Elena 299, 00161 Rome, Italy.     

F-specific RNA bacteriophages and sensitive host strains in faeces and wastewater of human and animal origin

Faeces of humans, pigs, cattle and chickens were investigated for the presence of somatic coliphages, F-specific bacteriophages and Escherichia coli strains sensitive to infection by F-specific phages. Attention was given to the possible effect of age and use of antibiotics on the prevalence of the FRNA phages and sensitive E . coli strains. Somatic coliphages were often detected in high numbers in all types of faeces. In contrast, FRNA phages were rarely detected in faeces from humans and cattle but more often in faeces from pigs and adult chickens. Samples from young chickens (with or without antibiotics) were consistently positive for FRNA phages (up to 3 × 10⁶ pfu/g). F-specific RNA phages were found in substantial numbers (> 10³ pfu/ml) in a variety of wastewaters: domestic, hospital, slaughterhouses and occasionally in 'grey water'. Their origin in wastewater was not clear. Strains from faeces usually belonged to serogroups I and IV. These types were also found in wastewater, as were group II and III strains. Serogroup II phages were abundant in wastewater of human origin but rare in faeces. Escherichia coli strains sensitive to infection by F-specific phages were common in faeces (overall 290/1081: 27%). No strains with fully derepressed F-pilus synthesis were detected among the sensitive strains. It is concluded that the occurrence of F-specific RNA bacteriophages in water points to sewage pollution rather than faecal pollution; the mechanism of replication of these organisms in wastewater is not understood.     

F-specific RNA bacteriophages and sensitive host strains in faeces and wastewater of human and animal origin

Faeces of humans, pigs, cattle and chickens were investigated for the presence of somatic coliphages, F-specific bacteriophages and Escherichia coli strains sensitive to infection by F-specific phages. Attention was given to the possible effect of age and use of antibiotics on the prevalence of the FRNA phages and sensitive E. coli strains. Somatic coliphages were often detected in high numbers in all types of faeces. In contrast, FRNA phages were rarely detected in faeces from humans and cattle but more often in faeces from pigs and adult chickens. Samples from young chickens (with or without antibiotics) were consistently positive for FRNA phages (up to 3 x 10(6) pfu/g). F-specific RNA phages were found in substantial numbers (greater than 10(3) pfu/ml) in a variety of wastewaters: domestic, hospital, slaughterhouses and occasionally in 'grey water'. Their origin in wastewater was not clear. Strains from faeces usually belonged to serogroups I and IV. These types were also found in wastewater, as were group II and III strains. Serogroup II phages were abundant in wastewater of human origin but rare in faeces. Escherichia coli strains sensitive to infection by F-specific phages were common in faeces (overall 290/1081: 27%). No strains with fully depressed F-pilus synthesis were detected among the sensitive strains. It is concluded that the occurrence of F-specific RNA bacteriophages in water points to sewage pollution rather than faecal pollution; the mechanism of replication of these organisms in wastewater is not understood.     

Distribution of the human faecal bacterium Bacteroides fragilis, its bacteriophages and their relationship to current sewage pollution indicators in bathing water

Although several bacteria are currently used as possible indicators of human pathogens in sewage-polluted sea water, they are often viewed as inadequate and especially inadequate as indicators of viral pathogens. This study investigates the distribution of Bacteroides fragilis and closely related Bacteroides spp. and their associated bacteriophages in sea water frequently used for recreational purposes. These organisms may provide a potentially more appropriate indicator. Bacteroides fragilis is one of about 10 species which are loosely placed together in the 'B. fragilis' group. Samples down-current from a sewage outfall were examined for the presence of B. fragilis group organisms and associated bacteriophages. Numbers were correlated with current bacterial and possible viral indicators at these sites. These B. fragilis group isolates were used as hosts to successfully isolate bacteriophages. The host range of these bacteriophages was investigated. It is hoped to expand this study by using these B. fragilis group hosts and their bacteriophages to identify a more suitable, European-wide, indicator of bacterial pathogens which can also be used to detect bacteriophages which are suitable as viral indicators.     

F-specific RNA bacteriophages are adequate model organisms for enteric viruses in fresh water.

Culturable enteroviruses were detected by applying concentration techniques and by inoculating the concentrates on the BGM cell line. Samples were obtained from a wide variety of environments, including raw sewage, secondary effluent, coagulated effluent, chlorinated and UV-irradiated effluents, river water, coagulated river water, and lake water. The virus concentrations varied widely between 0.001 and 570/liter. The same cell line also supported growth of reoviruses, which were abundant in winter (up to 95% of the viruses detected) and scarce in summer (less than 15%). The concentrations of three groups of model organisms in relation to virus concentrations were also studied. The concentrations of bacteria (thermotolerant coliforms and fecal streptococci) were significantly correlated with virus concentrations in river water and coagulated secondary effluent, but were relatively low in disinfected effluents and relatively high in surface water open to nonhuman fecal pollution. The concentrations of F-specific RNA bacteriophages (FRNA phages) were highly correlated with virus concentrations in all environments studied except raw and biologically treated sewage. Numerical relationships were consistent over the whole range of environments; the regression equations for FRNA phages on viruses in river water and lake water were statistically equivalent. These relationships support the possibility that enteric virus concentrations can be predicted from FRNA phage data.     

Use of yellow-pigmented enterococci as a specific indicator of human and nonhuman sources of faecal pollution

Antibiotic susceptibility tests and restriction enzyme analysis (REA) of genomic DNA were performed to characterize the relationship between sources of isolates of yellow-pigmented enterococci. Antibiotic susceptibility tests were conducted with 10 therapeutic antibiotics and 54 isolates grouped by source (wild and other) depending on their origin. In three antibiotics, cephalothin, erythromycin, and vancomycin, there was a significant (p < or = 0.05) association between susceptibility and source. Vancomycin resistance was significantly (p < or = 0.001) higher in isolates from wild sources compared with that in isolates from other sources. The REA technique was performed on genomic DNA obtained from 17 Enterococcus mundtii isolates from: human (3), dog (4), horse (4), Canada goose (4), domestic goose (1), and Enterococcus mundtii ATCC 43186. A total of 12 different DNA types (A-L) were identified. Except for type D, 11 DNA types were unique and were distributed among dog (A, B, and C), human (E), horse (F, G, and H), Canada goose (I, J, and K), and domestic goose (L). Results suggested that vancomycin-susceptibility testing of yellow-pigmented enterococci may have potential value in the identification of sources of faecal pollution, especially when combined with traditional quantitative methods.     

Relevance of bacteroidales and F-specific RNA bacteriophages for efficient fecal contamination tracking at the level of a catchment in France

The relevance of three host-associated Bacteroidales markers (HF183, Rum2Bac, and Pig2Bac) and four F-specific RNA bacteriophage genogroups (FRNAPH I to IV) as microbial source tracking markers was assessed at the level of a catchment (Daoulas, France). They were monitored together with fecal indicators (Escherichia coli and enterococci) and chemophysical parameters (rainfall, temperature, salinity, pH, and turbidity) by monthly sampling over 2 years (n = 240 water samples) and one specific sampling following an accidental pig manure spillage (n = 5 samples). During the 2-year regular monitoring, levels of E. coli, enterococci, total F-specific RNA bacteriophages, and the general Bacteroidales marker AllBac were strongly correlated with one another and with Rum2Bac (r = 0.37 to 0.50, P < 0.0001). Their correlations with HF183 and FRNAPH I and II were lower (r = 0.21 to 0.29, P < 0.001 to P < 0.0001), and HF183 and enterococci were associated rather than correlated (Fisher's exact test, P < 0.01). Rum2Bac and HF183 enabled 73% of water samples that had ≥ 2.7 log(10) most probably number (MPN) of E. coli/100 ml to be classified. FRNAPH I and II enabled 33% of samples at this contamination level to be classified. FRNAPH I and II complemented the water sample classification obtained with the two Bacteroidales markers by an additional 8%. Pig2Bac and FRNAPH III and IV were observed in a small number of samples (n = 0 to 4 of 245). The present study validates Rum2Bac and HF183 as relevant tools to trace fecal contamination originating from ruminant or human waste, respectively, at the level of a whole catchment.     

Detection of Helicobacter pylori DNA in human faeces and water with different levels of faecal pollution in the north-east of Spain

AIMS: To assess the role of water in the faecal transmission of Helicobacter pylori by detecting the DNA of this pathogen in human faecal samples and environmental water samples with a range of faecal pollution from the north-east of Spain. METHODS AND RESULTS: Semi-nested PCR was used to detect H. pylori in stools and water, both matrices with a complex biota. DNA was detected using highly specific primers of an ureA gene fragment. In addition, antigens were used to detect the bacteria in stools. Helicobacter pylori was detected in 33% of 36 human faecal samples and in 66% of wastewater samples, and 11% of river samples, but in none of the spring waters samples. Faecal pollution of the aquatic environment was tested analysing the presence of microbial indicators. CONCLUSIONS: We report the presence of H. pylori DNA in stools and in aquatic environments with different levels of faecal pollution, from the north-east of Spain. In this study a higher number of positive results were obtained in the more faecally polluted waters. SIGNIFICANCE AND IMPACT OF THE STUDY: These data indicate that water may be a vector of H. pylori in its faecal-oral route.     

Sorbitol-fermenting bifidobacteria as specific indicators of human faecal pollution

Bifidobacteria were consistently present in the faeces of both man and pigs but only occasionally in the faeces of cattle and sheep, and they were not isolated from faecal samples from other animals; total counts of bifidobacteria were obtained by membrane filtration with YN-17 medium, a modification of Resnick and Levin's YN-6 medium. Mannitol-fermenting strains of bifidobacteria were isolated from both human and animal faeces, but sorbitol-fermenting strains were obtained only from human samples. These sorbitol-fermenting strains were identified as either Bifidobacterium adolescentis or B. breve and their numbers were obtained by membrane filtration on Human Bifid Sorbitol agar (HBSA). Sorbitol-fermenting bifidobacteria are specific indicators of human faecal pollution of waters and wastewaters.     

Occurrence, survival, and persistence of human adenoviruses and F-specific RNA phages in raw groundwater

Detection of specific genetic markers can rapidly identify the presence of enteric viruses in groundwater. However, comparison of stability characteristics between genetic and infectivity markers is necessary to better interpret molecular data. Human adenovirus serotype 2 (HAdV2), in conjunction with MS2 phages or GA phages, was spiked into raw groundwater microcosms. Viral stability was periodically assessed by both infectivity and real-time PCR methods. The results of this yearlong study suggest that adenoviruses have the most stable persistence profile and an ability to survive for a long time in groundwater. According to a linear regression model, infectivity reductions of HAdV2 ranged from 0.0076 log(10)/day (4°C) to 0.0279 log(10)/day (20°C) and were significantly lower than those observed for phages. No adenoviral genome degradation was observed at 4°C, and the reduction was estimated at 0.0036 log(10)/day at 20°C. Occurrence study showed that DNA of human adenoviruses could be observed in groundwater from a confined aquifer (7 of the 60 samples were positive by real-time PCR), while no fecal indicators were detected. In agreement with the persistence of genetic markers, the presence of adenoviral DNA in groundwater may be misleading in term of health risk, especially in the absence of information on the infective status.     

The influence of rainfall on the incidence of microbial faecal indicators and the dominant sources of faecal pollution in a Florida river

AIMS: An assessment of microbial densities in an urbanized Florida watershed was performed during a period of changing rainfall patterns to investigate the role of climate coupled with urbanization in declining water quality. METHODS AND RESULTS: Concentrations of traditional and alternative faecal indicators were assessed by standard methods over 24 months. Sources of faecal contamination were determined by antibiotic resistance analysis (ARA) of faecal coliform (FC) bacteria. Composite indices of indicator organisms based on a suite of microbial measurements were used to quantify pollution impacts in the river. ARA results found that FC from wild animal sources dominated during the drought, and the relative frequency of FC from human sources increased after cumulative rainfall increased to near-normal levels. CONCLUSIONS: Changes observed in faecal indicator densities and in FC sources during changing rainfall patterns strongly suggest a role of precipitation on the sources and extent of microbial pollution in urbanized coastal watersheds. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial source tracking coupled with a composite index of microbial contamination resulted in a more complete picture of microbial pollution within the river, as opposed to the general practice of reliance on one indicator organism. Improved land use decisions in urban areas are necessary to insure maintenance of coastal environmental health under changing climate patterns and population density.     

Carbon source utilization profiles as a method to identify sources of faecal pollution in water

AIMS: Carbon source utilization profiles as a phenotypic fingerprinting methodology for determining sources of faecal pollution in water were evaluated. METHODS AND RESULTS: Three hundred and sixty-five Enterococcus isolates were collected from known faecal sources in four different geographical regions and were identified to species with the commercial Biolog system. Discriminant analysis (DA) was used to identify the substrate-containing wells that best classified the 365 isolates by source. By using 30 of the 95 wells for the analysis, the average rate of correct classification (ARCC) by source was 92.7% for a human vs non-human two-way classification when isolates from all regions were combined into one library. Corresponding ARCCs for other classification schemes were 81.9% for a four-way classification of human vs livestock vs wildlife vs domestic pets, and 85.7% for a three-way classification without human isolates. When three individual libraries were made based on classification of sources within Enterococcus species, the ARCC was 95.3% for the Ent. faecalis library, 95.8% for the Ent. gallinarum library and 94.7% for the Ent. mundtii library. Thirty Enterococcus isolates (unknown sources) were obtained from each of three stream sites where a specific source of pollution was apparent; 90.0% of the isolates from a human-suspected source were classified as human, 86.6% were classified as livestock from a livestock-suspected site, and 93.3% were classified as wildlife from a wildlife-suspected site. CONCLUSIONS: Phenotypic fingerprinting with carbon source utilization profiles provided levels of correct classification by sources from an Enterococcus library that were in the upper range of those reported in the literature. ARCCs for three Enterococcus species-specific libraries were very high and may be the best approach for further developing this concept and methodology. SIGNIFICANCE ANC IMPACT OF THE STUDY: The results, based on a modest Enterococcus library and a preliminary field validation test, demonstrated the potential for carbon source utilization profiles to be employed as a phenotypic method for determining sources of faecal pollution in water.     

Evaluation of antibiotic resistance analysis and ribotyping for identification of faecal pollution sources in an urban watershed

AIMS: The accuracy of ribotyping and antibiotic resistance analysis (ARA) for prediction of sources of faecal bacterial pollution in an urban southern California watershed was determined using blinded proficiency samples. METHODS AND RESULTS: Antibiotic resistance patterns and HindIII ribotypes of Escherichia coli (n = 997), and antibiotic resistance patterns of Enterococcus spp. (n = 3657) were used to construct libraries from sewage samples and from faeces of seagulls, dogs, cats, horses and humans within the watershed. The three libraries were analysed to determine the accuracy of host source prediction. The internal accuracy of the libraries (average rate of correct classification, ARCC) with six source categories was 44% for E. coli ARA, 69% for E. coli ribotyping and 48% for Enterococcus ARA. Each library's predictive ability towards isolates that were not part of the library was determined using a blinded proficiency panel of 97 E. coli and 99 Enterococcus isolates. Twenty-eight per cent (by ARA) and 27% (by ribotyping) of the E. coli proficiency isolates were assigned to the correct source category. Sixteen per cent were assigned to the same source category by both methods, and 6% were assigned to the correct category. Addition of 2480 E. coli isolates to the ARA library did not improve the ARCC or proficiency accuracy. In contrast, 45% of Enterococcus proficiency isolates were correctly identified by ARA. CONCLUSIONS: None of the methods performed well enough on the proficiency panel to be judged ready for application to environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Most microbial source tracking (MST) studies published have demonstrated library accuracy solely by the internal ARCC measurement. Low rates of correct classification for E. coli proficiency isolates compared with the ARCCs of the libraries indicate that testing of bacteria from samples that are not represented in the library, such as blinded proficiency samples, is necessary to accurately measure predictive ability. The library-based MST methods used in this study may not be suited for determination of the source(s) of faecal pollution in large, urban watersheds.     

Comparative resistance of phage isolates of four genotypes of f-specific RNA bacteriophages to various inactivation processes

The effect of natural inactivation in freshwater, chlorination, ammonia, extreme pHs, temperature, and salt content on phage inactivation was evaluated on mixtures of F-specific RNA bacteriophage isolates belonging to genotypes I, II, III, and IV. The bacteriophages studied were previously but recently isolated from natural samples, characterized as F-specific RNA bacteriophages and genotyped by plaque hybridization with genotype-specific probes. Natural inactivation in river water was modeled by in situ incubation of bacteriophages inside submerged dialysis tubes. After several days bacteriophages of genotype I showed the highest persistence, which was significantly different from that of bacteriophages of genotype II, IV, or III. The pattern of resistance of phages belonging to the various genotypes to extreme pHs, ammonia, temperature, salt concentration, and chlorination was similar. In all cases, phages of genotype I showed the highest persistence, followed by the phages of genotypes II, III, and IV. The phages of genotypes III and IV were the least resistant to all treatments, and resistance of genotypes III and IV to the treatments was similar. Bacteriophages of genotype II showed intermediate resistance to some of the treatments. The resistance of four phages of genotype I to natural inactivation and chlorination did not differ significantly. These results indicate that genotypes III and IV are much more sensitive to environmental stresses and to treatments than the other genotypes, especially than genotype I. This should be taken into consideration in future studies aimed at using genotypes of F-specific RNA bacteriophages to fingerprint the origin of fecal pollution.     

Sorbitol-fermenting bifidobacteria as indicators of diffuse human faecal pollution in estuarine watersheds

Sorbitol fermenting bifidobacteria were evaluated as indicators of non-point source human faecal pollution to three sub-estuaries with elevated faecal coliform densities. Human-specific bifidobacteria correlated with identifiable human sanitary deficiencies in feeder streams to estuarine creeks in two of three watersheds examined, one rural and one moderately developed. Sorbitol-fermenting bifidobacteria were recovered at densities ranging from 1 to 90 colony-forming-units 100 ml-1 in 11 of 258 water samples but were undetected in sediment (n = 68) and scat from resident wildlife (deer, muskrat and raccoon, n = 20). Failure to detect sorbitol-fermenting bifidobacteria in water samples during the summer months was consistent with laboratory microcosm results showing non-recoverability of Bifidobacterium adolescentis after 5-9 d in membrane-filtered estuarine water at 23 and 30 degrees C, but persistence for 4 weeks at 10 degrees C. Persistence of sewage-derived bifidobacteria in membrane-filtered freshwater at 15 degrees C was also observed. Recovery of sorbitol-fermenting bifidobacteria was complicated by high background levels of Gram-positive rods and cocci. Use of propionic acid and reduced pH (pH = 5.0), or use of a two-step resuscitation protocol using non-selective and selective media, did not improve recovery. Although human specific bifidobacteria hold promise as indicators of diffuse faecal contamination, methodological constraints now limit its application to situations of gross contamination, or sampling potential sources during environmental conditions conducive to bifid persistence.     

Evaluation of Bacteroides markers for the detection of human faecal pollution

Aims: This paper reports on the results of a study aimed at evaluating the specificity and sensitivity of human‐specific HF183 and HF134 Bacteroides markers in various host groups and their utility to detect human faecal pollution in storm water samples collected from nonsewered catchments in Southeast Queensland, Australia. Methods and Results: The specificity and sensitivity of the HF183 and HF134 Bacteroides markers was evaluated by testing 207 faecal samples from 13 host groups, including 52 samples from human sources (via sewage and septic tanks). Polymerase chain reaction analysis of these samples revealed the presence/absence of HF183 and HF134 across these host groups, demonstrating their suitability for distinguishing between human and animal faecal pollution. The HF183 marker was found to be more reliable than that of HF134, which was also found in dogs. Conclusions: Based on our data, it appears that the HF183 marker is specific to sewage and is a reliable marker for detecting human faecal pollution, while the use of HF134 marker alone may not be sufficient enough to provide the evidence of human faecal pollution. Significance and Impact of the Study: This is the first study in Australia that rigorously evaluated the specificity and sensitivity of Bacteroides markers. Based on our findings, we suggest that the HF183 marker could reliably be used to detect the sources of human faecal pollution in Southeast Queensland region.