Bacteriophages active against Bacteroides fragilis in sewage-polluted waters

Twelve strains of different Bacteroides species were tested for their efficiency of detection of bacteriophages from sewage. The host range of several isolated phages was investigated. The results indicated that there was a high degree of strain specificity. Then, by using Bacteroides fragilis HSP 40 as the host, which proved to be the most efficient for the detection of phages, feces from humans and several animal species and raw sewage, river water, water from lagoons, seawater, groundwater, and sediments were tested for the presence of bacteriophages that were active against B. fragilis HSP 40. Phages were detected in feces of 10% of the human fecal samples tested and was never detected in feces of the other animal species studied. Moreover, bacteriophages were always recovered from sewage and sewage-polluted samples of waters and sediments, but not from nonpolluted samples. The titers recovered were dependent on the degree of pollution in analyzed waters and sediments.     

Diversity of bacteroides fragilis strains in their capacity to recover phages from human and animal wastes and from fecally polluted wastewater

Great differences in capability to detect bacteriophages from urban sewage of the area of Barcelona existed among 115 strains of Bacteroides fragilis. The capability of six of the strains to detect phages in a variety of feces and wastewater was studied. Strains HSP40 and RYC4023 detected similar numbers of phages in urban sewage and did not detect phages in animal feces. The other four strains detected phages in the feces of different animal species and in wastewater of both human and animal origin. Strain RYC2056 recovered consistently higher counts than the other strains and also detected counts ranging from 10(1) to approximately 10(3) phages per ml in urban sewage from different geographical areas. This strain detected bacteriophages in animal feces even though their relative concentration with respect to the other fecal indicators was significantly lower in wastewater polluted with animal feces than in urban sewage.     

Diversity of Bacteroides fragilis Strains in Their Capacity To Recover Phages from Human and Animal Wastes and from Fecally Polluted Wastewater

Great differences in capability to detect bacteriophages from urban sewage of the area of Barcelona existed among 115 strains of Bacteroides fragilis. The capability of six of the strains to detect phages in a variety of feces and wastewater was studied. Strains HSP40 and RYC4023 detected similar numbers of phages in urban sewage and did not detect phages in animal feces. The other four strains detected phages in the feces of different animal species and in wastewater of both human and animal origin. Strain RYC2056 recovered consistently higher counts than the other strains and also detected counts ranging from 10¹ to approximately 10³ phages per ml in urban sewage from different geographical areas. This strain detected bacteriophages in animal feces even though their relative concentration with respect to the other fecal indicators was significantly lower in wastewater polluted with animal feces than in urban sewage.     

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.     

Human origin of Bacteroides fragilis bacteriophages present in the environment

Bacteroides fragilis HSP40 phages have been detected in waters with various levels of fecal contamination of human origin. The average numbers of B. fragilis phages present in sewage water reached 5.3 x 10(3) per 100 ml of water. We found a number 1,000 times lower in a river contaminated with domestic sewage only, in which the levels of fecal coliforms and fecal streptococci were 10,000 times lower than those found in raw sewage. In addition, B. fragilis phages were not found in significant numbers in slaughterhouse wastewaters. They were not present in fecal-polluted waters containing fecal contamination from wildlife only. Although the number of B. fragilis phages present in contaminated waters was lower than the number of coliphages, their presence indicated human fecal contamination. It is also shown that Bacteroides phages are only able to multiply under anaerobic conditions in the presence of nutrients, and they cannot multiply in natural waters and sediments.     

Human origin of Bacteroides fragilis bacteriophages present in the environment.

Bacteroides fragilis HSP40 phages have been detected in waters with various levels of fecal contamination of human origin. The average numbers of B. fragilis phages present in sewage water reached 5.3 x 10(3) per 100 ml of water. We found a number 1,000 times lower in a river contaminated with domestic sewage only, in which the levels of fecal coliforms and fecal streptococci were 10,000 times lower than those found in raw sewage. In addition, B. fragilis phages were not found in significant numbers in slaughterhouse wastewaters. They were not present in fecal-polluted waters containing fecal contamination from wildlife only. Although the number of B. fragilis phages present in contaminated waters was lower than the number of coliphages, their presence indicated human fecal contamination. It is also shown that Bacteroides phages are only able to multiply under anaerobic conditions in the presence of nutrients, and they cannot multiply in natural waters and sediments.     

Homogeneity of the Morphological Groups of Bacteriophages Infecting <Emphasis Type="Italic">Bacteroides fragilis</Emphasis> 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. Received: 7 March 2002 / Accepted: 5 August 2002     

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.     

Effect of distance from the polluting focus on relative concentrations of Bacteroides fragilis phages and coliphages in mussels.

Concentrations of fecal bacteria, somatic and F-specific coliphages, and phages infecting Bacteroides fragilis in naturally occurring black mussels (Mytilus edulis) were determined. Mussels were collected over a 7-month period at four sampling sites with different levels of fecal pollution. Concentrations of both fecal bacteria and bacteriophages in mussel meat paralleled the concentration of fecal bacteria in the overlying waters. Mussels bioaccumulated efficiently, although with different efficiencies, all of the microorganisms studied. Ratios comparing the levels of microorganisms in mussels were determined. These ratios changed in mussels collected at the different sites. They suggest that bacteriophages infecting B. fragilis and somatic coliphages have the lowest decay rates among the microorganisms studied, with the exception of Clostridium perfringens. On the contrary, concentrations of F-specific coliphages showed a greater rate of decay than the other bacteriophages at sites more distant from the focus of contamination. Additionally, levels of enteroviruses were studied in a number of samples, and in these samples, the B. fragilis bacteriophages clearly outnumbered the enteroviruses. The results of this study indicate that, under the environmental conditions studied, the fate of phages infecting B. fragilis released into the marine environment resembles that of human viruses more than any other microorganism examined.     

Phages of enteric bacteria in fresh water with different levels of faecal pollution

Levels of somatic and F-specific coliphages, and phages infecting Bacteroides fragilis were measured in 257 samples collected in different freshwater environments with different levels and characteristics of faecal pollution. In samples with recent pollution of domestic origin, the numbers of the three groups of phages were highly correlated, thus showing that their excretion is fairly constant. In this set of samples somatic coliphages, which were the most abundant, and F-specific coliphages outnumbered significantly Bact. fragilis phages. Normalized lines of the numbers of the three groups of phages in water samples and their sediments show that they settle similarly. The correlation between the values of the three groups of phages was not observed in waters with intermediate levels of pollution. An increase in the relative numbers of coliphages with respect to numbers of phages infecting Bact. fragilis was observed. In waters with persistent faecal pollution a dramatic change was recorded in the relative numbers of the different groups of phages. Phages infecting Bact. fragilis suffered the lowest reduction in numbers.     

Virological quality of the Ria de Aveiro: Validity of potential microbial indicators

The summer occurrence of human enteroviruses and rotaviruses in the bacteriologically clean area of the Ria de Aveiro, a coastal marine lagoon, prompted the question of the assessment of the virological quality of recreational waters and shellfish raising beds. Enteroviruses were present in surface water at a density of 3 pfu 10 l^–1 and were accumulated in sediments and, especially, in cockles where they reached concentrations 2 to 3¹⁰log units greater. Rotaviruses were detected at one¹⁰log unit below the density of enteroviruses in sediments and cockles and were not detected in water. Four bacteriophage systems were assayed as indicators of human enteric viruses: somatic coliphages ofE.coli C, sexual and sexual-RNA coliphages plated onSalmonella WG49 and phages againstBacteroides fragilis HSP40. The results obtained from 2 lagoon stations sampled in summer, autumn and winter showed that the four systems failed to indicate the presence of enteroviruses and rotaviruses in water, sediment and shellfish samples. The absence of phages ofB. fragilis HSP40 in all types of samples taken from the lagoon, but not from the residual waters of the treatment station, suggests that they may suffer a strong negative pressure in this ecosystem as their proportion to the coliphages in the cockles deviated strongly from the ratio of 1100 to 11000 observed at the sewage outfall. In fact, no correlation was observed between these phages and enteric viruses or coliphages. Alternatively, it is possible that the importance of diffuse faecal pollution and the interference of faecal pollutants of animal origin, including migratory sea birds which are abundant in winter, can alter the proportions of the faecal bacteriophages beyond recognition. It is apparent that bacteriophage monitoring of the health risk linked to the occurrence of viruses in the marine environment is not yet fully resolved, what may leave viral quality assessment dependent on direct detection of human enteric virus.     

Correlation between bacterial indicators and bacteriophages in sewage and sludge

The use of bacteriophages as potential indicators of faecal pollution has recently been studied. The correlation of the number of bacterial indicators and the presence of three groups of bacteriophages, namely somatic coliphages (SOMCPH), F-RNA-specific phages (FRNAPH) and phages of Bacteroides fragilis (BFRPH), in raw and treated wastewater and sludge is presented in this study. Raw and treated wastewater and sewage sludge samples from two wastewater treatment plants in Athens were collected on a monthly basis, over a 2-year period, and analysed for total coliforms, Escherichia coli, intestinal enterococci and the three groups of bacteriophages. A clear correlation between the number of bacterial indicators and the presence of bacteriophages was observed. SOMCPH may be used as additional indicators, because of their high densities and resistance to various treatment steps.     

A simple, rapid and sensitive presence/absence detection test for bacteriophage in drinking water

R. ARMON AND Y. KOTT. 1993. A rapid, simple and sensitive direct bacteriophage presence detection method for 500 ml drinking water samples has been developed. The method includes a glass device consisting of a jar containing the water sample and an immersible probe filled with solidified soft agar containing bacterial host cells. Host bacteria in logarithmic phase were added to the experimental volume and the probe was submerged. The entire device was incubated in a water bath at 36C.
Plaques of somatic bacteriophage infecting Escherichia coli strain CN₁₃, could be detected within 3 h. Male-specific bacteriophages infecting E. coli F+ amp were detected within 6 h. Bacteriophage infecting the anaerobe Bacteroides fragilis subsp. fragilis HSP40 were detected after 8 h. Application of this device and the associated technique, enabled a one-step detection of 1 pfu of E. coli or Bact. fragilis specific bacteriophage in 500 ml drinking water samples.     

Removal and inactivation of indicator bacteriophages in fresh waters

AIMS: The removal and inactivation of faecal coliform (FC) bacteria, enterococci (ENT), sulphite-reducing clostridia (SRC), somatic coliphages, F-specific RNA bacteriophages and bacteriophages infecting Bacteroides fragilis in fresh waters. METHODS AND RESULTS: Removal was studied in two areas of a river. The results showed different removal of each group of microbes. Faecal coliform bacteria were removed faster than any other, whereas SRC and bacteriophages infecting Bact. fragilis were the most persistent. Inactivation was measured by 'in situ' experiments, which showed significant differences in survival of the different groups of bacterial and bacteriophage indicators. The SRC and bacteriophages were more resistant than faecal coliforms and enterococci, with the exception of F-specific RNA bacteriophages in the summer. Inactivation experiments with pure cultures of bacteriophages confirmed that phage B40-8 of Bact. fragilis was the most resistant. CONCLUSIONS: Bacteria and bacteriophages show different resistance to natural inactivation. The use of phages allows information to be obtained in addition to that provided by bacterial indicators. Somatic coliphages and phages infecting Bact. fragilis might supply that indicator function. SIGNIFICANCE AND IMPACT OF THE STUDY: Confirmation was obtained that bacteriophages provided additional information to that provided by bacterial indicators to monitor the natural inactivation of viruses and/or pathogens.     

Usefulness of different groups of bacteriophages as model micro-organisms for evaluating chlorination

AIMS: To assess the usefulness of bacterial and viral indicators in chlorination processes and to collect quantitative information necessary for risk assessment analysis in water disinfection processes based on chlorination. METHODS AND RESULTS: Naturally occurring bacterial indicators, bacteriophages and enteroviruses were determined to evaluate the effect of chlorination in groundwater and secondary sewage effluents. Additionally, the effect of chlorinating on selected bacteriophages, enteroviruses and Escherichia coli was also tested in spiked samples of bottled water and sewage effluents. Results indicate that chlorination inactivates more efficiently bacteria than phages and enteroviruses. Among the human viruses, phages infecting Bacteroides fragilis and selected somatic coliphages belonging to the Siphoviridae family were the most persistent to chlorination. CONCLUSIONS: The three groups of bacteriophages studied were all more resistant to chlorination than bacteria and some of the phages were more resistant than enteroviruses. Results presented here indicate that it is very risky to generalize from information obtained with inactivation experiments done with single isolates of any phage or virus. If possible, inactivation studies should be done with naturally occurring populations. Phages offer a good opportunity for studying naturally occurring populations. Thus, the bacteriophages offer a range of resistance to chlorination that may represent most of the viruses that can be found in water. SIGNIFICANCE AND IMPACT OF THE STUDY: Data reported in this study support the inclusion of bacteriophages as additional indicators of the efficiency of water chlorination processes and water quality.     

Potential usefulness of bacteriophages that infect Bacteroides fragilis as model organisms for monitoring virus removal in drinking water treatment plants.

The presence of bacteriophages at different stages in three drinking water treatment plants was evaluated to study the usefulness of phages as model organisms for assessing the efficiency of the processes. The bacteriophages tested were somatic coliphages, F-specific coliphages, and phages infecting Bacteroides fragilis. The presence of enteroviruses and currently used bacterial indicators was also determined. Most bacteriophages were removed during the prechlorination-flocculation-sedimentation step. In these particular treatment plants, which include prechlorination, phages were, in general, more resistant to the treatment processes than present bacterial indicators, with the exception, in some cases, of clostridia. Bacteriophages infecting B. fragilis were found to be more resistant to water treatment than either somatic or F-specific coliphages or even clostridia. Enteric viruses were found only in untreated water in low numbers, and consequently, the efficiency of the plants in the removal of viruses could not be evaluated with precision. The numbers and frequencies of detection of the various microorganisms in water samples taken in the distribution network served by the three plants confirm the results found in the finished water at the plants.     

Occurrence and levels of phages proposed as surrogate indicators of enteric viruses in different types of sludges

A method based on the treatment of sludge with beef extract recovered, with similar efficiency, the three groups of bacteriophages studied from different kinds of sludges. The three groups of bacteriophages were found in high numbers in the different sludge types, the highest value being that of somatic coliphages in primary sludge of a biological treatment plant (1.1 x 10(5) pfu g-1) and the lowest being that of Bacteroides fragilis phages (110 pfu g-1) in de-watered, anaerobically, mesophilically-digested sludge. All phages studied accumulated in the sludges. In primary and activated sludges, all three types accumulated similarly but in lime-treated sludge and de-watered, anaerobically, mesophilically-digested sludge, the relative proportion of F-specific bacteriophages decreased significantly with respect to somatic coliphages and bacteriophages infecting B. fragilis. All phages survived successfully in stored sludge, depending on the temperature, and again, F-specific bacteriophages survived less successfully than the others.     

Survival of bacterial indicator species and bacteriophages after thermal treatment of sludge and sewage

The inactivation of naturally occurring bacterial indicators and bacteriophages by thermal treatment of a dewatered sludge and raw sewage was studied. The sludge was heated at 80 degrees C, and the sewage was heated at 60 degrees C. In both cases phages were significantly more resistant to thermal inactivation than bacterial indicators, with the exception of spores of sulfite-reducing clostridia. Somatic coliphages and phages infecting Bacteroides fragilis were significantly more resistant than F-specific RNA phages. Similar trends were observed in sludge and sewage. The effects of thermal treatment on various phages belonging to the three groups mentioned above and on various enteroviruses added to sewage were also studied. The results revealed that the variability in the resistance of phages agreed with the data obtained with the naturally occurring populations and that the phages that were studied were more resistant to heat treatment than the enteroviruses that were studied. The phages survived significantly better than Salmonella choleraesuis, and the extents of inactivation indicated that naturally occurring bacteriophages can be used to monitor the inactivation of Escherichia coli and Salmonella.     

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.