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.     

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.     

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.     

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     

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.     

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.     

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.     

Prevalence of the stx2 gene in coliform populations from aquatic environments

Shiga toxin-producing Escherichia coli strains are human pathogens linked to hemorrhagic colitis and hemolytic uremic syndrome. The major virulence factors of these strains are Shiga toxins Stx1 and Stx2. The majority of the genes coding for these toxins are borne by bacteriophages. Free Stx2-encoding bacteriophages have been found in aquatic environments, but there is limited information about the lysogenic strains and bacteria present in the environment that are susceptible to phage infection. The aim of this work was to study the prevalence and the distribution of the stx(2) gene in coliform bacteria in sewage samples of different origins. The presence of the stx(2) gene was monitored every 2 weeks over a 1-year period in a municipal sewage treatment plant. A mean value of 10(2) genes/ml was observed without significant variation during the study period. This concentration was of the same order of magnitude in raw municipal sewage of various origins and in animal wastewater from several slaughterhouses. A total of 138 strains carrying the stx(2) gene were isolated by colony hybridization. This procedure detected approximately 1 gene-carrying colony per 1,000 fecal coliform colonies in municipal sewage and around 1 gene-carrying colony per 100 fecal coliform colonies in animal wastewaters. Most of the isolates belonged to E. coli serotypes other than E. coli O157, suggesting a low prevalence of strains of this serotype carrying the stx(2) gene in the wastewater studied.     

A method for the enumeration of male-specific bacteriophages in sewage

H avelaar , A.H. & H ogeboom , W.M. 1984. A method for the enumeration of male-specific bacteriophages in sewage. Journal of Applied Bacteriology 56 , 439–447.
Male-specific bacteriophages adsorb to F-pili and thus can only infect male host strains. A method was developed for the selective enumeration of these phages, based on the observation that in sewage there are few phages capable of infecting F^--salmonellas—usually less than 10 pfu/ml. Using a male Salmonella strain, constructed by the introduction of the plasmid F'42 lac::Tn5 into Salmonella typhimu-rium phage type 3, plaque counts in secondary effluent were found to be in the range of 60–8200 pfu/ml. Practically all the phages detected had a host range restricted to male Salmonella or Escherichia coli strains, were resistant to chloroform and their infectivity was inhibited by RNase. Electron microscopy of lysates revealed phage particles that were morphologically identical to the male-specific single-strand RNA phages. Similar results were obtained with a strain of Salm. indiona carrying F'42 lac . A derivative of the Salm. typhimurium LT2 strain carrying an F-plasmid (F'42 lac fin P301) derepressed for fertility inhibition by the resident plasmid pSLT was equally sensitive to male-specific phages, but from sewage samples many other phages infecting F^- E. coli but not F^- Salmonella were isolated using this host strain.     

A method for the enumeration of male-specific bacteriophages in sewage

Male-specific bacteriophages adsorb to F-pili and thus can only infect male host strains. A method was developed for the selective enumeration of these phages, based on the observation that in sewage there are few phages capable of infecting F- -salmonellas--usually less than 10 pfu/ml. Using a male Salmonella strain, constructed by the introduction of the plasmid F'42 lac::Tn5 into Salmonella typhimurium phage type 3, plaque counts in secondary effluent were found to be in the range of 60-8200 pfu/ml. Practically all the phages detected had a host range restricted to male Salmonella or Escherichia coli strains, were resistant to chloroform and their infectivity was inhibited by RNase. Electron microscopy of lysates revealed phage particles that were morphologically identical to the male-specific single-strand RNA phages. Similar results were obtained with a strain of Salm. indiana carrying F'42 lac. A derivative of the Salm. typhimurium LT2 strain carrying an F-plasmid (F'42 lac fin P301) derepressed for fertility inhibition by the resident plasmid pSLT was equally sensitive to male-specific phages, but from sewage samples many other phages infecting F- E. coli but not F- Salmonella were isolated using this host strain.     

Reduction of bacterial indicators and bacteriophages infecting faecal bacteria in primary and secondary wastewater treatments

AIMS: To compare the suitability of various bacterial and viral indicators to assess the removal of faecal micro-organisms by primary and secondary wastewater treatment processes. METHODS AND RESULTS: The numbers of several bacterial indicators [faecal coliforms (FC), enterococci (ENT) and sulphite-reducing clostridia (SRC)] and bacteriophages (somatic coliphages, F-specific RNA phages and bacteriophages infecting Bacteroides fragilis strain RYC2056) were determined in incoming raw sewage and effluents from various primary and secondary wastewater treatment processes in several geographical areas. Reductions in the numbers of indicators were calculated as log10 reductions. Processes based on removal and mild disinfection, showed no significant differences in the elimination of any of the indicators tested or between geographical areas. In contrast, treatment processes that include strong microbial inactivation, such as lime-aided flocculation and lagooning, showed significant differences between the log10 reductions of the various micro-organisms studied, FC showing the highest reduction and spores of SRC and phages infecting B. fragilis the lowest. CONCLUSIONS: The microbial elimination performance of treatment processes based principally on removal and mild disinfection can be evaluated with a single indicator. In contrast, processes with additional disinfecting capabilities require more than one indicator for accurate evaluation of the treatment; bacteriophages are good candidates for use as second indicators. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriophages provide additional information for the evaluation of microbial elimination in some treatment plants. The easy, fast and cheap methods available for phage determination are feasible both in industrialized and developing countries.     

Distribution of ribonucleic acid coliphages in raw sewage from treatment plants in Japan.

To determine the transmission cycle of ribonucleic acid (RNA) coliphages in their natural habitats, we investigated the distribution patterns of RNA phages in raw sewage collected from treatment plants in various localities in Japan. Most of the sewage samples contained group II and III phages. Samples from treatment plants in Sapporo, Tokyo, and Toyama contained appreciable amounts of group I phages in addition to the group II and III phages. As a whole, raw sewage from treatment plants in Japan contained RNA phages of the three groups in the ratio 1:2:5, group I/II/III. Based on the distribution patterns of RNA phages in sewage from domestic drainage in Japan proper (group II/III, 3:1), in animal feces and sewage from slaughter houses (mostly group I), and in human feces (group II/III, 1:1), it can be reasonably said that group I phages tend to be introduced from animal sources and group II and III phages tend to be introduced from human sources. Raw sewage from treatment plants in Japan consists mainly of human feces, sewage from domestic drainage, and industrial wastewater, and, in part, from slaughter houses. In fact, sewage from slaughter houses together with that from human sources flowed into the treatment plants of Tokyo as far as we could confirm.     

Abundance in Sewage of Bacteriophages That Infect Escherichia coli O157:H7 and That Carry the Shiga Toxin 2 Gene

Shiga toxin-converting bacteriophages are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7, but data on the occurrence and distribution of such phages as free particles in nature were not available. An experimental approach has been developed to detect the presence of the Shiga toxin 2 (Stx 2)-encoding bacteriophages in sewage. The Stx 2 gene was amplified by PCR from phages concentrated from 10-ml samples of sewage. Moreover, the phages carrying the Stx 2 gene were detected in supernatants from bacteriophage enrichment cultures by using an Stx 2-negative E. coli O157:H7 strain infected with phages purified from volumes of sewage as small as 0.02 ml. Additionally, the A subunit of Stx 2 was detected in the supernatants of the bacteriophage enrichment cultures, which also showed cytotoxic activity for Vero cells. By enrichment of phages concentrated from different volumes of sewage and applying the most-probable-number technique, it was estimated that the number of phages infectious for E. coli O157:H7 and carrying the Stx 2 gene was in the range of 1 to 10 per ml of sewage from two different origins. These values were approximately 1% of all phages infecting E. coli O157:H7.     

Bacteriophages and indicator bacteria in human and animal faeces

In an attempt to explain the presence of F-specific (RNA) bacteriophages in waste-water, faecal material from humans and a variety of animals was examined. The phages were detected in appreciable numbers only in faeces from pigs, broiler chickens, sheep and calves but not from dogs, cows, horses and humans. Parallel examinations for somatic coliphages, thermotolerant coliforms, faecal streptococci and spores of sulphite-reducing clostridia revealed the consistent presence of these organisms in all types of samples, albeit in variable numbers. The number of F-specific bacteriophages was related to the total number of somatic coliphages, but phage counts were unrelated to bacterial counts. F-specific RNA phages were grouped by serotyping and all animal isolates were found to belong to either group I (MS2 subtype) or IV (four different subtypes). Among the group IV isolates, most belonged to well-known subtypes SP (24 isolates) or FI (18 isolates) but five isolates were related to phage ID2 and one isolate was a new subtype. In contrast with animal isolates, 19 isolates from hospital wastewater belonged to serogroups II or III.     

Bacteriophages and indicator bacteria in human and animal faeces

In an attempt to explain the presence of F-specific (RNA) bacteriophages in waste-water, faecal material from humans and a variety of animals was examined. The phages were detected in appreciable numbers only in faeces from pigs, broiler chickens, sheep and calves but not from dogs, cows, horses and humans. Parallel examinations for somatic coliphages, thermotolerant coliforms, faecal streptococci and spores of sulphite-reducing clostridia revealed the consistent presence of these organisms in all types of samples, albeit in variable numbers. The number of F-specific bacteriophages was related to the total number of somatic coliphages, but phage counts were unrelated to bacterial counts. F-specific RNA phages were grouped by serotyping and all animal isolates were found to belong to either group I (MS2 subtype) or IV (four different subtypes). Among the group IV isolates, most belonged to well-known subtypes SP (24 isolates) or FI (18 isolates) but five isolates were related to phage ID2 and one isolate was a new subtype. In contrast with animal isolates, 19 isolates from hospital wastewater belonged to serogroups II or III.     

Prevalence of the stx2 Gene in Coliform Populations from Aquatic Environments

Shiga toxin-producing Escherichia coli strains are human pathogens linked to hemorrhagic colitis and hemolytic uremic syndrome. The major virulence factors of these strains are Shiga toxins Stx1 and Stx2. The majority of the genes coding for these toxins are borne by bacteriophages. Free Stx2-encoding bacteriophages have been found in aquatic environments, but there is limited information about the lysogenic strains and bacteria present in the environment that are susceptible to phage infection. The aim of this work was to study the prevalence and the distribution of the stx₂ gene in coliform bacteria in sewage samples of different origins. The presence of the stx₂ gene was monitored every 2 weeks over a 1-year period in a municipal sewage treatment plant. A mean value of 10² genes/ml was observed without significant variation during the study period. This concentration was of the same order of magnitude in raw municipal sewage of various origins and in animal wastewater from several slaughterhouses. A total of 138 strains carrying the stx₂ gene were isolated by colony hybridization. This procedure detected approximately 1 gene-carrying colony per 1,000 fecal coliform colonies in municipal sewage and around 1 gene-carrying colony per 100 fecal coliform colonies in animal wastewaters. Most of the isolates belonged to E. coli serotypes other than E. coli O157, suggesting a low prevalence of strains of this serotype carrying the stx₂ gene in the wastewater studied.     

Bacterial host strains that support replication of somatic coliphages

Somatic coliphages detected by Escherichia coli strain WG5 have been proposed as potential indicators of water quality. Their potential replication in the water environment is considered a drawback for their use as indicators. However, the contribution of replication outside the gut to the total numbers has never been quantified. It has not been determined either the fraction of bacterial strains that might support replication of phages detected by strain WG5 in the water environment. We examined the sensitivity of 291 host strains to 25 phages by streaking slants of the presumptive host strain onto an agar layer that contains bacteriophages, which gives a total of 7275 combinations (sensitivity tests). Only a 3.02% of the tests showed sensitivity. Additionally, six environmental strains were used as hosts to count phages in sewage and seawater. Phages isolated on these strains were used to infect strain WG5. The environmental strains detected 1 log₁₀ fewer phages than strain WG5 in sewage and seawater. The fraction of phages that were detected by the six strains and that also infected strain WG5 ranged from < 0.07% to < 2.0% of the total amount of bacteriophages detected by strain WG5 in the same samples. Our results confirm that less than 3% of naturally occurring hosts support replication of phages infecting E. coli. We conclude that the contribution of replication to the number of somatic coliphages detected in the aquatic environment is negligible. This revised version was published online in June 2006 with corrections to the Cover Date.     

Occurrence of phages infecting Escherichia coli O157:H7 carrying the Stx 2 gene in sewage from different countries

Shiga toxin-converting bacteriophages are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7. Recent studies have demonstrated a relatively high presence of Shiga toxin 2 phages in sewage from Spain, but no data on sewage from other areas were available. In order to evaluate the presence of such phages in sewage from diverse geographical origins, 33 sewage samples, including samples from eight different European countries as well as from New Zealand and South Africa were analysed. Using an experimental approach based on the detection of Stx 2 gene by a phage enrichment culture followed by PCR, bacteriophages infecting E. coli O157:H7 carrying the Shiga toxin 2 gene were detected in 15 of the samples studied. Results presented here show that the presence of phages carrying the Stx 2 gene is common in sewage from developed countries.