Bacteroides Fragilis Phages Research Articles

Assessment of swine-specific bacteriophages of Bacteroides fragilis in swine farms with different antibiotic practices

We assessed the occurrence and specificity of bacteriophages of Bacteroides fragilis in swine farms for their potential application in microbial source tracking. A local B. fragilis host strain, SP25 (DSM29413), was isolated from a pooled swine feces sample taken from a non-antibiotic farm. This strain was highly specific to swine fecal materials because it did not detect bacteriophages in any samples from human sewage, sheep, goats, cattle, dogs, and cats. The reference B. fragilis strain, RYC2056, could detect phages in swine samples but also detected phages in most human sewage and polluted urban canal samples. Phages of SP25 exist in the proximity of certain swine farms, regardless of their antibiotic use (p > 0.05). B. fragilis strain SP25 exhibited relatively high resistance to most of the veterinary antimicrobial agents tested. Interestingly, most farms that were positive for SP25 phages were also positive for RYC2056 phages. In conclusion, the swine-specific SP25 strain has the potential to indicate swine fecal contamination in certain bodies of water. Bacterial isolates with larger distributions are being studied and validated. This study highlights the importance of assessing the abundance of phages in local swine populations before determining their potential applicability for source tracking in local surface waters.

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.

Disinfection of human enteric viruses on fomites.

The virucidal action of several commercially available disinfectant preparations was assayed against hepatitis A virus and human rotavirus dried on polystyrene. Overall, the level of virus disinfection achieved was very poor, usually inducing less than 3 log titre reduction. Suspension tests performed with the same disinfectants showed different virus inactivation rates, thus failing to provide a reliable indication of the actual virus disinfection on fomites. In our studies, bacteriophages of Bacteroides fragilis proved to be a simple, cheap and reliable screening tool for the evaluation of virus disinfection on non-porous surfaces. The same conclusion cannot be drawn for poliovirus.

Bacteriophages: update on application as models for viruses in water

Phages are valuable models or surrogates for enteric viruses because they share many fundamental properties and features. Amon g these are structure, composition, morphology, size and site of replication. Even though they use different host cells, colipha ges and Bacteroides fragilis phages predominantly replicate in the gastro-intestinal tract of humans and warm-blooded animals where enteric viruses also replicate. A major advantage of phages is that, compared to viruses, they are detectable by simple, inexp ensive and rapid techniques. In view of these features, phages are particularly useful as models to assess the behaviour and survival of enteric viruses in the environment, and as surrogates to assess the resistance of human viruses to water treatment and disinfec tion processes. Since there is no direct correlation between numbers of phages and viruses, phages cannot to a meaningful extent be used to indicate numbers of viruses in polluted water. The presence of phages typically associated with human and animal excre ta indicates the potential presence of enteric viruses. However, the absence of these phages from water environments is generally a meaningful indication of the absence of enteric viruses. This is because phages such as somatic coliphages, F-RNA coliphages a nd B. fragilis phages generally outnumber enteric viruses in water environments, and they are at least as resistant to unfavourable conditions including those in water treatment and disinfection processes. However, using highly sensitive molecular techniques viruses have been detected in drinking water supplies which yielded negative results in conventional tests for phages. Initial ly, data on phages were rather confusing because a wide variety of techniques was used. However, techniques for the detection of phages are being standardised internationally. This applies in particular to somatic and F-RNA coliphages, and B. fragilis phages, which are most commonly used in water quality assessment. Reliable and practical techniques now available include direct quantitativ e plaque assays on samples of water up to 100 ml, and qualitative tests on 500 m l or more using highly sensitive enrichment procedures.

Evaluation of bacteriophages during the treatment of sludge

The aim of this work was to determine the effect of liming and composting on the fate of three bacteriophages (somatic coliphages, F-RNA phages, Bacteroides fragilis phages) considered as potential indicators of viral contamination. It was shown that the three bacteriophages studied exhibited variable densities in sludge. Somatic coliphages were most abundant (10(4) to 10(5) x 10 g(-1) DM) then F-RNA bacteriophages (10(2) to 10(4) x 10 g(-1) DM) and Bacteroides fragilis phages (10(1) to 10(2) x 10 g(-1) DM). The efficacy of liming was found to be pH dependent but also sludge dependent. The pH allowing 99% elimination of somatic coliphage is close to 9 for solid sludges and close to 13.5 for liquid sludges. For composting, our findings clearly demonstrated that phage inactivation is very clearly temperature-dependent. For temperatures reaching 70 degrees, there is a 5 log reduction in somatic coliphages while for temperature in the 50-55 degrees C range, the drop off is only 2 log. Considering the efficacy of the treatment methods, it is clear that the well-established industrial procedures that reach temperatures in the 60-70 degrees C range totally inactivate all 3 phages tested and present in sludge before composting.

Fates of bacteriophages and bacterial indicators in the Moselle river (France)

It has been suggested that bacteriophages can provide useful information about the pathogenic microorganisms, particularly enteric viruses, present in water. This information is complementary to that obtained from bacterial indicators of faecal contamination, which would be of great value for evaluating the risks associated with the use of certain types of water. Before bacteriophages can be used as indicators of faecal contamination, we need to confirm that bacteriophages give a different response to that given by the well-known bacteria indicators and to determine what happens to bacteriophages in river water. Indeed, drinking water is often produced from river water, either by natural filtration through the soil or after undergoing various treatments. We collected 96 river water samples from six different sites between February and November 2000. The samples were analysed for three faecal indicator bacteria (thermotolerant coliforms, enterococci and spores of sulphite-reducing anaerobes) and three types of bacteriophages (somatic coliphages, F-specific phages and Bacteroides fragilis phages). The densities of thermotolerant coliforms and enterococci depended mainly on physical factors such as flow rate and water temperature. High temperature and low flow rate led to a decrease in the density of these microorganisms, especially in the absence of a major input of faecal pollution. Conversely, the densities of somatic coliphages, F-specific phages and spores of sulphite-reducing anaerobes remained constant regardless of the flow rate and temperature. The density of Bacteroides fragilis phages was too low for unambiguous determination of their fate in river water.

Bacteroides fragilis and Escherichia coli bacteriophages in human faeces

Some bacteriophages found in human faeces are being evaluated as possible indicators of viral contamination of water. These bacteriophages include somatic coliphages and Bacteroides fragilis phages. The aims of this study were to determine the occurrence and concentrations of somatic coliphages and Bacteroides fragilis phages in the stools of a human population residing in eastern France (n = 193). Somatic coliphages were detected in 68% of the stools at a mean concentration of 4.3 x 10(3) PFU.g-1 and Bacteroides fragilis phages were detected in 11% of the stools at a mean concentration of 7 x 10(1) PFU.g-1. Statistical analysis showed no correlation between the phage concentration and the age or sex of the human subject.

Removal of bacterial and viral faecal indicator organisms in a waste stabilization pond system in Choconta, Cundinamarca (Colombia)

A major objective for domestic wastewater treatment using waste stabilization pond systems is the removal of pathogenic microorganisms. Traditional evaluation parameters for faecal contamination are the total and faecal coliforms. However, epidemiological studies, environmental resistance and the behaviour in the treatment systems, show that viruses are an important disease agent and even more resistant to disinfection than bacteria. Therefore, it is important to introduce viruses as a faecal indicator and to compare them with the traditional bacterial indicators. A waste stabilization pond system was evaluated in the municipality of Chocontá, Cundinamarca (Colombia), for the removal of faecal indicators (such as Escherichia coli, Streptococcus faecalis, Clostridium perfringens) and viruses like F+, somatic and Bacteroides fragilis phages. The system includes two facultative ponds in series with a flow of 1555 m3/day. Samples were collected at the entrance of the system, in the two ponds and from the final effluent. Results show a decrease between 0.3 and 4.7 logarithmic units in the bacterial indicators and between 1 and 4.6 logarithmic units with viral indicators.

Faecal bacteria and bacteriophage inactivation in a full-scale UV disinfection system used for wastewater reclamation

A study was carried out to compare the inactivation of faecal bacteria and one type of bacteriophage in a full-scale UV disinfection system. The system is part of a water reclamation facility for effluent reuse in golf course and agricultural irrigation. Influent and effluent samples were taken over two sampling periods (three consecutive days in July and one day in August), with three different UV doses applied each day (ranging from 10 to 40 mW.s/cm2 and 20 to 80 mW.s/cm2 in July and August, respectively). Effluent samples were also taken from a chlorine disinfection channel (5 mg Cl2/L dose) operating in parallel to the UV system. Total coliforms (TC), faecal coliforms (FC), faecal streptoccoci (FS) and somatic coliphages (SC) were measured in each sample. F-specific RNA bacteriophages and bacteriophages of Bacteroides fragilis were also measured one day in July. The decay ratio observed for all the microorganisms was similar when UV doses applied were low (July), ranging from 1.15 to 1.25 log-units. This suggests that bacterial indicators may be suitable for virus inactivation control when low UV doses are applied; however, such low doses are inadequate to achieve effluent quality requirements for unrestricted irrigation. At higher UV doses (August), decay ratios for TC and FC were 3.1 and 2.8 log-units respectively, indicating that they were more susceptible to UV exposure than SC and FS, with decay ratios of 2.6 and 1.0 log-units, respectively. Nevertheless, these higher doses were also inadequate to achieve water quality requirements for unrestricted irrigation. The decay ratio of SC during chlorine disinfection was clearly lower than that of the other microorganisms. Bacteriophages of Bacteroides fragilis were more resistant to UV disinfection than SC and F-specific RNA. In fact, bacteriophages of Bacteroides fragilis were not affected during UV exposure. A UV dose ranging from 40 to 80 mW.s/cm2 marks the borderline beyond which inactivation rates of SC are clearly lower than those of bacterial indicators.

Bacteriological and virological quality of seawater bathing areas along the Tyrrhenian coast

Monitoring was carried out during summer 1997 along a selected area of the Tyrrhenian coast near the Tiber river mouth. Fifty-eight seawater samples, collected from 19 stations, were examined for coliforms, streptococci, Enteroviruses, Salmonellae, coliphages, Bacteroides fragilis phages, Pseudomonas, alophilic Vibrios, Aeromonas and yeasts. Salmonellae and coliphages were isolated in 3 and 12 out of 58 samples, respectively. Enteroviruses and Bacteroides fragilis phages were not isolated. Reoviruses were isolated only from 2 out of 58 samples. A limited number of samples of the northern stations located near the Tiber and other river mouths exceeded the guide values for bathing water by the EU Directive. All the southern stations, located near canals, were of very good microbiological quality. Pseudomonas, Vibrio, Aeromonas and yeasts were isolated from all stations and their values in 100 ml of seawater were 10-10 6 , 10-10 6 , 0-10 6 and 1-10 3 , respectively. An extensive disinfection practice carried out on domestic wastes, which are discharged in rivers and canals, probably brought pollution levels of most stations to values within the bacterial standards. The spread of Pseudomonas, Aeromonas, etc. showed that all the coastal area studied was characterized by the presence of organic matter coming from land that can support the presence of opportunistic pathogens and other microbial flora.

Occurrence and numbers of bacteriophages and bacterial indicators in faeces of yellow-legged seagull (Larus cachinnans).

Faeces from feral populations of yellow-legged seagulls from the northern coastal area of Catalonia (North-eastern Spain) contained variable amounts of faecal coliforms, faecal streptococci, somatic coliphages, F-specific bacteriophages and Bacteroides fragilis bacteriophages. Occurrence and numbers of bacterial indicators and bacteriophages in the faeces of yellow-legged seagulls are in the ranges described in the faeces of different animals. The ratios between numbers of bacterial indicators and numbers of bacteriophages are much higher in faeces of seagulls than in treated or raw sewage contributed by out-falls of the same area.

Genomic structure of phage B40-8 of Bacteroides fragilis.

Very few data are available on the molecular biology of Bacteroides fragilis bacteriophages, which have been considered in several studies as indicators of faecal contamination. Phage B40-8, initially isolated from an urban sewage sample using a strain of B. fragilis (HSP40) isolated from a clinical specimen, was chosen in this study as a prototype for morphological and molecular studies. Like most of the phages infective for B. fragilis, B40-8 belongs to the Siphoviridae family. Its genome has been found to be a double-stranded DNA molecule, of approximately 51.7 kb, containing a rather low percentage (38.9 mol%) of G + C. The ends of the molecule appeared not to be cohesive but permuted, with a terminal redundancy of 7.3%. A genomic map was constructed. Three major proteins (MP) out of 15 peptides in the SDS-PAGE profile were selected for N-terminal sequencing. From these data, degenerate probes were designed to locate the ORFs in the genomic map. Immunodetection by electron microscopy revealed that MP1 and MP3 were structural proteins of the phage head and that MP2 was a constituent of the tail. A genomic library of the phage was prepared, and a clone including the MP2 ORF was identified and sequenced.

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.

Comparative study of techniques used to recover viruses from residual urban sludge

Eight virus extraction techniques were compared on three types of residual urban sludge for simultaneous detection of infectious enteroviruses, somatic coliphages, F-specific RNA bacteriophages and Bacteroides fragilis bacteriophages. The highest virus counts were found in extracts obtained using three extraction techniques described by respectively using a 10% beef extract solution at pH 9 and sonication, using a 0.3 M NaCl/7% beef extract solution at pH 7.5 and freon, and finally using a 0.1 M borate buffer/3% beef extract solution at pH 9 and sonication.

Bacteriophages and bacteria as indicators of enteric viruses in oysters and their harvest waters

Reliable indicators are needed to detect enteric virus contamination of bivalve molluscan shellfish and their harvest waters. Concentrations of male-specific (F+) coliphages, Bacteroides fragilis phages, Salmonella phages and several indicator bacteria in wastewater, estuarine receiving water and its oysters were examined for their ability to predict the presence and levels of faecal contamination and enteric viruses in oysters. Enteric viruses in oysters were detected by cell culture and RT-PCR methods. F+ coliphages, Salmonella phages, B fragilis phages and faecal indicator bacteria (faecal coliforms, E coli, enterococci and Clostridium perfringens) were generally positively associated and were highest in raw sewage and progressively lower in sewage effluent and in receiving waters at increasing distance from the wastewater discharge. Indicator levels in oysters were highest for F+ coliphages and C perfringens. One F+ RNA coliphage serotype (Group II) predominated in the wastewater, receiving water and oysters. Human enteric viruses were detected in 17/31 oyster samples. The levels of most indicators in oysters and water were higher when oysters were enteric virus-positive and lower when oysters were enteric virus-negative. F+ coliphages and C perfringens were the only indicators significantly associated with the presence of enteric viruses in oysters. F+ coliphages and their serotypes are promising indicators of human enteric virus contamination in oysters and their harvest waters.

Detection of infectious enteroviruses, enterovirus genomes, somatic coliphages, and Bacteroides fragilis phages in treated wastewater.

In this study, three types of treated wastewater were tested for infectious enteroviruses, the enterovirus genome, somatic coliphages, and Bacteroides fragilis phages. The aim of this work was to determine whether the presence of the two types of bacteriophages or of the enterovirus genome was a good indicator of infectious enterovirus contamination. The enterovirus genome was detected by reverse transcription-polymerase chain reaction. Infectious enteroviruses were quantified by cell culturing (BGM cells), and the bacteriophages were quantified by plaque formation on the host bacterium (Escherichia coli or B. fragilis) in agar medium. Forty-eight samples of treated wastewater were analyzed. Sixteen samples had been subjected to a secondary treatment for 8 to 12 h (A), 16 had been subjected to a secondary treatment for 30 h (B1), and 16 had been subjected to both secondary and tertiary treatments (B2). The mean concentrations of somatic coliphages were 4.9 x 10(4) PFU . liter-1 for treatment line A, 9.8 x 10(3) PFU . liter-1 for B1, and 1.4 x 10(3) PFU . liter-1 for B2, with all the samples testing positive (100%). The mean concentrations of B. fragilis phages were 1.7 x 10(3) PFU . liter-1 for A (100% positive samples), 17 to 24 PFU . liter-1 for B1 (44% positive samples), and 0.8 to 13 PFU . liter-1 for B2 (6% positive samples). The mean concentrations of infectious enteroviruses were 4 most probable number of cytopathogenic units (MPNCU) . liter-1 for A (31% positive samples) and <1 MPNCU . liter-1 for B1 and B2 (0% positive samples). The percentages of samples testing positive for the enterovirus genome were 100% for A, 56% for B1, and 19% for B2. The percentages of samples testing positive for the enterovirus genome were significantly higher than those for infectious enteroviruses. This finding may have been due to the presence of noninfectious enteroviruses or to the presence of infectious enteroviruses that do not multiply in BGM cell cultures. However, under our experimental conditions, nondetection of the genome implies the absence of infectious viruses. There was a significant correlation between the concentration of somatic coliphages or B. fragilis phages and the presence of infectious enteroviruses or the presence of the enterovirus genome. However, the somatic coliphage concentration did not lead to fluctuations in the infectious enterovirus concentration, whereas the B. fragilis phage concentration did.

Bacteriophages and bacteria as indicators of enteric viruses in oysters and their harvest waters

Reliable indicators are needed to detect enteric virus contamination of bivalve molluscan shellfish and their harvest waters. Concentrations of male-specific (F+) coliphages, Bacteroides fragilis phages. Salmonella phages and several indicator bacteria in wastewater, estuarine receiving water and its oysters were examined for their ability to predict the presence and levels of faecal contamination and enteric viruses in oysters. Enteric viruses in oysters were detected by cell culture and RT-PCR methods. F+ coliphages. Salmonella phages, B fragilis phages and faecal indicator bacteria (faecal coliforms, E coli, enterococci and Clostridium perfringens) were generally positively associated and were highest in raw sewage and progressively lower in sewage effluent and in receiving waters at increasing distance from the wastewater discharge. Indicator levels in oysters were highest for F+ coliphages and C perfringens. One F+ RNA coliphage serotype (Group II) predominated in the wastewater, receiving water and oysters. Human enteric viruses were detected in 17/31 oyster samples. The levels of most indicators in oysters and water were higher when oysters were enteric virus-positive and lower when oysters were enteric virus-negative. F+ coliphages and C perfringens were the only indicators significantly associated with the presence of enteric viruses in oysters. F+ coliphages and their serotypes are promising indicators of human enteric virus contamination in oysters and their harvest waters.

Use of oxyrase enzyme (Oxyrase®) for the detection of bacteriophages of Bacteroides fragilis in aerobic incubating conditions

The biocatalytic oxygen reducing agent oxyrase enzyme (Oxyrase®) was shown to be useful for the detection of bacteriophages infecting the anaerobic bacteria Bacteroides fragilis, which have been proposed as potential indicators of human viruses and/or human faecal pollution in water. Oxyrase® provided very good conditions both for growing Bact. fragilis in liquid cultures in aerobic conditions and for the enumeration of bacteriophages by means of a triple agar layer for the detection of plaque-forming units.

Disinfection of human enteric viruses on fomites

The virucidal action of several commercially available disinfectant preparations was assayed against hepatitis A virus and human rotavirus dried on polystyrene. Overall, the level of virus disinfection achieved was very poor, usually inducing less than 3 log titre reduction. Suspension tests performed with the same disinfectants showed different virus inactivation rates, thus failing to provide a reliable indication of the actual virus disinfection on fomites. In our studies, bacteriophages of Bacteroides fragilis proved to be a simple, cheap and reliable screening tool for the evaluation of virus disinfection on non-porous surfaces. The same conclusion cannot be drawn for poliovirus.

Quantification of bacteriophages ofBacteroides fragilis in environmental water samples of Seine River

Virus detection in drinking water is very important to protect human health. The different methodologies for analysing human pathogenic virus are very time consuming and expensive, so until now only a few specialised laboratories carried out this analysis. Detection of bacteriophages may be possible by examining the aquatic virus, with advantages of easy and cheap. The bacteriophages of Bacteroides fragilis have been proven as specifically present in human faeces and have relationships with water contaminated by enterovirus. Our study, for the first time in France, discovered the B. fragilis phages present in sample of sewage, Seine river and raw water for water supply. Our results also presented that B. fragilis phages may be a better indicator for water bacteriology compared with classical bacteriological indicators in water treatment. On the other hand, our results demonstrated that MPN method (Most Probable Number) has more advantages than that of PFU (Plaque Forming Units).