Comparative Persistence of Subgroups of F-Specific RNA Phages in River Water

F-specific (F+) RNA phages are widely used as indicators for the presence of fecal contamination and/or enteric viruses in water, and identifying subgroups of F+ RNA phages provides an approach for microbial source tracking. Different survival characteristics of the F+ RNA phage subgroups result in a misinterpretation of their original proportion in water, thus giving misleading information when they are used for microbial source tracking. This study investigated the comparative persistence of subgroups of F+ RNA phages in river water under different conditions. Results suggested that temperature and pH are the major factors affecting the persistence of F+ RNA phages in river water, and organic substances promote phage survival. The comparative persistence patterns of subgroups of F+ RNA phages varied and may bias extrapolation of their initial proportions in surface water. Thus, the characteristics of water should be taken into consideration and the results should be carefully interpreted when F+ RNA phages are used for microbial source tracking.     

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.     

Distribution of ribonucleic acid coliphages in south and east Asia.

We investigated the distribution of ribonucleic acid (RNA) coliphages in the Philippines, Singapore, Indonesia, India, and Thailand by collecting sewage samples from domestic drainage in November 1976. Of the 221 samples collected from domestic drainage, 50 contained RNA phages (52 strains). By serological analysis, 46 of the 52 strains were found to belong to group III. It can thus be said that the most prevalent RNA phages in Southeast Asia (at least, in the Philippines, Singapore, and Indonesia) were group III phages. Investigations of sewage samples collected from domestic drainage in Japan indicate that the most prevalent RNA phages in mainland Japan (north of Kyushu) are group II phages, whereas group III phages are predominant in the southern part of Japan (south of Amamiohshima Island). We therefore propose a borderline between Kyushu and Amamiohshima Island for the geographical distribution of RNA coliphages in the domestic drainage of South and East Asia. Moreover, one strain (ID2) was inactivated to some extent with the antisera of four groups of RNA phages. This is thought to be significant from the evolutionary viewpoint.     

Synthesis of Indicator Strains and Density of Ribonucleic Acid-Containing Coliphages in Sewage

Escherichia coli strains freshly isolated from natural sources are inefficient indicators of coliphages present in sewage. Four E. coli strains recently isolated from clinical specimens were mutagenized to obtain lac(-) mutants. Such mutants were infected with an F'lac(+) sex factor of E. coli K-12. Pairs of isogenic lac(-) and lac(-)/F'lac(+) strains were used as indicators of coliphages present in sewage, and it was found that such strains can be effectively used for a direct and almost selective enumeration of F-specific coliphage contents of sewage samples. Serological tests were applied to a number of F-specific phages isolated. All the isolates that were tested fell into two distinguishable antigenic classes: members of one class being related to ribonucleic acid (RNA) phage MS2 and those of the other being related to another RNA phage, namely, Qbeta. MS2-related phages have been found to be more widely distributed than the Qbeta related phages. Most habitats sampled were found to yield only one or the other kind of phage. Single-stranded deoxyribonucleic acid-containing F-specific phages were not detectable by the methods employed by us.     

Concentration of coliphages from large volumes of water and wastewater.

Membrane filter adsorption-elution technology has been extensively used for the concentration and detection of animal viruses from large volumes of water. This study describes the development of positively charged microporous filters (Zeta Plus) for the concentration of coliphages from large volumes of water and wastewater. Four different coliphages were studied: MS-2, phi X174, T2, and T4. Positively charged microporous filters were found to efficiently adsorb these coliphages from tap water, sewage, and lake water at neutral pH. Adsorbed viruses were eluted with a 1:1 mixture of 8% beef extract and 1 M sodium chloride at pH 9. Using this method, coliphages could be concentrated from 17-liter volumes of tap water with recoveries ranging from 34 to 100%. Coliphages occurring naturally in raw and secondarily treated sewage were recovered with average efficiencies of 56.5 and 55.0%, respectively. This method should be useful in isolation of rare phages, the ecology of phages in natural waters, and the evaluation of water quality.     

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°C, and the sewage was heated at 60°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.     

Distribution of RNA coliphages in Senegal, Ghana, and Madagascar

The distribution patterns of RNA coliphages (phages) in Senegal, Ghana, and Madagascar were investigated by collecting sewage samples from domestic drainage in November, 1980. In Senegal, among 65 sewage samples collected mainly from Dakar and its vicinity, 14 (22%) contained RNA phages (16 strains). By serological analysis, 13 of 16 strains were found to belong to group III. This is consistent with the distribution pattern of RNA coliphages in tropical and subtropical regions of Asia. In Ghana, however, among 106 samples collected from Accra, Suhum, and their vicinities, only seven (7%) contained RNA phages (seven strains) (groups I, II, and III [1:3:3]). In Madagascar, among 124 samples collected from Antananarivo, Moramanga, and their vicinities, seven (6%) contained RNA phages (seven strains) (groups I, II, III, and IV [1:1:1:4]). In spite of the low isolation frequency, it can be said that Madagascar appears to have a unique distribution pattern (abundance of group IV phages) which differs from that of any other countries we have examined. The generality of the distribution pattern of RNA phages in the tropical region (abundance of group III phages) was thus verified at least in Senegal.     

Experimental evolution of RNA versus DNA viruses

Based on their extremely high mutation rates, RNA viruses have been traditionally considered as the fastest evolving entities in nature. However, recent work has revealed that, despite their greater replication fidelity, single-stranded (ss) DNA viruses can evolve fast in a similar way. To further investigate this issue, we have compared the rates of adaptation and molecular evolution of ssRNA and ssDNA viruses under highly controlled laboratory conditions using the bacteriophages ΦX174, G4, f1, Qβ, SP, and MS2 as model systems. Our results indicate that ssRNA phages evolve faster than ssDNA phages under strong selective pressure, and that their extremely high mutation rates appear to be optimal for this kind of scenario. However, their performance becomes similar to that of ssDNA phages over the longer term or when the population is moderately well-adapted. Interestingly, the roughly 100-fold difference between the mutation rates of ssRNA and ssDNA phages yields less than a fivefold difference in adaptation and nucleotide substitution rates. The results are therefore consistent with the observation that, despite their lower mutation rates, ssDNA viruses can sometimes match the evolvability of RNA viruses.     

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.     

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.     

Structural features in TAR RNA of human and simian immunodeficiency viruses: a phylogenetic analysis.

A comparative analysis of TAR RNA structures in human and simian immunodeficiency viruses reveals the conservation of certain structural features despite the divergence in sequence. Both the TAR elements of HIV-1 and SIV-chimpanzee can be folded into relatively simple one-stem hairpin structures. Chemical and RNAase probes were used to analyze the more complex structure of HIV-2 TAR RNA, which folds into a branched hairpin structure. A surprisingly similar RNA conformation can be proposed for SIV-mandrill, despite considerable divergence in nucleotide sequence. A third structural presentation of TAR sequences is seen for SIV-african green monkey. These results are generally consistent with the classification of HIV-SIV viruses in four subgroups based on sequence analyses (both nucleotide- and amino acid-sequences). However, some conserved TAR structures were detected for members of different virus subgroups. It is therefore proposed that RNA structure analysis might provide an additional tool for determining phylogenetic relationships among the HIV-SIV viruses.     

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.     

Bacteriophages LIMElight and LIMEzero of Pantoea agglomerans, belonging to the "phiKMV-like viruses"

Pantoea agglomerans is a common soil bacterium used in the biocontrol of fungi and bacteria but is also an opportunistic human pathogen. It has been described extensively in this context, but knowledge of bacteriophages infecting this species is limited. Bacteriophages LIMEzero and LIMElight of P. agglomerans are lytic phages, isolated from soil samples, belonging to the Podoviridae and are the first Pantoea phages of this family to be described. The double-stranded DNA (dsDNA) genomes (43,032 bp and 44,546 bp, respectively) encode 57 and 55 open reading frames (ORFs). Based on the presence of an RNA polymerase in their genomes and their overall genome architecture, these phages should be classified in the subfamily of the Autographivirinae, within the genus of the "phiKMV-like viruses." Phylogenetic analysis of all the sequenced members of the Autographivirinae supports the classification of phages LIMElight and LIMEzero as members of the "phiKMV-like viruses" and corroborates the subdivision into the different genera. These data expand the knowledge of Pantoea phages and illustrate the wide host diversity of phages within the "phiKMV-like viruses."     

Continuous survey of the distribution of RNA coliphages in Japan

In order to demonstrate the stability and continuity of RNA coliphages (phages) in their natural habitats, we investigated the amount and group types of RNA phages in sewage samples collected continuously from domestic drainage in Japan proper and islands in the seas adjacent to Japan (abbreviated simply as islands, hereafter) over a 5-yr period from 1973 to 1977. It was found that the frequencies of isolation of RNA phages were fairly high and constant. The group types of RNA phages isolated were also stable in the three cities. Choshi, Niigata, and Toyama in Japan proper. The average for the three cities was group II:III = 3:1. The investigation in islands revealed that the frequencies of isolation of RNA phages were fairly high as in the case of the above three cities in Japan proper and the group types of RNA phages isolated were also stable. That is to say, group II phages were predominant on Rishiri Island, Rebun I., Iki I., and Tsushima I., which are located relatively near to mainland Japan, while group III phages were predominant on Amamiohshima I., mainland Okinawa, Ishigakijima I., and Iriomotejima I., which are located south of Kyushu. It can thus be said that the RNA phages in the domestic drainage of Japan proper and islands remained more or less stable over at least the 5-yr period, and an apparent difference in the geographical distribution of RNA phages in Japan exists between Kyushu and Amamiohshima I.     

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.     

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.