Culture and decontamination methods affecting enumeration of phages infecting Bacteroides fragilis in sewage.

A new medium has been adapted for the growth of Bacteroides fragilis so that its phages can be recovered from environmental samples, and its efficiency has been assessed. Polyvinylidene difluoride membranes allow significantly higher recoveries among different membrane filters used to decontaminate the samples. In all cases, a number of phages remain in the filters and a percentage of them can be recovered by treatment with an eluant.     

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.     

Concentration of Marine Birnavirus from Seawater with a Glass Fiber Filter Precoated with Bovine Serum Albumin

In the present study an efficient method for sampling the marine birnavirus (MABV) gene from seawater was developed. MABV gene was monitored by a specific polymerase chain reaction. When Millipore filters were used, MABV was efficiently collected on a filter with 0.05-µm pore size. When both millipore and glass fiber filters were used, MABV was recovered from both filters. Use of plain glass fiber filters resulted in poor recovering efficiency. However, coating the glass fiber filters with 1% bovine serum albumin trapped MABV efficiently. Combining concentration on glass fiber filters with polymerase chain reaction is quantitative, economic and fast, suggesting that this method can be used to detect genetically identified fish disease viruses, algal viruses, and phages.     

Improved method for the isolation of Campylobacter jejuni and Campylobacter coli bacteriophages

A centrifugation and filtration method of isolating Campylobacter phages has been developed. Forty-nine Campylobacter phages were isolated from 272 effluent samples of which 42 produced lysis with Campylobacter jejuni strains and seven with C. coli strains. Phages were recovered from pig manure, abattoir effluents, human faeces, sewage and poultry manure. Phages were not isolated from water samples, cattle and sheep faeces or farm pasture soil.     

Lysogenic pneumococci and their bacteriophages.

About half of pneumococci recovered from pediatric patients and one-third of isolates from adult patients yielded bacteriophages active against one or more of four noncapsulated indicator strains of pneumococcus. Strains of capsular types most frequently causing pediatric infections were associated with lysogeny. Classical restriction-modification phenomena have been demonstrated in vivo with some of the temperate phages, and correlation of restriction with the presence of one or the other of the two known pneumococcal restriction endonucleases has been established. The temperate phages differ serologically and in several other characteristics from virulent pneumococcal phages previously described. All pneumococcal phages so far studied can be classified into a minimum of three serological groups.     

Variables Influencing Extraction of Nucleic Acids from Microbial Plankton (Viruses,Bacteria, and Protists) Collected on Nanoporous Aluminum Oxide Filters

Anodic aluminum oxide (AAO) filters have high porosity and can be manufactured with a pore size that is small enough to quantitatively capture viruses. These properties make the filters potentially useful for harvesting total microbial communities from water samples for molecular analyses, but their performance for nucleic acid extraction has not been systematically or quantitatively evaluated. In this study, we characterized the flux of water through commercially produced nanoporous (0.02 μm) AAO filters (Anotop; Whatman) and used isolates (a virus, a bacterium, and a protist) and natural seawater samples to test variables that we expected would influence the efficiency with which nucleic acids are recovered from the filters. Extraction chemistry had a significant effect on DNA yield, and back flushing the filters during extraction was found to improve yields of high-molecular-weight DNA. Using the back-flush protocol, the mass of DNA recovered from microorganisms collected on AAO filters was ≥100% of that extracted from pellets of cells and viruses and 94% ± 9% of that obtained by direct extraction of a liquid bacterial culture. The latter is a minimum estimate of the relative recovery of microbial DNA, since liquid cultures include dissolved nucleic acids that are retained inefficiently by the filter. In conclusion, we demonstrate that nucleic acids can be extracted from microorganisms on AAO filters with an efficiency similar to that achievable by direct extraction of microbes in suspension or in pellets. These filters are therefore a convenient means by which to harvest total microbial communities from multiple aqueous samples in parallel for subsequent molecular analyses.     

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of phages infecting Arthrobacter hosts has been limited, perhaps due to the low success rate of many previous isolation techniques, resulting in an underrepresented group of Arthrobacter phages available for study. The enrichment technique described here, unlike many others, uses a filtered extract free of contaminating bacteria as the base for indicator bacteria growth, Arthrobactersp. KY3901, specifically. By first removing soil bacteria the target phages are not hindered by competition with native soil bacteria present in initial soil samples. This enrichment method has resulted in dozens of unique phages from several different soil types and even produced different types of phages from the same enriched soil sample isolate. The use of this procedure can be expanded to most nutrient rich aerobic media for the isolation of phages in a vast diversity of interesting host bacteria.     

Small but sufficient: the Rhodococcus phage RRH1 has the smallest known Siphoviridae genome at 14.2 kilobases

Bacteriophages are considered to be the most abundant biological entities on the planet. The Siphoviridae are the most commonly encountered tailed phages and contain double-stranded DNA with an average genome size of ～50 kb. This paper describes the isolation from four different activated sludge plants of the phage RRH1, which is polyvalent, lysing five Rhodococcus species. It has a capsid diameter of only ～43 nm. Whole-genome sequencing of RRH1 revealed a novel circularly permuted DNA sequence (14,270 bp) carrying 20 putative open reading frames. The genome has a modular arrangement, as reported for those of most Siphoviridae phages, but appears to encode only structural proteins and carry a single lysis gene. All genes are transcribed in the same direction. RRH1 has the smallest genome yet of any described functional Siphoviridae phage. We demonstrate that lytic phage can be recovered from transforming naked DNA into its host bacterium, thus making it a potentially useful model for studying gene function in phages.     

利用噬菌体抗体库筛选U251细胞血清应答基因蛋白特异抗体

利用噬菌体表面展示抗体库对不同血清处理U251细胞吸附的抗体进行差异筛选,筛选获得血清饥饿细胞吸附的阳性噬菌体克隆96个和血清饥饿后恢复血清培养细胞吸附的阳性噬菌体克隆82个。细胞免疫组化检测发现应答反应差异较大的抗体2个,即血清饥饿培养细胞特异反应的抗体1个（11号抗体）和血清饥饿后恢复血清培养细胞特异反应的抗体1个（2号抗体）,其中2号抗体在恢复血清培养细胞中的应答反应强于血清饥饿培养细胞,是一个血清应答基因蛋白特异抗体,且在血清饥饿后恢复血清培养不同时间的U251细胞中具有一定的特异性反应。该研究为寻找与细胞周期调控有关的因子奠定了基础,同时对肿瘤的诊断和治疗研究也有重要意义。     

Campylobacters and bacteriophages in the surface waters of Canterbury (New Zealand)

Aim:  To determine the relationship between the presence of thermotolerant campylobacters and their bacteriophages (phages) in surface waters for the potential to use phages as an indicator of Campylobacter spp.
Methods and Results:  Thermotolerant campylobacters were enumerated in 53 water samples using a three tube most probable number (MPN) series in m-Exeter broth. The presence of phages in the same samples was tested using two approaches: qualitative enrichment with five different Campylobacter hosts and a quantitative membrane concentration method. Phages infecting an Escherichia coli O157:H7 isolate were also enumerated by the membrane concentration method. Campylobacter spp. were isolated from 45/53 (85%) of the samples at 0.4–110 MPN 100 ml⁻¹. No Campylobacter phages were isolated, but coliphages were present in 43/46 (93%) of samples.
Conclusions:  The membrane concentration method recovered >80% of Campylobacter phages from spiked samples. The absence of Campylobacter phages in environmental samples, from both enrichment and concentration methods, suggests that, if present, they are at very low titres.
Significance and Impact of the Study:  Testing for Campylobacter phages as an indicator of Campylobacter spp. presence is not effective. The quantitative data for Campylobacter spp. will be useful for risk assessment purposes.     

Filter-supported preparation of lambda phage DNA

A rapid and simple method is described for the isolation of DNA from phage lambda which requires neither special equipment nor expensive material such as cesium chloride for ultracentrifugation nor extractions with organic solvents or ethanol precipitation. Microgram quantities of lambda DNA are obtained in less than 2 h from 90-mm plate lysates or 5-ml liquid cultures. The method allows the simultaneous isolation of large numbers of probes, e.g., clones from phage libraries. Lambda phages are precipitated by polyethylene glycol/sodium chloride and recovered by low speed centrifugation onto glass fiber filters positioned in disposable syringes. The DNA of phages is released by a 50% formamide/4 M sodium perchlorate solution, washed in filter-bound form, eluted with a small volume of low-salt buffer or water, and finally recovered by centrifugation. Comparison of the DNA isolated by this method with that obtained by two conventional procedures reveals both a similar recovery and a similar suitability for restriction enzyme digestion and subcloning.     

Selection of scFv phages on intact cells under low pH conditions leads to a significant loss of insert-free phages

Display of functional antibody fragments on the surface of filamentous bacteriophages allows fast selection of specific phage antibodies against a variety of target antigens. However, enrichment of single chain variable fragment (scFv)-displaying phages is often hampered by the abundance of bacteriophages lacking antibody fragments. Moderate adhesive binding activities and production advantages of these "empty" phages results in their subsequent enrichment during selection on target cells. To date, very limited effort has been made to develop strategies removing nonspecific binding phages during the selection processes. To efficiently reduce insert-free phages when panning on intact cells, we increased the washing stringency by lowering the pH of the buffer with citric acid. Under standard washing procedures (pH 7.4), only approximately 73% of recovered phages were insert-free after three rounds of selection. Using stringent washing procedures (pH 5.0), approximately 12% of recovered phages contained no scFv. Using this protocol, we have cloned an antibody fragment from a mouse/human hybridoma cell line directed against the disialoganglioside GD2. This study confirms that selection of phage antibodies on cells is efficiently enhanced by assays augmenting the stringency to remove nonspecific binding phages.     

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.     

Temperate coliphages: classification and correlation with habitats

Temperate coliphages were recovered from sewage, mammalian feces, and lysogenic strains of Escherichia coli. A total of 32 phages of independent origin were divided into six groups by applying the criteria of host range, antigenic homology, and the ultraviolet inducibility of the prophage. The demonstration of genetic interactions in some cases has confirmed the classification scheme. Nine phages were assigned to the P2 family and 19 to the lambda family. The remaining four isolates may represent some novel phylogenetic types. Phages recovered from the lysogenic strains of E. coli were all found to be P2 related, whereas a majority of the phages recovered as cell-free plaque-forming units were assignable to the lambda family. It is proposed that the biological attributes of the phages belonging to the two principal families are reflected in the distribution patterns observed. The virions of phage HK256 show multiple tail fibers and may thus represent a "new" virion form among the temperate coliphages.     

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.     

Bacteriophage lambda cloning vehicles for studies of genetic recombination

A pair of bacteriophage lambda cloning vehicles has been constructed for use in studies of genetic recombination. These phages, lambda rva and lambda rvb, have the following properties: (1) Each vector has a single HindIII site in the immunity region, at which segments of DNA can be inserted. (2) These HindIII sites are flanked by selectable markers with the following phenotypes: Spi+/- (Fec+/-) to the left, and imm lambda or imm434 to the right. (3) There is essentially no sequence homology between the two phages in this region, so recombination of the markers at reasonable frequency depends on the presence of homologous inserts at the HindIII sites. As a consequence, recovered recombinants must have resulted from a crossover event within the insert DNA. Restriction enzyme maps of the vectors have been determined. Variants of the original vectors have been isolated which permit separate examination of the viral (Red) and bacterial (Rec) generalized recombination mechanisms, and which provide a standard interval to which frequencies of recombination in cloned DNAs can be compared.     

Characterization of Poultry Isolates of Staphylococcus aureus by a New Set of Poultry Phages

A set of phages has been isolated from strains of Staphylococcus aureus , non-typable with the International (human) phage set, recovered from processed poultry. This set of phages could distinguish three main groups of strains, and biochemical tests confirmed these divisions, members of each group exhibiting characteristics of both 'human'and 'animal'strains. A high proportion of strains from all three phage groups was enterotoxigenic.     

Survival of enteric viruses adsorbed on glass microfibers during postal transport

The survival of enteric viruses (poliovirus type 1, Mahoney strain, and indigenous viruses of waste waters) has been studied after adsorption of the viruses (pH 3.5) on glass microfiber filters. After postal transport, the presence of the viruses was checked on the filters being soaked in a 3% beef extract solution (pH 7.5) either frozen or without protection against heat. Viruses were recovered at a rate of 59 to 65%. For qualitative studies, postal shipment of samples adsorbed on fiberglass may allow extension of a control system for enteroviruses in water.     

Shiga Toxin 2-Encoding Bacteriophages in Human Fecal Samples from Healthy Individuals

Shiga toxin-converting bacteriophages (Stx phages) carry the stx gene and convert nonpathogenic bacterial strains into Shiga toxin-producing bacteria. Previous studies have shown that high densities of free and infectious Stx phages are found in environments polluted with feces and also in food samples. Taken together, these two findings suggest that Stx phages could be excreted through feces, but this has not been tested to date. In this study, we purified Stx phages from 100 fecal samples from 100 healthy individuals showing no enteric symptoms. The phages retrieved from each sample were then quantified by quantitative PCR (qPCR). In total, 62% of the samples carried Stx phages, with an average value of 2.6 × 10⁴ Stx phages/g. This result confirms the excretion of free Stx phages by healthy humans. Moreover, the Stx phages from feces were able to propagate in enrichment cultures of stx-negative Escherichia coli (strains C600 and O157:H7) and in Shigella sonnei, indicating that at least a fraction of the Stx phages present were infective. Plaque blot hybridization revealed lysis by Stx phages from feces. Our results confirm the presence of infectious free Stx phages in feces from healthy persons, possibly explaining the environmental prevalence observed in previous studies. It cannot be ruled out, therefore, that some positive stx results obtained during the molecular diagnosis of Shiga toxin-producing Escherichia coli (STEC)-related diseases using stool samples are due to the presence of Stx phages.     

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.