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.     

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.     

Detection of bacteriophage infection and prophage induction in bacterial cultures by means of electric DNA chips

Infections of bacterial cultures by bacteriophages are common and serious problems in many biotechnological laboratories and factories. A method for specific, quantitative, and quick detection of phage contamination, based on the use of electric DNA chip is described here. Different phages of Escherichia coli and Bacillus subtilis were analyzed. Phage DNA was isolated from bacterial culture samples and detected by combination of bead-based sandwich hybridization with enzyme-labeled probes and detection of the enzymatic product using silicon chips. The assay resulted in specific signals from all four tested phages without significant background. Although high sensitivity was achieved in 4h assay time, a useful level of sensitivity (10(7)-10(8) phages) is achievable within 25 min. A multiplex DNA chip technique involving a mixture of probes allows for detection of various types of phages in one sample. These analyses confirmed the specificity of the assay.     

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.     

New waterborne bacteriophages active on Yersinia enterocolitica.

Seven bacteriophages active on Yersinia enterocolitica (YE) were isolated from surface water samples collected in Granada, Spain. A comparison of the respective host ranges of these new phages and of reference phages used for YE phage typing showed that YE strains belonging to various phage types, grown at either 37 or 25 degrees C, expressed susceptibility to reference sewage water phages whereas susceptibility to new waterborne phages, as well as to reference phages from lysogenic YE, was only demonstrated in YE strains grown at 25 degrees C. A YE strain isolated by stool culture from a pig was lysogenic for a bacteriophage which behaved like waterborne phages and reference phages from lysogenic YE strains. The possibility that the isolation of waterborne bacteriophages might, in certain circumstances, reflect the presence of lysogenic YE was raised.     

An electron microscopic study of bacteriophages from marine waters

The morphology of 75 bacteriophage strains isolated from water samples collected in the North Sea or in the northern Atlantic was studied by electron microscopy. Only tailed phages were observed (bradley groups A, B, and C). According to structural similarities, the strains are ascribed to 12 groups, 5 of which comprise types of marine phages not reported before. Four of these 5 groups include phage types that have not been detected from any other source. Among the phages isolated from northern Atlantic water a high incidence was observed of strains the particles of which have long appendages. Certain types of the northern Atlantic phages investigated were derived only from samples collected either east or west of the Azores. This finding agrees with former observations pointing to the existence of different populations of closely related bacteria east and west, respectively, of the northern Mid-Atlantic Ridge.     

New bacteriophages active on strains of Hyphomicrobium

Fifty-five lytic bacteriophages isolated from water and soil samples were active on many strains of the genus Hyphomicrobium. The optimal isolation procedure was an adsorption method in which samples from a habitat similar to that of the respective host bacterium were used as the phage inoculum. According to the morphology and nucleic acid type these bacteriophages belonged to different families: Myoviridae (type A1: five phages); Styloviridae (type B1: 33 phages; type B2: eight phages) and Podoviridae (type C1: nine phages). The Styloviridae (type B1) appeared in two morphological variants (tails flexible or rigid). All phages investigated were specific for the genus Hyphomicrobium and were unable to lyse members of other genera of hyphal, budding bacteria (e.g. Hyphomonas, Pedomicrobium, genus D, genus T). The host specificity of 42 phages was tested with 156 Hyphomicrobium strains: 122 strains were lysed by at least one of these phages, but 34 Hyphomicrobium strains were not susceptible. Morphotype B1 phages with identical morphology could be distinguished according to their host-range properties on prophage-containing Hyphomicrobium strains. With regard to differences in morphology and host range, 25 phages were selected for more detailed investigations. From these phages DNA was isolated; the melting transition midpoints (Tm) ranged from 67 to 93 degrees C. The upper and higher values suggested the presence of DNA modifications. Six different adsorption patterns could be distinguished among the Hyphomicrobium phages. Preferred attachment sites were the proximal pole of the mother cell, the hyphal tip, the distal pole of the bud, and the distal pole of the swarmer cell.     

粘质沙雷菌裂解性噬菌体ФSM9-3Y的分离和鉴定

目的从医院污水中分离粘质沙雷菌噬菌体,并分析其生物学特性,为进一步研究针对耐药性粘质沙雷菌的噬菌体制剂提供依据。方法采用双层琼脂平板法分离纯化针对粘质沙雷菌的裂解性噬菌体,观察噬菌体对宿主菌的裂解特异性,通过负染法电镜观察噬菌体的形态结构,提取噬菌体核酸进行酶切电泳,测定噬菌体的最佳感染复数和一步生长曲线,SDS-PAGE电泳初步分析噬菌体的结构蛋白和非结构蛋白。结果从医院污水分离出7株可裂解粘质沙雷菌的噬菌体,对其中一株噬菌体(命名为ФSM9-3Y)的生物学特征进行了初步研究。电镜显示噬菌体呈蝌蚪状,头部为20面体立体对称、直径约70 nm;尾部长约50 nm。ФSM9-3Y的最佳感染复数为1。一步生长曲线表明;ФSM9-3Y的潜伏期约30 min,暴发时间70 min,暴发量为629 PFU/cell。凝胶电泳显示噬菌体基因组为双链DNA、大小约54 kb。SDS-PAGE呈现至少包括13种蛋白,相对分子质量范围在25～130 kD,其中主要蛋白的相对分子质量约为48 kD。结论此次分离的噬菌体ФSM9-3Y为裂解性噬菌体,根据形态和结构特征,粘质沙雷菌噬菌体ФSM9-3Y属于有尾病毒目,肌尾噬菌体科。     

Stability and infectivity of cytolethal distending toxin type V gene-carrying bacteriophages in a water mesocosm and under different inactivation conditions

Two cytolethal distending toxin (Cdt) type V-encoding bacteriophages (Φ62 and Φ125) were induced spontaneously from their wild-type Escherichia coli strains and from the lysogens generated in Shigella sonnei. The stability of Cdt phages was determined at various temperatures and pH values after 1 month of storage by means of infectivity tests using a plaque blot assay and analysis of phage genomes using real-time quantitative PCR (qPCR): both were highly stable. We assessed the inactivation of Cdt phages by thermal treatment, chlorination, UV radiation, and in a mesocosm in both summer and winter. The results for the two Cdt phages showed similar trends and were also similar to the phage SOM23 used for reference, but they showed a much higher persistence than Cdt-producing E. coli. Cdt phages showed maximal inactivation after 1 h at 70°C, 30 min of UV radiation, and 30 min of contact with a 10-ppm chlorine treatment. Inactivation in a mesocosm was higher in summer than in winter, probably because of solar radiation. The treatments reduced the number of infectious phages but did not have a significant effect on the Cdt phage particles detected by qPCR. Cdt phages were quantified by qPCR in 73% of river samples, and these results suggest that Cdt phages are a genetic vehicle and the natural reservoir for cdt in the environment.     

A Portable Device for Concentrating Bacteriophages from Large Volumes of Freshwater

A portable apparatus for concentrating bacteriophages from up to 200 1 of raw water is described. With this apparatus eight morphologically diverse phages were concentrated from 201 -to 10 ml with recoveries of 14–66%, representing concentration factors of x280-x1320. The apparatus was also used to concentrate indigenous phages from 251 samples of river water.     

Phage typing of Escherichia coli isolated from chickens.

Ten different bacteriophages were isolated from untreated city sewage water. These phages were stable at 57 degrees C for 40 min. A modified agar layer technique was used to obtain high titre phages. Ninety-four of a stock of 101 cultures of Escherichia coli, which were isolated from inflamed portions of intestines of chickens, were lysed by one or more of these phages. The E. coli of a known serological grouping were phage typed.     

Detection of phages carrying the Shiga toxin 1 and 2 genes in waste water and river water samples

AIMS: To evaluate the occurrence and abundance of phages that carry the stx(1) and stx(2) gene in water samples of different quality. METHODS AND RESULTS: Phages growing on the Shiga toxin-negative Escherichia coli O157:H7 (ATCC 43,888) strain were enumerated by a plaque assay in concentrated raw and treated waste water samples and river water samples. Plaques were investigated for the presence of stx(1) and stx(2) genes by a multiplex/nested PCR procedure. An overall number of 805 plaques were tested for the presence of stx-carrying phages. Stx genes could be demonstrated in 2% (stx(1)) and 16% (stx(2)) of the plaques. Stx-phages were eliminated with approximately the same efficiency in comparison with somatic coliphages during the waste water treatment process. CONCLUSIONS: Due to the low numbers of phages carrying the stx genes 1 and 2 in treated waste water and river water, the dilution and inactivation of host bacteria and the unsuitable conditions for the transduction of host organisms in aquatic environments, it is difficult to derive from the data the direct evidence for a public health problem. SIGNIFICANCE AND IMPACT OF THE STUDY: The results show the quantitative occurrence of stx-carrying phages in waste and river water and confirm the frequent circulation of these viruses in the aquatic environment.     

Antibiotic resistance genes in the bacteriophage DNA fraction of environmental samples

Antibiotic resistance is an increasing global problem resulting from the pressure of antibiotic usage, greater mobility of the population, and industrialization. Many antibiotic resistance genes are believed to have originated in microorganisms in the environment, and to have been transferred to other bacteria through mobile genetic elements. Among others, β-lactam antibiotics show clinical efficacy and low toxicity, and they are thus widely used as antimicrobials. Resistance to β-lactam antibiotics is conferred by β-lactamase genes and penicillin-binding proteins, which are chromosomal- or plasmid-encoded, although there is little information available on the contribution of other mobile genetic elements, such as phages. This study is focused on three genes that confer resistance to β-lactam antibiotics, namely two β-lactamase genes (blaTEM and blaCTX-M9) and one encoding a penicillin-binding protein (mecA) in bacteriophage DNA isolated from environmental water samples. The three genes were quantified in the DNA isolated from bacteriophages collected from 30 urban sewage and river water samples, using quantitative PCR amplification. All three genes were detected in the DNA of phages from all the samples tested, in some cases reaching 104 gene copies (GC) of blaTEM or 102 GC of blaCTX-M and mecA. These values are consistent with the amount of fecal pollution in the sample, except for mecA, which showed a higher number of copies in river water samples than in urban sewage. The bla genes from phage DNA were transferred by electroporation to sensitive host bacteria, which became resistant to ampicillin. blaTEM and blaCTX were detected in the DNA of the resistant clones after transfection. This study indicates that phages are reservoirs of resistance genes in the environment.     

The phages of halophilic vibrios and their use]

The range of the lytic activity of 46 phages of parahemolytic vibrios isolated from lysogenic strains, sea water samples, crabs and mussels has been studied. The phages are represented by virions belonging to morphological groups II, IV, V according to the phage classification currently used in Russia and to different serological groups. No relationship between the sensitivity of vibrio strains to the phages under study and the specificity of serotypes O and K has been established. The preparation of diagnostic phage [see text] suitable for the identification of 82% of strains of parahemolytic vibrios has been proposed.     

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.     

Application of Cation-Coated Filter Method to Detection of Noroviruses, Enteroviruses, Adenoviruses, and Torque Teno Viruses in the Tamagawa River in Japan

The occurrence of human enteric viruses in surface water in the Tamagawa River, Japan, was surveyed for 1 year, from April 2003 to March 2004. Sixty-four samples were collected from six sites along the river, and 500 ml of the sample was concentrated using the cation-coated filter method, which was developed in our previous study. This method showed recovery yields of 56% ± 32% (n = 37) for surface water samples inoculated with polioviruses. More than one kind of tested virus was detected in 43 (67%) of 64 samples by TaqMan PCR. Noroviruses and adenoviruses were detected in a high positive ratio; 34 (53%), 28 (44%), and 29 (45%) of 64 samples were positive for norovirus genotype 1 and genotype 2 and adenoviruses, respectively. The mean concentrations of norovirus genotype 1 or genotype 2 determined by real-time PCR were 0.087 and 0.61 genome/ml, respectively, showing much higher values in winter (0.21 genome/ml for genotype 1 and 2.3 genomes/ml for genotype 2). Enteroviruses were detected by both direct PCR (6 of 64 samples; 9%) and cell culture PCR (2 of 64 samples; 3%). Torque teno viruses, emerging hepatitis viruses, were also isolated in three samples (5%). The concentration of total coliforms and the presence of F-specific phages showed a high correlation with the presence of viruses, which suggested that the simultaneous use of total coliforms and F-specific phages as indicators of surface water may work to monitor viral contamination.     

Application of cation-coated filter method to detection of noroviruses, enteroviruses, adenoviruses, and torque teno viruses in the Tamagawa River in Japan

The occurrence of human enteric viruses in surface water in the Tamagawa River, Japan, was surveyed for 1 year, from April 2003 to March 2004. Sixty-four samples were collected from six sites along the river, and 500 ml of the sample was concentrated using the cation-coated filter method, which was developed in our previous study. This method showed recovery yields of 56% +/- 32% (n = 37) for surface water samples inoculated with polioviruses. More than one kind of tested virus was detected in 43 (67%) of 64 samples by TaqMan PCR. Noroviruses and adenoviruses were detected in a high positive ratio; 34 (53%), 28 (44%), and 29 (45%) of 64 samples were positive for norovirus genotype 1 and genotype 2 and adenoviruses, respectively. The mean concentrations of norovirus genotype 1 or genotype 2 determined by real-time PCR were 0.087 and 0.61 genome/ml, respectively, showing much higher values in winter (0.21 genome/ml for genotype 1 and 2.3 genomes/ml for genotype 2). Enteroviruses were detected by both direct PCR (6 of 64 samples; 9%) and cell culture PCR (2 of 64 samples; 3%). Torque teno viruses, emerging hepatitis viruses, were also isolated in three samples (5%). The concentration of total coliforms and the presence of F-specific phages showed a high correlation with the presence of viruses, which suggested that the simultaneous use of total coliforms and F-specific phages as indicators of surface water may work to monitor viral contamination.     

Practical Procedures for the Purification of Bacterial Viruses

The efficiencies of the various methods used for phage concentration have been compared. The two-phase concentration method (with polyethylene glycol and dextran sulfate) gave maximal recoveries of infectivity for coliphages of the T-even and T-odd series and for ribonucleic acid phages and single-stranded deoxyribonucleic acid phages. Precipitation of phages by acid gave high yields when applied to T2 and T4 phages but not with T3 and T7 coliphages. Differential centrifugation was efficient when sedimented phages were gently dispersed before repeating the centrifugation cycle. The efficiencies of the various methods have also been confirmed by electron microscope studies, which also show that the two-phase concentration method gave rise to intact phages. Zone centrifugations in sucrose gradients (12.5 to 52.5%) indicated that coliphages of the T-even series sediment faster than T-odd coliphages; they may thus be separated from each other and from empty ghosts by centrifugation at 100,000 x g for 40 min. Equilibrium centrifugation in preformed cesium chloride gradients was also useful for phage concentration and purification. This study also deals with some optical properties of purified phages; optical cross sections and absorbance ratios (at 260 and 280 nm) of the various preparations are given.     

Isolation of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Specific Phages with Broad Activity Spectra

The aim of the study was to screen various kinds of samples for Pseudomonas aeruginosa specific phages and to isolate and partially characterize those with broad activity spectra. The Pseudomonas specific phages were isolated using an enrichment procedure with single strains or the cocktail of P. aeruginosa strains as hosts. Using the described procedure, phages were successfully isolated only from water samples, while in soil and feces no Pseudomonas specific phages were detected. The lytic spectra of isolated phages were determined by spot method on lawns of 33 P. aeruginosa strains and five species belonging to family Enterobacteriaceae. The results showed that among isolated phages, 001A, δ, and I possessed the broad activity spectra, as were able to plaque on more than 50% of tested P. aeruginosa strains, while none of the phages were able to lyse the other tested species. Significant differences in phage activity spectra were not observed when P. aeruginosa cocktail was applied for sample enrichment. The most of the phages examined by electron microscopy belonged to family Siphoviridae, while the broad activity spectra isolates, except for 001A, possessed morphological characteristics of family Podoviridae. Digested DNA of the phages δ and I showed similar patterns, indicating the prevalence and success of this phage type in the environment.     

Isolation and characterization of Arthrobacter bacteriophages and their application to phage typing of soil arthrobacters.

Seventeen bacteriophages, active against 19 Arthrobacter soil isolates, were isolated from concentrated samples of river water and sewage. Attempts to isolate Arthrobacter bacteriophages from filtrates of broth cultures of the soil isolates or from ultraviolet light-irradiated cultures were unsuccessful. Bacteriophages were not detected in either concentrated or unconcentrated soil extracts. Electron microscopic studies of 11 phages showed morphologies characteristic of Bradley's groups B (exhibited by 9 phages) and C (exhibited by 2 phages). Moles percent guanine plus cytosine, calculated from the deoxyribonucleic acid density of three phages, ranged from 60.2 to 65.3. The phages were characterized by their plague and virion morphology, host range, and serological specificity.