Removal and inactivation of indicator bacteriophages in fresh waters

AIMS: The removal and inactivation of faecal coliform (FC) bacteria, enterococci (ENT), sulphite-reducing clostridia (SRC), somatic coliphages, F-specific RNA bacteriophages and bacteriophages infecting Bacteroides fragilis in fresh waters. METHODS AND RESULTS: Removal was studied in two areas of a river. The results showed different removal of each group of microbes. Faecal coliform bacteria were removed faster than any other, whereas SRC and bacteriophages infecting Bact. fragilis were the most persistent. Inactivation was measured by 'in situ' experiments, which showed significant differences in survival of the different groups of bacterial and bacteriophage indicators. The SRC and bacteriophages were more resistant than faecal coliforms and enterococci, with the exception of F-specific RNA bacteriophages in the summer. Inactivation experiments with pure cultures of bacteriophages confirmed that phage B40-8 of Bact. fragilis was the most resistant. CONCLUSIONS: Bacteria and bacteriophages show different resistance to natural inactivation. The use of phages allows information to be obtained in addition to that provided by bacterial indicators. Somatic coliphages and phages infecting Bact. fragilis might supply that indicator function. SIGNIFICANCE AND IMPACT OF THE STUDY: Confirmation was obtained that bacteriophages provided additional information to that provided by bacterial indicators to monitor the natural inactivation of viruses and/or pathogens.     

Longitudinal Study of Campylobacter jejuni Bacteriophages and Their Hosts from Broiler Chickens

A longitudinal study of bacteriophages and their hosts was carried out at a broiler house that had been identified as having a population of Campylobacter-specific bacteriophages. Cloacal and excreta samples were collected from three successive broiler flocks reared in the same barn. Campylobacter jejuni was isolated from each flock, whereas bacteriophages could be isolated from flocks 1 and 2 but were not isolated from flock 3. The bacteriophages isolated from flocks 1 and 2 were closely related to each other in terms of host range, morphology, genome size, and genetic content. All Campylobacter isolates from flock 1 were genotypically indistinguishable by pulsed-field gel electrophoresis (PFGE). PFGE and multilocus sequence typing indicated that this C. jejuni type was maintained from flock 1 to flock 2 but was largely superseded by three genetically distinct C. jejuni types insensitive to the resident bacteriophages. All isolates from the third batch of birds were insensitive to bacteriophages and genotypically distinct. These results are significant because this is the first study of an environmental population of C. jejuni bacteriophages and their influence on the Campylobacter populations of broiler house chickens. The role of developing bacteriophage resistance was investigated as this is a possible obstacle to the use of bacteriophage therapy to reduce the numbers of campylobacters in chickens. In this broiler house succession was largely due to incursion of new genotypes rather than to de novo development of resistance.     

First Evidence of Lysogeny in Propionibacterium freudenreichii subsp. shermanii

Dairy propionic acid bacteria, particularly the species Propionibacterium freudenreichii, play a major role in the ripening of Swiss type cheese. Isometric and filamentous bacteriophages infecting P. freudenreichii have previously been isolated from cheese. In order to determine the origin of these bacteriophages, lysogeny of P. freudenreichii was determined by isometric bacteriophage type analysis. The genomic DNA of 76 strains were hybridized with the DNA of nine bacteriophages isolated from Swiss type cheeses, and the DNA of 25 strains exhibited strong hybridization. Three of these strains released bacteriophage particules following UV irradiation (254 nm) or treatment with low concentrations of mitomycin C. A prophage-cured derivative of P. freudenreichii was readily isolated and subsequently relysogenized. Lysogeny was therefore formally demonstrated in P. freudenreichii.     

A highly efficient second-step concentration technique for bacteriophages and enteric viruses using ammonium sulfate and Tween 80

Addition of Tween 80 to a 1.5% solution of beef extract was found to enhance the elution of bacteriophages adsorbed to electronegative filters. When reconcentration of the eluate was attempted by ammonium sulfate precipitation, a floating layer containing most of the viruses was formed. This floating layer can be obtained with several nonionic detergents including Tween 80 and under a salt saturation of 55% with ammonium sulfate, potassium tartrate, and sodium phosphate. Virus recovery ranged from 91 to 103% and was obtained with several bacteriophage strains. With poliovirus type 1, coxsackievirus B-4, and rotavirus SA-11 the recoveries were 100, 20, and 80%, respectively, but toxicity to cell culture was encountered: after removal of the detergent by a second floating layer method the recovery was 32% for poliovirus. Compared with organic flocculation, this method also had both improved recovery for bacteriophages and protective properties for samples frozen at -70 degrees C.     

Prevalence of Broad-Host-Range Lytic Bacteriophages of Sphaerotilus natans, Escherichia coli, and Pseudomonas aeruginosa

Two bacteriophage collections were examined with regard to their ability to form plaques on multiple bacterial host species. Nine of 10 phages studied were found to be broad-host-range bacteriophages. These phages fell into two groups. Group 1, the SN series, was isolated from sewage treatment plant samples with Sphaerotilus natans ATCC 13338 as a host. The DNAs of these bacteriophages contained modified bases and were insensitive to cleavage by type I and II restriction endonucleases. The efficiency of plating of these bacteriophages was changed only slightly on the alternate host. Group 2, the BHR series, was isolated by a two-host enrichment protocol. These bacteriophages were sensitive to restriction, and their efficiency of plating was dramatically reduced on the alternate host. Our results suggest that a multiple-host enrichment protocol may be more effective for the isolation of broad-host-range bacteriophages by avoiding the selection bias inherent in single-host methods. At least two of the broad-host-range bacteriophages mediated generalized transduction. We suggest that broad-host-range bacteriophages play a key role in phage ecology and gene transfer in nature.     

Chromatography of Arbovirus Ribonucleic Acid Forms on Columns of Benzoylated-Diethylaminoethyl Cellulose

Benzoylated-diethylaminoethyl cellulose (BD-cellulose) column chromatography was found to be useful in resolving most of the ribonucleic acid (RNA) forms from the replicative cycle of group A arbovirus Semliki Forect virus (SFV). The elution patterns were independent of molecular weight and appeared to be related to the degree of secondary structure in the molecule. Fractions of RNA were taken from a sucrose density gradient of cytoplasmic extracts of SFV-infected chick cells pretreated with actinomycin D. In a linear salt gradient, 16S material cochromatographed with the rapidly eluted ribonuclease resistant core of the double-stranded SFV-RNA and with the homopolymer duplex polyinosinic acid: polycytidylic acid. This fraction, therefore, probably contains an SFV-RNA form similar to the completely double stranded replicative form (RF) of several RNA viruses and bacteriophages. Faster moving (>20S) sucrose gradient fractions eluted more slowly, suggesting a decreasing proportion of secondary structure with increasing sedimentation value. The fractions, therefore, seemed to contain replicative intermediate (RI) structures. The two single stranded forms of SFV-RNA (42S and 26S) could only be eluted from BD-cellulose in the presence of urea or dimethyl sulfoxide, suggesting the presence of minimal secondary structure. Under these conditions, the single-stranded viral RNA forms could not be resolved. Molecular sieve chromatography of the single-stranded RNA forms, performed by passage through an agarose column, also failed to resolve these forms. The viral RNA forms containing a high degree of secondary structure, probably the RF and the RI, could, therefore, be rapidly separated from each other and from the single-stranded forms.     

Sequencing and analysis of the cos region of the lactococcal bacteriophage c2

The cohesive termini of the DNA genome of the lactococcal bacteriophage c2 were directly sequenced and appeared to be complementary, non-symmetrical, 9-nucleotide single-stranded, 3′ extended DNAs, with the following sequence: 5′-GTTAGGCTT-3′ 3′-CAATCCGAA-5′. DNA located on either side of the cohesive ends was sequenced and several repeats and a region with the potential for a DNA bend were found. Previously sequenced cos regions of 13 other bacteriophages were also examined for similar sequence features. All of the bacteriophages from gram-positive hosts had 3′ extended DNA termini, in contrast to the bacteriophages from gram-negative hosts, which had 5′ extended DNA termini. All bacteriophages had a region of dyad symmetry close to the cohesive termini. A 7.3 kb DNA fragment of the c2 genome containing the cos sequences was cloned; transduction experiments demonstrated that these cloned sequences could act as a substrate for packaging enzymes of phage c2.     

Genotypic and Phenotypic Diversity among Induced, stx2-Carrying Bacteriophages from Environmental Escherichia coli Strains

Shiga toxin 2 (stx₂) gene-carrying bacteriophages have been shown to convert Escherichia coli strains to Shiga toxin-producing Escherichia coli (STEC). In this study, 79 E. coli strains belonging to 35 serotypes isolated from wastewaters of both human and animal origin were examined for the presence of stx₂-carrying bacteriophages in their genomes. The lytic cycle of the bacteriophages was induced by mitomycin, and the bacteriophage fraction was isolated and used for morphological and genetic characterization. The induced bacteriophages showed morphological diversity, as well as restriction fragment length polymorphism variation, in the different strains belonging to different serotypes. The ability to infect new hosts was highly variable, although most of the induced phages infected Shigella sonnei host strain 866. In summary, in spite of carrying either the same or different stx₂ variants and in spite of the fact that they were isolated from strains belonging to the same or different serotypes, the induced bacteriophages were highly variable. The high level of diversity and the great infectious capacity of these phages could enhance the spread of the stx₂ gene and variants of this gene among different bacterial populations in environments to which humans may be exposed.     

Characterization and Detection of Endolysin Gene from Three Acinetobacter baumannii Bacteriophages Isolated from Sewage Water

Acinetobacter baumannii is an opportunistic pathogen that exists in hospital environments. The emergence of multidrug resistant A. baumannii (MDRAB) has been reported worldwide. It is necessary to find a novel and effective treatment for MDRAB infection. In this study, three bacteriophages, designated as ØABP-01, ØABP-02 and ØABP-04 were selected for analysis. Transmission electron microscopy showed that bacteriophage ØABP-01 belonged to the Podoviridae family and bacteriophage ØABP-02 and ØABP-04 are classified into the family Myoviridae. ØABP-01 had the widest host range. ØABP-01, ØABP-02 and ØABP-04 exhibited a latent period of 15, 20 and 20 min. The burst sizes of the three bacteriophages were 110, 120 and 150 PFU/cell. DNA restriction analysis using EcoRI, HindIII, PstI, SphI, BamHI and SmaI showed different DNA fragment patterns between the three bacteriophages. ØABP-01 and ØABP-04 was positive for the endolysin gene as determined by PCR. In conclusion, bacteriophage ØABP-01 showed broad host-specificity, good lytic activity and a short latency period, making it an appropriate candidate for studying the control and diagnosis associated with MDRAB infections.     

Differential bacteriophage mortality on exposure to copper

Many studies report that copper can be used to control microbial growth, including that of viruses. We determined the rates of copper-mediated inactivation for a wide range of bacteriophages. We used two methods to test the effect of copper on bacteriophage survival. One method involved placing small volumes of bacteriophage lysate on copper and stainless steel coupons. Following exposure, metal coupons were rinsed with lysogeny broth, and the resulting fluid was serially diluted and plated on agar with the corresponding bacterial host. The second method involved adding copper sulfate (CuSO(4)) to bacteriophage lysates to a final concentration of 5 mM. Aliquots were removed from the mixture, serially diluted, and plated with the appropriate bacterial host. Significant mortality was observed among the double-stranded RNA (dsRNA) bacteriophages Φ6 and Φ8, the single-stranded RNA (ssRNA) bacteriophage PP7, the ssDNA bacteriophage ΦX174, and the dsDNA bacteriophage PM2. However, the dsDNA bacteriophages PRD1, T4, and λ were relatively unaffected by copper. Interestingly, lipid-containing bacteriophages were most susceptible to copper toxicity. In addition, in the first experimental method, the pattern of bacteriophage Φ6 survival over time showed a plateau in mortality after lysates dried out. This finding suggests that copper's effect on bacteriophage is mediated by the presence of water.     

Expression of Antisense RNA Targeted against Streptococcus thermophilus Bacteriophages

Antisense RNA complementary to a putative helicase gene (hel3.1) of a cos-type Streptococcus thermophilus bacteriophage was used to impede the proliferation of a number of cos-type S. thermophilus bacteriophages and one pac-type bacteriophage. The putative helicase gene is a component of the Sfi21-type DNA replication module, which is found in a majority of the S. thermophilus bacteriophages of industrial importance. All bacteriophages that strongly hybridized a 689-bp internal hel3.1 probe were sensitive to the expression of antisense hel3.1 RNA. A 40 to 70% reduction in efficiency of plaquing (EOP) was consistently observed, with a concomitant decrease in plaque size relative to that of the S. thermophilus parental strain. When progeny were released, the burst size was reduced. Growth curves of S. thermophilus NCK1125, in the presence of variable levels of bacteriophage κ3, showed that antisense hel3.1 conferred protection, even at a multiplicity of infection of approximately 1.0. When the hel3.1 antisense RNA cassette was expressed in cis from the κ3-derived phage-encoded resistance (PER) plasmid pTRK690::ori3.1, the EOP for bacteriophages sensitive to PER and antisense targeting was reduced to between 10⁻⁷ and 10⁻⁸, beyond the resistance conferred by the PER element alone (less than 10⁻⁶). These results illustrate the first successful applications of antisense RNA and explosive delivery of antisense RNA to inhibit the proliferation of S. thermophilus bacteriophages.     

An agarose derivative containing an arsenical for affinity chromatography of thiol compounds

An adsorbent for affinity chromatography of thiol compounds was prepared by covalently linking p-aminophenylarsine oxide to CNBr-activated Sepharose 6B. The adsorbent retained mono- and dithiols from acid and neutral solutions. Monothiols and 1,4-dithiols were eluted at pH 12, whereas more alkaline conditions were necessary for elution of 1,2- and 1,3-dithiols. The latter could also be eluted at pH 12 by 0.01 m phenylarsine oxide. Alternatively, thiol compounds may be eluted at neutral or acid conditions by other thiol compounds. Monothiols and 1,4-dithiols were thus eluted by cysteine, whereas 1,2- and 1,3-dithiols required dithiopropylamine for elution.     

Inactivation of bacteriophages with cis-platinum(II) diamminedichloride.

The ability of the cis-plantinum(II) diamminedichloride and its hydrolytic products to inactive DNA bacteriophages was examined on the models T2, T4, T4BO1, T3, and lambda. The inactivation of all bacteriophages under study increases gradually during the first 40-90 min of the action of neutral cis-Pt(II) and later passes into an exponential phase. The extent of the region of slower inactviation is larger for osmotically sensitive strains T2 and T4. Inactivation with the hydrolytic products for cis-Pt(II) proceeds exponentially starting from the very beginning and their inactivating effect is higher by 40-80 times than for a comparable concentration of the original complex. The extent of inactivation is not affected with the HCR marker of the host bacteria. The sensitivity to cis-Pt(II) is higher for bacteriophages with a head permeable to salts. An additional inactivation ("after-effect") was observed after dilution of the complex; it can be removed by adding S-aminoisothiuronium dihydrobromide (AET). The results obtained are in good accord with the assumption that inactivation is due to the hydrolytic products arising in the head of bacteriophage.     

Biofilm as an Environment for Dissemination of stx Genes by Transduction

Dissemination of Shiga toxin (Stx)-encoding bacteriophages is the most likely mechanism for the spread of Stx-encoding genes and the emergence of new Stx-producing Escherichia coli (STEC). Biofilm has been reported to be a place where horizontal gene transfer by plasmid conjugation and DNA transformation may occur, and in this study, horizontal gene transfer by transduction has been demonstrated. Transfer of Stx-encoding bacteriophages to potentially pathogenic E. coli in biofilm was observed at both 20°C and 37°C. The infection rates were higher at 37°C than at 20°C. To our knowledge, this study is the first to show lateral gene transfer in biofilm mediated by a temperate bacteriophage. The study shows that the biofilm environment can be suitable for transduction events and can thereby be an environment for the emergence of new pathogenic E. coli.     

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

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

Molecular characterization of the genome of Bacillus subtilis (natto) bacteriophage PM1, a phage associated with disruption of food production

“Natto”, regarded as a traditional food, is made by fermenting boiled soybeans with Bacillus subtilis (natto), which is a natto-producing strain related to B. subtilis. Natto production is disrupted by bacteriophage infection of B. subtilis (natto); thus, it is necessary to control bacteriophage infection. A bacteriophage of B. subtilis (natto), PM1, was isolated during interrupted natto production in a factory. As PM1 was shown to have a long non-contractile tail in a morphological study, it was believed to belong to the family Siphoviridae. The genome of PM1 was shown to be a linear double-stranded DNA of approximately 50 kb. Based on the results of studies using restriction endonucleases, PM1 DNA was found to be circularly permuted, similar to bacteriophage DNA without definite ends (e.g. bacteriophage T4). The nucleotide sequence of a 1.1 kb segment of PM1 was determined and used to design a PCR assay. A 0.5 kb product was amplified from eight of ten bacteriophage isolates that infect B. subtilis (natto), and the nucleotide sequences of the PCR-amplified products were identical to those of PM1, suggesting that PM1-related bacteriophages are the most prevalent infectious agents associated with the disruption of natto production. The PCR method might be useful to detect PM1-related bacteriophages and will help to control bacteriophage infection.     

Effect of nucleases on bacteria infected with bacteriophages

The effect of bacterial nucleases on bacteria infected by DNA- or RNA-containing bacteriophages with different serogroups was studied. Bacillary RNases have a strong inhibitory effect on RNA-containing bacteriophages. It was shown that nucleases suppressed the infection process of bacteria by bacteriophages M12, f2, PP7, and QB. The minimal inhibitory concentration ranged from 0.6 to 6 μg/mL. Bacterial ribonucleases have no impact on the development of DNA-containing bacteriophages PZ-A, PZ-B, P₃k, P118, and a lysogenic culture of Escherichia coli (λ) and Bacillus subtilis 168 (phi105). RNase from Bacillus pumilus did not inactivate bacteriophages Qβ and f2 in vitro and did not influence the adsorption on bacteriophages on the cell wall of the bacteria host E. coli AB301. The enzyme effect was shown at the level of bacteriophage infection of the host bacteria. Presumably, the phase between the adsorption and penetration of phage RNA into bacterial pili is the most sensitive to the effect of RNases.