Transduction of Porcine Enteropathogenic Escherichia coli with a Derivative of a Shiga Toxin 2-Encoding Bacteriophage in a Porcine Ligated Ileal Loop System

In this study, we have investigated the ability of detoxified Shiga toxin (Stx)-converting bacteriophages Φ3538 (Δstx₂::cat) (H. Schmidt et al., Appl. Environ. Microbiol. 65:3855-3861, 1999) and H-19B::Tn10d-bla (D. W. Acheson et al., Infect. Immun. 66:4496-4498, 1998) to lysogenize enteropathogenic Escherichia coli (EPEC) strains in vivo. We were able to transduce the porcine EPEC strain 1390 (O45) with Φ3538 (Δstx₂::cat) in porcine ligated ileal loops but not the human EPEC prototype strain E2348/69 (O127). Neither strain 1390 nor strain E2348/69 was lysogenized under these in vivo conditions when E. coli K-12 containing H-19B::Tn10d-bla was used as the stx1 phage donor. The repeated success in the in vivo transduction of an Stx2-encoding phage to a porcine EPEC strain in pig loops was in contrast to failures in the in vitro trials with these and other EPEC strains. These results indicate that in vivo conditions are more effective for transduction of Stx2-encoding phages than in vitro conditions.     

Persistence of Infectious Shiga Toxin-Encoding Bacteriophages after Disinfection Treatments

In Shiga toxin-producing Escherichia coli (STEC), induction of Shiga toxin-encoding bacteriophages (Stx phages) causes the release of free phages that can later be found in the environment. The ability of Stx phages to survive different inactivation conditions determines their prevalence in the environment, the risk of stx transduction, and the generation of new STEC strains. We evaluated the infectivity and genomes of two Stx phages (Φ534 and Φ557) under different conditions. Infectious Stx phages were stable at 4, 22, and 37°C and at pH 7 and 9 after 1 month of storage but were completely inactivated at pH 3. Infective Stx phages decreased moderately when treated with UV (2.2-log₁₀ reduction for an estimated UV dose of 178.2 mJ/cm²) or after treatment at 60 and 68°C for 60 min (2.2- and 2.5-log₁₀ reductions, respectively) and were highly inactivated (3 log₁₀) by 10 ppm of chlorine in 1 min. Assays in a mesocosm showed lower inactivation of all microorganisms in winter than in summer. The number of Stx phage genomes did not decrease significantly in most cases, and STEC inactivation was higher than phage inactivation under all conditions. Moreover, Stx phages retained the ability to lysogenize E. coli after some of the treatments.     

Genomic,Proteomic and Morphological Characterization of Two Novel Broad Host Lytic Bacteriophages ΦPD10.3 and ΦPD23.1 Infecting Pectinolytic Pectobacterium spp. and Dickeya spp.

Pectinolytic Pectobacterium spp. and Dickeya spp. are necrotrophic bacterial pathogens of many important crops, including potato, worldwide. This study reports on the isolation and characterization of broad host lytic bacteriophages able to infect the dominant Pectobacterium spp. and Dickeya spp. affecting potato in Europe viz. Pectobacterium carotovorum subsp. carotovorum (Pcc), P. wasabiae (Pwa) and Dickeya solani (Dso) with the objective to assess their potential as biological disease control agents. Two lytic bacteriophages infecting stains of Pcc, Pwa and Dso were isolated from potato samples collected from two potato fields in central Poland. The ΦPD10.3 and ΦPD23.1 phages have morphology similar to other members of the Myoviridae family and the Caudovirales order, with a head diameter of 85 and 86 nm and length of tails of 117 and 121 nm, respectively. They were characterized for optimal multiplicity of infection, the rate of adsorption to the Pcc, Pwa and Dso cells, the latent period and the burst size. The phages were genotypically characterized with RAPD-PCR and RFLP techniques. The structural proteomes of both phages were obtained by fractionation of phage proteins by SDS-PAGE. Phage protein identification was performed by liquid chromatography-mass spectrometry (LC-MS) analysis. Pulsed-field gel electrophoresis (PFGE), genome sequencing and comparative genome analysis were used to gain knowledge of the length, organization and function of the ΦPD10.3 and ΦPD23.1 genomes. The potential use of ΦPD10.3 and ΦPD23.1 phages for the biocontrol of Pectobacterium spp. and Dickeya spp. infections in potato is discussed.     

Characterization of the Genome of the Dairy Lactobacillus helveticus Bacteriophage ΦAQ113

The complete genomic sequence of the dairy Lactobacillus helveticus bacteriophage ΦAQ113 was determined. Phage ΦAQ113 is a Myoviridae bacteriophage with an isometric capsid and a contractile tail. The final assembled consensus sequence revealed a linear, circularly permuted, double-stranded DNA genome with a size of 36,566 bp and a G+C content of 37%. Fifty-six open reading frames (ORFs) were predicted, and a putative function was assigned to approximately 90% of them. The ΦAQ113 genome shows functionally related genes clustered together in a genome structure composed of modules for DNA replication/regulation, DNA packaging, head and tail morphogenesis, cell lysis, and lysogeny. The identification of genes involved in the establishment of lysogeny indicates that it may have originated as a temperate phage, even if it was isolated from natural cheese whey starters as a virulent phage, because it is able to propagate in a sensitive host strain. Additionally, we discovered that the ΦAQ113 phage genome is closely related to Lactobacillus gasseri phage KC5a and Lactobacillus johnsonii phage Lj771 genomes. The phylogenetic similarities between L. helveticus phage ΦAQ113 and two phages that belong to gut species confirm a possible common ancestral origin and support the increasing consideration of L. helveticus as a health-promoting organism.     

The Hybrid Pre-CTXΦ-RS1 Prophage Genome and Its Regulatory Function in Environmental Vibrio cholerae O1 Strains

The cholera toxin genes of Vibrio cholerae are encoded by CTXΦ, a lysogenic bacteriophage. Infection with this phage plays a determinant role in toxigenicity conversion and the emergence of new clones of pathogenic V. cholerae. Multiple phage alleles, defined by sequence types of the repressor gene rstR, have been found, showing the divergence of phage genomes. Pre-CTXΦ, which is characterized by the absence of toxin genes, is predicted to be the precursor of CTXΦ. We have found a new pre-CTXΦ prophage genome (named pre-CTX^ZJΦ for its novel rstR allele) in nontoxigenic V. cholerae O1 isolates that were obtained during surveillance of the estuary water of the Zhujiang River. A novel hybrid genome of the helper phage RS1 was identified in an environmental strain carrying pre-CTX^ZJΦ in this study. The chromosomal integration and genomic arrangement of pre-CTX^ZJΦ and RS1 were determined. The RS2 of pre-CTX^ZJΦ was shown to have a function in replication, but it seemed to have lost its ability to integrate. The RstR of pre-CTX^ZJΦ exerted the highest repression of its own rstA promoter compared to other RstRs, suggesting rstR-specific phage superinfection immunity and potential coinfection with other pre-CTXΦ/CTXΦ alleles. The environmental strain carrying pre-CTX^ZJΦ could still be infected by CTX^ETΦ, the most common phage allele in the strains of the seventh cholera pandemic, suggesting that this nontoxigenic clone could potentially undergo toxigenicity conversion by CTXΦ infection and become a new toxigenic clone despite already containing the pre-CTXΦ prophage.     

Beyond arbitrium: identification of a second communication system in Bacillus phage phi3T that may regulate host defense mechanisms

The evolutionary stability of temperate bacteriophages at low abundance of susceptible bacterial hosts lies in the trade-off between the maximization of phage replication, performed by the host-destructive lytic cycle, and the protection of the phage-host collective, enacted by lysogeny. Upon Bacillus infection, Bacillus phages phi3T rely on the “arbitrium” quorum sensing (QS) system to communicate on their population density in order to orchestrate the lysis-to-lysogeny transition. At high phage densities, where there may be limited host cells to infect, lysogeny is induced to preserve chances of phage survival. Here, we report the presence of an additional, host-derived QS system in the phi3T genome, making it the first known virus with two communication systems. Specifically, this additional system, coined “Rapφ-Phrφ”, is predicted to downregulate host defense mechanisms during the viral infection, but only upon stress or high abundance of Bacillus cells and at low density of population of the phi3T phages. Post-lysogenization, Rapφ-Phrφ is also predicted to provide the lysogenized bacteria with an immediate fitness advantage: delaying the costly production of public goods while nonetheless benefiting from the public goods produced by other non-lysogenized Bacillus bacteria. The discovered “Rapφ-Phrφ” QS system hence provides novel mechanistic insights into how phage communication systems could contribute to the phage-host evolutionary stability.Subject terms: Bacteriophages, Viral genetics     

Bacteriophage Prevalence in the Genus Azospirillum and Analysis of the First Genome Sequence of an Azospirillum brasilense Integrative Phage

The prevalence of bacteriophages was investigated in 24 strains of four species of plant growth-promoting rhizobacteria belonging to the genus Azospirillum. Upon induction by mitomycin C, the release of phage particles was observed in 11 strains from three species. Transmission electron microscopy revealed two distinct sizes of particles, depending on the identity of the Azospirillum species, typical of the Siphoviridae family. Pulsed-field gel electrophoresis and hybridization experiments carried out on phage-encapsidated DNAs revealed that all phages isolated from A. lipoferum and A. doebereinerae strains had a size of about 10 kb whereas all phages isolated from A. brasilense strains displayed genome sizes ranging from 62 to 65 kb. Strong DNA hybridizing signals were shown for most phages hosted by the same species whereas no homology was found between phages harbored by different species. Moreover, the complete sequence of the A. brasilense Cd bacteriophage (ΦAb-Cd) genome was determined as a double-stranded DNA circular molecule of 62,337 pb that encodes 95 predicted proteins. Only 14 of the predicted proteins could be assigned functions, some of which were involved in DNA processing, phage morphogenesis, and bacterial lysis. In addition, the ΦAb-Cd complete genome was mapped as a prophage on a 570-kb replicon of strain A. brasilense Cd, and a region of 27.3 kb of ΦAb-Cd was found to be duplicated on the 130-kb pRhico plasmid previously sequenced from A. brasilense Sp7, the parental strain of A. brasilense Cd.     

Antisense RNA Targeting of Primase Interferes with Bacteriophage Replication in Streptococcus thermophilus

The putative primase gene and other genes associated with the Sfi21-prototype genome replication module are highly conserved in Streptococcus thermophilus bacteriophages. Expression of antisense RNAs complementary to the putative primase gene (pri3.1) from S. thermophilus phage κ3 provided significant protection from κ3 and two other Sfi21-type phages. Expression of pri3.10-AS, an antisense RNA that covered the entire primase gene, reduced the efficiency of plaquing (EOP) of κ3 to 3 × 10⁻³ and reduced its burst size by 20%. Mutant phages capable of overcoming antisense inhibition were not recovered. Thirteen primase-specific antisense cassettes of different lengths (478 to 1,512 bp) were systematically designed to target various regions of the gene. Each cassette conferred some effect, reducing the EOP to between 0.8 and 3 × 10⁻³. The largest antisense RNAs (1.5 kb) were generally found to confer the greatest reductions in EOP, but shorter (0.5 kb) antisense RNAs were also effective, especially when directed to the 5′ region of the gene. The impacts of primase-targeted antisense RNAs on phage development were examined. The expression of pri3.10-AS resulted in reductions in target RNA abundance and the number of phage genomes synthesized. Targeting a key genome replication function with antisense RNA provided effective phage protection in S. thermophilus.     

Poly-γ-d-Glutamic Acid Capsule Interferes with Lytic Infection of Bacillus anthracis by B. anthracis-Specific Bacteriophages

The poly-γ-d-glutamic acid capsule of Bacillus anthracis is a barrier to infection by B. anthracis-specific bacteriophages. Capsule expression was found to completely inhibit lytic infection by γ phage, an observation supported by the demonstration that this phage does not elaborate a hydrolase that would facilitate penetration through the protective capsule outer layer.     

In Situ Population Dynamics of Bacterial Viruses in a Terrestrial Environment

Predation by bacteriophages is thought to control bacterial numbers and facilitate gene transfer among bacteria in the biosphere. A thorough understanding of phage population dynamics is therefore necessary if their significance in natural environments is to be fully appreciated. Here we describe the in situ population dynamics of three separate phage populations predating on separate bacterial species, living on the surface of field-grown sugar beet (Beta vulgaris var. Amethyst), as recorded over a 9-month period. The distributions of the three phage populations were different and fluctuated temporally in 1996 (peak density, ~10³ PFU g⁻¹). One of these populations, predating on the indigenous phytosphere bacterium Serratia liquefaciens CP6, consisted of six genetically distinct DNA phages that varied in relative abundance to the extent that an apparent temporal succession was observed between the two most abundant phages, ΦCP6-1 and ΦCP6-4.     

Isolation and Characterization of φkm18p,a Novel Lytic Phage with Therapeutic Potential against Extensively Drug Resistant Acinetobacter baumannii

Aims

To isolate phages against extensively drug resistant Acinetobacter baumannii (XDRAB) and characterize the highest lytic capability phage as a model to evaluate the potential on phage therapy.

Methods and Results

Eight phages were isolated from hospital sewage and showed narrow host spectrum. Phage φkm18p was able to effectively lyse the most XDRAB. It has a dsDNA genome of 45 kb in size and hexagonal head of about 59 nm in diameter and no tail. Bacterial population decreased quickly from 10⁸ CFU ml⁻¹ to 10³ CFU ml⁻¹ in 30 min by φkm18p. The 185 kDa lysis protein encoded by φkm18p genome was detected when the extracted protein did not boil before SDS-PAGE; it showed that the lysis protein is a complex rather than a monomer. Phage φkm18p improved human lung epithelial cells survival rates when they were incubated with A. baumannii. Combination of phages (φkm18p, φTZ1 and φ314) as a cocktail could lyse all genotype-varying XDRAB isolates.

Conclusion

Infections with XDRAB are extremely difficult to treat and development of a phage cocktails therapy could be a therapeutic alternative in the future. Phage φkm18p is a good candidate for inclusion in phage cocktails.     

Isolation and Characterization of Lytic Properties of Bacteriophages Specific for M. haemolytica Strains

ResultsEight bacteriophages were obtained, like typical of the families Myoviridae, Siphoviridae and Podoviridae. Most of the bacteriophages exhibited lytic properties against the M. haemolytica strains. Restriction analysis revealed similarities to the P2-like phage obtained from the strain M. haemolytica BAA-410. The most similar profiles were observed in the case of bacteriophages φA1 and φA5. All of the bacteriophages obtained were characterized by the presence of additional fragments in the restriction profiles with respect to the P2-like reference phage. In the analysis of PCR products for the P2-like reference phage phi-MhaA1-PHL101 ({"type":"entrez-nucleotide","attrs":{"text":"DQ426904","term_id":"90110542","term_text":"DQ426904"}}DQ426904) and the phages of the M. haemolytica serotypes, a 734-bp phage PCR product was obtained. The primers were programmed in Primer-Blast software using the structure of the sequence {"type":"entrez-nucleotide","attrs":{"text":"DQ426904","term_id":"90110542","term_text":"DQ426904"}}DQ426904 of reference phage PHL101.ConclusionsThe results obtained indicate the need for further research aimed at isolating and characterizing bacteriophages, including sequence analysis of selected fragments. Moreover, standardization of methods for obtaining them in order to eliminate M. haemolytica bacteria involved in the etiopathogenesis of BRDC is essential.     

CTXφ Replication Depends on the Histone-Like HU Protein and the UvrD Helicase

The Vibrio cholerae bacterium is the agent of cholera. The capacity to produce the cholera toxin, which is responsible for the deadly diarrhea associated with cholera epidemics, is encoded in the genome of a filamentous phage, CTXφ. Rolling-circle replication (RCR) is central to the life cycle of CTXφ because amplification of the phage genome permits its efficient integration into the genome and its packaging into new viral particles. A single phage-encoded HUH endonuclease initiates RCR of the proto-typical filamentous phages of enterobacteriaceae by introducing a nick at a specific position of the double stranded DNA form of the phage genome. The rest of the process is driven by host factors that are either essential or crucial for the replication of the host genome, such as the Rep SF1 helicase. In contrast, we show here that the histone-like HU protein of V. cholerae is necessary for the introduction of a nick by the HUH endonuclease of CTXφ. We further show that CTXφ RCR depends on a SF1 helicase normally implicated in DNA repair, UvrD, rather than Rep. In addition to CTXφ, we show that VGJφ, a representative member of a second family of vibrio integrative filamentous phages, requires UvrD and HU for RCR while TLCφ, a satellite phage, depends on Rep and is independent from HU.     

Isolation and Characterization of Two Lytic Bacteriophages, φSt2 and φGrn1; Phage Therapy Application for Biological Control of Vibrio alginolyticus in Aquaculture Live Feeds

Bacterial infections are a serious problem in aquaculture since they can result in massive mortalities in farmed fish and invertebrates. Vibriosis is one of the most common diseases in marine aquaculture hatcheries and its causative agents are bacteria of the genus Vibrio mostly entering larval rearing water through live feeds, such as Artemia and rotifers. The pathogenic Vibrio alginolyticus strain V1, isolated during a vibriosis outbreak in cultured seabream, Sparus aurata, was used as host to isolate and characterize the two novel bacteriophages φSt2 and φGrn1 for phage therapy application. In vitro cell lysis experiments were performed against the bacterial host V. alginolyticus strain V1 but also against 12 presumptive Vibrio strains originating from live prey Artemia salina cultures indicating the strong lytic efficacy of the 2 phages. In vivo administration of the phage cocktail, φSt2 and φGrn1, at MOI = 100 directly on live prey A. salina cultures, led to a 93% decrease of presumptive Vibrio population after 4 h of treatment. Current study suggests that administration of φSt2 and φGrn1 to live preys could selectively reduce Vibrio load in fish hatcheries. Innovative and environmental friendly solutions against bacterial diseases are more than necessary and phage therapy is one of them.     

Characterization of Poly-γ-Glutamate Hydrolase Encoded by a Bacteriophage Genome: Possible Role in Phage Infection of Bacillus subtilis Encapsulated with Poly-γ-Glutamate

Some Bacillus subtilis strains, including natto (fermented soybeans) starter strains, produce a capsular polypeptide of glutamate with a γ-linkage, called poly-γ-glutamate (γ-PGA). We identified and purified a monomeric 25-kDa degradation enzyme for γ-PGA (designated γ-PGA hydrolase, PghP) from bacteriophage ΦNIT1 in B. subtilis host cells. The monomeric PghP internally hydrolyzed γ-PGA to oligopeptides, which were then specifically converted to tri-, tetra-, and penta-γ-glutamates. Monoiodoacetate and EDTA both inhibited the PghP activity, but Zn²⁺ or Mn²⁺ ions fully restored the enzyme activity inhibited by the chelator, suggesting that a cysteine residue(s) and these metal ions participate in the catalytic mechanism of the enzyme. The corresponding pghP gene was cloned and sequenced from the phage genome. The deduced PghP sequence (208 amino acids) with a calculated M_r of 22,939 was not significantly similar to any known enzyme. Thus, PghP is a novel γ-glutamyl hydrolase. Whereas phage ΦNIT1 proliferated in B. subtilis cells encapsulated with γ-PGA, phage BS5 lacking PghP did not survive well on such cells. Moreover, all nine phages that contaminated natto during fermentation produced PghP, supporting the notion that PghP is important in the infection of natto starters that produce γ-PGA. Analogous to polysaccharide capsules, γ-PGA appears to serve as a physical barrier to phage absorption. Phages break down the γ-PGA barrier via PghP so that phage progenies can easily establish infection in encapsulated cells.     

Isolation and Characterization of Micromonospora Phage ΦHAU8 and Development into a Phasmid

ΦHAU8, a temperate Micromonospora phage, which is capable of infecting Micromonospora sp. strains 40027 and A-M-01, was isolated. The ΦHAU8 virion has a polyhedral head and a flexible tail and has a small genome (ca. 42.5 kb) with double-stranded DNA and cohesive ends. ΦHAU8 was most stable at 4°C in Difco nutrient broth within a pH range of 6 to 12. ΦHAU8 plaque formation on Micromonospora sp. strain 40027 was optimal with 32 mM Ca²⁺ and 30 mM Mg²⁺. A lysogen, LXH8, was isolated from turbid plaques, and a phasmid derivative that functions as a λ cosmid vector in Escherichia coli and as a phage in Micromonospora sp. strain 40027 was constructed. Pulsed-field gel electrophoresis of AseI-digested total DNA showed that ΦHAU8 DNA integrates into the 500-kb AseI fragment of Micromonospora sp. strain 40027.     

Genetic elements novel for Corynebacterium diphtheriae: specialized transducing elements and transposons

It was shown in an accompanying paper (Buck and Groman, J. Bacteriol. 148: 131-142, 1981) that γ-tsr-1 phage stocks produced by heat induction of lysogens are a mixture of two phages which differ in the content of their deoxyribonucleic acid (DNA). This difference is evidenced by the appearance of “heterogeneous” (HET) fragments in restriction enzyme digests of γ-tsr-1 phage DNA. It was estimated that 20 to 80% of the phage in these lysates produced HET fragments. The appearance of HET fragments correlated with the appearance of a DNA insertion (DI-1) in the γ phage genome as revealed in heteroduplexes of DNA from γ-tsr-1 and β corynebacteriophages. The HET fragments were seen in DNA from heat-induced lysates, but not in DNA from phage stocks produced by lytic infection. By DNA-DNA hybridization analysis it was shown that a fraction of γ-tsr-1 phages from heat-induced lysates carried an insertion of bacterial DNA in the vegetative phage attachment site (attP), and that this insertion was responsible for the formation of HET fragments. Since the phage produced by this event carried a complete phage genome plus a small segment of bacterial DNA, they were called transducing elements. On the basis of these facts it was concluded that heat-induced γ-tsr-1 prophage was excised at an abnormal site at a very high frequency. Abnormal excision was highly specific, and the change in excision specificity occurred simultaneously with the spontaneous mutation of the phage to heat inducibility. From this and other data it was postulated that a mutation in the immune repressor was reponsible for an alteration in the specificity of the normal excision process. This distinguishes the mechanism of formation of γ-tsr-1 transducing elements from that employed by other phages. A second DNA insertion (DI-2) in the tox (diphtheria toxin) gene of γ-tsr-1 and γ-tsr-2 was also identified as an insertion of bacterial DNA. The DI-2 insertion had a stem-and-loop structure similar to that seen in heteroduplexes visualizing transposons or insertion elements. It seems likely that γ wild-type phage, which is mutant for tox, was originally tox⁺, but that transposition of bacterial DNA into the gene inactivated it.     

Comparison of Five Bacteriophages as Models for Viral Aerosol Studies

Bacteriophages are perceived to be good models for the study of airborne viruses because they are safe to use, some of them display structural features similar to those of human and animal viruses, and they are relatively easy to produce in large quantities. Yet, only a few studies have investigated them as models. It has previously been demonstrated that aerosolization, environmental conditions, and sampling conditions affect viral infectivity, but viral infectivity is virus dependent. Thus, several virus models are likely needed to study their general behavior in aerosols. The aim of this study was to compare the effects of aerosolization and sampling on the infectivity of five tail-less bacteriophages and two pathogenic viruses: MS2 (a single-stranded RNA [ssRNA] phage of the Leviviridae family), Φ6 (a segmented double-stranded RNA [dsRNA] phage of the Cystoviridae family), ΦX174 (a single-stranded DNA [ssDNA] phage of the Microviridae family), PM2 (a double-stranded DNA [dsDNA] phage of the Corticoviridae family), PR772 (a dsDNA phage of the Tectiviridae family), human influenza A virus H1N1 (an ssRNA virus of the Orthomyxoviridae family), and the poultry virus Newcastle disease virus (NDV; an ssRNA virus of the Paramyxoviridae family). Three nebulizers and two nebulization salt buffers (with or without organic fluid) were tested, as were two aerosol sampling devices, a liquid cyclone (SKC BioSampler) and a dry cyclone (National Institute for Occupational Safety and Health two-stage cyclone bioaerosol sampler). The presence of viruses in collected air samples was detected by culture and quantitative PCR (qPCR). Our results showed that these selected five phages behave differently when aerosolized and sampled. RNA phage MS2 and ssDNA phage ΦX174 were the most resistant to aerosolization and sampling. The presence of organic fluid in the nebulization buffer protected phages PR772 and Φ6 throughout the aerosolization and sampling with dry cyclones. In this experimental setup, the behavior of the influenza virus resembled that of phages PR772 and Φ6, while the behavior of NDV was closer to that of phages MS2 and ΦX174. These results provide critical information for the selection of appropriate phage models to mimic the behavior of specific human and animal viruses in aerosols.