Presence Of Phage Research Articles

Bacteriophages in clinical samples can interfere with microbiological diagnostic tools.

Bacteriophages are viruses that infect bacteria, and they are found everywhere their bacterial hosts are present, including the human body. To explore the presence of phages in clinical samples, we assessed 65 clinical samples (blood, ascitic fluid, urine, cerebrospinal fluid, and serum). Infectious tailed phages were detected in >45% of ascitic fluid and urine samples. Three examples of phage interference with bacterial isolation were observed. Phages prevented the confluent bacterial growth required for an antibiogram assay when the inoculum was taken from an agar plate containing lysis plaques, but not when taken from a single colony in a phage-free area. In addition, bacteria were isolated directly from ascitic fluid, but not after liquid enrichment culture of the same samples, since phage propagation lysed the bacteria. Lastly, Gram-negative bacilli observed in a urine sample did not grow on agar plates due to the high densities of infectious phages in the sample.

Preliminary survey of local bacteriophages with lytic activity against multi-drug resistant bacteria.

Bacteriophages (phages) represent a potential alternative for combating multi-drug resistant bacteria. Because of their narrow host range and the ever emergence of novel pathogen variants the continued search for phages is a prerequisite for optimal treatment of bacterial infections. Here we performed an ad hoc survey in the surroundings of a University hospital for the presence of phages with therapeutic potential. To this end, 16 aquatic samples of different origins and locations were tested simultaneously for the presence of phages with lytic activity against five current, but distinct strains each from the ESKAPE-group (i.e., Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae). Phages could be isolated for 70% of strains, covering all bacterial species except S. aureus. Apart from samples from two lakes, freshwater samples were largely devoid of phages. By contrast, one liter of hospital effluent collected at a single time point already contained phages active against two-thirds of tested strains. In conclusion, phages with lytic activity against nosocomial pathogens are unevenly distributed across environments with the prime source being the immediate hospital vicinity.

Tandem mass spectrometry determination of the putative structure of a heterogeneous mixture of Lipid As isolated from the lipopolysaccharide of the Gram-negative bacteria Aeromonas liquefaciens SJ-19a.

We report herein the electrospray ionization mass spectrometry (ESI-MS) and low-energy collision-induced dissociation tandem mass spectrometry analysis (CID-MS/MS) of a mixture of lipid As isolated from the rough lipopolysaccharide (LPS) of the mutant wild strain of the Gram-negative bacteria Aeromonas liquefaciens (SJ-19a, resistant) grown in the presence of phages. The interaction between the phages and the Gram-negative bacteria regulates host specificity and the heterogeneity of the lipid A component of the LPS. The heterogeneous mixture of lipid As was isolated by the aqueous phenol method from the LPS of the rough wild strain of Gram-negative bacteria Aeromonas liquefaciens (SJ-19a). Hydrolysis of the LPS was with 1% acetic acid, and purification was by chromatography using Sephadex G-50 and Sephadex G-15. ESI-MS and low-energy CID-MS/MS analyses were performed with a triple-quadrupole (QqQ) and a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Preliminary analysis of the lipid As mixture was conducted by ESI-MS in the negative ion mode and the spectrum obtained suggested that the lipid A SJ-19a was composed of a heterogeneous mixture of different lipid A molecules. CID-MS/MS experiments confirmed the identities of the various mono-phosphorylated β-D-GlcpN-(1→6)-α-D-GlcpN disaccharide entities. This lipid As mixture was asymmetrically substituted with fatty acids such as ((R)-14:0(3-OH)), (14:0(3-(R)-(O-12:0)) and (14:0(3-(R)-O-(14:0)) located on the O-3, O-3', N-2 and N-2' positions, respectively. Low-energy collision-induced dissociation tandem mass spectrometry in-space (QqQ-MS/MS) and in-time (FTICR-MS/MS) allowed the exact determination of the fatty acid acylation positions on the H2 PO3 →4-O'-β-D-GlcpN-(1→6)-α-D-GlcpN disaccharide backbones of this heterogeneous mixture of lipid As , composed inter alia of seven different substituted lipid As , formed from the incomplete biosynthesis of their respective LPS.

Microbiological investigation into the cause of Equine Grass Sickness

A role for bacteriophage therapy was envisaged earlylast century; however, due to discovery of the antimicrobials, it fell out of research interest. Currently, bacteriophages are resurfacing as an alternative to antimicrobials in order to overcome the increasing incidence of antimicrobial resistance. Here, we report isolation of bacteriophages against Escherichia coli, Shigella spp., Aeromonas hydrophila and Citrobacter sedlakii isolates of equine origin. Phages were isolated from equine farm soil and sewage samples. For enrichment, sample aliquot was incubated overnight with host bacteria at 37 C with vigorous shaking. The crude lysate obtained was centrifuged and filtered and the presence of any phage in the suspension was detected by agar overlay technique. Appropriate dilutions of enriched samples were plated to obtain individual plaques and the most dominant plaque was transferred into SM buffer, serially diluted and plated for plaque re-isolation three times to ensure purity, followed by large scale preparation of phage stocks. Any host nucleic acids was degraded using pancreatic DNaseI and RNase and bacteriophage particles were precipitated using PEG8000. Phage titre was determined by plaque assay and phage concentrates were accessioned in the Veterinary Type Culture Collection (VTCC) repository. The phage concentrates were visualized by transmission electron microscopy (TEM). The temperature stability of bacteriophages was checked after incubating phage concentrates over the range of 4 C-8 0C temperature for one hour. A clear single plaque was obtained on nutrient agar against Shigella spp. and after purification and concentration, its analysis by electron microscopy revealed presence of multiple phages belonging to families Myoviridae, Siphoviridae and Podoviridae. However in case of Citrobacter sedlakii, a Siphoviridae phage (VTCCBPA61) with dimensions: 60 nm x 650 nm was observed. Against a pathogenic A. hydrophila isolate of equine origin (expressing aerolysin gene), a Myoviridae phage (VTCCBPA6) was isolated with dimensions: 62 nm x 138 nm. Against E. coli of equine origin, a Myoviridae phage (VTCCBPA9) of dimensions: 86 nm x 100 nm was obtained. Bacteriophage VTCCPBA61 against C. sedlakii was found to completely lose its biological activity at 65 C in vitro however the group of phages against Shigella spp. were found to be stable upto temperature as high as 80C. Thus we demonstrated the basic biological characteristics of phages, and some novel ones (such as against A. hyrophila and C. sedlakii) bacterial isolates of equine originwhich have never been reported till now. These lytic phages could find potential in phage therapy, as biocides, in biosensors and in phage ligand technology and are being explored by us further to depict their therapeutic value in small animal model. As more studies are reporting safety, tolerance and efficacy of phage therapy in humans and animals, their use in phage therapy has a promising future as an emerging alternative to chemical agents.

Isolation and characterization of bacteriophages against equine pathogens-novel phages revealed as phage therapy candidates

A role for bacteriophage therapy was envisaged early last century; however, due to discovery of the antimicrobials, it fell out of research interest. Currently, bacteriophages are resurfacing as an alternative to antimicrobials in order to overcome the increasing incidence of antimicrobial resistance. Here, we report isolation of bacteriophages against Escherichia coli, Shigella spp., Aeromonas hydrophila and Citrobacter sedlakii isolates of equine origin. Phages were isolated from equine farm soil and sewage samples. For enrichment, sample aliquot was incubated overnight with host bacteria at 37 C with vigorous shaking. The crude lysate obtained was centrifuged and filtered and the presence of any phage in the suspension was detected by agar overlay technique. Appropriate dilutions of enriched samples were plated to obtain individual plaques and themost dominant plaquewas transferred into SM buffer, serially diluted and plated for plaque re-isolation three times to ensure purity, followed by large scale preparation of phage stocks. Any host nucleic acids was degraded using pancreatic DNaseI and RNase and bacteriophage particles were precipitated using PEG8000. Phage titre was determined by plaque assay and phage concentrates were accessioned in the Veterinary Type Culture Collection (VTCC) repository. The phage concentrates were visualized by transmission electron microscopy (TEM). The temperature stability of bacteriophages was checked after incubating phage concentrates over the range of 4 C 80 C temperature for one hour. A clear single plaque was obtained on nutrient agar against Shigella spp. and after purification and concentration, its analysis by electron microscopy revealed presence of multiple phages belonging to families Myoviridae, Siphoviridae and Podoviridae. However in case of Citrobacter sedlakii, a Siphoviridae phage (VTCCBPA61) with dimensions: 60 nm x 650 nm was observed. Against a pathogenic A. hydrophila isolate of equine origin (expressing aerolysin gene), a Myoviridae phage (VTCCBPA6) was isolated with dimensions: 62 nm x 138 nm. Against E. coli of equine origin, a Myoviridae phage (VTCCBPA9) of dimensions: 86 nm x 100 nm was obtained. Bacteriophage VTCCPBA61 against C. sedlakiiwas found to completely lose its biological activity at 65 C in vitro however the group of phages against Shigella spp. were found to be stable upto temperature as high as 80 C. Thus we demonstrated the basic biological characteristics of phages, and some novel ones (such as against A. hyrophila and C. sedlakii) bacterial isolates of equine originwhich have never been reported till now. These lytic phages could find potential in phage therapy, as biocides, in biosensors and in phage ligand technology and are being explored by us further to depict their therapeutic value in small animal model. As more studies are reporting safety, tolerance and efficacy of phage therapy in humans and animals, their use in phage therapy has a promising future as an emerging alternative to chemical agents. Posters 006 Microbiological investigation into the cause of Equine Grass Sickness

Temperate phages promote colicin-dependent fitness of Salmonella enterica serovar Typhimurium.

Bacteria employ bacteriocins for interference competition in microbial ecosystems. Colicin Ib (ColIb), a pore-forming bacteriocin, confers a significant fitness benefit to Salmonella enterica serovar Typhimurium (S. Tm) in competition against commensal Escherichia coli in the gut. ColIb is released from S. Tm into the environment, where it kills susceptible competitors. However, colicin-specific release proteins, as they are known for other colicins, have not been identified in case of ColIb. Thus, its release mechanism has remained unclear. In the current study, we have established a new link between ColIb release and lysis activity of temperate, lambdoid phages. By the use of phage-cured S. Tm mutant strains, we show that the presence of temperate phages and their lysis genes is necessary and sufficient for release of active ColIb into the culture supernatant. Furthermore, phage-mediated lysis significantly enhanced S. Tm fitness in competition against a ColIb-susceptible competitor. Finally, transduction with the lambdoid phage 933W rescued the defect of E. coli strain MG1655 with respect to ColIb release. In conclusion, ColIb is released from bacteria in the course of phage lysis. Our data reveal a new mechanism for colicin release and point out a novel function of temperate phages in enhancing colicin-dependent bacterial fitness.

In-Vitro Adhesion and Invasion Properties of Salmonella Typhimurium Competing with Bacteriophage in Epithelial Cells and Chicken Macrophages

This study was designed to assess the role of bacteriophage P22 in the adhesion, invasion, intracellular survival of, and cellular immune response to Salmonella Typhimurium in intestinal epithelial INT-407 and chicken macrophage-like HD11 cells. The ability of S. Typhimurium to adhere, invade, and survive to INT-407 and HD11cells was evaluated under Salmonella infection alone (control), phage treatment followed by Salmonella infection (PS), Salmonella infection followed by phage treatment (SP), and a combination treatment with Salmonella and phage (S+P). The number of S. Typhimurium associated on INT-407 cells was reduced from 4.2 to 2.7 log cfu/cm2 by phage treatment (SP). The number of intracellular S. Typhimurium within INT-407 cells was significantly reduced to below the detection limit (0.7 log cfu/cm2) compared with the control (3.4 log cfu/cm2). S. Typhimurium remained inside HD11 cells at 49% and 17% levels in the absence and presence of phages, respectively, at 24 h post-infection (hpi). The expression levels of IFN-g, IL-10, IL-1b, IL-6, IL-8, iNOS, and IL-12 increased in HD11 cells regardless the absence and presence of phages, while those of IL-16, TLR2-1, TLR3, and TLR7 were decreased at 0 and 24 hpi. This study sheds new light on our understanding of the role of phages in Salmonella adhesion, invasion, survival, and cellular immune responses.

Phage selection for bacterial cheats leads to population decline.

While predators and parasites are known for their effects on bacterial population biology, their impact on the dynamics of bacterial social evolution remains largely unclear. Siderophores are iron-chelating molecules that are key to the survival of certain bacterial species in iron-limited environments, but their production can be subject to cheating by non-producing genotypes. In a selection experiment conducted over approximately 20 bacterial generations and involving 140 populations of the pathogenic bacterium Pseudomonas aeruginosa PAO1, we assessed the impact of a lytic phage on competition between siderophore producers and non-producers. We show that the presence of lytic phages favours the non-producing genotype in competition, regardless of whether iron use relies on siderophores. Interestingly, phage pressure resulted in higher siderophore production, which constitutes a cost to the producers and may explain why they were outcompeted by non-producers. By the end of the experiment, however, cheating load reduced the fitness of mixed populations relative to producer monocultures, and only monocultures of producers managed to grow in the presence of phage in situations where siderophores were necessary to access iron. These results suggest that public goods production may be modulated in the presence of natural enemies with consequences for the evolution of social strategies.

Plasmid carriage can limit bacteria-phage coevolution.

Coevolution with bacteriophages is a major selective force shaping bacterial populations and communities. A variety of both environmental and genetic factors has been shown to influence the mode and tempo of bacteria–phage coevolution. Here, we test the effects that carriage of a large conjugative plasmid, pQBR103, had on antagonistic coevolution between the bacterium Pseudomonas fluorescens and its phage, SBW25ϕ2. Plasmid carriage limited bacteria–phage coevolution; bacteria evolved lower phage-resistance and phages evolved lower infectivity in plasmid-carrying compared with plasmid-free populations. These differences were not explained by effects of plasmid carriage on the costs of phage resistance mutations. Surprisingly, in the presence of phages, plasmid carriage resulted in the evolution of high frequencies of mucoid bacterial colonies. Mucoidy can provide weak partial resistance against SBW25ϕ2, which may have limited selection for qualitative resistance mutations in our experiments. Taken together, our results suggest that plasmids can have evolutionary consequences for bacteria that go beyond the direct phenotypic effects of their accessory gene cargo.

Bacteriophages Isolated from Chicken Meat and the Horizontal Transfer of Antimicrobial Resistance Genes.

Antimicrobial resistance in microbes poses a global and increasing threat to public health. The horizontal transfer of antimicrobial resistance genes was thought to be due largely to conjugative plasmids or transposons, with only a minor part being played by transduction through bacteriophages. However, whole-genome sequencing has recently shown that the latter mechanism could be highly important in the exchange of antimicrobial resistance genes between microorganisms and environments. The transfer of antimicrobial resistance genes by phages could underlie the origin of resistant bacteria found in food. We show that chicken meat carries a number of phages capable of transferring antimicrobial resistance. Of 243 phages randomly isolated from chicken meat, about a quarter (24.7%) were able to transduce resistance to one or more of the five antimicrobials tested into Escherichia coli ATCC 13706 (DSM 12242). Resistance to kanamycin was transduced the most often, followed by that to chloramphenicol, with four phages transducing tetracycline resistance and three transducing ampicillin resistance. Phages able to transduce antimicrobial resistance were isolated from 44% of the samples of chicken meat that we tested. The statistically significant (P = 0.01) relationship between the presence of phages transducing kanamycin resistance and E. coli isolates resistant to this antibiotic suggests that transduction may be an important mechanism for transferring kanamycin resistance to E. coli. It appears that the transduction of resistance to certain antimicrobials, e.g., kanamycin, not only is widely distributed in E. coli isolates found on meat but also could represent a major mechanism for resistance transfer. The result is of high importance for animal and human health.

CRISPR immunity drives rapid phage genome evolution in Streptococcus thermophilus.

ABSTRACTMany bacteria rely on CRISPR-Cas systems to provide adaptive immunity against phages, predation by which can shape the ecology and functioning of microbial communities. To characterize the impact of CRISPR immunization on phage genome evolution, we performed long-term bacterium-phage (Streptococcus thermophilus-phage 2972) coevolution experiments. We found that in this species, CRISPR immunity drives fixation of single nucleotide polymorphisms that accumulate exclusively in phage genome regions targeted by CRISPR. Mutation rates in phage genomes highly exceed those of the host. The presence of multiple phages increased phage persistence by enabling recombination-based formation of chimeric phage genomes in which sequences heavily targeted by CRISPR were replaced. Collectively, our results establish CRISPR-Cas adaptive immunity as a key driver of phage genome evolution under the conditions studied and highlight the importance of multiple coexisting phages for persistence in natural systems.

Starter culture design to overcome phage infection during yogurt fermentation

Streptococcus thermophilus phages present hazards for yogurt processing. In order to avoid adverse effects, different component yogurt starters were designed. For 2-component yogurt starters consisting of an S. thermophilus sensitive and a Lactobacillus delbrueckii ssp. bulgaricus strain, phage infection was destructive. Phages resulted in increased fermentation time, unbalanced cocci to rod ratios, decreased viscosity values, and varied sensory properties. For 3-component yogurt starters consisting of S. thermophilus sensitive, S. thermophilus insensitive, and L. bulgaricus strains, phage invasion did not result in the undesirable effects of slow acidification, decreased viscosity values, and unbalanced cocci to rod ratios. The 3-component yogurt starter stabilized fermentation and improved the practical fermentation performance in the presence of phages. Combinations of different S. thermophilus strains in the design and use of yogurt starters should be considered.

Assessment of indicators of fecal contamination in soils treated with biosolids for growing grasses

Developing countries currently face challenges related to the management and disposal of biosolids that result from wastewater treatment. One of the most important issues they must deal with involves the maximum level of pathogens permitted and the effect they may have on the environment and human health once they are used for agricultural purposes. In order to learn about the risk generated by these kinds of practices, we assessed the behavior of indicators of fecal contamination in grasses used to feed dairy cattle. The study area was divided into two plots: an experimental plot in which diluted biosolids were applied and a control plot without the application of biosolids. We collected samples from soil, grass, surface water and groundwater and analyzed the presence of fecal coliforms, Salmonella, somatic phages and helminth eggs. Experimental and control plots showed a decrease in the concentration of bacterial indicators assessed in soil, and low concentrations in surface water, groundwater and grass. Concentrations of viral and parasite indicators tend to decrease more slowly. Our results show that this way of biosolid management allows a better use of its benefits as an organic amendment by lowering the sanitary risk due to the presence of pathogens.

Experimental approach for bacteriophage susceptibility testing of planktonic and sessile bacterial populations - Study protocol.

Antimicrobial resistance is a growing threat for all clinical branches. This phenomenon poses important challenges in controlling infectious diseases. However, multidrug resistance is not the only issue, as bacteria that are otherwise susceptible to common antibiotics express other patterns for evading antibiotherapy, for example they can aggregate within a self-produced matrix to form biofilm. We intend to perform a prospective laboratory study of the germs isolated from different samples collected from patients admitted with infectious pathology in reference hospitals in Romania. We will perform antibiotic resistance testing as well as phage testing, both on solid and liquid growth medium, for Staphylococcus spp., Enterococcus spp., and Pseudomonas spp. We intend to collect data for 150 patients with different infections with these identified pathogens. Phage susceptibility testing will be performed using 5 types of strain-specific bacteriophage mixtures: PYO, INTESTI, STAPHYLOCOCCAL (Eliava BioPreparations, Tbilisi, Georgia), PHAGYO, PHAGESTI (JSC "Biochimpharm", Tbilisi, Georgia). For phage-susceptible strains, we will evaluate biofilm formation in the presence of phages, as well as phage effect on already formed biofilm. Through this study, we intend to provide the first set of results on bacteriophage-susceptibility of bacteria isolated from patients with hard to treat infections, from reference hospitals in Romania. By evaluating a large number of bacterial strains we aim to predict and project biofilm kinetics, while adding binary phage dilutions at key timepoints during biofilm formation. POSDRU/159/1.5/S/141531; Carol Davila University of Medicine and Pharmacy, Young Researchers Grant no. 28341/2013.

Lytic phages obscure the cost of antibiotic resistance in Escherichia coli.

The long-term persistence of antibiotic-resistant bacteria depends on their fitness relative to other genotypes in the absence of drugs. Outside the laboratory, viruses that parasitize bacteria (phages) are ubiquitous, but costs of antibiotic resistance are typically studied in phage-free experimental conditions. We used a mathematical model and experiments with Escherichia coli to show that lytic phages strongly affect the incidence of antibiotic resistance in drug-free conditions. Under phage parasitism, the likelihood that antibiotic-resistant genetic backgrounds spread depends on their initial frequency, mutation rate and intrinsic growth rate relative to drug-susceptible genotypes, because these parameters determine relative rates of phage-resistance evolution on different genetic backgrounds. Moreover, the average cost of antibiotic resistance in terms of intrinsic growth in the antibiotic-free experimental environment was small relative to the benefits of an increased mutation rate in the presence of phages. This is consistent with our theoretical work indicating that, under phage selection, typical costs of antibiotic resistance can be outweighed by realistic increases in mutability if drug resistance and hypermutability are genetically linked, as is frequently observed in clinical isolates. This suggests the long-term distribution of antibiotic resistance depends on the relative rates at which different lineages adapt to other types of selection, which in the case of phage parasitism is probably extremely common, as well as costs of resistance inferred by classical in vitro methods.

Bacteriophage P22 to challenge Salmonella in foods

In this study we considered the influence of phage addition on the fate of Salmonella enterica serovar Typhimurium in different foods. Phage P22 was applied to the following: liquid eggs, energy drinks, whole and skimmed milk, apple juice, chicken breast and chicken mince all spiked with its host, whose growth was monitored for 24 and 48h at 4°C. Appreciable host inactivation, generally in the order of 2 log cycles, was achieved compared to phage-free controls in all food matrices when 104UFC/g host inoculum was used. Furthermore, wild food strains belonging to the serotypes Typhimurium, Enteritidis, Derby Give, Newport, Muenchen and Muenster were assayed towards phage P22. Only isolates of Salmonella Typhimurium as well as Salmonella Derby and Salmonella Enteritidis was inhibited by the presence of P22 phage. Additional challenge experiments were carried out by spiking liquid-eggs, chicken breast and chicken mince with mixes of wild Salmonella Typhimurium (at concentration of about 104UFC/g) strains along with their relative phage P22. The results showed a reduction of 2–3 log cycles after 48h at 4°C depending on both mix of strains and the specific food. Overall, the results indicate that phages may be useful in the control of food-borne pathogens. The food matrices considered, the liquid more than the solid, do not seem to affect the phage ability of infection compared to similar tests performed in vitro.

Identification and characterization of a peculiar vtx2-converting phage frequently present in verocytotoxin-producing Escherichia coli O157 isolated from human infections.

Certain verocytotoxin-producing Escherichia coli (VTEC) O157 phage types (PTs), such as PT8 and PT2, are associated with severe human infections, while others, such as PT21, seem to be restricted to cattle. In an attempt to delve into the mechanisms underlying such a differential distribution of PTs, we performed microarray comparison of human PT8 and animal PT21 VTEC O157 isolates. The main differences observed were in the vtx2-converting phages, with the PT21 strains bearing a phage identical to that present in the reference strain EDL933, BP933W, and all the PT8 isolates displaying lack of hybridization in some regions of the phage genome. We focused on the region spanning the gam and cII genes and developed a PCR tool to investigate the presence of PT8-like phages in a panel of VTEC O157 strains belonging to different PTs and determined that a vtx2 phage reacting with the primers deployed, which we named Φ8, was more frequent in VTEC O157 strains from human disease than in bovine strains. No differences were observed in the production of the VT2 mRNA when Φ8-positive strains were compared with VTEC O157 possessing BP933W. Nevertheless, we show that the gam-cII region of phage Φ8 might carry genetic determinants downregulating the transcription of the genes encoding the components of the type III secretion system borne on the locus of enterocyte effacement pathogenicity island.

Phages can constrain protist predation-driven attenuation of Pseudomonas aeruginosa virulence in multienemy communities

The coincidental theory of virulence predicts that bacterial pathogenicity could be a by-product of selection by natural enemies in environmental reservoirs. However, current results are ambiguous and the simultaneous impact of multiple ubiquitous enemies, protists and phages on virulence evolution has not been investigated previously. Here we tested experimentally how Tetrahymena thermophila protist predation and PNM phage parasitism (bacteria-specific virus) alone and together affect the evolution of Pseudomonas aeruginosa PAO1 virulence, measured in wax moth larvae. Protist predation selected for small colony types, both in the absence and presence of phage, which showed decreased edibility to protists, reduced growth in the absence of enemies and attenuated virulence. Although phage selection alone did not affect the bacterial phenotype, it weakened protist-driven antipredatory defence (biofilm formation), its associated pleiotropic growth cost and the correlated reduction in virulence. These results suggest that protist selection can be a strong coincidental driver of attenuated bacterial virulence, and that phages can constrain this effect owing to effects on population dynamics and conflicting selection pressures. Attempting to define causal links such as these might help us to predict the cold and hot spots of coincidental virulence evolution on the basis of microbial community composition of environmental reservoirs.

Enzyme Treatment Reverse Transcription-PCR To Differentiate Infectious and Inactivated F-Specific RNA Phages

F-specific (F+) RNA phages are recommended as indicators of fecal contamination and the presence of enteric viruses and as viral surrogates to elucidate the resistance of viruses to adverse conditions or to assess the effectiveness of inactivating processes. Reverse transcription (RT)-PCR methods have been used to detect, quantify, or identify subgroups of F+ RNA phages. However, these methods may overestimate the infectivity of F+ RNA phages in test samples, since the presence of both infectious and inactivated phages (or naked RNA) can lead to positive RT-PCR signals. In this study, we evaluated the ability of an enzyme treatment (ET) with proteinase K and RNase A prior to RNA extraction, followed by RT-PCR, to differentiate infectious and inactivated F+ RNA phages. The results indicated that ET RT-PCR reduced, but did not completely eliminate, false-positive signals encountered with RT-PCR alone. The two-step ET RT-PCR, in which the enzymes were added sequentially, was more effective at reducing false-positive signals than the one-step ET RT-PCR, which involved addition of both enzymes together. Despite its inability to completely eliminate false-positive signals, ET RT-PCR gave more reliable information on the infectivity of F+ RNA phages. Thus, the method is better than RT-PCR alone for detecting F+ RNA phages as indicators to assess the risk of fecal contamination by enteric pathogens or to evaluate the effectiveness of virus-inactivating processes.

Survey of lysogenic phages in the 72 strains of Escherichia coli collection of reference (ECOR) and identification of a phage derived from the ECOR52 strain

Escherichia coli collection of reference (ECOR) is a standard collection of 72 wild-type E. coli strains that represent natural E. coli populations found in various environments. Although these strains are widely used in experiments investigating the physiology and behavior of wild-type E. coli, their genetic features including accessory DNA have not been sufficiently studied. In this study, we surveyed for the presence of lysogenic phages in each ECOR strain under both inducing and non-inducing conditions. We found that 34 strains could produce plaque-forming phages; among them, 14 strains were newly discovered to harbor lysogenic phages capable of entering the lytic cycle. We isolated a new phage (designated as “MSU52-L1”) from the ECOR52 strain and identified it as a P22/lambda-like phage with homology to known phages, such as CUS-3, HK620, and HK140.