Bacteriophage-host arm race: an update on the mechanism of phage resistance in bacteria and revenge of the phage with the perspective for phage therapy.

Due to a constant attack by phage, bacteria in the environment have evolved diverse mechanisms to defend themselves. Several reviews on phage resistance mechanisms have been published elsewhere. Thanks to the advancement of molecular techniques, several new phage resistance mechanisms were recently identified. For the practical phage therapy, the emergence of phage-resistant bacteria could be an obstacle. However, unlike antibiotic, phages could evolve a mechanism to counter-adapt against phage-resistant bacteria. In this review, we summarized the most recent studies of the phage-bacteria arm race with the perspective of future applications of phages as antimicrobial agents.

Peculiarities of Staphylococcus aureus phages and their possible application in phage therapy.

Bacteriophage has become an attractive alternative for the treatment of antibiotic-resistant Staphylococcus aureus. For the success of phage therapy, phage host range is an important criterion when considering a candidate phage. Most reviews of S. aureus (SA) phages have focused on their impact on host evolution, especially their contribution to the spread of virulence genes and pathogenesis factors. The potential therapeutic use of SA phages, especially detailed characterizations of host recognition mechanisms, has not been extensively reviewed so far. In this report, we provide updates on the study of SA phages, focusing on host recognition mechanisms with the recent discovery of phage receptor-binding proteins (RBPs) and the possible applications of SA phages in phage therapy.

Analysis host-recognition mechanism of staphylococcal kayvirus ɸSA039 reveals a novel strategy that protects Staphylococcus aureus against infection by Staphylococcus pseudintermedius Siphoviridae phages.

Following the emergence of antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP), phage therapy has attracted significant attention as an alternative to antibiotic treatment. Bacteriophages belonging to kayvirus (previously known as Twort-like phages) have broad host range and are strictly lytic in Staphylococcus spp. Previous work revealed that kayvirus ɸSA039 has a host-recognition mechanism distinct from those of other known kayviruses: most of kayviruses use the backbone of wall teichoic acid (WTA) as their receptor; by contrast, ɸSA039 uses the ß-N-acetylglucosamine (ß-GlcNAc) residue in WTA. In this study, we found that ɸSA039 could switch its receptor to be able to infect S. aureus lacking the ß-GlcNAc residue by acquiring a spontaneous mutation in open reading frame (ORF) 100 and ORF102. Moreover, ɸSA039 could infect S. pseudintermedius, which has a different WTA structure than S. aureus. By comparison, with newly isolated S. pseudintermedius-specific phage (SP phages), we determined that glycosylation in WTA of S. pseudintermedius is essential for adsorption of SP phages, but not ɸSA039. Finally, we describe a novel strategy of S. aureus which protects the bacteria from infection of SP phages. Notably, glycosylation of ribitol phosphate (RboP) WTA by TarM or/and TarS prevents infection of S. aureus by SP phages. These findings could help to establish a new strategy for the treatment of S. aureus and S. pseudintermedius infection, as well as provide valuable insights into the biology of phage-host interactions.

Silviavirus phage ɸMR003 displays a broad host range against methicillin-resistant Staphylococcus aureus of human origin.

The emergence of life-threatening methicillin-resistant Staphylococcus aureus (MRSA) has led to increased interest in the use of bacteriophages as an alternative therapy to antibiotics. The success of phage therapy is greatly dependent on the selected phage possessing a wide host range. This study describes phage ɸMR003 isolated from sewage influent at a municipal wastewater treatment plant in Tokyo, Japan. ɸMR003 could infect 97% of 104 healthcare- and community-associated MRSA strains tested, compared with 73% for phage ɸSA012, which has a broad host range against bovine mastitis S. aureus. Genome analysis revealed that ɸMR003 belongs to the genus Silviavirus which has not been studied extensively. ɸMR003 recognizes and binds to wall teichoic acid (WTA) of S. aureus during infection. In silico comparisons of the genomes of ɸMR003 and ɸSA012 revealed that ORF117 and ORF119 of ɸMR003 are homologous to the putative receptor-binding proteins ORF103 and ORF105 of ɸSA012, with amino acid similarities of 75% and 72%, respectively. ORF104, which is an N-acetylglucosaminidase found in the ɸMR003 tail, may facilitate phage's infection onto the WTA-null S. aureus RN4220. The differences in tail and baseplate proteins may be key contributing factors to the different host specificities of ɸMR003 and ɸSA012. ɸMR003 showed strong adsorptivity, but not infectivity, against S. aureus SA003, which may be influenced by the bacterium's restriction modification system. This study expands our knowledge of the genomic diversity and host specificity of Silviavirus, which is a potential phage therapy candidate for MRSA infections.

Analysis of phage resistance in Staphylococcus aureus SA003 reveals different binding mechanisms for the closely related Twort-like phages ɸSA012 and ɸSA039.

We have previously generated strains of Staphylococcus aureus SA003 resistant to its specific phage ɸSA012 through a long-term coevolution experiment. However, the DNA mutations responsible for the phenotypic change of phage resistance are unknown. Whole-genome analysis revealed eight genes that acquired mutations: six point mutations (five missense mutations and one nonsense mutation) and two deletions. Complementation of the phage-resistant strains by the wild-type alleles showed that five genes were linked to phage adsorption of ɸSA012, and two mutated host genes were linked to the inhibition of post-adsorption. Unlike ɸSA012, infection by ɸSA039, a close relative of ɸSA012, onto early coevolved phage-resistant SA003 (SA003R2) was impaired drastically. Here, we identified that ɸSA012 and ɸSA039 adsorb to the cell surface S. aureus SA003 through a different mechanism. ɸSA012 requires the backbone of wall teichoic acids (WTA), while ɸSA039 requires both backbone and the ß-GlcNAc residue. In silico analysis of the ɸSA039 genome revealed that several proteins in the tail and baseplate region were different from ɸSA012. The difference in tail and baseplate proteins might be the factor for specificity difference between ɸSA012 and ɸSA039.

Fate of Escherichia coli in dialysis device exposed into sewage influent and activated sludge.

Tracing the fate of pathogens in environmental water, particularly in wastewater, with a suitable methodology is a demanding task. We investigated the fate of Escherichia coli K12 in sewage influent and activated sludge using a novel approach that involves the application of a biologically stable dialysis device. The ion concentrations inside the device could reach that of surrounding solution when it was incubated in phosphate buffered saline for 2 h. E. coli K12 above 107 CFU mL-1 (inoculated in distilled water, influent, activated sludge) were introduced into the device and incubated in influent and activated sludge for 10 days. Without indigenous microorganisms, E. coli K12 could survive even with the limited ions and nutrients concentrations in influent and activated sludge. E. coli K12 abundance in influent and activated sludge were reduced by 60 and 85%, respectively, after just 1 day. The establishment of microbial community in wastewater played an important role in reducing E. coli K12. Bacteriophage propagated in filtered influent or activated sludge when E. coli K12 was introduced, but not in raw influent or activated sludge. The methodology developed in this study can be applied in the actual environmental water to trace the fate of pathogens.

The Presence of Two Receptor-Binding Proteins Contributes to the Wide Host Range of Staphylococcal Twort-Like Phages.

UNLABELLED: Thanks to their wide host range and virulence, staphylococcal bacteriophages (phages) belonging to the genus Twortlikevirus (staphylococcal Twort-like phages) are regarded as ideal candidates for clinical application for Staphylococcus aureus infections due to the emergence of antibiotic-resistant bacteria of this species. To increase the usability of these phages, it is necessary to understand the mechanism underlying host recognition, especially the receptor-binding proteins (RBPs) that determine host range. In this study, we found that the staphylococcal Twort-like phage ΦSA012 possesses at least two RBPs. Genomic analysis of five mutant phages of ΦSA012 revealed point mutations in orf103, in a region unique to staphylococcal Twort-like phages. Phages harboring mutated ORF103 could not infect S. aureus strains in which wall teichoic acids (WTAs) are glycosylated with α-N-acetylglucosamine (α-GlcNAc). A polyclonal antibody against ORF103 also inhibited infection by ΦSA012 in the presence of α-GlcNAc, suggesting that ORF103 binds to α-GlcNAc. In contrast, a polyclonal antibody against ORF105, a short tail fiber component previously shown to be an RBP, inhibited phage infection irrespective of the presence of α-GlcNAc. Immunoelectron microscopy indicated that ORF103 is a tail fiber component localized at the bottom of the baseplate. From these results, we conclude that ORF103 binds α-GlcNAc in WTAs, whereas ORF105, the primary RBP, is likely to bind the WTA backbone. These findings provide insight into the infection mechanism of staphylococcal Twort-like phages. IMPORTANCE: Staphylococcus phages belonging to the genus Twortlikevirus (called staphylococcal Twort-like phages) are considered promising agents for control of Staphylococcus aureus due to their wide host range and highly lytic capabilities. Although staphylococcal Twort-like phages have been studied widely for therapeutic purposes, the host recognition process of staphylococcal Twort-like phages remains unclear. This work provides new findings about the mechanisms of host recognition of the staphylococcal Twort-like phage ΦSA012. The details of the host recognition mechanism of ΦSA012 will allow us to analyze the mechanisms of infection and expand the utility of staphylococcal Twort-like phages for the control of S. aureus.

Phage Therapy Is Effective in a Mouse Model of Bacterial Equine Keratitis.

UNLABELLED: Bacterial keratitis of the horse is mainly caused by staphylococci, streptococci, and pseudomonads. Of these bacteria, Pseudomonas aeruginosa sometimes causes rapid corneal corruption and, in some cases, blindness. Antimicrobial resistance can make treatment very difficult. Therefore, new strategies to control bacterial infection are required. A bacteriophage (phage) is a virus that specifically infects and kills bacteria. Since phage often can lyse antibiotic-resistant bacteria because the killing mechanism is different, we examined the use of phage to treat horse bacterial keratitis. We isolated Myoviridae or Podoviridae phages, which together have a broad host range. They adsorb efficiently to host bacteria; more than 80% of the ΦR18 phage were adsorbed to host cells after 30 s. In our keratitis mouse model, the administration of phage within 3 h also could kill bacteria and suppress keratitis. A phage multiplicity of infection of 100 times the host bacterial number could kill host bacteria effectively. A cocktail of two phages suppressed bacteria in the keratitis model mouse. These data demonstrated that the phages in this study could completely prevent the keratitis caused by P. aeruginosa in a keratitis mouse model. Furthermore, these results suggest that phage may be a more effective prophylaxis for horse keratitis than the current preventive use of antibiotics. Such treatment may reduce the use of antibiotics and therefore antibiotic resistance. Further studies are required to assess phage therapy as a candidate for treatment of horse keratitis. IMPORTANCE: Antibiotic-resistant bacteria are emerging all over the world. Bacteriophages have great potential for resolution of this problem. A bacteriophage, or phage, is a virus that infects bacteria specifically. As a novel therapeutic strategy against racehorse keratitis caused by Pseudomonas aeruginosa, we propose the application of phages for treatment. Phages isolated in this work had in vitro effectiveness for a broad range of P. aeruginosa strains. Indeed, a great reduction of bacterial proliferation was shown in phage therapy for mouse models of P. aeruginosa keratitis. Therefore, to reduce antibiotic usage, phage therapy should be investigated and developed further.

Persistence of antibiotic-resistant and -sensitive Proteus mirabilis strains in the digestive tract of the housefly (Musca domestica) and green bottle flies (Calliphoridae).

Synanthropic flies have been implicated in the rapid dissemination of antibiotic-resistant bacteria and resistance determinants in the biosphere. These flies stably harbor a considerable number of bacteria that exhibit resistance to various antibiotics, but the mechanisms underlying this phenomenon remain unclear. In this study, we investigated the persistence of antibiotic-resistant bacteria in the digestive tract of houseflies and green bottle flies, using Proteus mirabilis as a model microorganism. One resistant strain carried the blaTEM and aphA1 genes, and another carried a plasmid containing qnrD gene. Quantitative PCR and 454 pyrosequencing were used to monitor the relative abundance of the Proteus strains, as well as potential changes in the overall structure of the whole bacterial community incurred by the artificial induction of Proteus cultures. Both antibiotic-resistant and -sensitive P. mirabilis strains persisted in the fly digestive tract for at least 3 days, and there was no significant difference in the relative abundance of resistant and sensitive strains despite the lower growth rate of resistant strains when cultured in vitro. Therefore, conditions in the fly digestive tract may allow resistant strains to survive the competition with sensitive strains in the absence of antibiotic selective pressure. The composition of the fly-associated bacterial community changed over time, but the contribution of the artificially introduced P. mirabilis strains to these changes was not clear. In order to explain these changes, it will be necessary to obtain more information about bacterial interspecies antagonism in the fly digestive tract.

Seasonal variations in bacterial communities and antibiotic-resistant strains associated with green bottle flies (Diptera: Calliphoridae).

Green bottle flies occur frequently around human environments in Japan. Many species of green bottle flies have been studied with regard to their importance in forensic examinations or clinical therapies, but the bacterial communities associated with this group of flies have not been comprehensively investigated. In this research, 454 pyrosequencing was used to reveal the bacterial communities in green bottle flies collected in different seasons. Meanwhile, the bacteria were screened with selective media and tested for antibiotic susceptibility. Samples collected in three different seasons harbored distinctive bacterial communities. The predominant genera associated with green bottles flies were Staphylococcus in spring, Ignatzschineria in summer, and Vagococcus, Dysgonomonas, and an unclassified Acetobacteraceae in autumn. An upward trend in bacterial community diversity was observed from spring to autumn. Changes in climatic conditions could be the cause of these seasonal variations in fly-associated bacterial communities. The species of isolated antibiotic-resistant bacteria also differed across seasons, but it was difficult to correlate seasonal changes in antibiotic-resistant bacteria with changes in whole communities. A number of multiple-antibiotic-resistant bacteria were isolated, and some of these strains were closely affiliated with pathogens such as Enterococcus faecalis and Enterococcus faecium, which could cause serious threats to public health. Overall, this research provided us with information about the composition and seasonality of bacterial communities in green bottle flies, and highlighted the risks of fly-mediated dissemination of antibiotic-resistant pathogens.

Selective bacteriophages reduce the emergence of resistant bacteria in bacteriophage-antibiotic combination therapy.

Escherichia coli O157:H7 is a globally important foodborne pathogen with implications for food safety. Antibiotic treatment for O157 may potentially contribute to the exacerbation of hemolytic uremic syndrome, and the increasing prevalence of antibiotic-resistant strains necessitates the development of new treatment strategies. In this study, the bactericidal effects and resistance development of antibiotic and bacteriophage monotherapy were compared with those of combination therapy against O157. Experiments involving continuous exposure of O157 to phages and antibiotics, along with genetic deletion studies, revealed that the deletion of glpT and uhpT significantly increased resistance to fosfomycin. Furthermore, we found that OmpC functions as a receptor for the PP01 phage, which infects O157, and FhuA functions as a receptor for the newly isolated SP15 phage, targeting O157. In the glpT and uhpT deletion mutants, additional deletion in ompC, the receptor for the PP01 phage, increased resistance to fosfomycin. These findings suggest that specific phages may contribute to antibiotic resistance by selecting the emergence of gene mutations responsible for both phage and antibiotic resistance. While combination therapy with phages and antibiotics holds promise for the treatment of bacterial infections, careful consideration of phage selection is necessary.IMPORTANCEThe combination treatment of fosfomycin and bacteriophages against Escherichia coli O157 demonstrated superior bactericidal efficacy compared to monotherapy, effectively suppressing the emergence of resistance. However, mutations selected by phage PP01 led to enhanced resistance not only to the phage but also to fosfomycin. These findings underscore the importance of exercising caution in selecting phages for combination therapy, as resistance selected by specific phages may increase the risk of developing antibiotic resistance.

Comparative analysis of bacterial community and antibiotic-resistant strains in different developmental stages of the housefly (Musca domestica).

The housefly (Musca domestica) is an important host for a variety of bacteria, including some pathogenic and antibiotic-resistant strains. To further investigate the relationship between the housefly and the bacteria it harbors, it is necessary to understand the fate of microorganisms during the larval metamorphosis. The major bacterial communities in three developmental stages of the housefly (maggot, pupa, and adult fly) were investigated by a culture-independent method, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of 16S rRNA genes. The bacteria that were identified using DGGE analysis spanned phyla Proteobacteria, Firmicutes, and Bacteroidetes. Changes in the predominant genera were observed during the housefly development. Bacteroides, Koukoulia, and Schineria were detected in maggots, Neisseria in pupae, and Macrococcus, Lactococcus, and Kurthia in adult flies. Antibiotic-resistant bacteria were screened using a selective medium and tested for antibiotic susceptibility. Most resistant isolates from maggots and pupae were classified as Proteus spp., while those from adult flies were much more diverse and spanned 12 genera. Among 20 tested strains across the three stages, 18 were resistant to at least two antibiotics. Overall, we demonstrated that there are changes in the major bacterial communities and antibiotic-resistant strains as the housefly develops.

Iodine from bacterial iodide oxidization by Roseovarius spp. inhibits the growth of other bacteria.

Microbial activities in brine, seawater, or estuarine mud are involved in iodine cycle. To investigate the effects of the microbiologically induced iodine on other bacteria in the environment, a total of 13 bacteria that potentially participated in the iodide-oxidizing process were isolated from water or biofilm at a location containing 131 µg ml(-1) iodide. Three distinct strains were further identified as Roseovarius spp. based on 16 S rRNA gene sequences after being distinguished by restriction fragment length polymorphism analysis. Morphological characteristics of these three Roseovarius spp. varied considerably across and within strains. Iodine production increased with Roseovarius spp. growth when cultured in Marine Broth with 200 µg ml(-1) iodide (I(-)). When 10(6) CFU/ml Escherichia coli, Pseudomonas aeruginosa, and Bacillus pumilus were exposed to various concentrations of molecular iodine (I(2)), the minimum inhibitory concentrations (MICs) were 0.5, 1.0, and 1.0 µg ml(-1), respectively. However, fivefold increases in the MICs for Roseovarius spp. were obtained. In co-cultured Roseovarius sp. IOB-7 and E. coli in Marine Broth containing iodide (I(-)), the molecular iodine concentration was estimated to be 0.76 µg ml(-1) after 24 h and less than 50 % of E. coli was viable compared to that co-cultured without iodide. The growth inhibition of E. coli was also observed in co-cultures with the two other Roseovarius spp. strains when the molecular iodine concentration was assumed to be 0.52 µg ml(-1).

A method for evaluating the host range of bacteriophages using phages fluorescently labeled with 5-ethynyl-2'-deoxyuridine (EdU).

The evaluation of bacteriophage (phage) host range is a significant issue in understanding phage and prokaryotic community interactions. However, in conventional methods, such as plaque assay, target host strains must be isolated, although almost all environmental prokaryotes are recalcitrant to cultivation. Here, we introduce a novel phage host range evaluation method using fluorescently labeled phages (the FLP method), which consists of the following four steps: (i) Fluorescently labeled phages are added to a microbial consortium, and host cells are infected and fluorescently labeled. (ii) Fluorescent cells are sorted by fluorescence-activated cell sorting. (iii) 16S rRNA gene sequences retrieved from sorted cells are analyzed, and specific oligonucleotide probes for fluorescence in situ hybridization (FISH) are designed. (iv) Cells labeled with both fluorescently labeled phage and FISH probe are identified as host cells. To verify the feasibility of this method, we used T4 phage and Escherichia coli as a model. We first used nucleic acid stain reagents for phage labeling; however, the reagents also stained non-host cells. Next, we employed the Click-iT EdU (5-ethynyl-2'-deoxyuridine) assay kit from Invitrogen for phage labeling. Using EdU-labeled T4 phage, we could specifically detect E. coli cells in a complex microbial consortium from municipal sewage. We also confirmed that FISH could be applied to the infected E. coli cells. These results suggest that this FLP method using the EdU assay kit is a useful method for evaluating phage host range and may have a potential application for various types of phages, even if their prokaryotic hosts are currently unculturable.

Relationship between Phage Lytic Spectra and Sequence Types in Extended-Spectrum ß-Lactamase-Producing Escherichia coli Isolated in Japan.

The lytic spectrum of phages is usually limited to only a few strains of the same bacterial species that can lyse. In clinical molecular epidemiology, bacterial strains are commonly classified into sequence types (STs) using the multilocus sequence typing (MLST) approach. The aim of this study was to determine the association between the phage lytic spectrum and STs. MLST analysis of 11 extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli clinical isolates revealed that most belonged to ST73 or ST131, with four isolates each. Phages were isolated from sewage samples using various E. coli strains as hosts. The relationship between phage lytic spectra of ESBL-producing E. coli isolates ST73 and ST131 and STs was evaluated using Fisher's exact test. The lytic spectra of phages were significantly dependent on the ST classification of ST73 or ST131, suggesting that a phage lysing an isolate belonging to a particular ST could lyse other isolates of the same ST. We successfully isolated wide-host-range phages lysing all clinical isolates belonging to two clinically important ST types (ST73 and ST131).

Modification of the immune response by bacteriophages alters methicillin-resistant Staphylococcus aureus infection.

There is an urgent need to develop phage therapies for multidrug-resistant bacterial infections. However, although bacteria have been shown to be susceptible to phage therapy, phage therapy is not sufficient in some cases. PhiMR003 is a methicillin-resistant Staphylococcus aureus phage previously isolated from sewage influent, and it has demonstrated high lytic activity and a broad host range to MRSA clinical isolates in vitro. To investigate the potential of phiMR003 for the treatment of MRSA infection, the effects of phiMR003 on immune responses in vivo were analysed using phiMR003-susceptible MRSA strains in a mouse wound infection model. Additionally, we assessed whether phiMR003 could affect the immune response to infection with a nonsusceptible MRSA strain. Interestingly, wounds infected with both susceptible and nonsusceptible MRSA strains treated with phiMR003 demonstrated decreased bacterial load, reduced inflammation and accelerated wound closure. Moreover, the infiltration of inflammatory cells in infected tissue was altered by phiMR003. While the effects of phiMR003 on inflammation and bacterial load disappeared with heat inactivation of phiMR003. Transcripts of proinflammatory cytokines induced by lipopolysaccharide were reduced in mouse peritoneal macrophages. These results show that the immune modulation occurring as a response to the phage itself improves the clinical outcomes of phage therapy.

Biological properties of Staphylococcus virus ΦSA012 for phage therapy.

Staphylococcus virus ΦSA012 has a wide host range and efficient lytic activity. Here, we assessed the biological stability of ΦSA012 against temperature, freeze-thawing, and pH to clinically apply the phage. In addition, inoculation of ΦSA012 through i.p. and i.v. injections into mice revealed that phages were reached the limit of detection in serum and accumulated notably spleens without inflammation at 48 h post-inoculation. Furthermore, inoculation of ΦSA012 through s.c. injections in mice significantly induced IgG, which possesses neutralizing activity against ΦSA012 and other Staphylococcus viruses, ΦSA039 and ΦMR003, but not Pseudomonas viruses ΦS12-3 and ΦR18 or Escherichia viruses T1, T4, and T7 in vitro. Immunoelectron microscopic analysis showed that purified anti-phage IgG recognizes the long-tail fiber of staphylococcus viruses. Although S. aureus inoculation resulted in a 25% survival rate in a mouse i.p. model, ΦSA012 inoculation (i.p.) improved the survival rate to 75%; however, the survival rate of ΦSA012-immunized mice decreased to less than non-immunized mice with phage i.v. injection at a MOI of 100. These results indicated that ΦSA012 possesses promise for use against staphylococcal infections but we should carefully address the appropriate dose and periods of phage administration. Our findings facilitate understandings of staphylococcus viruses for phage therapy.

Increased bioclogging and corrosion risk by sulfate addition during iodine recovery at a natural gas production plant.

Iodine recovery at a natural gas production plant in Japan involved the addition of sulfuric acid for pH adjustment, resulting in an additional about 200 mg/L of sulfate in the waste brine after iodine recovery. Bioclogging occurred at the waste brine injection well, causing a decrease in well injectivity. To examine the factors that contribute to bioclogging, an on-site experiment was conducted by amending 10 L of brine with different conditions and then incubating the brine for 5 months under open air. The control case was exposed to open air but did not receive additional chemicals. When sulfate addition was coupled with low iodine, there was a drastic increase in the total amount of accumulated biomass (and subsequently the risk of bioclogging) that was nearly six times higher than the control. The bioclogging-associated corrosion rate of carbon steel was 84.5 µm/year, which is four times higher than that observed under other conditions. Analysis of the microbial communities by denaturing gradient gel electrophoresis revealed that the additional sulfate established a sulfur cycle and induced the growth of phototrophic bacteria, including cyanobacteria and purple bacteria. In the presence of sulfate and low iodine levels, cyanobacteria and purple bacteria bloomed, and the accumulation of abundant biomass may have created a more conducive environment for anaerobic sulfate-reducing bacteria. It is believed that the higher corrosion rate was caused by a differential aeration cell that was established by the heterogeneous distribution of the biomass that covered the surface of the test coupons.

Identification and characterization of a novel Enterococcus bacteriophage with potential to ameliorate murine colitis.

Increase of the enteric bacteriophages (phage), components of the enteric virome, has been associated with the development of inflammatory bowel diseases. However, little is known about how a given phage contributes to the regulation of intestinal inflammation. In this study, we isolated a new phage associated with Enterococcus gallinarum, named phiEG37k, the level of which was increased in C57BL/6 mice with colitis development. We found that, irrespective of the state of inflammation, over 95% of the E. gallinarum population in the mice contained phiEG37k prophage within their genome and the phiEG37k titers were proportional to that of E. gallinarum in the gut. To explore whether phiEG37k impacts intestinal homeostasis and/or inflammation, we generated mice colonized either with E. gallinarum with or without the prophage phiEG37k. We found that the mice colonized with the bacteria with phiEG37k produced more Mucin 2 (MUC2) that serves to protect the intestinal epithelium, as compared to those colonized with the phage-free bacteria. Consistently, the former mice were less sensitive to experimental colitis than the latter mice. These results suggest that the newly isolated phage has the potential to protect the host by strengthening mucosal integrity. Our study may have clinical implication in further understanding of how bacteriophages contribute to the gut homeostasis and pathogenesis.

Intercrossing of phage genomes in a phage cocktail and stable coexistence with Escherichia coli O157:H7 in anaerobic continuous culture.

The emergence of phage-resistant cells is the most serious problem for realizing phage therapy and is observed frequently if only one phage strain is used against a particular bacterium. By contrast, using multiple phages (phage cocktail) can delay or control the appearance of phage-resistant cells. Anaerobic continuous culturing of Escherichia coli O157:H7 and a cocktail of EP16, PP17, and SP22 phages were conducted. Comparison of the restriction fragment length polymorphism (RFLP) pattern of each phage genome showed a pattern different from wild type. Furthermore, the RFLP pattern of mutant phages consisted of fragments of PP17 and SP22 genome, suggesting both phages had infected the same host simultaneously (superinfection) and exchanged genomic DNA. Through observation of the binding of SYBR Gold-stained mutant phage to individual phage-resistant cells (RC), we found that clonal RC cultures were heterogeneous in their ability to bind mutant phage. The ratio of susceptibility was a few percent, which suggested that a minority of the RC population was susceptible to phage, and this heterogeneity contributes to the stable coexistence of RC and chimeric phages. The ratio of susceptible cells did not change appreciably from bacterial generation to generation.