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Extended-spectrum cephalosporin-resistant (ESC-R) Escherichia coli have disseminated in food-producing animals globally, attributed to horizontal transmission of blaCTX-M variants, as seen in the InCI1-blaCTX-M-1 plasmid. This ease of transmission, coupled with its demonstrated long-term persistence, presents a significant One Health antimicrobial resistance (AMR) risk. Bacteriophage (phage) therapy is a potential strategy in eliminating ESC-R E. coli in food-producing animals; however, it is hindered by the development of phage-resistant bacteria and phage biosafety concerns. Another alternative to antimicrobials is probiotics, with this study demonstrating that AMR-free commensal E. coli, termed competitive exclusion clones (CECs), can be used to competitively exclude ESC-R E. coli. This study isolated and characterized phages that lysed E. coli clones harboring the InCI1-blaCTX-M-1 plasmid, before investigation of the effect and synergy of phage therapy and competitive exclusion as a novel strategy for decolonizing ESC-resistant E. coli. In vitro testing demonstrated superiority in the combined therapy, reducing and possibly eliminating ESC-R E. coli through phage-mediated lysis coupled with simultaneous prevention of regrowth of phage-resistant mutants due to competitive exclusion with the CEC. Further investigation into this combined therapy in vivo is warranted, with on-farm application possibly reducing ESC-R prevalence, while constricting newly emergent ESC-R E. coli outbreaks prior to their dissemination throughout food-producing animals or humans. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is an opportunistic pathogen that causes a wide range of infections and food poisoning in humans with antibiotic resistance, specifically to methicillin, compounding the problem. Bacteriophages (phages) provide an alternative treatment strategy, but these only infect a limited number of circulating strains and may quickly become ineffective due to bacterial resistance. To overcome these obstacles, engineered phages have been proposed, but new methods are needed for the efficient transformation of large DNA molecules into S. aureus to “boot-up” (i.e., rescue) infectious phages. We presented a new, efficient, and reproducible DNA transformation method, NEST (non-electroporation Staphylococcus transformation), for S. aureus to boot-up purified phage genomic DNA (at least 150 kb in length) and whole yeast-assembled synthetic phage genomes. This method was a powerful new tool for the transformation of DNA in S. aureus and will enable the rapid development of engineered therapeutic phages and phage cocktails against Gram-positive pathogens. IMPORTANCE The continued emergence of antibiotic-resistant bacterial pathogens has heightened the urgency for alternative antibacterial strategies. Phages provide an alternative treatment strategy but are difficult to optimize. Synthetic biology approaches have been successfully used to construct and rescue genomes of model phages but only in a limited number of highly transformable host species. In this study, we used a new, reproducible, and efficient transformation method to reconstitute a functional nonmodel Siphophage from a constructed synthetic genome. This method will facilitate the engineering of Staphylococcus and Enterococcus phages for therapeutic applications and the engineering of Staphylococcus strains by enabling transformation of higher molecular weight DNA to introduce more complex modifications. [ABSTRACT FROM AUTHOR]

Bacterial endospores can serve as phage genome protection shells against various environmental stresses to enhance microbial control applications. The genomes of polyvalent lytic Bacillus phages PBSC1 and PBSC2, infecting both B. subtilis subsp. subtilis and B. cereus NRS 248, were incorporated into B. subtilis endospores (without integration into the host chromosome). When PBSC1 and PBSC2 were released from germinating endospores, they significantly inhibited the growth of the targeted opportunistic pathogen B. cereus. Optimal endospore entrapment was achieved when phages were introduced to the fast sporulating pre-spores at multiplicity of infection of 1. Longer endospore maturation (48 h vs 24 h) increased both spore yield and efficiency of entrapment. Compared with free phages, spore-protected phage genomes showed significantly higher resistance towards high temperatures (60 - 80 °C), extreme pH (pH2 or pH12) and copper ion (0.1 - 10 mg/L). Endospore germination is inducible by low concentrations of L-alanine or a germinant mixture (L-asparagine, D-glucose, D-fructose, and K+) to trigger the expression, assembly and consequent release of phage particles within 60 - 90 minutes. Overall, the superior resiliency of polyvalent phages protected by endospores might enable non-refrigerated phage storage and enhance phage applications after exposure to adverse environmental conditions. IMPORTANCE: Bacteriophages are being considered for the control of multi-drug resistant and other problematic bacteria in environmental systems. However, the efficacy of phage-based microbial control is limited by infectivity loss during phage delivery and/or storage. Here, we exploit the pseudolysogenic state of phages, involving incorporation of their genome into bacterial endospores (without integration into the host chromosome), to enhance survival in unfavorable environments. We isolated polyvalent (broad-host range) phages that efficiently infect both benign and opportunistic pathogenic Bacillus strains and encapsulated the phage genomes in B. subtilis endospores to significantly improve resistance to various environmental stressors. Encapsulation by spores also significantly enhanced phage genome viability during storage. We also show that endospore germination can be induced on-demand with nutrient germinants that trigger the release of active phages. Overall, we demonstrate that encapsulation of polyvalent phage genomes into benign endospores holds great promise for broadening the scope and efficacy of phage biocontrol. [ABSTRACT FROM AUTHOR]

Bacteriophage-based assays and biosensors rival traditional antibodybased immunoassays for detection of low-level Salmonella contaminations. In this study, we harnessed the binding specificity of the long tail fiber (LTF) from bacteriophage S16 as an affinity molecule for the immobilization, enrichment, and detection of Salmonella. We demonstrate that paramagnetic beads (MBs) coated with recombinant gp37-gp38 LTF complexes (LTF-MBs) are highly effective tools for rapid affinity magnetic separation and enrichment of Salmonella. Within 45 min, the LTF-MBs consistently captured over 95% of Salmonella enterica serovar Typhimurium cells from suspensions containing from 10 to 105 CFU · ml-1, and they yielded equivalent recovery rates (93% ± 5%, n = 10) for other Salmonella strains tested. LTF-MBs also captured Salmonella cells from various food sample preenrichments, allowing the detection of initial contaminations of 1 to 10 CFU per 25 g or ml. While plating of bead-captured cells allowed ultrasensitive but time-consuming detection, the integration of LTF-based enrichment into a sandwich assay with horseradish peroxidaseconjugated LTF (HRP-LTF) as a detection probe produced a rapid and easy-to-use Salmonella detection assay. The novel enzyme-linked LTF assay (ELLTA) uses HRP-LTF to label bead-captured Salmonella cells for subsequent identification by HRP-catalyzed conversion of chromogenic 3,3',5,5'-tetramethylbenzidine substrate. The color development was proportional for Salmonella concentrations between 102 and 107 CFU · ml-1 as determined by spectrophotometric quantification. The ELLTA assay took 2 h to complete and detected as few as 102 CFU · ml-1 S. Typhimurium cells. It positively identified 21 different Salmonella strains, with no cross-reactivity for other bacteria. In conclusion, the phage-based ELLTA represents a rapid, sensitive, and specific diagnostic assay that appears to be superior to other currently available tests. IMPORTANCE The incidence of foodborne diseases has increased over the years, resulting in major global public health issues. Conventional methods for pathogen detection can be laborious and expensive, and they require lengthy preenrichment steps. Rapid enrichment-based diagnostic assays, such as immunomagnetic separation, can reduce detection times while also remaining sensitive and specific. A critical component in these tests is implementing affinity molecules that retain the ability to specifically capture target pathogens over a wide range of in situ applications. The protein complex that forms the distal tip of the bacteriophage S16 long tail fiber is shown here to represent a highly sensitive affinity molecule for the specific enrichment and detection of Salmonella. Phage-encoded long tail fibers have huge potential for development as novel affinity molecules for robust and specific diagnostics of a vast spectrum of bacteria. [ABSTRACT FROM AUTHOR]

Bacteriophages are the source of many valuable tools for molecular biology and genetic manipulation. In Streptomyces, most DNA cloning vectors are based on serine integrase site-specific DNA recombination systems derived from phage. Because of their efficiency and simplicity, serine integrases are also used for diverse synthetic biology applications. Here, we present the genome of a new Streptomyces phage, ΦJoe, and investigate the conditions for integration and excision of the ΦJoe genome. ΦJoe belongs to the largest Streptomyces phage cluster (R4-like) and encodes a serine integrase. The attB site from Streptomyces venezuelae was used efficiently by an integrating plasmid, pCMF92, constructed using the ΦJoe int-attP locus. The attB site for ΦJoe integrase was occupied in several Streptomyces genomes, including that of S. coelicolor, by a mobile element that varies in gene content and size between host species. Serine integrases require a phage-encoded recombination directionality factor (RDF) to activate the excision reaction. The ΦJoe RDF was identified, and its function was confirmed in vivo. Both the integrase and RDF were active in in vitro recombination assays. The ΦJoe site-specific recombination system is likely to be an important addition to the synthetic biology and genome engineering toolbox. [ABSTRACT FROM AUTHOR]

The lactococcal abortive phage infection mechanism AbiQ recently was classified as a type III toxin-antitoxin system in which the toxic protein (ABIQ) is regulated following cleavage of its repeated noncoding RNA antitoxin (antiQ). In this study, we investigated the role of the antitoxin in antiphage activity. The cleavage of antiQ by ABIQ was characterized using 5= rapid amplification of cDNA ends PCR and was located in an adenine-rich region of antiQ. We next generated a series of derivatives with point mutations within antiQ or with various numbers of antiQ repetitions. These modifications were analyzed for their effect on the antiphage activity (efficiency of plaquing) and on the endoribonuclease activity (Northern hybridization). We observed that increasing or reducing the number of antiQ repeats significantly decreased the antiphage activity of the system. Several point mutations had a similar effect on the antiphage activity and were associated with changes in the digestion profile of antiQ. Interestingly, a point mutation in the putative pseudoknot structure of antiQ mutants led to an increased AbiQ antiphage activity, thereby offering a novel way to increase the activity of an abortive infection mechanism. [ABSTRACT FROM AUTHOR]

Lactococcal bacteriophages represent one of the leading causes of dairy fermentation failure and product inconsistencies. A new member of the lactococcal 949 phage group, named WRP3, was isolated from cheese whey from a Sicilian factory in 2011. The genome sequence of this phage was determined, and it constitutes the largest lactococcal phage genome currently known, at 130,008 bp. Detailed bioinformatic analysis of the genomic region encoding the presumed initiator complex and baseplate of WRP3 has aided in the functional assignment of several open reading frames (ORFs), particularly that for the receptor binding protein required for host recognition. Furthermore, we demonstrate that the 949 phages target cell wall phospho-polysaccharides as their receptors, accounting for the specificity of the interactions of these phages with their lactococcal hosts. Such information may ultimately aid in the identification of strains/strain blends that do not present the necessary saccharidic target for infection by these problematic phages. [ABSTRACT FROM AUTHOR]

Biofilms are major causes of impairment of wound healing and patient morbidity. One of the most common and aggressive wound pathogens is Staphylococcus aureus, displaying a large repertoire of virulence factors and commonly reduced susceptibility to antibiotics, such as the spread of methicillin-resistant S. aureus (MRSA). Bacteriophages are obligate parasites of bacteria. They multiply intracellularly and lyse their bacterial host, releasing their progeny. We isolated a novel phage, DRA88, which has a broad host range among S. aureus bacteria. Morphologically, the phage belongs to the Myoviridae family and comprises a large double-stranded DNA (dsDNA) genome of 141,907 bp. DRA88 was mixed with phage K to produce a high-titer mixture that showed strong lytic activity against a wide range of S. aureus isolates, including representatives of the major international MRSA clones and coagulase-negative Staphylococcus. Its efficacy was assessed both in planktonic cultures and when treating established biofilms produced by three different biofilm-producing S. aureus isolates. A significant reduction of biofilm biomass over 48 h of treatment was recorded in all cases. The phage mixture may form the basis of an effective treatment for infections caused by S. aureus biofilms. [ABSTRACT FROM AUTHOR]

Escherichia coli O157 is a food-borne pathogen whose major reservoir has been identified as cattle. Recent genetic information has indicated that populations of E. coli O157 from cattle and humans can differ genetically and that this variation may have an impact on their ability to cause severe human disease. In addition, there is emerging evidence that E. coli O157 strains from different geographical regions may also be genetically divergent. To investigate the extent of this variation, we used Shiga toxin bacteriophage insertion sites (SBI), lineage-specific polymorphisms (LSPA-6), multilocus variable-number tandem-repeat analysis (MLVA), and a tir 255T > A polymorphism to examine 606 isolates representing both Australian and U.S. cattle and human pop ulations. Both uni- and multivariate analyses of these data show a strong association between the country of origin and multilocus genotypes (P < 0.0001). In addition, our results identify factors that may play a role in virulence that also differed in isolates from each country, including the carriage of sfce, in the argW locus uniquely observed in Australian isolates and the much higher frequency of sfcc2-positive (also referred to as stx2J strains in the U.S. isolates (4% of Australian isolates versus 72% of U.S. iso lates). LSPA-6 lineages differed between the two continents, with the majority of Australian isolates belonging to lineage I/II (LI/ II) (LI, 2%; LI/II, 85%; LII, 13%) and the majority of U.S. isolates belonging to LI (LI, 60%; LI/II, 16%; LII, 25%). The results of this study provide strong evidence of phylogeographic structuring of E. coli 0157 populations, suggesting divergent evolution of enterohemorrhagic E. coli 0157 in Australia and the United States. [ABSTRACT FROM AUTHOR]

Malolactic fermentation (MLF) is a biochemical transformation conducted by lactic acid bacteria (LAB) that occurs in wine at the end of alcoholic fermentation. Oenococcus oeni is the main species responsible for MLF in most wines. As in other fermented foods, where bacteriophages represent a potential risk for the fermentative process, O. oeni bacteriophages have been reported to be a possible cause of unsuccessful MLF in wine. Thus, preparation of commercial starters that take into account the different sensitivities of O. oeni strains to different phages would be advisable. However, currently, no methods have been described to identify phages infecting O. oeni. In this study, two factors are addressed: detection and typing of bacteriophages. First, a simple PCR method was devised targeting a conserved region of the endolysin (lys) gene to detect temperate O. oeni bacteriophages. For this purpose, 37 O. oeni strains isolated from Italian wines during different phases of the vinification process were analyzed by PCR for the presence of the lys gene, and 25 strains gave a band of the expected size (1,160 bp). This is the first method to be developed that allows identification of lysogenic O. oeni strains without the need for time-consuming phage bacterial-lysis induction methods. Moreover, a phylogenetic analysis was conducted to type bacteriophages. After the treatment of bacteria with UV light, lysis was obtained for 15 strains, and the 15 phage DNAs isolated were subjected to two randomly amplified polymorphic DNA (RAPD)-PCRs. By combining the RAPD profiles and lys sequences, 12 different O. oeni phages were clearly distinguished. [ABSTRACT FROM AUTHOR]

A novel flagellatropic phage of Salmonella enterica serovar Typhimurium, called iEPS5, was isolated and characterized. iEPS5 has an icosahedral head and a long noncontractile tail with a tail fiber. Genome sequencing revealed a double-stranded DNA of 59,254 bp having 73 open reading frames (ORFs). To identify the receptor for iEPS5, Tn5 transposon insertion mutants of S. Typhimurium SL1344 that were resistant to the phage were isolated. All of the phage-resistant mutants were found to have mutations in genes involved in flagellar formation, suggesting that the flagellum is the adsorption target of this phage. Analysis of phage infection using the ΔmotA mutant, which is flagellated but nonmotile, demonstrated the requirement of flagellar rotation for LEPS5 infection. Further analysis of phage infection using the ΔcheY mutant revealed that iEPS5 could infect host bacteria only when the flagellum is rotating counterclockwise (CCW). These results suggested that the CCW-rotating flagellar filament is essential for phage adsorption and required for successful infection by iEPS5. In contrast to the well-studied flagellatropic phage Chi, iEPS5 cannot infect the ΔfliK mutant that makes a polyhook without a flagellar filament, suggesting that these two flagellatropic phages utilize different infection mechanisms. Here, we present evidence that iEPS5 injects its DNA into the flagellar filament for infection by assessing DNA transfer from SYBR gold-labeled iEPS5 to the host bacteria. [ABSTRACT FROM AUTHOR]

Here we present an entire temporal transcriptional profile of Lactococcus lactis subsp. cremoris UC509.9 undergoing lytic infection with two distinct bacteriophages, Tuc2009 and c2. Furthermore, corresponding high-resolution whole-phage genome tiling arrays of both bacteriophages were performed throughout lytic infection. Whole-genome microarrays performed at various time points postinfection demonstrated a rather modest impact on host transcription. The majority of changes in the host transcrip-tome occur during late infection stages; few changes in host gene transcription occur during the immediate and early infection stages. Alterations in the L. lactis UC509.9 transcriptome during lytic infection appear to be phage specific, with relatively few differentially transcribed genes shared between cells infected with Tuc2009 and those infected with c2. Despite the apparent lack of a coordinated general phage response, three themes common to both infections were noted: alternative transcription of genes involved in catabolic flux and energy production, differential transcription of genes involved in cell wall modification, and differential transcription of genes involved in the conversion of ribonucleotides to deoxyribonucleotides. The transcriptional profiles of both bacteriophages during lytic infection generally correlated with the findings of previous studies and allowed the confirmation of previously predicted promoter sequences. In addition, the host transcriptional response to lysogenization with Tuc2009 was monitored along with tiling array analysis of Tuc2009 in the lysogenic state. Analysis identified 44 host genes with altered transcription during lysogeny, 36 of which displayed levels of transcription significantly reduced from those for uninfected cells. [ABSTRACT FROM AUTHOR]

Cellular metagenomes are primarily used for investigating microbial community structure and function. However, cloned fosmids from such metagenomes capture phage genome fragments that can be used as a source of phage genomes. We show that fosmid cloning from cellular metagenomes and sequencing at a high coverage is a credible alternative to constructing metaviriomes and allows capturing and assembling novel, complete phage genomes. It is likely that phages recovered from cellular metagenomes are those replicating within cells during sample collection and represent "active" phages, naturally amplifying their genomic DNA and increasing chances for cloning. We describe five sets of siphoviral contigs (MEDS1, MEDS2, MEDS3, MEDS4, and MEDS5), obtained by sequencing fosmids from the cellular metagenome of the deep chlorophyll maximum in the Mediterranean. Three of these represent complete siphoviral genomes and two represent partial ones. This is the first set of phage genomes assembled directly from cellular metagenomic fosmid libraries. They exhibit low sequence similarities to one another and to known siphoviruses but are remarkably similar in overall genome architecture. We present evidence suggesting they infect picocyanobacteria, likely Synechococcus. Four of these sets also define a novel branch in the phylogenetic tree of phage large subunit terminases. Moreover, some of these siphoviral groups are globally distributed and abundant in the oceans, comparable to some known myoviruses and podoviruses. This suggests that, as more siphoviral genomes become available, we will be better able to assess the abundance and influence of this diverse and polyphyletic group in the marine habitat. [ABSTRACT FROM AUTHOR]

Bacterial biofilms are crucial to the pathogenesis of many important infections and are difficult to eradicate. Streptococcus suis is an important pathogen of pigs, and here the biofilm-forming ability of 32 strains of this species was determined. Significant biofilms were completely formed by 10 of the strains after 60 h of incubation, with exopolysaccharide production in the biofilm significantly higher than that in the corresponding planktonic cultures. S. suis strain SS2-4 formed a dense biofilm, as revealed by scanning electron microscopy, and in this state exhibited increased resistance to a number of antibiotics (ampicillin, amoxicillin, ciprofloxacin, kanamycin, and rifampin) compared to that of planktonic cultures. A bacteriophage lysin, designated LySMP, was used to attack biofilms alone and in combination with antibiotics and bacteriophage. The results demonstrated that the biofilms formed by S. suis, especially strains SS2-4 and SS2-H, could be dispersed by LySMP and with >80% removal compared to a biofilm reduction by treatment with either antibiotics or bacteriophage alone of less than 20%; in addition to disruption of the biofilm structure, the S. suis cells themselves were inactivated by LySMP. The efficacy of LySMP was not dose dependent, and in combination with antibiotics, it acted synergistically to maximize dispersal of the S. suis biofilm and inactivate the released cells. These data suggest that bacteriophage lysin could form part of an effective strategy to treat S. suis infections and represents a new class of antibiofilm agents. [ABSTRACT FROM AUTHOR]

Campylobacter jejuni continues to be the leading cause of bacterial food-borne illness worldwide, so improvements to current methods used for bacterial detection and disease prevention are needed. We describe here the genome and proteome of C. jejuni bacteriophage NCTC 12673 and the exploitation of its receptor-binding protein for specific bacterial detection. Remarkably, the 135-kb Myoviridae genome of NCTC 12673 differs greatly from any other proteobacterial phage genome described (including C. jejuni phages CP220 and CPt10) and instead shows closest homology to the cyanobacterial T4-related myophages. The phage genome contains 172 putative open reading frames, including 12 homing endonucleases, no visible means of packaging, and a putative trans-splicing intein. The phage DNA appears to be strongly associated with a protein that interfered with PCR amplification and estimation of the phage genome mass by pulsed-field gel electrophoresis. Identification and analyses of the receptor-binding protein (Gp48) revealed features common to the Salmonella enterica P22 phage tailspike protein, including the ability to specifically recognize a host organism. Bacteriophage receptor-binding proteins may offer promising alternatives for use in pathogen detection platforms. [ABSTRACT FROM AUTHOR]

The formation of stable foam in activated sludge plants is a global problem for which control is difficult. These foams are often stabilized by hydrophobic mycolic acid-synthesizing Actinobacteria, among which are Tsukamurella spp. This paper describes the isolation from activated sludge of the novel double-stranded DNA phage TPA2. This polyvalent Siphoviridae family phage is lytic for most Tsukamurella species. Whole-genome sequencing reveals that the TPA2 genome is circularly permuted (61,440 bp) and that 70% of its sequence is novel. We have identified 78 putative open reading frames, 95 pairs of inverted repeats, and 6 palindromes. The TPA2 genome has a modular gene structure that shares some similarity to those of Mycobacterium phages. A number of the genes display a mosaic architecture, suggesting that the TPA2 genome has evolved at least in part from genetic recombination events. The genome sequence reveals many novel genes that should inform any future discussion on Tsukamurella phage evolution. [ABSTRACT FROM AUTHOR]

The growth of Clostridium tyrobutyricum in developing cheese leads to spoilage and cheese blowing. Bacteriophages or their specific lytic enzymes may provide a biological control method for eliminating such undesirable organisms without affecting other microflora. We isolated the virulent bacteriophage ΦCTP1 belonging to the Siphoviridae and have shown that it is effective in causing lysis of sensitive strains. The double-stranded DNA genome of ΦCTP1 is 59,199 bp, and sequence analysis indicated that it has 86 open reading frames. orf29 was identified as the gene coding for the phage endolysin responsible for cell wall degradation prior to virion release. We cloned and expressed the ctp1l gene in E. coli and demonstrated that the partially purified protein induced lysis of C. tyrobutyricum cells and reduced viable counts both in buffer and in milk. The endolysin was inactive against a range of clostridial species but did show lysis of Clostridium sporogenes, another potential spoilage organism. Removal of the C-terminal portion of the endolysin completely abolished lytic activity. [ABSTRACT FROM AUTHOR]

Metagenomics can be used to determine the diversity of complex, often unculturable, viral communities with various nucleic acid compositions. Here, we report the use of hydroxyapatite chromatography to efficiently fractionate double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), dsRNA, and ssRNA genomes from known bacteriophages. Linker-amplified shotgun libraries were constructed to generate sequencing reads from each hydroxyapatite fraction. Greater than 90% of the reads displayed significant similarity to the expected genomes at the nucleotide level. These methods were applied to marine viruses collected from the Chesapeake Bay and the Dry Tortugas National Park. Isolated nucleic acids were fractionated using hydroxyapatite chromatography followed by linker-amplified shotgun library construction and sequencing. Taxonomic analysis demonstrated that the majority of environmental sequences, regardless of their source nucleic acid, were most similar to dsDNA viruses, reflecting the bias of viral metagenomic sequence databases. [ABSTRACT FROM AUTHOR]

The transfer range of phage genes was investigated at the single-cell level by using an in situ DNA amplification technique. After absorption of phages, a phage T4 gene was maintained in the genomes of non-plaque-forming bacteria at frequencies of 10-2 gene copies per cell. The gene transfer decreased the mutation frequencies in nonhost recipients. [ABSTRACT FROM AUTHOR]

Eleven Bacillus isolates from the surface and subsurface waters of the Gulf of Mexico were examined for their capacity to sporulate and harbor prophages. Occurrence of sporulation in each isolate was assessed through decoyinine induction, and putative lysogens were identified by prophage induction by mitomycin C treatment. No obvious correlation between ability to sporulate and prophage induction was found. Four strains that contained inducible virus-like particles (VLPs) were shown to sporulate. Four strains did not produce spores upon induction by decoyinine but contained inducible VLPs. Two of the strains did not produce virus-like particles or sporulate significantly upon induction. Isolate B14905 had a high level of virus-like particle production and a high occurrence of sporulation and was further examined by genomic sequencing in an attempt to shed light on the relationship between sporulation and lysogeny. In silico analysis of the B14905 genome revealed four prophage-like regions, one of which was independently sequenced from a mitomycin C-induced lysate. Based on PCR and transmission electron microscopy (TEM) analysis of an induced phage lysate, one is a noninducible phage remnant, one may be a defective phage-like bacteriocin, and two were inducible prophages. One of the inducible phages contained four putative transcriptional regulators, one of which was a SinR-like regulator that may be involved in the regulation of host sporulation. Isolates that both possess the capacity to sporulate and contain temperate phage may be well adapted for survival in the oligotrophic ocean. [ABSTRACT FROM AUTHOR]

Vibrio paralzaemolyticus is a common food-borne pathogen that is normally associated with seafood. In 1996, a pandemic O3:K6 strain abruptly appeared and caused the first pandemic of this pathogen to spread throughout many Asian countries, America, Europe, and Africa. The role of temperate bacteriophages in the evolution of this pathogen is of great interest. In this work, a new temperate phage, VP882, from a pandemic 03:K6 strain of V. parahaemolyticus was purified and characterized after mitomycin C induction. VP882 was a Myoviridae bacteriophage with a polyhedral head and a long rigid tail with a sheath-like structure. It infected and lysed high proportions of V. parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae strains. The genome of phage VP882 was sequenced and was 38,197 bp long, and 71 putative open reading frames were identified, of which 27 were putative functional phage or bacterial genes. YP882 had a linear plasmid-like genome with a putative protelomerase gene and cohesive ends. The genome does not integrate into the host chromosome but was maintained as a plasmid in the lysogen. Analysis of the reaction sites of the protelomerases in different plasmid-like phages revealed that VP882 and ΦHAP-1 were highly similar, while N15, FK02, and PY54 made up another closely related group. The presence of DNA adenine methylase and quorum-sensing transcriptional regulators in YP882 may play a specific role in this phage or regulate physiological or virulence-associated traits of the hosts. These genes may also be remnants from the bacterial chromosome following transduction. [ABSTRACT FROM AUTHOR]

Approximately 15% of overall Australian household water usage is in the laundry; hence, a significant reduction in household drinking water demand could be achieved if potable-quality water used for clothes washing is replaced with recycled water. To investigate the microbiological safety of using recycled water in washing machines, bacteriophages MS-2 and PRD-1, Escherichia coli, and Cryptosporidium parvum oocysts were used in a series of experiments to investigate the transfer efficiency of enteric microorganisms from washing machine water to objects including hands, environmental surfaces, air, and fabric swatches. By determining the transference efficiency, it is possible to estimate the numbers of microorganisms that the user will be exposed to if recycled water with various levels of residual microorganisms is used in washing machines. Results, expressed as transfer rates to a given surface area per object, showed that the mean transfer efficiency of E. coli, bacteriophages MS-2 and PRD-1, and C. parvum oocysts from seeded water to fabric swatches ranged from 0.001% to 0.090%. Greatest exposure to microorganisms occurred through direct contact of hands with seeded water and via hand contact with contaminated fabric swatches. No microorganisms were detected in the air samples during the washing machine spin cycle, and transfer rates of bacteriophages from water to environ- mental surfaces were 100-fold less than from water directly to hands. Findings from this study provide relevant information that can be used to refine regulations governing recycled water and to allay public concerns about the use of recycled water. [ABSTRACT FROM AUTHOR]

The genetic diversity of the natural freshwater community of cyanophages and its variations over time have been investigated for the first time in the surface waters of the largest natural lake in France. This was done by random screening of clone libraries for the g20 gene and by denaturing gradient gel electrophoresis (DGGE). Nucleotide sequence analysis revealed 35 distinct cyanomyovirus g20 genotypes among the 47 sequences analyzed. Phylogenetic analyses showed that these sequences fell into seven genetically distinct operational taxonomic units (OTUs). The distances between these OTUs were comparable to those reported between marine clusters. Moreover, some of these freshwater cyanophage sequences were genetically more closely related to marine cyanophage sequences than to other freshwater sequences. Both approaches for the g20 gene (sequencing and DGGE analysis) showed that there was a clear seasonal pattern of variation in the composition of the cyanophage community that could reflect changes in its biological, chemical, and/or physical environment. [ABSTRACT FROM AUTHOR]

Determines the DNA sequence of the replication module, part of the lysis module, and remnants of a lysogenic module from the lytic P335 species lactococcal bacteriophage. Investigation of its regulatory elements; Identification of an early expressed promoter; Putative helicase and primase.