Escherichia coli O157:H7 typing phage V7 is a T4-like virus.

The complete genome sequence of the Escherichia coli O157:H7 typing phage V7 was determined. Its double-stranded DNA genome is 166,452 bp long, encoding 273 proteins and including 11 tRNAs. This virus belongs to the genus T4-like viruses within the subfamily Tevenvirinae, family Myoviridae.

Survey of Pseudomonas aeruginosa and its phages: de novo peptide sequencing as a novel tool to assess the diversity of worldwide collected viruses.

A collection of 15 newly isolated (bacterio)phages infecting the opportunistic pathogen Pseudomonas aeruginosa was established to investigate their global diversity and potential in phage therapy. These phages were sampled in 14 different countries traversing four continents, from both natural environments and hospital sewage. They all display unique DNA and protein profiles and cluster morphologically into six groups within the three major families of the Caudovirales. Extensive host range studies on a library of 122 AFLP-genotyped clinical P. aeruginosa strains (of which 49 were newly isolated at the University Hospital of Leuven, Belgium) showed that the phages lysed 87% of the strains. Infection analysis of outer membrane mutants identified 10 phages as type IV pili-dependent. More detailed information about the evolutionary relatedness of the phages was gathered by de novo peptide sequencing of major virion proteins using tandem Matrix-Assisted Laser Desorption/Ionization Time of Flight technology. Applying this technique for the first time to viruses, seven groups of closely related phages were identified without the need of prior knowledge of genome content and/or electron microscopic imaging. This study demonstrates both the epidemic population structure of P. aeruginosa and the global spread of P. aeruginosa phage species, and points at the resistance of two clinically predominant, widespread P. aeruginosa strains against phage attack.

Quantitating Phage Efficacy in Ready-To-Eat Meats.

Bacteriophages are being applied in biocontrol of bacterial pathogens in foods and food processing environments. There is need for the development of standardized protocols to quantify the effectiveness of phage preparations in reducing food-borne pathogens on foods. Here, we present a procedure for the verification of the effectiveness of a phage preparation in reducing Listeria monocytogenes on ready-to-eat (RTE) meats. The protocol is designed taking into account real-world scenarios and avoiding common errors reported in previous phage decontamination assays.

Application of a Virucidal Agent to Avoid Overestimation of Phage Kill During Phage Decontamination Assays on Ready-to-Eat Meats.

We describe a method for assessing the effectiveness of tea extract based virucide (TeaF) application to remove phage LISTEX™ P100 not bound to Listeria monocytogenes from stomached rinses prior to direct plating and bacterial enumeration, where the phage is being used as a decontaminant to reduce L. monocytogenes levels on ready-to-eat meat.

Effect of salt types and concentrations on the high-pressure inactivation of Listeria monocytogenes in ground chicken.

National and international health agencies have recommended a significant reduction in daily intake of sodium by reducing the amount of NaCl in foods, specifically processed meats. However, sodium reduction could increase the risk of survival and growth of spoilage and pathogenic microorganisms on these products. Therefore, alternate processing technologies to improve safety of sodium reduced foods are necessary. This study examined the effects of three different salt types and concentrations on high-pressure inactivation of Listeria monocytogenes in pre-blended ground chicken formulations. Ground chicken formulated with three salt types (NaCl, KCl, CaCl2), at three concentrations (0, 1.5, 2.5%) and inoculated with a four strain cocktail of L. monocytogenes (10(8) CFU g(-1)) were subjected to four pressure treatments (0, 100, 300, 600 MPa) and two durations (60, 180 s) in an experiment with factorial design. Surviving cells were enumerated by plating on Oxford agar and analysed by factorial ANOVA. Pressure treatments at 100 or 300 MPa did not significantly (P=0.19-050) reduce L. monocytogenes populations. Neither salt type nor concentration had a significant effect on L. monocytogenes populations at these pressure levels. At 600 MPa, salt types, concentrations and duration of pressure treatment all had a significant effect on L. monocytogenes populations. Formulations with increasing concentrations of NaCl or KCl showed significantly lower reduction in L. monocytogenes, while increase in CaCl2 concentration resulted in a significantly higher L. monocytogenes reduction. For instance, increase in NaCl concentration from 0 to 1.5 or 2.5% resulted in a log reduction of 6.16, 2.49 and 1.29, respectively, when exposed to 600 MPa for 60s. In the case of CaCl2, increase from 0 to 1.5 or 2.5% resulted in a log reduction of 6.16, 7.28 and 7.47, respectively. These results demonstrate that high-pressure processing is a viable process to improve microbial safety of sodium reduced poultry products.

Efficacy of bacteriophage LISTEX™P100 combined with chemical antimicrobials in reducing Listeria monocytogenes in cooked turkey and roast beef.

The aim of this study was to verify the effectiveness of the commercially available anti-Listeria phage preparation LISTEX(™)P100 in reducing Listeria monocytogenes on ready-to-eat (RTE) roast beef and cooked turkey in the presence or absence of the chemical antimicrobials potassium lactate (PL) and sodium diacetate (SD). Sliced RTE meat cores at 4 and 10 °C were inoculated with cold-adapted L. monocytogenes to result in a surface contamination level of 10(3)CFU/cm(2). LISTEX(TM)P100 was applied at 10(7) PFU/cm(2) and samples taken at regular time intervals during the RTE product's shelf life to enumerate viable L. monocytogenes. LISTEX(™)P100 was effective during incubation at 4 °C with initial reductions of L. monocytogenes of 2.1 log10 CFU/cm(2) and 1.7 log10 CFU/cm(2), respectively, for cooked turkey and roast beef without chemical antimicrobials (there was no significant difference to the initial L. monocytogenes reductions in the presence of LISTEX(TM)P100 for cooked turkey containing PL and roast beef containing SD-PL). In the samples containing no chemical antimicrobials, the presence of phage resulted in lower L. monocytogenes numbers, relative to the untreated control, of about 2 log CFU/cm(2) over a 28-day storage period at 4 °C. An initial L. monocytogenes cell reduction of 1.5 log10 CFU/cm(2) and 1.7 log10 CFU/cm(2), respectively, for cooked turkey and roast beef containing no chemical antimicrobials was achieved by the phage at 10 °C (abusive temperature). At this temperature, the L. monocytogenes cell numbers of samples treated with LISTEX™ P100 remained below those of the untreated control only during the first 14 days of the experiment for roast beef samples with and without antimicrobials. On day 28, the L. monocytogenes numbers on samples containing chemical antimicrobials and treated with LISTEX(TM)P100 stored at 4 and 10 °C were 4.5 log10 CFU/cm(2) and 7.5 log10 CFU/cm(2), respectively, for cooked turkey, and 1.2 log10 CFU/cm(2) and 7.2 log10 CFU/cm(2), respectively, for roast beef. In both cooked turkey samples with and without chemical antimicrobials stored at 10 °C, the phage-treated samples had significantly lower numbers of L. monocytogenes when compared to the untreated controls throughout the 28-day storage period (P<0.0001). For roast beef and cooked turkey containing chemical antimicrobials treated with LISTEX(TM)P100 and stored at 4 °C, no more than a 2 log CFU/cm(2) increase of L. monocytogenes was observed throughout the stated shelf life of the product. This study shows that LISTEX(™)P100 causes an initial reduction of L. monocytogenes numbers and can serve as an additional hurdle to enhance the safety of RTE meats when used in combination with chemical antimicrobials.

Bacteriophages with the ability to degrade uropathogenic Escherichia coli biofilms.

Escherichia coli-associated urinary tract infections (UTIs) are among the most common bacterial infections in humans. UTIs are usually managed with antibiotic therapy, but over the years, antibiotic-resistant strains of uropathogenic E. coli (UPEC) have emerged. The formation of biofilms further complicates the treatment of these infections by making them resistant to killing by the host immune system as well as by antibiotics. This has encouraged research into therapy using bacteriophages (phages) as a supplement or substitute for antibiotics. In this study we characterized 253 UPEC in terms of their biofilm-forming capabilities, serotype, and antimicrobial resistance. Three phages were then isolated (vB_EcoP_ACG-C91, vB_EcoM_ACG-C40 and vB_EcoS_ACG-M12) which were able to lyse 80.5% of a subset (42) of the UPEC strains able to form biofilms. Correlation was established between phage sensitivity and specific serotypes of the UPEC strains. The phages' genome sequences were determined and resulted in classification of vB_EcoP_ACG-C91 as a SP6likevirus, vB_EcoM_ACG-C40 as a T4likevirus and vB_EcoS_ACG-M12 as T1likevirus. We assessed the ability of the three phages to eradicate the established biofilm of one of the UPEC strains used in the study. All phages significantly reduced the biofilm within 2-12 h of incubation.

A procedure for systematic identification of bacteriophage-host interactions of P. aeruginosa phages.

Immediately after bacteriophage infection, phage early proteins establish optimal conditions for phage infection, often through a direct interaction with host-cell proteins. We implemented a yeast two-hybrid approach for Pseudomonas aeruginosa phages as a first step in the analysis of these - often uncharacterized - proteins. A 24-fold redundant prey library of P. aeruginosa PAO1 (7.32x10(6) independent clones), was screened against early proteins (gp1 to 9) of phiKMV, a P. aeruginosa-infecting member of the Podoviridae; interactions were verified using an independent in vitro assay. None resembles previously known bacteriophage-host interactions, as the three identified target malate synthase G, a regulator of a secretion system and a regulator of nitrogen assimilation. Although at least two-bacteriophage infections are non-essential to phiKMV infection, their disruption has an influence on infection efficiency. This methodology allows systematic analysis of phage proteins and is applicable as an interaction analysis tool for P. aeruginosa.

The adsorption of Pseudomonas aeruginosa bacteriophage phiKMV is dependent on expression regulation of type IV pili genes.

Pseudomonas aeruginosa bacteriophage phiKMV requires type IV pili for infection, as observed from the phenotypic characterization and phage adsorption assays on a phage infection-resistant host strain mutant. A cosmid clone library of the host (P. aeruginosa PAO1) genomic DNA was generated and used to select for a clone that was able to restore phiKMV infection in the resistant mutant. This complementing cosmid also re-established type IV pili-dependent twitching motility. The correlation between bacteriophage phiKMV infectivity and type IV pili, along with its associated twitching motility, was confirmed by the resistance of a P. aeruginosa PAO1DeltapilA mutant to the phage. Subcloning of the complementing cosmid and further phage infection analysis and motility assays suggests that a common regulatory mechanism and/or interaction between the ponA and pilMNOPQ gene products are essential for bacteriophage phiKMV infectivity.

Genomic analysis of Pseudomonas aeruginosa phages LKD16 and LKA1: establishment of the phiKMV subgroup within the T7 supergroup.

Lytic Pseudomonas aeruginosa phages LKD16 and LKA1 were locally isolated and morphologically classified as Podoviridae. While LKD16 adsorbs weakly to its host, LKA1 shows efficient adsorption (ka = 3.9 x 10(-9) ml min(-1)). LKA1, however, displays a narrow host range on clinical P. aeruginosa strains compared to LKD16. Genome analysis of LKD16 (43,200 bp) and LKA1 (41,593 bp) revealed that both phages have linear double-stranded DNA genomes with direct terminal repeats of 428 and 298 bp and encode 54 and 56 genes, respectively. The majority of the predicted structural proteins were experimentally confirmed as part of the phage particle using mass spectrometry. Phage LKD16 is closely related to bacteriophage phiKMV (83% overall DNA homology), allowing a more thoughtful gene annotation of both genomes. In contrast, LKA1 is more distantly related, lacking significant DNA homology and showing protein similarity to phiKMV in 48% of its gene products. The early region of the LKA1 genome has diverged strongly from phiKMV and LKD16, and intriguing differences in tail fiber genes of LKD16 and LKA1 likely reflect the observed discrepancy in infection-related properties. Nonetheless, general genome organization is clearly conserved among phiKMV, LKD16, and LKA1. The three phages carry a single-subunit RNA polymerase gene adjacent to the structural genome region, a feature which distinguishes them from other members of the T7 supergroup. Therefore, we propose that phiKMV represents an independent and widespread group of lytic P. aeruginosa phages within the T7 supergroup.

Genetic and physical map of broad host range cosmid pRG930cm

We hereby present the complete sequence and annotation of pRG930cm, a spectinomycin/streptomycin/chloramphenicol-resistant cosmid vector. pRG930cm (17,256 bp; GenBank Accession No.: FM174471) has a broad host range, and is stably maintained by a number of Gram-negative bacteria including Pseudomonas spp, Escherichia coli, Agrobacterium tumefaciens and Azorhizobium caulinodans ORS571. pRG930cm is already widely used and its sequence will aid efficient construction and analysis of cosmid libraries.