Characterization of the Newly Isolated Lytic Bacteriophages KTN6 and KT28 and Their Efficacy against Pseudomonas aeruginosa Biofilm

We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants.     

Isolation and Characterization of Bacteriophages Infecting Staphylococcus epidermidis

Bacteriophages infecting Staphylococcus epidermidis were isolated by mitomycin C induction. Three distinct phages (vB_SepiS-phiIPLA5, vB_SepiS-phiIPLA6, and vB_SepiS-phiIPLA7)—defined by plaque morphology, structure, virion proteins pattern, DNA restriction bands, and host range—were obtained. One-step growth curves of bacteriophages under optimal growth conditions for S. epidermidis F12 revealed eclipse and latent periods of 5–10 and 10–15 min, respectively, with burst sizes of about 5 to 30 PFU per infected cell. Transmission electron microscopy revealed that the phages were of similar size and belonged to the Siphoviridae family. Phage phi-IPLA7 had the broadest host range infecting 21 out of 65 S. epidermidis isolates. Phage phi-IPLA5 seemed to be a virulent phage probably derived from phi-IPLA6. Phages phi-IPLA5 and phi-IPLA7 exhibited increasing plaques surrounded by a halo that could be indicative of a polysaccharide depolymerase activity. Viable counts, determined during the infection of S. epidermidis F12, confirmed that phi-IPLA5 had a potent lytic capability and reduced S. epidermidis population by 5.67 log units in 8 h of incubation; in the presence of the mixture of phi-IPLA6 and phi-IPLA7, however, a reduction of 2.27 log units was detected     

Characterization of Two Psychrophilic Pseudomonas Bacteriophages Isolated from Ground Beef

Characterization studies were performed on two psychrophilic phages which were isolated from ground beef samples. Phage inactivation by exposure to heat, low pH, osmotic shock conditions, and freezing showed that these two isolates were different. One-step growth experiments indicated that one isolate had a burst size five times as large (500) and a latent period two times as long (4 hr) as the other when tested at 7 C. Nucleic acid type was 2-deoxyribonucleic acid for both. Electron micrographs showed one to belong to Bradley's phage group A and the other to phage group C.     

Characterization of Newly Isolated Lytic Bacteriophages Active against Acinetobacter baumannii

Based on genotyping and host range, two newly isolated lytic bacteriophages, myovirus vB_AbaM_Acibel004 and podovirus vB_AbaP_Acibel007, active against Acinetobacter baumannii clinical strains, were selected from a new phage library for further characterization. The complete genomes of the two phages were analyzed. Both phages are characterized by broad host range and essential features of potential therapeutic phages, such as short latent period (27 and 21 min, respectively), high burst size (125 and 145, respectively), stability of activity in liquid culture and low frequency of occurrence of phage-resistant mutant bacterial cells. Genomic analysis showed that while Acibel004 represents a novel bacteriophage with resemblance to some unclassified Pseudomonas aeruginosa phages, Acibel007 belongs to the well-characterized genus of the Phikmvlikevirus. The newly isolated phages can serve as potential candidates for phage cocktails to control A. baumannii infections.     

Prevalence of Broad-Host-Range Lytic Bacteriophages of Sphaerotilus natans, Escherichia coli, and Pseudomonas aeruginosa

Two bacteriophage collections were examined with regard to their ability to form plaques on multiple bacterial host species. Nine of 10 phages studied were found to be broad-host-range bacteriophages. These phages fell into two groups. Group 1, the SN series, was isolated from sewage treatment plant samples with Sphaerotilus natans ATCC 13338 as a host. The DNAs of these bacteriophages contained modified bases and were insensitive to cleavage by type I and II restriction endonucleases. The efficiency of plating of these bacteriophages was changed only slightly on the alternate host. Group 2, the BHR series, was isolated by a two-host enrichment protocol. These bacteriophages were sensitive to restriction, and their efficiency of plating was dramatically reduced on the alternate host. Our results suggest that a multiple-host enrichment protocol may be more effective for the isolation of broad-host-range bacteriophages by avoiding the selection bias inherent in single-host methods. At least two of the broad-host-range bacteriophages mediated generalized transduction. We suggest that broad-host-range bacteriophages play a key role in phage ecology and gene transfer in nature.     

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

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

Isolation and Partial Characterization of Bacteriophages of the Phytopathogen Pseudomonas syringae

Bacteriophages isolated from culture supernatants of Pseudomonas syringae pv. syringae and from sewage were identified. The DNA from each phage was isolated and digested with the restriction endonuclease EcoRI. Eight isolates were determined to be different, with two phage isolates from sewage having restriction patterns identical to two phages from culture supernatants. The sizes of the phage DNA ranged from 24 to49 kilobases for isolates from sewage and from 39 to 52.5 kilobases for the isolates from culture supernatants. Buoyant densities of phage particles in CsCl varied from 1.498 to 1.507 g/cm³ for isolates from sewage and from 1.506 to 1.516 g/cm³ for isolates from culture supernatants. Electron microscopy revealed four morphological types. Based on plaque-forming ability of culture supernatants, 31 out of 47 strains of P. syringae are probably lysogenic.     

Isolation and Characterization of a Cyanophage Infecting the Toxic Cyanobacterium Microcystis aeruginosa

We isolated a cyanophage (Ma-LMM01) that specifically infects a toxic strain of the bloom-forming cyanobacterium Microcystis aeruginosa. Transmission electron microscopy showed that the virion is composed of anisometric head and a tail complex consisting of a central tube and a contractile sheath with helical symmetry. The morphological features and the host specificity suggest that Ma-LMM01 is a member of the cyanomyovirus group. Using semi-one-step growth experiments, the latent period and burst size were estimated to be 6 to 12 h and 50 to 120 infectious units per cell, respectively. The size of the phage genome was estimated to be ca. 160 kbp using pulse-field gel electrophoresis; the nucleic acid was sensitive to DNase I, Bal31, and all 14 restriction enzymes tested, suggesting that it is a linear double-stranded DNA having a low level of methylation. Phylogenetic analyses based on the deduced amino acid sequences of two open reading frames coding for ribonucleotide reductase alpha- and beta-subunits showed that Ma-LMM01 forms a sister group with marine and freshwater cyanobacteria and is apparently distinct from T4-like phages. Phylogenetic analysis of the deduced amino acid sequence of the putative sheath protein showed that Ma-LMM01 does not form a monophyletic group with either the T4-like phages or prophages, suggesting that Ma-LMM01 is distinct from other T4-like phages that have been described despite morphological similarity. The host-phage system which we studied is expected to contribute to our understanding of the ecology of Microcystis blooms and the genetics of cyanophages, and our results suggest the phages could be used to control toxic cyanobacterial blooms.     

Isolation and Characterization of Bacteriophages from Fermenting Sauerkraut

This paper presents the first report of bacteriophage isolated from commercial vegetable fermentations. Nine phages were isolated from two 90-ton commercial sauerkraut fermentations. These phages were active against fermentation isolates and selected Leuconostoc mesenteroides and Lactobacillus plantarum strains, including a starter culture. Phages were characterized as members of the Siphoviridae and Myoviridae families. All Leuconostoc phages reported previously, primarily of dairy origin, belonged to the Siphoviridae family.     

Isolation from Sewage Influent and Characterization of Novel Staphylococcus aureus Bacteriophages with Wide Host Ranges and Potent Lytic Capabilities

Staphylococcus aureus is a gram-positive pathogen that causes a variety of diseases, including bovine mastitis, which has severe economic consequences. Standard antibiotic treatment results in selection of resistant strains, leading to a need for alternative treatments, such as bacteriophage therapy. Forty-nine S. aureus isolates were obtained from the milk of mastitic cows for use in screening of staphylococcal phages. Fifteen isolates which were positive for both coagulase and hemolysin were assayed by PCR for variation in the X region and the immunoglobulin G-binding region of the protein A gene (spa) and in the carboxy terminus of the coagulase gene (coa) and for the presence of enterotoxin C, G, H, and I genes. The host ranges of 52 phages isolated from sewage influent were determined by performing spot tests with the 15 S. aureus isolates, and two phages were subsequently chosen for further analysis. ΦSA039 had the widest host range, producing clear plaques on 13 of the 15 isolates (87%), while ΦSA012 produced clear plaques on 8 isolates (53%) and was the only phage that produced a clear plaque on a nonmastitic S. aureus strain. Transmission electron microscopy revealed that the phages were similar sizes and belonged to the Myoviridae family. Measurement of optical densities during coculture with S. aureus isolates confirmed the breadth of the ΦSA039 host range and showed that ΦSA012 had potent lytic capability. ΦSA012-resistant bacteria did not appear for three of seven isolates tested (43%) after 65 h of incubation. These two phages are proposed as candidates for phage therapy of bovine mastitis.In the dairy industry, mastitis is a widespread problem responsible for important decreases in milk production. Economic losses of $100 million per year have been estimated for farms in Hokkaido, one of the largest milk-producing areas in Japan (28). Mastitis can be caused by over 150 different microorganisms, and one of the most important of these organisms is Staphylococcus aureus (22). After diagnosis of mastitis, the standard treatment regimen consists of isolating the diseased cow and treating it with antibiotics. However, this approach has drawbacks, such as its high cost and the eradication of harmless or beneficial organisms due to the lack of specificity of antibiotics. Additionally, the incidence of antibiotic-resistant bacteria has increased in recent years (4). As a result, there has been renewed interest in the use of other natural or engineered antimicrobial agents as an alternative or supplementary treatment for staphylococcal diseases such as mastitis (11, 21, 26). One group of alternatives with great potential involves bacteriophages (phages) and their derivatives, and a number of promising candidates have been described (2, 5, 7, 13, 17, 18, 27), notably bacteriophage K.One of the main obstacles to successful treatment of mastitis using phages is the fact that most phages are able to infect only a very narrow range of hosts. Given the plural etiology of many mastitis cases, it is desirable to find a phage-based treatment that targets a wide range of pathogens. One proposed method is a combination of two or more phages with different and broad host ranges in a cocktail (25, 29). This method has been shown to delay considerably the appearance of phage-resistant cells in Escherichia coli cultures, providing hope that similar success can be achieved using S. aureus phages.In this paper we describe the isolation of two novel phages, designated ΦSA012 and ΦSA039, that were found to have a lytic effect on a broad range of S. aureus isolates obtained from mastitic milk. ΦSA012 and ΦSA039 were selected after host range analysis from a total of 52 phages screened from sewage influent. These two phages were characterized morphologically by using electron microscopy, and their effects on representative strains of S. aureus were examined by coculture in milk.     

Construction and Characterization of T7 Bacteriophages Harboring Apidaecin-Derived Sequences

The global spread of multi- and pan-resistant bacteria has triggered research to identify novel strategies to fight these pathogens, such as antimicrobial peptides and, more recently, bacteriophages. In a proof-of-concept study, we have genetically modified lytic T7Select phages targeting Escherichia coli Rosetta by integrating DNA sequences derived from the proline-rich antimicrobial peptide, apidaecin. This allowed testing of our hypothesis that apidaecins and bacteriophages can synergistically act on phage-sensitive and phage-resistant E. coli cells and overcome the excessive cost of peptide drugs by using infected cells to express apidaecins before cell lysis. Indeed, the addition of the highly active synthetic apidaecin analogs, Api802 and Api806, to T7Select phage-infected E. coli Rosetta cultures prevented or delayed the growth of potentially phage-resistant E. coli Rosetta strains. However, high concentrations of Api802 also reduced the T7Select phage fitness. Additionally, plasmids encoding Api802, Api806, and Api810 sequences transformed into E. coli Rosetta allowed the production of satisfactory peptide quantities. When these sequences were integrated into the T7Select phage genome carrying an N-terminal green fluorescent protein (GFP-) tag to monitor the expression in infected E. coli Rosetta cells, the GFP–apidaecin analogs were produced in reasonable quantities. However, when Api802, Api806 and Api810 sequences were integrated into the T7Select phage genome, expression was below detection limits and an effect on the growth of potentially phage-resistant E. coli Rosetta strains was not observed for Api802 and Api806. In conclusion, we were able to show that apidaecins can be integrated into the T7Select phage genome to induce their expression in host cells, but further research is required to optimize the engineered T7Select phages for higher expression levels of apidaecins to achieve the expected synergistic effects that were visible when the T7Select phages and synthetic Api802 and Api806 were added to E. coli Rosetta cultures.     

Characterization of Salmonella Bacteriophages Isolated from Swine Lagoon Effluent

Four Salmonella bacteriophages that had been originally isolated from swine manure lagoons were characterized and compared to each other and to well-known Salmonella phages P22 and Felix 01. Host ranges of the lagoon phages were similar to each other in spot tests on reference strains of Salmonella, but differed slightly from each other on a panel of Salmonella lagoon strains. In single-step growth at 35°C the lagoon phages had latent periods of 15 to 20 min and burst sizes from 100 to 230. The lagoon phages and P22 were purified by cesium chloride (CsCl) gradient centrifugation and used to produce specific antisera and DNA. The lagoon phages were indistinguishable from each other but distinct from P22 and Felix 01 in immunodiffusion and infectivity neutralization tests and in restriction digest analysis. Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee, warranty, or endorsement by the USDA and does not imply its approval to the exclusion of other products that may be suitable.     

Phenotypic and Molecular Characterization of Multidrug Resistant Klebsiella pneumoniae Isolated from a University Teaching Hospital,China

The multidrug-resistant rate of Klebsiella pneumoniae has risen rapidly worldwide. To better understand the multidrug resistance situation and molecular characterization of Klebsiella pneumoniae, a total of 153 Klebsiella pneumoniae isolates were collected, and drug susceptibility test was performed to detect its susceptibility patterns to 13 kinds of antibiotics. Phenotypic tests for carbapenemases ESBLs and AmpC enzyme-producing strains were performed to detect the resistance phenotype of the isolates. Then PCR amplification and sequencing analysis were performed for the drug resistance determinants. The results showed that 63 strains harbored bla _CTX-M gene, and 14 strains harbored bla _DHA gene. Moreover, there were 5 strains carrying bla _KPC gene, among which 4 strains carried bla _CTX-M, bla _DHA and bla _KPC genes, and these 4 strains were also resistant to imipenem. Our data indicated that drug-resistant Klebsiella pneumoniae were highly prevalent in the hospital. Thus it is warranted that surveillance of epidemiology of those resistant isolates should be a cause for concern, and appropriate drugs should be chosen.     

BVPaP-3, a T7-Like Lytic Phage of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>: Its Isolation and Characterisation

The increasing emergence of antibiotic-resistant bacteria has produced a growing interest among scientists in bacteriophages as alternative antimicrobial agents. This article reports a lytic phage against an antibiotic-resistant strain of Pseudomonas aeruginosa. Phage BVPaP-3 is a member of the Podoviridae family and morphologically similar to the T7-like phage gh-1. The phage has a hexagonal head of 58–59 nm in diameter and a short tail of 10 × 8 nm. It is stable at a wide range of pH (6–10) and temperatures (4–40°C). Its optimal growth temperature is 37°C and the adsorption rate constant is 1.19 × 10⁻⁹. Latent and eclipse periods are 20 and 15 min, respectively, and the burst size is 44 after 35 min at 37°C. The phage has a DNA size of 41.31 kb and a proteome of 11 proteins. The major protein is 33 kDa in size.     

Isolation and Characterization of Campylobacter Bacteriophages from Retail Poultry

The ability of phages to survive processing is an important aspect of their potential use in the biocontrol of Campylobacter in poultry production. To this end, we have developed a procedure to recover Campylobacter bacteriophages from chilled and frozen retail poultry and have validated the sensitivity of the method by using a characterized Campylobacter phage (i.e., NCTC 12674). By using this method, we have shown that Campylobacter phages can survive on retail chicken under commercial storage conditions. Retail chicken portions purchased in the United Kingdom were screened for the presence of endogenous Campylobacter phages. Thirty-four Campylobacter bacteriophages were isolated from 300 chilled retail chicken portions, but none could be recovered from 150 frozen chicken portions. The phage isolates were characterized according to their lytic profiles, morphology, and genome size. The free-range products were significantly more likely to harbor phages (P < 0.001 by single-factor analysis of variance) than were standard or economy products. This study demonstrates that Campylobacter bacteriophages, along with their hosts, can survive commercial poultry processing procedures and that the phages exhibited a wide range of recovery rates from chicken skin stored at 4°C.     

Isolation and Characterisation of Lytic Bacteriophages of Klebsiella pneumoniae and Klebsiella oxytoca

Klebsiella bacteria have emerged as an increasingly important cause of community-acquired nosocomial infections. Extensive use of broad-spectrum antibiotics in hospitalised patients has led to both increased carriage of Klebsiella and the development of multidrug-resistant strains that frequently produce extended-spectrum β-lactamases and/or other defences against antibiotics. Many of these strains are highly virulent and exhibit a strong propensity to spread. In this study, six lytic Klebsiella bacteriophages were isolated from sewage-contaminated river water in Georgia and characterised as phage therapy candidates. Two of the phages were investigated in greater detail. Biological properties, including phage morphology, nucleic acid composition, host range, growth phenotype, and thermal and pH stability were studied for all six phages. Limited sample sequencing was performed to define the phylogeny of the K. pneumoniae- and K. oxytoca-specific bacteriophages vB_Klp_5 and vB_Klox_2, respectively. Both of the latter phages had large burst sizes, efficient rates of adsorption and were stable under different adverse conditions. Phages reported in this study are double-stranded DNA bacterial viruses belonging to the families Podoviridae and Siphoviridae. One or more of the six phages was capable of efficiently lysing ~63 % of Klebsiella strains comprising a collection of 123 clinical isolates from Georgia and the United Kingdom. These phages exhibit a number of properties indicative of potential utility in phage therapy cocktails.