Characterization of Two Virulent Phages of Lactobacillus plantarum

We characterized two Lactobacillus plantarum virulent siphophages, ATCC 8014-B1 (B1) and ATCC 8014-B2 (B2), previously isolated from corn silage and anaerobic sewage sludge, respectively. Phage B2 infected two of the eight L. plantarum strains tested, while phage B1 infected three. Phage adsorption was highly variable depending on the strain used. Phage defense systems were found in at least two L. plantarum strains, LMG9211 and WCSF1. The linear double-stranded DNA genome of the pac-type phage B1 had 38,002 bp, a G+C content of 47.6%, and 60 open reading frames (ORFs). Surprisingly, the phage B1 genome has 97% identity with that of Pediococcus damnosus phage clP1 and 77% identity with that of L. plantarum phage JL-1; these phages were isolated from sewage and cucumber fermentation, respectively. The double-stranded DNA (dsDNA) genome of the cos-type phage B2 had 80,618 bp, a G+C content of 36.9%, and 127 ORFs with similarities to those of Bacillus and Lactobacillus strains as well as phages. Some phage B2 genes were similar to ORFs from L. plantarum phage LP65 of the Myoviridae family. Additionally, 6 tRNAs were found in the phage B2 genome. Protein analysis revealed 13 (phage B1) and 9 (phage B2) structural proteins. To our knowledge, this is the first report describing such high identity between phage genomes infecting different genera of lactic acid bacteria.     

Characterization of Helicobacter pylori Bacteriophage KHP30

Helicobacter pylori inhabits the stomach mucosa and is a causative agent of stomach ulcer and cancer. In general, bacteriophages (phages) are strongly associated with bacterial evolution, including the development of pathogenicity. Several tailed phages have so far been reported in H. pylori. We have isolated an H. pylori phage, KHP30, and reported its genomic sequence. In this study, we examined the biological characteristics of phage KHP30. Phage KHP30 was found to be a spherical lipid-containing phage with a diameter of ca. 69 nm. Interestingly, it was stable from pH 2.5 to pH 10, suggesting that it is adapted to the highly acidic environment of the human stomach. Phage KHP30 multiplied on 63.6% of clinical H. pylori isolates. The latent period was ca. 140 min, shorter than the doubling time of H. pylori (ca. 180 min). The burst size was ca. 13, which was smaller than the burst sizes of other known tailed or spherical phages. Phage KHP30 seemed to be maintained as an episome in H. pylori strain NY43 cells, despite a predicted integrase gene in the KHP30 genomic sequence. Seven possible virion proteins of phage KHP30 were analyzed using N-terminal protein sequencing and mass spectrometry, and their genes were found to be located on its genomic DNA. The genomic organization of phage KHP30 differed from the genomic organizations in the known spherical phage families Corticoviridae and Tectiviridae. This evidence suggests that phage KHP30 is a new type of spherical phage that cannot be classified in any existing virus category.     

Bacteriophage-resistance mechanisms examined by transfection of Lactococcus lactis subsp. lactis 4513-5 mediated by high voltage electroporation

Summary Phage adsorption tests and transfection by electroporation were carried out to decide whether phage-resistance in Lactococcus lactis subsp. lactis strain 4513-5 is based on intracellular or extracellular mechanisms. Using high voltage (12.5 kV/cm) electroporation, untreated phage DNA was introduced into phage-sensitive and phage-resistant cells. Since phages showed low adsorption frequencies on resistant bacteria, resistance is localized in the cell wall preventing phage DNA from entering the cell. This is the only mechanism responsible for the resistance of L. lactis subsp. lactis 4513-5 against its homologous phage P4513-K12 and non-homologous phages P05M-13 and P05M-47, but not against phage P530-7 and phage P530-12. In the case of the latter two phage strains, intracellular resistance mechanisms are involved and discussed.     

Isolation and Partial Characterization of Two Aeromonas hydrophila Bacteriophages

Two Aeromonas hydrophila bacteriophages, Aeh1 and Aeh2, were isolated from sewage. Both phages showed binal symmetry. The dimensions of A. hydrophila phages Aeh1 and Aeh2 differed from those of the other Aeromonas phages. Also, phage Aeh2 was the largest Aeromonas phage studied to date. Phage Aeh1 formed small, clear plaques, and phage Aeh2 formed turbid plaques with clear centers. Both phages were sensitive to chloroform treatment, being totally inactivated after treatment for 1 h at 60°C at pH 3 and 11. However, the infectivity of Aeh1 phage stocks increased by approximately fivefold after they were treated at pH 10 for 1 h at 22°C. Phages Aeh1 and Aeh2 were serologically unrelated and had latent periods of 39 and 52 min, respectively. The average burst sizes of phages Aeh1 and Aeh2 were 17 and 92 PFU per cell, respectively. Phage Aeh1 infected 13 of 22 A. hydrophila strains tested, whereas phage Aeh2 infected only its original host. Phage Aeh1 infected some A. hydrophila strains only at or below 37°C. Neither phage infected the two A. (Plesiomonas) shigelloides strains used in this study.     

Epifluorescence and atomic force microscopy: Two innovative applications for studying phage-host interactions in Lactobacillus helveticus

Bacteriophages attacking lactic acid bacteria (LAB) still represent a crucial problem in industrial dairy fermentations. The consequences of a phage infection against LAB can lead to fermentation delay, alteration of the product quality and, in most severe cases, the product loss. Phage particles enumeration and phage-host interactions are normally evaluated by conventional plaque count assays, but, in many cases, these methods can be unsuccessful. Bacteriophages of Lactobacillus helveticus, a LAB species widely used as dairy starter or probiotic cultures, are often unable to form lysis plaques, thus impairing their enumeration by plate assay. In this study, we used epifluorescence microscopy to enumerate L. helveticus phage particles from phage-infected cultures and Atomic Force Microscopy (AFM) to visualize both phages and bacteria during the different stages of the lytic cycle. Preliminary, we tested the sensitivity of phage counting by epifluorescence microscopy. To this end, phage particles of ΦAQ113, a lytic phage of L. helveticus isolated from a whey starter culture, were stained by SYBR Green I and enumerated by epifluorescence microscopy. Values obtained by the microscopic method were 10 times higher than plate counts, with a lowest sensitivity limit of ≥ 6 log phage/ml. The interaction of phage ΦAQ113 with its host cell L. helveticus Lh1405 was imaged by AFM after 0, 2 and 5 h from phage-host adsorption. The lytic cycle was followed by epifluorescence microscopy counting and the concomitant cell wall changes were visualized by AFM imaging. Our results showed that these two methods can be combined for a reliable phage enumeration and for studying phage and host morphology during infection processes, thus giving a complete overview of phage-host interactions in L. helveticus strains involved in dairy productions.     

The Molecular and Genetic Basis of Repeatable Coevolution between Escherichia coli and Bacteriophage T3 in a Laboratory Microcosm

The objective of this study was to determine the genomic changes that underlie coevolution between Escherichia coli B and bacteriophage T3 when grown together in a laboratory microcosm. We also sought to evaluate the repeatability of their evolution by studying replicate coevolution experiments inoculated with the same ancestral strains. We performed the coevolution experiments by growing Escherichia coli B and the lytic bacteriophage T3 in seven parallel continuous culture devices (chemostats) for 30 days. In each of the chemostats, we observed three rounds of coevolution. First, bacteria evolved resistance to infection by the ancestral phage. Then, a new phage type evolved that was capable of infecting the resistant bacteria as well as the sensitive bacterial ancestor. Finally, we observed second-order resistant bacteria evolve that were resistant to infection by both phage types. To identify the genetic changes underlying coevolution, we isolated first- and second-order resistant bacteria as well as a host-range mutant phage from each chemostat and sequenced their genomes. We found that first-order resistant bacteria consistently evolved resistance to phage via mutations in the gene, waaG, which codes for a glucosyltransferase required for assembly of the bacterial lipopolysaccharide (LPS). Phage also showed repeatable evolution, with each chemostat producing host-range mutant phage with mutations in the phage tail fiber gene T3p48 which binds to the bacterial LPS during adsorption. Two second-order resistant bacteria evolved via mutations in different genes involved in the phage interaction. Although a wide range of mutations occurred in the bacterial waaG gene, mutations in the phage tail fiber were restricted to a single codon, and several phage showed convergent evolution at the nucleotide level. These results are consistent with previous studies in other systems that have documented repeatable evolution in bacteria at the level of pathways or genes and repeatable evolution in viruses at the nucleotide level. Our data are also consistent with the expectation that adaptation via loss-of-function mutations is less constrained than adaptation via gain-of-function mutations.     

Cloning of Genomic DNA of Lactococcus lactis That Restores Phage Sensitivity to an Unusual Bacteriophage sk1-Resistant Mutant

An unusual, spontaneous, phage sk1-resistant mutant (RMSK1/1) of Lactococcus lactis C2 apparently blocks phage DNA entry into the host. Although no visible plaques formed on RMSK1/1, this host propagated phage at a reduced efficiency. This was evident from center-of-infection experiments, which showed that 21% of infected RMSK1/1 formed plaques when plated on its phage-sensitive parental strain, C2. Moreover, viable cell counts 0 and 4 h after infection were not significantly different from those of an uninfected culture. Further characterization showed that phage adsorption was normal, but burst size was reduced fivefold and the latent period was increased from 28.5 to 36 min. RMSK1/1 was resistant to other, but not all, similar phages. Phage sensitivity was restored to RMSK1/1 by transformation with a cloned DNA fragment from a genomic library of a phage-sensitive strain. Characterization of the DNA that restored phage sensitivity revealed an open reading frame with similarity to sequences encoding lysozymes (β-1,4-N-acetylmuramidase) and lysins from various bacteria, a fungus, and phages of Lactobacillus and Streptococcus and also revealed DNA homologous to noncoding sequences of temperate phage of L. lactis, DNA similar to a region of phage sk1, a gene with similarity to tRNA genes, a prophage attachment site, and open reading frames with similarities to sun and to sequences encoding phosphoprotein phosphatases and protein kinases. Mutational analyses of the cloned DNA showed that the region of homology with lactococcal temperate phage was responsible for restoring the phage-sensitive phenotype. The region of homology with DNA of lactococcal temperate phage was similar to DNA from a previously characterized lactococcal phage that suppresses an abortive infection mechanism of phage resistance. The region of homology with lactococcal temperate phage was deleted from a phage-sensitive strain, but the strain was not phage resistant. The results suggest that the cloned DNA with homology to lactococcal temperate phage was not mutated in the phage-resistant strain. The cloned DNA apparently suppressed the mechanism of resistance, and it may do so by mimicking a region of phage DNA that interacts with components of the resistance mechanism.     

Transposon-Induced Mutations in Two Loci of Listeria monocytogenes Serotype 1/2a Result in Phage Resistance and Lack of N-Acetylglucosamine in the Teichoic Acid of the Cell Wall

Teichoic acid-associated N-acetylglucosamine and rhamnose have been shown to serve as phage receptors in Listeria monocytogenes serotype 1/2a. We generated and characterized two single-copy Tn916ΔE mutants which were resistant to phage A118 and several other serotype 1/2a-specific phages. In one mutant the insertion was immediately upstream of the recently identified ptsHI locus, which encodes two proteins of the phosphoenolpyruvate-dependent carbohydrate uptake system, whereas in the other the insertion was immediately upstream of an operon whose most distal gene was clpC, involved in stress responses and virulence. Transduction experiments confirmed the association of the phage-resistant phenotype of these mutants with the transposon insertion. Phage A118 resistance of the mutants could be attributed to inability of the phage to adsorb onto the mutant cells, and biochemical analysis of cell wall composition showed that the teichoic acids of both mutants were deficient in N-acetylglucosamine. Rhamnose and other teichoic acid and cell wall components were not affected.     

A Virulent Phage Infecting Lactococcus garvieae,with Homology to Lactococcus lactis Phages

A new virulent phage belonging to the Siphoviridae family and able to infect Lactococcus garvieae strains was isolated from compost soil. Phage GE1 has a prolate capsid (56 by 38 nm) and a long noncontractile tail (123 nm). It had a burst size of 139 and a latent period of 31 min. Its host range was limited to only two L. garvieae strains out of 73 tested. Phage GE1 has a double-stranded DNA genome of 24,847 bp containing 48 predicted open reading frames (ORFs). Putative functions could be assigned to only 14 ORFs, and significant matches in public databases were found for only 17 ORFs, indicating that GE1 is a novel phage and its genome contains several new viral genes and encodes several new viral proteins. Of these 17 ORFs, 16 were homologous to deduced proteins of virulent phages infecting the dairy bacterium Lactococcus lactis, including previously characterized prolate-headed phages. Comparative genome analysis confirmed the relatedness of L. garvieae phage GE1 to L. lactis phages c2 (22,172 bp) and Q54 (26,537 bp), although its genome organization was closer to that of phage c2. Phage GE1 did not infect any of the 58 L. lactis strains tested. This study suggests that phages infecting different lactococcal species may have a common ancestor.     

Lactococcal Bacteriophages Require a Host Cell Wall Carbohydrate and a Plasma Membrane Protein for Adsorption and Ejection of DNA

The mechanism of the initial steps of bacteriophage infection in Lactococcus lactis subsp. lactis C2 was investigated by using phages c2, ml3, kh, l, h, 5, and 13. All seven phages adsorbed to the same sites on the host cell wall that are composed, in part, of rhamnose. This was suggested by rhamnose inhibition of phage adsorption to cells, competition between phage c2 and the other phages for adsorption to cells, and rhamnose inhibition of lysis of phage-inoculated cultures. The adsorption to the cell wall was found to be reversible upon dilution of the cell wall-adsorbed phage. In a reaction step that apparently follows adsorption to the cell wall, all seven phages adsorbed to a host membrane protein named PIP. This was indicated by the inability of all seven phages to infect a strain selected for resistance to phage c2 and known to have a defective PIP protein. All seven phages were inactivated in vitro by membranes from wild-type cells but not by membranes from the PIP-defective, phage c2-resistant strain. The mechanism of membrane inactivation was an irreversible adsorption of the phage to PIP, as indicated by adsorption of [³⁵S] methionine-labeled phage c2 to purified membranes from phage-sensitive cells but not to membranes from the resistant strain, elimination of adsorption by pretreatment of the membranes with proteinase K, and lack of dissociation of ³⁵S from the membranes upon dilution. Following membrane adsorption, ejection of phage DNA occurred rapidly at 30°C but not at 4°C. These results suggest that many lactococcal phages adsorb initially to the cell wall and subsequently to host cell membrane protein PIP, which leads to ejection of the phage genome.     

Improving the Safety of Staphylococcus aureus Polyvalent Phages by Their Production on a Staphylococcus xylosus Strain

Team1 (vB_SauM_Team1) is a polyvalent staphylococcal phage belonging to the Myoviridae family. Phage Team1 was propagated on a Staphylococcus aureus strain and a non-pathogenic Staphylococcus xylosus strain used in industrial meat fermentation. The two Team1 preparations were compared with respect to their microbiological and genomic properties. The burst sizes, latent periods, and host ranges of the two derivatives were identical as were their genome sequences. Phage Team1 has 140,903 bp of double stranded DNA encoding for 217 open reading frames and 4 tRNAs. Comparative genomic analysis revealed similarities to staphylococcal phages ISP (97%) and G1 (97%). The host range of Team1 was compared to the well-known polyvalent staphylococcal phages phi812 and K using a panel of 57 S. aureus strains collected from various sources. These bacterial strains were found to represent 18 sequence types (MLST) and 14 clonal complexes (eBURST). Altogether, the three phages propagated on S. xylosus lysed 52 out of 57 distinct strains of S. aureus. The identification of phage-insensitive strains underlines the importance of designing phage cocktails with broadly varying and overlapping host ranges. Taken altogether, our study suggests that some staphylococcal phages can be propagated on food-grade bacteria for biocontrol and safety purposes.     

Identification of a phage-derived depolymerase specific for KL47 capsule of Klebsiella pneumoniae and its therapeutic potential in mice

Klebsiella pneumoniae is one of the major pathogens causing global multidrug-resistant infections. Therefore, strategies for preventing and controlling the infections are urgently needed. Phage depolymerase, often found in the tail fiber protein or the tail spike protein, is reported to have antibiofilm activity. In this study, phage P560 isolated from sewage showed specific for capsule locus type KL47 K. pneumoniae, and the enlarged haloes around plaques indicated that P560 encoded a depolymerase. The capsule depolymerase, ORF43, named P560dep, derived from phage P560 was expressed, purified, characterized and evaluated for enzymatic activity as well as specificity. We reported that the capsule depolymerase P560dep, can digest the capsule polysaccharides on the surface of KL47 type K. pneumoniae, and the depolymerization spectrum of P560dep matched to the host range of phage P560, KL47 K. pneumoniae. Crystal violet staining assay showed that P560dep was able to significantly inhibit biofilm formation. Further, a single dose (50 μg/mouse) of depolymerase intraperitoneal injection protected 90%–100% of mice from lethal challenge before or after infection by KL47 carbapenem-resistant K. pneumoniae. And pathological changes were alleviated in lung and liver of mice infected by KL47 type K. pneumoniae. It is demonstrated that depolymerase P560dep as an attractive antivirulence agent represents a promising tool for antimicrobial therapy.     

Mobilization of Genomic Islands of Staphylococcus aureus by Temperate Bacteriophage

The virulence of Staphylococcus aureus, in both human and animal hosts, is largely influenced by the acquisition of mobile genetic elements (MGEs). Most S. aureus strains carry a variety of MGEs, including three genomic islands (νSaα, νSaβ, νSaγ) that are diverse in virulence gene content but conserved within strain lineages. Although the mobilization of pathogenicity islands, phages and plasmids has been well studied, the mobilization of genomic islands is poorly understood. We previously demonstrated the mobilization of νSaβ by the adjacent temperate bacteriophage ϕSaBov from strain RF122. In this study, we demonstrate that ϕSaBov mediates the mobilization of νSaα and νSaγ, which are located remotely from ϕSaBov, mostly to recipient strains belonging to ST151. Phage DNA sequence analysis revealed that chromosomal DNA excision events from RF122 were highly specific to MGEs, suggesting sequence-specific DNA excision and packaging events rather than generalized transduction by a temperate phage. Disruption of the int gene in ϕSaBov did not affect phage DNA excision, packaging, and integration events. However, disruption of the terL gene completely abolished phage DNA packing events, suggesting that the primary function of temperate phage in the transfer of genomic islands is to allow for phage DNA packaging by TerL and that transducing phage particles are the actual vehicle for transfer. These results extend our understanding of the important role of bacteriophage in the horizontal transfer and evolution of genomic islands in S. aureus.     

Roles of Exopolysaccharides and Lipopolysaccharides in the Adsorption of the Siphovirus Phage NM8 to Rhizobium meliloti M11S Cells

The exopolysaccharides produced by Rhizobium meliloti M11S inhibited nonspecifically the adsorption of phage NM8 by coating the cells. But lipopolysaccharides (LPS) had a specific inhibitory effect. Only the polysaccharide moiety of LPS, composed of glucose, glucosamine, galactose, 3-deoxy-D-manno-octulosonic acid (KDO), and large amounts of sialic acid, inhibited phage adsorption; neither the lipid A moiety nor a cellular glucan was involved. Rhizobium strains lacking sialic acids did not bind phage NM8. Inhibition of phage binding by lectin specific for N-acetylneuraminic acid demonstrated that phage NM8 bound to sialic acids. Preincubation of the phage with monosaccharides showed that inactivation of phage was very stereospecific for N-acetylneuraminic acid. Phage adsorption was also strongly inhibited by N-acetylglucosamine, which is not present in the LPS. Therefore, the receptor for phage NM8 appears to be a saccharide site, probably involving the acetyl groups of sialic acids. Received: 8 March 1996 / Accepted: 29 June 1996     

Characterization of a temperate actinophage,MPphiWR-1, capable of infectingMicromonospora purpurea ATCC 15835

Summary A temperate actinophage was isolated from soil using the gentamicin-producing microorganism,Micromonospora purpurea ATCC 15835 as host. The characterization of the phage represents the initial step in its development as a cloning vector. The phage isolated, MPphiWR-1, formed red- to purple-pigmented turbid plaques. Cells isolated from these plaques were resistant to superinfection with lytic mutants of MPphiWR-1. Southern blots of genomic DNA from a resistant culture showed that MPphiWR-1 integrated into the host genome. The phage was UV- or Mitomycin C-inducible. The integration, resistance to superinfection and inducibility indicated a lysogenic relationship with the host.Using MPphiE-RCPM, a lytic derivative, the phage host range was demonstrated to include members of three genera: one species each ofAmpullariella andCatellatospora, and 12 species ofMicromonospora. The phage belonged to Ackerman's B1 morphotype having an isometric head and a flexible noncontractile tail. The density of the phage was 1.525 g/cc. Restriction site mapping demonstrated that the phage DNA was 57.9 kb long and had cohesive ends. Using EDTA enrichment, viable mutants with deletions of at least 3.5 kb were isolated and mapped. Phage adsorption, sensitivities and plating efficiency were investigated. Non-liposome PEG-mediated transfection was demonstrated.     

Structure and Functions of the Bacteriophage P22 Tail Protein

The product of gene 9 (gp9) of Salmonella typhimurium bacteriophage P22 is a multifunctional structural protein. This protein is both a specific glycosidase which imparts the adsorption characteristics of the phage for its host and a protein which participates in a specific assembly reaction during phage morphogenesis. We have begun a detailed biochemical and genetic analysis of this gene product. A relatively straightforward purification of this protein has been devised, and various physical parameters of the protein have been determined. The protein has an s_20,w of 9.3S, a D_20,w of 4.3 × 10⁻⁷ cm²/s, and a molecular weight, as determined by sedimentation equilibrium, of 173,000. The purified protein appears as a prolate ellipsoid upon electron microscopic examination, with an axial ratio of 4:1, which is similar to the observed shape when it is attached to the phage particle. The molecular weight is consistent with the tail protein being a dimer of gp9 and each phage containing six of these dimers. An altered form of the tail protein has been purified from supF cells infected with a phage strain carrying an amber mutation in gene 9. Phage “tailed” with this altered form of gp9 adsorb to susceptible cells but form infectious centers with a severely reduced efficiency (ca. 1%). Biochemical analysis of the purified wild-type and genetically altered tail proteins suggests that loss of infectivity correlates with a loss in the glycosidase activity of the protein (2.5% residual activity). From these results we propose that the glycosidic activity of the P22 tail protein is not essential for phage assembly or adsorption of the phage to its host but is required for subsequent steps in the process of infection.     

Interaction of the ΦHSIC Virus with Its Host: Lysogeny or Pseudolysogeny?

The marine phage ΦHSIC has been previously reported to enter into a lysogenic relationship with its host, HSIC, identified as Listonella pelagia. This phage produces a variety of plaques on its host, including turbid and haloed plaques, from which lysogens were previously isolated. These lysogens were unstable during long-term storage at −80^° C and were lost. When HSIC was reinfected with phage ΦHSIC, pseudolysogen-like interactions between the phage and its host were observed. The cells (termed HSIC-2 or HSIC-2e) produced high viral titers (10¹¹ ml⁻¹) in the absence of inoculating phage and yet reached culture densities of nearly 10⁹ ml⁻¹. Prophages were not induced by mitomycin C or the polyaromatic hydrocarbon naphthalene in cells harboring such infections. However, such cells were homoimmune to superinfection. Colonies hybridized strongly with a gene probe from a 100-bp fragment of the ΦHSIC genome, while the host did not. Analysis of chromosomal DNA preparations suggested the presence of a chromosomally integrated prophage. Phage adsorption experiments suggested that HSIC-2 was adsorption impaired. Because of the chromosomal prophage integration and homoimmunity, we interpret these results to indicate that ΦHSIC establishes a lysogenic relationship with its host that involves an extremely high level of spontaneous induction. This could be caused by a weak repressor of phage production. Additionally, poor phage adsorption of HSIC-2 compared to the wild type probably helped maintain this pseudolysogen-like relationship. In many ways, pseudolysogenic phage-host interactions may provide a paradigm for phage-host interactions in the marine environment.     

Phage typing,PCR amplification for mecA gene,and antibiotic resistance patterns as epidemiologic markers in nosocomial outbreaks of methicillin resistant Staphylococcus aureus

Staphylococcus aureus is one of the major causes of community and hospital-acquired infections. Bacteriophage considered as a major risk factor acquires S. aureus new virulence genetic elements. A total number of 119 S. aureus isolated from different specimens obtained from (RKH) were distinguished by susceptibility to 19 antimicrobial agents, phage typing, and PCR amplification for mecA gene. All of MRSA isolates harbored mecA gene, except three unique isolates. The predominant phage group is belonging to the (mixed group). Phage group (II) considered as an epidemiological marker correlated to β-lactamase hyper producer isolates. MRSA isolates indicated high prevalence of phage group (II) with highly increase for phage types (Ø3A), which were correlated to the skin. Phage types (Ø80/Ø81) played an important roll in Community Acquired Methicillin Resistant S. aureus (CAMRSA). Three outpatients MRSA isolates had low multiresistance against Bacitracin (Ba) and Fusidic acid (FD), considered as CAMRSA isolates. It was detected that group I typed all FD-resistant MSSA isolates. Phage groups (M) and (II) were found almost to be integrated for Gentamycin (GN) resistance especially phage type (Ø95) which relatively increased up to 20% in MRSA. Tetracycline (TE) resistant isolates typed by groups (II) and (III) in MSSA. Only one isolate resistant to Sulphamethoxazole/Trimethoprim (SXT) was typed by (III/V) alone in MSSA. MRSA isolates resistant to Chloramphenicol (C) and Ba were typed by all groups except (V). It could be concluded that (PERSA) S. aureus isolates from the wound that originated and colonized, and started to build up multi-resistance against the topical treatment antibiotics. In this study, some unique sporadic isolates for both MRSA and MSSA could be used as biological, molecular and epidemiological markers such as prospective tools.     

Detailed Characterization and Comparison of Four Lactic Streptococcal Bacteriophages Based on Morphology, Restriction Mapping, DNA Homology, and Structural Protein Analysis

Bacteriophages uc1001 and uc1002, which are lytic for Streptococcus cremoris UC501 and UC502, respectively, were characterized in detail. Comparisons were made with a previously characterized phage, P008, which is lytic for Streptococcus lactis subsp. diacetylactis F7/2, and uc3001, which is a lytic phage for S. cremoris UC503. Phages uc1001 and uc1002 had small isometric heads (diameters, 52 and 50 nm, respectively) and noncontractile tails (lengths, 152 and 136 nm, respectively), and uc1002 also had a collar. Both had 30.1 ± 0.6 kilobase pairs (kbp) of DNA with cross-complementary cohesive ends. Restriction endonuclease maps made with seven endonucleases showed no common fragments. Despite this there was a very high level of homology between uc1001 and uc1002, and results of cross-hybridization experiments showed that the organization of both phage genomes was similar. Heteroduplex analysis confirmed this and quantified the level of homology at 83%. The regions of nonhomology comprised 2.1−, 1.1−, and 1.0-kbp deletion loops and 13 smaller loops and bubbles. The sodium dodecyl sulfate-polyacrylamide gel electrophoretic structural protein profiles were related, with a major band of about 40,000 molecular weight and minor bands of 35,000 and 34,000 molecular weight in common. There were also differences, however, in that uc1001 had a second major band of 68,000 molecular weight and two extra minor bands. Except for the restriction maps, which were strain specific, phages uc1001, uc1002, and P008 were closely related by all the criteria listed above. Their DNAs also showed a very significant bias against the cleavage sites of 9 of 11 restriction endonucleases. Phage uc3001 was unrelated to uc1001, uc1002, or P008 in that it had a prolate head (53 by 39 nm) and a shorter tail (105 nm), contained approximately 22 kbp of DNA, had unrelated cohesive ends, showed no DNA homology with the isometric-headed phages, and displayed a very different structural protein profile.     

Phage Display of a Biologically Active Bacillus thuringiensis Toxin

Activated forms of Bacillus thuringiensis insecticidal toxins have consistently been found to form insoluble and inactive precipitates when they are expressed in Escherichia coli. Genetic engineering of these proteins to improve their effectiveness as biological pesticides would be greatly facilitated by the ability to express them in E. coli, since the molecular biology tools available for Bacillus are limited. To this end, we show that activated B. thuringiensis toxin (Cry1Ac) can be expressed in E. coli as a translational fusion with the minor phage coat protein of filamentous phage. Phage particles displaying this fusion protein were viable, infectious, and as lethal as pure toxin on a molar basis when the phage particles were fed to insects susceptible to native Cry1Ac. Enzyme-linked immunosorbent assay and Western blot analysis showed the fusion protein to be antigenically equivalent to native toxin, and micropanning with anti-Cry1Ac antibody was positive for the toxin-expressing phage. Phage display of B. thuringiensis toxins has many advantages over previous expression systems for these proteins and should make it possible to construct large libraries of toxin variants for screening or biopanning.