Isolation and Characterization of Novel Giant Stenotrophomonas maltophilia Phage φSMA5

Stenotrophomonas maltophilia is one of the most prevalent opportunistic bacteria causing nosocomial infections. It has become problematic because most of the isolates are resistant to multiple antibiotics, and therefore, development of phage therapy has attracted strong attention. In this study, eight S. maltophilia phages were isolated from clinical samples including patient specimens, catheter-related devices, and wastewater. These phages can be divided into four distinct groups based on host range and digestibility of the phage DNAs with different restriction endonucleases. One of them, designated SMA5, was further characterized. Electron microscopy showed it resembled Myoviridae, with an isometric head (90 nm in diameter), a tail (90 nm long), a baseplate (25 nm wide), and short tail fibers. The SMA5 double-stranded DNA, refractory to digestion by most restriction enzymes, was tested and estimated to be 250 kb by pulsed-field gel electrophoresis. This genome size is second to that of the largest phage, KZ of Pseudomonas aeruginosa. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 25 virion proteins were visualized. N-terminal sequencing of four of them suggested that each of them might have had its N terminus cleaved off. Among the 87 S. maltophilia strains collected in this study, only 61 were susceptible to SMA5, indicating that more phages are needed toward a phage therapy strategy. Since literature search yielded no information about S. maltophilia phages, SMA5 appears to be the first reported.     

Characterization of a novel T4-type Stenotrophomonas maltophilia virulent phage Smp14

Stenotrophomonas maltophilia (Sm), with most of the isolates being resistant to multidrugs, is an opportunistic bacterium causing nosocomial infections. In this study, a novel virulent Sm phage, Smp14, was characterized. Electron microscopy showed that Smp14 resembled members of Myoviridae and adsorbed to poles of the host cells during infection. It lysed 37 of 87 clinical Sm isolates in spot test, displayed a latent period of ca. 20 min, and had a burst size of ca. 150. Its genome (estimated to be 160 kb by PFGE), containing m4C and two unknown modified bases other than m5C and m6A as identified by HPLC, resisted to digestion with many restriction endonucleases except MseI. These properties indicate that it is a novel Sm phage distinct from the previously reported phiSMA5 which has a genome of 250 kb digestible with various restriction enzymes. Sequencing of a 16 kb region revealed 12 ORFs encoding structural proteins sharing 15-45% identities with the homologues from T4-type phages. SDS-PAGE displayed 20 virion proteins, with the most abundant one being the 39 kDa major capsid protein (gp23), which had the N-terminal 52 amino acids removed. Phylogenetic analysis based on gp23 classified Smp14 into a novel single-membered T4-type subgroup.     

Structure and function of a novel coliphage-associated sialidase

A coliphage named 63D, isolated previously, associated sialidase as a component of phage particles. In order to localize the enzyme in phage particles, phages were partially destroyed by sonication, and the disrupted particles were size fractionated using a sucrose density gradient. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme assay and electron micrography of the fractions revealed the enzyme to be composed of four identical subunits with a molecular mass of 90 kDa, and the subunits were cross-linked by disulfide bonds. Electron micrographic observation indicated that six enzyme molecules were localized in a phage tail plate as a hexagonal array.     

Morphological and genetic analysis of three bacteriophages of Serratia marcescens isolated from environmental water

Increases in multidrug-resistant strains of Serratia marcescens are of great concern in pediatrics, especially in neonatal intensive care units. In the search for bacteriophages to control infectious diseases caused by multidrug-resistant S. marcescens , three phages (KSP20, KSP90, and KSP100) were isolated from environmental water and were characterized morphologically and genetically. KSP20 and KSP90 belonged to morphotype A1 of the family Myoviridae , and KSP100 belonged to morphotype C3 of the family Podoviridae . Analysis of the DNA region coding virion proteins, together with their morphological features, indicated that KSP20, KSP90, and KSP100 were related to the P2-like phage (temperate), T4-type phage (virulent), and phiEco32 phage (virulent), respectively. Based on amino acid sequences of the major capsid protein, KSP90 formed a new branch with a Stenotrophomonas maltophilia phage, Smp14, in the T4-type phage phylogeny. Both Smp14 and phiEco32 have been reported as potential therapeutic phages. These results suggest that KSP90 and KSP100 may be candidate therapeutic phages to control S. marcescens infection.     

粘质沙雷菌裂解性噬菌体ФSM9-3Y的分离和鉴定

目的从医院污水中分离粘质沙雷菌噬菌体,并分析其生物学特性,为进一步研究针对耐药性粘质沙雷菌的噬菌体制剂提供依据。方法采用双层琼脂平板法分离纯化针对粘质沙雷菌的裂解性噬菌体,观察噬菌体对宿主菌的裂解特异性,通过负染法电镜观察噬菌体的形态结构,提取噬菌体核酸进行酶切电泳,测定噬菌体的最佳感染复数和一步生长曲线,SDS-PAGE电泳初步分析噬菌体的结构蛋白和非结构蛋白。结果从医院污水分离出7株可裂解粘质沙雷菌的噬菌体,对其中一株噬菌体(命名为ФSM9-3Y)的生物学特征进行了初步研究。电镜显示噬菌体呈蝌蚪状,头部为20面体立体对称、直径约70 nm;尾部长约50 nm。ФSM9-3Y的最佳感染复数为1。一步生长曲线表明;ФSM9-3Y的潜伏期约30 min,暴发时间70 min,暴发量为629 PFU/cell。凝胶电泳显示噬菌体基因组为双链DNA、大小约54 kb。SDS-PAGE呈现至少包括13种蛋白,相对分子质量范围在25～130 kD,其中主要蛋白的相对分子质量约为48 kD。结论此次分离的噬菌体ФSM9-3Y为裂解性噬菌体,根据形态和结构特征,粘质沙雷菌噬菌体ФSM9-3Y属于有尾病毒目,肌尾噬菌体科。     

Characterisation of four Leuconostoc bacteriophages isolated from dairy fermentations

Abstract Four bacteriophages (phages) growing on the same Leuconostoc strain were characterised. Electron micrographs showed these phages to be similar in morphology to the commonly isolated lactococcal phages with head diameters ranging from 49–55 nm and tail lengths of 117–131 nm. A distinctive base plate and collar were also present. From restriction enzyme analysis of purified phage DNA, the genome sizes were 23–29 kb. All four phages showed one major structural protein (of approximately 24 kDa) on SDS polyacrylamide gels. Hybridization experiments confirmed that the phages belonged to the same homology group. There was no homology between DNA from these phages and DNA from a prolate or small isometric lactococcal phage.     

Some Properties of Five New Salmonella Bacteriophages

Five bacteriophages were isolated from lysogenic strains of Salmonella potdam. On the basis of plaque morphology, thermostability, serology, host range, one-step growth parameters, and phage morphology, they were divided into three groups: group A, phages P4 and P9c; group B, phages P3 and P9a; and group C, phage P10. Group A phages had a hexagonal head 55 nm in diameter with a short tail 15 nm long. These phages were particularly characterized by high thermostability, lack of serological relationship with any of the other phages, and restriction of lysis to other Salmonella strains of Kauffmann-White group C(1). Group B phages had a head identical in size and shape to that of the A phages, but they possessed a tail 118 nm long with a contractile sheath. A unique feature was the occurrence of tail fibers at the end of the core rather than at the base of the sheath. These phages were considerably less thermostable, had extended host ranges, and were serologically distinct from each other but unrelated to the A phages. The group C phage, P10, had a head identical to that of the A and B phages. It had a tail 95 nm in length, with tail fibers attached to a base plate at the end of a contractile sheath. P10 was highly sensitive to heat, lysed only smooth strains of Salmonella, and showed a degree of serological relationship to both B phages. The relationship of these phage groups to previous Salmonella phage grouping schemes is discussed.     

Electron microscopy of virulent phages for Streptococcus lactis.

Electron microscopic studies were made on eight virulent Streptococcus lactis bacteriophages. These phages were taken as representative of eight host range groups established in a study of 75 phage isolates and 253 hosts (213 S. lactis, 22 S. cremoris, 18 S. diacetilactis). The phages studied were shown to have an isometric hexagonal head and noncontractile tails, usually several times longer than the head diameter. The virus heads were octahedral. The phages investigated represented three morphological types on the basis of head diameter , tail thickness, and tail length. These dimensions were approximately: for type I phages, 63, 172, and 11 nm, respectively; type II, 73, 200, and 20 nm, respectively; and type III, represented here by a single phage, 98, 551, and 12 nm, respectively. The tail surface revealed a different arrangment of the structural subunits which lent a helical appearance to the tails of type I and II phages and a guaffered tube appearance to the tail of type III phage. The number of turns along the tail axis, turn length, axial pitch, and helix angle were: type I, 32, 12 to 13 nm, 7.14 nm, and 11 degrees 43', respectively; type II, 24, 24, to 28 nm, 40.00 nm, and 32 degrees 30', respectively; and type III, 120, 12 nm, and no visible slope towards the axis. The morphology types showed complete correlation with serological groups, but not with groups based on host range pattern.     

Isolation and characterization of a new bacteriophage, Cp-1, infecting Streptococcus pneumoniae.

Several pneumococcal phages showing a morphology completely different from those of all other previously found pneumococcal bacteriophages have been isolated. Bacteriophage Cp-1, one of the phages isolated, showed an irregular hexagonal structure and a short tail of 20 nm. The virion density was 1.46 g/cm3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of nine polypeptides. The polypeptide showing a molecular weight of 39,000 accounted for more than the 90% of the total protein. The nucleic acid of Cp-1 was linear, double-stranded DNA with a mean length of 6.3 microns and a guanine-plus-cytosine content of 41%; its buoyant density was 1.699 and 1.422 g/cm3 in CsCl and CS2SO4, respectively. Its sedimentation coefficient (S20,w) was 19S. Cp-1 DNA showed a remarkable resistance to a large number of restriction endonucleases. A total of 12 fragments, ranging in molecular weight from 1.3 X 10(6) to 0.09 X 10(6), were produced by AluI, two fragments (molecular weight, 5.5 X 10(6) and 0.9 X 10(6)) were generated by HindIII, and two fragments (molecular weight, 6.0 X 10(6) and 5.7 X 10(6)) were produced by HaeIII. The easy visualization of th plaques produced by Cp-1, the small size of Cp-1 DNA (12 X 10(6) daltons), and other biological and physiochemical properties make this phage potentially useful for genetic studies.     

Isolation and Characterization of a Novel Bacteriophage φ4D Lytic Against Enterococcus faecalis Strains

In recent years, Enterococcus faecalis has emerged as an important opportunistic nosocomial pathogen capable of causing dangerous infections. Therefore, there is an urgent need to develop novel antibacterial agents to control this pathogen. Bacteriophages have very effective bactericidal activity and several advantages over other antimicrobial agents and so far, no serious or irreversible side effects of phage therapy have been described. The objective of this study was to characterize a novel virulent bacteriophage φ4D isolated from sewage. Electron microscopy revealed its resemblance to Myoviridae, with an isometric head (74 ± 4 nm) and a long contractile tail (164 ± 4 nm). The φ4D phage genome was tested using pulsed-field gel electrophoresis and estimated to be 145 ± 2 kb. It exhibited short latent period (25 min) and a relatively small burst size (36 PFU/cell). Tests were conducted on the host range, multiplicities of infection (MOI), thermal stability, digestion of DNA by restriction enzymes, and proteomic analyses of this phage. The isolated phage was capable of infecting a wide spectrum of enterococcal strains. The results of these investigations indicate that φ4D is similar to other Myoviridae bacteriophages (for example φEF24C), which have been successfully used in phagotherapy.     

Characterization of a temperate phage isolated fromLeuconostoc oenos strain 1002

A temperate phage was induced by mitomycin C fromLeuconostoc oenos strain 1002, a New Zealand isolate. The phage has an isometric head (52 ± 3 nm) with a tail (210 ± 10 nm) and gives a lytic burst size of 25 when grown on a sensitive strain. Three major structural proteins of 43, 30 and 14 kDa are visible by Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). A restriction map of the 36-kb genome was determined. Commercially availableL. oenos strains PSU-1, ML34 and Lco23 and 46.6% of New Zealand isolates were sensitive to the phage.     

Ten new temperate bacteriophages of<Emphasis Type="Italic">Citrobacter youngae</Emphasis>

In a cross-test, we examined 55 strains of Citrobacter youngae against each other as potential producers of temperate bacteriophages and as potential sensitive indicators for them. Ten strains (18.2 %) showed the production of phages. Seven different strain-specific spectra of activity (from 1 to 11 strains each) were found. Phage production by 6 strains was inducible with mitomycin C, in 4 strains it was not inducible. The plaques of the phages were more or less turbid, without a lytic halo, tiny to small, 0.2-1.3 mm in diameter. Using a polyclonal, specific anti-lambda serum, all 10 phages were found to be clearly distinct from E. coli lambda phage, the phage 31/47 showing the highest neutralization titre of all. Interspecific tests with 15 strains of 8 species of Enterobacteriaceae revealed not a single case of activity of Citrobacter phages towards any of them. Five phage-immune clones lysogenized with 5 of the phages kept their remaining phage sensitivity spectra, though extended by sensitivity to 1-3 phages; 2 of these strains acquired also sensitivity to phage lambda. The phages belong to the morphotypes of Myoviridae (6 phages) and Siphoviridae (4 phages), with head diameters of 51-58 nm and tail length of 97-173 nm. Three strains produced corpuscular bacteriocins.     

The morphology and nucleotide composition of DNA of Citrobacter phages

Citrobacter phages 38/37, 31/37, 40/1 and 8/5, isolated from lysogenic cultures, were concentrated and purified by 2 cycles of differential centrifugation. Electron microscopy of the phages has shown that their particles have similar morphology and that they relate to the morphological group A1. The heads of the phages are hexagonal, 50 +/- 2 nm in diameter. The tail of the phage is straight, 112-152 nm in length, with a contracting sheath 11.5-12.5 nm wide. The tails of the phages 38/37 and 40/1 were found to be slightly longer in comparison with the phages 31/37 and 8/5. Chromatographic investigation of DNA preparations of the phages revealed the presence of 4 nitrous bases. Identification of the latter permitted us to relate them to common nitrous bases. DNA of the phages is double-stranded and belongs to a weakly expressed guanine-cytosine type. The content of guanine and cytosine in DNA of the phage 38/37 amounts to 56.68%, that of the phage 31/37 to 56.75, of the phage 40/1 to 57.36% and of the phage 8/5 to 55.58%. No substantial variations were observed in the DNA composition of the phages.     

Classification of 17 newly isolated virulent bacteriophages of Pseudomonas aeruginosa

Seventeen virulent bacteriophages specific to Pseudomonas aeruginosa strains were isolated by screening various environmental samples. These isolated bacteriophages were grouped based on results obtained from restriction fragment analysis of phage genomes, random amplification of polymorphic DNA (RAPD) typing, morphology observations under transmission electron microscope, and host range analysis. All 17 bacteriophages are double-stranded DNA viruses and can be divided into 5 groups based on DNA restriction profiles. A set of 10-mer primers was used in RAPD typing of phages, and similar conclusions were obtained as for restriction fragment analysis. One phage was randomly selected from each of the 5 groups for morphology observations. Four of them had an icosahedral head with a long contractile tail, belonging to the Myoviridae family, and one phage had an icosahedral head with a short tail, thereby belonging to the Podoviridae family. Host range experiments were conducted on 7 laboratory strains and 12 clinical strains of P.?aeruginosa. The results showed that 13 phages had the same infection profile, killing 8 out of 19 tested P.?aeruginosa strains, and the remaining 4 phages had different and unique infection profiles. This study highlights the diversity of bacteriophages specific to P.?aeruginosa in the environment.     

Morphological,host range,and genetic characterization of two coliphages

Two coliphages, AR1 and LG1, were characterized based on their morphological, host range, and genetic properties. Transmission electron microscopy showed that both phages belonged to the Myoviridae; phage particles of LG1 were smaller than those of AR1 and had an isometric head 68 nm in diameter and a complex contractile tail 111 nm in length. Transmission electron micrographs of AR1 showed phage particles consisting of an elongated isometric head of 103 by 74 nm and a complex contractile tail 116 nm in length. Both phages were extensively tested on many strains of Escherichia coli and other enterobacteria. The results showed that both phages could infect many serotypes of E. coli. Among the enterobacteria, Proteus mirabilis, Shigella dysenteriae, and two Salmonella strains were lysed by the phages. The genetic material of AR1 and LG1 was characterized. Phage LG1 had a genome size of 49.5 kb compared to 150 kb for AR1. Restriction endonuclease analysis showed that several restriction enzymes could degrade DNA from both phages. The morphological, genome size, and restriction endonuclease similarities between AR1 and phage T4 were striking. Southern hybridizations showed that AR1 and T4 are genetically related. The wide host ranges of phages AR1 and LG1 suggest that they may be useful as biocontrol, therapeutic, or diagnostic agents to control and detect the prevalence of E. coli in animals and food.     

Comparative characterization of inducible and virulent Vibrio parahaemolyticus bacteriophages having unique head projections

Phage TP1, induced from Vibrio parahaemolyticus K-20 pilot strain by mitomycin C, exhibited a unique hexagonal head with knob-like projections which covered the whole capsid and a noncontractile tail. The appearance of this phage was very similar to those of phages VP3 and VP6, isolated from seawater. The host range of phage TP1 was similar to those of phages VP3 and VP6. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the three phage particles revealed almost identical patterns with four major polypeptides with apparent approximate molecular masses: 78, 42, 37 and 34.5 kDa. On the basis of HindIII digestion patterns on agarose gel electrophoresis, the lengths of phage TP1 and VP3 DNAs were estimated to be about 65 kilobase pairs (kbp) and that of VP6 DNA was about 74 kbp. The digestion patterns of all three phage DNAs by DraI, BamHI and MspI were very similar. The DNAs of TP1 and VP3 exhibited almost the same digestion patterns with HindIII and EcoRI, whereas the digestion patterns of VP6 DNA were significantly different from those of the former. From these findings, it seems likely that virulent phage VP3 is originated from a lysogenic phage, probably TP1, of V. parahaemolyticus.     

黏质沙雷菌短尾噬菌体中国分离株的鉴定

以黏质沙雷菌jn01株为宿主菌,从环境污水中分离噬菌体,经反复挑取噬菌斑,获得1株纯化的噬菌体,定名为SmPjn。SmPjn在双层琼脂平板上可形成直径约2mm,圆形、透明的噬菌斑,边缘清晰。透射电镜观察,该噬菌体有一短尾,长（7± 1.25）nm,头部长、宽分别为（58± 2.16）nm×（55±0.47）nm,属短尾噬菌体科（Podoviridae）。可裂解jn01以外的2株黏质沙雷菌;与宿主菌共培养4h后的最佳感染复数为1;一步生长曲线表明该噬菌体的潜伏期约为50min,平均爆发量约为1125pfu/cell。基因组为大于27 kb的DNA,可分别被HindⅢ、EcoRⅠ切成11和9个电泳片段。本报道为国内首次分离黏质沙雷菌短尾噬菌体。     

Molecular characterization of new group A streptococcal bacteriophages containing the gene for streptococcal erythrogenic toxin A (speA)

Summary Bacteriophage T12 is the prototype phage carrying the streptococcal erythrogenic toxin A (speA) gene. To examine more closely the phages involved in lysogenic conversion, we examined 300 group A streptococcal strains, and identified and isolated two new phages that carry the speA gene. The molecular sizes of these phage genomes were between 32 and 40 kb, similar to that of phage T12 (35 kb). However, as ascertained by restriction analysis, the physical maps of the new phage genomes were different from phage T12 and from each other. Hybridization analysis also showed that all of these phages were only partially related to one another and the speA gene was always located close to the phage attachment site. Additionally, colony hybridization showed that whereas phage T12 or one of its close relatives is the most common phage associated with the group A streptococci, phage 49 has a much stronger association with the speA gene. A defective phage was also found following pulsed field gel electrophoresis of total phage DNA. This phage appears to be a resident of strain T25₃c and is found only following induction of a T25₃c lysogen. Restriction enzyme analysis of the isolated defective phage DNA suggests that it is the source of the submolar amounts of DNA previously found in association with phage T12 digestion patterns. Additionally, the defective phage may serve as the site of integration of the speA gene-carrying phages described above.     

Characterization of the bacteriophage B2 of Lactobacillus plantarum ATCC 8014

Bacteriophage B2 of Lactobacillus plantarum ATCC 8014, isolated in 1971, belonged to Bradley's group B. Electron microscopy revealed an isometric head (110 nm) and a long non-contractile and flexible tail (500 nm) containing about 75 regularly aligned lateral striations. Burst size was 12-14 phages per infectious centre. The latent period for phage development was 75 min and the rise period approximately 90 min. The phage particle contained 5 major proteins. The buoyant density of the phage in CsCl was measured as 1.575 g/cm3. B2 genome was a linear double-stranded DNA molecule of 37 +/- 1% guanosine-cytosine. Its size was 73 kilobase pairs (kbp). Restriction analysis of the genome showed that 4 restriction enzymes (Xba I, Sac I, Bgl II and Sma I) gave single site cuts in the DNA, while Ava I and Sal I formed 2 and 5 cuts, respectively.     

Characterization of four lytic transducing bacteriophages of luminescent Vibrio harveyi isolated from shrimp (Penaeus monodon) hatcheries

Four lytic bacteriophages designated as φVh1, φVh2, φVh3, and φVh4 were isolated from commercial shrimp hatcheries, possessing broad spectrum of infectivity against luminescent Vibrio harveyi isolates, considering their potential as biocontrol agent of luminescent bacterial disease in shrimp hatcheries, and were characterized by electron microscopy, genomic analysis, restriction enzyme analysis (REA), and pulsed-field gel electrophoresis (PFGE). Three phages φVh1, φVh2, and φVh4 had an icosahedral head of 60-115 nm size with a long, noncontractile tail of 130-329 × 1-17 nm, belonged to the family Siphoviridae. φVh3 had an icosahedral head (72 ± 5 nm) with a short tail (27 × 12 nm) and belonged to Podoviridae. REA with DraI and PFGE of genomic DNA digested with ScaI and XbaI and cluster analysis of their banding patterns indicated that φVh3 was distinct from the other three siphophages. PFGE-based genome mean size of the four bacteriophages φVh1, φVh2, φVh3, and φVh4 was estimated to be about 85, 58, 64, and 107 kb, respectively. These phages had the property of generalized transduction as demonstrated by transduction with plasmid pHSG 396 with frequencies ranging from 4.1 × 10(-7) to 2 × 10(-9) per plaque-forming unit, suggesting a potential ecological role in gene transfer among aquatic vibrios.