Isometric Head Research Articles

Occurrence of Bacillus thuringiensis and their phages in Yemen soil.

Bacillus thuringiensis (Bt) isolates were found in all samples of soil in nine Governorates of Yemen. From 384 isolates of Bacillus recovered from these soil samples after acetate selection, 104 isolates (27.1%) were Bt. Five isolates of Bt were selected and insecticidal activity was tested against Culex pipiens, Callosobruchus maculatus and Spodoptera littoralis. The Bt isolate YH18 gave toxicity to all tested insects larvae. This study extended to isolate phages active against the selected Bt isolates. Five phages were isolated and classified into two groups of tailed phages. Four phages with long non-contractile tails and hexagonal heads (Siphoviridae) and one phage with very short tail and isometric head (Podoviridae). Susceptibility of selected Bt to infect by these phages was studied by spot-test technique. Also the Bt isolate no YH18 was resistant to all tested phages. This is the first report illustrates the diversity and the abundance of Bt and Bt phage in Yemen soil.

Characterization of Enterococcus faecalis Phage IME-EF1 and Its Endolysin

Enterococcus faecalis is increasingly becoming an important nosocomial infection opportunistic pathogen. E. faecalis can easily obtain drug resistance, making it difficult to be controlled in clinical settings. Using bacteriophage as an alternative treatment to drug-resistant bacteria has been revitalized recently, especially for fighting drug-resistant bacteria. In this research, an E. faecalis bacteriophage named IME-EF1 was isolated from hospital sewage. Whole genomic sequence analysis demonstrated that the isolated IME-EF1 belong to the Siphoviridae family, and has a linear double-stranded DNA genome consisting of 57,081 nucleotides. The IME-EF1 genome has a 40.04% G+C content and contains 98 putative coding sequences. In addition, IME-EF1 has an isometric head with a width of 35 nm to 60 nm and length of 75 nm to 90 nm, as well as morphology resembling a tadpole. IME-EF1 can adsorb to its host cells within 9 min, with an absorbance rate more than 99% and a latent period time of 25 min. The endolysin of IME-EF1 contains a CHAP domain in its N-terminal and has a wider bactericidal spectrum than its parental bacteriophage, including 2 strains of vancomycin-resistant E. faecalis. When administrated intraperitoneally, one dose of IME-EF1 or its endolysin can reduce bacterial count in the blood and protected the mice from a lethal challenge of E. faecalis, with a survival rate of 60% or 80%, respectively. Although bacteriophage could rescue mice from bacterial challenge, to the best of our knowledge, this study further supports the potential function of bacteriophage in dealing with E. faecalis infection in vivo. The results also indicated that the newly isolated bacteriophage IME-EF1 enriched the arsenal library of lytic E. faecalis bacteriophages and presented another choice for phage therapy in the future.

The first isolation of a cyanophage-Synechococcus system from the East China Sea

A cyanophage strain and its host Synechococcus were isolated from the East China Sea. The host Synechococcus sp. SJ01 was characterized by its 16S rRNA, ITS, and psbA gene sequences as well as by its morphological appearance and pigmentation. The cyanophage, strain S-SJ2, was able to cause a lytic infection of the coastal Synechococcus. TEM of negative-stained specimens showed that the phage isolate has an isometric head with a diameter of 68 nm and a long tail with a length of 280 nm. The cyanophage-Synechococcus system from the East China Sea shares many properties with other marine cyanophage-Synechococcus systems worldwide.

Isolation and Characterization of Bacteriophages of Helicobacter Pylori isolated from Egypt

Aim: The aim of this work was to isolate and characterize bacteriophages from wastewater for the pathogenic bacteria Helicobacter pylori. Materials & methods: In this study, we isolated and characterized two phages against H. pylori isolated from gastric biopsies. The specific phages were isolated by the single plaque isolation technique, propagated by the liquid enrichment method and purified by the polyethylene glycol–dextran sulfate two-phase system. Results: The phages were designated as φHPE1 and φHPE2; φHPE1 had a head with a diameter of approximately 62 nm and short noncontractile tail with a length and width of approximately 12 × 6 nm and φHPE2 had an isometric head with a diameter of approximately 92.5 nm and a tail with a length of approximately 180 nm and width of approximately 15 nm. The host ranges were investigated with four strains of Helicobacter and all strains were susceptible to either φHPE1 or φHPE2. Conclusion: These results suggest that it may be possible to use this phage to control the disease caused by H. pylori.

Genomic analysis of bacteriophage PBECO4 infecting Escherichia coli O157:H7

Escherichia coli O157:H7 is a human pathogen. We isolated a novel bacteriophage infecting this bacterium from a sewage water treatment facility. Phage PBECO4 belongs to the family Myoviridae, having an isometric head and a contractile tail. It has a linear double-stranded DNA genome of 348,113 base pairs in length with a GC content of 34.09 %. Whole-genome sequencing revealed that PBECO4 is distantly related to enterobacteria phage vB_KleM_RaK2, with 10 % similarity, and Cronobacter phage vB_CsaM_GAP32 with 6 % similarity. Five hundred fifty-one putative open reading frames (ORFs) and six tRNA genes were found. Eight ORFs are related to genes encoding structural proteins, nine to DNA packaging, two to DNA lysis activity, and 42 to replication and regulation. Four hundred ninety ORFs have not been functionally annotated.

Genome sequence of temperate bacteriophage Psymv2 from Antarctic Dry Valley soil isolate Psychrobacter sp. MV2

A temperate phage, Psymv2, was isolated from an Antarctic soil bacterium, Psychrobacter sp. MV2. The morphology of Psymv2 was typical of the Siphoviridae, with an isometric head and non-contractile tail. The Psymv2 genome was found to be 35,725 bp in length, had a G + C content of 44.5 %, with 49 protein-coding genes and one tRNA gene predicted. Integration of Psymv2 occurred at an ssrA gene, with the last 27 bases of this gene directly repeated at the prophage ends. The genome was organised in a modular fashion: integration, regulation, packaging, head assembly, tail assembly, host specificity and lysis. While the genome sequence had little similarity on a nucleotide level to previously reported phage sequences, the genome architecture resembled that of Siphoviridae of low G + C Gram-positive bacteria. The closest relatives to Psymv2 were uncharacterized putative prophages within the P. arcticus 273-4 and Acinetobacter baumannii 6013113 genomes. Global alignment of the Psymv2 genome and these prophages revealed significant conservation of the structural modules despite the large spatial divergence of their hosts. A number of unique ORFs were identified in the Psymv2 genome that may contribute to phage and lysogen fitness.

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.

Genome sequence of the phage clP1, which infects the beer spoilage bacterium Pediococcus damnosus

Pediococcus damnosus (P. damnosus) bacteriophage (phage) clP1 is a novel virulent phage isolated from a municipal sewage sample collected in Southern Ireland. This phage infects the beer spoilage strain P. damnosus P82 which was isolated from German breweries. Sequencing of the phage has revealed a linear double stranded DNA genome of 38,013 base pairs (bp) with an overall GC content of 47.6%. Fifty seven open reading frames (ORFs) were identified of which 30 showed homology to previously sequenced proteins, and as a consequence 20 of these were assigned predicted functions. The majority of genes displayed homology with genes from the Lactobacillus plantarum phage phiJL-1. All genes were located on the same coding strand and in the same orientation. Morphological characterisation placed phage clP1 as a member of the Siphoviridae family with an isometric head (59nm diameter) and non-contractile tail (length 175nm; diameter 10nm. Interestingly, the phage clP1 genome was found to share very limited identity with other phage genome sequences in the database, and was hence considered unique. This was highlighted by the genome organisation which differed slightly to the consensus pattern of genomic organisation usually found in Siphoviridae phages. With the genetic machinery present for a lytic lifecycle and the absence of potential endotoxin factors, this phage may have applications in the biocontrol of beer spoilage bacteria. To our knowledge, this study represents the first reported P. damnosus phage genome sequence.

Effect of a Novel Podophage AB7-IBB2 on Acinetobacter baumannii Biofilm

Biofilm formation in Acinetobacter baumannii is a common cause of nosocomial infections in humans. Clinical devices and abiotic surfaces are important sites of colonization leading to formation of biofilms. Such infections are often resistant to multiple antibiotic therapies, and hence there is need for an effective mode of control. Herein, we describe the isolation, characterization of a new lytic bacteriophage of A.baumannii and its effect on biofilm. The phage AB7-IBB2, with a genome size of about 170kb was identified to be of family Podoviridae as revealed by transmission electron microscopy. It had an isometric head (35nm) and a short tail (7nm). It lysed 19/39 (49%) clinical isolates of A.baumannii. Rapid adsorption (>99% adsorbed in 4min), a latency period of 25min and a burst size 22PFU/infected cell was observed. The phage could inhibit A.baumannii biofilm formation and disrupt preformed biofilm as well. The phage has promising potential to be considered as a candidate biocontrol agent for A.baumannii infections.

Characterization of a bacteriophage, isolated from a cow with mastitis, that is lytic against Staphylococcus aureus strains

Methicillin-resistant strains of Staphylococcus aureus (MRSA) are now the most commonly reported antibiotic-resistant bacterium in clinical settings. Therefore, there is an urgent need to develop novel antibacterial agents to control this pathogen. Bacteriophage therapy is a potential alternative treatment for MRSA infections. The objective of this study was characterization of a novel virulent bacteriophage (MSA6) isolated from a cow with mastitis. Electron microscopy showed its resemblance to members of the family Myoviridae, with an isometric head (66 nm) and a long contractile tail (173 nm). The genome of phage MSA6 was tested by pulsed-field gel electrophoresis and estimated to be about 143 kb. It exhibited rapid adsorption (>82% in 5 min), a short latent period (15 min) and a relatively small burst size (23 PFU/cell). Isolated phage was capable of infecting a wide spectrum of staphylococcal strains of both human and bovine origin. The results of this investigation indicate that MSA6 is similar to other bacteriophages belonging to the family Myoviridae (Twort, K, G1, 812) that have been successfully used in bacteriophage therapy.

Lytic Myophage Abp53 Encodes Several Proteins Similar to Those Encoded by Host Acinetobacter baumannii and Phage phiKO2

Acinetobacter baumannii is an important Gram-negative opportunistic pathogen causing nosocomial infections. The emergence of multiple-drug-resistant A. baumannii isolates has increased in recent years. Directed toward phage therapy, a lytic phage of A. baumannii, designated Abp53, was isolated from a sputum sample in this study. Abp53 has an isometric head and a contractile tail with tail fibers (belonging to Myoviridae), a latent period of about 10 min, and a burst size of approximately 150 PFU per infected cell. Abp53 could completely lyse 27% of the A. baumannii isolates tested, which were all multiple drug resistant, but not other bacteria. Mg(2+) enhanced the adsorption and productivity of, and host lysis by, Abp53. Twenty Abp53 virion proteins were visualized in SDS-polyacrylamide gel electrophoresis, with a 47-kDa protein being the predicted major capsid protein. Abp53 has a double-stranded DNA genome of 95 kb. Sequence analyses of a 10-kb region revealed 8 open reading frames. Five of the encoded proteins, including 3 tail components and 2 hypothetical proteins, were similar to proteins encoded by A. baumannii strain ACICU. ORF1176 (one of the tail components, 1,176 amino acids [aa]), which is also similar to tail protein gp21 of Klebsiella phage phiKO2, contained repeated domains similar to those within the ACICU_02717 protein of A. baumannii ACICU and gp21. These findings suggest a common ancestry and horizontal gene transfer during evolution. As phages can expand the host range by domain duplication in tail fiber proteins, repeated domains in ORF1176 might have a similar significance in Abp53.

Complete genomic sequence of the Lactobacillus temperate phage LF1

Bacteriophage LF1, a newly isolated temperate phage from a mitomycin-C-induced lysate of wild type Lactobacillus fermentum, was found to contain a double-strand DNA of 42,606 base pairs (bp) with a G+C content of 45%. Bioinformatic analysis of the phage genome revealed 57 putative open reading frames (ORFs). The predicted protein products of ORFs were determined and described. According to morphological analysis by transmission electron microscopy (TEM), LF1 has an isometric head and a non-contractile tail, indicating that it belongs to the family Siphoviridae. The temperate phage LF1 has a good genetic mosaic relationship with ΦPYB5 in the packaging module. To our knowledge, this is first report of genomic sequencing and characterization of temperate phage LF1 from wild-type L. fermentum isolated from Kimchi in Korea.

Sequence analysis of the Lactobacillus temperate phage Sha1

Bacteriophage Sha1, a newly isolated temperate phage from a mitomycin-C-induced lysate of Lactobacillus plantarum isolated from Kimchi, has an isometric head (58 nm × 60 nm) and a long tail (259 nm × 11 nm). The double-strand DNA genome of the phage Sha1 was 41,726 base pairs (bp) long, with a G+C content of 40.61%. Bioinformatic analysis of Sha1 shows that this phage contains 58 putative open reading frames (ORFs). Sha1 can be classified as a member of the large family Siphoviridae by genomic structure and morphology. To our knowledge, this is the first report of genomic sequencing and characterization of temperate phage Sha1 from wild-type L. plantarum isolated from kimchi in Korea.

Genomic sequence of temperate phage TEM126 isolated from wild type S. aureus

Bacteriophage TEM126, a newly isolated temperate phage from a mitomycin-C-induced lysate of wild-type Staphylococcus aureus isolated from food, has an isometric head, a noncontractile tail, and a double-stranded DNA genome with a length of 33,540bp and a G+C content of 33.94%. Bioinformatics analysis of the phage genome revealed 44 putative open reading frames (ORFs). Predicted protein products of the ORFs were determined and described. Temperate phage TEM126 can be classified as a member of the family Siphoviridae by morphology and genome structure. Temperate phage TEM126 showed 84% similarity with Staphylococcus phage phiNM1. To our knowledge, this is the first report of genomic sequencing and characterization of temperate phage TEM126 from a wild-type S. aureus isolated from foods in Korea.

The complete genome sequence and proteomics of Yersinia pestis phage Yep-phi

Yep-phi, a lytic phage of Yersinia pestis, was isolated in China and is routinely used as a diagnostic phage for the identification of the plague pathogen. Yep-phi has an isometric hexagonal head containing dsDNA and a short non-contractile conical tail. In this study, we sequenced the Yep-phi genome (GenBank accession no. HQ333270) and performed proteomics analysis. The genome consists of 38 ,616 bp of DNA, including direct terminal repeats of 222 bp, and is predicted to contain 45 ORFs. Most structural proteins were identified by proteomics analysis. Compared with the three available genome sequences of lytic phages for Y. pestis, the phages could be divided into two subgroups. Yep-phi displays marked homology to the bacteriophages Berlin (GenBank accession no. AM183667) and Yepe2 (GenBank accession no. EU734170), and these comprise one subgroup. The other subgroup is represented by bacteriophage ΦA1122 (GenBank accession no. AY247822). Potential recombination was detected among the Yep-phi subgroup.

Complete genome sequence of ΦMH1, a Leuconostoc temperate phage

Bacteriophage ΦMH1, a newly isolated temperate phage from a UV-induced lysate of Leuconostoc pseudomesenteroides, has an isometric head, a noncontractile tail, and a double-stranded DNA genome with a length of 38709 bp. Bioinformatic analysis of the phage genome revealed 65 putative open reading frames (ORFs). Predicted protein products of the ORFs were determined and described. ΦMH1 can be classified as a member of the family Siphoviridae by morphology and genome structure. The phage did not show any significant similarity to other previously reported bacteriophages of Leuconostoc species. To our knowledge, this is the first report of genomic sequencing and characterization of a L. pseudomesenteroides temperate phage.

Characterization of Bacillus phage-K2 isolated from chungkookjang, a fermented soybean foodstuff

An investigation of a virulent Bacillus phage-K2 (named Bp-K2) isolated from chungkookjang (a fermented soybean foodstuff) was made. Bp-K2 differed in infectivity against a number of Bacillus subtilis strains including starter strains of chungkookjang and natto, being more infectious to Bacillus strains isolated from the chungkookjang, but much less active against a natto strain. Bp-K2 is a small DNA phage whose genome size is about 21 kb. Bp-K2 is a tailed bacteriophage with an isometric icosahedral head (50 nm long on the lateral side, 80 nm wide), a long contractile sheath (85-90 nm × 28 nm), a thin tail fiber (80-85 nm long, 10 nm wide), and a basal plate (29 nm long, 47 nm wide) with a number of spikes, but no collar. The details of the structures of Bp-K2 differ from natto phage ϕBN100 as well as other known Bacillus phages such as SPO1-like or ϕ 29-like viruses. These data suggest that Bp-K2 would be a new member of the Myoviridae family of Bacillus bacteriophages.

Isolation and characterization of ϕAB2: a novel bacteriophage of Acinetobacter baumannii

Multidrug-resistant strains of Acinetobacter baumannii (MDRAB) are increasingly being reported worldwide. Bacteriophage therapy is a potential alternative treatment for MDR bacterial infections. Although A. baumannii infection has been experimentally treated with phages, no MDRAB-specific phage has been characterized. In this study, 10 phages with differing host ranges and lysis efficacy for MDRAB were isolated; one of these, ϕAB2, was further studied. Electron microscopy revealed ϕAB2 to have an isometric head (60 nm), a short tail (diameter, 9 nm; length, 11 nm) and a double-stranded DNA genome—which was resistant to digestion with several restriction endonucleases—estimated to be 40 kb by pulsed-field gel electrophoresis. Partial genome sequencing of a 2.1 kb region gave sequences resembling the tubular proteins A and B of Pseudomonas aeruginosa phage LKA1. These data suggest that ϕAB2 resembles ϕKMV-like phages and is a new member of the Podoviridae family. It exhibited rapid adsorption (>99% adsorbed in 8 min), a short latent period (<10 min) and a large burst size (ca. 200); moreover, it was capable of infecting a wide spectrum of A. baumannii strains, causing virtually complete lysis, indicating that ϕAB2 may be a good candidate as a therapeutic/disinfectant agent to control nosocomial infections caused by MDRAB.

Genome of a virulent bacteriophage Lb338-1 that lyses the probiotic Lactobacillus paracasei cheese strain

There is a lack of fundamental knowledge about the influence of bacteriophage on probiotic bacteria and other commensals in the gut. Here, we present the isolation and morphological and genetic characterization of a virulent narrow-host-range bacteriophage, φLb338-1. This phage was isolated from fresh sewage and was shown to infect the probiotic cheese strain Lactobacillus paracasei NFBC 338. Electron microscopy studies revealed that φLb338-1 is a member of the Myoviridae family, with an isometric head, a medium-sized contractile tail, and a complex base plate. Genome sequencing revealed a 142-kb genome with 199 open reading frames. Putative functions could be assigned to 22% of the open reading frames; these had significant homology to genes found in the broad-host-range SPO1-like group of phages which includes the Enterococcus faecalis phage φEF24C, Listeria phage A511, and Lactobacillus plantarum phage LP65. Interestingly, no significant genomic similarity was observed between the phage and the probiotic host strain. Future studies will determine if the presence of bacteriophage φLb338-1 or others in the human or animal gut plays an antagonistic role against the probiotic effect of beneficial bacteria.

Decreasing Enterobacter sakazakii (Cronobacter spp.) food contamination level with bacteriophages: prospects and problems

SummaryEnterobacter sakazakii (Cronobacter spp.) is an opportunistic pathogen, which can cause rare, but life‐threatening infections in neonates and infants through feeding of a contaminated milk formula. We isolated 67 phages from environmental samples and tested their lytic host range on a representative collection of 40 E. sakazakii strains. A cocktail of five phages prevented the outgrowth of 35 out of 40 test strains in artificially contaminated infant formula. Two E. sakazakii phages represented prolate head Myoviridae. Molecular tests identified them as close relatives of Escherichia coli phage T4. The remaining three phages represented isometric head Myoviridae with large genome size of 140 and 200 kb, respectively, which belonged to two different DNA hybridization groups. A high dose of 108 pfu ml−1 of phage could effectively sterilize a broth contaminated with both high and low pathogen counts (106 and 102 cfu ml−1). In contrast, broth inoculated with 104 phage and 102 bacteria per ml first showed normal bacterial growth until reaching a cell titre of 105 cfu ml−1. Only when crossing this threshold, phage replication started, but it could not reduce the contamination level below 100 cfu ml−1. Phages could be produced with titres of 1010 pfu ml−1 in broth culture, but they were not stable upon freeze‐drying. Addition of trehalose or milk formula stabilized the phage preparation, which then showed excellent storage stability even at elevated temperature.