Bacteriophage YeO3-12 is a lytic phage of Yersinia enterocolitica serotype O:3. The phage receptor is the lipopolysaccharide O chain of this serotype that consists of the rare sugar 6-deoxy-L-altropyranose. A one-step growth curve of YeO3-12 revealed eclipse and latent periods of 15 and 25 min, respectively, with a burst size of about 120 PFU per infected cell. In electron microscopy YeO3-12 virions showed pentagonal outlines, indicating their icosahedral nature. The phage capsid was shown to be composed of at least 10 structural proteins, of which a protein of 43 kDa was predominant. N-terminal sequences of three structural proteins were determined, two of them showing strong homology to structural proteins of coliphages T3 and T7. The phage genome was found to consist of a double-stranded DNA molecule of 40 kb without cohesive ends. A physical map of the phage DNA was constructed using five restriction enzymes. The phage infection could be effectively neutralized using serum from a rabbit immunized with whole YeO3-12 particles. The antiserum also neutralized T3 infection, although not as efficiently as that of YeO3-12. YeO3-12 was found to share, in addition to the N-terminal sequence homology, several common features with T3, including morphology and nonsubjectibility to F exclusion. The evidence conclusively indicated that YeO3-12 is the first close relative of phage T3 to be described.Yersinia enterocolitica is a Gram-negative species which contains several serotypes, some of which are pathogenic to humans. The major pathogens in Europe, Canada, Japan, and South Africa belong to serotypes O:3 and O:9, and those in the United States belong to serotype O:8 (11). The main reservoir in nature for Y. enterocolitica is pigs (15), and human infections usually take place after ingestion of contaminated foodstuffs.A number of yersiniophages have been described, but only a few have been characterized by electron microscopy and to our knowledge none have been studied in detail. In our laboratory a number of Yersinia-specific bacteriophages have been isolated, all originating from the raw incoming sewage of the Turku City sewage treatment plant, and the phages have been used as genetic tools (32). One of the phages, YeO3-12, was isolated as specific to Y. enterocolitica serotype O:3. The phage could infect Escherichia coli C600 expressing the cloned O antigen of Y. enterocolitica serotype O:3 and spontaneous phage-resistant Y. enterocolitica serotype O:3 strains were missing the O antigen, indicating that the O antigen is the phage receptor (4, 5). The serotype O:3 specificity makes the phage YeO3-12 a potential biotechnological tool, and therefore we have initiated its detailed characterization. Here we present the biological and physical properties of the phage and evidence suggesting that YeO3-12 is closely related to coliphages T3 and T7. MATERIALS AND METHODSCulture conditions. Bacterial strains, bacteriophages and plasmids used in this study are listed in Table 1. Virulence plasmid-cured Y. enterocolitica serotyp...

The challenges for successful launching of a profitable phage therapeutic product include intellectual property rights, safety issues, reproducibility, stability and robustness of the product. A successful and marketable product would be a highly purified bacteriophage preparation containing one or several fully characterized phages, accompanied by optimized methods of administration and backed up by properly controlled efficacy and safety studies.

Yersiniophage wR1-37 is a tailed bacteriophage having a 270 kb DNA genome with thymidine replaced by deoxyuridine Bacteriophage wR1-37 was isolated based on its ability to infect strain YeO3-R1, a virulenceplasmid-cured O antigen-negative derivative of Yersinia enterocolitica serotype O : 3. In this study, the phage receptor was found to be a structure in the outer core hexasaccharide of Y. enterocolitica O : 3 LPS. The phage receptor was present in the outer core of strains of many other Y. enterocolitica serotypes, but also in some Yersinia intermedia strains. Surprisingly, the receptor structure resided in the O antigen of Yersinia pseudotuberculosis O : 9. Electron microscopy demonstrated that wR1-37 particles have an icosahedral head of 88 nm, a short neck of 10 nm, a long contractile tail of 236 nm, and tail fibres of at least 86 nm. This implies that the phage belongs to the order Caudovirales and the family Myoviridae in the ICTV (International Committee for Taxonomy of Viruses) classification. wR1-37 was found to have a lytic life cycle, with eclipse and latent periods of 40 and 50 min, respectively, and a burst size of~80 p.f.u. per infected cell. Restriction digestions and PFGE showed that the wR1-37 genome was dsDNA and~270 kb in size. Enzymically hydrolysed DNA was subjected to HPLC-MS/MS analysis, which demonstrated that the wR1-37 genome is composed of DNA in which thymidine (T) is >99 % replaced by deoxyuridine (dU). The only organisms known to have similar DNA are the Bacillus subtilis-specific bacteriophages PBS1 and PBS2. N-terminal amino acid sequences of four major structural proteins did not show any similarity to (viral) protein sequences in databases, indicating that close relatives of wR1- 37 have not yet been characterized. Genes for two of the structural proteins, p24 and p46, were identified from the partially sequenced wR1-37 genome. INTRODUCTIONYersinia enterocolitica is a Gram-negative bacterium of the family Enterobacteriaceae. The species includes over 30 serotypes, a number of which are human pathogens (Wauters et al., 1991). While the major pathogenic serotypes in Europe, Canada, Japan and South Africa are O : 3 and O : 9; in the United States serotype O : 8 is more prevalent. Y. enterocolitica is widely distributed in nature, swine being the major reservoir of the pathogenic strains (Bottone, 1997).Bacteriophages are the most abundant organisms on Earth, and it is estimated that for each microbial isolate at least 10 different phages can be found (Hendrix, 2002;Pedulla et al., 2003). A number of phages infecting the members of the genus Yersinia have been isolated (Baker & Farmer, 1982;Popp et al., 2000;Stevenson & Airdrie, 1984), but relatively few have been characterized in more detail. Y. enterocolitica O : 3-specific phage wYeO3-12 has been shown to be closely related to Escherichia coli phages T3 and T7 (Pajunen et al., 2000(Pajunen et al., , 2001. Likewise, Yersinia pestis phage wA1122 was recently found to be a close relative of T7 (Garcia et al., 2003a). Bacteriophage...

Coxsackievirus A9 (CAV9) is a member of the human enterovirus B species within the Enterovirus genus of the family Picornaviridae. It has been shown to utilize ␣V integrins, particularly ␣V␤6, as its receptors. The endocytic pathway by which CAV9 enters human cells after the initial attachment to the cell surface has so far been unknown. Here, we present a systematic study concerning the internalization mechanism of CAV9 to A549 human lung carcinoma cells. The small interfering RNA (siRNA) silencing of integrin ␤6 subunit inhibited virus proliferation, confirming that ␣V␤6 mediates the CAV9 infection. However, siRNAs against integrinlinked signaling molecules, such as Src, Fyn, RhoA, phosphatidylinositol 3-kinase, and Akt1, did not reduce CAV9 proliferation, suggesting that the internalization of the virus does not involve integrin-linked signaling events. CAV9 endocytosis was independent of clathrin or caveolin-1 but was restrained by dynasore, an inhibitor of dynamin. The RNA interference silencing of ␤2-microglobulin efficiently inhibited virus infection and caused CAV9 to accumulate on the cell surface. Furthermore, CAV9 infection was found to depend on Arf6 as both silencing of this molecule by siRNA and the expression of a dominant negative construct resulted in decreased virus infection. In conclusion, the internalization of CAV9 to A549 cells follows an endocytic pathway that is dependent on integrin ␣V␤6, ␤2-microglobulin, dynamin, and Arf6 but independent of clathrin and caveolin-1.

A1122 is a T7-related bacteriophage infecting most isolates of

YeO3-12 is a T3-related lytic bacteriophage of Yersinia enterocolitica serotype O:3. The nucleotide sequence of the 39,600-bp linear double-stranded DNA (dsDNA) genome was determined. The phage genome has direct terminal repeats of 232 bp, a GC content of 50.6%, and 54 putative genes, which are all transcribed from the same DNA strand. Functions were assigned to 30 genes based on the similarity of the predicted products to known proteins. A striking feature of the YeO3-12 genome is its extensive similarity to the coliphage T3 and T7 genomes; most of the predicted YeO3-12 gene products were >70% identical to those of T3, and the overall organizations of the genomes were similar. In addition to an identical promoter specificity, YeO3-12 shares several common features with T3, nonsubjectibility to F exclusion and growth on Shigella sonnei D 2 371-48 (M. Pajunen, S. Kiljunen, and M. Skurnik, J. Bacteriol. 182:5114-5120, 2000). These findings indicate that YeO3-12 is a T3-like phage that has adapted to Y. enterocolitica O:3 or vice versa. This is the first dsDNA yersiniophage genome sequence to be reported.Yersinia enterocolitica is a gram-negative species which has ϳ70 serotypes, some of which are pathogenic to humans. The major pathogens in Europe, Canada, Japan, and South Africa belong to serotypes O:3 and O:9, and those in the United States belong to serotype O:8 (10) The main reservoir in nature for Y. enterocolitica is pigs (13), and infection usually occurs by ingestion of contaminated foodstuffs. Several yersiniophages have been described in the literature, but only a few have been characterized in detail. In our laboratory, a number of Yersinia-specific bacteriophages have been isolated, all originating from the raw incoming sewage of the Turku, Finland, city sewage treatment plant, and they have been used as genetic tools (44). One phage, YeO3-12, was isolated as specific to Y. enterocolitica serotype O:3 (YeO3). It infects the Escherichia coli C600 strain expressing the cloned O-antigen of YeO3, and spontaneous phage-resistant YeO3 strains were missing the O-antigen, thus indicating that Oantigen is the phage receptor (1, 2). The serotype O:3 specificity makes the YeO3-12 a potential biotechnological tool, and therefore we have initiated a detailed characterization; its biological and physical properties were reported previously (35). The dimensions of the icosahedral virion are ϳ57 nm in diameter for the head and 15 by 8 nm for the tail, and thus YeO3-12 belongs to the family Podoviridae. Based on an N-terminal sequence analysis of the major capsid protein and on its host requirements, nonsubjectibility to F exclusion, and growth on Shigella sonnei D 2 371-48, it was concluded that YeO3-12 belongs to the T7 group and that it is the first described close relative of bacteriophage T3.The T7 group comprises about 60 phages that have been divided into three subgroups on the basis of the promoter specificity of the phage-encoded RNA polymerase (RNAP).Bacteriophage T3 is the only member of one subgroup; BA14, B...

Introduction: Klebsiella is a clinically important pathogen causing a variety of antimicrobial resistant infections in both community and nosocomial settings, particularly pneumonia, urinary tract infection, and sepsis. Bacteriophage (phage) therapy is being considered a primary option for the treatment of drug-resistant infections of these types. Methods: We report the successful isolation and characterization of 30 novel, genetically diverse Klebsiella phages. Results: The isolated phages span six different phage families and nine genera, representing both lysogenic and lytic lifestyles. Individual Klebsiella phage isolates infected up to 11 of the 18 Klebsiella capsule types tested, and all 18 capsule-types were infected by at least one of the phages. Conclusions: Of the Klebsiella -infecting phages presented in this study, the lytic phages are most suitable for phage therapy, based on their broad host range, high virulence, short lysis period and given that they encode no known toxin or antimicrobial resistance genes. Phage isolates belonging to the Sugarlandvirus and Slopekvirus genera were deemed most suitable for phage therapy based on our characterization. Importantly, when applied alone, none of the characterized phages were able to suppress the growth of Klebsiella for more than 12 h, likely due to the inherent ease of Klebsiella to generate spontaneous phage-resistant mutants. This indicates that for successful phage therapy, a cocktail of multiple phages would be necessary to treat Klebsiella infections.

Staphylococcus aureus is a commensal and pathogenic bacterium that causes infections in humans and animals. It is a major cause of nosocomial infections worldwide. Due to increasing prevalence of multidrug resistance, alternative methods to eradicate the pathogen are necessary. In this respect, polyvalent staphylococcal myoviruses have been demonstrated to be excellent candidates for phage therapy. Here we present the characterization of the bacteriophage vB_SauM-fRuSau02 (fRuSau02) that was isolated from a commercial Staphylococcus bacteriophage cocktail produced by Microgen (Moscow, Russia). The genomic analysis revealed that fRuSau02 is very closely related to the phage MSA6, and possesses a large genome (148,464 bp), with typical modular organization and a low G+C (30.22%) content. It can therefore be classified as a new virus among the genus Twortlikevirus. The genome contains 236 predicted genes, 4 of which were interrupted by insertion sequences. Altogether, 78 different structural and virion-associated proteins were identified from purified phage particles by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The host range of fRuSau02 was tested with 135 strains, including 51 and 54 Staphylococcus aureus isolates from humans and pigs, respectively, and 30 coagulase-negative Staphylococcus strains of human origin. All clinical S. aureus strains were at least moderately sensitive to the phage, while only 39% of the pig strains were infected. Also, some strains of Staphylococcus intermedius, Staphylococcus lugdunensis, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus saprophyticus and Staphylococcus pseudointer were sensitive. We conclude that fRuSau02, a phage therapy agent in Russia, can serve as an alternative to antibiotic therapy against S. aureus.