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.

Staphylococcus aureus produces biofilm and this mode of colonization facilitates infections that are often difficult to treat and engender high morbidity and mortality. We have exploited bacteriophage Mu transposition methods to create an insertional mutant library in a highly biofilm-forming S. aureus clinical isolate. Our screen identified 38 insertions in 23 distinct genes together with one intergenic region that significantly reduced biofilm formation. Nineteen insertions were mapped in loci not previously known to affect biofilm in this organism. These include insertions in codY, srrA, mgrA, and fmtA, a putative DEAD-box helicase, two members of the zinc-metallo-␤ lactamase/␤-CASP family, and a hypothetical protein with a GGDEF motif. Fifteen insertions occurred in the icaADBC operon, which produces intercellular adhesion antigen (PIA) and is important for biofilm formation in many strains of S. aureus and Staphylococcus epidermidis. Obtaining a high proportion of independent Em-Mu disruptions in icaADBC demonstrated both the importance of PIA for biofilm formation in this clinical strain and the strong validation of the screening procedure that concomitantly uncovered additional mutants. All non-ica mutants were further analyzed by immunoblotting and biochemical fractionation for perturbation of PIA and wall teichoic acid. PIA levels were diminished in the majority of non-ica insertional mutants. Three mutant strains were chosen and were functionally complemented for restored biofilm formation by transformation with plasmids carrying the cloned wild-type gene under the control of a xylose-inducible promoter. This is a comprehensive collection of biofilm-defective mutants that underscores the multifactorial genetic program underlying the establishment of biofilm in this insidious pathogen.

Eukaryotic organisms are continuously exposed to bacteriophages, which are efficient gene transfer agents in bacteria. However, bacteriophages are considered not to pass the eukaryotic cell membrane and enter nonphagocytic cells. Here we report the binding and penetration of Escherichia coli PK1A2 bacteriophage into live eukaryotic neuroblastoma cells in vitro. The phage interacts with cell surface polysialic acid, which shares structural similarity with the bacterial phage receptor. Using fluorescence and electron microscopy, we show that phages are internalized via the endolysosomal route and persist inside the human cells up to one day without affecting cell viability. Phage capsid integrity is lost in lysosomes, and the phage DNA is eventually degraded. We did not detect the entry of phage DNA into the nucleus; however, we speculate that this might occur as a rare event, and propose that this potential mechanism could explain prokaryote–eukaryote gene flow.