Genomic analysis of Staphylococcus phage Stau2 isolated from medical specimen.

Stau2 is a lytic myophage of Staphylococcus aureus isolated from medical specimen. Exhibiting a broad host range against S. aureus clinical isolates, Stau2 is potentially useful for topical phage therapy or as an additive in food preservation. In this study, Stau2 was firstly revealed to possess a circularly permuted linear genome of 133,798 bp, with low G + C content, containing 146 open reading frames, but encoding no tRNA. The genome is organized into several modules containing genes for packaging, structural proteins, replication/transcription and host-cell-lysis, with the structural proteins and DNA polymerase modules being organized similarly to that in Twort-like phages of Staphylococcus. With the encoded DNA replication genes, Stau2 can possibly use its own system for replication. In addition, analysis in silico found several introns in seven genes, including those involved in DNA metabolism, packaging, and structure, while one of them (helicase gene) is experimentally confirmed to undergo splicing. Furthermore, phylogenetic analysis suggested Stau2 to be most closely related to Staphylococcus phages SA11 and Remus, members of Twort-like phages. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis showed 14 structural proteins of Stau2 and N-terminal sequencing identified three of them. Importantly, this phage does not encode any proteins which are known or suspected to be involved in toxicity, pathogenicity, or antibiotic resistance. Therefore, further investigations of feasible therapeutic application of Stau2 are needed.

Wide host range and strong lytic activity of Staphylococcus aureus lytic phage Stau2.

In searching for an alternative antibacterial agent against multidrug-resistant Staphylococcus aureus, we have isolated and characterized a lytic staphylophage, Stau2. It possesses a double-stranded DNA genome estimated to be about 134.5 kb and a morphology resembling that of members of the family Myoviridae. With an estimated latency period of 25 min and a burst size of 100 PFU/infected cell, propagation of Stau2 in liquid culture gave a lysate of ca. 6 × 10(10) PFU/ml. It was stable at pH 5 to 13 in normal saline at room temperature for at least 4 weeks and at -85°C for more than 2 years, while 1 × 10(9) out of 2 × 10(12) PFU/ml retained infectivity after 36 months at 4°C. Stau2 could lyse 80% of the S. aureus isolates (164/205) obtained from hospitals in Taiwan, with complete lysis of most of the isolates tested within 3 h; however, it was an S. aureus-specific phage because no lytic infection could be found in the coagulase-negative staphylococci tested. Its host range among S. aureus isolates was wider than that of polyvalent phage K (47%), which can also lyse many other staphylococcal species. Experiments with mice demonstrated that Stau2 could provide 100% protection from lethal infection when a multiplicity of infection of 10 was administered immediately after a challenge with S. aureus S23. Considering these results, Stau2 could be considered at least as a candidate for topical phage therapy or an additive in the food industry.

Importance of amino acid alterations and expression of penicillin-binding protein 5 to ampicillin resistance of Enterococcus faecium in Taiwan.

The importance of the amino acid sequence in the C-terminal domain of penicillin-binding protein 5 (PBP5) and the levels of PBP5 expression to ampicillin resistance of Taiwan clinical isolates of Enterococcus faecium were studied. Sequence data revealed the existence of 12 amino acid sequence variants within the C-terminal domain of PBP5 in the 33 tested isolates (ampicillin minimum inhibitory concentrations (MICs) 1 mg/L to >256 mg/L). Western blot analyses of the levels of PBP5 showed that, with few exceptions, lower amounts of PBP5 were present in the susceptible strains than in the resistant strains. More importantly, a significant correlation (P=0.004, Fisher's exact test) between the expression of PBP5 and ampicillin resistance was detected. Point mutations in PBP5, including addition of aspartic acid or serine after position 466 and change of methionine to alanine or threonine at position 485, alanine or isoleucine to threonine at position 499 and glutamate to valine at position 629, were found to be significantly associated with ampicillin resistance. A significant correlation was obtained for the combined mutation (alleles 10 and 11), suggesting that combined mutation of PBP5 can be a marker for ampicillin resistance of E. faecium.