Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 1/10/2008
Publication Date: 7/29/2008
Citation: Donovan, D.M., Abaev, I., Schischkova, N., Myakinina, V., Korobova, O., Pecherskich, E., Kopylov, P., Kiseleva, N. 2008. Genotyping Staphylococcus aureus allows one to identify bacteriophages harboring unknow endolysins. Meeting Abstract.
Interpretive Summary: The search for new bacteriophage endolysins is important in light of the need for novel antimicrobials against multi-drug resistant strains (methicillin resistant staphylococci). Most known genomes of Staphylococcus aureus strains contain two or more prophage genomes. We expect that S. aureus strains not harbouring the most common prophage genomes (phi11, phi 12 and phi 13 endolysins, or other known endolysins) are candidate sources for novel endolysins. Based on RFLP patterns of the gene coa from 167 clinical isolates of S. aureus, as well as the phi11, phi12 and phi13 endolysin genes, 42 different genotypes have been identified. 23 genotypes having been selected for further study as potentially harboring novel endolysins based on negative PCR results with primers specific for known endolysin genes. Mitomycin C induction of these 23 isolates have yielded nine lysates. The lytic effect of the crude phage lysates on exponentially growing S. aureus BAC170190 was assessed by turbidometry and zymogram. Crude lysates of three phage isolates appeared lytic for living S. aureus. Candidate lytic proteins near the size of 52 kD (expected of endolysins) have been observed in zymograms. Hopefully these efforts will identify novel phage endolysins for use in developing novel antimicrobials for multi-drug resistant staphylococci.
Technical Abstract: Background and Objectives. The search of new bacteriophage endolysins is important in view of the ability of staphylococci to acquire resistance to commonly used antibiotics. Most known genomes of Staphylococcus aureus strains contain two or more temperate bacteriophages. For example, the chromosome of S. aureus 8325 contains genomes of three temperate bacteriophages: phi 11, phi 12 and phi 13. Among all bacteriophage endolysins of S. aureus, lysis genes of these bacteriophages, i.e. phi11, phi 12 and phi 13 endolysin genes, are most common. We expect that S. aureus strains not harbouring phi11, phi 12 and phi 13 endolysins, or other known endolysins, would carry novel perspective endolysins. The objective of the research was to test a screening system to identify novel phage endolysins. Methods and Results. 167 clinical isolates of S. aureus from hospitals of Moscow and Yaroslavl’ (Russia) have been studied by restriction fragment length polymorphism (RFLP) of the coagulase (coa) gene, as well as by PCR with specific oligonucleotide primers for amplification of phi11, phi12 and phi13 endolysin genes. As a result of RFLP of the coagulase gene, 21 different genotypes have been identified. The isolates were typed by PCR using our own phi11, phi12 and phi13 endolysin primers. Based on RFLP patterns of the gene coa, as well as on presentation of phi11, phi12 and phi13 endolysin genes, 42 different genotypes among 167 isolates of S. aureus have been identified. Of them, four genotypes have demonstrated simultaneously negative PCR with phi11, phi12 and phi13 endolysin primers. 19 genotypes carried one of phi11, phi12 and phi13 endolysin genes only. In total, 23 genotypes have been selected for further identification of new endolysins. Selected isolates of each genotype were used for prophage induction with mitomycin C. As a result of the prophage induction we have obtained nine phage isolates from strains of different genotypes. Phage DNAs have been tested by our PCR approach allowing us to identify and to rule out known endolysin genes. Of nine phage isolates, eight were revealed as negative in PCR with LysK-, phi68P-, TWORT-, phi37-, phi2638a-, phiEW-specific endolysin primers. These phage isolates were used to prepare crude phage lysates. The lytic effect of the crude phage lysates on exponentially growing S. aureus BAC170190 was assessed by turbidometry and zymogram. Crude lysates of three phage isolates appeared lytic for living cells of S. aureus. In the zymogram, selected phage lysates formed a lysis zone (about 52 kDa) that was absent in control. DNAs of these phages were isolated and purified for the subsequent restriction analysis using AvaI and EcoRV endonucleases. Analyzed RFLP patterns of phage DNA differed. Conclusions. The proposed approach may be perspective for identifying novel endolysin genes. Our further research will be focused on identification of phage proteins possessing lytic activity against S. aureus. Acknowledgements. This research is supported by ARS-ISTC #499/3571 Project Grant.