Study On S. Aureus Bacteriophage IME-SA1and IME-SA2Genome And Novel S. Aureus Detection Method

Background and ObjectivesStaphylococcus aureus (S. aureus) is everywhere in nature, andit can be found in water, air, dust, and intestines of animals and human. S.aureus is a common human skin flora, but it is also a very importantpathogen, causing illnesses ranging from minor skin infections tolife-threatening diseases such as septicemia and sepsis. Like humans,livestock are susceptible to S. aureus, resulting in bovine mastitis andchicken arthritis, which seriously threatens the food safety and causesfood poisoning and significant economic losses. Notably, the increasingincidence of antibiotic-resistant S.aureus strains, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistantStaphylococcus aureus (VRSA), has attracted more attention, becauseS.aureus is one of the most common causes of nosocomial infections.Bacteriophages are widespread in nature. Where there is thepresence of bacteria, such as soil, water, etc., there is the presence of corresponding bacteriophages. The latest study of phage andphage-derived lysozymes in the treatment of bacterial infection revealsthat they are the potential substitutes for antibiotics to control or preventmulti-drug resistant bacterial infections. As the natural bacterium killer,phage owns incomparable advantages over antibiotics in the treatment ofbacterial infections especially multi-drug resistant infections:Bacteriophage is capable of self-replication in the host, and therefore verysmall doses of phage preparations can be used for therapeutic purposes；Bacteriophage specificity is strict. It can only lyse its host bacterium orwithin the same species of bacteria and have little impact on the body’sother normal microbial flora, and will not infect human cells, so will notcause any adverse side effects; Bacteriophages are unlikely to cause anallergic reaction when it is used in patients allergic to antibiotics;Bacteriophages can be used alone or in combination with otherbactericidal substances; Bacteriophages can mutate along with thepathogens, producing new phage which can crack the mutant bacteria;Bacteriophage lytic enzymes also have many advantages: They can behighly expressed by genetic engineering and can be purified, and havestrong substrate specificity; Compared with the bacteriophage,Bacteriophage lytic enzymes do not require infection and replication, andcan be used for faster sterilization; Bacteriophage lytic enzymes havedifferent binding domains and the catalytic domains, which are used inthe binding and cleaving of the cell wall respectively, and therefore it isnot easy to cause bacteria resistance.We determined genomic characteristics of two isolated strainsof S. aureus phages and carried homologous recombination modification of the genome of one of the isolated strains. Also, we established a newS.aureus detection method. This work laid the foundation forStaphylococcus aureus phage application and rapid detection ofStaphylococcus aureus.Methods and ResultsThe genomic DNA of Staphylococcus aureus phage IME-SA1and IME-SA2were sequenced by Ion Torrent sequencing method andthen the whole genome sequence was assembled. We conducted genomicanalysis and found that there are many repeat sequences in the genomictermini, and these repeats are variable, which leads to different PCRproduct length. Culture of phage clones with long terminal repeatsequences resulted in phage clones with short terminal repeats, while nophage clones with long terminal repeat were generated in the culture ofphages with short terminal repeats. Meanwhile, analysis of genomecharacteristics and biological characteristics of two strains ofbacteriophages, IME-SA1and IME-SA2, found that the two strains ofphage had very high genome homology but their lysis spectrum is verydifferent. Then we designed a homologous recombination experimentwhich we called “genome shuffling”, and with the “genome shuffling”the narrow spectrum phages were successfully converted into widespectrum phages.In order to establish a new specific quantitative detectionmethod of Staphylococcus aureus, we obtained from NCBI the gene oflysostaphin which can specific lyse Staphylococcus aureus; then weredesigned and made codon optimization of the lysostaphin gene, whichmakes it easier to carry out soluble expression in E. coli; thirdly, we synthesized lysostaphin gene and inserted it into prokaryotic expressionvector pQE30; finally, we succeeded in expressing the recombinantlysostaphin in E.coli expression system, and used recombinantlysostaphin combined ATP bioluminescence technology to establish aspecific quantitative detection approach of Staphylococcus aureus.ConclusionWith the rapid growing Staphylococcus aureus resistanceagainst antibiotics and pathogenicity, it is imperative to explore newtreatment alternative to antibiotics and to establish methods for rapiddetection of Staphylococcus aureus. Our research established a “genomeshuffling” technique based on homologous recombination, and by suingthis technique, efficiently converted narrow spectrum Staphylococcusaureus phages into wide spectrum Staphylococcus aureus. This study alsodeveloped a novel quantitative detection method by using recombinantlysostaphin combined with ATP chemiluminescence, which laidfoundation for the application of Staphylococcus aureus phage anddetection of Staphylococcus aureus.