Molecular Characteristics And Drug Related Resistant Molecular Markers Of Staphylococcus Aureus

Staphylococcus aureus has been one of the main pathogenic bacteria causing common human infection worldwide. Presently, the resistant mechanism of Methicillin-resistant S.aureus strains is attributed to the Staphyloccoccal Cassette Chromosome mec(SCCmec) obtained by the Methicillin-sensitive Staphylococcus aureus(MSSA). Clustered regularly interspaced short palindromic repeats(CRISPRs)is a bacterial immune system discovered recently targeted for foreign genes.CRISPR can protect the cell from attacking by invading DNA such as plasmids and phage.Objective1. To investigate the drug-resistant patterns, drug-resistant genes and molecular characteristics of Staphylococcus aureus strains.2. To understand the main prevalent spa types of S. aureus and MLST types of MRSA in order to provide foundation for the epidemiological characteristics of Staphylococcus aureus.3. To investigate the distribution of core genes(cas1 and cas2) of CRISPR and to explore preliminarily the relationship between S.aureus resistance and cas1 and cas2 gene.Methods1. Antibiotic susceptibility testing for 25 drugs were explicated based on minimum inhibitory concentration.2. A total of 11 antibiotic resistant genes(β-lactams resistance genes, aminogl ycosides-Resistance gene,macrolides-resistance genes,tetracyclines-Resistance gen e) and CRISPR-associated genes(cas1 and cas2) were amplified by traditional PCR, as well as the results of DNA sequencing were analyzed.3. The multiplex PCR were adopted to analyze the SCCmec genotype of isolated MRSA strains.4. Spa types were assigned by PCR and the amplified products were sequenced and analyzed determine the spa type of each isolate. The seven housekeeping genes of MRSA isolates were amplified and sequenced. The allelic number and profile of each gene were determined by comparing the sequences to the known alleles which deposited in the S.aureus MLST database.5. To describe the distribution of drug-resistant patterns, drug-resistant genes and cas1, cas2 by percentage. Differences in the drug-resistant patterns, drug-resistant genes between MRSA and MSSA were assessed using the chi-square test. The correlation of the drug-resistant patterns, drug-resistant genes and positive rate of cas1, cas2 was also assessed using the chi-square test or Fisher exact test. Results were obtained with SPSS software, version 21.0.6. To analyse the sequence homology and to construct the Phylogenetic trees by DNAstar7.0 software.Results1. A total of 86 S.aureus were identified, including 28 MRSA and 58 MSSA.Resistant rate of Penicillin is the highest(95.3%) and sensitive rates of 11 kinds of drugs were greater than 50%. MRSA presented higher resistant rates to all the 19 antibiotic drugs than MSSA.2. The positive rates of acc(6’)/aph(2’’), aph(3’)-III, ant(4’, 4"), ermA, ermB, ermC,msr A, tet M and tet K were 100.00%, 72.09%, 86.05%, 95.35%, 79.07%, 93.02%,58.14%, 97.67% and 81.40%, respectively. bla TEM was negative in all the 86 S.aureus strains.3. The 86 isolated strains were divided in 11 spa types. There were 4 MLST types and 3 SCCmec types among 28 MRSA strains.4. The results show that 44 strains carry cas1 gene and 30 carry cas2 gene. The differences of detection rate of cas1 gene between MRSA and MSSA as well as among SCCmec types were statistically significant(P<0.05). Resistance to 15 antibiotic drugs was associated with the positive rate of the cas1 gene. cas1 gene was also related to the positive rate of erm C、msr A and tet K and resistance to 2 antibiotic drugs was linked to the cas2 gene.Conclusions1. S. aureus is extensively drug-resistant, both drug resistant rates and positive rates of the resistant genes were high. MRSA has generally higher drug resistance rates than MSSA.2. There are 4 SCCmec gene types, including SCCmecⅢ, SCCmecⅠand SCCmecⅣ E as the three major ones. SCCmec is associated with multi-drug resistance of MRSA.3. There are 11 spa types and 4 MLST types.4. Cas1 gene and cas2 gene may be related to the resistance of S.aureus and cas1 gene may be connected with drug resistance, resistant genes and SCCmec typing.