Biological Characteristics And Resistance Mechanisms Of Vancomycin Intermediate Staphylococcus Aureus

Staphylococcus aureus is an important pathogen which causes human and animalinfections, and is frequently found in the human skin and nasal cavity. S. aureus can cause awide range of diseases, from minor skin and soft tissue purulent infections to bacteremia,meningitis, osteomyelitis and other serious systemic infections, which can be widelydistributed and spread in a hospital environment. Since penicillin used clinically in1940s,infections of S. aureus had been controlled a lot. However, with the extensive application ofpenicillin, the emergence of S. aureus strains to produce penicillinase, showing resistance topenicillin. So dimethoxyphenyl penicillin, namely methicillin, began to be used clinically in1959, two years later, the first case of methicillin-resistant S. aureus (MRSA) was isolatedin the United Kingdom. So far, MRSA infections have been found throughout the world,and MRSA becomes the most important pathogen associated with nosocomial infections.The MRSA strains offen exhibit multiple drug resistance and have strong abilities to adaptto the the environment, which make it be related to particularly serious infections. Theinfections caused by MRSA, HBV, and HIV have been considered the most three refractoryinfectious diseases worldwide. Glycopeptide antibiotic vancomycin is the final drugselected for treatment of MRSA infections, however, with increases in the clinicalapplication of vancomycin, S. aureus with reduced sensitivity to vancomycin has emergedin recent years. Heterogeneous vancomycin-intermediate S. aureus (hVISA),vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA)have been reported in the domestic and international, which make the treatment of clinicalMRSA infections face serious challenges. However, the mechanisms for S. aureus strains todecrease susceptibility to vancomycin have not been fully elucidated, several proposalsassociated with the reduced vancomycin sensitivity of MRSA have been established, including:(i) cell wall thickening;(ii) changes in penicillin-binding proteins;(iii) decreasedautolysis ability;(iv) mutations and changes in transcript levels; and (v) resistance genetransfer. In this study, a vancomycin intermediate S. aureus strain (XN108) was isolated andcharacterized. The biological characteristics and the possible vancomycin resistancemechanisms of XN108were also invested. The main results are as following:1. Bacterial isolation, identification and susceptibility testing.XN108was isolated from a steam burned patient with wound infection,and identifiedstrains as an MRSA by adopted automatic identification system and specific genes detection.The antibiogram determination revealed that XN108was muiltiple drug resistane and has avancomycin MIC of12μg/ml tested by agar dilution method, E-test strip method andpopulation analysis. The results demonstrated XN108is a VISA according to the ClinicalLaboratory Standard Insititute2011.2. Molecular typing of XN108.The molecular typing methods, such as multilocus sequence typing (MLST),pulsed-field gel electrophoresis (PFGE), X region encoding staphylococcal protein A (spatyping), staphylococcal cassette chromosome mec typing (SCCmec typing)were applied toXN108, and confirmed that XN108is an ST239-MRSA-SCCmec type III MRSA with a spatype of t037, the first VISA identified from the most prevalent clone (ST239-MRSA-III) inour country,.3. Biological characteristics determination of XN108.Growth curves showed XN108started growth about3.5h at the beginning of theculture growth. XN108displayed decreased hemolytic activity. Compared with VSSAATCC25923, the autolysis capacity of XN108was declined. All these biologicalcharacteristics were similar with the VISA reference strain Mu50.4. The mechanisms of XN108for vancomycin resistanceWe failed to detect the vancomycin resistance genes, such as vanA,vanB or vanC, inXN108with PCR using genomic DNA as template. Transmission electron microscopyobserved that the cell wall of XN108was more than two times thicker than that of VSSAATCC25923. The expression levels of cell wall biosynthetic genes, including pbpB, glmUand murG, in XN108cultured in the medium with8g/ml vancomycin were about sixtimes higher than that of ATCC25923. Congo red sensitivity test showed XN108is sensitive to Congo red.A transposon mutant library of XN108was constructed using a plasmid pID408carrying the transposon TN917, and the mutants were screened with a certain concentrationof vancomycin. Fortunately, we got three mutant strains that can be stably cultured in themedium containing16μg/ml，24μg/ml, and36μg/ml vancomycin, respectively. Thesemutant strains derived from XN108were designated as XN16+, XN24+, and XN36+,respectively.5. Genome sequencing of XN108and the related mutants.The whole genome sequences of XN108, XN16+, XN24+, and XN36+were carried outusing Ion torrent PGM sequencer with a300-bp single-end library and a4-kb matepairlibrary, The resulting sequences were de novo assembled in the Roche454Newbler2.5assembler, and6primary scaffolds were connected according to matepair relationships andthe internal gaps were filled using the GapFiller version1.11. The complete genome of S.aureus strain XN108contains a circular3,052,037bp chromosome with a32.76%G+Ccontent and a circular plasmid of31,500bp in size. Comparisons with the three mutantgenomes revealed that all the sequenced strains have a tranked graR gene, which may resultin the intermediate vancomycin resistance of XN108.