Molecular epidemiology and characteristic of virulence gene of community-acquired and hospital-acquired methicillin-resistant Staphylococcus aureus isolates in Sun Yat-sen Memorial hospital, Guangzhou, Southern China

Sun Yat-Sen Memorial Hospital is also known as the Second Affiliated Hospital of Sun Yat-Sen University. With more than 4200 staff and 2200 inpatient beds available, the hospital performs over 50,000 inpatient operations, discharges about 80,000 inpatients, and handles more than 3 million outpatient visits annually. A hospital-based retrospect study of 587 inpatients hospitalized for < 48 h infected by Staphylococcus aureus, included 67 inpatients with MRSA infection in Sun Yat-Sen Memorial Hospital between Jan.1, 2006 and Dec.31, 2011 were carried out. Five patients with MRSA infection were eliminated because the strains can’t recovery. Therefore, a total of 62 compete surveys were obtained, with an efficiency rate of 92.5 %. Based on the US Centers for Disease Control and Prevention criteria, we roughly defined HA-MRSA infections as those patients who fit any one of the following situations: surgery, haemodialysis, residence in a long-term care facility or treatment in hospital during the previous year, presence of a permanent catheter or percutaneous device at the time of culture, or previous isolation of MRSA. If the patients did not meet any of the above risk factors, had an infection at the time of admission, and the culture of the infection was taken within 48 h, then the infection was considered as CA-MRSA infections. Clinical data related to the type of infection and outcome (death, improvement, and relapse) of the patients, as well as other data, were collected for up to a period of 30 days after the diagnosis of infection by MRSA. All patients, parents or guardians signed informed consent approving the use of their specimen samples for research purposes and the study was approved by the Ethics Committee of Sun Yat-Sen Memorial Hospital. Ethical committee’s reference number: [2016]伦备第 (11)号 (Additional file 1).

According to the above definition, 39 HA-MRSA isolates and 23 CA-MRSA isolates were collected from the 62 patients. To avoid sample duplication, isolates that were consecutively isolated from the same individual were excluded. All isolates underwent phenotypic identification using the VITEK® 2 microbial identification system (bioMérieux, Marcy l’Etoile, France) according to the manufacturer’s instructions. Antibiotics used for susceptibility testing included penicillin, erythromycin, clindamycin, cefuroxime, ceftriaxone, cefoxitin, gentamicin, rifampicin, quinupristin/dalfopristin, trimethoprim/sulfamethoxazole, cefotaxime, tetracycline, imipenem, teicoplanin, vancomycin, ciprofloxacin, and moxifloxacin. Susceptibilities were determined using the disk diffusion method in accordance with the performance standards for antimicrobial susceptibility testing, 25^rd informational supplement (M100-S25), recommended by the Clinical and Laboratory Standards Institute. The inducible clindamycin resistance was determined by D-test. All disks were obtained from Oxoid Ltd (Oxoid, Basingstoke, England), and S. aureus ATCC 25923 and ATCC29213 were used as the quality control strain.

Bacterial DNA was extracted using DNA extracted kit(Tiangen Biotech, Beijing) with lysostaphin according to the manufacturer’s instructions. The sequence type (ST) was characterized by MLST, and the products of seven house-keeping gene fragments were sequenced (Sangon Biotech, Shanghai) and compared with allele profiles from database of S. aureus (www.​mlst.​net/​). SCCmec typing of MRSA strains was conducted by multiplex PCR method as previously described [13]. The genetic relationship of the isolates was determined by PFGE as previously described [18]. Cluster analysis was performed with the software program BioNumerics 5.0 (Maths, Belgium) using the Dice coefficient and the unweighted pair group method (UPGMA). The isolates with the similarity over 75 % were clustered in patterns. The presence of the genes that code for PVL (lukF-PV and lukS-PV), twelve staphylococcal enterotoxins (sea ~ see, seg ~ sej and sem-seo), two exfoliative toxins (eta and etb), four hemolysins (α-hemolysin, β-hemolysin, δ-hemolysin and γ-hemolysin), and the toxic shock syndrome toxin (tsst-1) and mecA gene was determined using PCR as previously reported methodologies, with some modifications [14, 15].

In descriptive statistics, frequency and proportions were calculated for categorical variables. The frequency of SCCmec type, specimen type and virulence genes were treated as categorical variables. The chi-square or two-sided Fisher’s exact test was used to discriminate whether the distributions of the studied genes or types were significantly different between HA-MRSA and CA-MRSA. The only continuous variable, age, was transformed into a categorical variable using the quartiles of the frequency distribution (~18, 19–60, >60). It was considered statistically significant if the two-side P-value < 0.05. All statistical analyses were carried out using SPSS 19.0 for Windows (IBM). All susceptibility data and molecular test results were analyzed using WHONET software, version 5.6.