Study On The Resistance Spectrum, Resistance Mechanism And Molecular Epidemiology For Staphylococcus Aureus

Staphylococcus aureus is one of the most common pathogenic bacteria. It is widely distributed in the hospital environment and is highly pathogenic. It can cause both common and severe infection, including suppurative skin inflammation, otitis media, arthritis, pneumonia, osteomyelitis, endocarditis, meningitis, sepsis, etc. This bacterium can survive for a long time in hospital environment because it is highly adaptive and can easily develop antibiotics resistance and disinfectant resistance. Thus, S. aureus is one of the most common bacteria in nosocomial infection.In recent years, as a result of the abuse of antibiotics, S. aureus has become more resistant to antibiotics and has greater hazard to people’s health. This phenomenon has attracted the worldwide attention. The main features of the S. aureus resistance are as follows: (1) Incidence of hospital-acquired methicillin-resistant S. aureus (HA-MRSA) has steadily increased and shows multi-drug resistance. (2) MRSA has disseminated from hospital to community. Community acquired meticillin resistant S. aureus (CA-MRSA) has more virulence than HA-MRSA and can infect healthy children and adults; (3) Emergence of S. aureus with reduced vancomycin susceptibility (SA-RVS) becomes a worrying phenomenon, which can cause treatment failure and an increased mortality.In this study, the drug resistant spectrum and resistant genes of S. aureus were detected and the resistance mechanism and molecular epidemiological characteristics were studied. The results will provide experimental evidence for the reasonable prevention and treatment for multi-drug resistant of S. aureus. The main contents are divided into four parts as follows: Part I: Analysis for the resistance spectrum phenotype and related genes of S. aureusObjective: To understand the antimicrobial resistance spectrum and resistance mechanism of S. aureus isolated in Hefei, Anhui, China.Methods: The susceptibility test of S. aureus strains to 13 antibiotics was carried out by using Vitek32 instrument. Resistance genes, including mecA, aac (6 ’) / aph (2’’), aph (3’)-III, ermA, ermC, tetM, tetK were detected by PCR. SCCmec types were determined by amplifying the key genetic elements using multiplex PCR.Results: (1) The resistant proportion of S. aureus isolated from inpatients to cefazolin, penicillin, clindamycin, erythromycin, oxacillin, gentamicin, tetracycline, and sulfonamides was significantly higher than that of strains isolated from outpatients. In the inpatients, methicillin resistance rate among S. aureus increased obviously from 2004 to 2006, which was 40.4%, 73.6% and 69.4%, respectively. And 83.9% of MRSA showed resistance to more than three types of antibiotics (Multi-drug resistant, MDR). (2) The coincidence rates of mecA gene detection with penicillin, ampicillin, oxacillin, cefazolin resistant, ermA/ermC genes with erythromycin and clindamycin resistance, tetM/tetK genes with tetracycline resistance, (6 ’) / aph (2’’) and aph (3’)-III genes with gentamicin were 64.71%, 64.22%, 94.11%, 99.50%, 88.24%, 89.22%, 77.45% and 77.21%, respectively. Positive predictive value was 100%, 100%, 99.6%, 93.3%, 98.60%, 91.58%, 96.59%, and 95.83%, respectively. Negative predictive value was 6.41%, 7.69%, 95.51%, 99.36%, 64.23%, 83.74%, 62.39% and 64.17%, respectively. (3) Among the 114 MRSA strains isolated in Hefei, the SCCmec types were as follows: 34(24.1%) were SCCmec type II, 102 (72.3%) were type III, and 5(3.5%) were type IV. SCCmec type IV was non-multi-resistant, and it was only resistant toβ-lactam antibiotics. Both type II and III were multi-drug resistant, however type III had higher resistant proportion than type II. Conclusion: (1) Sinec the resistant proportion of S. aureus isolated from inpatients is higher, it is necessary to take susceptibility test as early as possible so as to derect the selection of reasonable antibiotics; However, S. aureus isolated from outpatients is less resistant to oxacillin, cefazolin, levofloxacin, gentamicin and sulfonamides, then those antibiotics can be used in experience treatment. (2) The resistant genes detected in this study show higher coincidence rates with resistant spectrum of S. aureus. It is considered that mecA gene encoding PBP2a, ermA/ermC genes encoding inactivating enzymes, aac (6 ’) / aph (2’’) gene encoding acetyltransferase, tetK gene encoding efflux pump system and tetM gene encoding ribosomal protection protein are the main mechanism of S. aureus resistance toβ-lactam antibiotics, macrolide/clindamycin, aminoglycoside antibiotics and tetracycline. The higher positive predictive value of resistant genes detection indicates that S. aureus carried resistance genes can express as drug-resistant phenotype. The lower negative predictive value suggests that in addition to the main resistance mechanisms, there are other resistance mechanisms. (3) Multi-drug resistance of MRSA is related to the structure and molecular weight of SCCmec type. SCCmec typeⅢhas the most complex gene structure, the largest molecular weight, and it also has the highest resistant proportion in all SCCmec types. On the contrary, SCCmec type IV has the relatively simple structure and smaller molecular weight, and it was only resistant toβ-lactam antibiotics.Part II: Rapid detection of MRSA directly from clinical samples by an immunomagnetic bead-multiplex PCR assayObjective: To develop a method to detect MRSA rapidly and evaluate its sensitivity and specificity.Methods: An immunomagnetic bead-multiplex PCR (IB-MPCR) assay was developed. With this method, immunomagnetic bead was used to capture MRSA and multiplex PCR was used to identify bacterial species and antibiotic resistance. This method was also compared with some other methods to evaluate its clinical efficacy.Results: Comparing with“the gold standard methods”, the immunomagnetic beads- multiple PCR method has a high sensitivity (100%) and specificity (96.8%); detection time was significantly shorter (6 ~ 8h); but the cost of testing is higher.Conclusion: IB-MPCR is a rapid, sensitive and specific method for detecting MRSA. The only disadvantage is that it’s more expensive. How to reduce the testing cost will be studied in the future.Part III: Detection methods and resistance mechanisms of SA-RVSObjective: To investigate the prevalence of SA-RVS and reveal the resistance mechanism. To discuss the impact of resistance mutations on conventional bacterial identification and susceptibility tests.Methods: 331 S. aureus strains were screened by using brain heart infusion agar containing 4μg of vancomycin/ml. Screen-positive isolates were confirmed by using population analysis profiles. An h-VRSA strain (No. 10827) was picked and was exposed to gradually increasing concentrations of vancomycin and was induced to a series of vancomycin-resistant mutants. These mutants were used to evaluate methods of bacterial identification and susceptibility test.Results: Among 331 S. aureus, 14 strains were detected as h-VRSA (4.23%). The size and dyeing of h-VRSA were not uniform. Scanning electron microscopic photo showed that the cell wall surface of h-VRSA strain was rough with irregular uplifted spots. Transmission electron microscopy showed that the cell wall of h-VRSA was much thicker than that of its parental strain (10827-P). The size of colony was smaller. The hemolytic zones became narrowed or disappeared. The color changed from yellow to grey or colorless. The coagulase test and mannitol utilization became negative. By using the automated instrument for bacteria identification, out of the 14 hetero-VRSA strains, 11 had been wrongly identified as Staphylococcus haemolyticus. All of h-VRSA, VISA and VRSA strains were incorrectly judged as“susceptive”by disk diffusion method and Vitek32 instrument, which was considered as“very major discrepancy”.Conclusion: The h-VRSA has emerged in HeFei area. The resistant mechanism of h-VRSA is cell wall thickening, which may change the biological characteristics and then lead to miss identification and error susceptibility test result detected by clinical conventional methods. This scenario should bring to the attention.Part IV: Risk factors and molecular epidemiology of MRSA, h-VRSAObjective: To investigate the infection risk factors and molecular epidemiology background of MRSA, h-VRSA, and then develop the effective prevention and control measures.Methods: The risk factors associated with infection of MRSA and h-VRSA were established from the patients’medical files. MLST were used to assess the genetic relatedness of 95 S. aureus isolates (MSSA, MRSA, h-VRSA). RAPD(Random Amplifed Polymorphic DNA)was used to study the genetic homology of 31MRSA isolated from ICU.Results: (1) MRSA was found significantly more often in older patients (85.5%) than in children (16.3%). MRSA was primarily isolated from samples of sputum (88.1%) and pus (68.3%) and from wards of ICU (methicillin resistance rates was 90.9%). The risk factors associated with the infection of MRSA were invasive treatment, long-term hospitalization, and long-term combination using antibiotics, etc. (2) In 79 samples collected from 28 doctor-patient staffs, 24(30.4%) MRSA strains were identified. In 52 samples collected from the environment, 7(13.5%) MRSA strains were identified. And in 15 samples collected from air, no MRSA strain was identified. In all 31 MRSA strains, 20 strains belong to the same type detected by RAPD typing. (3) MLST typing showed that the prevalence genotypes of MSSA were differ from that of MRSA; and the prevalence genotype of h-VRSA was the same with MRSA. Among 62 MRSA, 27strains belong to the ST239-MRSA-III (43.5%) and 19 strains belong to the ST5-MRSA-II (30.6%); Among 13 h-VRSA, 7 strains belong to the ST239-MRSA-III (53.8%).Conclusion: (1) The MRSA monitoring should be strengthened on the elderly and patients with risk factors. (2) In the ICU, there is an MRSA clone spread between health care professionals, patients and the environment. (3) Most of MRSA infection among inpatients is from nosocomial infection of MRSA epidemic strains, not from MSSA. However h-VRSA infection originates in HA-MRSA, may be the result of antibiotic selective pressure.