目的 研究黄连水煎剂对体外建立的金黄色葡萄球菌生物膜(bacterial biofilm,BF)的作用,及黄连水煎剂联合苯唑西林钠对金黄色葡萄球菌作用。方法 由广西医科大学第一附属医院检验科提供金黄色葡萄球菌菌株34134,采用二倍稀释法分别测出黄连水煎剂和苯唑西林钠对金黄色葡萄球菌的最低抑菌浓度(minimum inhibitory concentration MIC),在体外使用腹膜透析管建立金黄色葡萄球菌生物膜模型,加入不同浓度的黄连水煎剂和苯唑西林钠作用3天和7天后,琼脂平板菌落计数法评估细菌粘附腹透管的能力,结晶紫染色法行载体表面BF半定量,银染法快速鉴定BF。结果 黄连水煎剂MIC为16 mg/mL,苯唑西林钠MIC为4 μg/mL,3天BF模型中,1/2MIC黄连水煎剂、1/4MIC黄连水煎剂、1/8MIC黄连水煎剂、1/16MIC黄连水煎剂、1/2MIC苯唑西林钠、1/4MIC苯唑西林钠作用下的金黄色葡萄球菌的菌落计数分别为(39.333 3±3.994 2)×10⁶ CFU/mL、(52.366 7±3.537 8)×10⁶ CFU/mL、(81.266 7±3.341 8)×10⁶ CFU/mL、(90.900 0±2.040 1)×10⁶ CFU/mL、(50.866 7±2.208 6)×10⁶ CFU/mL、(77.666 7±2.880 7)×10⁶ CFU/mL,空白组的菌落计数为(92.033 3±3.890 6)×10⁶ CFU/mL,与空白组相比较,1/2MIC黄连水煎剂,1/4MIC黄连水煎剂、1/8MIC黄连水煎剂、1/2MIC苯唑西林钠、1/4MIC苯唑西林钠的差异有统计学意义(P<0.001),1/16MIC黄连水煎剂与空白组比较,差异无统计学意义(P=0.173)。7天的BF模型中,1/2黄连水煎剂比1/2苯唑西林钠抑制金黄色葡萄球菌生物膜形成的作用更强(P<0.001),结晶紫、银染也得到相似的结果,进一步的研究发现在1/16MIC黄连水煎剂的作用下,加入不同浓度的苯唑西林钠,此时苯唑西林钠的最低抑菌浓度为0.25 μg/mL。结论 黄连水煎剂能抑制金黄色葡萄球菌生物膜的形成,并且在苯唑西林钠联合黄连水煎剂时,能有效地提高苯唑西林钠的疗效,为治疗金黄色葡萄球菌导致的腹膜透析相关性腹膜炎提供新的治疗方案,同时也提高了抗生素的疗效,为临床上减少耐药菌的产生提供了帮助。

Objective To study the effect of Coptis chinensis decoction on bacterial biofilm(BF)in vitro and the effect of Coptis chinensis decoction combined with oxacillin sodium on Staphylococcus aureus. Methods Staphylococcusaureus strain 34 134 was provided by the First Affiliated Hospital of Guangxi Medical University. Using doubling dilution method to measure the minimum inhibitory concentration of Coptis decoction and Oxacillin sodium on Staphylococcus aureus,respectively. In vitro, peritoneal dialysis tube was used to establish Staphylococcus aureus biofilm model after adding different concentrations of Coptis decoction and Oxacillin sodium for 3 days and 7 days, we used Agar plate count method to assess bacterial adhesion on the dialysis tube, crystal violet staining and semi quantitative method to measure the BF of the surface of the carrier and silver staining(light microscopy) rapid identification of BF. Results The MIC of Coptidis decoction was 16 mg/ml, and that of Oxacillin sodium was 4 μg/mL. In the BF model after 3 days, the colony counting of bacteria of 1/2MIC Coptis decoction, 1/4MIC Coptis decoction, 1/8MIC Coptis decoction,1/16MIC Coptis decoction, 1/2MIC Oxacillin sodium,1/4MIC Oxacillin sodium were(39.333 3+3.994 2)×10⁶(52.366 7+3.537 8),CFU/mL×10⁶, CFU/mL×10⁶(81.266 7+3.341 8), CFU/mL(90.900 0+2.040 1)×10⁶,CFU/mL(50.866 7+2.208 6)×10⁶, CFU/mL(77.666 7+2.880 7)×10⁶ CFU/mL, colony counting of blank group was(92.033 3+3.890 6)×10⁶ CFU/mL, respectively. Compared with the blank group, 1/2MIC Coptis decoction, 1/4MIC Coptis decoction,1/8MIC Coptis decoction,1/2MIC Oxacillin sodium,1/4MIC Oxacillin sodium all had differences in statistical significantce(P=0.001), but 1/16MIC Coptis decoction had no statistically significant(P=0.173). In the BF model after 7 days, colony counting bacteria, crystal violet and silver staining showed similar results. Further studies showed that under the effect of 1/16MIC Coptis decoction, the minimum inhibitory concentration of oxacillin sodium was 0.25 μg/mL. Conclusion Coptis decoction could inhibit the formation of Staphylococcus aureus biofilm, and oxacillin sodium combined with Coptidis decoction was effective to improve the curative effect of oxacillin sodium, providing new treatment for peritoneal dialysis related peritonitis that is caused by Staphylococcus aureus improving the efficacy of antibiotics, and providing help for the clinical on reducing the generation of antibiotic resistant bacteria.

目的 了解中山市7家医院金黄色葡萄球菌感染的临床分布,并对耐药基因进行检测,为临床经验治疗金黄色葡萄球菌感染提供用药及分子生物学依据。方法 收集2015年1月—2015年6月中山市7家医院分离到的金黄色葡萄球菌,使用ATB半自动细菌鉴定及药敏分析仪(法国梅里埃)对分离到的菌株进行鉴定及药敏试验,使用PCR技术对耐甲氧西林金黄色葡萄球菌(MRSA)的耐药基因进行检测。结果 7家医院共分离到89株金黄色葡萄球菌,其中MRSA检出33株,检出率为37.1%。金黄色葡萄球菌主要来源于呼吸内科(32株,36.0%)、骨科(20株,22.5%),主要分离自痰(41株,46.1%),伤口分泌物(16株,18%),对万古霉素、替考拉宁、奎奴普丁/达福普丁、复方新诺明、左氧氟沙星、诺氟沙星具有较高敏感性,MRSA对常用抗菌药物耐药率高于甲氧西林敏感金黄色葡萄球菌。共有32株MRSA检出bla_mecA基因,检出率为97%。结论 MRSA耐药情况较为严峻,临床科室应根据微生物培养报告合理使用抗菌药物。bla_mecA基因在MRSA检出较高,是MRSA主要的耐药机制。

Objective To analyze clinical distribution of Staphylococcus aureus infections from 7 hospitals in Zhongshan city, as well as to provide basis of empirical treatment and molecular biology for Staphylococcus aureus infections. Methods Staphylococcus aureus were collected from January 2015 to June 2015 in Zhongshan city, and then the strains were identified and tested antibiotic susceptibility by using ATB semiautomatic analyzer(Merieux). Resistance gene of methicillin-resistant Staphylococcus aureus(MRSA) was detected by polymerase chain reaction. Results 89 strains of Staphylococcus aureus were isolated from 7 hospitals and with prevalence of 33 strains of MRSA. Of all strains, 32(36.0%) were isolated from respiratory medicine and 20(22.5%) from orthopedics. 41(46.1%) strains of Staphylococcus aureus were isolated from sputum and 16(18.0%) from wound secretion. 89 strains of Staphylococcus aureus had highly susceptibility to vancomycin, teicoplanin, quinupristin/dalfopristin, cotrimoxazole, levofloxacin, norfloxacin. Resistance rates to commonly used antimicrobial drugs of MRSA were significantly higher than methicillin-sensitive. A total of 32 MRSA were detected carrying bla_mecA gene with the detection rate of 97%. Conclusion Clinical departments should be based on microbial culture report for rational use of antibiotics because of MRSA with more serious drug resistance. The gene of bla_mecA is the main mechanism of resistance for MRSA.

目的 了解广州地区儿童患者分离的社区获得性耐甲氧西林金黄色葡萄球菌(CA-MRSA)临床分离株的分子特征。方法 收集2009年—2014年我院分离获得的65株CA-MRSA临床分离株,PCR法检测杀白细胞素(PVL)基因阳性菌株,多位点基因序列类型(MLST)测定MRSA菌株的序列类型,多重PCR法对MRSA菌株进行葡萄球菌mec盒式染色体(SCCmec)分型。结果 65株CA-MRSA分离株中PVL基因阳性31株,阳性率47.69%;MLST分型表明以ST5933.84%(22/65)及ST8823.07%(15/65)为主;SCCmec分型中发现3种类型,分别为SCCmecⅡ 32.30%(21/65)、SCCmecⅣ 49.23%(32/65)及SCCmecⅤ 18.46%(12/65),未存在未能分型菌株。结论 广州地区儿童患者分离的CA-MRSA临床分离株的PVL基因阳性率相对较高,SCCmecIV型、V型可形成小范围内的流行,其基因表型存在多种ST分型。

Objective To investigate the molecular characteristic of Community-acquired Methicillin-resistant Staphylococcus aureus(CA-MRSA) isolates from pediatric patients in Guangzhou. Methods 65 non-duplicate strains of Community-acquired Methicillin-susceptible Staphylococcus aureus(CA-MSSA) isolated from our hospital in 2009-2014 were investigated. Panton-valentine leukocidin(PVL) gene was detected by polymerase chain reaction(PCR). The sequence type (ST) of each strain was determined by muhilocus sequence typing (MLST). The genotypes of staphylococcal cassette chromosome mec(SCCmec) of the CA-MRSA isolates were confirmed by multiplex PCR. Results Among 65 isolates of CA-MRSA, the positive rate of PVL genes was 47.69%(31／65). The most common MLST type was ST59 (n=22, 33.84%), followed by ST88 (n=15, 23.07%). Three SCCmec types were found. The most common type was SCCmecIV (49.23%, 32/65), followed by SCCmecⅡ (32.30%, 21/65) and SCCmecV (8.46%, 12/65). Conclusion The PVL gene-positive rate of CA-MRSA isolates in our hospitals is at a relatively high level. SCCmec type IV and SCCmec type V could spread among hospitals and cause a small scale epidemic. Their phenotypes have a variety of ST types.