摘要
金黄色葡萄球菌是临床重要的致病菌,耐甲氧西林金葡菌(MRSA)使得金葡菌感染的病死率增加,成为院内感染的最常见致病菌。社区获得性耐甲氧西林金黄色葡萄球菌(Community acquired methicillin-resistant Staphylococcusaureus,CA—MRSA)自上世纪末开始,相继出现报道。
目的:了解天津地区CA—MRSA临床株的遗传学特点,包括:检测它们的耐药表型、pvl基因的携带情况、SCCmec分型和MLST分型。为临床诊断、预防和治疗奠定基础。
方法:
(1)菌株收集与药敏检测:收集自2001年—2006年天津市四家三级甲等医院金葡菌,其中15株金葡菌鼻腔携带株、125株金葡菌临床株。依据CA—MRSA的概念将临床株分成CA—MRSA和医院获得MRSA(HA—MRSA)。采用头孢西丁纸片法,检测MRSA。应用微量肉汤稀释法检测金葡菌对12中抗生素(包括:青霉素、苯唑西林、氨苄西林、环丙沙星、莫西沙星、头孢唑啉、氯霉素、林可霉素、庆大霉素、头孢曲松、万古霉素和红霉素)的MIC,分析其耐药率。
(2)金葡菌的pvl筛检:应用PCR方法,对全部金葡菌检测pvl基因,并对PCR阳性的扩增产物进行DNA序列分析。
(3)金葡菌的SCCmec分型:参照Olivera等的文献报道合成引物。对CA—MRSA和HA—MRSA菌株应用多重PCR方法,进行SCCmec分型。
(4)金葡菌的MLST分析:扩增金葡菌的7个高度保守的看家基因,将扩增的序列片段进行DNA序列分析。将分析结果提交国际数据库网站http://saureus.mlst.net进行比对,最终获得菌株ST分型。
结果:
(1)MRSA的检测:132例健康查体者鼻腔携带菌株中,筛检出15例金葡菌携带株。金葡菌携带率为11.36%。其中,经头孢西丁纸片法检测,15例携带株均为MSSA。在健康查体者中,没有发现MRSA鼻腔携带株。在125株金葡菌临床株中MRSA54株,依据临床资料分成12株CA-MRSA和42株HA—MRSA。应用微量肉汤稀释法,检测对12种抗生素的MIC。其中,42株HA—MRSA除对万古霉素敏感外,对其他11种抗生素均有较高的耐药率,呈现多重耐药的表型。而12株CA—MRSA除对β—内酰胺类抗生素有很高的耐药率外,对非β—内酰胺类抗生素耐药率较低。在15株携带株中,对青霉素耐药率较高,对林可霉素和红霉素也有中等程度的耐药,对其他类抗生素仍较敏感。
(2)pvl基因PCR方法检测:检测125株临床株和15株携带株的pvl基因。结果仅检出pvl基因阳性菌株3株。pvl基因检出率较低(3/140)。其中1株是从CA—MRSA中检出,来自脓液,另外2株为MSSA,来自痰液。
(3)SCCmec分型:用多重PCR方法,对12株CA—MRSA和42株HA—MRSA进行SCCmec分型。在12株CA—MRSA中,除1株未定型外,其余11株均属于SCCmecⅣ型;在42株HA—MRSA中,以SCCmecⅢ型和Ⅲa型较多,分别为10株和16株,其次为SCCmecⅡ型7株和SCCmecⅢb型6株,还有3株未定型。没有发现SCCmecⅠ型菌株。
(4)MLST分型:对pvl基因阳性的CA—MRSA临床株6Saul,进行MLST分型。属于ST88。该菌株来自脓液。
结论
1、健康者鼻腔携带菌株中,没有发现CA—MRSA。在54株MRSA临床株中,发现12株CA—MRSA。
2、应用临床药敏实验、PCR法筛检pvl基因、SCCmec分型,及MLST分型,对12株CA—MRSA进行分析。其中1株携带pvl基因,为SCCmecⅣ型,MLST型别为ST88;另外11株pvl基因均阴性,除1株SCCmec分型属未定型外,均为SCCmecⅣ型。CA—MRSA的情况与国内外报道相符。本研究是天津地区首次应用遗传学方法,系统地对CA—MRSA临床株的流行情况、临床特点和基因型别进行综合研究。
Staphylococcus aureus is a clinical significant pathogenic bacteria. Methicillin-resistant Staphylococcus aureus(MRSA)increases the case fatality rate of infectious disease by Staphylococcus aureus.It becomes a major pathogen of hospital onset of infection.From the last end of the century,Community acquired methicillin-resistant Staphylococcus aureus(CA—MRSA)was reported one after another.
Object:To investigate genetic characteristic of CA-MRSA in Tianjin.Include as follows:detect their drug sensitivity,investigate the information of carrying pvl gene, typing by SCCmec and MLST.
Method:
1.Strains' collection and their drug sensitivity:Collect staphylococcus aureus from four hospitals in Tianjin during 2001 to 2006.15 of all are Staphylococcus aureus Nasal Carriage;125 of all are clinical strains.According to the concept of CA-MRSA,the clinical strains are classified HA-MRSA and CA-MRSA.screening MRSA by disc agar diffusion method with cefoxitin.Through broth dilution method, detect their MIC for 12 antibiotics,(including:penicillin,oxacillin,ampicillin, ciprofloxacin,moxifloxacin,cefazolin,chlormycetin,lincomycin,gentamycin, ceftriaxone,vancomycin and erythromycin).Analyze their drug tolerance ratio.
2.Screening for pvl gene:By PCR method,detect all strain for pvl gene.Then, Carry out DNA sequence analysis of PCR's product.
3.Typing by SCCmec:Reference Olivera's study and synthetic primer. Through multiplex PCR assay,typing HA-MRSA and CA-MRSA by SCCmec.
4.Typing by MLST:amplify seven house-keeping gene,and carry out DNA sequence analysis of PCR's product.Send analytic result to database's network (http://saureus.mlst.net).Get the strain's sequence type.
Result:
1.Screening MRSA:Among 132 Nasal Carriage of health people,obtain 15 staphylococcus aureus strains.The nasal carriage ratio is 11.36%.By disc agar diffusion method with cefoxitin,they are all MSSA.No MRSA was found among health people.There are 54 MRSA in 125 clinical strains,12 is CA-MRSA and 42 is HA-MRSA.Through broth dilution method,detect their MIC for 12 antibiotics. There are high drug tolerance ratios to 11 antibiotics except vancomycin,among 42 HA-MRSA.It shows multi-resistant feature.And,among 12 CA-MRSA,there are low drug tolerance ratios to nonβ-lactam antibiotics exceptβ-lactam antibiotics. There are high drug tolerance ratios to penicillin and middle drug tolerance ratios to lincomycin and erythromycin among 15 Nasal Carriage.They are sensitive to other antibiotics.
2.Detect pvl gene by PCR:In 125 clinical strains and 15 Nasal Carriage strains, detect pvl gene.Only 3 strains carry pvl gene.There is low carriage ratio for pvl gene (3/140).One belongs to CA-MRSA.It's from pus.The other belong to MSSA from sputum.
3.Typing by SCCmec:typing 42 HA-MRSA and 12 CA-MRSA through multiplex PCR assay by SCCmec.Among 12 CA-MRSA,they all belong to SCCmecⅣexcept one belong to unknow type.Among 42 HA-MRSA,there are more SCCmecⅢand SCCmecⅢa strains,10 and 16 strains,respectively.Otherwise,7 belong to SCCmecⅡ,and 6 belong to SCCmecⅢb.3 strains belong to unknow type.No SCCmecⅠstrain was found.
4.Typing by MLST:typing one CA-MRSA carried pvl gene,namely 6Saul,by MLST.It belongs to ST88 from pus.
Conclusion:
1.No CA-MRSA was found among health people Nasal Carriage.There 12 CA-MRSA in 54 MRSA.
2.Through drug sensitivity test,pvl gene by PCR,SCCmec typing and MLST, analysis 12 CA-MRSA.One carried pvl gene belongs to SCCmecⅣ.It's ST88 by MLST.Moreover,11 CA-MRSA don't carry pvl gene,and they all belong to SCCmecⅣexcept one strain.It is coincidence with reports abroad.This study is first and systemic investigation for epidemic,clinical feature and gene typing of CA-MRSA,by genetic analysis method,in Tianjin.
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