耐甲氧西林葡萄球菌的耐药模式和SCCmec分型研究
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摘要
目的:
葡萄球菌是引起医院内感染的重要病原菌,近年来,因其临床分离率逐年增高,耐药性日益严重,已成为全球关注的热点和焦点问题。本文旨在研究医院内感染葡萄球菌儿童株的临床分离情况和抗生素耐药状况,检测并分析耐药谱,为临床合理使用抗生素提供依据,并通过mecA基因检测和SCCmec基因分型,对耐甲氧西林葡萄球菌的耐药机制进行深入探讨,了解临床葡萄球菌感染的流行病学特征,为临床预测耐药菌株的发展趋势、抗感染治疗以及预防耐甲氧西林葡萄球菌的爆发流行提供对策和依据。
方法:
(1)对138株分离自天津某儿童医院院内感染病人的葡萄球菌进行菌种鉴定和临床资料分析。
(2)应用微量肉汤稀释法药敏板检测葡萄球菌对16种常用抗生素的耐药率,分析耐药谱;万古霉素平板筛选实验筛选出潜在的VISA,通过万古霉素E实验进行验证。
(3)应用Nitrocefin平板检测β-内酰胺酶活性,并用同样的方法检测经0.05μg/ml苯唑西林诱导后的葡萄球菌的β-内酰胺酶活性。
(4)采用PCR技术检测葡萄球菌的mecA基因携带状况,基因水平上验证MRS。
(5)对mecA阳性的葡萄球菌菌株采用两种经典的多重PCR快速简便地进行SCCmec基因分型,阐述其多重耐药产生的分子生物学基础。
结果:
(1)138株葡萄球菌经鉴定,分出金黄色葡萄球菌(SA)65株和凝固酶阴性葡萄球菌(CNS)73株,SA的主要分离自呼吸道痰液标本,而CNS主要分离自血液标本,两者的甲氧西林耐药率分别为10.8%、80.8%。
(2)CNS对苯唑西林、红霉素、庆大霉素、氨苄/舒巴坦、头孢拉定、环丙沙星、头孢吡肟、氨氯青霉素等抗生素的耐药率均高于SA(P＜0.05),差异具有统计学意义。两者除对万古霉素敏感外,对其他常用抗生素都有不同程度的耐药性。MRCNS对头孢拉定、头孢三嗪、氨氯青霉素、氨苄/舒巴坦、红霉素等的耐药率均高于MSCNS(P＜0.05),具有多重耐药的特点。万古霉素平板筛选试验和E试验证实目前尚未出现万古霉素耐药株。
(3)经0.05μg/ml苯唑西林诱导后,SA的β-内酰胺酶检出率从50.8%提高到89.2%,CNS的β-内酰胺酶检出率从61.6%提高到90.4%,临床实验室在检测β-内酰胺酶时可应用苯唑西林诱导以减少假阴性的产生。
(4)mecA基因的检出率为38%。在7株表型MRSA中,5株证实带有mecA;在59株表型MRCNS中,46株(78%)为mecA阳性,13株没有检测到mecA(22%的假阳性);在由表型分组的MSSA中,没有测到mecA阳性株;14株表型MSCNS中,仅1株mecA阳性(7%假阴性)。
(5)5株mecA阳性的金黄色葡萄球菌中检测出SCCmecⅠ、Ⅲ、Ⅳa型各一株,两株未能分型;47株(64.4%)mecA阳性的凝固酶阴性葡萄球菌中,共分出SCCmecⅠ型和ⅠA型6株,Ⅲ型和ⅢB型10株,Ⅳ型9株,Ⅴ型3株和变异的新型SCCmec类Ⅰ型4株,类ⅠA型5株,仍有10株未能分型。
结论:
医院内感染凝固酶阴性葡萄球菌的多重耐药率和mecA携带率高于金黄色葡萄球菌,需引起临床足够的重视,本医院尚未发现万古霉素耐药株或中介耐药株;进行β-内酰胺酶检测时可应用苯唑西林诱导以减少假阴性的产生;通过表型判断MRSA或MRCNS出现假阳性的可能大于出现假阴性的可能,需根据mecA基因检测来判断MRS;半数以上凝固酶阴性葡萄球菌的SCCmec可用现有多重PCR方法分型,但有两种新型SCCmec出现,疑为Ⅰ型变异株,暂定为类Ⅰ型和类ⅠA型,可作为进一步研究MRS耐药基因起源与变异的切入点。
Objectives:
Staphylococci played an important role in nosocomial infections.Recently,the infections of staphylococci isolates have become the focus of clinician around the world,for their increasing isolating rates and antibiotic resistance rates.In this study, the isolate rates and resistant rates of staphylococci isolated from children were investigated,and the spectrum of resistance was made to provide basic rule for proper drug administration in clinical practice.Moreover,the mechanism of methicillin resistance in staphylococci was studied through the detection of mecA gene and genotyping of SCCmec.To predict and prevent the development of MRS and provide information for anti-infective therapy,molecular epidemiology of MRS recovered from patients with nosocomial infection was also studied.
Methods:
(1)138 staphylococcal strains isolated from patients with nosocomial infection in Tianjin Children's Hospital were identified and the clinical data were collected.
(2)The antibiotic susceptibility pattern of the staphylococcal isolates to 16 clinical routing used antibiotics was studied,and then the strains were screened with vancomycin-containing plates and verified by E-test.
(3)The Nitrocefin plates were used for detectingβ-lactamase activity in two types of strains,the original isolates and the isolated induced by oxacillin-containing plates at the concentration of 0.05μg/ml.
(4)The status of gene mecA carrying was investigated with PCR amplification to identify MRS strains.
(5)The genotyping of SCCmec with multiplex PCR was progressed on the mecA gene positive strains,to explain the molecular epidemiological characteristic.
Results:
(1)The total of 138 staphylococcal isolates included 65 Staphylococcus aureus strains (SA)and 73 coagulase-negative staphylococci strains(CNS),the oxacillin-resistant rates were 10.8%and 80,8%,respectively.SA strains were mainly isolated from sputum of respiratory tract,and CNS strains were mostly from blood cultures.
(2)The CNS strains suggested higher resistance to oxacillin,erythromycin, cidomycin,benzyl-sulbactam,cefradine,ciprofloxacin,cefepime and rectocillin than SA strains(P＜0.05);The resistance of MRCNS strains to cefradine,cefatrizine, rectocillin,benzyl-sulbactam,erythromycin were higher than the MSCNS strains(P＜0.05).The MRCNS strains were characterized with multi-drug resistance.All isolates were sensitive to vancomycin.
(3)Theβ-lactamase isolating rates of SA and CNS strains changed after induced by oxacillin-containing plates.Theβ-lactamase positive rate of SA increased from 50.8% to 89.2%and CNS from 61.6%to 90.4%.It indicates we could reduce the false negative rate by oxacillin induced in clinical detection of theβ-lactamase.
(4)The mecA carrying rate was 38%.5 of 7 MRSA strains were carrying mecA.In 59 MRCNS isolates,47 isolates were mecA positive,while other 13 isolates were negative(false rate is 22%).No mecA positive strains were detected in phenotypic MSSA,and only one was mecA positive in phenotypic MSCNS.
(5)In 5 mecA positive SA isolates,three were carrying SCCmec typesⅠ,ⅢandⅣ, respectively.47 mecA positive CNS strains including 6 for typeⅠ,10 for typeⅢ,9 for typeⅣ,3 for typeⅤand novel variants of SCCmec 4 strains for typeⅠ-like,5 strains for typeⅠA-like,the rest 10 CNS strains and 2 SA strains were not-typeable.
Conclusions:
The antibiotic resistance of CNS is much more severe than that of SA,Which needs more attention from clinician.All the strains are sensitive to vancomycin. When detecting theβ-lactamase,the strains induced by oxacillin could decrease the false negation.More than half of the SCCmec in CNS could be typed by the current multiplex PCR assays,but there are two novel variants of SCCmec in the staphylococci isolates,tentatively designated typeⅠ-like and typeⅠA-like.
葡萄球菌是引起医院内感染的重要病原菌,近年来,因其临床分离率逐年增高,耐药性日益严重,已成为全球关注的热点和焦点问题。本文旨在研究医院内感染葡萄球菌儿童株的临床分离情况和抗生素耐药状况,检测并分析耐药谱,为临床合理使用抗生素提供依据,并通过mecA基因检测和SCCmec基因分型,对耐甲氧西林葡萄球菌的耐药机制进行深入探讨,了解临床葡萄球菌感染的流行病学特征,为临床预测耐药菌株的发展趋势、抗感染治疗以及预防耐甲氧西林葡萄球菌的爆发流行提供对策和依据。
方法:
(1)对138株分离自天津某儿童医院院内感染病人的葡萄球菌进行菌种鉴定和临床资料分析。
(2)应用微量肉汤稀释法药敏板检测葡萄球菌对16种常用抗生素的耐药率,分析耐药谱;万古霉素平板筛选实验筛选出潜在的VISA,通过万古霉素E实验进行验证。
(3)应用Nitrocefin平板检测β-内酰胺酶活性,并用同样的方法检测经0.05μg/ml苯唑西林诱导后的葡萄球菌的β-内酰胺酶活性。
(4)采用PCR技术检测葡萄球菌的mecA基因携带状况,基因水平上验证MRS。
(5)对mecA阳性的葡萄球菌菌株采用两种经典的多重PCR快速简便地进行SCCmec基因分型,阐述其多重耐药产生的分子生物学基础。
结果:
(1)138株葡萄球菌经鉴定,分出金黄色葡萄球菌(SA)65株和凝固酶阴性葡萄球菌(CNS)73株,SA的主要分离自呼吸道痰液标本,而CNS主要分离自血液标本,两者的甲氧西林耐药率分别为10.8%、80.8%。
(2)CNS对苯唑西林、红霉素、庆大霉素、氨苄/舒巴坦、头孢拉定、环丙沙星、头孢吡肟、氨氯青霉素等抗生素的耐药率均高于SA(P＜0.05),差异具有统计学意义。两者除对万古霉素敏感外,对其他常用抗生素都有不同程度的耐药性。MRCNS对头孢拉定、头孢三嗪、氨氯青霉素、氨苄/舒巴坦、红霉素等的耐药率均高于MSCNS(P＜0.05),具有多重耐药的特点。万古霉素平板筛选试验和E试验证实目前尚未出现万古霉素耐药株。
(3)经0.05μg/ml苯唑西林诱导后,SA的β-内酰胺酶检出率从50.8%提高到89.2%,CNS的β-内酰胺酶检出率从61.6%提高到90.4%,临床实验室在检测β-内酰胺酶时可应用苯唑西林诱导以减少假阴性的产生。
(4)mecA基因的检出率为38%。在7株表型MRSA中,5株证实带有mecA;在59株表型MRCNS中,46株(78%)为mecA阳性,13株没有检测到mecA(22%的假阳性);在由表型分组的MSSA中,没有测到mecA阳性株;14株表型MSCNS中,仅1株mecA阳性(7%假阴性)。
(5)5株mecA阳性的金黄色葡萄球菌中检测出SCCmecⅠ、Ⅲ、Ⅳa型各一株,两株未能分型;47株(64.4%)mecA阳性的凝固酶阴性葡萄球菌中,共分出SCCmecⅠ型和ⅠA型6株,Ⅲ型和ⅢB型10株,Ⅳ型9株,Ⅴ型3株和变异的新型SCCmec类Ⅰ型4株,类ⅠA型5株,仍有10株未能分型。
结论:
医院内感染凝固酶阴性葡萄球菌的多重耐药率和mecA携带率高于金黄色葡萄球菌,需引起临床足够的重视,本医院尚未发现万古霉素耐药株或中介耐药株;进行β-内酰胺酶检测时可应用苯唑西林诱导以减少假阴性的产生;通过表型判断MRSA或MRCNS出现假阳性的可能大于出现假阴性的可能,需根据mecA基因检测来判断MRS;半数以上凝固酶阴性葡萄球菌的SCCmec可用现有多重PCR方法分型,但有两种新型SCCmec出现,疑为Ⅰ型变异株,暂定为类Ⅰ型和类ⅠA型,可作为进一步研究MRS耐药基因起源与变异的切入点。
Objectives:
Staphylococci played an important role in nosocomial infections.Recently,the infections of staphylococci isolates have become the focus of clinician around the world,for their increasing isolating rates and antibiotic resistance rates.In this study, the isolate rates and resistant rates of staphylococci isolated from children were investigated,and the spectrum of resistance was made to provide basic rule for proper drug administration in clinical practice.Moreover,the mechanism of methicillin resistance in staphylococci was studied through the detection of mecA gene and genotyping of SCCmec.To predict and prevent the development of MRS and provide information for anti-infective therapy,molecular epidemiology of MRS recovered from patients with nosocomial infection was also studied.
Methods:
(1)138 staphylococcal strains isolated from patients with nosocomial infection in Tianjin Children's Hospital were identified and the clinical data were collected.
(2)The antibiotic susceptibility pattern of the staphylococcal isolates to 16 clinical routing used antibiotics was studied,and then the strains were screened with vancomycin-containing plates and verified by E-test.
(3)The Nitrocefin plates were used for detectingβ-lactamase activity in two types of strains,the original isolates and the isolated induced by oxacillin-containing plates at the concentration of 0.05μg/ml.
(4)The status of gene mecA carrying was investigated with PCR amplification to identify MRS strains.
(5)The genotyping of SCCmec with multiplex PCR was progressed on the mecA gene positive strains,to explain the molecular epidemiological characteristic.
Results:
(1)The total of 138 staphylococcal isolates included 65 Staphylococcus aureus strains (SA)and 73 coagulase-negative staphylococci strains(CNS),the oxacillin-resistant rates were 10.8%and 80,8%,respectively.SA strains were mainly isolated from sputum of respiratory tract,and CNS strains were mostly from blood cultures.
(2)The CNS strains suggested higher resistance to oxacillin,erythromycin, cidomycin,benzyl-sulbactam,cefradine,ciprofloxacin,cefepime and rectocillin than SA strains(P＜0.05);The resistance of MRCNS strains to cefradine,cefatrizine, rectocillin,benzyl-sulbactam,erythromycin were higher than the MSCNS strains(P＜0.05).The MRCNS strains were characterized with multi-drug resistance.All isolates were sensitive to vancomycin.
(3)Theβ-lactamase isolating rates of SA and CNS strains changed after induced by oxacillin-containing plates.Theβ-lactamase positive rate of SA increased from 50.8% to 89.2%and CNS from 61.6%to 90.4%.It indicates we could reduce the false negative rate by oxacillin induced in clinical detection of theβ-lactamase.
(4)The mecA carrying rate was 38%.5 of 7 MRSA strains were carrying mecA.In 59 MRCNS isolates,47 isolates were mecA positive,while other 13 isolates were negative(false rate is 22%).No mecA positive strains were detected in phenotypic MSSA,and only one was mecA positive in phenotypic MSCNS.
(5)In 5 mecA positive SA isolates,three were carrying SCCmec typesⅠ,ⅢandⅣ, respectively.47 mecA positive CNS strains including 6 for typeⅠ,10 for typeⅢ,9 for typeⅣ,3 for typeⅤand novel variants of SCCmec 4 strains for typeⅠ-like,5 strains for typeⅠA-like,the rest 10 CNS strains and 2 SA strains were not-typeable.
Conclusions:
The antibiotic resistance of CNS is much more severe than that of SA,Which needs more attention from clinician.All the strains are sensitive to vancomycin. When detecting theβ-lactamase,the strains induced by oxacillin could decrease the false negation.More than half of the SCCmec in CNS could be typed by the current multiplex PCR assays,but there are two novel variants of SCCmec in the staphylococci isolates,tentatively designated typeⅠ-like and typeⅠA-like.
引文
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[49] Katayama Y, Ito T, Hiramatsu K. A new class of genetic element staphylococcus cassette mec, encodes methicillin resistance in Staphylococcus aureus [J]. Antimicrob Agents Chemother, 2000,44(6): 1549-1555.
[50] Ito T, Okuna K, Ma XX, et al. Insights on antibiotic resistance of Staphylococcus aureus from it whole genome: genomic island SCC [J]. Drug Resistance Updates, 2003,6:41-52.
[51] Oliveira DC, Tomasa A, de Lencastre H. The evolution of pandemic clones of methicillin-resistant Staphylococcus aureus identification of two ancestral genetic backgrounds and the associated mec elements [J]. Microb Drug Resist, 2001,7(4):349.
[52] Daum RS, Ito T, Hiramatsu K, et al. A novel methicillin-resistance cassette in community-acquired methicillin-resistant Staphylococcus aureus isolates of diverse genetic backgrounds [J]. J Infect Dis, 2002, 186: 1344-7.
[53] Hanssen AM, Ericson Sollid JU. SCCmec in staphylococci: genes on the move [J]. FEMS Immunol Med Microbiol, 2006,46(1):8-20.
[54] Mombach Pinheiro Machado AB, Reiter KC, Paiva RM, et al. Distribution of staphylococcal cassette chromosome mec (SCCmec) types I, II, III and IV in coagulase-negative staphylococci from patients attending a tertiary hospital in southern Brazil[J].J Med Microbiol.2007,56(10):1328-1333.
[55]Wisplinghoff H,Rosato AE,Enright Me,et al.Related clones containing SCCmec type Ⅳ predominate among clinically significant Staphylococcus epidermidis isolates[J].Antimicrob Agents Chemother,2003,47(11):3574-3579.
[56]Oliveira,D.C.,and H.de Lencastre.Multiplex PeR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus[J].Antimicrob Agents Chemother,2002,46:2155-61.
[57]Shore A,Rossney AS,Keane CT,et al.Seven novel variants of staphylococcal chromosomal cassette mec in methicillin-resistant Staphylococcus aureus isolates from Ireland[J].Antimicrob Agents Chemother,2005,49(5):2070-2083.
[58]欧阳范献,卜平凤,黄惠琴,等.MRSA的7种新SCCmec型别及其抗药特性[J].微生物学报,2007,47(2):201-207.
[1]Livermore DM.Antibiotic resistance in staphylococci[J].Int J Antimicrob Agents,2000,16(Suppl1):S3-10.
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[1]Livermore DM.Antibiotic resistance in staphylococci[J].Int J Antimicrob Agents,2000,16(Suppl1):S3-10.
[2]Stefani S,Varaldo PE.Epidemiology of methicillin-resistant staphylococci in Europe[J].J Clin Microbiol Infect,2003,9(12):1179-1180.
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