长沙地区临床分离金黄色葡萄球菌的耐药特征及femB基因的克隆和原核表达
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摘要
目的(1)了解长沙地区临床分离金黄色葡萄球菌(以下简称金葡菌)对常用抗菌药物的耐药现状,探讨金黄色葡萄球菌对甲氧西林和万古霉素的耐药水平。(2)构建金葡菌甲氧西林耐药辅助基因femB的原核表达载体并在大肠杆菌中表达,为进一步研究金葡菌对甲氧西林的耐药机制奠定基础。
方法(1)收集长沙地区11家医院2009年11月-2010年11月临床分离的非重复金葡菌279株,应用Vitek-2全自动微生物分析系统进行鉴定,K-B法检测金葡菌对24种药物的敏感性,产色头孢菌素试验检测β-内酰胺酶以及D试验检测诱导型克林霉素耐药。(2)应用头孢西丁和苯唑西林纸片扩散法筛查耐甲氧西林的金葡菌(MRSA);琼脂稀释法检测头孢西丁,苯唑西林和万古霉素的最低抑菌浓度(MIC)。(3)提取金葡菌基因组DNA,并以此为模板进行femB基因的PCR扩增,构建重组质粒pGEX-4T-1-femB,并将重组质粒导入大肠杆菌BL21中进行表达。采用SDS-PAGE及Western blot分析对表达蛋白进行验证。
结果(1)279株分离的金葡菌对24种药物的耐药分析显示,敏感率>50%的药物为9种,敏感率最高的为万古霉素、替考拉宁和利奈唑胺,均为100%,其次为呋喃妥因(97.1%)、氯霉素(93.5%)和复方新诺明(87.1%)。耐药率>50%的抗菌药物有11种,其中以青霉素G和氨苄西林的耐药率最高,均为97.1%。MRSA的分离率为54.5%,除万古霉素、替考拉宁、利奈唑胺、呋喃妥因均高度敏感外,MRSA对常用的16种抗生素的耐药率均显著高于MSSA。(2) 279株金黄葡菌中,p-内酰胺酶阳性250株(89.6%),红霉素耐药而克林霉素敏感、中介的30株菌中,D试验阳性22株(73.3%)。(3)苯唑西林(OXA)和头孢西丁(FOX) MIC范围分别为O.125μg/ml～>256μg/ml和2μg/ml～>256μg/ml,苯唑西林的MIC50和MIC90分别为128μg/ml和256μg/ml,头孢西丁的MIC50和MIC90分别为64μg/ml和256μg/ml。万古霉素MIC范围为1～2μg/ml,MIC50和MIC90均为2μg/ml。(4)经PCR,双酶切以及测序验证,成功的构建了重组质粒pGEX-4T-1-femB;重组质粒转化大肠杆菌BL21经IPTG诱导后,SDS-PAGE和Western blot分析证实表达出49KDa目的蛋白。
结论(1)长沙地区临床分离的金葡菌p-内酰胺酶阳性率,克林霉素诱导型耐药率均较高,且呈多重耐药特征。(2)长沙地区临床分离的金葡菌不仅MRSA分离率高,而且对甲氧西林呈高水平耐药；所有菌株对万古霉素均敏感,但MIC已接近中介水平,应引起高度重视。(3)成功构建了重组质粒pGEX-4T-1-femB,并在大肠杆菌中高效的表达。
Objective (1) To investigate the characteristic of drug resistance and current situation of methicillin-resistance and vancomycin-resistance in clinical isolates of staphylococcus aureus in Changsha. (2) To construct prokaryotic expression vector of femB gene (Factors essential for the expression of methicillin resistance gene), and express it in E. coli and establish foundation for further investigations of methicillin-resistance in staphylococcus aureus.
Methods (1) Totally 279 non-duplicate clinical isolates of staphylococcus aureus were collected during November 2009 to November 2010 from 11 hospitals in Changsha and then identified by Vitek-2 system. K-B disk method was used to test drug sensitivity of 24 commonly used antibiotics, chromogenic cephalosporin spot test was applied to detectβ-lactamase and D-test were used to check inducible resistance of erythromycin to clindamycin. (2) MRSA was screened by oxacillin and cefoxitin disk diffusion methods. Agar dilution method was used to determine the minimal inhibitory concentrations (MICs) of oxacillin, cefoxitin and vancomycin to isolates of staphylococcus aureus. (3) Genomic DNA were extracted from staphylococcus aureus and used as templete for PCR amplification of femB gene fragment using special PCR primers. Recombinant plasmids pGEX-4T-1-femB were constructed and transformed into E.coli BL21. The expressed product was identified by SDS-PAGE and Western blot.
Results (1) Of the 279 S. aureus isolates, The sensitive rates to 9 of the 24 antibiotics tested were higher than 50%. All the isolates were susceptible to tecoplanin, vancomycin and linezolid, and the sensitive rate to Nitrofurantoin, chlormycetin and trimethoprim-sulfamethoxazole was 97.1%,93.5% and 87.1% respectively. The resistant rates to 11 of the 24 antibiotics tested were higher than 50%, resistant rates to penicillin and ampicillin were the highest (both 97.1%).Among 279 strains, MRSA accounted for 54.5%, which were high sensitive to vancomycin, tecoplanin and linezolid. The resistant rates of MRSA to 16 antibiotics were higher than MSSA. (2) Of the 279 strains, the positive rate ofβ-lactamase was 89.6%(250/279). Of the 30 isolates resistant to erythromycin but susceptible or intermediate to clindamycin, 22(73.3%)showed a positive result of D-test. (3) The MIC range of OXA and FOX on staphylococcus aureus was 0.125->256μg/ml and 2～>256μg/ml respectively. The MIC50 and MIC90 of OXA and FOX was 128μg/ml and 256μg/ml,64μg/ml and 256μg/ml respectively. The MIC range of vancomycin on staphylococcus aureus was 1～2μg/ml, both the MIC50 and MIC90 of vancomycin were 2μg/ml. (4) Verified by PCR, double-enzyme digested assessment and sequencing, recombinant plamid pGEX-4T-1-femB was successfully constructed. SDS-PAGE and Western blot analysis confirmed the recombinant plasmid pGEX-4T-1-femB expressed a 49KD target protein in E.coli BL21.
Conclusions (1) Clinical isolates of staphylococcus aureus in Changsha are multiple resistant to commonly used antimicrobial agents, positive rate ofβ-lactamase and rate of inducible resistance of erythromycin to clindamycin are high. (2) Clinical isolates of staphylococcus aureus in Changsha have a high isolated rate of MRSA, and are highly resistant to methicillin. All the strains are sensitive to vancomycin, but special attention should be paied to the incresed MICs which nearly reached intermediate level. (3) Recombinant plasmid pGEX-4T-1-femB is successfully constructed and high-effective expressed in E. Coli.
方法(1)收集长沙地区11家医院2009年11月-2010年11月临床分离的非重复金葡菌279株,应用Vitek-2全自动微生物分析系统进行鉴定,K-B法检测金葡菌对24种药物的敏感性,产色头孢菌素试验检测β-内酰胺酶以及D试验检测诱导型克林霉素耐药。(2)应用头孢西丁和苯唑西林纸片扩散法筛查耐甲氧西林的金葡菌(MRSA);琼脂稀释法检测头孢西丁,苯唑西林和万古霉素的最低抑菌浓度(MIC)。(3)提取金葡菌基因组DNA,并以此为模板进行femB基因的PCR扩增,构建重组质粒pGEX-4T-1-femB,并将重组质粒导入大肠杆菌BL21中进行表达。采用SDS-PAGE及Western blot分析对表达蛋白进行验证。
结果(1)279株分离的金葡菌对24种药物的耐药分析显示,敏感率>50%的药物为9种,敏感率最高的为万古霉素、替考拉宁和利奈唑胺,均为100%,其次为呋喃妥因(97.1%)、氯霉素(93.5%)和复方新诺明(87.1%)。耐药率>50%的抗菌药物有11种,其中以青霉素G和氨苄西林的耐药率最高,均为97.1%。MRSA的分离率为54.5%,除万古霉素、替考拉宁、利奈唑胺、呋喃妥因均高度敏感外,MRSA对常用的16种抗生素的耐药率均显著高于MSSA。(2) 279株金黄葡菌中,p-内酰胺酶阳性250株(89.6%),红霉素耐药而克林霉素敏感、中介的30株菌中,D试验阳性22株(73.3%)。(3)苯唑西林(OXA)和头孢西丁(FOX) MIC范围分别为O.125μg/ml～>256μg/ml和2μg/ml～>256μg/ml,苯唑西林的MIC50和MIC90分别为128μg/ml和256μg/ml,头孢西丁的MIC50和MIC90分别为64μg/ml和256μg/ml。万古霉素MIC范围为1～2μg/ml,MIC50和MIC90均为2μg/ml。(4)经PCR,双酶切以及测序验证,成功的构建了重组质粒pGEX-4T-1-femB;重组质粒转化大肠杆菌BL21经IPTG诱导后,SDS-PAGE和Western blot分析证实表达出49KDa目的蛋白。
结论(1)长沙地区临床分离的金葡菌p-内酰胺酶阳性率,克林霉素诱导型耐药率均较高,且呈多重耐药特征。(2)长沙地区临床分离的金葡菌不仅MRSA分离率高,而且对甲氧西林呈高水平耐药；所有菌株对万古霉素均敏感,但MIC已接近中介水平,应引起高度重视。(3)成功构建了重组质粒pGEX-4T-1-femB,并在大肠杆菌中高效的表达。
Objective (1) To investigate the characteristic of drug resistance and current situation of methicillin-resistance and vancomycin-resistance in clinical isolates of staphylococcus aureus in Changsha. (2) To construct prokaryotic expression vector of femB gene (Factors essential for the expression of methicillin resistance gene), and express it in E. coli and establish foundation for further investigations of methicillin-resistance in staphylococcus aureus.
Methods (1) Totally 279 non-duplicate clinical isolates of staphylococcus aureus were collected during November 2009 to November 2010 from 11 hospitals in Changsha and then identified by Vitek-2 system. K-B disk method was used to test drug sensitivity of 24 commonly used antibiotics, chromogenic cephalosporin spot test was applied to detectβ-lactamase and D-test were used to check inducible resistance of erythromycin to clindamycin. (2) MRSA was screened by oxacillin and cefoxitin disk diffusion methods. Agar dilution method was used to determine the minimal inhibitory concentrations (MICs) of oxacillin, cefoxitin and vancomycin to isolates of staphylococcus aureus. (3) Genomic DNA were extracted from staphylococcus aureus and used as templete for PCR amplification of femB gene fragment using special PCR primers. Recombinant plasmids pGEX-4T-1-femB were constructed and transformed into E.coli BL21. The expressed product was identified by SDS-PAGE and Western blot.
Results (1) Of the 279 S. aureus isolates, The sensitive rates to 9 of the 24 antibiotics tested were higher than 50%. All the isolates were susceptible to tecoplanin, vancomycin and linezolid, and the sensitive rate to Nitrofurantoin, chlormycetin and trimethoprim-sulfamethoxazole was 97.1%,93.5% and 87.1% respectively. The resistant rates to 11 of the 24 antibiotics tested were higher than 50%, resistant rates to penicillin and ampicillin were the highest (both 97.1%).Among 279 strains, MRSA accounted for 54.5%, which were high sensitive to vancomycin, tecoplanin and linezolid. The resistant rates of MRSA to 16 antibiotics were higher than MSSA. (2) Of the 279 strains, the positive rate ofβ-lactamase was 89.6%(250/279). Of the 30 isolates resistant to erythromycin but susceptible or intermediate to clindamycin, 22(73.3%)showed a positive result of D-test. (3) The MIC range of OXA and FOX on staphylococcus aureus was 0.125->256μg/ml and 2～>256μg/ml respectively. The MIC50 and MIC90 of OXA and FOX was 128μg/ml and 256μg/ml,64μg/ml and 256μg/ml respectively. The MIC range of vancomycin on staphylococcus aureus was 1～2μg/ml, both the MIC50 and MIC90 of vancomycin were 2μg/ml. (4) Verified by PCR, double-enzyme digested assessment and sequencing, recombinant plamid pGEX-4T-1-femB was successfully constructed. SDS-PAGE and Western blot analysis confirmed the recombinant plasmid pGEX-4T-1-femB expressed a 49KD target protein in E.coli BL21.
Conclusions (1) Clinical isolates of staphylococcus aureus in Changsha are multiple resistant to commonly used antimicrobial agents, positive rate ofβ-lactamase and rate of inducible resistance of erythromycin to clindamycin are high. (2) Clinical isolates of staphylococcus aureus in Changsha have a high isolated rate of MRSA, and are highly resistant to methicillin. All the strains are sensitive to vancomycin, but special attention should be paied to the incresed MICs which nearly reached intermediate level. (3) Recombinant plasmid pGEX-4T-1-femB is successfully constructed and high-effective expressed in E. Coli.
引文
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[1]Deurenberg RH, Stobberingh RE. The evolution of staphylococcus aureus. Infection Genetic Evol,2008,8:747-763
[2]Llarrull LI, Fisher JF, Mobashery S. Molecular basis and phenotype of methicillin resistance in staphylococcus aureus and insights into new β-Lactams that meet the challenge. J Antimicrob Agents Chemother,2009,53(10):4051-4063
[3]Subray SH, Thomas ES. FemABX family members are novel nonribosomal peptidyl transfcrases and important pathogen specific drugs target. J Biol Chem,2001, 276(10):6998-7003
[4]Li X,Xiong Y, Fan X, et al. A study of the regulating gene of femA from methicillin-resistant Staphylococcus aureus clinical isolates. J Int Med Res,2008, 36(3):420-33
[5]Berger-Bachi B, Rohrer S. Factors influencing methicillin resistance in staphylococci. J Arch Microbiol,2002,178:165-171
[6]Cha J, Vakulenko SB, Mobashery S. Characterization of the β-lactam antibiotic sensor domain of the MecRl signal sensor/transducer protein from methicillin-resistant staphylococcus aureus. Biochemistry,2007,46:7822-7831
[7]Akcam FZ, Tinaz GB, Kaya O, et al. Evaluation of methicillin resistance by cefoxitin disk diffusion and PBP2a latex agglutination test in mecA-positive staphylococcus aureus, and comparison of mecA with femA, femB, femX positivities. J Microbiological Res,2009,164(4):400-403
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[9]Maidhof H, Reinicke B, Blumed P, et al. femA, which encodes a factor essential for expression of methicillin resistance, affects glycine content of peptidoglycan in methicillin-resistant and methicillin-susceptible staphylococcus aureus strains. J Bacteriol,1991,173(11):3507-3513
[10]Mohanasoundaram KM, Lalitha MK. Comparison of phenotypic versus genotypic methods in the detection of methicillin resistance in staphylococcus aureus. J Indian J M Res,2008,127(1):78-84
[11]Ehlert K, Schroder W, Labischinski H. Specificities of FemA and FemB for different glycine residues: FemB cannot substitute for FemA in staphylococcal peptidoglycan pentaglycine side chain formation. J Bacteriol.1997,179(23): 7573-7576
[12]Rohrer S, Ehlert K, Tschiemke M, et al. The essential staphylococcus aureus gene fmhB is involved in the first step of peptidoglycan pentaglycine interpeptide formation. Proc Natl Acad Sci USA,1999,96,9351-9356
[13]Hong HJ, Hutchings MI, Hill LM, et al. The Role of the Novel Fem Protein VanK in Vancomycin Resistance in Streptomyces coelicolor. J Biol Chem,2005, 280(13):13055-13061
[14]Kopp U, Roos M, Wecke J, et al. Staphylococcus peptidoglycan interpeptide bridge biosynthesis:a novel antistaphylococcal target. J Microbiol Drug Resist,1996, 2:185-198
[15]Parvez MA, Shibata H, Nakano T, et al. No relationship exists between PBP 2a amounts expressed in different MRSA strains obtained clinically and their beta-lactam MIC values. J Med Invest,2008,55(34):246-253
[16]李艺,杨信怡,游雪甫,等.耐甲氧西林金葡菌的耐药分子机制研究.国外医药抗生素分册,2005.26(4)：173-177
[17]Henze U, Sidow T, Wecke J, et al. Influence of femB on methicillin resistance and peptidoglycan metabolism in staphylococcus aureus. J Bacteriol,1993, 175(6):1612-1620
[18]熊亚莉,范昕建,张磊,等.金黄色葡萄球菌耐药表型与femA表达水平关系研究.四川大学学报(医学版),2007.38(2)：268-271
[19]Ling B, Berger-Bachi B. Increased overall antibiotic susceptibility in Staphylococcus aureus femAB null mutants. J Antimicrob Agents Chemother,1998, 42:936-938.
[20]Schneider T, Senn MM, Berger-Bachi B, et al. In vitro assembly of a complete, pentaglycine interpeptide bridge containing cell wall precursor (lipid Ⅱ-Gly5) of Staphylococcus aureus. Mol Microbiol,2004,53(2):675-685
[21]Grundling A, Missiakas DM, Schneewind O. Staphylococcus aureus mutants with increased lysostaphin resistance. J Bacteriol,2006,188(17):6286-6297
[22]Hiibscher J, Berger-Bachi B, Kotte O, et al. Living with an imperfect cell wall: compensation of femAB inactivation in staphylococcus aureus. BMC Genomics 2007, 8(1):307-322
[23]Sharif S, Sung JK, Labischinski H, et al. Characterization of peptidoglycan in Fem-Deletion mutants of Methicillin-resistant Staphylococcus aureus by Solid-State NMR. Biochemistry,2009,48(14):3100-3108
[24]Kraus D, Kalbacher H, Berger-Bachi B, et al. Muropeptide modification-amidation of peptidoglycan D-glutamate does not affect the proinflammatory activity of staphylococcus aureus. J Infect Immun,2007, 75(4):2084-2087
[25]McAleese F, Wu SW, Sieradzki K, et al. Overexpression of genes of the cell wall stimulon in clinical isolates of Staphylococcus aureus exhibiting vancomycin-intermediate S.aureus-type resistance to vancomycin. J Bacteriol,2006, 188:1120-1133
[26]Komatsuzawa H, Ohta K, Labischinski H, et al. Characterization of fmtA:a gene that modulates the expression of methicillin resistance in staphylococcus aureus. J Antimicrob Agents Chemother,1999,43:2121-2125
[27]Yang SJ, Xiong YQ, Dunman PM. Regulation of mprF in daptomycin-nonsusceptible staphylococcus aureus strains. J Antimicrob Agents Chemother,2009,53(6):2636-2637
[28]Gardete S, Ludovice AM, Sobral RG, et al. Role of murE in the expression of β-Lactam antibiotic resistance in staphylococcus aureus. J Bacteriol,2004, 186(6):1705-1713
[29]Gardete S, Ludovice AM, Sobral RG, et al. Role of murF in cell wall biosynthesis isolation and characterization of a murF conditional mutant of staphylococcus aureus. J Bacteriol,2006,188(7):2543-2553
[30]Katayama Y, Zhang HZ, Chambem HF. PBP2a mutations producing very high-level resistance to beta-lactam. J Antimicrob Agents Chemother,2004, 48(2):453-459
[31]Rohrer S, Berger-Bachi B. FemABX peptidyl transferases:a link between branched-chain cell wall peptide formation and β-Lactam resistance in Gram-Positive Cocci. J Antimicrob Agents Chemother,2003,47(3):837-846