金葡菌的生长速度和凝固酶活性对巴马小型猪化脓性肝脓肿形成的影响
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摘要
目的探讨金葡菌的生长速度和外毒素活性对巴马小型猪化脓性肝脓肿形成的影响。方法采用培养时间和菌液OD450吸光度值法描记细菌生长曲线;采用RT-PCR法检测凝固酶(coa)、溶血酶α(hla)和耐热核酸酶A(nuc A)的mRNA表达;采用试管法检测游离凝固酶、溶血法检测游离溶血酶和甲苯胺蓝DNA酶琼脂法检测游离耐热核酸酶的活性。结果金葡菌ATCC29213的生长速度显著快于ATCC 25923。ATCC 29213的凝固酶和耐热核酸酶A mRNA表达量显著高于ATCC 25923,但溶血酶α表达两者无显著性差异。ATCC 29213的游离凝固酶活性显著高于ATCC 25923,但其游离耐热核酸酶和游离溶血酶的活性显著低于ATCC 25923。结论金葡菌ATCC 29213引起巴马小型猪化脓性肝脓肿的机制可能与其生长速度快和游离凝固酶活性高相关。
Objective To investigate the effect of the growth velocity and coagulase activity of S. aureus on pyogenic hepatic abscess in Bama minipig. Methods Growth curves of S. aureus were monitored by the spectrophotometry. Expressions of virulence protein mRNA were measured by RT-PCR method,and enzyme activities of virulence protein were detected by toluidine blue-DNA assay,tube coagulase test and hemolysin assay. Results The growth velocity of S. aureus ATCC 29213 was significantly quicker than that of ATCC 25923. The expressions of coagulase( coa) and thermostable nuclease A( nuc A) mRNA in ATCC 25213 were significantly higher than those in ATCC 25923. However,the expression of alphatoxin( hla) was not markedly different. The free coagulase activity of ATCC 29213 was markedly larger than that of ATCC 25923. However,its activities of free thermostable nuclease and free staphylolysin were notablely lower than those of ATCC 25923. Conclusion The reason why the pyogenic hepatic abscess is caused by S. aureus ATCC 29213 could be related to its quick growth velocity and high free coagulase activity secreted.
Objective To investigate the effect of the growth velocity and coagulase activity of S. aureus on pyogenic hepatic abscess in Bama minipig. Methods Growth curves of S. aureus were monitored by the spectrophotometry. Expressions of virulence protein mRNA were measured by RT-PCR method,and enzyme activities of virulence protein were detected by toluidine blue-DNA assay,tube coagulase test and hemolysin assay. Results The growth velocity of S. aureus ATCC 29213 was significantly quicker than that of ATCC 25923. The expressions of coagulase( coa) and thermostable nuclease A( nuc A) mRNA in ATCC 25213 were significantly higher than those in ATCC 25923. However,the expression of alphatoxin( hla) was not markedly different. The free coagulase activity of ATCC 29213 was markedly larger than that of ATCC 25923. However,its activities of free thermostable nuclease and free staphylolysin were notablely lower than those of ATCC 25923. Conclusion The reason why the pyogenic hepatic abscess is caused by S. aureus ATCC 29213 could be related to its quick growth velocity and high free coagulase activity secreted.
引文
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[12]ANDERSON J C,ADLAM C,KNIGHTS J M.The effect of staphylocoagulase in the mammary gland of the mouse[J].Br J Exp Pathol,1982,63(3):336-340.
[13]SCHMIDT K A,MANNA A C,GILL S,et al.Sar T,a repressor of alpha-hemolysin in Staphylococcus aureus[J].Infect Immun,2001,69(8):4749-4758.DOI:10.1128/IAI.69.8.4749-4758.2001.
[2]QIU J,WANG D,XIANG H,et al.Subinhibitory concentrations of thymol reduce enterotoxins A and B and alpha-hemolysin production in Staphylococcus aureus isolates[J].PLo S One,2010,5(3):e9736.DOI:10.1371/journal.pone.0009736.
[3]BRAKSTAD O G,AASBAKK K,MAELAND J A.Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene[J].J Clin Microbiol,1992,30(7):1654-1660.
[4]张汝钢,张修礼,杨云生.巴马小型猪化脓性肝脓肿模型的建立和鉴定[J].胃肠病学和肝病学杂志,2017,26(1):66-69.DOI:10.3969/j.issn.1006-5709.2017.01.018.ZHANG R G,ZHANG X L,YANG Y S.Establishment and identification of pyogenic hepatic abscess in Bama minipig[J].Chin J Gastroenterol Hepatol,2017,26(1):66-69.DOI:10.3969/j.issn.1006-5709.2017.01.018.
[5]ZHANG R G,WANG X D,ZHANG X L,et al.An experimental model for Staphylococcus aureus hepatic abscess in Bama minipig[J].Genet Mol Res,2014,13(3):7113-7122.DOI:10.4238/2014.February.21.12.
[6]DOMNGUEZ M C,DE LA ROSA M,BOROBIO M V.Application of a spectrophotometric method for the determination of post-antibiotic effect and comparison with viable counts in agar[J].J Antimicrob Chemother,2001,47(4):391-398.
[7]张汝钢,杨云生.OD450值与金黄色葡萄球菌活菌数目的相关性研究[J].军医进修学院学报,2011,32(4):343-344.ZHANG R G,YANG Y S.Correlation between OD450 value and viable number of Staphylococcus aureus[J].J Chinese PLA Postgrad Med Sch,2011,32(4):343-344.
[8]QIU J,FENG H,LU J,et al.Eugenol reduces the expression of virulence-related exoproteins in Staphylococcus aureus[J].Appl Environ Microbiol,2010,76(17):5846-5851.DOI:10.1128/AEM.00704-10.
[9]LACHICA R V,GENIGEORGIS C,HOEPRICH P D.Metachromatic agar-diffusion methods for detecting staphylococcal nuclease activity[J].Appl Microbiol,1971,21(4):585-587.
[10]TANG J,ZHOU R,SHI X,et al.Two thermostable nucleases coexisted in Staphylococcus aureus:evidence from mutagenesis and in vitro expression[J].FEMS Microbiol Lett,2008,284(2):176-183.DOI:10.1111/j.1574-6968.2008.01194.x.
[11]PHONIMDAENG P,O'REILLY M,O'TOOLE P W,et al.Molecular cloning and expression of the coagulase gene of Staphylococcus aureus8325-4[J].J Gen Microbiol,1988,134(1):75-83.DOI:10.1099/00221287-134-1-75.
[12]ANDERSON J C,ADLAM C,KNIGHTS J M.The effect of staphylocoagulase in the mammary gland of the mouse[J].Br J Exp Pathol,1982,63(3):336-340.
[13]SCHMIDT K A,MANNA A C,GILL S,et al.Sar T,a repressor of alpha-hemolysin in Staphylococcus aureus[J].Infect Immun,2001,69(8):4749-4758.DOI:10.1128/IAI.69.8.4749-4758.2001.