流式细胞术应用于金黄色葡萄球菌体外药敏试验的初步研究
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摘要
目的：传统的抗生素敏感试验包括试管肉汤稀释法和琼脂稀释法，相当费时费力；目前临床常用的纸片扩散法、VITEK仪器检测法及偶尔使用的微量稀释法和E-试验等方法，需要将细菌在药物作用下培养逾夜方能观察结果，且多是采用比浊法进行判断，所提供的是细菌群体生长的均值。流式细胞术可快速分析细胞个体的特征，已应用于生物医学的许多领域，但在微生物学上的研究应用相对较少。八十年代初国外学者已开始研究流式细胞术在微生物活性方面的应用，但国内至今未见相关报道。耐甲氧西林的金黄色葡萄球菌(MRSA)是医院内感染的主要菌种之一，加强对此类细菌的监测对于预防和治疗院内感染，减少多重耐药菌株的产生与传播都有重要意义。目前临床上常通过检测金黄色葡萄球菌对苯唑西林的耐药性来推断MRSA。
本研究选择能够穿过破损的细胞膜、着染核酸的荧光染料碘化丙啶(PI)，根据流式细胞术所检测到的荧光强度来判断经药物处理后的培养液中细菌的存活率，通过检测2株标准菌及56株临床分离的金黄色葡萄球菌对苯唑西林的敏感性，并与微量稀释法和VITEK仪检测结果进行比较，显示出流式细胞荧光
术抗生素敏感试验具有快速、敏感、准确和客观的优点，为该
方法的进一步批广积累了可靠的实验依据。
方法：本研究收集了 2000年 11 月至 2001年 10月浙江省
人民医院检验中心和浙江人学医学院附属第一医院细菌室临床
二 分离的金黄色葡萄球菌共56 株，并购买质控菌株ATCC29213
和 A T C C 2 5 9 2 3。将细菌转种于血琼脂平板，3 5 C孵育过夜。次
日，从平板上挑取1－2个菌落于M叫肉汤中，于37℃恒温水温
箱中培养2小时，用V二T【K比浊仪调整浓度至O．5麦氏浓度（二．5
XIO’C F U／’ml）。在测定管中加人苯哇西林使终浓度分别为 8、4、
2‘…··0．06卜卜］，然后丁各检测管中加入菌悬液并调整终浓度
至 l．SX 10‘’CFU／ml，37℃孵育 3 ’J’时，加染料 Plloyl（终浓度
2．5 ng／ml），探匀，室温避光 15 分钟，以流式细胞术检测 10‘
个菌细胞，根据荧光强度的变化与否判断菌株对苯哇西林的敏
感性。随着药物浓度的增加，若荧光阳性百分率（卜1％）或平均
荧光强度（Mnlx）均在一狭窄范围，无明显上升者，推断为菌
株耐药；若卜二％逐渐增至80％或90％以上，则此时的药物浓度判
为MIC，表示菌株对药物敏感，或者Mnlx值突然增加了40％以
上，则椎断此时的药物浓度为MIC，且菌株敏感。
结果：2株标准菌和 56株临床分离菌的流式细胞荧光术药
敏试验结果与临床VITEK仪及微量稀释法结果完全一致。
对于32株MRSA，FCM检测到PI＄te药物浓度增加术有明显
上升，每株凶的各个PI％在较窄范围内，所有菌株PI％平均值在
50％以卜。PI％均值在 30－50＄之间共有 9 株菌（28．l＄），余 23
株（71．9％）低于 30％。同时，Mnlx亦无明显增加，各株菌的最
高、最低值均处在非常有限范围中，均值低于4．0 者共26 株
（81．3％）。故两种数值均可推断细菌耐药。
2
而对于26株 回SSA，两种数值的判断略有差异。随着药物
浓度的成倍增高，26株菌的PI％均显著增加至80％或90％以上，
此时的药物浓度为 问IC，菌株对药物敏感。但仅22株菌的 问nlx
随钓物浓度的增加而明显上升，当 饲nlx值突然增加了40％以上
时的钓物浓度被定为MIC，而其它4株菌的Mnlx值则略有波动
或增加不明显，故难以作出明确判断。经x‘检验，p＞0．0 5，判
断纪果的两种力法并无显著差异。
纪论：1．本研究）戊功地建立了金茧色葡萄球流式细胞荧光
术药物敏感试验（FCST）的具体操作方法，并检测了 58株金黄
色葡酣球伯对苯！哇两林的敏感性，结果与微量稀邪法和 VIT E K
仪器检测完全一致，但FCST ＆其余两种方法更为快洼、准确和
客观。2．实验证明采用PI％推断问IC较Mnlx更为方便可靠。故
建议川PI％ 仆辅以Mnlx值计算来判断FCST药敏试验结果。
Objective:
Traditional antibiotic susceptibility testing methods include broth dilution and agar dilution which are time-consuming. In the clinical tests of disc diffusion and VITEK instrument, as well as micro-dilution or E-test, bacteria should be cultivated with drugs overnight. Judgments made are usually based on the culture turbidity, which indicates an average value of bacterial group growth. Flow cytometry (FCM) can be used to analyse the individual cell features. It has been applied in many fields of biomedical science, but is rarely used in microbiology. Although FCM was first employed to study the activities of microorganism abroad as early as in the 80s of the 20th century, in China there is no related report up to now. At present, the methicillin resistant Staphylococcus aureus (MRSA) is one of the major drug-resistant species of
germs. Monitoring the dynamical change of drug-resistant
4
bacteria will play an important role to prevent and cure
bacterial infection inside hospitals, and reduce the generation and spread of multi-resistant organisms. In clinical lab, MRSA is usually identified by the sensitivity test of S. aureus to oxacillin.
.In this study, the fluorescent substance presidium iodide ( PI) was chosen to penetrate the broken bacterial cell-membrane and bind nucleic acid. The exis' ence rate of live bacteria in the culture after drug-treatment was determined according to the fluorescence strength by FCM. The susceptibility to
,-
oxacillin of 2 standard strains and 56 isolates of S. aureus were tested using FCM, compared with micro-dilution and VITEK methods. It was demonstrated that flow cytoflurometric antibiotic susceptibility test (FCST) was more rapid, sensitive, accurate and objective than the other two tests. Method:
Two type strains of S. aureus ATCC29213 and ATCC25923 purchased from Beijing and 56 isolates from the laboratory center of ZheJiang provincial people's hospital and the first affiliated hospital, School of Medicine, Zhejiang University, collected from Nov. 2000 to Oct. 2001, were used in this study. Bacteria were grown on blood-agar plates at 35"C overnight. The next day, one colony or two was subcultured in M-H broth, kept in a water-bath box at 37"C for 2 hours. The bacterial suspension was prepared to a concentration of 0. SMcFland (about
1.5X108CFU/ml) by VITEK turbidity-instrument. Oxacillin was
5
put into the test tubes to make the concentration of 8> 4>
2......0.06 n g/ml respectively. Then the bacterial suspension was
added, the final concentration was adjusted to I. " 5 X 106CFU/ml. The tubes were incubated at 37"C for 3 hours. Ten M-l of PI was added (final concentration 2.5 Hg/ml), mixed well. After 15 min in dark, about 104 cells of each strain were detected by FCM, the sensitivity to oxacillin was judged according to the change of fluorescence strength. With the increase of drug concentration, if fluorescence's positive percentage (PI%) or its mean strength (Mnlx) remained in a narrow range, without remarkable increase, it could be concluded that the organisms were drug-resistant. If PI% rose to 80-90% or more, it meant that drug concentration was the MIC, indicating the strain was drug-sensitive. If Mnlx increased 40% above suddenly, it could be thought that this drug concentration was the MIC, and the germ was sensitive. Results:
In this study, the oxacillin-susceptibility result of 2 standard strains and 56 isolates of S. aureus by FCST was the same to that by the methods of VITEK instrument and micro-dilution. Among 32 strains of MRSA, the PI% detected by FCM did not rise with the increase of drug concentration. The PI% of each strain remained in a narrow range. The PI% average values of all strains were below 50%. 9 strains (28.1%) were between 30%-50%; the rest 23 strains (71.9%) were below 30%.
Meanwhile, their Mnlx also did not show significant increase.
6
Both the highest one and the lowe st one were in the very limited
range, with the average values of 26 strains (81.3%) were below 4.0. Thus, both the c
本研究选择能够穿过破损的细胞膜、着染核酸的荧光染料碘化丙啶(PI)，根据流式细胞术所检测到的荧光强度来判断经药物处理后的培养液中细菌的存活率，通过检测2株标准菌及56株临床分离的金黄色葡萄球菌对苯唑西林的敏感性，并与微量稀释法和VITEK仪检测结果进行比较，显示出流式细胞荧光
术抗生素敏感试验具有快速、敏感、准确和客观的优点，为该
方法的进一步批广积累了可靠的实验依据。
方法：本研究收集了 2000年 11 月至 2001年 10月浙江省
人民医院检验中心和浙江人学医学院附属第一医院细菌室临床
二 分离的金黄色葡萄球菌共56 株，并购买质控菌株ATCC29213
和 A T C C 2 5 9 2 3。将细菌转种于血琼脂平板，3 5 C孵育过夜。次
日，从平板上挑取1－2个菌落于M叫肉汤中，于37℃恒温水温
箱中培养2小时，用V二T【K比浊仪调整浓度至O．5麦氏浓度（二．5
XIO’C F U／’ml）。在测定管中加人苯哇西林使终浓度分别为 8、4、
2‘…··0．06卜卜］，然后丁各检测管中加入菌悬液并调整终浓度
至 l．SX 10‘’CFU／ml，37℃孵育 3 ’J’时，加染料 Plloyl（终浓度
2．5 ng／ml），探匀，室温避光 15 分钟，以流式细胞术检测 10‘
个菌细胞，根据荧光强度的变化与否判断菌株对苯哇西林的敏
感性。随着药物浓度的增加，若荧光阳性百分率（卜1％）或平均
荧光强度（Mnlx）均在一狭窄范围，无明显上升者，推断为菌
株耐药；若卜二％逐渐增至80％或90％以上，则此时的药物浓度判
为MIC，表示菌株对药物敏感，或者Mnlx值突然增加了40％以
上，则椎断此时的药物浓度为MIC，且菌株敏感。
结果：2株标准菌和 56株临床分离菌的流式细胞荧光术药
敏试验结果与临床VITEK仪及微量稀释法结果完全一致。
对于32株MRSA，FCM检测到PI＄te药物浓度增加术有明显
上升，每株凶的各个PI％在较窄范围内，所有菌株PI％平均值在
50％以卜。PI％均值在 30－50＄之间共有 9 株菌（28．l＄），余 23
株（71．9％）低于 30％。同时，Mnlx亦无明显增加，各株菌的最
高、最低值均处在非常有限范围中，均值低于4．0 者共26 株
（81．3％）。故两种数值均可推断细菌耐药。
2
而对于26株 回SSA，两种数值的判断略有差异。随着药物
浓度的成倍增高，26株菌的PI％均显著增加至80％或90％以上，
此时的药物浓度为 问IC，菌株对药物敏感。但仅22株菌的 问nlx
随钓物浓度的增加而明显上升，当 饲nlx值突然增加了40％以上
时的钓物浓度被定为MIC，而其它4株菌的Mnlx值则略有波动
或增加不明显，故难以作出明确判断。经x‘检验，p＞0．0 5，判
断纪果的两种力法并无显著差异。
纪论：1．本研究）戊功地建立了金茧色葡萄球流式细胞荧光
术药物敏感试验（FCST）的具体操作方法，并检测了 58株金黄
色葡酣球伯对苯！哇两林的敏感性，结果与微量稀邪法和 VIT E K
仪器检测完全一致，但FCST ＆其余两种方法更为快洼、准确和
客观。2．实验证明采用PI％推断问IC较Mnlx更为方便可靠。故
建议川PI％ 仆辅以Mnlx值计算来判断FCST药敏试验结果。
Objective:
Traditional antibiotic susceptibility testing methods include broth dilution and agar dilution which are time-consuming. In the clinical tests of disc diffusion and VITEK instrument, as well as micro-dilution or E-test, bacteria should be cultivated with drugs overnight. Judgments made are usually based on the culture turbidity, which indicates an average value of bacterial group growth. Flow cytometry (FCM) can be used to analyse the individual cell features. It has been applied in many fields of biomedical science, but is rarely used in microbiology. Although FCM was first employed to study the activities of microorganism abroad as early as in the 80s of the 20th century, in China there is no related report up to now. At present, the methicillin resistant Staphylococcus aureus (MRSA) is one of the major drug-resistant species of
germs. Monitoring the dynamical change of drug-resistant
4
bacteria will play an important role to prevent and cure
bacterial infection inside hospitals, and reduce the generation and spread of multi-resistant organisms. In clinical lab, MRSA is usually identified by the sensitivity test of S. aureus to oxacillin.
.In this study, the fluorescent substance presidium iodide ( PI) was chosen to penetrate the broken bacterial cell-membrane and bind nucleic acid. The exis' ence rate of live bacteria in the culture after drug-treatment was determined according to the fluorescence strength by FCM. The susceptibility to
,-
oxacillin of 2 standard strains and 56 isolates of S. aureus were tested using FCM, compared with micro-dilution and VITEK methods. It was demonstrated that flow cytoflurometric antibiotic susceptibility test (FCST) was more rapid, sensitive, accurate and objective than the other two tests. Method:
Two type strains of S. aureus ATCC29213 and ATCC25923 purchased from Beijing and 56 isolates from the laboratory center of ZheJiang provincial people's hospital and the first affiliated hospital, School of Medicine, Zhejiang University, collected from Nov. 2000 to Oct. 2001, were used in this study. Bacteria were grown on blood-agar plates at 35"C overnight. The next day, one colony or two was subcultured in M-H broth, kept in a water-bath box at 37"C for 2 hours. The bacterial suspension was prepared to a concentration of 0. SMcFland (about
1.5X108CFU/ml) by VITEK turbidity-instrument. Oxacillin was
5
put into the test tubes to make the concentration of 8> 4>
2......0.06 n g/ml respectively. Then the bacterial suspension was
added, the final concentration was adjusted to I. " 5 X 106CFU/ml. The tubes were incubated at 37"C for 3 hours. Ten M-l of PI was added (final concentration 2.5 Hg/ml), mixed well. After 15 min in dark, about 104 cells of each strain were detected by FCM, the sensitivity to oxacillin was judged according to the change of fluorescence strength. With the increase of drug concentration, if fluorescence's positive percentage (PI%) or its mean strength (Mnlx) remained in a narrow range, without remarkable increase, it could be concluded that the organisms were drug-resistant. If PI% rose to 80-90% or more, it meant that drug concentration was the MIC, indicating the strain was drug-sensitive. If Mnlx increased 40% above suddenly, it could be thought that this drug concentration was the MIC, and the germ was sensitive. Results:
In this study, the oxacillin-susceptibility result of 2 standard strains and 56 isolates of S. aureus by FCST was the same to that by the methods of VITEK instrument and micro-dilution. Among 32 strains of MRSA, the PI% detected by FCM did not rise with the increase of drug concentration. The PI% of each strain remained in a narrow range. The PI% average values of all strains were below 50%. 9 strains (28.1%) were between 30%-50%; the rest 23 strains (71.9%) were below 30%.
Meanwhile, their Mnlx also did not show significant increase.
6
Both the highest one and the lowe st one were in the very limited
range, with the average values of 26 strains (81.3%) were below 4.0. Thus, both the c
引文
1． Cohen. M. L. Epidemiology of drug resistance: implications for a post-antimicrobial era. Science. 1992 ; 257(5073) 1050-1055．
2． Kunin, C. M. Resistance to antimicrobial drugs worldwide calamity. Annals of Internal Medicine. 1993; 118(7) :557-561．
3． Neu, H. C. The crisis in antibiotic resistance. Science. 1992; 257(5073) :1064-1073．
4． Brumfitt, W. & Hamilton-Miller, J. Methicillin-resistance Staphylococcus aureus. New England Journal of Medicine. 1989; 320(18) : 1188-1196．
5． Kayser, F. H. Methicillin and glycopeptide resistance in staphylococci: a new threat? Current Opinion in Infectious Disease.1995; 8, Suppl. 1, S7-11．
6． National Committee for Clinical Laboratory Standard. Performance Standards for Antimicrobial Susceptibility Testing; Ninth Informational Supplement. NCCLS documents M100-S9． Pennsylrania: NCCL, 1999． 17-99
7． Granato, P. A. The impact of same-day tests versus traditional overnight testing. Diagnostic Microbiology and Infectious Disease. 1993; 16(3) :237-243．
8． Boye, E., Steen, H. B. & Skarstad, K. Flow cytometry or bacteria: a promising tool in experimental and clinical microbiology. Journal of General Microbiology. 1983; 129(Pt 4) 973-980．
9． Allman, R. Flow cytometry: principles and applications. Proceedings of the Royal Microscopical Society. 1993; 28(1) :45-54．
10． Steen HB, Boye E, Skarstad K., et al. Applications of flow cytometry on bacteria: cell-cycle kinetics, drug effects, and quantitation of antibody binding. Cytometry, 1 982;2(4) :249-257．
11． Shapiro HM. Practical Cytometry, 1988:2nd edn. Alan R. Liss, New York.
12． Boye E, Lobner-Olesen A. Bacterial growth control studied by flow cytometry. Research in Microbiology, 1991; 142(2-3) :131-135．
13． Green L, Petersen B, Steimel L. Rapid determination of antifungal activity by flow cytometry. Journal of Clinical Microbiology, 1994;32(4) : 1088-1091．
14． Norden MA, Kurzynski TA, Bownds SE et al. Rapid susceptibility testing of Mycobacterium tuberculosis(H37Ra) by flow cytometry. Journal of Clinical Microbiology, 1995;33(5) 1231-1237．
15． Suller MT, Stark JM, Lloyd D. A flow cytometric study of antibiotic-induced damage and evaluation as a rapid antibiotic susceptibility test for MRSA. Journal of Antimicrobial Chemother, 1997;40(1) :77-83．
16． Sutler MT, Lloyd D. Fluorescence Monitoring of Antibiotic-Induced Bacterial Damage Using Flow Cytometry. Cytometry 1999;35(2) :235-241．
17． Ordonez JV, Wehman NM. Rapid flow cytometric antibiotic susceptibility assay for Staphylococcus aureus. Cytometry, 1993:14(7) 811-818．
18． Mason DJ, Allman R, Srark JM et al. Rapid estimation of
bacterial antibiotic susceptibility with flow cytometry. Journal of Microscope, 1994; 176(1) :8-16．
19． Durodie J, Coleman K, Simpson ZN et al. Rapid detection of antimicrobial activity using flow cytometry. Cytometry, 1995(4) :374-377．
20． 中化人民共和国卫生部医政司编，全国临床检验操作规程，南京：东南大学出版社, 1997
21． Peyron F, Favel A, Guriraud-Daurial H et al. Evaluation of a flow cytometry method for rapid determination of amphotericin B susceptibility of yeast isolates. Antimicrob Agents Chemother, 1997;41(7) : 1537-1540．
22． Walberg M, Gaustad P, Steen HB. Rapid Assessment of Ceftazidime, Ciprofloxacin, and Gentamicin Susceptibility in Exponenitially-Growing E. Coli cells by means of Flow Cytometry. Cytometry, 1997;27(2) :169-178．
23． Lee W, Kwark Y. Antifungal susceptibility testing of Candida species by flow cytometry. Journal of Korean Medicine, 1999;14(1) :21-26．
2． Kunin, C. M. Resistance to antimicrobial drugs worldwide calamity. Annals of Internal Medicine. 1993; 118(7) :557-561．
3． Neu, H. C. The crisis in antibiotic resistance. Science. 1992; 257(5073) :1064-1073．
4． Brumfitt, W. & Hamilton-Miller, J. Methicillin-resistance Staphylococcus aureus. New England Journal of Medicine. 1989; 320(18) : 1188-1196．
5． Kayser, F. H. Methicillin and glycopeptide resistance in staphylococci: a new threat? Current Opinion in Infectious Disease.1995; 8, Suppl. 1, S7-11．
6． National Committee for Clinical Laboratory Standard. Performance Standards for Antimicrobial Susceptibility Testing; Ninth Informational Supplement. NCCLS documents M100-S9． Pennsylrania: NCCL, 1999． 17-99
7． Granato, P. A. The impact of same-day tests versus traditional overnight testing. Diagnostic Microbiology and Infectious Disease. 1993; 16(3) :237-243．
8． Boye, E., Steen, H. B. & Skarstad, K. Flow cytometry or bacteria: a promising tool in experimental and clinical microbiology. Journal of General Microbiology. 1983; 129(Pt 4) 973-980．
9． Allman, R. Flow cytometry: principles and applications. Proceedings of the Royal Microscopical Society. 1993; 28(1) :45-54．
10． Steen HB, Boye E, Skarstad K., et al. Applications of flow cytometry on bacteria: cell-cycle kinetics, drug effects, and quantitation of antibody binding. Cytometry, 1 982;2(4) :249-257．
11． Shapiro HM. Practical Cytometry, 1988:2nd edn. Alan R. Liss, New York.
12． Boye E, Lobner-Olesen A. Bacterial growth control studied by flow cytometry. Research in Microbiology, 1991; 142(2-3) :131-135．
13． Green L, Petersen B, Steimel L. Rapid determination of antifungal activity by flow cytometry. Journal of Clinical Microbiology, 1994;32(4) : 1088-1091．
14． Norden MA, Kurzynski TA, Bownds SE et al. Rapid susceptibility testing of Mycobacterium tuberculosis(H37Ra) by flow cytometry. Journal of Clinical Microbiology, 1995;33(5) 1231-1237．
15． Suller MT, Stark JM, Lloyd D. A flow cytometric study of antibiotic-induced damage and evaluation as a rapid antibiotic susceptibility test for MRSA. Journal of Antimicrobial Chemother, 1997;40(1) :77-83．
16． Sutler MT, Lloyd D. Fluorescence Monitoring of Antibiotic-Induced Bacterial Damage Using Flow Cytometry. Cytometry 1999;35(2) :235-241．
17． Ordonez JV, Wehman NM. Rapid flow cytometric antibiotic susceptibility assay for Staphylococcus aureus. Cytometry, 1993:14(7) 811-818．
18． Mason DJ, Allman R, Srark JM et al. Rapid estimation of
bacterial antibiotic susceptibility with flow cytometry. Journal of Microscope, 1994; 176(1) :8-16．
19． Durodie J, Coleman K, Simpson ZN et al. Rapid detection of antimicrobial activity using flow cytometry. Cytometry, 1995(4) :374-377．
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