| 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 ofgerms. Monitoring the dynamical change of drug-resistant4bacteria will play an important role to prevent and curebacterial 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 (about1.5X108CFU/ml) by VITEK turbidity-instrument. Oxacillin was5put into the test tubes to make the concentration of 8> 4>2......0.06 n g/ml respectively. Then the bacterial suspension wasadded, 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.6Both the highest one and the lowe st one were in the very limitedrange, with the average values of 26 strains (81.3%) were below 4.0. Thus, both the c... |
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