Medical Technology SA
Volume 24 No. 2 | December 2010
Peer reviewed SHORT COMMUNICATION
Antibiotic Susceptibility of Multi-drug resistant Mycobacterium tuberculosis using Flow Cytometry S Govender1 PhD, SJ du Plessis1 MSc, M van de Venter1 PhD, C Hayes2 HND Med.Tech 1 2
Department of Biochemistry and Microbiology, Nelson Mandela Metropolitan University, Port Elizabeth, South Africa Tuberculosis Laboratory, National Health Laboratory Services, Port Elizabeth
Corresponding author: Sharlene Govender email:
[email protected] | tel: +27 41 504 2347 | fax: +27 41 504 2814
Abstract Increased resistance of Mycobacterium tuberculosis (M. tuberculosis) to antibiotics has emphasized the need for rapid susceptibility testing. BACTEC MGIT 960 system is routinely used for drug susceptibility testing; however, the main drawback is the long incubation time. An alternative flow cytometry-based assay was evaluated for the assessment of drug susceptibility. Thirteen multi-drug resistant (MDR) M. tuberculosis cultures were tested against rifampicin (1 µg/ml), isoniazid (0.2 µg/ml), ethambutol (7.5 µg/ml), and ofloxacin (2 µg/ml) using flow cytometry by staining cells with SYTO16 and propidium iodide. Correlation rates of flow cytometry results with BACTEC MGIT 960 system were 28.5% (rifampicin); 15.4% (isoniazid), 61.5% (ethambutol) and 46.1% (ofloxacin). Findings indicate that while flow cytometry for tuberculosis (TB) drug susceptibility may reduce the length of time taken for diagnosis, there was low correlation with BACTEC MGIT 960. The high cost associated with implementation of the technique in diagnostic laboratories is a further disadvantage. Further studies with larger sample sizes are needed to confirm these data. Keywords M. tuberculosis, Flow cytometry, BACTEC MGIT 960, antibiotic susceptibility. Introduction The emergence of multi-drug resistant and extreme drug-resistant tuberculosis (TB) has emphasised the need for methods that will allow quick detection of the microbial agent, while simultaneously performing rapid antibiotic susceptibility tests. Due to the long generation time of Mycobacterium tuberculosis, antimicrobial susceptibility testing takes a few weeks, delaying treatment, which may negatively affect the patient’s health, and lead to an increase in disease transmission[1-2]. The conventional method of drug susceptibility testing relies on the culture of M. tuberculosis which is reported to be accurate, sensitive and specific. The introduction of automated systems, such as the BACTEC MGIT 960 in clinical laboratories, has shown significant correlation with conventional methods, such as the agar dilution method. The BACTEC MGIT 960 has been accepted by South African health laboratories for drug susceptibility testing of M. tuberculosis from clinical samples. A disadvantage of the BACTEC MGIT 960 is the relatively long time before results becomes available (7-12 days)[3-5]. Flow cytometry is a new technique, with many applications, and has been used for the detection and quantification of bacteria, and is becoming increasingly popular due to its ability to provide rapid, reliable and sensitive results[2, 5-7]. SYTO 16 belongs to a group of cell-permeable nucleic acid stains that show a characteristic increase in fluorescence when binding to nucleic acids. SYTO 16 dye stains all heat killed cells while drug susceptible cells have a reduced number of fluorescent particles in comparison with the drug free growth control and drug resistant cells. Propidium iodide, also a fluorescent dye, does not stain mycobacteria and is used to detect microbial contamination[2, 8].
Rifampicin and isoniazid testing using flow cytometry produced correlation rates of up to 97% with other drug susceptibility methods, such as the agar dilution method[11]. M. tuberculosis cells need to be destroyed without compromising their staining ability to ensure safety of the procedure, and the small size of M. tuberculosis and their tendency to aggregate makes analysis fairly challenging[1, 2, 4, 7, 10]. Thus far, four of the antibiotics (rifampicin, isoniazid, ethambutol and streptomycin) used to treat tuberculosis have been evaluated using flow cytometry[1-2, 5, 7, 10-11]. Other flow cytometry investigations have included pyrazinamide, but this antibiotic was found to be problematic for two reasons: (1) pyrazinamide requires an acidic pH for its activity, and it was found that the acidity of the medium (required for drug activity) inhibited the growth of M. tuberculosis and (2) use of a large inoculum of M. tuberculosis led to an increase in the pH of the medium and subsequently inactivated pyrazinamide[12]. The drawback of previous flow cytometry approaches for M. tuberculosis susceptibility testing is the biosafety issue. Inaccurate results were obtained after inactivation of the cells with paraformaldehyde, prior to analysis by flow cytometry and staining cells with flourescein diacetate[5, 7, 9, 13]. The aim of this study was to investigate whether the susceptibility test outlined by Pina-Vaz et al., (2005)[2], in which M. tuberculosis heat killed cells were stained with SYTO 16 dye would provide a safe, accurate way to determine susceptibility of M. tuberculosis. This study assessed the susceptibility of M. tuberculosis to rifampicin, isoniazid, ethambutol and the fluoroquinolone, ofloxacin. ISSN 1011 5528 | www.smltsa.org.za
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Medical Technology SA
Volume 24 No. 2 | December 2010
Table 1: Susceptibility results for 13 MDR-TB samples and M. tuberculosis ATCC 27294 towards rifampicin, isoniazid, ethambutol and ofloxacin, determined with the BACTEC MGIT 960 system and flow cytometry.
R: Resistant; S: Susceptible; #: Inconclusive Result; ND: Not determined
Materials and Methods Preparation of cultures and antibiotics
Scientific Group) [10 µM] for 30 min.
Thirteen MDR-TB isolates on Lowenstein-Jensen agar slants, from NHLS, were aseptically transferred to Middlebrook 7H9 broth. M. tuberculosis ATCC 27294, an avirulent strain, susceptible to all antibiotics was used as a control in this study. Prior to antibiotic susceptibility testing, broth cultures were incubated at 37°C for 10 days after which the turbidity of the culture was equivalent to a McFarland 0.5 standard (1.5 x 108 bacteria). Five hundred microlitres of this culture was inoculated into freshly prepared Middlebrook 7H9 broth, supplemented with different concentrations of antibiotics. The following antibiotics (SigmaAldrich) were tested at single concentrations: ofloxacin (2 µg/ ml), isoniazid (0.2 µg/ml), rifampicin (1 µg/ml) and ethambutol (7.5 µg/ml).
Prior to analysis, the heat-killed TB cell suspension was vortexed, passed through a Neomedic 25 gauge micro-emulsifying needle (Separations), and vortexed again to ensure complete separation of the cells, and to avoid clumping. The cells were then analysed using a flow cytometer (Beckman Coulter FC500) equipped with a 488 nm Argon ion laser. Emitted fluorescence was detected at 620 nm (red fluorescence for PI) and 525 nm (green fluorescence for SYTO 16). Samples were analysed within the flow cytometer for 60 sec at a low aspiration speed[2]. The number of fluorescent organisms per millilitre was determined, using the data obtained from flow cytometry after specific gates (a region drawn around a specific cell population and allowing all events occurring within this area to be measured) had been established to eliminate electronic noise and background interference. Forward (related to cell size) and side angle (related to complexity and granularity of the cell) light scattering was also used in the analysis of the M. tuberculosis cells. An isolate was considered sensitive whenever the number of fluorescent particles in the drug containing medium was reduced in comparison to the drug free growth control (ratio
