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Antimicrobial Agents and Chemotherapy, February 2005, p. 804-807, Vol. 49, No. 2
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.2.804-807.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Simple Fibroblast-Based Assay To Test the Pyrazinamide Susceptibility of Mycobacterium tuberculosis

Takemasa Takii,1* Sonomi Hamasaki,2 Kazue Hirano,2 Chiyoji Abe,2 and Kikuo Onozaki1

Department of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya,1 Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, Kiyose, Tokyo, Japan2

Received 4 May 2004/ Returned for modification 5 August 2004/ Accepted 11 October 2004


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ABSTRACT
 
A simple fibroblast-based assay (SFA) was found to be efficient in evaluating the susceptibilities of clinical isolates of Mycobacterium tuberculosis to pyrazinamide (PZA). Forty-five clinical isolates were examined. The MICs of PZA for susceptible strains in an SFA were between 3.13 and 12.5 µg/ml, and the MICs of PZA for resistant strains were more than 100 µg/ml.


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TEXT
 
The incidence of tuberculosis is still increasing in some countries, and control of the disease is threatened by the emergence of drug-resistant strains (23). At this time, pyrazinamide (PZA) is one of the first-line drugs in the standard treatment regimen used for tuberculosis patients, and it is especially indispensable to DOTS (directly observed treatment, short course) (5, 11, 12). In the past few years, considerable progress has been made in understanding the mechanism of action of PZA and the genetic basis of resistance to the compound (11, 24). Most of the resistance to PZA has been shown to be accompanied by the loss of pyrazinamidase (PZase) activity in Mycobacterium tuberculosis (9). Mutations in the pncA gene have been identified as the cause for acquired PZA resistance in M. tuberculosis, and the sequence alteration of this gene has also been reported in naturally resistant Mycobacterium bovis strains (16, 17, 18). However, the mutation sites in the pncA gene are diverse (1), and in addition, mechanisms other than mutation of pncA are operative. In order to detect PZA-resistant strains, in vitro testing of the susceptibility of M. tuberculosis to PZA is highly recommended. Unfortunately, conventional agar-based testing for PZA susceptibility often leads to ambiguous results because of insufficient growth in the acidified medium (21). Therefore, a new method for PZA susceptibility testing is necessary. We have previously reported that viable M. tuberculosis H37Rv exhibits cytotoxicity to a human lung fibroblast cell line (19, 20). As the cytotoxicity was counteracted by drugs, including isoniazide, rifampin, ethambutol, streptomycin, and PZA, a simple fibroblast cell-based assay (SFA) was developed to screen antimycobacterial drugs (20). In this study, we applied SFA to test the susceptibilities of clinical isolates of M. tuberculosis against PZA.

Clinical isolates. Forty-five clinical isolates of M. tuberculosis from Asian countries and Canada, which were characterized for PZase activity and susceptibility to PZA in acidic agar medium, were used for susceptibility testing by an SFA. For some isolates, mutation analysis of the pncA gene was also performed by sequencing a 561-bp region (8). In addition, three PZA-resistant mutants (KK-117, KK-118, and KK-123) obtained from M. tuberculosis clinical isolates by serial passage in Middlebrook 7H9 liquid medium (pH 5.5) with twofold increasing concentrations of PZA ranging from 50 to 3,200 µg/ml were also examined. These three mutants had no mutations in the pncA gene.

PZA susceptibility testing using acidic agar medium. The MIC (drug concentration required to inhibit more than 99% of colony formation) of PZA was determined on Middlebrook 7H11 agar at a pH value of 6.0 containing twofold concentrations of drug ranging from 25 to 800 µg/ml. The inoculum consisted of 0.1 ml of a 10–3 dilution of a 2-week-old liquid culture (107 to 108 CFU/ml) of mycobacteria. Cultures were incubated at 37°C with 5% CO2 for 3 weeks before the MIC was determined. The threshold concentration for the evaluation of PZA resistance in the acidic medium was defined as 200 µg of PZA per ml.

Susceptibility testing of M. tuberculosis by SFA. The human lung fibroblast cell line MRC-5 has been shown to be very sensitive to the cytotoxicity of live virulent bacilli of M. tuberculosis H37Rv, and the host cell viability was reflected by the state of the bacilli inside the host cells (19, 20). As MRC-5 is a healthy diploid cell line, the cells are difficult to propagate for long periods. Accordingly, in this study, we used the transformed strain MRC-5 SV TG1 (Cell Bank, Osaka, Japan), which is immortal. The cell line was susceptible to the cytotoxicity of live bacilli in the same manner as the parent MRC-5 cells (data not shown).

MRC-5 SV TG1 cells were plated as previously described (20), and the bacterial suspensions (up to 50 µl) of clinical isolates of M. tuberculosis were added. After incubation for 16 to 18 h, the host cells were washed with phosphate-buffered saline (PBS), and then 200 µl of fresh tissue culture medium with or without PZA (at twofold concentrations from 0.78 to 100 µg/ml) was added. After 3 days of culture, the host cells were stained with crystal violet, and the optical density at 595 nm was measured to determine the host cell viability.

In the SFA, MIC was defined as the lowest concentration of drug exhibiting a statistically significant inhibitory effect on the bacterial cytotoxicity as determined by examining cell viability (20). In this study, the MICs of PZA for sensitive clinical isolates of M. tuberculosis were between 3.13 and 12.5 µg/ml, and the MICs of PZA for the resistant isolates were more than 100 µg/ml. The PZA-sensitive and -resistant isolates could be clearly distinguished at the PZA concentration of 100 µg/ml. Therefore, the threshold value for PZA resistance in the SFA was defined as 100 µg/ml.

Forty-five clinical isolates of M. tuberculosis were used to determine the susceptibilities of the isolates to PZA in a SFA. Twenty isolates were susceptible to PZA, and 25 isolates were resistant (Table 1). The results obtained by SFA are based on the viability of the bacilli inside host cells. Using clinical isolates of M. tuberculosis, we attempted to confirm this fact (Table 2). The data show that PZA reduced the number of PZA-susceptible bacilli inside host cells, but not PZA-resistant bacilli. These results clearly indicate that the viability of clinical isolates inside host cells also correlates with the cytotoxicity against host cells. The concordance ratio between SFA and the PZase assay and/or growth test in acidic medium was 95.0 and 96.0% for susceptible and resistant strains, respectively. These ratios are comparable to or higher than those of other methods on the basis of PZase activity, drug susceptibility test in acidic agar, BACTEC system, and sequence analysis of the pncA gene (2, 3, 7, 10). All the resistant strains with mutations in pncA appeared to be resistant by SFA as well. Three PZA-resistant mutants (KK-117, KK-118, and KK-123), which do not have mutations in pncA, could also be defined as resistant strains by SFA. There was a discrepancy between the result obtained by PZase activity or acid agar test and SFA in two clinical isolates A.3.5 and A 4.30. No phenotypic abnormality was observed in these two strains, and the reason for this discrepancy is not clear.


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  		TABLE 1. Comparison of SFA and three methods in determining the susceptibilities of 45 clinical isolates of M. tuberculosis to PZA


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  		TABLE 2. Antimycobacterial activity of PZA to the PZA-susceptible and -resistant clinical isolates of M. tuberculosis in MRC-5 SV TG1 host cells

It is interesting that the PZA MICs for PZA-susceptible isolates were between 3.13 and 12.5 µg/ml by modified SFA (Table 1), which is close to the clinical concentration of PZA reported in the lungs of the patients (about 20 µg/ml) (15). On the other hand, the MICs of PZA for the PZA-resistant strains were more than 100 µg/ml. These results strongly indicate that a SFA could determine the susceptibility of clinical isolates to PZA and also provide us with the critical information of the drug dose that is effective in vivo.

Host cell-based methods using macrophages of either human or animal origin are documented (4, 6, 14, 15), but the drawback of the use of macrophages derived from experimental animals is the heterogeneity of different batches of animal cells and consequent problems of reproducibility (13). Also, it takes more than 2 weeks to obtain results by the conventional method, because the number of live bacilli has to be counted after growth on agar plates. The SFA yields results within 3 to 4 days, which is highly advantageous compared to other host cell-based assays. In our experience, about half of the clinical isolates cannot grow in the acidic conditions at pH 5.5. Therefore, in addition to the speed with which the assay can be conducted, SFA can evaluate the clinical strains that are difficult to grow in acidic medium. Also, the strains with no mutations in the pncA gene can be defined as resistant strains by the SFA.


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ACKNOWLEDGMENTS
 
This work was supported in part by grants from the Grant-in-Aids for Scientific Research on Priority Areas (C) from the Ministry of Education, Sciences, Sports and Culture of Japan, the U.S.-Japan Cooperative Medical Sciences Program, Ohyama Health Foundation, and Takeda Science Foundation.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-Dori, Mizuho-Ku, Nagoya 467-8603, Japan. Phone: 81-52-836-3421. Fax: 81-52-836-3419. E-mail: ttakii{at}phar.nagoya-cu.ac.jp. Back


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Antimicrobial Agents and Chemotherapy, February 2005, p. 804-807, Vol. 49, No. 2
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.2.804-807.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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