Antimicrobial Susceptibility of Standard Strains of Nontuberculous Mycobacteria by Microplate Alamar Blue Assay

In this study, 24 standard nontuberculous mycobacteria (NTM) species strains including 12 slowly growing mycobacteria strains and 12 rapidly growing mycobacteria strains were subjected to drug susceptibility testing using microplate Alamar Blue assay-based 7H9 broth. The most active antimicrobial agents against the 24 NTM strains were streptomycin, amikacin, the fluoroquinolones, and the tetracyclines. Mycobacterium chelonae, Mycobacterium abscessus, Mycobacterium bolletii, and Mycobacterium simiae are resistant to most antimicrobial agents. The susceptibility results of this study from 24 NTM standard strains can be referenced by clinicians before susceptibility testing for clinical isolates is performed or when conditions do not allow for susceptibility testing. The application of broth-based methods is recommended by the Clinical and Laboratory Standards Institute, and the documentation of the susceptibility patterns of standard strains of mycobacteria can improve the international standardization of susceptibility testing methods.


Introduction
Although the prevalence of tuberculosis is decreasing globally, increased numbers of nontuberculous mycobacteria (NTM) have been reported in human infections in recent years because of the growing number of immunosuppressed patients coupled with better diagnostic techniques [1]. Classified into rapidly growing mycobacteria (RGM) and slowly growing mycobacteria (SGM), NTM are opportunistic pathogens that can cause a wide variety of disseminated or localized diseases, particularly pulmonary, skin, and soft tissue infections. Due to the differences between even individual NTM strains, these organisms require individualized treatment that must be selected on the basis of results obtained from in vitro drug susceptibility tests (DST).
With the evolution of assay techniques, especially the wide application of a new commercially available DNA strip assay (GenoType Mycobacterium, Hain Lifescience, Nehren, Germany) [2,3], Mycobacteria can be easily identified to the species level; however, our knowledge about the susceptibility patterns of NTM is limited. Our results presented here about the susceptibilities of 15 antibiotic agents against standard NTM strains could be highly valuable for clinicians.
The methods for antimycobacterial susceptibility testing include the Clinical and Laboratory Standards Institute (CLSI) brothbased methodology [4], E-test [5], agar-based testing methods [6], and the disk elution and diffusion method [7,8]. The CLSI currently recommends Mueller-Hinton broth-based methods for RGM and Mueller-Hinton or 7H9 broth-based methods supplemented with OADC or acid-albumin-dextrose-catalase (ADC) for SGM [4]. Another broth-based method, the microplate Alamar Blue assay, has been used for years with favorable results for Mycobacterium tuberculosis complex isolates [9,10]. In this study, we used the microplate Alamar Blue assay to test the activities of 15 drugs against 24 standard NTM strains in China.

Strains
Twenty-four NTM standard strains including 12 RGM and 12 SGM were included in this study (Table 1).

Antibiotics and Chemicals
Middlebrook 7H9 broth and ADC supplement were purchased from Difco (Detroit, MI, USA). Powders of 15 antimicrobial agents including rifampicin, isoniazid, streptomycin, amikacin, kanamycin, ciprofloxacin, ofloxacin, levofloxacin, capreomycin, cefoxitin, doxycycline, minocycline, ethionamide, and p-aminosalicylic acid were purchased from Sigma-Aldrich Company (St. Louis, USA), while dipasic was purchased from Chongqing Huapont Pharmaceutical Company (Chongqing, China). Alamar Blue was purchased from AbD Serotec (Oxford, UK). All of the antibiotic solutions were prepared before the day of the experiment and stored at 270uC.

Antimicrobial Susceptibility Testing
Susceptibility testing was performed using the Middlebrook 7H9 broth microdilution method. All tests for each strain were carried out at least in duplicate. The isolates were grown on microplates. The inocula were prepared from actively growing bacteria collected from Lowenstein-Jensen slants. The strains were then adjusted with saline to a bacterial cell density of 3.0610 8 (McFarland 1.0 standard), and then adjusted to a 1:20 dilution with Middlebrook 7H9 Supplement (7H9-S) (7H9 broth +10% ADC + 0.5% glycerol). Antibiotics were serially diluted twofold in 100 mL of 7H9-S. The range of antibiotic concentrations was 256-0.5 mg/mL except for ciprofloxacin and ofloxacin, which were 128-0.25 mg/mL, and minocycline, which was 42.6-0.04 mg/mL. The final reaction volume was 200 mL (100 mL of antibiotic solution and 100 mL of bacterial suspension). Three negative controls were set in this study. Drug free control well(7H9-S+inoculum) was used to decide the time of adding alamar blue. The medium(7H9-S) without inoculum control well was used to decide the interference of 7H9-S to alamar blue and a series control wells of rifampicin concentration gradients of rifampicin and 7H9-S mixture were also used to decide the interference of the color of rifampicin-7H9-S mixture to alamar blue. The plates were sealed in individual Ziploc bags and then incubated at 37uC.
After 24 h, the first drug-free growth control wells were examined using indicator (20 mL of Alamar Blue and 50 mL of sterile 5% Tween-80). The plates were then re-incubated for 8 h. If the control well turned pink, all of the other wells received the indicator. After a further 24 h of incubation, the colors of all wells were recorded. If the first drug-free growth control well did not change to pink, the second drug-free control well received the indicator and the above steps were repeated. Each minimum inhibitory concentration (MIC) was read on the 3 rd to 6 th days. The MIC was defined as the lowest drug concentration that prevented a change in color. The final result of MIC of each drug for each strain was the mean value from two tests. The MIC breakpoints of the drugs indicating sensitivity, moderate susceptibility, and resistance were interpreted according to the approved guidelines established by the National Committee for Clinical Laboratory Standards [4] and World Health Organization guidance [11] (Table 2) except that minocycle was according to Vanitha et al [12].

Results
The results of the antimicrobial susceptibility testing of the 12 standard RGM and 12 standard SGM strains are shown in Table 1. All 24 strains were highly resistant to isoniazid (all MIC . 256 mg/mL). Nine and six of 12 RGM strains were resistant to rifampicin and streptomycin, respectively, especially Mycobacterium abscessus and Mycobacterium bolletii, which showed very high resistance, while seven and two of 12 SGM strains were resistant to rifampicin and streptomycin, respectively. Among members of the Mycobacterium chelonae-abscessus complex, M. abscessus was more resistant to rifampicin than M. chelonae.
All of the NTM strains were sensitive or moderately susceptible to amikacin. However, most of the standard NTM strains (19/23) were resistant to the aminoglycoside kanamycin. The 12 SGM strains were all susceptible to amikacin, whereas only one SGM (Mycobacterium senegalense) was susceptible to kanamycin.
All of the standard NTM strains were highly resistant to capreomycin (MIC . 256 mg/mL) except for M. scrofulaceum and Mycobacterium gordonae, which were low-level resistant (MIC = 4 mg/mL).
Twelve NTM strains (6/12 RGM strains and 6/12 SGM strains) were sensitive or moderately susceptible to the b-lactam antibiotic cefoxitin, while the other strains were highly resistant. Cefoxitin was the only antibiotic agent that M. chelonae was more resistant to than M. abscessus (.256 mg/mL vs. 64 mg/mL). Dipasic is a mixture of p-aminosalicylic acid and isoniazid, and its MIC breakpoints (mg/mL) corresponded to those of paminosalicylic acid in this study (the MIC of dipasic for these 24 strains were all . 256 mg/mL). As such, all 24 NTM strains were resistant to p-aminosalicylic acid and dipasic.

Discussion
Analyzing the susceptibility results of 24 standard NTM strains using the microplate Alamar Blue assay, it became evident that these findings yielded important clues for the optimization of NTM species-specific therapy. The results showed that streptomycin, amikacin, the fluoroquinolones, and the tetracyclines were the most active antimicrobial agents against the 12 RGM and 12 SGM strains. This is the first report of susceptibility patterns of standard NTM strains.
It is well know that most NTM strains are resistant to conventional anti-tuberculous agents [13][14][15][16], a fact that was further proven by the current findings. However, more SGM strains were susceptible to the drugs than RGM stains. Most of the SGM strains(10/12) were also susceptible to streptomycin. Several reports have shown that amikacin has good activity against RGM [7,[17][18]. In this study, 22 NTM strains were susceptible to and 2 NTM strains (M. abscessus and M. chelonae) were moderately susceptible to amikacin.
M. abscessus is naturally sensitive to amikacin, cefoxitin, and imipenem [19] and very resistant to many other chemotherapeutic agents [20]. In our study, M. abscessus was resistant to 13 antibiotics and moderately susceptible to amikacin and cefoxitin. These findings are comparable to those described in the other studies. A total of 38 (95%) isolates in a Taiwanese study (40 isolates of M. abscessus isolates obtained from January 2006 to December 2008) and 73 (99%) isolates in a Korean study (74 isolates of M. abscessus isolates obtained from July 2005 to December 2006) of in vitro antimicrobial susceptibility were sensitive to amikacin [21][22]. M. abscessus and M. chelonae are members of the M. chelonae-abscessus complex, the susceptibility patterns of which are similar, although M. abscessus was more resistant than M. chelonae to rifampicin, ciprofloxacin, ofloxacin, and levofloxacin. Cefoxitin was the only antibiotic agent tested here that M. chelonae was more resistant to than M. abscessus (.256 mg/mL vs. 64 mg/mL), suggesting that cefoxitin resistance could be a way to distinguish between M. chelonae and M. abscessus.
In summary, M. abscessus, M. chelonae, and M. bolletii were resistant to almost all 15 antimicrobial agents, while the other nine standard RGM strains were resistant to 4-11 drugs (Figure 1). A total of 11 SGM standard strains were resistant to 6-11 drugs, while M. simiae was resistant to 14 drugs (Figure 2).
The American Thoracic Society advocated the use of macrolide-based multidrug regimens for NTM treatment [13], and some studies have reported that most NTM strains are sensitive to the macrolide clarithromycin [6,27] and that rifabutin and tigecycline also showed high activity against many NTM strains [6,17]. As such, streptomycin, amikacin, the fluoroquinones, the tetracyclines, and the above three antibiotics were the alternative choices for the treatment of NTM infection. Studies have shown that the susceptibilities of clinical NTM isolates of a species were also quite different [21,27], so our results from NTM standard strains can only be referenced by clinicians before susceptibility testing for clinical isolates is performed or when conditions do not allow for susceptibility testing. Susceptibility testing for clinical isolates should always be performed prior to treatment unless conditions do not permit such. The technique described here can offer the MIC of antimicrobial agents within 6 days. The microplate Alamar Blue assay is inexpensive and reliable for the DST of NTM. Alternatively, the application of broth-based methods is recommended by the CLSI and the susceptibility patterns of standard strains of Mycobacterium can improve the international standardization of susceptibility testing methods.