Antibiotic susceptibility pattern of rapidly growing mycobacteriaR Gayathri, K Lily Therese, P Deepa, S Mangai, HN Madhavan
Larsen and Toubro Microbiology Research Centre, Vision Research Foundation, 18, College Road, Chennai - 600 006, India
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0022-3859.65278
Source of Support: None, Conflict of Interest: None
Background : The rapidly growing mycobacteria (RGM) causing human infections primarily consist of the Mycobacterium fortuitum group, Mycobacterium abscessus and Mycobacterium chelonae. The antibiotic susceptibility testing is important to determine the appropriate therapy as the antibiotics used to treat RGM are different from those used for treating infections caused by slow growers of mycobacteria. Aim : To determine antibiotic susceptibility of RGM using Kirby Bauer method and following Clinical and Laboratory Standards Institute (CLSI) guidelines. Settings and Design : Larsen and Toubro Microbiology Research Centre, Vision Research Foundation, Sankara Nethralaya, Chennai, Retrospective study. Materials and Methods : The antibiotic susceptibility testing was performed following CLSI method for the drugs Amikacin, Azithromycin, Tobramycin, Ceftazidime, Cephotaxime, Cefuroxime, Cefaperazone, Ceftriaxone, Ciprofloxacin, Ofloxacin, Norfloxacin, Gatifloxacin and Moxifloxacin. Results and Conclusions : Out of the 148 RGM isolates 146 (98%) were susceptible to amikacin, 138 (91%) to gatifloxacin, 132 (87%) to moxifloxacin, 122 (76%) to ciprofloxacin and 116 (74%) to Norfloxacin. Majority of the RGM were resistant to Ceftazidime, Cephotaxime and Cefaperazone. All the M. abscessus isolates were resistant to tobramycin. The in vitro antibiotic susceptibility testing by disc diffusion method showed that majority of the RGM were sensitive to Amikacin followed by Gatifloxacin, Moxifloxacin and Ciprofloxacin
Keywords: Amikacin, antibiogram, rapid growers of mycobacteria
The growing population of HIV-infected individuals and other immunosuppressed patients coupled with better diagnostic techniques has led to an increase in the number of non-tuberculous mycobacteria (NTM) being reported in human infections in recent years.  Relatively few drugs are effective against these NTM and new drugs potentially active against these bacteria are needed.  Mycobacterium fortuitum I (M. fortuitum) group, Mycobacterium chelonae (M. chelonae) and Mycobacterium abscessus bscessus) are the species of rapid growers of mycobacteria (RGM) most often associated with human diseases. , These organisms cause a wide variety of disseminated or localized diseases, particularly pulmonary infections as well as primary skin and soft tissue infections. Pseudo outbreaks of infections due to these organisms caused by contaminated medical equipment have been reported.  These organisms are resistant to the conventional anti-tuberculous agents and some investigators have reported that in vitro susceptibilities to several of these agents correlated with clinical response to therapy.  Considering the paucity of data on the subject, we decided to perform a study to determine the susceptibility of RGM to fluoroquinolones, macrolides, cephalosporins and amikacin.
RGM strains isolated from various clinical specimens received at our laboratory from January 2000-July 2008 were included in this retrospective study after obtaining the ethical clearance from the institutional ethical review board. Antibiotic susceptibility testing was performed by the Kirby Bauer Method following the Clinical and Laboratory Standards Institute (CLSI) guidelines and as described previously. ,,, Antibiotics tested and concentration of antibiotics is mentioned in [Table 1]. Susceptibility to Norfloxacin, Gatifloxacin and Moxifloxacin was not performed for 20 RGM isolated during 2000-01.
The distribution of the 148 RGM isolates from various clinical specimens is shown in [Table 2]. Majority of RGM were M. abscessus consisting of 77 (52%) strains, followed by 66 (44.6%) strains of M. fortuitum and one each of M. smegmatis, M. chelonae, M. immunogenum, M. farcinogenes, M. fortuitum III biovariant complex. All the isolates were identified to species level by conventional biochemical tests  and confirmed by polymerase Chain Reaction (PCR) based techniques using species-specific primers for M. fortuitum and M. chelonae,  PCR based Restriction fragment length polymorphism (RFLP)targeting hsp65 gene  and by PCR-based DNA sequencing technique targeting hsp65 gene.
The results of in vitro antimicrobial susceptibility testing of M. abscessus and M. fortuitum are given in [Table 1]. All the 77 M. abscessus were resistant to Tobramycin which is one of the important tests for differential identification of M. chelonae and M. abscessus.
Among the 77 M. abscessus isolates, 75 were sensitive to Amikacin and two of the M. abscessus isolates, one each from sputum and corneal scraping were resistant to Amikacin. Ofloxacin and Norfloxacin were active against M. chelonae, M. immunogenum and M. farcinogenes. Moxifloxacin and Azithromycin were active against M. chelonae, M. smegmatis, M. immunogenum and M. farcinogenes. Tobramycin was active only against M. chelonae, M. smegmatis and M. fortuitum III biovariant complex. M. chelonae, M. smegmatis and M. immunogenum were susceptible to Cefaperazone and Cefuroxime among the cephalosporins.
Majority of the RGM were resistant to cephalosporins. The resistance to cephalosporins ranged from 75-84% for M. abscessus and 71-86% for M. fortuitum. The highest percentage of resistance for M. abscessus and M. fortuitum was found with ceftriaxone (84 and 89% respectively) followed by cefaperazone (78 and 86% respectively).
Among the 66 isolates of M. fortuitum, all (100%) were susceptible to Amikacin, followed by Gatifloxacin (92%), Moxifloxacin (86%), Ciprofloxacin (86%), Norfloxacin (74%) and Azithromycin (64%).
The methods for antimycobacterial susceptibility testing include CLSI broth-based methodology, E-test, agar-based testing methods, disk elution method, disk diffusion method, flow cytometry and radiometric methods.  The above mentioned methods except the agar-based testing methods are not affordable to the routine laboratories as additional infrastructure facilities with sophisticated equipments are required. In order to find a suitable, reliable and economical method we attempted the disk diffusion method of antibiotic susceptibility testing on RGM.
Aminoglycosides are important parenteral antibiotics used in the treatment of M. abscessus infection. Traditionally, amikacin was the most active agent against RGM. According to the study by Swenson et al.,  95% of M. abscessus, 99% of M. fortuitum and 88% of M. chelonae were susceptible to Amikacin with a MIC of < 16 μg/ml. In the study by Park et al.,  99% of M. abscessus were susceptible to Amikacin. In the present study, more than 98% isolates were susceptible to amikacin. Among the fluoroquinolones, 91% of M. abscessus and 92% of M. fortuitum were susceptible to gatifloxacin followed by Moxifloxacin (88% of M. abscessus and 86% of M. fortuitum) and ciprofloxacin (82% of M. abscessus and M. fortuitum). Our results showed an increased susceptibility to fluoroquinolones when compared with the study by Park et al. 
M. abscessus is known to be susceptible to amikacin, cefoxitin and clarithromycin  . Among the macrolides, 70% of RGM were susceptible to Azithromycin. It is interesting to note that most of the RGM were resistant to cephalosporin groups of drugs in this study. In the study by Park et al.,  36% of M. abscessus isolates were susceptible to tobramycin, but in our study all the 77(100%) strains of M. abscessus were resistant to Tobramycin. When comparing the sensitivity pattern between ocular and respiratory isolates, ocular isolates of M. abscessus and M. fortuitum showed an increase in resistance to Ciprofloxacin, ofloxacin and Norfloxacin while respiratory isolates of M. abscessus and M. fortuitum showed an increase in resistance to gatifloxacin. There was no difference in the sensitivity pattern of the ocular and respiratory isolates of M. abscessus and M. fortuitum against moxifloxacin. Amikacin, ciprofloxacin and ofloxacin are considered the drugs of choice. 
NTM comprising of over 153 species are naturally seen as saprophytes  but are known to cause four different categories of infections in humans, such as pulmonary infections resembling tuberculosis, extra-pulmonary infections affecting lymph nodes, skin and soft tissue, multifocal disseminated infections and infections in immunocompromised individuals such as AIDS and transplant patients. 
Although there are many reports from India, the exact disease burden and the antibiotic susceptibility pattern of RGM infections still remains unclear in India. These infections are under-diagnosed in many laboratories due to the lack of facilities and expertise. These organisms can be misidentified as Corynebacterium spp. or Nocardia spp.  Recognition of colonies on blood agar, confirming any poorly staining gram-positive bacilli on gram stain with Ziehl Nielsen stain would be helpful in establishing the etiology. In addition molecular methods such as PCR-based RFLP and DNA sequencing help in the rapid identification of RGM.
The management of RGM infections includes medical treatment with various antimicrobial agents based on susceptibility patterns and surgical treatment as in the case of lymphadenitis, skin or soft tissue infections. Since most of these organisms are resistant to commonly used antimicrobial agents, susceptibility testing becomes mandatory before instituting an effective therapy. The RGM show varying degree of susceptibility to the commonly used antibiotics including the fluoroquinolones, aminoglycosides and erythromycin.  To conclude, the in vitro susceptibility testing results of RGM isolates indicate that Amikacin is the drug of choice followed by the newer generation of fluoroquinolones.
[Table 1], [Table 2]