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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 19  |  Issue : 1  |  Page : 6-10

Antimicrobial susceptibility testing of rapidly growing mycobacteria isolated from cases of surgical site infections by microbroth dilution method at a Tertiary Care Center


1 Department of Microbiology, AFMC, Pune, Maharashtra, India
2 Department of Microbiology, Shri Sathya Sai Medical College and Research Institute, Kancheepuram, Tamil Nadu, India

Date of Web Publication17-Aug-2017

Correspondence Address:
Raghu Sriram
Department of Microbiology, Diamond Jubilee Block, AFMC, Pune - 411 040, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jmms.jmms_19_17

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  Abstract 


Aim: The study was carried out to ascertain antimicrobial susceptibility pattern of rapidly growing mycobacteria (RGM) isolated from cases of surgical site infections from May 2013 to May 2014 at a tertiary care center in Pune. Materials and Methods: This study included the isolation of RGM from surgical site infections from May 2013 to May 2014 at a tertiary care center in Pune. The specimens were processed for microscopy (Ziehl–Neelsen stain) and culture (Lowenstein–Jensen media) using conventional methods. The minimum inhibitory concentrations of each of the isolates were determined by microbroth dilution, using Sensititre RAPMYCO manufactured by the firm TREK Diagnostic Systems from Thermo Scientific. Interpretations of results were done as per the Clinical and Laboratory Standards Institute M24-A. Results: Of the RGM isolated, Mycobacterium abscessus was the predominant isolate followed by Mycobacterium fortuitum and Mycobacterium chelonae. Of the 102 isolates, 64 (53.3%) were M. abscessus, 30 (25%) were M. fortuitum, 8 (6.67%) were M. chelonae, and 18 (15%) were other atypical Mycobacterium species. All 102 isolates of RGM were tested for antibiotic susceptibility testing by microbroth dilution. M. fortuitum and M. chelonae were sensitive to most of the antibiotics tested with few exceptions. All isolates (M. fortuitum and M. chelonae) were resistant to cefoxitin except few M. abscessus. Maximum resistance was seen with M. abscessus and they were sensitive to amikacin, tobramycin, tigecycline, and clarithromycin only. Out of 64 isolates, 32 (50%) were resistant to linezolid and imipenem. Conclusions: This study was carried out to highlight the importance of RGM and their susceptibility testing in all clinical microbiology laboratories.

Keywords: Antimicrobial susceptibility, broth microdilution, rapidly growing mycobacteria


How to cite this article:
Sriram R, Sarangan P. Antimicrobial susceptibility testing of rapidly growing mycobacteria isolated from cases of surgical site infections by microbroth dilution method at a Tertiary Care Center. J Mar Med Soc 2017;19:6-10

How to cite this URL:
Sriram R, Sarangan P. Antimicrobial susceptibility testing of rapidly growing mycobacteria isolated from cases of surgical site infections by microbroth dilution method at a Tertiary Care Center. J Mar Med Soc [serial online] 2017 [cited 2017 Nov 21];19:6-10. Available from: http://www.marinemedicalsociety.in/text.asp?2017/19/1/6/213096




  Introduction Top


Rapidly growing mycobacteria (RGM) are defined as nontuberculous species that grow within 7 days on laboratory media. They are important environmental pathogens and are capable of causing a broad spectrum of diseases.[1] At present, there are more than 130 known species of nontuberculous mycobacteria (NTM), of these seventy are species of RGM. There are currently six major groups of RGM based on pigmentation and genetic relatedness. Among these six groups, the most important species of nonpigmented groups are Mycobacterium fortuitum, Mycobacterium abscessus, and Mycobacterium chelonae responsible for 80% of diseases in humans. The rest are pigmented and of less importance clinically.[1],[2],[3]

RGM are ubiquitous organisms and can be found in municipal tap water and even form biofilms.[3],[4] They have been implicated as causative agents of a number of health care-associated outbreaks and pseudo-outbreaks.[5],[6],[7],[8],[9],[10] They cause skin and soft tissue infections, surgical site infections, pulmonary infections, bone and joint infections, and central nervous system infections. Pseudo-outbreaks of disease have been associated with contamination of equipment (bronchoscopes, automated endoscopic cleaning machines) with tap water as the source of the organism.[11],[12],[13]

It is very important to differentiate between RGM from other bacterial and mycobacterial infections because antituberculous therapy (ATT) and other empirical antibiotics are not useful for treating these organisms.[10],[11],[12],[13] The exact prevalence of diseases caused by these organisms is unclear even though there are many case reports and series from India on NTM. This study was carried out to highlight the importance of identification of different species of RGM, especially M. fortuitum, M. abscessus, and M. chelonae and also their susceptibility testing by microbroth dilution.


  Materials and Methods Top


This study included the isolation of RGM from surgical site infections from May 2013 to May 2014 at a tertiary care center in Pune. Specimens were collected in sterile containers using strict aseptic precautions. They were then transported to the laboratory and were processed immediately for microscopy (Ziehl–Neelsen [ZN] stain) and culture using standard procedures. Cultures were done by inoculation of the specimens on Lowenstein–Jensen (LJ) media and incubation at 37°C.

The cultures were examined daily during the 1st week and thereafter weekly for a further 8 weeks. Confirmation of any growth obtained within the 1st week was done by ZN staining (for demonstration of acid-fast bacilli). Identification of RGM to the species level was done using the following phenotypic characteristics; growth on MacConkey agar, growth on Mueller-Hinton agar with sodium chloride, reduction of nitrates, and susceptibility to polymyxin B (300 units) and cefoxitin.

Sensititre RAPMYCO manufactured by the firm TREK Diagnostic Systems from Thermo Scientific was used for susceptibility testing of RGM. This kit can be used for RGM including M. fortuitum group (M. fortuitum, Mycobacterium peregrinum, and M. fortuitum third biovariant complex), M. chelonae, M. abscessus, Mycobacterium mucogenicum), and Mycobacterium smegmatis group.

Suspensions for inoculation were prepared by sweeping four to five colonies with a loop and mixing in sterile water. The inoculum concentration was adjusted to 0.5 McFarland standard. Fifty microliter of the suspension was transferred into a tube of cation-adjusted Mueller-Hinton broth with TES buffer. The final inoculum was calculated to be 5 × 105 cfu/mL (range varying from 1 × 105 cfu/mL to 1 × 106 cfu/mL). From this suspension, 100 μl was transferred to each well of the Sensititre plate which was dosed with the following antimicrobial agents at appropriate dilutions (cotrimoxazole, linezolid, ciprofloxacin, imipenem, moxifloxacin, cefepime, cefoxitin, amoxicillin-clavulanic acid (AMC), amikacin, ceftriaxone, doxycycline, minocycline, tigecycline, clarithromycin, tobramycin, and positive control) [Figure 1]. The wells were covered with adhesive seal and incubation was done at 30°C. Results were read manually by visual reading of growth. Growth was ascertained by either turbidity or as a deposit of cells at the bottom of the well [Figure 2]. The interpretations of the minimum inhibitory concentrations (MICs) obtained were done according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.[14]
Figure 1: Microbroth dilution method (RAPMYCOI-Sensititre): An isolate of Mycobacterium abscessus.

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Figure 2: Reference card showing various antibiotics tested with concentration.

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  Results Top


Out of the total 102 isolates of RGM isolated from surgical site infections, M. abscessus was the predominant isolate followed by M. fortuitum and M. chelonae. Of the 102 isolates, 64 (62.74%) were M. abscessus, 30 (29.41%) were M. fortuitum, and 8 (7.84%) were M. chelonae. The RGM (M. fortuitum, M. abscessus, and M. chelonae) were tested for antibiotic susceptibility testing by microbroth dilution.

All 30 isolates of M. fortuitum (100%) were sensitive to clarithromycin, amikacin, tobramycin, cotrimoxazole, tigecycline, minocycline, and doxycycline and all 30 M. fortuitum (100%) were resistant to cefoxitin. Sensitivity to both the quinolones tested ciprofloxacin and moxifloxacin was 50% with the other 50% intermediate. Similar sensitivity was found to linezolid and imipenem [Figure 3].
Figure 3: Susceptibility pattern of Mycobacterium fortuitum.

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All 64 M. abscessus (100%) were sensitive to amikacin, tobramycin, and tigecycline. A high degree of sensitivity was also found to clarithromycin with 60 (93.75%) isolates sensitive and only 4 (6.25%) resistant. Reasonable sensitivity was seen with linezolid with 32 isolates (50%) sensitive and 32 (50%) intermediately sensitive to linezolid with no resistant strains.

Forty-three (77.2%) of the isolates were resistant to cefoxitin. The sensitivity to imipenem was very poor with only 8 (12.5%) being sensitive with 32 (50%) being intermediate and 24 (37.5%) resistant. There was poor sensitivity to ciprofloxacin with only 7 (10.9%) sensitive, 35 (54.6%) were intermediate, and 22 (34.3%) resistant to ciprofloxacin. The sensitivity was even poorer with the other quinolone tested, moxifloxacin, with only 6 (9.4%) of the isolates sensitive and 20 (31.3%) intermediate and 38 (59.3%) resistant. For the other antibiotics tested, there was a high degree of resistance to cotrimoxazole and doxycycline with 54 (84.4%) resistant to both [Figure 4].
Figure 4: Susceptibility pattern of Mycobacterium abscessus.

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All 8 M. chelonae (100%) were sensitive to clarithromycin, amikacin, tobramycin, ciprofloxacin, moxifloxacin, and tigecycline. The sensitivity to the antibiotics such as minocycline, doxycycline, cotrimoxazole, imipenem, and linezolid was identical with only four (50%) out of eight of these strains sensitive. All 8 (100%) M. chelonae isolates were resistant to cefoxitin [Figure 5].
Figure 5: Susceptibility pattern of Mycobacterium chelonae.

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  Discussion Top


RGM are being recognized as important human pathogens capable of causing a variety of diseases. The diseases they cause range from localized cutaneous infections to disseminated disease. Furthermore, they have been found to be responsible for a number of health care-associated outbreaks as well as pseudo-outbreaks.

NTM causes disease worldwide and its distribution also varies. In India, the isolation rate varies between 0.7% and 34%.[15] Among the RGM, three species, namely, M. fortuitum, M. abscessus, and M. chelonae are the most commonly reported agents causing skin and soft tissue infections, joint and bursal infections, wound infections, and injection abscesses.[12],[13] In our study, RGM constituted 102/120 (85%) of all clinically relevant NTM isolates. M. abscessus was the predominant isolate constituting 64 of the 102 isolates. In a study carried out by Jesudason and Gladstone in South India on NTM, reported 100 (2.23%) isolates of M. chelonae and M. fortuitum out of 4473 mycobacterial isolates.[16]

Antibiotic susceptibility testing of these RGM is of vital importance because of the marked difference in the susceptibility patterns not only between the different species but also in the subspecies and even in isolates. The difficulty most clinical microbiological laboratories face is that disc diffusion tests are not able to reliably ascertain the susceptibility pattern of these organisms,[17] and hence, the CLSI has recommended only the microbroth dilution method.[14]

Previous reports suggested that addition of clavulanic acid to amoxicillin enhances thein vitro activity against M. fortuitum because amoxicillin alone provides limited activity. However, contrary to this, in our study, all the isolates were resistant to AMC combination with MIC of > 64/32 μg/ml.

Recommended empirical therapy for respiratory and cutaneous infections caused by M. chelonae, M. abscessus, and by M. fortuitum usually involves the use of macrolides.[18] In our study, all 30 M. fortuitum isolates were sensitive to clarithromycin as were all eight isolates of M. chelonae. However, of the isolates of M. abscessus, 4 out of the 64 (6.25%) were resistant to clarithromycin.

Other oral options including a single dose of sulphonamides and doxycycline have been used successfully for treating cutaneous infections caused by M. fortuitum.[19] In our study, out of 30 M. fortuitum, all isolates were sensitive to cotrimoxazole and doxycycline. In contrast, out of 64 M. abscessus, isolates only 5 (7.8%) were sensitive, 5 (7.8%) were intermediate, and 54 (84.4%) resistant to cotrimoxazole and doxycycline. Out of 8 M. chelonae, 4 (50%) were sensitive and 4 (50%) were resistant to doxycycline and cotrimoxazole.

RGM do not respond significantly to agents such as ceftriaxone, cefepime, and antipseudomonal penicillins.[15] This was also borne out in our study where none of the isolates were sensitive to either cefepime or ceftriaxone by microbroth dilution method.

Cefoxitin has been used to differentiate M. chelonae which is always resistant (MIC >64 μg/ml) from M. abscessus (usual MIC range 16–32 μg/ml).[1],[2]

Recommendations also include starting empirical therapy with cefoxitin and amikacin for serious infections.[11] However, in our study, all 30 M. fortuitum (100%), 43 out of 64 M. abscessus (77.2%), and 8 M. Chelonae (100%) were resistant to cefoxitin (MIC >256 μg/ml).

All 102 isolates (M. fortuitum, M. chelonae, and M. abscessus) were sensitive to amikacin (MIC <4 μg/ml) though amikacin resistance has been reported in M. chelonae (MIC ranging from 1 to 32 μg/ml).

Few previous studies have addressed the susceptibility pattern of RGM to imipenem and linezolid. CLSI M24-A mentioned MIC of imipenem should not be reported for M. chelonae and M. abscessus. In our study, out of 30 M. fortuitum 15 (50%) were sensitive and 15 (50%) were intermediate to imipenem and linezolid. Similarly out of 64 M. abscessus, 32 (50%) were sensitive and 32 (50%) were intermediate to imipenem and linezolid. Out of 8 M. chelonae, 4 (50%) were sensitive and 4 (50%) were resistant to imipenem and linezolid.


  Conclusions Top


This study was carried out mainly to highlight the importance of identification of RGM into M. fortuitum, M. abscessus, and M. chelonae and their susceptibility testing by microbroth dilution. This is especially important due to M. abscessus being resistant to many of the available therapeutic options. RGM are also resistant to many routinely used empirical antibiotics and ATT is ineffective.

In our setting, we found that it was necessary to use multidrug therapy with macrolides as the backbone of the treatment; however, sensitivity to other oral antibiotics was very poor and sensitivity was high to injectable like amikacin. Many patients started with only a single antibiotic showed a poor response, and in some cases, a second surgery was required.

Therefore, identification of RGM up to the species level and reporting their susceptibility pattern in all clinical microbiology laboratories routinely is necessary. Recognition of these organisms as not uncommon causes of surgical site infections is vital. All samples obtained from discharging sinuses from surgical site infections should have a ZN stain and culture in LJ media done routinely.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Brown-Elliott BA, Wallace RJ Jr. Mycobacterium: Clinical and laboratory characteristics of rapidly growing mycobacteria. In: Versalovic J, editor. Manual of clinical Microbiology. 10th ed., Vol. 1. Washington, DC: ASM Press; 2011. p. 525-38.  Back to cited text no. 1
    
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Al Shaalan M, Law BJ, Israels SJ, Pianosi P, Lacson AG, Higgins R. Mycobacterium fortuitum interstitial pneumonia with vasculitis in a child with Wilms' tumor. Pediatr Infect Dis J 1997;16:996-1000.  Back to cited text no. 3
    
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Carson LA, Cusick LB, Bland LA, Favero MS. Efficacy of chemical dosing methods for isolating nontuberculous mycobacteria from water supplies of dialysis centers. Appl Environ Microbiol 1988;54:1756-60.  Back to cited text no. 4
    
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Wallace RJ Jr., Brown BA, Onyi GO. Susceptibilities of Mycobacterium fortuitum biovar. fortuitum and the two subgroups of Mycobacterium chelonae to imipenem, cefmetazole, cefoxitin, and amoxicillin-clavulanic acid. Antimicrob Agents Chemother 1991;35:773-5.  Back to cited text no. 11
    
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Wallace RJ Jr., Brown BA, Silcox VA, Tsukamura M, Nash DR, Steele LC, et al. Clinical disease, drug susceptibility, and biochemical patterns of the unnamed third biovariant complex of Mycobacterium fortuitum. J Infect Dis 1991;163:598-603.  Back to cited text no. 12
    
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Villanueva A, Calderon RV, Vargas BA, Ruiz F, Aguero S, Zhang Y, et al. Report on an outbreak of postinjection abscesses due to Mycobacterium abscessus, including management with surgery and clarithromycin therapy and comparison of strains by random amplified polymorphic DNA polymerase chain reaction. Clin Infect Dis 1997;24:1147-53.  Back to cited text no. 13
    
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