Identification and characterization of breakthrough contaminants associated with the conventional isolation of Mycobacterium tuberculosis

Abstract

Breakthrough contamination of tuberculosis (TB) cultures is a problem in that it allows the overgrowth of another bacterium present in the sputum specimen, which can potentially mask the presence of Mycobacterium tuberculosis. The aim of this study was to isolate and characterize the bacterial organisms responsible for such overgrowth and contamination, and to examine their susceptibility to (i) various chemical selective decontamination steps and (ii) antibiotics in liquid culture media, in an attempt to develop a method to help alleviate contamination problems associated with the conventional isolation of M. tuberculosis from routine patient sputum specimens. Bacterial contaminants from 102 routine sputum cultures were identified molecularly by 16S rRNA gene PCR and direct sequencing from contaminated Lö wenstein-Jensen (LJ) slopes and BacT/Alert liquid medium. It was found that the contaminants from LJ slopes belonged to 11 different genera and were composed largely of Gram-negative organisms (84.9 %; 45/53), whereas the liquid culture contaminants belonged to 13 different genera, with 37/66 isolates (56.1 %) being Gram-negative. Pseudomonas aeruginosa was the dominant contaminant in both media. The effect of six different selective decontamination protocols was examined. Four of the six methods were effective at eliminating all culturable organisms present; these were 5 % oxalic acid, 5 % oxalic acid/2 % NaOH, 5 % oxalic acid/4 % NaOH and 1 % chlorhexidine. NaOH at a concentration of 2 or 4 % was less effective as it was unable to eliminate all organisms of each species tested, with the exception of P. aeruginosa. In conclusion, breakthrough contamination of TB cultures is due to a diverse range of at least 17 different bacterial genera, with P. aeruginosa and Staphylococcus epidermidis accounting for the dominant contaminating flora. Employment of chemical decontaminating protocols solely involving NaOH may lead to higher rates of contamination. Where such contamination is encountered, TB laboratories should consider the reprocessing of such sputum samples with an alternative decontamination method such as 1 % chlorhexidine.

Evaluating decontamination protocols for the isolation of Mycobacterium ulcerans from swabs

“…Similar observations were made by McClean et al [11], where they also observed that P. aeruginosa w as the predominant contaminant associated with the conventional isolation of Mycobacterium tuberculosis . That study also reported that 5% oxalic acid alone and in combination with NaOH was effective in eliminating P. aeruginosa [11]. The challenge in eliminating Aspergillus niger with the chemical agents used in the current study could be attributed to the fact that, most vegetative fungi and fungal spores are more resistant to decontamination agent.…”

“…In spite of high activity recorded by some of the chemical agents, P. aeruginosa and A. niger proved to be the most challenging to inhibit among all the microbial isolates tested . This finding brings to the fore the intrinsic resistance properties exhibited by P. eruginosa to most antimicrobials [11]. Similar observations were made by McClean et al [11], where they also observed that P. aeruginosa w as the predominant contaminant associated with the conventional isolation of Mycobacterium tuberculosis .…”

“…This finding brings to the fore the intrinsic resistance properties exhibited by P. eruginosa to most antimicrobials [11]. Similar observations were made by McClean et al [11], where they also observed that P. aeruginosa w as the predominant contaminant associated with the conventional isolation of Mycobacterium tuberculosis . That study also reported that 5% oxalic acid alone and in combination with NaOH was effective in eliminating P. aeruginosa [11].…”

“…The culture process requires the use of diagnostic specimens from BU lesions. Faster growing microbial contaminants in BU lesions overgrow the M.ulcerans cultures, affecting their yield [11]. The reduction of these contaminants is usually achieved by processing the samples with chemical agents having selective antimicrobial properties in a process referred to as decontamination [12].…”

The Evaluations of Bleach as Decontaminant Solution to Promote The Positivity Rate of Mycobacterium Tuberculosis Culture for Sputum Specimen

“…4 Another study also found that 2% NALC-NaOH more ease to be contaminated rather than the 4% NaOH method. 6,9 NALC is a good agent as mucolytic, but not as decontaminant, this factor could lead the capability of NALC-NaOH in recovery rate is weaker than Bleach NaOH NALC Bleach Oxalic bleach or NaOH and mask the growth of Mycobacterium tuberculosis. 2,6,10 Based on the number of contamination rate, 5% oxalic acid that had low number of positivity rate was also had a great number in contamination.…”