Mycobacteriology
Evaluation of sputum decontamination methods for Mycobacterium tuberculosis using viable colony counts and flow cytometry

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Abstract

Continuous monitoring systems for the detection of Mycobacterium tuberculosis are reported to have higher contamination rates than traditional radiometric technologies. Multiple decontamination methods have recently been reported in an attempt to optimize contamination rates for these systems. In this study, several decontamination methods for sputum were evaluated using viable colony count and flow cytometry. The decontamination protocols evaluated include N-Acetyl-L-Cysteine-Sodium Hydroxide (NALC-NaOH), modified Petroffs’s method, and the Yamane procedure. Several parameters of the NALC-NaOH method were analyzed including final NaOH concentrations of 0.5-3%, NaOH exposure times of 0-30 min, and variations in resuspension media for the resultant pellet. All decontamination methods were performed on pooled and sterilized sputum seeded separately with either a mixture of common contaminating bacteria or M. tuberculosis H37Ra. Viability of organisms following decontamination was assessed by both colony counts and flow cytometric analysis. Flow cytometry viability assays utilized a combination of viability dyes and reference beads to determine viable organism concentrations. The results indicated that no decontamination method was clearly superior, however a concentration of 1-2% NaOH and an increase in the time of NaOH exposure to 30 min will effectively kill contaminating bacteria without significantly affecting the viability of M. tuberculosis H37Ra. While flow cytometry viability analysis did not directly correspond to viable colony counts, it was a useful tool for rapid viability analysis M. tuberculosis.

Introduction

New instrumentation for detection of Mycobacterium tuberculosis such as the MGIT 960 (Becton Dickinson, Sparks, MD), ESP II (Difco Laboratories, Detroit, Mich.) and the MB\BacT (Organon Teknika, Durham, NC) have several advantages over the traditional BACTEC 460 (Becton Dickinson) system including non-radioactivity and continuous monitoring Benjamin et al 1998, Hanna et al 1999, Kanchana et al 2000, Tortoli et al 1998, Tortoli et al 1999. However, there have been a number of reports indicating that the contamination rates of these new systems are higher than the established BACTEC 460 technology Benjamin et al 1998, Hanna et al 1999, Kanchana et al 2000, Tortoli et al 1998, Tortoli et al 1999.

Most clinical sputum samples contain a variety of microorganisms that may quickly overgrow M. tuberculosis (Nolte et al., 1995) and several methods for decontamination of sputum for the recovery of M. tuberculosis exist. The use of NaOH for the decontamination of specimens is widely used, however alkali conditions are toxic not only to contaminating organisms but to mycobacteria as well (Kent and Kubica, 1985). While the N-Acetyl-L-Cysteine-Sodium Hydroxide (NALC-NaOH) method is the accepted gold standard, others have published newer methods of decontamination. Examination and evaluation of these various methods of decontamination of clinical specimens may facilitate reduced contamination rates of these new detection systems. It is unclear in the literature which method provides maximum decontamination while maintaining the viability of M. tuberculosis. Many of these studies focus on decontamination methods involving clinical specimens where the exact number of contaminating bacteria and M. tuberculosis is unknown and may vary from sample to sample (Thorton et al., 1998), or examine positivity and contamination rates of seeded clinical samples Ratnam et al 1987, Thorton et al 1998, Whittier et al 1993. In both cases, the degree of loss of viability for M. tuberculosis has not been examined.

Viability studies using colony count methods are costly, time consuming, and laborious due to the slow growing nature of the organism. Flow cytometry is an alternative technology that may overcome these obstacles by rapidly and easily assessing viability of M. tuberculosis and other bacteria. Flow cytometry has been used previously to examine viability of many bacterial species for a variety of clinical and environmental applications Boulos et al 1999, Clarke and Pinder 1998, Duffey and Sheridan 1998, Jacobsen et al 1997, Virta et al 1999, including rapid susceptibility testing for M. tuberculosis Kirk et al 1998, Moore et al 1999, Norden et al 1995. A correlation of viability measured by viable cell counts and flow cytometry would enable this technology to be used for evaluating, comparing, and optimizing specific methodologies for new decontamination methods in the future.

In the present study, different decontamination methods are critically evaluated by assessing the loss of viability of both contaminating organisms and M. tuberculosis H37Ra with a viable colony count method and flow cytometry. The decontamination protocols evaluated include the N-Acetyl-L-Cysteine-Sodium Hydroxide (NALC-NaOH) method, the modified Petroffs’s method, and the Yamane procedure. For each method, formulations were varied to identify optimum conditions. Flow cytometric viability assays were performed using a combination of a bacteria counting kit (Molecular Probes, Eugene, OR) for determining cell concentrations and the LIVE/DEAD BacLight kit (Molecular Probes) for determining viability.

Section snippets

Strains

Mycobacterium tuberculosis H37Ra ATCC 25177, Staphylococcus aureus ATCC 29213 Staphylococcus epidermidis ATCC 27853, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, and clinical isolates of viridans streptococcus and Moraxella catarrhalis were used for the decontamination experiments. The bacterial species were chosen to be representative of common bacterial contaminants of sputum. P. aeruginosia was included due to the difficulty associated with decontamination of specimens

Results

Examples of flow cytometric analysis of contaminating bacteria and H37Ra post decontamination are presented in Fig. 1. Both bacteria and H37Ra were stained with the bacteria counting kit (Fig. 1A and C) and the two populations (R1- reference beads and R2–organisms) were easily distinguished. The LIVE/DEAD viability staining produced two populations (R1- live and R2-dead) with both the contaminating bacteria and H37Ra (Fig. 1B and D). Events were scored as either “live” or “dead” depending upon

Discussion

The results of the viable cell count experiments indicate that there is no clearly superior method between NALC-NaOH, modified Petroffs’ method, or the Yamane procedure. All of the evaluated decontamination methods are similar to the extent that they all rely on NaOH for decontamination and variations in NaOH conditions may equally affect all methods. With the NALC-NaOH method, the greatest amount of contaminating bacterial with the least reduction in H37Ra viability occurred at a concentration

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