Optimization of Procedures for Isolation of Mycobacteria from Soil and Water Samples Obtained in Northern India

For isolation of environmental mycobacteria, a decontaminationprocedure has been standardized by which treatment with 3% sodiumdodecyl sulfate plus 4% NaOH (15 and 30 min for rapid and slowgrowers, respectively) is followed by incubation with 2% cetrimide(5 and 15 min for fast- and slow-growing mycobacteria, respectively);this procedure was found to completely eliminate contaminationwith other organisms and resulted in the isolation of only mycobacteria.

INTRODUCTION

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Introduction

Several species of environmental mycobacteria have been knownto be important human pathogens (12). Further exposure to themis believed to alter immunity to vaccines like Mycobacteriumbovis BCG (11). Isolation of mycobacteria from environmentalsamples is difficult because other microbes are also presentin the environment. All mycobacterial species are not equallyresistant to the different decontamination procedures. For theisolation of mycobacteria from environmental samples, such assoil and water, different methods have been described by variousworkers (1-10). These are not universally applicable becauseof differences between floras. No studies of this issue hadpreviously been carried out in the northern parts of India.For this reason, the present study was undertaken to selector develop an improved, appropriate decontamination method(s)for the isolation of mycobacteria from the predominantly hot,dry environment of Agra, India (annual temperature, maximumof 16 to 47°C and minimum of 3 to 30°C; humidity, maximumof 49 to 100% and minimum of 28 to 70%).

After we tried different permutations and combinations in controlledexperiments, the following procedure was standardized (Fig.1). Wet soil samples of approximately 5 g were collected froma depth of 3 cm, and 50-ml water samples were collected fromditches, ponds, lakes, and rivers in the Agra region throughoutthe year. Soil was suspended in 20 ml of double-distilled autoclavedwater (D/W) in polycarbonate centrifuge tubes. After being shakenmanually for 60 s, the suspension was centrifuged at 600 x gfor 5 min at 4°C to pellet the soil particles. The turbidsupernatant (10 ml) was transferred into other sterile centrifugetubes and centrifuged at 8,000 x g for 15 min at 4°C. Watersamples were centrifuged at 8,000 x g for 15 min at 4°C.Pellets from the soil and water samples were resuspended in20 ml of treatment solution (3% sodium dodecyl sulfate [SDS]plus 4% NaOH) and then divided into two parts: A and B. PartA was incubated at room temperature (RT) for 15 min to obtainthe growth of rapid growers, and part B was incubated at RTfor 30 min to obtain the growth of slow growers. After incubation,both the suspensions were centrifuged at 8,000 x g for 15 minat 4°C, and then the supernatants were decanted. Sedimentswere processed for cetrimide treatment. In the initial pilotexperiments, various incubation periods with 2% cetrimide treatmentwere tried for slow and rapid growers. The pellets were resuspendedin 20 ml of 2% cetrimide. Part A was incubated at RT for 5 minto obtain the growth of rapid growers, and part B was incubatedat RT for 15 min to obtain the growth of slow-growing mycobacteria,following which the suspensions were centrifuged at 8,000 xg for 15 min at 4°C. Subsequently the pellets were washedtwice with 20 ml of D/W and finally resuspended in 0.5 ml ofD/W. A 0.1-ml sample of the suspension was inoculated on Lowenstein-Jensen(L-J) slants in duplicate and incubated at 30 and 37°C.

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FIG. 1. Flowchart showing decontamination procedure used for mycobacteria isolated from environmental samples.

Isolation of mycobacteria from water.

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Isolation of mycobacteria from...

When 3% SDS plus 1% NaOH was used with water samples, no mycobacteriacould be isolated because all of the samples showed contaminationwith other organisms. With 3% SDS plus 2% NaOH and with 3% SDSplus 4% NaOH, more than 50% of the samples were found to becontaminated. Finally, treatment with 3% SDS plus 4% NaOH followedby 2% cetrimide was found to be best, as it succeeded in totallyeliminating contamination, and both slow and rapid growers couldbe isolated. M. avium, M. kansasii, M. terrae, M. marinum, M.fortuitum, and M. chelonae were isolated from these specimens(Table 1).

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TABLE 1. Effects of different concentrations of NaOH and cetrimide with 3% SDS on isolation of mycobacteria from water samples

Isolation of mycobacteria from soil.

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As with the water samples, when 3% SDS plus 1% NaOH was used,all the soil samples showed contamination. Treatments with 3%SDS plus 2% NaOH and with 3% SDS plus 4% NaOH were found tobe successful for 62.5 and 75% of the samples, respectively.When the same eight samples were processed by treatment with3% SDS plus 4% NaOH and 1% cetrimide and with 3% SDS plus 4%NaOH and 2% cetrimide, the success rates were found to be 87.5and 100%, respectively, and both the slow-growing mycobacteriaM. avium and M. terrae and the fast-growing mycobacteria M.fortuitum and M. chelonae were isolated (Table 2).

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TABLE 2. Effects of different concentrations of NaOH and cetrimide with 3% SDS on isolation of mycobacteria from soil samples

It is known that all mycobacterial species are not equally resistantto the different decontamination procedures (5). Falkinham etal. (4) and Reznikov and Leggo (9) had originally developedthe methods for the isolation of mycobacteria, particularlythose belonging to the M. avium-M. intracellulare-M. scrofulaceumcomplex from soil. Engbaek et al. (2) have used five methodsfor decontamination, and the sodium lauryl sulfate method wasreported as most suitable. Kamala et al. (7) found treatmentwith 3% SDS in combination with 1% NaOH to be the most effectivedecontamination method for soil as well as water samples. Whenthis procedure was followed in our study, contamination couldnot be removed from any of specimens, which is obviously dueto the differences between the range of contaminants presentin the samples collected for the present study and that in samplesfrom southern India. In an attempt to further improve successrates, the technique was modified by trying different concentrationsof reagents (from 1 to 4% NaOH and 1 to 2% cetrimide) for decontamination.Finally, 3% SDS plus 4% NaOH with 2% cetrimide appears to bemore useful, at least in our cases. The 100% success rate obtainedby this procedure implies that either only mycobacteria wereisolated or no contamination occurred. However, different procedureswere followed for rapid and slow growers, because if the samemethod was followed for both, the rapid growers survived, theL-J slants were full with their growth, and no zone on the L-Jslants was left for the slow growers to survive. For this reason,the time of decontamination treatment was increased. When thetime of treatment with 3% SDS plus 4% NaOH was increased from15 to 30 min and the time of 2% cetrimide treatment was increasedfrom 5 to 15 min, the rapid growers were killed or inhibited,but the slow growers survived. One spiking experiment in whichwater and soil samples (which were positive for fast-growingmycobacteria) were spiked with M. avium (a slow grower) anddecontaminated with the method employing increased NaOH andcetrimide treatment times (30 and 15 min, respectively) yieldeda 100% rate of isolation of M. avium.

The experience of our study indicates that in-house methodsshould be developed for the efficient recovery of environmentalmycobacteria from various settings in different parts of theworld. In the case of water and soil samples, 3% SDS plus 4%NaOH followed by 2% cetrimide treatment yielded more mycobacterialisolates than 3% SDS plus 4% NaOH and 1% cetrimide. The informationgenerated from this study will have a wider application valuefor the development or optimization of methods for undertakingsuch studies of similar environmental conditions in other partsof the world.

ACKNOWLEDGMENTS

This work was supported by grants from the Department of Biotechnologyof the Government of India (grant no. BT/PR/1253/MED/09/203/98)and LEPRA, Colchester, United Kingdom.

We thank Sri Ram and Harishankar for their technical support.

FOOTNOTES

* Corresponding author. Mailing address: Central JALMA Institute for Leprosy and Other Mycobacterial Diseases (ICMR), Tajganj, Agra 28201, India. Phone: 91-562-2331756. Fax: 91-562-2331755. E-mail: jalma{at}sancharnet.in.

REFERENCES

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