Identification of environmental mycobacteria isolated from Agra, north India by conventional & molecular approaches.
OBJECTIVE
Several environmental mycobacteria have been shown to be important human pathogens linked to immunomodulation especially in relation to effect on vaccination. Hence identification of mycobacteria to the species level is not only relevant to patient management but also to understand epidemiology of mycobacterial diseases and effect on vaccination. We undertook this study to assess the usefulness of various conventional and molecular methods in identification of environmental mycobacterial species from Agra, north India.


METHODS
One hundred nineteen isolates of environmental mycobacteria were grown from 291 (116 soil and 175 water) samples. These isolates were identified by standard biochemical tests, and a simple, rapid and cost-effective in-house developed gene amplification restriction analysis targeting 16S-23S rRNA spacer and flanking region and 16S rRNA sequencing.


RESULTS
Biochemical tests could clearly identify only 68.1 per cent (81/119) of isolates to species level. An in-house developed gene amplification--restriction analysis method could confirm the identity of 102 of 119 (85.7%) isolates and the remaining 17 isolates (14.3%) were confirmed by 16S rRNA sequencing also. These 119 environmental mycobacterial isolates, included several potentially pathogenic species such as M. fortuitum, M. chelonae, M. avium, M. marinum, M. manitobense, M. kansasii and others belonged to nonpathogenic species, M. terrae, M. smegmatis and M. flavescens. M. chelonae was isolated from water samples only whereas M. fortuitum was isolated from both water as well as soil samples.


CONCLUSIONS
The in-house developed gene amplification restriction analysis method though failed to accurately identify 14.3 per cent of isolates, facilitated rapid differentiation of most of environmental mycobacteria including potential pathogens from this area and thus would have diagnostic potential in cases with NTM infections. This combination strategy using PCR-RFLP and 16S rRNA sequencing may be useful for characterization of mycobacteria from similar environmental settings from other parts of world.
Parashar
Deepti
D
National JALMA Institute for Leprosy & Other Mycobacterial Diseases, Agra, India.
Das
Ram
R
Chauhan
D S
DS
Sharma
V D
VD
Lavania
Mallika
M
Yadav
V S
VS
Chauhan
S V S
SV
Katoch
V M
VM
D054458:Amplified Fragment Length Polymorphism Analysis / Q000379:methods*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004783:Environmental Microbiology; D007194:India; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000145:classification / Q000235:genetics*; D012335:RNA, Ribosomal / Q000235:genetics; D017422:Sequence Analysis, DNA; D013045:Species Specificity
D017931:DNA Primers; D012335:RNA, Ribosomal