Molecular epidemiology of nontuberculous mycobacteria isolates from clinical and environmental sources of a metropolitan city

Abstract

Introduction: While NTM infection is mainly acquired from environmental exposure, monitoring of environmental niches for NTM is not a routine practice. This study aimed to find the prevalence of environmental NTM in soil and water in four highly populated suburbs of Tehran, Iran.

Material and methods: A total of 4014 samples from soil and water resources were collected and studied. Sediments of each treated sample were cultured in Lowenstein-Jensen medium and observed twice per week for growth rate, colony morphology, and pigmentation. Colonies were studied with phenotypic tests. Molecular analysis was performed on single colonies derived from subculture of original isolates. Environmental samples were compared with 34 NTM isolates from patients who were residents of the study locations.

Results: Out of 4014 samples, mycobacteria were isolated from 862 (21.4%) specimens; 536 (62.1%) belonged to slow growing mycobacteria (SGM) and 326 (37.8%) were rapid growing mycobacteria (RGM). The five most frequent NTM were M. farcinogens (105/862; 12.1%), M. fortuitum (72/862; 8.3%), M. senegalense (58/862; 6.7%), M. kansasii (54/862; 6.2%), and M. simiae (46/862; 5.3%). In total, 62.5% (539/862) of mycobacterial positive samples were isolated from water and only 37.4% (323/862) of them were isolated from soil samples (P<0.05). Out of 5314 positive clinical samples for mycobacteria, 175 (3.2%) isolates were NTM. The trend of NTM isolates increased from 1.2% (13 out of 1078) in 2004 to 3.8% (39 out of 1005) in 2014 (P = 0.0001). The major clinical isolates were M. simiae (51; 29.1%), M. kansasii (26; 14.8%), M. chelonae (28; 16%), and M. fortuitum (13; 7.4%).

Conclusions: Comparing the distribution pattern of environmental NTM isolates with clinical isolates suggests a possible transmission link, but this does not apply to all environmental NTM species. Our study confirms an increasing trend of NTM isolation from clinical samples that needs further investigation.

Figure 1. Comparison of HaeIII and BstEII RFLP patterns from isolates of M. conceptionense, M. senegalense and M. farcinogenes isolates from soil and water.

As it shown, digested pattern of PCR product by SP primer was different in these species. No digested pattern formed in M. conceptionense after digestion with HaeIII and cfoI; whereas the digested pattern for M. farcinogenes was 190–110 bp and 168 –132 bp, respectively. The pattern for M. senegalense was 148–109 bp, and 174–72 bp. But the digested pattern of PCR product by HSP primer produce single pattern i.e., 234-120-80 bp with BsteIII and 140-125-60-55 bp with HaeIII restriction enzyme.

Figure 2. Geographic distribution of the most common NTM species isolated from clinical and environmental samples.