Mycobacterial ecology of the Rio Grande

Abstract

This is the first study to characterize the environmental conditions which contribute to the presence and proliferation of environmental mycobacteria in a major freshwater river. Over 20 different species of environmental mycobacteria were isolated, including the pathogenic M. avium and M. kansasii. Species of the rapidly growing M. fortuitum complex were the most commonly isolated mycobacteria, and one-third of all isolates were not identified at the species level, even by 16S sequencing. PCR restriction analysis of the hsp65 gene was more accurate and rapid than biochemical tests and as accurate as yet less expensive than 16S sequencing, showing great promise as a new tool for species identification of environmentally isolated mycobacteria. Total environmental mycobacteria counts positively correlated with coliform and Escherichia coli counts and negatively correlated with chemical toxicity and water temperature. Environmental mycobacteria can survive in the alkaline conditions of the river despite previous reports that especially acidic conditions favor their presence. A representative river isolate (M. fortuitum) survived better than E. coli O157:H7 at pHs below 7 and above 8 in nutrient broth. The river strain also retained viability at 8 ppm of free chlorine, while E. coli was eliminated at 2 ppm and above. Thus, in vitro studies support environmental observations that a variety of extreme conditions favor the hardy environmental mycobacteria.

FIG. 1.

Number of species isolated by month and site. The raw number of each species group (colored sections of columns) isolated each month at all sites (panel A) and each site for all months (panel B). FC, M. fortuitum complex; scr, M. scrofulaceum; sem, M. semiae; gor, M. gordonae; IISA, a PRA pattern shared by M. avium, M. intracellulare, M. simiae, and M. intermedium; other, seven rare species combined (M. smegmatis, M.interjectium, M. lentiflavum, M. nonchromo, M. celatum, M. chitae, and M. pheli), int, M. intracellulare; unk, unknown; avi, M. avium; MM, M.malmoense or M. marinum (indistinguishable by PRA); and kan, M. kansasii.

FIG. 2.

Effect of treatment on mycobacteria. Data for site 4 are species plate counts from effluent from the Haskins Waste Water Treatment Plant. Data for all other sites are the mean ± standard deviation from the untreated river sites, excluding low-abundance sites 5 and 6. Abbreviations are the same as in Fig. 1.

FIG. 3.

pH coincubation cultures. Exponentially growing M. fortuitum strain 4G and E. coli O157:H7 strain Sakai were mixed into 7H9-ADC with the pH adjusted to 4 (=A), 5 (B), 6 (C), 7 (D), 8 (E), or 9 (F). Culture pH after 144 h is also indicated. Cultures were incubated at 150 rpm and 37°C, and dilutions were plated in triplicate for CFU determination at the indicated time points. E. coli was determined by LB plates, and M. fortuitum was determined on selective 7H11. Square symbols, mean E. coli viability; circles, mycobacterial viability; error bars, standard deviation.

FIG. 4.

Chlorine combination survival. Exponentially growing M.fortuitum strain 4G and E. coli O157:H7 strain Sakai were mixed into deionized water with 1, 2, 4, or 8 ppm of free chlorine added. Viability was determined as in Fig. 3. E. coli is only indicated at 1 ppm because no colonies were obtained at higher chlorine concentrations.