Spatio-temporal study of environmental nontuberculous mycobacteria isolated from Wardha district in Central India

During the last two decades, nontuberculous mycobacteria (NTM) have gained in importance but there is still a paucity of data, particularly for environmental isolates. We studied, over a period of two years, the spatio-temporal features of NTM isolates obtained from different environmental sources in Wardha district, India. A total of 1398 samples (699 each of soil and water) were tested and 170 (12.2%) yielded NTM isolates, including 123 from soil and 47 from water samples. Out of 170 NTM isolates, 107 (63%) belonged to potentially pathogenic mycobacteria (PPM) and 63 (37%) to the less pathogenic mycobacterial (LPM) group. Overall, maximum isolation was obtained in rainy season (20.3%) followed by winter (13.5%), post rainy (8.7%) and summer seasons (5.8%). Mycobacterium fortuitum, Mycobacterium gordonae and Mycobacterium avium complex (MAC) were common isolates followed by Mycobacterium flavescens, Mycobacterium scrofulaceum, Mycobacterium simiae and Mycobacterium marinum. From soil, isolation of NTM was highest from grounds used for community gatherings (42.8%) followed by soil from residential premises (27.7%) and near the wells (26.0%). From drinking water sources, highest NTM isolation was obtained from wells (15.4%) followed by treated water tanks (6.9%), household receptacles (6.3%), hand pumps (5.6%) and tap water supply (3.5%). Isolation from natural canal water was 6.6%, while from drainage and waste water ponds isolation was 8.3%. The results of the study revealed that in Wardha district, NTM are present both in the soil and drinking water. As NTM can be pathogenic, particularly in immune-compromised individuals, these can be of potential risk to the human population.     

Occurrence and molecular differentiation of environmental mycobacteria in surface waters

To investigate the occurrence and species diversity of mycobacteria in waters, surface water samples were collected monthly from the Han River and tap water samples at the terminal sites of the distribution system. Mycobacteria in each water sample were isolated by decontamination using cetylpyridinium chloride (CPC) and cultivation on Middlebrook 7H10 agar, and then identified by polymerase chain reaction-restriction fragment length polymorphism analysis (PRA) and sequencing of the 65-kDa heat-shock protein gene (hsp65 gene). Mycobacteria were detected in 59% of the surface water samples and 26% of the tap water samples. Over half of the 158 isolates could not be identified by hsp65 PRA and gene sequencing, and several identification discrepancies were observed between the two methods. The most frequently isolated species was Mycobacterium gordonae in surface water and M. lentiflavum in tap water. M. avium complex (MAC), the most important pathogen among environmental mycobacteria, was detected in the surface water samples but not found in the tap water samples. The result demonstrated that water is an important environmental source of mycobacteria and the combined application of hsp65 PRA and sequencing was more reliable than hsp65 PRA alone to accurately identify mycobacteria present in water.     

Activities of Linezolid against nontuberculous mycobacteria

The activity of linezolid (Pfizer, USA) was tested by broth microdilution against 53 clinical isolates of non-tuberculous mycobacteria (NTM), including the common disease producing species Mycobacterium avium, M. intracellulare, M. fortuitum, M. chelonae and M. abscessus, obtained from western Turkey. The isolates of M. abscessus and M. intracellulare were the least susceptible, M. mucogenicum, M. gordonae and M. avium were the most susceptible to linezolid of the common species of NTM. Linezolid showed a variable sensitivity in all strains; therefore, each species and strain must be individually evaluated, and it is always advisable to perform in vitro sensitivity tests before using the drug for human therapy.     

Prevalence of nontuberculous mycobacteria in water supplies of hemodialysis centers.

Infection of hemodialysis patients with nontuberculous mycobacteria (NTM) has been associated with water used in reprocessing hemodialyzers. This study was conducted to determine the prevalence of NTM and other bacteria in water samples collected over a 13-week period from 115 randomly selected dialysis centers in the United States. Total viable counts were determined by membrane filter assays; increased recovery of NTM was obtained by dosing a portion of each water sample with 1% formaldehyde (HCHO) before filtering. NTM were widely distributed and occurred with a high frequency in water supplies in dialysis centers. NTM were detected in water from 95 centers (83%), and 50% of all samples examined contained NTM. The results of this study support recommendations to use 4% HCHO or a chemical germicidal equivalent for disinfecting dialyzers that are to be reused.     

Shifting paradigms of nontuberculous mycobacteria in cystic fibrosis

Important paradigms of pulmonary disease with nontuberculous mycobacteria (NTM) are currently shifting based on an increasing attention within the field of cystic fibrosis (CF). These shifts are likely to benefit the management of all patients with pulmonary NTM, regardless of underlying pathology. Currently several key areas are being revised: The first outbreak of human NTM transmission has been proven and new evidence of biofilm growth in vivo has been demonstrated. A better understanding of the clinical impact of NTM infection has led to increased diagnostic vigilance and new recommendations for lung transplantation are under way. While recent changes have reinvigorated the interest in NTM disease, the challenge remains, whether such advances can be successfully translated into improved management and care.     

Prevalence of nontuberculous mycobacteria in water supplies of hemodialysis centers

Infection of hemodialysis patients with nontuberculous mycobacteria (NTM) has been associated with water used in reprocessing hemodialyzers. This study was conducted to determine the prevalence of NTM and other bacteria in water samples collected over a 13-week period from 115 randomly selected dialysis centers in the United States. Total viable counts were determined by membrane filter assays; increased recovery of NTM was obtained by dosing a portion of each water sample with 1% formaldehyde (HCHO) before filtering. NTM were widely distributed and occurred with a high frequency in water supplies in dialysis centers. NTM were detected in water from 95 centers (83%), and 50% of all samples examined contained NTM. The results of this study support recommendations to use 4% HCHO or a chemical germicidal equivalent for disinfecting dialyzers that are to be reused.     

Effect of freezing of water samples on viable counts of environmental mycobacteria

The effect of prolonged storage on mycobacteria and other heterotrophic bacteria in brook water samples was examined by determination of viable counts from fresh samples and again after water concentrates had been stored in nutrient broth at —75°C for 15 months. The counts of mycobacteria were on average three times higher after storage (range of ratio 0·9–10·4). In contrast, the viable counts of other heterotrophic bacteria were reduced by 69%. The increase in mycobacterial counts was probably due to break-up of microcolonies or release of attached bacteria from particles. The possibility of cultivating mycobacteria from frozen samples is of practical help in large-scale field surveys.     

Identification of nontuberculous mycobacteria by methods of molecular biology

A number of clinical and laboratory strains of microorganisms belonging to different species of mycobacteria of the nontuberculous complex were tested with the use of the polymerase chain reaction and the restriction analysis. The unique restriction profiles of the following species of mycobacteria have been obtained: M. fortuitum VI, M. kansasii I, M. intracellulare, M. avium.     

Identification of nontuberculous mycobacteria existing in tap water by PCR-restriction fragment length polymorphism

This paper presents the finding of the possible cause of the high false-positive rate in acid-fast staining in histological examinations. Using acid-fast staining, culture, and PCR, acid-fast bacilli were detected in 83.7% of 49 hospital tap water samples and nontuberculous mycobacteria (NTM) were detected in 20.4% of the same 49 samples. The 10 NTM isolates were also identified to the species level using PCR-restriction fragment length polymorphism. Our findings indicate that NTM in hospital tap water are the possible cause of false positives in acid-fast staining and of nosocomial infection in immunocompromised patients.     

Efficacy of chemical dosing methods for isolating nontuberculous mycobacteria from water supplies of dialysis centers.

Investigations of nontuberculous mycobacterium (NTM) infections associated with various environmental sources have been hampered by the lack of adequate techniques for selective isolation of these organisms from environmental fluids. This study compared chemical dosing techniques for recovery of NTM from water samples collected from 115 randomly selected dialysis centers. Cell suspensions of NTM group II and IV isolates and gram-negative bacteria were exposed to solutions containing sodium hypochlorite (0.2 micrograms/ml of free available chlorine), formaldehyde (1, 0.75, or 0.5%), oxalic acid (1.25%), cetylpyridinium chloride (25 micrograms/ml), or cetyltrimethylammonium bromide (100 micrograms/ml). Results of standard membrane filtration assays with laboratory test strains and water samples from dialysis centers showed that 5 min of exposure to 1% formaldehyde effectively reduced gram-negative bacterial populations and allowed increased recovery of NTM in environmental fluids containing mixed microbial populations.     

Recovery and survival of nontuberculous mycobacteria under various growth and decontamination conditions

The survival of microorganisms of the Mycobacterium avium, M. intracellulare, and M. scrofulaceum (MAIS) complex was evaluated after various soil and water decontamination regimens. Survival was reduced by growing cells in natural waters compared with laboratory media and by inclusion of malachite green in media as an antifungal agent. Decontamination with benzalkonium chloride, while reducing survival significantly less than 1% NaOH, failed to eliminate many fungi. Recovery from soil was further reduced by transfer losses and by irreversible cell adsorption onto particulates.     

Survival of environmental mycobacteria in Acanthamoeba polyphaga

Free-living amoebae in water are hosts to many bacterial species living in such an environment. Such an association enables bacteria to select virulence factors and survive in adverse conditions. Waterborne mycobacteria (WBM) are important sources of community- and hospital-acquired outbreaks of nontuberculosis mycobacterial infections. However, the interactions between WBM and free-living amoebae in water have been demonstrated for only few Mycobacterium spp. We investigated the ability of a number (n = 26) of Mycobacterium spp. to survive in the trophozoites and cysts of Acanthamoeba polyphaga. All the species tested entered the trophozoites of A. polyphaga and survived at this location over a period of 5 days. Moreover, all Mycobacterium spp. survived inside cysts for a period of 15 days. Intracellular Mycobacterium spp. within amoeba cysts survived when exposed to free chlorine (15 mg/liter) for 24 h. These data document the interactions between free-living amoebae and the majority of waterborne Mycobacterium spp. Further studies are required to examine the effects of various germicidal agents on the survival of WBM in an aquatic environment.     

Incidence of nontuberculous mycobacteria in four hot water systems using various types of disinfection

The objective of this study was to determine the incidence of nontuberculous mycobacteria (NTM) in hot water systems of 4 selected hospital settings. The hospitals provided the following types of disinfection for their hot water systems: hydrogen peroxide and silver, thermal disinfection, chlorine dioxide, and no treatment (control). In each building, 6 samples were collected from 5 sites during a 3 month period. NTM were detected in 56 (46.7%) of 120 samples; the CFU counts ranged from 10 to 1625 CFU/L. The detected NTM species were the pathogens Mycobacterium kansasii, Mycobacterium xenopi, and Mycobacterium fortuitum and the saprophyte Mycobacterium gordonae. The most common to be isolated was M. xenopi, which was present in 51 samples. The hot water systems differed significantly in the incidence of NTM. NTM were not detected in the system treated by thermal disinfection, and a relatively low incidence (20% positive samples) was found in the system disinfected with chlorine dioxide. However, a high incidence was found in the control system with no additional disinfection (70% positives) and in the system using hydrogen peroxide and silver (97% positives). Water temperatures above 50 degrees C significantly limited the occurrence of NTM.     

Persistence of nontuberculous mycobacteria in a drinking water system after addition of filtration treatment

There is evidence that drinking water may be a source of infections with pathogenic nontuberculous mycobacteria (NTM) in humans. One method by which NTM are believed to enter drinking water distribution systems is by their intracellular colonization of protozoa. Our goal was to determine whether we could detect a reduction in the prevalence of NTM recovered from an unfiltered surface drinking water system after the addition of ozonation and filtration treatment and to characterize NTM isolates by using molecular methods. We sampled water from two initially unfiltered surface drinking water treatment plants over a 29-month period. One plant received the addition of filtration and ozonation after 6 months of sampling. Sample sites included those at treatment plant effluents, distributed water, and cold water taps (point-of-use [POU] sites) in public or commercial buildings located within each distribution system. NTM were recovered from 27% of the sites. POU sites yielded the majority of NTM, with >50% recovery despite the addition of ozonation and filtration. Closely related electrophoretic groups of Mycobacterium avium were found to persist at POU sites for up to 26 months. Water collected from POU cold water outlets was persistently colonized with NTM despite the addition of ozonation and filtration to a drinking water system. This suggests that cold water POU outlets need to be considered as a potential source of chronic human exposure to NTM.     

Real-time PCR detection of environmental mycobacteria in house dust

Analysing the number and species of microbes in indoor dust is needed for assessment of human exposure to microbes in dwellings. Environmental mycobacteria are common heterotrophic bacteria in both natural and man-made environments and potential inducers of human immune system. Culture of mycobacteria from samples rich with other microbes is difficult due to the slow growth rate of mycobacteria and this has hampered the studies on their role in indoor human exposure. A quantitative, real-time 5'-nuclease (TaqMan) PCR assay was developed to detect environmental mycobacteria in indoor dust samples. The specificity of the primers and the probe targeting the 16S rDNA of mycobacteria, tested with 26 mycobacterial and 10 non-mycobacterial but related species, proved to be high. When tested on 20 indoor dust samples collected from five homes, the assay gave counts varying between 4.8 × 10⁴ and 7.2 × 10⁶ cell/g, being on average 1.1 × 10³ times higher than culture. Seasonal variation in the dust counts of mycobacteria was observed by both culture and qPCR. Total of 140 isolates considered as mycobacteria by Ziehl-Neelsen staining and GLC-analyses were subjected to PCR analysis with the mycobacterial primers, and 39 isolates to partial 16S rDNA sequencing. All proved to be mycobacteria and revealed high diversity of mycobacterial species in the dust samples. Majority of the sequences were related to M. terrae and M. avium complexes.     

Efficacy of chemical dosing methods for isolating nontuberculous mycobacteria from water supplies of dialysis centers

Investigations of nontuberculous mycobacterium (NTM) infections associated with various environmental sources have been hampered by the lack of adequate techniques for selective isolation of these organisms from environmental fluids. This study compared chemical dosing techniques for recovery of NTM from water samples collected from 115 randomly selected dialysis centers. Cell suspensions of NTM group II and IV isolates and gram-negative bacteria were exposed to solutions containing sodium hypochlorite (0.2 micrograms/ml of free available chlorine), formaldehyde (1, 0.75, or 0.5%), oxalic acid (1.25%), cetylpyridinium chloride (25 micrograms/ml), or cetyltrimethylammonium bromide (100 micrograms/ml). Results of standard membrane filtration assays with laboratory test strains and water samples from dialysis centers showed that 5 min of exposure to 1% formaldehyde effectively reduced gram-negative bacterial populations and allowed increased recovery of NTM in environmental fluids containing mixed microbial populations.     

Processing and transport of environmental virus samples.

Poliovirus-seeded tap water, conditioned with MgCl2 and passed through virus-adsorbing filters, gave better poliovirus recovery than water identically treated but conditioned with AlCl3. Elution of several filter types with beef extract yielded higher recoveries than did elution with glycine. Seeded samples filtered through various filters and stored showed considerable virus loss in 2 days when stored at 4 degrees C, whereas those stored at -70 degrees C gave stable virus recovery up to 4 days. Additionally, the use of antifoam during the elution process reduced foaming and increased virus recovery by 28%.