Environmental Nontuberculous Mycobacteria in the Hawaiian Islands

Lung disease caused by nontuberculous mycobacteria (NTM) is an emerging infectious disease of global significance. Epidemiologic studies have shown the Hawaiian Islands have the highest prevalence of NTM lung infections in the United States. However, potential environmental reservoirs and species diversity have not been characterized. In this cross-sectional study, we describe molecular and phylogenetic comparisons of NTM isolated from 172 household plumbing biofilms and soil samples from 62 non-patient households and 15 respiratory specimens. Although non-uniform geographic sampling and availability of patient information were limitations, Mycobacterium chimaera was found to be the dominant species in both environmental and respiratory specimens. In contrast to previous studies from the continental U.S., no Mycobacterium avium was identified. Mycobacterium intracellulare was found only in respiratory specimens and a soil sample. We conclude that Hawai’i’s household water sources contain a unique composition of Mycobacterium avium complex (MAC), increasing our appreciation of NTM organisms of pulmonary importance in tropical environments.     

Species Spectrum of Nontuberculous Mycobacteria Isolated from Clinical Specimens in Kuwait

Specific identification of mycobacteria is of clinical relevance since treatment varies according to the Mycobacterium species causing infection. All mycobacterial isolates are currently identified as M. tuberculosis (MTB) or nontuberculous mycobacteria (NTM) based on p-nitro-α-acetylamino-β-hydroxypropiophenone (NAP) test, and the species spectrum of NTM-causing infections in Kuwait remains unknown. This study identified all NTM strains isolated in Kuwait from 1 October 2003 to 31 March 2004 to the species level. The mycobacteria were cultured from various clinical specimens using the BACTEC 460 TB system and NAP test was performed to differentiate MTB from NTM strains. The INNO-LiPA MYCOBACTERIA v2 assay (LiPA) was used for species-specific identification of NTM strains and some randomly selected MTB strains. The LiPA results for selected isolates were confirmed by DNA sequencing of the 16S-23S internal transcribed spacer region. A total of 325 isolates of Mycobacterium species originating from 305 individual patients were recovered during the study period, with 307 and 18 isolates identified as MTB and NTM, respectively. The LiPA correctly identified all 18 MTB isolates analyzed. Seven different NTM species were identified among 18 NTM isolates originating from 14 patients, with M. fortuitum causing the majority of NTM infections in Kuwait. One patient was infected with two NTM species. Rapid species-specific identification of NTM may help with appropriate treatment regimens for proper patient management. The DNA sequencing data reported in this study are deposited in EMBL under accession numbers AM709724 to AM709731.     

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.     

Cooccurrence of Free-Living Amoebae and Nontuberculous Mycobacteria in Hospital Water Networks,and Preferential Growth of Mycobacterium avium in Acanthamoeba lenticulata

The incidence of lung and other diseases due to nontuberculous mycobacteria (NTM) is increasing. NTM sources include potable water, especially in households where NTM populate pipes, taps, and showerheads. NTM share habitats with free-living amoebae (FLA) and can grow in FLA as parasites or as endosymbionts. FLA containing NTM may form cysts that protect mycobacteria from disinfectants and antibiotics. We first assessed the presence of FLA and NTM in water and biofilm samples collected from a hospital, confirming the high prevalence of NTM and FLA in potable water systems, particularly in biofilms. Acanthamoeba spp. (genotype T4) were mainly recovered (8/17), followed by Hartmannella vermiformis (7/17) as well as one isolate closely related to the genus Flamella and one isolate only distantly related to previously described species. Concerning mycobacteria, Mycobacterium gordonae was the most frequently found isolate (9/17), followed by Mycobacterium peregrinum (4/17), Mycobacterium chelonae (2/17), Mycobacterium mucogenicum (1/17), and Mycobacterium avium (1/17). The propensity of Mycobacterium avium hospital isolate H87 and M. avium collection strain 104 to survive and replicate within various FLA was also evaluated, demonstrating survival of both strains in all amoebal species tested but high replication rates only in Acanthamoeba lenticulata. As A. lenticulata was frequently recovered from environmental samples, including drinking water samples, these results could have important consequences for the ecology of M. avium in drinking water networks and the epidemiology of disease due to this species.     

Occurrence of Nontuberculous Mycobacterial Pulmonary Infection in an Endemic Area of Tuberculosis

The majority of investigations of the epidemiology of nontuberculous mycobacteria (NTM) have focused on highly developed nations with a low prevalence of tuberculosis. In contrast, the Para state of north Brazil represents an area of high tuberculosis prevalence and increasing NTM incidence. Toward the goal of understanding the dynamics of infection by all Mycobacterium species, we report patient characteristics and the identification of NTM strains isolated from sputum samples from patients that were residents of Para, a state in the Amazon region, Northern of Brazil, over the period January 2010 through December 2011 (2 years). The 29 NTM patients comprised 13.5% of positive mycobacterial cultures over the 2-year period. A major risk factor for NTM pulmonary disease was previous tuberculosis (76%). Further, the average age of NTM patients (52 years) was significantly higher than that of tuberculosis patients (39 years) and more were female (72.4% vs. 37.4%). Unlike other Brazilian states, NTM pulmonary patients in Para were infected with a different spectrum of mycobacteria; primarily the rapidly growing Mycobacterium massiliense and Mycobacterium simiae complex.     

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.     

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.     

Culture-Independent Identification of Nontuberculous Mycobacteria in Cystic Fibrosis Respiratory Samples

Respiratory tract infections with nontuberculous mycobacteria (NTM) are increasing in prevalence and are a significant cause of lung function decline in individuals with cystic fibrosis (CF). NTM have been detected in culture-independent analyses of CF airway microbiota at lower rates than would be expected based on published prevalence data, likely due to poor lysing of the NTM cell wall during DNA extraction. We compared a standard bacterial lysis protocol with a modified method by measuring NTM DNA extraction by qPCR and NTM detection with bacterial 16S rRNA gene sequencing. The modified method improved NTM DNA recovery from spiked CF sputum samples by a mean of 0.53 log₁₀ copies/mL for M. abscessus complex and by a mean of 0.43 log₁₀ copies/mL for M. avium complex as measured by qPCR targeting the atpE gene. The modified method also improved DNA sequence based NTM detection in NTM culture-positive CF sputum and bronchoalveolar lavage samples; however, both qPCR and 16S rRNA gene sequencing remained less sensitive than culture for NTM detection. We highlight the limitations of culture-independent identification of NTM from CF respiratory samples, and illustrate how alterations in the bacterial lysis and DNA extraction process can be employed to improve NTM detection with both qPCR and 16S rRNA gene sequencing.     

Development of a Real-Time qPCR Method for Detection and Enumeration of Mycobacterium spp. in Surface Water

A real-time quantitative PCR method was developed for the detection and enumeration of Mycobacterium spp. from environmental samples and was compared to two other methods already described. The results showed that our method, targeting 16S rRNA, was more specific than the two previously published real-time quantitative PCR methods targeting another 16S rRNA locus and the hsp65 gene (100% versus 44% and 91%, respectively).Water exposure (15) is one source of human infection caused by nontuberculous mycobacteria (NTM). Nevertheless, the isolation and enumeration of NTM from water is difficult because other microorganisms overgrow NTM colonies (22). Consequently, the development of an alternative detection and enumeration method is essential for monitoring NTM sources in the environment.Two real-time quantitative PCR (qPCR) methods for NTM measurement have been described (7, 29). The primer pair used in the first real-time qPCR method (7) targets 16S rRNA and was previously used to track mycobacterial growth in industrial water samples by conventional PCR (31). It was presented as a sensitive test for members of the Mycobacterium genus because it detected 34 species of mycobacteria (19, 25). However, the primer specificity was only measured by conventional PCR against DNA of Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus (31) or by in silico analysis (7). The second real-time qPCR method, targeting the hsp65 gene (29), was also sensitive (detection of 34 out of 37 Mycobacterium spp. tested). Although the primers showed high specificity (no detection of 16 different nonmycobacterial species) by conventional PCR (21), their specificity combined with the qPCR probe was only tested against Candida albicans DNA (29).We sought to develop a reliable real-time qPCR method to detect Mycobacterium spp. in water samples. The development involved in silico primer screening followed by a specificity study by conventional PCR. Furthermore, the efficiency (Ef), correlation coefficient (r²), limit of quantification (LOQ), specificity (Sp), and sensitivity (Ss) of this new method targeting 16S rRNA were compared with those of the two previously described methods (7, 29).     

Delivery of Aerosolized Liposomal Amikacin as a Novel Approach for the Treatment of Nontuberculous Mycobacteria in an Experimental Model of Pulmonary Infection

Pulmonary infections caused by nontuberculous mycobacteria (NTM) are an increasing problem in individuals with chronic lung conditions and current therapies are lacking. We investigated the activity of liposomal amikacin for inhalation (LAI) against NTM in vitro as well as in a murine model of respiratory infection. Macrophage monolayers were infected with three strains of Mycobacterium avium, two strains of Mycobacterium abscessus, and exposed to LAI or free amikacin for 4 days before enumerating bacterial survival. Respiratory infection was established in mice by intranasal inoculation with M. avium and allowing three weeks for the infection to progress. Three different regimens of inhaled LAI were compared to inhaled saline and parenterally administered free amikacin over a 28 day period. Bacteria recovered from the mice were analyzed for acquired resistance to amikacin. In vitro, liposomal amikacin for inhalation was more effective than free amikacin in eliminating both intracellular M. avium and M. abscessus. In vivo, inhaled LAI demonstrated similar effectiveness to a ∼25% higher total dose of parenterally administered amikacin at reducing M. avium in the lungs when compared to inhaled saline. Additionally, there was no acquired resistance to amikacin observed after the treatment regimen. The data suggest that LAI has the potential to be an effective therapy against NTM respiratory infections in humans.     

Isolation of novel mycobacteria contaminating an aquarium fish tank in a Japanese university hospital

Aims: To better understand nontuberculous mycobacteria (NTM) contamination in a hospital setting, six freshwater fish gut homogenates and water in an aquarium fish tank placed on the reception counter of a nursing station were cultured for mycobacteria. Methods and Results: By direct sequencing of 16s rRNA, rpoB and hsp65, scotochromogenic and nonchromogenic Mycobacterium szulgai isolates containing hsp65 type II (GenBank accession nos. FJ384762 and FJ384764 , respectively), Mycobacterium gordonae isolates containing rpoB clusters B and E (GenBank accession no. FJ384766 ), and Mycobacterium kansasii isolates containing hsp65 type VI were collected from the gut homogenates and water from the fish tank. However, no isolates were obtained from the tap water used to refill the fish tank. A randomly amplified polymorphic DNA (RAPD) analysis using a 10‐mer primer (5′‐TGGTCGCGGC) showed that some NTM from the fish tank water were identical to those obtained from the gut homogenates. Conclusions: Fish and water in the tank were contaminated by the novel NTM. Significance and Impact of the Study: These findings could help to elucidate infection routes and contamination sources of novel NTM from water sources.     

Comparison of Culture Methods for Isolation of Nontuberculous Mycobacteria from Surface Waters

The environment is the likely source of most nontuberculous mycobacteria (NTM) involved in human infections, especially pulmonary, skin, and soft tissue infections. In order to measure the prevalence of NTM in different aquatic ecosystems, we tried to standardize the culture methods used for surface water testing since many procedures have been described previously. Cultivation of mycobacteria requires long-term incubation in rich media and inactivation of rapidly growing microorganisms whose growth impedes observation of mycobacterial colonies. Consequently, the two criteria used for evaluation of the methods examined were (i) the rate of inhibition of nontarget microorganisms and (ii) the efficiency of recovery of mycobacteria. We compared the competitive growth of Mycobacterium chelonae and M. avium with nontarget microorganisms on rich Middlebrook 7H11-mycobactin medium after treatment by several chemical decontamination methods that included acids, bases, detergent, or cetylpyridinium chloride (CPC) with and without an antibiotic cocktail, either PANTA (40 U/ml polymyxin, 4 μg/ml amphotericin B, 16 μg/ml nalidixic acid, 4 μg/ml trimethoprim, and 4 μg/ml azlocillin) or PANTAV (PANTA plus 10 μg/ml vancomycin). Our results showed that treatment for 30 min with CPC (final concentration, 0.05%) of water concentrated by centrifugation, followed by culture on a rich medium supplemented with PANTA, significantly decreased the growth of nontarget microorganisms (the concentrations were 6.2 ± 0.4 log₁₀ CFU/liter on Middlebrook 7H11j medium and 4.2 ± 0.2 log₁₀ CFU/liter on Middlebrook 7H11j medium containing PANTA [P < 0.001]), while the effect of this procedure on NTM was not as great (the concentrations of M. chelonae on the two media were 7.0 ± 0.0 log₁₀ CFU/liter and 6.9 ± 0.0 log₁₀ CFU/liter, respectively, and the concentrations of M. avium were 9.1 ± 0.0 log₁₀ CFU/liter and 8.9 ± 0.0 log₁₀ CFU/liter, respectively). We propose that this standardized culture procedure could be used for detection of NTM in aquatic samples.It is generally accepted that environmental exposure, particularly exposure through water, is the main source of most human infections caused by nontuberculous mycobacteria (NTM). The incidence of waterborne NTM skin and soft tissue infections in immunocompetent patients is increasing (31), as is the incidence of pulmonary infections that occur due to aerosol inhalation (15, 31). Ingestion or inhalation of contaminated water (while swimming, for instance) could also be a source of NTM infections in children (31). Because NTM are emerging pathogens for humans and domestic animals, it is important to identify their environmental sources and reservoirs and to measure their proliferation and persistence in freshwater ecosystems. A robust and standardized method for environmental detection of NTM is necessary to do this.NTM are ubiquitous and can be isolated from a variety of aquatic ecosystems, including natural water, wastewater, drinking water, recreational water, and industrial water (16, 51). Even hospital water has been reported to be contaminated by NTM (31). More precisely, aquatic plants, amoebae, and aquatic vertebrates and invertebrates could be considered NTM reservoirs in aquatic ecosystems in natural environments and in drinking water distribution systems or buildings and homes (19, 26, 37). Once present in a system, mycobacteria may proliferate and persist (4).Typically, the methods usually used for detection of NTM are methods that are used for clinical microbiology and have not been adapted for environmental samples. Surface water samples are quite different from clinical samples, since they may contain low levels of NTM but typically contain highly diverse bacterial communities in which the concentrations of bacteria range from 10⁴ to 10⁷ cells per ml (54). This microbial diversity makes it likely that nontarget species will overgrow NTM in nutrient-rich medium. Several studies have been conducted to determine the optimum decontamination method for inhibiting the growth of nontarget bacteria in NTM assays, although most of the methods were developed for clinical samples (2, 8, 20, 42, 56). Moreover, no clear consensus for treatment of environmental samples has emerged from these studies. The combination of chemical decontamination and addition of antibiotics to culture medium has not been studied previously for water surface samples.The aim of this study was to develop and validate an improved method for detecting and counting NTM in surface water. To do this, we compared the results for recovery of mycobacteria from water samples and inactivation of nontarget microorganisms (fungi and bacteria other than mycobacteria) when various antibiotics and chemical decontaminants were used.     

Profiling serum antibodies to Mycobacterium tuberculosis proteins in rhesus monkeys with nontuberculous Mycobacteria

Recent evidence indicates that the prevalence of diseases caused by nontuberculous mycobacteria (NTM) has been increasing in both human and animals. In this study, antibody profiles of NTM in rhesus monkeys (Macaca mulatta) were determined and compared with those of monkeys infected with Mycobacterium tuberculosis complex (MTBC). Antibodies against 10 M. tuberculosis proteins, purified protein derivative (PPD), and mammalian old tuberculin (MOT) were detected in 14 monkeys naturally infected with NTM by indirect ELISA. Sera from 10 monkeys infected with MTBC and 10 healthy monkeys were set as controls. All antigens showed high serological reactivities to MTBC infections and low reactivities in healthy monkeys. NTM infections showed strong antibody responses to MOT and PPD; moderate antibody responses to 16kDa, U1, MPT64L, 14kDa, and TB16.3; and low antibody responses to 38kDa, Ag85b, CFP10, ESAT-6, and CFP10-ESAT-6. According to the criteria of MTBC, only CFP10, ESAT-6, and CFP10-ESAT-6 showed negative antibody responses in all NTM infections. Taken together, these results suggest that positive results of a PPD/MOT-based ELISA in combination with results of antibodies to M. tuberculosis-specific antigens, such as CFP10 and ESAT-6, could discriminate NTM and MTBC infections. Two positive results indicate an MTBC infection, and a negative result for an M. tuberculosis-specific antigen may preliminarily predict an NTM infection.     

Antimicrobial Susceptibility of Standard Strains of Nontuberculous Mycobacteria by Microplate Alamar Blue Assay

In this study, 24 standard nontuberculous mycobacteria (NTM) species strains including 12 slowly growing mycobacteria strains and 12 rapidly growing mycobacteria strains were subjected to drug susceptibility testing using microplate Alamar Blue assay-based 7H9 broth. The most active antimicrobial agents against the 24 NTM strains were streptomycin, amikacin, the fluoroquinolones, and the tetracyclines. Mycobacterium chelonae, Mycobacterium abscessus, Mycobacterium bolletii, and Mycobacterium simiae are resistant to most antimicrobial agents. The susceptibility results of this study from 24 NTM standard strains can be referenced by clinicians before susceptibility testing for clinical isolates is performed or when conditions do not allow for susceptibility testing. The application of broth-based methods is recommended by the Clinical and Laboratory Standards Institute, and the documentation of the susceptibility patterns of standard strains of mycobacteria can improve the international standardization of susceptibility testing methods.     

Pyrosequence Analysis of the hsp65 Genes of Nontuberculous Mycobacterium Communities in Unchlorinated Drinking Water in the Netherlands

Studies have shown that certain opportunistic pathogenic species of nontuberculous mycobacteria (NTM) can be present in distributed drinking water. However, detailed information about NTM population composition in drinking water is lacking. Therefore, NTM communities in unchlorinated drinking water from the distribution system of five treatment plants in the Netherlands were characterized using 454 pyrosequencing of the hsp65 gene. Results showed high diversities in unchlorinated drinking water, with up to 28 different NTM operational taxonomic units (OTUs) in a single sample. Each drinking water sample had a unique NTM community, and most (81.1%) OTUs were observed only once. One OTU was observed in 14 of 16 drinking water samples, indicating that this NTM species is well adapted to unchlorinated drinking water conditions. A clear influence of season, source type (groundwater, surface water), easily assimilable organic carbon (AOC) concentration, biofilm formation rate, and active biomass in treated water on the establishment of an NTM community in drinking water was not observed. Apparently, local conditions are more important for the development of a specific NTM community in the drinking water distribution system. A low (4.2%) number of hsp65 gene sequences showed more than 97% similarity to sequences of the opportunistic pathogens M. avium, M. genavense, and M. gordonae. However, most (95.8%) NTM hsp65 gene sequences were related to not-yet-described NTM species that have not been linked to disease, indicating that most NTM species in unchlorinated drinking water from distribution systems in the Netherlands have a low public health significance.     

Complete Genome Sequence of Mycobacterium fortuitum subsp. fortuitum Type Strain DSM46621

Mycobacterium fortuitum is a member of the rapidly growing nontuberculous mycobacteria (NTM). It is ubiquitous in water and soil habitats, including hospital environments. M. fortuitum is increasingly recognized as an opportunistic nosocomial pathogen causing disseminated infection. Here we report the genome sequence of M. fortuitum subsp. fortuitum type strain DSM46621.     

Combining comparative genomic analysis with machine learning reveals some promising diagnostic markers to identify five common pathogenic non-tuberculous mycobacteria

Non-tuberculous mycobacteria (NTM) can cause various respiratory diseases and even death in severe cases, and its incidence has increased rapidly worldwide. To date, it’s difficult to use routine diagnostic methods and strain identification to precisely diagnose various types of NTM infections. We combined systematic comparative genomics with machine learning to select new diagnostic markers for precisely identifying five common pathogenic NTMs (Mycobacterium kansasii, Mycobacterium avium, Mycobacterium intracellular, Mycobacterium chelonae, Mycobacterium abscessus). A panel including six genes and two SNPs (nikA, benM, codA, pfkA2, mpr, yjcH, rrl C2638T, rrl A1173G) was selected to simultaneously identify the five NTMs with high accuracy (> 90%). Notably, the panel only containing the six genes also showed a good classification effect (accuracy > 90%). Additionally, the two panels could precisely differentiate the five NTMs from M. tuberculosis (accuracy > 99%). We also revealed some new marker genes/SNPs/combinations to accurately discriminate any one of the five NTMs separately, which provided the possibility to diagnose one certain NTM infection precisely. Our research not only reveals novel promising diagnostic markers to promote the development of precision diagnosis in NTM infectious, but also provides an insight into precisely identifying various genetically close pathogens through comparative genomics and machine learning.     

Isolation and Identification of Environmental Mycobacteria in the Waters of a Hemodialysis Center

The use of poorly treated water during hemodialysis may lead to contamination with nontuberculous mycobacteria (NTM). This study aimed to isolate and identify NTM species in the water of a Brazilian hemodialysis center. We collected 210 samples of water from the hydric system of the unit (post-osmosis system, hemodialysis rooms, reuse system, and hemodialysis equipment) and from the municipal supply network; we isolated the NTM by a classic microbiological technique and identified them by the PCR restriction enzyme pattern of the hsp65 gene (PRA). Fifty-one (24.3 %) of the collected samples tested positive for NTM; both the municipal supply network (2 samples, 3.2 %) and the hydric system of the hemodialysis center (49 samples, 96.1 %) contained NTM. We isolated and identified potentially pathogenic bacteria such as Mycobacterium lentiflavum (59.0 %) and M. kansasii (5.0 %), as well as rarely pathogenic bacteria like M. gordonae (24.0 %), M. gastri (8.0 %), and M. szulgai (4.0 %). The ability of NTM to cause diseases is well documented in the literature. Therefore, the identification of NTM in the water of a Brazilian hemodialysis center calls for more effective water disinfection procedures in this unit.     

Prevalence and Concentration of Non-tuberculous Mycobacteria in Cooling Towers by Means of Quantitative PCR: A Prospective Study

There is an increasing level of interest in non-tuberculous mycobacteria (NTM) due to the increasing reported rates of diseases caused by them. Although it is well known that NTM are widely distributed in the environment it is necessary to identify its reservoirs to prevent possible infections. In this study, we aimed to investigate the occurrence and levels of NTM in cooling towers to provide evidences for considering these settings as possible sources of respiratory infections. In the current study, we detected and quantified the presence of NTM by means of a rapid method in water samples taken from 53 cooling towers of an urban area (Barcelona, Spain). A genus-specific quantitative PCR (Q-PCR) assay with a quantification limit (QL) of 500?cells?l^?1 was used. 56% (30) of samples were positive with a concentration range from 4.6?×?10³ to 1.79?×?10⁶?cells?l^?1. In some cases (9/30), samples were positive but with levels below the QL. The colonization rate confirmed that cooling towers could be considered as a potential reservoir for NTM. This study also evaluated Q-PCR as a useful method to detect and quantify NTM in samples coming from environmental sources.     

Diversity of Nontuberculoid Mycobacterium Species in Biofilms of Urban and Semiurban Drinking Water Distribution Systems

Nontuberculous mycobacteria (NTM) are ubiquitous and have been isolated from a variety of environmental sources, including water. Various NTM were isolated from biofilms in drinking water distribution systems in two urban and two semiurban areas in South Africa. Most of the isolates belonged to opportunistic pathogenic species of the NTM group, but none belonged to the Mycobacterium avium complex.