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.     

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.     

Targeting the rpoB gene using nested PCR-restriction fragment length polymorphism for identification of nontuberculous mycobacteria in hospital tap water

Nontuberculous mycobacteria (NTM) are ubiquitous in the environment and can cause nosocomial infections in immunocompromised patients. Recently the presence of NTM in public drinking water and hospital water distribution systems has been reported. Their ability to form biofilms and their resistance to chlorine both contribute to their survival and colonization in water distribution systems. Here we analyzed thirty-two hospital tap water samples that were collected from different locations in three hospitals so as to evaluate the prevalence of NTM species. The water samples were concentrated by membrane filtration and then eluted with sterilized water following sonication. Two-step direct PCR targeting the rpoB gene, restriction fragment length polymorphism (RFLP) using the MspI restriction enzyme, and sequence analysis were performed for identification of NTM to the species level. The sequences of each PCR product were analyzed using BLASTN. Seven samples (7/32, 21.9%) were positive for NTM as determined by nested-PCR. The PCR-RFLP results indicated five different patterns among the seven positive PCR samples. The water-born NTM were identified, including M. peregrinum, M. chelonae (2 cases), M. abscessus, M. gordonae (2 cases), and Mycobacterium sp. JLS. The direct two-step PCR-RFLP method targeting the rpoB gene was effective for the detection and the differentiation of NTM species from hospital tap water.     

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.     

Nontuberculous mycobacteria: an emerging risk in engineered environmental habitats

Nontuberculous mycobacteria (NTM) are microorganisms commonly living in the environment. Nevertheless, most of them are opportunistic pathogens. To verify concentrations of NTM in some man-made habitats, analyses were performed on water and surface samples, and the data were correlated to the global microbiological quality of water. Most of the drinking water samples (98 %) complied with the microbial requirements established by the European Directive 98/83/EC on drinking water when Escherichia coli was considered. Low counts of heterotrophs were also obtained. NTM were isolated from 72 % of the analyzed samples. Tap water from private buildings, schools and hospitals provided positive results for NTM, with comparable densities ranging from 1 to 6?×10² CFU/L. NTM were also found in swimming pool water samples, with concentrations ranging from 29 to 3?×10⁴ CFU/L, as well as in 70 % of the surface sample. The most frequently isolated species were Mycobacterium mucogenicum, M. intracellulare and M. terrae. These yields confirm that no correlation exists between the monitoring controls carried out by law and the occurrence of these bacteria that may represent a potential risk, especially for immunocompromised people and vulnerable groups.     

Occurrence of nontuberculous mycobacteria in environmental samples.

Nontuberculous mycobacteria (NTM) are a major cause of opportunistic infection in immunocompromised hosts. Because there is no evidence of person-to-person transmission and NTM have been found in drinking water, the environment is considered a likely source of infection. In this study the widespread occurrence of NTM was examined in drinking water, bottled water, and ice samples. A total of 139 samples were examined for NTM by a membrane filtration culture technique followed by PCR amplification and 16S rRNA sequence determination to identify the isolates. NTM were not detected in bottled water or cisterns but were detected in 54% of the ice samples and 35% of the public drinking-water samples from 21 states. The most frequently occurring isolate was M. mucogenicum (formerly referred to as an M. chelonae-like organism).     

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.     

供水系统非结核分枝杆菌生长因素及控制措施研究进展

近年来,非结核分枝杆菌感染在世界范围内日益普遍,严重威胁公众健康。供水系统是非结核分枝杆菌重要环境来源和主要传播途径,但目前对供水系统非结核分枝杆菌生长因素及控制措施的认识仍有较多不足。本文介绍了供水系统非结核分枝杆菌的生长传播特征,探讨了多个工程环境因素(如消毒剂、有机碳、管材和温度)和生物因子(如生物膜、阿米巴原虫和细菌)对非结核分枝杆菌丰度和物种多样性特征的影响,分析了供水全流程不同阶段控制措施对非结核分枝杆菌的控制效用,提出了深化认识供水系统非结核分枝杆菌的研究需求。     

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.     

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.     

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.     

Aerosolization of mycobacteria and legionellae during dental treatment: low exposure despite dental unit contamination

Dental unit waterlines (DUWL) support growth of a dense microbial population that includes pathogens and hypersensitivity-inducing bacteria, such as Legionella spp. and non-tuberculous mycobacteria (NTM). Dynamic dental instruments connected to DUWL generate aerosols in the work environment, which could allow waterborne pathogens to be aerosolized. The use of the real-time quantitative polymerase chain reaction (qPCR) provides a more accurate estimation of exposure levels compared with the traditional culture approach. Bioaerosol sampling was performed 13 times in an isolated dental treatment room according to a standardized protocol that included four dental prophylaxis treatments. Inhalable dust samples were taken at the breathing zone of both the hygienist and patient and outside the treatment room (control). Total bacteria as well as Legionella spp. and NTM were quantified by qPCR in bioaerosol and DUWL water samples. Dental staff and patients are exposed to bacteria generated during dental treatments (up to 4.3 E + 05 bacteria per m(3) of air). Because DUWL water studied was weakly contaminated by Legionella spp. and NTM, their aerosolization during dental treatment was not significant. As a result, infectious and sensitization risks associated with legionellae and NTM should be minimal.     

Automated concentration and recovery of micro-organisms from drinking water using dead-end ultrafiltration

Aims:  Concentration of pathogens diluted in large volumes of water is necessary for their detection. An automated concentration system placed online in drinking water distribution systems would facilitate detection and mitigate the risk to public health.
Methods and Results:  A prototype concentrator based on dead-end hollow fibre ultrafiltration was used to concentrate Bacillus atrophaeus spores directly from tap water. Backflush was used to recover accumulated particulates for analysis. In field tests conducted on a water utility distribution system, 3·2 × 10⁴–1·4 × 10⁶ CFU ml⁻¹ (6·1 × 10⁶–3·0 × 10⁸ CFU) were recovered from the filter when 2·9 × 10⁷–1·0 × 10⁹ CFU were spiked into the system. Per cent recovery ranged from 21% to 68% for flow volumes of 15–21 l. Tests using spore influent levels <10 CFU l⁻¹ (spike < 1000 CFU) yielded 23–40% recovery for volumes >100 l.
Conclusions:  B. atrophaeus spores at levels <10 CFU l⁻¹ were concentrated directly from tap water using an automated dead-end hollow-fibre ultrafiltration system.
Significance and Impact of the Study:  The prototype concentrator represents a critical step towards an autonomous system that could be installed in drinking water distribution lines or other critical water lines to facilitate monitoring. Recovered samples can be analysed using standard or rapid biosensor methods.     

Interaction between phosphorus and biodegradable organic carbon on drinking water biofilm subject to chlorination

Aims: To examine whether phosphorus and biodegradable organic carbon interact to impact biofilm density and physiological function of biofilm‐forming bacteria under conditions relevant to chlorinated drinking water distribution systems. Materials and Results: The 2 × 2 factorial experiments with low and high levels of phosphorus and biodegradable organic carbon were performed on 4 ‐week‐old drinking water biofilms in four separate pipe systems in the presence of chlorine. Experimental results revealed that biofilm heterotrophic plate count levels increased with the increase in biodegradable organic carbon concentration, showed no response to increases in levels of phosphorus and was not affected by interaction between phosphorus and biodegradable organic carbon. However, a significant positive interaction between phosphorus and biodegradable organic carbon was found to exist on biofilm mass and physiological function and/or metabolic potentials of biofilm communities; the effects of biodegradable organic carbon on biofilm mass and physiological function of biofilm‐forming bacteria were accelerated in going from low to high level of phosphorus. Conclusions: Biodegradable organic carbon was found to be the primary nutrient in regulating biofilm formation in drinking water regardless of the presence of chlorine. It can be therefore concluded that the removal of an easily biodegradable organic carbon is necessary to minimize the biofilm growth potential induced by the intrusion of phosphorus. Significance and Impact of the Study: Phosphorus introduced to drinking water may interact with biodegradable organic carbon, thus leading to measurable impact on the biofilm formation.     

Detection and enumeration of haloacetic acid-degrading bacteria in drinking water distribution systems using dehalogenase genes

Aims:  To develop a PCR-based tracking method for the detection of a subset of bacteria in drinking water distribution systems capable of degrading haloacetic acids (HAAs).
Methods and Results:  Published degenerate PCR primers were used to determine that 54% of tap water samples (7/13) were positive for a deh gene, indicating that drinking water distribution systems may harbour bacteria capable of HAA degradation. As the published primer sets were not sufficiently specific for quantitative PCR, new primers were designed to amplify deh II genes from selected indicator strains. The developed primer sets were effective in directly amplifying deh II genes from enriched consortia samples, and the DNA extracted from tap water provided that an additional nested PCR step for detection of the deh II gene was used.
Conclusions:  This study demonstrates that drinking water distribution systems harbour microbes capable of degrading HAAs. In addition, a quantitative PCR method was developed to detect and quantify deh II genes in drinking water systems.
Significance and Impact of the Study:  The development of a technique to rapidly screen for the presence of dehalogenase genes in drinking water distribution systems could help water utilities determine if HAA biodegradation is occurring in the distribution system.     

The Tracing of Mycobacteria in Drinking Water Supply Systems by Culture, Conventional, and Real Time PCRs

Mycobacteria are widely present in diverse aquatic habitats, where they can survive for months or years while some species can even proliferate. The resistance of different mycobacterial species to disinfection methods like chlorination or ozonation could result in their presence in the final tap water of consumers. In this study, the culture method, Mycobacterium tuberculosis complex conventional duplex PCR for detection of non-tuberculous mycobacteria (NTM) and quantitative real-time PCR (qPCR) to detect three subspecies of M. avium species (M. a. avium, M. a. hominissuis, and M. a. paratuberculosis) were used to trace their possible path of transmission from the watershed through the reservoir and drinking water plant to raw drinking water and finally to households. A total of 124 samples from four drinking water supply systems in the Czech Republic, 52 dam sediments, 34 water treatment plant sludge samples, and 38 tap water household sediments, were analyzed. NTM of 11 different species were isolated by culture from 42 (33.9 %) samples; the most prevalent were M. gordonae (16.7 %), M. triplex (14.3 %), M. lentiflavum (9.5 %), M. a. avium (7.1 %), M. montefiorenase (7.1 %), and M. nonchromogenicum (7.1 %). NTM DNA was detected in 92 (76.7 %) samples. By qPCR analysis a statistically significant decrease (P < 0.01) was observed along the route from the reservoir (dam sediments), through water treatment sludge and finally to household sediments. The concentrations ranged from 10⁰ to 10⁴ DNA cells/g. It was confirmed that drinking water supply systems (watershed–reservoir–drinking water treatment plant–household) might be a potential transmission route for mycobacteria.     

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.     

Occurrence of Nontuberculous Mycobacteria in Environmental Samples

Nontuberculous mycobacteria (NTM) are a major cause of opportunistic infection in immunocompromised hosts. Because there is no evidence of person-to-person transmission and NTM have been found in drinking water, the environment is considered a likely source of infection. In this study the widespread occurrence of NTM was examined in drinking water, bottled water, and ice samples. A total of 139 samples were examined for NTM by a membrane filtration culture technique followed by PCR amplification and 16S rRNA sequence determination to identify the isolates. NTM were not detected in bottled water or cisterns but were detected in 54% of the ice samples and 35% of the public drinking-water samples from 21 states. The most frequently occurring isolate was M. mucogenicum (formerly referred to as an M. chelonae-like organism).     

Occurrence of mycobacteria in water treatment lines and in water distribution systems

The frequency of recovery of atypical mycobacteria was estimated in two treatment plants providing drinking water to Paris, France, at some intermediate stages of treatment. The two plants use two different filtration processes, rapid and slow sand filtration. Our results suggest that slow sand filtration is more efficient for removing mycobacteria than rapid sand filtration. In addition, our results show that mycobacteria can colonize and grow on granular activated carbon and are able to enter distribution systems. We also investigated the frequency of recovery of mycobacteria in the water distribution system of Paris (outside buildings). The mycobacterial species isolated from the Paris drinking water distribution system are different from those isolated from the water leaving the treatment plants. Saprophytic mycobacteria (present in 41.3% of positive samples), potentially pathogenic mycobacteria (16.3%), and unidentifiable mycobacteria (54.8%) were isolated from 12 sites within the Paris water distribution system. Mycobacterium gordonae was preferentially recovered from treated surface water, whereas Mycobacterium nonchromogenicum was preferentially recovered from groundwater. No significant correlations were found among the presence of mycobacteria, the origin of water, and water temperature.