Nontuberculous mycobacteria (NTM) refer to mycobacteria other than Mycobacterium tuberculosis and Mycobacterium leprae. Some NTM are known to cause pulmonary and skin diseases. As NTM are prevalent in water supply systems, the health burden of waterborne NTM is concerned. However, little is known about NTM in building plumbing. This study revealed the abundance and diversity of NTM in building plumbing by using culture-dependent and culture-independent approaches. Moreover, the chlorine susceptibility of NTM isolates in tap water was evaluated. The increase in the abundance of Mycobacterium spp. was observed in some taps after 24 h of stagnation, indicating that NTM could have the potential to regrow in building plumbing. While Mycobacterium mucogenicum and Mycobacterium avium were detected by cultivation, amplicon sequencing of NTM-specific hsp65 genes revealed that Mycobacterium gordonae was dominant in most of the samples, which was not detected by cultivation. The chlorine susceptibility experiment demonstrated that NTM strains related to M. avium and M. mucogenicum were 119 and 426 times more tolerant to chlorine than Escherichia coli, respectively. As M. mucogenicum, M. avium, and M. gordonae are regarded as opportunistic pathogens, intensive monitoring of NTM in tap water in building plumbing is necessary.

  • NTM in building plumbing had the potential to increase after water stagnation.

  • Potentially pathogenic M. avium and M. mucogenicum were isolated from building plumbing, which were more tolerant to chlorine than E. coli.

  • Integration of both culture-dependent and culture-independent approaches is necessary for a comprehensive understanding of NTM diversity in building plumbing.

Nontuberculous mycobacteria (NTM) are environmental mycobacteria in soil and water other than Mycobacterium tuberculosis and Mycobacterium leprae, and some species are known as opportunistic pathogens (Falkinham et al. 2015). Exposure to pathogenic NTM through inhalation, ingestion, and skin contact can result in pulmonary and skin/soft tissue infection, particularly for individuals with compromised immune systems (Hamilton et al. 2017). The incidence of NTM infection has increased in many countries, including Japan, leading to a significant healthcare burden, particularly among the elderly population (Harada et al. 2020). Waterborne transmission of NTM is a major concern, and the Centers for Disease Control and Prevention (CDC) has reported a high number of hospitalizations and deaths related to waterborne NTM infections in the U.S. between 2010 and 2015 (Collier et al. 2021). Nosocomial NTM infection was related to the occurrence of NTM in the hospital water supply system (Zlojtro et al. 2014). A significant correlation was also found between the abundance of pathogenic NTM species (e.g., Mycobacterium avium complex and Mycobacterium abscessus complex) in household showerhead biofilm and the prevalence of NTM disease from medicare beneficiaries and cystic fibrosis patients (Gebert et al. 2018). These studies highlighted the necessity to control NTM in water supply systems.

While culture-dependent methods have been established for several NTM species in drinking water (Le Dantec et al. 2002; Pfaller et al. 2022), NTM cultivation can be time-consuming, especially for slow-growing species. In contrast, culture-independent characterization using next-generation sequencing has emerged as a valuable tool to comprehensively assess the diversity of NTM communities in drinking water (van der Wielen et al. 2013; Gebert et al. 2018; Waak et al. 2019). For NTM characterization, the use of hsp65 genes instead of 16S rRNA genes has demonstrated enhanced resolution for species identification of NTM (van der Wielen et al. 2013). Amplicon sequencing of mycobacterial hsp65 genes has provided valuable insights, such as the predominance of Mycobacterium gordonae in a chlorinated drinking water distribution system (Waak et al. 2019). Considering that NTM is resistant to chlorine (Taylor et al. 2000; Wang et al. 2019) and has the potential for regrowth during water stagnation in building plumbing (Rahmatika et al. 2022), it is possible that disinfection conditions can influence the diversity of NTM in drinking water.

Despite M. avium and M. abscessus being listed on the contaminant candidate list (CCL-5) for drinking water (USEPA 2021), our understanding of the abundance, diversity, and characteristics of NTM in building plumbing is still limited due to the lack of routine monitoring for NTM. While the culture-independent method can provide valuable information on NTM, the throughput is low and it generally takes a long time for cultivation of NTM. On the other hand, culture-independent methods have the advantage of rapid and comprehensive analysis. Therefore, the integration of both culture-dependent and culture-independent approaches is useful for monitoring NTM. This study aims to characterize NTM in tap water in building plumbing by using culture-dependent and culture-independent methods. Additionally, the chlorine tolerance of indigenous NTM strains isolated from tap water was also evaluated.

Sample collection

Fresh water (FW) and stagnant water (SW) samples were collected from four taps (A1–A4) in the buildings of the National Institute of Public Health in Saitama Prefecture, Japan (site A) in December 2020 (Supplementary material, Table S1). NTM in the samples were analyzed with culture-dependent and culture-independent approaches. Hot water was supplied at A1, while cold water was supplied at the other taps. Site A receives mixed drinking water derived from two plants: one plant treats surface water, while the other plant treats groundwater. From April 2020 to March 2021, standard plate counts (37 °C for 24 h) and heterotrophic plate counts (HPC, 20 °C for 7 days) were 0 and Escherichia coli were not detected in the mixed finished water from these plants, according to an annual water quality report of the waterworks. The faucets were flushed for 5 min at a flow rate of approximately 5 L/min before collecting the FW samples (4 L). The faucets were subsequently closed for 24 h. After 24 h of stagnation, the first 4 L of SW samples were immediately collected from the same faucets with the same flow rate. For reference, the groundwater used for non-potable use at site A was also collected to reveal NTM diversity. Additional SW samples were collected in the same manner from three faucets (B1–B3) at The University of Tokyo in Tokyo, Japan (site B) in four different seasons: Summer (May 2018), Autumn (November 2018), Winter (January 2019), and Spring (April 2019) (Rahmatika et al. 2022). Tap water in site B was supplied from a water treatment plant that treats surface water by advanced treatment using ozonation and biologically activated carbon filtration. The first 1 L of SW samples was collected after 24 h of stagnation from each faucet. These samples were used only for analyzing the diversity of NTM communities using the culture-independent method.

Water temperatures were measured on-site by a thermometer. Free chlorine was measured by N,N-diethyl-p-phenylenediamine (DPD) colorimetric method (Hach, CO, USA) with a limit of quantification (LOQ) of 0.02 mg/L.

Cultivation and isolation of NTM

FW and SW samples as well as groundwater collected from site A were subjected to NTM cultivation. The samples were incubated with 0.04% cetylpyridinium chloride (CPC) (Sigma-Aldrich, MO, USA) for 30 min at room temperature to eliminate other microorganisms (Schulze-Röbbecke et al. 1991). Tap water samples (650 mL) and groundwater samples (1.3 L) were filtered through 0.45-μm EZ-Fit™ filtration unit (Merk-Millipore, MA, USA) in triplicate. The membrane filters were then rinsed with autoclaved Milli-Q water to remove the residual CPC. The first and second membrane filters were transferred to Middlebrook 7H10 agar (Becton, Dickinson and Company, NJ, USA) and R2A agar (Becton, Dickinson and Company), respectively. Middlebrook 7H10 agar contained 10% OADC enrichment (Becton, Dickinson, and Company) and 10% MGIT PANTA (Becton, Dickinson, and Company). These plates were incubated at 30–32 °C until the colonies were formed. The third membrane filter was preincubated in Middlebrook 7H9 broth (Becton, Dickinson, and Company) containing 10% OADC enrichment and 10% MGIT PANTA. The cultivation is conducted in aerobic conditions. After one week of enrichment, the broth was spread on 2% Ogawa medium (Kyokuto Pharmaceutical, Japan) and was incubated at 30–32 °C until the colonies were formed. Following 17–23 days of incubation, representative colonies were picked and subcultured using Middlebrook 7H10 agar for colony direct PCR and sequencing analysis.

Identification of NTM isolates

Colony-direct PCR was used to determine 16S rRNA genes and hsp65 genes of the isolates. Amplification of 16S rRNA genes was performed with the primer set of 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) (Heuer et al. 1997). Moreover, the primer set of Tb11 (5′-ACCAACGATGGTGTGTCCAT-3′) and Tb12 (5′-CTTGTCGAACCGCATACCCT-3′) were used to amplify hsp65 genes of NTM (Waak et al. 2019). Each PCR reaction mixture (50 μL) was prepared using TaKaRa EX Taq HS version (TaKaRa Bio, Japan), consisting of 40.6 μL of nuclease-free water (Ambion Nuclease-Free Water, Invitrogen, MA, USA), 5.0 μL of 10× PCR buffer, 4.0 μL of 10 mM dNTPs, 0.25 μL of TaKaRa EX Taq polymerase, and 0.1 μL of each primer (100 pmol/μL). A small amount of a single colony was directly added to the reaction mixture. The thermal conditions for 16S rRNA genes were set as follows: 94 °C for 2 min, followed by 30 cycles at 94 °C for 30 s, 53 °C for 30 s, and 72 °C for 1 min (Heuer et al. 1997). For hsp65 genes, the thermal condition consisted of initial denaturation at 98 °C for 2 min, followed by 30 cycles at 98 °C for 15 s, 60 °C for 30 s, and 72 °C for 1 min (Waak et al. 2019). The final extension step at 72 °C for 10 min was added for both reactions. PCR products were purified by a MinElute PCR Purification kit (QIAGEN, Germany) and submitted to FASMAC (Japan) for sanger sequencing. A sequence homology search was conducted using BLAST. Sequences of NTM isolates were aligned using MUSCLE (Edgar 2004), and the phylogenetic tree was generated using the Neighbor-Joining method by MEGAX software (Kumar et al. 2018). The reference sequences of characterized NTM obtained from the GenBank database were added to the phylogenetic tree. The sequences of 16S rRNA genes and hsp65 genes of the isolates have been deposited on DDBJ/EMBL/GenBank databases under accession numbers LC637767–LC637776 and LC637933–LC637942, respectively.

DNA extraction

To harvest bacteria in the samples, 2 L (tap and groundwater samples from site A) and 1 L (tap water samples from site B) of samples were filtered through 0.2-μm polycarbonate filters (Isopore, Merk-Millipore). DNA was extracted directly from the filter by FastDNA SPIN Kit for Soil following the manufacturer's instructions (MP Biomedicals, OH, USA). Before extraction, the filters were dissolved in phenol-chloroform-isoamyl alcohol (25:24:1) (Nippon Gene, Japan). The DNA concentration and purity (absorbance ratio at 260 nm/280 nm) were measured by Qubit Fluorometer (ThermoFischer Scientific, MA, USA) and NanoDrop spectrophotometer (ThermoFischer Scientific), respectively.

Real-time PCR

For FW and SW samples collected from site A, Myco637f (5′- GGGCGATACGGGCAGAC-3′), Myco841r (5′- GAAACCCACACCTAGTACC-3′), and Myco662p (5′-FAM-CAGGGGAGACTGGAATTCCTGGTGTA-MGBNFQ-3′) were used for quantification of 16S rRNA genes of Mycobacterium spp. with TaqMan-based real-time PCR (Feazel et al. 2009). The thermal condition consisted of initial denaturation at 95 °C for 5 min, followed by 45 cycles at 95 °C for 20 s and 60 °C for 1 min (Feazel et al. 2009). All reactions were prepared in triplicate by LightCycler® Probes Master solution (Roche, Switzerland) and analyzed using a LightCycler® LC480 system (Roche). Standards were prepared using an artificially synthesized plasmid containing 16S rRNA genes of Mycobacterium spp. with the concentration of 5.0 × 100–5.0 × 106 gene copies/μL. The LOQ was 2.5 × 10−1 gene copies/mL. To evaluate the significant difference (p < 0.05) in the abundance of Mycobacterium spp. between FW and SW, paired student's t-test was performed for log-transformed data by R statistical software (ver. 3.4.1).

Amplicon sequencing of hsp65 genes

The diversity of NTM communities in FW and SW samples from site A and SW samples from site B was characterized using amplicon sequencing targeting hsp65 genes. The fragments of hsp65 genes (441 bp) were amplified with Tb11 and Tb12 with an adapter sequence (Waak et al. 2019). Amplicon sequencing was conducted on an Illumina MiSeq platform at the Bioengineering Lab (Japan). Sequences generated from the Illumina MiSeq analysis were processed using Quantitative Insights into Microbial Ecology 2 (QIIME 2) (Bolyen et al. 2019). After eliminating noise and chimera sequences, the feature-classifier (QIIME 2 plugin) was used to affiliate OTUs to an in-house database of 149 different hsp65 genes of Mycobacterium spp. Operational taxonomic units (OTUs) were clustered with 100% identity. To evaluate the significant difference of NTM communities between samples in sites A and B, analysis of similarity (ANOSIM) was performed based on the Bray–Curtis dissimilarity index at the OTU level using vegan package (ver. 2.5-7) in R statistical software (ver. 3.4.1). The sequences of representative OTUs were analyzed using a phylogenetic tree together with the sequences of the isolates. All data obtained from amplicon sequencing were submitted to the DDBJ Sequence Read Archive under accession number DRA012200.

Chlorine tolerance test

Two NTM strains isolated from site A (T1 and T8) were subjected to a chlorine tolerance test. Strain T1 (Accession number: LC637941) and T8 (Accession number: LC637937) were incubated in Middlebrook 7H9 broth (Becton, Dickinson Company) containing 10% OADC enrichment 30 °C for 7 days. In addition, Escherichia coli strain K-12 as a reference was incubated in LB broth (Becton, Dickinson Company) overnight. The cultures were then collected by centrifugation (6,000× g for 10 min) and washed with phosphate-buffered saline (PBS) three times, then finally suspended in PBS. Triplicate sets of cell suspension in PBS (105 CFU/mL) were prepared in 300 mL chlorine demand-free Erlenmeyer flasks for chlorine disinfection test. Free chlorine with a concentration of 0.60–0.85 mg/L was added to the cell suspension. The experiments were performed at room temperature with gentle stirring (60 rpm). 10 mL of samples were collected during the experiment to measure free chlorine residual by DPD colorimetric method (Hach, Colorado, USA). In parallel, samples were also collected and quenched with sodium thiosulfate to enumerate NTM on Middlebrook 7H10 agar containing 10% OADC enrichment or E. coli on LB agar. Log reduction values of the NTM strains and E. coli were summarized with Ct value which is defined as the product of free chlorine concentration (mg/L) and contact time (min).

Changes in NTM abundance during water stagnation

The increase in temperature and decrease of free chlorine was observed after 24 h of water stagnation in the samples in site A (A1–A4) (Supplementary material, Table S1). 16S rRNA genes of Mycobacterium spp. were quantified for samples collected from A1 to A4 (Figure 1). FW contained 1.3 × 10–4.8 × 102 gene copies/mL. After 24 h of stagnation, the abundances were 3.9 × 10–2.5 × 102 gene copies/mL. The apparent increase of Mycobacterium spp. was observed at A1–A3 and the increases at A2 and A3 were significant (Student's t-test; p < 0.05). The abundance slightly decreased after stagnation at A4 where NTM abundance before stagnation was higher compared to the others. The initial abundance before stagnation and the regrowth potential of NTM after stagnation may depend on water usage frequency and various factors such as temperature and pipe condition. The abundance of 16S rRNA genes of NTM in the groundwater sample was 2.0 × 10 gene copies/mL, indicating that NTM abundance in groundwater was less than that in stagnant tap water. It is possible that NTM could adapt more in building plumbing environments. The concentration of NTM at site B before and after stagnation in different seasons was 2.5 × 100 and 8.8 × 10 gene copies/mL, respectively, indicating that regrowth after stagnation was reproducibly observed (Rahmatika et al. 2022). The increase in NTM abundance after stagnation can be attributed to their characteristics, such as resistance to chlorine, biofilm formation ability, and association with free-living amoeba (Falkinham et al. 2015). The level of Mycobacterium spp. after stagnation in site A and site B was almost equivalent to the abundance of 16S rRNA genes of Mycobacterium spp. in the chlorinated drinking water in Paris (average: 1.3 × 102 gene copies/mL) (Perrin et al. 2019).
Figure 1

The copy number of 16S rRNA genes of Mycobacterium spp. in the samples collected from site A. Error bars indicate the standard deviation of triplicate measurement of real-time PCR. FW, fresh water before stagnation; SW, stagnant water after 24 h stagnation.

Figure 1

The copy number of 16S rRNA genes of Mycobacterium spp. in the samples collected from site A. Error bars indicate the standard deviation of triplicate measurement of real-time PCR. FW, fresh water before stagnation; SW, stagnant water after 24 h stagnation.

Close modal

HPC as well as other opportunistic pathogens should be monitored to obtain a more comprehensive view of microbial regrowth in building plumbing. Investigating the relationship between NTM and HPC or other opportunistic pathogens can provide valuable information into the occurrence and dynamics of NTM.

Identification of NTM isolates

Eight colonies (T1–T8) were isolated from tap water samples in site A. T1–T3 were isolated from FW at A1. T4 and T5 were isolated from FW and SW at A2, respectively. T6 and T7 were isolated from FW and SW at A3, respectively. T8 was isolated from FW at A4. Two colonies (G1 and G2) were isolated from the groundwater sample. They were identified as Mycobacterium spp. by sequencing their 16S rRNA genes and hsp65 genes (Supplementary material, Table S2). Figures 2 and 3 show the phylogenetic trees based on 16S rRNA genes and hsp65 genes, respectively. The isolates obtained from tap water were closely related to Mycobacterium mucogenium for T2 and T4–T8 and M. avium for T1 and T3, which were supported by both 16S rRNA genes and hsp65 genes.
Figure 2

Phylogenetic tree of 16S rRNA gene sequences of NTM isolates using the Neighbor-Joining method. The tree is rooted with the outgroup (Escherichia coli). The bootstrap values higher than 50% are shown.

Figure 2

Phylogenetic tree of 16S rRNA gene sequences of NTM isolates using the Neighbor-Joining method. The tree is rooted with the outgroup (Escherichia coli). The bootstrap values higher than 50% are shown.

Close modal
Figure 3

Phylogenetic tree of hsp65 gene sequences of NTM isolates and the representative OTUs retrieved by next-generation sequencing using the Neighbor-Joining method. The tree is rooted with the outgroup (Streptomyces somaliensis). The bootstrap values higher than 50% are shown.

Figure 3

Phylogenetic tree of hsp65 gene sequences of NTM isolates and the representative OTUs retrieved by next-generation sequencing using the Neighbor-Joining method. The tree is rooted with the outgroup (Streptomyces somaliensis). The bootstrap values higher than 50% are shown.

Close modal

The closest relative of T1 and T3 was identified as M. avium JNBP_03535 (16S rRNA gene) and M. avium ATCC25291 (hsp65 gene). M. avium JNBP_03535 was isolated from the sputum of a patient in Japan (Mizuno et al. 2016), while M. avium ATCC25291 was a clinical isolate collected from Korea (Kim et al. 2005). M. avium is well-known as a major pathogenic NTM that causes pulmonary disease. In Japan, M. avium has been reported to be one of the most pathogenic agents that caused NTM disease (Morimoto et al. 2017). Interestingly, T1 and T3 were obtained from fresh tap water at A1, which supplied hot water with a temperature of 34.4–55.0 °C. M. avium is relatively resistant to elevated temperatures up to 50 °C for 60 min (Schulze-Röbbecke & Buchholtz 1992). Previous studies reported the occurrence of M. avium from shower water and bathtub water from the bathroom of patient with NTM disease in Japan (Nishiuchi et al. 2009), suggesting that a hot water environment could be preferable for M. avium.

T2 and T4–T8 were closely related to M. mucogenicum S12 (16S rRNA gene) and M. mucogenicum ATCC 49651 (hsp65 gene). M. mucogenicum-like strains were isolated from both FW and SW samples at A2 and A3. On the other hand, G1 and G2 isolated from groundwater were affiliated with Mycobacterium chelonae, which has been recently subclassified as new emended genera, Mycobacteroides chelonae (Gupta et al. 2018). M. mucogenicum is associated with catheter-related infections, respiratory infections, and skin and soft tissue infections (Adékambi 2009). These findings suggested that building a plumbing environment could affect culturable NTM diversity.

While M. mucogenium was isolated using R2A agar, M. avium and M. chelonae were recovered from Middlebrook 7H10 agar and 2% Ogawa medium after preincubation in Middlebrook 7H9 broth. Because M. mucogenicum and M. chelonae are rapid growers (van Ingen et al. 2009), it is possible that they could outcompete other NTM during cultivation. This result indicates that preferable cultivation conditions could be different for these NTM species.

Although the occurrence of NTM related to M. avium and M. mucogenicum in tap water was noteworthy, it is essential to support future studies with evidence of water-linked infections, by incorporating patient data on the incidence and prevalence of NTM infections to establish a connection between the isolated strains and their potential role in causing diseases. In addition, previous studies reported the occurrence of M. mucogenicum, M. avium, as well as other opportunistic pathogens (e.g., Legionella spp., and Pseudomonas aeruginosa) from the outlet of drinking water treatment plants (Dávalos et al. 2021; Huang et al. 2021b). Though their abundance at the outlet of the treatment plant was low, we should pay more attention to the persistence of these opportunistic pathogens along the treatment process. To reveal the comprehensive dynamics of NTM and other opportunistic pathogens, their occurrence at the outlet of drinking water treatment plants and pipe networks should be studied.

The diversity of NTM communities revealed by next-generation sequencing

NTM communities in tap water samples from site A and site B were analyzed using next-generation sequencing targeting hsp65 genes. In total, 41,073–60,974 reads were determined for each sample. After eliminating noise and chimera sequences, 26,168–45,748 qualified reads were used for further analysis. All of them were 86.8–100% identical to hsp65 genes of Mycobacterium spp. and were classified into 94 OTUs in Mycobacterium spp. ANOSIM revealed that NTM communities between samples in sites A and B were statistically different (R-value = 0.8253, p < 0.05). M. gordonae was the most dominant NTM species, with an average abundance of 75.6 ± 27.7 and 78.7 ± 28.8% of the total reads in the samples from site A and site B, respectively (Figure 4). M. avium and M. mucogenicum were not detected by next-generation sequencing, demonstrating that cultivation could have a great selection bias on the diversity of NTM communities. For practical monitoring of NTM, both culture-dependent and culture-independent methods should be implemented to comprehensively reveal NTM communities in fresh and stagnant drinking water. M. gordonae can cause pulmonary infection in immunocompromised patients (Mazumder et al. 2010). The dominance of M. gordonae was reported in chloraminated and chlorinated drinking water (Waak et al. 2019). Moreover, M. gordonae was frequently detected in the distribution system supplied by surface water (Le Dantec et al. 2002). Mycobacterium paraterrae was also abundant in some samples of site A (0–66.3%). While M. paraterrae was isolated from patients with symptomatic pulmonary infection in Korea (Lee et al. 2010), information on the occurrence of M. paraterrae in drinking water is limited. Mycobacterium lentiflavum was the second most dominant NTM in site B (0–84.7%). M. lentiflavum was also reported to cause pulmonary infection (Yagi et al. 2018). M. lentiflavum was detected in chlorinated drinking water distribution systems and chlorinated treated water (King et al. 2016). Different water sources and different building plumbing conditions between sites A and B could probably affect NTM diversity. Moreover, the relative abundance of M. gordonae, M. paraterrae, and M. lentiflavum differed among taps even in the same building, suggesting that NTM communities could be site-specific. The diversity of Mycobacterium spp. at site B were also analyzed using Nanopore sequencing targeting the full length of the 16S rRNA gene and only 18.7% of Mycobacterium-related sequences were assigned to species in Mycobacterium spp. (Rahmatika et al. 2022). This indicated that sequencing of hsp65 gene provided better resolution for Mycobacterium spp. Identification in tap water.
Figure 4

The composition of NTM communities identified by hsp65 genes from tap water samples in site A and site B. FW, fresh water before stagnation; SW, stagnant water after 24 h stagnation; SU, summer; AU, autumn; WI, winter; SP, spring.

Figure 4

The composition of NTM communities identified by hsp65 genes from tap water samples in site A and site B. FW, fresh water before stagnation; SW, stagnant water after 24 h stagnation; SU, summer; AU, autumn; WI, winter; SP, spring.

Close modal

Water stagnation not only increased NTM abundance (Figure 1), but also impacted the diversity of NTM communities at some faucets (Figure 4). For example, M. gordonae decreased from 75.7 to 42.5% and M. paraterrae increased from 24.2 to 57.0% after stagnation at site A2. However, the opposite trend was observed at A4. It is possible that changes in water quality could trigger the changes in NTM diversity and some selection may occur during water stagnation. On the other hand, M. gordonae was dominant in both fresh and SW samples at A1 and A3. Thus, changes in NTM communities during water stagnation could be dependent on the condition of each faucet. Seasonal variation of NTM communities was found in B1 in site B. In summer, M. lentiflavum was the most dominant, while M. gordonae was abundant in the other seasons.

M. gordonae, M. paraterrae, and M. lentiflavum were not detected by cultivation, probably because they are slow-growing NTM species (Lee et al. 2010; Mazumder et al. 2010; Yagi et al. 2018). It is possible that the growth of the slow-growing NTM species was outcompeted by rapidly growing NTM during cultivation. However, further evaluation is required to assess the selection pressure on slow-growing NTM during cultivation. Additionally, the decontamination process employed during cultivation should be evaluated to enhance the isolation of more NTM strains.

These findings highlighted the dynamics of NTM communities in building plumbing and the influence of various factors such as water stagnation on their abundance and diversity. However, it is important to note that the sample size in this study was limited, which could impact the overall understanding of the influence of water stagnation on the abundance and diversity of NTM. Future studies should consider collecting a larger number of tap water samples from various sites.

Chlorine tolerance of NTM

Chlorine tolerance of two NTM isolates (T1: M. avium-like, T8: M. mucogenicum-like) and E. coli were compared. As shown in Figure 5, NTM isolates were more resistant to chlorine compared to E. coli. The inactivation curves of T1, T8 and E. coli indicated that the inactivation rate constant was 0.091, 0.023, and 10.911 L mg−1 min−1 with the R2 of 0.996, 0.935, and 0.979, respectively. T8 is more resistant than T1. CT values required for 3-log reduction (CT99.9%) of T1 and T8 were 34.2 and 122.7 mg min/L, respectively, which were 119 and 426 times greater than that of E. coli. A previous study reported the CT99.9% of M. avium strain isolated from AIDS patients was 106 mg min/L (Taylor et al. 2000), which was higher than our finding. It was also reported that M. avium was resistant to other disinfectants, including monochloramine, chlorine dioxide and ozone (Taylor et al. 2000). CT99.9% of M. mucogenicum in our study was higher than that of M. mucogenicum strains isolated from other distribution systems reported by Wang et al. (59.2 mg min/L) (Wang et al. 2019) and Chen et al. (29.6 mg min/L) (Chen et al. 2012).
Figure 5

Chlorine inactivation of (a) NTM strains (T1 and T8) in this study and (b) E. coli K12.

Figure 5

Chlorine inactivation of (a) NTM strains (T1 and T8) in this study and (b) E. coli K12.

Close modal

The high hydrophobicity of the cell membrane of NTM could be one of the major factors influencing their tolerance to chlorine (Steed & Falkinham 2006; Wang et al. 2019). Furthermore, the biofilm formation could further increase the disinfectant resistance of NTM, including M. avium and Mycobacterium phlei (Steed & Falkinham 2006,). It is also reported that the dominance of NTM in building plumbing biofilm (Huang et al. 2021a). Although disinfection is essential for maintaining microbial safety, it can consequently select chlorine-tolerant NTM that can survive in drinking water (Falkinham et al. 2015). Those factors likely contribute to the regrowth and persistence of NTM in tap water. Further study is thus required to control chlorine-resistance NTM completely in building plumbing.

NTM in tap water were characterized using culture-dependent and culture-independent approaches. Regrowth of NTM was observed after water stagnation. The cultivation method using R2A and Middlebrook 7H10 agar/Ogawa medium detected M. mucogenicum and M. avium, respectively. On the other hand, amplicon sequencing of hsp65 genes of NTM could demonstrate that M. gordonae was the most prevalent in most samples, followed by M. paraterrae and M. lentiflavum. A significant difference between the results of culture-dependent and culture-independent methods indicated that NTM diversity should be carefully interpreted depending on the detection approaches. The chlorine tolerance test demonstrated that NTM strains related to M. avium and M. mucogenicum were 119 and 426 times more resistant to chlorine compared with E. coli, respectively. Since M. mucogenicum, M. avium, M. gordonae, M. paraterrae, and M. lentiflavum were reported as opportunistic pathogens causing pulmonary and respiratory infections as well as skin and soft tissue infections, we should pay attention to the occurrence of these NTM in building plumbing. Moreover, disinfection and residual disinfectant conditions should be revisited to control the occurrence of NTM in the water supply system.

The authors thank Ms Tomoko Inoue, The University of Tokyo, for her contribution to sampling.

This study was supported by JSPS KAKENHI (Grant Number 20H02282).

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.

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