Nontuberculous mycobacteria (NTM) are a large, diverse group of environment organisms that are ubiquitous in the soil, water, animals, human, and food (Griffith et al., 2007; Prevots and Marras, 2015). Despite of low pathogenicity to humans, NTM can cause a wide array of clinical illness, especially in human immune-deficiency virus (HIV) infected patients or those with severely compromised immune systems (Prevots and Marras, 2015). In recent years, increasing attention has been paid to disease caused by NTM because of its more prevalent incidence globally (Hoefsloot et al., 2013; Russell et al., 2014).

Only behind Mycobacterium avium complex, Mycobacterium kansasii is the second most common cause of NTM diseases in South American and some European countries (Hoefsloot et al., 2013). In addition to the most frequent pulmonary disease, M. kansasii is a contributor to various clinical manifestations, including extrapulmonary and disseminated diseases in patients with immunodeficiencies (Campo and Campo, 1997; Pintado et al., 1999). Previous epidemiological studies have demonstrated that patients with chronic lung disease, previous mycobacterial disease, malignancy, and alcoholism are at high risk for M. kansasii infection (Guay, 1996). M. kansasii is often recovered from tap water, and occasionally from natural water (McSwiggan and Collins, 1974; Steadham, 1980; Taillard et al., 2003). Due to wide exposure to the bacterium, human-to-human transmission of M. kansasii is traditionally thought to be uncommon (Ricketts et al., 2014). However, a recent report from Ricketts et al. (2014) observed the potential transmission of M. kansasii given the ideal circumstances, providing important insights for public health concern regarding M. kansasii.

PCR-restriction fragment analysis of the hsp65 gene (PRA) is a powerful technique of differentiating heterogeneous subtypes of M. kansasii (Telenti et al., 1993; Santin et al., 2004). To date, seven subtypes (I-VII) have been identified based on the PCR-RFLP profiles (Picardeau et al., 1997; Richter et al., 1999; Santin et al., 2004). Of these seven subtypes, subtype I represents the most frequent isolates contributing to human disease (Taillard et al., 2003), and subtype II is likely to be observed in HIV-infected patients, indicating that it may act as an opportunistic agent (Witzig et al., 1995). In contrast, the other five subtypes are generally isolated from environmental samples rather than human specimens (Alcaide et al., 1999). The niche heterogeneity indicates that there may be diverse pathogenicity and biological features among the M. kansasii subtypes (Santin et al., 2004). Considering drug susceptibility is an important indicator for predicting the clinical outcome of patients infected with M. kansasii, it is meaningful to investigate the assumed difference in drug susceptibility profiles of these seven subtypes. Unfortunately, limited data is known regarding this issue. The aim of the present study was to analyze M. kansasii subtypes isolated from patients in China, and to explore the antimicrobial susceptibility of the differentiation among these diverse subtypes.

To our best knowledge, the present study is the first provided fundamental information on the drug susceptibility and molecular analysis of M. kansasii isolates from China. In keeping with previous reports, our results demonstrated that AMK exhibits favorable in vitro activity against M. kansasii isolates, which supports its important role in the initial treatment of M. kansasii infections (van Ingen et al., 2010; Cowman et al., 2016). Notably, we found that the prevalence of CLA-resistance was 20.5%, which was higher than that from Spain (0%) (Guna et al., 2005), Netherlands (1%) (van Ingen et al., 2010), Brazil (3%) (da Silva Telles et al., 2005), and UK (13%) (Cowman et al., 2016). Due to the satisfactory antibacterial activity and rare adverse effect, macrolides have been used as the first-choice treatment for respiratory tract infection (Yezli and Li, 2012). Numerous literatures have demonstrated that the misuse of macrolides is associated with emergency and increased prevalence of CLA-resistant bacteria in China (Wang et al., 1998, 2001; Chen et al., 2013), which may also serve as a major contributor to the high prevalence of CLA-resistant M. kansasii isolates in China. In vitro susceptibility of non-tuberculous mycobacteria against CLA exhibits excellent correlation with clinical outcomes (Cowman et al., 2016), the high proportion of CLA-resistant M. kansasii in China thus highlights the urgent need to perform in vitro CLA susceptibility testing before the initial treatment with regimen containing CLA.

The common in vitro resistance to LFX and RIF was observed from our data, indicating the inclusion of these drugs in the therapeutic regimen is controversial. However, the good activity of MOX was detected in this study, which is consistent to previous results from van Ingen et al (van Ingen et al., 2010). A recent study revealed that the combination of MOX and CLA is associated with attenuated CLA activity in a murine model (Kohno et al., 2007). Nevertheless, MOX is an alternative antimicrobial agent for the patients infected with CLA-resistant M. kansasii.

Point mutations at either position 2058 or 2059 in the 23S rRNA gene have been reported to confer macrolide resistance phenotypes in NTM species (Inagaki et al., 2011; Zhao et al., 2014). For M. avium complex, almost 90% of CLA-resistant isolates harbor nucleotide substitutions in the 23S rRNA gene (Inagaki et al., 2011). In contrast, our data revealed that more than 40% of CLA-resistant M. kansasii isolates had no mutation at either position 2058 or 2059 of the 23S rRNA gene. The unsatisfactory correlation between in vitro CLA resistance and mutations of 23S rRNA indicates that other mechanisms, such as efflux pump and methylation of 23S rRNA, may be involved in CLA resistance for M. kansasii (Inagaki et al., 2011; Brown-Elliott et al., 2012).

Mycobacterium kansasii subtype I is the most frequent subtype isolated from humans in many geographic areas of the world (da Silva Telles et al., 2005). In agreement with previous reports, the subtype I is also the predominant genotype of M. kansasii in China, the prevalence of which is similar to that in Switzerland (68%) (Taillard et al., 2003), although it is lower than that in Spain (98%) (Santin et al., 2004) and Brazil (98%) (da Silva Telles et al., 2005), and higher than that in Germany (33%) (Richter et al., 1999), Poland (40%) (Bakula et al., 2013), and France (43%) (Alcaide et al., 1997). Hence, the distribution of subtype I exhibits geographical diversity, which may be associated with the existence of different ecosystems for these microorganisms (Taillard et al., 2003). In addition, only 14.1% of M. kansasii isolates in this study were classified as subtype II, lower than data from previous studies (Alcaide et al., 1997; Richter et al., 1999). Different from Subtype I, Subtype II is considered as an opportunistic agent, which seems to be more frequently isolated from HIV-infected patients (Taillard et al., 2003). Thus, the low prevalence of subtype II may be due to the low epidemic of HIV in China.

Another important finding of the current study is that M. kansasii subtype I is associated with CLA resistance in China. Subtype I is the most common cause responsible for human infection by M. kansasii, while the other subtypes are likely to be isolated from environmental samples (Taillard et al., 2003). We speculate that the different drug susceptibility profiles between subtype I and other subtypes may reflect their significant difference in ecological niches. Due to more frequent exposure to antibiotic selection pressure from human, subtype I emerges the higher proportion of drug resistance to increase adaptability in hosts. Given that CLA-based drug regimens are effective options for treatment of M. kansasii infection, the statistical difference observed in CLA-resistance between subtype I and other subtypes is reasonable. In line to our hypothesis, we also found that subtype I was more likely to be resistant to EMB, MOX, and GAT than other subtypes. Unfortunately, because the small sample size undermines the statistical power, the difference was not statistically significant. Further study with more than 200 isolates will be required to determine the potential relationship between M. kansasii subtypes and drug susceptibility profiles.

There were several obvious limitations in this study. First, although chronic pulmonary disease is the most frequent clinical manifestation of NTM infection, it also causes extrapulmonary diseases, such as lymphatic, skin and soft tissue diseases (Griffith et al., 2007). Considering the predominance of M. kansasii subtype I in the pulmonary diseases from our observations, it is important to investigate the distribution of difference subtypes in the extrapulmonary infections due to M. kansasii. Unfortunately, the isolates enrolled in this study were only collected from suspected pulmonary tuberculosis patients, which impedes the further analysis. Second, HIV status in patients, who are at a high risk for NTM diseases (Henkle and Winthrop, 2015), was not collected. Due to the relative low prevalence of HIV in China, routine HIV examination is not performed among TB patients. On the basis of our findings, future studies will be done to validate the distribution of M. kansasii subtypes in these special populations, which will extend our knowledge of host preference and bacterial pathogenicity among various M. kansasii subtypes.

Our data demonstrate that AMK is the most active agent against M. kansasii in vitro, while the high proportion of CLA resistance is noted in M. kansasii isolates from China. Further PRA analysis reveals that subtype I is the predominant genotype of M. kansasii in China. In addition, M. kansasii subtype I is associated with CLA resistance. Given the CLA-containing regimen frequently used in the treatment M. kansasii, the high proportion of CLA resistance in China highlights the urgent need to perform in vitro CLA susceptibility testing before the initial treatment with CLA.

YL, YP, YZ, and CW designed this study. YL, YP, and HZ performed experiments. YL, YP, and XT interpreted the data. YL, YP, and CW wrote the manuscript. All authors approved the final version of the paper.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.