It was shown in this study that there was 6.4% NTM pulmonary disease among all myco-bacteriological culture positive cases based on nationwide surveillance of drug-resistant tuberculosis. The pulmonary NTM infection was happened more frequently in southern China, especially, in southeastern regions coast areas with high humidity. The most prevalent SGM was M. avium-intracellulare complex which consist of seven subspecies, and the predominant subspecies is M. intracellulare. M. intracellulare was widely distributed in northern and southern China. The most prevalent RGM was M. abscessus complex which consist of three subspecies, and the predominant subspecies is M. abscessus that mainly distributed in southern China. The drug susceptibility testing results indicated that drug-resistant spectrum varied greatly in different strains subspecies. Macrolides and amikacin recommended to NTM treatment in China were showed lower resistant rates to NTM in vitro.
Distinguishing NTM from MTBC infection is of great significance in clinic, which can direct accurate and rapid clinical treatment [13–15]. The screening method used for NTM species are based on PNB which can inhibit growth of M. tuberculosis complex in most published paper in China [16], a method which is time-consuming and strenuous. The laboratory diagnosis methods used for mycobacterial species identification have evolved though the past decades [17].With the development of several extraction methods that enhance the amount of bacterial proteins available for MALDI-TOF MS identification and the increasing number of mycobacterial spectra in commercial databases, MALDI-TOF MS technology has been implemented for NTM identification in many laboratories [7, 18]. Several researches have proved this method achieved more than 95% agreement with the DNA sequencing of variable genomic regions (16S rRNA, hsp65, rpoB and ITS genes) [5, 19]. In our research, we obtained 98.4% NTM detection rate and achieved 93.4% agreement with 16S rRNA, hsp65, ITS and rpoB genes sequencing. Although we cannot identify the NTM strains that were not contained in the Bruker MBT strains database, MALDI-TOF MS is available to identify most clinically NTM in a rapid, reliable, and inexpensive manner.
The overall NTM pulmonary infection rate is about 6% in our study similar with a systematic review and meta-analysis of NTM infections which demonstrated that the prevalence of NTM infections among tuberculosis suspects was 6.3% in mainland China [20]. The geographic variability both in prevalence of NTM infections and in mycobacterial species composition is distinctly presented in our study. A research in southern-central China demonstrated that the NTM infection rate is 4.0%, and the two most prevalent species are M. avium-intracellulare complex and M. chelonae-abscessus Complex [6]. An article in Shanghai province proved that the overall rate of NTM isolated from mycobacterial culture-positive patients was 5.9% and the most frequently identified species is M. kansasii, with an increasing trend from 3.0% in 2008 to 8.5% in 2012 [21]. In our study, the most frequently species in Shanghai province is also M. kansasii (7/11), with an increasing NTM prevalence rate 11% in 2013. In another research from Guangdong and Shanghai province proved that M. intracellulare was the most commonly isolated NTM in Shanghai and the most frequently isolated species in Guangzhou is M. abscessus [22]. Some researches in eastern and northern region of China demonstrated the NTM prevalence rate is around 2.0%-3.0% and the predominated species is M. intracellulare, followed by M. abscessus [5, 21–23]. In our study, the NTM infection is more prevalent in southern than northern China and is more frequently in eastern than western China. The most epidemic NTM species are M. avium-intracellulare complex that is widely distributed and M. abscessus Complex which is mainly distributed in southeastern China. In addition, we isolated 16 M. marseillense strains of M. avium-intracellulare complex from sputum sample. Pulmonary disease caused by M. marseillense should be paid more attention as relevant report is less [24, 25].
Besides Mycobacteria spp., we identified some acid fast staining positive non-mycobacteria. As showed in Fig.S1, we randomly selected 60 non-mycobacteria from 286 polluted or other species for species identification, 15 Gordonia, 2 Nocardia, 2 Streptococcus and 1 Tsukamurella (File S6). The 15 Gordomia (8 G.sputi, 4 G. bronchialis and 3 G. rubripertincta) are distributed in 9 provinces. Interestingly, the occurrence of Gordonia is similar to some report in China [22]. Two Nocardia which often cause chronic lung diseases were isolated, as reported in the several article in China [23]. In addition, Tsukamurella infection case in Jiangxi province has previously been reported in in Southern-central China [6]. Besides NTM and MTBC infection, especially Gordomia and Nocardia species should be identified when using acid-fast staining to diagnosis pulmonary infection.
We evaluated the susceptibility of the RGM and SGM in China by measuring the MIC values of antimicrobials for bacteria using the RAPIDMYCOI and SLOWMYCOI Sensititre™ panel (Thermo Fisher Scientific, Waltham, MA, USA) according to CLSI protocol M24-A2. In China, no such simple MIC measurement commercial kit is available, despite the increase of patients with NTM infections, information on drug susceptibility of NTM isolates is still lacking. We mainly analyzed the susceptibility of the M. abscessus complex, which are the most common clinical RGM isolates. Inducible macrolide resistance affected differences of treatment outcome between M. abscessus and M. massiliense. The same with previous articles [26–28], we found that M. abscessus had a higher inducible resistance rate (65.67% vs 2.22%, p < 0.001) and acquired resistance rate (17.91% vs 8.89%, p = 0.2841) for clarithromycin than M. massiliense. These results further emphasized the importance of identification of subspecies for M. abscessus and M. massiliense to gain an accurate clinical treatment consequent by using different treatment strategies. Amikacin was the most active antimicrobial agent against M. abscessus complex species, showing a 94.74% overall susceptibility rate, a similar overall susceptible rate was observed in some researches from China or Australia [29, 30]. However, a higher resistant rate was observed in Japan and South Korea, from 28.2–76.0% [14, 31]. After amikacin, cefoxitin with a 16.67% resistant rate was the second most effective antimicrobial agent against M. abscessus complex, which is not the same with results in South Korea [31] where the second most effective antimicrobial agent is linezolid, but same with Japan [14]. The resistance rate to cefoxitin was higher in M. abscessus (19.40%) than in M. massiliense (11.11%). Linezolid with a resistant rate of 33.33% could be used as an alternative choice against RGM isolates. Considering a high resistant rate with the other drugs tested in our study, they may not be appropriate for M. abscessus complex, however, the clinical therapeutic effect need to be observed.
For SGM, we mainly analyzed the susceptibility of the M. avium-intracellulare complex and M. kansasii, the most two species in SGM. Consistent with previous studies[32], the macrolides and amikacin have shown excellent in vitro activity against MAC isolates with 90% susceptibility rates, as the first line therapeutic agent for lung diseases caused by M. avium-intracellulare complex. As we know, patients with M. avium-intracellulare complex pulmonary diseases are frequently administered a combination of clarithromycin, ethambutol, and rifampicin. A study suggests that treatment with clarithromycin and ethambutol is not inferior to treatment with clarithromycin, ethambutol, and rifampicin for M. avium-intracellulare complex lung disease [33]. We support the two treatment regimens as the resistant rates of ethambutol and rifampicin are 58.33% and 91.67% in vitro, respectively. Some researchers have reported differential drug susceptibility patterns of M. chimaera and other members of the M. avium-intracellulare complex [34]. In our study, we only obtained one M. chimaera strain. We compared the drug susceptibility patterns of the most two species of M. avium-intracellulare complex, the results showed no significant difference between M. intracellulare and M. marseillense. As M. marseillense infections are rare in humans [24, 25, 35], the drug susceptibility can facilitate our knowledge for the species.
M. kansasii which is the second most species of SGM infection is showed a higher susceptibility rate to majority first and second line antibiotics recommended by CLSI (M24, 3rd Edition). The drug susceptibility patterns presented a huge difference with a previous study which used a total of 78 M. kansasii strains from 13 provinces of China for drug susceptibility testing [36]. Except ethambutol (83.87% vs 20.5%), the resistant rate in our study is lower than previous study, clarithromycin (0 vs 20.5%), amikacin (0 vs 5.1%), rifampicin (6.45% vs 56.4%), rifabutin (3.23%% vs 34.6%), moxifloxacin (0 vs 16.7%) and linezolid (3.23%% vs 32.1%). Our result is similar with a report which used 85 M. kansassi obtained from 8 countries in Europe and Asia [37]. All isolates tested in that study were susceptible to rifampicin, amikacin, rifabutin, moxifloxacin, and linezolid. Resistance to ethambutol, ciprofloxacin, and clarithromycin was found in 83 (97.7%), 17 (20%), and 1 (1.2%) isolate, respectively. The drug susceptibility patterns in other studies associated with M. kansasii infection presented incompatibility [38–40]. Although all 31 M. kansasii in our study were isolated from 13 provinces in China, more isolates should be tested to quality drug susceptibility patterns of M. kansasii considering less strain number and regional disparity.
In this study, we obtained the isolates from all the 31 provinces in mainland China; unfortunately, we got recovery failure among strains collected from XinJiang and obtained only one strain from HaiNan province. We also identified no NTM in some provinces from northwestern region considering the small sample size. And we also analyzed the drug susceptibility pattern of all 317 NTM strains, but a limitation in strains number of each species. We have prepared to collect more samples from these regions for completing the NTM infection and drug resistant status in China and planned to analyze the NTM infection using the isolates in following nationwide surveillance of drug-resistant tuberculosis. The acquaintance of NTM pulmonary infection will facilitate the building of TB treatment and control targets.