Nationwide surveillance of antimicrobial susceptibility of 509 rapidly growing mycobacteria strains isolated from clinical specimens in Japan

This study aimed to identify effective treatments against rapidly growing mycobacteria (RGM) infections by investigating the minimum inhibitory concentrations (MIC) of 24 antimicrobial agents and their molecular mechanisms of resistance. In total, 509 clinical RGM isolates were identified by analyzing the sequences of three housekeeping genes (hsp65, rpoB, and sodA), and their susceptibilities to 24 antimicrobial agents were tested. We also performed sequencing analysis of antimicrobial resistance genes (rrl, rrs, gyrA, and gyrB). To identify Mycobacteroides abscessus group subspecies, we performed PCR-based typing and determined the sequevar of erm(41). We identified 15 RGM species, most of which were susceptible to amikacin and linezolid. Among these species, arbekacin and sitafloxacin had the lowest MIC among the same class of antimicrobials. The MIC of rifabutin for M. abscessus subsp. abscessus (MAB) was lower than that for M. abscessus subsp. massiliense (MMA). The proportion of MAB isolates with MIC ≤ 2 mg/L for rifabutin was significantly higher than that of MMA [MAB: 50/178 (28.1%) vs. MMA: 23/130 (17.7%); p = 0.041]. In summary, our study revealed the antimicrobial susceptibility profile of 15 RGM species isolated in Japan and indicated that arbekacin, sitafloxacin, and rifabutin may be possible therapeutic options for RGM infections.


Results
The details of 15 species that were identified are shown in Table 1. Eleven isolates, M. abscessus subsp. abscessus (MAB) (5), M. abscessus subsp. massiliense (MMA) (3), M. chelonae (2), and M. senegalense (1) grew poorly in the culture medium at five days after the start of susceptibility test, and thus we could not obtain MIC data for these isolates.
Characteristics of antimicrobial susceptibilities of RGM species. Other than MMA, which was susceptible to both amikacin and clarithromycin, MAB and M. abscessus subsp. bolletii (MBO) were susceptible to only amikacin ( Table 2). Although M. fortuitum was resistant to macrolides, it was susceptible to amika- Table 1. Distribution of rapidly growing mycobacteria (RGM) species by specimen type. PD Peritoneal dialysis, LRS lower respiratory specimens, NLRS non-lower respiratory specimens. www.nature.com/scientificreports/ cin, imipenem, fluoroquinolones, and trimethoprim/sulfamethoxazole (Table 3). Only three isolates were not susceptible to fluoroquinolones. Most M. chelonae isolates were susceptible to clarithromycin. However, the proportion of isolates intermediate and resistant to aminoglycosides, imipenem, cefoxitin, and fluoroquinolones was high. Additionally, we found that 46% of M. chelonae strains were susceptible to tobramycin (Table 3). M. mageritense isolates showed remarkably high resistance to clarithromycin and amikacin, but were susceptible to fluoroquinolones, imipenem, and cefoxitin ( Table 3). The results of the antimicrobial susceptibility test and  Table 4 and Table S2, respectively. Amikacin and linezolid were the most effective against the 15 isolated RGM species (Table 4). We also investigated the MICs against RGM for antibacterial drugs for which CLSI did not set breakpoints. The MIC50 of sitafloxacin was the lowest among all the fluoroquinolones for all RGM species (Tables 2, 3). Table 3. Antimicrobial susceptibility of major rapidly growing mycobacteria (RGM) strains other than M. abscessus group (MAG). A) Minimum inhibitory concentration (MIC) for ciprofloxacin and moxifloxacin could not be determined for one strain. B) Trimethoprim/sulfamethoxazole. C) Amoxicillin/clavulanic acid.
Quinolone resistance of M. fortuitum and its mechanism. Of the three isolates of M. fortuitum that were resistant to ciprofloxacin, only one had a mutation in gyrA. In the mutant strain, the gyrA gene resulted in S83W amino acid substitution (TCG → TGG). None of the ciprofloxacin-susceptible isolates had mutations in gyrA and gyrB. Table 4. Characteristics of antimicrobial susceptibility of rapidly growing mycobacteria (RGM) species. ◎Susceptible isolates > 75%, 〇 Susceptible isolates 51%-75%, △Susceptible isolates 25%-50%, ▲Susceptible isolates < 25% A) Trimethoprim/sulfamethoxazole.

Discussion
In this study, we accurately identified 15 species of RGM from clinical isolates obtained from different locations around Japan. We characterized the susceptibility of these isolates to 24 antimicrobials, including tigecycline, sitafloxacin, rifabutin, and cefmetazole; none have defined MIC breakpoints in the CLSI, but they may have potential as therapeutic agents for RGM infections. We investigated not only MAG antimicrobial susceptibility, but also several gene mutations involved in antimicrobial resistance and prepared a summary of the susceptibility of the remaining 14 species of RGM. The proportion of the C28 sequevar in MAB isolated from lower respiratory specimens (LRS) has been reported to be approximately 16-35% 4, 10, 16, 17 . However, in some previous Japanese reports, the ratio of the C28 sequevar among MAB from LRS was very low at 4.2% (2/48) 18 . In our survey, it was 12.2% (22/180), which is higher than that in the previous report 18 . In Japan, it is necessary to continue to evaluate whether the proportion of the C28 sequevar in MAB is lower than those in other countries.  MAB). A) These strains did not grow sufficiently in the control well, and MIC could not be determined. B) Other rare sequevar types. These types did not include types 3, 4, and 5. C) PCR was performed to obtain a product, but sufficient erm(41) gene sequence data could not be obtained.  Table 6. Frequency of rrl mutation in 37 Mycobacteroides abscessus group (MAG) strains. A) Acquired resistance = CAM minimum inhibitory concentration (MIC) ≥ 8 mg/L at early-reading-time. B) PCR was performed to obtain a product, but sufficient rrl gene sequence data could not be obtained. C) PCR was performed to obtain a product, but sufficient erm(41) gene sequence data could not be obtained. www.nature.com/scientificreports/ A previous report from the USA indicated that sequevar types 4, 6, 7, 8, 9, and 10 (all T28 sequevars) may be associated with macrolide-induced resistance 10 . However, similar assessments outside of the USA have not been conducted so far. Among the 180 MABs in our study, only 4 isolates with erm(41) sequevar types 6, 7, 8, and 10 were susceptible to clarithromycin. Our data were generally consistent with the previous report 10 . Therefore, it was suggested that these sequevars are macrolide-resistant. So far, CLSI has recommended the determination of the erm(41) sequevar type for evaluation of induced macrolide resistance in MAB 11 , and our results support this recommendation. Further investigations on the relationship between sequevar types and macrolide resistance in other regions are required.
The rrl gene mutation is more likely to occur in the MAB C28 sequevar and MMA than in the MAB T28 sequevar among clarithromycin-acquired resistant strains in MAG 4 . In our survey, we found a similar trend but could not show a significant difference. Among MAG, more than half of the macrolide-acquired resistance occurred by mechanisms other than rrl gene mutation. The exact mechanism remains to be investigated.
Additionally, none of the amikacin non-susceptible isolates in our survey had the rrs gene mutation. A previous French study of antimicrobial susceptibility in 165 isolates of MAG showed that 7/8 strains with amikacin MIC > 64 mg/L had a rrs A1408G gene mutation 16 , which suggested that amikacin MIC > 64 mg/L is a criterion to suspect amikacin-acquired resistance 16 . In our survey, only one isolate of MAG showed MIC > 64 mg/L, and none of the isolates showed rrs mutation. MAG isolated in Japan may have fewer amikacin-acquired resistant isolates than those isolated in France.
As reported previously 18,19 , M. fortuitum was resistant to clarithromycin; however, it was susceptible to aminoglycosides, carbapenems, and fluoroquinolones in our study. Previous reports suggest that, in M. fortuitum, a serine residue at the 83 rd position of gyrA constitutes QRDR and contributes to susceptibility to fluoroquinolones compared with other NTMs 6 . However, to date, only one report has shown quinolone resistance due to mutations in gyrA 19 . There has been no report of mutations in a serine residue at the 83 rd position of gyrA. Fluoroquinolone resistance was found in 3 of 85 (3.5%) isolates in our study, and the S83W amino acid substitution was present in one of the three isolates. Our result also suggests that fluoroquinolone resistance can occur based on genetic changes other than QRDR mutations, and it is necessary to clarify the resistance mechanism in the future. In Japan, fluoroquinolones are being overused 20 , and there is a concern regarding the increase of fluoroquinoloneresistant isolates in M. fortuitum. Because M. fortuitum shows induced resistance to macrolides, fluoroquinolones play an important role in the treatment of M. fortuitum infections as an oral antibiotic. There is a great concern regarding treatment efficacy with the increase in resistant isolates.
Among M. chelonae isolates, resistance to clarithromycin was found in approximately 10% of isolates, consistent with previous reports 18,21 . Previous reports seem to indicate regional variability in tobramycin susceptibility, ranging from 54% in the UK 21 to 83% and 17% in Japan 18,22 . In our study, approximately 40% of the strains were tobramycin-susceptible, an intermediate value between the values reported by the two previous reports from Japan. In addition, no rrs mutations were found in amikacin non-susceptible isolates. Arbekacin may be a potential therapeutic for isolates that are less susceptible to amikacin and tobramycin.
M. peregrinum was susceptible to most of the tested antimicrobials. M. mageritense isolates were resistant to clarithromycin, as has been previously reported 23 , and showed a low susceptibility to amikacin, although none had a rrs gene mutation (3 isolates showed an amikacin MIC > 64 mg/L). The mechanism of M. mageritense resistance to amikacin remains to be investigated. Conversely, it showed good susceptibility to quinolones, cefoxitin, and linezolid.
There are few reports on antimicrobial susceptibility for other rare RGM species using a sufficiently high number of clinical isolates. There is only one study involving M. mucogenicum and M. immunogenum reporting that most of the isolates were susceptible to linezolid, amikacin, and trimethoprim/sulfamethoxazole, while showing a poor susceptibility to clarithromycin 24 . In our study, although the number of isolates was small, we could show the tendency of antimicrobial susceptibility for rare RGM species. These rare RGM species tended to be susceptible to linezolid, quinolones, and trimethoprim/sulfamethoxazole.
Although there have been no reports regarding the MIC of arbekacin in RGM, this antimicrobial showed the lowest MIC among the aminoglycosides for almost all RGM species in this study (Tables 2, 3). The MIC50 value of sitafloxacin is reported to be lower than that of other fluoroquinolones in MAG, M. fortuitum, and M. chelonae 18 . However, in this study, we showed that the effect of sitafloxacin was similar on the 15 RGM species (Tables 2, 3). Cefmetazole and cefoxitin, cephamycin-based antimicrobials, had similar MICs, consistent with previous reports 25,26 (Tables 2, 3). In countries such as Japan, when patients cannot be administered cefoxitin, cefmetazole may be an option for RGM treatment. In recent years, rifabutin has attracted attention as an oral treatment for MAG 27,28 , but, so far, there have been few reports of MICs measured by micro-dilution using cation-adjusted Mueller-Hinton broth medium 28 . Here, we have not only measured rifabutin MICs for many isolates using this standard method, but also showed that MICs were lower for MAB than for MMA (Table 2). MAB has a very high resistance rate not only to clarithromycin but also to fluoroquinolone; thus, finding an alternative orally administrated therapeutic option is essential. A detailed evaluation is required in the future to determine whether rifabutin will be an effective orally administered therapeutic option.
There are some limitations to our study. It was unclear whether there was prior administration of antibacterial drugs before susceptibility testing for all isolates. Some of the RGM species isolated in this study were rarely isolated to evaluate drug susceptibility. However, despite these limitations, our study reveals important epidemiological information about RGM in Japan and suggests several drugs that can be investigated as new treatment candidates. It is therefore necessary to accumulate and evaluate data from a larger set of samples and to verify the correlation between the actual therapeutic effect and the MIC values of these drugs in clinical trials.
We showed antimicrobial susceptibility profiles of 15 RGM species isolated in Japan. Amikacin and linezolid were the most effective against the 15 isolated RGM species. Arbekacin, sitafloxacin, and cefmetazole may be www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.