Antimicrobial Susceptibility Testing Using the MYCO Test System and MIC Distribution of 8 Drugs against Clinical Isolates of Nontuberculous Mycobacteria from Shanghai

ABSTRACT Given the increased incidence and prevalence of nontuberculous mycobacterial (NTM) diseases and the natural resistance of NTM to multiple antibiotics, in vitro susceptibility testing of different NTM species against drugs from the MYCO test system and new applied drugs is required. A total of 241 NTM clinical isolates were analyzed, including 181 slowly growing mycobacteria (SGM) and 60 rapidly growing mycobacteria (RGM). The Sensititre SLOMYCO and RAPMYCO panels were used for testing susceptibility to commonly used anti-NTM antibiotics. Furthermore, MIC distributions were determined against 8 potential anti-NTM drugs, including vancomycin (VAN), bedaquiline (BDQ), delamanid (DLM), faropenem (FAR), meropenem (MEM), clofazimine (CLO), cefoperazone-avibactam (CFP-AVI), and cefoxitin (FOX), and epidemiological cutoff values (ECOFFs) were analyzed using ECOFFinder. The results showed that most of the SGM strains were susceptible to amikacin (AMK), clarithromycin (CLA), and rifabutin (RFB) from the SLOMYCO panels and BDQ and CLO from the 8 applied drugs, while RGM strains were susceptible to tigecycline (TGC) from the RAPMYCO panels and also BDQ and CLO. The ECOFFs of CLO were 0.25, 0.25, 0.5, and 1 μg/mL for the mycobacteria M. kansasii, M. avium, M. intracellulare, and M. abscessus, respectively, and the ECOFF of BDQ was 0.5 μg/mL for the same four prevalent NTM species. Due to the weak activity of the other 6 drugs, no ECOFF was determined. This study on the susceptibility of NTM includes 8 potential anti-NTM drugs and a large sample size of Shanghai clinical isolates and demonstrates that BDQ and CLO had efficient activities against different NTM species in vitro, which can be applied to the treatment of NTM diseases. IMPORTANCE We designed customized panel that contains 8 repurposed drugs, including vancomycin (VAN), bedaquiline (BDQ), delamanid (DLM), faropenem (FAR), meropenem (MEM), clofazimine (CLO), cefoperazone-avibactam (CFP-AVI), and cefoxitin (FOX) from the MYCO test system. To better understand the efficacy of these 8 drugs against different NTM species, we determined the MICs of 241 NTM isolates collected in Shanghai, China. We attempted to define the tentative epidemiological cutoff values (ECOFFs) for the most prevalent NTM species, which is an important factor in setting up the breakpoint for a drug susceptibility testing. We used the MYCO test system as an automatic quantitative drug sensitivity test of NTM and extended the method to BDQ and CLO in this study. The MYCO test system complements commercial microdilution systems that currently lack BDQ and CLO detection.

1) Line 71: Authors should include epidemiological data about NTM infectious to support their claim that those infections are increasing worldwide. The literature review in the introductory part is too short and general.
2) Line 72: What is the natural drug resistance for NTM? Authors should list the antibiotics to which NTMs are resistant.
3) Lines 72-75: What is the complex approach used to treat NTM? What are the most common treatments for these infections? What is the average treatment duration for these infections? What is the average percentile of efficacy? Authors should feed the introduction with more information, otherwise, it is too vague. 4) Lines 77-81: These sentences are unclear. The authors start by saying that "Based on the commonly used anti-NTM antibacterial drugs, 8 drugs were repurposed, namely... (list of drugs)...". Then, the second sentence says, "were selected in this study to evaluate drug sensitivity in vitro". Apparently, these two sentences are not linked. Also, why is bedaquiline and clofazimine considered to be repurposed drugs, if they are used to treat multidrug-resistant TB? 5) Line 82: I believe data is still limited. However, a quick search about this topic brings some insightful articles that could be included in the introduction or discussion sections. Below are examples of articles describing the use of the above-mentioned antibiotics against NTMs: In vitro activity of bedaquiline and delamanid against nontuberculous mycobacteria, including macrolide-resistant clinical isolates (2019). Bedaquiline as a potential agent in the treatment of Mycobacterium abscessus infections (2017). Successful bedaquiline-containing antimycobacterial treatment in post-traumatic skin and soft-tissue infection by Mycobacterium fortuitum complex: a case report (2020). Emergence of nontuberculous mycobacteria infections during bedaquiline-containing regimes in multidrug-resistant tuberculosis patients. A bedaquiline/clofazimine combination regimen might add activity to the treatment of clinically relevant non-tuberculous mycobacteria (2019). 6) Line 106: Mycobacterium abscessus. 7) Lines 105-108: I would suggest creating a figure or graph to display all species/clinical isolates included in this study. Overall, the manuscript does not have many figures that illustrate the methodologies and or results raised, and the inclusion of this figure could make it easier to appreciate your work. 8) Lines 117-118: Poor writing quality sentence. I suggest rewriting it. 9) Lines 139-143: Overall, the writing quality of this paragraph is very low. I strongly suggest rewriting it to make the reading clearer and smoother for the reader. 10) Line 145: Authors wrote "it can be recorded and observed in advance". This sentence is not properly connected to the previous one. I suggest rewriting it. 11) Line 161: In Table 1 REB is RFB (rifampicin). I couldn't find what this abbreviation stands for in the text. 12) Lines 262-264: Overall, the writing quality of this paragraph is very low. Also, isn't there a specific code for each strain? There are 6 M. abscessus species, which I assume are different strains. Authors should find a better way to describe these strains.
13) Lines 270-271: Low-quality writing. 14) Line 275: Species are sensitive to a given antibiotic, not the antibiotics themselves. 15) Lines 288-289: Any data to support this sentence? What is the concept of extensive drug resistance here? 16) Lines 293-294: Low-quality writing.
Minor comments: 1) Authors should include a space between the MIC value and the unit (16 µg/ml). In addition, the unit ug/ml is unacceptable.
2) The name of all species should be in italics, even when they appear in tables and figures.
Reviewer #2 (Comments for the Author): The study by Ying et al performed drug-susceptibility tests for 241 NTM isolates against antibiotics using a commercial system (Sensititre SLOMYCO or RAPMYCOI) and an in-house assay. The commercial system included 13 or 15 antibiotics and the selfdefined panel included 8 antibiotics. The NTM isolates were composed of different NTM species including M. kansasii, M. avium, M. intracellulare and M. abscessus. The authors reported that slow-growing NTMs had "good sensitivity" to amikacin, clarithromycin, and rifampin, while the rapidly growing NTM species only had sensitivity to amikacin and tigecycline. Bedaquiline and clofazimine had favorable activities against different NTM species. Similar studies have been done in other countries before (Carvalho, Int J Mycobacteriol, 2021;Litvinov, PLoS One, 2018), but such a dataset is scarce from China and would thus hold relevant significance for clinical reference.
Major comments 1. The overall impression is that the authors presented this work in a "raw data" format. There are too many tables but I didn't see the necessity of dividing them separately. Please try to combine those tables into one or two. Besides, please also consider reducing the raw text that just simply described the table contents -you don't need to repeat all the information that has already been presented in the table. Keep the text clear and concise, and highlight the major findings. 2. The methods section lacks a lot of details. a. How were those clinical isolates processed before the AST experiments? Besides, for the AST experiment, the authors wrote "100 μl of NTM suspension was added to each panel". Did the authors measure the number of bacterial cells in the inoculation and normalize different isolates to similar input? This is important because different inoculations might affect the interpretation of AST results. b. Eight drugs apart from those of the MYCO test system were selected to perform the antimicrobial susceptibility testing, namely, vancomycin (VA), bedaquiline (BDQ), delamanid (DLM), faropenem (FAR), meropenem (MPM), clofazimine (CFZ), avibactam (CAZ), and cefoxitin (FOX). Please provide the rationality of choosing the eight drugs. c. How were the MIC values determined? The lowest concentration of the drug that inhibited the visible growth of the isolates tested? How did you measure visible growth? Please specify. d. Please provide the details for epidemiological cut-off value (ECOFFs) determination of BDQ and CFZ, and please discuss why the ECOFFs values of BDQ differed from those adopted by Yu et al (Antimicrob Agents Chemother, 2019) (Line 295). 3. The authors concluded that "The increased MIC value of BDQ corresponds to the increased MIC value of CFZ", but the MIC values of M.abscessus against BDQ and CFZ as shown in Table 9 did not support this conclusion. Among the six M. abscessus isolates with MIC values of BDQ greater than the minimum MIC range, three (50%) with the MIC of 0.12 μg/ml, which is the minimum MIC value of M. abscessus isolates to CFZ.
Minor comments 1. This manuscript needs considerable language editing. For example, phrasing like "good sensitivity" is confusing. 1. Line 264, the authors stated nine of the eleven isolates with MIC values of CFZ increase to 1-to 3-fold, but the results shown in Figure 2 and Table 9 indicate six. 2. "REB" in the "MIC distribution of SGM species in SLOMYCO panel" section of results (Lines 159-187) should be "RFB". 3. Consider using figures to present Table 3 and Table 7

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Reviewer comments:
Reviewer #1 (Comments for the Author): Overview: The present manuscript aims to evaluate the susceptibility of nontuberculous mycobacteria (fast-and slow-growing) to different drug panels. The authors came to the conclusion that fast-or slow-growing NTMs show varied susceptibility to the antibiotics tested, and that bedaquiline and clofazimine showed broad activity among different NTM species.
Major comments: 1) Line 71: Authors should include epidemiological data about NTM infectious to support their claim that those infections are increasing worldwide. The literature review in the introductory part is too short and general.
As per the reviewer's suggestion, we have made the correction accordingly.
2) Line 72: What is the natural drug resistance for NTM? Authors should list the antibiotics to which NTMs are resistant.

Most NTM are intrinsically resistant or only partially sensitive to first-line anti-tuberculous drugs.
3) Lines 72-75: What is the complex approach used to treat NTM? What are the most common treatments for these infections? What is the average treatment duration for these infections? What is the average percentile of efficacy? Authors should feed the introduction with more information, otherwise, it is too vague.
Most NTM are intrinsically resistant or only partially sensitive to first-line anti-tuberculous drugs. Unlike tuberculosis (TB), treatment for NTM disease takes very long period of time, with a regimen consisting of a macrolides as the core drug and 2-4 other antibiotics until 12 months after sputum conversion. We thank the reviewer for the comments. We have included these in the discussion section and cited these papers. 6) Line 106: Mycobacterium abscessus.
As per the reviewer's suggestion, we have made the correction accordingly.

As per the reviewer's suggestion, we have made the correction accordingly.
9) Lines 139-143: Overall, the writing quality of this paragraph is very low. I strongly suggest rewriting it to make the reading clearer and smoother for the reader.

As per the reviewer's suggestion, we have made the correction accordingly.
10) Line 145: Authors wrote "it can be recorded and observed in advance". This sentence is not properly connected to the previous one. I suggest rewriting it. Table 1 REB is RFB (rifampicin). I couldn't find what this abbreviation stands for in the text.

As per the reviewer's suggestion, we have made the correction accordingly.
12) Lines 262-264: Overall, the writing quality of this paragraph is very low. Also, isn't there a specific code for each strain? There are 6 M. abscessus species, which I assume are different strains. Authors should find a better way to describe these strains.
As per the reviewer's suggestion, we have rewritten this paragraph. Because our mycobacterium identification kit does not support subdivision into subspecies, so our study focused on the four predominant prevalent species.

As per the reviewer's suggestion, we have made the correction accordingly.
14) Line 275: Species are sensitive to a given antibiotic, not the antibiotics themselves.

We thank the reviewer for the comments. As per the reviewer's suggestion, we have improved the description of the sentences.
15) Lines 288-289: Any data to support this sentence? What is the concept of extensive drug resistance here?

As per the reviewer's suggestion, we have improved the description of the sentences. Treatment of RGM infection is very difficult because this bacterium is resistant to a wider variety of drugs than SGM.
According to the reference 30(Guz L, Puk K. 2022), they are reach show that the drug sensitivity of NTM varies from species to species. KAN, AMK, CLR and SMX were the most active against RGM isolates, and these same four plus DOX and CIP were the best drugs against SGM isolates. 16) Lines 293-294: Low-quality writing.

As per the reviewer's suggestion, we have made the correction accordingly.
Minor comments: 1) Authors should include a space between the MIC value and the unit (16 µg/ml). In addition, the unit ug/ml is unacceptable.

As per the reviewer's suggestion, we have made the correction accordingly.
2) The name of all species should be in italics, even when they appear in tables and figures.

As per the reviewer's suggestion, we have made the correction accordingly.
Reviewer #2 (Comments for the Author): The study by Ying et al performed drug-susceptibility tests for 241 NTM isolates against antibiotics using a commercial system (Sensititre SLOMYCO or RAPMYCOI) and an in-house assay. abscessus. The authors reported that slow-growing NTMs had "good sensitivity" to amikacin, clarithromycin, and rifampin, while the rapidly growing NTM species only had sensitivity to amikacin and tigecycline. Bedaquiline and clofazimine had favorable activities against different NTM species. Similar studies have been done in other countries before (Carvalho, Int J Mycobacteriol, 2021;Litvinov, PLoS One, 2018), but such a dataset is scarce from China and would thus hold relevant significance for clinical reference.
Major comments 1. The overall impression is that the authors presented this work in a "raw data" format. There are too many tables but I didn't see the necessity of dividing them separately. Please try to combine those tables into one or two. Besides, please also consider reducing the raw text that just simply described the table contents -you don't need to repeat all the information that has already been presented in the table. Keep the text clear and concise, and highlight the major findings.
We thank the reviewer for the comments. As per the reviewer's suggestion, we have improved the description of the results obtained.  We detected an increase in MIC values for BDQ in 11 strains, followed by an increase in MIC values of CFZ (9/11), as shown in Table 7. Meanwhile, the same phenomenon on the contrary was not obvious. From this, we hypothesize that the development of resistance in BDQ usually commonly occur alongside resistance in CFZ The minimum MIC value of M. abscessus isolates to CFZ was <=0.06.

Minor comments
1. This manuscript needs considerable language editing. For example, phrasing like "good sensitivity" is confusing.
We thank the reviewer for the comments. As per the reviewer's suggestion, we have improved the description of the sentences.
1. Line 264, the authors stated nine of the eleven isolates with MIC values of CFZ increase to 1-to 3-fold, but the results shown in Figure 2 and Table 9 indicate six.
We didn't find this sentence at Line 264. We think it should be Line 277. Among the three most prevalent SGM (M. kansasii, M. avium and M. intracellulare), compared with the levels for M. kansasii, the MICs of more than half of the strains increased 1-to 2-fold for M. avium and M. intracellulare, as shown in Figure 5. Table 7 showed among the 241 patients, we detected an increase in MIC values for BDQ in 11 strains, followed by an increase in MIC values of CFZ (9/11).

The results in
2. "REB" in the "MIC distribution of SGM species in SLOMYCO panel" section of results (Lines 159-187) should be "RFB".
As per the reviewer's suggestion, we have made the correction accordingly. Table 3 and Table 7.

Consider using figures to present
As per the reviewer's suggestion, we have made the correction accordingly.

All Latin names of species should be italic.
As per the reviewer's suggestion, we have made the correction accordingly.
5. Reference 26 is incomplete, lacking volume, issue and pages.
As per the reviewer's suggestion, we have made the correction accordingly. In this regression study, 241 clinical isolates of NTM patients admitted to Shanghai Pulmonary Hospital from 2020 to 2021 were collected. The distribution of NTM species usually has regional characteristics. Mycobacterium intracellular was most isolated in clinical specimens, and Mycobacterium abscessus also accounted for a higher proportion.

5.there is no statistical design for the results.
Statistical design has been added in compliance with the reviewers' comments.
Excel (MS Office 2019) software was used for data statistics and GrahpPad Prism 8 (San Diego, CA) was used for graphical display.
The MS needs revision.