Omadacycline, Eravacycline, and Tigecycline Express Anti-Mycobacterium abscessus Activity In Vitro

ABSTRACT Mycobacterium abscessus infections are increasing worldwide necessitating the development of new antibiotics and treatment regimens. The utility of third-generation tetracycline antibiotics was reestablished; their anti-M. abscessus activity needs further study. The activities of omadacycline (OMC), eravacycline (ERC), tigecycline (TGC), and sarecycline (SAC) were tested against two reference strains and 193 clinical M. abscessus isolates at different temperatures (30°C and 37°C). The minimum bactericidal concentrations (MBCs) of the four drugs were determined to distinguish between their bactericidal and bacteriostatic activities. The MICs of OMC, ERC, and TGC for the reference strains and clinical isolates were summarized and compared. OMC, ERC, and TGC exhibited a high level of bacteriostatic activity against M. abscessus. The MICs of OMC and ERC for M. abscess remained stable, while the MICs of TGC for the isolates/strains increased with increasing temperature. Notably, the MICs of OMC for M. abscessus isolates obtained in the United States are lower than for those obtained in China. IMPORTANCE The antimicrobial activities of four third-generation tetracycline-class drugs, omadacycline (OMC), eravacycline (ERC), tigecycline (TGC), and sarecycline (SAC), were determined for 193 M. abscessus isolates. The activities of the four drugs at two different temperatures (30°C and 37°C) were also tested. OMC, ERC, and TGC exhibited significant activity against M. abscessus. The anti-M. abscessus activity of TGC increased when the temperature was increased from 30°C to 37°C; the activities of OMC and ERC, on the other hand, remained the same. We found that in vitro MICs of OMC against Chinese and American isolates were distinct. Evaluations in in vivo models of M. abscessus disease or in the clinical setting will provide more accurate insight into potency of OMC against distinct isolates.

The authors evaluated the in vitro activities of several third generation tetracyclines against M. abscessus. They found omadacycline, eravacycline, and tigecycline to have good in vitro activities against the panel of isolates they tested. These compounds did not have good bactericidal activity. Specific comments: lines 56-57: This statement needs to be redone too clarify their meaning. line 68: "improved or good" would be better than great oral line 113: Why was Middlebrook 7H10 agar used rather CAMH agar? line 116: It would be useful for authors to define MBC in terms of the log killing required. I have usually considered a 2 log reduction to be necessary for bactericidal activity. It seems that the authors are using a more stringent definition likely 3 log reduction. line 165: higher would be better than larger line 166: lowest would be better than smallest line 167: higher would be better than larger line 171: lower would be better than smaller line 173: consistent would be preferable to correlating line 174: supporting would be preferable to evidencing line 180: Combination or multidrug would be better than phase line 181: delete "that" line 208: higher would be preferable to larger line 216: measured would be better than "calculated" line 217: delete assays -"at random was determined" line 218: measured would be preferable to "calculated" line 226: lower would be better than "less" line 256: remarkably would be preferable to remarkable line 256: delete "separated" Reviewer #2 (Comments for the Author): 1. Abstract: M. abscessus -It should be Mycobacterium abscessus in the first mention. 2. Line 106: How were MIC values determined? If only visible growth was measured, how did the authors calculate 50% and 90% inhibition (as mentioned in Table 1)? The authors should clarify this. 3. The authors should also evaluate the activity of these drugs against strains in macrophages or mice. 4. The legend to figure 1 needs to be more elaborated. The x-and y-axis of the MIC graphs need to be uniform. 5. The authors should include a figure showing structures of drugs. A figure showing structure of 1st, 2nd and 3rd generation tetracyclines will be more informative. 6. There were few grammatical errors in the manuscript. The authors should check them thoroughly. 7. The authors should also discuss the plausible reasons for differential activity of these drugs against various clinical strains. Is there any correlation between sequencing data and differential susceptibility of these strains.

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The authors have determined the antimycobacterial activity of third generation tetracylines; omadacycline, ervacycline, tigecycline and sarecycline against 2 reference strains and 193 clinical isolates of M. abscessus with incubation at two different temperatures (30°C and 37°C). They have also reviewed and compared their activity with the activity reported in 5 previous studies. The authors also show that omadacycline is also less effective against Chinese isolates in comparison to American isolates. The study lacks novelty as similar studies have been performed earlier. The present study is about evaluating antimycobacterial activity of third generation tetracyclines and is more suited for publication in another Journal.
The authors may find the following comments useful to improve upon their manuscript. 3. The authors should also evaluate the activity of these drugs against strains in macrophages or mice.
4. The legend to figure 1 needs to be more elaborated. The x-and y-axis of the MIC graphs need to be uniform.

The authors should include a figure showing structures of drugs. A figure showing
structure of 1 st , 2 nd and 3 rd generation tetracyclines will be more informative.
6. There were few grammatical errors in the manuscript. The authors should check them thoroughly.
7. The authors should also discuss the plausible reasons for differential activity of these drugs against various clinical strains. Is there any correlation between sequencing data and differential susceptibility of these strains.

Editor comments:
Your manuscript was reviewed by two referees with expertise in mycobacterial experimental therapeutics. Please revise your manuscript as instructed by the two reviewers.
Also, note that all assessments of anti-M. abscessus activities were performed in vitro. As results from in vitro studies do not recapitulate in vivo conditions and MICs from in vitro studies have not been consistently informative for treating M. abscessus disease in the clinic, please include this as inherent limitation of the study. Because of this, strong statements such as in line 43: 'Notably, we found OMC was less likely effective against Chinese isolates than American isolates' should not be included. This claim is vague and inaccurate as it suggests that this study investigated actual activity of OMC of these two sets of isolates in the clinical setting. I suggest that you revise the statement to something like 'We found that in vitro MIC of OMC against Chinese and American isolates were distinct. Evaluations in in vivo models of M. abscessus disease or in the clinical setting will provide more accurate insight into potency of OMC against distinct isolates.'

Reviewer #1 (Comments for the Author):
1. lines 56-57: This statement needs to be redone too clarify their meaning.

Response: The statement was revised (revised manuscript, lines 57-59 ).
2. line 68: "improved or good" would be better than great oral Response: "improved" was substituted from great (revised manuscript, line 69).

Response: Middlebrook 7H10 agar is widely used for mycobacteria isolation, cultivation and sensitivity testing. It is also suggested as solid media for mycobacteria colony counting in CLSI standards. OADC enriched Middlebrook 7H10 agar was used for M. abscessus isolation and CFUs count for MBC determination in this study.
4. line 116: It would be useful for authors to define MBC in terms of the log killing required. I have usually considered a 2 log reduction to be necessary for bactericidal activity. It seems that the authors are using a more stringent definition likely 3 log reduction.