In vitro effects of the new oral β-lactamase inhibitor xeruborbactam in combination with oral β-lactams against clinical Mycobacterium abscessus isolates

ABSTRACT Non-tuberculosis mycobacteria (NTM), particularly Mycobacterium abscessus subsp. abscessus (M. abscessus), are increasingly being recognized as etiological agents of NTM pulmonary disease. However, treatment options for M. abscessus are limited owing to their natural resistance to most antibiotics, including β-lactams. M. abscessus produces a class A β-lactamase, whose activity is inhibited by cyclic boronic acid β-lactamase inhibitors. We aimed to evaluate the in vitro effects of xeruborbactam, a cyclic boronic acid β-lactamase inhibitor, against M. abscessus when combined with five β-lactams (amoxicillin, tebipenem, cefdinir, cefuroxime, and cefoxitin). The drug susceptibilities of 43 M. abscessus clinical isolates obtained from 43 patients between August 2005 and May 2014 were tested. The MIC results for each β-lactam with or without 4 µg/mL xeruborbactam were examined. Xeruborbactam lowered the MIC90 values of tebipenem, amoxicillin, cefuroxime, and cefdinir by 5, ≥4, 3, and 3 dilutions, respectively. The MIC90 values of cefoxitin without xeruborbactam were 32 µg/mL and did not change upon the addition of xeruborbactam. The lowest MIC90 value was obtained for tebipenem with xeruborbactam. Almost all isolates had an MIC of 4 µg/mL; one isolate had an MIC of 2 µg/mL. With respect to the susceptibility to the same family drug, the number of susceptible isolates increased from 1/43 (2%) to 43/43 (100%) for tebipenem with xeruborbactam. Combining tebipenem and xeruborbactam could be considered an effective all-oral regimen that benefits outpatient treatment of M. abscessus pulmonary disease. IMPORTANCE Mycobacterium abscessus subsp. abscessus (M. abscessus) disease is treated in two phases; injectable drugs for initial followed by others for continuation. There is a need to develop all-oral treatment methods for M. abscessus infection, especially in the continuation phase. However, treatment options for M. abscessus are limited owing to their natural resistance to most antibiotics. This is the first report to evaluate the in vitro effects of xeruborbactam, a cyclic boronic acid β-lactamase inhibitor capable of inhibiting the class A β-lactamase produced by M. abscessus, against 43 M. abscessus clinical isolates when combined with five β-lactam antibiotics. Xeruborbactam lowered the MIC90 values of tebipenem by five dilutions, and the number of susceptible isolates increased from 1/43 (2%) to 43/43 (100%). We showed that the tebipenem-xeruborbactam combination might be of interest to explore further as a potentially effective oral regimen for outpatient treatment of M. abscessus pulmonary disease.

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Thank you for submitting your paper to Spectrum.It is data of interest since M. abscessus are intrinsically resistant to nearly all antimicrobials and the need of novel antimicrobials or novel antibiotic combination is highly required.Beta-lactams are on the first line list of antimicrobial therapy for M. abscessus infections, particularly imipenem and cefoxitin.Since M. abscessus produces a beta-lactamase, several combinations with beta-lactamase inhibitors were tested, showing a decrease of some MIC dilutions.This study tested a novel inhibitor of beta-lactamase, xeruborbactam, which belongs to the cyclic boronate acid beta-lactamase inhibitors and has the interesting pharmacokinetic enabling an oral administration.Major comments and questions: -It is very interesting to have these data about a new combination of penem and inhibitor of beta lactamase that could be delivered orally to patients infected with M. abscessus.However, the paper contains limited data (MIC testing on 44 isolates for 5 beta-lactams combined with xeruborbactam), and consequently can be shorten into a note of a brief report.
-MIC protocol and description of the M. abscessus isolates results are according to the reference.However, we would expect to see results of repeatability and reproducibility at least on the reference strain.
-Results show interesting data with low MIC of the combination xeruborbactam and terbipenem.
-Bibliography is consistent with the topic.
Minor comments: 1. abstract : the results for cefoxitin are missing 2. MIC determination protocol: a. page 7, lines 118-121.Is the xeruborbactam water soluble, or is it dissolved in a specific solvent, e.g.DMSO? b.Page 8, line 134: waiting for 14 days could increase the MIC by degrading the inhibitor or the beta-lactam, which is often observed for penems.For how many isolates (for one isolate, the reading was done at D10), this extended incubation was used?If it is zero, better to suppress this sentence.3. Results: a. Tables: results shown in table 3 can be combined with those of the table 2. b.Figure: how do you explain that there is a nice Gauss distribution observed for terbipenem on the figure 1b, and the all MIC results are concentrated on the 4 µg/mL value when the xeruborbactam is added?4. Discussion: a. Page 12, line 208.Since xeruborbactam will be commercialized with ceftibuten as an oral form, it could have been worthwhile to test this combination.b.The authors showed that MIC on Rough isolates are higher than on the Smooth isolates?Page 13, Lines 230-231, the authors explained that the absence of GPL enhance the permeability cell wall.So consequently, the MIC should be lower for the R isolates?Do they have hypotheses for this unexpected result?

Re: In vitro effects of the new oral β-lactamase inhibitor xeruborbactam in combination with oral βlactams against Mycobacterium abscessus clinical isolates
The authors have studied the effect of five oral β-lactams against Mycobacterium abscessus isolates.There is an urgent need to improve the treatment of M.abscessus and oral treatment options are needed.The study is therefore of interest for the readers of Spectrum Microbiology.
The manuscript is clear, well-written and a joy to read, apart from some small mistakes.I do have some suggestions to improve the manuscript, including readability.

Major comments:
The introduction is rather lengthy, and parts of the text can be moved to the discussion part.
Line 46.How were the critical concentrations decided?There is no CC established for tebipenem so this needs to be noted in the methods section and possibly discussed in the discussion section.
Line 98.What is this Ogawa medium?Used outside Japan?Consists of?Add in M& M section about how colony morphology was determined.Any indeterminate?Add this as a secondary aim as it is an important finding and deserves more discussion about.Consider referring to previously published studies on the subject:

Minor comments:
In general, up to the authors of course, but the readability of the manuscript would be improved if abbreviations are kept to a minimum.I would not abbreviate the B-lactam antibiotics as all readers are not aware of these abbreviations.

Suggest rewording the title:
In vitro effect of the new oral β-lactamase inhibitor xeruborbactam in combination with oral βlactams against clinical Mycobacterium abscessus isolates Line 31.Rephrase -.Almost all isolates had an MIC of 4 mg/L, whereas one isolate had an MIC of 2 mg/L.Line 39. Change "it will be needed" to "There is a need to"….Line 42. "which is inhibited by cyclic boronate acid β-lactamase inhibitors."Add: …, amongst other.As the sentence reads now you can think that it is only cyclic boronate that inhibits it.Line 198 lina.I would recommend comparing your MICs with previous studies.References the systematic review is ok but also add info and reference specific studies, and discuss the difference in results (" the addition of avibactam to tebipenem showed a more pronounced reduction from 256 to 16 mg/L" Fröberg G, Ahmed A, Chryssanthou E, Davies Forsman L. 2023.The in vitro effect of new combinations of carbapenem-β-lactamase inhibitors for Mycobacterium abscessus .Antimicrob Agents Chemother 67:e00528-23.https://doi.org/10.1128/aac.00528-23Line 200.Add "Thus" or explain why as the sentence is not clear enough now.Line 204.Change to "may be" instead of "should be" as the latter is an opinion.

Response to Reviewers
We thank the Reviewers for their useful comments that helped us to make significant improvements to our manuscript, and we greatly appreciate the time and effort that they spent on our behalf.
Reviewer #1 1.The authors have studied the effect of five oral β-lactams against Mycobacterium abscessus isolates.There is an urgent need to improve the treatment of M. abscessus and oral treatment options are needed.The study is therefore of interest for the readers of Spectrum Microbiology.The manuscript is clear, well-written and a joy to read, apart from some small mistakes.I do have some suggestions to improve the manuscript, including readability.
Authors' response: Thank you for your helpful comments.
2. The introduction is rather lengthy, and parts of the text can be moved to the discussion part.
Authors' response: Thank you for your suggestions.We have moved the text about the details of novel β-lactamase inhibitors, in vivo studies, and a case report to the Discussion section.
3. Line 46 "the number of susceptible isolates increased from 1/43 (2%) to 43/43 (100%) for tebipenem with xeruborbactam".How were the critical concentrations decided?There is no CC established for tebipenem so this needs to be noted in the methods section and possibly discussed in the discussion section.
Authors' response: Thank you for your suggestion.We have added in the Methods and Discussion sections that there was no CC established for tebipenem, cefuroxime, cefdinir, and amoxicillin.We referred to the susceptibility of the same family drug, imipenem and meropenem (MIC ≤4 µg/mL is susceptible), and cefoxitin (MIC ≤16 µg/mL is susceptible).At that time, the comparison of individual MICs with peak plasma concentration, half-life, and plasma protein binding may be useful clinically; thus, we have added that information about each β-lactams.
Authors' response: Thank you for pointing this out.The 2% Ogawa medium is mainly used in Japan and some Asian coutries, instead of the Löwenstein-Jensen medium, which is an egg-based medium and consists of potassium dihydrogen phosphate, sodium glutamate, magnesium citrate, soluble starch, sodium pyruvate, glycerol, 2% malachite green, and homogenized whole egg.We have added this information in the Methods section.
5. Add in M& M section about how colony morphology was determined.Any indeterminate?Add this as a secondary aim as it is an important finding and deserves more discussion about.Consider referring to previously published studies on the subject: Hedin W, et al. J Infect Dis. 2023 Mar 28;227(6):820-827.
Authors' response: Thank you for your suggestion.We have added details on how colony morphology was determined in the Methods section, and there was no indeterminate colony morphotype in the Results section.We have discussed the clinical significance of colony morphology and set the secondary aim to evaluate the differences in the effect of colony morphology.
6.In general, up to the authors of course, but the readability of the manuscript would be improved if abbreviations are kept to a minimum.I would not abbreviate the B-lactam antibiotics as all readers are not aware of these abbreviations.
Reviewer #2 1.The authors determined Minimal inhibitory concentrations of various beta-lactams combined with a new inhibitor of beta-lactamase against Mycobacterium abscessus.It is data of interest since M. abscessus are intrinsically resistant to nearly all antimicrobials and the need of novel antimicrobials or novel antibiotic combination is highly required.Beta-lactams are on the first line list of antimicrobial therapy for M. abscessus infections, particularly imipenem and cefoxitin.Since M. abscessus produces a beta-lactamase, several combinations with beta-lactamase inhibitors were tested, showing a decrease of some MIC dilutions.This study tested a novel inhibitor of beta-lactamase, xeruborbactam, which belongs to the cyclic boronate acid beta-lactamase inhibitors and has the interesting pharmacokinetic enabling an oral administration.
Authors' response: Thank you for your helpful comments.
2. It is very interesting to have these data about a new combination of penem and inhibitor of beta lactamase that could be delivered orally to patients infected with M. abscessus.However, the paper contains limited data (MIC testing on 44 isolates for 5 beta-lactams combined with xeruborbactam), and consequently can be shorten into a note of a brief report.
Authors' response: Thank you for your helpful comments.However, because this report has two tables and one figure, and we have added some discussions according to the reviewers' comments, it was difficult to shorten the text into a brief report.We are very much grateful if you kindly understand our considerations.
3. MIC protocol and description of the M. abscessus isolates results are according to the reference.However, we would expect to see results of repeatability and reproducibility at least on the reference strain.
Authors' response: Thank you for your suggestion.However, we are running out of xeruborbactam and are unable to do the re-test immediately.It may take one more month to get xeruborbactam, in which case we will likely not be able to return our manuscript within 60 days.It is a last resort, but we confirmed the repeatability and reproducibility using each β-lactam alone at least on the QC strain.
4. Results show interesting data with low MIC of the combination xeruborbactam and terbipenem.
Authors' response: Thank you for your helpful comments.
5. Bibliography is consistent with the topic.
Authors' response: Thank you for your helpful comments.
6. abstract: the results for cefoxitin are missing Authors' response: Thank you for pointing this out.We have added the results of cefoxitin in the Abstract.
7. page 7, lines 118-121.Is the xeruborbactam water soluble, or is it dissolved in a specific solvent, e.g.DMSO?
Authors' response: According to the manufacturer's instructions, xeruborbactam can be dissolved in DMSO, and there is no data available about water solubility.We have added that information in the Methods section.In this study, we dissolved xeruborbactam and each β-lactam directly in CAMHB.When measuring the MIC values of each β-lactam alone, a two-fold dilution series of the β-lactam stock solution was prepared using CAMHB.When measuring the MIC values of each β-lactam and xeruborbactam combination, a two-fold dilution series of the β-lactam stock solution was prepared using CAMHB with xeruborbactam.
8. Page 8, line 134: waiting for 14 days could increase the MIC by degrading the inhibitor or the beta-lactam, which is often observed for penems.For how many isolates (for one isolate, the reading was done at D10), this extended incubation was used?If it is zero, better to suppress this sentence.
Authors' response: Extended incubation time beyond Day 5 was used for only one isolate (No. 7).We would like to keep this sentence.9. Tables: results shown in table 3 can be combined with those of the table 2.
Authors' response: We have combined Tables 2 and 3.
10. Figure : how do you explain that there is a nice Gauss distribution observed for terbipenem on the figure 1b, and the all MIC results are concentrated on the 4 µg/mL value when the xeruborbactam is added?
Authors' response: As previous study indicated that it appeared to be a lower limit for the MIC of carbapenem and avibactam combination, we have considered this as the reason for the convergence of the MIC values.We have added the information in the Discussion section.
11. Page 12, line 208.Since xeruborbactam will be commercialized with ceftibuten as an oral form, it could have been worthwhile to test this combination.
Authors' response: Thank you for your comment.As per your suggestions, we should have tested the combination of xeruborbactam and ceftibuten.When we started this study, we were not aware that xeruborbactam was being developed in combination with ceftibuten, and as we had run out of xeruborbactam, we could not test this combination this time, unfortunately.
12. The authors showed that MIC on Rough isolates are higher than on the Smooth isolates?
Page 13, Lines 230-231, the authors explained that the absence of GPL enhance the permeability cell wall.So consequently, the MIC should be lower for the R isolates?Do they have hypotheses for this unexpected result?
Authors' response: The absence of GPL may enhance the permeability of the cell wall and the accumulation of the drug inside the bacteria.We have considered that this might prevent β-lactams from acting on the bacterial cell wall, leading to the low susceptibility of the rough morphotypes, and added that in the Discussion section.
Comments for the Author): Spectrum00084-24 Yamatani et al.The authors determined Minimal inhibitory concentrations of various beta-lactams combined with a new inhibitor of beta-lactamase against Mycobacterium abscessus.
Line 45. Β-lactam antibiotic (add antibiotic) Line 54. MABC instead of MABS (MAB complex is more commonly used) Line 64.Add: The (The T28C) Line 81.Plural form -β-lactams 84.This is the first case of … Rephrase -you get the impression that this study will describe this as you use the present tense.Use past tense to describe previous studies.Make it clearer that you are referring to the case report mentioned before.Line 123.What is the half-life of XER?Is it stable during long incubation times?At least discuss this as a possible limitation.This has been discussed previously with clavulanic acid when MER-CLA has been tested.Line 157.QC -write out (quality control) at least in the subheading Line 168.Rephrase "slightly low".Very subjective.Compare to something or state the MIC Lina 171.Rephrase -not clear enough Line 176.Change to "upon the addition of…" Line 180.Suggest rephrasing to: This study demonstrated that XER restored the activity of certain βlactams against M. abscessus, most notably seen for tebipenem.
Schön T, Davies Forsman L. A Rough Colony Morphology of Mycobacterium abscessus Is Associated With Cavitary Pulmonary Disease and Poor Clinical Outcome.