Meropenem-resistant Burkholderia pseudomallei: a concerning single case in Australia with no prior meropenem exposure
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We report a case of cutaneous melioidosis in a 54-year-old male with a meropenem-resistant sub-population. He was empirically treated with episodic doxycycline and trimethoprim–sulfamethoxazole; however, the abscess re-accumulated. The patient had no prior exposure to meropenem. A sub-population of the isolate was meropenem resistant with an MIC >32 µg ml −1 and the identification was re-confirmed as Burkholderia pseudomallei . Whole-genome sequencing with ARDaP analysis only revealed a resistance determinant to doxycycline and did not reveal a resistance determinant to meropenem. Furthermore, no carbapenemases were detected through multiple bioinformatics tools. To date, this is the first reported case in Australia of a B. pseudomallei isolate resistant to meropenem without previous carbapenem exposure.
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Thank you for making the minor amendments and sign posting the data.
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I believe the additional methodology details describing the repeat testing to confirm original findings lessens the likelihood that the described phenotype arose in vitro during testing. Additionally the authors have added the caveat that in vitro development of resistance during the E-test procedure cannot be fully ruled out. Minor edits Line 197- in vivo should be in vitro The data summary needs to be changed to signpost the available sequencing data of the resistant isolate.
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Please address all reviewers comments and make amendments where required, including uploading the genome sequence data generated as part of this study into a publicly accessible repository.
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Comments to Author
The study reports an isolate of B. pseudomallei resistant to meropenem from a patient in Australia. It is interesting to observe the development of MEM resistance of B. pseudomallei in a patient. However, I wish to see this study have shown the comparison WGS data of MEM susceptible and resistance isolates. Many sentences in this manuscript need more clarification and explanation. Line 23: What is ARDaP analysis? What is a resistance determinant to DOX? Line 25: What is the Abricate program? What are in CARD and ResFinder databases? Line 36: It would be appropriate to cite data from a recent large study in Thailand for the mortality rate and readmission. See the following information from Abstract of Chantratital et al, Lancet Reg Health Southeast Asia 2023. Findings—2574 individuals hospitalised …
Comments to Author
The study reports an isolate of B. pseudomallei resistant to meropenem from a patient in Australia. It is interesting to observe the development of MEM resistance of B. pseudomallei in a patient. However, I wish to see this study have shown the comparison WGS data of MEM susceptible and resistance isolates. Many sentences in this manuscript need more clarification and explanation. Line 23: What is ARDaP analysis? What is a resistance determinant to DOX? Line 25: What is the Abricate program? What are in CARD and ResFinder databases? Line 36: It would be appropriate to cite data from a recent large study in Thailand for the mortality rate and readmission. See the following information from Abstract of Chantratital et al, Lancet Reg Health Southeast Asia 2023. Findings—2574 individuals hospitalised with culture-confirmed melioidosis were screened and 1352 patients were analysed. The median age was 55 years, 975 (72%) were male, and 951 (70%) had diabetes. 565 (42%) patients presented with lung infection, 1042 (77%) were bacteremic, 442 (33%) received vasopressors/inotropes and 547 (40%) received mechanical ventilation. 1307 (97%) received an intravenous antibiotic against B. pseudomallei. 335/1345 (25%) patients died within one month and 448/1322 (34%) of patients died within one year. Most patients had risk factors for melioidosis, but patients without identified risk factors did not have a reduced risk of death. Of patients discharged alive, most received oral trimethoprim-sulfamethoxazole, which was associated with decreased risk of post-discharge death; 235/970 (24%) were readmitted, and 874/1015 (86%) survived to one year. Line 49 and 115-132: It may be more update to add a reference for "To date the primary resistance of B. pseudomallei to carbapenems is extremely rare". See a recent information in Hii SYF et al. Antimicrobial Agents and Chemotherapy 2021. "All 1,317 primary isolates were IPM susceptible, but we observed two CAZ-resistant isolates and one CAZ-intermediate isolate, two MEM less-susceptible isolates, and one AMC-resistant and two AMC-intermediate isolates." Line 62: How to ensure that the patient know about meropenem exposure? This should be obtained from a Medical Record. Line 66: How to identify B. pseudomallei culture positive? Line 98: To determine potential genetic mechanism of meropenem resistance, WGS data should be compared between isolate 1 and isolate 2. Line 100: What was Galaxy using Kraken 2 program used for? Line 102: What are carbapemases in the ResFinder databases? Line104: What is B. pseudomallei resistance genes searched in the ARDaP database? Line 107: What is the predicted function of BPSL3085 gene? Line 143: What mutation in amrR gene was reported to increase meropenem MIC to 3 µg/ml? Does WGS data of isolate 2 show this mutation? Line 177-148: What is the role of other regulators that the authors want to mention? Do you mean mutations may confer the meropenem resistance? Does the WGS data reveal the mutations in these efflux pump genes?
Please rate the quality of the presentation and structure of the manuscript
Poor
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
The authors present an interesting melioidosis case study where they identified an isolate with high-level meropenem resistance. The work is well-written and overall, the study is reasonably well conducted. I do, however, have several major reservations with this study and think there are several critical points that need to be addressed. Major comments: Have the authors considered the possibility that the meropenem resistant strain may have evolved in-vitro, and only identified during the Etest procedure? Without the investigation of this possibility, the major conclusions of this manuscript are not well supported by the data. Given the brief description of the methodology, I was unable to determine if this possibility had been investigated. 1) Was a colony purification step undertaken prior to …
Comments to Author
The authors present an interesting melioidosis case study where they identified an isolate with high-level meropenem resistance. The work is well-written and overall, the study is reasonably well conducted. I do, however, have several major reservations with this study and think there are several critical points that need to be addressed. Major comments: Have the authors considered the possibility that the meropenem resistant strain may have evolved in-vitro, and only identified during the Etest procedure? Without the investigation of this possibility, the major conclusions of this manuscript are not well supported by the data. Given the brief description of the methodology, I was unable to determine if this possibility had been investigated. 1) Was a colony purification step undertaken prior to the first Etest or was the Etest performed directly on a (potentially) mixed population from a clinical sample? Clinical isolates cam be passed through a genetic bottleneck (in-part) to avoid confusion from any spontaneous mutants that may arise during antimicrobial sensitivity testing. The methods section of the manuscript doesn't contain sufficient detail to determine if this was performed. If the initial Etest was performed directly on a potential mixture of strains, this needs to be stated in the methodology. 2) Did the authors repeat the initial Etest to see if the meropenem resistant sub-population could be repeatedly identified? If the initial Etest was performed on a mixed population, then these resistant strains should be relatively easy to isolate upon a repeat test and their proportion within the population should remain consistent. 3) Did the authors whole-genome sequence the initial, meropenem susceptible isolate? Sequencing this isolate would determine the genetic relatedness of these two strains and potentially allow the identification of the mutation driving meropenem resistance in the latter isolate. Metagenomic sequencing of the clinical sample is also a possibility to identify a mixed strain population. The authors also need to mention this possibility as a limitation of their study, especially since their major conclusions rely heavily on in vivo evolution of this sub-population rather than the isolation of a spontaneous mutant. General comments: The authors need to make the whole-genome sequence data generated during the study publicly available in an online database (e.g. NCBI's Sequence Read Archive). Minor comments: Line 33 - suggest changing gram-negative to Gram-negative. Gram is a proper noun and should be capitalized unless this is a journal formatting requirement. Line 43 - The Schweizer et al reference here isn't ideal. It would be more appropriate to reference the current treatment guidelines rather than a review of resistance mechanisms. I would suggest the Dance, 2014 paper (10.1016/j.ijantimicag.2014.01.005). It would be worth mentioning the duration of each phase here too as the lengthy treatment is not necessarily appreciated by those outside of the melioidosis field. Line 46 - It might be worth explaining the difference between primary and secondary resistance here? Line 49 - The sentence is missing a reference here. Line 52 - The authors do not present any evidence that the isolate exhibits primary meropenem resistance. The isolate was only identified after several rounds of treatment and therefore the potential for cross-resistance (or indeed in-vitro selection, given the methods description. Suggest removing the word "primary" from this sentence. Line 64 - Is "denied" the best word choice here? Was there any investigation whether systemic symptoms were present? Line 66 - What were the culture conditions for the isolation of B. pseudomallei? Was selective media used? Line 67-68 - What was the Vitek 2 result if it didn't identify B. pseudomallei? What database version was used? The Vitek 2 system was known to misclassify B. pseudomallei, generally as B. cepacia, but this result is worthy of some investigation since these problems should have been addressed with newer database versions. Line 71- EUCAST has updated their guidelines to include B. pseudomallei (https://www.eucast.org/ast_of_bacteria/calibration_and_validation). Their breakpoints for meropenem (S2mg/mL), would suggest that the primary isolate in this study is already meropenem resistant. Line 71-72 - How was the isolate prepared prior to susceptibility testing? Was the isolate sub-cultured from a clinical specimen through a single-colony bottleneck or was a potentially mixed population of strains tested directly from the initial sub-culture? I would assume that since the authors claim to have identified a meropenem resistant sub-population, there was no single colony purification step? Line 101-102 - The versions of the CARD and Resfinder databases should be included here. Line 120 - "only few" should be "only a few" or just "few". Line 120 - Are these reports on primary carbapenem resistance? Line 136 - 143 - This study also noted meropenem resistant isolates in patients without any prior meropenem use. Lines 51-52 also incorrectly state that the current work is the first study to note meropenem resistance without prior exposure. Line 159 - Were any mutations noted in any of the efflux pump regulators?
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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