The impact of zinc supplementation on carbapenem MICs among bacteria expressing IMP metallo-beta-lactamase

  1. Susan Vaughn Grooters
  2. Dixie F. Mollenkopf
  3. Gregory A Ballash
  4. Thomas E Wittum

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Abstract

2. Abstract Antibiotic-resistant infections cause an estimated 2.8 million illnesses and 35,900 deaths annually in the US. Carbapenems are a class of antibiotics that are generally reserved to treat life-threatening invasive infections including sepsis. Accurate diagnosis of carbapenem-resistant infections is critical for early and appropriate treatment. blaIMP encodes bacterial production of the IMP metallo-beta-lactamase (MBL) which can confer resistance to all the beta-lactams including carbapenems. Zinc is an essential co-factor in the IMP MBL enzymatic hydrolysis of carbapenems. Tests for the presence of IMP carbapenemase, such as the Carba NP, include zinc sulfate (ZnSO4) although broth dilution methods for determining minimum inhibitory concentration (MIC) for carbapenems may vary. We hypothesized that ZnSO4 availability would improve the accuracy of carbapenem MIC determination for bacteria expressing blaIMP. Thus, the objective of this study was to determine if supplemental ZnSO4 affects the carbapenem MICs of Enterobacterales, Alteromonadales, and Moraxellales expressing blaIMP. Isolates utilized for this study were originally recovered from environmental samples collected at farms, wastewater treatment plants, and from surface water. They were selected based on phenotypic non-susceptibility to carbapenems and genetic confirmation of bacterial carriage of blaIMP. Cation-adjusted Mueller-Hinton broth suspensions of each isolate standardized to a 0.5 MacFarland standard were tested with and without ZnSO4 at 0.1 mmol/L concentration to determine MICs using standard extended-spectrum beta-lactamase micro-broth dilution MIC panels. Although we observed that Morganellaceae imipenem MICs were higher (P < 0.001) than those from other bacteria harboring blaIMP, the inclusion of supplemental ZnSO4 did not influence carbapenem MIC. This suggests that supplemental ZnSO4 will not improve the accuracy of carbapenem MICs in bacteria expressing IMP carbapenemase. Additional research will be required to identify important factors that may influence the expression of carbapenemase including IMP, and the accurate determination of clinical MICs which is critical to appropriate therapeutic decision-making.

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  1. Version published to 10.1099/acmi.0.000972.v2 on Access Microbiology
  3. Access Microbiology

    Comments to Author

    - The authors didn't mention the time frame of the study. - The reference number 17 which was used for antimicrobial susceptibility CLSI 2018 is too old. - The number of tested isolates is too few (25 isolates) to reach conclusions. - Moreover eight out of the 25 tested isolates belong to Morganellaceae that is likely to have mechanisms for carbapenem resistance other than the production of carbapenemases according to CLSI 2024 reference number 18. -

    Please rate the manuscript for methodological rigour

    Poor

    Please rate the quality of the presentation and structure of the manuscript

    Poor

    To what extent are the conclusions supported by the data?

    Not at all

    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

  4. Access Microbiology

    Comments to Author

    Principal Advantages 1. Relevance: Improving diagnosis and treatment requires addressing carbapenem resistance. 2. Clear Hypothesis: Given zinc's function in MBL activity, it makes sense to test its effect on MICs. 3. Data Transparency: Reproducibility is increased by sharing MIC values and sequencing data. Places to polish 1. Diversity in the Sample Is Important Although there are just two representatives of certain groups (such as Moraxellaceae), the 25 isolates are a decent start. More isolates, particularly those that are clinical. Recognizing that environmental strains may behave differently than human infections could increase complexity. 2. Measure Zinc Before Adding It Was there enough zinc in the original broth before you added ZnSO4? It's difficult to determine the significance of the supplement without examining baseline levels. This confusion might be cleared up with a fast zinc check in your media. 3. What Makes Morganellaceae Unique? It's interesting that their imipenem MICs are greater. However, referring to it as "intrinsic resistance" without examining its underlying mechanisms such as porin genes or efflux pumps raises concerns. Here, a little more information could transform conjecture into verifiable proof. 4. Mixed MIC Trends Require Clarification With zinc, certain isolates (such as G1) displayed significant MIC decreases. Even while statistics indicate "no overall effect," examining subgroups (for example, by bla IMP allele) may reveal unnoticed trends. 5. Figures and Tables: It's Important to be Clear It seems like the graphs are simplified. Boxplots or individual data points could be used to more effectively illustrate variability. Additionally, explain the meaning of the bold entries in Table 1, as readers may be confused. 6. Provide Evidence to Support Clinical Claims The clinical focus of the impact statement seems a little out of place because all isolates are environmental. Suggestions for Next Steps Minor Adjustments: Correct figure labels, provide context for table annotations, and define terms (e.g., "orders" vs. "families"). Larger Steps: Investigate the resistance mechanisms of Morganellaceae, evaluate baseline zinc, or examine clinical isolates if at all possible. Final Thoughts Your research sheds important light on the role zinc plays in carbapenem resistance diagnosis. This paper has the potential to be exceptional with a few changes.

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Good

    To what extent are the conclusions supported by the data?

    Strongly 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

  5. Access Microbiology

    Comments to Author

    I appreciate the opportunity to review this manuscript. I've reviewed the document. Here's a structured review, focusing on strengths, issues, and actionable suggestions: Strengths: 1. Clear Objective: The study is well-defined, investigating the impact of zinc on MIC determination for bacteria with blaIMP genes. 2. Relevant Research: It addresses a critical public health issue, antimicrobial resistance. 3. Methodological Rigor: The inclusion of various bacterial families, standard methods (e.g., CLSI guidelines), and robust statistical analysis adds credibility. 4. Detailed Results: Data tables and figures provide transparency and support conclusions. Concerns and Suggestions: 1. Abstract and Impact Statement * Concern: The abstract provides an overview but could better emphasize the broader implications of findings. * Suggestion: Highlight how the findings might influence clinical practices or future research. 2. Introduction * Concern: While informative, the introduction could better synthesize prior studies to set the stage for the hypothesis. * Suggestion: Explicitly link the study's objectives to gaps in the literature, emphasizing the novelty of zinc supplementation testing. 3. Methods * Concern: The description of zinc concentrations and their rationale is clear but lacks justification for why 0.1 mmol/L ZnSO4 was chosen. * Suggestion: Add a brief discussion on how this concentration compares to physiological zinc levels and its relevance to clinical settings. 4. Results * Concern: The results mention statistical findings without delving into potential biological explanations for observations. * Suggestion: Incorporate a brief discussion of why Morganellaceae showed higher imipenem MICs, connecting it to intrinsic resistance mechanisms. 5. Discussion * Concern: The discussion sometimes reiterates results instead of exploring broader implications or potential limitations. * Suggestions: o Reflect on the implications of intrinsic resistance in clinical diagnostics. o Address potential limitations, such as the environmental origins of isolates or the controlled lab setting versus in vivo conditions. o Consider how findings might guide diagnostic improvements or antibiotic stewardship policies. 6. Figures and Tables * Concern: Figures 1-3 are helpful but could benefit from more detailed legends explaining their significance. * Suggestion: Ensure figure legends include key observations, statistical significance, and implications. 7. Conclusion * Concern: The conclusion is succinct but could be more forward-looking. * Suggestion: Propose specific future research directions, such as exploring other cofactors affecting MIC determination or testing clinical isolates. 8. References * Concern: References are robust but heavily weighted toward foundational studies rather than recent advancements. * Suggestion: Consider adding recent reviews or studies on carbapenem resistance mechanisms and diagnostic innovations. Minor Edits: 1. Typos and Formatting: Ensure consistent formatting of bacterial names (italicization) and proper spacing between sections. 2. Acronyms: Define acronyms (e.g., MIC, ZnSO4) on first use for clarity. 3. Title: Consider simplifying the title to focus on the main findings, e.g., "Impact of Zinc Supplementation on Carbapenem MICs in blaIMP-Expressing Bacteria."

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Very good

    To what extent are the conclusions supported by the data?

    Strongly 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

  6. Version published to 10.1099/acmi.0.000972.v1 on Access Microbiology