Comparison of two phenotypic methods for the detection of carbapenemase production in Enterobacteriaceae

  1. Ramnath P
  2. Malavika B
  3. Veena Kumari Haradara Bahubali
  4. Srinivasa Sundara Rajan R

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Abstract

Carbapenemase production in Enterobacteriaceae constitutes the capacity of these bacteria to synthesize enzymes known as carbapenemases, which degrade carbapenem antibiotics, thus rendering them ineffective. This mechanism plays a pivotal role in promoting antibiotic resistance, therefore posing a formidable threat to public health by restricting therapeutic options for bacterial infections. The objective of the present study was to detect and compare the types of carbapenemase produced by two phenotypic methods: the modified carbapenem inactivation method (mCIM) combined with the EDTA carbapenem inactivation method (eCIM) and a combined disc test for carbapenem-resistant Enterobacteriaceae (CRE), carbapenem-resistant Klebsiella pneumoniae (CRKP) and carbapenem-resistant Escherichia coli (CREC). A total of 112 clinical isolates of CRE were obtained, of which 87 (77.6%) were metallo-beta lactamase (MBL) producers and 2 (1.8%) were serine carbapenemase producers. Among the tracheal isolates, predominantly K. pneumoniae produced MBL, whereas among the urine isolates, predominantly Escherichia coli were MBL producers. Since imipenem+EDTA (indigenous) is cost effective and easy to perform under laboratory conditions, it can be considered a reliable phenotypic method for the detection of carbapenemase production.

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  1. Version published to 10.1099/acmi.0.001035.v4 on Access Microbiology
  3. Access Microbiology

    Comments to Author

    Following re-review of the revised manuscript, the study is clearly improved in presentation and transparency. However, the statistical analysis remains the principal methodological weakness and continues to limit the interpretability and robustness of the findings, particularly given that the manuscript's central claim rests on comparative diagnostic performance. The analysis relies exclusively on p-values derived from Chi-square testing, without accompanying effect sizes, confidence intervals, or measures of agreement. While statistical significance is reported, the absence of magnitude estimates makes it difficult to judge whether observed differences between phenotypic methods are clinically or diagnostically meaningful. In a comparative diagnostic study, effect measures such as absolute differences in detection rates, risk ratios, odds ratios, or agreement statistics would provide substantially greater interpretive value than p-values alone. In addition, the assumptions underpinning the Chi-square test are not addressed. The manuscript does not discuss independence of observations, adequacy of expected cell counts, or whether contingency table structures were appropriate across the multiple subgroup analyses performed. Given the stratification by organism, specimen type, and ward location, it is plausible that some comparisons may violate these assumptions, potentially inflating statistical significance. The statistical framework also does not account for multiple comparisons, despite repeated testing across organisms, methods, and specimen categories. Without adjustment or explicit justification for an exploratory analytical approach, there is an increased risk of type I error. This is particularly relevant where statistical significance is used to support claims of methodological superiority. Finally, the statistical methods section remains under-described. There is no justification for test selection, no indication of statistical software used, and no explanation of how hypotheses were defined a priori. Together, these omissions weaken confidence in the analytical rigor supporting the study's conclusions. Beyond the statistical issues, the manuscript shows clear improvement in structure, transparency of limitations, and alignment between text and figures. The inclusion of explicit inclusion and exclusion criteria and the expanded limitations section are welcome. The Results are clearly presented, although still somewhat granular. The Discussion is better focused than in the original submission but remains largely descriptive, with limited analytical synthesis beyond restating results.

    Please rate the manuscript for methodological rigour

    Satisfactory

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

    Very good

    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

  4. Version published to 10.1099/acmi.0.001035.v3 on Access Microbiology
  6. Access Microbiology

    Comments to Author

    1. Title, Abstract, and Keywords The title clearly reflects the study's aim and retains appropriate specificity. The Abstract provides an adequate overview, yet remains overly descriptive in several places. Critical details such as total sample size, the two methods compared, and major findings are present, but the prose still reads as dense, with some sentences continuing to be longer than necessary. The Abstract also reports serine-carbapenemase producers (1.8%) but does not contextualise their relevance, which subtly misbalances emphasis given the study's primary focus on MBL detection. A stronger synthesis of what the findings imply for practice would enhance impact. 2. Introduction The Introduction covers the necessary conceptual ground: Enterobacteriaceae epidemiology, carbapenem resistance mechanisms, and justification for phenotypic detection methods. Scientific accuracy is largely sound, although the narrative remains long-winded in places. Several sections read as textbook summaries rather than a focused build-up to the study's rationale. A core conceptual issue persists: while the Introduction mentions phenotypic and genotypic detection strategies, it does not sufficiently explain why only phenotypic assays were selected for this study, nor does it contextualise how the chosen methods perform relative to molecular assays in low-resource settings. This limits the logical transition into the Methods. 3. Materials and Methods The Methods section is comprehensive and replicable, which is a strength. The procedural detail for mCIM/eCIM and CDT is meticulous. However: * Several steps include operational detail that does not add scientific value (e.g., repeated notes on pH adjustments, precise plate-drying times), which could be streamlined. * Statistical methodology remains minimal. The revision specifies that a Chi-square test was used, but there is no justification for the choice of test, no description of expected/observed comparisons, and no information on software used. * The study design would benefit from explicit mention of inclusion/exclusion criteria, which are implied but never formally stated. * The lack of genotypic testing is disclosed later but not here; noting this upfront would improve transparency. 4. Results The Results section presents a large volume of data across multiple specimen types. Key strengths include: * clear reporting of proportions of MBL producers across methods, * structured presentation of CREC and CRKP results, * integration of figures that align better with the textual descriptions. However, several limitations persist: * The logic and sequence of result reporting remain overly granular, leading to repetition. * Tables 3A and 3B are very dense; the number of subcategories within each table makes interpretation difficult. * The Venn diagrams (Fig. 1a-b) communicate comparative detection patterns well, but the legends remain somewhat verbose. 5. Statistical Analysis The statistical analysis is functional but limited. Only one test is applied (Chi-square), and the approach assumes: * independence of observations, * adequate expected frequencies, * categorical comparators with clear hypotheses. These assumptions are not discussed, raising questions about robustness. The choice of p-value thresholds and whether adjustments for multiple comparisons were considered is not stated. The Results reference only p-values without effect sizes or confidence intervals. Effect measures would provide a more nuanced comparison of performance between methods. 6. Discussion The Discussion shows better focus than before and integrates relevant literature. However, several limitations remain: * The authors overemphasise descriptive findings; the Discussion reads like a restatement of the Results. * One key finding—the substantial proportion of isolates negative by all methods—is acknowledged but not adequately explored. The Discussion stops short of proposing mechanistic explanations grounded in published evidence (e.g., specific porin mutations, efflux pumps, or OXA-48-like enzymes with low EDTA responsiveness). * While the authors contextualise MBL prevalence, they do not comment on potential sampling bias inherent in a single-centre ICU-dominated cohort. * Interpretation tends to generalise beyond the dataset. Statements such as "MBL is the most prevalent and difficult-to-treat carbapenemase" are true but detract from study-specific insight. 7. Study Limitation This section now clearly outlines the primary limitations: lack of genotypic confirmation, potential false negatives, and inability of phenotypic tests to differentiate all carbapenemase classes. However, the limitations could be strengthened by acknowledging: * lack of a gold-standard comparator, * absence of external quality assurance in the phenotypic assays, * limited generalisability given the short sampling window (January-May 2024), * unclear selection process for isolates (possible spectrum bias). 8. Conclusion The conclusion accurately reflects the study findings and avoids overclaiming. The emphasis on cost-effectiveness and feasibility of the indigenous EDTA-based CDT is appropriate for the study's context. However, the conclusion could more clearly differentiate between sensitivity for MBL detection vs. broader carbapenemase detection. Overall Strengths * Well-defined aim comparing phenotypic detection methods. * Large sample size for a single-site phenotypic study (n=112). * Clear improvement in nomenclature, figure placement, and limitations. * Practical relevance for resource-limited settings. Weaknesses * Statistical analysis remains insufficiently justified. * Overly descriptive narrative with limited critical synthesis. * Heavy reliance on phenotypic data without molecular corroboration. * Tables are difficult to navigate. * Some language and structural issues persist. * The rationale for choosing only phenotypic assays is still underdeveloped. Overall judgement The revised manuscript is significantly improved and addresses many of the earlier reviewer concerns. However, from a fresh scientific standpoint, it would still benefit from: * stronger statistical analysis, * more analytical rather than descriptive Discussion, * clearer justification for methodological choices, * tighter language editing and improved table readability.

    Please rate the manuscript for methodological rigour

    Very 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

  7. Version published to 10.1099/acmi.0.001035.v2 on Access Microbiology
  8. Access Microbiology

    Comments to Author

    This manuscript provides a relevant and timely evaluation of two phenotypic methods (mCIM + eCIM and combined disc test with EDTA) for the detection of carbapenemase production in Enterobacteriaceae. The use of indigenous EDTA-based CDT is particularly valuable for laboratories with limited resources. The study is well-structured, and the data presentation is mostly clear. However, there are several aspects that could be improved to enhance the clarity and impact of the manuscript: 1. The statistical analysis section is underdeveloped. Only a brief mention of a significant p-value is included, without clear explanation of what comparisons were performed. More detail is needed on the statistical methods used, variables compared, and any assumptions checked. 2. Although the phenotypic approaches are appropriate and practical, the absence of genotypic confirmation (e.g., PCR detection of specific carbapenemase genes) is a notable limitation. Confirming the presence of resistance genes would have significantly strengthened the conclusions. It is strongly recommended that this limitation be more explicitly acknowledged in the discussion section. 3. The language and phrasing throughout the manuscript require improvement to enhance clarity, conciseness, and academic tone. While the manuscript is technically understandable, it contains repetitive and awkward constructions. For example, the sentence "CDT indigenous was more common than other two methods were" is grammatically incorrect and stylistically weak. It is strongly recommended that the manuscript be reviewed by a native English-speaking editor or professional language service. 4. Some figures and tables require clearer integration and formatting within the manuscript. - Figure 1a and 1b (the Venn diagrams) are referenced in the text but not clearly placed or labeled, making them difficult to interpret. - Figure 2a and 2b (ward-wise distribution of MBL producers) are also referenced but not visible in the main text. - Table 3, although comprehensive, is very dense and difficult to follow. Splitting it into two separate tables or adding clearer subheadings (CRKP vs. CREC) would improve readability. - Figure legends should briefly explain what is shown to avoid requiring the reader to return to the results section. The manuscript reports that among 69 CRKP isolates, 36 (52.1%) were identified as MBL producers using mCIM/eCIM, and 49 (71.01%) using CDT with indigenous EDTA discs. For the 43 CREC isolates, 33 (76.7%) were identified as MBL producers via mCIM/eCIM, while 38 (88.4%) were detected using CDT with indigenous EDTA. These findings support the authors' conclusion that the indigenous version of the CDT shows higher sensitivity. However, 25 isolates remained negative by all three methods, and the manuscript suggests, without further validation, that these could be due to alternative resistance mechanisms. This point deserves a more cautious interpretation in the discussion. Additionally, while many references are appropriate, a few citations are quite dated (e.g., from the 1990s). Where possible, please consider replacing or supplementing these with more recent literature to strengthen the scientific context of the manuscript. Overall, the manuscript presents useful findings, especially for laboratories with limited diagnostic resources. With minor revisions focused on language, statistical explanation, clearer data visualization, and a more transparent discussion of limitations, this work would be suitable for publication.

    Please rate the manuscript for methodological rigour

    Good

    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

  9. Access Microbiology

    Comments to Author

    Revisions 1. Line 28: Keywords: Carbapenemase, Enterobacteriaceae, antibiotics, Escherichia coli, Klebsiella pneumoniae 2. Line 32: Klebsiella, Escherichia coli, Salmonella, … Klebsiella, E. coli, Salmonella, … 3. Line 34: among Klebsiella pneumoniae and … among K. pneumoniae and … 4. Line 34: Escherichia coli (E. coli) (4). … E. coli (4). 5. Line 54: (Klebsiella pneumoniae carbapenemase, NMC), … (K. pneumoniae carbapenemase, NMC), 6. Line 62: Klebsiella pneumoniae carbapenemase, … K. pneumoniae carbapenemase, 7. Line 86-7: carbapenem-resistant Klebsiella pneumoniae and Escherichia coli isolates …carbapenem-resistant K. pneumoniae and E. coli isolates … 8. Line 90-91: strains of Klebsiella pneumoniae and Escherichia coli via … strains of K. pneumoniae and E. coli via 9. Line 97-98: isolates of Klebsiella pneumoniae and Escherichia coli from … isolates of K. pneumoniae and E. coli from … 10. Line 107: control strain was Klebsiella pneumoniae ATCC BAA- 1705. control strain was K. pneumoniae ATCC BAA- 1705. 11. Line 115: Modified Carbapenem Inactivation Methods: Modified Carbapenem Inactivation Methods (mCIM): 12. Line 148: Klebsiella pneumoniae (CRKP) … K. pneumoniae (CRKP) 13. Line 162: Tables 1, 2 and 3 should be installed after this section.

    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

  10. Version published to 10.1099/acmi.0.001035.v1 on Access Microbiology