Analysis of a simple disk diffusion method to evaluate ceftazidime–avibactam/aztreonam combination synergism against New Delhi metallo-beta-lactamase-producing clinical isolates
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The rise of multidrug-resistant Enterobacterales and Pseudomonas aeruginosa has diminished the reliability of conventional antibiotics for treating Multidrug Resistant (MDR) infections. The combination of ceftazidime–avibactam with aztreonam has demonstrated in vitro synergism against multidrug-resistant organisms, notably metallo-beta-lactamase-producing strains. Treatment with the ceftazidime–avibactam/aztreonam combination may provide clinical benefits for patients with multidrug-resistant bacterial infections. The present study aimed to detect genes encoding carbapenem resistance in clinical strains and to determine the efficacy of ceftazidime–avibactam/aztreonam against carbapenemase co-producers. A cross-sectional research study was conducted on 62 carbapenem-resistant clinical isolates collected from November 2022 to February 2024. Ceftazidime–avibactam/aztreonam synergy against 55 carbapenemase producers [New Delhi metallo-beta-lactamase (NDM), imipenem-hydrolysing metallo-beta-lactamase (IMP), Verona Integron-encoded metallo-beta-lactamase (VIM) and oxacillinase-48 (OXA-48)] was determined using the disc diffusion method. Data analysis was performed by chi-square test. Ceftazidime–avibactam/aztreonam synergy was identified against 25 (64.1%) out of 39 isolates exhibiting the NDM gene, seven (77.8%) out of nine isolates that were co-producers of NDM and OXA-48 genes, two (50%) out of four isolates co-producing NDM and VIM carbapenemase genes and a single isolate (33.3%) out of three isolates with NDM, VIM and OXA-48 genes. A wide zone of 3–23 mm diameter was observed for Enterobacterales and 6–7 mm for P. aeruginosa with ceftazidime–avibactam/aztreonam in relative to ceftazidime–avibactam and aztreonam discs when tested alone. More than 30% of isolates showed a statistically significant difference in zone diameter for the ceftazidime–avibactam/aztreonam combination ( P <0.05), when compared with the zone size for ceftazidime–avibactam and aztreonam discs when tested alone. The present study showed the in vitro effectiveness of the ceftazidime–avibactam/aztreonam combination against 63.6% of carbapenem-resistant isolates studied. The disc diffusion method requires less technical expertise, and the test result aids in identifying true clinical synergy by observing the widening of the zone diameter that exceeds the aztreonam susceptibility breakpoint.
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I would like to thank you for addressing the reviewer comments and I am pleased to tell you that your article has now been accepted for publication in Access Microbiology. Note: During the production process I would like you to remove the titles from Figures 1, 2 and 3 and present them as legends similar to the format utilised for the other figures and tables in the manuscript.
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Thank you for addressing both reviewer comments in your submission of the revised manuscript. One of the original reviewers has reviewed the updated manuscript and is happy with the amendments made. They have, however, suggested a couple of minor changes related to the figures and identified a number of smaller corrections. I would specifically like to suggest that Figures 4 and 5 are revised and formatted in a similar style to aid in reader interpretation of the data. I'm looking forward to receiving the revised manuscript.
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Comments to Author
The manuscript has been substantially improved in both structure and clarity. The updates to the rationale and methodology strengthen the study considerably. There are, however, some remaining issues with formatting, consistency, and minor language details that should be addressed before publication. Throughout manuscript - - Follow correct conventions for species names. Write the full name (e.g. Klebsiella pneumoniae) on first mention, then abbreviate thereafter (e.g. K. pneumoniae). Apply this rule consistently to all species - Include a space before brackets and before units - Maintain consistency in terminology. Use the same form for "metallo-beta-lactamases" (or the abbreviation "MBL" if preferred), "Ceftazidime-avibactam", and all units (choose either "micrograms" or "µg" and use it throughout)
Comments to Author
The manuscript has been substantially improved in both structure and clarity. The updates to the rationale and methodology strengthen the study considerably. There are, however, some remaining issues with formatting, consistency, and minor language details that should be addressed before publication. Throughout manuscript - - Follow correct conventions for species names. Write the full name (e.g. Klebsiella pneumoniae) on first mention, then abbreviate thereafter (e.g. K. pneumoniae). Apply this rule consistently to all species - Include a space before brackets and before units - Maintain consistency in terminology. Use the same form for "metallo-beta-lactamases" (or the abbreviation "MBL" if preferred), "Ceftazidime-avibactam", and all units (choose either "micrograms" or "µg" and use it throughout) Introduction - All previous comments have been addressed well. The structure, rationale, and flow are much improved. Please proofread carefully to correct the remaining inconsistencies noted below. - Line 46 - Genus/species names should be italicised - Lines 58, 59 - Add a space between the number and bracket - Lines 62, 169, 212, 231 etc - Use P. aeruginosa after first mention - Line 84 - Clarify phrasing to "Carbapenem-resistant Gram negatives pose a significant public health threat due to the presence, acquisition, or production of carbapenemases, specifically metallo-beta-lactamases." - Line 94 - Use lower case 'm' in "metallo-beta-lactamases" - Line 96 - Consider rephrasing to "These metallo-beta-lactamases can deactivate nearly all beta-lactam drugs…" - Lines 102, 313 - Introduce the abbreviation (MBL) after first mention on line 85 and use only the abbreviation thereafter - Line 104 - Introduce "ESBL" after its first mention on line 88, not here - Line 105 - Use lower case for "carbapenem-resistant Enterobacterales" - Line 112, 212, 229, 249, 251 etc - Use K. pneumoniae after first mention - Lines 118, 175 - Write "minimum inhibitory concentration" in lower case - Line 119 - Add a reference to support this statement Methods - The requested additions (ethics statement, biochemical tests, and controls) have been well implemented. Only minor corrections remain. - Line 144 - Remove full stop after "Salem" - Line 150 - Remove space in "MacConkey" - Lines 157 to 162 - Use either "micrograms" or "µg" consistently - Line 162 - Format as E. coli ATCC 25922 (with ATCC before the number) Results - The written description is clear, and the statistical analysis is stronger. However, the figures require significant revision for clarity and accuracy. - Line 222 - move [Figure:1] to end of line 225 - Line 226 - Figure 1. a title is unnecessary if the legend is descriptive. Could this information be incorporated into Figure 2 instead, or is a separate figure required? - Line 237 - Figure 2. remove title if duplicated in legend. "Carbapenem" should not be capitalised - Line 256/257 - The referenced figures do not appear to match this section's content - Line 258 - Figure 4. This figure does not clearly convey the data. The text references 35 out of 55 isolates, but the figure appears to show more. Please clarify or revise - Line 262 - Figure 5. Again, the data presentation could be improved for clarity - Line 285 - Use lower case 's' in "seven" - Lines 288, 293 - Use lower case 't' in "three" Discussion - The discussion is well written and well referenced, with only a few clarifications and minor edits suggested. Adding a summarising sentence at the end of the paragraph on line 339 would strengthen the comparison with previous studies. Some data referenced in this section are not clearly visible in the results and should be checked for consistency. - Line 312 - Remove the abbreviation "CRE" as it is not used elsewhere - Lines 314-317 - Add a reference - Lines 322 and 387 - Use lower case 'p' in "polymyxins" - Lines 328-330 - Move the sentence beginning "The predominance of carbapenemase…" to the start of the paragraph for better flow - Line 347 - Add % for easier comparison with the % in line 346 - Lines 359-361 - The sentence listing enzyme combinations is hard to follow; consider simplifying for clarity - Line 389-391 - Clarify where this data is presented in the results This version shows substantial improvement in structure, rationale, and written clarity. The introduction and methods sections are now strong, and the study presents relevant findings. However, the figures currently do not align well with the results text and do not always effectively communicate the data. These need to be carefully reviewed, restructured, or redesigned before the manuscript can be considered ready for publication.
Please rate the manuscript for methodological rigour
Very 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
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This is a study that would be of interest to the field and community. The reviewers have highlighted major concerns with the work presented. Please ensure that you address their com
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Comments to Author
This manuscript addresses the important and clinically relevant challenge of managing infections caused by carbapenem-resistant Gram-negative bacteria. The aims are clearly formatted and understandable. The sample size is reasonable, and the diversity of organisms tested makes the findings more clinically applicable. A notable strength is the use of complementary approaches, with both phenotypic (disc diffusion, Vitek-2, synergy testing) and genotypic (PCR) methods included, which strengthens the study design. Adherence to CLSI guidelines for susceptibility testing is also appropriate. The evaluation of ceftazidime-avibactam/aztreonam synergy using a simple disc diffusion method is valuable, as this approach is feasible for routine diagnostic laboratories and could have practical implications for …
Comments to Author
This manuscript addresses the important and clinically relevant challenge of managing infections caused by carbapenem-resistant Gram-negative bacteria. The aims are clearly formatted and understandable. The sample size is reasonable, and the diversity of organisms tested makes the findings more clinically applicable. A notable strength is the use of complementary approaches, with both phenotypic (disc diffusion, Vitek-2, synergy testing) and genotypic (PCR) methods included, which strengthens the study design. Adherence to CLSI guidelines for susceptibility testing is also appropriate. The evaluation of ceftazidime-avibactam/aztreonam synergy using a simple disc diffusion method is valuable, as this approach is feasible for routine diagnostic laboratories and could have practical implications for therapy in resource-limited settings. The dataset is robust, linking genotypic and phenotypic resistance profiles, and the findings are significant in highlighting treatment options for regions with a high prevalence of carbapenemase producers. The quality of writing requires improvement. A proofreader is recommended to improve sentence structure and readability. Consistency in species names, gene nomenclature, formatting, and abbreviations also needs attention. Review of published literature - The introduction provides useful and relevant background information, and the article overall is an appropriate length. - Sentences are at times difficult to follow due to grammar, word choice, and flow. - The technical and scientific detail is strong, but the section could be made more reader-friendly by structuring it more clearly (progressing from problem → specific mechanisms → therapeutic challenges → rationale for the study). - References included are relevant and up-to-date but some claims (e.g. "various studies have introduced…") are under-referenced, with only a single supporting reference cited. Additional citations are needed. - The rationale for focusing specifically on NDM, VIM, and OXA-48 co-producers could be better supported with recent prevalence or patient outcome data. Methods - Sample size and range of organisms are appropriate and add to clinical applicability, but the description of sample collection is incomplete, as the source of isolates is not reported. - Ethical approval is reported, but the date (Nov 2022) falls after the start of sample collection (Oct 2022). This should be clarified - was approval retrospective, or was consent/waiver of consent obtained? - Identification of isolates is only briefly described (microscopic/colony morphology and biochemical reactions) without specifying which biochemical tests were performed. - The combination of phenotypic (disc diffusion, Vitek2, synergy testing) and genotypic (PCR) methods is well aligned to the study's aims. - Following CLSI guidelines for susceptibility testing is a strength, especially given the introduction highlighted a lack of standardisation for synergy testing. It is unclear whether controls were included for susceptibility and synergy testing, which should be clarified. - The wide range of antibiotics initially tested provides broad clinical relevance, and susceptibility testing cut-offs and synergy interpretation are described in good detail. More information is needed on culturing conditions. - Information on replicates is missing. - The PCR section provides cycling conditions, but the target genes could be explicitly listed (e.g., NDM, VIM, OXA-48) rather than "carbapenemase genes." - Only descriptive statistics ("frequencies and percentages") were used. While completely acceptable for a preliminary in vitro study, comparisons between groups (e.g., resistance rates, synergy outcomes) could be strengthened with further statistics. Results and discussion - The dataset is strong in both number and diversity of isolates. Combining phenotypic and genotypic data adds strength. The genotype-synergy link is clinically valuable. - Findings are significant in the context of limited therapeutic options and high prevalence of resistance. The use of a simple disc diffusion method for demonstrating synergy is practical for routine laboratories. - The novelty lies more in local epidemiology and methodological accessibility than in demonstrating a new therapeutic effect. - Results are generally interpreted correctly, but the presentation is sometimes dense and repetitive. Visual representation could be improved (bar charts for species distribution or resistance values, pie charts for specimen sources, heatmaps or Venn diagrams for gene-resistance combinations). - Tables are informative but Table 1 lacks measures of variability (e.g., ranges or SD). The narrative around Table 2 largely repeats what is shown and could be streamlined to highlight key patterns. - Some percentages are reported to two decimal places, which is unnecessary given the sample size. - No supplementary data are provided. Inclusion of raw VITEK2 results and initial antimicrobial susceptibility testing would add transparency, but should be placed in supplementary files to keep the main text concise. - The discussion includes a nice section on strengths (synergy in most Enterobacterales isolates) with limitations (small number of IMP producers, no co-production in P. aeruginosa). - The conclusions are supported by the data and reasonably referenced, but the discussion is overly descriptive. A clearer separation between in vitro findings and clinical implications, and a focus on key take-home messages and comparison with other published work would improve impact. Overall, this is an interesting and clinically relevant study, but I recommend major revisions to improve clarity, reproducibility, and impact.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
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
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Comments to Author
The study presents important observations on the synergistic activity of ceftazidime-avibactam/aztreonam against carbapenem-resistant organisms, particularly NDM-producing strains. However, several areas in the manuscript require revisions for clarity, consistency, scientific rigor, and improved presentation. 1. The abstract suddenly jumps at the start of the treatment without any background information. Even just one sentence stating the background should make the start better. 2. Please ensure grammatical errors throughout the manuscript. 3. The bacteria name should be italic, for example, Klebsiella pneumoniae in line 85. 4. Line 90, the start of the sentence should be uppercase- disc stacking. 5. In the introduction section, please explain why the work is important. Mention specifically the …
Comments to Author
The study presents important observations on the synergistic activity of ceftazidime-avibactam/aztreonam against carbapenem-resistant organisms, particularly NDM-producing strains. However, several areas in the manuscript require revisions for clarity, consistency, scientific rigor, and improved presentation. 1. The abstract suddenly jumps at the start of the treatment without any background information. Even just one sentence stating the background should make the start better. 2. Please ensure grammatical errors throughout the manuscript. 3. The bacteria name should be italic, for example, Klebsiella pneumoniae in line 85. 4. Line 90, the start of the sentence should be uppercase- disc stacking. 5. In the introduction section, please explain why the work is important. Mention specifically the novelty. 6. Method section- where did you collect the isolates from? Is it the same as the ethics committee? 7. Briefly explain the collection process- for example, how you collected and transported? 8. How did you specify the susceptibility and resistant zone of inhibition range? Please add an appropriate reference for the paragraph with lines 120-129. 9. Line 158- Percentages should be in lower case. 10. In Table 1, add another column stating the isolates as resistant/susceptible. 11. Imply statistical analysis and add p-values for the analysis included (for each Bacterial species). 12. Adding a comparative table with results before (when used alone) and after the combination of antibiotics would be great. This can even be stronger if comparative statistical analysis is done. 13. Same comment for discussion (as in point 1 recommendation for abstract). The start of the discussion is abrupt. The same goes for the ending of the discussion. 14. Explicitly highlight the novelty. What is new in your study? And why? 15. There is a separate novelty section but it is not to the point and does not clearly explain the specific novelty. There is no comparison with existing literature. Is this first time ? But there is already protocol for the same antibiotic synergistic effect, then what exactly makes your work novel? Instead of mentioning novelty in the end, adding it in intro and also explaining it in discussion is a better idea.
Please rate the manuscript for methodological rigour
Poor
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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