Evaluation of different methods for in vitro susceptibility testing of colistin in carbapenem resistant Gram-negative bacilli

Introduction. The increasing antibiotic resistance like the advent of carbapenem resistant Enterobactarales (CRE), Carbapenem Resistant Acinetobacter baumanii (CRAB), and Carbapenem Resistant Pseudomonas aeruginosa (CRPA) has led to to the use of toxic and older drugs like colistin for these organisms. But worldwide there is an increase in resistance even to colistin mediated both by chromosomes and plasmids. This necessitates accurate detection of resistance. This is impeded by the unavailability of a user-friendly phenotypic methods for use in routine clinical microbiology practice. The present study attempts to evaluate two different methods – colistin broth disc elution and MIC detection by Vitek two in comparison to CLSI approved broth microdilution (BMD) for colistin for Enterobactarales, Pseudomonas aeruginosa , and Acinetobacter baumanii clinical isolates. Methods. Colistin susceptibility of 6013 carbapenem resistant isolates was determined by BMD, Colistin Broth Disc Elution (CBDE), and Vitek two methods and was interpreted as per CLSI guidelines. The MIC results of CBDE, Vitek two were compared with that of BMD and essential agreement (EA), categorical agreement (CA), sensitivity, specificity, very major error (VME), major error (ME) and Cohen’s kappa (CK) was calculated. The presence of any plasmid-mediated colistin resistance (mcr-1, 2, 3, 4 and 5) was evaluated in all colistin-resistant isolates by conventional polymerase chain reaction. Results. Colistin resistance was found in 778 (12.9 %) strains among the carbapenem resistant isolates. Klebsiella pneumoniae had the highest (18.9 %) colistin resistance by the BMD method. MIC of Vitek two had sensitivity ranging from 78.2–84.8% and specificity of >92 %. There were 171 VMEs and 323 MEs by Vitek two method, much more than CLSI acceptable range. The highest percentage of errors was committed for Acinetobacter baumanii (27.8 % of VME and 7.9 % ME). On the other hand, the CBDE method performed well with EA, CA, VME and ME within acceptable range for all the organisms. The sensitivity of the CBDE method compared to gold standard BMD varied from 97.5–98.8 % for different strains with a specificity of more than 97.6 %. None of the isolated colistin resistant organisms harboured mcr plasmids. Conclusion. As BMD has many technical complexities, CBDE is the best viable alternative available for countries like India. A sensitive MIC reported by Vitek two needs to be carefully considered due high propensity for VMEs particularly for Klebsiella spp.


INTRODUCTION
In 2019, 4.95 million deaths were attributed to bacterial anti-microbial resistance (AMR) which is projected to rise further in the future [1].Increasing drug resistance among Gram-negative bacilli (GNB) worldwide has reduced the essential antibiotic arsenal [2].There is rapid expansion of carbapenemase producing Enterobacterales (CRE), Acinetobacter baumanii (CRAB), Pseudomonas aeruginosa (CRPA) and carbapenem heteroresistance in strains like Enterobacter cloacae.These bacteria are often resistant to other available classes of antibiotics.The high prevalence of metallo beta-lactamases presides against the use of less toxic alternatives like ceftazidime avibactam in India.Newer beta lactamase inhibitors and ceftiderocol are costly and not available in India.To counter this issue, older antibiotics like colistin were ameliorated and revived as a last resort toxic alternative.But now there are reports of colistin resistance mediated by chromosomal mutations and plasmid transfer [3].In India, colistin resistant rates have been reported to be as high as 40 % in carbapenem resistant K. pneumoniae and 10 % in carbapenem resistant E. coli [4].These rates seem to be increasing by the day with the added fear of plasmid (mcr) mediated resistance.
The cationic nature of the colistin molecule impedes the suitability of disc diffusion as well as E-test methods for susceptibility testing.Broth microdilution (BMD) performed with colistin sulphate in cation-adjusted Mueller-Hinton broth in polystyrene trays without any other added surfactant is the recommended method for testing [5].But this is often labour intensive in diagnostic settings.Another method which was put forth by Simner et al. [6], was the colistin broth disc elution (CBDE) method, which is easier to perform in a clinical microbiology lab.It is accepted by CLSI for Enterobactarales and Pseudomonas spp.but is not yet approved for Acinetobacter spp.[5].This method challenges a known concentration of test organism with a graded concentration of colistin (1, 2, 4 µg ml −1 ) achieved by eluting 10 µg colistin discs (1, 2, 4 respectively) into 10 ml of Cation Adjusted Mueller Hinton broth.Automated methods like Vitek two (bioMérieux, France) are the most convenient methods and often serve as a backbone of labs.Thus its use to perform colistin susceptibility needs to be evaluated.This present study focused on evaluating the performance of the Vitek two automated system (bioMérieux, France) and CBDE method for organisms of Enterobacterale family, Acinetobacter baumanii and Pseudomonas aeruginosa from ICUs, the harbinger places of colistin resistant bugs.Additionally the mcr 1-5 gene was detected in the colistin resistant isolates in a view to assess the performance of the testing methods in phenotypic detection of mcr based resistance.

Study settings
This study was conducted prospectively in samples received at the Central Laboratory of the Institute of Medical Science and SUM Hospital, Odisha, India from June 2020-June 2022.

Bacterial strains
During this study period, 6013 significant, non-repetitive carbapenem resistant Gram-negative bacilli were collected from various clinical samples of patients from the Intensive Care Unit (ICU).The samples included endotracheal aspirates, bronchoalveolar lavage fluid, blood, pus, wound swabs, tissue biopsy, urine, and sterile body fluids which were collected at bedside by trained personnel following the standard operating procedures.The samples were cultured on blood agar, chocolate agar, maconkey agar and colonies obtained on them were subjected to Gram-staining, motility, catalase and oxidase tests as per routine microbiological techniques.Further identification and sensitivity pattern of these bacteria was performed by Vitek two automated system (bioMérieux, France).Strains with MIC of imipenem >1 µg ml −1 , doripenem >1 µg ml −1 or, meropenem >1 µg ml −1 or ertapenem >0.5 µg ml −1 were considered as carbapenem resistant.On account of being intrinsic resistant Serratia marcescens, Proteus mirabilis, Morganella morganii, Proteus vulgaris, Providencia stuartii and Providencia rettgeri were excluded from the Enterobactarales in the study.Susceptibility testing for colistin was performed for all the isolates by three different methods -CBDE, BMD and Vitek two automated system.

Detection of colistin resistance by Vitek two
Manufacturer's instruction was used for determining colistin Minimum Inhibitory Concentrations (MIC) by commercial Vitek two AST system by using AST-N280 and AST-N281 cards.(bioMérieux, France).

Broth microdilution method for detection of colistin resistance
Broth microdilution with MIC range: 0.25-16 mg l −1 was performed based on ISO-20776-1 protocol [7] for all the 6013 isolates.Colistin sulphate powder (PCT1142-HiMedia Labs) was diluted with sterile distilled water as per the manufacturers' instructions.Then 25 µl each of the two-fold serially diluted colistin solutions was put into the different wells of flat bottomed 96 well microtitre plate (Tarson) and in each row.An overnight 37 °C incubated agar plate of the test organism was used and its colonies were directly suspended in normal saline to obtain turbidity standardized to 0.5 McFarland (1.5×10 8 c.f.u.ml −1 ) which was further diluted 1 in 75 to obtain the standardized inoculums (2×10 6 ).From this, 25 µl of the inoculum was added to each well within 15 min of its preparation and 50 µl of CAMHB media (Himedia Labs M1657) to arrive at the final inoculum in each well (5×10 5 c.f.u.ml −1 ).The inoculated microtitre plates were incubated at 35±2 °C for 16-18 h following which MIC was recorded by unaided eye.Each row contained a growth control to check the viability of the organism and a negative control for checking the broth media for any contamination.

Colistin broth disc elution method
Four tubes of Cation adjusted Muller Hinton Broth were taken for each of 6013 isolates and labelled as 0 (for control), 1, 2 and 4 µg ml −1 and to them one, two and four colistin sulphate 10 µg discs (Himedia Labs) were added respectively.These were vortexed and placed for 30 mins, following which 50 µl of the inoculums adjusted to 0.5 Mc Farland was added to each of these tubes to attain a final concentration of approximately 7.5×10 5 c.f.u.ml −1 .These tubes were further vortexed and incubated at 35 °C in ambient air for 16-20 h.A purity check was performed from inoculums and MIC was read as the lowest concentration of the antibiotic that completely inhibited the growth of the isolate.

Interpretation
CLSI MIC breakpoints of >2 mg l −1 for resistance and ≤2 mg l −1 for intermediate were utilized in the study [5].

DNA extraction and polymerase chain reaction for mobile colistin resistance (mcr) gene
Genomic DNA extraction performed for all 6013 samples was done using the boiling method [8].The extracted DNA was standardized using a nanodot to 40 ng µl −1 .Conventional PCR was done for all these samples using a QIAGEN Taq PCR master mix kit and primers from Eurofins for mcr-1, mcr-2, mcr-3, mcr-4, mcr-5 genes (Table 1).Gel electrophoresis was done to observe the amplification of the desired gene with 1.5 % agarose gel.

Quality control
Manufacturer's instructions were followed for quality control testing of the Vitek two instrument.E. coli NCTC 13846 (colistin MIC-4 µg ml −1 ) and E. coli ATCC 25922 were used in each batch of BMD and CBDE tests as quality control organisms.For each new bottle of procured cation adjusted muller hinton broth Pseudomonas aeruginosa ATCC 27853 with imipenem (MIC-1-4 µg ml −1 ) was used for assessing the media quality.

Statistical analysis
All three methods were conducted in parallel and results obtained by Vitek two and CBDE were compared with the gold standard BMD method.Essential agreement (EA) was the MIC of the test method that was varying by one log2 dilution of reference method expressed in percentage.Categorical Agreement was the similarity of categories (resistant or sensitive) by test and reference BMD method expressed in percentages.Major Error (ME) percentage was identified as a colistin-susceptible isolate (by BMD) that was misinterpreted as a colistin-resistant strain (by test method) divided by true susceptible (by BMD) isolates.Similarly, Very Major Error (VME) percentage was calculated as the number of colistin susceptible strains by the test method (CBDE and Vitek two) which is a colistin-resistant strain by BMD divided by true resistant (by BMD) isolates.Acceptable agreement for was defined as CA≥90 %, EA ≥90 %, VME≤1.5 % and ME ≤3 % as per CLSI guidelines [5].Sensitivity, measuring true positive and specificity measuring true negative portion [9] were calculated.Positive Predictive Value (PPV) is the strains giving resistant test results, which are true resistant (by BMD).Negative Predictive Value (NPV) is the isolates giving sensitive test results, which are true sensitive (by BMD) [9].Test reliability was calculated using Cohen's Kappa (CK) statistics that measure inter-rater agreement was calculated and interpreted as below (Table 2) [10].

Ethical approval
This study was conducted following approval from the Institute's ethics committee via-no.IEC/IMS.SH/SOA/2O22/290.All the samples were received during the purpose of diagnosis in the laboratory.The protocols followed for the tests were within patient care standards.No patient data was disclosed in any form and no diagnostic or therapeutic activity was hampered in this study.
When the results of Vitek two were compared to MICs obtained by BMD the sensitivity ranged from 78.2-84.8% and specificity was more than 92 % in case of all the organisms (Table 3).Thus Vitek two has a chance to report a large number of isolates as false susceptible to colistin while false resistance may not be as high.The PPV also varied from 58.4-78.37 %, while the NPV was between 93.4 and 98.48 %.EA of the Vitek two method varied between 71 % in Klebsiella spp. to 88 % in the case of E. coli, which is not acceptable as per CLSI standards of EA >90 %.Vitek two MIC for colistin also did not meet the CA standards for P. aeruginosa and Klebsiella pnuemoniae (Table 3).There were 171 VMEs and 323 MEs by the Vitek two method of which the highest percentage were committed for Acinetobacter baumanii (27.8 % of VME and 7.9 % ME).
The sensitivity of the CBDE method compared to gold standard BMD varied from 97.5-98.8% for different species with a specificity of more than 97.6 %.The PPV ranged from 76.82-95.09%, whereas the NPV was recorded as more than 99.4 %.The EA of the CBDE method ranged within acceptable limits for all the organisms.EA levels were highest in E. coli by both methods and lowest in Klebsiella pnuemoniae (71.18 %) for Vitek two and Pseudomonas aeruginosa (92.47 %) for CBDE.CA was met for the CBDE method for all the bacteria (Table 3).There were 14 VMEs and 107 MEs by the CBDE method.
The performance of CBDE and Vitek two in reference to BMD is shown in Fig. 1 determining the MICs of colistin.In general, the MIC values by BMD for all of the isolates examined were greater than those obtained by CBDE and Vitek two.Vitek two's correlation with reference MICs was weak, and a 45 degree correlation could not be achieved.Vitek two has a propensity of underestimating MICs for resistant isolates.Cohen's Kappa value suggests the reliability of the tests was strong for CBDE for all the isolates except for that of Klebsiella pneumoniae where it was moderate.None of the samples harboured mcr genes.
The susceptibility testing for colistin is challenging due to poor diffusion of the drug in agar, cationic property of colistin and heteroresistance in MDR organisms [26].There is no agreement between colistin susceptibility testing methodologies [27].BMD, the gold standard method adopted is time-and resource-consuming [18].The techniques frequently used in routine laboratories is disc diffusion and E-test, but have been shown to be inefficient for colistin [28].
Several laboratories depend on automated methods like the automated method Vitek two system.A recent study [18] found solid agreement between BD Phoenix automated system and BMD for colistin susceptibility testing.Thus it is critical to investigate the performance of Vitek two in this direction which we attempted with a large sample size including both colistin sensitive and resistant strains.In the present study when compared to the gold standard; the automated Vitek two approach had unacceptable EA for all the organisms.CA was unacceptable for Klebsiella pneumoniae and Pseudomonas aeruginosa.Previous studies have also shown ambiguous conclusions for Enterobacterales, with some showing a high rate of EA and CA without any VME [14,29].
Both VME and ME of Vitek two were not within acceptable standards in the present study as in other studies [30,31].In other studies VME is as high as 36 % [12,16,32].This high VME and ME may be due to the use of plastics in this automated system which is shown to absorb the drug to its surface.All the phenotypic methods performed better against E. coli in comparison to K. pneumoniae, as in previous studies [12].
All strains fulfilled the EA and CA of >90 % when tested with the CBDE method in the present study.A similar result of 88.9 and 97.8 % CA was noted in studies by Shams et al. [18] and Humphries et al. [33] was also obtained in previous studies.Disc elution is easier to perform, interpret and has higher affordability in comparison to other methods and is a CLSI endorsed test for Enterobactarales and Pseudomonas aeruginosa.Simner et al. [6] found 98 % CA, 99 % EA and no errors with this method.Koyuncu et al. found 99 % CA with 0.5 % very major errors [34].In our study, the EA and CA were acceptable with 14 VMEs and 97 MEs by CBDE.The dissimilarity in results may be caused due to discs produced by different manufacturers.A MIC of two may warrant further testing, particularly for Klebsiella pneumoniae.We found a PPV of >90 % of all bacteria except E. coli (76.8 %) which is comparable to that of Shams et al. [18] who had a PPV of 95.56 % for the CBDE method.But we had a much better NPV for the CBDE test (>93 %) than that of Shams et al. (80 %) [18].Although still not endorsed by CLSI, we found acceptable EA, CA and VMEs while using the CBDE method for Acinetobacter baumanii as well.
Plasmid mediated (mcr) colistin resistance was first detected in 2015 in food industries in China and since then has been reported in various other countries [35].But in our study no mcr 1-5 mediated resistance was detected similar to other studies from India [1,36].Simner et al. recommended that mcr related colistin resistance may be further confirmed by BMD [6].As mcr is not yet prevalent in clinical strains in India CBDE may be adopted as an easy colistin susceptibility testing method.One of the automated platforms BD Phoenix has been proposed as a reliable tool for the detection of mcr mediated colistin resistance [37], but Vitek two results in view of mcr genes need to be evaluated further.
The isolates were taken from one centre and susceptible isolates were much more than resistant strains.Due to a large number of isolates and a lack of adequate manpower triplicate testing of the isolates were not performed.There were no mcr positive strains in the present work and no positive control was run.Sequencing studies of the colistin resistant isolates were not undergone.These are the limitations of the present study.Heteroresistance in colistin and the clinical importance of mcr mediated resistance should be the topics for further research.
The panel of antibiotics that can be administered in case of carbapenem resistant bacteria is limited to a few like ceftazidime avibactam, ceftalozane tazobactam, tigecycline and colistin.As India has a predominance of ndm-1 metallo beta lacatamases, colistin often is the only drug at hand.Thus every laboratory should perform appropriate colistin susceptibility testing.As BMD has many technical complexities, CBDE is the best viable alternative available for countries like India.We also found good agreement between CBDE and BMD in susceptibility testing of Acinetobacter baumanii.A sensitive MIC reported by Vitek two needs to be carefully considered due high propensity for VMEs particularly for Klebsiella spp.
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Comments:
The authors have addressed previous review comments and I have no further concerns.

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

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
Reviewer The triplicate testing of isolates were not undertaken and is discussed as a limitation.
The quality control organisms were used in each batch of tests to ensure rigour.

Line-292
Please cite the ISO protocol Cited as reference 7 Line-123 Line 119-how was the broth cation adjusted?Please add a citation here for the methodology if it has been previously described.
The cation adjusted muller hinton broth was procured from Himedia Labs.(M 1657) Pseudomonas aerugi-nosaATCC27853 strain was used with each new lot of media procured.

Line 122-McFarland is one word corrected Line-128
The relevance of the mcr gene to this research should be explained in the introduction section so that the manuscript can flow.There is no rationale for the PCR testing before Line 129. Ensure that gene names are italicised.

Included in introduction Italics done
Line 99-100 Throughout the text Line 130-Please cite the boiling methodology and whether the DNA extracted was standardized to a certain concentration before being loaded into the PCR reaction.

Citation provided
The DNA concentration was standardized to 40ng/ µl using nanodot.
Reference Throughout the text Line 253-please add this information to the text rather than state a "similar high rate" was seen.Exact statistics will strengthen the narrative of the manuscript The percentages has been added Line-248-251 Line 274-this needs to be reworded as it does not flow well in its current form ""thus we propose…" The line has been omitted.

Line 287: please clarify "Indian Set up
The line has been modified Line 291 Adding more information about the difficulties of testing and access o medicines such as antibiotics in India would also strengthen the narrative of colistin being the only drug "at hand".This is important information for the reader as it justifies the rationale for these experiments and perhaps could be included in the introduction for readers to understand the antibiotic prescribing situation in India presently.

Added in introduction
Line 75-78 The results from the PCR have not been clearly addressed in the discussion

Recommendation: Major Revision
Comments: This research article seeks to evaluate varying methods for in vitro susceptibility testing of colistin in carbapenem resistant Gram negative bacilli.The Vitek system and broth microdilution methods are evaluated and data is presented.The number of isolates used is high in excess of 6000 isolates which allows for robustness and cross sectional analysis of the data.MIC results were obtained after using appropriate statistical analysis.The results demonstrate that colistin resistance is high across the isolates tested and are tabulated appropriately.The figure requires some improvements.More detailed comments are listed below.
The formatting should also be examined for consistency throughout.Ensure binomial nomenclature is used throughout the text, with species names and gene names (e.g.mcr) italicised.Specific comments: introduction 1. Line 70-This sentence could be phrased better to allow for readability.Specifically "shape like a pandemic".The citation used is not current for AMR (O'Neill 2016-more appropriate for diagnostics" and authors should seek to look at papers such as "https://www.thelancet.com/ journals/ lancet/ article/ PIIS0140-6736( 21) 02724-0/ fulltext" to find up to date statistics about AMR global burden.2. Line 72 -change "crushed down" to "reduced" 3. Line 75 "counter" rather than "cater"? 4. Line 76-77.Please add relevant citation to justify the point that there are "reports of colistin resistance" 5.In lines 81-92, could authors please provide citations that cover information regarding the effectiveness of these tests described i.e.PPV, NPV values in diagnostic settings (if available), or highlight data from previous studies that can support the rationale for undertaking these diagnostic evaluations.6. Line 90-Please state the manufacturer of Vitek 2 (Biomerieux) Methods 7. Can authors clarify whether ethical approval was required for use of samples and isolation of bacteria.In Line 98 "varying clinical samples" were collected.Can authors please state where the majority f these samples were isolated from?(i.e.UTI, stool?).How were these isolates isolated in the laboratory and identified/characterised? 8. Please use consistent formatting with antibiotic names-they do not have to be capitalised (e.g Meropenem, Ertapenem).9. Authors sue concentration unit of ug/dl.This is not conventional for MIC testing and we usually use ug/ml.please can authors convert this information into ug/ml.Line 253-please add this information to the text rather than state a "similar high rate" was seen.Exact statistics will strengthen the narrative of the manuscript.23.Line 274-this needs to be reworded as it does not flow well in its current form ""thus we propose…" 24.Line 287: please clarify "Indian Set up" 25.Adding more information about the difficulties of testing and access o medicines such as antibiotics in India would also strengthen the narrative of colistin being the only drug "at hand".This is important information for the reader as it justifies the rationale for these experiments and perhaps could be included in the introduction for readers to understand the antibiotic prescribing situation in India presently.26.The results from the PCR have not been clearly addressed in the discussion.

Please rate the quality of the presentation and structure of the manuscript Satisfactory
To what extent are the conclusions supported by the data?The study aims to achieve novel results by determining the prevalence of colistin resistance among carbapenem-resistant bacterial strains in the local region (Odisha, India), and by comparing the diagnostic usefulness of two test methods against a reference method.The Methods section is generally very good, and presents a scientifically sound and rigorous approach.The research goals are clearly stated and appear to have been met, but the details of the methodology has some omissions that would prevent an independent researcher from fully replicating the study (minor comments are detailed below).The statistical analysis described is appropriate for this study, however an unorthodox change is made by the authors to a standard reporting metric, and I recommend that this is modified before publication (major comments are detailed below).Relevant ethical guidelines appear to have been followed.The Results section is clear and informative, and clearly relates to the methods section.The main findings are presented concisely and effectively.The results provide novel information relating to the prevalence of colistin-resistant organisms in Odisha, India.The majority of the data appear to be interpreted correctly, but I recommend restating the VME and ME data with the more common definition.The colour code in the figure could be explained in the caption to ease understanding.The Discussion places the work in global context and reiterates the need for clinically available methods for detection of colistin resistance.The author's data supports their conclusions.Major comments: Line 110 and 119: information on number of biological replicates is not provided for these tests.This should be revised and the corresponding results restated, particularly as these results appear in the abstract and may be misrepresented for readers who do not note this distinction in the statistical analysis section.Minor comments: The grammar and formatting throughout should be checked.It may be beneficial to include brief details of the tests as described by Simner et al during the introduction, as the article does not introduce these details until the Methods section, and it is difficult for the reader to contextualise without prior knowledge of the CBDE assay.Line 111: requires a citation to this ISO standard and it must appear in the reference list.Line 112: diluent should be named.Line 114: standardised inoculum concentration stated but prior growth conditions absent, please present them in brief.No indication of the media composition for either the inoculum or the BMD assay.Line 130: boiling method details should be stated Figure 1: Colour-code should be stated in figure caption.Figure should be called in the text and accompanied by a text commentary.Line 196: the authors did not find any mcr genes in any isolates, but no data is presented against positive controls for these genes.It is unclear whether the absence of mcr-1-5 could be explained by improper primer selection/PCR conditions.This should be stated as a limitation of the work.

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

Fig. 1 .
Fig. 1.Scatter diagram obtained by plotting MIC obtained by reference BMD method (x-axis) with MIC by test method i.e.Vitek two/CBDE (y-axis).Bold lines represent the breakpoint MICs in micrograms per millilitre.The pink colour boxes are the isolates showing perfect agreement.The essential agreement is highlighted by both pink and white colour boxes. 1 a.MIC of BMD vs Vitek2 for E. coli (Escherichia coli), 1 b.MIC of BMD vs CBDE for E. coli 1 c.MIC of BMD vs Vitek2 for K.pn (Klebsiella pneumoniae), 1 d.MIC of BMD vs CBDE for K.pn 1e.MIC of BMD vs Vitek2 for A.bau (Acinetobacter baumanii), 1 f.MIC of BMD vs CBDE for A.bau 1 g.MIC of BMD vs Vitek2 for P.aer (Pseudomonas aeruginosa),1h.MIC of BMD vs CBDE for P.aer 1i.MIC of BMD vs Vitek2 for E.cl (Enterobacter cloacae), 1 j.MIC of BMD vs CBDE for E.cl.

Table 1 .
List of primers used for the detection of plasmid mediated colistin resistance

Table 2 .
Cohen's Kappa value and their interpretation

Table 3 .
Performance of CBDE and Vitek two in comparison to reference BMD method for detecting colistin resistance 16. Line 110-for robustness, were technical or biological repeats undertaken to ensure rigour?11.Please cite the ISO protocol 12. Line 119-how was the broth cation adjusted?Please add a citation here for the methodology if it has been previously described.13.Line 122-McFarland is one word 14. the relevance of the mcr gene to this research should be explained in the introduction section so that the manuscript can flow.There is no rationale for the PCR testing before Line 129. Ensure that gene names are italicised.15.Line 130-please cite the boiling methodology and whether the DNA extracted was standardised to a certain concentration before being loaded into the PCR reaction.16.Lines 138-153-please check word spacing.Some words are not spaced.Results 17.Line 157 Please use enclosed brackets around 2255.It could be assumed that this is an isolate species number, not the number of samples.18.Line 200, Figure1. Figure legends should be below the figure and should describe the figure without having to refer to the text.Can authors add the appropriate legends to this figure to enable readers to understand the data sets.What is this data showing?(summarize this).Discussion 19. please change "last ray of hope" to "last line antibiotic" 20.Line 245.Please review "A" in eth sentence "A reliable…" 21.Line 251, please italicise E. coli.22.

recommendation and comments https
://doi.org/10.1099/acmi.0.000595.v1.3 © 2023 Scimone A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.Comments to the Authors: This manuscript details an interesting and timely study comparing the effectiveness of the automated Vitek-2 platform and recently developed Colistin Broth Disc Elution methods against the CLSI-recommended Broth Microdilution Assay for the detection of colistin resistance in Odisha, India.In general, the article is well written and an appropriate length.The number of figures and tables is suitable for the data presented.The Introduction identifies the research need sufficiently, and existing literature is reviewed well and key articles from recent years are included.
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?YesReviewer 1