Comparative analysis of clinical breakpoints, normalized resistance interpretation and epidemiological cut-offs in interpreting antimicrobial resistance of Escherichia coli isolates originating from poultry in different farm types in Tanzania
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Introduction. Existing breakpoint guidelines are not optimal for interpreting antimicrobial resistance (AMR) data from animal studies and low-income countries, and therefore their utility for analysing such data is limited. There is a need to integrate diverse data sets, such as those from low-income populations and animals, to improve data interpretation.
Gap statement. There is very limited research on the relative merits of clinical breakpoints, epidemiological cut-offs (ECOFFs) and normalized resistance interpretation (NRI) breakpoints in interpreting microbiological data, particularly in animal studies and studies from low-income countries.
Aim. The aim of this study was to compare antimicrobial resistance in Escherichia coli isolates using ECOFFs, CLSI and NRI breakpoints.
Methodology. A total of 59 non-repetitive poultry isolates were selected for investigation based on lactose fermentation on MacConkey agar and subsequent identification and confirmation as E. coli using chromogenic agar and uidA PCR. Kirby Bauer disc diffusion was used for susceptibility testing. For each antimicrobial agent, inhibition zone diameters were measured, and ECOFFs, CLSI and NRI bespoke breakpoints were used for resistance interpretation.
Results. According to the interpretation of all breakpoints except ECOFFs, tetracycline resistance was significantly higher (TET) (67.8 –69.5 %), than those for ciprofloxacin (CIPRO) (18.6 –32.2 %), imipenem (IMI) (3.4 –35 %) and ceftazidime (CEF) (1.7 –45.8 %). Prevalence estimates of AMR using CLSI and NRI bespoke breakpoints did not differ for CEF (1.7 % CB and 1.7 % CO WT ), IMI (3.4 % CB and 4.0 % CO WT ) and TET (67.8 % CB and 69.5 % CO WT ). However, with ECOFFs, AMR estimates for CEF, IMI and CIP were significantly higher (45.8, 35.6 and 64.4 %, respectively; P <0.05). Across all the three breakpoints, resistance to ciprofloxacin varied significantly (32.2 % CB, 64.4 % ECOFFs and 18.6 % CO WT , P <0.05).
Conclusion. AMR interpretation is influenced by the breakpoint used, necessitating further standardization, especially for microbiological breakpoints, in order to harmonize outputs. The AMR ECOFF estimates in the present study were significantly higher compared to CLSI and NRI.
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Thank you very much for your efforts in applying all suggestions and comments to the manuscript. This work is now suitable for publication. Congratulations!
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Thank you very much for your efforts in including all the reviewers suggestions, the manuscript has undoubtedly improved in quality. However, there some changes that still need to be address: ·Please review the use of italics throughout the manuscript as some are missing (e.g. gene names such as uidA and the et al. abbreviation on the citations need italics) ·The comment of reviewer 2 on the differences between the archived and the stocked samples is still not addressed and the statement is a bit confusing. Please clarify. ·Supplementary tables have been provided, but they are not named appropriately following the platform's guidelines. Furthermore, they are not cited in the results section where it could help the reader's understanding. Also, please provide a Table Legend within the spreadsheets.
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Comments to Author
This manuscript compares 3 different phenotypic antimicrobial testing methods to calculate clinical or microbiological breakpoints of bacterial isolates, using a set of approximately 70 E. coli poultry isolates from various different farms and farm types in Tanzania. The main findings are that ECOFFS breakpoints tend to be higher than clinical breakpoints or the relatively new method of NRI. Also, the authors observe unexpected prevalence of imipenem and ceftazidime resistance in poultry isolates, which they speculate may be related to resistance genes present in the local water reservoirs. Overall, the manuscript is straightforward, well written and easy to understand. However, in some cases the Results section lacks clarity and the Methodology lacks detail - to address these issues I have a list of …
Comments to Author
This manuscript compares 3 different phenotypic antimicrobial testing methods to calculate clinical or microbiological breakpoints of bacterial isolates, using a set of approximately 70 E. coli poultry isolates from various different farms and farm types in Tanzania. The main findings are that ECOFFS breakpoints tend to be higher than clinical breakpoints or the relatively new method of NRI. Also, the authors observe unexpected prevalence of imipenem and ceftazidime resistance in poultry isolates, which they speculate may be related to resistance genes present in the local water reservoirs. Overall, the manuscript is straightforward, well written and easy to understand. However, in some cases the Results section lacks clarity and the Methodology lacks detail - to address these issues I have a list of minor corrections below that the authors should address before publication. Line 86: remove 'the' before Europe Line 117: what is meant by 'wards' here? I only know this term in a hospital context so please clarify. Also not clear if there are 10 wards total, or 10 for each district (presume the latter as this adds up to 800 total but please clarify). Lines 127 and 330: change gram to Gram Line 130: clarify what is meant by 'plain' plates Line 140/141: what is meant by 'one vial is archived' as opposed to 'the other preserved at -80 in glycerol'? What does the archiving entail? The results section lacks clarity. What isolates are used in the susceptibility testing in Table 1 and Fig. 2? Does this refer to the E. coli positive control? If so please indicate. Line 215/216: "In at least one antimicrobial, there was one isolate with a 6mm zone diameter". What do the authors mean? According to Fig. 2 there is at least one isolate with 6mm zone for each of the 4 tested antimicrobials. Line 216/217: "Except for tetracycline, the wild type cut-off (COWT) was lower than CB and ECOFFs". Where can I see this? Or if not visualized, please put between brackets the actual CB and ECOFFS values. Table 1: please indicate how the functional peak was determined. Also clarify the following in Table legend as this is not currently understandable "….in a range of inhibition zone diameters and output by Normalised Resistance Interpretation (COWT)". Fig. 2: enlarge font of the y-axis (values and axis title). Also enlarge values on x-axis (x-axis title font is fine). Fig. 3: is it possible to use different colors to indicate the CB, ECOFFS and COWT dashed lines, to avoid confusion with the colors used for the different antimicrobials? Table 2: why are ECOFFS values used for distinguishing %WT and %R? or should WT be S here? Lines 251-256: In line 254, is ECOFFS prevalence significantly different from both CB and COWT prevalence or only from one of the 2? Should a correction for multiple testing be applied here (because 2 comparisons were done)? What is the difference between the tests described in lines 251-254 and in line 255-256? Line 272: should ECOFF be CB here? As CB and COWT were not significantly different.. Line 273: add "from both COWT and CB" (if this is true; see my comment about lines 251-254 above)
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
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In this manuscript, Maganga et al. present an AMR epidemiology study of E. coli isolates in poultry farms and compare it with various clinical and microbiological AMR breakpoints. The premise of this study seems interesting, as the authors point out indications of a bias in the current standard breakpoints for different antibiotics. However, several concerns have emerged after the review process. Please, address the reviewers’ suggestions and comments thoroughly, especially those concerning: • Lack of clarity in the Results section and availability of the source material information. • Transparency of the methodology. • Preliminary explanations of some key concepts (for example, COwt, functional peak…) to make the text more readable for non-experts • Coherence of the points mentioned in the Discussion section and their …
In this manuscript, Maganga et al. present an AMR epidemiology study of E. coli isolates in poultry farms and compare it with various clinical and microbiological AMR breakpoints. The premise of this study seems interesting, as the authors point out indications of a bias in the current standard breakpoints for different antibiotics. However, several concerns have emerged after the review process. Please, address the reviewers’ suggestions and comments thoroughly, especially those concerning: • Lack of clarity in the Results section and availability of the source material information. • Transparency of the methodology. • Preliminary explanations of some key concepts (for example, COwt, functional peak…) to make the text more readable for non-experts • Coherence of the points mentioned in the Discussion section and their relation with the results. Please, provide a revised manuscript containing all suggestions and a point-by-point response to the reviewers’ comments within 1 month.
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Comments to Author
In this work, the authors identify a number of E. coli isolates from poultry farms and assess their antimicrobial resistance profile for four antibiotics, showing discrepancies on if they should be considered resistant or sensitive depending on the breakpoints (ECOFFS, CLSI, NRI). The premise is valid, since it is true that different standard uses will give different results. However, it is also true that the main standards (ECOFFS and CLSI) are used for different purposes (lines 82-85). Furthermore, I have my concerns on the applicability of using an NRI method as standard, as according to it, an isolate will be resistant or not depending on the sample, a criterion that cannot be applied in the clinic (which is the original point of these standards). Nevertheless, it is obvious that the ECOFFS and …
Comments to Author
In this work, the authors identify a number of E. coli isolates from poultry farms and assess their antimicrobial resistance profile for four antibiotics, showing discrepancies on if they should be considered resistant or sensitive depending on the breakpoints (ECOFFS, CLSI, NRI). The premise is valid, since it is true that different standard uses will give different results. However, it is also true that the main standards (ECOFFS and CLSI) are used for different purposes (lines 82-85). Furthermore, I have my concerns on the applicability of using an NRI method as standard, as according to it, an isolate will be resistant or not depending on the sample, a criterion that cannot be applied in the clinic (which is the original point of these standards). Nevertheless, it is obvious that the ECOFFS and CSLI breakpoints differ by far, so this is debate worth having. From the literature point of view, the reference format seems a bit messy, and should be eventually adjusted to the guidelines of this platform. I would also suggest that the authors improve the fluency of the manuscript and the readability. For example, in the results section, each paragraph should start with the objective of the experiment or an introduction linking to the previous section, and finish with the conclusion of the experiment and its meaning. This is missing in all the section and makes it difficult to read and to keep track of the information shown. I hope my recommendations help improving the quality of this manuscript. Some other minor amendments could be made, but the changes proposed are extensive enough to leave the small details for a future corrected version. Please see the following specific comments. Specific comments L78: in microbiology, usually a wild type is a strain as it is found in nature, no matter what phenotype or genotype it has. Here, this concept is different, but would only be explained several lines below. I would tackle this distinction at this point, or else it might confuse some readers. L82-85: this sentence seems to imply that the EUCAST standards do not have clinical applicability at all. More clarity is needed here. L86: change "the Europe" by "Europe". L89: At this point, please give a clear explanation of how the NRI method works and its applicability. As it is explained, it seems very limited and the results might not be comparable to other samples/laboratories/regions. L90: here the authors mention a distinction between WT and NWT. However, this definition is still different from the general use of this expression in microbiology. Please clarify. L105: change "current" to "present". L118: where in Glasgow and which analyses where performed there? L123: I assume "Caudell" refers to reference 15, however this is not in the reference list. L125: if coliforms were assessed as resistant according to CLSI standards, would this not be biased according to this work? I suggest that the authors give a justification for this or at least a reflection. L133: Please clarify where each experiment was performed. L137: Please explain the standardised protocol for the sake of reproducibility. L145-149: was this performed in the same experimental batch as in the previous section? Either way, if the methodology is the same, the authors can refer to that section rather than repeating. L153: Please explain the standardised protocol for the sake of reproducibility. L167-173: why use this method rather than 16S rRNA sequencing? L191: tigecycline belongs to the same antibiotic family as tetracycline, but it is not analogous to it. Please justify why the authors consider appropriate substituting one for the other. L207: Are the different isolates individually labelled/barcoded? As they are mentioned, the reader cannot know which of them correspond to each result. This information must be made available. L211: Figure 1 is not cited in the text. Furthermore, it merely shows a calibration curve. It would much more appropriate to show the actual qPCR results compared to a negative and positive control. L215-216: This is a clear example of the need to improve clarity. The sentence "In at least one antimicrobial, there was one isolate with a 6 mm zone diameter". These sentence does not offer any information and, as there is no individualised information of the isolates, the reader cannot be able to track it. Also, to the best of my knowledge, 6-7 mm is the usual size of the antibiotic discs used for disc diffusion assays. Does this mean this grew to the edge of the disc? L220: does this issue with the SD mean the results are not reliable? L225: what is the functional peak? L237: how would this compare to the CLSI breakpoints? L251: what is the prevalence estimate for cipro? L253: previously, the authors had mentioned that they would consider breakpoints for tigecycline as they are not available for tetracycline. However, here the authors consider all breakpoints except the one for tetracycline as it is not available. Please clarify this. L272: here ECOFFS should be CLSI breakpoint? L285-286: here the authors reach the idea that NRI interpretation might be affected by the dataset. Can the authors elaborate on how the application of this method can contribute to standardisation? L294-295: speaking of prevalence, the authors consider it high, low or moderate with respect to what? L323: I have my doubts that imipenem can be used informally. As a last resort antibiotic, not only it is restricted, but also rather expensive in comparison to other antibiotics. Anyway, can the authors give a reason why they think this antibiotic is informally used? Would there be any alternative explanation? L327-330: this sentence confuses me. According to my interpretation of Figure 3, all CB and COWT values are below the ECOFFS threshold, making them more restrictive in the definition of a resistant isolate. Can the authors clarify how those isolates could be misclassified as resistant according to ECOFFS in comparison to the other standards? L332-333: The authors should elaborate more on how the differences between the human and ruminant digestive tract relate to this study. L334: hyperefflux and antibiotic resistance are widely known to be linked. There are many more recent works in the literature the authors can refer to give a more comprehensive view of this. L334-338: the authors mention differences in the resistance prevalence in birds from pristine environments with respect to the results of this work, obtained from farm chickens, and justify it by methodological discrepancies. To me, it is very obvious that those two sample groups were not expected to give similar results, and that differences might be affected by the methodology, but I do not think that is the main cause of the discrepancies between these two particular studies.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Very 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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