Molecular Detection of Colistin-Resistant E. coli in Village Chickens from Kelantan, Malaysia

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Overview and general recommendation:

In the study, the authors describe the genotypic characterization of the colistin-resistant E. coli isolated from village chickens in Kelantan by using multiple methods such as minimum inhibitory concentration (MIC), antimicrobial susceptibility test. Especially, they extract the genomic DNA and determine the colistin resistance genes (mcr) in E. coli isolates, thus generating the phylogenetic grouping of E. coli isolates. Furthermore, the perform MLST analysis of specific isolates and show 10 different sequence type (ST).

I find the paper is organized in a proper way and the results are well described. And major methods are well described in the manuscript and properly used in the research. The figures are well organized and presented in an appropriate way. I think there is something can be improved.

 

Major comments:

1. How can this research benefit the local people health and how this research will help to improve the local poultry production systems?
2. The author only analyze colistin-resistant E.coli. How about other antibiotics? Please add this part in discussion.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Habiba. et al. This study found E. coli carrying multidrug-resistant genes (including the colistin-resistant mcr gene) in village chickens in Ken Landan, which showed resistance to multiple antibiotics, a finding that shows the threat of multidrug-resistant strains even in village poultry that are considered to rarely use antibiotics. Moreover, the result is technically sounded and worthy to be published in Bacteria.

The following are some comments and suggestions that are given to improve the manuscript:

Comment 1: Why colistin-resistant E. coli emerge in village flocks where antibiotics are rarely used theoretically. What might be the source of this resistance.

Comment 2: What is the mechanism of transmission of mcr genes (especially mcr-1, mcr-4, and mcr-9) in the environment. Why they can persist without direct antibiotic selection pressure.

Comment 3: Analysis of the sources of the different sequence types (STs) detected showed that they came from different countries, which illustrates what problems. What is the implications for the understanding of the global spread of drug-resistant bacteria.

Comment 4: The study found that the phylogenetic groups of E. coli in village flocks were mainly groups C, B2, and E, which differed from the findings in commercial chicken farms in other studies. What this difference means.

Comment 5: How to interpret high MAR index values for all mcr-positive strains in these village flocks in the absence of antibiotic use.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript by Lawal et al reports for the first time the isolation of mcr marker gene for colistin resistance among village chickens in Kelantan, Malaysia.

The manuscript is overall well written and structured. The methodologies are well described and supportive of their observations and conclusions. The introduction outlines well the significance of monitoring the spread of the overall resistance genes, and positions the study to support its significance.

I find the study important as it outlines that no space around the globe is spared when it comes to the antibiotic resistance, and mapping this resistance is important as countries use this information to determine their policies in curbing the spread of antibiotic resistance and the misuse and overuse of antibiotics in general.

I have outlines couple of minor points below, the clarification of which, I think, should strengthen the manuscript.

1. Line 92, please describe clearly how the samples were collected in order to determine the significance of these findings. Were the samples collected from different sites in the village? Was it one village or many villages in Kelantan area? It makes a huge difference, whether these findings came from one village or one spot in Kelantan, or many villages or spots around Kelantan; these answers are important to justify the discussion section. Section 2.1, line 92, refers to “In a previous study…”. Is this a published study, I do not see a reference for it? Please be clearer.
2. Line 92, refers to 26 E. coli samples, however, later the authors talk about 60 E. coli isolates (line 201). How were these E. coli samples isolated? What are the stock cultures. Also, in line 182 it is written “specific isolates” and in Table 1 is written “Sample identification, C5, C6 etc”. It is not clear what “E. coli samples” and E. coli isolates” refer to. Please clarify this. I think by clarifying the method used to collect environmental samples (soil, or faecal, or something else) it will become easier to understand the above and appreciate the conclusions.
3. Line 135, I think the best term to use for the experiments carried out in this section is “Genetic Characterization of E. coli” as the authors have done specific gene identification on the E. coli isolates, not genomic characterizations; the term “genomics” implies they sequenced the entire genomes of these isolates.
4. Section 2.5.1, from my experience this method cane give false positive, did the authors face any issues with this method?
5. Line 146, there is mistake is spelling of “species”.
6. Line 182, the labeling of the isolates (C5, C6 etc) has to be introduced first, otherwise it is confusing.
7. Line 200, I’m perplexed where did 60 E. coli isolates came from. As mentioned above, clarify the issue with the terminology, what refers to “sample” and “isolates”. Line 92, describes 26 E.coli samples.
8. Line 202, talks about 10 out 60 E. coli isolates are mcr positive. These are shown in Table 1 as C samples. However, in the discussion, Line 268, it is written that 4 (not 10) out 19 village chicken samples were found to be positive for colistin-resistant E. coli. This seems to be a BIG discrepancy. Nowhere in the results it is mentioned this piece of information (4/19 village chicken samples). Needs explanation. The line 92 talks about 26 E. coli samples, not 19. Also, in the same line, it is not clear whether colistin-resistant E. coli means phenotypically, and/or genotypically. The authors have brought the point that colistin-resistant E. coli can be only a genotype not necessarily a phenotype (Line 290).
9. Line 217, The sentence that starts in this line is not correct the way it is written as it does not match with the data presented in Table 2. Ceftiofur, shows a 0% resistance rate, not 100%. Please check carefully the data presented on Table 2.
10. Line 228, I don’t see any E. coli strain with MIC below 2microg/mL in Table 1. Please check and correct accordingly.
11. Line 262, please clarify how sample were collected, as it is not clear whether the “village chicken” refers to a single village sampled, or many villages, around Kelantan were sampled.
12. Line 268 to 273, the number here don’t quite make sense, e.g 4/10 village chicken samples, I am not sure where this nr came as it was not mentioned in the Result section. Hence, it is not clear how the following results in lines 269 and 270 (25%) were calculated.
13. Line 277, when was use of colistin in food animal production banned in Malasyia? This information is important to understand the dominance of the mcr genes in the environment.
14. Lines 284 to 288, this paragraph seems confusing. Do you mean that mcr genes facilitate the accumulation of other resistance genes?
15. Start a new paragraph on line 287, put the sentence, “ A strong correlation was …” into a new line. This entire page looks very condensed. Use paragraphs to ease the reading.
16. Same for sentence in Line 297, start a new paragraph with the sentence “ Livestock, particularly…”
17. Line 301, it is not clear what references does “Recent reports” refers to, do you mean references 19, 20 and 24?
18. Line 319, the word “widespread” is not strongly supported from the study, as the authors make reference to only “village chicken samples” without defining/describing how the samples were collected, form one single spot/village or from many spots/many villages in the Kelantan area.

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf