Antimicrobial Dilution Susceptibility Testing of Erysipelothrix rhusiopathiae According to CLSI Document VET06 Reveals High Resistance against Penicillin G, Erythromycin and Enrofloxacin

Round 1

Reviewer 1 Report

The short manuscript of Hess et al. describes the analysis of the antibiotic resistance of 30 strains of the bacterium E. rhusiopathiae isolated from chickens. The subject is interesting, although in my opinion the study would have deserved to be a little more complete, for example by investigating by sequencing the genes, plasmids and transposons responsible for resistance. Here are some comments: 

1. Were the strains isolated by the authors? No ethical mention is present in the manuscript.

 2. It would be interesting to indicate the individual resistance for each of the strains, not just a compilation. 

3. Several italics are missing throughout the manuscript.

Author Response

Reviewer 1

We thank reviewer 1 for the support to improve the quality of the manuscript with the critical and helpful comments which are addressed below.

Comments and Suggestions for Authors

The short manuscript of Hess et al. describes the analysis of the antibiotic resistance of 30 strains of the bacterium E. rhusiopathiae isolated from chickens. The subject is interesting, although in my opinion the study would have deserved to be a little more complete, for example by investigating by sequencing the genes, plasmids and transposons responsible for resistance. 

The present study aimed to provide phenotypic susceptibility data in regard to E. rhusiopathiae which might be helpful to optimize targeted therapy.  Actually, not only limited availability of data is a problem, but also the application of different methods to determine the susceptibility to antibiotics limits the comparison between studies. Therefore, we applied the method according the CLSI document VET06 which was developed for the purpose of providing guidance to veterinary diagnostic, clinic, or public health microbiology laboratories regarding the performance of standardized susceptibility testing of infrequently isolated or fastidious bacteria. We did not intend to investigate the genetic causes of resistance.

Here are some comments: 

1. Were the strains isolated by the authors? No ethical mention is present in the manuscript.

The strains were isolated by the authors during diagnostics performed at the laboratory at the Clinic. Diagnostics were performed in cooperation with the veterinarians in charge of the respective flocks having agreements for the application of veterinary procedures with the farmers. This information is now included in lines 68-72.

No approval from the authorized ethical commission is necessary in such cases.

2. It would be interesting to indicate the individual resistance for each of the strains, not just a compilation. 

Based on the reviewer’s suggestion we included in Table 1 the individual resistance for each strain.

3. Several italics are missing throughout the manuscript.

We thank the reviewer for spotting this, we revised the manuscript accordingly.

 

Reviewer 2 Report

The manuscript is well prepared and it woud be contribute to the field of antibiotic resistance in poultry.

line 59, E. rhusiopathiae should be italic, also check throughout the manuscript

Table 1, layer hens to laying hens, unified expression in the manuscript

Table 2, in the AMR test experiment, why used these five antibiotics?

Why did not detect the expression of antimicrobial resistance genes?

Author Response

Reviewer 2

We thank reviewer 2 for the helpful comments and the positive feedback. All comments are addressed below and helped to improve the quality of the manuscript.

Comments and Suggestions for Authors

The manuscript is well prepared and it woud be contribute to the field of antibiotic resistance in poultry.

line 59, E. rhusiopathiae should be italic, also check throughout the manuscript

We thank the reviewer for spotting this. We revised the manuscript accordingly.

Table 1, layer hens to laying hens, unified expression in the manuscript

We revised layer hens to laying hens in Table 1.

Table 2, in the AMR test experiment, why used these five antibiotics?

These six antibiotics and their MICs are listed in the CLSI document VET06 to be used for susceptibility tests for E. rhusiopathiae.

Why did not detect the expression of antimicrobial resistance genes?

The present study aimed to provide phenotypic susceptibility data in regard to E. rhusiopathiae which might be helpful to optimize targeted therapy.  Actually, not only limited availability of data is a problem, but also the application of different methods to determine the susceptibility to antibiotics limits the comparison between studies. Therefore, we applied the method according the CLSI document VET06 which was developed for the purpose of providing guidance to veterinary diagnostic, clinic, or public health microbiology laboratories regarding the performance of standardized susceptibility testing of infrequently isolated or fastidious bacteria. We did not intend to investigate the genetic causes of resistance.

 

Reviewer 3 Report

Revision of manuscript poultry-2178645

 

 

Dear Authors,

 

Your manuscript entitled “High antimicrobial resistance of Erysipelothrix rhusiopathiae isolates obtained from poultry against penicillin G, a major substance used for treatment” reports data on antimicrobial resistance in a subset of E. rhusiopathiae isolated from poultry. E. rhusiopathiae is a neglected pathogen and not a lot of studies are available on this topic. For this, the results are interesting and should be shared with scientific communities. The manuscript is well written; the data clearly presented and well discussed.

However, I have some considerations/questions:

1.      Title did not reflect the content of the manuscript; it looks like you found only, or mainly resistance against penicillin G and this is not right, even if penicillin G has a great importance for treatment; indeed, penicillin G resulted more effective than erythromycin and enrofloxacin, for example. Please evaluate an alternative title.

2.      Material and methods section should be divided in sub-sections, like “strains origin, E. rhusiopathiae identification, antimicrobial susceptibility tests” or similar.

3.      Table 1: add year of isolation (even if I suppose that the number after PA is the year).

4.      Lines 59-70 and Table 1: did all the strains come from different farms? did you isolate E. rhusiopathiae from the same farm more time?

5.      The manuscript should be presented as “Communication” because it has the following limitations (but the work is goo!):

a.      The number of strains is limited;

b.      You can not be sure you did not include and test multiple copies of the same strains; they come from different outbreaks, but you did not perform specific test for this (MLST, PFGE, or similar).

c.       To know antimicrobial resistance of strains isolated 20 years ago for can not be “helpful and necessary to optimize a targeted therapy.”, it could be useful to see the resistance trends.

d.      No molecular approach was adopted; for this kind of survey phenotypic resistance evaluation is enough, but your study is not complete.

6.      Carefully check al bacteria and gene names for italic; below I report some of them.

 

·         Abstract

o   Lines 12 and 12: E. rhusiopathiae in italics.

·         Introduction

o   .

·         Material and methods

o   Lines 59 and 12: E. rhusiopathiae in italics.

·         Results

o   .

·         Discussion

o   Lines 168-169: Enterococcus faecalis in italics.

o   Lin 177: gyrA and parC in italics.

·         Conclusions

o   .

 

I sincerely hope that these suggestions will enhance this manuscript. However, if I have made any errors or misinterpretations, I apologize in advance.

Sincerely

The Reviewer

Author Response

Reviewer 3

We thank reviewer 3 for the critical review of our manuscript, the helpful suggestions and comments are all addressed below. They helped to improve the quality of the manuscript.

Dear Authors,

 

Your manuscript entitled “High antimicrobial resistance of Erysipelothrix rhusiopathiae isolates obtained from poultry against penicillin G, a major substance used for treatment” reports data on antimicrobial resistance in a subset of E. rhusiopathiae isolated from poultry. E. rhusiopathiae is a neglected pathogen and not a lot of studies are available on this topic. For this, the results are interesting and should be shared with scientific communities. The manuscript is well written; the data clearly presented and well discussed.

However, I have some considerations/questions:

1. Title did not reflect the content of the manuscript; it looks like you found only, or mainly resistance against penicillin G and this is not right, even if penicillin G has a great importance for treatment; indeed, penicillin G resulted more effective than erythromycin and enrofloxacin, for example. Please evaluate an alternative title.

Based on the reviewer’s comment we revised the title to “Antimicrobial dilution susceptibility testing of Erysipelothrix rhusiopathiae poultry isolates according to CLSI document VET06 reveals high resistance against penicillin G, erythromycin and enrofloxacin”

2. Material and methods section should be divided in sub-sections, like “strains origin, E. rhusiopathiae identification, antimicrobial susceptibility tests” or similar.

We thank the reviewer for this comment, and included now sub-sections accordingly (lines 65, 82, 104).

3. Table 1: add year of isolation (even if I suppose that the number after PA is the year).

As supposed by the reviewer the year is included in the isolates name after PA. We agree with the reviewer the need for an explanation which is now included in the legend of Table 1.

4. Lines 59-70 and Table 1: did all the strains come from different farms? did you isolate E. rhusiopathiae from the same farm more time?

We do not have any data from isolate no. 11, but all other isolates were derived from different farms. We included this information now in lines 72-73.

5. The manuscript should be presented as “Communication” because it has the following limitations (but the work is goo!):

The reviewer’s opinion to present this study as “Communication” is in contrast to the other three reviewers. We also believe that it is suitable as research article.

 

1. The number of strains is limited;

In poultry erysipelas is isolated infrequently, but gained more importance during the last decade due to the increase of birds kept in alternative husbandry systems.  Most antimicrobial susceptibility investigations from E. rhusiopathiae are reported from pigs which often include several hundred isolates. This is not comparable with studies performed on poultry isolates. The only comparable study to the presented was performed on 45 poultry isolates in 2009 (Eriksson et al., 2009).

1. You can not be sure you did not include and test multiple copies of the same strains; they come from different outbreaks, but you did not perform specific test for this (MLST, PFGE, or similar).

The isolates were derived from different outbreaks/different poultry species over several years. We did not see any epidemiological context between them. Furthermore, we found a great heterogeneity in antimicrobial resistance patterns, the 30 isolates spread into 11 patterns. With the presented study we did not intend to investigate phylogenetic relationships between the isolates.

 

1. To know antimicrobial resistance of strains isolated 20 years ago for can not be “helpful and necessary to optimize a targeted therapy.”, it could be useful to see the resistance trends.

Due to the low amount of isolates in some of the years (e.g. 2003, 2011, 2014, 2020 – none in 2005, 2007-2010, 2015, 2018-2019) a trend assessment was not possible. Recent field survey data on E. rhusiopathiae poultry isolates were provided 2009 (Eriksson et al., 2009). Compared to this study, the present data revealed an increased number of penicillin G and enrofloxacin resistant isolates and a comparable high resistance rate to erythromycin. Therefore, we are convinced to contribute to an optimization of therapy.

 

1. No molecular approach was adopted; for this kind of survey phenotypic resistance evaluation is enough, but your study is not complete.

The present study aimed to provide phenotypic susceptibility data in regard to E. rhusiopathiae which might be helpful to optimize targeted therapy.  Actually, not only limited availability of data is a problem, but also the application of different methods to determine the susceptibility to antibiotics limits the comparison between studies. Therefore, we applied the method according the CLSI document VET06 which was developed for the purpose of providing guidance to veterinary diagnostic, clinic, or public health microbiology laboratories regarding the performance of standardized susceptibility testing of infrequently isolated or fastidious bacteria. We did not intend to investigate the genetic causes of resistance.

 

6. Carefully check al bacteria and gene names for italic; below I report some of them.

We revised this accordingly throughout the manuscript.

 

• Abstract
• Lines 12 and 12: E. rhusiopathiae in italics.

Revised accordingly.

• Introduction

o   .

• Material and methods
• Lines 59 and 12: E. rhusiopathiae in italics.

Revised accordingly.

• Results

o   .

• Discussion

o   Lines 168-169: Enterococcus faecalis in italics.

• Lin 177: gyrA and parC in italics.

Revised accordingly.

• Conclusions

o   .

 

I sincerely hope that these suggestions will enhance this manuscript. However, if I have made any errors or misinterpretations, I apologize in advance.

Sincerely

The Reviewer

Reviewer 4 Report

Dear editors and authors:

In this study, the antimicrobial susceptibility of 30 E. rhusiopathiae isolates derived from field outbreaks in poultry flocks towards penicillins, macrolides, lincosamides and fluoroquinolones were evaluated. The results will assist targeted treatments of affected poultry flocks. However, there are still lots of problems. Some suggestions have been listed as follow:

 

1. The experimental method should be briefly described in the Abstract, such as “...by broth microdilution susceptibility testing.”

2. Authors need to indicate the background information and main objective of the study clearer and precisely.

3. The author should explain the reasons for choosing these five drugs in this experiment, and indicate the manufacturer and batch number of each drug.

4. The description of L64-L67 should be put in the results, and adding pictures of typical symptoms.

5. "." should be used in percentages and decimals of the full text, not ","

6. In Table 1, add the year of each sample.

7. The explanations of the table contents cannot be placed directly in the header, and should be listed as table notes separately, especially Table2 and Table3.

8. In the table, it is recommended to use another way to express intermediate and resistance, because italics and bold are difficult to distinguish, especially for numbers.

9. In L118-L125, check the description of Table 2 carefully.

10. There are only 28 E. rhusiopathiae isolates in Table 3, not 30. If 2 isolates are non-resistant to all antimicrobials tested, they should be marked in the table.

11. In Table 3, the specific isolates corresponding to each antimicrobial resistance patterns should be marked , not just numbers.

12. The author should analyze the experimental data from multiple perspectives and mine more conclusions rather than listing the data simply. For example, is the change of resistance pattern correlate with the year? Or for penicillin G, does the MIC increase with the increase of years? These trends can be presented in a variety of forms such as line charts.

13. In the discussion, it was mentioned in many places that the current resistance data was in clear contrast with that before. Can you add specific tables to explain it in the results ?

 

Totally, the research results are reliable, but the presentation of the results is not standardized, and the degree of data mining is not enough. The manuscript needs to be revised and resubmitted.

Comments for author File: Comments.docx

Author Response

Reviewer 4

We thank reviewer 4 for his critical comments and helpful suggestions for the revision of the manuscript. They helped to improve the manuscript. Please find our answers below.

Comments and Suggestions for Authors

Dear editors and authors:

In this study, the antimicrobial susceptibility of 30 E. rhusiopathiae isolates derived from field outbreaks in poultry flocks towards penicillins, macrolides, lincosamides and fluoroquinolones were evaluated. The results will assist targeted treatments of affected poultry flocks. However, there are still lots of problems. Some suggestions have been listed as follow:

 

1. The experimental method should be briefly described in the Abstract, such as “...by broth microdilution susceptibility testing.”

Based on the reviewer’s suggestion we included this information in the abstract (lines 14-15).

2. Authors need to indicate the background information and main objective of the study clearer and precisely.

We revised this accordingly (lines 59-63).

3. The author should explain the reasons for choosing these five drugs in this experiment, and indicate the manufacturer and batch number of each drug.

We followed the guidelines of the CLSI document VET 06 which comprises the testing of the six drugs used the present study. There seems to be a misunderstanding: the susceptibility tests were performed by using a commercially available with antimicrobials pre-coated plate (MICRONAUT-S Lifestock/Equines GP). This information is given in lines 105-108.

4. The description of L64-L67 should be put in the results, and adding pictures of typical symptoms.

We would like to keep this as background information to the isolates in Material & Methods, as none of the other three reviewers regarded this to be revised. The focus of our study is set on antimicrobial susceptibility of the E. rhusiopathiae, not on the presentation of the clinical course of infection. Therefore, we decided not to include pictures of gross pathological lesions.

5. "." should be used in percentages and decimals of the full text, not ","

We thank the reviewer for this comment. The manuscript was revised accordingly.

6. In Table 1, add the year of each sample.

The year of isolation is included in the isolate name. To make this clear we included an explanation in the legend of Table 1.

7. The explanations of the table contents cannot be placed directly in the header, and should be listed as table notes separately, especially Table2 and Table3.

The Tables were revised accordingly.

8. In the table, it is recommended to use another way to express intermediate and resistance, because italics and bold are difficult to distinguish, especially for numbers.

We revised the marking of intermediate and resistant in Table 2, and hope to improve the readability of the data.

9. In L118-L125, check the description of Table 2 carefully.

We thank the reviewer for this comment, and revised line 45-46 accordingly.

10. There are only 28 E. rhusiopathiae isolates in Table 3, not 30. If 2 isolates are non-resistant to all antimicrobials tested, they should be marked in the table.

Based on a comment from reviewer 1 we included now the individual susceptibility data from all isolates in Table 1. Here, the 2 pan-susceptible isolates No. 1 and No. 3 are demonstrated. Therefore, we would like to keep Table 3 as it is comprising the isolates with the resistance patterns.

11. In Table 3, the specific isolates corresponding to each antimicrobial resistance patterns should be marked , not just numbers.

This goes in line with the previous comment. By including the individual susceptibility data of each isolate in Table 1 they can now be related with their resistance patterns.

12. The author should analyze the experimental data from multiple perspectives and mine more conclusions rather than listing the data simply. For example, is the change of resistance pattern correlate with the year? Or for penicillin G, does the MIC increase with the increase of years? These trends can be presented in a variety of forms such as line charts.

Due to the low amount of isolates in some of the years (e.g. 2003, 2011, 2014, 2020) or none in 2005, 2007-2010, 2015, 2018-2019) a trend assessment was not possible. Recent field survey data on E. rhusiopathiae poultry isolates were provided 2009 (Eriksson et al., 2009). Compared to this study, the present data reveal an increased number of penicillin G and enrofloxacin resistant isolates and a comparable high resistance rate to erythromycin.

13. In the discussion, it was mentioned in many places that the current resistance data was in clear contrast with that before. Can you add specific tables to explain it in the results ?

We think that including such tables are appropriate in case of a review study. Therefore, we would like to keep this data in discussion.

 

Totally, the research results are reliable, but the presentation of the results is not standardized, and the degree of data mining is not enough. The manuscript needs to be revised and resubmitted.

 

Round 2

Reviewer 1 Report

The authors responded to my comments.

Reviewer 4 Report

It's the same problem. I think the explanations for light, bold and dark gray in Table 2 should be included in the table notes, not in the header, like Table 3.