Evaluation of the Antimicrobial Resistance of Different Serotypes of Salmonella enterica from Livestock Farms in Southern Italy

Round 1

Reviewer 1 Report

Dear Editor and Authors, I appreciate for inviting me to review this paper.

 

The paper approaches a subject of great relevance when related to One Health, however some points must be rewritten so that the work does not present incomplete information and is clearer for the readers. In general, the results were not well explored, leaving gaps for the reader.

 

Introduction

The authors claim that antibiotic resistance is primarily attributed to improper and reckless use for animals, but this is not true. The livestock is important for this problem, but the use of antibiotics in humans, incorrect disposal (both veterinary and human) and use in the agricultural also have the same important impact by contributing to antibiotic resistance and permanence of these strains in the environment. It is wrong to assign this responsibility to just one niche. 

The antimicrobial resistance is a One Health problem. Therefore, I believe it would be better for the authors to rewrite this part.

The phrase "Currently, livestock production uses 47 more than 63,151 tons of antibiotics worldwide, and this demand is forecast to rise by 67% 48 in the near future, making this sector the principal antibiotic consumer [6,7]": These references, despite being from 2015, are old. Nowadays it has a more current reference. Furthermore, several countries are already implementing actions to reduce the use of antibiotics in this sector, as recommended by the WHO and OIE Global Action Plan.

 

Material and Methods

It would be better if the authors separated into sub-items:

Experimental design (outline), colletect, analyzes. 

I could not understand wicth was the N (numbert total) of samples collected. 

Was the skin collected on the farm or at the slaughterhouse? 

Did you follow any documents or not? 

What technique is used for Salmonella research? 

It would be better to describe the phenotypic assays first (describe the control used in the MICs) and then to describe the genotypic assays (boil extraction - use a reference; and the PCRs - also include primer references in the table). It is important to inform if positive controls were used for PCRs and what they are.

 

Results

If you improve the material and methods, automatically the result will be improved. First describe the positive N of Salmonella spp. by matrix and after opening this result for the serovars.

I didn't understand table 2. Maybe you could have a complementary file with the raw data, which was the MIC of each isolate and leave the table with the average, but put the N of each serovar. Most items are greater and equal, does this mean that higher concentrations have not been tested? This has to be described (it can be described in the material and methods that the concentrations used are X to X).

 

The work is very important and rich, so the results can be explored further. It does not have the visible result of the PCRs. According to the experimental design, is it not possible to do some kind of statistical analysis? Or just the analysis described? Regardless, the result should be further explored and described.

Author Response

Dear Editor,

 

Please find attached the revised version of our manuscript entitled “Evaluation of antimicrobial resistance of different serotypes of Salmonella enterica from livestock farms in Southern Italy”. We revised our manuscript following reviewers’ comments and answered to all their comments in the dedicated sessions.

We attached a word file with the track changes made to ease your perusal of our manuscript changes.

We hope that this version of the manuscript will be accepted for the publication.

Could you modify Giorgia Schirò’s email address as follow “giorgia.schiro91@gmail.com”? I’ve already changed it in the manuscript as well.

Thank you again for your consideration.

Best regards

Elisabetta Giudice

Department of Veterinary Sciences, University of Messina, Polo Universitario dell’Annunziata, 98168

Email: egiudice@unime.it.

Tel.: +39090676830

 

 

Reviewer #1: Comments to the Author

 

The paper approaches a subject of great relevance when related to One Health, however some points must be rewritten so that the work does not present incomplete information and is clearer for the readers. In general, the results were not well explored, leaving gaps for the reader.

1. Introduction

The authors claim that antibiotic resistance is primarily attributed to improper and reckless use for animals, but this is not true. The livestock is important for this problem, but the use of antibiotics in humans, incorrect disposal (both veterinary and human) and use in the agricultural also have the same important impact by contributing to antibiotic resistance and permanence of these strains in the environment. It is wrong to assign this responsibility to just one niche.

The antimicrobial resistance is a One Health problem. Therefore, I believe it would be better for the authors to rewrite this part.

We’d like to thank the reviewer for this helpful comment. According to that, we’ve improved this section of text.

The phrase "Currently, livestock production uses 47 more than 63,151 tons of antibiotics worldwide, and this demand is forecast to rise by 67% 48 in the near future, making this sector the principal antibiotic consumer [6,7]": These references, despite being from 2015, are old. Nowadays it has a more current reference. Furthermore, several countries are already implementing actions to reduce the use of antibiotics in this sector, as recommended by the WHO and OIE Global Action Plan.

We have changed this sentence with a more recent reference of Tiseo et al, 2020.

2. Material and Methods

It would be better if the authors separated into sub-items: Experimental design (outline), colletect, analyzes.

We thank the reviewer for his/her comment. The authors divided this section into sub-items.

I could not understand wicth was the N (numbert total) of samples collected.

The authors specified in the text the number of samples collected.

Was the skin collected on the farm or at the slaughterhouse? 

The authors have no information about where the skin samples were collected.

Did you follow any documents or not? What technique is used for Salmonella research?

We thank the reviewer for his/her comment. The authors specified in the text the methods used for Salmonella research.

It would be better to describe the phenotypic assays first (describe the control used in the MICs) and then to describe the genotypic assays (boil extraction - use a reference; and the PCRs - also include primer references in the table). It is important to inform if positive controls were used for PCRs and what they are.

We thank the reviewer for his/her suggestion. We modified the text as suggested: we described first the phenotypic assays, for which controls are present in the kit used, and then the genotypic assays for which one gene from each type was sequenced and used as positive control.

 

3. Results

If you improve the material and methods, automatically the result will be improved. First describe the positive N of Salmonella spp. by matrix and after opening this result for the serovars.

 

We thank the reviewer for his/her suggestion. We modified the text as suggested.

 

I didn't understand table 2. Maybe you could have a complementary file with the raw data, which was the MIC of each isolate and leave the table with the average, but put the N of each serovar. Most items are greater and equal, does this mean that higher concentrations have not been tested? This has to be described (it can be described in the material and methods that the concentrations used are X to X).

We thank the reviewer for his/her comment. We have improved table 2 and reported for each isolate: host species, sampling source, MIC. We have also reported the EUKAST breakpoints in red. Moreover, we described in the material and methods the concentrations used.

 

The work is very important and rich, so the results can be explored further. It does not have the visible result of the PCRs. According to the experimental design, is it not possible to do some kind of statistical analysis? Or just the analysis described? Regardless, the result should be further explored and described.

We thank the reviewer for his/her comment and suggestion. We have improved the results sections.

 

Author Response File: Author Response.docx

Reviewer 2 Report

P82: What method did you use to test for Salmonella positivity? How did you select the positive colonies for future identification, selected all, or randomly picked some?

Author Response

Dear Editor,

 

Please find attached the revised version of our manuscript entitled “Evaluation of antimicrobial resistance of different serotypes of Salmonella enterica from livestock farms in Southern Italy”. We revised our manuscript following reviewers’ comments and answered to all their comments in the dedicated sessions.

We attached a word file with the track changes made to ease your perusal of our manuscript changes.

We hope that this version of the manuscript will be accepted for the publication.

Could you modify Giorgia Schirò’s email address as follow “giorgia.schiro91@gmail.com”? I’ve already changed it in the manuscript as well.

Thank you again for your consideration.

Best regards

Elisabetta Giudice

Department of Veterinary Sciences, University of Messina, Polo Universitario dell’Annunziata, 98168

Email: egiudice@unime.it.

Tel.: +39090676830

 

 

 

Reviewer #2: Comments to the Author

 

P82: What method did you use to test for Salmonella positivity? How did you select the positive colonies for future identification, selected all, or randomly picked some?

We thank the reviewer for his/her suggestion. We have reported the requested information in the materials and methods section.

 

Author Response File: Author Response.docx

Reviewer 3 Report

The Authors presented a nice study focused on the problem of antimicrobial resistance in Salmonella enterica collected from farms in South Italy. This kind of regional data is of significant scientific value. The title is consistent with the subject of the manuscript. The appropriate methodology was applied. The whole manuscript has fitting composition. The conclusions are mostly supported by the results. However, some of the aspects of the manuscript need to be corrected:

·         In the chapter “Introduction” there is a lot of very general information about antimicrobial resistance. In the reviewer’s opinion, the paragraph (lines: 34-47) should be more compressed.

·         Also, in the “Introduction”, the Authors should elaborate on how frequently Salmonella isolates harbor antimicrobial resistance genes (AMR). It could additionally underline the importance of the presented results.

·         The sampling process in methodology was described too generally. How many samples were collected from each sample type and farm? How many samples were collected in total?  This paragraph should be rearranged in order to present to the Readers full data on the sampling process and the range of performed surveys.

·         Similarly, more information should be added to the description of the results, to establish the baseline of resistance occurrence. Based on the presented data it is impossible to evaluate the scale of antimicrobial resistance increase and its timeline.

·         Line 113: “Some of the biological samples collected from the selected farms tested positive to for Salmonella spp.” – The Authors should use precise language when describing the results. How many biological samples were positive?

·         The results of sensitivity tests and molecular biology are described very vaguely. Are the results obtained by both methods consistent with each other? The Authors should consider adding a table, that would summarize the results of resistance genes detection by PCR. Because no additional data is supporting the description in the text.

·         Line 187-189 – The Authors should consider adding specific dates from cited publication, as time references for when the increase in antimicrobial resistance started to occur.

·         Line 200-206  – The paragraph is a little disjointed from the article, there should stronger references to the results and research scope.

·         In the Reviewer’s opinion, in the chapter “Discussion”, more focus should be put on discussing the results of resistance detection and its occurrence in South Italy and Europe, rather than the description of some of the resistance mechanisms. This deviates from the main scope of the manuscript.

·         Conclusions should be more compact and to the point.

Comments for author File: Comments.pdf

Author Response

Dear Editor,

 

Please find attached the revised version of our manuscript entitled “Evaluation of antimicrobial resistance of different serotypes of Salmonella enterica from livestock farms in Southern Italy”. We revised our manuscript following reviewers’ comments and answered to all their comments in the dedicated sessions.

We attached a word file with the track changes made to ease your perusal of our manuscript changes.

We hope that this version of the manuscript will be accepted for publication.

Thanks for your consideration,

Best regards

Elisabetta Giudice

Department of Veterinary Sciences, University of Messina, Polo Universitario dell’Annunziata, 98168

Email: egiudice@unime.it.

Tel.: +39090676830

The Authors presented a nice study focused on the problem of antimicrobial resistance in Salmonella entericacollected from farms in South Italy. This kind of regional data is of significant scientific value. The title is consistent with the subject of the manuscript. The appropriate methodology was applied. The whole manuscript has fitting composition. The conclusions are mostly supported by the results. However, some of the aspects of the manuscript need to be corrected:

In the chapter “Introduction” there is a lot of very general information about antimicrobial resistance. In the reviewer’s opinion, the paragraph (lines: 34-47) should be more compressed. Also, in the “Introduction”, the Authors should elaborate on how frequently Salmonella isolates harbor antimicrobial resistance genes (AMR). It could additionally underline the importance of the presented results.

We have improved the paragraph (lines: 34-47) as follows: “Antibiotic resistance is the ability of some microorganisms to survive and multiply in the presence of various antimicrobials and represents a health problem that has afflicted the world population for years [1]. Various bacterial species are naturally resistant, while others acquire, through vertical and horizontal transfer, characteristics that make them resistant to the action of some antibiotics. Antimicrobial resistance is a serius global problem in 21 century, strickly due to several factors linked with the growth and lifestyle of popolation, excretion of incompletely metabolized antibiotics by humans and animals, disposal of the unusued drugs and waste from pharmaceutical processess [2]. The livestock sector constitutes one of many focal points for the evolution and dissemination of antibiotic-resistant bacteria [3]. As a matter of facts, antibiotics are widely used in food-producing animals for therapeutic, prophylactic and growth-promoting purposes [4-6]. In addition, global sale of antimicrobial is in continuously increase: the sale of 93,309 tonnes of antimicrobial is estimated in 2017 and the global sale is expected to rise by 11,5% in 2023 to 104,079 tonnes [7]. Antimicrobial resistance represents a problem of worldwide interest, which including animals and humans. In particular a study found resistance to two or more antimicrobials of 85% in food and animals strains and 77.4% in human strains [8].”

 

 

The sampling process in methodology was described too generally. How many samples were collected from each sample type and farm? How many samples were collected in total? This paragraph should be rearranged in order to present to the Readers full data on the sampling process and the range of performed surveys.

Similarly, more information should be added to the description of the results, to establish the baseline of resistance occurrence. Based on the presented data it is impossible to evaluate the scale of antimicrobial resistance increase and its timeline.

Line 113: How many biological samples were positive?

The results of sensitivity tests and molecular biology are described very vaguely. Are the results obtained by both methods consistent with each other? The Authors should consider adding a table, that would summarize the results of resistance genes detection by PCR. Because no additional data is supporting the description in the text.

We thank the reviewer for his/her comment and suggestion. We have improved the materials and methods and results sections by reporting the required information and have also added Table 3 as suggested.

 

Line 187-189 – The Authors should consider adding specific dates from cited publication, as time references for when the increase in antimicrobial resistance started to occur.

We have added specific dates from cited publication as follow “A previous epidemiological study carried out on Sicilian territory from 1999 to 2002 reported that Salmonella Enteritidis (39%) was the most prevalent serovar followed by S. Typhimurium (16%), S. newport (6%), S. Salamae (5%) and others.”

 

Line 200-206– The paragraph is a little disjointed from the article, there should stronger references to the results and research scope.

We thank the reviewer for his/her comment and suggestion. We have removed the initial sentence of this paragraph.

 

In the Reviewer’s opinion, in the chapter “Discussion”, more focus should be put on discussing the results of resistance detection and its occurrence in South Italy and Europe, rather than the description of some of the resistance mechanisms. This deviates from the main scope of the manuscript.

New references (Di Marcantonio et al, 2022) were added to show the occurrence of antimicrobial resistance in South Italy.

 

Conclusions should be more compact and to the point.

“Consciousness of the consequences of indiscriminate and unnecessary use of antimicrobials is slowly growing but numerous studies still report highly virulent and resistant bacteria in areas of human activity. This study reported the results on the antibiotic resistance profiles of 10 different serotypes of Salmonella spp. in circulation in livestock-related samples in Sicily. Also, the Salmonella serotypes analyzed here exhibited the presence of blaTEM and blaCTX-M I. The results gathered in the current study confirm the scientific evidences available in the literature, according to which, the resistance towards antibiotics is continuously increasing. This represents a worrying perspective since they should be usually used as last chance of therapy against bacterial infections.  As a matter of facts, the current study highlights the need of consolidation of surveillance activities for public health, with the integration of veterinary data with those of clinical origin, and the definition of analysis protocols that provide for precise selection criteria of the serotypes to be subjected to molecular typing tests, since it is neither possible nor reasonable to extend the execution of very large and expensive analysis panels to all isolates. These informations are advocated to implement surveillance systems and disseminate guidelines on the correct use of antibiotics in both human and veterinary medicine also in view of the ever more current holistic approach of One Health concept.”