Review Reports

Comment

Response

Action

1.    The last paragraph is unclear regarding the specific objective of the study. It should be rewritten to clearly define the study aim, as it is currently too general.

This study was designed to investigate the prevalence and antimicrobial resistance profiles of key livestock-associated bacterial pathogens, including Escherichia coli, Salmonella spp., and Staphylococcus aureus, along with their molecular characterization and phylogenetic analysis to assess genetic diversity and potential zoonotic transmission of antimicrobial resistance.

Corrections are made

2.    The title refers to “prevalence,” but prevalence data are not clearly reported. While the detected bacterial species are listed, the manuscript does not indicate the proportion or frequency of each species. If prevalence is mentioned in the title, corresponding quantitative information must be included.

Specie specific data has mentioned in Table#01 with detailed quantitative representation. Percentage prevalence is also mentioned in table #3.

Suggestions have been incorporated.

3.    The selection criterion states: “Due to ever-changing resistance patterns and current obstacles in treatment, Salmonella sp., E. coli, and S. aureus were selected for this Antibiogram study.” If this is the selection criterion, other relevant species, such as Klebsiella sp. and Pseudomonas sp., should be included, as both are listed in the WHO Global Priority List of Antibiotic-Resistant Bacteria under the CRITICAL category (Pseudomonas aeruginosa – carbapenem-resistant, and Enterobacteriaceae including Klebsiella spp.).

We appreciate the reviewer’s valuable suggestion regarding the inclusion of other clinically important pathogens such as Klebsiella spp. and Pseudomonas spp. While these organisms are recognized in the WHO Global Priority List of antibiotic-resistant bacteria, the selection of Salmonella spp., Escherichia coli, and Staphylococcus aureus in this study was based on their higher frequency of isolation in our sample population.

During the study period, the majority of samples received and analyzed were predominantly positive for Salmonella spp., E. coli, and S. aureus, whereas Klebsiella spp. and other species were detected at very low frequencies. Therefore, to ensure statistical relevance and meaningful interpretation of antibiogram data, we focused on the most commonly isolated pathogens.

However, we acknowledge the importance of Klebsiella spp. and Pseudomonas spp., and their inclusion will be considered in future studies with a larger and more diverse sample set.

Comment has been addressed

4.     In Figure 1, bacterial names are not italicized; please correct.

Bacterial names italicized

Correction have been made in figure 01.

5.      It is unclear how 542 samples yielded 850 isolates. How many isolates of the same species were obtained from each plate? If multiple isolates were taken from the same sample, there is a risk of analyzing clones, which could bias results—this must be clarified.

We thank the reviewer for highlighting this important point. The higher number of isolates compared to samples is due to the use of multiple selective and differential culture media for each sample, which allowed the recovery of different bacterial species present within a single specimen. Specifically, samples were cultured on multiple media targeting distinct bacterial groups, resulting in the isolation of more than one organism per sample where applicable. It is important to note that this study focused only on Salmonella spp., Escherichia coli, and Staphylococcus aureus. From the total isolates obtained (n = 831), only representative isolates of these target species were selected for further analysis. Care was taken to avoid duplicate analysis of morphologically identical colonies from the same sample to minimize the risk of clonal bias.

Comment has been addressed with proper description.

6.    All bacterial names throughout the manuscript should be italicized.

Bacterial names have been italicized

Corrections have been made

7.    Section 4.6 (Antimicrobial Sensitivity Test): Include the tested antibiotics and their respective concentrations.

Antibiotics with concentrations have been included

Suggestions have been incorporated in section 4.6 line 8

8.    Indicate whether tests were performed in duplicate or triplicate, and specify the controls used.

All the tests were performed in duplicate under laboratory positive and negative control isolates.

Comment has been addressed in section 4.5 line 10

9.    Section 4.7 (Molecular Identification): Only 14 samples were molecularly identified. How can the authors be confident about the identity of the remaining isolates?

We thank the reviewer for this important observation. Due to resource and cost constraints associated with sequencing, only a subset of representative isolates (n = 14; 5 Escherichia coli, 5 Salmonella spp., and 4 Staphylococcus aureus) was selected for molecular identification. The selection was performed to include representative isolates based on colony morphology, biochemical profiles, and distribution across samples. All remaining isolates were identified using standard microbiological methods, including selective culture, morphological characterization, and biochemical testing, which are widely accepted for routine identification of these bacterial species.

The molecular identification results showed complete concordance with the phenotypic identification, thereby validating the reliability of the conventional methods used for the remaining isolates. We will clarify this selection strategy and its justification in the revised manuscript.

Comment has been addressed

10.   Figures 3 and 4 lack complete legends and require clarification.

Legends have been added and clarification made in on the respective figures.

Comment has been addressed.

11.   Section 2.4 (Multi-Drug-Resistant strains): Clearly define the concept of multidrug resistance (MDR), as it is evolving; e.g., is MDR defined as resistance to more than two antibiotic classes?

Defined MDR in section 2.4

Suggestions have been incorporated in section 2.4 line 01

12.   The statement “Norfloxacin and Colistin were found effective against some MDR strains tested” requires clarification regarding the method used. If Kirby-Bauer disk diffusion was employed, this method is outdated; microdilution should be used instead. Please include in Materials and Methods.

We studied this disk diffusion Methods for the differentiation between susceptible and resistant isolates. However, we didn’t perform any mics against any antibiotics. The suggested technique will be used for MICs, in next study

Comment has been addressed

13.   The concept of MDR must be consistently clarified throughout the manuscript

MDR concept has been successfully discussed throughout the manuscript

Suggestions have been incorporated

14.   It would strengthen the discussion to contextualize the results by comparing them with previous studies conducted in the same country, even from earlier years, to better understand the current AMR landscape

Few recent and relavent studies have been added to compare the results  in the discussion.

Suggestion has been incorporated and reference has been added

1.    The authors should revise the formatting of microorganism names, which must be written in italics according to scientific nomenclature standards.

Formatting revised completely

Suggestions have been incorporated.

2.    It would be valuable to include a discussion on the interaction between the human, animal, and environmental microbiomes (soil), as this perspective is essential within the One Health framework for understanding antimicrobial resistance dynamics.

One health frame work with recently published articles are added in the discussion..

Suggestions have been incorporated.

3.    The table should clearly specify the origin of each sample or the type of sample analyzed, indicating the animal species from which it was obtained.

Table No.01 and 02 has been updated which give an idea about origin of sample.

Suggestions have been incorporated.

4.      Microbiological identification
Although selective and differential media were used, the authors should clearly describe the criteria used to differentiate bacterial species, for example:

·        A. Staphylococcus aureus vs. coagulase-negative Staphylococcus

·        B. Escherichia coli vs. Citrobacter spp.

·        C. Salmonella spp. vs. Shigella spp.

These criteria should be explicitly described in the methodology section.

·       Both are reported pathogenic in nature.

·       IMVIC test/EMB agar

·       On SS agar

·       Also API kit was used

Comment has been addressed section 4.5 line 09

5.    The authors should clarify the criteria used to select the 14 strains subjected to molecular analysis, specifying whether the selection was random or based on specific microbiological or epidemiological criteria.

We thank the reviewer for this important observation. Due to resource and cost constraints associated with sequencing, only a subset of representative isolates (n = 14; 5 Escherichia coli, 5 Salmonella spp., and 4 Staphylococcus aureus) was selected for molecular identification. The selection was performed to include representative isolates based on colony morphology, biochemical profiles, and distribution across samples. All remaining isolates were identified using standard microbiological methods, including selective culture, morphological characterization, and biochemical testing, which are widely accepted for routine identification of these bacterial species.

The molecular identification results showed complete concordance with the phenotypic identification, thereby validating the reliability of the conventional methods used for the remaining isolates. We will clarify this selection strategy and its justification in the revised manuscript.

Questioned have been addressed.

6.    It would be advisable to include additional antibiotics, particularly carbapenems, due to their importance in public health and antimicrobial resistance surveillance.

Carbapenems are not authorized or commonly used in poultry production because they are considered last-resort antibiotics for human medicine.

Explanation have been provided.

7.    The results section reports the detection of several microorganisms; however, the methodology only describes identification procedures for some of them (S. aureus, E. coli, and Salmonella). The methods used to identify the other reported microorganisms (Klebsiella, Shigella, Pseudomonas, Streptococcus, Mycoplasma, Clostridium, and Pasteurella) should be clarified.

We appreciate the reviewer’s valuable suggestion regarding the inclusion of other clinically important pathogens such as Klebsiella spp. and Pseudomonas spp. While these organisms are recognized in the WHO Global Priority List of antibiotic-resistant bacteria, the selection of Salmonella spp., Escherichia coli, and Staphylococcus aureus in this study was based on their higher frequency of isolation in our sample population.

During the study period, the majority of samples received and analyzed were predominantly positive for Salmonella spp., E. coli, and S. aureus, whereas Klebsiella spp. and other species were detected at very low frequencies. Therefore, to ensure statistical relevance and meaningful interpretation of antibiogram data, we focused on the most commonly isolated pathogens.

However, we acknowledge the importance of Klebsiella spp. and Pseudomonas spp., and their inclusion will be considered in future studies with a larger and more diverse sample set.

Explanations have been provided.

8.      Figure 1
Please review the use of italics for microorganism names. Additionally, the 3.9% value should be explained, specifying the criteria used to classify those isolates as different, in order to support the methodological approach.

Named have been italicized.

3.9% isolates include: Klebsiella sp, Shigella sp, Pseudomonas sp, Streptococcus sp, Mycoplasma sp, and Pasteurella sp.
Mainly used Bergey’s reference for this.

Comments have been addressed.

9.      Section 2.3.1. Antibiotic-resistant patterns of Salmonella spp.

Please revise the formatting of microorganism names.

Revised the formatting pattern

Correction have been made

10.   Figure 2, The X and Y axes should be clearly labeled, indicating whether they correspond to antibiotic names or percentage of resistance.

Figure 2 changed.

Suggestion has been incorporated.

11.   Figure 3
Please specify the meaning of the X and Y axes.

Figure 3, clearly mentioned X and Y

Figure has been explained

12.   Section 2.3.2. Antibiotic-resistant patterns of E.coli.

Please revise the formatting of microorganism names

Revised the formatting pattern

Correction have been made

13.   Figure 4
The X and Y axes should be clearly explained.

Figure 4, clearly mentioned X and Y

Figure has been explained

14.   Section 2.3.3. Antibiotic-resistant patterns of S. aureus
Please correct the formatting of the microorganism’s name.

Revised the formatting pattern

Correction have been made

15.   Figures 5, 6, and 7
The X and Y axes should be clearly defined, specifying whether they represent resistance percentages or antibiotic names.

Figure 5,6 and 7 clearly mentioned X and Y

Figure 5,6 and 7 has been explained

·       Please state clearly the prevalence of antimicrobial resistance pathogens across the livestock species sampled, and also state how many samples were collected from each livestock species.

·       Also state the summary of the PCR findings and the phylogenetic analysis results here.

·       Please rewrite the result part of the abstract, it only contains the antibiogram for each bacterial isolate and no mention of the livestock spp.

The majority of the pathogens isolated were Salmonella spp. 417/831 (50.2%), followed by E. coli 336/831 (40.4%), and Staphylococcus aureus 43/831 (5.17%).

(We have to explain this in abstract)

Please describe the burden of AMR among livestock in Pakistan to provide a better understanding of the problem and justify this study.

Few studies have been discussed in discussion and introduction portion

Suggestions have been incorporated.

Please state the study design used in this study.

we have isolated and identified the organisms in the disease of poultry, rumants and large animals secondly, to identified the resistance to each isolate against different antibiotics by disk diffusion methods.

Comments have been addressed. In intro para 4 line 01

·       Please state clearly what samples were taken from live animals and what samples were taken at postmortem.

Live animal: Swabs, blood, urine stool
Dead: organs after post mortem

Comments have been addressed in table 02.

Please explain in detail the sampling procedure.

The sampling procedure was carried out in accordance with internationally recognized guidelines for animal health surveillance and disease investigation, particularly those recommended by the World Organization for Animal Health (WOAH, formerly OIE).

Explanation have been made.

Please state if a positive control was used to standardize the procedure.

Confirmed positive and negative samples from laboratory were used as control.

Comments have been addressed. In section 4.5 line 11

·        Please, what is the rationale for taking only 14 samples, and what criteria were used to select them?

We thank the reviewer for this important observation. Due to resource and cost constraints associated with sequencing, only a subset of representative isolates (n = 14; 5 Escherichia coli, 5 Salmonella spp., and 4 Staphylococcus aureus) was selected for molecular identification. The selection was performed to include representative isolates based on colony morphology, biochemical profiles, and distribution across samples. All remaining isolates were identified using standard microbiological methods, including selective culture, morphological characterization, and biochemical testing, which are widely accepted for routine identification of these bacterial species.

The molecular identification results showed complete concordance with the phenotypic identification, thereby validating the reliability of the conventional methods used for the remaining isolates. We will clarify this selection strategy and its justification in the revised manuscript.

Explanation have been provided.

·        Why were the selected samples not taken across animal species? These 14 isolates can be argued to have been taken from a single animal host, which will not give a true reflection of other animals. Please clarify.

Our main focus was on poultry as majority samples were obtained from poultry region, though few samples were from other species as well.

Explanation have been provided.

·        Please state whether the agarose Gel was stained with a dye, if yes, state the name of the dye.

We used Ethidium Bromide which is an intercalating dye.

Comments have been addressed.

·        Also describe the phylogenetic analysis method

NJ method (described in the paper)

Comments have been addressed.

Statistical Analysis

The comparison done was just for the isolated bacterial pathogens. This result won’t make much sense unless it is corroborated with the corresponding animal (host) from which the isolates were recovered.

Statistical analysis was done on bacterial isolates rather than species because our study main focus is on isolates.

Comments have been addressed.

Result

2.1 Prevalence of bacterial pathogens in animals:

·        Please relocate the first paragraph to the methodology section (Study design).

·        The second paragraph did not say anything about the prevalence of bacterial pathogens in animals from this study. I suggest you please rewrite this section by providing prevalence either in percentages or fractions, or both.

The paragraph is relocated to material & methods.

Suggestions have been incorporated.

In the methodology and also the opening paragraph of this section, you stated the total samples as 542, but for the calculation of pathogen occurrence, 885 was used as the denominator. Please clarify this.

We thank the reviewer for highlighting this important point. The higher number of isolates compared to samples is due to the use of multiple selective and differential culture media for each sample, which allowed the recovery of different bacterial species present within a single specimen. Specifically, samples were cultured on multiple media targeting distinct bacterial groups, resulting in the isolation of more than one organism per sample where applicable. It is important to note that this study focused only on Salmonella spp., Escherichia coli, and Staphylococcus aureus. From the total isolates obtained (n = 831), only representative isolates of these target species were selected for further analysis. Care was taken to avoid duplicate analysis of morphologically identical colonies from the same sample to minimize the risk of clonal bias.

Clarifications have been made.

2.3. Antibiotic-resistant patterns in pathogens isolated from animals:

·        The first paragraph should also be moved to the methodology as it describes the rationale for the selection of the antimicrobials.

·        Was a questionnaire used in this study? How did you come about with the following assertion: “Among these antibiotics, Florfenicol, Flumequine, and Enrofloxacin are commonly used against bacterial infections in domestic and farming animals, like cows, poultry, in aquaculture, and others. However, farmers and veterinarians also use most broad-spectrum antibiotics, recommended in human infections”. If not, move to methodology and also provide a reference for this claim in the study state or country.

·        Please provide the resistance profile across animals sampled.

·        3.1 to 2.3.3, please rename the subheadings as “Antibiotic resistant profile”, not pattern.

·        Please provide the antibiotic-resistant pattern for this work. This will help to see if similar patterns of resistance exist in the study area and across the animals sampled.

2.4 Multidrug-resistant strains:

·        Please rewrite this section and present it in tables.

·        Also, present the result for the multiple antibiotic resistance index (MARI), which is a very important finding in this type of work. The index will guide and tell the readers what is the level of abuse or misuse of antibiotics in the environment.

Paragraph shifted to MM and Reference is inserted

Multidrug-resistant bacteria are bacteria that are resistant to three or more classes of antimicrobial drugs, making them hard to treat. MDR bacteria have seen an increase in prevalence in recent years and pose serious risks to public health.

Multidrug-resistant (MDR) bacteria are resistant to three or more classes of antimicrobial drugs, making treatment difficult. The prevalence of MDR has become a serious problem to treat man and animals. The Multiple Antibiotic Resistance Index (MARI) is widely used as a surveillance tool for monitoring the prevalence of multidrug-resistant (MDR) strains and the evaluation of appropriate treatment, particularly with  Salmonella, E. coli, and Staphylococcus aureus strains. MARI was found to be 79.7% of Salmonella, including all MDR strains (n = 332); 55.59 of E. coli (n=189), and 88.37% of Staphylococcus aureus (n=39) with indexes greater than 0.2.

Inserted at appropriate place

Suggestion has been made

·        Table 3 should include one additional column for animal species so the reader can understand where each strain was isolated from.

Thank You for the suggestion, we have added in paragraph before the table to make it clear

Suggestion has been incorporated

·        Figure 8, please label the gel image correctly (the calibration of the molecular DNA ladder, the corresponding band size, etc).

Figure has been modified

Suggestions have been incorporated.

Figure 9 did not show clustering of the said E. coli strains from the previous study, but rather the study isolates show clustering together, inferring the same ancestry.

The isolates collected in this study have “PX” initials which are thoroughly distributed in the tree suggesting different ancestry

Clarifications have been made.

Also, why was 100 bootstrapping used instead of 500 or 1000? Please note that the higher the bootstrap, the more reliable the result

Your suggestion is highly appreciated but due to limited resourcing we could only manage 100 bootstrap.

Clarifications have been made.

The Latin names of bacteria should always be written in italics. The authors should carefully review the entire manuscript and correct this consistently. Errors such as “S. Aureus” should be corrected to “S. aureus”.

Corrections have been made carefully.

Suggestions have been incorporated.

The names of antibiotic active substances should not be capitalized

Corrections have been made carefully.

Suggestions have been incorporated.

“E. coli is the main culprit in AMR list and become the primary indicator to detect AMR.” Why? This statement requires explanation, as it currently appears unsupported for the reader

E.coli Presence in almost every environment food waster animal that’s why it is consider to bre the common indicator in AMR. REFERENCE?

Explanation have been given.

“Resistance profiles for S. aureus vary significantly by host” – this also requires explanation. What are the main reasons for this?

Reasons have been addressed.

Explanation have been given introduction

“These organisms then spread in poultry and environments, threatening both public health and food safety” – this should be placed within a One Health context.

Thank You for your valuable suggestion, already discussed in One health

Suggestion has been incorporated.

Figure 1: Bacterial species names should be written in italics. In the case of Escherichia coli, the second part should not be capitalized.

Corrections have been made carefully.

Suggestions have been incorporated.

The use of the expression “mainly poultry” is not appropriate. Were results from different animal species evaluated together? At a minimum, results should be analyzed separately by animal species, or the authors should clearly justify why a combined analysis was performed.

Our institute is  focused on poultry industry that’s why we received more samples from this sector though we have also received samples from other species.  

Explanation have been given

Figure 3 and Figure 4: Why are there ellipses (“…”) after the names of the active substances?

Figures have placed again after correction

Corrections have been made

Figure 5: The figure legend should clearly explain what is shown. Currently, it is not described what the numbers on the horizontal axis represent. This also applies to Figure 6.

Figures have placed again after correction

Corrections have been made

“16S bacterial identification” – did the authors mean 16S rRNA?

Yes 16s rRNA. We have added the proper term in the text.

Explanation have given

“Pakistan is also facing drug-resistant problems…” – What measures is Pakistan taking to address this issue? What regulatory or policy actions are in place?

National Action Plan for AMR 2024-2028 (NAP 2.0) is a comprehensive guideline/roadmap for the next five years to catter against this anitibiotic resistance problem. Our study followed the guideline of NAP 2.0.

“switched to alternative methods like the use of effective vaccines” – this represents only one aspect. There are several other alternative approaches that should be mentioned to provide a more comprehensive overview.

Following explaination has been added in discussion section“Furthermore, other methods may be explored as alternatives to antimicrobials, such as phages, probiotics, antibodies, etc”.

Addition have been made to justify

The Discussion lacks a proper consideration of the study’s limitations, which should be addressed in detail

The following lines have been added in the discussion section, focusing the limitation of this study “Continues funding, Animal proper history un availability, sample transportation and unhygienic condition of farms and utilities, training of farmers were not suitable”.

Explanations have been given.