Impact of Parenteral Ceftiofur on Developmental Dynamics of Early Life Fecal Microbiota and Antibiotic Resistome in Neonatal Lambs

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes/Can be improved/Must be improved/Not applicable

Thank you again for your constructive comments. We agreed with all the comments, please find the following point by point responses to your valuable comments.

Are all the cited references relevant to the research?

Yes/Can be improved/Must be improved/Not applicable

Is the research design appropriate?

Yes/Can be improved/Must be improved/Not applicable

Are the methods adequately described?

Yes/Can be improved/Must be improved/Not applicable

Are the results clearly presented?

Yes/Can be improved/Must be improved/Not applicable

Are the conclusions supported by the results?

Yes/Can be improved/Must be improved/Not applicable

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: (general comment): The language throughout the manuscript is mostly clear but could benefit from minor proofreading for grammar and style.]

Response 1: [Thank you for pointing this out. We agree with this comment and therefore the whole manuscript has been revised for any minor grammar proofreading using Grammarly, offered by the University of Illinois at Urbana-Champaign.]

Comments 2: (abstract): Improve language for conciseness (e.g., the conclusion should be clear for readers to highlight the outcome of this study for piratical implementation).

Response 2: “Thank you for your valuable comments. The revised version of the abstract has been checked for language and typos. The conclusion has been updated. The outcome and recommendation of the study for piratical implementation have been stated.” Page: 1 (abstract)

Comments 3: (introduction): The author generally provides the sufficient background regarding the role of microbiome for the growth and development in animals. Authors not specifically discus the role of microbiome in lamb or small ruminants. Several factors can promote and inhibit the bacterial colonization including nutrition and others. It was recommended to provide the background to address the objective of this study.

Response 3: Thank you for this piece of valuable information and comments. The introduction has been improved to include a discussion on the role of microbiome in lambs and small ruminants” The edits were as follow

” Beyond the influence of host genetics, microbial colonization in newborn lambs is shaped by maternal factors, early nutrition, and the surrounding environment. At birth, lambs encounter microbes from maternal sources such as the vaginal canal, feces, and teat skin, each providing early exposure to beneficial bacteria, including Lactobacillus and Bifidobacterium. These microbes contribute to the early development of gut structure and immune competence. Colostrum and milk play a central role as well, not only delivering immunological components like antibodies and antimicrobial peptides but also introducing microbial species that help establish the neonatal gut flora. As lambs grow, environmental exposures to microbes from bedding, housing surfaces, and the air further increase the diversification of their microbiota [4]. Nutritional transitions, particularly the shift from milk to solid feed, promote the rise of anaerobic fermenters such as Prevotella and Ruminococcus, key contributors to short-chain fatty acid production and rumen maturation. However, when nutrition is suboptimal, such as with low-quality milk replacers, or when hygiene is compromised, these early colonization processes can be disrupted [5,6]. This may result in microbial imbalances that favor pathogenic species, potentially hindering growth and health outcomes” Page 2, lines 68 to 83

Comments 4 (introduction): Last para of introduction clearly mentions the hypothesis of this study and highlight the impotence, novelty and applicability of this study

Response 4 “thank you for this comment ,:the last paragraph has been updated as follows to highlight the importance, novelty, and applicability of this study as follow

“In this study, we hypothesized that early-life exposure to parenteral antibiotics disrupts the natural progression of gut microbiota development and promotes the emergence of antimicrobial resistance genes (ARGs) in neonatal lambs. Therefore, we investigated the impact of a single dose of Ceftiofur Crystalline-Free Acid (CCFA) on the fecal microbiome composition, diversity, and resistome during two critical developmental stages: the nursing period and the transition to grazing within the first two months of life. We utilized the whole-genome shotgun metagenomic sequencing (WGSS) to characterize microbiota dynamics over time and assess microbial resilience following CCFA intervention” Page 3, line 102-107

Comments 5 (results): Impact of CCFA on the lamb’s body weight: Where is the data. ?????

Response5 : We apologize for missing the data; the data of lamb’s body weight in the material and methods has been reported as mean ± SD.

“On Day 1, female lambs had an average body weight of 3.88 ± 0.19 kg in the control group and 4.09 ± 0.23 kg in the treated group. Male lambs averaged 3.74 ± 0.28 kg in the control group and 4.12 ± 0.30 kg in the treated group. By Day 56, average weights for female lambs reached 26.60 ± 0.97 kg (control) and 27.10 ± 1.21 kg (treated), while male lambs reached 25.30 ± 1.30 kg (control) and 27.60 ± 1.36 kg (treated). Although males tended to be slightly heavier than females by Day 56, no statistically significant differences in body weight were detected between the sexes or treatment groups during the experiment time.” Page 3, lines 112- 118.

Comments 6 (results): Whole genome shotgun sequencing summary: This chapter is not clear to understand, need to revise and improve readability and fluency.

Response 6 : Response: Thank you. We rephrased the summary of sequencing results to make it clearer.

“Whole-genome shotgun metagenomic sequencing generated a total of 24,627,178 raw reads from fecal samples collected across all time points. The sequencing depth per sample was adequate, with a mean of 139,803 reads, a median of 152,468 reads, and a maximum of 207,217 reads. The distribution of reads was comparable between experimental groups, with 12,222,322 reads derived from control animals and 12,079,860 reads from the CCFA-treated group. The quality of sequencing data was consistently high, as indicated by an average Phred quality score of 36.7. In addition, less than 1% of total reads had a Phred score below 22, which confirms the inclusion of minimal low-quality sequence reads and supports the reliability of downstream analyses. Taxonomic classification was performed using the MG-RAST® platform, enabling the annotation of microbial communities across all samples. In total, 28 bacterial phyla and 587 genera were identified, underscoring the microbial diversity present in the gastrointestinal tract of neonatal lambs during the first two months of life”. Page 3, lines 120-132

Comments 7(results): Figure 4 only showed the title of figure, however the detail description of table is important to understand the outcomes of study and improve the understanding of readers and researchers. It is advised to add description of all figures in figure legends.

Response 7: Response: All the figure legends have been edited to include a description of the figures (each figures caption has been updated throughout the manuscript)

Comments 8 (results): Minor language and typing errors should be corrected during thorough reading

Response 8: The whole manuscript has been revised using Grammarly, offered by the University of Illinois at Urbana Champaign

Comments 9 (discussion): Discussion should be enriched by providing the scientific reasons, why CCFA alter the GIT microbial population.

Response 9:: Thank you for your comment; this was also a hidden piece that should be very clear to the reader. The discussion has been revised, and scientific reasons have been added to address why CCFA alters the GIT microbial population.

“Another key finding in this study was the transient impact of a single injection of CCFA on the microbiota of the neonatal lamb gastrointestinal tract. Ceftiofur (CCFA) was selected as the antibiotic of interest in our study because of its widespread use in the livestock industry and its importance as a human therapeutic. It is a third-generation cephalosporin with an extended duration of action, effective against a wide range of Gram-positive and Gram-negative bacteria. Although the main route of its administration is parenteral, its active derivatives are partially eliminated via bile into the intestinal tract, where they can influence gut microbial populations. This biliary excretion enables CCFA to impact intestinal microbiota despite its systemic administration. Sensitive microbial taxa, particularly those involved in early colonization, maybe transiently suppressed. Moreover, the low but sustained presence of the drug due to enterohepatic circulation can prolong its effects in the intestinal lumen and potentially promote conditions that favor the survival of resistant organisms. The longevity of low-level exposure of the gastrointestinal microbial community to CCFA following a single parenteral injection matches the temporal pattern of change observed in the treated lambs in our study”. Page 10, lines 283-294

Comments 10 (material and methods): In abstract author mentioned that they used 16 animals each group contains 8 animals, but in Materials and Methods section author used 24 animals for experiment. Please clarify why.????.

Response 10: We do apologize for this typo mistake. The correct number is 24 lambs (12 in each group), as it is stated in the material and methods. The number of animals has been corrected in the abstract. Page 1 (abstract)

I was working on another paper simultaneously, and the number was mixed up in my head during the abstract revision.

Comments 11 (material and methods): In abstract author mentioned that they used 16 animals each group contains 8 animals, but in Materials and Methods section author used 24 animals for experiment. Please clarify why.????.

Response 11: References were cited as required at specific sites of the material and methods such as

 1: The treated lambs have received a single intramuscular dose of ceftiofur crystalline free acid (CCFAa, Excede®) at a dose of 5.0 mg /kg 24 hrs after birth .

2: Qubit DNA quantifier (Life Technologies, Grand Island, NY, United States)

Page 12, lines 389 and Page 13 line 416

4. Response to Comments on the Quality of English Language

Point 1:

Response 1: The whole manuscript has been revised for any minor grammar proofreading using Grammarly, offered by the University of Illinois at Urbana-Champaign.)

5. Additional clarifications

none