Acinetobacter baumannii Sampled from Cattle and Pigs Represent Novel Clones

ABSTRACT Acinetobacter baumannii is a very important human pathogen. Nonetheless, we know very little about nonhuman isolates of A. baumannii. Here, we determine the genomic identity of 15 Scottish cattle and pig isolates, as well as their antibiotic and virulence genetic determinants, and compare them with 148 genomes from the main human clinical international clones. Our results demonstrate that cattle and pig isolates represent novel clones well separated from the major international clones. Furthermore, these new clones showed fewer antibiotic resistance genes and may have fewer virulence genes than human clinical isolates. IMPORTANCE Over the last decades, huge amounts of information have been obtained for clinical isolates of A. baumannii and the clones they belong to. In contrast, very little is known about the genomic identity and the genomic basis for virulence and resistance of animal isolates. To fulfil this gap, we conducted a genomic epidemiology study of 15 Scottish cattle and pig isolates in the context of almost 150 genomes belonging to the main international clones of A. baumannii. Our findings show that these animal isolates represent novel clones clearly different from the major international clones. Furthermore, these new clones are distinct in nature considering both antibiotic resistance and virulence when compared with their human clinical counterparts.

these isolates is interesting, a main criticism to the Manuscript is that sequencing is limited to only 16 isolates, of which one was discarded from analysis due to contamination. The 15 isolates were genetically compared to a collection of 148 human-related A. baumannii isolates. Results highlighted that animal isolates i) formed separate clusters genetically distant from human isolates, and ii) showed a reduced number of antimicrobial resistance and virulence genes compared to human isolates. I have some minor suggestions to improve the quality of the manuscript. 1. Please submit whole-genome sequencing data for the 15 A. baumannii isolates to a publicly available repository of high throughput sequencing data (i.e., NCBI SRA or ENA). 2. Please consider changing the title to "Acinetobacter baumannii sampled from Scottish cattle and pigs represent novel clones" to better represent the main focus of the study 3. Lines 32, 43-44, 150-152. Please change the term "lineages" with "clones". 4. Line 45. Please spell "antibiotic resistance and virulence genes". 5. Lines 33-34. Please change to "these new clones showed less antibiotic resistance and virulence genes". 6. Line 40. Please change "to alleviate this" with "to fulfil this gap". 7. Line 55. Please clarify what "natural home" refers to or rephrase. 8. Line 66. It is unclear from which site the 16 strains were isolated. Please specify. 9. Line 83. Please provide additional information on the 148 A. baumannii isolates selected for comparison, especially isolation sites as "human-related" is too generic. Data can be provided as supplementary material. 10. Lines 86-88. Which is the size of pan and core genome? Please express the 759 concatenated genes as proportion of core genome. 11. Line 91. It is unclear if assembled genomes or raw reads were screened against CARD. Please specify. The same applies to the virulence factor analysis. 12. Lines 139-141. Please specify how the 15 isolates from humans were selected for virulence factor analysis, especially because this selection could have biased the following statistical analysis. 13. Lines 150-152. Please change the term "lineages" with "clones". 14. Reference 15 is incomplete. Please revise. 15. Figure 2 legend. How distances between isolates (top tree) and genes (side tree) were calculated? Please explain what the dashed line on the side tree refers to. 16. Please revise Affiliation no. 3, which at present is "Birmingham, UK".
Reviewer #3 (Public repository details (Required)): The genome sequencing data need to be deposited and accession provided.

Reviewer #3 (Comments for the Author):
The study is experimentally/methodologically solid. I only have two points of criticism: 1. The genome sequencing data need to be deposited and accession provided. 2. The in silico analysis of virulence factors does not justify statements such as "These new lineages have less...virulence..." in the abstract. Rather, it is as written at the end of the manuscript that "...these data suggest that the animal isolates may have fewer virulence factors than human clinical isolates." Staff Comments:

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Reply: We sincerely thank both reviewers for the time invested in reviewing our manuscript and their constructive comments. The lines mentioned in the answers refer to the Mark-Up copy of the manuscript.
Reviewer #2 (Public repository details (Required)): Whole-genome sequencing data for the 15 Acinetobacter baumannii isolates should be submitted to a publicly available repository of high throughput sequencing data (i.e., NCBI SRA or ENA).
Reply: We thank both reviewers for noticing this. We have submitted the 16 isolates to the NCBI. The BioProject number has been included in the "Data availability" section at the end of the manuscript and the BioSample ID for each isolate have been included in Supplementary  Table 1.

Reviewer #2 (Comments for the Author):
In the paper entitled "Acinetobacter baumannii sampled from cattle and pigs represent novel clones", Authors sequenced and analysed A. baumannii isolates collected from Scottish cattle and pigs. While the rationale of studying the genetic background of these isolates is interesting, a main criticism to the Manuscript is that sequencing is limited to only 16 isolates, of which one was discarded from analysis due to contamination. The 15 isolates were genetically compared to a collection of 148 human-related A. baumannii isolates. Results highlighted that animal isolates i) formed separate clusters genetically distant from human isolates, and ii) showed a reduced number of antimicrobial resistance and virulence genes compared to human isolates. I have some minor suggestions to improve the quality of the manuscript.
Reply: We do thank the reviewer for taking the time to carefully review our work. Her/his comments about our work have been included in the revised version of the manuscript.
1. Please submit whole-genome sequencing data for the 15 A. baumannii isolates to a publicly available repository of high throughput sequencing data (i.e., NCBI SRA or ENA).
Reply: We thank both reviewers for noticing this. We have submitted the 16 isolates to the NCBI. The BioProject number has been included in the "Data availability" section at the end of the manuscript and the BioSample ID for each isolate have been included in Supplementary  Table 1. 2. Please consider changing the title to "Acinetobacter baumannii sampled from Scottish cattle and pigs represent novel clones" to better represent the main focus of the study Reply: The size of the pangenome is 15,374 gene families and the core genome is 1588 gene families. The 759 non-recombinant families represent 47.8% of the core genome; this last point has been added to the manuscript (see line 105).
11. Line 91. It is unclear if assembled genomes or raw reads were screened against CARD. Please specify. The same applies to the virulence factor analysis.
Reply: Thanks for noticing this. This has been clarified.
12. Lines 139-141. Please specify how the 15 isolates from humans were selected for virulence factor analysis, especially because this selection could have biased the following statistical analysis.
Reply: These 15 isolates represent the total available in the VFanalyser tool. They represent 6 different MLST STs, with a bias towards ST1 (3 genomes) and ST2 (8 genomes), the most widespread clinical STs. Text to this effect has been added to the manuscript (lines 173 -175).
Reply: Thanks for the suggestion. We have replaced "lineages" with "clones" 14. Reference 15 is incomplete. Please revise.
Reply: Thanks for noticing this. We have revised reference 15. 15. Figure 2 legend. How distances between isolates (top tree) and genes (side tree) were calculated? Please explain what the dashed line on the side tree refers to.
Reply: Thanks for the suggestion. For clarity sake, we have simplified the figure, taking out the clusterings on the top and the right of the figure. The clusterings were according to gene presence and absence (right) and strains (top) but they are not relevant to the points that me make in the manuscript.