Pharyngeal Communities and Antimicrobial Resistance in Pangolins in Gabon

ABSTRACT Wildlife can be a reservoir and source of zoonotic pathogens for humans. For instance, pangolins were considered one of the potential animal reservoirs of SARS-CoV-2. The aim of this study was to assess the prevalence of antimicrobial-resistant species (e.g., extended-spectrum β-lactamase [ESBL]-producing Enterobacterales) and Staphylococcus aureus-related complex and to describe the bacterial community in wild Gabonese pangolins. The pharyngeal colonization of pangolins sold in Gabon (n = 89, 2021 to 2022) was analyzed using culture media selective for ESBL-producing Enterobacterales, S. aureus-related complex, Gram-positive bacteria and nonfermenters. Phylogenetic analyses of ESBL-producing Enterobacterales was done using core-genome multilocus sequence typing (cgMLST) and compared with publicly available genomes. Patterns of cooccurring species were detected by network analysis. Of the 439 bacterial isolates, the majority of species belonged to the genus Pseudomonas (n = 170), followed by Stenotrophomonas (n = 113) and Achromobacter (n = 37). Three Klebsiella pneumoniae isolates and one Escherichia coli isolate were ESBL-producers, which clustered with human isolates from Nigeria (MLST sequence type 1788 [ST1788]) and Gabon (ST38), respectively. Network analysis revealed a frequent cooccurrence of Stenotrophomonas maltophilia with Pseudomonas putida and Pseudomonas aeruginosa. In conclusion, pangolins can be colonized with human-related ESBL-producing K. pneumoniae and E. coli. Unlike in other African wildlife, S. aureus-related complex was not detected in pangolins. IMPORTANCE There is an ongoing debate if pangolins are a relevant reservoir for viruses such as SARS-CoV-2. Here, we wanted to know if African pangolins are colonized with bacteria that are relevant for human health. A wildlife reservoir of antimicrobial resistance would be of medical relevance in regions were consumption of so-called bushmeat is common. In 89 pangolins, we found three ESBL-producing Klebsiella pneumoniae strains and one ESBL-producing Escherichia coli strains, which were closely related to isolates from humans in Africa. This points toward either a transmission between pangolins and humans or a common source from which both humans and pangolins became colonized.

investigated with 3 ESBL-producing K. pneumoniae and 1 ESBL-producing E. coli being described. Having investigated 89 animals and 439 isolates, the sample size is one big strength of this study. However more emphasis could have been given to the species found in the samples investigated, as studies on bacterial communities in pangolins' pharynx are lacking. Additionally, the discussion could benefit from further discussing public health risks (including AMR dissemination) associated with dealing with wild pangolins, in my perspective.
Please see my comments below: a) Generic comments -Title: Please consider rephrasing the title to better reflect the study conducted (bacteria in pharyngeal swabs and selective media) -Importance: It may be relevant to mention the role of wildlife as potential reservoirs of resistant bacteria and give more emphasis to AMR. -Throughout the manuscript the authors use "sp." Can the authors confirm if they mean species as plural "spp.", please? b) Specific comments Abstract: Line 32: Can the author use "Gram" instead of "gram", please?
Introduction: -Can the authors add more information on the importance of pharyngeal bacteria and how these bacteria can/may spread to humans, please? -Can the authors give more emphasis to the AMR issue, please? The study design of this study has been tailored by the SARS-CoV-2 study however the authors can focus more on AMR. Lines 59 and 60: Please consider using "(...) viral genomes with a nucleotide identity ranging from 85.5% to 92.4% were detected (...)" Line 64: Can the authors use "extended" instead of "Extended", please? Line 65: Can the authors replace "unresolved" with "unknown", please? Line 76: Can the authors use "public health", please? Lines 75-77: The authors mention "Therefore, from a public-health perspective, there is reasonable interest in having a clearer picture of bacterial colonization in African wildlife". Can the authors add a sentence explaining the relationship between human population and pangolins/wildlife, please? Line 80: Can the authors consider using "project" instead of "investigation", please? Line 82: Can the authors remove "ancillary", please? Line 83: Can the authors please add "(...) and S. aureus-related complex in the pharynx of pangolins, and to (...)"?

Methods:
Line 87: Can the authors confirm if this refers to ethical approval, please? Line 97: Can the authors add "(...) were included in this study (...)", please? Line 105: Can the authors clarify if the cotton tips were sterile, please? Line 107: Can the authors clarify for how long these samples were stored at 4C, please? Lines 110 and 111: Can the authors please confirm if the samples were sent at room temperature, please? Additionally, how long did it take on average? Lines 113 to 118: Can the authors confirm if only selective media chromID ESBL, cetrimide agar and colistin-nalidixin agar were used, please? Line 117: Can the authors use "cetrimide", please? Line 118: Can the authors confirm how many isolates were identified by MALDI-TOF, please? Can the authors also add some information on the quality of the identification, please? Line 119: Can the authors confirm if 4.2 is the version, please? Lines 122 to 131: Can the authors confirm if they took into consideration the intrinsic resistance of some species when performing the AST, please? Lines 122 to 134: Can the authors add references, please? Line 139: Can the authors add the number (n=), please? Line 142 and 145: Can the authors add a reference, please? Line 152: Can the authors expand/justify this choice, please?
Results: Line 164: Can the authors clarify why 89 out of 100 pangolins were included in this study, please? Line 170: Can the authors use ""adult"" or "adults", please? Line 176: The authors mention that 439 isolates were included. Out of how many? It could be useful to provide a list of all species/genus isolated in this study (potentially as supplemental material). Lines 178 to 180: Can the authors clarify if they mean antimicrobial susceptibility rates or resistance rates, please? Line 181 to 182: Can the authors consider " Resistance rate against piperacillin in Pseudomonas putida isolates were up to 28%" Line 189: Can the authors clarify how many E. coli and Klebsiella isolates were found in this study, please? Can the authors also clarify if the selection of the 4 isolates for WGS was based on the ESBL phenotype provided by Vitek2, please? Lines 198 and 202: Can the authors add "the" before "UK", please? Lines 209 to 211: Can the authors consider using "and the cgMLST allelic distance between the two isolates was 414 different alleles (excluding a closer relationship) using the Enterobase E. coli cgMLST scheme", please?
Discussion: Line 225: Can the authors remove "is", please? Line 239 to 244: Can the authors please consider other limitations including the number of isolates subjected to WGS, testing limitations associated with using selective media, storage/transport, non-culturable bacteria etc.? Additionally, can the authors discuss the public health risks associated with dealing with wild pangolins. Can pangolins contact with humans and get AMR isolates from humans due to management practices, food habits etc.? Isolates were mainly susceptible which is an important finding that can be further discussed.
Tables: Line 355: Can the authors please confirm if the issues with table 1 are due to formatting at editing stage? Can the authors provide an improved version of the table, please? Line 356: The authors mention that 439 isolates were investigated in this study however in Table 1, 465 isolates are referred. Can the authors clarify this, please? Lines 356 to 357: Can the authors clarify the following, please? The authors mention that for some species, it was not possible to perform AST with VITEK2. Was this because of limitations of the VITEK cards used and was disk diffusion performed? Or was this an issue due to the lack of breakpoints in the literature for certain species? Line 366: Can the authors use "cephalosporin-resistance", please?
Reviewer #3 (Comments for the Author): Importance and Introduction: 1. Care should be taken to differentiate this work from the SARS-CoV-2 work performed on these same specimen. Too often the SARS-CoV-2 work was mentioned unnecessarily. Results: 1. Initial results confusing as presented. Would be beneficial to provide total list of organisms identified by MALDI-TOF prior to discussion of AMR.
2. Result would benefit from analysis of trends for AMR: more AMR/species found in dead animals? Live animals? Region?
3. Were all strains identified by MALDI-TOF and then sequenced? Unclear as presented.
4. Why were the specific antibiotics chosen for each genus? 5. The networking analysis for co-occurance is very interesting.
6. Overall, Results could be better organized to more effectively show process and information Discussion 1. Discussion is rushed and should be better organized; discussion on totality of data, not just Klebsiella and E. coli.

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Spectrum00664-23
Münster, 05.06.2023 Dear Editor, we thank you and the two reviewers for thoroughly reviewing our manuscript. We appreciate the constructive suggestions and fair evaluation. In the following paragraphs, we reply point-by-point to the reviewers suggestions.

Reviewer #2
It would be beneficial if the authors reported all the species/genus identified in the study. The exact number of isolates identified by MALDI-TOF needs to be clarified by the authors. Reply: We now upload the database with all species and corresponding antimicrobial test results.
The manuscript is well written and provides sufficient background information. It reports the levels of antimicrobial resistance (AMR) in bacteria isolated from the pharynx of pangolins in Gabon. The authors report low levels of AMR in the 439 isolates investigated with 3 ESBL-producing K. pneumoniae and 1 ESBL-producing E. coli being described. Having investigated 89 animals and 439 isolates, the sample size is one big strength of this study. However, more emphasis could have been given to the species found in the samples investigated, as studies on bacterial communities in pangolins' pharynx are lacking. Additionally, the discussion could benefit from further discussing public health risks (including AMR dissemination) associated with dealing with wild pangolins, in my perspective.
Reply: Thank you for your kind evaluation. We will address the issues in detail below.
a) Generic comments -Title: Please consider rephrasing the title to better reflect the study conducted (bacteria in pharyngeal swabs and selective media) Reply: We changed the title better reflect the fact that we looked at the pharyngeal flora. However, we feel that the title would be too extended if we also address the use of the media.
-Importance: It may be relevant to mention the role of wildlife as potential reservoirs of resistant bacteria and give more emphasis to AMR. Reply: We now added a sentence on the importance of AMR in wildlife particularly in regions where the consumption of bushmeat is common.
-Throughout the manuscript the authors use "sp." Can the authors confirm if they mean species as plural "spp.", please? Reply: Thank you for pointing out this issue. We now corrected the manuscript accordingly.

b) Specific comments
Abstract: Line 32: Can the author use "Gram" instead of "gram", please? Reply: Corrected as suggested.
Introduction: -Can the authors add more information on the importance of pharyngeal bacteria and how these bacteria can/may spread to humans, please? Reply: We now provide an explanation why we consider the pharyngeal flora as the most relevant flora for a transmission of bacterial species from wildlife to humans. This is, also now supported by two additional references.