The resistomes of rural and urban pigs and poultry in Ghana

ABSTRACT There is limited knowledge on the bacteriome and resistome in livestock in Africa and the potential influence of the animal husbandry practices and scale has also been scantly explored. We quantified and characterized the antimicrobial resistance gene (ARG) pools (resistomes) and bacteriome in 30 pigs and 60 poultry samples (free-range: rural and urban, and industrialized) across Ghana using Resfinder and Silva databases and compared them to similar data from pigs and poultry from nine European countries. The pig and poultry were very different in ARG and bacterial abundance and composition. The bacterial communities in the Ghanaian samples also differed substantially from the European samples, especially driven by a much higher abundance of Subdoligranulum in both animals. We found lower ARG loads in Ghanaian pigs compared to European counterparts. Among poultry, urban free-range samples exhibited lower ARG abundances compared to the lowest European levels, while rural free-range samples were comparable to the European average, and industrialized samples showed higher ARG abundances. Despite major differences in abundance, the resistome composition of Ghanaian urban, rural, and industrialized poultry samples was similar. Contrasts with European samples were mainly driven by increased abundances of different tetracycline resistance genes in Ghanaian samples, and increased abundances of ARGs encoding resistance to macrolides, beta-lactams, and trimethoprim in Europe. Among pig samples, the main differences were caused by ARGs encoding resistance to nitroimidazoles, beta-lactams, and macrolides in European samples. This study is, to the best of our knowledge, the first report on the resistome measured using metagenomics in livestock from Sub-Saharan Africa. IMPORTANCE To the best of our knowledge, this is the first report on the resistomes that are measured using metagenomics in livestock from Sub-Saharan Africa. We find notable differences in the microbiomes between both pigs and poultry, and those also varied markedly compared to similar samples from Europe. However, for both animal species, the same bacterial taxa drove such differences. In pigs and urban free-range poultry, we find a very low abundance of antimicrobial resistance genes (ARGs), whereas rural free-range poultry displayed similarity to the European average, and industrialized poultry exhibited higher levels. These findings show how different African livestock bacterial communities and resistomes are from their European counterparts. They also underscore the importance of continued surveillance and investigation into antimicrobial resistance across diverse ecosystems, contributing significantly to global efforts toward combating the threat of antibiotic resistance.


Main points:
It is unfortunate that there are no studies that the authors can use for relevant comparisons using poultry and pig samples from other countries besides Ghana and the 9 EU countries.It is certainly understandable that comparisons using metagenomic samples from different studies will introduce a significant bias rendering it impossible to draw any solid conclusions.However, maybe the authors could discuss a little more results from previous metagenomic studies in the resistome of poultry and pigs from other parts of the world, especially since Africa is considered a continent with the lowest antimicrobials usage in the world.
Throughout the figures and the manuscript text, there are interesting comparisons between the resistomes of the EU countries and the industrialized, rural, and urban resistomes of Ghana, but there is no info about the EU countries' resistomes.Were they collected from industrialized facilities or ecological farms?Considering the details about the Ghana sampling, the manuscript would benefit greatly if the authors provided some details about the origin of the EU samples.I understand these details may exist in the cited literature (and the effort website), but it would be nice if that information is also readily available in the manuscript.In short, this information should include the type of facilities (free range, industrialized etc), the type of feed (whenever possible), and the antibiotics usage (again, depending on the availability of information).If the metagenomic samples from the EU countries came from different types of facilities, maybe, it would make sense to separate and present these samples to industrialized/rural/urban, similarly to what was done for the Ghana samples.
Minor comments: L79: do the authors have any details about the age of the animals?L83: are any details available about the composition of the commercial feed and how it may differ from the European?L89: do you know how frequently they were allowed to scavenge outside?L92: what does "self-medication" entail?L96: collected in sterile containers?falcon tubes?L102: please remove "apparently"; either they were purchased from the markets or not L107-109: sentence is a bit too colloquial, can you replace it with something like "Slaughtered birds arrive in central markets via intermediaries, who typically transport them from rural farms."L115: please specify how long the interruptions were L119: this is the first mention, please specify which nine countries L159: please specify which versions or when were the databases downloaded L207: I may have missed that, but how exactly did you estimate the number of genera per sample?Based on the 16S?Because, if yes, that is not ideal.... (I would suggest sth like MicrobeCensus for such estimations) L410-411: My point again, regarding more information about the diets (whenever possible) Reviewer #2 (Comments for the Author): In their manuscript "The resistome of rural and urban pigs and poultry in Ghana", the authors determine metagenomic data in livestock from Sub-Saharan Africa with a focus on antimicrobial resistance genes and bacterial composition.The authors identify differences between livestock microbiota in Ghana and Europe, which contributes to a better understanding of the global microbiome diversity and warrants further investigation to complete a microbiota catalogue encompassing livestock species and breeds, countries, and different production systems.The data are solid, well-presented and support the conclusions drawn by the authors.I have only a few comments to the manuscript.
Results, lines 269, 270: tetA(P) and tetB(P) are frequently associated with Clostridium and blaACI also with anaerobic bacteria.Can the authors comment on this, as Clostridia were identified as being among the more abundant genera in the pig samples (Table S4)?Discussion, Effects of host breed and diet on gut microbiome community and resistome: The bacterial composition and the resistomes differ strongly between pigs and poultry in Ghana and between the Ghanaian samples and the European samples.Differences in diet and in breeds present in the animal populations surely contribute the differences in microbiota and resistomes.Due to the means of poultry acquisition, there is no metadata as to antimicrobial usage and diet.This should be mentioned as a limitation to the study.Future studies should try to include such data.
Discussion: Was or will a metabolic reconstruction of the metagenome be attempted.This is beyond the scope of this study, but it would be interesting to compare metabolic profiles of European and Ghanaian samples to identify differences and try to backtrace dietary influences.

Minor points
Title: Since the authors stress that the pig and poultry metagenomes differ strongly in bacterial composition and in antibiotic resistance genes from each other, they might want to change the title to "The resistomes of rural and urban pigs and poultry in Ghana".
Introduction, line 56: change spacity to sparsity or to scarcity.M&M, Pig samples, lines 84-87: change Netting to netting, Veterinary to veterinary and medications to medications.M&M, Poultry samples: were the chickens from rural and urban sources also layers or broilers?Is there any information on the chicken breed or breeds that were slaughtered?M&M, Antimicrobial resistance gene quantification, line 154: which scheme was used to classify the resistance genes?
Results, Summary of the data, line 203: the standard deviation of the sample is quite high, although there is only one sample each with a very low and one with a very high sample count.It might be easier to evaluate the homogeneity of the sample if a size distribution plot of the fragment counts were given in the supplemental material.Results, line 267: change resistant to resistance.

Reviewer #1 (Comments for the Author):
This work by EEB Jensen et al is an excellent manuscript that will be of great interest to scientists working with animal resistomes and microbiomes, as well as other researchers, including microbial ecologists and genomicists.I enjoyed reading it, and I believe the core technical analysis (on which all of the important biology in this type of manuscript rests) has been carefully designed and performed.Also, I have to congratulate the authors on the excellent and self-explanatory figures they designed, which unfortunately is not always the case.I have read the manuscript thoroughly and have only a few suggestions for the authors.

Main points:
It is unfortunate that there are no studies that the authors can use for relevant comparisons using poultry and pig samples from other countries besides Ghana and the 9 EU countries.It is certainly understandable that comparisons using metagenomic samples from different studies will introduce a significant bias rendering it impossible to draw any solid conclusions.However, maybe the authors could discuss a little more results from previous metagenomic studies in the resistome of poultry and pigs from other parts of the world, especially since Africa is considered a continent with the lowest antimicrobials usage in the world.
"We thank the reviewer for their general positive feedback.We agree with the reviewer that the comparison part of our study could be expanded a bit.We therefore have discussed our output in comparison not only to those 9 European countries, but also compared to the metagenomic study of all available shotgun metagenomic sequencing data from NCBI SRA by Lawther et al, 2022 (doi: 10.3389/fmicb.2022.897905)(Line 414).We would like to stress, as mentioned in the manuscript, the lack of data on such topic from Africa, therefore we are constrained by limiting the comparison and discussion to non-African regions." Throughout the figures and the manuscript text, there are interesting comparisons between the resistomes of the EU countries and the industrialized, rural, and urban resistomes of Ghana, but there is no info about the EU countries' resistomes.Were they collected from industrialized facilities or ecological farms?Considering the details about the Ghana sampling, the manuscript would benefit greatly if the authors provided some details about the origin of the EU samples.I understand these details may exist in the cited literature (and the effort website), but it would be nice if that information is also readily available in the manuscript.In short, this information should include the type of facilities (free range, industrialized etc), the type of feed (whenever possible), and the antibiotics usage (again, depending on the availability of information).If the metagenomic samples from the EU countries came from different types of facilities, maybe, it would make sense to separate and present these samples to industrialized/rural/urban, similarly to what was done for the Ghana samples.
"We thank the reviewer for their comment.We have now added more details about farm animals and their details in the EFFORT samples (Lines 146-147)." Minor comments: L79: do the authors have any details about the age of the animals?"Farmers do not keep records of age and it was therefore not available.However, we ensured that all categories ( piglets, weaners, growers, and adults) were sampled on each farm if available."L83: are any details available about the composition of the commercial feed and how it may differ from the European?"Home-prepared feed for pigs, wheat bran (60%), soybean, maize chaff, and fish in proportions that are dependent on the availability of feed ingredients and the budget of the farmer."L89: do you know how frequently they were allowed to scavenge outside?"For rural pigs, they scavenge till evening until the time when owners drive them back into their pens." L92: what does "self-medication" entail?"When farmers treat the sick or give prophylactic medication to animals without recourse to trained veterinary personnel."L96: collected in sterile containers?falcon tubes?"All samples were collected in a sterile fashion in sterile falcon tubes.The manuscript has been adjusted accordingly (Line 121)."L102: please remove "apparently"; either they were purchased from the markets or not "We thank the reviewer for commenting on this, as we realize this sentence could be misleading.The "apparently" in the sense was meant to refer to the health status of the chickens and not if they were purchased or not.We realize this sentence could be misleading and have rephrased it (Line 102)." L107-109: sentence is a bit too colloquial, can you replace it with something like "Slaughtered birds arrive in central markets via intermediaries, who typically transport them from rural farms.""We thank the reviewer for pointing this out.We have adjusted the sentence (Line 110-111)."L115: please specify how long the interruptions were "After collecting the first and second batches, the third batch was delayed for a week.The fourth batch was collected a month later; therefore, the number had to be increased to make up for time lost.Finally, the last batch was collected after seven weeks, with the number increased to complete the sampling.This has now been included in the manuscript."L119: this is the first mention, please specify which nine countries "The manuscript has been adjusted accordingly (Line 142-143)."L159: please specify which versions or when were the databases downloaded "We thank the reviewer for this observation.The download date has been added to each database mention (Line 167)."L207: I may have missed that, but how exactly did you estimate the number of genera per sample?Based on the 16S?Because, if yes, that is not ideal.... (I would suggest sth like MicrobeCensus for such estimations) "We thank the reviewer for their comment.Yes, the number of unique genera per sample was estimated based on 16S.We realize that it is not ideal, and we have added the average number of genome equivalents per sample identified with MicrobeCensus to the manuscript (Line 218)."L410-411: My point again, regarding more information about the diets (whenever possible) "Unfortunately this is not available."

Reviewer #2 (Comments for the Author):
In their manuscript "The resistome of rural and urban pigs and poultry in Ghana", the authors determine metagenomic data in livestock from Sub-Saharan Africa with a focus on antimicrobial resistance genes and bacterial composition.The authors identify differences between livestock microbiota in Ghana and Europe, which contributes to a better understanding of the global microbiome diversity and warrants further investigation to complete a microbiota catalogue encompassing livestock species and breeds, countries, and different production systems.The data are solid, well-presented and support the conclusions drawn by the authors.I have only a few comments to the manuscript.

Major points
Results, lines 269, 270: tetA(P) and tetB(P) are frequently associated with Clostridium and blaACI also with anaerobic bacteria.Can the authors comment on this, as Clostridia were identified as being among the more abundant genera in the pig samples (Table S4)?
"We thank the reviewer for their general positive feedback and their comment.As we already mention in the discussion the bacterial composition can have a major effect on the resistome.As correctly observed by the reviewer Clostridium was more abundant in pigs from Ghana compared to Europe.However, the higher abundance of tetA(P) and tetB(P) was when comparing pigs and poultry from Ghana and with a higher abundance in poultry.We do agree that further studies into co-abundance and gene synteny are needed, but would at this stage prefer not to become too speculative regarding two genes only."Discussion, Effects of host breed and diet on gut microbiome community and resistome: The bacterial composition and the resistomes differ strongly between pigs and poultry in Ghana and between the Ghanaian samples and the European samples.Differences in diet and in breeds present in the animal populations surely contribute the differences in microbiota and resistomes.Due to the means of poultry acquisition, there is no metadata as to antimicrobial usage and diet.This should be mentioned as a limitation to the study.Future studies should try to include such data.
"We thank the reviewer for their comment, and we agree such metadata should be included.We have tried to address this in the discussion."Discussion: Was or will a metabolic reconstruction of the metagenome be attempted.This is beyond the scope of this study, but it would be interesting to compare metabolic profiles of European and Ghanaian samples to identify differences and try to backtrace dietary influences.
"We thank the reviewer for this very valid comment.While metabolite predictions and functional analyses are extremely interesting and important for those data (metagenomics), we believe this is out of the scope of our current manuscript (as mentioned by the reviewer).However, we completely agree that it would be extremely interesting to compare how such changes between the locations and the animal populations might also translate into various functions (metabolomics) to show the adaptations to different area/locations.This is particularly interesting to investigate the functional differences between the bacterial taxa that are differently abundant between the populations like Enorma and Subdoligranulum).However, this is outside the current manuscript focus and aim, but we do plan to carry out such analyses in combination with other datasets we currently have."

Minor points
Title: Since the authors stress that the pig and poultry metagenomes differ strongly in bacterial composition and in antibiotic resistance genes from each other, they might want to change the title to "The resistomes of rural and urban pigs and poultry in Ghana"."The manuscript has been adjusted."M&M, Poultry samples: were the chickens from rural and urban sources also layers or broilers?Is there any information on the chicken breed or breeds that were slaughtered?"All the rural and urban chickens were local (Ghanaian) breeds.The industrial chickens were foreign breeds, though the exact breed is not known."M&M, Antimicrobial resistance gene quantification, line 154: which scheme was used to classify the resistance genes?"Classification of resistance genes was made from the official annotation given by the ResFinder database documentation (https://bitbucket.org/genomicepidemiology/resfinder_db/src/master/).We have added this information to the manuscript (Line 161-162)." Results, Summary of the data, line 203: the standard deviation of the sample is quite high, although there is only one sample each with a very low and one with a very high sample count.It might be easier to evaluate the homogeneity of the sample if a size distribution plot of the fragment counts were given in the supplemental material."We thank the reviewer for this suggestion.A size distribution plot of the fragments counts has been added to the manuscript (Figure S2)."Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication.For your reference, ASM Journals' address is given below.Before it can be scheduled for publication, your manuscript will be checked by the mSystems production staff to make sure that all elements meet the technical requirements for publication.They will contact you if anything needs to be revised before copyediting and production can begin.Otherwise, you will be notified when your proofs are ready to be viewed.
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Good point.has been done."Introduction, line 56: change spacity to sparsity or to scarcity."The manuscript has been adjusted."M&M, Pig samples, lines 84-87: change Netting to netting, Veterinary to veterinary and medications to medications.
Results, line 267: change resistant to resistance."The manuscript has been adjusted (line 277)."Results, line 292 replace cfc(C) with cfr(C)."The manuscript has been adjusted (Line 281)."-23R1 (The resistomes of rural and urban pigs and poultry in Ghana) Dear Prof. Frank M. Aarestrup: Thank you for submitting your paper to mSystems.