Uncovering changes in microbiome profiles across commercial and backyard poultry farming systems

ABSTRACT The microbiome profiles of poultry production systems significantly impact bird health, welfare, and the environment. This study investigated the influence of broiler-rearing systems on the microbiome composition of commercial and backyard chicken farms and their environment over time. Understanding these effects is vital for optimizing animal growth, enhancing welfare, and addressing human and environmental health implications. We collected and analyzed various samples from commercial and backyard farms, revealing significant differences in microbial diversity measurements between the two systems. Backyard farms exhibited higher alpha diversity measurements in soil and water samples, while commercial farms showed higher values for litter and feeder samples. The differences in microbial diversity were also reflected in the relative abundance of various microbial taxa. In backyard farms, Proteobacteria levels increased over time, while Firmicutes levels decreased. Campilobacterota, including the major poultry foodborne pathogen Campylobacter, increased over time in commercial farm environments. Furthermore, Bacteroides, associated with improved growth performance in chickens, were more abundant in backyard farms. Conversely, pathogenic Acinetobacter was significantly higher in backyard chicken fecal and feeder swab samples. The presence of Brevibacterium and Brachybacterium, associated with low-performing broiler flocks, was significantly higher in commercial farm samples. The observed differences in microbial composition and diversity suggest that farm management practices and environmental conditions significantly affect poultry health and welfare and have potential implications for human and environmental health. Understanding these relationships can inform targeted interventions to optimize poultry production, improve animal welfare, and mitigate foodborne pathogens and antimicrobial resistance risks. IMPORTANCE The microbiome of poultry production systems has garnered significant attention due to its implications on bird health, welfare, and overall performance. The present study investigates the impact of different broiler-rearing systems, namely, commercial (conventional) and backyard (non-conventional), on the microbiome profiles of chickens and their environment over time. Understanding the influence of these systems on microbiome composition is a critical aspect of the One-Health concept, which emphasizes the interconnectedness of animal, human, and environmental health. Our findings demonstrate that the type of broiler production system significantly affects both the birds and their environment, with distinct microbial communities associated with each system. This study reveals the presence of specific microbial taxa that differ in abundance between commercial and backyard poultry farms, providing valuable insights into the management practices that may alter the microbiome in these settings. Furthermore, the dynamic changes in microbial composition over time observed in our study highlight the complex interplay between the poultry gut microbiome, environmental factors, and production systems. By identifying the key microbial players and their fluctuations in commercial and backyard broiler production systems, this research offers a foundation for developing targeted strategies to optimize bird health and welfare while minimizing the potential risks to human and environmental health. The results contribute to a growing body of knowledge in the field of poultry microbiome research and have the potential to guide future improvements in poultry production practices that promote a sustainable and healthy balance between the birds, their environment, and the microbial communities they host.


Introduction
Line 66: minor: antibiotics are not only used for growth promotion but for treatment, prevention and control.Also, please consider revising "antibiotic use could result in gut dysbiosis in chickens.

Materials and methods
Line 104: major -these are all required for context relevant to the results.This section requires a brief overview of the farm-level demographics, at the minimum, breed (genetics was mentioned in the discussion), the population size (number of barns, total birds raised), basic biosecurity (presence of other animals on farm) and litter/manure disposal (on-farm composting, re-used litter, hauled away?) average rearing period, cleaning and disinfection prior to chick placement (+antimicrobial use) that could potentially impact the microbiome.Was a data collection/questionnaire not used here?As for a baseline data, was there no day 0 (chick placement) sampling (reflective of starting point microbiome status before chick placement)?Rationale for the timepoints/farm visits?Results: 210 to 212 -these sentences belong to the discussions.Discussions Lines 249-250 -"so what?" aspects need to addressed, what is the significance of this study to poultry rearing and how is this relevant to food safety and public health?Which poultry production type should be monitored more closely (rearing conditions affecting microbiome) from a food safety and public health perspective based on the predominance of certain phyla/genera?Lines 290-291 -what are these farm management practices?Conclusion Line 346 to 347 -implications on food safety/public health.
Reviewer #2 (Comments for the Author): The header "Sample collection" requires more information than what has been provided.State how the farms were selected.Line 107-109: please clarify if the number of each sample presented here is per farm.How each sample type was obtained: more information is needed.Line 163-164 and elsewhere: please present the statement(s) in the form of "higher" or "lower" instead, for better understanding .Line 193-194: I think you need to include backyard and commercial farms in the statement.Italicize all the genera names throughout the manuscript as you did 206-213.Discussions were presented in the results and vice versa: example line 210-213.Table 1: The asterisks (*) for significance, for some measurements it was shown in both production types; for others it was shown only for one of them.To avoid confusion, please present the "*" sign only for one of the production types choosing "higher" or "lower" and state this in the caption.All

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex.Go to Author Tasks and click the appropriate manuscript title to begin the revision process.The information that you entered when you first submitted the paper will be displayed.Please update the information as necessary.Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER.
• Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file.
• Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file.For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process.Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript." Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me.If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail.Arrangements for payment must be made before your article is published.For a complete list of Publication Fees, including supplemental material costs, please visit our website.
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees.Need to upgrade your membership level?Please contact Customer Service at Service@asmusa.org.
Thank you for submitting your paper to Microbiology Spectrum.

1
Response to Reviewer #1 1. Line 66: minor: antibiotics are not only used for growth promotion but for treatment, prevention and control.Also, please consider revising "antibiotic use could result in gut dysbiosis in chickens.
The sentence in the manuscript is changed accordingly.
2. Line 104: major -these are all required for context relevant to the results.This section requires a brief overview of the farm-level demographics, at the minimum, breed (genetics was mentioned in the discussion), the population size (number of barns, total birds raised), basic biosecurity (presence of other animals on farm) and litter/manure disposal (on-farm composting, re-used litter, hauled away?) average rearing period, cleaning and disinfection prior to chick placement (+antimicrobial use) that could potentially impact the microbiome.Was a data collection/questionnaire not used here?As for a baseline data, was there no day 0 (chick placement) sampling (reflective of starting point microbiome status before chick placement)?Rationale for the timepoints/farm visits?All these comments are addressed by adding a new section "Farm demographics and operational practices" under "Materials and Methods."

Farm demographics and operational practices
The study involved sampling from 5 commercial farms and 5 backyard farms, adhering to the minimum inclusion criteria of raising at least 100 chickens without the usage of antibiotics as growth promoters.The anonymous data was safeguarded by a coding system, assuring confidentiality of farm information.Our sampling protocol varied according to the type of farm; backyard farms were visited thrice during the production cycle, specifically on days 10, 31, and 52, while commercial farms were visited on days 10, 24, and 38, reflecting the more rapid pace of broiler production in these environments.The chosen timepoints likely represent key stages in the broiler's growth and health, allowing the study to capture meaningful changes in the microbiome across their accelerated growth period.The commercial farms, employing an intensive production system, reared thousands of birds indoors, with new litter added prior to each stocking cycle.Composting was not implemented in these settings.Conversely, backyard farms which usually raise between 100-1000 birds at a time, practiced on-site composting and daily rotation of pens on the pasture.Furthermore, multiple livestock species were maintained in the backyard farms.A marked contrast was observed in biosecurity measures between the two farm types.Commercial farms implemented rigorous procedures such as tire washes, foot dips, and personal protective equipment usage.However, biosecurity practices in backyard farms were more varied, with gum boots being the most common protective gear used.Lastly, the typical production duration in commercial farms was 5-6 weeks, with backyard farms taking a slightly longer time of 8-10 weeks.This may reflect the differences in breed genetics and production strategies between the two settings.However, data collection did not include a baseline microbiome sample at day 0 (chick placement), an aspect that could have provided valuable insight into initial conditions.The specific rationale for the chosen timepoints of farm visits pertained to the different production schedules of commercial and backyard farms.
3. Results: 210 to 212 -these sentences belong to the discussions.

Lines 249-250 -"so what?" aspects need to addressed, what is the significance of this study to poultry rearing and how is this relevant to food safety and public health? Which poultry production type should be monitored more closely (rearing conditions affecting microbiome) from a food safety and public health perspective based on the predominance of certain phyla/genera?
The comments are addressed as below in the manuscript.The population of Proteobacteria in backyard farms demonstrated a rising trend over time, whereas the number of Firmicutes showed a decreasing trajectory.In commercially raised poultry, there was an upward trend in the prevalence of Campilobacterota, which includes Campylobacter, a primary pathogen causing foodborne diseases originating from poultry.Interestingly, a greater abundance of Bacteroides, which is associated with enhanced growth rates in chickens, was observed in backyard farms.However, a drawback was the significant increase in the concentration of potentially harmful Acinetobacter in samples from both backyard chicken feces and feeder swabs.Commercial farms, on the other hand, saw a marked increase in Brevibacterium and Brachybacterium, bacteria associated with broiler flocks that perform poorly.The implications of these findings can significantly impact the poultry farming industry.The increasing levels of Proteobacteria and Bacteroides in backyard farms might suggest a potential for improved growth performance in these environments.However, the higher presence of Acinetobacter poses a health risk that needs to be managed.On the commercial side, the increasing presence of the foodborne pathogen Campylobacter requires vigilance and proactive strategies to prevent potential outbreaks.The presence of Brevibacterium and Brachybacterium, indicators of low-performing broiler flocks, might suggest a need for improved farming practices to enhance the productivity and overall health of the flocks in commercial farm environments.

Lines 290-291 -what are these farm management practices?
The sentence modified to include the management practices such as feeding, housing, waste management, irrigation practices, antibiotic usage, use of pesticide, and rotation of livestock.

Line 346 to 347 -implications on food safety/public health.
A new section "Farm demographics and operational practices" under "Materials and Methods" is added.

Farm demographics and operational practices
The study involved sampling from 5 commercial farms and 5 backyard farms, adhering to the minimum inclusion criteria of raising at least 100 chickens without the usage of antibiotics as growth promoters.The anonymous data was safeguarded by a coding system, assuring confidentiality of farm information.Our sampling protocol varied according to the type of farm; backyard farms were visited thrice during the production cycle, specifically on days 10, 31, and 52, while commercial farms were visited on days 10, 24, and 38, reflecting the more rapid pace of broiler production in these environments.The chosen timepoints likely represent key stages in the broiler's growth and health, allowing the study to capture meaningful changes in the microbiome across their accelerated growth period.The commercial farms, employing an intensive production system, reared thousands of birds indoors, with new litter added prior to each stocking cycle.Composting was not implemented in these settings.Conversely, backyard farms which usually raise between 100-1000 birds at a time, practiced on-site composting and daily rotation of pens on the pasture.Furthermore, multiple livestock species were maintained in the backyard farms.A marked contrast was observed in biosecurity measures between the two farm types.Commercial farms implemented rigorous procedures such as tire washes, foot dips, and personal protective equipment usage.However, biosecurity practices in backyard farms were more varied, with gum boots being the most common protective gear used.Lastly, the typical production duration in commercial farms was 5-6 weeks, with backyard farms taking a slightly longer time of 8-10 weeks.This may reflect the differences in breed genetics and production strategies between the two settings.However, data collection did not include a baseline microbiome sample at day 0 (chick placement), an aspect that could have provided valuable insight into initial conditions.The specific rationale for the chosen timepoints of farm visits pertained to the different production schedules of commercial and backyard farms.
2. Line 107-109: please clarify if the number of each sample presented here is per farm.How each sample type was obtained: more information is needed.
Figure have poor resolution and at times very difficult to read the texts.Please provide publication quality figures.

Figure 2 :
Although color keys for backyard vs commercial were presented, these were not reflected in the figure.OR please explain what the keys means.The same for Figure3.

Figure 4 :
Resolution is low.The colors for the visits not reflected in the graph.It is not clear what the bar charts indicate.
• Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred