F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.131679.1 Study Protocol Articles Vegetables as a vehicle for antimicrobial resistance (vAMR): An agroecosystem exploration from the One Health perspective in India

[version 1; peer review: 1 approved with reservations, 1 not approved]

 Kalpana Pachillu Conceptualization Data Curation Formal Analysis Funding Acquisition Investigation Methodology Project Administration Resources Supervision Validation Writing – Original Draft Preparation https://orcid.org/0000-0002-3847-221X a 1 2 Falkenberg Timo Conceptualization Data Curation Methodology Supervision Writing – Review & Editing 1 3 Yasobant Sandul Conceptualization Data Curation Investigation Methodology Project Administration Resources Supervision Validation Writing – Review & Editing https://orcid.org/0000-0003-1770-8745 1 3 4 5 Saxena Deepak Conceptualization Data Curation Project Administration Resources Supervision Writing – Review & Editing 4 5 Schreiber Christiane Conceptualization Data Curation Funding Acquisition Methodology Supervision Validation Writing – Review & Editing 3 One Health Graduate School, Center for Development Research (ZEF), University of Bonn, Bonn, NRW, 53113, Germany Department of Pharmacy, Faculty of Mathematics and Natural Sciences, University of Bonn, Bonn, NRW, 53113, Germany Institute for Hygiene and Public Health (IHPH), Universitätsklinikum Bonn (University Hospital Bonn), Bonn, NRW, 53127, Germany Centre for One Health Education, Research & Development (COHERD), Indian Institute of Public Health Gandhinagar (IIPHG), Gandhinagar, Gujarat, 382042, India School of Epidemiology & Public Health, Datta Meghe Institute of Medical Sciences (DMIMS), Wardha, Maharastra, 442004, India pachillukalpana@gmail.com

No competing interests were disclosed.

 22 3 2023 2023 12 316 7 3 2023 Copyright: © 2023 Kalpana P et al. 2023 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction: Antimicrobial resistance (AMR) has emerged as one of the leading threats to public health. AMR possesses a multidimensional challenge that has social, economic, and environmental dimensions that encompass the food production system, influencing human and animal health. The One Health approach highlights the inextricable linkage and interdependence between the health of people, animals, agriculture, and the environment. Antibiotic use in any of these One Health areas can potentially impact the health of other areas. There is a dearth of evidence on AMR from the natural environment, such as the plant-based agriculture sector. Antibiotics, antibiotic-resistant bacteria (ARB), and related AMR genes (ARGs) are assumed to present in the natural environment and disseminate resistance to fresh produce/vegetables and thus to human health upon consumption. Therefore, this study aims to investigate the role of vegetables in the spread of AMR through an agroecosystem exploration from a One Health perspective in Ahmedabad, India.

Protocol: The present study will be executed in Ahmedabad, located in Gujarat state in the Western part of India, by adopting a mixed-method approach. First, a systematic review will be conducted to document the prevalence of ARB and ARGs on fresh produce in South Asia. Second, agriculture farmland surveys will be used to collect the general farming practices and the data on common vegetables consumed raw by the households in Ahmedabad. Third, vegetable and soil samples will be collected from the selected agriculture farms and analyzed for the presence or absence of ARB and ARGs using standard microbiological and molecular methods.

Discussion: The analysis will help to understand the spread of ARB/ARGs through the agroecosystem. This is anticipated to provide an insight into the current state of ARB/ARGs contamination of fresh produce/vegetables and will assist in identifying the relevant strategies for effectively controlling and preventing the spread of AMR.

 Antimicrobial resistance antibiotic-resistant bacteria antibiotic-resistant genes vegetables fresh produce agroecosystem One Health Ministerium für Kultur und Wissenschaft des Landes Nordrhein-Westfalen OneHealthandUrbanTransformation This work was supported by the University of Bonn, the University of Applied Sciences Bonn-Rhein-Sieg, and the Ministry of Culture and Science of North Rhine-Westphalia, Germany, through the Forschungskolleg “One Health and Urban Transformation”. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. List of abbreviations AMR

Antimicrobial resistance

 ARB

Antibiotic resistant bacteria

 ARGs

AMR genes

 WHO

World Health Organization

 AMC

Ahmedabad Municipality Corporation

 AUDA

Ahmedabad Urban Development Authority

 E. coli

Escherichia coli

 XLD Agar

Xylose Lysine Deoxycholate

 AST

Antimicrobial Susceptibility Test

 CLSI

Clinical Laboratory Standards Institute

 qPCR

Quantitative Polymerase Chain Reaction

 LOQ

Limit of Quantification

 Introduction

The worldwide emergence of antimicrobial resistance (AMR), altering lifesaving antibiotics, is a significant public health challenge. 1 The estimation exhibited that AMR is accountable for 700,000 mortalities annually worldwide and as per the current rate of resistance-related deaths, it is projected to rise to about 10 million mortalities annually by 2050. 2  The impact of AMR is not confined to human mortalities but is also extended to related economic losses, estimated at around $100-210 trillion in healthcare-related expenses. 3 A dearth of reliable data and evidence from low-middle income countries such as India contributes to underestimating the rate of AMR and related threats. Conversely, the recent Global Antimicrobial Resistance and Use Surveillance System (GLASS)-AMR 2021 report consisting of data from the national surveillance has reported the average increased burden of AMR with a significant focus on human clinical settings in India. 4

The major contributing factors to the AMR issue are not limited to the overuse and increased misuse of antibiotics (prescription and consumption) in human settings. 5 The prophylactic use and practice of sub-lethal doses of antibiotics in livestock as growth promoters to maintain health and productivity contribute to a significant part. 6 Most antibiotics used in human and animal sectors are not entirely metabolized in the bodies; a high percentage of administered drugs are discharged into water and soil through municipal wastewater, animal manure, sewage sludge, and biosolids. 7 The relationship between the consumption of antibiotics is directly associated with the emergence and promotion of antibiotic resistance in the microbial population of multiple sectors, including humans, animals, and the environment. 8

The environmental dimension of AMR has received comparatively less focus than AMR in human or animal health. Directly connecting antibiotics, animal production, and the environment is the practice of recycling animal waste as manure for reintroducing organic materials and nutrients to the soil to improve crop productivity. 9 At the same time, it presents a major pathway for the dissemination of antibiotic residues, antibiotic-resistant bacteria (ARB), and AMR genes (ARGs). 10 Thus, agricultural sources are considered a critical source and dissemination route for AMR. 11 The spread of antibiotic resistance in the agricultural ecosystem is a major concern because agriculture is closely related to food safety and human health. The exposure of humans to foodborne bacterial pathogens, including ARB, through the food chain, has been reported in recent decades in foodborne outbreaks. 12

The enrichment of ARB and ARGs in agricultural soil, even without the apparent application of antibiotics via human agriculture practices, has raised concerns because soil ARB and ARGs can be disseminated to plants, leading to the potential spread from farms to consumers. 13 There are many potential pathways by which soil-associated ARB and ARGs can transfer to plant microbiomes contributing to the emergence and spread of plant resistome to the human food chain. 14 , 15 Food consumption represents a major route for human exposure to ARGs in environmental microbiomes. 16 Some studies indicate that the consumption of raw fruit and vegetable is a potential pathway for disseminating ARGs to human microbiomes. 17 It has been suggested that the long-term use of organic fertilizers causes ARGs enrichment not only of soils but also of plant phyllosphere microbiomes. 14 There are many potential pathways by which soil-associated ARB and ARGs can be transferred to plant microbiomes contributing to the emergence and spread of plant resistome to the human food chain. Fresh produce can be contaminated with bacterial pathogens at multiple points throughout its production by direct contact with fecal waste during farming, wastewater irrigation, use of biosolids or animal manure as fertilizer. 11 , 18 While these potential contamination pathways have been studied well for traditional pathogens, their relative contributions to the contamination of fresh produce with ARB and ARGs have not been quantified. This is essential to capture the unexplored factors contributing towards the issue of AMR to hinder the spread. Therefore, this study findings will contribute towards the data from the foods of plant origin which connects with a link of dietary exposure to humans.

The study aim is to investigate the presence of antibiotic-resistant bacteria and the distribution of antibiotic-resistant genes across the vegetable and soil at the farm level in alignment with a one health approach in Ahmedabad city, India to provide an evidence about resistance spread for future intervention strategies.

 Protocol Ethical statement

Ethics approval has been obtained from the Research Ethics Committee, Center for Development Research (ZEF), University of Bonn, Germany (17c_22), and the Institutional Ethics Committee of the Indian Institute of Public Health Gandhinagar, India (TRC/2022-23/10). All details collected in the study will be kept confidential and complies with the Declaration of Helsinki. Written informed consent will be taken from each participant. All methods will be carried out in accordance with relevant guidelines and regulations. 33

Aim

This study aims to provide insights into the knowledge gap and investigate the role of vegetables in spread of antimicrobial resistance through an agroecosystem exploration from a One Health perspective in one of the western communities of India. More specifically, it aims to investigate the presence of antibiotic resistant bacteria and the distribution of antibiotic resistant genes across the soil-vegetable continuum at the farm level with a One Health approach in Ahmedabad city, India.

The specific research questions are:

Which antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARGs) are already published or known to (or can be supposed to) be widespread in soil-plant-fruit continuum of the South Asia region?

What is the risk of AMR spread across soil-plant-fruit continuum in agriculture farms of Ahmedabad, India?

How can the general agriculture practices of farmers in the region of Ahmedabad, India be characterized and which potential risk factors can be identified?

Can ARB and/or ARGs be detected in the fresh produce/vegetables that are consumed raw or/and in the soil where these plants grow in the region of Ahmedabad, India?

Study design

This present study is an interdisciplinary social-ecological study with the prime aim to investigate and characterize the antimicrobial resistance in vegetables and soil on harvest, which network in an intimate proximity and their possible transmission pathway along the environment-human direction using a One Health approach. A mixed-method approach is adopted: secondary data analysis (review) and primary data collection including a survey of the general agricultural practices and laboratory analysis (microbiological and molecular), which will be merged in a triangulation concept to converge the findings and generate outcomes related to the possibility of vegetables as vehicles for AMR.

 Study settings

The proposed study will be conducted in one of the largest and most populous cities of the Western state, Gujarat, India, known as Ahmedabad ( Figure 1). Ahmedabad is located in the central part of Gujarat on the banks of the Sabarmati river, with a population of 5,633,927. 19 Ahmedabad's population has a combination of both vegetarian (60%) and non-vegetarian (40%) consumption patterns. 20 Gujarat state has an outstanding contribution to the country’s horticulture sector. 21

vAMR Project study site indicating the Ahmedabad city in Gujarat, India.

Ahmedabad city is located in the north-central part of the Ahmedabad district in Gujarat. Ahmedabad municipality was established in 1873 and later upgraded to Ahmedabad Municipality Corporation (AMC), divided into six municipal zones (north, south, east, central, west and new west) consisting of a total of 64 wards. Ahmedabad Urban Development Authority (AUDA) was established on 1st February 1978 with the prime objective to carry out the sustained, planned development of the area falling outside the periphery of Ahmedabad Municipal Corporation. The AUDA encompasses an area of 186,600 hectares, including Ahmedabad City (Municipal Corporation of 44,950 Hectares). The AUDA developed a comprehensive development plan for Ahmedabad city and the district, including the current general agriculture zone and the prime agriculture zone. The general agricultural zone is intended to provide areas with large parcels that are conducive to growing crops and the keeping of farm animals and poultry for the production of milk, wool, eggs, and other farm products. This zone is also intended to permit limited commercial and light industrial activities that support agriculture or are connected to the agricultural industry. The prime agriculture zones represent large, generally contiguous blocks of land that enable current and future opportunities for agriculture.

The increasing urbanization of Ahmedabad city observably led to developing more residential areas in the central part, shifting the agriculture practitioners involved in both livestock and plant production to the outskirts of the urban and peri-urban areas of the city. A large part of the general agriculture zones of the city is located adjacent to the city's sewage treatment plant. The general agriculture zone in the east of the city is known as a hotspot for livestock farmers. This area produces a significant amount of animal waste, the primary fertilizer source of the farms in this agriculture zone. The agriculture zone on the city's west side is located far from the sewage treatment plant and only small-scale livestock farming is being practiced.

This study is planned to be implemented in the general agriculture zones of Ahmedabad city because of the established and in-process use of land for agriculture purposes.

 Research design

The study starts with a large-scale literature-based analysis of the information on the qualitative resistance data, abundances, and patterns on ARB and ARGs on fresh produce and food production environment in context of South Asia (Research question 1). The field exploration starts with recruitment of farms, followed by a survey of the general agricultural practices in the study area and for the identification of vegetables that are produced by the farmers and also consumed raw, to identify potential risk factors for the spread of resistances (objective 2.1). The sampling of the raw consumed vegetables and the agricultural soil will be performed at the farm-level. The sampled vegetables and soil will then be microbiologically analyzed for the presence and quantity of ARB and ARGs. The gained information on the current state of ARB and ARGs on fresh produce and the agricultural soil will be reported from Ahmedabad, India (objective 2.2). Further, the findings of all the three objectives will result in the identification of possible factors that may play a role in the spread of AMR through plant-based food to human due to consumption of ARB/ARGs contaminated vegetables. Recommendations of intervention strategies will be developed to support involved actors and decision-makers of Ahmedabad, India.

Methods for research question 1

To understand the AMR background in the South Asia region in general, a systematic review methodology will be used. This systematic review aims to screen, assemble, summarize and evaluate the peer-reviewed and published evidence on the transmission of antibiotic resistances from differently fertilized soil to plants in the South Asia context, and to assess the evidence for possible human health related risks associated with consumption of antibiotic resistance contaminated vegetables produced on such fertilized soils.

The search strategy ( Table 1) is designed to identify published studies on ARB, ARGs in the soil, plants, and plants parts produced on manure amended soil and associated human health related issues. The interest is in identifying studies which looked upon the ARB and ARGs contamination of plants and plant produce (i.e., vegetables) through any pathway of spread from soil to plants. The identification includes both lab-based and field-based studies. The systematic literature review will be conducted using two scientific online databases: PubMed and Web of Science. The time limitation will be set to the last 10 years so that the studies selected are up-to-date and to represent knowledge that is scientifically state-of-the-art, or allow identification of recent trends in resistance spread, respectively. The key search terms are generated to encapsulate the four main concepts and therefore the review pertains to: Antibiotic resistance; bacteria and genes; fresh produce/vegetables/plants/fruits; health effects; South Asia. The table below shows the terms and how they are combined.

Search terms for systematic review to be undertaken under the vAMR project, India.
Antimicrobial Resistance- related terms Agriculture and fresh produce-related terms Location
antimicrobial resistan$, antibiotic-resistan$ bacteria, antibiotic resistan$ genes, antimicrobial- resistant$ organisms, antibiotic -resistant pathogen$ agriculture$, farming$, fresh agriculture produc$, fresh produc$, fresh vegetable$, vegetable$, raw vegetable$, salad vegetable$, leafy green$, salad$, fruit$ South Asia$, Afghanistan$, Bangladesh$, Bhutan$, India$, Maldives$, Nepal$, Pakistan$, Sri Lanka$
Criteria for inclusion and exclusion

The published peer-reviewed literature that documents information regarding the ARB and/or ARGs presence in plants, plant produce, vegetables, and the plant-fruit-soil continuum will be included in the review. Studies documenting the impact of plants and plant produce containing ARB and/or ARGs on human health will also be included. The review will be restricted to studies that explicitly explored the South Asia region's ARB and ARGs plant and soil environment context. The geographical restriction will involve the field site of the study conducted in South Asia, irrespective of the authors' affiliations. There will be no restriction on the design of the study. Studies published in languages other than English will be excluded. The outcomes targeted by this review will include the incidence of ARB and ARGs on plants and plant produce, from the soil environment, and the possible related human health risks due to consumption of such plant produce to define/plan next research steps more precisely.

 Data extractions

Data will be extracted from the eligible studies to summarize and tabulate important findings. Data will be extracted and tabulated about the type and prevalence of ARB/ARGs, type of food source, study design, location of the study, methodological approach, key findings. The results form an important evidence base for the selection of ARB and ARGs for the next objective of the study.

Method for research question 2

Objective 2.1

The AMR field investigation is intended to capture a baseline for the further exploration of trends in vegetables and the soil used for the production of vegetables. It includes selective bacteria cultivation and identification, antibiotic resistance (pattern), and resistance genes. According to Masterton (2008), the basic protocol includes microbiological culturing and a validated antimicrobial susceptibility testing method for the first step. 22 The basic protocol includes methods to gain better insights into the collection of ARGs that may circulate in the environment, leading to new bacterial serotype-resistance gene combinations and can be transmitted to humans through food consumption. 23 The molecular methodology thus will be followed with the basic protocol to quantify the selected ARGs.

Sampling procedure for agriculture farms

The agriculture farm areas for this study will be selected from the general agriculture zones of Ahmedabad city. A preliminary screening will be performed in Ahmedabad city to identify the main areas involved in agricultural practices located in the urban part of the general agriculture zones, i.e., within the city’s six municipal zones to be included in this study. This screening is important given the unavailability of a database of agricultural farms in Ahmedabad city. Snowball sampling will be used to recruit the farms engaged with agriculture production. The researchers will visit the farmers physically for recruitment process in the study. Farmers will be informed about the purpose of the study and written informed consent will be obtained from the farmers. The farmers will also be informed about their right to withdraw consent at any point of time during the study period. Consent will be confirmed again before initiating the interviews and surveys. Two copies of the consent form will be prepared along with the signatures or thumb impression of farmer. One copy will remain with the farmer and one with the researcher. The farmer and farm households will be asked to list six nearby farms engaged in agriculture production activity. This process will be repeated until the desired sample size is reached. About 300 odd farms based on the type of agricultural product is aimed to be recruited from different zones of the city. The second screening of farms will be done with the principal inclusion criteria for the recruitment as vegetable production. Thus, depending on the number of farms engaged in agricultural production in a given locality, the equal number of representative farms from the different zones of the city will be surveyed and enrolled in the study. The enrolled 300 farms which will be surveyed for the general agricultural practices will then be assessed for the vegetable producing farms. 150 vegetable producing farms from the total vegetable producing farms will be selected from the different zones of the city based on their vegetable production for the next investigation step.

Source of data and data collection methods

A set of questions will be asked to each 300 farms engaged in agriculture production. The structured questionnaire for interviewing the farmers will be divided into five main sections: demographic characteristics, general farming practices, agriculture inputs, irrigation water and post-harvest practice. The questionnaire will also have aspects on information on the different types of crops as well as vegetables that are cultivated and those that are consumed raw by the household. This cross-sectional survey aims to identify the vegetable producing farms and the five major vegetables produced by the farms and also consumed raw by the households. The field observation will be used to validate the general farming system, fertilizer and pesticide use along with the questionnaire during the farm visits. This survey will be focused in collecting and documenting data on the general agriculture practices of farmers to explore factors, such as the use of fertilizers, pesticides, and irrigation water in farming system.

Selection of bacteria for microbiological analysis

The selection of bacteria is based on the recommendation of the World Health Organization (WHO). 23 The guidance mentions that the food of plant origin commonly intended to be consumed by humans, either fresh, minimally processed, or consumed after cooking, in that the consideration should be given to the pathogens of importance to human health and where AMR may be a hazard. The list of bacteria consists as below:

Escherichia coli (E. coli) as a measure of hygienic production and faecal indicator in general

Salmonella species as a pathogenic representative of human- and animal-derived contamination

Pseudomonas species as a common environmental bacterial contaminant serving as a sentinel for AMR

In addition to these bacteria recommended for screening by the WHO, selected species of other important bacteria can further be included in the study according to the initial systematic review of this study.

Selection of AMR genes (ARGs) for molecular analysis

The choice of ARGs to be included in this study will be based on the systematic review conducted for research question 1. At least two ARGs will be selected based on the most found ARGs in the context of vegetables through the systematic review. This process is important as it will provide an overview of the major ARGs which are circulating in the selected geographical area and thus comparing the presence of these genes also in Ahmedabad, India.

Sampling procedure for microbiological analysis

The samples for resistance investigations will be biological samples for microbiological and molecular analysis. Sampling will include soil and vegetable samples grown on the selected agricultural farms as they are considered significant vehicles for the spread of AMR. Samples will be collected when vegetables reach the marketing stage. The farm households' top five vegetables produced and consumed raw will be identified through the first survey. Five vegetable samples and the consecutive soil sample in duplicate will be collected from each selected farm. A simple random sampling method will be used for collecting vegetable samples from each farm.

 Sample collection

Vegetable sampling: The vegetable samples will be collected when they reach their commercial size through simple random sampling from each selected farm. A total of one vegetable sample in duplicates will be collected from each selected farm, thus it will be two vegetable samples from each farm. All the vegetable samples will be collected aseptically by utilizing a sterile zipper bag. After collecting samples, they will be transported to the laboratory within two hours in an icebox. 24

Soil sampling: There will be only one soil sample collection from each selected farm. The one soil sample in duplicates will be collected for the corresponding above selected vegetable sampling. The soil samples will be collected near the roots of the plants at a depth of 10 cm. The sampling will be taken out by utilizing a sterile shovel, and the collection of the sample will be in a sterile falcon tube. 25

Sample processing:

Vegetable sample: Vegetable samples collected from each farm will be analysed for selected bacterial contamination. The farms' samples will be first freed from any adhering soil particles. The samples will then be processed following safe handling procedures recommended for human consumption purposes (e.g., handled after hand washing, trimmed of spoiled parts, and washed thoroughly under sterile water). Vegetable samples will be chopped into small pieces with a sterile knife. 25 g of each sample will be added to 225 ml of 0.1% peptone water and will then be shaken using a horizontal shaker for 30 min at 200 rpm to get a homogenous suspension, equal to an initial dilution of 10 0. Finally, a dilution series ranging from 10 -1-10 -10 will be prepared from this initial dilution for further processing. 26

Soil sample: The soil samples collected in the falcon tubes will be processed for further investigation. 10 g of soil will be taken from the collected soil and mixed with 90 ml of 0.1% peptone water in a beaker. 26 The mixture will be mixed well using a horizontal shaker for 30 min at 200 rpm to have the initial dilution for processing. The 10-fold serial dilution will be prepared from the initial dilution to have the dilution ranging from 10 -1-10 -10 for identification and enumeration of selected bacteria.

Identification and enumeration of bacteria

E. coli: A selective HiCrome TM E. coli Agar (Himedia, India) 27 will be used for cultivation of E. coli. The dilution range of 10 -1 – 10 -10 will be prepared and five consecutive dilutions of 100 μl will be spread on the agar plates. The plates will be incubated at 44°C for 24 hours. After incubation, E. coli will grow as blue-green colonies, and the results of the total bacterial count will be expressed as a colony-forming unit (CFU)/g of sample. The colonies will be subjected to biochemical confirmation, including an Indole production test to confirm the positive or negative isolates. The positive results for indole production isolates will be confirmed as E. coli. 28

Salmonella species: A similar procedure as for E. coli will be carried out to identify and enumerate Salmonella species. Briefly, each 100 μl from the dilution range of 10 -1 – 10 -10will be spread on the selective media Xylose Lysine Deoxycholate (XLD) Agar. The plates will be incubated at 37°C for 24 h. After incubation, the bacterial colonies will appear pink to red-colored with or without centers. The typical colonies will be further subjected to an array of biochemical tests such as oxidase, indole production, and catalase for further confirmation 26

Pseudomonas species: A selective and differential medium, Cetrimide Agar (Himedia, India), will be prepared to identify and enumerate Pseudomonas spp. Each 100 μl from the dilution range 10 -1 – 10 -10 will be spread on the selective media Cetrimide agar. 29 The plates will be incubated at 42°C for 48h. After incubation, the presumptive colonies will be identified further by a classical biochemical method, including triple sugar iron and oxidase tests. 29

Antimicrobial susceptibility testing

The antimicrobial resistance pattern of the bacteria isolated from the vegetable and soil samples will be performed by antimicrobial susceptibility testing (AST). The determination of antimicrobial susceptibility will be performed by Kirby-Bauer disk diffusion technique using Muller Hinton Agar. 30 To check the sensitivity of the drugs, different panel of antibiotic-impregnated discs recommended for the bacterial isolates will be used as per the Clinical and Laboratory Standards Institute (CLSI) guidelines. 31

Procedure for antimicrobial susceptibility testing

For pure subculture isolation, two bacterial colonies will be taken from each the vegetable and soil sample plates. The isolated colonies will be separately mixed well under aseptic conditions in physiological saline and dissolved to get a homogenous mixture comparable to 0.5 McFarland standard. The diluted bacterial suspension will then be transferred to Muller-Hinton agar plate using sterile cotton swab and the plates will be seeded uniformly rubbing the swab against the entire agar surface. After the inoculums get dried, antibiotic impregnated disks will be applied aseptically to the surface of the inoculated plates at an appropriate distance using sterile forceps. The plates will then be incubated at 37°C for 24h. 32 The same procedure will be followed for all the bacterial isolates included in this study: E. coli, Salmonella species and Pseudomonas species.

 Analysis for antimicrobial susceptibility testing

The analysis of the antibiotic susceptibility tests will be determined by the zone of inhibition. The inhibition zone will be measured for the different antibiotics used as per the bacterial isolates. The interpretation of susceptible, intermediate and resistant categories will be assigned on the basis of the critical points recommended by the CLSI guidelines. 31

Sample processing for molecular analysis

DNA extraction:

Soil sample: The soil sample collected will be processed for DNA extraction. The HiPurA TM Soil DNA purification kit will be used for the isolation of DNA from soil samples. The protocol provided with the kit will be followed for isolation of complete DNA from the soil samples. The extracted DNA will be stored in elution buffer. The eluate containing the pure soil DNA will be stored at 2-8°C if processed within short-term (24-48 hours), otherwise for long-term duration -20°C or lower temperature (-80°C) is recommended by the manufacturer.

Plant sample: The plant parts sample collected will be processed for DNA extraction. The isolation of plant genomic DNA will be processed using HiPer® Plant Genomic DNA Extraction kit. The whole plant genomic DNA will be extracted following the protocol provided with the kit. The eluate containing pure extracted genomic DNA will be stored for short term storage (24-48 h) at 2-8°C as recommended. For long-term storage, -20°C or lower temperature (-80°C) is recommended. The Elution Buffer will help to stabilize the DNA at these temperatures.

Detection and quantification of ARGs

The extracted DNA of both, soil and plant samples, will be analyzed for the presence and the quantification of ARGs. The standardized protocol for the detection and quantification after standardization will be performed by Quantitative Polymerase Chain Reaction (qPCR). Melting curves will obtained to confirm the amplification specificity. The qPCR reactions will be performed by triplicates, using a fixed dilution of raw DNA extract.

Molecular analysis

The quantification standards will be obtained using TaqMan TM Comprehensive Microbiota control. Standard curves (Ct per log copy number) for quantification of all genes will be obtained for each run. Gene copy number of standards will be determined via the genomic DNA concentration, applying the DNA copy number and the Dilution calculator algorithms. Three technical replicates will be run for the standard curves; complete reaction mixes plus nuclease free water, but without DNA sample were used as a negative control for qPCR reactions. ARGs concentration in samples will be calculated using the standard curve equation and the measure Ct value. The quality control of raw Ct values for standard curve and unknown samples will be performed before the analysis. The limit of quantification (LOQ) will be defined as the lowest point on the linear part of the standard curve: 100 gene copies per reaction for ARGs for 16S rDNA. Copy numbers of all ARGs will be normalized to gram of sample (gene copies g −1).

 Availability of data and materials

Data from this study will be available at the Center for Development Research (ZEF), Bonn, Germany, after the completion of this study. Researchers who meet the criteria for access to confidential data are encouraged to approach Ms. Ana Maria, Coordinator Fortschrittskolleg ‘One Health’, Center for Development Research (ZEF), Bonn, Genscherallee 3, 53113 Bonn, Germany. Email: health@uni-bonn.de

Conclusion

The benefit and expected outcome of this study will be the collection of baseline data for the AMR in the fresh produce and soil in the Ahmedabad, India. This will be the first of its kind study bringing exploration of the agroecosystem and One Health approach together in India AMR loads in vegetables and corresponding soil will be linked to the agricultural practices followed by the farmers. With the objectives of this study, it will not only develop evidence on the possible routes of AMR spread and risk factors, but also will facilitate recommended WHO AMR surveillance in the plant-based agriculture through One Health approach. This vAMR study targets two broad level of dissemination. First the results will be published in the scientific journals as part of the evidence contribution from India. Second the key findings will be disseminated through the regional workshops with the policy makers, program managers, and other relevant stakeholders engaged in the concerned sectors. Recommendations from this study could be a potential source for the future surveillance of AMR for benchmarking its status, developing intervention strategies and prioritizing effective evidence-based actions. This will also provide the evidence for including the different reservoirs of environment as one of the major AMR surveillance point in the Indian National Action Plan for AMR surveillance.

 Data availability

No data are associated with this article.

Reporting guidelines

Repository: PRISMA-P checklist for ‘Vegetables as vehicle for antimicrobial resistance (vAMR): An agroecosystem exploration from the One Health perspective in India’. https://doi.org/10.6084/m9.figshare.22188334. 33

Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).

 Acknowledgements

We wish to thank the team of experts from the Ahmedabad Municipal Corporation, Department of Health & Family Welfare, Department of Animal Husbandry, Government of Gujarat, India, and selected agricultural farmers from Ahmedabad city, India for their valuable inputs and suggestions in the development phase of the study. We would also like to thank our colleagues at the Centre for One Health Education, Research & Development, Indian Institute of Public Health Gandhinagar, India, for facilitating the study permission and providing all the administrative support for the same.

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Total Environ. 2017;581-582:32–39. 10.1016/j.scitotenv.2016.12.138 He Y Yuan Q Mathieu J : Antibiotic resistance genes from livestock waste: occurrence, dissemination, and treatment. npj Clean Water. 2020;3:1–11. 10.1038/s41545-020-0051-0 Zhang L Kinkelaar D Huang Y : Acquired antibiotic resistance: Are we born with it? Appl. Environ. Microbiol. 2011;77:7134–7141. 21821748 10.1128/AEM.05087-11 PMC3194877 Ondon BS Li S Zhou Q : Sources of Antibiotic Resistant Bacteria (ARB) and Antibiotic Resistance Genes (ARGs) in the Soil: A Review of the Spreading Mechanism and Human Health Risks. Rev. Environ. Contam. Toxicol. 2021;256:121–153. 33948742 10.1007/398_2020_60 Chen QL Cui HL Su JQ : Antibiotic Resistomes in Plant Microbiomes. Trends Plant Sci. 2019;24:530–541. 10.1016/j.tplants.2019.02.010 Zhang YJ Hu HW Chen QL : Transfer of antibiotic resistance from manure-amended soils to vegetable microbiomes. Environ. Int. 2019;130:104912. 31220751 10.1016/j.envint.2019.104912 Bhunia AK Liu Y Kniel K : Characteristics and Global Occurrence of Human Pathogens Harboring Antimicrobial Resistance in Food Crops: A Scoping Review. 2022;6:824714. Rahman M Alam M-U Luies SK : Contamination of Fresh Produce with Antibiotic-Resistant Bacteria and Associated Risks to Human Health: A Scoping Review. Int. J. Environ. Res. Public Health. 2021;19:360. 35010620 10.3390/ijerph19010360 PMC8744955 Alegbeleye OO Singleton I Sant’Ana AS : Sources and contamination routes of microbial pathogens to fresh produce during field cultivation: A review. Food Microbiol. 2018;73:177–208. 29526204 10.1016/j.fm.2018.01.003 PMC7127387 Ministry of Home Affairs: Census of India Website: Office of the Registrar General & Census Commissioner, India. 2011. Accessed 25 Nov 2021. Reference Source Times of India: “Veg” Gujarat has 40% non-vegetarians|Ahmedabad News - Times of India. 2016. Accessed 25 Nov 2021. Reference Source Gujarat Livelihood Promotion: Horticulture - Sector Overview|Sector wise Development|Activities|Gujarat Livelihood Promotion Company. 2011. Accessed 25 Nov 2021. Reference Source Masterton R : The importance and future of antimicrobial surveillance studies. Clin. Infect. Dis. 2008;47:S21–S31. 10.1086/590063 WHO: Joint FAO/WHO expert meeting in collaboration with OIE on foodborne antimicrobial resistance: role of the environment, crops and biocides: meeting report. 2018. Accessed 17 Nov 2021. Reference Source Schwaiger K Helmke K Hölzel CS : Antibiotic resistance in bacteria isolated from vegetables with regards to the marketing stage (farm vs. supermarket). Int. J. Food Microbiol. 2011;148:191–196. 21700353 10.1016/j.ijfoodmicro.2011.06.001 Cerqueira F Matamoros V Bayona JM : Antibiotic resistance gene distribution in agricultural fields and crops. A soil-to-food analysis. Environ. Res. 2019;177:108608. 10.1016/j.envres.2019.108608 Abdus Sobur M Momen Sabuj A al Sarker R : Antibiotic-resistant Escherichia coli and Salmonella spp. associated with dairy cattle and farm environment having public health significance. Vet World. 2019;12:984–993. 31528022 10.14202/vetworld.2019.984-993 PMC6702575 Antony AC Paul MK Silvester R : Comparative evaluation of EMB agar and hicrome E. coli agar for differentiation of green metallic sheen producing non E. Coli and typical E. Coli colonies from food and environmental samples. J. Pure Appl. Microbiol. 2016;10:2863–2870. 10.22207/JPAM.10.4.48 Kannan MN Badoni A Chamoli V : Advances in Agriculture and Natural Sciences for Sustainable Agriculture (October 12 &13, 2018) Isolation and characterization of bacterial isolates from agriculture field soil of Roorkee region. ~ 108 ~. J. pharmacogn. phytochem. 2018;5:108–10. Ruiz-Roldán L Rojo-Bezares B Lozano C : Occurrence of pseudomonas spp. In raw vegetables: Molecular and phenotypical analysis of their antimicrobial resistance and virulence-related traits. Int. J. Mol. Sci. 2021;22:12626. 34884433 10.3390/ijms222312626 PMC8657893 EUCAST: Disk Diffusion Method for Antimicrobial Susceptibility Testing-Antimicrobial susceptibility testing EUCAST disk diffusion method. 2021. Weinstein MP Patel JB Bobenchik AM : M100 Performance Standards for Antimicrobial Susceptibility Testing A CLSI supplement for global application. Performance Standards for Antimicrobial Susceptibility Testing Performance Standards for Antimicrobial Susceptibility Testing. 2020. Garedew L Hagos Z Addis Z : Prevalence and antimicrobial susceptibility patterns of Salmonella isolates in association with hygienic status from butcher shops in Gondar town, Ethiopia. Antimicrob. Resist. Infect. Control. 2015;4:1–7. Kalpana P Falkenberg T Yasobant S : PRISMA-P-checklist_vAMR_Kalpana P et al, 2023.doc.figshare. Online resource.2023. 10.6084/m9.figshare.22188334.v1 10.5256/f1000research.144543.r195948 Reviewer response for version 1 Calero-Cáceres William 1 Referee Department of Food and Biotechnology Science and Engineering, Technical University of Ambato, Ambato, Tungurahua, Ecuador

Competing interests: No competing interests were disclosed.

 25 9 2023 Copyright: © 2023 Calero-Cáceres W 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation reject

The manuscript by Kalpana et al. offers a compilation of methodologies intended to address two research questions: "1. Which antibiotic-resistant bacteria (ARB) and antibiotic-resistant genes (ARGs) are prevalent, or presumed to be widespread, within the soil-plant-fruit continuum in the South Asia region?" and "2. What is the risk of AMR spreading across the soil-plant-fruit continuum in agricultural farms of Ahmedabad, India?" The article is classified as a "Study Protocol"-type and is supported by F1000Research.

However, the title "Vegetables as a vehicle for antimicrobial resistance (vAMR): An agroecosystem exploration from the One Health perspective in India" implies that conclusive research has been conducted, leading readers to expect evidence within the article supporting the notion that vegetables act as vectors of AMR determinants. I recommend modifying the title to more accurately reflect the content, emphasizing that this manuscript primarily proposes protocols for similar studies.

The criteria for the "Selection of AMR genes (ARGs) for molecular analysis" are vague. Why only two ARGs? Most studies often include common ARGs like sul1, tetA, ermB. The rationale should be modified to emphasize clinically relevant or emerging ARGs.

Concerning the source of data and data collection methods, the reasoning behind choosing 300 samples needs a thorough explanation.

In the section on the identification and enumeration of bacteria, standardized methodologies like ISO or BAM should be detailed. The mentioned methodologies lack validation. For instance, direct plating is suggested for Salmonella enumeration, but ISO 6579 recommends pre-enrichment and enrichment steps. The methodological limitation for Salmonella does not allow direct quantification from the sample.

In procedures for antimicrobial susceptibility testing, the rationale for selecting specific antibiotics should be included. As it stands, the description does not significantly contribute to current knowledge or provide guidance for conducting studies as suggested.

Regarding molecular analysis, it is mentioned that qPCR will be applied for the quantification of specific ARGs. However, the selection rationales for these genes are missing. Please include references detailing the initial validation of the techniques you intend to develop.

Based on my experience, this article is currently incomplete and requires more in-depth analysis to serve as a foundational resource for studies about AMR following the One Health approach. I earnestly recommend publishing your article once the protocols are described in greater detail and, ideally, results are included.

Is the study design appropriate for the research question?

No

Is the rationale for, and objectives of, the study clearly described?

Partly

Are sufficient details of the methods provided to allow replication by others?

No

Are the datasets clearly presented in a useable and accessible format?

Not applicable

Reviewer Expertise:

Antibiotic resistance, bacteriophages, molecular analysis, eDNA, Salmonella, Whole-genome sequencing

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

Kalpana Pachillu University of Bonn, Germany

Competing interests: No competing interests were disclosed.

 14 3 2024

Comment 1: The manuscript by Kalpana et al. offers a compilation of methodologies intended to address two research questions: "1. Which antibiotic-resistant bacteria (ARB) and antibiotic-resistant genes (ARGs) are prevalent, or presumed to be widespread, within the soil-plant-fruit continuum in the South Asia region?" and "2. What is the risk of AMR spreading across the soil-plant-fruit continuum in agricultural farms of Ahmedabad, India?" The article is classified as a "Study Protocol"-type and is supported by F1000Research.

Response: Thanks for your time reviewing the work and your kind reflections.

Changes: No change

Comment 2: However, the title "Vegetables as a vehicle for antimicrobial resistance (vAMR): An agroecosystem exploration from the One Health perspective in India" implies that conclusive research has been conducted, leading readers to expect evidence within the article supporting the notion that vegetables act as vectors of AMR determinants. I recommend modifying the title to more accurately reflect the content, emphasizing that this manuscript primarily proposes protocols for similar studies.

Response: You are totally right; thanks for the advice. The title has been changed to:

''Agroecosystem exploration for Antimicrobial Resistance in Ahmedabad, India: A Study Protocol.''

Changes: The title of the manuscript has been revised.

Comment 3: The criteria for the "Selection of AMR genes (ARGs) for molecular analysis" are vague. Why only two ARGs? Most studies often include common ARGs like sul1, tetA, ermB. The rationale should be modified to emphasize clinically relevant or emerging ARGs.

Response: Thank you for pointing this out. The sentence reflected the need to select at least two; however, if the review finds more, they will also be included in the molecular analysis process. To clear up any misunderstanding, the sentence has been rephrased. Also, with the updated investigation plan, a greater number of ARGs can also be analysed.

Changes: The manuscript is updated from lines 268-270 in track changes mode. 

Comment 4: Concerning the source of data and data collection methods, the reasoning behind choosing 300 samples needs a thorough explanation.

Response: Thanks for your kind reflections.

The manuscript has been updated to clarify the idea of the samples. In the first step, 300 farms will be selected for the survey, and in the second step, 300 samples, including 150 vegetable samples and 150 soil samples, will be collected. Choosing 300 samples for the survey depends heavily on funding availability and the time frame. We have added the same limitation.

“The limitation is because of funding and time frame available.”

Changes: The manuscript is updated from lines 221-222 and 226-231 in track changes mode.

Comment 5: In the section on the identification and enumeration of bacteria, standardized methodologies like ISO or BAM should be detailed. The mentioned methodologies lack validation. For instance, direct plating is suggested for Salmonella enumeration, but ISO 6579 recommends pre-enrichment and enrichment steps. The methodological limitation for Salmonella does not allow direct quantification from the sample.

Response: Thank you for your suggestion. The reason for the identification and enumeration of bacteria not according to standardized methods like ISO and BAM is that those are developed and optimized for human samples or other samples containing low background flora, such as drinking water or food. Environmental samples, e.g., soil and plants, as investigated here, or even wastewater or manure, contain much more background flora, which interferes with the culture assays up to non-analyzability, especially if target bacteria concentrations are low.

For medical/hygienic surveillance purposes, pre-enrichment is okay, but that leads to non-quantifiable results concerning the original concentrations in a sample.

Choosing a methodology that is known to work with environmental samples and, moreover, is adapted to them seems to be more useful for our research objective, and the intended results are more likely to be yielded. Therefore, the published articles that have reported the data on the same have been used as reference for the methodology.

Changes: The manuscript has been updated, and a note has been added from lines 346-357 in track changes mode.

Comment 6: In procedures for antimicrobial susceptibility testing, the rationale for selecting specific antibiotics should be included. As it stands, the description does not significantly contribute to current knowledge or provide guidance for conducting studies as suggested.

Response: You are right; we missed that information in part.

The antibiotic panel selection is mainly based on the pre-selected bacteria. We added information about the selection of antibiotics for susceptibility testing into the chapter “Method for research question 2.”

Concerning the testing method, we are not sure if the sentence was not clearly describing the rationale.

We revised the text of the three sub-chapters dealing with antimicrobial susceptibility testing.

Changes: The manuscript is updated from lines 260-265 in track changes mode.

Comment 7: Regarding molecular analysis, it is mentioned that qPCR will be applied for the quantification of specific ARGs. However, the selection rationales for these genes are missing. Please include references detailing the initial validation of the techniques you intend to develop.

Response: The manuscript has been updated concerning molecular analysis to improve the data and answer the objectives. We switched to whole metagenomic analysis instead of qPCR because of a discussion with the labs working in the field of AMR. It has been argued that the qPCR method is more sensitive and suitable for absolute quantification of target genes, while whole metagenomics has the potential to provide an overview of the genes as well as of the respective relative abundance of the antibiotic resistance genes. This switch will help focus on the selected genes through review and also find the presence of new resistant genes.

The rationale behind the selection of ARGs depends on the first objective, which will provide evidence-based and reported data on the presence of ARGs in Southeast Asia for better geographical comparisons.

Changes: The manuscript is updated from lines 416-527 in track changes mode.

Comment 8: Based on my experience, this article is currently incomplete and requires more in-depth analysis to serve as a foundational resource for studies about AMR following the One Health approach. I earnestly recommend publishing your article once the protocols are described in greater detail and, ideally, results are included.

Response: Thanks for your reflection. The idea of having a protocol paper prior to the real field data collection is to provide insight into the suggested study methods. We value your suggestion and will prepare a separate manuscript once the data collection and evaluation are completed. We hope that the recent changes made to the manuscript will now be sufficient to provide a resource for conducting studies in this field of AMR.

Changes: The whole manuscript has been updated.

 10.5256/f1000research.144543.r187877 Reviewer response for version 1 Schwaiger Karin 1 Referee https://orcid.org/0000-0001-5317-6892 Unit of Food Hygiene and Technology, Institute for Food Safety, Food Technology and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria

Competing interests: No competing interests were disclosed.

 11 8 2023 Copyright: © 2023 Schwaiger K 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

The authors describe a study design to determine the extent to which vegetables are a vehicle for antimicrobial resistance in Ahmedabad, India. For this purpose, they first intend to conduct a systematic review on the occurrence of antibiotic resistant bacteria (ARB) and AR genes (ARGs) on vegetables (and in soil) in South Asia. This is followed by agriculture farmland surveys to find out, among other things. about general agricultural practices and prevailing household consumption patterns. Based on the results of the upstream research, vegetable and soil samples will be collected from selected farms and analyzed for the presence of ARB and ARGs using bacteriological and molecular biological techniques.

Overall, the study is logically and consecutively structured and explained in an understandable way. It should be emphasized that the authors apparently already have practical experience, as they are aware of some difficulties (e.g., lack of databases of agricultural farms), but found solutions for these (e.g., snowball system). It is also very good that an ethical statement is already available. The rationale for, and objectives of, the study are clearly described – although I think the term “One Health” is somewhat overused or lofty. It is true that antibiotic-resistant bacteria may play an important role for humans along the food chain. This study aims to determine prevalences, which of course can lead to a better understanding and consequently (indirectly by implementing appropriate measures/intervention strategies) also to a risk reduction. Humans themselves or samples of them, however, are not taken into account in the investigations. In this respect, I would recommend using the term "One Health" in a somewhat less inflationary way.

I have a few more comments regarding the wording of the methodology:

Introduction: The authors state that “The prophylactic use and practice of sub-lethal doses of antibiotics in livestock as growth promoters to maintain health and productivity contribute to a significant part.” to the AMR issue. I would be careful with this wording: In many countries, growth promoters have long been banned. Of course, this does not mean that antibiotics can no longer be used at all in animals, but only for therapeutic purposes (not prophylactic).

Correctly, the authors recognize that organic fertilizers (e.g., manure) may play an important role in ARB transmission. They also seem to take this important point into account in their own research (important data would be e.g. animal species, storage time, temperature, hygienization measures). When later comparing the results of the present study with those of other countries, it should be recognized that in vegetable cultivation often no organic fertilizers (only mineral fertilizers) are used for hygienic reasons (e.g. in Germany). This might then explain (besides irrigation water) differences in the prevalences of certain (AR) bacteria (especially faecal indicators such as E. coli or Salmonella).

Methods for research question 1: The authors will conduct a systematic review. They state that data will be extracted and the results will form an evidence base for the selection of ARB and ARGs for the next objective of the study. This is basically a valid approach. However, since the review focuses on Southeast Asia and not much data is available here yet, this may miss some important ARB and ARGs. Therefore, I would suggest expanding the search in this regard by including studies from other countries as well. In any case, antibiotics important in human medicine should be considered (e.g. according to WHO/Ranking of medically important antimicrobials).

Methods for research question 2: The AMR field investigation seems to be very well thought out. A personal visit to the farms is also very important in my experience to inform the farmers about the basic idea and to arouse interest in participating. The selection of bacteria is also sensible in principle (faecal indicators, pathogens, environmental bacteria), but I am not sure if a sufficient number of E. coli and Salmonella will be isolated to do a statistical analysis afterwards (the prevalence in other studies is quite low). However, since the authors state that they will include the results of their systematic review anyway, I assume this will not be a problem (but then, not only the occurrence of ARBs should be searched but also that of bacteria on vegetables in general).

Selection of AMR genes: See comment in “Methods for research question 1”.

Sampling procedure: It is not clear to me how many samples are actually to be taken: first it says "Five vegetable samples....will be collected", but then at "Vegetable sampling" it says “A total of one vegetable sample…will be collected”. How does that come about? Is it related to the 5 different vegetable types (top five)? Please clarify/correct/rephrase.

Identification and enumeration of bacteria: Possibly, one could consider investigating the aerobic mesophilic bacterial count as well, if bacteriology is performed anyway. This is only a small additional effort (even if the soil samples probably have to be diluted more than to 10^-10) and provides information about the general hygiene status of vegetables from this region (after following safe handling procedures). E. coli: the authors state that E. coli will be confirmed by a positive indole test. Since other Enterobacteriaceae are indole positive as well, I assume that the mentioned “biochemical confirmation” is able to differentiate these species from E. coli? Salmonella: I think that for Salmonella detection in this highly heterogeneous microbiota in the sample material, pre-enrichment is mandatory. It is also important to use not only peptone water but buffered peptone water to neutralize the acid produced by other competing bacteria. Please follow established protocols.

Antimicrobial susceptibility testing: The authors will use the disk diffusion technique. This is basically a valid method – but why not using microdilution? In my opinion, this method would be better suited for the intended high throughput. Anyhow, it should follow an established protocol. In the description of the procedure it is not quite clear if it is intended to take the two bacterial colonies directly from the selective agar plates (it is important to take the colonies from neutral agar plates incubated in consistent times and temperatures). Since a biochemical confirmation of the colonies was conducted beforehand, I think my concern is unfounded, but please clarify. It is also not clear if a total of two bacterial colonies are taken or two bacterial colonies from each duplicate.

Molecular analysis: Plant sample: Why using a plant genomic DNA extraction kit if it is intended to investigate bacterial DNA? Of course, there might be some bacterial endophytes in the plant cells, but that is certainly not the overwhelming majority – most of the bacteria will be contaminants on the surface of the vegetables. Detection and quantification of ARGs: This part of the investigation seems a bit separated from the rest of the study. Does that mean that the isolated bacteria will not be tested for ARGs, only the sample material (soil, vegetables) itself? This can be done, but it should be kept in mind that this will limit the informative value: Since many resistance genes are found in different bacterial species and, additionally, the microbiota differ greatly among different vegetable species, the results should be interpreted very carefully (e.g., only a very small fraction of the tetracyline resistance genes will origin from E. coli, but no definite statement is possible). It is also not possible to determine multidrug resistances, and it cannot be differentiated between opportunists and pathogens (e.g. for risk estimation). Standard curve: If only the sample material itself will be investigated for the presence of ARGs: I assume that the standard curve is created in the respective DNA-free (e.g. irradiated) sample material (vegetables, soil suspensions) in order to take any matrix effects into account?

Is the study design appropriate for the research question?

Yes

Is the rationale for, and objectives of, the study clearly described?

Yes

Are sufficient details of the methods provided to allow replication by others?

Partly

Are the datasets clearly presented in a useable and accessible format?

Not applicable

Reviewer Expertise:

Microbiology, antibiotic resistance, bacteriology, molecular biology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Kalpana Pachillu University of Bonn, Germany

Competing interests: No competing interests were disclosed.

 14 3 2024

Comment 1: The authors describe a study design to determine the extent to which vegetables are a vehicle for antimicrobial resistance in Ahmedabad, India. For this purpose, they first intend to conduct a systematic review on the occurrence of antibiotic resistant bacteria (ARB) and AR genes (ARGs) on vegetables (and in soil) in South Asia. This is followed by agriculture farmland surveys to find out, among other things. about general agricultural practices and prevailing household consumption patterns. Based on the results of the upstream research, vegetable and soil samples will be collected from selected farms and analyzed for the presence of ARB and ARGs using bacteriological and molecular biological techniques.

Response: Thanks for your time and reviewing the draft in detail.

Changes: NA

Comment 2: Overall, the study is logically and consecutively structured and explained in an understandable way. It should be emphasized that the authors apparently already have practical experience, as they are aware of some difficulties (e.g., lack of databases of agricultural farms), but found solutions for these (e.g., snowball system). It is also very good that an ethical statement is already available.

Overall, the study is logically and consecutively structured and explained in an understandable way. It should be emphasized that the authors apparently already have practical experience, as they are aware of some difficulties (e.g., lack of databases of agricultural farms), but found solutions for these (e.g., snowball system). It is also very good that an ethical statement is already available.

The rationale for, and objectives of, the study are clearly described – although I think the term “One Health” is somewhat overused or lofty. It is true that antibiotic-resistant bacteria may play an important role for humans along the food chain. This study aims to determine prevalence, which of course can lead to a better understanding and consequently (indirectly by implementing appropriate measures/intervention strategies) also to a risk reduction. Humans themselves or samples of them, however, are not taken into account in the investigations. In this respect, I would recommend using the term "One Health" in a somewhat less inflationary way.

Response: Thank you very much for your kind review. Yes, as a part of the study, the base information was gathered before planning for the study and to be aware of the difficulties during the process. The ethical clearance was obtained from both institutions for conducting the study.

Thank you. You also very well mentioned the importance of ARB and its role in humans through the food chain, which describes it well as a One Health concept. But we agree completely that if we are not able to approach all the sectors of One Health, it is better to use the term, however, keeping it less inflammatory. Taking that into account, the manuscript has been updated.

Changes: The whole manuscript is updated.

Comment 3: I have a few more comments regarding the wording of the methodology:

Introduction: The authors state that “The prophylactic use and practice of sub-lethal doses of antibiotics in livestock as growth promoters to maintain health and productivity contribute to a significant part.” to the AMR issue. I would be careful with this wording: In many countries, growth promoters have long been banned. Of course, this does not mean that antibiotics can no longer be used at all in animals, but only for therapeutic purposes (not prophylactic).

Response: Thank you. I agree that many developed countries banned the use of antibiotics as growth promoters and, therefore, reframed the sentence to make it much more globally sound. There are countries such as India where, due to a lack of policies for antibiotic use in animals, prophylactic use still plays a very prominent role.

Changes: The manuscript is updated from lines 15-23 in track changes mode.

Comment 4: Correctly, the authors recognize that organic fertilizers (e.g., manure) may play an important role in ARB transmission. They also seem to take this important point into account in their own research (important data would be e.g. animal species, storage time, temperature, hygienization measures). When later comparing the results of the present study with those of other countries, it should be recognized that in vegetable cultivation often no organic fertilizers (only mineral fertilizers) are used for hygienic reasons (e.g. in Germany). This might then explain (besides irrigation water) differences in the prevalences of certain (AR) bacteria (especially faecal indicators such as E. coli or Salmonella).

Response: Thank you for taking this point into account. This will be a good point of view to see during analysis. During the comparison of results, this might be a good explanation point for the results.

Changes: No changes

Comment 5: Methods for research question 1: The authors will conduct a systematic review. They state that data will be extracted and the results will form an evidence base for the selection of ARB and ARGs for the next objective of the study. This is basically a valid approach. However, since the review focuses on Southeast Asia and not much data is available here yet, this may miss some important ARB and ARGs. Therefore, I would suggest expanding the search in this regard by including studies from other countries as well. In any case, antibiotics important in human medicine should be considered (e.g. according to WHO/Ranking of medically important antimicrobials).

Response: Yes, we agree that the scope of review in only including the published literature from Southeast Asia might lead to skip other important ARB and ARGs reported from other countries. However, one of the major reasons for only selecting these areas is to have a focus on scarcity of data from this region and concentrate attention on the need for more studies in this area. This is also decided as per the mandate of the funded project.

Regarding the WHO suggestion, the selection of bacteria is according to the WHO guidance for the importance of their role in plant-based food and their role in spread of AMR.

Changes: No changes

Comment 6: Methods for research question 2: The AMR field investigation seems to be very well thought out. A personal visit to the farms is also very important in my experience to inform the farmers about the basic idea and to arouse interest in participating. The selection of bacteria is also sensible in principle (faecal indicators, pathogens, environmental bacteria), but I am not sure if a sufficient number of E. coli and Salmonella will be isolated to do a statistical analysis afterwards (the prevalence in other studies is quite low). However, since the authors state that they will include the results of their systematic review anyway, I assume this will not be a problem (but then, not only the occurrence of ARBs should be searched but also that of bacteria on vegetables in general).

Response: Thank you for appreciating the investigation plans. The review will provide us with the idea of prevalence, as you have also quite clearly mentioned, which will help us in the process. I agree with you about the importance of including the total occurrence of bacteria on vegetables, which can have added value. However, the limited time and resources available to complete the project led to the compromise of this idea, focusing on some specific bacteria with much more in focus according to the WHO guidelines.  Indeed, our final analysis method does not only include ARB because identification is done in two steps: first, cultivation of bacteria species of interest, and second, resistance tests of these isolates. That was a result of the review and practical possibilities in the labs, therefore not clear when writing this study concept.

Changes: No changes

Comment 7: Selection of AMR genes: See comment in “Methods for research question 1”.

Sampling procedure: It is not clear to me how many samples are actually to be taken: first it says "Five vegetable samples....will be collected", but then at "Vegetable sampling" it says “A total of one vegetable sample…will be collected”. How does that come about? Is it related to the 5 different vegetable types (top five)? Please clarify/correct/rephrase.

Response: I hope the reply to the comment for research question 1 clarifies this one here.

The top five vegetables will be selected after the survey. During the sample collection, one vegetable sample and its soil sample will be collected in duplicate from every selected vegetable-producing farm. The collected samples from all investigated farms together will represent the top five vegetables. The sentence has been rephrased for clarification.

Changes: The manuscript is updated from lines 275-286 in track changes mode.

Comment 8:Identification and enumeration of bacteria: Possibly, one could consider investigating the aerobic mesophilic bacterial count as well, if bacteriology is performed anyway. This is only a small additional effort (even if the soil samples probably have to be diluted more than to 10^-10) and provides information about the general hygiene status of vegetables from this region (after following safe handling procedures). E. coli: the authors state that E. coli will be confirmed by a positive indole test. Since other Enterobacteriaceae are indole positive as well, I assume that the mentioned “biochemical confirmation” is able to differentiate these species from E. coli? Salmonella: I think that for Salmonella detection in this highly heterogeneous microbiota in the sample material, pre-enrichment is mandatory. It is also important to use not only peptone water but buffered peptone water to neutralize the acid produced by other competing bacteria. Please follow established protocols.

Response: I hope the reply to the comment for research question 1 clarifies this one here.

The top five vegetables will be selected after the survey. During the sample collection, one vegetable sample and its soil sample will be collected in duplicate from every selected vegetable-producing farm. The collected samples from all investigated farms together will represent the top five vegetables. The sentence has been rephrased for clarification.

We agree that investigating the aerobic mesophilic bacterial count will be a small additional effort, but as you mentioned, soil samples will need to be diluted more. Additionally, we use selective agar for coliforms. Therefore, we have considered not only reporting E. coli but also accounting for the total coliform count for the samples, which is often an indicator of cleanliness and hygiene.

Regarding the biochemical confirmation of E. coli with indole due to the very selective media that will be used for the isolation of E. coli. Although the manufacturer says there could be growth of Salmonella in very rare cases. The bacterial isolates can be differentiated with their color on the agar plates but indole positive E. coli and Salmonella which is an indole negative bacterium can be a confirmatory test that while proceeding further there is no contamination. However, with the consultation with labs, we considered going with IMViC tests to differentiate the Enterobacteriaceae family members further.

The pre-enrichment process for Salmonella detection is well understood. The suggestion provided is well taken and the buffered peptone water will be used for the process. The BPA will be used for the non- selective enrichment process which will be followed by the very selective media for isolation of Salmonella.  The authors thought of following the published articles protocol due to the non-sample specific protocol for Salmonella detection in established protocols such as BAM during the manuscript preparation. But doing pre-enrichment, we are not able to count real Salmonella concentrations. The researchers of the articles which has been referenced in the manuscript have used the same media and method for the isolation of Salmonella from vegetable samples as we choose, and they were able to find relevant numbers of Salmonella. 

Changes: The manuscript is updated from lines 321-331 in track changes mode.

Comment 9: Antimicrobial susceptibility testing: The authors will use the disk diffusion technique. This is basically a valid method – but why not using microdilution? In my opinion, this method would be better suited for the intended high throughput. Anyhow, it should follow an established protocol. In the description of the procedure it is not quite clear if it is intended to take the two bacterial colonies directly from the selective agar plates (it is important to take the colonies from neutral agar plates incubated in consistent times and temperatures). Since a biochemical confirmation of the colonies was conducted beforehand, I think my concern is unfounded, but please clarify. It is also not clear if a total of two bacterial colonies are taken or two bacterial colonies from each duplicate.

Response: The disk diffusion method is been chosen due to its much use and less expense in manual work regarding less technical equipment as compared to the microdilution method. You can test more than one antibiotic in parallel on the same plate, whereas microdilution needs a single dilution series for every antibiotic.

In the beginning of the study, during the conceptual phase, it was not clear how many bacterial colonies will be found during the process. The idea is to test several, but eventually not all bacterial colonies (depending on their numbers found and the workload we can afford) from both, the vegetable and the respective soil, to compare their resistance patterns. The text is revised.

Changes: The manuscript is updated from lines 360-363 in track changes mode.

Comment 10: Molecular analysis: Plant sample: Why using a plant genomic DNA extraction kit if it is intended to investigate bacterial DNA? Of course, there might be some bacterial endophytes in the plant cells, but that is certainly not the overwhelming majority – most of the bacteria will be contaminants on the surface of the vegetables. Detection and quantification of ARGs: This part of the investigation seems a bit separated from the rest of the study. Does that mean that the isolated bacteria will not be tested for ARGs, only the sample material (soil, vegetables) itself? This can be done, but it should be kept in mind that this will limit the informative value: Since many resistance genes are found in different bacterial species and, additionally, the microbiota differ greatly among different vegetable species, the results should be interpreted very carefully (e.g., only a very small fraction of the tetracyline resistance genes will origin from E. coli, but no definite statement is possible). It is also not possible to determine multidrug resistances, and it cannot be differentiated between opportunists and pathogens (e.g. for risk estimation). Standard curve: If only the sample material itself will be investigated for the presence of ARGs: I assume that the standard curve is created in the respective DNA-free (e.g. irradiated) sample material (vegetables, soil suspensions) in order to take any matrix effects into account?

Response: The protocol has been updated concerning the extraction kit that will ultimately be used for DNA extraction in the study. This results from a methodical harmonization with the other researchers who are working in this field, and it was previously shown that the kit worked on both types of samples selected in this study.

The disk diffusion method will be used to test the isolated bacteria for AMR susceptibility. Gene detection of ARG within individual isolates will not be done. We know that, thus, we underestimate phenotypically expressed resistances and do not detect non-expressed multi-resistances, but we thought that ARG occurrence in the environment (soil and samples) does tell us more about the resistance spread of human-relevant antibiotics.

ARG analysis in both the isolates and samples is out of our monetary budget and available time frame. Thus, we had to restrict and select the better analysis for our research objectives and keep in mind that this will limit the informative value, as you stated. However, the sample will be investigated by isolating the DNA from the complete sample using the whole metagenomic sequencing method. The AST patterns will provide us with information regarding the specific bacteria selected as per WHO suggestions, and the Whole metagenomic sequencing will provide us with more in-depth information of ARGs. This can also help the researchers compare the much better methodology to be followed in the near future and according to the budget. Whole metagenomic sequencing will help identify and cluster many gene fragments into groups. The resistant genes will be identified using the AMR databases and the potential connections of metagenomic results to phenotypically observed data. With the changed investigation plan, I hope the standard curve will no longer be required.

Changes: The manuscript is updated from lines 399-411 and 416-527  in track changes mode.

Thank you very much for your comments and suggestions and for reviewing the manuscript. I hope we clarified them and updated the manuscript for better understanding and adaptability.