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

Abstract

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, animal, agriculture, and the environment. Antibiotic use in any of these areas can potentially impact the health of others. 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 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.

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 exploitation of antibiotics in food animals which is not limited to their therapeutic use but also for metaphylaxis (administration of antibiotics when contact with diseased animal), prophylaxis (mass administration of antibiotics to animals when risk is established to prevent disease) and as antibiotic growth promoters (administration of antibiotics to animals to boost feed efficiency and increase weight gain) has amplified the extend of AMR globally. Despite many of the developed countries banned the use of antibiotics as growth promoters, in other countries such as India because of the lack of policies for antibiotic use in animals the prophylactic use still marks a very prominent role. 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 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. ³⁵

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 in Ahmedabad city, India.

The specific research questions are:

1.

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?

2.

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

2.1.

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

2.2.

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

Figure 1.

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.

Table 1.

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 limitation has to be done because of funding plan and time frame available. 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 maximum enrolled 300 farms which will be surveyed for the general agricultural practices will then be assessed for the vegetable producing farms. About 150 vegetable producing farms (maximum half of total farms surveyed) 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 of sample collection and further microbiological and molecular analysis.

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:

a.

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

b.

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

c.

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.

The selection of antibiotics for susceptibility testing of the selected three bacteria is based on the CLSI guidelines. ²⁴ The following antibiotics selected for testing in this study can be found in Table 2.

Table 2.

Selection of antibiotics for susceptibility testing of ARB isolates.

Antibiotic class	Antibiotic	Concentration (mcg)	Abbreviation
Aminoglycosides	Amikacin	30	AK
	Gentamycin	10	GEN
	Tobramycin	10	TOB
Beta-lactam combination agent (BLCA)	Amoxicillin-clavulanate	20/10	AMC
Carbapenems	Ertapenem	10	ETP
	Doripenem	10	DOR
	Imipenem-relebactam	10/750	IE
	Meropenem	10	MRP
Cephalosporins	Cefazolin	30	CZ
	Cefepime	50	CPM
	Cefotaxime	5	CTX
	Ceftriaxone	10	CTR
	Cefuroxime	30	CXM
Fluoroquinolones	Ciprofloxacin	1	CIP
	Levofloxacin	5	LE
Folate pathway	Trimethoprim	5	TR
Monobactam	Aztreonam	50	AT
Penicillins	Ampicillin	10	AMP
	Piperacillin	30	PI
	Ticarcillin-clavulanate	75/10 mcg	TCC
Phenicol	Chloramphenicol	10	C
Tetracyclines	Tetracycline	10	TE

Supplementary antibiotics to be tested may result on relevant resistances found in of the systematic review to be conducted for research question 1.

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. The selection of ARGs will be based on the reported ARGs in the context of vegetables and its producing environment 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. The 150 farms identified as vegetable producing farms from the first survey step will be used for collecting a total of 300 samples, which will include 150 vegetable and respective 150 soil samples. 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. In anticipation that every farm produces only limited numbers of vegetables, othe top five vegetables selected as the major produced and consumed raw within the study area will be sampled along with the consecutive soil sample in duplicate from each selected farm. A simple random sampling method will be used for collecting vegetable samples from each farm in such a way, that in the end all samples from all investigated farms together will represent the top five vegetables.

Sample collection

a)

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. ²⁵

b)

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. ²⁶

Sample processing:

a)

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. ²⁷

b)

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. ²⁷ 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) ²⁸ 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. As per manufacturer information, after incubation, E. coli will grow as blue-green colonies, other coliform bacteria which can grow on the selected agar Salmonella enteritidis will grow different with showing colorless colonies aiding in proper differentiation process. The isolates will be confirmed biochemically by the use of IMViC (Indole, Methyl red, Voges Proskauer and Citrate utilization) tests and to differentiate members of the family Enterobacteriaceae. The results of the total and confirmed E. coli count will be expressed as a colony-forming unit (CFU)/g of sample. ²⁹

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. ²⁷ 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. ²⁷

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. ³⁰ 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. ³⁰

Note: The reason of doing 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 interfere 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 which is known to work with environmental samples, and moreover, is adopted to them, seems to be more useful for our research objective, and that intended results are more likely to be yielded. Therefore, the published articles which have reported the data on the same has been used as reference for the methodology.

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). It is planned to test equal percentage of selected species for e.g., 20% of positive samples of each species, but this is expected to vary during the analysis process depending on sample contamination and number of positive samples and colonies grown. The determination of antimicrobial susceptibility will be performed by Kirby-Bauer disk diffusion technique using Muller Hinton Agar. ³¹ To check the sensitivity of the drugs, different panel of antibiotic-impregnated discs recommended for the target bacteria of this study will be used as suggested by the Clinical and Laboratory Standards Institute (CLSI) guidelines. ²⁴

Procedure for antimicrobial susceptibility testing

The bacterial colonies will be further streaked on a nutrient agar for gaining pure cultures of the isolates, and as pre-culturing for initiating the susceptibility testing. The bacterial colonies will be taken from vegetable and the respective soil samples. 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. Selection of antibiotics based on target species tested, as stated above in detail. The interpretation of susceptible, intermediate and resistant categories will be assigned on the basis of the critical points recommended by the CLSI guidelines. ²⁴

Sample processing for molecular analysis

I.

DNA extraction for microbiome and ARG analysis:

a)

Soil sample: The soil sample collected will be processed for DNA extraction. The Qiagen DNeasy PowerSoil 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. ³³

b)

Plant sample: The plant parts sample collected will be processed for DNA extraction. The Qiagen DNeasy PowerSoil kit will be used for the isolation of DNA from plant samples. Studies have shown that the kit also worked for extraction of bacterial DNA from plant samples, and that it resulted in less background of harvested plant DNA in DNA detection. Thus, harvested plant DNA did not distort the analysis. ³⁴ This will aid the process of harvesting not only bacterial DNA from the plant surfaces, which always can’t be done completely, but reliably all bacterial DNA from plant surface including bacterial DNA existing within the plant. 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. ³³

II.

Quantitative analysis of DNA

The total DNA extracted from both soil and plant samples will be amplified with 16sRNA Specific Primer (8F and 1492R). The total DNA concentration will be determined using Qubit ^® 4.0 fluorometer and amplicon quality will be analyzed using 1.8% agarose gel.

III.

Preparation of library

The pair-end sequencing library will be prepared using Twist Bioscience DNA Library Kit for Illumina ^® (CAT No./ID 104119). The library preparation process will be initiated with 50 ng input. DNA will be enzymatically fragmented and continuous step of end-repair and A-tailing where an ‘A’ is added to the 3′ ends making the DNA fragments ready for adapter ligation. Following this step, illumina specific adapters are ligated to both ends of the DNA fragments. These adapters contain sequences essential for binding barcoded libraries to a flow cell for sequencing, allowing for PCR amplification of adapter-ligated fragments, and binding standard Illumina sequencing primers. To ensure maximum yields from limited amounts of starting material, a high-fidelity amplification step will be performed using HiFi PCR Master Mix.

The libraries will be sequenced on Illumina Novaseq6000 platform (2 × 150 bp chemistry) to generate 5GB data/Sample.

IV.

Quantity and quality check (QC) of library

The amplified libraries will be analyzed on TapeStation 4150 (Agilent Technologies) using RNA ScreenTape ^® as per manufacturer’s instructions.

V.

Cluster Generation and Sequencing

After obtaining the Qubit concentration for the library and the mean peak size from Tape Station profile, library will be loaded onto illumina Novaseq 6000 for cluster generation and sequencing. Paired-End sequencing will allow the template fragments to be sequenced in both the forward and reverse directions. The library molecules will bound to complementary adapter oligos on paired-end flow cell. The adapter will be designed to allow selective cleavage of the forward strands after re-synthesis of the reverse strand during sequencing. The copied reverse strand will be then used to sequence from the opposite end of the fragment.

VI.

Bioinformatic analysis

Data generation

Data generated from Novoseq6000 will be demultiplexed.

•

Metagenome Assembly

Before initiating denovo assembly, data will be filtered to remove sequencing adapters and low-quality sequences below QV20. Assembly of the clean reads will be carried out using MEGAHIT v1.2.9 which is a standalone metagenome assembly program for assembling large and complex metagenomics data. Fasta sequences will be generated and processed further. The statistical elements of the assemblies will be calculated by using in-house Pearl scripts.

•

Gene Prediction

The assembled scaffolds for the sample were subjected to gene prediction using Prodigal (v2.6.3) considering the metagenome gene prediction method. These predicted genes will be then taken further for taxonomic and functional analysis.

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Taxomonic Annotation using Kaiju

Kaiju is a fast, free, sensitive and standalone metagenome classifier which will find maximum exact matches on the protein-level using the Burrows Wheeler transform algorithm. Kaiju will classify individual metagenomic reads using a reference database comprising the annotated protein-coding genes of a set of microbial genomes. By default, Kaiju uses either the available complete genomes from NCBI RefSeq or the microbial subset of the non-redundant protein database nr used by NCBI BLAST, optionally also including fungi and microbial eukaryotes. It employs a search strategy, which finds maximal exact matching substrings between query and database using a modified version of the backwards search algorithm in the BWT. Kaiju translates metagenomic sequencing reads into the six possible reading frames and searches for maximum exact matches (MEMs) of amino acid sequences in a given database of annotated proteins from microbial reference genomes and also optionally perform searches using GREEDY algorithm. If matches to one or more database sequences are found for a read, Kaiju outputs the taxonomic identifier of the corresponding taxon, or it determines the LCA in the case of equally good matches to different taxa. The above mentioned final gene sequences obtained from prodigal will be taken as a input in Standalone Kaiju. The results of taxonomic hits distribution at the phylum, class, order, family, genus, species level of the samples analysed will be obtained for further process.

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Functional annotation using COGNIZER

COGNIZER (v0.9b) will be used at default parameters to assess the functional capacities of microbial communities present in the samples. Cognizer is a comprehensive stand-alone framework which is enabled to simultaneously provide COG, KEGG, Pfam, GO and FIGfams annotations to individual sequences constituting metagenomic datasets. The predicted genes will be taken here as an input in Cognizer for the final downstream analysis.

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AMR genes (ARGs) identification analysis

A homology search will be conducted between the proteins assembled genome and antibiotic resistance genes retrieved from Comprehensive Antibiotic Resistance Database (CARD) using BLASTP algorithm with an e-value threshold of 1e-10. The AMR genes will be identified for each sample.

Gene detection of ARG within individual isolates will not be done. It is known, that neglecting not phenotypically expressed resistances results in an underestimation of antibiotic resistant target bacteria and although of multi-resistant bacteria, but general occurrence of target ARG in the environment (soil and samples) give more information about the dimension of dissemination of resistances against human relevant antibiotics, because indirectly spread risk via horizontal gene transfer and other than the target species is included. The factor of unknown species origin of detected ARG will be taken into account during data interpretation and risk estimation.

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: [email protected]

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.