Screening process

Search results from each academic and internet database will be exported to the Covidence systematic review management software. The management software will remove any duplicates present. No manual curation will be used to remove duplicates. Two reviewers will independently screen the title and abstracts of exported articles. Any disagreements or ambiguity will be identified through the Covidence software and resolved by review from a senior third party prior to full-text screening. Reviewers will independently complete a second level of screening to review the full text of the titles and abstracts meeting the eligibility criteria. Any disagreements or ambiguity will be identified through the Covidence software and resolved by review from a senior third party. Records of the number of articles excluded and reasons for their exclusion, at full-text level, will be provided as Additional file 1.

Eligibility criteria

For a study to be included in the systematic review, it must meet the followingcriteria:

Eligible population: plant and root-associated microbiota: soil, endosphere, rhizosphere, or root.

Eligible exposure: aminoglycosides: kanamycin, gentamicin, neomycin, tobramycin, streptomycin, kasugamycin, amikacin, dihydrostreptomycin, apramycin, paromomycin.

Eligible comparator: no aminoglycoside.

Eligible outcome: Changes in overall species richness and diversity (microbial composition), change of the resistome (i.e. the quantification of resistance genes), and the ability to suppress plant pathogens (e.g., Biomass reported as mg/kg; changes in banding patterns, or richness expressed as H’ and S’ indices, and abundance of resistance genes or suppressive pathogens reported as percentages).

Eligible study types: Both field and laboratory control-interventions studies that include aminoglycoside exposure and show comparison to comparator i.e., no aminoglycoside application to soil.

Eligible types of articles: peer-reviewed research articles, conference presentations, other grey literature from the specified websites.

Study validity assessment

Studies deemed eligible will be subjected to critical appraisal by two reviewers during the full-text screening. The CEE Critical Appraisal Tool Version 0.2 (Prototype) [19], modified to our review question, will be used for study validity. The CEE Critical Appraisal Tool will be used to assess and categorize each study’s susceptibility to bias. Any disagreements will be resolved by a senior-level reviewer. For procedural independence, none of the reviewers will review any articles they have authored.

Data coding and extraction strategy

Using Covidence, evidence tables of meta-data and data extraction (i.e., study findings) will be produced. For each screened study that fits the inclusion criteria and meets the study validity criteria, data will be extracted according to predetermined codes (Table 2). The following data will be coded for:

*Bibliographic information (author, year, title, source of publication).

*Study location (country).

*Study site (field/laboratory).

*Population (Soil, root, endosphere, rhizosphere).

*Soil type (i.e. sandy, loamy, clay, silt).

*Soil use (agricultural, natural).

*Type of tillage (no-till, deep (deeper than 10 inches), medium-depth (5–10 inches), shallow (1–4 inches)).

*Plant species (name).

*Experimental conditions (light vs dark, temperature, duration).

*Properties of soil (pH, organic matter content, moisture, oxygen status, compost use).

*Antibiotic characteristics (name, chemical formula, concentration, method of application, frequency of application, previous history of antibiotic application, and limits of detection).

*Method for evaluating microbial composition, resistance genes, and soil suppressiveness (e.g., Phospholipid Fatty Acid Analysis (PFLA), Terminal Restriction Fragment Length Polymorphism (T-RFLP), Denaturing Gradient Gel Electrophoresis (DGGE), Shotgun Sequencing, High throughput sequencing).

*Reported mean and standard deviation of microbial composition, resistance genes, and soil pathogen suppression for the control and intervention/experimental groups (e.g., Biomass reported as mg/kg; changes in banding patterns, or richness expressed as H’ and S’ indices, and abundance of resistance genes or suppressive pathogens reported as percentages).

*Comparator (description of the control with no exposure, i.e. no aminoglycosides).

Two reviewers will simultaneously and independently extract data from all studies deemed eligible based on the inclusion criteria. Any disagreements between the two reviewers will be resolved by a senior level third reviewer. If relevant data is either missing or ambiguous, the corresponding authors of those studies will be contacted. In the event of no response, we will indicate no response in our metadata and only report available data in our final analysis. If the amount of missing data is deemed to be substantial (> 50%), the study will be excluded from our final analysis.

Potential effect modifiers and reasons for heterogeneity

The identified potential effect modifiers and sources for heterogeneity are listed below. Also mentioned are the methods of testing.

*Soil type (i.e. sandy, loamy, clay, silt) [subgroup analysis].

*Soil usage (agricultural, natural) [subgroup analysis].

* Type of tillage (no-till, deep (deeper than 10 inches), medium-depth (5–10 inches), shallow (1–4 inches)) [subgroup analysis].

*Plant species (name) [subgroup analysis].

*Experimental conditions (light vs dark), the method used [subgroup analysis], and temperature and duration [meta-regression].

*Properties of the soil (pH, organic matter content, moisture, oxygen content status, compost use) [meta-regression].

*Antibiotic characteristics (name), method of application, previous application of antibiotics [subgroup analysis] and concentration, half-life, sorption coefficient, frequency of application [meta-regression].

This list was developed based on a preliminary literature search performed by KB, BJ, and NC with input from JC. MA provided overall scientific expertise. If additional effect modifiers are identified during the review process, we will document any changes in a final list of effect modifiers.

Data synthesis and presentation

After data extraction from all eligible studies, a narrative descriptive synthesis will be conducted on those demonstrating a low and medium risk of bias (based on study validity assessment), summarizing information in tables and figures. Summaries will be descriptive, outlining bibliographic information, study location, and site, population, soil characteristics, aminoglycoside characteristics, and experimental methods and conditions.

To assess the risk of publication bias, effects from individual studies will be visualized in funnel plots. Where evidence allows, efforts will be made to estimate the effect size of the outcome (i.e. microbial composition, antibiotic resistance genes, and soil suppression after aminoglycoside exposure relative to the comparator). We will attempt to estimate the standardized between-group mean difference (SMDbetween) and the standard error of SMDbetween of each outcome previously described for every article deemed eligible by our inclusion criteria. We will pool the effect size using a frequentist random-effects statistical model and perform a meta-regression analysis to understand the relationship between aminoglycoside concentration and the SMDbetween. Meta-regressions or subgroup analyses of studies will be performed where studies report sources of heterogeneity. To test the robustness of reported findings, sensitivity analysis will be performed by including or excluding studies of unclear or high risk of bias. If data does not allow us to estimate a pooled effect size, we will report a narrative synthesis of the included articles and report the SMDbetween point estimates for the individual studies.

In conclusion, the overall aim of this review is to identify, evaluate, and summarize all impacts of aminoglycoside exposure on soil and plant root-associated microbial composition, resistance genes, and soil suppressive species. The review will make the available evidence more accessible to researchers, farmers and environmental agencies invested in this information.

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