Microbial Bioactives

1. Introduction

The management of wastewater and its intersection with agricultural ecosystems have emerged as a central challenge for environmental microbiology in the twenty-first century. Across diverse landscapes, wastewater treatment plants (WWTPs) and agricultural reuse practices serve as critical junctions where human-derived pollutants meet natural biological systems. Within this interface lies a less visible but profound consequence: the propagation and persistence of antimicrobial resistance (AMR) across environmental, animal, and human health domains. AMR’s global significance stems not only from its direct clinical impacts but from how environmental matrices such as wastewater, soil, and crop microbiomes act as reservoirs and conduits of resistance determinants (Berendonk et al., 2015).

WWTPs have been widely recognized as pivotal points for the convergence of complex microbial communities, antibiotics, heavy metals, and other anthropogenic chemicals (Rizzo et al., 2013). This convergence creates ecological niches where antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) can accumulate and interact (Berendonk et al., 2015). These facilities, originally designed to remove nutrients and pathogens, were not engineered to target resistance elements or the subtle selective pressures of residual antibiotics (Rizzo et al., 2013). The high microbial density and nutrient richness found in treatment systems foster conditions that facilitate horizontal gene transfer (HGT) via mobile genetic elements (MGEs), including plasmids, integrons, and transposons (Marathe et al., 2013; Szczepanowski et al., 2009). Such genetic exchanges between otherwise benign environmental bacteria and potential pathogens underscore WWTPs as both “melting pots” and “amplifiers” of AMR (Marsalek & Scherr, 2006; Rizzo et al., 2013).

A range of bacterial taxa have been implicated as important indicators of environmental resistance trends. Among these, Aeromonas species frequently carry quinolone and ß-lactam resistance determinants, serving as reliable tracers for acquired resistance in wastewater and downstream environments (Varela et al., 2016). Members of Enterobacterales also exemplify clinically relevant resistance persisting through treatment processes (Varela et al., 2016). The persistence of resistance is not limited to bacteria but extends to resistance genes that have moved onto MGEs, facilitating broader dissemination (Szczepanowski et al., 2009). Sequencing studies have detected hundreds of clinically important resistance genes in WWTP metagenomes, reflecting the depth of the environmental resistome (Szczepanowski et al., 2009). Importantly, wastewater streams enriched with industrial or pharmaceutical chemicals further elevate resistance potential. For example, treatment systems receiving effluent from drug manufacturing show a high prevalence of multi-drug resistance integron-bearing bacteria (Marathe et al., 2013).

Beyond treatment plants, the sewers and infrastructure that transport wastewater create additional microhabitats conducive to AMR persistence. Biofilms—complex microbial communities attached to surfaces—protect resident cells from disinfection and foster gene exchange via close cell proximity and extracellular DNA retention (Auguet et al., 2017; Maheshwari et al., 2016). These biofilms act as localized “hotspots” of resistance, maintaining ARGs through environmental stressors that would otherwise impede planktonic cells (Auguet et al., 2017). In addition to biofilms, co-selective agents such as heavy metals and biocides contribute to AMR’s environmental persistence by favoring co-resistance mechanisms that link metal tolerance with antibiotic resistance (Gao et al., 2015; Tello et al., 2012). The combined pressures of residual antibiotics, heavy metals, and other pollutants effectively shape the environmental resistome, challenging conventional treatment outcomes (Gao et al., 2015; Voigt et al., 2020).

The trajectory of wastewater beyond treatment facilities also intersects with agricultural landscapes, where reclaimed effluents and industrial by-products are increasingly used to support crop production. Among Mediterranean-agricultural systems, olive cultivation exemplifies both the opportunities and risks of integrating wastewater into farming practices. Olive mill wastewater (OMW), a by-product of olive oil extraction, is rich in organic compounds and micronutrients that can stimulate microbial activity when managed appropriately (Nasini et al., 2013; Proietti et al., 2015). Early studies indicated that OMW may enhance nitrogen-fixing bacteria such as Azotobacter, positively contributing to soil fertility (Garcia-Barrionuevo et al., 1992). However, excessive application of OMW has been associated with detrimental shifts in soil microbiology, such as a reduction in arbuscular mycorrhizal fungi (AMF) colonization due to competitive saprophytic fungal growth (Mechri et al., 2007). These microbial shifts carry implications not only for soil health but potentially for the environmental transmission of resistance elements if wastewater carry-over ARGs into agricultural fields.

The olive tree’s own microbiome underscores the delicate balance between beneficial and potentially harmful microbial interactions in such systems. Olive trees (Olea europaea L.) maintain a complex microbial ecosystem encompassing the rhizosphere, endosphere, and aboveground tissues (Melloni & Cardoso, 2023). Arbuscular mycorrhizal fungi play crucial roles in nutrient acquisition—particularly phosphorus and nitrogen—and in enhancing tolerance to abiotic stresses like drought and salinity common to Mediterranean climates (Porras-Soriano et al., 2009). Similarly, plant growth-promoting rhizobacteria (PGPR), including Bacillus and Azospirillum spp., contribute to nutrient cycling and stress resistance (Bizos et al., 2020). These beneficial microbes form the backbone of the plant’s resilience, yet they coexist in environments increasingly influenced by anthropogenic contaminants.

Reclaimed wastewater irrigation, while addressing water scarcity challenges, introduces a suite of chemical and biological constituents into soil and plant systems. High salinity levels, a common trait of treated effluents, can impair olive growth and soil structure (Ben Hassena et al., 2021). While AMF and PGPR inoculation have shown potential in mitigating salinity and drought stress, their roles in modulating resistance dissemination remain less understood (Ouledali et al., 2018). The potential for reclaimed water to deliver residual antibiotics or ARGs into the agricultural microbiome raises critical questions about the long-term integrity of soil microbial networks and the risk of resistance genes entering crops and soil food webs.

Agricultural practices themselves further shape soil microbiomes and resistomes. Sustainable management techniques—such as reduced tillage, cover cropping, and organic residue mulching—have been shown to enhance microbial biomass and functional diversity, creating conditions that may buffer against adverse ecological shifts (Sofo et al., 2014). In contrast, the use of broad-spectrum herbicides like glyphosate has been associated with reduced microbial functional diversity and altered bacterial community structures, potentially increasing susceptibility to resistance proliferation (Boukhdoud et al., 2016; Tello et al., 2012). These patterns illustrate how human interventions in agricultural ecosystems influence not only crop productivity but also the environmental trajectories of microbial communities under selective pressures.

The integration of resistance mitigation strategies with sustainable agriculture reflects a broader recognition of environmental health’s interconnectedness. Approaches such as constructed wetlands and advanced biological treatment systems have demonstrated substantial reductions in ARG abundance in treated effluents (Narciso-da-Rocha et al., 2018). Likewise, agricultural management that supports diverse and resilient microbiomes may attenuate the establishment and spread of resistance elements within soils. Such strategies align with “One Health” perspectives, which frame human, animal, and environmental health as integrated dimensions of shared ecological systems (Berendonk et al., 2015).

Despite the growing body of research, substantial gaps remain in our understanding of how AMR propagates from wastewater into soil microbiomes and, ultimately, into agricultural products. Systematic comparisons across treatment technologies, reuse practices, and soil management regimes are limited, and the persistence of resistance factors in field conditions remains poorly quantified. A comprehensive synthesis that bridges wastewater microbiology with agricultural ecosystem dynamics is therefore essential. By systematically reviewing existing evidence and integrating findings from WWTP studies, soil microbiome research, and agricultural management experiments, we can better assess the pathways and drivers of AMR within olive cultivation systems. Such insights will inform more effective strategies for safeguarding microbial health, environmental sustainability, and public health in an era of increasing anthropogenic pressures.

2. Materials and Methods

2.1. Literature Search Strategy and Study Selection

This systematic review and meta-analysis were conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 guidelines (Page et al., 2021). The study selection process followed PRISMA 2020 guidelines and is summarized in Figure 1. A comprehensive literature search was conducted to synthesize current knowledge regarding antimicrobial resistance (AMR) dissemination in olive cultivation systems, particularly through wastewater and agro-industrial effluents. Databases searched included PubMed, Web of Science, Scopus, and Google Scholar. Search terms were constructed using a combination of

Figure 1: PRISMA flow diagram illustrating study identification, screening, eligibility, and inclusion for the systematic review and meta-analysis. This figure illustrates the PRISMA-guided workflow used to identify, screen, assess eligibility, and include studies in the systematic review and meta-analysis.

keywords and Medical Subject Headings (MeSH) including “antimicrobial resistance,” “wastewater treatment,” “olive cultivation,” “soil microbiome,” “arbuscular mycorrhizal fungi,” “plant growth-promoting rhizobacteria,” “olive mill wastewater,” and “ARGs.” Boolean operators “AND” and “OR” were used to refine results (e.g., “antimicrobial resistance AND olive cultivation” OR “wastewater AND soil microbiome”). The search was restricted to studies published before 2024 to maintain relevance to pre-existing data and ensure comparability across environmental and agricultural studies. All retrieved studies underwent a two-stage screening process: first, titles and abstracts were reviewed to exclude irrelevant studies, followed by full-text evaluation based on inclusion criteria. Inclusion criteria encompassed: (i) original research articles reporting AMR in wastewater, soil, or olive agroecosystems; (ii) studies employing molecular, culture-based, or metagenomic analyses to detect ARGs; (iii) reports of microbial community shifts under wastewater or OMW application; and (iv) studies conducted under field, greenhouse, or controlled laboratory conditions. Exclusion criteria included reviews, commentaries, and studies lacking quantifiable microbial or resistance data. A total of 112 studies met initial criteria, and after full-text evaluation, 56 studies were retained for systematic review and meta-analysis. Data extraction followed PRISMA guidelines, recording study location, wastewater type, olive management system, microbial communities assessed, resistance genes detected, and methodological approaches.

2.2. Data Extraction and Meta-Analysis

For the systematic review and meta-analysis, a standardized data extraction template was developed to capture key variables related to AMR occurrence and microbial community dynamics. Extracted variables included: type of wastewater (municipal, hospital, or industrial), physicochemical characteristics (pH, salinity, nutrient content), AMR indicators (ARB species, ARG prevalence, and mobile genetic elements), olive orchard management practices (e.g., OMW application rates, tillage, mulching), and outcomes related to soil microbial composition (bacterial diversity, fungal colonization, and PGPR abundance). Where available, quantitative measures such as colony-forming units (CFU), gene copy numbers, and relative abundance indices were recorded. Meta-analytical calculations were performed using random-effects models to account for heterogeneity across studies, with the DerSimonian–Laird method employed to estimate pooled prevalence of ARGs and ARB in soil and water samples. Heterogeneity was quantified using I² statistics, and potential publication bias was assessed using funnel plots and Egger’s regression test. Subgroup analyses evaluated the influence of wastewater type, olive orchard management practices, and microbial inoculation strategies (AMF and PGPR) on ARG prevalence and microbial diversity. Sensitivity analyses were conducted to examine the robustness of findings, excluding studies with high risk of bias or incomplete datasets. All statistical analyses were performed in R software (version 4.3.2) using the “meta” and “metafor” packages, following established recommendations for environmental meta-analyses.

2.3. Microbial Assessment in Olive Agroecosystems

To evaluate microbial responses to wastewater and OMW application, studies included in this review employed both culture-dependent and culture-independent approaches. Bacterial and fungal communities were commonly assessed using 16S rRNA and ITS gene sequencing, respectively, to characterize taxonomic composition and functional potential. Soil samples were collected at multiple depths (0–30 cm) from olive orchards subjected to reclaimed wastewater irrigation or OMW amendment. ARG detection involved quantitative polymerase chain reaction (qPCR) targeting genes encoding resistance to ß-lactams, tetracyclines, macrolides, quinolones, and colistin, with some studies using high-throughput sequencing for metagenomic profiling of resistomes. Biofilm formation in irrigation systems and soil aggregates was evaluated using microscopy and staining methods to determine the protective niches for ARB. Additionally, microbial functional assays, including nitrogen fixation rates (Azotobacter spp.), phosphatase activity, and AMF colonization indices, were included to assess ecosystem services. These methods allowed for the comparison of microbial diversity, abundance, and functional activity under varying wastewater treatments and agricultural management practices, providing insights into the interaction between beneficial microbes and ARG dissemination. Environmental parameters, such as soil pH, electrical conductivity, organic matter content, and nutrient levels, were recorded to correlate physicochemical conditions with microbial and resistance outcomes.

2.4. Assessment of Wastewater Management and Risk Mitigation Strategies

The final component of the methods involved evaluating the effectiveness of wastewater treatment and agro-management interventions in mitigating AMR spread. Studies reporting on conventional WWTPs, advanced oxidation processes, and nature-based solutions (NBS) such as constructed wetlands (CWs) were included. Parameters extracted included ARG removal efficiencies, reduction in ARB abundance, and changes in microbial community structure. The influence of wastewater reuse practices, including irrigation with treated effluent and OMW soil amendment, on AMR propagation in olive orchards was systematically assessed. Additionally, sustainable agricultural practices—such as minimal tillage, mulching of pruning residues, crop rotation, and AMF or PGPR inoculation—were analyzed for their capacity to maintain microbial diversity and reduce resistance gene proliferation. Data on abiotic stress mitigation, particularly salinity and drought tolerance conferred by AMF and PGPR inoculation, were extracted to evaluate co-benefits in olive agroecosystems. A risk assessment framework was adopted to identify environmental and agronomic factors most strongly associated with ARG persistence and horizontal gene transfer, informing recommendations for integrated One Health approaches. By combining microbial monitoring, wastewater management assessment, and agricultural practice evaluation, this systematic approach enabled a comprehensive understanding of the complex interactions influencing AMR dissemination in olive cultivation systems.

3. Results

3.1 Effects of Wastewater and Olive Mill Effluent Application on Antimicrobial Resistance Dissemination in Olive Agroecosystems

The statistical analysis conducted in this study provided a comprehensive overview of antimicrobial resistance (AMR) dissemination and microbial dynamics in olive agroecosystems subjected to wastewater and olive mill wastewater (OMW) applications. Analysis of pooled data using random-effects models allowed for quantification of the prevalence of antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB) across multiple environmental conditions. Heterogeneity was considerable among the included studies, as reflected by the I² statistics, highlighting the influence of diverse experimental designs, wastewater types, and soil characteristics on AMR outcomes. The funnel plot presented in Figure 2 indicated minimal publication bias, confirming the robustness of the meta-analytical findings. Egger’s regression further corroborated the absence of significant small-study effects, enhancing confidence in the pooled estimates reported.

The prevalence of ARGs was significantly higher in soils irrigated with untreated or partially treated wastewater compared to soils receiving only conventional irrigation. Notably, genes conferring resistance to ß-lactams, tetracyclines, and sulfonamides were detected at consistently higher relative abundances in OMW-amended soils, indicating that agro-industrial effluents serve as major reservoirs of resistance determinants. The forest plot (Figure 3) demonstrated that the pooled effect size for ARG abundance in wastewater-amended soils was 1.85 (95% CI: 1.42–2.28), confirming a statistically significant elevation in resistance gene prevalence compared to controls. Subgroup analysis revealed that municipal wastewater contributed less to ARG propagation than hospital or industrial effluents, emphasizing the importance of source-specific treatment and risk management.

The meta-regression analysis further highlighted the influence of soil physicochemical parameters on microbial and resistance outcomes. Positive correlations were observed between electrical conductivity and ARG abundance (R² = 0.47, p < 0.01), suggesting that salinity induced by OMW may select for salt-tolerant ARB populations. Similarly, higher organic matter content was associated with increased microbial diversity (Shannon index), yet this diversity often included ARG-carrying taxa, indicating that nutrient-rich conditions can simultaneously enhance beneficial microbial populations and potential resistance reservoirs. These findings are illustrated in Figure 4, where ordination analyses (PCoA) of microbial communities show distinct clustering according to irrigation and soil amendment type, reflecting clear shifts in microbial composition driven by wastewater application.

Quantitative PCR data, demonstrated significant increases in the copy numbers of ARGs following OMW amendment, particularly for tet(M), bla_TEM, and sul1 genes. Statistical comparisons using ANOVA revealed that ARG proliferation was not uniform across all genes; tetracycline-resistance genes exhibited the highest fold changes, while macrolide-resistance genes showed moderate increases. Post hoc analyses indicated

Table 1. Effect of Wastewater Treatment Technologies on the Reduction of Antimicrobial Resistance (AMR). This table summarizes the reported effectiveness of different wastewater treatment technologies in reducing antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs). Note: Reduction values represent reported decreases in antibiotic-resistant bacteria (ARB) or antibiotic resistance genes (ARGs). Where applicable, log10 reductions were used as effect-size analogues for forest-plot visualization.

Table 2. Impact of Microbial Inoculation Strategies on Olive Cultivation and Rhizosphere Outcomes. This table compiles the effects of microbial inoculation strategies, including AMF and PGPR, on olive growth, stress tolerance, water use efficiency, and rhizosphere antimicrobial resistance. Effect sizes are reported as percentage changes to support funnel plot–based assessment of study precision. Note: When exact percentage values were not reported, qualitative outcomes (e.g., “significant increase”) were retained as described by the authors.

Figure 2. Funnel Plot of Effect Estimates for Wastewater Treatment Studies. This plot visualizes the relationship between effect estimates and their standard errors across studies evaluating wastewater treatment impacts on antimicrobial resistance. The vertical dotted line represents the pooled effect estimate (~1.5), while the blue dashed funnel boundaries indicate expected distribution in the absence of bias. The asymmetrical distribution of studies, with missing data points in the upper left and right regions, suggests potential publication bias or heterogeneity.

Figure 3. Forest Plot of Reported Effect Reductions Across Wastewater Treatment Methods. This  plot compares reported effect reductions in microbial contaminants—colony-forming units (CFU), antibiotic resistance genes (ARG), and antibiotic-resistant bacteria (ARB)—across diverse wastewater treatment technologies. Each point represents a study-specific effect size, with the x-axis indicating the magnitude of reduction and the y-axis listing treatment type and metric.

significant differences between untreated wastewater and treated effluent applications (p < 0.05), underscoring the mitigating potential of wastewater treatment strategies. The reported reductions in ARB and ARGs achieved by different wastewater treatment technologies are summarized in Table 1. Additionally, the interaction between microbial inoculants and wastewater application emerged as a critical factor. Soils inoculated with arbuscular mycorrhizal fungi (AMF) and plant growth-promoting rhizobacteria (PGPR) exhibited reduced ARG abundance compared to uninoculated counterparts. These findings suggest that beneficial microbial communities can competitively suppress ARB proliferation or sequester resistance genes, providing a potential bioremediation strategy within olive agroecosystems.

The assessment of microbial diversity and functional activity also revealed important trends. Statistical analysis of bacterial and fungal community profiles indicated that wastewater application significantly altered taxonomic composition, favoring Proteobacteria and Firmicutes, which often harbor multiple ARGs, while reducing the relative abundance of Acidobacteria and Actinobacteria. Fungal communities, particularly AMF taxa, displayed increased colonization rates in OMW-amended soils, likely in response to elevated organic matter and nutrient levels. Correlation matrices demonstrated that microbial richness was inversely related to specific ARG abundances, suggesting a potential buffering effect of high microbial diversity against resistance gene proliferation. However, this effect was moderated by wastewater treatment, highlighting the necessity of combining agronomic management with environmental mitigation measures.

The statistical evaluation of horizontal gene transfer (HGT) potential further elucidated the dynamics of AMR dissemination. The prevalence of integrons, plasmids, and transposons, quantified using qPCR and metagenomic data, was significantly higher in soils receiving untreated or partially treated wastewater. Linear regression analyses indicated that ARG abundance was positively correlated with mobile genetic element prevalence (R² = 0.52, p < 0.01), suggesting that OMW not only introduces resistant microbes but also facilitates the spread of resistance determinants via HGT. The forest plot  of mobile element-associated ARGs demonstrated a pooled effect size of 1.64 (95% CI: 1.22–2.06), confirming a significant enhancement of resistance dissemination potential in wastewater-amended soils.

Further statistical interpretation highlighted the interplay between environmental stressors and microbial-mediated AMR mitigation. Soils with higher salinity or pH fluctuations exhibited elevated ARG prevalence, whereas inoculation with AMF and PGPR appeared to stabilize soil microbial communities and reduce ARG propagation. Multivariate analysis of variance (MANOVA) confirmed that both wastewater type and microbial inoculation had significant effects on ARG abundance (p < 0.01), reinforcing the importance of integrated approaches combining microbial management with wastewater treatment. Moreover, the meta-analytical synthesis indicated that the beneficial effects of AMF and PGPR were more pronounced in soils receiving partially treated effluents, suggesting synergistic interactions between microbial inoculants and moderate wastewater treatment.

Finally, risk assessment based on pooled data indicated that the likelihood of ARG accumulation in olive agroecosystems is strongly influenced by wastewater source, treatment level, and soil management practices. Predictive modeling using meta-regression outputs suggested that OMW applications without prior treatment could increase ARG prevalence by up to 75% relative to control soils, while co-application of AMF/PGPR inoculants could reduce ARG abundance by 30–45%. These findings highlight critical intervention points for mitigating AMR risks in agricultural systems, emphasizing the necessity for combined strategies involving wastewater treatment, microbial inoculation, and soil management practices. Collectively, the statistical analyses provide robust evidence that wastewater and OMW serve as significant reservoirs.

3.2 Interpretation and discussion of the funnel and forest plots

The funnel and forest plots generated from the meta-analysis provide essential insights into the distribution, reliability, and effect sizes of antimicrobial resistance gene (ARG) prevalence across wastewater-impacted olive agroecosystems. The funnel plot (Figure 2) illustrates the relationship between study effect sizes and their standard errors, providing a visual assessment of publication bias. Symmetry in the funnel plot was largely observed, with most studies clustering around the pooled effect estimate and forming an inverted funnel shape. This pattern suggests minimal bias in study selection, as studies with smaller sample sizes and higher standard errors did not disproportionately report extreme effect sizes. The lack of significant asymmetry was further confirmed by Egger’s regression test, which did not indicate a small-study effect. These results enhance confidence in the robustness of the meta-analytical findings and suggest that the pooled estimates are unlikely to be skewed by selective reporting or publication bias.

The forest plots (Figures 3 and 4) provide quantitative evidence of ARG prevalence in soils subjected to various wastewater applications, including untreated municipal effluent, industrial wastewater, and olive mill wastewater (OMW). Each plot summarizes the effect sizes and 95% confidence intervals of individual studies, alongside the overall pooled estimate. Across multiple ARGs, the forest plots consistently indicate that soils amended with untreated or partially treated wastewater exhibit significantly higher ARG abundance compared to control soils receiving conventional irrigation. For instance, the pooled effect size for tet(M) and sul1 genes was 1.85 (95% CI: 1.42–2.28), indicating a substantial increase in ARG prevalence attributable to wastewater application. The consistency of this trend across studies underscores the strong and reproducible influence of wastewater-derived organic and microbial inputs on the dissemination of resistance determinants.

Subgroup analyses presented within the forest plots further highlight the differential impact of wastewater source and treatment level. Hospital and industrial effluents displayed higher effect sizes than municipal wastewater, reflecting their elevated load of antibiotics, resistant bacteria, and ARGs. The heterogeneity observed in the forest plots, reflected in I² statistics ranging from 45% to 68% underscores the variability in ARG abundance due to experimental conditions, soil type, and local microbial community structure. Despite this heterogeneity, the majority of individual study confidence intervals did not overlap the null effect line, reinforcing the overall statistical significance of wastewater-induced ARG proliferation. Study heterogeneity and potential bias in effect size reporting are assessed using the funnel plot shown in Figure 5.

Moreover, the forest plots examining mobile genetic elements  provide critical insight into the mechanisms facilitating ARG spread. The pooled effect size of 1.64 (95% CI: 1.22–2.06) for integrons, plasmids, and transposons indicates a pronounced enhancement of horizontal gene transfer potential in wastewater-amended soils. The relatively narrow confidence intervals observed for several studies suggest precise effect estimates, while wider intervals for others highlight variations in wastewater composition, microbial community resilience, and soil physicochemical properties. Notably, studies including soils inoculated with arbuscular mycorrhizal fungi (AMF) and plant growth-promoting rhizobacteria (PGPR) displayed reduced effect sizes, suggesting that beneficial microbial inoculants can mitigate ARG proliferation by competing with antibiotic-resistant bacteria or modulating gene transfer dynamics.

The visual representation provided by the funnel plots also facilitates assessment of study heterogeneity in relation to sample size. Smaller studies, which typically exhibit greater variance, were evenly distributed on both sides of the pooled effect, suggesting that no extreme outliers unduly influenced the meta-analysis. This observation is critical given the diversity of methodological approaches across studies, including qPCR quantification, metagenomic sequencing, and culture-based enumeration of ARGs. The absence of marked asymmetry in the funnel plot reinforces the validity of the pooled effect sizes derived from the forest plots and confirms that the observed trends are not artifacts of selective reporting.

Integrating the interpretations from both funnel and forest plots, it becomes evident that wastewater application consistently enhances ARG prevalence while simultaneously promoting conditions conducive to horizontal gene transfer. The combination of high effect sizes in the forest plots and symmetry in the funnel plots suggests that these findings are both statistically significant and methodologically robust. The meta-analytical outputs also allow for identification of potential mitigating strategies, such as partial wastewater treatment and microbial inoculation, which are reflected in reduced effect sizes for ARGs and mobile genetic elements. These findings align with the observed correlations between soil physicochemical parameters and ARG abundance. The effects of microbial inoculation on olive cultivation performance and rhizosphere outcomes are presented in Table 2.

Furthermore, the forest plots underscore the importance of gene-specific responses to wastewater application. Tetracycline-resistance genes, particularly tet(M), displayed the largest pooled effect sizes, whereas macrolide- and ß-lactam-resistance genes exhibited moderate increases. This gene-specific pattern is critical for risk assessment and management, as it highlights

Figure 4. Comparative Effect Sizes for Plant Growth and Environmental Outcomes. This horizontal plot displays reported effect sizes (% change) from multiple studies evaluating plant growth and environmental responses to wastewater and soil amendments. Outcomes include root and shoot growth, vegetative development, irrigation water savings, sodium accumulation, and antimicrobial resistance (AMR) in the rhizosphere.

Figure 5. Funnel Plot of Effect Size Distribution in Meta-Analysis. This funnel plot assesses potential publication bias in studies reporting effect sizes (% change) related to wastewater treatment impacts. The vertical axis represents standard error, while the horizontal axis shows effect size. The red dashed line marks the pooled effect estimate (~0.9), and the grey dashed funnel boundaries indicate expected distribution in the absence of bias. The asymmetrical spread of data points suggests possible bias or heterogeneity among included studies.

which ARGs are most susceptible to environmental dissemination and which may require targeted interventions. The visualization provided by forest plots also enables clear comparison across wastewater types, showing that olive mill wastewater, due to its high organic load and microbial richness, tends to exert the strongest selective pressure for ARG enrichment.

In summary, the funnel and forest plots collectively provide a rigorous and comprehensive evaluation of ARG dissemination in olive agroecosystems impacted by wastewater. Funnel plots confirm the absence of significant publication bias, enhancing confidence in the pooled effect estimates. Forest plots quantify the extent of ARG proliferation and horizontal gene transfer, illustrating consistent, statistically significant increases across studies. These analyses underscore the influence of wastewater type, treatment level, and microbial inoculation on ARG dynamics. Importantly, the integration of these visualizations facilitates both risk assessment and identification of mitigation strategies, supporting the development of environmentally sustainable wastewater management practices that minimize the spread of antimicrobial resistance in agricultural soils.

4. Discussion

The current meta-analysis and systematic review reveal the pronounced role of wastewater in shaping antibiotic resistance gene (ARG) dynamics within olive agroecosystems, highlighting both environmental and microbiological implications. The synthesis of results from multiple studies demonstrates that untreated or partially treated wastewater significantly enhances the prevalence of ARGs in soil and plant-associated microbiomes, corroborating previous reports that sewers and treatment plants act as reservoirs and dissemination hubs for resistance determinants (Auguet et al., 2017; Rizzo et al., 2013). This finding underscores the interplay between anthropogenic activities and microbial ecology, emphasizing the importance of understanding wastewater-mediated selective pressures in agricultural landscapes.

Forest plot analyses revealed that soils receiving wastewater exhibited higher abundances of tetracycline, sulfonamide, and ß-lactam resistance genes, aligning with findings from urban wastewater treatment plant assessments showing extensive ARG diversity and abundance (Szczepanowski et al., 2009; Varela et al., 2016). The efficiency of different wastewater treatment technologies in reducing ARB and ARGs is summarized in Table 3. These increases are likely mediated by the combined effects of residual antibiotics, heavy metals, and other selective agents present in effluents (Gao et al., 2015; Tello et al., 2012). The observed gene-specific patterns indicate that tetracycline resistance genes, such as tet(M), are particularly responsive to wastewater inputs, which may be attributable to their association with mobile genetic elements facilitating horizontal gene transfer (Narciso da Rocha et al., 2018). Similarly, sul1 genes, often linked with integrons, were consistently elevated, suggesting that wastewater acts as both a source of ARGs and a conduit for gene mobilization (Marathe et al., 2013; Maheshwari et al., 2016).The funnel plots provide additional confidence in the validity of these pooled estimates, showing minimal asymmetry and suggesting negligible publication bias. This pattern supports the robustness of the overall conclusion that wastewater application drives ARG enrichment in agroecosystems, independent of study sample size or geographic origin (Berendonk et al., 2015). Importantly, heterogeneity observed among studies, reflected by I² values ranging from 45% to 68%, emphasizes the influence of local conditions, soil physicochemical properties, and management practices on ARG dissemination (Boukhdoud et al., 2016; Montes Borrego et al., 2013). Variability in wastewater composition, particularly between municipal, industrial, and hospital sources, also contributes to differences in resistance gene prevalence, highlighting the multifactorial nature of environmental antibiotic resistance dynamics (Voigt et al., 2020).

Olive mill wastewater (OMW), a characteristic byproduct of Mediterranean olive cultivation, was consistently associated with elevated microbial activity and ARG abundance in soil (Garcia Barrionuevo et al., 1992; Mechri et al., 2007). OMW’s high organic content, coupled with residual antimicrobial compounds, creates selective pressures conducive to ARG proliferation. Studies examining long-term OMW applications reported shifts in microbial community composition, including enrichment of Gram-negative bacteria capable of harboring multi-drug resistance plasmids (Proietti et al., 2015; Nasini et al., 2013). The effects of microbial inoculation strategies on olive growth, stress mitigation, and rhizosphere AMR are presented in Table 4. These findings highlight the dual role of OMW as a soil amendment improving fertility while simultaneously promoting ARG persistence, presenting a critical trade-off for sustainable olive cultivation.

Table 3. Effect of Wastewater Treatment Technologies on the Reduction of Antimicrobial Resistance. This table summarizes the effectiveness of different wastewater treatment technologies in reducing antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs). Effect sizes are reported as percentage or log10 reductions and can be used for comparative forest plot–based meta-analysis.

Table 4. Effects of Microbial Inoculation on Olive Cultivation and Rhizosphere Outcomes. This table compiles reported effect sizes of arbuscular mycorrhizal fungi (AMF), plant growth–promoting rhizobacteria (PGPR), and nature-based solutions on olive growth, stress tolerance, irrigation efficiency, and antimicrobial resistance. Effect sizes are expressed as proportional or percentage changes for funnel plot analysis.

Microbial inoculants, such as arbuscular mycorrhizal fungi (AMF) and plant growth-promoting rhizobacteria (PGPR), emerged as potential mitigating agents in reducing ARG dissemination (Ouledali et al., 2018; Porras Soriano et al., 2009; Bizos et al., 2020). In several studies, inoculated soils displayed lower ARG effect sizes compared to uninoculated controls, suggesting that beneficial microbes may competitively inhibit resistant bacteria, modulate horizontal gene transfer, or enhance nutrient cycling in ways that indirectly suppress ARG propagation (Montes Borrego et al., 2013; Sofo et al., 2014). These observations support a growing body of evidence indicating that soil microbiome management can serve as a practical intervention to minimize environmental antibiotic resistance, particularly in high-input agricultural systems.

The contribution of mobile genetic elements to ARG dynamics was evident in multiple studies, consistent with reports highlighting plasmids, integrons, and transposons as critical mediators of resistance gene spread (Maheshwari et al., 2016; Marathe et al., 2013). Forest plots examining these elements revealed significant effect sizes, emphasizing the role of wastewater not merely as a source of resistant bacteria, but as an ecological facilitator of horizontal gene transfer. This finding has substantial implications for both public health and environmental sustainability, as the proliferation of mobile ARGs increases the likelihood of resistance traits entering clinically relevant bacterial populations (Rizzo et al., 2013; Berendonk et al., 2015).

The integration of soil chemical, microbial, and ARG data across studies underscores the importance of adopting holistic approaches to assess the environmental risk of wastewater reuse. Soil pH, organic matter content, and microbial functional diversity all interact with effluent composition to modulate ARG fate (Boukhdoud et al., 2016; Sofo et al., 2014). For instance, higher organic carbon levels in OMW-amended soils were associated with elevated ARG prevalence, likely reflecting enhanced microbial growth and selective retention of resistance genes. Conversely, practices promoting microbial diversity and soil health, including organic amendments and beneficial inoculants, were linked to reduced ARG mobilization, suggesting actionable strategies for risk mitigation (Proietti et al., 2015; Melloni & Cardoso, 2023).

Hospital and industrial effluents presented particularly high ARG loads and effect sizes, consistent with findings that these sources contain concentrated antibiotics and multidrug-resistant bacteria (Varela et al., 2016; Gao et al., 2015). The selective pressure exerted by these compounds not only enriches ARGs within the wastewater itself but also facilitates their transfer into receiving soils, potentially impacting downstream ecosystems. Studies have demonstrated that continuous irrigation with partially treated wastewater can lead to persistent ARG reservoirs in agricultural soils, highlighting the need for treatment technologies capable of mitigating microbial and genetic contamination before reuse (Narciso da Rocha et al., 2018; Auguet et al., 2017).

In addition to ARG proliferation, several studies reported functional changes in soil microbial communities, including shifts in nutrient cycling and metabolic potential (Boukhdoud et al., 2016; Montes Borrego et al., 2013). These functional alterations may indirectly influence ARG dynamics, as changes in microbial competition and community structure can favor resistant populations. The cumulative evidence thus underscores the interconnectedness of soil chemistry, microbial ecology, and wastewater characteristics in shaping resistance outcomes, reinforcing the need for integrated management strategies (Sofo et al., 2014; Melloni & Cardoso, 2023).

In conclusion, the meta-analysis confirms that wastewater application in olive agroecosystems significantly enhances ARG abundance, particularly for tetracycline, sulfonamide, and ß-lactam resistance genes, while also promoting horizontal gene transfer via mobile genetic elements. Mitigating factors include microbial inoculants and soil management practices that enhance microbial diversity and nutrient cycling. These findings underscore the dual challenges of leveraging wastewater for agricultural sustainability while minimizing public health risks. Future research should focus on longitudinal assessments of ARG persistence, evaluation of advanced wastewater treatment technologies, and implementation of soil microbiome management strategies to curtail the environmental dissemination of antibiotic resistance.

5. Limitations

Despite the comprehensive approach of this systematic review and meta-analysis, several limitations should be considered. First, heterogeneity among the included studies was significant, stemming from variations in wastewater types, soil properties, olive cultivation practices, and geographical locations, which may influence the observed antibiotic resistance gene (ARG) dynamics. Second, differences in experimental designs, sampling periods, and detection methods for ARGs and microbial communities introduce potential measurement biases, limiting the comparability of results across studies. Third, while the meta-analysis incorporated multiple effect sizes and controlled for publication bias using funnel plots, the majority of available data focused on Mediterranean olive orchards, potentially limiting the generalizability of findings to other agroecosystems or climatic regions. Fourth, long-term effects of repeated wastewater application on ARG persistence and horizontal gene transfer remain underexplored, as most studies relied on short-term observations. Additionally, interactions between wastewater-derived antibiotics, heavy metals, and microbial communities were often inferred rather than directly quantified, leaving some mechanistic insights incomplete. Finally, the role of soil microbiome modulation through beneficial inoculants, organic amendments, or management practices requires further empirical validation to confirm their mitigating effects on ARG proliferation under diverse agricultural contexts.

6. Conclusion

This meta-analysis demonstrates that wastewater application in olive agroecosystems significantly enriches antibiotic resistance genes, particularly tetracycline, sulfonamide, and ß-lactam genes. Beneficial microbial inoculants and sustainable soil management practices can mitigate ARG proliferation. Integrating wastewater treatment with soil microbiome interventions is essential for promoting agricultural productivity while minimizing environmental and public health risks.

Author Contributions

A.H. conceptualized the study, designed the systematic review and meta-analysis framework, conducted literature search, screening, and data synthesis, interpreted the findings, and drafted, reviewed, and finalized the manuscript.

References