Unearthing the soil-borne microbiome of land plants.

Plant-soil biodiversity interactions are fundamental for the functioning of terrestrial ecosystems. Yet, the existence of a set of globally distributed topsoil microbial and small invertebrate organisms consistently associated with land plants (i.e., their consistent soil-borne microbiome), together with the environmental preferences and functional capabilities of these organisms, remains unknown. We conducted a standardized field survey under 150 species of land plants, including 58 species of bryophytes and 92 of vascular plants, across 124 locations from all continents. We found that, despite the immense biodiversity of soil organisms, the land plants evaluated only shared a small fraction (less than 1%) of all microbial and invertebrate taxa that were present across contrasting climatic and soil conditions and vegetation types. These consistent taxa were dominated by generalist decomposers and phagotrophs and their presence was positively correlated with the abundance of functional genes linked to mineralization. Finally, we showed that crossing environmental thresholds in aridity (aridity index of 0.65, i.e., the transition from mesic to dry ecosystems), soil pH (5.5; i.e., the transition from acidic to strongly acidic soils), and carbon (less than 2%, the lower limit of fertile soils) can result in drastic disruptions in the associations between land plants and soil organisms, with potential implications for the delivery of soil ecosystem processes under ongoing global environmental change.

Increases in the soil ammonia oxidizing phylotypes and their rechange due to long-term irrigation with wastewater.

Wastewater irrigation is a common practice for agricultural systems in arid and semiarid zones, which can help to overcome water scarcity and contribute with nutrient inputs. Ammonia-oxidizing bacteria (AOB) and archaea (AOA) are key in the transformation of NH4+-N in soil and can be affected by variations in soil pH, EC, N and C content, or accumulation of pollutants, derived from wastewater irrigation. The objective of this study was to determine the changes in the ammonia oxidizing communities in agricultural soils irrigated with wastewater for different periods of time (25, 50, and 100 years), and in rainfed soils (never irrigated). The amoA gene encoding for the catalytic subunit of the ammonia monooxygenase was used as molecular reporter; it was quantified by qPCR and sequenced by high throughput sequencing, and changes in the community composition were associated with the soil physicochemical characteristics. Soils irrigated with wastewater showed up to five times more the abundance of ammonia oxidizers (based on 16S rRNA gene relative abundance and amoA gene copies) than those under rainfed agriculture. While the amoA-AOA: amoA-AOB ratio decreased from 9.8 in rainfed soils to 1.6 in soils irrigated for 100 years, indicating a favoring environment for AOB rather than AOA. Further, the community structure of both AOA and AOB changed during wastewater irrigation compared to rainfed soils, mainly due to the abundance variation of certain phylotypes. Finally, the significant correlation between soil pH and the ammonia oxidizing community structure was confirmed, mainly for AOB; being the main environmental driver of the ammonia oxidizer community. Also, a calculated toxicity index based on metals concentrations showed a correlation with AOB communities, while the content of carbon and nitrogen was more associated with AOA communities. The results indicate that wastewater irrigation influence ammonia oxidizers communities, manly by the changes in the physicochemical environment.

A comparative analysis of regional infection risk due to wastewater recontamination in the Mezquital Valley, Mexico.

Urban wastewater reuse for agriculture provides reliable nutrient-rich water, reduces water stress, and strengthens food systems. However, wastewater reuse also presents health risks and characterizing the spatial dynamics of wastewater can help optimize risk mitigation. We conducted comparative risk analysis of exposure to wastewater in irrigation canals, where we compared those exposed to a) treated vs. untreated wastewater, and b) wastewater upstream vs. downstream from communities in the Mezquital Valley. The canal system with treated wastewater was sampled prior to being treated, directly after treatment, as well as before and after it flowed through a community. Along the canal system that carried untreated wastewater, we sampled before and after a community. We quantified the concentrations of bacterial, protozoal, and viral pathogens in the wastewater. Pathogen concentration data were used to calculate measures of relative risk between sampling points. Wastewater treatment reduced predicted bacterial pathogen infection risk in post-treatment locations (RR = 0.73, 95 % CI 0.61, 0.87), with no evidence of similar reductions in Giardia or viral pathogens (RR = 1.02, 95 % CI 0.56, 1.86 and RR = 1.18, 95 % CI 0.70, 2.02 respectively). Although infection risk decreased further down the canals, infection risk increased for bacterial pathogens after our sentinel community (RR = 1.94, 95 % 1.34, 2.86). For Giardia and viral pathogens infection risk was elevated but not significantly. We found similar evidence for increases in risk when comparing the treated section of the canal system with a canal section whose wastewater was not treated, i.e., the risk benefits of wastewater treatment were lost after our sentinel community for bacteria (RR = 5.27 vs. 2.08 for sampling points before and after our sentinel community respectively) and for Giardia (RR = 6.98 vs. 3.35 respectively). The increase in risk after transit through communities could have resulted from local community recontamination of the treated wastewater stream.

Soil contamination in nearby natural areas mirrors that in urban greenspaces worldwide.

Soil contamination is one of the main threats to ecosystem health and sustainability. Yet little is known about the extent to which soil contaminants differ between urban greenspaces and natural ecosystems. Here we show that urban greenspaces and adjacent natural areas (i.e., natural/semi-natural ecosystems) shared similar levels of multiple soil contaminants (metal(loid)s, pesticides, microplastics, and antibiotic resistance genes) across the globe. We reveal that human influence explained many forms of soil contamination worldwide. Socio-economic factors were integral to explaining the occurrence of soil contaminants worldwide. We further show that increased levels of multiple soil contaminants were linked with changes in microbial traits including genes associated with environmental stress resistance, nutrient cycling, and pathogenesis. Taken together, our work demonstrates that human-driven soil contamination in nearby natural areas mirrors that in urban greenspaces globally, and highlights that soil contaminants have the potential to cause dire consequences for ecosystem sustainability and human wellbeing.

Soil biodiversity supports the delivery of multiple ecosystem functions in urban greenspaces.

While the contribution of biodiversity to supporting multiple ecosystem functions is well established in natural ecosystems, the relationship of the above- and below-ground diversity with ecosystem multifunctionality remains virtually unknown in urban greenspaces. Here we conducted a standardized survey of urban greenspaces from 56 municipalities across six continents, aiming to investigate the relationships of plant and soil biodiversity (diversity of bacteria, fungi, protists and invertebrates, and metagenomics-based functional diversity) with 18 surrogates of ecosystem functions from nine ecosystem services. We found that soil biodiversity across biomes was significantly and positively correlated with multiple dimensions of ecosystem functions, and contributed to key ecosystem services such as microbially driven carbon pools, organic matter decomposition, plant productivity, nutrient cycling, water regulation, plant-soil mutualism, plant pathogen control and antibiotic resistance regulation. Plant diversity only indirectly influenced multifunctionality in urban greenspaces via changes in soil conditions that were associated with soil biodiversity. These findings were maintained after controlling for climate, spatial context, soil properties, vegetation and management practices. This study provides solid evidence that conserving soil biodiversity in urban greenspaces is key to supporting multiple dimensions of ecosystem functioning, which is critical for the sustainability of urban ecosystems and human wellbeing.

The global distribution and environmental drivers of the soil antibiotic resistome.

BACKGROUND: Little is known about the global distribution and environmental drivers of key microbial functional traits such as antibiotic resistance genes (ARGs). Soils are one of Earth's largest reservoirs of ARGs, which are integral for soil microbial competition, and have potential implications for plant and human health. Yet, their diversity and global patterns remain poorly described. Here, we analyzed 285 ARGs in soils from 1012 sites across all continents and created the first global atlas with the distributions of topsoil ARGs. RESULTS: We show that ARGs peaked in high latitude cold and boreal forests. Climatic seasonality and mobile genetic elements, associated with the transmission of antibiotic resistance, were also key drivers of their global distribution. Dominant ARGs were mainly related to multidrug resistance genes and efflux pump machineries. We further pinpointed the global hotspots of the diversity and proportions of soil ARGs. CONCLUSIONS: Together, our work provides the foundation for a better understanding of the ecology and global distribution of the environmental soil antibiotic resistome. Video Abstract.

Metallo-beta-lactamase-producing Escherichia coli in the sewage of Mexico City: where do they come from?

While monitoring the presence of antibiotic resistance in municipal wastewater bacteria from Mexico City, five Escherichia coli isolates were found to be resistant to carbapenems, antibiotics of "last resort" used mostly in hospitals. Further analysis revealed that these carbapenem-resistant isolates carried the gene encoding a metallo-beta-lactamase, NDM-5. The gene was found to be beared by a large, ∼145 kb conjugative plasmid, which also carries putative genes encoding resistance to sulfonamides, trimethoprim, tetracycline, ciprofloxacin, and chloramphenicol (although no phenotypic chloramphenicol resistance was detected) and quaternary-ammonium compounds. The plasmid also carried gene mobility determinants, such as integron integrase and two transposases. In addition to the direct public health threat posed by the presence of such multi-resistant organisms in wastewater released into the environment and used for crop irrigation; it is particularly concerning that carbapenem-resistant E. coli is rather rare in Mexican hospitals (<1%), but was found in small, 100 mL samples of municipal wastewater. This suggests that these organisms are under-reported by clinical microbiology laboratories, underlining the usefulness of wastewater monitoring, or that there is an unknown source of such carbapenem-resistant organisms that are being dumped into the wastewater. The source of these bacteria must be assessed and controlled to prevent further spread of this multi-resistance plasmid among other environmental and clinical microorganisms.

Global homogenization of the structure and function in the soil microbiome of urban greenspaces.

The structure and function of the soil microbiome of urban greenspaces remain largely undetermined. We conducted a global field survey in urban greenspaces and neighboring natural ecosystems across 56 cities from six continents, and found that urban soils are important hotspots for soil bacterial, protist and functional gene diversity, but support highly homogenized microbial communities worldwide. Urban greenspaces had a greater proportion of fast-growing bacteria, algae, amoebae, and fungal pathogens, but a lower proportion of ectomycorrhizal fungi than natural ecosystems. These urban ecosystems also showed higher proportions of genes associated with human pathogens, greenhouse gas emissions, faster nutrient cycling, and more intense abiotic stress than natural environments. City affluence, management practices, and climate were fundamental drivers of urban soil communities. Our work paves the way toward a more comprehensive global-scale perspective on urban greenspaces, which is integral to managing the health of these ecosystems and the well-being of human populations.

The influence of soil age on ecosystem structure and function across biomes.

The importance of soil age as an ecosystem driver across biomes remains largely unresolved. By combining a cross-biome global field survey, including data for 32 soil, plant, and microbial properties in 16 soil chronosequences, with a global meta-analysis, we show that soil age is a significant ecosystem driver, but only accounts for a relatively small proportion of the cross-biome variation in multiple ecosystem properties. Parent material, climate, vegetation and topography predict, collectively, 24 times more variation in ecosystem properties than soil age alone. Soil age is an important local-scale ecosystem driver; however, environmental context, rather than soil age, determines the rates and trajectories of ecosystem development in structure and function across biomes. Our work provides insights into the natural history of terrestrial ecosystems. We propose that, regardless of soil age, changes in the environmental context, such as those associated with global climatic and land-use changes, will have important long-term impacts on the structure and function of terrestrial ecosystems across biomes.

Temporal analysis of the microbial communities in a nitrate-contaminated aquifer and the co-occurrence of anammox, n-damo and nitrous-oxide reducing bacteria.

Groundwater-N pollution derives from agricultural and urban activities, and compromises water quality in shallow aquifers, putting human and environmental health at risk. Nonetheless, subsurface microbiota can transform dissolved inorganic nitrogen into N2. In this study, we surveyed the microbial community of a shallow aquifer by sampling one well, one piezometer and a spring within an agricultural area that receives N-inputs of more than 700 kg/ha per year through irrigation with wastewater. The survey was conducted during a year with a 16S rRNA next-gen approach. In parallel, we quantified the number of gene copies and transcripts related to anaerobic ammonium oxidation (anammox, hzo), nitrite-dependent anaerobic methane oxidation (n-damo, nod and pmoA) and nitrous oxide reduction (last step of denitrification, nosZ), during the dry and rainy seasons. Our results showed that the groundwater samples had 17.7 to 22.5 mg/L of NO3--N. The bacterial and archaeal community structure was distinctive at each site, and it remained relatively stable over time. We verified the co-occurrence of N-transforming bacteria, which was correlated with the concentration of NO2-/NO3- and ORP/DO values (DO: ~3.0 mg/L). Our analyses suggest that these conditions may allow the presence of nitrifying microorganisms which can couple with anammox, n-damo and denitrifying bacteria in interrelated biogeochemical pathways. Gene density (as the number of gene copies per litre) was lower in the rainy season than in the dry season, possibly due to dilution by rainwater infiltration. Yet, the numbers of hzo gene copies here found were similar to those reported in oceanic oxygen minimum zones and in a carbonate-rock aquifer. The transcript sequences showed that Candidatus Brocadia spp. (anammox), Candidatus Methylomirabilis spp. (n-damo) and autotrophic denitrifying Betaproteobacteria coexist in the groundwater environment, with the potential to attenuate the concentration of dissolved inorganic nitrogen by reducing it to N2 rather than N2O; delivering thus, an important ecosystem service to remove contaminants.

Modeling Spatial Risk of Diarrheal Disease Associated with Household Proximity to Untreated Wastewater Used for Irrigation in the Mezquital Valley, Mexico.

BACKGROUND: Reusing wastewater for irrigation is a longstanding practice that enhances crop yields and improves climate resilience. Without treatment, however, wastewater contains harmful pathogens and chemicals. Reuse of untreated wastewater has been shown to be harmful to the health of nearby communities, but the routes of exposure are unknown and do not appear to be occupational. Some routes occur throughout entire communities, such as food contamination. Other routes may be spatially dependent, such as spread by domestic animals or through aerosolization. OBJECTIVES: To examine whether those wastewater exposure routes with a spatial dependency affect health, we estimated the risks of diarrheal disease in children under age 5 associated with living near wastewater canals, while adjusting for potential individual- and household-level confounders. METHODS: We conducted three surveys over 1 y in the Mezquital Valley, Mexico, to measure diarrhea in children. The distance between each participating household and a wastewater canal was measured using GPS coordinates. The association between proximity and diarrhea was estimated with a multilevel logistic regression model accounting for spatial autocorrelation. RESULTS: A total of 564 households completed one to three surveys, resulting in 1,856 survey observations of 646 children. Children living 100m from a canal had 45% lower odds of diarrhea than those living within 10m of a canal, and children living 1000m away had 70% lower odds of diarrhea [100m vs. 10m adjusted odds ratio (OR)=0.55, 95% credible interval (CI): 0.33, 0.91; 1000m vs. 10m adjusted OR=0.30, 95% CI: 0.11, 0.82]. DISCUSSION: The estimated decline in diarrheal prevalence with household distance from a canal persisted after controlling for occupational exposure. Identifying the specific routes of exposure that drive this relationship will help identify which interventions, such as upstream treatment, can reduce health risks for entire communities where wastewater exposure occurs. https://doi.org/10.1289/EHP6443.

N2O emission factors from a wastewater irrigated land in a semiarid environment in Mexico.

Irrigation with wastewater is an increasing practice around the world triggered by the large needs of water for agriculture. Many times this source of water is added by flooding to the fields, creating short temporal oxygen-depleted environments, where nitrous oxide (N2O) emissions are promoted. Agriculture is responsible for up to 60% of the global annual emissions of this gas, and its emission factors (EF) must be reported. During 21 months, we installed closed chambers to measure the N2O emissions from a wastewater irrigated plot in the Mezquital Valley. Here, alfalfa, rye grass, and maize were grown in succession, receiving mainly organic N contained in the untreated wastewater and the residues of the previous crop; only maize received extra mineral fertilization of 100 kg of N ha-1. We obtained a time series where the gaps were filled by linear interpolation. EF and N2O direct emissions were estimated by the Tier 1 approach of the IPCC. We found that the alfalfa and rye grass, which only receive organic nitrogen, produced minor N2O emissions, that reflected in lower EF (0.004 and 0.001, respectively), compared with the default value of the IPCC (0.01). In contrast, maize, which receives organic and inorganic N, lost 1% of this N input as N2O, the same as the default value (0.01). It seems that the form of N, the crop age and type influence greatly the N dynamics in this wastewater irrigated land-use system. Therefore, the Tier I approach of the IPCC seems to underestimate the N efficiency of this agroecosystem and overestimates the N2O direct emissions from alfalfa and rye grass.

Global ecological predictors of the soil priming effect.

Identifying the global drivers of soil priming is essential to understanding C cycling in terrestrial ecosystems. We conducted a survey of soils across 86 globally-distributed locations, spanning a wide range of climates, biotic communities, and soil conditions, and evaluated the apparent soil priming effect using 13C-glucose labeling. Here we show that the magnitude of the positive apparent priming effect (increase in CO2 release through accelerated microbial biomass turnover) was negatively associated with SOC content and microbial respiration rates. Our statistical modeling suggests that apparent priming effects tend to be negative in more mesic sites associated with higher SOC contents. In contrast, a single-input of labile C causes positive apparent priming effects in more arid locations with low SOC contents. Our results provide solid evidence that SOC content plays a critical role in regulating apparent priming effects, with important implications for the improvement of C cycling models under global change scenarios.

Land Use Change and Water Quality Use for Irrigation Alters Drylands Soil Fungal Community in the Mezquital Valley, Mexico.

Soil fungal communities provide important ecosystem services, however, some soil borne representatives damage agricultural productivity. Composition under land-use change scenarios, especially in drylands, is rarely studied. Here, the soil fungal community composition and diversity of natural shrubland was analyzed and compared with agricultural systems irrigated with different water quality, namely rain, fresh water, dam-stored, and untreated wastewater. Superficial soil samples were collected during the dry and rainy seasons. Amplicon-based sequencing of the ITS2 region was performed on total DNA extractions and used the amplicon sequence variants to predict specific fungal trophic modes with FUNGuild. Additionally, we screened for potential pathogens of crops and humans and assessed potential risks. Fungal diversity and richness were highest in shrubland and least in the wastewater-irrigated soil. Soil moisture together with soil pH and exchangeable sodium were the strongest drivers of the fungal community. The abundance of saprophytic fungi remained constant among the land use systems, while symbiotic and pathogenic fungi of plants and animals had the lowest abundance in soil irrigated with untreated wastewater. We found lineage-specific adaptations to each land use system: fungal families associated to shrubland, rainfed and part of the freshwater were adapted to drought, hence sensitive to exchangeable sodium content and most of them to N and P content. Taxa associated to freshwater, dam wastewater and untreated wastewater irrigated systems show the opposite trend. Additionally, we identified potentially harmful human pathogens that might be a health risk for the population.

Changes in belowground biodiversity during ecosystem development.

Belowground organisms play critical roles in maintaining multiple ecosystem processes, including plant productivity, decomposition, and nutrient cycling. Despite their importance, however, we have a limited understanding of how and why belowground biodiversity (bacteria, fungi, protists, and invertebrates) may change as soils develop over centuries to millennia (pedogenesis). Moreover, it is unclear whether belowground biodiversity changes during pedogenesis are similar to the patterns observed for aboveground plant diversity. Here we evaluated the roles of resource availability, nutrient stoichiometry, and soil abiotic factors in driving belowground biodiversity across 16 soil chronosequences (from centuries to millennia) spanning a wide range of globally distributed ecosystem types. Changes in belowground biodiversity during pedogenesis followed two main patterns. In lower-productivity ecosystems (i.e., drier and colder), increases in belowground biodiversity tracked increases in plant cover. In more productive ecosystems (i.e., wetter and warmer), increased acidification during pedogenesis was associated with declines in belowground biodiversity. Changes in the diversity of bacteria, fungi, protists, and invertebrates with pedogenesis were strongly and positively correlated worldwide, highlighting that belowground biodiversity shares similar ecological drivers as soils and ecosystems develop. In general, temporal changes in aboveground plant diversity and belowground biodiversity were not correlated, challenging the common perception that belowground biodiversity should follow similar patterns to those of plant diversity during ecosystem development. Taken together, our findings provide evidence that ecological patterns in belowground biodiversity are predictable across major globally distributed ecosystem types and suggest that shifts in plant cover and soil acidification during ecosystem development are associated with changes in belowground biodiversity over centuries to millennia.

Drylands soil bacterial community is affected by land use change and different irrigation practices in the Mezquital Valley, Mexico.

Dryland agriculture nourishes one third of global population, although crop irrigation is often mandatory. As freshwater sources are scarce, treated and untreated wastewater is increasingly used for irrigation. Here, we investigated how the transformation of semiarid shrubland into rainfed farming or irrigated agriculture with freshwater, dam-stored or untreated wastewater affects the total (DNA-based) and active (RNA-based) soil bacterial community composition, diversity, and functionality. To do this we collected soil samples during the dry and rainy seasons and isolated DNA and RNA. Soil moisture, sodium content and pH were the strongest drivers of the bacterial community composition. We found lineage-specific adaptations to drought and sodium content in specific land use systems. Predicted functionality profiles revealed gene abundances involved in nitrogen, carbon and phosphorous cycles differed among land use systems and season. Freshwater irrigated bacterial community is taxonomically and functionally susceptible to seasonal environmental changes, while wastewater irrigated ones are taxonomically susceptible but functionally resistant to them. Additionally, we identified potentially harmful human and phytopathogens. The analyses of 16 S rRNA genes, its transcripts and deduced functional profiles provided extensive understanding of the short-term and long-term responses of bacterial communities associated to land use, seasonality, and water quality used for irrigation in drylands.

Health risks from exposure to untreated wastewater used for irrigation in the Mezquital Valley, Mexico: A 25-year update.

Wastewater reuse for agriculture is common worldwide; wastewater treatment, however, is rare in many countries, leading to high potential for exposure to harmful pathogens. Mexico City, one of the largest producers of untreated wastewater for agricultural use worldwide, was the site of key epidemiologic studies conducted in the 1990s. We both reviewed the literature on and conducted a cross-sectional study of diarrheal risk and wastewater contamination to provide an updated assessment of health risks and to inform an upcoming update of the 2006 WHO guidelines on wastewater reuse. We surveyed communities in the Mezquital Valley that use wastewater for irrigation and communities that use well water to compare the prevalence of self-reported diarrheal disease in children under five years old. Wastewater, well water, household environmental samples, and stool samples were collected and analyzed. Communities exposed to wastewater had a higher one-week prevalence of diarrhea (10%) compared to unexposed communities (5%). This association remained in an adjusted modified Poisson regression model (PR = 2.31, 95% CI 1.00, 5.31), but not when limited to households engaged in agriculture. Water quality indicators document differences between irrigation water from the two community groups. These results are in agreement with 25 population studies identified by our review that were conducted since or not included in the 2006 WHO guidelines and show consistent negative impacts of wastewater exposure on health. While overall diarrheal prevalence has declined when compared to studies conducted over 25 years ago in the same region, the association of diarrheal disease and wastewater exposure has remained and possibly increased. With rising urbanization worldwide, attention to these risks and wastewater treatment is becoming increasingly important.

Accumulation and Distribution of Lead and Chromium in Laboratory-Scale Constructed Wetlands Inoculated with Metal-Tolerant Bacteria.

The accumulation and distribution of lead and chromium was tested in a laboratory-scale constructed wetland (CW) inoculated with metal-tolerant bacteria. Two non-inoculated systems also were evaluated, one planted and the other unplanted. Mass balances indicated that 57% of chromium input was accumulated into inoculated CW after 151 days of operation. The distribution was similar in support media and vegetation, in which 78% was transferred to aerial part. Similarly Pb was accumulated 29% in the support media and 39% in vegetation, which was distributed 52% in rhizome and 48% in aerial part. Significantly lower amounts of heavy metals were accumulated in non-inoculated systems than in the inoculated wetlands (p < 0.005). In addition, a markedly higher proportion of chromium in aerial vegetation and of lead in the suspended fraction of the effluent was exhibited, which raises a subsequent recovery of the metal by harvest and settling, respectively. Results indicate that CW inoculated with metal-tolerant bacteria might be a suitable option for treating wastewater with content of lead and chromium.

Effects of 100 years wastewater irrigation on resistance genes, class 1 integrons and IncP-1 plasmids in Mexican soil.

Long-term irrigation with untreated wastewater can lead to an accumulation of antibiotic substances and antibiotic resistance genes in soil. However, little is known so far about effects of wastewater, applied for decades, on the abundance of IncP-1 plasmids and class 1 integrons which may contribute to the accumulation and spread of resistance genes in the environment, and their correlation with heavy metal concentrations. Therefore, a chronosequence of soils that were irrigated with wastewater from 0 to 100 years was sampled in the Mezquital Valley in Mexico in the dry season. The total community DNA was extracted and the absolute and relative abundance (relative to 16S rRNA genes) of antibiotic resistance genes (tet(W), tet(Q), aadA), class 1 integrons (intI1), quaternary ammonium compound resistance genes (qacE+qacEΔ1) and IncP-1 plasmids (korB) were quantified by real-time PCR. Except for intI1 and qacE+qacEΔ1 the abundances of selected genes were below the detection limit in non-irrigated soil. Confirming the results of a previous study, the absolute abundance of 16S rRNA genes in the samples increased significantly over time (linear regression model, p < 0.05) suggesting an increase in bacterial biomass due to repeated irrigation with wastewater. Correspondingly, all tested antibiotic resistance genes as well as intI1 and korB significantly increased in abundance over the period of 100 years of irrigation. In parallel, concentrations of the heavy metals Zn, Cu, Pb, Ni, and Cr significantly increased. However, no significant positive correlations were observed between the relative abundance of selected genes and years of irrigation, indicating no enrichment in the soil bacterial community due to repeated wastewater irrigation or due to a potential co-selection by increasing concentrations of heavy metals.