The global spread of antibiotic resistance genes (ARGs) poses a significant threat to human and animal health. Addressing this challenge requires a "One Health" approach, integrating human, veterinary, and environmental health perspectives. The environmental component is particularly complex, necessitating a thorough understanding of ecological dispersal barriers to limit AMR propagation. ARGs are naturally occurring in environmental bacteria, but anthropogenic activities, such as sewage discharge and manure application, have significantly increased the frequency of ARB and ARG invasion events. Even in environments without direct anthropogenic impact, low-frequency invasion events occur through wildlife and aerial dispersal. Successful invasion requires overcoming the habitat's biotic resistance, a process that becomes increasingly challenging with rising biodiversity. Higher diversity reduces exploitable niches for invaders, thus lowering their establishment success. This study hypothesized that the prevalence of AMR in environmental microbiomes is inversely correlated with community diversity. To test this, the researchers collected 167 environmental samples from various European countries, with half from forest soils (static environment) and half from river sediments and biofilms (dynamic environment). Bacterial diversity was assessed using 16S rRNA gene sequencing, and ARG diversity and abundance were determined using high-throughput qPCR. The abundance of mobile genetic elements (MGEs) and the fecal pollution indicator crAssphage were also quantified.

Existing literature highlights the crucial role of biodiversity in resisting invasions, both at macro and micro scales. Studies have shown that increased biodiversity reduces available niches for invaders, hindering their establishment and spread. However, small-scale disturbances can enhance invasion success. In microbial communities, the invasion process is often stochastic when invaders are closely related to existing community members. The influence of diversity on ARG spread has been explored in laboratory settings, with diverse microcosms demonstrating lower invasiveness of ARBs. In aquatic systems, stress conditions can increase the invasiveness of resistant strains into biofilms. High-density bacterial communities and abundant resources can promote horizontal gene transfer of mobile ARGs. This study aimed to bridge the gap in knowledge by investigating the relationship between microbial diversity and ARG prevalence in real-world environmental microbiomes.

The study involved a pan-European sampling campaign conducted during fall/winter 2020/21. A total of 167 samples were collected from seven countries, with 83 samples from forest soils and 84 from river sediments and biofilms. Sampling aimed to minimize anthropogenic impact. For river samples, either epilithic biofilms or sediments were collected; five individual samples were combined to create composite samples. Soil samples were collected similarly, combining five soil cores for each composite. Samples were processed to extract DNA, and bacterial diversity was assessed using 16S rRNA gene sequencing on an Illumina MiSeq platform. The researchers utilized DADA2 for amplicon sequence variant (ASV) inference and filtering. High-throughput qPCR was performed to quantify 27 ARGs and 5 MGEs, along with the 16S rRNA gene and crAssphage (a fecal pollution indicator). Alpha diversity indices (Chao1 richness, Shannon diversity, and Pielou evenness) were calculated to assess diversity within each sample. Beta diversity (Bray-Curtis dissimilarity) was also analyzed. Statistical analyses included PERMANOVA, one-way ANOVA with post-hoc Tukey HSD tests, Pearson and Spearman correlations, and t-tests.

The river and soil datasets showed significant differences in bacterial community composition. Soil samples were dominated by Acidobacteria, Actinobacteriota, and Bacteroidota, while river samples were dominated by Proteobacteria, Bacteroidota, and Actinobacteriota. CrAssphage was absent from soil samples and present at low levels in most river samples. Both datasets exhibited a wide range of ARG diversity, but soil samples had significantly fewer ARGs per sample than river samples. The cadA3-V1 gene was the most abundant ARG in both datasets. In the soil dataset, higher diversity (Pielou evenness and Shannon diversity) was significantly and negatively correlated with the number of detected ARGs and the relative abundance of many individual ARGs. This negative correlation was less pronounced or absent in the river dataset. The analysis of mobile genetic elements (MGEs) revealed no significant correlations between MGE abundance and diversity metrics in either dataset. Finally, communities with high total ARG abundance showed higher phylogenetic similarity compared to those with low abundance, indicating that high ARG hosting is a specialist trait, whereas low ARG abundance is a more general community trait.

The results support the hypothesis that microbial diversity acts as a barrier to ARG accumulation, at least in stable, structured environments like soil. The stronger negative correlation observed in soil compared to river environments likely reflects the difference in environmental dynamics. Soil microbiomes are more stable and competitive, allowing for the development of diversity-based resilience against ARG invasion. The dynamic nature of river environments, with frequent disturbances, may mask the impact of diversity on long-term ARG establishment. The positive correlation between blaCTX-M-2 abundance and diversity in both datasets suggests that this ARG might have a different origin than those predominantly linked to anthropogenic pollution. The study also highlights that the invasion success of ARGs depends not only on host establishment but also on the duration of host presence, which can influence horizontal gene transfer. In diverse communities, reduced horizontal gene transfer and increased competition might lead to less successful ARG persistence.

This study demonstrates that high bacterial diversity can limit the abundance and diversity of ARGs in soil environments. This finding highlights the importance of maintaining diverse microbial communities in environmental management strategies, potentially creating natural barriers against the spread of AMR. Future research should investigate the underlying mechanisms driving this relationship and explore how to leverage this natural barrier effect in practical applications, such as wastewater treatment and aquifer recharge.

The study's cross-sectional design limits the ability to determine causality. The relatively small sample size for each country could limit the statistical power. The focus on a limited set of ARGs might not fully capture the overall AMR landscape. Future research with larger sample sizes, longitudinal data, and a broader range of ARGs would strengthen the findings.