Curiosity at the beginning: why is antibiotic resistance ancient and ubiquitous in nature?

The antibiotic resistance - a global health concern in the 21^st century – occurs when microorganisms acquire genetic information (e.g., antibiotic resistance genes (ARGs)) from the bacterial gene pool. The rapid dissemination of the global antibiotic resistance elevates risks to from One Health to Global Health across different countries or continents (1). However, the antibiotic resistance is a natural and ancient phenomenon. In our review article published in 2020, we have noticed substantial evidence of antibiotic resistant determinants such as ARGs, mobile genetic elements and antibiotic resistant bacteria (ARB) detected from thousand-to-million-year-old permafrost samples - before human antibiotic usage - to soils or sediments in pristine environments (e.g., isolated jungles or caves, Arctic and Antarctica) as reported in many previous studies (2). Surprisingly, these samples harbour diverse ARGs and ARB, which confer resistance against a wide array of modern antibiotics such as β-lactam, tetracycline, macrolides and aminoglycosides. Our comprehension of the fundamental mechanisms governing antibiotic resistance remains limited. This curiosity leads us to ask, “What truly occurs in natural habitats?” The understanding of how biological interactions between bacteria and other microorganisms contribute to the antibiotic resistance in natural ecosystems is pivotal in tackling the global challenge of antibiotic resistance.

Less attention to predators of bacteria

Effects of microbiome predators of bacteria on the evolution and spread of antibiotic resistance have been overlooked, compared to anthropogenic factors like antibiotics, heavy metals, or biocides, as well as antagonists of bacteria. In natural settings, bacteria face a wide range of biotic and abiotic stressors including competitors, predators and environmental stressors. The production of antibiotics and/or the evolution of the antibiotic resistance system are the key strategies of bacteria to safeguard their cells and withstand different stressors. The influence of antagonists of bacteria (i.e., fungi and other bacteria) on the antibiotic resistance is widely recognized. Specifically, the competition between bacteria and their antagonists induces the antibiotic production to kill others (3). The bacterial–fungal antagonism also significantly correlates with the relative abundance of global ARGs (4). Given that protists – primary predators of bacteria and fungi – are highly abundant and diverse in soil ecosystems (5-8), sharing similar soil pores with bacteria, we speculate that, in natural settings, bacteria produce antibiotics as weapons for targeting not only their competitors but their predators - protists. Our hypothesis suggests that the protistan predation can exert a selective pressure on bacterial communities, leading to antimicrobial production and ARG enrichment in natural soils.

To test the hypothesis, we designed a soil microcosm incubation aimed at elucidating roles of indigenous protists in bacterial antibiotic resistance in a natural forest soil. We maintained the original moisture content throughout the incubation period to mimic the natural condition. After 90 days inoculating with low, medium and high protist concentrations, our results indicated that an increasing pressure of the protistan predation was strongly associated with higher abundance and diversity of soil ARGs (9).

Interestingly, our findings revealed that high protist concentrations significantly enriched the abundance of ARGs encoding primary mechanisms of antibiotic resistance — specifically, those involved in antibiotic deactivation and efflux pumps. This increase was particularly evident in the abundance of multidrug (oprJ and ttgB genes) and tetracycline (tetV) efflux pump genes by 608%, 724% and 3052%, respectively.  Notably, many antibiotic-producing bacteria (APB) and other bacterial taxa, such as Mycobacterium, Nocardia and Streptomycetaceae, were more abundant in the presence of higher protist levels. These APB possessing intrinsic resistance also play as antibiotic-resistant bacteria.

Strikingly, we found strong association between many protistan genera of the orders Glissomonadida and Spongomonadidae (bacterivores), and Euglyphida and Cercomonadida (omnivores), with bacterial genera, particularly APB (Bacillus, Streptomyces, Mycobacterium, Nocardia and Streptomycetaceae) and enriched ARGs (tetV, aadA1, oprJ, ttgB, vanWG). The most abundant gene, blaTEM, which encodes antibiotic deactivation, was negatively associated with bacterivorous Cercomonadidae (Cercomonadida), suggesting the antibiotic production of bacteria to fight against the predation of bacterivorous protists.

The novel findings about the importance of soil protists to soil resistome from our study prompt future research on unexplored roles of other soil predators - such as nematodes, rotifers, tardigrades, and various other animals - in the evolution and dissemination of the bacterial antibiotic resistance.

For details, please refer to our paper at https://www.nature.com/articles/s41396-023-01524-8.

References

1. Hernando-Amado S, Coque TM, Baquero F, Martínez JL. Defining and combating antibiotic resistance from One Health and Global Health perspectives. Nature microbiology. 2019;4(9):1432-42.
2. Nguyen B-AT, Chen Q-L, He J-Z, Hu H-W. Microbial regulation of natural antibiotic resistance: Understanding the protist-bacteria interactions for evolution of soil resistome. Science of the Total Environment. 2020;705:135882.
3. Hibbing ME, Fuqua C, Parsek MR, Peterson SB. Bacterial competition: surviving and thriving in the microbial jungle. Nature reviews microbiology. 2010;8(1):15-25.
4. Bahram M, Hildebrand F, Forslund SK, Anderson JL, Soudzilovskaia NA, Bodegom PM, et al. Structure and function of the global topsoil microbiome. Nature. 2018;560(7717):233-7.
5. Nguyen B-AT, Chen Q-L, He J-Z, Hu H-W. Oxytetracycline and ciprofloxacin exposure altered the composition of protistan consumers in an agricultural soil. Environmental Science & Technology. 2020;54(15):9556-63.
6. Nguyen B-AT, Chen Q-L, He J-Z, Hu H-W. Livestock manure spiked with the antibiotic tylosin significantly altered soil protist functional groups. Journal of Hazardous Materials. 2022;427:127867.
7. Nguyen B-AT, Chen Q-L, Yan Z-Z, Li C, He J-Z, Hu H-W. Distinct factors drive the diversity and composition of protistan consumers and phototrophs in natural soil ecosystems. Soil Biology and Biochemistry. 2021;160:108317.
8. Nguyen TBA, Chen QL, Yan ZZ, Li C, He JZ, Hu HW. Trophic interrelationships of bacteria are important for shaping soil protist communities. Environmental Microbiology Reports. 2023.
9. Nguyen TB-A, Bonkowski M, Dumack K, Chen Q-L, He J-Z, Hu H-W. Protistan predation selects for antibiotic resistance in soil bacterial communities. The ISME Journal. 2023:1-8.