Transfer dynamics of antibiotic resistance determinants across urban water systems: The free-floating genetic perspective

Antibiotic resistance is one of the biggest threats to global health, food security, and development today, leading to a growing number of difficult-to-treat infections and an economic burden. It can affect anyone of any age and in any country. It is mainly accelerated by the misuse and abuse of antibiotics, poor hygiene, and a lack of sanitation infrastructure. From the One Health concept, water is the main link connecting all the compartments where antibiotic resistance has primarily developed (human, animal, and natural environments). It carries microorganisms, pharmaceuticals such as antibiotics, floating genetic information in the form of mobile genetic elements (MGEs), and genes conferring antibiotic resistance. It is thought that bacteria found in anthropogenic barriers such as wastewater and drinking water treatment plants could play a role in transferring and disseminating resistant bacteria into the natural environment. However, the mechanisms by which bacteria can exchange via horizontal gene transfer (HGT) to further disseminate antibiotic resistance genes (ARGs) in such compartments are unknown. Natural transformation is one of the main HGT phenomena by which competent bacteria pull extracellular DNA into their cytoplasm. Still, it remains widely unknown which bacteria can use such a mechanism and under which circumstances. Unraveling the composition of such free-floating extracellular DNA (exDNA) fraction in complex systems such as wastewater is crucial to identify the environmental conditions promoting gene transfer. This thesis aims to understand further the role of exDNA in the transfer and development of antibiotic-resistant bacteria (ARBs) from complex systems. The status of released DNA from different model microorganisms after different sterilization procedures was evaluated in Chapter 2. The results showed that current sterilization methods are effective in microorganism inactivation. However, stable DNA is released from microbial cultures and ends up in sewage streams with genetic information from microorganisms originating from human and animal discharges. In Chapter 3, a method using chromatography to isolate and enrich exDNA without causing cell lysis from complex wastewater matrices like influent (9 μg exDNA was obtained out of 1 L), activated sludge (5.6 μg out of 1 L), and treated effluent (4.3 μg out of 1 L) was developed. Thus, this was necessary to profile its genetic composition. Surprisingly, results highlighted that exDNA is mainly comprised of MGEs (65%), posing a risk as the prevalence of MGEs in the extracellular fraction can indirectly promote antibiotic resistance development mainly via natural transformation. In the two field investigation chapters (Chapters 4 and 5), the transfer of ARGs and MGEs and their removal capacity in a full-scale Nereda® reactor removing nutrients with aerobic granular sludge and in chlorine-free drinking water treatment plants were evaluated. These two chapters summarize the journey that antibiotic-resistant bacteria follow toward water sanitation. Resistance determinants decreased their load reaching effluents from wastewater (1.1 log gene copies mL−1) and drinking water treatment plants (2.5 log gene copies mL−1), at least when inside active bacterial cells. It is less clear regarding exDNA since the treatment process involves cell decay and lysis that releases exDNA into the environment. After profiling the exDNA both in lab-scale and full-scale experiments, the effect of environmental factors such as increasing antibiotic concentrations was evaluated on exDNA transformation in an activated sludge enrichment in Chapter 6. We showed the feasibility of distantly-related microorganisms for DNA uptake when strong environmental pressures (≥50 mg L−1) were applied. Thus, it shows that natural transformation under environmental antibiotic concentrations may not be the driving force by which bacteria take up exDNA in complex systems. However, the focus should be on other compartments such as research facilities and pharmaceutical industrial discharges. Finally, strategies to remediate ARGs (intracellular and extracellular) and ARBs from wastewater effluents were evaluated in Chapter 7. We showed how byproducts from wastewater and drinking water treatment plants, such as sewage-sludge biochar and iron-oxide coated sands, were effective at removing ARBs and exDNA from effluent waters. Collectively, this thesis shows that the exDNA fraction from water matrices is an overlooked pool of genetic fragments containing MGEs and ARGs. Thus, these could be used as genetic material to transform competent bacteria and develop ARBs. However, exDNA transformation under environmental antibiotic concentrations is not the main mechanism by which bacteria evolve and adapt in mixed cultures. It is important to highlight that anthropogenic barriers are effective at remediating ARBs, which should redirect the focus from wastewater treatment plants and tackle the antibiotic resistance issue from multiple compartments simultaneously.