Control of Antibiotic-Resistant Bacteria in Activated Sludge Using Polyvalent Phages in Conjunction with a Production Host

Bacteriophage-based microbial control could help address a growing need to attenuate the proliferation of antibiotic-resistant bacteria (ARB) in wastewater treatment plants (WWTPs). However, the infectivity of commonly isolated narrow-host-range phages decreases quickly upon addition to activated sludge (i.e., plaque-forming units had a half-life of 0.63 h). Here, we show that polyvalent (broad-host-range) phages proliferate and thrive in activated sludge microcosms, especially when added along with their production hosts. Polyvalent phage cocktails (PER01 and PER02) were significantly more effective than narrow-host-range coliphage cocktails (MER01 and MER02) in suppressing a model ARB [β-lactam-resistant Escherichia coli NDM-1, initially present at 6.2 ± 0.1 log 10 colony-forming units (CFU)/mL]. After 5 days, the NDM-1 concentration significantly decreased to 3.8 ± 0.2 log 10 CFU/mL in the presence of the polyvalent phage cocktail, compared to 4.7 ± 0.3 log 10 CFU/mL for the coliphage cocktail treatment. Because of the presence of alternative hosts, polyvalent phages reached greater densities, which increased the probability of ARB infection. The fraction of surviving E. coli harboring the bla NDM-1 resistance gene was also significantly lower for the polyvalent phage cocktail treatment (0.57 ± 0.07) than for the control (0.74 ± 0.08). Therefore, polyvalent phages safely produced with nonpathogenic hosts could offer a novel approach to controlling problematic ARB in WWTPs and mitigating the propagation and discharge of associated resistance genes to the environment.