Bacterial strains isolated from biofilms of drinking water distribution systems: disinfectant tolerance and antibiotic resistance profile

ORTEGA, BA; CORIMAYO, SN; RAJAL, VB; CRUZ, MC

Congreso; Congreso Conjunto de la Sociedad Argentina de Investigación Bioquímica (SAIB) y la Asociación Civil de Microbiología General (SAMIGE); 2021

Drinking water is not sterile; indeed, it has many microorganisms that resisted the disinfection process and could deteriorate the water quality due to the unrestrained microbial activities. The interactions between these microorganisms caused the formation of biofilms on the inner wall of pipes, even in the presence of disinfectants, providing a refuge for pathogens and a high risk of waterborne diseases transmission. Furthermore, the presence of disinfectant and the low nutrient load make drinking water distribution systems (DWDS) unique environments, with bacteria in the sessile phase (biofilms) behaving differently than in the planktonic phase. It is hypothesized that selection pressure for disinfection is related to the presence of antibiotic resistance genes and their biofilm-forming capacity could promote that resistance. In this study, we therefore aimed to isolate bacterial strains from biofilms in the DWDS pipes and assess their disinfectant tolerance, ability to form biofilms and resistance to antibiotics. Biofilms were aseptically collected during pipe replacement works at the university campus. More than 50 bacterial strains were isolated from biofilms. Bacteria that grew on specific media for opportunistic pathogens were selected: SRCO01 (Pseudomonas sp.), SRTQ01 (E. coli), and SRCD02 that grew on non-selective agar. Tolerance to the disinfectant was determined by exposing them to two sodium hypochlorite concentrations: 2 mg/l (high) and 0.2 mg/l (low) corresponding to the minimum concentration required by legislation. Biofilm formation was determined using 96-well polystyrene plates in two media: with high nutrients concentration (Luria Bertani, LB, Britania) and low nutrient broth (Reasoner?s 2A, R2A, Oxoid). To promote biofilm formation, the plates were incubated aerobically on a shaker at 150 rpm at room temperature for 24, 48, and 72 h. Every 24 h, the growth medium was carefully discarded and replaced by a fresh one. The disk diffusion test was used to determine the susceptibility of the strains to eight different antibiotics. All three strains tolerated the low concentration of disinfectant (0.2 mg/l), meanwhile only SRCO01 also tolerated the highest concentration (2 mg/l). Strain SRCO01 demonstrated to form biofilm and resisted to amoxicillin, clavulanic acid, and levofloxacin. SRTQ01 was found to be biofilm-forming and resistant to erythromycin and tetracycline. The SRCD02 strain did not form biofilms in either of the tested broths and resisted only to erythromycin. The characterization of isolated strains represents a great advance in improving monitoring systems and disinfection strategies to provide safe water and thus, reducing waterborne diseases transmission.