| Congresso Brasileiro de Microbiologia 2023 | Resumo: 981-2 | ||||
Resumo:It has been suggested that phage therapy is a promising alternative treatment option for antibiotic-resistant pathogens. Nonetheless, the emergence of phage resistant phenotype can compromise the success of the therapy. Therefore, this study evaluated the emergence of phage-resistant clones after short and long-term combined phage-antibiotic therapy. Endotracheal tube specimens (Ø 5 mm) received 1.5 mL of Tryptic Soy Broth (TSB) inoculated with 107 colony-forming units per milliliter (CFU/mL) of Pseudomonas aeruginosa (ATCC 27853) in 24-well tissue culture plates. The plates were incubated at 37oC for 48 h, and after biofilm maturation, the specimens were exposed for 2, 4, 24, 48 and 72 h to combined phage-antibiotic therapy. Endotracheal tube (ETT) associated biofilm was exposed to 108 plaque-forming units per milliliter (PFU/mL) of phage vB_PaM_USP_2, phage vB_PaM_USP_18 or 5 μg/mL of ceftolozane/tazobactam (CT), alone or in combination. The sequential combination of the two different phages and CT was also tested. To evaluate the sequential combination of two different phages and antibiotic, the virus type was switched at 24 h, and only the 48 h of treatment was considered. After each point time, the specimens were removed from the plates, transferred to microtubes containing 1 mL of Phosphate Buffered Saline and sonicated for 5 min at 40 kHz in a ultrasound-cleaning equipment. The ETT-biofilm suspension was added to TSB soft (45oC) and poured onto Petri plates with a layer of Tryptic Soy agar (1.5% agar). Twenty milliliters of ten-fold dilutions (starting at 109 PFU/mL) of the phages vB_PaM_USP_2 and vB_PaM_USP_18 were spotted to each bacterial lawn and incubated at 37°C for 24 h. The presence of clear zones indicating viral lysis of bacterial cells were scored as (2) complete clearing, no turbidity; (1) partial clearing, opaque or turbidity; and (0) no clearing. In the control and antibiotic groups, the spot assay revealed clear lysis zones on the agar plate during all evaluated periods. In the groups with phage-antibiotic combined treatment complete clearing was also observed after 2, 4, 24, 48 and 72 h of treatment. Such aspect was also observed after 2 and 4 h in the groups treated with phages vB_PaM_USP_2 and vB_PaM_USP_18 alone. Nonetheless, the infectivity of both phages was extensively reduced after 24 h of treatment, indicating the emergence phage-resistant phenotype. It was also found that P. aeruginosa biofilms insensitive to phages vB_PaM_USP_2 were partially sensitive to vB_PaM_USP_18 and vice versa. The findings regarding the emergence of phage-resistant phenotype emphasize that the exclusive use of phages in the treatment of ETT-biofilm is not a realistic approach. Palavras-chave: Pseudomonas aeruginosa, Biofilm, Phage-therapy, Phage-resistant phenotype, Phage-antibiotic therapy Agência de fomento:Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) - (2018/09757-0; 2021/00510-5; 2022/01992-6); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) - (405622/2018-0); Pró-Reitoria de Pesquisa e Inovação da USP. | |||||