Estudo comparativo da formação de biofilme de cepas de Clostridioides difficile MLST CLADO 2 (ICC45/CE e NAP1/027)

Abstract

One of the difficulties in treating Clostridioides difficile infection (CDI) is that this bacterium forms biofilms, an important virulence mechanism known to promote antibiotic resistance and, as a result, consequently, a greater recurrence of the disease. The goal of this study was to compare the in vitro ability of three MLST Clade 2: ICC-45 (ribotype SLO231/UK[CE]821) a ST41 toxinotype IXb isolated in Brazil, to epidemic NAP1/027/ST01 strains NAP1/027/ST01 (LIBA5756), isolated during a 2010 outbreak in Costa Rica, and the reference epidemic strain NAP1/027/ST01 (R20291). ATCC700057, a non-toxigenic strain, was used as a control strain. Total biofilm biomass estimated by crystal violet staining was used to calculate strains’ ability to form biofilm. In addition, samples were stained with the Film Tracer biofilm matrix (Invitrogen®) and the thickness of the biofilm matrix was measured using confocal microscopy. Scanning electron microscopy was used to determine matrix architecture. Confocal microscopy was used to detect the presence of toxin A (TcdA) using an anti-Clostridioides difficile TcdA antibody. We also looked at the expression of virulence genes (tcdA, tcdB, tcdC, cdtA, cdtB, spo0A, slpA, cwp66 and cwp84), as well as the effect of antibiotics metronidazole (MTZ) and vancomycin (VAN) on biofilm growth. All the strains tested formed a moderate biofilm 1.1 <DO570nm>3.5. After 72h, the epidemic NAP1/027/ST01 strains (LIBA5756 and R20291) biofilm biomass were significantly higher than ICC-45 and ATCC 700057 biofilm, which was corroborated by electron and confocal microscopy. At 120h, the LIBA5756 biofilm biomass decreased compared to other strains. ICC-45 had higher expression of genes tcdA, tcdB, tcdC, cdtA, slpA and spo0A than the toxigenic strains R20291 or LIBA 5756, however there were no significant difference in the expression levels of cdtB, cwp66 and cwp84. VAN and MTZ caused an inhibitory effect in biofilm formation in epidemic strains, however for ICC-45 strain, minimal inhibitory concentration (MIC) of VAN and MIC and 4MIC of MTZ did not inhibit biofilm formation. In conclusion, the three MLST Clade 2 from different rybotipes, two of them isolated from Latin America, are o competent biofilm-forming bacterium, indicating a bacterial fitness that may favor the recurrence of C. difficile infection, making treatments more difficult.