Genome sequence and characterization of a Hypervirulent BI/NAP1/027 Clostridioides difficile (CDC20121308)

ESPAÑOL LA; PALUMBO MC; SERRAL F; BARBERO AM; PALMA S; RUGGERI D; FERNÁNDEZ DO PORTO D; HERNÁNDEZ DEL PINO RE; PASQUINELLI V

Mar del Plata

Congreso; Reunión Anual de Sociedades de Biociencias (SAIC, SAI y FAIC, SAFIS) - LXX Reunión Anual de la Sociedad Argentina de Inmunología (SAI; 2022

SAIC, SAI y FAIC, SAFIS

Clostridioides difficile (C. difficile) is a Gram-positive, obligate anaerobic and spore-forming bacteria that is widespread in the environment. C. difficile infection is an important cause of antimicrobial-associated diarrhea, life-threatening pseudomembranous colitis and toxic megacolon. Since 2000, the rapid emergence of the hyper- virulent PCR ribotype (RT) 027 complex has been associated with increases in the incidence and severity of disease and mortality. We characterized a BI/NAP1/027 C. difficile strain CDC20121308. The genome sequence was obtained using a whole-genome shotgun strategy. The draft genome was 4,188,514 bases in length and the G+C content of the genomic DNA was 29.3 mol%. A total of 3,971 coding sequences (CDS) and 55 tRNAs were predicted. We detected the presence of RT 027 lineage markers (thyA, cdtA, cdtB and tcdC 18bp-deletion), previously characterized by MALDI-TOF. The annotation identified 25% of CDS into RAST subsystems. The genome of CDC20121308 had 11 genes devoted to resistance to toxic compounds, antibiotics (Tetracycline (Tet) and Vancomycin (Van)) and disinfecting agents as predicted using CARD. Indeed, by broth microdilution assay we found that C. difficile CDC20121308 is resistant to Van and Tet with minimum inhibitory concentrations of 4 mg/ ml and 8 mg/ml, respectively. Crystal violet staining demonstrated biofilm formation, which could be associated with antibiotic resistance and pathogenicity. Moreover, we observed a spreading diffuse growth away from the inoculum stab at 12, 24 and 48 h, suggesting a motile phenotype. In conclusion, our results allowed the characterization of the BI/NAP1/027 C. difficile CDC20121308 strain. We demonstrated the presence of several genes associated with pathogenesis that were validated by functional assays. This study provides additional data for the use of this highly virulence commercial strain of epidemiological relevance worldwide in research work involving in vitro and in vivo assays.