Nathália L. Andrade, Ana Carolina C. Campos, Claudio Querido Fortes, Alex W.Friedrich, John W.A. Rossen, Ana Cláudia P. Rosa, Paulo V. Damasco
Infective endocarditis (IE) is a systemic infection and life threatening disease with high mortality (1–4). The incidence of IE caused by gram-negative bacteria has been increasing (5). These cases are rare and the few reported cases include IE caused by Enterobacteriacea, such as Serratia marcescens, Enterobacter spp., E. coli, K. Pneumoniae, Salmonella spp. and Pseudomonas aeruginosa (5,6). We had reported in the 27th ECCMID that our patients with EE have a higher mortality rate in Brazil. Hereby, we will explore virulence mechanisms and resistance of blood isolated Escherichia coli (E. coli) and Klebsiella pneumonia (K. pneumonia), in two EE cases. The qualitative biofilm formation was performed with isolates of E. coli and K. pneumoniae incubation within D-MEM (Dulbecco´s Minimal Essential Medium, Gibco-BRL), onto polystyrene coverlips for 18 hrs. The biofilm formation was evaluated with optical microscope. The adherence of both isolates in Vero and HEp-2 cell line was performed through incubations of 3h and 6h within MEM (Minimal Essential Medium, Gibco- BRL). After incubation, the interaction between bacteria and cell was evaluated with optical microscope. The molecular assays were performed by WGS (Whole Genome Sequencing) where total bacterial DNA was extracted from each isolate using the Ultraclean® microbial DNA isolation kit (MO BIO Laboratories, Carlsbad, CA, US). A DNA library was prepared using the Illumina Nextera XT kit. Biofilm could be observed after the period of incubation. The E. coli DO7785 showed strong biofilm formation while K. pneumonia isolates did moderate biofilm. In adherence assays with Vero and HEP-2 cells line, E. coli presented the same adherence pattern. Kp showed the same outcome in both cell lines.The molecular assays revealed that the E.coli strain belongs to ST69 and serotype O153:H2. All K. pneumoniae isolates belong to ST76. The genotyping assay determined that the resistance genes of E. coli were positive to strA, aadA5, strB, blaTEM-1B, sul1, sul2, dfrA17 while KP isolates were positive to genes blaSHV-1, oqxB,oqxA and fosA. The E. coli was resistant to Trimethoprim, Trimethoprim/ sulfamethoxazole and Ampicillin. All Kp isolates were resistance to ampicillin, nitrofurantoin and fosfomycin. The plasmid replicon type FIA was found in all K. pneumoniae. Virulence genes were investigated, all isolates were found to mrkD, urea, uge, pgaC, fimC, ompA and hgpA genes. The strains isolated showed resistance genes and consequently express resistance against important antibiotics. They also showed virulence genes and phenotypic aspects that prove their pathogenicity.