Caracterização fenotípica e molecular de Escherichia coli uropatogênica pic+ isoladas de pacientes com infecção do trato urinário

Abstract

Urinary tract infection (UTI) is characterized by the presence of microorganisms that invade the urinary tissues, with the presence of bacteria in the urine in amounts equal to or greater than 105 CFU/ml. It is mainly caused by enterobacteria, among which the uropathogenic Escherichia coli (UPEC) stands out, due to the presence of several virulence factors, such as the production of serine proteases. In Gram negative bacteria, proteases belonging to the Enterobacteriaceae family are known as SPATEs (Serino protease autotransporter of Enterobacteriaceae). Among SPATEs, the protein involved in colonization (Pic) is of great relevance and is produced both by E. coli pathotypes and by Shigella flexneri and Citrobacter rodentium. Pic is described in the literature for causing hemagglutination, degrading mucin, cleaving complement system molecules, among others. Thus, this work aims to detect the pic gene in uropathogenic Escherichia coli isolated from patients with urinary tract infection and perform the phenotypic characterization of these isolates. In a two-month period, the laboratory collected 9,961 UTI samples, of which 369 were selected for the antimicrobial susceptibility profile, followed by PCR to characterize the virulence genes associated with the production of SPATEs. The ability of these isolates to form a biofilm was verified using the crystal violet technique and later a survival test using invertebrate animals (Tenebrio mollitor larvae) was evaluated for 10 days. Of the 369 samples analyzed in the study, four had only the pic gene, among the SPATEs genes researched. The sensitivity test of the samples indicated resistance to antibiotics used in cases of UTI, which belong to the class of beta-lactams, sulfonamides and quinolones. The presence of the pic gene may be related to the ability of UPEC to degrade mucus and promote colonization in the urinary tract mucosa. The tests carried out showed that the strains containing pic produced different amounts of biofilm from each other, with the so-called ITU032 being considered a weak biofilm former, while the ITU015 strain produced more biofilm than the positive control 042, demonstrating that the pathogenicity of the isolate possibly it is not related to the amount of biofilm it can produce. In the survival test using T. molitor larvae, it was possible to observe a survival rate of 40% in larvae infected with strains ITU014 and ITU040, with the highest mortality rate in those infected with strain ITU015, with survival of only 10%. The UTI32 strain demonstrated a 70% lethality. This method is faster and more economical than murine models and presents more relevant results than cell-based assays and can be used to verify if any natural compound has antibacterial capacity. In general, this study demonstrates that UPEC strains have shown resistance to several antimicrobials, in addition to expressing the pic virulence gene characteristic of EAEC. New trials are nearing completion with the aim of understanding the role of these serine proteases in urinary infections and thereby seeking new mechanisms for treating these complications.