Resistência antimicrobiana, genes e propriedades de virulência em cepas de <i>Salmonella enterica</i> sorotipo Enteritidis isoladas de fontes animais, alimento e de doença humana no Brasil

Abstract

<i>Salmonella</i> Enteritidis is a major cause of foodborne illnesses worldwide. Birds are the main reservoir and poultry-derived foods are important sources of human infection. Many chromosomal and plasmid genes are required for virulence, and invasion of the intestinal epithelial cells is an essential step for the success of the infection. Considering that few studies on <i>S.</i> Enteritidis virulence were conducted in Brazil, we investigated the occurrence of virulence genes, production of biofilm, adhesins and cellulose, flagella expression and cell invasiveness of <i>S.</i> Enteritidis and its correlation with plantonic and biofilm growing conditions, still evaluating the differential expression of genes involved in cell invasion. All strains were isolated between 2006 and 2010 in different regions of Brazil from humans, food and animals. The presence of 14 virulence genes was tested by PCR, the invasion of human epithelial cells (Caco-2 and HEp-2) was evaluated by a quantitative test, the antimicrobial resistance was assessed by standard susceptibility tests, the production of biofilm was investigated by a quantitative test and differences in cell-association ability and cellular invasion were compared after plantonic- and biofilm-growing conditions, with the differential expression of genes involved in invasion examined by RT-qPCR. From 163 <i>S.</i> Enteritidis strains (human, 55; food, 75; animals, 33) 98% have all tested positive for 14 virulence genes (<i>invE/A, orgA, hilA, sipA, sseA, ssrB, mgtC, siiE, sopB, sefA, slyA, phoP/Q, stn</i> and <i>spvC</i>). From 85 strains, only 2.3% showed susceptibility to all antimicrobials tested and 71% were resistant to quinolone. Overall, 13% were multi-drug resistant (MDR for &#8805; 3 classes of antimicrobials). Two strains isolated from cases of human gastroenteritis carried the ESBL gene <i><SUB>blaCTX-M-2</SUB></i>. From 40 strains (human, 25; birds, 8; poultry-derived food, 7) 87.5% invaded human enterocytes (Caco-2). From 72 strains, 92% were strong or moderately biofilm-producers, occurring coexpression of curli pili and cellulose in 83% of the strong biofilm-producing strains. Strains growing in plantonic condition were more efficient in enterocyte invasion than strains growing in biofilms, and the expression of <i>sipA, invA</i> and <i>hilA</i> genes, located in the SPI-1 island (associated with cell invasion) was reduced in micro-organisms recovered from the biofilm. Most of the strains studied, besides of carrying a wide range of chromosomal genes and the virulence megaplasmid, is capable of adhering and efficiently invade human enterocytes, suggesting a pathogenic potential for the strains that circulate in the animal reservoir and animal foods. There was also a high frequency of MDR strains, especially among human isolates. We detected significant rates of resistance to quinolones (71%) and fluoroquinolones (7%) in addition to the resistance to cephalosporins (6%) and the aminoglycosides (2%). It was identified for the first time in Brazil, two CTX-M-2 ESBL-producing strains isolated from two epidemiologically unrelated cases of human disease. Striking biofilm production capability was found for most of the strains tested. We also observed reduced cell invasiveness after bacterial growth in biofilms and characterized that such defect results from differential regulation of genes found in the SPI-1 island.