Diagnóstico molecular e detecção de genes de virulência de Campylobacter jejuni em crianças com diarreia moderada a severa na cidade de Fortaleza – CE, Brasil.

Abstract

Campylobacter spp. infections are considered one of the most common causes of bacterial gastroenteritis caused by contamination of water and food. Campylobacter jejuni is the most characterized specie, and the investigation of the epidemiology and virulence genes can elucidate some aspects of the microorganism pathogenicity. The aim of this study was to diagnose and identify the presence of virulence genes related to Campylobacter jejuni in children with moderate to severe diarrhea in the city of Fortaleza – CE, Brazil. This work is part of a project entitled “Diarrhea Enteric Card (DEC)”, which goal is to develop PCR-based multiplex diagnostic assays for bacterial enteric pathogens. That project was approved by the local and national ethical committees in Brazil (HIAS 80/06 and CONEPE 13523/2007, respectively). DNA was extracted directly from fecal samples isolated from 436 childrenwith moderate to severe diarrhea from May 2008 to April 2009, in Fortaleza, Ceará, Brazil. The diagnosis of C. jejuni was performed by conventional PCR using hipO gene. The detection of genes that encode proteins associated with virulence of C.jejuni was performed by uniplex and multiplex PCR techniques. C. jejuni was diagnosed in 14% (61/436) of the samples, presenting significant association among children aged 0-12 months (P = 0.0001) and children aged 12, 1-24 months. 51 positive samples for C. jejuniwere used for the detection of the virulence genes. The prevalence of theC. jejuni’s virulence-associated genes were detected in the following proportions of C. jejuni-positive DNA samples: flgE, (92.2%, 47/51) and flaA, (76.5%, 39/51), related to motility;cheW, (90.2%, 46/51); (cheA, 82.4%, 42/51) andcheR, (66.6%, 34/51), related to bacterial chemiotaxis; cadF, (100%, 51/51) andjlpA, (43.1%, 22/51), related to bacterial adhesion; ciaB, (96.1%, 49/51); iamA, (90.2%, 46/51); pldA, (45.1%, 23/51) and pVir(0%, 0/51), related to invasion; cdtABC, (94.1%, 48/51), related to cytolethal distending toxin (CDT); fur, (66.6%, 34/51); cfrA, (31.4%, 16/51) and ceuE, (21.7%, 11/51), related to bacterial iron transport and regulation; racR, (100%, 51/51); sodB, (96.1%, 49/51); dnaJ, (88.2%, 45/51) andkatA, (66.6%, 34/51), related to oxidative stress. The distribution profiles of C. jejuni’s virulence did not correspond to the patient’s clinical presentation of abdominal pain. But the presence of cfrA and dnaJ genes was correlated with fever (P=0,0214)and the presence of jlpA e katA gene were correlated with vomiting (P=0,0211) and pldA and ceuE genes was correlated with the presence of blood in stool (P=0,0013), this data suggests that relationships might be related to the severity of infection by this microorganism. New studies about the expression of proteins associated with the virulence genes must be carried out to better understand the pathobiology mechanisms of Campylobacter jejuni infections.