Molecular identification of intracellular survival related Brucella melitensis virulence factors

Abstract

Introduction and Aim: Brucellosis is an important zoonotic disease caused by Brucella spp. which is an intracellular gram-negative bacterium. Brucella melitensis lacks the "traditional" virulence factors such as exotoxins or cytolysins, but is capable of persisting intracellularly and evading the immune system. This study aims to identify B. melitensis using PCR and discover genes associated with its severity for early detection and therapy. Materials and Methods: Ten ml of unclotted blood sample was collected from each patient (n=100) suspected to be infected with brucellosis. The Castaneda technique was used to inoculate blood samples onto Brucella Basel agar with a selective supplement and tryptone soy broth in a diphasic flask. Biochemical tests were used in identifying the isolated colonies. B. melitensis isolates were further confirmed by the polymerase chain reaction (PCR) technique using, primers targeting a specific region (IS711 gene) of the genome. Multiplex PCR was used to determine the four virulence related genes (lps B, mgtA, omp25, CBG) in all positive samples. Results: Brucella melitensis was detected in 9% (9/100) of the blood samples. Among the virulence factors, Lps B and mgtA, were detected in all the isolates while, the genes omp25 and CBG were detected in 66.6% and 55.5% of the isolates, respectively. Conclusion: Brucellosis could be diagnosed rapidly using molecular techniques. PCR technique could also be used in identifying the Brucella virulence related genes lpsB, mgtA, CBG, and omp25 that are crucial to the bacterium's pathogenicity in the intracellular environment.