The effect of rifampicin and gentamicin on toxin release and the SOS response in Escherichia coli O104:H4 -

Abstract

Background: Escherichia coli O104:H4, a new pathotype Enteroaggregative Hemorrhagic Escherichia coli (EAHEC), was the cause of a 2011 severe outbreak affecting European countries especially Germany. Its high virulence and novel pathophysiology seem to be due to the acquisition of a Shiga toxin 2 (Stx2) encoding prophage by horizontal gene transfer. Treatment of Shiga toxin producing E.coli (STEC) with antimicrobial agents is currently refrained from since it may lead to bacterial lysis, enhanced release of toxins, and exacerbation of the disease. To assess whether antimicrobial agents can be used in the treatment of the outbreak Escherichia coli O104:H4 strain infections, the effects of rifampicin and gentamicin were evaluated at the levels of the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). They were also evaluated at two sub MIC levels to assess the SOS response, a bacterial DNA damage response, and its effect on toxin release.Methods: The MIC and MBC of rifampicin and gentamicin for the novel pathotype outbreak strain from Germany (D3774-C22711) were determined using the broth dilution method. Pulsed field gel electrophoresis (PFGE) was carried out on the new E. coli O104:H4 pathotype and two pre-outbreak E. coli O104:H4 strains (2009 EL-2050 and 2009 EL-2071 from the Republic of Georgia), to determine their genomic relatedness. Polymerase Chain reaction (PCR) analysis of the stx2 gene in the outbreak and pre-outbreak strains was performed. The transcription level of the stx2 gene was assessed in the 3 strains using Real time polymerase chain reaction (RT-qPCR). In addition, the transcription levels of the stx2 and recA (SOS response inducer) genes were assessed using RT-qPCR in the outbreak E.coli O104:H4 samples treated with different regimens of rifampicin and gentamicin. Subsequently, Reverse Passive Latex Agglutination (RPLA) was used to determine the Stx2 titers in supernatants of bacterial cultures subjected to different doses and combinations of the antimicrobia