Analysis of heterogeneity of Corynebacterium diphtheriae toxin gene, tox, and its regulatory element, dtxR, by direct sequencing

The largest diphtheria outbreak in the developed world since the 1960s is in progress in the Russian Federation. Seventy-two Corynebacterium diphtheriae strains from throughout Russia and the Ukraine, selected for temporal and geographic diversity, and 6 reference and control strains were assayed by DNA direct sequencing, and DNA sequences of their diphtheria toxin gene, fox and the regulatory dtxR gene, were compared to those of the Park-Williams no. 8 strain (PW8). Twenty-eight C. diphtheriae strains had entire fox sequences identical to that of the PW8 strain. Among the remaining 40 strains which were toxigenic, 4 point mutations were detected in the fox gene, one within the A and three within the B subunit gene. All four were silent mutations, indicating that diphtheria toxin is highly conserved at the amino acid sequence level; therefore, changes in the efficacy of the current vaccines would be unlikely to occur. Within the open reading frame of the regulatory dtxR gene, 35 point mutations were detected. Only 15 strains had entire dtxR sequences identical to that of the PW8 strain. Nine amino acid substitutions were found in the carboxyl half of dtxR: 22 and 25 strains differed from the PW8 strain in one and two amino acids, respectively. Given that naturally occurring variations of dtxR might be associated with increased diphtheria toxin production, studies to investigate the association of these point mutations and amino acid substitutions with quantified toxin production in the strains causing the current epidemic are under way.