ABSTRACT
We report the complete genome sequence and analysis of an invasive Corynebacterium diphtheriae strain that caused endocarditis in Rio de Janeiro, Brazil. It was selected for sequencing on the basis of the current relevance of nontoxigenic strains for public health. The genomic information was explored in the context of diversity, plasticity and genetic relatedness with other contemporary strains.
Subject(s)
Corynebacterium diphtheriae/genetics , DNA, Bacterial/genetics , Genome, Bacterial/genetics , Brazil , Corynebacterium diphtheriae/classification , Corynebacterium diphtheriae/pathogenicity , Diphtheria/genetics , Phylogeny , VirulenceABSTRACT
The construction of a hexahistidine-tagged version of the B fragment of diphtheria toxin (DTB) represents an important step in the study of the biological properties of DTB because it will permit the production of pure recombinant DTB (rDTB) in less time and with higher yields than currently available. In the present study, the genomic DNA of the Corynebacterium diphtheriae Park Williams 8 (PW8) vaccine strain was used as a template for PCR amplification of the dtb gene. After amplification, the dtb gene was cloned and expressed in competent Escherichia coli M15 cells using the expression vector pQE-30. The lysate obtained from transformed E. coli cells containing the rDTB PW8 was clarified by centrifugation and purified by affinity chromatography. The homogeneity of the purified rDTB PW8 was confirmed by immunoblotting using mouse polyclonal anti-diphtheria toxoid antibodies and the immune response induced in animals with rDTB PW8 was evaluated by ELISA and dermonecrotic neutralization assays. The main result of the present study was an alternative and accessible method for the expression and purification of immunogenically reactive rDTB PW8 using commercially available systems. Data also provided preliminary evidence that rabbits immunized with rDTB PW8 are able to mount a neutralizing response against the challenge with toxigenic C. diphtheriae.
Subject(s)
Animals , Male , Mice , Rabbits , Corynebacterium diphtheriae/genetics , Diphtheria Toxin/genetics , Gene Expression Regulation, Bacterial/genetics , Corynebacterium diphtheriae/classification , DNA, Bacterial , Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
BACKGROUND & OBJECTIVE: Diphtheria infections caused by the different toxigenic biotypes of Corynebacterium diphtheriae are endemic in Delhi. Information on biochemical identification, toxigenicity and antimicrobial susceptibility to this bacterium is scanty. This retrospective study was carried out to identify isolated Corynebacteria biochemically, determine their toxigenicity, drug sensitivity and some epidemiological characteristics of diphtheria cases from Delhi and adjoining States for the period 1998-2004. METHODS: A total of 1118 throat and 585 nasal swabs were used to detect human pathogenic corynebacteria. WHO recommended methods were used for the detection, screening, toxigenicity and antibiogram pattern of the isolates. RESULTS: Among 493 (44.1%) cases detected positive for corynebacteria 71.8 per cent were pharyngeal, 20.9 per cent nasopharyngeal and rest 7.3 per cent nasal diphtheria cases. Biochemical identification revealed two species i.e., C. diphtheriae and C. pseudodiphtheriticum. In C. diphtheriae three biotypes were detected viz., intermedius (95.5%), gravis (3.4%) and mitis (1.1%). Toxin was expressed by 96 per cent isolates of C. diphtheriae. Cases were recorded from Delhi and four adjoining States. Sex ratio among male to female was 1.6:1. Prime victims were less than 9 yr old children (93.3%). Unvaccinated children (70.2%) were the main sufferers. Fatality rate was highest in Delhi cases (16.8%) followed by UP (14.6%) and Haryana (5.9%). INTERPRETATION & CONCLUSION: Standard methods revealed the replacement of C. diphtheriae var mitis with var intermedius and occurrence of diphtheria infections due to other human pathogenic corynebacteria. It is imperative to have good bacteriological facilities to have better surveillance with regular monitoring in the endemic areas to keep the disease under control.
Subject(s)
Child , Child, Preschool , Corynebacterium/classification , Corynebacterium diphtheriae/classification , Diphtheria/epidemiology , Female , Humans , India/epidemiology , Infant , Male , Retrospective Studies , SeasonsABSTRACT
Throat swabs from 1000 clinical cases of diphtheria were studied for isolation of C. diphtheria; only 21.4% were found to be positive. Most of the cases were between 1 to 4 years of age followed by 5 to 8 years. No cases were found below 8 months of age. The throat swabs taken from another 22 clinical diphtheria patients were immediately cultured at patients bed side and 63.6% were found to be positive for C. diphtheria. Time between the collection and plating the specimen was considered to be one of the main factors in the variation of the percentage isolation of C. diphtheria. From all KLB positive cases on direct smear, C. diphtheria could not be isolated on culture. Also, from all cases having definite patch(es) over tonsil(s), C. diphtheria could not be isolated. All strains were found to be sensitive against commonly used antibiotics.