Persistence of Nontoxigenic Corynebacterium diphtheriae Biotype Gravis Strains in Pondicherry, Southern India

Diphtheria infections caused by the different toxigenic biotypes of Corynebacterium diphtheriae have shown to be endemic in many parts of the world, including India (1, 7). Among the total number of cases reported worldwide during the year 2005, 80% of cases were from India (1). Diseases due to nontoxigenic C. diphtheriae strains in vaccinated individuals are now being increasingly reported globally (2, 9). However, their incidence is very rare in India, as only one instance of nontoxigenic strains causing diphtheria has been documented earlier, from Pondicherry, India (6). We report here two more similar cases from the same place with pharyngeal and nasopharyngeal etiology. The second such occurrence reveals that nontoxigenic strains may be circulating in this region, thus forming a potential reservoir.

DtxR is a global regulator involved in the regulation of expression of diphtheria toxin, and nontoxigenic strains may represent a potential reservoir for the emergence of toxigenic strains if they possess functional dtxR and tox genes. Hence, it is important to determine the carriage status of tox and dtxR genes in our strains and to ascertain whether they are functional. A comparison of dtxR sequences of our isolates with the sequences of worldwide dtxR genotype strains was undertaken to detect any similarity or other patterns. Sequence variations in dtxR can serve as potential molecular subtyping markers for the surveillance of the spread of particular strains across any geographical area.

Of the two cases admitted during September 2008, the first patient was suffering from high fever with throat pain and the second one had fever with a history of nasal bleeding. Cultures (IR74 and IR125) obtained from throat swab and membrane from the tonsillar area of patients were identified as C. diphtheriae biotype Gravis by standard biochemical tests. A toxigenicity test by Elek's gel precipitation (3) was negative in both cases. The first patient had been immunized earlier with a full course of diphtheria-pertussis-tetanus (DPT) vaccine, whereas the second one was not able to recollect the history of immunization. Both patients recovered after antibiotic treatment without any clinical complications.

C. diphtheriae strains were analyzed by PCR (Veriti; Applied Biosystems), and direct sequencing was performed to determine the presence and intactness of the tox and dtxR genes. PCR primers were derived from earlier studies (2, 7, 8). Of the two strains, only IR74 showed positive for the tox gene while both were positive for dtxR amplification. Purified amplicons were custom sequenced (Macrogen). Raw chromatography data were edited using GeneTool software and aligned using CLUSTAL W, and the sequences were compared with previously published dtxR sequences of C. diphtheriae, namely, NCTC13129, UK-NT95-407, strain 1030, consensus sequence (2), Russian NTTB, Thailand, Uzbekistan, PW8, and C7hm723 (2, 9). There was no correlation between the PCR results and the Elek toxigenicity tests. Sequence analysis of dtxR alleles showed that they were identical to the published sequences of strain variants and found to be identical to the majority of nontoxigenic strains of the United Kingdom strain, the Russian NTTB strain, and an Uzbekistan strain and also to the toxigenic PW8 strain. Amino acid sequence analysis of the amplified dtxR genes from the two strains revealed variations of the predicted DtxR protein. Our strains were identical with variant 4, obtained in the United Kingdom (2), wherein there were two amino acid changes (alanine to valine at residue 147 and leucine to isoleucine at residue 214) compared with strain NCTC13129.

An increase of systemic diseases due to nontoxigenic strains has been observed recently in many parts of the world (9). However, nontoxigenic diphtheria cases have not been reported in India except in Pondicherry (5). Of the two nontoxigenic strains studied, only one showed the presence of the tox gene. Groman et al. (4) showed previously that nontoxigenic strains can carry DNA sequences for the tox gene, whereby such positive PCR cases were generally a consequence of amplification of DNA sequences that are part of a mutated or partial, and therefore nonfunctional, tox gene. Data from our study and other studies indicate that tox-bearing, nontoxigenic C. diphtheriae strains are rarely isolated from human clinical specimens (4, 6, 9).

Toxigenic strains have shown heterogeneity in sequences of the toxin-regulatory element dtxR. Previously, it was shown that mutations in the dtxR gene affect the functioning of DtxR and that even a single amino acid substitution is capable of severely diminishing or abolishing repressor activity (10). Hence, it is essential to explore whether variation in dtxR has any inhibitory effect on diphtheria toxin production. Investigation of our dtxR genotypes revealed that they were identical to dtxR genotype variant 4 from the United Kingdom (2). However, the exact association of dtxR variants with increased or decreased toxin production is still unknown and needs to be further evaluated. Nevertheless, one can speculate that naturally occurring variants of dtxR may be due to point mutations having their association with increased or decreased levels of toxin production and that such variations may not necessarily result in amino acid substitutions. Moreover, such variants can be potentially used for molecular subtyping (8).

The source of our strains is unknown; however, a large multicenter study involving large numbers of nontoxigenic strains from different countries may be helpful in deducing the source. The present and previous studies from Pondicherry demonstrated the presence of nontoxigenic strains and their potential to spread in this region. Although the limited number of strains encountered may not enable us to draw any conclusions, their persistence in this region is certainly evident. In conclusion, diseases due to nontoxigenic strains of C. diphtheriae are least often reported in India, unlike the rest of the world. Two nontoxigenic strains from Pondicherry reported here were shown to be identical to global nontoxigenic dtxR genotypes and the toxigenic PW8 strain.

Nucleotide sequence accession numbers.

Nucleotide sequences of DtxR variants from strains IR74 and IR125 have been deposited in GenBank under accession numbers HM231328 and HM231329, respectively.