Pathogenicity factors of Corynebacterium diphtheriae circulating in the Primorski region under conditions of mass immunization of population

The study of the main pathogenicity factors of C. diphtheriae (adhesive activity, toxigenicity, detection of tox+ gene) circulating in the Primorski Territory has been made. As revealed in this study, at the period of declined epidemic process due to mass immunization of the adult and child population against diphtheria the selection of C. diphtheriae strains with weak toxigenicity and low adhesiveness was observed. No strains having tox+ genes have been detected among C. diphtheriae nontoxigenic strains circulating in the Primorski Territory.     

Pangenomic study of Corynebacterium diphtheriae that provides insights into the genomic diversity of pathogenic isolates from cases of classical diphtheria, endocarditis, and pneumonia

Corynebacterium diphtheriae is one of the most prominent human pathogens and the causative agent of the communicable disease diphtheria. The genomes of 12 strains isolated from patients with classical diphtheria, endocarditis, and pneumonia were completely sequenced and annotated. Including the genome of C. diphtheriae NCTC 13129, we herewith present a comprehensive comparative analysis of 13 strains and the first characterization of the pangenome of the species C. diphtheriae. Comparative genomics showed extensive synteny and revealed a core genome consisting of 1,632 conserved genes. The pangenome currently comprises 4,786 protein-coding regions and increases at an average of 65 unique genes per newly sequenced strain. Analysis of prophages carrying the diphtheria toxin gene tox revealed that the toxoid vaccine producer C. diphtheriae Park-Williams no. 8 has been lysogenized by two copies of the ω(tox)(+) phage, whereas C. diphtheriae 31A harbors a hitherto-unknown tox(+) corynephage. DNA binding sites of the tox-controlling regulator DtxR were detected by genome-wide motif searches. Comparative content analysis showed that the DtxR regulons exhibit marked differences due to gene gain, gene loss, partial gene deletion, and DtxR binding site depletion. Most predicted pathogenicity islands of C. diphtheriae revealed characteristics of horizontal gene transfer. The majority of these islands encode subunits of adhesive pili, which can play important roles in adhesion of C. diphtheriae to different host tissues. All sequenced isolates contain at least two pilus gene clusters. It appears that variation in the distributed genome is a common strategy of C. diphtheriae to establish differences in host-pathogen interactions.     

Identification of Corynebacterium diphtheria biovar belfanti among circulating C. diphtheriae strains in Ukraine

The biochemical test of the reduction of nitrates to nitrites made it possible to identify 5.2% of strains belonging to biovar belfanti among 135 C. diphtheriae strains, initially classified within biovar mitis. Out of 7 identified C. diphtheriae belfanti strains, 2 toxigenic strains were isolated from multiple foci diphtheria. According to the results of the polymerase chain reaction, 1 out of 5 non-toxigenic strains had tox gene. All C. diphtheriae belfanti strains were found to have pronounced capacity for adhesion to sheep and human red blood cells. At the stage of the extinction of diphtheria epidemic the practical identification of C. diphtheriae belfanti strains is necessary, as increased adhesion in combination with toxigenic properties may probably promote for bacteria of this biovar to take the leading role at the period of sporadic morbidity.     

Epiedmiologic significance of the distribution of corynephages in different collectives]

Corynephage distribution was studied in the nasopharyngeal washings of 252 persons infected with C. diphtheriae of gravis type, toxigenic (21 patients and 147 carriers) and non-toxigenic ones (84 carriers), and in 468 uninfected persons in collective bodies under different epidemic conditions. Corynephages were isolated from the nasopharyngeal washings only in persons infected with toxigenic C. diphtheriae--in 4 (of 21) patients, and in 21% (of 147) carriers. Phages tox+ (4--6.2%) were revealed only in carriers of toxigenic C. diphtheriae with numerous bacteria in the nasopharynx and in diphtheria patients. Carriers of nontoxigenic diphtheria bacilli can become infected with phage tox+ only together with the toxigenic strains (reinfection). The data obtained indicated that toxigenic and nontoxigenic C. diphtheriae strains were individual variants.     

Point mutations in tox promoter/operator and diphtheria toxin repressor (DTXR) gene associated with the level of toxin production by Corynebacterium diphtheriae strains isolated in Belarus

DNA fragments 129 bp in length containing promoter region of the tox gene from 81 toxigenic strains Corynebacterium diphtheriae were analyzed using the SSCP (single strand conformational polymorphism). We found that only two strains had mutations; the strains also had highest levels of toxin production (over 5120 Vero CD50/ml). Other strains were characterized either as high-level toxin-producing (640-5120 Vero CD50/ml, 41 strains) or low-level toxin-producing (40-320 Vero CD50/ml, 38 strains). Nucleotide sequence analysis revealed single T to C mutations at positions -54 and -184 within -232 - +85 region of tox operon. The first mutation at the -184 position was mapped outside the tox promoter/operator, whereas the second substitution at the -54 position modified the 9-base-pair interrupted palindromic sequence of the tox promoter/operator from ATAATTAGG in the wild-type bacteriophage (to ACAATTAGG in strains with enhanced level of toxin production. Nucleotide sequence analysis of -76 - +681 region of diphtheria toxin repressor (dtxR) gene from 15 strains of C. diphtheriae revealed two missense mutations resulting in amino acid substitutions A 147 V; and L 214 I in the C-terminal region of the DtxR protein. Seven of these strains were identified as high-level toxin-producing and 4 strains, as low-level toxin-producing. In addition, one low-level toxin-producing strain was shown to contain a missense mutation leading to amino acid substitution I 221 T. Three strains, including two highest-level toxin producing strains contained no nucleotide substitutions, as well as the C7(-) strain. The 10 strains belonging to the Sankt-Peterburg and Rossija epidemic ribotypes as well as NCTC 13129 strain (etiologic agent of the diphtheria epidemic outbreak in the Eastern Europe) was shown to contain two mutations A 147 V and L 214 I in the C-terminal region of the DtxR protein.     

Corynebacterium diphtheriae nontoxigenic strain carrying the gene of diphtheria toxin

Among 828 C. diphtheriae nontoxigenic cultures isolated in different region of Russia in 1994-2002, 114 cultures (13.8%) had the gene of diphtheria toxin (gene tox) and were thus called nontoxigenic tox-carrying (NTTC) strains. All NTTC strains were found to belong to biovar mitis and formed neither normal, nor "defective" diphtheria toxin. The most of NTTC strains (94%) belonged to ribotype "Moskva", not occurring among C. diphtheriae toxigenic strains. The incapacity of NNTC strains of forming diphtheria toxin was caused by mutation: the deletion of one nucleotide which led to the shift of the open reading frame and to the formation of the stop codon. The results of these studies are indicative of the fact that a sufficiently homogeneous and isolated group of C. diphtheriae nontoxigenic strains is spread in Russia. These strains carry the nonexpressing gene of diphtheria toxin and are of no epidemic importance in diphtheria infection.     

Integration of corynebacteriophages beta tox+, omega tox+, and gamma tox- into two attachment sites on the Corynebacterium diphtheriae chromosome.

The bacterial attachment sites of independently isolated Corynebacterium diphtheriae strains C7s and (belfanti)1030 lysogenic for corynebacteriophages beta tox+, omega tox+, and gamma tox- were determined by Southern blot analysis. Both corynebacterial strains contained two distinct bacterial attachment sites (attB1 and attB2). We found that infection by any of the three closely related corynebacteriophages may give rise to single, double, and triple lysogens. In the case of toxigenic C. diphtheriae strains C7s(beta tox+) and C7s(omega tox+), the final yields of diphtheria toxin produced under optimal conditions were equivalent and varied by one-, two-, or threefold depending upon the number of integrated prophage.     

DNA homology study of Corynebacterium diphtheriae v. gravis groups I, II and III, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis (ovis)

The homology of genomes within Krylova 's groups I, II and III of C. diphtheriae, including toxigenic C. diphtheriae and their nontoxigenic precursors within the same group, was confirmed by the method of DNA/DNA molecular hybridization; the homology of DNA within the groups was 89-103%, the thermostability of heteroduplexes being high (on the level of homoduplexes ). The heterogeneity of genomes within these 3 groups of cultivar gravis was confirmed, which made it possible to consider C. diphtheriae, groups I, II and III, to belong to different, though closely related species; in intergroup hybridization the homology of DNA varied, as a rule, between 66% and 73%, while the thermostability of heteroduplexes was low: delta T50 was -3 degrees C to -6 degrees C. The differences in genomes (on the level of different species) between 3 groups of C. diptheriae v. gravis on one hand and C. diphtheriae v. mitis C7 (-) tox- and its convertant C7 (beta) tox+ of phage tox+ on the other hand (DNA homology being 56-62%), as well as between C. diphtheriae v. intermedius No. 328 tox+ on one hand and the representatives of 3 groups of C. diphtheriae v. gravis and C. diphtheriae v. mitis, strain C7 (beta) tox+, on the other hand (DNA homology being 42-43%) were revealed. The heterogeneity of genomes (on the level of different genera) was revealed between C. diphtheriae strains, cultivars gravis (groups I, II and III), mitis (C7(-) tox- and C7 (beta) tox+) and intermedius (No. 328 tox+) on one hand and C. ulcerans and C. pseudotuberculosis (ovis) strains on the other hand; DNA homology was 11-17% for C. ulcerans and 22-26% for C. pseudotuberculosis (ovis), the thermostability of heteroduplexes being at the lowest level (delta T50 was -11 degrees C to -13 degrees C). As a result, C. diphtheriae, classified by Bergey as a single species, was found to comprise 5 species detected by means of marking in accordance with their phenotypical features and genome structure, carried out by the method of DNA/DNA molecular hybridization; among these species were group I, II and III strains of cultivar gravis, strain C7 of cultivar mitis and strain No. 328 of cultivar intermedius. C. ulcerans and C. pseudotuberculosis (ovis) strains investigated in this study can possibly be placed outside the genus including 5 C. diphtheriae species.     

Pathogenicity of Corynebacterium diphtheriae circulating in Rostov-on-Don City and Rostov region during interepidemic period

Main pathogenic characteristics (toxin production, tox-gene detection, adhesiveness) of 59 strains of C. diphtheriae circulating in Rostov-on-Don city and Rostov region in 2004-2005 were studied. Study of toxigenicity of 15 tox+ C.di phtheriae strains showed full coincidence of Elek immunoprecipitation test and polymerase chain reaction (PCR). Presence of part of A-fragment of tox-gene was detected in 5 (11.4%) of 44 C. diphtheriae strains that were negative in Elek test. Hemagglutinating activity of toxin producing strains was intermediate (40%) or high (60%). Among non-toxigenic strains those with intermediate adhesiveness were predominated (45,5%), the intermediate or high adhesiveness was detected in strains positive in PCR. Obtained characteristics of C. diphtheriae can be useful for surveillance for diphtheria infection during interepidemic period.     

Improvement of the preliminary classification of Corynebacterium diphtheriae v. gravis

In this study the previously published preliminary scheme for the subdivision of toxigenic and nontoxigenic Corynebacterium diphtheriae, classified with cultivar gravis, is made more precise. 3 groups remain in this scheme: I, II and III; each of them contains toxigenic C. diphtheriae (subgroup a) and nontoxigenic precursors of C. diphtheriae (subgroup b). For the first time nontoxigenic analogs of C. diphtheriae, phagovar OPQSTg, have been introduced into group I and newly discovered toxigenic C. diphtheriae, phagovar K, with their nontoxigenic precursors converted by phages 5 tox+, 6 tox+ and W tox+ have been introduced into group III. Group IV has been provisionally excluded from the scheme because this group comprises a small number of strains (3 strains). This classification can already be used in research practice for a finer differentiation of strains classified with cultivar gravis and for correct epidemic orientation.     

Comparative analysis of the DNA of Corynebacterium diphtheriae phages and the cloning of the gene determining diphtheria toxin synthesis

The analysis of the DNA of one nontoxigenic C. diphtheriae phage and two toxigenic ones has revealed that phage phi 984tox+ belongs to omega-like tox+ phages, phage phi 9tox+ is a representative of a new group of phages and phage B (Freeman) tox is a deletion mutant of phage beta. The location of this deletion on the physical map of this phage has been established. To obtain the physical map of phage phi 984tox+, the complete library of internal DNA fragments has been constructed in vector pBR 322. The gene of native diphtheria toxin has been cloned in vectors pBR 322 and pUR 250. Plasmids pUR 250 with the inserts of the toxin gene have been shown to be unstable if tox and lac promoters are located in tandem before the body of the toxin gene. The prolonged cultivation of clones having such structure leads to the formation of a spontaneous mutation located in the region coding the C-end part of the A-fragment of the toxin.     

Restriction endonuclease map of corynebacteriophage omega ctox+ isolated from the Park-Williams no. 8 strain of Corynebacterium diphtheriae.

The toxigenic corynebacteriophage omega tox+ was isolated from the hypertoxigenic Park-Williams no. 8 (PW8) strain of Corynebacterium diphtheriae and compared with the toxigenic corynebacteriophage beta tox+. The physical size and host range of both phages were found to be identical. An endonuclease restriction map of omega tox+ was constructed, and the locations of the cohesive ends (cos), phage attachment site (attP), and the diphtheria tox operon were identified. The genome of omega tox+ was found to differ from that of beta tox+ in three regions. In addition, omega tox+ was shown to be integrated into two nontandem corynebacterial phage attachment sites (attB1, attB2) in the PW8 chromosome. The differences in the restriction endonuclease digestion maps of omega tox+ and beta tox+ and the contribution of double lysogeny are discussed in relation to the hypertoxigenicity of the PW8 strain.     

Isolation and partial characterization of a corynebacteriophage beta, tox operator constitutive-like mutant lysogen of Corynebacterium diphtheriae.

We have isolated and partially characterized a beta-phage mutant lysogen of Corynebacterium diphtheriae, C7(betatoxct1+), which is partially insensitive to iron inhibition of diphtheria toxin production. tox expression by C7(betatoxct1+) was found to be partially constitutive. In the presence of concentrations of iron that almost completely inhibit the expression of diphtheria toxin by the wild type, C7(beta), the level of toxin production by C7(betatoxct1+) was found to be at least 25 times that of the parent. The purified tox gene product of C7(betatoxct1+) was immunologically and electrophoretically identical to, and equally as toxic as, diphtheria toxin purified from C7(beta). In addition, the partial N-terminal amino acid sequence was found to be identical to diphtheria toxin. This data strongly suggests that the mutation allowing for the constitutive expression of tox in C7(betatoxct1+) is outside of the structural gene. Furthermore, the constitutive expression of diphtheria toxin was found to be cis dominant in the double lysogen C7(betacrm45+/betatoxct1+). The data presented is consistent with the existence of a tox operator locus.     

Detection and physical map of a omega tox+-related defective prophage in Corynebacterium diphtheriae Belfanti 1030(-)tox-.

A library of chromosomal DNA from Corynebacterium diphtheriae Belfanti 1030(-)tox- was cloned in the lambda phage vector EMBL4 and screened for sequences homologous to corynephage omega tox+ and the attB1-attB2 region of the C7(-)tox- chromosome. Two portions of the 1030(-)tox- chromosome, 35 and 30.5 kilobases long which contain, respectively, the entire region homologous to corynephage omega tox+ and the attB1-attB2 sites, were mapped with the restriction endonucleases BamHI and EcoRI. Chromosomal DNA from 1030(-)tox- was shown to contain a 15.5-kilobase region that was homologous to ca. 42% of the corynephage omega tox+ genome. These sequences were found to hybridize to three regions of the phage genome and do not contain either the diphtheria tox operon or the attP site. These sequences are distant from the chromosomal region that contains the attB1-attB2 sites. Moreover, unlike other known defective prophages, the physical map of this prophage starts at the cos site and is colinear with the vegetative phage map. The 30.5-kilobase region of the 1030(-)tox- chromosome, which contains the attB1-attB2 sites, has a central core region that is almost identical to the corresponding region of the C7(-)tox- chromosome; however, the flanking sequences in these two strains of C. diphtheriae are different.     

DNA element of Corynebacterium diphtheriae with properties of an insertion sequence and usefulness for epidemiological studies.

The segment of DNA which is inserted within the tox gene of bacteriophage gamma and is responsible for its Tox- phenotype was found to be present and repeated approximately 30 times in the chromosome of Corynebacterium diphtheriae Belfanti 1030. Other C. diphtheriae strains contained a variable number of copies (1 to 25) of the same element. Sequence analysis showed that this repeated and interspersed DNA element was flanked by 9-base-pair direct repeats and that the 5' and 3' ends of the insertion contained sequences forming an imperfect inverted repeat. Therefore, the DNA segment here described has most of the typical structural features of a bacterial insertion sequence element. We show that different C. diphtheriae isolates derived from the same outbreak of diphtheria have an identical genomic distribution of this DNA element and that such DNA can be useful for epidemiological studies.