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.     

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.     

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.     

Restriction endonuclease map of the nontoxigenic corynephage gamma c and its relationship to the toxigenic corynephage beta c.

Clear-plaque-forming mutant gamma tox- corynephages were isolated independently from nontoxigenic lysogenic Corynebacterium diphtheriae strains C7s(gamma tox-) and C4(gamma tox-). A physical map was constructed by using restriction endonucleases BamHI, EcoRI, HindIII, and KpnI. A comparison of nontoxigenic gamma c with toxigenic corynephage beta c revealed large areas of homology, including common regions for cohesive ends (cos) and attachment sites (att). Localization of the att sites on the beta prophage and correlation of the physical and genetic maps defined the orientation of the diphtheria tox operon. Diphtheria tox sequence homologies were mapped on gamma c by hybridizing 32P-labeled diphtheria tox mRNA to restriction fragments of gamma c DNA. Two regions of heterogeneity between phages beta c and gamma c were localized and these regions accounted for the 3-kilobase larger molecular size of gamma c compared with beta c. One change occurs near the tox promoter and may explain the nontoxigenic phenotype of corynephage gamma tox-.     

Adhesion in Corynebacterium diphtheriae

The study of 8 C. diphtheriae strains of different origin revealed that these strains were capable of inducing the agglutination of trypsinized sheep red blood cells (SRBC). Toxigenic strains gravis isolated from diphtheria patients were more active in their adhesion to SRBC than toxigenic strains gravis isolated from carriers. The latter were, in their turn, more active than nontoxigenic strains mitis. No fimbriae were detected on the cell surface by electron microscopy.     

The adhesive activity of diphtheritic strains in relation to the characteristics of the infectious process they induce]

The degree of adhesiveness of 602 C. diphtheriae strains isolated from patients with different forms of diphtheria was studied on trypsinized sheep red blood cells (SRBC) used as an experimental model. The titer of bacterial suspension, i.e. its highest dilution ensuring the agglutination of 50% of SRBC, was assumed to be the index of adhesive activity. The toxigenic strains were more homogeneous with respect to the degree of their activity and proved to be moderately and highly adhesive, while among the nontoxigenic strains faintly and moderately adhesive ones prevailed. The degree of adhesiveness was not linked with the cultural biological strains variants, but depended on the form of C. diphtheriae infection. The toxigenic strains isolated from diphtheria patients were essentially more active than those isolated from carriers. Both toxigenic and nontoxigenic strains isolated in cases of prolonged carrier state (more than 4 weeks) did not differ in the degree of their adhesiveness and were essentially more active than the strains isolated from short-term carriers. The strains circulating in 10 closed groups with a high proportion of pronounced cases of carrier state (70.6% to 86.7%) were essentially more active than those circulating in 10 similar groups, but having a low proportion of pronounced cases of carrier state (6.7% to 23.8%). The conclusion was made that the degree of adhesiveness proved to be an important factor of C. diphtheriae pathogenicity, responsible for the formation of carrier state. Along with pathogenicity, this factor should be taken into consideration in the evaluation of the epidemiological importance of different sources of infection.     

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.     

The use of polymerase chain reaction in the diagnosis of diphtheria infection

624 Corynebacterium diphtheriae strains, newly isolated from patients and carriers, were studied with the use of the methods of gel immunodiffusion (Elek's test) and polymerase chain reaction (PCR). In the evaluation of 388 C. diptheriae strains, found to be toxigenic in PCR, the results of Elek's test coincided with those of PCR on 98% of cases. In 38 out of 143 strains (26.5%), nontoxigenic according to the results of Elek's test, the presence of the A-fragment of the tox-gene was established. Subculturing in nutrient media made it possible to determine the presence of toxin in 19 out of 38 of these strains; the remaining strains, isolated mainly from carriers, were found to have the "silent" gene. The advantage of using PCR for the detection of toxigenic and nontoxigenic C. diphtheriae strains of different origin was shown.     

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.     

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.     

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.     

Non-opsonic phagocytosis of homologous non-toxigenic and toxigenic Corynebacterium diphtheriae strains by human U-937 macrophages

As interactions between bacteria and macrophages dictate the outcome of most infectious diseases, analyses of molecular mechanisms of non-opsonic phagocytosis should lead to new approaches for the prevention of diphtheria and systemic Corynebacterium diphtheriae infections. The present study aimed to evaluate human macrophage–bacteria interactions in the absence of opsonin antibodies and the influence of the tox gene on this process. Homologous C. diphtheriae tox + and tox – strains were evaluated for adhesion, entering and survival within U-937 human macrophages at different incubation periods. Higher numbers of viable bacteria associated with and internalized by macrophages were demonstrated for the tox + strain. However, viable intracellular bacteria were detected at T-24 hr only for the tox – strain. Cytoskeletal inhibitors, cytochalasin E, genistein and colchicine, inhibited intracellular viability of both strains at different levels. Bacterial replication was evidenced at T-24 hr in supernatants of monolayers infected with the tox – strain. Host cell death and nuclear alterations were evidenced by the Trypan blue exclusion assay and DAPI fluorescence microscopy. ELISA of histone-associated DNA fragments allowed detection of apoptosis and necrosis induced by tox + and tox – strains at T-1 hr and T-3 hr. In conclusion, human macrophages in the absence of opsonins may not be promptly effective at killing diphtheria bacilli. The presence of the tox gene influences the susceptibility of C. diphtheriae to human macrophages and the outcome of non-opsonic phagocytosis. C. diphtheriae strains exhibit strategies to survive within macrophages and to exert apoptosis and necrosis in human phagocytic cells, independent of the tox gene.     

Characteristics of antitoxic and antibacterial immune response in nontoxic forms of oropharyngeal diphtheria

The enzyme immunoassay system (EIA) for differentiation of antibodies in therapeutic heterogeneous antitoxic serum and antibodies to Corynebacterium diphtheriae toxigenic strains in patients and carriers was developed. The use of EIA permitted the dynamic evaluation of the characteristics of humoral antitoxic and antibacterial immune response in 50 patients with the localized and disseminated forms of stomatopharyngeal diphtheria and 14 "healthy" carriers of toxigenic C. diphtheriae. As revealed in this study, the symptoms of the disease in patients with disseminated forms of stomatopharyngeal diphtheria developed in the presence of statistically significant low quantitative values of antitoxic and antibacterial antibodies to C. diphtheriae antigens. In the group of patients with the localized forms of the disease the initially low level of antitoxic antibodies was detected with the concentration of antibacterial antibodies remaining unchanged. During the period of convalescence the levels of antitoxic antibodies in both groups reached those of healthy persons. In case of localized forms of the disease the level of antibacterial antibodies decreased as compared with healthy persons, starting from the second week of the disease. The period of convalescence in the disseminated forms was characterized by the low concentration of antibacterial antibodies. Carrier state was formed in the presence of high levels of antitoxic antibodies and significantly low levels of antibacterial ones.     

The study of agglutination of trypsin-treated sheep red cells by Corynebacterium diphtheriae strains

Abstract 620 Corynebacterium diphtheriae strains from 472 sick and healthy persons were studied for their adhesive activity (AA) in direct agglutination of trypsin-treated sheep erythrocytes. Toxigenic strains had more active AA than non-toxigenic ones which was not dependent on the presence of toxin in the culture. Neither biotype nor serotype of the strains correlated with their AA. Several lysotypes among toxigenic and non-toxigenic strains were more active than others. Toxigenic strains from patients had higher AA than those from carriers. Both toxigenic and non-toxigenic strains isolated from the prolonged carriers possessed the highest AA. It was concluded that AA measured in this way was an important colonization factor for all diphtheria strains and a pathogenicity factor for toxigenic strains.     

Effect of lysogenic conversion of Corynebacterium diphtheriae strain on its iron assimilation and spectrum of cellular proteins

Growth of the isogenic pair of C. diphtheriae strains, the nontoxigenic strain C7 (-) and the toxigenic strain C7 (beta), was studied under conditions of limited availability of the iron source. The growth of the toxigenic strain was shown to depend on the concentration of iron in the medium to a lesser degree than the nontoxigenic one. Lysogenic conversion results in the synthesis of additional iron-dependent proteins, absent in C. diphtheriae initial nontoxigenic strain C7 (-). Special attention was paid to proteins with a mol. wt. 66 kD, synthesized by the toxigenic strain irrespective of the concentration of iron in the medium, while in the toxigenic strain these proteins were detected only under conditions of iron deficiency.     

Indices of diphtheria (antitoxic) immunity in the dynamics of the disease and its treatment in adults

A total of 1,034 serum samples from 618 persons, including patients with different forms of diphtheria, carriers of the toxigenic forms of Corynebacterium diphtheriae, and angina patients, were studied. Analysis of the incidence of antibodies to C. diphtheriae toxin and their titers revealed that in more than half of all diphtheria patients no antibodies to C. diphtheriae toxin were detected upon admission to hospital. At the same time in 26% of the patients no antibodies were detected during the whole period of the disease; in such patients the toxic and subtoxic forms of diphtheria were registered twice as often as in seropositive patients. In 31% of the patients seronegative by the moment of hospitalization a rapid increase in the titers of antibodies C. diphtheriae toxin was observed in the course of the disease, which was indicative of the secondary character of immune response in patients who had been immunized earlier.     

Results of conducting an epidemiological survey of diphtheria in 21 territories of the RSFSR

As the result of epidemiological survey of diphtherial infection, carried out in conformity with the unified methodological recommendations in 21 regions of the RSFSR during 1980-1981, the expediency of such experiment was established. Immunity to diphtheria in children aged up to 14 years was high: children with negative Schick tests constituted 96.9-99.1%. No biological changes in Corynebacterium diphtheriae occurred during the term of observation. Toxigenic C. diphtheriae showed a high level of pathogenicity. The epidemiological survey contributed to a more thorough detection of diphtheria patients and carriers releasing toxigenic C. diphtheriae. The quality of clinical bacteriological diagnosis improved. In rare cases angina with the concomitant carriership of toxigenic C. diphtheriae could be diagnosed with the indispensable serological examination of the patients by Jensen's method.     

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.     

Interconversion of Type C and D Strains of Clostridium botulinum by Specific Bacteriophages

These studies show that Clostridium botulinum types C and D cultures can be cured of their prophages and converted to either type C or D depending on the specific phage used. Strains of types C and D were cured of their prophages and simultaneously ceased to produce their dominant toxins designated as C(1) and D, respectively. Cured nontoxigenic cultures derived from type C strain 162 were sensitive to the phages from the toxigenic type C strain 162 and type D strain South African. When cured nontoxigenic cultures derived from strain 162 were infected with the tox(+) phages from the 162 strain of type C and the South African strain of type D, they then produced toxin neutralized by types C and D antisera, respectively. Cured nontoxigenic cultures isolated from the type D South African strain were only sensitive to the parent phage, and, when reinfected with the tox(+) phage, they produced toxin neutralized by type D antiserum. Type C strain 153 and type D strain 1873, when cured of their respective prophages, also ceased to produce toxins C(1) and D, but, unlike strain 162 and the South African strain, they continued to produce a toxin designated as C(2). When the cured cultures from strains 153 and 1873 were infected with the tox(+) phage from type D strain 1873, the cultures simultaneously produced toxin that was neutralized by type D antiserum. When these cured cultures were infected with the tox(+) phage from type C strain 153, the cultures produced toxin that was neutralized by type C antiserum. These studies with the four strains of C. botulinum confirm that the toxigenicity of types C and D strains requires the continued participation of tox(+) phages. Evidence is presented that types C and D cultures may arise from a common nontoxigenic strain.     

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.