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.     

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.     

Antimicrobial resistance among Brazilian Corynebacterium diphtheriae strains

The increasing problems with multidrug resistance in relation to Corynebacterium, including C. diphtheriae, are examples of challenges confronting many countries. For this reason, Brazilian C. diphtheriae strains were evaluated by the E-Test for their susceptibility to nine antibacterial drugs used in therapy. Resistance (MIC < 0.002; 0.38 microg/ml) to penicillin G was found in 14.8% of the strains tested. Although erythromycin (MIC90 0.75 microg/ml) and azithromycin (MIC90 0.064 microg/ml) were active against C. diphtheriae in this study, 4.2% of the strains showed decreased susceptibility (MIC 1.0 microg/ml) to erythromycin. Multiple resistance profiles were determined by the disk diffusion method using 31 antibiotics. Most C. diphtheriae strains (95.74%) showed resistance to mupirocin, aztreonam, ceftazidime, and/or oxacillin, ampicillin, penicillin, tetracycline, clindamycin, lincomycin, and erythromycin. This study presents the antimicrobial susceptibility profiles of Brazilian C. diphtheriae isolates. The data are of value to practitioners, and suggest that some concern exists regarding the use of penicillin.     

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.     

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.     

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.     

Genetic structure of Corynebacterium diphtheriae strains isolated in Russia during epidemics of various intensity

The genetic structure of C. dipthteriae toxigenic strains isolated in Russia during the period of more than 50 years was analysed. The use of the method of ribotyping made it possible to register 17 C. diphtheriae ribotypes. The study revealed that the genetic structure of C. diphtheriae population varied in the dynamics of the epidemic process: each epidemic cycle characterized by predominant spread of epidemic strains of definite biovars and ribotypes. Thus, C. diphtheriae strains of biovar gravis, ribotype M11, dominated in the 40-60 years and C. diphtheriae strains of biovar mitis, closely related ribotypes M1 and M1v, dominated in the 80 years. During the last epidemic rise of diphtheriae morbidity in the 90 s C. diphtheriae strains of biovar gravis, closely related ribotypes G1 and G4, dominated among circulating strains. The proportion of these ribotypes began to increase 3 years before the rise of morbidity. The data of microbiological monitoring are recommended for use in the prognostication of the development of the epidemic process of diphtheria infection.     

Polyacrylamide Gel Electrophoresis of Corynebacterium diphtheriae: a Possible Epidemiological Aid

In this preliminary study, the use of polyacrylamide gel electrophoresis as an aid in the characterization of Corynebacterium diphtheriae was evaluated and a standardized method was developed. The electrophoretic patterns of 17 gravis, 14 mitis, and 2 intermedius types of C. diphtheriae were compared with the electrophoretic patterns of 5 Robinson and Peeney stock gravis serotype strains. Each of the 5 stock serotype strains had different electrophoretic patterns, although some common bands were present. The 17 gravis strains isolated in the United States showed patterns identical to those of the stock gravis serotype II strain. The 14 mitis strains examined produced 6 different electrophoretic patterns, irrespective of geographical location. One mitis pattern corresponded with the pattern of gravis serological type II. The two intermedius strains examined had identical electrophoretic patterns that resembled the pattern of gravis serotype IV. Polyacrylamide gel electrophoresis of C. diphtheriae strains may prove to be a useful epidemiologic tool in establishing the distribution and occurrence of various C. diphtheriae types.     

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.     

Feasibility of decreasing the adhesive activity of Corynebacterium diphtheriae by biopolymers of natural origin]

Possible decreasing of the Corynebacterium diphtheriae adhesive activity by natural biopolymers was studied. It was shown that the strains of C.diphtheriae circulating on the Primorye Territory had middle, low or minimal adhesive activity. Natural biopolymers were found to decrease the adhesive properties of C.diphtheriae. The results of the study are promising for further investigation of natural biopolymers as agents preventing C.diphtheriae colonization on the stomatopharynx mucosa.     

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.     

Antibiotic Susceptibility Patterns of Recent Isolates of Corynebacterium diphtheriae

A variety of factors which might affect zone sizes were studied with strains of Corynebacterium diphtheriae; a standard disc method for antimicrobial sensitivity testing was used. Moderate variations in inoculum size, inoculum preparation, and pH of Mueller Hinton agar (MHA) did not appreciably affect zone sizes. The addition of blood to MHA was necessary to insure the growth of all C. diphtheriae strains on all lots of MHA. Zone diameters on MHA with blood were consistently 4 to 9 mm smaller than on plain MHA; however, zone diameters were within the sensitive range for seven antibiotic discs used on both media. Minimal inhibitory concentration (MIC) values for penicillin, erythromycin, and rifampin were determined by a plate dilution method. The geographical source, toxigenicity, and type of the strains showed no significant correlation with MIC values or zone diameters for eight antibiotic discs. When MIC values were compared to obtainable blood levels, all of the strains appeared to be sensitive with MIC values of 相似文献   

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.     

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.     

Pathogenic properties of Corynebacterium diphtheriae

The main pathogenic properties of 73 C. diphtheriae strains (their adhesive, invasive and cytotoxic activity) were characterized in the cultures of cells HEp-2 and Vero. The quantitative determination of the toxigenicity of 381 strains in the indirect hemagglutination test was made, and the strains were distributed by the degree of their toxigenicity. The characteristics of C. diphtheriae obtained with the use of in vitro experimental models, coincided with the severity of clinical manifestations of the diphtheria in humans, which made it possible to regard the models used in this study as adequate. On the basis of the chosen criteria the characterized strains could be subdivided into highly, moderately and low virulent and the degree of their potential epidemic danger could be determined.     

The effect of the nature of the iron sources on the growth of different Corynebacterium diphtheriae strains

In the study of the influence of organic and inorganic sources of iron on the growth of 5 C. diphtheriae clinical isolates bacterial growth was found to depended on the nature of the source of iron and its concentration. Differences between the strains in the level of growth, observed when ferric sulfate was used as the only source of iron in the medium, were established. Quantitative differences in the concentrations of inorganic and organic sources of iron, necessary for growth, were determined. The influence of three chemical chelators on the growth of C. diphtheriae under the conditions of iron deficiency in the culture medium was studied. The results of the study are indicative of the possibility of the differentiation of C. diphtheriae isolated according to the level of iron consumption.     

The use of MLVA for Corynebacterium diphtheriae genotyping--preliminary studies

The complete genome sequence of strain NCTC 13129 C. diphtheriae were investigated in order to identify tandem repeats (VNTR). From 75 VNTR loci identified in the genome 14 were selected. Primers were designed and PCR conditions were optimized for amplification of the selected VNTR markers. Preliminary studies of usefulness of selected VNTR markers were conducted using a group of 28 C. diphtheriae strains. From 14 markers 8 were regarded as potentially useful. The diversity of individual markers ranged from 1 to 6 alleles (Simpson index from 0 to 0,746). No diversity were observed for 3 VNTR markers but it could be a results of too small group of strains analyzed in the tests. Simpson diversity index calculated for all the markers tested on 28 strains was 0,87. Results of the preliminary studies showed usefulness of MLVA for C. diphtheriae genotyping. Nevertheless, confirmation of reliability of the method should be done using a large group of strains. Moreover, the method should be compared with other genotyping methods.     

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.