Chemical composition of a preparation of the cell walls of Corynebacterium diphtheriae

The chemical characteristics of 6 batches of the preparation, obtained from the cell wall of C. diphtheriae grown in liquid and solid culture media, with respect to their content of nitrogen, hexoses, pentoses, total amino sugars, lipids and to the possible admixture of nucleic acids are presented. From the results of the chemical analysis of these batches their standardization according to the ratio of total amino sugars and pentoses to total nitrogen in C. diphtheriae cell-wall preparation is proposed.     

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.     

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.     

Evidence that the regulation of diphtheria toxin production is directed at the level of transcription.

It has been known for several decades that iron inhibits the production of diphtheria toxin by Corynebacterium diphtheriae by preventing expression at maximal levels. We examined the inhibition kinetics of toxin production after the addition of either iron or rifampin to iron-limited cultures of C7 (betatox+). Iron-mediated inhibition of toxin production was found to be linear within the range of 16 nM to 16 micron. The inhibition kinetics following the addition of iron or rifampin was almost identical. [3H]RNA extracted from iron-limited toxigenic C. diphtheriae was found to hybridize to a greater extent to corynephage beta DNA than either [3H]RNA extracted from toxigenic C. diphtheriae before the onset of toxin production or [3H]RNA extracted from nonlysogenic, nontoxigenic C. diphtheriae.     

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.     

Detection of toxin and evaluation of the degree of toxin-formation of Corynebacterium diphtheriae using immunoenzyme analysis

Toxin production and the intensity of toxin formation in 265 C. diphtheriae strains circulating in different areas of the USSR have been studied by the method of the enzyme-linked immunosorbent assay (ELISA). This study has been carried out with the use of the assay system consisting of monoclonal antibodies to the COOH-area of the B-fragment of the toxin molecule adsorbed onto the surface of polystyrene plates, affinity-purified polyclonal antidiphtheria antibodies labeled with horse-radish peroxidase and substrate indicator mixture (5-aminosalicylic acid and hydrogen peroxide). Some specific features of using ELISA for the detection of C. diphtheriae toxin directly in liquid culture medium are presented. High sensitivity, specificity and good reproducibility of this method permitting the detection of C. diphtheriae toxin and the determination of the intensity of toxin formation in the C. diphtheriae strains under study are shown. The method may be recommended for practical use at health institutions.     

The amino acid requirements of Corynebacterium diphtheriae PW 8 substrain CN 2000

AIMS: To determine the amino acid requirement and utilization pattern of Corynebacterium diphtheriae during growth and toxin production. METHODS AND RESULTS: Comparing across different batches of beef-based media, the growth and toxin yield were correlated significantly with nine of the amino acids. The amino acid utilization pattern during growth of C. diphtheriae further showed that only four of the nine amino acids, namely cystine, histidine, aspartate and methionine, were critical for growth of the vaccine strain. Further investigations using synthetic media with combinations of amino acid supplements demonstrated that among the four, cystine was the most growth limiting. CONCLUSIONS: Only certain amino acids are critical for growth and toxin production by C. diphtheriae, cystine being the single most important. SIGNIFICANCE AND IMAPCT OF THE STUDY: Owing to the potential threat from Bovine Spongiform Encephalopathy (BSE), a need is recognized by vaccine manufacturers to substitute beef-based production media. An understanding of the specific amino acid requirements would help to develop and optimize alternative production media.     

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.     

Utilization of host iron sources by Corynebacterium diphtheriae: identification of a gene whose product is homologous to eukaryotic heme oxygenases and is required for acquisition of iron from heme and hemoglobin.

Corynebacterium diphtheriae was examined for the ability to utilize various host compounds as iron sources. C. diphtheriae C7(-) acquired iron from heme, hemoglobin, and transferrin. A siderophore uptake mutant of strain C7 was unable to utilize transferrin but was unaffected in acquisition of iron from heme and hemoglobin, which suggests that C. diphtheriae possesses a novel mechanism for utilizing heme and hemoglobin as iron sources. Mutants of C. diphtheriae and Corynebacterium ulcerans that are defective in acquiring iron from heme and hemoglobin were isolated following chemical mutagenesis and streptonigrin enrichment. A recombinant clone, pCD293, obtained from a C7(-) genomic plasmid library complemented several of the C. ulcerans mutants and three of the C. diphtheriae mutants. The nucleotide sequence of the gene (hmuO) required for complementation was determined and shown to encode a protein with a predicted mass of 24,123 Da. Sequence analysis revealed that HmuO has 33% identity and 70% similarity with the human heme oxygenase enzyme HO-1. Heme oxygenases, which have been well characterized in eukaryotes but have not been identified in prokaryotes, are involved in the oxidation of heme and subsequent release of iron from the heme moiety. It is proposed that the HmuO protein is essential for the utilization of heme as an iron source by C. diphtheriae and that the heme oxygenase activity of HmuO is involved in the release of iron from heme. This is the first report of a bacterial gene whose product has homology to heme oxygenases.     

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.     

Draft Genome Sequence of Corynebacterium diphtheriae Biovar Intermedius NCTC 5011

We report an annotated draft genome of the human pathogen Corynebacterium diphtheriae bv. intermedius NCTC 5011. This strain is the first C. diphtheriae bv. intermedius strain to be sequenced, and our results provide a useful comparison to the other primary disease-causing biovars, C. diphtheriae bv. gravis and C. diphtheriae bv. mitis. The sequence has been deposited at DDBJ/EMBL/GenBank with the accession number AJVH01000000.     

Analysis of secreted protein profile and enzymatic activities from Corynebacterium diphtheriae and Bordetella pertussis on production batch media using peptide quenched fluorescent substrates

Proteases were identified and characterized from the culture supernatant of the C. diphtheriae and B. pertussis bacteria. The proteases were secreted in the media and detected at the end of the exponential growth phase. Activity was detected in some fluorescent substrates, based on selected protein sequences such as insuline beta-chain, bradykinin, and synaptobrevin. The proteases were purified by means of gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified proteins indicated, for the main secreted proteins, an estimated molecular mass of 30 kDa in C. diphtheriae and 69 kDa in B. pertussis culture media. The proteases were stable and presented enzymatic activity at 37 degrees C. These proteases were not related to the main toxic compounds described in these two bacteria, but could represent good markers for the fermentation process when the enzyme activity was measured with the fluorescent substrates.     

The interrelationship of biomass and toxin accumulation to the dynamics of the physicochemical parameters of Corynebacterium diphtheriae PW8 cultivation]

As established in the process the controlled cultivation of C. diphtheriae PW8, the rate of the accumulation of biomass and antigenic material increases with higher values of the redox potential of the nutrient medium (exceeding 11.0 mV). The data obtained in this study are indicative of the expediency of using the value of redox potential as an additional criterion in the evaluation of the quality of the nutrient media. The direct relationship between the productivity of the process of C. diphtheriae PW8 cultivation as regards the accumulation of biomass and antigenic material and the economic coefficients of the accumulation of biomass and antigenic material as determined by the utilized substrate has been established. The above relationship may facilitate the prognostication of the course of the process of microbial cultivation and the accumulation of diphtheria toxin, evaluated by antigenic activity.     

A new dried nutrient medium for the isolation of Corynebacterium diphtheriae (its development and experimental study)

A new dried diagnostic medium for the isolation of C. diphtheriae has been developed on the basis of aminopeptide. This new aminopeptide-based medium compares favorably with Buchin's medium in its growth and inhibitory properties.     

Use of paper indicator systems for the rapid identification of Corynebacterium diphtheriae, Neisseria meningitidis and the genus Staphylococcus

The possibility of using paper indicator discs with glucose, saccharose, lactose and urea, manufactured at the Gorky Research Institute of Epidemiology and Microbiology, has been studied and the methods for the preparation of indicator discs with fructose, maltose, starch and sodium phenolphthalein-phosphate for the biochemical identification of C. diphtheriae, N. meningitidis and staphylococci have been proposed. The study of 395 C. diphtheriae strains, 98 N. meningitidis strains and 328 staphylococcal strains has shown that paper indicator systems are highly sensitive and specific, which makes it possible to recommend their introduction in laboratory practice for the rapid identification of the above-mentioned organisms.     

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