Strains Of Corynebacterium Diphtheriae Research Articles

Experimental model of infection with non-toxigenic strains of Corynebacterium diphtheriae and development of septic arthritis

Corynebacterium diphtheriae is a well-known cause of localized respiratory tract infections. However, this micro-organism can also be associated with invasive infections, such as endocarditis, septic arthritis and osteomyelitis. Invasive infections are often caused by non-toxigenic strains. To set up an in vivo experimental model of C. diphtheriae infection, mice were infected intravenously with different doses (ranging from 1 x 10(7) to 5 x 10(8) bacteria per mouse) of three non-toxigenic strains, namely ISS-4749, ISS-4746 and ISS-3319. Similar mortality rates were observed with the three strains, with an LD50 ranging from 9 x 10(7) to 1.2 x 10(8). All strains were arthritogenic, although to different extents. ISS-4749 and ISS-4746 infection resulted in a maximum of 60 and 50 %, respectively, of animals with articular lesions, while in the ISS-3319-infected group only 25 % were positive. There were differences in systemic and joint cytokine production in the three experimental groups. ISS-4749- and ISS-4746-infected mice exhibited higher local levels of interleukin (IL)-6 and IL-1beta than ISS-3319-infected animals. At systemic levels, ISS-3319 was able to induce early and sustained production of interferon-gamma (IFN-gamma), but not IL-6. Conversely, infection with the other strains resulted in high IL-6, but not IFN-gamma, production. In conclusion, an experimental model of C. diphtheriae infection was set up, with development of septic arthritis. This model could be useful in studies on the pathogenicity and characterization of virulence factors other than toxin production.

Eagle Effect inCorynebacterium diphtheriae

The in vivo relevance of the paradoxical bactericidal effect (the Eagle effect) is not evident. We found in vitro a paradoxical bactericidal effect of amoxicillin on 2 strains of nontoxigenic Corynebacterium diphtheriae. Then, using an experimental rabbit model of endocarditis, we evaluated the in vivo relevance of this phenomenon. Rabbits were assigned to the following groups: no treatment (control group), continuous amoxicillin infusion simulating a dosage of 200 mg/kg/day in humans, and continuous amoxicillin infusion simulating a dosage of 20 mg/kg/day in humans. The low dosage (20 mg/kg/day) was significantly more effective than the high dosage (200 mg/kg/day) against both strains (P<.025), confirming the paradoxical bactericidal effect observed in vitro.

The first case of septicemia due to nontoxigenic Corynebacterium diphtheriae in Poland: case report

BackgroundToxigenic strains of Corynebacterium diphtheriae are well known agent of diphtheria. Nontoxigenic strains can cause atypical course of the disease. Invasive diseases caused by C. diphtheriae occur very rare.Case presentationWe have described the first case of septicemia and endocarditis due to nontoxigenic C. diphtheriae biotype gravis in Poland. The patient has not belonged to any group of risk such infection.ConclusionThe case presented in this article shows unusual case of infection connected with nontoxigenic C. diphtheriae that took place in the area where have been no cases of diphtheria and other C. diphtheriae infections for near ten years. It shows the importance of identifying Corynebacterium isolates at the species level especially when the strain has been isolated from normally sterile sites.

Infections with Corynebacterium diphtheriae: six years’ experience at an inner London teaching hospital

Recently, a preponderance of non-toxigenic strains of Corynebacterium diphtheriae has been reported, as has the broadening spectrum of disease caused by these strains. This study presents data on 85 isolates of C. diphtheriae over a six year period (1998–2003). Eighty were non-toxigenic isolates from patients with sore throat, and five (one toxigenic) were from cutaneous ulcers in travellers returning from endemic areas. When examined in relation to denominator data provided by the Health Protection Agency (HPA) for the whole of England and Wales, 1998–2002, 75% of all notifications of C. diphtheriae for England and Wales originated from the laboratories at University College London Hospitals (UCLH). In some years (1999 and 2001) 95–100% of isolates came from UCLH. We believe that national data do not reflect true incidence, as universal screening for these organisms is not routine policy in many laboratories. The results presented suggest the need for increased clinical and laboratory awareness of this important pathogen.

Electrocardiographic abnormalities in patients with diphtheria: a prospective study

Purpose To determine the incidence of and risk factors for electrocardiographic (ECG) abnormalities in adults with diphtheria. Methods A prospective study was conducted involving 122 adult patients with respiratory tract diphtheria. Diphtheria was confirmed by isolation of a toxin-producing strain of Corynebacterium diphtheriae. Patients had serial clinical evaluations and ECGs for a minimum of 21 days. Results Cardiac involvement was detected in 25 (28%) of 88 evaluable patients, with a median time from symptom onset to an abnormal ECG of 9 days (range, 4 to 24 days). In a logistic regression analysis, age (odds ratio [OR] = 4.1; 95% confidence interval [CI]: 1.6 to 11.0), shared accommodation (OR = 2.9; 95% CI: 1.0 to 8.6), fever (OR = 4.2; 95% CI: 1.1 to 16.6), and extensive respiratory tract infection with subcutaneous edema (OR = 7.0; 95% CI: 1.2 to 42.2) were independent risk factors for cardiac involvement. Conclusion Cardiac involvement is a common complication of respiratory tract infection with C. diphtheriae, and occurs more often among older patients, those with lower socioeconomic status, and those with severe respiratory tract involvement.

Patterns of adherence to HEp-2 cells and actin polymerisation by toxigenic Corynebacterium diphtheriae strains

Corynebacterium diphtheriae strains displayed different degrees of attachment to HEp-2 cell monolayers with two distinct adherence patterns, termed localised (LA) and diffuse (DA). The LA phenotype predominated over the DA phenotype. The non-sucrose fermenting strains expressing DA pattern adhered mostly with high index values (≥10 bact/cell). Low adhesion index (<10 bact/cell) was mainly observed among sucrose fermenting strains. The fluorescein isothiocyanate (FITC)-labelled phalloidin assay (fluorescent-actin staining test) showed positive results for microorganisms of both LA and DA phenotypes. The FITC-labelled C. diphtheriae non-fimbrial surface proteins 67–72p interacted directly with HEp-2 cell membranes. Therefore, toxigenic C. diphtheriae exhibited LA and DA adherence patterns and ability to induce actin polymerisation. The experimental evidences also pointed to 67–72p as putative adhesins of C. diphtheriae to HEp-2 cells.

Influence of stannous chloride on the adhesive properties of Corynebacterium diphtheriae strains

Stannous ion, as a chloride salt, influenced on the survival and adhesive properties of two toxigenic Corynebacterium diphtheriae of the sucrose-fermenting (241 strain) and non-sucrose-fermenting (CDC-E8392 strain) biotypes. Differences in survival fractions suggested differences in susceptibility of strains to bactericidal effect of stannous chloride (SnCl2). A number of 0.3% bacterial cells of 241 strain and 0.02% of CDC-E8392 strain survived after 220 micro l ml(-1) SnCl2 treatment. Results of polystyrene and spontaneous autoaggregation tests showed an increase in hydrophobicity of SnCl2 treated-bacteria. Spontaneous bacterial autoaggregation was induced in the presence of SnCl2. Stannous chloride also induced adherence to glass and totally inhibited the haemagglutinating activity of the non-sucrose-fermenting CDC-E8392 strain (original titer 32). Decrease in haemagglutination was dependent on SnCl2 concentration used. The presence of SnCl2 exerted differences in the expression of diphtheria bacilli surface carbohydrates possibly related with differences in degrees of haemagglutination and adherence to glass. Lectin-binding assays showed increase in the expression of cell surface receptors to the lectin Canavalia ensiformis (Con A) with affinity for mannose-like residues. The occurrence of cell filamentation suggests genotoxicity of SnCl2 to diphtheria bacilli. SnCl2 treatment was capable of modifying cell morphology, hydrophobins and adhesin expression, suggesting ability of C. diphtheriae to withstand oxidative stressing environment. Therefore, the SnCl2, widely used in nuclear medicine as reducing agent in the 99mTc-labelling process, may influence the outcome of bacterial infections.

Activité de l’amoxicilline in vivo dans l’endocardite expérimentale du lapin à Corynebacterium diphtheriae non toxinogène

The mortality of non-toxigenic Corynebacterium diphtheriae endocarditis is high (27%). One explanation could be tolerance to amoxicillin. The aim of this work was to evaluate in vivo the tolerance phenomenon, in a rabbit endocarditis experimental model. Two strains of non-toxigenic Corynebacterium diphtheriae were compared: a tolerant one and a non-tolerant one. Each animal was randomly assigned to one of the following groups: no treatment, continuous infusion of amoxicillin simulating 200 mg/kg/24 h in humans, or fractionated infusion of amoxicillin simulating 66 mg/kg/8 h in humans. Surviving bacteria were counted in vegetations after one or three days of treatment. The 24 h fractionated amoxicillin infusion was more efficacious on the non-tolerant strain than on the tolerant strain. On the tolerant strain, 24 h amoxicillin was more efficacious as a continuous infusion than as a fractionated one.

Intracellular viability of toxigenic Corynebacterium diphtheriae strains in HEp-2 cells

Corynebacterium diphtheriae, generally considered an extracellular coloniser, was evaluated for its ability to enter and survive within HEp-2 monolayers by gentamicin protection assay. Intracellular viability of HC01 strain, isolated from endocarditis, was more expressive (2.59%) than observed in 241 (0.21%) and CDC-E8392 (1.93%) strains. Electron microscopy of C. diphtheriae-infected HEp-2 cells revealed intracellular bacteria inside membrane-bound vacuoles. Bacterial internalisation was totally inhibited by 5 μM cytochalasin E and significantly inhibited by 100 μM genistein ( P<0.05). Therefore, C. diphtheriae presents the ability to survive within cultured epithelial cells and signalling cascade as well as actin polymerisation are required for entry of diphtheria bacilli into HEp-2 cells.

Molecular characterization of Corynebacterium diphtheriae isolates, Russia, 1957-1987.

In the 1990s, the Newly Independent and Baltic States of the former Soviet Union experienced the largest diphtheria outbreak since the 1960s; it was caused by Corynebacterium diphtheriae strains of a unique clonal group. To address its origin, we studied 47 clinical isolates from Russia and demonstrated that this clonal group was an integral part of the endemic reservoir that existed in Russia at least 5 years before the epidemic began.

Study of Corynebacterium diphtheriae strains isolated in Romania, northwestern Russia and the Republic of Moldova

A selection of 167 Corynebacterium diphtheriae strains isolated in Romania, the Russian Federation and the Republic of Moldova were analysed by biotyping, phage typing, the toxin production test and by molecular techniques such as ribotyping, pulsed field gel electrophoresis and random amplified polymorphic DNA, in order to establish the epidemiological relatedness, genetic divergence and strain circulation within and between the bordering countries. Using a set of five digoxigenin-labeled oligonucleotides and BstEII digestion, 34 ribotypes were identified. The strains isolated in the epidemic areas (Russia and Moldova) were very closely related but different from those isolated in Romania. C1 and C5 were the main ribotypes identified in these areas. Neither ribotype was found in Romania, where the main circulating types were C3 and C7. Field inversion gel electrophoresis was more discriminative than ribotyping and revealed 54 macrorestriction profiles after SfiI restriction. Both methods showed a significant homogeneity of the strains from epidemic areas and a large diversity among the Romanian strains. Random amplification was useful as an identification method for the epidemic strains, but not for the Romanian ones which displayed a large number of amplification profiles. The phenotypic methods associated with molecular typing techniques enabled distinguishing between strains, detecting the epidemic clone, and sustaining the absence of transmission across borders.

Corynebacterium diphtheriae surface proteins as adhesins to human erythrocytes

Corynebacterium diphtheriae strains express non-fimbrial surface proteins able to recognize and bind to specific host cells receptors. Protein extracts were obtained from bacterial cells by mechanical process and ammonium sulfate precipitation at 25 and 45% (w/v) saturation. SDS-PAGE analysis of the extracts detected two polypeptide bands of 67 and 72 kDa, named 67-72 p. The 67-72 p, rabbit anti-67-72 p IgG antibodies as well as human gastric mucin, N-acetylneuraminic acid and N-acetyl D-glucosamine molecules were able to inhibit bacterial hemagglutination. Hemagglutination assays using 67-72 p-coated latex beads and Western blot analysis of biotin-labeled 67-72 p and erythrocyte receptors demonstrated the binding of 67-72 p to human erythrocyte membranes. Immunolabeled colloidal gold-A protein transmission electron microscopy using anti-67-72 p revealed a diffuse distribution of non-fimbrial 67-72 p on the surface of C. diphtheriae strains of both sucrose-fermenting and non-fermenting biotypes. Non-fimbrial lectin-like surface 67-72 p may play a role as adhesins in bacterial attachment thereby facilitating the early steps in pathogenesis of both toxigenic and non-toxigenic C. diphtheriae.

Corynebacterium diphtheriae surface proteins as adhesins to human erythrocytes.

Corynebacterium diphtheriae strains express non-fimbrial surface proteins able to recognize and bind to specific host cells receptors. Protein extracts were obtained from bacterial cells by mechanical process and ammonium sulfate precipitation at 25 and 45% (w/v) saturation. SDS-PAGE analysis of the extracts detected two polypeptide bands of 67 and 72 kDa, named 67-72 p. The 67-72 p, rabbit anti-67-72 p IgG antibodies as well as human gastric mucin, N-acetylneuraminic acid and N-acetyl D-glucosamine molecules were able to inhibit bacterial hemagglutination. Hemagglutination assays using 67-72 p-coated latex beads and Western blot analysis of biotin-labeled 67-72 p and erythrocyte receptors demonstrated the binding of 67-72 p to human erythrocyte membranes. Immunolabeled colloidal gold-A protein transmission electron microscopy using anti-67-72 p revealed a diffuse distribution of non-fimbrial 67-72 p on the surface of C. diphtheriae strains of both sucrose-fermenting and non-fermenting biotypes. Non-fimbrial lectin-like surface 67-72 p may play a role as adhesins in bacterial attachment thereby facilitating the early steps in pathogenesis of both toxigenic and non-toxigenic C. diphtheriae.

Epidemiology of Diphtheria in the 1990s

Introduction Respiratory diphtheria, a severe systemic illness caused by toxigenic strains of Corynebacterium diphtheriae, was one of the most common causes of death among children in the pre-vaccine era. Diphtheria toxoid vaccine was developed in the 1920s. Widespread immunization led to the virtual elimination of diphtheria in developed countries by the 1960s and to a marked reduction of diphtheria cases in developing countries in the 1980s. The control of diphtheria has resulted in a larger proportion of adults lacking immunity as a result of failure to vaccinate or waning immunity among the vaccinated in the absence of exposure to infection. In the early 1990s, epidemic diphtheria re-emerged in the Newly Independent States (NIS) of the former Soviet Union after 30 years of control and caused thousands of deaths, primarily among adults. This paper briefly reviews the epidemiology of diphtheria in developed and developing countries, focusing on the 1990s, and comments on recent laboratory developments.

Diphtheria in the Russian Federation in the 1990s.

A resurgence of diphtheria spread throughout the Russian Federation in the early 1990s; diphtheria had been well controlled, but circulation of toxigenic strains of Corynebacterium diphtheriae had persisted since the implementation of universal childhood vaccination in the late 1950s. More than 115,000 cases and 3,000 deaths were reported from 1990 to 1997, and, in contrast to the situation in the prevaccine era, most of the cases and deaths occurred among adults. Contributing factors included the accumulation of susceptible individuals among both adults and children and probably the introduction of new strains of C. diphtheriae. Vaccine quality, vaccine supply, or access to vaccine providers did not significantly contribute to the epidemic. Mass vaccination of adults and improved childhood immunization controlled the epidemic. High levels of population immunity, especially among children, will be needed to prevent and control similar outbreaks in the future.

Direct polymerase chain reaction for detection of toxigenic Corynebacterium diphtheriae strains from the Republic of Georgia after prolonged storage.

A total of 226 paired nose and throat swab specimens from 113 clinical diphtheria cases from the republic of Georgia were analyzed by direct polymerase chain reaction targeting both A and B subunits of the diphtheria toxin gene, tox. Even after prolonged transport and extensive storage (7-14 months) of the clinical specimens in silica gel packages, direct polymerase chain reaction detected the diphtheria tox gene in 54% of the specimens. Specimens obtained by throat swab were three times more likely than those obtained by nose swab to be positive for Corynebacterium diphtheriae.

Diphtheria in Lithuania, 1986-1996.

Diphtheria reappeared in Lithuania in 1986 and rose to epidemic levels by 1992. Between 1991 and 1996, 110 cases of diphtheria were registered, with an incidence of 0.03-1.15/100,000 population. Most cases (84%) and all 17 deaths occurred among persons >/=15 years, most of whom had never been vaccinated. Persons 40-49 years old had the highest average annual age-specific morbidity (1.70/100,000) and mortality (0.53/100,000) rates. Low levels of immunity among individuals 40-49 years old and migration to epidemic areas in Russia and Belarus contributed to the epidemic's occurrence. Between 1991 and 1995, toxigenic Corynebacterium diphtheriae strains were isolated from 84 of all registered patients (76%), and nontoxigenic strains were isolated from 13 (12%). By 1996, two mass vaccination campaigns, which provided one dose of vaccine to individuals 25-30 years old and three doses of vaccine to persons 31-60 years old, helped reduce the number of cases. The first campaign achieved 69% coverage; the second achieved 48% coverage.

The European Laboratory Working Group on Diphtheria: A global microbiologic network.

Increasing international travel, migration to and from epidemic areas, and the emergence of epidemic Corynebacterium diphtheriae strains globally have highlighted the need for better microbiologic surveillance and greater clinical and laboratory awareness. As part of the strategy developed by the World Health Organization, one major goal is particularly applicable to the microbiology and epidemiology of diphtheria: to improve surveillance for early detection of emerging and reemerging diseases by establishing a network of national and international laboratories. The European Laboratory Working Group on Diphtheria (ELWGD) was therefore formed in 1993 as a result of the epidemic situation in Eastern Europe. Currently, there is participation from 20 different countries from Western and Eastern Europe, the United States, Australia, and Southeast Asia. ELWGD is a prime example of a collaborative and coordinated approach to the microbiology of diphtheria and should serve as a potential model for other infectious diseases networks.

Characterization of surface saccharides in two Corynebacterium diphtheriae strains

Two Corynebacterium diphtheriae strains were analyzed by assays employing a battery of highly purified fluorescent lectins. From 22 lectins tested only seven with affinity to receptor molecules containing N-acetylglucosamine ( d-GlcNAc), N-acetylgalactosamine ( d-GalNAc), galactose ( d-Gal), mannose-like ( d-Man-like) and sialic acid residues showed positive fluorescent labeling. A higher reactivity of Triticum vulgaris (WGA), which binds to sialic acid and/or β- d-GlcNAc-containing residues, and Bandeiraea simplicifolia II (BS-II), which recognizes α and β- d-GlcNAc units, was shown by the sucrose- fermenting strain. Ricinus communis (RCA-I), which recognizes d-Gal units in addition to both Glycine max (SBA) and Artocarpus integrifolia (Jacaline) agglutinins that bind to d-GalNAc-containing residues, reacted preferentially with the sucrose-negative strain. Canavalia ensiformis (Con A), which recognizes d-Man-like receptors, reacted with both sucrose-fermenting and non-sucrose-fermenting C. diphtheriae biotypes. However, higher interaction was observed with the non-sucrose-fermenting strain. Fluorescence of WGA binding was significantly decreased by neuraminidase treatment suggesting the presence of an exposed sialic acid moiety on C. diphtheriae surfaces. Binding assay using radiolabeled [ 125I]WGA essentially confirmed the lectin fluorescence studies. N-Acetylneuraminic acid moieties were detected in whole cell hydrolysates as assessed by thin-layer and gas-liquid chromatography. The data indicate differences on the cell surface saccharide ligands between the sucrose-fermenting and the non-sucrose-fermenting C. diphtheriae strains.

Characterization of surface saccharides in two Corynebacterium diphtheriae strains.

Two Corynebacterium diphtheriae strains were analyzed by assays employing a battery of highly purified fluorescent lectins. From 22 lectins tested only seven with affinity to receptor molecules containing N-acetylglucosamine (D-GlcNAc), N-acetylgalactosamine (D-GalNAc), galactose (D-Gal), mannose-like (D-Man-like) and sialic acid residues showed positive fluorescent labeling. A higher reactivity of Triticum vulgaris (WGA), which binds to sialic acid and/or beta-D-GlcNAc-containing residues, and Bandeiraea simplicifolia II (BS-II), which recognizes alpha and beta-D-GlcNAc units, was shown by the sucrose-fermenting strain. Ricinus communis (RCA-I), which recognizes D-Gal units in addition to both Glycine max (SBA) and Artocarpus integrifolia (Jacaline) agglutinins that bind to D-GalNAc-containing residues, reacted preferentially with the sucrose-negative strain. Canavalia ensiformis (Con A), which recognizes D-Man-like receptors, reacted with both sucrose-fermenting and non-sucrose-fermenting C. diphtheriae biotypes. However, higher interaction was observed with the non-sucrose-fermenting strain. Fluorescence of WGA binding was significantly decreased by neuraminidase treatment suggesting the presence of an exposed sialic acid moiety on C. diphtheriae surfaces. Binding assay using radiolabeled [125I]WGA essentially confirmed the lectin fluorescence studies. N-Acetylneuraminic acid moieties were detected in whole cell hydrolysates as assessed by thin-layer and gas-liquid chromatography. The data indicate differences on the cell surface saccharide ligands between the sucrose-fermenting and the non-sucrose-fermenting C. diphtheriae strains.