Diphtheriae Strains Research Articles

Internalization of non-toxigenic Corynebacterium diphtheriae by cultured human respiratory epithelial cells

Although infection by Corynebacterium diphtheriae is a model of extracellular mucosal pathogenesis, and diphtheria is one of the most worried diseases, this microorganism can be associated also with invasive infections such as endocarditis, septic arthritis, and osteomyelitis. Invasive infections are usually caused by non-toxigenic C. diphtheriae strains. Over the last years severe pharyngitis/tonsillitis associated with the isolation of non-toxigenic C. diphtheriae have been described. Penicillin treatment failure of these infections could only partially be explained by penicillin tolerance of the causing strain. Thus, we examined the in vitro ability of non-toxigenic C. diphtheriae throat clinical isolates to adhere to, and enter human respiratory epithelial cells. Trasmission and scanning electron microscopy demonstrated intracellular C. diphtheriae in laryngeal (HEp-2 cells) and pharyngeal (Detroit D562 cells) tissue culture. Live intracellular bacteria were detectable up to 48 h post-infection. Using a variety of compound that act on eukariotic cell structures, the internalization of C. diphtheriae seems to occur via a zipper-like mechanism. It is likely that internalization of C. diphtheriae can be involved in throat colonization contributing to bacterial eradication failure and asymptomatic carriage.

Molecular composition of diphtheria toxoid produced using semi-synthetic and meat extract-based broths

The use of semi-synthetic broths for cultivation of Corynebacterium diphtheriae instead of a meat extract-based broth avoids the presence of highly undesirable bovine meat antigens in the diphtheria toxoid. As information on the composition of casein digest-based broths used for the production of diphtheria toxoid is scarce, we have now developed one. The composition of a casein-based medium that supports vigorous bacterial growth as well as high toxin production is described below. The comparative analysis of the toxoids, produced using the meat-based Pope–Lingood and the casein digest-based broths, showed considerable differences in their molecular composition. The variance of weight distribution of toxoid-containing molecular complexes was smaller when the semi-synthetic broth was used. Normal human therapeutic IgG recognizes some of the proteins in the meat-based medium but does not react with any components of the semi-synthetic medium. While precipitation at the isoelectric point of the diphtheria toxoid produced by culturing the C. diphtheriae strain in the semi-synthetic medium resulted in a preparation meeting the requirement for purity (more than 1500 limit floculation Lf/mg protein nitrogen PN), the toxoid produced in the Pope–Lingood broth failed to meet this requirement in some cases, even after a second purification step using ultrafiltration.

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.

Microbiography and resistance in the human culture medium.

Writing at the very beginning of the antibiotic era, Hans Zinsser observed signs of bacterial resistance to human efforts at fighting disease. He identified a rise in "excessively toxic and deadly" strains of diphtheria in response to "modern bacteriological methods" such as vaccination with serum antitoxin (p.67). This observation leads him to try to recount the control of epidemic disease from a perspective wider than that of a single human generation. To convey this perspective, he takes the biomedical concept of immunization and makes it a metaphor for the evolutionary relationship between nature, which he figures as a kind of physician, and its patient, the human race. The effect of this metaphor is to diminish the perspective of the individual human. In order to avoid disease's long-term revenge on the species, Zinsser implies, humans should become patients, relinquishing the project of active disease prevention to another agent, a personified nature. My interest here is in how the human role in infection narratives seems [End Page 84] constantly threatened with this kind of displacement, both by the infectious agent's microscopic spatial scale and by the disease's evolutionary temporal scale. Thinking about microbes can pose challenges to anthropocentrism. How do stories about microbial disease respond to these challenges?

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.

Epidemic Diphtheria in the Newly Independent States of the Former Soviet Union: Implications for Diphtheria Control in the United States

The re-emergence of diphtheria in the Newly Independent States of the former Soviet Union in the 1990s raised global awareness of the potential for resurgent disease in countries with long-standing immunization programs. In the United States, the large population of susceptible adults and the possibility of a reintroduction of toxigenic strains of diphtheria create a setting in which diphtheria could spread. In addition, at least one focus of continued circulation of endemic toxigenic Corynebacterium diphtheriae has been identified. Few physicians now have expertise in the diagnosis and treatment of persons with diphtheria, and laboratory capacity is lacking throughout the country. These concerns highlight the importance of maintaining high levels of age-appropriate diphtheria toxoid vaccination, surveillance, accessible and reliable laboratory testing, and training of health care providers. Although the risk of resurgence of diphtheria in the United States is low, public health authorities must ensure that the capacity to recognize, diagnose, and control diphtheria is maintained.

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 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.

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.

Trans-Sialidase activity for sialic acid incorporation on Corynebacterium diphtheriae

A rapid and sensitive assay for neuraminidase using peanut lectin hemagglutination was used to study the prevalence of neuraminidase activity among sucrose-fermenting and non-sucrose-fermenting toxigenic Corynebacterium diphtheriae strains. Neuraminidase activity was found in 15 (100%) isolates regardless of biotype, hemagglutinating activity and site of isolation of bacteria. Besides expressing the neuraminidase activity that hydrolyzes sialic acid from glycoconjugates, C. diphtheriae was also capable of transferring sialic acid residues from a sialyl-lactose donor. A single molecule probably expresses both neuraminidase and trans-sialidase activity. The trans-sialidase activity was documented by observations of the interactions of bacterial cells with wheat germ agglutinin and peanut lectins. C. diphtheriae expressed a trans-sialidase activity located on the cell surface that produced asialoglycoconjugates from a sialyl donor substrate and at the same time generated bacterial sialyl derivatives of β-Gal acceptors.

Trans-sialidase activity for sialic acid incorporation on Corynebacterium diphtheriae.

A rapid and sensitive assay for neuraminidase using peanut lectin hemagglutination was used to study the prevalence of neuraminidase activity among sucrose-fermenting and non-sucrose-fermenting toxigenic Corynebacterium diphtheriae strains. Neuraminidase activity was found in 15 (100%) isolates regardless of biotype, hemagglutinating activity and site of isolation of bacteria. Besides expressing the neuraminidase activity that hydrolyzes sialic acid from glycoconjugates, C. diphtheriae was also capable of transferring sialic acid residues from a sialyl-lactose donor. A single molecule probably expresses both neuraminidase and trans-sialidase activity. The trans-sialidase activity was documented by observations of the interactions of bacterial cells with wheat germ agglutinin and peanut lectins. C. diphtheriae expressed a trans-sialidase activity located on the cell surface that produced asialoglycoconjugates from a sialyl donor substrate and at the same time generated bacterial sialyl derivatives of beta-Gal acceptors.

Use of Random Amplified Polymorphic DNA for Rapid Molecular Subtyping of Corynebacterium diphtheriae

A total of 210 Corynebacterium diphtheriae strains isolated worldwide were assayed by random amplified polymorphic DNA (RAPD) assay. RAPD was as discriminating as standard ribotyping, and in some cases, even further differentiation was obtained. RAPD can rapidly aid clinical and molecular epidemic studies in a simple and cost-effective manner.

Corynebacterium imitans sp. nov. isolated from patients with suspected diphtheria

A 5-month-old boy of a Romanian family traveling via Ukraine to Poland developed a respiratory disease that resembled and that was initially diagnosed as pharyngeal diphtheria. The child recovered after treatment with antidiphtheria antitoxin. A coryneform bacterium had been isolated from a nasopharyngeal specimen from the child and was initially identified as an atypical Corynebacterium diphtheriae strain. Seven adults who had contact with either the child or an adult contact person also developed symptoms of pharyngeal diphtheria, were also treated with antitoxin, and recovered uneventfully. Coryneform bacteria similar to that originating from the index patient were also isolated from the throat swabs of three adults. Detailed biochemical and chemotaxonomic investigations revealed that the coryneform bacteria belonged to the genus Corynebacterium and could be differentiated from all other defined species of this genus. Ribotyping and pulsed-field gel electrophoresis demonstrated that all four patients' isolates were of clonal origin. The diphtheria toxin gene and its product were not detected either by PCR assays or by the Elek test, making a possible disease association of the Corynebacterium more unlikely. Comparative 16S rRNA gene sequence analysis revealed that the coryneform bacterium represented a new subline within the genus Corynebacterium, for which the name Corynebacterium imitans sp. nov. is proposed. The type strain is NCTC 13015 (DSM 44264; CCUG 36877).