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.     

Growth of the surface of Corynebacterium diphtheriae

Surface structure and growth of the surface of Corynebacterium diphtheriae mitis strain were investigated by scanning electron microscopy and the immunofluorescence technique. The surface of the cell revealed by the scanning electron microscope showed a few elevated circular zones which encompassed the cell. The cell diameter increased at this zone and this gave the club-shaped appearance to this species. The cell surface labeled with specific antibodies against the whole bacterial cell and tagged with ferritin remained at a constant length during cell division cycles and the new cell surface emerged from the polar ends of the cell. This new wall surface was completely devoid of the ferritin particles indicating that the cell wall component on the old preexistent wall was completely conserved. A similar finding was obtained by immunofluorescence microscopy. C. diphtheriae, unlike Bacillus spp., showed apical growth as has been observed in fungal cells.     

Assembly of pili on the surface of Corynebacterium diphtheriae

Pili of Gram-negative pathogens are formed from pilin precursor molecules by non-covalent association within the outer membrane envelope. Gram-positive microbes employ the cell wall peptidoglycan as a surface organelle for the covalent attachment of proteins, however, an assembly pathway for pili has not yet been revealed. We show here that pili of Corynebacterium diphtheriae are composed of three pilin subunits, SpaA, SpaB and SpaC. SpaA, the major pilin protein, is distributed uniformly along the pilus shaft, whereas SpaB is observed at regular intervals and SpaC seems positioned at the pilus tip. Assembled pili are released from the bacterial surface by treatment with murein hydrolase, suggesting that the pilus fibres may be anchored to the cell wall envelope. All three pilin subunit proteins are synthesized as precursors carrying N-terminal signal peptides and C-terminal sorting signals. Some, but not all, of the six sortase genes encoded in the genome of C. diphtheriae are required for precursor processing, pilus assembly or cell wall envelope attachment. Pilus assembly is proposed to occur by a mechanism of ordered cross-linking, whereby pilin-specific sortase enzymes cleave precursor proteins at sorting signals and involve the side chain amino groups of pilin motif sequences to generate links between pilin subunits. This covalent tethering of adjacent pilin subunits appears to have evolved in many Gram-positive pathogens that encode sortase and pilin subunit genes with sorting signals and pilin motifs.     

Cell surface components and adhesion in Corynebacterium diphtheriae

Main primary approaches and new developments in the study of the molecular basis of the adhesive process of Corynebacterium diphtheriae are reviewed along with a discussion of the potential importance of hemagglutinins, exposed sugar residues, hydrophobins and trans-sialidase enzymes as adhesins of strains of the sucrose fermenting and non-fermenting biotypes.     

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.     

Isolation and characterization of extragenic suppressor strains of Corynebacterium diphtheriae.

The isolation and characterization of two different nonsense suppressor strains of Corynebacterium diphtheriae C7 sup+(-)tox- are described. Appropriate lysogens of these strains with corynephage beta, carrying known class II tox premature polypeptide chain termination mutations [C7sup-1(betatox-30) and C7sup-2(betatox-45)], each produce a 62,000-dalton polypeptide with nicotinamide adenine dinucleotide: elongation factor-2 adenosine diphosphate ribosyltransferase activity in addition to a chain-terminated polypeptide of 30,000 or 45,000 daltons, respectively. In addition, purified protein of 62,000 daltons, resulting from the suppression of the nonsense mutations tox-30 and tox-45, will react with antisera purified against the terminal 17,000 daltons of the toxin molecule and are immunologically identical to toxin by radial immunodiffusion. The suppression pattern of lysogenic derivatives of C7sup-1(-)tox- and C7sup-2(-)tox- with other class II and III mutants of corynephage beta was determined.     

Assembly of distinct pilus structures on the surface of Corynebacterium diphtheriae

Different surface organelles contribute to specific interactions of a pathogen with host tissues or infectious partners. Multiple pilus gene clusters potentially encoding different surface structures have been identified in several gram-positive bacterial genomes sequenced to date, including actinomycetales, clostridia, corynebacteria, and streptococci. Corynebacterium diphtheriae has been shown to assemble a pilus structure, with sortase SrtA essential for the assembly of a major subunit SpaA and two minor proteins, SpaB and SpaC. We report here the characterization of a second pilus consisting of SpaD, SpaE, and SpaF, of which SpaD and SpaE form the pilus shaft and SpaF may be located at the pilus tip. The structure of the SpaDEF pilus contains no SpaABC pilins as detected by immunoelectron microscopy. Neither deletion of spaA nor sortase srtA abolishes SpaDEF pilus formation. The assembly of the SpaDEF pilus requires specific sortases located within the SpaDEF pilus gene cluster. Although either sortase SrtB or SrtC is sufficient to polymerize SpaDF, the incorporation of SpaE into the SpaD pili requires sortase SrtB. In addition, an alanine in place of the lysine of the SpaD pilin motif abrogates pilus polymerization. Thus, SpaD, SpaE, and SpaF constitute a different pilus structure that is independently assembled and morphologically distinct from the SpaABC pili and possibly other pili of C. diphtheriae.     

Similarity of rpoB gene sequences of sucrose-fermenting and non-fermenting Corynebacterium diphtheriae strains

During the last decades, the majority of Brazilian Corynebacterium diphtheriae isolates were shown to be capable to metabolize sucrose, sometimes leading to erroneous identification as a non-diphtheric Corynebacterium species. The sequencing of the polymorphic region of the RNA polymerase beta subunit-encoding gene (rpoB) is an important taxonomic tool for identification of corynebacteria. The present study aimed to investigate the rpoB gene polymorphic features of sucrose-fermenting and non sucrose-fermenting strains. The results showed that sucrose-fermenting strains presented rpoB gene polymorphic regions with more than 98% similarity with the sequences deposited in the gene bank corresponding to non sucrose-fermenting strains. Data indicate that sucrose-fermenting isolates may act as a variant of C. diphtheriae biotype mitis. In addition we alert that sucrose-fermenting strains should not be discarded as contaminants mainly in countries where the possibility of isolation of this variant is higher.     

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

We report the identification and genetic analysis of mutants in the antitoxin of the parD (kis, kid) killer system of plasmid R1. Missense mutants placed at codons 10, 11, 12 and 18 maintained the antitoxin activity of Kis, but not the ability of this protein to co-regulate the parD system together with the Kid toxin. Deletion of the last 33 amino acids of Kis inactivated the antitoxin activity of the protein and reduced substantially, but not completely, its regulatory activity. These results define two functional regions in Kis: an amino-terminal region which is specifically involved in regulation, and a carboxy-terminal region of the protein, which is important both for its regulatory and antitoxin activities.     

Adhesion of Corynebacterium diphtheriae

The conditions for the direct hemagglutination test performed to determine the degree of adhesion of C. diphtheriae were defined. For this test sheep red blood cells, trypsin-treated ex tempore, were used. Only newly isolated cultures, subcultured for not more than 2-5 times and stored for not more than 2-7 days or freeze-dried, were employed. The culture to be tested was grown in nutrient agar with 10% of normal horse serum. The test was made in microtitrator round-bottom wells. The mixture of different dilutions of the culture was incubated for 2 hours at 37 degrees C, then left overnight at 4 degrees C. All 147 newly isolated or freeze-dried C. diphtheriae strains under test had different degrees of adhesion. Their adhesive activity was unrelated to their biovar. Toxigenic strains were significantly more active in hemagglutination (53.5 +/- 3.0%) than nontoxigenic ones (23.5 +/- 3.9%). The strains isolated from the nose, irrespective of their biological properties, were more active than those isolated from the pharynx.     

Neuraminidase of Corynebacterium diphtheriae

Neuraminidase activity has been found in a variety of strains of Corynebacterium diphtheriae, both toxinogenic and nontoxinogenic. The enzyme has been shown to be intracellular, possibly associated with the cytoplasmic membrane. Toxinogenic strains of the diphtheria bacillus, grown under conditions unsuitable for maximal toxin production, produce neuraminidase, and the enzyme has been purified from cells of the Park Williams no. 8 strain grown under such conditions. Diphtherial toxin and diphtherial neuraminidase have similar molecular weights and remain associated during column chromatography; immunochemically, and in their electrophoretic behavior, they appear distinct.