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Modified culture technique for Corynebacterium diphtheriae isolation from desiccated swabs.

Corynebacterium diphtheriae was isolated from pyoderma and ulcerative skin lesions with a modified delayed culture procedure as late as 9 weeks after field collection of silica gel-desiccated swabs. Biotypes gravis and mitis were identified. Most isolates were nontoxigenic. Todd-Hewitt broth enrichment enhanced recovery of C. diphtheriae by 70%.

Bacteriological Techniques

Corynebacterium diphtheriae endocarditis.

Two patients with Corynebacterium diphtheriae endocarditis have been observed in the past ten years. Both cases occurred sporadically. In one case toxigenic C. diphtheriae caused endocarditis in an immunized child with congenital heart disease. The second patient developed rapidly progressive valve destruction caused by a nontoxigenic strain of C. diphtheriae. These cases illustrate that C. diphtheriae may produce endocarditis in addition to its previously described diseases spectrum.

Child, Preschool

Analysis of novel zinc-binding proteins in the cell wall of Corynebacterium diphtheriae.

UNLABELLED: Zinc is a critical nutrient for all living organisms, including bacterial pathogens such as Corynebacterium diphtheriae, the causative agent of the severe human respiratory disease diphtheria. As such, zinc acquisition is essential for many pathogens to cause disease. We previously showed that the zinc-regulated ABC transporter encoded by the znu locus is one of several zinc uptake systems that support the growth of C. diphtheriae in zinc-limited medium. In this study, we examine the function and cellular localization of components encoded by the znu gene cluster, which includes the ZnuABC transporter and the novel membrane and cell wall proteins ZnuE, ZnuF, and ZnuG. Deletion of znuE results in reduced growth in zinc-limited media, suggesting a role in zinc uptake. While the growth of znuF and znuG mutants was not affected in zinc-limited medium, deletion of these two genes in a mutant also lacking the znuABC transporter restored growth to wild-type levels, suggesting that ZnuF and ZnuG exert an unusual modulating effect on zinc import. ZnuE, ZnuF, and ZnuG possess a unique motif that is associated with Zn binding as demonstrated by thermal shift assays, targeted mutagenesis, and structural analysis. Although ZnuF and ZnuG are both present in the cell wall, only ZnuG contains a sortase recognition signal that is critical for localizing proteins to the cell wall. Furthermore, ZnuF localization does not require any of the six known sortase enzymes in C. diphtheriae, suggesting a novel localization mechanism. IMPORTANCE: Zinc is a critical nutrient required by many bacterial pathogens. While the function of multiple zinc importer systems has been previously characterized in Corynebacterium diphtheriae, the transporter encoded by the znu gene cluster includes components not found in other metal transport systems. In this report, we examined the roles of three components of the znu gene cluster, ZnuE, ZnuF, and ZnuG, and show that these proteins all possess a putative zinc-binding domain and have varying effects on growth in zinc-limited medium. Additionally, ZnuF uses a novel mechanism for cell wall localization. This study further expands our understanding of C. diphtheriae zinc import and points to a potentially novel mechanism for the localization of cell wall proteins.

Corynebacterium diphtheriae

Isolation, structural studies and chemical synthesis of a 'palmitone lipid' from Corynebacterium diphtheriae.

The isolation of a 'palmitone lipid' from Corynebacterium diphtheriae is described. The use of a temporary hydrophobic protecting group allows the obtaining of the lipid in free and pure form. Structural studies by chemical degradation and mass spectrometry allow one to propose structure Ic for this compound, namely 6-(2-tetradecyl 3-keto octadecanoyl)-alpha-D-trehalose. This structure was confirmed by chemical synthesis.

Corynebacterium diphtheriae

Bacteriophage typing of Corynebacterium diphtheriae.

Bacteriophage types of over 3,000 strains of Corynebacterium diphtheriae isolated in Canada have been determined. The typing scheme used involved the use of nine phages. Results indicated that phage types correlate with biotypes to a large degree. Corynecin types were also determined for a limited number of cultures, and results indicated that the indicator strains presently available are unsuitable for the typing of C. diphtheriae strains isolated in North America. The distribution of phage types is similar throughout Canada, and the types present correspond to types reported from other countries. Phage typing could be of value in the study of circumscribed outbreaks and in epidemiological surveillance of types over long periods.

Bacterial Toxins

Erythromycin and clindamycin resistance in Corynebacterium diphtheriae from skin lesions.

Erythromycin- and clindamycin-resistant Corynebacterium diphtheriae isolates were recovered from skin lesions. Resistance to erythromycin and clindamycin was induced by a subinhibitory concentration (0.03 microgram/ml) of erythromycin. Clindamycin (0.07 microgram/ml) was a more effective inducer of its own resistance than of erythromycin resistance. Erythromycin-inducible cross-resistance to vernamycin B alpha was demonstrated in disk diffusion tests.

Clindamycin

Bacteriophage production by doubly lysogenic Corynebacterium diphtheriae.

Parental and recombinant phage production by tandem, double lysogens of Corynebacterium diphtheriae was studied in strains in which the coupling of prophage markers and the order of prophage was established. The results from studies of mass lysates and single bursts showed that the recombinant class of phage, designated R1, was predominant in UV-induced lysates followed by the parental, P1 class and to a lesser extent the P2 and R2 classes. Single bursts of UV-treated cells contained phage from one to all four of the phage classes, and this appeared to reflect the action of two excision processes. The data indicate that recombinant phages R1 and R2 are formed by a process of general recombinational excision and that this is the primary event leading to phage production in both UV-irradiated and spontaneously induced double lysogens. This process, which depends on exchange between homologous genes and is reciprocal, accounts for the excision of R1 phage from the host chromosome. A second excision process, probably site-specific excision, also occurs in many of the same cells and accounts for the excision of P1, P2, and R2 phages. The significance of these results for the spread of toxinogenicity in strains of C. diphtheriae is discussed.

Bacteriophages

Non-toxigenic Corynebacterium diphtheriae in a boarding school.

A non-toxigenic strain of Corynebacterium diphtheriae var. gravis was isolated from 31 boys during an outbreak of sore throats at a boarding school of 15 staff and 134 pupils. Despite the non-toxigenicity of the strain involved, conventional isolation procedures were observed and all boys at the school were treated with antibiotics. Restrictions on the school were only relaxed when no further carriers were detected. The rationale of such management of an episode involving a non-toxigenic strain is discussed.

Adolescent

Molecular epidemiology and antimicrobial resistance determinants of Corynebacterium diphtheriae causing infections in Karachi, Pakistan, 2023-2024.

OBJECTIVES: Diphtheria remains endemic in Pakistan, with cases increasing following the COVID-19 pandemic despite ongoing vaccination programs. This study analyzes the genomic diversity, virulence, and antimicrobial resistance patterns of pharyngeal diphtheria strains collected during the Karachi outbreak. METHODS: Corynebacterium diphtheriae isolates from a tertiary care hospital laboratory in Karachi (August 2023-October 2024) were included. Antimicrobial susceptibility testing and whole-genome sequencing of phenotypically confirmed isolates were performed. Phylogenetic and bioinformatics analyses were performed using diphtOscan and AMRFinderPlus tools. RESULTS: A total of 47 pharyngeal C. diphtheriae isolates were included. The median age of patients was 7 years, and the male-to-female ratio was 1.6:1. The tox gene was present in 89.4% of isolates, while only 29% (n = 13/45) demonstrated toxin production. Genomic analysis identified 10 sequence types; ST384 and ST698 were most prevalent. Phenotypically, 34% (n = 16) were resistant to both erythromycin and penicillin, and 49% (n = 23) were multidrug-resistant. The most prevalent resistance genes were sul1 (100%), erm(X) (76.6%), and pbp2m (51.1%). CONCLUSION: Circulation of diverse C. diphtheriae strains with alarming antimicrobial resistance underscores the need for genomic surveillance to evaluate transmission trends. We further highlight the limitations of the Elek test in detecting toxin production and the need for improved diagnostics in low- and middle-income countries.

Antimicrobial resistance

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.

Bacteriophages

Determination of Corynebacterium diphtheriae toxigenicity by a colorimetric tissue culture assay.

Chinese hamster ovary (CHO) cell cultures in microtiter wells are sensitive to growth inhibition and killing by picogram quantities of diphtheria toxin. In the absence of biologically active toxin, the CHO cell culture produces sufficient acidic metabolites to change the phenol red pH indicator from pink to yellow within 56 h. In the presence of 10 pg of toxin per well, growth inhibition can be observed microscopically within 24 h. Diphtheria toxin can be qualitatively assayed from culture supernatants of Corynebacterium diphtheriae or from beta-phage agar plaque plugs. The colorimetric CHO cell assay method for determining toxigenicity allows for the large-scale screening of either diphtheria toxigenicity or antitoxin titration of sera.

Animals

Isolation and partial characterization of a corynebacteriophage beta, tox operator constitutive-like mutant lysogen of Corynebacterium diphtheriae.

We have isolated and partially characterized a beta-phage mutant lysogen of Corynebacterium diphtheriae, C7(betatoxct1+), which is partially insensitive to iron inhibition of diphtheria toxin production. tox expression by C7(betatoxct1+) was found to be partially constitutive. In the presence of concentrations of iron that almost completely inhibit the expression of diphtheria toxin by the wild type, C7(beta), the level of toxin production by C7(betatoxct1+) was found to be at least 25 times that of the parent. The purified tox gene product of C7(betatoxct1+) was immunologically and electrophoretically identical to, and equally as toxic as, diphtheria toxin purified from C7(beta). In addition, the partial N-terminal amino acid sequence was found to be identical to diphtheria toxin. This data strongly suggests that the mutation allowing for the constitutive expression of tox in C7(betatoxct1+) is outside of the structural gene. Furthermore, the constitutive expression of diphtheria toxin was found to be cis dominant in the double lysogen C7(betacrm45+/betatoxct1+). The data presented is consistent with the existence of a tox operator locus.

Bacteriophages

Interaction of toxin of Corynebacterium diphtheriae with phagocytes from susceptible and resistant species.

The interaction of the toxin of Corynebacterium diphtheriae with leukocytes from sensitive and resistant animal species was examined by determining the ability of toxin to inhibit protein synthesis by several types of phagocytic cell. Small amounts of toxin (25 minimal lethal doses) impaired protein synthesis in both polymorphonuclear leukocytes and mononuclear cells from humans and guinea pigs, whereas large amounts (2,000 minimal lethal doses) were required for minimal inhibition of mouse phagocytes. Peritoneal macrophages from hyperimmunized guinea pigs exhibited the same high degree of sensitivity to diphtheria toxin as did those from unimmunized animals. Prolonged incubation with toxin resulted in a 75% reduction in phagocytosis of polystyrene latex spheres but had no effect on transport of the glucose analogue 2-deoxy-D-glucose by guinea pig macrophages. Thus phagocytic cells, although they are endowed with a high level of phagocytic cell appear to reflect the native resistance or sensitivity of the host species of origin.

Animals