PMC8742586_01
Microbiological features of penicillin-resistant 

Corynebacterium diphtheriae

 strain isolated of infected skin ulcer from a Brazilian diabetic elderly female patient. (a) Colonial morphology on 5 % sheep blood agar plate. (b) Gram-staining (original magnification,x1000) showing pleomorphic Gram-positive bacillary forms. (c) MALDI-TOF MS spectrum. (d) Amplification profile by multiplex PCR assay for differentiation between 

C. diphtheriae

 (including 

Corynebacterium belfantii

 and Coryrnebacterium rouxii), 

Corynebacterium ulcerans

 and 

Corynebacterium pseudotuberculosis

 strains and detection of diphtheria toxin gene (tox): Lane 1, molecular weight (1 kb DNA ladder); Lanes 2 and 3, 

C. diphtheriae

 clinical isolate (tox-
); Lane 4, negative control (reaction without template DNA); Lane 5, 

C. diphtheriae

 ATCC 27012 (tox+
); Lane 6, 

C. diphtheriae

 ATCC 27010 (tox-
); Lane 7, 

C. ulcerans

 809 (tox-
); Lane 8, 

C. pseudotuberculosis

 ATCC 19410 (tox-
).
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F1
After incubation at 37  C for 48 h, the growth of white, opaque colonies, showing slight hemolysis, was observed (Fig. 1a). Gram-stained optical microscopy of these colonies showed Gram-positive bacillary forms, arranged in pallid shapes with angular formations between cells (Fig. 1b). Identification System (SARAMIS , bioMerieux , France) database, which in turn uses common peaks of strains of the same species (between 15-20) to build a 'super spectrum'. Finally, agreement values above 60 % mean the species was identified. The MALDI-TOF MS spectrum of   C. diphtheriae   clinical isolate can be observed in Fig. 1(c). In addition, two other colonies grown in the primary culture were identified by MS MALDI-TOF as   Staphylococcus aureus   and   Pseudomonas putida  . Adolfo Lutz Institute and Laboratory of Diphtheria and Corynebacteria of Clinical Relevance (LDCIC), in order to confirm   C. diphtheriae   identification, investigate the DT-production by multiplex Polymerase Chain Reaction (mPCR) assays and determine the susceptibility to antimicrobials. mPCR assays were carried out as previously described with primers pairs targeting the following genes: rpoB - beta subunit of RNA polymerase - of   C. diphtheriae   (including the novel species   Corynebacterium belfantii   and   Corynebacterium rouxii  ),   Corynebacterium ulcerans   and   Corynebacterium pseudotuberculosis   (C2700F and C3130R, 446 bp); 16S rRNA - 16S ribosomal RNA - of both   C. pseudotuberculosis   and   C. ulcerans   (16SF and 16 SR; 816 bp); dtxR - diphtheria toxin repressor - of   C. diphtheriae   (DtxR1F and DtxR1R, 258 bp); pld - sphingomyelinase - of   C. pseudotuberculosis   (pldF and pldR2, 203 bp); tox - diphtheria toxin (Dipht 4F and Dipht 4R, 303 bp). As illustrated in Fig. 1(d), mPCR results confirmed that the clinical isolate was a non-DT producing   C. diphtheriae   strain (positive for rpoB and dtxR genes; negative for tox, 16S rRNA and pld genes).