Table 1.
Arcanobacterium haemolyticum strains used in this study.
Table 2.
Oligonucleotide primers used in this study.
Fig 1.
Colony morphology and hemolysis of clinical isolates of A. haemolyticum is enhanced on Todd-Hewitt media supplemented with 6% horse blood.
Growth of clinical isolates for 48 hours in 5% CO2 showcases the differences between colony morphology and hemolysis of A. haemolyticum. (A) Distinct colony morphologies between smooth (Left-S1) and rough (Right-R44) isolates visualized after 48 h incubation. Line represents 1.0 mm. (B) Hemolysis variation between smooth (Left-S1 and S22) and rough (Right-R49 and R30) isolates.
Fig 2.
PCR and sequence analysis of the aln-coding region yields an insertion sequence (IS) element in some rough isolates of A. haemolyticum.
The aln-coding region of rough and smooth clinical isolates of A. haemolyticum was PCR amplified using primers DM1014 and DM1015. (A) DNA gel electrophoresis of smooth isolate ATCC9345 (Lane 2) and rough isolate AhR30 (Lane 3) amplify the predicted 2.0 kb product compared to rough isolate, AhR28 (Lane 4), which amplifies a 3.2 kb product. (B) Features of the IS element found in the aln coding region of some rough isolates of A. haemolyticum. (C) Statistical analysis of A. haemolyticum isolates from Finland or United States (Nebraska).
Fig 3.
Hemolytic activity of smooth and rough isolates of A. haemolyticum incubated with horse erythrocytes.
Hemolysis assay on washed horse erythrocytes (1%) incubated with smooth or rough live strains of A. haemolyticum. A. haemolyticum smooth and rough isolates vary in hemolytic activity when bacteria are incubated with horse erythrocytes for one hour. Representative of at least six individual experiments. Shown is an average of three replicates ± standard deviation. Smooth isolates tend to have more hemolytic activity than rough isolates.
Fig 4.
Smooth and rough clinical isolates of A. haemolyticum can be distinguished by sequence analysis of the upstream region of aln.
The upstream region of aln from 73 clinical isolates was PCR-amplified using primers DM1078 and DM1080 to generate an ~830 bp product. Following sequence analysis, ~ 50 bp of the 5’ end and ~70 bp of the 3’ end were trimmed, yielding a ~715 bp upstream region for further alignments and analysis. An unrooted tree (generated with TreeView) generated from multi-sequence alignment of the intergenic region of aln from 62 isolates demonstrates that nearly all of the rough isolates phylogenetically cluster, and are distinct from smooth isolates of A. haemolyticum. * Represent outliers. Previously designated as smooth, strain S21 was shown in our laboratory to be a rough isolate and was re-designated as R21 and phylogenetically clusters with other rough isolates. Black line separates rough isolates (top) from smooth isolates (bottom). 11 isolates not shown in the tree are rough isolate R31, smooth isolate B5366 and the nine isolates from Denmark and Germany. These isolates also cluster in the tree as rough or smooth (data not shown for 11 isolates to prevent overcrowding of figure).
Fig 5.
Smooth and rough clinical isolates of A. haemolyticum can be distinguished by differential restriction enzyme analysis of the upstream region of aln.
The upstream region of aln was PCR-amplified using primers DM1078 and DM1080 to generate an ~830 bp product. Purified PCR products were cleaved with ClaI or XcmI. Representative of 21 strains tested. In silico results of all 73 strains shown in Table 3. U, undigested; C, ClaI, X, XcmI. Both single digests yield products of about 295 bp and 535 bp. Undigested product is ~830 bp.
Table 3.
Summary of restriction enzyme patterns of the aln upstream region of 73 A. haemoluyticum clinical isolates based on in silico sequence analysis.
Fig 6.
Nucleotide polymorphisms found in the intergenic region of aln.
Multi-sequence alignment of the ~715 bp intergenic region of aln from clinical isolates of A. haemolyticum shows 40 polymorphisms. Nucleotides 39–41 are the translation stop codon for the upstream gene pgm, encoding phosphoglycerate mutase. Nucleotides 151–223 encode the tRNA-ala gene. Nucleotides 633–647 contain the Shine-Dalgarno (SD) sequence, and nucleotides 650–652 are the translation start codon for aln. * Denote polymorphisms detailed in Table 4.
Table 4.
Nucleotide polymorphisms in A. haemolyticum aln upstream region.
Numbering based on ATCC9345. Nucleotides 39–41 are the translation stop codon for the upstream gene, pgm, encoding a phosphoglucomutase. Nucleotides 151–223 encode the tRNA-ala, which is completely conserved in all isolates. Nucleotides 633–647 contain the Shine-Dalgarno sequence, and 650–652 is the translation start codon for aln.