Further Studies on Arcanobacterium phocisimile: a Novel Species of Genus Arcanobacterium

Arcanobacterium phocisimile, a newly described species with the type strain A. phocisimile 2698T isolated from a vaginal swab of a harbour seal and four additional A. phocisimile strains also isolated from four harbour seals could reliably be identified by phenotypic properties, by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), and by sequencing the genomic targets 16S rDNA and 16S-23S rDNA intergenic spacer region and the genes rpoB and gap. The A. phocisimile strains investigated in the present study were isolated together with several other bacterial species indicating that the pathogenic importance of A. phocisimile remains unclear. However, the detection of peptidic spectra by MALDI-TOF MS and the presented phenotypic and genotypic approach might help to identify A. phocisimile in future.


Introduction
Genus Arcanobacterium comprises the species Arcanobacterium haemolyticum, Arcanobacterium hippocoleae, Arcanobacterium pluranimalium, and Arcanobacterium phocae [1]. More recently A. canis and A. phocisimile were described as novel species of this genus [2,3]. Arcanobacterium pyogenes together with Arcanobacterium bernardiae, Arcanobacterium bonasi, and Arcanobacterium bialowiezense was reclassified to the newly described species Trueperella [1]. The original description of A. phocisimile was based on physiological and biochemical characteristics, chemotaxonomic analysis, and 16S rDNA sequencing results of two strains isolated with several other bacterial species from a vaginal swab and an anal swab of two free living harbour seals of the German North Sea [3].
In the present study both initially described A. phocisimile strains and three additional strains obtained from three harbour seals were identified and further characterized phenotypically by MALDI-TOF MS analysis and genotypically by amplification and sequencing of various molecular targets.

Phenotypic and Genotypic
Identification. All three newly investigated A. phocisimile strains were initially characterized phenotypically and by 16S rDNA sequencing [3,4]. Both A. phocisimile strains previously mentioned in the species description [3] and the three A. phocisimile strains of the present study were further analysed by MALDI-TOF MS [5] and genotypically by amplification and sequencing of the previously described molecular target 16S-23S rDNA intergenic spacer region (ISR) and the genes rpoB and gap [4, 6, 7].   The primer sequences and the thermocycler programs are given in Table 1.

Results and Discussion
All three strains newly characterized in the present study could reliably be identified as A. phocisimile by phenotypic properties and by 16S rDNA sequencing. The phenotypic properties appeared to be almost identical to both previously characterized A. phocisimile strains (Table 2). However, a positive pyrazinamidase reaction of A. phocisimile seems to be the only reliable biochemical property for differentiation of A. phocisimile from pyrazinamidase negative A. phocae.
As shown by numerous authors MALDI-TOF MS is a powerful tool for species characterization of a broad spectrum of gram-positive and gram-negative bacteria [8][9][10]. This technique had previously been successfully used for rapid and reliable identification of bacteria of genera Arcanobacterium and Trueperella [5,11]. The MALDI-TOF MS analysis of the present study revealed that by using the current Bruker data base, all five strains of this hitherto unknown species could not be identified to species level. However, using the MALDI Biotyper 3.  Figure 1.
The genotypic classification by 16S rDNA sequencing revealed that the three novel A. phocisimile strains of the present study yielded 100% identity to both A. phocisimile strains described previously [3], also including the type strain A. phocisimile 2698 T (Figure 2).
Comparable to previously described A. canis [11] all five A. phocisimile from the present study could additionally be classified by amplification and sequencing of ISR (FN563000, FN563002, HG316083, HG316084, and HG316085), gene rpoB (HG316078, HG316079, HG316080, HG316081, and HG316082), and gene gap (HF679531, HG316074, HG316075, HG316076, and HG316077) yielding for all three molecular targets an identity of ≥99.4%, ≥99.8%, and ≥99.8%, respectively, for all five strains among each other. A typical dendrogram using the sequencing results of the target genes rpoB and gap is shown in Figure 3.
The results of the present study revealed that phenotypic properties, the determination of peptidic spectra by MALDI-TOF MS, and the various genotypic targets allow for a reliable identification of A. phocisimile and a further differentiation of A. phocisimile from closely related A. phocae which could also be isolated from marine mammals [12]. However, all A. phocisimile strains of the present study were isolated together with various other bacteria, partly from obviously healthy animals, indicating that the pathogenic importance of this species for marine mammals remains unclear.

Interpretive Summary
Arcanobacterium phocisimile type strain and four additional A. phocisimile strains isolated from harbour seals were identified phenotypically, by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), by sequencing 16S rDNA, and, as novel molecular targets, by sequencing 16S-23S rDNA intergenic spacer region and the genes rpoB and gap indicating that MALDI TOF MS and the molecular targets might help to identify this novel species.