# Further Studies onArcanobacterium phocisimile: a Novel Species of Genus Arcanobacterium

Authors: Osama Sammra; Anna Balbutskaya; Muaz Hijazin; Samy Nagib; Jörg Alber; Christoph Lämmler; Amir Abdulmawjood; Ellen Prenger-Berninghoff; Markus Timke; Markus Kostrzewa; Ursula Siebert
Journal: Journal of Veterinary Medicine
 (2014)

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## Abstract

Arcanobacterium phocisimile, a newly described species with the type strain A. phocisimile 2698
T 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.


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## Body

## 1. Introduction

GenusArcanobacterium 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 describedA. 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.


## 2. Materials and Methods

### 2.1. Bacterial Strains

TheA. phocisimile strains used in the present study included the previously described type strains A. phocisimile  2698
T (LMG 27073
T; CCM 8430
T) and A. phocisimile 4112 [3]. Additionally investigated A. phocisimile 3047 was isolated (post mortem) together with Bacillus spp., Enterococcus spp., Erysipelothrix rhusiopathiae, and  α-haemolytic streptococci in the year 2005 from the lung of a female harbour seal with bronchopneumonia and perforation of stomach. The harbour seal was found dead in Rantum, Sylt of the German North Sea. Also investigated A. phocisimile 4113 was recovered together with Pasteurella spp. and α-haemolytic streptococci from an anal swab and A. phocisimile 4125 together with Pseudomonas spp. and α-haemolytic streptococci also from an anal swab of two apparently healthy female harbour seals, respectively. A. phocisimile 4113 and A. phocisimile 4125 were isolated in 2007 during a monitoring program of free living harbour seals of the German North Sea.


### 2.2. Phenotypic and Genotypic Identification

All three newly investigatedA. 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.Table 1
Oligonucleotide primer sequences and PCR conditions of the target genes used in the present study.
Oligonucleotide primers
Sequence
Program*
Expected size of PCR product (bp)
References
(1) 16S rDNA UNI-L(2) 16S rDNA UNI-R (amplification primer)
5′-AGAGTTTGATCATGGCTCAG-3′5′-GTGTGACGGGCGGTGTGTAC-3′
1
1,403
[4]
(3) 16S rDNA-533F(4) 16S rDNA-907R (sequencing primer)
5′-GTGCCAGCMGCCGCGGTAA-3′5′-CCGTCAATTCMTTTGAGTTT-3′
—
—
[4]
(5) Gap-F(6) Gap-R (gap primer)
5′-TCGAAGTTGTTGCAGTTAACGA-3′5′-CCATTCGTTGTCGTACCAAG-3′
2
830
[4]
(7) ISR-23S-F(8) ISR-23S-R (intergenic spacer region primer)
5′-CCTAGCCTGGTGGTTGGGTAG-3′5′-GTGCGGGTAACCAGAAATAACTCTG-3′
3
345
[6]
(9) C2700F(10) C3130R (rpoB primer)
5′-CGWATGAACATYGGBCAGGT-3′5′-TCCATYTCRCCRAARCGCTG-3′
4
406
[7]
*PCR program 1: x1 (95°C, 600 sec), x30 (95°C, 30 sec, 58°C, 60 sec, 72°C, 60 sec), and x1 (72°C, 420 sec). 2: x1 (94°C, 180 sec), x30 (94°C, 30 sec, 50°C, 40 sec, 72°C, 60 sec), and x1 (72°C, 300 sec). 3: x1 (95°C, 240 sec), x30 (95°C, 8 sec, 66°C, 10 sec, 72°C, 10 sec), x1 (72°C, 420 sec). 4: x1 (95°C, 600 sec), x35 (94°C, 30 sec, 50°C, 30 sec, 72°C, 120 sec), and x1 (72°C, 600 sec).


## 2.1. Bacterial Strains

TheA. phocisimile strains used in the present study included the previously described type strains A. phocisimile  2698
T (LMG 27073
T; CCM 8430
T) and A. phocisimile 4112 [3]. Additionally investigated A. phocisimile 3047 was isolated (post mortem) together with Bacillus spp., Enterococcus spp., Erysipelothrix rhusiopathiae, and  α-haemolytic streptococci in the year 2005 from the lung of a female harbour seal with bronchopneumonia and perforation of stomach. The harbour seal was found dead in Rantum, Sylt of the German North Sea. Also investigated A. phocisimile 4113 was recovered together with Pasteurella spp. and α-haemolytic streptococci from an anal swab and A. phocisimile 4125 together with Pseudomonas spp. and α-haemolytic streptococci also from an anal swab of two apparently healthy female harbour seals, respectively. A. phocisimile 4113 and A. phocisimile 4125 were isolated in 2007 during a monitoring program of free living harbour seals of the German North Sea.


## 2.2. Phenotypic and Genotypic Identification

All three newly investigatedA. 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.Table 1
Oligonucleotide primer sequences and PCR conditions of the target genes used in the present study.
Oligonucleotide primers
Sequence
Program*
Expected size of PCR product (bp)
References
(1) 16S rDNA UNI-L(2) 16S rDNA UNI-R (amplification primer)
5′-AGAGTTTGATCATGGCTCAG-3′5′-GTGTGACGGGCGGTGTGTAC-3′
1
1,403
[4]
(3) 16S rDNA-533F(4) 16S rDNA-907R (sequencing primer)
5′-GTGCCAGCMGCCGCGGTAA-3′5′-CCGTCAATTCMTTTGAGTTT-3′
—
—
[4]
(5) Gap-F(6) Gap-R (gap primer)
5′-TCGAAGTTGTTGCAGTTAACGA-3′5′-CCATTCGTTGTCGTACCAAG-3′
2
830
[4]
(7) ISR-23S-F(8) ISR-23S-R (intergenic spacer region primer)
5′-CCTAGCCTGGTGGTTGGGTAG-3′5′-GTGCGGGTAACCAGAAATAACTCTG-3′
3
345
[6]
(9) C2700F(10) C3130R (rpoB primer)
5′-CGWATGAACATYGGBCAGGT-3′5′-TCCATYTCRCCRAARCGCTG-3′
4
406
[7]
*PCR program 1: x1 (95°C, 600 sec), x30 (95°C, 30 sec, 58°C, 60 sec, 72°C, 60 sec), and x1 (72°C, 420 sec). 2: x1 (94°C, 180 sec), x30 (94°C, 30 sec, 50°C, 40 sec, 72°C, 60 sec), and x1 (72°C, 300 sec). 3: x1 (95°C, 240 sec), x30 (95°C, 8 sec, 66°C, 10 sec, 72°C, 10 sec), x1 (72°C, 420 sec). 4: x1 (95°C, 600 sec), x35 (94°C, 30 sec, 50°C, 30 sec, 72°C, 120 sec), and x1 (72°C, 600 sec).


## 3. Results and Discussion

All three strains newly characterized in the present study could reliably be identified asA. 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.Table 2
Phenotypical properties of threeA. phocisimile strains investigated in the present study and A. phocisimile 4112 and A. phocisimile
2698
T described previously.
Phenotypic properties
3047
4113
4125
4112
*
*
2698
T
*
*
Hemolysis on sheep blood agar
+
+
+
+
+
Hemolysis on rabbit blood agar
+
+
+
+
+
CAMP-like reaction with:*
Staphylococcus aureus
β-hemolysin
−
−
−
−
−
Streptococcus agalactiae
+
+
+
+
+
Rhodococcus equi
+
+
+
+
+
Arcanobacterium haemolyticum
−
−
−
−
−
Reverse CAMP reaction
+
+
+
+
+
Nitrate reduction
−1
−1
−1
−1
−1
Pyrazinamidase
(+)1
(+)1
+1
+1
+1
Pyrrolidonyl arylamidase
−1
−1
(+)1
−1
−1
Alkaline phosphatase
−1
−1
−1
(+)1
(+)1
β-Glucuronidase (β-GUR)
-
1
,
3
-
1
,
3
-
1
,
3
-
1
,
3
-
1
,
3
β-Galactosidase (β-GAL)
+
1
,
3
+
1
,
3
+
1
,
3
+
1
,
3
+
1
,
3
α-Glucosidase (α-GLU)
+
1,2
,
3
+
1,2
,
3
+
1,2
,
3
+
1,2
,
3
+
1,2
,
3
β-Glucosidase (β-GLU)
−2
−2
−2
−2
−2
N-Acetyl-β-glucosaminidase (β-NAG)
−1, +3
−1, +3
−1, +3
−1, +3
−1, +3
Esculin (β-glucosidase)
−1
−1
−1
−1
−1
Urease
−1
−1
+1
−1
−1
Gelatine
−1
−1
−1
−1
−1
Fermentation of:
D-Glucose
+1
+1
+1
+1
+1
D-Ribose
−1
+1
+1
+1
+1
D-Xylose
−1
−1
−1
−1
−1
D-Mannitol
−1
−1
−1
−1
−1
D-Maltose
+1
+1
+1
+1
+1
D-Lactose
+1
+1
+1
+1
+1
D-Saccharose
+1
+1
+1
+1
+1
Glycogen
+1
+1
+1
+1
+1
α-Mannosidase
+2
+2
+2
+2
+2
Catalase
+
+
+
+
+
Serolysis on Loeffler agar
−
−
−
−
−
Caseinase
−
−
−
−
−
Starch hydrolysis
+
+
+
+
+
The reactions are shown as follows:*synergistic CAMP-like reaction with indicator strains; *
*results mostly obtained from Hijazin et al., 2013 [3]; +: positive reaction; (+): weak positive reaction; −: negative reaction; 1: Api Coryne test system (Biomerieux, Nürtingen, Germany); 2: tablets containing substrates (Rosco Diagnostica A/S, Taastrup, Denmark); 3: 4-methylumbelliferyl conjugated substrates (Sigma, Steinheim, Germany).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 [8–10]. 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 3.1 software package the log (score) values of A. phocisimile 4112, A. phocisimile 3047, A. phocisimile 4113, and A. phocisimile 4125 matched against A. phocisimile  2698
T with log (score) values between 2.69 and 2.74 indicating that all five strains belong to this newly described species. Inclusion of A. phocisimile in the Bruker reference database will allow for the identification of this new species in future. A dendrogram analysis of the MALDI-TOF MS results is presented in Figure 1.Figure 1
MALDI-TOF MS spectra fromA. phocisimile4113, A. phocisimile 4125, type strain A. phocisimile  2698
T, A. phocisimile4112, A. phocisimile 3047, and all other species of genus Arcanobacterium.The genotypic classification by 16S rDNA sequencing revealed that the three novelA. 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).Figure 2
Dendrogram analysis of 16S rDNA sequences of theA. phocisimile strains of the present study and reference strains of genus Arcanobacteriumobtained from NCBI GenBank.Comparable to previously describedA. 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.Dendrogram analysis of sequences of the genesrpoB (a) and gap (b) of the A. phocisimile strains of the present study and all other species of genus Arcanobacterium obtained from NCBI GenBank.
(a)
(b)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 ofA. 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.


## 4. 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.



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*Source: 923592-2014-02-06.xml*