Identification of Arcanobacterium pyogenes isolated by post mortem examinations of a bearded dragon and a gecko by phenotypic and genotypic properties.

The present study was designed to identify phenotypically and genotypically two Arcanobacterium (A.) pyogenes strains isolated by post mortem examinations of a bearded dragon and a gecko. The A. pyogenes strains showed the typical biochemical properties and displayed CAMP-like synergistic hemolytic activities with various indicator strains. The species identity could be confirmed genotypically by amplification and sequencing of the 16S rDNA gene and, as novel target gene, by sequencing of the beta subunit of RNA polymerase encoding gene rpoB, of both strains and of reference strains representing nine species of the genus Arcanobacterium. The species identity of the two A. pyogenes strains could additionally be confirmed by PCR mediated amplification of species specific parts of the 16S-23S rDNA intergenic spacer region, the pyolysin encoding gene plo and by amplification of the collagen-binding protein encoding gene cbpA. All these molecular targets might help to improve the future identification and further characterization of A. pyogenes which, as demonstrated in the present study, could also be isolated from reptile specimens.

Arcanobacterium (A.) pyogenes is a well-known pathogen of domestic ruminants and pigs causing mastitis, abortion and a variety of pyogenic infections [10]. As summarized by Jost and Billington [7] this bacterial pathogen is also able to cause disease in a large number of various animal species. However, at present little is known about A. pyogenes isolated from reptiles.

A total of 13 bacterial cultures were used in this study. The cultures included 11 reference strains of nine species of the genus Arcanobacterium [5,6,12], and the strains A. pyogenes 734 and A. pyogenes 4984 described in the present study. A. pyogenes 734 was isolated from the lung of a bearded dragon (Pogona vitticeps) together with γ-hemolytic streptococci, Pseudomonas spp. and coliform bacteria; A. pyogenes 4984 was isolated from the intestine of a gecko together with α and β-hemolytic streptococci, Acinetobacter spp. and yeasts. Both animals were investigated by post mortem examinations. A. pyogenes was isolated in large numbers from the lung and kidney of the bearded dragon and from the intestine, lung, liver and kidney of the gecko. The gecko showed signs of enteritis. The post mortem isolation of A. pyogenes in relatively large numbers from various organs led to a diagnosis of septicemia for both animals with A. pyogenes deemed the major causative agent. The A. pyogenes strains were investigated for cultural and biochemical properties using the Api-Coryne test system (bioMérieux, Germany), tablets containing various substrates (Inverness Medical, Germany), and 4-methylumbelliferyl-conjugated substrates (Sigma, Germany). The strains were also investigated for CAMP-like synergistic hemolytic activities, as previously described [6,12].

For molecular identification both A. pyogenes strains were investigated by 16S rDNA amplification and sequencing [6]. In addition, the beta subunit of RNA polymerase encoding gene rpoB of nine species of genus Arcanobacterium and both A. pyogenes strains were amplified using the oligonucleotide primers described by Khamis et al. [8] (Table 1). DNA extraction, sequencing and the alignment studies were performed as described previously [5,6]. In addition, both A. pyogenes strains were investigated by PCR-mediated amplification of a species specific region of the 16S-23S rDNA intergenic spacer region (ISR), by amplification of the pyolysin encoding gene plo described in the present study and by Ertas et al. [3], and by amplification of the collagen-binding protein encoding gene cbpA. The oligonucleotide primers used in this study were synthesized by MWG Biotech (Germany) and Operon (Germany). The oligonucleotide primer sequences, the thermal cycler PCR programs and the expected amplicon sizes are summarized in Table 1.

Table 1

Oligonucleotide primer sequences and PCR conditions

*PCR Program 1: ×1 (95℃, 600 sec), ×30 (95℃, 30 sec, 58℃, 60 sec, 72℃, 60 sec), ×1 (72℃, 420 sec). 2: ×1 (95℃, 600 sec), ×35 (94℃, 30 sec, 37℃, 30 sec, 72℃, 120 sec), ×1 (72℃, 600 sec). 3: ×1 (95℃, 600 sec), ×35 (94℃, 30 sec, 50℃, 30 sec, 72℃, 120 sec), ×1 (72℃, 600 sec). 4: ×1 (95℃, 600 sec), ×30 (95℃, 30 sec, 64℃, 15 sec, 72℃, 30 sec), ×1 (72℃, 420 sec). 5: ×1 (95℃, 600 sec), ×30 (95℃, 60 sec, 62℃, 60 sec, 72℃, 60 sec), ×1 (72℃, 420 sec). 6: ×1 (95℃, 600 sec), ×35 (94℃, 60 sec, 55℃, 60 sec, 72℃, 60 sec), ×1 (72℃, 300 sec). 7: ×1 (94℃, 600 sec), ×30 (95℃, 30 sec, 58℃, 30 sec, 72℃, 60 sec), ×1 (72℃, 420 sec). †For Arcanobacterium (A.) bialowiezense DSM 17162, A. bonasi DSM 17163, A. haemolyticum DSM 20595, A. hippocoleae DSM 15539, A. phocae DSM 10002, A. phocae DSM 10003. ‡For A. abortisuis DSM 19515, A. bernardiae DSM 9152, A. pyogenes DSM 20630, A. pyogenes DSM 20594 and both A. pyogenes strains of the present study. §16S-23S rDNA intergenic spacer region.

Both bacterial strains investigated in the present study were identifiable phenotypically and genotypically as A. pyogenes. In similarity to the A. pyogenes reference strains and to previous findings [12] both A. pyogenes strains displayed hemolytic properties on sheep and rabbit blood agar, a synergistic CAMP-like hemolysis with staphylococcal β-hemolysin, Rhodococcus equi and Arcanobacterium hemolyticum indicator strains and, described for the first time in the present study, a synergistic CAMP-like hemolysis with A. phocae.

Both A. pyogenes strains of the present study were additionally identifiable genotypically by amplification and sequencing of the 16S rRNA gene (GenBank accession number, FN394982 and FN394983) and by amplification and sequencing of the rpoB gene of both strains (FN550376, FN550377) which show an almost complete identity to the rpoB sequences of A. pyogenes DSM 20630 (FN550375) and A. pyogenes DSM 20594 (FN550374). The A. pyogenes rpoB sequences differed clearly from the rpoB sequences of reference strains of eight additionally sequenced species of the genus Arcanobacterium (FN550365-FN550373). Dendrogram analysis of the 16S rDNA and rpoB sequencing results are shown in Fig. 1. 16S rDNA and ISR sequencing had already been used to characterize A. haemolyticum isolates from horses [6] and ISR sequencing had been used to design ISR specific oligonucleotide primers for PCR mediated identification of A. bialowiezense and A. bonasi [5]. Comparable to 16S rDNA and ISR, rpoB gene sequencing of bacteria of the genus Arcanobacterium might represent a novel target for molecular characterization of bacteria of this genus.

Fig. 1

Dendrogram analysis of 16S rRNA gene sequences (A) and rpoB gene sequences (B) of the A. pyogenes strains of the present study and of various Arcanobacterium spp. obtained from the NCBI GenBank and of sequencing results of the present study.

In addition both strains reacted positively with the A. pyogenes ISR-specific oligonucleotide primers described in the present study (data not shown). A. pyogenes expresses a cholesterol-dependent cytolysin, designated as pyolysin, that is a well known major virulence factor of this species [1]. As a constant characteristic feature of A. pyogenes, the pyolysin encoding gene plo could be used for molecular identification of this species [2,3].

The A. pyogenes plo specific oligonucleotide primers described in the present study and by Ertaş et al. [3] could be used to amplify a corresponding gene of both A. pyogenes strains of the present study (data not shown) indicating that all three species specific oligonucleotide primer sets could successfully be used for molecular identification of this bacterial species.

Summarizing the potential virulence factors of A. pyogenes, Jost and Billington [7] described a collagen-binding protein cbpA which seems to promote the adhesion of these bacteria to collagen-rich tissues. As shown in the present study the virulence factor cbpA encoding gene cbpA seems to also be present in both A. pyogenes strains isolated from reptiles and in reference strain A. pyogenes DSM 20630 (ATCC 19411) of porcine origin. According to Esmay et al. [4] cbpA was found in A. pyogenes of bovine (49%), porcine (40%) and avian (100%) origin. Isolates from canines and felines did not carry cbpA. Silva et al. [11] were able to detect cpbA in all 57 investigated A. pyogenes strains of bovine origin.

To our knowledge the present study is the first detailed phenotypic and genotypic characterization of A. pyogenes strains isolated from specimens of a bearded dragon and a gecko. However, the usefulness of the rpoB gene as a target gene for identification of bacteria of the genus Arcanobacterium and the usefulness of the various species specific oligonucleotide primers for molecular identification and further characterization of A. pyogenes need to be investigated with a larger number of A. pyogenes strains of various origins.