Studies on Trueperella pyogenes isolated from an okapi (Okapia johnstoni) and a royal python (Python regius).

BACKGROUND: The present study was designed to characterize phenotypically and genotypically two Trueperella pyogenes strains isolated from an okapi (Okapia johnstoni) and a royal python (Python regius). CASE
PRESENTATION: The species identity could be confirmed by phenotypic properties, by MALDI-TOF MS analysis and by detection of T. pyogenes chaperonin-encoding gene cpn60 with a previously developed loop-mediated isothermal amplification (LAMP) assay. Furthermore, sequencing of the 16S ribosomal RNA (rRNA) gene, the 16S-23S rDNA intergenic spacer region (ISR), the target genes rpoB encoding the β-subunit of bacterial RNA polymerase, tuf encoding elongation factor tu and plo encoding the putative virulence factor pyolysin allowed the identification of both T. pyogenes isolates at species level.
CONCLUSIONS: Both strains could be clearly identified as T. pyogenes. The T. pyogenes strain isolated in high number from the vaginal discharge of an okapi seems to be of importance for the infectious process; the T. pyogenes strain from the royal python could be isolated from an apparently non-infectious process. However, both strains represent the first isolation of T. pyogenes from these animal species.

Background

Trueperella pyogenes is worldwide considered as part of the commensal biota of skin and mucous membranes of the upper respiratory and urogenital tract of animals [1]. However, T. pyogenes is also an important opportunistic pathogen that causes mastitis, abortion and a variety of diverse pyogenic infections in livestock, including cattle, sheep, goats, horses, and pigs [2-4]. In cattle, T. pyogenes appears to be responsible for infections of the reproductive tract [5] and the mammary gland [6], as well as cases of pneumonia and liver abscessation of large and small ruminants [7]. In swine, T. pyogenes is well known as a causative agent of different types of inflammation in various organs including the lung, heart, joints, mammary glands, and in the reproductive tract [8, 9]. Furthermore, T. pyogenes could be found in companion animals [4]. One of the first reported cases in companion animals was an otitis externa detected in a cat and cystitis in a dog [10]. More recently, Wareth et al. [11] described a co-infection case of T. pyogenes with Brucella abortus in a cat and dog. Additionally, various wildlife animals could harbour T. pyogenes [3]. In 2010, Ülbegi-Mohyla et al. [12] characterized two T. pyogenes strains isolated from a bearded dragon and a gecko. Additionally, T. pyogenes infections were reported from a bison and from camels [13, 14], from goitered gazelles [15] and from a white-tailed deer [16]. Likewise, some other sporadic cases of infectious diseases associated with T. pyogenes were described in a galago [17], in gray slender lorises [18, 19] and in a eurasian lynx [20].

Besides conventional bacteriological methods for identifying T. pyogenes isolates, other new, fast and reliable techniques were described and utilized in this study: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [20-23], a loop-mediated isothermal amplification (LAMP) assay [24] and 16S rRNA gene sequencing [25, 26].

The present study was designed to identify and further characterize T. pyogenes isolated from wildlife animals phenotypically and genotypically. To the best of our knowledge, the present study provides a first detailed description of T. pyogenes recovered from an okapi and a royal python.

Case presentation

As part of routine examination and diagnostics performed on zoo animals at Frankfurt Zoo (Frankfurt am Main, Germany) in 2019, T. pyogenes 24398 was isolated from a vaginal discharge of an okapi (Okapia johnstoni) in high numbers (+++), together with Enterobacter cloacae (+) and Pasteurella spp. (+). The initial bacteriology analysis for T. pyogenes 24398 was performed at Hessian State Laboratory (LHL) Gießen, Germany. As a result of post-mortem examination conducted in 2017, T. pyogenes 171003246 was recovered in low numbers (+) from a kidney of a seven-year-old female royal python (Python regius). The python was found dead in a bird park in Hesse (Germany) and was 107 cm in length and weighted 1.23 kg. In addition, Escherichia coli (+), α-hemolytic streptococci (+), Corynebacterium spp. (+) and Clostridium sardiniense (+) were cultured from the python specimen. The post-mortem analysis of the royal python revealed a good body condition and in the throat and head area a 15 cm lung edema and swelling, possibly caused by traumatic reasons. The post-mortem examination and the initial bacteriology analysis were also performed at Hessian State Laboratory. Both T. pyogenes strains were further investigated phenotypically and genotypically.

Phenotypic characterization

A phenotypic characterization was performed using conventional cultural and biochemical assays as previously described [12, 18, 20, 27] and the API-Coryne test System (BioMérieux, Nürtingen, Germany) in accordance with the manufacturer’s instructions. Furthermore, the bacterial isolates were identified by MALDI-TOF MS using a Microflex LT (Bruker Daltonik GmbH, Bremen, Germany) instrument and MBT Compass Explorer 4.1 software (Bruker Daltonik GmbH). Sample preparation was carried out in accordance with the manufacturer’s instructions using the direct transfer method. Briefly, one microbial colony was first smeared in duplicate onto spots of the MALDI target MSP 96 target (MicroScout Target plate; Bruker Daltonik GmbH) with sterile toothpicks. The air-dried bacteria were overlaid with 1 µl of an α-cyan 4-hydroxycinnamic acid matrix solution (HCCA, in 50% acetonitrile and 2.5% trifluoroacetic acid in pure water) followed by drying and loading into the mass spectrometer.

Genotypic properties

The genomic DNA of both isolates and the type strains T. pyogenes DSM 20630T (pig), T. abortisuis DSM 19515T (placenta of sow after abortion), T. bernardiae DSM 9152T (human blood) and T. bonasi DSM 17163T (european bison) were extracted using the DNeasy blood and tissue kit (Qiagen GmbH, Hilden, Germany), in accordance with the manufacturer’s instructions. The concentration and purity of DNA were measured by utilizing a Nano Drop spectrophotometer (ND1000; Thermo Fisher Scientific GmbH, Dreieich, Germany).

The detection of gene cpn60 of T. pyogenes was performed using a previously designed loop-mediated isothermal amplification (LAMP) assay [24] with a portable real-time fluorometer (Genie II®, OptiGene Ltd, Horsham, UK) and the reference strains T. pyogenes DSM 20630T, T. abortisuis DSM 19515T, T. bernardiae DSM 9152T and T. bonasi DSM 17163T.

Both T. pyogenes isolates were also evaluated by PCR for the presence of five genomic targets: 16S rRNA gene (16S), 16S-23S rDNA intergenic spacer region (ISR), the β-subunit of bacterial RNA polymerase encoding gene rpoB, the elongation factor tu encoding gene tuf, and pyolysin encoding gene plo. The sequence of the oligonucleotide primers and PCR conditions were previously described by Hassan et al. [25], Ülbegi-Mohyla et al. [12], Hijazin et al. [27], Eisenberg et al. [18], Wickhorst et al. [28], Wickhorst et al. [23], Alssahen et al. [20].

The PCR products were purified and sequenced by Eurofins Umwelt Nord GmbH (Göttingen, Germany). The obtained sequences were analyzed via the cluster method of the MegAlign program (DNASTAR Inc., ver. 15, Madison, WI, USA) by comparing with the nucleotide sequences of 16S rRNA, ISR, rpoB, tuf and plo from different Trueperella reference strains. Moreover, the resulting amino acid sequences of pyolysin of both T. pyogenes isolates were compared with the respective sequences of pyolysin of T. pyogenes DSM 20630T, closely related pore-forming toxins of genus Arcanobacterium and with other bacterial pore-forming toxins. All the nucleotide and amino acid sequences were obtained from the NCBI GenBank.

Discussion and Conclusion

Both T. pyogenes strains investigated in the present study showed a narrow zone of complete hemolysis on 5% sheep blood agar and CAMP-like reactions in the staphylococcal β-hemolysin zone with Rhodococcus hoagii as an indicator strain. The conventional biochemical properties and the results of the commercial identification system revealed almost identical results to previously investigated T. pyogenes of various origins and T. pyogenes DSM 20630T [12, 18, 20, 27] (Table. 1). The T. pyogenes isolates yielded positive reactions for pyrrolidonyl arylamidase, alkaline phosphatase, β-glucuronidase, β-galactosidase, α-glucosidase and N-acetyl-β-glucosaminidase and negative reactions for nitrate reduction and pyrazinamidase. Additionally, the isolates hydrolyzed gelatine, but not esculin and urea. The isolates also fermented D-glucose, D-ribose, D-xylose, D-maltose, D-lactose and glycogen, but not D-mannitol. T. pyogenes 24398 fermented D-saccharose; however, T. pyogenes 171003246 was D-saccharose negative. In addition, both isolates showed a negative catalase reaction and a positive reaction on Löffler agar (Table 1). A postitive reaction on Löffler agar is typical for T. pyogenes and widely used for phenotypic identification of this species [2, 18].

Table 1

Biochemical properties of T. pyogenes 24398 (okapi), T. pyogenes 171003246 (royal python) and type strain T. pyogenes DSM 20630T

Biochemical properties	T. pyogenes24398(okapi)	T. pyogenes171003246(royal python)	T. pyogenesa DSM 20630T
Haemolysis on SBAb	+	+	+
CAMP-like hemolytic reaction withc:
Staphylococcus aureus	+	+	+
Streptococcus agalactiae	−	−	−
Rhodococcus hoagii	+	+	+
Reverse CAMP reaction	−	−	−
Nitrate reduction	−	−	−
Pyrazinamidase	−	−	−
Pyrrolidonyl Arylamidase	+	+	+
Alkaline phosphatase	+	+	+
β-Glucuronidase (β-GUR)	+	+	+
β-Galactosidase (β-GAL)	+	+	+
훼α-Glucosidase (훼α-GLU)	+	+	+
N-Acetyl-β-glucosaminidase (β-NAG)	+	+	+
Esculin (β-glucosidase)	−	−	−
Urease	−	−	−
Gelatine	+	+	+
Fermentation of:
Glucose	+	+	+
Ribose	+	+	+
Xylose	+	+	+
Mannitol	−	−	−
Maltose	+	+	+
Lactose	+	+	+
Saccharose	−	+	−
Glycogen	+	+	+
Catalase	−	−	−
Serolysis on Loeffler agar	+	+	+
Identification % according to  API-Coryne test System	99.9	99.9	99.9

aResults taken from (Ülbegi-Mohyla et al. 2010 [12]; Hijazin et al. 2011 [27]; Eisenberg et al. 2012 [18]; Alssahen et al. 2020 [20])

bSBA: Sheep Blood Agar

csynergistic or reverse CAMP-like reaction with indicator strains

+: positive reaction, −: negative reaction, :type strain

Moreover, MALDI-TOF MS identified T. pyogenes 24398 and T. pyogenes 171003246 with log score values of 2.35 and 2.29 for the first hit and log score values of 2.28 and 1.9 for the second hit, respectively (data not shown). These log score values confirmed, in accordance with the current decision rules of the manufacturer, the species designation. Comparable to the present results, MALDI-TOF MS had already been shown to be a rapid and reliable technique for identifying bacteria of genera Arcanobacterium and Trueperella, including T. pyogenes [20, 21].

The previously described cpn60-specific LAMP assay could successfully be used to identify the species-specific gene cpn60 of T. pyogenes 24,398 and T. pyogenes 171,003,246 in the present investigation. This was comparable to the LAMP assay for detecting gene cpn60 of the previously described T. pyogenes of various origins [24], a T. pyogenes strain isolated from an adult roebuck (Capreolus capreolus) [23], and a T. pyogenes strain isolated from a eurasian lynx (Lynx lynx) [20]. The results of the cpn60 LAMP assay are shown in Fig. 1; Table 2.

Fig. 1

Positive LAMP assay of T. pyogenes 24398 (okapi), T. pyogenes 171003246 (royalpython), T. pyogenes DSM 20630T, the LAMP negative control strains T. abortisuis DSM 19515T, T. bernardiaeDSM 9152Tand T. bonasi DSM 17163T, and a negative control

Table 2

Results of LAMP including detection time and annealing temperature of the tested isolate, positive and negative control

Sample ID	T. pyogenes 24398	T. pyogenes171003246	T. pyogenes DSM 20630T	T. abortisuis DSM 19515T	T. bernardiaeDSM 9152T	T. bonasi DSM 17163T	HPLC water and Master mix
Result	+ve	+ve	+ve	-ve	-ve	-ve	-ve
Detection time hh:mm:ss	00:11:00	00:08:30	00:10:00	0	0	0	0
Annealing	89.6	89.4	89.9	0	0	0	0

+ve: Positive, -ve: Negative, :type strain

The oligonucleotide primers, 16SUNI-L and 16SUNI-R, were used for amplifying of 16S rRNA gene of the investigated T. pyogenes isolates. The nucleotide sequence data of T. pyogenes 24398 (GenBank accession numbers: MN946520) and T. pyogenes 171003246 (MN712476) were compared with type strain T. pyogenes DSM 20630T (AAC45754) and with the previously described strain T. pyogenes S 1276/1/18 isolated from a eurasian lynx (MN135984), T. abortisuis DSM 19515T (FN667628), T. bernardiae DSM 9152T (X79224), T. bialowiezensis DSM 17162T (EU194569), and T. bonasi DSM 17163T (EU194570). The nucleotide sequence data of T. pyogenes 24398 and T. pyogenes 171003246 revealed a sequence homology of 98.9% among both strains, a sequence homology of 99.5% and 98.7% with T. pyogenes DSM 20,630T, and a sequence homology of 99.9% and 99.1% with T. pyogenes S1276/1/18, respectively. The control strains of genus Trueperella yielded a sequence homology to both T. pyogenes isolates ≤ 98.7% (Fig. 2).

Fig. 2

Phylogenetic analysis based on nucleotide sequences of 16S rRNA gene of the investigated T. pyogenes 24398 and T. pyogenes 171003246 isolated from okapi and royal python compared with the type strain T. pyogenes DSM 20630T and T. pyogenes S 1276/1/18 isolated from a eurasian lynx, T. abortisuis DSM 19515T, T. bernardiae DSM 9152T, T. bialowiezensis DSM 17162T, and T. bonasi DSM 17163T

Both strains T. pyogenes 24398 and T. pyogenes 171003246 were further identified by sequencing ISR, the genes tuf and rpoB and the putative virulence factor pyolysin encoding gene plo. T. pyogenes 24398 and T. pyogenes 171003246 showed sequence similarities of ISR (MN947249, MN724920) of 99.8% and 98.9% with T. pyogenes DSM 20630T (EU194563) and 100% and 99.8% with T. pyogenes S 1276/1/18 (MN164031), respectively with 98.5% identity between both strains. The additionally investigated gene tuf (MN956808, MN741111) showed a sequence similarity of 99.6% and 99.7% with T. pyogenes DSM 20630T (HG941716), and 99.6% and 99.7% with T. pyogenes S 1276/1/18 (MN163266), respectively; gene rpoB (MN956807, MN741109), a sequence similarity of 99.8% and 98.3% with T. pyogenes DSM 20630T (FN550375), and 98.8% and 98.3% with T. pyogenes S 1276/1/18 (MN163265), respectively, and gene plo (MN956806, MN741110), a sequence similarity of 99.5% with T. pyogenes DSM 20630T (U84782) for both isolates, and 99.1% with T. pyogenes S 1276/1/18 (MN163264) for both isolates.

Dendrograms of the ISR, tuf and rpoB genes are presented in Fig. 3.

Fig. 3

Phylogenetic analyses based on ISR (a), tuf (b) and ropB (c) nucleotide sequences of the investigated T. pyogenes 24398 and T. pyogenes 171003246 and T. pyogenes S 1276/1/18 isolated from a eurasian lynx and the control strains, T. pyogenes DSM 20630T, T. abortisuis DSM 19515T, T. bernardiae DSM 9152T, T. bialowiezensis DSM 17162T, and T. bonasi DSM 17163T

A phylogenetic analysis of the amino acid sequences of pyolysin (PLO) encoded by gene plo of T. pyogenes 24398 (MN956806), and T. pyogenes 171003246 (MN741110) PLO of type strain T. pyogenes DSM 20630T (AAC45754), PLO of T. pyogenes S 1276/1/18 (MN163264), arcanolysin (ALN) of Arcanobacterium haemolyticum (ACV96715), phocaelysin (PHL) of Arcanobacterium phocae 10002T (SMR98720), listeriolysin O (HLY) of Listeria monocytogenes (NP_463733), intermedilysin (ILY) of Streptococcus intermedius (BAA89790), pneumolysin (PLY) of Streptococcus pneumoniae (ADF28298) and streptolysin O (SLO) of Streptococcus pyogenes (BAB41212). The results showed an amino acid similarity of 99.5% for both T. pyogenes 24,398 and T. pyogenes 171003246 with PLO of T. pyogenes DSM 20630T and 99.1% with PLO of T. pyogenes S 1276/1/18 (Fig. 4).

Fig. 4

Phylogenetic relationships among amino acid sequences of PLO of the investigated T. pyogenes 24398 and T. pyogenes 171003246, PLO of type strain T. pyogenes 20630T, T. pyogenes S 1276/1/18 isolated from a eurasian lynx, ALN of A. haemolyticum, PHL of A. phocae, HLY of L. monocytogenes, PLY of S. pneumoniae

T. pyogenes 24398 was isolated in high numbers from vaginal discharge of an okapi and seems to be responsible for the infectious process; T. pyogenes 171003246 was isolated from a non-infectious process of a royal python suffering from a throat swelling, possibly caused by trauma. Both T. pyogenes isolates were identified by a biochemical test, LAMP and MALDI-TOF MS. The genomic targets of the two isolates, 16S rRNA gene, ISR, tuf, rpoB and plo were sequenced and compared to the respective targets of reference and other strains. Thus, the report is the first to provide a detailed characterization of T. pyogenes strains of these origin.