Phenotypic and genotypic approach to characterize a Trueperella pyogenes strain isolated from an Eurasian lynx (Lynx lynx)

Within genus Trueperella (T.) five species were distinguished currently: T. pyogenes, T. abortisuis, T. bernardiae, T. bonasi and T. bialowiezensis (Yassin et al. 2011). T. pyogenes is a worldwide known pathogen of domestic ruminants and pigs, causing mastitis, abortion and a variety of pyogenic infections (Lämmler and Hartwigk 1995). As summarized by Jost and Billington (2005), this bacterial pathogen is also able to cause diseases in a large number of various animal species including antelopes, wildebeest, gazelles, deer, reindeer, bison, camels, elephants, horses, macaws, chicken and turkeys and also in companion animals such as dogs and cats (Billington et al. 2002). T. pyogenes infections in humans are rare (Gahrn-Hansen and Frederiksen 1992) and these infections are often a result of occupational exposure.

In 2010, Ülbegi-Mohyla et al. characterized phenotypically and genotypically two T. pyogenes strains isolated from septicaemia of a gecko and a bearded dragon, Eisenberg et al. (2012) a T. pyogenes strain isolated in pure culture from a facial abscess of a grey slender loris of Frankfurt Zoo (Frankfurt am Main, Germany), Al-Tarazi et al. (2012) T. pyogenes recovered from lung abscesses of one-humped camels and Wickhorst et al. (2017a) a T. pyogenes strain isolated from a brain abscess of an adult roebuck.

T. pyogenes possesses a number of known and putative virulence factors that may contribute to its pathogenic potential. A well characterized virulence factor is pyolysin, a haemolysin which is also cytolytic for immune cells (Jost and Billington 2005). The present study was designed to identify and further characterize a T. pyogenes strain isolated from a Eurasian lynx (Lynx lynx).

A male 11-year-old Eurasian lynx (Lynx lynx) kept in a game park did not appear at the feeding place for three days and was subsequently found dead in its enclosure. The necropsy was performed at the Chemisches und Veterinäruntersuchungsamt Westfalen, Bochum (CVUA-Westfalen). The animal was in moderate body condition weighing 20.2 kg. Gross examination revealed an exudative pyogranulomatous pleuropneumonia with approximately two litres of reddish cloudy pleural exudation in both pleural cavities containing sulfur granules (Fig. 1A, B). Both lung lobes showed severe compression atelectasis. A traumatic lesion could neither detected in the thoracic wall nor in the oesophagus. T. pyogenes was isolated from lung in mixed culture with Pasteurella multocida and Escherichia coli. The T. pyogenes lung isolate was further characterized in detail.

Phenotypic analyses were performed by conventional biochemical assays and the API-Coryne System (BioMérieux, Nürtingen, Germany) as described (Al-Tarazi et al. 2012, Hijazin et al. 2011, Nagib et al. 2014, Ülbegi-Mohyla et al. 2010). In addition, the bacterial strain was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS, Bruker Biotyper) using the commercial database version MBT 7,854 (Wickhorst et al. 2019) and by Fourier Transform Infrared Spectroscopy (FT-IR) (Nagib et al. 2014).

The presence of T. pyogenes chaperonin-encoding gene cpn60 was determined with a previously described loop-mediated isothermal amplification (LAMP) assay. This was performed using a real-time fluorometer (Genie II^®, OptiGene, Horsham, UK) (Abdulmawjood et al. 2016, Wickhorst et al. 2017a).

A further genotypic analysis was conducted by sequencing the 16S ribosomal RNA (rRNA) gene (Hassan et al. 2009), the 16S-23S rDNA intergenic spacer region (ISR) (Hassan et al. 2009, Wickhorst et al. 2017a), by sequencing the elongation factor tu-encoding gene tuf (Wickhorst et al. 2017a, b) and by sequencing the β subunit of bacterial RNA polymerase encoding gene rpoB (Ülbegi-Mohyla et al. 2010, Wickhorst et al. 2017a). The bacterial strain was also characterized by amplification and sequencing of pyolysin-encoding gene plo (Eisenberg et al. 2012, Hijazin et al. 2011, Ülbegi-Mohyla et al. 2010).

T. pyogenes S 1276/1/18 investigated in the present study showed a narrow zone of haemolysis on 5% sheep blood agar and CAMP-like reactions in the zone of staphylococcal β-haemolysin and with Rhodococcus hoagii as indicator strain. In addition, the isolate could be identified phenotypically with a commercial identification system. The biochemical properties were almost identical to T. pyogenes DSM 20630^T and T. pyogenes DSM 20594 described previously (Eisenberg et al. 2012, Hijazin et al. 2011, Ülbegi-Mohyla et al. 2010). The isolate gave positive reactions for pyrrolidonyl arylamidase, alkaline phosphatase, β-glucuronidase, β-galactosidase, α-glucosidase, N-acetyl-β-glucosaminidase and negative reactions for nitrate reduction and pyrazinamidase. T. pyogenes S 1276/1/18 hydrolysed gelatine and esculin, but not urea, fermented D-glucose, D-ribose, D-xylose, D-maltose, D-lactose, D-saccharose, but not D-mannitol and glycogen. In addition, the isolate showed a negative catalase reaction and a positive reaction on Löffler agar. A positive reaction on Löffler agar is typical for T. pyogenes and widely used for phenotypic identification of this species (Bisping and Amtsberg 1988, Eisenberg et al. 2012, Hijazin et al. 2011, Lämmler 1990, Lämmler and Hartwigk 1995, Wickhorst et al. 2017a). The species identification of T. pyogenes S 1276/1/18 could be confirmed by MALDI-TOF MS analysis with a log (score) value of 2,14 for the first, and 2,108 for the second hit, according to the current decision rules of the manufacturer. Similar to the present results MALDI-TOF MS had already been shown to be a rapid and reliable technique for identification of bacteria of genera Arcanobacterium and Trueperella, also including T. pyogenes (Hijazin et al. 2012, Wickhorst et al. 2017a).

FT-IR spectroscopy, a promising technique for rapid and reliable identification of bacterial microorganisms, had already been used as tool for classification of Listeria (Janbu et al. 2008) and Yersinia species (Kuhm et al. 2009) and for a large number of other clinically relevant pathogens (Contzen et al. 2011, Grunert et al. 2013, Samuels et al. 2009), also including T. pyogenes isolated from bovine mastitis (Nagib et al. 2014). The infrared spectra of T. pyogenes S 1276/1/18 of the present study were analysed by the method described before (Nagib et al. 2014). Cluster analyses revealed spectra which were closely related to the type strain T. pyogenes DSM 20630^T and to T. pyogenes DSM 20594 and clearly separated from available spectra of other species of the genus Trueperella and the genus Arcanobacterium (Fig. 2).

Comparable to the previously described LAMP assay for detection of gene cpn60 of T. pyogenes of various origins (Abdulmawjood et al. 2016, Wickhorst et al. 2017a), the species-specific gene cpn60 of the T. pyogenes strain of the present investigation could be successfully identified by using this cpn60 specific LAMP assay, indicating that this method allows a low-cost and reliable identification of T. pyogenes.

The cpn60 LAMP product could be amplified using a real-time fluorometer (Fig. 3).

T. pyogenes S 1276/1/18 was also identified by sequencing the 16S rRNA gene, ISR, the genes tuf and rpoB and the putative virulence factor pyolysin encoding gene plo. T. pyogenes S 1276/1/18 and the type strain T. pyogenes DSM 20630^T showed sequence similarities of the 16S rRNA gene (acc. no.: MN135984, X79225), ISR (MN164031, EU194563) and for the genes tuf (MN163266, HG941716), rpoB (MN163265, FN550375) and plo (MN163264, U84782) of 99.6%, 100%, 100%, 98.5%, and 99.8%, respectively. A typical dendrogram analyses of ISR and for gene rpoB is presented in Figure 4.

Gene plo expresses the cholesterol-dependent pyolysin, which is well known as a major virulence factor of this species (Billington et al. 1997, Ding and Lämmler 1996, Jost and Billington 2005) and could be used for molecular identification of this species, because it is generally present in all T. pyogenes isolates (Billington et al. 2002, Ertaş et al. 2005, Ülbegi-Mohyla et al. 2010). A phylogenetic analysis of the amino acid sequences of pyolysin (PLO) of T. pyogenes S1276/1/18 of the present study, PLO of the reference strain T. pyogenes DSM 20630^T (AAC45754), phocaelysin (PHL) of A. phocae (SMR98720), arcanolysin (ALN) of A. haemolyticum (ACV96715) and of other pore forming toxins including streptolysin O (SLO) of Streptococcus pyogenes (BAB41212), intermedilysin (ILY) of Streptococcus intermedius (BAA89790), pneumolysin (PLY) of Streptococcus pneumoniae (ADF28298) and listeriolysin O (HLY) of Listeria monocytogenes (NP_463733) obtained from NCBI GenBank revealed a close relationship of PLO of T. pyogenes S 1276/1/18 to PLO of T. pyogenes DSM 20630^T (99.5% amino acid similarity) and a less pronounced relation to the other pore forming toxins (Fig. 5).

T. pyogenes S 1276/1/18 was isolated from a lung of an adult lynx, which suffered from an exudative pleuropneumonia. The strain was identified phenotypically, by MALDI-TOF MS and FT-IR analyses, and genotypically by detection of various species-specific targets. However, the importance of the T. pyogenes strain, which was isolated in mixed culture, for the clinical picture of the adult lynx and the route of infection of this bacterial pathogen remains unclear. The MALDI TOF mass-spectrum of T. pyogenes S 1276/1/18 and further information for the isolates used for comparison are available via the MALDI-TOF user platform (http://www.maldi-up.ua-bw.de; Rau et al. 2016).

The authors declare no conflict of interest.

Authors assure to have met common international ethical guidelines during the genesis of the above work, the underlying research and its publication.

There was no funding.

Konzeption oder Design der Arbeit: MA, CL.
Datenerhebung: MA, MP, JR, AH, MPlö, AA.
Datenanalyse und Interpretation: MA, MP, JR, AH, OS, EP-B.
Manuskriptentwurf: MA, OS, CL.
Kritische Revision des Artikels: MA, MP, JR, AH, OS, CL, MPlö, AA.
Endgültige Zustimmung der für die Veröffentlichung vorgesehen Version: MA, MP, JR, AH, OS, CL, EP-B, MPlö, AA.

Prof. Dr. Christoph Lämmler
Institut für Hygiene und Infektionskrankheiten der Tiere
Justus-Liebig-Universität Gießen
Frankfurter Str. 85–91
35392 Gießen
Germany
Christoph.Laemmler@vetmed.uni-giessen.de

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