Identification of Trueperella bernardiae isolated from peking ducks (Anas platyrhynchos domesticus) by phenotypical and genotypical investigations and by a newly developed loop-mediated isothermal amplification (LAMP) assay

Trueperella (T.) bernardiae is a well-known bacterial pathogen in infections of humans, rarely in animals. In the present study, five T. bernardiae isolates, isolated from five Peking ducks of four different farms, were identified by phenotypic properties, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, and genotypically by sequencing the 16S ribosomal RNA (rRNA) gene, the superoxide dismutase A encoding gene sodA, and the glyceraldehyde-3-phosphate dehydrogenase encoding gene gap. In addition, the T. bernardiae isolates could be identified with a newly developed loop-mediated isothermal amplification (LAMP) assay based on the gyrase encoding housekeeping gene gyrA. All these tests clearly identified the T. bernardiae isolates to the species level. However, the detection of the specific gene gyrA with the newly designed LAMP assay appeared with a high sensitivity and specificity, and could help to identify this bacterial species in human and animal infections in future. The importance of the T. bernardiae isolates for the clinical condition of the ducks and for the problems at farm level remains unclear.

The first characterization of T. bernardiae of animal origin (3-day-old piglet) was made by Hijazin et al. (2012c). In a second case, Arnafia et al. (2017) described a T. bernardiae strain recovered from a purulent thelitis of a 12-year-old male dog.
These previously isolated T. bernardiae isolates of animal origin were identified and further characterized by matrixassisted desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and by sequencing various genomic targets (Hijazin et al. 2012c;Arnafia et al. 2017).
At present, no further data are available concerning the isolation of T. bernardiae from animals or animal infections.
In the present investigation, five T. bernardiae isolates recovered from post mortem samples of Peking ducks (Anas platyrhynchos domesticus) could be identified phenotypically and genotypically, and with a newly described loopmediated isothermal amplification (LAMP) assay based on the housekeeping gene gyrA.

Bacterial strains
The five T. bernardiae isolates investigated in the present study (T. bernardiae D12-0613-1-4-3, T. bernardiae D13-1622-5-3-2, T. bernardiae D14-1577-4-8-1, T. bernardiae D13-1772-748-1-2, T. bernardiae D14-1481-2029-1-1) were isolated from five Peking ducks (Anas platyrhynchos domesticus) from three different farms in Germany (n = 3) and one in Thailand (n = 2). The Geman samples were collected from the heart, lung, and joint, while the Thailand samples were delivered as swabs after post-mortem examination; unfortunately, the source organs were not defined. All five isolates were collected during post-mortem examination in the period between 2012 and 2014. Further details about the five isolates are given in Table 1. The bacterial cultivation and a preliminary identification of the bacteria were performed at Ripac-Labor GmbH, Potsdam, Germany. The bacterial culturing of the T. bernardiae isolates was carried out on sheep blood agar plates (Oxoid GmbH, Wesel, Germany) for 48 h at 37 °C under a microaerophilic gas atmosphere using a candle jar.

Phenotypic identification
A phenotypic identification was performed using conventional cultural and biochemical assays as previously shown Ülbegi-Mohyla et al. 2009) and with the API-Coryne test system (BioMérieux Deutschland GmbH, 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 following the manufacturer's instructions using the direct transfer method. Briefly, one microbial colony was first smeared in duplicate onto spots of the MALDI MSP 96 target plate (MicroScout Target plate; Bruker Daltonik GmbH) with sterile toothpicks. The air-dried bacteria were overlaid with 1µL 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. The analysis of the spectra was carried out by MBT Compass Explorer 4.1 software (Bruker Daltonik GmbH).

DNA extraction
The genomic DNA of the five isolates, type strain T. bernardiae DSM 9152 T , and various other strains of genus Trueperella and genus Arcanobacterium (A.) were extracted using the DNeasy Blood and Tissue kit (Qiagen GmbH, Hilden, Germany), in conformance with the manufacturer's instructions. The concentration and purity of DNA were measured by means of a NanoDrop spectrophotometer (ND1000; Thermo Fisher Scientific GmbH, Dreieich, Germany).

Sequencing the molecular targets
The five T. bernardiae isolates were also investigated by sequencing the following molecular targets: 16S rRNA gene, superoxide dismutase A encoding gene sodA, and glyceraldehyde-3-phosphate dehydrogenase encoding gene gap. The sequences of the oligonucleotide primers and the PCR conditions were used as previously described for 16S rRNA gene ), gene sodA (Hijazin et al. 2011(Hijazin et al. , 2012c, and gene gap (Wickhorst et al. 2019). The PCR products were purified and sequenced by Eurofins Genomics Germany GmbH (Ebersberg, Germany). The obtained sequences of the different genes of the T. bernardiae isolates were aligned and further analyzed using the clustal w method of the MegAlign program version 15 (DNASTAR, Inc., Madison, WI, USA) and compared with the nucleotide sequences of the targets 16S rRNA gene, sodA, and gap of type strain T. bernardiae DSM 9152 T , and type strain of T. pyogenes DSM 20630 T obtained from the NCBI Gen-Bank, and for control purposes from A. haemolyticum DSM 20595 T also obtained from the NCBI GenBank.

Design of oligonucleotide primers for LAMP assay
Oligonucleotide primers for the T. bernardiae-specific LAMP assay were developed using the gyrase subunit A encoding gene gyrA of T. bernardiae (LNIZ01000002).

LAMP reaction and amplification conditions
In accordance with the manufacturer's instructions, the LAMP assay based on gene gyrA was carried out with the five T. bernardiae isolates, type strain T. bernardiae DSM 9152 T , and with control strains of genus Trueperella and closely related genus Arcanobacterium. A total volume of 25 µL for each reaction included 15 µL GspSSD isothermal master mix (ISO-001) (OptiGene Ltd., Horsham, UK) and 2.5 µL primer mix (ISO-001; OptiGene Ltd.), gyrA-F3 primer, and gyrA-B3 primer with a final concentration equivalent to 0.2 µmol/L, gyrA-FIP primer, and gyrA-BIP primer with final concentration equivalent to 0.8 µmol/L and gyrA-LoopF Primer and gyrA-LoopB Primer with a final concentration equivalent to 0.4 µmol/L. Subsequently, 5 µL DNA was added as a template. The LAMP assay was run at 70 °C for 20 min with a melting curve analysis step (annealing curve 98 to 80 °C ramping at 0.05 °C/s) in a real-time fluorometer GenieII® (OptiGene Ltd.).

Analytical sensitivity and specificity of the LAMP assay
Determination of the analytic sensitivity of the LAMP assay was performed seven times using a serially diluted DNA (10 −1 -10 −6 ) isolated from type strain T. bernardiae DSM 9152 T in AE buffer (10 mM Tris-Cl, 0.5 mM EDTA; pH 9.0) with the conditions mentioned above. DNA isolation and concentration were performed as stated above. The amount of DNA ranged from 3.0 ng/µL (10 −0 ) to 3.0 fg/µL (10 −6 ) bacterial DNA. The colony-forming unit (cfu/mL) was subsequently estimated.
The specificity of the LAMP assay was determined using the DNA of T. bernardiae DSM 9152 T and closely related species of genus Trueperella and Arcanobacterium. These

Results and discussion
The phenotypic properties of the five T. bernardiae isolates of duck origin investigated in the present study were almost identical to those of type strain T. bernardiae DSM 9152 T ,   All T. bernardiae isolates gave positive reactions for pyrazinamidase, pyrrolidonyl arylamidase, and α-glucosidase, and reacted negatively in nitrate reduction and for alkaline phosphatase, β-glucuronidase, β-galactosidase, and N-acetyl-βglucosaminidase. Also, all isolates did not hydrolyze esculin, urea, and gelatine. The isolates also fermented D-glucose, except T. bernardiae D14-1481-2029-1-1 and type strain T. bernardiae DSM 9152 T , D-ribose, D-maltose, and glycogen, but not D-xylose, D-mannitol, D-lactose, and D-saccharose.
In addition, all isolates showed a negative catalase reaction (Table 3).
With the additionally performed MALDI-TOF MS analysis, all five isolates were identified to the species level as T. bernardiae with log-score values varying between 1.87 and 2.2 (data not shown). MALDI-TOF MS appeared to be a fast, accurate, and less expensive tool for microbial identification of bacteria, viruses, and fungi (Singhal et al. 2015), also including T. bernardiae (Hijazin et al. 2012a) and various other species of genera Trueperella and Arcanobacterium (Hijazin et al. 2012a, b).
The additionally used T. bernardiae gyrA-specific LAMP assay could successfully be used to identify the speciesspecific gene gyrA of all five T. bernardiae isolates in the present investigation. This newly established assay demonstrated a specificity for T. bernardiae DSM 9152 T with an annealing temperature between 91.9 and 92.4 °C. No crossreactivity with any other related species of genus Trueperella or genus Arcanobacterium could be observed (Table 4).
The application of LAMP assays as being a rapid and reliable method for detecting species of genus Trueperella and Arcanobacterium has been previously published. In 2013, Zhang et al. developed a LAMP assay using the gene encoding pyolysin, the plo gene, for a specific identification   (Zhang et al. 2013). Furthermore, a pla LAMP assay was used for identifying A. pluranimalium (Abdulmawjood et al. 2015), and a cpn60 LAMP assay for identifying T. pyogenes from different animal origins (Abdulmawjood et al. 2016;Ahmed et al. 2020;Alssahen et al. 2020). The present study gives a reliable phenotypic and genotypic characterization of T. bernardiae of duck origin, also including a newly developed LAMP assay. To our knowledge, the study gives the first detailed characterization of this bacterial species isolated from Peking ducks. However, the pathogenic importance of T. bernardiae, which was partly isolated together with various other bacteria from apparently healthy animals, for the high mortality rate or joint infections of the Peking ducks at farm level remains unclear. The described LAMP assay might help to identify this bacterial species in future and might elucidate the role this species plays in human and animal infections. Funding Open Access funding enabled and organized by Projekt DEAL. This publication was supported by Deutsche Forschungsgemeinschaft and University of Veterinary Medicine Hannover, Foundation within the funding program Open Access Publishing.

Availability of data and materials
The data that support the findings of this study are available on NCBI's Genbank and are accessible through the accession numbers listed in the manuscript.

Declarations
Ethics approval and consent to participate This study did not require official or institutional ethical approval. The material was collected post-mortem and/or during routine diagnosis. According to competent authorities, this kind of research does not require ethics approval or general approval with respect to German law.

Conflict of interest The authors declare no competing interests.
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