Identification of Arcanobacterium pluranimalium by matrix-assisted laser desorption ionization-time of flight mass spectrometry and, as novel target, by sequencing pluranimaliumlysin encoding gene pla.

In the present study 13 Arcanobacterium pluranimalium strains isolated from various animal origin could successfully be identified phenotypically by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and genotypically by sequencing 16S rDNA and the pluranimaliumlysin encoding gene pla. The detection of mass spectra by MALDI-TOF MS and the novel genotypic approach using gene pla might help to identify A. pluranimalium in future and might elucidate the role this species plays in infections of animals.

Further characteristics of Arcanobacterium canis, a novel species of genus Arcanobacterium.

Comparable to previously conducted phenotypical and genotypical investigations characterizing Arcanobacterium canis, a newly described species with the type strain A. canis DSM 25104 isolated from an otitis externa of a dog, four additional A. canis strains isolated from infections of three dogs and one cat 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, 16S-23S rDNA intergenic spacer region, 23S rDNA, and the genes rpoB and gap. All four A. canis investigated in the present study were isolated from the infected animals together with several other bacterial species indicating that the pathogenic importance of A. canis remains unclear. However, the detection of peptidic spectra by MALDI-TOF MS and the presented phenotypic and genotypic approaches might help to identify A. canis in future and might elucidate the role this species plays in infections of dogs and cats.

First description of Trueperella (Arcanobacterium) bernardiae of animal origin.

In the present study a Trueperella (Arcanobacterium) bernardiae strain isolated from an anal swab of a three-day-old piglet could be identified phenotypically, by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and genotypically by sequencing the 16S rDNA, the 16S-23S rDNA intergenic spacer region (ISR) and by sequencing the superoxide dismutase A encoding gene sodA. The present study gives the first information about the presence of T. (A.) bernardiae in specimen of animals.

Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for species identification of bacteria of genera Arcanobacterium and Trueperella.

In the present study matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was evaluated for species identification of 98 bacteria previously classified phenotypically and genotypically to genera Arcanobacterium and Trueperella. Species identification was carried out by comparing the main spectra of each strain with the main spectra of reference strains of both genera and 3740 database entries included in the MALDI Biotyper 2.0 software package (Bruker Daltonik GmbH, Bremen, Germany). MALDI-TOF MS correctly identified (log (score) values ≥ 2.0) all investigated strains of the species A. (T.) bialowiezense (n=3), A. (T.) bonasi (n=7), A. haemolyticum (n=10), A. pluranimalium (n=1) and A. (T.) pyogenes (n=77). According to the present results MALDI-TOF MS had a comparable discriminating power than previously conducted tests on DNA level. Further studies with strains isolated from human infections would show the robustness of MALDI-TOF MS for identification of bacteria of these genera.

Genetic relatedness of methicillin-resistant Staphylococcus pseudintermedius (MRSP) isolated from a dog and the dog owner.

In the present study four methicillin-resistant Staphylococcus pseudintermedius (MRSP) strains isolated from a dog (n=3) and the anterior nares of the dog owner (n=1) were investigated by conventional and molecular methods. The species identity of the four S. pseudintermedius strains was confirmed by conventional methods, by PCR mediated amplification of S. intermedius/S. pseudintermedius specific segments of thermonuclease encoding gene nuc and by restriction fragment length polymorphism analysis of phosphoacetyltransferase encoding gene pta. Investigation of the four S. pseudintermedius for toxinogenic potential revealed that all four strains were positive for the exfoliative toxin encoding gene siet and the leukotoxin encoding genes lukS, lukF. The oxacillin and penicillin resistance of the four S. pseudintermedius strains could be determined by cultivation of the strains on oxacillin resistant screening agar base, ChromID MRSA Agar and Brilliance MRSA Agar and by multiplex PCR detecting the resistance genes mecA and blaZ. The genetic relatedness of the strains was studied by macrorestriction analysis of their chromosomal DNA using pulsed field gel electrophoresis (PFGE). According to PFGE all four S. pseudintermedius strains represent an identical bacterial clone indicating a cross transmission between the dog and the dog owner.

Molecular identification and further characterization of Arcanobacterium pyogenes isolated from bovine mastitis and from various other origins.

The present study was designed to identify phenotypically and genotypically 61 Arcanobacterium pyogenes isolated from bovine mastitis and from various other origins. The A. pyogenes isolates showed the typical cultural and biochemical properties of this species and displayed CAMP-like synergistic hemolytic activities with various indicator strains. The species identity could be confirmed genotypically by amplification and sequencing of the superoxide dismutase A encoding gene sodA of reference strains representing 8 species of genus Arcanobacterium and subsequent design of A. pyogenes sodA gene-specific oligonucleotide primer. The A. pyogenes sodA gene-specific oligonucleotide primer allowed, together with previously described A. pyogenes 16S-23S rDNA intergenic spacer region-specific oligonucleotide primer, a reliable molecular identification of all 61 A. pyogenes of various origins. The additionally performed PCR-mediated amplification of 5 known and putative virulence factor encoding genes revealed that 100, 20, 87, 75, and 98% of the A. pyogenes carried the genes plo, cbpA, nanH, nanP, and fimA, which allowed an individual strain characterization. This might help to elucidate the role the putative virulence factors play in bovine mastitis and in various other infections caused by this bacterial pathogen.

Characterization of Arcanobacterium abortisuis by phenotypic properties and by sequencing the 16S-23S rDNA intergenic spacer region.

The present study was designed to characterize phenotypically and genotypically nine Arcanobacterium abortisuis strains collected from specimen of pigs in a period of nine years. All nine A. abortisuis strains and A. abortisuis reference strain DSM 19515 displayed a synergistic hemolytic reaction with Staphylococcus aureus ß-hemolysin, Rhodococcus equi, and Arcanobacterium haemolyticum indicator strains and showed the typical biochemical properties of this species. The species identity could be confirmed by identification and sequencing of the 16S-23S rDNA intergenic spacer region (ISR), which appeared to be a useful target for genotypic characterization of this bacterial species. The A. abortisuis strains of the present study were isolated from specimen of pigs together with various other bacterial species indicating that the pathogenic importance of this newly described species remains to be elucidated.

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.

Identification of Arcanobacterium haemolyticum isolated from postcastrational complications of a horse.

An Arcanobacterium haemolyticum strain isolated from a postcastrational lesion of a horse was identified phenotypically and genotypically. The latter was performed by sequencing the 16S-23S rDNA intergenic spacer region (ISR), by amplification of the gene encoding A. haemolyticum phospholipase D, by amplification of A. haemolyticum specific parts of ISR-23S rDNA and by amplification of the newly described CAMP factor family protein encoding gene of A. haemolyticum. This indicates (as described previously for seven additional A. haemolyticum strains; Hassan et al. 2009) that A. haemolyticum seems to occur also in infections of horses.

Pentoxifylline is not a promising treatment for multiple sclerosis in progression phase.

Fourteen MS patients took pentoxifylline at varying doses for up to 24 months. In vitro production of tumor necrosis factor alpha was reduced in patients taking 2,400 to 3,200 mg/day of pentoxifylline for 12 weeks or more. Twelve of the 14 patients experienced worsening of the disease during the study according to clinical, MRI, or visual evoked potential criteria. These results provide no hint of efficacy for pentoxifylline as a treatment for MS in progression phase.