M. Hijazin

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.

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.

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.

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.

Arcanobacterium phocisimile sp. nov., isolated from harbour seals.

A polyphasic taxonomic study was performed on two previously unidentified Arcanobacterium-like Gram-positive strains isolated from harbour seals. Comparative 16S rRNA gene sequencing showed that both bacteria belonged to the genus Arcanobacterium and were most closely related to Arcanobacterium haemolyticum CIP 103370(T) (98.4% 16S rRNA gene sequence similarity), A. canis P6775(T) (97.4%), A. phocae DSM 10002(T) (97.4%), A. pluranimalium M430/94/2(T) (95.7%) and A. hippocoleae CCUG 44697(T) (95.5%). The presence of the major menaquinone MK-9(H4) supported the affiliation of the isolates with the genus Arcanobacterium. The polar lipid profile consisted of major amounts of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, an unidentified phospholipid and two unidentified glycolipids. The major fatty acids were C16:0, C18:0, C18:1ω9c and summed feature 5 (comprising C18:2ω6,9c and/or anteiso-C18:0). Physiological and biochemical tests clearly distinguished the isolates from other members of the genus Arcanobacterium. Based on the common origin and various physiological properties comparable to those of A. phocae, it is proposed that the isolates are classified as members of a novel species with the name Arcanobacterium phocisimile sp. nov. The type strain is 2698(T) (=LMG 27073(T) =CCM 8430(T)).

Identification of Trueperella (Arcanobacterium) bernardiae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis and by species-specific PCR.

Trueperella (Arcanobacterium) bernardiae was first described as a probable opportunistic pathogen in 1987. It was among isolates from a variety of clinical specimens in the Centers for Disease Control and Prevention described as group 2 coryneform bacteria (Na’Was et al., 1987). It was finally classified in the genus Arcanobacterium by Ramos et al. (1997). However, Yassin et al. (2011) proposed that Arcanobacterium bernardiae together with Arcanobacterium abortisuis, Arcanobacterium pyogenes, Arcanobacterium bialowiezense and Arcanobacterium bonasi should be reclassified into the newly described genus Trueperella. T. bernardiae has been reported as causative agent of urinary tract infections (Ieven et al., 1996; Lepargneur et al., 1998), from a patient suffering from septic arthritis (Adderson et al., 1998), from necrotizing fasciitis (Clarke et al., 2010), from coinfection with Staphylococcus aureus from chronic osteitis (Bemer et al., 2009) and more recently from bacteraemia of a patient with a deep soft tissue infection (Weitzel et al., 2011). However, the latter could be identified by partial sequencing of the 16S rRNA gene but not by classical biochemical tests, indicating that additional species characteristics would assist a future identification of this bacterial species.

Arcanobacterium canis sp. nov., isolated from otitis externa of a dog, and emended description of the genus Arcanobacterium Collins et al. 1983 emend. Yassin et al. 2011.

A polyphasic taxonomic study was performed on an unidentified Arcanobacterium-like Gram-stain-positive bacterium isolated from otitis externa of a dog. Comparative 16S rRNA gene sequencing showed that the bacterium belonged to the genus Arcanobacterium and was most closely related to the type strains of Arcanobacterium haemolyticum (97.2 %), Arcanobacterium hippocoleae (96.5 %) and Arcanobacterium phocae (96.4 %). The presence of the major menaquinone MK-9(H(4)) supported the affiliation of this strain to the genus Arcanobacterium. The polar lipid profile contained the major lipids phosphatidylcholine, diphosphatidylglycerol, phosphatidylinositol mannoside and an unidentified phospholipid (PL2). Major fatty acids were C(14 : 0), C(16 : 0), C(18 : 0), C(18 : 1)ω9c and C(18 : 2)ω6,9c/anteiso-C(18 : 0) (detected as a summed feature). C(10 : 0) and C(12 : 0) were present in minor amounts. The results of physiological and biochemical testing clearly distinguished the unknown bacterium from other species of the genus Arcanobacterium. Based on these tests, it is proposed that the unknown bacterium should be classified in the novel species Arcanobacterium canis sp. nov. The type strain of Arcanobacterium canis is P6775(T) (= CCM 7958(T) = CCUG 61573(T) = CIP 110339(T)). An emended description of the genus Arcanobacterium is also provided.

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.

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.

Actinomyces weissii sp. nov., isolated from dogs.

Two Gram-positive, rod-shaped, non-spore-forming bacteria were isolated from the oral cavities of two dogs. On the basis of 16S rRNA gene sequence similarities both strains were shown to belong to the genus Actinomyces and were most closely related to Actinomyces bovis (97.3% and 97.5%, respectively). The polyamine profile of the two isolates and Actinomyces bovis DSM 43014(T) was composed of spermidine and spermine as the major components. Menaquinone MK-9 was the major compound in the quinone system of the two strains and Actinomyces bovis. The polar lipid profiles of strains 2298(T) and 4321 were almost identical, containing diphosphatidylglycerol as the major compound, and moderate to trace amounts of phosphatidylcholine, phosphatidylinositol, phosphatidylinositol-mannoside, phosphatidylglycerol and several unidentified lipids. A highly similar polar lipid profile was detected in Actinomyces bovis DSM 43014(T) supporting the affiliation of strains 2298(T) and 4321 to the genus Actinomyces. The typical major fatty acids were C(16:0), C(18:0) and C(18:1)ω9c. Fatty acids C(14:0) and C(18:2)ω6,9c were found in minor amounts. The results of physiological and biochemical analyses revealed clear differences between both strains and the most closely related species of the genus Actinomyces. Thus, strains 2298(T) and 4321 represent a novel species, for which the name Actinomyces weissii sp. nov., is proposed, with strain 2298(T) ( = CIP 110333(T) = LMG 26472(T) = CCM 7951(T) = CCUG 61299(T)) as the type strain.