- Email: [email protected]
Characterization of Arcanobacterium abortisuis by phenotypic properties and by sequencing the 16S–23S rDNA intergenic spacer region
Veterinary Microbiology 148 (2011) 431–433
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
Characterization of Arcanobacterium abortisuis by phenotypic properties and by sequencing the 16S–23S rDNA intergenic spacer region ¨ lbegi-Mohyla a, A.A. Hassan b, M. Hijazin a, J. Alber a, C. La¨mmler a,*, A. Abdulmawjood c, H. U E. Prenger-Berninghoff d, R. Weiß d, M. Zscho¨ck e a
Institut fu¨r Pharmakologie und Toxikologie, Justus-Liebig-Universita¨t Gießen, Frankfurter Str. 107, 35392 Gießen, Germany GD Animal Health Service, Department of Bacteriology and Parasitology, Postbus 9, 7400 AA Deventer, The Netherlands Institut fu¨r Tiera¨rztliche Nahrungsmittelkunde, Justus-Liebig-Universita¨t Gießen, Frankfurter Str. 92, 35392 Gießen, Germany d Institut fu¨r Hygiene und Infektionskrankheiten der Tiere, Justus-Liebig-Universita¨t Gießen, Frankfurter Str. 85-91, 35392 Gießen, Germany e Landesbetrieb Hessisches Landeslabor, Schubertstr. 60, 35392 Gießen, Germany b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 March 2010 Received in revised form 23 August 2010 Accepted 30 August 2010
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 b-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. ß 2010 Elsevier B.V. All rights reserved.
Keywords: Arcanobacterium abortisuis Pig Phenotypic properties 16S–23S rDNA intergenic spacer region
1. Introduction At present genus Arcanobacterium (A) consist of nine species namely A. haemolyticum and A. bernardiae which generally cause infections in humans (Collins et al., 1982; Funke et al., 1995) and A. pyogenes, A. phocae, A. bonasi, A. bialowiezense, A. hippocoleae, A. pluranimalium and A. abortisuis which could mainly be recovered from infections of various animals (La¨mmler and Hartwigk, 1995; Ramos et al., 1997; Lawson et al., 2001; Hoyles et al., 2002; Lehnen et al., 2006; Azuma et al., 2009). In 2009, Azuma et al., characterized the novel species A. abortisuis. The species description was based on a single strain isolated from a placenta of a sow following an abortion. According to a
* Corresponding author. Tel.: +49 6419938406; fax: +49 6419938409. E-mail address: [email protected] (C. La¨mmler). 0378-1135/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2010.08.026
proposal of Yassin et al. (in press) A. abortisuis should be reclassified in the newly described genus Trueperella as T. abortisuis. The aim of the present study was to further characterize this novel species A. abortisuis by investigating nine A. abortisuis strains isolated from specimen of nine pigs over a period of nine years during routine microbiological diagnostic by phenotypic properties and by sequencing the 16S–23S rDNA intergenic spacer region (ISR). 2. Materials and methods 2.1. Bacterial cultures and phenotypic properties A total of 10 bacterial cultures were used in this study. The cultures included the reference strain A. abortisuis DSM 19515 and nine additional strains described in the present study. Further data about the origin of the nine strains are summarized in Table 1. The nine strains were investigated
H. U¨lbegi-Mohyla et al. / Veterinary Microbiology 148 (2011) 431–433
432
Table 1 Origin of the nine investigated strains, isolated from nine pigs. Year of isolation
Strain designation
Origin
Additionally isolated microorganisms
Additional information
1999 2000 2002
P7403 P8609 P7069
Vagina Vagina Vagina
Vaginitis, vaginal discharge No data available Vaginal discharge, fertility problems
2002 2004
P6572 1142
Vagina Kidney
2006 2006
5041 4257
Cervix Urine
2007 2007
815 3064
Vagina Vagina
Anhemolytic streptococci, Escherichia coli No data available Acinetobacter spp., Corynebacterium spp., E. coli, Flavobacterium spp. E. coli, Staphylococcus epidermidis a-Hemolytic streptococci., Acinetobacter spp., Branhamella spp., E. coli, Staphylococcus hyicus Enterococcus spp., E. coli a-Hemolytic streptococci, Acinetobacter spp., Bacillus spp., Corynebacterium spp., E. coli, Staphylococcus chromogenes a-Hemolytic streptococci, Bacillus spp., E. coli a-Hemolytic streptococci, Corynebacterium spp., E. coli, S. epidermidis
for cultural and biochemical properties and for CAMP-like ¨ lbegi-Mohyla activities as described (Hassan et al., 2009; U et al., 2009, 2010). 2.2. Genotypic properties For molecular identification the bacterial strains of the present study were investigated by amplification and sequencing of the ISR as described recently (Hassan et al., 2008, 2009). 3. Results and discussion All nine bacterial cultures investigated in the present study could be identified phenotypically and genotypically as A. abortisuis. The A. abortisuis strains, also including reference strain A. abortisuis DSM 19515, could be cultivated under aerobic conditions, microaerobic conditions in a candle jar and under anaerobic conditions and produced a narrow zone of complete hemolysis on sheep and rabbit blood agar. Cultivation of the bacteria under anaerobic conditions and by microaerobic conditions in a candle jar, compared to aerobic conditions, resulted in a slightly enhanced growth of the bacteria. However, according to the information given by Azuma et al. (2009) A. abortisuis is strictly anaerobic. The degree of hemolysis of the A. abortisuis strains and the reference strain A. abortisuis DSM 19515 on sheep blood agar appeared to be slightly enhanced after cultivation under anaerobic conditions. By determination of synergistic CAMP-like activities A. abortisuis displayed synergistic hemolytic reactions in close proximity of b-hemolytic Staphylococcus aureus, Rhodococcus equi and A. haemolyticum indicator strains. The synergistic and antagonistic hemolytic reactions of the remaining eight species of genus Arcanobacterium had ¨ lbegi-Mohyla et al., 2009). In been described in detail (U addition A. abortisuis did not cause liquefaction of Loeffler agar and showed no cross reaction with group G Streptococcus antisera. Both properties are well known for A. pyogenes (Bisping and Amtsberg, 1988; La¨mmler and Hartwigk, 1995). The biochemical properties of the A. abortisuis strains of the present study, determined with the Api–Coryne test system, with tablets containing various substrates and with methylumbelliferyl conjugated sub-
Vaginitis, vaginal discharge Male pig, respiratory problem, post mortem Vaginitis Vaginal discharge, urinary gravel Blood discharge from vagina Purulent discharge from vagina 3 days post partum
trates generally corresponded to the findings of Azuma et al. (2009) and are summarized in Table 2. In addition all A. abortisuis strains investigated appeared to be positive for DNase and negative for catalase, hyaluronidase and acetoin (Table 2). The result for catalase also confirmed the findings of Azuma et al. (2009). The nine A. abortisuis strains and A. abortisuis DSM 19515 could be identified genotypically by amplification of ISR and part of the 16S rDNA and 23S rDNA yielding for all 10 A. abortisuis strains an identical amplicon size of approximately 580 bp. According to Hassan et al. (2008) a comparable ISR-PCR of the other eight species of genus Arcanobacterium displayed amplicons with approximate Table 2 Biochemical properties of nine A. abortisuis strains and A. abortisuis DSM 19515 investigated in the present study. Biochemical properties
A. abortisuis (n = 9)
A. abortisuis DSM 19515
Nitrate reduction Pyrazinamidase Pyrrolidonyl arylamidase Alkaline phosphatase b-Glucuronidase b-Galactosidase a-Glucosidase N-acetyl-b-glucosaminidase Esculin (b-glucosidase) Urease Gelatine Fermentation of: Glucose Ribose Xylose Mannitol Maltose Lactose Saccharose Glycogen a-Mannosidase Catalase Hyaluronidase DNase Voges-Proskauer
+1 +(6*)1, (+)(3)1 1 ; +(1)2, (8)2
+1
1
1,2
1,2
1,2
+1,3 +1,3 +1,3
+1,3 +1,3 +1,3
1,3
1,3
+1
+1
1
1
1
1
+(8)1, (+)(1)1 +(3)1, (+)(2)1,
(4)1
+1 (+)1
1
+(5)1, (+)(1)1, +1 +(2)1, (+)(1)1, +(4)1, (5)1 +(7)1, (+)(1)1,
1
(3)1 (6)1 (1)1
1
+1 +1 +1 +1
2
+
2
+
+ = Positive reaction; (+) = weak reaction; = negative reaction; * = number of strains; 1 = Api–Coryne test system (Biomerieux); 2 = tablets containing substrates (Inverness Medical); 3 = 4-methylumbelliferyl conjugated substrates (Sigma).
[(Fig._1)TD$IG]
H. U¨lbegi-Mohyla et al. / Veterinary Microbiology 148 (2011) 431–433
433
3A. abortisuis 3046 (FN667634) 4A. abortisuis 4257 (FN667633)
A. abortisuis 1142 (FN667635) 5A. abortisuis 5041 (FN667632)
A. abortisuis 7069 (FN667628) 8A. abortisuis 815 (FN667629) 6A. abortisuis 6572 (FN667631) A. abortisuis DSM 19515 (FN667627) A
A. abortisuis P7403 (FN667630) A. abortisuis P8609 (FN667626) A. bernardiae DSM 9152 (EU194562) A. pyogenes DSM 20630 (EU194563) A. bonasi DSM 17163 (EU194570) A. bialowiezense DSM 17162 (EU194569) A. haemolyticum DSM 20595 (EU194564) A. phocae DSM 10003 (EU194566) A. phocae DSM 10002 (EU194565) A. pluranimalium DSM 13483 (EU194567) A. hippocoleae DSM 15539 (EU194568)
10.9 10
8
6
4
2
0
Nucleotide Substitutions (X100) Fig. 1. Dendrogram analysis of ISR sequences of the A. abortisuis strains of the present study and of various Arcanobacterium spp. obtained from NCBI GenBank.
sizes of 580–620 bp and an ISR sequence length between 333 and 380 bp. Sequencing ISR of the A. abortisuis strains of the present study revealed an ISR sequence length between 333 and 335 bp. A dendrogram analysis of the ISR sequences of the nine A. abortisuis strains, A. abortisuis DSM 19515 and the remaining eight species of genus Arcanobacterium is shown in Fig. 1. All nine A. abortisuis strains, isolated from the urogenital tract of pigs with varying clinical symptoms, were recovered together with various other bacteria indicating that the pathogenic importance of A. abortisuis remains questionable. None of the strains was isolated from an abort which was originally described by Azuma et al. (2009). However, the phenotypic and genotypic properties described in the present study might help to improve a future diagnostic of A. abortisuis and might elucidate the role this species plays in infections of pigs, other animals and possibly in humans. References Azuma, R., Murakami, S., Ogawa, A., Okada, Y., Miyazaki, S., Makino, T., 2009. Arcanobacterium abortisuis sp. nov., isolated from a placenta of a sow following an abortion. Int. J. Syst. Evol. Microbiol. 59, 1469–1473. Bisping, W., Amtsberg, G., 1988. Grampositive, sporenlose Sta¨bchen. In: Bisping, W., Amstberg, G. (Eds.), Farbatlas zur Diagnose bakterieller Infektionserreger der Tiere. Verlag Paul Parey, Berlin and Hamburg, pp. 45–48. Collins, M.D., Jones, D., Schofield, G.M., 1982. Reclassification of Corynebacterium haemolyticum (MacLean Liebow & Rosenberg) in the genus Arcanobacterium gen. nov. as Arcanobacterium haemolyticum nom. rev., comb. nov. J. Gen. Microbiol. 128, 1279–1281. Funke, G., Ramos, C.P., Ferna´ndez-Garayza´bal, J.F., Weiss, N., Collins, M.D., 1995. Description of human-derived centers for disease control Coryneform group 2 bacteria as Actinomyces bernardiae sp. nov. Int. J. Syst. Bacteriol. 45, 57–60. Hassan, A.A., Mohyla, H., Kanbar, T., Alber, J., La¨mmler, C., Abdulmawjood, A., Speck, S., Zscho¨ck, M., Weiss, R., 2008. Molecular identification of
Arcanobacterium bialowiezense and Arcanobacterium bonasi based on 16S-23S rRNA intergenic spacer region sequences. Vet. Microbiol. 130, 410–414. ¨ lbegi-Mohyla, H., Kanbar, T., Alber, J., La¨mmler, C., AbdulHassan, A.A., U mawjood, A., Zscho¨ck, M., Weiss, R., 2009. Phenotypic and genotypic characterization of Arcanobacterium haemolyticum isolates from infections of horses. J. Clin. Microbiol. 47, 124–128. Hoyles, L., Falsen, E., Foster, G., Rogerson, F., Collins, M.D., 2002. Arcanobacterium hippocoleae sp. nov., from the vagina of a horse. Int. J. Syst. Evol. Microbiol. 52, 617–619. Lawson, P.A., Falsen, E., Weiss, N., Collins, M.D., 2001. Arcanobacterium pluranimalium sp. nov., isolated from porpoise and deer. Int. J. Syst. Evol. Microbiol. 51, 55–59. La¨mmler, C., Hartwigk, H., 1995. Actinomyces pyogenes und Arcanobacterium haemolyticum. In: Blobel, H., Schließer, T. (Eds.), Handbuch der bakteriellen Infektionen bei Tieren, Band II/3. 2 edition. Gustav Fischer Verlag, Jena, Stuttgart, Germany, pp. 196–240. Lehnen, A., Busse, H.J., Fro¨lich, K., Krasinska, M., Ka¨mpfer, P., Speck, S., 2006. Arcanobacterium bialowiezense sp. nov., and Arcanobacterium bonasi sp. nov., isolated from the prepuce of European bison bulls (Bison bonasus) suffering from balanoposthitis, and emended description of the genus Arcanobacterium Collins et al., 1983. Int. J. Syst. Evol. Microbiol. 56, 861–866. Ramos, C.P., Foster, G., Collins, M.D., 1997. Phylogenetic analysis of the genus Actinomyces based on 16S rRNA gene sequences: description of Arcanobacterium phocae sp. nov., Arcanobacterium bernardiae comb. nov., and Arcanobacterium pyogenes comb. nov. Int. J. Syst. Bacteriol. 47, 46–53. ¨ Ulbegi-Mohyla, H., Hassan, A.A., Kanbar, T., Alber, J., La¨mmler, C., PrengerBerninghoff, E., Weiss, R., Siebert, U., Zscho¨ck, M., 2009. Synergistic and antagonistic hemolytic activities of bacteria of genus Arcanobacterium and CAMP-like hemolysis of Arcanobacterium phocae and Arcanobacterium haemolyticum with Psychrobacter phenylpyruvicus. Res. Vet. Sci. 87, 186–188. ¨ Ulbegi-Mohyla, H., Hassan, A.A., Alber, J., La¨mmler, C., Prenger-Berninghoff, E., Weiss, R., Zscho¨ck, M., 2010. Identification of Arcanobacterium pluranimalium isolated from of a dog by phenotypic properties and by PCR mediated characterization of various molecular targets. Vet. Microbiol. 142, 458–460. Yassin, A.F., Hupfer, H., Siering, C., Schumann, P., in press. Comparative chemotaxonomic and phylogenetic studies on the genus Arcanobacterium Collins et al., 1982 emend Lehnen et al., 2006 proposal for Trueperella gen. nov and emended description of the genus Arcanobacterium. Int. J. Syst. Evol. Microbiol.
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
Characterization of Arcanobacterium abortisuis by phenotypic properties and by sequencing the 16S–23S rDNA intergenic spacer region ¨ lbegi-Mohyla a, A.A. Hassan b, M. Hijazin a, J. Alber a, C. La¨mmler a,*, A. Abdulmawjood c, H. U E. Prenger-Berninghoff d, R. Weiß d, M. Zscho¨ck e a
Institut fu¨r Pharmakologie und Toxikologie, Justus-Liebig-Universita¨t Gießen, Frankfurter Str. 107, 35392 Gießen, Germany GD Animal Health Service, Department of Bacteriology and Parasitology, Postbus 9, 7400 AA Deventer, The Netherlands Institut fu¨r Tiera¨rztliche Nahrungsmittelkunde, Justus-Liebig-Universita¨t Gießen, Frankfurter Str. 92, 35392 Gießen, Germany d Institut fu¨r Hygiene und Infektionskrankheiten der Tiere, Justus-Liebig-Universita¨t Gießen, Frankfurter Str. 85-91, 35392 Gießen, Germany e Landesbetrieb Hessisches Landeslabor, Schubertstr. 60, 35392 Gießen, Germany b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 March 2010 Received in revised form 23 August 2010 Accepted 30 August 2010
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 b-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. ß 2010 Elsevier B.V. All rights reserved.
Keywords: Arcanobacterium abortisuis Pig Phenotypic properties 16S–23S rDNA intergenic spacer region
1. Introduction At present genus Arcanobacterium (A) consist of nine species namely A. haemolyticum and A. bernardiae which generally cause infections in humans (Collins et al., 1982; Funke et al., 1995) and A. pyogenes, A. phocae, A. bonasi, A. bialowiezense, A. hippocoleae, A. pluranimalium and A. abortisuis which could mainly be recovered from infections of various animals (La¨mmler and Hartwigk, 1995; Ramos et al., 1997; Lawson et al., 2001; Hoyles et al., 2002; Lehnen et al., 2006; Azuma et al., 2009). In 2009, Azuma et al., characterized the novel species A. abortisuis. The species description was based on a single strain isolated from a placenta of a sow following an abortion. According to a
* Corresponding author. Tel.: +49 6419938406; fax: +49 6419938409. E-mail address: [email protected] (C. La¨mmler). 0378-1135/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2010.08.026
proposal of Yassin et al. (in press) A. abortisuis should be reclassified in the newly described genus Trueperella as T. abortisuis. The aim of the present study was to further characterize this novel species A. abortisuis by investigating nine A. abortisuis strains isolated from specimen of nine pigs over a period of nine years during routine microbiological diagnostic by phenotypic properties and by sequencing the 16S–23S rDNA intergenic spacer region (ISR). 2. Materials and methods 2.1. Bacterial cultures and phenotypic properties A total of 10 bacterial cultures were used in this study. The cultures included the reference strain A. abortisuis DSM 19515 and nine additional strains described in the present study. Further data about the origin of the nine strains are summarized in Table 1. The nine strains were investigated
H. U¨lbegi-Mohyla et al. / Veterinary Microbiology 148 (2011) 431–433
432
Table 1 Origin of the nine investigated strains, isolated from nine pigs. Year of isolation
Strain designation
Origin
Additionally isolated microorganisms
Additional information
1999 2000 2002
P7403 P8609 P7069
Vagina Vagina Vagina
Vaginitis, vaginal discharge No data available Vaginal discharge, fertility problems
2002 2004
P6572 1142
Vagina Kidney
2006 2006
5041 4257
Cervix Urine
2007 2007
815 3064
Vagina Vagina
Anhemolytic streptococci, Escherichia coli No data available Acinetobacter spp., Corynebacterium spp., E. coli, Flavobacterium spp. E. coli, Staphylococcus epidermidis a-Hemolytic streptococci., Acinetobacter spp., Branhamella spp., E. coli, Staphylococcus hyicus Enterococcus spp., E. coli a-Hemolytic streptococci, Acinetobacter spp., Bacillus spp., Corynebacterium spp., E. coli, Staphylococcus chromogenes a-Hemolytic streptococci, Bacillus spp., E. coli a-Hemolytic streptococci, Corynebacterium spp., E. coli, S. epidermidis
for cultural and biochemical properties and for CAMP-like ¨ lbegi-Mohyla activities as described (Hassan et al., 2009; U et al., 2009, 2010). 2.2. Genotypic properties For molecular identification the bacterial strains of the present study were investigated by amplification and sequencing of the ISR as described recently (Hassan et al., 2008, 2009). 3. Results and discussion All nine bacterial cultures investigated in the present study could be identified phenotypically and genotypically as A. abortisuis. The A. abortisuis strains, also including reference strain A. abortisuis DSM 19515, could be cultivated under aerobic conditions, microaerobic conditions in a candle jar and under anaerobic conditions and produced a narrow zone of complete hemolysis on sheep and rabbit blood agar. Cultivation of the bacteria under anaerobic conditions and by microaerobic conditions in a candle jar, compared to aerobic conditions, resulted in a slightly enhanced growth of the bacteria. However, according to the information given by Azuma et al. (2009) A. abortisuis is strictly anaerobic. The degree of hemolysis of the A. abortisuis strains and the reference strain A. abortisuis DSM 19515 on sheep blood agar appeared to be slightly enhanced after cultivation under anaerobic conditions. By determination of synergistic CAMP-like activities A. abortisuis displayed synergistic hemolytic reactions in close proximity of b-hemolytic Staphylococcus aureus, Rhodococcus equi and A. haemolyticum indicator strains. The synergistic and antagonistic hemolytic reactions of the remaining eight species of genus Arcanobacterium had ¨ lbegi-Mohyla et al., 2009). In been described in detail (U addition A. abortisuis did not cause liquefaction of Loeffler agar and showed no cross reaction with group G Streptococcus antisera. Both properties are well known for A. pyogenes (Bisping and Amtsberg, 1988; La¨mmler and Hartwigk, 1995). The biochemical properties of the A. abortisuis strains of the present study, determined with the Api–Coryne test system, with tablets containing various substrates and with methylumbelliferyl conjugated sub-
Vaginitis, vaginal discharge Male pig, respiratory problem, post mortem Vaginitis Vaginal discharge, urinary gravel Blood discharge from vagina Purulent discharge from vagina 3 days post partum
trates generally corresponded to the findings of Azuma et al. (2009) and are summarized in Table 2. In addition all A. abortisuis strains investigated appeared to be positive for DNase and negative for catalase, hyaluronidase and acetoin (Table 2). The result for catalase also confirmed the findings of Azuma et al. (2009). The nine A. abortisuis strains and A. abortisuis DSM 19515 could be identified genotypically by amplification of ISR and part of the 16S rDNA and 23S rDNA yielding for all 10 A. abortisuis strains an identical amplicon size of approximately 580 bp. According to Hassan et al. (2008) a comparable ISR-PCR of the other eight species of genus Arcanobacterium displayed amplicons with approximate Table 2 Biochemical properties of nine A. abortisuis strains and A. abortisuis DSM 19515 investigated in the present study. Biochemical properties
A. abortisuis (n = 9)
A. abortisuis DSM 19515
Nitrate reduction Pyrazinamidase Pyrrolidonyl arylamidase Alkaline phosphatase b-Glucuronidase b-Galactosidase a-Glucosidase N-acetyl-b-glucosaminidase Esculin (b-glucosidase) Urease Gelatine Fermentation of: Glucose Ribose Xylose Mannitol Maltose Lactose Saccharose Glycogen a-Mannosidase Catalase Hyaluronidase DNase Voges-Proskauer
+1 +(6*)1, (+)(3)1 1 ; +(1)2, (8)2
+1
1
1,2
1,2
1,2
+1,3 +1,3 +1,3
+1,3 +1,3 +1,3
1,3
1,3
+1
+1
1
1
1
1
+(8)1, (+)(1)1 +(3)1, (+)(2)1,
(4)1
+1 (+)1
1
+(5)1, (+)(1)1, +1 +(2)1, (+)(1)1, +(4)1, (5)1 +(7)1, (+)(1)1,
1
(3)1 (6)1 (1)1
1
+1 +1 +1 +1
2
+
2
+
+ = Positive reaction; (+) = weak reaction; = negative reaction; * = number of strains; 1 = Api–Coryne test system (Biomerieux); 2 = tablets containing substrates (Inverness Medical); 3 = 4-methylumbelliferyl conjugated substrates (Sigma).
[(Fig._1)TD$IG]
H. U¨lbegi-Mohyla et al. / Veterinary Microbiology 148 (2011) 431–433
433
3A. abortisuis 3046 (FN667634) 4A. abortisuis 4257 (FN667633)
A. abortisuis 1142 (FN667635) 5A. abortisuis 5041 (FN667632)
A. abortisuis 7069 (FN667628) 8A. abortisuis 815 (FN667629) 6A. abortisuis 6572 (FN667631) A. abortisuis DSM 19515 (FN667627) A
A. abortisuis P7403 (FN667630) A. abortisuis P8609 (FN667626) A. bernardiae DSM 9152 (EU194562) A. pyogenes DSM 20630 (EU194563) A. bonasi DSM 17163 (EU194570) A. bialowiezense DSM 17162 (EU194569) A. haemolyticum DSM 20595 (EU194564) A. phocae DSM 10003 (EU194566) A. phocae DSM 10002 (EU194565) A. pluranimalium DSM 13483 (EU194567) A. hippocoleae DSM 15539 (EU194568)
10.9 10
8
6
4
2
0
Nucleotide Substitutions (X100) Fig. 1. Dendrogram analysis of ISR sequences of the A. abortisuis strains of the present study and of various Arcanobacterium spp. obtained from NCBI GenBank.
sizes of 580–620 bp and an ISR sequence length between 333 and 380 bp. Sequencing ISR of the A. abortisuis strains of the present study revealed an ISR sequence length between 333 and 335 bp. A dendrogram analysis of the ISR sequences of the nine A. abortisuis strains, A. abortisuis DSM 19515 and the remaining eight species of genus Arcanobacterium is shown in Fig. 1. All nine A. abortisuis strains, isolated from the urogenital tract of pigs with varying clinical symptoms, were recovered together with various other bacteria indicating that the pathogenic importance of A. abortisuis remains questionable. None of the strains was isolated from an abort which was originally described by Azuma et al. (2009). However, the phenotypic and genotypic properties described in the present study might help to improve a future diagnostic of A. abortisuis and might elucidate the role this species plays in infections of pigs, other animals and possibly in humans. References Azuma, R., Murakami, S., Ogawa, A., Okada, Y., Miyazaki, S., Makino, T., 2009. Arcanobacterium abortisuis sp. nov., isolated from a placenta of a sow following an abortion. Int. J. Syst. Evol. Microbiol. 59, 1469–1473. Bisping, W., Amtsberg, G., 1988. Grampositive, sporenlose Sta¨bchen. In: Bisping, W., Amstberg, G. (Eds.), Farbatlas zur Diagnose bakterieller Infektionserreger der Tiere. Verlag Paul Parey, Berlin and Hamburg, pp. 45–48. Collins, M.D., Jones, D., Schofield, G.M., 1982. Reclassification of Corynebacterium haemolyticum (MacLean Liebow & Rosenberg) in the genus Arcanobacterium gen. nov. as Arcanobacterium haemolyticum nom. rev., comb. nov. J. Gen. Microbiol. 128, 1279–1281. Funke, G., Ramos, C.P., Ferna´ndez-Garayza´bal, J.F., Weiss, N., Collins, M.D., 1995. Description of human-derived centers for disease control Coryneform group 2 bacteria as Actinomyces bernardiae sp. nov. Int. J. Syst. Bacteriol. 45, 57–60. Hassan, A.A., Mohyla, H., Kanbar, T., Alber, J., La¨mmler, C., Abdulmawjood, A., Speck, S., Zscho¨ck, M., Weiss, R., 2008. Molecular identification of
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