- Email: [email protected]
Molecular identification of Arcanobacterium bialowiezense and Arcanobacterium bonasi based on 16S–23S rRNA intergenic spacer region sequences
Available online at www.sciencedirect.com
Veterinary Microbiology 130 (2008) 410–414 www.elsevier.com/locate/vetmic
Short communication
Molecular identification of Arcanobacterium bialowiezense and Arcanobacterium bonasi based on 16S–23S rRNA intergenic spacer region sequences A.A. Hassan a, H. Mohyla b, T. Kanbar b, J. Alber b, C. La¨mmler b,*, A. Abdulmawjood c, S. Speck d, M. Zscho¨ck a, R. Weiss e a Landesbetrieb Hessisches Landeslabor, Schubertstraße 60, 35392 Gießen, Germany Institut fu¨r Pharmakologie und Toxikologie, Justus-Liebig-Universita¨t Gießen, Frankfurter Straße 107, 35392 Gießen, Germany c Institut fu¨r Tiera¨rztliche Nahrungsmittelkunde, Justus-Liebig-Universita¨t Gießen, Frankfurter Straße 92, 35392 Gießen, Germany d Leibniz-Institut fu¨r Zoo- und Wildtierforschung, Alfred-Kowalke-Straße 17, P.O. Box 601103, 10252 Berlin, Germany e Institut fu¨r Hygiene und Infektionskrankheiten der Tiere, Justus-Liebig-Universita¨t Gießen, Frankfurter Straße 85-91, 35392 Gießen, Germany b
Received 25 January 2008; received in revised form 8 February 2008; accepted 14 February 2008
Abstract In the present study, the 16S–23S rDNA intergenic spacer region (ISR) of Arcanobacterium (A.) bialowiezense DSM 17162, A. bonasi DSM 17163, A. bernardiae DSM 9152, A. haemolyticum DSM 20595, A. hippocoleae DSM 15539, A. phocae DSM 10002, A. pluranimalium DSM 13483 and A. pyogenes DSM 20630 was amplified, sequenced and compared with the corresponding 16S rRNA gene sequences yielding comparable phylogenetic relationships. The ISR sequence of A. bialowiezense and A. bonasi allowed the design of species-specific oligonucleotide primers which could successfully be used for PCRmediated identification of previously characterized A. bialowiezense and A. bonasi isolated from infections of the European bison. The presented molecular identification might help to improve a future diagnosis of both newly described bacterial pathogens. # 2008 Elsevier B.V. All rights reserved. Keywords: Genus Arcanobacterium; Arcanobacterium bialowiezense; Arcanobacterium bonasi; 16S–23S DNA intergenic spacer region; Species-specific PCR
1. Introduction * Corresponding author. Tel.: +49 641 9938406; fax: +49 641 9938409. E-mail address: [email protected] (C. La¨mmler).
Initially, genus Arcanobacterium (A.) consisted of the single human pathogenic species A. haemolyticum, formerly designated as Corynebacterium haemolyticum (Collins et al., 1982). In the following years the
0378-1135/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2008.02.008
A.A. Hassan et al. / Veterinary Microbiology 130 (2008) 410–414
well known mainly animal pathogenic species Actinomyces pyogenes and human pathogenic Actinomyces bernardiae were reclassified to this genus as A. pyogenes and A. bernardiae and A. phocae, isolated from sea mammals, was described as new species (Ramos et al., 1997). In more recent phylogenetic studies four new members were assigned to this genus. These included A. pluranimalium, isolated from a dead harbour porpoise and a dead fallow deer (Lawson et al., 2001), A. hippocoleae, isolated from the vagina of a horse (Hoyles et al., 2002), and A. bialowiezense and A. bonasi, isolated from European bison bulls (Bison bonasus) suffering from balanoposthitis (Lehnen et al., 2006). The latter had been isolated from a chronic disease of the prepuce and penis of male European bisons on the Polish side of Bialowieza Forest, originally described by Kita et al. (1994). The taxonomic position of this two new Arcanobacterium species and their relation to the above-mentioned Arcanobacterium species was mainly based on sequence analysis of the 16S rRNA gene and chemotaxonomic characteristics. In the present study, sequence analyses were performed by comparatively investigating the 16S– 23S rDNA intergenic spacer region (ISR) of all eight Arcanobacterium species leading to the design of species-specific oligonucleotide primers allowing a PCR-mediated identification of the newly described species A. bialowiezense and A. bonasi.
2. Material and methods 2.1. Bacterial cultures and DNA extraction A total number of 18 bacterial cultures were used in this study. The cultures included the reference strains A. bialowiezense DSM 17162, A. bonasi DSM 17163, A. bernardiae DSM 9152, A. haemolyticum DSM 20595, A. hippocoleae DSM 15539, A. phocae DSM 10002, A. pluranimalium DSM 13483, A. pyogenes DSM 20630 and three A. bialowiezense and seven A. bonasi strains described previously (Lehnen et al., 2006). For DNA extraction, a single colony of each isolate was cultivated and incubated for 48 h on sheep blood agar. Three to five colonies of the freshly subcultured strain were subsequently suspended in 180 ml TE
411
buffer (10 mmol/l Tris–HCl, 1 mmol/l EDTA, pH 7.2) containing 5 ml mutanolysin (10 U/ml, Sigma, Taufkirchen, Germany). After incubation for 1 h at 37 8C, 25 ml proteinase K (Qiagen, Hilden, Germany) and 200 ml AL lysis buffer (Qiagen) was added and the suspension incubated for additional 2 h at 56 8C. The DNA was subsequently isolated by using DNeasy1 Tissue Kit according to the manufacturer’s instruction (Qiagen). 2.2. Sequence analysis of ISR The ISR of all eight Arcanobacterium reference strains was amplified using the oligonucleotide primers c (TTG TAC ACA CCG CCC GTC A) and b (GGT ACC TTA GAT GTT TCA GTT C). The primers were derived from conserved regions within the 16S rRNA (primer c) and the 23S rRNA (primer b) as described by Kostman et al. (1995) and Chanter et al. (1997). PCR amplification was performed with the following reaction mixture (30 ml): 1 ml (10 pmol/ ml) of each primer, 0.6 ml (10 mM) of dNTPs (MBI Fermentas, St. Leon-Rot, Germany), 3 ml GeneAmp 10 PCR Gold Buffer (150 mM Tris–HCl, 500 mM KCL, pH 8.0) (Applied Biosystem, Darmstadt, Germany), 1.8 ml MgCl2 (25 mM) (Applied Biosystem), 0.2 ml AmpliTaq Gold1 polymerase (5 U/ml, Applied Biosystem) and 19.9 ml sterile aqua dest. Finally, 2.5 ml DNA template was added to this reaction mixture. The PCR was carried out for 1 cycle at 95 8C for 10 min and then 30 cycles at 95 8C for 70 s, 45 8C for 70 s, and 72 8C for 70 s, followed by 1 cycle at 72 8C for 7 min using Biometra T3000 thermocycler (Biometra, Go¨ttingen, Germany) or Gene Amp PCR System 2400 (PerkinElmer, Rodgau Ju¨gesheim, Germany). The PCR products (8 ml) were mixed with 2 ml loading dye solution (MBI Fermentas) and separated by 2% agarose gel electrophoresis (Biozym, Hessisch-Oldendorf, Germany) at 120 V in 1 TBE buffer and a 100 bp DNA Molecular Weight Marker XIV (Roche Diagnostics, Mannheim, Germany) as molecular standard followed by staining for 5 min with 5 ml/ml ethidium bromide solution (Sigma). The amplicons were then visualized under a UV trans-illuminator (Biorad, Mu¨nchen, Germany). In parallel the PCR products were purified using a commercial PCR purification kit (QIAquick PCR Purification Kit) as recommended by the manufacturer
412
A.A. Hassan et al. / Veterinary Microbiology 130 (2008) 410–414
were synthesized by MWG Biotech (Ebersberg, Germany). The PCR reaction mixture was used as described above. The PCR program for A. bialowiezense was carried out for 1 cycle at 95 8C for 10 min and then 30 cycles at 95 8C for 10s, 59 8C for 10 s, and 72 8C for 15 s, followed by 1 cycle at 72 8C for 7 min. For A. bonasi the PCR was performed for 1 cycle at 95 8C for 10 min and then 30 cycles at 95 8C for 30 s, 54 8C for 30 s, and 72 8C for 30 s, followed by 1 cycle at 72 8C for 7 min.
(Qiagen). The purified DNA was sequenced by SEQLAB Sequence Laboratories (Go¨ttingen, Germany). The obtained sequences of ISR of the eight reference strains were aligned and further analyzed using the cluster method of MegAlign program (DNASTAR Inc., Madison, USA). 2.3. Analysis of 16S rRNA gene sequences of genus Arcanobacterium The nucleotide sequences of the 16S rRNA gene of all eight Arcanobacterium species were obtained from NCBI GenBank: A. bialowiezense (accession number AJ879696), A. bonasi (AJ879697), A. bernardiae (X79224), A. haemolyticum (AJ234059), A. hippocoleae (AJ300767), A. phocae (X97049), A. pluranimalium (AJ250959) and A. pyogenes (X79225). The gene sequences were aligned and analyzed using the cluster method of MegAlign program.
3. Results and discussion The oligonucleotide primers c and b used in the present investigation were originally described by Kostman et al. (1995) for amplification of ISR of different bacteria for molecular epidemiological analysis. The sequence diversity and unequal length of ISR obtained by PCR amplification with these primers were used for identification of Staphylococcus aureus, Enterococcus faecium, Escherichia coli and Enterobacter species (Kostman et al., 1995). In addition Chanter et al. (1997) and Hassan et al. (2003a) used these oligonucleotide primers for amplification of ISR of streptococci of various species. Species-specific oligonucleotide primers for PCR-mediated identification were designed from ISR of genus Streptococcus for S. agalactiae, S. uberis, S. parauberis, S. dysgalactiae, S. phocae and S. canis and of genus Staphylococcus for S. aureus, S. chromogenes, S. epidermidis, S. simulans, S. xylosus and S. hyicus (Forsman et al., 1997; Hassan et al., 2001, 2003a,b,c, 2008; Khan et al., 2003; Voytenko et al., 2006).
2.4. Species-specific PCR for A. bialowiezense and A. bonasi The A. bialowiezense and A. bonasi ISR-specific oligonucleotide primers were designed by comparison of internal parts of ISR of all eight Arcanobacterium reference strains with the help of OLIGO 4 primer analysis software (ver. 4.0). The forward primer sequence for A. bialowiezense was CAC CGT TGT GGC CCT CGA and the reverse sequence ACC ACC ACA AAC AGG CAG TAC G (expected size 138 bp). For A. bonasi the forward sequence was CCA CTG TTT CTT GCG CAT G and the reverse sequence ACA CAC AAC AAC ACA AAC CAT GTC (expected size 178 bp). All oligonucleotide primers used in this study Table 1 Similarity matrix of 16S–23S rRNA ISR sequences Arcanobacterium species
1 2 3 4 5 6 7 8
A. A. A. A. A. A. A. A.
bialowiezense DSM 17162 bonasi DSM 17163 haemolyticum DSM 20595 hippocoleae DSM 15539 phocae DSM 10002 pluranimalium DSM 13483 bernardiae DSM 9152 pyogenes DSM 20630
For accession numbers see Fig.1A.
ISR-PCR product (bp)
ISR sequence length (bp)
Percent similarity 8
7
6
5
4
3
2
1
580 600 600 600 620 580 580 580
399 425 419 431 441 405 401 395
77.8 78.0 76.3 75.9 77.8 75.3 84.2 100
77.2 78.7 76.0 73.0 75.6 74.4 100
73.3 71.1 78.3 76.2 80.3 100
74.3 73.2 78.1 69.9 100
72.2 70.3 71.7 100
74.3 71.9 100
79.6 100
100
A.A. Hassan et al. / Veterinary Microbiology 130 (2008) 410–414
413
Fig. 1. Dendrogram analysis of the ISR sequences of the present study (A) and 16S rDNA sequences obtained from NCBI GenBank (B).
In the present study, the oligonucleotide primers described by Kostman et al. (1995) could successfully be used for characterization of ISR of all eight Arcanobacterium species. The ISR-PCR displayed a variety of amplicons with approximate sizes of 580– 620 bp and an ISR sequence length between 395 and 441 bp. The relation between the ISR sequences of all eight Arcanobacterium species could be demonstrated by a similarity matrix and by dendrogram analysis. Comparing ISR and the 16S rRNA gene sequences by dendrogram analysis yielded comparable phylogenetic relationships (Table 1, Fig. 1 A and B). The ISR sequences of A. bialowiezense and A. bonasi could be used for the design of species-specific oligonucleotide primers. Both oligonucleotide primers could successfully be used for PCR-mediated amplification of ISR of both newly described Arcanobacterium species. These oligonucleotide primers allowed
Fig. 2. Typical PCR products of A. bialowiezense (1 and 2) and A. bonasi (4 and 5) using A. bialowiezense (1–3) and A. bonasi (4–6) ISR specific oligonucleotide primers, respectively. Negative reaction of A. bonasi (3) and A. bialowiezense (6). M = DNA Molecular Weight Marker XIV (Roche Diagnostics, Mannheim, Germany).
414
A.A. Hassan et al. / Veterinary Microbiology 130 (2008) 410–414
a molecular identification of A. bialowiezense and A. bonasi reference strains and confirmed the species identity of the ten previously characterized isolates of both species, respectively (Lehnen et al., 2006). Typical amplicons are shown in Fig. 2. The use of the species-specific oligonucleotide primers designed in the present study, might help to improve a future diagnosis of A. bialowiezense and A. bonasi and might elucidate the role both species play in infections of the European bison and possibly in other animal species.
References Chanter, N., Collin, N., Holmes, N., Binns, M., Mumford, J., 1997. Characterization of the Lancefield group C streptococcus 16S– 23S RNA gene intergenic spacer and its potential for identification and sub-specific typing. Epidemiol. Infect. 118, 125–135. 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. Forsman, P., Tilsala-Timisja¨rvi, A., Alatossava, T., 1997. Identification of staphylococcal and streptococcal causes of bovine mastitis using 16S–23S rRNA spacer regions. Microbiol. 143, 3491–3500. Hassan, A.A., Khan, I.U., Abdulmawjood, A., La¨mmler, C., 2001. Evaluation of PCR methods for rapid identification and differentiation of Streptococcus uberis and Streptococcus parauberis. J. Clin. Microbiol. 39, 1618–1621. Hassan, A.A., Khan, I.U., Abdulmawjood, A., La¨mmler, C., 2003a. Inter- and intra-species variations of the 16S–23S rDNA intergenic spacer region of various streptococcal species. Syst. Appl. Microbiol. 26, 97–103. Hassan, A.A., Khan, I.U., Abdulmawjood, A., La¨mmler, C., 2003b. Development of PCR assays for detection of Streptococcus canis. FEMS Microbiol. Lett. 219, 209–214. Hassan, A.A., Khan, I.U., La¨mmler, C., 2003c. Identification of Streptococcus dysgalactiae strains of Lancefield’s group C, G and L by polymerase chain reaction. J. Vet. Med. 50, 161–165.
Hassan, A.A., Vossen, A., La¨mmler, C., Siebert, U., Ferna´ndezGarayza´bal, J.F., 2008. PCR amplification of species specific sequences of 16S rDNA and 16S–23S rDNA intergenic spacer region for identification of Streptococcus phocae. Microbiol. Res. 163, 132–135. 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. Khan, I.U., Hassan, A.A., Abdulmawjood, A., La¨mmler, C., Wolter, W., Zscho¨ck, M., 2003. Identification and epidemiological characterization of Streptococcus uberis isolated from bovine mastitis using conventional and molecular methods. J. Vet. Sci. 4, 213–224. Kita, J., Dziaba, K., Piusinski, W., Kowalski, B., Lenartowicz, Z., Kingston, N., Drozdz, J., Rutkowska, M., Krasinski, Z., Krupa, J., Lesniewski, S., 1994. Preliminary studies on the diagnosis of a disease of the genital organs of male European bison in the Bialowieza Forest of Poland. IUCN Species survival commission. Vet. Spec. Gr. Newslett. 8, 8–9. Kostman, J.R., Alden, M.B., Mair, M., Edlind, T.D., LiPuma, J.J., Stull, T.L., 1995. A universal approach to bacterial molecular epidemiology by polymerase chain reaction ribotyping. J. Infect. Dis. 171, 204–208. Lawson, P.A., Falsen, E., Foster, G., Eriksson, E., Weiss, N., Collins, M.D., 2001. Arcanobacterium pluranimalium sp. nov., isolated from porpoise and deer. Int. J. Syst. Evol. Microbiol. 51, 55–59. 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. Voytenko, A.V., Kanbar, T., Alber, J., La¨mmler, C., Weiss, R., ¨ ., Hassan, Prenger-Berninghoff, E., Zscho¨ck, M., Akineden, O A.A., Dmitrenko, O.A., 2006. Identification of Staphylococcus hyicus by polymerase chain reaction mediated amplification of species specific sequences of superoxide dismutase A encoding gene sodA. Vet. Microbiol. 116, 211–216.
Veterinary Microbiology 130 (2008) 410–414 www.elsevier.com/locate/vetmic
Short communication
Molecular identification of Arcanobacterium bialowiezense and Arcanobacterium bonasi based on 16S–23S rRNA intergenic spacer region sequences A.A. Hassan a, H. Mohyla b, T. Kanbar b, J. Alber b, C. La¨mmler b,*, A. Abdulmawjood c, S. Speck d, M. Zscho¨ck a, R. Weiss e a Landesbetrieb Hessisches Landeslabor, Schubertstraße 60, 35392 Gießen, Germany Institut fu¨r Pharmakologie und Toxikologie, Justus-Liebig-Universita¨t Gießen, Frankfurter Straße 107, 35392 Gießen, Germany c Institut fu¨r Tiera¨rztliche Nahrungsmittelkunde, Justus-Liebig-Universita¨t Gießen, Frankfurter Straße 92, 35392 Gießen, Germany d Leibniz-Institut fu¨r Zoo- und Wildtierforschung, Alfred-Kowalke-Straße 17, P.O. Box 601103, 10252 Berlin, Germany e Institut fu¨r Hygiene und Infektionskrankheiten der Tiere, Justus-Liebig-Universita¨t Gießen, Frankfurter Straße 85-91, 35392 Gießen, Germany b
Received 25 January 2008; received in revised form 8 February 2008; accepted 14 February 2008
Abstract In the present study, the 16S–23S rDNA intergenic spacer region (ISR) of Arcanobacterium (A.) bialowiezense DSM 17162, A. bonasi DSM 17163, A. bernardiae DSM 9152, A. haemolyticum DSM 20595, A. hippocoleae DSM 15539, A. phocae DSM 10002, A. pluranimalium DSM 13483 and A. pyogenes DSM 20630 was amplified, sequenced and compared with the corresponding 16S rRNA gene sequences yielding comparable phylogenetic relationships. The ISR sequence of A. bialowiezense and A. bonasi allowed the design of species-specific oligonucleotide primers which could successfully be used for PCRmediated identification of previously characterized A. bialowiezense and A. bonasi isolated from infections of the European bison. The presented molecular identification might help to improve a future diagnosis of both newly described bacterial pathogens. # 2008 Elsevier B.V. All rights reserved. Keywords: Genus Arcanobacterium; Arcanobacterium bialowiezense; Arcanobacterium bonasi; 16S–23S DNA intergenic spacer region; Species-specific PCR
1. Introduction * Corresponding author. Tel.: +49 641 9938406; fax: +49 641 9938409. E-mail address: [email protected] (C. La¨mmler).
Initially, genus Arcanobacterium (A.) consisted of the single human pathogenic species A. haemolyticum, formerly designated as Corynebacterium haemolyticum (Collins et al., 1982). In the following years the
0378-1135/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2008.02.008
A.A. Hassan et al. / Veterinary Microbiology 130 (2008) 410–414
well known mainly animal pathogenic species Actinomyces pyogenes and human pathogenic Actinomyces bernardiae were reclassified to this genus as A. pyogenes and A. bernardiae and A. phocae, isolated from sea mammals, was described as new species (Ramos et al., 1997). In more recent phylogenetic studies four new members were assigned to this genus. These included A. pluranimalium, isolated from a dead harbour porpoise and a dead fallow deer (Lawson et al., 2001), A. hippocoleae, isolated from the vagina of a horse (Hoyles et al., 2002), and A. bialowiezense and A. bonasi, isolated from European bison bulls (Bison bonasus) suffering from balanoposthitis (Lehnen et al., 2006). The latter had been isolated from a chronic disease of the prepuce and penis of male European bisons on the Polish side of Bialowieza Forest, originally described by Kita et al. (1994). The taxonomic position of this two new Arcanobacterium species and their relation to the above-mentioned Arcanobacterium species was mainly based on sequence analysis of the 16S rRNA gene and chemotaxonomic characteristics. In the present study, sequence analyses were performed by comparatively investigating the 16S– 23S rDNA intergenic spacer region (ISR) of all eight Arcanobacterium species leading to the design of species-specific oligonucleotide primers allowing a PCR-mediated identification of the newly described species A. bialowiezense and A. bonasi.
2. Material and methods 2.1. Bacterial cultures and DNA extraction A total number of 18 bacterial cultures were used in this study. The cultures included the reference strains A. bialowiezense DSM 17162, A. bonasi DSM 17163, A. bernardiae DSM 9152, A. haemolyticum DSM 20595, A. hippocoleae DSM 15539, A. phocae DSM 10002, A. pluranimalium DSM 13483, A. pyogenes DSM 20630 and three A. bialowiezense and seven A. bonasi strains described previously (Lehnen et al., 2006). For DNA extraction, a single colony of each isolate was cultivated and incubated for 48 h on sheep blood agar. Three to five colonies of the freshly subcultured strain were subsequently suspended in 180 ml TE
411
buffer (10 mmol/l Tris–HCl, 1 mmol/l EDTA, pH 7.2) containing 5 ml mutanolysin (10 U/ml, Sigma, Taufkirchen, Germany). After incubation for 1 h at 37 8C, 25 ml proteinase K (Qiagen, Hilden, Germany) and 200 ml AL lysis buffer (Qiagen) was added and the suspension incubated for additional 2 h at 56 8C. The DNA was subsequently isolated by using DNeasy1 Tissue Kit according to the manufacturer’s instruction (Qiagen). 2.2. Sequence analysis of ISR The ISR of all eight Arcanobacterium reference strains was amplified using the oligonucleotide primers c (TTG TAC ACA CCG CCC GTC A) and b (GGT ACC TTA GAT GTT TCA GTT C). The primers were derived from conserved regions within the 16S rRNA (primer c) and the 23S rRNA (primer b) as described by Kostman et al. (1995) and Chanter et al. (1997). PCR amplification was performed with the following reaction mixture (30 ml): 1 ml (10 pmol/ ml) of each primer, 0.6 ml (10 mM) of dNTPs (MBI Fermentas, St. Leon-Rot, Germany), 3 ml GeneAmp 10 PCR Gold Buffer (150 mM Tris–HCl, 500 mM KCL, pH 8.0) (Applied Biosystem, Darmstadt, Germany), 1.8 ml MgCl2 (25 mM) (Applied Biosystem), 0.2 ml AmpliTaq Gold1 polymerase (5 U/ml, Applied Biosystem) and 19.9 ml sterile aqua dest. Finally, 2.5 ml DNA template was added to this reaction mixture. The PCR was carried out for 1 cycle at 95 8C for 10 min and then 30 cycles at 95 8C for 70 s, 45 8C for 70 s, and 72 8C for 70 s, followed by 1 cycle at 72 8C for 7 min using Biometra T3000 thermocycler (Biometra, Go¨ttingen, Germany) or Gene Amp PCR System 2400 (PerkinElmer, Rodgau Ju¨gesheim, Germany). The PCR products (8 ml) were mixed with 2 ml loading dye solution (MBI Fermentas) and separated by 2% agarose gel electrophoresis (Biozym, Hessisch-Oldendorf, Germany) at 120 V in 1 TBE buffer and a 100 bp DNA Molecular Weight Marker XIV (Roche Diagnostics, Mannheim, Germany) as molecular standard followed by staining for 5 min with 5 ml/ml ethidium bromide solution (Sigma). The amplicons were then visualized under a UV trans-illuminator (Biorad, Mu¨nchen, Germany). In parallel the PCR products were purified using a commercial PCR purification kit (QIAquick PCR Purification Kit) as recommended by the manufacturer
412
A.A. Hassan et al. / Veterinary Microbiology 130 (2008) 410–414
were synthesized by MWG Biotech (Ebersberg, Germany). The PCR reaction mixture was used as described above. The PCR program for A. bialowiezense was carried out for 1 cycle at 95 8C for 10 min and then 30 cycles at 95 8C for 10s, 59 8C for 10 s, and 72 8C for 15 s, followed by 1 cycle at 72 8C for 7 min. For A. bonasi the PCR was performed for 1 cycle at 95 8C for 10 min and then 30 cycles at 95 8C for 30 s, 54 8C for 30 s, and 72 8C for 30 s, followed by 1 cycle at 72 8C for 7 min.
(Qiagen). The purified DNA was sequenced by SEQLAB Sequence Laboratories (Go¨ttingen, Germany). The obtained sequences of ISR of the eight reference strains were aligned and further analyzed using the cluster method of MegAlign program (DNASTAR Inc., Madison, USA). 2.3. Analysis of 16S rRNA gene sequences of genus Arcanobacterium The nucleotide sequences of the 16S rRNA gene of all eight Arcanobacterium species were obtained from NCBI GenBank: A. bialowiezense (accession number AJ879696), A. bonasi (AJ879697), A. bernardiae (X79224), A. haemolyticum (AJ234059), A. hippocoleae (AJ300767), A. phocae (X97049), A. pluranimalium (AJ250959) and A. pyogenes (X79225). The gene sequences were aligned and analyzed using the cluster method of MegAlign program.
3. Results and discussion The oligonucleotide primers c and b used in the present investigation were originally described by Kostman et al. (1995) for amplification of ISR of different bacteria for molecular epidemiological analysis. The sequence diversity and unequal length of ISR obtained by PCR amplification with these primers were used for identification of Staphylococcus aureus, Enterococcus faecium, Escherichia coli and Enterobacter species (Kostman et al., 1995). In addition Chanter et al. (1997) and Hassan et al. (2003a) used these oligonucleotide primers for amplification of ISR of streptococci of various species. Species-specific oligonucleotide primers for PCR-mediated identification were designed from ISR of genus Streptococcus for S. agalactiae, S. uberis, S. parauberis, S. dysgalactiae, S. phocae and S. canis and of genus Staphylococcus for S. aureus, S. chromogenes, S. epidermidis, S. simulans, S. xylosus and S. hyicus (Forsman et al., 1997; Hassan et al., 2001, 2003a,b,c, 2008; Khan et al., 2003; Voytenko et al., 2006).
2.4. Species-specific PCR for A. bialowiezense and A. bonasi The A. bialowiezense and A. bonasi ISR-specific oligonucleotide primers were designed by comparison of internal parts of ISR of all eight Arcanobacterium reference strains with the help of OLIGO 4 primer analysis software (ver. 4.0). The forward primer sequence for A. bialowiezense was CAC CGT TGT GGC CCT CGA and the reverse sequence ACC ACC ACA AAC AGG CAG TAC G (expected size 138 bp). For A. bonasi the forward sequence was CCA CTG TTT CTT GCG CAT G and the reverse sequence ACA CAC AAC AAC ACA AAC CAT GTC (expected size 178 bp). All oligonucleotide primers used in this study Table 1 Similarity matrix of 16S–23S rRNA ISR sequences Arcanobacterium species
1 2 3 4 5 6 7 8
A. A. A. A. A. A. A. A.
bialowiezense DSM 17162 bonasi DSM 17163 haemolyticum DSM 20595 hippocoleae DSM 15539 phocae DSM 10002 pluranimalium DSM 13483 bernardiae DSM 9152 pyogenes DSM 20630
For accession numbers see Fig.1A.
ISR-PCR product (bp)
ISR sequence length (bp)
Percent similarity 8
7
6
5
4
3
2
1
580 600 600 600 620 580 580 580
399 425 419 431 441 405 401 395
77.8 78.0 76.3 75.9 77.8 75.3 84.2 100
77.2 78.7 76.0 73.0 75.6 74.4 100
73.3 71.1 78.3 76.2 80.3 100
74.3 73.2 78.1 69.9 100
72.2 70.3 71.7 100
74.3 71.9 100
79.6 100
100
A.A. Hassan et al. / Veterinary Microbiology 130 (2008) 410–414
413
Fig. 1. Dendrogram analysis of the ISR sequences of the present study (A) and 16S rDNA sequences obtained from NCBI GenBank (B).
In the present study, the oligonucleotide primers described by Kostman et al. (1995) could successfully be used for characterization of ISR of all eight Arcanobacterium species. The ISR-PCR displayed a variety of amplicons with approximate sizes of 580– 620 bp and an ISR sequence length between 395 and 441 bp. The relation between the ISR sequences of all eight Arcanobacterium species could be demonstrated by a similarity matrix and by dendrogram analysis. Comparing ISR and the 16S rRNA gene sequences by dendrogram analysis yielded comparable phylogenetic relationships (Table 1, Fig. 1 A and B). The ISR sequences of A. bialowiezense and A. bonasi could be used for the design of species-specific oligonucleotide primers. Both oligonucleotide primers could successfully be used for PCR-mediated amplification of ISR of both newly described Arcanobacterium species. These oligonucleotide primers allowed
Fig. 2. Typical PCR products of A. bialowiezense (1 and 2) and A. bonasi (4 and 5) using A. bialowiezense (1–3) and A. bonasi (4–6) ISR specific oligonucleotide primers, respectively. Negative reaction of A. bonasi (3) and A. bialowiezense (6). M = DNA Molecular Weight Marker XIV (Roche Diagnostics, Mannheim, Germany).
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a molecular identification of A. bialowiezense and A. bonasi reference strains and confirmed the species identity of the ten previously characterized isolates of both species, respectively (Lehnen et al., 2006). Typical amplicons are shown in Fig. 2. The use of the species-specific oligonucleotide primers designed in the present study, might help to improve a future diagnosis of A. bialowiezense and A. bonasi and might elucidate the role both species play in infections of the European bison and possibly in other animal species.
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