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Lim, Hong, Jo, Kim, and Chang: Genospecies Classification of Acinetobacter calcoaceticus-Acinetobacter baumannii Complex by Restriction Fragment Length Polymorphism
Original Article

J Bacteriol Virol 2011;41(1):37-45.

Published online: 18 January 2011

DOI: https://doi.org/10.4167/jbv.2011.41.1.37

Genospecies Classification of Acinetobacter calcoaceticus-Acinetobacter baumannii Complex by Restriction Fragment Length Polymorphism

Hyo Sun Lim, Hyo Jung Hong, Hyun Jung Jo, Do Hee Kim, Kyung Soo Chang∗

Department of Clinical Laboratory Science, Catholic University of Pusan, Busan, Korea

∗Corresponding author: Kyung Soo Chang. Department of Clinical Laboratory Science, Catholic University of Pusan, Busan 609-757, Korea. Phone: +82-51-510-0565, Fax: +82-51-510-0568 e-mail: kschang@cup.ac.kr

∗∗ This research was supported by the research fund of the Catholic University of Pusan.

      Revised 30 January 201115 February 2011       Accepted 18 February 2011

Copyright © 2011 Korean Society for Legal Medicine

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Acinetobacter calcoaceticus-Acinetobacter baumannii (A. calcoaceticus-A. baumannii) complex, which includes A. calcoaceticus (genospecies 1), A. baumannii (genospecies 2), Acinetobacter genospecies 3 and 13, has been identified as A. baumannii by automated bacteria identification system. The purpose of this study is to develop rapid genospecies classification of A. calcoaceticus-A. baumannii complex by molecular techniques. Random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) were determined for 4 reference strains and 80 isolates of A. calcoaceticus-A. baumannii complex from clinical sources. Four and eleven RAPD patterns were observed among the reference strains and the isolates, respectively. RAPD might be useful for genomic typing but not for genospecies classification of Acinetobacter spp. RFLP of 16S-23S rRNA intergenic spacer gene with three selected restriction enzymes (ApaLI, SwaI, and SalI) showed only four RFLP patterns in the reference and the isolates. Of 80 isolates, 10 of A. calcoaceticus (12.5%), 50 of A. baumannii (62.5%), 11 of A. genospecies 3 (13.75%), and 9 of A. genospecies 13 (11.25%) were classified by RFLP. This result suggests that RFLP of 16S-23S rRNA intergenic spacer gene of A. calcoaceticus-A. baumannii complex might be useful for genospecies classification.

Keywords: Genospecies classification, A. calcoaceticus-Acinetobacter baumannii complex, RAPD, RFLP, 16S-23S rRNA intergenic spacer gene

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Figure 1.
RAPD patterns of reference A. calcoaceticus-A. baumannii complex strains by PCR using the six RAPD primers. (A) RAPD patterns using RAPD primer 1, (B) RAPD patterns using RAPD primer 2, (C) RAPD patterns using RAPD primer 3, (D) RAPD patterns using RAPD primer 4, (E) RAPD patterns using RAPD primer 5, (F) using RAPD patterns using RAPD primer 6. M: marker, 2: A. baumannii (genospecies type 2), 1: A. calcoaceticus (genospecies type 1), 3: A. genospecies type 3, 13: A. genospecies type 13.
jbv-41-37f1.tif
Figure 2.
RAPD patterns of representative clinical A. calcoaceticus-A. baumannii complex isolates by PCR using the six RAPD primers. (A) RAPD patterns using RAPD primer 1, (B) RAPD patterns using RAPD primer 2, (C) RAPD patterns using RAPD primer 3, (D) RAPD patterns using RAPD primer 4, (E) RAPD patterns using RAPD primer 5, (F) using RAPD patterns using RAPD primer 6. M: marker, 2-2d: A. baumannii (genospecies 2) subtype 2, subtype 2a, subtype 2b, subtype 2c, subtype 2d, respectively, 1: A. calcoaceticus (genospecies 1) subtype 1, 3-3a: A. genospecies 3 subtype 3, subtype 3a, respectively, 13-13b: A. genospecies 13 subtype 13, subtype 13a, subtype 13b, respectively.
jbv-41-37f2.tif
Figure 3.
DNA fragments obtained by amplification of the 16S-23S rRNA intergenic spacer gene of A. baumannii and other bacteria. M: marker, 1: A. baumannii (genospecies type 2) 2: Staphylococcus aureus, 3: Streptococcus pyogenes, 4: Enteroccus faecalis, 5: Escherichia coli 0157, 6: Salmomella typhimurium, 7: Shigella dysenteriae.
jbv-41-37f3.tif
Figure 4.
RFLP patterns of the 16S-23S rRNA intergenic spacer gene of reference A. calcoaceticus-A. baumannii complex strains treated with selected restriction enzymes. (A) not treated with restriction enzyme, (B) treated with ApaLI, (C) treated with SwaI, (D) treated with SalI. M: marker, 2: A. baumannii (genospecies type 2), 1: A. calcoaceticus (genospecies type 1), 3: A. genospecies type 3, 13: A. genospecies type 13.
jbv-41-37f4.tif
Figure 5.
RFLP patterns of the 16S-23S rRNA intergenic spacer gene of representative clinical A. calcoaceticus-A. baumannii complex isolates with selected restriction enzymes. (A) not treated with restriction enzyme, (B) treated with ApaLI, (C) treated with SwaI, (D) treated with SalI. M: marker, 2-2d: A. baumannii (genospecies 2) subtype 2, subtype 2a, subtype 2b, subtype 2c, subtype 2d, respectively, 1: A. calcoaceticus (genospecies 1) subtype 1, 3-3a: A. genospecies 3 subtype 3, subtype 3a, respectively, 13-13b: A. genospecies 13 subtype 13, subtype 13a, subtype 13b, respectively.
jbv-41-37f5.tif
Table 1.
Sequences of the six primers for RAPD analysis
RAPD primers DNA sequences (5′ to 3′)
RAPD1 GGTGCGGGAA
RAPD2 GTTTCGCTCC
RAPD3 GTAGACCCGT
RAPD4 AAGAGCCCGT
RAPD5 AACGCGCAAC
RAPD6 CCCGTCAGCA
Table 2.
Sequences of primers to amplify 16S-23S rRNA intergenic spacer gene for RFLP assay
Primer names DNA sequences (5′ to 3′) Size
Spacer-1 TTG TAC ACA CCG CCC GTC A 989 bp
Spacer-2 GGT ACT TAG ATG TTT CAG TTC  
Table 3.
Genospecies classification of eighty clinical A. calcoaceticus-A. baumannii complex isolates by RFLP
A. genospecies No. of population Isolation frequency (%)
A. calcoaceticus 10 12.5
A. baumannii 50 62.5
A. genospecies 3 11 13.75
A. genospecies 13 9 11.25
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