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A Comparison of Genospecies of Clinical Isolates in the Acinetobacter Spp. Complex Obtained from Hospitalized Patients in Busan, Korea

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A Comparison of Genospecies of Clinical Isolates in the Acinetobacter Spp. Complex Obtained from Hospitalized Patients in Busan, Korea Biomedical Science Letters 2019, 25(1): 40 ~53 Original Article https://doi.org/10.15616/BSL.2019.25.1.40 eISSN : 2288-7415 A Comparison of Genospecies of Clinical Isolates in the Acinetobacter spp. Complex Obtained from Hospitalized Patients in Busan, Korea Gyu-Nam Park 1,*, Hye-Sook Kang 2,** , Hye-Ran Kim 3,** *, Bo-Kyung Jung 1,*, Do-Hee Kim 4,* and Kyung-Soo Chang 1, †,*** 1Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan, Busan 46252, Korea 2Department of Laboratory Medicine, Maryknoll Medical Center, Busan 48972, Korea 3Department of Clinical Laboratory Science, College of Health and Therapy, Daegu Haany University, Gyeongsangbuk-Do 38610, Korea 4Department of Laboratory Medicine, Busan Veterans Hospital, Busan 46996, Korea Of the Acinetobacter spp., A. baumannii (genospecies 2) is the most clinically significant in terms of hospital-acquired infections worldwide. It is difficult to perform Acinetobacter -related taxonomy using phenotypic characteristics and routine laboratory methods owing to clusters of closely related species. The ability to accurately identify Acinetobacter spp. is clinically important because antimicrobial susceptibility and clinical relevance differs significantly among the different genospecies. Based on the medical importance of pathogenic Acinetobacter spp., the distribution and characterization of Acinetobacter spp. isolates from 123 clinical samples was determined in the current study using four typically applied bacterial identification methods; partial rpoB gene sequencing, amplified rRNA gene restriction analysis (ARDRA) of the intergenic transcribed spacer (ITS) region of the 16 ~23S rRNA, the VITEK ® 2 system (an automated microbial identification system) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). A. baumannii isolates (74.8%, 92/123) were the most common species, A. nosocomialis (10.6%, 13/123) and A. pittii isolates (7.5%, 9/123) were second and third most common strains of the A. calcoaceticus -A. baumannii (ACB) complex, respectively. A. soli (5.0%, 6/123) was the most common species of the non-ACB complex. RpoB gene sequencing and ARDRA of the ITS region were demonstrated to lead to more accurate species identification than the other methods of analysis used in this study. These results suggest that the use of rpoB genotyping and ARDRA of the ITS region is useful for the species-level identification of Acinetobacter isolates. Key Words: Acinetobacter spp., Genomic species, RpoB genotyping, ARDRA of the ITS region, MALDI-TOF MS, Colony morphology dales and family Moraxellaceae . It comprises Gram-negative, INTRODUCTION non-motile, oxidase-negative, glucose non-fermenting, strictly aerobic and catalase-positive coccobacilli rods. To date, more The genus Acinetobacter belongs to the order Pseudomona- than 40 Acinetobacter species have been described. Of these, Received: December 18, 2018 / Revised: January 10, 2019 / Accepted: January 14, 2019 *Graduate student, ** Researcher, *** Professor. † Corresponding author: Kyung-Soo Chang. Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan, Busan 46252, Korea. Tel: +82-51-510-0565, Fax: +82-51-510-0568, e-mail: [email protected] ○C The Korean Society for Biomedical Laboratory Sciences. All rights reserved. ○CC 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. - 40 - A. baumannii is the most clinically significant because it is mass spectrometry (MALDI-TOF MS), a rapid, accurate, closely associated with nosocomial infections. It is also most and low-cost technique, is successfully utilized to achieve frequently found in clinical samples. A. baumannii is asso- microbial characterization and identification. This technology ciated with a wide range of infections, including bacteraemia, is applied to identify diverse microorganisms, including bac- meningitis, pneumonia, urinary tract infection, ventilator teria, fungi and viruses in clinical microbiology laboratories associated pneumonia, and wound infections. In recent years, (Croxatto et al., 2012; Dingle and Butler-Wu, 2013; Jeong infection due to other Acinetobacter spp. that belong to the et al., 2016). A. calcoaceticus -A. baumannii (ACB) complex (including A. The study objective was to compare the identification calcoaceticus , A. nosocomialis and A. pittii ) has raised major accuracy of four typically applied bacterial identification concerns worldwide (Vaneechoutte et al., 1995; Cisneros et methods; partial rpoB gene sequencing, ARDRA of the ITS al., 2002; Lee et al., 2011; Visca et al., 2011; Tien et al., 2012; region of the 16 ~23S rRNA gene, the VITEK ® 2 system (an Almasaudi, 2018). automated microbial identification system), and MALDI- The rapid and accurate identification of pathogens, and TOF MS. Although the epidemiology of pathogenic Acineto- in particular, Acinetobacter spp., is a clinically important bacter spp. has been described, there are few data on patho- objective in clinical microbiology. This is primarily because genic Acinetobacte r spp. in Korea. Motivated by on the different genospecies have differing biological characteristics medical importance of pathogenic Acinetobacter spp., an and pathogenicity, and this can be relevant when seeking to evaluation was performed in the current study of the iso- ensure treatment efficacy. However, identification at the in- lation frequency and characterization of Acinetobacter spp. dividual species level of Acinetobacter is difficult because clinical isolates from various clinical samples collected from Acinetobacter spp. are very similar phenotypically, while general hospitals in Busan, Korea, between 2013 and 2015. being closely related genetically. As a result, the traditional approaches used to for identify non-baumannii Acinetobacter MATERIALS AND METHODS species often result in the latter being falsely identified as A. Bacterial isolates baumannii . Therefore, the development of reliable identifi- cation methods has been the focus of recent studies. Several One hundred and twenty-three non-duplicate clinical iso- genotypic methods have been found to be effective when lates of Acinetobacter strains, which were collected from seeking to differentiate the Acinetobacter spp. isolates from two general hospitals in Busan, Korea (between March 2013 one another. Of the molecular techniques employed, sequ- and February 2015); 68 isolates from sputum (55.3%), 17 ence analysis of the 16 ~23S rRNA or rpoB genes is widely isolates from blood (13.8%), 14 isolates from urine (11.4%), used in clinical microbiology laboratories. However, these 7 isolates bronchial fluid (5.7%), 6 isolates from wound approaches are not sufficiently polymorphic to successfully (4.9%), 5 isolates from foley catheter (4.1%), and 3 isolates differentiate between the different Acinetobacter species. A from pus and ear, respectively (2.4%). All the isolates were number of genomic fingerprinting methods have been propo- identified as Acinetobacter spp. using an automated microbial sed for genospecies typing. Amplified rRNA gene restriction identification system (VITEK ®2; bioMerieux, Marcy l'Etoile, analysis (ARDRA) and amplified fragment length polymorp- France) utilized in conjunction with the VITEK ®2 GN-ID hism (AFLP) analysis are examples of useful genomic finger- card. Reference strain American Type Culture Collection printing techniques that are used for clinical identification. [ATCC] 19606 ( A. baumannii , genospecies 2) was purchased. However, these methods are laborious, especially when a The clinical isolates and reference strain were stored at large group of isolates is under consideration (Koeleman et -70 ℃ in a freezer prior to use. al., 1998; Cisneros et al., 2002; Lee et al., 2014; Wang et al., Cultures and isolation 2014). Matrix-assisted laser desorption/ionization time-of-flight The clinical isolates were recovered by using blood agar - 41 - Table 1. Primer pair used in the experiment for rpoB gene amplification Gene Sequence (5' to 3') Product size (bp) Reference Forward : TAC CGT AAA GAC TTG AAA GAA G rpoB 380 La Scola et al., 2006 Reverse : CAA CAC CTT TGT TCC CGT GA that containing 5% sheep blood. The agar plates were in- reference data in the Genbank database using Basic Local cubated in the dark at 37 ℃ under a humid conditions for Alignment Search Tool (BLAST) to determine species iden- 24 ~48 hrs. The plates were examined daily to search for the tification. appearance of pure colonies. The colonies were sub-cultured Amplified rRNA gene restriction analysis of the ITS on MacConkey agar for the pure isolation of Acinetobacter region isolates. ARDRA of the ITS region of the 16 ~23S rRNA was per- Bacterial genomic DNA extraction of the isolates formed to identify genospecies of the isolates. The sequences The clinical isolates were incubated in a brain-heart in- of the primers were 5'-TGG CTC AGA TTG AAC GCT fusion (BHI) broth at 37 ℃ for 18 hrs for genotyping. The GGC GGC-3' (forward) and 5'-TAC CTT GTT ACG ACT genomic DNA of each of the 123 Acinetobacter clinical TCA CCC CA-3' (reverse). isolates was extracted using the Accuprep ® Genomic DNA PCR was performed using the following optimum con- extraction kit (Bioneer, Daejeon, Korea).
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