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Jeong, Kim, Chang, and Lee: Identification of Mycobacteria Species by HPLC and Species Distribution during Five Years at Ulsan University Hospital
Original Article

Korean J Leg Med 2008;28(1):24-33.

Published online: 14 February 2008

DOI: https://doi.org/10.3343/kjlm.2008.28.1.24

Identification of Mycobacteria Species by HPLC and Species Distribution during Five Years at Ulsan University Hospital

Joseph Jeong, M.D.1, Sung-Ryul Kim, M.D.1, Chulhun L. Chang, M.D.2, Seon Ho Lee, M.D.1

1Department of Laboratory Medicine, Ulsan University College of Medicine, Ulsan, Korea

2Department of Laboratory Medicine, Pusan National University School of Medicine, Busan, Korea

Received 12 October 200723 October 2007       Accepted 7 November 2007

Copyright © 2008 by the 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

Background

Infections caused by mycobacteria have been significantly increasing. Due to the difficulty of making a decision about the pathogenicity of mycobacteria, species-level identification is very important for patients’ diagnosis and treatment. The purpose of this study was to identify mycobacteria species using a high performance liquid chromatography (HPLC) method and to provide an initial database for the distribution of mycobacteria in Korea.

Methods

Acid fast bacteria isolated from 3,107 clinical specimens were identified by mycolic acid analysis using HPLC. The HPLC patterns were compared with those of standard mycobacteria species.

Results

The HPLC patterns were divided into single, double, and triple cluster groups, each group comprising 9, 20, and 4 species, respectively. Mycobacteria and non-tuberculous mycobacteria (NTM) were identifies by HPLC at the rates of 99.5% and 95.6%, respectively. NTM was isolated in 12.4% of the mycobacteria positive specimens. This study also found that there were 20 different NTM species with the distribution of each species ranging from 0.3% to 15.9% of the total NTM. While the rate of NTM has been increasing in Korea, M. avium-intracellulare, M. fortuitum, and M. chelonae are relatively decreasing, and M. kansasii and M. gordonae are relatively increasing.

Conclusions

HPLC method was highly discriminative for the identification of NTM in clinical specimens.

Keywords: Mycobacteia, M. tuberculosis, Nontuberculous mycobacteria, HPLC

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Fig. 1.
Characteristic HPLC chromatograms of standard Mycobacterium species with single-cluster peak patterns. (A) M. asiaticum. (B) M. bovis. (C) M. gastri. (D) M. gordonae. (E) M. kansasii. (F) M. marinum. (G) M. szulgai. (H) M. trivial and (I) M. tuberculosis.
kjlm-28-24f1.tif
Fig. 2.
Characteristic HPLC chromatograms of standard Mycobacterium species with double-cluster peak patterns. (A) M. abscessus. (B) M. acapulsensis. (C) M. agri. (D) M. avium. (E) M. celatum. (F) M. chelonae. (G) M. diernhoferi. (H) M. flavescens. (I) M. fortuitum. (J) M. gilvum. (K) M. intracellulare. (L) M. mucogenicum. (M) M. nonchromogenicum. (N) M. peregrinum. (O) M. phlei. (P) M. porcinum. (Q) M. scrofulaceum. (R) M. smegmatis. (S) M. terrae and (T) M. xenopi.
kjlm-28-24f2.tif
Fig. 3.
Characteristic HPLC chromatograms of standard Mycobacterium species with triple-cluster peak patterns. (A) M. austroafricanum. (B) M. pulveris. (C) M. simiae and (D) M. vaccae.
kjlm-28-24f3.tif
Table 1.
Standard mycobacterial isolates used in this study
Mycobacterium species Strain number Mycobacterium species Strain number
M. abscessus ATCC 19977 M. marinum ATCC 927
M. acapulsensis KTCC 9501 M. mucogenicum ATCC 49650
M. agri ATCC 27406 M. nonchromogenicum ATCC 19530
M. asiaticum ATCC 25276 M. peregrinum ATCC 14467
M. austroafricanum ATCC 33464 M. phlei ATCC 354
M. avium ATCC 25291 M. porcinum KTCC 9517
M. bovis ATCC 19210 M. pulveris KTCC 9518
M. celatum ATCC 51131 M. scrofulaceum ATCC 19981
M. chelonae ATCC 35752 M. simiae ATCC 25275
M. diernhoferi KTCC 9506 M. smegmatis ATCC 21701
M. flavescens ATCC 14474 M. szulgai ATCC 35799
M. fortuitum ATCC 6841 M. terrae ATCC 15755
M. gastri ATCC 15754 M. triviale ATCC 23292
M. gilvum KTCC 9512 M. tuberculosis H37Rv ATCC 27294
M. gordonae ATCC 14470 M. vaccae ATCC 15483
M. intracellulare ATCC 13950 M. xenopi ATCC 19250
M. kansasii ATCC 12478    
Table 2.
Isolation rates of M. tuberculosis and nontuberculous mycobacteria in AFB culture positive specimens using HPLC method
M. tuberculosis Nontuberculous mycobacteria
Patients Specimens Patients Specimens
N (%) N (%) N (%) N (%)
1,141 (85.0) 2,723 (87.6) 201 (15.0) 384 (12.4)
Table 3.
Distribution of nontuberculous Mycobacterium species in clinical specimens
Specimen Sputum BA Urine Pleural fluid Skin tissue Lymph node Pus CSF GA BM JF Total N (%)
Mycobacterium species
M. kansasii (%) 39 (10.16) 9 (2.34) 0 (0) 5 (1.30) 0 (0) 6 (1.56) 1 (0.26) 1 (0.26) 0 (0) 0 (0) 0 (0) 61 (15.89)
M. intracellurare (%) 33 (8.59) 6 (1.56) 0 (0) 2 (0.52) 2 (0.52) 3 (0.78) 0 (0) 1 (0.26) 0 (0) 2 (0.21) 0 (0) 49 (12.76)
M. gordonae (%) 21 (5.47) 3 (0.78) 15 (3.91) 2 (0.52) 0 (0) 1 (0.26) 0 (0) 0 (0) 1 (0.26) 0 (0) 1 (0.26) 44 (11.46)
M. avium (%) 25 (6.51) 2 (0.52) 0 (0) 2 (0.52) 2 (0.52) 3 (0.78) 3 (0.78) 0 (0) 0 (0) 1 (0) 0 (0) 38 (9.90)
M. fortuitum (%) 24 (6.25) 1 (0.26) 2 (0.52) 3 (0.78) 1 (0.26) 1 (0.26) 1 (0.26) 0 (0) 1 (0.26) 0 (0) 0 (0) 34 (8.85)
M. abscessus (%) 19 (4.95) 2 (0.52) 0 (0) 2 (0.52) 0 (0) 0 (0) 0 (0) 2 (0.52) 0 (0) 0 (0) 0 (0) 25 (6.51)
M. peregrinum (%) 16 (4.17) 1 (0.26) 2 (0.52) 1 (0.26) 0 (0) 2 (0.52) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 22 (5.73)
M. szlugai (%) 14 (3.65) 0 (0) 0 (0) 0 (0) 0 (0) 1 (0.26) 2 (0.52) 2 (0.52) 0 (0) 0 (0) 0 (0) 19 (4.95)
M. marinum (%) 4 (1.04) 0 (0) 0 (0) 0 (0) 8 (2.08) 0 (0) 2 (0.52) 0 (0) 0 (0) 0 (0) 0 (0) 14 (3.65)
M. mucogenicum (%) 9 (2.34) 1 (0.26) 2 (0.52) 1 (0.26) 0 (0) 1 (0.26) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 14 (3.65)
M. xenopi (%) 2 (0.56) 0 (0) 0 (0) 0 (0) 8 (2.08) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 10 (2.60)
M. terrae (%) 6 (1.56) 1 (0.26) 0 (0) 1 (0.26) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 8 (2.08)
M. scrofulaceum (%) 5 (1.30) 1 (0.26) 0 (0) 1 (0.26) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 7 (1.82)
M. chelonae (%) 4 (1.04) 1 (0.26) 0 (0) 1 (0.26) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 6 (1.56)
M. triviale (%) 4 (1.04) 0 (0) 0 (0) 1 (0.26) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 5 (1.30)
M. simiae (%) 3 (0.78) 0 (0) 1 (0.26) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 4 (1.04)
M. acpulsensis (%) 2 (0.52) 0 (0) 1 (0.26) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 3 (0.78)
M. asiaticum (%) 2 (0.52) 0 (0) 1 (0.26) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 3 (0.78)
M. nonchromogenicum (%) 1 (0.26) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1 (0.26)
unclassified NTM (%) 11 (2.86) 1 (0.26) 1 (0.26) 1 (0.26) 1 (0.26) 1 (0.26) 0 (0) 0 (0) 1 (0.26) 0 (0) 0 (0) 17 (4.43)
Total 244 (63.54) 29 (7.55) 25 (6.51) 23 (5.99) 22 (5.73) 19 (4.95) 9 (2.34) 6 (1.56) 3 (0.78) 3 (0.78) 1 (0.26) 384 (100)

Abbreviations: BA, Bronchial aspirates; CSF, Cerebrospinal fluid; GA, Gastric aspirates; BM, Bone marrow; JF, Joint fluid.

Table 4.
Annual incidence of species comprising more than 5% of total nontuberculous mycobacteria
Years 2002 N 2003 N 2004 N 2005 N 2006 Total NN
Mycobacterium species
M. kansasii 12 12 14 11 12 61
M. intracellurare 9 10 9 9 12 49
M. gordonae 10 9 7 10 8 44
M. avium 7 8 8 9 7 38
M. fortuitum 6 6 8 7 7 34
M. abscessus 5 4 5 5 6 25
M. peregrinum 3 5 4 4 6 22
M. szlugai 5 4 2 4 4 19
Total 57 58 57 59 62 293
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