Mycolic acid analysis by high-performance liquid chromatography for identification of Mycobacterium species

Abstract

Mycobacterium tuberculosis is the etiologic agent of tuberculosis and can be accurately detected by laboratories using commercial genetic tests. Nontuberculosis mycobacteria (NTM) causing other mycobacterioses can be difficult to identify. The identification processes are confounded by an increasing diversity of newly characterized NTM species. The ubiquitous nature of NTM, combined with their potential to be opportunistic pathogens in immunocompromised as well as nonimmunodeficient patients, further complicates the problem of their identification. Since clinical case management varies depending on the etiologic agent, laboratories must identify the species in a timely manner. However, only a few identification methods can detect the species diversity within the Mycobacterium genus. Over the last decade, high-performance liquid chromatography analysis of the mycolic acids has become an accepted method for identification of mycobacteria. In this review, we assess its development and usefulness as an identification technique for Mycobacterium species.

FIG. 1

Representative HPLC system with dual pumps for gradient elution of solvents.

FIG. 2

Flowchart for isolation and detection of mycolic acids.

FIG. 3

Characteristic UV-HPLC chromatograms of Mycobacterium species with late-emerging, simple, single-cluster peak patterns. (A) M. asiaticum ATCC 25276^T; (B) M. bovis BCG Pasteur; (C) M. gastri ATCC 15754^T; (D) M. gordonae ATCC 14470^T, UV-HPLC chromotype I; (E) M. kansasii, ATCC 12478^T; (F) Mycobacterium leprae, ‘armadillo’; (G) M. tuberculosis complex (includes M. africanum ATCC 25420^T, M. bovis ATCC 19210^T, “M. canettii” NZM 217/94, M. caprae CIP 105776^T, M. microti ATCC 19422^T, and M. tuberculosis ATCC 27294^T); (H) M. szulgai ATCC 35799^T.

FIG. 3

Characteristic UV-HPLC chromatograms of Mycobacterium species with late-emerging, simple, single-cluster peak patterns. (A) M. asiaticum ATCC 25276^T; (B) M. bovis BCG Pasteur; (C) M. gastri ATCC 15754^T; (D) M. gordonae ATCC 14470^T, UV-HPLC chromotype I; (E) M. kansasii, ATCC 12478^T; (F) Mycobacterium leprae, ‘armadillo’; (G) M. tuberculosis complex (includes M. africanum ATCC 25420^T, M. bovis ATCC 19210^T, “M. canettii” NZM 217/94, M. caprae CIP 105776^T, M. microti ATCC 19422^T, and M. tuberculosis ATCC 27294^T); (H) M. szulgai ATCC 35799^T.

FIG. 4

Characteristic UV-HPLC chromatograms of Mycobacterium species with late-emerging, complex, single-cluster peak patterns. (A) Mycobacterium confluentis ATCC 49920^T; (B) M. haemophilum ATCC 29548^T; (C) M. intermedium ATCC 51848^T; (D) M. malmoense ATCC 29571^T; (E) M. marinum CDC 875; (F) M. thermoresistibile CDC 7305; (G) M. ulcerans ATCC 19423^T.

FIG. 4

Characteristic UV-HPLC chromatograms of Mycobacterium species with late-emerging, complex, single-cluster peak patterns. (A) Mycobacterium confluentis ATCC 49920^T; (B) M. haemophilum ATCC 29548^T; (C) M. intermedium ATCC 51848^T; (D) M. malmoense ATCC 29571^T; (E) M. marinum CDC 875; (F) M. thermoresistibile CDC 7305; (G) M. ulcerans ATCC 19423^T.

FIG. 5

Characteristic UV-HPLC chromatograms of Mycobacterium species with late-emerging, simple, single-cluster peak patterns with unresolved shoulder peaks. (A) Mycobacterium brumae ATCC 51384^T; (B) M. fallax ATCC 35219^T; (C) M. triviale ATCC 23292^T.

FIG. 5

Characteristic UV-HPLC chromatograms of Mycobacterium species with late-emerging, simple, single-cluster peak patterns with unresolved shoulder peaks. (A) Mycobacterium brumae ATCC 51384^T; (B) M. fallax ATCC 35219^T; (C) M. triviale ATCC 23292^T.

FIG. 6

Characteristic UV-HPLC chromatograms of Mycobacterium species with two-peak clusters that emerge late and close together. (A) Mycobacterium agri ATCC 27406^T; (B) M. chelonae ATCC 35751 (includes M. abscessus ATCC 19977^T); (C) Mycobacterium chitae ATCC 19629; (D) Mycobacterium farcinogenes ATCC 35753^T; (E) M. fortuitum complex (includes M. goodii ATCC 700504^T, M. peregrinum ATCC 14467^T, M. smegmatis ATCC 14468, and M. wolinskyi ATCC 700010^T); (F) M. porcinum, ATCC 33775^T; (G) M. senegalense ATCC 35796.

FIG. 6

Characteristic UV-HPLC chromatograms of Mycobacterium species with two-peak clusters that emerge late and close together. (A) Mycobacterium agri ATCC 27406^T; (B) M. chelonae ATCC 35751 (includes M. abscessus ATCC 19977^T); (C) Mycobacterium chitae ATCC 19629; (D) Mycobacterium farcinogenes ATCC 35753^T; (E) M. fortuitum complex (includes M. goodii ATCC 700504^T, M. peregrinum ATCC 14467^T, M. smegmatis ATCC 14468, and M. wolinskyi ATCC 700010^T); (F) M. porcinum, ATCC 33775^T; (G) M. senegalense ATCC 35796.

FIG. 7

Characteristic UV-HPLC chromatograms of Mycobacterium species that have widely separated, double-peak clusters with prominent peaks in the early cluster that emerge prior to 5.0 min. (A) Mycobacterium aichiense ATCC 27280^T; (B) Mycobacterium chlorophenolicum ATCC 49826^T; (C) Mycobacterium diernhoferi ATCC 19340^T; (D) M. flavescens ATCC 14474^T (underlined peaks are not present in all strains); (E) Mycobacterium gadium ATCC 27726^T; (F) Mycobacterium gilvum ATCC 43909; (G) M. hiberniae ATCC 49874^T; (H) Mycobacterium komossense ATCC 33013^T; (I) Mycobacterium madagascariense ATCC 49865^T; (J) M. mucogenicum ATCC 49651; (K) M. neoaurum ATCC 25795^T; (L) Mycobacterium phlei ATCC 11758^T; (M) Mycobacterium rhodesiae ATCC 27024^T; (N) Mycobacterium sphagni ATCC 33027.

FIG. 7

Characteristic UV-HPLC chromatograms of Mycobacterium species that have widely separated, double-peak clusters with prominent peaks in the early cluster that emerge prior to 5.0 min. (A) Mycobacterium aichiense ATCC 27280^T; (B) Mycobacterium chlorophenolicum ATCC 49826^T; (C) Mycobacterium diernhoferi ATCC 19340^T; (D) M. flavescens ATCC 14474^T (underlined peaks are not present in all strains); (E) Mycobacterium gadium ATCC 27726^T; (F) Mycobacterium gilvum ATCC 43909; (G) M. hiberniae ATCC 49874^T; (H) Mycobacterium komossense ATCC 33013^T; (I) Mycobacterium madagascariense ATCC 49865^T; (J) M. mucogenicum ATCC 49651; (K) M. neoaurum ATCC 25795^T; (L) Mycobacterium phlei ATCC 11758^T; (M) Mycobacterium rhodesiae ATCC 27024^T; (N) Mycobacterium sphagni ATCC 33027.

FIG. 7

Characteristic UV-HPLC chromatograms of Mycobacterium species that have widely separated, double-peak clusters with prominent peaks in the early cluster that emerge prior to 5.0 min. (A) Mycobacterium aichiense ATCC 27280^T; (B) Mycobacterium chlorophenolicum ATCC 49826^T; (C) Mycobacterium diernhoferi ATCC 19340^T; (D) M. flavescens ATCC 14474^T (underlined peaks are not present in all strains); (E) Mycobacterium gadium ATCC 27726^T; (F) Mycobacterium gilvum ATCC 43909; (G) M. hiberniae ATCC 49874^T; (H) Mycobacterium komossense ATCC 33013^T; (I) Mycobacterium madagascariense ATCC 49865^T; (J) M. mucogenicum ATCC 49651; (K) M. neoaurum ATCC 25795^T; (L) Mycobacterium phlei ATCC 11758^T; (M) Mycobacterium rhodesiae ATCC 27024^T; (N) Mycobacterium sphagni ATCC 33027.

FIG. 8

Characteristic UV-HPLC chromatograms of Mycobacterium species that have widely separated, double-peak clusters with prominent peaks in the early cluster that emerge after 5.0 min. (A) M. avium ATCC 19698^T; (B) Mycobacterium branderi ATCC 51789^T; (C) M. celatum ATCC 51131^T; (D) M. gordonae ATCC 35759 (UV-HPLC chromotype II); (E) M. interjectum ATCC 51457^T (underlined peaks are not present in all strains); (F) M. intracellulare ATCC 13950^T; (G) M. scrofulaceum ATCC 19981^T; (H) M. terrae ATCC 15755^T; (I) M. xenopi ATCC 19250.

FIG. 8

Characteristic UV-HPLC chromatograms of Mycobacterium species that have widely separated, double-peak clusters with prominent peaks in the early cluster that emerge after 5.0 min. (A) M. avium ATCC 19698^T; (B) Mycobacterium branderi ATCC 51789^T; (C) M. celatum ATCC 51131^T; (D) M. gordonae ATCC 35759 (UV-HPLC chromotype II); (E) M. interjectum ATCC 51457^T (underlined peaks are not present in all strains); (F) M. intracellulare ATCC 13950^T; (G) M. scrofulaceum ATCC 19981^T; (H) M. terrae ATCC 15755^T; (I) M. xenopi ATCC 19250.

FIG. 8

Characteristic UV-HPLC chromatograms of Mycobacterium species that have widely separated, double-peak clusters with prominent peaks in the early cluster that emerge after 5.0 min. (A) M. avium ATCC 19698^T; (B) Mycobacterium branderi ATCC 51789^T; (C) M. celatum ATCC 51131^T; (D) M. gordonae ATCC 35759 (UV-HPLC chromotype II); (E) M. interjectum ATCC 51457^T (underlined peaks are not present in all strains); (F) M. intracellulare ATCC 13950^T; (G) M. scrofulaceum ATCC 19981^T; (H) M. terrae ATCC 15755^T; (I) M. xenopi ATCC 19250.

FIG. 9

Characteristic UV-HPLC images of Mycobacterium species that have triple-peak clusters, close together that emerge later than 6.0 min. (A) M. genavense CDC 892120; (B) Mycobacterium lentiflavum 2186/92; (C) M. simiae ATCC 25275^T; (D) M. triplex ATCC 700071^T.

FIG. 10

Characteristic UV-HPLC chromatograms of Mycobacterium species that have triple-peak clusters with peaks in the early cluster that emerge before 5.0 min. (A) Mycobacterium alvei ATCC 51304^T; (B) Mycobacterium aurum ATCC 25793; (C) M. austroafricanum ATCC 33464^T; (D) Mycobacterium chubuense ATCC 27278^T; (E) Mycobacterium cookii ATCC 49103^T; (F) Mycobacterium duvalii ATCC 43910^T; (G) Mycobacterium obuense ATCC 27023^T; (H) Mycobacterium parafortuitum ATCC 19686^T; (I) M. shimoidei ATCC 27964; (J) M. tokaiense ATCC 27282^T; (K) M. vaccae ATCC 15483^T.

FIG. 10

Characteristic UV-HPLC chromatograms of Mycobacterium species that have triple-peak clusters with peaks in the early cluster that emerge before 5.0 min. (A) Mycobacterium alvei ATCC 51304^T; (B) Mycobacterium aurum ATCC 25793; (C) M. austroafricanum ATCC 33464^T; (D) Mycobacterium chubuense ATCC 27278^T; (E) Mycobacterium cookii ATCC 49103^T; (F) Mycobacterium duvalii ATCC 43910^T; (G) Mycobacterium obuense ATCC 27023^T; (H) Mycobacterium parafortuitum ATCC 19686^T; (I) M. shimoidei ATCC 27964; (J) M. tokaiense ATCC 27282^T; (K) M. vaccae ATCC 15483^T.

FIG. 10

Characteristic UV-HPLC chromatograms of Mycobacterium species that have triple-peak clusters with peaks in the early cluster that emerge before 5.0 min. (A) Mycobacterium alvei ATCC 51304^T; (B) Mycobacterium aurum ATCC 25793; (C) M. austroafricanum ATCC 33464^T; (D) Mycobacterium chubuense ATCC 27278^T; (E) Mycobacterium cookii ATCC 49103^T; (F) Mycobacterium duvalii ATCC 43910^T; (G) Mycobacterium obuense ATCC 27023^T; (H) Mycobacterium parafortuitum ATCC 19686^T; (I) M. shimoidei ATCC 27964; (J) M. tokaiense ATCC 27282^T; (K) M. vaccae ATCC 15483^T.