Identification of Mycobacterium species using reversed-phase high performance liquid chromatographic analysis of mycolic acid

Abstract

A method for the identification of Mycobacterium species using reversed-phase high performance liquid chromatography (RP-HPLC) was developed. The fingerprints library was constructed on the basis of RP-HPLC chromatograms of the mycolic acids derivatives from 49 Mycobacterium species cultures. Two inoculation loops of Mycobacterium, for fast growth with one week incubation or for slow growth with three weeks incubation, were saponified for 1 h and stocked at 4 degrees C. The mycolic acids from each culture of Mycobacterium species were acidified, extracted, derivatized, and analyzed by the RP-HPLC method. On the basis of the HPLC patterns of relative retention time and relative peak height, the identifications of Mycobacterium species were performed. This established method has a good precision of retention times with the relative standard deviations (RSDs) ranging from 0.13% to 1.07%. The mycolic acids fingerprints library of HPLC patterns was set up, including 49 species that were recorded in "Bergey's Manual of Systematic Bacteriology". Three patterns were observed from the chromatographic behaviors of mycolic acids derivatives, including single peak-cluster, double peak-clusters, triple and multiple peak-clusters. Forty-one species were successfully identified according to the different relative retention times of the peaks and the relative peak heights. The established method can identify Mycobacterium species with rapidity and high reliability.

Application of High Performance Liquid Chromatography for Identification of Mycobacterium spp

“…Authors performed some modifications in a CDC protocol for HPLC identification of MAs. Du et al [9] tested a column with different dimensions (15.0 cm × 4.6 mm, 5 µm) and also a different elution program (run time 30 min and 1.5 mL min -1 flow rate) from the CDC specifications [14]. However, they obtained chromatograms quite similar to those from the CDC protocol.…”

“…Different species can display distinct antibiotic resistances and require different prescriptions for treatment. For this reason it is important to identify Mycobacterium species in a rapidly and accurately way [8,9] Complex high-molecular-weight β-hydroxyl fatty acids with a 22-or 24-carbon alkyl chain at the α-position are structural characteristics of mycolic acids (MAs), a type of fatty acid found in the Mycobacterium spp. cell wall.…”