Different chromatographic methods to
1. Introduction
Since 1896, when Lehmann and Neumann described the bacterium responsible for causing tuberculosis and leprosy, about 150 species of
Some strategies employed to identify
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
In this review are presented the procedures to saponification, extraction (chloroform), derivatization (p-bromophenacyl), separation (C18 column and a gradient of methanol and methylene chloride) and detection (ultraviolet spectrophotometer) of MAs. Also is explored the importance of built a pattern chromatogram library for successful identification of clinical samples based on comparison of the relative retention times (RRT) of the chromatogram patterns with those obtained from reference strains and with those available in external databases. HPLC is necessary for separation of MAs due to their large size and complexity that requires the use of different columns and solvents. Initial methods required manual interpretation of chromatograms with eventual development of automated systems.
2. Mycobacterium species and mycolic acids
The analysis of lipid fractions has contributed significantly to the knowledge of
Structurally similar substances to MAs have been found in all mycobacterial species, with very few exceptions (e.g.,
3. High performance liquid chromatography methodology
Reverse-phase high-performance liquid chromatography of MAs esters has been demonstrated to be a rapid, reproducible, species-specific method for the identification of mycobacterial species. Also this method is relatively inexpensive and has been found to be more rapid alternative laboratory technique than the use of commercial nucleic acid probes [20]. Different methods have been developed for the detection of mycobacteria in clinical samples (e.g., blood, sputum) but they can also be applied to detection in other sources such as water [21] and milk [22]. Standard procedures to HPLC identification of mycobacterial species and most common steps used for different researchers are showed in the Figure 2 and Table 1, respectively.
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25% KOH in 50% ethanol | Chloroform | C18 | Chloroform (C) + methanol (M): 10 % C:90%M: 13 min 25% C:75%M: 14 min 70% C:30%M: 20 min Gradient elution: 0.6 mL/min |
p-bromophenacyl/UV (254nm) | [13] |
25% KOH in 50% methanol | Chloroform | C18 | Methanol (M) + methylene chloride (MC): 98% M:2% MC: Initial 80% M:20% MC:1 min 35% M:65% MC: 10 min 98% M:2% MC: 10.5 98% M:2% MC: 12 min Gradient elution: 2.5 mL/min |
p-bromophenacyl/UV (260nm) | [20] |
50% aqueous potassium hydroxide Autoclaved: 1 h /121 ºC |
Chloroform | C18 | Methanol (M) + methylene chloride (MC): 98% M:2% MC: Initial 85% M:15% MC:1 min 55% M:45% MC: 1.75 min 55% M:45% MC: 8.75 min 30% M:70% MC: 11.75 98% M:2% MC: 12 min Gradient elution: 2.5 mL/min. |
4-bromomethyl- 6,7-dimethoxycoumarin/ FL (Emission: 418nm; Excitation: 240-420nm) | [26] |
Methanolic potassium hydroxide | Chloroform | C18 | Methanol-methylene chloride Gradient elution |
p-bromophenacyl/UV (254nm) | [34] |
25% of KOH in 50% ethanol Autoclaved: 1 h /121 °C |
Chloroform | C18 | Methanol (M) + methylene chloride (MC): 98% M:2% MC: Initial 80% M:20% MC:1 min 35% M:65% MC: 10 min 98% M:2% MC: 10.5 98% M:2% MC: 12 min Gradient elution: 2.5 mL/min |
p-bromophenacyl /UV (254-260nm) | [35] |
25% KOH in a water methanol (1:1) mixture. Autoclaved: 1 h /121 °C. |
Chloroform | C18 | Not referenced | p-bromophenacyl /UV | [36] |
2 ml KOH 25% in 50% ethanol Autoclaved: 1 h /121 °C. |
Chloroform | C18 | Methanol (M) + methylene chloride (MC): 80% M:20% MC: Initial 60% M:40% MC:1 min 40% M:60% MC: 5.5 min 40% M:60% MC: 6 min 80% M:20% MC: 8 min Gradient elution: 3 mL/min |
p-bromophenacyl /UV (254nm) | [37] |
2 ml KOH 25% in methanol:H2O (v:v) Autoclaved: 1 h /121 °C, 15 psi |
Chloroform | C18 | Methanol (M) + methylene chloride (MC): 98% M:2% MC: Initial 80% M:20% MC:1 min 35% M:65% MC: 2 min 98% M:2% MC: 17.5 min 98% M:2% MC: 20 min Gradient elution: 2 mL/min |
p-bromophenacyl /UV (260nm) | [22] |
25% KOH in 50% ethanol | Chloroform | C18 | Chloroform (C) + methanol (M): 9 % C:91%M: Initial 70% C:30%M: 65 min Gradient elution: 2 mL/min |
p-bromophenacyl /UV (254nm) | [38] |
50% KOH in methanol solution | Chloroform | C18 | Methanol (M) + 2-propanol (P): 60 % M: 40% P: Initial 60 % M: 40% P: 3 min 6 % M: 94% P: 21 min 30 % M: 70% P: 25 min Gradient elution: 1.5 mL/min |
p-bromophenacyl /UV (260nm) | [25] |
3.1. Bacterial culture
HPLC still requires initial culture of isolates on solid medium before analysis. This can be problematic because the slow growth rate of mycobacteria delays full identification and leaves treating physicians with little useful information after the initial report of an acid-fast bacilli (AFB)–positive broth culture [23]. The identification is achieved when Mycobacteriae are grown under standardized culture medium conditions such as Lowenstein-Jensen (L-J) slant, which may be supplemented with additional growth factors for those strains of Mycobacteriae that are unable to grow on L-J. A carbol fuchsin/phenol or fluorochrome stain is performed to verify the presence of AFB. Another common solid medium used for mycobacterial species is the Middlebrook 7 H10 or 7 H11 at 35–37 °C. Currently available databases have been developed which incorporate mycobacterial species which require different growth conditions such as
3.2. Saponification
The autoclaving-saponification steps in the HPLC procedure is performed for two reasons: frees MAs and kills the mycobacteria, assuring laboratory safety. Also this step is important because it will determine the amount of MAs that will be extracted. MAs are covalently linked to the cell wall arabinogalactan matrix. Removal of the MAs requires saponification with potassium hydroxide (50 % w/v), which is often performed in an autoclave to accelerate the process and provide for the safety of laboratory personnel working with Biosafety Level III mycobacterial species. Once autoclaved, the organisms are killed by the procedure and the mycolic acids released from the cell wall [24]. A standard protocol for HPLC identification of mycobacteria of CDC suggest transfer 1–2 loops of bacterium culture to a glass tube (13 by 100 mm) and add 2 mL of methanolic saponification reagent (25% potassium hydroxide in 50% methanol). The tube is capped tightly, homogenised and autoclaved for 1 h at 121°C and 15 psi [14].
3.3. Extraction
MAs exist in the cell in two basic forms: covalently bound to the cell wall, and loosely associated with an insoluble matrix esterified to a variety of carbohydrate containing molecules. Treatment of intact cells with mixtures of chloroform and methanol is suitable for extracting the smaller quantity of non-covalently attached mycolate [16]. Once autoclaving has been completed, samples are cooled to room temperature, acidified, and extracted into chloroform. Free MAs are extracted by acidifying with 1.5 ml of a 50% solution of concentrated HCl and H20 (v/v) and 2 mL chloroform. The chloroform layer is dried under air at 80-100 °C, and 2 mg of potassium bicarbonate is added [14].
3.4. Derivatization
The preparation from extraction step is resuspended in 1.0 mL chloroform, and a derivatize reagent (
Some experiences make done using fluorescence-labeling compounds. According to Butler and Guthertz [6] these compounds evolved as a follow: 4-bromomethyl-7-methoxycoumarin (Br-Mmc), performed in TLC analysis for detection of picomolar amounts; 4-bromomethyl-7-acetoxycoumarin (Br-Mac), suggested for femtomolar detection and finally 4-bromomethyl-6,7-dimethoxycoumarin and 3-bromomethyl-7-methoxy-1,4-benzoxazin-2-1 and 4-bromomethyl-7-acetoxycoumarin. All fluorophores produced mycolic acid patterns similar to the patterns for
3.5. HPLC conditions
MAs are analyzed using a HPLC apparatus, in a gradient elution, and a UV detector set at 260 nm. Samples are separated in a C-18 reverse-phase column. The mobile phase is a mixture of methanol and methylene chloride in a flow rate of 2 mL min-1. 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. On the other hand, Figueiredo et al. [22] use a C-18 column 33% taller than those used in the CDC protocol (7.5 cm), increased the run time to 20 min. With these changes they observed a superior resolution in an adapted protocol and could be an alternative to discriminate between species with homologous HPLC chromatogram patterns. Special careful must be taken when manual injection is performed. Its is recommended cleaned the syringe at least five times with HPLC-grade methylene chloride and the injection loop be cleaned one time with 1 mL of the mobile phase solvent; it is also recommended that a blank injection be used between samples when the prior MAs signal is high [27].
4. Identification of mycobacteria species
There is a wide range of structures and also concentrations of types or classes of MAs (α, methoxy, keto, epoxy mycolates, etc.) among mycobacterial species. The HPLC methodology is unable to separate all the homologous series of MAs, and for this reason the chemical composition of the chromatogram components could not be precisely identified. Although the individual mycolate cannot be identified, this is not necessary for identification of mycobacteria, since a species-specific chromatographic pattern is generated [10, 28].
In order to identify unknown mycobacteria specimens using HPLC, the laboratory maintains chromatograms of mycobacteria commonly seen in the laboratory. HPLC profiles of unknown mycobacteria are compared to the patterns contained in this spectral library. The chromatographic pattern for each strain is examined for differences in the heights for pairs of peaks. HPLC patterns are grouped according to species, and the values calculated for each ratio are combined, sorted in numerical order, and examined for their ability to discriminate species, using the range of the relative standard deviation (RSD) of the absolute retention times (ART) and the relative retention times (RRT). RRTs are adjusted by comparison with external mycobacterial MA peaks [29]. The
The visual pattern recognition method employs only chromatographic criteria, although when available, other identification test results should be included in the decision-making processes. The initial step for identifying a species is determining the overall complexity and number of MA peak clusters. These clusters may consist of a few peaks or many peaks and are further defined as single-, double-, and triple-or multiple-peak clusters [6]. The amount of MA present is related to the amount of light emitted and the structure of the MA is related to the time of elution off the column. Pattern recognition is performed by visual comparison of sample results with MA patterns from reference species of known Mycobacteriae; however, a correct pattern of interpretation requires training. For that reason computer-assisted pattern recognition software, which utilizes retention time, peak width, and peak amount to provide a peak name which can then be compared to a library database, was developed [24].
5. Chromatogram profile database from Mycobacterium spp.
Due to the interpretation of chromatographic data can become tedious and time consuming for laboratories that process large numbers of samples, some studies recommend the construction of computer-based file (library) of
Figueiredo
5.1. Single-peak cluster patterns
Members of the MTC such as
5.2. Double-peak cluster patterns
5.3. Triple-peak cluster patterns
6. Application of high performance liquid chromatography
According to Figueiredo
7. Conclusion
HPLC procedure for MAs separation is a rapid, reproducible and easily way to
Acronyms
AFB=Acid-fast bacilli
ART=Absolute retention time
CDC=Centers for Disease Control and Prevention
CFU=Colony forming unit
DNA=Deoxyribonucleic acid
GC=Gas chromatography
HPLC=High-performance liquid chromatography
L-J=Lowenstein-Jensen
MAs=Mycolic acids
MTC=
NTM=Non-tuberculous mycobacteria
RRT=relative retention times
RSD=Relative standard deviation
SMIS=Sherlock Mycobacterial Identification System
TLC=Thin-layer chromatography
Acknowledgments
The authors thank the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (process no. E-26/103.003/2012, FAPERJ, Brazil) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (processes no. 311361/2013-7, 313917/2013-2 and 401922/2013-8, CNPq, Brazil) for the financial support.
References
- 1.
Brasil. Ministério da Saúde. Secretaria de Vigilância em Saúde. Manual Nacional de Vigilância Laboratorial da Tuberculose e Outras Micobactérias. In: Epidemiológica. V, editor. Brasilia2008. p. 436. - 2.
List of Prokaryotic names with Standing in Nomenclature-Genus Mycobacterium. [Internet]. 2011. Available from: http://www.bacterio.cict.fr/m/mycobacterium.htm. - 3.
WHO. Response to the global TB epidemic. Global Health Observatory (GHO). 2014. - 4.
Dye C. Global epidemiology of tuberculosis. The Lancet. 2006;367(9514):938-40. - 5.
Griffith DE, Aksamit T, Brown-Elliott BA, Catanzaro A, Daley C, Gordin F, et al. An Official ATS/IDSA Statement: Diagnosis, Treatment, and Prevention of Nontuberculous Mycobacterial Diseases. American Journal of Respiratory and Critical Care Medicine. 2007;175(4):367-416. - 6.
Butler WR, Guthertz LS. Mycolic Acid Analysis by High-Performance Liquid Chromatography for Identification of Mycobacterium Species. Clinical Microbiology Reviews. 2001;14(4):704-26. - 7.
Furlanetto LV, Figueiredo EES, Conte Júnior CA, Carvalho RCT, Silva FGS, Silva JT, et al. Use of complementary methods in post mortem inspection of carcasses with suspected bovine tuberculosis. Pesquisa Veterinária Brasileira. 2012;32:1138-44. - 8.
Furlanetto LV, Figueiredo EES, Conte Júnior CA, Silva FGS, Duarte RS, Silva JT, et al. Prevalence of bovine tuberculosis in herds and animals slaughtered in 2009 in the State of Mato Grosso, Brazil. Arquivo Brasileiro de Medicina Veterinária e Zootecnia. 2012;64:274-80. - 9.
Du R, Chen B, Guo L, Li Y, Xie J, G. W, et al. Identification of Mycobacterium species using reversed-phase high performance liquid chromatogram analysis of mycolic acid. Chinese Journal of Chromatography. 2008(26):234-9. - 10.
Butler WR, Jost KC, Kilburn JO. Identification of mycobacteria by high-performance liquid chromatography. Journal of Clinical Microbiology. 1991;29(11):2468-72. - 11.
Hagen SR, Thompson JD. Analysis of mycolic acids by high-performance liquid chromatography and fluorimetric detection implications for the identification of mycobacteria in clinical samples. Journal of Chromatography A. 1995;692(1–2):167-72. - 12.
Tiwari RP, Hattikudur NS, Bharmal RN, Kartikeyan S, Deshmukh NM, Bisen PS. Modern approaches to a rapid diagnosis of tuberculosis: Promises and challenges ahead. Tuberculosis. 2007;87(3):193-201. - 13.
Butler WR, Kilburn JO. Identification of major slowly growing pathogenic mycobacteria and Mycobacterium gordonae by high-performance liquid chromatography of their mycolic acids. Journal of Clinical Microbiology. 1988;26(1):50-3. - 14.
CDC. Standardized Method for HPLC Identification of Mycobacteria . Atlanta: U.S. Department of Health and Human Services. Public Health Service; 1996. p. 99. - 15.
Garza-González E, Guerrero-Olazarán M, Tijerina-Menchaca R, Viader-Salvadó JM. Determination of drug susceptibility of Mycobacterium tuberculosis through mycolic acid analysis. Journal of Clinical Microbiology. 1997;35(5):1287-9. - 16.
Barry Iii CE, Lee RE, Mdluli K, Sampson AE, Schroeder BG, Slayden RA, et al. Mycolic acids: structure, biosynthesis and physiological functions. Progress in Lipid Research. 1998;37(2–3):143-79. - 17.
Marrakchi H, Lanéelle M-A, Daffé M. Mycolic Acids: Structures, Biosynthesis, and Beyond. Chemistry & Biology. 2014;21(1):67-85. - 18.
Minnikin DE. Mycolic acids. In: Mukherjee KD, Weber N, editors. CRC Handbook of Chromatograpy: Analysis of Lipids. Boca Raton: WILEY-VCH Verlag; 1994. p. 339-48. - 19.
Rafidinarivo E, Lanéelle M-A, Montrozier H, Valero-Guillén P, Astola J, Luquin M, et al. Trafficking pathways of mycolic acids: structures, origin, mechanism of formation, and storage form of mycobacteric acids. Journal of Lipid Research. 2009;50(3):477-90. - 20.
Glickman SE, Kilburn JO, Butler WR, Ramos LS. Rapid identification of mycolic acid patterns of mycobacteria by high-performance liquid chromatography using pattern recognition software and a Mycobacterium library. Journal of Clinical Microbiology. 1994;32(3):740-5. - 21.
Galassi L, Donato R, Tortoli E, Burrini D, Santianni D, Dei R. Nontuberculous mycobacteria in hospital water systems: application of HPLC for identification of environmental mycobacteria . Journal of water health. 2003;1(3):133-9. - 22.
Figueiredo EES, Conte-Junior CA, Furlaneto LV, Silvestre FG, Duarte R, Lilenbaum W, et al. Molecular Techniques for Identification of Species of the Mycobacterium tuberculosis complex: The use of multiplex PCR and an adapted HPLC method for identification ofMycobacterium bovis and diagnosis of bovine tuberculosis. In: Pere-Joan C, editor. Understanding Tuberculosis-Global Experiences and Innovative Approaches to the Diagnosis. Rijeta: InTech-Open Access Publisher; 2012. p. 411-32. - 23.
Buchan BW, Riebe KM, Timke M, Kostrzewa M, Ledeboer NA. Comparison of MALDI-TOF MS With HPLC and Nucleic Acid Sequencing for the Identification of Mycobacterium Species in Cultures Using Solid Medium and Broth. American Journal of Clinical Pathology. 2014;141(1):25-34. - 24.
Parrish N, Riedel S. Cellular Fatty Acid-Based Microbial Identification and Antimicrobial Susceptibility Testing. In: Tang Y-W, Stratton CW, editors. Advanced Techniques in Diagnostic Microbiology: Springer US; 2013. p. 177-85. - 25.
Vilchèze C, Jacobs WR. Isolation and Analysis of Mycobacterium tuberculosis Mycolic Acids. Current Protocols in Microbiology: John Wiley & Sons, Inc.; 2005. - 26.
Jost KC, Dunbar DF, Barth SS, Headley VL, Elliott LB. Identification of Mycobacterium tuberculosis andM. avium complex directly from smear-positive sputum specimens and BACTEC 12B cultures by high-performance liquid chromatography with fluorescence detection and computer-driven pattern recognition models. Journal of Clinical Microbiology. 1995;33(5):1270-7. - 27.
Viader-Salvadó JM, Garza-González E, Valdez-Leal R, de los Angeles del Bosque-Moncayo M, Tijerina-Menchaca R, Guerrero-Olazarán M. Mycolic Acid Index Susceptibility Method for Mycobacterium tuberculosis . Journal of Clinical Microbiology. 2001;39(7):2642-5. - 28.
Butler WR, Thibert L, Kilburn JO. Identification of Mycobacterium avium complex strains and some similar species by high-performance liquid chromatography. Journal of Clinical Microbiology. 1992;30(10):2698-704. - 29.
CDC. Mycolic Acid Pattern Standards for HPLC Identification of Mycobacteria . Atlanta: U.S. Department of Health and Human Services. Public Health Service; 1999. p. 99. - 30.
Kellogg JA, Bankert DA, Withers GS, Sweimler W, Kiehn TE, Pfyffer GE. Application of the Sherlock Mycobacteria Identification System Using High-Performance Liquid Chromatography in a Clinical Laboratory. Journal of Clinical Microbiology. 2001;39(3):964-70. - 31.
Duarte RS, Lourenço MCS, Fonseca LdS, Leão SC, Amorim EdLT, Rocha ILL, et al. Epidemic of Postsurgical Infections Caused by Mycobacterium massiliense . Journal of Clinical Microbiology. 2009;47(7):2149-55. - 32.
Leao SC, Tortoli E, Viana-Niero C, Ueki SYM, Lima KVB, Lopes ML, et al. Characterization of Mycobacteria from a Major Brazilian Outbreak Suggests that Revision of the Taxonomic Status of Members of theMycobacterium chelonae-M. abscessus Group Is Needed. Journal of Clinical Microbiology. 2009;47(9):2691-8. - 33.
Mondragón-Barreto M, Vázquez-Chacón CA, Barrón-Rivero C, Acosta-Blanco P, Jost Jr KC, Balandrano S, et al. Comparison among three methods for mycobacteria identification. Salud Pública de México. 2000;42:484-9. - 34.
Thibert L, Lapierre S. Routine application of high-performance liquid chromatography for identification of mycobacteria . Journal of Clinical Microbiology. 1993;31(7):1759-63. - 35.
Tortoli E, Bartoloni A. High-performance liquid chromatography and identification of mycobacteria . Reviews in Medical Microbiology. 1996;7:207-19. - 36.
Jeong J, Kim S-R, Lee SH, Lim J-H, Choi JI, Park JS, et al. The Use of High Performance Liquid Chromatography to Speciate and Characterize the Epidemiology of Mycobacteria . Lab Medicine. 2011;42(10):612-7. - 37.
Floyd MM, Silcox VA, Jones WD, Butler WR, Kilburn JO. Separation of Mycobacterium bovis BCG from Mycobacterium tuberculosis andMycobacterium bovis by using high-performance liquid chromatography of mycolic acids. Journal of Clinical Microbiology. 1992;30(5):1327-30. - 38.
Aravindhan V, Sulochana S, Narayanan S, Paramasivan CN, Narayanan PR. dentification & differentiation of Mycobacterium avium &M. intracellulare by PCR-RFLP assay using the groES gene. Indian Journal of Medical Research. 2007;126:575-9.