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Identification & differentiation of Mycobacterium avium & M. intracellulare by PCR-RFLP assay using the groES gene.

Background & objectives: We report a new polymerase chain reaction (PCR)--restriction fragment length polymorphism (RFLP) assay using mycobacterial groES as a target to identify Mycobacterium avium and M. intracellulare in clinical samples.

Methods: The assay was standardized using M. avium and M. intracellulare standard strains obtained from ATCC and was tested with 45 M. avium-M, intracellulare complex (MAC) clinical isolates (Of which 31 were from [HIV.sup.+] individuals). The standard and clinical strains were typed with HPLC based mycolic acid fingerprinting.

Results: Three polymorphisms (BamHI, BstNI and HgaI) were identified for inter-species differentiation among standard strains; of which, only HgaI was found to be useful in clinical isolates. Of the 45 isolates, 25 were M. avium and 20 were M. intracelluare. MAC isolates, which could not be differentiated by HPLC analysis, were also typed by this method.

Interpretation & conclusions: The use of mycobacterial groES as a PCR-RFLP target for M. avium and M. intracellulare is a simple and rapid method that can complement HPLC in their differentiation.

Key words groES--HPLC--MAC--Mycobacterium avium--M. intracellulare--PCR-RFLP

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Mycobacterium avium--M. intracellulare complex (MAC) comprises two genetically distinct but difficult to discriminate species namely M. avium, which predominates (87-98%) in AIDS patients with no antiretroviral therapy and M. intracellulare, which is more frequent among non-AIDS patients (1). Identifying clinical isolates as M. avium and M. intracellulare would have both clinical and epidemiological implications. MAC strains occasionally cause human disease that is indistinguishable from tuberculosis. The organisms infect lung, lymph nodes, skin, bones, gastrointestinal and genitourinary tracts. Conventional techniques fail to discriminate M. avium and M. intracellulare. The assays used to identify these two species include both genotypic methods like polymerase chain reaction (PCR) (mig), PCR with restriction fragment length polymorphism (PCR-RFLP) (hsp65), probe hybridization and sequencing (16S rRNA) and phenotypic methods like high performance liquid chromatography (HPLC) and serotyping (2). In general, genotypic assays have proved far superior to conventional methods in terms of specificity, sensitivity and rapidity (3,4). Of the several genotypic assays, PCR-RFLP analysis offers an easy, rapid and an inexpensive way to identify several mycobacterial species (5).

The groESL operon that encodes 10 kDa (GroES) and 60 kDa (GroEL) heat shock proteins is ubiquitous and evolutionarily conserved among bacteria (6). The Hsp65 (GroEL2) gene of mycobacteria is well documented for its utility in species identification (7,8). But the Hsp10 (groES) gene (Rv3418c), though well conserved like the Hsp65 gene, has not been utilized for species identification, probably due to its small size (9). Despite its highly conserved amino acid sequence, immunodominant species-specific T- and B-cell epitopes have been identified (10-12) and have been utilized for immunodiagnosis (13). In the present study, we attempted M. avium and M. intracellulare species differentiation using groES-based PCR-RFLP assay. This assay was also validated using MAC clinical isolates obtained from HIV positive individuals.

Material & Methods

Bacterial strains: All the reference strains of mycobacteria (M. avium- ATCC no. 25291, 43015 and M. intracellulare--ATCC no. 23434, 23435 and 23436) were from American Tissue Culture Collection (ATCC), USA. The MAC clinical isolates from HIV patients and from non HIV patients (n=45) were collected from the Department of Bacteriology, Tuberculosis Research Centre (ICMR), Chennai. Species identification by high performance liquid chromatography (HPLC) and HIV testing were also carried out at the same centre. Of the 45 MAC clinical isolates, 32 were from HIV positive subjects. The study was conducted during 2002-2005 and most of the samples were collected during this period and a few were from the archival collection.

PCR amplification: DNA extraction was carried out following the standard Cetyl trimethyl ammonium bromide (CTAB)-NaCl method (14, 15). Mycobacterial groES specific primers (MAF- 5' C C T T G A G T A C TAGCACTCTC A T G 3'; MAR-5' A T C A G C T T G C T C A T C A G G C T C C 3') (Invitrogen, USA) were used for PCR amplification (16). PCR reaction was carried out in a final volume of 25 [micro]l that contains 5-10ng DNA, 20pM each of forward and reverse primers, 1.5mM Mg[Cl.sub.2]; 125 [micro]M dNTPs, 2.5U Taq polymerase (Amersham, Singapore), 10mM Tris-HCl (pH 9.0) and 50mM KCl. The PCR conditions were: 5 min initial denaturation at 95[degrees]C followed by 40 cycles of 1 min denaturation at 95[degrees]C, 1 min annealing at 55[degrees]C, 1 min extension at 72[degrees]C which is followed by a final extension at 72[degrees]C for 5 min. PCR products were visualized on 2 per cent agarose gel.

PCR-RFLP: Equal quantity of PCR products were digested for 3h with BamHI, BstNI or HgaI (New England Biolabs, USA) at 37[degrees]C for BamHI and HgaI and 60[degrees]C for BstNI following manufactures instruction. The digested products were visualized on a 2 per cent agarose gel.

HPLC analysis: The use of HPLC to separate the bromophenacyl esters of mycobacterial mycolic acids was as described by Butler et al (15). HPLC was conducted on a Beckman model 330 liquid chromatograph (Beckman Instruments, Inc., Berkeley, CA) equipped with an Altex/Hitachi model 155 UV detector for measuring A254, two Beckman model 110A solvent pumps, and a model 420 pump controller. The mycolic acid p-bromophenacyl esters (5-p.l samples) were applied to a 5-ri. particle size, C 18 reverse-phase column (4.6 nun by 25 cm) equilibrated in 91 per cent methanol-9 per cent chloroform. After injection of the sample, the gradient was changed linearly to 30 per cent methanol-70 per cent chloroform over a period of 65 min at a total flow rate of 2 ml/min.

Results & Discussion

PCR was attempted using conserved primers for groES, as reported by Cobb and Forthinham (16) on two M. avium and three M. intracellulare standard strains obtained from ATCC. This yielded a single band of 600bp in both species. The PCR products were gel purified and sequenced (data not shown). Sequence analysis revealed the presence of BamHI, BstNI (both sites present in M. avium and absent in M. intracellulare) and HgaI (absent in M. avium and present in M. intracellulare) polymorphisms which could be used for PCR-RFLP analysis. The PCR products were digested with BamHI, BstNI or HgaI. BamHI digestion yielded a 400bp and 200bp doublet in M. avium and an undigested band in M. intracellulare. Several other non tuberculous mycobacteria (NTM) species yielded the same doublet pattern obtained for M. intracellulare and thus this polymorphism was not found to be useful for species identification (results not shown). Upon BstNI digestion, M. avium yielded two distinct bands (500 and 100bp) while the 600bp fragment of M. intracellulare remained intact due to the absence of this restriction site. Upon HgaI digestion, M. avium fragment remained undigested due to the lack of the HgaI site (600bp) while M. intracellulare yielded a doublet of 300bp. Thus, out of BamHI, BstNI and HgaI, only HgaI and BstNI were found to be useful for species differentiation.

To validate this assay on clinical specimens, 45 MAC clinical isolates were subjected to PCR-RFLP assay. These isolates were selected based upon their HPLC pattern. HPLC analysis showed three distinct chromatographic patterns for these isolates: those resembling M. avium, M. intracellulare and an intermediate pattern (Fig. la, b and c) indicating significant phenotypic heterogeneity. Based on HPLC results, 14 M. avium, 18 M. intracellulare and 13 MAC isolates were subjected to PCR-RFLP assay. Upon BstNI and BamHI digestion all the 45 MAC clinical isolates gave identical band pattern resembling M. intracellulare (Fig. 2a, data not shown for BamHI digestion) irrespective of their HPLC based phenotype. This indicates the inability of these polymorphisms in permitting species identification among clinical isolates. This also shows that not all polymorphisms seen in standard laboratory strains would be useful for diagnosis. When the same samples were subjected to HgaI digestion, clear species discrimination was seen (Fig. 2b). Twelve out of 14 (85.7%) and 14 out of 18 (77.7%) isolates were correctly identified as M. avium and M. intracellulare respectively by both HPLC and PCR-RFLP (Table). Two were M. avium by HPLC and M. intracellulare by PCR-RFLP. Similarly 4 were M. intracellulare by HPLC and M. avium by PCR-RFLP. More importantly, 13 isolates which could not be typed by HPLC, were identified as M. avium (9 isolates) and M. intracellulare (4 isolates). Thus, of the 45 isolates, 25 were found to be M. avium and 20 were M. intracellulare by the PCR-RFLP assay. Of the 25 M. avium and 20 M. intracellulare isolates, 22 and 10 isolates were from HIV-positive subjects respectively. These results were in good accordance with previous reports showing the preponderance of M. avium infection among HIV-positive subjects (17,18).

[FIGURE 1 OMITTED]

M. tuberculosis and MAC species are the most important mycobacterial pathogens since they account for more than 90 per cent of all the mycobacterial infections (11). Among these three species, M. avium and M. intracellulare have overlapping phenotypic properties that make their identification difficult by conventional methods. But these two species differ markedly at the genome level as evidenced by unique polymorphisms in otherwise highly conserved genes (3,16,17). Because of the scarceness of biochemical differences between M. avium and M. intracellulare, additional techniques such as HPLC, serotyping and genotypic assays have been attempted with the aim of discriminating them. HPLC is time-consuming and not readily available to most clinical laboratories. On the other hand, serotyping has several drawbacks such as producing inconsistent data among laboratories and inability to type all isolates. PCR-RFLP assay is a simple genotypic method which has been used worldwide for mycobacterial species identification.

[FIGURE 2 OMITTED]

Earlier Delvallois et al (19), have reported limited utility of Hsp65 based PCR-RFLP in species identification among the south Indian isolates, while the same and other reports (7) have shown the usefulness of this assay among isolates from other countries. Thus, there is a need to use other mycobacterial genes as PCR-RFLP targets, since a single assay may be inadequate in discriminating M. avium and M. intracellulare strains from diverse geographical locations. Therefore, we have developed an indigenous groES based PCR-RFLP assay which was found to be useful for species identification among the south Indian clinical isolates. This assay was also validated using MAC clinical isolates from HIV-positive individuals.

In conclusion, our data showed that HgaI based groES PCR-RFLP is a simple and rapid method for the identification and differentiation of M. avium and M. intracellulare among strains that are refractory to HPLC based species identification. The use of DNA amplification based techniques for the diagnosis of MAC is essential for the timely onset of chemotherapy, which can lead to improvement of symptoms, clearing of bacilli and in some instances an increased rate of survival in patients with AIDS suffering from MAC disease.

Acknowledgment

The first author (VA) acknowledges the Council of Scientific and Industrial Research (CSIR), New Delhi, for senior research fellowship.

Received March 28, 2006

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Reprint requests: Dr P.R. Narayanan, Director, Tuberculosis Research Centre (ICMR), Mayor V.R. Ramanathan Road, Chetput, Chennai 600031, India e-mail: prnarayanan@trcchennai.in

V. Aravindhan, S. Sulochana, Sujatha Narayanan, C.N. Paramasivam, P.R. Narayanan

Tuberculosis Research Centre (ICMR), Chennai, India
Table. Summary of comparison between the newly developed PCR-
RFLP assay and the well established HPLC based mycolic acid
analysis. MAC clinical isolates (n=45) were used to validate the
groES based PCR-RFLP assay. These isolates were also subjected
to HPLC analysis for species identification

 HPLC analysis
 M. intracel-
 M. avium lulare MAC
 (n=14) (n=18) (n=13)

 M. avium 12 4 9
 (n=25)

PCR- M. intracellulare 2 14 4
RFLP (n=20)
assay
 Total 14+18+13= 45

MAC refers to strains showing an intermediate HPLC profile
between M. avium and M. intracellulare. This group was
discriminated by the PCR-RFLP assay
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Title Annotation:polymerase chain reaction-restriction fragment length polymorphism
Author:Aravindhan, V.; Sulochana, S.; Narayanan, Sujatha; Paramasivam, C.N.; Narayanan, P.R.
Publication:Indian Journal of Medical Research
Article Type:Report
Geographic Code:9INDI
Date:Dec 1, 2007
Words:2543
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