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Journal of Clinical Microbiology, October 2005, p. 5369-5371, Vol. 43, No. 10
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.10.5369-5371.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Use of Specific rRNA Oligonucleotide Probes for Microscopic Detection of Mycobacterium tuberculosis in Culture and Tissue Specimens

Allison L. St. Amand,1 Daniel N. Frank,1 Mary Ann De Groote,1,2 Randall J. Basaraba,3 Ian M. Orme,3 and Norman R. Pace1*

Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347,1 Department of Medicine, Division of Infectious Diseases, University of Colorado Health Sciences Center, Denver, Colorado 80262,2 Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado 80523-16193

Received 5 May 2005/ Returned for modification 20 July 2005/ Accepted 31 July 2005


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ABSTRACT
 
Mycobacterium tuberculosis infections are a major global health problem. Fast and accurate detection of M. tuberculosis is clearly needed at both the clinical level and the research level. We report a rapid and reliable in situ hybridization technique using rRNA oligonucleotide probes for the identification of M. tuberculosis in tissues and cultures.


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TEXT
 
Mycobacterium tuberculosis is the causative agent of tuberculosis. Mycobacterium tuberculosis infections often result in pulmonary disease but also can cause skin infections, lymphadenitis, meningitis, and other manifestations (1, 3). Detection of Mycobacterium tuberculosis generally relies on a combination of staining of acid-fast bacilli (AFB) and in vitro cultivation (7). However, AFB staining is unable to identify the species of different mycobacteria, and at different growth stages, some mycobacteria are reported to lose their "acid fastness" (2, 15, 16). After cultivation, a variety of methods are used to identify Mycobacterium tuberculosis, including PCR of IS6110 or 16S rRNA genes (17). Several commercial products that use 16S rRNA sequences as probes or amplification primers are approved for molecular identification of mycobacteria (5, 13, 18). These assays either require cultivation of the organism or are approved only for sputum samples.

A more rapid methodology for the detection of mycobacteria in both culture and tissue is in situ hybridization (ISH) (11). The ISH technique allows for direct observation of bacterial distribution and morphology in the context of the histopathology of the tissue. Specific oligonucleotide probes that distinguish between closely related organisms can be developed based on comparisons of rRNA gene sequences (6, 8). In this paper, we report the development of Mycobacterium tuberculosis-specific rRNA probes and techniques for reproducible ISH on Mycobacterium tuberculosis in culture, sputum, and tissue.

Cultures were acquired from Leonid Heifets (National Jewish Medical and Research Center, Denver, CO) and Mark Hernandez (University of Colorado, Boulder, CO) and were fixed in 10% formalin for 4 to 10 h. Denver Health Medical Center (Denver, CO) provided acid-fast-positive sputum from a patient not identified but with a history of tuberculosis. Mycobacterium avium subsp. paratuberculosis-infected bovine mesenteric lymph node tissue and lung tissue sections from guinea pigs and mice infected with Mycobacterium tuberculosis H37Rv were fixed and processed as previously described (12, 14). Tissue and cultures were pretreated with xylene, lysozyme, and achromopeptidase according to the method of St. Amand et al. (11).

Probes used were EUB338 (4), MTB770 (5'-CACTATTCACACGCGCGT-3'), MTB226 (5'-CCACACCGCTAAAG-3'), MTB187 (5'-TGCATCCCGTGGTCCTATCC-3'), and MAVP187 (11). Oligonucleotides were obtained from Integrated DNA Technologies, Inc., and labeled at the 5' and 3' ends with either 6'-carboxyfluorescein (FAM), Cy3, or Cy5. All ISH protocols were performed according to St. Amand et al. (11) with the following exceptions: specimens were incubated in a 20% formamide hybridization buffer at either 38°C (MTB770, MTB226, and MTB187) or 40°C (MAVP187 and EUB338) for 6 to 12 h. Following hybridization, slides were washed with the appropriate wash buffer for 20 min at 39°C or 41°C. Colorimetric detection of fluorescein probes by using anti-fluorescein-alkaline phosphatase antibodies and INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride)-BCIP (5-bromo-4-chloro-3-indolylphosphate) was carried out as previously described (11).

MTB-specific ISH oligonucleotide probe sequences were chosen by using the "Probe Design" function of the ARB software package (9) and an alignment of rRNA sequences obtained from GenBank. As a first test of specificity, fluorescent probes were applied to pure cultures, including Mycobacterium tuberculosis H37Rv, M. avium, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium abscessus, Mycobacterium parafortuitum, Mycobacterium phlei, Escherichia coli, Bacillus subtilis, Rhodococcus sp., and Corynebacterium sp. The bacterial 16S probe EUB338-FAM was included in each ISH experiment to monitor the effectiveness of permeabilization and hybridization protocols.

Cy3-conjugated probes MTB770, MTB226, and MTB187 (MTB probes) hybridized with Mycobacterium tuberculosis H37Rv culture (Fig. 1E) but not to M. intracellulare (Fig. 1F), M. avium (Fig. 1G), M. kansasii (Fig. 1H), or other bacterial species (Table 1). Hybridization with probe EUB338-FAM clearly labeled all the cultures tested (Fig. 1A through D; Table 1), indicating that MTB probe-negative cells were sufficiently permeabilized.



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  		FIG. 1. Fluorescent in situ hybridization of the M. tuberculosis. (A and E) M. tuberculosis dual hybridized with EUB338-FAM (green) (A) and MTB-Cy3 (red) (E) probes. (B and F) M. intracellulare dual hybridized with EUB338-FAM (green) (B) and MTB-Cy3 (red) (F) probes. (C and G) M. avium dual hybridized with EUB338-FAM (green) (C) and MTB-Cy3 (red) (G) probes. (D and H) M. kansasii dual hybridized with EUB338-FAM (green) (D) and MTB-Cy3 (red) (H) probes. Pictures were taken under epifluorescence with a Nikon Eclipse E600 microscope. Original magnification, x1,000. (I) Fluorescent in situ hybridization utilizing three oligonucleotide probes: MTB-FAM probes (green), MAVP187-Cy3 (red), and EUB338-Cy5 (pseudoblue), hybridized with a mixed culture which contains M. tuberculosis, M. avium, M. intracellulare, and Corynebacterium sp. The micrograph was taken by a Leica confocal laser scanning microscope in which all three channels (FAM, Cy3, and Cy5) were merged. EUB338-Cy5 hybridized with all the bacteria (pseudoblue). Long M. tuberculosis rods are fluorescent green-blue. Short M. avium rods are fluorescent red-blue. Long M. intracellulare rods and short Corynebacterium sp. rods are fluorescent pseudoblue, since they are detected by the EUB338-Cy5 probe but not by the MTB-FAM probe or the MAVP187-Cy3 probe. Original magnification, x630. (J) Sputum (from a patient with a history of tuberculosis) hybridized with MTB-FAM probes. The arrow indicates a positive Mycobacterium tuberculosis bacillus (green) positively identified by MTB-ISH. (N) A phase-contrast micrograph of the same field shown in panel J. The arrow indicates the same bacillus shown in panel J. (K and L) Brightfield micrographs from AFB staining of Mycobacterium tuberculosis H37Rv-infected guinea pig lung (K) and mouse lung (L) sections. Nuclei were counterstained with methylene blue. (M) Brightfield micrograph from AFB staining of a M. avium subsp. paratuberculosis-infected bovine lymph node. Nuclei counterstained with methylene blue. (O) MTB-FAM probes hybridize with multiple Mycobacterium tuberculosis bacilli (green) in a representative granuloma of the guinea pig lung. Nuclei were counterstained with DAPI (4 ' ,6 ' -diamidino-2-phenylindole) (cyan). (P) A representative micrograph of a mouse lung specimen probed with the MTB-FAM oligos and visualized with enzymatic amplification of a colorimetric dye INT-BCIP (reddish-brown bacilli). Nuclei were counterstained with methylene blue. (Q) MTB-FAM probes did not hybridize with the bacilli in the M. avium subsp. paratuberculosis-infected bovine lymph node (fuzzy green background). MAVP187-Cy3 did hybridize to the M. avium subsp. paratuberculosis within the tissue (red bacilli). (K, L, M, and P) Brightfield micrographs taken with a Nikon Eclipse E600 microscope. Original magnification, x1,000. (O and Q) Epifluorescent micrograph taken with a Leica confocal laser scanning microscope. Original magnification, x630. Bar = 1 micrometer (for all micrograph scales).


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  		TABLE 1. Specificity of MTB ISH probes

The MTB probes were also tested against a mixed culture containing Mycobacterium tuberculosis, M. avium, M. intracellulare, and Corynebacterium sp. (Fig. 1I). Probes EUB338-Cy5 and MAVP187-Cy3 (M. avium subspecies specific) were also included in the hybridization experiments. EUB338-Cy5 hybridized to all bacteria in the culture showing cell permeabilization. MTB-FAM hybridized to long Mycobacterium tuberculosis rods, while the MAVP187-Cy3 hybridized to short M. avium rods. We tested the MTB probes on a clinical sputum sample that was AFB positive but culture negative. Figures 1J and N show the ability of the MTB probes to detect bacilli in a sputum sample. The sputum's MTB-ISH-positive result was confirmed by IS6110 PCR with extracted genomic DNA from the sputum (data not shown).

To assess MTB ISH in tissue samples, we applied the probes to well-characterized Mycobacterium tuberculosis H37Rv-infected guinea pig and mouse lungs. M. avium subsp. paratuberculosis-infected bovine lymph node was used to establish the specificities of the MTB probes when hybridized to tissue sections. Guinea pig and mouse tissue sections were probed with a combination of MTB-FAM probes and EUB338-Cy3. The bovine lymph node was probed with both the MTB-FAM and MAVP187-Cy3.

The presence of mycobacteria in tissue was initially demonstrated by AFB staining (10). Figures 1K, L, and M show the results of AFB staining on mycobacterium-infected guinea pig lung, mouse lung, and bovine lymph node, respectively. Figure 1O is a representative micrograph of positive hybridization of the MTB-FAM probes to the Mycobacterium tuberculosis bacilli located in the granulomas of guinea pig lung tissue. Although tissue autofluorescence did not generally impact the detection of mycobacteria, occasionally it was necessary to visualize the MTB-FAM probes with a nonfluorescent approach. This method utilizes anti-fluorescein-alkaline phosphatase-conjugated antibodies that bind to the FAM moieties on the probe. The colorimetric dye INT-BCIP was used as a substrate for the alkaline phosphatase. A representative micrograph of a mouse lung probed with the MTB-FAM probes and visualized with INT-BCIP is shown in Fig. 1P (reddish-brown bacilli). Additionally, the MTB probes did not hybridize with the M. avium subsp. paratuberculosis present in the bovine lymph node tissue while the MAVP187-Cy3 did hybridize to the M. avium subsp. paratuberculosis (Fig. 1Q), as expected.

By our methodology, the ISH probes specifically labeled the Mycobacterium tuberculosis organisms in culture, sputum, and tissue. ISH probes offer researchers and clinicians a fast and accurate method for identifying members of the MTB complex in tissue and culture. Future work with diseased and normal specimens from humans will be needed to further validate the sensitivities and usefulness of the MTB probes.


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ACKNOWLEDGMENTS
 
We thank Leonid Heifets, Sarah Wilson, Robert Belknap, and Mark Hernandez for providing strains, clinical specimens, and valuable feedback.

This research was conducted under the University of Colorado at Boulder Human Research Committee exemption of protocol 0799.14 (Molecular Analysis of Microbes in Human and Animal Diseased Tissue) for analysis of human and animal samples.

This work was supported by grants from the National Institutes of Health to N.R.P. (AI-51298) and to I.M.O. (AI-054697).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309-0347. Phone: (303) 735-1864. Fax: (303) 492-7744. E-mail: nrpace{at}colorado.edu. Back


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Journal of Clinical Microbiology, October 2005, p. 5369-5371, Vol. 43, No. 10
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.10.5369-5371.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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