Method for Rapid Identification and Differentiation of the Species of the Mycobacterium chelonae Complex Based on 16S-23S rRNA Gene Internal
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微生物临床杂志 2005年第9期
Environmental Genetics and Molecular Toxicology Division, Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0056
ABSTRACT
Members of the Mycobacterium chelonae complex (MCC), including M. immunogenum, M. chelonae, and M. abscessus, have been associated with nosocomial infections and occupational hypersensitivity pneumonitis due to metalworking fluid (MWF) exposures. In order to minimize these health hazards, an effective and rapid assay for detection of MCC species and differentiation of MCC species from other species of rapidly growing mycobacteria (RGM) and from one another is warranted. Here we report such a method, based on the variable 16S-23S rRNA gene internal transcribed spacer (ITS) region. Mycobacterium genus-specific primers derived from highly conserved sequences in the ITS region and the flanking 16S rRNA gene were used. Specificity of the primers was verified using the MCC member species, 11 non-MCC RGM species, 3 slow-growing mycobacterial (SGM) species (two strains each), and 19 field isolates, including 18 MCC isolates (from in-use MWF) and one non-MCC isolate (from reverse osmosis water). The ITS amplicon size of M. immunogenum varied from those of M. chelonae and M. abscessus. Sequencing of the 250-bp-long ITS amplicons of the three MCC member species showed differences in 24 to 34 bases, thereby yielding variable deduced restriction maps. ITS PCR-restriction analysis using the in silico-selected restriction enzyme MaeII or HphI differentiated the three MCC members from one another and from other RGM and SGM species without sequencing. The enzyme MaeII discriminated all three member species; however, HphI could only differentiate M. immunogenum from M. chelonae and M. abscessus. Use of an optimized rapid DNA template preparation step based on direct cell lysis in the PCR tube added to the simplicity and adaptability of the developed assay.
INTRODUCTION
Nontuberculous mycobacteria, which are ubiquitously present in the environment, have the potential to cause occupational respiratory illnesses and nosocomial infections (6). Among the rapidly growing nontuberculous mycobacteria, members of the Mycobacterium chelonae complex (MCC), which includes three closely related species, i.e., M. chelonae, M. abscessus, and the newly described M. immunogenum, have been implicated in pseudo-outbreaks involving contaminated hospital washing equipment and bronchoscopes (7). Of these, M. immunogenum and M. chelonae in particular are associated with metalworking fluids (MWF) (8, 26) used in machine industries for cooling and lubrication and are considered causative agents for hypersensitivity pneumonitis and other respiratory illnesses, including bronchitis, in machine workers exposed to these fluids and their aerosols (15, 18). Increasing incidences of nosocomial infections and occupational respiratory illnesses caused by the MCC species and the need for species-specific differences in patient management (24) have made it clinically important to rapidly and unambiguously identify individual species of this group in order to design appropriate treatment regimens and control strategies (25).
PCR has proven to be a useful tool for the rapid diagnosis of bacterial pathogens. Existing PCR-based assays for detection or species differentiation of mycobacteria rely on primers targeting the genes encoding 16S rRNA, 23S rRNA, and the 65-kDa heat shock protein (hsp65) (21, 22, 23). In addition, the internal transcribed spacer (ITS) region, a stretch of DNA that lies between the 16S and 23S rRNA subunit genes, has proved to be more variable than the adjacent 16S and 23S ribosomal genes. Hence, this region may allow efficient identification of species due to its enhanced variability within a genus (10). The ITS region in the genus Mycobacterium is approximately 270 to 360 bp in size, depending on the species (17).
PCR-restriction analysis of Mycobacterium-specific PCR products is a useful tool in yielding species-specific patterns. Due to few base pair differences, the restriction pattern analysis of 16S or 23S rRNA sequences has limitations in differentiating the three members of MCC group from other rapidly growing mycobacterial (RGM) species and from each other (13, 14). Analysis of the variable part of the hsp65 gene (441 bp) by PCR-restriction analysis has been widely used for diagnosis of several species of Mycobacterium (5, 20, 21). However, for MCC species the currently used hsp65 441-bp restriction analysis protocol is complicated, as it requires multiple enzymes in various combinations to differentiate the three member species in a two-step restriction digestion. Moreover, intraspecies polymorphism in the 441-bp target region observed in the individual MCC species, such as M. chelonae, was shown to cause different restriction patterns for individual isolates of the same species, thereby increasing the ambiguity of the method (25). This hsp65-based protocol yields inefficient amplification in M. immunogenum due to sequence variability in the primer-binding region and often yields insufficient amplicon for restriction analysis (12). In view of these limitations and constraints, there is a need to develop methods based on alternative genomic targets with better resolving ability for species differentiation. The present study was designed to develop a procedure for efficient identification and differentiation of the three MCC species based on the ITS region. The method involves the use of newly designed Mycobacterium genus-specific ITS primers coupled with PCR-restriction enzyme pattern analysis based on a selected set of restriction enzymes. In order to increase the speed of analysis, the DNA template preparation step was based on optimized direct cell lysis in the PCR tube instead of a lengthy DNA extraction protocol (12). The developed method, designated ITS-PCR restriction analysis (ITSPRA), is therefore a simple, rapid, and adaptable assay for differentiating the three member species of the MCC group with a single-step restriction analysis.
(Part of this study was presented at the 104th General Meeting of the American Society for Microbiology, New Orleans, La., 22-27 May 2004.)
MATERIALS AND METHODS
Mycobacterial strains and isolates. Different Mycobacterium reference strains originally isolated from both clinical and environmental sources were used in this study. These included the member species of the MCC obtained from the American Type Culture Collection (ATCC), viz., Mycobacterium immunogenum ATCC 700506 (from metalworking fluid), M. chelonae ATCC 35752T (from tortoise), and M. abscessus ATCC 19977T (from a knee abscess) and ATCC 23006 (from human sputum). For validation of the developed ITSPRA protocol, we used the reference species and isolates of other nonpigmenting RGM, including M. fortuitum ATCC 6841T (from a cold abscess), M. mageritense ATCC 700351T (from human sputum), M. peregrinum ATCC 14467T (from bronchial aspiration), M. mucogenicum ATCC 49650T (from an infected thyroglossal duct cyst), M. senegalense ATCC 35796T (from a bovine farcy lesion), M. smegmatis ATCC 19420T (from endothelial cells), M. wolinskyi ATCC 700010T (from a human facial wound), and M. septicum ATCC 700731T (from a venous catheter tip); of the pigmenting RGM, including M. phlei ATCC 11758T, M. vaccae ATCC 15483T (from cow milk), and Mycobacterium sp. strain RJGII.135 (from soil); and of slow-growing mycobacteria (SGM), including M. avium strain W144, M. avium strain W359, M. avium subsp. paratuberculosis strain 202, M. avium subsp. paratuberculosis strain 1112, M. intracellulare strain W253st, and M. intracellulare strain HO3AN5st. In addition to these known reference species/isolates, 19 mycobacterial field isolates collected over a period of 3 years, comprising 18 isolates from MWF samples obtained from different occupational settings located in different regions of the country and 1 isolate from a reverse osmosis water source used for MWF dilution, were included. The MWF isolates included in this study were obtained by culturing on Middlebrook 7H10 agar supplemented with oleic acid-albumin-dextrose-catalase enrichment, using incubation at 37 and 30°C, respectively. Putative mycobacterial colonies were selected based on their colony morphology, growth behavior, and acid-fast staining reactions, and their identity as mycobacteria was confirmed by PCR-based analyses (12, 25).
DNA template preparation. The DNA template for PCR amplification was prepared for all strains by a rapid direct cell lysis protocol recently developed in our laboratory (12), except for the SGM strains M. avium W144, M. avium W359, M. intracellulare W253st, and M. intracellulare HO3AN5st, where the extracted genomic DNA was used. Briefly, a single colony of each culture was taken with a sterile pipette tip in a 0.2-ml PCR tube containing 5 μl lysis solution (2% sodium dodecyl sulfate and 10% Triton-X-100 in Tris-EDTA buffer, pH.8.0), followed by gentle mixing. The PCR tube was preheated at 98°C for 5 min and kept at 4°C for 1 min, using the GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA). The tube containing the cell lysate was directly used for the PCR amplification reaction. The DNA template for generating the ITS amplicons for sequencing was prepared as described previously (12).
PCR amplification of ITS region. A pair of primers was designed from the conserved regions by alignment of all available ITS region sequences and the flanking distal region sequences of the 16S rRNA genes of different mycobacteria, using the MegAlign program 1997 to 2001 (DNASTAR Inc., Madison, WI). Amplification of the ITS region (ranging from 254 to 296 bp in size) with the newly designed primers ITS-forward (5'-CCT TTC TAA GGA GCA CC-3') and ITS-reverse (5'-GAT GCT CGC AAC CAC TAT CC-3') was performed using the GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA). The reaction mixture (50 μl) contained 5 μl of template DNA, 2.5 units of PfuUltra high-fidelity DNA polymerase (Stratagene, La Jolla, CA), 1x PfuUltra reaction buffer with MgCl2, 200 μM of each of the four deoxynucleoside triphosphates (Panvera, Madison, WI), and 100 ng each of the forward and reverse primers. The amplification regimen included 30 cycles (each cycle using 94°C for 30 s, 50°C for 30 s, and 72°C for 30 s) followed by 5 min of incubation at 72°C. All PCR products except those from SGM species (10 μl each) were resolved on 1% agarose using the Trevigel gel matrix (Trevigen, Gaithersburg, MD) with 1x Tris-acetate/EDTA electrophoresis buffer (0.04 mol/liter Tris, 0.001 mol/liter EDTA, pH. 7.8) with 5 μl of a 100-bp DNA size marker (Invitrogen, Carlsbad, CA, or PGC Scientific, Gaithersburg, MD); the SGM PCR products were resolved on a 12% polyacrylamide gel using 1x Tris-borate/EDTA electrophoresis buffer (0.089 mol/liter Tris-borate, 0.002 mol/liter EDTA, pH 8.0). The gels were stained with ethidium bromide (0.5 μg/ml) and photographed under UV light, using the Kodak Edas 290 gel documentation system (Kodak, Rochester, NY).
Amplicon sequencing. The ITS amplicons (250 to 260 bp) from the three MCC member reference species (M. immunogenum, M. chelonae, and M. abscessus) and the 19 field isolates were purified using the Gene Clean II kit (Bio 101 Systems, Vista, CA) and cloned using the TOPO 2.1 cloning kit (Invitrogen Corporation, Carlsbad, CA) according to the manufacturer's recommendations. The ITS inserts were sequenced by the DNA Core Facility at the University of Cincinnati. The sequence data were further analyzed by multiple alignment using MegAlign, 1997 to 2001 (DNASTAR Inc., Madison, WI).
Development of ITSPRA. In order to determine the appropriate species-specific restriction sites and to select appropriate commercial restriction enzymes, deduced restriction maps of the ITS amplicon sequences were generated using the Gene Runner program (Hastings Software Inc., Hudson, NY). The PCR amplicons were purified by removing inhibitors such as cell debris, salts, and other chemicals, including sodium dodecyl sulfate, and Triton X-100, to enhance the restriction enzyme activity. In order to pellet the cell debris, the PCR tube was centrifuged at full speed for 15 s and the supernatant was further processed using Montage PCR centrifugal filters (Millipore Corp., Bedford, MA) according to the manufacturer's instructions.
Amplicons (8 μl each) of all reference species used in this study and the 19 field isolates were digested in a 20-μl reaction mixture with 10 units of either of the two in silico-identified restriction enzymes, HphI (New England Biolabs, Beverly, MA) and MaeII (Roche Applied Sciences, Indianapolis, IN), using appropriate restriction buffers supplied by the respective manufacturer. The short restriction fragments were resolved by polyacrylamide gel electrophoresis using a 12% acrylamide gel matrix at 150 V for 3 h, and the sizes were confirmed using a 50-bp DNA ladder (Promega, Madison, WI). The gels were stained with ethidium bromide (0.5 μg/ml) and photographed under UV light. Restriction fragment sizes for the MCC isolates and non-MCC species were visually compared with those of the three MCC member species M. immunogenum, M. chelonae, and M. abscessus run in parallel.
Nucleotide sequence accession numbers. The amplicon sequences have been submitted to GenBank under the following accession numbers: M. immunogenum, AY497531; M. chelonae, AY498739; and M. abscessus, AY498740.
RESULTS
PCR amplification of the ITS region with the designed genus-specific oligonucleotide primers and optimized PCR conditions yielded a single amplicon for all tested Mycobacterium reference species (RGM and SGM) and the 19 field isolates. However, M. avium subsp. paratuberculosis showed no amplification due to lack of binding of the MCC-specific primers, as discussed below. Since the primers were derived from a conserved ITS region and the flanking 16S rRNA gene, the amplified products contained an approximately 7-bp-long terminal region of the 16S rRNA gene in addition to the ITS region. Based on comparison with the DNA size marker, the estimated sizes of the amplified products of all MCC species and control, non-MCC species varied from 215 to 296 bp (Table 1). The amplicon size estimate for M. immunogenum and 10 field isolates (M-JY3, M-JY4, M-JY10, and M-JY12 through M-JY18) was approximately 265 bp, whereas that for M. chelonae, M. abscessus, and eight field isolates (M-JY1, M-JY2, M-JY6, M-JY7, M-JY8, M-JY9, M-JY11, and M-JY19) was 255 bp. The non-MCC species yielded a product size range of 215 to 296 bp, as shown in Table 1. The results indicated a minor heterogeneity in PCR amplicon size among individual MCC species but detectable size differences between MCC and non-MCC species or isolates (Fig. 1A and B).
Sequencing of the amplicons for MCC reference species allowed the determination of the exact size of the amplified ITS region, which was 247 bp for M. chelonae and M. abscessus and 256 bp for M. immunogenum, when counted after subtracting the seven nucleotide bases of the flanking 16S rRNA gene. Multiple alignment of the amplicon sequences for the three species revealed that M. immunogenum differed in 27 and 34 bases from M. chelonae and M. abscessus, respectively, while the latter two species differed from each other in 24 bases (Fig. 2).
Considering the resolving ability of the ITS amplicon sequencing, we used this approach for independent preidentification of all 19 putative mycobacterial isolates from MWF. Of the 19 isolates sequenced, 8 showed closest homology to M. chelonae and 10 matched M. immunogenum, whereas the water isolate M-JY5 showed significant nonhomology with any of the three MCC species. Further comparison of the consensus ITS amplicon sequences of the field isolates revealed two strains for M. chelonae isolates; strain 1 included M-JY1, M-JY2, M-JY7, M-JY8, M-JY9, and M-JY19, which had identical sequences, whereas strain 2 included M-JY6 and M-JY11, which had three base differences from strain 1 (Fig. 2). Similarly, the 10 isolates of M. immunogenum represented two different strains; strain 1 (which included the MWF isolates M-JY13, M-JY14, M-JY15, M-JY16, M-JY17, and M-JY18) differed from strain 2 (which included M-JY3, M-JY4, M-JY10, and M-JY12) by one base difference (Fig. 2). The water isolate (M-JY5) had a relatively longer ITS sequence with a variable region extending over 119 bases and was identified as M. diernhoferi based on BLAST homology comparison of the ITS sequence with the available gene database.
Restriction analysis of the ITS amplicons by using the in silico-selected endonucleases HphI and MaeII yielded fragments of various length for different species, as listed in Table 1. The MCC member species differed from the other RGM species in their restriction pattern, thereby enabling their identification and differentiation from other mycobacteria. Five of the RGM species, namely, M. fortuitum, M. mageritense, M. mucogenicum, M. vaccae, and M. wolinskyi, showed distinctly different restriction patterns (Fig. 3A; Table 1). The other non-MCC RGM species, namely M. peregrinum, M. phlei, M. senegalense, M. smegmatis, Mycobacterium sp. strain RJGII.135, and M. septicum, and the SGM species M. avium and M. intracellulare did not show any ITSPRA pattern with HphI, indicating the lack of this restriction site in these species, unlike in the MCC group (Fig. 3A). Similarly, the RGM species M. mageritense, M. peregrinum, M. senegalense, M. vaccae, Mycobacterium sp. strain RJGII.135, and M. septicum and the SGM species M. avium and M. intracellulare did not respond to MaeII endonuclease, whereas the RGM species M. fortuitum, M. phlei, M. mucogenicum, M. smegmatis, and M. wolinskyi showed multiple restriction patterns with this enzyme, typically different from those observed for MCC species (Fig. 3B; Table 1). In addition, the whole-genome sequences of strains of the other three SGM species, M. leprae, M. tuberculosis strain H37Rv, M. tuberculosis strain CDC1551, and M. avium subsp. paratuberculosis, which are available in the GenBank sequence database, were analyzed in silico for the presence of the primer-binding sequences and the restriction sites used in the ITSPRA method developed in this study. The results showed no match for both primers in M. avium subsp. paratuberculosis, no match for the reverse primer in M. leprae, and lack of the restriction sites (HphI and MaeII) in both M. tuberculosis strains, thereby further confirming the specificity of the developed ITSPRA method for the MCC group (data not shown).
All three reference species of the MCC group, M. immunogenum, M. chelonae, and M. abscessus, could be differentiated from one another by the unique ITSPRA patterns based on the MaeII enzyme; however, HphI yielded a common pattern for M. chelonae and M. abscessus but a distinct pattern for M. immunogenum. With HphI, M. immunogenum was differentiated from the other two species by the presence of its unique 174-bp fragment and absence of the 65-bp and 99-bp fragments characteristic of the other two species (Fig. 3A). However, due to size fractionation limitations under the gel electrophoresis conditions used, the 99-bp bands (Table 1) of M. chelonae and M. abscessus was not well resolved from their 89-bp bands and visually migrated in line with the 89-bp fragment of M. immunogenum. The three MCC species had the following MaeII restriction fragments: M. immunogenum, 139 and 124 bp; M. chelonae, 129 and 125 bp; and M. abscessus, 129, 68, and 57 bp (Fig. 3B). Of the 18 MWF isolates, 8 isolates (M-JY1, M-JY2, M-JY6, M-JY7, M-JY8, M-JY9, M-JY11, and M-JY19) were identified as M. chelonae and the remaining 10 isolates (M-JY3, M-JY4, M-JY10, and M-JY12 through M-JY18) were identified as M. immunogenum based on the ITSPRA patterns. Representative patterns are shown in Fig. 3A and B. The water isolate M-JY5, previously identified as M. diernhoferi, did not respond to either of the enzymes, further confirming it as a non-MCC isolate. In terms of the rapidity of the developed method, a schematic work flow of the protocol is shown in Fig. 4, indicating the time required for each step of the protocol.
DISCUSSION
In comparison to conventional biochemical methods such as sodium chloride tolerance, utilization of citrate (19), or high-performance liquid chromatography-based mycolic acid profiling (2), PCR-based methods provide a time-saving and cost-effective alternative for detection and identification of mycobacteria to the species level. Several attempts have been made to differentiate various mycobacterial species by using PCR target genes encoding 16S rRNA, 23S rRNA, or the 65-kDa heat shock protein. However, these targets have had limited impact in differentiating mycobacterial species due to their insufficient sequence variability, among other limitations as discussed in the preceding sections (3, 21, 22, 23, 25). In contrast, the ITS region has been considered a suitable target for differentiating species and potentially can be used to distinguish clinically relevant mycobacterial species (16). Such efforts have been reported for species differentiation in different groups of SGM with little interspecies 16S rRNA sequence variation, such as among species of the M. avium-M. intercellulare complex (4, 9) and between M. gastri and M. kansasii (17). The use of this variable target to differentiate RGM species such as the M. chelonae complex has not been reported to date. In this study, the ITS-based amplification utilized a newly designed genus-specific primer pair based on the ITS region and the flanking 16S rRNA region. Variable-size amplicons were observed in MCC and non-MCC species, but the amplicon size variation among the member species of the MCC was too small to resolve in agarose electrophoresis (Fig. 1A and B). This limited variation in amplicon size led us to sequence individual amplicons for the three MCC species to examine specific sequence variations in size and bases. Indeed, the amplicon sequencing results revealed a high degree of variability among the three MCC species (Fig. 2), indicating the potential use of ITS sequencing in species differentiation. In earlier studies, ITS sequencing of M. tuberculosis and several nontuberculous species (11, 16) had indicated a high degree of ITS variability among these different species. However, amplicon sequencing, although useful in differentiating different species, is not very adaptable for practical diagnostic or analytical applications.
In order to circumvent the need for amplicon sequencing, we developed and optimized an amplicon-restriction analysis assay, designated ITSPRA, which could rapidly and efficiently identify and differentiate the individual MCC species. The accuracy of the developed ITSPRA method was confirmed based on the fact that similar results on identification of the 19 isolates were obtained using ITS amplicon sequencing. With an aim to increase the rapidity of the developed assay, we used direct cell lysis in the PCR tube, instead of using conventional, time-consuming DNA extraction before PCR (12). The assay involves a single-step restriction analysis. A 12% polyacrylamide gel electrophoresis-based amplicon separation method was applied in the present study, which provided more precise estimates than those obtainable by agarose gel electrophoresis and allowed identification of the mycobacterial species whose ITSPRA patterns were characterized by <60-bp fragments. It showed better resolution than agarose gel electrophoresis, particularly for MaeII patterns, with bands differing by ±5 bp. Use of a 12% instead of the 10% concentration of polyacrylamide used earlier (1) enhanced the resolution in the developed assay. In view of our results, HphI-based restriction analysis can be useful for differentiation between M. immunogenum and the other two MCC species, whereas MaeII can be used for differentiating all three MCC species from one another. The present study offers an advantage over the available hsp-based restriction analysis method because of increased rapidity and specificity for identification of MCC species. The amplicon generation and clarification steps require <2 h, and confirmation of the PCR amplicon for MCC isolates by restriction digestion and analysis may require an additional 5 h (Fig. 4). Moreover, the assay involves a single-step restriction digestion of the amplicon, in contrast to a two-step digestion required for the current hsp65-based assay. Considering the simple and rapid nature of the developed ITSPRA method, it could be adapted not only in reference laboratories but also in analytical laboratories, such as industrial exposure assessment laboratories, for efficient identification and differentiation of MCC species isolated from clinical and occupational environments.
ACKNOWLEDGMENTS
This study was supported by grant 1R01OH007364 (to J.S.Y.) from the National Institute of Occupational Safety and Health, Centers for Disease Control and Prevention (CDC).
Thanks are due to Stacy Pfaller of the U.S. Environmental Protection Agency, Cincinnati, Ohio, for providing cells or genomic DNAs of the slow-growing mycobacteria used in this study.
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Yadav, J. S., I. U. H. Khan, F. Fakhari, and M. B. Soellner. 2003. DNA-based methodologies for rapid detection, quantification, and species- or strain-level identification of respiratory pathogens (Mycobacteria and Pseudomonads) in metalworking fluids. Appl. Occup. Environ. Hyg. 18:966-975.(Izhar U. H. Khan, Suresh )
ABSTRACT
Members of the Mycobacterium chelonae complex (MCC), including M. immunogenum, M. chelonae, and M. abscessus, have been associated with nosocomial infections and occupational hypersensitivity pneumonitis due to metalworking fluid (MWF) exposures. In order to minimize these health hazards, an effective and rapid assay for detection of MCC species and differentiation of MCC species from other species of rapidly growing mycobacteria (RGM) and from one another is warranted. Here we report such a method, based on the variable 16S-23S rRNA gene internal transcribed spacer (ITS) region. Mycobacterium genus-specific primers derived from highly conserved sequences in the ITS region and the flanking 16S rRNA gene were used. Specificity of the primers was verified using the MCC member species, 11 non-MCC RGM species, 3 slow-growing mycobacterial (SGM) species (two strains each), and 19 field isolates, including 18 MCC isolates (from in-use MWF) and one non-MCC isolate (from reverse osmosis water). The ITS amplicon size of M. immunogenum varied from those of M. chelonae and M. abscessus. Sequencing of the 250-bp-long ITS amplicons of the three MCC member species showed differences in 24 to 34 bases, thereby yielding variable deduced restriction maps. ITS PCR-restriction analysis using the in silico-selected restriction enzyme MaeII or HphI differentiated the three MCC members from one another and from other RGM and SGM species without sequencing. The enzyme MaeII discriminated all three member species; however, HphI could only differentiate M. immunogenum from M. chelonae and M. abscessus. Use of an optimized rapid DNA template preparation step based on direct cell lysis in the PCR tube added to the simplicity and adaptability of the developed assay.
INTRODUCTION
Nontuberculous mycobacteria, which are ubiquitously present in the environment, have the potential to cause occupational respiratory illnesses and nosocomial infections (6). Among the rapidly growing nontuberculous mycobacteria, members of the Mycobacterium chelonae complex (MCC), which includes three closely related species, i.e., M. chelonae, M. abscessus, and the newly described M. immunogenum, have been implicated in pseudo-outbreaks involving contaminated hospital washing equipment and bronchoscopes (7). Of these, M. immunogenum and M. chelonae in particular are associated with metalworking fluids (MWF) (8, 26) used in machine industries for cooling and lubrication and are considered causative agents for hypersensitivity pneumonitis and other respiratory illnesses, including bronchitis, in machine workers exposed to these fluids and their aerosols (15, 18). Increasing incidences of nosocomial infections and occupational respiratory illnesses caused by the MCC species and the need for species-specific differences in patient management (24) have made it clinically important to rapidly and unambiguously identify individual species of this group in order to design appropriate treatment regimens and control strategies (25).
PCR has proven to be a useful tool for the rapid diagnosis of bacterial pathogens. Existing PCR-based assays for detection or species differentiation of mycobacteria rely on primers targeting the genes encoding 16S rRNA, 23S rRNA, and the 65-kDa heat shock protein (hsp65) (21, 22, 23). In addition, the internal transcribed spacer (ITS) region, a stretch of DNA that lies between the 16S and 23S rRNA subunit genes, has proved to be more variable than the adjacent 16S and 23S ribosomal genes. Hence, this region may allow efficient identification of species due to its enhanced variability within a genus (10). The ITS region in the genus Mycobacterium is approximately 270 to 360 bp in size, depending on the species (17).
PCR-restriction analysis of Mycobacterium-specific PCR products is a useful tool in yielding species-specific patterns. Due to few base pair differences, the restriction pattern analysis of 16S or 23S rRNA sequences has limitations in differentiating the three members of MCC group from other rapidly growing mycobacterial (RGM) species and from each other (13, 14). Analysis of the variable part of the hsp65 gene (441 bp) by PCR-restriction analysis has been widely used for diagnosis of several species of Mycobacterium (5, 20, 21). However, for MCC species the currently used hsp65 441-bp restriction analysis protocol is complicated, as it requires multiple enzymes in various combinations to differentiate the three member species in a two-step restriction digestion. Moreover, intraspecies polymorphism in the 441-bp target region observed in the individual MCC species, such as M. chelonae, was shown to cause different restriction patterns for individual isolates of the same species, thereby increasing the ambiguity of the method (25). This hsp65-based protocol yields inefficient amplification in M. immunogenum due to sequence variability in the primer-binding region and often yields insufficient amplicon for restriction analysis (12). In view of these limitations and constraints, there is a need to develop methods based on alternative genomic targets with better resolving ability for species differentiation. The present study was designed to develop a procedure for efficient identification and differentiation of the three MCC species based on the ITS region. The method involves the use of newly designed Mycobacterium genus-specific ITS primers coupled with PCR-restriction enzyme pattern analysis based on a selected set of restriction enzymes. In order to increase the speed of analysis, the DNA template preparation step was based on optimized direct cell lysis in the PCR tube instead of a lengthy DNA extraction protocol (12). The developed method, designated ITS-PCR restriction analysis (ITSPRA), is therefore a simple, rapid, and adaptable assay for differentiating the three member species of the MCC group with a single-step restriction analysis.
(Part of this study was presented at the 104th General Meeting of the American Society for Microbiology, New Orleans, La., 22-27 May 2004.)
MATERIALS AND METHODS
Mycobacterial strains and isolates. Different Mycobacterium reference strains originally isolated from both clinical and environmental sources were used in this study. These included the member species of the MCC obtained from the American Type Culture Collection (ATCC), viz., Mycobacterium immunogenum ATCC 700506 (from metalworking fluid), M. chelonae ATCC 35752T (from tortoise), and M. abscessus ATCC 19977T (from a knee abscess) and ATCC 23006 (from human sputum). For validation of the developed ITSPRA protocol, we used the reference species and isolates of other nonpigmenting RGM, including M. fortuitum ATCC 6841T (from a cold abscess), M. mageritense ATCC 700351T (from human sputum), M. peregrinum ATCC 14467T (from bronchial aspiration), M. mucogenicum ATCC 49650T (from an infected thyroglossal duct cyst), M. senegalense ATCC 35796T (from a bovine farcy lesion), M. smegmatis ATCC 19420T (from endothelial cells), M. wolinskyi ATCC 700010T (from a human facial wound), and M. septicum ATCC 700731T (from a venous catheter tip); of the pigmenting RGM, including M. phlei ATCC 11758T, M. vaccae ATCC 15483T (from cow milk), and Mycobacterium sp. strain RJGII.135 (from soil); and of slow-growing mycobacteria (SGM), including M. avium strain W144, M. avium strain W359, M. avium subsp. paratuberculosis strain 202, M. avium subsp. paratuberculosis strain 1112, M. intracellulare strain W253st, and M. intracellulare strain HO3AN5st. In addition to these known reference species/isolates, 19 mycobacterial field isolates collected over a period of 3 years, comprising 18 isolates from MWF samples obtained from different occupational settings located in different regions of the country and 1 isolate from a reverse osmosis water source used for MWF dilution, were included. The MWF isolates included in this study were obtained by culturing on Middlebrook 7H10 agar supplemented with oleic acid-albumin-dextrose-catalase enrichment, using incubation at 37 and 30°C, respectively. Putative mycobacterial colonies were selected based on their colony morphology, growth behavior, and acid-fast staining reactions, and their identity as mycobacteria was confirmed by PCR-based analyses (12, 25).
DNA template preparation. The DNA template for PCR amplification was prepared for all strains by a rapid direct cell lysis protocol recently developed in our laboratory (12), except for the SGM strains M. avium W144, M. avium W359, M. intracellulare W253st, and M. intracellulare HO3AN5st, where the extracted genomic DNA was used. Briefly, a single colony of each culture was taken with a sterile pipette tip in a 0.2-ml PCR tube containing 5 μl lysis solution (2% sodium dodecyl sulfate and 10% Triton-X-100 in Tris-EDTA buffer, pH.8.0), followed by gentle mixing. The PCR tube was preheated at 98°C for 5 min and kept at 4°C for 1 min, using the GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA). The tube containing the cell lysate was directly used for the PCR amplification reaction. The DNA template for generating the ITS amplicons for sequencing was prepared as described previously (12).
PCR amplification of ITS region. A pair of primers was designed from the conserved regions by alignment of all available ITS region sequences and the flanking distal region sequences of the 16S rRNA genes of different mycobacteria, using the MegAlign program 1997 to 2001 (DNASTAR Inc., Madison, WI). Amplification of the ITS region (ranging from 254 to 296 bp in size) with the newly designed primers ITS-forward (5'-CCT TTC TAA GGA GCA CC-3') and ITS-reverse (5'-GAT GCT CGC AAC CAC TAT CC-3') was performed using the GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA). The reaction mixture (50 μl) contained 5 μl of template DNA, 2.5 units of PfuUltra high-fidelity DNA polymerase (Stratagene, La Jolla, CA), 1x PfuUltra reaction buffer with MgCl2, 200 μM of each of the four deoxynucleoside triphosphates (Panvera, Madison, WI), and 100 ng each of the forward and reverse primers. The amplification regimen included 30 cycles (each cycle using 94°C for 30 s, 50°C for 30 s, and 72°C for 30 s) followed by 5 min of incubation at 72°C. All PCR products except those from SGM species (10 μl each) were resolved on 1% agarose using the Trevigel gel matrix (Trevigen, Gaithersburg, MD) with 1x Tris-acetate/EDTA electrophoresis buffer (0.04 mol/liter Tris, 0.001 mol/liter EDTA, pH. 7.8) with 5 μl of a 100-bp DNA size marker (Invitrogen, Carlsbad, CA, or PGC Scientific, Gaithersburg, MD); the SGM PCR products were resolved on a 12% polyacrylamide gel using 1x Tris-borate/EDTA electrophoresis buffer (0.089 mol/liter Tris-borate, 0.002 mol/liter EDTA, pH 8.0). The gels were stained with ethidium bromide (0.5 μg/ml) and photographed under UV light, using the Kodak Edas 290 gel documentation system (Kodak, Rochester, NY).
Amplicon sequencing. The ITS amplicons (250 to 260 bp) from the three MCC member reference species (M. immunogenum, M. chelonae, and M. abscessus) and the 19 field isolates were purified using the Gene Clean II kit (Bio 101 Systems, Vista, CA) and cloned using the TOPO 2.1 cloning kit (Invitrogen Corporation, Carlsbad, CA) according to the manufacturer's recommendations. The ITS inserts were sequenced by the DNA Core Facility at the University of Cincinnati. The sequence data were further analyzed by multiple alignment using MegAlign, 1997 to 2001 (DNASTAR Inc., Madison, WI).
Development of ITSPRA. In order to determine the appropriate species-specific restriction sites and to select appropriate commercial restriction enzymes, deduced restriction maps of the ITS amplicon sequences were generated using the Gene Runner program (Hastings Software Inc., Hudson, NY). The PCR amplicons were purified by removing inhibitors such as cell debris, salts, and other chemicals, including sodium dodecyl sulfate, and Triton X-100, to enhance the restriction enzyme activity. In order to pellet the cell debris, the PCR tube was centrifuged at full speed for 15 s and the supernatant was further processed using Montage PCR centrifugal filters (Millipore Corp., Bedford, MA) according to the manufacturer's instructions.
Amplicons (8 μl each) of all reference species used in this study and the 19 field isolates were digested in a 20-μl reaction mixture with 10 units of either of the two in silico-identified restriction enzymes, HphI (New England Biolabs, Beverly, MA) and MaeII (Roche Applied Sciences, Indianapolis, IN), using appropriate restriction buffers supplied by the respective manufacturer. The short restriction fragments were resolved by polyacrylamide gel electrophoresis using a 12% acrylamide gel matrix at 150 V for 3 h, and the sizes were confirmed using a 50-bp DNA ladder (Promega, Madison, WI). The gels were stained with ethidium bromide (0.5 μg/ml) and photographed under UV light. Restriction fragment sizes for the MCC isolates and non-MCC species were visually compared with those of the three MCC member species M. immunogenum, M. chelonae, and M. abscessus run in parallel.
Nucleotide sequence accession numbers. The amplicon sequences have been submitted to GenBank under the following accession numbers: M. immunogenum, AY497531; M. chelonae, AY498739; and M. abscessus, AY498740.
RESULTS
PCR amplification of the ITS region with the designed genus-specific oligonucleotide primers and optimized PCR conditions yielded a single amplicon for all tested Mycobacterium reference species (RGM and SGM) and the 19 field isolates. However, M. avium subsp. paratuberculosis showed no amplification due to lack of binding of the MCC-specific primers, as discussed below. Since the primers were derived from a conserved ITS region and the flanking 16S rRNA gene, the amplified products contained an approximately 7-bp-long terminal region of the 16S rRNA gene in addition to the ITS region. Based on comparison with the DNA size marker, the estimated sizes of the amplified products of all MCC species and control, non-MCC species varied from 215 to 296 bp (Table 1). The amplicon size estimate for M. immunogenum and 10 field isolates (M-JY3, M-JY4, M-JY10, and M-JY12 through M-JY18) was approximately 265 bp, whereas that for M. chelonae, M. abscessus, and eight field isolates (M-JY1, M-JY2, M-JY6, M-JY7, M-JY8, M-JY9, M-JY11, and M-JY19) was 255 bp. The non-MCC species yielded a product size range of 215 to 296 bp, as shown in Table 1. The results indicated a minor heterogeneity in PCR amplicon size among individual MCC species but detectable size differences between MCC and non-MCC species or isolates (Fig. 1A and B).
Sequencing of the amplicons for MCC reference species allowed the determination of the exact size of the amplified ITS region, which was 247 bp for M. chelonae and M. abscessus and 256 bp for M. immunogenum, when counted after subtracting the seven nucleotide bases of the flanking 16S rRNA gene. Multiple alignment of the amplicon sequences for the three species revealed that M. immunogenum differed in 27 and 34 bases from M. chelonae and M. abscessus, respectively, while the latter two species differed from each other in 24 bases (Fig. 2).
Considering the resolving ability of the ITS amplicon sequencing, we used this approach for independent preidentification of all 19 putative mycobacterial isolates from MWF. Of the 19 isolates sequenced, 8 showed closest homology to M. chelonae and 10 matched M. immunogenum, whereas the water isolate M-JY5 showed significant nonhomology with any of the three MCC species. Further comparison of the consensus ITS amplicon sequences of the field isolates revealed two strains for M. chelonae isolates; strain 1 included M-JY1, M-JY2, M-JY7, M-JY8, M-JY9, and M-JY19, which had identical sequences, whereas strain 2 included M-JY6 and M-JY11, which had three base differences from strain 1 (Fig. 2). Similarly, the 10 isolates of M. immunogenum represented two different strains; strain 1 (which included the MWF isolates M-JY13, M-JY14, M-JY15, M-JY16, M-JY17, and M-JY18) differed from strain 2 (which included M-JY3, M-JY4, M-JY10, and M-JY12) by one base difference (Fig. 2). The water isolate (M-JY5) had a relatively longer ITS sequence with a variable region extending over 119 bases and was identified as M. diernhoferi based on BLAST homology comparison of the ITS sequence with the available gene database.
Restriction analysis of the ITS amplicons by using the in silico-selected endonucleases HphI and MaeII yielded fragments of various length for different species, as listed in Table 1. The MCC member species differed from the other RGM species in their restriction pattern, thereby enabling their identification and differentiation from other mycobacteria. Five of the RGM species, namely, M. fortuitum, M. mageritense, M. mucogenicum, M. vaccae, and M. wolinskyi, showed distinctly different restriction patterns (Fig. 3A; Table 1). The other non-MCC RGM species, namely M. peregrinum, M. phlei, M. senegalense, M. smegmatis, Mycobacterium sp. strain RJGII.135, and M. septicum, and the SGM species M. avium and M. intracellulare did not show any ITSPRA pattern with HphI, indicating the lack of this restriction site in these species, unlike in the MCC group (Fig. 3A). Similarly, the RGM species M. mageritense, M. peregrinum, M. senegalense, M. vaccae, Mycobacterium sp. strain RJGII.135, and M. septicum and the SGM species M. avium and M. intracellulare did not respond to MaeII endonuclease, whereas the RGM species M. fortuitum, M. phlei, M. mucogenicum, M. smegmatis, and M. wolinskyi showed multiple restriction patterns with this enzyme, typically different from those observed for MCC species (Fig. 3B; Table 1). In addition, the whole-genome sequences of strains of the other three SGM species, M. leprae, M. tuberculosis strain H37Rv, M. tuberculosis strain CDC1551, and M. avium subsp. paratuberculosis, which are available in the GenBank sequence database, were analyzed in silico for the presence of the primer-binding sequences and the restriction sites used in the ITSPRA method developed in this study. The results showed no match for both primers in M. avium subsp. paratuberculosis, no match for the reverse primer in M. leprae, and lack of the restriction sites (HphI and MaeII) in both M. tuberculosis strains, thereby further confirming the specificity of the developed ITSPRA method for the MCC group (data not shown).
All three reference species of the MCC group, M. immunogenum, M. chelonae, and M. abscessus, could be differentiated from one another by the unique ITSPRA patterns based on the MaeII enzyme; however, HphI yielded a common pattern for M. chelonae and M. abscessus but a distinct pattern for M. immunogenum. With HphI, M. immunogenum was differentiated from the other two species by the presence of its unique 174-bp fragment and absence of the 65-bp and 99-bp fragments characteristic of the other two species (Fig. 3A). However, due to size fractionation limitations under the gel electrophoresis conditions used, the 99-bp bands (Table 1) of M. chelonae and M. abscessus was not well resolved from their 89-bp bands and visually migrated in line with the 89-bp fragment of M. immunogenum. The three MCC species had the following MaeII restriction fragments: M. immunogenum, 139 and 124 bp; M. chelonae, 129 and 125 bp; and M. abscessus, 129, 68, and 57 bp (Fig. 3B). Of the 18 MWF isolates, 8 isolates (M-JY1, M-JY2, M-JY6, M-JY7, M-JY8, M-JY9, M-JY11, and M-JY19) were identified as M. chelonae and the remaining 10 isolates (M-JY3, M-JY4, M-JY10, and M-JY12 through M-JY18) were identified as M. immunogenum based on the ITSPRA patterns. Representative patterns are shown in Fig. 3A and B. The water isolate M-JY5, previously identified as M. diernhoferi, did not respond to either of the enzymes, further confirming it as a non-MCC isolate. In terms of the rapidity of the developed method, a schematic work flow of the protocol is shown in Fig. 4, indicating the time required for each step of the protocol.
DISCUSSION
In comparison to conventional biochemical methods such as sodium chloride tolerance, utilization of citrate (19), or high-performance liquid chromatography-based mycolic acid profiling (2), PCR-based methods provide a time-saving and cost-effective alternative for detection and identification of mycobacteria to the species level. Several attempts have been made to differentiate various mycobacterial species by using PCR target genes encoding 16S rRNA, 23S rRNA, or the 65-kDa heat shock protein. However, these targets have had limited impact in differentiating mycobacterial species due to their insufficient sequence variability, among other limitations as discussed in the preceding sections (3, 21, 22, 23, 25). In contrast, the ITS region has been considered a suitable target for differentiating species and potentially can be used to distinguish clinically relevant mycobacterial species (16). Such efforts have been reported for species differentiation in different groups of SGM with little interspecies 16S rRNA sequence variation, such as among species of the M. avium-M. intercellulare complex (4, 9) and between M. gastri and M. kansasii (17). The use of this variable target to differentiate RGM species such as the M. chelonae complex has not been reported to date. In this study, the ITS-based amplification utilized a newly designed genus-specific primer pair based on the ITS region and the flanking 16S rRNA region. Variable-size amplicons were observed in MCC and non-MCC species, but the amplicon size variation among the member species of the MCC was too small to resolve in agarose electrophoresis (Fig. 1A and B). This limited variation in amplicon size led us to sequence individual amplicons for the three MCC species to examine specific sequence variations in size and bases. Indeed, the amplicon sequencing results revealed a high degree of variability among the three MCC species (Fig. 2), indicating the potential use of ITS sequencing in species differentiation. In earlier studies, ITS sequencing of M. tuberculosis and several nontuberculous species (11, 16) had indicated a high degree of ITS variability among these different species. However, amplicon sequencing, although useful in differentiating different species, is not very adaptable for practical diagnostic or analytical applications.
In order to circumvent the need for amplicon sequencing, we developed and optimized an amplicon-restriction analysis assay, designated ITSPRA, which could rapidly and efficiently identify and differentiate the individual MCC species. The accuracy of the developed ITSPRA method was confirmed based on the fact that similar results on identification of the 19 isolates were obtained using ITS amplicon sequencing. With an aim to increase the rapidity of the developed assay, we used direct cell lysis in the PCR tube, instead of using conventional, time-consuming DNA extraction before PCR (12). The assay involves a single-step restriction analysis. A 12% polyacrylamide gel electrophoresis-based amplicon separation method was applied in the present study, which provided more precise estimates than those obtainable by agarose gel electrophoresis and allowed identification of the mycobacterial species whose ITSPRA patterns were characterized by <60-bp fragments. It showed better resolution than agarose gel electrophoresis, particularly for MaeII patterns, with bands differing by ±5 bp. Use of a 12% instead of the 10% concentration of polyacrylamide used earlier (1) enhanced the resolution in the developed assay. In view of our results, HphI-based restriction analysis can be useful for differentiation between M. immunogenum and the other two MCC species, whereas MaeII can be used for differentiating all three MCC species from one another. The present study offers an advantage over the available hsp-based restriction analysis method because of increased rapidity and specificity for identification of MCC species. The amplicon generation and clarification steps require <2 h, and confirmation of the PCR amplicon for MCC isolates by restriction digestion and analysis may require an additional 5 h (Fig. 4). Moreover, the assay involves a single-step restriction digestion of the amplicon, in contrast to a two-step digestion required for the current hsp65-based assay. Considering the simple and rapid nature of the developed ITSPRA method, it could be adapted not only in reference laboratories but also in analytical laboratories, such as industrial exposure assessment laboratories, for efficient identification and differentiation of MCC species isolated from clinical and occupational environments.
ACKNOWLEDGMENTS
This study was supported by grant 1R01OH007364 (to J.S.Y.) from the National Institute of Occupational Safety and Health, Centers for Disease Control and Prevention (CDC).
Thanks are due to Stacy Pfaller of the U.S. Environmental Protection Agency, Cincinnati, Ohio, for providing cells or genomic DNAs of the slow-growing mycobacteria used in this study.
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