Use of PCR and reverse line blot hybridization macroarray based on 16S-23S rRNA gene internal transcribed spacer sequences for rapid identification of 34 mycobacterium species

The aim of this study was to develop a PCR and reverse line blot hybridization (PCR-RLB) macroarray assay based on 16S-23S rRNA gene internal transcribed spacer sequences for the identification and differentiation of 34 mycobacterial species or subspecies. The performance of the PCR-RLB assay was assessed and validated by using 78 reference strains belonging to 55 Mycobacterium species, 219 clinical isolates which had been identified as mycobacteria by high-performance liquid chromatography or gas chromatography, three skin biopsy specimens from patients with suspected leprosy which had been shown to contain acid-fast bacilli, and isolates of 14 nonmycobacterial species. All mycobacteria were amplified in the PCR and hybridized with a genus-specific probe (probe MYC). The 34 species-specific probes designed in this study hybridized only with the corresponding Mycobacterium species. The mycobacterial PCR-RLB assay is an efficient tool for the identification of clinical isolates of mycobacteria; it can reliably identify mixed mycobacterial cultures and M. leprae in skin biopsy specimens.     

Genus-specific polymerase chain reaction for the mycobacterial dnaJ gene and species-specific oligonucleotide probes.

Identification of tuberculous and nontuberculous mycobacteria by biochemical methods is a long-term process that takes up to 8 weeks for completion and requires expertise to interpret the results. In order to detect and differentiate the major pathogenic mycobacterial species, we developed genus-specific primers that amplify the dnaJ gene from the broad spectrum of mycobacterial species and determined the nucleotide sequences within the dnaJ genes from 19 mycobacterial species (Mycobacterium tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. microti, M. kansasii, M. marinum, M. gastri, M. simiae, M. scrofulaceum, M. szulgai, M. gordonae, M. avium, M. intracellulare, M. xenopi, M. fortuitum, M. chelonei, M. haemophilum, and M. paratuberculosis). On the basis of the dnaJ gene sequences, we developed dot blot hybridization analysis with species-specific oligonucleotide probes for the M. tuberculosis complex. M. avium, M. intracellulare, and M. kansaii, allowing a rapid identification of these species following polymerase chain reaction for the dnaJ gene. We conclude that polymerase chain reaction with the genus-specific primer that amplifies the dnaJ genes and subsequent dot blot analysis with species-specific oligonucleotide probes are most useful for differential diagnosis of tuberculosis and nontuberculous mycobacterial infections.     

Development and evaluation of a novel multiplex probe array for rapid differential identification of Mycobacterium in clinical specimens

Rapid differential identification of Mycobacterium species is essential for effective diagnosis and management of mycobacteriosis. The aim of this study was to develop a novel multiplex probe array based on the 16S-23S rRNA gene internal transcribed spacer sequence for the genotyping of mycobacteria to the species level. A pair of primers and a set of genus- and species-specific probes were designed from the conserved and polymorphic regions of the 16S rRNA gene, internal transcribed spacer, and 23S rRNA gene sequences of mycobacteria. We used a novel multiplex probe array for identification of 266 clinical specimens obtained from patients with mycobaterial infection. The results showed that the overall specificity and sensitivity of our novel probe array were both 100% for the genus-specific probe and Mycobacterium tuberculosis complex-specific probe. There were 79.3% (23/29) of nontuberculous mycobacteria which could be identified to the species level directly in the specimens from China. Some intraspecies heterogeneity in M. avium, M. intracellulare , M. chelonae and M.abscessus was observed. With the increase of sequences of internal transcribed spacer and numbers of whole microbial genomes, and further optimization of probes, the multiplex probe array will become a promising tool for the rapid and accurate identification of mycobacteria in ordinary clinical laboratories.     

Genotypic identification of pathogenic Mycobacterium species by using a nonradioactive oligonucleotide probe.

Commercial DNA hybridization assays (Syngene, Inc., San Diego, Calif.) utilizing alkaline phosphatase-labeled oligonucleotide probes for the identification of Mycobacterium tuberculosis complex and M. avium complex (MAC) were evaluated with 261 isolates of mycobacteria. On the basis of biochemical criteria, the test for MAC was 98% specific and more sensitive (95 of 99, 95%) than Gen-Probe (88 of 99, 89% sensitivity); the major difference in sensitivity noted between the two systems was related to the hybridization of seven MAC strains to the SNAP X probe. The M. tuberculosis complex probe correctly identified all 62 isolates of M. tuberculosis and all 11 isolates of M. bovis, for a sensitivity of 100%. There were two discrepant reactions with mycobacteria other than M. tuberculosis complex isolates.     

Multilocus variable-number tandem repeat typing of Mycobacterium ulcerans

The apparent genetic homogeneity of Mycobacterium ulcerans contributes to the poorly understood epidemiology of M. ulcerans infection. Here, we report the identification of variable number tandem repeat (VNTR) sequences as novel polymorphic elements in the genome of this species. A total of 19 potential VNTR loci identified in the closely related M. marinum genome sequence were screened in a collection of 23 M. ulcerans isolates, one Mycobacterium species referred to here as an intermediate species, and five M. marinum strains. Nine of the 19 loci were polymorphic in the three species (including the intermediate species) and revealed eight M. ulcerans and five M. marinum genotypes. The results from the VNTR analysis corroborated the genetic relationships of M. ulcerans isolates from various geographical origins, as defined by independent molecular markers. Although these results further highlight the extremely high clonal homogeneity within certain geographic regions, we report for the first time the discrimination of the two South American strains from Surinam and French Guyana. These findings support the potential of a VNTR-based genotyping method for strain discrimination within M. ulcerans and M. marinum.     

Species-specific assessment of Mycobacterium leprae in skin biopsies by in situ hybridization and polymerase chain reaction.

Conventional histopathologic diagnosis of mycobacterial infections are limited to the determination of "acid-fast bacilli". A species-specific diagnosis is thus far impossible. In addition, routine microbiologic assessments of mycobacteria suffer from the major drawback that a species-specific diagnosis is extremely time-consuming and in several cases even impossible. As Mycobacterium leprae cannot be cultured in vitro, we tried to specifically target this obligate intracellular parasite by in situ hybridization and polymerase chain reaction (PCR) techniques. For this purpose we used a 22 mer oligonucleotide probe recognizing a species-specific sequence of the 16S rRNA of Mycobacterium leprae. Using an immunoenzymatic detection method for in situ hybridization we were able to specifically assess Mycobacterium leprae (a) in long-term cultured macrophages in vitro infected with different mycobacteria species and (b) in frozen sections of skin biopsies obtained from patients suffering from lepromatous leprosy. These results could be confirmed and extended by PCR experiments in which we used conserved oligonucleotide primers for 16S rRNA to amplify bacterial DNA isolated from different eubacterial species and from fresh-frozen as well as from formalin-fixed, paraffin-embedded and routinely processed mycobacteria-infected tissues. Upon Southern blot analysis, the Mycobacterium leprae-specific oligonucleotide probe exclusively hybridized with PCR products obtained from Mycobacterium leprae-containing samples (including paraffin sections), but not with PCR products obtained from samples containing other mycobacterial species. As species-specific oligonucleotide probes targeted at rRNA are described for a variety of mycobacterial species, these methods may be generally applied for a rapid species-specific assessment of mycobacteria in histologic material.     

Multicenter Evaluation of a Pathogenic Mycobacterium Screening Probe

The introduction of nucleic acid amplification assays into the clinical laboratory has reduced the time needed to diagnose diseases caused by members of the Mycobacterium tuberculosis complex (MTBC). However, several mycobacterial species other than those of the MTBC are known to cause disease, especially in immunocompromised individuals. A screening assay has been developed for the detection of the major pathogenic mycobacterial species. The assay utilizes pan-genus primers to amplify mycobacterial DNA and a screening probe (KY493) that detects all major pathogenic mycobacteria. A multicenter European study was conducted to assess the performance of the screening probe in the clinical laboratory. The screening probe was evaluated against individual probes specific for M. tuberculosis, M. avium, and M. intracellulare, a genus-specific probe with broader species coverage, and culture. The screening probe had a sensitivity equivalent to that of the species-specific probes; all specimens positive with any of the species-specific probes were also positive with the screening probes. Compared to culture, the sensitivity of the screening probe was 89% (154 of 173) for all culture-positive specimens tested. This value was 89.6% for the genus-specific probe. The screening probe was more specific than the genus-specific probe. Specificity was 93.9% (661 of 704) compared to culture results alone. The comparable specificity value for the genus-specific probe was 84.8%. When clinical data were taken into consideration, the sensitivity of the screening assay was similar to that of culture (81% versus 76.2%) but the positive predictive value of the test was lower (76.2% versus 100% for culture). However, the screening probe was more sensitive than smear and may be a useful tool in the rapid diagnosis of mycobacterial disease.     

Globally distributed mycobacterial fish pathogens produce a novel plasmid-encoded toxic macrolide, mycolactone F

Mycobacterium ulcerans and Mycobacterium marinum are closely related pathogens which share an aquatic environment. The pathogenesis of these organisms in humans is limited by their inability to grow above 35 degrees C. M. marinum causes systemic disease in fish but produces localized skin infections in humans. M. ulcerans causes Buruli ulcer, a severe human skin lesion. At the molecular level, M. ulcerans is distinguished from M. marinum by the presence of a virulence plasmid which encodes a macrolide toxin, mycolactone, as well as by hundreds of insertion sequences, particularly IS2404. There has been a global increase in reports of fish mycobacteriosis. An unusual clade of M. marinum has been reported from fish in the Red and Mediterranean Seas and a new mycobacterial species, Mycobacterium pseudoshottsii, has been cultured from fish in the Chesapeake Bay, United States. We have discovered that both groups of fish pathogens produce a unique mycolactone toxin, mycolactone F. Mycolactone F is the smallest mycolactone (molecular weight, 700) yet identified. The core lactone structure of mycolactone F is identical to that of M. ulcerans mycolactones, but a unique side chain structure is present. Mycolactone F produces apoptosis and necrosis on cultured cells but is less potent than M. ulcerans mycolactones. Both groups of fish pathogens contain IS2404. In contrast to M. ulcerans and conventional M. marinum, mycolactone F-producing mycobacteria are incapable of growth at above 30 degrees C. This fact is likely to limit their virulence for humans. However, such isolates may provide a reservoir for horizontal transfer of the mycolactone plasmid in aquatic environments.     

Detection of phospholipase C in nontuberculous mycobacteria and its possible role in hemolytic activity

Phospholipase C plays a key role in the pathogenesis of several bacterial infections, for example, those caused by Clostridium perfringens and Listeria monocytogenes. Previous studies have reported multiple copies of plc genes homologous to Pseudomonas aeruginosa plcH and plcN genes encoding the hemolytic and nonhemolytic phospholipase C enzymes in the genomes of Mycobacterium tuberculosis, M. marinum, M. bovis, and M. ulcerans. In this study we analyzed the possible relationship between phospholipase C and hemolytic activity in 21 strains of nontuberculous mycobacteria representing nine different species. Detection of phospholipase C enzymatic activity was carried out using thin-layer chromatography to detect diglycerides in the hydrolysates of radiolabeled phosphatidylcholine. DNA sequences of M. kansasii and M. marinum homologous to the genes encoding phospholipase C from M. tuberculosis and M. ulcerans were identified by DNA-DNA hybridization and sequencing. Finally, we developed a direct and simple assay to detect mycobacterial hemolytic activity. This assay is based on a modified blood agar medium that allows the growth and expression of hemolysis of slow-growing mycobacteria. Hemolytic activity was detected in M. avium, M. intracellulare, M. ulcerans, M. marinum, M. tuberculosis, and M. kansasii mycobacteria with phospholipase C activity, but not in M. fortuitum. No hemolytic activity was detected in M. smegmatis, M. gordonae, and M. vaccae. Whether or not phospholipase C enzyme plays a role in the pathogenesis of nontuberculous mycobacterial diseases needs further investigation.     

Direct detection and identification of Mycobacterium ulcerans in clinical specimens by PCR and oligonucleotide-specific capture plate hybridization.

We compared various diagnostic tests for their abilities to detect Mycobacterium ulcerans infection in specimens from patients with clinically active disease. Specimens from 10 patients from the area of Zangnanado (Department of Zou, Benin) with advanced, ulcerated active M. ulcerans infections were studied by direct smear, histopathology, culture, PCR, and oligonucleotide-specific capture plate hybridization (OSCPH). A total of 27 specimens, including 12 swabs of exudate collected before debridement and 15 fragments of tissue obtained during debridement, were submitted to bacteriologic and histopathologic analysis. The histopathologic evaluation of tissues from all six patients so tested revealed changes typical of those caused by M. ulcerans infection. Five specimens were contaminated, and M. ulcerans was cultivated on Löwenstein-Jensen medium from 12 of the remaining 22 (54.5%) specimens. Detection of mycobacteria was performed by PCR, and M. ulcerans was detected by OSCPH with a new probe (5'-CACGGGATTCATGTCCTGT-3') reacting with M. ulcerans and Mycobacterium marinum. In 10 of 22 (45.5%) specimens, M. ulcerans was identified by PCR-OSCPH. There was no statistically significant difference between the detection of M. ulcerans by culture and by PCR-OSCPH (P > 0.05). This is the first demonstration of an amplification system (PCR-OSCPH) with a sensitivity similar to that of culture for the direct and rapid recognition of M. ulcerans in clinical specimens. This system is capable of identifying M. ulcerans, even in paucibacillary lesions. Our findings suggest that PCR-OSCPH should be used in the quest for the elusive environmental reservoir(s) of M. ulcerans.     

Characterization of an unusual Mycobacterium: a possible missing link between Mycobacterium marinum and Mycobacterium ulcerans

In an attempt to characterize an unusual mycobacterial isolate from a 44-year-old patient living in France, we applied phenotypic characterizations and various previously described molecular methods for the taxonomic classification of mycobacteria. The results of the investigations were compared to those obtained in a previous study with a set of temporally and geographically diverse Mycobacterium ulcerans (n = 29) and Mycobacterium marinum (n = 29) isolates (K. Chemlal, G. Huys, P.-A. Fonteyne, V. Vincent, A. G. Lopez, L. Rigouts, J. Swings, W. M. Meyers, and F. Portaels, J. Clin. Microbiol. 39:3272-3278, 2001). The isolate, designated ITM 00-1026 (IPP 2000-372), is closely related to M. marinum according to its phenotypic properties, lipid pattern, and partial 16S rRNA sequence. Moreover, fingerprinting by amplified fragment length polymorphism (AFLP) analysis unequivocally classified this strain as a member of the species M. marinum, although it lacked two species-specific AFLP marker bands. However, PCR and restriction fragment length polymorphism analysis based on M. ulcerans-specific insertion sequence IS2404 showed the presence of this element in a low copy number in isolate ITM 00-1026. In conclusion, the designation of this isolate as a transitional species further supports the recent claim by Stinear et al. (T. Stinear, G. Jenkin, P. D. Johnson, and J. K. Davies, J. Bacteriol. 182:6322-6330, 2000) that M. ulcerans represents a relatively recent phylogenetic derivative of M. marinum resulting from the systematic acquisition of foreign DNA fragments.     

The superoxide dismutase gene, a target for detection and identification of mycobacteria by PCR.

The superoxide dismutase gene has been identified as a target in screening for the presence of mycobacteria on the genus level and differentiating relevant mycobacterial species from one another by PCR. Consensus primers deduced from known superoxide dismutase gene sequences allowed the amplification of DNAs from a variety of bacteria, fungi, and protozoa. Selected amplicons from Actinomyces viscosus, Corynebacterium diphtheriae, Corynebacterium pseudodiphtheriticum, Mycobacterium avium, M. fortuitum, M. gordonae, M. intracellulare, M. kansasii, M. scrofulaceum, M. simiae, M. tuberculosis, M. xenopi, and Nocardia asteroides were subsequently cloned and sequenced. The alignment of those sequences facilitated the selection of primers targeting conserved regions present in myobacterial species but absent in nonmycobacterial species and thus allowed the genus-specific amplification of all 28 different mycobacterial species tested. A pool of genus-specific allowed the genus-specific amplification of all 28 different mycobacterial species tested. A pool of genus-specific probes recognized 23 of the 28 mycobacterial species and did not cross-react with any of the 96 nonmycobacterial species tested. In addition, probes recognizing species-specific variable regions within the superoxide dismutase genes of M. avium, M. fortuitum, M. gordonae, M. intracellulare, M. kansasii, M. scrofulaceum, M. simiae, the M. tuberculosis complex, and M. xenopi were identified. All probes recognized only the species from which they were derived and did not cross-react with any other mycobacterial species or with any of the nonmycobacterial species tested. We conclude that the superoxide dismutase gene is a suitable target for amplifying mycobacteria by PCR on the genus level, confirming correct amplification by genus-specific probes, and differentiating relevant species from one another by a set of species-specific probes.     

Evaluation of acridinium-ester-labeled DNA probes for identification of Mycobacterium tuberculosis and Mycobacterium avium-Mycobacterium intracellulare complex in culture.

The detectability of mycobacteria in culture by the use of nonisotopic, chemiluminescent DNA probes for Mycobacterium tuberculosis and the M. avium-M. intracellulare complex (MAC) was evaluated and compared with that by the use of 125I-labeled DNA probes for the same mycobacteria. In the assay, rRNA-directed DNA probes labeled with acridinium ester (AE-DNA probes) were used. Unhybridized probes were chemically degraded, and the esterified acridinium on the hybridized probes was hydrolyzed by the addition of alkaline hydrogen peroxide solution, resulting in the production of visible light which was measured with a luminometer. The detection limits of the AE-DNA probes were almost the same as those of the 125I-labeled DNA probes. A total of 107 clinical isolates of mycobacteria (47 isolates of M. tuberculosis, 36 MAC, and 24 atypical mycobacteria other than MAC) were tested. The sensitivity and specificity of the AE-DNA probes for M. tuberculosis were 100% both for the conventional method and with the 125I-labeled DNA probe. The sensitivity and specificity of the AE-DNA probes for MAC were 97.2 and 100%, respectively, for the conventional method and were both 100% with the 125I-labeled DNA probes. Because the procedure is simple, reliable, rapid (it can be completed within an hour), and safe (it does not use radioisotopes), it can easily be performed in any clinical laboratory.     

Detection and identification of mycobacteria by DNA amplification and oligonucleotide-specific capture plate hybridization.

We have developed an easy and rapid detection and identification system for the diagnosis of mycobacterial diseases. The system is based on selective amplification by PCR of mycobacteria with primers based on the genes coding for 16S rRNA. During PCR, a label (digoxigenin-11-dUTP) is incorporated with biotinylated species-specific oligonucleotides (oligonucleotide-specific capture plate hybridization [OSCPH]. One oligonucleotide specific for the genus Mycobacterium and seven species-specific (Mycobacterium tuberculosis, M. avium, M. intracellulare, M. scrofulaceum, M. xenopi, M. genavense, and M. chelonae) oligonucleotides were designed as capturing probes. After specific hybridization, an enzyme immunoassay reveals the specifically bound complexes and thus permits identification of the mycobacterium. A total of 70 mycobacterial strains were tested. For 69 strains, results concordant with conventional identification were obtained. One M. chelonae strain was negative with the M. chelonae probe and was later reidentified as M. fortuitum. Moreover, for 15 clinical samples suspected of harboring nontuberculous mycobacteria, OSCPH was able to confirm all culture results and could identify one M. genavense infection for which standard culture results were negative. PCR-OSCPH is easily applicable and much faster than culture. It could become a valuable alternative approach for the diagnosis of mycobacterial infections.     

PCR结合寡核苷酸探针杂交检测临床常见真菌的实验研究

目的 建立PCR结合生物标记的寡核甘酸探针斑点杂交技术,鉴定临床常见的真菌。方法 首先用真菌通用引物扩增白念球菌、热带念球菌、假热带念球菌、近平滑念球菌、光滑念球菌、解脂念球菌、克鲁斯念球菌、季也蒙念球菌、黄曲 霉、烟曲霉的核糖体大亚单位基因的保守区序列,然后用生物素标记的种特异性寡核苷酸探针与扩增产物杂交,并将此方法用于临床标本和临床分离菌株的检测。结果 通用引物可以扩增上述11种临床常见真菌的DNA,扩增片段长度在260bp左右。9种特异性探针分别与11种真菌标准菌株的PCR扩增产物杂交,结果表明每种探针都具有高度特异性。斑点杂交法和Southerm杂交法检测敏感性相同,为100fg;琼脂糖凝胶电泳法检测敏感性为1pg。通过69例临床标本和31例临床分析菌株的检测,PCR－杂交法的结果和真菌培养法的结果基本一致。结论 PCR结合生物素标记的寡核苷酸探针杂交技术可将9种临床常见真菌鉴定到种,方法快速、敏感、特异。     

Rapid, species-specific detection of uropathogen 16S rDNA and rRNA at ambient temperature by dot-blot hybridization and an electrochemical sensor array

Development of rapid molecular approaches for pathogen detection is key to improving treatment of infectious diseases. For this study, the kinetics and temperature-dependence of DNA probe hybridization to uropathogen species-specific sequences were examined. A set of oligonucleotide probes were designed based on variable regions of the 16S gene of the Escherichia coli, Proteus mirabilis, Klebsiella oxytoca, and Pseudomonas aeruginosa. A universal bacterial probe and probes-specific for gram-positive and gram-negative organisms were also included. The oligonucleotide probes discriminated among 16S genes derived from 11 different species of uropathogenic bacteria applied to nylon membranes in a dot-blot format. Significant binding of oligonucleotide probes to target DNA and removal of nonspecific binding by membrane washing could both be achieved rapidly, requiring as little as 10 min. An oligonucleotide probe from the same species-specific region of the E. coli 16S gene was used as a capture probe in a novel electrochemical 16-sensor array based on microfabrication technology. Sequence-specific hybridization of target uropathogen 16S rDNA was detected through horseradish peroxidase acting as an electrochemical transducer via a second, detector probe. The sensor array demonstrated rapid, species-specific hybridization in a time course consistent with the rapid kinetics of the dot-blot hybridization studies. As in the dot-blot hybridization studies, species-specific detection of bacterial nucleic acids using the sensor array approach was demonstrated both at 65 degrees C and at room temperature. These results demonstrate that molecular hybridization approaches can be adapted to rapid, room temperature conditions ideal for an electrochemical sensor array platform.     

Detection and identification of Mycobacterium species isolates by DNA microarray

Rapid identification of Mycobacterium species isolates is necessary for the effective management of tuberculosis. Recently, analysis of DNA gyrase B subunit (gyrB) genes has been identified as a suitable means for the identification of bacterial species. We describe a microarray assay based on gyrB gene sequences that can be used for the identification of Mycobacteria species. Primers specific for a gyrB gene region common to all mycobacteria were synthesized and used for PCR amplification of DNA purified from clinical samples. A set of oligonucleotide probes for specific gyrB gene regions was developed for the identification of 14 Mycobacterium species. Each probe was spotted onto a silylated glass slide with an arrayer and used for hybridization with fluorescently labeled RNA derived from amplified sample DNA to yield a pattern of positive spots. This microarray produced unique hybridization patterns for each species of mycobacteria and could differentiate closely related bacterial species. Moreover, the results corresponded well with those obtained by the conventional culture method for the detection of mycobacteria. We conclude that a gyrB-based microarray can rapidly detect and identify closely related mycobacterial species and may be useful in the diagnosis and effective management of tuberculosis.     

Absence of Mycobacterium intracellulare and Presence of Mycobacterium chimaera in Household Water and Biofilm Samples of Patients in the United States with Mycobacterium avium Complex Respiratory Disease

Recent studies have shown that respiratory isolates from pulmonary disease patients and household water/biofilm isolates of Mycobacterium avium could be matched by DNA fingerprinting. To determine if this is true for Mycobacterium intracellulare, household water sources for 36 patients with Mycobacterium avium complex (MAC) lung disease were evaluated. MAC household water isolates from three published studies that included 37 additional MAC respiratory disease patients were also evaluated. Species identification was done initially using nonsequencing methods with confirmation by internal transcribed spacer (ITS) and/or partial 16S rRNA gene sequencing. M. intracellulare was identified by nonsequencing methods in 54 respiratory cultures and 41 household water/biofilm samples. By ITS sequencing, 49 (90.7%) respiratory isolates were M. intracellulare and 4 (7.4%) were Mycobacterium chimaera. In contrast, 30 (73%) household water samples were M. chimaera, 8 (20%) were other MAC X species (i.e., isolates positive with a MAC probe but negative with species-specific M. avium and M. intracellulare probes), and 3 (7%) were M. avium; none were M. intracellulare. In comparison, M. avium was recovered from 141 water/biofilm samples. These results indicate that M. intracellulare lung disease in the United States is acquired from environmental sources other than household water. Nonsequencing methods for identification of nontuberculous mycobacteria (including those of the MAC) might fail to distinguish closely related species (such as M. intracellulare and M. chimaera). This is the first report of M. chimaera recovery from household water. The study underscores the importance of taxonomy and distinguishing the many species and subspecies of the MAC.     

Evaluation of PCR-restriction profile analysis and IS2404 restriction fragment length polymorphism and amplified fragment length polymorphism fingerprinting for identification and typing of Mycobacterium ulcerans and M. marinum

Mycobacterium ulcerans and M. marinum are emerging necrotizing mycobacterial pathogens that reside in common reservoirs of infection and exhibit striking pathophysiological similarities. Furthermore, the interspecific taxonomic relationship between the two species is not clear as a result of the very high phylogenetic relatedness (i.e., >99.8% 16S rRNA sequence similarity), in contrast to only 25 to 47% DNA relatedness. To help understand the genotypic affiliation between these two closely related species, we performed a comparative analysis including PCR restriction profile analysis (PRPA), IS2404 restriction fragment length polymorphism (RFLP), and amplified fragment length polymorphism (AFLP) on a set of M. ulcerans (n = 29) and M. marinum (n = 28) strains recovered from different geographic origins. PRPA was based on a triple restriction of the 3' end region of 16S rRNA, which differentiated M. ulcerans into three types; however, the technique could not distinguish M. marinum from M. ulcerans isolates originating from South America and Southeast Asia. RFLP based on IS2404 produced six M. ulcerans types related to six geographic regions and did not produce any band with M. marinum, confirming the previous findings of Chemlal et al. (K. Chemlal, K. DeRidder, P. A. Fonteyne, W. M. Meyers, J. Swings, and F. Portaels, Am. J. Trop. Med. Hyg. 64:270-273, 2001). AFLP analysis resulted in profiles which grouped M. ulcerans and M. marinum into two separate clusters. The numerical analysis also revealed subgroups among the M. marinum and M. ulcerans isolates. In conclusion, PRPA appears to provide a rapid method for differentiating the African M. ulcerans type from other geographical types but is unsuitable for interspecific differentiation of M. marinum and M. ulcerans. In comparison, whole- genome techniques such as IS 2404-RFLP and AFLP appear to be far more useful in discriminating between M. marinum and M. ulcerans, and may thus be promising molecular tools for the differential diagnosis of infections caused by these two species.     

Comparison of three methods for rapid identification of mycobacterial clinical isolates to the species level

A new PCR-reverse dot blot hybridization (RDBH) assay was developed for the rapid identification of Mycobacterium species in clinical isolates. The assay, which targets the 16S rRNA, was evaluated for 27 mycobacterial reference strains and 340 clinical isolates that were simultaneously identified by DNA sequencing and conventional methods, including growth characteristics, pigment production, colony morphology, and biochemical tests. All reference strains and clinical isolates hybridized to the Mycobacterium genus probe (probe M) on the membrane (100% sensitivity). Each probe had only one hybridization signal with the corresponding Mycobacterium species or complex (100% specificity). Compared with DNA sequencing, the RDBH assay correctly identified 337 (99.1% accuracy) of the 340 isolates tested. One M. asia isolate and one M. neoaurum isolate were not identified by the RDBH assay due to the absence of specific probes for the two species on the membrane. Three isolates with different nucleotide sequences from M. intracellulare reference strains had a negative hybridization signal with probe c, which is specific for M. intracellulare. The whole procedure can be completed within 2.5 h post-PCR processing. A total of 32 of 340 isolates were erroneously identified by conventional methods (90.6% accuracy). Molecular identification based on the 16S rRNA sequence was superior to the conventional approaches in speed, sensitivity, and specificity. Therefore, the RDBH assay can be considered a rapid, simple, and reliable method for routine identification of frequently occurring and clinically relevant mycobacteria.