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A nonpigmented rapidly growing mycobacterium was isolated from wound liquid outflow, bone tissue biopsy, and excised skin tissue from a 31-year-old woman who suffered an accidental open right tibia fracture and prolonged stay in a river. The three isolates grew in 3 days at 24 to 37°C. 16S rRNA sequence analyses over 1,483 bp showed that they were identical and shared 99.7% (4-bp difference) sequence similarity with that of Mycobacterium porcinum, the most closely related species. Partial rpoB (723 bp) sequence analyses showed that the isolates shared 97.0% sequence similarity with that of M. porcinum. Further polyphasic approaches, including biochemical tests, antimicrobial susceptibility analyses, and hsp65, sodA, and recA gene sequence analysis, as well as % G+C determination and cell wall fatty acid composition analysis supported the evidence that these isolates were representative of a new species. Phylogenetic analyses showed the close relationship with M. porcinum in the Mycobacterium fortuitum group. The isolates were susceptible to most antibiotics and exhibited evidence for penicillinase activity, in contrast to M. porcinum. We propose the name Mycobacterium conceptionense sp. nov. for this new species associated with posttraumatic osteitis. The type strain is D16T (equivalent to CIP 108544T and CCUG 50187T).
Mycobacterium fortuitum group species are, among rapidly growing mycobacteria (RGM), encountered as emerging opportunistic pathogens (8, 36). Reports of new M. fortuitum group species have steadily increased during the last decade (23, 24). The M. fortuitum group now comprises M. fortuitum, Mycobacterium peregrinum, Mycobacterium senegalense, Mycobacterium alvei, Mycobacterium houstonense, Mycobacterium neworleansense, Mycobacterium boenickei, Mycobacterium septicum, and Mycobacterium porcinum (2, 4, 24). Emerging infections include skin and soft tissue infection (usually following penetrating trauma) characterized by slowly progressive granulomatous inflammation, lymphadenitis, skeletal and pulmonary infections, and catheter-related, disseminated infection in immunocompromised patients (8, 24, 25, 35). Water has been shown as an important source for these opportunistic mycobacteria (9, 10, 36, 37). This fact was illustrated by Mycobacterium fortuitum furunculosis following footbaths (41) and disseminated infection in leukemia patient (12). We herein report on a clinically significant case of posttraumatic osteitis caused by a novel species of RGM that presents a unique combination of genotypic and phenotypic characteristics.
A previously healthy 31-year-old woman suffered an accidental open right tibia fracture when canyoning on Reunion Island, close to Madagascar Island in the Indian Ocean, in September 2002. She remained in the river for 2 h before rescue. She underwent osteosynthesis with a centromedullar lock nail and treatment with amoxicillin combined with clavulanic acid 3 g/day, and she was then transferred to our hospital. Wound cicatrization evolved favorably over 3 months, and antibiotic treatment was stopped in December 2002. In January 2003, the wound opened and wound liquid began to outflow. Surgical drainage proved necessary, and three additional surgical samples obtained from wound liquid outflow, bone tissue biopsy, and excised skin tissue were submitted for routine bacteriology and mycology. Gram staining revealed no bacteria, but numerous polymorphonuclear leukocytes and standard culture remained sterile. Microscopic analysis after Ziehl-Neelsen staining revealed a few acid-fast bacilli in each specimen, which formed colonies in pure culture after 3 days of inoculation.
The three isolates were recovered from wound liquid, bone tissue biopsy, and excised skin tissue specimen after direct inoculation into BACTEC 9000MB broth according to the manufacturer's instructions (BD Biosciences, Sparks, Md.). They were subcultured on Middlebrook 7H10 agar, egg-based Lowenstein-Jensen slants (BioMérieux, La Balme-les-Grottes, France), and 5% sheep blood agar (BioTechnologie Appliquée, Dinan, France) at 37°C under a 5% CO2 atmosphere. One of these isolates, designated D16T, has been deposited in the Pasteur Institute Collection and the Culture Collection of the University of Göteborg under the accession numbers CIP 108544T and CCUG 50187T, respectively. We observed colony morphology, pigmentation, and the ability of the isolate to grow at various temperatures (24, 30, 37, 42°C) on 5% sheep blood agar, Middlebrook 7H10 agar, and Lowenstein-Jensen slants in the presence of 5% sodium chloride (NaCl). We tested the activities of arylsulfatase and catalase, iron uptake, and degradation of p-aminosalicylic acid (13, 34). Additional biochemical tests were performed by inoculation of API Coryne and API 20E strips (BioMérieux) (3) according to the manufacturer's instructions with an incubation time of 5 days at 30°C under a highly humidified atmosphere.
The MIC of rifampin, ciprofloxacin, ofloxacin, sparfloxacin, doxycycline, minocycline, erythromycin, clarithomycin, azithromycin, amikacin, penicillin, amoxicillin, imipenem, cefotaxime, ceftriaxone, metronidazole, teicoplanin, and vancomycin was determined by incubation with the respective E-test (AB Biodisk, Solna, Sweden) at 30°C for 3 days (42). For all drugs, the MIC was recorded as the point of intersection between the zone edge and the E-test strip. Since the breakpoints for determining the susceptibility of RGM using the E-test method have not been standardized or approved by the Clinical Laboratory Standards Institute (CLSI; formerly NCCLS), the breakpoints for susceptibility used were those of the CLSI for broth microdilution interpretative criteria (NCCLS M100-S12 and M24-A) (18, 19) and those proposed by Brown-Elliott and Wallace (8). The disk diffusion method on 5% sheep blood agar for 3 days at 30°C was used to determine the susceptibility to pipemidic acid (30 μg) (38), cephalothin (30 μg) (39), and tobramycin (10 μg) (7) as previously described and to amoxicillin-clavulanate (disk 20 μg plus 10 μg) and trimethoprim-sulfamethoxazole (disk 1.25 μg plus 23.75 μg). Every test was done three times on three separate days to ensure reproducibility of the results.
DNA was extracted from colonies grown on 5% sheep blood agar using the Fast-prep device and the FastDNA kit according to the recommendations of the manufacturer (BIO 101, Inc., Carlsbad, Calif.). We performed the amplification and sequencing of the 16S rRNA (40), sodA (2), hsp65 (28), recA (5), and rpoB (1) genes. The partial 764-bp rpoB gene was amplified using primer pair Myco-F (5′-GGCAAGGTCACCCCGAAGGG-3′) and Myco-R (5′-AGCGGCTGCTGGGTGATCATC-3′), and the 723-bp sequence (excepting 41 nucleotides at both ends of the amplicon, corresponding to primer binding sites) was derived from that amplicon by using the same primer pair in both directions (1). Products of sequencing reactions were recorded with an ABI Prism 3100 DNA sequencer following the standard protocol of the supplier (Perkin Elmer Applied Biosystems, Foster City, Calif.). The percentages of similarity between the sequences were determined using the Clustal W program supported by the PBIL website. For phylogenetic analyses, sequences were trimmed to start and finish at the same nucleotide position for all the isolates. Multisequence alignment was performed by using the Clustal X program, version 1.81, in the PHYLIP software package (29). A phylogenetic tree was obtained from DNA sequences by using the neighbor-joining method with Kimura's two-parameter distance correction model with 1,000 bootstrap replications in the MEGA, version 2.1, software package (15) and was rooted by using Mycobacterium tuberculosis and Mycobacterium leprae.
The mol% G+C content of DNA was determined by high-performance liquid chromatography according to the work of Mesbah et al. (17), except that a Waters 625 LC system with a Waters 486 tenable absorbance detector and a Waters 746 data module (Millipore, Saint Quentin en Yvelines, France) were used. Three determinations were done. Total fatty acid methyl esters were extracted and analyzed by gas chromatography (MIDI Sherlock, Newark, NJ) as previously described (4).
The sequences determined for M. conceptionense (D16T = CIP 108544T = CCUG 50187T) in this study have been deposited into GenBank under the following accession numbers: 16S rRNA, AY859684; rpoB, AY859695; hsp65, AY859678; sodA, AY859708; recA, AY859690.
A group of isolates or species reported as M. fortuitum third biovariant sorbitol-negative group consists of at least 4 species, i.e., M. porcinum, M. septicum, M. boenickei, and M. neworleansense (8, 24). Phenotypic features of species of this group include that they utilize mannitol and inositol but not sorbitol (d-glucitol) and rhamnose or citrate as sole carbon sources (24, 37, 38). Isolates herein reported as representative of a new species exhibited a profile similar to that of M. porcinum (Table (Table1).1). In our hands, M. porcinum did not utilize mannitol as a sole carbon source, although this characteristic was reported positive among 47 M. porcinum isolates, including the type strain, using standard techniques (37). This discrepancy may be due to the use of the Api 20E strip as an alternative identification strip. Therefore, this new species is suspected to belong to this group. This new species exhibited positive activity for acetoin and differed from its closest relative, M. porcinum, by exhibiting positive nitrate reductase activity (Table (Table1).1). A negative nitrate reductase activity appears to be specific for the porcine type strain of M. porcinum, since all human isolates tested have been positive (37). The three isolates herein described were susceptible to imipenen, minocycline, doxycycline, clarithromycin, erythromycin, azithromycin, amikacin, ciprofloxacin, ofloxacin, and sparfloxacin using E-test (Table (Table2).2). MICs determined using E-tests were found to be higher than those obtained by reference broth microdilution in RGM (42). MICs determined for M. porcinum in this study using E-test are similar to that reported using the reference broth microdilution method (37). The antibiotic susceptibility profile is helpful for the identification of the new species. However, they should not be extrapolated to clinical management of patients. In contrast to M. porcinum, the isolates were resistant to penicillin and amoxicillin but susceptible to amoxicillin-clavulanate. They exhibited a positive cefinase test, suggestive of a penicillinase activity.
Isolate D16T exhibited a fatty acid profile diagnostic for members of the genus Mycobacterium with similarities of 95.8% with M. porcinum and 93.4% with M. fortuitum. These results suggest that the D16T strain is different from the closely related species. It was composed of straight chain saturated and unsaturated fatty acids including 16:0 (27.1%), 18:1 w9c (24.7%), 16:1 w7c/15:0 ISO (14.4%), 14:0 (10.8%), and TBSA 10Me18:0 (8.9%). Isolate D16T was characterized by small amounts of 18:2 w6,9c/18:0 ANTE (2.1%) and 16:1 w7c (1.3%) (Table (Table3).3). The % G+C value was 64% ± 2%.
Isolate D16T shared with M. porcinum and M. fortuitum, respectively, 99.7% (4-bp difference) and 99.4% (8-bp difference) similarity in the 16S rRNA gene, 97.0% and 95.7% similarity in 723 bp of the rpoB gene, 96.4% (15-bp difference) and 98.3% (7-bp difference) similarity in the hsp65 gene, 96.0% and 94.8% similarity in the sodA gene, and 95.0% and 94.8% similarity in the recA gene. Complete rpoB gene sequence analyses of isolate D16T provided similar homologies (97.2% and 96.8% with M. porcinum and M. fortuitum, respectively) as the partial rpoB sequence analysis, as previously described (1). An rpoB phylogenetic tree was created that included 24 sequences from 21 established RGM species tested in our laboratory in addition to that of isolate D16T (Fig. (Fig.1).1). This analysis suggested that isolate D16T belongs to the M. fortuitum group and was recently derived from M. porcinum. A bootstrap value of 90% in the neighbor-joining tree supported the fork separating isolate D16T from M. porcinum. The isolate D16T lineage was clearly different from that of closely related species and quite distant from other recognized RGM.
16S rRNA gene sequencing has been used as the first line method for identifying unusual mycobacterial isolates (6, 21, 30, 31, 33). This approach resulted in the description of 45 new Mycobacterium species isolated from clinical sources during the last 10 years (4, 30). sodA (44), dnaJ (27), 32-kDa protein encoding gene (26), hsp65 (22), recA (5), internal transcribed spacer 16S-23S rRNA (20), DNA gyrase (11), and secA1 (43) gene analyses were proposed as alternative tools for the molecular identification of RGM isolates. However, a validated criterion for the delineation of new RGM species was disponible only for the rpoB gene (1, 3, 4). This single-copy gene was previously the basis for the molecular identification of Mycobacterium spp. (1, 14, 16). We herein found that isolate D16T exhibited 3% rpoB gene sequence divergence with the closest species, M. porcinum. We previously showed that different RGM isolates belonged to a new species if they exhibited >3% rpoB sequence divergence from established species using the partial 723-bp rpoB sequence (1, 3, 4). We further found that isolate D16T shared 95.0% recA sequence similarity with M. porcinum when a 98.7 to 100% intraspecies similarity was found over 915 bp (5). As for hsp65 gene analysis, our data are comparable to those reported for the description of M. neworleansense, which differed from M. porcinum by 1 bp in 16S rRNA gene sequence and by 11 bp in the hsp65 gene sequence (24, 37). Also, an hsp65 gene sequence-based analysis of M. porcinum isolates disclosed 4 sequevars differing by 2 to 3 bp (37). We now found that the isolate D16T hsp65 gene sequence differed from that of the M. porcinum type strain by 15 bp. Sequences herein determined were made public by deposition in GenBank, allowing clinical microbiologists to develop an alternative molecular technique for identification, such as profile restriction analysis (37).
These findings, in addition to unique phenotypic characteristics and cell wall fatty acid composition, allow us to propose isolate D16T as representative of a new species within the Mycobacterium genus.
The pathogenic role of the three isolates was supported by several lines of evidence. First, they were microscopically observed in three different surgical purulent specimens following Ziehl-Nielsen staining. Also, no such isolate was made in our laboratory during the same period of time. Second, the 3 isolates were isolated in pure culture. No other mycobacterium had been isolated from the orthopedic service patients for more than 1 year around the time of the isolation of the 3 isolates and no other organism similar to isolate D16T was isolated in our mycobacteriological laboratory during this period. Third, the isolates were made from diseased tissues in a patient with clinical and radiological signs of osteitis. Fourth, wound cicatrization evolved favorably over 3 months and antibiotic treatment was stopped. A few RGM species have been previously associated with postsurgical infections (8, 37, 38) or infection following exposure of open fractures to natural waters (32). It was not possible to determine whether the infections represented by the isolates herein reported were contracted during the long immersion of the fractured leg in the river water or during initial surgical operation on Reunion Island. Bibliographic data are in favor of the first hypothesis (8, 38). Indeed, this new species is a member of the M. fortuitum third biovariant complex, and osteomyelitis after open fracture is common with this group of opportunistic pathogens (38). This case illustrates that all clinical isolates of RGM should be accurately identified to the species level. This goal is better achieved by using molecular identification, and present data indicate that selected partial rpoB gene sequencing contributes to the accurate identification of emerging RGM in humans.
The organisms are acid-fast and gram-positive bacilli. Colonies are nonpigmented and appear on 5% sheep blood agar, Middlebrook 7H10 agar, and egg-based Lowenstein-Jensen slants in 2 to 5 days at temperatures between 25 and 37°C, optimally at 30°C. No growth occurs at 42°C. This species is associated with posttraumatic osteitis. It is susceptible in vitro to imipenem, minocycline, doxycycline, clarithromycin, erythromycin, azithromycin, amikacin, ciprofloxacin, ofloxacin, sparfloxacin, and amoxicillin-clavulanate and resistant to penicillin, amoxicillin, and vancomycin. It is positive for 3-day arylsulfatase, penicillinase, pyrazimidase, phosphatase alkaline, iron uptake, and acetoin production activities and negative for urease. It utilizes inositol but not sorbitol (d-glucitol), rhamnose, or citrate as sole carbon sources. It shares 99.7% 16S rRNA (4-bp difference) and 97.0% rpoB gene sequence similarity with M. porcinum, the nearest species. The G+C content of DNA is 64 ± 2 mol%. The type strain, which was recovered from wound liquid, is strain D16T (equivalent to CIP 108544T and CCUG 50187T).
We thank Christian de Fontaine for technical assistance and Esther Platt for expert reviewing of the manuscript.