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1 National Reference Centre for Mycobacteriology, National Microbiology Laboratory, Health Canada, Winnipeg, Manitoba, Canada
2 Division of TB Control, BC Centre for Disease Control Society, Vancouver, British Columbia, Canada
3 Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada
4 Laboratoire de santé publique du Québec, Institut national de santé publique du Québec, Sainte-Anne-de-Bellevue, Québec, Canada
5 Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada
Correspondence
C. Y. Turenne
cturenne{at}hc-sc.gc.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Published online ahead of print on 4 June 2004 as DOI 10.1099/ijs.0.02940-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence of strain HSC-68T and the ITS1 and partial hsp65 gene representing each sequevar are AY337273–AY337283.
Sequence identity and Hamming distances between M. parascrofulaceum and its closest established relatives, ITS1 sequence analysis and sequence alignment of sequevars I–V are available as supplementary material in IJSEM Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The purpose of this study was to establish as a novel species a group of representative mycobacterial strains commonly found, not only in human clinical isolates obtained from our laboratory, but also in several past publications where evidence of its existence has been found. This species has shown similarities to several known NTM species using single identification methods, which may have been the cause for the delay in its establishment, but it is clearly a unique species as determined from a polyphasic approach. In one symptomatic patient, this organism was isolated in multiple respiratory samples, and associated computed tomography (CT) scan and chest X-ray showed evidence of respiratory disease, as described below. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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In February 2002, her annual screening chest X-ray showed a small peripheral cavitary lesion in the right mid-lung region. She denied symptoms of TB except for a dry cough. Sputum samples were repeated for AFB smear and culture. A CT scan of the chest showed multiple calcified granulomas with scarring in the right upper lobe and calcified pre-tracheal lymph nodes. An 18 mm thick-walled cavity in the superior segment of the right lower lobe associated with smaller satellite nodules (the largest of which measured 12 mm) was also visible. No air–fluid level was visible. Fibre-optic bronchoscopy and bronchoalveolar lavage (BAL) was performed. Samples were negative for AFB on smear. However, both sputum and bronchoscopy specimens grew an atypical mycobacterium, later identified by 16S rRNA gene sequencing as Mycobacterium sp. strain MCRO 33 (described herein as Mycobacterium parascrofulaceum sp. nov.). Bronchoscopy specimens were negative for malignancy.
Repeat chest X-rays in October 2002 confirmed an enlarging cavity in the right lower lobe. Sputum samples were obtained and empirical anti-TB treatment was instituted: daily isoniazid 300 mg, rifampicin 600 mg, ethambutol 800 mg, pyrazimamide 1 g, levofloxacin 500 mg and vitamin B6 25 mg, a regimen chosen due to concerns for atypical mycobacteria and known associated drug resistance. Her human immunodeficiency virus status was negative. After 2 months of treatment, the cavity continued to increase in size and wall thickness. Medication side-effects included oral dryness and persistent arthalgias. Liver tests remained within normal limits. Discontinuation of pyrazinamide led to resolution of her joint symptoms. The scotochromogenic mycobacterium was again isolated from her sputum and, in January 2003, the final identification and sensitivities of the organism became available. The organism was resistant to streptomycin, isoniazid, low-dose ethambutol and levofloxacin and was sensitive to rifampicin, high-dose ethambutol, clarithromycin, kanamycin, ethionamide and amikacin. On this basis, the treatment regimen was adjusted to include clarithromycin 500 mg twice daily and high-dose ethambutol 1200 mg and rifampicin 600 mg once daily, a regimen that was well tolerated and which resulted in resolution of her cough.
A follow-up CT scan of the chest was performed after 3 months and showed a decrease in the thickness of the cavity wall although the overall size of the cavity had increased from 18 to 22 mm in maximal dimension. The dominant satellite nodule had reduced in size from 12 mm to less than 5 mm. The other surrounding satellite nodules measuring less than 5 mm were still present and remained unchanged. Sputum cultures are now negative for M. parascrofulaceum.
Strain selection.
Strains used in this study were obtained in three ways. Table 1 lists 12 strains obtained from patient samples. Five of these, designated ‘HSC’, were identified by 16S rRNA gene sequencing as part of a retrospective analysis of Accuprobe-negative slowly growing NTM strains isolated at the Health Sciences Centre Mycobacteriology Laboratory, Winnipeg, Manitoba, from 1981 until 1999 (Pauls et al., 2003). These five strains had been previously identified as Mycobacterium scrofulaceum by conventional phenotypic testing and morphology. The other seven strains, designated ‘U’, were submitted to the National Reference Centre for Mycobacteriology for identification by other provincial public health laboratories from 2001 to 2003. Table 2 lists reference strains from the ATCC and TMC at our disposal, designated M. scrofulaceum or Mycobacterium simiae, and deemed potential candidates for belonging to this novel species group due to phenotypic similarities. Strains M. scrofulaceum ATCC 19981T and M. simiae ATCC 25275T were also included for comparative purposes. All strains had been stored at –80 °C in skimmed milk with glycerol and were subcultured to Bactec12B liquid medium and Middlebrook 7H10 agar at 37 °C for testing.
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; these characteristics are listed in Table 3.
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, Brown-Elliott et al. (2003), NCCLS (2003) and Wallace et al. (1986). Microdilution plates were prepared in-house using Middlebrook 7H9 broth (Difco Laboratories) supplemented with 5 % oleic acid/albumin/glucose enrichment. Seven or eight concentrations were tested for each antimicrobial agent. Stock concentrations of the following antimicrobial agents were prepared from manufacturer's powders: rifampicin, ethambutol, isoniazid, amikacin and sulfamethoxazole (Sigma), ciprofloxacin and moxifloxacin (Bayer), rifabutin and linezolid (Pharmacia), streptomycin (ICN), clarithromycin (Abbott) and levofloxacin (Jansen-Othro). The isolates were tested at a final inoculum of 103–104 c.f.u. ml–1. Microdilution plates were sealed in plastic bags and incubated at 37 °C until macroscopic growth was sufficient for interpretation (7–14 days). Minimum inhibitory concentration (MIC) was defined as the lowest concentration of drug to inhibit visible growth with the exception of sulfamethoxazole, for which the MIC is determined at 80 % inhibition of growth compared to the growth control well. Quality control was performed using Mycobacterium avium ATCC 700898, Mycobacterium marinum ATCC 927T (Wallace et al., 1986) and M. avium NJ-9141 (Heifets et al., 1993). Staphylococcus aureus ATCC 29213 was used as a quality control strain in cation-supplemented Mueller–Hinton broth when MIC ranges for mycobacteria were not available.
HPLC.
Analysis of the cell wall mycolic acids was performed by HPLC with UV detection according to standard methods (Butler et al., 1996). Briefly, the mycolic acids were cleaved from the cells by saponification, extracted with chloroform and then derivatized to UV-absorbing bromophenacyl esters. The esters were separated in a column, where they were eluted using a graded series of methanol and methylene chloride. For identification of the species, visual comparison of patterns with those of recognized species, in-house and published data, is still the most reliable procedure. The reference strain of Mycobacterium intracellulare ATCC 13950T was used as a control as well as a comparative strain.
Sequencing of the 16S rRNA gene and ITS1 region.
A loopful of cells from pure culture was suspended in sterile distilled water, boiled for 10 min and then subjected to mechanical lysis using the Mini Bead-Beater (Biospec Products). The lysates were then centrifuged for 2 min at 12 000 g and the supernatant was used for PCR. Amplification of the 16S rRNA gene (Rogall et al., 1990; Turenne et al., 2001) and the ITS1 region (Harmsen et al., 2002; Turenne et al., 2002) was performed as previously described and the PCR products were directly sequenced using forward and reverse primers and loaded on an ABI PRISM 310 Genetic Analyzer capillary sequencer (Applied Biosystems). Sequence editing and phylogenetic analyses by CLUSTAL V (neighbour-joining method) were performed using the SEQMAN and MEGALIGN components of LASERGENE 5 (DNASTAR).
Sequencing and PCR-restriction enzyme analysis (PRA) of the hsp65 gene.
A 441 bp fragment of the hsp65 gene was amplified from the DNA lysates prepared above using the protocol of Telenti et al. (1993). The resulting PCR product was sequenced using both the forward and the reverse PCR primers TB11 and TB12. Computer-generated PRA profiles of 441 bp sequences with enzymes HaeIII and BstEII were determined using MAPDRAW (dnastar) and were compared with available pra profiles from PRASITE (http://www.hospvd.ch/prasite).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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-glucosidase, acid phosphatase and 3-day arylsulfatase tests. Results were variable for the semi-quantitative catalase test and 14-day arylsulfatase test and were not attributed to any specific hsp65 or ITS1 sequevars.
Susceptibility testing
According to current NCCLS guidelines for susceptibility testing of M. avium complex and resistance breakpoints for Mycobacterium kansasii (NCCLS, 2003), all M. parascrofulaceum isolates were sensitive to rifampicin, clarithromycin, amikacin and, with the exception of one isolate (U02307), sulfamethoxazole (Table 4). Seventy-six per cent were susceptible to a streptomycin breakpoint of 10 µg ml–1. All isolates were resistant to isoniazid. Eighty-eight per cent of isolates were resistant to an ethambutol breakpoint of 5 µg ml–1 and 70 % were resistant to the ciprofloxacin breakpoint of 2 µg ml–1. If proposed breakpoints for linezolid and slowly growing mycobacteria are followed, all isolates are susceptible to this antimicrobial (Brown-Elliott et al., 2003). For moxifloxacin, 71 % of isolates had an MIC 
1·0 µg ml–1. M. scrofulaceum ATCC 19981T revealed a susceptibility pattern identical to the consensus profile for M. parascrofulaceum sp. nov. (data not shown).
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with the reference control strain M. intracellulare, the pattern produced by this particular isolate of M. parascrofulaceum overlaps with and is indistinguishable from patterns seen with isolates of M. avium complex and some M. scrofulaceum isolates (Butler et al., 1999).
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), from E. coli bp 54–510, was [M. simiae] ATCC 15080, with 100 % identity. This strain is not the type strain of M. simiae and the RIDOM results prompted us to include this strain among the misidentified reference strains suspected of belonging to M. parascrofulaceum sp. nov. The second closest match by RIDOM was M. simiae DSM 44165T with seven mismatches. Complete 16S rRNA gene sequence comparisons with M. simiae revealed a 19 bp mismatch (98·6 % identity).
Final sequence analysis was done using our in-house sequence database (in combination with GenBank sequences) and evaluating from bases 54 to 1470 on the E. coli 16S rRNA gene (Fig. 2). Sequence identity and Hamming distances against the closest established species are available as Supplementary Table A in IJSEM Online. An alignment of hypervariable regions A and B reveals signature sequences in the A region (Fig. 3), with the B region being identical to that of M. simiae.
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BLAST search analysis of sqv. I revealed closest similarity with various sequences designated M. scrofulaceum. Highest scores were for M. scrofulaceum KPM 2027 (AB026702) and M. scrofulaceum ATCC 19981T (=TMC 1323T) (L15622), both with five mismatches over 
350 bases, and identical to M. scrofulaceum ATCC 19981T determined in-house. Shorter GenBank entries (280 bases) resulting in a lower score were in fact 100 % identical to sqv. I: M. scrofulaceum C58 (AJ314885) and M. scrofulaceum ATCC 19073 (AJ314884). As these are not representative of the type strain, we assume that these also belong to M. parascrofulaceum. RIDOM analysis results indicated 100 % identity over 280 bases with [M. simiae] ATCC 15080, followed by M. scrofulaceum S343 (four mismatches) and M. scrofulaceum DSM 43992T (five mismatches) and finally sequences of M. intracellulare, with 28 nt differences and above.
Analysis of sqv. IV revealed a closest match with M. scrofulaceum DSM 43992T (11 mismatches) using RIDOM and the same M. scrofulaceum sequences as determined for sqv. I using BLAST, but with 12–15 mismatches from entries corresponding to the type strain of M. scrofulaceum or 6–7 mismatches from entries corresponding to sqv. I.
Sequencing and PRA of the hsp65 gene
Sequences of the hsp65 441 bp region of strains of M. parascrofulaceum revealed five distinct sequevars, sqv. I–V (Table 5; Supplementary Fig. B), with one or two base variations from each other, with the exception of strains U02532 (sqv. II) and U02127 (sqv. V), differing by 17 and 23 bases from sqv. I, respectively. Sequevar I was the most prevalent, followed by sqv. II, which differed from sqv. I by one base at position 129 of the 441 bp fragment. Sequevar IV, represented by strain U01260 alone, differed from sqv. I by two bases at positions 189 and 327 of the 441 bp fragment. Sequevars I, II and IV are most closely related to M. scrofulaceum ATCC 19981T, which differs from sqv. I by 16 bases. The partial protein sequences of the 441 bp fragments reveal two groups, the first consisting of sqv. I, II and IV and the other consisting of sqv. III and V. They differ from each other by three amino acids.
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Computerized restriction maps of the 441 bp hsp65 fragment as determined by MAPDRAW (LaserGene) using enzymes BstEII and HaeIII revealed a unique pattern for each sequevar, with the exception of sqv. IV, whose pattern is close to that of Mycobacterium lentiflavum type 4 as determined by PRASITE. These patterns are listed in Table 5
; fragments smaller than 50 bp were not taken into account.
M. scrofulaceum was first described by Prissick & Masson (1956) and was often grouped with M. avium, M. intracellulare and related organisms to form the so-called MAIS complex. It has long since been established as a distinct species, with its main characteristics being orange or yellow pigmentation, positive reaction for urease, negative for Tween, semi-quantitative catalase >45 mm and negative nitrate results. M. scrofulaceum is found in the environment and is an opportunistic pathogen associated with childhood lymphadenitis, disseminated infections, pulmonary disease and skin infection (Aikin & Johnston, 1978; Corbett et al., 1999; Dustin et al., 1980; Hsueh et al., 1996; Joshi et al., 1989; Murray-Leisure et al., 1987; Prissick & Masson, 1956; Sanders et al., 1995; Wolinsky, 1979, 1995). However, in most of these clinical cases, identification of the organism was obtained via conventional methods, such as pigmentation and biochemical testing. A recent increase in novel mycobacterial species due to molecular-based techniques, including scotochromogenic slowly growing species, suggests that these species probably always existed in clinical samples but were unidentifiable by conventional methodology. This idea was built upon by Tortoli (2003), who stated a possible link between a decrease in cases of M. scrofulaceum lymphadenitis and the recent discovery of several novel species of scotochromogenic slowly growing mycobacterial species.
In our study, most isolates of M. parascrofulaceum were obtained from respiratory samples. Determining the role of the organism in the disease process was difficult owing to a lack of medical records. Medical charts were reviewed for two of the Winnipeg isolates, but the isolates could not indisputably be associated with disease (Pauls et al., 2003). However, the clinical case described here represents a clear example of the pathogenic potential of this organism.
Similar species reported in other studies
We found that a great proportion of reference strains from ATCC and TMC, and also clinical isolates identified as M. scrofulaceum prior to sequence-based testing, were deemed to belong to the novel species M. parascrofulaceum. This is not surprising given the phenotypic similarities between the two species. However, molecular data, such as from sequencing of the 16S rRNA gene, hsp65 and ITS1, clearly distinguish this species from M. scrofulaceum. Springer et al. (1996) first documented this organism (which they designated MCRO 33) and described it as a unique slowly growing species related to M. simiae based on 16S rRNA gene analysis but most closely resembling M. avium–M. intracellulare–M. scrofulaceum (or MAIS complex) based on biochemical testing. Since then, other laboratories that use sequence-based methods for identification of mycobacterial species have shown the presence of this previously unidentified species in clinical samples, either by recognition of a 16S rRNA gene sequence similarity with MCRO 33 or through our own analyses of their sequences (published or deposited in GenBank).
A BLAST analysis using only the first 500 bases of the 16S rRNA gene revealed additional GenBank submissions with identical sequences to that of ‘MCRO 33’. These consisted of unspeciated strains Mycobacterium sp. N228 (AY215339) (Hall et al., 2003) and Mycobacterium sp. N625 (AY215248) (Hall et al., 2003), assumed to be derived from clinical specimens. Although using only the partial gene for analysis is satisfactory for clinical strains in a routine laboratory, it is important to bear in mind that analysis using the nearly complete 16S rRNA gene remains the best strategy for accurate eubacterial speciation and is essential for taxonomic studies.
Holberg-Petersen et al. (1999) identified a clinical isolate (strain M.16/89) as M. scrofulaceum by conventional testing, but with a clearly distinct 16S rRNA gene sequence closer to M. simiae and a lipid profile of MAIS. They suggested the strain may not belong to either M. simiae or M. scrofulaceum (Holberg-Petersen et al., 1999). Our evaluation of their 16S rRNA gene hypervariable regions A and B indicated perfect identity with M. parascrofulaceum. Tortoli et al. (2001), using a polyphasic approach to decipher the taxonomic status of unusual mycobacteria, described two strains (FI-12100 and FI-25197) identified by conventional testing as M. lentiflavum, by HPLC as MAC and by 16S rRNA gene sequencing as MCRO 33. Strain FI-25197 was obtained from blood of an AIDS patient and was therefore deemed of probable clinical significance.
Swanson et al. (1996) recognized five isolates of M. scrofulaceum as having a distinct hsp65 gene sequence (corresponding to our sqv. 1) along with a 16S rRNA gene sequence corresponding to MCRO 33, and suggested that this group probably represented a novel species. One of these strains, Lane 3081, was also part of a past study that identified this strain as having the ‘MAC-G’ ITS1 sequevar (corresponding to our ITS1 sqv. I) (Frothingham & Wilson, 1993) and has been described as ‘exhibiting cultural, biochemical and serological properties characteristic of M. scrofulaceum’ (Saito et al., 1990; Tomioka et al., 1993). Four of five clinical isolates in Brazil identified as M. scrofulaceum by da Silva Rocha et al. (2002) revealed a ‘new’ pattern similar to our sqv. II: 235/115/85-125/95, as determined by gel electrophoresis.
Roth et al. (2000), using the ITS1 spacer for mycobacterial identification, revealed five taxonomically uncertain strains closest to M. scrofulaceum. Based on a partial ITS1 alignment of these strains, we determined that one corresponds to our ITS1 sqv. III and the other four to our sqv. IIb. Roth et al. (2000) also determined that these strains had a distinctive 16S rRNA gene sequence that was identical to that of MCRO 33. Because many researchers have already detected this organism in past studies and with sequence-based methods being commonplace in laboratories, it seems inevitable that this organism be described as a distinct species. We propose the name Mycobacterium parascrofulaceum sp. nov. for this unique Mycobacterium species.
Description of Mycobacterium parascrofulaceum sp. nov.
Mycobacterium parascrofulaceum (pa.ra.scro.fu.la'ce.um. Gr. prep. para like; N.L. neut. adj. scrofulaceum specific epithet of a bacterial species; N.L. neut. adj. parascrofulaceum referring to the phenotypic resemblance of the isolate to M. scrofulaceum).
Microscopy shows pleomorphic bacillary forms of AFB with moderate beading. Strains are slowly growing and scotochromogenic and grow on Löwenstein–Jensen medium at 25–37 °C, optimally at 37 °C. Isolates are positive for urease, 68 °C catalase and pyrazinamidase; negative for Tween 80 hydrolysis, nitrate reductase, iron uptake, tolerance to 5 % NaCl, growth on MacConkey agar, niacin, -glucosidase, acid phosphatase and 3-day arylsulfatase; variable results seen for the semi-quantitative catalase test and 14-day arylsulfatase test. Morphology and biochemical profile indistinguishable from M. scrofulaceum. Isolates show in vitro susceptibility to rifampicin, clarithromycin, amikacin and sulfamethoxazole and resistance to isoniazid. The organism is almost identical to MAC and some M. scrofulaceum by HPLC. Various molecular targets confirm this organism as a distinct species. 16S rRNA gene sequence analysis reveals that the organism contains the characteristic short helix 18 of the M. simiae group and whose closest established species is the recently described M. palustre. Five sequevars each of hsp65 and ITS1 have been observed among isolates of M. parascrofulaceum, none corresponding to established strains of known species in public sequence databases.
The type strain, HSC-68T (=ATCC BAA-614T=DSM 44648T), was the first isolate studied in our laboratory and was representative of the most common hsp65 or ITS1 sequevar (I) of M. parascrofulaceum. Most isolates were from human respiratory samples and, in at least one case, was felt to be clinically significant, requiring anti-mycobacterial therapy.
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  S.-H. Wang, J. E. Mangino, K. Stevenson, M. A. Yakrus, R. Cooksey, W. R. Butler, M. Healy, M. G. Wise, L. S. Schlesinger, and P. Pancholi Characterization of "Mycobacterium paraffinicum" Associated with a Pseudo-Outbreak J. Clin. Microbiol., May 1, 2008; 46(5): 1850 - 1853. [Abstract] [Full Text] [PDF]
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 H.-S. Mun, J.-H. Park, H. Kim, H.-K. Yu, Y.-G. Park, C.-Y. Cha, Y.-H. Kook, and B.-J. Kim Mycobacterium senuense sp. nov., a slowly growing, non-chromogenic species closely related to the Mycobacterium terrae complex Int J Syst Evol Microbiol, March 1, 2008; 58(3): 641 - 646. [Abstract] [Full Text] [PDF]
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 R. Santos, J. Fernandes, N. Fernandes, F. Oliveira, and M. Cadete Mycobacterium parascrofulaceum in Acidic Hot Springs in Yellowstone National Park Appl. Envir. Microbiol., August 1, 2007; 73(15): 5071 - 5073. [Abstract] [Full Text] [PDF]
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 D. E. Griffith, T. Aksamit, B. A. Brown-Elliott, A. Catanzaro, C. Daley, F. Gordin, S. M. Holland, R. Horsburgh, G. Huitt, M. F. Iademarco, et al. An Official ATS/IDSA Statement: Diagnosis, Treatment, and Prevention of Nontuberculous Mycobacterial Diseases Am. J. Respir. Crit. Care Med., February 15, 2007; 175(4): 367 - 416. [Full Text] [PDF]
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J. L. Cloud, J. J. Meyer, J. I. Pounder, K. C. Jost Jr, A. Sweeney, K. C. Carroll, and G. L. Woods Mycobacterium arupense sp. nov., a non-chromogenic bacterium isolated from clinical specimens Int J Syst Evol Microbiol, June 1, 2006; 56(6): 1413 - 1418. [Abstract] [Full Text] [PDF]
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M. Drancourt and D. Raoult Sequence-Based Identification of New Bacteria: a Proposition for Creation of an Orphan Bacterium Repository J. Clin. Microbiol., September 1, 2005; 43(9): 4311 - 4315. [Full Text] [PDF]
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E. Tortoli, L. Chianura, L. Fabbro, A. Mariottini, N. Martin-Casabona, G. Mazzarelli, C. Russo, and M. Spinelli Infections Due to the Newly Described Species Mycobacterium parascrofulaceum J. Clin. Microbiol., August 1, 2005; 43(8): 4286 - 4287. [Abstract] [Full Text] [PDF]
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E. Tortoli, L. Rindi, K. S. Goh, M. L. Katila, A. Mariottini, R. Mattei, G. Mazzarelli, S. Suomalainen, P. Torkko, and N. Rastogi Mycobacterium florentinum sp. nov., isolated from humans Int J Syst Evol Microbiol, May 1, 2005; 55(3): 1101 - 1106. [Abstract] [Full Text] [PDF]
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