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1 Laboratorio de Micobacterias, Servicio de Bacteriología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain
2 Servicio de Microbiología, Hospital de Gran Canaria Dr Negrin, Las Palmas de Gran Canaria, Spain
3 Unitat de Microbiología, Departament de Genetica i de Microbiología, Universitat Autónoma de Barcelona, Bellaterra, Barcelona, Spain
4 Departamento de Medicina Preventiva, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain
Correspondence
M. Soledad Jiménez
msjimenz{at}isciii.es
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- and '-mycolates in their cell wall. Sequence analysis of the hsp65 and 16S rRNA genes identified the isolates as rapidly growing mycobacteria. Sequences of both genes were unique within the mycobacteria. DNA–DNA hybridization showed that the isolates had less than 15 % reassociation with 13 other recognized rapidly growing mycobacteria. The name Mycobacterium canariasense sp. nov. is proposed for this novel opportunistic pathogen, which is most closely related to Mycobacterium diernhoferi. The type strain is 502329T (=CIP 107998T=CCUG 47953T).
Some characteristics of strain 502329T (Table A), TLC of methyl mycolates of M. canariasense strains and related Mycobacterium species (Fig. A), GC analysis of fatty acid methyl esters of strain 502329T (Fig. B), hybridization dot-blot assays (Fig. C) and DNA–DNA relatedness between strain 502329T and other Mycobacterium species (Table B) are available as supplementary material in IJSEM Online.
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). Members of this group, especially the three major species Mycobacterium fortuitum, Mycobacterium chelonae and Mycobacterium abscessus, have been reported as aetiological agents of a variety of infections, including bacteraemia and disseminated disease in patients with long-term venous catheters (Raad et al., 1991). They have also been involved in nosocomial outbreaks or pseudo-outbreaks related to contamination of hospital water supplies and reagents (Ashford et al., 1997; Chadha et al., 1998; LaBombardi et al., 2002). In this study, a novel mycobacterium involved in nosocomial infection is described; the name proposed for this species is Mycobacterium canariasense sp. nov. During the period January 2000 to September 2002, a mycobacterium that could not be identified by conventional procedures was isolated from blood specimens of 17 patients with suspected nosocomial acquisition. Patients were admitted to a tertiary care hospital in the Canary Islands, Spain. Most of them (n=15) had a malignant disease and all of them carried at that time a central venous catheter. Mycobacteria were considered to be the cause of the febrile syndrome in 12 cases. Ten patients were treated with specific antibiotic therapy and the central catheter was removed in nine of them. Catheters were left in two patients, who later relapsed. The same mycobacterium grew in their subsequent blood cultures. Most of the patients recovered after treatment. However, two patients died as a consequence of their disease status. All bacterial isolates had homogeneous biochemical characteristics and antimicrobial susceptibility patterns. The first isolate and four other randomly selected strains were sent to the reference Laboratory of Mycobacteria (Centro Nacional de Microbiología, Madrid, Spain) for identification. These five isolates were examined for several phenotypic and genotypic characteristics and results were compared with those displayed by other rapidly growing mycobacterial species.
Acid–alcohol-fastness, colony morphology and pigment production, as well as the ability to grow at various temperatures (22–45 °C), on Löwenstein–Jensen medium (LJ), in the presence of 5 % NaCl and on MacConkey agar without crystal violet (Kubica & David, 1980) were examined. A total of 12 biochemical tests was performed (Marks & Trollope, 1960), including the use of single carbon sources and acid formation (Silcox et al., 1981; Tsukamura, 1967). Hydrolysis of seven different amides was also tested (Vestal, 1975). Sensitivities to eight different antimycobacterial drugs were tested by the proportion method in LJ (Canetti et al., 1963). E-test strips on Mueller–Hinton medium were used to determine sensitivity to 13 further antibiotics.
Bacterial cells were partially acid-fast rods that grew on LJ, initially as non-pigmented colonies, but later developing a more yellowish, smooth, moist and shiny appearance. Growth on LJ occurred after 2–3 days at 30 and 37 °C; growth did not occur at the other temperatures tested. The main phenotypic characteristics of isolates are indicated in Table 1 (see supplementary material in IJSEM Online for a complete set of data). All isolates of M. canariasense exhibited identical characteristics. The novel species could be differentiated from the M. chelonae group by single carbon source testing, from M. fortuitum and M. diernhoferi by the nitrate reduction test and from Mycobacterium frederiksbergense and other related species by pigmentation development (Table 1).
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). Mycolic acid methyl esters were studied by analytical one-dimensional TLC using silica gel 60 TLC plates (Merck) as described previously (Luquín et al., 1991a). Fatty acid methyl esters and methyl mycolate cleavage products were determined by GC-MS (Luquin et al., 1991b).
TLC analysis of the mycolic acid methyl esters revealed that the five M. canariasense strains contained - and '-mycolates (see supplementary material available in IJSEM Online). To date, this mycolate pattern has only been described in M. chelonae and M. abscessus (see Table 1
) (Hinrikson & Pfyffer, 1994) and species within the genus Tsukamurella (Hamid et al., 1993). This mycolate pattern allows M. canariasense to be differentiated from related species such as Mycobacterium mucogenicum and M. diernhoferi (see Table 1; Muñoz et al., 1997). GC-MS analysis of M. canariasense strains indicated the presence of fatty acid methyl esters with 14–24 carbon atoms, of which hexadecanoate (C16 : 0) and octadecenoate (C18 : 1) were the most prominent. All strains also showed appreciable amounts of tuberculostearate. The primary methyl ester derived from thermal cleavage of methyl mycolates was tetracosanoate (C24 : 0; see supplementary material available in IJSEM Online), which allows M. canariasense to be differentiated from Tsukamurella. Species of this genus released C20 and C22 esters from pyrolysis of mycolates, but not C24 (Goodfellow et al., 1978). Unlike M. canariasense, strains of M. diernhoferi, M. frederiksbergense, M. mucogenicum and Mycobacterium neoaurum revealed secondary alcohols by GC-MS analysis; thus, the absence of these compounds in M. canariasense strains enables them to be clearly differentiated from these closely related species.
PCR was performed on DNA isolated from bacterial LJ cultures using the boiling method and centrifugation. A 439 bp region of the hsp65 gene was subjected to PCR amplification followed by restriction analysis using the primers and conditions described by Telenti et al. (1993). Amplicons were also sequenced in triplicate according to Ringuet et al. (1999) using the BigDye Terminator sequencing kit and the ABI Prism 3700 automated sequencer (Applied Biosystems). In addition, 1514 bp of the 16S rRNA gene were sequenced from PCR amplicons produced as described by Springer et al. (1995). Sequences of hsp65 and 16S rRNA genes were aligned against previously described sequences from rapidly growing mycobacterial species, using EDITSEQ and MEGALIGN software (Lasergene MegAlign; DNAstar) (Altschul et al., 1997). Sequences were clustered using CLUSTAL W and weightings were used to construct a phylogenetic dendrogram (Kimura, 1980).
The hsp65 gene of M. canariasense strains contained a PCR-RFLP pattern that differed from those published previously or compiled in the PRAsite database (http://www.hospvd.ch:8005). Patterns consisted of two fragments of 325 and 130 bp by BstEII digestion and three fragments of 140, 90 and 80 bp by HaeIII digestion. The sequence of the M. canariasense hypervariable region (Ringuet et al., 1999) was significantly different from those of other closely related rapidly growing species. Phylogenetic analysis of 441 bp of this gene demonstrated that M. diernhoferi and M. mucogenicum are the closest non-pigmented relatives (94·3 and 93·5 % similarity, respectively), whereas M. neoaurum and M. frederiksbergense were the most closely related pigmented species (94·4 and 93·6 % similarity, respectively) (Fig. 1).
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). The complete sequence of the 16S rRNA gene of M. canariasense shows substantial differences when compared with those corresponding to other rapidly growing mycobacteria. Fig. 2 shows a comparison of the M. canariasense 16S rRNA gene sequence with 10 other closely related mycobacteria; Tsukamurella paurometabola was used as an outgroup. The dendrogram shows that M. diernhoferi is the closest non-pigmented relative (99·2 % similarity) to M. canariasense, followed by M. mucogenicum (97·0 %) and, as seen previously with the hsp65 gene, M. neoaurum was the most closely related pigmented rapidly growing mycobacteria (98·7 %). The similarity between M. canariasense and T. paurometabola was 91·2 %.
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). DNA–DNA hybridization experiments were performed on membrane filters, using a novel dot-blot-based procedure. Genomic DNA from M. canariasense strain 502329T (300 ng) was labelled in vitro using the Megaprime DNA labelling system (Amersham) and 25 µCi (925 kBq) [-32P]dCTP (Amersham). Portions (500 ng) of each unlabelled DNA were dot-blotted and bound to nylon membrane filters (Hybond-N+; Amersham). Hybridization and washes were carried out as described previously (Brown et al., 1999), except that ECL buffer (Amersham) was used both for pre-hybridization and hybridization steps. The relative binding ratios (expressed as percentages) for each species were calculated from the counts of homologous bound DNA, as measured using an Instant Imager counter (Izasa Instruments). In addition, the blots were stripped and rehybridized with a PCR amplicon from the 16S rRNA gene as the new probe, as described by Domenech et al. (1997). This procedure has several advantages compared to previous methods (Keswani & Whitman, 2001), mainly because the amount of DNA fixed in each dot can be determined by measuring the amount of radioactivity when using the 16S rRNA gene as the second probe. Due to the high sequence conservation within mycobacterial species inherent within this probe (Menendez et al., 2002), the radioactivity level can be considered to be proportional to the amount of target molecules on the filter. The number of copies of the 16S rRNA gene was already known for the mycobacterial species included in this work (Menendez et al., 2002; M. J. Garcia, unpublished results). The levels of DNA–DNA hybridization between M. canariasense strains were greater than 90 %. On the other hand, the level of reassociation between the M. canariasense type strain and 13 other related rapidly growing species corresponded to less than 15 %. These results strongly suggest that our isolates represent a novel species (Lan & Reeves, 2001). Results of DNA–DNA hybridization analysis are available as supplementary material in IJSEM Online.
RFLP analysis of the 16S rRNA gene was also performed to complement DNA–DNA hybridization data (Domenech et al., 1994, 1997; Brown et al., 1999). A previously described experimental procedure (Domenech et al., 1997) was used, with the exception that ECL buffer (Amersham) was used for both the pre-hybridization and hybridization steps. Fig. 3 shows the RFLP patterns from BamHI-digested genomic DNAs from M. canariasense strains, eight other rapidly growing mycobacterial species and Nocardia asteroides. Patterns of different species were different, but the patterns of all four M. canariasense strains were identical. All species tested, with the exception of M. abscessus, produced a pattern with two bands, indicating the presence of two copies of the 16S rRNA gene. These results indicate that M. canariasense belongs to the II-s mycobacterial class, i.e. species with two rrn operons per genome and short helix 18 in the 16S rRNA gene coding region (Menendez et al., 2002).
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Description of Mycobacterium canariasense sp. nov.
Mycobacterium canariasense (ca.na.ri.a.sen'se. L. gen. adj. canariasense referring to the Latin adjective of the Spanish islands where all strains were isolated).
Cells are partially acid-fast rods. Visible growth appears in 2–3 days as smooth, moist, shiny, non-pigmented colonies on Löwenstein–Jensen medium. Growth occurs at 30 and 37 °C, but not at 22, 42 or 45 °C. Grows on MacConkey agar without crystal violet, but does not grow in the presence of 5 % NaCl. Positive for arylsulfatase activity (3 days) and Tween 80 hydrolysis. Produces a low level of heat-stable catalase and is negative for reduction of nitrates. These characteristics allow this novel species to be differentiated from other closely related species such as M. diernhoferi and M. mucogenicum. The inability to utilize citrate as a single carbon source allows differentiation from M. chelonae. Lipid composition of the cell wall is characterized by the presence of - and '-mycolates, similar to M. chelonae and M. abscessus. Results of DNA analyses such as PCR-RFLP of the hsp65 gene, sequences of conserved genes and DNA–DNA hybridization define M. canariasense as a distinct genomic mycobacterial species most closely related to M. diernhoferi and M. mucogenicum. The 16S rRNA and hsp65 gene sequences of M. canariasense are unique. This species belongs to the II-s mycobacterial class according to the classification of Menendez et al. (2002).
The type strain is 502329T (=CIP 107998T=CCUG 47953T).
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ACKNOWLEDGEMENTS |
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