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1 Regional Reference Center for Mycobacteria, Microbiology and Virology Laboratory, Careggi Hospital, 50134 Florence, Italy
2 Department of Experimental Pathology, Medical Biotechnologies, Infectivology and Epidemiology, University of Pisa, 56127 Pisa, Italy
3 Unité de la Tuberculose et des Mycobactéries, Institut Pasteur de Guadeloupe, 97165 Pointe-à-Pitre, Guadeloupe, France
4 Department of Clinical Microbiology, Kuopio University Hospital, 70211 Kuopio, Finland
5 Genetics and Cytogenetics Unit, Careggi Hospital, 50134 Florence, Italy
6 Clinical Laboratory, Campo di Marte Hospital, 55100 Lucca, Italy
7 Microbiological and Virological Serum-immunology Laboratory, Careggi Hospital, 50134 Florence, Italy
8 Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland
9 Laboratory of Environmental Microbiology, National Public Health Institute, 70211 Kuopio, Finland
Correspondence
Enrico Tortoli
e.tortoli{at}libero.it
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ABSTRACT |
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Published online ahead of print on 9 December 2004 as DOI 10.1099/ijs.0.63485-0.
The GenBank/EMBL/DDBJ accession number of 16S rRNA gene and ITS region sequence of strain FI-93171T is AJ616230.
Antimicrobial susceptibility data for three strains of M. florentinum are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). A group genetically related to Mycobacterium terrae has been recognized within the slowly growing mycobacteria, and a group of thermotolerant mycobacteria has been characterized within the rapid growers (Tortoli, 2003). However, the more striking new taxonomic entity is the one related to Mycobacterium simiae, which presents genetic features typical of rapid growers despite the fact that its members unquestionably belong to slowly growing species, requiring at least 2 weeks of incubation (Tortoli, 2003). The only species known initially to present such unique features was M. simiae; however, since 1993, this species has been joined by many others to embrace 14 taxa at present (Böttger et al., 1993; Fanti et al., 2004; Floyd et al., 1996, 2000; Haas et al., 1997; Levi et al., 2003; Meier et al., 1993; Selvarangan et al., 2004; Springer et al., 1993, 1996; Torkko et al., 2002; Turenne et al., 2004a, b). Eight mycobacterial strains isolated from clinical human samples drew our attention as they presented an identical lipid profile by HPLC, which differed from known HPLC patterns. Genetic characterization showed that they belonged to a novel species characterized by the genetic properties of the M. simiae group. We propose the name Mycobacterium florentinum sp. nov. for this species.
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METHODS |
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) hospitalized in Italy (seven cases) or in Finland (one case). Six strains were isolated, two or more times, from the sputum of elderly patients. The others were grown from the stools of an AIDS patient and from the excised cervical lymph node of a young girl. Media used for the primary growth varied according to the procedures adopted by different isolating laboratories. Four of the strains were grown on Löwenstein–Jensen medium and in broth (radiometric Bactec, MGIT or BacT Alert), three in liquid medium only and one on Löwenstein–Jensen medium only.
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). Biochemical tests included niacin accumulation, nitrate reduction, Tween 80 hydrolysis, urea hydrolysis, tellurite reduction, 
-glucosidase, 3 day arylsulfatase, and semi-quantitative and thermostable catalase. Cultural tests were performed on Löwenstein–Jensen medium to investigate the growth rate, the ability to grow at different temperatures (25, 37 and 45 °C) and the possible production of pigmentation. The ability to grow on MacConkey's agar and the inhibitory effect of several substances [5 % (w/v) NaCl, 1 µg isoniazid ml–1, 500 µg hydroxylamine ml–1, 250 µg oleic acid ml–1, 500 µg p-nitrobenzoic acid ml–1, 10 µg thiacetazone ml–1] were also tested.
Chromatographic investigation of the cell-wall lipids.
The lipid composition was investigated with different chromatographic approaches. TLC was run on silica gel after methyl esterification (Minnikin et al., 1984) and spots corresponding to mycolic acids were identified by comparison with those of authentic reference strains run in parallel.
Mycolic acid cleavage products, saturated and unsaturated fatty acids and secondary alcohols were investigated with GLC. The fatty acids were extracted as methyl esters by means of methanolysis and injected in a temperature-controlled column where the pyrolysis of mycolic acid occurred (Torkko et al., 2003). GLC and TLC were performed on four strains only (FI-93171T, FI-93188, FI-94029 and 1696).
All the test strains were also subjected to HPLC. Cell-wall mycolic acids, once saponified and chloroform-extracted, were derivatized to UV-adsorbing mycolic esters and then separated with a gradient of methanol and methylene chloride (Butler et al., 1996).
Antimicrobial susceptibility testing.
Antimicrobial susceptibility was investigated in three strains (FI-93171T, FI-93199, FI-94029) using the macrodilution radiometric method developed for Mycobacterium avium complex (MAC). This was made possible as the growth rate of the test strains, very similar to that of MAC, meant that all the requirements of the technique were fulfilled (Siddiqi et al., 1993). Antimicrobial agents are listed in a supplementary table available in IJSEM Online.
Genetic sequencing and phylogenetic investigations.
Complete sequences of the 16S rRNA gene and the internal transcribed spacer (ITS) were determined using primers and PCR protocols described previously (Kirschner et al., 1993; Roth et al., 1998). The PCR products were sequenced with an automated apparatus (ALFexpress DNA sequencer; Pharmacia Biotech) using the Thermo Sequenase fluorescent labelled primer cycle-sequencing kit with 7-deaza-dGTP and the Thermo Sequenase Cy5 dye terminator kit (Amersham Pharmacia Biotech). Both the forward and the reverse strands of each strain were sequenced.
The nucleotide sequences were compared with those present in the GenBank and RIDOM (Harmsen et al., 1999) databases. Furthermore, they were aligned with sequences from all other species that were characterized by genetic relatedness to M. simiae by using the CLUSTAL W program (European Bioinformatics Institute; http://www.ebi.ac.uk/clustalw/).
The neighbour-joining method was used for the construction of a phylogenetic tree (Felsenstein, 1993). The tree was rooted using Mycobacterium tuberculosis as the outgroup. Tree branches were reproduced by performing 100 bootstrap replicates.
PCR restriction analysis (PRA).
PRA of the 65 kDa heat-shock protein gene (hsp65) was performed as previously reported using the enzymes BstEII and HaeIII and separating the digestion products by agarose gel electrophoresis (Telenti et al., 1993). A third enzyme, HhaI, was used to differentiate species with overlapping PRA patterns.
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RESULTS AND DISCUSSION |
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Phenotypic features
The strains grew in about 2 weeks at 25 and 37 °C, but not at 45 °C and formed smooth, creamy, non-chromogenic colonies. They reduced tellurite and hydrolysed urea, were unable to accumulate niacin or to hydrolyse Tween 80 and did not possess -glucosidase or arylsulfatase. Nitrate reduction was positive in five of the strains and negative or weakly positive in the others; catalase too was variable. Mycobacterial growth was inhibited on MacConkey's agar and on Löwenstein–Jensen medium supplemented with NaCl. No inhibition was observed on media containing isoniazid, hydroxylamine, oleic acid, p-nitrobenzoic acid or thiacetazone. The above phenotypic tests did not reveal features helpful in differentiating the organisms from M. triplex or (apart from colony morphology) from M. lentiflavum.
TLC revealed the presence of alpha-, methoxy- and keto-mycolates, a pattern very common within the genus Mycobacterium, presented also by M. tuberculosis.
GLC analysis revealed patterns close to those reported for M. lentiflavum and M. triplex. However, the patterns of the novel strains differed from those of both of these species in having the peak for 24 : 0 higher than that for 26 : 0, and 18 : 0 higher than that for tuberculostearic acid (10-Me-18 : 0, TBSA); they also differed from M. triplex in having 20 : 0 higher than 22 : 0 (Fig. 1).
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Genotypic features
The number of nucleotide mismatches with the most closely related species, M. triplex, was 22 (1·2 %) in the 1788 bp stretch including the 16S rRNA gene (8 mismatches) and the ITS (14 mismatches). Such a number of mismatches is clearly higher than that differentiating many of the other recognized species. Within the 16S rRNA gene, four mismatches occurred in the first third of the gene, one of them in hypervariable region A, thus allowing differentiation from all other species by studying this region alone.
In the RIDOM database, consisting of the 5' end of the 16S rRNA gene sequences (corresponding to positions 54–510 of the Escherichia coli 16S rRNA gene) of strictly controlled mycobacterial strains, the most closely related species was M. triplex (ATCC 700071T) which differed by 4 bp. In GenBank, the complete sequence of the 16S rRNA gene, and the ITS as well, was 100 % identical to that of a strain tentatively assigned to the species M. triplex, isolate 23 (Suomalainen et al., 2001); however, the sequences of the latter strain and our novel strains differed by 22 bp from the reference strain of that species (ATCC 700071T).
The position of our strains in the phylogenetic tree was close to M. lentiflavum and the recently described species Mycobacterium montefiorense (Fig. 3). Interestingly, its position was closer to M. montefiorense and M. lentiflavum than to M. triplex.
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).
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). The second, responsible for disseminated infection in an AIDS patient in France (Zeller et al., 2003), presents four mismatches in a 229-nucleotide 16S rRNA gene fragment. However, the third, isolated in Finland and responsible for a pulmonary infection (Table 1
), is identical to our strains, as strongly supported by the sequence identity of both the 16S rRNA gene and the ITS (Suomalainen et al., 2001). This latter strain was retrieved and was tested for chromatographic patterns of cell-wall lipids by running in parallel with the test strains. Interestingly, it was found to be identical to the novel strains investigated here.
Description of Mycobacterium florentinum sp. nov.
Mycobacterium florentinum (flo'ren.ti.num. N.L. neut. adj. florentinum of the Italian city of Florence, where the majority of the strains were collected and investigated).
Non-motile, acid-fast bacillus devoid of spores or capsule. Grows in about 2 weeks at temperatures ranging from 25 to 37 °C, forming unpigmented colonies. The biochemical profile, characterized by positive urease and negative Tween 80 hydrolysis and 3 day arylsulfatase, is not suitable for unambiguous differentiation of strains from several other non-chromogenic species, in particular from M. triplex and M. lentiflavum. Among lipid investigations, GLC can differentiate M. florentinum sp. nov. from all other related species and the HPLC pattern is unique, although it roughly follows the three-clustered motif characteristic of many M. simiae-related species. M. lentiflavum is the only species that presents a PRA pattern that overlaps with that of M. florentinum sp. nov.; distinction is possible by digesting the DNA with HhaI, however. Finally, the sequences of both the 16S rRNA gene and of the ITS are unique. These data, in addition to its position in the phylogenetic tree, strongly support the novel species status of M. florentinum sp. nov.
The type strain, strain FI-93171T (=DSM 44852T=CIP 108409T), was isolated from an excised lymph node of a young girl in Italy.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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