Mycobacterium chimaera sp. nov.

1
Mycobacterium chimaera sp. nov.

• Enrico Tortoli
• Regional Reference Center for Mycobacteria
• Florence - Italy

presented at 24 Congress of ESM Tartu, July 2,
2003
2
M. avium complex

• M. avium
• M. avium subsp. avium
• M. avium subsp. silvaticum
• M. avium subsp. paratuberculosis
• M. avium subsp. hominissuis
• M. intracellulare
• mycobacteria other than avium and intracellulare

3
Strains and patients

• Isolated from 12 independent patients in 4 years
• 11 out of 12 isolated from respiratory specimens
• multiple isolate in 6 cases, single isolate in 6
  cases
• strains clinically non significant 5
• strains clinically significant 7
• immunocompetent patients (4 M and 3 F), 56-82
  year old
• pulmonary cavitations and emoptysis pulmonary
  abscess chronic obstructive pulmonary disease
  bronchiectasis
• dead 1 treated 6, recovered 3

4
Conventional tests

• Slow growth at 25-37C
• Colonies unpigmented or slightly yellow pigmented
• Negative tests
• niacin nitrate reduction ß-glucosidase Tween
  80 hydrolysis 3-day arylsulfatase urease
  semiquantitative catalase NaCl tolerance
  MacConkey growth
• Positive tests
• 68C catalase tiacetazone and isoniazid
  tolerance

5
Lipid analysis

• TLC
• a-, keto- and wax ester-mycolates
• GLC
• best resemblance M. intracellulare (50
  similarity)
• HPLC
• two clusters of peaks

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HPLC pattern
M. avium
M. intracellulare
???
7
DNA probe

• Accuprobe
• Positive MAC, M. intracellulare
• Negative M. avium
• INNO LiPA v.1
• Positive Mycobacterium genus, MAIS
• Negative M. avium, M. intracellulare, M.
  scrofulaceum
• INNO LiPA v. 2
• Positive Mycobacterium genus, MAIS, M.
  intracellulare 2
• Negative M. avium, M. intracellulare 1, M.
  scrofulaceum

8
Genetic sequencing

• 16S rRNA
• One nucleotide mismatch with M. intracellulare
  (GenBank)
• Full match with M. intracellulare sequevar v
  (RIDOM)
• ITS
• Full match with MAC-A

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Insertion elements

• IS900 absent
• IS901 absent
• IS1245 absent

10
Philogeny
11
Philogeny
12
Philogeny
13
Philogeny
14
Philogeny
15
Philogeny
16
Philogeny
17
Philogeny
MAC-A
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The 16S-ITS region variations

• M. avium includes sequevars differing for up to 4
  bases
• M. intracellulare includes sequevars differing
  for up to 7 bases
• Our strains differs
• from M. avium for at least 20 bases
• from M. intracellulare for at least 21 bases
• from any other MAC for at least 15 bases
• Unique markers to our strains
• ITS position 149 adenosine replaced by thymidine
• ITS position 234 guanosine replaced by adenosine
• ITS position 230 one nucleotide deletion

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Antimicrobial pattern

• Resistance to ethambutol
• Variable susceptibility to other drugs
• Four strains present very low MICs to almost all
  drugs

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M. chimaera sp. nov.

• Accuprobe M. intracellulare LiPA 1 MAIS (non
  M. avium non M. intracellulare) LiPA 2 M.
  intracellulare 2
• Two-clustered HPLC profile
• Unique genetic sequence in the 16S-ITS trait and
  unique phylogenetic position
• Resistance to ethambutol
• Isolated from the respiratory tract of
  immunocompetent, elderly patients with various
  pulmonary disorders never isolated from the
  environment or from animals
• High rate of clinical significance

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Collaborations
Regional Reference Center for Mycobacteria,
Careggi Hospital, Florence Alessandro Mariottini,
Gianna Mazzarelli Department of Experimental
Pathology, University of Pisa, Pisa Carlo
Garzelli, Nicoletta Lari, Laura
Rindi Microbiology and Virology Laboratory, S.
Camillo-Forlanini Hospitals, Rome Patrizia
Chiaradonna Department of Public Health,
University of Florence, Florence Rosanna
Dei German Collection of Microorganisms and Cell
Cultures, Braunschweig Reiner Kroppenstedt Region
al Reference Center for Mycobacteria, S. Bortolo
Hospital, Vicenza Paola Piccoli, Claudio Scarparo