This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rodríguez-Nava, V.
Right arrow Articles by Laurent, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rodríguez-Nava, V.
Right arrow Articles by Laurent, F.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, December 2005, p. 6167-6170, Vol. 43, No. 12
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.12.6167-6170.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Nocardia ignorata, a New Agent of Human Nocardiosis Isolated from Respiratory Specimens in Europe and Soil Samples from Kuwait

Verónica Rodríguez-Nava,1 Andrée Couble,1 Z. U. Khan,2 M. Pérouse de Montclos,3 L. Brasme,4 Cruz Villuendas,5 Claudie Molinard,1 Patrick Boiron,1 and Frédéric Laurent1*

UMR CNRS 5557, Center for Microbial Ecology, Opportunistic Pathogens and Environment Research Group, Observatoire Français des Nocardioses, Department of Fundamental and Applied Mycology, Faculty of Pharmacy, Claude Bernard Lyon I University, 8 Avenue Rockefeller, 69393 Lyon, France,1 Department of Microbiology, Faculty of Medicine, Kuwait University, Safat, 13110, Kuwait,2 Department of Bacteriology, Lyon Sud University Hospital, 69495 Pierre-Bénite Cedex, France,3 Department of Microbiology, Robert Debre University Hospital, 51092 Reims Cedex, France,4 Department of Microbiology, Miguel Servet University Hospital, Zaragoza, Paseo Isabel la Catolica No. 1-3, 50009, Zaragoza, Spain5

Received 30 March 2005/ Returned for modification 3 May 2005/ Accepted 27 September 2005


arrow
ABSTRACT
 
Nocardia ignorata is a recently described species identified on the basis of a single isolate of unknown origin. Here we describe the epidemiological, phenotypic, and phylogenetic characteristics of this new species, based on five new clinical and soil isolates.


arrow
TEXT
 
Nocardia ignorata is a new species first described in 2001 on the basis of a single isolate (26). This isolate was mistakenly used by a Microbiological Quality Control Laboratory and was dispatched to German laboratories labeled as a Mycobacterium sp. during a quality control exercise. Among the 400 isolates collected between 2000 and 2003 by the French Nocardiosis Observatory, five were distinguished by their atypical orange-then-black colony color on Bennett agar plates and aspect identical to that of the N. ignorata type strain, DSM 44496T. Four of the five isolates were recovered from clinical specimens in Europe (see Table 1 for clinical data) and one was a soil isolate from the Ahmadi oil field in Kuwait (13).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Epidemiological dataa

The five isolates had the ecological and epidemiological characteristics typical of Nocardia spp. (18). Recovery of the Kuwaiti soil isolate (OFN N21) indicates that N. ignorata is present in soil, which is thought to be the primary Nocardia reservoir. The recovery site had been contaminated by crude oil spilled during the Gulf War, and it is noteworthy in this respect that actinomycetes can saprophytically digest and recycle several sources of carbon present in natural environments. Isolates OFN 27.3837 and OFN 00.69 were considered to be of uncertain clinical significance, and the patients involved were not treated, despite signs of lung disease and predisposing factors. Their clinical manifestations were attributed to cocultured microorganisms (Aspergillus fumigatus and Candida sp. in one patient and Mycobacterium intracellulare in the other patient). Furthermore, as Nocardia spp. are ubiquitous in the environment, their isolation from clinical samples may be spurious or simply reveal respiratory colonization (18). In contrast, isolates OFN 03.01.4 and OFN 00.18 were both clearly responsible for multiple necrotizing pulmonary abscesses. Direct examination of sputum (patient 1) and bronchoalveolar lavage fluid (patient 2) showed gram-positive branching filamentous organisms, and culture yielded numerous colonies. The two patients had chronic obstructive pulmonary disease and were receiving steroid therapy, a well-documented predisposing factor for nocardiosis (2, 12). Patient 1, who was receiving palliative care, died rapidly after diagnosis of nocardiosis.

All the isolates had initially been identified as N. asteroides or other Nocardia spp. Different molecular methods were used here for species identification. DNA was extracted with Chelex resin (17). Genus-specific 16S rRNA PCR (17) confirmed that the five isolates belonged to the genus Nocardia. By hsp65 pattern restriction analysis (24), all the isolates yielded the same specific band pattern, which was identical to that obtained with N. ignorata DSM 44496T: BstE II (320 and 80 bp), MspI (130, 120, 115, and 70 bp), and Hinf I (250 and 190 bp) (data not shown). Definitive taxonomic classification of the five isolates was obtained by multilocus sequencing (5, 20). Each gene was partially amplified, as follows: (i) a 606-nucleotide fragment of 16S rRNA, using primers Noc1 (5'-GCTTAACACATGCAAGTCG-3'; positions 46 to 64, Escherichia coli numbering) and Noc2 (5'-GAATTCCAGTCTCCCCTG-3'; positions 663 to 680, E. coli numbering) (20); (ii) a 441-nucleotide fragment of the hsp65 gene, using primers TB11 and TB12 (24); and (iii) a 438-nucleotide fragment encoding part of the RNA polymerase ß-subunit, using primers Kim1 (5'-CGACCACTTCGGCAACCG-3') and TBB2 (5'-TACGGCGTCTCGATGAASCC-3') (5).

PCR products were purified and sequenced on both strands. The resulting sequences were aligned with the corresponding sequences of representative Nocardia species in the GenBank (16S rRNA) or Bioinformatic Bacterial Identification (BIBI) database (hsp65 and rpoB) (6) by using Clustal W software (25). Using Mega and Phylo_win software (8, 16), evolutionary trees were inferred with three treeing algorithms, the maximum-likelihood (7), maximum-parsimony (15), and neighbor-joining (22) methods, and the Kimura two-parameter model (14). The robustness of the trees was tested by bootstrap resampling (1,000 replicates each).

The 16S rRNA, hsp65, and rpoB nucleotide sequences determined in the present study have been deposited in GenBank under accession numbers AY756554, AY769533, AY769532, AY769535, AY769531, AY769534, AY756526, AY769528, AY769527, AY769527, AY769530, AY769526, AY769529, AY885686, AY882016, AY882015, AY882018, AY882014, andAY882017.

Analysis of the phylogenetic trees constructed with Mega 2.1 software (16) showed that the five isolates and the type strain N. ignorata DSM 44496T clustered together, separately from all other nocardial species (Fig. 1). The 16S rRNA sequences showed more than 99.3% similarity to N. ignorata DSM 44496T. The question of how similar a given strain must be to the reference strain before it can be assigned to the same species is controversial. A reporting criterion of >99% similarity for nocardial 16S rRNA (in the same region as that used here) with the MicroSeq 500 kit has been proposed by Cloud et al. (4), whereas Mellmann et al. calculated a criterion of <99.12% similarity in the same region for distinct species, with reference to the RIDOM database (19).



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 1. Phylogenetic trees based upon 16S rRNA, hsp65, and rpoB sequences from Nocardia type strains and study isolates. The trees were based on a comparison of stretches of 606 nucleotides of 16S rRNA, 441 nucleotides of hsp65, and 438 nucleotides of rpoB. Evolutionary trees were inferred by neighbor joining (22) with bootstrap resampling (1,000 replicates).

Whatever the criterion used, the 16S rRNA patterns we obtained clearly grouped the five isolates together and identified them as N. ignorata. We also applied the same approach to the hsp65 gene and the rpoB gene, with reference to the BIBI database (6, 20), which, on 2 October 2004, contained all validated Nocardia species. This approach yielded similar results: the six strains clustered together with similarities of >99.5% and bootstrap values of 99 (Fig. 1). The combined use of sequences of several genes (16S rRNA, hsp65, and rpoB) refined the phylogenetic analysis and reinforced the 16S rRNA data. The agreement of the multilocus study strengthened the taxonomic and phylogenetic position of the six isolates, and also showed the low intraspecies variability of each gene. This multigene approach complies with the recommendations of the ad hoc committee reevaluating the definition of bacterial species (23). All our molecular results confirmed that the five new isolates belonged to N. ignorata.

The isolates were also characterized phenotypically by using conventional procedures (1, 9, 10, 11), in order to further analyze intraspecies variability and to characterize the new species (Table 2). This approach also complies with the conclusions of the above-mentioned ad hoc committee, which recommended analysis of at least five isolates for definitive description of a new species (23). Our biochemical and physiological results corresponded to previously published data on N. ignorata DSM 44496T except for variable xylose and mannitol utilization as sole carbon sources.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Characteristics of N. ignorata isolatesa

MICs were determined by using the broth microdilution method according to the Clinical Laboratory Standards Institute guidelines for aerobic actinomycetes (3). The six isolates showed very similar susceptibility profiles, with the following MICs: amikacin, <0.06 µgml–1; amoxicillin, 16 to 32 µg ml–1; cefotaxim, 128 to 256 µgml–1; ciprofloxacin, 1 to 2 µgml–1; imipenem, 1 to 4 µgml–1; and trimethoprim-sulfamethoxazole, 0.05/1 to 0.1/2 µgml–1. Imipenem, amikacin, and trimethoprim/sulfamethoxazole thus appear to be the drugs of choice for therapeutic use. Initial intravenous combination therapy with imipenem and amikacin may be recommended, followed by oral trimethoprim-sulfamethoxazole. High-level cefotaxime resistance must be taken into account, as this drug is often used to treat nocardiosis. N. ignorata must be added to the list of the rare cefotaxime-resistant nocardial species.

In conclusion, this report shows the clinical significance of N. ignorata, ranging from colonization to multiple necrotizing pulmonary abscesses in immunodepressed patients. N. ignorata appears to be present in soil and human environments worldwide. Our findings highlight the value of multigene sequencing for rapidly confirming the identification of new pathogenic Nocardia species.


arrow
ACKNOWLEDGMENTS
 
We thank Grégory Devulder for his considerable assistance with this project.

This work was financed by a grant from Institut Fédératif de Recherche 41 (IFR41), Université Claude Bernard Lyon 1, CNRS, Lyon, France. Verónica Rodríguez-Nava thanks Consejo Nacional de Ciencia y Tecnologia (CONACYT), Mexico City, Mexico, and Société Française d'Exportation des Ressources Educatives (SFERE), Paris, France, for financial support.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Laboratoire de Mycologie Fondamentale et Appliquée aux Biotechnologies Industrielles, UMR CNRS 5557, Faculté de Pharmacie, UCB Lyon I, 8 Avenue Rockefeller, 69373 Lyon Cedex 08, France. Phone: (33) (0) 4 78 77 70 00. Fax: (33) (0) 4 78 77 72 12. E-mail: flaurent{at}ch-bourg01.fr. Back


arrow
REFERENCES
 
    1
  1. Boiron, P., F. Provost, and B. Dupont. 1993. Technical protocols, p. 107-126. In Methodes de laboratoire pour le diagnostic de la nocardiose. Institut Pasteur, Paris, France.
    2. 2
  2. Boiron, P., F. Provost, G. Chevrier, and B. Dupont. 1992. Review of nocardial infections in France 1987 to 1990. Eur. J. Clin. Microbiol. 11:709-714.
    3. 3
  3. Clinical and Laboratory Standards Institute. 2005. Susceptibility testing of Mycobacteria, Nocardia and other aerobics actinomycetes. Approved Standard M24-A, vol. 23, no. 18. Clinical and Laboratory Standards Institute, Wayne, Pa.
    4. 4
  4. Cloud, J. L., P. S. Conville, A. Croft, D. Harmsen, F. G. Witebsky, and K. C. Carroll. 2004. Evaluation of partial 16S ribosomal DNA sequencing for identification of Nocardia species by using the MicroSeq 500 system with an expanded database. J. Clin. Microbiol. 42:578-584.[Abstract/Free Full Text]
    5. 5
  5. Devulder, G., M. Pérouse de Montclos, and J. P. Flandrois. 2005. A multigenic approach to phylogenetic analysis using the genus Mycobacterium as a model. Int. J. Syst. Evol. Microbiol. 55:293-302.[Abstract/Free Full Text]
    6. 6
  6. Devulder, G., G. Perriere, F. Baty, and J. P. Flandrois. 2003. BIBI, a bioinformatics bacterial identification tool. J. Clin. Microbiol. 41:1785-1787.[Abstract/Free Full Text]
    7. 7
  7. Felsenstein, J. 1981. Evolutionary trees from DNA sequences: a maximum likelihood approach. J. Mol. Evol. 17:368-376.[CrossRef][Medline]
    8. 8
  8. Galtier, N., M. Gouy, and C. Gautier. 1996. SeaView and Phylo_win, two graphic tools for sequence alignment and molecular phylogeny. Comput. Appl. Biosci. 12:543-548.[Abstract/Free Full Text]
    9. 9
  9. Goodfellow, M., and M. P. Lechevalier. 1989. Genus Nocardia Trevisan, p. 2350-2361. In S. T. Williams, M. E. Sharpe, and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 4. The Williams & Wilkins Co., Baltimore, Md.
    10. 10
  10. Goodfellow, M. 1992. The family Nocardiaceae, p. 1188-1213. In A. Balows, H. G. Truper, M. Dworkin, W. Harder, and K. H. Schleifer (ed.), The prokaryotes, 2nd ed. Springer, New York, N.Y.
    11. 11
  11. Goodfellow, M. 1998. The genus Nocardia Trevisan 1889, p. 464-489. In A. Balows and B. I. Duerden (ed.), Topley and Wilson's microbiology and microbial infections, vol. 2. Edward Arnold, London, United Kingdom.
    12. 12
  12. Kageyama, A., K. Yazawa, J. Ishikawa, K. Hotta, K. Nishimura, and Y. Mikami. 2004. Nocardial infections in Japan from 1992 to 2001, including the first report of infection by Nocardia transvalensis. Eur. J. Epidemiol. 19:383-389.[CrossRef][Medline]
    13. 13
  13. Khan, Z. U., L. Neil, R. Chandy, T. D. Chugh, H. Al-Sayer, F. Provost, and P. Boiron. 1997. Nocardia asteroides in the soil of Kuwait. Mycopathologia. 137:159-163.[CrossRef][Medline]
    14. 14
  14. Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16:111-120.[CrossRef][Medline]
    15. 15
  15. Kluge, A. G., and F. S. Farris. 1969. Quantitative phyletics and the evolution of anurans. Syst. Zool. 18:1-32.
    16. 16
  16. Kumar, S., K. Tamura, and M. Nei. 1994. MEGA: Molecular Evolutionary Genetics Analysis software for microcomputers. Comput. Appl. Biosci. 10:189-191.[Abstract/Free Full Text]
    17. 17
  17. Laurent, F., F. Provost, and P. Boiron. 1999. Rapid identification of clinically relevant Nocardia species to genus level by 16S rRNA gene PCR. J. Clin. Microbiol. 37:99-102.[Abstract/Free Full Text]
    18. 18
  18. McNeil, M. M., and J. M. Brown. 1994. The medically important aerobic actinomycetes: epidemiology and microbiology. Clin. Microbiol. Rev. 7:357-417.[Abstract/Free Full Text]
    19. 19
  19. Mellmann, A., J. L. Cloud, S. Andrees, K. Blackwood, K. C. Carroll, A. Kabani, A. Roth, and D. Harmsen. 2003. Evaluation of RIDOM, MicroSeq, and Genbank services in the molecular identification of Nocardia species. Int. J. Med. Microbiol. 293:359-370.[CrossRef][Medline]
    20. 20
  20. Rodríguez-Nava, V., A. Couble, G. Devulder, J-P. Flandrois, P. Boiron, and F. Laurent. hsp65 gene-based identification of Nocardia species: use of pattern restriction analysis and sequencing database. Submitted for publication.
    21. 21
  21. Roth, A., S. Andrees, R. M. Kroppenstedt, D. Harmsen, and H. Mauch. 2003. Phylogeny of the genus Nocardia based on reassessed 16S rRNA gene sequences reveals underspeciation and division of strains classified as Nocardia asteroides into three established species and two unnamed taxons. J. Clin. Microbiol. 41:851-856.[Abstract/Free Full Text]
    22. 22
  22. Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425.[Abstract]
    23. 23
  23. Stackebrandt, E., W. Frederiksen, G. M. Garrity, P. A. Grimont, P. Kampfer, M. C. Maiden, X. Nesme, R. Rossello-Mora, J. Swings, H. G. Truper, L. Vauterin, A. C. Ward, and W. B. Whitman. 2002. Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int. J. Syst. Evol. Microbiol. 52:1043-1047.[Abstract]
    24. 24
  24. Steingrube, V. A., R. W. Wilson, B. A. Brown, K. Jost, J. L. Gibson, J. Brown, Z. Blacklock, J. L. Gibson, and R. J. Wallace, Jr. 1997. Rapid identification of clinically significant species and taxa of aerobic actinomycetes, including Actinomadura, Gordona, Nocardia, Rhodococcus, Streptomyces, and Tsukamurella isolates, by DNA amplification and restriction endonuclease analysis. J. Clin. Microbiol. 35:817-822.[Abstract]
    25. 25
  25. Thompson, J. D., D. G Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.[Abstract/Free Full Text]
    26. 26
  26. Yassin, A. F., F. A. Rainey, and U. Steiner. 2002. Nocardia ignorata sp. nov. Int. J. Syst. Evol. Microbiol. 51:2127-2131.

Journal of Clinical Microbiology, December 2005, p. 6167-6170, Vol. 43, No. 12
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.12.6167-6170.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Rodriguez-Nava, V., Khan, Z. U., Potter, G., Kroppenstedt, R. M., Boiron, P., Laurent, F. (2007). Nocardia coubleae sp. nov., isolated from oil-contaminated Kuwaiti soil. Int. J. Syst. Evol. Microbiol. 57: 1482-1486 [Abstract] [Full Text]  
  • Conville, P. S., Witebsky, F. G. (2007). Analysis of Multiple Differing Copies of the 16S rRNA Gene in Five Clinical Isolates and Three Type Strains of Nocardia Species and Implications for Species Assignment. J. Clin. Microbiol. 45: 1146-1151 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rodríguez-Nava, V.
Right arrow Articles by Laurent, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rodríguez-Nava, V.
Right arrow Articles by Laurent, F.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»