JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2005, p. 6167–6170 0095-1137/05/$08.00⫹0 doi:10.1128/JCM.43.12.6167–6170.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 43, No. 12
Nocardia ignorata, a New Agent of Human Nocardiosis Isolated from Respiratory Specimens in Europe and Soil Samples from Kuwait Vero ´nica Rodrı´guez-Nava,1 Andre´e Couble,1 Z. U. Khan,2 M. Pe´rouse de Montclos,3 L. Brasme,4 Cruz Villuendas,5 Claudie Molinard,1 Patrick Boiron,1 and Fre´de´ric Laurent1* UMR CNRS 5557, Center for Microbial Ecology, Opportunistic Pathogens and Environment Research Group, Observatoire Franc¸ais des Nocardioses, Department of Fundamental and Applied Mycology, Faculty of Pharmacy, Claude Bernard Lyon I University, 8 Avenue Rockefeller, 69393 Lyon, France1; Department of Microbiology, Faculty of Medicine, Kuwait University, Safat, 13110, Kuwait2; Department of Bacteriology, Lyon Sud University Hospital, 69495 Pierre-Be´nite Cedex, France3; Department of Microbiology, Robert Debre University Hospital, 51092 Reims Cedex, France4; and 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
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. 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). 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 ab-
scesses. 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⬘-GCTTAACAC ATGCAAGTCG-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 -subunit, using primers Kim1 (5⬘-CGA CCACTTCGGCAACCG-3⬘) and TBB2 (5⬘-TACGGCGTC TCGATGAASCC-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-
* Corresponding author. Mailing address: Laboratoire de Mycologie Fondamentale et Applique´e aux Biotechnologies Industrielles, UMR CNRS 5557, Faculte´ 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:
[email protected]. 6167
a Abbreviations: AN: amikacin; BAL: bronchoalveolar lavage fluid; CIP: ciprofloxacin; CLA: clarithromycin; COPD: chronic obstructive pulmonary disease; ETH: ethambutol; GPBF: gram-positive branching filamentous; IMP: imipenem; OFN: Observatoire Franc¸ais des Nocardioses; RIF: rifabutin; S: sulfamethoxazole; TA: tracheal aspirate; TMP: trimethoprim.
Contaminant None OFN 00.69 4
Reims (France)
71
COPD ⫹ panacimar emphysema
Chest pain, dyspenia
Blood culture: GPBF
TA: M. intracellulare ⫹ Nocardia spp. Nocardia sp. (A. fumigatus and Candida sp. isolated in)
Mycobacteriosis ⫹ colonization of upper airways by Nocardia sp. BAL: Mycobacterium intracellulare BAL ⫹ TA: negative Steroids Chronic pulmonary sarcoı¨dosis 65 OFN 27.3837 3
Lyon (France)
Steroids COPD 74 OFN 00.18 2
Firminy (France)
None
Cure
Cure
IMP 3 g/day, CIP 1,500 mg/day, AN 800 mg/day (45 days) RIF 600 mg/day, ETH 1.2 g/day ⫹ CLA 1.5 g/day (6 months) Nocardiosis Nocardia sp. ⫹ Pseudomonas aeruginosa BAL: GPBF
TMP 320 mg/day, S 1,600 mg/day (1 month) Nocardiosis Sputum: GPBF OFN 03.1.4.sp 1
Zaragoza (Spain)
79
Lung cancer
Steroids
Asthenia ⫹ anxiety ⫹ persistent cough Increased spastic emphysema ⫹ pulmonary abscesses Pulmonary infection
Nocardia sp.
Therapy Diagnosis Smear Reference strain
Origin
Age (yr)
Predisposing disease
Predisposing treatment
Symptoms
Culture
J. CLIN. MICROBIOL.
Patient no.
TABLE 1. Epidemiological dataa
Death
NOTES Outcome
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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, and AY882017. 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). 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. 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 g 䡠 ml⫺1; amoxicillin, 16 to 32 g 䡠 ml⫺1; cefotaxim, 128 to 256 g 䡠 ml⫺1; ciprofloxacin, 1 to 2 g 䡠 ml⫺1; imipenem, 1 to 4 g 䡠 ml⫺1; and trimethoprimsulfamethoxazole, 0.05/1 to 0.1/2 g 䡠 ml⫺1. Imipenem, amikacin, and trimethoprim/sulfamethoxazole thus appear to be the
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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).
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
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NOTES
J. CLIN. MICROBIOL.
TABLE 2. Characteristics of N. ignorata isolatesa Strain no. Parameter
Growth on carbon sources L-Arabinose D-Fructose D-Fucose D-Galactose D-Glucose Maltose D-Mannitol Mannose Raffinose L-Rhamnose D-Ribose Saccharose Sorbitol D-Xylose Growth on Bennett agar at: 25°C 37°C 45°C Arylsulfatase production at: 3 days 14 days Decomposition of: Caseine Hypoxanthine Testosterone Tyrosine Uric acid Xanthine
1
2
3
4
5
6
7
8
9
10 11
⫺ ⫹ ⫺ ⫺ ⫹ ⫹ w ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺
⫺ ⫹ ⫺ ⫺ ⫹ ⫹ w ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺
⫺ ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫹
⫺ ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺
⫺ ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺
⫺ ⫹ ⫺ ⫺ ⫹ ⫹ w ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺
⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺
⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ⫹ ⫹ ⫺ ⫺ ⫺
⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺
⫺ ⫹ ⫺ ⫹ ⫹ ⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺
⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹
⫹ ⫹ ⫺
⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
⫺ ⫺
⫺ ⫺ ⫹ ⫺ ⫺ ⫺
⫹ ⫹ ⫺ ⫹ ⫺ ⫺
⫺ ⫺ ⫹ ⫺ ⫺ ⫺
⫺ ⫺ ⫹ ⫺ ⫺ ⫺
⫺ ⫺ ⫹ ⫺ ⫺ ⫺
⫺ ⫺ ⫹ ⫺ ⫺ ⫺
⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺
⫺ ⫺ ⫹ ⫺ ⫺ ⫺
a Physiological characteristics of N. ignorata isolates DSM 44496T, OFN 03.1.4.sp, OFN 27.3837, OFN 00.18, OFN 00.69, and OFN N21 and Nocardia reference strains. Strain numbering: 1, OFN 03.1.4.sp; 2, OFN 27.3837; 3, OFN 00.18; 4, OFN 00.69; 5, OFN N21; 6, N. ignorata DSM 44496T; 7, N. soli DSM 44488T; 8, N. cummidelens DSM 44490T; 9, N. asteroides ATCC 19247T; 10, N. farcinica DSM 43578T; 11, N. pseudobrasiliensis DSM 44290T. Reactions: ⫺, negative; ⫹, positive; w, weak.
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