Use Of PCR-Restriction Enzyme Pattern Analysis And Sequencing Database For Hsp65 Gene-Based Identification Of Nocardia Species

J Clin Microbiol. 2006 February; 44(2): 536–546.

doi: 10.1128/JCM.44.2.536-546.2006

PMCID: PMC1392680

Use of PCR-Restriction Enzyme Pattern Analysis and Sequencing Database for hsp65 Gene-Based Identification of Nocardia Species

Verónica Rodríguez-Nava,1 Andrée Couble,1 Gregory Devulder,2 Jean-Pierre Flandrois,2 Patrick Boiron,1 and Frédéric Laurent1,*

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ABSTRACT

Nocardia identification required laborious and time-consuming phenotypic and chemotaxonomic methods until molecular methods were developed in the mid-1990s. Here we reassessed the capacity of PCR-restriction enzyme pattern analysis (PRA) of the hsp65 gene to differentiate Nocardia species, including 36 new species. Our results confirm that hsp65 PRA must no longer be used for Nocardia species identification, as many species have the same restriction pattern. We then compared sequencing-based strategies using an hsp65 database and a 16S rRNA database and found that the hsp65 region contained sufficient polymorphisms for comprehensive Nocardia species identification.

Nocardia species are gram-positive, weakly acid-fast, strictly aerobic bacteria that form filamentous branched cells which fragment into pleomorphic rod-shaped or coccoid elements. Nocardia species are essentially soil saprophytes involved in the decomposition of plant material (12, 16, 18). However, some species can infect both immunocompromised and immunocompetent individuals (18). Genus and species identification is necessary to predict antimicrobial susceptibility and for epidemiological purposes and also for environmental investigations (biodiversity, ecological niches, etc.).

Nocardia identification used to be based on laborious and time-consuming phenotypic and chemotaxonomic methods. Molecular methods were developed in the 1990s, including a 16S rRNA gene PCR-based method capable of distinguishing the genus Nocardia among aerobic actinomycetes (15). PCR-restriction enzyme pattern analysis (PRA) of a 441-bp fragment of the 65-kDa heat shock protein (hsp65) gene was developed to identify individual Nocardia species (28, 29). Sequential use of the two techniques provided rapid and simplified identification of Nocardia isolates from molecular dichotomous decision trees based on amplification/no amplification and the number and size of restriction fragments.

The genus Nocardia has undergone a taxonomic revolution during the last 10 years. Only 12 species were described between 1888, when the genus was first isolated by Nocard (20), and 1996, whereas more than 40 species are now recognized to exist. Some have been collected from clinical specimens; other have been isolated only from environmental specimens. PCR methods developed during the last decade have not yet been tested on the full range of known Nocardia species. For example, no data are available on the hsp65-PRA patterns of the 36 new species. Moreover, sequencing methods are increasingly important identification tools, and those based on 16S rRNA gene polymorphism have been applied to Nocardia (7, 19, 21). The MicroSeq 500 16S rRNA gene kit (PE Applied Biosystems) and the RIDOM database and BIBI database based on this methodology have recently been applied to the species identification of Mycobacterium and Nocardia isolates (6, 7, 9, 19, 21, 32). These approaches proved to be as efficient as conventional methods (biochemical tests, high-pressure liquid chromatography, and molecular probes) for many but not all Nocardia species (7). The latter authors underlined that public databases which are not monitored (no standard annotation, no control of strain identification, etc.) should be used with caution. Moreover, in order to overcome the strong similarity of 16S rRNA gene sequences within the genus Mycobacterium (e.g., M. gastri and M. kansasii) and microheterogeneity within a given species (e.g., M. gordonae), the study of other genes such as hsp65 (23), rpoB (13), sod (35), recA (1), and 16S-23S ITS (24) may be used. Similar problems arise with Nocardia (19, 21).

Here we reevaluated the accuracy of the hsp65-PRA method and compared it with an alternative strategy based on partial hsp65 gene sequences for Nocardia species identification.

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MATERIALS AND METHODS

Type and reference strains.

Forty-four strains corresponding to 44 species of Nocardia were studied (Table (Table1).1). N. asteroides ATCC 49872, representative of “N. asteroides type IV,” was also included, as it corresponded to clearly individualized clusters (which have not yet been named) (4, 17). The strain ATCC 19247T, previously used as a representative of N. asteroides, was included in this study. The taxonomic position of this strain has given rise to much controversy. This strain was in fact representative only of a rare unnamed subgroup of the former N. asteroides complex (21). In the same way, the strain ATCC 14759 was proposed as the reference strain for the type VI drug susceptibility pattern. But some authors indicated that N. cyriacigeorgica may be the same as the major group of isolates (i.e., type VI) within the N. asteroides complex (21, 25). In the absence of information (especially DNA-DNA homology and decision by taxonomic committees) (21) allowing a definitive conclusion, we decided to include the two species in our study and to present separately the data for the two representative strains. Streptomyces somaliensis DSM 41612T was used as the outgroup for phylogenetic analysis. The strains were obtained from international collections and grown on Bennett agar at 37°C for 3 to 15 days.

TABLE 1.

Strains of Nocardia studied

Clinical isolates.

We also studied 21 clinical isolates sent for identification to the Observatoire Français des Nocardioses (Lyon, France). We confirmed that they belonged to the genus Nocardia by analyzing basic phenotypic characteristics such as culture morphology, mesodiaminopimelic acid, lysozyme resistance, substrate use (2), and also PCR (15).

DNA extraction.

DNA was extracted with achromopeptidase. Colonies were picked off with a loop, and one loopful was suspended in 250 μl of sterile pyrolyzed water and vortexed for 1 minute. The bacterial filaments were crushed manually with conical plastic crushers. The mixture was then incubated for 15 min at 70°C. Fifty microliters of the suspension plus 1.5 μl of achromopeptidase (10 U/ml; Sigma, Steinheim, Germany) was incubated at 55°C for 15 min. The suspensions were then centrifuged for 3 min at 13,000 rpm. The supernatants were stored at −20°C until use.

hsp65 amplification and PRA identification of Nocardia species.

A 441-bp fragment

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