Characterization of Nocardia asteroides Isolates from Different Ecological Habitats on the Basis of Repetitive Extragenic Palindromic-PCR Fingerprinting

Thirteen isolates of Nocardia asteroides from both soils andaquatic samples (lake and moat sediments, as well as scum fromactivated sludge), together with a type strain and two knownclinical isolates of this species, were characterized by repetitiveextragenic palindromic-PCR fingerprinting with the BOX-A1R primer.The resulting DNA fingerprint patterns proved to be strain specific,and cluster analysis distinguished the soil isolates, the aquaticisolates, and the known strains as being in separate groups.

INTRODUCTION

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Introduction

Nocardia asteroides is a gram-positive, partially acid-fast,aerobic mesophile that characteristically produces primary amycelium that often fragments into bacillary and coccoid elements(7). An aerial mycelium with short chains of arthrospores isusually formed. This actinomycete has been implicated as anopportunistic pathogen in a range of pulmonary diseases, includingnocardiosis (6). Therefore, subtyping, or differentiation ofisolates belonging to this taxon, is of epidemiological importancefor investigating outbreaks of infection, determining the sourceof the infection, and recognizing especially virulent strains(12). DNA-based typing methods, including pulsed-field gel electrophoresisand randomly amplified polymorphic-DNA analyses, have been usedto characterize clinically related N. asteroides strains (10,11) and have shown that clinical isolates of N. asteroides aregenetically heterogeneous.

N. asteroides is also involved in the saprophytic digestionand recycling of plant material in natural environments (5-7).The primary reservoir of N. asteroides is thought to be thesoil, but this organism can also be found in lake and marinesediments. However, there is little published information onthe genetic diversity of N. asteroides strains associated withthese natural habitats (6). The purpose of the present studywas to characterize and differentiate N. asteroides strainsisolated from both terrestrial and aquatic environments by usingthe repetitive extragenic palindromic (REP)-PCR fingerprintingtechnique, in which PCR amplification of the DNA between adjacentrepetitive extragenic elements is used to obtain DNA fingerprints(19). REP-PCR has recently proven to be highly discriminatoryfor subtyping or strain classification of several bacterialspecies (12).

N. asteroides isolates.

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N. asteroides isolates.

Sixteen N. asteroides strains were included in this study (Table1). Of these, three strains used as reference strains for REP-PCRanalysis were obtained from the Japan Collection of Microorganisms,RIKEN, Saitama Prefecture (JCM; n = 1) and the Institute forFermentation, Osaka Prefecture (IFO; n = 2), Japan. All otherstrains were isolated from soil, lake, or moat sediments orfrom scum of activated sludge. Soil samples were collected fromvarious locations in Yamanashi Prefecture, Japan. Sediment sampleswere taken from Lake Suwa, Nagano Prefecture, and from the moatsurrounding Takeda Shrine, Yamanashi Prefecture, Japan, by useof an Ekman-Birge sediment surface sampler (Watanabe Keiki Co.,Ltd., Tokyo, Japan). Lake Suwa is a eutrophic lake with a maximumdepth of 7.2 m and a surface area of 13 km². The Takeda Shrinemoat is about 18 m wide and has a total length of 1,000 m anda maximum depth of 1 m. A scum sample was obtained from activatedsludge at a sewage treatment plant in Saitama Prefecture, Japan.The Nocardia isolation method is described in detail in a previouspaper (21).

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TABLE 1. N. asteroides strains used in this study

The identities of N. asteroides isolates were confirmed by morphologicaland chemotaxonomic characterization, restriction fragment lengthpolymorphism (RFLP) analysis of 16S rRNA genes (rDNAs), andsequencing of 16S rDNA signature sequences (2) between positions591 and 648 (Escherichia coli numbering) (1). Details of thetaxonomic tests employed have been previously described (9,13, 21). All 13 test isolates consistently developed a fragmentingsubstrate mycelium and a relatively short aerial mycelium withchains of smooth-surfaced arthrospores when inoculated ontooatmeal agar (16). All 13 test isolates were found to containmeso-diaminopimelic acid as a cell wall diamino acid and tohave galactose and arabinose as characteristic whole-cell sugars.The predominant menaquinone component of these strains is MK-8(H₄, ${omega}$ cyclized), and they also contain phosphatidylethanolamineand nocardomycolic acid. These morphological and chemotaxonomicproperties of the test strains are consistent with those ofthe genus Nocardia (6, 7). The 13 test isolates, together withthree reference strains, were evaluated by amplification andrestriction endonuclease analysis of a portion (1 kb; positions30 to 1049) of the 16S rDNA (3, 21). PCR products were subjectedto digestion with HhaI, NdeI, BstEII and HindIII, all of whichhave been recommended for use in identification of Nocardiaspecies on the basis of RFLP analysis of 16S rDNA (3, 21). All13 test isolates had RFLP patterns identical to those of N.asteroides (strains JCM 3384^T, IFO 3423, and IFO 3424) and couldbe differentiated from all other known Nocardia species, includingthe closely related species N. abscessus, N. cyriacigeorgica,N. farcinica, and N. nova (3, 10, 14, 21). For sequence analysis,a portion of 16S rDNA (0.8 kb; positions 9 to 802) was amplifiedby PCR and sequenced by a procedure described previously (21).It was confirmed that 16S rDNA nucleotide signatures typicalof N. asteroides (2) were present between positions 591 and648 in all test strains.

REP-PCR.

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Rep-pcr.

Total genomic DNA was prepared from the 16 test N. asteroidesstrains by the method of Torres et al. (18) and used as a templatein REP-PCR performed with the BOX-A1R primer (5'-CTACGGCAAGGCGACGCTGACG-3')(20). The reaction mixture (50 µl) contained 1x PCR buffer(Takara Shuzo Co., Ltd., Kyoto, Japan), a 2.5 mM concentrationof each deoxynucleoside triphosphate (Takara), a 50 pM concentrationof the BOX-A1R primer, 100 ng of genomic DNA, and 1 U of Taqpolymerase. Amplification was performed in a Takara PCR ThermalCycler PERSONAL as follows: after an initial denaturation step(95°C, 8 min), 30 cycles of denaturation (95°C, 1 min),annealing (53°C, 1 min) and extension (65°C, 4 min)were performed, followed by a single final extension (65°C,8 min). PCR products were separated by horizontal gel electrophoresison a 1.5% Seakem GTG agarose gel (FMC Bioproducts, Rockland,Maine) containing 0.5 µg of ethidium bromide/ml. The amplimerswere visualized and photographed under UV light.

Photographs of REP fingerprinting patterns were scanned, convertedto digitized images, and analyzed by the fragment analysis softwareDiversity Database (pdi, Inc., New York, N.Y.). Figure 1 showstypical fingerprints for N. asteroides test isolates and referencestrains, generated by REP-PCR with primer BOX-A1R. The profilesof the 16 strains revealed multiple DNA bands correspondingto sizes ranging from approximately 0.2 to 2.5 kb. The bandingpatterns were diverse and strain specific. However, it was evidentthat some of the strains are related to each other. Isolatesfrom aquatic habitats (lake and moat sediments, as well as scumof activated sludge) showed a conserved major band at a positioncorresponding to a molecular size of about 1,085 bp. Most ofthe soil isolates showed major conserved bands at positionscorresponding to molecular sizes of about 535 and 590 bp. Similarly,reference strains (a type strain and known clinical isolates)showed major conserved bands at about 230 and 485 bp. To determinethe stability and reproducibility of DNA fingerprint patterns,DNA was extracted from three single-colony isolates of N. asteroidesJCM 3384^T and analyzed by REP-PCR with the BOX-A1R primer. Whenthe resulting patterns of DNA fragments were compared with thatof the original N. asteroides JCM 3384^T culture, no significantdifference in the DNA fingerprints was observed (data not shown).Similar results were obtained for other test N. asteroides strains,including SO 008-2 and SO 031-3.

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FIG. 1. Fingerprint patterns of Nocardia asteroides strains, obtained by REP-PCR with the BOX-A1R primer. Lanes: 1, 100-bp DNA ladder; 2, LS 001-3; 3, LS 001-4; 4, LS 002-1; 5, LS 002-2; 6, LS 002-5; 7, MS 002-1; 8, AS 004-5; 9, SO 031-3; 10, SO 008-1; 11, SO 008-2; 12, SO 020-4; 13, SO 026-3; 14, SO 027-1; 15, N. asteroides IFO 3384; 16, N. asteroides IFO 3424; 17, N. asteroides JCM 3384^T; 18, 100-bp DNA ladder.

Similarity between strains was determined by the Pearson productmoment correlation coefficient with clustering by the unweightedpair group method with arithmetic mean (UPGMA) algorithm. Adendrogram (Fig. 2), constructed by performing a cluster analysisof the BOX-A1R primer DNA fingerprint patterns with the Pearsonsimilarity coefficient and UPGMA algorithm, produced three clusters(A, B, and C) defined at the 57% similarity level. Cluster Aincludes the type strain and two clinically related, known strainsof N. asteroides. Cluster B is composed of five strains originatingfrom soil. Cluster C includes all aquatic isolates and one soilisolate.

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FIG. 2. Dendrogram of REP-PCR genomic fingerprints of N. asteroides strains. Percent similarity between patterns was calculated using Pearson coefficients. The clustering pattern was generated using the UPGMA method.

Data generated by the REP-PCR DNA fingerprint technique withthe BOX-A1R primer in the present study show that genetic diversityexists among N. asteroides isolated from natural habitats andthat N. asteroides isolates cluster by environment; most ofthe soil isolates and aquatic isolates of this taxon were assignedto separate groups (Fig. 2). Therefore, REP-PCR performed withthe BOX-A1R primer could be useful for determining and characterizingN. asteroides strains endemic to specific natural habitats.This technique could also be useful for investigating an outbreakof N. asteroides infection. However, additional comparativestudies involving larger numbers of judiciously selected environmentaland virulent clinical N. asteroides strains are needed to devisemethods for acute strain differentiation in epidemiologicalstudies.

In conclusion, REP-PCR DNA fingerprint analysis can be usedfor differentiating N. asteroides isolates obtained from differentecological habitats. REP-PCR has already been successfully usedto classify and differentiate strains of E. coli (4), Bacillussporothermodurans (8), Streptomyces spp. (15), and several otherbacteria (12), but the method has not been previously appliedto Nocardia strain classification. REP-PCR DNA fingerprintingis simple and easy to perform and, thus, may prove to be a usefultool for rapidly determining N. asteroides strain identity andtracking specific strains for epidemiological and ecologicalstudies.

ACKNOWLEDGMENTS

We thank T. Kohno (University of Yamanashi, Kofu, Japan) forhelpful suggestions, F. Komatsu (Nihon Hels Industry Corp.,Tokyo, Japan) for providing the scum sample from activated sludge,and H. Iino (University of Yamanashi, Kofu, Japan) for technicalassistance.

FOOTNOTES

* Corresponding author. Mailing address: Division of Applied Biological Sciences, University of Yamanashi, Takeda-4, Kofu 400-8511, Japan. Phone: 81 (55) 220-8542. Fax: 81 (55) 220-8543. E-mail: hayakawa{at}ab11.yamanashi.ac.jp.

REFERENCES

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