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Journal of Clinical Microbiology, December 2008, p. 4111-4113, Vol. 46, No. 12
0095-1137/08/$08.00+0     doi:10.1128/JCM.01604-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Cross-Reaction of Stenotrophomonas and Xanthomonas Species in a 23S rRNA Gene-Directed PCR for Detection of S. maltophilia{triangledown}


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LETTER
 
Stenotrophomonas maltophilia is an opportunistic pathogen that occupies a variety of niches both inside and outside hospitals, mostly in moist environments (1). Whitby et al. recently developed species-specific PCR (SS-PCR) primers directed at a region of the S. maltophilia 23S rRNA gene to overcome problems with definitive identification of the species in the clinical laboratory (5). Several pulmonary pathogens, including Alcaligenes xylosoxidans, Burkholderia sp., Elizabethkingia meningosepticum, Haemophilus influenzae, Klebsiella pneumoniae, Moraxella catarrhalis, Proteus mirabilis, Pseudomonas sp., and Ralstonia sp. were tested in this (5) and other (3) studies. In addition, environmental strains of E. meningosepticum, Pseudomonas sp., Sphingomonas paucimobilis, and Wautersia paucula have been tested and no cross-reactivity was reported (2). In the present study, we examined cross-reactions with other Stenotrophomonas species and closely related genera occupying similar environmental niches.

Crude bacterial DNA extracts from Burkholderia cepacia genomovar I ATCC 25416, Pseudomonas aeruginosa NCTC 10662, Stenotrophomonas acidaminiphila ATCC 700916T, S. maltophilia ATCC 13637T, Stenotrophomonas nitritireducens ATCC BAA-12T, Stenotrophomonas rhizophila ATCC BAA-473T, Xanthomonas axonopodis pv. citri DAR 65864, and Xanthomonas campestris pv. campestris DAR 65808 (Xanthomonas strains were provided by the Australian Collection of Plant Pathogenic Bacteria, Orange, Australia) were prepared as described elsewhere (2). The SS-PCR protocol described by Whitby et al. (5) was used with modifications. The reaction mixture (50 µl) contained 1x reaction buffer II (Applied Biosystems), 3 mM MgCl2, 200 µM of each deoxyribonucleoside triphosphate, 1.25 U AmpliTaq Gold polymerase (Applied Biosystems), 1 µM of each primer (SM1 and SM4), and 5 µl of DNA extract. Amplifications were performed on a GeneAmp PCR system 2700 thermocycler (Applied Biosystems) as follows: 95°C for 10 min and then 45 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 60 s, with final extension at 72°C for 7 min. A product of approximately 531 bp was expected.

All Stenotrophomonas and Xanthomonas species tested gave a positive reaction in the S. maltophilia SS-PCR. Pseudomonas aeruginosa NCTC 10662 and Burkholderia cepacia ATCC 25416 were PCR negative; clinical isolates of P. aeruginosa and B. cepacia genomovar I were PCR negative elsewhere (5). Results were reproducible over two or more replicates. Sequencing of products was performed (ABI PRISM 3100 Avant genetic analyzer; Applied Biosystems) and the S. acidaminiphila ATCC 700916T, Stenotrophomonas nitritireducens ATCC BAA-12T, Stenotrophomonas rhizophila ATCC BAA-473T, Xanthomonas axonopodis pv. citri DAR 65864, and Xanthomonas campestris pv. campestris DAR 65808 sequences have the GenBank accession no. EU878276 to EU878280. All were highly homologous to the 23S rRNA partial gene sequence of S. maltophilia ATCC 13637 (GenBank accession no. AF273255) (5) (Table 1). The partial nucleotide sequence of the product from ATCC 13637 (481 bp) was 99% homologous to AF273255, with a single polymorphism (A567G).


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  		TABLE 1. Sequence similarity of SS-PCR products of Stenotrophomonas and Xanthomonas spp. to the S. maltophilia ATCC 13637 23S rRNA partial gene sequencea

The S. maltophilia 23S target sequence from GenBank sequence AF273255 (nucleotide positions 62 to 593) was used to query the GenBank database to identify other possible cross-reactivity of the PCR. S. maltophilia K279a genome (AM743169), and 23S rRNA genes from three other S. maltophilia strains had 97 to 99% identity to the target sequence (532 nucleotides) (E value, 0.0). The next highest homology (93% identity; 99% query coverage; E value, 0.0) was in the 23S rRNA gene from complete genome sequences of X. axonopodis pv. citri (one strain), X. campestris pv. campestris (3), X. campestris pv. vesicatoria (1), and X. oryzae pv. oryzae (3). A single mismatch (A-C [primer-template]) exists at the 3' terminus of the forward primer (SM1) among Xanthomonas spp., while this mismatch and a second internal mismatch with the reverse primer (SM4) (T-G) exist in Xylella fastidiosa (23S rRNA gene from complete genome sequences of four strains; 89% identity; 99% query coverage; E value, 0.0) (Fig. 1). Single internal mismatches and single A-C mismatches at the 3'-terminal nucleotide of a primer may not reduce amplification efficiency (4). Although 23S rRNA gene sequences from P. aeruginosa, Pseudomonas fluorescens, and Pseudomonas stutzeri (PCR-negative species) (5) were of the next highest identity to the query sequence (83 to 85% identity; 99% query coverage; E value, 2e–163 to 6e–158), they had more significant mismatches in their primer binding regions.


Figure 1
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  		FIG. 1. Alignment of the SM1 (A) and SM4 (B) primer sequences with high-scoring sequences from a BLASTn query (June 2008) of the AF273255 SS-PCR target sequence. Sequences known to be from the same strain were not included. Dots indicate identity with the primer sequence.

S. maltophilia is the only Stenotrophomonas species to cause infection in humans. With the potential for use of Stenotrophomonas species as biological control agents (1, 6), it is important to be able to distinguish between S. maltophilia and other apparently less harmful Stenotrophomonas species sharing the same habitat and possessing similar biological control capacity. Molecular methods are increasingly used to identify or confirm the identity of bacterial isolates. Our findings emphasize the need to thoroughly evaluate SS-PCR methods using related species, particularly when such species may share an ecological niche.


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FOOTNOTES
 
{triangledown} Published ahead of print on 8 October 2008. Back


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REFERENCES
 
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  1. Denton, M., and K. G. Kerr. 1998. Microbiological and clinical aspects of infection associated with Stenotrophomonas maltophilia. Clin. Microbiol. Rev. 11:57-80.[Abstract/Free Full Text]
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  2. Foster, N. F., B. J. Chang, and T. V. Riley. 2008. Evaluation of a modified selective differential medium for the isolation of Stenotrophomonas maltophilia. J. Microbiol. Methods 75:153-155.[CrossRef][Medline]
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  3. Giordano, A., A. Magni, M. Trancassini, P. Varesi, R. Turner, and C. Mancini. 2006. Identification of respiratory isolates of Stenotrophomonas maltophilia by commercial biochemical systems and species-specific PCR. J. Microbiol. Methods 64:135-138.[CrossRef][Medline]
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  4. Kwok, S., D. E. Kellogg, N. McKinney, D. Spasic, L. Goda, C. Levenson, and J. J. Sninsky. 1990. Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucleic Acids Res. 18:999-1005.[Abstract/Free Full Text]
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  5. Whitby, P. W., K. B. Carter, J. L. Burns, J. A. Royall, J. J. LiPuma, and T. L. Stull. 2000. Identification and detection of Stenotrophomonas maltophilia by rRNA-directed PCR. J. Clin. Microbiol. 38:4305-4309.[Abstract/Free Full Text]
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  6. Wolf, A., A. Fritze, M. Hagemann, and G. Berg. 2002. Stenotrophomonas rhizophila sp. nov., a novel plant-associated bacterium with antifungal properties. Int. J. Syst. Evol. Microbiol. 52:1937-1944.[Abstract]
Niki F. Foster*
Discipline of Microbiology & Immunology
School of Biomedical, Biomolecular and Chemical Sciences
The University of Western Australia
Queen Elizabeth II Medical Centre
Nedlands, Australia

Gerald B. Harnett
Division of Microbiology and Infectious Diseases
PathWest Laboratory Medicine WA
Queen Elizabeth II Medical Centre
Nedlands, Australia

Thomas V. Riley
Barbara J. Chang
Discipline of Microbiology & Immunology
School of Biomedical, Biomolecular and Chemical Sciences
The University of Western Australia
Queen Elizabeth II Medical Centre
Nedlands, Australia

* Phone: 61-8-9346-2250, Fax: 61-8-9346-2912, E-mail: nfoster{at}cyllene.uwa.edu.au

Journal of Clinical Microbiology, December 2008, p. 4111-4113, Vol. 46, No. 12
0095-1137/08/$08.00+0     doi:10.1128/JCM.01604-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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