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Journal of Clinical Microbiology, April 2003, p. 1740-1742, Vol. 41, No. 4
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.4.1740-1742.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Enterobacterial Repetitive Intergenic Consensus 1R PCR Assay for Detection of Raoultella sp. Isolates among Strains Identified as Klebsiella oxytoca in the Clinical Laboratory

Sophie A. Granier,1,2 Véronique Leflon-Guibout,1,3 Fred W. Goldstein,2 and Marie-Hélène Nicolas-Chanoine1,3*

Service de Microbiologie-Hygiène, Hôpital Ambroise Paré AP-HP, Boulogne,1 Faculté Paris-Ile-de-France-Ouest, Université Versailles-Saint-Quentin-en-Yvelines, Versailles,3 Fondation Hôpital Saint-Joseph, Laboratoire de Microbiologie Médicale, Paris, France2

Received 16 September 2002/ Returned for modification 15 November 2002/ Accepted 10 January 2003


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ABSTRACT
 
The enterobacterial repetitive intergenic consensus 1R PCR method, which provided recognizable profiles for reference strains of the three species of Raoultella and the two genetic groups of Klebsiella oxytoca, was applied to 19 clinical isolates identified as K. oxytoca. By this method, as confirmed by species-specific gene sequencing, two Raoultella ornithinolytica and two unclassifiable K. oxytoca isolates were identified.


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TEXT
 
The genus Klebsiella has recently been reanalyzed with regard to its phylogenetic structure, and all studies performed have shown the taxonomic heterogeneity of this genus (1, 2, 4). Thus, Drancourt et al. suggested on the basis of 16S ribosomal DNA (rDNA) and rpoB gene sequences that the genus Klebsiella be divided into two genera, Klebsiella and Raoultella (Raoultella planticola, Raoultella ornithinolytica, and Raoultella terrigena), and that Klebsiella oxytoca be left as a monophyletic taxon (2). For our part, we have demonstrated that the K. oxytoca taxon is divided into two clades which correspond to two genetic groups, called oxy-1 and oxy-2. These genetic groups are recognizable by three independent molecular characters: (i) group-specific nucleotides located both in a 23-bp fragment of the 16S rDNA and in a 512-bp fragment of the rpoB gene, (ii) characteristic bands in the profiles generated by the enterobacterial repetitive intergenic consensus 1R (ERIC-1R) PCR method, and (iii) the type of blaOXY gene (blaOXY-1 or blaOXY-2) (3, 4). In spite of this taxonomic advance, it remains very difficult to distinguish between isolates of Klebsiella, particularly K. oxytoca, and isolates of Raoultella by routine chemical analysis. As the ERIC-1R PCR method was able to differentiate the two K. oxytoca genetic groups and as it is a simple method accessible to the majority of microbiologists, we decided to test its ability to differentiate Raoultella species from K. oxytoca.

The ERIC-1R PCR assay was first applied, as previously described (4), to eight reference strains, including two reference strains of K. oxytoca representative of the two genetic groups (strain SL781 [oxy-1] and strain SL911 [oxy-2]), three American Type Culture Collection (ATCC) strains (ATCC 31898T, ATCC 33531T, and ATCC 33257T), and three bioMérieux collection strains (BM85.01.101, BM85.01.092, and BM85.01.095) of R. ornithinolytica, R. planticola, and R. terrigena, respectively. As indicated in Fig. 1, the two strains of K. oxytoca were distinguishable from each other as previously described (4). Briefly, strain SL781 had a profile marked by two intensive bands of approximately 1,400 and 1,600 bp, whereas strain SL911 had a profile marked by a single or a double intensive band of approximately 500 bp. Regarding Raoultella spp., Fig. 1 shows that the two strains of each species displayed profiles close to each other, whereas a distinguishable profile was observed for each species on the basis of the presence of species-specific bands. Thus, the profile obtained from R. ornithinolytica strains was marked by two intensive bands of approximately 1,300 and 2,300 bp, that obtained from R. planticola strains was marked by two intensive bands of approximately 400 and 900 bp, and that obtained from R. terrigena strains was marked by three bands, two with high intensity and sizes of approximately 800 and 1,500 bp and one with moderate intensity and a size of approximately 2,100 bp. The ERIC-1R PCR method was then applied to 19 clinical isolates (Table 1) identified as K. oxytoca by the routine method (API system; bioMérieux, Marcy l'Etoile, France). They had been obtained over a 2-month period from 16 inpatients and 2 outpatients in Saint-Joseph hospital in Paris, one inpatient having had two different specimens positive for K. oxytoca in a 1-month period (strains SG251 and SG273). Their ERIC-1R profiles (Fig. 2) were analyzed and compared with those of the reference strains of K. oxytoca and of Raoultella species. By this procedure, strains SG245, SG246, SG248, SG249, SG250, SG252, SG254, SG258, SG261, SG264, and SG265 were identified as members of the genetic group oxy-2 of K. oxytoca because their profiles displayed one or two intensive bands of 500 bp, whereas strains SG251, SG255, SG259, and SG273 were identified as members of the genetic group oxy-1 because their profiles displayed the two intensive bands of 1,400 and 1,600 bp. Strains SG251 and SG273 displayed identical ERIC-1R profiles. Of the four remaining strains, two, SG247 and SG269, displayed a profile with two intensive bands of 1,300 and 2,300 bp which strongly resembled that displayed by the two reference strains of R. ornithinolytica (Fig. 1), and the other two, SG266 and SG271, displayed two profiles which did not include the characteristic bands defined for the different species of Raoultella and the two genetic groups of K. oxytoca.



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  		FIG. 1. ERIC-1R profiles of R. ornithinolytica, R. planticola, R. terrigena, and K. oxytoca strains. Lanes 1 and 10, molecular size markers (100-bp ladder; Amersham Biosciences, Little Chalfont, United Kingdom), with the most intense band corresponding to 800 bp; lanes 2 and 3, R. ornithinolytica ATCC 31898T and BM85.01.101, respectively; lanes 4 and 5, R. planticola ATCC 33531T and BM85.01.092, respectively; lanes 6 and 7, R. terrigena ATCC 33257T and BM85.01.095, respectively; lanes 8 and 9, K. oxytoca strains SL781 (genetic group oxy-1) and SL911 (genetic group oxy-2), respectively.


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  		TABLE 1. Molecular analysis of clinical isolates identified by routine chemical analysis as K. oxytoca



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  		FIG. 2. ERIC-1R profiles of 19 clinical isolates identified as K. oxytoca and reference strains of K. oxytoca. (A) Lanes 1 and 12, molecular size markers (100-bp ladder; Amersham Biosciences), with the most intense band corresponding to 800 bp; lanes 2 to 9, isolates SG245, SG246, SG247, SG248, SG249, SG250, SG251, and SG252, respectively; lanes 10 and 11, K. oxytoca strains SL781 (genetic group oxy-1) and SL911 (genetic group oxy-2), respectively. (B) Lanes 1 and 15, molecular size markers (100-bp ladder, Amersham Biosciences), with the most intense band corresponding to 800 bp; lanes 2 to 12, isolates SG254, SG255, SG258, SG259, SG261, SG264, SG265, SG266, SG269, SG271, and SG273, respectively; lanes 13 and 14, K. oxytoca strains SL781 (genetic group oxy-1) and SL911 (genetic group oxy-2), respectively.

In order to confirm the classification of the clinical isolates made on the basis of the ERIC-PCR method, blaOXY gene amplification and sequencing were performed as previously described (4). blaOXY gene amplification was obtained for all clinical isolates except for strains SG247 and SG269, which had been recognized by the ERIC-1R typing system as R. ornithinolytica. Furthermore, no amplification was obtained for the reference strains of Raoultella, meaning that the primers used are specific for the bla gene of K. oxytoca. By amplifying and sequencing the 16S rDNA and rpoB genes of strains SG247 and SG269 as previously described (4), we observed 100% identity with the corresponding genes of R. ornithinolytica strain ATCC 31898T (GenBank accession numbers AF129441 and AF129447, respectively). This result proves that the ERIC-1R PCR method is a reliable method for identifying R. ornithinolytica isolates (10.5%) among isolates identified as K. oxytoca in the clinical laboratory. Unfortunately, there were neither R. planticola nor R. terrigena strains among our clinical isolates, although these two species and particularly R. planticola were previously found (2 to 10%), by the use of fastidious biochemical tests, among isolates conventionally identified as K. oxytoca (5-10). For the 17 clinical isolates proved to be K. oxytoca by blaOXY gene amplification, the comparison of the sequence of the amplified fragments (GenBank accession numbers in Table 1) with the blaOXY-1 and blaOXY-2 reference genes (GenBank accession numbers Z30177 and Z49084, respectively) allowed us to confirm the classification into the two genetic groups made on the basis of the ERIC-1R profiles. This comparison also showed that the blaOXY gene of strain SG266 had nucleotide sequence identities of 95 and 86.3% with blaOXY-1 and blaOXY-2 genes, respectively, whereas that of strain SG271 had identities of 84.5 and 84.4% with these two reference genes, respectively. In fact, these blaOXY genes do not display the level of gene identity (>=99%) required for classification in one of the two blaOXY gene groups (3, 4). Because of these two K. oxytoca strains, particularly with regard to ERIC-1R profile and blaOXY gene sequence, additional molecular analyses are currently being performed.

In conclusion, this study demonstrates that R. ornithinolytica isolates can be differentiated from K. oxytoca isolates by ERIC-1R PCR. It suggests that the other species of Raoultella could also be differentiated, as the reference strains of these species display distinguishable ERIC-1R profiles. Finally, although the number of isolates tested was small, the ERIC-1R PCR method allowed us to detect two K. oxytoca isolates which apparently do not belong to the two genetic groups defined to date.

Nucleotide sequence accession numbers. Sequences being reported here for the first time have been deposited in GenBank under accession no. AY208274, AY210427, AY208273, AY208272, AY210429, AY210432, AY207376, AY210431, AY210428, AY210425, AY210430, AY208276, AY210426, AY208275, AY077481, AF491278, and AY210433.


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ACKNOWLEDGMENTS
 
This work was supported by a grant from the French ß-Lactamase Network (Ministère de la Recherche, Paris, France).


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FOOTNOTES
 
* Corresponding author. Mailing address: Microbiology Department, Hôpital A. Paré, 9 avenue Charles de Gaulle, 92100 Boulogne-Billancourt, France. Phone: 33-1-49 09 55 40. Fax: 33-1-49 09 59 21. E-mail: marie-helene.nicolas-chanoine{at}apr.ap-hop-paris.fr. Back


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Journal of Clinical Microbiology, April 2003, p. 1740-1742, Vol. 41, No. 4
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.4.1740-1742.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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