A PCR-mediated method for discrimination of Klebsiella oxytoca between closely related bacteria in environmental and clinical specimens

A specific detection method was developed to discriminate Klebsiella oxytoca between other species of the genus Klebsiella on the basis of PCR amplification of the unique DNA sequences within the polygalacturonase-encoding (pehX) gene. Four primers have been designed for performing PCRs gaining amplicons of 282, 344, 45J and 513 bp. The specificity of the test was verified by the lack of PCR products in case of related K. pneumoniae, K. planticola, and polygalacturonate-degrading species of the genus Erwinia. The PCR-mediated test gives a rapid answer, concerning the presence of K. oxytoca in a sample, or in differentiating this bacterium from other species, such as K. pneumoniae, with which they can be confused. The diagnostic test can be used in ecological monitoring of К oxytoca as well as in medical laboratories.

Introduction.The bacteria K. oxytoca and K. pneu moniae are controversial species of the genus Kleb siella.On the one hand, they are known as nitrogenfixing organisms beneficial in agriculture [1 ].Be sides, they are concerned with pioneer work on genetics [2,3] and enzymology of nitrogen (N 2 ) fixation [4].The nitrogen-fixing K. oxytoca strains have been isolated from the rhizosphere of rice [5], or from the interior of rice and sweet potato roots [6,7 ].K. pneumoniae has been isolated from the interior of maize roots [8 ].At the same time, representatives of these two species are described as causal agents of human diseases [9].
The taxonomic position of K. oxytoca is still not clear, this bacterium is often in «the shadow» of К pneumoniae as it was considered for a long time as a subspecies of the latter [10], and was separated only according to the DNA-DNA hybridization and phenotype characteristics [11].According to the worldwide predominant Orskov's classification [10] К oxytoca is an independent species of the genus, however, within the classification of Cowan [9 ] which is also valid, there is no such a name.The lack of a consistent nomenclature results sometimes in mistakes in differentiation of the two species.Nitrogen-fixing isolates of Klebsiella, possessing distinguishing chara cteristics of K. oxytoca, have been designated as К pneumoniae (for example, strain К pneumoniae M5al) [1].Consequently, the name K. pneumoniae instead of K. oxytoca is probably used in many publications improperly.Nowadays, due to the rapid development of bioinformatics, DNA sequences of improperly named bacteria being deposited in public gene banks and spread quickly, determine the ap pearance of more errors.
Another reason for the misidentification of К oxytoca is that detecting discriminative trait of К oxytoca such as pectate degradation appears too labour-consuming to be taken into account in the identification process.The Biolog system and com mercial biochemical tests (API 20E) designed for the Enterobacteriaceae identification are unreliable being used for the Klebsiella species differentiation.A new identification system designed on the basis of colony color and morphology on CHROMagar orientation medium in combination with simple biochemical tests such as indole, lysine decarboxylase, and ornithine decarboxylase does not discriminate properly between K. oxytoca and K. pneumoniae as well [12].
Various DNA probes have been used in several hybridization and PCR procedures to distinguish К oxytoca [13][14][15][16][17][18].However, none of these tech niques is completely satisfactory since the sequences used are not present exclusively in K. oxytoca (e. g. gyrA, parC, infB, rrs, and bid).This can lead to cross-reactions between the probe and DNA sequ ences from other species of the genus Klebsiella and from other enterobacteria resulting in a large number of false negatives or false positives.
The aim of this study was to devise a rapid and sensitive test for the discrimination between K. oxy toca and close relatives and detection of this bacte rium in the environmental and clinical specimens.The idea was to exploit a pectate degradation capability of K. oxytoca, which distinguishes the bacterium from other species.More specifically, we used a unique sequence of the gene encoding the enzyme poly galacturonase that cleaves a polygalacturonic chain of demethoxylated pectin.
Materials and Methods.Bacteria are listed in Table 1.
Biochemical tests.Bacterial strains were consi dered as belonging to the genus Klebsiella on confi dent identification as «K.oxytoca or K. pneumoniae* after biochemical key tests such as indole (positive for К oxytoca, negative for К pneumoniae), melezitose (positive for some strains of K. oxytoca, negative for K. pneumoniae), gas production from lactose at 44.5 °С (positive for К pneumoniae, negative for К oxytoca), growth at 10 °С (positive for K. oxytoca, negative for К pneumoniae) [19 ].The degradation of pectate [20] was used to analyze collection strains along with type ones.The cultures were spotted on pectate-containing semisolid PEC-SSA medium, and pectolytic ones exhibited indenting the agar.
PCR and primer design.In this study, the tests were carried out by using lysed cells of approximately 1.0 10 7 ~8 cfu/ml in PCR mixtures with annealing temperature of 58 °С and MgCl 2 concentration of 2.5 mM.The primer sequences PEH-A (5'-ggactacgccgtctatcgtcaag-3'), PEH-B (5'-aatatccagggtcatatcgctgtg-3'), PEH-C (5'-gatacggagtatgcctttacggtg-3'), PEH-D (5'-tagcctttatcaagcggatactgg-3') were chosen in the coding part of the gene.The primer and available  [21 ].The sequence was compared with the other peh gene sequences available in GenBank, and the comparison did not display essential homology in the DNA sequences of pectinolytic bacteria.However, alignment of the encoded amino acids revealed 52-71 % homology with the Yersinia enterocolitica, E. chrysanthemi, and Ralstonia solanacearum exopolygalacturonases.PCR-mediated method for detection of K. oxyto ca.Our approach utilizes the PCR-mediated tech nique that allows determination of a specific gene in a living organism or a sample.The genetic marker pehX having been identified, permits the specific detection of K. oxytoca in any sample and dis crimination of the bacterium from related strains.The set of strains, such as type strains of the genus Klebsiella (K.oxytoca ATCC 13183, K. planticola ATCC 33531 and K. pneumoniae ATCC 13883), well characterized strains K. pneumoniae M5al and K. oxytoca VN13 [1,6], as well as strains of the genus Erwinia, and E. coli JM109 analyzed previously by the method of pectate degradation, were used in experiments to verify specificity of the primers de signed.All strains of Erwinia sank into the medium, as well as the K. oxytoca strains and K. pneumoniae M5al (Fig. 1).Other strains of Klebsiella grew but did not indent the medium.The K. oxytoca VN13 and K. oxytoca ATCC 13183 strains produced the 344 and 513 bp amplicons in PCR with a pair of primers, PEH-C, D, and PEH-A, D (Fig. 2, A, B).The K.

Fig. 1. Capability of Klebsiella oxytoca to indent a polygalacturonate semisolid agar as evidence for pectinase production
pneumoniae ATCC 13883, E. carotovora subsp.caro tovora, E, carotovora subsp.atroceptica, E. chrysanthemi, K. planticola ATCC 33531 and E. coli JM109 were negative in the PCR tests.The nitrogenfixing strain of K. pneumoniae M5al possessing characteristics of K. oxytoca, also generated specific amplicons of 344 and 513 bp (Fig. 2, C).DNA of diazotrophic bacterium K. pneumoniae 342, correctly identified by molecular methods [8 ], was used as a reference in experiments to detect the pekX specific sequence in bacterial DNA.The primers designed for the pehX gene did not anneal to DNAs of K. pneumoniae 342, K. pneumoniae ZMVSY and the clinical isolate K. pneumoniae MGH78578.Amplification of the unique DNA fragments in the PCR test revealed high level of specificity of the primers designed for the pehX gene.The negative results in E. carotovora and E. chrysanthemi strains were expected because there was practically no homo logy between the polygalacturonase-encoding gene of K. oxytoca and analogous genes of Erwinia [21 ].The absence of amplicons in K. pneumoniae, K. planticola and E. coli JM109 can be explained by the absence of specific gene encoding the hydrolytic enzyme.
The method presented can be used for the detection of specific target bacteria in a complex sample mixture.The latter may contain a variety of components including non-target or background mic roorganisms.The method proceeds by first culturing the complex sample mixture in a non-selective growth medium followed by the PCR-mediated detection of a target bacterial DNA.The target DNA is detected via a DNA amplification protocol with a primer pair selected to amplify a specific portion of the target bacterial DNA.A control DNA is amplified in parallel with the target bacterial DNA.The control DNA is  specifically designed to be amplified with the primers that are identical to the primers used in the ampli fication of the target genomic DNA.Use of this control validates the amplification reaction.Detection of the amplified target DNA and the control is accomplished by gel electrophoresis.

Screening collection of Klebsiella by a molecular method.
A set of bacterial strains of the genus Klebsiella, deposited in the collection of Kyiv Re search Institute of Epidemiology and Infection Disea ses, both type strains gathered from international collections and clinical isolates have been screened for the availability of the pehX gene.Five strains, depo sited as K. pneumoniae (type strains K41, K66, K69, K75, K76), generated specific K. oxytoca amplicons with the primers PEH-A, В and PEH-C, D (see Fig. 2, C).DNA of K. pneumoniae PZH (K79) did give a 344 bp fragment, but did not give a 451 bp one which may be explained either by some deletion in the gene, or by a lack of homology between strains VN13 and PZH (K79) within sites of the primers PEH-A and PEH-B recognition.
Identification of K. oxytoca or K. pneumoniae by biochemical tests.Six Peri strains were examined by the biochemical key tests listed above that differen tiate К oxytoca from K. pneumoniae.In parallel six type strains of K. pneumoniae (K10, K21, K28, K30, K37, K55) that did not possess the peAX-specific sequence in their genomes were included in bioche mical testing.The results of these tests correlated with the data of the peh DNA amplification and confirmed the taxonomic positions of six strains previously identified by molecular methods as K. oxytoca and six strains identified as K. pneumoniae (Table 2).This means that six type strains of K. pneumoniae deposited in international collections we re identified earlier according to an outdated taxo nomic system.
Conclusions.The rapid and sensitive PCR test for the discrimination of K. oxytoca between closely related bacteria and detection of this bacterium in environmental and clinical samples has been elabo rated.The test is based on the recognition of unique DNA sequence within the gene encoding the polyga lacturonase that cleaves a polygalacturonic chain of demethoxylated pectin.The use of different pairs of primers specific to the pehX gene helps to avoid mistakes in differentiation of K. oxytoca.The diag nostic test gives a rapid answer, regarding the pre sence of K. oxytoca in a sample, or in differentiating this bacterium from some other bacteria, such as K. pneumoniae, with which they can be confused.The specific PCR test overcomes the handicap of timeconsuming microbiological and biochemical methods and can be used for ecological monitoring K. oxytoca and in medical laboratories.
Acknowledgements.We are grateful to Eric Triplett (Wisconsin University) for providing the K. pneumoniae DNA and to Mike Merrik (John Innes Centre) for critical reading of the manuscript.