Molecular serotyping of Klebsiella species isolates by restriction of the amplified capsular antigen gene cluster

Abstract

The objective of the present work was to develop a molecular method that would enable determination of the capsular serotypes of Klebsiella isolates without the use of antiserum. PCR amplification of the capsular antigen gene cluster (cps) was followed by digestion with the restriction enzyme HincII (cps PCR-restriction fragment length polymorphism [RFLP] analysis). The profiles (C patterns) obtained for 224 strains representing the 77 known K serotypes showed 3 to 13 fragments ranging in size from 0.2 to 4.4 kb. A total of 97 distinct C patterns were obtained; 100% of 61 pairs of samples tested twice showed reproducible C patterns. The C patterns were K-type specific; i.e., the C pattern(s) of any K serotype was distinct from the C patterns of all other K serotypes, with the only exceptions being serotypes K22 and K37, which are known to cross-react. For 12 of 17 K types for which at least two strains were included, C-pattern variations were found among strains with the same K serotype. Therefore, cps PCR-RFLP analysis has a higher discriminatory power than classical K serotyping. C-pattern identity was observed among strains with a given K type that were collected many years apart and from distinct sources, indicating C-pattern stability. Only 4.5% of the strains were nontypeable, because of unsuccessful PCR amplification (whereas 8 to 23% are nontypeable by classical K serotyping). Three of four noncapsulated strains analyzed showed recognizable C patterns. The K serotypes of 18 (82%) of 22 recent Klebsiella pneumoniae clinical isolates could be deduced from their C patterns. In conclusion, cps PCR-RFLP analysis allows determination of the K serotype, while it is easier to perform and more discriminatory than classical serotyping.

FIG. 1.

cps PCR-RFLP analysis of 12 Klebsiella strains. Lanes M, molecular size markers (in base pairs). The C-pattern codes are indicated above the lanes, and the K serotypes and strain names are given in parentheses. Small differences can be observed between patterns C2d and C2e, on the one hand, and between patterns C4a, C4b, and C4c, on the other. C pattern C4f shares only two fragments with the other C patterns of K4 strains.

FIG. 2.

Overview of the 102 distinct C patterns found during this study. The five C patterns obtained for clinical strains whose K serotypes were initially unknown and which did not exactly match a reference C pattern are given. The patterns obtained with primer rCPS2 (Fig. 3) are not included. C patterns were ordered by similarity by the unweighted pair group method with arithmetic averages procedure, based on the Pearson coefficient, by using a 1% optimization parameter. Pearson coefficients were converted to percentages with BioNumerics software. Uncertain bands, which were weak and which were not taken into account for pattern comparison, are indicated by dashes.

FIG. 3.

Comparison of the patterns obtained after amplification with the alternative primer, gnd-specific primer rCPS2, with closely matching C patterns obtained with primer rCPS (and BioNumerics software). In most cases, the patterns obtained with primer rCPS2 differed by one or two bands from the corresponding C pattern obtained for strains of the same K serotype. The only exceptions are for K68 strain 1578 and K6 strain 1283; the C pattern of the latter strain nevertheless shares four fragments with pattern C6a.