Evidence for Clonal Dissemination of the Serotype K1 Klebsiella pneumoniae Strain Causing Invasive Liver Abscesses in Korea

Abstract

Seventy-three liver abscess isolates of serotype K1 Klebsiella pneumoniae from a nationwide collection in Korea were genotypically characterized using pulsed-field gel electrophoresis and multilocus sequence typing. We found that serotype K1 K. pneumoniae strains that caused liver abscesses in Korea were genotypically related and that most were sequence type 23.

Klebsiella pneumoniae has become a major etiologic agent of liver abscess in recent decades, and such an epidemiologic change has been most prominent in Asian countries, including Taiwan and Korea (4, 13, 18). Meta-analysis of the etiology of pyogenic liver abscesses in Korea has clearly documented that the etiology of pyogenic liver abscesses has continuously changed in this country during the last half-century (4). A recent nationwide Korean study showed that K. pneumoniae accounted for 78.2% of community-acquired liver abscesses in Korea (4). The K. pneumoniae liver abscess has distinctive clinical characteristics, such as a strong association with diabetes mellitus (10, 12) and a high tendency for other metastatic infectious foci (2, 4, 9). Previous studies have shown that K1 is the predominant serotype among K. pneumoniae isolates that cause liver abscesses in Taiwan and Korea, accounting for around 60% of cases (4, 9). However, the K1 serotype had been uncommon among the clinical isolates before the 1990s (1, 5, 6). Furthermore, infection by this serotype has been more widespread in the Asian countries even though there have been reports addressing a recent increasing role of K. pneumoniae in liver abscess in the United States (8, 15, 16). To date, there is no explanation for the epidemiological changes and global differences observed.

In order to elucidate the reasons for the emergence of serotype K1 K. pneumoniae as a major causative organism for liver abscesses in some Asian countries, molecular characterization of the isolates is essential. In this study, we determined the genotypes of serotype K1 K. pneumoniae liver abscess isolates collected through a prospective nationwide study in Korea and investigated the possibility of regional spread of these invasive strains in Korea.

Serotype K1 K. pneumoniae isolates that were collected from community-acquired liver abscesses during a nationwide prospective study by the Korean Study Group for Liver Abscess (4) were included. The residences of the patients were widely distributed throughout the entire country of South Korea. Community-acquired infection was defined as an infection diagnosed within 48 h of admission to the hospital. The K antigen serotype was determined using both slide agglutination testing with antisera (Denka Seiken, Tokyo, Japan) and magA PCR, as previously described (4). Seventy-three liver abscess isolates were randomly selected and were genotyped. Five ATCC reference strains of serotype K1 (ATCC 8044, ATCC 8045, ATCC 8047, ATCC 35593, and ATCC 13886) were also typed for comparison. For pulsed-field gel electrophoresis (PFGE), agarose-embedded bacterial genomic DNA was digested with 20 U of XbaI. The restriction fragments were separated by electrophoresis in 0.5× Tris-borate-EDTA buffer. Electrophoresis was conducted using a CHEF Mapper XA (Bio-Rad Laboratories, Hercules, CA). The PFGE patterns were analyzed by using BioNumerics software, version 4.01 (Applied Maths, Belgium). Clustering of patterns was done by the unweighted-pair group method with arithmetic averaging and the Dice coefficient. The Dice similarity coefficient was used with optimization and position tolerance settings of 1.0%. A PFGE-based clonal group (CG) was defined as a group of isolates with ≥70% similarity on a dendrogram.

In order to better characterize the evolutionary genetic relationships among the isolates, multilocus sequence typing (MLST) was performed on the isolates of serotype K1 K. pneumoniae by determining the nucleotide sequences of seven housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB, and tonB), as described previously (7). ATCC reference strains of serotype K1 were compared with our strains. The internal fragments of those genes were amplified by PCR, and the nucleotide sequences were determined for both strands. Sequence types (STs) were assigned by reference to the K. pneumoniae MLST website (http://pubmlst.org/kpneumoniae/) developed by Keith Jolley and sited at the University of Oxford (11). For phylogenetic analysis of the strains, the eBURST algorithm, available at http://eburst.mlst.net/, was used. STs were clustered into groups, employing the group definition where all members assigned to the same group share identical alleles at ≥5 of the 7 loci.

The PFGE patterns of the 73 K. pneumoniae isolates of the K1 serotype were analyzed (Fig. 1). Sixty-nine (94.5%) out of 73 isolates were clustered into a major group with >70% similarity and were designated CG1. Within this group, eight subgroups were further clustered with >80% similarity. All five ATCC strains of the K1 serotype showed closely related PFGE patterns within each other; however, their PFGE patterns were different from those of our isolates.

FIG. 1.

Dendrogram of PFGE profiles and MLST STs of the K1 serotype K. pneumoniae liver abscess isolates.

Strikingly, MLST showed that 71 (97.3%) out of 73 isolates had the same ST, ST23 (Fig. 1). The other two isolates were assigned to ST163 and ST217 and were single-locus variants of ST23. All five ATCC strains were classified as ST82. By the group definition in eBURST, all of the liver abscess isolates found in our study were grouped as a single clonal complex that contrasted with the ATCC strains of ST82. Bootstrap resampling of the eBURST analysis yielded a value of 91% for prediction of ST23 as the primary founder of this clonal complex.

The results of this study provide evidence that the K1 serotype K. pneumoniae strains, responsible for the increasing prevalence of invasive liver abscesses in Korea, are genetically homogeneous. Our findings showed that most isolates of serotype K1 K. pneumoniae from liver abscess cases in Korea showed very similar PFGE patterns; most isolates belonged to the same ST, ST23. This is the first report demonstrating that the recent increasing prevalence of serotype K1 K. pneumoniae liver abscesses is attributable to the nationwide dissemination of the ST23 strain throughout the entire country. The K. pneumoniae strains of ST23 were originally registered in the database by a European investigator from three blood isolates from Belgium, The Netherlands, and Spain (7). Furthermore, a recent report from England suggested the possibility that this genotype (ST23) is geographically widespread, based on the results of PFGE and MLST evaluations of 16 K. pneumoniae isolates from the United Kingdom, Hong Kong, Israel, Taiwan, and Australia (17). Although previous studies using PFGE pattern analysis in Taiwan showed controversial results regarding the clonality of the K. pneumoniae liver abscess isolates (3, 14, 19), some isolates showed similar PFGE patterns with the Korean isolates in our study (CG1) (14). Many factors, including different definitions of clonality, limit the interpretation and comparison of the PFGE patterns from different laboratories in many countries. In this view, comparison with the MLST data from other regions will be valuable for further understanding of the worldwide epidemiology of K1 K. pneumoniae infection.

These findings suggest the possibility of global dissemination of the virulent K1 serotype K. pneumoniae strains of ST23 and its association with invasive diseases, including liver abscesses. Although the spread of virulent strains has contributed to the increasing prevalence of K. pneumoniae liver abscesses in Korea, host factors, such as ethnicity, dietary practices, and the local food chain, must also be considered a contributing factor. Further research is necessary to understand the distinct global epidemiology of invasive disease caused by K. pneumoniae.

In conclusion, serotype K1 K. pneumoniae isolates causing liver abscesses in Korea are genotypically closely related, and most isolates identified were ST23 or its single-locus variants. These findings suggest that nationwide clonal dissemination of these virulent strains of ST23 may be associated with an increasing prevalence of invasive liver abscesses caused by the K1 type K. pneumoniae in Korea.