Diversity of Capsular Polysaccharide Gene Clusters in Kpc-Producing Klebsiella pneumoniae Clinical Isolates of Sequence Type 258 Involved in the Italian Epidemic

Strains of Klebsiella pneumoniae producing KPC-type beta-lactamases (KPC-Kp) are broadly disseminating worldwide and constitute a major healthcare threat given their extensively drug resistant phenotypes and ability to rapidly disseminate in healthcare settings. In this work we report on the characterization of two different capsular polysaccharide (CPS) gene clusters, named cps BO-4 and cps 207-2, from two KPC-Kp clinical strains from Italy belonging in sequence type (ST) 258, which is one of the most successful ST of KPC-Kp spreading worldwide. While cps BO-4 was different from known 78 K-types according to the recently proposed typing schemes based on the wzi or wzc gene sequences, cps 207-2 was classified as K41 by one of these methods. Bioinformatic analysis revealed that they were represented in the genomic sequences of KPC-Kp from strains of ST258 from different countries, and cps BO-4 was also detected in a KPC-Kp strain of ST442 from Brazil. Investigation of a collection of 46 ST258 and ST512 (a single locus variant of ST258) clinical strains representative of the recent Italian epidemic of KPC-Kp by means of a multiplex PCR typing approach revealed that cps BO-4 was the most prevalent type, being detected both in ST258 and ST512 strains with a countrywide distribution, while cps 207-2 was only detected in ST258 strains with a more restricted distribution.


Introduction
The capsular polysaccharide (CPS or K-antigen) is a recognized virulence factor of Klebsiella pneumoniae [1,2]. This component exhibits a remarkable intra-specific structural diversity which translates into different antigenic properties that may be relevant to bacterial virulence [2][3][4].
CPS diversity has classically been detected by serotyping techniques [5], but genotyping systems have recently been developed, offering several advantages vs. the conventional serotyping approach [6][7][8][9][10]. Among systems that do not require a sequencing step, a PCR-based typing system has been proposed for the detection of isolates of the K1, K2, K5, K20, K54 and K57 capsular types, that are commonly associated with invasive diseases or having a prominent pathogenicity [6]. Conversely, two systems based on amplification and sequencing of the conserved wzi and wzc genes were recently proposed to determine the K-type of K. pneumoniae [9,10].
Infections caused by these strains pose a major challenge due to their extended antibiotic resistance phenotypes and ability to rapidly disseminate in healthcare settings, and are associated with high mortality rates [19][20]. Detailed knowledge on the CPSs of these strains, however, is still limited. A ST258 KPC-Kp strain from Greece has recently been reported to express a K41 serotype CPS [21], while the chemical structure of the CPS of two representatives of an outbreak clone of ST258 KPC-Kp from USA has recently been described [22].
In this work we have characterized two different cps gene clusters from two KPC-Kp clinical strains of ST258 from Italy, and report on their distribution in a collection of KPC-Kp isolates of ST258 and ST512 representative of the recent Italian epidemic. We also propose a modification to a previously established PCRbased CPS typing system [6], to include recognition of these CPS types.

Characterization of Two Different CPS Gene Clusters in ST258 KPC-Kp Strains of Clinical Origin
The CPS gene cluster of two KPC-Kp strains of clinical origin, KKBO-4 and KK207-2, were characterized by an HTGS approach. The two strains had been isolated in 2010 from bloodstream infections of inpatients in two different Italian hospitals and produced either KPC-2 (KK207-2) or KPC-3 (KKBO-4). They were both of ST258, and exhibited a related although not identical XbaI PFGE profile [12] (difference of two bands, data not shown).
Comparison of the draft genomes using GGDC 2.0 confirmed the close relatedness between the two strains at the genomic level (intergenomic distance of 0.0015). Despite this close relatedness, however, the cps gene clusters of the two strains were significantly different from each other.
The CPS gene cluster of KKBO-4 (named cps BO-4 ) was found to be 26,587 bp-long, consisting of 20 ORFs (from galF to wzy), and was characterized by the presence of the K-antigen flippase-and polymerase-encoding genes (wzx and wzy, respectively) at the 39end, and by the presence of the rmlBADC operon for the synthesis of dTDP-L-rhamnose in the central region (Fig. 1).
The cps BO-4 gene cluster was identical or very similar to those present in a number of ST258 K. pneumoniae strains from different countries, whose genome sequences are available in the public domain, and also very similar to that previously described in a ST442 KPC-Kp strain (Kp13) that caused an outbreak in Brazil [23] (Table 1 and Fig. 1). Compared to the CPS gene cluster of K. pneumoniae HS11286 (ST11, a single locus variant of ST258) [29], cps BO-4 exhibited significant similarities in some regions (e. g. from galF to orf8 and from gnd to uge-1, comprising the rmlBADC operon), but also substantial differences in the central and the 39-region of the gene cluster ( Fig. 1).
According to the cps-typing protocol based on sequencing of the wzi gene [9] cps BO-4 showed a single nucleotide difference with the wzi81-K81 reference amplicon. According to the cps-typing protocol based on sequencing of the wzc gene [10] cps BO-4 was ,80% identical to that of any other reference sequence.
The CPS gene cluster of strain KK207-2 (named cps 207-2 ) was found to be 23,994 bp-long, consisting of 19 ORFs (from galF to ugd). It did not contain the rmlBADC operon but contained original genes, of which some encode putative glycosyltransferases, located between the wzy and wcaJ genes (Fig. 1). The cps 207-2 gene cluster was very similar to those present in ST258 K. pneumoniae strains from USA, whose genome sequences are available in the public domain (Table 1). It also exhibited regions of similarity with the CPS gene clusters of K. pneumoniae strains 1996/49 and 8238, producing CPS of the K22 and K37 serotype, respectively, and with both cps BO-4 and cps HS11286 (Fig. 1).
According to the cps-typing protocol based on sequencing of the wzi gene [9] cps 207-2 was identical to the wzi29-K41 reference amplicon. According to the cps-typing protocol based on sequencing of the wzc gene [10] cps 207-2 was 93% identical to the K22_ref and K37_ref reference sequences.
Taken together, these results suggested that the CPS composition of KK207-2 was different from that of KKBO-4, demonstrating that at least two different types of CPS gene clusters may be found in KPC-Kp of ST258, and that cps BO-4 -like gene clusters can also be found in KPC-Kp of unrelated STs such as ST442. The CPS gene cluster of strain 8238 (K-type 37) (accession number AB819894), differing from cps K22 by a single nucleotide deletion resulting in a frameshift mutation located in a putative acetyltransferase downstream gnd, is not included for simplicity. Homologous regions are connected by areas of different colors reflecting the degree of nucleotide identity (from 67% to 100%). Open reading frames encoding transposases are colored in red, while those encoding hypothetical glycosyltransferases are colored in yellow. The locations of synonymous, nonsynonymous and intergenic single nucleotide variations (SNVs) occurring between the CPS gene clusters of KKBO-4 and Kp13 are indicated by green, red and black stars, respectively. The cps 207-2 gene cluster exhibited regions of similarity to cps BO-4 including the conserved galF-wzc region (83.2% of nucleotide identity), and the conserved gnd and ugd genes (95.5% and 96.8% nucleotide identity, respectively). doi:10.1371/journal.pone.0096827.g001

Analysis of the cps Gene Clusters in a Contemporary Collection of ST258 and ST512 KPC-Kp Strains from the Italian Epidemic
A multiplex PCR protocol derived from that originally proposed by Turton et al. [6], modified to detect the cps BO-4 and cps 207-2 gene clusters, was used to analyze a collection of 46 nonreplicate KPC-Kp clinical strains of ST258 or ST512 isolated from 19 different centers (Fig. 2) during the first Italian countrywide survey on carbapenem-resistant Enterobacteriaceae [12] and selected as representatives of the recent Italian KPC-Kp epidemic. Nine additional carbapenem-resistant but KPC-negative K. pneumoniae strains with different carbapenem-resistance mechanisms (production of VIM-1, of OXA-48, or of an extended-spectrum beta-lactamase in presence of a permeability defect), isolated during the same survey, were also analyzed for comparison.

Discussion
Results of this work showed that KPC-Kp belonging to ST258, which have largely contributed to the epidemic dissemination of  The first two characters of each strain ID identify the center from which the isolate was obtained. Identifiers are as reported in the legend to Fig. 2 the KPC-type beta-lactamases in Italy and elsewhere [12,20], can be equipped with at least two different types of CPS gene clusters, here named cps BO-4 and cps 207-2 . The former type was more prevalent in a collection of representative isolates from the recent Italian epidemic, being also present in strains of ST512. The differences in the nature of these CPS gene clusters could be related with differences in the ability of spreading and virulence of different clones, which will deserve further investigation. The detailed chemical structure of cps BO-4 was recently solved for two representatives of the outbreak clone of KPC-Kp found at the Clinical Center of the U.S. National Institutes of Health [22,24]. The Authors demonstrated that this CPS type is structurally different from any other published K. pneumoniae CPS, even if similarity to K. pneumoniae K19 and K34 antigens was observed, possibly explaining the cross-reactivity of this CPS with the K34 antiserum [22]. These results corroborate the hypothesis that cps BO-4 -like gene clusters belong to a novel capsular type, as also suggested by the results obtained for cps BO-4 using the typing methods based on wzi and wzc gene sequences.
On the other hand, results obtained for cps 207-2 using the above genotyping methods were not in agreement between each other. In fact, while according to the wzc-based method [10] cps 207-2 corresponded to a new K-type, according to the wzi-based method [9] this gene cluster corresponds to the known K41 K-type. The result obtained with the wzi-based method could be consistent with the finding that a strain of KPC-Kp of ST258, representative of the dominant clone circulating in Greece during 2009-2011, was serotyped as K41 [21]. This finding also suggests that this K-type has achieved a significant distribution in some settings, and it would therefore be interesting to further investigate the nature of the whole CPS gene cluster in KPC-Kp strains of K-type 41.
Data presented here also confirmed that the CPS gene cluster do not unambiguously correlate with any particular ST, confirming the notion that CPS gene clusters can be exchanged between different strains of Enterobacteriaceae species [30][31][32].

Bacterial Strains
Two KPC-Kp strains of ST258, KKBO-4 and KK207-2, isolated in 2010 from two different Italian hospitals and epidemiologically unrelated with each other, were used for highthroughput genome sequencing (HTGS) analysis and characterization of their cps gene clusters.
Forty-six additional KPC-Kp strains of ST258 or ST512 plus nine carbapenem-resistant but KPC-negative K. pneumoniae strains of different STs were investigated by the modified multiplex PCR for CPS genotyping developed in this work. These strains were selected as representative of the recent Italian epidemic of carbapenem-resistant K. pneumoniae from a collection of clinical isolates obtained during the first nationwide survey on carbapenem-resistant Enterobacteriaceae carried out in Italy in 2011 [12].

High-Throughput Genome Sequencing and Analysis of Sequence Data
HTGS was performed using a HiSeq 2000 Illumina platform and a paired-ends protocol with an average insert size of 300 bp.
Reads were assembled using ABySS [33]. GGDC software was used to assess the genomic diversity of the investigated isolates [34]. HTGS for strain KKBO-4 has been described previously [35]. The web interface of BLAST available at the NCBI website was used to compare the CPS gene clusters of the two strains with homologues in the nr or wgs databases [36]. CPS gene clusters sequences were aligned with ClustalX [37]. Structural comparisons of KKBO-4, KK207-2 and other published cps gene clusters were performed with EasyFig. [38].
The nucleotide sequences of the cps gene clusters of KKBO-4 and KK207-2 were deposited in the DDBJ/EMBL/GenBank databases under accession numbers HE866751 and HE866752, respectively.

Multiplex PCR for CPS Typing
CPS gene clusters were genotyped using a multiplex PCR approach as previously described [6], modified by including two additional primer pairs designed to amplify specific targets in the cps gene clusters described in this paper: wziBO-4F (59-CGGTTTCCTGATGCAGCGG-39) and wziBO-4R (59-AT-CATGTGCTTCCAGGTACC-39), targeting the wzi gene of the cps BO-4 gene cluster, and hgt207-2F (59-GCAGCTGATTCCA-GAAATATTG-39) and hgt207-2R (59-CATATGCTCTAATAC-CAAAGCC-39), targeting a hypothetical glycosyltransferase gene of the cps 207-2 gene cluster (orf9 in Fig. 1). These two additional primer pairs yielded amplicons of 478 and 352 bp, respectively, being suitable for the inclusion in the multiplex PCR because of the unique band sizes. Primers K.pneumoniae Pf and K.pneumoniae Pr1, designed for the identification at the species level of K. pneumoniae and included in the original multiplex PCR protocol, were not included in the reaction mix.

Addendum in Proof
After the revised version of this manuscript had been submitted, two articles have been published reporting the occurrence of two distinct cps gene clusters in Klebsiella pneumoniae isolates belonging to the ST258 clonal lineage [39], and the development of a PCRbased assay for their detection [40]. The two gene clusters, named cps-1 and cps-2, correspond to cps 207-2 and cps BO-4 described here, respectively, while the PCR assay targets the different wzy genes of the two clusters. At the same time, an additional article has been published reporting that K. pneumoniae isolates of ST258 are characterized by cps gene clusters carrying a novel wzi allele (wzi-154) [41], that is identical to the wzi allelic variant of cps BO-4 .