Multidrug resistant (MDR) Klebsiella pneumoniae clinical isolates: a zone of high heterogeneity (HHZ) as a tool for epidemiological studies

Abstract

Comparison of genome-wide, high-resolution restriction maps of Klebsiella pneumoniae clinical isolates, including an NDM-1 producer, and in silico-generated restriction maps of sequenced genomes revealed a highly heterogeneous region we designated the “high heterogeneity zone” (HHZ). The HHZ consists of several regions including a “hot spot” prone to insertions and other rearrangements. The HHZ is a characteristic genomic area that can be used in the identification and tracking of outbreak-causing strains.

The ability of K. pneumoniae to easily spread together with the rise in the occurrence of multidrug resistant (MDR) strains make this bacterium an important public health problem [1]. Molecular genotyping methods using amplified fragment length polymorphism, repetitive extragenic palindromic PCR, or pulsed field gel electrophoresis are utilized to decipher the molecular epidemiology of K. pneumoniae and facilitate the identification of the source as well as the prevalence of strains isolated from outbreaks [2–4]. Here, we carry out a genome-wide, high-resolution restriction map comparison of clinically important K. pneumoniae strains.

To better understand the epidemiology of MDR outbreak strains we hypothesized that precise, high-resolution (HR) fingerprinting of strains would reveal novel insights not previously evident. HR fingerprinting of strains can offer unique insights into strain relatedness, genome assembly and comparative genomics [5, 6].

The characteristics of the K. pneumoniae strains JHCK1, VA 360, and 1.53 are summarized in Table 1. These strains represent well-studied isolates and embody problematic resistance phenotypes that are emerging among K. pneumoniae [1, 7, 8]. In particular, K. pneumoniae 1.53 carries the bla_NDM-1 gene. The genomes of K. pneumoniae isolates deposited in GenBank were used for the comparative studies (MGH 78578 [http://genome.wustl.edu/genomes/view/klebsiella_pneumoniae/], NTUH-K2044 [9], and KCTC 2242 [10], GenBank accession numbers CP000647, AP006725, and CP002910). Strain MGH 78578 was isolated in 1994 from the sputum of a patient with pneumonia in the intensive care unit. This strain harbors five plasmids and it is resistant to several antibiotics, including ampicillin, ticarcillin, trimethoprim-sulfamethoxazole, and gentamicin. MGH 78578 is susceptible to amikacin, ciprofloxacin, and imipenem. Strain NTUH-K2044 harbors a large plasmid and was isolated from the blood of a patient with primary liver abscess and meningitis [9]. HR restriction maps (optical maps) of strains with no available genome sequence were generated at OpGen Technologies, Inc. Analyses were done comparing the AflII restriction maps using the MapSolver software (version 3.2.0; OpGen Technologies, Inc.). Blastn analyses were done at the National Center for Biotechnology Information site [11]. Comparison of syntenies of genomes was performed using the Artemis Comparison Tool (ACT) (www.sanger.ac.uk/software/artemis/ACT) [12]. Susceptibility to different antibiotics was determined by the disk diffusion method following CLSI guidelines [13].

Table 1.

Characteristics of K. pneumoniae strains

Strain	Year isolated	Country	β-lactamase	Antibiotic					Ref
				AMP CFZ CEF CHL GEN	CIP CAZ CRO CTX CXM FEP FOX IPM LVX MEM NAL SXT	TET	AMK	AMC
JHCK1	1986	Argentina	TEM-1	R	S	R	R	I	[8]
VA 360	2006	USA	TEM-1, KPC-2, SHV-11, SHV-12	R	R	S	I	R	[4]
1.53	2009	India	NDM-1, CTX-M-15	R	R	R	R	R	[22]

Amikacin, AMK; amoxicillin-clavulanic acid, AMC; ampicillin, AMP; cefazolin, CFZ; cefepime, FEP; cefoxitin, FOX; cephalothin, CEF; chloramphenicol, CHL; cefotaxime, CTX; ceftazidime, CAZ; ceftriaxone, CRO; cefuroxime, CXM; ciprofloxacin, CIP; gentamicin, GEN; imipenem, IPM; levofloxacin, LVX; meropenem, MEM; nalidixic acid, NAL; tetracycline, TET; trimethoprim-sulfamethoxazole, SXT. R, resistant; I, intermediate; S, susceptible. Ref, reference.

Whole genome comparisons of the HR restriction maps of K. pneumoniae strains revealed that the genomes are mostly homogeneous as determined by the extensive blocks of regions in blue, the color that indicates matching DNA segments (Fig. 1A). However, we detected a distinctive region of heterogeneity colored in white, which indicates not matching sequences. We designated this region “high heterogeneity zone” (HHZ) (Figs. 1A and 1B). This region is readily evident within all genomes indicating that it is specific to each of the compared genomes. Using the annotations of the genomes of strain MGH 78578 the HHZ region was operationally defined as that one limited by the hypothetical gene yedA (starting at nucleotide 2648600) and metG (ending at nucleotide 2807698), the gene coding for methionine-tRNA ligase (Fig. 1C).

FIG 1.

Genomic comparison. A. The K. pneumoniae JHCK1, VA 360, and 1.53 optical maps and the restriction maps of K. pneumoniae strains NTUH-K2044, KCTC 2242, and MGH 78578 AflII obtained in silico were compared using the MapSolver 3.2.0 software. The blue and white regions represent matching and non-matching DNA fragments, respectively. The genomic comparison was done between strains JHCK1, 1.53, and MGH 78578 vs. strains VA360, KCTC 2242, and NTUH-K2044, a combination that maximized the detection of the HHZ region among all strains using the default settings of MapSolver v.3.2.0. Vertical lines represent AflII restriction sites. B. Zoom-in the HHZ region. C. Synteny among K. pneumoniae MGH 78578, KCTC 2242, and NTUH-K2044 strains at the HHZ regions. The genetic maps and comparison have been generated using the ACT [12] to visualize the Blastn output. Colinear regions are represented in red, colinear regions that have been inverted are represented in blue, and deletions/insertions are represented in white. The HHZs have been colored yellow in MGH 78578, KCTC 2242 and NTUH-K2044 strains, the coordinate numbers are 2648600..2807698, 3122007.. 3313398 and 3356452..3613107 respectively.

The availability of complete sequenced genomes permitted us to further analyze the nature of the HHZ in those strains. A comparison of the HHZ regions in strains MGH 78578, NTUH-K2044 as well as the recently sequenced strain KCTC 2242 revealed the presence of some homologous DNA segments, as well as inversions and insertions (Fig. 1C). In addition, four identical copies of the tRNA-Asn gene were identified in this region and numbered accordingly. tRNA genes are known to act as spots for insertion and other recombinational events [14]. On the basis of this comparative analysis we subdivided the HHZ in 5 highly relevant regions (Fig. 1C).

Region 1 is the location of insertion of two fragments upstream and downstream of a LysR-family transcriptional regulator gene present in all three strains and that it is located near the 3′ end of tRNA-Asn1 (Fig. S1). The insertion upstream of this gene in strain NTUH-K2044 codes for a possible translation initiation inhibitor as annotated in GenBank. Whether this insertion is just an exceptional event or it occurs with many other strains will be known when more genomes are available.

Region 2 consists of an inverted segment found in strain NTUH-K2044 that includes seven genes (Fig. S2). One of them, pmrD, which codes for a small basic protein called PmrD that may be involved in resistance to polymyxin B in some gram negatives and connects the two component systems PhoP–PhoQ and PmrA–PmrB [15, 16]. This inversion is likely the result of homologous recombination between 2 identical tRNAs (tRNA-Asn2 and -3) at its flanking regions (Fig. S2).

Region 3 is a “hot spot” that also exists in other Enterobacteriaceae [17]. In strain NTUH-K2044 the asn3 tRNA gene is the point of insertion of ICEKp1, a ~76 kbp integrative and conjugative element that includes a high-pathogenicity island similar to that found in Yersinia pestis and Y. pseudotuberculosis containing the siderophore yersiniabactin suggesting active exchange among common virulence mechanisms [18, 19] (Fig. S3A and S3B). In strain KCTC 2242 the insertion occurred at the same location, i.e. immediately downstream of tRNA-Asn4 (see Figs. S3A and S3B, and Fig. S5A), but the inserted sequence is not homologous to ICEKp1 and showed few hits in the Blastn analysis (genes KPN_2242_15200 to KPN2242_15275) (Fig. S4A and S4B). K. pneumoniae plasmid pKP91 [20] shows homology at both ends of the fragment (see Fig. S4A, S4B, S5A and S5B, genes in blue) and towards the 3′ end there is a stretch of homology with K. oxytoca (Fig. S4A and S4B). At the 5′ and 3′ ends of the inserted fragment there are numerous hits to enterobacterial integrase and transposase genes, which may have played a role in the process of insertion of the fragment found in strain KCTC-2242 and other fragments in their respective genomes (Fig. S4A, red and yellow arrows, and S5A, genes in red and yellow). Interestingly, both KCTC 2242 and NTUH-K2044 encode the same integrase gene at the 5′ end of the inserted fragment, which might suggest that both insertions occurred through similar mechanisms (Fig S5A, yellow arrows).

Region 4 is composed of three subregions that are unique to MGH 58758 (see white regions in Fig. S6). Subregions “a” and “b” share homology in strains NTUH-K2044 and KCTC 2242 but region “c” is different in all three strains at the nucleotide level (see Fig. S6). Strain KCTC 2242 houses two transposases in this subregion (Fig. S6, yellow arrows). In strain NTUH-K2044 this region includes genes responsible for capsular serotype K1 including magA (mucoviscosity-associated gene A), which directs the secretion of polysaccharide capsule and confers resistance to serum killing and phagocytosis [21]. Table S1 shows a list of unique genes in each subregion.

Region 5 consists of another insertion location at the gene coding for the universal stress protein A9 (Fig. S7). A segment from KP1_3770 to KP1_3775 has been inserted in strain NTUH-K2044 to completely replace the gene coding for the universal stress protein A9 (Fig. S7). Most likely, this insertion and replacement were mediated by the IS element found at the 5′ end of the inserted fragment. Further upstream of this location there are two identical genes coding for a D-arabinitol transporter in opposite orientations in each of the strains compared (Fig. S7, blue).

In conclusion, comparison of HR restriction maps of newly isolated strains for which the genome sequence is not available and in silico–generated maps of known genomes may be a very useful approach for epidemiologic studies and for an initial characterization of bacterial strains. In the particular case of K. pneumoniae clinical isolates, the maps show a high degree of similarity, which contrasts with the high variability exhibited by HHZ. This region is easily discernible when doing comparisons of restriction maps and could serve as signature to assist in the identification and tracking of outbreak-causing strains. A limitation of comparison of HHZ regions by HR restriction maps is that small differences may be missed. Future studies will permit us to assess with more precision the contribution of HHZ comparisons to the epidemiological characterization of K. pneumoniae isolates.

Supplementary Material

Supp Fig S1. Figure S1.

Region 1 synteny among Klebsiella pneumoniae MGH 78578, KCTC 2242, and NTUH-K2044 strains. The genetic maps and comparison have been generated using the ACT [12] to visualize the Blastn output. Homologous regions are indicated in red. Copies of tRNA genes (tRNA-Ser and tRNA-Asn1) are colored in green.

Supp Fig S2. Figure S2.

Region 2 synteny among K. pneumoniae MGH 78578, KCTC 2242, and NTUH-K2044 strains. The genetic maps and comparison have been generated using the ACT [12] to visualize the Blastn output. Homologous regions in the same or opposite direction are indicated in red or blue color respectively. Two identical copies of tRNA genes (tRNA-Asn 2 & 3) at its flanking region are colored in green and pointed by green arrows.

Supp Fig S3. Figure S3.

Region 3 synteny among K. pneumoniae MGH 78578, KCTC 2242, and NTUH-K2044 strains. The genetic maps and comparison have been generated using the ACT [12] to visualize the Blastn output. Although more than region 3 is shown, the graphic is intended to focus on the point of insertion and inserted sequences/genes found in strain KCTC 2242 and NTUH-K2044 strain, which are highlighted in green color bars. Homologous regions in the same or opposite direction are indicated in red or blue color respectively. Panel A and B represent the 5′ end and 3′ end of flanking sequence alignment of inserted segments.

Supp Fig S4. Figure S4.

A. Graphic presentation of Blastn output of the inserted segment in K. pneumoniae KCTC 2242 (from 3160195 bp to 3179506 bp) against Genbank NT database. B. Top 4 Blastn hits of the unique region within the inserted fragment. The 1^st hit is matching to itself. The 2^nd hit is K. pneumoniae 342 plasmid pKP91 with 44% query coverage.

Supp Fig S5. Figure S5.

A. Zoom-in view of region 3 synteny among K. pneumoniae MGH 78578, KCTC 2242, and NTUH-K2044 strains. Homologous sequences are indicated in red color. Identical copies of tRNA-Asn, integrase, and transposase genes at its boundary region are colored in green, yellow and red, and also pointed by the same color-coded arrows. Unique genes to strain KCTC 2242 are colored white, genes homologous to those found in pKP91 are colored in blue. B. Genbank annotation of KCTC 2242 unique insertion region. All coding genes are using the same color – coded.

Supp Fig S6. Figure S6.

Region 4 synteny among K. pneumoniae MGH 78578, KCTC 2242, and NTUH-K2044 strains. The genetic maps and comparison have been generated using the ACT [12] to visualize the Blastn output. The letters “a”, “b”, and “c” indicate the subregions mentioned in the text and its coordinate numbers are listed in table S1. Homologous regions are indicated in red color. Transposase genes in this sub-region are colored and pointed in yellow and arrows.

Supp Fig S7. Figure S7.

Region 5 synteny among K. pneumoniae MGH 78578, KCTC 2242, and NTUH-K2044 strains. A. Homologous regions in the same and opposite direction are indicated in red and blue color respectively. Transposase genes are colored in yellow and also pointed by the same color-coded arrows. Two duplicated D-arabinitol transporter genes in each strain are in opposite direction shown as blue crosses. The universal stress protein A gene that was replaced by the insertion event in NTUH-K2044 is colored in purple. B. Genbank annotation of NTUH-K2044 unique insertion region.

Supp Table S1. Table S1.

Unique genes in subregions of region 4.