Accepted Manuscript

Molecular epidemiology of Klebsiella pneumoniae K1 and K2

isolates in Japan

Sohei Harada, Yoshikazu Ishii, Tomoo Saga, Kotaro Aoki,

Kazuhiro Tateda

PII: S0732-8893(18)30100-7
DOI: doi:10.1016/j.diagmicrobio.2018.03.010
Reference: DMB 14562
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Revised date: 3 March 2018
Accepted date: 15 March 2018

Please cite this article as: Sohei Harada, Yoshikazu Ishii, Tomoo Saga, Kotaro Aoki,
Kazuhiro Tateda , Molecular epidemiology of Klebsiella pneumoniae K1 and K2 isolates
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authors. Please check if appropriate. Dmb(2018), doi:10.1016/j.diagmicrobio.2018.03.010

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 ACCEPTED MANUSCRIPT

Molecular epidemiology of Klebsiella pneumoniae K1 and K2 isolates in Japan

Sohei Harada,a, b* Yoshikazu Ishii,a Tomoo Saga,c Kotaro Aoki,a and Kazuhiro Tateda a

a
Department of Microbiology and Infectious Diseases, Toho University School of Medicine,

5-21-16 Omori-nishi, Ota-ku 143-8540, Tokyo, Japan

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b
Department of Infectious Diseases, Cancer Institute Hospital, Japanese Foundation for

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Cancer Research, 3-8-31 Ariake, Koto-ku 135-8550, Tokyo, Japan
c

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Central Laboratory Division, Akita University Hospital 1-1-1 Hondo, Akita 010-8543,

Japan.
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*Corresponding author: Sohei Harada, M.D., Ph.D.
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Department of Microbiology and Infectious Diseases, Toho University School of Medicine,

5-21-16 Omori-nishi, Ota-ku 143-8540, Tokyo, Japan

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Phone: +81-3-3762-4151, ext. 2396.

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Fax: +81-3-5493-5415.
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E-mail: haradas-tky@umin.ac.jp
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Running title: Molecular epidemiology of K. pneumoniae

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Abstract

Although severe infections caused by hypervirulent Klebsiella pneumoniae isolates,

such as K1 isolates belonging to sequence type (ST) 23, have been a significant problem in

Asian countries, epidemiology of these isolates in Japan remains unclear. We performed a

nationwide molecular epidemiological study of K. pneumoniae K1 and K2 isolates in Japan.

Of the 259 K. pneumoniae isolates collected, 14 and 16 isolates were identified as capsular

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genotypes K1 and K2, respectively. All K1 isolates were ST23 or its closely related clones

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and showed high genetic similarity by pulsed-field gel electrophoresis (PFGE) and the

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DiversiLab system (DL). K2 isolates, belonging to ST14, ST25, ST65, ST86, and ST110,

were more genetically diverse than K1 isolates. Isolates belonging to a specific ST showed
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identical virulence gene profiles with a few exceptions. PFGE and DL results using K1 and

K2 isolates were generally in agreement.

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Keywords: Klebsiella pneumoniae, capsular genotype, MLST, virulence genes

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1. Introduction

Klebsiella pneumoniae is responsible for a variety of nosocomial infections, such as

urinary tract infections, pneumonia, and bloodstream infections, mainly in high-risk patients

with underlying conditions or medical devices. On the other hand, K. pneumoniae

occasionally causes severe community-acquired infections in immunocompetent or low-risk

individuals (Podschun et al., 1998). Previous studies identified hypervirulent isolates, which

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have a higher potential to cause these infections (Paczosa et al., 2016). Although many of the

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virulence factors of K. pneumoniae, such as lipopolysaccharide, fimbriae, and siderophores,

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have been identified, the capsule is the most thoroughly investigated virulence factor. The

capsule of K. pneumoniae is classified into 78 K antigen types by the serological antigenic

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property of the capsular polysaccharides in each strain (Paczosa et al., 2016).

Community-acquired liver abscesses occasionally accompanied by metastatic

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meningitis or endophthalmitis in healthy populations had become recognized as a newly

appearing severe K. pneumoniae infection in the 1990s in Taiwan. Molecular analysis of the
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causative organisms demonstrated that magA, which is located on the operon for capsular
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serotype K1, is the critical virulence factor of these hypervirulent strains (Chuang et al, 2006;
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Fang et al., 2004; Fang et al., 2007). The fact that the proportion of K1 isolates among
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clinical isolates of K. pneumoniae was extremely high in Taiwan compared with those

observed in the United States and European countries supported the notion that this group of
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isolates is associated with the severe community-acquired infections, which were observed at

an exceptionally high frequency in Taiwan (Cryz et al., 1986; Fung et al., 2000; Turton et al.,

in press). Recently, increase in the frequency of K. pneumoniae as the causative organisms of

community-acquired liver abscesses, with the involvement of K1 isolates, has been reported

from South Korea, China, and other Asian countries (Chung et al., 2007; Siu et al., 2011;

Zhang et al., 2016). Severe infections caused by K1 isolates have also been reported
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sporadically from countries outside of Asia, albeit at a reduced frequency (Decré et al., 2011;

Sachdev et al., 2013; Turton et al., 2007). Interestingly, K1 isolates causing

community-acquired liver abscesses showed high genetic similarity and most were

determined to be sequence type (ST) 23 by multilocus sequence typing (MLST) (Brisse et al.,

2009; Chung et al., 2008). In a study analyzing environmental, animal, and human K.

pneumoniae isolates collected globally, K1 isolates were divided into two clones, clonal

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complex (CC) 23 and CC82, with CC23 carrying the virulence genes allS and mrkD, whereas

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the CC82 organisms lacked these virulence genes (Brisse et al., 2009).

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The K. pneumoniae K2 serotype was classically considered to be the most common

among organisms isolated from patients with urinary tract infections, pneumonia, and
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bacteremia (Podschun et al., 1998). Several studies suggested that K2 isolates were the major

causative organisms of primary K. pneumoniae liver abscesses in Asia and had phagocytic
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resistance and high virulence, similar to K1 isolates (Yeh et al., 2007). As was observed in K1

isolates, K2 isolates of each ST had characteristic patterns of virulence genes and several STs,
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such as ST25, ST65, ST86, ST375, and ST380, carried many virulence genes and have been
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considered to be hypervirulent clones (Bialek-Davenet et al., 2014; Brisse et al., 2009;

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Cubero et al., 2016; Decré et al., 2011). Experiments with a mouse peritoneal infection model
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showed that K2 isolates of a hypervirulent clone had higher lethality in infected mice

compared to other K2 isolates (Brisse et al., 2009). Recently, lethal infections caused by these
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virulent clones of K2 isolates have been reported (Decré et al., 2011; Zhang et al., 2015).

The prevalence and molecular epidemiology of K1 and K2 isolates in K. pneumoniae

clinical cases in Japan remain unclear. A surveillance study performed in the 1980s showed

13 (5.1%) and 9 (3.5%) K1 and K2 isolates, respectively, out of 257 K. pneumoniae clinical

isolates collected (Mori et al., 1989). This was a regional surveillance study performed more

than 30 years ago. Therefore, whether this figure matches the current situation in all of Japan
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is unknown. Although another regional study showed 9 (7.5%) and 13 (10.8%) K1 and K2

isolates, respectively, out of 120 K. pneumoniae clinical isolates collected mainly in 2010,

only isolates recovered from blood of patients with pneumonia were collected in the study

(Ito et al., 2015). Japan is located near Taiwan and South Korea, where K. pneumoniae

K1-ST23 isolates have been endemic and sporadic cases of community-acquired liver

abscesses caused by the clone have been reported (Harada et al., 2011). Thus, the

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epidemiology of virulent clones of K. pneumoniae including K1-ST23 should be clarified.

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The DiversiLab system (DL) (bioMérieux, Marcy l'Etoile , France) is an automated,

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repetitive sequence-based PCR (rep-PCR) system and has increasingly gained acceptance as

a molecular tool for strain typing because of its convenience and standardized methodology
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compared to the manual performance of rep-PCR (Healy et al., 2005). In addition, DL

requires a shorter time for the analysis compared to pulsed-field gel electrophoresis (PFGE),
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which has been the gold standard for strain typing in outbreak investigations. The

discriminatory ability of DL has been reported to be different for each bacterial species.
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While the results of Klebsiella spp. typing correlated well with those obtained by PFGE in
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one study, another study showed that the discriminatory ability of DL is higher than that of
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PFGE for K. pneumoniae and DL may miss genetic relatedness in outbreak investigations
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(Brolund et al., 2010; Fluit et al., 2010).

In this study, we collected clinical isolates of K. pneumoniae from various areas of

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Japan and clarified the prevalence of K1 and K2 isolates. In addition, K1 and K2 isolates

were typed with MLST, PFGE, and DL, and the presence of virulence genes was assessed.
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2. Materials and methods

2.1. Bacterial strains

Clinical isolates of Klebsiella spp. from various body sites were collected from 51

health-care facilities located in various areas in Japan from July through September 2008 and

sent to the Department of Microbiology and Infectious Diseases, Toho University School of

Medicine (Ishii et al., 2011). Bacterial species were identified by various automated methods

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in each health-care facility and the first ten isolates of Klebsiella spp. were cryopreserved at

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80°C at the Microbank (Iwaki. Co., Tokyo, Japan). Multiple isolates were not collected from

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the same patient.
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2.2. Confirmation of bacterial species

Confirmation of bacterial species for collected Klebsiella spp. isolates were performed
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with PCR amplification of 16S–23S rDNA internal transcribed spacer (ITS) of K.

pneumoniae, Voges–Proskauer reactions, and malonate reactions. Primer pairs of Pf/Pr1 and
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Pf/Pr2 were used for the amplification of 16S-23S ITS of K. pneumoniae (Liu et al., 2008).
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Because Klebsiella ozaenae was demonstrated to be positive in the PCR, Voges–Proskauer

and malonate reactions, both of which are positive for most of the K. pneumoniae isolates and
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negative for most of K. ozaenae isolates, were added for species confirmation (Turton et al,

2010). Isolates were identified as K. pneumoniae when PCR amplification of 16S-23S ITS,
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and Voges–Proskauer and malonate reactions were all positive.

2.3. Capsular genotyping PCR

K1 and K2 isolates were identified with capsular genotyping PCR (Fang et al., 2007).

Positive PCR results were confirmed with Klebsiella antisera Seiken (Denka Seiken, Tokyo,

Japan). K1 and K2 isolates of K. pneumoniae were used for subsequent analysis.

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2.4. MLST

MLST was performed according to the protocol described in INSTITUT PASTEUR

MLST on a website (http://bigsdb.pasteur.fr/klebsiella/primers_used.html).

2.5. PCR for virulence genes

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Virulence gene profiles were determined for fimH, coding for a type 1 fimbriae subunit,

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kfu coding for an ABC iron transport system, uge, coding for a UDP-galacturonate

4-epimerase, wabG, coding for a GalA transferase, allS, coding for the activator of the

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allantoin regulon, mrkD, coding for the type 3 fimbriae adhesion gene, ureA, coding for a
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subunit of urease, rmpA, coding for the regulator of the mucoid phenotype, and genes for

aerobactin, a citrate-hydroxamate siderophore, by PCR with primers used in previous studies

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(Brisse et al., 2009; Zhang et al., 2016).

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2.6. PFGE
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PFGE was performed with CHEF Mapper (Bio-Rad, Hercules, CA, USA) using

genomic DNA from K. pneumoniae K1 and K2 isolates digested with XbaI (Bio-Rad),
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according to the standard protocol for K. pneumoniae provided by the manufacturer. The

sizes of the fragments were determined by comparison with Lambda Ladder PFG Marker
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(New England Biolabs, Hertfordshire, United Kingdom). Patterns of restriction fragments

were analyzed with Fingerprinting II software (Bio-Rad), using the Dice coefficient and the

unweighted pair group method with averages. Position tolerance and optimization were both

set to 1%. Bands within the size range 50-700 kb were included in the analysis.

2.7. Typing by DL
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DNA extraction was performed with the UltraClean Microbial DNA Isolation Kit (Mo

Bio Laboratories Inc., Solana Beach, CA, USA). The purity and concentrations of the DNA

preparations were measured using a Biospec-Nano spectrophotometer (Shimadzu, Kyoto,

Japan). The DiversiLab Klebsiella kit was used, following the manufacturer's instructions.

The electrophoresis data were analyzed by internet-based DiversiLab (version. 3.4) software

using the Pearson’s correlation coefficient.

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2.8. Comparison of PFGE and DL results

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PFGE and DL results were compared using the Comparing Partitions website

(http://www.comparingpartitions.info/).
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3. Results

3.1. Prevalence of isolates with K1 and K2 genotypes

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Although 510 Klebsiella spp. isolates were sent to the Department of Microbiology and

Infectious Diseases, Toho University School of Medicine, 48 isolates could not be recovered
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from the Microbank vials. Of the 462 isolates recovered, 259 isolates were identified as K.
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pneumoniae with positive results for PCR amplification of 16S-23S ITS, the
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Voges–Proskauer reactions, and the malonate reactions. Fourteen (5.4%) and sixteen (6.2%)
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isolates were determined to be genotypes K1 and K2, respectively, using capsular genotyping

PCR. The capsular genotyping PCR results agreed with the capsular serotyping results using
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the Klebsiella antisera Seiken for all K1 and K2 isolates. K1 and K2 isolates were isolated

from various body sites and patients were from a variety of geographical areas in Japan

(Table 1, 2, 3).

3.2. ST and virulence gene profiles

Among 14 K1 isolates, 12 isolates were classified as ST23 by MLST (Table 2).

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TUM6698 and TUM7040 presented the novel STs, ST2732 and ST2733, respectively.

ST2732 is a single locus variant of ST23, with a single nucleotide difference in tonB. ST2733

is a double locus variant of ST23, with a single nucleotide difference in rpoB and pgi. All K1

isolates, except one, had identical virulence gene profiles, which were positive for all

virulence genes. One of the K1-ST23 isolates, TUM7031, lacked rmpA. K2 isolates were

separated into a variety of STs, including ST14 (n=4), ST25 (n=4), ST65 (n=3), ST86 (n=4),

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and ST110 (n=1) (Table 3). ST65 is a single locus variant of ST25, with a single nucleotide

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difference in mdh. Isolates belonging to an identical ST carried the same virulence genes

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except for TUM6871, which carried allS, rmpA, and genes for aerobactin and these were not

identified in other K2-ST25 isolates.

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3.3. Clonality of K1 and K2 isolates by PFGE
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All K1 isolates showed a similarity of 65% by PFGE (Figure 1). Nevertheless, no

pairs of K1 isolates, even from the same hospitals (TUM7020 and TUM7021, and TUM7031
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and TUM7040), had a similarity of 90%. K2 isolates were more diverse than K1 isolates.
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While isolates belonging to ST86 and those belonging to ST65 showed a relatively high

similarity at 70% despite the collection from various areas of Japan, other isolates belonging
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to the same ST had lower similarity.

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3.4. Results of DL in comparison to those with PFGE

The results of DL generally agreed with those of PFGE (Figure 2). All K1 isolates,

except three, showed a similarity of 95%. Although the three K1 isolates had a similarity of

~90% to the main cluster of K1 isolates, five K2 isolates belonging to ST86 or ST14 showed

higher similarity to the main cluster of K1 isolates. Although similarity of K2 isolates as a

whole was lower compared to K1 isolates, isolate of K2-ST86 and those of K2-ST65 showed
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a similarity of 95%. Simpson’s index of diversity (95% CI) results were 0.830 (0.731-0.928),

0.821 (0.704-0.938), and 0.963 (0.937-0.989) for DL using a cutoff of >95% similarity, for

PFGE using a cutoff of >70% similarity, and PFGE using a cutoff of >80% similarity,

respectively. Typing by PFGE using a cutoff of >70% similarity showed reasonable

agreement with results by DL using a cutoff of >95% similarity (Adjusted Rand's coefficient:

0.649, Adjusted Wallace's coefficient 1: 0.629, Adjusted Wallace's coefficient 2: 0.671).

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4. Discussion

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In this study, we analyzed the molecular characteristics of K. pneumoniae clinical

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isolates collected from patients at health-care facilities located in various areas in Japan. Of

the 259 K. pneumoniae clinical isolates, 14 and 16 isolates were identified as capsular
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genotypes K1 and K2, respectively. All K1 isolates belonged to ST23 or its closely related

clones and showed high genetic similarity by PFGE and DL. K2 isolates were more
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genetically diverse and were divided into several STs.

Among the K. pneumoniae clinical isolates there were 5.4% K1 isolates in our study
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and this corresponds to those reported in previous regional studies in Japan (Ito et al., 2015;
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Mori et al., 1989). This suggests that the prevalence of K1 isolates is higher in Japan than in

the United States and European countries, but lower than in Taiwan (Cryz et al., 1986; Fung
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et al., 2000; Turton et al., 2017). While clinical isolates of K. pneumoniae K1 were divided

into CC23 and CC82 in a previous study using globally collected isolates, all K1 isolates in
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our study belonged to ST23 or its closely related clones (Brisse et al., 2009). Two studies

performed in South Korea also demonstrated that almost all K. pneumoniae K1 isolates

recovered from stool cultures of healthy persons and those from abscesses or blood cultures

from patients with community-acquired liver abscesses belonged to ST23 or its closely

related clones (Chung et al., 2012; Chung et al., 2008). The similarity of K1 isolates analyzed

by PFGE was 65% in our study and this also corresponds well with those of K1 isolates
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causing community-acquired liver abscess in South Korea (Chung et al., 2008). These

findings suggest the possibility that K. pneumoniae K1 clinical isolates in East Asian

countries have high genetic similarity and most of them belong to CC23. Thirteen of fourteen

K1 isolates in our study had virulence gene profiles identical to those of K1-ST23 isolates

causing community-acquired liver abscesses in Taiwan and Japan (Brisse et al., 2009; Harada

et al., 2011). While the remaining one isolates lacked rmpA, the clinical significance of the

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difference remains unknown.

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The K. pneumoniae K2 isolates in our study showed higher genetic diversity than the

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K1 isolates. Isolates belonging to ST86 and ST110 were identified together with ST14, ST25,

and ST65, which were the major K2 clones among the K. pneumoniae isolates collected
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globally (Brisse et al., 2009). In recent years, isolates of ST25, ST65, and ST86 have been

recognized as hypervirulent strains together with K1-ST23 (Bialek-Davenet et al., 2014;

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Cubero et al., 2016; Decré et al., 2011). Although the isolates of ST65 showed high similarity

by PFGE and DL and the same applied to the isolates of ST86, the generalizability of this
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finding is unclear because only a limited number of isolates were investigated. K2 isolates
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belonging to a specific ST showed the presence of identical virulence genes, with only one
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isolate (TUM6871) as the exception, and this finding agrees to that of a previous study
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(Brisse et al., 2009). While it is possible that TUM6871 may obtain the virulence genes by

conjugative transfer of a virulence plasmid, which occurred in the carbapenem-resistant

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hypervirulent K. pneumoniae ST11 isolates in China, plasmid analysis was beyond the scope

of our study (Gu et al., 2018).

There was general agreement between PFGE and DL results in our study. DL is an

attractive method for the analysis of genetic similarity of bacterial strains because it is less

time consuming and labor intensive compared to PFGE, provided that it yields results similar

to PFGE (Healy et al., 2005). Nevertheless, some discordance between the results obtained
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with PFGE and DL was observed. For example, several K2 strains showed higher similarity

to the major groups of isolates of K1 than other K1 isolates (TUM6684, TUM7021, and

TUM7073) when using DL and this was not observed in the PFGE results. It is natural that

some differences were observed between the results of PFGE and DL because these two

methods employ different methodology to determine the similarity of bacterial isolates. The

results of DL should be interpreted with clinical information, phenotypic data of the isolates,

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and the results of other typing methods if available. Usefulness of DL should continue to be

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validated based on a comparison to PFGE and SNP-based phylogenetic analysis using

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whole-genome sequencing data, which has emerged as a novel tool in recent years (Quainoo

et al., 2017).
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Our study has several limitations. First, we did not obtain clinical information for the

patients from whom K. pneumoniae were isolated. Therefore, the clinical relevance of the
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isolates, including the hypervirulent strains, remains unclear. Second, the number of K.

pneumoniae isolates collected from each geographical area was small. Thus, it is difficult to
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determine the difference in the prevalence of K. pneumoniae hypervirulent strains in different

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areas in Japan. Third, the isolates analyzed in this study were collected about 10 yeas ago.
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Epidemiology of the hypervirulent K. pneumoniae strains might have changed during the
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period. Nevertheless, this is the first nationwide molecular epidemiological study of

hypervirulent K. pneumoniae clinical isolates recovered from various types of specimens in

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Japan and could be utilized as baseline data.

This is the first nationwide molecular epidemiological study of K. pneumoniae in Japan

and it demonstrated that hypervirulent isolates, such as K1-ST23, K2-ST25, K2-ST65, and

K2-ST86, were not rare.

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Conflicts of interest

All authors report no conflicts of interest relevant to this article.

Acknowledgements

This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of

Education, Culture, Sport and Technology of Japan to SH (No. 23971143). The collection of

the isolates was supported by a grant from Bristol-Myers Squibb Inc.

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Figure 1.

Results of PFGE for K. pneumoniae K1 and K2 isolates.

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Figure 2.

DL Results for K. pneumoniae K1 and K2 isolates.

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Table 1. Number of K. pneumoniae isolates from each geographic area of Japan

Number of facilities Number of K. pneumoniae Number of Number of

Geographic area
participated isolates collected K1 isolates
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K2 isolates

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Hokkaido
Tohoku
1
6
7
28
0
3
R I 0
1

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Kanto 9 46 3 7
Chubu 9 29 2 1
Kinki
Chugoku
8
5
54
22
N U0
2
1
1
Shikoku
Kyushu
Okinawa
4
8
1
18
46
9 M A 1
2
1
2
3
0

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Table 2. Profiles of K1 isolates

Isolate Facility Geographic Type of Virulence genes

ST
No. No. area sample fim kfu uge wabG allS mrkD ureA rmpA Aerobactin
TUM7020 23
23
2 Tohoku Respiratory +
+
+
+
+
+
+
+
+
+
P T +
+
+
+
+
+
+
+

I
TUM7021 2 Tohoku Respiratory
TUM7073 23 3 Tohoku Respiratory + + + + + + + + +
TUM6892
TUM6911
23
23
8
9
Kanto
Kanto
Blood
Urinary tract
+
+
+
+
+
+
C
+
+
R +
+
+
+
+
+
+
+
+
+
TUM6932
TUM7031
23
23
10
17
Kanto
Chubu
Gastrointestinal
Respiratory
+
+
+
+
+
+
U S +
+
+
+
+
+
+
+
+

+
+
TUM6880
TUM6956
23
23
34
35
Chugoku
Chugoku
Respiratory
Urinary tract
+
+
+

A
+ N +
+
+
+
+
+
+
+
+
+
+
+
+
+
TUM6684
TUM6994
23
23
39
43
Shikoku
Kyushu
Respiratory
Respiratory
+
+ M+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
TUM6971
TUM6698
23
2732
51
44
Okinawa
Kyushu
Blood
Respiratory
E D +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
TUM7040 2733 17 Chubu
T
Gastrointestinal

P
+ + + + + + + + +

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Table 3. Profiles of K2 isolates

Isolate Facility Geographic Type of Virulence genes

ST
No.
TUM6889 14
No.
8
area
Kanto
sample
Urinary tract
fim
+
kfu
+
uge
+
wabG
+
allS

P T mrkD
+
ureA
+
rmpA

Aerobactin

TUM6918
TUM7076
14
14
11
12
Kanto
Kanto
Urinary tract
Abscess/Drain fluid
+
+
+
+
+
+
+
+
R I 

+
+
+
+




TUM6945
TUM6666
14
25
40
13
Shikoku
Kanto
Respiratory
Urinary tract
+
+
+

+
+
S C +
+


+
+
+
+




TUM6871
TUM6763
25
25
14
18
Kanto
Chubu
Gastrointestinal
Abscess/Drain fluid
+
+


N U
+
+
+
+
+

+
+
+
+
+

+

TUM6769
TUM6913
TUM6732
25
65
65
45
15
36
Kyushu
Kanto
Chugoku
Blood
Respiratory
Blood
+

M
+
+
A



+
+
+
+
+
+



+
+
+
+
+
+

+
+

+
+
TUM6803
TUM6682
65
86
41
4
Shikoku
Tohoku
E D
Urinary tract
Urinary tract
+
+


+
+
+
+


+
+
+
+
+
+
+
+
TUM6903
TUM6986
86
86
9
46
Kanto
Kyushu
P T
Abscess/Drain fluid
Respiratory
+
+


+
+
+
+


+
+
+
+
+
+
+
+
TUM6987
TUM6721
86
110
46
26
Kyushu

C
Kinki E Respiratory
Respiratory
+
+


+
+
+
+


+
+
+
+
+

+


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Highlights
• Fourteen K1 isolates and sixteen K2 isolates were found among 259 K. pneumoniae isolates in Japan.
• All K1 isolates were ST23 or its closely related clones.
• Seven of Sixteen K2 isolates were hypervirulent clones, such as ST65 and ST86.
• Pulsed-field gel electrophoresis and the DiversiLab system results were generally in agreement.

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