International Journal of Infectious Diseases 37 (2015) 107–112

Contents lists available at ScienceDirect

International Journal of Infectious Diseases

journal homepage: www.elsevier.com/locate/ijid

Clinical and molecular characteristics of multi-clone

carbapenem-resistant hypervirulent (hypermucoviscous) Klebsiella
pneumoniae isolates in a tertiary hospital in Beijing, China
Bei Yao a, Xiumei Xiao a, Fei Wang b, Lei Zhou a, Xiaowei Zhang a, Jie Zhang a,*
a
Department of Laboratory Medicine, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing, 100191, China
b
Department of Respiratory Medicine, Peking University Third Hospital, Haidian District, Beijing, China

A R T I C L E I N F O S U M M A R Y

Article history: Objectives: To provide the clinical and molecular characteristics of carbapenem-resistant hypervirulent
Received 1 April 2015 (hypermucoviscous) Klebsiella pneumoniae (cr-hvKP) in a tertiary hospital in Beijing, China.
Received in revised form 24 June 2015 Methods: The clinical characteristics of four patients with cr-hvKP isolates and 29 patients with
Accepted 25 June 2015
carbapenem-resistant classic K. pneumoniae (cr-cKP) infections were analyzed retrospectively. The
Corresponding Editor: Eskild Petersen, molecular characteristics of cr-hvKP and cr-cKP isolates were compared.
Aarhus, Denmark.
Results: The KPC-2 gene was detected in all cr-hvKPs except for cr-hvKP6. The cr-hvKPs belonged to
three sequence types (STs; ST25, ST65, and ST11), with three pulsed-field gel electrophoresis patterns (I,
Keywords: II, and III) and two capsular serotypes (K2 and non-typeable). Although cr-hvKP1–7 did not cause
Klebsiella pneumoniae invasive clinical syndromes such as community-acquired liver abscess with or without extrahepatic
Hypervirulent complications, they were all nosocomially acquired; cr-hvKP1–5 were clones disseminated between
Hypermucoviscous
patients A and B. Compared with cr-cKPs, pLVPK-related loci, repA, iroN, and K2 capsular serotype were
Carbapenem-resistant
more prevalent in cr-hvKPs, although no significant difference was found in clinical characteristics
between patients with cr-hvKP and cr-cKP infection.
Conclusions: The hypervirulent ST65 and ST25 K. pneumoniae, along with carbapenem-resistant clonal
populations ST11, appear to have evolved into cr-hvKP strains. The evidence of bi-directional evolution
and emergence of hospital-acquired multi-clone cr-hvKP indicates a confluence of virulence and
carbapenem resistance, which might pose major problems in the management of K. pneumoniae
infection.
ß 2015 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).

1. Introduction
Classic Klebsiella pneumoniae (cKP) is an important pathogen,
causing various infections including pneumonia, bacteremia,
septicemia, purulent infections, and urinary tract infections, all
of which may occur in either a community or hospital setting.1
In 1986, a new and hypervirulent (hypermucoviscous) clinical
variant of K. pneumoniae (hvKP) was identified in Taiwan, and
hvKP strains are increasingly being reported in the USA,
Argentina, Scandinavia, Korea, and Australia.2–7 hvKP may cause
an invasive clinical syndrome, which has been defined as an
infection presenting as community-acquired liver abscess with
or without extrahepatic complications, such as endophthalmitis
* Corresponding author. Tel.: +86 1082265719; fax: +86 1082265719.
E-mail address: zhangjiebjmu15@163.com (J. Zhang).
and necrotizing fasciitis.8 Further, hvKP infection has the ability
to spread metastatically in the absence of host compromise.8
With regard to the bacterial phenotypic features, hvKP
isolates differ from the classic strains in that colonies grown
on agar plates appear hypermucoviscous, which contributes
significantly to the virulence of K. pneumoniae in invasive
infections and appears to be a surrogate marker for the presence
of hvKP.9–11
Different to hvKP strains, most of which are resistant only to
ampicillin,12 increasing resistance to carbapenems among cKP
strains represents a real threat worldwide.13–16 Infections caused
by carbapenem-resistant K. pneumoniae pose an increasing
therapeutic dilemma because of their extended antibiotic resis-
tance phenotypes and ability to disseminate rapidly. As a result,
there are few available therapeutic options, and these strains are
associated with high mortality rates.17,18

http://dx.doi.org/10.1016/j.ijid.2015.06.023
1201-9712/ß 2015 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
108 B. Yao et al. / International Journal of Infectious Diseases 37 (2015) 107–112
A more complicated clinical situation concerns the emergence
of carbapenem-resistant hypervirulent (hypermucoviscous) K.
pneumoniae (cr-hvKP) in America and Brazil, and the increasing
antimicrobial resistance of hvKP in China, which are a cause for
concern.12,19,20 To better understand the incidence and character-
istics of cr-hvKP strains in China, multi-clone cr-hvKP isolates from
a single medical center in China were characterized in the present
study.
2. Materials and methods
2.1. Study setting and design
Peking University Third Hospital (PUTH) is a university-
affiliated medical center with a 1498-bed capacity and 79 000 hos-
pital admissions per year. It provides both primary and tertiary
referral care to patients from Beijing, and to patients referred from
various outside institutions throughout China.
From 2010, the identification and antimicrobial susceptibility
testing of all K. pneumoniae isolates from patients admitted to
PUTH was performed using Vitek panels (bioMérieux, Marcy
l’Etoile, France); isolates were stored at 80 8C. To identify cr-hvKP
isolates for the current study, the computerized databases of the
microbiology laboratories were searched retrospectively. All K.
pneumoniae isolates exhibiting an imipenem or meropenem
minimum inhibitory concentration (MIC) 4 mg/ml by Vitek
2 were examined for the hypermucoviscosity phenotype (de-
scribed below). Non-repetitive putative cr-hvKP isolates were
selected, with only the first isolate from each different anatomical
site for each patient included in the study. If frozen samples
contained both a cKP and an hvKP isolate, then the cKP isolate was
also included in the study. For comparison of clinical and molecular
characteristics, all the carbapenem-resistant classic K. pneumoniae
(cr-cKP) strains isolated during the same study period were
included. Different from hvKP, only the first cr-cKP strain from the
first anatomical site of each patient was included in the study.
The medical records of all patients colonized or infected with
cr-hvKP and cr-cKP were reviewed retrospectively by an indepen-
dent physician. For the purposes of this study, colonization implied
that the patient had a sufficiently high concentration of cr-hvKP
and cr-cKP at a site to allow detection, yet the organism was not
causing any symptoms. The standard US Centers for Disease
Control and Prevention definitions were used to define infections
at different anatomical sites.21
For cr-hvKP infection cases, the treatment outcome was
evaluated on day 7. Cure was defined as no clinical or laboratory
evidence of infection. Improvement was defined as partial resolution
of signs, symptoms, and laboratory parameters of infection. Cure and
improvement were characterized as a successful outcome; all other
outcomes were characterized as failures. For colonization cases,
treatment outcomes were defined as ‘not assessable’.
2.2. Detection of the hypermucoviscosity phenotype
The frozen carbapenem-resistant isolates were subcultured
overnight on blood agar at 37 8C. Isolates were considered positive
for the hypermucoviscosity phenotype if an inoculation loop
touched to the surface of the colony generated a viscous string of
5 mm in length when pulled away from the colony.22 Carbapenem-
resistant K. pneumoniae strains with a positive string test were
designated cr-hvKP.
2.3. Antimicrobial susceptibility testing
Susceptibility of the isolates to 18 antimicrobial agents
(ampicillin, ampicillin/sulbactam, piperacillin, piperacillin/
tazobactam, cefazolin, cefuroxime, cefuroxime axetil, cefotetan,
ceftriaxone, cefepime, aztreonam, gentamicin, tobramycin, nitro-
furantoin, trimethoprim/sulfamethoxazole, cefoxitin, ertapenem,
and tigecycline) was examined using Vitek panels (bioMérieux), in
accordance with the manufacturer’s instructions. For cr-hvKP, the
Etest method was used for further determination of MICs for
minocycline, amikacin, cefotaxime, ceftazidime, moxifloxacin,
ciprofloxacin, levofloxacin, meropenem, and imipenem on Muel-
ler–Hinton agar plates, in accordance with manufacturer’s
instructions (bioMérieux). Results were interpreted according to
the interpretive standards of the Clinical and Laboratory Standards
Institute (CLSI).23 Breakpoints for tigecycline were as defined by
the US Food and Drug Administration (susceptible, 2 mg/ml;
resistant, 8 mg/ml).24 Escherichia coli ATCC 25922 was used as a
quality control strain for susceptibility testing.
2.4. Modified Hodge test and PCR detection of carbapenemase genes
The modified Hodge test (MHT) was performed for the
detection of carbapenemases, as described previously.23 PCR
detection and sequencing of carbapenemase genes (blaIMP, blaVIM,
blaSPM, blaNDM, blaKPC, blaBIC, blaAIM, blaGIM, blaSIM, and blaDIM) were
performed as described previously.25 For the blaKPC-positive strain,
the subtype of KPC was identified.13
2.5. Capsular polysaccharide (CPS) typing and detection of virulence
factors
K1, K2, K5, K20, K54, and K57 capsular serotypes were
identified by PCR, as described previously.26 The presence of
genes encoding virulence factors (mrkD, kfuBC, fimH, uge, wabG,
ureA, ycfM, entB, ybtS, iroN, and allS), pLVPK-related loci (terW–
iutA–rmpA–silS), and repA was determined by PCR using primers
documented previously.27–29 The first strain with a DNA sequence
of the PCR product that was identical to the published sequence
was selected as a positive control for the subsequent PCR
experiments.
2.6. Multilocus sequence typing (MLST) and pulsed-field gel
electrophoresis (PFGE) analysis of cr-hvKP
MLST was performed by amplifying and sequencing seven
housekeeping genes according to protocols provided on the MLST
website for K. pneumoniae (http://bigsdb.web.pasteur.fr/klebsiella/
klebsiella.html). Isolates were typed by PFGE of XbaI-digested total
genomic DNA, and DNA patterns were interpreted according to
Tenover et al.30,31 Strains were considered to be the same clone
(type) if they showed 85% genetic identity, or fewer than four
fragment differences in the PFGE profiles.
2.7. Statistical analysis
Data were analyzed using the statistical package SPSS for
Windows version 17.0 (SPSS Inc., Chicago, IL, USA). The Chi-square
test or Fisher’s exact test was used for categorical variables. All
statistical tests were two-tailed and a p-value of 0.05 was
considered to be statistically significant.
3. Results
3.1. Clinical characteristics of patients with cr-hvKP infection
From January 2010 to August 2014, a total of four patients
admitted to PUTH tested positive for cr-hvKP and 53 tested
positive for cr-cKP. Seven non-replicate cr-hvKP isolates
were obtained from patients A (cr-hvKP1, cr-hvKP4), B
 B. Yao et al. / International Journal of Infectious Diseases 37 (2015) 107–112 109

(cr-hvKP2, cr-hvKP3, cr-hvKP5), C (cr-hvKP6), and D (cr-hvKP7).
Clinical characteristics of these four patients are shown in
Table 1. A non-hypermucoviscous K. pneumoniae was also
isolated from the sample containing cr-hvKP6. The four patients
had multiple co-morbidities and had required the use of
invasive devices. For patient A, cr-hvKP isolates 1 and 4 were
isolated from tracheal secretion and urine, respectively, and
were not the cause of clinical disease. As a result, patient A was
not treated with antimicrobials. Isolates cr-hvKP2 and cr-hvKP3
were the cause of a urinary tract infection and secondary
bloodstream infection, respectively, in patient B. In addition, cr-
hvKP5 was isolated from the tracheal secretion of patient B, but
was considered to be a colonizer only. cr-hvKP 6 and 7 were the
cause of pneumonia in patients C and D, respectively. While
patients B and C were treated successfully with antimicrobial
drugs, patient D died from heart failure. Patient A remained
persistently colonized with cr-hvKP, with positive results
obtained from both urine and tracheal secretion specimens.
All isolates were acquired nosocomially, with a median duration
of hospitalization of 769 days (range 11–1171 days) prior to
testing positive for the organism.
3.2. Antimicrobial susceptibility and the presence of carbapenemases
of cr-hvKP
The antimicrobial susceptibility testing results for cr-hvKP
isolates 1–7 and cKP1 are summarized in Table 2. cr-hvKP
isolates 1–5 had very similar susceptibility profiles for the
27 antimicrobials tested, with all five isolates showing
sensitivity to cefepime, trimethoprim/sulfamethoxazole, genta-
micin, amikacin, minocycline, tigecycline, and quinolones, while
cr-hvKP7 was sensitive only to trimethoprim/sulfame. Although
cr-hvKP6 and cKP1 were isolated from the same tracheal
secretion sample, they exhibited different sensitivity profiles to
piperacillin/tazobactam, ceftriaxone, cefepime, aztreonam, cef-
tazidime, trimethoprim/sulfame, minocycline, and carbape-
nems. Results of the carbapenemase assays showed that cr-
hvKP isolates 1–5 and cr-hvKP7 were MHT-positive, with only
the blaKPC-2 gene being detected.
3.3. Molecular characteristics of cr-hvKP
Capsular serotyping showed that cr-hvKP isolates 1–6 and cKP1
were serotype K2. cr-hvKP7 was non-typeable. Isolates cr-hvKP1–
5 from patients A and B produced identical PFGE profiles and were
typed as ST65, suggesting a clonal origin. Isolates cr-hvKP6 and
cKP1 from patient C also showed identical PFGE types, and both
belonged to the clonal group ST25. Isolate cr-hvKP7 had a distinct
PFGE profile and belonged to clonal group ST11 (Figure 1, Table 3).
The presence/absence of virulence factors in each isolate is
outlined in Table 3. Of the virulence genes tested, wabG, fimH, entB,
uge, and ureA were present in all of the isolates. ycfM and mrkD
were present in all cr-hvKP isolates, but were not amplified from
cKP1. pLVPK-related loci and iroN were amplified from all isolates
apart from cr-hvKP7. None of the isolates contained kfuBC, while
ybtS was only detected in cr-hvKP7, and allS was only found in cr-
hvKP6 and cKP1.
3.4. Comparison of the clinical and molecular characteristics between
the cr-hvKP infection group and cr-cKP infection group
Among the four cr-hvKPs, three strains were considered to be
infection, and among the 53 cr-cKPs, 29 were considered to be
infection. There were no differences in clinical characteristics
between the cr-cKP infection group (n = 29) and the cr-hvKP
infection group (n = 3) (Table 4). The virulence genes uge, wabG,
fimH, entB, ybtS, kfuBC, mrkD, ycfM, ureA, and allS were distributed
equally in the two groups, whereas pLVPK-related loci, repA, and
iroN differed significantly between the two groups (p = 0.006). For
the capsular serotypes, significant differences were found in K2
and non-typeable serotypes between the two groups (p  0.05).
There were no differences in the carbapenemase genes between
the groups, and only the KPC-2 gene was positive in both groups
(Table 5).
4. Discussion
In this study, ST65 K2, ST25 K2, and ST11 non-typeable cr-hvKP
isolates from patients at PUTH were characterized; this appears to

Table 1
Clinical characteristics of the four patients with cr-hvKP-positive samples

Patient Age/sex Comorbidities Use of invasive Date of first isolation at the Antimicrobial therapy Treatment outcome
devices different anatomical (final outcome)
sites (infection/ colonization)
Before ID/AST After ID/AST

A 78/M RF, PKD, CVA, Bc, CVC, MV, 2/4/2013, urine (colonization); None None Not assessable
DU, HTN, CHC, TPN, Trac, NFT cr-hvKP1
UTI, PAF
1/20/2014, tracheal secretion None None
(colonization); cr-hvKP4
B 85/M COPD, RF, UTI, Bc, CVC, MV, 1/7/2014, urine (UTI); cr-hvKP2 IPM BIN, IPM, ISP Success for UTI and
HTN, SE, CVA TPN, Trac, NFT BSI, not assessable
for tracheal secretion
isolate
1/8/2014, blood (secondary BSI); IPM, ISP BIN, IPM, ISP
cr-hvKP3
2/17/2014, tracheal secretion None None
(colonization); cr-hvKP5
C 68/M RF, CVA, HTN, SE Bc, NFT 3/26/2014, tracheal secretion IPM, VA SCF, ISP Success (discharge)
(pneumonia); cr-hvKP6; cKP1
D 91/F RF, COPD, colon Bc, TPN, MV, 8/28/2014, tracheal secretion SCF ETP Failure (death); died
CA, PE,CAD Trac, CVC, NFT (pneumonia); cr-hvKP7 from heart failure

Bc, bladder catheter; BIN, bladder irrigation with nitrofurazone solution (0.02%); BSI, bloodstream infection; CA, carcinoma; CAD, coronary artery disease; CHC, chronic
hepatitis C; cKP, classic Klebsiella pneumoniae; COPD, chronic obstructive pulmonary disease; cr-hvKP, carbapenem-resistant hypervirulent (hypermucoviscous) Klebsiella
pneumoniae; CVA, cerebrovascular accident; CVC, central vascular catheter; DU, duodenal ulcer; ETP, ertapenem; F, female; HTN, hypertension; ID/AST, identification/
antimicrobial susceptibility; IPM, imipenem; ISP, isepamicin; M, male; MV, mechanical ventilation; NFT, nasogastric feeding tube; PAF, paroxysmal atrial fibrillation; PE,
pleural effusion; PKD, Parkinson’s disease; RF, respiratory failure; SCF, cefoperazone–sulbactam; SE, secondary epilepsy; TPN, total parenteral nutrition; Trac, tracheostomy;
UTI, urinary tract infection; VA, vancomycin.
110 B. Yao et al. / International Journal of Infectious Diseases 37 (2015) 107–112

AK, amikacin; AMP, ampicillin; ATM, aztreonam; CI, ciprofloxacin; cKP, classic Klebsiella pneumoniae; cr-hvKP, carbapenem-resistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae; CRO, ceftriaxone; CT, cefotaxime; CTT,
cefotetan; CXA, cefuroxime axetil; CXM, cefuroxime; CZO, cefazolin; ETP, ertapenem; FEP, cefepime; FOX, cefoxitin; GEN, gentamicin; IP, imipenem; LE, levofloxacin; MC, minocycline; MIC, minimum inhibitory concentration; MP,
Carbapenemase MHT

+
+
+
+
+

meropenem; MX, moxifloxacin; NIT, nitrofurantoin; PIP, piperacillin; SAM, ampicillin/sulbactam; SXT, trimethoprim/sulfamethoxazole; TGC, tigecycline; TOB, tobramycin; TZ, ceftazidime; TZP, piperacillin/tazobactam.
gene

KPC
KPC
KPC
KPC
KPC

KPC
-

-
>32
>32
>32
>32
>32
>32
>32
IP

3 0.75
>32
>32
>32
>32
>32
>32
>32
MP

0.19

0.75
2
2
2
2
2

32
LE

0.25

0.75
>32
3
3
3
3
3
CI

0.25

0.75
>32 >256 >32 >256 >32
3
4
3
3
4
MX

192
32
128
192
96
4 >32 >256

48
TZ

24 >32
24 >32
24 >32
24 >32
24 >32

4 >32
Etest MIC (mg/l)

CT
AK

12
3
3
3
3
3
4
MC

0.5
0.5
0.5
0.5
0.5
0.5

0.5
TGC

8
Antimicrobial susceptibility, presence of carbapenemase genes, and modified Hodge test (MHT) results for cr-hvKP1–7 and cKP1

8 0.25

Figure 1. Pulsed-field gel electrophoresis profiles of Klebsiella pneumoniae isolates

FOX ETP

8 8
8 8
8 8
8 8
8 8
8 8
2/38 64 8

following digestion with XbaI. Lanes 1–3, lane 6, and lane 8 correspond to cr-hvKP1–
5. Lanes 4 and 5 correspond to cr-hvKP6 and cKP1. Lane 7 corresponds to cr-hvKP7.
1/19
1/19
1/19
1/19
1/19
1/19

4/76
SXT

be the first report of multi-clonal cr-hvKP isolates from a single

medical center in China. The first reported cr-hvKP isolate
64
64
64
64
64
256
16 512
1 512
ATM GEN TOB NIT

belonged to clonal complex 65 (CC65), which includes ST65 and

ST25, and has been identified as a virulent clone that mostly
1
16
16
16
16
16

corresponds to capsular serotype K2.27 In the current study, cr-

hvKP isolates 1–5 were classified as K2/ST65, and were shown to
1
1
1

16
1
1
1

1

have the same PFGE type, demonstrating the potential spread of cr-
hvKP1 from patient A to patient B. At the end of the study period,
64
64
64
64
64
64
64
4

patient A, who was a long-term urinary catheterization patient,

16
64 64 64
2
2
2
2
2

16 1

was still colonized with cr-hvKP1. This may explain the persistent
CRO FEP

colonization and potential for dissemination to patient B. The

16
16
16
16
16
64 64

process of colonization and dissemination can be lengthy, yet

isolates cr-hvKP1–5 all contained the same virulence factors,
64
64
64
64
64

64
CZO CXM CXA CTT

showing the stability of the virulence genes and the possibility of

continued evolution. cr-hvKP1 had identical virulence factors to a
64
64
64
64
64
64
64
64

previously reported non-cr-hvKP isolate, demonstrating that

acquisition of KPCs does not result in a loss of virulence.27,29,32,33
64
64
64
64
64
64
64
64

The KPC plasmid can be conjugated and retained by virulent K2/

64
64
64
64
64
64
64
64

ST65 K. pneumoniae while still maintaining a high serum resistance

and murine lethality.34 The present study has confirmed for the
4/4
128/4
128/4
128/4
128/4
128/4
128/4
128/4

first time that a KPC gene has disseminated into hypervirulent K.

TZP

pneumoniae K2/ST65, and that the drug resistance and virulence

can be maintained long-term.
Vitek 2 MIC (mg/l)

32
128
128
128
128
128
128
128

Isolate cr-hvKP6 was also found to belong to CC65 and to have

AMP SAM PIP

virulence factors consistent with the previous report discussed

32
32
32
32
32
32
32
32

above.27,29,32 However, it was determined that cKP1, also K2/ST25

with the same PFGE profile as cr-hvKP6, was isolated from the
32
32
32
32
32
32
32
32

same sample. Both isolates were negative for carbapenemase

genes and the MHT. Based on these data, it is speculated that cr-
cr-hvKP1
cr-hvKP2
cr-hvKP3
cr-hvKP4
cr-hvKP5
cr-hvKP6
cr-hvKP7

hvKP6 may have evolved from cKP1, although a whole genome

Isolate
Table 2

cKP1

comparative analysis is required to further evaluate this

possibility.
 B. Yao et al. / International Journal of Infectious Diseases 37 (2015) 107–112 111

Table 3
Molecular characteristics of cr-hvKP1–7 and cKP1

Isolate K type/ST PFGE type pLVPK-related loci repA uge wabG fimH entB iroN ybtS kfuBC mrkD ycfM ureA allS
terW–iutA–rmpA–silS

cr-hvKP1 K2/ST65 I + + + + + + + + + + + + +
cr-hvKP2 K2/ST65 I + + + + + + + + + + + + +
cr-hvKP3 K2/ST65 I + + + + + + + + + + + + +
cr-hvKP4 K2/ST65 I + + + + + + + + + + + + +
cr-hvKP5 K2/ST65 I + + + + + + + + + + + + +
cr-hvKP6 K2/ST25 II + + + + + + + + + + + + + +
cr-hvKP7 N/ST11 III + + + + + + + +
cKP1 K2/ST25 II ++++ + + + + + + + +

cKP, classic Klebsiella pneumoniae; cr-hvKP, carbapenem-resistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae; PFGE, pulsed-field gel electrophoresis; ST,
sequence type.

Although ST11, associated with multi-drug resistance, has been
demonstrated to be the prevalent clone associated with the spread
of KPC-producing K. pneumoniae in China, there are no previous
reports of hvKP belonging to ST11, let alone cr-hvKP.35 In China,
isolate cr-hvKP7 identified in this study is the first ST11 clone that
combines a KPC gene with the hyperviscous phenotype, and this
was shown to be pan drug-resistant to all antimicrobials tested
except trimethoprim/sulfame. Although the patient was never
treated with tigecycline, cr-hvKP7 showed resistance to tigecycline
in vitro, and this mechanism of resistance needs further
investigation. The virulence of K. pneumoniae has been associated
with clone rather than with serotype, which could explain the
Table 4
Comparison between the cr-hvKP group and cr-cKP group for clinical characteristics
Characteristics cr-cKP cr-hvKP
infection infection
group group
(n = 29) (n = 3)
difference in virulence factors between ST11 and ST25/ST65.27 The
pLVPK-derived terW–iutA–rmpA–silS loci are significantly corre-
lated with abscess formation and prevalence of K1 and K2 serotype
strains,29,32 but these virulence loci have not previously been
detected in ST11 K. pneumoniae. Compared with previously
reported ST11 strains,20 cr-hvKP7 has more virulence factors,
including the hypermucoviscosity phenotype.10 In contrast to a cr-
hvKP isolate reported in Brazil, which did not contain either magA
or rmpA,20 the hypermucoviscous phenotype of cr-hvKP7 persisted
without decrease following several rounds of subculturing.
Several reports have shown that aminoglycosides are effective
for the clearance of urinary tract infections caused by aminoglyco-
side-susceptible carbapenem-resistant K. pneumoniae.36 In the
present study, cr-hvKP isolates 2, 3, and 6 were treated successfully
with a regimen that included isepamicin, indicating that amino-
glycosides may also be effective in treating cr-hvKP. Although the
p-Valuea
four patients included in this study had a variety of underlying
diseases and use of invasive devices, none had invasive liver
abscess syndrome or extra-hepatic metastatic infections.8 Further

Age, >60 years 25 3 1.000

Sex, n 0.552 Table 5
Male 11 2 Comparison between the cr-hvKP group and cr-cKP group for molecular
Female 18 1 characteristics
Hepatitis B virus 1 0 1.000
cr-cKP cr-hvKP p-Value
Hepatitis C virus 0 0 NA
infection infection
Alcoholic hepatitis 0 0 NA
group group
Fatty liver 1 0 1.000
(n = 29) (n = 3)
Gallbladder stone 3 0 1.000
Liver abscess 0 0 NA K serotype
Respiratory tract disorder 6 2 0.147 K1 0 0 NA
Cardiovascular disease 21 1 0.224 K2 0 3 0.000a
Diabetes 7 0 1.000 K5 0 0 NA
Tuberculosis 1 0 1.000 K20 0 0 NA
Pancreatitis 4 0 1.000 K54 0 0 NA
Nephritis 3 0 1.000 K57 0 0 NA
Non-hepatic malignancy 4 1 0.410 K non-typeable 29 1 0.006a
Non-hepatic abscess 1 0 1.000 Virulence gene
Duodenal ulcer 0 0 NA pLVPK-related loci 0 2 0.006a
Parkinson’s disease 2 0 1.000 repA 0 2 0.006a
Cerebrovascular accident 9 2 0.266 uge 29 3 NA
Secondary epilepsy 2 2 0.035 wabG 29 3 NA
Hypertension 17 2 1.000 fimH 29 3 NA
Respiratory failure 12 3 0.092 entB 29 3 NA
Thyroid disorder 1 0 1.000 iroN 0 2 0.006a
Use of invasive devices ybtS 21 1 0.224
Nasogastric feeding tube 27 3 1.000 kfuBC 0 0 NA
Total parenteral nutrition 20 2 1.000 mrkD 25 3 1.000
Bladder catheter 27 2 0.263 ycfM 29 3 NA
Endotracheal intubation 16 2 1.000 ureA 29 3 NA
Central vascular catheter 12 2 0.568 allS 0 1 0.094
Pleural effusion 2 1 0.263 blaKPC 28 3 1.000
Mortality 15 1 1.000 Other carbapenemase genes 0 0 NA

cr-cKP, carbapenem-resistant classic Klebsiella pneumoniae; cr-hvKP, carbapenem- cr-cKP, carbapenem-resistant classic Klebsiella pneumoniae; cr-hvKP, carbapenem-
resistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae; NA, not resistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae; NA, not
assessable. assessable.
a a
cr-cKP vs. cr-hvKP. A p-value 0.05 was considered to be statistically significant.
112 B. Yao et al. / International Journal of Infectious Diseases 37 (2015) 107–112
to these clinical observations, the ability of cr-hvKP to cause
invasive infection should be evaluated further in vitro, as well as in
phagocytosis assays and murine lethality tests.37 The length of stay
prior to isolation of cr-hvKP in the current study differed markedly,
with the shortest stay being 11 days (cr-hvKP6) and the longest
being 1171 days (cr-hvKP5). Combined with differences in
molecular characteristics and resistance patterns, it is concluded
that cr-hvKP isolates 1, 6, and 7 represent three separate
evolutionary lineages. The emergence of regional polyclonal cr-
hvKP isolates is of significant concern and may herald the cr-hvKP
era.
It has been reported that patients with hvKP infection tend to
have different underlying illnesses and more invasive infections
compared to those with cKP.38 However, in the present study, no
differences in clinical characteristics were found between cr-hvKP
and cr-cKP infected patients. Reports on the virulence genes and K
serotype of cr-cKP, let alone cr-hvKP, are scarce. The present study
showed that pLVPK-related loci, iroN, and K2 serotype were more
prevalent in cr-hvKP than in cr-cKP, but the significance of these
differences needs to be studied further.
In summary, this study provides the first clinical and molecular
characterization of ST65, ST25, and ST11 cr-hvKP isolates in China.
The clonal populations of invasive infection (ST65 and ST25) have
evolved to become multidrug-resistant, while a related multidrug-
resistant clonal population (ST11) has evolved to become
hypermucoviscous. The confluence of virulence and carbapenem
resistance and the bi-directional evolution might pose major
problems in the future for the management of K. pneumoniae
infections.
Conflict of interest/funding: None.
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