Infection and Drug Resistance Dovepress

open access to scientific and medical research

Open Access Full Text Article

ORIGINAL RESEARCH

Clinical and Molecular Characteristics of

Carbapenem-Resistant Hypervirulent Klebsiella
pneumoniae Isolates in a Tertiary Hospital in Shanghai,
China

Cong Zhou 1 Background: The convergence of carbapenem-resistance and hypervirulence in Klebsiella

Qiang Wu 1 pneumoniae has led to a significant public health challenge. In recent years, there have been
Leqi He 1 more and more reports on carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP)
Hui Zhang 1 isolates.
Materials and Methods: Clinical data of patients infected with CR-hvKP from January 2019 to
Maosuo Xu 1
December 2020 in a tertiary hospital were retrospectively evaluated. The number of isolates of
Baoyu Yuan 2
Klebsiella pneumoniae, hypermucoviscous Klebsiella pneumoniae (hmKP), carbapenem-resistant
Zhi Jin 3
hypermucoviscous Klebsiella pneumoniae (CR-hmKP) and carbapenem-resistant hypervirulent
Fang Shen 1 Klebsiella pneumoniae (CR-hvKP) collected during the period of 2 years was calculated. The
1
Department of Clinical Laboratory antimicrobial resistance gene, virulence-associated gene, capsular serotype gene and multilocus
Medicine, Shanghai Fifth People’s
sequence typing (MLST) of CR-hvKP isolates were detected by PCR.
Hospital, Fudan University, Shanghai,
People’s Republic of China; 2Department Results: During the study period, a total of 1081 isolates of non-repeat Klebsiella pneumo­
of Clinical Laboratory, Shanghai niae were isolated, including 392 isolates of hypermucoviscous Klebsiella pneumoniae
Children’s Hospital, Shanghai Jiao Tong
University, Shanghai, People’s Republic of
(36.3%), 39 isolates of CR-hmKP (3.6%), and 16 isolates of CR-hvKP (1.5%). About
China; 3Department of Neurology, 31.2% (5/16) of CR-hvKP were isolated from 2019, and 68.8% (11/16) of CR-hvKP were
Shanghai Fifth People’s Hospital, Fudan isolated from 2020. Among the 16 isolates of CR-hvKP, 13 isolates were ST11 and serotype
University, Shanghai, People’s Republic of
China K64, 1 isolate was ST11 and serotype K47, 1 isolate was ST23 and serotype K1, and 1
isolate was ST86 and serotype K2. The virulence-associated genes entB, fimH, rmpA2, iutA,
iucA were present in all of 16 CR-hvKP isolates, followed by mrkD (n=14), rmpA (n=13),
aerobactin (n=2), allS (n=1). Sixteen CR-hvKP isolates all carry carbapenemase gene
blaKPC-2 and extended-spectrum β-lactamase gene blaSHV. ERIC-PCR DNA fingerprinting
results showed that 16 CR-hvKP isolates were highly polymorphic, and there were signifi­
cant differences in bands among the isolates, presenting a sporadic state.
Conclusion: Although CR-hvKP was sporadically distributed, it showed an increasing
trend year by year. Therefore, clinical attention should be paid, and necessary measures
should be taken to avoid the cloning and transmission of superbacterium CR-hvKP.
Keywords: Klebsiella pneumoniae, carbapenem-resistant, hypervirulent, hypermucoviscous,
epidemiology

Correspondence: Fang Shen

Department of Clinical Laboratory
Medicine, Shanghai Fifth People’s Hospital, Introduction
Fudan University, No. 128, Ruili Road,
Minhang District, Shanghai, 200240, Klebsiella pneumoniae is an opportunistic pathogen that causes a wide range of
People’s Republic of China infections, including pneumonia, urinary tract infections, bacteremia, and
Tel +86 18021073261
Email [email protected] meningitis.1 In the past three decades, unlike classical Klebsiella pneumoniae

Infection and Drug Resistance 2021:14 2697–2706 2697

Received: 25 May 2021 © 2021 Zhou et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php
Accepted: 1 July 2021 and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work
you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For
Published: 14 July 2021 permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php).
 Zhou et al Dovepress

(cKP), a new hypervirulent Klebsiella pneumoniae (hvKP) CR-hvKP in a tertiary hospital in Shanghai, eastern
with hypermucoviscosity has emerged as a clinically China. This study is of significance for understanding the
important pathogen that can cause highly invasive infec­ molecular epidemiology of CR-hvKP in Shanghai.
tions such as liver abscess in healthy and immunocompro­
mised individuals.2 It is important to note that these
Materials and Methods
infections are often accompanied by destructive dissemi­
nated infections, including endophthalmitis and Collection and Identification of
meningitis.3 The hypermucoviscous phenotype of hvKP K. pneumoniae Isolates
is usually due to increased production of capsular poly­ A retrospective study was conducted to collect non-
saccharides and the presence of specific virulence genes, repeated Klebsiella pneumoniae isolates from Shanghai
such as rmpA and rmpA2.4 The hypermucoviscous pheno­ Fifth People’s Hospital affiliated to Fudan University
type is generally determined by the “string test”. from January 2019 to December 2020. The percentage
K. pneumoniae colonies grown on blood agar plates over­ of hmKP, CRKP, CR-hmkp and CR-hvKP was calcu­
night were stretched with a loop, and the “string test” is lated. All isolates were identified by VITEK-2 compact
positive when a viscous string >5mm in length is formed.5 automated microbiology analyzer (Biomerieux, Marcy
A recent study showed that peg-344, iroB, iucA, rmpA and L‘Etoile, France). The Maldi-Tof mass spectrometry
rmpA2 are biomarkers that can accurately identify hvkp.6 (Bruker Daltonics, Billerica, MA, USA) was used to
In this study, hypervirulent K. pneumoniae is defined as recheck the identification of the bacterial strain. The
having a hypermucoviscous phenotype (a positive string hypermucoviscous phenotype was determined by the
test result) and carrying K. pneumoniae virulence plasmid- “string test”. Carbapenem resistance was determined
associated loci (rmpA2, iutA, iucA). In the 1980s, case when imipenem or meropenem was resistant by antimi­
reports from Taiwan first described community-acquired
crobial susceptibility test. Hypervirulent Klebsiella
liver abscesses caused by hvKP with severe terminal organ
pneumoniae is defined as having a hypermucoviscous
damage, such as meningitis and endophthalmitis.7,8 The
phenotype (a positive string test result) and carrying
sporadic spread of hvKP has occurred in many countries in
K. pneumoniae virulence plasmid-associated loci
Asia, Europe and the Americas. Although several cases of
(rmpA2, iutA, iucA).6
hvKP have been reported in Europe and the Americas, the
prevalence of hvKP mainly occurs in Asian countries,
especially in China.9 String Test
In general, hvKP has a high sensitivity to antibiotics, A single Klebsiella pneumoniae colony was inoculated on
while carbapenem-resistant K. pneumoniae (CRKP) has 5% sheep blood agar plate. After overnight culture at
a low toxicity. However, with the spread of drug-resistant 37°C, the bacterial colony was gently pulled up with the
and virulence plasmids, CR-hvKP was first described by inoculation loop and repeated for 3 times. If a viscous
Zhang et al in 2015, and more and more reports have been string formed in all 3 times and the length was longer
described in China.10 Because CR-hvKP can cause severe than 5mm, it was considered positive for the “string test”,
and hard-to-treat infections, it could become the next and the strain was hypermucoviscous phenotype.
“superbug” if a pandemic clone emerges. So far, most CR-
hvKP-induced infections have occurred in sporadic cases,
and small outbreaks are rare.11,12 Antimicrobial Susceptibility Testing
At present, the detection rate of CR-hvKP is low, and Antimicrobial susceptibility to several commonly used
the relevant studies are few. The molecular epidemiology antibiotics was detected by broth microdilution in the
of CR-hvKP is different in different regions, so it is VITEK-2 compact automated microbiology analyzer
necessary to study the clinical distribution and molecular (Biomerieux, Marcy L‘Etoile, France). The results were
epidemiological characteristics of CR-hvKP in this region. interpreted according to the guideline document estab­
In this study, antimicrobial resistance genes, virulence- lished by Clinical and Laboratory Standards Institute
associated genes and MLST of CR-hvKP were analyzed (CLSI, 2019). Escherichia coli ATCC 25922 and
comprehensively. We attempted to investigate the preva­ Klebsiella pneumoniae ATCC 700603 were used as con­
lence and molecular epidemiological characteristics of trols for antimicrobial susceptibility testing.

2698 https://doi.org/10.2147/IDR.S321704 Infection and Drug Resistance 2021:14

DovePress

Powered by TCPDF (www.tcpdf.org)

 Dovepress Zhou et al

PCR Detection of Antimicrobial Statistical Analysis

Resistance Genes, Virulence-Associated Data were analyzed using the statistical package SPSS for
Windows version 22.0. Data were described as means
Genes and Capsule Serotype (CPS) Gene
±standard deviation (SD). Categorical variables were
Genomic DNA of all Klebsiella pneumoniae isolates was
assessed by Chi-square test or Fisher’s exact test. All
extracted by TIANamp Bacteria Genomic DNA Kit
statistical tests were 2 tailed and a P-value <0.05 was
(TiangenBiotechCo. Ltd., Beijing, China). The extended-
considered to be statistically significant.
spectrum β-lactamase genes (blaCTX-M, blaSHV and blaTEM)
, carbapenemase genes (blaKPC, blaNDM, blaVIM, blaIMP, and
blaOXA-48) and 9 representative virulence-associated genes Results
including the pLVPK-like plasmid genetic loci (allS, fimH, Prevalence of Carbapenem-Resistant
mrkD, entB, iutA, rmpA, rmpA2, iucA, and aerobactin) were
hvKP Isolates
amplified by PCR as previously described.13,14 Capsular
A total of 1081 Klebsiella pneumoniae isolates were col­
serotype-specific genes (K1, K2, K5, K20, K54, and K57)
lected from Shanghai Fifth People’s Hospital affiliated to
were amplified by PCR as previously described.14 If nega­
Fudan University from January 1, 2019 to December 31,
tive, wzi loci were amplified and sequenced to determine
2020, and the duplicate isolates of the same patient were
capsular serotype-specific genes.15 The primers used in this
excluded. Among them, 392 isolates (36.3%) were hmKP,
study are listed in Table S1. The positive PCR products were
341 isolates (31.5%) were CRKP, 39 isolates (3.6%) were
sequenced by the NextSeq 500 sequencing platform
CR-hmKP and 16 isolates (1.5%) were CR-hvKP. Notably,
(Illumina, San Diego, CA, USA). Nucleotide sequences
33.3% (13/39) of CR-hmKP and 31.2% (5/16) of CR-
were compared by running BLAST at NCBI website
hvKP were isolated from 2019, 66.7% (26/39) of CR-
(http://blast.ncbi.nlm.nih.gov/Blast.cgi).
hmKP and 68.8% (11/16) of CR-hvKP were isolated
from 2020. Thirty-nine CR-hmKP were recovered from
Multilocus Sequence Typing (MLST) sputum (17 isolates), urine (12 isolates), drainage fluid (4
Multilocus sequence typing (MLST) was performed as isolates), blood (2 isolates), pus (2 isolates), bile (1 isolate)
described on the Pasteur Institute MLST website (https:// and pleural fluid (1 isolate), respectively. Sixteen CR-
bigsdb.pasteur.fr/klebsiella/klebsiella.html). Seven house­ hvKP were recovered from sputum (9 isolates), urine (5
keeping genes gapA, infB, mdh, pgi, phoE, rpoB, and isolates), blood (1 isolate) and pleural fluid (1 isolate).
tonB were amplified by PCR and sequenced. The sequence
types (STs) were determined by comparing the sequencing
results with the MLST database.
Clinical Characteristics of 16 Patients
Infected with CR-hvKP Isolates
Sixteen isolates of CR-hvKP were screened out through
Enterobacterial Repetitive Intergenic strain identification, antimicrobial susceptibility testing,
Consensus Polymerase Chain Reaction string test and virulence-associated gene detection.
(ERIC-PCR) DNA Fingerprint Clinical characteristics of 16 patients infected with CR-
The homology of Klebsiella pneumoniae was analyzed. hvKP isolates are summarized in Table 1. Thirteen
Genomic DNA of Klebsiella pneumoniae was extracted (81.3%) of the 16 patients were males, and all patients
as template, and ERIC primer was listed in Table S1. were older than 62 years (the mean age: 83.1±10.5 y).
Genomic DNA was amplified by PCR, and the fingerprint They came from 8 wards, more than half from the
of genomic DNA was constructed.16 The PCR products central ICU (9 cases). Underlying conditions included
were detected by 2% agarose gel electrophoresis. The cerebrovascular diseases (75%, 12/16), hypertension
DNA fingerprinting results were identified by band identi­ (50%, 8/16), chronic obstructive pulmonary disease
fication using QuantityOne software, and genetic analysis (50%, 8/16), etc. Invasive procedures included mechan­
was performed by unweighted pair group method with ical ventilation (62.5%, 10/16), urinary catheter (37.5%,
arithmetic mean (UPGMA). The isolates with similarity 6/16), gastrictube (18.8%, 3/16), surgery (12.5%, 2/16)
> 75% was regarded as the same genotype, and those with and venous catheter (6.3%, 1/16). Nine of the 16 patients
similarity < 75% as different genotypes. died, and seven patients improved and were discharged.

Infection and Drug Resistance 2021:14 https://doi.org/10.2147/IDR.S321704

2699
DovePress

Powered by TCPDF (www.tcpdf.org)

Powered by TCPDF (www.tcpdf.org)
2700
Zhou et al

Table 1 Clinical Characteristics of 16 Patients Infected with CR-hvKP Isolates

Isolates Age Sex Ward Underlying Conditions Treatment Invasive Procedures Outcome

DovePress
(Years)

KP3 83 F Neurology Department Cerebrovascular disease, type 2 diabetes mellitus, ventilator-associated SCF, TZP Mechanical ventilation Died
pneumonia, epilepsy

https://doi.org/10.2147/IDR.S321704
KP58 86 F Central ICU Cerebrovascular disease, coronary heart disease, hypertension, urinary tract TZP, TGC Urinary catheter, Gastric tube Survived
infection

KP63 89 M Central ICU Cerebrovascular disease, hypertension, Parkinson’s disease, renal malignancy, TGC Mechanical ventilation Died
pulmonary infection

KP104 74 M Traditional Chinese Medicine Cerebrovascular disease, hypertension, urinary tract infection, pulmonary TZP, CRO Mechanical ventilation, Urinary catheter Survived
Department embolism

KP129 67 M Central ICU Cerebrovascular disease, type 2 diabetes, hypertension, coronary heart CAZ Mechanical ventilation, Urinary catheter, Died
disease, colon tumors Gastric tube

KP178 96 M Geriatric Ward Cerebrovascular disease, COPD, coronary heart disease TZP, SCF, MER Mechanical ventilation Died

KP188 91 M Respiratory Intensive Care Unit Chronic obstructive pulmonary disease, coronary heart disease, hypertension TZP Mechanical ventilation Survived

KP194 87 M Central ICU Pulmonary infection,hypertension, cerebrovascular disease TZP, SCF, MER, Venous catheter Died
CMZ

KP284 90 M Central ICU Cerebrovascular disease,Chronic obstructive pulmonary disease SCF Mechanical ventilation Died

KP290 63 M Central ICU Cerebrovascular disease,hypertension,type 2 diabetes SCF, TGC, Mechanical ventilation, Urinary catheter Died
CAZ-AVI

KP298 83 M Central ICU Cerebrovascular disease,hypertension, respiratory failure IPM, AN Mechanical ventilation, Urinary catheter Died

KP302 87 M Gastroenterology Department Chronic obstructive pulmonary disease,Sigmoid colon cancer CMZ Surgery Survived

KP358 75 M General Surgery Department Coronary heart disease, gallstone with cholecystitis, COPD SCF, CMZ, SXT Gastric tube Died

KP359 85 F Central ICU Cerebrovascular disease,hypertension SCF, AN Mechanical ventilation Survived

KP374 102 M Endocrinology Department Cerebrovascular disease, Parkinson’s disease, coronary heart disease TZP Urinary catheter Survived

KP393 71 M Central ICU Rectum cancer, COPD, cerebrovascular disease, hypertension CMZ Surgery Survived

Abbreviations: M, male; F, female; COPD, chronic obstructive pulmonary disease; SCF, cefoperazone/sulbactam; TZP, piperacillin/tazobactam; AN, amikacin; CMZ, cefmetazole; TGC, tigecycline; SXT, trimethoprim-sulfamethoxazole;
CRO, ceftriaxone; CAZ, ceftazidime; MER, meropenem; IPM, imipenem; CAZ-AVI, ceftazidime/avibactam.

Infection and Drug Resistance 2021:14

Dovepress
 Dovepress Zhou et al

Table 2 Virulence-Associated Gene Positive Rate of 39 CR- Table 3 The Antimicrobial Resistance Profile of 16 CR-hvKP
hmKP Isolates Isolates [n(%)]
Virulence- Viscous String Viscous String P value Antimicrobials CR-hvKP(n=16)
Associated Length ≤25mm Length>25mm
Susceptible Resistant
Gene (n=20) (n=19)
Ampicillin 0 16(100)
rmpA 6(30%) 7(36.8%) 0.651
Ampicillin/sulbactam 0 16(100)
rmpA2 8(40%) 8(42.1%) 0.894
Cefoperazone/sulbactam 0 16(100)
iutA 8(40%) 8(42.1%) 0.894
Piperacillin/tazobactam 0 16(100)
iucA 8(40%) 8(42.1%) 0.894
Cefazolin 0 16(100)
Cefuroxime 0 16(100)
Ceftazidime 0 16(100)
Virulence-Associated Gene Positive Rate Ceftriaxone 0 16(100)
of 39 CR-hmKP Isolates Cefepime 0 16(100)
Cefoxitin 0 16(100)
Thirty-nine CR-hmKP isolates were divided into two
Imipenem 0 16(100)
groups according to the length of viscous string. Among
Meropenem 0 16(100)
them, 20 CR-hmKP isolates with viscous string length Amikacin 6(37.5) 10(62.5)
≤25mm were divided into one group, and 19 CR-hmKP Gentamicin 5(31.2) 11(68.8)
isolates with viscous string length >25mm were divided Ciprofloxacin 2(12.5) 14(87.5)
into the other group. The positive rates of virulence- Trimethoprim-sulfamethoxazole 9(56.2) 7(43.8)

associated genes rmpA, rmpA2, iutA and iucA were

detected by PCR method. The positive rates of virulence-
the most predominant ST (87.5%, 14/16), followed by ST23
associated genes of CR-hmKP in the two groups are
(6.25%, 1/16) and ST86 (6.25%, 1/16). According to the
shown in Table 2. The positive rate of virulence-
results of the wzi typing, a total of 4 different capsular ser­
associated genes in CR-hmKP strain showed no statistical
otypes were identified (Figure 1). Among the 16 Carbapenem-
difference between the two groups.
Resistant hvKP isolates, K64 was the most common serotype
(n=13), followed by K1 (n=1), K2 (n=1), and K47 (n=1). In
The Antimicrobial Resistance Profile of addition, the strain of capsular serotype K1 is ST23, the strain
16 CR-hvKP Isolates of capsular serotype K2 is ST86, and the remaining 13 isolates
The detailed antimicrobial resistance profile of the 16 drugs is of K64 and 1 strain of K47 were all ST11. The positive rates of
listed in Table 3. Sixteen isolates of CR-hvKP showed multi­ the 9 virulence genes in 16 CR-hvKP isolates are shown in
ple drug resistance. All isolates were resistant to ampicillin, Figure 1, the virulence-associated genes entB, fimH, rmpA2,
ampicillin/sulbactam, cefoperazone/sulbactam, piperacillin/ iutA, iucA were present in all of 16 CR-hvKP isolates, fol­
tazobactam, cefazolin, cefuroxime, ceftazidime, ceftriaxone, lowed by mrkD (n=14), rmpA (n=13), aerobactin (n=2), allS
cefepime, cefoxitin, imipenem, meropenem. The antimicro­ (n=1). Sixteen CR-hvKP isolates all carried carbapenemase
bial resistance rate of Trimethoprim-sulfamethoxazole was the gene blaKPC-2 and extended-spectrum β-lactamase gene
lowest (43.8%), followed by amikacin (62.5%), gentamicin blaSHV. Sixteen CR-hvKP isolates did not carry carbapenem
(68.8%) and ciprofloxacin (87.5%). genes blaNDM, blaVIM, blaIMP, blaOXA-48 and extended-
spectrum β-lactamase genes blaTEM, blaCTX-M-2 group,
Virulence-Associated Genes, blaCTX-M-8 group. Among 16 CR-hvKP isolates, 5 isolates
carried extended-spectrum β-lactamase gene blaCTX-M-1
Antimicrobial Resistance Genes, Capsule
group, and 6 isolates carried extended-spectrum β-lactamase
Serotype Genes and MLST gene blaCTX-M-9 group.
The distribution of virulence-associated genes, antimicrobial
resistance genes, capsular serotype genes and MLST of 16
CR-hvKP isolates is shown in Figure 1. Agarose gel electro­ ERIC-PCR DNA Fingerprint Analysis of
phoresis results of some virulence-associated genes, antimi­ Klebsiella pneumoniae Isolates
crobial resistance genes and capsule serotype genes are shown ERIC-PCR was used to analyze the homology of 16 CR-
in Figure 2. MLST analysis revealed a total of 3 STs, ST11 was hvKP isolates. After PCR amplification and agarose gel

Infection and Drug Resistance 2021:14 https://doi.org/10.2147/IDR.S321704

2701
DovePress

Powered by TCPDF (www.tcpdf.org)

 Zhou et al Dovepress

Figure 1 Virulence-associated genes, antimicrobial resistance genes, capsular serotype genes and MLST of 16 CR-hvKP isolates.
Note: The black color indicated the presence of a gene in the corresponding strain.

electrophoresis, there were 3–9 DNA fragments. The fin­ hospital in Shanghai area from 2019 to 2020 were studied
gerprint results showed that 16 CR-hvKP isolates were to evaluate whether there is a risk of CR-hvKP outbreak
highly polymorphic, and there were obvious differences and its development trend in this area. At the same time,
among isolates (Figure 3). this study can provide a more comprehensive clinical
assessment of infectivity, which is of great importance to
prevent the further spread of such isolates.
Discussion This study retrospectively analyzed the clinical distri­
In recent years, there have been more and more reports on
bution and change trend of CR-hvKP from 2019 to 2020.
CR-hvKP isolates. The emergence of CR-hvKP isolates
The CR-hvKP isolates showed an increasing trend from
poses a significant threat to public health because they can
2019 to 2020. About 31.2% (5/16) of CR-hvKP were
cause serious, hard-to-treat infections in healthy popula­ isolated in 2019, and 68.8% (11/16) of CR-hvKP were
tions. In this study, the prevalence and molecular epide­ isolated in 2020. This is consistent with the increasing
miological characteristics of CR-hvKP in a tertiary trend of CR-hvKP literature reports. Since the first
description of CR-hvKP by Zhang et al in 2015,10 more
and more CR-hvKP literature has been reported,17–20
mainly in the Asia-Pacific region, especially in China. CR-
hvKP is a super bacterium with hypervirulence and multi-
drug resistance, which has great harm to people’s health
and high mortality rate. Therefore, attention should be paid
to it, and measures should be taken to prevent its spread.
Figure 2 Agarose gel electrophoresis of some virulence-associated genes, antimi­
Antimicrobial resistance analysis of 16 isolates of CR-
crobial resistance genes and capsule serotype genes. hvKP showed high resistance rate to antibiotics. All isolates
Notes: M, DNA marker; 1, blaKPC(893bp); 2, entB(400bp); 3, rmpA2(609bp); 4,
rmpA(429bp); 5, iucA(239bp); 6, iutA(880bp); 7, aerobactin(556bp); 8, K1(1283bp); 9, were resistant to ampicillin, ampicillin/sulbactam, cefopera­
K2(641bp); 10, allS(508bp); 11, mrkD(340bp); 12, fimH(609bp). zone/sulbactam, piperacillin/tazobactam, cefazolin,

2702 https://doi.org/10.2147/IDR.S321704 Infection and Drug Resistance 2021:14

DovePress

Powered by TCPDF (www.tcpdf.org)

 Dovepress Zhou et al

Figure 3 ERIC-PCR DNA fingerprint of 16 CR-hvKP isolates.

cefuroxime, ceftazidime, ceftriaxone, cefepime, cefoxitin, correlated with the length of viscous string (or did not increase
imipenem, meropenem. The antimicrobial resistance rate of with the length of viscous string).
Trimethoprim-sulfamethoxazole was the lowest (43.8%), In this study, the ERIC PCR fingerprint was pleomorphic
followed by amikacin (62.5%), gentamicin (68.8%) and and there was no clinical intersection between patients, so 16
ciprofloxacin (87.5%). The antimicrobial resistance rate of patients infected with CR-hvKP were sporadic cases. In the
CR-hmkp studied by Lingling Zhan et al is similar to this past, the majority of CR-hvKP-induced infections have been
study.12 Patients infected with CR-hvKP have many under­ reported as isolated or sporadic cases,23,24 and small out­
lying diseases, low immunity and weak ability of indepen­ breaks of CR-hvKP are rare in the literature.11,25 ST11 is
dent bacteria elimination. Therefore, timely treatment the most prevalent ST among CRKP and CR-hvKP isolates
according to the results of antimicrobial sensitivity test is in China.26,27 Although ST11 CR-hvKP accounted for 87.5%
very important. If necessary, infection site can be found and (14/16) of the 16 CR-hvKP isolates in this study, it could not
treated by means of drainage and debridement. be assumed that the 14 ST11 CR-hvKP were from the same
Thirty-nine CR-hmKP isolates were divided into two clone strain, so it was necessary to conduct homology ana­
groups according to the length of viscous string. Among lysis by ERIC PCR fingerprint.
them, 20 CR-hmKP isolates with viscous string length In this study, all 16 patients infected with CR-hvKP
≤25mm were divided into one group, and 19 CR-hmKP iso­ underwent invasive procedures. An outbreak of fatal venti­
lates with viscous string length >25mm were divided into the lator-associated pneumonia caused by CR-hvKP has been
other group. The positive rate of virulence-associated gene of reported,11 suggesting that invasive procedures may increase
CR-hmKP was compared between the two groups, and there the risk of CR-hvKP infection. Meanwhile, all 16 patients
was no statistical significance in the positive rate of virulence infected with CR-hvKP had underlying conditions, among
gene between the two groups. The study of Lin Z et al revealed which cerebrovascular disease was the most common.
that the virulence gene positive rate of hypermucoviscous A previous study showed that cerebrovascular disease is
Klebsiella pneumoniae was significantly higher than that of a significant independent risk factor for CR-hvKP
classical Klebsiella pneumoniae.21 However, it is not clear infection.28 The reason for this phenomenon may be that
whether the virulence gene positive rate is positively corre­ patients with cerebrovascular diseases have low immunity
lated with the length of viscous string. Other studies have and cannot independently exclude pathogenic bacteria, and
shown that classical Klebsiella pneumoniae may also be only rely on the bactericidal effect of antibiotics, which will
hypervirulent Klebsiella pneumoniae, with a high virulence lead to the phenomenon of multiple drug resistance com­
gene positive rate.22 In this study, it was found that the viru­ bined with hypervirulence in the long run. Of the 16 patients,
lence gene positive rate of CR-hmKP was not positively 9 patients died, with a mortality rate of 56.3% (9/16). The

Infection and Drug Resistance 2021:14 https://doi.org/10.2147/IDR.S321704

2703
DovePress

Powered by TCPDF (www.tcpdf.org)

 Zhou et al Dovepress

mortality rate was higher than that reported by previous Although the infection caused by CR-hvKP was sporadic,
studies10,12 and lower than that reported by previous it showed an increasing trend year by year. The results
studies.11,21 The mean age of 16 patients was 83.1±10.5 y, support previous studies indicating that the ST11 CR-
which showed that the elderly were more susceptible to CR- hvKP is the most prevalent CR-hvKP in China. ST23
hvKP infection. Previous study has showed that young peo­ and ST86 CR-hvKP showed higher virulence than ST11
ple are susceptible to hypervirulent Klebsiella pneumoniae.29 CR-hvKP, despite both being hypervirulent Klebsiella
However, other studies have showed that the elderly are pneumoniae. With the increase of the percentage of hyper­
susceptible to hypervirulent Klebsiella pneumoniae,24,28 virulent Klebsiella pneumoniae, the antimicrobial resis­
and the present study is consistent with that. tance rate of Klebsiella pneumoniae may decrease, which
Among the 16 isolates of CR-hvKP, except 1 isolate of will lead to blind optimism in clinical practice. Therefore,
ST23 CR-hvKP and 1 isolate of ST86 CR-hvKP, the other it is necessary to study the virulence of Klebsiella pneu­
14 isolates were all ST11 CR-hvKP. The capsular serotype moniae as well as its antimicrobial resistance.
corresponding to ST23 CR-hvKP is K1, and the capsular
serotype corresponding to ST86 CR-HVKP is K2, which
Ethics Approval and Informed
is similar to previous studies.30–32 Patients infected with
ST23 (K1) CR-hvKP or infected with ST86 (K2) CR- Consent
hvKP died, with a mortality rate (100%) significantly The medical ethics council of Shanghai Fifth People’s
higher than that of patients infected with ST11 CR-hvKP Hospital approved this study (Approval No. 104, 2020).
(50%). As shown in Figure 1, the virulence-associated The clinical samples were part of the routine hospital
gene positive rate of ST23 (K1) or ST86 (K2) isolates laboratory procedure.
were higher than that of ST11 (K64) isolates, and the
mortality rate might be related to the virulence-associated Acknowledgments
gene positive rate. In this study, 16 isolates of CR-hvKP We thank all the staff of the Central Laboratory of
all carried the carbapenemase gene blaKPC-2 and extended- Shanghai Fifth People’s Hospital for their technical gui­
spectrum β-lactamase gene blaSHV. blaKPC-2 is the most dance in this study.
prevalent carbapenemase gene in CR-hvKP in China.33 In
the study of Zhao, Y.et al,25 blaSHV is the extended-
Funding
spectrum β-lactamase gene with the highest positive rate.
This work was supported by the Minhang District Natural
The virulence genes entB, fimH, rmpA2, iutA, iucA were
Science Foundation of Shanghai (Grant No. 2020MHZ039).
present in all of 16 CR-hvKP isolates, followed by mrkD
(n=14), rmpA (n=13), aerobactin (n=2), allS (n=1), which
were similar to previous study.34 Some studies have shown Disclosure
that rmpA and rmpA2 (regulator of the mucoid phenotype The authors report no conflicts of interest in this work.
gene) can promote capsule polysaccharide secretion,
resulting in the hypermucoviscosity phenotype and
References
increase in virulence.35 Aerobactin is encoded by the
1. Navon-Venezia S, Kondratyeva K, Carattoli A. Klebsiella pneumo­
iucABCD gene, and its cognate receptor is encoded by niae: a major worldwide source and shuttle for antibiotic resistance.
the iutA gene, they had higher virulence levels in FEMS Microbiol Rev. 2017;41(3):252–275. doi:10.1093/femsre/
G. mellonella infection assays. allS is a marker for K1- fux013
2. Prokesch BC, TeKippe M, Kim J, et al. Primary osteomyelitis caused
ST23 and is not in pLVPK, which is a virulence plasmid by hypervirulent. Lancet Infect Dis. 2016;16(9):e190–e195.
from a K2 hypervirulent type. allS is a HTH-type tran­ doi:10.1016/S1473-3099(16)30021-4
3. Shon AS, Bajwa RPS, Russo TA. Hypervirulent (hypermucoviscous).
scriptional activator. These virulence genes were known to Klebsiella Pneumoniae Virulence. 2014;4(2):107–118. doi:10.4161/
contribute to virulence and are responsible for coloniza­ viru.22718
tion, invasion, and pathogenicity.36 4. Paczosa MK, Mecsas J. Klebsiella pneumoniae: going on the Offense
with a Strong Defense. Microbiol Mol Biol Rev. 2016;80(3):629–661.
doi:10.1128/MMBR.00078-15
Conclusion 5. Fang C, Chuang Y, Shun C, et al. A Novel Virulence Gene in
Klebsiella pneumoniae Strains Causing Primary Liver Abscess and
This study described the prevalence and molecular epide­ Septic Metastatic Complications. J Exp Med. 2004;199(5):697–705.
miological characteristics of CR-hvKP in Shanghai, China. doi:10.1084/jem.20030857

2704 https://doi.org/10.2147/IDR.S321704 Infection and Drug Resistance 2021:14

DovePress

Powered by TCPDF (www.tcpdf.org)

 Dovepress Zhou et al

6. Russo TA, Olson R, Fang CT, et al. Identification of Biomarkers for 22. Krapp F, Morris AR, Ozer EA, et al. Virulence Characteristics of
Differentiation of Hypervirulent Klebsiella pneumoniae from Carbapenem-Resistant Klebsiella pneumoniae Strains from Patients
Classical K. Pneumoniae J Clin Microbiol. 2018;56(9):e00776. with Necrotizing Skin and Soft Tissue Infections. Sci Rep. 2017;7(1).
7. Y C L, Cheng DL, Lin CL. Klebsiella pneumoniae liver abscess doi:10.1038/s41598-017-13524-8.
associated with septic endophthalmitis. Arch Intern Med. 1986;146 23. Yang Q, Jia X, Zhou M, et al. Emergence of ST11-K47 and
(10):1913–1916. doi:10.1001/archinte.1986.00360220057011 ST11-K64 hypervirulent carbapenem-resistant Klebsiella pneumo­
8. Chiu C, Lin D, Liaw Y. Metastatic Septic Endophthalmitis in niae in bacterial liver abscesses from China: a molecular, biological,
Pyogenic Liver Abscess. J Clin Gastroenterol. 1988;10(5):524–527. and epidemiological study. Emerg Microbes Infect. 2020;9
doi:10.1097/00004836-198810000-00009 (1):320–331. doi:10.1080/22221751.2020.1721334
9. Guo Y, Wang S, Zhan L, et al. Microbiological and Clinical 24. Liu C, Shi J, Guo J. High prevalence of hypervirulent Klebsiella
Characteristics of Hypermucoviscous Klebsiella pneumoniae pneumoniae infection in the genetic background of elderly patients in
Isolates Associated with Invasive Infections in China. Front Cell two teaching hospitals in China. Infect Drug Resist.
Infect Microbiol. 2017;7. 2018;11:1031–1041. doi:10.2147/IDR.S161075
10. Zhang Y, Zeng J, Liu W, et al. Emergence of a hypervirulent 25. Zhao Y, Zhang X, Torres VVL, et al. An Outbreak of
carbapenem-resistant Klebsiella pneumoniae isolate from clinical Carbapenem-Resistant and Hypervirulent Klebsiella pneumoniae in an
infections in China[J]. J Infection. 2015;71(5):553–560. Intensive Care Unit of a Major Teaching Hospital in Wenzhou, China.
doi:10.1016/j.jinf.2015.07.010 Front Public Health. 2019;7:229. doi:10.3389/fpubh.2019.00229
11. Gu D, Dong N, Zheng Z, et al. A fatal outbreak of ST11 26. Zhang R, Liu L, Zhou H, et al. Nationwide Surveillance of Clinical
carbapenem-resistant hypervirulent Klebsiella pneumoniae in Carbapenem-resistant Enterobacteriaceae (CRE) isolates in China.
a Chinese hospital: a molecular epidemiological study. Lancet Infect EBioMedicine. 2017;19(C):98–106. doi:10.1016/j.ebiom.2017.04.032
Dis. 2018;18(1):37–46. doi:10.1016/S1473-3099(17)30489-9 27. Zhou K, Xiao T, David S, et al. Novel Subclone of
12. Zhan L, Wang S, Guo Y, et al. Outbreak by Hypermucoviscous Carbapenem-Resistant Klebsiella pneumoniae Sequence Type 11
Klebsiella pneumoniae ST11 Isolates with Carbapenem Resistance with Enhanced Virulence and Transmissibility, China. Emerg Infect
in a Tertiary Hospital in China. Front Cell Infect Microbiol. 2017;7. Dis. 2020;26(2):289–297. doi:10.3201/eid2602.190594
13. F R E, Messai Y, Alouache S, et al. Virulence profiles and antibiotic 28. Liu C, Du P, Xiao N, et al. Hypervirulent Klebsiella pneumoniae is
susceptibility patterns of Klebsiella pneumoniae strains isolated from emerging as an increasingly prevalent K. pneumoniae pathotype
different clinical specimens[J]. Pathol Biol. 2013;61(5):209–216. responsible for nosocomial and healthcare-associated infections in
doi:10.1016/j.patbio.2012.10.004 Beijing, China. Virulence. 2020;11(1):1215–1224. doi:10.1080/
14. Turton JF, Perry C, Elgohari S, et al. PCR characterization and typing
21505594.2020.1809322
of Klebsiella pneumoniae using capsular type-specific, variable num­
29. Paczosa MK, Mecsas J. Klebsiella pneumoniae: going on the Offense
ber tandem repeat and virulence gene targets[J]. J Med Microbiol.
with a Strong Defense. Microbiol Mol Biol Rev. 2016;80(3):629–661.
2010;59(Pt 5):541–547. doi:10.1099/jmm.0.015198-0
30. Liu Z, Chu W, Li X, et al. Genomic Features and Virulence
15. Brisse S, Passet V, Haugaard AB, et al. wzi Gene Sequencing,
Characteristics of a Community-Acquired Bloodstream
a Rapid Method for Determination of Capsular Type for Klebsiella
Infection-Causing Hypervirulent Klebsiella pneumoniae ST86 Strain
Strains[J]. J Clin Microbiol. 2013;51(12):4073–4078. doi:10.1128/
Harboring KPC-2-Encoding IncX6 Plasmid. Microbial Drug Resist.
JCM.01924-13
2021;27(3):36–368. doi:10.1089/mdr.2019.0394
16. Ranjbar R, Tabatabaee A, Behzadi P, et al. Enterobacterial Repetitive
31. Li D, Liao W, Huang H, et al. Emergence of Hypervirulent
Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR)
Ceftazidime/Avibactam-Resistant Klebsiella pneumoniae Isolates in
Genotyping of Escherichia coli Strains Isolated from Different
a Chinese Tertiary Hospital. Infect Drug Resist. 2020;13:2673–2680.
Animal Stool Specimens[J]. Iran J Pathol. 2017;12(1):25–34.
doi:10.2147/IDR.S257477
doi:10.30699/ijp.2017.21506
32. Chen Y, Marimuthu K, Teo J, et al. Acquisition of Plasmid with
17. Yao B, Xiao X, Wang F, et al. Clinical and molecular characteristics
Carbapenem-Resistance Gene blaKPC2 in Hypervirulent Klebsiella
of multi-clone carbapenem-resistant hypervirulent (hypermucovis­
pneumoniae, Singapore. Emerg Infect Dis. 2020;26(3):549–559.
cous) Klebsiella pneumoniae isolates in a tertiary hospital in
doi:10.3201/eid2603.191230
Beijing, China. Int J Infect Dis. 2015;37:107–112. doi:10.1016/j.
33. Zhang Y, Wang Q, Yin Y, et al. Epidemiology of
ijid.2015.06.023
Carbapenem-Resistant Enterobacteriaceae Infections: report from
18. Solgi H, Shahcheraghi F, Bolourchi N, et al. Molecular characteriza­
tion of carbapenem-resistant serotype K1 hypervirulent Klebsiella the China CRE Network. Antimicrob Agents Chemother. 2018;62
pneumoniae ST11 harbouring blaNDM-1 and blaOXA-48 carbapene­ (2). doi:10.1128/AAC.01882-17.
mases in Iran. Microb Pathog. 2020;149:104507. doi:10.1016/j. 34. Xu M, Fu Y, Fang Y, et al. High prevalence of KPC-2-producing hyper­
micpath.2020.104507 virulent Klebsiella pneumoniae causing meningitis in Eastern China.
19. Li P, Liang Q, Liu W, et al. Convergence of carbapenem resistance and Infect Drug Resist. 2019;12:641–653. doi:10.2147/IDR.S191892
hypervirulence in a highly-transmissible ST11 clone of K. pneumoniae: 35. Liao W, De Wang L, Li D, et al. High Prevalence of 16s rRNA
an epidemiological, genomic and functional study. Virulence. 2021;12 Methylase Genes Among Carbapenem-Resistant Hypervirulent
(1):377–388. doi:10.1080/21505594.2020.1867468 Klebsiella pneumoniae Isolates in a Chinese Tertiary Hospital.
20. Zhang X, Ouyang J, He W, et al. Co-occurrence of Rapid Gene Gain Microb Drug Resist. 2021;27(1):44–52. doi:10.1089/mdr.2019.0482
and Loss in an Interhospital Outbreak of Carbapenem-Resistant 36. Rastegar S, Moradi M, Kalantar-Neyestanaki D, et al. Virulence
Hypervirulent ST11-K64 Klebsiella pneumoniae. Front Microbiol. Factors, Capsular Serotypes and Antimicrobial Resistance of
2020;11::579618. doi:10.3389/fmicb.2020.579618 Hypervirulent Klebsiella pneumoniae and Classical Klebsiella pneu­
21. Lin Z, Zheng J, Bai B, et al. Characteristics of Hypervirulent moniae in Southeast Iran. Infect Chemother. 2019;51. doi:10.3947/
Klebsiella pneumoniae: does Low Expression of rmpA Contribute ic.2019.0027
to the Absence of Hypervirulence? Front Microbiol. 2020;11.

Infection and Drug Resistance 2021:14 https://doi.org/10.2147/IDR.S321704

2705
DovePress

Powered by TCPDF (www.tcpdf.org)

 Zhou et al Dovepress

Infection and Drug Resistance Dovepress

Publish your work in this journal
Infection and Drug Resistance is an international, peer-reviewed open- antibiotic resistance and the mechanisms of resistance development and
access journal that focuses on the optimal treatment of infection diffusion in both hospitals and the community. The manuscript manage­
(bacterial, fungal and viral) and the development and institution of ment system is completely online and includes a very quick and fair peer-
preventive strategies to minimize the development and spread of resis­ review system, which is all easy to use. Visit http://www.dovepress.com/
tance. The journal is specifically concerned with the epidemiology of testimonials.php to read real quotes from published authors.

Submit your manuscript here: https://www.dovepress.com/infection-and-drug-resistance-journal

2706 DovePress Infection and Drug Resistance 2021:14

Powered by TCPDF (www.tcpdf.org)