Clinical Microbiology and Infection 27 (2021) 583e589

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Clinical Microbiology and Infection

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Original article

Detection of multiple hypervirulent Klebsiella pneumoniae strains in a

New York City hospital through screening of virulence genes
A.M. Parrott 1, *, J. Shi 1, J. Aaron 2, D.A. Green 1, S. Whittier 1, F. Wu 1
1)
Department of Pathology & Cell Biology, Columbia University Irving Medical Center, New York, NY, USA
2)
Department of Medicine, Division of Infectious Diseases, Columbia University Irving Medical Center, New York, NY, USA

a r t i c l e i n f o a b s t r a c t

Article history: Objectives: The ‘hypervirulent’ variant of Klebsiella pneumoniae (hvKp) is a predominant cause of
Received 25 November 2019 community-acquired pyogenic liver abscess in Asia, and is an emerging pathogen in Western countries.
Received in revised form hvKp infections have demonstrated ‘metastatic’ dissemination in immunocompetent hosts, an unusual
9 May 2020
mode of infection associated with severe complications. Two cases alerted us to the possible presence of
Accepted 13 May 2020
Available online 24 May 2020
hvKp at our hospital, both involving elderly Hispanic males who presented with recurrent fever, bac-
teraemia, epigastric pain and liver abscesses/phlegmon, thus prompting an assessment of hvKp
Editor: P.T. Tassios prevalence.
Methods: A surveillance of K. pneumoniae blood, body fluid and wound isolates was conducted using
Keywords: real-time PCR to detect virulence-associated genes (uni-rmpA, iucA and peg344). Positive isolates were
Aerobactin further characterized by wzi gene sequencing to determine capsular types (K-type) and by multilocus
Endemics sequence typing and pulsed-field gel electrophoresis to determine strain relatedness.
Epidemiology Results: Four-hundred and sixty-three K. pneumoniae isolates, derived from 412 blood, 21 body fluids and
Infection
30 abdominal wound specimens, were screened over a 3-year period. Isolates included 98 multidrug-
Klebsiella pneumoniae
resistant strains. Eighteen isolates from 17 patients, including two from the index patient, screened
Metastatic
RmpA protein positive for all three virulence genes. Sixteen of 18 positive isolates had K-types associated with hvKp,
Surveillance and isolates from different patients were unrelated strains, indicating likely community acquisition. Of 13
patients with significant morbidity, five died; eight patients had co-existing hepatobiliary disease, and
six had diabetes mellitus.
Conclusions: Multiple strains of hvKp are emerging in New York City and are associated with high
mortality relative to multidrug-resistant and classical Klebsiella infections. Co-existing hepatobiliary
disease appears to be a potential risk factor for these infections. A.M. Parrott, Clin Microbiol Infect
2021;27:583
© 2020 The Authors. Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and
Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction K. pneumoniae strains, and is termed ‘hypervirulent Klebsiella

From the mid-1980s, an increasing number of reports from the
Asian Pacific rim, in particular Taiwan, described a surge in
community-acquired liver abscesses [1]. The lesions were mono-
microbial and the responsible pathogen was identified as Klebsiella
pneumoniae. Defining clinical and phenotypic traits heralded the
emergence of a distinct variant [2] that has evolved from ‘classical’
* Corresponding author. Andrew M. Parrott, Columbia University Irving Medical
Center, Department of Pathology & Cell Biology, 630 West 168th Street, New York,
NY, 10032, USA.
E-mail address: ap3436@cumc.columbia.edu (A.M. Parrott).
pneumoniae’ (hvKp). Characteristic features include hyper-
mucoviscous colonies on agar [2], acquisition from the community,
infection of previously healthy hosts and a propensity for meta-
static dissemination to distant sites, leading to high morbidity and
mortality [3,4]. At present, hvKp is undergoing a global spread to
Western hemisphere [3e12]. Efforts to detect this emerging path-
ogen in low prevalence areas have been hampered by a lack of a
definitive test for its detection. Identification has historically been
reliant on unique clinical manifestations and phenotypic traits,
which are neither specific nor sensitive for hvKp [13]. Advances in
genomic analysis revealed that drug-resistant K. pneumoniae are
highly genetically diverse, frequently undergoing chromosomal

https://doi.org/10.1016/j.cmi.2020.05.012
1198-743X/© 2020 The Authors. Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases. This is an open access article under
the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
584 A.M. Parrott et al. / Clinical Microbiology and Infection 27 (2021) 583e589

Table 1
hvKp case demographics, clinical history, phenotypic and genotypic characteristics

Case Age/sex/ History Source Phenotypic PCR results wzi gene MLST
ethnicity results sequencing

String test CAS test crmpA prmpA prmpA2 Uni-rmpA iucA peg344 Allele K-types
(halo zone)

1 77/M/His Index case. Travelled to Peru 9/ Blood and POS NA N POS POS POS POS POS 115 K54 ST4057
2015. GORD, HTN, BPH. abscess
Presented with recurrent
hepatic abscesses from 1/12/
2015, CBD stricture, cholangitis
2 61/M/His Travelled to DR 8 years prior. Blood and POS þþþ N POS POS POS POS POS 72 K2 ST65
HBV, HLD. Presented with fever, wound
abdominal pain and
leucocytosis in 06/2015.
Hepatic phlegmon identified on
d1, s/p partial hepatectomy
3 88/F/As Choledocholithiasis, HTN. Blood POS þþþ N POS POS POS POS POS 1 K1 ST23
Presented in 01/2016 with
cholangitis and bacteraemia
4 74/F/Wh CAD, HLD, pancreatic Blood POS þþþ N POS POS POS POS POS 72 K2 ST375
adenocarcinoma. Presented in
1/2016 with septic shock
5 35/M/His DM2 (HbA1c 12.3). Presented in Blood POS þþ POS N N POS POS POS 5 K5 ST60
12/2015 with bacteraemia and
UTI
6 52/F/His Travelled to DR late 2015. HTN, Blood POS þþ N POS N POS POS POS 2 K2 ST4058
alcohol abuse, DM2 (HbA1c
10.8) and ampullary
adenocarcinoma s/p Whipple
02/2016. Re-admitted post
surgery with sepsis e died of
septic shock (K. pneumoniae,
E. faecalis, S. anginosus),
K. pneumoniae surgical wound
infection
7 61/M/Wh CHF, asthma, alcoholic Blood POS þþ N POS N POS POS POS 257 NT ST66
hepatitis. Presented 3/2016
with bacteraemia
8 60/M/His Alcoholic cirrhosis, refractory Ascites fluid POS þþþ N POS POS POS POS POS 72 K2 ST65
ascites, chronic pancreatitis,
CKD, CAD, HTN and DM2.
Admitted 4/2016 for worsening
ascites, peritonitis
9 4mo/F/Wh Delivered at ~37 weeks in- Blood, urine N þþ N POS POS POS POS POS 1 K1 ST23
house, with complex congenital and tracheal
heart disease, imperforate anus, aspirate
dysplastic kidney and chylous
effusion. Developed sepsis
DOL42 (blood and urine), with
recurrent infection on DOL65
(urine), and DOL96
(respiratory). Deceased DOL126
of cardiorespiratory failure
10 72/M/His Vascular dementia, CVA, Blood & N þþþ N POS POS POS POS POS 77 K57 ST218
seizures, HTN, HLD, drug abuse. bronchial
Presented 12/2016 with lavage
dyspnoea and chest pain.
Diagnosed with atrial
fibrillation and collapsed left
lower lung. Developed lobar
pneumonia and sepsis on d3
11 94/F/Wh CAD, aortic stenosis s/p TAVR, Blood N þþ N POS POS POS POS POS 2 K2 ST86
COPD, CHF. Presented 7/2017
with respiratory failure and
septic shock, likely due to
pneumonia e deceased d3
12 52/M/His HTN, alcohol abuse and Blood POS þ N POS N POS POS POS 2 K2 ST3690
depression. Presented 9/2017
with intoxication, chest pain,
vomiting, fever and headache
due to trauma. Found to be in
septic shock, likely due to
pneumonia
 A.M. Parrott et al. / Clinical Microbiology and Infection 27 (2021) 583e589 585

Table 1 (continued )

Case Age/sex/ History Source Phenotypic PCR results wzi gene MLST
ethnicity results sequencing

String test CAS test crmpA prmpA prmpA2 Uni-rmpA iucA peg344 Allele K-types
(halo zone)

13 68/M/AA CKD, HIV, DVT s/p inferior vena Blood N þþ N POS POS POS POS POS 1 K1 ST23
cava filter. Presented 9/2017
with fever in setting of
antiretroviral non-compliance
(CD4 T cell 2 cells/mL; HIV RNA
32648 copies/mL). Chest
cavitary lesions and
polymicrobial bacteraemia
(K. pneumoniae and E. faecalis)
on admission
14 61/M/His CVA, HTN, HLD, DM2, CAD, BPH. Blood POS N N POS POS POS POS POS 2 K2 ST86
Presented 11/2017 with chest
pain and septic shock, likely
due to urosepsis e deceased d1
15 84/M/Wh CVA, DM2 (HbA1c 7.1), Blood N þþþ N POS POS POS POS POS 1 K1 ST680
dementia. Presented with
emaciation, AMS, UTI (E. coli)
and dysphagia 4/2018. Became
tachypnoeic on d16, likely due
to aspiration, resulting in septic
shock (K. pneumoniae) e
deceased d17
16 56/F/His Visited Ecuador 2 years prior. Blood and N þþ N POS POS POS POS POS 1 K1 ST23
DM2 (HbA1c 9.9), HTN. abscess
Presented with fever, nausea,
chills and fatigue 6/2018.
Hepatic abscess identified on d2
17 65/F/As Small bowel myopathy and Small bowel POS N N POS POS POS POS POS 503 NT ST4582
motility disorder s/p multiple aspirate
resections for obstruction,
chronic anaemia. Outpatient
endoscopy 5/2018

All cases were PCR positive for peg344, iucA and rmpA genes. Bold denotes isolate tested in this study. AA, African-American; As, Asian; His, Hispanic; Wh, White; AMS, altered
mental status; BPH, benign prostatic hyperplasia; CAD, coronary artery disease; CBD, common biliary duct; CHF, congestive heart failure; CKD, chronic kidney disease; COPD,
chronic obstructive pulmonary disease; CVA, cerebrovascular accident; d, days post admission; DM2, diabetes mellitus type 2; DOL, day of life; DR, Dominican Republic; DVT,
deep vein thrombosis; GORD, gastro-oesophageal reflux disease; HbA1c, haemoglobin A1c; HBV, hepatitis B virus; HIV, human immunodeficiency virus; HLD, hyper-
lipidaemia; HTn, hypertension; s/p, status post; TAVR, transcatheter aortic valve replacement; UTI, urinary tract infection; MLST, multi-locus sequence typing. POS, positive; N,
negative; þþ/þþþ halo zone >10 mm; þ halo zone <8 mm; NA, not available for the test; NT, non-typeable.

recombination and plasmid acquisition, whereas hvKp strains
show lower gene content diversity and limited gene acquisition
[14]. Individual strains vary in their virulence potential due to the
presence of mobile accessory genes. The majority of K. pneumoniae
clones found to be hypervirulent carry virulence plasmids encoding
aerobactin siderophore biosynthesis (iucA), regulator of mucoid
phenotype A (rmpA) [15], and the putative metabolite transporter
peg344 [16]. The three genotypic markers, iucA, rmpA and peg344
loci are usually collocated on a large virulence plasmid, although
genetic diversity of the plasmid has been observed [17]. Clinical
research has increasingly employed genotypic markers to quickly
screen for key virulence genes as a more reliable identifier of
hypervirulence [18].
Two cases alerted us to the possible presence of hvKp at our
hospital. The index case (Case 1, Table 1) involved a 78-year-old
Hispanic male who presented to our hospital on six different oc-
casions in 2015 with recurrent fever, bacteraemia, epigastric pain,
liver abscesses and cholangitis. In the second case (Case 2), a 61-
year-old Hispanic male presented with fever, bacteraemia and
liver phlegmon, necessitating partial right hepatectomy. Identifi-
cation of the virulence genes in the pathogenic strains and capsule
K-typing studies confirmed hvKp infection in both cases. To assess
hvKp prevalence, we conducted surveillance of K. pneumoniae
isolates collected over a 3-year period at our hospital, based in
New York City, a sentinel city for the introduction of emerging
pathogens [19].
Materials and methods
Patients and specimens
A total of 463 K. pneumoniae isolates were recovered from 462
critically ill patients between 30 November 2015 and 10 August
2018, including 412 blood cultures, 30 abdominal wounds and 21
sterile body fluid cultures (Table S1). Each bacterial isolate was
collected from a separate individual except two isolates from the
index case (Table S1).
Bacterial identification and antimicrobial susceptibility testing
All isolates were identified to the species level with matrix-
assisted laser desorption/ionization time-of-flight mass spectrom-
etry (Bruker Daltonics, Billerica, MA). Antimicrobial susceptibility
was determined by MicroScan WalkAway with Neg MIC 42 Panel
(Beckman Coulter, Atlanta, GA) for the following agents: amikacin,
ampicillinesulbactam, aztreonam, cefepime, cefoxitin, ceftazidime,
ceftriaxone, gentamicin, levofloxacin, meropenem, piperacillin-
tazobactam, tobramycin and trimethoprimesulfamethoxazole.
Phenotypic assays
All isolates were tested for the hypermucoviscous phenotype
by the ‘String test’, the formation of a viscous string >5 mm in
586 A.M. Parrott et al. / Clinical Microbiology and Infection 27 (2021) 583e589
length when fresh bacterial colonies are stretched by an inocu-
lation loop [2]. To demonstrate excessive production of side-
rophore by hvKp, the 17 iucA (aerobactin) positive strains and ten
randomly selected iucA negative strains underwent testing by a
qualitative plate siderophore production assay. Kings B agar plates
containing chromeazurol S dye (CAS) were prepared as detailed in
Louden et al. [20]. Fresh K. pneumoniae isolates were spotted onto
the media, incubated at 37 C and evaluated after 48 hr. The
diameter of the yellow to light orange halo surrounding the col-
ony, which is indicative of siderophore production, was measured
(Fig. S1).
Molecular methods
All isolates were screened by qPCR targeting iucA [15], rmpA and
peg344 [16] virulence genes. Initially, we used uni-rmpA primers
(Table S2) that target the homologous region of all variant rmpA
genes reported. Uni-rmpA-positive strains were further genotyped
for plasmid derived prmpA or prmpA2 and chromosomal derived
crmpA, allowing investigation of rmpA genotype with capsular
type and virulence [21]. For locus-based typing, bacterial DNA was
amplified using wzi [22] and seven K. pneumoniae multilocus
sequence typing (MLST) primers [23] (please see supplementary
material for further details).
Risk and outcome assessment
A patient's type 2 diabetes status was defined as positive by
prior clinical history or a haemoglobin A1c measurement 6.5%.
Ethnicity was determined by self-declared patient questionnaire
or clinical notes. The presence of sepsis was determined by
clinical documentation. Hepatobiliary disease was defined by
imaging studies or prior clinical history. K. pneumoniae-related
mortality was defined as death within 30 days of a positive
K. pneumoniae culture. A value of p  0.05 was considered sta-
tistically significant.
Ethics approval
This retrospective study was approved by the Columbia Uni-
versity Institutional Review Board (protocol number AAAS5378).
Results
Virulence gene screening
A retrospective screening of the three virulence genes (uni-
rmpA, iucA and peg344) led to the identification of 18 hvKp strains
from 17 patients, including two from the index patient (Table 1). Of
462 invasive K. pneumoniae infection cases, 17 patients were
infected by a hypervirulent strain (3.7%). All triple virulence gene
positive (trigenic positive) isolates were susceptible to all tested
antibiotics. The remaining isolates, including all multidrug-
resistant (MDR) isolates, screened negative for all three bio-
markers, with the exception of a single specimen derived from a
perihepatic abscess that tested positive for rmpA alone. Seventeen
uni-rmpA-positive strains were further differentiated; 16 isolates
were derived from plasmids including 13 isolates positive for both
prmpA and prmpA2, and three isolates positive for only prmpA. Only
one strain carried the virulence gene derived from chromosomal
crmpA (Table 1). There were no apparent correlations between
rmpA origin and phenotypic characteristics or genotype.
Phenotypic characteristics
Of the 463 K. pneumoniae isolates, 98/463 (21.2%) were MDR as
defined by Magiorakos et al. [24], 33/463 (7.1%) displayed a
hypermucoviscous phenotype and were String test positive
(Table S1), including 12 among the 18 trigenic positive isolates
(66.7%). The qualitative siderophore production assay showed
siderophore-indicative halo production of hvKp strains on blue
agar plates (Fig. S1). Twelve of the 16 (75%) trigenic-positive iso-
lates tested produced yellow-orange halo zone >10 mm (þþ/þþþ),
indicating a high level siderophore production, while the ten
trigenic-negative and the remaining four trigenic-positive isolates
showed none or <8 mm (þ) halo zone (Table 1).
Genotypic characteristics
Capsule K-typing by wzi locus sequencing identified seven iso-
lates to be genotype K2, five isolates to be genotype K1, the index
case to be K54 and the remaining isolates to be K5, K57 and two
undetermined genotypes (Table 1). Capsule K-types K1 and K2
account for the majority of hvKp isolates (70.6%) in this study and
previously reported [25], but hvKp strains with K5, K54 and K57
genotypes have also been documented [26]. Of note, K2 and K54
isolates that were negative for both hypermucoviscosity and
peg344/iucA/rmpA genes were identified, with the ‘negative’ K54
isolate having an identical wzi allele to the index case (Table S1).
These results demonstrate capsule serotyping alone is insufficient
to identify hvKp strains [27]. Further analysis of the positive isolates
by MLST sequencing revealed four out of five of the K1 genotype
isolates to be sequence type ST23, the archetypal hvKp clone of the
K1 genotype (Table 1; [25]). K2 genotypes were identified as ST65,
ST86, ST375 and ST3690, which have also been previously reported
as hvKp clonal groups (Table 1; [28]). Three new MLST genotypes,
ST4057, ST4058 and ST4582, were recognized in this study (Table 1)
and deposited into the database http://www.pasteur.fr/mlst/.
Hence, the chosen trigenic PCR screening methodology correctly
identified hvKp pathotype. Analysis of incidence of hvKp infections
over the collection period reveals a largely uniform distribution,
without clustering, and incidence of the different genotypes was
scattered chronologically, making a strain-specific ‘outbreak’ un-
likely (Fig. S2). Pulsed-field gel electrophoresis (PFGE) typing
established 17 hvKp isolates to be distinct clones according to the
interpretive criteria of Tenover et al. [29]. Interestingly, strains
carrying the same sequence type, i.e. two strains of ST86, two
strains of ST65 and three strains of ST23 shared at least 70% simi-
larity, but also classify as distinct clones (Fig. 1). Furthermore, a
blood isolate collected in January 2015 and an abscess isolate
collected in December 2015 from the index case showed indistin-
guishable PFGE patterns (Fig. 1), indicating a failure to eradicate the
pathogen despite multiple courses of antibiotics.
Clinical manifestations
Our hospital serves a diverse ethnic population which includes
recent and established immigrants. A good approximation to the
ethnic diversity of the population can be gleaned from Fig. 2A,
which details the ethnicity of a large sample set of patients infected
with cKp and MDR. However, there are no recent immigrants in our
cohort of hvKp-infected patients, all are long term residents in the
United States. Details of their most recent travel (if any) are noted in
Table 1. Hypervirulent infection was most frequently seen in pa-
tients of Hispanic ethnicity (nine Hispanic, five White, one African-
American, two Asian), but this apparent bias was statistically
insignificant compared with the ethnic compositions of cKp and
MDR infections (p < 0.1; Fig. 2A). All K. pneumoniae infections had
 A.M. Parrott et al. / Clinical Microbiology and Infection 27 (2021) 583e589 587

Fig. 1. Pulsed-field gel electrophoresis (PFGE) profiles of Klebsiella pneumoniae isolates were generated using the XbaI restriction enzyme digestion, and dendrograms were obtained
using Dice similarity coefficient and UPGMA. Eighteen hypervirulent K. pneumoniae isolates and two reference strains (ATCC 35593 and ATCC 43816) were included. C (1, 2, 3 …):
Case number (1, 2, 3 …) according to Table 1. ST, sequence type; B, blood; W, wound.

an apparent bias toward male predominance (Fig. 2A) and to affect
older adults (Fig. 2B). Accordingly, patients infected with hvKp
showed statistically insignificant apparent gender bias (ten male,
seven female) and ranged in age from 4 months to 94 years
(average age 62.4 years, Fig. 2B).
Review of the clinical record in Table 1, determined at least eight
patients had co-existing hepatobiliary disease (47.1%; Cases 1e4,
6e8 and 16), including three patients with hepatic abscess/phleg-
mon (Cases 1, 2 and 16). Six patients had a history of diabetes
(35.3%; Cases 5, 6, 8 and 14e16), a previously reported risk factor for
hvKp infection [1,7,30]. Most patients (15 of 17) had acquired hvKp
infection prior to admission (isolates were recovered <72 hr from
admission), and, to our knowledge, none had recently travelled to a
known hvKp endemic area, such as Asia or South America, indi-
cating a local community-acquired source. Examples of hvKp
morbidity included peritonitis (Case 8), hepatic abscess/phlegmon
(Cases 1, 2 and 16), cavitary pulmonary lesions (Case 13), recurrent
multisite infections (Case 9) and at least 11 cases of sepsis (Cases 4,
6, 10e12, 14 and 15). Five patients infected with hvKp died within
30 days of a positive K. pneumoniae culture (29.4%; n ¼ 5/17), three
as a direct consequence of hvKp sepsis (Cases 11, 14 and 15), one
due to polymicrobial sepsis that included concurrent hvKp wound
infection (Case 6), and one due to heart failure following recurrent
hvKp infection (Case 9). In comparison, classical (cKp) and MDR

Fig. 2. Patient demographics. Data are compiled from Table S1. (A) Ethnicity and gender of patients infected with multidrug resistant (MDR), classical (cKp), and hypervirulent
(hvKp) strains of Klebsiella pneumoniae. Odds ratios (OR) and p values are given for comparison of patients that identify as Hispanic, that were infected with hvKp versus cKp or
MDR. (B) Mean age of patients in years. Standard deviation bars are shown.
588 A.M. Parrott et al. / Clinical Microbiology and Infection 27 (2021) 583e589
K. pneumoniae infections in this study were associated with 65
(18.7%, n ¼ 65/347) and 23 (23.5%, n ¼ 23/98) deaths, respectively
(Fig. 3 and Table S1).
Discussion
This study confirmed the findings of Russo and colleagues, who
demonstrated that certain virulence genes have high specificity and
sensitivity for the identification of hvKp strains in areas of low
prevalence [18]. Our study employs the PCR amplification of three
such virulence genes (rmpA, iucA and peg344), and similarly finds
these biomarkers to be superior to culture-based phenotypic
methods in the detection of hvKp isolates. The remaining
K. pneumoniae isolates, including all MDR isolates, screened nega-
tive for all three biomarkers, consistent with the known current
restricted distribution of hvKp drug-resistant strains to the Far East
[14,31]. There are case-based studies of hvKp infection in the
United States [3e10,32,33], but to our knowledge only three North
American surveillance studies using genotypic biomarkers have
been reported: the first study collected data from 2001 through
2007 in Calgary, Canada [11], the second study was from 2009
through 2010 in Houston, Texas [12], and the third study conducted
from 2009 through 2013 in Montreal, Canada [18]; they reported
prevalence of 7.5% (n ¼ 10/134), 6.3% (n ¼ 4/64) and 0.9% (n ¼ 1/
110), respectively. These studies analysed K. pneumoniae blood
isolates but did not elaborate on their antimicrobial susceptibility.
The prevalence estimate in the first study was based on String test
positivity, thus may have overestimated hypervirulence (see dis-
cussion below). The second study relied on detection of two viru-
lence biomarkers, rmpA and wzy genotype K1 (MagA), but was
limited by a small sample set [12]. In contrast, the present study
utilized three virulence biomarkers and identified 17 hvKp strains
from 462 patients (3.7% prevalence) over a 2 year and 9 month
period, which is considerably increased from the previous estimate
of 0.9% established from specimens collected over 5 years ago [18].
Our study also employed a classical phenotypic measure of
hypermucoviscosity, the String test. This test was positive in only
Fig. 3. Patient risk factors. Data is derived from Table S1. Mortality is defined as death within 30 days of a positive Klebsiella pneumoniae culture. Odds ratios (OR) and p values are
given for pertinent results.
two-thirds of rmpA/iucA/peg344 gene-positive isolates (n ¼ 12/18),
hence in our hands, this test had a sensitivity of 66.7% and a
specificity of 95.2%. Russo and colleagues recently concluded that
the String test has inferior accuracy, sensitivity and specificity for
hvKp detection when compared to several virulence plasmid bio-
markers [18]. This study supports those claims, and perhaps the
String test would best serve as a negative predictive value (NPV)
test in areas of low prevalence (NPV ¼ 98.6% in this study).
Siderophore production is a major factor in the ability of path-
ogens to cause disease. HvKp strains have the capability to produce
multiple types of siderophores including enterobactin, salmochelin,
yersiniabactin and aerobactin. Although the CAS assay is a universal
colorimetric method that detects siderophores independent of
their type, aerobactin has been shown to account for >80e90% of
total siderophore production under iron-limiting conditions [34]. In
this study, 75% of the trigenic positive isolates produced high levels
of siderophore, without any false positives. Therefore, siderophore
production may serve as a hvKp confirmatory test in the clinical
laboratory setting.
Septic metastases are associated with hvKp, and have prompted
case studies [3,4] or have been found in specimens enriched for
hvKp, such as in liver abscess studies, where they occur in a mi-
nority of patients (10e12% [30,35]). However, septic metastatic foci
were not identified in our patients. We surmise that patients with
hvKp infection may not always develop metastatic disease, or that
metastatic disease may be a result of prolonged infection, indi-
cating a potential window period for intervention. Nevertheless,
this study highlights the high disease burden of hvKp infection and
its lethality: five patients (29.4%) infected with hvKp died within
30 days of its detection, suggesting increased mortality relative to
classical (19.0%) and even MDR (23.5%) K. pneumoniae infections.
Although it is established that patients with diabetes are at
increased risk of hvKp infection [1,7,30], this study found that
diabetic patients (and those with sepsis) are at equal risk of cKp,
MDR or hvKp infection (Fig. 3). Interestingly, patients with co-
existing hepatobiliary disease had a statistically higher risk of
having hvKp infection than those with MDR (p < 0.05) infections
 A.M. Parrott et al. / Clinical Microbiology and Infection 27 (2021) 583e589
(Fig. 3). This latter finding may be pertinent to future screening
studies aimed at the early detection of hvKp disease and its time-
frame of pathogenesis.
Through the techniques of virulence gene PCR, PFGE and
sequence-based genotyping coupled with the clinical record, we
concluded that patients acquired unique hvKp clones (Fig. 1) from
the community. Hence multiple clones of the hvKp pathotype are
now established in New York City. New York City remains one of the
foremost ports of entry into the United States for immigrant pop-
ulations, and as such, represents a sentinel city for foreign patho-
gens. Although this study is limited in scope, and derives results
from a single centre, its findings show the successful introduction
of an invasive pathogen into a North American city, highlighting a
pressing need for further epidemiological studies of hvKp in similar
settings.
Transparency declaration
The authors declare that they have no conflicts of interest. No
external funding was received.
Author contributions
Conceptualization: A.P./F.W. equal. Methodology: F.W. (lead),
A.P. (supporting). Validation: J.S. (lead), A.P./F.W. (supporting).
Formal analysis: A.P./F.W. equal. Investigation: J.S. (lead), A.P./F.W.
(supporting). Resources: F.W. (lead), S.W. (supporting). Data cura-
tion: A.P./F.W. equal, J.S. (supporting). Writingdoriginal draft: A.P.
Writingdreview and editing: A.P./F.W. (lead), D.G./JA./S.W. (sup-
porting). Visualization: A.P. (lead), F.W. (supporting). Supervision:
F.W. Project Administration: F.W. (lead), A.P. (supporting). Funding
acquisition: F.W. (lead), S.W. (supporting).
Acknowledgement
We would like to thank Dr Thomas Russo and team, Department
of Medicine, University at Buffalo, who gave us technical assistance
for the siderophore production assay.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.cmi.2020.05.012.
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