Colistin Heteroresistance in Klebsiella Pneumoniae Isolates and Diverse Mutations of PmrAB and PhoPQ in Resistant Subpopulations

Abstract

Heteroresistance may pose a threat to the prognosis of patients following colistin treatment. We investigated colistin heteroresistance in Klebsiella pneumoniae isolates from South Korea. Among 252 K. pneumoniae blood isolates, 231 were susceptible to polymyxins. Heteroresistance to colistin was determined using population analysis profiles, disk diffusion assays, and E-test strip tests for the susceptible isolates. As a result, we identified three colistin-heteroresistant K. pneumoniae isolates belonging to separate clones (ST11, ST461, and ST3217) by multilocus sequence typing analysis. Two colistin-resistant subpopulations were selected from each heteroresistant isolate in either disk diffusion testing or E-testing. Two resistant subpopulations from the same isolate exhibited different amino acid substitutions in the two-component regulatory systems PmrAB and PhoPQ. An in vitro time–kill assay showed that meropenem combined with colistin had a 1× minimum inhibitory concentration bactericidal effect against a multidrug-resistant, colistin-heteroresistant isolate.

Full text

Journal of
Clinical Medicine
Article
Colistin Heteroresistance in Klebsiella Pneumoniae
Isolates and Diverse Mutations of PmrAB and
PhoPQ in Resistant Subpopulations
Hae Suk Cheong 1, †, So Yeon Kim 2,†, Yu Mi Wi 3, Kyong Ran Peck 4and Kwan Soo Ko 2, *
1Division of Infectious Disease, Department of Internal Medicine, Kangbuk Samsung Hospital,
Sungkyunkwan University School of Medicine, Seoul 03181, Korea
2
Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 16419, Korea
3Division of Infectious Diseases, Samsung Changwon Hospital,
Sungkyunkwan University School of Medicine, Changwon 51353, Korea
4Division of Infectious Disease, Samsung Medical Center, Sungkyunkwan University School of Medicine,
Seoul 06351, Korea
*Correspondence: ksko@skku.edu; Tel.: +82-31-299-6223; Fax: +82-31-299-6229
†Cheong H.S. and Kim S.Y. contributed equally to this work.
Received: 22 August 2019; Accepted: 9 September 2019; Published: 11 September 2019


Abstract:
Heteroresistance may pose a threat to the prognosis of patients following colistin treatment.
We investigated colistin heteroresistance in Klebsiella pneumoniae isolates from South Korea. Among 252
K. pneumoniae blood isolates, 231 were susceptible to polymyxins. Heteroresistance to colistin was
determined using population analysis profiles, disk diffusion assays, and E-test strip tests for the
susceptible isolates. As a result, we identified three colistin-heteroresistant K. pneumoniae isolates
belonging to separate clones (ST11, ST461, and ST3217) by multilocus sequence typing analysis.
Two colistin-resistant subpopulations were selected from each heteroresistant isolate in either disk
diffusion testing or E-testing. Two resistant subpopulations from the same isolate exhibited different
amino acid substitutions in the two-component regulatory systems PmrAB and PhoPQ. An
in vitro
time–kill assay showed that meropenem combined with colistin had a 1
×
minimum inhibitory
concentration bactericidal effect against a multidrug-resistant, colistin-heteroresistant isolate.
Keywords:
Klebsiella pneumoniae; colistin; heteroresistance; population analysis profiles;
PmrAB; PhoPQ
1. Introduction
Klebsiella pneumoniae is one of the most clinically significant pathogens belonging to the
family Enterobacteriaceae. It is an important pathogen in community- and hospital-acquired
infections [
1
]. Carbapenems have been used for infections caused by extended-spectrum
β
-lactamase
(ESBL)-producing K. pneumoniae, and carbapenem-resistant K. pneumoniae first emerged in 1985 [
2
].
The prevalence of carbapenem-resistant K. pneumoniae has continued to increase globally, and such
infections pose a critical threat to human health, with high mortality rates owing to the limited
treatment options available [
3
–
5
]. Colistin and polymyxin B are important therapeutic options for
treating infections caused by carbapenem-resistant K. pneumoniae [6,7].
Colistin exerts bactericidal action against Gram-negative pathogens, targeting the lipid A moiety
of lipopolysaccharide (LPS) and leading to cell membrane disruption [
8
]. Unfortunately, colistin
resistance has been reported in surveillance studies as well as in clinical case reports [
9
]. Resistance to
colistin generally involves mutations in chromosomal genes. Acquired resistance to polymyxins in
J. Clin. Med. 2019,8, 1444; doi:10.3390/jcm8091444 www.mdpi.com/journal/jcm

J. Clin. Med. 2019,8, 1444 2 of 9
strains such as K. pneumoniae and Escherichia coli involves mutations in the two-component regulatory
systems PmrAB and PhoPQ or alterations to the negative regulator of PhoPQ, MgrB [10,11].
Antibiotic heteroresistance is a phenomenon where subpopulations of seemingly isogenic bacteria
exhibit a range of susceptibilities to a particular antibiotic [
12
]. It has only been reported in
a limited number of studies because it cannot be assessed using ordinary minimum inhibitory
concentration (MIC) testing methods. There have been many studies on the heteroresistance to various
antibiotics for specific bacterial species, which also encompass colistin heteroresistance in K. pneumoniae
isolates [13–18]. Although little is known about the clinical significance of antibiotic heteroresistance,
instances of treatment failure associated with colistin heteroresistance in K. pneumoniae have been
reported [19]. In addition, clinicians may be prompted to investigate the most efficient use of colistin
on multidrug-resistant K. pneumoniae, including antibiotic combinations [20].
In this study, we investigated the incidence rate and genomic variation of colistin heteroresistance in
K. pneumoniae blood isolates and examined the
in vitro
efficacy of colistin and meropenem combination
treatment against colistin-heteroresistant K. pneumoniae.
2. Materials and Methods
2.1. Bacterial Strains and Antibiotic Susceptibility Testing
In total, 252 nonduplicated K. pneumoniae blood isolates were collected from January to December
2017 from Samsung Medical Center (Seoul, Korea). Species identification was performed using
a VITEK-2 system (BioMérieux, Hazelwood, MO, USA).
In vitro
antimicrobial susceptibility testing was performed using the broth microdilution method
outlined in the Clinical and Laboratory Standards Institute (CLSI) guidelines [
21
]. For all isolates,
the MICs of four antibiotic agents—imipenem and meropenem (carbapenems) as well as colistin
and polymyxin B (polymyxins)—were determined. The MICs of seven other antibiotics (cefotaxime,
ceftazidime, cefepime, ciprofloxacin, amikacin, tigecycline, and piperacillin–tazobactam) were also
determined for three colistin-heteroresistant isolates and their resistant subpopulations. Most of the
antibiotics, except tigecycline, were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA),
and tigecycline (Tygacil
®
Injection) was provided from Pfizer (Korea). E. coli ATCC 25922 and
Pseudomonas aeruginosa ATCC27853 were employed as quality control strains.
2.2. Detection of Colistin-Heteroresistant Isolates
Colistin heteroresistance was detected using a colistin disk diffusion assay (BD BBL
™
Sensi-Disc
™
antimicrobial susceptibility test disks, colistin 10
µ
g) or a colistin E-test strip (bioM
é
rieux SA, France)
on Mueller–Hinton agar (Difco BBL, USA) plates, where colonies were observed within the clear zone
of inhibition. Subpopulations were separated by subculture, and their colistin MIC was assessed by
the broth microdilution method and interpreted according to CLSI guidelines [
21
]. To confirm the
presence of colistin heteroresistance, population analysis profiles (PAPs) were obtained. Full 24 h
cultures (~10
8
CFU/mL) were employed. Bacterial cell suspension samples (50
µ
L) (corresponding
to a 0.5 McFarland standard for K. pneumoniae cultures) were plated on Mueller–Hinton agar plates
containing 0, 0.5, 1, 2, 4, 6, 8, or 10 mg/L of colistin sulfate (Sigma-Aldrich, St. Louis, MO, USA).
After 24 h of incubation at 37
◦
C, the number of colonies were counted. Colistin heteroresistance
was defined as the presence of a colistin-susceptible isolate in which the detectable colistin-resistant
subpopulations were able to grow in the presence of
≥
10 mg/L of colistin sulfate. The detection limit of
colistin-resistant subpopulations was 20 CFU/mL and the lower limit of quantification (LOQ) was 400
CFU/mL (i.e., 2.6 log10 CFU/mL) [16].
2.3. Genotyping and Sequence Analysis of Genes Associated with Colistin Resistance
Multilocus sequence typing (MLST) was performed for three heteroresistant isolates and
their resistant subpopulations using a previously described protocol (www.pasteur.fr/recherche/

J. Clin. Med. 2019,8, 1444 3 of 9
genopole/PF8/mlstKpneumoniae.html) [
22
]. Genomic DNAs were isolated from overnight cultures
in Luria–Bertani agar at 37
◦
C using the G-spin
™
genomic DNA extraction kit for bacteria G-spin
™
Genomic DNA Extraction Mini Kit (for Bacteria)G-spin
™
Genomic DNA Extraction Mini Kit (for
Bacteria) (iNtRON Biotechnology, Korea).
Polymerase chain reaction (PCR) and DNA sequencing were performed to identify nucleotide
and resultant amino acid alterations in PhoPQ, PmrAB, and MgrB of parental colistin-heteroresistant
isolates and their resistant subpopulations [
23
]. The presence of the mcr-1 gene was investigated by
PCR [24].
2.4. Time–Kill Assays
We examined the time–kill kinetics of colistin and/or meropenem against a colistin-heteroresistant
K. pneumoniae blood isolate (S1703-112), which is also multidrug-resistant. Colistin was added to
a logarithmic-phase broth culture of approximately 10
6
CFU/mL to yield concentrations that were 0-,
0.25-, 1-, and 4-fold of the MIC. Samples were collected at 0, 4, 8, 12, 16, 20, and 24 h after adding
antibiotics, and a viable cell count was performed by spirally plating the bacterial cell suspension
on Mueller–Hinton agar plates after appropriate dilutions. Time–kill curves were constructed by
plotting mean colony counts (log
10
CFU/mL) versus time. Bactericidal activity was defined as a
≥
3
log
10
CFU/mL reduction in the total CFU/mL from the original inoculum [
25
]. Synergy was defined as
a≥2 log10 CFU/mL decrease between the combination and the most efficient agent alone at 24 h [26].
3. Results
Among 252 K. pneumoniae blood isolates, 13 and 12 isolates (5.1% and 4.7%) were resistant to
colistin and polymyxin B (MICs, >4 mg/L), respectively. Eight and nine isolates (3.2% and 3.6%)
showed intermediate resistance toward colistin and polymyxin B. The others (231 isolates, 91.7%)
were susceptible to both colistin and polymyxin B. Only three and one isolates were resistant to
meropenem and imipenem, respectively, whereas nine and three isolates exhibited intermediate
resistance. The others were susceptible to meropenem and imipenem (240 and 248; 95.2% and 98.4%,
respectively).
We assayed colistin heteroresistance for 231 susceptible K. pneumoniae isolates. As a result,
we identified three isolates (1.3%) being heteroresistant to colistin using a disk diffusion test or E-test.
They showed typical bactericidal patterns in population analysis profiling (Figure 1A). For each isolate,
we obtained two colonies growing within the zone of inhibition in disk diffusion or E-test (Figure 1B).
They were separated by subculture and were named RP1 and RP2 after the isolate number. All the
colistin-resistant subpopulations of the three heteroresistant isolates showed colistin MICs of
≥
64
µ
g/mL, whereas the colistin MICs of parental K. pneumoniae isolates were 0.25 or 1 mg/L (Table 1).
The colistin MICs of
≥
64
µ
g/mL in the resistant subpopulations persevered after serial subculture in
colistin-free media, indicating their stable feature of colistin resistance. The MICs of the other antibiotics
tested in this study were not significantly different, except for cefepime and tigecycline in some resistant
subpopulations (Table 1). Particularly, the isolate S1703-112 was nonsusceptible to most antibiotics
except gentamicin and tigecycline. Thus, we selected this isolate for time–kill assays to investigate
the efficacy of a combination of meropenem and colistin. The isolate S1703-112 produced CTX-M-15,
an ESBL. According to MLST analysis, the three colistin-heteroresistant K. pneumoniae blood isolates
belonged to different clones—ST3217 (S1703-35), ST461 (S1703-109), and ST11 (S1703-112)—which
were clones not be strictly associated with colistin resistance. The resistant subpopulations showed the
same STs as those of their parental isolates. All isolates were negative for the mcr-1 gene.

J. Clin. Med. 2019,8, 1444 4 of 9
Figure 1.
(
A
) Population analysis profiles of three colistin-heteroresistant Klebsiella pneumoniae blood
isolates and Escherichia coli ATCC 25922. LOQ, limit of quantification. The three isolates—S1703-35,
S1703-109, and S1703-112—grew in the presence of colistin at concentrations of 4–10 mg/L. (
B
) The
results of disk diffusion test or E-test. Resistant subpopulations (each two in three isolates) analyzed
further are indicated; 35-RP, 109-RP, and 112-RP indicate the resistant subpopulations of S1703-35,
S1703-109, and S1703-112, respectively. For S1703-35, no resistant colonies were detected in the E-test;
thus, we selected resistant colonies in independent disk diffusion tests.
Table 1.
Antibiotic susceptibility against three colistin-heteroresistant K. pneumoniae isolates and their
resistant populations.
Antibiotics
MIC (mg/L) a, b
S1703-35 S1703-109 S1703-112
P RP1 RP2 P RP1 RP2 P RP1 RP2
Colistin 1 (S) 128 (R) 64 (R) 0.25 (S) 64 (R) 64 (R) 1 (S) 256 (R) 128 (R)
Polymyxin B 1 (S) 64 (R) 32 (R) 0.25 (S) 64 (R) 32 (R) 1 (S) 64 (R) 64 (R)
Meropenem 0.06 (S)
0.125 (S) 0.125 (S)
0.06 (S) 0.06 (S) 0.06 (S) 4 (R) 4 (R) 2 (I)
Imipenem 1 (S) 1 (S) 0.5 (S) 0.25 (S) 0.25 (S) 0.25 (S) 2 (I) 1 (S) 1 (S)
Cefotaxime 0.125 (S) 0.25 (S) 0.25 (S) 0.25 (S) 0.25 (S)
0.125 (S)
>128 (R) >128 (R) >128 (R)
Ceftazidime 0.5 (S) 1 (S) 1 (S) 1 (S) 1 (S) 1 (S) >64 (R) >64 (R) >64 (R)
Cefepime 0.25 (S) 1 (S) 1 (S)
0.125 (S)
1 (S) 1 (S) >64 (R) >64 (R) >64 (R)
Amikacin 4 (S) 4 (S) 4 (S) 2 (S) 2 (S) 2 (S) 32 (I) 32 (I) 32 (I)
Gentamicin 1 (S) 1 (S) 1 (S) 0.5 (S) 0.5 (S) 0.5 (S) 2 (S) 2 (S) 1 (S)
Ciprofloxacin 0.25 (S) 0.25 (S) 0.25 (S) 0.06 (S) 0.06 (S) 0.06 (S) >64 (R) >64 (R) >64 (R)
Aztreonam 0.125 (S)
0.125 (S) 0.125 (S) 0.125 (S) 0.125 (S) 0.125 (S)
>64 (R) >64 (R) >64 (R)
Tigecycline 2 (S) 1 (S) 1 (S) 2 (S) 0.5 (S) 0.5 (S) 1 (S) 1 (S) 1 (S)
Piperacillin–tazobactam
16/4 (S) 8/4 (S) 8/4 (S) 8/4 (S) 8/4 (S) 8/4 (S) >256/4 (R) >256/4 (R) >256/4 (R)
a
MIC, minimal inhibitory concentration; P, parental; RP, resistant population; S, susceptible; I, intermediate; R,
resistant.
b
Data are underlined when the MIC increased more than 2-fold in the RP compared with the parental
isolate (P).
We investigated the amino acid alterations of the two-component regulatory systems PmrAB
and PhoPQ, which are known to be associated with colistin resistance in K. pneumoniae (Table 2).
We identified amino acid variations in 18 sites, where 11 were likely not associated with colistin
resistance because the amino acids in the resistant subpopulations could be found in other parental
isolates. As a result, it was assumed that seven amino acid substitutions may be associated with
colistin resistance in resistant subpopulations: two in PmrA, one in PmrB, two in PhoP, and two in

J. Clin. Med. 2019,8, 1444 5 of 9
PhoQ. Of note, two resistant subpopulations from the same parental isolate did not show amino acid
variations in PmrAB and PhoPQ. Two variations in PhoP (Arg198His and Lys199Asn) and one in PhoQ
(Leu414Agr) were identified in S1703-35-RP1 but not in S1703-35-RP2. Further, Asp152Asn in PhoQ
was identified only in S1703-35-RP2. For resistant subpopulations of S1703-109, Ile178Phe in PmrA
and Asp150Asn in PmrB were found in different resistant subpopulations. In addition, Leu414Agr in
PhoQ was identified in S1703-112-RP2 but not in S1703-112-RP1. No changes were found in MgrB.
The time–kill assays were performed for the multidrug-resistant and colistin-heteroresistant K.
pneumoniae isolate S1703-112. While 4- and 1-fold MICs of meropenem showed complete killing efficacy
after 12 and 24 h, respectively (Figure 2A), colistin did not eradicate the colistin-heteroresistant isolate
even at 4
×
MIC (Figure 2B). Although the combination of 0.25
×
MICs of meropenem and colistin did
not kill the heteroresistant isolate, the combination of 1
×
and 4
×
MICs demonstrated a rapid killing
effect compared with a single regimen of meropenem (Figure 2C).
Figure 2.
Time–kill curves for meropenem (
A
), colistin (
B
), and combination of meropenem and colistin
(C) against a colistin-heteroresistant K. pneumoniae isolate (S1703-112) that is multidrug-resistant.

J. Clin. Med. 2019,8, 1444 6 of 9
Table 2. Amino acid substitutions in PmrA, PmrB, PhoP, and PhoQ in three colistin-heteroresistant K. pneumoniae isolates and their resistant subpopulations.
Isolatea
Amino Acid Substitutions in:
PmrA PmrB PhoP PhoQ
178 203 43 150 163 185 186 198 199 216 152 154 359 414 421 423 429 430
S1703-35
P
Iso Arg Glu Asp
Arg
Arg Lys Arg Lys Gln Asp Lys Arg Leu Asp Ala Val Phe
RP1
Lys Asn Leu Glu His
Asn
Lys
Arg
Pro Ala Val
RP2
Lys Asn Glu Asn Lys Pro Ala Val
S1703-109
P
Ile Gly
Arg
Asp Cys Thr Gly Gly Cys Gly Asp Ser Lys Leu Gly Pro Ala Val
RP1
Phe Glu
RP2
Glu Asn
S1703-112
P
Ile Arg Pro Leu Glu Arg Lys
Arg
Asp Gln Lys Leu Gly Pro Ala Val
RP1
Lys
Arg
Arg Gln Lys Arg
RP2
Lys
Arg
Arg Gln Lys Arg
Arg
aP, parental; RP, resistant population. bAmino acid alterations that are supposed to be associated with colistin resistance are indicated as white letters with a grey background.

J. Clin. Med. 2019,8, 1444 7 of 9
4. Discussion
Heteroresistance has been recognized in both Gram-positive and -negative bacteria and is
a phenomenon in which a subpopulation of seemingly isogenic bacteria exhibits a range of
susceptibilities to a particular antibiotic [
16
]. Heteroresistance may have an effect on the outcome of
clinical infection, particularly because of limitations in detection by routine microbiological susceptibility
testing [
12
]. This study showed that heteroresistance among apparently susceptible isolates forms
a reservoir for the emergence of colistin resistance during treatment.
In this study, only a few K. pneumoniae isolates were heteroresistant to colistin. They were
clonally unrelated to each other. The rate of colistin heteroresistance found here was lower than
that in a previous study [
13
], in which it was reported that 12 among the 16 colistin-susceptible,
carbapenemase-producing K. pneumoniae isolates from Greece were heteroresistant to colistin. The rates
of colistin heteroresistance vary according to locality, isolation source, treatment of colistin, and so
forth. In addition, undetected colistin heteroresistance has been reported in K. pneumoniae [
19
,
27
],
suggesting the possibility that the rate of colistin heteroresistance may be higher than that identified in
this study.
We identified amino acid alterations that are supposed to be associated with colistin resistance
in resistant subpopulations, but it was not known if the genetic changes were induced by colistin
treatment. The amino acid alterations have not been previously reported, and it is not known if
the changes affect the function of PmrAB or PhoPQ. Of note, two resistant subpopulations from the
same isolate showed different amino acid substitutions in the two-component regulatory systems
PmrAB and PhoPQ. To our knowledge, variations between colistin-resistant subpopulations have not
been reported thus far. However, diverse genetic variations between colistin-resistant K. pneumoniae
mutants derived from the same parental strain after treatment have been reported [
23
]. Our results
may indicate that diverse subpopulations with resistance to colistin coexist in the heteroresistant or
susceptible isolates, which may develop into resistant strains with diverse mutations associated with
colistin resistance.
The combination of meropenem and colistin has been suggested to treat multidrug-resistant K.
pneumoniae infections [
28
]. The results of our time–kill assays showed that monotherapy with colistin
may be problematic for the treatment of infections caused by colistin-heteroresistant K. pneumoniae.
Although meropenem alone was effective at killing the heteroresistant isolate, the combination
of meropenem and colistin allowed rapid eradication at 1
×
MICs. Meropenem combined with
colistin at the appropriate dosage intervals might be a therapeutic option for infections caused by
colistin-heteroresistant K. pneumoniae. However, the effectiveness of the combination should be
investigated for carbapenemase-producing K. pneumoniae isolates.
5. Conclusions
We identified three colistin-heteroresistant K. pneumoniae isolates. The resistant populations of the
same isolate showed different amino acid alterations in PmrAB and PhoPQ. Meropenem combined
with colistin would be a suitable therapeutic option for infections caused by multidrug-resistant,
colistin-heteroresistant K. pneumoniae isolates.
Author Contributions:
Conceptualization, H.S.C., S.Y.K., Y.M.W. and K.S.K.; methodology, S.Y.K.; software,
S.Y.K. and K.S.K.; validation, Y.M.W. and K.R.P.; formal analysis, H.S.C. and S.Y.K.; investigation, H.S.C. and
S.Y.K.; resources, Y.M.W and K.R.P.; data curation, K.S.K.; writing—original draft preparation, H.S.C. and S.Y.K.;
writing—review and editing, Y.M.W., K.R.P. and K.S.K.; visualization, K.S.K.; supervision, K.R.P. and K.S.K.;
project administration, K.S.K.; funding acquisition, Y.M.W.
Funding:
This research was funded by the Basic Science Research Program through the National Research
Foundation of Korea (NRF), funded by the Ministry of Science and ICT (grant no. 2018R1D1A1B07049433).
Acknowledgments:
The Klebsiella pneumoniae isolates used in this study were obtained from the Asian Bacterial
Bank (ABB) of the Asia Pacific Foundation for Infectious Diseases (APFID) (Seoul, South Korea).
Conflicts of Interest: The authors declare no conflict of interest.

J. Clin. Med. 2019,8, 1444 8 of 9
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