Fukuzawa et al. Ann Clin Microbiol Antimicrob
https://doi.org/10.1186/s12941-023-00610-1(2023) 22:60

Annals of Clinical Microbiology
and Antimicrobials

Open Access

RESEARCH

High prevalence of colistin heteroresistancein specific species and lineages of Enterobactercloacae complex derived from human clinicalspecimens
Shota Fukuzawa1,2, Toyotaka Sato1,3,4,5*, Kotaro Aoki6, Soh Yamamoto1, Noriko Ogasawara1, Chie Nakajima7,8,9,
Yasuhiko Suzuki7,8,9, Motohiro Horiuchi3,4,5, Satoshi Takahashi10,11 and Shin‑ichi Yokota1

Abstract
Background Colistin (CST) is a last-line drug for multidrug-resistant Gram-negative bacterial infections. CST-heterore‑sistant Enterobacter cloacae complex (ECC) has been isolated. However, integrated analysis of epidemiology and resist‑ance mechanisms based on the complete ECC species identification has not been performed.
Methods Clinical isolates identified as “E. cloacae complex” by MALDI-TOF MS Biotyper Compass in a university
hospital in Japan were analyzed. Minimum inhibitory concentrations of CST were determined by the broth microdilu‑
tion method. The population analysis profiling (PAP) was performed for detecting the heteroresistant phenotype. The
heat shock protein 60 (hsp60) cluster was determined from its partial nucleotide sequence. From the data of wholegenome sequencing, average nucleotide identity (ANI) for determining ECC species, multilocus sequence type, coregenome single-nucleotide-polymorphism-based phylogenetic analysis were performed. phoPQ-, eptA-, and arnTdeleted mutants were established to evaluate the mechanism underlying colistin heteroresistance. The arnT mRNA
expression levels were determined by reverse transcription quantitative PCR.
Results Thirty-eight CST-resistant isolates, all of which exhibited the heteroresistant phenotype by PAP, were
found from 138 ECC clinical isolates (27.5%). The prevalence of CST-resistant isolates did not significantly differ
among the origin of specimens (29.0%, 27.8%, and 20.2% for respiratory, urine, and blood specimens, respectively).
hsp60 clusters, core genome phylogeny, and ANI revealed that the CST-heteroresistant isolates were found in allor most of Enterobacter roggenkampii (hsp60 cluster IV), Enterobacter kobei (cluster II), Enterobacter chuandaensis(clusters III and IX), and Enterobacter cloacae subspecies (clusters XI and XII). No heteroresistant isolates were foundin Enterobacter hormaechei subspecies (clusters VIII, VI, and III) and Enterobacter ludwigii (cluster V). CST-induced mRNA
upregulation of arnT, which encodes 4-amino-4-deoxy-l-arabinose transferase, was observed in the CST-heteroresist‑
ant isolates, and it is mediated by phoPQ pathway. Isolates possessing mcr-9 and mcr-10 (3.6% and 5.6% of total ECC
isolates, respectively) exhibited similar CST susceptibility and PAP compared with mcr-negative isolates.
Conclusions Significant prevalence (approximately 28%) of CST heteroresistance is observed in ECC clinical isolates,
and they are accumulated in specific species and lineages. Heteroresistance is occurred by upregulation of arnTmRNA induced by CST. Acquisition of mcr genes contributes less to CST resistance in ECC.
*Correspondence:
Toyotaka Satosato.t@vetmed.hokudai.ac.jp
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Fukuzawa et al. Ann Clin Microbiol Antimicrob(2023) 22:60

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Keywords Enterobacter cloacae complex, Colistin, Heteroresistance, arnT, PhoPQ, Average nucleotide identity (ANI)

Background
Enterobacter cloacae complex (ECC) and Enterobacteraerogenes (currently as Klebsiella aerogenes) represent
the most frequently isolated Enterobacter spp. in respiratory, urinary tract, and bloodstream infections [1,2]. According to recent literatures, following species,
Enterobacter asburiae, Enterobacter cancerogenus, Enterobacter cloacae, Enterobacter hormaechei, Enterobacterkobei, Enterobacter ludwigii, Enterobacter mori, Enterobacter nimipressuralis, Enterobacter roggenkampii, andEnterobacter soli are assigned to ECC [3–5]. However,the classification has been expanding due to the application of whole-genome sequencing (WGS) to bacterial
species identification. The clinical relevancy and antimicrobial resistance of individual ECC species are unclear
because routine bacterial identification procedures, suchas matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS) and conventional biochemical testing with automated systems, usedin clinical laboratories have not stay in step with classification of ECC into individual species [3]. Whereas heatshock protein 60 gene (hsp60) clustering have been used
to classify ECC clinical isolates [6], DNA–DNA hybridization and average nucleotide identity (ANI) are the mostreliable bacterial identifications at present [7].
Significant numbers of multidrug-resistant ECC haveemerged [8, 9]. Thus, the choice of antimicrobials in thetreatment of their infections appears to be becoming
difficult. Colistin (CST) is a polycationic antimicrobial
peptide that is effective and used as a last-line drug for
multidrug-resistant Gram-negative bacteria infections,
including ECC; however, CST-resistant ECC has emergedin clinical settings worldwide [6, 10, 11].
CST resistance is acquired by upregulation of the
intrinsic lipid A-modifying enzyme gene, eptA (alsoknown as pmrC) or arnT (present in the arn operon,arnBCADTEF), which encode the lipid A-modifying
enzymes, phosphoethanolamine (pEtN) transferase and4-amino-4-deoxy-l-arabinose (l-Ara4N) transferase,
respectively [12]. Modification of lipid A decreases thenegative charge of the outer membrane to repulse thepositive charge of CST, leading to CST resistance [13].
Activation of two-component regulatory systems, PhoPQ
and PmrAB, upregulates expression of the eptA and arn
operon, and MgrB negatively regulates the two component system [12]. The mutations of phoPQ, mgrB, and/or pmrAB mediate CST resistance [14]. In addition, theworldwide spread of plasmid-mediated CST resistancegenes, mcr-1 to mcr-10 has also been reported [15].
The CST resistance mechanism of ECC reported in
the E. cloacae subsp. cloacae ATCC 13047 strain, whichdisplays the CST-heteroresistant phenotype, involves
the PhoPQ two-component system and arn operon [16].
CST heteroresistance refers to a bacterial clone containing both CST-susceptible and -resistant subpopulations,and causes therapeutic failure [17]. Thus, it necessitatesassessment of CST-resistant subpopulations during clinical diagnosis of ECC infections.
In daily clinical laboratory investigations of antimicrobial susceptibility testing, the skip-well phenomenon
with the broth microdilution method and the appearanceof colonies in the inhibitory zone with the disc diffusion
test and E-test are indications of the heteroresistant phenotype [11, 18]. Population analysis profiling (PAP) is themost reliable method to identify CST heteroresistance[19]. Unfortunately, identifications for ECC species and/or the CST heteroresistance in previous studies were
inadequate [6, 10, 11, 16, 17].
This study aimed to elucidate the relationship between
the genetic features and CST resistance mechanism byusing the integrated analysis with molecular epidemiology [hsp60 clustering, multilocus sequence typing(MLST), core genome analysis, and species identification by ANI] and the CST-heteroresistance [evaluationsof skip-well phenomenon, PAP test, bacterial growth, andcontributions of phoPQ, mgrB, pmrAB, eptA, and arnT]
to comprehensively understand CST resistance in ECC
clinical isolates.

Methods
Bacterial isolates

ECC isolates (n = 138) were derived from clinical specimens of different patients at Sapporo Medical University
Hospital (Sapporo, Japan) from May 2017 to May 2018.
These isolates were identified as “E. cloacae complex” by
MALDI-TOF MS Biotyper Compass (software version
4.1.80.24), bacterial library version 8 (Bruker, Billerica,MA) in clinical laboratory testing. We also collected K.
aerogenes (formerly, E. aerogenes) isolates (n = 30). These
clinical specimens were respiratory (n = 78, 46.4%), urine(n = 22, 13.1%), blood (n = 12, 7.2%), and others (n = 56,
33.3%).

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Antimicrobial susceptibility testing, monitoringof bacterial growth, and detection of the skip‑wellphenomenon(from 1 × ­1010 to 1 × ­103 CFU/mL) of the suspension werespread onto cation-adjusted Mueller–Hinton II agar
(MHII agar; Becton, Dickinson and Co.) plates containing 0, 0.125, 1, or 8 mg/L CST and cultured for 20 h at
37 ℃. The occurrence frequencies of the CST-resistant
subpopulations were calculated by dividing the numbersof CFU on CST-containing MHII agar plates by those on
plain MHII agar plates. CST heteroresistance was determined using a previously published method based on
the following criteria [19, 21]: (1) bacterial growth was
confirmed in the presence of > 2 mg/L CST by the brothmicrodilution method, and (2) the CST concentration in
the presence of which the CST-resistant subpopulations
grew (within a frequency range from 1 × ­10–7 to 1 × ­10–1)
were at least eightfold higher (≥ 1 mg/L CST) than thehighest CST concentration that did not affect the growthof CST-susceptible subpopulations (0.125 mg/L CST).
Data are expressed as the means ± standard deviations
from three independent experiments.

CST, ampicillin, meropenem, gentamicin, and amikacin were purchased from FUJIFILM Wako Pure Chemical Corp. (Osaka, Japan). Cefotaxime and ciprofloxacin
were purchased from Tokyo Chemical Industry (Tokyo,
Japan). Minimum inhibitory concentration (MIC)s were
determined by the broth microdilution method using
cation-adjusted Mueller–Hinton II broth (MHII broth;
Becton, Dickinson and Co., Franklin Lakes, NJ) according to the guidelines of the Clinical and Laboratory
Standards Institute (CLSI) [20]. Isolates with a CST MIC
of ≥ 4 mg/L were defined as CST-resistant according to
CLSI M100-S32.
Bacterial growth was measured by assessing turbidity­(OD600) after cultivation for 20 h under the same condition as used for the susceptibility test in the presenceof CST (0–128 mg/L). Turbidity was measured using an
Infinite M200 PRO instrument (Tecan, Kawasaki, Japan).
The skip-well phenomenon was defined as no growth­(OD600 < 0.1) at a certain CST concentration(s) andgrowth ­(OD600 > 0.5) at higher CST concentrations. Data
are expressed as the means ± standard deviations fromindependent triplicate experiments.
PAP

Heteroresistance was determined by PAP as previously
described [19]. Briefly, ECC clinical isolates were inoculated into 10 mL of Trypticase Soy Broth (TSB) (Beckton,
Dickinson and Co.) and cultured overnight at 37 ℃. The
bacterial cell culture (approximately 1 × ­109 CFU/mL)
was centrifuged and resuspended in 1 mL of sterile 0.85%
NaCl. One hundred microliters of tenfold serial dilutions

hsp60 clustering

The 273 nucleotides of hsp60 fragment was amplified by
PCR with specific primers as described previously [22].
The nucleotide sequences of the hsp60 fragment werealigned in MAFFT [23] (http://​www.​ebi.​ac.​uk/​Tools/​
msa/​mafft/). The phylogenetic tree was generated by
MEGA X [24].
WGS

To determine the draft whole-genome sequences of
95 ECC clinical isolates (Table 1), genomic DNA wasextracted from bacteria using DNeasy Blood & Tissue Kit(Qiagen, Hulsterweg, The Netherlands), and DNA library

Table 1 Species determined by ANI, hsp60 cluster classification, and CST susceptibility of ECC clinical isolates
hsp60
cluster

ECC species (No. of isolates)
CST heteroresistant (38)

CST susceptible (57)I

E. asburiae (7), E. cancerogenus (1)E. asburiae (16), E. soli (1)II

E. kobei (14)

E. kobei (1)III

E. chuandaensisa (1)E. hormaechei subsp. hoffmannii (2)IV

E. roggenkampii (9)

–

V

–

E. ludwigii (15)VI–

E. hormaechei subsp. xiangfangensis(6), E. hormaechei subsp. oharae (1)

VIII

–

E. hormaechei subsp. steigerwaltii (13)IX

E. chuandaensisa (3)E. chuandaensisa (1)XI

E. cloacae subsp. cloacae (1)–XII

E. cloacae subsp. dissolvens (2)

–

XIII

–

E. mori (1)a

ANI value 94.1%

Fukuzawa et al. Ann Clin Microbiol Antimicrob(2023) 22:60

for WGS was prepared using Nextera XT DNA Library
Preparation Kit (Illumina, San Diego, CA) according
to the manufacturer’s instructions. The libraries weresequenced on MiSeq Sequencing System (Illumina) with300 bp paired-end reads according to the manufacturer’s
protocol.
MLST

MLST was performed using ECC MLST databases in
PubMLST.org(https://​pubml​st.​org/​organ​isms/​enter​obact​er-​cloac​ae/) and the Center for Genomic Epidemiology MLST 2.0 Web tool (https://​cge.​cbs.​dtu.​dk/​servi​
ces/​MLST/).
ECC species identification

ECC species were identified by ANI analysis using
fastANI of the draft WGS data compared with the typestrain genomes obtained from GenBank [25]. A cutoff of
98% of the ANI value compared with the type strains wasused for species and subspecies identification [7, 26].
Phylogenetic analysis of core genome single nucleotide
polymorphisms (SNPs), annotation, and mcr gene
identification

Core genome SNPs-based phylogenetic analysis was performed with WGS data. MiSeq read data were mapped to
the reference genome, E. cloacae ATCC 13047 (accession
number: NC_014121.1), using bwa version 0.7.17. The
core genome of the applied strain was extracted using
mpileup of SAMtools version 1.9 and the “cns” optionof VarScan version 2.3.9 [27, 28]. A phylogenetic tree
based on SNPs in the core genome was generated using
the default parameter of FastTree version 2.1.10 [29].
Genome annotation was performed with DFAST v1.1.0
and standard settings [30] (https://​doi.​org/​10.​1093/​bioin​
forma​tics/​btx713). DNA sequences were compared using
Clustal W [31] (https://​www.​genome.​jp/​tools-​bin/​clust​alw). mcr genes were identified using assembled genome
data by Resfinder (https://​www.​genom​icepi​demio​logy.​org).

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Reverse transcription quantitative PCR (RT‑qPCR)

Overnight cultures with TSB were diluted 1:100 in
MHII broth, and cultivated at 37 ℃ for 0, 5, 15, 30, 60,
and 120 min in the presence of CST (0, 0.25, 1, 4, 16,
and 64 mg/L). Total RNA was extracted using a RNeasy Plus Mini Kit (Qiagen). cDNA was synthesizedfrom total RNA (100 ng) using ReverTra Ace RT-qPCR
Master Mix with Genomic DNA (gDNA) Remover(Toyobo). RT-qPCR was performed on LightCycler 480
II system (Roche, Mannheim, Germany) using KOD
SYBR qPCR Mix (Toyobo) and primer pairs for arnT,
which are shown in Additional file 1: Table S1. Gene
expression levels relative to the housekeeping gene
gyrB were determined using the ΔΔCt method. Data
are expressed as the means ± standard deviations fromthree independent experiments.
Construction of phoPQ‑, eptA‑, and arnT‑deleted mutants

Deletion mutants of phoPQ, eptA, and arnT were generated by the λ red recombinase method using pKD46hyg [32, 33]. Mini-cassette genes containing 50 bases
corresponding to the upstream and downstream
regions of the target genes and kanamycin resistance cassettes (Gene Bridges, Heidelberg, Germany)
were amplified by PCR and the gene deletion was confirmed by PCR using specific primers (Additional file 1:
Table S1).
Statistical analysis

Mann–Whitney and a one-way ANOVA with the
Kruskal–Wallis tests were used to examine the significance of differences between two and multiple groups,
respectively. Occurrence rates of phenotypes amonggroups were examined by χ2-test. p < 0.05 was considered significant. Pearson’s correlation coefficients werealso calculated.

(See figure on next page.)
Fig. 1 Classification of ECC and K. aerogenes into hsp60 clusters. A Neighbor-joining unrooted tree based on hsp60 clustering of ECC and K.
aerogenes clinical isolates. CST-resistant and -susceptible isolates are indicated by red and black circles, respectively. The GenBank accession
numbers of the reference genes are AJ417128 (I), AJ417141 (I), AJ567895 (I), AJ417135 (I), AJ417140 (I), AJ567899 (II), AJ417126 (II), AJ543882 (III),
AJ543804 (III), AJ543864 (III), AJ417112 (IV), AJ417125 (IV), AJ866524 (V), AJ417132 (V), AJ417115 (V), AJ417114 (V), AJ417118 (V), HG764321 (V),HG764343 (V), AJ567878 (VI), AJ567885 (VI), AJ417108 (VII), AJ543857 (VIII), AJ543908 (VIII), AJ543821 (VIII), AJ417127 (IX), AJ567900 (X), AJ417138(XI), AJ417139 (XI), CP001918 (XI), AJ417142 (XI), AJ417143 (XII), AJ567881 (XIII), AJ543837 (XIII), CP016762 (unclassified), CP009072 (unclassified),
AJ567897 (unclassified), AJ567901 (unclassified), AJ567902 (unclassified), AJ567894 (unclassified), AJ567898 (unclassified), AJ567896 (unclassified),
AB375469 (unclassified), and AB375468 (unclassified). B Comparison of hsp60 cluster types of ECC and K. aerogenes clinical isolates according
to the origin of clinical specimens. Significant differences of the occurrence rates of hsp60 clusters did not observe among the origins of specimens

Fukuzawa et al. Ann Clin Microbiol Antimicrob

Fig. 1 (See legend on previous page.)(2023) 22:60

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Results

bias in the distribution of hsp60 cluster types according
to the origin of the clinical specimens (Fig. 1B).

Classification of ECC and K. aerogenes clinical isolates
into hsp60 clusters

We analyzed ECC isolates and K. aerogenes isolates.
Because K. aerogenes was formerly classified in genus
Enterobacter, and CST-resistant K. aerogenes wasreported [3, 11]. The 138 ECC isolates were classified
into 11 hsp60 clusters, and 30 K. aerogenes isolates
were defined as unclassified (Fig. 1A, Additional file 1:
Table S2). The most abundant cluster was VIII, followed by V, I, and VI (Table 2). There was no significant

CST susceptibility of ECC and K. aerogenes, and its
relationships with the origin of the clinical specimens
and hsp60 clustering

The MIC of CST was determined for the 138 ECC and
30 K. aerogenes clinical isolates (Table 3). It ranged
from 0.5 to > 128 mg/L, and ­MIC50 and M­ IC90 were 1
and > 128 mg/L, respectively. Thirty-eight (27.5%) of ECC
isolates were CST-resistant (MIC ≥ 8 mg/L), whereas
none of CST-resistant isolates were observed in K.
aerogenes.

Table 2 hsp60 cluster classification and prevalence of CST-resistant ECC clinical isolates
hsp60 cluster

No. of ECC isolates (percentage among ECC isolates)

No. of CST-resistant isolates(percentage among eachcluster)I

25 (18.1%)

8 (32.0%)II

15 (10.9%)

14 (93.3%)

III

3 (2.2%)

1 (33.3%)IV

9 (6.5%)9 (100%)V

27 (19.6%)

0 (0%)

VI

17 (12.3%)

0 (0%)VIII

34 (24.6%)

0 (0%)IX

4 (2.9%)3 (75.0%)

XI

1 (0.7%)

1 (100%)XII

2 (1.4%)2 (100%)XIII

1 (0.7%)0 (0%)

Table 3 CST susceptibility testing of 138 ECC and 30 K. aerogenes clinical isolates
Bacterial species

CST MIC (mg/ L)
0

0.125

0.25

0.5

1

2

4

8

16

32

64

128

> 128

No. of ECC isolates

–

–

–

44

56

–

–

1

2 (2)

2 (1)

–

5

28 (7)

No. of K. aerogenes isolates

–

–

–

18

12

–

–

–

–

–

–

–

–

Breakpoint, 2 mg/L of CST. Parentheses indicates No. of isolates displaying the skip-well phenomenon(See figure on next page.)
Fig. 2 Comparison of CST-resistant and -susceptible ECC. A Comparison of CST MICs of ECC clinical isolates according to the origin of clinicalspecimens. Percentages indicate the prevalence of CST-resistant isolates. Numbers in parentheses indicate the number of ECC isolates. CST
susceptibility did not significantly differ according to the origin of clinical isolates. B hsp60 cluster classification of CST-susceptible and -resistant
ECC clinical isolates. Significant difference was observed between the groups (*p < 0.05). C Growth of CST-susceptible and -resistant (withand without the skip-well phenomenon) ECC clinical isolates in the presence of various CST concentrations. Bacterial growth was measured
by assessing turbidity ­(OD600) after cultivation for 20 h based on the broth microdilution method in 96-well plates. The skip-well phenomenon
was defined as no growth ­(OD600 < 0.1) at a certain CST concentration(s) and growth ­(OD600 > 0.1) at higher CST concentrations. Representative
ECC clinical isolate data are shown: CST-susceptible isolate (SMEnt52 strain; E. hormaechei subsp. steigerwaltii, hsp60 cluster VIII), CST-resistant isolate
displaying the skip-well phenomenon (SMEnt513 strain; E. kobei, hsp60 cluster II), and CST-resistant isolate not displaying the skip-well phenomenon(none-skip-well) (SMEnt1102 strain; E. cloacae subsp. cloacae, hsp60 cluster XI). Bacterial growths (­ OD600 values) in the presence of CST werecompared with the bacterial growth in the absence of CST of the respective strains (*p < 0.05 and **p < 0.001)

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Fig. 2 (See legend on previous page.)(2023) 22:60

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The prevalence of CST-resistant ECC isolates did not
significantly differ according to the origin of the clinical specimens (Fig. 2A). The distribution of hsp60 cluster types significantly differed between CST-susceptible
and -resistant isolates (p < 0.01) (Fig. 2B). CST-resistantisolates comprised seven clusters, and clusters II, IV,
and I were dominant (Table 2). All isolates in clusters IV,
XI, and XII, almost all isolates in clusters II and IX, andapproximately one-third of isolates in clusters I and III
were CST-resistant.
Skip‑well phenomenon and PAP

Ten CST-resistant isolates exhibited the skip-well phenomenon (26.3% of CST-resistant isolates and 7.2% of
total ECC isolates) (Table 3, Additional file 1: Table S2).
Their growth was almost completely inhibited in wells
containing 0.5 or 1 mg/L CST, but cells grew in wells
containing higher CST concentrations (higher than
2 mg/L) (Fig. 2C). Although the remaining 28 CSTresistant isolates did not display the skip-well phenomenon, they exhibited weak growth ­(OD600 of 0.2–0.6) inwells containing 0.5 or 1 mg/L CST and strong growth­(OD600 > 0.8) in wells containing higher CST concentrations (≥ 2 mg/L) (Fig. 2C).
To analyze the CST-heteroresistant phenotype by
PAP, we measured the occurrence frequencies of CSTresistant subpopulations in the presence of various CST
concentrations (Fig. 3A). Growth of all isolates was
unaffected in the presence of 0.125 mg/L CST. For all
CST-susceptible isolates, the occurrence frequency of
CST-resistant subpopulations was lower than 1 × ­10–7(ranged from 1.6 × ­10–9 to 8.5 × ­10–8) in the presenceof 1 and 8 mg/L CST. For all CST-resistant isolates,
the occurrence frequency of CST-resistant subpopulations in the presence of 1 and 8 mg/L CST (more thaneightfold higher than 0.125 mg/L) exceeded 1 × ­10–7(ranged from 2.2 × ­10–6 to 7.1 × ­10–3 at 1 mg/L and from
1.3 × ­10–4 to 6.4 × ­10–2 at 8 mg/L). These results indicatethat all CST-resistant ECC clinical isolates were CST
heteroresistant.
The occurrence frequencies of CST-resistant subpopulations in CST-resistant isolates with high(≥ 128 mg/L) and moderate (8–32 mg/L) MICs were

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significantly higher than those in CST-susceptible isolates (Fig. 3B). They also tended to be higher in CSTresistant isolates with high MICs than in those withmoderate MICs, but this difference was not significant.
The occurrence frequencies of CST-resistant subpopulations in the presence of 1 mg/L CST were significantly
higher in CST-resistant isolates that did not exhibit theskip-well phenomenon than in those that did (p < 0.05)(Fig. 3C), whereas there is no significant differences in
0.125 and 8 mg/L of CST. The occurrence frequencies
of CST-resistant subpopulations correlated with the
CST MICs (r = 0.95) (Fig. 3D).
We evaluated the relationship between the occurrence
frequencies of CST-resistant subpopulations and hsp60
cluster types. In agreement with the results of CST susceptibility testing (Table 3), CST-heteroresistant isolateswith higher occurrence frequencies of CST-resistant
subpopulations (> 1 × ­10–5) were found in certain hsp60
clusters (I, II, III, IV, IX, XI, and XII) but not in otherhsp60 clusters (p < 0.001) (Fig. 4A), and in certain species,
such as E. kobei, E. roggenkampii, and E. cloacae subsp.
(p < 0.001) (Fig. 4B).
MLST analysis and antimicrobial susceptibilities
of CST‑heteroresistant ECC clinical isolates

MLST analysis of the CST-heteroresistant isolates was
performed. Sequence type (ST)s, ST1939, ST125, ST997,
and ST1953 were the dominant in hsp60 clusters I, II, IV,
and XII, respectively. In antimicrobial susceptibility testing, 12 (31.6%) CST-heteroresistant isolates were resistant to cefotaxime and one was resistant to meropenem(Additional file 1: Table S3). No CST-heteroresistant isolate was resistant to aminoglycosides and ciprofloxacin.
ECC species identification by ANI and generationof a phylogenetic tree based on core genome SNPs

Ninety-five clinical isolates identified as ECC by the
MALDI Biotyper were analyzed by ANI, and a phylogenetic tree was generated using core genome SNPs
(Fig. 5, Additional file 1: Table S2). All 38 CST-heteroresistant isolates and 57 CST-susceptible isolates randomly selected to include half the isolates in each hsp60(See figure on next page.)
Fig. 3 PAP of ECC. A Occurrence frequencies of CST-resistant subpopulations in the presence of various CST concentrations (0, 0.125, 1,
and 8 mg/L). B Occurrence frequencies of CST-resistant subpopulations in isolates with low, moderate, and high CST MICs. Number in parentheses
indicates the number of ECC isolates. *p < 0.05 and **p < 0.001. C Comparison of the occurrence frequencies of CST-resistant subpopulations
between CST-resistant isolates displaying and not displaying the skip-well phenomenon in the presence of 0.125, 1, and 8 mg/L CST. Number
in parentheses indicates the number of ECC isolates. *p < 0.05. D Correlation between CST MICs and occurrence frequencies of CST-resistant
subpopulations

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Fig. 4 ECC species and occurrence frequencies of CST-resistant subpopulations. A Occurrence frequencies of CST-resistant subpopulations
according to hsp60 cluster classification. B Occurrence frequencies of CST-resistant subpopulations according to ECC species determined by ANI.
CST-heteroresistant and -susceptible isolates are indicated by red and black circles, respectively. aE. cloacae subsp. including E. cloacae subsp. cloacae(n = 1) and E. cloacae subsp. dissolvens (n = 2). bE. hormaechei subsp. including E. hormaechei subsp. hoffmannii (n = 2), E. hormaechei subsp. oharae(n = 1), E. hormaechei subsp. steigerwaltii (n = 13), and E. hormaechei subsp. xiangfangensis (n = 6)

Fukuzawa et al. Ann Clin Microbiol Antimicrob(2023) 22:60

cluster were examined. Ten species were identified by
ANI (Additional file 1: Table S2). The CST-heteroresistant isolates were identified as one of six species: E.
asburiae, E. cancerogenus, E. chuandaensis, E. cloacae(including subsp. cloacae and subsp. dissolvens), E. kobei,
and E. roggenkampii (Table 1). The 57 CST-susceptible
isolates were identified as one of seven species: E. asburiae, E. chuandaensis, E. hormaechei (including subsp.
hoffmannii, subsp. oharae, subsp. steigerwaltii, and subsp.
xiangfangensis), E. kobei, E. ludwigii, E. mori, and E. soli
(Table 1). The occurrence frequencies of CST-resistant
subpopulations were closely related with the results ofspecies identification, phylogeny, and hsp60 clustering(Fig. 4A, B).
In CST-susceptible ECC, 10 isolates of E. hormaechei
subsp. (including subsp. hoffmannii, subsp. oharae, subsp.
steigerwaltii, and subsp. xiangfangensis) lacked the arn
operon in their genomes according to annotation analysis, whereas other chromosomal CST resistance determinants (phoPQ, mgrB, pmrAB, and eptA) were present(Fig. 5). mcr-9 and mcr-10 were detected in five isolates(two E. kobei and one each of E. asburiae, E. hormaechei
subsp. xiangfangensis, and E. ludwigii) and eight isolates(five E. roggenkampii, two E. asburiae, and one E. kobei),
respectively (Fig. 5). The CST MIC and occurrence frequency of CST-resistant subpopulations analyzed by PAP
did not significantly differ among the none-mcr, mcr9-positive, and mcr-10-positive groups (Additional file 1:
Table S4, Additional file 2: Fig. S1).
arnT mRNA expression levels in CST‑heteroresistantand ‑susceptible ECC clinical isolates

We measured the arnT mRNA expression levels in the
CST-heteroresistant strain SMEnt513 (E. kobei, hsp60
cluster II which mostly includes CST-heteroresistant isolates) and the CST-susceptible strain SMEnt52 (E. hormaechei subsp. steigerwaltii, hsp60 cluster VIII which

Page 11 of 18

mostly includes CST susceptible isolates) in the presence and absence of CST. CST induced arnT mRNA
expression in SMEnt513, but not in SMEnt52 (Fig. 6A).
arnT mRNA expression in SMEnt513 was significantly
increased by treatment with 0.25 mg/L CST and peaked(approximately 18-fold) upon treatment with 16 mg/L
CST for 45 min (Fig. 6B).
Evaluation of the CST heteroresistance mechanism in ECC

To evaluate the mechanism underlying CST heteroresistance in individual ECC species, we selected one
CST-heteroresistant strain from each of the seven species. No mutation involved in CST resistance was foundin phoPQ, mgrB, pmrAB, arnT, and eptA between these
CST-heteroresistant and CST-susceptible isolates. We
generated phoPQ-, eptA-, and arnT-deleted mutants ofthe seven isolates. All parent strains and eptA-deleted
mutants exhibited the typical CST-heteroresistant phenotype in the presence of CST (growth was decreasedin the presence of 0.5 or 1 mg/L CST but was observedin the presence of higher CST concentrations) (Fig. 7A).
All phoPQ- and arnT-deleted mutants did not growin the presence of ≥ 1 mg/L CST. In PAP analysis, theoccurrence frequencies of CST-resistant subpopulations were high (> 1 × ­10–3) in parent strains and eptAdeleted mutants in the presence of 8 mg/L CST, but weremarkedly decreased (< 1 × ­10–8) and below the criteriaof heteroresistance (ranged from 1 × ­10–7 to 1 × ­10–1)
in phoPQ- and arnT-deleted mutants (Fig. 7A). In thesusceptibility test, the phenotype of phoPQ- and arnTdeleted mutants changed from CST-resistant to CSTsusceptible (Additional file 1: Table S5). Treatment with
16 mg/L CST induced arnT mRNA expression more thantenfold in the parent strains of all ECC species, whereas
arnT mRNA induction by CST was abolished in all
phoPQ-deleted mutants (Fig. 7B).
In the presence of 16 mg/L CST, arnT mRNA
expression levels were increased more than eightfold(See figure on next page.)
Fig. 5 Phylogenetic tree based on core genome SNPs of ECC. Core genome SNP-based phylogenetic analysis was performed with WGS dataof 95 ECC clinical isolates. CST-susceptible strains, CST-heteroresistant strains, strains harboring mcr-9, strains harboring mcr-10, and strains
lacking the arn operon are indicated by black, red, blue, green, and yellow circles to the right of their names. aE. hormaechei (subsp. hoffmannii,
subsp. xiangfangensis, subsp. steigerwaltii, and subsp. oharae). bE. cloacae (subsp. cloacae and subsp. dissolvens). The GenBank accession numbersof the reference genes are NZ_CP011863 (E. asburiae), NZ_LEDH00000000 (E. asburiae), NZ_FYBA00000000 (E. cancerogenus), NZ_CP043318(E. chengduensis), NZ_QZCS00000000 (E. chuandaensis), NC_014121 (E. cloacae subsp. cloacae), NZ_LEDL00000000 (E. cloacae subsp. cloacae),
NC_018079 (E. cloacae subsp. dissolvens), NZ_VTTY00000000 (E. dykesii), NZ_CP017186 (E. hormaechei subsp. hoffmannii), NZ_AFHR01000000 (E.
hormaechei subsp. hormaechei), NZ_CP017180 (E. hormaechei subsp. oharae), NZ_CP017179 (E. hormaechei subsp. steigerwaltii), NZ_AMGJ01000000(E. hormaechei subsp. steigerwaltii), NZ_CP017183 (E. hormaechei subsp. xiangfangensis), NZ_LABA01000000 (E. hormaechei subsp. xiangfangensis),
NZ_CP043342 (E. huaxiensis), NZ_CP01718 (E. kobei), NZ_LEDC01000000 (E. kobei), NZ_JTLO00000000 (E. ludwigii), NC_016514 (E. ludwigii), NZ_
GL890773 (E. mori), NZ_AP019007 (E. oligotrophicus), NZ_SJON00000000 (E. quasihormaechei), NZ_CP017184 (E. roggenkampii), NZ_LXES00000000(E. soli), NZ_FCOP00000000 (E. timonensis), NZ_VTUC00000000 (E. vonholyi), NZ_SJOO00000000 (E. wuhouensis), NZ_JZZB01000000 (ECC), NZ_
LEEP01000000 (ECC), and NZ_AXLJ01000000 (ECC)Fukuzawa et al. Ann Clin Microbiol Antimicrob

Fig. 5 (See legend on previous page.)(2023) 22:60

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Fukuzawa et al. Ann Clin Microbiol Antimicrob(2023) 22:60

Page 13 of 18

Fig. 6 arnT mRNA expression levels in CST-susceptible and -heteroresistant ECC. SMEnt52 (E. hormaechei subsp. steigerwaltii, hsp60 cluster
VIII) and SMEnt513 (E. kobei, hsp60 cluster II) were used as CST-susceptible and -resistant isolates, respectively. A arnT mRNA expression levels
during cultivation of SMEnt52 and SMEnt513 in the presence (16 mg/L) and absence of CST. The y axis shows relative expression levels normalized
by that at 0 min (set to 1.0). *p < 0.05 and **p < 0.001. B arnT mRNA expression levels in SMEnt52 and SMEnt513 cultivated in the presence of various
CST concentrations. Total RNA was isolated after cultivation for 45 min at 37 ℃. The y axis shows relative mRNA expression levels normalized
by that in SMEnt52 in the absence of CST (set to 1.0). *p < 0.05 and **p < 0.001(9.1–56.7-fold) in all CST-heteroresistant isolates, butless than fourfold in CST-susceptible isolates (Fig. 8A, B).
The arnT mRNA expression levels were positively relatedwith CST MICs (Fig. 8C).

Discussion
In the present study, 27.5% of ECC clinical isolates
obtained from a university hospital in Japan was CST
resistant showing heteroresistant phenotype. In the previous studies, the prevalence of CST resistance in ECC
clinical isolates was 23.9% in Korea (2012–2014) [11],11.3–13.3% in UK and Ireland (2017) [10], and 34.5% in
China (2020) [34]. These observations indicate that CSTresistant ECC is present in clinical specimens of various origins. Although no multidrug-resistant ECC was
detected in our study, the CST resistance in ECC should

be an international concern because multidrug-resistant
ECC has emerged [9]. However, CST-heteroresistancephenotype has not been adequately examined for the
CST-resistant isolates in previous studies [9–11, 16, 17,34].
The CST-heteroresistant isolates were assigned to certain species and lineages similar to a previous French
report [6]. However, no CST-heteroresistant isolates were
found in clusters V and VIII in the present study, whereas
100% and 79% of the clusters were CST-heteroresistant in
the report, respectively [6] suggesting different CST-heteroresistant lineages have been spread in other counties.
We found several mcr-9/10-positive isolates in various lineages regardless of CST susceptibility. Acquisition of the plasmid-mediated CST resistance genes mcr-9
and mcr-10 contributed little to CST heteroresistance in
ECC because CST MICs and occurrence frequencies of(See figure on next page.)
Fig. 7 CST-heteroresistant phenotype of phoPQ-, eptA-, and arnT-deleted mutants derived from various species of ECC. Deletion mutants wereprepared for the following seven representative species of ECC clinical isolates: SMEnt819 (E. asburiae by ANI, hsp60 cluster I), SMEnt494 (E.
cancerogenus, hsp60 cluster I), SMEnt1102 (E. cloacae subsp. cloacae, hsp60 cluster XI), SMEnt835 (E. cloacae subsp. dissolvens, hsp60 cluster XII),
SMEnt513 (E. kobei, hsp60 cluster II), SMEnt1062 (E. roggenkampii, hsp60 cluster IV), and SMEnt1003 [E. chuandaensis (ANI value 94.1%), hsp60
cluster IX]. A Heat maps representing bacterial growth and occurrence of CST-resistant subpopulations in the presence of CST. Bacterial growth
was measured by assessing turbidity ­(OD600) after cultivation for 20 h based on the broth microdilution method in 96-well plates. O­ D600 < 0.1
was defined as no growth. A CST MIC ≥ 4 mg/L was defined as CST resistance. The occurrence frequencies of CST-resistant subpopulations
were measured in the presence of 1 or 8 mg/L CST. Color shading was used to visualize the turbidities (­ OD600) and frequencies of CST-resistant
subpopulations at each CST concentration. Turbidities ­(OD600) decrease from yellow to purple and occurrence frequencies of CST-resistant
subpopulations in PAP decrease from red to blue. B Comparison of arnT mRNA expression levels in the presence of CST (16 mg/L) between parent
strains and their phoPQ-deleted mutants. The y axis shows relative mRNA expression levels normalized by those in the absence of CST in each strain(set to 1.0). *p = 0.002

Fukuzawa et al. Ann Clin Microbiol Antimicrob

Fig. 7 (See legend on previous page.)(2023) 22:60

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Fukuzawa et al. Ann Clin Microbiol Antimicrob(2023) 22:60

CST-resistant subpopulations did not obviously differ
between mcr-9/10-positive and -negative isolates. Thisobservation strongly indicates that CST heteroresistanceof ECC was mainly mediated by intrinsic genes, namely,
CST-inducible arnT, not by exogenous genes.
The STs of CST-heteroresistant isolates were diverse,
but E. asburiae ST1939 (hsp60 cluster I), E. kobei ST125(hsp60 cluster II), and E. roggenkampii ST997 (hsp60
cluster IV) accounted for more than 40% of each species.
E. kobei ST125 and E. roggenkampii ST997 have beenclinically isolated in many countries [35–38]. These findings suggest that certain CST-heteroresistant ECC lineages have spread worldwide.
We evaluated the skip-well phenomenon with thebroth microdilution method, which is expected to be aneasy and useful approach to screen heteroresistant isolates [11]. However, only 26.3% of CST-heteroresistantisolates exhibited the skip-well phenomenon. The isolates with a higher occurrence frequency of CST-resistant subpopulations (from 1 × ­10–4 to 1 × ­10–2) by PAP did
not display the skip-well phenomenon because this frequency exceeded the inoculating cell number used in thesusceptibility test of 5 × ­105 CFU/mL (5 × ­104 CFU/100µL in each well of a 96-well plate). Thus, monitoring
the skip-well phenomenon was an unreliable method todetect CST heteroresistance in ECC clinical isolates. PAP
is a gold standard for detecting heteroresistance [19], but
is laborious. We found CST-heteroresistant isolates grewless or not at all at a certain CST concentration (0.5 or
1 mg/L) and grew proficiently at higher CST concentrations. Thus, measuring bacterial growth by assessing
turbidity ­(OD600) in the presence of various CST concentrations should be convenient method for screening CSTheteroresistant isolates in routine clinical laboratory. It
did not require any additional experiments other thanthose performed to determine CST MICs by the brothmicrodilution method (Additional file 1: Table S2).

Page 15 of 18

Why do CST-resistant subpopulations appear? The
present study demonstrated that the PhoPQ-arnT pathway was involved in CST heteroresistance in certain ECC
species, E. asburiae, E. cancerogenus, E. chuandaensis, E.
cloacae (subsp. cloacae and subsp. dissolvens), E. kobei,
and E. roggenkampii. Contribution of this pathway has
been shown in a reference strain E. cloacae subsp. cloacae
ATCC 13047 [16]. In addition, we revealed that this pathway is activated by CST, and the resulting arnT induction occurred in the presence of CST at a concentration
around the MIC. Thus, CST-heteroresistant phenomenon, namely decreased bacterial growth in the presenceof 0.5 or 1 mg/L CST, was attributed to inadequate (orunresponsive) arnT induction at lower CST concentrations (< 2 mg/L), while induced ArnT overcame the bactericidal activity of CST at higher CST concentrations(≥ 2 mg/L). These findings suggest that the appearance of
CST-resistant subpopulations in a strain displaying CST
heteroresistance reflects the ability of arnT induction by
CST.

Conclusion
This study demonstrates that CST-heteroresistant ECC
is present in clinical specimens from various infection
sites. Inducible CST heteroresistance is mediated via the
PhoPQ-arnT pathway in CST-resistant isolates of certain ECC species, such as E. roggenkampii, E. kobei, E.
chuandaensis, E. cloacae, and a part of E. asburiae. On
the other hand, no CST-resistant isolates were found in
E. hormaechei and E. ludwigii. Therefore, attention isrequired to be paid to CST heteroresistance at all infection sites of these ECC species, which have a common
intrinsic gene background, such as species, hsp60 clustering, MLST, and/or core genomic features. Exogenous mcr
genes, such as mcr-9 and mcr-10, found in ECC clinicalisolates [34, 39, 40] contribute less to CST resistance.

(See figure on next page.)
Fig. 8 Induction of arnT mRNA expression in the presence of CST in ECC. A arnT mRNA induction by CST in various species found in ECC clinicalisolates. The y axis shows relative mRNA expression levels following culture in the presence of 16 mg/L CST for 120 min. The values were normalized
by those in the absence of CST in each strain (set to 1.0). CST-heteroresistant and -susceptible isolates are indicated by red and black circles,
respectively. Isolates of E. hormaechei subsp. hoffmannii, E. hormaechei subsp. oharae, and E. hormaechei subsp. xiangfangensis, which lack the arn
operon, were not examined. a, E. cloacae subsp. cloacae (n = 1) and E. cloacae subsp. dissolvens (n = 2). B Comparison of arnT mRNA induction by CST
between CST-susceptible and -heteroresistant ECC clinical isolates. The y axis shows relative mRNA expression levels in ECC clinical isolates cultured
in the presence of 16 mg/L CST for 120 min. Number in parentheses indicates the number of ECC isolates. The values were normalized by thosein the absence of CST in each strain (set to 1.0). *p < 0.001. C arnT mRNA induction by CST in ECC clinical isolates with low, moderate, and high CST
MICs. The y axis shows relative mRNA expression levels in the presence of 16 mg/L CST normalized by those in the absence of CST in each strain (setto 1.0). Number in parentheses indicates the number of ECC isolates. *p < 0.05 and **p < 0.001

Fukuzawa et al. Ann Clin Microbiol Antimicrob

Fig. 8 (See legend on previous page.)(2023) 22:60

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Fukuzawa et al. Ann Clin Microbiol Antimicrob(2023) 22:60

Abbreviations
ANI	Average nucleotide identity
CLSI	Clinical and Laboratory Standards Institute
CST	Colistin
ECC	Enterobacter cloacae complex
hsp60	Heat shock protein 60 gene
L-Ara4N	4-Amino-4-deoxy-l-arabinose
MALDI-TOF MS	Matrix-assisted laser desorption/ionization time-of-flightmass spectrometry
MHII agar	Mueller–Hinton II agar
MHII broth	Mueller–Hinton II broth
MIC	Minimum inhibitory concentration
MLST	Multilocus sequence typing
PAP	Population analysis profilingpEtN	Phosphoethanolamine
RT-qPCR	Reverse transcription quantitative PCR
SNPs	Single nucleotide polymorphisms
ST	Sequence type
TSB	Trypticase soy broth
WGS	Whole-genome sequencing

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12941-​023-​00610-1.
Additional file 1: Table S1. Sequences of primers used in this study.
Table S2. Characteristics of ECC and K. aerogenes clinical isolates used inthis study. Table S3. Characteristics of CST-heteroresisatnt ECC clinical iso‑lates. Table S4. CST MICs of none-mcr, mcr-9-positive, and mcr-10-positive
ECC isolates. Table S5. Comparison of CST susceptibility of phoPQ-, eptA-,
and arnT-deleted mutants of different ECC species.
Additional file 2: Fig. S1. Contribution of mcr-9 and mcr-10 to CST
heteroresistance of ECC clinical isolates. A Comparison of the occur‑
rence frequencies of CST-resistant subpopulations among none-mcr,
mcr-9-positive, and mcr-10-positive ECC clinical isolates. There was no sig‑
nificant difference among the groups. B–G Comparison of the occurrence
frequencies of CST-resistant subpopulations between mcr-9-positive and-negative B–D and between mcr-10-positive and -negative E–G isolates.
These isolates were selected from the same node of the core genome
phylogeny. PAP was performed with the following CST concentrations:
0.125 (B and E), 1 (C and F), and 8 (D and G) mg/L. There were no signifi‑
cant differences among the groups. a, E. hormaechei subsp. xiangfangensis.
Acknowledgements
Not applicable.
Author contributions
SF participated in experimental works, statistical analyses, and preparing themanuscript. TS conceived the objectives and designed the study protocol
and revised the manuscript. KA performed ANI, MLST, and core genome
SNPs-based phylogenetic analysis. SYamamoto and NO made kind discussion,and revised the manuscript. TS, CN and YS conducted WGS MH supervised
the study ST collected the clinical isolates. SY supervised the study, criticallyrevised and edited the manuscript. All authors approved the final manuscript.
Funding
This work was supported by the Japan Agency for Medical Researchand Development (AMED) (JP20ak0101118h0002, JP223fa627005,
JP22wm0125008, and 22jk021004h0001), JSPS KAKENHI (JP21H03622), Takeda
Science Foundation, the Ministry of Education, Culture, Sports, Science, andTechnology (MEXT), and the Joint Research Program of the International
Institute for Zoonosis Control, Hokkaido University. All funders had no role instudy design, data collection and analysis, decision to publish, or preparationof the manuscript.

Page 17 of 18

Availability of data and materials
Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank
databases under accession numbers from DRX398430 to DRX398524.

Declarations
Ethics approval and consent to participate
This study was approved by the Sapporo Medical University Hospital Institu‑
tional Review Board (IRB No. 272-70).
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Microbiology, Sapporo Medical University School of Medi‑cine, Sapporo, Japan. 2 Clinical Laboratory, National Hospital Organization
Hokkaido Cancer Center, Sapporo, Japan. 3 Laboratory of Veterinary Hygiene,
Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan. 4 Gradu‑
ate School of Infectious Diseases, Hokkaido University, Sapporo, Japan. 5 One
Health Research Center, Hokkaido University, Sapporo, Japan. 6 Departmentof Microbiology and Infectious Diseases, Toho University School of Medicine,Tokyo, Japan. 7 Division of Bioresources, Hokkaido University International
Institute for Zoonosis Control, Sapporo, Japan. 8 International Collaboration
Unit, Hokkaido University, International Institute for Zoonosis Control, Sapporo,
Japan. 9 Institute for Vaccine Research and Development (HU‑IVReD), Hokkaido
University, Sapporo, Japan. 10 Department of Infection Control and Laboratory
Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan.
11
Division of Laboratory Medicine, Sapporo Medical University Hospital, Sap‑poro, Japan.
Received: 14 April 2023 Accepted: 5 July 2023

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