Biological and Genetic Characteristics of Clinically Isolated Enterobacter cloacae with Multidrug Resistance

Jae-Jung Kim; Sun Hoe Koo

doi:10.3343/lmo.2018.8.3.99

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Kim and Koo: Biological and Genetic Characteristics of Clinically Isolated Enterobacter cloacae with Multidrug Resistance

Original Article

Lab Med Online 2018;8(3):99-106.

Published online: 19 July 2018

DOI: https://doi.org/10.3343/lmo.2018.8.3.99

Biological and Genetic Characteristics of Clinically Isolated Enterobacter cloacae with Multidrug Resistance

Jae-Jung Kim, M.T.¹, Sun Hoe Koo, M.D.²

¹Department of Laboratory Medicine, St. Mary's Hospital, Daejeon Catholic University, Daejeon, Korea

²Department of Laboratory Medicine, Chungnam National University Hospital, Daejeon, Korea

Corresponding author: Sun Hoe Koo Department of Laboratory Medicine, Chungnam National University Hospital, 282 Munhwa-ro, Joong-gu, Daejeon 35015, Korea Tel: +82-42-280-7798, Fax: +82-42-257-5365, E-mail: shkoo@cnu.ac.kr

Received 22 November 2017 Revised 27 November 2017 Accepted 27 November 2017

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background:

From January 2014 to December 2015, 69 clones of Enterobacter cloacae showing multidrug resistance to six classes of antimicrobial agents were collected from two medical centers in Korea.

Methods:

Minimum inhibitory concentrations were determined using the E-test method, and 17 genes were detected using polymerase chain reaction (PCR). The epidemiological relatedness of the strains was identified using repetitive element sequence-based PCR and multilocus sequence typing.

Results:

The 69 E. cloacae clones produced extended spectrum β lactamase (ESBL) and AmpC and showed multidrug resistance to cefotaxime, ceftazidime, and aztreonam. We identified the following sequence types: ST56 of type VI for ESBL SHV (N=12, 17.4%); ST53, ST114, ST113, and ST550 of types I, IV, VI, and VII, respectively, for CTX-M (N=11, 15.9%); and ST668 of type III for the carbapenemase NDM gene (N=1, 1.5%). The AmpC DHA gene (N=2, 2.89%) was confirmed as ST134, although its type was not identified, whereas EBC (MIR/ACT; N=18, 26.1%) was identified as ST53, ST24, ST41, ST114, ST442, ST446, ST484, and ST550 of types V, I, III, IV, VII, and VI, respectively. The formed subclasses were blaCTX-M-3 and blaCTX-M-22 by CTX-M-1, blaCTX-M-9 and blaCTX-M-125 by CTX-M-9, blaDHA-1 by DHA, and blaMIR-7 and blaACT-15,17,18,25,27,28 by EBC (MIR/ACT).

Conclusions:

There were no epidemiological relationships between the gene products and the occurrence of resistance among the strains.

Keywords: Enterobacter cloacae, Extended spectrum β lactamase, AmpC, Repetitive element sequence-based polymerase chain reaction, Multilocus sequence typing, Multidrug resistance

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Fig. 1.

REP-PCR patterns of E. cloacae isolates. Lane M 0.05–2.5 kb molecular size marker. Lane 2, 8, 11, 14, 23–25, 30-31, 43, 70: I type. Lane 1, 48–49, 51, 53, 56–57, 59: II type. Lane 33, 37, 61, 63, 65–68: III type. Lane 38–40, 42, 44, 47, 52: IV type. Lane 4, 18–19, 26, 34, 64, 69: V type. Lane 12, 20, 50, 54, 60, 46: VI type. Lane 9–10, 13, 17, 28: VII type. Lane 35–36, 62: VIII type. Lane 3, 22, 32: IX type. Lane 45, 55: X type.

Table 1.

Sequences of primers for the amplification of the β-lactamase and AmpC genes of Enterobacter cloacae

Primer		Primer sequence (5’–3’)	Size	Ref
CTXM-1	F	CCGTCACGCTGTTGTTAGG	693	26
	R	GACGATTTTAGCCGCCGAC
CTXM-2	F	CGGTGCTTAAACAGAGCGAG	684
	R	CCATGAATAAGCAGCTGATTGCCC
CTXM-8	F	ACGCTCAACACCGCGATC	695
	R	CGTGGGTTCTCGGGGATAA
CTXM-9	F	GATTGACCGTATTGGGAGTTT	683
	R	CGGCTGGGTAAAATAGGTCA
TEM-1	F	ATGAGTATTCAACATTTCCGT	997	27
	R	TTACCAATGCTTAATCAGTGA
SHV-12	F	CCGGGTTATTCTTATTTGTCGCT	936
	R	TAGCGTTGCCAGTGCTCG
NDM-1	F	GCCCAATATTATGCACCCGG	738	28
	R	CTCATCACGATCATGCTGGC
IMP-1	F	AAGGCGTTTATGTTCATACTTCG	605
	R	TTTAACCGCCTGCTCTAATGTAA
OXA-48	F	GATTATCGGAATGCCTGCGG	845
	R	CTACAAGCGCATCGAGCATCA
IMI-1	F	AGAGTTCYATTCACCCATCACA	803	29
	R	TCTCCAATCGACCGCATGAA
VIM-1	F	TGGGCCATTCAGCCAGATC	749	31
	R	TGGGCCATTCAGCCAGATC
FOX	F	AACATGGGGTATCAGGGAGATG	190	32
	R	CAAAGCGCGTAACCGGATTGG
EBC	F	TCGGTAAAGCCGATGTTGCGG	302
	R	CTTCCACTGCGGCTGCCAGTT
CIT	F	TGGCCAGAACTGACGGCAAA	462
	R	TTTCTCCTGAACGTGGCTGGC
ACC	F	AACAGCCTCAGCAGCCGGTTA	346
	R	TTCGCCGCAATCATCCCTAGC
DHA	F	AACTTTCACAGG TGTGCTGGGT	405
	R	CCGTACGCATACTGGCTTTGC
MOX	F	GCTGCTCAAGGAGCACAGGAT	520
	R	CACATTGACATAGGTGTGGTGC

Abbreviations: F, forward; R, reverse.

Table 2.

Primers for the multilocus sequence typing analysis of En terobacter cloacae

Primer			Sequence (5’–3’)	Position in the target gene	Ref
A	dnaA	F	AYAACCCGCTGTTCCTBTATGGCGGCAC	500–527∗	33
		R	KGCCAGCGCCATCGCCATCTGACGCGG	1222–1248∗
	fusA	F	TCGCGTTCGTTAACAAAATGGACCGTAT	413–440∗
		R	TCGCCAGACGGCCCAGAGCCAGACCCAT	1291–1318
	gyrB	F	TCGACGAAGCGCTCGCGGGTCACTGTAA	143–170
		R	GCAGAACCGCCCGCGGAGTCCCCTTCCA	1268–1295
	leuS	F	GATCARCTSCCGGTKATCCTGCCGGAAG	1342–1369∗
		R	ATAGCCGCAATTGCGGTATTGAAGGTCT	2159–2186∗
	pyrG	F	AYCCBGAYGTBATTGCRCAYMAGGCGAT	56–83∗
		R	GCRCGRATYTCVCCCTSHTCGTCCCAGC	563–590∗
	rplB	F	GTAAACCGACATCTCCGGGTCGTCGCCA	17–44∗
		R	ACCTTTGGTCTGAACGCCCCACGGAGTT	735–762∗
	rpoB	F	CCGAACCGTTCCGCGAACATCGCGCTGG	252–280∗
		R	CCAGCAGATCCAGGCTCAGCTCCATGTT	973–1000∗
S	gyrB	F	AAAACCGGTACYATGGTGCGTTTCTGG	484–510∗
		R	GCAGAACCGCCCGCGGAGTCCCCTTCC	1269–1295∗
	fusA	R	ATCTCTTCACGYTTGTTAGCGTGCATCT	1094–1121∗

^∗ These primers were used for sequencing respective amplicons. Abbreviations: A, amplification; F, forward; R, reverse; S, sequencing.

Table 3.

Characteristics of multidrug-resistant Enterobacter cloacae isolates based on repetitive element sequence-based polymerase chain reaction and multilocus sequence typing

Isolate	Specimen source	REP-PCR	MLST (STs)	) β-lactamase	AmpC	GM	ETP	CT	TZ	PM	CL	AT	TS
K1	W	II	668	NDM-1	.	0.5	0.25	>32	16	0.25	0.064	12	0.064
K2	S	I	24	.	DHA, EBC	0.5	0.25	>32	16	0.25	0.064	12	0.064
K3	W	IX	134	.	DHA	1.5	0.064	1	4	0.047	0.38	0.5	0.5
K4	O	V	41	.	EBC	>256	1	>48	>32	24	1.5	128	>32
K5	U	.	280	.	.	0.5	3	>32	>256	1	0.064	128	0.125
K6	U	.	.	.	.	0.5	0.25	>32	32	0.05	0.047	24	0.064
K7	S	.	78	.	.	0.38	0.75	>32	>256	2	0.047	128	0.125
K8	B	I	.	.	.	0.5	0.032	_>32	3	16	_>32	16	_>32
K9	U	VII	.	.	.	0.5	0.19	_>32	_>256	1.5	0.032	24	0.064
K10	O	VII	279	.	.	0.5	0.125	_>32	128	1.5	0.032	16	0.064
K11	O	I	45	SHV-12	.	0.05	0.75	_>32	_>256	2	0.38	64	0.5
K12	U	VI	41	.	EBC	2	0.25	_>32	128	2	1.5	_>256	_>256
K13	S	VII	53	.	EBC	0.5	0.38	_>32	_>256	1	0.47	128	0.064
K14	BF	I	.	.	.	0.38	0.5	_>32	_>256	0.075	0.5	64	0.32
K15	U	.	190	.	.	0.75	0.008	0.19	0.5	0.064	0.064	0.25	0.064
K17	O	VII	245	.	.	>256	0.032	>32	2	8	0.047	8	0.38
K18	U	V	78	.	.	1	0.19	1	1	0.75	1.5	0.38	0.25
K19	U	V	422	.	EBC	>256	1	>32	32	48	>32	64	0.25
K20	B	VI	133	CTX-M-9	.	0.25	0.19	>32	48	0.38	0.023	24	0.064
K21	N.S	.	148	.	.	6	0.38	>32	1.5	3	1	2	>32
K22	Ctip	IX	244	.	.	0.38	0.25	>32	128	1.5	0.047	24	0.094
K23	W	I	.	.	.	0.5	0.25	>32	>256	3	0.064	128	0.047
K24	S	I	53	CTX-M-9	EBC	0.5	0.19	>32	>256	2	0.32	24	0.064
K25	S	I	53	.	EBC	4	0.5	>32	256	3	0.75	64	>32
K26	O	V	484	.	EBC	0.38	0.25	>32	256	1	0.75	96	0.047
K27	S	.	584	.	.	2.5	0.38	>32	64	0.5	0.047	24	0.094
K28	U	VII	550	CTX-M-9	EBC	0.25	0.5	>32	>256	1	0.064	96	0.064
K29	W	.	.	.	.	6	1.5	>32	>256	6	4	128	>32
K30	U	I	144	.	.	1	0.19	>32	>256	1.5	0.032	24	0.064
K31	B	I	604	.	.	1	1.5	>32	>256	16	0.19	256	0.5
K32	S	IX	78	.	.	0.5	0.25	>32	>256	2	0.032	32	0.094
K33	W	III	.	.	.	0.38	0.047	>32	3	16	24	16	>32
K34	U	V	466	.	EBC	0.75	0.023	0.5	1	0.38	3	0.25	1.5
K35	U	VIII	477	.	.	0.75	0.5	>32	16	0.125	0.16	8	0.094
K36	U	VIII	477	.	.	0.75	0.125	>32	256	0.75	0.047	16	0.125
K37	S	III	148	.	.	0.5	0.064	>32	>256	0.5	0.047	24	0.094
K38	U	IV	114	.	.	0.38	0.38	>32	256	4	0.064	48	0.064
K39	S	IV	114	SHV-12	EBC	0.38	0.19	>32	>256	3	0.047	48	0.064
K40	S	IV	114	.	EBC	0.38	0.25	>32	256	4	0.047	48	0.064
K41	BF	.	.	CTX-M-1	EBC	0.5	0.38	_>32	_>256	4	0.047	48	0.094
K42	S	IV	114	.	EBC	0.5	0.19	_>32	64	0.75	0.064	32	0.064
K43	U	I	51	CTX-M-9, SHV-12	.	0.5	0.25	_>32	_>256	3	0.047	48	0.094
K44	U	IV	114	.	.	6	0.5	>32	256	3	0.19	48	0.064
K45	S	X	.	CTX-M-9,	.	0.5	0.25	>32	>256	3	0.047	48	0.094
				SHV-12
K46	BF	VI	125	.	.	0.5	0.125	>32	64	0.5	0.047	32	0.064
K47	U	IV	114	SHV-12	EBC	0.38	0.38	_>32	48	0.38	0.032	32	0.047
K48	U	II	.	CTX-M-9,	EBC	0.38	0.38	>32	>256	3	0.032	48	0.094
				SHV-12
K49	U	II	175	CTX-M-9, SHV-12	.	0.5	0.32	0.125	0.19	0.064	0.016	0.047	0.094
K50	S	VI	56	SHV-12	.	0.5	0.032	0.19	0.38	0.047	0.016	0.064	0.094
K51	S	II	782	.	.	0.5	0.023	6	16	0.5	0.125	32	>32
K52	PF	IV	114	CTX-M-9	EBC	3	0.25	>32	>256	16	0.75	48	0.094
K53	U	II	.	.	.	0.5	0.25	>32	256	3	0.032	32	0.094
K54	U	VI	.	.	.	0.5	0.047	0.19	0.25	0.047	0.016	0.047	0.125
K55	S	X	.	.	.	0.5	0.008	0.064	0.19	0.047	0.006	0.047	0.064
K56	S	II	.	.	.	0.5	0.38	>32	128	0.5	0.064	32	0.125
K57	BW	II	51	.	.	0.5	0.064	0.75	0.25	0.064	0.023	0.064	0.19
K58	S	XI	350	CTX-M-9,	.	0.5	0.125	>32	64	2	0.032	24	0.064
				SHV-12
K59	U	II	175	CTX-M-9, SHV-12	.	0.38	0.094	>32	48	1.5	0.016	24	0.094
K60	S	VI	56	SHV-12	.	6	0.19	24	16	0.1	0.19	16	>32
K61	S	III	114	.	.	6	0.064	>32	32	0.5	0.19	32	>32
K62	O	VIII	477	SHV-12	.	0.5	0.25	>32	>256	3	0.32	48	0.094
K63	U	III	148	.	.	0.5	0.25	>32	256	0.5	0.032	24	0.064
K64	U	V	133	.	.	0.25	0.75	>32	>256	3	0.064	64	0.064
K65	BF	III	24	OXA-48	EBC	128	0.38	>32	32	1	0.023	24	>32
K66	U	III	114	.	.	0.38	0.004	0.125	0.125	0.032	0.032	0.047	0.047
K67	S	III	53	.	EBC	12	0.75	>32	64	32	12	128	4
K68	U	III	114	.	.	0.38	0.19	>32	64	0.75	0.5	48	0.047
K69	U	V	61	.	.	48	0.19	>32	24	24	>32	48	48
K70	U	I	329	.	.	0.75	0.19	>32	96	1.5	0.032	24	24

Abbreviations: ATM, aztreonam; B, blood; BF, bile fluid; BW, bronchial wash; CIP, ciprofloxacin; CTX, cefotaxime; Ctip, catheter tip; ETP, ertapenem; GM, gentamicin; NS, nasal swab; O, other; PF, pleural fluid; FEP, cefepime; S, sputum; TS, cotrimoxazole; CAZ, ceftazidime; U, urine; W, wound; “.”, Not detected.

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