Yun-Yi Yang; Min-Ho Suh

doi:10.4167/jbv.2019.49.3.95

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Yang and Suh: Diversity of Genetic Environment of blaCTX-M Genes and Antimicrobial Susceptibility in Extended-spectrum β-lactamase producing Escherichia coli Isolated in Korea

Original Article

J Bacteriol Virol 2019;49(3):95-114.

Published online: 9 September 2019

DOI: https://doi.org/10.4167/jbv.2019.49.3.95

Diversity of Genetic Environment of bla_CTX-M Genes and Antimicrobial Susceptibility in Extended-spectrum β-lactamase producing Escherichia coli Isolated in Korea

Yun-Yi Yang, Min-Ho Suh^∗

Department of Microbiology, Keimyung University School of Medicine, 1035 Dalgubeol-daero, Dalseo-Gu, Daegu, 42601, Korea

Corresponding Min-Ho Suh Department of Microbiology, Keimyung University School of Medicine, 1035 Dalgubeol-daero, Dalseo-Gu, Daegu, 42601, Korea. Phone: +82-53-258-7371 Fax: +82-53-258-7363 E-mail: minho@dsmc.or.kr

Received 30 July 201927 August 2019 Accepted 28 August 2019

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Increasing resistance due to the production of extended-spectrum β-lactamase (ESBL) in Escherichia coli is a major problem to public health and CTX-M enzymes have become the most prevalent ESBL worldwide. In this study, resistance profiles of E. coli isolated in Korea and the genetic environments of bla_CTX-M genes were analyzed by PCR and direct sequencing to clarify the mechanisms of spread of CTX-M. Resistance rates of CTX-M-producing E. coli, including β-lactams, fluoroquinolones and aminoglycosides, were significantly higher than that of CTX-M-non-producers (p<0.01). Of 41 tested, 39 (95.1%) isolates of CTX-M-producing E. coli showed resistance transfer by conjugation. All the transconjugants harboured large plasmids of 118~172 megadalton. Insertion sequence ISEcp1B was detected in the upstream of the bla_CTX-M in 38 (92.7%) isolates with bla_CTX-M. ISEcp1B was disrupted by IS26 in 16(39.0%) isolates with bla _CTX-M. ISEcp1B carried -35 and -10 promoter components between right inverted repeat (IRR) and the start codon of bla_CTX-M. orf477 or IS903D was observed in the downstream of the bla_CTX-M in all the isolates with bla_{CTX-M-3/15/55} or with bla _CTX-M-14/27, respectively. Sequence similar to IRR of ISEcp1B was located downstream of orf477. Target duplication sequences were detected both upstream of IRL and downstream of IRR. These results showed the involvement of ISEcp1B in the mobilization of the resistance genes. In conclusion, the surrounding DNAs of bla_CTX-M genes were very diverse, and the spread and the expression of CTX-M may be deeply related with ISEcp1B. These informations will provide important knowledge to control the increase in CTX-M-ESBLs.

Keywords: ESBL, CTX-M, ISEcp1B, IS26, Escherichia coli

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Figure 1.

Resistance to increasing numbers of different antibiotics in CTX-M-producing (░) and CTX-M-non-producing (■) isolates.

Figure 2.

Schematic representation of the surrounding DNA sequences of the bla_CTx-_m-3 genes of four multidrug resistant Escherichia coli isolates. Row1, JE120: 2, JE125: 3, JE64: 4, JE62. TD, Target duplication. IRL and IRR, left and right inverted repeat sequences for ISEcp1B or IS26. tnpA, transposase gene. ATG, start codon. TAA, stop codon. P, promotor.

Figure 3.

Nucleotide sequence of DNA region of CTX-M-3-producing Escherichia coli JE1 20. The deduced amino acid sequence is indicated in single-letter code below the nucleotide sequence. Horizontal arrows, start codons. Asterisks, stop codons. TD, target duplication. IRL and IRR, left and right inverted repeat sequences, respectively. RBS, ribosomal binding site. The -35 and -10 signals, the promoter sequences of the bla_cTx-M-3. tnpA, transposase gene.

Figure 4.

Schematic representation of the surrounding DNA sequences of the blacTx-M-15 and blacTx-M-55 genes of five multidrug resistant Escherichia coli isolates. Row1, JE36: 2, JE56 and JE11: 3, JE77: 4, JE28. TD, Target duplication. IRL and IRR, left and right inverted repeat sequences for IS26 or ISEcp1B. tnpA, transposase gene. ATG, start codon. TAA, stop codon P, promotor. a. TTCCT in JE11 (CTX-M-55). b. 45 bp in JE11 (CTX-M-55).

Figure 5.

Schematic representation of the surrounding DNA sequences of the blacTx-M-14 genes of four multidrug resistant Escherichia coli isolates. Row1, JE45: 2, JE121: 3, JE123: 4, JE124. TD, Target duplication. IRL and IRR, left and right inverted repeat sequences for ISEcp/Bor IS26. tnpA, transposase gene. ATG and GTG, start codon. TAA, stop codon. P, promotor.

Figure 6.

Schematic representation of the surrounding DNA sequences of the blactx-m-27 genes of three multidrug resistant E scherichia coli isolates. Row1, JE22: 2, JE78: 3, JE80. TD, Target duplication. IRL and IRR, left and right inverted repeat sequences for ISEcp/Bor IS26. tnpA, transposase gene. iron, iron transport gene. ATG and GTG, start codon. TAA, stop codon. P, promotor.

Table 1.

Primer sequences used for PCR and DNA sequencing

Primer name	Primer sequence (5’ to 3’)	GenBank accession no.	Position of primer
CTX-M3-S	CGT CAC GCT GTT GTT AGG AA	AM003906	1520-1539
CTX-M3-AS	ACG GCT TTC TGC CTT AGG TT	AM003906	2300-2281
CTX-M3-F	CTT CCA GAA TAA GGA ATC CCA T	AM003906	1453-1474
CTX-M3-R1	CCC ATT CCG TTT CCG CTA	AM003906	2366-2349
CTXM914F	GCT GGA GAA AAG CAG CGGAG	AJ972955	1751-1770
CTXM914R	GTA AGC TGA CGC AAC GTC TG	AJ972955	2224-2205
CTX-M9-S	TAT TGG GAG TTT GAG ATG GT	AJ972955	1620-1639
CTX-M9-AS	TCC TTC AAC TCA GCA AAA GT	AJ972955	2552-2533
CTX-M19-Rf	CCG TTG CAC TCT CTT TGT CA	AJ972955	1658-1639
TEM-S	ATA AAA TTC TTG AAG ACG AAA	AB103506	166-186
TEM-AS	GAC AGT TAC CAA TGC TTA ATC A	AB103506	1245-1225
SHV/F	CGC CGG GTT ATT CTT ATT TGT CGC	X04515	215-238
SHV/R	TCT TTC CGA TGC CGC CGC CAG TCA	X04515	1223-1204
ISEcp1-F	ATC TAA CAT CAA ATG CAG G	AF458080	4958-4976
ISEcp1-R	AGA CTG CTT CTC ACA CAT	AF458080	6338-6321
ISEcp1-M	TGT CGT ATC TCG CGT ACT GAA	AF458080	5754-5774
ISEcp1-R-P2	TTT CCG CAG CAC CGT TTG C	AF458080	5497-5479
PROM-P4	TGC TCT GTG GAT AAC TTG C	AF458080	6218-6236
ISEcp1-TNPF	TTG GGC GAA TGA AGC CGT GT	AF550415	2680-2699
IS903-F	CAC ATG AAA TCA TCT GCG C	AF458080	7863-7881
IS903-R	CCG TAG CGG GTT GTG TTT TC	AF458080	8335-8316
IS903-R2	G CGC AGA TGA TTT CAT GTG	AF458080	7881-7863
IS903-5′-F	CTA CGG CAC CAC CAA TGA TA	AJ972954	2360-2379
IS903-5′-R	CAT CAT CCA GCC AGA AAG TT	AJ972954	2680-2661
IS26tnpA-3′F	AGC GGT AAA TCG TGG AGT GA	AM003906	540-559
IS26-2F	CCG GCC TTT GAA TGG GTT CAT	AM003906	667-687
IS26-2R	ATG AAC CCA TTC AAA GGC CGG	AM003906	687-667
Orf477-F	ACT TCA AAA ATT ATG CCA CC	AM003906	2531-2550
Orf477-R	GG TGG CAT AAT TTT TGA AGT	AM003906	2550-2531
mucA lower-R	GGC ATC AGG CAG GGG TAA GG	AM003906	3312-3293
PemK-up	AAC GAG AAT GGC TGG ATG C	AF550415	968-986

Table 2.

Antibiotic susceptibility of CTX-M-producing and CTX-M-non-producing Escherichia coliisolated in Korea

Antibiotic	CTX-M-producing isolates (n=41)						CTX-M-non-producing isolates (n=80)						p value
Antibiotic	MIC₅₀ (mg/L)	MIC₉₀ (mg/L)	MIC range (mg/L)	S n(%)	I n(%)	R n(%)	MIC₅₀ (mg/L)	MIC₉₀ (mg/L)	MIC range (mg/L)	S n(%)	I n(%)	R n(%)	S	I	R
cefotaxime	512	>512	32->512	0(0)	0(0)	41(100)	0.064	3	0.023-32	60(75.0)	12(15.0)	8(10.0)	<0.001	0.008^a	<0.001
cefotaxime-clavulanate	2	4	025->256	10(24.4)	30(73.2)	1(2.4)	0.064	3	0.023-256	65(81.3)	7(8.7)	8 (10.0)	<0.001	<0.001	0.270^a
ceftazidime	2	64	0.064->512	23(56.1)	3(7.3)	15(36.6)	1	8	0.016-64	71(88.8)	4(5.0)	5(6.2)	<0.001	0.688^a	<0.001
ceftazidime- clavulanate	0.5	1	0.125->512	38(92.7)	2(4.9)	1(2.4)	0.5	4	0.023-32	72(90.0)	4(5.0)	4(5.0)	0.748^a	0.661^a	1.000^a
imipenem	0.5	0.5	0.125-2	39(95.1)	2(4.9)	0(0)	0.5	1	0.023-2	74(92.5)	6(7.5)	0(0)	0.715^a	0.715^a
aztreonam	8	256	0.064->256	13(31.7)	9(22.0)	19(46.3)	0.5	2	0.064-16	75(93.8)	3(3.7)	2(2.5)	<0.001	0.003^a	<0.001
ampicillin	>512	>512	1->512	2(4.9)	0(0)	39(95.1)	256	>512	0.125->512	36(45.0)	2(2.5)	42(52.5)	<0.001	0.548^a	<0.001
nalidixic acid	>256	>256	4->256	4(9.8)	0(0)	37(90.2)	16	>256	4->256	39(48.8)	2(2.5)	39(48.8)	<0.001	0.548^a	<0.001
ciprofloxacin	>64	>64	0.008->64	9(22.0)	0(0)	32(78.0)	0.004	>64	0.004->64	54(67.5)	3(3.7)	23(28.8)	<0.001	0.550^a	<0.001
chloramphenicol	2	>512	0.25->512	34(82.9)	2(4.9)	5(12.2)	2	4	0.5->512	76(93.1)	1(1.1)	3(5.8)	0.043^a	0.265^a	0.119^a
tetracycline	8	>512	1->512	20(48.8)	3(7.3)	18(43.9)	8	>512	0.5->512	37(46.3)	26(32.5)	17(21.2)	0.792	0.002	0.009
streptomycin	32	>256	4->256	11(26.8)	8(19.5)	22(53.7)	8	128	1->256	48(60.0)	15(18.8)	17(21.2)	0.001	0.919	<0.001
amikacin	8	64	0.25->512	33(80.5)	2(4.9)	6(14.6)	4	8	0.25-8	80(100)	0(0)	0(0)	<0.001^a	0.113	0.001^a
tobramycin	1	128	0.064->512	22(53.7)	1(2.4)	18(43.9)	0.5	8	0.125->512	67(83.8)	8(10.0)	5(6.2)	<0.001	0.270^a	<0.001
kanamycin	32	>512	2->512	17 (41.5)	8(19.5)	16(39.0)	16	32	4->512	61(76.3)	14(17.5)	5(6.2)	<0.001	0.786	<0.001
gentamicin	1	>512	0.125->512	21(51.2)	2(4.9)	18(43.9)	0.5	>512	0.064->512	66(82.6)	1(1.1)	13(16.3)	<0.001	0.265^a	0.001
sulfamethoxazol-trimethoprim	>320	>320	20->320	18(43.9)	1(2.4)	22(53.7)	20	>320	10->320	56(70.0)	1(1.1)	23(28.8)	0.005	1.000^a	0.007

S, susceptible; I, intermediate; R, resistant. The p value was computed using the chi-square test;

^a Fisher's exact test.

Table 3.

Antibiotic susceptibility of CTX-M-14-producing and CTX-M-27-producing Escherichia coliisolated in Korea

Antibiotic	CTX-M-14-producing isolates (n=18)						CTX-M-27-producing isolates (n=5)						p value^a
Antibiotic	MIC₅₀ (mg/L)	MIC₉₀ (mg/L)	MIC range (mg/L)	S n(%)	I n(%)	R n(%)	MIC₅₀ (mg/L)	MIC₉₀ (mg/L)	MIC range (mg/L)	S n(%)	I n(%)	R n(%)	S	I	R
cefotaxime	256	>512	63->512	0(0)	0(0)	18(100)	>512	>512	>512	0(0)	0(0)	5(100)	.	.	.
cefotaxime-clavulanate	2	4	05->256	5(27.8)	13(72.2)	0(0)	2	4	2-4	1(20)	4(80.0)	0(0)	1.000	1.000	.
ceftazidime	2	16	0.25-128	14(77.8)	2(11.1)	2(11.1)	32	64	0.5-64	1(20.0)	1(20.0)	3(60.0)	0.033	0.539	0.048
ceftazidime- clavulanate	1	8	0.125-64	16(88.9)	1(5.6)	1(5.6)	0.5	1	0.25-1	5(100)	0(0)	0(0)	1.000	1.000	1.000
imipenem	0.5	2	0.125-2	16(88.9)	2(11.1)	0(0)	0.25	0.5	0.125-0.5	5(100)	0 (0)	0(0)	1.000	1.000	.
aztreonam	8	128	0.125->256	8(44.4)	6(33.3)	4(22.2)	32	64	0.25-64	1(20.0)	1(20.0)	3(60.0)	0.611	1.000	0.142
ampicillin	>512	>512	1->512	2(11.1)	0(0)	16(88.9)	>512	>512	>512	0(0)	0(0)	5(100)	1.000	.	1.000
nalidixic acid	>256	>256	4->256	3(16.7)	0(0)	15(83.3)	>256	>256	128->256	0(0)	0(0)	5(100)	1.000	.	1.000
ciprofloxacin	16	>64	0.008->64	6(33.3)	0(0)	12(66.7)	>64	>64	0.25->64	2(40.0)	0(0)	3(60.0)	1.000	.	1.000
chloramphenicol	2	>512	0.25->512	16(88.9)	0(0)	2(11.1)	1	2	0.5-2	5(100)	0(0)	0(0)	1.000		1.000
tetracycline	4	>512	1->512	11(61.1)	2(11.1)	5(27.8)	>512	>512	4->512	2(40.0)	0(0)	3(60.0)	0.618	1.000	0.297
streptomycin	16	>256	4->256	5(27.8)	5(27.8)	8(44.4)	>256	>256	32- >256	0(0)	0(0)	5(100)	0.545	0.545	0.046
amikacin	4	8	0.25->512	17(94.4)	0(0)	1(5.6)	8	8	2-8	5(100)	0(0)	0(0)	1.000	.	1.000
tobramycin	2	32	0.064- 32	10(55.6)	0(0)	8(44.4)	0.5	1	0.25-1	5(100)	0(0)	0(0)	0.122	.	0.122
kanamycin	32	64	2->512	9 (50.0)	7(38.9)	2 (11.1)	16	16	8-16	5(100)	0(0)	0(0)	0.116	0.272	1.000
gentamicin	128	>512	0.125->512	8(44.4)	1 (5.6)	9(50.0)	0.5	1	0.25-1	5(100)	0(0)	0(0)	0.046	1.000	0.116
sulfamethoxazol-trimethoprim	>320	>320	10->320	9(50.0)	0 (0)	9(50.0)	>320	>320	40->320	1(20.0)	0(0)	4(80.0)	0.339	.	0.339

S, susceptible; I, intermediate; R, resistant.

^a The p value was computed using the Fisher's exact test.

Table 4.

Antibiotic susceptibility of CTX-M group 1-producing and CTX-M group 9-producing Escherichia coliisolated in Korea

Antibiotic	CTX-M group 1-producing isolates (n=18)						CTX-M group 9-producing isolates (n=23)						p value
Antibiotic	MIC₅₀ (mg/L)	MIC₉₀ (mg/L)	MIC range (mg/L)	S n(%)	I n(%)	R n(%)	MIC₅₀ (mg/L)	MIC₉₀ (mg/L)	MIC range (mg/L)	S n(%)	I n(%)	R n(%)	S	I	R
cefotaxime	>512	>512	32- >512	0(0)	0(0)	18(100)	256	>512	64- >512	0(0)	0(0)	23(100)	.	.	.
cefotaxime-clavulanate	2	4	0.25-256	4(22.2)	13(72.2)	1(5.5)	2	2	0.5- 256	6(26.1)	17(73.9)	0 (0)	1.000^a	1.000^a	0.44^a
ceftazidime	32	128	0.064->512	8(44.4)	0(0)	10(55.6)	2	32	0.25-128	15(65.2)	3(13.0)	5(21.7)	0.219	0.243^a	0.049
ceftazidime- clavulanate	0.5	1	0.25-1	17(94.4)	1(5.5)	0(0)	0.5	1	0.125-64	21(91.3)	1(4.3)	1(4.3)	1.000^a	1.000^a	1.000^a
imipenem	0.5	0.5	0.125- 0.5	18(100)	0(0)	0(0)	0.25	0.5	0.125-2	21(91.3)	2 (8.7)	0(0)	0.495^a	0.495^a	.
aztreonam	64	>256	0.064->256	4(22.2)	2(11.1)	12(66.7)	8	64	0.125-256	9(39.1)	7(30.4)	7(30.4)	0.321	0.254^a	0.030
ampicillin	>512	>512	>512	0(0)	0(0)	18(100)	>512	>512	1->512	2(8.7)	0(0)	21(91.3)	0.495^a	.	0.495^a
nalidixic acid	>256	>256	4->256	1(5.5)	0(0)	17(94.4)	>256	>256	4->256	3(13.0)	0(0)	20(87.0)	0.618^a	.	0.618^a
ciprofloxacin	>64	>64	0.012->64	3(16.7)	0(0)	15(83.3)	12	>64	0.008->64	8(34.8)	0(0)	15(65.2)	0.291^a	.	0.291^a
chloramphenicol	2	>512	1->512	13(72.2)	2(11.1)	3(16.7)	1	4	0.25->512	21(91.3)	0(0)	2(8.7)	0.209^a	0.187^a	0.638^a
tetracycline	>512	>512	1->512	7(38.9)	1(5.5)	10(55.5)	4	>512	1->512	13(56.5)	2(8.7)	8(34.8)	0.262	1.000^a	0.183
streptomycin	16	>256	4->256	6(33.3)	3(16.7)	9(50.0)	32	>256	4- >256	5(21.7)	5(21.7)	13(56.5)	0.489^a	1.000^a	0.678
amikacin	8	>512	0.25->512	11(61.1)	2(11.1)	5(27.8)	4	8	0.25->512	22(95.7)	0(0)	1(4.3)	0.013^a	0.187^a	0.070^a
tobramycin	16	>512	0.064->512	7 (38.9)	1(5.5)	10(55.5)	0.5	32	0.064-32	15(65.2)	0(0)	8(34.8)	0.093	0.439^a	0.183
kanamycin	128	>512	8->512	3 (16.7)	1(5.5)	14 (77.8)	16	32	2- >512	14(60.9)	7(30.4)	2(8.7)	0.004^a	0.059^a	<0.001
gentamicin	8	>512	0.125->512	8(44.4)	1 (5.5)	9(50.0)	0.5	>512	0.125->512	13(56.5)	1(4.3)	9(39.1)	0.443	1.000^a	0.486
sulfamethoxazol-trimethoprim	60	>320	10->320	8(44.4)	1 (5.5)	9(50.0)	>320	>320	10->320	10(43.5)	0(0)	13(56.5)	0.951	0.439^a	0.678

S, susceptible; I, intermediate; R, resistant. The p value was computed using the chi-square test.

^a Fisher's exact test.

Table 5.

Genotypic characterization of bla _CTX-M surrounding DNAs of CTX-M group 1-producing Escherichia coliisolated in Korea

Strain	CTX-M identified by sequencing	Upstream bla _CTX-M							Downstream bla _CTX-M
		tnpA	pemK	TD	IRL	tnpA	Promoter	IRR	Space^a	Size (bp)	Space^a	orf477	IRR-like	TD	mucA
		IS26	start		ISEcp1B	ISEcp1B	bla _CTX-M	ISEcp1B	(bp)	bla _CTX-M	(bp)		ISEcp1B
JE120	CTX-M-3	-	+	+	+	+	+	+	127	876	46	+	+	+	+
JE125	CTX-M-3	-	+	+	+	+	+	+	127	876	46	+	+	+	-
JE64	CTX-M-3	+	-	-	-	T	+	+	127	876	46	+	+	+	-
JE62	CTX-M-3	-	-	-	-	T	+	+	127	876	46	+	+	+	-
JE36	CTX-M-15	+	-	-	-	T	+	+	48	876	46	+	+	+	-
JE56	CTX-M-15	-	-	+	+	+	+	+	48	876	46	+	+	+	-
JE77	CTX-M-15	-	-	-	-	T	+	+	48	876	46	+	+	+	-
JE28	CTX-M-15	-	-	-	-	T	+	+	48	876	46	+	+	+	-
JE11	CTX-M-55	-	-	+	+	+	+	+	45	876	46	+	+	+	-

TD, Target duplication of ISEcp1B; IRL, Inverted repeat left; IRR, Inverted repeat right; T, Truncated in upstream sequence of the gene

^a Space between IRR and start codon of bla _CTX-M gene

^b Space between stop codons of bla _CTX-M gene and orf477

Table 6.

Genotypic characterization of bla _CTX-M surrounding DNAs of CTX-M group 9-producing Escherichia coliisolated in Korea

Strain	CTX-M identified by sequencing	Upstream bla _CTX-M								Downstream bla _CTX-M
		IRR	tnpA	TD	IRL	tnpA	Promoter	IRR	Space^a	Size (bp)	Space^b	IS903D	iron	IRR-like	TD	tnpA
		IS26	IS26		ISEcp1B	ISEcp1B	bla _CTX-M	ISEcp1B	(bp)	bla CTX-M	(bp)			ISEcp1B		IS26
JE45	CTX-M-14	-	-	+	+	+	+	+	42	876	79	T^d	-	-	-	-
JE121	CTX-M-14	-	-	-	-	T^c	+	+	42	876	79	T^d	-	-	-	-
JE123	CTX-M-14	+	+	-	-	T^c	+	+	42	876	79	T^d	-	-	-	-
JE124	CTX-M-14	+	+	-	-	T^c	+	+	42	876	79	T^d	-	-	-	-
JE22	CTX-M-27	+	+	-	-	T^c	+	+	42	876	79	T^d	-	-	-	+
JE78	CTX-M-27	+	+	-	-	T^c	+	+	42	876	79	+	+	+	+	T^d
JE80	CTX-M-27	+	+	-	-	T^c	+	+	42	876	79	T^d	-	-	-	-

TD, Target duplication of ISEcp1B; IRL, Inverted repeat left; IRR, Inverted repeat right

^a Space between IRR and start codon of bla _CTX-M gene

^b Space between stop codon of bla _CTX-M gene and start codon of IS903DtnpA

^c Truncated in upstream sequence of the gene

^d Truncated in downstream sequence of the gene.

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