So Youn Shin; Kye Chul Kwon; Jong Woo Park; Jeong Hoon Song; Young Hyun Ko; Ji Youn Sung; Hae Won Shin; Sun Hoe Koo

doi:10.3343/kjlm.2009.29.6.541

Journal List > Korean J Lab Med > v.29(6) > 1011596

Go to TopGo to Top Go to BottomGo to Bottom

TOOLS

Shin, Kwon, Park, Song, Ko, Sung, Shin, and Koo: Characteristics of aac(6′)-Ib-cr Gene in Extended-Spectrum β-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae Isolated from Chungnam Area

Original Article

Korean J Leg Med 2009;29(6):541-550.

Published online: 14 January 2009

DOI: https://doi.org/10.3343/kjlm.2009.29.6.541

Characteristics of aac(6′)-Ib-cr Gene in Extended-Spectrum β-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae Isolated from Chungnam Area

So Youn Shin, M.D.¹, Kye Chul Kwon, M.D.¹, Jong Woo Park, M.D.¹, Jeong Hoon Song, M.D.¹, Young Hyun Ko, M.D.¹, Ji Youn Sung, M.T.¹, Hae Won Shin, MPH.², Sun Hoe Koo, M.D.¹

¹Department of Laboratory Medicine, Chungnam National University College of Medicine, Daejeon, Korea

²Health Insurance Review & Assessment Service, Health Technology Assessment Department Research Team, Seoul, Korea

Corresponding author: Sun Hoe Koo, M.D. Department of Laboratory Medicine, Chungnam National University Hospital, 640 Daesa-dong, Jung-gu, Daejeon 301-721, Korea Tel: +82-42-280-7798, Fax: +82-42-257-5365 E-mail: shkoo@cnu.ac.kr

Revised 31 March 200926 July 2009 Accepted 13 November 2009

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

Background:

Concomitant quinolone resistance in extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae is a crucial problem in the clinical management of infections. In foreign countries, the fluoroquinolone acetylating aminoglycoside-(6)-N-acetyltransferase (aac[6′]-Ib-cr) gene, a novel plasmid-mediated quinolone resistance determinant has been reported to occur in conjunction with qnr. We aim to investigate the prevalence and characteristics of concomitant aac(6′)-Ib-cr and qnr expression in ESBL-producing Escherichia coli and Klebsiella pneumoniae in Korea.

Methods:

Between December 2007 and April 2008, we collected 60 and 69 clonally unrelated non-repetitive clinical isolates of ESBL-producing E. coli and K. pneumoniae, respectively. We studied the expressions of 11 types of ESBL-encoding genes, 4 types of 16s rRNA methylase genes; rmtA, rmtB, rmtC and armA, 3 types of qnr genes; qnrA, qnrB, qnrS and aac(6′)-Ib. The presence of aac(6′)-Ib-cr variants was detected by sequencing. The involvement of integrons was studied using multiplex PCR and sequencing of gene-cassette arrays. Conjugation experiments were performed to confirm plasmid-mediated resistance and the relationships among coharbored genes.

Results:

We observed a high prevalence of the cr variant (61.1%) of aac(6′)-Ib, and the prevalence of this variant in qnr and aac(6′)-Ib-coharboring isolates (67.4%) was higher than in qnr-negative isolates (51.7%). The high prevalence of the cr variant was significantly related to the high minimum inhibitory concentrations (MICs) of ciprofloxacin, tobramycin, and amikacin and indicated the statistically significant roles of qnrB, qnrS, rmtA, and rmtB in quinolone and aminoglycoside resistance.

Conclusions:

The aac(6′)-Ib-cr variants were widespread and showed significant relation to the high-level quinolone and aminoglycoside resistance in ESBL-producing E. coli and K. pneumoniae. (Korean J Lab Med 2009;29:541-50)

Keywords: aac(6′)-Ib-cr, E. coli, K. pneumoniae, ESBL, Quinolone resistance

REFERENCES

1.Podschun R., Ullmann U. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods and pathogenicity factors. Clin Microbiol Rev. 1998. 11:589–603.

2.Garau J., Xercavins M., Rodríguez-Carballeira M., Gómez-Vera JR., Coll I., Vidal D, et al. Emergence and dissemination of quinolone-resistant Escherichia coli in the community. Antimicrob Agents Chemother. 1999. 43:2736–41.

3.Lautenbach E., Strom BL., Bilker WB., Patel JB., Edelstein PH., Fishman NO. Epidemiological investigation of fluoroquinolone resistance in infections due to extended-spectrum β-lactamase-producing Escherichia Coli and Klebsiella pneumoniae. Clin Infect Dis. 2001. 33:1288–94.

4.Ling TK., Xiong J., Yu Y., Lee CC., Ye H., Hawkey PM. Multicenter antimicrobial susceptibility survey of gram-negative bacteria isolated from patients with community-acquired infections in the People's Republic of China. Antimicrob Agents Chemother. 2006. 50:374–8.

5.Jacoby GA., Chow N., Waites KB. Prevalence of plasmid-mediated quinolone resistance. Antimicrob Agents Chemother. 2003. 47:559–62.

6.Martínez-Martínez L., Pascual A., Jacoby GA. Quinolone resistance from a transferable plasmid. Lancet. 1998. 351:797–9.

7.Klugman KP., Levin BR. One enzyme inactivates two antibiotics. Nat Med. 2006. 12:19–20.

8.Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing: seventeenth informational supplement M100-S17. Wayne, PA: CLSI. 2007.

9.Kang JH., Bae IK., Kwon SB., Jeong SH., Lee JW., Lee WG. Prevalence of Ambler Class A extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates in Korea. Korean J Clin Microbiol. 2005. 8:17–25. (강지혜, 배일권, 권수봉, 정석훈, 이종욱, 이위교등. Ambler Class A extended-spectrum β-lactamase 생성 Escherichia coli 와 Klebsiella pneumoniae 의 국내 분리 현황. 대한임상미생물학회지 2005;8:17-25.).

10.Cattoir V., Weill FX., Poirel L., Fabre L., Soussy CJ., Nordmann P. Prevalence of qnr genes in Salmonella in France. J Antimicrob Chemother. 2007. 59:751–4.

11.Bogaerts P., Galimand M., Bauraing C., Deplano A., Vanhoof R., De Mendonca R, et al. Emergence of ArmA and RmtB aminoglycoside resistance 16S rRNA methylases in Belgium. J Antimicrob Chemother. 2007. 59:459–64.

12.Park YJ., Lee S., Yu JK., Woo GJ., Lee K., Arakawa Y. Co-production of 16S rRNA methylases and extended-spectrum β-lactamases in AmpC-producing Enterobacter cloacae, Citrobacter freundii and Serratia marcescens in Korea. J Antimicrob Chemother. 2006. 58:907–8.

13.Fihman V., Lartigue MF., Jacquier H., Meunier F., Schnepf N., Raskine L, et al. Appearance of aac(6′)-Ib-cr gene among extended-spectrum β-lactamase-producing Enterobacteriaceae in a French hospital. J Infect. 2008. 56:454–9.

14.Dillon B., Thomas L., Mohmand G., Zelynski A., Iredell J. Multiplex PCR for screening of integrons in bacterial lysates. J Microbiol Methods. 2005. 62:221–32.

15.White PA., McIver CJ., Rawlinson WD. Integrons and gene cassettes in the enterobacteriaceae. Antimicrob Agents Chemother. 2001. 45:2658–61.

16.Jeong JY., Yoon HJ., Kim ES., Lee Y., Choi SH., Kim NJ, et al. Detection of qnr in clinical isolates of Escherichia coli from Korea. Antimicrob Agents Chemother. 2005. 49:2522–4.

17.Kim MH., Sung JY., Park JW., Kwon GC., Koo SH. Coproduction of qnrB and armA from extended-spectrum β-lactamase-producing Klebsiella pneumoniae. Korean J Lab Med. 2007. 27:428–36. (김문희, 성지연, 박종우, 권계철, 구선회. Extended-spectrum β-lactamase 를생성하는 Klebsiella pneumoniae 에서의 qnrB 와 armA 유전자의동시생성.대한진단검사의학회지 2007;27:428-36.).

18.Versalovic J., Koeuth T., Lupski JR. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res. 1991. 19:6823–31.

19.Poirel L., Pitout JD., Calvo L., Rodriguez-Martinez JM., Church D., Nordmann P. In vivo selection of fluoroquinolone-resistant Escherichia coli isolates expressing plasmid-mediated quinolone resistance and expanded-spectrum β-lactamase. Antimicrob Agents Chemother. 2006. 50:1525–7.

20.Robicsek A., Jacoby GA., Hooper DC. The worldwide emergence of plasmid-mediated quinolone resistance. Lancet Infect Dis. 2006. 6:629–40.

21.Jiang Y., Zhou Z., Qian Y., Wei Z., Yu Y., Hu S, et al. Plasmid-mediated quinolone resistance determinants qnr and aac(6′)-Ib-cr in extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae in China. J Antimicrob Chemother. 2008. 61:1003–6.

22.Park CH., Robicsek A., Jacoby GA., Sahm D., Hooper DC. Prevalence in the United States of aac(6′)-Ib-cr encoding a ciprofloxacin-modifying enzyme. Antimicrob Agents Chemother. 2006. 50:3953–5.

23.Szabó D., Kocsis B., Rókusz L., Szentandrássy J., Katona K., Kristóf K, et al. First detection of plasmid-mediated, quinolone resistance determinants qnrA, qnrB, qnrS and aac(6′)-Ib-cr in extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae in Budapest, Hungary. J Antimicrob Chemother. 2008. 62:630–2.

24.Robicsek A., Strahilevitz J., Jacoby GA., Macielag M., Abbanat D., Park CH, et al. Fluoroquinolone-modifying enzyme: a n adaptation of a common aminoglycoside acetyltransferase. Nat Med. 2006. 12:83–8.

25.Robicsek A., Strahilevitz J., Sahm DF., Jacoby GA., Hooper DC. qnr prevalence in ceftazidime-resistant Enterobacteriaceae isolates from the United States. Antimicrob Agents Chemother. 2006. 50:2872–4.

26.Fluit AC., Schmitz FJ. Class 1 integrons, gene cassettes, mobility, and epidemiology. Eur J Clin Microbiol Infect Dis. 1999. 18:761–70.

27.Collis CM., Hall RM. Expression of antibiotic resistance genes in the integrated cassettes of integrons. Antimicrob Agents Chemother. 1995. 39:155–62.

28.Park YJ., Yu JK., Lee S., Oh EJ., Woo GJ. Prevalence and diversity of qnr alleles in AmpC-producing Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii and Serratia marcescens: a multicentre study from Korea. J Antimicrob Chemother. 2007. 60:868–71.

Table 1.

Oligonucleotides of the primers used for amplification

Enzymes	Primer pairs	Target	Sequence (5′-3′)	Amplicon size (bp)	Reference
b-lactamase	TEM F	bla_TEM & variants	ATG AGT ATT CAA CAT TTC CGT	861	[9], In this study
	TEM R		TTA CCA ATG CTT AAT CAG TGA
	SHV F	bla_SHV & variants	CCG GGT TAT TCT TAT TTG TCG CT	831
	SHV R		TAG CGT TGC CAG TGC TCG
	PER-F	bla_PER	GTT AAT TTG GGC TTA GGG CAG	855
	PER-R		CAG CGC AAT CCC CAC TGT
	GES/IBC F	bla_GES-1, bla_GES-2, IBC-1	GTT AGA CGG GCG TAC AAA GAT AAT	903
	GES/IBC R		TGT CCG TGC TCA GGA TGA GT
	CTX-MF	bla_CTX-M-1,2,9 cluster	GAT TGA CCG TAT TGG GAG TTT	947
	CTX-MR		CGG CTG GGT AAA ATA GGT CA
	CTX-M3F	bla_CTX-M-3	AGT TCA CGC TGA TGG CGA CG	602
	CTX-M3R		AAC CCA GGA AGC AGG CAG CG
	CTX-M8F	bla_CTX-M-8	ACG CTC AAC ACC GCG ATC	954
	CTX-M8R		CGT GGG TTC TCG GGG ATA A
	VEB F	bla_VEB	CGA CTT CCA TTT CCC GAT GC	650
	VEB R		GGA CTC TGC AAC AAA TAC GC
Qnr	qnrA F	qnrA	GGG TAT GGA TAT TAT TGA TAA AG	660	[10]
	qnrA R		CTA ATC CGG CAG CAC TAT TA
	qnrB F	qnrB1-6 variants	GGM ATH GAA ATT CGC CAC TG	264
	qnrB R		TTT GCY GYY CGC CAG TCG AA
	qnrS F	qnrS	AGT GAT CTC ACC TTC ACC GC	550
	qnrS R		CAG GCT GCA ATT TTG ATA CC
16S rRNA methylase	armA F	armA	AGG TTG TTT CCA TTT CTG AG	776	[11,12]
	armA R		TCT CTT CCA TTC CCT TCT CC
	rmtA F	rmtA	CTAGCGTCCATCCTTTCCTC	635
	rmtA R		TTT GCT TCC ATG CCC TTG CC
	rmtB F	rmtB	CCC AAA CAG ACC GTA GAG GC	769
	rmtB R		CTC AAA CTC GGC GGG CAA GC
	rmtC F	rmtC	CGA AGA AGT AAC AGC CAA AG	700
	rmtC R		ATC CCA ACA TCT CTC CCA CT
aac(6′)-Ib	aac(6′)F	aac(6′)-Ib	TGA CCA ACA GCA ACG ATT CC	500	[13]
	aac(6′)R		TTA GGC ATC ACT GCG TGT TC

Abbreviations: F, forward; R, reverse.

Table 2.

Species-specific distributions of antibiotic resistance genes

Genes	Escherichia coli N=60 (%)	Klebsiella pneumoniae N=69 (%)	Total N=129	P value∗
bla_TEM	41 (68.3)	33 (47.8)	74 (57.0)	0.03
bla_SHV	5 (8.3)	65 (94.2)	70 (54.0)	≤0.0001
blaCTX-M-1	34 (56.7)	11 (15.9)	45 (35.0)	≤0.0001
blaCTX-M-2	2 (3.3)	11 (15.9)	13 (10.0)	0.02
blaCTX-M-3	18 (30.0)	12 (17.4)	30 (23.0)	0.1384
blaCTX-M-8	6 (10.0)	4 (5.8)	10 (8.0)	0.5127
blaCTX-M-9	34 (56.7)	8 (11.6)	42 (33.0)	≤0.0001
bla_GES	0 (0)	5 (7.2)	5 (4.0)	0.0607
bla_VEB	0 (0)	1 (1.4)	1 (1.0)	1
rmtA	4 (6.7)	7 (10.1)	11 (9.0)	0.5424
rmtB	37 (61.7)	7 (10.1)	44 (34.0)	≤0.0001
armA	14 (23.3)	46 (66.7)	60 (47.0)	≤0.0001
aac(6′)-Ib	39 (65.0)	33 (47.8)	72 (55.8)	0.0748
aac(6′)-Ib-cr^†	25/39 (64.1)	19/33 (57.6)	44 (61.1)
wild-type^†	14/39 (35.9)	14/33 (42.4)	28 (38.9)
qnrA	0 (0)	1 (1.4)	1 (1.0)	1
qnrB	1 (1.7)	49 (71.0)	50 (39.0)	≤0.0001
qnrS	24 (40.0)	5 (7.2)	29 (22.0)	≤0.0001

^∗ Statistical analysis for categorical variables and incidence across series were performed using Pearson's chi test and Fisher's exact tests;

^† The ratio of cr-variants to the isolates carrying aac(6′)-Ib.

Table 3.

Prevalence of the qnr subtypes and the cr-variant in the 43 strains coharboring qnr and aac(6′)-Ib

Strain (N [%])	qnrA positive	qnrB positive	qnrS positive	aac(6′)-Ib-cr
Escherichia coli 19	0 (0)	0 (0)	19 (100)	12 (63.2)
Klebsiella pneumoniae 24	1 (0.04)	21 (87.5)	3 (12.5)	17 (70.8)
Total 43	1 (0.02)	21 (48.8)	22 (51.2)	29 (67.4)

Table 4.

Cassette arrays of the type-1 integrons detected in the 43 strains coharboring qnr and aac(6′)-Ib

Type of cassette array	Sequence of cassette	Size (kb)	Isolates
Type of cassette array	Sequence of cassette	Size (kb)	Escherichia coli	Klebsiella pneumoniae
1	dfr17-aadA5	1.8	5	0
2	aadA2	1.1	0	12
3	aadB-aadA2	1.6	0	1
4	aac(6′)-Ib-cm1a1	2.5	1∗	0
5	aac(6′)-Ib-bla_OXA4-aadA2	3.0	0	1
6	aadA6-OfrD	1.2	0	1
Total			5	15

^∗ Simultaneously harbored type 1 cassette array in the isolate.

Table 5.

Minimum inhibitory concentration values and resistance gene profiles of the 11 strains showing high-level resistances to quinolone or aminoglycoside

Species	No. strain	Array type	aac(6′)	qnrA	qnrB	qnrS	armA	rmtA	rmtB	NOR	CIP	TOB	GEN	AMK
Escherichia coli	100	.	Ib	N	N	P	P	N	P	>128	>32	16	>128	64
	23	1,4	Ib	N	N	P	N	N	N	>128	>32	16	16	8
	21	1	Ib	N	N	P	P	N	P	>128	>32	4	16	≤2
Klebsiella pneumoniae	126	.	cr	N	P	N	N	N	N	>128	>32	>128	>128	>512
	37	.	cr	N	P	N	P	N	N	>128	>32	>128	32	>512
	42	.	cr	N	P	N	N	N	N	>128	8	>128	>128	128
	28	2	cr	N	P	N	P	N	N	>128	2	>128	>128	>512
	31	.	cr	N	P	N	P	N	N	>128	4	64	>128	>512
	33	6	cr	N	P	N	P	N	N	>128	4	>128	>128	64
	45	2	cr	N	P	N	P	N	N	>128	2	>128	64	64
	44	2	cr	N	P	N	P	N	N	>128	2	>128	16	64

Abbreviations: CIP, ciprofloxacin; TOB, tobramycin; GEN, gentamicin; AMK, amikacin; NOR, norfloxacin; N, negative; P, positive.

Table 6.

Candidate resistance genes showing statistical association with the minimum inhibitory concentration values of quinolones and aminoglycosides in the 43 strains cohaboring qnr and wild-type aac(6′)-Ib

		N	NOR	CIP	TOB	GEN	AMK
qnrB	Positive	21			53.4±55.3	40.5±51.9	136±190
	Negative	22			11.5±5.6	15.5±26.6	14.2±21.3
	P value∗				≤0.001	0.0044	≤0.001
qnrS	Positive	22				14.8±26.8	11.6±18
	Negative	21				41.2±51.4	138.6±188.5
	P value∗					0.0054	≤0.001
rmtA	Positive	4	6±2.3	2.1±2.0	9.2±7.8	9.2±7.8	19.5±29.7
	Negative	39	41.8±55.7	7±9.7	34.9±45.8	30±44.4	81.0±153.3
	P value∗		0.0002	0.025	0.0139	0.0153	0.0199
rmtB	Positive	14			10±6.2		14.7±22.4
	Negative	29			42.5±50.2		102.2±170.2
	P value∗				≤0.001		≤0.001
cr-variant	Positive	29		5.2±7.7	42.2±51.6		97.4±175.6
	Negative	14		9±12.5	12.9±5.2		30.5±30.2
	P value∗			0.0324	≤0.001		≤0.001

^∗ The Wilcoxon rank-sum test was used to compare the group mean (negative vs positive) values; P≤0.05 was considered to indicate statistical significance.

Abbreviations: CIP, ciprofloxacin; TOB, tobramycin; GEN, gentamicin; AMK, amikacin; NOR, norfloxacin.

Table 7.

Amplified association between the candidate genes and the minimum inhibitory concentration values of quinolones and amino-glycosides in the 29 strains co-harboring qnr and the cr-variant

		N	NOR	CIP	TOB	GEN	AMK
qnrB	Positive	16	67±63.0	6.0±10.0	65.1±58.9	50.1±56.3	161.5±212.3
	Negative	13	7±1.8	3.5±1.7	11.7±5.5	10.2±7.1	12.0±17.3
	P value∗		≤0.001	≤0.001	≤0.001	≤0.001	≤0.001
qnrS	Positive	13	6.6±1.9	6.6±10.0	65.1±58.9		161.5±212.2
	Negative	16	67.2±62.5	3.5±1.1	11.7±5.5		12±17.3
	P value∗		≤0.001	≤0.001	≤0.001		≤0.001
rmtA	Positive	3				7±7.9
	Negative	26				36.2±48.6
	P value∗					0.0526
rmtB	Positive	8			9.8±6.2		7.1±6.4
	Negative	21			53.0±55.6		127.5±194.5
	P value∗				≤0.001		≤0.001

^∗ The Wilcoxon rank-sum test was used to compare the group mean (negative vs positive) values; P≤0.05 was considered to indicate statistical significance.

Abbreviations: See Table 6.

TOOLS

Similar articles