Occurrence of CTX-M-3, CTX-M-15, CTX-M-14, and CTX-M-9 Extended-Spectrum β-Lactamases in Enterobacteriaceae Clinical Isolates in Korea

Abstract

Among 603 isolates of Enterobacteriaceae collected between June and November 2003 from three university hospitals within Korea, bla_CTX-M-3, bla_CTX-M-15, bla_CTX-M-14, and bla_CTX-M-9 were detected in 41 isolates of species from five different genera of Enterobacteriaceae, Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Enterobacter spp., and Serratia marcescens.

Although most extended-spectrum β-lactamases (ESBL) belong to the TEM- and SHV-type ESBL families, the members of a novel ESBL family, CTX-M, are increasingly being reported in gram-negative bacilli (1). Here, we examined the presence of CTX-M enzymes and the predominant type of CTX-M enzyme in Korea.

Between June and November 2003, 603 consecutive nonduplicate nosocomial isolates of Enterobacteriaceae were collected from three university hospitals located in three different cities—Daegu, Daejun, and Cheonan—in Korea. Among the 603 isolates collected, 163 (27%) were grown on Mueller-Hinton agar plates containing 2 μg of cefotaxime (Sigma)/ml, and they were subjected to PCR for detecting bla_CTX-M with primers listed in Table 1, designed for detection of enzymes from the CTX-M-1, CTX-M-2, and CTX-M-9 groups. As a result of the PCR experiment, 41 of 163 isolates (25.2%) have been shown to carry bla_CTX-M: 28 strains were positive for the PCR of the CTX-M-1 group, and 13 strains were positive for the PCR of the CTX-M-9 group. Further determination of bla_CTX-M alleles was performed by nucleotide sequencing of PCR products on both strands with primers used for PCR. Sequencing was carried out with the Taq DyeDeoxyTerminal cycle-sequencing kit using primers used for PCR, and the sequence was analyzed by using an automatic DNA sequencer (377 ABI Prism; Perkin Elmer). Of the 28 strains positive for the CTX-M-1 group, 17 were confirmed to carry bla_CTX-M-3, and the remaining 11 strains carried bla_CTX-M-15. Of the 13 strains positive for the CTX-M-9 group, 9 were confirmed to carry bla_CTX-M-14, and the remaining 4 strains carried bla_CTX-M-9 (Table 2). In Escherichia coli isolates, all four kinds of bla_CTX-M were demonstrated. bla_CTX-M-3 was identified in species from four different genera of Enterobacteriaceae, Citrobacter freundii (one isolate), E. coli (three isolates), Klebsiella pneumoniae (four isolates), and Serratia marcescens (nine isolates), indicating horizontal transfer and wide dissemination of bla_CTX-M-3 among the family Enterobacteriaceae. bla_CTX-M-14 and bla_CTX-M-15 were detected in all three university hospitals located in three different cities. bla_CTX-M-3 and bla_CTX-M-9 were not detected in the hospital located in Daejun and in the hospital located in Cheonan, respectively.

TABLE 1.

Oligonucleotide primers used for detection of β-lactamase genes

Primer	Tempa	Nucleotide sequence	GenBank accession no.	Nucleotide position	Expected amplicon size (bp)
CTX-M-2-S	58°C	5′-TTAATGATGACTCAGAGCATTC-3′	X92507	3-24	901
CTX-M-2-AS		5′-GATACCTCGCTCCATTTATTG-3′	X92507	884-904
CTX-M-9-S	50°C	5′-TAT TGG GAG TTT GAG ATG GT-3′	AF4546633.2	742-761	932
CTX-M-9-AS		5′-TCC TTC AAC TCA GCA AAA GT-3′	AF4546633.2	1655-1674
CTX-M-1-S	55°C	5′-CGT CAC GCT GTT GTT AGG AA-3′	AJ632119.1	180-209	780
CTX-M-1-AS		5′-ACG GCT TTC TGC CTT AGG TT-3′	AJ632119.1	941-960
TEM-S	50°C	5′-ATA AAA TTC TTG AAG ACG AAA-3′	AB103506	166-186	1,080
TEM-AS		5′-GAC AGT TAC CAA TGC TTA ATC-3′	AB103506	1225-1245
SHV-S	55°C	5′-TGG TTA TGG GTT ATA TTC GCC-3′	AY223863	166-186	865
SHV-AS		5′-GGT TAG CGT TGC CAG TGC T-3′	AY223863	1015-1031
OXA-1-S	55°C	5′-AGC CGT TAA AAT TAA GCC C-3′	AV162283.2	1052-1070	908
OXA-1-AS		5′-CTT GAT TGA AGG GTT GGG CG-3′	AV162283.2	1941-1960
CMY-1-S	60°C	5′-GAG CAG ACC CTG TTC GAG AT-3′	X92508	570-589	846
CMY-1-AS		5′-GAT TGG CCA GCA TGA CGA TG-3′	X92508	1397-1416
DHA-1-S	50°C	5′-GTT ACT CAC ACA CGG AAG GT-3′	AY205600	75-94	869
DHA-1-AS		5′-TTT TAT AGT AGC GGG TCT GG-3′	AY205600	925-944

^aAnnealing temperature used for PCR.

TABLE 2.

Phenotypic and genotypic characterization of 41 isolates carrying the bla_CTX-M gene

Species	Strain	Hospitala	CTX-M group	pI(s)b	Other β-lactamase gene product(s)	MICc (μg/ml)					Antimicrobial resistance patternd
						CTX	CAZ	ATM	FEP	FOX
C. freundii	03K892	K	CTX-M-3	5.4, 7.4, 8.4, 8.7	TEM-1, OXA-30, AmpC	256	8	32	64	512	AMP AMK GEN KAN CHL TET STR SXT TMP
E. coli	EC25	D	CTX-M-3	5.4, 8.4	TEM-1	128	2	16	16	8	AMP AMK GEN KAN TET STR SXT TMP
E. coli	03K681	K	CTX-M-3	5.4, 8.4	TEM-1	512	8	32	128	256	AMP AMK GEN KAN CHL TET STR SXT TMP
E. coli	03K203	K	CTX-M-3	7.4, 8.0, 8.4	OXA-30, CMY-1	128	128	256	256	≥512	AMP AMK GEN KAN STR SXT
K. pneumoniaee	KP2	D	CTX-M-3	7.4, 7.8, 8.4	OXA-30, DHA-1	256	128	256	32	256	AMP AMK KAN CHL STR SXT
K. pneumoniae	KP7	D	CTX-M-3	8.4		256	8	32	128	16	AMP AMK GEN KAN SXT TMP
K. pneumoniae	KP9	D	CTX-M-3	8.4		256	8	32	128	8	AMP AMK GEN KAN SXT TMP
K. pneumoniae	03K839	K	CTX-M-3	5.4, 7.4, 7.8, 8.4	TEM-1, OXA-30, DHA-1	≥512	256	512	256	≥512	AMP AMK GEN KAN CHL STR SXT TMP
S. marcescens	03K188	K	CTX-M-3	7.4, 8.4, 8.7	OXA-30, AmpC	≥512	128	256	512	≥512	AMP AMK GEN KAN STR SXT
S. marcescens	03K196	K	CTX-M-3	7.4, 8.4, 8.7	OXA-30, AmpC	≥512	128	128	512	≥512	AMP AMK GEN KAN STR SXT
S. marcescens	03K201	K	CTX-M-3	7.4, 8.4, 8.7	OXA-30, AmpC	≥512	64	128	512	≥512	AMP AMK GEN KAN STR SXT
S. marcescens	03K205	K	CTX-M-3	7.4, 8.4, 8.7	OXA-30, AmpC	≥512	128	128	512	≥512	AMP AMK GEN KAN STR SXT
S. marcescens	03K168	K	CTX-M-3	7.4, 8.4, 8.7	OXA-30, AmpC	≥512	64	128	>512	≥512	AMP AMK GEN KAN STR SXT
S. marcescens	03K921	K	CTX-M-3	8.4, 8.7	AmpC	≥512	32	128	512	256	AMP AMK GEN KAN STR SXT
S. marcescens	03K980	K	CTX-M-3	8.4, 8.7	AmpC	≥512	64	256	512	≥512	AMP AMK GEN KAN STR SXT
S. marcescens	SM 16	D	CTX-M-3	8.4		64	≥1	32	32	8	AMP AMK GEN KAN STR SXT TMP
S. marcescens	SM 3	D	CTX-M-3	8.4		512	4	64	64	16	AMP AMK GEN KAN STR SXT TMP
Enterobacter aerogenes	EA11	D	CTX-M-15	5.4, 7.4, 7.6, 8.6	TEM-1, OXA-30, SHV-1	512	128	128	256	4	AMP STR SXT TMP
Enterobacter aerogenes	EA6	D	CTX-M-15	5.4, 7.4, 7.6, 8.6	TEM-1, OXA-30, SHV-1	512	128	128	256	4	AMP TET STR SXT TMP
Enterobacter aerogenes	EA7	D	CTX-M-15	5.4, 7.4, 7.6, 8.6	TEM-1, OXA-30, SHV-1	512	64	128	256	4	AMP TET STR SXT TMP
E. coli	03K969	K	CTX-M-15	5.4, 7.4, 8.6	TEM-1, OXA-30	256	16	32	16	4	AMP GEN TET STR SXT TMP
E. coli	03K865	K	CTX-M-15	5.4, 7.4, 8.6	TEM-54, OXA-30	256	8	16	4	2	AMP GEN KAN TET
E. coli	03K582	K	CTX-M-15	5.4, 7.4, 8.6	TEM-54, OXA-30	256	32	64	128	8	TET
E. coli	J133	E	CTX-M-15	5.4, 7.4, 8.6	TEM-54, OXA-30	256	32	64	128	8	AMP GEN KAN STR SXT TMP
E. coli	J144	E	CTX-M-15	5.4, 7.4, 8.6	TEM-1, OXA-30	512	128	256	256	128	AMP GEN KAN TET SXT TMP
E. coli	J158	E	CTX-M-15	5.4, 7.4, 8.6	TEM-1, OXA-30	512	128	256	256	16	AMP GEN KAN TET
E. coli	J159	E	CTX-M-15	5.4, 8.6	TEM-1	512	64	256	256	32	AMP GEN TET SXT TMP
E. coli	EC35	D	CTX-M-15	5.4, 7.4, 8.6	TEM-1, OXA-30	≥512	128	256	128	16	AMP GEN KAN TET
E. cloacae	J187	E	CTX-M-9	8.0, 8.5	AmpC	32	4	4	4	≥512	AMP KAN CHL TET STR SXT TMP
E. cloacae	03K42	K	CTX-M-9	8.0, 8.2, 8.5	SHV-12, AmpC	32	64	128	4	512	AMP KAN CHL TET SXT TMP
E. coli	03K380	K	CTX-M-9	8.0, 8.2, 8.5	SHV-12, AmpC	8	32	32	<1	128	AMP KAN CHL TET STR SXT TMP
E. coli	03K776	K	CTX-M-9	8.0, 8.5	AmpC	8	≤1	1	<1	256	AMP KAN TET STR SXT TMP
E. coli	EC27	D	CTX-M-14	8.0		128	2	8	64	8	AMPTET
E. coli	ECS	D	CTX-M-14	5.4, 8.0	TEM-1	512	8	32	64	32	AMP GEN KAN CHL TET STR SXT
E. coli	J167	E	CTX-M-14	5.4, 8.0	TEM-1	128	2	16	8	8	AMP GEN TET STR SXT TMP
E. coli	03K319	K	CTX-M-14	5.4, 8.0, 8.5	TEM-1	32	≤1	8	4	4	TET STR SXT
K. pneumoniae	03K930	K	CTX-M-14	8.0		512	8	32	128	32	AMP KAN STR SXT
K. pneumoniae	J22	E	CTX-M-14	6.2,f8.0, 8.2	OXA, SHV-12	256	8	32	64	32	AMP KAN STR
K. pneumoniae	J64	E	CTX-M-14	6.2,f8.0, 8.2	OXA, SHV-12	256	8	512	64	8	AMP KAN STR
K. pneumoniae	J1	E	CTX-M-14	6.2,f8.0, 8.2	OXA, SHV-12	128	256	512	16	8	AMP KAN STR
K. pneumoniae	J112	E	CTX-M-14	6.2,f8.0, 8.2	OXA, SHV-12	256	512	512	32	8	AMP KAN STR

^aHospital K is located in the city of Daegu, hospital D is in Cheonan, and hospital E is in Daejun in Korea.

^bThe pI of β-lactamase as determined by IEF; underlined pIs correspond to the CTX-M enzymes.

^cMIC as determined by the agar dilution method. Abbreviations: CTX, cefotaxime; CAZ, ceftazidime; ATM, aztreonam; FEP, cefepime; FOX, cefoxitin.

^dAbbreviations: AMP, ampicillin; CHL, chloramphenicol; TET, tetracycline; STR, streptomycin; SXT, sulfisoxazole; TMP, trimethoprim; KAN, kanamycin; GEN, gentamicin; AMK, amikacin.

^eAll K. pneumoniae isolates were positive for the PCR with the SHV primer sets, and further sequence determination revealed that these isolates have SHV-11, the β-lactamase located on the chromosome of K. pneumoniae, although the β-lactamase with a pI of 7.6 corresponding to the SHV-11 was not detected by the IEF analysis.

^fThe β-lactamase with a pI of 6.2 was not inhibited by either 0.3 mM clavulanic acid or 0.3 mM cloxacillin, indicating an OXA-type β-lactamase. We did not perform further characterization of this β-lactamase.

Characterization of 41 isolates carrying bla_CTX-M was performed via antimicrobial susceptibility testing, an isoelectric focusing (IEF) assay (2), PCR, and nucleotide sequencing for β-lactamase genes. MICs were measured using a standard agar dilution method according to the approved method of the National Committee for Clinical Laboratory Standards (3). E. coli ATCC 25922 was used as a quality reference strain. Isoelectric focusing and inhibition assays with 0.3 mM clavulanic acid or cloxacillin were performed as described previously (2, 4).

As shown in Table 2, most isolates expressing CTX-M enzyme were found to produce additional β-lactamases. The β-lactamase with a pI of 5.4 was confirmed as TEM-1 or TEM-54, inhibitor-resistant TEM, by TEM-specific PCR and sequencing. The β-lactamases with pIs of 7.6 and 8.2 and whose activity was inhibited by 0.3 mM clavulanic acid were SHV-1 and SHV-12, respectively. The β-lactamase with a pI of 7.4 whose activity was not inhibited by either 0.3 mM clavulanic acid or 0.3 mM cloxacillin was OXA-30, confirmed by OXA-1-specific PCR and subsequent sequencing. The β-lactamases with pIs of 8.0 and 7.8 whose activity was inhibited by 0.3 mM cloxacillin were CMY-1 and DHA-1, respectively.

For almost all strains expressing CTX-M enzyme, except five strains which coexpressed SHV-12 or CMY-1, the MICs of cefotaxime were higher than those of ceftazidime (Table 2). The cefotaxime MICs for such strains were two- to sevenfold higher dilutions than those of ceftazidime. Ratios of cefotaxime MIC to ceftazidime MIC for isolates expressing CTX-M-15 were lower than those for isolates expressing CTX-M-3, as demonstrated by other reports (6, 7). Although there is only one amino acid difference between CTX-M-3 and CTX-M-15 (Asp²⁴⁰→Gly), Poirel et al. (6) demonstrated that the amino acid difference in the omega loop region of CTX-M-15 results in increased ceftazidime hydrolysis and antibiotic resistance compared to those for CTX-M-3. For four strains expressing CTX-M-9 (two E. coli strains and two Enterobacter cloacae strains), the MICs of cefotaxime were lower, ranging from 8 to 32 μg/ml, than those for strains expressing CTX-M-3, CTX-M-15, or CTX-M-14.

Some strains demonstrated high levels of resistance to cefoxitin, and these strains were found to produce additional chromosomal AmpC enzyme or plasmid-mediated AmpC enzymes, such as CMY-1 and DHA-1.

Transferability of cefotaxime resistance was determined by conjugation experimentation using E. coli J53 Azide^R (confers resistance to sodium azide) as a recipient. Donor and recipient strains at logarithmic phase were grown in 4 ml of Trypticase soy broth (Difco Laboratories) and were mixed at a ratio of 4 (recipient) to 1 (donor) at 37°C for 20 h. Transconjugants were selected on Mueller-Hinton agar plates (Difco Laboratories) supplemented with sodium azide (150 μg/ml) and cefotaxime (4 μg/ml). By conjugation, cefotaxime resistance was transferred in 29 isolates, and the bla_CTX-M gene was confirmed in all 29 transconjugants by PCR (Table 3). Some other bla genes, such as bla_OXA-30, bla_TEM, bla_DHA-1, and bla_SHV-12, were cotransferred with bla_CTX-M to transconjugants. Especially, bla_OXA-30 was cotransferred with bla_CTX-M-3 in almost all strains, indicating that bla_CTX-M-3 and bla_OXA-30 might be located on the same transferable plasmid.

TABLE 3.

Transfer of resistance for cefotaxime and other antimicrobial agents of clinical isolates carrying bla_CTX-M

Transconjugant	Donor strain	Transferred bla gene(s)	MICa (μg/ml)					Antimicrobial resistance patternb
			CTX	CAZ	ATM	FEP	FOX
P1-1J	03K892	blaCTX-M-3, blaOXA-30	32	<1	2	<1	2	AMP AMK GEN KAN SXT
11-1J	03K681	blaCTX-M-3, blaOXA-30	128	4	16	16	8	AMP AMK GEN KAN SXT
15-1J	EC25	blaCTX-M-3	64	<1	16	8	2	AMP AMK GEN KAN SXT TMP
35-1J	KP7	blaCTX-M-3	32	<1	2	<1	4	AMP AMK GEN KAN SXT TMP
36-1J	KP9	blaCTX-M-3	32	<1	2	<1	4	AMP AMK GEN KAN SXT TMP
34-2J	KP2	blaCTX-M-3, blaOXA-30	32	<1	2	<1	2	AMP AMK GEN KAN SXT
34-1J	KP 2	blaCTX-M-3, blaSHV-12, blaDHA-1	32	16	32	<1	32	AMP AMK GEN KAN CHL STR SXT
24-1J	03K839	blaCTX-M-3, blaOXA-30, blaTEM-1	128	16	32	8	4	AMP GEN KAN SXT TMP
38-1J	03K188	blaCTX-M-3, blaOXA-30	32	<1	2	<1	4	AMP AMK GEN KAN SXT
39-1J	03K196	blaCTX-M-3, blaOXA-30	64	<1	8	4	4	AMP AMK GEN KAN SXT
40-1J	03K201	blaCTX-M-3, blaOXA-30	32	<1	2	<1	4	AMP AMK GEN KAN SXT
41-1J	03K205	blaCTX-M-3, blaOXA-30	32	2	16	8	8	AMP AMK GEN KAN SXT
37-1J	03K168	blaCTX-M-3, blaOXA-30	32	<1	2	<1	4	AMP AMK GEN KAN SXT
43-1J	03K921	blaCTX-M-3	32	2	8	32	2	AMP KAN STR
44-1J	03K980	blaCTX-M-3	4	2	8	32	4	AMP AMK KAN STR
45-1J	SM 16	blaCTX-M-3	4	2	16	32	8	AMP AMK GEN KAN STR SXT TMP
46-1J	SM 3	blaCTX-M-3	8	2	16	16	8	AMP AMK GEN KAN STR SXT TMP
13-1J	03K865	blaCTX-M-15	128	<1	8	2	4	AMP
14-1J	03K969	blaCTX-M-15, blaOXA-30, blaTEM-1	512	8	16	8	4	AMP SXT TMP
20-1J	J144	blaCTX-M-15	256	8	16	8	4	AMP SXT
22-1J	J159	blaCTX-M-15	128	<1	4	8	32	AMP
19-1J	J133	blaCTX-M-15, blaTEM-54	256	4	16	4	4	AMP
06-1J	J187	blaCTX-M-9	8	<1	2	2	32	AMP CHL TET SXT TMP
05-1J	03K42	blaCTX-M-9, blaSHV-12	4	4	8	<1	2	AMP CHL SXT TMP
09-2J	03K380	blaCTX-M-9, blaSHV-12	8	64	64	<1	2	AMP CHL SXT TMP
18-1J	18-1J	blaCTX-M-14, blaOXA-30, blaTEM-1	512	2	8	16	4	AMP TET STR SXT
16-1J	EC27	blaCTX-M-14	128	2	16	16	8	AMP
23-1J	J167	blaCTX-M-14	64	<1	8	8	8	AMP STR SXT
25-1J	03K930	blaCTX-M-14	64	<1	2	4	4	AMP STR SXT
	E. coli J53 AzideR recipient		<1	<1	<1	<1	4

^aMIC as determined by the agar dilution method. Abbreviations: CTX, cefotaxime; CAZ, ceftazidime; ATM, aztreonam; FEP, cefepime; FOX, cefoxitin.

^bAbbreviations: AMP, ampicillin; CHL, chloramphenicol; TET, tetracycline; STR, streptomycin; SXT, sulfisoxazole; TMP, trimethoprim; KAN, kanamycin; GEN, gentamicin; AMK, amikacin.

Resistance to chloramphenicol, tetracycline, aminoglycosides, and co-trimoxazole was found in most strains carrying bla_CTX-M, and the resistance was also found in most transconjugants (Table 3). Interestingly, a high level of amikacin resistance (MIC, ≥512 μg/ml) was demonstrated in all 17 isolates carrying bla_CTX-M-3 but not in isolates carrying another subtype of bla_CTX-M, and the amikacin resistance was transferred to transconjugants.

In conclusion, the occurrence of CTX-M-3, CTX-M-15, CTX-M-9, and CTX-M-14 in species from five different genera of Enterobacteriaceae, C. freundii, E. coli, Enterobacter spp., K. pneumoniae, and S. marcescens was demonstrated. This finding indicates horizontal transfer and wide dissemination of these enzymes in Korea and would suggest that CTX-M enzymes have existed for several years and have evolved in Korean hospital environments. Although CTX-M-14 was identified in one isolate of Shigella sonnei, two of K. pneumoniae, and one of E. coli in Korea in 2001 (5), to our knowledge this study represents the first identification of CTX-M-3, CTX-9, and CTX-M-15 in Korea.