Epidemiology of Nalidixic Acid Resistance and TEM-1- and TEM-52-Mediated Ampicillin Resistance of Shigella sonnei Isolates Obtained in Korea between 1980 and 2000

Abstract

The resistance to ampicillin and nalidixic acid in Shigella sonnei isolates obtained in Korea during the period 1998 to 2000 was characterized. Recently (J. Y. Oh, H. S. Yu, S. K. Kim, S. Y. Seol, D. T. Cho, and J. C. Lee, J. Clin. Microbiol. 41:421-423, 2003) ampicillin and nalidixic acid resistance was found in 49 and 70%, respectively, of the 67 S. sonnei isolates obtained during this period. We analyzed 138 S. sonnei isolates collected during the same period. Ampicillin and nalidixic acid resistance was found in 30 and 86% of the isolates, respectively. The ampicillin resistance was mediated by a TEM-1 β-lactamase, and TEM-52 extended-spectrum β-lactamase was identified in one sporadic S. sonnei isolate from 1999. bla_TEM-1 and bla_TEM-52 were located in conjugative R-plasmids. Tn3 was detected in 41% of the ampicillin-resistant isolates. The R-plasmids from the transconjugants that transferred resistance to ampicillin exhibited different restriction fragment length polymorphism patterns, and a bla_TEM-1 probe was hybridized with the different fragments. The nalidixic acid resistance was exclusively associated with an amino acid substitution, Ser83→Leu (TCG→TTG), in gyrA. These findings indicate that the genetically related S. sonnei strains readily acquire resistance to ampicillin, streptomycin, trimethoprim, and sulfamethoxazole but not nalidixic acid through conjugative R-plasmids from difference sources when confronted by antibiotic selective pressures.

Shigellosis is an important cause of acute diarrheal disease in both developing and industrialized countries (16). Shigella flexneri was the most predominant Shigella species in Korea before 1990, but Shigella sonnei has been the most predominant Shigella species since 1990. Epidemic outbreaks of S. sonnei infection occurred in Korea after 1998 (9). In a previous study, the phenotypic and genotypic similarities among S. sonnei isolates during epidemic periods revealed that this explosive increase of S. sonnei infection was mainly due to the spread of a specific clone (5, 11).

Antimicrobial therapy against shigellosis has become very limited because of antimicrobial resistance (10, 15). Among the S. sonnei isolates obtained in Korea during the last 2 decades, the most striking change in antimicrobial susceptibility has been a marked increase in resistance to ampicillin and nalidixic acid, while a marked decrease in resistance to chloramphenicol has been observed in S. sonnei isolates from 1998 to 2000 (6, 11). Since the S. sonnei isolates obtained in Korea during the period 1998 to 2000 were genetically related, the increase of resistance to ampicillin and nalidixic acid raises the question of whether this was due to the spread of an epidemic clone that already carried resistance determinants or whether the ampicillin resistance determinants were acquired when the isolates were confronted by antibiotic selective pressures. The aim of this study was to characterize the resistance to ampicillin and nalidixic acid of S. sonnei isolates obtained between 1998 and 2000.

MATERIALS AND METHODS

Bacterial strains.

A total of 138 S. sonnei isolates, 30 isolates from two defined outbreaks and 108 isolates from sporadic cases, were collected from stool samples in different parts of Korea during the period 1998 to 2000. One hundred sixty-one S. sonnei isolates obtained during the period 1980 to 1986 and 13 isolates obtained during the period 1990 to 1997 were also included for a comparison of the mechanisms of antimicrobial susceptibility and resistance to nalidixic acid.

Determination of MICs.

The MICs of the antimicrobial agents were determined by the agar dilution method in accordance with the guidelines of the National Committee for Clinical Laboratory Standards (8). The inoculated plates were incubated at 37°C for 20 h. The MIC was defined as the lowest concentration of antimicrobial agent that completely inhibited the growth of the organism. Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality control strains. The antimicrobial agents included were ampicillin, carbenicillin, cefazolin, cefoxitin, cefamandole, cefuroxime, ceftriaxone, cefotaxime, cefoperazone, kanamycin, gentamicin, streptomycin, nalidixic acid, norfloxacin, chloramphenicol, sulfamethoxazole, tetracycline, and trimethoprim (all from Sigma); aztreonam (Difco Laboratories); amoxicillin-clavulanic acid (Kunhil Pharmaceutical Co., Seoul, Korea); ceftazidime (Glaxo Group Research Ltd., Greenford, United Kingdom); imipenem (Choongwae Pharmaceutical Co., Seoul, Korea), and ciprofloxacin (Bayer HealthCare, Seoul, Korea).

Transfer of antibiotic resistance.

All the ampicillin-resistant isolates were included as putative donors in a conjugation experiment to detect the transfer of R-plasmids to E. coli RG488 Rif^r and RG176 Nal^r (5). Donor and recipient strains at logarithmic phase were grown in Trypticase soy broth (Difco Laboratories) and were mixed and incubated at 37°C for 20 h. Transconjugants were selected on Mueller-Hinton medium (Difco Laboratories) supplemented with ampicillin (50 μg/ml) and rifampin (50 μg/ml) or nalidixic acid (50 μg/ml).

Plasmid preparation and Southern hybridization.

The plasmid DNA from clinical isolates or their transconjugants was isolated by the alkaline extraction method (1). The extracted DNA was digested with either the EcoRI or SmaI restriction enzyme and separated by electrophoresis in 0.8% agarose gels for restriction fragment length polymorphism (RFLP) analysis. For Southern hybridization, the digested DNA fragments were transferred to a positively charged nylon membrane (Amersham, Freiburg, Germany) and hybridized with a bla_TEM-1 probe, prepared using 296 bp of PstI/HincII-digested fragments of pBR322. A digoxigenin DNA labeling and detection kit (Boehringer Mannheim, Mannheim, Germany) was used according to the manufacturer's instructions.

IEF studies of β-lactamases.

Crude preparations of β-lactamases from clinical isolates or their transconjugants were obtained from sonic extracts prepared in 0.1 M phosphate buffer (pH 7.0). Isoelectric focusing (IEF) was performed by the method of Matthew et al. (7) with a precast vertical IEF gel (Novex). Enzyme activities were detected by overlaying the gel with chromogenic cephalosporin nitrocefin (0.5 mM) in a 0.1 M phosphate buffer (pH 7.0). Strains carrying the β-lactamase of TEM-1, TEM-4, SHV-1, and SHV-5 were used as the controls for isoelectric points (pIs) of 5.4, 5.9, 7.6, and 8.2, respectively.

PCR amplification and DNA sequencing.

A PCR was performed to amplify the entire bla_TEM gene as previously described (6). The primers gyr-A (5′-TACACCGGTCAACATTGACC-3′) and gyr-B (5′-TTAATGATTGCCGCCGTCGG-3′) were used for the amplification of the quinolone resistance-determining region (QRDR) in gyrA (14). A specific primer (5′-GTCTGACGCTCAGTGGAACG-3′), an internal fragment of tnpR, was designed on the basis of published nucleotide sequences of Tn3 in E. coli (4) and used for the amplification of Tn3-specific regions. The amplification reactions for Tn3-specific regions were performed in a GenAmp 9600 thermal cycler (Perkin-Elmer Cetus) with the Expand Long Template PCR system (Boehringer Mannheim). The amplification reaction consisted of 10 cycles of denaturation at 94°C for 10 s, annealing at 67°C for 30 s, and extension at 68°C for 4 min, followed by 15 cycles of denaturation at 94°C for 10 s, annealing at 67°C for 30 s, and extension at 68°C for 4 min, with cycle elongation times of 20 s in each cycle. The PCR was completed with a final elongation step of 7 min at 68°C. The PCR products of bla_TEM and the QRDR of gyrA were ligated with pGEM T-easy vector (Promega) and introduced into E. coli DH5α cells. Sequencing reactions were performed with double-stranded plasmid preparation by dideoxy chain termination with T7 and Sp6 primers.

RESULTS AND DISCUSSION

Antimicrobial susceptibility.

Resistance to ampicillin emerged in 30% (41 of 138) of S. sonnei isolates (Table 1), while no isolates have exhibited resistance to cefazolin, cefoxitin, cefamandole, ceftriaxone, cefotaxime, ceftazidime, aztreonam, and imipenem. One sporadic isolate (99KT183) from 1999 was resistant to cefuroxime and cefoperazone (Table 2). Overall, 100% of S. sonnei isolates were resistant to streptomycin and trimethoprim, 95% were resistant to sulfamethoxazole and tetracycline, and 86% were resistant to nalidixic acid. Only three isolates were resistant to chloramphenicol. No isolates were resistant to ciprofloxacin and norfloxacin. All S. sonnei isolates showed multiple antimicrobial resistances. The resistance to ampicillin increased sharply from 2% of the isolates during the period 1980 to 1986 to 30% of the isolates during the period 1998 to 2000 (Table 1). Ampicillin has been one of the most commonly used drugs for the treatment of shigellosis in Korea during the last 2 decades. This may account for the emergence of resistance to ampicillin and the selection of ampicillin-resistant strains

TABLE 1.

Antimicrobial resistance of S. sonnei isolates obtained in Korea during the last 2 decades

Antimicrobial agent	% of isolates that were resistant
	1998-2000 (n = 138)	1990-1997 (n = 13)	1980-1986 (n = 161)
Ampicillin	30	54	2
Chloramphenicol	2	0	99
Kanamycin	18	38	1
Nalidixic acid	86	8	6
Streptomycin	100	100	99
Sulfamethoxazole	95	100	99
Tetracycline	95	85	98
Trimethoprim	100	100	94

TABLE 2.

MICs of β-lactams for ampicillin-resistant S. sonnei isolates and their transconjugants that transferred resistance to ampicillin

Strain	MIC (μg/ml)a
	AP	AMCb	CB	CZ	CN	CU	CA	CT	CD	AZ	CX	IP	CP
99KT183c	>512	8	>512	16	16	32	4	4	4	2	2	0.25	64
H140(pKY1402)d	>512	8	>512	8	8	16	2	1	4	1	4	1	32
99KT036	>512	8	>512	2	2	2	<0.06	<0.06	<0.06	<0.06	2	0.25	4
H110(pKY1101)	>512	8	>512	2	2	2	<0.06	<0.06	0.25	<0.06	2	1	4
99KT174	>512	8	>512	8	2	2	<0.06	<0.06	<0.06	<0.06	2	0.25	32
H138(pKY1382)	>512	8	>512	2	2	2	<0.06	<0.06	0.25	<0.06	4	1	4
99SS2	>512	8	>512	4	8	2	<0.06	<0.06	<0.06	<0.06	2	0.25	32
H232(pKY2322)	>512	8	>512	2	4	4	<0.06	<0.06	0.25	<0.06	4	1	8
99KE014	>512	8	>512	4	4	4	<0.06	<0.06	<0.06	<0.06	2	0.25	16
H213(pKY2131)	>512	8	>512	2	2	2	<0.06	<0.06	0.25	<0.06	4	1	4
99KT111	>512	16	>512	2	4	2	<0.06	<0.06	<0.06	<0.06	2	0.25	16
H130(pKY1302)	>512	8	>512	2	2	2	<0.06	<0.06	0.25	<0.06	4	1	4
00CNSM2	>512	64	>512	2	0.5	2	<0.06	<0.06	<0.06	<0.06	2	0.25	8
H252(pKY2524)	>512	32	>512	2	0.5	2	<0.06	<0.06	0.25	<0.06	4	1	4
98KS048	>512	16	>512	4	4	8	<0.06	<0.06	<0.06	0.125	8	0.5	8
H025(pKY251)	>512	16	>512	4	4	2	<0.06	<0.06	0.5	0.125	4	1	8

^aAbbreviations: AP, ampicillin; AMC, amoxicillin-clavulanic acid; CB, carbenicillin; CZ, cefazolin; CN, cefamandole; CU, cefuroxime; CA, ceftriaxone; CT, cefotaxime; CD, ceftazidime; AZ, aztreonam; CX, cefoxitin; IP, imipenem; CP, cefoperazone.

^bThe amoxicillin-clavulanic acid combination was a 2:1 ratio of amoxicillin to clavulanic acid.

^cIsolate of S. sonnei.

^dTransconjugant E. coli RG176 harbored an ampicillin resistance plasmid.

β-Lactamases.

According to antimicrobial susceptibility assays and Southern blotting of R-plasmids, the 41 ampicillin-resistant S. sonnei isolates could be classified into eight different groups (Table 3). IEF was used to detect the production of β-lactamases in each isolate. All the ampicillin-resistant isolates produced a β-lactamase with a pI of 5.4, consistent with TEM-1, which was also corroborated by nucleotide sequencing of the PCR products. One isolate (99KT183) produced β-lactamases with pIs of 5.4 and 5.9. To identify a β-lactamase with a pI of 5.9, TEM-specific PCR and the nucleotide sequencing of the cloned PCR product were performed for the transconjugant of 99KT183. DNA sequencing and deduced amino acid sequence analysis revealed that the β-lactamase with a pI of 5.9 was TEM-52, which differed from TEM-1 by three point mutations; Glu104→Lys (GAG→AAG), Met182→Thr (ATG→ACG), and Gly238→Ser (GGT→AGT) (http://www.lahey.org/studies/temtable.asp).

TABLE 3.

Characteristics of ampicillin-resistant S. sonnei isolates and their transconjugants that transferred resistance to ampicillin

Strain	Antimicrobial resistancea	Isoelectric point(s)	Tn3 PCR result	No. of strains
99KT183b	Ap Cu Cp Tc Sm Su Tp Na Gm	5.4, 5.9	+	1
H140(pKY1402)c	Ap Cp Cu Sm Su Tp Gm	5.4, 5.9	+
99KT036	Ap Tc Sm Su Tp Na Km	5.4	−	2
H110(pKY1101)	Ap Sm Su Km	5.4	−
99KT174	Ap Tc Sm Su Tp Na	5.4	+	3
H138(pKY1382)	Ap Sm Su Tp	5.4	+
99SS2	Ap Tc Sm Su Tp Na	5.4	+	10d
H232(pKY2322)	Ap Sm Su Tp	5.4	+
99KE014	Ap Tc Sm Su Tp Na	5.4	−	1
H213(pKY2131)	Ap Tc Sm Su	5.4	−
99KT111	Ap Tc Sm Su Tp Na Km	5.4	+	3
H130(pKY1302)	Ap Km	5.4	+
00CNSM2	Ap Tc Sm Su Tp Km	5.4	−	20d
H252(pKY2524)	Ap Km	5.4	−
98KS048	Ap Tc Sm Su Tp Na	5.4	−	1
H025(pKY251)	Ap	5.4	−

^aAbbreviations: Ap, ampicillin; Cu, cefuroxime; Cp, cefoperazone; Tc, tetracycline; Sm, streptomycin; Su, sulfamethoxazole; Tp, trimethoprim; Na, nalidixic acid; Km, kanamycin; Gm, gentamicin.

^bIsolates of S. sonnei.

^cTransconjugant E. coli RG 176 harbored an Ap^r plasmid.

^dThe isolates were obtained from defined outbreaks.

This study has revealed that resistance to ampicillin in S. sonnei isolates obtained in Korea during the periods 1998 to 2000 was mediated by a TEM-1 β-lactamase. In the previous study, S. sonnei isolates obtained during the period 1980 to 1997 produced a TEM-1 β-lactamase (6). Accordingly, when previous results are combined with the present results, the production of TEM-1 β-lactamase appears to be the most frequent mechanism of resistance to ampicillin found in Korea. However, several outbreak-related S. sonnei isolates obtained in 2000, from Cheju Island in Korea, have been found to be resistant to expanded-spectrum cephalosporins (12). Among these isolates, a CTX-M-14 extended-spectrum β-lactamase (ESBL) was described to occur in one S. sonnei isolate, and this is the first report of ESBL-producing Shigella strains in Korea. Furthermore, in this study, TEM-52 ESBL was identified in one sporadic S. sonnei isolate. This is the first report of TEM-type ESBL-producing Shigella strains in the world and the second report of ESBL-producing Shigella strains in Korea. Although more than 70 ESBLs have been found, there have been few reports of ESBL-producing Shigella species. The incidence of resistance to extended-spectrum cephalosporins is increasing among Enterobacteriaceae in Korea. Of the E. coli isolates obtained in 1997, 4.8% were found to produce ESBLs, and TEM-52 was the most prevalent ESBL (13). The emergence of TEM-52 ESBLs in S. sonnei might be explained by the high prevalence of TEM-52 ESBLs among Enterobacteriaceae in Korea.

Genetic location of bla_TEM.

The resistance to ampicillin was conjugally transferable to a recipient E. coli strain (Table 3), indicating that bla_TEM-1 was located in conjugative R-plasmids. bla_TEM-52 was also transferred by conjugation. Both bla_TEM-1 and bla_TEM-52 were located on the same plasmid and cotransferred to the recipient. The transconjugants that transferred resistance to ampicillin carried one large plasmid, approximately 75.1 kb, yet exhibited six different antimicrobial resistance patterns. To determine whether bla_TEM-1 was located in Tn3, Tn3-specific PCR was performed. The Tn3-specific amplification confirmed that the PCR products were digested with RsaI, and the digested-fragment patterns were compared to those of S. sonnei 91NH13, the Tn3 region of which was previously sequenced (unpublished data). A total of 17 (41%) out of the 41 ampicillin-resistant S. sonnei isolates gave the expected product of approximately 5.0 kb upon PCR amplification, indicating the occurrence of bla_TEM-1 in Tn3 (Fig. 1).

FIG. 1.

Tn3-specific PCR amplification of R-plasmids from transconjugants. Lanes: 1, lambda DNA size marker; 2, pKY1402; 3, pKY1101; 4, pKY1382; 5, pKY2322; 6, pKY2131; 7, pKY1302; 8, pKY2524; 9, pKY251.

Characterization of conjugative R-plasmids.

The identification of bla_TEM-1 and bla_TEM-52 in conjugative R-plasmids indicated that these genes could be acquired by other bacteria. Thus, to determine whether the R-plasmids carrying bla_TEM-1 originated from single or different sources, an RFLP analysis of R-plasmids and Southern hybridization with a bla_TEM-1 probe were performed. The conjugative R-plasmids exhibited different RFLP patterns according to the combination of EcoRI and SmaI used (Fig. 2A). Furthermore, bla_TEM-1 was located in different digested fragments of the R-plasmids (Fig. 2B). Tn3 was detected in 41% of the ampicillin-resistant isolates. These results indicated that the R-plasmids from S. sonnei isolates were derived from different sources. The resistance to ampicillin, streptomycin, trimethoprim, and sulfamethoxazole was conjugally transferable, but resistance to nalidixic acid was not (Table 3). It has been shown that S. sonnei isolates obtained during the period 1998 to 2000 originated from a single clone and propagated to different parts of Korea (11). This finding indicated that the genetically related S. sonnei isolates readily acquired resistance to ampicillin, streptomycin, trimethoprim, and sulfamethoxazole but not nalidixic acid through conjugative R-plasmids when they were confronted with antibiotic selective pressures.

FIG. 2.

(A) RFLP patterns of R-plasmids extracted from transconjugants by using EcoRI and SmaI. (B) Southern transfer of gel hybridized with 296 bp of the PstI/HincII-digested pBR322 plasmid consisting of bla_TEM-1. (A and B) Lanes: 1, lambda DNA size marker; 2, pKY1402; 3, pKY1101; 4, pKY1382; 5, pKY2322; 6, pKY2131; 7, pKY1302; 8, pKY2524; 9, pKY251; P, pBR322.

Resistance to nalidixic acid.

The resistance to nalidixic acid underwent a marked increase from 6% of the isolates obtained during the period 1980 to 1986 to 86% of the isolates obtained during the period 1998 to 2000 (Table 1). The MICs of nalidixic acid at which 50% of the isolates tested were resistant were 1, 0.5, and 128 μg/ml in the isolates obtained during the periods 1980 to 1986, 1990 to 1997, and 1998 to 2000, respectively. Nalidixic acid has not been used to treat shigellosis but has been widely used to treat urinary tract infections caused by Enterobacteriaceae since the early 1970s in Korea. This fact raises the question of whether an increase in the use of fluoroquinolones is associated with the resistance of S. sonnei strains to nalidixic acid. The MICs of ciprofloxacin were less than 0.03 μg/ml in the isolates obtained during the period 1980 to 1997, while they ranged from 0.06 to 0.13 μg/ml in the isolates obtained during the period 1998 to 2000. Although the MICs of ciprofloxacin for S. sonnei isolates increased slightly during the last 2 decades, it has not been determined that the use of fluoroquinolones was directly associated with the emergence of nalidixic acid-resistant S. sonnei strains.

Of the 119 nalidixic acid-resistant S. sonnei isolates obtained during the period 1998 to 2000, 21 isolates with different phenotypic and genotypic characteristics were selected, and their mechanism of resistance to nalidixic acid was investigated. A 648-bp fragment of the QRDR in gyrA was amplified and sequenced, which revealed a single point mutation, Ser83→Leu (TCG→TTG), in all the isolates tested, while nine nalidixic acid-resistant isolates obtained from 1980 to 1986 only showed a single point mutation at Asp87→Asn (GAC→GGC). As such, this result suggests that the S. sonnei isolates obtained from 1998 to 2000 originated from a genetically related clone, which was also verified by previously reported pulsed-field gel electrophoresis profiles and biotypes (5, 11). Such point mutations in the QRDR of gyrA have already been reported to occur in nalidixic acid-resistant Shigella species. For example, epidemic nalidixic acid-resistant S. sonnei isolates from Israel were found to carry the mutation at Ser83→Phe in gyrA (3), while nalidixic acid-resistant S. flexneri isolates from Hong Kong were associated with a mutation in gyrA encoding a Ser83 alteration (2).

Conclusion.

S. sonnei isolates readily acquired resistance to commonly used antimicrobial agents through conjugative R-plasmids when they were confronted with antibiotic selective pressures. The TEM-52 ESBL was first identified in an S. sonnei isolate obtained in Korea. Therefore, appropriate control and careful monitoring of antimicrobial resistance may prevent the emergence and spread of specific clones of multiresistant S. sonnei isolates.