Abstract

Objectives: This study was conducted to investigate the occurrence of 16S rRNA methylases that confer high-level aminoglycoside resistance in Klebsiella pneumoniae and Escherichia coli isolates from two Taiwanese hospitals and the characteristics of these isolates.

Methods: A total of 1624 K. pneumoniae and 2559 E. coli isolates consecutively collected over an 18 month period from a university hospital and seven E. coli and eight K. pneumoniae isolates that were resistant to amikacin from a district hospital were analysed. Two 16S rRNA methylase genes, armA and rmtB, were detected by PCR-based assays. β-Lactamase characteristics were determined by phenotypic and genotypic methods.

Results: Overall, 28 armA-positive and seven rmtB-positive isolates were identified, and extended-spectrum β-lactamases (ESBLs) were detected in 33 (94.3%) isolates. The prevalence rates of armA and rmtB at the university hospital were 0.9% (n=15) and 0.3% (n=5) in K. pneumoniae and 0.4% (n=10) and 0.04% (n=1) in E. coli. CTX-M-3, CTX-M-14, SHV-5-like ESBLs, and CMY-2 were detected alone or in combination in 21, 6, 11, and 2, respectively, of the 28 armA-positive isolates. CTX-M-14 was detected in six of the seven rmtB-positive isolates. Fingerprinting of conjugative plasmids revealed the dissemination of closely related plasmids containing both armA and blaCTX-M-3. PFGE suggests that armA and rmtB spread by both horizontal transfer and clonal spread.

Conclusions: This is the first report of the emergence of 16S rRNA methylases in Enterobacteriaceae in Taiwan. The spread of the multidrug-resistant isolates producing both ESBLs and 16S rRNA methylases may become a clinical problem.

Introduction

Aminoglycosides are among the most commonly used broad-spectrum antibiotics for the treatment of infectious diseases caused by Gram-negative bacilli. They inhibit bacterial protein synthesis by binding irreversibly to the bacterial 30S ribosomal subunit, thereby leading to cell death.1 Resistance to aminoglycosides is frequently due to the acquisition of modifying enzymes such as acetyltransferases, phosphorylases and adenylyltransferases.1,2 Other mechanisms of resistance include ribosomal alteration, impaired uptake of the antibiotics, and ribosomal protection by methylation of 16S rRNA.1,3,4 16S rRNA methylases are intrinsically produced by some aminoglycoside-producing organisms such as Streptomyces spp. and Micromonospora spp.59 Methylation of bases involved in the binding between 16S rRNA and aminoglycosides leads to a loss of affinity, thereby resulting in high-level resistance to aminoglycosides.4,10

Recently, several plasmid-encoded 16S rRNA methylases have emerged in clinical isolates of Gram-negative bacilli.1114 In Japan, the RmtA methylase has been detected in Pseudomonas aeruginosa strains,13,14 and the RmtB methylase has been identified from a Serratia marcescens strain.12 A gene suggested to encode a 16S rRNA methylase on a plasmid from a Polish Citrobacter freundii strain has been deposited in the EMBL and GenBank databases since 2002 (accession number AF550415). The same gene was cloned from a plasmid of a clinical Klebsiella pneumoniae strain from France, then designated armA, and characterized by Galimand et al.11 Both ArmA-encoding plasmids from the C. freundii and K. pneumoniae strains were found to carry blaCTX-M-3, an extended-spectrum β-lactamase (ESBL) gene.11 Unlike aminoglycoside-modifying enzymes that vary in their substrate ranges,3 the acquired 16S rRNA methylases can confer high-level resistance to almost all clinically important aminoglycosides.1114 Thus, the spread of such resistance determinants has become a great concern. The prevalence of high-level aminoglycoside resistance mediated by 16S rRNA methylases among clinical isolates of Gram-negative bacilli in Taiwan has not been reported. Thus, the aims of this study were to investigate the occurrence of 16S rRNA methylases in K. pneumoniae and Escherichia coli isolates from two hospitals in southern Taiwan and to characterize these isolates.

Materials and methods

Clinical isolates

Between January 2001 and June 2002, 2559 non-replicate clinical isolates of E. coli and 1624 non-replicate clinical isolates of K. pneumoniae were consecutively collected from the National Cheng Kung University Hospital (NCKUH), a 900-bed university hospital in southern Taiwan. Among these isolates, 692 E. coli isolates and 155 K. pneumoniae isolates demonstrated resistance to gentamicin (inhibition zone diameter, ≤12 mm) based on the NCCLS criteria for the disc diffusion method,15 and 21 E. coli isolates and 45 K. pneumoniae isolates demonstrated resistance to amikacin (inhibition zone diameter, ≤14 mm). Since plasmid-mediated 16S rRNA methylases can confer high-level resistance to gentamicin and amikacin, we selected the amikacin-resistant isolates for further investigation. Moreover, seven non-replicate E. coli isolates and eight non-replicate K. pneumoniae isolates that were randomly collected between January and June 2002 from Tainan Municipal Hospital (a district hospital in southern Taiwan) due to amikacin resistance were also included for analysis.

Antimicrobial susceptibility testing

MICs of various antimicrobial agents were determined by the agar dilution method in accordance with the NCCLS guidelines.16E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as reference strains.

Detection of methylase genes

The armA and rmtB genes were detected by PCR and colony hybridization.17 A fresh bacterial colony was suspended in 100 μL of sterile distilled water and boiled at 100°C for 10 min. After centrifugation, the supernatant was removed for PCR assays. armA (774 bp) was amplified with primers 5′-CCGAAATGACAGTTCCTATC-3′ and 5′-GAAAATGAGTGCCTTGGAGG-3′, which are specific for the flanking regions of the gene,11 to produce a 846 bp product. The whole rmtB gene (756 bp) was amplified with primers 5′-ATGAACATCAACGATGCCCT-3′, which corresponds to nucleotide positions 1 to 20 of the structural gene,12 and 5′-CCTTCTGATTGGCTTATCCA-3′ to produce a 769 bp product. Reactions for both genes were run on a GeneAmp PCR system 480 (PE Applied Biosystems, Foster City, CA, USA) with the GeneAmp DNA amplification kit containing AmpliTaq polymerase (PE Applied Biosystems) under the following conditions: 12 min at 95°C; 35 cycles of 1 min at 94°C, 1 min at 55°C, and 2 min at 72°C; and finally, 7 min at 72°C. PCR products were electrophoresed in 1.5% agarose gels and visualized under UV light. PCR products were then purified with a commercial kit and both strands of the amplicons were sequenced on an ABI PRISM 310 automated sequencer (PE Applied Biosystems). The sequences were compared with the sequences of the armA gene from K. pneumoniae strain BM4536 (GenBank AY220558) and the rmtB gene from S. marcescens strain S-95 (GenBank AB103506). The PCR products were also used as templates to make the digoxigenin-11-dUTP-labelled armA and rmtB probes. Colony hybridization was carried out with the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche Applied Science, Mannheim, Germany) according to the manufacturer's instructions.

β-Lactamase characterization

ESBL production was detected by the confirmatory disc diffusion tests recommended by the NCCLS.15,18 Crude β-lactamase extracts were prepared using sonication as described previously.19 We carried out isoelectric focusing (IEF) by the method of Matthew et al.20 with an LKB Multiphor apparatus on prepared PAGplate gels (pH 3.5–9.5; Amersham Biosciences, Hong Kong, China) as described previously.21 β-Lactamase activities were detected by overlaying the gel with 0.5 mM nitrocefin (Oxoid, Basingstoke, UK) in 0.1 M phosphate buffer, pH 7.0. PCR detection of blaTEM, blaSHV, blaCTX-M-1-related, blaCTX-M-9-related and blaCMY-2 genes was carried out with the previously reported oligonucleotide primers.2225 PCR products of blaSHV obtained from K. pneumoniae isolates were subjected to the PCR-NheI method to discriminate between blaSHV-ESBL and blaSHV-non-ESBL genes.23 The other amplicons were all subjected to direct sequencing. Sequence alignments and analyses were carried out online using the BLAST program (www.ncbi.nlm.nih.gov).

Conjugation experiments and plasmid analysis

Conjugation experiments were carried out by the liquid mating-out assay with streptomycin-resistant E. coli C600 as the recipient as described previously.21,26,27 Transconjugants were selected on tryptic soy agar plates supplemented with 512 mg of streptomycin (Sigma Chemical Co., St Louis, MO, USA) and 256 mg of amikacin (Sigma Chemical Co.) per litre. Plasmids from transconjugants were extracted using a rapid alkaline lysis procedure.28 We analysed restriction fragment length polymorphism of transferred plasmids using agarose gel electrophoresis of plasmid DNA samples treated with the restriction endonuclease EcoRI (Roche Applied Science).

PFGE analysis

PFGE was carried out with a CHEF-DR III apparatus (Bio-Rad Laboratories, Hercules, CA, USA) according to the instruction manual. Chromosomal DNA was digested with XbaI (New England Biolabs, Beverly, MA, USA) and was separated on 1% agarose gels. A bacteriophage lambda DNA ladder (GibcoBRL, Gaithersburg, MD, USA) was used as a size marker. PFGE patterns were interpreted in accordance with the criteria of Tenover et al.29

Results and discussion

Prevalence of methylase genes

Of the 81 amikacin-resistant isolates from the two hospitals, 14 E. coli isolates and 21 K. pneumoniae isolates demonstrated high-level resistance to amikacin (MICs, >256 mg/L) in the agar dilution tests. Among these 35 isolates, armA was detected in 12 E. coli isolates and 16 K. pneumoniae isolates, and rmtB was detected in two E. coli isolates and five K. pneumoniae isolates by PCR and nucleotide sequencing. The MICs of amikacin for the 46 isolates negative for armA and rmtB in the PCR assays ranged between 16 and 64 mg/L. The results of colony hybridization were consistent with the PCR results. Twenty-five (15 K. pneumoniae and 10 E. coli) of the 28 armA-positive isolates and six (five K. pneumoniae and one E. coli) of the seven rmtB-positive isolates were isolated at NCKUH from 29 patients. Thus, among the 1624 K. pneumoniae isolates and 2559 E. coli isolates collected from NCKUH, the overall prevalence rates of 16S rRNA methylases were 1.2% in K. pneumoniae and 0.4% in E. coli, the prevalence rates of armA were 0.9% in K. pneumoniae and 0.4% in E. coli, and the prevalenace rates of rmtB were 0.3% in K. pneumoniae and 0.04% in E. coli. Since rmtA has not been detected in Enterobacteriaceae and P. aeruginosa isolates collected between 2000 and 2002 at NCKUH (J.-J. Yan, S.-H. Tsai & C.-L. Chuang, unpublished data), our study indicates that clinical E. coli and K. pneumoniae isolates that produced 16S rRNA methylases remained rare at NCKUH and suggests that armA is more prevalent than rmtB amongst Enterobacteriaceae isolates in Taiwan.

Of the 29 patients with armA-positive or rmtB-positive isolates recovered at NCKUH, nine (31.0%) patients acquired infections 6–462 days (median, 30 days) after admission. Of these nine patients, seven patients stayed at six different wards when the armA-positive isolates were recovered. The data indicate that armA-positive isolates have spread widely in the university hospital. Three (10.3%) of the 29 patients had been discharged from the university hospital within 1 month of the isolations of the multidrug-resistant organisms. The remaining 17 (58.6%) patients were suspected to have acquired infections before admission to NCKUH. Three of them had not been admitted to any hospitals within 6 months of infections, and 14 patients were transferred from or had been hospitalized recently at other healthcare settings including a medical centre in China, a university hospital in northern Taiwan, a district hospital in central Taiwan, six other hospitals in southern Taiwan, and three nursing homes in southern Taiwan. Although only bacterial strains isolated from two hospitals were tested in this study, these data indicate that armA and rmtB have been disseminated widely in Taiwan.

The sites of infection or colonization by the armA-positive and rmtB-positive isolates recovered at NCKUH from the 29 patients were as follows: skin and soft tissue or surgical wound, in 11 (37.9%) patients; respiratory, in 10 (34.5%) patients; urinary, in seven (24.1%) patients; and blood, in two (6.9%) patients. The commonest comorbid conditions associated with the infections were diabetes mellitus and cerebrovascular disease, which were found in 14 (48.3%) and seven (24.1%) patients, respectively.

β-Lactamase characterization

ESBL production was suggested by the NCCLS confirmatory tests in 33 (94.3%) of the 35 isolates that showed high-level amikacin resistance. The pIs of β-lactamases expressed by these isolates on IEF gels and the consistent β-lactamase genes detected by PCR assays are shown in Table 1. In the PCR-NheI tests, six of the 21 K. pneumoniae isolates showed an undigested band and two digested bands on the agarose gel, and the six isolates contained pI 7.6 and 8.2 β-lactamases (data not shown). The results suggest that the six isolates co-produced an SHV-1-related non-ESBL and an SHV-5-related ESBL.23,30 The remaining 15 K. pneumoniae isolates had a pI 7.6 β-lactamase and showed only one undigested band in the PCR-NheI tests, suggesting that they produced an intrinsic SHV-1-related non-ESBL. Of the two isolates negative for ESBLs, the armA-positive E. coli isolate expressed no β-lactamase, and the rmtB-positive K. pneumoniae isolate produced two narrow-spectrum β-lactamases, TEM-1 and an SHV-1-related β-lactamase. One or two ESBL genes were detected in each of the 33 ESBL-producing isolates, and CTX-M-type enzymes were identified in all ESBL-producing isolates. Among the 16 armA-positive ESBL-producing K. pneumoniae isolates, TEM-1 and SHV-1-related narrow-spectrum β-lactamases were detected in all 16 isolates, and CTX-M-3, CTX-M-14, and SHV-5-related ESBLs were detected in 15, one, and six isolates. Among the 11 armA-positive ESBL-producing E. coli isolates, TEM-1 was detected in all 11 isolates, CTX-M-3, CTX-M-14, and SHV-12 ESBLs were detected in six, five, and five isolates (SHV-12 is a variant of SHV-5), and the CMY-2 cephalosporinase was detected in two isolates. All six rmtB-positive ESBL-producing isolates were found to harbour blaCTX-M-14 and blaTEM-1.

Antimicrobial susceptibility testing

The resistance patterns of the 35 armA-positive and rmtB-positive isolates are summarized in Table 1. All 35 isolates displayed high-level resistance (MICs, >256 mg/L) to gentamicin, kanamycin, and tobramycin in addition to amikacin, were resistant to trimethoprim/sulfamethoxazole (MICs, >256 mg/L), and were susceptible to imipenem (MICs, ≤1 mg/L). All 35 isolates except one non-ESBL-producing isolate (strain 903/01) showed resistance to amoxicillin (MICs, >256 mg/L). Of the 28 armA-positive isolates, 18 (64.3%) and 19 (67.9%) isolates were resistant to chloramphenicol (MICs, >256 mg/L) and tetracycline (MICs, >256 mg/L), respectively, and all seven (100%) rmtB-positive isolates were resistant to these two drugs. The 33 ESBL-producing isolates demonstrated reduced susceptibilities to cefotaxime (MICs, 16–256 mg/L), ceftazidime (MICs, 4–256 mg/L), aztreonam (MICs, 4–128 mg/L), and cefepime (MICs, 4–64 mg/L). Two CMY-2-producing isolates were resistant to cefoxitin (MICs, 128 and 256 mg/L), and the remaining 33 isolates were susceptible to this drug (MICs, 4–16 mg/L). Nine (32.1%) of the 28 armA-positive isolates and six (85.7%) of the seven rmtB-positive isolates were resistant to ciprofloxacin (MICs, ≥4 mg/L). Since 13 (37.1%) of the 35 isolates with high-level aminoglycoside resistance were ESBL producers and ciprofloxacin-resistant, the spread of such multidrug-resistant organisms may pose a formidable challenge in the management of seriously ill patients. Therefore, continuous surveillance of such organisms is needed.

Conjugation experiments and plasmid analysis

Plasmid transfer of high-level aminoglycoside resistance to E. coli C600 was successful for 15 of the 28 armA-positive isolates and three of the seven rmtB-positive isolates. The clinical isolates for which plasmid transfer was unsuccessful demonstrated high-level resistance to streptomycin (MICs, ≥256 mg/L). The resistance patterns of the transconjugants and the β-lactamases expressed by the transconjugants are summarized in Table 1. ESBL production was detected by the NCCLS confirmatory tests in all 15 armA-positive E. coli and K. pneumoniae transconjugants and none of the three rmtB-positive transconjugants. PCR and sequence analyses revealed that blaCTX-M-3, blaCTX-M-14, and blaSHV-12 were co-transferred with armA to 13, one, and one transconjugants. All three rmtB-positive transconjugants expressed TEM-1. The armA gene was found to be flanked by putative transposable elements and on the plasmids with blaTEM-1 and blaCTX-M-3 in Europe.11 The complete nucleotide sequences of the ArmA-encoding plasmid from the Polish C. freundii strain have been determined (accession number AF550415), and armA was found to be downstream from a type I integron. In Japan, rmtB was found to be immediately downstream from the right end of transposon Tn3, including blaTEM-1, and to be upstream of a putative transposase gene.12 In this study, blaCTX-M-3 and blaTEM-1 were co-transferred with armA and blaTEM-1 was co-transferred with rmtB to E. coli recipients. Whether the plasmids with the same resistance determinants in different countries occurred by coincidence or are closely related is not known and deserves further investigation.

The restricted patterns of the conjugative plasmids are shown in Figure 1. The 15 armA-positive plasmids gave four major patterns. The pattern 1 plasmids were subtyped into patterns 1a (n=7), 1b (n=1), 1c (n=1), 1d (n=1), and 1e (n=1). Patterns 1b to 1e differed from pattern 1a by one to three bands. The pattern 2 plasmids were subtyped into two patterns, which had two band differences and of which each was represented by a single plasmid. The three rmtB-positive plasmids showed two major patterns, patterns 5 and 6. The major pattern 5 was subtyped into two patterns, which had two band differences. These data indicate that armA and rmtB might be mobilized between different plasmids and that the high rate of ESBL production among the isolates that displayed high-level aminoglycoside resistance in this study resulted in part from the spread of closely related plasmids containing both blaCTX-M-3 and armA.

PFGE analysis

Of the 35 isolates that displayed high-level resistance to aminoglycosides, 20 K. pneumoniae isolates and 11 E. coli isolates were successfully typed by PFGE. Thirteen major profiles were obtained among the K. pneumoniae isolates, and 11 of them were represented by a single isolate (2a). Six profile K-II isolates were subgrouped into five subclones, and three profile K-III isolates were subgrouped into three subclones. Eight different profiles were obtained among the 11 E. coli isolates, and seven of them were represented by a single isolate. Profile E-I was shared by four isolates. The PFGE results indicate that armA and rmtB spread mainly by horizontal transfer in Taiwan. The four armA-positive profile E-I E. coli isolates were suspected to be acquired at four different healthcare facilities, including Tainan Municipal Hospital, NCKUH, a nursing home, and a local district hospital in southern Taiwan. Among the six armA-positive profile K-II-related K. pneumoniae isolates, the K-IIb and K-IIe isolates were associated with community-acquired infections, and the K-IIa and K-IIc isolates were suspected of being acquired at a local district hospital and a nursing home in southern Taiwan. Among the three rmtB-positive profile K-III-related K. pneumoniae isolates, the K-IIIa isolate was suspected to be acquired at a medical centre in northern Taiwan. These data indicate that the two resistance genes may also spread by strain dissemination among different healthcare facilities.

Seven armA-positive conjugative plasmids with the restriction pattern 1a were obtained from one E. coli isolate and six K. pneumoniae isolates. The six K. pneumoniae isolates might be acquired by the patients in NCKUH, three other hospitals, and the community. PFGE profiles obtained for five of six of these K. pneumoniae isolates were different. In addition, four armA-positive conjugative plasmids showed restriction patterns similar to pattern 1a. These data indicate the spread of closely related armA-positive plasmids among different Enterobacteriaceae isolates and among different healthcare facilities in Taiwan.

In conclusion, this is the first report of the occurrence of plasmid-mediated 16S rRNA methylases that confer high-level aminoglycoside resistance in human pathogens in Taiwan. armA was found to be more prevalent than rmtB among E. coli and K. pneumoniae isolates. A high rate of ESBL production among the isolates that exhibited high-level aminoglycoside resistance may result in part from the spread of closely related plasmids containing both blaCTX-M-3 and armA. armA and rmtB may spread by horizontal transfer of the resistance determinants as well as by clonal spread of some resistant strains.

Figure 1.

EcoRI restriction patterns of conjugative plasmids for 15 armA-positive and three rmtB-positive isolates. Lane 1, a 1 kb ladder (Promega Co., Madison, WI, USA); lanes 2 and 16, transconjugants of E. coli isolates 124/01 and 149/02; lanes 3 to 15, transconjugants of K. pneumoniae isolates 690/01, 1430/01, 244/02, 276/02, 197/01, 1178/01, 776/01, 994/01, 1592/01, TM-118, 211/02, 298/02, and 654/01; lanes 17 to 19, transconjugants of K. pneumoniae isolates 433/02, 486/02, and 654/02; lane 20, DNA molecular size marker III (Roche Applied Science). Numbers designating restriction patterns are shown below the gel.

Figure 1.

EcoRI restriction patterns of conjugative plasmids for 15 armA-positive and three rmtB-positive isolates. Lane 1, a 1 kb ladder (Promega Co., Madison, WI, USA); lanes 2 and 16, transconjugants of E. coli isolates 124/01 and 149/02; lanes 3 to 15, transconjugants of K. pneumoniae isolates 690/01, 1430/01, 244/02, 276/02, 197/01, 1178/01, 776/01, 994/01, 1592/01, TM-118, 211/02, 298/02, and 654/01; lanes 17 to 19, transconjugants of K. pneumoniae isolates 433/02, 486/02, and 654/02; lane 20, DNA molecular size marker III (Roche Applied Science). Numbers designating restriction patterns are shown below the gel.

Figure 2.

PFGE of XbaI-digested genomic DNA for 20 K. pneumoniae isolates (a) and 11 E. coli isolates (b). Numbers designating PFGE profiles are shown below the gels. (a) Lanes 1 and 22, a lambda ladder; lanes 2 to 21, K. pneumoniae isolates 197/01, 211/02, 338/02, 1178/02, 654/01, 291/02, 298/02, 289/01, 433/02, 654/02, 690/01, 486/02, 1430/01, 244/02, 276/02, 1046/01, TM-118, 776/01, 1592/01, and 6-3/01. (b) Lanes 1 and 13, a lambda ladder; lanes 2 to 12, E. coli isolates 234/01, TM-37, 589/01, 773/01, 1686/01, 903/01, 1045/01, TM-107, 324/02, 501/01, and TM-115.

Figure 2.

PFGE of XbaI-digested genomic DNA for 20 K. pneumoniae isolates (a) and 11 E. coli isolates (b). Numbers designating PFGE profiles are shown below the gels. (a) Lanes 1 and 22, a lambda ladder; lanes 2 to 21, K. pneumoniae isolates 197/01, 211/02, 338/02, 1178/02, 654/01, 291/02, 298/02, 289/01, 433/02, 654/02, 690/01, 486/02, 1430/01, 244/02, 276/02, 1046/01, TM-118, 776/01, 1592/01, and 6-3/01. (b) Lanes 1 and 13, a lambda ladder; lanes 2 to 12, E. coli isolates 234/01, TM-37, 589/01, 773/01, 1686/01, 903/01, 1045/01, TM-107, 324/02, 501/01, and TM-115.

Table 1.

β-Lactamase characteristics and resistance patterns of the 28 armA-positive and seven rmtB-positive isolates of K. pneumoniae and E. coli and their transconjugants

Species pI(s)/β-lactamases(s) Resistance pattern Straina 
armA-positive    
    KP 7.9, 7.6, 5.4/CTX-M-14, SHV-1-related, TEM-1 HA ESBL CIP SXT CHL 1592/01 
 8.4, 7.6, 5.4/CTX-M-3, SHV-1-related, TEM-1 HA ESBL SXT CHL TET 6-3/01, 291/02 
  HA ESBL SXT CHL 276/02 
  HA ESBL SXT TET 244/02, 298/02, TM-118 
  HA ESBL SXT 1430/01 
  HA ESBL CIP SXT CHL  TET 690/01, 776/01 
 8.4, 8.2, 7.6, 5.4/CTX-M-3, SHV-5-related, HA ESBL SXT CHL TET 994/01, 1178/01 
     SHV-1-related, TEM-1 HA ESBL SXT CHL 197/01, 211/02, 338/02 
  HA ESBL SXT 654/01 
    EC 8.2, 7.9, 5.4/SHV-12, CTX-M-14, TEM-1 HA ESBL CIP SXT CHL  TET 234/01, 589/01, 773/01, TM-37 
 8.4, 5.4/CTX-M-3, TEM-1 HA ESBL SXT CHL TET 124/01 
  HA ESBL SXT TET 1045/01 
  HA ESBL SXT 1686/01, 340/02 
  HA ESBL CIP SXT TET 324/02 
 9.0, 8.2, 7.9, 5.4/CMY-2, SHV-12,  CTX-M-14, TEM-1 HA ESBL FOX CIP SXT  CHL TET 149/02 
 9.0, 8.4, 5.4/CMY-2, CTX-M-3, TEM-1 HA ESBL FOX SXT CHL  TET TM-107 
 negative/negative HA CIP SXT TET 903/01 
    TC 7.9, 5.4/CTX-M-14, TEM-1 HA ESBL TC of KP 1592/01 
 8.4, 5.4/CTX-M-3, TEM-1 HA ESBL SXT TCs of KP 1430/01, 244/02,  276/02, TM-118, 690/01, 776/01, 197/01,  654/01, 994/01 and 1178/01, and  TC of EC 124/01 
  HA ESBL TCs of KP 298/02 and  211/02 
 8.2/SHV-12 HA ESBL TC of KP 149/02 
rmtB-positive    
    KP 7.9, 7.6, 5.4/CTX-M-14, SHV-1-related, TEM-1 HA ESBL CIP SXT CHL  TET 289/01, 1046/01, 433/02, 654/02 
 7.6, 5.4/SHV-1-related, TEM-1 HA AMX CIP SXT CHL  TET 486/02 
    EC 7.9, 5.4/CTX-M-14, TEM-1 HA ESBL SXT CHL TET 501/01 
  HA ESBL CIP SXT CHL  TET TM-115 
    TC 5.4/TEM-1 HA AMX SXT CHL TET TCs of KP 433/02, 654/02  and 486/02 
Species pI(s)/β-lactamases(s) Resistance pattern Straina 
armA-positive    
    KP 7.9, 7.6, 5.4/CTX-M-14, SHV-1-related, TEM-1 HA ESBL CIP SXT CHL 1592/01 
 8.4, 7.6, 5.4/CTX-M-3, SHV-1-related, TEM-1 HA ESBL SXT CHL TET 6-3/01, 291/02 
  HA ESBL SXT CHL 276/02 
  HA ESBL SXT TET 244/02, 298/02, TM-118 
  HA ESBL SXT 1430/01 
  HA ESBL CIP SXT CHL  TET 690/01, 776/01 
 8.4, 8.2, 7.6, 5.4/CTX-M-3, SHV-5-related, HA ESBL SXT CHL TET 994/01, 1178/01 
     SHV-1-related, TEM-1 HA ESBL SXT CHL 197/01, 211/02, 338/02 
  HA ESBL SXT 654/01 
    EC 8.2, 7.9, 5.4/SHV-12, CTX-M-14, TEM-1 HA ESBL CIP SXT CHL  TET 234/01, 589/01, 773/01, TM-37 
 8.4, 5.4/CTX-M-3, TEM-1 HA ESBL SXT CHL TET 124/01 
  HA ESBL SXT TET 1045/01 
  HA ESBL SXT 1686/01, 340/02 
  HA ESBL CIP SXT TET 324/02 
 9.0, 8.2, 7.9, 5.4/CMY-2, SHV-12,  CTX-M-14, TEM-1 HA ESBL FOX CIP SXT  CHL TET 149/02 
 9.0, 8.4, 5.4/CMY-2, CTX-M-3, TEM-1 HA ESBL FOX SXT CHL  TET TM-107 
 negative/negative HA CIP SXT TET 903/01 
    TC 7.9, 5.4/CTX-M-14, TEM-1 HA ESBL TC of KP 1592/01 
 8.4, 5.4/CTX-M-3, TEM-1 HA ESBL SXT TCs of KP 1430/01, 244/02,  276/02, TM-118, 690/01, 776/01, 197/01,  654/01, 994/01 and 1178/01, and  TC of EC 124/01 
  HA ESBL TCs of KP 298/02 and  211/02 
 8.2/SHV-12 HA ESBL TC of KP 149/02 
rmtB-positive    
    KP 7.9, 7.6, 5.4/CTX-M-14, SHV-1-related, TEM-1 HA ESBL CIP SXT CHL  TET 289/01, 1046/01, 433/02, 654/02 
 7.6, 5.4/SHV-1-related, TEM-1 HA AMX CIP SXT CHL  TET 486/02 
    EC 7.9, 5.4/CTX-M-14, TEM-1 HA ESBL SXT CHL TET 501/01 
  HA ESBL CIP SXT CHL  TET TM-115 
    TC 5.4/TEM-1 HA AMX SXT CHL TET TCs of KP 433/02, 654/02  and 486/02 

EC, E. coli; KP, K. pneumoniae; TC, E. coli transconjugant; HA, high-level resistance (MICs, >256 mg/L) to gentamicin, tobramycin, kanamycin and amikacin. ESBL, ESBL production detected by the NCCLS confirmatory test and reduced susceptibilities to ceftazidime, cefotaxime, aztreonam, or cefepime. AMX, amoxicillin; CHL, chloramphenicol; CIP, ciprofloxacin; FOX, cefoxitin; SXT, trimethoprim/sulfamethoxazole; TET, tetracycline.

a

Isolates TM-37, TM-107, TM-115 and TM-118 were collected from Tainan Municipal Hospital. The remainder were collected from NCKUH.

This work was supported by grant NSC 92-2320-B-006-088 from the National Science Council, Taiwan.

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Author notes

1Department of Pathology, National Cheng Kung University Hospital, No. 138, Sheng-Li Rd, Tainan 70428; Departments of 2Pathology, 3Medical Technology and 4Internal Medicine, College of Medicine, National Cheng Kung University, No. 1, University Rd, Tainan 70101; 5Department of Laboratory Medicine, Tainan Municipal Hospital, No. 670, Chongde Rd, Tainan City 70173, Taiwan