1Department of Pathology, National Cheng Kung University Hospital, No. 138, Sheng-Li Rd, Tainan 70428; Departments of 2 Pathology, 3 Medical Technology and 4 Internal Medicine, College of Medicine, National Cheng Kung University, No. 1, University Rd, Tainan 70101; 5 Department of Laboratory Medicine, Tainan Municipal Hospital, No. 670, Chongde Rd, Tainan City 70173, Taiwan
Received 30 June 2004; returned 30 July 2004; revised 3 September 2004; accepted 11 September 2004
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Abstract |
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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. ß-Lactamase characteristics were determined by phenotypic and genotypic methods.
Results: Overall, 28 armA-positive and seven rmtB-positive isolates were identified, and extended-spectrum ß-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.
Keywords: resistance genes , multidrug resistance , armA , rmtB , extended-spectrum ß-lactamases
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Introduction |
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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 ß-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.
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Materials and methods |
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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.16 E. 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.
ß-Lactamase characterization
ESBL production was detected by the confirmatory disc diffusion tests recommended by the NCCLS.15,18 Crude ß-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 3.59.5; Amersham Biosciences, Hong Kong, China) as described previously.21 ß-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
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Results and discussion |
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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 6462 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.
ß-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 ß-lactamases expressed by these isolates on IEF gels and the consistent ß-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 ß-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 ß-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 ß-lactamase, and the rmtB-positive K. pneumoniae isolate produced two narrow-spectrum ß-lactamases, TEM-1 and an SHV-1-related ß-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 ß-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.
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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, 16256 mg/L), ceftazidime (MICs, 4256 mg/L), aztreonam (MICs, 4128 mg/L), and cefepime (MICs, 464 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, 416 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 ß-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.
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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.
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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.
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Acknowledgements |
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Footnotes |
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References |
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2 . Miller, G. H., Sabatelli, F. J., Hare, R. S. et al. (1997). The most frequent aminoglycoside resistance mechanismschanges with time and geographic area: a reflection of aminoglycoside usage patterns? Clinical Infectious Diseases 24, Suppl. 1, S46S62.[ISI][Medline]
3 . Quintiliani, R., Jr, Sahm, D. F. & Courvalin, P. (1999). Mechanisms of resistance to antimicrobial agents. In Manual of Clinical Microbiology, 7th edn. (Murray, P. R., Baron, E. J., Pfaller, M. A. et al., Eds), pp. 150525. American Society for Microbiology, Washington, DC, USA.
4 . Davies, J. & Wright, G. D. (1997). Bacterial resistance to aminoglycoside antibiotics. Trends in Microbiology 5, 23440.[CrossRef][ISI][Medline]
5 . Demydchuk, J., Oliynyk, Z. & Fedorenko, V. (1998). Analysis of a kanamycin resistance gene (kmr) from Streptomyces kanamyceticus and a mutant with increased aminoglycoside resistance. Journal of Basic Microbiology 38, 2319.[CrossRef][ISI][Medline]
6 . Holmes, D. J. & Cundliffe, E. (1991). Analysis of a ribosomal RNA methylase gene from Streptomyces tenebrarius which confers resistance to gentamicin. Molecular and General Genetics 229, 22937.[CrossRef][Medline]
7 . Kelemen, G. H., Cundliffe, E. & Financsek, I. (1991). Cloning and characterization of gentamicin-resistance genes from Micromonospora purpurea and Micromonospora rosea. Gene 98, 5360.[CrossRef][ISI][Medline]
8 . Kojic, M., Topisirovic, L. & Vasiljevic, B. (1992). Cloning and characterization of an aminoglycoside resistance determinant from Micromonospora zionensis. Journal of Bacteriology 174, 786872.[Abstract]
9 . Skeggs, P. A., Thompson, J. & Cundliffe, E. (1985). Methylation of 16S ribosomal RNA and resistance to aminoglycoside antibiotics in clones of Streptomyces lividans carrying DNA from Streptomyces tenjimariensis. Molecular and General Genetics 200, 41521.[CrossRef][Medline]
10 . Beauclerk, A. A. & Cundliffe, E. (1987). Sites of action of two ribosomal RNA methylases responsible for resistance to aminoglycosides. Journal of Molecular Biology 193, 66171.[ISI][Medline]
11
.
Galimand, M., Courvalin, P. & Lambert, T. (2003). Plasmid-mediated high-level resistance to aminoglycosides in Enterobacteriaceae due to 16S rRNA methylation. Antimicrobial Agents and Chemotherapy 47, 256571.
12
.
Doi, Y., Yokoyama, K., Yamane, K. et al. (2004). Plasmid-mediated 16S rRNA methylase in Serratia marcescens conferring high-level resistance to aminoglycosides. Antimicrobial Agents and Chemotherapy 48, 4916.
13 . Yokoyama, K., Doi, Y., Yamane, K. et al. (2003). Acquisition of 16S rRNA methylase gene in Pseudomonas aeruginosa. Lancet 362, 188893.[CrossRef][ISI][Medline]
14
.
Yamane, K., Doi, Y., Yokoyama, K. et al. (2004). Genetic environments of the rmtA gene in Pseudomonas aeruginosa clinical isolates. Antimicrobial Agents and Chemotherapy 48, 206974.
15 . National Committee for Clinical Laboratory Standards. (2004). Performance Standards for Antimicrobial Susceptibility TestingFourteenth Informational Supplement: Approved Standard M100-S14. NCCLS, Wayne, PA, USA.
16 . National Committee for Clinical Laboratory Standards. (2003). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallySixth Edition: Approved Standard M7-A6. NCCLS, Wayne, PA, USA.
17 . Grunstein, M. & Hogness, D. S. (1975). Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proceedings of the National Academy of Sciences, USA 72, 39615.[Abstract]
18 . National Committee for Clinical Laboratory Standards. (2003). Performance Standards for Antimicrobial Disk Susceptibility TestsEighth Edition: Approved Standard M2-A8. NCCLS, Wayne, PA, USA.
19 . Bauernfeind, A., Grimm, H. & Schweighart, S. (1990). A new plasmidic cefotaximase in a clinical isolate of Escherichia coli. Infection 18, 2948.[ISI][Medline]
20 . Mathew, A., Harris, A.M., Marshall, M. J. et al. (1975). The use of analytical isoelectric focusing for detection and identification of ß-lactamases. Journal of General Microbiology 88, 16978.[ISI][Medline]
21
.
Yan, J.-J., Ko, W.-C., Tsai, S.-H. et al. (2000). Dissemination of CTX-M-3 and CMY-2 ß-lactamases among clinical isolates of Escherichia coli in southern Taiwan. Journal of Clinical Microbiology 38, 43205.
22 . Mabilat, C. & Goussard, S. (1993). PCR detection and identification of genes for extended-spectrum ß-lactamases. In Diagnostic Molecular Microbiology: Principles and Applications (Persing, D. H., Smith, T. F., Tenover, F. C. et al., Eds), pp. 5539. American Society for Microbiology, Washington, DC, USA.
23 . Nüesch-Inderbinen, M. T., Hächler, H. & Kayser, F. H. (1996). Detection of genes coding for extended-spectrum SHV ß-lactamases in clinical isolates by a molecular genetic method, and comparison with the E test. European Journal of Clinical Microbiology and Infectious Diseases 15, 398402.[ISI][Medline]
24 . Saladin, M., Cao, V. T. B., Lambert, T. et al. (2002). Diversity of CTX-M ß-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals. FEMS Microbiology Letters 209, 1618.[CrossRef][ISI][Medline]
25
.
Winokur, P. L., Brueggemann, A., Desalvo, D. L. et al. (2000). Animal and human multidrug-resistant, cephalosporin-resistant Salmonella isolates expressing a plasmid-mediated CMY-2 AmpC ß-lactamase. Antimicrobial Agents and Chemotherapy 44, 277783.
26 . Provence, D. L. & Curtiss, R., III (1994). Gene transfer in gram-negative bacteria. In Methods for General and Molecular Bacteriology (Gerhardt, P., Murray, R. G. E., Wood, W. A. et al., Eds), pp. 31947. American Society for Microbiology, Washington, DC, USA.
27 . Bachmann, B. J. & Low, K. B. (1980). Linkage map of Escherichia coli K-12, edition 6. Microbiology Reviews 44, 14516.
28 . Kado, C. I. & Liu, S. T. (1981). Rapid procedure for detection and isolation of large and small plasmids. Journal of Bacteriology 145, 136573.[ISI][Medline]
29
.
Tenover, F. C., Arbeit, R. D., Goering, R. V. et al. (1995). Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. Journal of Clinical Microbiology 33, 22339.
30
.
Bush, K., Jacoby, G. A. & Medeiros, A. A. (1995). A functional classification scheme for ß-lactamases and its correlation with molecular structure. Antimicrobial Agents and Chemotherapy 39, 121133.