a CIISA, Faculty of Veterinary Medicine, Universidade Técnica de Lisboa, Rua Prof. Cid dos Santos, 1300-477, Lisboa b Antibiotic Resistance Unit, National Institute of Health Dr. Ricardo Jorge, Av. Padre Cruz, 1649-016 Lisboa, Portugal
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Abstract |
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Introduction |
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Materials and methods |
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Seventy-two E. coli isolates from dogs with significant bacteriuria and clinical symptoms of UTI were collected from urine specimens at the Veterinary Medical School Teaching Hospital and at private practices in the Lisbon area. Isolates were identified with the API 20E System (bioMérieux, Marcy l'Étoile, France).
E. coli ATCC 25922 was used as a reference control isolate for disc diffusion and microdilution susceptibility testing. E. coli R111 (TEM-1, pI 5.4), E. coli RP4 (TEM-2, pI 5.6), E. coli Sal (IRT-1, pI 5.2), E. coli Guer (IRT-2, pI 5.2), E. coli P37 (IRT-14, pI 5.2), E. coli K12 (SHV-1, pI 7.6), Salmonella typhimurium (OXA-1, pI 7.4) and E. coli Solar (AmpC, pI 9.2) were used as reference isolates for MIC determination and for isoelectric focusing (IEF) assays. The PCR-positive controls included the above isolates and Pseudomonas aeruginosa (OXA-3), E. coli K12 transconjugants (OXA-4, SHV-1, SHV-3, SHV-4, SHV-5) and E. coli K12 (AmpC). S. typhimurium NCTC 74, Klebsiella pneumoniae NCTC 418, Proteus mirabilis ATCC 43071, P. aeruginosa (OXA-3) and S. typhimurium (OXA-1) were used as negative controls for the ampC PCR.
Susceptibility testing
Agar disc diffusion was carried out according to standard procedures.7 The MIC microdilution assay was based on standard procedures,7,8 and an inoculum of 5 x 105 cfu/mL was used. Susceptibility patterns were interpreted according to the recommendations of the Antibiogram Committee of the French Society for Microbiology.9 Antibiotic discs (Oxoid, Basingstoke, UK) included: amoxicillin (25 µg), co-amoxiclav (20/10 µg), ticarcillin (75 µg), piperacillin (75 µg), mecillinam (10 µg), cefalothin (30 µg), cefuroxime (30 µg), cefoxitin (30 µg), ceftazidime (30 µg), ceftriaxone (30 µg), cefotaxime (30 µg) and aztreonam (30 µg). Antibiotic and ß-lactamase-inhibitor powders for MIC assays were provided by the following manufacturers: clavulanic acid and ticarcillin (GlaxoSmithKline, Oeiras, Portugal), mecillinam (Leo Pharmaceutical Products, Lisbon, Portugal), cefalothin (Lilly Farma, Oeiras, Portugal), ceftazidime (GlaxoWellcome, Oeiras, Portugal), ceftriaxone (Roche Pharmaceuticals, Amadora, Portugal), cefotaxime (Hoechst Marion Roussel, Sintra, Portugal), aztreonam (Bristol-Myers Squibb, Oeiras, Portugal). Amoxicillin was purchased from Sigma (Lisbon, Portugal). Clavulanate was used at a fixed concentration of 2 mg/L with serial two-fold dilutions of amoxicillin.9 Extended-spectrum ß-lactamase production was screened for using the double-disc synergy test.10
Preparation and electrofocusing of ß-lactamases
Amoxicillin-resistant isolates were grown overnight in 40 mL of tryptone soya broth (Oxoid), with constant shaking at 37°C, and harvested by centrifugation. Crude cell extracts were obtained by sonicating the cells for 7 min at 20 kHz (Vibra-cell; Bioblock Scientific), under optimum conditions of cooling. Cellular debris was removed by centrifugation and the supernatants were then subjected to IEF with a constant voltage of 350 V, current of 15 mA and power of 1 W for 5 h at 4°C (Multiphor II; Amersham Pharmacia Biotech, Lisbon, Portugal), on polyacrylamide gels containing ampholites with a pH range of 3.59.5 (Amersham Pharmacia Biotech), as described previously by Matthew et al.11 The ß-lactamases were located on the gels with nitrocefin (500 µg/mL) (Oxoid). Positive control ß-lactamases and an IEF marker (Bio-Rad, Hemel Hempstead, UK) allowed pI to be calculated.
DNA extraction and PCR for blaTEM, blaOXA-1, blaSHV and ampC genes
Total DNA was extracted by a rapid boiling procedure. Briefly, bacteria were harvested from 2 mL of overnight culture, suspended in 100 µL sterile distilled water and lysed by heating at 100°C for 10 min. The DNA supernatant obtained by centrifugation was used immediately or stored at -20°C.
Oligonucleotides B and K were used to amplify the blaTEM gene, and shvf and shvr were used to amplify the blaSHV gene.12,13 Primers for the amplification of blaOXA-1 and ampC genes were designed using published sequences14,15 and by use of online software Primer 3 from the MIT Center for Genome Research:16 oxa-1 forward (oxa-1f), 5'-TATCTACAGCAGCGCCAGTG-3'; oxa-1 reverse (oxa-1r), 5'-CGCATCAAATGCCATAAGTG-3'; ampC forward (ampCf), 5'-CCCCGCTTATAGAGCAACAA-3'; ampC reverse (ampCr), 5'-TCAATGGTCGACTTCACACC-3'. The oxa-1f/oxa-1r and ampCf/ ampCr primer pairs yield a 199 bp fragment (nucleotide positions 9291127 bp) and a 634 bp fragment (nucleotide positions 157791 bp), respectively.
Each gene was amplified separately in a total volume of 50 µL containing the following components: 2 µL DNA; 10 pmol each of the B/K, oxa1f/oxa1r or ampCf/ampCr primer pairs, or 30 pmol of the shvf/shvr primer pair; 0.2 mM of each deoxynucleoside triphosphate (Amersham Pharmacia Biotech); 10 mM TrisHCl pH 8.8, 50 mM KCl and 1.25 U Taq DNA polymerase (MBI Fermentas, Lisbon, Portugal). The ampC and blaSHV gene reactions also included 5% (v/v) dimethylsulphoxide and 2.0 mM MgCl2, whereas the blaTEM and blaOXA-1 gene reactions also included 3.5 mM MgCl2. The reaction mixture was overlaid with 25 µL of mineral oil and cycled in an Eppendorf Mastercycler (Eppendorf AG, Hamburg, Germany). PCR amplification comprised a first cycle of 7 min denaturation at 94°C, 5 min annealing at 60°C and 60 s extension at 72°C, followed by 30 cycles of 60 s at 94°C, 2 min at 60°C and 60 s at 72°C and a final extension step of 5 min at 72°C.
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Results |
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The resistance patterns of 72 uropathogenic E. coli isolates to 11 ß-lactams and one ß-lactamß-lactamase-inhibitor combination are shown in Table 1. Twenty-six (36%) of these isolates were amoxicillin resistant, but only 19% were resistant to the combination of amoxicillin plus 2 mg/L clavulanate. We found that 35, 4, 14 and 25% of isolates were resistant to ticarcillin, piperacillin (disc diffusion method only), mecillinam and cefalothin, respectively. Just 4% of isolates were resistant to cefoxitin and 3% to cefuroxime. Only 1.4% of the isolates were resistant to the third-generation cephalosporins (ceftazidime and ceftriaxone). None of the isolates was fully resistant to cefotaxime and aztreonam and only 1.4% and 4%, respectively, showed intermediate susceptibility (Table 1
).
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The MIC90s of amoxicillin and ticarcillin were 2048 mg/L, revealing the high level of resistance towards these drugs. Fourteen isolates were not inhibited by 2 mg/L clavulanate and were also resistant to amoxicillin (MIC90 128 mg/L). The MIC90s of cefalothin and mecillinam were 128 and 32 mg/L, respectively, but resistance to ceftazidime, ceftriaxone, cefotaxime and aztreonam was low with MIC90 values of 0.5, 0.06, 0.25 and 0.5 mg/L, respectively (Table 2
).
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IEF (Table 4) revealed ß-lactamases with pIs of 5.4, 7.4, 7.6 and
9.0. Approximately one-quarter of the isolates (27%) encoded simultaneously two different types of ß-lactamase and the remaining 73% encoded a single enzyme. TEM-1 ß-lactamase was the most frequently detected enzyme (77%), followed by AmpC (31%), SHV (11.5%) and OXA-1 (7.7%).
Twenty isolates producing TEM-1 ß-lactamases with a pI of 5.4, either alone (14 of 20) or in addition to enzymes focusing at 9.0 (six of 20), were positive for the presence of the blaTEM gene (Table 4
). IEF showed that one isolate produced AmpC alone with a pI of 9.0. The remaining ß-lactamases belonged to the SHV family or were OXA-1 (Table 4
). Briefly, three ß-lactamase-producing isolates with pIs of 7.6, which aligned with SHV-1, were positive for the blaSHV gene. One of those SHV-producing isolates also showed an AmpC enzyme with a pI > 9.6. Two isolates co-focusing with the OXA-1 enzyme at pI 7.4 were positive for the blaOXA-1 gene. All amoxicillin-resistant E. coli isolates were positive for the ampC gene. Two ß-lactam-susceptible uropathogenic E. coli isolates, the E. coli K12 and all E. coli K12 transconjugants used as controls, were also positive for the ampC gene. However, other Gram-negative bacilli, such as S. typhimurium, P. mirabilis, P. aeruginosa and K. pneumoniae, were negative for the ampC gene.
Six of the 14 clavulanate-resistant isolates with MICs ranging from 32 to 2048 mg/L produced TEM-1 ß-lactamase alone. Four clavulanate-resistant isolates with MICs ranging from 512 to >2048 mg/L produced both TEM-1 and AmpC enzymes. Of the remaining four clavulanate-resistant isolates, one was an SHV producer (MIC 32 mg/L), two were OXA-1 producers (MIC 128 mg/L) and one was an AmpC producer (MIC 256 mg/L) (Tables 3 and 4). SHV enzymes showed high MICs of amoxicillin (between 1024 and >2048 mg/L) and of ticarcillin (>2048 mg/L). Isolates producing OXA-1 or AmpC ß-lactamase showed lower levels of resistance to amoxicillin, with MICs of 512 and 256 mg/L, respectively.
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Discussion |
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In the Portuguese human population, Sousa et al.23 and Caniça et al.24 found a higher level of resistance to aminopenicillins (51.2% to ampicillin and 57.8% to amoxicillin, respectively), a lower level of resistance to co-amoxiclav combination (3.9% and 6.8%, respectively) and to cefalothin (4.5% and 17.1%, respectively) in E. coli UTI isolates and in E. coli isolates from in-patients, respectively. Resistance to second- and third-generation cephalosporins was very low in clinical isolates, as in Portuguese canine uropathogenic E. coli isolates. Worldwide, the level of resistance towards aminopenicillins and co-amoxiclav combination in human urinary tract E. coli isolates is also of great concern.2527
In this study, the most frequent mechanism of resistance to ß-lactams in uropathogenic E. coli was the presence of TEM-1 ß-lactamase. This is consistent with previous reports in human clinical E. coli isolates from Portugal23,24 and other countries worldwide.2830 TEM-1 alone mediated resistance in 14 out of 20 isolates. Eight TEM-1-producing isolates showed a diversity of resistance phenotypes. Six isolates (194, 224, 226, 291, 505 and 553) were resistant to the ß-lactamase inhibitor, susceptible or resistant to mecillinam and intermediate or resistant to cefalothin. These six resistance phenotypes indicate TEM-1 hyperproduction, but sequencing studies are required to exclude the possibility that this resistance to co-amoxiclav combination is due to inhibitor-resistant TEM ß-lactamase production.31 TEM-1 and AmpC enzymes were produced simultaneously in six isolates. This association of ß-lactamases in enteropathogenic E. coli isolates of bovine origin was also reported previously.5 The characterization of the AmpC enzymes in this study relied on electrofocusing results with the rapid appearance of strong bands at pI 9.0 or above, rather than on the detection of the ampC gene. In fact, ampC genes were amplified in all amoxicillin-resistant E. coli isolates, in agreement with their almost ubiquitous distribution in enterobacteria, in particular in E. coli.30,32 However, the resistance mechanism present in the six isolates cannot be attributed to one enzyme alone as both types of ß-lactamases may demonstrate different resistance phenotypes.32,33 Isolate 434, which also encoded both TEM-1 and AmpC ß-lactamases, was clearly resistant to extended-spectrum cephalosporins, with an MIC of 64 mg/L of ceftazidime and ceftriaxone, as well as resistance to clavulanate. It is possible that in this isolate AmpC overexpression plays a significant role in the resistance phenotype to ß-lactams. The ß-lactam resistance phenotype of isolate 615 is due to the presence of an AmpC enzyme alone focusing at 9.0. This phenotype resembles perman-ent AmpC hyperproduction, similar to that found in derepressed Enterobacter spp., except in terms of genetic organization. It showed a reduced susceptibility to ß-lactams and clavulanate, and only a moderate resistance to aztreonam, with a MIC of 8 mg/L. However, although the AmpC producer isolates from group 1 of the Bush JacobyMedeiros classification34 are clavulanate resistant, we found three clavulanate-susceptible isolates (isolates 91, 238 and 386). The pI of these isolates indicates that they are AmpC producers. It is possible that these clavulanate-susceptible ß-lactamases are low-level plasmid-mediated AmpC-type enzymes.33,35
Two isolates encoding the OXA-1 enzyme exhibited resistance phenotypes comparable to those of TEM-1- producing isolates. However, they showed lower MICs than TEM-1 producers of amoxicillin and co-amoxiclav, with MICs of 512 and 128 mg/L, respectively. Similarly, elevated MICs of ticarcillin were obtained for TEM-1 or OXA-1 producers.
The three ß-lactamases from the SHV family, with pI 7.6 and positive for the blaSHV gene, all conferred resistance to amoxicilin and ticarcillin, one also mediated low resistance to clavulanate (MIC 32 mg/L) and another to cefalothin (MIC 64 mg/L). Isolate 239 may be less susceptible to clavulanate due to a hyperproduction mechanism, as described previously.36 Given the pI of these three SHV enzymes, SHV-1 or SHV-11 may be the ß-lactamase involved.37
In our study the double-disc synergy test used to screen for the presence of ESBLs presented some limitations as already described,38,39 with a non-specific result for isolate 434. The MIC assay was more accurate in the level of resistance characterization than the disc diffusion assay, allowing the pharmacological breakpoint to be established more exactly. These criteria define resistance relative to the drug concentration in vivo and are essential in these uropathogens that are exposed to elevated antimicrobial concentrations in urine.
There was good agreement between the ß-lactamase identifications inferred from electrofocusing and the corresponding encoding gene detected for all isolates. However, there was poor agreement between ß-lactamase types and resistance phenotypes. In fact, the MICs for ESBLs expressed low-level resistance, which may reflect great permeability in E. coli isolates, low enzyme quantity or low-level activity against those drugs. Overall, we observed similar susceptibility patterns between clavulanateresistant E. coli isolates producing high levels of plasmid-mediated TEM-1 ß-lactamases, and isolates producing OXA-1 or AmpC enzymes. Laboratories worldwide are faced with the difficulty of distinguishing between these patterns,40,41 and this problem needs to be resolved.
This is the first study of resistance to ß-lactamß-lactamase inhibitor combinations in animals in Portugal. We found an important pattern of resistance to amoxicillin and co-amoxiclav, which are first-line and overused drugs for UTI therapy in dogs in this country. The production of TEM-1, SHV, AmpC and OXA-1 enzymes in uropathogenic canine E. coli is of concern. This study confirms that, even in animals, ß-lactamase detection is important. The data indicate that the clinical use of ß-lactamß-lactamase inhibitor combinations in canine therapy imposes a strong selection pressure for the emergence of resistant bacterial isolates. Thus, the need for antimicrobial resistance surveillance in veterinary medicine is applicable to farm animals and pets, which may be in direct contact with humans. The establishment of veterinary antibiotic policies is of prime importance to safeguard the future efficacy of antimicrobial therapy in animals and to protect public health. It would be interesting to use molecular typing methods to explore the relationships between these animal uropathogenic isolates and human E. coli populations.
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Acknowledgements |
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Notes |
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References |
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2 . Medeiros, A. A. (1997). Evolution and dissemination of ß-lactamases accelerated by generations of ß-lactam antibiotics. Clinical Infectious Diseases 24, Suppl. 1, S1945. [ISI][Medline]
3 . Sanders, C. C. & Sanders W. E., Jr (1992). ß-Lactam resistance in Gram-negative bacteria: global trends and clinical impact. Clinical Infectious Diseases 15, 82439. [ISI][Medline]
4 . Hunter, J. E. B., Corkill, J. E., McLennan, A. G., Fletcher, J. N. & Hart, C. A. (1993). Plasmid encoded ß-lactamases resistant to inhibition by clavulanic acid produced by calf faecal coliforms. Research in Veterinary Science 55, 36770. [ISI][Medline]
5
.
Bradford, P. A., Petersen, P. J., Fingerman, I. M. & White, D. G. (1999). Characterization of expanded-spectrum cephalosporin resistance in E. coli isolates associated with bovine calf diarrhoeal disease. Journal of Antimicrobial Chemotherapy 44, 60710.
6
.
Teshager, T., Domínguez, L., Moreno, M. A., Saénz, Y., Zarazaga, M., Torres, C. et al. (2000). Isolation of an SHV-12 ß-lactamase-producing Escherichia coli strain from a dog with recurrent urinary tract infections. Antimicrobial Agents and Chemotherapy 44, 34834.
7 . Courvalin, P., Goldstein, F., Phillipon, A. & Sirot, S. (1985). Antibiogramme, 1st edn. Mpc-Vidéon, Brussels.
8 . American Society for Microbiology. (1992). Section 5.2Broth microdilution MIC testing. In Clinical Microbiology Procedures Handbook, 1st edn, (Isenberg, H. D., Ed.). American Society for Microbiology, Washington, DC.
9 . Soussy, C. J., Carret, G., Cavallo, J. D., Chardon, H., Chidiac, C., Choutet, P. et al. (2000). Antibiogram Committee of the French Society for Microbiology 20002001, Statement, pp. 256.
10 . Jarlier, V., Nicolas, M. H., Fournier, G. & Philippon, A. (1988). Extended broad-spectrum ß-lactamases conferring resistance to newer ß-lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns. Reviews of Infectious Diseases 10, 86778. [ISI][Medline]
11 . Matthew, M., Harris, A. M., Marshall, M. J. & Ross, G. W. (1975). The use of analytical isoelectric focusing for detection and identification of ß-lactamases. Journal of General Microbiology 88, 16978. [ISI][Medline]
12 . Belaaouaj, A., Lapoumeroulie, C., Caniça, M., Vedel, G., Névot, P., Krishnamoorthy, R. et al. (1994). Nucleotide sequences of the genes coding for the TEM-like ß-lactamases IRT-1 and IRT-2 (formerly called TRI-1 and TRI-2). FEMS Microbiology Letters 120, 7580. [ISI][Medline]
13 . M'Zali, F., Gascoyne-Binzi, D. M., Heritage, J. & Hawkey, P. M. (1996). Detection of mutations conferring extended-spectrum activity on SHV ß-lactamases using polymerase chain reaction single strand conformational polymorphism (PCR-SSCP). Journal of Antimicrobial Chemotherapy 37, 797802. [Abstract]
14 . Ouellete, M., Bissonnette, L. & Roy, P. H. (1987). Precise insertion of antibiotic resistance determinants into Tn21-like transposons: nucleotide sequence of the OXA-1 ß-lactamase gene. Proceedings of the National Academy of Sciences, USA 84, 737882. [Abstract]
15 . Jaurin, B. & Grundström, T. (1981). AmpC cephalosporinase of Escherichia coli K-12 has a different evolutionary origin from that of the ß-lactamases of the penicillinase type. Proceedings of the National Academy of Sciences, USA 78, 4897901. [Abstract]
16 . MIT Center for Genome Research. Software Primer 3. [On-line.] http://www.genome.w1.mit.edu/cgi-bin/primer/primer3 (25 January 2001, date last accessed).
17 . Féria, C., Correia, J. D., Machado, J., Vidal, R. & Gonçalves, J. (2000). Urinary tract infection in dogs, analysis of 419 urocultures carried out in Portugal. In Genes and Proteins Underlying Microbial Urinary Tract Virulence, (Emödy, L., Pál, T., Blurn-Oehler, G. & Hacker, I. H., Eds), pp. 3014. Kluwer Academic/Plenum Publishers, New York, USA.
18 . Normand, E. H., Gibson, N. R., Reid, S. W. J., Carmichael, S. & Taylor, D. J. (2000). Antimicrobial-resistance trends in bacterial isolates from companion-animal community practice in the UK. Preventive Veterinary Medicine 46, 26778. [ISI][Medline]
19 . Rohrich, P. J., Ling, G. V., Ruby, A. L., Jang, S. S. & Johnson, D. L. (1983). In vitro susceptibilities of canine urinary bacteria to selected antimicrobial agents. Journal of the American Veterinary Medical Association 8, 86367.
20 . Kilgore, W. R., Simmons, R. D. & Jackson, J. W. (1986). ß-Lactamase inhibition: a new approach in overcoming bacterial resistance. Compendium on Continuing Education for the Practicing Veterinarian 8, 32531. [ISI]
21 . Franklin, A. & Mörner, A. P. (1996). Antibiotic sensitivity of bacterial isolates from urinary tract infections and metritis in dogs. Supplement to Compendium on Continuing Education for the Practicing Veterinarian 18, 96.
22
.
Orden, J. A., Ruiz-Santa-Quiteria, J. A., García, S., Cid, D. & De La Fuente, R. (1999). In vitro activities of cephalosporins and quinolones against Escherichia coli strains isolated from diarrhoeic dairy calves. Antimicrobial Agents and Chemotherapy 43, 5103.
23 . Sousa, J. C., Carneiro, G., Peixe, M. L., Queirós, M. L. & Rebelo, I. (1991). Characterization of ß-lactamases encoded by pathogenic strains of Escherichia coli from Portugal. Journal of Antimicrobial Chemotherapy 27, 43740. [Abstract]
24 . Caniça, M., Ferreira, M., Vaz-Pato, V., Ferreira, E. & Grupo de Estudo Multicêntrico de Vigilância da Susceptibilidade aos Antibióticos. (2000). Mecanismos de resistência aos ß-lactâmicos em estirpes de Escherichia coli de origem clínica. Arquivos de Medicina 14, 71.
25
.
Gales, A. C., Jones, R. N., Gordon, K. A., Sader, H. S., Wilke, W. W., Beach, M. L. et al. (2000). Activity and spectrum of 22 antimicrobial agents tested against urinary tract infection pathogens in hospitalized patients in Latin America: report from the second year of the SENTRY Antimicrobial Surveillance Program (1998). Journal of Antimicrobial Chemotherapy 45, 295303.
26 . Winstanley, T. G., Limb, D. I., Eggington, R. & Hancock, F. (1997). A 10 year survey of the antimicrobial susceptibility of urinary tract isolates in the UK: the Microbe Base project. Journal of Antimicrobial Chemotherapy 40, 5914. [Abstract]
27
.
Zhanel, G. G., Karlowsky, J. A., Harding, G. K. M., Carrie, A., Mazzulli, T., Low, D. E. et al. (2000). A Canadian national surveillance study of urinary tract isolates from outpatients: comparison of the activities of trimethoprimsulfamethoxazole, ampicillin, mecillinam, nitrofurantoin and ciprofloxacin. Antimicrobial Agents and Chemotherapy 44, 108992.
28 . Cooksey, R., Swenson, J., Clark, N., Gay, E. & Thornsberry, C. (1990). Patterns and mechanisms of ß-lactam resistance among isolates of Escherichia coli from hospitals in the United States. Antimicrobial Agents and Chemotherapy 34, 73945. [ISI][Medline]
29 . Liu, P. Y. F., Gur, D., Hall, L. M. C. & Livermore, D. M. (1992). Survey of the prevalence of ß-lactamases amongst 1000 Gramnegative bacilli isolated consecutively at the Royal London Hospital. Journal of Antimicrobial Chemotherapy 30, 42947. [Abstract]
30 . Sirot, D. (1995). Extended-spectrum plasmid-mediated ß-lactamases. Journal of Antimicrobial Chemotherapy 36, Suppl. A, 1934. [ISI][Medline]
31 . Caniça, M. M., Lu, C. Y., Krishnamoorthy, R. & Paul, G. C. (1997). Molecular diversity and evolution of blaTEM genes encoding ß-lactamases resistant to clavulanic acid in clinical E. coli. Journal of Molecular Evolution 44, 5765. [ISI][Medline]
32 . Livermore, D. M. (1995). ß-Lactamases in laboratory and clinical resistance. Clinical Microbiology Reviews 8, 55784. [Abstract]
33
.
Coudron, P. E., Moland, E. S. & Thonson, K. S. (2000). Occurrence and detection of AmpC ß-lactamases among Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates at a Veterans Medical Center. Journal of Clinical Microbiology 38, 17916.
34
.
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.
35 . Matsumoto, Y., Ikeda, F., Kamimura, T., Yokota, Y. & Mine, Y. (1988). Novel plasmid-mediated ß-lactamase from Escherichia coli that inactivates oxyimino-cephalosporins. Antimicrobial Agents and Chemotherapy 32, 12436. [ISI][Medline]
36 . Miró, E., Cuerpo, M., Navarro, F., Sabaté, M., Mirelis, B. & Prats, G. (1998). Emergence of clinical Escherichia coli isolates with decreased susceptibility to ceftazidime and synergic effect with co-amoxiclav due to SHV-1 hyperproduction. Journal of Antimicrobial Chemotherapy 42, 5358. [Abstract]
37
.
Heritage, J., M'Zali, F. H., Gascoyne-Binzi, D. & Hawkey, P. M. (1999). Evolution and spread of SHV extended-spectrum ß-lactamases in Gram-negative bacteria. Journal of Antimicrobial Chemotherapy 44, 30918.
38 . Thomson, K. S. & Sanders, C. C. (1992). Detection of extended spectrum ß-lactamases in members of the family Enterobacteriaceae: comparison of the double-disk and three dimensional tests. Antimicrobial Agents and Chemotherapy 36, 187782. [Abstract]
39 . Jacoby, G. A. & Han, A. (1996). Detection of extendedspectrum ß-lactamases in clinical isolates of Klebsiella pneumoniae and Escherichia coli. Journal of Clinical Microbiology 3, 90811.
40
.
Thomson, K. S., Sanders, C. C. & Moland, E. S. (1999). Use of microdilution panels with and without ß-lactamase inhibitors as a phenotypic test for ß-lactamase production among Escherichia coli, Klebsiella spp., Enterobacter spp., Citrobacter freundii, and Serratia marcescens. Antimicrobial Agents and Chemotherapy 43, 1393400.
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Tenover, F. C., Mohammed, M. J., Gorton, T. S. & Dembek, Z. F. (1999). Detection and reporting of organisms producing extended-spectrum ß-lactamases: survey of laboratories in Connecticut. Journal of Clinical Microbiology 37, 406570.
Received 6 June 2001; returned 18 August 2001; revised 18 September 2001; accepted 27 September 2001