ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1994, p. 494-498 Vol. 38, No. 3 0066-4804/94/$04.00+0 Copyright © 1994, American Society for Microbiology Effect of Hyperproduction of TEM-1 1-Lactamase on In Vitro Susceptibility of Escherichia coli to 3-Lactam Antibiotics PEI-JUN WU,t KEVIN SHANNON,*t AND IAN PHILLIPS Department of Microbiology, United Medical and Dental Schools, St. Thomas' Hospital, London SE1 7EH, United Kingdom Received 21 July 1993/Returned for modification 4 November 1993/Accepted 4 January 1994 The susceptibility of 173 TEM-1-producing isolates of Escherichia coli was assessed by determination of MICs by the agar dilution method. MICs of amoxicillin, mezlocillin, cephaloridine, and, to a smaller extent, amoxicillin-clavulanic acid (but not cephalexin, cefuroxime, cefotaxime, ceftazidime, or imipenem) were higher for isolates that produced large amounts of f8-lactamase than for isolates that produced smaller amounts. The effect of fixed concentrations of clavulanic acid on resistance to amoxicillin was assessed for 34 selected TEM-1-producing isolates. Low concentrations of the inhibitor (0.5 to 1 ,ug/ml) reduced the amoxicillin MICs substantially for almost all the isolates, although the reductions were not sufficient to render any of the isolates amoxicillin susceptible. Higher concentrations of clavulanic acid had progressively greater effects on amoxicillin MICs, but even at 8 ,ug/ml some of the isolates with high P-lactamase activities remained resistant or only moderately susceptible to amoxicillin. All the isolates were inhibited by clavulanic acid (in the absence of amoxicillin) at concentrations of 16 to 32 ,ug/ml. TEM-1 ,-lactamase activity was inhibited in vitro by clavulanic acid, but not totally, with approximately 2% of the initial activity remaining at 2 ,ug/ml and 0.4% remaining at 8 ,guml. These findings suggest that the amount of ,-lactamase activity is a major determinant of the degree of resistance to several ,-lactam antibiotics and can make the difference between susceptibility and resistance to some compounds, notably the combination of amoxicillin with clavulanic acid. Clavulanic acid [Z-(3R,5R)-2-,-hydroxyethylidene clavam-3- resistant to amoxicillin has increased during the past 20 years carboxylic acid] is an inhibitor of many P-lactamases, particu- (18, 20). larly the plasmid-determined enzymes (11, 12). One of these This study was undertaken to examine in more detail the enzymes, TEM-1, is responsible for 80% or more of resistance effect of production of different amounts of TEM-1 P-lacta- to amoxicillin and other P-lactamase-sensitive antibiotics in mase on the susceptibility of E. coli to a range of f-lactam Escherichia coli (reviewed by Wiedemann et al. [24]). antibiotics and also to study the effects of different concentra- It has become apparent that the amount of ,B-lactamase tions of clavulanic acid on the degrees of resistance to amoxi- produced by the bacterium affects the resistance profile, since cillin. there have been a number of reports of E. coli resistant to ,B-lactam agents expected to have activity against strains that produce TEM-1 or related 3-lactamases. Although some have MATERIALS AND METHODS involved extended-spectrum enzymes that confer resistance to cephalosporins such as ceftazidime and cefotaxime (see the Collection and identification of organisms. Consecutive work of Jacoby and Medeiros [5] for a review) or ,3-lactamase isolates of E. coli were collected from urine specimens submit- inhibitor-resistant enzymes (1, 22), there have also been re- ted to the Department of Microbiology, St. Thomas' Hospital, of of amounts of TEM-2, or ports production large TEM-1, between March and May 1991. The organisms were identified resistance to and to com- SHV-1 conferring ureidopenicillins with API 20E strips, and antibiotic susceptibility was tested by binations of a ,B-lactamase inhibitor with a 3-lactamase-sensi- the comparative disc method (26) in the clinical laboratory. tive antibiotic (2, 8, 13, 16, 17, 21, 23, 25). The existence of such Organisms that appeared resistant to amoxicillin were saved strains is not a new phenomenon; Page et al. (10) demon- for further study on nutrient agar slants. strated 3-lactamase and resistance to amoxi- hyperproduction Determination of MICs. MICs were determined by inocula- cillin-clavulanic acid by TEM-1-producing strains isolated in tion, with a multiple inoculator (Denley, Billingshurst, Sussex, 1967 to 1974 but gave no indication of how abundant such United Kingdom), of about 104 CFU per spot suspended in strains were in those days. There is no evidence that hyperpro- brain heart infusion broth (Oxoid CM 225) on diagnostic ducers have increased as a proportion of P-lactamase-produc- sensitivity test agar (Oxoid CM 261) containing serial doubling ing E. coli in recent years since Seetulsingh et al. (19) examined amoxicillin-resistant, TEM-1-producing strains of E. coli and concentrations of the antibiotic. E. coli NCTC 10418 was used as the control strain. The MIC was defined as the lowest reported that there was no significant difference in the distri- bution of 3-lactamase activities between 50 isolates collected in concentration on which there was no visible growth after 46 collected in 1989. Nevertheless, there is clear overnight incubation at 37°C. Antibiotics were kindly given, as 1982 and powders of known potency, by the following companies: amoxi- evidence that the proportion of isolates of E. coli that are cillin and clavulanic acid by SmithKline Beecham, Betchworth, Surrey, United Kingdom; cephaloridine, cephalexin, cefu- * Corresponding author. Phone: 44 71-955 4572. Fax: 44 71-955 roxime, and ceftazidime by Glaxo Group Research, Greenford, 4295. Middlesex, United Kingdom; cefotaxime by Roussel, Ux- t'Present address: Department of Microbiology, UMDS, Guy's bridge, Middlesex, United Kingdom; and imipenem by Merck, Hospital, London SEI 9RT, United Kingdom. Sharp and Dohme, Hoddesdon, Hertfordshire, United King- 494 VOL. 38, 1994 HYPERPRODUCTION OF TEM-1 1-LACTAMASE 495 dom. Except where explicitly stated otherwise, amoxicillin was 60 combined with clavulanic acid in the ratio 2:1 by weight. The MIC for 10% of strains tested (MIC10), MIC25, M'C50, M'C75, and MIC90 were calculated as described by Hamilton- 50 - Miller (3). The MICo was defined as the concentration imme- diately below the concentration at which at least one isolate 4040 was inhibited. Confidence intervals of the MIC50 were calcu- 0 lated by the method described by Martin and his colleagues co (7). National Committee for Clinical Laboratory Standards 0 30- criteria for susceptibility and resistance were applied (9) and were as follows: amoxicillin (without or with clavulanic acid), cefuroxime, and cephalexin, <8 jig/ml (susceptible), 16 ,ug/ml E-2020 (moderately susceptible), and -32,ug/ml (resistant); mezlocil- lin, <16 ,ug/ml (susceptible), 32 to 64 ,ug/ml (moderately 10 susceptible), and .128,ug/ml (resistant); cefotaxime and cefta- zidime, <8 ,ug/ml (susceptible), 16 to 32 ,ug/ml (moderately susceptible), and .64 ,ug/ml (resistant); and imipenem, s4 ,ug/ml (susceptible), 8 ,ug/ml (moderately susceptible), and 0 100 200 300 400 500 600 700 800 -16 j.g/ml (resistant). 50 150 250 350 450 550 650 750 Identification of P-lactamases and measurement of j8-lacta- activity mase activity. Cells were harvested from overnight brain heart 0-lactamase infusion broth cultures by centrifugation and resuspended in FIG. 1. Distribution of 13-lactamase activity for TEM-1-producing 0.5 ml of sodium phosphate buffer (0.1 M, pH 7), and isolates of E. coli. ,B-Lactamase activity is expressed as nanomoles of P-lactamase was released by sonication (two bursts of 30 s in nitrocefin hydrolyzed per minute per milligram of protein. ice at an amplitude of 12 ,um in an MSE Ultrasonic Disinte- grator Mark II). Enzymes were identified by isoelectric focus- ing in Agarose-IEF (Pharmacia) gels containing Pharmalyte shown in Fig. 2 and 3. All the isolates were resistant to (pH range, 3 to 10; Pharmacia). Preparations of strains known amoxicillin, but there was an increase in the degree of resis- to produce TEM-1, TEM-2, OXA-1, SHV-1, or PSE-4 were tance as the 1-lactamase activity increased, with the 95% used as standards. After the focusing, ,B-lactamases were confidence intervals of the MIC50 (786 to 1,192 ,ug/ml) for the detected by spreading a solution of nitrocefin (100 ,ug/ml) on group with the lowest activity being significantly lower than the the surface of the gel. Isolates that produced TEM-1 as the values for the group with the highest activity (3,419 to >4,096 sole detectable ,-lactamase were included in the collection of ,ug/ml). MICs of mezlocillin and cephaloridine were lower, but TEM-1 producers. Strains that produced TEM-1 and also again there was an increase as the ,-lactamase activity in- apparently overproduced the chromosomal 1-lactamase (i.e., creased. In the case of mezlocillin the 95% confidence intervals showed a significant band of activity at alkaline pH on isoelec- of the MIC50 for the group with the lowest 3-lactamase activity tric focusing) were excluded from the collection. was 19.1 to 28.9 ,ug/ml compared with 99.2 to 151 ,ug/ml for the f-Lactamase activity was measured spectrophotometrically group with highest activity; for cephaloridine the ranges were against 50 ,uM nitrocefin at 482 nm. The buffer used was 0.1 M 5.49 to 8.33 compared with 19.8 to 30.2 ,Ig/ml for these two sodium phosphate (pH 7), the light path was 1 cm, and all groups. The changes were less marked for the combination of assays were performed at room temperature. Enzyme activity amoxicillin with clavulanic acid; however, there was a signifi- was standardized against the total protein concentration in the cant difference between the group with the lowest 3-lactamase enzyme preparation, as estimated by the biuret method (4).