Beta-Lactamases of Type Culture Strains of the Bacteroides Fragilis Group and of Strains That Hydrolyse Cefoxitin, Latamoxef and Imipenem
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J. Med. Microbiol. -Vol. 21 (1986), 49-57 0 1986 The Pathological Society of Great Britain and Ireland Beta-lactamases of type culture strains of the Bacteroides fragilis group and of strains that hydrolyse cefoxitin, latamoxef and imipenem A. ELEY and D. GREENWOOD Department of Microbiology and Public Health Laboratory, University Hospital, Queen's Medical Centre, Nottingham NG7 2UH Summary. Susceptibilities to P-lactam antibiotics and P-lactamase content of two groups of Bacteroides strains were compared. Type cultures produced low levels of p- lactamase and were susceptible to cefoxitin, latamoxef, imipenem and the combination of benzylpenicillin and clavulanic acid. Other Bacteroides strains that produced higher levels of P-lactamase were generally less susceptible to these antibiotics; this resistance was more closely related to enzyme type than to the amount of enzyme present. The P-lactamases produced by the test strains fell into three broad groups on the basis of antibiotic degradation and inhibitor profiles: (i) those that inactivated benzylpenicillin, but not cefoxitin, latamoxef or imipenem, and were susceptible to inhibition by P-lactamase inhibitors; (ii) those that hydrolysed benzylpenicillin, cefoxitin and latamoxef, but not imipenem, and which were less susceptible to inhibition by P-lactamase inhibitors; (iii) an enzyme that inactivated all the antibiotics and was not inhibited by p-lactamase inhibitors. Introduction tin, latamoxef and imipenem. The P-lactamases of these strains have been characterised with respect to Production of P-lactamase is the major factor the kinetics of antibiotic breakdown and the pattern involved in the resistance of many bacteria to p- of inhibition by various enzyme inhibitors. lactam antibiotics and the Bacteroides fragilis group of organisms is no exception to this general finding. The resistance is highly dependent upon the level of Materials and methods P-lactamase activity and is not associated with any particular enzyme type (Simpson et al., 1982). There Bacterial strains are at least four types of P-lactamase in the B. fragilis group. The most common type possessed by B. fragilis NCTC 9344, B. ovatus NCTC 11 153, B. thetaiotaomicron NCTC 10582 and B. uulgatus NCTC most strains has cephalosporinase activity (Ander- 10583 were used as reference strains. B. fragilis 119 was son and Sykes, 1973); other enzymes have been isolated in this laboratory from a wound swab and described that inactivate cefoxitin (Cuchural et al., identified by procedures recommended by Holdeman et 1983) or imipenem (Yotsuji et al., 1983). A different al. (1977). enzyme, with penicillinase rather than cephalospor- Other strains were kindly made available to us as inase activity, has been described by Sat0 et al. follows: B. fragilis 2013E by I.N. Simpson (Glaxo Ltd, (1 982). Greenford, Middlesex). B. distasonis R939 by J. Brazier It has been suggested that the P-lactamases of (Luton Public Health Laboratory) and strains B. fragilis Bacteroides spp. may be classified according to their 0423 and B. thetaiotaomicron 0456 by V.L. Sutter (Wads- substrate profiles and susceptibility to various worth Veterans Administration Medical Center, Los Angeles, CA, USA). enzyme inhibitors (Tajima et al., 1983) but such studies have generally been carried out on strains within the B. fragilis group that show normal Antibiotics susceptibility to P-lactam antibiotics. Solutions of benzylpenicillin (Glaxo Laboratories We have selected for investigation type cultures Ltd), clavulanic acid (Beecham Pharmaceuticals), cefoxi- of Bacteroides and strains able to inactivate cefoxi- tin (Merck Sharp and Dohme Ltd) and latamoxef (Eli Lilly and Co. Ltd) were freshly prepared as required in Received 18 Mar. 1985; accepted 29 Apr. 1985. sterile distilled water. Appropriate concentrations of 49 50 A. ELEY AND D. GREENWOOD imipenem (Merck Sharp and Dohme Ltd) were freshly at 3000 rpm for 20 min. Supernates were removed and prepared in 0.01~phosphate buffer (PH 7-0) according to assayed by a well-diffusion technique (Eley and Green- the manufacturers instructions. wood, 1981). Culture medium Viable counts Brain-heart-infusion broth supplemented with yeast Samples of broth cultures were removed during turbi- extract 5 mg/ml, haemin 5 pg/ml and menadione 1 pg/ml dimetry experiments and were diluted in fresh BHIS (BHIS), was used throughout. Agar 1.0% and lysed horse broth; further dilutions in BHIS agar were made with the blood 5% were added for minimum inhibitory concentra- Colworth ‘Droplette’ dispenser (A.J. Seward Ltd, Lon- tion (MIC) titrations. All culture media except those used don). Colonies in the agar droplets were counted with the for viable counting were pre-reduced by incubation illuminated magnifier and counter unit incorporated in overnight in a mixture of N2 SO%, C02 10%’ Hz 10%. the ‘Droplette’ device. Viable counts were expressed in terms of the mean number of cfu/ml. Sensitivity tests Preparation of crude P-lactamase MICs of antibiotics were determined by spot inocula- tion of 0.001 ml of 1 in 10 and 1 in 1000 dilutions of The /3-lactamase preparations were obtained from overnight cultures in BHIS on to plates containing serial ultrasonicated bacteria as follows: surface growth from twofold dilutions of antibiotics; the lowest concentration blood-agar cultures was scraped off into 4 ml of 0.02 M that completely inhibited growth after incubation for 48 h phosphate buffer (PH 7-0) containing 1 mM dithiothreitol was taken as the MIC. and the cells were disrupted with an ultrasonicator (MSE PG- 100, MSE Scientific Instruments, Crawley, Sussex). Cell debris was removed by centrifugation at 5000 g for Turbidimetr ic studies 30 min at 4°C (MSE ‘Coolspin’). The clear supernates Bacteria from overnight broth cultures were seeded were stored at - 20°C until required. into 9-ml volumes of prereduced BHIS to achieve an Protein content of sonicates was estimated by the inoculum of c. lo6 cfu/ml. Antibiotic was added either method of Lowry et al. (1 95 1) with bovine serum albumin immediately after inoculation or when bacterial growth as the standard. had raised the opacity of the cultures to 30% of that Isoelectric focusing of crude /3-lactamase extracts was obtained with a fully grown broth culture. At this point performed on cellulose acetate membranes according to the viable count was approximately lo8cfu/ml. The tubes the method previously described by Eley et al. (1983). were incubated anaerobically in a six-channel turbidi- meter (O’Grady and Eley, 1983). In this system, antibiotic Quantitation of #I-lactamaseand eflect of inhibitors can be added during growth, or samples removed without disturbing anaerobiosis so that antibiotic-induced mor- Enzyme activity and enzyme inhibition analyses were phological changes can be observed and viable counting performed in a centrifugal fast analyser (Centrifichem procedures performed. To detect antibiotic breakdown, 400, Union Carbide, Terrytown, New York) by following cultures exposed to antibiotic overnight were centrifuged a colour change in the chromogenic cephalosporin, Table I. Conditions used for high performance liquid chromatography Flow- Injection rate volume Wavelength Sensitivity Antibiotic M obile-phase (ml/min) (Pl) (nm) (A.U.F.S.)* Benzyl ’ CH3CN: 0.0 1 M Ammonium 2 25 227 0.01 penicillin acetate Clavulanic 20: 80 acid 2.5 25 31 1 0.08 Imipenem , 2 25 300 0.04 * Absorbance unit full scale. BETA-LACTAMASES OF BACTEROIDES 51 nitrocefin, at 500 nm. The total reaction volume was 300 Results pl which included 250 pl of nitrocefin solution (to give a final concentration of 50 pg/ml), 25 pl of distilled water, Min imum inhibitory con cen t r a t ions 15 pl of the enzyme preparation and 10 pl of inhibitor. Assays were routinely performed at 37'C and the nitroce- MICs of cefoxitin, latamoxef, imipenem and fin solution and enzyme + inhibitor mixture were tem- benzylpenicillin (in the presence and absence of perature equilibrated for 15 min before mixing. clavulanic acid) for the nine test strains are shown in The in hi bi tors tested were para-chloromercuriben- table 11. Type-culture strains of B. fragilis and B. zoate (pcmb), cloxacillin, clavulanic acid, sulbactam, vulgatus were more susceptible to cefoxitin, lata- cefoxitin, latamoxef and imipenem at concentrations (p~)moxef and imipenem than were those of B. ovatus of 0. I, 1, 10 and 100. Enzyme preparations from strains and B. thetaiotaomicron. The presence of clavulanic that produced high levels of p-lactamase were standar- dized to give relative activities similar to those of the type acid 4 pg/ml (a concentration which alone had no cultures by making appropriate dilutions with phosphate inhibitory effect) reduced the MIC of benzylpenicil- buffer. Appropriate enzyme and substrate controls were lin for these four strains from 16-32 pg/ml to 0.25- included with each experiment. 0-5pg/ml. The other five strains had raised MICs in titrations of all the test compounds; an exception was B.fragilis 2013E, which was as sensitive as the High performance liquid chromatography ( HPLC) type-culture B.fragilis strain to the combination of The kinetics of breakdown of latamoxef, cefoxitin, benzylpenicillin and clavulanic acid. imipenem and a combination of benzylpenicillin and clavulanic acid were determined by HPLC on a 100 x 4 Turbidime tric experiments mm diameter ODS-Hypersil 5 p column. The conditions used are shown in table I. Clavulanic acid was derivatised The response of all the test strains to antibiotics with imidazole by the method of Foulstone and Reading was observed by continuous turbidimetric monitor- (1982) before injection on to the column. The kinetics of ing. Antibiotic was added at the same time as an /I-lactamase-mediated inactivation of the p-lactam anti- inoculum of c. lo6cfu/ml, or when bacterial growth biotics were determined as follows: crude p-lactamase c. extract 0.1 ml and antibiotic solution 50 pg/ml were pre- had raised the inoculum to lo* cfu/ml.