J. Med. Microbiol. - Vol. 43 (1999, 30G309 0 1995 The Pathological Society of Great Britain and Ireland ANTI MICROBIAL AGENTS Mechanisms of resistance to p-lactam antibiotics amongst Pseudomonas aeruginosa isolates collected in the UK in 1993 H. Y. CHEN, ME1 YUAN and D. M. LIVERMORE Department of Medical Microbiology, The London Hospital Medical College, Turner Street, London El 2AD Summary. Antimicrobial resistance among 199 1 Pseudomonas aeruginosa isolates collected at 24 UK hospitals during late 1993 was surveyed. Three-hundred and seventy-two of the isolates were resistant, or had reduced susceptibility, to some or all of azlocillin, carbenicillin, ceftazidime, imipenem and meropenem, and the mechanisms underlying their behaviour were examined. Only 13 isolates produced secondary p-lactamases : six possessed PSE- 1 or PSE-4 enzymes and seven had novel OXA enzyme types. Those with PSE types were highly resistant to azlocillin and carbenicillin whereas those with OXA enzymes were less resistant to these penicillins. Chromosomal p-lactamase derepression was demonstrated in 54 isolates, most of which were resistant to ceftazidime and azlocillin although susceptible to carbenicillin and carbapenems. p-Lactamase-independent “ intrinsic” resistance occurred in 277 isolates and is believed to reflect some combination of impermeability and efflux. Two forms were seen : the classical type, present in 195 isolates, gave carbenicillin resistance (MIC > 128 mg/L) and reduced susceptibility to ciprofloxacin and to all p-lactam agents except imipenem; a novel variant, seen in 82 isolates, affected only azlocillin, ceftazidime and, to a small extent, meropenem. Resistance to imipenem was largely dissociated from that to other p-lactam agents, and probably reflected loss of D2 porin, whereas resistance to meropenem was mostly associated with intrinsic resistance to penicillins and cephalosporins. Comparison of the present results with those of a similar study in 1982 revealed significant increases in the proportions of isolates with intrinsic resistance or stable derepression (p < 0.01, x2 test). Isolates with secondary p-lactamases appeared significantly rarer than in 1982 (p < 0-01, x2 test), but this comparison was distorted by outbreak strains. Introduction hospitals (see Acknowledgements) collected up to 100 consecutive non-replicate isolates between 1 Sept. and Most Pseudornonas aeruginosa isolates are susc- 31 Dec., 1993, and sent them to this laboratory. Of eptible to ureido- and carboxy-penicillins, ceftazidime, 21 84 isolates received, 1991 were confirmed as viable cefoperazone, cefsulodin, aztreonam, imipenem and P. aeruginosa. Resistance rates were 10.4% to meropenem. Nevertheless, resistance to these agents azlocillin 16 mg/L, 11.7 YOto carbenicillin 128 mg/L, can arise by various mechanisms, including mutational 9.6% to ceftazidime 4mg/L, 2.5% to imipenem derepression of the AmpC chromosomal p- 4mg/L and 1.1% to meropenem 4mg/L. For lactamase,l, ’ acquisition of secondary plasmid- or carbenicillin and ceftazidime, these frequencies were transposon-mediated p-lactama~es,~,~reduced per- increased significantly (p < 0.05) compared to those meability5. or multi-drug efflux’’ ’ (see Discussion) found in a similar study in 1982.12Although resistance or, finally, in the case of imipenem, via loss of the D2 to azlocillin, 16 mg/L was not increased significantly porin.’, lo (p > 0.05) there was a significant rise (p < 0.01) in the During 1993 the incidence of antibiotic resistance proportion of isolates resistant to the drug at amongst P. aeruginosa isolates in the UK was 32 mg/L.12 Imipenem and meropenem were not tested surveyed.” Microbiology laboratories in each of 24 in 1982. In the present study the mechanisms under- lying these resistances were investigated and their current incidences were compared to those found in Received 8 Dec. 1994; revised version accepted 25 March 1995. 1982. 300 DRUG RESISTANCE IN P. AERUGINOSA 301 Materials and methods enzyme detection tests described above, except that 0.01 M, rather than 0.1 M, phosphate buffer was Selection of isolates used. After centrifugation for 2 min at 12000 (1, the supernates were subjected to electrofocusing at Of the 1991 P. aeruginosa isolates collected in the 15 W constant power in polyacrylamide gels 1993 survey," 372 were examined, being selected (220 x 100 x 1 mm) containing ampholines (Pharmacia, because they were resistant to one or more of the Milton Keynes, Buckinghamshire) 2 % v/v of various following drug concentrations: azlocillin 16 mg/L; carbenicillin 128 mg/L; ceftazidime 4 mg/L; pH ranges.15 Enzyme activities were detected by overlaying the gel with 0.5 mM nitrocefin in 0.1 M imipenem 4 mg/L, meropenem 4 mg/L. These con- phosphate buffer, pH 7-0, and observing for the centrations were selected to facilitate discrimination of appearance of pink bands. Identification of p-lacta- normal isolates from those with abnormally reduced mases was by comparison to reference enzymes, run susceptibility, and do not invariably coincide with in tracks adjacent to the test samples. clinical breakpoints." The MIC tests allowing this categorisation were performed on IsoSensitest Agar Assays of /I-lactamase actiziity (Chipath) with inocula of lo4 cfufspot, and have been described already." Reference P. aeruginosa strains Cultures were grown overnight at 37°C in 25-ml included NCTC10662, as the control in the suscep- volumes of nutrient broth with orbital shaking at tibility testing, R20, as a control with known 150 rpm, then diluted into warm (37°C) 250-ml inducibility of AmpC P-lactamase,13and 1405-con and volumes of the same broth. Incubation was continued 2297-con, with derepressed AmpC p-lactamase ex- for 4 h under the previous conditions. Subsequently, pre~sion.'~Transconjugants of P. aeruginosa PU2 1 the cells were harvested by centrifugation at 5000y, and Escherichia coli K-12 553 and J622114served as washed once in 0.1 M phosphate buffer, pH 7.0, re- sources of reference plasmid- and transposon- suspended in 8 ml of the same buffer and subjected to mediated enzymes. two alternate cycles of freezing and thawing. Debris and residual cells were removed by ultracentrifugation Antimicrobial agents for 30 min at 100000 g and 4°C. The supernates were assayed against 0.5 mM p-lactam solutions in 0.1 M Antimicrobial agents were from suppliers as phosphate buffer, pH 7.0, by UV spectrophotometry follows : azlocillin sodium and ciprofloxacin, Bayer, at 37°C. Substrates and wavelengths were : ampicillin, Newbury, Berkshire ; ampicillin sodium, carbenicillin benzylpenicillin and carbenicillin, 23 5 nm ; oxacillin, disodium, clavulanate lithium and cloxacillin sodium, 265 nm; and cephaloridine, 295 nm. SmithKline Beecham, Brockham Park, Surrey; cephaloridine and oxacillin, Sigma ; cefoxitin and P-Lactamase induction assays imipenem, Merck, Hoddesdon, Hertfordshire ; ceftazidime, Glaxo, Greenford, Middlesex ; mero- Sonicates were prepared from logarithmic phase penem, Zeneca, Macclesfield, Cheshire; and nitrocefin, cells in nutrient broth cultures that had or had not BBL, Cockeysville, MD, USA. been exposed to cefoxitin 500 mg/L for 4 h and were assayed against 0.1 mM nitrocefin in 0.1 mM phosphate /3- Lac tamase de tee tion and typing buffer, pH 7.0.13 Activity was standardised against protein concentration, assayed by the method of Cultures were grown overnight on nutrient agar Lowry et plates at 37"C, then harvested by washing into 1.5-ml volumes of 0.1 M phosphate buffer, pH 7.0. The Data handling and analysis washings were transferred to Eppendorf tubes and sonicated for 2 x 30 s at an amplitude of 12 pm, with Data analysis was with the programs Statview intermediate cooling on ice. Three 25-pl samples of SE + Graphics and STATXACT. Comparisons of each sonicate were transferred to wells in a susceptibility data for different groups of isolates were microtitration plate. One well was supplemented with by x2 or Fisher's exact tests, as appropriate to the 25 pl of 0.3 mM cloxacillin in 0.1 M phosphate buffer, sample numbers. pH 7.0, one with 25 pl of 0.3 mM clavulanate in 0.1 M phosphate buffer, pH 7.0 and the third with drug-free Results buffer. After 10 min at room temperature, 25-pl of 0.3 mM nitrocefin in 0.1 M phosphate buffer, pH 7.0, Categorisation of isolates resistant to penicillins or were added to each well. The appearance of a red ceftazidime colour, indicating p-lactamase activity, was noted Nitrocefin tests in the presence and absence of in comparison to the controls (R20, 1405-con and 0.1 mM cloxacillin or clavulanate were performed on 2297-con). 344 isolates selected as resistant to one or more of azlocillin 16 mg/L, carbenicillin 128 mg/ L or Iso-electric focusing of p-lactamases ceftazidime 4 mg/L. Most (274), like the p-lactamase- P-Lactamase extracts were prepared as for the inducible control strain R20, gave a pink colour in the 302 H. Y. CHEN, M. YUAN AND D. M. LIVERMORE Table I. Properties of isolates with secondary P-lactamases and of their enzymes Relative hydrolysis rates tls 0.5 mM B-Lactamase antibiotic Cb Az Caz Imp Mem PI Alignment PenG Amp Cb Oxa Cld 85 4096 512 4 2 5.7 = PSE-1 100 115 86 10 9 723 4096 512 4 0.125 5.7 = PSE- 1 100 106 93 10 10 I294 4096 512 4 1 5-7 = PSE- 1 100 127 118 11 11 1856 4096 256 2 1 5.3 = PSE-4 100 106 105 10 12 2223 4096 512 4 1 5.7 = PSE- 1 100 101 84 8 8 2312 4096 512 8 2 5.7 = PSE- 1 100 100 101 9 16 PSE- 1 * ... .., 5.7 ... 100 107 99 10 10 PSE-4* ... ... ... ... ... 5.3 ... 100 127 89 9 11 520 512 128 4 1 0-5 7.5 = OXA-4 100 874 79 537 67 543 256 64 2 2 0.25 7.5 = OXA-4 100 914 101 453 115 556 512 128 4 1 0.5 7.5 = OXA-4 100 1293 52 549 72 OXA-4* ..