1466 DEVELOPMENT OF RESISTANCE TO BETA-LACTAM ANTIBIOTICS DURING THERAPY OF PSEUDOMONAS AERUGINOSA INFECTIONS
SIR,—Variants of Pseudomonas aeruginosa NCTC 10662 that show reduced susceptibility to both carbenicillin and azlocillin can be selected in the laboratory if carbenicillin is used as the selecting agent. However, variants selected for resistance to azlocillin do not show reduced susceptibility to carbenicillin.These changes do not depend on the acquisition of plasmids, and therefore differ from those described in burns patients in Birmingham in 1969.2 We describe here the development of resistance by isolates of Ps. aeruginosa from patients treated with azlocillin or ticarcillin to add to previous reports of development of resistance during therapy with carbenicillin,3 ticarcillin,4 or piperacillin.5 Results for isolates from three patients are shown in the table. All organisms were isolated from sputum. In addition the first two organisms from patient B and the organisms from patient C were isolated from blood. Minimum inhibitory concentrations (MICs) of antibiotics were determined by agar dilution on ’Oxoid Diagnostic Sensitivity Test Agar’ with an inoculum of about 104 colonyforming units. &bgr;-lactamase activity was measured by monitoring the rate of change of absorbance of nitrocefin (100 µmol/1 in 100 mmol/l phosphate buffer pH 7) at 37°C in the presence of an ultrasonically disintegrated suspension of bacterial cells. Protein contents of the cell extracts were measured by the biuret method. The organisms isolated before therapy resembled NCTC 10662 in being susceptible to carbenicillin and azlocillin and producing only small, basal amounts of &bgr;-lactamase unless synthesis of the Id enzyme (in the terminology of Richmond and Sykes6) was induced by certain &bgr;-lactams, such as cefoxitin. The organism isolated from patient A after treatment with ticarcillin showed increased resistance to both carbenicillin and azlocillin-analogous to the selection of resistant variants by carbenicillin in vitro. In contrast, the organisms isolated after treatment of patients B and C with azlocillin resembled those selected in vitro by azlocillin in that susceptibility to azlocillin was reduced but with little or no change for carbenicillin. Increased &bgr;-lactamase synthesis was detected but, except for the third isolate from patient B, not to the same extent as that induced by cefoxitin. There was no difference in either pyocine type or in the isoelectric focusing pattern of the &bgr;-lactamases between the organisms isolated before and after treatment. Similar azlocillin-resistant clinical isolates of Ps. aeruginosa are reported by Dr Livermore and his colleagues on this page. We have no explanation for the different amounts of &bgr;-lactamase induced in the isolates by cefoxitin, though it could be that the second isolate from patient A was relatively impermeable to cefoxitin thus rendering this compound less effective as a &bgr;-lactamase inducer. We were able to confirm the previous reportthat the Id &bgr;-lactamase has some activity on azlocillin but little on carbenicillin. We used the ultraviolet spectrophotometric method at a wavelength of 235 nm and a substrate concentration of 100 µmol/1.8 Under these conditions the &bgr;-lactamase from the third isolate from patient B hydrolysed azlocillin at about 5% of the rate of hydrolysis of benzylpenicillin: no hydrolysis of carbenicillin was detected. Thus Gwynne MN, Rolinson GN.
Selection of variants of Pseudomonas aeruginosa resistant beta-lactam antibiotics. Infection 1980; 8: 73-80. 2. Lowbury EJL, Lilly HA, Kidson A, Ayliffe GAJ, Jones RJ. Sensitivity of Pseudomonas aeruginosa to antibiotics emergence of strains highly resistant to carbenicillin. Lancet 1969; ii. 448-52. 3. Darrell JH, Waterworth PM Carbenicillin resistance in Pseudomonas aeruginosa from clinical material. Br Med J 1969; iii: 141-43. 4. Ervin FR, Bullock WE Clinical and pharmacological studies of ticarcillin in gramnegative infections. Antimicrob Ag Chemother 1976, 9: 94-101. 5. Marier RL, Marinaccio AT, Sanders CV, Aldridge KE, Mitchell JW Susceptibility patterns of bacteria following therapy with piperacillin J Antimicrob Chemother 1982; 9 suppl B: 85-91 6. Richmond MH, Sykes RB. The &bgr;-lactamases of gram-negative bacteria and their possible physiological role In Rose AH, Tempest DW, eds. Advances in microbial physiology: Vol IX. London and New York- Academic Press, 1973: 31-88. 7 Basker MJ, Edmonson RAE, Sutherland R Comparative antibacterial activity of azlocillin, mezlocillin, carbenicillin and ticarcillin and relative stability to betalactamases of Pseudomonas aeruginosa and Klebsiella aerogenes. Infection 1979; 7: 67-73 8. King A, Shannon K, Phillips I In vitro antibacterial activity and susceptibility of cefsulodin, an antipseudomonal cephalosporin, to beta-lactamases. Antimicrob Ag Chemother 1980, 17: 165-69. 1.
-LACTAMASE ACTIVITY AND MICS OF CARBENICILLIN AND AZLOCILLIN FOR ISOLATES OF PS. AERUGINOSA
*In relation to therapy. µmol nitrocefin hydrolysed/min/mg protein of bacterial extract $Induced by cefoxitin (500 mg/1) §After further therapy.
it seems probable that the enhanced synthesis of Id &bgr;-lactamase is responsible for the reduced susceptibility to azlocillin in the later isolates from patients B and C. The mechanism of this enhanced synthesis of &bgr;-lactamase is not clear. Except for the third isolate from patient B the amounts are too low to suggest complete derepression. Neither is it clear whether azlocillin selects a pre-existing minority of cells that produce larger amounts of &bgr;-lactamase or in some way induces increased synthesis of the enzyme. However, it does not seem to be analagous to what might be called "classical induction", since the continued presence of azlocillin is not necessary. Fortunately, emergence of resistance
of this kind appears to be relatively uncommon unless there is sump of infection that the antibiotic cannot clear rapidly. Department of Microbiology, St Thomas’s Hospital Medical School, London SE1 7EH
KEVIN SHANNON ANNA KING IAN PHILLIPS
PSEUDOMONAS AERUGINOSA ISOLATES WITH MODIFIED BETA-LACTAMASE INDUCIBILITY: EFFECTS ON BETA-LACTAM SENSITIVITY
SIR,-Typical Pseudomonas aeruginosa are more susceptible to the beta-lactams than they are to carbenicillin.1,2 This report concerns five clinical isolates with the opposite susceptibility pattern. They were collected from separate centres during a national survey of antibiotic resistance in Ps. aeruginosa. Minimum inhibitory concentrations (MICs) were determined by agar dilution, with an inoculum of 105 colony-forming units. The results are shown in table I together with those for Ps. aeruginosa R201 which has a typical beta-lactam sensitivity pattern. All five new
isolates were, unlike R20, more susceptible to carbenicillin than to cefotaxime and ceftriaxone. Strains 1405, 1511, and 84 were at least as sensitive to carbenicillin as they were to azlocillin or mezlocillin. Cefoperazone, piperacillin, and ceftazidime MICs for these organisms were also higher than those for R20. Beta-lactamase involvement was investigated with nitrocefin after disruption of cells in 0 1 mmol/1 EDTA in 0’ 1 mol/1 borate buffer pH 7 1. All five strains, but not R20, were strongly positive. The beta-lactamases were extracted and compared by isoelectric focusing with standard plasmid-mediated and Ps. aeruginosa chromosomally mediated Sabath and Abrahams (SA) enzymes. Extracts from all five strains focused in the pH range 7-7-8-2, distinct from all the plasmid-mediated beta-lactamases except DM, Williams RJ, Williams JD. Comparison of the beta-lactamase stability and the in-vitro activity of cefoperazone, cefotaxime, cefsulodin, ceftazidime, moxalactam and ceftriaxone against Pseudomonas aeruginosa. J Antimicrob Chemother 1981; 8: 323-31. Phillips I, Warren C, Shannon K, King A, Hanslo D Ceftazidime: In vitro antibacterial and susceptibility to beta-lactamases compared with that of cefotaxime, moxalactam and other beta-lactam antibiotics. J Antimicrob Chemother 1981; 8
(suppl B) 23-31.