AmpC induction by ceftaroline.

Sir, Ceftaroline is a novel, parenteral, broad-spectrum oxyimino cephalosporin active against staphylococci, including methicillinresistant strains, and pneumococci, including penicillinand cefotaxime-resistant strains. It also has anti-Enterobacteriaceae activity, but this is constrained by lability to b-lactamases, including AmpC, extended-spectrum, KPC and metallo types. It has recently completed Phase III trials in complicated skin and skin structure infections and in moderate to severe communityacquired pneumonia, with better outcomes than seen with ceftriaxone in the latter setting. Like available oxyimino cephalosporins, ceftaroline has nearequal activity against AmpC-inducible Enterobacteriaceae and their AmpC-basal mutants, and the potential for these enzymes to cause resistance is revealed only when they are manufactured copiously without induction, as in derepressed mutants, or by strains with plasmid-determined expression. This behaviour, along with a propensity to select AmpC-derepressed mutants from AmpC-inducible populations in vitro, implies that ceftaroline, like other oxyimino cephalosporins, is a weak inducer of AmpC enzymes at sub-MIC concentrations. We attempted to confirm or refute this inference by direct enzyme assays. The test strains (two per species) comprised the b-lactamase-inducible parent organisms from the AmpC inducibility mutant series previously used for MIC comparisons. All except the Proteus vulgaris strains have AmpC-type enzymes; P. vulgaris has a class A (or 2e) chromosomal b-lactamase, which is inhibited by clavulanate. We also examined preand post-therapy Enterobacter cloacae isolates (designated 615 and 616, respectively) from the sole patient in the Phase III clinical trials in whom resistant variants were selected during ceftaroline therapy. This patient had a skin structure infection. MICs were determined by CLSI agar dilution, and AmpC induction was measured using logarithmic-phase cells exposed to MIC multiples of ceftaroline, cefotaxime, ceftriaxone and cefoxitin for 2 h, with shaking, in nutrient broth at 378C. b-Lactamase activity was assayed versus 0.1 mM nitrocefin in 0.1 M phosphate buffer pH 7.0 by spectrophotometry at 482 nm and 378C, and standardized against protein concentration, measured by the Bradford method (Bio-Rad Protein Assay, Bio-Rad, Oxford, UK). b-Lactamase profiles of E. cloacae 615 and 616 were examined by isoelectric focusing (IEF) and their relatedness was assessed by PFGE of XbaI-restricted DNA. All the reference strains had minimal b-lactamase activity in the absence of inducers but showed strong induction by cefoxitin at 1 the MIC, with induction ratios (b-lactamase specific activity with inducer/specific activity without inducer) averaging 116-fold, and ranging up to 457-fold (Table 1). In contrast, ceftaroline, cefotaxime and ceftriaxone induced weakly at 1 the MIC, with geometric mean induction ratios for all tested strains of 1.81-, 1.78and 2.95-fold, respectively, and with no ratio exceeding 5-fold. Stronger induction was seen at 4 to 16 the MIC but, even then, the induction ratios remained lower than for cefoxitin. IEF showed that E. cloacae 615 and 616 lacked other b-lactamases besides AmpC, which gave a cloxacillin-inhibited band at a pI.8.0, above any of the reference standards. Both isolates had identical PFGE profiles, confirming that isolate 616 was a cephalosporin-resistant mutant of 615, not a case of superinfection. Neither organism showed total derepression of AmpC; nevertheless isolate 616 had an 5-fold higher uninduced b-lactamase specific activity compared with isolate 615 and, with ceftaroline at 1 the MIC, had 12.8-fold greater specific activity, implying stronger inducibility. The present data confirm that ceftaroline resembles other oxyimino cephalosporins in being a weak inducer of AmpC b-lactamases at or below the MIC, thus agreeing both with the relative MIC data for AmpC-inducible, AmpC-basal and AmpCderepressed Enterobacteriaceae and with the propensity to select AmpC-derepressed or hyperinducible mutants. Such selection is a significant concern with oxyimino cephalosporins in some settings, occurring in 20%–30% of Enterobacter bacteraemias treated with these agents and leading to increased mortality, length of stay and cost. – 7 Nevertheless, in vivo selection by ceftaroline should be limited by two factors: first, Research letters