Currently, CTX-M ␤-lactamases are among the most prevalent and most heterogeneous extended-spectrum ␤-lactamases (ESBLs). In general, CTX-M enzymes are susceptible to inhibition by ␤-lactamase inhibitors. However, it is unknown if the pathway to inhibition by ␤-lactamase inhibitors for CTX-M ESBLs is similar to TEM and SHV ␤-lactamases and why bacteria possessing only CTX-M ESBLs are so susceptible to carbapenems. Here, we have performed a kinetic analysis and timed electrospray ionization mass spectrometry (ESI-MS) studies to reveal the intermediates of inhibition of CTX-M-9, an ESBL representative of this family of enzymes. CTX-M-9 ␤-lactamase was inactivated by sulbactam, tazobactam, clavulanate, meropenem, doripenem, ertapenem, and a 6-methylidene penem, penem 1. CTX-M enzymes are becoming one of the most prevalent extended-spectrum ␤-lactamases (ESBLs) (3,8,9,(30)(31)(32) in the world. The widespread dissemination of CTX-M ␤-lactamases, especially Escherichia coli ST131 possessing CTX-M-15, has had a significant impact on the treatment of hospital-and community-acquired infections caused by E. coli and other enteric bacilli (6, 7, 13, 23, 36, 41, 44-49, 55, 59).As class A family ␤-lactamases, CTX-Ms are the most genetically heterogeneous (5 major divisions, CTX-M-1, -2, -8, -25, and -9-like groups) (1, 24, 35, 44-46, 58, 60). Most CTX-M enzymes expressed in E. coli provide a high level of resistance to the oxyimino-cephalosporins, cefotaxime and ceftriaxone, and variable levels of resistance to cefepime and cefpirome (3,43). Depending on the type of CTX-M expressed by the isolates, the MICs of ceftazidime are also increased (43). In addition, the MICs of combinations of clavulanate with amoxicillin or ticarcillin vary, and in some cases, low-level resistance has been observed (3).As a consequence of their clinical importance, the reaction mechanism of CTX-M ESBLs has been the subject of intense study (10-12, 14, 16, 42). However, the molecular properties and characteristics of CTX-M that determine the level of susceptibility and resistance to ␤-lactam-␤-lactamase inhibitor combinations and carbapenems are still unknown. Of the currently available inhibitors, tazobactam is the most active (50% inhibitory concentrations [IC 50 s] are 2 to 10 nM for tazobactam and 9 to 90 nM for clavulanate), and sulbactam is the least active (IC 50 s are 0.1 to 4.5 M) (3).In order to develop more effective and broader-spectrum ␤-lactamase inhibitors (18), detailed kinetic and biochemical measurements are needed to reveal the important intermediates in the inactivation of the CTX-M family. In TEM-1 and SHV-1, Arg244 is important in the mechanism of inactivation of carbapenems (imipenem and meropenem), clavulanic acid, sulbactam, and tazobactam (27,28,51,53,63). CTX-M-9 does not contain Arg244, and mutagenesis of a potential corresponding position, Arg276, does not firmly establish this amino acid as an "Arg244 equivalent" (42). Given the differences among class A enzymes, we wondered what the intermediates of inactivation by ...