The Reaction Mechanism of Metallo-ß-Lactamases Is Tuned by the Conformation of an Active-Site Mobile Loop.

Carbapenems are "last resort" ß-lactam antibiotics used to treat serious and life-threatening health care-associated infections caused by multidrug-resistant Gram-negative bacteria. Unfortunately, the worldwide spread of genes coding for carbapenemases among these bacteria is threatening these life-saving drugs. Metallo-ß-lactamases (MßLs) are the largest family of carbapenemases. These are Zn(II)-dependent hydrolases that are active against almost all ß-lactam antibiotics. Their catalytic mechanism and the features driving substrate specificity have been matter of intense debate. The active sites of MßLs are flanked by two loops, one of which, loop L3, was shown to adopt different conformations upon substrate or inhibitor binding, and thus are expected to play a role in substrate recognition. However, the sequence heterogeneity observed in this loop in different MßLs has limited the generalizations about its role. Here, we report the engineering of different loops within the scaffold of the clinically relevant carbapenemase NDM-1. We found that the loop sequence dictates its conformation in the unbound form of the enzyme, eliciting different degrees of active-site exposure. However, these structural changes have a minor impact on the substrate profile. Instead, we report that the loop conformation determines the protonation rate of key reaction intermediates accumulated during the hydrolysis of different ß-lactams in all MßLs. This study demonstrates the existence of a direct link between the conformation of this loop and the mechanistic features of the enzyme, bringing to light an unexplored function of active-site loops on MßLs.

Defining Substrate Specificity in the CTX-M Family: the Role of Asp240 in Ceftazidime Hydrolysis.

The natural diversification of CTX-M ß-lactamases led to the emergence of Asp240Gly variants in the clinic that confer reduced susceptibility to ceftazidime (CAZ). In this study, we compared the impact of this substitution on CAZ and ceftazidime-avibactam (CZA) MICs against isogenic Escherichia coli strains with different porin deficiencies. Our results show a noticeable increase in CAZ resistance in clones expressing Asp240Gly-harboring CTX-M when combined with OmpF porin deficiency. Kinetic analysis revealed that the kcat/Km for CAZ was 5- to 15-fold higher for all Asp240Gly variants but remained 200- to 725-fold lower than that for cefotaxime (CTX). In vitro selection of CAZ-resistant clones yielded nonsusceptible CTX-M producers (MIC of >16 µg/ml) only after overnight incubation; the addition of avibactam (AVI) decreased MICs to a susceptible range against these variants. In contrast, the use of CZA as a selective agent did not yield resistant clones. AVI inactivated both CTX-M-12 and CTX-M-96, with an apparent inhibition constant comparable to that of SHV-2 and 1,000-fold greater than that of PER-2 and CMY-2, and k2/K for CTX-M-12 was 24- and 35-fold higher than that for CTX-M-96 and CTX-M-15, respectively. Molecular modeling suggests that AVI interacts similarly with CTX-M-96 and CTX-M-15. We conclude that the impact of Asp240Gly in resistance may arise when other mechanisms are also present (i.e., OmpF deficiency). Additionally, CAZ selection could favor the emergence of CAZ-resistant subpopulations. These results define the role of Asp240 and the impact of the -Gly substitution and allow us to hypothesize that the use of CZA is an effective preventive strategy to delay the development of resistance in this family of extended-spectrum ß-lactamases.

[Comparison of three microbiological methods for detection of expanded-spectrum betalactamases in Enterobacteriaceae isolated in Santa Fe (Argentina)]. / Comparación de tres métodos microbiológicos para la detección de betalactamasas de espectro extendido en enterobacterias aisladas en Santa Fe (Argentina).

INTRODUCTION: Expanded-spectrum betalactamases (ESBLs) are the main source of resistance to oxyimino cephalosporins and monobactams in Enterobacteriaceae. Most of them derive from TEM or SHV, however the incidence of other families like CTX-M, OXA and PER has increased. In Argentina, the most frequent ESBL in Enterobacteriaceae is CTX-M-2. This specific circumstance, which differs from the situation in the Northern Hemisphere, motivated us to study new diagnostic strategies for the detection of ESBLs in our region. METHOD: Microbiological ESBL detection was performed by double-disk synergy tests, cefotaxime and ceftazidime disks with and without clavulanic acid (NCCLS), and cefotaxime and ceftazidime disks in Müeller-Hinton agar supplemented with lithium clavulanate (MH-cla). Betalactamases were characterized by isoelectric focusing, hydrolysis profile and PCR amplification. RESULTS: Among 575 clinical isolates of Enterobacteriaceae, 14% were oxyimino cephalosporin-resistant. Two different ESBLs were detected in 31 resistant strains: CTX-M-2 (28) and PER-2 groups (3). The double-disk synergy test was the least sensitive method for ESBL detection. ESBLs were detected by the other two methods in all isolates with the use of cefotaxime disks, but not with ceftazidime disks. CONCLUSION: The microbiological method employing MH-cla with cefotaxime disks had a sensitivity and specificity comparable to the referral test using the same antibiotic proposed by the NCCLS for the detection of ESBLs.

Efficacy of inhaled amikacin as adjunct to intravenous combination therapy (ceftazidime and amikacin) in cystic fibrosis.

Eighty-seven patients with cystic fibrosis were admitted to hospital with an acute exacerbation of pulmonary symptoms associated with isolation of Pseudomonas aeruginosa from sputum. The patients were randomly allocated to receive intravenously administered ceftazidime (250 mg/kg/day) and amikacin (33 mg/kg/day) alone or with inhaled amikacin (100 mg twice a day). Other aspects of the 2-week treatment were constant. The two therapy groups were comparable in all aspects. At the completion of therapy, the addition of aerosolized amikacin produced temporary eradication of P. aeruginosa in 70% of the patients, compared with 41% in the intravenous therapy only group (P less than 0.02). Suppression of P. aeruginosa in sputum cultures was correlated with the amikacin sputum concentrations. However, both regimens resulted in similar improvements in clinical, radiologic, laboratory, and pulmonary function measurements, and within 4 to 6 weeks most patients were recolonized with P. aeruginosa. There was no serious toxicity or adverse effect. In patients with cystic fibrosis, the addition of aerosol aminoglycoside to systemic antipseudomonal combination therapy is not clinically beneficial.