Slater Cole L, Winogrodzki Judith, Fraile-Ribot Pablo A, Oliver Antonio, Khajehpour Mazdak, Mark Brian L
Department of Microbiology, University of Manitoba, Winnipeg, Canada.
Red Española de Investigación en Patología Infecciosa (REIPI), Instituto de Salud Carlos III, Madrid, Spain.
Antimicrob Agents Chemother. 2020 Aug 20;64(9). doi: 10.1128/AAC.00894-20.
is a leading cause of nosocomial infections worldwide and notorious for its broad-spectrum resistance to antibiotics. A key mechanism that provides extensive resistance to β-lactam antibiotics is the inducible expression of AmpC β-lactamase. Recently, a number of clinical isolates expressing mutated forms of AmpC have been found to be clinically resistant to the antipseudomonal β-lactam-β-lactamase inhibitor (BLI) combinations ceftolozane-tazobactam and ceftazidime-avibactam. Here, we compare the enzymatic activity of wild-type (WT) AmpC from PAO1 to those of four of these reported AmpC mutants, bearing mutations E247K (a change of E to K at position 247), G183D, T96I, and ΔG229-E247 (a deletion from position 229 to 247), to gain detailed insights into how these mutations allow the circumvention of these clinically vital antibiotic-inhibitor combinations. We found that these mutations exert a 2-fold effect on the catalytic cycle of AmpC. First, they reduce the stability of the enzyme, thereby increasing its flexibility. This appears to increase the rate of deacylation of the enzyme-bound β-lactam, resulting in greater catalytic efficiencies toward ceftolozane and ceftazidime. Second, these mutations reduce the affinity of avibactam for AmpC by increasing the apparent activation barrier of the enzyme acylation step. This does not influence the catalytic turnover of ceftolozane and ceftazidime significantly, as deacylation is the rate-limiting step for the breakdown of these antibiotic substrates. It is remarkable that these mutations enhance the catalytic efficiency of AmpC toward ceftolozane and ceftazidime while simultaneously reducing susceptibility to inhibition by avibactam. Knowledge gained from the molecular analysis of these and other AmpC resistance mutants will, we believe, aid in the design of β-lactams and BLIs with reduced susceptibility to mutational resistance.
是全球医院感染的主要原因，并且因其对抗生素的广泛耐药性而臭名昭著。赋予对β-内酰胺抗生素广泛耐药性的一个关键机制是AmpCβ-内酰胺酶的诱导表达。最近，已发现一些表达AmpC突变形式的临床分离株对抗假单胞菌β-内酰胺-β-内酰胺酶抑制剂（BLI）组合头孢洛扎-他唑巴坦和头孢他啶-阿维巴坦具有临床耐药性。在此，我们将来自PAO1的野生型（WT）AmpC的酶活性与四个报道的AmpC突变体（携带E247K突变（第247位的E变为K）、G183D、T96I和ΔG229-E247（从第229位到247位的缺失））的酶活性进行比较，以深入了解这些突变如何规避这些临床上至关重要的抗生素-抑制剂组合。我们发现这些突变对AmpC的催化循环有双重影响。首先，它们降低了酶的稳定性，从而增加了其灵活性。这似乎提高了酶结合的β-内酰胺的脱酰化速率，导致对头孢洛扎和头孢他啶的催化效率更高。其次，这些突变通过增加酶酰化步骤的表观活化能垒来降低阿维巴坦对AmpC的亲和力。这对头孢洛扎和头孢他啶的催化周转没有显著影响，因为脱酰化是这些抗生素底物分解的限速步骤。值得注意的是，这些突变提高了AmpC对头孢洛扎和头孢他啶的催化效率，同时降低了对阿维巴坦抑制的敏感性。我们相信，从这些和其他AmpC耐药突变体的分子分析中获得的知识将有助于设计对突变耐药性敏感性降低的β-内酰胺和BLI。
