Structural/mechanistic insights into the efficacy of nonclassical β‐lactamase inhibitors against extensively drug resistant Stenotrophomonas maltophilia clinical isolates

Abstract

Clavulanic acid and avibactam are clinically deployed serine β-lactamase inhibitors, important as a defence against antibacterial resistance. Bicyclic boronates are recently discovered inhibitors of serine and some metallo β-lactamases. Here, we show that avibactam and a bicyclic boronate inhibit L2 (serine β-lactamase) but not L1 (metallo β-lactamase) from the extensively drug resistant human pathogen Stenotrophomonas maltophilia. X-ray crystallography revealed that both inhibitors bind L2 by covalent attachment to the nucleophilic serine. Both inhibitors reverse ceftazidime resistance in S. maltophilia because, unlike clavulanic acid, they do not induce L1 production. Ceftazidime/inhibitor resistant mutants hyperproduce L1, but retain aztreonam/inhibitor susceptibility because aztreonam is not an L1 substrate. Importantly, avibactam, but not the bicyclic boronate is deactivated by L1 at a low rate; the utility of avibactam might be compromised by mutations that increase this deactivation rate. These data rationalize the observed clinical efficacy of ceftazidime/avibactam plus aztreonam as combination therapy for S. maltophilia infections and confirm that aztreonam-like β-lactams plus nonclassical β-lactamase inhibitors, particularly avibactam-like and bicyclic boronate compounds, have potential for treating infections caused by this most intractable of drug resistant pathogens.

TonB‐dependent uptake of β‐lactam antibiotics in the opportunistic human pathogen Stenotrophomonas maltophilia

“…It was collected as part of the SENTRY antimicrobial surveillance program (Toleman, Bennett, Bennett, Jones, & Walsh, ). Remarkably, isolate number 31 also carries an ampD loss of function mutation and hyperproduces both the L1 and L2 β‐lactamases, which is enough to give pan β‐lactam resistance without any additional mechanism (Calvopiña et al, ; Gould et al, ; Talfan et al, ). We have reported, however, that isolate number 31 is unusual in its resistance to ceftazidime/β‐lactamase inhibitor combinations (Calvopiña et al, ), so combination therapy including a β‐lactam/β‐lactamase inhibitor might have selected for this mutation even in a background of β‐lactamase hyperproduction.…”

“…Remarkably, isolate number 31 also carries an ampD loss of function mutation and hyperproduces both the L1 and L2 β‐lactamases, which is enough to give pan β‐lactam resistance without any additional mechanism (Calvopiña et al, ; Gould et al, ; Talfan et al, ). We have reported, however, that isolate number 31 is unusual in its resistance to ceftazidime/β‐lactamase inhibitor combinations (Calvopiña et al, ), so combination therapy including a β‐lactam/β‐lactamase inhibitor might have selected for this mutation even in a background of β‐lactamase hyperproduction. Whatever the specifics of selection in this case, we have demonstrated the existence of S. maltophilia clinical isolates with mutations in the TonB energy transducer Smlt0009, which have reduced susceptibility to β‐lactams, fluoroquinolones and siderophore‐conjugated antimicrobials.…”

“…For example, OmpK36 loss causes meropenem resistance, but does not confer resistance to the closely related carbapenem, imipenem (Findlay et al, ). Because of the importance of carbapenems as last‐resort antimicrobials, porin loss conferring carbapenem resistance in clinical isolates was quickly identified but S. maltophilia is intrinsically resistant to all carbapenems because of the production of an inducible carbapenemase, named L1 (Calvopiña et al, ). Indeed, there are only six agents for which the United States Clinical and Laboratory Standards Institute (CLSI) provides resistance/susceptibility breakpoints; indicating potential clinical efficacy (CLSI, ): two β‐lactams, ceftazidime and ticarcillin–clavulanate; a fluoroquinolone, levofloxacin; a tetracycline derivative, minocycline; a phenicol, chloramphenicol and the drug of choice (and the only agent for which breakpoints are provided by the European Committee on Antimicrobial Susceptibility Testing) trimethoprim–sulfamethoxazole.…”

“…Production of L1 and L2 is coordinately controlled by AmpR, a LysR‐type transcriptional activator and induced during β‐lactam challenge of cells (Okazaki & Avison, ). Despite this, many S. maltophilia clinical isolates remain susceptible to the β‐lactam ceftazidime because it is a relatively poor substrate for these two enzymes (Calvopiña et al, ). However, mutants that have acquired ceftazidime resistance can easily be identified in the laboratory and ceftazidime‐resistant isolates are commonly encountered in the clinic.…”

Escape mutations circumvent a tradeoff between resistance to a beta-lactam and a beta-lactamase inhibitor

“…While mutations that increase beta-lactamase expression can increase resistance to the drug alone and in combination with the inhibitor, resistance to the drug-inhibitor treatment via structural intragenic mutations is inherently constrained: due to the structural similarity between the drug and the inhibitor, mutations that increase drug degradation also tend to increase the affinity of the enzyme to the inhibitor 7,[33][34][35][36][37] . Of course, this functional tradeoff between resistance to the drug and the inhibitor does not completely preclude mutations that increase resistance to the combination [38][39][40][41] . Indeed, even without escaping this tradeoff, a mutation providing strong resistance to one compound, even at the cost of mild sensitivity to the other, can provide an overall increased resistance to the combination 34,40,41 .…”