Institut d'Investigació Sanitària Illes Balears (IdISBa) Research Group.

Role of Enzymatic Activity in the Biological Cost Associated with the Production of AmpC β-Lactamases in Pseudomonas aeruginosa

Isabel M Barceló# 1 2 3, Elena Jordana-Lluch# 1 2, María Escobar-Salom1 2 3, Gabriel Torrens1 2 3 4 5, Pablo A Fraile-Ribot1 2 3, Gabriel Cabot 1 2 3, Xavier Mulet1 2 3, Laura Zamorano1 2 3 , Carlos Juan1 2 3 , Antonio Oliver1 2 3

1Research Unit, University Hospital Son Espases – Health Research Institute of tne Balearic Islands (IdISBa), Palma, Spain.
2Microbiology Department, University Hospital Son Espases, Palma, Spain.
3Centro de Investigación Biomédica en Red, Enfermedades Infecciosas, Madrid, Spain.
4Department of Molecular Biology, Umeå Centre for Microbial Research (UCMR), Umeå University, Umeå, Sweden
5Laboratory for Molecular Infection Medicine Sweden, Umeå Centre for Microbial Research (UCMR), Umeå University, Umeå, Sweden
#Contributed equally.


In the current scenario of growing antibiotic resistance, understanding the interplay between resistance mechanisms and biological costs is crucial for designing therapeutic strategies. In this regard, intrinsic AmpC β-lactamase hyperproduction is probably the most important resistance mechanism of Pseudomonas aeruginosa, proven to entail important biological burdens that attenuate virulence mostly under peptidoglycan recycling alterations. P. aeruginosa can acquire resistance to new β-lactam-β-lactamase inhibitor combinations (ceftazidime-avibactam and ceftolozane-tazobactam) through mutations affecting ampC and its regulatory genes, but the impact of these mutations on the associated biological cost and the role that β-lactamase activity plays per se in contributing to the above-mentioned virulence attenuation are unknown. The same questions remain unsolved for plasmid-encoded AmpC-type β-lactamases such as FOX enzymes, some of which also provide resistance to new β-lactam-β-lactamase inhibitor combinations. Here, we assessed from different perspectives the effects of changes in the active center and, thus, in the hydrolytic spectrum resistance to inhibitors of AmpC-type β-lactamases on the fitness and virulence of P. aeruginosa, using site-directed mutagenesis; the previously described AmpC variants T96I, G183D, and ΔG229-E247; and, finally, blaFOX-4 versus blaFOX-8. Our results indicate the essential role of AmpC activity per se in causing the reported full virulence attenuation (in terms of growth, motility, cytotoxicity, and Galleria mellonella larvae killing), although the biological cost of the above-mentioned AmpC-type variants was similar to that of the wild-type enzymes. This suggests that there is not an important biological burden that may limit the selection/spread of these variants, which could progressively compromise the future effectiveness of the above-mentioned drug combinations.

IMPORTANCE The growing antibiotic resistance of the top nosocomial pathogen Pseudomonas aeruginosa pushes research to explore new therapeutic strategies, for which the resistance-versus-virulence balance is a promising source of targets. While resistance often entails significant biological costs, little is known about the bases of the virulence attenuations associated with a resistance mechanism as extraordinarily relevant as β-lactamase production. We demonstrate that besides potential energy and cell wall alterations, the enzymatic activity of the P. aeruginosa cephalosporinase AmpC is essential for causing the full attenuation associated with its hyperproduction by affecting different features related to pathogenesis, a fact exploitable from the antivirulence perspective. Less encouraging, we also show that the production of different chromosomal/plasmid-encoded AmpC derivatives conferring resistance to some of the newest antibiotic combinations causes no significantly increased biological burdens, which suggests a free way for the selection/spread of these types of variants, potentially compromising the future effectiveness of these antipseudomonal therapies.

Microbiol Spectr. 2022 Oct 10; e0270022 doi: 10.1128/spectrum.02700-22. [Online ahead of print]

Link to Microbiol Spectr.

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Dr. Bartolome Moyà

Instituto de Investigación Sanitaria Illes Balears

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