Transferable AmpCs in
Klebsiella pneumoniae
: interplay with peptidoglycan recycling, mechanisms of hyperproduction, and virulence implications
Authors: Barceló Isabel M. , Escobar-Salom Maria , Cabot Gabriel , Perelló-Bauzà Pau , Jordana-Lluch Elena , Taltavull Biel , Torrens Gabriel , Rojo-Molinero Estrella , Zamorano Laura , Pérez Astrid , Oliver Antonio , Juan Carlos 
Published: March 2024

ABSTRACT

Chromosomal and transferable AmpC β-lactamases represent top resistance mechanisms in different gram-negatives, but knowledge regarding the latter, mostly concerning regulation and virulence-related implications, is far from being complete. To fill this gap, we used
Klebsiella pneumoniae
(KP) and two different plasmid-encoded AmpCs [DHA-1 (AmpR regulator linked, inducible) and CMY-2 (constitutive)] as models to perform a study in which we show that blockade of peptidoglycan recycling through AmpG permease inactivation abolished DHA-1 inducibility but did not affect CMY-2 production and neither did it alter KP pathogenic behavior. Moreover, whereas regular production of both AmpC-type enzymes did not attenuate KP virulence, when
bla
DHA-1 was expressed in an
ampG
-defective mutant,
Galleria mellonella
killing was significantly (but not drastically) attenuated. Spontaneous DHA-1 hyperproducer mutants were readily obtained
in vitro
, showing slight or insignificant virulence attenuations together with high-level resistance to β-lactams only mildly affected by basal production (e.g., ceftazidime, ceftolozane/tazobactam). By analyzing diverse DHA-1-harboring clinical KP strains, we demonstrate that the natural selection of these hyperproducers is not exceptional (>10% of the collection), whereas mutational inactivation of the typical AmpC hyperproduction-related gene
mpl
was the most frequent underlying mechanism. The potential silent dissemination of this kind of strains, for which an important fitness cost-related contention barrier does not seem to exist, is envisaged as a neglected threat for most β-lactams effectiveness, including recently introduced combinations. Analyzing whether this phenomenon is applicable to other transferable β-lactamases and species as well as determining the levels of conferred resistance poses an essential topic to be addressed.

IMPORTANCE

Although there is solid knowledge about the regulation of transferable and especially chromosomal AmpC β-lactamases in Enterobacterales, there are still gaps to fill, mainly related to regulatory mechanisms and virulence interplays of the former. This work addresses them using
Klebsiella pneumoniae
as model, delving into a barely explored conception: the acquisition of a plasmid-encoded inducible AmpC-type enzyme whose production can be increased through selection of chromosomal mutations, entailing dramatically increased resistance compared to basal expression but minor associated virulence costs. Accordingly, we demonstrate that clinical
K. pneumoniae
DHA-1 hyperproducer strains are not exceptional. Through this study, we warn for the first time that this phenomenon may be a neglected new threat for β-lactams effectiveness (including some recently introduced ones) silently spreading in the clinical context, not only in
K. pneumoniae
but potentially also in other pathogens. These facts must be carefully considered in order to design future resistance-preventive strategies.