Mutation-driven evolution of Pseudomonas aeruginosa in the presence of either ceftazidime or ceftazidime/avibactam

Abstract

12Ceftazidime/avibactam is a combination of beta-lactam/beta-lactamases inhibitor, which 13 use is restricted to some clinical cases including cystic fibrosis patients infected with 14 multidrug resistant Pseudomonas aeruginosa, in which mutation is the main driver of 15 resistance. This study aims to predict the mechanisms of mutation-driven resistance that 16 are selected for when P. aeruginosa is challenged with either ceftazidime or 17 ceftazidime/avibactam. For this purpose, P. aeruginosa PA14 was submitted to 18 experimental evolution in the absence of antibiotics and in the presence of increasing 19 concentrations of ceftazidime or ceftazidime/avibactam for 30 consecutive days. Final 20 populations were analysed by whole-genome sequencing. All evolved populations 21 reached similar levels of ceftazidime resistance. Besides, all of them were more 22 susceptible to amikacin and produced pyomelanin. A first event in the evolution was the 23 selection of large chromosomal deletions containing hmgA (involved in pyomelanin 24 production), galU (involved in β-lactams resistance) and mexXY-oprM (involved in 25 aminoglycoside resistance). Besides mutations in mpl and dacB that regulate β-26 lactamase expression, mutations related to MexAB-OprM overexpression were 27 prevalent. Ceftazidime/avibactam challenge selected mutants in the putative efflux 28 pump PA14_45890-45910 and in a two-component system (PA14_45870-45880), likely 29 regulating its expression. All populations produce pyomelanin and were more 30 susceptible to aminoglycosides likely due to the selection of large chromosomal 31 deletions. Since pyomelanin-producing mutants, presenting similar deletions are 32 regularly isolated from infections, the potential aminoglycosides hyper-susceptiblity and 33 reduced β-lactams susceptibility of pyomelanin-producing P. aeruginosa should be 34 taken into consideration for treating infections by these isolates. 35 42 including those encoding multidrug (MDR) efflux pumps (4) and beta-lactamases. In 43 addition, an increasing number of P. aeruginosa isolates has acquired several resistance 44 genes through horizontal gene transfer (HGT), including different classes of 45carbapenemases. Finally, P. aeruginosa is able to develop resistance through mutation, 46 particularly when causing chronic infections, to nearly any available antibiotic. Under 47 this situation, the emergence and spread of MDR resistant global clones is of special 48 concern (5). 49The use of β-lactam/β-lactamase inhibitor combinations, such as amoxicillin/clavulanic 50 acid or ceftolozane/tazobactam, has proven to be effective against class A β-lactamases 51 (which include narrow and extended-spectrum β-lactamases and some 52 carbapenemases); whereas effective combinations against class B, C (extended 53 spectrum cephalosporinases) and D β-lactamases (6-8) have not been available until 54 recently. One of them is the ceftazidime/avibactam combination, whose use was 55 approved in 2015 by the FDA (9). 56 Avibactam, formerly known as NXL104, be...

Phylogenetic analysis of resistance to ceftazidime/avibactam, ceftolozane/tazobactam and carbapenems in piperacillin/tazobactam-resistant Pseudomonas aeruginosa from cystic fibrosis patients

“…We identified only one mobile genetic element associated with resistance in three isolates This is supported by the emergence of mutations (in some cases identical) in phylogenetically distant lineages against a backdrop of phenotypic variations in sequential isolates from the same patient. These observations confirm the strong contribution of mutation-driven evolution to the population structure of P. aeruginosa (17).…”

“…Development of multidrug resistance in P. aeruginosa lung isolates from cystic fibrosis patients has been mainly attributed to its ability to adapt to the cystic fibrosis airway microenvironment by multiple genotypic changes, hence the emphasis on intra-patient evolutionary isolate analyses (16). However, its ability to develop mutational resistance in the context of high selective pressure is also well known (17).…”

Rapid and robust evolution of collateral sensitivity in Pseudomonas aeruginosa antibiotic-resistant mutants

“…In a previous study, we observed that P. aeruginosa PA14 populations experimentally evolved in the presence of ceftazidime displayed a robust collateral sensitivity to amikacin (25), as a consequence of selection of large chromosomal deletions upon 1 day of adaptive laboratory evolution (ALE) (25). The chromosomal deletions included hmgA, which encodes an enzyme whose lack of activity leads to the hyperproduction of the brown pigment pyomelanin (26); galU, whose inactivation reduces ceftazidime susceptibility (27); and mexXY, which encodes a multidrug resistance (MDR) efflux pump that contributes to P. aeruginosa intrinsic aminoglycosides resistance (28).…”

“…To determine whether chromosomal deletions containing mexXY would be early selected during P. aeruginosa evolution in presence of ceftazidime in the set of mutants mentioned above, as it was the case in the wild-type strain PA14 (25), four biological replicates of each single (nfxB177, parR87, mexZ43, orfN50, and nuoD184) and multiple mutants (MDR6 and MDR12), and the wild-type PA14 strain, were subjected to ALE in presence or absence of ceftazidime (a total of 64 populations) for 3 days. Upon 1 day of experimental evolution, almost every P. aeruginosa population challenged with antibiotic hyperproduced pyomelanin (27 of 32 populations; Fig.…”

Antimicrobial resistance: A multifaceted problem with multipronged solutions

“…These results highlight the fact that the acquisition of resistance to one antibiotic can modify the susceptibility to others, either reducing (cross‐resistance) or increasing (collateral sensitivity) bacterial susceptibility to them. While the analysis of cross‐resistance phenotypes gives clues for avoiding the use of some specific antibiotics when bacteria have acquired resistance to another one, the analysis of the collateral sensitivity networks can help to develop combined or cyclic therapeutic regimes (Podnecky et al, ; Sanz‐Garcia, Hernando‐Amado, & Martinez, ). Indeed, Pseudomonas aeruginosa mutants selected in the presence of carbapenems presented decreased susceptibility to doripenem, meropenem, and imipenem, while their susceptibility to aminoglycosides, fluoroquinolones, and noncarbapenem β‐lactams was higher than that of the parental clinical isolate from which these mutants derive (Harrison, Fowler, Abdalhamid, Selmecki, & Hanson, ).…”