Mutation-Driven Evolution of Pseudomonas aeruginosa in the Presence of either Ceftazidime or Ceftazidime-Avibactam

Abstract

Ceftazidime-avibactam is a combination of β-lactam/β-lactamase inhibitor, the use of which is restricted to some clinical cases, including cystic fibrosis patients infected with multidrug-resistant , in which mutation is the main driver of resistance. This study aims to predict the mechanisms of mutation-driven resistance that are selected for when is challenged with either ceftazidime or ceftazidime-avibactam. For this purpose, PA14 was submitted to experimental evolution in the absence of antibiotics and in the presence of increasing concentrations of ceftazidime or ceftazidime-avibactam for 30 consecutive days. Final populations were analyzed by whole-genome sequencing. All evolved populations reached similar levels of ceftazidime resistance. In addition, they were more susceptible to amikacin and produced pyomelanin. A first event in this evolution was the selection of large chromosomal deletions containing (involved in pyomelanin production), (involved in β-lactams resistance), and (involved in aminoglycoside resistance). Besides mutations in and that regulate β-lactamase expression, mutations related to MexAB-OprM overexpression were prevalent. Ceftazidime-avibactam challenge selected mutants in the putative efflux pump and and in a two-component system ( and ), likely regulating its expression. All populations produced pyomelanin and were more susceptible to aminoglycosides, likely due to the selection of large chromosomal deletions. Since pyomelanin-producing mutants presenting similar deletions are regularly isolated from infections, the potential aminoglycoside hypersusceptiblity and reduced β-lactam susceptibility of pyomelanin-producing should be taken into consideration for treating infections caused by these isolates.

Dynamic Emergence of Mismatch Repair Deficiency Facilitates Rapid Evolution of Ceftazidime-Avibactam Resistance in Pseudomonas aeruginosa Acute Infection

“…BepR/NalD is a member of the TetR helix-turn-helix (HTH) family of transcription factors that contain an N-terminal DNA binding domain. The corresponding C22R amino acid substitution in the CZA-resistant isolates is located in the HTH domain, suggesting the possibility that DNA binding might be disrupted, resulting in transcriptional derepression of MexAB-OprM, as has been noted in other studies (53,54).…”

“…Among the remaining 12 mutations in the CZA-resistant isolates, three other genes known to be associated with the development of antimicrobial resistance-mexB, bepR, and clpA-contained single nonsynonymous substitutions (53,54). MexB is an MDR family efflux pump that, when overexpressed, can confer resistance to multiple classes of antimicrobials in P. aeruginosa, including CZA, and numerous studies have demonstrated the evolution of resistance in association with mutations in the mexB gene (53)(54)(55)(56). A P326L substitution was present in the MexB protein in a position adjacent to the channel domain in the crystal structure (PDB accession no.…”

“…An E531G substitution was observed in ClpA, an AAA family ATPase and a motor subunit of the ClpP protease that is involved in a broad variety of cellular functions in bacteria, including virulence and bacterial persistence (60). Mutations in Clp family proteins have been associated with cephalosporin resistance and have been observed to develop under monobactam, CAZ, and CZA selection (53,54,61). Although the exact impact of the observed substitution in ClpA is unclear, the above evidence suggests the potential involvement in CZA resistance observed in this case.…”

Identification of Drug Resistance Determinants in a Clinical Isolate of Pseudomonas aeruginosa by High-Density Transposon Mutagenesis

“…While deletion of mepM1 leads to reduced MIC values of ␤-lactam antibiotics, deletion of ctpA leads to hyperresistance, probably by deregulating the levels of its substrates. The role of deleted or nonfunctional CtpA in mediating hyperresistance is further supported by the findings of a study by Sanz-García et al, who showed that upon ceftazidime-avibactam treatment, mutations in the ctpA gene emerge and that these lead to resistance (49). Additional deletion of mepM1 in the ctpA mutant reduces MIC values compared to those for the ΔctpA mutant for PIP and ATM but results in still higher MIC values compared to those for the mepM1 deletion mutant, indicating that other CtpA-dependent EPs also contribute to the upregulation of ampC expression.…”

Analysis of the Pseudomonas aeruginosa Aminoglycoside Differential Resistomes Allows Defining Genes Simultaneously Involved in Intrinsic Antibiotic Resistance and Virulence

“…In addition to genes already known to be involved in P. aeruginosa intrinsic resistance to aminoglycosides, we identified two novel loci (glnD and mucD) in the chromosome of P. aeruginosa PA14 that contribute to intrinsic resistance to at least one aminoglycoside (see Table S1 in the supplemental material), although inactivation of mucD increases susceptibility to one aminoglycoside and reduces susceptibility to another. Interestingly, both genes have been proposed to play a potential role in ␤-lactam resistance (26,36). Further, 14 novel loci could potentially be involved in the acquisition of mutational resistance to at least one aminoglycoside, since their inactivation increases the MICs of such aminoglycosides by at least 3-fold compared with the wild-type parental strain.…”