Pseudomonas aeruginosa OprD-Family Porins Facilitate Imipenem Translocation Resulting in AmpC β-Lactamase Induction Contributing to the Effectiveness of Imipenem/Relebactam
Background: Multidrug-resistant Pseudomonas aeruginosa is a serious concern in the United States and considered an urgent priority pathogen globally. Due to multiple chromosomal resistance factors, P. aeruginosa can emerge resistant to most available antibiotics. Carbapenem antibiotics are a last line of defense antibiotic of choice for P. aeruginosa; however, emergence of resistance has increased in hospital onset cases since 2019. The introduction of the β-lactamase inhibitor relebactam in combination with the carbapenem imipenem has restored susceptibility in some isolates, yet the mechanism remains unknown. Imipenem resistance has been tied to the depletion of the outer membrane porin, OprD and overproduction of AmpC chromosomal β-lactamase. It is not known how relebactam potentiates imipenem when AmpC levels are low, nor how imipenem traffics into the cell in the absence of OprD. The purpose of this dissertation was to address these questions using three approaches. First, determine the contribution of the chromosomal β-lactamases, AmpC, Pib-1, and OXA-50 on imipenem susceptibility. Second, determine how relebactam potentiates imipenem in the absence of OprD. Third, to determine the contribution other OccD porins: OpdP, OpdB, and OpdB in the translocation of imipenem into the cell in the absence of OprD. Method: A panel of laboratory strains of P. aeruginosa and various clinical isolates were evaluated. Methods used in this dissertation included RT-qPCR, western analysis, whole genome sequencing, RNAseq analysis and linear regression analysis. Results: Basal levels of ampC were found to contribute very little to imipenem MICs and were not an accurate predictor of the impact relebactam had on imipenem MICs. However, loss of OprD correlated with imipenem nonsusceptibility. Protein production was the strongest indicator and resolved discordant phenotypes observed in isolates when only transcript levels were taken into consideration. AmpC induction occurred upon exposure to imipenem in all isolates and was determined to provide a substrate for relebactam. The transcript level for ampC following induction was found to correlate with the reduction in imipenem MICs in the presence of relebactam. Competition with nutrient substrates revealed OpdP to be a strong candidate for imipenem translocation in the absence of OprD. However, OpdC and OpdB did not appear to traffic imipenem efficiently. Cross reactivity between substrates was likely to have impacted imipenem traffic. Discussion: This work demonstrates that imipenem resistance involves the interplay of multiple mechanisms in P. aeruginosa. AmpC β-lactamase contributes to imipenem MIC after it has been induced by imipenem; however, OprD protein loss is required for nonsusceptibility. In the isolates examined, the presence of the other chromosomal β-lactamases did not impact imipenem MICs due to low expression of the genes. OpdP was the only alternate porin in the OccD family with a strong link for imipenem translocation and OprD and OpdP are often coregulated. Other porins within the OccD family may play much smaller roles or serve as redundant nutrient transporters so that OprD loss does not impact fitness. This dissertation revealed the role AmpC induction plays in imipenem and impenem/relebactam MICs and the ability for OpdP to translocate imipenem.
Beta-Lactamase, Carbapenem, Pseudomonas, Resistance
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