Outer membrane permeability of Pseudomonas aeruginosa through β-lactams: new evidence on the role of OprD and OpdP porins in antibiotic resistance

Abstract

Gram-negative bacteria are a major concern for public health, particularly due to the continuous rise of antibiotic resistance. A major factor that helps the development of resistance is the outer membrane that is essential since it acts as a strong permeability barrier to many antibiotics that are effective against other bacteria. In this study, we determine the specific permeability coefficients for various antibiotics in Pseudomonas aeruginosa strains, which differ from each other for their porin expressions. We showed that OprD and OpdP porins contribute both to internalize meropenem and biapenem. Using qRT-PCR, we demonstrated that their expression is dependent of the various phases of cellular growth. We were able to show how the OpdP porin is less expressed in exponential growth phases, while it tends to be produced when the bacterial culture enters into the latent phase, in an inversely proportional way compared to the OprD porin. The deletion of the OpdP porin, in the presence of meropenem at concentrations equivalent to the MIC values, contributes to the selection of carbapenem-resistant strains. Therefore, the presence of mutations/deletions of the OpdP porin should receive greater consideration from a clinical point of view as the use of meropenem at nonoptimal concentrations could lead to the appearance of resistance phenotypes.IMPORTANCECarbapenem-resistant strains of Pseudomonas aeruginosa are among the major threats to public health. The permeability of the outer membrane for the β-lactam antibiotics is one of the major factors that reduce the activity of the antibiotics. In this study, we measure the low permeability coefficient of the P. aeruginosa outer membrane to β-lactams. The methodology we develop to determine the permeability can be applied to other antibiotic families and/or pathogens.

Keywords: OpdP porin; Pseudomonas aeruginosa; external membrane permeability; porins.

Fig 1

Circular visualization of the genome assembly obtained using the software Geneious (version R10). (A) The genome sequence of TNP004 was compared to that of its parental strain PAO1-Jap. The mutations between the genomes are shown. (B) Genome sequence of LG03 was compared with that of its parental strain ARC545. The opdP deletion is highlighted, and the other observed mutations are shown.

Fig 2

OprD detection performed by Western Blot in the following strains: (A). 1, protein marker; 2,PAO1; 3,TNP004; 4, ARC5990 (PAO1.ΔoprD); 5, ARC5170 (PAO1ΔopdP); 6, ARC5782 (PAO1ΔoprD, ΔopdP) and 7, ARC5998 (PAO1ΔoprD, ΔopdP,ΔораB, ΔopdC, and ΔopdT). (B). 1, protein marker; 2, PAO1; 3, LG01; 4, ARC5170 (PAO1ΔopdP); 5, LG02; 6, LG03; 7, LG04; 8, LG05; 9, LG06 and 10, LG07. The porin OprD is marked by an arrow. Protein markers’ pictures were put aside the chemiluminescence scans.

Fig 3

Growth curves of P. aeruginosa strains in LB cultures.

Fig 4

Relative expressions of (A) oprD, (B) opdP, (C) opdB, (D) opdC, and (E) opdT mRNAs in P. aeruginosa PAOI and four porin(s) mutant strains; mRNAs were extracted at four different points of bacterial growth, reported in the x-axis as the absorbance at 600 nm, and the relative expression reported on the y-axis is the mean of the transcription of three indipendent reference genes (PA3340, gyrA, and cysG). Data were analyzed by two-way analysis of variance (ANOVA), followed by Bonferroni multiple comparison post-test.

Fig 5

Relative expression of oprD mRNA in P. aeruginosa PAOI, LG01, and LG03; mRNAS were extracted at A600 = 1.6, and the relative expression reported on the y axis is the mean of the transcriptions of three indipendent reference genes (PA3340, gyrA, and cysG) mentioned above. Data were analyzed by two-way analysis of variance (ANOVA), followed by Bonferroni multiple comparison post-test.

Fig 6

(A) Densitometric analysis of the fluorescence signal of the BlaR-CTD-Bocillin adduct, shown as an example; samples from 1 to 7 represent different aliquots, taken every 5 minutes for determining meropenem uptake in ARC5782 (PAO1ΔoprD, ΔopdP); samples 8 and 9 are aliquots taken before the addition of meropenem to the culture, representing the total quantity of produced BlaR-CTD; sample 10 is a not sonicated aliquot, used to quantify possible BlaR-CTD release in the medium. (B) Graph representing the increase of the meropenem-BlaR-CTD adduct as a function of time; this quantification was made possible by subtracting the values obtained during the experiment (samples 1-7 of panel A) from the total BlaR-CTD produced in an aliquot (samples 8 or 9 of panel A). The slope of the line represents the antibiotic flux passing through the outer membran.