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, 8 (1), 16463

Interplay Between MexAB-OprM and MexEF-OprN in Clinical Isolates of Pseudomonas Aeruginosa


Interplay Between MexAB-OprM and MexEF-OprN in Clinical Isolates of Pseudomonas Aeruginosa

Gertrudis Horna et al. Sci Rep.


MexAB-OprM and MexEF-OprN are Pseudomonas aeruginosa efflux pumps involved in the development of antibiotic resistance. Several studies developed with laboratory strains or using a few clinical isolates have reported that the regulation system of MexEF-OprN is involved in the final levels of MexAB-OprM expression. Therefore, this study was aimed to determine the interplay between MexAB-OprM and MexEF-OprN in 90 out of 190 P. aeruginosa clinical isolates with an efflux pump overexpression phenotype. Regarding oprD, 33% (30/90) of isolates displayed relevant modifications (RM) defined as frameshift or premature stop, both related to carbapenem resistance. On the other hand, 33% of the isolates displayed RM in nalC, nalD or mexR, which were significantly associated with multidrug resistance (MDR), non-susceptibility to carbapenems, OprD alterations and strong biofilm production. Meanwhile, the RM in MexS were associated with presence of pigment (p = 0.004). Otherwise, when all the regulators were analysed together, the association between RM in MexAB-OprM regulators and MDR was only significant (p = 0.039) when mexS was the wild type. These data show the modulatory effect of MexEF-OprN on MexAB-OprM in a clinical population of P. aeruginosa. Further studies may contribute to design of novel molecules acting on this interplay to fight against antimicrobial resistance.

Conflict of interest statement

The authors declare no competing interests.


Figure 1
Figure 1
Association of EPO phenotype with biofilm formation, swarming, pigment and antibiotic resistance. SBP: Strong biofilm producer; Swarming: Microorganisms showing swarming motility; Pigment: Presence of pigment; LVX: Resistance to levofloxacin; Carbapenem: resistance to carbapenems; MDR: Multidrug resistance; EPO: Efflux Pump Overexpression. In 2 isolates the MICI was >256 mg/L while the MICPABN was 256 mg/L, therefore the EPO phenotype was not determined. *p = 0.0093; **p = 0.0013; ***p = 0.006.
Figure 2
Figure 2
Association of MexAB-OprM regulators and MexEF-OprN regulators with biofilm formation, swarming, pigment, oprD gene and antibiotic resistance. SBP: Strong biofilm producer; Swarming: Microorganisms showing swarming motility; Pigment: Presence of pigment; OprD (RM): Presence of relevant modifications in OprD (frameshifts + premature STOPs + no amplification); Carbapenem: resistance to carbapenems; MDR: Multidrug resistance. (a) MexAB-OprM regulators. IM: Irrelevant modifications (amino acid substitution + amino acid insertions + amino acid deletions); RM. Relevant Modifications (frameshifts + premature STOPs + no amplification); *p: 0.006; **p < 0.0001. (b) MexEF-OprN regulators. PA14: Sequence identical to PA14; NA: no amplification. *p: 0.004. Only the isolates presenting the genotypes PA14/PA14 (47 isolates) and NA/PA14 (34 isolates) were analysed.
Figure 3
Figure 3
PCR strategy used in the analysis of mexS gene. In all figure sections is first presented the scenario which explain the obtained results, and just below the scheme of the PCR reactions. When in grey a positive amplification was obtained, and the amplicon size is within the rectangle, when in blank, no amplification was obtained. In all cases the primers used are represented by thin arrows and numbered following the same numeration presented in Table 4. All positions arbitrarily refer to the first base of the mexS gene. The figure is not made to scale. Furthermore, in scenarios b and c, the presence of internal modifications in DNA sequence (affecting or not mexS functionality) may lead to DNA secondary structures which obstacle PCR amplification. (a) PCR amplification of mexS gene. (b) No amplification of mexS gene and amplification of N- and C-terminal regions. (c) No amplification of mexS gene and amplification of N-terminal regions. Two scenarios are considered. Scenario 1: a DNA sequence (represented with a weft filling rectangle) disrupt mexS after base position 650, allowing the amplification on the N-terminal region but avoiding that of full mexS gene as well as that of the C-terminal region. Scenario 2: a polymorphism (represented with “xxx”) avoid the annealing of primer 2. (d) No amplification of Mex S gene and no amplification of N- and C- terminal regions. Two scenarios are considered. Scenario 1: the mexS gene has been deleted or is absent. Scenario 2: polymorphisms are present in both annealing position of primers 1 and 2. If this late option was right, the most probable is the presence of additional differences in the sequence.

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