In our study, a mass spectrometry–based proteomic analysis was used to comparatively analyze the proteomic profiles of MDR9 versus PAO1. Efflux pumps were among the proteins that were identified as significantly increased. Bacterial efflux pumps are proteins that are embedded in the bacterial plasma membrane. They function to recognize noxious agents that have penetrated the cell wall, and expel them.
38 In this study, hyperexpression of efflux pumps of the resistance-nodulation-cell division (RND)–type chromosomally encoded by mexAB-oprM, mexCD-oprJ, mexEF-oprN, and mexXY (-oprA) were detected and have been shown to contribute to multidrug resistance phenotypes.
39 PAO1, the reference strain used in our study, has 12 efflux systems of the RND type.
40,41 Four of these contribute most significantly to antibiotic resistance: MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM.
42 MexB transports β-lactams, including but not limited to β-lactamase inhibitors, carbapenems, aminoglycides, and fluoroquinolones. Fluoroquinolones like levofloxacin, ciprofloxacin, norfloxacin, and others, have been reported to be most efficiently exported by MexEF-OprN and MexCD-OprJ and with less efficiency by MexAB-OprM and MexXY-OprM (or OprA).
40,41 Susceptibility of PA toward many antibiotics has been restored when four systems (MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM) have been deleted.
42,43 In this regard, GLY decreased mRNA expression of RND efflux pumps in both isolates (exception = MexX in B1045) which is consistent with our in vivo data showing increased sensitivity of MDR9 to ciprofloxacin when combined with GLY. To test activity of MDR9 efflux pumps, EB, a common substrate, and a DNA-intercalating agent that fluoresces only when bound to DNA,
44 was used. We found that higher fluorescence, indicating EB is intracellular
24 was observed after treatment of MDR9 with GLY, indicating a change in efflux activity. In fact, quantitation of the data showed that with GLY, fluorescence for MDR9 was similar to the PAO1 positive control. What cannot be determined from these data is whether GLY altered uptake or retention of EB or had other deleterious effects to bacterial membranes, but the latter was provided by SEM. SEM data confirmed that bacterial membranes were perturbed, but also showed that some bacteria remained viable after GLY treatment. We hypothesize that it is this group that would be responsive to having their efflux pump systems modulated by GLY (as shown in our EB data). In this regard, uptake of compounds can be achieved by control of decreasing porins in the outer membrane and/or by increasing the effectiveness of the efflux of antibiotics, usually by increasing the number of pumps.
45,46 In regard to efflux pumps, in gram-negative bacteria, the predominant form of resistance is through the efflux pump mechanism,
47 leading to decreased bioavailability of antibiotic allowing the bacteria to survive, despite the noxious stimulus.
48 Of course, overexpression of the main efflux pumps is one among many mechanisms, resulting in a MDR phenotype. Our mRNA data supports GLY reduction of the majority of efflux pumps (RND), but we cannot rule out that other contributors to MDR such as biofilm, production of antibiotic inactivating enzymes, etc., are involved. We also do not know if uptake was pH or temperature dependent. To resolve this, future approaches including, but not limited to flow cytometry, could be used to interrogate mechanisms involved in the change in efflux evoked by GLY.
24,49