The carbon source influences the efflux pump-mediated antimicrobial resistance in clinically important Gram-negative bacteria

J Antimicrob Chemother 2012; 67: 921 – 927 doi:10.1093/jac/dkr573 Advance Access publication 17 January 2012 The carbon source influences the efflux pump-mediated antimicrobial resistance in clinically important Gram-negative bacteria Nicolás A. Villagra1, Juan A. Fuentes1, Matı́as R. Jofré1, Alejandro A. Hidalgo2, Patricia Garcı́a3 and Guido C. Mora4* 1 Laboratorio de Microbiologı́a, Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago, Chile; 2Department of Biochemistry, State University of New York, University of Buffalo, NY, USA; 3Laboratorio de Microbiologı́a, Departamentos de Laboratorios Clı́nicos, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile; 4Unidad de Microbiologı́a, Facultad de Medicina, Universidad Andres Bello, Santiago, Chile *Corresponding author. Tel: +56-2-6618373; E-mail: Received 1 October 2011; returned 7 November 2011; revised 9 December 2011; accepted 14 December 2011 Objectives: Multidrug efflux pumps are proteins known to play an important role in resistance in bacteria. These proteins are located in the inner membrane (IM), together with many other proteins, including inducible permeases that participate in the uptake of non-phosphotransferase system (PTS) carbohydrates (i.e. carbohydrates uptaken by mechanisms other than the PTS). However, lipid bilayer space in the IM is limited. Therefore, we examined whether the overexpression of unrelated IM proteins is able to interfere with the efflux-mediated resistance mechanism, consequently increasing the susceptibility towards different antimicrobial compounds. Methods: We cultured bacteria under different conditions that increase the synthesis of unrelated IM proteins, either by using a non-PTS carbohydrate as the sole carbon source or by artificially overexpressing IM proteins, prior to determining the resistance to different antimicrobial compounds by disc diffusion assays. Results: We observed that efflux-pump-mediated resistance is affected by the carbon source in all the strains tested, exhibiting increased susceptibility when a non-PTS carbohydrate was used as the sole carbon source. Moreover, when we artificially overexpressed an unrelated IM protein, we also observed decreased effluxmediated resistance. Conclusions: These results strongly suggest that overexpression of IM proteins, by using a non-PTS carbohydrate as the sole carbon source, or by artificially introducing a high number of copies of an unrelated IM protein, competes with the antibiotic efflux systems, thereby decreasing the efflux-mediated resistance to different antimicrobial compounds. This sort of competition arises because of the limited available space in the bacterial IM, or by an unknown mechanism. Keywords: alternative carbon source, permeases, antibiotics, resistance mechanisms Introduction Bacteria are recognized as the aetiological agents of many important infectious diseases in humans, resulting in death and economic loss.1 The introduction of antibiotic therapy in 1940 strongly contributed to saving the lives of millions of people. Nevertheless, the problem of antimicrobial drug-resistant pathogens was recognized shortly after antimicrobial drugs began to be used. In fact, it is not uncommon to observe the emergence of antibiotic resistance in bacteria when they encounter a new, commercially produced antibiotic.2 – 5 Over the years, the selective pressure exerted by different new antibiotics has resulted in bacteria bearing different kinds of resistance mechanisms, leading to the multidrug resistant (MDR) phenotype.3 The appearance of MDR is one of the most important concerns in the clinical and public-health management of nearly every type of infectious disease.1,6,7 Some authors have defined the MDR phenotype as resistance to two or more antibiotic classes.3,8 Bacteria exhibit different strategies to develop resistance to antibiotics,9 – 11 and prevention of antibiotic accumulation in the bacterial cytoplasm (either by decreasing influx or, most importantly, by increasing efflux) has been considered a major contributor to the emergence of MDR pathogens.3,12 – 17 Antibiotic efflux is efficiently achieved by efflux pumps—proteins located in the inner membrane of Gram-negative bacteria, and in the cytoplasmic # The Author 2012. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: 921 Villagra et al. membrane of Gram-positive bacteria.15 These pumps have been described as promiscuous/polyselective proteins, able to recognize and expel a large variety of antimicrobial agents, including antibiotics, biocides and detergents, thereby yielding their role in the development of the MDR phenotype.12,13,15,17 It has been reported that the protein content in the bacterial inner membrane is diverse, including the efflux pumps and more than 100 other different types of proteins.18 Moreover, in Gramnegative bacteria 20% –30% of total protein is located at the inner membrane, whereas only 2% are outer membrane proteins.19,20 Nevertheless, the membrane lipid bilayer occupies less than 4% of total bacterial volume, strongly suggesting that the available space for the overproduction of inner membrane proteins is limited.21 Accordingly, many inner membrane proteins, including several efflux pumps, are especially difficult to overproduce in vivo,22 and are tightly regulated in order to prevent excessive production.23,24 Besides the efflux pumps, the inner membrane also harbours other proteins, including transporters/permeases for the uptake of solutes, including carbohydrates.24 – 33 In Salmonella, Escherichia coli and other enterobacteria, the phosphotransferase system (PTS), also located in the inner membrane, performs the translocation of glucose with concomitant phosphorylation.21,25 When bacteria grow under glucose-depleted conditions, other inner membrane permeases, normally repressed by catabolite repression, are expressed in order to take up alternative carbon sources.24 – 33 Given that the inner membrane space is limited, it is plausible that overproduction of carbohydrate permeases due to the presence of a non-PTS carbohydrate as the sole carbon source (or the overproduction of any inner-membrane protein) might alter the protein composition of the inner membrane, thereby affecting efflux-pump-mediated antibiotic resistance. In this paper it is demonstrated that efflux-pump-mediated antibiotic resistance can be subverted by culturing bacteria with non-PTS sugars as the sole carbon source, or by artificially overproducing unrelated inner-membrane proteins. Materials and methods Bacterial strains, media and growth conditions A total of 60 clinical strains, including Pseudomonas aeruginosa (50 clinical strains) and Acinetobacter baumannii (10 clinical strains), which had been collected at the Hospital of Pontificia Universidad Católica de Chile and from other h (...truncated)