Xylose Improves Antibiotic Activity of Chloramphenicol and Tetracycline against K. pneumoniae and A. baumannii in a Murine Model of Skin Infection

Hindawi Canadian Journal of Infectious Diseases and Medical Microbiology Volume 2018, Article ID 3467219, 6 pages https://doi.org/10.1155/2018/3467219 Research Article Xylose Improves Antibiotic Activity of Chloramphenicol and Tetracycline against K. pneumoniae and A. baumannii in a Murine Model of Skin Infection Alejandro A. Hidalgo,1,2 Ángel J. Arias,1 Juan A. Fuentes,3 Patricia Garcı́a,4 Guido C. Mora,1 and Nicolás A. Villagra 1 1 Laboratorio de Patogénesis Molecular y Antimicrobianos, Facultad de Medicina, Universidad Andres Bello, Santiago, Chile Escuela de Quı́mica y Farmacia, Facultad de Medicina, Universidad Andres Bello, Santiago, Chile 3 Laboratorio de Genética y Patogénesis Bacteriana, Facultad de Ciencias de la Vida, Universidad Andres Bello, Santiago, Chile 4 Servicio de Laboratorios Clı́nicos Laboratorio de Microbiologı́a, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile 2 Correspondence should be addressed to Nicolás A. Villagra; Received 4 October 2017; Accepted 9 May 2018; Published 18 July 2018 Academic Editor: Jorge Garbino Copyright © 2018 Alejandro A. Hidalgo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Increased resistance to antimicrobials in clinically important bacteria has been widely reported. The major mechanism causing multidrug resistance (MDR) is mediated by efflux pumps, proteins located in the cytoplasmic membrane to exclude antimicrobial drug. Some efflux pumps recognize and expel a variety of unrelated antimicrobial agents, while other efflux pumps can expel only one specific class of antibiotics. Previously, we have reported that xylose decreases the efflux-mediated antimicrobial resistance in Salmonella typhimurium, Pseudomonas aeruginosa, and Acinetobacter baumannii in vitro. In this work, we assessed the effectiveness of combining xylose with antibiotics to kill resistant Acinetobacter baumannii and Klebsiella pneumoniae in a murine model of skin infection. Skin infections were established by seeding 109 bacteria onto eroded skin of mice. Mice treated with the antibiotic alone or with a mixture of glucose and antibiotics or xylose and antibiotics were compared to a control group that was infected but received no further treatment. We observed that the mixtures xylose-tetracycline and xylose-chloramphenicol produced a decrease of at least 10 times viable Acinetobacter baumannii and Klebsiella pneumoniae recovered from infected skin, compared with mice treated with the antibiotic alone. Our results show that xylose improves the antibiotic activity of tetracycline and chloramphenicol against efflux-mediated resistance Acinetobacter baumannii and Klebsiella pneumoniae, in a murine model of skin infection. We envision these combined formulations as an efficient treatment of skin infections with bacteria presenting efflux-mediated resistance, in both humans and animals. 1. Introduction Skin infections are one of the most common infections [1]. Breaks in the skin, such as leg ulcers and surgical or traumatic wounds, constitute a perfect environment for infections by a broad range of bacteria [2]. Most skin infections are caused by Gram-positive bacteria, commonly Staphylococcus aureus and group A β-haemolytic Streptococcus [1]. However, Gramnegative bacteria such as Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae may also cause skin infections [2]. The incidence of skin infections has increased due to ageing of the general population, increased number of critically ill patients, increased number of immunocompromised patients, and recent emergence of multidrug-resistant pathogens [3]. Multidrug resistance (MDR) is defined as the resistant phenotype to antibiotics belonging to two or more classes of antibiotics and represents a serious problem in healthcare settings [4, 5]. Drug-resistant bacteria are responsible for more than 30,000 deaths per year in the UK and Europe, and it is estimated that 23,000 people 2 Canadian Journal of Infectious Diseases and Medical Microbiology in the United States die from pathogens that are not responsive to treatments with current antibiotic therapies [6]. Bacteria exhibit different strategies to resist antibiotics. One of the most important mechanisms, considered a major contributor to the emergence of MDR pathogens, is the antibiotic efflux achieved by efflux pumps [7]. Efflux pumps are proteins located in the inner membrane of Gramnegative bacteria and in the cytoplasmic membrane of Gram-positive bacteria [7]. The continuous onset of MDR in bacterial strains limits the clinical efficacy of most available antibiotics. Therefore, there is an urgent need to introduce novel antimicrobial molecules that may be active by themselves or potentiate current available antibiotics [8]. In a previous in vitro study, we found that xylose decreases the efflux-mediated antimicrobial resistance in S. typhimurium, P. aeruginosa, and A. baumannii. Although the mechanism behind sensitization remains elusive, it has been speculated that either competitions for limited space in the inner membrane or interference with the translocon systems may affect translocation of efflux pumps into membrane, thereby affecting efflux-mediated resistance [9]. Because the in vitro potentiation of actively expelled antimicrobials was fairly significant in the presence of xylose, we ought to find whether this potentiation can be reproduced in vivo. Therefore, in this work, we assessed the effectiveness of combining xylose with antibiotics in vivo. Our results show that xylose increases the antibiotic activity of tetracycline and chloramphenicol against efflux-dependent resistant A. baumannii and K. pneumoniae, in a model of skin infection in mice. 2. Materials and Methods 2.1. Bacterial Strains and Growth Conditions. Clinical strains of A. baumannii and K. pneumoniae were collected from different healthcare facilities throughout Santiago, Chile, and collected at Servicio de Laboratorios Clı́nicos, Escuela de Medicina, Pontificia Universidad Católica de Chile in Santiago, between 2014 and 2015. The A. baumannii strains were isolated from tracheal secretions from patients with respiratory infection. The K. pneumoniae strains were isolated from urine of patients with urinary infection. Strains were grown in LB broth at 37°C with aeration. Solid media (LB agar) included Bacto agar (15 g/L). 2.2. Antimicrobial Susceptibility Test. We used a modification of the disc diffusion assay previously described [9, 10]. Briefly, cultures were grown for 16 h in LB broth; bacteria were washed three times and resuspended in PBS. 106 cells were spread on M9 plates supplemented with glucose or xylose (2 mg/mL) [9]. When required, the medium was supplemented with 12.5 μM carbonyl cyanide-mchlorophenylhydrazone (CCCP), an indirect efflux pumps inhibitor that ac (...truncated)