Carbohydrate Polymers 157 (2017) 146–155 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Development of in vitro resistance to chitosan is related to changes in cell envelope structure of Staphylococcus aureus Dina Raafat a,∗ , Nicole Leib a , Miriam Wilmes a , Patrice Franc ¸ ois b , Jacques Schrenzel b , Hans-Georg Sahl a a Institute for Medical Microbiology, Immunology and Parasitology (IMMIP), Pharmaceutical Microbiology Unit, University of Bonn, D-53115 Bonn, Germany b Genomic Research Laboratory, Division of Infectious Diseases, University of Geneva Hospitals, CH-1211 Geneva, Switzerland article info Article history: Received 22 July 2016 Received in revised form 23 September 2016 Accepted 23 September 2016 Available online 26 September 2016 Keywords: Chitosan Antibiotic resistance Phospholipids analysis Microarray analysis abstract The bacterial cell envelope is believed to be a principal target for initiating the staphylocidal pathway of chitosan. The present study was therefore designed to investigate possible changes in cell surface phenotypes related to the in vitro chitosan resistance development in the laboratory strain S. aureus SG511-Berlin. Following a serial passage experiment, a stable chitosan-resistant variant (CRV) was identified, exhibit- ing >50-fold reduction in its sensitivity towards chitosan. Our analyses of the CRV identified phenotypic and genotypic features that readily distinguished it from its chitosan-susceptible parental strain, includ- ing: (i) a lower overall negative cell surface charge; (ii) cross-resistance to a number of antimicrobial agents; (iii) major alterations in cell envelope structure, cellular bioenergetics and metabolism (based on transcriptional profiling); and (iv) a repaired sensor histidine kinase GraS. Our data therefore suggest a close nexus between changes in cell envelope properties with the in vitro chitosan-resistant phenotype in S. aureus SG511-Berlin. © 2016 Elsevier Ltd. All rights reserved. 1. Introduction Chitosan is a linear high molecular weight heteropolysac- charide, consisting of N-acetyl-d-glucosamine and d-glucosamine units, linked together by ˇ-(1 → 4) glycosidic bonds; it is pro- duced from chitin by exhaustive alkaline deacetylation (Kumar, 2000). Since the relative amount of the two monosaccharides may vary, the term chitosan usually refers to a family of copolymers with various fractions of acetylated units (Singla & Chawla, 2001; Abbreviations: AMP, antimicrobial peptide; CAMHB, cation-adjusted Mueller–Hinton II broth; CL, cardiolipin; CRV, chitosan-resistant variant; FDR, False Discovery Rate; HMG-CoA, Hydroxymethylglutaryl-CoA; L-PG, lysyl- phosphotidylglycerol; MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration; OD, optical density; ORF, Open Reading Frame; PG, phos- phatidylglycerol; PL, phospholipid; RT, room temperature; TCRS, two-component regulatory system; TEM, Transmission electron microscope; WT, wild-type. ∗ Corresponding author. Present address: Institute of Immunology and Trans- fusion Medicine, Immunology Department, University Medicine Greifswald, F. Sauerbruch-Straße DZ 7, D-17475 Greifswald, Germany. E-mail addresses: dina.raafat@uni-greifswald.de, dina raafat@yahoo.com (D. Raafat), Nicole.Leib1@gmx.de (N. Leib), mwilmes@uni-bonn.de (M. Wilmes), patrice.francois@genomic.ch (P. Franc ¸ ois), jacques.schrenzel@hcuge.ch (J. Schrenzel), hgsahl@uni-bonn.de (H.-G. Sahl). Tharanathan & Kittur, 2003). In contrast to most of the naturally- occurring polysaccharides, chitosan is an example of a highly basic polysaccharide with a high charge density. Its unique chemical structure, combined with its physico-chemical and biological char- acteristics, allow for a wide range of applications ranging from pharmaceutical, cosmetic, medical, food and textile to agricultural applications (Raafat & Sahl, 2009). Recent developments in the field of biomaterials have led to a renewed interest in this biopolymer, especially with an increas- ing number of publications describing the antimicrobial potentials of chitosan and its derivatives against filamentous fungi, yeasts and bacteria (Champer et al., 2013; Galván Márquez et al., 2013). It is generally assumed that the polycationic nature of chitosan contributes to its interaction with anionic microbial cell surface components, resulting in random multiple detrimental events which may each contribute to the overall efficacy (Je & Kim, 2006; Raafat, Bargen, von Haas, & Sahl, 2008; Torr, Chittenden, Franich, & Kreber, 2005; Zakrzewska, Boorsma, Brul, Hellingwerf, & Klis, 2005). Our previous data clearly indicate that the initial contact between chitosan and the negatively-charged cell wall polymers (teichoic acids) is indeed driven by electrostatic interactions. This leads to impairment and destabilization of membrane function with subsequent leakage of cellular components, and ultimately to http://dx.doi.org/10.1016/j.carbpol.2016.09.075 0144-8617/© 2016 Elsevier Ltd. All rights reserved.