Elsevier

Carbohydrate Polymers

Volume 174, 15 October 2017, Pages 369-376
Carbohydrate Polymers

Investigation of chitosan’s antibacterial activity against vancomycin resistant microorganisms and their biofilms

https://doi.org/10.1016/j.carbpol.2017.06.087Get rights and content

Highlights

  • Chitosan inhibited VRSA and VREF planktonic growth.

  • VREF was inhibited at 0.025 mg/mL of chitosan.

  • VREF counts were reduced by 6 log of CFU in 30 min.

  • Chitosan inhibited VRSA and VREF biofilm formation and growth.

  • Chitosan inhibited dual-species biofilms of VRSA and S. aureus.

Abstract

Vancomycin-resistant microorganisms are a hurdle that traditional antibiotics struggle to overcome. These difficulties have led to search for new solutions based on natural products. Chitosan has been recognized as an effective antibacterial agent against a vast array of microorganisms including antibiotic resistant ones. As such, this work aimed to evaluate chitosan as an alternative to traditional antibiotics in the management/control of two vancomycin-resistant microorganisms, VRSA and VREF, in planktonic and sessile settings. The results obtained showed that chitosan was highly effective in inhibiting VRSA and VREF planktonic growth and reduced VREF viable counts by 6 log CFU in 30 min. Additionally, chitosan was active upon several phases of VRSA and VREF sessile growth inhibiting adhesion, biofilm formation and dual-species biofilms at concentrations as low as 0.0125 mg/mL. In lieu of these results chitosan shows great potential as a possible alternative for the control of vancomycin-resistant microorganisms in recalcitrant wound infections.

Introduction

The enterococci have an intrinsic resistance to most of the commonly used antibiotics as well as the ability to acquire resistance to current available antibiotics and therefore pose a major therapeutic challenge. Nowadays, Enterococcus spp. have become the second most commonly recovered microorganisms from wound infections and the third most common cause of nosocomial bacteraemia in the United States. Vancomycin resistance is an example of resistance that emerged first in enterococci and has become a major health concern, particularly as the horizontal transfer of vancomycin resistance genes to other Gram-positive organisms led to the emergence of other vancomycin-resistant strains such as vancomycin-resistant Staphylococcus aureus (VRSA) (Cetinkaya, Falk, & Mayhall, 2000; Munita et al., 2013).

Vancomycin resistant Staphylococcus aureus, emerged from methicillin resistant S. aureus (MRSA) clinical isolates, was first detected in 2002 (Sievert et al., 2008). These new strains possessed plasmidic-borne copies of the transposon Tn1546 that were acquired from vancomycin-resistant Enterococcus faecalis (VREF) (Limbago et al., 2014). This link between VRSA and VRSA strains is further accentuated by the unique epidemiological scenario of VRSA, as it is typically found in diabetic wounds were VREF is also present.

The management of skin infections caused by vancomycin-resistant microorganisms is an important clinical challenge since they typically exhibit intrinsic resistance to many other antimicrobial compounds. Moreover, this emergence and dissemination of multidrug-resistant (MDR) strains has led clinics to combine the use of several licensed antibiotics in the hope that they will act synergistically, fact that could lead to further antibiotic resistances and therefore demonstrates the need for new solutions (Adler, Krausz, & Friedman, 2014; Apisarnthanarak et al., 2014, Boucher et al., 2013, Hiraki et al., 2013, Latibeaudiere et al., 2015, Thandar et al., 2016).

Chitosan, given its recognized potential as an antibacterial agent could be an interesting solution for this problem. This polysaccharide is obtained through partial deacetylation of chitin, creating a polysaccharide composed of glucosamine (2-amino-2-deoxy-d-glucose) and N-acetyl glucosamine (2-acetamido-2-deoxy-D–glucose) units linked by β(1  4) bonds. The amino groups in its structure confers chitosan a cationic nature which, at low pH values, grants it biological activity. An example of this is chitosan’s capacity to interact with negatively charged compounds such as proteins or anionic polysaccharides. Despite possessing some practical limitations, namely its insolubility in water, high viscosity and tendency to coagulate proteins at high pH, chitosan’s high biological potential in conjunction with its biocompatibility makes it an apt contender as a localized antibacterial alternative to traditional treatments (Kumar, 2000; Raafat & Sahl, 2009; Rabea et al., 2003).

While S. aureus and E. faecalis susceptibility to chitosan has been previously established (Benhabiles et al., 2012, Chen, Chiang et al., 2012, Fernandes et al., 2008), to the best of our knowledge, no work has focused on chitosan’s activity upon vancomycin resistant strains, which are notorious for their intrinsic resistance. As such, the aim of this work was to assess the antibacterial potential of two chitosan molecular weights (MW) against two vancomycin resistant microorganisms in planktonic and sessile settings and to evaluate if the nature of the antimicrobial resistance mechanism influenced the microorganisms’ sensitivity to chitosan.

Section snippets

Materials

High and low molecular weight chitosan (HMW and LMW, respectively) were obtained from Sigma-Aldrich (St. Louis, USA). High molecular weight chitosan presented a Deacetylation Degree (DD) >75% and a Molecular Weight (MW) of 624 kDa. Low molecular weight chitosan presented a DD between 75 and 85% and a MW of 107 kDa.

Microorganisms

Vancomycin-resistant Staphylococcus aureus and VREF were obtained from American Type Culture Collection (ATCC 700699 and BAA-2365, respectively). VRSA inoculum for antibacterial assays

MIC and MBC determination

When regarding chitosan’s inhibitory activity upon planktonic VRSA and VREF the results obtained (Table 1) showed that chitosan inhibited bacterial growth of both microorganisms at low concentrations, with the average MIC for HMW chitosan being 0.175 mg/mL and for LMW being 0.150 mg/mL. When comparing the sensibility to chitosan of VREF and VRSA, it is possible to see that VREF was significantly (p < 0.05) more sensitive to chitosan than VRSA, as it presented an average MIC of 0.075 mg/mL in

Discussion

Infections caused by vancomycin resistant microorganisms are difficult to manage and treat using only traditional antimicrobials. However, natural products have been employed, either as synergist or as antibacterial agents, with remarkable success in the treatment of vancomycin resistant microorganisms (Akinpelu et al., 2016; Brown, Lister, & May-Dracka, 2014; Hemaiswarya, Kruthiventi, & Doble, 2008).

The existing work relating chitosan and its capacity to inhibit vancomycin resistant strains is

Conclusions

Overall, chitosan showed potential as a possible topical alternative in the management of vancomycin related recalcitrant wound infections, as it was capable of inhibiting both VRSA and VREF in planktonic and sessile settings. However, while promising, these results require further work as the impact of chitosan upon VRSA and VREF single and polymicrobial populations still requires further elucidation.

Acknowledgements

The present work was supported by National Funds from FCT, through project UID/Multi/50016/2013, and from QREN-ANI, through project 17819. Additionally, the author E.M. Costa would like to acknowledge FCT and Aquitex S.A. for his Ph.D. grant SFRH/BDE/103957/2014.

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