Chitosan coated catheters alleviates mixed species biofilms of Staphylococcus epidermidis and Candida albicans

Highlights

• Extracted Chitosan coated catheters disrupts mixed biofilms.

• Bioprospecting marine biowaste to tackle device mediated infections.

• Marine polysaccharides as anti-infectives to mitigate Antimicrobial Resistance.

Abstract

Microorganisms which adhere to the surfaces of indwelling medical implants develop into a sessile microbial community to form monomicrobial or polymicrobial biofilms. Staphylococcus epidermidis and Candida albicans are the most common pathogens co-isolated from device mediated infections. Hence development of catheters coated with anti-fouling substances is of great interest. In this current study, chitosan, extracted from the shells of marine crab Portunus sanguinolentus was coated over the surface of the urinary catheters and checked for its efficacy to inhibit the adherence of both mono and mixed species biofilms. The Extracted Chitosan (EC) coated catheters showed profound activity in reducing the preformed biofilms and the other virulence factors of the pathogens like slime production in S. epidermidis and yeast to hyphal swtich in C. albicans. Furthermore, qPCR analysis showed that EC could downregulate the virulence genes in both the pathogens when grown as monospecies and mixed species biofilms.

Introduction

Indwelling medical implants like urinary catheters, contact lens, stents, prosthetic heart valves and orthopedic joint replacements are colonized by different type of microorganism that forms an intact biofilm on the surface of the devices (Adam, Baillie, & Douglas, 2002). Recent development in the widespread usage of medical devices has given rise to the emergence of biofilm infections in patient with these indwelling medical devices (Wu, Moser, Wang, Hoiby, & Song, 2015). Studies have reported that most of the devices which is used for medical applications may end up causing biofilm mediated infections caused by bacteria which accounts for about 65 % of bacterial infections (Pletzer & Hancock, 2015). Biofilms are the communities of cells which are enclosed inside the extracellular polymeric substances (EPS) (Donlan & Costerton, 2002). Biofilm cells are phenotypically different from those of planktonic cells wherein they display high resistance to host defense mechanisms and it also shows decreased susceptibility to antimicrobial agents (Gebreyohannes, Nyerere, Bii, & Sbhatu, 2019). The cells that are present inside the biofilm matrix are highly resistant to antibiotics because of various types of mechanism adopted by the pathogens. These include phenotypic changes that release secreted virulence factors, cellular communication, complex biofilm architecture and expression of drug efflux pumps (Singh, Kumar singh, Chowdhry, & Singh, 2017).

Human microbiota comprises of a diverse group of microorganisms and henceforth the biofilms generated by these microorganisms are mostly polymicrobial that involves either members of different genera or members of different kingdoms like bacteria and fungi (Donlan & Costerton, 2002). These microorganisms which adhere to the surfaces of medical implants develop into sessile microbial communities as polymicrobial biofilms. It has been reported that due to the diverse population and complexity of multiple pathogens, polymicrobial biofilms significantly contributes to several clinical manifestations (Ribeiro et al., 2016). The interaction between the organisms present in polymicrobial biofilms will lead to co-aggregation, host colonization, antibiotic resistance and quorum sensing phenomenon (Harriott & Noverr, 2011). Among a wide variety of human pathogens, Staphylococcus epidermidis and Candida albicans are the most common bacterial and fungal pathogens often co-isolated from patients suffering with urinary tract infections.

S. epidermidis generally a commensal organism, which is present on the human skin is now considered as one of the opportunistic pathogens and it is also responsible for the frequent cause of nosocomial infections (Centers for Disease Control and Prevention (CDC) (2004)). Among various types of device associated infections, S. epidermidis is a major pathogen responsible for catheter-related urinary tract infections (Khal, Becker, & Loffler, 2016). Attachment of S. epidermidis to abiotic surfaces like catheters is mediated by bacterial cell surface hydrophobicity (Vacheethasanee et al., 1998). Various types of proteins are involved in the surface adhesion of S. epidermidis that includes AltE (adhesion/autolysin protein) and bhp (Biofilm formation protein) and these proteins are likely to contribute to the hydrophobic property of the cell surface (Heilmann, Hussain, Peters, & Gotz, 1997; Tormo, Knecht, Gotz, Lasa, & Penades, 2005).

Candida is one of the opportunistic pathogens that cause systematic infections by colonizing the oral cavity, gastrointestinal tract and the urinary tract (Nobile & Johnson, 2016). Among all the fungal pathogens, Candida albicans has the ability to form biofilms on almost any medical device that includes urinary catheter, prosthetic joints, cardiac valves, pacemakers and urinary stents (Sardi, Scorzoni., Bernardi, Fusco-Almeida, & Giannini, 2012). The formation of C. albicans biofilms on the surface of indwelling urinary catheters are complex in nature due to the presence of micro colonies like yeast, hyphae and pseudo hyphae and these biofilms are also highly resistant to antibiotics like fluconazole and amphotericin B (Rishpana & Kabbin, 2015). Various factors that attribute to the pathogenicity of C. albicans in CAUTIs include adhesion and invasion of the host cells, yeast to hyphal transition, drug efflux, biofilm formation and phenotypic switching (Majumder, Ahmed, Ahemd, & Rahman Khan, 2018).

Among various types of nosocomial infections about 27 % of nosocomial candidal infections are polymicrobial and S. epidermidis is the most common organism isolated from patients suffering by CAUTIs which is found in association with C. albicans (Harriott & Noverr, 2009). It has also been reported that bacteria play vital role in pathogenesis of C. albicans during UTIs by facilitating the adherence of C. albicans to bladder mucosa (Fisher, Kavanagh, Sobel, Kauffman, & Newmann, 2011). It has also been demonstrated that polymicrobial biofilms of C. albicans and S. epidermidis enhances the growth of S. epidermidis and it also increase the resistivity of S. epidermidis to antibiotics (Adam et al., 2002; Azizi, Starks, & Khardori, 2004). The physical interaction of fungi by its hyphal structure with the bacteria increases the tolerance of the polymicrobial biofilms to antimicrobial therapies (Jenkinson & Douglas, 2002). During the polymicrobial infections different types of suppressive and inhibitory interaction are mediated by different molecules depending upon the environment (Nogueira, Sharghi, Kuchler, & Lion, 2019). Studies have demonstrated that polymicrobial infections are more severe and they result in high mortality rate when compared to that of mono-species infections (Kim & Guthmiller, 2003; Peters, Jabra-Rizk, May, Costerton, & Shirtliff, 2012; Wargo & Hogan, 2006). Hence new therapeutic strategies are in need of the hour to target the interaction between the bacterial-fungal virulence factors without developing drug resistance (Dong, Xu, Li, & Zhang, 2000).

Usages of indwelling medical devices like catheters are highly prone to infections due to the entry of the pathogens through the preurethral opening that colonize the lumen of the urinary catheter or along the catheter-urethral interface which is present in the bladder by using various quorum sensing (QS) mediated virulence factors(Warren, 2001).Urinary catheters also facilitate the attachment of the pathogens by providing the surface for attachment to the host cell receptors which are recognized by bacterial adhesion molecules (Jacobsen, Stickler, Mobley, & Shirtliff, 2008). Further the presence of urinary catheter will result in disruption of the normal host defense mechanism which leads to over distension of bladder, and this causes the presence of residual urine in the bladder to support the microbial biofilm growth (Hashmi, Kelly, Rogers, & Gates, 2003). So, in order to overcome biofilm mediated infections that arises due to catheterization, development of catheters with surface modification is of great importance (Ahmed, Zhai, & Gao, 2019).The surface of the catheter can be impregnated with antifouling substances that have hydrophobic, non-charged properties which prevents the attachment of bacteria to the surface (Campoccia, Montanaro, & Arciola, 2013).

In recent days anti-fouling biomaterials coated with natural compounds are of great choice because of its various biological properties like biocompatibility, biodegradability which is required for biomedical applications (Sionkowska et al., 2006). Chitosan a marine polysaccahride is one such natural polymer derived from chitin, which is obtained from the shells of crustaceans by various chemical treatments (Islam, Masum, Mahbub, & Haque, 2011; Sarbon, Sandanamsamy, Kamaruzaman, & Ahmad, 2015). Chitosan can be used for the development of biomaterials because of its properties like hydrophilicity and broad- spectrum antibacterial and antifungal activity (Husain et al., 2017). Chitosan has also proven for its high degree of biocompatibility in animal models and hence it is considered as a versatile compound for the development of implantable biomaterials (Konovalova et al., 2017) Portunus sanguinolentus which is discarded as a waste.

Owing to the alarming issue of antimicrobial resistance among pathogens, natural compounds are actively being pursued to be used as anti-biofilm agents. Chitosan being a natural polymer can be used as an anti-infective that targets the bacterial virulence alone without killing the cells which is considered as a viable option to tackle drug resistant pathogens (Saurav, Costantino, Venturi, & Steindler, 2017). In our previous studies we have shown the antibiofilm and quorum sensing inhibition (QSI) activity of chitosan extracted from the shells of the marine crab against Methicillin Resistant Staphylococcus aureus (MRSA) and urinary tract infection causing pathogens (Rubini et al., 2018, 2019; Rubini, Vishnu varthan, Jayasankari, Vedahari, & Nithyanand, 2020). Formation of mixed species biofilms on urinary catheters is the most common cause for CAUTIs which leads to increased morbidity and mortality. In the present study we hypothesized that modifying the surface of catheters by fabricating the Extracted chitosan (EC) over the surface of urinary catheter will reduce the risk of infections at the site of infection as it will prevent adhesion, aggregation and consequently biofilm formation of the pathogen over the surface of the indwelling device. We further evaluated the antibiofilm activity of chitosan fabricated catheters against mixed species biofilms in a simulated urine milieu. Moreover, chitosan coated catheters will disrupt the bacterial virulence, and hence development of natural anti-infective coated biomaterial will reduce the chance of the pathogens to develop drug resistance.

The most important novelty of this present work lies in providing a site-specific treatment option for UTI without the implementation of antibiotics. Further this study is first of its kind to unravel the molecular interaction and the gene expression pattern in mixed species biofilms in a simulated urine milieu by using qPCR analysis. Another highlight of the present study is its use of chitosan derived from a cheap waste source i.e. (Shells of crustaceans which are discarded as marine biowaste) as an anti-infective. We envision the easy and cheap transformation of a waste into anti-infective compounds would be a solution to two problems at once; (i) exploiting the huge amount of recalcitrant marine biowaste, (ii) development of natural non-antibiotic biomaterial in an era of rising antimicrobial resistance.