Antibacterial, Antibiofilm, and Antiadhesive Properties of Different Quaternized Chitosan Derivatives

Abstract

In the era of antimicrobial resistance, the identification of new antimicrobials is a research priority at the global level. In this regard, the attention towards functional antimicrobial polymers, with biomedical/pharmaceutical grade, and exerting anti-infective properties has recently grown. The aim of this study was to evaluate the antibacterial, antibiofilm, and antiadhesive properties of a number of quaternized chitosan derivatives that have displayed significant muco-adhesive properties and wound healing promotion features in previous studies. Low (QAL) and high (QAH) molecular weight quaternized chitosan derivatives were synthetized and further modified with thiol moieties or pendant cyclodextrin, and their antibacterial activity evaluated as minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC). The ability of the derivatives to prevent biofilm formation was assessed by crystal violet staining. Both QAL and QAH derivatives exerted a bactericidal and/or inhibitory activity on the growth of P. aeruginosa and S. epidermidis. The same compounds also showed marked dose-dependent anti-biofilm activity. Furthermore, the high molecular weight derivative (QAH) was used to functionalize titanium plates. The successful functionalization, demonstrated by electron microscopy, was able to partially inhibit the adhesion of S. epidermidis at 6 h of incubation. The shown ability of the chitosan derivatives tested to both inhibit bacterial growth and/or biofilm formation of clinically relevant bacterial species reveals their potential as multifunctional molecules against bacterial infections.

Keywords: antibiofilm; bacteria adhesion; chitosan; chitosan derivatives; functionalized titanium; implant-associated infection; quaternized chitosan.

Scheme 1

Representative structure of the quaternized Ch-derivatives QAH and QAL further functionalized with either pendant thiols protected with 6-mercaptonicotinamide (QAH-Pro and QAL-Pro) or pendant methylated cyclodextrins (QAH-CD and QAL-CD.

Figure 1

Absorbance values recorded after 20 h of incubation of bacterial cells with different concentrations of various Ch-derivatives. Exponentially growing P. aeruginosa (a) or S. epidermidis (b) were incubated in Mueller Hinton broth (MHB) at 37 °C in static conditions for 20 h prior of measuring optical density at 590 nm. Graphs show mean values ± SEM from three independent experiments.

Figure 2

MBC determination of Ch-derivatives against S. epidermidis and P. aeruginosa. Exponentially growing cultures of P. aeruginosa (a) and S. epidermidis (b) were exposed to different concentrations of Ch-derivatives (white numbers; mg/mL) for 24 h. An aliquot of 10 µL from each well was then spot-plated on the surface of agar blood plates and incubated for overnight at 37 °C. MBC was determined as the lower concentration of each compound resulting in the growth of 5 colonies or less per spot. Results obtained in a representative experiment for QAL are shown.

Figure 3

Inhibition of P. aeruginosa ATCC 27853 (a) and S. epidermidis ATCC 35984 (b) biofilm formation by different chitosan derivatives. Overnight cultures of each bacterial species were diluted 1:100 and incubated with different concentrations of four chitosan derivatives. After 18 h of incubation at 37 °C, biofilm formation was quantified by crystal violet staining. The Figure depicts the mean values ± SEM of at least two independent experiments performed in duplicate. Dotted lines indicate a 50% reduction in biofilm formation. Concentrations are in mg/mL. K-: wells without bacteria.

Figure 4

FE-SEM (100,000×) micrographs of titanium samples. (a) Untreated titanium (Ti) and (b) titanium subjected to etching, Ti-Ox, with inset showing nanopits.

Figure 5

FE-SEM (10,000× and 30,000×) images of titanium samples. (a) and (b) titanium as such, Ti; (c) and (d) titanium subjected to etching, Ti-Ox; (e) and (f) titanium with grafted QAH, Ti-QAH.

Figure 6

Micro-ATR of Ti-QAH sample. (a) ATR/FT-IR of the QAH polymer grafted to the titanium surface. (b) Distribution map of the matrix based on the band at 1639 cm⁻¹.

Figure 7

Ability of titanium functionalized with QAH to inhibit adhesion of S. epidermidis. Titanium plates (7 × 7 mm) functionalized with QAH (Ti-QAH) or just etched (Ti-Ox) were incubated with S. epidermidis for 6 h. After gentle washing and sonication to detach adhered bacteria, suspensions were serially diluted and plated for CFU count. ** p < 0.01, Student’s t-test for paired samples.