pharmaceutics Article Dehydroabietic Acid Microencapsulation Potential as Biofilm-Mediated Infections Treatment Iris Neto 1,2, Eva María Domínguez-Martín 1,3 , Epole Ntungwe 1,3 , Catarina P. Reis 2 , Milica Pesic 4 , Célia Faustino 2,* and Patrícia Rijo 1,2,* 1 CBIOS-Research Center for Biosciences & Health Technologies, Universidade Lusófona de Humanidades e Tecnologias, Campo Grande 376, 1749-024 Lisboa, Portugal; [email protected] (I.N.); [email protected] (E.M.D.-M.); [email protected] (E.N.) 2 Instituto de Investigação do Medicamento (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, 1649-003 Lisboa, Portugal; [email protected] 3 Pharmacology Area (Pharmacognosy Laboratory), New Antitumor Compounds: Toxic Action on Leukemia Cells Research Group. Ctra. A2, Department of Biomedical Sciences, Faculty of Pharmacy, Km 33.100—Campus Universitario, University of Alcalá de Henares, Alcalá de Henares, 28805 Madrid, Spain 4 Institute for Biological Research “Sinisa Stankovic”-National Institute of Republic of Serbia, University of Belgrade 142, 11060 Belgrade, Serbia; [email protected] * Correspondence: [email protected] (C.F.); [email protected] (P.R.) Abstract: The antimicrobial activity of dehydroabietic acid (DHA) for its use as an antibiofilm agent was tested in this work. DHA was assayed against a collection of Gram-positive, Gram-negative sensitive and resistant bacteria and yeasts through the minimum inhibitory concentration (MIC), MIC Citation: Neto, I.; with Bioburden challenge, minimum bactericidal concentration (MBC), minimum biofilm inhibitory Domínguez-Martín, E.M.; Ntungwe, concentration (MBIC), MBIC with Bioburden challenge and growth curve studies. Toxicological E.; Reis, C.P.; Pesic, M.; Faustino, C.; studies (Artemia salina, sulforhodamine B (SRB) assay) were done to assess if the compound had Rijo, P. Dehydroabietic Acid Microencapsulation Potential as antimicrobial and not cytotoxic properties. Furthermore, microencapsulation and stability studies Biofilm-Mediated Infections were carried out to evaluate the chemical behavior and stability of DHA. On MIC results, Gram- Treatment. Pharmaceutics 2021, 13, 825. positive bacteria Staphylococcus aureus ATCC 1228 and Mycobacterium smegmatis ATCC 607 presented https://doi.org/10.3390/ a high efficiency (7.81 µg/mL), while on Gram-negative bacteria the highest MIC value of 125 µg/mL pharmaceutics13060825 was obtained by all Klebsiella pneumoniae strains and Escherichia coli isolate strain HSM 303. Bioburden challenge showed that MIC, MBIC and percentage biofilm inhibition (BI) values suffered alterations, Academic Editors: Maria therefore, having higher concentrations. MBIC values demonstrated that DHA has a higher efficiency Camilla Bergonzi, Javier Garcia-Pardo against S. aureus ATCC 43866 with a percentage of BI of 75.13 ± 0.82% at 0.49 µg/mL. Growth curve and Charles M. Heard kinetic profiles of DHA against S. aureus ATCC 25923 were observed to be bacteriostatic. DHA- alginate beads had a average size of 2.37 ± 0.20 and 2.31 ± 0.17 × 103 µm2 with an encapsulation Received: 15 April 2021 efficiency (EE%) around 99.49 ± 0.05%, a protection percentage (PP%) of 60.00 ± 0.05% in the Accepted: 31 May 2021 ± Published: 2 June 2021 gastric environment and a protection efficiency (PE%) around 88.12 0.05% against UV light. In toxicological studies DHA has shown IC50 of 19.59 ± 7.40 µg/mL and a LC50 of 21.71 ± 2.18%. The Publisher’s Note: MDPI stays neutral obtained results indicate that DHA is a promising antimicrobial candidate against a wide range of with regard to jurisdictional claims in bacteria and biofilm formation that must be further explored. published maps and institutional affil- iations. Keywords: dehydroabietic acid; antimicrobial resistance; biofilm; infection; microencapsulation Copyright: © 2021 by the authors. 1. Introduction Licensee MDPI, Basel, Switzerland. Antimicrobial resistance (AMR) is one of the current worldwide problems in develop- This article is an open access article ing and developed countries, because of the misuse and overuse of antibiotics [1]. In fact, distributed under the terms and among the main causes of morbidity and mortality in developing countries are infectious conditions of the Creative Commons diseases, causing the healthcare-acquired infections (HAI) high rates of deaths globally [2]. Attribution (CC BY) license (https:// Actually, it is estimated that there are 700,000 deaths worldwide related to AMR, and that creativecommons.org/licenses/by/ number will rise in 2050 approximately to 10 million deaths [3]. 4.0/). Pharmaceutics 2021, 13, 825. https://doi.org/10.3390/pharmaceutics13060825 https://www.mdpi.com/journal/pharmaceutics Pharmaceutics 2021, 13, x 2 of 18 Currently, some microorganisms’ strains have become common inhabitants in the hospital environments, such as methicillin-resistant Staphylococcus aureus (MRSA), vanco- mycin-resistant S. aureus (VRSA), vancomycin-resistant Enterococcus (VRE) and multi- drug-resistant (MDR) [4–6]. The problem aggravates considering the diminution of new antibiotics development, raising the infections specter that once were treatable may soon become untreatable [7]. This justifies why the World Health Organization (WHO) identi- Pharmaceutics 2021, 13, 825 fied antimicrobial resistance as a public health concern just at the beginning of the2 of21st 18 century [8], as a consequence of the quickly arise and spread of resistant microorganisms to commonly used antibiotics. AMRCurrently, to regularly some microorganisms’ employed antimicrobial strains have agents become is primarily common inhabitants a result of inthe the enzy- hos- pitalmatic environments, inactivation of such the asdrug, methicillin-resistant a shift of its receptorStaphylococcus or target aureussite, altered(MRSA), membrane vancomycin- per- meabilityresistant S. (which aureus (VRSA),prevents vancomycin-resistant the drug to access toEnterococcus the inner of(VRE) the bacteria) and multidrug-resistant or the overex- (MDR)pression [4 of–6 ].efflux The problempumps [9–11]. aggravates Moreover, considering in the thepast, diminution many antibiotics of new antibioticswere developed devel- consideringopment, raising the microorganisms the infections specter grew thatin planktonic once were cultures, treatable but may currently soon become it is clear untreat- that mostable [ 7of]. Thisthe bacteria justifies live why as the complex World Health communi Organizationties known (WHO) as biofilms. identified Undoubtedly, antimicrobial as currentresistance published as a public works health evidenced, concern microbial just at the biofilms beginning are the of theresponsible 21st century for the [8], treat- as a mentconsequence failure to of conventional the quickly ariseantimicrobial and spread ther ofapy, resistant due to microorganismsthe difficulty of these to commonly drugs to penetrateused antibiotics. and destroy biofilm matrix [12–14]. Therefore, to contain and to solve the global increasingAMR toof regularlyAMR, it is employed necessary antimicrobial to invest on agentsnovel isclasses primarily of antimicrobial a result of the agents enzymatic with betterinactivation efficacy of theand drug, mechanisms a shift of of its action, receptor to or be target used site,alone altered or as membrane combination permeability regimens (whichwith another prevents antibiotics. the drug Isolated to access compound to the inners from of theherbal bacteria) medicines or the can overexpression enrich our AMR of therapeuticefflux pumps arsenal [9–11]. used Moreover, solely or in in the combination past, many antibioticswith the current were developedantibiotics consideringavailable in the market. microorganisms grew in planktonic cultures, but currently it is clear that most of the bacteriaOur research live as group complex has recently communities isolated known several as antimicrobial biofilms. Undoubtedly, diterpenes asfrom current Plec- publishedtranthus plants, works namely evidenced, royleanones microbial and biofilms coleons, are the which responsible belong forto the treatmentlargest group failure of naturallyto conventional occurring antimicrobial abietanes therapy,[15–17]. The due abietanes to the difficulty possess of a these characteristic drugs to penetratearomatic ring and C,destroy being biofilm an example, matrix the [12– dehydroabietic14]. Therefore, toac containid (DHA) and (Figure to solve 1). the The global abietic increasing acids are of AMR,strong itantibacterial is necessary agents to invest with on DHA novel generally classes of being antimicrobial the most agentspotent with[18]. better efficacy and mechanismsAntimicrobial of diterpenes action, to like be used DHA alone target or cellular as combination membranes regimens by a combination with another of hydrophobicantibiotics. Isolated and electrostatic compounds adsorption from herbal effects medicines at the membrane/water can enrich our AMRinterface, therapeutic leading toarsenal membrane usedsolely destabilization or in combination and enhanced withthe permeability. current antibiotics Subsequently available, disruption in the market. of the physicalOur integrity research groupof the hasmembrane recently or isolated translocation several into antimicrobial the cell occurs, diterpenes compromising from Plec- cellulartranthus processesplants, namely such as royleanones DNA replication, and coleons, protein which folding belong and synthesis. to the largest This groupmode ofof actionnaturally has occurring been shown abietanes