From mono-functional enzymatic coatings to bi-functional coatings to impair Staphylococci adhesion

Despite the remarkable advances in modern healthcare, there are some drawbacks associated to the extended use of medical devices and biomaterial implants when microorganisms are able to reach their surface, forming biofilms and becoming the focus of biomaterial-associated infections (BAI) which are hardly to treat. The growing number of BAI has led to the need of developing novel antibacterial coatings for medical devices. The use of enzymes able to degrade biofilm matrix components, such as proteins and extracellular DNA, represents a promising approach to fight these infections. The first aim of this study was to apply a biologically inspired strategy for covalent immobilization of different enzymes (lysozyme, proteinase K and DNAse I) on clinically relevant substrata (silicone) to obtain mono-functional coatings able to prevent staphylococci adhesion or to kill the adhered bacteria, depending on the enzyme used. The coating developed with the best anti-adhesive properties was afterwards combined with an antimicrobial peptide (colistin), generating a bi-functional coating. Compounds immobilization was mediated by a polydopamine (pDA) coating and the anti-adhesive and antimicrobial performances of the generated surfaces were investigated for a clinical isolate of Staphylococcus aureus using fluorescence microscopy. Results showed that unmodified silicone allowed the adhesion of bacteria without compromising their viability. Silicone modified with polydopamine coating had no significant effect on bacterial attachment and viability. Lysozyme immobilization was not able to reduce bacterial attachment or compromise their viability. On the other hand, proteinase K was able to reduce the percentage of bacterial attachment and a significant fraction of these adhered bacteria was found dead. Regarding the functionalization with DNAse I, these coatings presented the best anti-adhesive properties and since it is know that this enzyme is not cytotoxic, it was further combined with colistin and the bi-functional coating obtained proved to be more effective on reducing the fraction of bacterial attachment. The overall results suggest that the use of coatings functionalized with enzymes is able to degrade biofilm matrix components and their conjugation with antimicrobial peptides presents a promising strategy for creating antibacterial surfaces to be applied in biomaterials for medical devices and implants

Effects of the interactions between glutaraldehyde and the polymeric matrix on the efficacy of the biocide against pseudomonas fluorescens biofilms

Glutaraldehyde (GTA) is a widely used biocide due to its high effectiveness. The experimental work reported here was carried out to assess the effectiveness of GTA in controlling biofilms formed by Pseudomonas fluorescens on stainless steel slides, and to compare efficacy against both planktonic and sessile microrganisms. The tests were performed using two concentrations of GTA (50 and 100mg L-1), biofilms of two ages (7 and 15 d), several pH values (5, 7 and 9) and a range of exposure times (from 0 (control) to 1, 3, 7 and 24 h). The action of GTA on biofilm and planktonic populations was assessed by means of activity tests, zeta potential, and the wet weight of the biofilms. Biofilms were not completely removed after treatment with GTA in any of the conditions studied. The higher GTA concentration was more effective in reducing the bacterial activity of the biofilm. The biocide proved to be more effective for longer exposure times. GTA showed good antimicrobial activity against P. fluorescens in suspension, with higher activity at pH 9. The findings of this study suggest that when GTA is used to control biofilms, it reacts with one of the components of the matrix, the proteins, thereby reducing its antimicrobial action.Instituto de Biotecnologia e Química Fina (IBQF)

Detection of resistant mutants within Pseudomonas aeruginosa colony morphology variants in lung cystic fibrosis environment

Bacterial infections caused mainly by P. aeruginosa are typical of cystic fibrosis (CF) lung disease. Despite the long and aggressive antibiotic therapy, CF patients still died because of these chronic infections. The deprived bacterial eradication is mainly due to several strategies adopted by bacteria to achieve CF airways adaptation and tolerance to antibiotics. Biofilm formation and phenotypic switching are among the most relevant adaptive biological processes. Triggering those processes bacteria have the potential to better survive to CF conditions and antibiotics action. Phenotypic switching provides a source of microbial diversity through switch between two phenotypic states, analogue to a mechanism ON/OFF, without the fitness costs of irreversible mutations. This interchange of states, visible by differential colony morphology, can have serious impact on bacterial virulence, antimicrobial resistance and persistence. The present work aims to investigate the specific colony variants-forming bacteria responsible by typical CF chronic infections. Through isolation and deep characterization of those colony variants, including discriminatory antibiotic susceptibility profiles and virulence characterization, it is intended to determine the mechanisms underlying the inefficiency of antimicrobial therapies of airway CF. P. aeruginosa strains (collection and clinical isolated) and LB medium were used to simulate airway CF infections. Clonal diversification of P. aeruginosa was checked after 24, 48, 72 and 120 hours after media inoculation (or initial infection) by colony morphology observation. The different colonies were further deep characterized in terms of resistance to in-use antibiotics including piperacillin (PRL), aztreonam (ATM), cefepime (FEP) and ceftazidime (CAZ) in Mueller-Hinton agar plates. Biofilm formation was also tested since it is one of the most relevant virulence factors in airway CF context. Data obtained revealed that new colony variants arisen according infection development stage. These new variants were generally more resistant than the normal P. aeruginosa colony morphology. Interestingly, the population were generally composed by colony variants with distinct resistance profiles. Some colony variants exhibited high specific resistance to CAZ, other variants to PRL or FEP. Some colony variants multi-resistant were as well observed. Such heterogeneous behaviours were also observed concerning biofilm formation ability. These results seemed to corroborate the “insurance hypothesis” that posits biodiversity as a mechanism to ensure population survival. In addition, data also highlight novel evidences since it was noted that single and well-differentiated colonies seemed to encompass bacteria with distinct phenotypes. Such evidences emerged when a single colony was used to test its susceptibility and it was observed resistant mutants within the inhibition zones. The inclusion of resistant mutants was not exclusive of small colony variants, well known for its high and diverse antibiotic resistance. Several other colony morphologies showed to encompass resistant mutants in their populations. This evidence might revolutionize the actual colony morphology knowledge. Colony variants have been so far studied and considered as an amount of identical bacteria. So, it is not just need the simple profiling of colony variants but the deep knowledge about bacteria that composed them. The ignorance or under valorisation of these mutants may be the reason of recalcitrance of CF infections. CF antimicrobial therapies must consider those mutants

Role of the polymeric matrix constituents on the performance of a biocide against pseudomonas fluorescens biofilms

The effectiveness of glutaraldehyde – a very common biocide – to control biofilms formed by Pseudomonas fluorescens on stainless steel slides, was investigated. The tests were performed using two concentrations of the biocide (50 and 100 mg Lˉ¹), biofilms of two ages (7 and 15 days), several pH values (5, 7 and 9) and a range of exposure times (from 0 to 1, 3, 7 and 24 hours). The GTA action on biofilm and planktonic populations was assessed by means of activity tests and wet weight of the biofilms. The results showed that biofilms were not completely removed after the treatment with biocide, in all the situations studied. The higher concentration was more effective in reducing the bacterial activity of the biofilm. The biocide proved to be more effective for longer exposure times. GTA showed good antimicrobial activity against P. fluorescens in suspension, with higher activity for pH 9. The findings of this study suggest that when GTA is used to control biofilms, it reacts with one of the components of the matrix – the proteins – thereby reducing its antimicrobial action.Instituto de Biotecnologia e Química Fina (IBQF)

A prospect of current microbial diagnosis methods

An accurate identification and characterization of pathogens is crucial in disease management. The appropriateness and effectiveness of the microbial diagnosis method influence the choice of the antimicrobial agent to be used in the treatment of infection. Traditionally, bacterial diagnosis is based on conventional and culturing-dependent approaches, such as culture and counting methods, generally coupled to morphological and physiological characterization. Currently, rapid technological advances in bacterial identification methods are occurring providing a bewildering wide range of techniques to detect, identify and differentiate bacteria. Molecular methods, such as ELISA and PCR, had introduced great improvements in bacterial identification as they contributed to speed up the analysis and the reduction of handling. However, it has been demonstrated that heterogeneous microbial communities are the main cause of several human infections. This genetic and phenotypic heterogeneity is crucial to microorganisms achieving adaptation to human host, and it might reflect distinct pathogenicity potential. The aforementioned molecular methods and new emergent methods, such as MALDI-TOF MS, have still limitations in full identification and differentiation of microbial heterogeneity. Therefore, a new generation of diagnosis methods able to detect and characterize microbial heterogeneity should be developed. Microbial infections are like dynamic systems and it is essential that diagnosis methods and technologies rapidly evolve to detect and measure changes occurring at individual and population level. This new kind of methods will allow a relevant shift about infection development understanding, as well about microbial mechanisms of resistance to antibiotics and human defences and persistence ability in human host that culminate in better medical decisions about antimicrobial therapy

Control of biofilms using surfactants: persistence and regrowth

The action of the cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS), respectively, a cationic and an anionic surfactant were investigated to control mature biofilms formed under turbulent and laminar flow, by P.fluorescens. The sanitizer action of the surfactants on biofilms was assessed by means of respiratory activity and variation of biofilm mass, immediately, 3, 7 and 12 h after the treatment of the chemicals. The latter experimental times were tested in order to assess the biofilm regrowth. The structure of the biofilms was assessed before and after surfactant treatment by SEM. The results showed that, laminar biofilms were more susceptible to the action of CTAB than those formed under turbulent flow. Concerning SDS, both biofilms showed analogous susceptibility to the surfactants. However, total inactivation of the cells within the biofilms was not achieved for both types of biofilms. CTAB application by itself did not promoted the detachment of biofilms from the surface. Regarding SDS, higher concentrations applied promoted significant biofilm inactivation. Turbulent and laminar flow had analogous susceptibility to SDS application. However, SDS did not promoted the detachment of biofilms from the metal surfaces. The structure of the biofilms was changed after the application of both surfactants. It was found that after CTAB and SDS application, the biofilms recovered its respiratory activity, reaching, in same situations, higher values than the ones found before chemical treatment. The CTAB application promoted similar recovery in the respiratory activity for both biofilms. Concerning biofilm behaviour after SDS treatment, turbulent biofilms showed a higher potential to recover their metabolic activity than laminar biofilms. Biofilm mass did not experienced any significant variation after the treatment, for both surfactants tested. This study highlights the need of care in choosing the correct procedure for biofilm control and the recalcitrant properties of biofilms.Fundação para a Ciência e a Tecnologia (FCT

Motility and biofilm formation ability of isolated vs collection P. aeruginosa : effect of single and combined antimicrobial application

IBB-CEB and FC

OMP proteomic analysis of benzalkonium chloride and ciprofloxacin adapted biofilm cells

Adaptive resistance to antimicrobials has been widely reported in planktonic studied trough phenotypic characterization and proteomic analysis. Concerning biofilm adaptation, the response of biofilm-entrapped cells to chemical stress conditions is not yet well studied. There is evidence that proteins involved in oxidative stress response, cell envelope synthesis, as well as in synthesis of EPS become up- or down-regulated in biofilms, indicating that these altered phenotypes might contribute to antimicrobial tolerance. This work aimed to examine whether exposure of Pseudomonas aeruginosa biofilms to benzalkonium chloride (BC) and ciprofloxacin (CIP) could induce an adaptive response in bacteria. This was attained by inspection of proteome alterations of the outer membrane (OMP) in biofilm cells. Biofilms were formed in 6-well plates for 24h being after submitted to the presence of 0.9 mM BC and 6.0 ug/ml CIP, during 13 days. The obtained biofilm-cells were separated and the OMP extracted. Protein patterns were analysed by 2-DE and gels by the SameSpot software. Biofilm-proteome showed that P. aeruginosa adaptation to BC promoted the down-regulation of 36 OMP and the up-regulation of only one. OMP 2DE of P. aeruginosa adapted to CIP revealed the down-regulation of 29 OMP. Six OMPs were changed in common by both antimicrobials, revealing a possible similar stress response. Proteins identification is in progress. This study highlighted that there might be an OMP regulation when bacteria within biofilms are submitted to chemical adaptation. This particular response to the environment can be one of the causes of the well-known biofilm resistance phenotype

Novel nanoparticle-based therapy to eradicate Pseudomonas aeruginosa infections in cystic fibrosis lungs: dual drug release by cubosomes

Book of Abstracts of CEB Annual Meeting 2017info:eu-repo/semantics/publishedVersio