Effects of antimicrobial photodynamic therapy with photodithazine® on methicillin-resistant Staphylococcus aureus (MRSA): Studies in biofilms and experimental model with Galleria mellonella.

Staphylococcus aureus infections are a severe health problem due to the high mortality rate. Conventional treatment of these infections is via the administration of antibiotics. However, its indiscriminate use can select resistant microorganisms. Thus, it is necessary to develop alternatives for antibiotic therapy. Antimicrobial Photodynamic Therapy (aPDT), a therapeutic method that associates a photosensitizer (PS), a light source with adequate wavelength to the PS, interacts with molecular oxygen generating reactive oxygen species responsible for cell inactivation, is a viable alternative. This work aimed to analyze, in vitro and in vivo, the action of aPDT with PS Photodithazine® (PDZ) on the methicillin-resistant S. aureus (MRSA) strain. In the in vitro method, the S. aureus biofilm was incubated with PDZ at 50 and 75 µg.mL-1 for 15 min, adopting the light dose of 25, 50, and 100 J/cm2. In addition, PS interaction, formation of reactive oxygen species (ROS), bacterial metabolism, adhesion, bacterial viability, and biofilm structure were evaluated by scanning electron microscopy. Subsequently, the strain was inoculated into models of Galleria mellonella, and the survival curve, health scale, blood cell analysis, and CFU/mL of S. aureus in the hemolymph were analyzed after aPDT. In the in vitro results, bacterial reduction was observed in the different PDZ concentrations, highlighting the parameters of 75 µg.mL-1 of PDZ and 100 J/cm2. As for in vivo results, aPDT increased survival and stimulated the immune system of G. mellonella infected by S. aureus. aPDT proved effective in both models, demonstrating its potential as an alternative therapy in treating MRSA bacterial infections.

Different Impact of Suspended Al2O3 Nanoparticles on Microbial Communities: Formation of 2D-Networks (Without Humic Acids) or 3D-Colonies (With Humic Acids).

The use of metal-based and, particularly, Al2O3 nanoparticles (Al2O3-NP) for diverse purposes is exponentially growing. However, the growth of such promissory market is not accompanied by a parallel extensive investigation related to the impact of this pollution on groundwater and biological systems. Pseudomonas species, ubiquitous, environmentally critical microbes, frequently respond to stress conditions with diverse strategies that generally include extracellular polymeric substances (EPS) formation. The aim of this study is to report that changes in the aqueous environment, particularly, the addition of Al2O3-NP without and with humic acids, induce different adaptive strategies of Pseudomonas aeruginosa early biofilms. To this purpose, early biofilms were incubated in diluted culture media without (control) and with Al2O3-NP, and with humic acids (HA-control, HA-Al2O3-NP) for 24 h. 3D colonies with EPS strings and isolated bacteria in their surroundings were detected in the control biofilms. Unlikely, an unusual adaptive behaviour was developed in the presence of Al2O3-NP. Bacteria opt to disassemble the 3D arrangements and to implement a 2D network promoting morphological and size changes of bacterial cells (small coccoid shapes). Remarkably, this strategy allows their temporarily non-EPS-depending survival without decreasing the number of cells. This behaviour was not observed with ZnO-NP, HA-Al2O3-NP, or HA-ZnO-NP. Physicochemical analysis revealed that HA were adsorbed on Al2O3-NP and promoted the Al(III) ions complexation. This supports the hypothesis that the reduction of toxicity of Al ions and the 3D colony formation in the presence of HA-Al2O3-NP is promoted by the complexation of the metal ions with HA components.

Antimicrobial photodynamic therapy with curcumin on methicillin-resistant Staphylococcus aureus biofilm.

Healthcare-Associated Infections (HAI) effect approximately 1.5 million individuals worldwide. Among the causes of HAIs in Latin America, Staphylococcus aureus presents a severe danger due to its rapid spread and ease of developing antibiotic resistance. Upon acquiring methicillin resistance, it receives the classification Methicillin-Resistant Staphylococcus aureus (MRSA), responsible for 40 to 60% of HAIs. The increase in resistant microorganisms led to the search for alternative methods, such as antimicrobial Photodynamic Therapy (aPDT), forming Reactive Oxygen Species (ROS), leading bacterial cells to death. The objective of this work was to evaluate in vitro the antimicrobial action of PDT with curcumin in MRSA biofilm. The strains were induced to form biofilm and incubated with curcumin for 20 min, irradiated with LED (Light Emitting Diode) 450 nm, at 110 mW/cm2, 50 J/cm2 for 455 s, subsequently counting the Colony Forming Units, Scanning Electron Microscopy (SEM) micrographs, Confocal Microscopy images, Resazurin dye test, ROS quantification to assess the effect of PDT on biofilm. The results show that PDT with curcumin reduced the biofilm growth of the MRSA strain. In addition, confocal microscopy showed that curcumin was internalized by S. aureus in the cells at the concentration used, and when isolated, curcumin and the irradiation parameter did not show cytotoxicity. The study demonstrated that the PDT in the established parameters reduced the growth of the MRSA strain biofilm, making it a relevant alternative possibility for the inactivation of this strain.

Efficiency of Antimicrobial Photodynamic Therapy with Photodithazine® on MSSA and MRSA Strains.

Staphylococccus aureus is a ubiquitous and opportunistic bacteria associated with high mortality rates. Antimicrobial photodynamic therapy (aPDT) is based on the application of a light source and a photosensitizer that can interact with molecular oxygen, forming Reactive Oxygen Species (ROS) that result in bacterial inactivation. This study aimed to analyze, in vitro, the action of aPDT with Photodithazine® (PDZ) in methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) strains. The strains were incubated with PDZ at 25, 50, 75, and 100 mg/L for 15 min and irradiated with fluences of 25, 50, and 100 J/cm2. The internalization of PDZ was evaluated by confocal microscopy, the bacterial growth by counting the number of colony-forming units, as well as the bacterial metabolic activity post-aPDT and the production of ROS. In both strains, the photosensitizer was internalized; the production of ROS increased when the aPDT was applied; there was a bacterial reduction compared to the control at all the evaluated fluences and concentrations; and, in most parameters, it was obtained complete inactivation with significant difference (p < 0.05). The implementation of aPDT with PDZ in clinical strains of S. aureus has resulted in its complete inactivation, including the MRSA strains.

Environmentally Induced Changes of Commercial Carbon Nanotubes in Aqueous Suspensions. Adaptive Behavior of Bacteria in Biofilms.

The effects of environmental factors such as sunlight irradiation and the presence of humic acid (HA) on the physicochemical properties of commercial multiwall carbon nanotubes (MWCNT) suspended in a simulated inorganic matrix (SIM) and their impacts on bacteria growing in biofilms were evaluated. Both solar irradiation and the presence of HA lead to the dissolution of adsorbed metals on the MWCNT, which are residues of synthesis catalysts. Also, preferential adsorption of certain HA components on the MWCNT induces important modifications in the aliphatic/aromatic relationship of HA components in solution and the generation and release of new moieties. Results demonstrated that the variation of such physicochemical parameters strongly affects the interactions of MWCNT with Pseudomonas aeruginosa sessile bacteria. Thus, the number of attached bacteria increased, and stress responses such as decrease in bacterial size were found in the presence of sunlight-irradiated MWCNT with a particular distribution of extracellular polymeric substances (EPS) strands. A shielding effect was observed when HA was added. It was concluded that physicochemical alterations caused by environmental conditions (with/without irradiation, presence/absence of HA) on MWCNT-containing SIM trigger distinctive adaptive behavior of bacteria in biofilms. This information must be taken into account in the development of biologically assisted treatments for organic metal co-contamination of MWCNT-containing media since MWCNT discharge alters the physicochemical properties and composition of the aqueous environment and the response of the biofilms that interact with it.

Immobilization of alkyl-pterin photosensitizer on silicon surfaces through in situ SN2 reaction as suitable approach for photodynamic inactivation of Staphylococcus aureus.

The tuning of surface properties through functionalization is an important field of research with a broad spectrum of applications. Self-assembled monolayers (SAMs) allow the surface tailoring through the adsorption of molecular layers having the appropriate functional group or precursor group enabling in situ chemical reactions and thus to the incorporation of new functionalities. The latter approach is particularly advantageous when the incorporation of huge groups is needed. In this study, we report the immobilization of pterin moieties on 11-bromoundecyltrichlorosilane-modified silicon substrates based on the in situ replacement of the bromine groups by pterin (Ptr), the parent derivative of pterins, by means of a nucleophilic substitution reaction. The modified surface was structurally characterized through a multi-technique approach, including high-resolution XPS analysis, contact angle measurements, and AFM. The designed synthesis method leads to the functionalization of the silicon surface with two compounds, O-undecyl-Ptr and N-undecyl-Ptr, with a higher proportion of the N-derivative (1:8 ratio). The alkyl-pterins immobilized via the proposed strategy, retain their photochemical properties, being able to inhibit Staphylococcus aureus growth under irradiation (84.3 ± 15.6 % reduction in viable cells). Our results open the possibility for the modification of several materials, such as glass and metal, through the formation of SAMs having the proper head group, thus allowing the design of photosensitive surfaces with potential microbiological self-cleaning properties.

Staphylococcus aureus biofilm eradication by the synergistic effect exerted by PEG-coated silicon dots immobilized in silica films and light irradiation.

Immobilization of PEG-covered silicon dots, PEGSiDs, on glass substrates was performed following a simple strategy involving particle embedding by a sol-gel process forming a silica film on glass slides. The obtained films, denoted as fSiO x -PEGSiD, constitute a water-wettable, strongly supported, photoluminescent glass coating. The films showed high capacity for photosensitizing singlet oxygen (1O2) in the UVA when immersed in water. Staphylococcus aureus colonies formed on fSiO x -PEGSiDs modified glasses revealed the inhibition of bacterial adhesion and bacterial growth leading to the formation of loosely-packed and smaller S. aureus colonies. Upon 350 nm light irradiation of the biofilmed fSiO x -PEGSiDs -modified glasses, S. aureus growth was inhibited and bacteria killed reducing the number of living bacteria by three orders of magnitude. Eradication of attached bacteria was achieved by the synergistic effect exerted by a less adherent fSiO x -PEGSiDs surface that inhibits biofilm formation and the ability of the surface to photosensitize 1O2 to kill bacteria.

Characterization and antimicrobial effect of a bioinspired thymol coating formed on titanium surface by one-step immersion treatment.

OBJECTIVE: To develop an antimicrobial and anti-adherent thymol (TOH)-containing coating on titanium (Ti) by a bioinspired one-step biocompatible method. METHODS: A nanolayer of adsorbed TOH (TOH-NL-Ti) was formed by an easy deep coating method on Ti surface. The treatment consists in a simple one-step immersion process in a TOH-containing solution. Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR), potentiodynamic electrochemical technique, open circuit potential records, Atomic Force Microscopy (AFM) and measurements of TOH release were used to characterize TOH-NL-Ti. Live/Dead staining and plate counting were employed to quantify attached and living adhered bacteria, respectively. Biocompatibility and cytotoxicity in fibroblastic and pre-osteoblastic cell lines were evaluated by acridine orange staining and MTT assay, respectively. RESULTS: TOH adsorbed on TOH-NL-Ti was detected by ATR-FTIR and electrochemical techniques. ATR-FTIR results showed that TOH nanofilms development involves spontaneous production of ketonic structures on Ti surface. AFM analysis revealed that the thickness of the TOH-NL was below 80 nm. Finally, microbiological assays confirmed that TOH-NL-Ti can inhibit the adhesion and kill attached bacteria leading to the eradication of leaving cells on its surface. After 24 h of biocidal release, the antimicrobial effect is also significant (a decrease of 3 orders in the number of attached bacteria). SIGNIFICANCE: The formation of TOH-NL-Ti nanolayer is a simple strategy able to be applied by not specially trained personnel, to reduce implant infection risks, ensure highly effective antimicrobial action and inhibition of bacterial adhesion on Ti surfaces without showing toxic effects for pre-osteoblastic and fibroblastic cells.

Synergistic effect of carboxypterin and methylene blue applied to antimicrobial photodynamic therapy against mature biofilm of Klebsiella pneumoniae.

The control of multidrug-resistant (MDR) bacteria is a growing public health problem, and new strategies are urgently needed for the control of the infections caused by these microorganisms. Notoriously, some MDR microorganisms generate complex structures or biofilms, which adhere to surfaces and confer extraordinary resistance properties that are fundamental challenges to control infections. One of the promising strategies for the control of MDR bacteria is antimicrobial photodynamic therapy (aPDT), which takes advantage of suitable photosensitizers (PS), oxygen and radiation to eradicate microorganisms by the generation of highly reactive species, including reactive oxygen species (ROS) that cause cytotoxic damage and cell death. Habitual aPDT treatments use only methylene blue (MB), but MDR microorganism eradication is not completely achieved. The key result of this study revealed that a combination of two known PSs, 6-carboxypterin (Cap, 100 µM) and MB (2.5-10 µM) exposed to ultraviolet and visible radiation, presents a synergistic effect on the eradication of a MDR Klebsiella pneumoniae strain. Similar effect was observed when the treatment was performed either with planktonic or biofilm growing cells. Moreover, it was found that after treatment the killing action continues in the absence of irradiation leading to the eradication of the microorganisms growing in biofilm. Therefore, the combined aPDT represents a promising strategy for the management of clinical contact surfaces, disinfection of surgical instruments, biofouling and even antimicrobial wastewater treatment.

How Functionalized Surfaces Can Inhibit Bacterial Adhesion and Viability.

Device-associated infections (DAI) remain a serious concern in modern healthcare. Bacterial attachment to a surface is the first step in biofilm formation, which is one of the main causes of DAIs. The development of materials capable of preventing or inhibiting bacterial attachment constitutes a promising approach to deal with this problem. The multifactorial nature of biofilm maturation and antibiotic resistance directs the research for multitargeted or combinatorial therapeutic approaches. One attractive strategy is the modification or the engineering of surfaces in order to provide antiadhesive and/or antimicrobial properties. Currently, several different approaches that involve physical and chemical surface modification deliver some possible alternatives to achieve this goal. The engineered surfaces can be coated with molecules capable of inhibiting the bacterial adhesion or with active agents that kill microorganisms. In addition, surfaces can also be modified in order to be stimuli-responsive, responding to a particular trigger and then delivering the consequent antimicrobial outcome. Here, we review the prevailing strategies to modify surfaces in order to create an antimicrobial surface and discuss how different surface functionalization can affect bacterial adhesion and/or viability.

Highly Stabilized Nanoparticles on Poly-l-Lysine-Coated Oxidized Metals: A Versatile Platform with Enhanced Antimicrobial Activity.

The increasing incidence of infections in implantable devices has encouraged the search for biocompatible antimicrobial surfaces. To inhibit the bacterial adhesion and proliferation on biomaterials, several surface functionalization strategies have been developed. However, most of these strategies lead to bacteriostatic effect and only few of these are able to reach the bactericidal condition. In this work, bactericidal surfaces were designed through the functionalization of titanium surfaces with poly-l-lysine (PLL) as the mediator for the incorporation of antimicrobial silver nanoparticles (AgNPs). This functionalization influences the adsorption of the particles on the substrate impeding the agglomeration observed when bare titanium surfaces are used, leading to a homogeneous distribution of AgNPs on the surfaces. The antimicrobial activity of this surface has been tested against two different strains, namely, Staphylococcus aureus and Pseudomonas aeruginosa. For both strains and different AgNPs sizes, the surface modified with PLL and AgNPs shows a much enhanced antimicrobial activity in comparison with AgNPs deposited on bare titanium. This enhanced antibacterial activity is high enough to reach bactericidal effect, a condition hard to achieve in antimicrobial surfaces. Importantly, the designed surfaces are able to decrease the bacterial viability more than 5 orders with respect to the initial bacterial inoculum. That means that a relative low load of AgNPs on the PLL-modified titanium surfaces reaches 99.999% bacterial death after 24 h. The results of the present study are important to avoid infections in indwelling materials by reinforcing the preventive antibiotic therapy usually dosed throughout the surgical procedure and during the postoperative period.

Self-sterilizing ormosils surfaces based on photo-synzthesized silver nanoparticles.

Medical device-related infections represent a major healthcare complication, resulting in potential risks for the patient. Antimicrobial materials comprise an attractive strategy against bacterial colonization and biofilm proliferation. However, in most cases these materials are only bacteriostatic or bactericidal, and consequently they must be used in combination with other antimicrobials in order to reach the eradication condition (no viable microorganisms). In this study, a straightforward and robust antibacterial coating based on Phosphotungstate Ormosil doped with core-shell (SiO2@TiO2) was developed using sol-gel process, chemical tempering, and Ag nanoparticle photoassisted synthesis (POrs-CS-Ag). The coating was characterized by X-ray Fluorescence Spectroscopy (XRF), Field Emission Scanning Electron Microscopy (FE-SEM), Atomic Force Microscopy (AFM) and X-ray Photoelectron Microscopy (XPS). The silver free coating displays low antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa, in opposition to the silver loaded ones, which are able to completely eradicate these strains. Moreover, the antimicrobial activity of these substrates remains high until three reutilization cycles, which make them a promising strategy to develop self-sterilizing materials, such as POrs-CS-Ag-impregnated fabric, POrs-CS-Ag coated indwelling metals and polymers, among other materials.

Resveratrol enhancement staphylococcus aureus survival under levofloxacin and photodynamic treatments.

Reactive oxygen species (ROS) are an efficient tool to eradicate micro-organisms owing to the capacity of these species to damage almost all types of biomolecules and to kill cells. The increase in mechanisms of antimicrobial resistance has led to the exploration of new strategies to eliminate micro-organisms that involve the production of ROS such as superoxide anion (O2•-) and hydrogen peroxide (H2O2). ROS are produced during several antimicrobial treatments, including antibiotic and photodynamic therapies. Among the natural antioxidants, resveratrol (RSV) is efficient at preventing damage from ROS, and every day more people incorporate it as a dietary or cosmetic supplement. However, the consequences of the administration of RSV during antimicrobial treatment are unknown. To investigate possible antagonistic or synergistic effects of RSV during antibiotic therapy (levofloxacin) or photodynamic therapy (visible radiation and methylene blue), killing of planktonic Staphylococcus aureus bacteria was evaluated in the presence of RSV. The results showed that the antimicrobial capacity of these therapies is significantly diminished when levofloxacin or methylene blue are co-administered with RSV, indicating that consumption of RSV during antimicrobial treatment must be, at least, cautioned. Moreover, considering the ROS antimicrobial activity of antibacterial agents, the topical addition of RSV may also affect the control of pathogens of the human body. The results presented in this article highlight the importance of the evaluation of possible antagonistic effects when an antimicrobial agent with ROS-mediated action is co-administrated with RSV.

Polyethylene glycol-coated blue-emitting silicon dots with improved properties for uses in aqueous and biological environments.

Grafting of polyethylene glycol (PEG) to ultrasmall photoluminescent silicon dots (SiDs) is expected to improve and expand the applications of these particles to aqueous environments and biological systems. Herein we report a novel one-pot synthesis of robust, highly water compatible PEG-coated SiDs (denoted as PEG-SiDs) of (3.3 ± 0.5) nm size. The nanoparticles' synthesis is based on the liquid phase oxidation of magnesium silicide using PEG as reaction media and leading to high PEG density grafting. PEG-SiDs enhanced photophysical, photosensitising, and solution properties in aqueous environments are described and compared to those of 2 nm size PEG-coated SiDs with low PEG density grafting (denoted as PEG-NHSiDs) obtained from a multistep synthesis strategy. PEG-SiDs form highly dispersed suspensions in water showing stable photoluminescence and quantum yields of Φ = 0.13 ± 0.04 at 370 nm excitation in air-saturated suspensions. These particles exhibited the capacity of photosensitising the formation of singlet molecular oxygen, not observed for PEG-NHSiDs. PEG robust shielding of the silicon core luminescent properties is further demonstrated in bio-imaging experiments stressing the strong interaction between PEG-SiDs and Staphylococcus aureus smears by observing the photoluminescence of particles. PEG-SiDs were found to be nontoxic to S. aureus cells at concentrations of 100 mg ml-1, though a bacteriostatic effect on S. aureus biofilms was observed upon UV-A irradiation under conditions where light alone has no effect.

Delivery of fluorophores by calcium phosphate-coated nanoliposomes and interaction with Staphylococcus aureus biofilms.

The delivery capacity and mechanical stability of calcium phosphate (CaP) coated 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) liposomes free and adsorbed on bacterial surface was investigated introducing either acridine orange (AO) or 5,10,15,20-Tetrakis(1-methyl-4-pyridinio)porphyrin (TMP) in the aqueous core of the liposomes. The obtained nanomaterials were thoroughly characterized by electron and optical microscopy and by fluorescence techniques. Distribution of the AO and TMP molecules between the aqueous liposomes core and the outer solution was demonstrated by the band shifts and broadening of the excitation-emission matrices and the modified Stern-Volmer model for fluorescence quenching. In aqueous suspensions, c.a. 40% of AO was released to the outer solution while only a small percentage of TMP was observed to reach the outer liposome surface. The nanoliposomes adhesion capacity and the leaking of fluorophore molecules to Staphylococcus aureus (S. aureus) biofilms were further evaluated. A close interaction between liposomes and S. aureus biofilm was evidenced by TEM and SEM imaging. Epifluorescence experiments demonstrated that CaP-coated liposomes have good biofilm staining capability after two hours incubation of the biofilms with the liposomes, thus supporting an important release of the fluorophores when in contact with the biofilm. Altogether, the obtained results strongly suggest that CaP-coated liposomes are capable of activating drug release when in presence of S. aureus biofilms and smears. The studies herein presented, indicate that CaP-coated liposomes are potential vehicles for the selective delivery of drugs to S. aureus biofilms, as is the case of the singlet oxygen photosensitizer TMP, a well known photodynamic antibacterial agent.

Photodynamic inactivation induced by carboxypterin: a novel non-toxic bactericidal strategy against planktonic cells and biofilms of Staphylococcus aureus.

Microbial related contamination is of major concern and can cause substantial economic losses. Photodynamic inactivation (PDI) has emerged as a suitable approach to inhibit microorganism proliferation. In this work, PDI induced by 6-carboxypterin (Cap), a biocompatible photosensitizer (PS), was analyzed. The growth inhibition of Staphylococcus aureus exposed to artificial UV-A radiation and sunlight in the presence of Cap was investigated. After UV-A irradiation, 50 µM Cap was able to decrease by three orders (with respect to the initial value) the number of S. aureus cells in early biofilms. However, this concentration was 500 times higher than that needed for eradicating planktonic cells. Importantly, under solar exposure, 100 µM Cap was able to suppress sessile bacterial growth. Thus, this strategy is able to exert a bactericidal effect on sessile bacteria and to eradicate planktonic cells by exposing the Cap-containing sample to sunlight.

Self-assembly of flagellin on Au(111) surfaces.

The adsorption of flagellin monomers from Pseudomonas fluorescens on Au(111) has been studied by Atomic Force Microscopy (AFM), Scanning Tunneling Microscopy (STM), X-ray Photoelectron Spectroscopy (XPS), Surface Plasmon Resonance (SPR), and electrochemical techniques. Results show that flagellin monomers spontaneously self-assemble forming a monolayer thick protein film bounded to the Au surface by the more hydrophobic subunit and exposed to the environment the hydrophilic subunit. The films are conductive and allow allocation of electrochemically active cytochrome C. The self-assembled films could be used as biological platforms to build 3D complex molecular structures on planar metal surfaces and to functionalize metal nanoparticles.

Citrate-capped silver nanoparticles showing good bactericidal effect against both planktonic and sessile bacteria and a low cytotoxicity to osteoblastic cells.

A common problem with implants is that bacteria can form biofilms on their surfaces, which can lead to infection and, eventually, to implant rejection. An interesting strategy to inhibit bacterial colonization is the immobilization of silver (Ag) species on the surface of the devices. The aim of this paper is to investigate the action of citrate-capped silver nanoparticles (AgNPs) on clinically relevant Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria in two different situations: (i) dispersed AgNPs (to assess the effect of AgNPs against planktonic bacteria) and (ii) adsorbed AgNPs on titanium (Ti) substrates, a material widely used for implants (to test their effect against sessile bacteria). In both cases, the number of surviving cells was quantified. The small amount of Ag on the surface of Ti has an antimicrobial effect similar to that of pure Ag surfaces. We have also investigated the capability of AgNPs to kill planktonic bacteria and their cytotoxic effect on UMR-106 osteoblastic cells. The minimum bactericidal concentration found for both strains is much lower than the AgNP concentration that leads to cytotoxicity to osteoblasts. Planktonic P. aeruginosa show a higher susceptibility to Ag than S. aureus, which can be caused by the different wall structures, while for sessile bacteria, similar results are obtained for both strains. This can be explained by the presence of extracellular polymeric substances in the early stages of P. aeruginosa biofilm formation. Our findings can be important to improving the performance of Ti-based implants because a good bactericidal action is obtained with very small quantities of Ag, which are not detrimental to the cells involved in the osseointegration process.

Rapid identification of Burkholderia cepacia complex species including strains of the novel Taxon K, recovered from cystic fibrosis patients by intact cell MALDI-ToF mass spectrometry.

Two approaches based on intact cell matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (IC-MALDI-ToF MS) have been evaluated in order to discriminate and identify nine former Burkholderia cepacia complex (Bcc) species, Burkholderia contaminans belonging to the novel Taxon K, Burkholderia gladioli, and the most relevant non-fermentative (NF) Gram-negative rods recovered from cystic fibrosis (CF) sputum cultures. In total, 146 clinical isolates and 26 reference strains were analysed. IC mass spectra were obtained with high reproducibility applying a recently developed inactivation protocol which is based on the extraction of microbial proteins by trifluoroacetic acid (TFA). In a first approach, spectral analysis was carried out by means of a gel-view representation of mass spectra, which turned out to be useful to recognize specific identifying biomarker proteins (SIBPs). A series of prominent mass peaks, mainly assigned to constitutively expressed proteins, were selected as SIBPs for identifications at the genus and species level. Two distinctive mass peaks present in B. contaminans spectra (7501 and 7900 Da) were proposed as SIBPs for the identification of this novel species. A second approach of spectral analysis based on data reduction, feature selection and subsequent hierarchical cluster analysis was used to obtain an objective discrimination of all species analysed. Both complementary modalities of analyzing complex IC-MALDI-ToF MS data open the path towards a rapid, accurate and objective means of routine clinical microbiology diagnosis of pathogens from sputum samples of CF patients.

Fourier transform infrared spectroscopy for rapid identification of nonfermenting gram-negative bacteria isolated from sputum samples from cystic fibrosis patients.

The accurate and rapid identification of bacteria isolated from the respiratory tract of patients with cystic fibrosis (CF) is critical in epidemiological studies, during intrahospital outbreaks, for patient treatment, and for determination of therapeutic options. While the most common organisms isolated from sputum samples are Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, in recent decades an increasing fraction of CF patients has been colonized by other nonfermenting (NF) gram-negative rods, such as Burkholderia cepacia complex (BCC) bacteria, Stenotrophomonas maltophilia, Ralstonia pickettii, Acinetobacter spp., and Achromobacter spp. In the present study, we developed a novel strategy for the rapid identification of NF rods based on Fourier transform infrared spectroscopy (FTIR) in combination with artificial neural networks (ANNs). A total of 15 reference strains and 169 clinical isolates of NF gram-negative bacteria recovered from sputum samples from 150 CF patients were used in this study. The clinical isolates were identified according to the guidelines for clinical microbiology practices for respiratory tract specimens from CF patients; and particularly, BCC bacteria were further identified by recA-based PCR followed by restriction fragment length polymorphism analysis with HaeIII, and their identities were confirmed by recA species-specific PCR. In addition, some strains belonging to genera different from BCC were identified by 16S rRNA gene sequencing. A standardized experimental protocol was established, and an FTIR spectral database containing more than 2,000 infrared spectra was created. The ANN identification system consisted of two hierarchical levels. The top-level network allowed the identification of P. aeruginosa, S. maltophilia, Achromobacter xylosoxidans, Acinetobacter spp., R. pickettii, and BCC bacteria with an identification success rate of 98.1%. The second-level network was developed to differentiate the four most clinically relevant species of BCC, B. cepacia, B. multivorans, B. cenocepacia, and B. stabilis (genomovars I to IV, respectively), with a correct identification rate of 93.8%. Our results demonstrate the high degree of reliability and strong potential of ANN-based FTIR spectrum analysis for the rapid identification of NF rods suitable for use in routine clinical microbiology laboratories.