Study of the Resistance of Staphylococcus aureus Biofilm, Biofilm-Detached Cells, and Planktonic Cells to Microencapsulated Carvacrol Used Alone or Combined with Low-pH Treatment.

Microbial biofilms pose severe problems in the medical field and food industry, as they are the cause of many serious infections and food-borne diseases. The extreme biofilms' resistance to conventional anti-microbial treatments presents a major challenge to their elimination. In this study, the difference in resistance between Staphylococcus aureus DSMZ 12463 biofilms, biofilm-detached cells, and planktonic cells against microcapsules containing carvacrol was assessed. The antimicrobial/antibiofilm activity of low pH disinfection medium containing the microencapsulated carvacrol was also studied. In addition, the effect of low pH on the in vitro carvacrol release from microcapsules was investigated. The minimum inhibitory concentration of microencapsulated carvacrol was 0.625 mg mL-1. The results showed that biofilms exhibited greater resistance to microencapsulated carvacrol than the biofilm-detached cells and planktonic cells. Low pH treatment alone, by hydrochloric acid addition, showed no bactericidal effect on any of the three states of S. aureus strain. However, microencapsulated carvacrol was able to significantly reduce the planktonic cells and biofilm-detached cells below the detection limit (no bacterial counts), and the biofilm by approximatively 3 log CFU mL-1. In addition, results showed that microencapsulated carvacrol combined with low pH treatment reduced biofilm by more than 5 log CFU mL-1. Thus, the use of microencapsulated carvacrol in acidic environment could be a promising approach to combat biofilms from abiotic surfaces.

Antimicrobial and Antibiofilm Potential of Flourensia retinophylla against Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive bacteria with the greatest impact in the clinical area, due to the high rate of infections and deaths reaching every year. A previous scenario is associated with the bacteria's ability to develop resistance against conventional antibiotic therapies as well as biofilm formation. The above situation exhibits the necessity to reach new effective strategies against this pathogen. Flourensia retinophylla is a medicinal plant commonly used for bacterial infections treatments and has demonstrated antimicrobial effect, although its effect against S. aureus and bacterial biofilms has not been investigated. The purpose of this work was to analyze the antimicrobial and antibiofilm potential of F. retinophylla against S. aureus. The antimicrobial effect was determined using an ethanolic extract of F. retinophylla. The surface charge of the bacterial membrane, the K+ leakage and the effect on motility were determined. The ability to prevent and remove bacterial biofilms was analyzed in terms of bacterial biomass, metabolic activity and viability. The results showed that F. retinophylla presents inhibitory (MIC: 250 µg/mL) and bactericidal (MBC: 500 µg/mL) activity against S. aureus. The MIC extract increased the bacterial surface charge by 1.4 times and the K+ concentration in the extracellular medium by 60%. The MIC extract inhibited the motility process by 100%, 61% and 40% after 24, 48 and 72 h, respectively. The MIC extract prevented the formation of biofilms by more than 80% in terms of biomass production and metabolic activity. An extract at 10 × MIC reduced the metabolic activity by 82% and the viability by ≈50% in preformed biofilms. The results suggest that F. retinophylla affects S. areus membrane and the process of biofilm formation and removal. This effect could set a precedent to use this plant as alternative for antimicrobial and disinfectant therapies to control infections caused by this pathogen. In addition, this shrub could be considered for carrying out a purification process in order to identify the compounds responsible for the antimicrobial and antibiofilm effect.

Antibacterial and anti-biofilm activities of antibiotic-free phosphatidylglycerol/docosahexaenoic acid lamellar and non-lamellar liquid crystalline nanoparticles.

Infectious diseases, particularly those associated with biofilms, are challenging to treat due to an increased tolerance to commonly used antibiotics. This underscores the urgent need for innovative antimicrobial strategies. Here, we present an alternative simple-by-design approach focusing on the development of biocompatible and antibiotic-free nanocarriers from docosahexaenoic acid (DHA) that has the potential to combat microbial infections and phosphatidylglycerol (DOPG), which is attractive for use as a biocompatible prominent amphiphilic component of Gram-positive bacterial cell membranes. We assessed the anti-bacterial and anti-biofilm activities of these nanoformulations (hexosomes and vesicles) against S. aureus and S. epidermidis, which are the most common causes of infections on catheters and medical devices by different methods (including resazurin assay, time-kill assay, and confocal laser scanning microscopy on an in vitro catheter biofilm model). In a DHA-concentration-dependent manner, these nano-self-assemblies demonstrated strong anti-bacterial and anti-biofilm activities, particularly against S. aureus. A five-fold reduction of the planktonic and a four-fold reduction of biofilm populations of S. aureus were observed after treatment with hexosomes. The nanoparticles had a bacteriostatic effect against S. epidermidis planktonic cells but no anti-biofilm activity was detected. We discuss the findings in terms of nanoparticle-bacterial cell interactions, plausible alterations in the phospholipid membrane composition, and potential penetration of DHA into these membranes, leading to changes in their structural and biophysical properties. The implications for the future development of biocompatible nanocarriers for the delivery of DHA alone or in combination with other anti-bacterial agents are discussed, as novel treatment strategies of Gram-positive infections, including biofilm-associated infections.

Growth of the Nitrosomonas europaea cells in the biofilm and planktonic growth mode: Responses of extracellular polymeric substances production and transcriptome.

Nitrosomonas europaea, an aerobic ammonia oxidizing bacterium, is responsible for the first and rate-limiting step of the nitrification process, and their ammonia oxidation activities are critical for the biogeochemical cycling and the biological nitrogen removal of wastewater treatment. In the present study, N. europaea cells were cultivated in the inorganic or organic media (the NBRC829 and the nutrient-rich, NR, media, respectively), and the cells proliferated in the form of planktonic and biofilm in those media, respectively. The N. europaea cells in the biofilm growth mode produced larger amounts of the extracellular polymeric substances (EPS), and the composition of the EPS was characterized by the chemical analyses including Fourier transform infrared spectroscopy (FT-IR) and 1H-nuclear magnetic resonance (NMR) measurements. The RNA-Seq analysis of N. europaea in the biofilm or planktonic growth mode revealed that the following gene transcripts involved in central nitrogen metabolisms were abundant in the biofilm growth mode; amo encoding ammonia monooxygenase, hao encoding hydroxylamine dehydrogenase, the gene encoding nitrosocyanine, nirK encoding copper-containing nitrite reductase. Additionally, the transcripts of the pepA and wza involved in the bacterial floc formation and the translocation of EPS, respectively, were also abundant in the biofilm-growth mode. Our study was first to characterize the EPS production and transcriptome of N. europaea in the biofilm and planktonic growth mode.

Untargeted Metabolomics for Unraveling the Metabolic Changes in Planktonic and Sessile Cells of Salmonella Enteritidis ATCC 13076 after Treatment with Lippia origanoides Essential Oil.

Nontyphoidal Salmonella species are one of the main bacterial causes of foodborne diseases, causing a public health problem. In addition, the ability to form biofilms, multiresistance to traditional drugs, and the absence of effective therapies against these microorganisms are some of the principal reasons for the increase in bacterial diseases. In this study, the anti-biofilm activity of twenty essential oils (EOs) on Salmonella enterica serovar Enteritidis ATCC 13076 was evaluated, as well as the metabolic changes caused by Lippia origanoides thymol chemotype EO (LOT-II) on planktonic and sessile cells. The anti-biofilm effect was evaluated by the crystal violet staining method, and cell viability was evaluated through the XTT method. The effect of EOs was observed by scanning electron microscopy (SEM) analysis. Untargeted metabolomics analyses were conducted to determine the effect of LOT-II EO on the cellular metabolome. LOT-II EO inhibited S. Enteritidis biofilm formation by more than 60%, without decreasing metabolic activity. Metabolic profile analysis identified changes in the modulation of metabolites in planktonic and sessile cells after LOT-II EO treatment. These changes showed alterations in different metabolic pathways, mainly in central carbon metabolism and nucleotide and amino acid metabolism. Finally, the possible mechanism of action of L. origanoides EO is proposed based on a metabolomics approach. Further studies are required to advance at the molecular level on the cellular targets affected by EOs, which are promising natural products for developing new therapeutic agents against Salmonella sp. strains.

Antibiofilm action using water-soluble tetra-cationic porphyrin and antibacterial photodynamic therapy against Moraxella spp. from cattle.

Infectious bovine keratoconjunctivitis (IBK) is the most important eye disease in ruminants worldwide. Moraxella bovis and Moraxella bovoculi can form biofilm and are frequently isolated from affected animals. Antimicrobials are used worldwide to treat clinical cases of IBK, although they have limited success in clearing the infection. Therefore, photodynamic therapy using porphyrins as photosensitizing molecules is an alternative method to eliminate microorganisms, including biofilms. We evaluated the antibacterial activity of a zinc(II) metalloporphyrin (ZnTMeP) against M. bovis and M. bovoculi biofilms since this compound can efficiently inactivate planktonic Moraxella spp. This study was carried out with two reference strains of Moraxella spp. (M. bovis: ATCC® 10900 and M. bovoculli: ATCC® BAA1259). The antibacterial activity of 4.0 µM of the ZnTMeP porphyrin was evaluated on forming and consolidate biofilms with three 30-min cycles of white-light exposure for three days. The ZnTMeP porphyrin reduced M. bovis and M. bovoculi biofilm formation. In addition, ZnTMeP partially destroyed consolidated M. bovoculi biofilms in the second white-light irradiation cycle, although the porphyrin had no effect against the consolidated biofilm of M. bovis. Despite the biofilm still not being completely inactivated, our findings are promising and encourage further experiments using the phototherapy protocol.

Anti-Biofilm Effects of Rationally Designed Peptides against Planktonic Cells and Pre-Formed Biofilm of Pseudomonas aeruginosa.

Biofilms are resistant to antibiotics and are a major source of persistent and recurring infections by clinically important pathogens. Drugs used for biofilm-associated infections are limited because biofilm-embedded or biofilm-matrix bacteria are difficult to kill or eradiate. Therefore, many researchers are developing new and effective antibiofilm agents. Among them, antimicrobial peptides have an attractive interest in the development of antibiofilm agents. The present study evaluated the effects of 10 synthetic peptides on growth inhibition, inhibition of biofilm formation, and biofilm elimination in drug-resistant Pseudomonas aeruginosa. The planktonic cell growth and biofilm formation were dose-dependently inhibited by most of the peptides. WIK-14 eliminated preformed biofilm masses by removing carbohydrates, extracellular nucleic acids, proteins, and lipids constituting extracellular polymeric substances. The results demonstrated that WIK-14 and WIKE-14 peptides might provide novel therapeutic drugs to overcome multidrug resistance in biofilm-associated infections.

Metabolomic Profiling of the Responses of Planktonic and Biofilm Vibrio cholerae to Silver Nanoparticles.

Vibrio cholerae causes cholera and can switch between planktonic and biofilm lifeforms, where biofilm formation enhances transmission, virulence, and antibiotic resistance. Due to antibiotic microbial resistance, new antimicrobials including silver nanoparticles (AgNPs) are being studied. Nevertheless, little is known about the metabolic changes exerted by AgNPs on both microbial lifeforms. Our objective was to evaluate the changes in the metabolomic profile of V. cholerae planktonic and biofilm cells in response to sublethal concentrations of AgNPs using MS2 untargeted metabolomics and chemoinformatics. A total of 690 metabolites were quantified among all groups. More metabolites were significantly modulated in planktonic cells (n = 71) compared to biofilm (n = 37) by the treatment. The chemical class profiles were distinct for both planktonic and biofilm, suggesting a phenotype-dependent metabolic response to the nanoparticles. Chemical enrichment analysis showed altered abundances of oxidized fatty acids (FA), saturated FA, phosphatidic acids, and saturated stearic acid in planktonic cells treated with AgNPs, which hints at a turnover of the membrane. In contrast, no chemical classes were enriched in the biofilm. In conclusion, this study suggests that the response of V. cholerae to silver nanoparticles is phenotype-dependent and that planktonic cells experience a lipid remodeling process, possibly related to an adaptive mechanism involving the cell membrane.

UVA as environmental signal for alginate production in Pseudomonas aeruginosa: role of this polysaccharide in the protection of planktonic cells and biofilms against lethal UVA doses.

Pseudomonas aeruginosa is an extremely versatile microorganism that survives in a wide variety of niches. It is capable to respond rapidly to changes in the environment by producing secondary metabolites and virulence factors, including alginate. Alginate is an extracellular polysaccharide that protects the bacteria from antibiotics and oxidative agents, and enhances cell adhesion to solid surfaces in the process of biofilm formation. In the present study, we analyzed the role of alginate in the response of P. aeruginosa to lethal doses of ultraviolet-A (UVA) radiation, the major fraction of solar UV radiation reaching the Earth's surface. We also studied the role of alginate in the context of the adaptive responses generated when P. aeruginosa is exposed to sublethal doses of UVA radiation. The survival studies demonstrated that alginate has a key role in the resistance of P. aeruginosa to the oxidative stress generated by lethal UVA doses, both in planktonic cells and in static biofilms. In addition, the presence of alginate proved to be essential in the occurrence of adaptive responses such as induction of biofilm formation and cross-protection against hydrogen peroxide and sodium hypochlorite, both generated by exposure to low UVA doses. Finally, we demonstrated that the increase of biofilm formation is accompanied by an increase in alginate concentration in the biofilm matrix, possibly through the ppGpp-dependent induction of genes related to alginate regulation (algR and algU) and biosynthesis (algD operon). Given the importance of alginate in biofilm formation and its protective roles, better understanding of the mechanisms associated to its functions and synthesis is relevant, given the normal exposure of P. aeruginosa to UVA radiation and other types of oxidative stresses.

Inhibitory effects of lactobionic acid on Vibrio parahaemolyticus planktonic cells and biofilms.

Vibrio parahaemolyticus is the primary pathogenic bacteria associated with seafood-borne illnesses. Lactobionic acid (LBA) is an organic acid with multiple biological activities that has recently been a focus of interest as an antibacterial agent. The aim of this study was to investigate the inhibitory effects of LBA on Vibrio parahaemolyticus planktonic cells and biofilms. The minimum inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of LBA against Vibrio parahaemolyticus were both identified as 4 mg/mL. LBA exerted antimicrobial effects against planktonic Vibrio parahaemolyticus cells by damaging their membranes, as revealed by reduced intracellular ATP concentrations, increased protein leakage, abnormal cell morphology and diminished membrane integrity after treatment. At 1/16 × MIC and 1/8 × MIC, LBA inhibited biofilm formation and downregulated the expression of some biofilm-related genes, which was confirmed by crystal violet staining, field-emission scanning electron microscopy (FESEM) observations, and real-time quantitative PCR analysis. Moreover, LBA inactivated Vibrio parahaemolyticus cells within biofilms (on polystyrene and stainless steel surfaces), destroyed biofilm structures on stainless steel surfaces, and also reduced the levels of polysaccharides and proteins in the biofilms. Therefore, LBA is a potential low-cost agent that can be used to control planktonic Vibrio parahaemolyticus and its biofilms.

Characterization of biofilm formation and reduction of hexavalent chromium by bacteria isolated from tannery sludge.

Biofilm formation ability of bacteria makes them potential in the field of tannery effluent treatment. However, the hazardous nature of effluent and environmental conditions may disturb the biofilm formation ability of bacteria which ultimately affects their effluent treatment efficiency. Accordingly, we isolated and characterized biofilm-forming bacteria Bacillus vallismortis (MT027009), Bacillus haynesii (MT027008), and Alcaligenes aquatilis (MT027005) from tannery sludge and examined them for biofilm formation under variable environmental conditions. Biofilm formation in tryptic soy broth (TSB) at different incubation times (24-120 h) revealed that the biofilm formation activity of the strain B. haynesii was not affected by incubation time, whereas the increase in biofilm formation was observed in the case of B. vallismortis (28 %) and A. aquatilis (52 %) after 48 h. The medium pH (pH 5.0-9.0) had a limited effect on biofilm formation except in the case of A. aquatilis at pH 5.0 (94 %) and pH 9.0 (80 %). Furthermore, compared to the controls (only TSB), the strains B. vallismortis, B. haynesii, and A. aquatilis showed enhanced biofilm formation in undiluted tannery effluent (28, 33, and 21 %) and 25 mg L-1 Cr(VI) (23 %, 48 % 32 %). The biofilm structure was influenced by Cr(VI) as revealed by scanning electron microscopy (SEM) analysis. The results of Cr(VI) bioreduction studies suggest that bacterial biofilm (60-99 %) has a greater potential to remove Cr(VI) than planktonic cells (43-94 %). The results of the study provide important data on biofilm formation by indigenous bacteria in effluent environment conditions, making them potential isolates for tannery effluent treatment.

codY and pdhA Expression Is Induced in Staphylococcus epidermidis Biofilm and Planktonic Populations With Higher Proportions of Viable but Non-Culturable Cells.

Staphylococcus epidermidis biofilm cells can enter a physiological state known as viable but non-culturable (VBNC), where, despite being alive, they do not grow in conventional laboratory media. As such, the presence of VBNC cells impacts the diagnosis of S. epidermidis biofilm-associated infections. Previous transcriptomics analysis of S. epidermidis strain 9142 biofilms with higher proportions of VBNC cells suggested that the genes pdhA, codY and mazEF could be involved in the induction of the VBNC state. However, it was previously demonstrated that VBNC induction is strain-dependent. To properly assess the role of these genes in VBNC induction, the construction of mutant strains is necessary. Thus, herein, we assessed if VBNC cells could be induced in strain 1457, a strain amenable to genetic manipulation, and if the previously identified genes were involved in the modulation of the VBNC state in this strain. Furthermore, we evaluated the formation of VBNC cells on planktonic cultures. Our results showed that despite being commonly associated with biofilms, the proportion of VBNC cells can be modulated in both biofilm and planktonic cultures and that the expression of codY and pdhA was upregulated under VBNC inducing conditions in both phenotypes. Overall, our study revealed that the formation of VBNC cells in S. epidermidis is independent of the mode of growth and that the genes codY and pdhA seem to be relevant for the regulation of this physiological condition.

Antimicrobial Peptides With Antibiofilm Activity Against Xylella fastidiosa.

Xylella fastidiosa is a plant pathogen that was recently introduced in Europe and is causing havoc to its agriculture. This Gram-negative bacterium invades the host xylem, multiplies, and forms biofilm occluding the vessels and killing its host. In spite of the great research effort, there is no method that effectively prevents or cures hosts from infections. The main control strategies up to now are eradication, vector control, and pathogen-free plant material. Antimicrobial peptides have arisen as promising candidates to combat this bacterium due to their broad spectrum of activity and low environmental impact. In this work, peptides previously reported in the literature and newly designed analogs were studied for its bactericidal and antibiofilm activity against X. fastidiosa. Also, their hemolytic activity and effect on tobacco leaves when infiltrated were determined. To assess the activity of peptides, the strain IVIA 5387.2 with moderate growth, able to produce biofilm and susceptible to antimicrobial peptides, was selected among six representative strains found in the Mediterranean area (DD1, CFBP 8173, Temecula, IVIA 5387.2, IVIA 5770, and IVIA 5901.2). Two interesting groups of peptides were identified with bactericidal and/or antibiofilm activity and low-moderate toxicity. The peptides 1036 and RIJK2 with dual (bactericidal-antibiofilm) activity against the pathogen and moderate toxicity stand out as the best candidates to control X. fastidiosa diseases. Nevertheless, peptides with only antibiofilm activity and low toxicity are also promising agents as they could prevent the occlusion of xylem vessels caused by the pathogen. The present work contributes to provide novel compounds with antimicrobial and antibiofilm activity that could lead to the development of new treatments against diseases caused by X. fastidiosa.

The combination of ultrasound and chlorogenic acid to inactivate Staphylococcus aureus under planktonic, biofilm, and food systems.

This study aimed to investigate the mechanism of different treatments, namely, ultrasound (US), chlorogenic acid (CA), and ultrasound combined with chlorogenic acid (US plus CA) on the inactivation of Staphylococcus aureus planktonic and biofilm cells. Results showed that the combined treatment of US and CA exhibited remarkable synergistic antibacterial and antibiofilm effects. Scanning electron microscopy images indicated that the combined treatment of US and CA caused the most serious damage to the cell morphology. Confocal laser scanning microscopy images revealed that the combined treatment led to sharp increase and severe damage to the permeability of the cell membrane, causing the release of ATP and nucleic acids and decreasing the exopolysaccharide contents in S. aureus biofilm. Finally, the combined treatment of US plus 1% CA for 60 min inactivated S. aureus cells by 1.13 lg CFU/g on mutton. Thus, the combined treatment of US and CA had synergistic effect against S. aureus under planktonic, biofilm, and food systems.

Planktonic and Biofilm-Associated Pseudomonas aeruginosa and Staphylococcus epidermidis Elicit Differential Human Peripheral Blood Cell Responses.

Despite the considerable progress made in recent years, our understanding of the human immune response to microbial biofilms is still poor. The aim of the present study was to compare the in vitro response of human peripheral blood mononuclear cells (PBMC) to biofilms and planktonic cells of Pseudomonas aeruginosa and Staphylococcus epidermidis, two bacterial species particularly relevant in patients with cystic fibrosis or undergoing endovascular catheterization, respectively. PBMC isolated from healthy donors were co-cultured with 24 h-old biofilms or with exponentially growing cells of both species. Following 24 h of co-culture, the expression of early activation markers and the levels of cytokines in the culture supernatants were assessed by flow cytometry, while biofilm biomass and architecture were evaluated by crystal violet staining, CFU count, and confocal microscopy. Around 20% of PBMC was activated in response to both biofilms and planktonic cells of P. aeruginosa. In contrast, planktonic cells of S. epidermidis induced a statistically higher degree of activation than their biofilm counterpart (25% versus 15%; p < 0.01). P. aeruginosa biofilms stimulated pro-inflammatory (TNF-α, IL-1ß, IFN-γ, and IL-6) and anti-inflammatory (IL-10) cytokine production at statistically significant levels higher than its planktonic counterpart, while an opposite trend was observed with S. epidermidis. Differences in the architecture of the biofilms and in the number of PBMC infiltrating the biofilms between the two bacterial species may at least partially explain these findings. Collectively, the results obtained highlighted marked differences in the host-cell response depending on the species and the mode of growth (biofilms versus planktonic cultures), allowing speculations on the different strategies adopted by P. aeruginosa and S. epidermidis to persist in the host during the course of chronic infections.

Effect of Gaseous Ozone on Listeria monocytogenes Planktonic Cells and Biofilm: An In Vitro Study.

Among food-borne pathogens, Listeria monocytogenes continues to pose concerns to food business operators due to its capacity to form biofilm in processing environments. Ozone may be an eco-friendly technology to control microbial contaminations, but data concerning its effect on Listeria monocytogenes biofilm are still limited. In this study, the effect of gaseous ozone at 50 ppm on planktonic cells and biofilm of reference and food-related Listeria monocytogenes strains was evaluated. Ozone caused a reduction in microbial loads of 3.7 ± 0.4 and 3.9 ± 0.4 Log10 CFU/mL after 10 and 30 min, respectively. A complete inactivation of planktonic cells after 6 h of treatment was observed. Biofilm inhibition and eradication treatments (50 ppm, 6 h) resulted in a significant decrease of the biofilm biomass for 59% of the strains tested, whilst a slight dampening of live cell loads in the biofilm state was observed. In conclusion, gaseous ozone is not sufficient to completely counteract Listeria monocytogenes biofilm, but it may be useful as an additional tool to contrast Listeria monocytogenes free-living cells and to improve the existing sanitization procedures in food processing environments.

S. aureus Biofilm Protein Expression Linked to Antimicrobial Resistance: A Proteomic Study.

Antimicrobial resistance (AMR) represents one of the most critical challenges that humanity will face in the following years. In this context, a "One Health" approach with an integrated multidisciplinary effort involving humans, animals and their surrounding environment is needed to tackle the spread of AMR. One of the most common ways for bacteria to live is to adhere to surfaces and form biofilms. Staphylococcus aureus (S. aureus) can form biofilm on most surfaces and in a wide heterogeneity of environmental conditions. The biofilm guarantees the survival of the S. aureus in harsh environmental conditions and represents an issue for the food industry and animal production. The identification and characterization of biofilm-related proteins may provide interesting insights into biofilm formation mechanisms in S. aureus. In this regard, the aims of this study were: (i) to use proteomics to compare proteomes of S. aureus growing in planktonic and biofilm forms in order to investigate the common features of biofilm formation properties of different strains; (ii) to identify specific biofilm mechanisms that may be involved in AMR. The proteomic analysis showed 14 differentially expressed proteins among biofilm and planktonic forms of S. aureus. Moreover, three proteins, such as alcohol dehydrogenase, ATP-dependent 6-phosphofructokinase, and fructose-bisphosphate aldolase, were only differentially expressed in strains classified as high biofilm producers. Differentially regulated catabolites metabolisms and the switch to lower oxygen-related metabolisms were related to the sessile conformation analyzed.

Efficacy of Toluidine Blue-Mediated Antimicrobial Photodynamic Therapy on Candida spp. A Systematic Review.

The effectiveness of antimicrobial photodynamic therapy (aPDT) in the treatment of oral yeast infections was examined many times in recent years. The authors of this review tried to address the question: "Should TBO (toluidine blue ortho)-mediated aPDT be considered a possible alternative treatment for oral candidiasis?". PubMed/Medline and the Cochrane Central Register of Controlled Trials (CEN-TRAL) databases were searched from 1997 up to the 27th of October 2020 using a combination of the following keywords: (Candida OR Candidiasis oral OR Candidosis oral OR denture stomatitis) AND (toluidine blue OR photodynamic therapy OR aPDT OR photodynamic antimicrobial chemotherapy OR PACT OR photodynamic inactivation OR PDI). Animal studies or in vitro studies involving Candida albicans (C. albicans) and/or nonalbicans stain, randomized clinical trials (RCT) involving patients with oral candidiasis or denture stomatitis published solely in English language were included. Candida elimination method in animal, in vitro studies and RCT used was TBO-mediated aPDT. Exactly 393 studies were taken into consideration. Then, after analyzing titles and abstracts of said studies, 361 were excluded. Only 32 studies ended up being selected for in-depth screening, after which 21 of them were included in this study. All studies reported the antifungal effectiveness of aPDT with TBO against C. albicans and non-albicans Candida. In studies conducted with planktonic cells, only one study showed eradication of C. albicans. All others showed partial elimination and only one of them was not statistically significant. Experiments on yeast biofilms, in all cases, showed partial, statistically significant cell growth inhibition and weight reduction (a reduction in the number of cells-mainly hyphae) and the mass of extracellular polymeric substance (EPS). In vivo aPDT mediated by TBO exhibits antifungal effects against oral Candida spp.; however, its clinical effectiveness as a potent therapeutic strategy for oral yeast infections requires further investigation.

In Vitro Newly Isolated Environmental Phage Activity against Biofilms Preformed by Pseudomonas aeruginosa from Patients with Cystic Fibrosis.

As disease worsens in patients with cystic fibrosis (CF), Pseudomonas aeruginosa (PA) colonizes the lungs, causing pulmonary failure and mortality. Progressively, PA forms typical biofilms, and antibiotic treatments determine multidrug-resistant (MDR) PA strains. To advance new therapies against MDR PA, research has reappraised bacteriophages (phages), viruses naturally infecting bacteria. Because few in vitro studies have tested phages on CF PA biofilms, general reliability remains unclear. This study aimed to test in vitro newly isolated environmental phage activity against PA isolates from patients with CF at Bambino Gesù Children's Hospital (OBG), Rome, Italy. After testing in vitro phage activities, we combined phages with amikacin, meropenem, and tobramycin against CF PA pre-formed biofilms. We also investigated new emerging morphotypes and bacterial regrowth. We obtained 22 newly isolated phages from various environments, including OBG. In about 94% of 32 CF PA isolates tested, these phages showed in vitro PA lysis. Despite poor efficacy against chronic CF PA, five selected-lytic-phages (Φ4_ZP1, Φ9_ZP2, Φ14_OBG, Φ17_OBG, and Φ19_OBG) showed wide host activity. The Φ4_ZP1-meropenem and Φ14_OBG-tobramycin combinations significantly reduced CF PA biofilms (p < 0.001). To advance potential combined phage-antibiotic therapy, we envisage further in vitro test combinations with newly isolated phages, including those from hospital environments, against CF PA biofilms from early and chronic infections.

Novel antifungal activity of oligostyrylbenzenes compounds on Candida tropicalis biofilms.

As sessile cells of fungal biofilms are at least 500-fold more resistant to antifungal drugs than their planktonic counterparts, there is a requirement for new antifungal agents. Olygostyrylbenzenes (OSBs) are the first generation of poly(phenylene)vinylene dendrimers with a gram-positive antibacterial activity. Thus, this study aimed to investigate the antifungal activity of four OSBs (1, 2, 3, and 4) on planktonic cells and biofilms of Candida tropicalis. The minimum inhibitory concentration (MIC) for the planktonic population and the sessile minimum inhibitory concentrations (SMIC) were determined. Biofilm eradication was studied by crystal violet stain and light microscopy (LM), and confocal laser scanning microscopy (CLSM) was also utilized in conjunction with the image analysis software COMSTAT. Although all the OSBs studied had antifungal activity, the cationic OSBs were more effective than the anionic ones. A significant reduction of biofilms was observed at MIC and supraMIC50 (50 times higher than MIC) for compound 2, and at supraMIC50 with compound 3. Alterations in surface topography and the three-dimensional architecture of the biofilms were evident with LM and CLSM. The LM analysis revealed that the C. tropicalis strain produced a striking biofilm with oval blastospores, pseudohyphae, and true hyphae. CLSM images showed that a decrease occurred in the thickness of the mature biofilms treated with the OSBs at the most effective concentration for each one. The results obtained by microscopy were supported by those of the COMSTAT program. Our results revealed an antibiofilm activity, with compound 2 being a potential candidate for the treatment of C. tropicalis infections. LAY SUMMARY: This study aimed to investigate the antifungal activity of four OSBs (1, 2, 3, and 4) on planktonic cells and biofilms of Candida tropicalis. Our results revealed an antibiofilm activity, with compound 2 being a potential candidate for the treatment of C. tropicalis infections.