OBJECTIVE: The antifungal activity was studied on sessile and persister cells (PCs) of Candida tropicalis biofilms of gold nanoparticles (AuNPs) stabilized with cetyltrimethylammonium bromide (CTAB-AuNPs) and those conjugated with cysteine, in combination with Amphotericin B (AmB).
METHODS: The PC model was used and synergistic activity was tested by the checkerboard assay. Biofilms were studied by crystal violet and scanning electron microscopy.
CONCLUSIONS: After the combination of both AuNPs and AmB the biofilm biomass was reduced, with significant differences in architecture being observed with a reduced biofilm matrix. In addition, the CTAB-AuNPs-AmB combination significantly reduced PCs. Understanding how these AuNPs aid in the fight against biofilms and the development of new approaches to eradicate PCs has relevance for chronic infection treatment.

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.

In this study cell surface hydrophobicity and the ability to adhere on abiotic surfaces (polystyrene plates, stainless steel and oak chips) of 10 Candida zemplinina strains were assessed. Moreover, the impact of C. zemplinina cells adhered on oak surface on fermentation kinetics and volatile profile of Montepulciano d\'Abruzzo organic wines was evaluated. All strains showed a hydrophobic nature with a certain affinity for the apolar solvents tested (hexadecane and decane). In agreement with this data strains were able to adhere on abiotic surfaces in a strain dependent way. On polystyrene plates all strains mainly grew as planktonic cells. On stainless steel surfaces sessile cells ranged from 2.6 Log CFU/mL (SB2) to 4.1 Log CFU/mL (SB8), while on oak chips were about 2 log higher ranging from 4.3 Log CFU/mL (SB8) to 6.1 Log CFU/mL (SB10). Candida zemplinina sessile state resulted in an increase of glycerol (from 6.98 g/L to 11.92 g/L) and esters amount (from 55.47 g/L to 91.5 mg/L), and a reduction of ethanol content (from 14.13% to 9.12% v/v). As for esters, methyl vanillate, ethyl isobutyrate, and ethyl isovalerate were present only when C. zemplinina was adhered on oak chips. This study revealed that changes of concentrations in esters and glycerol content reflected the fermentation bioactivity of yeast cells attached on oak chips. Surface-adhered behaviours should be considered in the improvement of strategies for the development of high-quality organic wines and eventually obtain novel wine styles.

Candida tropicalis can undergo multiple forms of phenotypic switching. We have reported a switching system in C. tropicalis that is associated with changes in virulence attributes. We aimed to assess biofilm formation by distinct switch states of C. tropicalis and evaluate whether their sessile cells exhibit altered virulence traits. C. tropicalis strains included the parental phenotype (a clinical isolate) and four switch phenotypes (crepe, rough, revertant of crepe and revertant of rough). Biofilm formation and adhesion capability of sessile cells on polystyrene were assessed through quantification of total biomass. Filamentous forms were characterized by direct counting of sessile cells. A virulence assay was conducted using the Galleria mellonella infection model. Switch variants (crepe and rough) and their revertant counterparts produced higher biofilm biomass (P < 0.05) than the parental strain. Additionally, filamentous forms were enriched among sessile cells of switched strains compared to those observed for sessile cells of the parental strain, with the exception of the revertant of rough. Sessile cells of switched strains showed higher adhesion to polystyrene compared to the parental strain. Sessile cells of the crepe variant and its revertant strain (RC) exhibited higher virulence against G. mellonella larvae than sessile cells of the parental strain. Our findings indicate that switching events in C. tropicalis affect biofilm development and that sessile cells of distinct switch states may exhibit increased adhesion ability and enhanced virulence towards G. mellonella larvae.

Four wild strains of Saccharomyces cerevisiae and the collection strain S. cerevisiae var. boulardii ATCC MYA-796 were used as test organisms to study the effect of some environmental conditions on the formation of biofilm by potentially probiotic yeasts. In a first step, the formation of biofilm was studied in four different media (YPD-Yeast Peptone Glucose; diluted YPD; 2% BP, a medium containing only bacteriological peptone; 2% GLC, a medium containing only glucose). Then, the dilution of YPD was combined with pH and temperature through a mixture design to assess the weight of the interaction of the variables; the experiments were done on S. boulardii and on S. cerevisiae strain 4. The dilution of nutrients generally determined an increased biofilm formation, whereas the effect of pH relied upon the strain. For S. cerevisiae strain 4, the highest level of sessile cells was found at pH 4-5, while S. boulardii experienced an enhanced biofilm formation at pH 6.0. Concerning temperature, the highest biofilm formation was found at 25-30 °C for both strains. The importance of this work lies in its extension of our knowledge of the effect of different environmental conditions on biofilm formation by potentially probiotic S. cerevisiae strains, as a better understanding of this trait could be an important screening tool into the selection of new multifunctional yeasts.

Bacterial biofilms can cause medical problems and issues in technical systems. While a large body of knowledge exists on the phenotypes of planktonic and of sessile cells in mature biofilms, our understanding of what happens when bacteria change from the planktonic to the sessile state is still very incomplete. Fundamental questions are unanswered: for instance, how do bacteria sense that they are in contact with a surface, and what are the very initial cellular responses to surface contact. Here, we review the current knowledge on the signals that bacteria could perceive once they attach to a surface, the signal transduction systems that could be involved in sensing the surface contact and the cellular responses that are triggered as a consequence to surface contact ultimately leading to biofilm formation. Finally, as the main obstacle in investigating the initial responses to surface contact has been the difficulty to experimentally study the dynamic response of single cells upon surface attachment, we also review recent experimental approaches that could be employed to study bacterial surface sensing, which ultimately could lead to an improved understanding of how biofilm formation could be prevented.

Surface disinfection is of utmost importance in the prevention of bacterial infections. This study aims to assess the ability of ten phytochemicals and related derivatives as potentiators of two commonly used biocides-cetyltrimethylammonium bromide (CTAB) and lactic acid (LA). LA in combination with cinnamic, hydrocinnamic, α-methylcinnamic, and α-fluorocinnamic acids had a factional inhibitory concentration index (FICI) ≤ 1 for Escherichia coli and Staphylococcus aureus. Several phytochemicals/derivatives in combination with biocides improved the biocidal efficacy against early sessile bacteria. The most effective combination was LA with allyl cinnamate (2.98 ± 0.76 log CFU.cm-2 reduction) against E. coli. The combination with CTAB was successful for most phytochemicals/derivatives with a maximum bactericidal efficacy against sessile E. coli when combined with allyl cinnamate (2.20 ± 0.07 log CFU.cm-2 reduction) and for S. aureus when combined with α-methylcinnamic acid (1.68 ± 0.30 log CFU.cm-2 reduction). This study highlights the potential of phytochemicals and their derivatives to be used in biocide formulations.

This study compared the outcome of photosensitization on the viability of four different cariogens in planktonic form as well as biofilms in human dentine. Photodynamic therapy was carried out with a gallium aluminium arsenide laser (670 nm wavelength) using Toluidine blue O (TBO) as the photosensitizer. Cariogenic bacteria ( Streptococcus mutans , Lactobacillus casei , Streptococcus salivarius and Actinomyces viscosus ) were exposed to TBO and then to the laser for 1 minute in planktonic suspension. Then, tooth slices previously incubated for 24 hours with broth cultures of broth culture of the four cariogenic organisms were exposed to antimicrobial photosensitization. The control samples consisted of planktonic and sessile cells that were exposed to TBO alone, laser alone and the bacterial cells that were not treated with TBO or laser. The results showed significant reductions in the viability of S. mutans , L. casei and A. viscosus in both planktonic form (to 13%, 30%, and 55%, respectively) and sessile form hosted in dentinal tubules (to 19%, 13% and 52%, respectively), relative to the controls. S. salivarius was the least affected in planktonic (94% viability) and sessile form (86% viability). In conclusion, sensitivity to photosensitization is species-dependent and sessile biofilm cells are affected to the same extent as their planktonic counterparts.
This study compared the outcome of photosensitization on the viability of four different cariogens in planktonic form as well as biofilms in human dentine. Photodynamic therapy was carried out with a gallium aluminium arsenide laser (670 nm wavelength) using Toluidine blue O (TBO) as the photosensitizer. Cariogenic bacteria (Streptococcus mutans, Lactobacillus casei, Streptococcus salivarius and Actinomyces viscosus) were exposed to TBO and then to the laser for 1 minute in planktonic suspension. Then, tooth slices previously incubated for 24 hours with broth cultures of broth culture of the four cariogenic organisms were exposed to antimicrobial photosensitization. The control samples consisted of planktonic and sessile cells that were exposed to TBO alone, laser alone and the bacterial cells that were not treated with TBO or laser. The results showed significant reductions in the viability of S. mutans, L. casei and A. viscosus in both planktonic form (to 13%, 30%, and 55%, respectively) and sessile form hosted in dentinal tubules (to 19%, 13% and 52%, respectively), relative to the controls. S. salivarius was the least affected in planktonic (94% viability) and sessile form (86% viability). In conclusion, sensitivity to photosensitization is species-dependent and sessile biofilm cells are affected to the same extent as their planktonic counterparts.

Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease and rainbow trout fry syndrome, and hence this bacterium is placed among the most important salmonid pathogens in the freshwater aquaculture industry. Since bacteria in biofilms differ substantially from free-living counterparts, this study sought to find the main differences in gene expression between sessile and planktonic states of F. psychrophilum LM-02-Fp and NCMB1947T, with focus on stress-related changes in gene expression occurring during biofilm formation. To this end, biofilm and planktonic samples were analyzed by RNA sequencing to detect differentially expressed candidate genes (DECGs) between the two growth states, and decreasing the effects of interstrain variation by considering only genes with log2-fold changes ≤ -2 and ≥ 2 at Padj-values ≤ 0.001 as DECGs. Overall, 349 genes accounting for ~15% of total number of genes expressed in transcriptomes of F. psychrophilum LM-02-Fp and NCMB1947T (n = 2327) were DECGs between biofilm and planktonic states. Approximately 83 and 81% of all up- and down-regulated candidate genes in mature biofilms, respectively, were assigned to at least one gene ontology term; these were primarily associated with the molecular function term \"catalytic activity.\" We detected a potential stress response in mature biofilms, characterized by a generalized down-regulation of DECGs with roles in the protein synthesis machinery (n = 63, primarily ribosomal proteins) and energy conservation (seven ATP synthase subunit genes), as well as an up-regulation of DECGs involved in DNA repair (ruvC, recO, phrB1, smf, and dnaQ) and oxidative stress response (cytochrome C peroxidase, probable peroxiredoxin, and a probable thioredoxin). These results support the idea of a strategic trade-off between growth-related processes and cell homeostasis to preserve biofilm structure and metabolic functioning. In addition, LDH-based cytotoxicity assays and an intraperitoneal challenge model for rainbow trout fry agreed with the transcriptomic evidence that the ability of F. psychrophilum to form biofilms could contribute to the virulence. Finally, the reported changes in gene expression, as induced by the plankton-to-biofilm transition, represent the first transcriptomic guideline to obtain insights into the F. psychrophilum biofilm lifestyle that could help understand the prevalence of this bacterium in aquaculture settings.

Candida spp. is able to form a biofilm, which is considered resistant to the majority of antifungals used in medicine. The aim of this study was to evaluate the in vitro activity of micafungin against Candida spp. biofilms at different stages of their maturation (2, 6, and 24 h). We assessed the inhibitory effect of micafungin against 78 clinical isolates of Candida spp., growing as planktonic or sessile cells, by widely recommended broth microdilution method. The in vitro effect on sessile cells viability was evaluated by colorimetric reduction assay. All examined strains were susceptible or intermediate to micafungin when growing as planktonic cells. At the early stages of biofilm maturation, from 11 (39.3%) to 20 (100%), tested strains, depending on the species, exhibited sessile minimal inhibitory concentrations (SMICs) of micafungin at ≤ 2 mg/L. For 24-h-old Candida spp. biofilms, from 3 (10.7%) to 20 (100%) of the tested strains displayed SMICs of micafungin at ≤ 2 mg/L. Our findings confirm that micafungin exhibits high potential anti-Candida-biofilm activity. However, this effect does not comprise all Candida species and strains. All strains were susceptible or intermediate to micafungin when growing as planktonic cells, but for biofilms, micafungin displays species- and strain-specific activity. Paradoxical growth of C. albicans and C. parapsilosis was observed. Antifungal susceptibility testing of Candida spp. biofilms would be the best solution, but to date, no reference method is available. The strongest antibiofilm activity of micafungin is observed at early stages of biofilm formation. Possibly, micafungin could be considered as an effective agent for prevention of biofilm-associated candidiasis, especially catheter-related candidaemia.