Fluconazole and biogenic silver nanoparticles-based nano-fungicidal system for highly efficient elimination of multi-drug resistant Candida biofilms

BackgroundAlthough the most widely accepted mechanism of action for polymyxins is related to bacterial lysis via disruption, we hypothesized that this antimicrobial drug class could have other effects on Pseudomonas aeruginosa planktonic and sessile cells. Little is known regarding oxidative burst and zeta potential (ZP) data associated with the interaction between polymyxin B and P. aeruginosa cells. The present study evaluated endogenous reactive oxygen species (ROS) production and changes in the net charges of biofilm and planktonic cells in response to polymyxin B.ResultsPolymyxin B induced concentration-dependent killing at all concentrations tested in planktonic and sessile cells from P. aeruginosa strains. Sublethal concentrations of polymyxin B induced oxidative burst. ROS production was higher in resistant planktonic cells than in biofilm cells but this was not observed for susceptible cells. Moreover, no net surface charge alterations were observed in planktonic cells from a susceptible strain treated with polymyxin B, but a significant increase of ZP was noted in planktonic cells from a resistant strain.ConclusionOxidative burst generated by planktonic and sessile cells from P. aeruginosa strains against polymyxin B indicates that ROS may have an important role in the mechanism of action of this drug. ZP data revealed that electrostatic interactions of the cationic peptide with the anionic surface of the cells are strain-dependent. Therefore, we suggested that the intracellular effects of polymyxin B should be further investigated to understand polymyxin B-induced stress in P. aeruginosa.

The aim of this study was to evaluate miltefosine and four synthetic compounds (TCAN26, TC19, TC106 and TC117) for their in vitro inhibitory activity against Candida albicans planktonic and biofilm cells and investigate whether these compounds are able to inhibit the biofilm formation and to reduce the viability of mature C. albicans biofilm cells. The XTT reduction assay and transmission and scanning electron microscopy were employed to determine the inhibitory effects of the test compounds in comparison with amphotericin B and fluconazole against both planktonic cells and sessile cells in biofilms. C. albicans planktonic cells were susceptible to miltefosine, TCAN26 and TC19, all alkylphospholipid compounds. Miltefosine and TCAN26 present a fungicidal activity with similar values of MIC and minimum fungicidal concentration (MFC), ranging from 2 to 8 mg/L. Cell treatment with sub-inhibitory concentrations of alkylphospholipids induced several ultrastructural alterations. In relation to biofilms, miltefosine reduced formation (38%-71%) and mature biofilms viability (32%-44%), at concentrations of 64 mg/L. TCAN26 also reduced biofilm formation (24%-30%) and mature biofilm viability (15%-20%), at concentrations of 64 mg/L. Although amphotericin B reduced biofilm formation similarly to miltefosine (51%-74%), its activity was lower on mature biofilms (24%-30%). Miltefosine antibiofilm activity was significantly higher than amphotericin B, on both formation and mature biofilms (P<0.05 and P<0.0001, respectively). Fluconazole was the least effective compound tested. Promising antibiofilm activity was displayed by miltefosine and other alkylphosphocholine compounds, which could be considered a putative option for future treatment of candidaemia associated with biofilm formation, although further evaluation in in vivo systems is required.

Silver nanoparticles (AgNPs) have been extensively studied because of their anti-microbial potential. Here, we evaluated the effect of biologically synthesized silver nanoparticles (AgNPbio) alone and in combination with fluconazole (FLC) against planktonic cells and biofilms of FLC-resistant Candida albicans AgNPbio exhibited a fungicidal effect, with a minimal inhibitory concentration (MIC) and fungicidal concentration ranging from 2.17 to 4.35 μg/ml. The combination of AgNPbio and FLC reduced the MIC of FLC around 16 to 64 times against planktonic cells of allC. albicans There was no significant inhibitory effect of AgNPbio on biofilm cells. However, FLC combined with AgNPbio caused a significant dose-dependent decrease in the viability of both initial and mature biofilm. All concentrations of AgNPbio, alone or in combination with FLC, were not cytotoxic to mammalian cells.The results highlight the effectiveness of the combination of AgNPbio with FLC against FLC-resistant C. albicans.

An in vitro overview of the inhibitory effects of selected fluoroquinolones against planktonic and biofilm cells of the methicillin-resistant Staphylococcus aureus (MRSA) strain American type culture collection (ATCC) 43300 and the Pseudomonas aeruginosa strain ATCC 27853 was carried out. Biofilm cells of both strains were less susceptible to the selected antibiotics than their planktonic counterparts. In addition, certain antibiotics were more effective against biofilm cells, while others performed better on the planktonic cells. Against P. aeruginosa, ciprofloxacin was the most potent on both planktonic and biofilm cells, whereas ofloxacin was the least potent on both biofilm and planktonic cells. Moxifloxacin and gatifloxacin were the most potent against both planktonic and biofilm MRSA bacteria, however, not in the same order of activity. Norfloxacin was the least active when tested against both planktonic and biofilm cells. The results of this work are expected to provide insight into the efficacy of various fluoroquinolones against MRSA and Pseudomonas aeruginosa biofilms. This study could form the basis for future clinical studies that could recommend special guidelines for the management of infections that are likely to involve bacteria in their biofilm state.

Biofilm-specific surface properties and protein expression in oral Streptococcus sanguis

Diatoms are abundant in biofilms developed on surfaces immersed in sunlit waters. In both the planktonic and the biofilm mode of growth, diatoms produce carbohydrate polymers which perform several functions including motility, protection, production of macro-aggregates and detoxification. However, little is known about the differences, if any, in the production and characterization at the molecular level of carbohydrates in planktonic and biofilm cells. In order to identify the differences in these two modes of growth, the concentration of total carbohydrates, carbohydrate fractions, neutral carbohydrates, uronic acids and amino sugars in planktonic and biofilm cells of Amphora rostrata were measured. The results showed that the distribution of carbohydrate fractions, uronic acids and amino sugars was different in biofilm and planktonic cells. Cell normalized concentrations of these components were two to five times greater in planktonic cells compared with biofilm cells. The concentrations of glucose and glucosamine decreased, whereas fucose increased in planktonic cells over the period of cultivation. Conversely, the concentrations of glucose and glucosamine increased while that of fucose decreased in attached cells. The study suggests that marked differences exist between the carbohydrates of the planktonic and the biofilm cells of A. rostrata.

Acinetobacter baumannii has emerged as a dangerous opportunistic pathogen, with many strains able to form biofilms and thus cause persistent infections. The aim of the present study was to use high-throughput sequencing techniques to establish complete transcriptome profiles of planktonic (free-living) and sessile (biofilm) forms of A . baumannii ATCC 17978 and thereby identify differences in their gene expression patterns. Collections of mRNA from planktonic (both exponential and stationary phase cultures) and sessile (biofilm) cells were sequenced. Six mRNA libraries were prepared following the mRNA-Seq protocols from Illumina. Reads were obtained in a HiScanSQ platform and mapped against the complete genome to describe the complete mRNA transcriptomes of planktonic and sessile cells. The results showed that the gene expression pattern of A . baumannii biofilm cells was distinct from that of planktonic cells, including 1621 genes over-expressed in biofilms relative to stationary phase cells and 55 genes expressed only in biofilms. These differences suggested important changes in amino acid and fatty acid metabolism, motility, active transport, DNA-methylation, iron acquisition, transcriptional regulation, and quorum sensing, among other processes. Disruption or deletion of five of these genes caused a significant decrease in biofilm formation ability in the corresponding mutant strains. Among the genes over-expressed in biofilm cells were those in an operon involved in quorum sensing. One of them, encoding an acyl carrier protein, was shown to be involved in biofilm formation as demonstrated by the significant decrease in biofilm formation by the corresponding knockout strain. The present work serves as a basis for future studies examining the complex network systems that regulate bacterial biofilm formation and maintenance.

Formation of viable, but putatively non-culturable (VPNC) cells of beer-spoilage lactobacilli growing in biofilms

Toluene degradation kinetics by biofilm and planktonic cells of Pseudomonas putida 54G were compared in this study. Batch degradation of 14C toluene was used to evaluate kinetic parameters for planktonic cells. The kinetic parameters determined for toluene degradation were: specific growth rate, μmax = 10.08 ± 1.2/day; half-saturation constant, KS = 3.98 ± 1.28 mg/L; substrate inhibition constant, KI = 42.78 ± 3.87 mg/L. Biofilm cells, grown on ceramic rings in vapor phase bioreactors, were removed and suspended in batch cultures to calculate 14C toluene degradation rates. Specific activities measured for planktonic and biofilm cells were similar based on toluene degrading cells and total biomass. Long-term toluene exposure reduced specific activities that were based on total biomass for both biofilm and planktonic cells. These results suggest that long-term toluene exposure caused a large portion of the biomass to become inactive, even though the biofilm was not substrate limited. Conversely, specific activities based on numbers of toluene-culturable cells were comparable for both biofilm and planktonically grown cultures. Planktonic cell kinetics are often used in bioreactor models to model substrate degradation and growth of bacteria in biofilms, a procedure we found to be appropriate for this organism. For superior bioreactor design, however, changes in cellular activity that occur during biofilm development should be investigated under conditions relevant to reactor operation before predictive models for bioreactor systems are developed. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 535–546, 1997.

Toluene degradation kinetics by biofilm and planktonic cells of Pseudomonas putida 54G were compared in this study. Batch degradation of (14)C toluene was used to evaluate kinetic parameters for planktonic cells. The kinetic parameters determined for toluene degradation were: specific growth rate, micro(max) = 10.08 +/- 1.2/day; half-saturation constant, K(S) = 3.98 +/- 1.28 mg/L; substrate inhibition constant, K(I) = 42.78 +/- 3.87 mg/L. Biofilm cells, grown on ceramic rings in vapor phase bioreactors, were removed and suspended in batch cultures to calculate (14)C toluene degradation rates. Specific activities measured for planktonic and biofilm cells were similar based on toluene degrading cells and total biomass. Long-term toluene exposure reduced specific activities that were based on total biomass for both biofilm and planktonic cells. These results suggest that long-term toluene exposure caused a large portion of the biomass to become inactive, even though the biofilm was not substrate limited. Conversely, specific activities based on numbers of toluene-culturable cells were comparable for both biofilm and planktonically grown cultures. Planktonic cell kinetics are often used in bioreactor models to model substrate degradation and growth of bacteria in biofilms, a procedure we found to be appropriate for this organism. For superior bioreactor design, however, changes in cellular activity that occur during biofilm development should be investigated under conditions relevant to reactor operation before predictive models for bioreactor systems are developed.

Biofilm characteristics and transcriptomic profiling of Acinetobacter johnsonii defines signatures for planktonic and biofilm cells

Potential spoilage of extended shelf-life (ESL) milk by Bacillus subtilis and Bacillus velezensis

The objective of the present study was to determine the antimicrobial resistance profile of planktonic and biofilm cells of Staphylococcus aureus and coagulase-negative staphylococci (CoNS). Two hundred Staphylococcus spp. strains were studied, including 50 S. aureus and 150 CoNS strains (50 S. epidermidis, 20 S. haemolyticus, 20 S. warneri, 20 S. hominis, 20 S. lugdunensis, and 20 S. saprophyticus). Biofilm formation was investigated by adherence to polystyrene plates. Positive strains were submitted to the broth microdilution method to determine the minimum inhibitory concentration (MIC) for planktonic and biofilm cells and the minimal bactericidal concentration for biofilm cells (MBCB). Forty-nine Staphylococcus spp. strains (14 S. aureus, 13 S. epidermidis, 13 S. saprophyticus, 3 S. haemolyticus, 1 S. hominis, 3 S. warneri, and 2 S. lugdunensis) were biofilm producers. These isolates were evaluated regarding their resistance profile. Determination of planktonic cell MIC identified three (21.4%) S. aureus strains that were resistant to oxacillin and six (42.8%) that were resistant to erythromycin. Among the CoNS, 31 (88.6%) strains were resistant to oxacillin, 14 (40%) to erythromycin, 18 (51.4%) to gentamicin, and 8 (22.8%) to sulfamethoxazole/trimethoprim. None of the planktonic isolates were resistant to vancomycin or linezolid. MICs were 2-, 4-, 8-, and up to 16-fold higher for biofilm cells than for planktonic cells. This observation was more common for vancomycin and erythromycin. The MBCB ranged from 8 to >256 µg/mL for oxacillin, 128 to >128 µg/mL for vancomycin, 256 to >256 µg/mL for erythromycin and gentamicin, >64 µg/mL for linezolid, and 32/608 to >32/608 µg/mL for sulfamethoxazole/trimethoprim. The results showed considerably higher MICs for S. aureus and CoNS biofilm cells compared to planktonic cells. Analysis of MBCM confirmed that even high concentrations of vancomycin were unable to eliminate the biofilms of S. aureus and CoNS species. Linezolid was the most effective drug in inhibiting staphylococci in the biofilm, without an increase in the MIC, when compared to planktonic cells. None of the isolates were resistant to this drug.

Pseudomonas aeruginosa is a versatile opportunistic pathogen which causes a variety of acute and chronic human infections, some of which are associated with the biofilm phenotype of the pathogen. We hypothesize that defining the intracellular metabolome of biofilm cells, compared to that of planktonic cells, will elucidate the metabolic pathways and biomarkers indicative of biofilm inception. Disc-shaped stainless-steel coupons (12.7 mm diameter) were employed as a surface for static biofilm establishment. Each disc was immersed in a well, of a 24-well microtiter plate, containing a 1-mL Lysogeny broth (LB) suspension of P. aeruginosa ATCC 9027, a strain known for its biofilm prolificacy. This setup underwent oxygen-depleted incubation at 37°C for 24 hours to yield hypoxic biofilms and the co-existing static planktonic cells. In parallel, another planktonic phenotype of ATCC 9027 was produced in LB under shaking (200 rpm) incubation at 37°C for 24 hours. Planktonic and biofilm cells were harvested, and the intracellular metabolites were subjected to global untargeted metabolomic analysis using LC-MS technology, where small metabolites (below 1.5 kDa) were selected. Data analysis showed the presence of 324 metabolites that differed (p < 0.05) in abundance between planktonic and biofilm cells, whereas 70 metabolites did not vary between these phenotypes (p > 0.05). Correlation, principal components, and partial least square discriminant analyses proved that the biofilm metabolome is distinctly clustered away from that of the two planktonic phenotypes. Based on the functional enrichment analysis, arginine and proline metabolism were enriched in planktonic cells, but butanoate metabolism was enriched in biofilm cells. Key differential metabolites within the butanoate pathway included acetoacetate, 2,3-butandiol, diacetyl, and acetoin, which were highly upregulated in the biofilm compared to the planktonic cells. Exogenous supplementation of acetoin (2 mM), a critical metabolite in butanoate metabolism, augmented biofilm mass, increased the structural integrity and thickness of the biofilm, and maintained the intracellular redox potential by balancing NADH/NAD+ ratio. In conclusion, P. aeruginosa hypoxic biofilm has a specialized metabolic landscape, and butanoate pathway is a metabolic preference and possibly required for promoting planktonic cells to the biofilm state. The butanoate pathway metabolites, particularly acetoin, could serve as markers for biofilm development.

The human pathogen Listeria monocytogenes forms biofilms that are relatively resistant to chemical sanitizing treatments. Ionizing radiation effectively inactivates planktonic Listeria, but no information is available on the relative efficacy of the process against biofilm-associated Listeria. The irradiation sensitivity of planktonic or biofilm cells was determined for L. monocytogenes ATCC 43256 and ATCC 49594 and a commonly used surrogate Listeria innocua ATCC 51742. Biofilms were formed on sterile glass slides incubated for 48 h at 22°C, 28°C, or 37°C. The cultures were gamma irradiated and the irradiation D 10 value was calculated for each combination of isolate/culture/temperature. The effect of temperature of cultivation on the irradiation sensitivity of both planktonic cells and biofilm cells varied for each of the isolates. Depending on isolate and temperature, biofilm cells were equally sensitive or more sensitive (P < 0.05) to irradiation. D 10 values overall tended to increase with temperature of cultivation for L. monocytogenes 49594 and L. innocua 51742, but tended to decrease with increasing temperature for L. monocytogenes 43256. The D 10 values of the various culture/temperature combinations differed significantly among the isolates examined. Irradiation effectively eliminates both planktonic and biofilm-associated cells. The extent to which the biofilm habitat modifies the antimicrobial efficacy of irradiation is dependent on the specific isolate examined and the temperature at which it forms. This study is the first inquiry to show that biofilm Listeria cells are as sensitive or more sensitive to irradiation compared with planktonic cells and that this response is dependent on biofilm formation conditions.