Diffusible signal factor signaling controls bioleaching activity and niche protection in the acidophilic, mineral-oxidizing leptospirilli

Bioleaching of metal sulfide ores involves acidophilic microbes that catalyze the chemical dissolution of the metal sulfide bond that is enhanced by attached and planktonic cell mediated oxidation of iron(II)-ions and inorganic sulfur compounds. Leptospirillum spp. often predominate in sulfide mineral-containing environments, including bioheaps for copper recovery from chalcopyrite, as they are effective primary mineral colonizers and oxidize iron(II)-ions efficiently. In this study, we demonstrated a functional diffusible signal factor interspecies quorum sensing signaling mechanism in Leptospirillum ferriphilum and Leptospirillum ferrooxidans that produces (Z)-11-methyl-2-dodecenoic acid when grown with pyrite as energy source. In addition, pure diffusible signal factor and extracts from supernatants of pyrite grown Leptospirillum spp. inhibited biological iron oxidation in various species, and that pyrite grown Leptospirillum cells were less affected than iron grown cells to self inhibition. Finally, transcriptional analyses for the inhibition of iron-grown L. ferriphilum cells due to diffusible signal factor was compared with the response to exposure of cells to N- acyl-homoserine-lactone type quorum sensing signal compounds. The data suggested that Leptospirillum spp. diffusible signal factor production is a strategy for niche protection and defense against other microbes and it is proposed that this may be exploited to inhibit unwanted acidophile species.

Activity of Amphotericin B Formulations and Voriconazole, alone or in combination, against Biofilms of Scedosporium and Fusarium spp.

Scedosporium and Fusarium species are emerging opportunistic pathogens, causing invasive fungal diseases in humans, particularly in immunocompromised patients. Biofilm-related infections are associated with increased morbidity and mortality. We herein assessed the ability of Scedosporium apiospermum ( SA ) and Fusarium solani species complex ( FSSC ) isolates to form biofilms and evaluated the efficacy of deoxycholate amphotericin B (D-AMB), liposomal amphotericin B (L-AMB) and voriconazole (VRC), alone or in combination, against mature biofilms. Biofilm formation was assessed by safranin staining and spectrophotometric measurement of optical density. Planktonic and biofilm damage was assessed by XTT reduction assay. Planktonic cell and biofilm MIC50’s were determined as the minimum concentrations that caused ≥50% fungal damage compared to untreated controls. The combined activity of L-AMB (0.5-32 mg/L) with VRC (0.125-64 mg/L) against biofilms was determined by the checkerboard microdilution method and analyzed by the Bliss independence model. Biofilm MIC50’s of D-AMB and L-AMB against SA isolates were 1 and 2 mg/L and against FSSC isolates were 0.5 and 1 mg/L, respectively. Biofilm MIC50’s of VRC against SA and FSSC were 32 mg/L and >256 mg/L, respectively. Synergistic effects were observed at 2-4 mg/L of L-AMB combined with 4-16 mg/L of VRC against SA biofilms (mean ΔE±standard error: 17% ± 3.7%). Antagonistic interactions were found at 0.5-4 mg/L of L-AMB combined with 0.125-16 mg/L of VRC against FSSC isolates with -28% ± 2%. D-AMB and L-AMB were more efficacious against SA and FSSC biofilms than VRC.

Antibacterial and Antibiofilm Activities of Chloroindoles Against Vibrio parahaemolyticus

Vibrio parahaemolyticus is a food-borne pathogen recognized as the prominent cause of seafood-borne gastroenteritis globally, necessitating novel therapeutic strategies. This study examined the antimicrobial and antivirulence properties of indole and 16 halogenated indoles on V. parahaemolyticus. Among them, 4-chloroindole, 7-chloroindole, 4-iodoindole, and 7-iodoindole effectively inhibited planktonic cell growth, biofilm formation, bacterial motility, fimbrial activity, hydrophobicity, protease activity, and indole production. Specifically, 4-chloroindole at 20 μg/mL inhibited more than 80% of biofilm formation with a minimum inhibitory concentration (MIC) of 50 μg/mL against V. parahaemolyticus and Vibrio harveyi. In contrast, 7-chloroindole inhibited biofilm formation without affecting planktonic cell growth with a MIC of 200 μg/mL. Both chlorinated indoles caused visible damage to the cell membrane, and 4-chloroindole at 100 μg/mL had a bactericidal effect on V. parahaemolyticus within 30 min treatment, which is superior to the effect of tetracycline at the same dose. The quantitative structure-activity relationship (QSAR) analyses revealed that chloro and bromo at positions 4 or 5 of the indole are essential for eradicating the growth of V. parahaemolyticus. These results suggest that halogenated indoles have potential use in antimicrobial and antivirulence strategies against Vibrio species.

Leptothrix cholodnii Response to Nutrient Limitation

Microorganisms are widely utilized for the treatment of wastewater in activated sludge systems. However, the uncontrolled growth of filamentous bacteria leads to bulking and adversely affects wastewater treatment efficiency. To clarify the nutrient requirements for filament formation, we track the growth of a filamentous bacterium, Leptothrix cholodnii SP-6 in different nutrient-limited conditions using a high aspect-ratio microfluidic chamber to follow cell-chain elongation and sheath formation. We find that limitations in Na+, K+, and Fe2+ yield no observable changes in the elongation of cell chains and sheath formation, whereas limitations of C, N, P, or vitamins lead to more pronounced changes in filament morphology; here we observe the appearance of partially empty filaments with wide intercellular gaps. We observe more dramatic differences when SP-6 cells are transferred to media lacking Mg2+ and Ca2+. Loss of Mg2+ results in cell autolysis, while removal of Ca2+ results in the catastrophic disintegration of the filaments. By simultaneously limiting both carbon and Ca2+ sources, we are able to stimulate planktonic cell generation. These findings paint a detailed picture of the ecophysiology of Leptothrix, which may lead to improved control over the unchecked growth of deleterious filamentous bacteria in water purification systems.

Pure-culture and mixed community biofilm responses to carbon-starvation and UV-C exposure /

Biofilms are known to contribute to disease through inherent protective mechanisms and propagation strategies. These multi-cellular systems also play essential roles in numerous environmental processes. The current study investigated the responses of a mixed community biofilm to carbon-starvation, and measured the effects of UV-C on pure-culture biofilms at different stages of maturity by monitoring metabolic and cell yield responses. Carbon dioxide production and biofilm-derived planktonic cell yield were used at the measurement parameters. The mixed community rapidly responded to induced carbon-starvation under continuous flow conditions by remaining metabolically inactive throughout the 96 and 120 h starvation periods, only to promptly return to a metabolically active state upon the reintroduction of carbon. The effects of UV-C on pure-culture biofilms was negligible, with no log activation being achieved, and metabolic activity remaining static.

Pure-culture and mixed community biofilm responses to carbon-starvation and UV-C exposure /

Biofilms are known to contribute to disease through inherent protective mechanisms and propagation strategies. These multi-cellular systems also play essential roles in numerous environmental processes. The current study investigated the responses of a mixed community biofilm to carbon-starvation, and measured the effects of UV-C on pure-culture biofilms at different stages of maturity by monitoring metabolic and cell yield responses. Carbon dioxide production and biofilm-derived planktonic cell yield were used at the measurement parameters. The mixed community rapidly responded to induced carbon-starvation under continuous flow conditions by remaining metabolically inactive throughout the 96 and 120 h starvation periods, only to promptly return to a metabolically active state upon the reintroduction of carbon. The effects of UV-C on pure-culture biofilms was negligible, with no log activation being achieved, and metabolic activity remaining static.

Biofilms' Role in Planktonic Cell Proliferation

The detachment of single cells from biofilms is an intrinsic part of this surface-associated mode of bacterial existence. Pseudomonas sp. strain CT07gfp biofilms, cultivated in microfluidic channels under continuous flow conditions, were subjected to a range of liquid shear stresses (9.42 mPa to 320 mPa). The number of detached planktonic cells was quantified from the effluent at 24-h intervals, while average biofilm thickness and biofilm surface area were determined by confocal laser scanning microscopy and image analysis. Biofilm accumulation proceeded at the highest applied shear stress, while similar rates of planktonic cell detachment was maintained for biofilms of the same age subjected to the range of average shear rates. The conventional view of liquid-mediated shear leading to the passive erosion of single cells from the biofilm surface, disregards the active contribution of attached cell metabolism and growth to the observed detachment rates. As a complement to the conventional conceptual biofilm models, the existence of a biofilm surface-associated zone of planktonic cell proliferation is proposed to highlight the need to expand the traditional perception of biofilms as promoting microbial survival, to include the potential of biofilms to contribute to microbial proliferation.

Biofilms' Role in Planktonic Cell Proliferation

The detachment of single cells from biofilms is an intrinsic part of this surface-associated mode of bacterial existence. Pseudomonas sp. strain CT07gfp biofilms, cultivated in microfluidic channels under continuous flow conditions, were subjected to a range of liquid shear stresses (9.42 mPa to 320 mPa). The number of detached planktonic cells was quantified from the effluent at 24-h intervals, while average biofilm thickness and biofilm surface area were determined by confocal laser scanning microscopy and image analysis. Biofilm accumulation proceeded at the highest applied shear stress, while similar rates of planktonic cell detachment was maintained for biofilms of the same age subjected to the range of average shear rates. The conventional view of liquid-mediated shear leading to the passive erosion of single cells from the biofilm surface, disregards the active contribution of attached cell metabolism and growth to the observed detachment rates. As a complement to the conventional conceptual biofilm models, the existence of a biofilm surface-associated zone of planktonic cell proliferation is proposed to highlight the need to expand the traditional perception of biofilms as promoting microbial survival, to include the potential of biofilms to contribute to microbial proliferation.

Anticariogenic Activity of Oregano against Streptococcus Mutans Biofilm for Investigating its Potential Use as an Oral Care Agent

Being consumed as a spice, Origanum species are also utilized for alternative medical treatment and are therefore of great economic importance. This study examined the effect of Origanum vulgare L. subsp. gracile (OVG) essential oil (EO) against planktonic cell growth and biofilm formation of cariogenic Streptococcus mutansas well as its cytotoxic activity. The antimicrobial potential of EO was screened by broth microdilution method. Crystal violet (CV) biofilm assay method has been used to determine the antimicrobial activitiy and the biofilm formation was also displayed through scanning electron microscopy (SEM). The minimum inhibitory concentration (MIC) value of the EO was determined as 2.5±0.31 mg/mL. The EO acted as an anti-biofilm agent by preventing biofilm formation at sub-MIC concentrations (27.15 to 33.2%). The SEM imaging of biofilms treated with EO showed density and morphological changes when compared to the untreated group. To determine the cytotoxicity, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay wascarried out using the NIH-3T3 murine fibroblast cells andand exhibited IC50 value of 427±1.05 μg/mL.This study has shown that with strong antibacterial and antibofilm capacity, the EO of the OVG can be used as a potentialanticariogenicagent in the oral care products.

Impact of γ-irradiation on biofilm-formation by corrosion-relevant heterotrophic bacteria

At nuclear hazard sites, such as the Chernobyl reactor sarcophagus or Fukushima Nuclear Power Plant, radiation is one of the main factors influencing microbial communities including those involved in microbially influenced corrosion (MIC) of metal structures. By studying the impact of radiation on corrosion-relevant bacteria it may be possible in the future to predict changes in MIC. We believe that the composition and function of natural multi-species biofilms will change when exposed to the stress of ionizing radiation. To address this possibility, biofilm formation by Pseudomonas pseudoalcaligenes and Stenotrophomonas maltophilia were studied after exposure to a range of radiation dosages. Altered planktonic cell morphologies and biofilm architectures on submerged glass surfaces were noted 3 – 7 days after low-doasage sub-lethal irradiation (5.3 Gy) of samples at the micro-colony, macro-colony and mature biofilm stages of development. Furthermore, significant differences in the percentage area covered by biofilms and the release of viable planktonic cells was also noted. These observations suggested that exposure, considered as insignificant levels of irradiation, can be enough to alter biofilm formation of corrosion-relevant bacteria. Such low dosage radiation may have significant impact on soil microbial communities in nuclear hazard sites, potentially altering the MIC of exposed metal structures, their stability and service life of underground metal constructions.