Application of biofilm dispersion-based nanoparticles in cutting off reinfection.

Bacterial biofilms commonly cause chronic and persistent infections in humans. Bacterial biofilms consist of an inner layer of bacteria and an autocrine extracellular polymeric substance (EPS). Biofilm dispersants (abbreviated as dispersants) have proven effective in removing the bacterial physical protection barrier EPS. Dispersants are generally weak or have no bactericidal effect. Bacteria dispersed from within biofilms (abbreviated as dispersed bacteria) may be more invasive, adhesive, and motile than planktonic bacteria, characteristics that increase the probability that dispersed bacteria will recolonize and cause reinfection. The dispersants should be combined with antimicrobials to avoid the risk of severe reinfection. Dispersant-based nanoparticles have the advantage of specific release and intense penetration, providing the prerequisite for further antibacterial agent efficacy and achieving the eradication of biofilms. Dispersant-based nanoparticles delivered antimicrobial agents for the treatment of diseases associated with bacterial biofilm infections are expected to be an effective measure to prevent reinfection caused by dispersed bacteria. KEY POINTS: • Dispersed bacteria harm and the dispersant's dispersion mechanisms are discussed. • The advantages of dispersant-based nanoparticles in bacteria biofilms are discussed. • Dispersant-based nanoparticles for cutting off reinfection in vivo are highlighted.

Impacts of microplastic and seawater acidification on unicellular red algae: Growth response, photosynthesis, antioxidant enzymes, and extracellular polymer substances.

Microplastics (MPs) pollution and seawater acidification have increasingly become huge threats to the ocean ecosystem. Their impacts on microalgae are of great importance, since microalgae are the main primary producers and play a critical role in marine ecosystems. However, the impact of microplastics and acidification on unicellular red algae, which have a unique phycobiliprotein antenna system, remains unclear. Therefore, the impacts of polystyrene-MPs alone and the combined effects of MPs and seawater acidification on the typical unicellular marine red algae Porphyridium purpureum were investigated in the current study. The result showed that, under normal seawater condition, microalgae densities were increased by 17.75-41.67 % compared to the control when microalgae were exposed to small-sized MPs (0.1 µm) at concentrations of 5-100 mg L-1. In addition, the photosystem II and antioxidant enzyme system were not subjected to negative effects. The large-sized MPs (1 µm) boosted microalgae growth at a low concentration of MPs (5 mg L-1). However, it was observed that microalgae growth was significantly inhibited when MPs concentration increased up to 50 and 100 mg L-1, accompanied by the remarkably reduced Fv/Fm value and the elevated levels of SOD, CAT enzymes, phycoerythrin (PE), and extracellular polysaccharide (EPS). Compared to the normal seawater condition, microalgae densities were enhanced by 52.11-332.56 % under seawater acidification, depending on MPs sizes and concentrations, due to the formed CO2-enrichment condition and appropriate pH range. PE content in microalgal cells was significantly enhanced, but SOD and CAT activities as well as EPS content markedly decreased under acidification conditions. Overall, the impacts of seawater acidification were more pronounced than MPs impacts on microalgae growth and physiological responses. These findings will contribute to a substantial understanding of the effects of MPs on marine unicellular red microalgae, especially in future seawater acidification scenarios.

The extracellular polymeric substances (EPS) accumulation of Spirulina platensis responding to Cadmium (Cd2+) exposure.

Spirulina platensis can secrete extracellular polymeric substances (EPS) helping to protect damage from stress environment, such as cadmium (Cd2+) exposure. However, the responding mechanism of S. platensis and the secreted EPS to exposure of Cd2+ is still unclear. This research focuses on the effects of Cd2+ on the composition and structure of the EPS and the response mechanism of EPS secretion from S. platensis for Cd2+ exposure. S. platensis can produce 261.37 mg·g-1 EPS when exposing to 20 mg·L-1 CdCl2, which was 2.5 times higher than the control group. The S. platensis EPS with and without Cd2+ treatment presented similar and stable irregularly fibrous structure. The monosaccharides composition of EPS in Cd2+ treated group are similar with control group but with different monosaccharides molar ratios, especially for Rha, Gal, Glc and Glc-UA. And the Cd2+ treatment resulted in a remarkable decline of humic acid and fulvic acid content. The antioxidant ability of S. platensis EPS increased significantly when exposed to 20 mg·L-1 CdCl2, which could be helpful for S. platensis protecting damage from high concentration of Cd2+. The transcriptome analysis showed that sulfur related metabolic pathways were up-regulated significantly, which promoted the synthesis of sulfur-containing amino acids and the secretion of large amounts of EPS.

Metal Complexes-A Promising Approach to Target Biofilm Associated Infections.

Microbial biofilms are represented by sessile microbial communities with modified gene expression and phenotype, adhered to a surface and embedded in a matrix of self-produced extracellular polymeric substances (EPS). Microbial biofilms can develop on both prosthetic devices and tissues, generating chronic and persistent infections that cannot be eradicated with classical organic-based antimicrobials, because of their increased tolerance to antimicrobials and the host immune system. Several complexes based mostly on 3D ions have shown promising potential for fighting biofilm-associated infections, due to their large spectrum antimicrobial and anti-biofilm activity. The literature usually reports species containing Mn(II), Ni(II), Co(II), Cu(II) or Zn(II) and a large variety of multidentate ligands with chelating properties such as antibiotics, Schiff bases, biguanides, N-based macrocyclic and fused rings derivatives. This review presents the progress in the development of such species and their anti-biofilm activity, as well as the contribution of biomaterials science to incorporate these complexes in composite platforms for reducing the negative impact of medical biofilms.

Strategies and Approaches for Discovery of Small Molecule Disruptors of Biofilm Physiology.

Biofilms, the predominant growth mode of microorganisms, pose a significant risk to human health. The protective biofilm matrix, typically composed of exopolysaccharides, proteins, nucleic acids, and lipids, combined with biofilm-grown bacteria's heterogenous physiology, leads to enhanced fitness and tolerance to traditional methods for treatment. There is a need to identify biofilm inhibitors using diverse approaches and targeting different stages of biofilm formation. This review discusses discovery strategies that successfully identified a wide range of inhibitors and the processes used to characterize their inhibition mechanism and further improvement. Additionally, we examine the structure-activity relationship (SAR) for some of these inhibitors to optimize inhibitor activity.

Alternatives to Conventional Antibiotic Therapy: Potential Therapeutic Strategies of Combating Antimicrobial-Resistance and Biofilm-Related Infections.

Antibiotics have been denoted as the orthodox therapeutic agents for fighting bacteria-related infections in clinical practices for decades. Nevertheless, overuse of antibiotics has led to the upsurge of species with antimicrobial resistance (AMR) or multi-drug resistance. Bacteria can also grow into the biofilm, which accounts for at least two-thirds of infections. Distinct gene expression and self-produced heterogeneous hydrated extracellular polymeric substance matrix architecture of biofilm contribute to their tolerance and externally manifest as antibiotic resistance. In this review, the difficulties in combating biofilm formation and AMR are introduced, and novel alternatives to antibiotics such as metal nanoparticles and quaternary ammonium compounds, chitosan and its derivatives, antimicrobial peptides, stimuli-responsive materials, phage therapy and other therapeutic strategies, from compounds to hydrogel, from inorganic to biological, are discussed. We expect to provide useful information for the readers who are seeking for solutions to the problem of AMR and biofilm-related infections.

Study of the impact of cultivation conditions and peg surface modification on the in vitro biofilm formation of Staphylococcus aureus and Staphylococcus epidermidis in a system analogous to the Calgary biofilm device.

Introduction. Staphylococcus aureus (SA) and Staphylococcus epidermidis (SE) are the most common pathogens from the genus Staphylococcus causing biofilm-associated infections. Generally, biofilm-associated infections represent a clinical challenge. Bacteria in biofilms are difficult to eradicate due to their resistance and serve as a reservoir for recurring persistent infections.Gap Statement. A variety of protocols for in vitro drug activity testing against staphylococcal biofilms have been introduced. However, there are often fundamental differences. All these differences in methodical approaches can then be reflected in the form of discrepancies between results.Aim. In this study, we aimed to develop optimal conditions for staphylococcal biofilm formation on pegs. The impact of peg surface modification was also studied.Methodology. The impact of tryptic soy broth alone or supplemented with foetal bovine serum (FBS) or human plasma (HP), together with the impact of the inoculum density of bacterial suspensions and the shaking versus the static mode of cultivation, on total biofilm biomass production in SA and SE reference strains was studied. The surface of pegs was modified with FBS, HP, or poly-l-lysine (PLL). The impact on total biofilm biomass was evaluated using the crystal violet staining method and statistical data analysis.Results. Tryptic soy broth supplemented with HP together with the shaking mode led to crucial potentiation of biofilm formation on pegs in SA strains. The SE strain did not produce biofilm biomass under the same conditions on pegs. Preconditioning of peg surfaces with FBS and HP led to a statistically significant increase in biofilm biomass formation in the SE strain.Conclusion. Optimal cultivation conditions for robust staphylococcal biofilm formation in vitro might differ among different bacterial strains and methodical approaches. The shaking mode and supplementation of cultivation medium with HP was beneficial for biofilm formation on pegs for SA (ATCC 29213) and methicillin-resistant SA (ATCC 43300). Peg conditioning with HP and PLL had no impact on biofilm formation in either of these strains. Peg coating with FBS showed an adverse effect on the biofilm formation of these strains. By contrast, there was a statistically significant increase in biofilm biomass production on pegs coated with FBS and HP for SE (ATCC 35983).

Destruction of Staphylococcus aureus biofilms by combining an antibiotic with subtilisin A or calcium gluconate.

In S. aureus biofilms, bacteria are embedded in a matrix of extracellular polymeric substances (EPS) and are highly tolerant to antimicrobial drugs. We thus sought to identify non-antibiotic substances with broad-spectrum activity able to destroy the EPS matrix and enhance the effect of antibiotics on embedded biofilm bacteria. Among eight substances tested, subtilisin A (0.01 U/mL) and calcium gluconate (CaG, Ca2+ 1.25 mmol/L) significantly reduced the biomass of biofilms formed by at least 21/24 S. aureus isolates. Confocal laser scanning microscopy confirmed that they both eliminated nearly all the proteins and PNAG from the matrix. By contrast, antibiotics alone had nearly no effect on biofilm biomass and the selected one (oxytetracycline-OTC) could only slightly reduce biofilm bacteria. The combination of OTC with CaG or subtilisin A led to an additive reduction (average of 2 log10 CFU/mL) of embedded biofilm bacteria on the isolates susceptible to OTC (MBC < 10 µg/mL, 11/24). Moreover, these two combinations led to a reduction of the embedded biofilm bacteria higher than 3 log10 CFU/mL for 20-25% of the isolates. Further studies are now required to better understand the factors that cause the biofilm produced by specific isolates (20-25%) to be susceptible to the combinations.

Interactions of plasma-activated water with biofilms: inactivation, dispersal effects and mechanisms of action.

Biofilms have several characteristics that ensure their survival in a range of adverse environmental conditions, including high cell numbers, close cell proximity to allow easy genetic exchange (e.g., for resistance genes), cell communication and protection through the production of an exopolysaccharide matrix. Together, these characteristics make it difficult to kill undesirable biofilms, despite the many studies aimed at improving the removal of biofilms. An elimination method that is safe, easy to deliver in physically complex environments and not prone to microbial resistance is highly desired. Cold atmospheric plasma, a lightning-like state generated from air or other gases with a high voltage can be used to make plasma-activated water (PAW) that contains many active species and radicals that have antimicrobial activity. Recent studies have shown the potential for PAW to be used for biofilm elimination without causing the bacteria to develop significant resistance. However, the precise mode of action is still the subject of debate. This review discusses the formation of PAW generated species and their impacts on biofilms. A focus is placed on the diffusion of reactive species into biofilms, the formation of gradients and the resulting interaction with the biofilm matrix and specific biofilm components. Such an understanding will provide significant benefits for tackling the ubiquitous problem of biofilm contamination in food, water and medical areas.

Effects of biotin on promoting anammox bacterial activity.

Anaerobic ammonium oxidation (anammox) bacteria significantly improve the efficiency and reduce cost of nitrogen removal in wastewater treatment plants. However, their slow growth and vulnerable activity limit the application of anammox technology. In this paper, the enhancement of biotin on the nitrogen removal activity of anammox bacteria in short-term batch experiments was studied. We found that biotin played a significant role in promoting anammox activity within a biotin concentration range of 0.1-1.5 mg/L. At a biotin concentration of 1.0 mg/L, the total nitrogen removal rate (NRR) increased by 112%, extracellular polymeric substance (EPS) secretion and heme production significantly improved, and anammox bacterial biomass increased to maximum levels. Moreover, the predominant genus of anammox bacteria was Candidatus Brocadia.

Weaponizing volatiles to inhibit competitor biofilms from a distance.

The soil bacterium Bacillus subtilis forms beneficial biofilms that induce plant defences and prevent the growth of pathogens. It is naturally found in the rhizosphere, where microorganisms coexist in an extremely competitive environment, and thus have evolved a diverse arsenal of defence mechanisms. In this work, we found that volatile compounds produced by B. subtilis biofilms inhibited the development of competing biofilm colonies, by reducing extracellular matrix gene expression, both within and across species. This effect was dose-dependent, with the structural defects becoming more pronounced as the number of volatile-producing colonies increased. This inhibition was mostly mediated by organic volatiles, and we identified the active molecules as 3-methyl-1-butanol and 1-butanol. Similar results were obtained with biofilms formed by phylogenetically distinct bacterium sharing the same niche, Escherichia coli, which produced the biofilm-inhibiting 3-methyl-1-butanol and 2-nonanon. The ability of established biofilms to inhibit the development and spreading of new biofilms from afar might be a general mechanism utilized by bacterial biofilms to protect an occupied niche from the invasion of competing bacteria.

Nosocomial pathogen biofilms on biomaterials: Different growth medium conditions and components of biofilms produced in vitro.

BACKGROUND/PURPOSE (S): Nosocomial pathogens can develop biofilms on hospital surfaces and medical devices; however, few studies have focused on the evaluation of mono-and dual-species biofilms developed by nosocomial pathogens under different growth conditions. METHODS: This study investigated biofilm development by nosocomial pathogens (Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa) on biomaterials in different culture media and their components of the extracellular matrix biofilm. RESULTS: The mono-species biofilms showed cell densities from 7.50 to 9.27 Log10 CFU/cm2 on natural rubber latex type I (NLTI) and from 7.58 to 8.79 Log10 CFU/cm2 on stainless steel (SS). Dual-species biofilms consisted of S. aureus + P. aeruginosa (7.87-8.27 Log10 CFU/cm2 in TSBP and TSBME onto SS; p < 0.05), E. coli + P. aeruginosa (8.32-8.86 Log10 CFU/cm2 in TSBME onto SS and TSBP onto NLTI; p < 0.05), and S. aureus + E. coli (7.82 Log10 CFU/cm2 in TSBME onto SS; p < 0.05). Furthermore, biofilm detachment after proteinase K treatment was 5.54-32.81% compared to 7.95-24.15% after DNase I treatment in the mono-dual species biofilm matrix. Epifluorescence microscopy and scanning electron microscopy (SEM) enabled visualizing the bacteria and extracellular polymeric substances of biofilms on SS and NLTI. CONCLUSION: Nosocomial pathogens can develop biofilms on biomaterials. Mono-species biofilms of Gram-negative bacteria showed lower densities than dual-species biofilms in TSBME and TSBP. Additionally, dual-species biofilms showed a higher concentration of proteins and eDNA in the extracellular matrix.

Common plant flavonoids prevent the assembly of amyloid curli fibres and can interfere with bacterial biofilm formation.

Like all macroorganisms, plants have to control bacterial biofilm formation on their surfaces. On the other hand, biofilms are highly tolerant against antimicrobial agents and other stresses. Consequently, biofilms are also involved in human chronic infectious diseases, which generates a strong demand for anti-biofilm agents. Therefore, we systematically explored major plant flavonoids as putative anti-biofilm agents using different types of biofilms produced by Gram-negative and Gram-positive bacteria. In Escherichia coli macrocolony biofilms, the flavone luteolin and the flavonols myricetin, morin and quercetin were found to strongly reduce the extracellular matrix. These agents directly inhibit the assembly of amyloid curli fibres by driving CsgA subunits into an off-pathway leading to SDS-insoluble oligomers. In addition, they can interfere with cellulose production by still unknown mechanisms. Submerged biofilm formation, however, is hardly affected. Moreover, the same flavonoids tend to stimulate macrocolony and submerged biofilm formation by Pseudomonas aeruginosa. For Bacillus subtilis, the flavonone naringenin and the chalcone phloretin were found to inhibit growth. Thus, plant flavonoids are not general anti-biofilm compounds but show species-specific effects. However, based on their strong and direct anti-amyloidogenic activities, distinct plant flavonoids may provide an attractive strategy to specifically combat amyloid-based biofilms of some relevant pathogens.

Enhanced bacterial killing by vancomycin in staphylococcal biofilms disrupted by novel, DMMA-modified carbon dots depends on EPS production.

Alternatives for less and less effective antibiotic treatment of bacterial infections, are amongst others based on nanotechnological innovations, like carbon-dots. However, with a focus on chemistry, important characteristics of bacterial strains, like (in-)ability to produce extracellular-polymeric-substances (EPS) are often neglected. EPS is the glue that certain bacterial strains produce to keep a biofilm together. Here we report on synthesis of novel, pH-responsive, 2,3-dimethylmaleic-anhydride modified carbon-dots (CDMMA-dots). CDMMA-dots, like unmodified C-dots without DMMA, were little bactericidal. However, CDMMA-dots reduced volumetric-bacterial-density within the acidic-environment of a biofilm for a non-EPS-producing Staphylococcus epidermidis strain, indicative for a more open structure. Such a structural disruption was not observed for an EPS-producing strain. Disrupted biofilms of the non-EPS-producing strain pre-exposed to CDMMA-dots at pH 5.0, were more amenable to vancomycin penetration and killing of their inhabitants than biofilms of EPS-producing-staphylococci. Herewith, we describe a new role of carbon-dots as synthetic disruptants of biofilm structure. It is a partial success story, identifying the challenge of making carbon-dots that act as a universal disruptant for biofilms of strains with different microbiological characteristics, most notably the ability to produce or not-produce EPS. Such carbon-dots, will enable more effective clinical treatment of bacterial infections combined with current antibiotics.

Impact of additive application on the establishment of fast and stable aerobic granulation.

Aerobic granular sludge (AGS) is a microbial biofilm self-aggregation, which is effective for nutrient and pollutant removal, through the development of dense microbial layers bound together with extracellular polymeric substances (EPSs). However, long start-up times and granule disintegration are still challenges ahead. An array of external additives, including ion chelating agents, sludge-based enhancers, and magnetic influence have been tested to overcome these barriers. The application of such additives may promote enhanced EPS production, neutralization of charges on the bacterial surface, acts as a core-induced agent, or as a bridge to connect EPSs and cell surfaces. Although additives may improve the granule formation without reducing treatment efficiencies, there are still environmental concerns due to the fate and toxicity of discharged excess sludge. This mini-review identifies an array of external additives and their mechanisms to improve granulation properties, and proposes discussion about the technical and economic viability of these additives. KEY POINTS: • Additives reduce granulation time and repair granule disintegration. • Biopolymer-based additives fulfill technical and environmental requirements. • Sludge-based additives are cheap and in line with the resource recovery concept. • The need for environmental-friendly additives for aerobic granular sludge process. • External additives affect granular biomass size distribution.

Simvastatin decreases the silver resistance of E. faecalis through compromising the entrapping function of extracellular polymeric substances against silver.

Enterococcus faecalis (E. faecalis) is a Gram-positive bacterium closely related to many refractory infections of human and shows the resistant ability against the antibacterial effects of silver. Simvastatin is a semisynthetic compound derived from lovastatin and a hydroxymethyl glutaryl coenzyme A(HMG-COA) reductase inhibitor showing certain inhibitive effects on bacteria. The main purpose of this study was to establish and characterize the Ag+/silver nanoparticles (AgNPs)-resistant E. faecalis, and further evaluate the function of extracellular polymeric substances (EPS) in the silver resistance and the effect of simvastatin on the silver-resistance of E. faecalis. The results showed that the established silver-resistant E. faecalis had strong resistance against both Ag+ and AgNPs and simvastatin could decrease the silver-resistance of both original and Ag+/AgNPs-resistant E. faecalis. The Transmission electron microscopy (TEM), High-angle annular dark-field (HAADF) and mapping images showed that the silver ions or particles aggregated and confined in the EPS on surface areas of the cell membrane when the silver-resistant E. faecalis were incubated with Ag+ or AgNPs. When the simvastatin was added, the silver element was not confined in the EPS and entered the bacteria. These findings may indicate that the silver resistance of E. faecalis was derived from the entrapping function of EPS, but simvastatin could compromise the function of EPS to decrease the silver resistant ability of E. faecalis.

N-Acetyl-cysteine and Mechanisms Involved in Resolution of Chronic Wound Biofilm.

Chronic wounds are a major global health problem with the presence of biofilm significantly contributing to wound chronicity. Current treatments are ineffective in resolving biofilm and simultaneously killing the bacteria; therefore, effective biofilm-resolving drugs are needed. We have previously shown that, together with α-tocopherol, N-acetyl-cysteine (NAC) significantly improves the healing of biofilm-containing chronic wounds, in a diabetic mouse model we developed, by causing disappearance of the bacteria and breakdown of the extracellular polymeric substance (EPS). We hypothesize that NAC creates a microenvironment that affects bacterial survival and EPS integrity. To test this hypothesis, we developed an in vitro biofilm system using microbiome taken directly from diabetic mouse chronic wounds. For these studies, we chose mice in which chronic wound microbiome was rich in Pseudomonas aeruginosa (97%). We show that NAC at concentrations with pH < pKa causes bacterial cell death and breakdown of EPS. When used before biofilm is formed, NAC leads to bacterial cell death whereas treatment after the biofilm is established NAC causes biofilm dismantling accompanied by bacterial cell death. Mechanistically, we show that NAC can penetrate the bacterial membrane, increase oxidative stress, and halt protein synthesis. We also show that low pH is important for the actions of NAC and that bacterial death occurs independently of the presence of biofilm. In addition, we show that both the acetyl and carboxylic groups play key roles in NAC functions. The results presented here provide insight into the mechanisms by which NAC dismantles biofilm and how it could be used to treat chronic wounds after debridement (NAC applied at the start of culture) or without debridement (NAC applied when biofilm is already formed). This approach can be taken to develop biofilm from microbiome taken directly from human chronic wounds to test molecules that could be effective for the treatment of specific biofilm compositions.

Inhibiting bacterial cooperation is an evolutionarily robust anti-biofilm strategy.

Bacteria commonly form dense biofilms encased in extracellular polymeric substances (EPS). Biofilms are often extremely tolerant to antimicrobials but their reliance on shared EPS may also be a weakness as social evolution theory predicts that inhibiting shared traits can select against resistance. Here we show that EPS of Salmonella biofilms is a cooperative trait whose benefit is shared among cells, and that EPS inhibition reduces both cell attachment and antimicrobial tolerance. We then compare an EPS inhibitor to conventional antimicrobials in an evolutionary experiment. While resistance against conventional antimicrobials rapidly evolves, we see no evolution of resistance to EPS inhibition. We further show that a resistant strain is outcompeted by a susceptible strain under EPS inhibitor treatment, explaining why resistance does not evolve. Our work suggests that targeting cooperative traits is a viable solution to the problem of antimicrobial resistance.

New insights into aniline toxicity: Aniline exposure triggers envelope stress and extracellular polymeric substance formation in Rubrivivax benzoatilyticus JA2.

Aniline is a major environmental pollutant of serious concern due to its toxicity. Although microbial metabolism of aniline is well-studied, its toxic effects and physiological responses of microorganisms to aniline are largely unexplored. Rubrivivax benzoatilyticus JA2, an aniline non-degrading bacterium, tolerates high concentrations of aniline and produces extracellular polymeric substance(EPS). Surprisingly, strain JA2 forms EPS only when exposed to aniline and other toxic compounds like organic solvents and heavy metals indicating that EPS formation is coupled to cell toxicity. Further, extensive reanalysis of the previous proteomic data of aniline exposed cells revealed up-regulation of envelope stress response(ESR) proteins such as periplasmic protein folding, envelope integrity, transmembrane complex, and cell-wall remodelling proteins. In silico analysis and molecular modeling of three highly up-regulated proteins revealed that these proteins were homologous to CpxARP proteins of ESR signalling pathway. Furthermore, EPS formation to known ESR activators(Triton-X-100, EDTA) suggests that envelope stress possibly regulating the EPS production. The present study suggests that aniline triggers envelope stress; to counter this strain JA2 activates ESR pathway and EPS production. Our study revealed the hitherto unknown toxic effects of aniline as an acute envelope stressor thus toxicity of aniline may be more profound to life-forms than previously thought.

'Biofilm Clippers'- enzyme formulation for bovine mastitic biofilm therapy.

Mastitis is one of the most important diseases that are threatening modern dairy farms. Biofilms of mastitic teat canal have serious clinical implications because of colonized pathogens having the ability to construct an extracellular polymeric substance (EPS) with increased tolerance to antimicrobials leads to difficulty in eradicating the infection. In this study, we investigated the synergistic biofilm disruptive effect of a combination of carbohydrate hydrolases targeting extracellular polysaccharides of biofilm matrix and we termed it as 'Biofilm Clippers (BC)'. Our findings demonstrate that the BC formulation exhibits intense biofilm-disrupting activity against Staphylococcus aureus biofilms. The results of the study showed that BC enables activity equivalent to physiologically achievable concentrations in disrupting biofilms of S. aureus in vitro. The synergistic anti-biofilm activities of BC on S. aureus biofilms demonstrated that the biofilm matrix is predominant of complex polysaccharides. Further, the confocal microscopic analysis demonstrates that the BC formulation is highly effective compared to the single treatment of either of the enzymes in disrupting the biofilm. To the best of our knowledge, this is the first report on the synergistic anti-biofilm activity of a class of enzyme formulation against mastitic biofilm mass. Even though a small study showed a promising effect on mastitic teat canal, further extensive investigation on a large number of bovines for mastitis therapeutic potential of this BC-derived product is now warranted.