Fungal infections: Pathogenesis, antifungals and alternate treatment approaches.

Increasing incidence of fungal infections of recent times requires immediate intervention. Fungal infections are seldom construed at initial stages that intensify the severity of infections and complicate the treatment procedures. Fungal pathogens employ various mechanisms to evade the host immune system and to progress the severity of infections. For the treatment of diverse superficial and systemic infections, antifungal drugs from the available repertoire are administered. However, well documented evidence of fungal resistance to most of the antifungal drugs hampers disease control and poses challenges in antifungal therapy. Several physiological adaptations and genetic mutations followed by their selection in presence of antifungal agents drive the resistance development in fungi. The availability of limited antifungal arsenal, emergence of resistance and biofilm-conferred resistance drives the need for development of novel drugs and alternate approaches for the better treatment outcome against mycoses. This graphical review explicitly shed light on various fungal infections and causative organisms, pathogenesis, different antifungal drugs and resistance mechanisms including host immune response and evasion strategies. Here, we have highlighted recent developments on novel antifungal agents and other alternate approaches for fighting against fungal infections.

In Vitro Studies of Jatropha curcas L. Latex Spray Formulation for Wound Healing Applications.

OBJECTIVES: There is an increasing demand for wound healing products of natural origin. Our objective was to develop a spray formulation from Jatropha curcas (J. curcas) L. latex extracts for wound healing applications. MATERIALS AND METHODS: J. curcas L. latex was subjected to solvent extraction. The phytochemical structure was elucidated by 1H-NMR and confirmed by liquid chromatography-mass spectrometer spectrometry. A topical spray formulation prepared from J. curcas latex extracts was evaluated in terms of its antimicrobial activity and radical scavenging activity. The toxicity of the formulation on fibroblast cell lines, collagen production, and wound healing activities were tested. RESULTS: The 1H-NMR and mass spectrometric analyses revealed the pure compound as curcacycline A. The J. curcas latex extract formulation had radical scavenging and antibacterial activities. Moreover, the formulation was not toxic to the human fibroblast cells and it stimulated collagen production and healed cell injury in 24 h. CONCLUSION: The J. curcas latex extract promoted wound healing after cell injury. Our findings indicate the possibility of utilizing the J. curcas latex extract spray formulation as a potential antibacterial, antioxidant, and wound healing product from nature.

Impediment to growth and yeast-to-hyphae transition in Candida albicans by copper oxide nanoparticles.

The effects of two prominent copper oxide nanoparticles (CuO-NP and Cu2O-NP), with the oxidation state of Cu++ (cupric) and Cu+ (cuprous), on Candida albicans were evaluated. CuO-NP and Cu2O-NP were synthesized and characterized by XRD, FESEM, HR-TEM and Zeta potential. At sub-MIC (50 µg ml-1), both cupric and cuprous oxide NPs prevented yeast-to-hyphae switching and wrinkling behaviour in C. albicans. The mechanism for the antifungal action of the two NPs differed; CuO-NP significantly elicited reactive oxygen species, whereas membrane damage was more pronounced with Cu2O-NP. Real time PCR analysis revealed that CuO-NP suppressed the morphological switching of yeast-to-hyphae by down-regulating cph1, hst7 and ras1 and by up-regulation of the negative regulator tup1. In comparison, Cu2O-NP resulted in down-regulation of ras1 and up-regulation of the negative regulators nrg1 and tup1. Between the two NPs, CuO exhibited increased antifungal activity due to its stable oxidation state (Cu++) and its smaller dimensions compared with Cu2O-NP.

Copper oxide nanoparticles as an effective anti-biofilm agent against a copper tolerant marine bacterium, Staphylococcus lentus.

Biofilm formation on antifouling coatings is a serious concern in seawater cooling systems and the maritime industry. A prolific biofilm forming strain (Staphylococcus lentus), possessing high tolerance (>1,000 µg ml-1) to dissolved copper ions (Cu++) was isolated from titanium coupons exposed in the coastal waters of Kalpakkam, east coast of India. S. lentus formed increased biofilm (p < 0.05) at 100 µg ml-1 of Cu++ ions, when compared with the untreated control. To combat biofilm formation of this strain, the efficacy of copper oxide nanoparticles synthesized from copper nitrate by varying the concentrations of hexamine and cetyl trimethyl ammonium bromide (CTAB), was investigated. Complete (100%) inhibition of biofilm formation was observed with plain CuO NP (0.5 M hexamine, uncapped) at 1,000 µg ml-1. Capping with CTAB, influenced the morphology and the purity of the synthesized CuO NPs but did not alter their surface charge. Capping reduced metal ion release from CuO NPs and their antibacterial and anti-biofilm property against S. lentus. Overall, uncapped CuO NPs were effective in controlling biofilm formation of S. lentus. Concurrent release of copper ions and contact mediated physical damage by CuO NPs offer a promising approach to tackle metal tolerant biofilm bacteria.

Anti-biofilm properties of a mupirocin spray formulation against Escherichia coli wound infections.

Mupirocin ointment is a widely used topical drug for the treatment of bacterial skin infections. However, ointments have some limitations which motivated the development of a film forming spray of mupirocin. Mupirocin spray (2%) was formulated with Eudragit E100 as a film forming agent and tested for its antibacterial and anti-biofilm activities against Escherichia coli, a skin pathogen causing wound and surgical site infections. Treatment with mupirocin spray resulted in significant antibacterial and anti-biofilm activities (inhibition and disruption) with single spray and sub-actual dose concentrations at par with the commercial ointment concentration. The spray formulation was found to be non-toxic to fibroblast cells and greatly resisted removal from the site of application upon washing, in contrast to the ointment which was significantly removed after a single wash. This is the first study to develop and evaluate a spray formulation for mupirocin that forms a stable thin film for sustained release of the drug.

Evaluation of Proinflammatory Cytokines and Adverse Events in Healthy Volunteers upon Inhalation of Antituberculosis Drugs.

Inhalation therapy is a promising drug delivery approach for tuberculosis treatment. However, there is always concern about the safety of the dosage form by inhalation as it may induce inflammation. Developing a new dosage form for inhalation must include tests for its safety especially for the tumor necrosis factor (TNF)-α and interleukine (IL)-1ß. The safety of four anti-tuberculosis (anti-TB) drugs administered via inhalation was assessed in healthy volunteers. Four anti-TB drugs; isoniazid, rifampicin, pyrazinamide and levofloxacin were prepared as dry powder and evaluated for uniformity of delivered dose and in vitro drug deposition. These four anti-TB dry powder formulations for inhalation met the criteria of uniformity of delivered dose and exhibited suitable size for lung delivery. Forty healthy volunteers were recruited and each was sequentially challenged with isoniazid, rifampicin, pyrazinamide and levofloxacin in different orders. Safety was monitored by measuring the pro-inflammatory cytokines in their sputum, lung function test, blood chemistry and adverse events. This study proves that all four anti-TB dry powders did not provoke inflammatory cytokines and are safe to healthy volunteers.

Effect of 2, 4-di-tert-butylphenol on growth and biofilm formation by an opportunistic fungus Candida albicans.

Candida albicans, an opportunistic pathogen, has been known to form hypoxic biofilms on medical devices which in turn confers resistance towards antifungals, resulting in subsequent therapeutic failures. Inclusion of anti-biofilm agents in the control of infections is a topic of current interest in developing potential anti-infectives. The in vitro anti-fungal and anti-biofilm efficacy of 2,4-di-tert-butyl phenol [DTBP] was evaluated in this study, which revealed the potential fungicidal action of DTBP at higher concentrations where fluconazole failed to act completely. DTBP also inhibited the production of hemolysins, phospholipases and secreted aspartyl proteinase which are the crucial virulence factors required for the invasion of C. albicans. Various anti-biofilm assays and morphological observations revealed the efficacy of DTBP in both inhibiting and disrupting biofilms of C. albicans. Inhibition of hyphal development, a key process that aids in initial adhesion of C. albicans, was observed, and this could be a mechanism for the anti-biofilm activity of DTBP.

Assessment of 2,4-Di-tert-butylphenol induced modifications in extracellular polymeric substances of Serratia marcescens.

Extracellular polymeric substances (EPS) play crucial roles in biofilm formation and biocorrosion resulting in heavy economic loss in an industrial setup. Hence, in an attempt to develop an agent to control the EPS across the hosts, the ability of 2,4-Di-tert-butylphenol (DTBP), a potent antioxidant, to modify the EPS of Serratia marcescens has been investigated in this study using biophysical methods. Protein, polysaccharides and eDNA components of EPS were inhibited significantly (p < 0.05) upon exposure to DTBP. DTBP treatment reduced the crystallite size and crystallinity index of EPS and increased the dislocation density of crystallites without inducing stress, besides increasing the hydration of EPS which reduced its thermal stability. On the whole, this study highlights the efficacy of DTBP to modulate secreted EPS of S. marcescens which in turn could facilitate the disruption of biofilms besides favouring the diffusion of antimicrobials into the cell aggregates resulting eradication of persistent biofilms.

Phenol, 2,4-bis(1,1-dimethylethyl) of marine bacterial origin inhibits quorum sensing mediated biofilm formation in the uropathogen Serratia marcescens.

Intercellular communication in bacteria (quorum sensing, QS) is an important phenomenon in disease dissemination and pathogenesis, which controls biofilm formation also. This study reports the anti-QS and anti-biofilm efficacy of seaweed Gracilaria gracilis associated Vibrio alginolyticus G16 against Serratia marcescens. Purification and mass spectrometric analysis revealed the active principle as phenol, 2,4-bis(1,1-dimethylethyl) [PD]. PD affected the QS regulated virulence factor production in S. marcescens and resulted in a significant (p < 0.05) reduction in biofilm (85%), protease (41.9%), haemolysin (69.9%), lipase (84.3%), prodigiosin (84.5%) and extracellular polysaccharide (84.62%) secretion without hampering growth, as evidenced by XTT [2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay. qPCR analysis confirmed the down-regulation of the fimA, fimC, flhD and bsmA genes involved in biofilm formation. Apart from biofilm inhibition and disruption, PD increased the susceptibility of S. marcescens to gentamicin when administered synergistically, which opens another avenue for combinatorial therapy where PD can be used to enhance the efficacy of conventional antibiotics.

Antibiofilm activity of biosurfactant producing coral associated bacteria isolated from gulf of mannar.

Coral Associated Bacteria (CAB) (N = 22) isolated from the mucus of the coral Acropora digitifera were screened for biosurfactants using classical screening methods; hemolysis test, lipase production, oil displacement, drop collapse test and emulsifying activity. Six CAB (U7, U9, U10, U13, U14, and U16) were found to produce biosurfactants and were identified by 16S ribosomal RNA gene sequencing as Providencia rettgeri, Psychrobacter sp., Bacillus flexus, Bacillus anthracis, Psychrobacter sp., and Bacillus pumilus respectively. Their cell surface hydrophobicity was determined by Microbial adhesion to hydrocarbon assay and the biosurfactants produced were extracted and characterized by Fourier Transform Infrared spectroscopy. Since the biosurfactants are known for their surface modifying capabilities, antibiofilm activity of positive isolates was evaluated against biofilm forming Pseudomonas aeruginosa ATCC10145. Stability of the active principle exhibiting antibiofilm activity was tested through various temperature treatments ranging from 60 to 100 °C and Proteinase K treatment. CAB isolates U7 and U9 exhibited stable antibiofilm activity even after exposure to higher temperatures which is promising for the development of novel antifouling agents for diverse industrial applications. Further, this is the first report on biosurfactant production by a coral symbiont.