Overexpression of efflux pump transporter genes and mutations in ERG11 pave the way to fluconazole resistance in Candida tropicalis: A study from a North India region.

INTRODUCTION: Fluconazole resistance in Candida tropicalis health care-associated infections is increasing. We investigated the role of efflux pump and mutations in ERG11p conferring fluconazole resistance in C. tropicalis. MATERIALS AND METHODS: A total of 17 C. tropicalis clinical isolates, including fluconazole-resistant and fluconazole-susceptible/fluconazole-susceptible dose-dependent isolates, were collected from a tertiary care centre in a North India region between 2015 and 2018. Antifungal susceptibility, reversal of fluconazole resistance by tacrolimus, ERG11 amplification and its sequencing and a quantitative polymerase chain reaction (PCR) assay for expression analysis of ERG11, MDR1 and CDR1 genes were performed. RESULTS AND DISCUSSION: Synergism between fluconazole and tacrolimus was observed in all resistant C. tropicalis isolates. Overexpression of all the three genes, MDR1, ERG11 and CDR1, was observed in resistant isolates (P = 0.05). Among resistant isolates, mutations leading to amino acid substitution were seen in two, Ct10 (glysine464serine) and Ct16 (tyrosine132phenylalanine; serine154phenylalanine). CONCLUSION: Overexpression in efflux pump transporter genes, together with mutations in ERG11, lead to fluconazole resistance among C. tropicalis. To the best of our knowledge, this is the first study on the C. tropicalis fluconazole resistance mechanism from the North India region.

Antibiofilm Potential of Silver Sulfadiazine-Loaded Nanoparticle Formulations: A Study on the Effect of DNase-I on Microbial Biofilm and Wound Healing Activity.

Biofilm resistance is one of the severe complications associated with chronic wound infections, which impose extreme microbial tolerance against antibiotic therapy. Interestingly, deoxyribonuclease-I (DNase-I) has been empirically proved to be efficacious in improving the antibiotic susceptibility against biofilm-associated infections. DNase-I hydrolyzes the extracellular DNA, a key component of the biofilm responsible for the cell adhesion and strength. Moreover, silver sulfadiazine, a frontline therapy in burn wound infections, exhibits delayed wound healing due to fibroblast toxicity. In this study, a chitosan gel loaded with solid lipid nanoparticles of silver sulfadiazine (SSD-SLNs) and supplemented with DNase-I has been developed to reduce the fibroblast cytotoxicity and overcome the biofilm-imposed resistance. The extensive optimization using the Box-Behnken design (BBD) resulted in the formation of SSD-SLNs with a smooth surface as confirmed by scanning electron microscopy and controlled release (83%) for up to 24 h. The compatibility between the SSD and other formulation excipients was confirmed by Fourier transform infrared, differential scanning calorimetry, and powder X-ray diffraction studies. Developed SSD-SLNs in combination with DNase-I inhibited around 96.8% of biofilm of Pseudomonas aeruginosa as compared to SSD with DNase-I (82.9%). In line with our hypothesis, SSD-SLNs were found to be less toxic (cell viability 90.3 ± 3.8% at 100 µg/mL) in comparison with SSD (Cell viability 76.9 ± 4.2%) against human dermal fibroblast cell line. Eventually, the results of the in vivo wound healing study showed complete wound healing after 21 days' treatment with SSD-SLNs along with DNase-I, whereas marketed formulations SSD and SSD-LSNs showed incomplete healing after 21 days. Data in hand suggest that the combination of SSD-SLNs with DNase-I is an effective treatment strategy against the biofilm-associated wound infections and accelerates wound healing.

Alginate lyase immobilized chitosan nanoparticles of ciprofloxacin for the improved antimicrobial activity against the biofilm associated mucoid P. aeruginosa infection in cystic fibrosis.

Dense colonization of mucoid Pseudomonas aeruginosa within the self-secreted extracellular matrix (mainly alginate), called biofilm, is a principal reason for the failure of antimicrobial therapy in cystic fibrotic patients. Alginate is a key component in the biofilm of mucoid P. aeruginosa and responsible for surface adhesion and stabilization of biofilm. To overcome this problem, alginate lyase functionalized chitosan nanoparticles of ciprofloxacin were developed for the effective treatment of P. aeruginosa infection in cystic fibrosis patients. The developed nanoparticles were found to have desired quality attributes and demonstrated sustained release following the Higuchi release kinetics. Drug compatibility with the chitosan was confirmed by FTIR while powder X-ray diffraction analysis confirmed the entrapment of drug within the nanoparticle matrix. Lactose adsorbed NPs showed promising aerodynamic property. Nanoparticles showed prolonged MIC and significant reduction in biofilm aggregation and formation in planktonic bacterial suspension. Nanoparticles exhibited significantly higher inhibitory effect against biofilm of P. aeruginosa and reduced the biomass, thickness and density confirmed by confocal microscopy. Furthermore, developed nanoparticles were haemocompatible and did not exhibit any toxicity in vitro MTT assay and in vivo on lungs male Wistar rats. The data in hand collectively suggest the proposed strategy a better alternative for the effective treatment of cystic fibrosis infections.

Concomitant and discrete expressions of aldose reductase and sorbitol dehydrogenase in the male reproductive tract.

This study aimed at investigating the expression and localization of the polyol pathway enzymes; aldose reductase (AR) and sorbitol dehydrogenase (SDH), in the male reproductive tract of rat. Gene expression analysis showed maximum expression of AR and SDH in the coagulating glands. Western blot analysis showed a coordinated presence of the two enzymes in the coagulating glands, seminal vesicle and epididymis. Immunohistochemistry showed a concordant expression of the two enzymes in the coagulating gland, which goes well with its function of fructose production in rats. A less concordant expression of the two enzymes in the seminal vesicle was also seen. Discrete expression of AR was seen in the Sertoli cells without SDH. Germ cells including sperm in the seminiferous tubules lacked AR, but SDH was present in all stages of developing germ cells including sperm present in the seminiferous tubules. The epithelial layer of epididymis showed the presence of AR, but it was negligible in vas deferens and prostate. SDH was not seen in the epithelial layer of epididymis, vas deferens or prostate. Though sperm in the seminiferous tubules lacked AR, sperm extracted from cauda showed the presence of both AR and SDH. Immunofluorescence localization of AR and SDH on sperm showed the presence of both the enzymes all over sperm. Discrete expression of AR in the Sertoli cells may be linked to detoxification of a number of metabolism by-products. Similarly, the presence of polyol enzymes on sperm in epididymis and beyond may be to tackle toxic metabolites they may encounter during their journey along the male or female reproductive tract.

Mucuna pruriens and its major constituent L-DOPA recover spermatogenic loss by combating ROS, loss of mitochondrial membrane potential and apoptosis.

BACKGROUND: The Ayurvedic medicinal system claims Mucuna pruriens (MP) to possess pro-male fertility, aphrodisiac and adaptogenic properties. Some scientific evidence also supports its pro-male fertility properties; however, the mechanism of its action is not yet clear. The present study aimed at demonstrating spermatogenic restorative efficacy of MP and its major constituent L-DOPA (LD), and finding the possible mechanism of action thereof in a rat model. METHODOLOGY/FINDINGS: Ethinyl estradiol (EE) was administered at a rate of 3 mg/kg body weight (BW)/day for a period of 14 days to generate a rat model with compromised spermatogenesis. MP and LD were administered in two separate groups of these animals starting 15(th) day for a period of 56 days, and the results were compared with an auto-recovery (AR) group. Sperm count and motility, testis histo-architecture, level of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), apoptosis, peripheral hormone levels and testicular germ cell populations were analysed, in all experimental groups. We observed efficient and quick recovery of spermatogenesis in MP and LD groups in comparison to the auto-recovery group. The treatment regulated ROS level, apoptosis, and mitochondrial membrane potential (MMP), recovered the hypothalamic-pituitary-gonadal axis and the number of testicular germ cells, ultimately leading to increased sperm count and motility. CONCLUSION/SIGNIFICANCE: M. pruriens efficiently recovers the spermatogenic loss induced due to EE administration. The recovery is mediated by reduction in ROS level, restoration of MMP, regulation of apoptosis and eventual increase in the number of germ cells and regulation of apoptosis. The present study simplified the complexity of mechanism involved and provided meaningful insights into MP/LD mediated correction of spermatogenic impairment caused by estrogens exposure. This is the first study demonstrating that L-DOPA largely accounts for pro-spermatogenic properties of M. pruriens. The manuscript bears CDRI communication number 8374.