Efficacy of a rhamnolipid biosurfactant to inhibit Trichophyton rubrum in vitro and in a mice model of dermatophytosis.

Dermatophytic infections caused by Trichophyton rubrum are the most prevalent superficial mycoses worldwide. The present study was aimed at evaluating the anti-dermatophytic effect of a rhamnolipid biosurfactant (RL) produced by Pseudomonas aeruginosa SS14 (GenBank Accession no. KC866140) against T. rubrum in experimentally induced dermatophytosis in mice models. The purified RL could effectively suppress spore germination and hyphal proliferation of T. rubrum at 500 µg/mL. Ultramicroscopic observations involving SEM and AFM studies revealed severely altered hyphal morphology in the RL-treated mycelia. The membrane disruptive effect of RL was measured by the uptake of propidium iodide (PI) as visualized by CLSM. The absorbance of the RL-treated cell suspension at 260 nm showed concentration-dependant exudation of nucleic acid due to loss of cell membrane integrity. On evaluation of the therapeutic efficacy of RL on experimentally induced cutaneous dermatophytosis in mice models, we observed that the RL, when applied topically at a concentration of 500 µg/mL, was effective in completely curing dermatophytosis at the end of 21-day treatment period. The results were statistically similar to those obtained using the standard drug terbinafine as control. Apart from macroscopic observation, the results were confirmed by culture of skin scrapings and histopathological examination. The results are suggestive of the effectiveness of RL in control of dermatophytosis caused by T. rubrum.

Production, characterization, and antifungal activity of a biosurfactant produced by Rhodotorula babjevae YS3.

BACKGROUND: Sophorolipids are one of the most promising glycolipid biosurfactants and have been successfully employed in bioremediation and various other industrial sectors. They have also been described to exhibit antimicrobial activity against different bacterial species. Nevertheless, previous literature pertaining to the antifungal activity of sophorolipids are limited indicating the need for further research to explore novel strains with wide antimicrobial activity. A novel yeast strain, Rhodotorula babjevae YS3, was recently isolated from an agricultural field in Assam, Northeast India. This study was primarily emphasized at the characterization and subsequent evaluation of antifungal activity of the sophorolipid biosurfactant produced by R. babjevae YS3. RESULTS: The growth kinetics and biosurfactant production by R. babjevae YS3 was evaluated by cultivation in Bushnell-Haas medium containing glucose (10% w/v) as the sole carbon source. A reduction in the surface tension of the culture medium from 70 to 32.6 mN/m was observed after 24 h. The yield of crude biosurfactant was recorded to be 19.0 g/l which might further increase after optimization of the growth parameters. The biosurfactant was characterized to be a heterogeneous sophorolipid (SL) with both lactonic and acidic forms after TLC, FTIR and LC-MS analyses. The SL exhibited excellent oil spreading and emulsifying activity against crude oil at 38.46 mm2 and 100% respectively. The CMC was observed to be 130 mg/l. The stability of the SL was evaluated over a wide range of pH (2-10), salinity (2-10% NaCl) and temperature (at 120 °C for time intervals of 30 up to 120 min). The SL was found to retain surface-active properties under the extreme conditions. Additionally, the SL exhibited promising antifungal activity against a considerably broad group of pathogenic fungi viz. Colletotrichum gloeosporioides, Fusarium verticilliodes, Fusarium oxysporum f. sp. pisi, Corynespora cassiicola, and Trichophyton rubrum. CONCLUSIONS: The study reports, for the first time, the biosurfactant producing ability of R. babjevae, a relatively lesser studied yeast. The persistent surface active properties of the sophorolipid in extreme conditions advocates its applicability in diverse environmental and industrial sectors. Further, antifungal activities against plant and human pathogens opens up possibilities for development of efficient and eco-friendly antifungal agents with agricultural and biomedical applications.

Rhamnolipid exhibits anti-biofilm activity against the dermatophytic fungi Trichophyton rubrum and Trichophyton mentagrophytes.

Dermatophytes are responsible for a majority of fungal infections in humans and other vertebrates, causing dermatophytosis. Treatment failures are often associated with biofilm formation, making dermatophytes resistant to antifungals. In this study, effects of a rhamnolipid (RL-SS14) produced by Pseudomonas aeruginosa SS14 on planktonic cells of Trichophyton rubrum and Trichophyton mentagrophytes, their biofilm formation, and disruption of mature biofilms were assessed. The composition of RL-SS14 was analysed using FTIR, HPLC-ESI-MS, and GC-MS. Minimum inhibitory concentrations against the planktonic forms of T. rubrum and T. mentagrophytes were 0.5 mg/mL and 0.125 mg/mL, respectively. Crystal-violet (biofilm biomass) and safranin (extracellular matrix) staining revealed that RL-SS14 significantly inhibited biofilm formation and also reduced preformed biofilms in a dose-dependent manner. Microscopic visualization of treated biofilms via SEM, AFM, and CLSM revealed marked morphological damage, cell death, and reduced extracellular matrix. The results indicate the potential of RL-SS14 as an anti-biofilm agent against dermatophytes.

Sophorolipid Biosurfactant Can Control Cutaneous Dermatophytosis Caused by Trichophyton mentagrophytes.

Trichophyton mentagrophytes, a zoophilic species, is one of the most frequently isolated dermatophytes in many parts of the world. This study investigated the efficacy of a sophorolipid (SL-YS3) produced by Rhodotorula babjevae YS3 against dermatophytosis caused by T. mentagrophytes. SL-YS3 was characterized by gas chromatography-mass spectrometry (GC-MS) and ultra-performance liquid chromatography, coupled with electrospray mass spectrometry (UPLC-ESI-MS). SL-YS3 comprised of six different fatty acids as the hydrophobic components of constituent congeners and sophorose as the hydrophilic component. Inhibitory effects of purified SL-YS3 against hyphal growth was found to be 85% at a 2 mg ml-1 concentration, and MIC was 1 mg ml-1. Microscopic examination with scanning electron microscopy (SEM), atomic force microscopy, and confocal laser scanning microscopy (CLSM) revealed that SL-YS3 exerts its effect by disrupting cell membrane integrity causing cell death. SL-YS3 was also effective in reducing the biofilms formed by T. mentagrophytes, which was observed spectrophotometrically with crystal-violet staining and further validated with SEM and CLSM studies of treated biofilms. In vivo studies in a mouse model of cutaneous dermatophytosis involving macroscopic observations, percent culture recovery from skin samples, and histopathological studies showed that SL-YS3 could effectively cure the infected mice after 21 days of topical treatment. Terbinafine (TRB) was used as a standard drug in the experiments. We demonstrate, for the first time, the antidermatophytic activity of a sophorolipid biosurfactant. The findings are suggestive that SL-YS3 can be formulated as a novel antifungal compound to treat cutaneous mycoses caused by T. mentagrophytes.

Rice based distillers dried grains with solubles as a low cost substrate for the production of a novel rhamnolipid biosurfactant having anti-biofilm activity against Candida tropicalis.

In this study, rhamnolipid (RL) production by Pseudomonas aeruginosa SS14 utilizing rice based Distillers Dried Grains with Solubles (rDDGS) as the sole carbon source was evaluated and the production parameters were optimized using response surface methodology. Highest RL (RL-rDDGS) yield was 14.87 g/L in a culture medium containing 12% (w/v) rDDGS and 11% (v/v) inoculum concentration after 48 h of fermentation at 35 °C. RL-rDDGS was produced as a mixture of mono and di-RL congeners with four novel homologues Rha-C18:2, Rha-C19, Rha-C9, and Rha-Rha-C19. The RL reduced the surface tension of water to 34.8 mN/m at a critical micelle concentration (CMC) value of 100 mg/L, exhibited high stability at a wide range of pH (6-12), heating time (0-120 min), and salinity (2-12% NaCl). Furthermore, RL-rDDGS demonstrated appreciable biofilm disruptive property against Candida tropicalis. This is the first report on the usage of rDDGS for sustainable and low cost production of RL.

Incidence of metallo-beta-lactamase producing clinical isolates of Escherichia coli and Klebsiella pneumoniae in central Nepal.

BACKGROUND: The increasing and rapid spread of metallo-beta-lactamase (MBL) producing Enterobacteriaceae, particularly Escherichia coli and Klebsiella pneumoniae represents an emerging public health threat. However, limited data is available on MBL production in clinical isolates of E. coli and K. pneumoniae from Nepal. We have therefore undertaken this study to ascertain the incidence of MBL production in clinical isolates of E. coli and K. pneumoniae at a tertiary care teaching hospital in central Nepal. METHODS: A total of 401 consecutive, non-duplicate isolates of E. coli (n = 216) and K. pneumoniae (n = 185) were recovered from various clinical samples between July and December, 2012. These isolates were screened for the detection of carbapenemase production on the basis of their reduced susceptibility to meropenem or ertapenem by the disc diffusion method. The screened isolates were further phenotypically studied for carbapenemase production by modified Hodge test (MHT). MBL production was detected by performing combined disc test by using imipenem discs with and without ethylenediaminetetraacetic acid (EDTA), which chelates zinc required for MBL activity. RESULTS: Out of 216 E. coli isolates, a total of 41 isolates (18.98%) and out of 185 K. pneumoniae isolates, a total of 39 isolates (21.08%) were suspected to be carbapenemase- producers on the basis of their reduced susceptibility to meropenem or ertapenem. Interestingly, all the initially suspected isolates of E. coli and K. pneumoniae for carbapenemase production were found to be positive in both MHT and combined disc test. However, few weakly positive reactions were observed in MHT. All the MBL producing isolates were multidrug-resistant (MDR). In addition, 75.60% E. coli and 71.79% of K. pneumoniae isolates producing MBL were found to be "pandrug- resistant". CONCLUSIONS: Our findings showed MBL production in a considerable number of E. coli and K. pneumoniae isolates with MDR and pandrug-resistant phenotypes. Combined disc method can provide a sensible choice for phenotypic detection of MBL production in clinical microbiology laboratories as detection of MBL in bacterial isolates is indispensable for establishing the effective antibiotic policies and infection control strategies in the hospital setting.

Prevalence of blaTEM , blaSHV and blaCTX-M genes in clinical isolates of Escherichia coli and Klebsiella pneumoniae from Northeast India.

AIM: This study was carried out to determine the presence of blaTEM , blaSHV and blaCTX-M genes in extended-spectrum ß-lactamase (ESBL) producing Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) at a tertiary care referral hospital in Northeast India. MATERIALS AND METHODS: A total of 270 E. coli and 219 K. pneumoniae isolates were recovered during the period between August 2009 and July 2010. Kirby-Bauer disk diffusion method was performed to determine the antibiotic resistance pattern. Screening and phenotypic confirmatory test for ESBL production were performed using standard disc diffusion methods. Each of the initial ESBL screening test isolate was investigated for the presence of blaTEM , blaSHV and blaCTX-M genes via polymerase chain reaction (PCR) using gene-specific primers. RESULTS: Phenotypic confirmatory test able to detect ESBL production in 73.58% of E. coli and 67.24% of K. pneumoniae. However, PCR amplification showed the presence of one or more ESBL genes in each of the initial ESBL screening positive isolate. Among three ESBL genotypes, the most prevalent genotype was found to be blaCTX-M in E. coli (88.67%) and blaTEM in K. pneumoniae (77.58%) ESBL producing isolates. Majority of ESBL producing isolates possess more than one ESBL genes. CONCLUSION: This study constituted a primer report on high prevalence of blaTEM and blaCTX-M genes in ESBL producing isolates of E. coli and K. pneumoniae and denotes the need of more extensive studies on these antibiotic genes to determine the magnitude of the problem of antibiotic resistance exiting in this locality.