Antifungal peptides: a potential new class of antifungals for treating vulvovaginal candidiasis caused by fluconazole-resistant Candida albicans.

Vulvovaginal candidiasis/candidosis is a common fungal infection afflicting approximately 75% of women globally caused primarily by the yeast Candida albicans. Fluconazole is widely regarded as the antifungal drug of choice since its introduction in 1990 due to its high oral bioavailability, convenient dosing regimen and favourable safety profile. However, its widespread use has led to the emergence of fluconazole-resistant C. albicans, posing a universal clinical concern. Coupled to the dearth of new antifungal drugs entering the market, it is imperative to introduce new drug classes to counter this threat. Antimicrobial peptides (AMPs) are potential candidates due to their membrane-disrupting mechanism of action. By specifically targeting fungal membranes and being rapidly fungicidal, they can reduce the chances of resistance development and treatment duration. Towards this goal, we conducted a head-to-head comparison of 61 short linear AMPs from the literature to identify the peptide with the most potent activity against fluconazole-resistant C. albicans. The 11-residue peptide, P11-6, was identified and assayed against a panel of clinical C. albicans isolates followed by fungicidal/static determination and a time-kill assay to gauge its potential for further drug development. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Rapid Detection of Carbapenemase-Producing Enterobacteriacae Based on Surface-Enhanced Raman Spectroscopy with Gold Nanostars.

The emergence and rapid spread of antibiotic resistance poses a serious threat to healthcare systems across the globe. The existence of carbapenemase-producing Enterobacteriaceae (CPE) such as Klebsiella pneumoniae renders the use of carbapenems, the last-resort class of ß-lactam antibiotics, ineffective against bacterial infections, often leading to CPE-associated mortalities. Current methods of detection such as the Carba NP test and modified Hodge's test require hours to days to detect, which delays the response to isolate patients for rapid intervention. Here, we developed a surface-enhanced Raman scattering (SERS)-based detection scheme which utilizes gold nanostars conjugated to a ß-lactam antibiotic ceftriaxone (CRO) as a beacon for rapid detection of bacterial ß-lactamase secreted by Delhi metalloproteinase (NDM)-producing Escherichia coli as our CPE model with carbapenemase activity. The cleavage of ß-lactam ring in CRO by NDM (Class B ß-lactamase) caused a detectable reduction in SERS intensities at 722, 1358, and 1495 cm-1 within 25 min. Ratiometric analysis of the SERS peaks at 722, 1358, and 1495 cm-1 normalized against the Raman peak of polystyrene cuvette at 620 cm-1 showed the peak at 1358 cm-1 having the most significant change in intensity upon CPE detection. This reduced detection time has not been reported to date for CPE detection, and our novel approach using SERS could be extended to detect the activity of other classes of ß-lactamases to broaden its clinical utility.

Cartography of opportunistic pathogens and antibiotic resistance genes in a tertiary hospital environment.

Although disinfection is key to infection control, the colonization patterns and resistomes of hospital-environment microbes remain underexplored. We report the first extensive genomic characterization of microbiomes, pathogens and antibiotic resistance cassettes in a tertiary-care hospital, from repeated sampling (up to 1.5 years apart) of 179 sites associated with 45 beds. Deep shotgun metagenomics unveiled distinct ecological niches of microbes and antibiotic resistance genes characterized by biofilm-forming and human-microbiome-influenced environments with corresponding patterns of spatiotemporal divergence. Quasi-metagenomics with nanopore sequencing provided thousands of high-contiguity genomes, phage and plasmid sequences (>60% novel), enabling characterization of resistome and mobilome diversity and dynamic architectures in hospital environments. Phylogenetics identified multidrug-resistant strains as being widely distributed and stably colonizing across sites. Comparisons with clinical isolates indicated that such microbes can persist in hospitals for extended periods (>8 years), to opportunistically infect patients. These findings highlight the importance of characterizing antibiotic resistance reservoirs in hospitals and establish the feasibility of systematic surveys to target resources for preventing infections.

Detection and genetic characterisation of qnrB in hospital isolates of Klebsiella pneumoniae in Singapore.

Polymerase chain reaction (PCR) screening of 116 ciprofloxacin-resistant Klebsiella pneumoniae hospital isolates for the presence of qnr genes that mediate plasmid quinolone resistance revealed that none were positive for qnrA or qnrS. However, qnrB was detected in ca. 5.2% of the isolates. Southern hybridisation demonstrated that the qnrB-hybridising plasmids were large (>70kb) and capable of transferring quinolone resistance by conjugation. Sequence analysis of the qnrB genes detected in this study showed that they were identical to previously identified qnrB1, qnrB4 and qnrB6 genes, although a novel variant designated qnrB20 was also identified. Analysis of the genetic environment around the cloned qnrB genes showed that they were present in diverse plasmid backbones, sometimes within novel genetic contexts, but always associated with mobile or transposable elements.

Delay in effective therapy in anidulafungin-resistant Candida tropicalis fungaemia: Potential for rapid prediction of antifungal resistance with whole-genome-sequencing.

OBJECTIVES: This study investigated the feasibility of using whole-genome sequencing (WGS) for the prediction of antifungal resistance in anidulafungin-resistant Candida tropicalis candidaemia isolates. METHODS: Next-generation sequencing was performed for three anidulafungin-resistant C. tropicalis isolates on an Illumina MiSeq system with in-house bioinformatics analysis. RESULTS: Mutations in Fks1p associated with anidulafungin resistance were identified. Other mutations associated with varying levels of phenotypic resistance to fluconazole were also identified. CONCLUSIONS: These data demonstrate the potential to predict antifungal resistance using WGS. With improving technology, real-time WGS may be used for tailoring effective antifungal therapy in patients with candidaemia.

Structure-activity relationship studies of ultra-short peptides with potent activities against fluconazole-resistant Candida albicans.

Vulvovaginal candidiasis (VVC) is a genital fungal infection afflicting approximately 75% of women globally and is primarily caused by the yeast Candida albicans. The extensive use of fluconazole, the first-line antifungal drug of choice, has led to the emergence of fluconazole-resistant C. albicans, creating a global clinical concern. This, coupled to the lack of new antifungal drugs entering the market over the past decade, has made it imperative for the introduction of new antifungal drug classes. Peptides with antifungal properties are deemed potential drug candidates due to their rapid membrane-disrupting mechanism of action. By specifically targeting and rapidly disrupting fungal membranes, they reduce the chances of resistance development and treatment duration. In a previous screening campaign involving an antimicrobial peptide library, we identified an octapeptide (IKIKIKIK-NH2) with potent activity against C. albicans. Herein, we report a structure-activity relationship study on this peptide with the aim of designing a more potent peptide for further development. The lead peptide was then tested against a panel of fluconazole-resistant C. albicans, subjected to a fungicidal/static determination assay, a human dermal fibroblast viability assay and a homozygous profiling assay to gain insights into its mechanism of action and potential for further development as a topical antifungal agent.