Antibacterial and antifungal activities of natural deep eutectic solvents.

The increasing antibiotic resistance towards a panel of microorganisms is one of the public health concerns. For this reason, the search for alternatives to the widely used antibiotic has been undertaken. In the era of sustainable chemistry, deep eutectic solvents (DESs) have emerged as promising antimicrobial agents. These solvents possess several advantages such as low volatility, low flammability, ease of preparation, and typically low cost of production. These properties make DES suitable for various applications, including extraction of biomolecules and preparation of cosmetics. Natural DESs (NADESs) are special category of DESs prepared from natural sources, which matched the recent trends of leaning back to nature, and decreasing dependence on synthetic precursors. NADES can be prepared by heating and stirring, freeze-drying, evaporation, grinding, and ultrasound-assisted and microwave-assisted synthesis. Utilizing NADESs as an alternative to traditional antibiotics, which become ineffective over time due to bacterial resistance, holds great promise for these reasons. This review aims to discuss the antimicrobial properties of multiple NADESs, including antibacterial and antifungal activities. To the best of our knowledge, this review is the first literature survey of the antimicrobial activities of NADESs. KEY POINTS: • Natural deep eutectic solvents are promising antimicrobial alternative to antibiotics • NADES holds high potential for their activity against bacterial resistance • NADES have also substantial antifungal activities.

Oropharyngeal Candidiasis among Egyptian COVID-19 Patients: Clinical Characteristics, Species Identification, and Antifungal Susceptibility, with Disease Severity and Fungal Coinfection Prediction Models.

The study aimed to investigate the causative species, antifungal susceptibility, and factors associated with oropharyngeal candidiasis (OPC) among Egyptian COVID-19 patients. This is an observational, case-controlled, single-center study that included three groups: COVID-19 patients (30), COVID-19 patients with OPC (39), and healthy individuals (31). Patients' demographic data (age, sex), laboratory tests, comorbidities, treatment, and outcomes were included. Candida species were isolated from COVID-OPC patient's oropharyngeal swabs by convenient microbiological methods. Isolated strains were tested for antimicrobial susceptibility, biofilm production, aspartyl protease, and phospholipase activities. The most common respiratory symptoms reported were dyspnea (36/39; 92.4%) and cough (33/39; 84.7%). Candida albicans was the most common isolated species, accounting for 74.36% (29/39), followed by Candida tropicalis and Candida glabrata (15.38% and 10.26%, respectively). Amphotericin was effective against all isolates, while fluconazole was effective against 61.5%. A total of 53.8% of the isolates were biofilm producers. The phospholipase activity of C. albicans was detected among 58.6% (17/29) of the isolates. Significant variables from this study were used to create two equations from a regression model that can predict the severity of disease course and liability to fungal infection, with a stativity of 87% and 91%, respectively. According to our findings, COVID-19 patients with moderate to severe infection under prolonged use of broad-spectrum antibiotics and corticosteroids should be considered a high-risk group for developing OPC, and prophylactic measures are recommended to be included in the treatment protocols. In addition, due to the increased rate of fluconazole resistance, other new antifungals should be considered.

Gold nanoparticles in the clinical laboratory: principles of preparation and applications.

In order to meet the challenges of effective healthcare, the clinical laboratory is constantly striving to improve testing sensitivity while reducing the required time and cost. Gold nanoparticles (AuNPs) are proposed as one of the most promising tools to meet such goals. They have unique optophysical properties which enable sensitive detection of biomarkers, and are easily amenable to modification for use in different assay formats including immunoassays and molecular assays. Additionally, their preparation is relatively simple and their detection methods are quite versatile. AuNPs are showing substantial promise for effective practical applications and commercial utilization is already underway. This article covers the principles of preparation of AuNPs and their use for development of different diagnostic platforms.

Development of an Immunochromatographic Strip Using Conjugated Gold Nanoparticles for the Rapid Detection of Klebsiella pneumoniae Causing Neonatal Sepsis.

Neonatal sepsis is a leading cause of death among newborns and infants, especially in the developing world. The problem is compounded by the delays in pinpointing the causative agent of the infection. This is reflected in increasing mortality associated with these cases and the spread of multi-drug-resistant bacteria. In this work, we deployed bioinformatics and proteomics analyses to determine a promising target that could be used for the identification of a major neonatal sepsis causative agent, Klebsiella pneumoniae. A 19 amino acid peptide from a hypothetical outer membrane was found to be very specific to the species, well conserved among its strains, surface exposed, and expressed in conditions simulating infection. Antibodies against the selected peptide were conjugated to gold nanoparticles and incorporated into an immunochromatographic strip. The developed strip was able to detect as low as 105 CFU/mL of K. pneumoniae. Regarding specificity, it showed negative results with both Escherichia coli and Enterobacter cloacae. More importantly, in a pilot study using neonatal sepsis cases blood specimens, the developed strip selectively gave positive results within 20 min with those infected with K. pneumoniae without prior sample processing. However, it gave negative results in cases infected with other bacterial species.

Formulation and Characterization of Sertaconazole Nitrate Mucoadhesive Liposomes for Vaginal Candidiasis.

PURPOSE: The aim of this study is to develop efficient localized therapy of sertaconazole nitrate for the treatment of vaginal candidiasis. METHODS: Sertaconazole nitrate-loaded cationic liposomes were prepared by thin-film hydration method and coated with different concentrations of pectin (0.05%, 0.1% and 0.2%) to develop mucoadhesive liposomes. The formulated mucoadhesive vesicles were characterized in terms of morphology, entrapment efficiency, particle size, zeta value, mucoadhesive properties and drug release. The selected formula was incorporated into a gel base and further characterized by an ex vivo permeation study in comparison with conventional sertaconazole gel. Also, the in vivo study was performed to assess the efficacy of sertaconazole mucoadhesive liposomal gel in treating rats with vaginal candidiasis. RESULTS: The mucoadhesive liposomes were spherical. Coating liposomes with pectin results in increased entrapment efficiency and particle size compared with uncoated vesicles. On the contrary, zeta values were reduced upon coating liposomes with pectin indicating efficient coating of liposomes with pectin. Mucoadhesive liposomes showed a more prolonged and sustained drug release compared with uncoated liposomes. Ex vivo study results showed that mucoadhesive liposomal gel increased sertaconazole tissue retention and reduced drug tissue penetration. In the invivo study, the mucoadhesive liposomal gel showed a significant reduction in the microbial count with a subsequent reduction in inflammatory responses with the lowest histopathological change compared with conventional gel. CONCLUSION: The study confirmed the potentiality of employing mucoadhesive liposomes as a successful carrier for the vaginal delivery of antifungal drugs.

A Novel Surface-Exposed Polypeptide Is Successfully Employed as a Target for Developing a Prototype One-Step Immunochromatographic Strip for Specific and Sensitive Direct Detection of Staphylococcus aureus Causing Neonatal Sepsis.

Neonatal sepsis is a life-threatening condition and Staphylococcus aureus is one of its major causes. However, to date, no rapid and sensitive diagnostic tool has been developed for its direct detection. Bioinformatics analyses identified a surface-exposed 112-amino acid polypeptide of the cell wall protein NWMN_1649, a surface protein involved in cell aggregation and biofilm formation, as being a species-specific and highly conserved moiety. The polypeptide was cloned, purified, and used to immunize mice to raise specific immunoglobulins. The purified antibodies were conjugated to gold nano-particles and used to assemble an immunochromatographic strip (ICS). The developed prototype ICS detected as low as 5 µg purified polypeptide and 102 CFU/mL S. aureus within 15 min. The strip showed superior ability to directly detect S. aureus in neonatal sepsis blood specimens without prior sample processing. Moreover, it showed no cross-reaction in specimens infected with two other major causes of neonatal sepsis; coagulase-negative staphylococci and Klebsiella pneumoniae. The selected NWMN_1649-derived polypeptide demonstrates success as a promising biomolecule upon which a prototype ICS has been developed. This ICS provides a rapid, direct, sensitive, and specific option for the detection of S. aureus causing neonatal sepsis. Such a tool is urgently needed especially in resources-limited countries.

A Study of the Virulence Traits of Carbapenem-Resistant Klebsiella pneumoniae Isolates in a Galleria mellonella Model.

The increasing incidence of carbapenem-resistant Klebsiella pneumoniae (CRKP) strains is considered as a terrifying public health concern. This study target was to gain a further insight into the virulence traits of CRKP isolates in Egypt. The study was carried out by using 43 clinical K. pneumoniae isolates. Antibiotic susceptibility testing, biofilm formation assay, and molecular characterization of carbapenemase and virulence genes were done for all isolates. In addition, the genotypic relationship between CRKP isolates was identified by using enterobacterial repetitive intergenic consensus-polymerase chain reactions (ERIC-PCRs). A Galleria mellonella survival assay was adopted for in vivo testing of virulence of the CRKP. Carbapenem resistance was exhibited among 58% (25/43) isolates. Minimum inhibitory concentration values of carbapenem-resistant K. pneumoniae (CRKP) ranged from 32 to 128 µg/mL. Biofilm assay has revealed that 21 isolates (49%) had moderate biofilm formation and 11 isolates (25.5%) were strong biofilm producers. BlaNDM-1 was recognized in 20.9% (9/43) of the isolates, while blaOXA-48 was observed in 18.5% (8/43). Type 3 fimbriae (mrkD) and entB were addressed among 72.1% and 62.8% of K. pneumoniae isolates, respectively. The ybtS and iutA genes were detected among 44.2% and 37.2% of the isolates, respectively. ERIC-PCR showed 23 genetic profiles among CRKP isolates. CRKP biofilm producers were virulent according to the G. mellonella model, which indicates the importance of biofilm as a virulence trait among CRKP. This study indicates the emergence of CRKP with increased virulence traits, especially biofilm formation, in Egypt. This alarming report highlights the ongoing need for effective screening procedures and strict infection control measures.

Unmodified gold nanoparticles for direct and rapid detection of Mycobacterium tuberculosis complex.

OBJECTIVES: This work aims to develop rapid nano-gold assay prototypes for specific detection of Mycobacterium tuberculosis complex (MTBC). DESIGN AND METHODS: Spherical gold nanoparticles (AuNPs, 14nm) were synthesized by citrate reduction method and characterized by spectrophotometry and SEM. MTB 16s rDNA regions were amplified by PCR and amplicons were detected using genus- and species-specific oligotargeters and AuNPs. In a second prototype, MTBC unamplified genomic DNA was directly detected using species-specific oligo-targeters and AuNPs. RESULTS: Detection limits were 1ng for PCR product and 40ng for genomic DNA. The nano-gold prototype detected 45 positive genomic DNA samples which were also positive with automated liquid culture system (BACTEC™ MGIT™) and semi-nested PCR (100% concordance). Following DNA extraction, using standard procedures, the TB nano-gold prototype turnaround time is about 1h. CONCLUSIONS: We have developed nano-gold assay prototype for direct and inexpensive detection of MTBC. The developed prototypes are simple, sensitive, rapid and can substitute PCR-based detection. The developed assay may show potential in the clinical diagnosis of TB especially in developing countries.

Quantum dots: heralding a brighter future for clinical diagnostics.

Quantum dots (QDs) are semiconductor nanocrystals that possess unique optical properties including broad-range excitation, size-tunable narrow emission spectra and high photostability, giving them considerable value in various biomedical applications. The size and composition of QDs can be varied to obtain the desired emission properties and make them amenable to simultaneous detection of multiple targets. Furthermore, numerous surface functionalizations can be used to adapt QDs to the needed application. The successful use of QDs has been reported in the areas of in vitro diagnostics and imaging. There is also potential for multimodal applications for simultaneous imaging. Toxicity issues are still a prime concern with regards to in vivo applications on account of the toxic constituents of QDs.