The effect of antifungal combination on transcripts of a subset of drug-resistance genes in clinical isolates of Candida species induced biofilms.

Biofilm formation is often associated with increased Candida resistance toward antifungal agents. Therefore, the current study aimed to assess the incidence of biofilm formation among Candida isolates and to investigate the effect of high doses of fluconazole {FLC}, voriconazole {VOC} and amphotericin B {AMB}, singly and in combination on mature biofilms. Moreover, it aimed to assess the expression of selected genes (CDR1, KRE1 and SKN1) responsible for Candida biofilm resistance. The study included 49 patients; samples were collected from the King Khalid Hospital, Riyadh, Saudi Arabia. Isolates were prepared for biofilm formation and quantification using 0.4% (w/v) crystal violet. Minimum Inhibitory concentration (MIC) and fractional inhibitory concentration (FIC) were conducted by the broth microdilution method. Biofilm eradication was evaluated using counting, XTT stain intensity and observed under the inverted microscope. Selected genes were evaluated in Candida biofilms under the effect of antifungal exposure using QPCR. The major isolates were Candida albicans (65.3%) followed by Candida tropicalis and Candida glabrata. 77.6% of the strains were biofilm formers. AMB showed susceptibility in 87.8% of isolates, followed by VOC (77.6%) and FLC (67.3%). MIC50 and MIC90 were (0.03, 0.125), (0.5, 8), (2, >128) µg/ml for AMB, VOC and FLC, respectively. 34.7% and 18.4% of the isolates were antagonistic to AMB/FLC and AMB/VOC, respectively. Mature biofilms of ten selected isolates were found resistant to FLC (1000 µg/ml). VOR and AMB concentration required to inhibit biofilm formation was 16-250 fold higher than the MIC for planktonic cells. Isolates showed significant reduction with antifungal combination when compared with the untreated controls (p value â©½ 0.01), or using fluconazole alone (p value â©½ 0.05). High doses of the antifungals were employed to assess the effect on the persisters' selected gene expression. Marked over expression of SKN1 and to a lesser extent KRE1 was noticed among the mature biofilms treated with AMB alone or in combination after 1 h of exposure, and SKN1 expression was even more sharply induced after 24 h. No statistically significant over expression of CDR1 was observed in biofilms after exposure to high doses of FLC, VOC or any of the combinations used.

Use of the single cell gel electrophoresis (comet assay) for comparing apoptotic effect of conventional antibodies versus nanobodies.

The large molecular size of antibodies is considered one major factor preventing them from becoming more efficient therapeutically. It is well established that all camelids have unique antibodies circulating in their blood called heavy-chain antibodies (HcAbs). Unlike antibodies from other species, these HcAbs contain a single variable domain and two constant domains (CH2 and CH3). HcAbs are a novel type of immunoglobulin-like, antigen binding protein with beneficial pharmacokinetic properties that are ideally suited to targeting cellular antigens for molecular imaging or therapeutic purposes. Since the antigen-binding site of dromedary HcAb is comprised in one single domain, it was referred to as nanobody. In the present work, the different IgG subclasses from immunized camel (Camelus dromedairus) were purified employing their different affinity for protein A column (PA) and protein G column (PG). Characterization of IgG subclasses was done by using 12% SDS-PAGE under reducing conditions. Protein bands were visualized after staining with Coomassie Brilliant Blue, showing two bands at 50 kDa and 30 kDa in case of IgG1 while IgG2 and IgG3 produce only one band at 46 kDa and 43 kDa respectively. The induction of apoptosis by either conventional or nanobodies was evaluated on two different cell lines, Colon and Hepatic cancer cell (HCT116 and HepG2), using the comet assay. Induced apoptosis were confirmed by visualizing DNA fragmentation bands on 2% agarose gel, and the gel was photographed under UV light. This study demonstrates the successful targeting of human cancer colon cell lines by nanobodies in vitro. It may open perspectives for their future use as tumor target vehicle, due to their small size, soluble behavior and they interact with epitopes that are less antigenic for conventional antibodies.

Incidence of Helicobacter pylori infection and their clarithromycin-resistant strains in otitis media with effusion regarding phenotypic and genotypic studies.

Helicobacter pylori (H. pylori) are pathogenic bacteria that infect a half of the human population, colonize gastric mucosa and can be found in gastric juice. Reflux of gastric juice has been suggested to be associated with glue ear in children. It has been suggested that tonsil and adenoid tissues are potential reservoirs of H. pylori infection. These observations raise the question as to whether H. pylori infection might have a role in otitis media with effusion (OME) in children. The objectives of this research were to evaluate the incidence and possible role of H. pylori in the pathogenesis of OME in children and to evaluate the clarithromycin-resistant strains. Molecular assessment was done to evaluate the culture results vs. molecular study. A total of 60 children, who were prone to ventilation tube insertion, adenoidectomy and/or tonsillectomy were included in the study. The control group consisted of 40 children who underwent adenoidectomy and/or tonsillectomy without the history of OME. Samples of the middle ear fluid and mucosa, adenoid tissue, tonsillar tissue and gastric lavage were cultured and underwent polymerase chain reaction (PCR) analysis then were assembled by using QIAxcel System as capillary electrophoresis for H. pylori detection. There was significant difference between the results of cultures and PCR (P < 0.05). Middle ear fluid culture was positive for H. pylori in 40% of the patients vs. 56.7% PCR results while middle ear mucosa culture was positive in 20% vs. 26.7% PCR results. Gastric lavage culture was positive in 46.6% of the patients and PCR was positive in 63.3% of the patients. Adenoid culture and PCR were positive in 56.3% for each, while tonsil culture was positive in 70% and PCR was positive in 90%. H. pylori presence in the gastric lavage, the tonsillar and adenoid tissues by culture and PCR was significantly more frequent in the study group compared to the control group. The minimum inhibitory concentration (MIC) values of clarithromycin-resistant isolates ranged from 1.5 to 8 µg/ml. This study showed the presence of H. pylori in around 50% of the patients with OME. PCR revealed its sensitivity than culture techniques. The incidence of clarithromycin resistance was found to be high among the isolates (39.6%).