Reduction of biofilm formation of Escherichia coli by targeting quorum sensing and adhesion genes using the CRISPR/Cas9-HDR approach, and its clinical application on urinary catheter.

BACKGROUND: Escherichia coli is a common cause of biofilm-associated urinary tract infections (UTIs). Biofilm formation in E. coli is responsible for various indwelling medical device-associated infections, including catheter-associated urinary tract infections (CAUTIs). This study aimed to reduce biofilm formation of E. coli ATCC 25922 by knocking out genes involved in quorum sensing (QS) (luxS) and adhesion (fimH and bolA) using the CRISPR/Cas9-HDR approach. METHOD: Single-guide RNAs (sgRNAs) were designed to target luxS, fimH and bolA genes. Donor DNA for homologous recombination was constructed to provide accurate repairs of double-strand breaks (DSBs). A biofilm quantification assay (crystal violet assay) was performed to quantify the biofilm formation of mutant and wild-type strains. Morphological changes in biofilm architecture were confirmed by scanning electron microscopy (SEM). Further application of the biofilm formation of mutant and wild-type strains on urinary catheter was tested. RESULTS: Crystal violet assay showed that the biofilm formation of ΔfimH, ΔluxS, and ΔbolA strains was significantly reduced compared to the wild-type strain (P value<0.001). The percentage of biofilm reduction of mutant strains was as follows: ΔluxS1 77.51 %, ΔfimH1 78.37 %, ΔfimH2 84.17 %, ΔbolA1 78.24 %, and ΔbolA2 75.39 %. Microscopic analysis showed that all mutant strains lack extracellular polymeric substances (EPS) production compared to the wild-type strain, which was embedded in its EPS matrix. The adherence, cell aggregation, and biofilm formation of wild-type strain on urinary catheters were significantly higher compared to ΔfimH, ΔluxS and ΔbolA strains. CONCLUSION: Altogether, our results demonstrated that the knockout of luxS, fimH, and bolA genes reduced EPS matrix production, which is considered the main factor in the development, maturation, and maintenance of the integrity of biofilm. This pathway could be a potential strategy to disrupt E. coli biofilm-associated UTIs. This study suggests that CRISPR/Cas9-HDR system may provide an efficient and site-specific gene editing approach that exhibits a possible antibiofilm strategy through intervention with the QS mechanism and adhesion property to suppress biofilm formation associated with UTI catheter infections.

Dizziness in Saudi Arabia: An epidemiologic study.

Introduction: Dizziness is one of the most common and recurring complaints in adults presenting at the clinic. However, its prevalence in the population of the Kingdom of Saudi Arabia remains unclear. We aimed to examine the prevalence and correlates of dizziness in a large sample of the Saudi population. Methods: In this is cross-sectional study, we used an electronic survey, which was completed by 1.478 respondents, with a response rate of 84% across five regions of Saudi Arabia. The online survey was launched on the Qualtrics website and distributed via social media channels to obtain heterogeneous responses. The study included adults aged ≥18 years who resided in Saudi Arabia during data collection. We used t-test and chi-square test for descriptive analysis and multiple logistic regression model to assess prevalence and predictors of dizziness. Results: More than half of the participants were aged between 26 years and 45 years (58.66%). Of the participants, 42.97% reported having dizziness at the time of taking the survey. Women were less likely than men to report dizziness (OR = 0.65; CI, 0.49, 0.87; p = 0.003). A description of the type of dizziness by age revealed that vertigo slightly decreased with age. Unclear vision with movement or blurry vision was common in young adults, whereas imbalance was common in older adults. A multiple regression model adjusted for demographic characteristics revealed a statistically significant association between dizziness and age group. Participants in the age group of 46-55 years were 1.83 times more likely to report dizziness compared to those aged >65 years (odds ratio = 1.83; confidence interval, 0.62, 5.41; p = 0.0009). Discussion: Dizziness is a common complaint in Saudi Arabia. Future studies should elucidate the risk factors for and mechanisms of dizziness to help prevent falls and reduced quality of life.

Identification and Characterization of Plasmids and Genes from Carbapenemase-Producing Klebsiella pneumoniae in Makkah Province, Saudi Arabia.

Klebsiella pneumoniae (K. pneumoniae) is involved in several hospital and community-acquired infections. The prevalence of K. pneumoniae-producing-carbapenemase (KPC) resistance genes rapidly increases and threatens public health worldwide. This study aimed to assess the antibiotic resistance level of K. pneumoniae isolates from Makkah Province, Saudi Arabia, during the Islamic 'Umrah' ritual and to identify the plasmid types, presence of genes associated with carbapenem hydrolyzing enzymes, and virulence factors. The phenotypic and genotypic analyses based on the minimum inhibitory concentration (MIC), biofilm formation, PCR, and characterization of KPC-encoding plasmids based on the replicon typing technique (PBRT) were explored. The results showed that most isolates were resistant to carbapenem antibiotics and other antibiotics classes. This study identified sixteen different replicons of plasmids in the isolates and multiple genes encoding carbapenem factors, with blaVIM and blaOXA-48 being the most prevalent genes identified in the isolates. However, none of the isolates exhibited positivity for the KPC production activity. In addition, this study also identified six virulence-related genes, including kfu, wabG, uge, rmpA, fimH, and a capsular polysaccharide (CPS). Together, the data reported in this study indicate that the isolated K. pneumoniae during the pilgrimage in Makkah were all resistant to carbapenem antibiotics. Although the isolates lacked KPC production activity, they carried multiple carbapenem-resistant genes and virulence factors, which could drive their resistant phenotype. The need for specialized methods for KPC detection, monitoring the possibility of nosocomial transmission, and diverse therapeutic alternatives are necessary for controlling the spreading of KPC. This study can serve as a reference for clinicians and researchers on types of K. pneumoniae commonly found during religious gathering seasons in Saudi Arabia.

Polynuclear aromatic anthracene biodegradation by psychrophilic Sphingomonas sp., cultivated with tween-80.

Anthracene is a low molecular weight polynuclear aromatic hydrocarbons (PAHs) being identified as a precedence toxic contaminant in the ecosystem. Thus, the present work was designed to evaluate anthracene biodegradation efficiency by selected marine bacteria. From the marine isolates, the most effective anthracene biodegrading strain was identified as Sphingomonas sp., KSU05. Time course batch growth results indicated that the isolate KSU05 was capable of surviving up to 500 mg/L of anthracene. The influence of various nutrient sources were screened for enhanced growth and pyrene degradation, based on results glucose and tween-80 were used for further optimization studies. Batch experimental analysis showed maximum biodegradation (70.5%) of anthracene (50 mg/L) with enhanced survival of Sphingomonas sp. KSU05 was observed at 96 h of cultivation. Box-Behnken design optimization results showed that the culture conditions enhanced the anthracene biodegradation (90.0%) at pH 7.0, 0.3 mM of tween-80 concentration, and 5.5% of glucose concentration. In addition, the isolate Sphingomonas sp. KSU05 was found to rapidly degrade anthracene within 96 h. The anthracene intermediates was analyzed using Gas chromatography mass spectrophotometer (GC-MS). Overall, this research shown that the Sphingomonas sp., cultivated with suggested optimum conditions could provide an effective prospective for the degradation of anthracene from contaminated environment.

Characterization of biofilm formed by multidrug resistant Pseudomonas aeruginosa DC-17 isolated from dental caries.

The present work reports with the screening of biofilm-producing bacteria from the dental caries. The dental pathogens showed resistance against various antibiotics and biofilm forming ability at various levels. Among the bacterial strain, Pseudomonas aeruginosa DC-17 showed enhanced biofilm production. Extracellular polymeric substance (EPS) was synthesized by the selected bacterial isolate considerably and contributed as the major component of biofilm. EPS composed of eDNA, proteins and lipids. The total protein content of the EPS was found to be 1.928 mg/mL and was the major component than carbohydrate and DNA. Carbohydrate content was 162.3 mg/L and DNA content of EPS was 4.95 µg/mL. These macromolecules interacted in the matrix to develop dynamic and specific interactions to signalling biofilm to differentiating various environments. Also, the isolated bacteria showed resistant against various commercially available antibiotics. The isolates showed more resistance against penicillin (98%) and were sensitive against amoxicillin. Among the factors, temperature, pH and sugar concentration influenced biofilm formation. Biofilm forming ability of the selected bacterial stain was tested at various pH values and alkaline pH was favoured for biofilm production. Biofilm production was found to be maximum at 40 °C and 8% sucrose enhanced biofilm formation. Biofilm formed by P. aeruginosa DC-17 was resistant against various tested antimicrobials and chemicals.

Anti-biofilm activity of plant derived extracts against infectious pathogen-Pseudomonas aeruginosa PAO1.

BACKGROUND: Biofilm forming ability of Pseudomonas aeruginosa make them vulnerable, because it makes them recalcitrant against various antibiotics. Quorum sensing (QS) is cell density based signaling that helps in bacterial cell-cell communication, which regulated various virulence factors such as pigment and biofilm formation that contribute in the establishment of chronic infections. The interruption of QS is one of the effective approach to control various virulence factors. Present study was intended with the aim to authenticate antibiofilm potential in different solvents based extracts of selected medicinal plant species viz. Berginia ciliata, Clematis grata and Clematis viticella traditionally used by the inhabitants of Himalayan region of Pakistan to treat various pathogenic diseases. P. aeruginosa PAO1, an opportunistic pathogen and involves in various life-threatening infections specifically in immune deficient patients was used as a model pathogen. METHODS: Plants were extracted in various organic (ethanol, methanol, acetone, ethyl acetate, hexane, chloroform) as well as in aqueous solvents and their ability to inhibit biofilm was measured. Biofilm of PAO1 was grown in Jensen's medium while growing at 30°C and crystal violet assay was performed to assess the biofilm inhibiting activity of plant extracts. RESULTS: Solvents play a vital role in extraction of plant components and it was found that the plants in various solvents exhibit different activity against the PAO1 biofilm. Comparatively, 1% methanolic extract of B. ciliata (rhizome with skin), showed more than 80% inhibition of biofilm formation without effecting on the growth of the bacterium. Significant correlation between flavonoids content and antibiofilm activity in methanolic extract revealed the contribution of secondary metabolites in P. aeruginosa (PAO1) biofilm inhibition. CONCLUSION: Our study revealed that plants under investigation more specifically B. ciliata could be a potential candidate for drug discovery to treat P. aeruginosa PAO1, induced infectious diseases especially for its biofilm treatment.

Exploring the potential of halotolerant bacteria for biodegradation of polycyclic aromatic hydrocarbon.

The present study aimed to determine the degradation and transformation of three-ring PAHs phenanthrene and anthracene by Cryptococcus sp. MR22 and Halomonas sp. BR04 under halophilic conditions. The growth progress of Cryptococcus sp. MR22 and Halomonas sp. BR04 on anthracene and phenanthrene was monitored by colony-forming unit (CFU) technique. The growth of the bacteria was maintained at a maximum concentration of 200 mg/L of all tested hydrocarbon, indicating that Cryptococcus sp. MR22 and Halomonas sp. BR04 significantly perform in the removal of the PAH-contaminated medium at low concentrations. The fit model to represent the biodegradation kinetics of both PAHs was first-order rate equation The extract prepared from cells supplemented with three different substrates exhibited some enzymes such as hydroxylase, dioxygenase, laccase and peroxidase. The results suggest that both strains had an impressive ability in the degradation of aromatic and aliphatic hydrocarbon but also could tolerate in the extreme salinity condition.

Incidence of Clostridium perfringens and its toxin genes in the gut of children with autism spectrum disorder.

This study was designed to determine the incidence of Clostridium perfringens and their toxin genes in children with autism spectrum disorder (ASD), and determine the antimicrobial susceptibility of C. perfringens isolates. A hundred and fourteen fecal samples were obtained from children aged 3-12 years old (57 samples from ASD children and 57 from healthy controls). Children were divided into four groups based on their gastrointestinal (GI) symptoms as follows: ASD group with and without GI symptoms, and control group with and without GI symptoms. Selective anaerobic media and VITEK®2 ANC ID card were used for isolation and identification of C. perfringens from fecal samples. Antimicrobial susceptibility of C. perfringnes isolates were performed using (E-Test) strips against clindamycin, penicillin and metronidazole antibiotics. Conventional PCR was used to detect the alpha toxin gene (Cpa) and the beta-2 toxin gene (Cpb2). Genetic Analyzer 3130Xi was used to confirm the sequencing of Cpb2 gene. Our findings indicated that 38.6% of ASD group samples had a significantly (p = 0.003) higher incidence of C. perfringens than that of the control group (14%). The highest incidence of C. perfringens was found in the ASD group with GI symptoms (53.8%; p = 0.001). C. perfringens isolated from the ASD group (54.5%) were significantly (p = 0.047) more resistant to clindamycin than those isolated from the control group (12.5%). The C. perfringens isolates from the ASD and the control group showed 95.5% and 100% susceptibility to penicillin, respectively. All C. perfringens isolates of ASD and control group were susceptible to metronidazole. The Cpa toxin gene was also detected in all the C. perfringens isolates of ASD and control group, both with and without GI symptoms. Cpb2 toxin gene showed 100% incidence in ASD samples with GI symptoms and in the control groups both with or without GI symptoms, while it was present at significantly lower levels (25%) in the ASD samples without GI symptoms (p = 0.001). Our findings suggests that a high incidence of C. perfringens and its toxin gene (Cpb2) are associated with the GI complications in ASD which may affect the severity of the disease.