Combining RSPH9 founder mutation screening and next-generation sequencing analysis is efficient for primary ciliary dyskinesia diagnosis in Saudi patients.

Primary ciliary dyskinesia (PCD) is a clinically and genetically heterogeneous ciliopathy. Dysfunction of motile respiratory and nodal cilia results in sinopulmonary symptoms associated with laterality defects (LD) found in half of the patients. The molecular basis of the disease is insufficiently investigated in patients originating from the Arabian Peninsula. In a group of 16 unrelated Saudi patients clinically suspected of PCD and among whom only 5 (31%) had LD, we first screened by PCR-RFLP two founder mutations, RSPH9 c.804_806del and CCDC39 c.2190del previously identified in patients from the Arabian Peninsula and Tunisia, respectively. When negative, targeted panel or whole-exome sequencing was performed. Three patients were homozygous for the mutation in RSPH9, which encodes an axonemal protein that is absent from nodal cilia. None of the patients carried the CCDC39 founder mutation frequent in Tunisia. NGS analysis showed that nine patients had homozygous mutations in PCD genes. In total, sequential RFLP and NGS analysis solved 75% (12/16) of cases and identified ten distinct mutations, among which six are novel, in nine different genes. These results, which highlight the genetic heterogeneity of PCD in Saudi Arabia, show that the RSPH9 c.804_806del mutation is a prevalent mutation among Saudi patients, whereas the CCDC39 c.2190del ancestral allele is most likely related to the Berber population. This study shows that RSPH9 founder mutation first-line screening and NGS analysis is efficient for the genetic exploration of PCD in Saudi patients. The RSPH9 founder mutation accounts for the low rate of LD among Saudi patients.

Antibacterial and Biofilm Inhibitory Activity of Medicinal Plant Essential Oils Against Escherichia coli Isolated from UTI Patients.

Urinary tract infections (UTIs), caused by Escherichia coli 80% to 85% of the time, are one of the most important causes of morbidity and health care spending affecting persons of all ages. These infections lead to many difficult problems, especially increasing resistance to antibiotic drugs. Bacterial biofilms play an important role in UTIs, responsible for persistent infections leading to recurrences and relapses. In this study, we have investigated the antibacterial activity of five medicinal plant essential oils against UTIs caused by E. coli using disc diffusion and minimal inhibition concentration (MIC) methods. In addition, biofilm inhibitory action of oils was realized by crystal violet. Gas chromatography⁻mass spectrometry (GC⁻MS) analysis showed a variability between oils in terms of compound numbers as well as their percentages. Antibacterial activity was observed only in cases of Origanum majorana, Thymus zygis and Rosmarinus officinalis, while Juniperus communis and Zingiber officinale did not showed any effect towards E. coli isolates. T. zygis essential oil demonstrated the highest antibacterial activity against E. coli isolates, followed by O. majorana and R. officinalis. Further, oils showed high biofilm inhibitory action with a percentage of inhibition that ranged from 14.94% to 94.75%. R. officinalis oil had the highest antibiofilm activity followed by T. zygis and O. majorana. Accordingly, tested oils showed very effective antibacterial and antibiofilm activities against E. coli UTIs and can be considered as good alternative for antibiotics substitution.

Effect of combined long-term starvation and γ-irradiation on membrane fatty acids and cell surface hydrophobicity of Salmonella enterica serovar Typhimurium.

This study was carried out to explore the adaptive mechanisms of Salmonella enterica serovar Typhimurium, in particular the implication of fatty acids (FA) in the remodeling of membrane lipid composition to overcome the combined effects of long-term starvation and γ-irradiation stresses. In addition, cell surface hydrophobicity was also evaluated. The bacterial strains (control and starved) were treated with a nonlethal γ-irradiation dose of 0.5 kGy and sublethal doses of 1 kGy. Gas chromatography analysis showed that the FA composition of starved and γ-irradiated cells was modified. However starvation combined with γ-irradiation induced more modifications in the FA composition than γ-irradiation or starvation alone. Indeed, the unsaturated FA-to-saturated FA ratio decreased significantly for both strains compared with γ-irradiated cells, as main consequence of the cyclic FA formation. Our results showed that starvation, irradiation, or combined stresses significantly influenced the hydrophobicity, and this may have affected the virulence state of Salmonella Typhimurium cells. This study represents one of the few to demonstrate the modifications on bacterial membrane as a cellular response to survive to the ionizing radiation combined with long-term starvation stress.

Evaluating aflatoxins and Sudan dyes contamination in red chili and turmeric and its health impacts on consumer safety of Lahore, Pakistan.

Spices are contaminated with aflatoxins (AFs) and Sudan dyes which are classified as class Group 1 and Group 3 human carcinogens by the International Agency for Research on Cancer (IARC) respectively and their prolonged exposure may raise a human health concern. A total of 474 samples of red chili and turmeric were collected from Lahore city and were subjected to quantitative and qualitative AFs and Sudan dyes analysis by thin layer chromatography (TLC) respectively. The number of red chili and turmeric samples with ≥10 µg/kg of total AFs (European Union standard limit) were 70% and 33% respectively and considered unfit for human consumption. The presence of Sudan dyes in red chili and turmeric samples was 67% and 27% respectively. The mean estimated daily intake (EDI) among females and males was 0.0019 µg/kg bw/day, 0.0012 µg/kg bw/day for red chili, and 0.0008 µg/kg bw/day, 0.0006 µg/kg bw/day for turmeric respectively. The mean value of margin of exposure (MOE) among females and males for ingestion of AFs-contaminated red chili and turmeric was 210.25, 332.13, 501.02, and 699.31 respectively. Therefore, the current study demands a continuous monitoring plan and the implementation of novel techniques to enhance the product's quality and protect public health.

Biofilm formation, cell surface hydrophobicity, and fatty acids analysis of starved Salmonella enterica serovar Typhimurium in seawater.

Salmonella is an international foodborne pathogen widely disseminated in seawater that regularly causes large outbreaks of food poisoning. In this study, we have investigated the effect of starvation on the ability of Salmonella enterica serovar Typhimurium cells to adhere to polystyrene microplate and Hep2 cells in seawater microcosms after incubation for 3 years. Cell surface hydrophobicity was evaluated. Effect of stress on fatty acids composition was also established. Our results showed that after incubation in seawater, the ability of starved cells to adhere to polystyrene microplate was decreased significantly. However, the adhesion values to Hep2 cells have increased. In addition, cells surface hydrophobicity was decreased. The fatty acids composition of starved cells was modified.

Primary ciliary dyskinesia associated with multiple drug intolerance syndrome: a case report.

INTRODUCTION: The term multiple drug intolerance syndrome is used for patients who express adverse drug reactions to three or more drugs without a known immunological mechanism. It is a distinct clinical entity, different from cross-reactivity. The symptoms can range from a benign rash to life threatening syndromes like drug reaction with eosinophilia and systemic symptoms. CASE REPORT: We report the case of an 8-year-old child with primary ciliary dyskinesia complicated by bronchiectasis who presented multiple drug intolerance syndrome.Through this observation; we discuss the diagnostic elements of this syndrome. CONCLUSION: In the absence of validated criteria for diagnosing multiple drug intolerance syndrome, a detailed history is essential, especially to identify the warning signs and the risk factors.

Molecular characterization of multidrug resistant Klebsiella pneumoniae clinical isolates recovered from King Abdulaziz Specialist Hospital at Taif City, Saudi Arabia.

Klebsiella pneumoniae is an opportunistic pathogen responsible for a significant proportion of nosocomial and community-acquired infections. Genotypic variation in K. pneumoniae populations is a major barrier to control public health risk associated with pathogen. In this work, thirty K. pneumoniae were recovered from hospital and were tested for their resistance to antibiotics. Genetic variability of the isolates was performed using PCR based on genes coding for porins and efflux pumps, (GTG)5 and BOX repetitive sequences. K. pneumoniae showed heterogenicity of resistance to antibiotics based on gender or specimen type. Further, out of 30 isolates, 25 different profiles were found and 83.33% are multidrug-resistant. PCR detection of genes coding for porins and efflux pumps revealed seven different genotypes and strong correlation between antibiotics resistance profiles and investigated genes. PCR genomic fingerprinting showed high genetic diversity of K. pneumoniae. BOX-PCR and (GTG)5 generated 18 and 19 clusters with discriminatory indexes 0.97 and 0.98, respectively at 80% of similarity. K. pneumoniae clinical isolates showed high phenotypic and genetic variability, and many strains can be circulating simultaneously. This genetic variability should be taken into consideration when designing strategies for controlling K. pneumoniae outbreaks. In addition, a significant correlation, was detected for the first time, between (GTG)5-genotyping and antibiotic resistance patterns of K. pneumoniae and could be valuable in the prediction of antibiotic resistance profiles of K. pneumoniae.

Molecular Investigation of Multidrug-Resistant Escherichia coli Clinical Isolates from Patients with Urinary Tract Infections.

<b>Background and Objective:</b> Urinary tract infections believe to be one of the main acquainted infections by <i>Escherichia coli</i> in hospitals with an excessive incidence of illness. This study aimed to analyze the antibiotic resistance profile and molecular characteristics of <i>E. coli</i> isolates recovered from patients with urinary tract infection at different hospitals in Taif Governorate, Saudi Arabia. <b>Materials and Methods:</b> Out of 143 isolates collected for 11 months, from February-December 2019, 24 isolates were identified as <i>E. coli</i> by API system and 16S rRNA sequences techniques. An antibiotic sensitivity test was performed using the disk diffusion method. Besides, the repetitive sequence repeat-PCR (Rep-PCR) technique was used for genotyping the 24 isolates. <b>Results:</b> Almost all isolates were resistant to most tested antibiotics such as ampicillin, ceftazidime, cefepime, trimethoprim/sulfamethoxazole, amox/clavulanic. The PCR results show that virulence genes <i>kpsII</i> and <i>yaiO</i> were detected in all <i>E. coli</i> isolates. <i>Stx1</i>, <i>fimH</i>, <i>hly</i> and <i>uidA</i> were moderate detected in all isolates. <b>Conclusion:</b> The high frequencies of antibiotic-resistant <i>E. coli</i> isolates in patients with urinary tract infections in the current study suggest that continuous surveillance of the use of appropriate antibiotics is required and that control of infections is necessary.

Biofilm Inhibition and Eradication Properties of Medicinal Plant Essential Oils against Methicillin-Resistant Staphylococcus aureus Clinical Isolates.

Methicillin-resistant Staphylococcus aureus is a major human pathogen that poses a high risk to patients due to the development of biofilm. Biofilms, are complex biological systems difficult to treat by conventional antibiotic therapy, which contributes to >80% of humans infections. In this report, we examined the antibacterial activity of Origanum majorana, Rosmarinus officinalis, and Thymus zygis medicinal plant essential oils against MRSA clinical isolates using disc diffusion and MIC methods. Moreover, biofilm inhibition and eradication activities of oils were evaluated by crystal violet. Gas chromatography-mass spectrometry analysis revealed variations between oils in terms of component numbers in addition to their percentages. Antibacterial activity testing showed a strong effect of these oils against MRSA isolates, and T. zygis had the highest activity succeeded by O. majorana and R. officinalis. Investigated oils demonstrated high biofilm inhibition and eradication actions, with the percentage of inhibition ranging from 10.20 to 95.91%, and the percentage of eradication ranging from 12.65 to 98.01%. O. majorana oil had the highest biofilm inhibition and eradication activities. Accordingly, oils revealed powerful antibacterial and antibiofilm activities against MRSA isolates and could be a good alternative for antibiotics substitution.

Alterations of outer membrane proteins and virulence genes expression in gamma-irradiated Vibrio parahaemolyticus and Vibrio alginolyticus.

Gamma-irradiation technology sterilizes microorganisms and thereby prevents decay and improves the safety and shelf stability of food products. In this study we treated the foodborne pathogens Vibrio parahaemolyticus and Vibrio alginolyticus with gamma-irradiation (0.5 kGy) to evaluate their adaptative response. Outer membrane protein patterns of irradiated bacteria were found altered when analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. These modifications were manifested by the appearance and/or disappearance of bands as well as in the expression level of certain proteins. In addition, we searched for the presence of eight Vibrio cholerae virulence genes, toxR, toxS, toxRS, ctxA, zot, ace, toxT, and virulence pathogenicity island (VPI), in the genome of investigated strains. The expression of toxR, toxS, VPI, and ace genes in gamma-irradiated bacteria, studied by reverse transcriptase polymerase chain reaction, was altered. These variations were manifested by an increase and/or a decrease in the expression level of tested virulence genes.

Adherence assays and Slime production of Vibrio alginolyticus and Vibrio parahaemolyticus.

In this study we investigated the phenotypic slime production of Vibrio alginolyticus and Vibrio parahaemolyticus strains, food-borne pathogens, using a Congo red agar plate assay. Furthermore, we studied their ability to adhere to abiotic surfaces and Vero cells line. Our results showed that only V. alginolyticus ATCC 17749 was a slime-producer developing almost black colonies on Congo red agar plate. Adherence to glace tube showed that all V. alginolyticus strains were more adherent than V. parahaemolyticus. Only V. alginolyticus ATCC 17749 was found to be able to form biofilm on polystyrene microplate wells (OD570 = 0.532). Adherence to Vero cells showed that all tested strains were non adherent after 30 min, however after 60 min all the studied strains become adherent. The percentage of adherence ranged from1.23% to 4.66%.

Detection of cell surface hydrophobicity, biofilm and fimbirae genes in salmonella isolated from tunisian clinical and poultry meat.

BACKGROUND: The aim of this study was to evaluate the ability of 15 serotypes of Salmonella to form biofilm on polystyrene, polyvinyl chloride (PVC) and glass surfaces. . METHODS: Initially slime production was assessed on CRA agar and hydrophobicity of 20 Salmonella strains isolated from poultry and human and two Salmonella enterica serovar Typhimurium references strains was achieved by microbial adhesion to n-hexadecane. In addition, biofilm formation on polystyrene, PVC and glass surfaces was also investigated by using MTT and XTT colorimetric assay. Further, distribution of Salmonella enterotoxin (stn), Salmonella Enteritidis fimbrial (sef) and plasmid encoded fimbrial (pef) genes among tested strains was achieved by PCR. RESULTS: Salmonella strains developed red and white colonies on CRA and they are considered as hydrophilic with affinity values to n-hexadecane ranged between 0.29% and 29.55%. Quantitative biofilm assays showed that bacteria are able to form biofilm on polystyrene with different degrees and 54.54% of strains produce a strong biofilm on glass. In addition, all the strains form only a moderate (54.54%) and weak (40.91%) biofilm on PVC. PCR detection showed that only S. Enteritidis harbour Sef gene, whereas Pef and stn genes were detected in S. Kentucky, S. Amsterdam, S. Hadar, S. Enteritidis and S. Typhimurium. CONCLUSION: Salmonella serotypes are able to form biofilm on hydrophobic and hydrophilic industrial surfaces. Biofilm formation of Salmonella on these surfaces has an increased potential to compromise food safety and potentiate public health risk.

Identification of outer membrane proteins of Vibrio parahaemolyticus and Vibrio alginolyticus altered in response to γ-irradiation or long-term starvation.

Vibrio parahaemolyticus and Vibrio alginolyticus were subjected to γ-irradiation (0.5 kGy) or starvation by incubation for 8 months in seawater to study modifications in their outer membrane protein patterns. After treatment, outer membrane protein profiles of starved or γ-irradiated bacteria were found to be altered when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Altered proteins were identified by mass spectrometry (MS and MS/MS) and analyses revealed that OmpU can be considered a starvation stress-induced protein. In addition, expression of OtnA, OmpW, OmpA and peptidoglycan-associated lipoprotein decreased to non-detectable levels in starved cells. Furthermore, MltA-interacting protein MipA appeared under γ-irradiation or starvation conditions. Thus, it can be considered to be a γ-irradiation, long-term starvation stress protein in some vibrios.

Preliminary safety assessment of Yarrowia lipolytica extracellular lipase: results of acute and 28-day repeated dose oral toxicity studies in rats.

Interest in extracellular lipase sourced from the non conventional yeast Yarrowia lipolytica has increased over the last decade. The enzyme was recently suggested as a good candidate for pancreatic exocrine insufficiency treatment. However, there is still a lack of oral safety evaluation data. In this work, we conducted acute and 28-day repeated dose toxicity studies in rats. Both male and female rats were first orally treated with fungal lipase at either single or repeated doses. The results demonstrated that neither single dose nor chronic administration of lipase was associated with mortality or abnormalities in general conditions, behavior and growth. Except a decrease in urine pH and a dose-unrelated increase of triglycerides observed in males, chronic administration of lipase resulted in similar hematological, blood biochemical and urine parameters to those of untreated animals. Minor histopathological changes were observed in lungs and livers of treated and untreated animals but they were considered of no toxicological significance. This study provides, for the first time, safety data on Yarrowia lipolytica extracellular lipase that support its use as a pharmaceutical.