Molecular detection of different virulence factors genes harbor pslA, pelA, exoS, toxA and algD among biofilm-forming clinical isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) is considered as the foremost cause of hospital-acquired infections due to its innate and plasmid-mediated resistance to multiple antibiotics making it a multi-drug resistant (MDR) pathogen. This study aimed to determine the biofilm formation ability and the presence of different virulence factors genes (pslA, pelA, exoS, toxA and algD) among biofilm-forming strains of P. aeruginosa clinical isolates from burn units in Ismailia Hospitals, Egypt. In our cross-sectional study, one hundred and twenty-six (126) non-duplicate clinical P. aeruginosa isolates were recovered from 450 clinical specimens from burn units in Ismailia Hospitals. The antibiotic sensitivity of strong and moderate biofilm producer isolates was investigated using the disc diffusion method. The isolated bacteria were tested for their ability to form biofilm using a microtiter plate assay. The expression of (pslA, pelA, exoS, toxA and algD) genes in biofilm producers isolates was detected using PCR. The MPA detected 80% (95 /126) isolates as biofilm producers, 18% (22/126) were strong biofilm producers, 34% (43/126) were moderate biofilm producers, 28% (35/126) were weak biofilm producers and 20% (31/126) non-biofilm producers. Susceptibility pattern analysis of biofilm-forming P. aeruginosa isolates (95) detected that 60% (68/ 95) were multi-drug resistant isolates (MDR). Resistance to all used antibiotics and multidrug resistance was higher among biofilm-producing than non-biofilm-producing strains, but the difference was statistically non-significant. Investigation of virulence factors associated genes revealed that 96%, 94%, 86.4%, 80.0% and 74% of the biofilm producers isolates were harboring algD, pslA, pel A, toxA and exoS gene, respectively. The present study confirmed that antimicrobial resistance and virulence genes were more prominent in biofilm-producing P. aeruginosa than in non-biofilm-producers.

Characterization of enterohemorrhagic Escherichia coli from diarrhoeic patients with particular reference to production of Shiga-like toxin.

Enterohemorrhagic Escherichia coli (EHEC) is a subtype of pathogenic E. coli that causes diarrhea or hemorrhagic colitis in humans, which can progresses to hemolytic uremic syndrome (HUS), a leading cause of acute renal failure in children, and morbidity and mortality in adults. Stool samples (n = 273) of patients (1 day-40 years old) suffered from bloody diarrhea and abdominal cramps, were examined bacteriologically and molecularly for the presence and pathogenicity of EHEC with phylogenetic analysis of the obtained stx1, stx2, and eaeA virulence genes' sequences. Overall, 71 (26.01%) E. coli isolates were identified as EHEC with the following serogroupes: O1:H11 (3), O128:H2 (9), O26:H11 (6), O157:H7 (3), O25:H2 (7), O145:H328 (2), O125:H6 (9), O86:H8 (5), O18:H15 (11) and untypable (16). The highest isolation rate were in samples belonged to infants below two years old (42.25%). Antimicrobial susceptibility testing showed that all isolates were highly sensitive to ciprofloxacin, nitrofurantoin, gentamycin, imipenem and vancomycin (100% each), however, they were resistant to ampicillin, cephalexin, penicillin and tetracycline (100% each). In-vitro pathogenicity testing of the isolates revealed that 67 (94.37%) isolates were positive for Congo red test, 47 (66.20%) isolates possessed P fimbriae (MRHA) and 17 (23.94%) possessed type 1 fimbriae (MSHA). Moreover, 46 (64.79%) isolates exhibited hemolysis and 42 (59.15%) isolates showed distinct cytopathic effect to Vero cells. Molecular detection of enterohemorrhagic E. coli (EHEC) pathotype virulence genes, confirmed the presence of stx1 gene in O157:H7 (MA2), O26:H11, O145:H328 and O125:H6 serogroups; stx2 gene in (O157:H7 (MA1), O128:H2 and O25:H2; while all serogroups except (O125:H6) carried the eaeA intimin virulence gene. A phylogenetic tree, based on the nucleotide sequences of toxin-encoding genes, demonstrates that Shiga toxin E. coli (STEC) isolates have considerable genetic variation and belong to various phylogenetic groupings.

Investigation of TLR2 and TLR4 Polymorphisms and Sepsis Susceptibility: Computational and Experimental Approaches.

Toll-like receptors (TLR) play an eminent role in the regulation of immune responses to invading pathogens during sepsis. TLR genetic variants might influence individual susceptibility to developing sepsis. The current study aimed to investigate the association of genetic polymorphisms of the TLR2 and TLR4 with the risk of developing sepsis with both a pilot study and in silico tools. Different in silico tools were used to predict the impact of our SNPs on protein structure, stability, and function. Furthermore, in our prospective study, all patients matching the inclusion criteria in the intensive care units (ICU) were included and followed up, and DNA samples were genotyped using real-time polymerase chain reaction (RT-PCR) technology. There was a significant association between TLR2 Arg753Gln polymorphisms and sepsis under the over-dominant model (p = 0.043). In contrast, we did not find a significant difference with the TLR4 Asp299Gly polymorphism with sepsis. However, there was a significant association between TLR4 Asp299Gly polymorphisms and Acinetobacter baumannii infection which is quite a virulent organism in ICU (p = 0.001) and post-surgical cohorts (p = 0.033). Our results conclude that the TLR2 genotype may be a risk factor for sepsis in adult patients.

Optimized polydopamine coating and DNA conjugation onto gold nanorods for single nanoparticle bioaffinity measurements.

Gold nanorods (NRs) have attracted a great deal of interest for a variety of biomedical and sensing applications. However, developing robust methods for biofunctionalizing NRs has continued to be challenging, especially for NR-DNA conjugates. This is due to the presence of cetyltrimethylammonium bromide (CTAB), which plays an essential role in controlling the anisotropic particle growth. In this article, we systematically explore the growth of a polydopamine (PDA) layer on a range of NR surfaces, comparing different polyelectrolyte and alkanethiol coatings as well as direct CTAB displacement. This revealed that the PDA layer thickness and growth rate is strongly dependent on the underlying nanorod functionalization chemistry and allowed us to establish a preferred route for the creation of stable, non-aggregated suspensions of PDA-coated NRs. The utility of this platform was then demonstrated by self-assembling packed monolayers of single-stranded DNA on the outer surface. Both the surface attachment and bioactivity of the resulting NR-DNA conjugates was then demonstrated by performing bulk solution and single nanoparticle imaging fluorescence measurements.

Light-Triggered Inactivation of Enzymes with Photothermal Nanoheaters.

A universal method for inactivating enzymes on demand is introduced, which involves irradiating nanorod-bound enzymes with near-infrared light. The subsequent generation of plasmonic heat denatures the enzymes selectively without damaging other proteins or cell membranes present in the same solution.

Antifungal activity of silver nanoparticles on Fluconazole resistant Dermatophytes identified by (GACA)4 and isolated from primary school children suffering from Tinea Capitis in Ismailia - Egypt.

Fungal infections caused by dermatophytes recently became more common. Available antifungal drugs are limited because of emergence of resistant strains due to prophylaxis with them, so there is an urgent need for novel antifungals. This study is aimed to detect the antifungal activity of silver nanoparticles (SNPs) on Fluconazole resistant dermatophytes isolated from primary school children clinically suffering from tinea capitis and attending El-Sheikh Zaid Dermatology Center in Ismailia. The study was done on 112 clinical cases. Examination with potassium hydroxide(KOH) of hair samples was done, followed by routine identification using culturing, macroscopical and microscopical examination and biochemical tests, finally molecular identification using Polymerase Chain Reaction (PCR) with (GACA) 4 was done. Fluconazole resistance of these dermatophytes was detected by different methods including agar disc diffusion method and broth microdilution susceptibility testing. Silver nanoparticles susceptibility testing was carried out on these Fluconazole resistant dermatophytes. The Ubiquitin 1 (Ub 1) gene was detected in samples which were Fluconazole resistant but SNPs susceptible. In this study dermatophytes were found only in 70 samples (62.5%). They were belonged to 3 species: Trichophyton violaceum, Microsporum gypseum and Microsporum canis. Fluconazole resistance was found in 58 samples (82.85%). Both M. canis and M. gypseum were resistant to all used concentrations of SNPs, while T. violaceum was susceptible to 50 µg/ml SNPs solution. The Ub1 gene was detected in 1 sample (4.8%). Therefore SNPs can be used for treatment of T. violaceum, while they can't be used for treatment of M. canis or M. gypseum.

Spectrofluorimetric and stability-indicating thin layer chromatographic methods for determination of cabergoline, a prolactin inhibitor in pharmaceuticals.

Simple, Economic, and selective spectrofluorimetric and stability-indicating thin layer chromatographic (TLC) with fluorescence detection methods were developed for the determination of Cabergoline, a potent prolactin inhibitor, and long-acting dopamine receptor agonist, in bulk drug and pharmaceutical dosage forms based on its native fluorescence. Method A was based on measuring the fluorescence intensity at 338 nm after excitation at 280 nm. The measured fluorescence was directly proportional to the concentration of the drug over the range of 50.0-450.0 ng/mL with a limit of detection of 14.4 and a limit of quantification of 43.7 ng/mL. The TLC method (method B) was employed on TLC silica gel 60 F254 aluminum sheets previously exposed to concentrated (30-34 %) hydrochloric acid vapor. Ethyl acetate: n-hexane: diethylamine system with a ratio of (10: 3: 1, v/v/v) developing system was used. The retention factor (Rf) of Cabergoline was 0.58 ± 0.03. Linearity was found to be in the range of 100.0-1500.0 ng/band. The LOD and LOQ were 25.4 and 76.9 ng/band, respectively. The methods were validated successfully according to ICH guidelines.

Association of TLR7 and TLR9 genes polymorphisms in Egyptian patients with systemic lupus erythematosus.

Introduction: Systemic lupus erythematosus (SLE) is a chronic, inflammatory, multiorgan, systemic autoimmune disease. It is characterized by the high production of autoantibodies against nuclear compounds. TLRs (toll-like receptors 7/9) are pattern-recognition receptors that recognize nucleic acids and induce proinflammatory responses by activating NF-kB and producing type I interferon, which play a role in eliciting innate/adaptive immune responses and developing chronic inflammation. TLR7 and TLR9 single nucleotide polymorphisms (SNPs) have been linked to systemic lupus erythematosus in numerous studies (SLE). In this work, we wanted to evaluate and analyze single nucleotide polymorphisms (SNPs) in the TLR7 (rs3853839) and TLR9 (rs187084) genes among Egyptian SLE patients and healthy controls. Method: Whole blood samples were taken from 100 SLE patients and 100 controls; DNA was extracted and then processed for TLR7 rs3853839 and TLR9 rs187084 single nucleotide polymorphisms analysis by real-time polymerase chain reaction technology and restriction fragment-length polymorphism. We also assessed the association between TLR 7 and TLR 9 genes polymorphism with SLE clinical parameters. Results: Our results showed that TLR7 rs3853839 CG genotypes and G allele were significantly associated with SLE. Also, TLR7 rs3853839 genotypes and alleles were significantly associated with nephritis, arthritis, oral ulcers, and thrombocytopenia.Whereas genotypes and alleles of TLR9 were not significantly associated with the risk nor the clinical characteristics of SLE except for malar rash. Conclusion: In the investigated Egyptian cohort, our findings suggest that TLR7 rs3853839 gene polymorphisms increase the risk for SLE development and play a role in developing clinical characteristics, especially nephritis.

Role of MBL2 Polymorphisms in Sepsis and Survival: A Pilot Study and In Silico Analysis.

Sepsis is a serious infection-induced syndrome with serious ramifications, especially in intensive care units. Global concern motivated the investigation of the role of related genes' polymorphism in predicting the liability to infection, sepsis, septic shock and survival. Among these genes is the gene encoding mannose-binding lectin (MBL), with its remarkable importance in the immune system. However, the previous studies showed conflicting results and ambiguity that urged us to engage with this issue in the Egyptian population. Prediction of functional and structural impacts of single nucleotide polymorphisms (SNPs) was done using in silico methods. A prospective observational study was conducted in intensive care units; one hundred and thirty patients were followed up. Genotyping was performed using real-time polymerase chain reaction (RT-PCR) technology. MBL SNPs showed a remarkable high frequency in our population, as well. No significant association was found between MBL2 genotypes and any of our analyses (sepsis, septic shock and survival). Only septic shock and age were independently associated with time of survival by Cox regression analysis. Our study may confirm the redundancy of MBL and the absence of significant impact on sepsis liability and mortality in adult patients.

In silico analysis of missense variants of the C1qA gene related to infection and autoimmune diseases.

Objectives: C1q is a key activator of the classical pathway of the complement system and exerts consequences relating to opsonization and phagocytosis. The C1qA gene is one of three genes encoding the C1q molecule. Defects in C1q, and especially in C1qA, have been linked to an increased susceptibility to infection, sepsis, and systemic lupus erythematosus. These defects could arise from missense single nucleotide polymorphisms (SNPs) and their deleterious impacts on protein structure and function. Thus, identifying high-risk missense SNPs in C1qA has become a necessity if we are to identify appropriate measures for prevention and management of affected patients. Methods: A comprehensive in silico study was conducted to screen the 184 missense SNPs in the C1qA gene using different tools with different algorithms and approaches. We investigated the impact of SNPs on protein function, stability, and structure. In addition, we identified the location of the SNPs on protein domains, secondary structure alignment, and the phylogenetic conservation of their positions. Results: Of the 184 missense SNPs, 10 SNPs were predicted to be the most damaging to protein function and structure. Conclusion: Ten missense SNPs were predicted to have the highest risk of damaging protein function and structure, thus leading to infection, sepsis, and systemic lupus erythematosus. These 10 SNPs constitute the best candidates for further experimental investigations.

Newly Emerging MDR B. cereus in Mugil seheli as the First Report Commonly Harbor nhe, hbl, cytK, and pc-plc Virulence Genes and bla1, bla2, tetA, and ermA Resistance Genes.

Background: Bacillus cereus is a common food poisoning pathogen in humans. This study aimed to investigate the prevalence, molecular typing, antibiogram profile, pathogenicity, dissemination of virulence and antibiotic resistance genes associated with natural B. cereus infection among Mugil seheli. Methods: Consequently, 120 M. seheli (40 healthy and 80 diseased) were obtained from private fish farms in Port-said Governorate, Egypt. Afterward, samples were processed for clinical, post-mortem, and bacteriological examinations. The recovered isolates were tested for antimicrobial susceptibility, phenotypic assessment of virulence factors, pathogeneicity, and PCR-based detection of virulence and antibiotic resistance genes. Results: B. cereus was isolated from 30 (25%) examined fish; the highest prevalence was noticed in the liver (50%). The phylogenetic and sequence analyses of the gyrB gene revealed that the tested B. cereus isolate displayed a high genetic similarity with other B. cereus strains from different origins. All the recovered B. cereus isolates (n =60, 100%) exhibited ß-hemolytic and lecithinase activities, while 90% (54/60) of the tested isolates were biofilm producers. Using PCR, the tested B. cereus isolates harbor nhe, hbl, cytK, pc-plc, and ces virulence genes with prevalence rates of 91.6%, 86.6%, 83.4%, 50%, and 33.4%, respectively. Moreover, 40% (24/60) of the tested B. cereus isolates were multidrug-resistant (MDR) to six antimicrobial classes and carried the bla1, bla2, tetA, and ermA genes. The experimentally infected fish with B. cereus showed variable mortality in direct proportion to the inoculated doses. Conclusion: As far as we know, this is the first report that emphasized the existence of MDR B. cereus in M. seheli that reflects a threat to the public health and the aquaculture sector. Newly emerging MDR B. cereus in M. seheli commonly carried virulence genes nhe, hbl, cytK, and pc-plc, as well as resistance genes bla1, bla2, tetA, and ermA.

Sequence Analysis, Antibiogram Profile, Virulence and Antibiotic Resistance Genes of XDR and MDR Gallibacterium anatis Isolated from Layer Chickens in Egypt.

Background: Gallibacterium anatis is incriminated frequently in severe economic losses and mortalities in the poultry industry. This study aimed to detect the prevalence of G. anatis in layer chickens, sequence analysis, the antibiogram profiles, and PCR screening of virulence determinants and antibiotic resistance genes. Methods: Accordingly, 300 samples (tracheal swabs, ovary and oviduct, and lung) were randomly collected from 100 diseased layer chickens from private commercial layer farms at Elsharkia Governorate, Egypt. The bacteriological examination was carried out. The retrieved isolates were tested for 16S rRNA-23S rRNA gene sequencing, antibiogram profiling, PCR screening of virulence (gtxA, fifA, and gyrB), and antibiotic resistance genes (bla ROB, aphA1, tetB, and tetH). Results: The prevalence of G. anatis was 25% in the examined diseased layer chickens. The sequence analyses emphasized that the tested strains derived from a common ancestor and exhibited a notable genetic similarity with other G. anatis strains from USA, China, and Denmark. The isolated G. anatis strains were highly resistant to sulfamethoxazole-trimethoprim, oxytetracycline, penicillin, ampicillin, kanamycin, neomycin, and erythromycin. The PCR revealed that the retrieved G. anatis strains carried gtxA, gyrB, and fifA virulence genes with a prevalence of 100%, 100%, and 38.3%, respectively. Approximately 30.1% of the retrieved G. anatis isolates were XDR to six antimicrobial classes and harbored bla ROB, aphA1, and tetB resistance genes. Moreover, 20.5% of the isolated G. anatis strains were MDR to three different classes and carried bla ROB and tetH resistance genes. Conclusion: Briefly, this study emphasized the existence of XDR and MDR G. anatis strains in poultry. Florfenicol and norfloxacin displayed a promising antimicrobial effect against the emerging XDR and MDR G. anatis in poultry. The emergence of XDR and MDR G. anatis is considered a public health alarm.

Detection and quantification of warfarin in pharmaceutical dosage form and in spiked human plasma using surface enhanced Raman scattering.

Analytical approaches for the quantitation of warfarin in plasma are high in demand. In this study, a novel surface enhanced Raman scattering (SERS) technique for the quantification of the widely used anticoagulant warfarin sodium in pharmaceutical dosage form and in spiked human plasma was developed. The colloidal-based SERS measurements were carefully optimized considering the laser wavelength, the type of metal nanoparticles, their surface functionalization and concentration as well as the time required for warfarin to associate with the metal surface. Poly(diallyldimethylammonium chloride) coated silver nanoparticles (PDDA-AgNPs) were established as a substrate which greatly enhanced the weak warfarin Raman signal with high reproducibility. The limit of detection was calculated in both water and human plasma to be 0.56 nM (0.17 ngmL-1) and 0.25 nM (0.08 ngmL-1) respectively, with a high degree of accuracy and reproducibility. The proposed method is simple, economical, and easily applied for routine application requiring only small plasma samples and also could be potentially useful for pharmacokinetic research on warfarin.

Stability-indicating micellar enhanced spectro-fluorometric determination of Daclatasvir in its tablet and spiked human plasma.

A fast, simple and sensitive micellar enhanced spectrofluorimetric method is performed for the determination of Daclatasvir dihydrochloride (DAC) in its pharmaceutical dosage form and in spiked human plasma. The fluorescence intensity (FI) was measured at 367 nm after excitation at 300 nm. In aqueous solution, the FI of DAC was greatly enhanced by >110% in the presence of sodium dodecyl sulphate (SDS). The detection method was linear over the range of 12.93 to 161.60 ng/mL, with a limit of detection of 1.75 ng/mL. The proposed method was successfully applied to the determination of DAC in its pharmaceutical dosage form and the mean % recovery of DAC in spiked human plasma was 95.42 ±â€¯2.52. The developed methodology was also extended to stress studies of DAC after exposure to different forced degradation conditions including acidic, alkaline, photolytic, thermal and oxidative environments.