Draft genome sequence of novel Candidatus Ornithobacterium hominis carrying antimicrobial resistance genes in Egypt.

BACKGROUND: Candidatus Ornithobacterium hominis (O. hominis), which was identified in nasopharyngeal swabs from Egypt, has been associated with respiratory disorders in humans. O. hominis, a recently identified member of the Flavobacteriaceae family, belongs to the largest family within the Bacteroidetes phylum. This family includes hundreds of species and 90 genera, including major human pathogens such as Capnocytophaga canimorsus and Elizabethkingia meningoseptica. Herein, we presented two draft genome assemblies of O. hominis that were extracted from metagenomic data using the Illumina sequencing method. The alignment of reads against the O. hominis genome was accomplished using BLASTN, and the reads with significant hits were extracted using Seqtk and assembled using SPAdes. The primary goal of this study was to obtain a more profound understanding of the genomic landscape of O. hominis, with an emphasis on identifying the associated virulence, antimicrobial genes, and distinct defense mechanisms to shed light on the potential role of O. hominis in human respiratory infections. RESULTS: The genome size was estimated to be 1.84 Mb, including 1,931,660 base pairs (bp), with 1,837 predicted coding regions and a G+C content of 35.62%. Genes encoding gliding motility, antibiotic resistance (20 genes), and the toxA gene were all included in the genome assembly. Gliding motility lipoproteins (GldD, GldJ, GldN, and GldH) and the gliding motility-associated ABC transporter substrate-binding protein, which acts as a crucial virulence mechanism in Flavobacterium species, were identified. The genome contained unique genes encoding proteins, such as the ParE1 toxin that defend against the actions of quinolone and other antibiotics. The cobalt-zinc-cadmium resistance gene encoding the protein CzcB, which is necessary for metal resistance, urease regulation, and colonization, was also detected. Several multidrug resistance genes encoding proteins were identified, such as MexB, MdtK, YheI, and VanC. CONCLUSION: Our study focused on identifying virulence factors, and antimicrobial resistance genes present in the core genome of O. hominis. These findings provide valuable insights into the potential pathogenicity and antibiotic susceptibility of O. hominis.

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.

The Eradication of Helicobacter pylori Was Significantly Associated with Compositional Patterns of Orointestinal Axis Microbiota.

BACKGROUND: Helicobacter pylori (H. pylori) is significantly linked to various diseases that seriously impact human health, such as gastric ulcers, chronic gastritis and gastric adenocarcinoma. METHODS: The compositional shifts in bacterial communities of the orointestinal axis were surveyed pre/post-eradication of H. pylori. In total, 60 samples, including stool and salivary specimens, were collected from 15 H. pylori-positive individuals (HPP) before beginning and 2 months after receiving the eradication therapy. The V3-V4 regions of the 16S rRNA gene were sequenced using MiSeq. RESULTS: Overall, oral microbiomes were collectively more diverse than the gut microbiomes (Kruskal-Wallis; p = 3.69 × 10-5). Notably, the eradication of H. pylori was associated with a significant reduction in the bacterial diversity along the orointestinal axis (Wilcoxon rank sum test; p = 6.38 × 10-3). Interestingly, the oral microbiome of HPP showed a positive correlation between Proteobacteria and Fusobacteria, in addition to a significant predominance of Streptococcus, in addition to Eubacterium_eligens, Haemophilus, Ruminococcaceae, Actinomyces and Staphylococcus. On the other hand, Fusobacterium, Veillonella, Catenibacterium, Neisseria and Prevotella were significantly enriched upon eradication of H. pylori. Generally, Bacteroidetes and Fusobacteria positively coexisted during H. pylori infection along the orointestinal axis (r = 0.67; p = 0.0006). The eradication of H. pylori was positively linked to two distinctive orotypes (O3 and O4). Orotype O4 was characterized by a robust abundance of Veillonella and Fusobacteria. The gut microbiomes during H. pylori infection showed a remarkable predominance of Clostridium_sensu_stricto_1 and Escherichia_Shigella. Likewise, Bifidobacterium and Faecalibacterium were significantly enriched upon eradication of H. pylori. CONCLUSIONS: Finally, the impact of eradication therapy clearly existed on the representation of certain genera, especially in the oral microbiome, which requires particular concern in order to counteract and limit their subsequent threats.

Comparative analysis of the ocular surface microbiome in type-1, type-2 diabetes mellitus and healthy individuals.

AIM: Ocular health greatly impacts the quality of life, and diabetes mellitus (DM) is a major cause of several visual diseases. Likewise, microbiomes have a pivotal role in eye health. The aim was to study the effect of DM, both type-1 (T1DM) and type-2 (T2DM) on the ocular microbiome. METHODS AND RESULTS: A total of 70 subjects were recruited for this study and divided into two main groups healthy nondiabetic (n = 18) and diabetic (28 T1DM and 24 T2DM). The ocular surface (OS) microbiome was more diverse in the healthy group than in the diabetic one. Taxonomic analysis revealed Proteobacteria as the main phylum (healthy nondiabetic 41.8%, T1DM 50.6%, and T2DM 52.5%), besides Streptococcus (healthy nondiabetic 16%, T1DM 26.75%, and T2DM 29.20%) and Paracoccus (healthy nondiabetic 17%, T1DM 34.85%, and T2DM 37.47%) as the main genera. No significant diversity was found between T1DM and T2DM on both phylum and genus levels; yet genera Brevundimonas and Leptotrichia were more significantly predominant in T1DM. CONCLUSION: Two pathogenic genera, Streptococcus and Paracoccus, were more predominant in the DM group than in the healthy one.

Compositional Changes in the Vaginal Bacterial Microbiome of Healthy Pregnant Women across the Three Gestational Trimesters in Ismailia, Egypt.

The composition of the vaginal microbiome may lead to adverse pregnancy outcomes. Normal pregnancy is associated with changes in the vaginal bacterial community composition, which tend to be more enriched with one or two Lactobacillus species promoting a healthy vagina and favorable birth outcomes. The aim of the current study was to determine compositional changes in the healthy vaginal microbiome composition during the three trimesters of pregnancy in Ismailia, Egypt using Illumina MiSeq sequencing of the V3-V4 region of the 16S rRNA. The phylum Firmicutes and the genus Lactobacillus dominated across the three trimesters of pregnancy. L. iners was the most abundant species. However, L. coleohominis and L. reuteri represented the least dominant vaginal lactobacilli. Core microbiome analyses showed the Lactobacillus genus and L. iners species to have the highest prevalence in all the samples of our study groups. The phylum Firmicutes was found to be negatively correlated with almost all other vaginal phyla during pregnancy. Likewise, a negative correlation between Lactobacillus and almost all other genera was detected, including significant negative correlations with Dialister and Prevotella. Furthermore, negative correlations of L. iners were detected with almost all other species, including a significant negative correlation with L. helveticus, G. vaginalis, S. anginosus, and S. agalactiae.

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.

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.

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.

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.

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.

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.

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.

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.

Changes in Vaginal Microbiome in Pregnant and Nonpregnant Women with Bacterial Vaginosis: Toward Microbiome Diagnostics?

Bacterial vaginosis (BV) is highly common, adversely affecting the health of millions of women. New therapeutic targets and diagnostics are urgently needed for BV. Microbiome research offers new prospects in this context. We report here original findings on changes in the vaginal microbiome in pregnant and nonpregnant women with BV. Reproductive age women were recruited for this study after a clinical examination. The total sample (N = 33) included four study groups: (1) healthy nonpregnant women (n = 9), (2) nonpregnant women with symptomatic BV (n = 11), (3) healthy pregnant women without BV (n = 6), and (4) pregnant women with symptomatic BV (N = 7). The vaginal microbiota in healthy women was less diverse, with dominance of a single genus, Lactobacillus. Six major phyla appeared upon taxonomic analysis of the bacterial sequences: Firmicutes, Actinobacteria, Proteobacteria, Tenericutes, Bacteroidetes, and Fusobacteria. For instance, Firmicutes had a significantly higher abundance (98.3%) in the nonpregnant healthy group and 94.3% in pregnant healthy group, compared with nonpregnant (49.7%) and pregnant (67%) women with BV (p = 0.003). Moreover, women with BV had significant increases in representation of Actinobacteria, Fusobacteria, and Bacteroidetes (p = 0.0003, 0.004, and 0.01, respectively). Although the Lactobacillus genus was predominant in healthy women, Gardnerella, Atopobium, Sneathia, and Prevotella significantly increased in nonpregnant women with BV (p = 0.001, 0.014, 0.004, and 0.012, respectively). Dysbiosis of Lactobacillus in pregnant women with BV was accompanied by increased prevalence of the Streptococcus genus. These findings contribute new insights toward microbiome diagnostics and therapeutics innovation in BV.

Proteomic analysis of bacterial communities associated with atopic dermatitis.

Atopic dermatitis (AD) is a relapsing, chronic, and inflammatory skin disorder. Its causes remain unclear. Here, we reported the first proteome study of the bacterial community in AD patients. Bacterial community in 7 patients and 1 healthy control using bottom-up proteomics were examined starting with in-solution digestion followed by purification steps with subsequent analysis using LC-MS/MS and ended with data processing and bioinformatic analysis. Overall, great bacterial changes between patient samples and healthy one were noticed with the presence of Staphylococcus aureus, Aeromonas hydrophila, and Shewanella species, and others that were present uniquely in patient samples suggesting their role in AD. Additionally, detection of some important proteins that trigger bacterial pathogenesis and the immune system such as enolase, glyceraldehyde-3-phosphate, Chaperone proteins DnaK and HtpG beside protein pathways needed for bacterial growth and pathogenesis like chaperones and folding catalysts; and Energy metabolism. These new findings of the microbiome and detected proteins could start a new era of proteomics to study the bacterial community as a whole and detect the way it interacts with each other and with the host. SIGNIFICANCE: This paper would represent a reference work for investigations on microbiota that present on AD, from both a microbiological and a functional proteomic point of view. We focused on analysisng bacteria community and proteins produced and its role in the disease, highlighting some functional characteristics of certain proteins and discussing its potential role in AD.

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.

New Insights on Obesity and Diabetes from Gut Microbiome Alterations in Egyptian Adults.

Obesity and diabetes are reaching epidemic levels globally. Metagenomics and microbiome science have recently emerged as new tools for studying common complex human diseases. We report in this study notable differences in gut microbiome in adult patients with obesity and diabetes in Egypt. The experimental design was based on comparisons of four study groups: (1) Controls (C) with a normal body mass index, without obesity or diabetes, (2) Obese adults (O) without diabetes, (3) adults with diabetes (D) who are not obese, and (4) Adults who are both obese and diabetic (OD). In a total study sample of 60 participants, we sequenced the 16S ribosomal RNA (rRNA) gene using the Illumina MiSeq platform. Alpha diversity analysis revealed greater diversity in bacterial communities of (D) than controls. Phylum-level analysis identified a trend for overrepresentation of Bacteroidetes (p < 0.07) in (O) and (D) than controls. The ratio of Firmicutes/Bacteroidetes (F/B) displayed a remarkable increase in (OD) than controls. At genus level, Faecalibacterium (p < 0.05) and Akkermansia (p < 0.001) distinguished (O) from controls, while Fusobacterium (p < 0.001) and Bacteroides (p < 0.001) was significantly more abundant in (OD) compared with D. Surprisingly, isoquinoline, quinone and ubiquinone alkaloid biosynthesis were overrepresented in controls compared with other three study groups. Presumably, the latter observation might potentially suggest an antihyperglycemic activity of the gut microbiota. In conclusion, the health state of the adults in our study defined the composition of the gut microbiota. Moreover, obesity and diabetes were associated with remarkably enriched populations of Firmicutes and Bacteroidetes. The abundance of Fusobacterium is worth further research and exploration as a candidate biomarker for prediabetes especially in obese individuals. The potential antihyperglycemic activity of the gut microbiota is also noteworthy for future studies in other world populations.

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.

Illumina MiSeq Sequencing for Preliminary Analysis of Microbiome Causing Primary Endodontic Infections in Egypt.

The use of high throughput next generation technologies has allowed more comprehensive analysis than traditional Sanger sequencing. The specific aim of this study was to investigate the microbial diversity of primary endodontic infections using Illumina MiSeq sequencing platform in Egyptian patients. Samples were collected from 19 patients in Suez Canal University Hospital (Endodontic Department) using sterile # 15K file and paper points. DNA was extracted using Mo Bio power soil DNA isolation extraction kit followed by PCR amplification and agarose gel electrophoresis. The microbiome was characterized on the basis of the V3 and V4 hypervariable region of the 16S rRNA gene by using paired-end sequencing on Illumina MiSeq device. MOTHUR software was used in sequence filtration and analysis of sequenced data. A total of 1858 operational taxonomic units at 97% similarity were assigned to 26 phyla, 245 families, and 705 genera. Four main phyla Firmicutes, Bacteroidetes, Proteobacteria, and Synergistetes were predominant in all samples. At genus level, Prevotella, Bacillus, Porphyromonas, Streptococcus, and Bacteroides were the most abundant. Illumina MiSeq platform sequencing can be used to investigate oral microbiome composition of endodontic infections. Elucidating the ecology of endodontic infections is a necessary step in developing effective intracanal antimicrobials.

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.