Production of highly cytotoxic and low immunogenic L-asparaginase from Stenotrophomonas maltophilia EMCC2297.

L-asparaginase is an important therapeutic enzyme that is frequently utilized in the chemotherapy regimens of adults as well as pediatric patients with acute lymphoblastic leukemia. However, a high rate of hypersensitivity with prolonged use has limited its utilization. Stenotrophomonas maltophilia (S. maltophilia) EMCC2297 isolate was reported as a novel and promising source for L- asparaginase. The present study aimed at the production, purification, and characterization of L- asparaginase from S. maltophilia EMCC2297 isolate. The microbial production of L-asparaginase by the test isolate could be increased by pre-exposure to chloramphenicol at 200 µg/ml concentration. S. maltophilia EMCC2297 L-asparaginase could be purified to homogeneity by ammonium sulphate precipitation and the purified form obtained by gel exclusion chromatography showed total activity of 96.4375 IU/ml and specific activity of 36.251 IU/mg protein. SDS-PAGE analysis revealed that the purified form of the enzyme is separated at an apparent molecular weight of 17 KDa. Michaelis-Menten constant analysis showed a Km value of 4.16 × 10- 2 M with L-asparagine as substrate and Vmax of 10.67 IU/ml. The antitumor activity of the purified enzyme was evaluated on different cell lines and revealed low IC50 of 2.2 IU/ml and 2.83 IU/ml for Hepatocellular cancer cell line (HepG-2), human leukemia cancer cell line (K-562), respectively whereas no cytotoxic effect could be detected on normal human lung fibroblast cells (MRC-5). However, mice treated with native L-asparaginase showed lower IgG titre compared to commercial L-asparaginase. This study highlights the promising characteristics of this enzyme making it a valuable candidate for further research and development to be an adduct in cancer chemotherapy.

Characterization of vitamin D3 biotransformation by the cell lysate of Actinomyces hyovaginalis CCASU-A11-2.

A former work conducted in our Lab, lead to in a effective scale up of vitamin D3 bioconversion into calcitriol by Actinomyces (A.) hyovaginalis isolate CCASU-A11-2 in Lab fermenter (14 L) resulting in 32.8 µg/100 mL of calcitriol. However, the time needed for such a bioconversion process was up to 5 days. Therefore, the objective of this study was to shorten the bioconversion time by using cell-free lysate and studying different factors influencing bioconversion. The crude cell lysate was prepared, freeze-dried, and primarily fractionated into nine fractions, of which, only three fractions, 50, 100, and 150 mM NaCl elution buffers showed 22, 12, and 2 µg/10 mL, calcitriol production, respectively. Ammonium sulfate was used for protein precipitation, and it did not affect the bioconversion process except at a concentration of 10%w/v. Secondary fractionation was carried out using 80 mL of the 50 mM NaCl elution buffer and the results showed the 80 mL eluent volume was enough for the complete elution of the active protein. The pH 7.8, temperature 28 °C, and 6 h reaction time were optimum for maximum calcitriol production (31 µg/10 mL). In conclusion, the transformation of vitamin D3 into calcitriol was successfully carried out within 6 h and at pH 7.8 and 28 °C using fractionated cell lysate. This process resulted in a 10-fold increase in calcitriol as compared to that produced in our previous study using a 14 L fermenter (32.8 µg/100 mL). Therefore, cell-free lysate should be considered for industrial and scaling up vitamin D3 bioconversion into calcitriol.

Bioconversion of vitamin D3 into calcitriol by Actinomyces hyovaginalis isolate CCASU- A11-2.

Vitamin D3 is a fat-soluble prohormone that is activated inside the liver to produce 25-hydroxyvitamin D3 (calcidiol), and in the kidney to produce the fully active 1α, 25-dihydroxy vitamin D3 (calcitriol). A previous work piloted in our laboratory, resulted in a successful recovery of a local soil-promising Actinomyces hyovaginalis isolate CCASU-A11-2 capable of converting vitamin D3 into calcitriol. Despite the rising amount of research on vitamin D3 bioconversion into calcitriol, further deliberate studies on this topic can significantly contribute to the improvement of such a bioconversion process. Therefore, this work aimed to improve the bioconversion process, using the study isolate, in a 14 L laboratory fermenter (4 L fermentation medium composed of fructose (15 g/L), defatted soybean (15 g/L), NaCl (5 g/L), CaCO3 2 g/L); K2HPO4, (1 g/L) NaF (0.5 g/L) and initial of pH 7.8) where different experiments were undertaken to investigate the effect of different culture conditions on the bioconversion process. Using the 14 L laboratory fermenter, the calcitriol production was increased by about 2.5-fold (32.8 µg/100 mL) to that obtained in the shake flask (12.4 µg/100 mL). The optimal bioconversion conditions were inoculum size of 2% v/v, agitation rate of 200 rpm, aeration rate of 1 vvm, initial pH of 7.8 (uncontrolled); addition of vitamin D3 (substrate) 48 h after the start of the main culture. In conclusion, the bioconversion of vitamin D3 into calcitriol in a laboratory fermenter showed a 2.5-fold increase as compared to the shake flask level where, the important factors influencing the bioconversion process were the aeration rate, inoculum size, the timing of substrate addition, and the fixed pH of the fermentation medium. So, those factors should be critically considered for the scaling-up of the biotransformation process.

Transferable IncX3 plasmid harboring blaNDM-1, bleMBL, and aph(3')-VI genes from Klebsiella pneumoniae conferring phenotypic carbapenem resistance in E. coli.

BACKGROUND: The dissemination of carbapenem resistance via carbapenemases, such as the metallo-ß-lactamase NDM, among Enterobacterales poses a public health threat. The aim of this study was to characterize a plasmid carrying the blaNDM-1 gene, which was extracted from a clinical Klebsiella pneumoniae uropathogen from an Egyptian patient suffering from a urinary tract infection. METHODS AND RESULTS: The recovered plasmid was transformed into competent E. coli DH5α which acquired phenotypic resistance to cefoxitin, ceftazidime, and ampicillin/sulbactam, and intermediate sensitivity to ceftriaxone and imipenem (a carbapenem). Whole plasmid sequencing was performed on the extracted plasmid using the DNBSEQ™ platform. The obtained forward and reverse reads were assembled into contigs using the PRINSEQ and PLACNETw web tools. The obtained contigs were uploaded to PlasmidFinder and ResFinder for in silico plasmid typing and detection of antimicrobial resistance genes, respectively. The final consensus sequence was obtained using the Staden Package software. The plasmid (pNDMKP37, NCBI accession OK623716.1) was typed as an IncX3 plasmid with a size of 46,160 bp and harbored the antibiotic resistance genes blaNDM-1, bleMBL, and aph(3')-VI. The plasmid also carried mobile genetic elements involved in the dissemination of antimicrobial resistance including insertion sequences IS30, IS630, and IS26. CONCLUSIONS: This is Egypt's first report of a transmissible plasmid co-harboring blaNDM-1 and aph(3')-VI genes. Moreover, the respective plasmid is of great medical concern as it has caused the horizontal transmission of multidrug-resistant phenotypes to the transformant. Therefore, new guidelines should be implemented for the rational use of broad-spectrum antibiotics, particularly carbapenems.

Phenotypic and genotypic characterization of linezolid resistance and the effect of antibiotic combinations on methicillin-resistant Staphylococcus aureus clinical isolates.

BACKGROUND: Methicillin-Resistant Staphylococcus aureus (MRSA) causes life-threatening infections, with narrow therapeutic options including: vancomycin and linezolid. Accordingly, this study aimed to characterize phenotypically and genotypically, the most relevant means of linezolid resistance among some MRSA clinical isolates. METHODS: A total of 159 methicillin-resistant clinical isolates were collected, of which 146 were indentified microscopically and biochemically as MRSA. Both biofilm formation and efflux pump activity were assessed for linezolid-resistant MRSA (LR-MRSA) using the microtiter plate and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) methods, respectively. Linezolid resistance was further characterized by polymerase chain reaction (PCR) amplification and sequencing of domain V of 23 S rRNA; rplC; rplD;and rplV genes. Meanwhile, some resistance genes were investigated: cfr; cfr(B); optrA; msrA;mecA; and vanA genes. To combat LR-MRSA, the effect of combining linezolid with each of 6 different antimicrobials was investigated using the checkerboard assay. RESULTS: Out of the collected MRSA isolates (n = 146), 5.48% (n = 8) were LR-MRSA and 18.49% (n = 27) were vancomycin-resistant (VRSA). It is worth noting that all LR-MRSA isolates were also vancomycin-resistant. All LR-MRSA isolates were biofilm producers (r = 0.915, p = 0.001), while efflux pumps upregulation showed no significant contribution to development of resistance (t = 1.374, p = 0.212). Both mecA and vanA genes were detected in 92.45% (n = 147) and 6.92% (n = 11) of methicillin-resistant isolates, respectively. In LR-MRSA isolates, some 23 S rRNA domain V mutations were observed: A2338T and C2610G (in 5 isolates); T2504C and G2528C (in 2 isolates); and G2576T (in 1 isolate). Amino acids substitutions were detected: in L3 protein (rplC gene) of (3 isolates) and in L4 protein (rplD gene) of (4 isolates). In addition, cfr(B) gene was detected (in 3 isolates). In 5 isolates, synergism was recorded when linezolid was combined with chloramphenicol, erythromycin, or ciprofloxacin. Reversal of linezolid resistance was observed in some LR-MRSA isolates when linezolid was combined with gentamicin or vancomycin. CONCLUSIONS: LR-MRSA biofilm producers' phenotypes evolved in the clinical settings in Egypt. Various antibiotic combinations with linezolid were evaluated in vitro and showed synergistic effects.

Dual Role of Mitogen-Activated Protein Kinase 8 Interacting Protein-1 in Inflammasome and Pancreatic ß-Cell Function.

Inflammasomes have been implicated in the pathogenesis of type 2 diabetes (T2D). However, their expression and functional importance in pancreatic ß-cells remain largely unknown. Mitogen-activated protein kinase 8 interacting protein-1 (MAPK8IP1) is a scaffold protein that regulates JNK signaling and is involved in various cellular processes. The precise role of MAPK8IP1 in inflammasome activation in ß-cells has not been defined. To address this gap in knowledge, we performed a set of bioinformatics, molecular, and functional experiments in human islets and INS-1 (832/13) cells. Using RNA-seq expression data, we mapped the expression pattern of proinflammatory and inflammasome-related genes (IRGs) in human pancreatic islets. Expression of MAPK8IP1 in human islets was found to correlate positively with key IRGs, including the NOD-like receptor (NLR) family pyrin domain containing 3 (NLRP3), Gasdermin D (GSDMD) and Apoptosis-associated speck-like protein containing a CARD (ASC), but correlate inversely with Nuclear factor kappa ß1 (NF-κß1), Caspase-1 (CASP-1), Interleukin-18 (IL-18), Interleukin-1ß (IL-1ß) and Interleukin 6 (IL-6). Ablation of Mapk8ip1 by siRNA in INS-1 cells down-regulated the basal expression levels of Nlrp3, NLR family CARD domain containing 4 (Nlrc4), NLR family CARD domain containing 1 (Nlrp1), Casp1, Gsdmd, Il-1ß, Il-18, Il-6, Asc, and Nf-κß1 at the mRNA and/or protein level and decreased palmitic acid (PA)-induced inflammasome activation. Furthermore, Mapk8ip1-silened cells substantially reduced reactive oxygen species (ROS) generation and apoptosis in palmitic acid-stressed INS-1 cells. Nonetheless, silencing of Mapk8ip1 failed to preserve ß-cell function against inflammasome response. Taken together, these findings suggest that MAPK8IP1 is involved in regulating ß-cells by multiple pathways.

Expression Silencing of Mitogen-Activated Protein Kinase 8 Interacting Protein-1 Conferred Its Role in Pancreatic ß-Cell Physiology and Insulin Secretion.

Mitogen-activated protein kinase 8 interacting protein-1 (MAPK8IP1) gene has been recognized as a susceptibility gene for diabetes. However, its action in the physiology of pancreatic ß-cells is not fully understood. Herein, bioinformatics and genetic analyses on the publicly available database were performed to map the expression of the MAPK8IP1 gene in human pancreatic islets and to explore whether this gene contains any genetic variants associated with type 2 diabetes (T2D). Moreover, a series of functional experiments were executed in a rat insulinoma cell line (INS-1 832/13) to investigate the role of the Mapk8ip1 gene in ß-cell function. Metabolic engineering using RNA-sequencing (RNA-seq) data confirmed higher expression levels of MAPK8IP1 in human islets compared to other metabolic tissues. Additionally, comparable expression of MAPK8IP1 expression was detected in sorted human endocrine cells. However, ß-cells exhibited higher expression of MAPK8IP1 than ductal and PSC cells. Notably, MAPK8IP1 expression was reduced in diabetic islets, and the expression was positively correlated with insulin and the ß-cell transcription factor PDX1 and MAFA. Using the TIGER portal, we found that one genetic variant, "rs7115753," in the proximity of MAPK8IP1, passes the genome-wide significance for the association with T2D. Expression silencing of Mapk8ip1 by small interfering RNA (siRNA) in INS-1 cells reduced insulin secretion, glucose uptake rate, and reactive oxygen species (ROS) production. In contrast, insulin content, cell viability, and apoptosis without cytokines were unaffected. However, silencing of Mapk8ip1 reduced cytokines-induced apoptosis and downregulated the expression of several pancreatic ß-cell functional markers including, Ins1, Ins2, Pdx1, MafA, Glut2, Gck, Insr, Vamp2, Syt5, and Cacna1a at mRNA and/or protein levels. Finally, we reported that siRNA silencing of Pdx1 resulted in the downregulation of MAPK8IP1 expression in INS-1 cells. In conclusion, our findings confirmed that MAPK8IP1 is an important component of pancreatic ß-cell physiology and insulin secretion.

Optimization of feather degradation by a Bacillus thuringiensis isolate using response surface methodology and investigation of the feather protein hydrolysate structure.

Valorization of chicken feather is a long-sought approach for its sustainable disposal. Being protein rich, hydrolyzed chicken feather has a wide range of applications, not limited to formulation of microbiological culture media, animal feed, and biofertilizers, but extends to synthesis of bioplastic films, cosmetics, and biomedicals. In this study, a potent keratinolytic isolate was recovered from soil and identified by 16S rRNA as Bacillus thuringiensis. Feather degradation by the isolate was optimized through response surface methodology. First, one-variable-at-a-time technique to assign the factors that affect feather degradation, then Box-Behnken central composite design model were employed. The model, involving three independent variables (initial pH, inoculum size, and concentration of supplementary glucose), was significant (R2  = 0.9716). According to the model, complete feather degradation is obtained at an inoculum size of B. thuringiensis B4 equal to 1 × 1010  CFU/ml, when feather meal broth is supplemented with 1.5% (w/v) glucose and pH adjusted to 8.5. Protein content of the lysate was 327.8 ± 25 µg/ml, and no carbohydrates were detected. SEM/EDX analysis has shown that the hydrolysate consisted mainly of O, P, S, and Se in addition to carbon, while FTIR images assured the presence of carboxyl and amino groups characteristic of peptides and amino acids.

Ramucirumab combination with sorafenib enhances the inhibitory effect of sorafenib on HepG2 cancer cells.

Sorafenib, an oral multiple kinase inhibitor, is the standardized treatment for hepatocellular carcinoma (HCC). One strategy to improve HCC therapy is to combine agents that target key signaling pathways. In this study we set out to investigate the effect of combining sorafenib with either bevacizumab (anti-VEGF), panitumumab (anti-EGFR) or ramucirumab (anti-VEGFR2) on HepG2 cancer cell line with the aim of improving efficacy and possibility of therapeutic dose reduction of sorafenib.: HepG2 cancer cell line was treated with sorafenib alone or in combination with either bevacizumab, panitumumab or ramucirumab. Cell proliferation; apoptosis and cell cycle distribution; gene expression of VEGFR2, EGFR, MMP-9 and CASPASE3; the protein levels of pVEGFR2 and pSTAT3 and the protein expression of CASPASE3, EGFR and VEGFR2 were determined. Combined treatments of sorafenib with ramucirumab or panitumumab resulted in a significant decrease in sorafenib IC50. Sorafenib combination with ramucirumab or bevacizumab resulted in a significant arrest in pre-G and G0/G1 cell cycle phases, significantly induced apoptosis and increased the relative expression of CASPASE3 and decreased the anti-proliferative and angiogenesis markers´ MMP-9 and pVEGFR2 or VEGFR2 in HepG2 cells. A significant decrease in the levels of pSTAT3 was only detected in case of sorafenib-ramucirumab combination. The combined treatment of sorafenib with panitumumab induced a significant arrest in pre-G and G2/M cell cycle phases and significantly decreased the relative expression of EGFR and MMP-9. Sorafenib-ramucirumab combination showed enhanced apoptosis, inhibited proliferation and angiogenesis in HepG2 cancer cells. Our findings suggest that ramucirumab can be a useful as an adjunct therapy for improvement of sorafenib efficacy in suppression of HCC.

Isolation, Propagation and Genotyping of Human Rotaviruses Circulating among Children with Gastroenteritis in Two Egyptian University Hospitals.

The most prevalent cause of infectious neonatal diarrhea is Group A rotavirus (RVA). Unfortunately, there is a dearth of data on the incidence of rotavirus-associated infections among Egyptian children. The present study aimed to isolate, propagate, and genotype human rotaviruses circulating among Egyptian children with acute gastroenteritis admitted to two main university pediatric hospitals, Abo El-Reesh and El-Demerdash, over two consecutive winters, 2018-2020. Diarrheal samples (n = 230) were screened for Group A rotavirus RNA using RT-PCR assay. In positive samples (n = 34), multiplex semi-nested PCR was utilized to determine G and P genotypes. Thirty-four (14.8%) of the collected samples tested positive. The genotype distribution revealed that G1P[8] was the predominant rotavirus genotype throughout the current study. All rotavirus-positive fecal samples were passaged twice on human colorectal adenocarcinoma cell line (Caco-2) and rhesus monkey kidney epithelial cell line (MA104). Both cell lines could successfully isolate 14.7% (n = 5 out of 34) of the identified strains; however, Caco-2 cell line was shown to be more efficient than MA104 in promoting the propagation of human rotaviruses identified in Egyptian children's feces.

Hypolipidemic activity of lactic acid bacteria: Adjunct therapy for potential probiotics.

BACKGROUND: Individuals with hyperlipidemia are two times more likely to develop atherosclerotic cardiovascular disease (ASCVD) as opposed to those with controlled serum total cholesterol (TC) levels. Considering the documented adverse events of the current lipid-lowering medications which ultimately affect patient's compliance, substantial efforts have been made to develop new therapeutic strategies. Probiotics, on the other hand, are reported to have lipid-lowering activity with the added benefit of being generally well-tolerated making it an appealing adjuvant therapy. METHODS: A total of fifty Lactic acid bacteria (LAB) were isolated from raw milk (human and animal) and dairy products. Isolates demonstrating promising in vitro cholesterol removal capabilities were morphologically and biochemically characterized. Lastly, two bacterial candidates were selected for evaluation of their potential hypolipidemic activity using a laboratory animal model. Statistical differences between the means were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test. A p-value < 0.05 was considered statistically significant. RESULTS: Most of the isolates demonstrated an in vitro cholesterol removal activity. The six LAB isolates showing the highest cholesterol removal activity (36.5-55.6%) were morphologically and biochemically identified as Lactobacillus, Pediococcus, and Lactococcus species. The results demonstrated two promising antihyperlipidemic candidates, a Lactococcus lactis ssp. lactis with an in vivo significant reduction of serum triglycerides (TG) levels by 34.3%, and a Pediococcus sp. that was able to significantly reduce both the serum TC and TG levels by 17.3% and 47.0%, respectively, as compared to the diet-induced hyperlipidemic animal group. CONCLUSION: This study further supports the growing evidence regarding the antihyperlipidemic activity among probiotics, presenting them as a promising therapeutic approach for the management of hyperlipidemia.

Evaluation of lyophilized royal jelly and garlic extract emulgels using a murine model infected with methicillin-resistant Staphylococcus aureus.

The limited therapeutic options associated with methicillin-resistant Staphylococcus aureus (MRSA) necessitate search for innovative strategies particularly, use of natural extracts such as lyophilized royal jelly (LRJ) and garlic extract (GE). Therefore, out study aimed to formulate emulgels containing different concentrations of both LRJ and GE and to evaluate their activities using a murine model infected with MRSA clinical isolate. Four plain emulgel formulas were prepared by mixing stearic acid/yellow soft paraffin-based O/W emulsion formulae based on Carbopol 940, Na alginate, Na carboxymethylcellulose or Hydroxypropyl methyl cellulose E4. Sodium alginate-based emulgel was selected for preparation of four medicated emulgel formulations combining LRJ and GE at four different concentrations. The selected medicated emulgels were used for the in vivo studies. The emulgel formulated with Na alginate and HPMC (MF3) exhibited optimum smooth homogeneous consistency, neutral pH, acceptable viscosity, spreadability, extrudability values and best storage stability properties. In vivo results revealed that, the wounds infected with MRSA isolate in rates were wet (oozing) and showed pus formation when compared to injured uninfected wounds. MF3 formula containing 4% LRJ and 50% GE showed the maximum wound healing properties, both in the apparent physical wound healing measurements and in the histopathological examination. In conclusion, the medicated emulgel formulation (MF3) prepared with Na alginate was found optimum for topical application. MF3 formula containing 4% LRJ and 50% GE has shown the highest in vivo wound healing capacities. Further clinical studies should be conducted to prove both its safety and efficacy and the potential use in human.

Evaluation and Correlation of Rabies Vaccine Potency Using the National Institute of Health, Rapid Focus Fluorescent Inhibition, and Passive Hemagglutination Tests.

Rabies vaccine preparations are quantitatively assayed for potency using the in-vivo challenge National Institute of Health (NIH), the main test that consumes a high number of animals, takes a long time, and has wide variability. The Rapid focus fluorescent inhibition (RFFIT) and the passive hemagglutination (PHA) tests, the two serologically based tests, were also used for such purpose. In this study, we aimed to evaluate and correlate the potency of the NIH, RFFIT, and PHA tests according to the World Health Organization (WHO) validity criteria, aiming to validate the use of RFFIT or PHA test as a substitute to the NIH test for determining the potency of commercially available Rabies vaccine preparations. The results showed that, the three tests can be successfully used; however, a higher correlation between RFFIT and NIH than PHA and NIH was recorded (Pearson correlation = 1). The potency of rabies vaccine preparations using NIH, RFFIT, and PHA were 3.73, 3.51, and 4.50, respectively. NIH is the main test for the determination of vaccine potency carried out by conducting 25 experiments and consuming about 5,000 mice compared to 1,200 mice used with RFFIT and 1,000 mice used with PHA test. Taken together, we concluded that (i) in some tested preparations, both RFFIT and PHA tests gave comparable results, and they can be used interchangeably; (ii) RFFIT could successfully replace NIH test, but not PHA; (iii) RFFIT and PHA tests are faster, more accurate, more economic, and more sensitive than NIH; nevertheless, PHA needs further investigations; and (iv) both RFFIT and NIH tests complement and reinforce each other as they provide a comprehensive picture of the product potency.

Bacterial cyclomodulins: types and roles in carcinogenesis.

Various studies confirmed that bacterial infections contribute to carcinogenesis through the excessive accumulation of reactive oxygen species (ROS) and the expression of toxins that disrupt the cell cycle phases, cellular regulatory mechanisms and stimulate the production of tumorigenic inflammatory mediators. These toxins mimic carcinogens which act upon key cellular targets and result in mutations and genotoxicities. The cyclomodulins are bacterial toxins that incur cell cycle modulating effects rendering the expressing bacterial species of high carcinogenic potentiality. They are either cellular proliferating or cell cycle arrest cyclomodulins. Notably, cyclomodulins expressing bacterial species have been linked to different human carcinomas. For instance, Escherichia coli species producing the colibactin were highly prevalent among colorectal carcinoma patients, CagA+ Helicobacter pylori species were associated with MALT lymphomas and gastric carcinomas and Salmonella species producing CdtB were linked to hepatobiliary carcinomas. These species stimulated the overgrowth of pre-existing carcinomas and induced hyperplasia in in vivo animal models suggesting a role for the cyclomodulins in carcinogenesis. Wherefore, the prevalence and mode of action of these toxins were the focus of many researchers and studies. This review discusses different types of bacterial cyclomodulins highlighting their mode of action and possible role in carcinogenesis.

Impact of target site mutations and plasmid associated resistance genes acquisition on resistance of Acinetobacter baumannii to fluoroquinolones.

Among bacterial species implicated in hospital-acquired infections are the emerging Pan-Drug Resistant (PDR) and Extensively Drug-Resistant (XDR) Acinetobacter (A.) baumannii strains as they are difficult to eradicate. From 1600 clinical specimens, only 100 A. baumannii isolates could be recovered. A high prevalence of ≥ 78% resistant isolates was recorded for the recovered isolates against a total of 19 tested antimicrobial agents. These isolates could be divided into 12 profiles according to the number of antimicrobial agents to which they were resistant. The isolates were assorted as XDR (68; 68%), Multi-Drug Resistant (MDR: 30; 30%), and PDR (2; 2%). Genotypically, the isolates showed three major clusters with similarities ranging from 10.5 to 97.8% as revealed by ERIC-PCR technique. As a resistance mechanism to fluoroquinolones (FQs), target site mutation analyses in gyrA and parC genes amplified from twelve selected A. baumannii isolates and subjected to sequencing showed 12 profiles. The selected isolates included two CIP-susceptible ones, these showed the wild-type profile of being have no mutations. For the ten selected CIP-resistant isolates, 9 of them (9/10; 90%) had 1 gyrA/1 parC mutations (Ser 81 → Leu mutation for gyrA gene and Ser 84 → Leu mutation for parC gene). The remaining CIP-resistant isolate (1/10; 10%) had 0 gyrA/1 parC mutation (Ser 84 → Leu mutation for parC gene). Detection of plasmid-associated resistance genes revealed that the 86 ciprofloxacin-resistant isolates carry qnrA (66.27%; 57/86), qnrS (70.93%; 61/86), aac (6')-Ib-cr (52.32%; 45/86), oqxA (73.25%; 63/86) and oqxB (39.53%; 34/86), while qepA and qnrB were undetected in these isolates. Different isolates were selected from profiles 1, 2, and 3 and qnrS, acc(6,)-ib-cr, oqxA, and oqxB genes harbored by these isolates were amplified and sequenced. The BLAST results revealed that the oqxA and oqxB sequences were not identified previously in A. baumannii but they were identified in Klebsiella aerogenes strain NCTC9793 and Klebsiella pneumoniae, respectively. On the other hand, the sequence of qnrS, and acc(6,)-ib-cr showed homology to those of A. baumannii. MDR, XDR, and PDR A. baumannii isolates are becoming prevalent in certain hospitals. Chromosomal mutations in the sequences of GyrA and ParC encoding genes and acquisition of PAFQR encoding genes (up to five genes per isolate) are demonstrated to be resistance mechanisms exhibited by fluoroquinolones resistant A. baumannii isolates. It is advisable to monitor the antimicrobial resistance profiles of pathogens causing nosocomial infections and properly apply and update antibiotic stewardship in hospitals and outpatients to control infectious diseases and prevent development of the microbial resistance to antimicrobial agents.

OXA-48 Carbapenemase-Encoding Transferable Plasmids of Klebsiella pneumoniae Recovered from Egyptian Patients Suffering from Complicated Urinary Tract Infections.

Gram-negative bacteria are common causes of urinary tract infections (UTIs). Such pathogens can acquire genes encoding multiple mechanisms of antimicrobial resistance, including carbapenem resistance. The aim of this study was to detect the carbapenemase-producing ability of some Gram-negative bacterial isolates from urine specimens of patients suffering from complicated UTIs at two vital tertiary care hospitals in Cairo, Egypt; to determine the prevalence of carbapenemase genes among plasmid-bearing isolates; and explore the possibility of horizontal gene transfer to other bacterial species. The collected isolates were subjected to antimicrobial susceptibility testing, phenotypic analysis of carbapenemase production, and molecular detection of plasmid-borne carbapenemase genes, then the extracted plasmids were transformed into competent E. coli DH5α. A total of 256 Gram-negative bacterial clinical isolates were collected, 65 (25.4%) isolates showed carbapenem resistance of which 36 (55.4%) were carbapenemase-producers, and of these 31 (47.7%) harbored plasmids. The extracted plasmids were used as templates for PCR amplification of blaKPC, blaNDM, blaVIM, blaOXA-48, and blaIMP carbapenemase genes. The blaOXA-48 gene was detected in 24 (77.4%) of the tested isolates while blaVIM gene was detected in 8 (25.8%), both blaKPC and blaNDM genes were co-present in 1 (3.2%) isolate. Plasmids carrying the blaOXA-48 gene from 4 K. pneumoniae clinical isolates were successfully transformed into competent E. coli DH5α. The transformants were carbapenemase-producers and acquired resistance to some of the tested antimicrobial agents as compared to untransformed E. coli DH5α. The study concluded that the rate of carbapenem resistance among Gram-negative bacterial uropathogens in Cairo, Egypt is relatively high and can be transferred horizontally to other bacterial host(s).

Biofilm formation by Streptococcus mutans and its inhibition by green tea extracts.

Dental Caries is considered one of the most existing and worldwide common diseases related to the oral cavity affecting both children and adults. Streptococcus mutans is the main cariogenic microorganism involved in the dental caries progression. Natural products such as herbal plants were found to have less side effects and economic value than those of the chemically synthesized antibiofilm agents. This study aimed to isolate Streptococcus mutans from different oral samples taken from saliva and dental plaques specimens to determine their capability for biofilm formation and to evaluate the antibiofilm activity of aqueous and alcoholic green tea extracts. The results revealed that 35, 4 and 1% of recovered dental plaque isolates exhibited strong, moderate and weak biofilm formation capabilities versus 26, 12 and 2% for those recovered from saliva. Two green tea extracts (aqueous and alcoholic) were tested for their antibiofilm formation activity against some selected S. mutans isolates. The results showed that the minimum biofilm inhibitory concentrations (MBICs) of the alcoholic and aqueous green tea extracts were in the range of 3.1 to 12.5 mg/ml and 6.5 to 50 mg/ml, respectively. Accordingly, green tea extracts can be incorporated in various oral preparations for preventing dental caries.

Trastuzumab immunogenicity development in patients' sera and in laboratory animals.

BACKGROUND: Immunogenicity is a major challenge in drug development and patient care. Clinicians and regulators are familiar with immunogenicity concerns of monoclonal antibody (mAb) therapeutics, growth factors and enzyme replacements. Although most small therapeutic molecules are unlikely to trigger undesirable immunogenic responses against themselves upon their administration, the biological therapeutic agents are likely to induce such kind of immunogenicity. This imparts a problem that has to be considered upon judging their risk-benefit ratio. In this article, we tested the immunogenicity developed in patients' sera due to the use of trastuzumab and that developed in laboratory animals injected with this recombinant humanized IgG1 monoclonal antibody. METHODS: We studied trastuzumab immunogenicity by: I in vitro detection of anti-trastuzumab antibody (Ab) levels in patient's serum samples withdrawn at different points during trastuzumab treatment course; I.1 using an Affinity Capture Elution (ACE) assay, the assay is both sensitive and highly tolerant to free drug; I.2 using MTT cytotoxicity method against MCF-7 cell line as confirmatory method used in sample showed high level of anti-trastuzumab Ab and to determine neutralizing activity of the anti-trastuzumab Ab. II in vivo immunogenicity testing of trastuzumab in lab animals. RESULTS: In vitro analysis of patients' sera for antibodies developed against trastuzumab revealed that this monoclonal antibody has low immunogenicity since most samples showed low levels of anti-trastuzumab antibodies that decreased progressively along the treatment course. Only 1% of samples showed high levels of anti-trastuzumab antibodies which might affect treatment course. In vivo immunogenicity testing in mice showed also low immunogenicity of trastuzumab that could support the in vitro clinical assessment applied in our study. CONCLUSIONS: The study gives an evidence for the low trastuzumab immunogenicity when assessed in Egyptian patients under treatment with this biological therapeutic agent. This supports its prescription and continuous use across the approved indications as biological therapeutic agent.

"Effect of Subinhibitory Concentrations of Some Antibiotics and Low Doses of Gamma Radiation on the Cytotoxicity and Expression of Colibactin by an Uropathogenic Escherichia coli isolate".

Colibactin and cytotoxic necrotizing factor 1 (Cnf 1) are cyclomodulins secreted by uropathogenic E. coli. In this study, uropathogenic E. coli expressing colibactin and Cnf 1 was exposed to antibiotics subMICs and gamma radiation to investigate their effects on its cytotoxicity and expression of colibactin. The test isolate was exposed to three subMIC levels of levofloxacin, ciprofloxacin, trimethoprim/sulfamethoxazole and ceftriaxone and irradiated with gamma rays at 10 and 24.4 Gy. The cytotoxicity for either antibiotic or gamma rays treated cultures was measured using MTT assay and the expression of colibactin encoding genes was determined by RT-PCR. Treatment with fluoroquinolones nearly abolished the cytotoxicity of E. coli isolate and significantly downregulated clbA gene expression at the tested subMICs (P ≤ 0.05) while trimethoprim/sulfamethoxazole treated cultures exerted significant downregulation of clbA and clbQ genes at 0.5 MIC only (P ≤ 0.05). Ceftriaxone treated cultured exhibited reduction in the cytotoxicity and insignificant effects on expression of clbA, clbQ and clbM genes. On contrast, significant upregulation in the expression of clbA and clbQ genes was observed in irradiated cultures (P ≤ 0.05). Fluoroquinolones reduced both the cytotoxicity of UPEC isolate and colibactin expression at different subMICs while ceftriaxone at subMICs failed to suppress the expression of genotoxin, colibactin, giving an insight to the risks associated upon their choice for UTI treatment. Colibactin expression was enhanced by gamma irradiation at doses resembling these received during pelvic radiotherapy which might contribute to post-radiotherapy complications.

Structural and Physicochemical Characterization of Rhamnolipids produced by Pseudomonas aeruginosa P6.

Rhamnolipids are important biosurfactants for application in bioremediation, enhanced oil recovery, pharmaceutical, and detergent industry. In this study, rhamnolipids extracted from P. aeruginosa P6 were characterized to determine their potential fields of application. Thin-layer chromatographic analysis of the produced rhamnolipids indicated the production of two homologues: mono- and di-rhamnolipids, whose structures were verified by 1H and 13C nuclear magnetic resonance spectroscopy. Additionally, high performance liquid chromatography-mass spectrometry identified seven different rhamnolipid congeners, of which a significantly high proportion was di-rhamnolipids reaching 80.16%. Rha-Rha-C10-C10 was confirmed as the principal compound of the rhamnolipid mixture (24.30%). The rhamnolipids were capable of lowering surface tension of water to 36 mN/m at a critical micelle concentration of 0.2 g/L, and exhibited a great emulsifying activity (E24 = 63%). In addition, they showed excellent stability at pH ranges 4-8, NaCl concentrations up to 9% (w/v) and temperatures ranging from 20 to 100 °C and even after autoclaving. These results suggest that rhamnolipids, produced by P. aeruginosa P6 using the cheap substrate glycerol, are propitious for biotechnology use in extreme and complex environments, like oil reservoirs and hydrocarbon contaminated soil. Moreover, P. aeruginosa P6 may be considered, in its wild type form, as a promising industrial producer of di-RLs, which have superior characteristics for potential applications and offer outstanding commercial benefits.