Regulation of overexpressed efflux pump encoding genes by cinnamon oil and trimethoprim to abolish carbapenem-resistant Acinetobacter baumannii clinical strains.

Resistance mechanisms are a shelter for Acinetobacter baumannii to adapt to our environment which causes difficulty for the infections to be treated and WHO declares this organism on the top of pathogens priority for new drug development. The most common mechanism that develops drug resistance is the overexpression of the efflux pump, especially Resistance-nodulation-cell division (RND) family, to almost most antibiotics. The study is designed to detect RND efflux pump genes in A. baumannii, and its correlation to multidrug resistance, in particular, the carbapenems resistance Acinetobacter baumannii (CRAB), and using different inhibitors that restore the antibiotic susceptibility of imipenem. Clinical A. baumannii isolates were recovered from different Egyptian hospitals in Intensive care unit (ICU). The expression of genes in two strains was analyzed using RT-PCR before and after inhibitor treatment. About 100 clinical A. baumannii isolates were recovered and identified and recorded as MDR strains with 75% strains resistant to imipenem. adeB, adeC, adeK, and adeJ were detected in thirty- seven the carbapenems resistance Acinetobacter baumannii (CRAB) strains. Cinnamomum verum oil, Trimethoprim, and Omeprazole was promising inhibitor against 90% of the carbapenems resistance Acinetobacter baumannii (CRAB) strains with a 2-6-fold decrease in imipenem MIC. Downregulation of four genes was associated with the addition of those inhibitors to imipenem for two the carbapenems resistance Acinetobacter baumannii (CRAB) (ACN15 and ACN99) strains, and the effect was confirmed in 24 h killing kinetics. Our investigation points to the carbapenems resistance Acinetobacter baumannii (CRAB) strain's prevalence in Egyptian hospitals with the idea to revive the imipenem activity using natural and chemical drugs as inhibitors that possessed high synergistic activity.

Prevalence and In Vivo Assessment of Virulence in Shiga Toxin-Producing Escherichia coli Clinical Isolates from Greater Cairo Area.

Background: Shiga toxin-producing Escherichia coli (STEC) has been identified as an important etiologic agent of human disease in Egypt. Aims: To investigate the occurrence and describe the characterization as well as prevalence of STEC in Greater Cairo hospitals as well as molecular characterization of virulence and resistance genes. Methods: Four hundred seventy E. coli clinical isolates were collected from eight hospitals and analyzed by genotypic and phenotypic methods for STEC, followed by histopathological examination and scoring of different organs lesions. Results: The highest proportion of isolates was from urine (151 isolates), whereas the lowest was from splenic drain (3 isolates). In tandem, when serogrouping was performed, 15 serogroups were obtained where the most prevalent was O157 and the least prevalent was O151. All isolates were positive when screened for identity gene gad A, while only typable strains were screened for seven virulence genes stx1 (gene encoding Shiga toxin 1), stx2 (gene encoding Shiga toxin 2), tsh (gene encoding thermostable hemagglutinin), eaeA (gene encoding intimin), invE (gene encoding invasion protein), aggR (gene encoding aggregative adherence transcriptional regulator), and astA (aspartate transaminase) where the prevalence was 48%, 30%, 50%, 57%, 7.5%, 12%, and 58%, respectively. Of 254 typable isolates, 152 were STEC carrying stx1 or stx2 genes or both. Conclusions: Relying on in vivo comparison between different E. coli pathotypes via histopathological examination of different organs, E. coli pathotypes could be divided into mild virulent, moderate virulent, and high virulent strains. Statistical analysis revealed significant correlation between different serogroups and presence of virulence genes.

New typhoid vaccine using sponge-like reduced protocol: development and evaluation.

Purpose: Typhoid remains a major health problem, especially in the developing world. Furthermore, the emergence of multidrug-resistant and extensively drug-resistant strains of Salmonella typhi added a sense of urgency to develop more effective typhoid vaccines, one of which is bacterial ghosts (BGs), prepared by both genetic and chemical means. The chemical method includes incubation with numerous agents for a short time at their minimum inhibitory or minimum growth concentrations. This study included the preparation of BGs by a sponge-like reduced protocol (SLRP). Materials and Methods: Critical concentrations of sodium dodecyl sulfate, NaOH, and H2O2 were used. Moreover, high-quality BGs were visualized by scanning electron microscope (SEM). Subculturing was used to confirm the absence of vital cells. Besides, the concentrations of the released DNA and protein were estimated spectrophotometrically. In addition, the integrity of cells was proved by visualizing Gram-stained cells using a light microscope. Furthermore, a comparison between the immunogenicity and safety of the prepared vaccine and the available whole-cell killed vaccine was established. Results: Improved preparation of high-quality BGs of S. typhi, visualized by SEM, revealed punctured cells with intact outer shells. Moreover, the absence of vital cells was confirmed by subculturing. At the same time, the release of respective amounts of proteins and DNA is another evidence of BGs' production. Additionally, the challenge test provided evidence that the prepared BGs are immunogenic and have the same efficacy as the whole cell vaccine. Conclusion: The SLRP provided a simple, economical, and feasible method for BGs preparation.

Advanced approaches for endotoxin detection and removal from snake antivenoms.

A comprehensive assessment of the literature on strategies for the detection and removing endotoxin from biotechnological preparations was conducted. This study highlighted the brief history of endotoxin. After that, a review of endotoxin's chemical and physical features, as well as its pathophysiological consequences when the body is exposed to LPS excessively or systemically, is presented. The procedures for determining endotoxin and the interaction of endotoxin with proteins are also discussed, considering both known approaches and cutting-edge technology in this sector. This review presented the endotoxin detection and removal approaches from antisera with an economical approach using several processes documented in the literature (e.g., adsorption, ultrafiltration, and chromatography). Different methods with relatively high protein recoveries are mentioned. This review concludes that heat activation at 70 °C-80 °C for 10 min and rehydration of the LAL reagent with endotoxin-specific buffer solution is the best technique to control the enhancement problem when testing polyvalent snake venom antiserum samples by the LAL method. The most efficient method for eliminating endotoxins has proven to be affinity resin-based chromatography.

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.

Evaluation of MMR live attenuated vaccine oncolytic potential using Ehrlich ascites carcinoma in a murine model.

MMR vaccine is a common vaccine that contains oncolytic viruses (Measles, Mumps, and Rubella) and could be used as a potential anti-cancer treatment. In this study, we assessed the anti-tumor activity of the MMR vaccine against Ehrlich ascites carcinoma (EAC) solid tumor induced in mice. The in vitro assay showed that vaccine IC50 in EAC was approximately 200 CCID50. The vaccine was intratumorally administrated twice weekly in EAC-bearing mice. The antitumor response of the vaccine was measured by tumor growth, survival rate, histopathologic examination, flow cytometry analysis, and body biochemical parameters. The MMR vaccine demonstrated a substantial reduction of tumor growth and prolongation of life span as well. The proliferation marker was significantly lower in the vaccine-treated group. Moreover, the apoptosis key parameter Casp-3 was also higher in the vaccine-treated group. The vaccine somewhat restored the deterioration of the biochemical parameters (LDH, GOT, GPT, MDA, NO, and PON-1) in the tumor-bearing mice. Finally, this study indicated the potential antitumor effect of MMR vaccine via anti­proliferative, apoptotic activities, and modulating the antioxidant parameters. This study opens a new field of inquiry for future research on the vaccine's anti-cancer properties.

Antivenoms, hepatitis B vaccine and oral polio vaccine can be considered for storage and handling outside the cold chain following the innovative 'controlled temperature chain' approach.

Possible applicability of controlled temperature chain (CTC) for selected antisera and vaccines was evaluated. Bivalent oral polio vaccine (OPV), hepatitis B vaccine (HepB vaccine; monovalent and combined) and antisera (lyophilized and liquid scorpion-antivenom and liquid snake-antivenom) were tested. Samples were stored at accelerated (35 ± 5 °C) and freezing (-25 ± 5 °C) conditions for 24 h, one week and one month in addition to recommended storage condition (2-8 °C), except OPV samples that were tested at accelerated and refrigerated (2-8 °C) conditions compared to recommended storage conditions (-25 ± 5 °C). All samples were tested for potency. Protein content and composition were determined for antisera samples. All vaccine vial-monitors were evaluated. HepB vaccine was subjected to aluminum-content assay, shake test and microscopical examination. No significant change in antisera potency was detectable under accelerated condition for a week. OPV stored in refrigerator for a month and at accelerated condition for 48 h maintained acceptable potency. Monovalent and combined HepB vaccine maintained acceptable potency under accelerated condition for a month and a week, respectively. Freezing adversely affected HepB vaccine. In conclusion, reevaluation of storage conditions of tested products is urgently required; this can reduce storage costs and improves their availability. Other products should be tested for possible CTC applicability.

Expression of full and fragment-B of diphtheria toxin genes in Escherichia coli for generating of recombinant diphtheria vaccines.

PURPOSE: In the present study, whole diphtheria toxin (dt) and fragment B (dtb) genes from Corynebacterium diphtheriae Park William were cloned into Escherichia coli, the purified expressed proteins were evaluated for ultimately using as a candidate vaccine. MATERIALS AND METHODS: The dt and dtb genes were isolated from bacterial strain ATCC (American Type Culture Collection) no. 13812. Plasmid pET29a+ was extracted by DNA-spin TM plasmid purification kit where genes were inserted using BamHI and HindIII-HF. Cloned pET29a+dt and pET29a+dtb plasmids were transformed into E. coli BL21(DE3)PlysS as expression host. The identity of the sequences was validated by blasting the sequence (BLASTn) against all the reported nucleotide sequences in the NCBI (National Center for Biotechnology Information) GenBank. Production of proteins in high yield by different types and parameters of fermentation to determine optimal conditions. Lastly, the purified concentrated rdtx and rdtb were injected to BALB/c mice and antibody titers were detected. RESULTS: The genetic transformation of E. coli DH5α and E. coli BL21 with the pET-29a(+) carrying the dt and dtb genes was confirmed by colony polymerase chain reaction assay and were positive to grow on Luria-Bertani/kanamycin medium. The open reading frame of dt and dtb sequences consisted of 1,600 bp and 1,000 bp, were found to be 100% identical to dt and dtb sequence of C. diphtheriae (accession number KX702999.1 and KX702993.1) respectively. The optimal condition for high cell density is fed-batch fermentation production to express the rdtx and rdtb at 280 and 240 Lf/mL, dissolved oxygen was about 24% and 22% and the dry cell weight of bacteria was 2.41 g/L and 2.18 g/L, respectively. CONCLUSION: This study concluded with success in preparing genetically modified two strains for the production of a diphtheria vaccine, and to reach ideal production conditions to achieve the highest productivity.

Quorum sensing inhibitory effect of bergamot oil and aspidosperma extract against Chromobacterium violaceum and Pseudomonas aeruginosa.

Quorum sensing (QS) represents a major target for reducing bacterial pathogenicity and antibiotic resistance. This study identifies bergamot and aspidosperma as new potential sources of anti-QS agents. We investigated the anti-QS activity of plant materials on both Chromobacterium violaceum and Pseudomonas aeruginosa. Initially, we determined the minimum inhibitory concentrations (MICs) of plant materials using a broth microdilution method. Subsequently, we tested the effect of sub-MIC concentrations on QS-regulated traits and virulence factors production in test bacteria. Results revealed that bergamot and aspidosperma inhibited the ability of C. violaceum to produce violacein. Other QS-controlled phenotypes of C. violaceum, namely chitinolytic activity, motility, and biofilm formation, were also reduced by both plant materials. Moreover, QS-linked traits of P. aeruginosa were also reduced. Bergamot inhibited swarming but not swimming motility, while aspidosperma diminished both motility types in P. aeruginosa. Both plant materials also demonstrated antibiofilm activity and inhibited the production of protease and pyocyanin in P. aeruginosa. Furthermore, we tested the anti-QS effect of plant materials on the transcriptional level using RT-qPCR. Bergamot dramatically downregulated the C. violaceum autoinducer synthase gene cviI and the vioB gene involved in violacein biosynthesis, confirming the phenotypic observation on its anti-QS activity. Aspidosperma also reduced the expression of cviI and vioB but less drastically than bergamot. In P. aeruginosa, downregulation in the transcripts of the QS genes lasI, lasR, rhlI, and rhlR was also achieved by bergamot and aspidosperma. Therefore, data in the present study suggest the usefulness of bergamot and aspidosperma as sources of antivirulence agents.

A Study on Multidrug-Resistant Escherichia coli Clinical Isolates from Different Hospitals in Greater Cairo.

The biological fitness cost of antibiotic resistance is a key parameter in determining the rate of appearance and spread of antibiotic-resistant bacteria in Egypt. Our study aimed to investigate the prevalence of antibiotic resistance among Escherichia coli clinical isolates from Greater Cairo area hospitals. A total of 537 clinical isolates were recovered from samples of urine, diarrheal specimen, pus, wound culture, gastric wound, blood, drain culture, sputum, high vaginal swab, abscess, amniotic fluid, ventilator, burn swab, splenic drain culture, and unknown site of infection during different seasons. All isolates were subjected to phenotypic and genotypic susceptibility testing for colistin, nitrofurantoin, fosfomycin, and trimethoprim, quinolones, and ß-lactam resistance. Our results revealed that 42.7% of the isolates harbored at least one resistance encoding gene, 10% harboring 2, 0.6% harboring 3, and 0.85% harboring 4 resistance-encoding genes. PCR reported the prevalence of resistance genes as follows: bla-SHV 13.4%, mcr-1 0.6%, qnr-A 23.8%, fos-A 1.06%, nfs-A 3.6%, and dfr-A 25.5%. We reported that three isolates carried the mcr-1 gene encoding colistin resistance from three different hospitals. Upon performing sequencing and phylogenetic analysis on the three positive mcr-1 isolates (MT890587, MT890588, and MT890589), the three isolates showed 100% identity with themselves, with some strains from Egypt and Japan, and 99.9% identity with an isolate from China.

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.

Kojic acid repurposing as a pancreatic lipase inhibitor and the optimization of its production from a local Aspergillus oryzae soil isolate.

BACKGROUND: Obesity and its related diseases are increasing worldwide. One of the best therapeutic strategies for obesity management is through the inhibition of pancreatic lipase (PL) enzyme. So far orlistat is the only FDA approved PL inhibitor, but with unpleasant side effects. New efficacious anti-obesity drugs are needed to achieve a successful reduction in the incidence and prevalence of obesity. Many microbial metabolites have PL inhibitory activity. Screening soil inhabitants for PL inhibitors could help in increasing the available anti-obesity drugs. We aimed to isolate and identify alternative PL inhibitors from soil flora. RESULTS: We screened the crude mycelial methanolic extracts of 39 soil samples for PL inhibitory activity by the quantitative lipase colorimetric assay, using the substrate p-nitrophenyl palmitate and orlistat as positive control. AspsarO, a PL inhibitor producer, was isolated from an agricultural field soil in Giza, Egypt. It was identified as Aspergillus oryzae using colony morphology, microscopical characteristics, 18S rDNA sequencing, and molecular phylogeny. Increasing the PL inhibitor activity, in AspsarO cultures, from 25.9 ± 2% to 61.4 ± 1.8% was achieved by optimizing the fermentation process using a Placket-Burman design. The dried 100% methanolic fraction of the AspsarO culture had an IC50 of 7.48 µg/ml compared to 3.72 µg/ml for orlistat. It decreased the percent weight gain, significantly reduced the food intake and serum triglycerides levels in high-fat diet-fed Sprague-Dawley rats. Kojic acid, the active metabolite, was identified using several biological guided chromatographic and 1H and 13C NMR techniques and had an IC50 of 6.62 µg/ml. Docking pattern attributed this effect to the interaction of kojic acid with the key amino acids (Lys80, Trp252, and Asn84) in PL enzyme binding site. CONCLUSION: Combining the results of the induced obesity animal model, in silico molecular docking and the lipase inhibitory assay, suggests that kojic acid can be a new therapeutic option for obesity management. Besides, it can lower serum triglycerides in obese patients.

A Validation Study of the Limulus Amebocyte Lysate Test as an End-Product Endotoxin Test for Polyvalent Horse Snake Antivenom.

The only definitive management of snake envenoming is the use of snake antivenom. Endotoxin contamination is a serious threat to the safe use of parenteral drugs. A greater understanding of the nature of limulus amebocyte lysate (LAL) test interference and use of permissible dilutions has minimized enhancement problems. Common interference issues include suboptimal pH, enzyme or protein modification, and nonspecific LAL activation. This study aimed at determining the interference factors associated with validating the antivenom sera preparations to avoid false-positive results when testing snake antivenom serum samples by the LAL method. Phase I (preliminary screening/interference assay) was performed to determine a compatible test dilution, which was then used in Phase II (inhibition-enhancement/validation study). The best approach to resolve interference issues was dilution by 1:80 (maximum valid dilution) plus a specific treatment as heat-activation at 70°C-80°C for 10 min with rehydration of LAL reagent with endotoxin-specific buffer solution.LAY ABSTRACT: Snake antivenom sera are produced by immunizing horses with repeated nonlethal doses of snake venom. Bacterial endotoxins constitute one of the major problems in the formulation of pharmaceutical products. One such method for detecting endotoxin levels is the bacterial endotoxin test (BET). However, some substances show strong interfering action with the BET that cannot be avoided by simply diluting the sample solution. In this work, the test for interfering factors was performed as two identical series of product dilutions-one spiked with 2λ and one left unspiked. The result of the interference test revealed the noninterfering dilution (NID) of the product, which was used for the actual validation. Our results showed that after treating the samples using different procedures, such as heat activation at 70-80°C for 10 min followed by centrifugation at 2000 rpm for 10 min and dilution of samples in BD100 (biodispersing agent), inhibition and enhancement up to 1:100 maximum valid dilution (MVD) were observed. Finally, to resolve this inhibition/enhancement problem, the activated sample was heated at 70-80°C for 10 min with rehydration of the Endosafe LAL reagent in an endotoxin-specific buffer solution (BG120) to block ß-d-glucans and limulus amebocyte lysate (LAL) reactive material (LAL-RM).

Mining of Egypt's Red Sea invertebrates for potential bioactive producers.

OBJECTIVE: The objective of this work was to isolate bacteria from Red Sea invertebrates, determine their antimicrobial activity, and screen for the biosynthetic gene clusters [polyketides (PKs) and nonribosomal peptides (NRPs)] which could be involved in the production of bioactive secondary metabolites. RESULT: Eleven different samples of marine invertebrates' were collected from Egypt's Red Sea (El-Tor-Sharm El-Sheikh and Hurghada) by scuba diving, and a total 80 isolates of the associated microorganisms were obtained from the cultivation on six different cultural medium. Seven isolates of them showed an antimicrobial activity against five pathogenic reference strains, while the most active antimicrobial agent was isolate number HFF-8 which was 99% identical to Bacillus amyloliquefaciens. HFF-8's extract showed positive results against Gram negative bacteria, Gram positive bacteria and yeast. Moreover, the isolates gave positive bands when screened for the presence of PK synthase (PKS) I and II and NRP synthetase (NRPS) I and II biosynthetic genes, those biosynthetic fragments when cloned and sequenced were primitively predicted as biosynthetic fragments for kirromycin and leinamycin production by NaPDoS program with 56 and 55%, respectively. CONCLUSION: The Red Sea can provide a sustainable solution to combat bacterial resistance. The contribution of this work is that B. amyloliquefaciens was isolated from Heteroxenia fuscescens, Red Sea, Egypt. Moreover, the bacterial extract showed a broad spectrum with a potent antimicrobial activity.

Synthesis, cloning and expression of a novel pre-miniproinsulin analogue gene in Escherichia coli.

In the present study, a novel pre-miniproinsulin analogue was designed to have a short 9 residue sequence replacing the 35 residue C-chain, one lysine and one arginine added to the C-terminus of the B-chain in combination with glycine and arginine substitution at A21 and B29, respectively, and a 16-residue fusion partner comprising the pentapeptide sequence (PSDKP) of the N-terminus of human tumor necrosis factor-α (TNF-α), 6 histidine residues for Ni(2+) chelated affinity purification and a pentapeptide ending with methionine for ease of chemical cleavage fused at the N-terminus. Homology modeling of the designed protein against miniproinsulin (protein databank file 1 efeA) as a template showed that the distance between the α-carbons of the C-terminus of the B-chain and the N-terminus of the A-chain did not change; the root-mean-square deviation of the backbone atoms between the structures of modeled miniproinsulin and miniproinsulin template was 0.000 Å. DNA sequencing of the synthesized gene showed 100% identity with theoretical sequence. The gene was constructed taking into account the codon preference of Escherichia coli (CAI value 0.99) in order to increase the expression rate of the DNA in the host strain. The designed gene was synthesized using DNA synthesis technology and then cloned into the expression plasmid pET-24a(+) and propagated in E. coli strain JM109. Gene expression was successful in two E. coli strains: namely JM109(DE3) and BL21(DE3)pLysS. SDS-PAGE analysis was carried out to check protein size and to check and optimize expression. Rapid screening and purification of the resulting protein was carried out by Ni-NTA technology. The identity of the expressed protein was verified by immunological detection method of western blot using polyclonal rabbit antibody against insulin.

Quality Control Testing for Tracking Endotoxin-Producing Gram-Negative Bacteria during the Preparation of Polyvalent Snake Antivenom Immunoglobulin.

UNLABELLED: Snake bites represent a serious public health problem, particularly in rural areas worldwide. Antitoxic sera preparations are antibodies from immunized animals and are considered to be the only treatment option. The purification of antivenom antibodies should aim at obtaining products of consistent quality, safety, efficacy, and adherence to good manufacturing practice principles. Endotoxins are an integral component of the outer cell surface of Gram-negative bacteria. They are common contaminates of the raw materials and processing equipment used in the manufacturing of antivenoms. In this work, and as a part of quality control testing, we establish and examine an environmental monitoring program for identification of potential sources of endotoxin-producing Gram-negative bacteria throughout the whole steps of antivenom preparation. In addition, we follow all the steps of preparation starting from crude plasma till finished product using a validated sterility and endotoxin testing.Samples from air, surface, and personnel were collected and examined through various stages of manufacturing for the potential presence of Gram-negative bacteria. A validated sterility and endotoxin test was carried out in parallel at the different production steps. The results showed that air contributed to the majority of bacterial isolates detected (48.43%), followed by surfaces (37.5%) and then personnel (14%). The most common bacterial isolates detected were Achromobacter xylosoxidans, Ochrobactrum anthropi, and Pseudomonas aeruginosa, which together with Burkholderia cepacia were both also detected in cleaning water and certain equipment parts. A heavy bacterial growth with no fungal contamination was observed in all stages of antivenom manufacturing excluding the formulation stage. All samples were positive for endotoxin including the finished product.Implementation and continued evaluation of quality assurance and quality improvement programs in aseptic preparation is essential in ensuring the safety and quality of these products. LAY ABSTRACT: Antitoxic sera preparations are the only treatment option for snake bites worldwide. They are prepared by immunizing animals, usually horses, with snake venom and collecting horse plasma, which is then subjected to several purification steps in order to finally prepare the purified immunoglobulins. Components of the bacterial cell wall known as endotoxins can constitute a potential hazardous contamination known as pyrogen in antisera, which can lead to fever and many other adverse reactions to the person subjected to it.In this work, we monitored the environment associated with the different steps of production and purification of snake antivenom prepared from immunized horses. We examined the air quality, surface, and personnel for possible sources of contamination, particularly the presence of Gram-negative bacteria, which is the major source of endotoxin presence. We also monitored all stages of preparation by sterility and endotoxin testing. Our results showed that air contributed to the majority of bacterial isolates. Sterility testing revealed the presence of bacterial contamination in all the intermediate steps, as only the final preparation after filtration was sterile. Endotoxin was present in all tested samples and the final product. Good manufacturing practice procedures are essential in any facility involved in antisera production.

Fluoroquinolone resistant mechanisms in methicillin-resistant Staphylococcus aureus clinical isolates in Cairo, Egypt.

INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) is a persistent problem in community and health care settings. Fluoroquinolones are among the drugs of choice used to treat MRSA infections. This study aims to identify different mechanisms of fluoroquinolne resistance in local MRSA random sampling isolates in Cairo, Egypt. METHODOLOGY: A total of 94 clinical isolates of S. aureus were collected from two major University hospitals in Cairo. Identification was confirmed by appropriate morphological, cultural, and biochemical tests. The antibiotic susceptibility pattern was determined for all isolates. The possible involvement of efflux pumps in mediating fluoroquinolone resistance as well as changes in the quinolone resistance determining region (QRDR) of gyrA and gyrB genes were investigated RESULTS: A total of 45 isolates were found to be MRSA, among which 26 isolates were found to be fluoroquinolone-resistant. The MIC values of the tested fluoroquinolones in the presence of the efflux pump inhibitors omeprazole and piperine were reduced. Measuring the uptake of ciprofloxacin upon the addition of the efflux pump inhibitor omeprazole, an increased level of accumulation was observed. Non-synonymous and silent mutations were detected in the QRDR of gyrA and gyrB genes. CONCLUSIONS: These results shed light on some of the resistance patterns of MRSA strains isolated from local health care settings in Cairo, Egypt. The resistance of these MRSA towards fluoroquinolones does not depend only on mutation in target genes; other mechanisms of resistance such as the permeability effect, efflux pumps and decreased availability of quinolones at the target site can also be involved.