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.

Biofilm Inhibitory Activity of Actinomycete-Synthesized AgNPs with Low Cytotoxic Effect: Experimental and In Silico Study.

The emergence of resistance by biofilm-forming bacteria has reached alarming and dangerous levels that threaten human civilization. The current study sought to investigate the antibiofilm potential of green-synthesized silver nanoparticles, mediated by a new Streptomyces strain. Zeta potential, transmission electron microscopy (TEM), and UV-Vis spectroscopy were used to analyze the biosynthesized AgNPs. Results revealed that silver nanoparticles had a size of (5.55 and 45.00 nm) nm and a spherical shape, with surface plasmon resonance (SPR) absorption at 400-460 nm in the UV-vis spectra establishing the formation of Streptomyces-Ag-NPs. The biosynthesized AgNPs showed a pronounced antibacterial efficacy against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus. Moreover, the obtained Streptomyces-AgNPs exerted biofilm inhibition activity against nosocomial hospital-resistant bacteria, including Bacillus subtilis, Staphylococcus aureus, and Escherichia coli. The mechanism of biogenic AgNPs antibacterial action was visualized using TEM, which indicated the AgNPs accumulation and disruption of bacterial cell membrane function. Additionally, a molecular docking study was conducted to evaluate the binding mode of AgNPs with an Escherichia coli outer membrane. Furthermore, the cytotoxic profile of the AgNPs was evaluated toward three cell lines (MCF-7, HepG2 & HCT 116), and the low cytotoxic effects of the obtained nanoparticles indicated their possible medical application with low risks to human health.

atpD gene sequencing, multidrug resistance traits, virulence-determinants, and antimicrobial resistance genes of emerging XDR and MDR-Proteus mirabilis.

Proteus mirabilis is a common opportunistic pathogen causing severe illness in humans and animals. To determine the prevalence, antibiogram, biofilm-formation, screening of virulence, and antimicrobial resistance genes in P. mirabilis isolates from ducks; 240 samples were obtained from apparently healthy and diseased ducks from private farms in Port-Said Province, Egypt. The collected samples were examined bacteriologically, and then the recovered isolates were tested for atpD gene sequencing, antimicrobial susceptibility, biofilm-formation, PCR detection of virulence, and antimicrobial resistance genes. The prevalence of P. mirabilis in the examined samples was 14.6% (35/240). The identification of the recovered isolates was confirmed by the atpD gene sequencing, where the tested isolates shared a common ancestor. Besides, 94.3% of P. mirabilis isolates were biofilm producers. The recovered isolates were resistant to penicillins, sulfonamides, ß-Lactam-ß-lactamase-inhibitor-combinations, tetracyclines, cephalosporins, macrolides, and quinolones. Using PCR, the retrieved strains harbored atpD, ureC, rsbA, and zapA virulence genes with a prevalence of 100%, 100%, 94.3%, and 91.4%, respectively. Moreover, 31.4% (11/35) of the recovered strains were XDR to 8 antimicrobial classes that harbored blaTEM, blaOXA-1, blaCTX-M, tetA, and sul1 genes. Besides, 22.8% (8/35) of the tested strains were MDR to 3 antimicrobial classes and possessed blaTEM, tetA, and sul1genes. Furthermore, 17.1% (6/35) of the tested strains were MDR to 7 antimicrobial classes and harbored blaTEM, blaOXA-1, blaCTX-M, tetA, and sul1 genes. Alarmingly, three strains were carbapenem-resistant that exhibited PDR to all the tested 10 antimicrobial classes and shared blaTEM, blaOXA-1, blaCTX-M, tetA, and sul1 genes. Of them, two strains harbored the blaNDM-1 gene, and one strain carried the blaKPC gene. In brief, to the best of our knowledge, this is the first study demonstrating the emergence of XDR and MDR-P.mirabilis in ducks. Norfloxacin exhibited promising antibacterial activity against the recovered XDR and MDR-P. mirabilis. The emergence of PDR, XDR, and MDR-strains constitutes a threat alarm that indicates the complicated treatment of the infections caused by these superbugs.

Molecular and HPLC-based approaches for detection of aflatoxin B1 and ochratoxin A released from toxigenic Aspergillus species in processed meat.

BACKGROUND: Meat-products are considered an enriched media for mycotoxins. This study aimed to investigate the prevalence of toxigenic Aspergillus species in processed meat samples, HPLC-quantitative measurement of aflatoxin B1 and ochratoxin A residues, and molecular sequencing of aflR1 and pks genes. One hundred and twenty processed beef meat specimens (basterma, sausage, and minced meat; n = 40 for each) were collected from Ismailia Province, Egypt. Samples were prepared for total mold count, isolation, and identification of Aspergillus species. All samples were analyzed for the production of both Aflatoxin B1 and Ochratoxin A mycotoxins by HPLC. Molecular identification of Aspergillus flavus and Aspergillus ochraceus was performed using PCR amplification of the internal transcribed spacer (ITS) region; furthermore, the aflR1 and pks genes were sequenced. RESULTS: The total mold count obtained from sausage samples was the highest one, followed by minced meat samples. The prevalence of A. flavus was (15%), (7.5%), and (10%), while the prevalence of A. ochraceus was (2.5%), (10%), and (0%) in the examined basterma, sausage, and minced meat samples, respectively. Using PCR, the ITS region was successfully amplified in all the tested A. flavus and A. ochraceus strains. Aflatoxin B1 was detected in six basterma samples (15%). Moreover, the ochratoxin A was detected only in four sausage samples (10%). The aflR1 and pks genes were amplified and sequenced successfully and deposited in the GenBank with accession numbers MF694264 and MF694264, respectively. CONCLUSIONS: To the best of our knowledge, this is the first report concerning the HPLC-Molecular-based approaches for the detection of aflatoxin B1 and ochratoxin A in processed beef meat in Egypt. The production of aflatoxin B1 and ochratoxin A in processed meat constitutes a public health threat. Aflatoxin B1 is commonly associated with basterma samples. Moreover, ochratoxin A was detected frequently in sausage samples. The routine inspection of mycotoxins in processed meat products is essential to protect human consumers.

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).

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.

High-throughput molecular identification of Staphylococcus spp. isolated from a clean room facility in an environmental monitoring program.

BACKGROUND: The staphylococci are one of the most common environmental isolates found in clean room facility. Consequently, isolation followed by comprehensive and accurate identification is an essential step in any environmental monitoring program. FINDINGS: We have used the API Staph identification kit (bioMérieux, France) which depends on the expression of metabolic activities and or morphological features to identify the Staphylococcus isolates. The API staphylococci showed low sensitivity in the identification of some species, so we performed molecular methods based on PCR based fingerprinting of glyceraldehyde-3-phosphate dehydrogenase encoding gene as useful taxonomic tool for examining Staphylococcus isolates. CONCLUSIONS: Our results showed that PCR protocol used in this study which depends on genotypic features was relatively accurate, rapid, sensitive and superior in the identification of at least 7 species of Staphylococcus than API Staph which depends on phenotypic features.