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.

Benchmarking long-read aligners and SV callers for structural variation detection in Oxford nanopore sequencing data.

Structural variants (SVs) are one of the significant types of DNA mutations and are typically defined as larger-than-50-bp genomic alterations that include insertions, deletions, duplications, inversions, and translocations. These modifications can profoundly impact the phenotypic characteristics and contribute to disorders like cancer, response to treatment, and infections. Four long-read aligners and five SV callers have been evaluated using three Oxford Nanopore NGS human genome datasets in terms of precision, recall, and F1-score statistical metrics, depth of coverage, and speed of analysis. The best SV caller regarding recall, precision, and F1-score when matched with different aligners at different coverage levels tend to vary depending on the dataset and the specific SV types being analyzed. However, based on our findings, Sniffles and CuteSV tend to perform well across different aligners and coverage levels, followed by SVIM, PBSV, and SVDSS in the last place. The CuteSV caller has the highest average F1-score (82.51%) and recall (78.50%), and Sniffles has the highest average precision value (94.33%). Minimap2 as an aligner and Sniffles as an SV caller act as a strong base for the pipeline of SV calling because of their high speed and reasonable accomplishment. PBSV has a lower average F1-score, precision, and recall and may generate more false positives and overlook some actual SVs. Our results are valuable in the comprehensive evaluation of popular SV callers and aligners as they provide insight into the performance of several long-read aligners and SV callers and serve as a reference for researchers in selecting the most suitable tools for SV detection.

Evaluation of clinically relevant serum proteins as biomarkers for monitoring COVID-19 severity, and end-organ damage among hospitalized unvaccinated patients.

BACKGROUND: The extensive variability and conflicting information in Coronavirus Disease 2019 (COVID-19) patient data have made it difficult for the medical community to gain a comprehensive understanding and develop clear, reliable guidelines for managing COVID-19 cases. As the world uncovers the diverse side effects of the pandemic, the pursuit of knowledge about COVID-19 has become crucial. The present study aimed to evaluate some clinically relevant serum proteins, providing analysis of the obtained results to employ them in the diagnosis, prognosis, and disease monitoring among COVID-19 patients. METHODS: Samples were collected from 262 COVID-19 unvaccinated hospitalized patients. Measurement of certain serum proteins, namely C-reactive protein (CRP), ferritin, D-dimer, procalcitonin, interleukin-6 (IL-6), serum creatinine (SCr), alanine transaminase (ALT), aspartate transaminase (AST) was done using standard methods. Statistical analysis was performed on the obtained data and the results were correlated to the severity and prognosis. RESULTS: The calculated Mortality rate was found to be 30% with a higher percentage observed among females. The results showed elevation in serum CRP, ferritin, D-dimer, and procalcitonin in most of the patients, also some patients had elevated SCr, ALT, and AST levels indicating end-organ damage. The statistical analysis displayed a strong correlation between serum levels of CRP and ferritin, between D-dimer and ferritin, and between ferritin and procalcitonin. No significant difference was observed between male and female patients' serum levels of the tested serum proteins. A significant correlation between increased serum procalcitonin and mortality was observed. CONCLUSION: The levels of measured serum proteins were impacted by SARS-CoV-2 infection. Serum ferritin, CRP, D-dimer, and procalcitonin are good predicting tools for end-organ damage and acute kidney impairment in COVID-19. Procalcitonin is a strong indicator of severity and mortality in hospitalized COVID-19 patients.

Genomic characterization, in vitro, and preclinical evaluation of two microencapsulated lytic phages VB_ST_E15 and VB_ST_SPNIS2 against clinical multidrug-resistant Salmonella serovars.

BACKGROUND: Salmonella infections continue to be one of the essential public health issues threatening millions of people. With the increasing occurrence of resistance against conventionally used antibiotics, the search for alternatives has become crucial. In this study, we aimed to isolate, characterize, and evaluate two lytic bacteriophages against clinically isolated multidrug-resistant (MDR) Salmonella serovars. METHODS: Screening for the phage lytic activity was performed using a spot test. Characterization of the isolated phages was done by determining the host range, longevity test, and the effect of temperature, pH, organic solvents, and morphological characterization using a transmission electron microscope. Genomic analysis was performed using Oxford nanopore sequencing. The lytic activities of the free phage lysates and formulated phage as microencapsulated were evaluated both in vitro and in vivo. RESULTS: Two phages (VB_ST_E15 and VB_ST_SPNIS2) were successfully isolated and showed lytic strong activities against MDR Salmonella (S.) Typhimurium ATCC 14,028, S. Paratyphi A, and S. Typhi. The two phages survived at the tested temperatures, maintained their infectivity for 90 days, and retained their activity until 60 °C with thermal inactivation at 65 °C. They were lytic at a pH range from 3 to 11 but lost their activities at extremely acidic or alkaline pH. The phages could withstand the organic solvents but were completely inactivated by 100% ethanol. Both phages were classified under the order Caudoviricetes, and Genus: Uetakevirus. Their genomic sequences were assembled, annotated, and submitted to the NCBI GenBank database (OR757455 and OR757456). The preclinical evaluation using the murine animal model revealed that the two-phage cocktail managed MDR Salmonella infection as evidenced by the reduction in the bacterial burden, increased animal weight, and histopathological examination. CONCLUSION: The two encapsulated phage formulas could be considered promising candidates for the management of MDR Salmonella-associated infections and clinical analysis should be undertaken to evaluate their potential use in humans.

Nanosponge hydrogel of octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propanoate of Alcaligenes faecalis.

Octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propanoate (ODHP) was extracted in a previous study from the culture broth of soil isolate Alcaligenes faecalis MT332429 and showed a promising antimycotic activity. This study was aimed to formulate ODHP loaded ß-cyclodextrins (CD) nanosponge (NS) hydrogel (HG) to control skin fungal ailments since nanosponges augment the retention of tested agents in the skin. Box-Behnken design was used to produce the optimized NS formulation, where entrapment efficiency percent (EE%), polydispersity index (PDI), and particle size (PS) were assigned as dependent parameters, while the independent process parameters were polyvinyl alcohol % (w/v %), polymer-linker ratio, homogenization time, and speed. The carbopol 940 hydrogel was then created by incorporating the nanosponges. The hydrogel fit Higuchi's kinetic release model the best, according to in vitro drug release. Stability and photodegradation studies revealed that the NS-HG remained stable under tested conditions. The formulation also showed higher in vitro antifungal activity against Candida albicans compared to the control fluconazole. In vivo study showed that ODHP-NS-HG increased survival rates, wound contraction, and healing of wound gap and inhibited the inflammation process compared to the other control groups. The histopathological examinations and Masson's trichrome staining showed improved healing and higher records of collagen deposition. Moreover, the permeability of ODHP-NS-HG was higher through rats' skin by 1.5-folds compared to the control isoconazole 1%. Therefore, based on these results, NS-HG formulation is a potential carrier for enhanced and improved topical delivery of ODHP. Our study is a pioneering research on the development of a formulation for ODHP produced naturally from soil bacteria. KEY POINTS: • Octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propanoate was successfully formulated as a nanosponge hydrogel and statistically optimized. • The new formula exhibited in vitro good stability, drug release, and higher antifungal activity against C. albicans as compared to the fluconazole. • Ex vivo showed enhanced skin permeability, and in vivo analysis showed high antifungal activity as evidenced by measurement of various biochemical parameters and histopathological examination.

Innovative approaches for mycotoxin detection in various food categories.

Mycotoxins (MTs), produced by filamentous fungi, represent a severe hazard to the health of humans and food safety, affecting the quality of various agricultural products. They can contaminate a wide range of foods, during any processing phase before or after harvest. Animals and humans who consume MTs-contaminated food or feed may experience acute or chronic poisoning, which may result in serious pathological consequences. Accordingly, developing rapid, easy, and accurate methods of MTs detection in food becomes highly urgent and critical as a quality control and to guarantee food safety and lower health hazards. In this review, we highlighted and discussed innovative approaches like biosensors, fluorescent polarization, capillary electrophoresis, infrared spectroscopy, and electronic noses for MT identification pointing out current challenges and future directions. The limitations, current challenges, and future directions of conventional detection methods versus innovative methods have also been highlighted and discussed.

Electrospun cellulose nanofibers immobilized with anthocyanin extract for colorimetric determination of bacteria.

A novel smart biochromic textile sensor was developed by immobilizing anthocyanin extract into electrospun cellulose acetate nanofibers to detect bacteria for numerous potential uses, such as healthcare monitoring. Red-cabbage was employed to extract anthocyanin, which was then applied to cellulose acetate nanofibers treated with potassium aluminum sulfate as a mordant. Thus, nanoparticles (NPs) of mordant/anthocyanin (65-115 nm) were generated in situ on the surface of cellulose acetate nanofibrous film. The pH of a growing bacterial culture medium is known to change when bacteria multiply. The absorbance spectra revealed a bluish shift from 595 nm (purple) to 448 nm (green) during the growth of Gram-negative bacteria (E. coli) owing to the discharge of total volatile basic amines as secretion metabolites. On the other hand, the absorption spectra of a growing bacterial culture containing Gram-positive bacteria (L. acidophilus) showed a blue shift from 595 nm (purplish) to 478 nm (pink) as a result of releasing lactic acid as a secretion metabolite. Both absorbance spectra and CIE Lab parameters were used to determine the color shifts. Various analytical techniques were utilized to study the morphology of the anthocyanin-encapsulated electrospun cellulose nanofibers. The cytotoxic effects of the colored cellulose acetate nanofibers were tested.

Statistical optimization and gamma irradiation on cephalosporin C production by Acremonium chrysogenum W42-I.

Most antibiotics now used in clinical practice are cephalosporins. Acremonium (A.) chrysogenum W42-I is an intermediate strain out of W42 strain improvement program whose productivity is above that of the wild-type strain to produce the broad-spectrum antibacterial cephalosporin C (CPC). As a result, fermentation process optimization is considered because it offers the ideal environment for strains to reach their full potential. Our research aimed to combine a rational design to regulate the fermentation process environment and culture media as well as to develop mutants with high productivity. Different media were tested to obtain maximum CPC production. To maximize the production of CPC, some environmental parameters were experimentally optimized via the Box-Behnken design used for response surface methodology (RSM). There were 17 tests conducted, and each experiment's reaction was recorded. Improvement of the CPC production was further achieved via mutagenesis using gamma radiation. Results revealed that a pH of 4, an incubation period of 4 days, and an inoculum size of 1% v/v using the optimized media (CPC2) were the optimum conditions for enhancing the CPC production by 4.43-fold. In addition, gamma irradiation further enhanced production to reach 3.46-fold using an optimum dose of 2 KGy. In conclusion, in comparison to initial production levels, CPC production increased 4.43-fold because of nutritional and environmental optimization. The mutant AC8 demonstrated a roughly 3.46-fold increase in activity against its parent type. Moreover, subsequent AC8 mutant culture demonstrated excellent genetic stability.

Identification, Characterization, and Production Optimization of 6-Methoxy-1H-Indole-2-Carboxylic Acid Antifungal Metabolite Produced by Bacillus toyonensis Isolate OQ071612.

Fungal infections currently pose a real threat to human lives. In the current study, soil bacterial isolates were screened for the production of antifungal compounds to combat human fungal pathogens. Notably, the bacterial F1 isolate exhibited antimycotic action towards the Candida albicans ATCC 10231 and Aspergillus niger clinical isolates. By employing phenotypic and molecular techniques, we identified the F1 isolate as the Bacillus toyonensis isolate OQ071612. The purified extract showed stability within a pH range of 6-7 and at temperatures of up to 50 °C. It demonstrated potential antifungal activity in the presence of various surfactants, detergents, and enzymes. The purified extract was identified as 6-methoxy-1H-Indole-2-carboxylic acid using advanced spectroscopic techniques. To optimize the antifungal metabolite production, we utilized response surface methodology (RSM) with a face-centered central composite design, considering nutritional and environmental variables. The optimal conditions were as follows: starch (5 g/L), peptone (5 g/L), agitation rate of 150 rpm, pH 6, and 40 °C temperature. A confirmatory experiment validated the accuracy of the optimization process, resulting in an approximately 3.49-fold increase in production. This is the first documented report on the production and characterization of 6-methoxy-1H-Indole-2-carboxylic acid (MICA) antifungal metabolite from Bacillus toyonensis.

Evaluation of ELISA and immunoaffinity fluorometric analytical tools of four mycotoxins in various food categories.

Mycotoxins (MTs) are secondary toxic metabolites that can contaminate food, impacting quality and safety, leading to various negative health effects and serious pathological consequences conferring urgent need to evaluate and validate the currently standard methods used in their analysis. Therefore, this study was aimed to validate ELISA and VICAM immunoaffinity fluorometric, the two common methods used to monitor the level of MTs according to the Egyptian Organization for Standardization and Quality Control. A total of 246 food samples were collected and tested for Aflatoxins (196 samples), Ochratoxin A (139), Zearalenone (70), and Deoxynivalenol (100) using both analytical methods. Results showed that aflatoxins exceeded limits in 42.9, 100, and 13.3% of oily seeds, dried fruits, and chili and spices, respectively. For ochratoxin A, 3.9% of Gramineae and 8% of spices and chili (locally sourced) exceeded the limits, while 17.6% of imported pasta and noodles exceeded the limits for deoxynivalenol. Significant differences for the aflatoxins and ochratoxin A detection among different categories of chocolate, dried fruits, and oily seeds (p-value < 0.05). No zearalenone contamination was detected in the exported, imported, and locally sourced categories. No deoxynivalenol contamination was detected in the tested Gramineae category. In contrast, for pasta and noodles, the imported samples exhibited the highest contamination rate (above the upper limit of 750 µg/kg) with 17.6% of the samples testing positive for deoxynivalenol with no significant difference among different sample categories of Gramineae, pasta, and noodles (p-value > 0.05). In conclusion, our study found no significant differences between the ELISA and immunoaffinity fluorometric analysis in the detection of the respective MTs in various food categories and therefore, they can substitute each other whenever necessary. However, significant differences were observed among different food categories, particularly the local and imported ones, highlighting the urgent need for strict and appropriate control measures to minimize the risk of MTs adverse effects.

New Insights on Biological Activities, Chemical Compositions, and Classifications of Marine Actinomycetes Antifouling Agents.

Biofouling is the assemblage of undesirable biological materials and macro-organisms (barnacles, mussels, etc.) on submerged surfaces, which has unfavorable impacts on the economy and maritime environments. Recently, research efforts have focused on isolating natural, eco-friendly antifouling agents to counteract the toxicities of synthetic antifouling agents. Marine actinomycetes produce a multitude of active metabolites, some of which acquire antifouling properties. These antifouling compounds have chemical structures that fall under the terpenoids, polyketides, furanones, and alkaloids chemical groups. These compounds demonstrate eminent antimicrobial vigor associated with antiquorum sensing and antibiofilm potentialities against both Gram-positive and -negative bacteria. They have also constrained larval settlements and the acetylcholinesterase enzyme, suggesting a strong anti-macrofouling activity. Despite their promising in vitro and in vivo biological activities, scaled-up production of natural antifouling agents retrieved from marine actinomycetes remains inapplicable and challenging. This might be attributed to their relatively low yield, the unreliability of in vitro tests, and the need for optimization before scaled-up manufacturing. This review will focus on some of the most recent marine actinomycete-derived antifouling agents, featuring their biological activities and chemical varieties after providing a quick overview of the disadvantages of fouling and commercially available synthetic antifouling agents. It will also offer different prospects of optimizations and analysis to scale up their industrial manufacturing for potential usage as antifouling coatings and antimicrobial and therapeutic agents.

Phenotypic and Genotypic Analysis of Bacterial Pathogens Recovered from Patients Diagnosed with Fever of Unknown Origin in Egypt.

Fever of unknown origin (FUO) is a medical term describing fever that lasts for at least three weeks without a diagnosis being reached after extensive diagnostic evaluation. Therefore, this study aimed to identify the common pathogens causing FUO in patients admitted to Abbasia Fever Hospital in Egypt from January 2020 to December 2022, their antimicrobial susceptibility profiles, and associated resistance genes. The study also aimed to investigate the burden of multidrug-resistant (MDR) pathogens and the priority pathogens nominated by the World Health Organization (WHO) for posing the greatest threat to human health due to antibiotic resistance. During the study period, about 726 patients were diagnosed with FUO. After extensive investigations, the cause of the FUO was found to be infectious diseases in 479/726 patients (66.0%). Of them, 257 patients had positive bacterial cultures, including 202 Gram-negative isolates that comprised Klebsiella pneumoniae (85/202; 42.1%), Escherichia coli (71/202; 35.1%), Acinetobacter baumannii (26/202; 12.9%), and Pseudomonas aeruginosa (14/202; 6.9%) and 55 Gram-positive isolates, including Staphylococcus aureus (23/55; 41.8%), Streptococcus pneumoniae (7/55; 12.7%), and Enterococcus spp. (25/55; 45.5%). The MDR phenotype was shown by 68.3% and 65.5% of the Gram-negative and Gram-positive isolates, respectively. Carbapenem resistance (CR) was shown by 43.1% of the Gram-negative isolates. Of the 23 S. aureus isolates obtained from research participants, 15 (65.2%) were methicillin-resistant S. aureus (MRSA). A high-level aminoglycoside resistance (HLAR) phenotype was found in 52.0% of the Enterococcus sp. isolates. The PCR screening of resistance genes in the MDR isolates showed that blaOXA-48 was the most prevalent (84%) among the carbapenemase-coding genes, followed by blaVIM (9%) and then blaIMP (12%). The ESBL-coding genes blaTEM, blaCTX-M,aac(6')-Ib, and blaSHV, were prevalent in 100%, 93.2%, 85,% and 53.4% of the MDR isolates, respectively. This study updates the range of bacteria that cause FUO and emphasizes the burden of multidrug resistance and priority infections in the region. The obtained data is of relevant medical importance for the implementation of evidence-based antimicrobial stewardship programs and tailoring existing empirical treatment guidelines.

Correlation of CRISPR/Cas and Antimicrobial Resistance in Klebsiella pneumoniae Clinical Isolates Recovered from Patients in Egypt Compared to Global Strains.

The CRISPR/Cas system has been long known to interfere with the acquisition of foreign genetic elements and was recommended as a tool for fighting antimicrobial resistance. The current study aimed to explore the prevalence of the CRISPR/Cas system in Klebsiella pneumoniae isolates recovered from patients in Egypt in comparison to global strains and correlate the CRISPR/Cas to susceptibility to antimicrobial agents. A total of 181 clinical isolates were PCR-screened for cas and selected antimicrobial resistance genes (ARGs). In parallel, 888 complete genome sequences were retrieved from the NCBI database for in silico analysis. CRISPR/Cas was found in 46 (25.4%) isolates, comprising 18.8% type I-E and 6.6% type I-E*. Multidrug resistance (MDR) and extensive drug resistance (XDR) were found in 73.5% and 25.4% of the isolates, respectively. More than 95% of the CRISPR/Cas-bearing isolates were MDR (65.2%) or XDR (32.6%). No significant difference was found in the susceptibility to the tested antimicrobial agents among the CRISPR/Cas-positive and -negative isolates. The same finding was obtained for the majority of the screened ARGs. Among the published genomes, 23.2% carried CRISPR/Cas, with a higher share of I-E* (12.8%). They were confined to specific sequence types (STs), most commonly ST147, ST23, ST15, and ST14. More plasmids and ARGs were carried by the CRISPR/Cas-negative group than others, but their distribution in the two groups was not significantly different. The prevalence of some ARGs, such as blaKPC, blaTEM, and rmtB, was significantly higher among the genomes of the CRISPR/Cas-negative strains. A weak, nonsignificant positive correlation was found between the number of spacers and the number of resistance plasmids and ARGs. In conclusion, the correlation between CRISPR/Cas and susceptibility to antimicrobial agents or bearing resistance plasmids and ARGs was found to be nonsignificant. Plasmid-targeting spacers might not be naturally captured by CRISPR/Cas. Spacer match analysis is recommended to provide a clearer image of the exact behavior of CRISPR/Cas towards resistance plasmids.

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.

Insights on monkeypox disease and its recent outbreak with evidence of nonsynonymous missense mutation.

The 2022 monkeypox outbreak has created a new global health threat and pandemic. Monkeypox virus is a descendant of the genus Orthopoxvirus, producing a febrile skin rash disease in humans. Monkeypox is zoonotic transmitted and transmitted from human to human in several ways. Even though this disease is self-limited, it creates important community health worries due to its inconvenience and widespread complications. Herein, we discussed the up-to-date current situation of monkeypox regarding its epidemiology, clinical manifestations, current in-use therapeutics, necessary protective measures, and response to potential occurrences considering the recent pandemic. Also, in this review, a comparative genomic analysis of the recent circulating strains that have been recovered from various countries including, Egypt, USA, Spain, Japan and South Africa has been investigated.

The recent monkeypox outbreaks in 2022 have created a new global health threat because of their high number of cases and the speed of infection. Infection of monkeypox is characterized by a febrile rash disease in humans and characteristic skin lesions. Monkeypox is transmitted to humans via various routes including, direct contact with infected humans or animals, or vial droplet means. Although, the disease is self-limited; however, it can cause important public health consequences, particularly in pediatric, immunocompromised individuals and during pregnancy. This review introduces the monkeypox illness epidemiology, clinical manifestations, genomic mutation of the circulating viral strains, and management.

Propranolol restores susceptibility of XDR Gram-negative pathogens to meropenem and Meropenem combination has been evaluated with either tigecycline or amikacin.

BACKGROUND: Infection with extensive-drug-resistant (XDR) carbapenem-resistant (CR) Gram-negative bacteria (GNB) are viewed as a serious threat to human health because of the limited therapeutic options. This imposes the urgent need to find agents that could be used as adjuvants or combined with carbapenems to enhance or restore the susceptibility of XDR CR- GNB. Therefore, this study aimed to examine the effect of propranolol (PR) in combination with Meropenem (MEM) on the susceptibility profile of XDR CR-GNB recovered from severely infected patients as well as to evaluate combining MEM with either tigecycline (TGC) or amikacin (AK). METHODS: A total of 59 non-duplicate CR- GNB were investigated for carbapenemase production by the major phenotypic methods. Molecular identification of five major carbapenemase-coding genes was carried out using polymerase chain reactions (PCR). Antimicrobial susceptibility tests were carried out using standard methods. Phenotypic and genotypic relatedness was carried out using the heatmap and ERIC PCR analysis. PR, 0.5 -1 mg/mL against the resulting non-clonal XDR CR-GNB pathogens were evaluated by calculating the MIC decrease factor (MDF). A combination of MEM with either AK or TGC was performed using the checkerboard assay. RESULTS: A total of 21 (35.6%) and 38 (64.4%) CR-GNB isolates were identified as enterobacterial isolates (including 16 (27.1%) Klebsiella Pneumoniae and 5 (8.5%) Escherichia coli) and non-fermentative bacilli (including, 23 (39%), Acinetobacter baumannii, and 15 (25.4%) Pseudomonas aeruginosa). The heatmap and ERIC PCR analysis resulted in non-clonal 28 XDR CR isolates. PR, at a concentration of 0.5 mg /ml, decreased MICs values of the tested XDR CR isolates (28; 100%) and restored susceptibility of only 4 (14.3%) isolates. However, PR (1 mg/mL) when combined with MEM has completely (28; 100%) restored the susceptibility of the tested XDR CR- GNB to MEM. The MEM + AK and MEM + TGC combination showed mostly additive effects (92.8% and 71.4%, respectively). CONCLUSION: PR at a concentration of 1 mg/mL restored the susceptibility of XDR CR- GNB to MEM which is considered a promising result that should be clinically investigated to reveal its suitability for clinical use in patients suffering from these life-threatening pathogens.

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.

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.

Statistical optimization of a podoviral anti-MRSA phage CCASU-L10 generated from an under sampled repository: Chicken rinse.

Introduction: The insurgence of antimicrobial resistance is an imminent health danger globally. A wide range of challenging diseases are attributed to methicillin-resistant Staphylococcus aureus (MRSA) as it is weaponized with a unique array of virulence factors, and most importantly, the resistance it develops to most of the antibiotics used clinically. On that account, the present study targeted the optimization of the production of a bacteriophage active against MRSA, and evaluating some of its characters. Methods and results: The bacteriophage originated from a quite peculiar environmental source, raw chicken rinse and was suggested to belong to Podoviridae, order Caudovirales. It withstood a variety of extreme conditions and yield optimization was accomplished via the D-optimal design by response surface methodology (RSM). A reduced quadratic model was generated, and the ideal production conditions recommended were pH 8, glycerol 0.9% v/v, peptone 0.08% w/v, and 107 CFU/ml as the host inoculum size. These conditions led to a two-log fold increase in the phage titer (1.17x10¹² PFU/ml), as compared to the regular conditions. Discussion: To conclude, statistical optimization successfully enhanced the output of the podoviral phage titer by two-log fold and therefore, can be regarded as a potential scale-up strategy. The produced phage was able to tolerate extreme environmental condition making it suitable for topical pharmaceutical preparations. Further preclinical and clinical studies are required to ensure its suitability for use in human.