Screening of microorganisms for hydrolyases with commercial potential

Advancement in green chemistry has increased the use of microbial hydrolyases in various industries and chemical processes because of high catalytic efficiency, specificity, cost-effectiveness and eco-friendly nature. Bioconversion of tannins such as tannic acid is achieved by tannin acyl hydrolase, also known as tannase. It converts tannic acid into glucose and gallic acid by catalyzing the hydrolysis of ester and depside linkages in tannic acid. Tyrosinase is monophenol and O-diphenol oxidase a copper containing enzyme catalyzes the oxidation of tyrosine and generates different types of pigment such as melanin. Xylanases hydrolyze xylan into its constituent sugar with the help of several debranching enzymes. Microbial strains isolated from various sources were screened for these hydrolyases: Bhavnagar marine salterns (Bacillus megaterium BVUC_01 and Bacillus licheniformis BVUCh_02); Okhamadhi marine salterns Aspergillus versicolor; Spoiled/infected pomegranate (Xenoacremonium falcatum, two strains PGF1 and PGF4, Bacillus velezensisPGF2 and Candida freyschussiiPGF3. The other laboratory maintained bacterial cultures namely, Bacillus subtilis, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi were also used in this study. Asp. versicolor and Xen. falcatum (PGF1) produced all the three enzymes (tannase, tyrosinase and xylanase). B. licheniformis, B. megaterium, B. subtilis, B. velezensis produced tyrosinase and xylanase. Xen. falcatum (PGF4) and PGF2 produced tannase and xylanase. PGF3 produced tannase and tyrosinase. While, Bacillus megaterium and Salmonella typhi showed only tyrosinase activity. Candida freyschussii showed tannase activity. Staphylococcus aureus did not produce any of these enzymes.

Antibacterial Activity of Manuka Honey Against Azithromycin Resistant Extensively Drug Resistant Salmonella Typhi Clinical Isolates: In-Vitro Study

Typhoid fever is a significant health problem in developing countries like Pakistan. Salmonella Typhi the causative agent of typhoid has developed resistant to almost all recommended antibiotics. Emergence of resistance to third generation cephalosporins has further complicated the situation and such strains are called as extensively drug resistant (XDR) Salmonella Typhi. Currently only available options are azithromycin and cabapenems. Recently few reports of azithromycin resistance have emerged from countries like Pakistan, India, Bangladesh and Nepal. As azithromycin is the only oral option available to treat XDR Typhoid, development of resistance may change treatment strategy altogether from out patient management to hospitalization of every patient. This may increase the burden on already weak health care system of countries like Pakistan. So there is dire need to look for the alternative treatment options. Manuka honey is well known for its therapeutic potential against wide range of bacteria including Salmonella Typhimurium. In this study 3 azithromycin resistant isolates were isolated and identified using disc diffusion, E-test and broth micro dilution methods and antibacterial activity, MIC and MBC of manuka honey was performed by agar well diffusion assay and broth micro dilution assay respectively. Manuka honey manifested significant antibacterial activity against all test isolates with zone of inhibition ranging from 7.3mm to 7.5mm, MIC and MBC values were between 10 to 15% v/v Here, we conclude that Manuka honey possess potent antibacterial activity and might be used as an alternative treatment option against azithromycin resistant XDR Typhid. However, further clinical trials are mandatory to validate our initial findings.

Case of Mycotic Pseudoaneurysm of the Common Carotid Artery due to Salmonella typhi Bacteremia

Aneurysm of the extracranial carotid artery is a rare disease, mycotic pseudoaneurysms being even less common. They are a life-threatening complication of systemic infection and atherosclerosis. Immunocompromised people, including patients with HIV, uncontrolled diabetes melltus, those on immunosuppressants like high-dose steroids, and chemotherapy, are at a higher risk for development of mycotic pseudoaneurysms. Due to the high risk of potential complications like rupture and thromboembolic events, mycotic aneurysms always require surgical management. Early detection followed by restoration of blood flow is critical to minimize a fatal outcome. Here we report the case of a 52-year-old man with a past history of hypertension and dyslipidemia who presented with a pulsatile painful neck swelling. On evaluation, the patient was diagnosed to have Salmonella typhi bacteremia, HIV infection, and a mycotic aneurysm of the left common carotid artery.

Evaluation of seaweed sulfated polysaccharides as natural antagonists targeting Salmonella typhi OmpF: molecular docking and pharmacokinetic profiling

Background Salmonella belongs to the Enterobacteriaceae family, a gram-negative, non-spore-forming, rod-shaped, motile, and pathogenic bacteria that transmit through unhygienic conditions. It is estimated that 21 million new infections arise every year, resulting in approximately 200,000 deaths. It is more prevalent among children, the old aged, and immunocompromised individuals. The frequent usage of classical antimicrobials has begun the increasing emergence of various drug-resistant pathogenic bacterial strains. Hence, this study was intended to evaluate the bioactive seaweed sulfated polysaccharides (SSPs) against the ompF (outer membrane porin F) protein target of Salmonella typhi. SSP is the sulfated compound with a wide range of biological activities, such as anti-microbial, anti-allergy, anti-cancer, anti-coagulant, anti-inflammation, anti-oxidant, and anti-viral. Results In this study, eleven compounds were targeted against S. typhi OmpF by the molecular docking approach and were compared with two commercially available typhoid medications. The SSP showed good binding affinity compared to commercial drugs, particularly carrageenan/MIV-150, carrageenan lambda, fucoidan, and 3-phenyllactate, ranked as top antagonists against OmpF. Further, pharmacokinetics and toxicology (ADMET) studies corroborated that SSP possessed drug-likeness and highly progressed in all parameters. Conclusions AutoDockTools and Schrodinger's QikProp module results suggest that SSP could be a promising drug for extensively drug-resistant (XDR) S. typhi. To the best of our knowledge, this is the first report on in silico analysis of SSP against S. typhi OmpF, thus implying the capabilities of SSPs especially compounds like carrageenans, as a potential anti-microbial agent against Salmonella typhi infections. Eventually, advanced studies could corroborate SSPs to the next level of application in the crisis of XDR microbial diseases. Graphical Abstract

The human bile salt sodium deoxycholate induces metabolic and cell envelope changes in Salmonella Typhi leading to bile resistance

Introduction. Salmonella enterica serovar Typhi (S. Typhi) is the etiological agent of typhoid fever. To establish an infection in the human host, this pathogen must survive the presence of bile salts in the gut and gallbladder. Hypothesis. S. Typhi uses multiple genetic elements to resist the presence of human bile. Aims. To determine the genetic elements that S. Typhi utilizes to tolerate the human bile salt sodium deoxycholate. Methodology. A collection of S. Typhi mutant strains was evaluated for their ability to growth in the presence of sodium deoxycholate and ox-bile. Additionally, transcriptomic and proteomic responses elicited by sodium deoxycholate on S. Typhi cultures were also analysed. Results. Multiple transcriptional factors and some of their dependent genes involved in central metabolism, as well as in cell envelope, are required for deoxycholate resistance. Conclusion. These findings suggest that metabolic adaptation to bile is focused on enhancing energy production to sustain synthesis of cell envelope components exposed to damage by bile salts.

Convolution Neural Networks for Point-of-Care Diagnostics of Bacterial Infections in Blood

ABSTRACTA droplet of blood, when evaporated on a surface, leaves dried residue—the fractal patterns formed on the dried residues can act as markers for infection present in the blood. Exploiting the unique patterns found in the residues of a naturally dried droplet of blood, we propose a Point-of-Care (POC) diagnostic tool for detecting broad-spectrum of bacterial infections (such as Enterobacter aerogenes, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa, Salmonella Typhi) in blood. The diagnosis process we propose is straightforward and can be performed with the following steps: A droplet of blood (healthy or infected) of volume range 0.5 to 2 μl is allowed to dry on a clean glass surface and is imaged using a conventional optical microscope. A computer algorithm based on the framework of convolution neural network (CNN) is used to classify the captured images of dried blood droplets according to the bacterial infection. In total, our multiclass model reports an accuracy of 92% for detecting six bacterial species infections in the blood (with control being the uninfected or healthy blood). The high accuracy of detecting bacteria in the blood reported in this article is commensurate with the standard bacteriological tests. Thus, this article presents a proof-of-concept of a potential futuristic tool for a rapid and low-cost diagnosis of bacterial infection in the blood.

Exogenous Production of N-acetylmuramyl-L Alanine Amidase (LysM2) from Siphoviridae Phage Affecting Anti-Gram-Negative Bacteria: Evaluation of Its Structure and Function

Background: To obtain endolysin with impact(s) on gram-negative bacteria as well as gram-positive bacteria, N-acetylmuramyl L-alanine-amidase (MurNAc-LAA) from a Bacillus subtilis-hosted Siphoviridae phage (SPP1 phage, Subtilis Phage Pavia 1) was exogenously expressed in Escherichia coli (E. coli). Methods: The sequences of MurNAc-LAA genes encoding peptidoglycan hydrolases were obtained from the Virus-Host database. The sequence of MurNAc-LAA was optimized by GenScript software to generate MurNAc-LAA-MMI (LysM2) for optimal expression in E. coli. Furthermore, the structure and function of LysM2 was evaluated in silico. The optimized gene was synthesized, subcloned in the pET28a, and expressed in E. coli BL21(DE3). The antibacterial effects of the protein on the peptidoglycan substrates were studied. Results: LysM2, on 816 bp gene encoding a 33 kDa protein was confirmed as specific SPP1 phage enzyme. The enzyme is composed of 271 amino acids, with a half-life of 10 hr in E. coli. In silico analyses showed 34.2% alpha-helix in the secondary structure, hydrophobic N-terminal, and lysine-rich C-terminal, and no antigenic properties in LysM2 protein. This optimized endolysin revealed impacts against Proteus (sp) by turbidity, and an antibacterial activity against Klebsiella pneumoniae, Salmonella typhi-murium, and Proteus vulgaris in agar diffusion assays. Conclusion: Taken together, our results confirmed that LysM2 is an inhibiting agent for gram-negative bacteria.

Comparative antimicrobial activity of Cymbopogon citratus essential oil and thiosemicarbazones derived from this oil

Introduction: The presence of microbes in our environment is always a permanent public health problem. In this context, research on natural treatment, less expensive and accessible to fight these microbial germs would be beneficial. Methods: During this work, molecules of thiosemicarbazones due to their numerous biological activities were hemi-synthesized in situ in the essential oil of Cymbopogon citratus in order to evaluate their antimicrobial activities. Results: Analysis of the essential oil extracted by hydrodistillation revealed the presence of 72.91% of citral. Citralthiosemicarbazone (CThio) and citral 4-phenyl-3-thiosemicarbazone (CPthio) were hemi-synthesized in this oil with interesting yields of 83% and 91%, respectively. After purification and confirmation of the structures of these molecules, the three substances were tested on eleven strains of microbes. Determination of the inhibition diameters showed that the activity of the essential oil is best in over 80% of strains. However, the largest diameter of inhibition (26 mm) was noted with CPthio against Salmonella typhi R 30951401. The determination of the minimum inhibitory concentrations showed that the oil remains more active with the smallest value of 0.3125 mg / ml against Micrococcus luteus. The lower value of minimum bactericidal concentrations was also obtained with the essential oil against Enterococcus foecalis ATCC 29212. Conclusion: The essential oil of C. citratus remains more active in the majority cases. It could be a great alternative in the fight against bacteria, and the advantage is that it remains a natural substance.