Molecular Assessments of Antimicrobial Protein Enterocins and Quorum Sensing Genes and Their Role in Virulence of the Genus Enterococcus.

Enterococcus has emerged as an opportunistic pathogen because of its antibiotic resistance and virulence profile, which makes it a causative agent of several diseases like endocarditis, surgical site, and urinary tract infections. Currently, species of this genus are the 2nd most frequently isolated microorganisms from hospital-acquired infections. Significant association with hospitals and unhygienic conditions of the environments has made them resistant to a wide range of antibiotics. On the brighter side, enterococci have the ability to produce antimicrobial proteins (i.e., enterocins) that exhibit wide antagonistic activity, thus making them useful microbes in the food and pharmaceutical industries. Enterocins are also involved in niche control in gut microbiota which is regulated by the quorum sensing (QS) system. A bacterial communication system that is controlled by the fsr operon in enterococci consists of FsrABDC, ef1097, and GelE/SprE genes. Hence, the present study was conducted for molecular assessment of enterocins and quorum sensing genes, inter-environmental correlation, and species prevalence of enterococci isolated from different environmental niches of Karachi, Pakistan. Obtained results revealed the highest prevalence of E. faecium and E. faecalis in all environments. Bacterial antagonism and enterocin genes were observed significantly high in poultry environments. The inter-environmental correlation indicated a strong positive correlation of freshwater with sewage and soil environments. Similarly, the fsr regulatory system was mostly identified in poultry-related environments, and a significant correlation between QS system and biofilm formation was established. In conclusion, this study confirmed the high prevalence of E. faecium in all tested sources, high enterocin production in non-clinical environments, and more fsr regulatory genes in poultry-related environments.

Molecular Assessment and Validation of the Selected Enterococcal Strains as Probiotics.

Probiotics are live microorganisms which confer health benefits to the host. Lactic acid bacteria (LAB) are used as probiotics since decades. Enterococci being the member of LAB have proven probiotic strains; therefore, this study was aimed at finding out the potential probiotic candidates from the pool of locally isolated strains. For initial screening, one hundred and twenty-two strains were selected and subjected to different confirmatory and phenotypic tests to choose the best strains that have potential probiotic criteria, i.e., no potential virulence traits, antibiotic resistance, and having tolerance properties. Keeping this criterion, only eleven strains (n = 11) were selected for further assessment. All virulence traits such as production of hemolysin, gelatinase, biofilm, and DNase were performed and not found in the tested strains. The molecular assessment indicates the presence of few virulence-associated genes in Enterococcus faecalis strains with variable frequency. The phenotypic and genotypic assessments of antibiotic resistance profile indicate that the selected strain was susceptible to ten commonly used antibiotics, and there were no transferrable antibiotic resistance genes. The presence of CRISPR-Cas genes also confirmed the absence of antibiotic resistance genes. Various enterocin-producing genes like EntP, EntB, EntA, and EntQ were also identified in the selected strains which make them promising probiotic lead strains. Different tolerance assays like acid, NaCl, and gastric juice tolerance that mimic host conditions was also evaluated by providing artificial conditions. Cellular adhesion and aggregation properties like auto- and co-aggregation were also checked and their results reflect all in the favor of lead probiotic strains.

Mining therapeutic targets from the antibiotic-resistant Campylobacter coli and virtual screening of natural product inhibitors against its riboflavin synthase.

Campylobacter coli resides in the intestine of several commonly consumed animals, as well as water and soil. It leads to campylobacteriosis when humans eat raw/undercooked meat or come into contact with infected animals. A common manifestation of the infection is fever, nausea, headache, and diarrhea. Increasing antibiotic resistance is being observed in this pathogen. The increased incidence of C. coli infection, and post-infection complications like Guillain-Barré syndrome, make it an important pathogen. It is essential to find novel therapeutic targets and drugs against it, especially with the emergence of antibiotic-resistant strains. In the current study, genomes of 89 antibiotic-resistant strains of C. coli were downloaded from the PATRIC database. Potent drug targets (n = 36) were prioritized from the core genome (n = 1,337 genes) of this species. Riboflavin synthase was selected as a drug target and pharmacophore-based virtual screening was performed to predict its inhibitors from the NPASS (n = ~ 30,000 compounds) natural product library. The top three docked compounds (NPC115144, NPC307895, and NPC470462) were selected for dynamics simulation (for 50 ns) and ADMET profiling. These identified compounds appear safe for targeting this pathogen and can be further validated by experimental analysis before clinical trials.

An in silico hierarchal approach for drug candidate mining and validation of natural product inhibitors against pyrimidine biosynthesis enzyme in the antibiotic-resistant Shigella flexneri.

Shigella flexneri is the main causative agent of the communicable diarrheal disease, shigellosis. It is estimated that about 80-165 million cases and > 1 million deaths occur every year due to this disease. S. flexneri causes dysentery mostly in young children, elderly and immunocompromised patients, all over the globe. Recently, due to the emergence of S. flexneri antibiotic resistance strains, it is a dire need to predict novel therapeutic drug targets in the bacterium and screen natural products against it, which could eliminate the curse of antibiotic resistance. Therefore, in current study, available antibiotic-resistant genomes (n = 179) of S. flexneri were downloaded from PATRIC database and a pan-genome and resistome analysis was conducted. Around 5059 genes made up the accessory, 2469 genes made up the core, and 1558 genes made up the unique genome fraction, with 44, 34, and 13 antibiotic-resistant genes in each fraction, respectively. Core genome fraction (27% of the pan-genome), which was common to all strains, was used for subtractive genomics and resulted in 384 non-homologous, and 85 druggable targets. Dihydroorotase was chosen for further analysis and docked with natural product libraries (Ayurvedic and Streptomycin compounds), while the control was orotic acid or vitamin B13 (which is a natural binder of this protein). Dynamics simulation of 50 ns was carried out to validate findings for top-scored inhibitors. The current study proposed dihydroorotase as a significant drug target in S. flexneri and 4-tritriacontanone & patupilone compounds as potent drugs against shigellosis. Further experiments are required to ascertain validity of our findings.

Differential analysis of Orientia tsutsugamushi genomes for therapeutic target identification and possible intervention through natural product inhibitor screening.

Orientia tsutsugamushi (Ott) is a causative agent of scrub typhus, and one of the emerging pathogens that could affect a large human population. It is one of the misdiagnosed and under-reported, febrile illnesses that infects various body organs (skin, heart, lung, kidney, and brain). The control of this infection is hampered due to the lack of drugs or vaccine against it. This study was undertaken to identify potential drug targets from the core genome of Ott and investigate novel natural product inhibitors against them. Hence, the available genomes for 22 strains of Ott were downloaded from the PATRIC database, and pan-genomic analysis was performed. Only 202 genes were present in the core region. Among these, 94 were identified as essential, 32 non-homologous to humans, nine non-homologous to useful gut flora and a single gene dapD as a drug target. Product of this gene (2,3,4,5-tetrahydropyridine-2-carboxylate N-succinyltransferase) was modeled and docked against traditional Indian (Ayurvedic) and Chinese phytochemical libraries, with best hits selected for docking, based on multiple target-drug/s interactions and minimum energy scores. ADMET profiling and molecular dynamics simulation was performed for top three compounds from each library to assess the toxicity and stability, respectively. We presume that these compounds (ZINC8214635, ZINC32793028, ZINC08101133, ZINC85625167, ZINC06018678, and ZINC13377938) could be successful inhibitors of Ott. However, in-depth experimental and clinical research is needed for further validation.

Pan-Genome Reverse Vaccinology Approach for the Design of Multi-Epitope Vaccine Construct against Escherichia albertii.

Escherichia albertii is characterized as an emerging pathogen, causing enteric infections. It is responsible for high mortality rate, especially in children, elderly, and immunocompromised people. To the best of our knowledge, no vaccine exists to curb this pathogen. Therefore, in current study, we aimed to identify potential vaccine candidates and design chimeric vaccine models against Escherichia albertii from the analysis of publicly available data of 95 strains, using a reverse vaccinology approach. Outer-membrane proteins (n = 4) were identified from core genome as vaccine candidates. Eventually, outer membrane Fimbrial usher (FimD) protein was selected as a promiscuous vaccine candidate and utilized to construct a potential vaccine model. It resulted in three epitopes, leading to the design of twelve vaccine constructs. Amongst these, V6 construct was found to be highly immunogenic, non-toxic, non-allergenic, antigenic, and most stable. This was utilized for molecular docking and simulation studies against six HLA and two TLR complexes. This construct can therefore be used for pan-therapy against different strains of E. albertii and needs to be tested in vitro and in vivo.