Bioassay- and metabolomics-guided screening of bioactive soil actinomycetes from the ancient city of Ihnasia, Egypt

Literature surveys, taxonomical differences, and bioassay results have been utilized in the discovery of new natural products to aid in Actinomycetes isolate-selection. However, no or less investigation have been done on establishing the differences in metabolomic profiles of the isolated microorganisms. The study aims to utilise bioassay- and metabolomics-guided tools that included dereplication study and multivariate analysis of the NMR and mass spectral data of microbial extracts to assist the selection of isolates for scaling-up the production of antimicrobial natural products. A total of 58 actinomycetes were isolated from different soil samples collected from Ihnasia City, Egypt and screened for their antimicrobial activities against indicator strains that included Bacillus subtilis, Escherichia coli, methicillin-resistant Staphylococcus aureus and Candida albicans. A number of 25 isolates were found to be active against B. subtilis and/or to at least one of the tested indicator strains. Principal component analyses showed chemical uniqueness for four outlying bioactive actinomycetes extracts. In addition, Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) and dereplication study led us to further select two outlying anti-MRSA active isolates MS.REE.13 and 22 for scale-up work. MS.REE.13 and 22 exhibited zones of inhibition at 19 and 13 mm against MRSA, respectively. A metabolomics-guided approach provided the steer to target the bioactive metabolites (P<0.01) present in a crude extract or fraction even at nanogram levels but it was a challenge that such low-yielding bioactive natural products would be feasible to isolate. Validated to occur only on the active side of OPLSDA loadings plot, the isolated compounds exhibited medium to weak antibiotic activity with MIC values between 250 and 800 μM. Two new compounds, P_24306 (C10H13N2) and N_12799 (C18H32O3) with MICs of 795 and 432 μM, were afforded from the scale-up of MS.REE. 13 and 22, respectively

Heterologous expression of the avirulence gene ACE1 from the fungal rice pathogen Magnaporthe oryzae

The ACE1 and RAP1 genes from the avirulence signalling gene cluster of the rice blast fungus Magnaporthe oryzae were expressed in Aspergillus oryzae and M. oryzae itself. Expression of ACE1 alone produced a polyenyl pyrone (magnaporthepyrone), which is regioselectively epoxidised and hydrolysed to give different diols, 6 and 7, in the two host organisms. Analysis of the three introns present in ACE1 determined that A. oryzae does not process intron 2 correctly, while M. oryzae processes all introns correctly in both appressoria and mycelia. Co-expression of ACE1 and RAP1 in A. oryzae produced an amide 8 which is similar to the PKS-NRPS derived backbone of the cytochalasans. Biological testing on rice leaves showed that neither the diols 6 and 7, nor amide 8 was responsible for the observed ACE1 mediated avirulence, however, gene cluster analysis suggests that the true avirulence signalling compound may be a tyrosine-derived cytochalasan compound.Government of Egypt ScholarshipThe School of Chemistry at the University of Bristol and the Mark Evans ScholarshipKano State Government NigeriaMacArthur FoundationBayero UniversityNigerian Petroleum Technology FundMalaysian Govenment ScholarshipEP/F066104/

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P &lt; 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Association of Carbapenem and Colistin Resistance in Pathogenic Gram Negative Bacteria

Diseases caused by multidrug-resistant (MDR) bacteria continue to challenge physicians and endanger their patients’ lives. Polymyxins, including colistin, are the last resort antibiotics to treat serious infections caused by carbapenem-resistant bacteria. The aim of this study is to explore the resistance of Gram negative isolates recovered from 200 clinical specimens to carbapenem and colistin antibiotics, and the prevalence of plasmid-mediated mcr-1 gene in the resistant isolates. Clinical specimens were collected from two teaching hospitals and two private clinical laboratories in Cairo, Egypt. We identified one hundred and thirty isolates as Gram negative. These isolates were screened for their susceptibility to β-lactams antibiotics, carbapenems, colistin, polymyxin B, levofloxacin and amikacin. Thirty isolates were found to be resistant to the tested carbapenems. Of these, five isolates were found to be resistant to both carbapenem and colistin. They were tested for the presence of mcr-1, pmrB and pmrA genes; known to be among the reasons for colistin resistance. One isolate showed the presence of pmrA while three isolates showed the presence of pmrA and pmrB. Only one isolate showed the presence of mcr-1, pmrA and pmrB. This was tested by real time PCR to ensure the activity of this plasmid-mediated gene. Using 16S rRNA sequencing, the isolate showed 100% similarity to Escherichia coli strain K12 (MG1655). Here, we report a carbapenem-resistant and colistin-resistant Escherichia coli strain producing mcr-1gene that is the first to be reported in Egypt between human

Molecular Characterization of Multiple Antibiotic-Resistant Acinetobacter baumannii Isolated from Egyptian Patients

Acinetobacter baumannii is an opportunistic microorganism commonly found in intensive care units (ICUs), and it is responsible for a broad span of hospital-acquired infections. Persistence of nosocomial infection caused by multidrug-resistant (MDR) A. baumannii is an alarming health care issue in Egypt, and at present, colistin remains the treatment of choice for the management of MDR A. baumannii infections. A. baumannii possesses great capacity to develop and acquire resistance to a broad range of antibiotics. The acquisition and dissemination of antibiotic-resistant determinants in A. baumannii strains are mediated by integrons, especially class I integrons. This study focuses on the characterization of some genetic mechanisms underlying the multidrug-resistant phenotypes of A. baumannii isolates in Egypt. Forty-eight A. baumannii specimens were isolated from different hospitalized patients; least resistance was observed against amikacin and tigecycline, with 60% and 58.5% of the isolates resistant, respectively, whereas 62.5% of the isolates were resistant to imipenem and meropenem. The highest sensitivity was found for colistin. Genetic analysis revealed that blaoxa-51 was detected in all isolates, the blaoxa-23-like gene was detected in 80% of the isolates, and blaoxa-24 and blaoxs-58 were not detected in any isolate. Finally, PCR analysis revealed that 6.6% of isolates carried the class I integron gene

Comparative structural analysis of different mycobacteriophage-derived mycolylarabinogalactan esterases (Lysin B)

Mycobacteriophage endolysins have emerged as a potential alternative to the current antimycobacterial agents. This study focuses on mycolylarabinogalactan hydrolase (LysB) enzymes of the α/β-hydrolase family, which disrupt the unique mycolic acid layer of mycobacterium cell wall. Multiple sequence alignment and structural analysis studies showed LysB-D29, the only enzyme with a solved three-dimensional structure, to share several common features with esterases (lacking lid domain) and lipases (acting on long chain lipids). Sequence and structural comparisons of 30 LysB homology models showed great variation in domain organizations and total protein length with major differences in the loop-5 motif harboring the catalytic histidine residue. Docking of different p-nitrophenyl ligands (C4-C18) to LysB-3D models revealed that the differences in length and residues of loop-5 contributed towards wide diversity of active site conformations (long tunnels, deep and superficial funnels, shallow bowls, and a narrow buried cave) resembling that of lipases, cutinases, and esterases. A set of seven LysB enzymes were recombinantly produced; their activity against p-nitrophenyl esters could be related to their active site conformation and acyl binding site. LysB-D29 (long tunnel) showed the highest activity with long chain p-nitrophenyl palmitate followed by LysB-Omega (shallow bowl) and LysB-Saal (deep funnel)

Fungal secondary metabolites as effectors of pathogenicity:role in the complex interplay between rice and Magnaporthe grisea

International audienc

Secondary metabolites as effectors: fungal secondary metabolism is an essential component of the complex interplay between rice and Magnaporthe grisea

National audienc

Exploration of Chemical Diversity and Antitrypanosomal Activity of Some Red Sea-Derived Actinomycetes Using the OSMAC Approach Supported by LC-MS-Based Metabolomics and Molecular Modelling

In the present study, we investigated the actinomycetes associated with the Red Sea-derived soft coral Sarcophyton glaucum in terms of biological and chemical diversity. Three strains were cultivated and identified to be members of genera Micromonospora, Streptomyces, and Nocardiopsis; out of them, Micromonospora sp. UR17 was putatively characterized as a new species. In order to explore the chemical diversity of these actinobacteria as far as possible, they were subjected to a series of fermentation experiments under altering conditions, that is, solid and liquid fermentation along with co-fermentation with a mycolic acid-containing strain, namely Nocardia sp. UR23. Each treatment was found to affect these actinomycetes differently in terms of biological activity (i.e., antitrypanosomal activity) and chemical profiles evidenced by LC-HRES-MS-based metabolomics and multivariate analysis. Thereafter, orthogonal projections to latent structures discriminant analysis (OPLS-DA) suggested a number of metabolites to be associated with the antitrypanosomal activity of the active extracts. The subsequent in silico screenings (neural networking-based and docking-based) further supported the OPLS-DA results and prioritized desferrioxamine B (3), bafilomycin D (10), and bafilomycin A1 (11) as possible antitrypanosomal agents. Our approach in this study can be applied as a primary step in the exploration of bioactive natural products, particularly those from actinomycetes