Metagenomic nanopore sequencing for exploring the nature of antimicrobial metabolites of Bacillus haynesii.

Multidrug-resistant (MDR) pathogens are a rising global health worry that imposes an urgent need for the discovery of novel antibiotics particularly those of natural origin. In this context, we aimed to use the metagenomic nanopore sequence analysis of soil microbiota coupled with the conventional phenotypic screening and genomic analysis for identifying the antimicrobial metabolites produced by promising soil isolate(s). In this study, whole metagenome analysis of the soil sample(s) was performed using MinION™ (Oxford Nanopore Technologies). Aligning and analysis of sequences for probable secondary metabolite gene clusters were extracted and analyzed using the antiSMASH version 2 and DeepBGC. Results of the metagenomic analysis showed the most abundant taxa were Bifidobacterium, Burkholderia, and Nocardiaceae (99.21%, followed by Sphingomonadaceae (82.03%) and B. haynesii (34%). Phenotypic screening of the respective soil samples has resulted in a promising Bacillus isolate that exhibited broad-spectrum antibacterial activities against various MDR pathogens. It was identified using microscopical, cultural, and molecular methods as Bacillus (B.) haynesii isolate MZ922052. The secondary metabolite gene analysis revealed the conservation of seven biosynthetic gene clusters of antibacterial metabolites namely, siderophore lichenicidin VK21-A1/A2 (95% identity), lichenysin (100%), fengycin (53%), terpenes (100%), bacteriocin (100%), Lasso peptide (95%) and bacillibactin (53%). In conclusion, metagenomic nanopore sequence analysis of soil samples coupled with conventional screening helped identify B. haynesii isolate MZ922052 harboring seven biosynthetic gene clusters of promising antimicrobial metabolites. This is the first report for identifying the bacteriocin, lichenysin, and fengycin biosynthetic gene clusters in B. haynesii MZ922052.

The Moraxella catarrhalis AdhC-FghA system is important for formaldehyde detoxification and protection against pulmonary clearance.

Multidrug-resistant clinical isolates of Moraxella catarrhalis have emerged, increasing the demand for the identification of new treatment and prevention strategies. A thorough understanding of how M. catarrhalis can establish an infection and respond to different stressors encountered in the host is crucial for new drug-target identification. Formaldehyde is a highly cytotoxic compound that can be produced endogenously as a by-product of metabolism and exogenously from environmental sources. Pathways responsible for formaldehyde detoxification are thus essential and are found in all domains of life. The current work investigated the role of the system consisting of the S-hydroxymethyl alcohol dehydrogenase (AdhC), a Zn-dependent class III alcohol dehydrogenase, and the S-formyl glutathione hydrolase (FghA) in the formaldehyde detoxification process in M. catarrhalis. Bioinformatics showed that the components of the system are conserved across the species and are highly similar to those of Streptococcus pneumoniae, which share the same biological niche. Isogenic mutants were constructed to study the function of the system in M. catarrhalis. A single fghA knockout mutant did not confer sensitivity to formaldehyde, while the adhC-fghA double mutant is formaldehyde-sensitive. In addition, both mutants were significantly cleared in a murine pulmonary model of infection as compared to the wild type, demonstrating the system's importance for this pathogen's virulence. The respective phenotypes were reversed upon the genetic complementation of the mutants. To date, this is the first study investigating the role of the AdhC-FghA system in formaldehyde detoxification and pathogenesis of M. catarrhalis.

Metagenomic nanopore sequencing versus conventional diagnosis for identification of the dieback pathogens of mango trees.

Dieback is one of the most dangerous fungal diseases affecting mango trees. In this study, nanopore metagenome sequencing of the root-soil samples and infected plant tissues was conducted to identify the fungal pathogens present. Soil analysis of the infected mango trees showed the abundance of the Dikarya subkingdom (59%) including Lasiodiplodia theobromae (15%), Alternaria alternata (6%), Ceratocystis huliohia and Colletotrichum gloeosporioides. Analysis of the infected plant tissues revealed the presence of A. alternata (34%). The data were deposited in the National Center of Biotechnology Information (PRJNA767267). In conclusion, nanopore metagenome sequencing analysis was a valuable tool to rapidly identify dieback-associated fungal pathogens.

A Metagenomic Nanopore Sequence Analysis Combined with Conventional Screening and Spectroscopic Methods for Deciphering the Antimicrobial Metabolites Produced by Alcaligenes faecalis Soil Isolate MZ921504.

The continuous development of multidrug resistance pathogens with limited therapeutic options has become a great problem globally that impose sever health hazards. Accordingly, searching for of new antimicrobials became an urgent demand and great challenge. Soil significantly have been associated with several species that are antibiotic producers. In this study, combination of conventional screening methods with Liquid chromatography- Mass spectroscopy (LC/MS) and metagenomic nanopore sequence analysis have been conducted for the deciphering the active metabolites produced by soil isolate(s). Preliminary soil screening resulted in a Gram-negative isolate identified via 16S ribosomal RNA as Alcaligenes faecalis isolate MZ921504 with promising antimicrobial activities against wide range of MDR gram-positive and gram-negative pathogens. The LC/MS analysis of the metabolites of A. faecalis isolate MZ921504 confirmed the presence of ectoine, bacillibactin, quinolobactin and burkholderic acid. Metagenomics sequence analysis of the soil sample (NCBI GenBank accession PRJNA771993) revealed the presence of conserved biosynthetic gene clusters of ectoine, bacteriocin, bacillibactin, quinolobactin, terpene and burkholderic acid of A. faecalis. In conclusion, A. faecalis isolate MZ921504 is a promising source for antimicrobial metabolites. LC/MS spectral analysis and third generation sequencing tools followed by secondary metabolite gene clusters analysis are useful methods to predict the nature of the antimicrobial metabolites.

Exploring the Nature of the Antimicrobial Metabolites Produced by Paenibacillus ehimensis Soil Isolate MZ921932 Using a Metagenomic Nanopore Sequencing Coupled with LC-Mass Analysis.

The continuous emergence of multidrug-resistant (MDR) pathogens poses a global threat to public health. Accordingly, global efforts are continuously conducted to find new approaches to infection control by rapidly discovering antibiotics, particularly those that retain activities against MDR pathogens. In this study, metagenomic nanopore sequence analysis coupled with spectroscopic methods has been conducted for rapid exploring of the various active metabolites produced by Paenibacillus ehimensis soil isolate. Preliminary soil screening resulted in selection of a Gram-positive isolate identified via 16S ribosomal RNA gene sequencing as Paenibacillus ehimensis MZ921932. The isolate showed a broad range of activity against MDR Gram-positive, Gram-negative, and Candida spp. A metagenomics sequence analysis of the soil sample harboring Paenibacillus ehimensis isolate MZ921932 (NCBI GenBank accession PRJNA785410) revealed the presence of conserved biosynthetic gene clusters of petrobactin, tridecaptin, locillomycin (ß-lactone), polymyxin, and macrobrevin (polyketides). The liquid chromatography/mass (LC/MS) analysis of the Paenibacillus ehimensis metabolites confirmed the presence of petrobactin, locillomycin, and macrobrevin. In conclusion, Paenibacillus ehimensis isolate MZ921932 is a promising rich source for broad spectrum antimicrobial metabolites. The metagenomic nanopore sequence analysis was a rapid, easy, and efficient method for the preliminary detection of the nature of the expected active metabolites. LC/MS spectral analysis was employed for further confirmation of the nature of the respective active metabolites.

Nanobiotic formulations as promising advances for combating MRSA resistance: susceptibilities and post-antibiotic effects of clindamycin, doxycycline, and linezolid.

Antimicrobial activity and post-antibiotic effects (PAEs) are both important parameters in determination of the dosage regimen of antimicrobial agents. In the present study, antimicrobial activity and PAEs of clindamycin, doxycycline, linezolid, and their nanobiotic formulations were evaluated against two methicillin resistant Staphylococcus aureus clinical isolates (MRSA) encoded (MRSA-S1 and MRSA-S2). Nanobiotic formulations increased the susceptibility of MRSA isolates by 4-64 folds as compared to their conventional ones. The PAE values were determined after exposure of MRSA isolates for 1 h to 10× the MICs of the tested antibiotics. The duration of PAEs were recorded after bacterial growth in Mueller Hinton broth (MHB) free from antibiotic has been restored. The PAE values for MRSA-S1 were 2.5 h for the conventional antibiotics. However, the PAEs for nanobiotics were 4 h for both clindamycin and linezolid, while 3 h for doxycycline. For MRSA-S2, linezolid and linezolid nanobiotics PAEs were 3 h. PAEs of clindamycin and clindamycin nanobiotics were 3.75 h and 4 h, respectively. Doxycycline and doxycycline nanobiotics revealed the same PAEs patterns of 3.5 h. The findings of the current study may positively influence the pharmacodynamics of the antibiotics and consequently the dosage regimen of nanobiotics as well as on their clinical outcome.

Staphylococcal Enterotoxins and Toxic Shock Syndrome Toxin-1 and Their Association among Bacteremic and Infective Endocarditis Patients in Egypt.

PURPOSE: Infective endocarditis (IE) is a major complication in patients with bacteremia of Staphylococcus (S.) aureus infection. Our aim was to determine the association of the major Staphylococcal superantigens (SAgs), including Staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin-1 (TSST-1), among hospitalized patients diagnosed with bacteremia and those with IE. METHODS: This study was conducted on 88 patients; of these, 84 (95.5%) had two positive blood cultures. Eighteen out of the 84 patients (21.4%) were diagnosed based on the modified Duke criteria by a cardiologist to have IE. The recovered isolates were screened phenotypically using ELISA followed by molecular analysis of sea, seb, sec, sed, see, and tsst-1, the major SAg coding genes, and the obtained findings were statistically analyzed. RESULTS: Phenotypic screening for SE production of 26 selected Staphylococci (15 isolated from the IE patients (10 S. aureus and 5 coagulase negative staphylococci (CoNS)) and 11 from bacteremic patients (10 S. aureus and 1 CoNS)) using ELISA revealed that 12/26 (46%) isolates were SE producers. PCR analysis showed that 19 (73%) isolates were PCR positive for SAg genes with the highest prevalence of the sea gene (79%), followed by seb (63%) and tsst-1 (21%). The least frequent gene was sed (5.3%). Statistical correlations between bacteremic and IE isolates with respect to prevalence of SAgs showed no significant difference (P value = 0.139, effect size = 0.572) indicating no specific association between any of the detected SAgs and IE. CONCLUSION: There is high prevalence of SEs among clinical isolates of Staphylococci recovered from patients suffering bacteremia and those with IE. No significant difference was found among Staphylococcal isolates recovered from patients with bacteremia or IE regarding both phenotypic and genotypic detection of the tested SAgs.

In Vitro Evaluation of Antimicrobial Activity and Cytotoxicity of Different Nanobiotics Targeting Multidrug Resistant and Biofilm Forming Staphylococci.

Antibiotic-resistant and biofilm-forming bacteria have surprisingly increased over recent years. On the contrary, the rate of development of new antibiotics to treat these emerging superbugs is very slow. Therefore, the aim of this study was to prepare novel nanobiotic formulations to improve the antimicrobial activity of three antibiotics (linezolid, doxycycline, and clindamycin) against Staphylococci. Antibiotics were formulated as nanoemulsions and evaluated for their antimicrobial activities and cytotoxicities. Cytotoxicity of the conventional antibiotics and nanobiotics was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on rat hepatocytes. Half-maximal inhibitory concentration (IC50) was estimated from an experimentally derived dose-response curve for each concentration using GraphPad Prism software. Upon quantitative assessment of Staphylococcus biofilm formation, eighty-four isolates (66.14 %) were biofilm forming. Linezolid and doxycycline nanobiotics exhibited promising antibacterial activities. On the contrary, clindamycin nanobiotic exhibited poor antibacterial activity. Minimum biofilm inhibitory concentrations showed that 73.68 %, 45.6%, and 5.2% of isolates were sensitive to linezolid, doxycycline, and clindamycin nanobiotics, respectively. Results of this study revealed that antibiotics loaded in nanosystems had a higher antimicrobial activity and lower cytotoxicities as compared to those of conventional free antibiotics, indicating their potential therapeutic values.