Genome-Scale Investigation of the Regulation of azoR Expression in Escherichia coli Using Computational Analysis and Transposon Mutagenesis.

Bacterial azoreductases are enzymes that catalyze the reduction of ingested or industrial azo dyes. Although azoreductase genes have been well identified and characterized, the regulation of their expression has not been systematically investigated. To determine how different factors affect the expression of azoR, we extracted and analyzed transcriptional data from the Gene Expression Omnibus (GEO) resource, then confirmed computational predictions by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results showed that azoR expression was lower with higher glucose concentration, agitation speed, and incubation temperature, but higher at higher culture densities. Co-expression and clustering analysis indicated ten genes with similar expression patterns to azoR: melA, tpx, yhbW, yciK, fdnG, fpr, nfsA, nfsB, rutF, and chrR (yieF). In parallel, constructing a random transposon library in E. coli K-12 and screening 4320 of its colonies for altered methyl red (MR)-decolorizing activity identified another set of seven genes potentially involved in azoR regulation. Among these genes, arsC, relA, plsY, and trmM were confirmed as potential azoR regulators based on the phenotypic decolorization activity of their transposon mutants, and the expression of arsC and relA was confirmed, by qRT-PCR, to significantly increase in E. coli K-12 in response to different MR concentrations. Finally, the significant decrease in azoR transcription upon transposon insertion in arsC and relA (as compared to its expression in wild-type E. coli) suggests their probable involvement in azoR regulation. In conclusion, combining in silico analysis and random transposon mutagenesis suggested a set of potential regulators of azoR in E. coli.

Pharmacomicrobiomics-Guided Precision Oncology: A New Frontier of P4 (Predictive, Personalized, Preventive, and Participatory) Medicine and Microbiome-Based Therapeutics.

Pharmacomicrobiomics is a rapidly developing field that promises to make significant contributions to predictive, personalized, preventive, and participatory (P4) medicine. This is becoming evident particularly in the field of precision (P4) oncology by taking seriously the crucial role microbiome plays in health and disease. Several studies have already shown that clinicians can harness insights from the microbiome to better predict treatment response, reduce side effects, and improve overall outcomes for cancer patients. Furthermore, pharmacomicrobiomics will undoubtedly play a crucial role in shaping the future of cancer treatment in the era of P4 oncology as we continue to unravel the intricate relationships between the microbiome and cancer. This perspective and innovation analysis discusses the emerging intersection of P4 medicine and P4 oncology, as seen through a lens of pharmacomicrobiomics. A key promise of pharmacomicrobiomics is the development of personalized microbiome-based therapeutics. In all, we suggest that optimizing cancer treatment and prevention by harnessing pharmacomicrobiomics has vast potentials for precision oncology, and personalized medicine using the right drug, at the right dose, for the right patient, and at the right time.

The curious case of Prevotella copri.

Prevotella copri is an abundant member of the human gastrointestinal microbiome, whose relative abundance has curiously been associated with positive and negative impacts on diseases, such as Parkinson's disease and rheumatoid arthritis. Yet, the verdict is still out on the definitive role of P. copri in human health, and on the effect of different diets on its relative abundance in the gut microbiome. The puzzling discrepancies among P. copri studies have only recently been attributed to the diversity of its strains, which substantially differ in their encoded metabolic patterns from the commonly used reference strain. However, such strain differences cannot be resolved by common 16S rRNA amplicon profiling methods. Here, we scrutinize P. copri, its versatile metabolic potential, and the hypotheses behind the conflicting observations on its association with diet and human health. We also provide suggestions for designing studies and bioinformatics pipelines to better research P. copri.

Integrating Microbiome Analysis, Metabolomics, Bioinformatics, and Histopathology to Elucidate the Protective Effects of Pomegranate Juice against Benzo-alpha-pyrene-Induced Colon Pathologies.

Polycyclic aromatic hydrocarbons, e.g., benzo[a]pyrene (BaP), are common dietary pollutants with potential carcinogenic activity, while polyphenols are potential chemopreventive antioxidants. Although several health benefits are attributed to polyphenol-rich pomegranate, little is known about its interaction with BaP. This study integrates histochemical, microbiomic, and metabolomic approaches to investigate the protective effects of pomegranate juice from BaP-induced pathologies. To this end, 48 Sprague-Dawley rats received, for four weeks, either pomegranate, BaP, both, or neither (n = 12 rats per group). Whereas histochemical examination of the colon indicated tissue damage marked by mucin depletion in BaP-fed animals, which was partially restored by administration of pomegranate juice, the fecal microbiome and metabolome retained their resilience, except for key changes related to pomegranate and BaP biotransformation. Meanwhile, dramatic microbiome restructuring and metabolome shift were observed as a consequence of the elapsed time (age factor). Additionally, the analysis allowed a thorough examination of fecal microbiome-metabolome associations, which delineated six microbiome clusters (marked by a differential abundance of Lactobacillaceae and Prevotellaceae, Rumincococcaceae, and Erysipelotrichaceae) and two major metabolome clusters (a sugar- and amino-acids-dominated metabotype vs. a cluster of fatty acids and hydrocarbons), with sugar alcohols maintaining a unique signature. In conclusion, using paired comparisons to minimize inter-individual animal variations allowed the dissection of temporal vs. treatment-derived variations. Microbiome-metabolome association clusters may be further exploited for metabotype prediction and gut-health biomarker discovery.

In Silico and In Vitro Investigation of the Distribution and Expression of Key Genes in the Fucose Operon of Escherichia coli.

Many gut bacteria degrade polysaccharides, providing nutritional advantages to their hosts. Fucose, a mucin degradation product, was suggested as a communication molecule between the resident microbiota and external pathogens. However, the precise role and variants of the fucose utilization pathway remain to be elucidated. Here, we computationally and experimentally investigated the fucose utilization operon of E. coli. While the operon is conserved among E. coli genomes, a variant pathway, in which an ABC transporter system replaces the fucose permease gene (fucP), was computationally identified in 50 out of 1058 genomes. Comparative genomics and subsystems analysis results were confirmed by polymerase chain reaction-based screening of 40 human E. coli isolates, which indicated the conservation of fucP in 92.5% of the isolates (vs. 7.5% of its suggested alternative, yjfF). The in silico predictions were confirmed by in vitro experiments comparing the growth of E. coli strains K12, BL21, and isogenic fucose-utilization K12 mutants. Additionally, fucP and fucI transcripts were quantified in E. coli K12 and BL21, after in silico analysis of their expression in 483 public transcriptomes. In conclusion, E. coli utilizes fucose by two pathway variants, with measurable transcriptional differences. Future studies will explore this variation's impact on signaling and virulence.

Abolishment of morphology-based taxa and change to binomial species names: 2022 taxonomy update of the ICTV bacterial viruses subcommittee.

This article summarises the activities of the Bacterial Viruses Subcommittee of the International Committee on Taxonomy of Viruses for the period of March 2021-March 2022. We provide an overview of the new taxa proposed in 2021, approved by the Executive Committee, and ratified by vote in 2022. Significant changes to the taxonomy of bacterial viruses were introduced: the paraphyletic morphological families Podoviridae, Siphoviridae, and Myoviridae as well as the order Caudovirales were abolished, and a binomial system of nomenclature for species was established. In addition, one order, 22 families, 30 subfamilies, 321 genera, and 862 species were newly created, promoted, or moved.

Phenotypic characterization of phage vB_vcM_Kuja.

Bacteriophage therapy targeting the increasingly resistant Vibrio cholerae is highly needed. Hence, studying the phenotypic behavior of potential phages under different conditions is a prerequisite to delivering the phage in an active infective form. The objective of this study was to characterize phage VP4 (vB_vcM_Kuja), an environmental vibriophage isolated from River Kuja in Migori County, Kenya in 2015. The phenotypic characteristics of the phage were determined using a one-step growth curve, restriction digestion profile, pH, and temperature stability tests. The results revealed that the phage is stable through a wide range of temperatures (20-50°C) and maintains its plaque-forming ability at pH ranging from 6 to 12. The one-step growth curve showed a latent period falling between 40 and 60 min, while burst size ranged from 23 to 30 plaque-forming units/10 µl at the same host strain. The restriction digestion pattern using EcoRI, SalI, HindIII, and XhoI enzymes showed that HindIII could cut the phage genome. The phage DNA could not be restricted by the other three enzymes. The findings of this study can be used in future studies to determine phage-host interactions.

In Vitro and Pre-Clinical Evaluation of Locally Isolated Phages, vB_Pae_SMP1 and vB_Pae_SMP5, Formulated as Hydrogels against Carbapenem-Resistant Pseudomonas aeruginosa.

The inadequate therapeutic opportunities associated with carbapenem-resistant Pseudomonas aeruginosa (CRPA) clinical isolates impose a search for innovative strategies. Therefore, our study aimed to characterize and evaluate two locally isolated phages formulated in a hydrogel, both in vitro and in vivo, against CRPA clinical isolates. The two phages were characterized by genomic, microscopic, phenotypic characterization, genomic analysis, in vitro and in vivo analysis in a Pseudomonas aeruginosa-infected skin thermal injury rat model. The two siphoviruses belong to class Caudovirectes and were named vB_Pae_SMP1 and vB_Pae_SMP5. Each phage had an icosahedral head of 60 ± 5 nm and a flexible, non-contractile tail of 170 ± 5 nm long, while vB_Pae_SMP5 had an additional base plate containing a 35 nm fiber observed at the end of the tail. The hydrogel was prepared by mixing 5% w/v carboxymethylcellulose (CMC) into the CRPA propagated phage lysate containing phage titer 108 PFU/mL, pH of 7.7, and a spreadability coefficient of 25. The groups were treated with either Phage vB_Pae_SMP1, vB_Pae_SMP5, or a two-phage cocktail hydrogel cellular subepidermal granulation tissues with abundant records of fibroblastic activity and mixed inflammatory cell infiltrates and showed 17.2%, 25.8%, and 22.2% records of dermal mature collagen fibers, respectively. In conclusion, phage vB_Pae_SMP1 or vB_Pae_SMP5, or the two-phage cocktails formulated as hydrogels, were able to manage the infection of CRPA in burn wounds, and promoted healing at the injury site, as evidenced by the histopathological examination, as well as a decrease in animal mortality rate. Therefore, these phage formulae can be considered promising for clinical investigation in humans for the management of CRPA-associated skin infections.

Gene Networks and Pathways Involved in Escherichia coli Response to Multiple Stressors.

Stress response helps microorganisms survive extreme environmental conditions and host immunity, making them more virulent or drug resistant. Although both reductionist approaches investigating specific genes and systems approaches analyzing individual stress conditions are being used, less is known about gene networks involved in multiple stress responses. Here, using a systems biology approach, we mined hundreds of transcriptomic data sets for key genes and pathways involved in the tolerance of the model microorganism Escherichia coli to multiple stressors. Specifically, we investigated the E. coli K-12 MG1655 transcriptome under five stresses: heat, cold, oxidative stress, nitrosative stress, and antibiotic treatment. Overlaps of transcriptional changes between studies of each stress factor and between different stressors were determined: energy-requiring metabolic pathways, transport, and motility are typically downregulated to conserve energy, while genes related to survival, bona fide stress response, biofilm formation, and DNA repair are mainly upregulated. The transcription of 15 genes with uncharacterized functions is higher in response to multiple stressors, which suggests they may play pivotal roles in stress response. In conclusion, using rank normalization of transcriptomic data, we identified a set of E. coli stress response genes and pathways, which could be potential targets to overcome antibiotic tolerance or multidrug resistance.

Bioprospecting the microbiome of Red Sea Atlantis II brine pool for peptidases and biosynthetic genes with promising antibacterial activity.

BACKGROUND: The search for novel antimicrobial agents is crucial as antibiotic-resistant pathogens continue to emerge, rendering the available antibiotics no longer effective. Likewise, new anti-cancer drugs are needed to combat the emergence of multi-drug resistant tumors. Marine environments are wealthy sources for natural products. Additionally, extreme marine environments are interesting niches to search for bioactive natural compounds. In the current study, a fosmid library of metagenomic DNA isolated from Atlantis II Deep Lower Convective Layer (ATII LCL), was functionally screened for antibacterial activity as well as anticancer effects. RESULTS: Two clones exhibited antibacterial effects against the marine Bacillus Cc6 strain, namely clones 102-5A and 88-1G and they were further tested against eleven other challenging strains, including six safe relatives of ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter  spp.), a safe relative to Mycobacterium tuberculosis and four resistant clinical isolates. Clone 88-1G resulted in clear zones of inhibition against eight bacterial strains, while clone 102-5A resulted in zones of inhibition against five bacterial strains. The whole cell lysates of clone 88-1G showed 15% inhibition of Mtb ClpP protease -Mycobacterium tuberculosis drug target-, while whole cell lysates of clone 102-5A showed 19% inhibition of Mtb ClpP protease. Whole cell lysates from the selected clones exhibited anticancer effects against MCF-7 breast cancer cells (cell viability at 50% v/v was 46.2% ± 9.9 for 88-1G clone and 38% ± 7 for 102-5A clone), U2OS osteosarcoma cells (cell viability at 50% v/v was 64.6% ± 12.3 for 88-1G clone and 28.3% ± 1.7 for 102-5A clone) and 1BR hTERT human fibroblast cells (cell viability at 50% v/v was 74.4% ± 5.6 for 88-1G clone and 57.6% ± 8.9 for 102-5A clone). Sequencing of 102-5A and 88-1G clones, and further annotation detected putative proteases and putative biosynthetic genes in clones 102-5A and 88-1G, respectively. CONCLUSIONS: The ATII LCL metagenome hosts putative peptidases and biosynthetic genes that confer antibiotic and anti-cancer effects. The tested clones exhibited promising antibacterial activities against safe relative strains to ESKAPE pathogens and Mycobacterium tuberculosis. Thus, searching the microbial dark matter of extreme environments is a promising approach to identify new molecules with pharmaceutical potential use.

Novel Salmonella Phage, vB_Sen_STGO-35-1, Characterization and Evaluation in Chicken Meat.

Salmonellosis is one of the most frequently reported zoonotic foodborne diseases worldwide, and poultry is the most important reservoir of Salmonella enterica serovar Enteritidis. The use of lytic bacteriophages (phages) to reduce foodborne pathogens has emerged as a promising biocontrol intervention for Salmonella spp. Here, we describe and evaluate the newly isolated Salmonella phage STGO-35-1, including: (i) genomic and phenotypic characterization, (ii) an analysis of the reduction of Salmonella in chicken meat, and (iii) genome plasticity testing. Phage STGO-35-1 represents an unclassified siphovirus, with a length of 47,483 bp, a G + C content of 46.5%, a headful strategy of packaging, and a virulent lifestyle. Phage STGO-35-1 reduced S. Enteritidis counts in chicken meat by 2.5 orders of magnitude at 4 °C. We identified two receptor-binding proteins with affinity to LPS, and their encoding genes showed plasticity during an exposure assay. Phenotypic, proteomic, and genomic characteristics of STGO-35-1, as well as the Salmonella reduction in chicken meat, support the potential use of STGO-35-1 as a targeted biocontrol agent against S. Enteritidis in chicken meat. Additionally, computational analysis and a short exposure time assay allowed us to predict the plasticity of genes encoding putative receptor-binding proteins.

Bacteriophages: from Isolation to Application

Bacteriophages are considered as a potential alternative to fight pathogenic bacteria during the antibiotic resistance era. With their high specificity, they are widely used in various applications: medicine, food industry, agriculture, animal farms, biotechnology, diagnosis, etc. Many techniques have been designed by different researchers for phage isolation, purification, and amplification, each of which has strengths and weaknesses. However, all aim at having a reasonably pure phage sample that can be further characterized. Phages can be characterized based on their physiological, morphological or inactivation tests. Microscopy, in particular, opened a wide gate, not only for visualizing phage morphological structure, but also for monitoring biochemistry and behavior. Meanwhile, computational analysis of phage genomes provides more details about phage history, lifestyle, and the potential for toxigenic or lysogenic conversion, which translate to safety in biocontrol and phage therapy applications. This review article summarizes phage application pipelines at different levels, and addresses specific restrictions and knowledge gaps in the field. Recently developed computational approaches, which are used in phage genome analysis, are critically assessed. We hope that this assessment provides researchers with useful insights for the selection of suitable approaches for phage-related research aims and applications.

Compositional variation of the human fecal microbiome in relation to azo-reducing activity: a pilot study.

BACKGROUND: Through an arsenal of microbial enzymes, the gut microbiota considerably contributes to human metabolic processes, affecting nutrients, drugs, and environmental poisons. Azoreductases are a predominant group of microbiota-derived enzymes involved in xenobiotic metabolism and drug activation, but little is known about how compositional changes in the gut microbiota correlate with its azo-reducing activity. RESULTS: To this end, we used high-throughput 16S rRNA amplicon sequencing, with Illumina MiSeq, to determine the microbial community composition of stool samples from 16 adults with different azo-reducing activity. High azo-reducing activity positively correlated with the relative abundance of phylum Firmicutes (especially genera Streptococcus and Coprococcus) but negatively with phylum Bacteroidetes (especially genus Bacteroides). Typical variations in the Firmicutes-to-Bacteroidetes and Prevotella-to-Bacteroides ratios were observed among samples. Multivariate analysis of the relative abundance of key microbial taxa and other diversity parameters confirmed the Firmicutes proportion as a major variable differentiating high and non-azo-reducers, while Bacteroidetes relative abundance was correlated with azo-reduction, sex, and BMI. CONCLUSIONS: This pilot study showed that stool samples with higher azo-reducing activity were enriched in Firmicutes but with relatively fewer Bacteroidetes. More samples and studies from different geographical areas are needed to bolster this conclusion. Better characterization of different azoreductase-producing gut microbes will increase our knowledge about the fate and differential human responses to azodye-containing drugs or orally consumed chemicals, thus contributing to efforts towards implementing microbiome testing in precision medicine and toxicology.

Draft genome sequence of a prodigiosin-hyperproducing Serratia marcescens strain isolated from Cairo, Egypt.

Serratia marcescens is a Gram-negative bacterium with both environmental and host-associated strains. Pigmentation is reportedly inversely correlated with infection frequency, and prodigiosin is one of Serratia pigments that has medical and industrial applications. Here, we report the draft genome sequence of prodigiosin-hyperproducing Serratia marcescens strain N2, isolated from Cairo, Egypt. The sequence is assembled into 142 contigs, with a combined size of 5,570,793 bp. The assembled genome carries typical S. marcescens genes, with potential prodigiosin-biosynthesizing genes detected.

Profiling the Human Oral Mycobiome in Tissue and Saliva Using ITS2 DNA Metabarcoding Compared to a Fungal-Specific Database.

The advent of high-throughput sequencing has caused a paradigm shift from the one-pathogen one-disease model to the significance of dysbiosis of the oral microbiome, including the oral mycobiome. The oral mycobiome can be profiled by a method modified from that used to profile the bacteriome with 16S rRNA gene primers. The first modification is to include an initial fungus lysis step that ensures representative yields of fungal DNA. The second step is to use a reliable target, the ITS1 and/or ITS2 regions of the 23S rRNA, to define the oral fungal population, and modifications of library preparation required to deal with the variable sized amplicons generated. In this chapter, a proven microbiomic approach to identify fungal populations in oral tissue samples associated with cancer is described. This approach is also applicable to the study of the salivary mycobiome in both healthy and diseased individuals.

Novel PhoH-encoding vibriophages with lytic activity against environmental Vibrio strains.

Cholera is a devastating diarrheal disease that accounts for more than 10% of children's lives worldwide, but its treatment is hampered by a rise in antibiotic resistance. One promising alternative to antibiotic therapy is the use of bacteriophages to treat antibiotic-resistant cholera infections, and control Vibrio cholera in clinical cases and in the environment, respectively. Here, we report four novel, closely related environmental myoviruses, VP4, VP6, VP18, and VP24, which we isolated from two environmental toxigenic Vibrio cholerae strains from river Kuja and Usenge beach in Kenya. High-throughput sequencing followed by bioinformatics analysis indicated that the genomes of the four bacteriophages have closely related sequences, with sizes of 148,180 bp, 148,181 bp, 148,179 bp, and 148,179 bp, and a G + C content of 36.4%. The four genomes carry the phoH gene, which is overrepresented in marine cyanophages. The isolated phages displayed a lytic activity against 15 environmental, as well as one clinical, Vibrio cholerae strains. Thus, these novel lytic vibriophages represent potential biocontrol candidates for water decontamination against pathogenic Vibrio cholerae and ought to be considered for future studies of phage therapy.

Bacterial Viruses Subcommittee and Archaeal Viruses Subcommittee of the ICTV: update of taxonomy changes in 2021.

In this article, we - the Bacterial Viruses Subcommittee and the Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) - summarise the results of our activities for the period March 2020 - March 2021. We report the division of the former Bacterial and Archaeal Viruses Subcommittee in two separate Subcommittees, welcome new members, a new Subcommittee Chair and Vice Chair, and give an overview of the new taxa that were proposed in 2020, approved by the Executive Committee and ratified by vote in 2021. In particular, a new realm, three orders, 15 families, 31 subfamilies, 734 genera and 1845 species were newly created or redefined (moved/promoted).

Traces of SARS-CoV-2 RNA in Peripheral Blood Cells of Patients with COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third virus that caused coronavirus-related outbreaks over the past 20 years. The outbreak was first reported in December 2019 in Wuhan, China, but rapidly progressed into a pandemic of an unprecedented scale since the 1918 flu pandemic. Besides respiratory complications in patients with COVID-19, clinical characterization of severe infection cases showed several other comorbidities, including multiple organ failure, and septic shock. To better understand the systemic pathogenesis of COVID-19, we interrogated the virus's presence in the peripheral blood cells, which might provide a form of trafficking or hiding to the virus. By analyzing >2 billion sequence reads of high-throughput transcriptome sequence data from 180 samples of patients with active SARS-CoV-2 infection or healthy controls collected from 6 studies, we found evidence of traces of SARS-CoV-2 RNA in peripheral blood mononuclear cells in two samples from two independent studies. In contrast, the viral RNA was abundant in bronchoalveolar lavage specimens from the same patients. We also devised a "viral spike-to-actin" RNA normalization as a metric to compare across various samples and minimize errors caused by intersample variability in total human RNA abundance. Our observation suggests immune presentation and discounts the possibility of extensive viral infection of lymphocytes or monocytes.

Genomics-guided identification of a conserved CptBA-like toxin-antitoxin system in Acinetobacter baumannii.

Introduction: Toxin-antitoxin (TA) systems are widespread among bacteria, archaea and fungi. They are classified into six types (I-VI) and have recently been proposed as novel drug targets. Objectives: This study aimed to screen the pathogen Acinetobacter baumannii, known for its alarming antimicrobial resistance, for TA systems and identified a CptBA-like type IV TA, one of the least characterized systems. Methods: In silico methods included secondary structure prediction, comparative genomics, multiple sequence alignment, and phylogenetic analysis, while in vitro strategies included plasmid engineering and expression of the TA system in Escherichia coli BL21, growth measurement, and transcription analysis with quantitative reverse-transcription polymerase chain reaction. Results: Comparative genomics demonstrated the distribution of CptBA-like systems among Gram-negative bacteria, while phylogenetic analysis delineated two major groups, in each of which Acinetobacter spp. proteins clustered together. Sequence alignment indicated the conservation of cptA and cptB in 4,732 strains of A. baumannii in the same syntenic order. Using A. baumannii recombinant cptA and cptB, cloned under different promoters, confirmed their TA nature, as cptB expression was able to reverse growth inhibition by CptA in a dose-time dependent manner. Furthermore, transcriptional analysis of cptBA in clinical and standard A. baumannii strains demonstrated the downregulation of this system under oxidative and antibiotic stress. Conclusion: Combining in silico and in vitro studies confirmed the predicted TA nature of a cptBA-like system in A. baumannii . Transcriptional analysis suggests a possible role of cptBA in response to antibiotics and stress factors in A. baumannii, making it a promising drug target.