Brevibacterium Bacteremia in the Setting of Pyogenic Liver Abscess: A Case Report with Accompanying Literature Review

A 71-year-old Pakistani man with poorly controlled type 2 diabetes mellitus presenting with worsening mental status, abdominal pain, and oral intake for the past seven days was found to have pyogenic hepatic abscess with unculturable bacteria and subsequently found to have rare Brevibacterium bacteremia.

Directed Evolution and Discovery of Nitroreductase Enzymes for Targeted Cell Ablation

<p>Nitroreductase enzymes are a superfamily of bacterial flavoproteins that can catalyze the reduction of aromatic nitro groups. The reduction of an aromatic nitro group, a highly electronegative functionality, causes a large electronic shift that can profoundly affect the activity of other substituents on the aromatic ring. For example, upon nitroreduction, initially non-toxic compounds known as prodrugs can be converted into a cytotoxic form. The ability of nitroreductases to alter the activity of compounds has lead to their development as tools for multiple biotechnological applications. Of particular note is the use of nitroreductase enzymes in combination with a nitroaromatic prodrug to study the role of specific cell populations in zebrafish (Danio rerio). Zebrafish are used as model organisms to study processes such as embryonic development and tissue regeneration. By expressing a nitroreductase enzyme in a specific tissue of a zebrafish, it is possible to selectively ablate that tissue upon administration of a prodrug. The subsequent phenotypic change induced by the ablation can provide information on the physiological role of the ablated tissue, or of the regenerative processes that can be recruited to repair the damage. The goal of this thesis was to engineer or discover new nitroreductase enzymes that could expand the capabilities of cell ablation studies in zebrafish. In particular, this work sought to develop a system that would enable the dual, or multiplexed, ablation of two tissues independently within the same organism. Control over the ablation of two distinct tissues could be useful for studying tissue interactions during developmental or regenerative processes. For this to be achievable, two different nitroreductase enzymes, each possessing distinct and non-overlapping prodrug selectivities would be required. Previous studies in the Ackerley lab had identified NfsA from Escherichia coli (NfsA_Ec) and NfsA from Pseudomonas putida (NfsA_Pp) as nitroreductase enzymes that were slightly more selective for the prodrug tinidazole compared than metronidazole. In contrast the NfsB nitroreductase from Vibrio vulnificus (NfsB_Vv) was substantially more selective for metronidazole than tinidazole. To further improve the tinidazole selectivity of the NfsA enzymes, directed evolution was employed as a tool to further enhance the substrate selectivity of each enzyme. The primary outcome of this work was the evolution of an NfsA_Ec mutant that was 12 fold more selective for tinidazole over metronidazole than wild type NfsA_Ec. In addition to engineering new enzymes for cell ablation experiments, this work also sought to discover new nitroreductase enzymes from unculturable bacteria, a previously unplumbed source. The genes and gene products of unculturable bacteria can be identified and studied by expressing fragments of their DNA in a readily culturable host such as E. coli. A variety of different screening methodologies were tested for identifying nitroreductase enzymes from eDNA inserts. The compound 4-nitroimidazole was found to be capable of detecting nitroreductase expression at the level of a single colony. While no novel nitroreductase enzymes were discovered in the scope of this work, the preliminary results are encouraging that a screening strategy centred on 4-nitroimidazole in particular could successfully do so in the near future.</p>

Directed Evolution and Discovery of Nitroreductase Enzymes for Targeted Cell Ablation

<p>Nitroreductase enzymes are a superfamily of bacterial flavoproteins that can catalyze the reduction of aromatic nitro groups. The reduction of an aromatic nitro group, a highly electronegative functionality, causes a large electronic shift that can profoundly affect the activity of other substituents on the aromatic ring. For example, upon nitroreduction, initially non-toxic compounds known as prodrugs can be converted into a cytotoxic form. The ability of nitroreductases to alter the activity of compounds has lead to their development as tools for multiple biotechnological applications. Of particular note is the use of nitroreductase enzymes in combination with a nitroaromatic prodrug to study the role of specific cell populations in zebrafish (Danio rerio). Zebrafish are used as model organisms to study processes such as embryonic development and tissue regeneration. By expressing a nitroreductase enzyme in a specific tissue of a zebrafish, it is possible to selectively ablate that tissue upon administration of a prodrug. The subsequent phenotypic change induced by the ablation can provide information on the physiological role of the ablated tissue, or of the regenerative processes that can be recruited to repair the damage. The goal of this thesis was to engineer or discover new nitroreductase enzymes that could expand the capabilities of cell ablation studies in zebrafish. In particular, this work sought to develop a system that would enable the dual, or multiplexed, ablation of two tissues independently within the same organism. Control over the ablation of two distinct tissues could be useful for studying tissue interactions during developmental or regenerative processes. For this to be achievable, two different nitroreductase enzymes, each possessing distinct and non-overlapping prodrug selectivities would be required. Previous studies in the Ackerley lab had identified NfsA from Escherichia coli (NfsA_Ec) and NfsA from Pseudomonas putida (NfsA_Pp) as nitroreductase enzymes that were slightly more selective for the prodrug tinidazole compared than metronidazole. In contrast the NfsB nitroreductase from Vibrio vulnificus (NfsB_Vv) was substantially more selective for metronidazole than tinidazole. To further improve the tinidazole selectivity of the NfsA enzymes, directed evolution was employed as a tool to further enhance the substrate selectivity of each enzyme. The primary outcome of this work was the evolution of an NfsA_Ec mutant that was 12 fold more selective for tinidazole over metronidazole than wild type NfsA_Ec. In addition to engineering new enzymes for cell ablation experiments, this work also sought to discover new nitroreductase enzymes from unculturable bacteria, a previously unplumbed source. The genes and gene products of unculturable bacteria can be identified and studied by expressing fragments of their DNA in a readily culturable host such as E. coli. A variety of different screening methodologies were tested for identifying nitroreductase enzymes from eDNA inserts. The compound 4-nitroimidazole was found to be capable of detecting nitroreductase expression at the level of a single colony. While no novel nitroreductase enzymes were discovered in the scope of this work, the preliminary results are encouraging that a screening strategy centred on 4-nitroimidazole in particular could successfully do so in the near future.</p>

Portable and Rapid Sequencing Device with Microbial Community-Guided Culture Strategies for Precious Field and Environmental Samples

Culturing unculturable bacteria is a classic microbiology challenge; to successfully culture unculturable bacteria, microbiologists work hard to create hundreds of culture conditions. To improve the success rate and efficiency of culturing a broad spectrum of environmental microbes, it is helpful to know more about the microbial community composition.

Prevalence of unculturable bacteria in the periapical abscess: A systematic review and meta-analysis

Objective To assess the prevalence of unculturable bacteria in periapical abscess, radicular cyst, and periapical granuloma. Methods PubMed, Scopus, Science Direct, and Ovid databases were systematically searched from January 1990 to May 2020. All the included studies were cross-sectional design. The risk of bias was assessed using Joanna Briggs Institute check-list. Heterogeneity was described using meta-regression and mixed-effects model for lesion, country, and sequence technique moderators. Funnel plot and unweighted Egger’s regression test were used to estimate the publication bias. Microbiome data on diversity, abundance, and frequency of unculturable bacteria in the periapical lesions were reviewed, analysed, and the principal component analysis (PCA) was performed. Results A total of 13 studies out of 14,780, were selected for the final analysis. These studies focused on the prevalence of unculturable bacteria in periapical abscesses and related lesions. Approximately 13% (95% CI: 7–23%) of the cumulative number of bacteria derived from periapical abscesses was unculturable. Country moderator significantly (P = 0.05) affects the diversity summary proportion. While the pooled frequency of unculturable bacteria was 8%; 95% CI: 5, 14%, the estimate of the pooled abundance of unculturable bacteria was 5%; 95% CI: 2, 12% with a significant (P = 0.05) country moderator that affects the abundance summary proportion. Of the 62 unculturable bacteria, 35 were subjected to PCA and Peptostreptococcus sp. oral clone CK035 was the most abundant species in periapical abscesses. Hybridization techniques were found to be the most reliable molecular methods in detecting the abundance and frequency of unculturable bacteria. Conclusion The significant prevalence of unculturable bacteria in the periapical abscess, suggests that they are likely to play, a yet unknown, critical role in the pathogenesis and progression of the disease. Further research remains to be done to confirm their specific contributions in the virulence and disease progression.

Candidatus Phytoplasma australiense.

Phytoplasmas are wall-less, phloem-limited unculturable bacteria that are naturally spread by sap-sucking insects. 'Candidatus Phytoplasma australiense', subgroup 16SrXII-B, is associated with a wide range of diseases in Australia and New Zealand. Important commercial crop hosts of 'Ca. Phytoplasma australiense' include grapevine, papaya and strawberry. This phytoplasma is associated with rapid death of its papaya and cabbage tree hosts. In New Zealand, the insect vectors have been confirmed to be the endemic Cixiid planthoppers, Zeoliarus atkinsoni and Z. oppositus, while in Australia no vector has yet been determined, although the leafhopper, Orosius argentatus, has been implicated. Long distance spread of the phytoplasma is possible through infected vegetative propagating material. 'Ca. Phytoplasma australiense' is on the A1 list of regulated organisms for Canada and Bahrain, and is listed as a quarantine pest for the USA.

Candidatus Phytoplasma australiense.

Phytoplasmas are wall-less, phloem-limited unculturable bacteria that are naturally spread by sap-sucking insects. 'Candidatus Phytoplasma australiense', subgroup 16SrXII-B, is associated with a wide range of diseases in Australia and New Zealand. Important commercial crop hosts of 'Ca. Phytoplasma australiense' include grapevine, papaya and strawberry. This phytoplasma is associated with rapid death of its papaya and cabbage tree hosts. In New Zealand, the insect vectors have been confirmed to be the endemic Cixiid planthoppers, Zeoliarus atkinsoni and Z. oppositus, while in Australia no vector has yet been determined, although the leafhopper, Orosius argentatus, has been implicated. Long distance spread of the phytoplasma is possible through infected vegetative propagating material. 'Ca. Phytoplasma australiense' is on the A1 list of regulated organisms for Canada and Bahrain, and is listed as a quarantine pest for the USA.

In silico analysis of phylogeny, structure, and function of arsenite oxidase from unculturable microbiome of arsenic contaminated soil

Background Arsenite oxidase (EC 1.20.2.1) is a metalloenzyme that catalyzes the oxidation of arsenite into lesser toxic arsenate. In this study, 78 amino acid sequences of arsenite oxidase from unculturable bacteria available in metagenomic data of arsenic-contaminated soil have been characterized by using standard bioinformatics tools to investigate its phylogenetic relationships, three-dimensional structure and functional parameters. Results The phylogenetic relationship of all arsenite oxidase from unculturable microorganisms was revealed their closeness to bacterial order Rhizobiales. The higher aliphatic content showed that these enzymes are thermostable and could be used for in situ bioremediation. A representative protein from each phylogenetic cluster was analysed for secondary structure arrangements which indicated the presence of α-helices (~63%), β-sheets (57–60%) and turns (13–15%). The validated 3D models suggested that these proteins are hetero-dimeric with two chains whereas alpha chain is the main catalytic subunit which binds with arsenic oxides. Three representative protein models were deposited in Protein Model Database. The query enzymes were predicted with two conserved motifs, one is Rieske 3Fe-4S and the other is molybdopterin protein. Conclusions Computational analysis of protein interactome revealed the protein partners might be involved in the whole process of arsenic detoxification by Rhizobiales. The overall report is unique to the best of our knowledge, and the importance of this study is to understand the theoretical aspects of the structure and functions of arsenite oxidase in unculturable bacteria residing in arsenic-contaminated sites.

Impact of endometrial microbiome on fertility

Background Next-generation sequencing isolates culturable and unculturable bacteria from the female reproductive tract. Current literatures surrounding the impact of endometrial microbiome on fertility are reviewed. Main body An abnormal endometrial microbiota has been associated with implantation failure pregnancy loss and other gynecological and obstetrical conditions. Identification of endometrial dysbiosis as a new cause of infertility opens a new microbiological field in the evaluation of endometrial factor, highlighting the relevance of assessing the uterine microbiota in infertile patients to restore a favorable endometrial flora in those patients with altered uterine microbiota to improve and personalize the clinical care of infertile patients. Understanding the significance of microbiome in the endometrium may completely change the therapeutic approach in the treatment of this part of the reproductive tract. Conclusion Investigation of the endometrial microbiota may be a future tool for improving reproductive outcomes in infertile patients. Further well-designed studies are required to establish its role in the evaluation and treatment of infertile patients