Identification of genes required for the survival of B. fragilis using massive parallel sequencing of a saturated transposon mutant library

BACKGROUND: Bacteroides fragilis is a Gram-negative anaerobe that is normally a human gut commensal; it comprises a small percentage of the gut Bacteroides but is the most frequently isolated Bacteroides from human infections. Identification of the essential genes necessary for the survival of B. fragilis provides novel information which can be exploited for the treatment of bacterial infections. RESULTS: Massive parallel sequencing of saturated transposon mutant libraries (two mutant pools of approximately 50,000 mutants each) was used to determine the essential genes for the growth of B. fragilis 638R on nutrient rich medium. Among the 4326 protein coding genes, 550 genes (12.7%) were found to be essential for the survival of B. fragilis 638R. Of the 550 essential genes, only 367 genes were assigned to a Cluster of Orthologous Genes, and about 290 genes had Kyoto Encyclopedia of Genes and Genomes orthologous members. Interestingly, genes with hypothetical functions accounted for 41.3% of essential genes (227 genes), indicating that the functions of a significant percentage of the genes used by B. fragilis 638R are still unknown. Global transcriptome analysis using RNA-Seq indicated that most of the essential genes (92%) are, in fact, transcribed in B. fragilis 638R including most of those coding for hypothetical proteins. Three hundred fifty of the 550 essential genes of B. fragilis 638R are present in Database of Essential Genes. 10.02 and 31% of those are genes included as essential genes for nine species (including Gram-positive pathogenic bacteria). CONCLUSIONS: The essential gene data described in this investigation provides a valuable resource to study gene function and pathways involved in B. fragilis survival. Thorough examination of the B. fragilis-specific essential genes and genes that are shared between divergent organisms opens new research avenues that will lead to enhanced understanding of survival strategies used by bacteria in different microniches and under different stress situations. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi: 10.1186/1471-2164-15-429) contains supplementary material, which is available to authorized users

Antimicrobial resistance and prevalence of resistance genes in intestinal Bacteroidales strains

OBJECTIVE: This study examined the antimicrobial resistance profile and the prevalence of resistance genes in Bacteroides spp. and Parabacteroides distasonis strains isolated from children's intestinal microbiota. METHODS: The susceptibility of these bacteria to 10 antimicrobials was determined using an agar dilution method. β-lactamase activity was assessed by hydrolysis of the chromogenic cephalosporin of 114 Bacteriodales strains isolated from the fecal samples of 39 children, and the presence of resistance genes was tested using a PCR assay. RESULTS: All strains were susceptible to imipenem and metronidazole. The following resistance rates were observed: amoxicillin (93%), amoxicillin/clavulanic acid (47.3%), ampicillin (96.4%), cephalexin (99%), cefoxitin (23%), penicillin (99%), clindamycin (34.2%) and tetracycline (53.5%). P-lactamase production was verified in 92% of the evaluated strains. The presence of the cfiA, cepA, ermF, tetQ and nim genes was observed in 62.3%, 76.3%, 27%, 79.8% and 7.8% of the strains, respectively. CONCLUSIONS: Our results indicate an increase in the resistance to several antibiotics in intestinal Bacteroides spp. and Parabacteroides distasonis and demonstrate that these microorganisms harbor antimicrobial resistance genes that may be transferred to other susceptible intestinal strains

Manual / Issue 13 / Storage

Manual, a journal about art and its making. Storage. Manual 13 opens with an introduction by Fred Wilson, who confides, “You can look at all the opulence on display in a museum and begin to understand that something nefarious might be behind it. Storage, for me, is where the action is.” Museums usually make choices for viewers, their curators presenting what they think most important within a category. They can be so good at doing this that visitors sometimes don’t realize there’s anything else to see: they don’t realize the nature of the decisions behind an exhibition, and they accept that the elites have made a judgment about which shoe is the shoe to see. Visitors can learn about what’s great, but they don’t necessarily consider the process of discernment. –– Fred Wilson The RISD Museum’s thirteenth issue of Manual unpacks the idea and reality of storage—objects museums don’t put on view, works made as containers of various sorts, and more metaphorical considerations about how meanings and narratives are stored. This issue serves as a companion to the Raid the Icebox Now series of exhibitions on view at the RISD Museum through November 2020, in which nine contemporary artists and design collectives use the museum and its collections as a site for critical creative production and presentation. Raid the Icebox Now marks the 50th anniversary of Raid the Icebox 1 with Andy Warhol, held in 1970 at the RISD Museum. Softcover, 120 pages. Published Fall/Winter 2019 by the RISD Museum. Manual 13 (Storage) contributors include: Christina Alderman, Issac M. Alderman, A.H. Jerriod Avant, Hannah Carlson, Wai Yee Chiong, John Dunnigan, Maria Morris Hambourg, David Hartt, Elaine Tyler May, Claire McCardell, Denise Murrell, Ingrid Schaffner, Holly Shaffer, Tanya Sheehan, John W. Smith, Mimi Smith, Sassan Tabatabai, Allen Wexler, and Fred Wilson.https://digitalcommons.risd.edu/risdmuseum_journals/1039/thumbnail.jp

Changing practices: The specialised domestic violence court process

Specialised domestic violence courts, initially developed in the United States of America, have been recognised by other jurisdictions including Canada, Australia and the United Kingdom. This article presents a case study of K Court in Toronto, drawing upon documentary evidence, direct observations and interviews with key informants. It is argued that the specialised domestic violence court process includes changing practices of some of the key stakeholders. Learning lessons from abroad can offer jurisdictions insights that can steer implementation of appropriate practices in the field

Fludarabine, cytarabine, granulocyte colony-stimulating factor, and idarubicin with gemtuzumab ozogamicin improves event-free survival in younger patients with newly diagnosed aml and overall survival in patients with npm1 and flt3 mutations

Purpose To determine the optimal induction chemotherapy regimen for younger adults with newly diagnosed AML without known adverse risk cytogenetics. Patients and Methods One thousand thirty-three patients were randomly assigned to intensified (fludarabine, cytarabine, granulocyte colony-stimulating factor, and idarubicin [FLAG-Ida]) or standard (daunorubicin and Ara-C [DA]) induction chemotherapy, with one or two doses of gemtuzumab ozogamicin (GO). The primary end point was overall survival (OS). Results There was no difference in remission rate after two courses between FLAG-Ida + GO and DA + GO (complete remission [CR] + CR with incomplete hematologic recovery 93% v 91%) or in day 60 mortality (4.3% v 4.6%). There was no difference in OS (66% v 63%; P = .41); however, the risk of relapse was lower with FLAG-Ida + GO (24% v 41%; P < .001) and 3-year event-free survival was higher (57% v 45%; P < .001). In patients with an NPM1 mutation (30%), 3-year OS was significantly higher with FLAG-Ida + GO (82% v 64%; P = .005). NPM1 measurable residual disease (MRD) clearance was also greater, with 88% versus 77% becoming MRD-negative in peripheral blood after cycle 2 (P = .02). Three-year OS was also higher in patients with a FLT3 mutation (64% v 54%; P = .047). Fewer transplants were performed in patients receiving FLAG-Ida + GO (238 v 278; P = .02). There was no difference in outcome according to the number of GO doses, although NPM1 MRD clearance was higher with two doses in the DA arm. Patients with core binding factor AML treated with DA and one dose of GO had a 3-year OS of 96% with no survival benefit from FLAG-Ida + GO. Conclusion Overall, FLAG-Ida + GO significantly reduced relapse without improving OS. However, exploratory analyses show that patients with NPM1 and FLT3 mutations had substantial improvements in OS. By contrast, in patients with core binding factor AML, outcomes were excellent with DA + GO with no FLAG-Ida benefit

CRISPR-Cas Systems in Bacteroides fragilis, an Important Pathobiont in the Human Gut Microbiome

Background: While CRISPR-Cas systems have been identified in bacteria from a wide variety of ecological niches, there are no studies to describe CRISPR-Cas elements in Bacteroides species, the most prevalent anaerobic bacteria in the lower intestinal tract. Microbes of the genus Bacteroides make up ~25% of the total gut microbiome. Bacteroides fragilis comprises only 2% of the total Bacteroides in the gut, yet causes of &gt;70% of Bacteroides infections. The factors causing it to transition from benign resident of the gut microbiome to virulent pathogen are not well understood, but a combination of horizontal gene transfer (HGT) of virulence genes and differential transcription of endogenous genes are clearly involved. The CRISPR-Cas system is a multi-functional system described in prokaryotes that may be involved in control both of HGT and of gene regulation.Results: Clustered regularly interspaced short palindromic repeats (CRISPR) elements in all strains of B. fragilis (n = 109) with publically available genomes were identified. Three different CRISPR-Cas types, corresponding most closely to Type IB, Type IIIB, and Type IIC, were identified. Thirty-five strains had two CRISPR-Cas types, and three strains included all three CRISPR-Cas types in their respective genomes. The cas1 gene in the Type IIIB system encoded a reverse-transcriptase/Cas1 fusion protein rarely found in prokaryotes. We identified a short CRISPR (3 DR) with no associated cas genes present in most of the isolates; these CRISPRs were found immediately upstream of a hipA/hipB operon and we speculate that this element may be involved in regulation of this operon related to formation of persister cells during antimicrobial exposure. Also, blood isolates of B. fragilis did not have Type IIC CRISPR-Cas systems and had atypical Type IIIB CRISPR-Cas systems that were lacking adjacent cas genes.Conclusions: This is the first systematic report of CRISPR-Cas systems in a wide range of B. fragilis strains from a variety of sources. There are four apparent CRISPR-Cas systems in B. fragilis—three systems have adjacent cas genes. Understanding CRISPR/Cas function in B. fragilis will elucidate their role in gene expression, DNA repair and ability to survive exposure to antibiotics. Also, based on their unique CRISPR-Cas arrays, their phylogenetic clustering and their virulence potential, we are proposing that blood isolates of B. fragilis be viewed a separate subgroup

Bacteroides: the Good, the Bad, and the Nitty-Gritty

Summary: Bacteroides species are significant clinical pathogens and are found in most anaerobic infections, with an associated mortality of more than 19%. The bacteria maintain a complex and generally beneficial relationship with the host when retained in the gut, but when they escape this environment they can cause significant pathology, including bacteremia and abscess formation in multiple body sites. Genomic and proteomic analyses have vastly added to our understanding of the manner in which Bacteroides species adapt to, and thrive in, the human gut. A few examples are (i) complex systems to sense and adapt to nutrient availability, (ii) multiple pump systems to expel toxic substances, and (iii) the ability to influence the host immune system so that it controls other (competing) pathogens. B. fragilis, which accounts for only 0.5% of the human colonic flora, is the most commonly isolated anaerobic pathogen due, in part, to its potent virulence factors. Species of the genus Bacteroides have the most antibiotic resistance mechanisms and the highest resistance rates of all anaerobic pathogens. Clinically, Bacteroides species have exhibited increasing resistance to many antibiotics, including cefoxitin, clindamycin, metronidazole, carbapenems, and fluoroquinolones (e.g., gatifloxacin, levofloxacin, and moxifloxacin)

Efflux Pump Overexpression in Multiple-Antibiotic-Resistant Mutants of Bacteroides fragilis

Multidrug-resistant mutants of a wild-type Bacteroides fragilis strain (strain ADB77) and a quadruple resistance nodulation division family efflux pump deletion mutant (ADB77 ΔbmeB1 ΔbmeB3 ΔbmeB12 ΔbmeB15) were selected with antimicrobials. Ampicillin, doripenem, imipenem, levofloxacin, and metronidazole selected for mutants from both strains; cefoxitin selected for mutants from strain ADB77 only; and sodium dodecyl sulfate selected mutants from ADB77ΔbmeB1 ΔbmeB3 ΔbmeB12 ΔbmeB15 only. The mutants overexpressed one or more efflux pumps