Lipid-Lowering Efficacy of Ezetimibe in Patients with Atherosclerotic Cardiovascular Disease: A Systematic Review and Meta-Analyses.

INTRODUCTION: Patients with atherosclerotic cardiovascular disease (ASCVD), especially those with recent (< 1 year) acute coronary syndrome (ACS), are at high risk for recurrent cardiovascular events. This risk can be reduced by lowering low-density lipoprotein cholesterol (LDL-C) levels. A comprehensive meta-analysis on the LDL-C-lowering efficacy of ezetimibe is lacking. This study attempts to address this gap. METHODS: A systematic literature review of randomized controlled trials evaluating the LDL-C-lowering efficacy of ezetimibe in the ASCVD population was conducted. MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials were searched for publications from database inception to August 2018 and for conference abstracts from 2015 to August 2018. Meta-analyses were conducted to evaluate the LDL-C-lowering efficacy of ezetimibe in the ASCVD population and the recent ACS subgroup. RESULTS: In total, 12 studies were eligible for the meta-analyses. Treatment with combination ezetimibe plus statin therapy showed greater absolute LDL-C reduction than statin monotherapy (mean difference - 21.86 mg/dL; 95% confidence interval [CI] - 26.56 to - 17.17; p < 0.0001) after 6 months of treatment (or at a timepoint closest to 6 months). Similarly, in patients with recent ACS, combination ezetimibe plus statin therapy was favorable compared with statin monotherapy (mean treatment difference - 19.19 mg/dL; 95% CI - 25.22 to - 13.16; p < 0.0001). CONCLUSIONS: Ezetimibe, when added to statin therapy, provided a modest additional reduction in LDL-C compared with statin monotherapy. However, this may not be sufficient for some patients with ASCVD who have especially high LDL-C levels despite optimal statin therapy.

Transposon Mutagenesis of Bacteroides fragilis.

Bacteroides fragilis is Gram-negative obligatory anaerobe which usually resides in the gut of humans and animals. As an important member of the human gut microbiota it plays a vital role in digestion and absorption of nutrients as well as shaping of host immune system. B. fragilis is also infamous for causing serious infections. Treatment of B. fragilis infections caused emergence of multidrug-resistant strains. Molecular biology tools such as transposon mutagenesis help to decipher and understand commensal and pathogenic faces of B. fragilis. Using two mariner transposon vectors we describe the detailed methodology for the transposon mutagenesis of B. fragilis. We also describe two methods for the identification of transposon integration site (TIS) in transposon mutants. Transposon mutagenesis methods described in this chapter serve as a great tool for studying B. fragilis.

Determining Ligand Path Through a Major Drug Transporter, AcrB, in Escherichia coli.

An experimental approach to detect the path a substrate takes through a complex membrane protein is described with emphasis on technical approach and theoretical considerations. The protocols for bacterial culture preparation, membrane protein purification, fluorescent assay standardization, data collection, and data analysis are provided. Useful software tools are recommended.

Novel large-scale chromosomal transfer in Bacteroides fragilis contributes to its pan-genome and rapid environmental adaptation.

Bacteroides fragilis, an important component of the human gastrointestinal microbiota, can cause lethal extra-intestinal infection upon escape from the gastrointestinal tract. We demonstrated transfer and recombination of large chromosomal segments from B. fragilis HMW615, a multidrug resistant clinical isolate, to B. fragilis 638R. In one example, the transfer of a segment of ~435 Kb/356 genes replaced ~413 Kb/326 genes of the B. fragilis 638R chromosome. In addition to transfer of antibiotic resistance genes, these transfers (1) replaced complete divergent polysaccharide biosynthesis loci; (2) replaced DNA inversion-controlled intergenic shufflons (that control expression of genes encoding starch utilization system outer membrane proteins) with more complex, divergent shufflons; and (3) introduced additional intergenic shufflons encoding divergent Type 1 restriction/modification systems. Conjugative transposon-like genes within a transferred segment and within a putative integrative conjugative element (ICE5) ~45 kb downstream from the transferred segment both encode proteins that may be involved in the observed transfer. These data indicate that chromosomal transfer is a driver of antigenic diversity and nutrient adaptation in Bacteroides that (1) contributes to the dissemination of the extensive B. fragilis pan-genome, (2) allows rapid adaptation to a changing environment and (3) can confer pathogenic characteristics to host symbionts.

The Ellis Island Effect: A novel mobile element in a multi-drug resistant Bacteroides fragilis clinical isolate includes a mosaic of resistance genes from Gram-positive bacteria.

Objectives: Bacteroides fragilis, a Gram-negative anaerobic bacterium, is alternately a gut commensal or virulent pathogen and is an important reservoir for horizontal gene transfer (HGT) of bacterial resistance and virulence genes in the human gastrointestinal tract. We identified a unique conjugative transposon (CTn) in a multidrug resistant clinical isolate of B. fragilis (BF-HMW615); we named this element CTnHyb because it included a hybrid mosaic of foreign elements. This study reports the characterization of CTnHyb and discusses the potential impact on horizontal spread of resistance genes. Results: CTnHyb contains several efflux pump genes and several genes that confer or may confer antibiotic resistance to tetracycline, kanamycin, metronidazole and spectinomycin (truncated gene). CTnHyb also contains a mosaic of mobile elements from Gram-positive organisms. CTnHyb is easily transferred from BF-HMW615 (the original isolate) to BF638R (lab strain) and integrated into the BF638R chromosome. The "foreign" (from Gram-positive bacteria) nucleotide sequences within CTnHyb were > 99% preserved indicating that the gene acquisition from the Gram-positive bacteria was very recent. Conclusion: CTnHyb is a novel CTn residing in a multidrug resistant strain of B. fragilis. The global nature and wide phylogenetic reach of HGT means that any gene in any bacterium can potentially be mobilized. Understanding the mechanisms that drive the formation and transfer of these elements and, potentially, ways to limit the transfer are necessary to prevent a devastating spread of resistance elements.

Deficiency of the ferrous iron transporter FeoAB is linked with metronidazole resistance in Bacteroides fragilis.

BACKGROUND: Metronidazole is the most commonly used antimicrobial for Bacteroides fragilis infections and is recommended for prophylaxis of colorectal surgery. Metronidazole resistance is increasing and the mechanisms of resistance are not clear. METHODS: A transposon mutant library was generated in B. fragilis 638R (BF638R) to identify the genetic loci associated with resistance to metronidazole. RESULTS: Thirty-two independently isolated metronidazole-resistant mutants had a transposon insertion in BF638R_1421 that encodes the ferrous transport fusion protein (feoAB). Deletion of feoAB resulted in a 10-fold increased MIC of metronidazole for the strain. The metronidazole MIC for the feoAB mutant was similar to that for the parent strain when grown on media supplemented with excess iron, suggesting that the increase seen in the MIC of metronidazole was due to reduced cellular iron transport in the feoAB mutant. The furA gene repressed feoAB transcription in an iron-dependent manner and disruption of furA resulted in constitutive transcription of feoAB, regardless of whether or not iron was present. However, disruption of feoAB also diminished the capacity of BF638R to grow in a mouse intraperitoneal abscess model, suggesting that inorganic ferrous iron assimilation is essential for B. fragilis survival in vivo. CONCLUSIONS: Selection for feoAB mutations as a result of metronidazole treatment will disable the pathogenic potential of B. fragilis and could contribute to the clinical efficacy of metronidazole. While mutations in feoAB are probably not a direct cause of clinical resistance, this study provides a key insight into intracellular metronidazole activity and the link with intracellular iron homeostasis.

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.

Two multidrug-resistant clinical isolates of Bacteroides fragilis carry a novel metronidazole resistance nim gene (nimJ).

Two multidrug-resistant Bacteroides fragilis clinical isolates contain and express a novel nim gene, nimJ, that is not recognized by the "universal" nim primers and can confer increased resistance to metronidazole when introduced into a susceptible strain on a multicopy plasmid. HMW615, an appendiceal isolate, contains at least two copies of nimJ on its genome, while HMW616, an isolate from a patient with sepsis, contains one genomic copy of nimJ. B. fragilis NimJ is phylogenetically closer to Prevotella baroniae NimI and Clostridium botulinum NimA than to the other known Bacteroides Nim proteins. The predicted protein structure of NimJ, based on fold recognition analysis, is consistent with the crystal structures derived for known Nim proteins, and specific amino acid residues important for substrate binding in the active site are conserved. This study demonstrates that the "universal" nim primers will not detect all nim genes with the ability to confer metronidazole resistance, but nimJ alone cannot account for the very high metronidazole MICs of these resistant clinical isolates.

Transposon mutagenesis of Bacteroides fragilis using a mariner transposon vector.

The mariner transposon vector pYV07 was tested for use in the mutagenesis of Bacteroides fragilis 638R. The transposon vector efficiently generated mutants in B. fragilis 638R. The transposon disrupted genes were scattered throughout the genome of B. fragilis 638R. This method serves as a powerful tool to study B. fragilis.

Transposon mutagenesis of the anaerobic commensal, Bacteroides fragilis, using the EZ::TN5 transposome.

Genetic analysis of Bacteroides fragilis (BF) is hindered because of the lack of efficient transposon mutagenesis methods. Here, we describe a simple method for transposon mutagenesis using EZ::TN5, a commercially available system that we optimized for use in BF638R. The modified EZ::TN5 transposon contains an Escherichia coli conditional origin of replication, a kanamycin resistance gene for E. coli, an erythromycin resistance gene for BF , and 19 basepair transposase recognition sequences on either ends. Electroporation of the transposome (transposon-transposase complex) into BF638R yielded 3.2 ± 0.35 × 10(3) CFU µg(-1) of transposon DNA. Modification of the transposon by the BF638R restriction/modification system increased transposition efficiency sixfold. Electroporation of the EZ::TN5 transposome results in a single-copy insertion of the transposon evenly distributed across the genome of BF638R and can be used to construct a BF638R transposon library. The transposon was also effective in mutating a BF clinical isolate and a strain of the related species, Bacteroides thetaiotaomicron. The EZ::TN5-based mutagenesis described here is more efficient than other transposon mutagenesis approaches previously reported for BF.

Vestibules are part of the substrate path in the multidrug efflux transporter AcrB of Escherichia coli.

The path of substrates in the multidrug efflux pump AcrB of Escherichia coli was examined by using labeling with a lipophilic substrate mimic, Bodipy FL maleimide. Four (out of eight) residues in the vestibule bound the dye, suggesting its role in substrate transport, whereas only one (out of nine) residue in the central cavity tested positive.

Substrate path in the AcrB multidrug efflux pump of Escherichia coli.

A major tripartite multidrug efflux pump of Escherichia coli, AcrAB-TolC, confers resistance to a wide variety of compounds. The drug molecule is captured by AcrB probably from the periplasm or the periplasm/inner membrane interface, and is passed through AcrB and then TolC to the medium. Currently, there exist numerous crystallographic and mutation data concerning the regions of AcrB and its homologues that may interact with substrates. Starting with these data, we devised fluorescence assays in whole cells to determine the entire substrate path through AcrB. We tested 48 residues in AcrB along the predicted substrate path and 25 gave positive results, based on the covalent labelling of cysteine residues by a lipophilic dye-maleimide and the blocking of Nile red efflux by covalent labelling with bulky maleimide reagents. These residues are all located in the periplasmic domain, in regions we designate as the lower part of the large external cleft, the cleft itself, the crystallographically defined binding pocket, and the gate between the pocket and the funnel. Our observations suggest that the substrate is captured in the lower cleft region of AcrB, then transported through the binding pocket, the gate and finally to the AcrB funnel that connects AcrB to TolC.

Interaction between the TolC and AcrA proteins of a multidrug efflux system of Escherichia coli.

This paper provides the biochemical evidence for physical interactions between the outer membrane component, TolC, and the membrane fusion protein component, AcrA, of the major antibiotic efflux pump of Escherichia coli. Cross-linking between TolC and AcrA was independent of the presence of any externally added substrate of the efflux pump or of the pump protein, AcrB. The biochemical demonstration of a TolC-AcrA interaction is consistent with genetic studies in which extragenic suppressors of a mutant TolC strain were found in the acrA gene.

Antibiotic-sensitive TolC mutants and their suppressors.

The TolC protein of Escherichia coli, through its interaction with AcrA and AcrB, is thought to form a continuous protein channel that expels inhibitors from the cell. Consequently, tolC null mutations display a hypersensitive phenotype. Here we report the isolation and characterization of tolC missense mutations that direct the synthesis of mutant TolC proteins partially disabled in their efflux role. All alterations, consisting of single amino acid substitutions, were localized within the periplasmic alpha-helical domain. In two mutants carrying an I106N or S350F substitution, the hypersensitivity phenotype may be in part due to aberrant TolC assembly. However, two other alterations, R367H and R390C, disrupted efflux function by affecting interactions among the helices surrounding TolC's periplasmic tunnel. Curiously, these two TolC mutants were sensitive to a large antibiotic, vancomycin, and exhibited a Dex(+) phenotype. These novel phenotypes of TolC(R367H) and TolC(R390C) were likely the result of a general influx of molecules through a constitutively open tunnel aperture, which normally widens only when TolC interacts with other proteins during substrate translocation. An intragenic suppressor alteration (T140A) was isolated from antibiotic-resistant revertants of the hypersensitive TolC(R367H) mutant. T140A also reversed, either fully (R390C) or partially (I106N and S350F), the hypersensitivity phenotype of other TolC mutants. Our data suggest that this global suppressor phenotype of T140A is the result of impeded antibiotic influx caused by tapering of the tunnel passage rather than by correcting individual mutational defects. Two extragenic suppressors of TolC(R367H), mapping in the regulatory region of acrAB, uncoupled the AcrR-mediated repression of the acrAB genes. The resulting overexpression of AcrAB reduced the hypersensitivity phenotype of all the TolC mutants. Similar results were obtained when the chromosomal acrR gene was deleted or the acrAB genes were expressed from a plasmid. Unlike the case for the intragenic suppressor T140A, the overexpression of AcrAB diminished hypersensitivity towards only erythromycin and novobiocin, which are substrates of the TolC-AcrAB efflux pump, but not towards vancomycin, which is not a substrate of this pump. This showed that the two types of suppressors produced their effects by fundamentally different means, as the intragenic suppressor decreased the general influx while extragenic suppressors increased the efflux of TolC-AcrAB pump-specific antibiotics.