Neha Shah

Characterizing a model human gut microbiota composed of members of its two dominant bacterial phyla

The adult human distal gut microbial community is typically dominated by 2 bacterial phyla (divisions), the Firmicutes and the Bacteroidetes. Little is known about the factors that govern the interactions between their members. Here, we examine the niches of representatives of both phyla in vivo. Finished genome sequences were generated from Eubacterium rectale and E. eligens, which belong to Clostridium Cluster XIVa, one of the most common gut Firmicute clades. Comparison of these and 25 other gut Firmicutes and Bacteroidetes indicated that the Firmicutes possess smaller genomes and a disproportionately smaller number of glycan-degrading enzymes. Germ-free mice were then colonized with E. rectale and/or a prominent human gut Bacteroidetes, Bacteroides thetaiotaomicron, followed by whole-genome transcriptional profiling, high-resolution proteomic analysis, and biochemical assays of microbial–microbial and microbial–host interactions. B. thetaiotaomicron adapts to E. rectale by up-regulating expression of a variety of polysaccharide utilization loci encoding numerous glycoside hydrolases, and by signaling the host to produce mucosal glycans that it, but not E. rectale, can access. E. rectale adapts to B. thetaiotaomicron by decreasing production of its glycan-degrading enzymes, increasing expression of selected amino acid and sugar transporters, and facilitating glycolysis by reducing levels of NADH, in part via generation of butyrate from acetate, which in turn is used by the gut epithelium. This simplified model of the human gut microbiota illustrates niche specialization and functional redundancy within members of its major bacterial phyla, and the importance of host glycans as a nutrient foundation that ensures ecosystem stability.

Genome sequence of Cronobacter sakazakii BAA-894 and comparative genomic hybridization analysis with other Cronobacter species.

Background The genus Cronobacter (formerly called Enterobacter sakazakii) is composed of five species; C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, and C. dublinensis. The genus includes opportunistic human pathogens, and the first three species have been associated with neonatal infections. The most severe diseases are caused in neonates and include fatal necrotizing enterocolitis and meningitis. The genetic basis of the diversity within the genus is unknown, and few virulence traits have been identified. Methodology/Principal Findings We report here the first sequence of a member of this genus, C. sakazakii strain BAA-894. The genome of Cronobacter sakazakii strain BAA-894 comprises a 4.4 Mb chromosome (57% GC content) and two plasmids; 31 kb (51% GC) and 131 kb (56% GC). The genome was used to construct a 387,000 probe oligonucleotide tiling DNA microarray covering the whole genome. Comparative genomic hybridization (CGH) was undertaken on five other C. sakazakii strains, and representatives of the four other Cronobacter species. Among 4,382 annotated genes inspected in this study, about 55% of genes were common to all C. sakazakii strains and 43% were common to all Cronobacter strains, with 10–17% absence of genes. Conclusions/Significance CGH highlighted 15 clusters of genes in C. sakazakii BAA-894 that were divergent or absent in more than half of the tested strains; six of these are of probable prophage origin. Putative virulence factors were identified in these prophage and in other variable regions. A number of genes unique to Cronobacter species associated with neonatal infections (C. sakazakii, C. malonaticus and C. turicensis) were identified. These included a copper and silver resistance system known to be linked to invasion of the blood-brain barrier by neonatal meningitic strains of Escherichia coli. In addition, genes encoding for multidrug efflux pumps and adhesins were identified that were unique to C. sakazakii strains from outbreaks in neonatal intensive care units.

L-5F, an apolipoprotein A-I mimetic, inhibits tumor angiogenesis by suppressing VEGF/basic FGF signaling pathways.

We recently reported that apolipoprotein A-I (apoA-I) and apoA-I mimetic peptides inhibit tumor growth and improve survival in a mouse model of ovarian cancer. The current study was designed to examine whether inhibition of angiogenesis is one of the mechanisms for the observed anti-tumorigenic effects. The apoA-I mimetic peptide L-5F had no affect on proliferation and cell viability of human umbilical vascular endothelial cells (HUVECs) in the basal state; however, treatment with L-5F at 1, 3, and 10 μg ml(-1), dose-dependently inhibited both vascular endothelial growth factor (VEGF)- and basic fibroblast growth factor (bFGF)-induced proliferation, cell viability, migration, invasion and tube formation in HUVECs. L-5F inhibited VEGF- and bFGF-induced activation of their corresponding receptors, VEGFR2 and FGFR1, as well as downstream signaling pathways, including Akt and ERK1/2. MicroCT scanning and immunohistochemistry staining demonstrated that daily injection of L-5F (10 mg kg(-1)) decreased both the quantity and size of tumor vessels in mice. L-5F treated mice showed significantly reduced levels of VEGF in both tumor tissue and the circulation, which is consistent with in vitro data showing that L-5F inhibited production and secretion of VEGF from mouse and human ovarian cell lines in the absence and presence of exogenously added lysophosphatidic acid, a potent tumor promoter. In conclusion, our data that L-5F inhibits angiogenesis suggests that apoA-I mimetic peptides may serve as novel anti-angiogenesis agents for the treatment of angiogenesis-associated diseases, including cancer.

Optimization of MicroCT Imaging and Blood Vessel Diameter Quantitation of Preclinical Specimen Vasculature with Radiopaque Polymer Injection Medium

Vascular networks within a living organism are complex, multi-dimensional, and challenging to image capture. Radio-angiographic studies in live animals require a high level of infrastructure and technical investment in order to administer costly perfusion mediums whose signals metabolize and degrade relatively rapidly, diminishing within a few hours or days. Additionally, live animal specimens must not be subject to long duration scans, which can cause high levels of radiation exposure to the specimen, limiting the quality of images that can be captured. Lastly, despite technological advances in live-animal specimen imaging, it is quite difficult to minimize or prevent movement of a live animal, which can cause motion artifacts in the final data output. It is demonstrated here that through the use of postmortem perfusion protocols of radiopaque silicone polymer mediums and ex-vivo organ harvest, it is possible to acquire a high level of vascular signal in preclinical specimens through the use of micro-computed tomographic (microCT) imaging. Additionally, utilizing high-order rendering algorithms, it is possible to further derive vessel morphometrics for qualitative and quantitative analysis.

Epidemiology of Human Mycobacterium bovis Disease, California, USA, 2003–2011

Disease was associated with the Hispanic binational population and immunosuppressive conditions, including diabetes.

Small Animal Imaging and Examination by Micro-CT

Micro-computed tomography imaging technology allows for the whole-mount investigation of skeletal structures in preclinical specimens at both fetal and postnatal time points. The imaging process is nondestructive to the specimen and can be performed at various resolutions to derive the region of interest information most relevant to individual researchers. In addition to qualitative imaging of skeletal samples, accurate and desirable metrics such as bone mineral density (BMD), discrete cortical and trabecular bone analysis, and milligrams hydroxyapatite per unit volume (mgHA/cc) are also achievable depending upon scanner platform. Additional benefits to the method include digitally archivable files, in vivo and ex vivo scanning options, and volumetric or slice-thru presentation of data in standard histological or oblique orientations. Some drawbacks to the method include long scan times at higher spatial resolutions, large file sizes, and limitation to imaging of highly dense biological structures (i.e., bone), though several groups have attempted to expand the modality to include soft tissue imaging in ex vivo specimens.

High Rate of Treatment Completion in Program Settings With 12-Dose Weekly Isoniazid and Rifapentine for Latent Mycobacterium tuberculosis Infection

Background Randomized controlled trials have demonstrated that the newest latent tuberculosis (LTBI) regimen, 12 weekly doses of directly observed isoniazid and rifapentine (3HP), is as efficacious as 9 months of isoniazid, with a greater completion rate (82% vs 69%); however, 3HP has not been assessed in routine healthcare settings. Methods Observational cohort of LTBI patients receiving 3HP through 16 US programs was used to assess treatment completion, adverse drug reactions, and factors associated with treatment discontinuation. Results Of 3288 patients eligible to complete 3HP, 2867 (87.2%) completed treatment. Children aged 2-17 years had the highest completion rate (94.5% [155/164]). Patients reporting homelessness had a completion rate of 81.2% (147/181). In univariable analyses, discontinuation was lowest among children (relative risk [RR], 0.44 [95% confidence interval {CI}, .23-.85]; P = .014), and highest in persons aged ≥65 years (RR, 1.72 [95% CI, 1.25-2.35]; P < .001). In multivariable analyses, discontinuation was lowest among contacts of patients with tuberculosis (TB) disease (adjusted RR [ARR], 0.68 [95% CI, .52-.89]; P = .005) and students (ARR, 0.45 [95% CI, .21-.98]; P = .044), and highest with incarceration (ARR, 1.43 [95% CI, 1.08-1.89]; P = .013) and homelessness (ARR, 1.72 [95% CI, 1.25-2.39]; P = .001). Adverse drug reactions were reported by 1174 (35.7%) patients, of whom 891 (76.0%) completed treatment. Conclusions Completion of 3HP in routine healthcare settings was greater overall than rates reported from clinical trials, and greater than historically observed using other regimens among reportedly nonadherent populations. Widespread use of 3HP for LTBI treatment could accelerate elimination of TB disease in the United States.

Completion Rate and Side-Effect Profile of Three-Month Isoniazid and Rifapentine Treatment for Latent Tuberculosis Infection in an Urban County Jail

In an urban jail population, 3 months of isoniazid and rifapentine (3HP) was associated with an 85% latent tuberculosis infection treatment completion rate compared with 18% in a standard 9-month isoniazid treatment group. Among the 91 patients who started 3HP therapy, there were 2 treatment discontinuations from adverse drug reactions.

Clinical Impact on Tuberculosis Treatment Outcomes of Discordance Between Molecular and Growth-Based Assays for Rifampin Resistance, California 2003-2013.

Background. Data from international settings suggest that isolates of Mycobacterium tuberculosis with rpoB mutations testing phenotypically susceptible to rifampin (RIF) may have clinical significance. We analyzed treatment outcomes of California patients with discordant molecular-phenotypic RIF results. Methods. We included tuberculosis (TB) patients, during 2003–2013, whose specimens tested RIF susceptible phenotypically but had a rpoB mutation determined by pyrosequencing. Demographic data were abstracted from the California TB registry. Phenotypic drug-susceptibility testing, medical history, treatment, and outcomes were abstracted from medical records. Results. Of 3330 isolates tested, 413 specimens had a rpoB mutation (12.4%). Of these, 16 (3.9%) had molecular-phenotypic discordant RIF results. Seven mutations were identified: 511Pro, 516Phe, 526Asn, 526Ser (AGC and TCC), 526Cys, and 533Pro. Fourteen (88%) had isoniazid (INH) resistance, 6 of whom were also phenotypically resistant to ethambutol (EMB) and/or pyrazinamide (PZA). Five patients (25%), 1 with 511Pro and 4 with 526Asn, relapsed or failed treatment. The initial regimen for 3 patients was RIF, PZA, and EMB; 1 patient received RIF, PZA, EMB, and a fluoroquinolone (FQN); and 1 patient received RIF, EMB, FQN, and some second-line medications. Upon retreatment with an expanded regimen, 3 (75%) patients completed treatment, 1 patient moved before treatment completion, and 1 patient continues on treatment. The remaining 11 patients had a successful outcome with 9 having received a FQN and/or a rifamycin. Conclusions. Rifampin molecular-phenotypic discordance was rare, and most isolates had INH resistance. Patients who did not receive an expanded regimen had poor outcomes. These mutations may have clinical importance, and expanded treatment regimens should be considered.

Performance and comparison of self-reported STI symptoms among high-risk populations – MSM, sex workers, persons living with HIV/AIDS – in El Salvador

Summary Resource-limited countries have limited laboratory capability and rely on syndromic management to diagnose sexually transmitted infections (STIs). We aimed to estimate the sensitivity, specificity and positive predictive value (PPV) of STI syndromic management when used as a screening method within a study setting. Men who have sex with men (MSM), female sex workers (FSWs) and people living with HIV/AIDS (PLWHA) participated in a behavioural surveillance study. Data were obtained on demographics, sexual behaviours, STI history and service utilisation. Biological specimens were tested for genital inflammatory infections (Neisseria gonorrhoeae [GC], Chlamydia trachomatis [CT], Mycoplasma genitalium [MG], Trichomonas vaginalis [TV]) and genital ulcerative infection (syphilis and Herpes simplex virus-2). There was a high prevalence of Herpes simplex virus-2 (MSM 48.1%, FSW 82.0% and PLWHA 84.4%). Most participants reported no ulcerative symptoms and the majority of men reported no inflammatory symptoms. Sensitivity and PPV were poor for inflammatory infections among PLWHA and MSM. Sensitivity in FSWs for inflammatory infections was 75%. For ulcerative infections, sensitivity was poor, but specificity and PPV were high. Reliance on self-reported symptoms may not be an effective screening strategy for these populations. STI prevention studies should focus on symptom recognition and consider routine screening and referral for high-risk populations.