Publications by authors named "Rembert Pieper"

68 Publications

Differences in plasma proteomes for active tuberculosis, latent tuberculosis and non-tuberculosis mycobacterial lung disease patients with and without ESAT-6/CFP10 stimulation.

Proteome Sci 2020 Oct 31;18(1):10. Epub 2020 Oct 31.

J. Craig Venter Institute, Rockville, MD, USA.

Background: Tuberculosis (TB) is one of the world's most problematic infectious diseases. The pathogen Mycobacterium tuberculosis (Mtb) is contained by the immune system in people with latent TB infection (LTBI). No overt disease symptoms occur. The environmental and internal triggers leading to reactivation of TB are not well understood. Non-tuberculosis Mycobacteria (NTM) can also cause TB-like lung disease. Comparative analysis of blood plasma proteomes from subjects afflicted by these pathologies in an endemic setting may yield new differentiating biomarkers and insights into inflammatory and immunological responses to Mtb and NTM.

Methods: Blood samples from 40 human subjects in a pastoral region of Ethiopia were treated with the ESAT-6/CFP-10 antigen cocktail to stimulate anti-Mtb and anti-NTM immune responses. In addition to those of active TB, LTBI, and NTM cohorts, samples from matched healthy control (HC) subjects were available. Following the generation of sample pools, proteomes were analyzed via LC-MS/MS. These experiments were also performed without antigen stimulation steps. Statistically significant differences using the Z-score method were determined and interpreted in the context of the proteins' functions and their contributions to biological pathways.

Results: More than 200 proteins were identified from unstimulated and stimulated plasma samples (UPSs and SPSs, respectively). Thirty-four and 64 proteins were differentially abundant with statistical significance (P < 0.05; Benjamini-Hochberg correction with an FDR < 0.05) comparing UPS and SPS proteomic data of four groups, respectively. Bioinformatics analysis of such proteins via the Gene Ontology Resource was indicative of changes in cellular and metabolic processes, responses to stimuli, and biological regulations. The m7GpppN-mRNA hydrolase was increased in abundance in the LTBI group compared to HC subjects. Charged multivesicular body protein 4a and platelet factor-4 were increased in abundance in NTM as compared to HC and decreased in abundance in NTM as compared to active TB. C-reactive protein, α-1-acid glycoprotein 1, sialic acid-binding Ig-like lectin 16, and vitamin K-dependent protein S were also increased (P < 0.05; fold changes≥2) in SPSs and UPSs comparing active TB with LTBI and NTM cases. These three proteins, connected in a STRING functional network, contribute to the acute phase response and influence blood coagulation.

Conclusion: Plasma proteomes are different comparing LTBI, TB, NTM and HC cohorts. The changes are augmented following prior blood immune cell stimulation with the ESAT-6/CFP-10 antigen cocktail. The results encourage larger-cohort studies to identify specific biomarkers to diagnose NTM infection, LTBI, and to predict the risk of TB reactivation.
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http://dx.doi.org/10.1186/s12953-020-00165-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7603755PMC
October 2020

Molecular epidemiology of clinical Mycobacterium tuberculosis complex isolates in South Omo, Southern Ethiopia.

BMC Infect Dis 2020 Oct 13;20(1):750. Epub 2020 Oct 13.

Aklilu Lemma Institute of Pathobiology, Addis Ababa University, PO. Box 1176, Addis Ababa, Ethiopia.

Background: Tuberculosis (TB) is caused by Mycobacterium tuberculosis complex (MTBC). Mapping the genetic diversity of MTBC in high TB burden country like Ethiopia is important to understand principles of the disease transmission and to strengthen the regional TB control program. The aim of this study was to investigate the genetic diversity of Mycobacterium tuberculosis complex (MTBC) isolates circulating in the South Omo, southern Ethiopia.

Methods: MTBC isolates (N = 156) were genetically analyzed using spacer oligotyping (spoligotyping) and mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) typing. Major lineages and lineages were identified using MTBC databases. Logistic regression was used to correlate patient characteristics with strain clustering.

Results: The study identified Euro-American (EA), East-African-Indian (EAI), Indo-Oceanic (IO), Lineage_7/Aethiops vertus, Mycobacterium bovis and Mycobacterium africanum major lineages in proportions of 67.3% (105/156), 22.4% (35/156), 6.4% (10/156), 1.9% (3/156), 1.3% (2/156) and 0.6% (1/156), respectively. Lineages identified were Delhi/CAS 23.9% (37/155), Ethiopia_2 20.6% (32/155), Haarlem 14.2% (22/155), URAL 14.2%(22/155), Ethiopia_3 8.4% (13/155), TUR 6.5% (10/155), Lineage_7/Aethiops vertus 1.9% (3/155), Bovis 1.3% (2/155), LAM 1.3% (2/155), EAI 0.6% (1/155), X 0.6% (1/155) and Ethiopia HRv-like strain 0.6% (1/155). Of the genotyped isolates 5.8% (9/155) remained unassigned. The recent transmission index (RTI) was 3.9%. Orphan strains compared to shared types (AOR: 0.09, 95% CI: 0.04-0.25) were associated with reduced odds of clustering. The dominant TB lineage in pastoral areas was EAI and in non-pastoral areas was EA.

Conclusion: The epidemiological data, highly diverse MTBC strains and a low RTI in South Omo, provide information contributing to the TB Control Program of the country.
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http://dx.doi.org/10.1186/s12879-020-05394-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7557052PMC
October 2020

Protein signatures from blood plasma and urine suggest changes in vascular function and IL-12 signaling in elderly with a history of chronic diseases compared with an age-matched healthy cohort.

Geroscience 2020 Sep 24. Epub 2020 Sep 24.

J. Craig Venter Institute, 9605 Medical Center Drive, Rockville, MD, 20850, USA.

Key processes characterizing human aging are immunosenescence and inflammaging. The capacity of the immune system to adequately respond to external perturbations (e.g., pathogens, injuries, and biochemical irritants) and to repair somatic mutations that may cause cancers or cellular senescence declines. An important goal remains to identify genetic or biochemical, predictive biomarkers for healthy aging. We recruited two cohorts in the age range 70 to 82, one afflicted by chronic illnesses (non-healthy aging, NHA) and the other in good health (healthy aging, HA). NHA criteria included major cardiovascular, neurodegenerative, and chronic pulmonary diseases, diabetes, and cancers. Quantitative analysis of forty proinflammatory cytokines in blood plasma and more than 500 proteins in urine was performed to identify candidate biomarkers for and biological pathway implications of healthy aging. Nine cytokines revealed lower quantities in blood plasma for the NHA compared with the HA groups (fold change > 1.5; p value < 0.025) including IL-12p40 and IL-12p70. We note that, sampling at two timepoints, intra-individual cytokine abundance patterns clustered in 86% of all 60 cases, indicative of person-specific, highly controlled multi-cytokine signatures in blood plasma. Twenty-three urinary proteins were differentially abundant (HA versus NHA; fold change > 1.5; p value < 0.01). Among the proteins increased in abundance in the HA cohort were glycoprotein MUC18, ephrin type-B receptor 4, matrix remodeling-associated protein 8, angiopoietin-related protein 2, K-cadherin, and plasma protease C1 inhibitor. These proteins have been linked to the extracellular matrix, cell adhesion, and vascular remodeling and repair processes. In silico network analysis identified the regulation of coagulation, antimicrobial humoral immune responses, and the IL-12 signaling pathway as enriched GO terms. To validate links of these preliminary biomarkers and IL-12 signaling with healthy aging, clinical studies using larger cohorts and functional characterization of the genes/proteins in cellular models of aging need to be conducted.
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http://dx.doi.org/10.1007/s11357-020-00269-yDOI Listing
September 2020

Gut Microbial Changes in Diabetic db/db Mice and Recovery of Microbial Diversity upon Pirfenidone Treatment.

Microorganisms 2020 Sep 3;8(9). Epub 2020 Sep 3.

J. Craig Venter Institute, 9605 Medical Center Drive, Suite 150, Rockville, MD 20850, USA.

The leptin receptor-deficient db/db mouse model is an accepted in vivo model to study obesity, type 2 diabetes, and diabetic kidney disease. Healthy gastrointestinal (GI) microbiota has been linked to weight loss, improved glycemic control, and physiological benefits. We investigated the effect of various drugs on the GI microbiota of db/db mice as compared to control db/m mice. Treatment with long-acting pirfenidone (PFD) increased gut microbial diversity in diabetic db/db mice. Firmicutes, the most abundant phylum in db/m mice, decreased significantly in abundance in db/db mice but showed increased abundance with long-acting PFD treatment. Several bacterial taxa, including and some , were less abundant in db/db mice and more abundant in long-acting-PFD-treated db/db mice. Long-acting PFD treatment reduced the abundance of (5%) as compared to db/db mice (~15%). We conclude that gut microbial dysbiosis observed in db/db mice was partially reversed by long-acting PFD treatment and hypothesize that PFD has beneficial effects, in part, via its influence on the gut microbial metabolite profile. In quantitatively assessing urine metabolites, we observed a high abundance of diabetic ketoacidosis biomarkers, including 3-hydroxybutyric acid and acetoacetic acid in db/db mice, which were less abundant in the long-acting-PFD-treated db/db mice.
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http://dx.doi.org/10.3390/microorganisms8091347DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564638PMC
September 2020

Persistent Gut Microbial Dysbiosis in Children with Acute Lymphoblastic Leukemia (ALL) During Chemotherapy.

Microb Ecol 2020 May 21;79(4):1034-1043. Epub 2019 Nov 21.

J. Craig Venter Institute (JCVI), Rockville, MD, USA.

Prophylactic or therapeutic antibiotic use along with chemotherapy treatment potentially has a long-standing adverse effect on the resident gut microbiota. We have established a case-control cohort of 32 pediatric and adolescent acute lymphoblastic leukemia (ALL) patients and 25 healthy siblings (sibling controls) to assess the effect of chemotherapy as well as antibiotic prophylaxis on the gut microbiota. We observe that the microbiota diversity and richness of the ALL group is significantly lower than that of the control group at diagnosis and during chemotherapy. The microbiota diversity is even lower in antibiotics-exposed ALL patients. Although the gut microbial diversity tends to stabilize after 1-year post-chemotherapy, their abundances were altered because of chemotherapy and prophylactic antibiotic treatments. Specifically, the abundances of mucolytic gram-positive anaerobic bacteria, including Ruminococcus gnavus and Ruminococcus torques, tended to increase during the chemotherapy regimen and continued to be elevated 1 year beyond the initiation of chemotherapy. This dysbiosis may contribute to the development of gastrointestinal complications in ALL children following chemotherapy. These findings set the stage to further understand the role of the gut microbiome dynamics in ALL patients and their potential role in alleviating some of the adverse side effects of chemotherapy and antibiotics use in immunocompromised children.
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http://dx.doi.org/10.1007/s00248-019-01448-xDOI Listing
May 2020

Gastro-intestinal and oral microbiome signatures associated with healthy aging.

Geroscience 2019 12 16;41(6):907-921. Epub 2019 Oct 16.

J. Craig Venter Institute, 9605 Medical Center Drive, Rockville, MD, 20850, USA.

The human oral and gut microbiomes influence health via competition for a distinct niche in the body with pathogens, via metabolic capabilities that increase host digestive capacity and generate compounds engaged in signaling pathways and modulation of immune system functions. Old age alters our metabolic and regenerative capacity. Following recruitment of 65 human subjects in the age range of 70 to 82, we discerned healthy aging (HA) and non-healthy aging (NHA) cohorts discordant in the occurrence of one or more major diseases: (1) cancer, (2) acute or chronic cardiovascular diseases, (3) acute or chronic pulmonary diseases, (4) diabetes, and (5) stroke or neurodegenerative disorders. We analyzed these cohorts' oral microbiomes (saliva) and gut microbiomes (stool) to assess diversity and identify microbial biomarkers for HA. In contrast to the gut microbiome where no change was observed, we found that the saliva microbiome had higher α-diversity in the HA compared with the NHA group. We observed the genus Akkermansia to be significantly more abundant in the gut microbiota of the HA group. Akkermansia muciniphila is a colonic mucin-degrading bacterium believed to have beneficial effects on gastrointestinal health, particularly in the context of diabetes and obesity. Erysipelotrichaceae UCG-003 was a taxon increased in abundance in the HA cohort. Streptococcus was the only genus observed to be significantly decreased in abundance in both the gut and oral microbiomes of the HA cohort compared with the NHA cohort. Our data support the notion that these microbes are potential probiotics to decrease the risks of non-healthy aging.
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http://dx.doi.org/10.1007/s11357-019-00098-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925087PMC
December 2019

and Persist in Polymicrobial Urethral Catheter Biofilms Examined in Longitudinal Profiles at the Proteomic Level.

Biochem Insights 2019 19;12:1178626419875089. Epub 2019 Sep 19.

J. Craig Venter Institute, Rockville, MD, USA.

() and () are gram-positive bacteria belonging to the family Aerococcaceae and colonize the human immunocompromised and catheterized urinary tract. We identified both pathogens in polymicrobial urethral catheter biofilms (CBs) with a combination of 16S rDNA sequencing, proteomic analyses, and microbial cultures. Longitudinal sampling of biofilms from serially replaced catheters revealed that each species persisted in the urinary tract of a patient in cohabitation with 1 or more gram-negative uropathogens. The and proteomes revealed active glycolytic, heterolactic fermentation, and peptide catabolic energy metabolism pathways in an anaerobic milieu. A few phosphotransferase system (PTS)-based sugar uptake and oligopeptide ABC transport systems were highly expressed, indicating adaptations to the supply of nutrients in urine and from exfoliating squamous epithelial and urothelial cells. Differences in the vs metabolisms pertained to citrate lyase and utilization and storage of glycogen (evident only in proteomes) and to the enzyme Xfp that degrades d-xylulose-5'-phosphate and the biosynthetic pathways for 2 protein cofactors, pyridoxal 6'-phosphate and 4'-phosphopantothenate (expressed only in proteomes). A predicted ZnuA-like transition metal ion uptake system was identified for while expressed 2 LPXTG-anchored surface proteins, one of which had a predicted pilin D adhesion motif. While these proteins may contribute to fitness and virulence in the human host, it cannot be ruled out that and fill a niche in polymicrobial biofilms without being the direct cause of injury in urothelial tissues.
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http://dx.doi.org/10.1177/1178626419875089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753514PMC
September 2019

Using Proteomics to Identify Inflammation During Urinary Tract Infection.

Methods Mol Biol 2019 ;2021:259-272

The J. Craig Venter Institute, Medical Center Drive, Rockville, MD, USA.

Urinary tract infections (UTIs) are one of the most common bacterial infections. Conventional approaches to diagnose these infections rely on microbial urine culture, urine sediment microscopy and basic molecular urinalysis tests, in combination with assessments of patient symptoms that are indicative of UTI. The last decade has seen a more widespread clinical use of standardized MALDI-TOF methods to identify UTI-causing microbial agents. Shotgun proteomics methods to determine the extent of inflammation and types of immune cell effectors in urine have not become part of routine clinical tests. However, such methods are useful to investigate UTI pathogenesis, identify difficult-to-culture pathogens and understand antimicrobial effector mechanisms. The present chapter describes these approaches in order to gain quantitative and qualitative insights into inflammation and immune responses in patients with UTI and simultaneously profile the causative agents. The methods are also applicable to examine catheter-associated UTIs and vaginal infections from urine samples. Protocols provided here pertain to direct analyses of clinical specimens including urine sediments and urethral catheter biofilms.
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http://dx.doi.org/10.1007/978-1-4939-9601-8_22DOI Listing
March 2020

Detection of Neutrophil Extracellular Traps in Urine.

Methods Mol Biol 2019 ;2021:241-257

The J. Craig Venter Institute, Rockville, MD, USA.

Neutrophils are important mediators of the antimicrobial defense during urinary tract infections (UTIs). When activated at the site of infection, these innate immune cells phagocytose and neutralize an invading pathogen. Another neutrophil defense strategy is the release of effectors, such as antimicrobial peptides and proteins stored in neutrophil granules and reactive oxygen species. Their release can be facilitated by cellular signals that trigger chromatic decondensation and the disruption of nuclear membranes, followed by granule and plasma membrane disintegration, DNA release into the extracellular milieu, and neutrophil cell death. Neutrophil extracellular traps (NETs) form. If microbial pathogens are the cause of neutrophil infiltration, they are entrapped in the network of DNA fibers that characterize NETs and are exposed to antimicrobial granule effectors and histones that bind to the extracellular DNA fibers. Here, we describe nonmicroscopic methods applied to clinical (urine sediment) samples to identify and characterize NETs associated with UTI. A stepwise extraction procedure using PBS, deoxyribonuclease I digestion and SDS-based solubilization is described. This is followed by native gel analysis to visualize protein-DNA macromolecular assemblies and proteomic analysis to identify signature proteins and their quantities in NETs. Microbes observed to be entrapped in NETs in the process of the innate immune response to the infection are Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Staphylococcus aureus, and Enterococcus faecalis.
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http://dx.doi.org/10.1007/978-1-4939-9601-8_21DOI Listing
March 2020

Proteome Profiled in Polymicrobial Urethral Catheter Biofilms.

Proteomes 2018 Dec 9;6(4). Epub 2018 Dec 9.

J. Craig Venter Institute, 9605 Medical Center Drive, Rockville, MD 20850, USA.

, a Gram-positive anaerobic coccoid rod colonizing the human urinary tract, belongs to the taxonomic class of Actinobacteria. We identified as a cohabitant of urethral catheter biofilms (CB). The CBs also harbored more common uropathogens, such as and , supporting the notion that is adapted to a life style in polymicrobial biofilms. We isolated a clinical strain from a blood agar colony and used 16S rRNA gene sequencing and shotgun proteomics to confirm its identity as . We characterized this species by quantitatively comparing the bacterial proteome derived from in vitro growth with that of four clinical samples. The functional relevance of proteins with emphasis on nutrient import and the response to hostile host conditions, showing evidence of neutrophil infiltration, was analyzed. Two putative subtilisin-like proteases and a heme/oligopeptide transporter were abundant in vivo and are likely important for survival and fitness in the biofilm. Proteins facilitating uptake of xylose/glucuronate and oligopeptides, also highly expressed in vivo, may feed metabolites into mixed acid fermentation and peptidolysis pathways, respectively, to generate energy. A polyketide synthase predicted to generate a secondary metabolite that interacts with either the human host or co-colonizing microbes was also identified. The product of the PKS enzyme may contribute to fitness and persistence in the CBs.
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http://dx.doi.org/10.3390/proteomes6040052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314084PMC
December 2018

Phenotypic and genotypic drug sensitivity of complex isolated from South Omo Zone, Southern Ethiopia.

Infect Drug Resist 2018 25;11:1581-1589. Epub 2018 Sep 25.

Aklilu Lemma Institute of Pathobiology, Addis Ababa University, Addis Ababa, Ethiopia,

Background: Knowledge of drug-sensitivity patterns of complex (MTBC) strains isolated from patients is an important aspect of TB control strategy. This study was conducted to evaluate the drug sensitivity of MTBC isolates in South Omo, southern Ethiopia.

Materials And Methods: A total of 161 MTBC isolates (153 from new cases and eight re-treatment TB cases) were isolated using Lowenstein Jensen medium of which 126 isolates were able to be tested for drug sensitivity by BACTEC™MGIT™ 960 system, while all the 161 isolates were tested by GenoType MTBDR VER 2.0. Descriptive statistics and logistic regression were used to express and present results.

Results: On the basis of MGIT 960 system, the prevalence of mono-resistance was 9.2% (11/119) in the new cases, although neither poly-resistance nor multidrug resistance (MDR) was recorded in these cases. On the basis of GenoType MTBDR assay, two of the 153 isolates (1.3%) of the new cases were mono-resistant for rifampicin (RIF) and one of these isolates had known gene mutation (H526D). One of the eight (12.5%) isolates obtained from the re-treatment cases was MDR with gene mutation (D516V) and gene mutation (S315T2). Taking MGIT 960 system as a gold standard, the sensitivities of the MTBDR assay were 33.3%, 100% and 100% for detection of resistance to isoniazid, RIF and MDR, respectively. On the other hand, its specificities were 99.2%, 100% and 100% for detection of resistance to RIF, isoniazid and MDR, respectively.

Conclusion: The magnitude of drug resistance was relatively low in the new TB cases of South Omo as compared to the reports from the other regions of the country. This is encouraging and hence the TB Control Program in the Zone should strengthen its program so that the emergence of drug resistance is inhibited.
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http://dx.doi.org/10.2147/IDR.S165088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6161742PMC
September 2018

ProteoStorm: An Ultrafast Metaproteomics Database Search Framework.

Cell Syst 2018 10 26;7(4):463-467.e6. Epub 2018 Sep 26.

Department of Computer Science and Engineering, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address:

Shotgun metaproteomics has the potential to reveal the functional landscape of microbial communities but lacks appropriate methods for complex samples with unknown compositions. In the absence of prior taxonomic information, tandem mass spectra would be searched against large pan-microbial databases, which requires heavy computational workload and reduces sensitivity. We present ProteoStorm, an efficient database search framework for large-scale metaproteomics studies, which identifies high-confidence peptide-spectrum matches (PSMs) while achieving a two-to-three orders-of-magnitude speedup over popular tools. A reanalysis of a urinary tract infection (UTI) dataset of 110 individuals revealed a complex pattern of polymicrobial expression, including sub-types of UTIs, cases of bacterial vaginosis, and evidence of no underlying disease. Importantly, compared to the initial UTI study that restricted the search database to a manually curated list of 20 genera, ProteoStorm identified additional genera that were previously unreported, including a case of infection with the rare pathogen Propionimicrobium.
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http://dx.doi.org/10.1016/j.cels.2018.08.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6231400PMC
October 2018

S-Trap, an Ultrafast Sample-Preparation Approach for Shotgun Proteomics.

J Proteome Res 2018 09 28;17(9):2917-2924. Epub 2018 Aug 28.

J. Craig Venter Institute , 9605 Medical Center Drive , Rockville , Maryland 20850 , United States.

The success of shotgun proteomic analysis depends largely on how samples are prepared. Current approaches (such as those that are gel-, solution-, or filter-based), although being extensively employed in the field, are time-consuming and less effective with respect to the repetitive sample processing, recovery, and overall yield. As an alternative, the suspension trapping (S-Trap) filter has been commercially available very recently in the format of a single or 96-well filter plate. In contrast to the conventional filter-aided sample preparation (FASP) approach, which utilizes a molecular weight cut-off (MWCO) membrane as the filter and requires hours of processing before digestion-ready proteins can be obtained, the S-Trap employs a three-dimensional porous material as filter media and traps particulate protein suspensions with the subsequent depletion of interfering substances and in-filter digestion. Due to the large (submicron) pore size, each centrifugation cycle of the S-Trap filter only takes 1 min, which significantly reduces the total processing time from approximately 3 h by FASP to less than 15 min, suggesting an ultrafast sample-preparation approach for shotgun proteomics. Here, we comprehensively evaluate the performance of the individual S-Trap filter and 96-well filter plate in the context of global protein identification and quantitation using whole-cell lysate and clinically relevant sputum samples.
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http://dx.doi.org/10.1021/acs.jproteome.8b00505DOI Listing
September 2018

Microbial metagenome of urinary tract infection.

Sci Rep 2018 03 12;8(1):4333. Epub 2018 Mar 12.

J. Craig Venter Institute, La Jolla, CA, 92037, USA.

Urine culture and microscopy techniques are used to profile the bacterial species present in urinary tract infections. To gain insight into the urinary flora, we analyzed clinical laboratory features and the microbial metagenome of 121 clean-catch urine samples. 16S rDNA gene signatures were successfully obtained for 116 participants, while metagenome sequencing data was successfully generated for samples from 49 participants. Although 16S rDNA sequencing was more sensitive, metagenome sequencing allowed for a more comprehensive and unbiased representation of the microbial flora, including eukarya and viral pathogens, and of bacterial virulence factors. Urine samples positive by metagenome sequencing contained a plethora of bacterial (median 41 genera/sample), eukarya (median 2 species/sample) and viral sequences (median 3 viruses/sample). Genomic analyses suggested cases of infection with potential pathogens that are often missed during routine urine culture due to species specific growth requirements. While conventional microbiological methods are inadequate to identify a large diversity of microbial species that are present in urine, genomic approaches appear to more comprehensively and quantitatively describe the urinary microbiome.
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http://dx.doi.org/10.1038/s41598-018-22660-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5847550PMC
March 2018

Latent tuberculosis infection and associated risk indicators in pastoral communities in southern Ethiopia: a community based cross-sectional study.

BMC Public Health 2018 02 17;18(1):266. Epub 2018 Feb 17.

Aklilu Lemma Institute of Pathobiology (ALIPB), Addis Ababa University, P O Box 1176, Addis Ababa, Ethiopia.

Background: Research pertaining to the community-based prevalence of latent tuberculosis infection (LTBI) is important to understand the magnitude of this infection. This study was conducted to estimate LTBI prevalence and to identify associated risk factors in the Omo Zone of Southern Ethiopia.

Methods: A community-based cross-sectional study was conducted in six South Omo districts from May 2015 to February 2016. The sample size was allocated to the study districts proportional to their population sizes. Participants were selected using a multi-stage sampling approach. A total of 497 adult pastoralists were recruited. Blood samples were collected from the study participants and screened for LTBI using a U.S. Food and Drug Administration approved interferon-gamma release assay (IGRA). Logistic regression was used to model the likelihood of LTBI occurrence and to identify risk factors associated with LTBI.

Results: The prevalence of LTBI was 50.5% (95% CI: 46%, 55%) with no significant gender difference (49.8% among males versus 51.2% among females; Chi-square (χ) = 0.10; P = 0.41) and marginally non-significant increasing trends with age (44.6% among those below 24 years and 59.7% in the age range of 45-64 years; χ = 6.91; P = 0.075). Being residence of the Dasanech District (adjusted odds ratio, AOR = 2.62, 95% CI: 1.30, 5.28; P = 0.007) and having a habit of eating raw meat (AOR = 2.89, 95% CI: 1.09, 7.66; P = 0.033) were significantly associated with an increased odds of being positive for LTBI. A large family size (size of 5 to 10) has significant protective effect against associated a reduced odds of being positive for LTBI compared to a family size of below 5 (AOR = 0.65, 95% CI: 0.42, 0.99; P = 0.045).

Conclusions: A high prevalence of LTBI in the South Omo Zone raises the concern that elimination of TB in the pastoral communities of the region might be difficult. Screening for and testing individuals infected with TB, independent of symptoms, may be an effective way to minimize the risk of disease spread.
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http://dx.doi.org/10.1186/s12889-018-5149-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5816385PMC
February 2018

Potential Immunological Biomarkers for Detection of Mycobacterium tuberculosis Infection in a Setting Where M. tuberculosis Is Endemic, Ethiopia.

Infect Immun 2018 04 22;86(4). Epub 2018 Mar 22.

Aklilu Lemma Institute of Pathobiology, Addis Ababa University, Addis Ababa, Ethiopia.

Accurate diagnosis and early treatment of tuberculosis (TB) and latent TB infection (LTBI) are vital to prevent and control TB. The lack of specific biomarkers hinders these efforts. This study's purpose was to screen immunological markers that discriminate infection outcomes in a setting where it is endemic, Ethiopia. Whole blood from 90 participants was stimulated using the ESAT-6/CFP-10 antigen cocktail. The interferon gamma (IFN-γ)-based QuantiFERON diagnostic test was used to distinguish between LTBI and uninfected control cases. Forty cytokines/chemokines were detected from antigen-stimulated plasma supernatants (SPSs) and unstimulated plasma samples (UPSs) using human cytokine/chemokine antibody microarrays. Statistical tests allowed us to identify potential biomarkers that distinguish the TB, LTBI, and healthy control groups. As expected, the levels of IFN-γ in SPSs returned a high area under the receiver operating characteristic curve (AUC) value comparing healthy controls and LTBI cases (Z = 0.911; < 0.001). The SPS data also indicated that interleukin 17 (IL-17) abundance discriminates LTBI from healthy controls (Z = 0.763; = 0.001). RANTES and MIP-1β were significantly elevated in SPSs of TB-infected compared to healthy controls ( < 0.05), while IL-12p40 and soluble tumor necrosis factor receptor II (sTNF-RII) were significantly increased in active TB cases compared to the combined LTBI and control groups ( < 0.05). Interestingly, quantitative changes for RANTES were observed using both SPSs and UPSs, with values of 0.013 and 0.012, respectively, in active TB versus LTBI cases and 0.001 and 0.002, respectively, in active TB versus healthy controls. These results encourage biomarker verification studies for IL-17 and RANTES. Combinations of these cytokines may complement IFN-γ measurements to diagnose LTBI and distinguish active TB from LTBI cases.
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http://dx.doi.org/10.1128/IAI.00759-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5865051PMC
April 2018

Mechanisms of action of Coxiella burnetii effectors inferred from host-pathogen protein interactions.

PLoS One 2017 27;12(11):e0188071. Epub 2017 Nov 27.

Department of Defense Biotechnology High Performance Computing Software Applications Institute, Telemedicine and Advanced Technology Research Center, U.S. Army Medical Research and Materiel Command, Fort Detrick, Maryland, United States of America.

Coxiella burnetii is an obligate Gram-negative intracellular pathogen and the etiological agent of Q fever. Successful infection requires a functional Type IV secretion system, which translocates more than 100 effector proteins into the host cytosol to establish the infection, restructure the intracellular host environment, and create a parasitophorous vacuole where the replicating bacteria reside. We used yeast two-hybrid (Y2H) screening of 33 selected C. burnetii effectors against whole genome human and murine proteome libraries to generate a map of potential host-pathogen protein-protein interactions (PPIs). We detected 273 unique interactions between 20 pathogen and 247 human proteins, and 157 between 17 pathogen and 137 murine proteins. We used orthology to combine the data and create a single host-pathogen interaction network containing 415 unique interactions between 25 C. burnetii and 363 human proteins. We further performed complementary pairwise Y2H testing of 43 out of 91 C. burnetii-human interactions involving five pathogen proteins. We used the combined data to 1) perform enrichment analyses of target host cellular processes and pathways, 2) examine effectors with known infection phenotypes, and 3) infer potential mechanisms of action for four effectors with uncharacterized functions. The host-pathogen interaction profiles supported known Coxiella phenotypes, such as adapting cell morphology through cytoskeletal re-arrangements, protein processing and trafficking, organelle generation, cholesterol processing, innate immune modulation, and interactions with the ubiquitin and proteasome pathways. The generated dataset of PPIs-the largest collection of unbiased Coxiella host-pathogen interactions to date-represents a rich source of information with respect to secreted pathogen effector proteins and their interactions with human host proteins.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0188071PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5703456PMC
December 2017

Type 1 Diabetes: Urinary Proteomics and Protein Network Analysis Support Perturbation of Lysosomal Function.

Theranostics 2017 7;7(10):2704-2717. Epub 2017 Jul 7.

J. Craig Venter Institute, 9714 Medical Center Drive, Rockville, MD 20850.

While insulin replacement therapy restores the health and prevents the onset of diabetic complications (DC) for many decades, some T1D patients have elevated hemoglobin A1c values suggesting poor glycemic control, a risk factor of DC. We surveyed the stool microbiome and urinary proteome of a cohort of 220 adolescents and children, half of which had lived with T1D for an average of 7 years and half of which were healthy siblings. Phylogenetic analysis of the 16S rRNA gene did not reveal significant differences in gut microbial alpha-diversity comparing the two cohorts. The urinary proteome of T1D patients revealed increased abundances of several lysosomal proteins that correlated with elevated HbA1c values. protein network analysis linked such proteins to extracellular matrix components and the glycoprotein LRG1. LRG1 is a prominent inflammation and neovascularization biomarker. We hypothesize that these changes implicate aberrant glycation of macromolecules that alter lysosomal function and metabolism in renal tubular epithelial cells, cells that line part of the upper urinary tract.
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http://dx.doi.org/10.7150/thno.19679DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5558563PMC
April 2018

Nontuberculosis mycobacteria are the major causes of tuberculosis like lesions in cattle slaughtered at Bahir Dar Abattoir, northwestern Ethiopia.

BMC Vet Res 2017 Aug 15;13(1):237. Epub 2017 Aug 15.

Aklilu Lemma Institute of Pathobiology, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia.

Background: The main cause of bovine tuberculosis (bTB) is believed to be Mycobacterium bovis (M. bovis). Nontuberculosis mycobacteria (NTM) are neglected but opportunistic pathogens and obstacles for bTB diagnosis. This study aimed to isolate and characterize the mycobacteria organisms involved in causing TB-like lesions in cattle in northwestern Ethiopia.

Results: A total of 2846 carcasses of cattle were inspected for TB lesions. Ninety six tissues (including lymph nodes such as submandibular, retropharyngeal, tonsilar, mediatinal, bronchial and mesenteric, and organs such as lung, liver and kidney) with suspicious TB lesion(s) were collected and cultured on Lowenstein-Jensen medium. Twenty one showed culture growth, of which only 17 were identified containing acid fast bacilli (AFB) by Ziehl-Neelsen staining. Among the 17 AFB isolates 15 generated a polymerase chain reaction product of 1030 bp by gel electrophoresis based on the 16S ribosomal RNA gene amplification. No M. tuberculosis complex species were isolated. Further characterization by Genotype Mycobacterium CM assay showed 6 isolates identified as M. peregrinum. Eight isolates represented by mixed species, which includes M. fortuitum-peregrinum (3 isolates), M. gordonae-peregrinum (3 isolates) and M. fortuitum-gordonae-peregrinum (2 isolates). One NTM could not be interpreted.

Conclusion: A significant number of NTM species were isolated from TB-like lesions of grazing cattle slaughtered at Bahir Dar Abattoir. Such finding could suggest the role of NTM in causing lesions in cattle. Further investigations are recommended on the pathogenesis of the reported NTM species in cattle, and if they have public health significance.
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http://dx.doi.org/10.1186/s12917-017-1168-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5558754PMC
August 2017

Quick 96FASP for high throughput quantitative proteome analysis.

J Proteomics 2017 08 29;166:1-7. Epub 2017 Jun 29.

J. Craig Venter Institute, 9714 Medical Center Drive, Rockville, MD 20850, United States.

Filter aided sample preparation (FASP) is becoming a central method for proteomic sample cleanup and peptide generation prior to LC-MS analysis. We previously adapted this method to a 96-well filter plate, and applied to prepare protein digests from cell lysate and body fluid samples in a high throughput quantitative manner. While the 96FASP approach is scalable and can handle multiple samples simultaneously, two key advantages compared to single FASP, it is also time-consuming. The centrifugation-based liquid transfer on the filter plate takes 3-5 times longer than single filter. To address this limitation, we now present a quick 96FASP (named q96FASP) approach that, relying on the use of filter membranes with a large MWCO size (~30kDa), significantly reduces centrifugal times. We show that q96FASP allows the generation of protein digests derived from whole cell lysates and body fluids in a quality similar to that of the single FASP method. Processing a sample in multiple wells in parallel, we observed excellent experimental repeatability by label-free quantitation approach. We conclude that the q96FASP approach promises to be a promising cost- and time-effective method for shotgun proteomics and will be particularly useful in large scale biomarker discovery studies.

Significance: High throughput sample processing is of particular interests for quantitative proteomics. The previously developed 96FASP is high throughput and appealing, however it is time-consuming in the context of centrifugation-based liquid transfer (~1.5h per spin). This study presents a truly high throughput sample preparation method based on large cut-off 96-well filter plate, which shortens the spin time to ~20min. To our knowledge, this is the first multi-well method that is entirely comparable with conventional FASP. This study thoroughly examined two types of filter plates and performed side-by-side comparisons with single FASP. Two types of samples, whole cell lysate of a UTI (urinary tract infection)-associated Klebsiella pneumoniae cell and human urine, were tested which demonstrated its capability for quantitative proteomics. The q96FSAP approach makes the filter plate-based approach more appealing for protein biomarker discovery projects, and could be broadly applied to large scale proteomics analysis.
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http://dx.doi.org/10.1016/j.jprot.2017.06.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6029622PMC
August 2017

Molecular typing of Mycobacterium tuberculosis complex isolated from pulmonary tuberculosis patients in central Ethiopia.

BMC Infect Dis 2017 03 1;17(1):184. Epub 2017 Mar 1.

Aklilu Lemma Institute of Pathobiology, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia.

Background: Identification of the types of strains of Mycobacterium tuberculosis (M. tuberculosis) complex causing tuberculosis (TB) could contribute to TB control program of specific geographic region as well as it could add knowledge onto the existing literature on TB worldwide. The objective of the present study was to identify the species and strains of M. tuberculosis complex causing pulmonary tuberculosis in central Ethiopia.

Methods: A health institution- based cross-sectional study was conducted on 338 smear positive TB cases visiting three hospitals between October 2012 and September 2013. Morning and spot sputum samples were collected before the starting of treatment regimens. Thus, a total of 338 pooled sputum samples collected from these cases. Samples were cultured on Löwenstein Jensen media and the isolates were identified by the region of difference (RD) 9 based polymerase chain reaction (PCR) and spoligotyping.

Result: Of the total isolates 98.6% of the isolates were identified to be M. tuberculosis while the remaining 1.4% were identified as M. africanum. Further, typing of M. tuberculosis using spoligotyping lead to the identification of 90 different strains of M. tuberculosis. Of these strains, 32 were clustered consisting of more than one isolate while the remaining 58 strains were unique consisting of single isolate. Thus, 79.3% (223/281) of the isolates were found in the clustered while only 20.6% (58/281) of the strains were unique. Forty-five of the spolgotyping patterns were registeredin the SITVIT2 or SpolDB4 database in while the remaining 45 were notfound in the database and hence were orphan strains. The dominant strains were SIT53, SIT149, and SIT54, consisting of 43, 37 and 34 isolates, respectively. Classification of the spoligotype patterns using TB-insight RUN TB-Lineage showed that 86.8, 6.4, 5, 1.4% ofthe isolatesbelonged to the Euro-American lineage, East-African-Indian, Indo-oceanic and M. africanum, respectively.

Conclusion: The identification of clustered and new strains using spolygotyping in present study does not give conclusive finding as spoligotyping has low discriminatory power. Thus, further identification of these isolates using mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VENTR) and or whole genome sequencing (WGS) recommended.
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http://dx.doi.org/10.1186/s12879-017-2267-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5333391PMC
March 2017

Characterization of Early-Phase Neutrophil Extracellular Traps in Urinary Tract Infections.

PLoS Pathog 2017 01 27;13(1):e1006151. Epub 2017 Jan 27.

The J. Craig Venter Institute, Rockville, MD, United States of America.

Neutrophils have an important role in the antimicrobial defense and resolution of urinary tract infections (UTIs). Our research suggests that a mechanism known as neutrophil extracellular trap (NET) formation is a defense strategy to combat pathogens that have invaded the urinary tract. A set of human urine specimens with very high neutrophil counts had microscopic evidence of cellular aggregation and lysis. Deoxyribonuclease I (DNase) treatment resulted in disaggregation of such structures, release of DNA fragments and a proteome enriched in histones and azurophilic granule effectors whose quantitative composition was similar to that of previously described in vitro-formed NETs. The effector proteins were further enriched in DNA-protein complexes isolated in native PAGE gels. Immunofluorescence microscopy revealed a flattened morphology of neutrophils associated with decondensed chromatin, remnants of granules in the cell periphery, and myeloperoxidase co-localized with extracellular DNA, features consistent with early-phase NETs. Nuclear staining revealed that a considerable fraction of bacterial cells in these structures were dead. The proteomes of two pathogens, Staphylococcus aureus and Escherichia coli, were indicative of adaptive responses to early-phase NETs, specifically the release of virulence factors and arrest of ribosomal protein synthesis. Finally, we discovered patterns of proteolysis consistent with widespread cleavage of proteins by neutrophil elastase, proteinase 3 and cathepsin G and evidence of citrullination in many nuclear proteins.
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http://dx.doi.org/10.1371/journal.ppat.1006151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5298345PMC
January 2017

Preliminary investigation of the transmission of tuberculosis between farmers and their cattle in smallholder farms in northwestern Ethiopia: a cross-sectional study.

BMC Res Notes 2017 Jan 7;10(1):31. Epub 2017 Jan 7.

Aklilu Lemma Institute of Pathobiology, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia.

Background: The feeding habits and close physical contact between Ethiopian farmers and their cattle promote the transmission of tuberculosis (TB) between the farmers and their cattle. This study aimed to investigate the transmission of TB between farmers and their cattle in smallholder farms in northwestern Ethiopia.

Results: A total of 70 human TB lymphadenitis (TBLN) cases visiting the Felegehiwot Comprehensive Specialized Hospital in Bahir Dar City and 660 cattle were investigated. Half of the cattle were owned by households with TB cases, and the remaining half by TB free households. Among the 70 human TBLN patients interviewed, 65.7% (46 out of 70) of the respondents were not aware of zoonotic TB, and 67.1% (47/70) of them consumed raw milk. Positive cultures of TB were obtained in 40 of the 70 cases where TBLN tests were positive with fine needle aspiration cytology. Spoligotyping resulted in 31 different patterns, of which 25 isolates were Mycobacterium (M.) tuberculosis, and the remaining were M. africanum (4 isolates) and M. bovis (2 isolates). None of the animals showed positive test results for bovine TB by comparative intradermal tuberculin test.

Conclusions: Based on the identification of M. bovis from two patients diagnosed with TBLN, we obtained preliminary evidence of zoonotic transmission of TB in northwestern Ethiopia. We did not identify a direct route of transmission between cattle and its owners. This is the objective of further investigations.
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http://dx.doi.org/10.1186/s13104-016-2349-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5219749PMC
January 2017

Comprehensive Metaproteomic Analyses of Urine in the Presence and Absence of Neutrophil-Associated Inflammation in the Urinary Tract.

Theranostics 2017 1;7(2):238-252. Epub 2017 Jan 1.

J. Craig Venter Institute, 9714 Medical Center Drive, Rockville, MD 20850.

Inflammation in the urinary tract results in a urinary proteome characterized by a high dynamic range of protein concentrations and high variability in protein content. This proteome encompasses plasma proteins not resorbed by renal tubular uptake, renal secretion products, proteins of immune cells and erythrocytes derived from trans-urothelial migration and vascular leakage, respectively, and exfoliating urothelial and squamous epithelial cells. We examined how such proteins partition into soluble urine (SU) and urinary pellet (UP) fractions by analyzing 33 urine specimens 12 of which were associated with a urinary tract infection (UTI). Using mass spectrometry-based metaproteomic approaches, we identified 5,327 non-redundant human proteins, 2,638 and 4,379 of which were associated with SU and UP fractions, respectively, and 1,206 non-redundant protein orthology groups derived from pathogenic and commensal organisms of the urogenital tract. Differences between the SU and UP proteomes were influenced by local inflammation, supported by respective comparisons with 12 healthy control urine proteomes. Clustering analyses showed that SU and UP fractions had proteomic signatures discerning UTIs, vascular injury, and epithelial cell exfoliation from the control group to varying degrees. Cases of UTI revealed clusters of proteins produced by activated neutrophils. Network analysis supported the central role of neutrophil effector proteins in the defense against invading pathogens associated with subsequent coagulation and wound repair processes. Our study expands the existing knowledge of the urinary proteome under perturbed conditions, and should be useful as reference dataset in the search of biomarkers.
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http://dx.doi.org/10.7150/thno.16086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5197061PMC
October 2017

Evaluation of the GenoType MTBDRplus assay for detection of rifampicin- and isoniazid-resistant Mycobacterium tuberculosis isolates in central Ethiopia.

Int J Mycobacteriol 2016 12 27;5(4):475-481. Epub 2016 Jun 27.

Aklilu Lemma Institute of Pathobiology, Addis Ababa University, Addis Ababa, Ethiopia.

Objective/background: Multidrug-resistant tuberculosis (MDR-TB) is growing globally and becoming a major challenge for national TB control programs. Therefore, rapid identification of MDR strains of Mycobacterium tuberculosis and monitoring their transmission could contribute significantly to the control of TB. The GenoType MTBDRplus assay has been recommended by the World Health Organization to identify rifampicin (RIF)- and isoniazid (INH)-resistant M. tuberculosis isolates. This study was carried out to evaluate the performance of the GenoType MTBDRplus assay for the detection of RIF- and INH-resistant M. tuberculosis isolates in central Ethiopia.

Methods: A total of 279 M. tuberculosis strains isolated from active TB cases in central Ethiopia were evaluated for their drug sensitivity by the conventional drug-susceptibility test (DST) and compared with data derived from the GenoType MTBDRplus assay. The DST served as the gold standard for evaluating the GenoType MTBDRplus assay.

Results: The sensitivity and specificity of the GenoType MTBDRplus assay for the detection of RIF-resistant M. tuberculosis isolates were 80.0% and 99.6%, respectively. Its sensitivity and specificity for the detection of INH-resistant M. tuberculosis isolates were 82.7% and 99.6%, respectively, whereas they were 75.0% and 100%, respectively, for the detection of MDR M. tuberculosis strains. The concordances of the GenoType MTBDRplus assay and the conventional DST for the detection of RIF and INH susceptibility were 80% (8/10) and 86.2% (25/29), respectively. Furthermore, the concordance of the two tests for the detection of MDR M. tuberculosis strains was 75%. Specific mutations were detected in 55.6% (5/9) of the RIF-resistant isolates, with the highest mutation rate (33.3%) for the rpoB gene (Codon S531L). For INH-resistant isolates, the highest mutation rate (88.8%) related to a katG mutation (Codon S315T1).

Conclusion: The findings of this study revealed that the GenoType MTBDRplus assay has high sensitivity and specificity for the detection of RIF and INH resistance. These preliminary data support the notion that the assay should be considered as an alternative to the DST for the characterization of MDR in M. tuberculosis isolates and the control of TB.
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http://dx.doi.org/10.1016/j.ijmyco.2016.06.005DOI Listing
December 2016

Gastrointestinal microbial populations can distinguish pediatric and adolescent Acute Lymphoblastic Leukemia (ALL) at the time of disease diagnosis.

BMC Genomics 2016 08 15;17(1):635. Epub 2016 Aug 15.

J. Craig Venter Institute (JCVI), 9714 Medical Center Drive, Rockville, MD, 20850, USA.

Background: An estimated 15,000 children and adolescents under the age of 19 years are diagnosed with leukemia, lymphoma and other tumors in the USA every year. All children and adolescent acute leukemia patients will undergo chemotherapy as part of their treatment regimen. Fortunately, survival rates for most pediatric cancers have improved at a remarkable pace over the past three decades, and the overall survival rate is greater than 90 % today. However, significant differences in survival rate have been found in different age groups (94 % in 1-9.99 years, 82 % in ≥10 years and 76 % in ≥15 years). ALL accounts for about three out of four cases of childhood leukemia. Intensive chemotherapy treatment coupled with prophylactic or therapeutic antibiotic use could potentially have a long-term effect on the resident gastrointestinal (GI) microbiome. The composition of GI microbiome and its changes upon chemotherapy in pediatric and adolescent leukemia patients is poorly understood. In this study, using 16S rRNA marker gene sequences we profile the GI microbial communities of pediatric and adolescent acute leukemia patients before and after chemotherapy treatment and compare with the microbiota of their healthy siblings.

Results: Our study cohort consisted of 51 participants, made up of matched pediatric and adolescent patients with ALL and a healthy sibling. We elucidated and compared the GI microbiota profiles of patients and their healthy sibling controls via analysis of 16S rRNA gene sequencing data. We assessed the GI microbiota composition in pediatric and adolescent patients with ALL during the course of chemotherapy by comparing stool samples taken before chemotherapy with stool samples collected at varying time points during the chemotherapeutic treatment. The microbiota profiles of both patients and control sibling groups are dominated by members of Bacteroides, Prevotella, and Faecalibacterium. At the genus level, both groups share many taxa in common, but the microbiota diversity of the patient group is significantly lower than that of the control group. It was possible to distinguish between the patient and control groups based on their microbiota profiles. The top taxa include Anaerostipes, Coprococcus, Roseburia, and Ruminococcus2 with relatively higher abundance in the control group. The observed microbiota changes are likely the result of several factors including a direct influence of therapeutic compounds on the gut flora and an indirect effect of chemotherapy on the immune system, which, in turn, affects the microbiota.

Conclusions: This study provides significant information on GI microbiota populations in immunocompromised children and opens up the potential for developing novel diagnostics based on stool tests and therapies to improve the dysbiotic condition of the microbiota at the time of diagnosis and in the earliest stages of chemotherapy.
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http://dx.doi.org/10.1186/s12864-016-2965-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986186PMC
August 2016

Using host-pathogen protein interactions to identify and characterize Francisella tularensis virulence factors.

BMC Genomics 2015 Dec 29;16:1106. Epub 2015 Dec 29.

Department of Defense Biotechnology High Performance Computing Software Applications Institute, Telemedicine and Advanced Technology Research Center, U.S. Army Medical Research and Materiel Command, Fort Detrick, MD, 21702, USA.

Background: Francisella tularensis is a select bio-threat agent and one of the most virulent intracellular pathogens known, requiring just a few organisms to establish an infection. Although several virulence factors are known, we lack an understanding of virulence factors that act through host-pathogen protein interactions to promote infection. To address these issues in the highly infectious F. tularensis subsp. tularensis Schu S4 strain, we deployed a combined in silico, in vitro, and in vivo analysis to identify virulence factors and their interactions with host proteins to characterize bacterial infection mechanisms.

Results: We initially used comparative genomics and literature to identify and select a set of 49 putative and known virulence factors for analysis. Each protein was then subjected to proteome-scale yeast two-hybrid (Y2H) screens with human and murine cDNA libraries to identify potential host-pathogen protein-protein interactions. Based on the bacterial protein interaction profile with both hosts, we selected seven novel putative virulence factors for mutant construction and animal validation experiments. We were able to create five transposon insertion mutants and used them in an intranasal BALB/c mouse challenge model to establish 50 % lethal dose estimates. Three of these, ΔFTT0482c, ΔFTT1538c, and ΔFTT1597, showed attenuation in lethality and can thus be considered novel F. tularensis virulence factors. The analysis of the accompanying Y2H data identified intracellular protein trafficking between the early endosome to the late endosome as an important component in virulence attenuation for these virulence factors. Furthermore, we also used the Y2H data to investigate host protein binding of two known virulence factors, showing that direct protein binding was a component in the modulation of the inflammatory response via activation of mitogen-activated protein kinases and in the oxidative stress response.

Conclusions: Direct interactions with specific host proteins and the ability to influence interactions among host proteins are important components for F. tularensis to avoid host-cell defense mechanisms and successfully establish an infection. Although direct host-pathogen protein-protein binding is only one aspect of Francisella virulence, it is a critical component in directly manipulating and interfering with cellular processes in the host cell.
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http://dx.doi.org/10.1186/s12864-015-2351-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4696196PMC
December 2015

Genetic Diversity of Mycobacterium tuberculosis Complex Isolated from Tuberculosis Patients in Bahir Dar City and Its Surroundings, Northwest Ethiopia.

Biomed Res Int 2015 28;2015:174732. Epub 2015 Sep 28.

Aklilu Lemma Institute of Pathobiology, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia.

The knowledge of the diversity of strains of Mycobacterium tuberculosis complex (MTBC) species in a specific geographical region can contribute to the control of tuberculosis (TB). This study was conducted to identify the MTBC isolates to the species and spoligotype international type (SIT) level by spoligotyping. A total of 168 MTBC isolates were recovered from TB patients, spoligotyped, and their patterns were compared with those of the strains registered in the SITVIT2 database. Of 168 isolates spoligotyped, 89 patterns were identified. Ninety-eight isolates were clustered into 19 strain groups with clustering percentage of 58.3%. Forty-four strains matched the preexisting SITs in the SITVIT2 database. The dominant strains were SIT289, SIT134, and SIT3411, comprising 16.7% (28/168), 7.14% (12/168), and 4.76% (8/168) of the isolates, respectively. Euro-American (51.2%), East-African-Indian (34.5%), and M. africanum (9.52%) were the major lineages identified. Two strains of M. bovis were isolated from TB lymphadenitis cases. The high percentage of clustered strains of M. tuberculosis could suggest that a small number of lineages of M. tuberculosis are causing the disease in the area while isolation of M. bovis could suggest its zoonotic potential. Additionally, identification of M. africanum requires further confirmation by tools with a better discriminatory power.
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http://dx.doi.org/10.1155/2015/174732DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4600926PMC
August 2016

A prospective analysis of urinary tract infections among elderly trauma patients.

J Trauma Acute Care Surg 2015 Oct;79(4):638-42

From the Mayo Clinic (M.D.Z., M.M.K., S.F.P., A.B., H.N., M.A.K., D.H.J., S.H., K.V.B.), Rochester, Minnesota; and J. Craig Venter Institute (R.P.), Baltimore, Maryland.

Background: Catheter-associated urinary tract infections (CAUTIs) have been deemed "reasonably preventable" by the Centers for Medicare and Medicaid, thereby eliminating reimbursement. Elderly trauma patients, however, are at high risk for developing urinary tract infections (UTIs) given their extensive comorbidities, immobilization, and environmental changes in the urine, which provide the ideal environment for bacterial overgrowth. Whether these patients develop CAUTI as a complication of their hospitalization or have asymptomatic bacteriuria (ASB) or UTI at admission must be determined to justify the "reasonably preventable" classification. We hypothesize that a significant proportion of elderly patients will present with ASB or UTI at admission.

Methods: Institutional review board permission was obtained to perform a prospective, observational clinical trial of all elderly (≥65 years) patients admitted to our Level I trauma center as a result of injury. Urinalysis (UA) and culture (UCx) were obtained at admission, 72 hours, and, if diagnosed with UTI, at 2 weeks after injury. Mean cost of UTI was calculated based on Centers for Disease Control and Prevention estimates of $862 to $1,007 per UTI.

Results: Of 201 eligible patients, 129 agreed to participate (64%). Mean (SD) age was 81 (8.6) years. All patients had a blunt mechanism of injury (76% falls), with a mean Injury Severity Score (ISS) of 13.8 (7.6). Of the 18 patients (14%) diagnosed with CAUTI, 14 (78%) were present at admission. In addition, there were 18 patients (14%) with ASB at admission. The most common bacterial species present at admission urine culture were Escherichia coli (24%) and Enterococcus (16%). Clinical features associated with bacteriuria at admission included a history of UTI, positive Gram stain result, abnormal microscopy, and pyuria. The estimated loss of reimbursement for 18 UTIs at admission was $15,516 to $18,126; however, given an estimated cost of $1,981 to screen all patients with UA and UCx at admission, up to $16,144 savings was realized.

Conclusion: Many elderly trauma patients present with UTI. Screening UA and UCx at admission for elderly trauma patients identifies these UTIs and is cost-effective.

Level Of Evidence: Epidemiologic study, level II.
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http://dx.doi.org/10.1097/TA.0000000000000796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4582427PMC
October 2015

Similar Neutrophil-Driven Inflammatory and Antibacterial Responses in Elderly Patients with Symptomatic and Asymptomatic Bacteriuria.

Infect Immun 2015 Oct 3;83(10):4142-53. Epub 2015 Aug 3.

The J. Craig Venter Institute, Rockville, Maryland, USA

Differential diagnosis of asymptomatic bacteriuria (ASB) and urinary tract infection (UTI) is based on the presence of diverse symptoms, including fever (≥38.5°C), rigors, malaise, lethargy, flank pain, hematuria, suprapubic discomfort, dysuria, and urgent or frequent urination. There is consensus in the medical community that ASB warrants antibiotic treatment only for patients undergoing urological procedures that lead to mucosal bleeding, catheterized individuals whose ASB persists for more than 48 h after catheter removal, and pregnant women. Pyuria is associated with UTI and implicates host immune responses via release of antibacterial effectors and phagocytosis of pathogens by neutrophils. Such responses are not sufficiently described for ASB. Metaproteomic methods were used here to identify the pathogens and evaluate molecular evidence of distinct immune responses in cases of ASB compared to UTI in elderly patients who were hospitalized upon injury. Neutrophil-driven inflammatory responses to invading bacteria were not discernible in most patients diagnosed with ASB compared to those with UTI. In contrast, proteomic urine analysis for trauma patients with no evidence of bacteriuria, including those who suffered mucosal injuries via urethral catheterization, rarely showed evidence of neutrophil infiltration. The same enzymes contributing to the synthesis of leukotrienes LTB4 and LTC4, mediators of inflammation and pain, were found in the UTI and ASB cohorts. These data support the notion that the pathways mediating inflammation and pain in most elderly patients with ASB are not quantitatively different from those seen in most elderly patients with UTI and warrant larger clinical studies to assess whether a common antibiotic treatment strategy for elderly ASB and UTI patients is justified.
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http://dx.doi.org/10.1128/IAI.00745-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4567619PMC
October 2015