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Real-time analysis of nanopore-based metagenomic sequencing from orthopaedic device infection

Prosthetic joint infections are clinically difficult to diagnose and treat. Previously, we demonstrated metagenomic sequencing on an Illumina MiSeq replicates the findings of current gold standard microbiological diagnostic techniques. Nanopore sequencing offers advantages in speed of detection over MiSeq. Here, we compare direct-from-clinical-sample metagenomic Illumina sequencing with Nanopore sequencing, and report a real-time analytical pathway for Nanopore sequence data, designed for detecting bacterial composition of prosthetic joint infections.\n\nDNA was extracted from the sonication fluids of seven explanted orthopaedic devices, and additionally from two culture negative controls, and was sequenced on the Oxford Nanopore Technologies MinION platform. A specific analysis pipeline was assembled to overcome the challenges of identifying the true infecting pathogen, given high levels of host contamination and unavoidable background lab and kit contamination.\n\nThe majority of DNA classified (>90%) was host contamination and discarded. Using negative control filtering thresholds, the species identified corresponded with both routine microbiological diagnosis and MiSeq results. By analysing sequences in real time, causes of infection were robustly detected within minutes from initiation of sequencing.\n\nWe demonstrate initial proof of concept that metagenomic MinION sequencing can provide rapid, accurate diagnosis for prosthetic joint infections. We demonstrate a novel, scalable pipeline for real-time analysis of MinION sequence data. The high proportion of human DNA in extracts prevents full genome analysis from complete coverage, and methods to reduce this could increase genome depth and allow antimicrobial resistance profiling.

microbiology

A Metagenomic Comparison of Tracheal Aspirate and Mini-Bronchial Alveolar Lavage for Assessment of Respiratory Microbiota

Lower respiratory tract infections (LRTI) lead to more deaths each year than any other infectious disease category. Despite this, etiologic LRTI pathogens are infrequently identified due to limitations of existing microbiologic tests. In critically ill patients, non-infectious inflammatory syndromes resembling LRTI further complicate diagnosis. To address the need for improved LRTI diagnostics, we performed metagenomic next-generation sequencing (mNGS) on tracheal aspirates from 92 adults with acute respiratory failure and simultaneously assessed pathogens, the airway microbiome and the host transcriptome. To differentiate pathogens from respiratory commensals, we developed rules-based and logistic regression models (RBM, LRM) in a derivation cohort of 20 patients with LRTI or non-infectious acute respiratory illnesses. When tested in an independent validation cohort of 24 patients, both models achieved accuracies of 95.5%. We next developed pathogen, microbiome diversity, and host gene expression metrics to identify LRTI-positive patients and differentiate them from critically ill controls with non-infectious acute respiratory illnesses. When tested in the validation cohort, the pathogen metric performed with an AUC of 0.96 (95% CI = 0.86 - 1.00), the diversity metric with an AUC of 0.80 (95% CI = 0.63 - 0.98), and the host transcriptional classifier with an AUC of 0.88 (95% CI = 0.75 - 1.00). Combining these achieved a negative predictive value of 100%. This study suggests that a single streamlined protocol offering an integrated genomic portrait of pathogen, microbiome and host transcriptome may hold promise as a novel tool for LRTI diagnosis.\n\nSIGNIFICANCE STATEMENTLower respiratory tract infections (LRTI) are the leading cause of infectious disease-related death worldwide yet remain challenging to diagnose because of limitations in existing microbiologic tests. In critically ill patients, non-infectious respiratory syndromes that resemble LRTI further complicate diagnosis and confound targeted treatment. To address this, we developed a novel metagenomic sequencing-based approach that simultaneously interrogates three core elements of acute airway infections: the pathogen, airway microbiome and host response. We studied this approach in a prospective cohort of critically ill patients with acute respiratory failure and found that combining pathogen, microbiome and host gene expression metrics achieved accurate LRTI diagnosis and identified etiologic pathogens in patients with clinically identified infections but otherwise negative testing.

microbiology

Speciation by Symbiosis: The Microbiome and Behavior

Species are fundamental units of comparison in biology. The newly discovered importance and ubiquity of host-associated microorganisms is now stimulating work on the roles that microbes can play in animal speciation. We previously synthesized the literature and advanced concepts of speciation by symbiosis with notable attention to hybrid sterility and lethality. Here, we review recent studies and relevant data on microbes as players in host behavior and behavioral isolation, emphasizing the patterns seen in these analyses and highlighting areas worthy of additional exploration. We conclude that the role of microbial symbionts in behavior and speciation is gaining exciting traction, and the holobiont and hologenome concepts afford an evolving intellectual framework to promote research and intellectual exchange between disciplines such as behavior, microbiology, genetics, symbiosis and speciation. Given the increasing centrality of microbiology in macroscopic life, microbial symbiosis is arguably the most neglected aspect of animal and plant speciation, and studying it should yield a better understanding of the origin of species.

Evolutionary Biology

Metagenomic Next-Generation Sequencing Detects Pulmonary Pathogens in Hematopoietic Cellular Transplant Patients with Acute Respiratory Illnesses

RATIONALECurrent microbiologic diagnostics often fail to identify the etiology of lower respiratory tract infections (LRTI) in hematopoietic cellular transplant recipients (HCT), which precludes the implementation of targeted therapies.\n\nOBJECTIVESTo address the need for improved LRTI diagnostics, we evaluated the utility of metagenomic next generation sequencing (mNGS) of bronchoalveolar lavage (BAL) to detect microbial pathogens in HCT patients with acute respiratory illnesses.\n\nMETHODSWe enrolled 22 post-HCT adults ages 19-69 years with acute respiratory illnesses who underwent BAL at the University of Michigan between January 2012 and May 2013. mNGS was performed on BAL fluid to detect microbes and simultaneously assess the host transcriptional response. Results were compared against conventional microbiologic assays.\n\nMEASUREMENTS & MAIN RESULTSmNGS demonstrated 100% sensitivity for detecting respiratory microbes (human metapneumovirus, respiratory syncytial virus, Stenotrophomonas maltophilia, human herpesvirus 6 and cytomegalovirus) when compared to standard testing. Previously unrecognized LRTI pathogens were identified in six patients for whom standard testing was negative (human coronavirus 229E, human rhinovirus A, Corynebacterium propinquum and Streptococcus mitis); findings were confirmed by independent PCR and 16S rRNA sequencing. Relative to patients without infection, patients with infection had increased expression of immunity related genes (p=0.022) and significantly lower diversity of their respiratory microbiome (p=0.017).\n\nCONCLUSIONSCompared to conventional diagnostics, mNGS enhanced detection of pathogens in BAL fluid from HCT patients. Furthermore, our results suggest that combining unbiased microbial pathogen detection with assessment of host gene biomarkers of immune response may hold promise for enhancing the diagnosis of post-HCT respiratory infections.

genomics

An outbreak of Pseudomonas aeruginosa infection linked to a Black Friday piercing event

BackgroundOutbreaks linked to cosmetic piercing are rare, but can cause significant illness. We report the investigation and management of a point-source outbreak that occurred during a \"Black Friday\" event in North West England.\n\nMethodsOutbreak investigation was led by Public Health England, and included active case finding among individuals pierced at a piercing premises between 25/11/2016 (\"Black Friday\") and 7/12/2016. Detailed epidemiological, environmental (including inspection and sampling), and microbiological investigation was undertaken.\n\nResultsDuring the \"Black Friday\" event (25/11/2016), 45 people were pierced (13 by a newly-appointed practitioner). Eleven cases were identified (7 microbiologically-confirmed, 2 probable, and 2 possible). All cases had clinical signs of infection around piercing sites, and five required surgical intervention, with varying degrees of post-operative disfigurement. All confirmed and probable cases had a \"scaffold piercing\" placed with a guide bar by the newly-appointed practitioner. Pseudomonas aeruginosa, indistinguishable at nine-locus variable-number tandem repeat loci, was isolated from four of the confirmed cases, and from pre- and post-flush samples from five separate water taps (three sinks) in the premises. Water samples taken after remedial plumbing work confirmed elimination of Pseudomonas contamination.\n\nConclusionsAlthough high levels of Pseudomonas water contamination and some poor infection control procedures were identified, infection appeared to require additional exposure to an inexperienced practitioner, and the more invasive scaffold piercing. A proactive collaborative approach between piercers and health and environmental officials is required to reduce outbreak risk, particularly when unusually large events are planned.

epidemiology

Trends in Escherichia coli bloodstream infection, urinary tract infections and antibiotic susceptibilities in Oxfordshire, 1998-2016: an observational study

BackgroundThe incidence of Escherichia coli bloodstream infections (EC-BSIs), particularly those caused by antibiotic-resistant strains, is increasing in the UK and internationally. This is a major public health concern but the evidence base to guide interventions is limited.\n\nMethodsIncidence of EC-BSIs and E. coli urinary tract infections (EC-UTIs) in one UK region (Oxfordshire) were estimated from anonymised linked microbiological and hospital electronic health records, and modelled using negative binomial regression based on microbiological, clinical and healthcare exposure risk factors. Infection severity, 30-day allcause mortality, and community and hospital co-amoxiclav use were also investigated.\n\nFindingsFrom 1998-2016, 5706 EC-BSIs occurred in 5215 patients, and 228376 EC-UTIs in 137075 patients. 1365(24%) EC-BSIs were nosocomial (onset >48h post-admission), 1863(33%) were community (>365 days post-discharge), 1346(24%) were quasi-community (31-365 days post-discharge), and 1132(20%) were quasi-nosocomial ([&le;]30 days postdischarge). 1413(20%) EC-BSIs and 36270(13%) EC-UTIs were co-amoxiclav-resistant (41% and 30%, respectively, in 2016). Increases in EC-BSIs were driven by increases in community (10%/year (95% CI:7%-13%)) and quasi-community (8%/year (95% CI:7%-10%)) cases. Changes in EC-BSI-associated 30-day mortality were at most modest (p>0{middle dot}03), and mortality was substantial (14-25% across groups). By contrast, co-amoxiclav-resistant EC-BSIs increased in all groups (by 11%-19%/year, significantly faster than susceptible EC-BSIs, pheterogeneity<0{middle dot}001), as did co-amoxiclav-resistant EC-UTIs (by 13%-29%/year, pheterogeneity*0{middle dot}001). Co-amoxiclav use in primary-care facilities was associated with subsequent co-amoxiclav-resistant EC-UTIs (p=0{middle dot}03) and all EC-UTIs (p=0{middle dot}002).\n\nInterpretationCurrent increases in EC-BSIs in Oxfordshire are primarily community-associated, with high rates of co-amoxiclav resistance, nevertheless not impacting mortality. Interventions should target primary-care facilities with high co-amoxiclav usage.\n\nFundingNational Institute for Health Research.\n\nResearch in contextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed for publications from inception up until October 26, 2017, with the terms \"Escherichia coli\", \"E. coli\", \"bacteraemia\", \"bloodstream infection\", restricting the search to English language articles, and also reviewed references from retrieved articles. Escherichia coli (E. coli) is the most common cause of bloodstream infection, and the incidence of E. coli bloodstream infection, and particularly antibiotic-resistant infections, is increasing in the UK and internationally. Although the UK government aims to reduce healthcare-associated E. coli bloodstream infection, there is only limited evidence to inform appropriate interventions.\n\nAdded value of this studyWe investigated potential drivers for these increases in incidence by exploiting available linked electronic health records over 19 years for ~5200 patients with E. coli bloodstream infection and ~140000 with E. coli urinary tract infection, together with community antimicrobial prescribing data for the most recent six years. Our study identified several findings with significant implications for health policy and patient care: O_LIIncreases in the incidence of E. coli bloodstream infections were driven mainly by non-hospital-associated cases; however, neither patients with previous urinary tract infections nor having previously had urine specimens sent from catheters appeared to be driving the increases\nC_LIO_LICo-amoxiclav-resistant bloodstream infections rose significantly faster than co-amoxiclav-susceptible bloodstream infections, with the greatest number of co-amoxiclav-resistant bloodstream infections in 2016 being in patients discharged more than a month previously (i.e. community-associated)\nC_LIO_LIHigher co-amoxiclav use in primary care was associated with higher rates of both co-amoxiclav-resistant E. coli urinary tract infections and E. coli urinary tract infections overall, supporting drives to reduce broad-spectrum and inappropriate antibiotic use in primary care\nC_LIO_LIDespite substantial increases in co-amoxiclav-resistant bloodstream infections there was no evidence that mortality was increasing in these cases; this does not support moving to broader empiric antibiotic prescribing in hospitals (i.e. carbapenems, piperacillin-tazobactam)\nC_LI\n\nImplications of all available adviceThis suggests that government strategies to effectively reduce E. coli bloodstream infections should target community settings, as well as healthcare-associated settings. The absence of an increased mortality signal suggests that co-amoxiclav resistant E. coli infections are either being successfully treated by dual empiric therapy in severe cases (e.g. with concomitant gentamicin), can be \"rescued\" once isolate susceptibilities become available, or currently deployed phenotypic susceptibility testing breakpoints do not adequately correlate with clinical outcome.

epidemiology

The effectiveness of glass beads for plating cell cultures

Cell plating, the spreading out of a liquid suspension of cells on a surface followed by colony growth, is a common laboratory procedure in microbiology. Despite this, the exact impact of its parameters on colony growth has not been extensively studied. A common protocol involves the shaking of glass beads within a petri dish containing solid growth media. We investigated the effects of multiple parameters in this protocol - the number of beads, the shape of movement, and the number of movements. Standard suspensions of Escherichia coli were spread while varying these parameters to assess their impact on colony growth. Results were assessed by a variety of metrics - the number of colonies, the mean distance between closest colonies, and the variability and uniformity of their spatial distribution. Finally, we devised a mathematical model of shifting billiard to explain the heterogeneities in the observed spatial patterns. Exploring the parameters that affect the most fundamental techniques in microbiology allows us to better understand their function, giving us the ability to precisely control their outputs for our exact needs.

biophysics

Integrating Host Response and Unbiased Microbe Detection for Lower Respiratory Tract Infection Diagnosis in Critically Ill Adults

Lower respiratory tract infections (LRTI) lead to more deaths each year than any other infectious disease category(1). Despite this, etiologic LRTI pathogens are infrequently identified due to limitations of existing microbiologic tests(2). In critically ill patients, non-infectious inflammatory syndromes resembling LRTI further complicate diagnosis. To address the need for improved LRTI diagnostics, we performed metagenomic next-generation sequencing (mNGS) on tracheal aspirates from 92 adults with acute respiratory failure and simultaneously assessed pathogens, the lung microbiome and the host transcriptome. To differentiate pathogens from respiratory commensals, we developed rules-based and logistic regression models (RBM, LRM) in a derivation cohort of 20 patients with LRTI or non-infectious acute respiratory illnesses. When tested in an independent validation cohort of 24 patients, both models achieved accuracies of 95.5%. We next developed pathogen, microbiome diversity, and host gene expression metrics to identify LRTI-positive patients and differentiate them from critically ill controls with non-infectious acute respiratory illnesses. When tested in the validation cohort, the pathogen metric performed with an AUC of 0.96 (95% CI = 0.86 - 1.00), the diversity metric with an AUC of 0.80 (95% CI = 0.63 - 0.98), and the host transcriptional classifier with an AUC of 0.91 (95% CI = 0.80 - 1.00). Combining all three achieved an AUC of 0.99 (95% CI = 0.97 - 1.00) and negative predictive value of 100%. This study suggests that a single streamlined protocol offering an integrated genomic portrait of pathogen, microbiome and host transcriptome may hold promise as a novel tool for LRTI diagnosis.\n\nSIGNIFICANCE STATEMENTLower respiratory tract infections (LRTI) are the leading cause of infectious disease-related death worldwide yet remain challenging to diagnose because of limitations in existing microbiologic tests. In critically ill patients, non-infectious respiratory syndromes that resemble LRTI further complicate diagnosis and confound targeted treatment. To address this, we developed a novel metagenomic sequencing-based approach that simultaneously interrogates three core elements of acute airway infections: the pathogen, lung microbiome and host response. We studied this approach in a prospective cohort of critically ill patients with acute respiratory failure and found that combining pathogen, microbiome and host gene expression metrics achieved accurate LRTI diagnosis and identified etiologic pathogens in patients with clinically identified infections but otherwise negative testing.\n\nFundingNHLBI K12HL119997 (Langelier C), NHLBI K23HL123778 (Christensen S), NIAID P01AI091575 and the Chan Zuckerberg Biohub (DeRisi JL), NHLBI K23 HL136844 (Moazed F), NHLBI R01HL110969, K24HL133390, R35HL140026 (Calfee C), Gladstone Institutes (Pollard KS).

genomics

Selection of an Appropriate Empiric Antibiotic Regimen in Culture-Negative Hematogenous Vertebral Osteomyelitis

The aim of this study was to determine which antibiotic combinations are appropriate for culture-negative hematogenous vertebral osteomyelitis (HVO), based on the antibiotic-susceptibility pattern of organisms isolated from cases of culture-proven HVO. We conducted a retrospective chart review of adult patients with microbiologically proven HVO in five tertiary-care hospitals over a 7-year period. The appropriateness of empiric antibiotic regimens was assessed based on the antibiotic susceptibility profiles of isolated bacteria. In total, 358 cases of microbiologically proven HVO were identified. The main causative pathogens identified were methicillin-susceptible Staphylococcus aureus (33.5%), followed by methicillin-resistant S. aureus (MRSA) (24.9%), aerobic gram-negative bacteria (21.8%), and Streptococcus species (11.7%). Extended spectrum {beta}-lactamase (ESBL)-producing Enterobacteriaceae and anaerobes accounted for only 1.7% and 1.4%, respectively, of the causative pathogens. Based on the susceptibility results of isolated organisms, levofloxacin plus rifampicin was appropriate in 73.5%, levofloxacin plus clindamycin in 71.2%, and amoxicillin-clavulanate plus ciprofloxacin in 64.5% of cases. These oral combinations were more appropriate for treating community-acquired HVO (85.8%, 84.0%, and 80.4%, respectively) than healthcare-associated HVO (52.6%, 49.6%, and 37.6%, respectively). Vancomycin combined with ciprofloxacin, ceftriaxone, ceftazidime, or cefepime was similarly appropriate (susceptibility rates of 93.0%, 94.1%, 95.8%, and 95.8%, respectively). In conclusion, in a setting with a high prevalence of MRSA HVO, oral antibiotic combinations may be suboptimal for treatment of culture-negative HVO and should be used only in patients with community-acquired HVO. Vancomycin combined with fluoroquinolone or a broad-spectrum cephalosporin was appropriate in most cases of HVO in this study.

epidemiology

The Integrated Rapid Infectious Disease Analysis (IRIDA) Platform

Whole genome sequencing (WGS) is a powerful tool for public health infectious disease investigations owing to its higher resolution, greater efficiency, and cost-effectiveness over traditional genotyping methods. Implementation of WGS in routine public health microbiology laboratories is impeded by a lack of user-friendly automated and semi-automated pipelines, restrictive jurisdictional data sharing policies, and the proliferation of non-interoperable analytical and reporting systems. To address these issues, we developed the Integrated Rapid Infectious Disease Analysis (IRIDA) platform (irida.ca), a user-friendly, decentralized, open-source bioinformatics and analytical web platform to support real-time infectious disease outbreak investigations using WGS data. Instances can be independently installed on local high-performance computing infrastructure, enabling private and secure data management and analyses according to organizational policies and governance. IRIDAs data management capabilities enable secure upload, storage and sharing of all WGS data and metadata. The core platform currently includes pipelines for quality control, assembly, annotation, variant detection, phylogenetic analysis, in silico serotyping, multi-locus sequence typing, and genome distance calculation. Analysis pipeline results can be visualized within the platform through dynamic line lists and integrated phylogenomic clustering for research and discovery, and for enhancing decision-making support and hypothesis generation in epidemiological investigations. Communication and data exchange between instances are provided through customizable access controls. IRIDA complements centralized systems, empowering local analytics and visualizations for genomics-based microbial pathogen investigations. IRIDA is currently transforming the Canadian public health ecosystem and is freely available at https://github.com/phac-nml/irida and www.irida.ca.\n\nImpact StatementWhole genome sequencing (WGS) is revolutionizing infectious disease analysis and surveillance due to its cost effectiveness, utility, and improved analytical power. To date, no \"one-size-fits-all\" genomics platform has been universally adopted, owing to differences in national (and regional) health information systems, data sharing policies, computational infrastructures, lack of interoperability and prohibitive costs. The Integrated Rapid Infectious Disease Analysis (IRIDA) platform is a user-friendly, decentralized, open-source bioinformatics and analytical web platform developed to support real-time infectious disease outbreak investigations using WGS data. IRIDA empowers public health, regulatory and clinical microbiology laboratory personnel to better incorporate WGS technology into routine operations by shielding them from the computational and analytical complexities of big data genomics. IRIDA is now routinely used as part of a validated suite of tools to support outbreak investigations in Canada. While IRIDA was designed to serve the needs of the Canadian public health system, it is generally applicable to any public health and multi-jurisdictional environment. IRIDA enables localized analyses but provides mechanisms and standard outputs to enable data sharing. This approach can help overcome pervasive challenges in real-time global infectious disease surveillance, investigation and control, resulting in faster responses, and ultimately, better public health outcomes.\n\nDATA SUMMARYO_LIData used to generate some of the figures in this manuscript can be found in the NCBI BioProject PRJNA305824.\nC_LI

bioinformatics

Comparison Between Non-Catheterized And Catheter Associated Urinary Tract Infections Caused By Extended Spectrum Β-Lactamase Producing Escherichia Coli And Klebsiella Pneumoniae

AbstractCatheter associated urinary tract infections (CAUTI) account for more than 80% of all healthcare associated infections (HAIs) as compared to non-catheterized urinary tract infections. Catheter associated urinary tract infections occur on the third day after insertion of catheter in patients having urinary tract infection (UTI). In long term catheter use, bio-film form along the catheter which increases the risk of antibiotic resistant pathogens. Most common pathogens involved were Escherichia coli and Klebsiella pneumoniae which produce the enzymes Extended Spectrum {beta}-Lactamases (ESBLs).\n\nObjectiveTo compare the frequency of ESBLs in catheterized and non-catheterized UTI infections.\n\nMaterials and MethodsThis comparative study was conducted at the Microbiology Department, Allama Iqbal Medical College, Lahore, from June 2014 to January 2015. Urine samples were cultured according to WHO protocol and antimicrobial Susceptibility testing was performed by Modified Kirby-Bauer disc diffusion Method. Escherichia coli and Klebsiella pneumoniae were tested for ESBL production by phenotypic confirmatory method of disk diffusion synergy using a disc of amoxicillin-clavulonate (30g) and ceftrixone (30g), cefotaxime (30g) and aztreonam (30g) discs.\n\nResultsOut of 300 positive urinary isolates of Escherichia coli and Klebsiella pneumoniae from CAUTI, 65.33% were ESBL producing isolates whereas out of 300 positive urinary isolates of Escherichia coli and Klebsiella pneumoniae from non-catheterized UTI, 47.66% were ESBL producing isolates. The results were highly significant (p < 0.001).\n\nConclusionResults showed that frequency of ESBLs were higher in catheterized patients as compared to non-catheterized patients. This is suggestive of a need for regular screening and surveillance for ESBL producing organisms. Patients infected with these organisms should be nursed with contact precautions to avoid the spread of nosocomial infection.

Microbiology

Divorcing strain classification from species names

Confusion about strain classification and nomenclature permeates modern microbiology. Although taxonomists have traditionally acted as gatekeepers of order, the numbers of and speed at which new strains are identified has outpaced the opportunity for professional classification for many lineages. Furthermore, the growth of bioinformatics and database fueled investigations have placed metadata curation in the hands of researchers with little taxonomic experience. Here I describe practical challenges facing modern microbial taxonomy, provide an overview of complexities of classification for environmentally ubiquitous taxa like Pseudomonas syringae, and emphasize that classification and nomenclature need not be the one in the same. A move toward implementation of relational classification schemes based on inherent properties of whole genomes could provide sorely needed continuity in how strains are referenced across manuscripts and data sets.

Microbiology

THE PATTERN OF BACTERIA AND ITS RESISTANCE ON ADULT SEPSIS PATIENT AT Dr. MOEWARDI GENERAL HOSPITAL, INDONESIA

BackgroundSepsis incidence which related with morbidity and mortality, is globally increasing. Sepsis occurs because of severe infections. Sepsis has life-threatening potential with organ disfungction complication, septic shock, and death. Alteration of resistance pattern always change at certain period. The aim of the study is to investigate the bacteria pattern and its resistance from adult sepsis patient at Dr. Moewardi Regional General Hospital.\n\nMethodsIsolated bacteria from adult sepsis patients blood at Dr. Moewardi Regional General Hospital. 7 bacteria isolates were taken from blood of adult sepsis patient (September - October 2014) and 46 bacteria isolates of secondary data (January - March 2014) at Dr. Moewardi Regional General Hospital. Bacteria isolation was performed based on standard Laboratory of Microbiology of Dr. Moewardi Regional General Hospital. 7 bacteria isolates were tested by using disc diffusion antibiotic on Mueller Hinton and coupled by 46 secondary data.\n\nResultsThe bacteria pattern from adult sepsis patients were Staphylococcus haemolyticus (15,09%), Staphylococcus hominis (15,09%), Escherichia coli (13,21%), and Acinetobacter baumannii (11,32%). Resistance pattern of bacteria on adult sepsis with level of resistant more than 50% as Staphylococcus haemolyticus (ciprofloxacin, erythromycin, levofloxacin, moxifloxacin, and gentamicin), Escherichia coli (gentamicin, ciprofloxacin, and levofloxacin), and Acinetobacter baumannii (gentamicin, ciprofloxacin, levofloxacin, and ceftazidime)\n\nConclutionsBased on this research, revealed that the most common bacteria caused sepsis was Gram negative about 64,64% of all bacteria on sepsis patient. The most frequent cause of sepsis was Staphylococcus hominis, Staphylococcus haemolyticus, and Escherichia coli.

Microbiology

Complex molecular mixtures under cycling gradients as basis for lifes origins

We consider life as a cyclic physicochemical process that makes heredity and Darwinian evolution observable through living cells. We elaborate four principles that constrain current speculations about lifes emergence to natural processes driven by diurnal physicochemical gradients, primarily of temperature, water activity and electromagnetic radiation. First, Earths prebiotic chemical evolution is historically continuous with Darwinian evolution; second, cycling energies of solar radiation are primary drivers of chemical evolution; third, environmental molecular complexity must be high at the origin of life; and fourth, non-covalent molecular forces determine molecular recognition and cellular organization. Under normal physiological conditions of high ionic strength and high macromolecular crowding, hydration interactions (hydrogen bonding), screened electrostatic forces and excluded volume repulsions act over a commensurate distance of about one nanometer. This intermolecular distance governs chemical coevolution of proto-biomacromolecular surfaces (nucleic acids, proteins and membranes) toward Darwinian thresholds and living states. The above physicochemical principles of lifes emergence are consistent with the second law of thermodynamics, and with the current facts of molecular microbiology and planetary sciences. New kinds of experimentation with crowded molecular mixtures under oscillating temperature gradients - a PCR-like mechanism of lifes origins - can further illuminate how living states come about.\n\nGraphical abstractLifes emergence follows from chemical and Darwinian evolution, a high degree of molecular complexity and a high crowdedness, and non-covalent molecular forces that determine molecular recognition and cellular organization. The macromolecules divide the cytoplasm into dynamically crowded macromolecular regions and topologically complementary electrolyte pools. Small ions and ionic metabolites are transported vectorially between the electrolyte pools and through the (semi-conducting) electrolyte pathways of the crowded macromolecular regions.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC=\"FIGDIR/small/050740_fig1.gif\" ALT=\"Figure 1\">\nView larger version (36K):\norg.highwire.dtl.DTLVardef@1963682org.highwire.dtl.DTLVardef@d49be0org.highwire.dtl.DTLVardef@45ac46org.highwire.dtl.DTLVardef@312efc_HPS_FORMAT_FIGEXP M_FIG C_FIG

Microbiology

Millions of Boreal Shield Lakes can be used to Probe the Evolution of Archaean Ocean Life

Life originated in Archaean oceans, almost 4 billion years ago, in the absence of oxygen and the presence of high dissolved iron concentrations. Early Earth oxidation is marked globally by extensive banded iron formations but the contributing processes and timing remain controversial. Very few aquatic habitats have been discovered that match key physico-chemical parameters of the early Archaean Ocean. All previous whole ecosystem Archaean analogue studies have been confined to rare, low sulfur, and permanently stratified lakes. Here we provide first evidence that millions of Boreal Shield lakes with natural anoxia offer the opportunity to constrain biogeochemical and microbiological aspects of early Archaean life. Specifically, we combined novel isotopic signatures and nucleic acid sequence data to examine processes in the anoxic zone of stratified boreal lakes that are naturally low in sulfur and rich in ferrous iron, hallmark characteristics predicted for the Archaean Ocean. Anoxygenic photosynthesis was prominent in total water column biogeochemistry, marked by distinctive patterns in natural abundance isotopes of carbon, nitrogen, and iron. These processes are robust, returning reproducibly after water column re-oxygenation following lake turnover. Evidence of coupled iron oxidation, iron reduction, and methane oxidation affect current paradigms of both early Earth and modern aquatic ecosystems.

Microbiology

A dormant microbial component in the development of pre-eclampsia

Pre-eclampsia (PE) is a complex, multi-system disorder that remains a leading cause of morbidity and mortality in pregnancy. Four main classes of dysregulation accompany PE, and are widely considered to contribute to its severity. These are abnormal trophoblast invasion of the placenta, anti-angiogenic responses, oxidative stress, and inflammation. What is lacking, however, is an explanation of how these themselves are caused.\n\nWe here develop the unifying idea, and the considerable evidence for it, that the originating cause of PE (and of the four classes of dysregulation) is in fact microbial infection, that most such microbes are dormant and hence resist detection by conventional (replication-dependent) microbiology, and that by occasional resuscitation and growth it is they that are responsible for all the observable sequelae, including the continuing, chronic inflammation. In particular, bacterial products such as lipopolysaccharide (LPS), also known as endotoxin, are well known as highly inflammagenic and stimulate an innate (and possibly trained) immune response that exacerbates the inflammation further. The known need of microbes for free iron can explain the iron dysregulation that accompanies PE. We describe the main routes of infection (gut, oral, urinary tract infection) and the regularly observed presence of microbes in placental and other tissues in PE. Every known proteomic biomarker of \"pre-eclampsia\" that we assessed has in fact also been shown to be raised in response to infection. An infectious component to PE fulfils the Bradford Hill criteria for ascribing a disease to an environmental cause, and suggests a number of treatments, some of which have in fact been shown to be successful.\n\nPE was classically referred to as endotoxaemia or toxaemia of pregnancy, and it is ironic that it seems that LPS and other microbial endotoxins really are involved. Overall, the recognition of an infectious component in the aetiology of PE mirrors that for ulcers and other diseases that were previously considered to lack one.\n\nInsight, innovation, integrationMany descriptors of pre-eclampsia are widely accepted (e.g. abnormal trophoblast invasion, oxidative stress, inflammation and altered immune response, and anti-angiogenic responses). However, without knowing what causes them, they do not explain the syndrome. The Biological Insight of this manuscript is that there is considerable evidence to the effect that each of these phenomena (hence PE) are caused by the resuscitation of dormant bacteria that shed (known and potent) inflammagens such as LPS, often as a consequence of iron availability. PE is thus seen as a milder form of sepsis. The Technological Innovations come from the use of molecular markers (of microbes and omics more generally, as well as novel markers of coagulopathies) to measure this. The Benefit of Integration comes from bringing together a huge number of disparate observations into a unifying theme.

Microbiology

Genomic dissection of an Icelandic epidemic of equine respiratory disease

The native horse population of Iceland has remained free of major infectious diseases. Between May and July 2010 an epidemic of respiratory disease swept through the population. Initial microbiological investigations ruled out known equine viral agents as the cause of the infections, but identified the opportunistic pathogen Streptococcus zooepidemicus as being frequently isolated from diseased animals. This diverse bacterial species has a broad host range and is usually regarded as a commensal of horses. By genome sequencing S. zooepidemicus recovered from horses during the epidemic we show that although multiple clones of S. zooepidemicus were present in the population, one particular clone, ST209, was responsible for the epidemic. Concurrent with the epidemic, ST209 caused zoonotic infections, highlighting the pathogenic potential of this clone. Phylogenetic analysis suggests that the original ST209 strain entered Iceland in late 2008 or early 2009. Epidemiological investigation revealed that the incursion of this strain into a training yard that utilized a submerged treadmill between the 5th and 19th of February 2010 was a critical trigger for the ensuing epidemic of disease, provided a nidus for the infection of multiple horses, and subsequent distribution of these animals to multiple sites in Iceland.

Microbiology

General calibration of microbial growth in microplate readers

Optical density (OD) measurements of microbial growth are one of the most common techniques used in microbiology, with applications ranging from antibiotic efficacy studies, studies of growth under different nutritional or stress environments, to studies of different mutant strains, including those harbouring synthetic circuits. OD measurements are performed under the assumption that the OD value obtained is proportional to the cell number, i.e. the concentration of the sample. However, the assumption holds true in a limited range of conditions and calibration techniques that determine that range are currently missing. Here we present a set of calibration procedures and considerations that are necessary to successfully estimate the cell concentration from OD measurements.

Microbiology