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Profiling Hospital-Acquired Pathogens and Antibiotic Resistance Genes Webinar

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Hospital-acquired infections (HAIs) are caused by bacterial, viral and fungal pathogens that easily spread through the body. The most common HAIs include urinary tract infections, bloodstream infections and pneumonia. HAIs are becoming more virulent and more resistant to the antibiotics typically used to fight them, making antibiotic resistance a serious public health concern. In this webinar, we will provide an overview of hospital-acquired pathogens and antibiotic resistance. We will also present tools to help you identify and characterize hospital-acquired bacterial species and antibiotic resistance genes in your research samples.

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Profiling Hospital-Acquired Pathogens and Antibiotic Resistance Genes Webinar

  1. 1. Sample to Insight Profiling hospital-acquired pathogens and antibiotic resistance genes Miranda Hanson-Baseler, Ph.D. Miranda.Hanson-Baseler@qiagen.com
  2. 2. Sample to Insight Legal disclaimer 2 • QIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention or treatment of a disease. • For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.QIAGEN.com or can be requested from QIAGEN Technical Services or your local distributor. Hospital Acquired Infection
  3. 3. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial analysis Hospital-acquired infections Antibiotic resistance QIAGEN’s microbial qPCR products Questions 3 1 2 3 4 5 6 Hospital Acquired Infection
  4. 4. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial analysis Hospital-acquired infections Antibiotic resistance QIAGEN’s microbial qPCR products Questions 4 1 2 3 4 5 6 Hospital Acquired Infection
  5. 5. Sample to Insight Microbiome: Definition and background 5 “The microbiome is defined as the collective genomes of the microbes (composed of bacteria, bacteriophage, fungi, protozoa, and viruses) that live inside and on the human body.” -NIH, 2012 Microbiota refers to the collection of microbial organisms that inhabits a certain environment Metagenomics is the study of the collective genomes of microorganisms from a sample without cultivation (Lederberg and McCray 2001, The NIH HMP Working Group) What does “microbiome” mean? Kuczynski et al. Nature Reviews Genetics 13, 47-58 (January 2012) Hospital Acquired Infection
  6. 6. Sample to Insight Human Microbiome Project 6 Microorganisms cluster by body site Cataloguing efforts by the NIH Human Microbiome Project suggest: • ~10,000 organisms live with us • ~ 8 ×106 genes in this “second genome” Identifying microbiota in healthy individuals revealed: • Different body sites have unique communities • Race, age, gender, weight or ethnicity have an effect 1 Hoffmann A.R., et al. “The Microbiome: The Trillions of Microorganisms That Maintain Health and Cause Disease in Humans and Companion Animals.” Vet Pathol. 2015 2 http://commonfund.nih.gov/hmp/index 3 Structure, function and diversity of the health human microbiome. The Human Microbiome Project Consortium. Nature, 486, 207-214 (14 June 2012). doi: 10.1038/nature11234 Hospital Acquired Infection
  7. 7. Sample to Insight Human microbiota and disease 7 Gut • Intestinal infections • Obesity • Inflammatory Bowel Disease Airway • Pneumonia and other respiratory infections • Chronic Obstructive Pulmonary Disease • Cystic Fibrosis Urogenital • Bacterial vaginosis • Urinary Tract Infections • Sexually Transmitted Disease Blood • Sepsis/bloodstream infections Oral • Periodontitis • Gingivitis http://commonfund.nih.gov/hmp/index Hospital Acquired Infection
  8. 8. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial analysis Hospital-acquired infections Antibiotic resistance QIAGEN’s microbial qPCR products Questions 8 1 2 3 4 5 6 Hospital Acquired Infection
  9. 9. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial analysis Hospital-acquired infections Antibiotic resistance QIAGEN’s microbial qPCR products Questions 9 1 2 3 4 5 6 Hospital Acquired Infection
  10. 10. Sample to Insight Technologies to analyze microbial communities 10 • Culture • Gene cloning (Pan 16S rRNA) and Sanger sequencing • Microarray • MALDI • Next generation sequencing • 16S rRNA sequencing • Whole genome sequencing • qPCR - target dependent • 16S rRNA gene • Other relevant gene (antibiotic resistance gene, virulence factor gene) Hospital Acquired Infection
  11. 11. Sample to Insight Limitations of current pathogen detection methods • Time consuming • (Involve multiple steps, 5-7 days) • Cannot identify all pathogens • Majority are non-culturable • Culture conditions are different • Require extensive microbiological training and expertise • Varying protocols for identification • Waste generation 11Hospital Acquired Infection
  12. 12. Sample to Insight 12 Rapid • Detection in less than 3 hours • Amenable to routine testing Sensitive • Can detect low copy numbers Standardized • Automated protocols • Stable chemical design Specific • Only detects target sequence Benefits of real-time PCR for detection of microorganisms Hospital Acquired Infection
  13. 13. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial analysis Hospital-acquired infections Antibiotic resistance QIAGEN’s microbial qPCR products Questions 13 1 2 3 4 5 6 Hospital Acquired Infection
  14. 14. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial analysis Hospital-acquired infections Antibiotic resistance QIAGEN’s microbial qPCR products Questions 14 1 2 3 4 5 6 Hospital Acquired Infection
  15. 15. Sample to Insight Hospital-acquired infections (HAIs) • Also called nosocomial or healthcare-associated infections • CDC: Infections that patients acquire during the course of receiving treatment for other conditions, or acquired by healthcare workers while performing their duties in healthcare settings • Infection was NOT present nor incubating at time of admission 3 main contributing factors of HAIs 1. Agent: Variety of microorganisms (viruses, bacteria, fungi and parasites) 2. Host factors: Suppressed immune system 3. Environment: Transmission of pathogens between staff and patients and among patients Hospital Acquired Infection 15
  16. 16. Sample to Insight Sources of infection 1. Exogenous: Outside the body • Caused by organisms acquired by exposure to hospital personnel, medical devices or hospital environment 2. Endogenous: By normal human flora • Caused by organisms that are present as a part of the normal flora of the patient • Organisms which are harmless in one site can be pathogenic when transferred to another site • Bacteria from other patients, visitors, hospital personnel • Contamination from medical equipment • Ventilation ducts, bacteria from outside Modes of transmission: • Contact • Airborne • Oral route • Parenteral route • Vector borne Hospital Acquired Infection 16
  17. 17. Sample to Insight Steps to infections 1. Source of microorganism • Infected person or other sources 2. Method of transmission • Medical instruments/devices, hands, clothing, sneezing, coughing, etc. 3. Point of entry • Orifices, mucus membranes, skin 4. Susceptible host • Immunocompromised; low resistance to infection Hospital Acquired Infection 17
  18. 18. Sample to Insight Pathogens that cause HAIs Majority of HAIs in the U.S. are caused by “ESKAPE” pathogens: Enterococcus faecium Staphylococcus aureus Klebsiella sp. Acinetobacter baumannii Pseudomonas aeruginosa Enterobacter sp. Viruses: • Blood borne infections – HBV, HCV, HIV • Rubella, varicella, SARS Fungi: • Candida • Aspergillus Hospital Acquired Infection 18
  19. 19. Sample to Insight Types of HAIs Urinary tract infections • Very common • ~80% of infections are associated with use of bladder catheter Sepsis (blood stream infections) • Represent small proportion of HAIs • High fatality rates Respiratory infections • Cough, purulent sputum, infiltrate on chest x-ray • Pneumonia Intestinal infections • Mostly caused by Clostridium difficile which is linked to 14,000 deaths in the U.S per year • Symptoms range from diarrhea to life-threatening inflammation of the colon Surgical site infections • Usually acquired during an operation • Purulent discharge, abscess, spreading cellulitis at surgical site within a month after the operation Hospital Acquired Infection 19
  20. 20. Sample to Insight Urinary tract infections: Microbial identification or profiling Urinary Tract Infections Microbial DNA qPCR Array • Research tool used to screen for pathogenic bacteria from the urogenital tract • Quickly detects the presence of pathogenic microorganisms from urine, urogenital swabs or other samples that originate from the urogenital tract • Can be used to monitor the frequency of various urinary tract infections in epidemiology research studies • Contains assays for the following bacterial pathogens, designed to target the 16S rRNA gene: • Streptococcus agalactiae • Pseudomonas aeruginosa • Morganella morganii • Escherichia coli • Enterococcus faecium • Enterococcus faecalis • Burkholderia cepacia • Aerococcus urinae • Acinetobacter baumannii Hospital Acquired Infection 20
  21. 21. Sample to Insight Sepsis: Microbial identification or profiling Sepsis Microbial DNA qPCR Array • Research tool used to screen for pathogenic bacteria and fungi associated with bloodstream infections • Quickly detects the presence of pathogenic bacteria from sepsis-associated samples such as blood, blood culture or isolated bacterial colonies • Can be used to monitor the frequency of sepsis-related microbial infections in epidemiology research studies • Contains 89 assays for the following bacterial pathogens, designed to target the 16S rRNA gene: Actinobacteria: Bifidobacterium longum, Brevibacterium casei, Corynebacterium diphtheriae, Kocuria kristinae, Leifsonia aquatica, Micrococcus luteus, Nocardia farcinica, Propionibacterium acnes, Streptomyces bikiniensis, Streptomyces griseus Bacteriodetes: Bacteroides fragilis, Prevotella bivia, Prevotella intermedia, Prevotella melaninogenica Firmicutes: Aerococcus viridans, Bacillus anthracis, Bacillus cereus, Bacillus licheniformis, Butyrivibrio fibrisolvens, Clostridium perfringens, Clostridium sordellii, Enterococcus faecalis, Enterococcus faecium, Erysipelothrix rhusiopathiae, Exiguobacterium aurantiacum, Geobacillus stearothermophilus, Paenibacillus larvae, Paenibacillus macerans, Paenibacillus thiaminolyticus, Pediococcus acidilactici, Pediococcus pentosaceus, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus anginosus, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus sanguinis, Weissella confusa Fusobacteria: Fusobacterium mortiferum, Fusobacterium necrophorum, Fusobacterium nucleatum, Fusobacterium varium Proteobacteria: Achromobacter xylosoxidans, Acinetobacter baumannii, Acinetobacter calcoaceticus, Aeromonas hydrophila, Aeromonas sobria, Alcaligenes faecalis, Brevundimonas diminuta, Brevundimonas vesicularis, Burkholderia cepacia, Burkholderia mallei, Citrobacter freundii, Desulfovibrio desulfuricans, Enterobacter cloacae, Escherichia coli, Haemophilus influenzae, Hafnia alvei, Helicobacter pylori, Methylobacterium fujisawaense, Methylobacterium zatmanii, Morganella morganii, Neisseria meningitidis, Ochrobactrum anthropi, Pantoea agglomerans, Plesiomonas shigelloides, Proteus mirabilis, Pseudomonas aeruginosa, Rahnella aquatilis, Ralstonia pickettii, Stenotrophomonas maltophilia, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Yersinia enterocolitica, Yersinia pestis Fungi: Aspergillus flavus, Aspergillus fumigatus, Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis. Antibiotic Resistance Marker: mecA. Virulence Factors: lukF, spa. Hospital Acquired Infection 21
  22. 22. Sample to Insight Respiratory infections: Microbial identification or profiling Respiratory Tract Infections Microbial DNA qPCR Array • Research tool used to screen for pathogenic bacteria and fungi from air-way derived samples • Quickly detects the presence of pathogenic bacteria or fungi from bronchoalveolar lavage fluid, sputum or other samples that originate from the lung or airway • Can be used to monitor the frequency of various respiratory infections in epidemiology research studies • Contains assays for the following bacterial pathogens, designed to target the 16S rRNA gene: • Acinetobacter baumannii • Acinetobacter calcoaceticus • Actinobacillus hominis • Aspergillus flavus • Aspergillus fumigatus • Bacillus anthracis • Bordetella parapertussis • Burkholderia cepacia • Burkholderia gladioli • Burkholderia mallei • Chlamydia trachomatis • Chlamydophila pneumoniae • Chlamydophila psittaci • Clostridium sordellii • Corynebacterium diphtheriae • Coxiella burnetii • Francisella tularensis • Haemophilus influenzae • Legionella pneumophila • Moraxella catarrhalis • Mycobacterium africanum • Mycobacterium avium • Mycobacterium kansasii • Mycobacterium tuberculosis • Mycoplasma pneumoniae • Neisseria meningitidis • Nocardia asteroides • Peptostreptococcus anaerobius • Pneumocystis jirovecii • Prevotella bivia • Prevotella oris • Proteus mirabilis • Pseudomonas aeruginosa • Rhodococcus equi • Stenotrophomonas maltophilia • Streptobacillus moniliformis • Streptococcus agalactiae • Streptococcus pneumoniae • Streptococcus pyogenes • Yersinia pestis • Staphylococcus aureus Hospital Acquired Infection 22
  23. 23. Sample to Insight Intestinal infections: Microbial identification or profiling Intestinal Infections Microbial DNA qPCR Array • Research tool used to screen for pathogenic bacteria from gut-derived samples • Quickly detects the presence of pathogenic bacteria in stool or other samples that originate from the gut and are associated with intestinal infections or gastroenteritis • Can be used to monitor the frequency of intestinal infections in epidemiology research studies • Contains 45 assays for the following bacterial pathogens, designed to target the 16S rRNA gene: Actinobacteria: Mycobacterium avium, Mycobacterium intracellulare. Bacteriodetes: Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides vulgatus. Firmicutes: Acidaminococcus fermentans, Anaerococcus lactolyticus, Anaerostipes caccae, Anaerotruncus colihominis, Bacillus cereus, Blautia hydrogenotrophica, Brevibacillus brevis, Butyricicoccus pullicaecorum, Clostridium difficile, Clostridium perfringens, Clostridium septicum, Enterococcus casseliflavus, Enterococcus faecalis, Enterococcus faecium, Enterococcus italicus, Listeria monocytogenes, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus suis. Proteobacteria: Aeromonas hydrophila, Aeromonas sobria, Campylobacter jejuni, Campylobacter fetus, Campylobacter upsaliensis, Citrobacter freundii, Enterobacter cloacae, Francisella tularensis, Helicobacter cinaedi, Helicobacter fennelliae, Helicobacter pylori, Morganella morganii, Plesiomonas shigelloides, Shigella dysenteriae, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Yersinia enterocolitica, Yersinia pestis. Verrucomicrobia: Akkermansia muciniphila Intestinal Infections 2 Microbial DNA qPCR Array (11 assays) • Aeromonas hydrophila • Plesiomonas shigelloides • Yersinia enterocolitica • Blastocystis hominis • Campylobacter spp. • Cryptosporidium spp. • Dientamoeba fragilis • Entamoeba histolytica • Giardia intestinalis • Salmonella spp. • ipaH Hospital Acquired Infection 23
  24. 24. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial analysis Hospital-acquired infections Antibiotic resistance QIAGEN’s microbial qPCR products Questions 1 2 3 4 5 6 Hospital Acquired Infection 24
  25. 25. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial analysis Hospital-acquired infections Antibiotic resistance QIAGEN’s microbial qPCR products Questions 1 2 3 4 5 6 Hospital Acquired Infection 25
  26. 26. Sample to Insight Antibiotic resistance Antibiotic-resistant genes: How can we take control? • CDC estimates: Causes sickness in 2 million people and 23,000 deaths per year • March 2015, Obama Administration Releases National Action Plan to Combat Antibiotic-Resistant Bacteria1 • June 2015, The National Cattlemen’s Beef Association participated in the White House Forum on Antibiotic Stewardship2 1) https://www.whitehouse.gov/the-press-office/2015/03/27/fact-sheet-obama-administration-releases-national-action-plan-combat-ant 2) https://www.beefusa.org/newsreleases1.aspx?NewsID=4966 Hospital Acquired Infection 26
  27. 27. Sample to Insight The rise of the superbugs • Antibiotics have been in use for the last 80 years • Prevalent use has led microbes to adapt to them, eliminating their effectiveness • Superbugs carry several resistance genes and are multi-antibiotic-resistance microbes (MRSA, VRSA, CRE) • Acquire resistance genes by a transfer of DNA from a bacterium already resistant (transformation, conjugation or transduction) or through a genetic mutation that helps the bacteria survive • Genetic mutations can enable bacteria to: • Express efflux systems that remove the drug from the cells (A) • Produce antibiotic-inactivating enzymes like carbapenemase (B) • Modify the drug’s target site or activate an alternative metabolic pathway (C) Hospital Acquired Infection 27
  28. 28. Sample to Insight Beta-lactam resistance • Beta-lactams belong to a family of antibiotics characterized by a beta-lactam ring • Penicillins, cephalosporins, clavans, cephamycins and carbapenems • Resistance to beta-lactams is primarily due to the hydrolysis of the antibiotic by a beta-lactamase • Mutations resulting in the modification of PBPs (penicillin binding proteins) or cellular permeability can lead to resistance [e.g, methicillin resistance in Staphylococcus aureus (MRSA)] • These genes are classified as groups based on function and classes based on sequence similarity Hospital Acquired Infection 28
  29. 29. Sample to Insight Aminoglycoside resistance • Aminoglycosides are characterized by the presence of an aminocyclitol ring linked to aminosugars in their structure • Resistance genes encode for aminoglycoside acetyltransferases, adenyltransferases and phosphotransferases • Streptomycin, kanamyin, tobramycin, amikacin • Bactericidal activity is attributed to the irreversible binding to the ribosomes • Have a broad antimicrobial spectrum • Active against aerobic and facultative aerobic Gram-negative bacilli and some Gram-positive bacteria • 3 mechanisms of resistance: Ribosome alteration, decreased permeability and inactivation of drugs by aminoglycoside modifying enzymes Hospital Acquired Infection 29
  30. 30. Sample to Insight Tetracycline resistance • Tetracyclines inhibit the bacterial growth by stopping protein synthesis • Tetracycline, doxycycline, minocycline • Most common antibiotic resistance encountered • 3 different mechanisms of resistance have been identified • Tetracycline efflux • Ribosome protection • Tetracycline modification Hospital Acquired Infection 30
  31. 31. Sample to Insight Macrolide, lincosamide and streptogramin resistance • Macrolides inhibit protein synthesis by stimulating dissociation of the peptidyl-tRNA molecule from the ribosomes during elongation • Erythromycin, clarithromycin, azithromycin • Intrinsic resistance is due to low permeability of outer membrane protein • Drug inactivation and active efflux may also occur Hospital Acquired Infection 31
  32. 32. Sample to Insight Quinolone and fluoroquinolone resistance • Quinolones and fluoroquinolones have diverse molecular mechanisms • Altering DNA topology • Performing enzymatic modifications • Acting as drug efflux pumps • Ciprofloxacin, gemifloxacin, moxifloxacin Hospital Acquired Infection 32
  33. 33. Sample to Insight Colistin-resistant bacteria in the U.S. • Researchers at the Walter Reed Army Institute of Research and the Walter Reed National Military Medical Center tested drug-resistant E.coli bacteria isolated from U.S. patients • Identified first instance of mcr-1-mediated colistin resistance in the U.S. • Sample collected from a woman treated for a UTI • mcr-1 is located on a plasmid that is capable of easily moving between bacterial species • Plasmid: an independent, circular, self-replicating DNA molecule that carries only a few genes McGann, P. et al. (2016) Escherichia coli Harboring mcr-1 and blaCTX-M on a Novel IncF Plasmid: First report of mcr-1 in the USA. Antimicrob. Agents Chemother. 60, 6. Hospital Acquired Infection 33
  34. 34. Sample to Insight Antibiotic resistance genes from food and fertilizer sources Antibiotic resistance genes in our food supply? • One potential source of acquiring antibiotic resistance genes is through the food-supply • Both livestock and feed may acquire antibiotic resistant bacteria through different mechanisms • Food can be exposed to antibiotic resistant bacteria through fertilizer originating from waste-water treatment plants. This, in addition to increasing administration of antibiotics to livestock can lead to food as being a potential source of antibiotic resistant genes • This may then lead to horizontal gene transfer to pathogenic enteropathogens leading to drug resistance in humans, therefore highlighting the importance of surveillance and prevention of antibiotic resistant genes in food Hospital Acquired Infection 34
  35. 35. Sample to Insight Antibiotic Resistance Genes Microbial DNA qPCR Array • Research tool used for the detection and relative profiling of antibiotic resistance genes • Quickly detects the presence of antibiotic resistance genes that may be present in isolated bacterial colonies, bacteria from blood culture, metagenomic samples or other sample types • Contains assays for 87 antibiotic resistance genes belonging to aminoglycoside, β-lactam, erythromycin, fluoroquinolone, macrolide-lincosamide-streptogramin B, tetracycline, vancomycin, and multidrug resistance classifications Hospital Acquired Infection 35
  36. 36. Sample to Insight Antibiotic Resistance Gene Screening Microbial qPCR Array Hospital Acquired Infection 36
  37. 37. Sample to Insight Microbial PCR array method Genomic DNA from stool samples was extracted using a QIAGEN QIAamp® DNA Stool Mini Kit. 500 ng of genomic DNA from stool samples were mixed with microbial qPCR probe mastermix and microbe-free water. This mixture was then uniformly dispensed into a 96-well PCR plate containing dried- down primers and 5’-hydrolysis probes for each of the antibiotic resistance genes tested. Each PCR plate was run on a Roche LightCycler 480 using the following cycling conditions: After the PCR run, raw CT values were exported to the microbial qPCR analysis software to detect the presence of antibiotic resistance genes. The identification criteria were as follows: CT<32 was identified as positive, CT>35 was identified as negative and a 32<CT<35 was inconclusive. In addition, the control assay PPC (Positive PCR Control) had to have a CT=22±2 to show that the PCR instrument and mastermix performed properly and there were no PCR inhibitors in the sample. Hospital Acquired Infection 37
  38. 38. Sample to Insight Screening of sewage samples and gut microbiota Hospital Acquired Infection 38
  39. 39. Sample to Insight Validation of specificity for PCR array by pyrosequencing Hospital Acquired Infection 39
  40. 40. Sample to Insight Screening using Antibiotic Resistance Genes qPCR Array Hospital Acquired Infection 40
  41. 41. Sample to Insight Summary • Microbial qPCR arrays are a collection of sensitive and specific qPCR assays for the detection of antibiotic resistance genes from both bacterial isolates and metagenomic samples • Detection of 87 antibiotic resistance genes can be performed simultaneously in one 3-hour PCR run • ermB and mefA were found in all meat and stool samples suggesting that acquisition of these antibiotic resistance genes may come from consumption of meat • The Antibiotic Resistance Gene Microbial PCR Array is an effective tool for monitoring potential outbreaks of antibiotic resistant bacteria, detection in food samples and potential sources of fertilizer, and identifying new sources of antibiotic resistance genes Hospital Acquired Infection 41
  42. 42. Sample to Insight In the literature: Antibiotic Resistance Genes Microbial DNA qPCR Array Vandini, A. et al. (2014) Hard surface biocontrol in hospitals using microbial-based cleaning products. PLoS One. 9, 9. • Determined the efficacy of a probiotic-based cleaning procedure by assessing the presence and survival of a number of bacteria responsible for HAIs • Study conducted in 3 different hospitals with ~20,000 microbial surface samples • Used the ARG Microbial DNA qPCR Array to analyze bacillus strains used in the cleaning products and on different isolates from the various hospital settings • Conclusion: Probiotic-based cleaning procedure is more effective in lowering the number of HAI-related microorganisms on surfaces, when compared to conventional cleaning products. Hospital Acquired Infection 42
  43. 43. Sample to Insight In the literature: Antibiotic Resistance Genes Microbial DNA qPCR Array Han, X-M. et al. (2015) Impacts of reclaimed water irrigation on soil antibiotic resistome in urban parks of Victoria, Australia. Environmental Pollution. 211. • Determined the impact of reclaimed water irrigation on the patterns of antibiotic resistance genes and the soil bacteria community • Used the ARG Microbial DNA qPCR Array to identify antibiotic resistance genes in 12 urban parks with and without reclaimed water irrigation • Conclusion: Irrigation of urban parks with reclaimed water could have an effect on the abundance, diversity and composition of a wide variety of soil antibiotic resistance genes of clinical relevance Hospital Acquired Infection 43
  44. 44. Sample to Insight In the literature: Antibiotic Resistance Genes Microbial DNA qPCR Array Agga, G.E. et al. (2015) Antimicrobial-resistant bacterial populations and antimicrobial resistance genes obtained from environments impacted by livestock and municipal waste. PLoS One. 10, 7. • Compared antimicrobial-resistant bacteria and antimicrobial resistance genes from environments associated with municipal sewage treatment plant runoff, cattle feedlot runoff ponds, swine waste lagoons and environments with minimal direct fecal impact • Liquid and solid samples were collected and tested for antibiotic resistance genes using the ARG Microbial DNA qPCR array • Conclusion: Antimicrobial-resistant bacteria and antimicrobial resistance genes exist in cattle, human and swine waste streams, but a higher diversity of ARGs are present in treated human waste discharged fro municipal wastewater treatment plants than in livestock environments Hospital Acquired Infection 44
  45. 45. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial analysis Hospital-acquired infections Antibiotic resistance QIAGEN’s microbial qPCR products Questions 1 2 3 4 5 6 Hospital Acquired Infection 45
  46. 46. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial analysis Hospital-acquired infections Antibiotic resistance QIAGEN’s microbial qPCR products Questions 1 2 3 4 5 6 Hospital Acquired Infection 46
  47. 47. Sample to Insight Overview of QIAGEN’s Microbial qPCR products Microbiology: From identification to characterization 16S rRNA gene - Conserved region - Variable region Microbial qPCR assays and arrays for identification and profiling use probes and primers against 16srRNA variable region. Content: Largest microbiome portfolio; experimentally verified 580 assays Custom: Select 8 to 384 microbial species for simultaneous detection/profiling Control: Integrated controls ensure reliability of results Sensitivity: Can detect as low as 10 copy numbers; data available Hospital Acquired Infection 47
  48. 48. Sample to Insight Overview of QIAGEN’s Microbial qPCR products Microbial DNA qPCR Array: Pre-printed assays profile up to 90 different species/genes PCR plates (either 96-well or 384-well) are pre-printed with primers and probes. Each numbered well is a separate assay that tests the same sample. Integrated control assays: • Host assays detect genomic DNA to test sample collection • Pan A/C is a pan- Aspergillus/Candida assay that detects the presence of fungal rRNA • PanB1 and PanB2 detect bacterial 16S rRNA to determine bacterial load in the sample • PPC is a positive PCR control reaction that tests if the PCR reactions failed from PCR inhibitors from the sample, etc. Hospital Acquired Infection 48
  49. 49. Sample to Insight Overview of QIAGEN’s Microbial qPCR products Layout of a Microbial DNA qPCR Array: Different arrays have different number of assays and samples Hospital Acquired Infection 49
  50. 50. Sample to Insight Overview of QIAGEN’s Microbial qPCR products Microbial DNA qPCR Arrays and Assays Profile or identify the presence of microbial DNA (from bacteria, fungi, virus, protist, antibiotic resistance and virulence factors) Identification experiment answers the following question: Are any of these microbes or genes present in the sample? • Must be compared against a known negative sample • Run NTC as one sample • Answers are Yes or No Profiling experiment answers the following question: Have the amounts of any of these microbes or genes changed? • Must be compared against a reference sample • Answers are fold change Hospital Acquired Infection 50
  51. 51. Sample to Insight Overview of QIAGEN’s Microbial qPCR products Sample to Insight: Microbial qPCR Assays and Arrays QIAsymphony/QIAcube/QIAcube HT QIAgility Rotor-Gene Q DNA Isolation Assays and Arrays Data Analysis • QIAamp DNA Microbiome Kit • Mericon Bacteria Kit • QIAamp UCP Pathogen Mini Kit • QIAamp DNA Stool Mini Kit • QIAamp UCP PurePathogen Blood Kit • QIAamp DNA Mini Kit • MagAttract HMW kit • Microbial DNA qPCR Arrays • Microbial DNA qPCR Assay Kits • Microbial DNA qPCR Assays • Microbial qPCR Multi-Assay Kits • Custom Microbial DNA qPCR Arrays • GeneGlobe Data Analysis Center Sample Insight Hospital Acquired Infection 51
  52. 52. Sample to Insight Overview of QIAGEN’s Microbial qPCR products Microbial NGS (microbiome/pathogen): QIAGEN Tools Sample Disruption Sample Preparation Library construction NGS run Data analysis Validation by PCR • TissueLyserII; TissueLyser LT; TissueRuptor • Pathogen Lysis Tubes • QIAamp DNA Microbiome Kit (human microbiome NGS) • QIAamp UCP Pathogen Mini Kit (If depletion of human gDNA is not necessary) • MagAttract HMW DNA Kit (For genome finishing, starting with culture) • Repli-g Single Cell Kit (Limited primary sample material) • QIAamp Fast DNA Stool Mini Kit (If inhibitors are present) Data Analysis Software • CLC Bio Genomics workbench • Microbial Genome Finishing module Predesigned & custom arrays / assays for verification and focused microbiome analyses • Microbial DNA qPCR Arrays • Microbial DNA qPCR Multi-Assay Kits • Microbial DNA qPCR Assay Kits • Microbial DNA qPCR Assays • QC assays kits to detect species specific gDNA and microbial DNA: o Pan bacteria, Pan fungal, Pan aspergillus, hgDNA, mgDNA etc. • QIAseq FX DNA Library Kit (ILMN) • GeneRead DNA Library Prep Kits (Life, ILMN) • GeneRead Size Selection Kit • GeneRead Library Quant System Hospital Acquired Infection 52
  53. 53. Sample to Insight Want to stay up-to-date? Visit the Biomarker Insights Blog for NGS, PCR, Liquid Biopsy, Microbiome updates and more biomarkerinsights.qiagen.com/ @QIAGENscience Follow us: QIAGEN Life Sciences QIAGENscience Hospital Acquired Infection 53
  54. 54. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial analysis Hospital-acquired infections Antibiotic resistance QIAGEN’s microbial qPCR products Questions 1 2 3 4 5 6 Hospital Acquired Infection 54
  55. 55. Sample to Insight Agenda Introduction to the microbiome Technologies for microbial analysis Hospital-acquired infections Antibiotic resistance QIAGEN’s microbial qPCR products Questions 1 2 3 4 5 6 Hospital Acquired Infection 55
  56. 56. Sample to Insight Thank you for coming Any questions? Contact us Telephone: 888-503-3187 Email: brcsupport@QIAGEN.com qiawebinars@QIAGEN.com Hospital Acquired Infection 56

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