PHAR4813: Detection of Pathogenic Bacteria
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What is the primary purpose of the enzyme-targeting portion linked to the chromogen in detection methods?

  • To change the substrate’s chemical structure
  • To ensure stability in the detection medium
  • To increase fluorescence intensity
  • To mute the color until the specific bacterium is present (correct)

    • Which of the following distinguishes between Gram negative and Gram positive bacteria?

  • Color retention in biofilms
  • 9-(4-N ‘-L-Ala-L-Ala-L-Ala-aminophenyl)-10-methylacridinium bis(trifluoroacetate) (correct)
  • Enzyme substrate stability
  • Specific bacterial biofilm formation

    • Which characteristic of Pseudomonas aeruginosa complicates its infection treatment?

  • Natural production of antibiotics
  • Resistance to fluoride-based treatments
  • Rapid reproduction rate
  • Generation of biofilm (correct)

    • What property of 1,2-substituted-7-aminophenoxazinones alters its color detection?

    <p>Hydrolysis of amides</p> Signup and view all the answers

    What is a major advantage of detecting Pseudomonas aeruginosa early?

    <p>Greater clinical success in treatment</p> Signup and view all the answers

    In the context of fluorogenic substrates, what role does a chromophore or fluorophore play?

    <p>It signals the presence of specific bacteria through color change</p> Signup and view all the answers

    What is a distinguishing feature of natural phenoxazinones found in certain fungi?

    <p>They produce a distinct red color</p> Signup and view all the answers

    What is the function of the linker in enzyme-targeting assays?

    <p>To connect the chromogen with the chromophore</p> Signup and view all the answers

    What is the significance of a chromophore in the context of electronic transitions?

    <p>It is the part of a molecule that contains electrons involved in electronic transitions.</p> Signup and view all the answers

    Which attributes must a chromogen possess to be effective in bacterial detection?

    <p>Mutated color or absence when linked to targeting molecule.</p> Signup and view all the answers

    What is lmax in relation to chromophores?

    <p>The wavelength of the maximum absorption in a chromophore.</p> Signup and view all the answers

    How do specific bacterial enzymes contribute to chromogen detection?

    <p>They break the link between the targeting molecule and chromogen.</p> Signup and view all the answers

    What characteristic must a fluorogenic substrate exhibit for effective bacterial detection?

    <p>It should be colorless when attached to the targeting molecule.</p> Signup and view all the answers

    Which type of enzyme is commonly targeted in the detection of Pseudomonas Aeruginosa?

    <p>Peptidases and Glycosidases</p> Signup and view all the answers

    What visual change indicates the presence of a specific bacterium when using a chromogenic detection method?

    <p>Release of color from the chromogen.</p> Signup and view all the answers

    In colorimetric detection techniques, which condition is necessary for reliable results?

    <p>Specific receptor interaction with the chromogen.</p> Signup and view all the answers

    What is the purpose of using linker molecules in chromogenic detection?

    <p>To connect the chromogen to the targeting agent while maintaining its inactive state.</p> Signup and view all the answers

    What wavelength range corresponds to the absorption of green light?

    <p>491–570 nm</p> Signup and view all the answers

    Which of the following characteristics is NOT part of the WHO criteria for an ideal Point of Care (POC) diagnostic test?

    <p>Requires complex procedures</p> Signup and view all the answers

    Which biorecognition element among the following is known for its portability in POC devices?

    <p>Aptamers</p> Signup and view all the answers

    What is a common drawback associated with multiplexing in biosensors?

    <p>Poor cross reactivity</p> Signup and view all the answers

    When considering bacterial enzyme characterization, which of the following factors is crucial for POC detection?

    <p>Reproducibility in production</p> Signup and view all the answers

    Which of the following techniques would NOT be classified as a colorimetric detection method?

    <p>Electrochemiluminescence</p> Signup and view all the answers

    In the context of rapid POC tests, which of the following is a characteristic of fluorogenic substrates?

    <p>Releases signals in the presence of enzymes</p> Signup and view all the answers

    Which of the following is an essential feature of an ideal POC diagnostic test for bacterial detection?

    <p>User-friendly operation</p> Signup and view all the answers

    Which method is primarily used for detecting an antibody sandwich complex in POC diagnostics?

    <p>Electrochemiluminescence</p> Signup and view all the answers

    What is a limitation associated with nucleic acids as biosensors in bacterial identification?

    <p>Degradation by nucleases</p> Signup and view all the answers

    Which process is essential for maintaining the stability of biosensors used in POC diagnostics?

    <p>Stability over temperature and pH ranges</p> Signup and view all the answers

    Which method is characterized by rapid and sensitive identification of microorganisms in bacterial surveillance?

    <p>Real time polymerase chain reaction (PCR)</p> Signup and view all the answers

    What essential characteristic ensures that a bacterial surveillance method identifies only the microorganism of interest?

    <p>Specificity</p> Signup and view all the answers

    Which organization published 'The Health and Social Care Act 2008' aimed at controlling healthcare associated infections?

    <p>National Health Service</p> Signup and view all the answers

    Which of the following is a key requirement for a bacterial surveillance method?

    <p>Simple to perform</p> Signup and view all the answers

    What is the primary advantage of using phenotypic methods in microbial identification?

    <p>They are less time-consuming than genotypic methods.</p> Signup and view all the answers

    What is the main benefit of using real-time PCR over traditional PCR assays?

    <p>It provides results in under an hour without the need for agarose gels.</p> Signup and view all the answers

    Which feature of molecular beacons is crucial for the detection of specific DNA sequences in qPCR?

    <p>The combination of quencher and fluorescent dye.</p> Signup and view all the answers

    What percentage of MRSA isolates were successfully detected by the real-time PCR technique, according to the data provided?

    <p>98.7%</p> Signup and view all the answers

    For which type of bacteria is the BD GeneOhm VanR assay designed to detect?

    <p>Vancomycin-resistant enterococci</p> Signup and view all the answers

    What mutation is associated with the detection of oxazolidinone-resistant E. faecalis and E. faecium in real-time PCR methods?

    <p>G to U mutation at residue 2576 of 23S ribosomal DNA</p> Signup and view all the answers

    What percentage of acute care hospital patients in the US are estimated to have at least one Healthcare Associated Infection on any given day?

    <p>4%</p> Signup and view all the answers

    Which of the following is a major concern regarding multidrug-resistant organisms (MRO)?

    <p>Transmission of resistance between organisms</p> Signup and view all the answers

    What was the primary goal of the Longitude Prize re-instituted by the UK Government in 2014?

    <p>Create a diagnostic test for bacterial infections</p> Signup and view all the answers

    How many patients are estimated to be affected by Healthcare Associated Infections in Europe annually?

    <p>4 million</p> Signup and view all the answers

    What can ultimately lead to strains of bacteria with limited or no susceptibility to antibacterial agents?

    <p>Transmission of resistance between MROs</p> Signup and view all the answers

    What is the significance of the Longitude Prize's 5-year timeline for entries?

    <p>Provides a structured assessment schedule</p> Signup and view all the answers

    What is the average prevalence of Healthcare Associated Infections in Europe per 100 patients?

    <p>7.1</p> Signup and view all the answers

    What monetary value is associated with the Longitude Prize for developing a diagnostic test?

    <p>£10M</p> Signup and view all the answers

    What must occur for a chromogen to confirm the presence of specific bacteria?

    <p>The bacterial enzyme must cleave the targeting molecule from the chromogen.</p> Signup and view all the answers

    Which statement best describes the chromophore's role in electronic transitions?

    <p>Chromophores contain electrons that can be excited to higher energy levels.</p> Signup and view all the answers

    What is the significance of lmax in the context of chromophores?

    <p>It indicates the wavelength of maximum absorption in an absorption spectrum.</p> Signup and view all the answers

    In the context of chromogen requirements, which of the following is NOT a requirement?

    <p>The chromogen must be brightly colored when attached.</p> Signup and view all the answers

    What general trend is observed in relation to HOMO and LUMO as conjugation increases?

    <p>The difference in energy between HOMO and LUMO decreases.</p> Signup and view all the answers

    What is the key characteristic of the free chromogenic molecule in bacterial detection?

    <p>It must exhibit strong color to be effective in detection.</p> Signup and view all the answers

    Which of the following best describes what happens to the chromophore as a specific bacterial enzyme cleaves the targeting molecule?

    <p>The chromophore is released, confirming the presence of the bacteria.</p> Signup and view all the answers

    What distinguishes between different visible light wavelengths absorbed by substances?

    <p>The type of chromophore present in the molecule.</p> Signup and view all the answers

    Which specific type of enzyme is commonly involved in bacterial detection methods?

    <p>Peptidases that cleave amino acids from peptides.</p> Signup and view all the answers

    What is the target of the capture antibody S4-20 in the detection method?

    <p>Lipoarabinomannan (LAM)</p> Signup and view all the answers

    What role does the Ru(bpy)32+ component play in the detection process?

    <p>It generates electrochemical luminescence.</p> Signup and view all the answers

    What is the clinical sensitivity of the optimized antibody pair according to the detection method?

    <p>93%</p> Signup and view all the answers

    What detection method utilizes 3,3′,5,5′-tetramethylbenzidene (TMB) in its process?

    <p>Colorimetric detection using biotin-labeled aptamer</p> Signup and view all the answers

    What is the quantitation limit for the aptamer-based Latent TB Infection Test?

    <p>10,000 CFU/mL</p> Signup and view all the answers

    What visual change indicates the release of nanoparticles in the S.aureus protease detection method?

    <p>Color change from black to gold</p> Signup and view all the answers

    What is the purpose of using horseradish peroxidase (HRP) in the aptamer-based detection method?

    <p>To oxidize the TMB substrate</p> Signup and view all the answers

    What type of nanoparticles are used in the detection of S.aureus protease?

    <p>Black coloured magnetic nanoparticles with a terminal carboxyl group</p> Signup and view all the answers

    Which characteristic is NOT a criterion for an ideal Point of Care (POC) diagnostic test?

    <p>High power consumption</p> Signup and view all the answers

    What is a common advantage of using antibodies as biorecognition elements in POC devices?

    <p>Reproducible production</p> Signup and view all the answers

    Which of the following is a limitation associated with the use of nucleic acids in biosensors for POC diagnostics?

    <p>Degradation by nucleases</p> Signup and view all the answers

    Which biorecognition element is known for its ability to operate without instrumentation in POC devices?

    <p>Aptamers</p> Signup and view all the answers

    What aspect of biosensors allows for identification of a significant number of species simultaneously?

    <p>Multiplexing</p> Signup and view all the answers

    Which property is essential for a POC diagnostic test to be considered rapid?

    <p>No pre-concentration steps</p> Signup and view all the answers

    What is a primary challenge associated with using antibodies in POC diagnostics?

    <p>Challenging preparation of Ab pairs</p> Signup and view all the answers

    Which feature of POC diagnostic tests is most critical for user interaction?

    <p>User-friendliness</p> Signup and view all the answers

    Which feature of biosensors allows for stable performance across varying conditions?

    <p>No need for refrigeration</p> Signup and view all the answers

    Study Notes

    Copyright Regulations 1969 - Warning

    • This material has been copied and distributed by the University of Sydney.
    • Copying or further communication of this material may be subject to copyright protection.
    • Do not remove this notice.

    Acknowledgement of Country

    • Traditional Owners of Australia and their continuing connection to land, water, and culture are acknowledged.
    • Acknowledgement is made of the Gadigal people and their Elders (past, present and emerging).
    • Traditional Owners of the land on which the user is located are also acknowledged (past, present, and future).

    PHAR4813 - Methods for the Detection of Pathogenic Bacteria

    • Course taught by Professor Paul Groundwater.
    • Email address provided.
    • Course name: Methods for the Detection of Pathogenic Bacteria.
    • Course code: PHAR4813.

    The Post Antibiotic Age?

    • Antibiotic resistance is making gonorrhoea treatment harder, sometimes impossible.
    • Gonorrhoea bacteria evolve to resist antibiotics.
    • Widespread resistance to older, cheaper antibiotics is reported globally.
    • Untreatable gonorrhoea cases are reported in higher-income countries, where surveillance is better; lacking in lower-income countries where infection is common.
    • Estimated 78 million people are infected with gonorrhoea annually.
    • Complications include pelvic inflammatory disease, ectopic pregnancy, infertility, and increased risk of HIV.
    • Factors contributing to the rise in resistance include decreased condom use, increased urbanization, travel, poor detection rates, and inadequate or failed treatment.

    Antimicrobial Treatment

    • Traditional advice is to complete the course of antibiotics.
    • Optimizing antibiotic use is a concern.

    Australian Pharmacy Council - Pharmacy Learning Domains

    • Learning domain 1: Contribution to good health/disease prevention, symptom recognition/management, differential diagnosis, and medical terminology.
    • Learning domain 4: Medicines (types, regulation, and use), medical devices, and physiological function/delivery.
    • Learning domain 6: Wider context of pharmacy, encompassing scientific, clinical, health services, and social services research; relevant applications to practice.

    Overview

    • Bacterial detection is needed.
    • Surveillance techniques are necessary.
    • Methods include microscopy and staining; chromogenic/fluorogenic methods, and molecular diagnostic methods.

    "The World is facing an Antibiotic Apocalypse" - Prof Sally Davies, UK CMO

    • Antibiotic resistance is a global problem.
    • Data from 77 countries shows the issue.
    • Antimicrobial resistance could lead to up to 10 million deaths by 2050.
    • Estimated global cases of antibiotic-resistant infections are high.

    Predicted Annual Deaths (and Mortality Rates) Due to AMR by 2050

    • Estimated deaths due to antimicrobial resistance vary by region globally.

    Multidrug Resistant Organisms (MROs) and Drug Resistance

    • Multidrug-resistant organisms (MROs) are of particular concern, as are potentially widespread strains resistant or with limited susceptibility to antibiotics.
    • Healthcare-associated infections (HAIs) are a substantial global concern.
    • Estimates indicate that a large number of patients annually experience HAIs globally.

    AMR is a Global Problem: Global Initiatives

    • The Longitude Prize was established to address antibiotic resistance, with a $10M prize for the best solution.
    • A 5-year period has been established for proposing and evaluating solutions.

    Evidence-Based Prescribing Should be Informed by Diagnostic Testing

    • WHO's global action plan on antimicrobial resistance emphasizes the critical role of rapid, accurate diagnostics before prescribing.
    • Evidence-based prescribing relies on effective, low-cost diagnostic tests.
    • Clinicians should perform rapid diagnostic tests before prescribing antimicrobials.

    WHO Global Action Plan on AMR

    • Broad plan for combating Antimicrobial Resistance.
    • Aimed at strengthening knowledge and evidence, reducing the incidence of infection, and optimizing antibiotic use.
    • Includes elements like improved surveillance, international information-sharing, and economic case development for investment.

    We Need Rapid Surveillance Techniques for Pathogenic Bacteria to Inform the Use of Antimicrobial Agents

    • Surveillance data is used to identify colonized/infected patients and inform their treatment (rather than empirical treatment).
    • Surveillance should inform organism-specific transmission-based precautions.

    Antimicrobial Stewardship

    • The aim is to optimize antimicrobial use in hospitals.
    • Guidelines, formulary restriction, and staff training are key components.

    MRSA

    • MRSA is susceptible to few antimicrobial agents.
    • Cases of methicillin-resistant strains and resistant to quinupristin-dalfopristin are already reported in Europe.
    • Resistance occurs via mechanisms affecting the ligase activity.

    Does Universal Surveillance Work?

    • A study demonstrated reduced rates of MRSA clinical disease in a 3-hospital organization via universal surveillance/testing after baseline data collection.
    • Universal surveillance led to a reduction in post-discharge disease of 30+ days.
    • Further transmissions would have occurred if surveillance wasn't implemented.
    • A national audit (2011), revealed low compliance rates with infection control guidelines amongst English hospital organizations.
    • Many patients positive for MRSA were not isolated or treated.
    • A revised approach is recommended: checklist-activated screening of patients with high risk-factors

    Australia - Healthcare Associated Infections

    • List of common healthcare-associated infections (HAIs)

    Elsewhere (UK)

    • The Health and Social Care Act 2008 Code of Practice included relevant healthcare-associated infection (HAI) strategies and guidelines.
    • Healthcare-associated infections policies are required for outbreaks/infections, including screenings.

    Requirements of a Bacterial Surveillance Method

    • Requirements of reliable surveillance methods: sensitivity, specificity, speed, reliability, cost-effectiveness, and simplicity.

    Current Detection Methods

    • The goal is to provide concise, accurate, and timely summaries of current detection methods for pathogenic bacteria.
    • Methods include phenotypic methods (Biochemical testing, Chromogenic media, MALDI-TOF MS) and genotypic methods (PCR, WGS, and multi-omics).

    Microscopy / Staining

    • Types of microorganisms (cocci, bacilli, spirillum) and imaging, staining methods.
    • Classifications of gram-positive and gram-negative cocci and coccobacilli are considered.
    • Gram-positive cocci may or may not grow aerobically, potentially correlating to catalase activity.

    Chromophore/Fluorophore Detection

    • Chromophore release in the presence of specific bacteria confirms identification and localization.

    The Visible Region

    • Wavelengths and perceived colours.
    • Chromogen/Fluorogen absorbs light at specific wavelengths, enabling colour-based identification.

    Chromophores

    • Description of chromophores and conjugation.
    • Understanding the relationship of electronic transitions to observed colours.
    • Relation to absorption maxima and the role in UV-Vis spectroscopy

    UV-VIS Spectrum of β-Carotene in Ethanol

    • Visible region data for the β-Carotene sample and wavelength.

    Phenolphthalein

    • Chemical structures and absorption data are summarized to demonstrate the principle of chromogens shifting colour to indicate particular conditions.

    Requirements for Chromogen

    • Criteria for chromogenic molecules for rapid detection.

    Specific Bacterial Enzyme Targets

    • Overview of enzymes/targets for specific pathogenic bacteria.

    Chromophore/Fluorophore Detection

    • Chromophore/fluorophore release in the presence of specific bacterial enzyme, is crucial.

    9-(4-N'-L-Ala-L-Ala-L-Ala-aminophenyl)-10-methylacridinium bis(trifluoroacetate)

    • Structural features are summarized to identify an appropriate chromophore.

    9-Aminophenyl-10-methylacridine

    • Structural features are summarized to identify an appropriate chromophore.

    Natural Phenoxazinones

    • Overview of natural products with chromogenic properties, including structural features.
    • Their use in detection, and applications to particular organisms is highlighted.

    Target Molecules: 1,2-Substituted-7-aminophenoxazinones

    • Compounds, highlighting structure.
    • Uses in identification.

    Pseudomonas aeruginosa

    • Pseudomonas aeruginosa characterization and its clinical relevance.
    • Difficulties in treatment are acknowledged.
    • Early detection and treatment are stated as critical aspects of managing this pathogen.

    Why β-Ala-Containing Substrates?

    • Rationale for using β-alanine-containing substrates for differentiating Gram-positive and Gram-negative bacteria.
    • Focus is on L-alanyl aminopeptidase and its presence in Gram-negative bacteria.

    7-N-(β-Alanyl)amino-1-pentylresorufamine (β-Ala-1-PRF)

    • Results of testing for β-Ala-1-PRF assay and the results of testing this against a range of different microorganisms using this diagnostic method.

    Origin of Colour

    • Mechanism for colour generation in presence of specific bacterial enzyme.

    Molecular Diagnostic Methods

    • Methods are based on PCR or mass spectrometry.
    • These are complex but offer speed and specificity

    PCR Technology

    • PCR technology provides multiple DNA copies.
    • Denaturation, annealing, and extension are steps of PCR testing.

    Real-time PCR Detection of MRSA in a Mixture of Staphylococci

    • Specific primers/probes for detecting MRSA strains among Staphylococci.
    • High accuracy rate for MRSA identification.

    Drawbacks with quantitative PCR (qPCR)

    • Concerns about contamination are stated.
    • The difference between asymptomatic colonization and clinically relevant infections relies on cut-off values and relationships between microorganisms and disease.

    PCR Technology

    • Traditional vs real-time PCR detection methods, and use of molecular beacons.

    Real-time PCR Detection of Other Organisms

    • PCR assays for other organisms (including vancomycin-resistant enterococci, oxazolidinone-resistant bacteria, and Pseudomonas aeruginosa).

    Vancomycin (Vancocin)

    • Vancomycin mechanism of action highlighting hydrophobic nature and interaction with the D-alanine-D-alanine sequence (prevention of cross-links).
    • Importance of ligase and its alteration due to antimicrobial resistance mechanisms is highlighted.

    MALDI-TOF Mass Spectrometry

    • Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) methods.
    • Analysis for identification including structural/molecular weight of microbial components.

    MALDI-TOF MS Detection of MRSA

    • MALDI-TOF MS distinguishing features between MRSA and MSSA strains.

    MALDI-TOF MS Detection of Carbapenemase Activity

    • MALDI-TOF MS is used to detect carbapenemase activity of different bacterial strains.

    Whole Genome Sequencing (WGS)

    • WGS approach for providing comprehensive pathogen and antimicrobial-resistance profiling.
    • Its applications in outbreak investigation and developing PCR probes are described.

    Whole Genome Sequencing (WGS) - Sanger method

    • Sanger method in WGS.

    Point-of-Care (POC) Tests / Devices

    • POC tests performed near patients for swift diagnosis; reducing time and necessity for central labs.

    WHO Criteria for an Ideal Point of Care (POC) Diagnostic Test

    • Requirements for rapid, accessible, accurate, and cost-effective diagnostics for patient care.

    Biorecognition Elements Used as Biosensors in POC Devices

    • Biorecognition elements include Antibodies, Aptamers, Nucleic Acids, and Proteins.
    • Advantages and disadvantages of each element, including considerations for point-of-care (POC) devices (e.g., sensitivity, stability, and cost), are highlighted.

    Electrochemiluminescence (ECL) detection of antibody sandwich complex

    • ECL method for antibody detection and its features; including sensitivity, specificity, and limitations.

    Aptamer-based Latent TB Infection Test

    • Colorimetric approach using aptamers to detect latent TB infection.

    Magnetic Nanoparticles for Detection of S. aureus Protease

    • Use of black-colored magnetic nanoparticles, allowing detection with the naked eye.

    Requirements of a Bacterial Surveillance Method

    • Table summarizing the key requirements of chromogenic media, PCR, and MALDI-TOF MS methods in bacterial surveillance.

    WORKSHOP - The Specific Detection of Pathogenic Bacteria using MALDI-TOF

    • Overview of workshop, including topics assigned per group.

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    PHAR4813 Methods for the Detection of Pathogenic Bacteria PDF

    Description

    Explore the critical methods for detecting pathogenic bacteria as covered in the PHAR4813 course taught by Professor Paul Groundwater. This quiz delves into the challenges posed by antibiotic resistance, particularly in the treatment of gonorrhoea. Understand the implications and evolving nature of bacterial resistance in clinical settings.

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