PHAR4813: Detection of Pathogenic Bacteria

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• Traditional Owners of Australia and their continuing connection to land, water, and culture are acknowledged.
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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.

Universal MRSA Surveillance is No Longer Recommended

• 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.

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.