Assay Development

BCARE personnel have considerable in depth knowledge in the development of new high pressure liquid chromatography (HPLC) and LC/MS assays for antibacterials, antivirals and antifungals. Immunoassay technologies have also been extensively trialled prior to introduction into clinical practice. In recent times the BCARE staff have developed HPLC assays for anti viral and anti fungal agents and we are keen to pursue pharmacokinetic studies on antivirals. Our expertise in HPLC assay has also lead to an active research interest in antimicrobial metabolites and/or breakdown products.

Pharmacokinetics

The staff within BCARE have a considerable expertise in antimicrobial pharmacokinetics both in patients and healthy volunteers. In the recent past pharmacokinetic studies have been performed in ITU patients, patients with severe sepsis (bacteraemia, pulmonary infection, after surgery) and orthopaedics.

These pharmacokinetic interests overlap with our knowledge of antimicrobial assay development and pharmacodynamics. In addition to performing pharmacokinetic studies BCARE supports pharmacokinetic research in other centers by the provision of assay services and external quality assurance.

Pharmacodynamics

Pharmacodynamics is an expanding and well established area, and it complements our strong track record in the basic laboratory aspects of antimicrobial chemotherapy and pharmacokinetic evaluations. All pharmacodynamic activities are laboratory based; animals are not used. Expertise exists to perform simple investigations such as post-antibiotic effect through to complex antimicrobial dose fractionation studies using continuous bacterial culture in-vitro models. These models enable intravenous, intramuscular and oral drug concentration serum profiles to be modelled. Hollow fibre in-vitro pharmacokinetic models are also used in preclinical antibacterial drug evaluation. At present we have experience with β-lactams, quinolones, glycopeptides, aminoglycoside and other antimicrobials classes in our in-vitro model systems.

Many major pathogen groups can be studied using these systems, for example, staphylococci, streptococci, enterobacteriacae, haemophilus, pseudomonas and anaerobes.

Antimicrobial Susceptibility Testing Techniques

Expertise exists to perform susceptibility tests by agar dilution and micro-and macro-broth dilution techniques, and E strips. BSAC, EUCAST or CLSI methodologies are used. In addition, β-lactamase induction tests can be performed. Confirmation of hGISA/GISAs by population analysis profiles (PAPs) are available.

Bactericidal-time kill curves can be performed and bacterial killing with single agents and combinations studied, using novel, computer generated curve fitting techniques.

Current work:

• in vitro susceptibility testing.
• determination of minimum inhibitory concentrations (MICs).
• bactericidal studies on antimicrobials.
• antibacterial – antibacterial interactions.

Within the Department of Medical Microbiology at Southmead is a collection of >40,000 bacterial strains, mostly isolated from clinical specimens, about 3000 of which were isolated from blood cultures. The collection contains significant numbers of all the following species:

Gram positive aerobic cocci:

Enterococcus faecalis, Enterococcus faecium; Staphylococcus aureus, penicillinase negative, penicillinase positive, methicillin resistant; coagulase negative staphylococci (various species); Staphylococcus saprophyticus; Streptococci of Lancefield Groups A, B, C, F and G; penicillin-susceptible and penicillin resistant Streptococcus pneumoniae; viridans streptococci.

Gram positive aerobic bacilli: Bacillus spp:

Corynebacteria spp; Listeria monocytogenes; other Listeria spp.

Gram negative aerobic cocci:

Moraxella catarrhalis; Neisseria meningitidis; Neisseria gonorrhoeae.

Gram negative aerobic bacilli:

Haemophilus influenzae, β-lactamase-negative and β-lactamase-positive; Citrobacter spp; Enterobacter spp; Escherichia coli; (ESBL producers and others); Klebsiella spp; Morganella morgani; Proteus spp; Providencia spp; Salmonella typhi, other Salmonella spp; Serratia spp; Shigella spp; Hafnia alvei; Aeromonas spp; Acinetobacter spp; Campylobacter spp; Pseudomonas aeruginosa (with multiple resistance mechanisms) other Pseudomonas spp; Stentotrophomonas maltophilia.

Anaerobes: Actinomyces spp:

Bacteroides fragilis, other Bacteroides spp; Bifidobacterium spp; Clostridium difficile, Clostridium perfringens, other Clostridium spp; Eubacterium spp; Fusobacterium spp; Lactobacillis spp; Peptostreptococcus spp; Prevotella spp; Propionibacterium spp; Porphromonas spp; Veilonella spp.

BCARE has a record of research and development in the field of antimicrobial chemotherapy and resistance going back more than 35 years. These research activities inter-relate, support and augment several other activities such as running the UK National External Quality Assurance Scheme (UK NEQAS) for antibiotic assays and the provision of specialist antimicrobial assays for health providers in the UK.

Southmead and Frenchay Hospitals, situated in North Bristol, is the largest NHS trust in the South West of England with over 1000 beds and is a University of Bristol teaching trust which is one of two medical schools in the South West.

The Antimicrobial Reference Laboratory provides a comprehensive assay service for the purposes of therapeutic monitoring for a wide range of antimicrobial, antifungal and antiviral agents using both liquid chromatography and LC/MS methodology. This is the only such service provided in the UK and includes the provision of consultative advice on technical aspect of TDM and the clinical interpretation of assay results. The service has been provided since the mid 1980s and year-on-year there have been increases in both the number of laboratories using the service and the total number of samples assayed, with currently over 240 laboratories using the service each year. The full range of analytes assayed and some of the conditions under which they may need to be assayed are detailed in the assay book which is circulated annually.

The Antimicrobial Reference Laboratory also offers a comprehensive bacteriology service for both routine diagnostic and research purposes. These services include undertaking specialist susceptibility testing, such as minimum inhibitory and bactericidal concentrations, population analysis profiles for hVISA, serum cidal assays and bacterial kill curves. This is backed up with an expanding molecular diagnostics service which is able to undertake molecular detection of a range of pathogenicity and antimicrobial resistance genes, along with typing of organisms in epidemiological studies.

Alasdair MacGowan BMedBiol (Hons), MD, FRCP, FRC.Path Professor of Clinical Microbiology & Antimicrobial Therapeutics, University of Bristol and North Bristol NHS Trust.

Alasdair MacGowan is Professor of Clinical Microbiology and Antimicrobial Therapeutics at the University of Bristol and Honorary Consultant at North Bristol NHS Trust. He graduated in Medicine at the University of Aberdeen, and trained in Infection in Aberdeen, Birmingham and Bristol. At present, he is Head of Research and Specialist Services in the Department of Medical Microbiology, North Bristol NHS Trust. His main research interests are in antibacterial pharmacokinetic/pharmacodynamics, resistance epidemiology in the community and understanding risk factors for poor outcome in hospitalised patients. He is a former President of the British Society for Antimicrobial Chemotherapy, and Chair of the Journal of Antimicrobial Chemotherapy Editor Board. Presently, he is Chairman of the BSAC Working Party on Antibiotic Resistance Surveillance, a member of the BSAC Working Party on Antimicrobial Susceptibility Testing, and a member of the European Committee on Antimicrobial Susceptibility Testing (EUCAST).

Andrew Lovering, BSc, PhD Consultant Clinical Scientist, North Bristol NHS Trust

Andrew Lovering is a Consultant Clinical Scientist and the scientific lead for the Antimicrobial Reference Laboratory at Southmead Hospital in Bristol. He graduated from the University of Reading and trained as a Clinical Scientist in Bristol, where he undertook his PhD. His main area of scientific interest is in the field of antimicrobial chemotherapy and many of the research studies that he has been involved with relate to the assay of antimicrobials, their pharmacokinetic/pharmacodynamic evaluation or the surveillance of antimicrobial resistance. He is a former editor of the Journal of Antimicrobial Chemotherapy. Currently, he is a member of the UKNEQAS Steering Committees for both Microbiology and Drug Monitoring and a member of the UKNEQAS Executive Committee. He is also the Clinical Lead for Infection at the Western Comprehensive Local Research Network and a member of the British Society for Antimicrobial Chemotherapy Working Party on Therapeutic Drug Monitoring.

Karen Bowker, PhD Principal Clinical Scientist, North Bristol NHS Trust

Karen Bowker has been an active research Clinical Scientist at the North Bristol NHS Trust since 1992 where she trained as a clinical scientist and undertook her PhD. She is scientific lead for the pharmacokinetic/pharmacodynamic (PK/PD) laboratory.  Her other main area of research is antimicrobial chemotherapy, antibacterial susceptibility testing and, assay of antimicrobials. She is currently a member of the BSAC working party on antimicrobial susceptibility testing. She is the lead organiser of the Antibiotic Assay Course held annually in Bristol. She is the author of over 80 peer-reviewed scientific publications and approximately 50 scientific abstracts and is a reviewer for several current journals.

John Leeming, BSc PhD Principal Clinical Scientist, North Bristol NHS Trust.

John joined the team in 2010, with a remit to lead molecular investigations. His academic training was undertaken at the University of Leeds and he then moved to Bristol become a clinical scientist. His interests include exploiting molecular biology techniques in the detection and characterisation of microbial pathogens, control of hospital infection, pneumococcal disease and the microbiology of skin. He is an honorary lecturer at the University of Bristol and has published over 50 peer-reviewed papers.

Alan Noel, MSc, Clinical Scientist, Antimicrobial Reference Unit, North Bristol NHS Trust

Alan Noel has been an active research Clinical Scientist at North Bristol NHS Trust since 2001. He has trained as a clinical scientist, with a focus on the Therapeutic Drug Monitoring of antibiotic, antifungal and antiviral agents. He has recently started a PhD – looking at the use of in-vitro pharmacokinetic infection models in anti-infective drug development. He is deputy scientific lead for the pharmacokinetic/pharmacodynamic (pK/pD) laboratory.  He has a keen interest in laboratory quality and has been the UK NEQAS for Antibiotic Assays Scheme Manager for 10 years. He is the deputy organiser of the Antibiotic Assay Course held annually in Bristol. He is the author of over 20 peer-reviewed scientific publications and approximately 50 scientific abstracts.

Members of BCARE have a considerable commitment to teaching undergraduate and postgraduate health care and associated workers in the area of antimicrobial chemotherapy. These include:-

• BSc and MSc courses and supervision of PhD students at the University of Bristol and the University of the West of England (UWE). Various staff are registered for MD, PhD or MSc degrees. Tutorials for medical students, registrars, infection control nurses and laboratory staff are regularly held at Southmead and Frenchay Hospitals
• ‘The Antibiotic Testing Course’ is held annually at University of the West of England in July. This covers all aspects of susceptibility testing, antibiotic assays, quality assurance / control / assessment, antibiotic/prescribing/guidelines/usage.

Molecular analysis

Molecular analysis is becoming increasingly important in the activities of the Laboratory. We are interested in the application of molecular technology for the diagnosis of infection and for characterization of bacterial isolates (detection of antibiotic resistance and virulence markers, sub-species typing). In particular we have introduced, or are working on, assays for:

• Direct detection of Mycobacterium tuberculosis in samples and characterization of mycobacteria in liquid cultures
• Typing and detection of virulence and antimicrobial resistance markers in Staphylococcus aureus
• Detection and typing of Clostridium difficile.

We have also undertaken gene sequencing for determination of resistance mechanisms in Streptococcus pneumoniae and detection of resistance markers in Gram negative bacilli by microarray analysis.

Disc susceptibility testing using BSAC and EUCAST methodologies are performed. Blactamase induction and MLSb testsare available.

MIC determination using BSAC, CLSI or ISO broth and agar dilution methodologies are performed. Determination of hGISA and GISAs are available.

Assay Development

BCARE personnel have considerable in depth knowledge in the development of new high pressure liquid chromatography (HPLC) and LC/MS assays for antibacterials, antivirals and antifungals. Immunoassay technologies have also been extensively trialled prior to introduction into clinical practice. In recent times the BCARE staff have developed HPLC assays for anti viral and anti fungal agents and we are keen to pursue pharmacokinetic studies on antivirals. Our expertise in HPLC assay has also lead to an active research interest in antimicrobial metabolites and/or breakdown products.

Pharmacokinetics

The staff within BCARE have a considerable expertise in antimicrobial pharmacokinetics both in patients and healthy volunteers. In the recent past pharmacokinetic studies have been performed in ITU patients, patients with severe sepsis (bacteraemia, pulmonary infection, after surgery) and orthopaedics.

These pharmacokinetic interests overlap with our knowledge of antimicrobial assay development and pharmacodynamics. In addition to performing pharmacokinetic studies BCARE supports pharmacokinetic research in other centers by the provision of assay services and external quality assurance.

Pharmacodynamics

Pharmacodynamics is an expanding and well established area, and it complements our strong track record in the basic laboratory aspects of antimicrobial chemotherapy and pharmacokinetic evaluations. All pharmacodynamic activities are laboratory based; animals are not used. Expertise exists to perform simple investigations such as post-antibiotic effect through to complex antimicrobial dose fractionation studies using continuous bacterial culture in-vitro models. These models enable intravenous, intramuscular and oral drug concentration serum profiles to be modelled. Hollow fibre in-vitro pharmacokinetic models are also used in preclinical antibacterial drug evaluation. At present we have experience with β-lactams, quinolones, glycopeptides, aminoglycoside and other antimicrobials classes in our in-vitro model systems.

Many major pathogen groups can be studied using these systems, for example, staphylococci, streptococci, enterobacteriacae, haemophilus, pseudomonas and anaerobes.

Sensitivity Testing:

Techniques

Expertise exists to perform susceptibility tests by agar dilution and micro-and macro-broth dilution techniques, and gradient strips. BSAC, EUCAST, CLSI or ISO methodologies are used. In addition, β-lactamase induction tests can be performed. Determination of GISAs, hGISAs are also available.

Bactericidal-time kill curves can be performed and bacterial killing with single agents and combinations studied, using novel, computer generated curve fitting techniques.

Current work:

• In vitro susceptibility testing.
• Determination of minimum inhibitory concentrations (MICs).
• Bactericidal studies on antimicrobials.
• Antibacterial – antibacterial interactions.

How long does the intervention take?

Are there cost implications with regard to staffing resource and the cost of the electronic patient record?

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