Academy of Clinical Science and Laboratory Medicine President’s Prize

The President’s Prize Competition entrants presented their research at LabCon2019. The research carried out by the students was of an exceptionally high standard. There was a large attendance and our plenary session speakers, Prof. Jo Martin and Dr Karin Denton both attended, in spite of the early morning start and their own presentation later on in the morning. They were seriously impressed with the standard.
The judges had a tough decision.
Congratulations to Katherine Dever and her supervisor Dr Debbie Corcoran. Katherine’s work, “Antibiotic Resistance Reversal in Multi-Drug Resistant Klebsiella pneumoniae Exploring the soxS Dependent Regulon” goes forward to the European Association for Professions in Biomedical Science (EPBS) meeting in October to participate in the poster competition with other student across the EU for the Martin Nicholson Award.

President’s Prize 2019 Judging Panel

CONGENITAL MACROTHROMBOCYTOPAENIA; DO DEVIATIONS IN THE CALCIUM BINDING AFFINITY OF ACTININ-1 MUTANTS CONTRIBUTE TO ITS PATHOGENESIS?

Aidan Murphy
Authors: Murphy A, Young P
Topic: Haematology
Institution: University College Cork
Address of First Author: Haematology Department, University
Hospital Waterford,

Biography: I am currently doing my student placement at University Hospital Waterford, having recently graduated with a
Biomedical Science degree from University College Cork/ Cork Institute of Technology. I have always expressed a keen interest in Science, as is reflected in the numerous scholarship funded research projects that I have partaken in throughout my years in college. My previous research at the APC Microbiome Institute with Dr. David Clarke and Dr. Susan Joyce was Microbiology based and involved studying bile resistance mechanisms in Bacteroides species. My Final Year Project with Dr. Paul Young was haematology based and involved identifying
pathogenic mechanisms for Congenital Macrothrombocytopaenia. I am dedicated and passionate about my work and I am enthusiastic about seeing what challenges are in store for me as a Medical Scientist in the future.

Actinin-1 is a cytoskeletal protein which crosslinks actin filaments, however recent studies have shown that mutations in actinin-1 cause a rare platelet disorder known as congenital macrothrombocytopenia (CMTP). My project attempts to identify a potential pathological mechanism for this disease by studying several disease-associated mutations localised within the calmodulin domain of actinin-1. Calcium plays a critical role in
regulating actin crosslinking by interacting with the calmodulin domain in actinin-1 (Drmota Prebil et al., 2016). This regulatory role of calcium in actin crosslinking is necessary for many platelet production and platelet activation events (Machlus, Thon and Italiano, 2014). Hence, it has been hypothesised that mutations in the calmodulin domain of actinin-1 may alter its calcium binding affinity, which may consequently interfere with normal platelet processes, thereby contributing to the pathogenesis of CMTP. To test this hypothesis wild type actinin-1 and CMTP associated
actinin-1 mutant proteins (G764S and E769K) were expressed using recombinant E.coli BL21 cells, and purified using techniques such as nickel column and GST column purification, as well as size exclusion fast protein liquid chromatography. The use of novel techniques such as microscale thermophoresis and isothermal calorimetry is then used on these purified actinin-1 proteins to provide a new insight into the binding interactions and thermodynamic events that occur between the calmodulin domain of actinin-1 CMTP associated mutants and calcium.

The results obtained showed that the G764S mutated actinin-1 had no difference in calcium binding affinity (KD) against wild type actinin-1, while the E769K mutation showed a noticeable difference in calcium binding affinity against wild type actinin-1. The results provided a new structural and mechanistic insight into the basis of actinin-1 dysfunction in CMTP and helped elude to the pathogenies of CMTP in individuals carrying the mutations
studied. The results of the project elucidate that the changes in calcium binding affinity in mutant actinin-1 proteins affect the strength in which actinin-1 can secure actin filaments, which can therefore alter normal platelet processes, thereby resulting in the occurrence of CMTP.

References:
Machlus, K., Thon, J. and Italiano, J. (2014). Interpreting the developmental dance of the megakaryocyte: a review of the cellular and molecular
processes mediating platelet formation. British Journal of Haematology, 165(2), pp.227-236. Drmota Prebil, S., Slapša, U., Pavšic, M., Ilc, G., Puž,
V., de Almeida Ribeiro, E., Anrather, D., Hartl, M., Backman, L., Plavec, J., Lenarcic, B. and Djinoviç-Carugo, K. (2016). Structure and calcium-binding studies of calmodulin-like domain of human non-muscle ·-actinin-1. Scientific Reports, 6(1).15-e518.

CARBAPENEM AND COLISTIN RESISTANCE DETECTION: A CHALLENGE FOR THE DIAGNOSTIC MICROBIOLOGY LABORATORY.

Kate Byrne
Topic/Discipline: Medical Microbiology
Byrne, K.1, Drudy, D.1, Keating, D.2, Coyle, E.2, Donoghue, O.2.
1Technological University Dublin
2Microbiology, St. Vincent’s University Hospital

Biography: I graduated in 2018 with a BSc in Biomedical Science (TUD). My undergraduate research project involved the evaluation of molecular assays for the detection of carbapenem resistant Enterobacterales in screening specimens. This was followed by the evaluation of a rapid method for the detection of colistin resistance in Enterobacterales. I am currently working as a medical scientist in the Microbiology department of the Mater Misericordiae University Hospital.

Background: The global dissemination of carbapenemase producing Enterobacterales (CPE) represents a significant public health issue. Rapid and accurate detection of CPE colonisation through screening is essential to enable effective infection control strategies. Carbapenem resistance detection remains a challenge for the diagnostic microbiology laboratory (1). The EntericBio realtime® CPE assay and the GeneXpert® Carba-R assay are molecular assays designed for the in vitro diagnostic testing and qualitative detection of Enterobacterale produced, carbapenemase genes: KPC, OXA-48, NDM, VIM and IMP . Colistin resistant Enterobacterales are being increasingly reported worldwide. Colistin susceptibility testing also represents a challenge for the diagnostic microbiology laboratory. The Rapid Polymyxin NP kit detects colistin resistance in Enterobacterales (2).

The aim of this study was to evaluate the EntericBio realtime® CPE assay for direct use, the GeneXpert® Carba-R assay and the Rapid Polymyxin NP kit for use with enterobacterial isolates in a diagnostic
microbiology laboratory.

Methods: A total of 277 rectal swabs were tested directly using the EntericBio realtime® CPE assay. A total of ten enterobacterial isolates were processed on the GeneXpert Carba-R assay and twelve enterobacterial isolates using the Rapid Polymyxin NP kit. Results: The EntericBio realtime® CPE assay demonstrated a sensitivity, specificity, positive predicted value (PPV) and negative predicted value (NPV) of 100%, 98.95%, 95.1% and 100% respectively. The GeneXpert® Carba-R assay and the Rapid Polymyxin NP kit exhibited sensitivities of 100% and 66.7% and specificities of 80% respectively.

Conclusion: The EntericBio realtime® CPE assay and the GeneXpert® Carba-R assay are rapid and accurate molecular assays for the detection of CPE that facilitate the improvement of infection control strategies. The Rapid Polymyxin NP kit is a rapid and easy to use method for the detection of colistin resistance. However, further study is required to determine its utility in a diagnostic microbiology laboratory.

References:
(1) Humphries, R. M. and McKinnell, J. A. Continuing Challenge for the Clinical Laboratory for Detection of Carbapenem-Resistant Enterobacteriaceae. J Clin Microbiol. 2015; 53(12):3712-3714.
(2) Poirel, L., Jayol, A. and Nordmann, P. Polymyxins: Antibacterial Activity, Susceptibility Testing, and Resistance Mechanisms Encoded by Plasmids or Chromosomes. Clinical Microbiology Reviews. 2017;30(2): 558-570

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RESISTANCE REVERSAL IN MULTI-DRUG RESISTANT KLEBSIELLA PNEUMONIAE: EXPLORING THE SOXS DEPENDENT REGULON

Kate Dever
Topic: Medical Microbiology
Centre for Food Safety University College Dublin,
Wyckham Way, Dundrum, Dublin
Dever, K, Srikumar, S, Anes, J, Fanning, S, Corcoran, D.

Biography: I completed my undergraduate degree in the Galway Mayo Institute of Technology. I have since been lucky enough to work in the University College Dublin Centre for Food Safety, where I completed my thesis, as a research technician in molecular microbiology. In September of 2018 I was offered a permanent position as a medical scientist in the microbiology laboratory of St. Vincent’s University Hospital Dublin, where I continue to work today.

The overuse and misuse of antibiotics has led to the emergence and dissemination of multi-drug resistance in Gram-negative pathogens, jeopardising the advances of modern medicine. The SoxRS regulon plays a key role in bacterial defence against oxidative stress (1). The aim of this project was to determine the role of the transcription factor, SoxS, as a regulator of antibiotic resistance under oxidative stress conditions (2). This was achieved by employing the multi-drug resistant (MDR) wildtype Klebsiella pneumoniae MGH 78578, the deletion mutant Klebsiella
pneumoniae MGH 78578 δsoxS (with soxS deleted from the bacterial chromosome), and the complementation Klebsiella pneumoniae MGH
78578 δsoxS pBADsoxS isolate (where soxS was over-expressed from the plasmid pBAD using an arabinose inducible promoter).

The project involved the interpretation of RNA (ribonucleic-acid)- sequencing data obtained from a broader study, conducted on MDR Klebsiella pneumoniae placed under oxidative stress conditions using paraquat, at the University College Dublin-Centre for Food Safety (UCDCFS). In order to determine if genotypic observations from this data translated into distinct phenotypes, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays were performed using six antibiotics namely, colistin, gentamicin, kanamycin, cefotaxime, tetracycline and rifampicin. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was employed for the confirmation of soxS over-expression in the complemented Klebsiella  pneumoniae MGH 78578 δsoxS pBADsoxS strain. Finally, the project incorporated the objective to determine the 5’transcription-start-site (5’TSS) of soxS using random amplification of cDNA (complementarydeoxyribonucleic-acid) ends.

The RNA-sequencing data outlined that under oxidative stress conditions certain antibiotic resistance genes were down-regulated in the absence of soxS. This was reflected in the phenotypic MIC and MBC assays which showed antibiotic resistance reversal in the Klebsiella pneumoniae MGH 78578 δsoxS isolate for the antibiotics cefotaxime and tetracycline.

These results highlighted that regulatory molecules, such as soxS, may provide novel targets to mitigate multi-drug resistance in the future.

References:
(1) Seo SW, Kim D, Szubin RO, Palsson B. Genome-wide reconstruction of OxyR and SoxRS transcriptional regulatory networks under oxidative stress in Escherichia coli K-12 MG1655. Cell Reports. 2015;12(8): 1289-1299.
(2) Kohanski MA, Dwyer DJ, Collins JJ. How antibiotics kill bacteria: from targets to networks. Nature Reviews Microbiology. 2010;8(6): 423-435.

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READY-TO-EAT DAIRY FOODS: A POTENTIALLY IMPORTANT VECTOR OF ANTIBIOTIC-RESISTANT BACTERIA

Shannon Diggin
Discipline: Medical Microbiology.
Institution: Department of Biological Sciences, Cork Institute of
Technology.
Authors: Diggin, S., Sleator, RD., Culligan, EP.

Biography: Biomedical Science student from Listowel, Co. Kerry. Currently undertaking clinical laboratory placement at University Hospital Kerry.

A perceived boost in nutritional value and flavour has seen the market for minimally processed, ready-to-eat (RTE) foods grow considerably in recent years. However, mounting food safety concerns have accompanied this demand, with RTE produce being increasingly recognised as a potential vehicle to transfer not only foodborne pathogens, but also antibiotic-resistant bacteria and their associated resistance genes to the consumer (1). This study examined the microbiological quality of RTE, unpasteurised milk and cheeses and investigated the antibiotic-resistance profiles of the bacteria present. Enumeration of the levels of bacteria was determined in CFU/mL and revealed that three out of the four samples tested contained unacceptably high quantities of bacteria (2), indicating poor standards of food hygiene and safety. Five species of bacteria were isolated from the dairy products; Hafnia spp. (cheese), Klebsiella oxytoca, Kluyvera cryocrescens, Escherichia coli and Hafnia paralvei (milk). All isolates were resistant to a minimum of three antibiotics (ampicillin, cephradine and fusidic acid), when subjected to antimicrobial susceptibility test using the agar disc diffusion method. The antimicrobial resistance profile of Hafnia spp. was particularly alarming; displaying resistance
to 8 out of 11 antibiotics tested and PCR revealed positive carriage of the ampC beta-lactamase gene. Moreover, bacterial isolates were shown to be capable of surviving conditions that simulated the human gastrointestinal tract and were resistant to a number of disinfectants, following stress survival testing. These findings suggest that individuals consuming unpasteurised dairy may be routinely inoculated with resistant bacteria and further reinforces the knowledge that raw milk and cheeses may represent a significant environment for the evolution of antibiotic-resistant bacteria and their spread to humans (3). The potential health implications of consuming RTE foods, therefore, need to be carefully re-evaluated. Limiting the spread of resistant bacteria throughout the food chain remains a crucial strategy to combat the global antibiotic resistance crisis.

References:
(1) Hudson, JA., Frewer, LJ., Jones, G., Brereton, PA., Whittingham, MJ., Stewart, G. The agri-food chain and antimicrobial resistance: A review. Trends Food Sci. Technol. 2017; 69:131–147. https://doi.org/10.1016/j.tifs.2017.09.007
(2) European Commission. Regulation (EC) No. 92/46/EEC of 16 June 1992 laying down the health rules for the production and placing on the market of raw milk, heattreated milk and milk-based products. Official Journal of the European Union. 1992; 268, (14):1–32
(3) Verraes, C., Van Boxstael, S., Van Meervenne, E., Van Coillie, E., Butaye, P., Catry, B., de Schaetzen, MA., Van Huffel, X., Imberechts, H., Dierick, K., Daube, G., Saegerman, C., De Block, J., Dewulf, J., Herman, L. Antimicrobial Resistance in the Food Chain: A Review. Int. J. Environ. Res. Public. Health. 2013; 10: 2643–2669. https://doi.org/10.3390/ijerph10072643

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EQUIVALENCE STUDY OF THE LYOPHILISED ENTERICBIO® NOROVIRUS ASSAY (EBVP2) TO THE ORIGINAL CE-IVD (EUROPEAN CONFORMITY – IN VITRO DIAGNOSTICS) MARKED ENTERICBIO® NOROVIRUS ASSAY (EBNOV) ON THE MARKET

Alanna Maunsell
Topic – Medical Microbiology
Institution – Serosep Ltd., Co. Limerick
Authors – Maunsell, A., O’Leary, J., Corcoran, D.”

Introduction: Norovirus is a major cause of inflammatory gastroenteritis. It peaks during the winter months and early spring giving it the name ‘Winter Vomiting Disease’. This study was carried out to assess the equivalence of the lyophilised EntericBio® Norovirus assay (EBVP2) to the CE-IVD marked EntericBio® Norovirus assay (EBNoV). These assays detect
Norovirus genogroup I and Norovirus genogroup II directly from faecal sample. The equivalence of the assays was assessed under analytical sensitivity, clinical sensitivity and clinical specificity.

Methods: The analytical sensitivity of the EBVP2 assay was examined by testing RNA solution diluted to the concentrations relevant to the limit of detection of the EBNoV assay – 100 genome copies for Norovirus genogroup I and 50 genome copies for Norovirus genogroup II. 24 replicates of each genogroup were tested. The clinical sensitivity was assessed by testing 30 faecal samples using both assays that had previously tested positive for Norovirus. The clinical specificity was assessed by testing 80 samples using both assays that had previously tested negative.

Results: The EBVP2 assay had an analytical sensitivity of 100% at the limit of detection (LOD) previously established for the EBNoV. Both assays had a clinical sensitivity and specificity of 100%. However, there was a difference in Cp values of positive samples in the assays. Reducing the time between heat treatment and testing improved this difference.

Conclusions: These results show that the assays are equivalent but poses questions about the stability of RNA in heated stool preparation solution.

THE PRODUCTION OF FORS1-POSITIVE CELLS AND PREVALENCE OF ANTI-FORS ANTIBODIES IN A PORTUGUESE POPULATION

Alyssa Corpuz
Topic: Transfusion and Transplantation Science
Department Biomedical Laboratory Sciences, ESTESC- Coimbra
Health School, Polytechnic Institute of Coimbra, Coimbra, Portugal
Authors: Corpuz, A, Mourato, C, Galvao, S, Jesus, C, Mendes, F.

Biography: I am a 1st Class Honours Graduate in Biomedical Sciences from Dublin Institute of Technology specialising in Blood Transfusion and Medical Microbiology. I completed my research project in Coimbra Health School, Portugal in the area of Transfusion Medicine. Since the completion of my project I have been working fulltime as a medical scientist in Beaumont Hospital Blood Transfusion Department.

In 2012, the FORS system was accepted by International Society of Blood Transfusion as the 31st blood group system. Forssman (Fs) antigen (Ag) expression is rare in human red blood cells (RBCs) and most commonly found on sheep RBCs. Anti-Fs antibodies (Abs) are naturally occurring in human sera and are predominantly IgM but can also be IgG. To this day the global prevalence of the FORS system is unknown. Currently, there is a lack of natural FORS1- positive RBCs available to use for anti-Fs screening in large scale populations. This study was designed to produce FORS1-positive cells viable for use in the screening and classification of anti-Fs in a Portuguese population.
A 3-5% FORS1-positive cell suspension was produced using sheep’s blood in CellStab stabilizer solution. The quality of the FORS1-positive cells was investigated through three independent experiments of AB0 titration, osmotic fragility test and supernatant haemolysis. For each batch of FORS1-positive cells produced, an extended antibody panel was performed. Demonstrating that the FORS1-positive cells can be used for up to 40 days, anti-Fs screening and classification was carried out in a patient (1115) and donor (1400) population. Antigenic expression and membrane integrity of FORS1-positive cells remained stable for 40 days. Good Fs Ag preservation was established and minimal haemolysis was observed. With this novel reagent, a large population screen was carried out providing evidence that FORS is a rare blood group system (99.97% anti-
Fs prevalence). Classification of anti-Fs Abs verified that it is predominantly IgM but may also be IgG in class.

In conclusion, a novel and easy-to-produce reagent has been developed and submitted to patent with stable Fs Ag expression. With this FORS1-positive cell suspension, it is now possible to screen and classify anti-Fs Abs in large scale populations.