President’s Prize Competition Seminars

13:00 - 13:15

Persister Formation In Escherichia coli.

Persister Formation In Escherichia coli.

Discipline: Microbiology.

Institution: School of Microbiology and APC Microbiome Ireland, University College Cork.

Authors: Dunlea, AD, Clarke, DC.

Address of first author: Pathology Laboratory, Cork University Hospital Co. Cork.

In bacteria, persister cells represent a subset of cells within a population that have the ability to survive potentially lethal stresses including antibiotic treatment. Current dogma suggests that the slow growing or dormant nature of persister cells enables them to avoid antibiotic killing while the majority of the cell population is killed (1). The clinical relevance of bacterial persistence lies with the contribution of these cells to antibiotic failure and the recurrence of infections. The aim of this study was to investigate the relationship between metabolism and persister cell formation in Escherichia coli HM605, a colonic mucosal isolate associated with Crohn’s disease. During antibiotic stress, metabolism plays a central role in establishing and maintaining a persister state (2). The level of persister formation in E. coli HM605 was assessed using a gentamicin persistence assay (3). Persistence of HM605 was found to be enhanced in the presence of glucose. High performance liquid chromatography analysis revealed the production of various organic acids by persister cells under antibiotic stress. Using mutants defective in the production of specific organic acids it was shown that the production of acetate, a byproduct of glucose metabolism, was not required for persister formation in E. coli HM605. However, there was a strong correlation between HM605 persister formation and the production of lactate. These findings suggest that persister cells exist in a metabolically-active state, contradicting previous beliefs that persister cells are in a metabolically inactive or dormant state. Moreover, these studies indicate that a better understanding of metabolic flux during antibiotic stress and persister cell formation could determine how wild type HM605 persister cells adapt to favor survival during antibiotic exposure. With this knowledge, the discovery of additional persister elimination strategies could be achieved so that the problem of antibiotic failure caused by persister cells could potentially be solved.

References:

[1] Lewis K. Persister cells. Annual Review of Microbiology. 2010;64(1): 357–372.

[2] Balaban N, Merrin J, Chait R, Kowalik L and Leibler S. Bacterial persistence as a phenotypic switch. Science. 2004;305(5690): 1622–1625.

[3] Hemsley C, Luo J, Andreae C, Butler C, Soyer O and Titball R. Bacterial Drug Tolerance under Clinical Conditions Is Governed by Anaerobic Adaptation but not Anaerobic Respiration. Antimicrobial Agents and Chemotherapy. 2014;58(10): 5775-5783.

Speaker

13:15 - 13:30

The Association Of The Human Papillomavirus (HPV) With P16ink4a In Head And Neck Cancer

The Association Of The Human Papillomavirus (HPV) With P16ink4a In Head And Neck Cancer

 Foy-Stones, H., Nor, N., Donegan, L., Murtagh, F., Muldoon, T.

Department of Biopharmaceutical and Medical Science, GMIT.

 Department of Histopathology, MRHT.

 

Introduction: The Human Papillomavirus (HPV) has emerged from anonymity as a carcinogenic agent in head and neck squamous cell carcinomas (HNSCCs). This sexually transmitted virus principally targets the oropharynx, which has recently led Ireland’s department of health to consider extending the HPV vaccine to Irish men.(1) There is no gold standard for HPV testing, but HPV in situ hybridization (ISH) is employed to directly detect HPV DNA.(2) The over expression of the tumour suppressor protein p16INK4a is detected using p16INK4a immunohistochemistry (IHC), which indirectly represents the presence of HPV.(3)

Materials and Methods: In total, thirty-five HNSCC samples from the pharynx (n=2), oropharynx (n=18), larynx (n=14) and salivary gland (n=1) were retrospectively selected and tested using the Ventana BenchMark Ultra staining system. The detection of p16INK4a involved using the CINtec p16INK4a antibody for IHC staining and the high-risk HPVs were detected using the Ventana HPV III Family 16 Probe for ISH staining.

Results: Analysis of the thirty-five cases demonstrated that 34.3% tested positive for HPV and p16INK4a (n=12/35). HPV tested negative in 65.7% of cases (n=23/35) and of these, p16INK4a tested both negative (n=17/23) and positive (n=6/23). The HPV ISH and p16 INK4a IHC correlated in 82.9% of cases. A statistical relationship between HPV and p16INK4a in the oropharynx was observed (p<0.05) with 61.1% of the selected oropharyngeal samples exhibiting positivity (n=11/18). The HPV/p16INK4a positive cases were prominent in males aged 60-69 but no statistical relationship was observed (p>0.05).

Conclusion: The correlation between HPV and p16INK4a in the thirty-five HNSCC cases highlights that p16INK4a IHC shows potential as a screening tool that would prompt HPV ISH testing in oropharyngeal cancers cases. The link between HPV and p16INK4a in cancers of the oropharynx should be highlighted to all health care systems throughout Ireland to promote routine histopathology testing. This would allow prevalence statistics to be generated and reinforce the importance of vaccinating both males and females to reduce HPV-associated oropharyngeal cancers for future generations.

References:

  1. Cancer Trends 33 - HPV-associated cancers | National Cancer Registry Ireland [Internet]. Ncri.ie. 2018 [cited 7 May 2017]. Available from: https://www.ncri.ie/publications/cancer-trends-and-projections/cancer-trends-33-hpv-associated-cancers
  2. Ventana, INFORM HPV III Family 16 Probe (B). [Pamphlet]. Arizona USA: Ventana. 2017.
  3. Ventana, CINtec p16 Histology. [Pamphlet]. Arizona USA: Ventana. 2016.

Speaker

13:30 - 13:45

 Optimisation Of Recombinant GP41 Protein For Use In HIV Diagnostic Assay

Optimisation Of Recombinant GP41 Protein For Use In HIV Diagnostic Assay

Molecular Diagnostics

School of Biological Sciences, Dublin Institute of Technology, Kevin Street, Dublin 2

Gilligan M, Beale S, Chia H, Beck I, Naughton JA, Goosen C

Human Immunodeficiency Virus (HIV) has been long established as the causative agent for Acquired Immune Deficiency Syndrome (AIDS). Recommendations are that initial testing for HIV should be carried out using an antigen/antibody combination immunoassay, such as an enzyme-linked immunosorbent assay (ELISA) (1). The project objective was to assess the functionality of two variants of the reference recombinant HIV protein Gp41 used in a commercial ELISA kit; a wild-type variant with a poly-His tag fused to its C-terminus and a mutant variant without cysteines produced using splicing by overlap extension PCR.

High-throughput cloning and transformation methods were used to establish the optimal expression vectors for both constructs via expression screening (2). The best expression vectors, determined by SDS-PAGE and Western Blot analysis, were selected for large-scale expression testing. The protein was purified by Immobilised Metal Affinity Chromatography (IMAC) (3). The wild-type and mutant protein products were biotinylated and their reactivity assessed in ELISA.

Although both constructs were observed to be functional in ELISA, neither the wild-type nor the mutant construct performed as well as the reference construct. The mutant construct performed comparatively well with an average reactivity of 54% of the reference standard. The wild-type protein product had an average reactivity determined to be less than 30% of the reference standard. However after use of automated purification techniques and optimisation of the biotinylation process, the mutant product was observed to be more reactive in ELISA than the current reference standard.

References

(1) Branson B, et al. Laboratory Testing for the Diagnosis of HIV Infection Updated Recommendations. Centers for Disease Control and Prevention. 2014: p. 8-14.
(2) Bryksin A, Matsumura I. Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids. BioTechniques. 2010;48(6):463-465.
(3) De Costa F, et al. Purification of a Recombinant Polyhistidine-Tagged Glucosyltransferase Using Immobilized Metal-Affinity Chromatography (IMAC). Methods in Molecular Biology. 2016;1405:91–97.

Speaker

13:45 - 14:00

CIRP As A Therapeutic Vaccination Platform Against Cancer

CIRP As A Therapeutic Vaccination Platform Against Cancer

Topic: Immunology

Centre for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain

Address of first author: Ellen Keane, 1 John Field Rd., Dublin 8, County Dublin

Authors: Keane, E, Villanueva, L, Neylan, D, Sarobe, P

 

The approval of immune checkpoint inhibitors (ICIs) was a major development in the treatment of certain cancers. However, response to ICI treatment is linked to high numbers of tumour infiltrating T-cells which are not always present(1). Therefore, it is of interest to produce a therapeutic vaccination capable of stimulating tumour infiltrating T-cells in order to maximise effectiveness of ICIs.

This study explored the production of a therapeutic vaccination platform based on cold-inducible RNA binding protein (CIRP), an endogenous TLR4 ligand capable of eliciting a targeted immune response when fused to tumour antigen.  CIRP-linked constructs were cloned in bacterial vectors. The protein constructs were then produced and purified via affinity chromatography. Immunogenicity was tested in vivo by Enzyme-Linked ImmunoSpot (ELISPOT).

Five separate CIRP-linked constructs were successfully cloned. Two of these constructs (OVA-CIRP and Spy-CIRP) were selected for protein production and purification. The immunogenicity of the OVA-CIRP product was tested in vivo, however, no statistically significant response was observed when compared to the OVA antigen alone. Overcoming limitations in purity in future studies may improve immunogenicity of OVA-CIRP.

Additionally, the effectiveness of a previously established construct SIIN-CIRP was tested in combination with ICIs (anti-CTLA4 and anti-PD1) against tumours in murine subjects. Tumour volume and survival rates were measured over 56 days.

The treatment group receiving SIIN-CIRP and ICIs in combination resulted in delayed tumour growth and improved survival rates overall, although results failed to reach statistical significance when compared with ICI treatment alone. Repeating the study with improved homogeneity of tumour size at treatment initiation and increased treatment group size may yield significant results.

Further study is warranted to fully appreciate the potential of CIRP in combination therapy. However, its ability to be combined with tumour antigen and elicit an immune response remains promising.

 

References:

  1. Tumeh PC, Harview CL, Yearley JH, Shintaku IP, Taylor EJ, Robert L, Chmielowski B, Spasic M, Henry G, Ciobanu V, et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature. 2014;515:568-71.

 

Speaker

14:00 - 14:15

Assessment Of The Comparability Of CLSI, EUCAST And Stokes Antimicrobial Susceptibility Test Methods For Escherichia Coli Uropathogenic Isolates

Assessment Of The Comparability Of CLSI, EUCAST And Stokes Antimicrobial Susceptibility Test Methods For Escherichia Coli Uropathogenic Isolates

Topic: Microbiology

Institution: Cork Institute of Technology

Address of First Author: Pathology Department, University Hospital Limerick.

Authors: O’Halloran, C, Lucey, B.

Abstract: Nowadays, as many clinical laboratories transition between Stokes, CLSI (Clinical and Laboratory Standards Institute) and EUCAST (European Committee on Antimicrobial Susceptibility Testing) methods for susceptibility testing of uropathogens, the problem of comparing differently-derived sets of antimicrobial susceptibility testing data with each other arises, owing to a scarcity of knowledge of inter-method comparability.

The purpose of the current study was to establish the potential degree of discrepancy associated with each of these three methods and their respective interpretive breakpoint guidelines, for six oral antimicrobial agents commonly used to treat urinary tract infection, namely ampicillin, amoxicillin-clavulanate, trimethoprim, ciprofloxacin, nitrofurantoin and cephradine/cephalexin.

A total of 100 uropathogenic Escherichia coli isolates obtained from boric acid-treated urine samples of GP patients were investigated. For the EUCAST and CLSI testing, the Kirby-Bauer method of disk diffusion and associated interpretive guidelines was employed. For the Stokes method, direct susceptibility testing was performed on the urine samples, and results were interpreted using the Stokes guidelines.

When comparing the interpretive results, only 45% of isolates showed accordance among all three methods across the 6 antibiotics tested. Certain antibiotics had high levels of accordance (trimethoprim – 2% discrepancy), while other antibiotics had large discrepancy levels, particularly amoxicillin-clavulanate (40% discrepancy rate) and ciprofloxacin (11% discrepancy rate). The annual adjustments to EUCAST and CLSI breakpoint guidelines (from 2011-2017) also affected the comparability of the 3 methods

Our novel data indicate that the discrepancies generated through using different AST methods and different interpretive guidelines may result in confusion and inaccuracies when prescribing treatment for urinary tract infection. These results also show that care should be taken to compare susceptibility test data between studies only when the same method has been used in each case.

Speaker

14:15 - 14:30

Confirmation of Clostridium difficile Toxin Expression in tcdB Gene Positive Stool Samples using an Enzyme Immunoassay and Antimicrobial Susceptibility Testing of Clostridium difficile Isolates in an Irish Hospital

Confirmation of Clostridium difficile Toxin Expression in tcdB Gene Positive Stool Samples using an Enzyme Immunoassay and Antimicrobial Susceptibility Testing of Clostridium difficile Isolates in an Irish Hospital

Clostridium difficile infection (CDI) is the leading cause of infectious nosocomial diarrhoea. The organism’s virulence is related to its toxin production. The laboratory must differentiate between non-toxigenic and toxigenic strains of C. difficile (Polage et al, 2016). C. difficile AST is essential for the monitoring of emerging resistance. C. difficile AST provides invaluable epidemiological data relating to ribotype prevalence (Solomon et al, 2011). A total of 26 tcdB positive stools were tested for the presence of detectable toxin using an EIA. Patients were divided into EIA toxin positive and EIA toxin negative groups. The white cell count (WCC), CRP value and length of stay (LOS) were recorded for each patient. The Bristol Stool Chart (BSC) score was also used to describe stools submitted in both groups. Antimicrobial susceptibility testing (AST) was performed on 27 isolates in accordance with bioMérioux Etest® guidelines. Ribotype data was available for 25 of these isolates. CLSI breakpoints were followed for the antibiotics used in the study (metronidazole, clindamycin and moxifloxacin). Study findings, showed that the WCC (U=11.5), Ct value(U=36) and BSC score (U=44) were statistically higher in the EIA toxin positive group. The CRP (U=48.5) and LOS (34.5) were not statistically higher in the EIA toxin positive group. AST results demonstrated all isolates were susceptible to metronidazole. Clindamycin resistance appeared to be associated with ribotype prevalence. In conclusion, the introduction of a second-step EIA test for tcdB positive stools was useful for identifying those with CDI. The reliability of metronidazole as the drug of choice for CDI was reaffirmed. The study provided further insight into the relationship between broad-spectrum antibiotic susceptibility and ribotype prevalence.

 

 

References

Polage, C.R., Gyorke, C.E., Kennedy, M.A., Leslie, J.L., Chin, D.L., Wang, S., Nguyen, H.H., Huang, B., Tang, Y.W., Lee, L.W., Kim, K., Taylor, S., Romano, P.S., and Cohen, S.H. (2016) Overdiagnosis of Clostridium difficile Infection in the Molecular Era. JAMA Internal Medecine, (11), 1798-1801.

Solomon, K., Fanning, S., McDermott, S., Murray, S., Scott, L., Martin, A., Skally, M., Burns, K., Kuijper, E., Fitzpatrick, F., Fenelon, L., and Kyne, L. (2011) PCR Ribotype Prevalence and Molecular Basis of Macrolide-Lincosamide-Streptogramin B (MLSB) and Fluoroquinolone Resistance in Irish Clinical Clostridium difficile Isolates. Journal of Antimicrobial Chemotherapy, (66), 1976-1982.

 

Speaker