Persister Formation in Escherichia coli.
Dunlea, AD, School of Microbiology and APC Microbiome Ireland, UCC.
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.
Optimisation of Recombinant GP41 Protein For Use In HIV Diagnostic Assay
Gilligan M, School of Biological Sciences, Dublin Institute of Technology
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.
Assessment of the Comparability Of CLSI, EUCAST and Stokes Antimicrobial Susceptibility Test Methods for Escherichia Coli uropathogenic isolates
O’Halloran. Cork Institute of Technology; Pathology Department, University Hospital Limerick.
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.
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
P. Ryan, Galway Mayo Institute of Technology
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.