Vyvyan Salisbury

Evaluation of the efficacy of electrochemically activated solutions against nosocomial pathogens and bacterial endospores.

Aims:  Electrochemically activated solutions (ECAS) are generated from halide salt solutions via specially designed electrolytic cells. The active solutions are known to possess high biocidal activity against a wide range of target microbial species, however, literature revealing the kill‐kinetics of these solutions is limited. The aim of the study was to identify the kill‐rate and extent of population kill for a range of target species (including endospores) using ECAS generated at the anode (anolyte).

Use of bioluminescent Escherichia coli O157:H7 to study intra-protozoan survival of bacteria within Tetrahymena pyriformis

A method was developed that enabled real-time monitoring of the uptake and survival of bioluminescent Escherichia coli O157 within the freshwater ciliate Tetrahymena pyriformis. Constitutively bioluminescent E. coli O157 pLITE27 was cocultured with T. pyriformis in nutrient-deficient (Chalkleys) and in nutrient-rich (proteose peptone, yeast extract) media. Non-internalised bacteria were inactivated by addition of colistin, indicated by a decline in bioluminescence. Protozoa were subsequently lysed with Triton X-100 which lead to a further drop in bioluminescence, consistent with release of live internal bacteria from T. pyriformis into the colistin-containing environment. Bioluminescence measurements for non-lysed cultures indicated that internalised E. coli O157 pLITE27 cells were only slowly digested by T. pyriformis, in both media, over the time period studied. The results suggest that bioluminescent bacteria are useful tools in the study of bacterial intra-protozoan survival.

Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria

BackgroundThe Gateway recombinatorial cloning system allows easy and rapid joining of DNA fragments. Here we report the construction and evaluation of three different Gram-positive vectors that can be used with the Multisite Gateway cloning system to rapidly produce new gene arrangements in plasmid constructs for use in a variety of Gram-positive bacteria.ResultsComparison of patterns of reporter gene expression with conventionally constructed clones show that the presence of residual recombination (att) sites does not have an effect on patterns of gene expression, although overall levels of gene expression may vary. Rapid construction of these new vectors allowed vector/gene combinations to be optimized following evaluation of plasmid constructs in different bacterial cells and demonstrates the benefits of plasmid construction using Gateway cloning.ConclusionThe residual att sites present after Gateway cloning did not affect patterns of promoter induction in Gram-positive bacteria and there was no evidence of differences in mRNA stability of transcripts. However overall levels of gene expression may be reduced, possibly due to some post-transcriptional event. The new vectors described here allow faster, more efficient cloning in range of Gram-positive bacteria.

The Use of Bioluminescence for Monitoring in planta Growth Dynamics of a Pseudomonas syringae Plant Pathogen

The use of bioluminescence was evaluated as a tool to study Pseudomonas syringae population dynamics in susceptible and resistant plant environments. Plasmid pGLITE, containing the luxCDABE genes from Photorhabdus luminescens, was introduced into Pseudomonassyringae pv. phaseolicola race 7 strain 1449B, a Gram-negative pathogen of bean (Phaseolus vulgaris). Bacteria recovered from plant tissue over a five-day period were enumerated by counting numbers of colony forming units and by measurement of bioluminescence. Direct measurement of bioluminescence from leaf disc homogenates consistently reflected bacterial growth as determined by viable counting, but also detected subtle effects of the plant resistance response on bacterial viability. This bioluminescence procedure enables real time measurement of bacterial metabolism and population dynamics in planta, obviates the need to carry out labour intensive and time consuming traditional enumeration techniques and provides a sensitive assay for studying plant effects on bacterial cells.

Bacterial Bioluminescent Biosensors: Applications in Food and Environmental Monitoring

Abstract Bioluminescence has emerged as an extremely useful and versatile reporter technology. It provides a sensitive, non‐destructive, and real‐time assay that allows for temporal and spatial measurement. The ability to emit light is dependent on the reducing power of the organism; hence, only metabolically active cells can produce light. The direct relationship between viability and light emission allows the use of bioluminescent bacteria to assess the effect of various chemical, biological, and physical signals. We have constructed a number of recombinant plasmids carrying the lux operon expressed constitutively in many Gram negative and Gram positive bacteria. These plasmids were used to transform a wide variety of bacteria, giving rise to a range of versatile and robust whole cell biosensors with varied applications. The clinical isolate Escherichia coli 16906 was transformed with the pUC19‐derived pLITE27 plasmid to study the antibacterial effect of xanthine oxidase found in bovine and human milk. The self‐bioluminescent strains Salmonella enterica serovar Typhimurium DT104 and E. coli O157 (tox‐) were used to monitor bacterial inactivation and recovery on food surfaces during and after heat treatment. Escherichia coli O157, transformed with the pLITE27 plasmid, was successfully employed to assess the uptake and survival of bacteria within the freshwater protozoa Tetrahymena pyriformis.

Abstract 2526: Detection of Ara-CTP following exposure to CPX-351 and potentiation of action by fludarabine in leukemia cell lines using a bioluminescent bacterial biosensor

Background: Cytarabine (ara-C) is a well-established agent widely used for treating AML. At relapse its use clinically has been combined with the purine analogue fludarabine plus idarubicin and G-CSF (FLAG-Ida). Fludarabine is known to potentiate intracellular accumulation of the active cytotoxic metabolite ara-CTP from ara-C via inhibition of ribonucleotide reductase and the normal formation of endogenous dCTP. CPX-351 is a liposomal formulation of a synergistic 5:1 molar ratio of ara-C and DNR, currently in a Phase III trial versus 7+3 ara-C/DNR in newly diagnosed elderly high risk (secondary) AML patients. The aim of the study here was to utilize the well-established Escherichia coli HA1 whole-cell biosensor to measure intracellular ara-C and ara-CTP following in vitro exposure of human immortalised leukemic cell lines to CPX-351, and to assess the ability of fludarabine to potentiate intracellular ara-CTP formation. Methods: Biosensor E. coli HA1 has been engineered to produce a bioluminescent output in response to ara-C. The bacterial biosensor is able to monitor both ara-C and ara-CTP by measuring signals in the absence and presence of alkaline phosphatase, respectively. A range of in vivo-relevant ara-C concentrations (5-50 μM) and incubation times (1-4 hours) were tested. Results: Free ara-C+DNR produced an increase in bioluminescence (and hence ara-CTP) at each time-point tested in line with previous results. Incubation with CPX-351 required a longer time-period to result in similar levels of ara-CTP, but a dose-dependent increase was noted, with the highest increase produced using 4-hour incubation of CPX-351 at a concentration of 25 μM. This delay was consistent with previously observed kinetics of liposomal drug uptake by leukemia cells. Following optimisation of in vitro dosing, potentiation of ara-CTP generation was assessed following 4-hour incubation with fludarabine (5 μM), a dose previously shown to increase ara-CTP accumulation in susceptible cell lines. Potentiation of ara-CTP generation from CPX-351 was observed in three of the four cell lines tested, with the largest increase observed in THP-1 cells. No potentiation was observed in CCRF-CEM cells, in line with previous findings with ara-C. Conclusions: This study demonstrates the ability of fludarabine to potentiate formation of ara-CTP upon exposure of leukemia cells to CPX-351, suggesting a favourable impact on efficacy for this combination. Additional testing in ex vivo blasts from AML patients may be warranted to support the further investigation of this combination. Citation Format: Lawrence D. Mayer, Elizabeth Anderson, Heather Bone, Gareth Robinson, Garreth Reynolds, Vyvyan Salisbury. Detection of Ara-CTP following exposure to CPX-351 and potentiation of action by fludarabine in leukemia cell lines using a bioluminescent bacterial biosensor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2526. doi:10.1158/1538-7445.AM2015-2526

Abstract 3534: Detection of intracellular ara-C/ara-CTP following treatment with the new anti-leukemic drug elacytarabine using a bioluminescent bacterial biosensor.

Cytarabine (ara-C) is an analogue of deoxycytidine with an established role in treating AML. Cytarabine depends on the human equilibrative nucleoside transporter-1 (hENT-1) for cellular entry (1). Loss of hENT-1 function in vitro has been shown to result in ara-C resistance (2), and reduced hENT-1 expression and activity has been associated with adverse therapeutic outcomes in patients treated with cytarabine (3). The lipophilic ara-C-5’ elaidic acid ester of cytarabine (elacytarabine) has shown nucleoside transport independent uptake and long retention of the active nucleotide ara-CTP in a leukemic cell line (4) and may have the ability to overcome resistance due to insufficient transport in the clinical setting. (1). The whole cell bioluminescent biosensor, Escherichia coli HA1 was engineered to produce a measurable bioluminescent output in response to the nucleoside analogue ara-C (5). This was achieved through reduced expression of cytidine deaminase and the insertion of the human deoxycytidine kinase gene and the full lux operon from the Photorhabdus luminescens. The bacterial biosensor is able to monitor both ara-C and ara-CTP. The aim of this study was to evaluate the feasibility of using the Escherichia coli HA1 biosensor to measure intracellular ara-C and ara-CTP following ex vivo exposure of human cells to elacytarabine and cytarabine. CCRF-CEM cells or blast cells from patients were treated with ara-C or elacytarabine for up to 4 hours in the presence or absence of the hENT inhibitor dipyridamole (DPM). Cells were washed, lysed and the lysate incubated with E. coli HA1 and monitored on a plate reader luminometer. A cell viability assay was also performed to confirm biosensor results. E. coli HA1 produced a measurable response to lysate from CCRF-CEM cells treated with either ara-C or elacytarabine. In the case of ara-C, this response was reduced when cells were incubated with DPM. No such reduction was observed for elacytarabine. When used to treat blast cells derived from leukemic patients, elacytarabine treatment produced a stronger signal from E. coli HA1 than an equivalent concentration of ara-C. Furthermore, the efficacy of elacytarabine was not susceptible to hENT inhibition in patient samples, whereas the efficacy of ara-C was. The findings of this project suggest that the E. coli HA1 biosensor is compatible with elacytarabine and that elacytarabine is able to bypass the effect of hENT inhibition on CCRF-CEM cells and patient derived blast cells