Benjamin
Crane
,
Jack P.
Hughes
,
Samuel J.
Rowley Neale
,
Mamun
Rashid
,
Patricia E.
Linton
,
Craig E.
Banks
* and
Kirsty J.
Shaw
*
Faculty of Science and Engineering, Manchester Metropolitan University, Chester Street, Manchester, M1 5GD, UK. E-mail: C.Banks@mmu.ac.uk; k.shaw@mmu.ac.uk
First published on 26th July 2021
Urinary tract infections (UTIs) are one of the most common types of bacterial infection. UTIs can be associated with multidrug resistant bacteria and current methods of determining an effective antibiotic for UTIs can take up to 48 hours, which increases the chances of a negative prognosis for the patient. In this paper we report for the first time, the fabrication of resazurin bulk modified screen-printed macroelectrodes (R-SPEs) demonstrating them to be effective platforms for the electrochemical detection of antibiotic susceptibility in complicated UTIs. Using differential pulse voltammetry (DPV), resazurin was able to be detected down to 15.6 μM. R-SPEs were utilised to conduct antibiotic susceptibility testing (AST) of E. coli (ATCC® 25922) to the antibiotic gentamicin sulphate using DPV to detect the relative concentrations of resazurin between antibiotic treated bacteria, and bacteria without antibiotic treatment. Using R-SPEs, antibiotic susceptibility was determined after a total elapsed time of 90 minutes including the inoculation of the artificial urine, preincubation and testing time. The use of electrochemistry as a phenotypic means of identifying an effective antibiotic to treat a complicated UTI offers a rapid and accurate alternative to culture based methods for AST with R-SPEs offering an inexpensive and simpler alternative to other AST methods utilising electrochemical based approaches.
The majority of uncomplicated and complicated UTIs are caused by Escherichia coli (E. coli).1,5 The E. coli associated with complicated UTIs can be either single- or multidrug-resistant which limits effective antibiotic treatment options.3 The principle issue with progressive colonisation of the urinary tract by antibiotic resistant bacteria is that it may lead to the infection of the kidneys which can progress to bacteraemia.1 Bacteraemia results in urosepsis which accounts for 25% of all adult sepsis cases and carries a morality rate of 25 to 60%.6 This highlights the importance of identifying effective antibiotics to treat UTIs, especially regarding complicated UTIs.
Following the diagnosis of a UTI through the presentation of clinical symptoms,7–9 or the use of a dipstick,10 antibiotic susceptibility testing (AST) is performed in order to assess the type of antibiotic a pathogenic bacteria may be susceptible to. Whilst AST is being conducted, it is common for patients to be treated with general broad-spectrum antibiotics until the results are available.11 This can lead to further deterioration in cases caused by strains resistant to such antibiotics, potentially leading to poor clinical outcomes for patients.12 Additionally this practice in prescribing a potentially ineffective antibiotic is a major contributor to the further development of antibiotic resistance in pathogenic bacterial strains.13,14
AST can be phenotypic or genotypic. Phenotypic testing directly determines antibiotic susceptibility and typically uses a culture-based methodology to monitor the response in bacterial growth to different antibiotics. Although accurate, culture-based methods are time consuming, taking between one to three days to sufficiently grow the bacterial culture and for AST to be conducted.15,16 Genotypic testing offers an alternative and utilises amplification and identification of known genes, which confer antibiotic resistance, to accurately identify which antibiotics would be ineffective against a bacterial strain type. One of the limitations of genotypic testing is that it relies on a time consuming pre-incubation step which can take up to four hours to grow the bacterial population in addition to the testing time.16 Additionally, it is impossible to identify all of the resistance genes which are numerous and subject to frequent changes due to mutations,17 in addition the presence of a resistance gene may not confer phenotypic resistance making genotypic testing potentially unreliable.18 A faster method of conducting AST could potentially help reduce or eliminate the prescription of ineffective broad-spectrum antibiotics.
Resazurin is a redox indicator that has a documented history of use in cell viability assays.19 Resazurin can be biologically reduced by metabolically active aerobic bacteria to form resorufin, and then reduced again to form dihydroresorufin.16,20 Conventional resazurin AST assays confirm the presence of live cells based off the detection of resorufin21–23 and can be carried out optically (using absorbance or fluorescence) or electrochemically. For example, Avesar et al., created a biosensor containing a stationary nanolitre droplet array for measuring production of the fluorescent resorufin product at 30 minutes time intervals for AST of different bacteria strains.23
The electrochemical mechanism of resazurin has been extensively studied in both aqueous solutions and ionic liquids using glassy carbon electrodes.20 An electrochemically based approach for resazurin based phenotypic AST offers a more sensitive method as resazurin can be detected directly rather than relying on an extended incubation period to enable resorufin concentrations to increase to detectable levels. Rapid AST has been demonstrated using organic redox-active crystalline layers on pyrolytic graphite sheets which enables accurate determination on minimum inhibitory concentrations (MIC) of antibiotics in as little as 60 mins.25 Microfabricated systems, for example, a three-dimensional interdigitated electrode array with impedimetric transducer, have shown how integration of the components required for AST can be achieved.19 While resazurin is heavily used in such systems for AST, other indicators are also available, such as ferricyanide which is reduced to ferrocyanide during bacterial respiration and has been investigated for amperometric assays.26
Screen-printed macroelectrodes (SPEs) present a variety of useful qualities such as being versatile, highly reproducible with the ability to be mass produced and can be utilised in either surface modified or bulk modified configurations26–28 For example, gold SPEs, modified with hydrogels containing antibiotics and growth media showed bacterial growth could be monitored using both electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV).29 SPEs have been utilised as part of a surface modified resazurin based detection method however,30 the use of SPEs as a platform for resazurin based AST has not yet been fully explored. The use of a simple method for the bulk-modification of SPEs that is part of the SPE fabrication process would reduce the time and complexity in the steps required for the produce of a set of modified SPEs whilst maintaining effectivity. Consequently, in this paper, we report for the first time, the bulk modification of SPEs with resazurin which are applied to the AST of UTI causative bacteria. Fig. 1 shows a schematic overview of the phenotypic AST using an electrochemical based sensing methodology.
![]() | ||
Fig. 1 An overview schematic of phenotypic AST using an electrochemical based sensing methodology. Resazurin can be reduced intracellularly by metabolically active bacteria or electrochemically at the surface of the working electrode which allows the antibiotic susceptible and antibiotic resistant bacteria to be determined.20,24 |
For overnight cultures, single strength nutrient broth was also made up according to manufacturer's instructions (Oxoid, 13 g to 1 L distilled water) and autoclaved. Artificial urine broth was made up using an equal volume of surine and double strength nutrient broth which was adjusted to a pH of 6. Artificial urine was autoclaved at 121 °C for 20 minutes to sterilise the solution before use. A stock solution of the antibiotic gentamicin sulphate was made up at a concentration of 96 mM in sterile deionised water before being filter sterilised using a 45 nm syringe filter. All experiments were performed at room temperature without oxygen removal.
E. coli (ATCC® 25922) was grown on nutrient agar from a stock kept in freezer storage and incubated at 37 °C for 24 hours. Overnight cultures were prepared from these plates using one colony taken from the plates to inoculate 10 mL of single strength nutrient broth and incubated at 37 °C for a further 24 hours.
In order to test the R-SPEs using artificial urine and to conduct the limit of detection experiments a micro-dropping (μDrop) methodology was used to emulate the type of volume that would be used for the AST method. μDrop involved pipetting a 60 μL volume of the chosen sample onto the SPE to cover the working, counter, and reference electrodes completely.
The R-SPEs were made by modifying the carbon-graphitic ink via modification with resazurin. This was carried out using a weight percentage of MP to MI, where MP is the mass of particulate (the mass of resazurin) and MI is the total mass of the ink including the base graphitic ink and the mass of the particulate.31–34 This was thoroughly mixed into the ink and screen-printed on top of the carbon-graphite working electrode. The equation (MP/MI) × 100 was used to formulate the 10 wt% R-SPEs. A 10 wt% of resazurin was used to ensure consistent printing of the analyte in the fabrication process and to test viability of the proposed electrochemical AST system.
Next, the effect of pH was explored, where the voltammetric profiles were explored as a function of pH. A plot the resazurin reduction peak position (Ep) using the voltametric peak at C1 was plotted vs. pH. Noting that the pKa of resazurin is 6.71 (at 25 °C),35 a linear trend was observed after which with an increase in pH causing a shift in Ep to a more negative value. The shift in the Ep value is due to the participation of protons in the electrochemical reduction of resazurin to resorufin which can be described with the following equation:
![]() | (1) |
Fig. 3A shows typical DPV responses of both the R-SPEs and bare SPEs. Both electrode types produce a single cathodic peak (C1), which is likely due to the resazurin and resorufin reduction peak have become amalgamated which has been previously observed when using DPV and gold working, counter and reference electrodes to detect resazurin in artificial urine where the bulk of the reduction peak height corresponds to resazurin reduction.16 In comparing the DPV as shown in Fig. 3, note that in the case of the R-SPEs compared to the bare SPEs, both are different electrochemical systems. In the later, resazurin is added into the solution such that the concentration is high resulting in a large voltammetric signal. However, in the case of the latter, the peak is smaller and not as well-defined due to the fact that the resazurin is lower in concentration within the bulk of the SPE and likely a dual processes of dissolution and surface electrochemistry is in operation. That said, the R-SPE simplify the system.
Next, DPV was explored within artificial urine. Artificial urine was used in place of a BSES to better emulate the conditions of a urine sample and thus gain better insight into how sensitive DPV is in detecting resazurin in real world urine samples. Artificial urine of pH 6 was used as the normal pH range of urine is pH 4–8 with an average skewed towards acidic values,38 therefore pH 6 was chosen as the physiological pH value. DPV was chosen as the electroanalytical technique due to its sensitivity,37 and rapid speed of analysis.
Fig. 3B shows the DPV peak height response of bare SPEs to a range of resazurin concentrations (0.0156–1 mM) using artificial urine and μDrop. A plot of analytical signal (Ip, C1) vs. resazurin concentration revealed that as the concentration was increased the magnitude of the analytical signal increased (see ESI Fig. S2†). The lowest tested concentration (LTC) of resazurin was 15.6 μM. Plotting the reduction peak height as a function of concentration produced a nonlinear trend, as such the following equations were used to estimate the limit of detection:39
LOB = Meanblank + 1.645 × SDblank | (2) |
LOD = LOB + 1.645 × SDLTC | (3) |
To determine the viability of using R-SPEs in combination with DPV as a method of conducting AST, R-SPEs were used to determine the antibiotic susceptibility of E. coli to gentamicin sulphate. Gentamicin sulphate concentrations in the range of 0.194–2.71 μM were used, with the antibiotic being added after the initial 70-minute preincubation step. After antibiotic loading, the artificial urine was added to the R-SPEs via μDrop and DPV was applied to produce an initial resazurin reduction peak. Subsequent readings were taken every hour, for up to four hours. This timing window was chosen to determine how quickly a significant difference in the resazurin reduction peak values could be determined to establish feasibility for use in a point of care setting. Statistically significant results were obtained at antibiotic concentrations of 1.36 μM and above. Fig. 4A shows the DPV peak height response of the R-SPEs using 1.55 and 0 μM concentrations of gentamicin sulphate. The trend shows that the DPV peak height response of the antibiotic loaded artificial urine is higher than the control as significantly less resazurin has been biologically reduced.
The reported MIC of gentamicin sulphate is 0.968 μM (see ESI Fig. S3†).40,41 MIC testing, however, relies on visual identification of microbial growth through liquid or agar based culturing not electrochemical detection of an redox marker. Gentamicin sulphate is an aminoglycoside type antibiotic and induces bacterial cell death via inhibition of protein synthesis.42 Although cell growth is inhibited at a concentration of 0.968 μM, the presence of residual protein synthesis can produce enzymes causing intracellular resazurin reduction.24 Therefore, a higher concentration of gentamicin sulphate than the MIC was required to inhibit the residual protein synthesis. For subsequent experiments, a concentration of 1.55 μM was used to ensure that bacterial protein synthesis was reliably inhibited. As part of a control we tested if gentamicin sulphate has a significant effect on the reduction of resazurin as the sulphate group may be electrochemically active in the applied potential range.43 Using the proposed method, the inhibitory concentration of gentamicin sulphate 1.55 μM was run against a control of 0 μM in sterile artificial urine using a one-hour time interval between electrochemical readings. This control experiment also served to determine if the significant difference that is observed between resazurin reduction peak heights between the gentamicin sulphate treated and the untreated artificial urine was due to bacterial metabolism and subsequent inhibition by the gentamicin, or just due to the potential electrochemical properties of the sulphate group. Fig. 4B shows the DPV response of R-SPEs using 1.55 and 0 μM concentrations of gentamicin sulphate in the absence of bacteria. No statistical significance was determined to occur at any of the time points. Therefore, at a dose of 1.55 μM the antibiotic gentamicin sulphate had no significant effect on the electrochemical reduction of resazurin and any statistically significance between the gentamicin sulphate treated and untreated artificial urine was caused by the metabolic activity of bacteria.
To determine if statistical significance could be determined faster and to minimise any effect that residual protein synthesis may have on the concentration of resazurin, the use of a shorter time interval between DPV readings was explored. Fig. 5 shows the difference in the DPV responses using R-SPEs and gentamicin sulphate concentrations of 1.55 and 0 μM using 20-minute time intervals. A statistically significant difference (paired T test; t = 6.3626, df = 4, p-value = 0.003128) between the resazurin reduction peak heights generated was found after 90-minutes of total elapsed time (70-minute preincubation + 20-minute time interval). Using this proposed method, the most rapid means of determining statistical significance using R-SPEs was to take a reading 20-minute after the 70-minute preincubation step.
Reported in the literature there have been other studies utilising resazurin or electrochemical AST methods (Table 1). The proposed method is significantly faster in terms of time to analysis than the disk diffusion assay which is the current gold standard for culture-based AST as well as the resazurin assay. The time to analysis is also comparable, or faster than many of the other electrical and electrochemical methods. The current limitation is that the proposed method has only been tested versus E. coli and the antibiotic gentamicin sulphate, however future work is being undertaken to test the method against other bacteria and antibiotics. The method is compatible with any bacteria that can synthesise dehydrogenase enzymes to biologically reduce resazurin,24 which are ubiquitous amongst aerobic bacteria, giving the proposed method a high potential for versatility in AST. Although the presented time to analysis is slower than some of the methods contemporaries, the simplicity in the fabrication and performance of the R-SPEs makes them a powerful analytical tool.
Electrode material | Method | Bacteria tested | Antibiotics tested | Time to analysis (Minutes) | Advantages | Disadvantages | |
---|---|---|---|---|---|---|---|
Disk diffusion assay | N/A | Zones of inhibition44 | Any Isolates | Any | 960–1440 | Can be used with any culturable bacteria | Slow |
Resazurin assay | N/A | Fluorescent detection of resorufin23 | Clinical isolates: | Freeze dried, multiplexed: | 330 | High throughput | Slow relative to electrochemical methods |
E. coli | Ampicillin | ||||||
K. pneumoniae | Ciprofloxacin | ||||||
S. aureus | Colistin | ||||||
A. baumannii | Erythromycin | ||||||
C. freundii | Gentamicin | ||||||
S. haemolyticus | |||||||
Label free | Planar pyrolytic graphite | CV detection of pH change via redox-active crystalline layer25 | E. coli (K-12) | Added to solution: Ampicillin Kanamycin | 60 | No dye | Extensive modification process |
Tantalum silicide | Impedance45 | E. coli ATCC 25922 | Added to solution: Ampicillin | 60–120 | High throughput | Specific to one bacterium | |
N/A | Impedance46 | E. coli ATCC 25922 (Susceptible) & ATCC 700609 (Resistant) | Added to solution: Nalidixic acid | N/A | Fastest AST method discussed | Requires adaptation for non-motile bacteria | |
Silver interdigitated carbon WE, CE & RE | Impedance47 | E. coli K-12 (ATCC 700891) | Added to solution: | < 90 | No dye to interfere with cellular processes. | Specific to one bacterium | |
Methicillin-resistant SRM551 | Ampicillin Erythromycin | ||||||
Ciprofloxacin | |||||||
Methicillin | |||||||
Daptomycin | |||||||
Gentamicin | |||||||
Labeled | Carbon WE, CE & RE | DPV detection of resazurin | E. coli ATCC 25922 | Added to solution: Gentamicin | 90 | Single step modification | Requires preincubation step. |
Gold WE &CE, silver RE | Impedance or DPV detection of oxidation using potassium ferrocyanide29 | S. aureus (ATCC 29213) Methicillin-resistant S. aureus (ATCC 43300) | Added to hydrogel: Amoxicillin Oxacillin | < 45 | Low cost, reagents encapsulated in a hydrogel | Electrodes require maintenance | |
Platinum WE, CE, silver, silver chloride RE | DPV detection of resazurin48 | E. coli ATCC 25922 | Added to solution: | 180 | Reusable biosensor | Electrode type incompatible with miniaturization | |
K. pneumoniae ATCC 700603 | Ampicillin | ||||||
Kanamycin | |||||||
Tetracycline | |||||||
Carbon WE & CE, silver, silver chloride RE | DPV detection of resazurin30 | S. gallinarum | Added to solution: Ofloxacin Penicillin | 60 | Single step modification | Modification process currently incompatible with bulk modification | |
Gold WE, CE & RE. | DPV detection of resazurin16 | E. coli ATCC 700928 | Added to solution: | 30 | High throughput | Reagents must be added to reaction chambers manually | |
K. pneumoniae ATCC 700603 | Ciprofloxacin | ||||||
Ampicillin | |||||||
Labeled | Platinum WE (3 mm) & CE, silver, silver chloride RE. | Amperometric detection of oxidation using potassium ferrocyanide49 | E. coli DH5α | Added to solution: | 180 | Use of a rotating WE to prevent loss of electrode sensitivity | Reliance on detection of a biological reduction product |
Clinical sample, B. pseudomallei | Ciprofloxacin | ||||||
Cefepime | |||||||
Ceftazidime | |||||||
Dicloxacillin | |||||||
Co-trimoxazole | |||||||
Carbon WE & CE, silver, silver chloride RE. | Cyclic voltammetry detection of didodecyldimethylammonium bromide50 | E. coli JM109 | Added to solution: | 120–300 | Single modification step | High variance in time to analysis | |
Cefepime | |||||||
Ampicillin | |||||||
Amikacin | |||||||
Erythromycin | |||||||
Carbon WE & CE, silver, silver chloride RE | Amperometric detection of potassium ferricyanide and dichlorophenolindophenol51 | E. coli JM109 | Added to solution: | 20 | Antifouling coating | Several off sensor preparatory steps add 20 minutes to the analysis time. | |
Bacitracin | |||||||
D-Cycloserin | |||||||
Erythromycin | |||||||
Geneticin | |||||||
Hygromycin | |||||||
Kanamycin | |||||||
Neomycin | |||||||
Paromomycin | |||||||
Rifampicin | |||||||
Streptomycin | |||||||
Trimethoprim | |||||||
Vancomycin | |||||||
Chloramphenicol | |||||||
Nystatin | |||||||
Carbenicillin | |||||||
Cefotaxime | |||||||
Nalidixic acid |
The proposed method offers an inexpensive and rapid alternative to other resazurin based electrochemical detection systems for antibiotic susceptibility testing with an application towards urinary tract infections. This proposed system utilises 10 wt% R-SPEs, the percentage of which can be varied to potentially further decrease the time taken to achieve statistical significance to further optimise the system and reduce cost; such work is currently being undertaken.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/d1an00850a |
This journal is © The Royal Society of Chemistry 2021 |