Methods for field measurement of antibiotic concentrations: limitations and outlook

Methods for field measurement of antibiotic concentrations: limitations and outlook ABSTRACT The growing prevalence of antibiotic resistance poses an increasingly serious threat to human health. Although an important driver of antibiotic resistance is the continuous exposure of bacteria to sublethal concentrations of antibiotics in natural environments, antibiotic pollutants are not currently tracked globally or systematically. This limits the international capacity to address the rise of antibiotic resistance at its source. To address this lack of data, the development of methods to measure antibiotic concentrations on-site is essential. These methods, ideally, must be sensitive to sublethal concentrations of antibiotics and require minimal technical expertise. Furthermore, factors such as cost, selectivity, biosafety and the ability to multiplex must be evaluated in the context of field use. Based on these criteria, we provide a critical review of current methods in antibiotic detection and evaluate their adaptability for use on-site. We categorize these methods into microbiological assays, physical and chemical assays, immunoassays, aptasensors and whole-cell biosensors. We recommend continued development of a dipstick or microfluidics approach with a bacterial promoter-based mechanism and colorimetric output. This technique would incorporate the advantageous aspects of existing methods, maximize shelf-life and ease-of-use, and require minimal resources to implement in the field. antibiotic resistance, detection limits, environmental pollutants, on-site monitoring, subinhibitory concentrations, dipstick sensor BACKGROUND Antibiotic resistance is an international problem that poses a direct threat to global health, agriculture and biosecurity (reviewed in Holmes et al.2016). The rise of antibiotic resistance in microorganisms is complex and multifactorial, but a significant source of antibiotic resistance acquisition is exposure to sublethal and subinhibitory concentrations of antibiotics in natural environments such as soil and water (Andersson and Hughes 2014; Long et al.2016; Le Page 2017; Sommer et al.2017). Sublethal concentrations are below the threshold for lethality in bacteria, while subinhibitory concentrations are below the threshold for growth inhibition (Hughes and Andersson 2012; Bengtsson-Palme and Larsson 2016; Long et al.2016). The presence of antibiotics at concentrations as low as nanograms per milliliter has been shown to promote the development of resistance in the laboratory (Hughes and Andersson 2012; Bengtsson-Palme and Larsson 2016; Long et al.2016; Almakki et al.2017). Due to the deleterious effects of low levels of antibiotics in soil and water, antibiotics are now regarded as a significant environmental pollutant (Li et al.2016; Jha et al.2017; Zhou et al.2017). However, the effect of antibiotic pollutants on microorganisms in the environment has not been extensively characterized (Brandt et al.2015). In particular, no global model exists to track and predict the distribution of antibiotic pollutants and their effects worldwide. In most existing antibiotic distribution studies, samples are obtained and taken to laboratories for analysis in a costly or time-consuming pipeline that limits capacity for widespread monitoring of antibiotic pollutant levels (Managaki et al.2007; Graham et al.2011; Hoa et al.2011; Zhang et al.2013; Tang et al.2015). While localized studies are clearly valuable, understanding the effects of antibiotics in a broader environmental context will improve efforts to combat the rise of antibiotic resistance (reviewed in Aminov 2009; Le Page et al.2017). The availability of quick, field-ready techniques would increase the convenience of on-site measurement, meaning that samples may be taken and analyzed more frequently, leading to more data and better predictive models of antibiotic pollutant entry and persistence in local environments and the rise of antibiotic resistance (Tang et al.2018). On-site measurement will allow researchers to assess levels of antibiotic pollutants in rural areas, where antibiotic pollution has been shown to be correlated with antibiotic resistance development (reviewed in Gothwal and Shashidhar 2014; McConnell et al.2018), without incurring the cost of transportation to laboratories. In addition, such a tool would benefit other researchers seeking on-site diagnostic tools and may draw on many similar methodological advances (Garg et al.2016). This paper reviews the status of the development of a first-line technique for preliminary, on-site detection of antibiotic pollutants by assessing the advantages and limitations of existing methods and highlights the most promising techniques for use in the field. Here, we evaluate the major classes of methods in the context of field use: microbiological assays, physical and chemical assays, immunoassays, aptasensors, and whole-cell biosensors. We then discuss future directions and criteria for the development of a first-line assay for use in the field to allow environmental monitoring of antibiotic concentrations. METHODS Microbiological assays A classic method for detecting the presence of antibiotics is the use of microbiological assays that employ antibiotic-sensitive species of bacteria to determine whether specific antibiotics are present in a given sample and, with limited sensitivity, their concentration. Since the 1950s, microbiological assays have identified aberrations in known growth patterns to detect the presence of antibiotics in a given sample (Kersey et al.1954). Current commercial assays rely on one of two techniques to identify antibiotic presence in a sample. Disc diffusion uses discs coated in the sample of interest that are placed on plates on which bacteria are grown; the presence of a halo of cleared growth around the disc indicates that the bacteria were killed by an antibiotic in the disc. This test can identify specific antibiotics or classes of antibiotics depending on the species of bacteria used and has been optimized for use in milk samples with a limit of detection (LOD) for β-lactams between 30 and 80 ng/mL (Kukurova and Hozova 2007; Piech et al.2016). A second technique uses specific bacteria with known sensitivity to antibiotics that have been engineered to yield a colorimetric output. These color-change reactions can be quantitatively analyzed with spectrophotometry to determine the concentration of antibiotic present in the sample and can be as sensitive as 1 ng/mL, but require sterile technique and laboratory facilities to co-incubate the sample with bacteria (Le Breton, Savoy-Perroud and Diserens 2007; Kalunke et al.2018). As a standard technique, commercially available microbiological assays have been optimized for testing food and drink samples, specifically milk and serum samples, to determine whether physiologically relevant levels of antibiotics are present (Kukurova and Hozova 2007; Le Breton, Savoy-Perroud and Diserens 2007; Beltrán et al.2015; Kalunke et al.2018). Other microbiological methods have qualitative or semi-quantitative outputs, and either provide a binary response or a range of concentrations (Kukurova et al.2007; Le Breton, Savoy-Perroud and Diserens 2007; Kalunke et al.2018). These methods are able to function in complex matrices, but due to their output, determining precise concentrations would be difficult to ascertain. Many microbiological assays are focused exclusively on the β-lactam family of antibiotics, commonly used in veterinary medicine, and have poor sensitivity outside this category; a review of commercial tests found that while they can detect concentrations as low as 4 ng/mL within the β-lactam family, the tests are limited to 100 ng/mL or more in other families (reviewed in Beltrán et al.2015). For some microbiological assays, the requirement for laboratory facilities is mitigated by microfluidics and paper-based techniques that function reliably with non-sterile samples and in field conditions (Sun et al.2011; Deiss et al.2014). Microfluidics analysis relies on changes in bacterial morphology when sublethal, but not subinhibitory, concentrations of antibiotics are present in the sample (Sun et al.2011). This technique requires some expertise to implement but shows promise for field adaptation. Droplet microfluidics exhibits greater sensitivity but current techniques have focused on identifying the presence of antibiotic resistance genes rather than measuring antibiotic concentrations (Kaminski, Scheler and Garstecki 2016). In summary, microbiological assays typically require significant equipment and sterile laboratory facilities and are often optimized for specific antibiotics, making them relatively ill-suited for on-site field analysis, although the development of microfluidics or paper-based techniques have expanded the range of these assays. A challenge for field use of paper-based microbial assays is the requirement for live or lyophilized bacteria that can be reconstituted on-site without introducing contamination. Due to these concerns as well as portability, incubation and sterility requirements, microbiological assays are not presently optimal for on-site field testing. Physical and chemical assays In contrast to microbiological assays, which are limited by bacterial sensitivity to antibiotics, physical and chemical assays target specific properties of the molecule such as size, charge, binding characteristics or reactive properties to identify antibiotics in a diverse sample. Physical purification entails separation of the antibiotic away from other impurities in the sample to allow isolation of the desired element through repeated fractionation. The final fraction for antibiotic purification is analyzed using spectrophotometry and has an LOD of approximately 25 ng/mL (reviewed in González de la Huebra and Vincent 2005). The prepared sample can additionally be analyzed using spectrometry, an equipment-intensive capability sensitive down to a single-molecule resolution. Tandem mass-spectrometry analyzes samples for molecules of interest, including antibiotics, with an LOD below 0.1 ng/mL (Zhang et al.2013). Liquid Chromatography/Mass-spectrometry (LC/MS) has been adapted to quantify antibiotic concentrations in milk, serum, meat, soil and water samples (Luo et al.2010; Hoa et al.2011; Zhang et al.2013 Bayen et al.2014; Kostich, Batt and Lazorchak 2014; Tang et al.2015; Jha et al.2017). LC/MS has also been implemented to analyze samples from the field, where matrix effects were determined to be insignificant after purification (Luo et al.2010; Hoa et al.2011; Zhang et al.2013 Kostich, Batt and Lazorchak 2014; Tang et al.2015; Jha et al.2017). Some commonly used techniques to combat the complications of soil matrices are isotope dilution, standard addition, manual calibration or changing the column (Aga et al.2005; Bayen et al.2014). Physical techniques have successfully been optimized for environmental samples, indicating that they do not require sterile inputs and are an excellent tool for laboratory analysis (Luo et al.2010; Bayen et al.2014; Chen and Zhou 2014; Kostich, Batt and Lazorchak 2014; Begou et al. 2017). However, both purification and spectrometry require a significant degree of user expertise and supporting infrastructure and involve challenging procedures or sensitive technology not easily transported to field sites; this technique is best adapted for processing field samples in fully equipped laboratories (Soprani et al.2016; Pan and Chu 2017). Alternatively, Surface-Enhanced Raman Spectroscopy (SERS) allows screen-printed electrodes to be used for spectroscopic analysis. This technique offers high sensitivity and portable, rapid detection that can be easily adapted to fieldwork (Li et al.2014). With a procedure length of less than 10 min, SERS has been used to determine antibiotic concentrations in water samples (Li et al.2013). However, SERS also requires sample purification to enrich antibiotics relative to other pollutants or solutes in the sample, increasing the technical knowledge required to test on-site. Eliminating pre-processing of the sample and automating data analysis of the output could optimize SERS for use on-site. Field-ready chemical assays have exploited microfluidics or paper-based approaches to be both portable and scalable. One group designed a microfluidic system that allows chemical interaction between aminoglycoside antibiotics and copper ions, with a measurable chemiluminescent output; this on-chip method is portable and has an LOD under 1 ng/mL (Sierra-Rodero, Fernández-Romero and Gómez-Hens 2012). While these features are promising for on-site use, this method requires some user expertise to microinject the sample onto the chip. Paper-based chemical solutions, in which chemical modifications to the paper induce colorimetric outputs in response to antibiotic presence, also show promise for fieldwork and require less technical expertise. Gomes, Goreti and Sales (2015) demonstrated that by adding a metal ion-responsive amine layer to a paper-based medium, oxytetracyclines could be detected in water with an LOD of around 30 ng/mL. However, paper-based methods can currently detect only a few predetermined compounds of interest. Further research should broaden the capacity of paper-based techniques to identify a wider range of compounds in a single test. Immunoassays Immunoassays provide highly sensitive, specific detection of antibiotics in complex liquid or processed solid samples, relying on specificity of interactions between antibodies and the molecule of interest (reviewed in Ahmed et al.2017). Immunoassays are typically colorimetric in output (Aga et al.2005; Cháfer-Pericás, Maquieira and Puchades 2010; Graham et al.2011) but some have fluorescent or chemiluminescent outputs (Meyer et al.2016), depending on the reporter molecule used. Immunoassays for antibiotic detection, specifically ELISAs, have been adapted for soil, food, and water samples (Aga et al.2005; Cháfer-Pericás, Maquieira and Puchades 2010; Graham et al.2011; Garg et al.2016; Meyer et al.2016). In addition, commercially available ELISAs developed for food samples have been utilized in field studies monitoring waste water from rivers and soil samples taken from experimental plots treated with manure (Aga et al.2005; and Graham et al.2011). Due to the cross-reactivity of antibodies detecting isomers and degradation products, ELISAs may overestimate concentrations and fail to show a decline over time (Aga et al.2005). Immunoassays can provide sensitive detection of antibiotics in samples using secondary antibodies to amplify the signal, and typically have an LOD below 1 ng/mL (reviewed in Cháfer-Pericás, Maquieira and Puchades 2010). However, adding secondary antibodies increases the cost per sample, limiting the utility of immunoassays as an inexpensive, field-ready technique. Another challenge limiting the use of immunoassays for on-site analysis is their reliance on spectrophotometers to quantify test results. These assays additionally require a moderate level of scientific expertise to complete the procedure, precluding untrained users from participating in the data collection process. The use of antibodies generally restricts one immunoassay to a single or relatively few antibiotics depending on the specificity of antibodies used in the assay (Graham et al.2011). Cross-reactivity of antibodies lessens the specificity of the test, as antibodies may react with non-biologically active components of antibiotic degradation pathways (Aga et al.2005). Concentration measures from a cross-reactive immunoassay would overestimate the level of antibiotic present in a given sample and impede efforts to understand antibiotic resistance in an ecological context. Despite these limitations, recent developments of miniaturized, user-friendly immunoassays offer some potential for the adaptation of this technique for the field. A diverse array of techniques has been developed that adapt immunoassays into a multiplex format, thus enhancing the efficiency and high-throughput ability of detection by localizing different antibodies to distinct parts of a chip or strip (Jiang et al.2013; Song et al.2015; Han et al.2016; Wang et al.2017a). One group has created a regenerable, chemiluminescent microarray that allows detection of up to 13 antibiotics at once in milk samples and is aiming to scale the product for low-cost use in the field (Meyer et al.2016). Other novel immunoassays have further improved usability by offering a multiplex assay with a quantifiable, colorimetric output. In 2013, Jiang and colleagues developed a 'dual-colorimetric' assay that employed two different enzyme substrate systems to simultaneously detect 13 fluoroquinolone and 22 sulfonamide antibiotics (Jiang et al.2013). More recently, a multiplex assay was created by Han et al. (2016) that enabled the simultaneous detection of three different antibiotics on a dipstick format; this technique offers several features that enhance usability, including a quantifiable, colorimetric output and a procedure time of 10 min with no pretreatment required for the device. In a similar device, which was specifically designed for high-throughput, on-site measurements, latex beads were utilized for colorimetric output on a single strip multiplex test for three antibiotic classes. When tested on spiked milk samples, this method demonstrated sensitivity of 0.3–10 ng/mL, with negligible crosstalk between the antibiotic classes and a procedure time of 10 min (Wang et al.2017a). These multiplexed immunoassays, which have been specifically designed with on-site, high-throughput detection in mind, show promise for first-line use in the field. Aptasensors A more recent technique for antibiotic detection uses aptamer sensors (aptasensors). Aptamers are DNA or RNA oligonucleotides that can bind with high affinity to specific targets (Yang et al.2017). Aptasensors developed for antibiotic detection fall into three categories: electrical, fluorescent and colorimetric. Electrical aptasensors typically immobilize an aptamer onto the surface of an electrode or a nanoparticle such that the introduction of the target molecule causes a conformational change in the aptamer. This change releases a probe or exposes the electrode to a redox probe. Abnous et al. designed an electrical aptasensor for fluoroquinolones that is representative of the electrode-based technique: a target-specific aptamer was immobilized on a gold electrode. In the absence of the target, single-stranded binding (SSBs) proteins bound to the aptamer and inhibited access of a redox probe to the electrode surface. In the presence of the target, aptamer-specific binding decreased the binding affinity of the SSBs, increasing access to the redox probe and inducing a detectable shift in current with a sensitivity of .0871 ng/mL (Abnous et al.2017). Other electrical aptasensors utilize different materials for the electrode (Li et al.2017; Wang et al.2018), complementary single stranded DNA to block a redox probe (Danesh et al.2016; Taghdisi et al.2016) or different probes such as ions or quantum dots (Li et al.2016; Yan et al.2016), but achieve the same output with comparable sensitivity (Liu et al. 2017a,b). Other electrical aptasensors utilize nanoparticles as a signal probe (Chen et al.2017b) or to immobilize the aptamer (Chen et al.2017a). Chen et al. designed an aptasensor where aptamers specific for kanamycin and chloramphenicol were immobilized on amino-modified magnetic beads and complementarily bound to DNA signal probes with a nanoscale metal organic framework (NMOF) carrying a specific ion. When the target was introduced, it bound to the aptamer, replacing and releasing the signal probe and generating a detectable current of ions in the NMOF (Chen et al.2017b). Fluorescent and colorimetric aptasensors employ a diverse array of mechanisms. Introduction of the target causes a conformational change that can expose a nanoparticle surface to a color-changing reagent (Emrani et al.2016; Lavaee et al.2017). Lavaee et al. immobilized complementary ssDNA on the surface of gold nanoparticles that, in the absence of the target, form arches with ciprofloxacin specific aptamers, blocking the surface of the nanoparticle and inhibiting the reduction of 4-nitrophenol, and therefore the color change. In the presence of the target, the surface is exposed, reducing the 4-nitrophenol and causing a measurable change in absorbance (Lavaee et al.2017). The presence of the target may also disassemble a complementary complex, releasing or quenching a fluorophore (Emrani et al.2016; Babaei et al.2017; He, Li and Hu 2017; Ha et al.2017; Wu et al.2017; Chen et al.2017d). Chen et al. utilized Waston-Crick and Hoogsten base pairing to form a triple helix between two arms flanking a tetracycline specific aptamer and a signal transduction probe (STP), which, in the presence of the target, disassembles and releases the STP. The STP then forms a quadruplex and binds to thioflavin T, generating a fluorescent output (Chen et al.2017d). Other fluorescent and colorimetric aptasensors may utilize hairpin loops that form a quadruplex in the presence of the target (Cui et al.2018) or an exonuclease to amplify a signal (He, Li and Hu 2017; Wu et al.2017). Aptasensors may also form hairpin loops that can then be detected using qRT-PCR (Duan et al.2017) or microchip electrophoresis (Wang et al.2017b). Paper-based aptasensors have been designed to make aptasensors easier to transport. Zhang, Zuo and Ye (2015) present a paper-based microfluidic device incorporating ssDNA aptamers for the detection of aminoglycoside antibiotics, with a sensitivity of around 300 ng/mL, while Sharma et al. (2017) designed a screen-printed electrode that detects kanamycin with a sensitivity of 0.11 ng/mL. Aptasensors are highly sensitive and have an LOD in the range of micrograms to nanograms per liter. The more sensitive aptasensors tend to be electrical and can have an LOD of 1.07 e – 5 ng/mL (Chen et al.2017a,b), which is ideal for detecting trace quantities of antibiotics in the field. In addition, aptasensors have relatively short procedures, ranging from 30 min to 2 h, which is useful for taking multiple samples in the field (Zhang, Zuo and Ye 2015; Li et al.2016; Yang et al.2017). Adapted to test antibiotic concentrations in milk, serum and water samples, aptasensors are able to detect antibiotics effectively in complex matrices (Hoa et al.2011). A significant limitation for the application of aptasensors in the field is that they are generally singleplex and therefore unable to identify multiple classes of antibiotics simultaneously. While multiplex aptasensors have been described, most have only tested simultaneous detection of up to two antibiotics (Yan et al.2016; Chen et al.2017a,b; Huang et al.2018; Li et al.2018; Zhou et al.2018). In 2016, Hao and colleagues implemented a cDNA aptamer system with chemiluminescent output to simultaneously detect oxytetracycline, tetracycline and kanamycin at a sensitivity of 0.05 ng/mL in milk samples. While this method was designed for usability and demonstrates high sensitivity, the chemiluminescent output may be a limitation for field use due to the need for detection equipment (Hao, Gu and Duan 2016). Aptasensors provide a highly sensitive, relatively fast and robust detection method; however, they have significant limitations in their ability to test a broad range of antibiotics simultaneously. In addition, aptasensors currently require significant training and detection reagents and equipment. While there are portable models of spectrophotometers and electrochemical workstations, they may be difficult to use in low-resource, isolated locations. Aptasensors would be a suitable secondary detection method to verify results with higher selectivity and specificity but are not ideal for widespread, high-throughput detection. However, the development of reagent-less or label-free aptasensors that would minimize training and require fewer resources is a promising direction for field use (Yang et al.2017; Wang et al.2017b; Chen et al.2017d). Whole-cell biosensors Another method of antibiotic detection uses synthetic biology to harness the internal machinery of bacterial cells to determine local concentrations of antibiotics. Rather than simply monitoring a cell’s response to antibiotic presence, bacterial cells can be genetically engineered to respond predictably to differing concentrations of antibiotics. This may be accomplished by inserting engineered plasmid constructs containing antibiotic-sensitive DNA sequences into host bacterial organisms. The product is known as a whole-cell biosensor (Korpela et al.1998; Gui et al.2017; Chen et al.2017c). Evolution has effectively primed bacteria to respond to potential damage; this response is often a change in gene expression driven by bacterial promoters, induced by the presence of a small molecule, including antibiotics (Goh et al.2002). This intrinsic cellular response to the presence of antibiotics can be detected by fusing the antibiotic-sensitive promoter to a reporter construct that transduces the signal to a visual response detectable by the user (Chen et al.2017c). The construction of whole-cell bacterial biosensors for antibiotic detection is not a new concept. In 1998, Korpela and colleagues genetically modified a strain of Escherichia coli by combining genetic elements responsive to tetracycline with a luciferase operon, creating a biosensor that responded to tetracycline with bioluminescence (Korpela et al.1998). Since then, dozens of iterations of similar designs have been tested and improved upon. The choice of promoter is perhaps the most vital aspect of biosensor design, as this choice determines the specificity as well as the sensitivity of the sensor (Gui et al.2017). Goh and colleagues found that after screening a library of 6500 bacterial promoters native to Salmonella typhimurium, up to 5% of these promoters demonstrated a change in expression in response to subinhibitory antibiotic concentrations. Many such promoters have been well-characterized in an array of bacterial species, providing hundreds of potential biosensor targets and bacterial hosts; furthermore, by using a panel of promoter-reporter fusions, it allows the classification of unknown antibiotics based on the response of each promoter (Goh et al.2002; Hutter et al.2004; Mascher et al.2004; Urban et al.2007; Staroń, Finkeisen and Mascher 2011; Melamed et al.2012). Whole-cell bacterial biosensors offer several advantages over other detection techniques. Bacteria require relatively few and low-cost resources to remain viable, and their rapid replication makes high-throughput testing easily achievable. Furthermore, bacterial sensors yield information about bioavailability of substrates which, in the context of tracking the development of bacterial resistance, may offer a more relevant metric than directly measuring concentrations in water or soil samples (Aga et al.2016; Gui et al.2017). Biosensors have been adapted for milk, water, serum and meat samples (Link, Weber and Fussenegger 2007; Pikkemaat et al.2010; Roda et al.2011; Zhang, Zuo and Ye 2015; Duyen et al.2017; Chen et al.2017c; Suárez et al.2009) and have also been implemented in field studies to detect antibiotics (Chen et al.2017c). While biosensors with qualitative binary outputs work consistently in spiked samples (Duyen et al.2017), more quantitative biosensors have shown recoveries of 95%–101%, and consistent results with HPLC and LC/MS (Pikkemaat et al.2010; Zhang, Zuo and Ye 2015; Chen et al.2017c). Most traditional biosensor techniques for detection of antibiotics have relied on fluorescent or bioluminescent output as a metric for local antibiotic concentrations, using plate readers or luminometers to determine output levels (Korpela et al.1998; Kurittu, Karp and Korpela 2000; Bahl, Hansen and Sørenson 2005; Eltzov et al.2008; Pikkemaat et al.2010). However, the threshold of detection for fluorescent output of biosensors remains a challenge. To address this, Melamed and colleagues present an innovative method for detection and identification of different types of antibiotics; they provide a panel of 12 bacterial reporter strains optimized for highly sensitive fluorescent output, paired with a computational algorithm which analyzes the dose-response curves for each fluorescent reporter strain in response to an unknown antibiotic and identifies the type of antibiotic based on mode of action (Melamed et al.2012; Melamed, Naftaly and Belkin 2014). While these analytical methods may improve antibiotic identification from biosensor output, fluorescence and bioluminescence remain difficult to assess precisely in the field. To address this issue, biosensors have been constructed with colorimetric output that can be qualitatively assessed by the naked eye or quantitatively assessed with a digital camera and online image analysis software (Fantino, Barras and Denizot 2009; Duyen et al.2017). While these solutions are still limited in their quantitative sensitivity, their design shows promise for applicability in the field. Another obstacle to field implementation is the portability of bacterial biosensors outside of the lab. Since these biosensors rely on healthy bacteria, the function of many existing biosensors is optimal at 37°C and therefore requires an incubator. Recent developments in cell immobilization and miniaturized devices, however, have improved prospects for storage of live cells. It has been demonstrated that lyophilization (freeze drying) and reconstitution has negligible effect on the functionality of bacterial biosensors (Kurittu, Karp and Korpela 2000); this technique has been successfully tested in a non-laboratory field setting for the detection of tetracyclines (Pikkemaat et al.2010). Another storage technique that takes advantage of the natural abilities of some bacterial cells is a 'sposensor,' a user-friendly, field-ready device that contains immobilized bacterial spores of genetically engineered Bacillus subtilis that act as whole-cell biosensors. While only tested on bacitracin with an LOD at around 50 ng/mL, it was designed specifically to be easily usable in the field, using only three tubes and limited reagents, and therefore may be a model for future work using these techniques (Fantino, Barras and Denizot 2009). The sensitivity of whole-cell biosensors remains a more challenging obstacle due to limitations in sensitivity in the detection of biosensor output, rather than the response of the biosensor itself. While colorimetric sensors provide a reliable equipment-free qualitative approach, they are not ideal for in-depth quantitative analysis. The field of bioengineering offers a powerful alternative: the biochip or 'lab-on-a-chip' design, which integrates bioluminescent whole-cell biosensors with miniaturized photomultipliers and charge-coupled detectors (CCDs) to amplify and convert the signal. This output may then be analyzed with online software to provide a quantitative result (Roggo and van der Meer 2017). In 2011, Roda and colleagues performed three different bioluminescent assays by implementing CCD imaging on a miniaturized device with engineered bacterial and yeast cells. While they did not attempt to detect antibiotic compounds using their device, the success of three different assays suggests that this technique could be adapted to antibiotic detection by using different engineered bacterial strains. This technique was shown to have a robust shelf life of up to 35 days with refrigeration, making it suitable for transport and use in the field (Roda et al.2011). While biochips are undergoing rapid improvements in size and usability, the most cost-effective field solution is paper based. Paper-based, colorimetric whole-cell biosensors including the 'sporosensor' have demonstrated innovative design and promising functionality and usability, but still work best at 37°C since they use living bacterial cells (Fantino, Barras and Denizot 2009). As an alternative, cell-free systems may be used that employ the same cellular machinery as a typical biosensor, hosted on a paper-based matrix rather than inside a living cell. Duyen and colleagues developed a paper-based, in vitro transcription/translation system for the detection of antibiotics that inhibit protein synthesis. While the device functioned most efficiently at 37°C (2-h incubation), detection at a comparable sensitivity could consistently be achieved at temperatures as low as 15°C by varying incubation time. With improvement to sensitivity that is currently in the range of 500–6100 ng/mL, this paper-based system shows potential for field use (Duyen et al.2017). FUTURE DIRECTIONS In this review, we have identified criteria for an effective device that is able to execute high throughput, on-site testing for detecting antibiotics in the environment. An optimal device would be highly sensitive, capable of multiplexing and detecting compounds from complex matrices, and would require minimal equipment, electricity, procedure time, and technical training for the user. Additionally, it would be cost effective, have extended shelf life under a range of environmental conditions, and meet biosafety standards. As discussed in this review, there are now many sensitive techniques for antibiotic detection and quantification; however, none currently satisfy all these criteria. While significant obstacles remain in adapting any of the existing devices for use in front-line, on-site implementation in the field, several do show promise. Perhaps the most convenient solution in terms of cost-effectiveness, shelf-life and usability for the field is a bacterial promoter-based approach using a strip or dipstick detector. A diverse array of dipstick-style solutions for antibiotic detection have been designed, covering a range of sensitivities and quantification ability (Link, Weber and Fussenegger 2007; Fantino, Barras and Denizot 2009; Gomes, Goreti and Sales 2015; Han et al.2016; Duyen et al.2017; Wang et al.2017a). In terms of device output, a colorimetric assay would provide user-friendly easily readable output that does not require expensive equipment for detection or analysis. The technique of Link, Weber and Fussenegger (2007) incorporates many of these field-ready criteria. Integrating a cell-free, inducible promoter system with an antibody-based reporter mechanism, this cell-free dipstick approach was shown to be highly sensitive, detecting a range of classes of antibiotics at concentrations of 2–40 ng/mL. Individual dipsticks were designed for the specific recognition of different antibiotics, without any signs of significant crosstalk between the targets. Finally, these dipsticks possess a shelf-life of up to 6 weeks at room temperature. The need for a dipstick-like technique, which is easy to use on-site and provides relatively quick, visual output, is well-justified within the relevant literature with that of Link et al. cited repeatedly a model of such a technique (Adrian et al.2012; Cháfer-Pericás, Maquieira and Puchades 2010; Chen et al.2012; Jiang et al.2013; Dincer et al.2017; Kamakoti et al.2018). The dipstick method was compared to other biosensor assays and was noted for its advantageous usability and sensitivity for antibiotic detection in food samples (Cháfer-Pericás, Maquieira and Puchades 2010). A recent review of future directions in biosensing and personalized drug therapy identified the dipstick method as the model exemplifying low cost and portability in biosensor design (Dincer et al.2017). However, despite its promise, significant problems remain barring its implementation in the field. When compared to other antibiotic detection techniques, the dipstick technique has been criticized for its lack of selectivity (Kim et al.2010; Hou et al.2013). While the dipstick assay can distinguish among three classes of antibiotics, tetracyclines, macrolides and streptogramins, it lacks further specificity due to the structural similarities of antibiotic molecules within these classes (Link, Weber and Fussenegger 2007). To distinguish between subclasses of antibiotics, therefore, an assay with higher specificity such as an aptasensor is advantageous. The dipstick assay has also been criticized for the length of the required incubation times, often requiring up to 1 h (Link, Weber and Fussenegger 2007; Cháfer-Pericás, Maquieira and Puchades 2010). However, enhancements to the dipstick assay constructed by Link et al. have already been published (Kling et al.2016; Dincer et al.2016). The dipstick detection mechanism was adapted onto a microfluidic platform to enable multiplexed detection on a miniaturized device. Furthermore, the output was modified to yield an electrochemical rather than colorimetric signal, enabling a more precise quantifiable output. This biosensor was shown to have a robust shelf-life of up to 3 months and a rapid output time of 15 min (Kling et al.2016). While it maintains limitations in specificity and has only been tested using spiked human serum, it offers the added advantages of a more rapid protocol time and multiplex capability. Ultimately, as an on-site, first-line evaluation technique, the broader detection and identification of antibiotic classes has been deemed sufficient (Cháfer-Pericás, Maquieira and Puchades 2010; Aga et al.2016; Kling et al.2016). Moreover, additional screening of natural promoters and the ability to design synthetic promoters has enormous potential to address specificity issues (Goh et al.2002; Hutter et al.2004; Mascher et al.2004; Urban et al.2007; Yagur-Kroll, Bilic and Belkin 2010; Staroń, Finkeisen and Mascher 2011; Melamed et al.2012; Wang, Tang and Yang 2017). CONCLUDING REMARKS All techniques described in this paper are viable methods for laboratory-based, in-depth testing of high-interest sites; however, this review focused on identifying the benefits and drawbacks of each method for use in high-throughput, on-site testing of environmental samples. For an antibiotic detection technique to be practical for field use it must be sensitive, easily usable with minimal training or equipment, and capable of detecting multiple antibiotic types from complex samples in a high-throughput manner; furthermore, it should be cost-effective, relatively quick, and possess a robust shelf life under diverse environmental conditions. A biosensor implementing an engineered bacterial promoter mechanism in a dipstick or microfluidic format meets many of these criteria and may be a favorable model for future development of on-site techniques. However, further testing in complex matrices is essential before this technique can be implemented in the field. The development and implementation of a field-ready antibiotic detection device that requires minimal scientific training will also enable crowdsourcing and local monitoring of antibiotic pollution data, in an analogous manner to crowdsourced monitoring of air pollution (Gurney and O’Keeffe 2013). The ability to use these methods on-site adds to the convenience for citizen scientists to provide time-series data on antibiotic concentrations at a local level. Compiling data on antibiotic pollutants at the national level will allow monitoring and modeling of antibiotic distributions similar to the successful global monitoring of mercury as an environmental pollutant (D’Amore et al. 2015). After collection of limited data, Pacyna and Pacyna (2002) presented an estimate of global mercury emissions, which was later modeled in a geospatial manner by Wilson et al. (2006) to determine patterns of mercury pollution and project environmental impact. Subsequently, Depew et al.(2013) compiled regional datasets to determine national mercury distribution in Canada, and researchers in the United States mapped mercury contamination in the Everglades to assess spatial and temporal trends in mercury bioaccumulation (Axelrad et al.2011; Depew et al. 2013). An analogous database of antibiotic contaminants could be assembled allowing international efforts to model the distribution of these pollutants and assess their impact (Travnikov et al.2015). Such a model will allow for the tracking and prediction of antibiotic resistance development on a global scale. SUPPLEMENTARY DATA Supplementary data are available at FEMSEC online. ACKNOWLEDGEMENTS The authors would like to recognize Sarah Jo McGeady, Meadow Parrish, and Cecilia Zheng for their significant contributions to the planning and preliminary literature search for this article. While not part of the project team, the authors also wish to thank the U.S. Department of State’s Diplomacy Lab program, and its Office of International Health and Biodefense, for working with them to identify this problem and consider solutions. Conflict of interest. None declared. REFERENCES Abnous K , Danesh NM , Alibolandi M et al. 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Methods for field measurement of antibiotic concentrations: limitations and outlook

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Blackwell
Copyright
© FEMS 2018.
ISSN
0168-6496
eISSN
1574-6941
D.O.I.
10.1093/femsec/fiy105
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Abstract

ABSTRACT The growing prevalence of antibiotic resistance poses an increasingly serious threat to human health. Although an important driver of antibiotic resistance is the continuous exposure of bacteria to sublethal concentrations of antibiotics in natural environments, antibiotic pollutants are not currently tracked globally or systematically. This limits the international capacity to address the rise of antibiotic resistance at its source. To address this lack of data, the development of methods to measure antibiotic concentrations on-site is essential. These methods, ideally, must be sensitive to sublethal concentrations of antibiotics and require minimal technical expertise. Furthermore, factors such as cost, selectivity, biosafety and the ability to multiplex must be evaluated in the context of field use. Based on these criteria, we provide a critical review of current methods in antibiotic detection and evaluate their adaptability for use on-site. We categorize these methods into microbiological assays, physical and chemical assays, immunoassays, aptasensors and whole-cell biosensors. We recommend continued development of a dipstick or microfluidics approach with a bacterial promoter-based mechanism and colorimetric output. This technique would incorporate the advantageous aspects of existing methods, maximize shelf-life and ease-of-use, and require minimal resources to implement in the field. antibiotic resistance, detection limits, environmental pollutants, on-site monitoring, subinhibitory concentrations, dipstick sensor BACKGROUND Antibiotic resistance is an international problem that poses a direct threat to global health, agriculture and biosecurity (reviewed in Holmes et al.2016). The rise of antibiotic resistance in microorganisms is complex and multifactorial, but a significant source of antibiotic resistance acquisition is exposure to sublethal and subinhibitory concentrations of antibiotics in natural environments such as soil and water (Andersson and Hughes 2014; Long et al.2016; Le Page 2017; Sommer et al.2017). Sublethal concentrations are below the threshold for lethality in bacteria, while subinhibitory concentrations are below the threshold for growth inhibition (Hughes and Andersson 2012; Bengtsson-Palme and Larsson 2016; Long et al.2016). The presence of antibiotics at concentrations as low as nanograms per milliliter has been shown to promote the development of resistance in the laboratory (Hughes and Andersson 2012; Bengtsson-Palme and Larsson 2016; Long et al.2016; Almakki et al.2017). Due to the deleterious effects of low levels of antibiotics in soil and water, antibiotics are now regarded as a significant environmental pollutant (Li et al.2016; Jha et al.2017; Zhou et al.2017). However, the effect of antibiotic pollutants on microorganisms in the environment has not been extensively characterized (Brandt et al.2015). In particular, no global model exists to track and predict the distribution of antibiotic pollutants and their effects worldwide. In most existing antibiotic distribution studies, samples are obtained and taken to laboratories for analysis in a costly or time-consuming pipeline that limits capacity for widespread monitoring of antibiotic pollutant levels (Managaki et al.2007; Graham et al.2011; Hoa et al.2011; Zhang et al.2013; Tang et al.2015). While localized studies are clearly valuable, understanding the effects of antibiotics in a broader environmental context will improve efforts to combat the rise of antibiotic resistance (reviewed in Aminov 2009; Le Page et al.2017). The availability of quick, field-ready techniques would increase the convenience of on-site measurement, meaning that samples may be taken and analyzed more frequently, leading to more data and better predictive models of antibiotic pollutant entry and persistence in local environments and the rise of antibiotic resistance (Tang et al.2018). On-site measurement will allow researchers to assess levels of antibiotic pollutants in rural areas, where antibiotic pollution has been shown to be correlated with antibiotic resistance development (reviewed in Gothwal and Shashidhar 2014; McConnell et al.2018), without incurring the cost of transportation to laboratories. In addition, such a tool would benefit other researchers seeking on-site diagnostic tools and may draw on many similar methodological advances (Garg et al.2016). This paper reviews the status of the development of a first-line technique for preliminary, on-site detection of antibiotic pollutants by assessing the advantages and limitations of existing methods and highlights the most promising techniques for use in the field. Here, we evaluate the major classes of methods in the context of field use: microbiological assays, physical and chemical assays, immunoassays, aptasensors, and whole-cell biosensors. We then discuss future directions and criteria for the development of a first-line assay for use in the field to allow environmental monitoring of antibiotic concentrations. METHODS Microbiological assays A classic method for detecting the presence of antibiotics is the use of microbiological assays that employ antibiotic-sensitive species of bacteria to determine whether specific antibiotics are present in a given sample and, with limited sensitivity, their concentration. Since the 1950s, microbiological assays have identified aberrations in known growth patterns to detect the presence of antibiotics in a given sample (Kersey et al.1954). Current commercial assays rely on one of two techniques to identify antibiotic presence in a sample. Disc diffusion uses discs coated in the sample of interest that are placed on plates on which bacteria are grown; the presence of a halo of cleared growth around the disc indicates that the bacteria were killed by an antibiotic in the disc. This test can identify specific antibiotics or classes of antibiotics depending on the species of bacteria used and has been optimized for use in milk samples with a limit of detection (LOD) for β-lactams between 30 and 80 ng/mL (Kukurova and Hozova 2007; Piech et al.2016). A second technique uses specific bacteria with known sensitivity to antibiotics that have been engineered to yield a colorimetric output. These color-change reactions can be quantitatively analyzed with spectrophotometry to determine the concentration of antibiotic present in the sample and can be as sensitive as 1 ng/mL, but require sterile technique and laboratory facilities to co-incubate the sample with bacteria (Le Breton, Savoy-Perroud and Diserens 2007; Kalunke et al.2018). As a standard technique, commercially available microbiological assays have been optimized for testing food and drink samples, specifically milk and serum samples, to determine whether physiologically relevant levels of antibiotics are present (Kukurova and Hozova 2007; Le Breton, Savoy-Perroud and Diserens 2007; Beltrán et al.2015; Kalunke et al.2018). Other microbiological methods have qualitative or semi-quantitative outputs, and either provide a binary response or a range of concentrations (Kukurova et al.2007; Le Breton, Savoy-Perroud and Diserens 2007; Kalunke et al.2018). These methods are able to function in complex matrices, but due to their output, determining precise concentrations would be difficult to ascertain. Many microbiological assays are focused exclusively on the β-lactam family of antibiotics, commonly used in veterinary medicine, and have poor sensitivity outside this category; a review of commercial tests found that while they can detect concentrations as low as 4 ng/mL within the β-lactam family, the tests are limited to 100 ng/mL or more in other families (reviewed in Beltrán et al.2015). For some microbiological assays, the requirement for laboratory facilities is mitigated by microfluidics and paper-based techniques that function reliably with non-sterile samples and in field conditions (Sun et al.2011; Deiss et al.2014). Microfluidics analysis relies on changes in bacterial morphology when sublethal, but not subinhibitory, concentrations of antibiotics are present in the sample (Sun et al.2011). This technique requires some expertise to implement but shows promise for field adaptation. Droplet microfluidics exhibits greater sensitivity but current techniques have focused on identifying the presence of antibiotic resistance genes rather than measuring antibiotic concentrations (Kaminski, Scheler and Garstecki 2016). In summary, microbiological assays typically require significant equipment and sterile laboratory facilities and are often optimized for specific antibiotics, making them relatively ill-suited for on-site field analysis, although the development of microfluidics or paper-based techniques have expanded the range of these assays. A challenge for field use of paper-based microbial assays is the requirement for live or lyophilized bacteria that can be reconstituted on-site without introducing contamination. Due to these concerns as well as portability, incubation and sterility requirements, microbiological assays are not presently optimal for on-site field testing. Physical and chemical assays In contrast to microbiological assays, which are limited by bacterial sensitivity to antibiotics, physical and chemical assays target specific properties of the molecule such as size, charge, binding characteristics or reactive properties to identify antibiotics in a diverse sample. Physical purification entails separation of the antibiotic away from other impurities in the sample to allow isolation of the desired element through repeated fractionation. The final fraction for antibiotic purification is analyzed using spectrophotometry and has an LOD of approximately 25 ng/mL (reviewed in González de la Huebra and Vincent 2005). The prepared sample can additionally be analyzed using spectrometry, an equipment-intensive capability sensitive down to a single-molecule resolution. Tandem mass-spectrometry analyzes samples for molecules of interest, including antibiotics, with an LOD below 0.1 ng/mL (Zhang et al.2013). Liquid Chromatography/Mass-spectrometry (LC/MS) has been adapted to quantify antibiotic concentrations in milk, serum, meat, soil and water samples (Luo et al.2010; Hoa et al.2011; Zhang et al.2013 Bayen et al.2014; Kostich, Batt and Lazorchak 2014; Tang et al.2015; Jha et al.2017). LC/MS has also been implemented to analyze samples from the field, where matrix effects were determined to be insignificant after purification (Luo et al.2010; Hoa et al.2011; Zhang et al.2013 Kostich, Batt and Lazorchak 2014; Tang et al.2015; Jha et al.2017). Some commonly used techniques to combat the complications of soil matrices are isotope dilution, standard addition, manual calibration or changing the column (Aga et al.2005; Bayen et al.2014). Physical techniques have successfully been optimized for environmental samples, indicating that they do not require sterile inputs and are an excellent tool for laboratory analysis (Luo et al.2010; Bayen et al.2014; Chen and Zhou 2014; Kostich, Batt and Lazorchak 2014; Begou et al. 2017). However, both purification and spectrometry require a significant degree of user expertise and supporting infrastructure and involve challenging procedures or sensitive technology not easily transported to field sites; this technique is best adapted for processing field samples in fully equipped laboratories (Soprani et al.2016; Pan and Chu 2017). Alternatively, Surface-Enhanced Raman Spectroscopy (SERS) allows screen-printed electrodes to be used for spectroscopic analysis. This technique offers high sensitivity and portable, rapid detection that can be easily adapted to fieldwork (Li et al.2014). With a procedure length of less than 10 min, SERS has been used to determine antibiotic concentrations in water samples (Li et al.2013). However, SERS also requires sample purification to enrich antibiotics relative to other pollutants or solutes in the sample, increasing the technical knowledge required to test on-site. Eliminating pre-processing of the sample and automating data analysis of the output could optimize SERS for use on-site. Field-ready chemical assays have exploited microfluidics or paper-based approaches to be both portable and scalable. One group designed a microfluidic system that allows chemical interaction between aminoglycoside antibiotics and copper ions, with a measurable chemiluminescent output; this on-chip method is portable and has an LOD under 1 ng/mL (Sierra-Rodero, Fernández-Romero and Gómez-Hens 2012). While these features are promising for on-site use, this method requires some user expertise to microinject the sample onto the chip. Paper-based chemical solutions, in which chemical modifications to the paper induce colorimetric outputs in response to antibiotic presence, also show promise for fieldwork and require less technical expertise. Gomes, Goreti and Sales (2015) demonstrated that by adding a metal ion-responsive amine layer to a paper-based medium, oxytetracyclines could be detected in water with an LOD of around 30 ng/mL. However, paper-based methods can currently detect only a few predetermined compounds of interest. Further research should broaden the capacity of paper-based techniques to identify a wider range of compounds in a single test. Immunoassays Immunoassays provide highly sensitive, specific detection of antibiotics in complex liquid or processed solid samples, relying on specificity of interactions between antibodies and the molecule of interest (reviewed in Ahmed et al.2017). Immunoassays are typically colorimetric in output (Aga et al.2005; Cháfer-Pericás, Maquieira and Puchades 2010; Graham et al.2011) but some have fluorescent or chemiluminescent outputs (Meyer et al.2016), depending on the reporter molecule used. Immunoassays for antibiotic detection, specifically ELISAs, have been adapted for soil, food, and water samples (Aga et al.2005; Cháfer-Pericás, Maquieira and Puchades 2010; Graham et al.2011; Garg et al.2016; Meyer et al.2016). In addition, commercially available ELISAs developed for food samples have been utilized in field studies monitoring waste water from rivers and soil samples taken from experimental plots treated with manure (Aga et al.2005; and Graham et al.2011). Due to the cross-reactivity of antibodies detecting isomers and degradation products, ELISAs may overestimate concentrations and fail to show a decline over time (Aga et al.2005). Immunoassays can provide sensitive detection of antibiotics in samples using secondary antibodies to amplify the signal, and typically have an LOD below 1 ng/mL (reviewed in Cháfer-Pericás, Maquieira and Puchades 2010). However, adding secondary antibodies increases the cost per sample, limiting the utility of immunoassays as an inexpensive, field-ready technique. Another challenge limiting the use of immunoassays for on-site analysis is their reliance on spectrophotometers to quantify test results. These assays additionally require a moderate level of scientific expertise to complete the procedure, precluding untrained users from participating in the data collection process. The use of antibodies generally restricts one immunoassay to a single or relatively few antibiotics depending on the specificity of antibodies used in the assay (Graham et al.2011). Cross-reactivity of antibodies lessens the specificity of the test, as antibodies may react with non-biologically active components of antibiotic degradation pathways (Aga et al.2005). Concentration measures from a cross-reactive immunoassay would overestimate the level of antibiotic present in a given sample and impede efforts to understand antibiotic resistance in an ecological context. Despite these limitations, recent developments of miniaturized, user-friendly immunoassays offer some potential for the adaptation of this technique for the field. A diverse array of techniques has been developed that adapt immunoassays into a multiplex format, thus enhancing the efficiency and high-throughput ability of detection by localizing different antibodies to distinct parts of a chip or strip (Jiang et al.2013; Song et al.2015; Han et al.2016; Wang et al.2017a). One group has created a regenerable, chemiluminescent microarray that allows detection of up to 13 antibiotics at once in milk samples and is aiming to scale the product for low-cost use in the field (Meyer et al.2016). Other novel immunoassays have further improved usability by offering a multiplex assay with a quantifiable, colorimetric output. In 2013, Jiang and colleagues developed a 'dual-colorimetric' assay that employed two different enzyme substrate systems to simultaneously detect 13 fluoroquinolone and 22 sulfonamide antibiotics (Jiang et al.2013). More recently, a multiplex assay was created by Han et al. (2016) that enabled the simultaneous detection of three different antibiotics on a dipstick format; this technique offers several features that enhance usability, including a quantifiable, colorimetric output and a procedure time of 10 min with no pretreatment required for the device. In a similar device, which was specifically designed for high-throughput, on-site measurements, latex beads were utilized for colorimetric output on a single strip multiplex test for three antibiotic classes. When tested on spiked milk samples, this method demonstrated sensitivity of 0.3–10 ng/mL, with negligible crosstalk between the antibiotic classes and a procedure time of 10 min (Wang et al.2017a). These multiplexed immunoassays, which have been specifically designed with on-site, high-throughput detection in mind, show promise for first-line use in the field. Aptasensors A more recent technique for antibiotic detection uses aptamer sensors (aptasensors). Aptamers are DNA or RNA oligonucleotides that can bind with high affinity to specific targets (Yang et al.2017). Aptasensors developed for antibiotic detection fall into three categories: electrical, fluorescent and colorimetric. Electrical aptasensors typically immobilize an aptamer onto the surface of an electrode or a nanoparticle such that the introduction of the target molecule causes a conformational change in the aptamer. This change releases a probe or exposes the electrode to a redox probe. Abnous et al. designed an electrical aptasensor for fluoroquinolones that is representative of the electrode-based technique: a target-specific aptamer was immobilized on a gold electrode. In the absence of the target, single-stranded binding (SSBs) proteins bound to the aptamer and inhibited access of a redox probe to the electrode surface. In the presence of the target, aptamer-specific binding decreased the binding affinity of the SSBs, increasing access to the redox probe and inducing a detectable shift in current with a sensitivity of .0871 ng/mL (Abnous et al.2017). Other electrical aptasensors utilize different materials for the electrode (Li et al.2017; Wang et al.2018), complementary single stranded DNA to block a redox probe (Danesh et al.2016; Taghdisi et al.2016) or different probes such as ions or quantum dots (Li et al.2016; Yan et al.2016), but achieve the same output with comparable sensitivity (Liu et al. 2017a,b). Other electrical aptasensors utilize nanoparticles as a signal probe (Chen et al.2017b) or to immobilize the aptamer (Chen et al.2017a). Chen et al. designed an aptasensor where aptamers specific for kanamycin and chloramphenicol were immobilized on amino-modified magnetic beads and complementarily bound to DNA signal probes with a nanoscale metal organic framework (NMOF) carrying a specific ion. When the target was introduced, it bound to the aptamer, replacing and releasing the signal probe and generating a detectable current of ions in the NMOF (Chen et al.2017b). Fluorescent and colorimetric aptasensors employ a diverse array of mechanisms. Introduction of the target causes a conformational change that can expose a nanoparticle surface to a color-changing reagent (Emrani et al.2016; Lavaee et al.2017). Lavaee et al. immobilized complementary ssDNA on the surface of gold nanoparticles that, in the absence of the target, form arches with ciprofloxacin specific aptamers, blocking the surface of the nanoparticle and inhibiting the reduction of 4-nitrophenol, and therefore the color change. In the presence of the target, the surface is exposed, reducing the 4-nitrophenol and causing a measurable change in absorbance (Lavaee et al.2017). The presence of the target may also disassemble a complementary complex, releasing or quenching a fluorophore (Emrani et al.2016; Babaei et al.2017; He, Li and Hu 2017; Ha et al.2017; Wu et al.2017; Chen et al.2017d). Chen et al. utilized Waston-Crick and Hoogsten base pairing to form a triple helix between two arms flanking a tetracycline specific aptamer and a signal transduction probe (STP), which, in the presence of the target, disassembles and releases the STP. The STP then forms a quadruplex and binds to thioflavin T, generating a fluorescent output (Chen et al.2017d). Other fluorescent and colorimetric aptasensors may utilize hairpin loops that form a quadruplex in the presence of the target (Cui et al.2018) or an exonuclease to amplify a signal (He, Li and Hu 2017; Wu et al.2017). Aptasensors may also form hairpin loops that can then be detected using qRT-PCR (Duan et al.2017) or microchip electrophoresis (Wang et al.2017b). Paper-based aptasensors have been designed to make aptasensors easier to transport. Zhang, Zuo and Ye (2015) present a paper-based microfluidic device incorporating ssDNA aptamers for the detection of aminoglycoside antibiotics, with a sensitivity of around 300 ng/mL, while Sharma et al. (2017) designed a screen-printed electrode that detects kanamycin with a sensitivity of 0.11 ng/mL. Aptasensors are highly sensitive and have an LOD in the range of micrograms to nanograms per liter. The more sensitive aptasensors tend to be electrical and can have an LOD of 1.07 e – 5 ng/mL (Chen et al.2017a,b), which is ideal for detecting trace quantities of antibiotics in the field. In addition, aptasensors have relatively short procedures, ranging from 30 min to 2 h, which is useful for taking multiple samples in the field (Zhang, Zuo and Ye 2015; Li et al.2016; Yang et al.2017). Adapted to test antibiotic concentrations in milk, serum and water samples, aptasensors are able to detect antibiotics effectively in complex matrices (Hoa et al.2011). A significant limitation for the application of aptasensors in the field is that they are generally singleplex and therefore unable to identify multiple classes of antibiotics simultaneously. While multiplex aptasensors have been described, most have only tested simultaneous detection of up to two antibiotics (Yan et al.2016; Chen et al.2017a,b; Huang et al.2018; Li et al.2018; Zhou et al.2018). In 2016, Hao and colleagues implemented a cDNA aptamer system with chemiluminescent output to simultaneously detect oxytetracycline, tetracycline and kanamycin at a sensitivity of 0.05 ng/mL in milk samples. While this method was designed for usability and demonstrates high sensitivity, the chemiluminescent output may be a limitation for field use due to the need for detection equipment (Hao, Gu and Duan 2016). Aptasensors provide a highly sensitive, relatively fast and robust detection method; however, they have significant limitations in their ability to test a broad range of antibiotics simultaneously. In addition, aptasensors currently require significant training and detection reagents and equipment. While there are portable models of spectrophotometers and electrochemical workstations, they may be difficult to use in low-resource, isolated locations. Aptasensors would be a suitable secondary detection method to verify results with higher selectivity and specificity but are not ideal for widespread, high-throughput detection. However, the development of reagent-less or label-free aptasensors that would minimize training and require fewer resources is a promising direction for field use (Yang et al.2017; Wang et al.2017b; Chen et al.2017d). Whole-cell biosensors Another method of antibiotic detection uses synthetic biology to harness the internal machinery of bacterial cells to determine local concentrations of antibiotics. Rather than simply monitoring a cell’s response to antibiotic presence, bacterial cells can be genetically engineered to respond predictably to differing concentrations of antibiotics. This may be accomplished by inserting engineered plasmid constructs containing antibiotic-sensitive DNA sequences into host bacterial organisms. The product is known as a whole-cell biosensor (Korpela et al.1998; Gui et al.2017; Chen et al.2017c). Evolution has effectively primed bacteria to respond to potential damage; this response is often a change in gene expression driven by bacterial promoters, induced by the presence of a small molecule, including antibiotics (Goh et al.2002). This intrinsic cellular response to the presence of antibiotics can be detected by fusing the antibiotic-sensitive promoter to a reporter construct that transduces the signal to a visual response detectable by the user (Chen et al.2017c). The construction of whole-cell bacterial biosensors for antibiotic detection is not a new concept. In 1998, Korpela and colleagues genetically modified a strain of Escherichia coli by combining genetic elements responsive to tetracycline with a luciferase operon, creating a biosensor that responded to tetracycline with bioluminescence (Korpela et al.1998). Since then, dozens of iterations of similar designs have been tested and improved upon. The choice of promoter is perhaps the most vital aspect of biosensor design, as this choice determines the specificity as well as the sensitivity of the sensor (Gui et al.2017). Goh and colleagues found that after screening a library of 6500 bacterial promoters native to Salmonella typhimurium, up to 5% of these promoters demonstrated a change in expression in response to subinhibitory antibiotic concentrations. Many such promoters have been well-characterized in an array of bacterial species, providing hundreds of potential biosensor targets and bacterial hosts; furthermore, by using a panel of promoter-reporter fusions, it allows the classification of unknown antibiotics based on the response of each promoter (Goh et al.2002; Hutter et al.2004; Mascher et al.2004; Urban et al.2007; Staroń, Finkeisen and Mascher 2011; Melamed et al.2012). Whole-cell bacterial biosensors offer several advantages over other detection techniques. Bacteria require relatively few and low-cost resources to remain viable, and their rapid replication makes high-throughput testing easily achievable. Furthermore, bacterial sensors yield information about bioavailability of substrates which, in the context of tracking the development of bacterial resistance, may offer a more relevant metric than directly measuring concentrations in water or soil samples (Aga et al.2016; Gui et al.2017). Biosensors have been adapted for milk, water, serum and meat samples (Link, Weber and Fussenegger 2007; Pikkemaat et al.2010; Roda et al.2011; Zhang, Zuo and Ye 2015; Duyen et al.2017; Chen et al.2017c; Suárez et al.2009) and have also been implemented in field studies to detect antibiotics (Chen et al.2017c). While biosensors with qualitative binary outputs work consistently in spiked samples (Duyen et al.2017), more quantitative biosensors have shown recoveries of 95%–101%, and consistent results with HPLC and LC/MS (Pikkemaat et al.2010; Zhang, Zuo and Ye 2015; Chen et al.2017c). Most traditional biosensor techniques for detection of antibiotics have relied on fluorescent or bioluminescent output as a metric for local antibiotic concentrations, using plate readers or luminometers to determine output levels (Korpela et al.1998; Kurittu, Karp and Korpela 2000; Bahl, Hansen and Sørenson 2005; Eltzov et al.2008; Pikkemaat et al.2010). However, the threshold of detection for fluorescent output of biosensors remains a challenge. To address this, Melamed and colleagues present an innovative method for detection and identification of different types of antibiotics; they provide a panel of 12 bacterial reporter strains optimized for highly sensitive fluorescent output, paired with a computational algorithm which analyzes the dose-response curves for each fluorescent reporter strain in response to an unknown antibiotic and identifies the type of antibiotic based on mode of action (Melamed et al.2012; Melamed, Naftaly and Belkin 2014). While these analytical methods may improve antibiotic identification from biosensor output, fluorescence and bioluminescence remain difficult to assess precisely in the field. To address this issue, biosensors have been constructed with colorimetric output that can be qualitatively assessed by the naked eye or quantitatively assessed with a digital camera and online image analysis software (Fantino, Barras and Denizot 2009; Duyen et al.2017). While these solutions are still limited in their quantitative sensitivity, their design shows promise for applicability in the field. Another obstacle to field implementation is the portability of bacterial biosensors outside of the lab. Since these biosensors rely on healthy bacteria, the function of many existing biosensors is optimal at 37°C and therefore requires an incubator. Recent developments in cell immobilization and miniaturized devices, however, have improved prospects for storage of live cells. It has been demonstrated that lyophilization (freeze drying) and reconstitution has negligible effect on the functionality of bacterial biosensors (Kurittu, Karp and Korpela 2000); this technique has been successfully tested in a non-laboratory field setting for the detection of tetracyclines (Pikkemaat et al.2010). Another storage technique that takes advantage of the natural abilities of some bacterial cells is a 'sposensor,' a user-friendly, field-ready device that contains immobilized bacterial spores of genetically engineered Bacillus subtilis that act as whole-cell biosensors. While only tested on bacitracin with an LOD at around 50 ng/mL, it was designed specifically to be easily usable in the field, using only three tubes and limited reagents, and therefore may be a model for future work using these techniques (Fantino, Barras and Denizot 2009). The sensitivity of whole-cell biosensors remains a more challenging obstacle due to limitations in sensitivity in the detection of biosensor output, rather than the response of the biosensor itself. While colorimetric sensors provide a reliable equipment-free qualitative approach, they are not ideal for in-depth quantitative analysis. The field of bioengineering offers a powerful alternative: the biochip or 'lab-on-a-chip' design, which integrates bioluminescent whole-cell biosensors with miniaturized photomultipliers and charge-coupled detectors (CCDs) to amplify and convert the signal. This output may then be analyzed with online software to provide a quantitative result (Roggo and van der Meer 2017). In 2011, Roda and colleagues performed three different bioluminescent assays by implementing CCD imaging on a miniaturized device with engineered bacterial and yeast cells. While they did not attempt to detect antibiotic compounds using their device, the success of three different assays suggests that this technique could be adapted to antibiotic detection by using different engineered bacterial strains. This technique was shown to have a robust shelf life of up to 35 days with refrigeration, making it suitable for transport and use in the field (Roda et al.2011). While biochips are undergoing rapid improvements in size and usability, the most cost-effective field solution is paper based. Paper-based, colorimetric whole-cell biosensors including the 'sporosensor' have demonstrated innovative design and promising functionality and usability, but still work best at 37°C since they use living bacterial cells (Fantino, Barras and Denizot 2009). As an alternative, cell-free systems may be used that employ the same cellular machinery as a typical biosensor, hosted on a paper-based matrix rather than inside a living cell. Duyen and colleagues developed a paper-based, in vitro transcription/translation system for the detection of antibiotics that inhibit protein synthesis. While the device functioned most efficiently at 37°C (2-h incubation), detection at a comparable sensitivity could consistently be achieved at temperatures as low as 15°C by varying incubation time. With improvement to sensitivity that is currently in the range of 500–6100 ng/mL, this paper-based system shows potential for field use (Duyen et al.2017). FUTURE DIRECTIONS In this review, we have identified criteria for an effective device that is able to execute high throughput, on-site testing for detecting antibiotics in the environment. An optimal device would be highly sensitive, capable of multiplexing and detecting compounds from complex matrices, and would require minimal equipment, electricity, procedure time, and technical training for the user. Additionally, it would be cost effective, have extended shelf life under a range of environmental conditions, and meet biosafety standards. As discussed in this review, there are now many sensitive techniques for antibiotic detection and quantification; however, none currently satisfy all these criteria. While significant obstacles remain in adapting any of the existing devices for use in front-line, on-site implementation in the field, several do show promise. Perhaps the most convenient solution in terms of cost-effectiveness, shelf-life and usability for the field is a bacterial promoter-based approach using a strip or dipstick detector. A diverse array of dipstick-style solutions for antibiotic detection have been designed, covering a range of sensitivities and quantification ability (Link, Weber and Fussenegger 2007; Fantino, Barras and Denizot 2009; Gomes, Goreti and Sales 2015; Han et al.2016; Duyen et al.2017; Wang et al.2017a). In terms of device output, a colorimetric assay would provide user-friendly easily readable output that does not require expensive equipment for detection or analysis. The technique of Link, Weber and Fussenegger (2007) incorporates many of these field-ready criteria. Integrating a cell-free, inducible promoter system with an antibody-based reporter mechanism, this cell-free dipstick approach was shown to be highly sensitive, detecting a range of classes of antibiotics at concentrations of 2–40 ng/mL. Individual dipsticks were designed for the specific recognition of different antibiotics, without any signs of significant crosstalk between the targets. Finally, these dipsticks possess a shelf-life of up to 6 weeks at room temperature. The need for a dipstick-like technique, which is easy to use on-site and provides relatively quick, visual output, is well-justified within the relevant literature with that of Link et al. cited repeatedly a model of such a technique (Adrian et al.2012; Cháfer-Pericás, Maquieira and Puchades 2010; Chen et al.2012; Jiang et al.2013; Dincer et al.2017; Kamakoti et al.2018). The dipstick method was compared to other biosensor assays and was noted for its advantageous usability and sensitivity for antibiotic detection in food samples (Cháfer-Pericás, Maquieira and Puchades 2010). A recent review of future directions in biosensing and personalized drug therapy identified the dipstick method as the model exemplifying low cost and portability in biosensor design (Dincer et al.2017). However, despite its promise, significant problems remain barring its implementation in the field. When compared to other antibiotic detection techniques, the dipstick technique has been criticized for its lack of selectivity (Kim et al.2010; Hou et al.2013). While the dipstick assay can distinguish among three classes of antibiotics, tetracyclines, macrolides and streptogramins, it lacks further specificity due to the structural similarities of antibiotic molecules within these classes (Link, Weber and Fussenegger 2007). To distinguish between subclasses of antibiotics, therefore, an assay with higher specificity such as an aptasensor is advantageous. The dipstick assay has also been criticized for the length of the required incubation times, often requiring up to 1 h (Link, Weber and Fussenegger 2007; Cháfer-Pericás, Maquieira and Puchades 2010). However, enhancements to the dipstick assay constructed by Link et al. have already been published (Kling et al.2016; Dincer et al.2016). The dipstick detection mechanism was adapted onto a microfluidic platform to enable multiplexed detection on a miniaturized device. Furthermore, the output was modified to yield an electrochemical rather than colorimetric signal, enabling a more precise quantifiable output. This biosensor was shown to have a robust shelf-life of up to 3 months and a rapid output time of 15 min (Kling et al.2016). While it maintains limitations in specificity and has only been tested using spiked human serum, it offers the added advantages of a more rapid protocol time and multiplex capability. Ultimately, as an on-site, first-line evaluation technique, the broader detection and identification of antibiotic classes has been deemed sufficient (Cháfer-Pericás, Maquieira and Puchades 2010; Aga et al.2016; Kling et al.2016). Moreover, additional screening of natural promoters and the ability to design synthetic promoters has enormous potential to address specificity issues (Goh et al.2002; Hutter et al.2004; Mascher et al.2004; Urban et al.2007; Yagur-Kroll, Bilic and Belkin 2010; Staroń, Finkeisen and Mascher 2011; Melamed et al.2012; Wang, Tang and Yang 2017). CONCLUDING REMARKS All techniques described in this paper are viable methods for laboratory-based, in-depth testing of high-interest sites; however, this review focused on identifying the benefits and drawbacks of each method for use in high-throughput, on-site testing of environmental samples. For an antibiotic detection technique to be practical for field use it must be sensitive, easily usable with minimal training or equipment, and capable of detecting multiple antibiotic types from complex samples in a high-throughput manner; furthermore, it should be cost-effective, relatively quick, and possess a robust shelf life under diverse environmental conditions. A biosensor implementing an engineered bacterial promoter mechanism in a dipstick or microfluidic format meets many of these criteria and may be a favorable model for future development of on-site techniques. However, further testing in complex matrices is essential before this technique can be implemented in the field. The development and implementation of a field-ready antibiotic detection device that requires minimal scientific training will also enable crowdsourcing and local monitoring of antibiotic pollution data, in an analogous manner to crowdsourced monitoring of air pollution (Gurney and O’Keeffe 2013). The ability to use these methods on-site adds to the convenience for citizen scientists to provide time-series data on antibiotic concentrations at a local level. Compiling data on antibiotic pollutants at the national level will allow monitoring and modeling of antibiotic distributions similar to the successful global monitoring of mercury as an environmental pollutant (D’Amore et al. 2015). After collection of limited data, Pacyna and Pacyna (2002) presented an estimate of global mercury emissions, which was later modeled in a geospatial manner by Wilson et al. (2006) to determine patterns of mercury pollution and project environmental impact. Subsequently, Depew et al.(2013) compiled regional datasets to determine national mercury distribution in Canada, and researchers in the United States mapped mercury contamination in the Everglades to assess spatial and temporal trends in mercury bioaccumulation (Axelrad et al.2011; Depew et al. 2013). An analogous database of antibiotic contaminants could be assembled allowing international efforts to model the distribution of these pollutants and assess their impact (Travnikov et al.2015). Such a model will allow for the tracking and prediction of antibiotic resistance development on a global scale. SUPPLEMENTARY DATA Supplementary data are available at FEMSEC online. ACKNOWLEDGEMENTS The authors would like to recognize Sarah Jo McGeady, Meadow Parrish, and Cecilia Zheng for their significant contributions to the planning and preliminary literature search for this article. While not part of the project team, the authors also wish to thank the U.S. Department of State’s Diplomacy Lab program, and its Office of International Health and Biodefense, for working with them to identify this problem and consider solutions. Conflict of interest. None declared. REFERENCES Abnous K , Danesh NM , Alibolandi M et al. 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FEMS Microbiology EcologyOxford University Press

Published: Jun 5, 2018

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