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Lyophilized Engineered Phages for Escherichia coli Detection in Food Matrices Juhong Chen, Rachael A Picard, Danhui Wang, and Sam R. Nugen ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00561 • Publication Date (Web): 18 Oct 2017 Downloaded from http://pubs.acs.org on October 19, 2017
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Lyophilized Engineered Phages for Escherichia coli Detection in Food Matrices Juhong Chen,† Rachael A. Picard,‡ Danhui Wang,† and Sam R. Nugen*† †
Department of Food Science, Cornell University, Stocking Hall, Ithaca, New York 14853, USA Department of Food Science, University of Massachusetts, 102 Holdsworth Way, Amherst, Massachusetts 01003, USA Supporting Information Placeholder ‡
Keywords: freeze-drying, genetically engineered phage, beta-galactosidase, bacteria detection, resource-limited setting ABSTRACT: Easy-to-use, low-cost, and convenient-of-
transport are the key requirements for a commercial bacteria detection kit designed for resource-limited settings. Here, we report the colorimetric detection of Escherichia coli (E. coli) in food samples using freeze-dried engineered bacteriophages (phages). In this approach, we have engineered T7 phages to carry the lacZ operon driven by T7 promoter to overexpress reporter enzyme. The engineered phages were freeze-dried in a watersoluble polymer for storage and transportation. When used for the detection of E. coli cells, the intracellular enzyme [β-galactosidase (β-gal)] was overexpressed and released into the surrounding media, providing an enzyme-amplified colorimetric signal. Using this strategy, we were able to detect E. coli cells at the concentration of 102 CFU•mL-1 in food samples without the need for sophisticated instruments or skilled operators.
Water and foodborne pathogenic bacteria can cause severe bacterial infection in humans which account for approximately one-third of global mortality, especially for children in developing countries.1-3 Current bacteria detection methods (e.g. plate counting and polymerase chain reaction) are time-consuming and/or take several days to receive results.3-4 In order to circumvent this issue, numerous strategies have been developed to detect bacteria with a pragmatic response time.5-8 Unfortunately, sensitive and specific results require sophisticated instruments and skilled operators, limiting their application in resource-limited settings. Thus, there is a continual and urgent need for the rapid and effective detection of pathogens in resource-limited settings. Recent technological advances have enabled the detection of bacteria without the requirement of skilled operators or sophisticated instruments. For example, advanced lateral flow assays have been reported to specifically detect pathogenic bacteria, including Listeria monocytogenes, Escherichia coli, and Staphylococcus
aureus.9-11 Additionally, personal glucose meters have been adapted as an analytical platform to detect pathogens.12-13 However, sample pretreatment, required of addition reagents, and the need for refrigeration during shipping and storage limit their application in resourcelimited settings. Ideally, a pragmatic bacteria detection kit should be amenable to large-scale industrial manufacture. Bacteriophages (phages), are viruses which infect bacteria and have been widely employed to detect pathogens in the area of food safety and environmental monitoring.14-18 After infecting a bacterial host, the newly replicated phages, or intracellular components (e.g. adenosine triphosphate, adenylate kinase, and βgalactosidase) can be used to estimate the original bacterial concentration.4, 19-21 The phage hosts specificity depends on the tail fiber and can range from narrow to broad. Most importantly, phages have the ability to distinguish viable bacteria from inactive bacteria cells, because phages can only replicate using the molecular machinery of viable bacteria cells.22-23 Although phages offer significant advantages when used for bacteria detection, there is significant room for the improvement of sensitivity. To achieve the aforementioned goal, the development of the detection kit should consider portability and simplicity of design. Here, we used lyophilization to store detection components (e.g. engineered phages, a colorimetric subtract, and Luria-Bertani broth) in a watersoluble polymer at ambient temperature. Several key features for freeze-drying technology are addressed, including (1) retention of engineered phage lytic activity following rehydration,24-26 (2) combination of all reagents in one tube by freeze-drying for end-user simplicity, (3) reduction of transportation cost through the removal of water from freeze-drying, and (4) extension of shelf life under atmospheric conditions from the storage of reagents in lyophilizing powder. In this study, we developed a colorimetric detection test of Escherichia coli (E. coli) using freeze-dried engi-
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Figure 1. Schematic representation for the detection of E. coli using engineered phage in freeze-dried polymer. Four steps were involved: (1) freeze-drying the reagent kit in centrifuge tubes; (2) addition of E. coil solution to dissolve freeze-dried polymer; (3) engineered phage infection of E. coli cells to overexpress β-gal; and (4) enzymatic reaction to hydrolysis CRPG to generate readout signal. neered phages in water-soluble polymers (Figure 1). The T7 phages were genetically engineered to carry the lacZ operon according to reported methods with slight modification.27-30 After the engineered phages attached to the surface of host bacteria cell, the engineered genomes containing the lacZ operon were inserted into the host bacteria cells. Within the cells, the phage genome took over bacteria machinery to transcribe and translate genes including the lacZ operon resulting in β-galactosidase (β-gal). Thus, the modified T7lacZ phages enabled the overexpression of β-gal during the phage infection of E. coli cells. Upon the phage lysis, the intracellular components including overexpressed β-gal were released into environmental media. In the presence of a colorimetric substrate [chlorophenol red-β-D-galactopyranoside (CRPG)], the released β-gal catalyzed CPRG resulting in a red color, thus providing a visual colorimetric signal. This system has been demonstrated to detect E. coli in
drinking water, skim milk, and orange juice. Prior to the detection of E. coli, the reagent kit with engineered phages was freeze-dried for storage. Compared with the conventional liquid storage of phages, freeze-drying can retain infectivity, as well as reduce transportation and storage costs.24 Thus, freeze-drying of engineered phages with the appropriate bulking agents and stabilizer is of significant importance for long-term storage.25 Here, engineered phages were added into water-soluble polyvinylpyrrolidone (PVP) solution containing trehalose as stabilizer, then freeze-dried.25 The lyophilization powders were evaluated using scanning electron microscopy (SEM). As shown in Figure S1, the sheet structure of PVP polymer was observed. A highmagnification SEM image is shown in Figure S1b. These PVP polymer-protected engineered phages were packed in vacuum bags and stored at room temperature
Figure 2. (a) The relationship between absorbance intensity (575 nm) against different bacterial concentrations within different detection times; (b) Photographs of detection solution against different bacterial concentrations within different detection time. ACS Paragon Plus Environment
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Figure 3. (a) The absorbance intensity towards storage time (60 days) for the detection of 106 CFU·mL-1 E. coli cells using our freeze-dried reagent kit; and (b) the relationship between phage concentration and storage time (60 days). Error bars represent the standard deviation of three replicates. One asterisk (0.01 < P value < 0.05) indicates difference and two asterisks (P value < 0.01) indicate significant difference. until further use. In order to demonstrate that our freeze-dried reagent kit can be used for E. coli detection, E. coli BL21 was used as a model analyte. The sensitivity and quantitative range of E. coli cells were investigated against various bacterial concentrations (from 101 to 106 CFU·mL-1, PBS without bacteria cells was used as a negative control). At each detection time, the absorbance intensities (575 nm) increased with the increasing of E. coli concentration (Figure 2a). For each E. coli concentration, the absorbance intensities also increased with the increasing of detection time (Figure 2a). The images of each detection solution against different E. coli concentrations within different detection times were shown in Figure 2b. The color of the detection solution changed from yellow to red, indicating the presence of E. coli cells in the solution. With the increase of detection time, the color change was observed at lower E. coli cell concentrations. Furthermore, the relationship between detection limit (distinguished color change) and E. coli concentration was plotted in Figure S2. The detection limits of 106, 105, 104, 103, and 102 CFU·mL-1 were obtained after the incubation of 3, 4, 5, 6, and 7 hours, respectively. Additionally, we investigated the stability of our freeze-dried reagent kit during a long-time storage. Trehalose and gelatin as stabilizer were used to retain engineered phage infectivity. The recipes and lyophilization conditions were investigated in our previous publications.24, 26 When not in use, the freeze-dried reagent kits were stored at room temperature and used to intermittently detect E. coli cells at the concentration of 106 CFU·mL-1. PBS without E. coli cells was again used as a negative control. The absorbance intensity was plotted against storage time (0-60 days). On selected days, the control and sample were tested. And, the difference between two sets of data was determined using student t-
test. As shown in Figure 3a, there was no significant change observed in the absorbance intensity for both tested and control samples after storage of 60 days. During the two-month storage study, the absorbance intensity of tested samples was statistically different (P value < 0.05) from that of control samples on each day test except day 40 (P value = 0.058), indicating that our freezedried reagent kit can be used to detect E. coli cells during the storage time. Furthermore, no reduction in active phage numbers was observed after storage for 60 days, indicating that phage infectivity was retained (Figure 3b). It is promising that our freeze-dried reagent kit could provide long-time stability for E. coli cell detection, suggesting an extended shelf-life at room temperature without a significant loss of functionality. To further investigate the application possibilities of our freeze-dried reagent kit for colorimetric detection of E. coli cells in resource-limited settings, food samples (drinking water, skim milk, and orange juice) were tested using our freeze-dried reagent kit. Prior to measurement, the E. coli BL21 cells were inoculated into drinking water, skim milk, and orange juice (these samples were used without pretreatment), respectively. As shown in Figure 4a, the color of the detection solution was more red with increasing of E. coli cell concentration in all the food samples. Compared to the control sample, a distinguished red color was observed at the E. coli concentration of 102 CFU·mL-1, indicating that we were able to detect E. coli cells at 102 CFU·mL-1 using the naked eye. An image of all the detection solutions in a 96-well plate is shown in Figure 4b. Due to the inference of the background color from orange juice, the color change was not as obvious. Furthermore, the color of each detection solution was analyzed using ImageJ V1.48 software (Wayne Rasband, Madison, WI, USA). The R/B values were plotted against E. coli concentrations in Figure 4c-e. The statistical analysis was con-
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Figure 4. (a) Photograph of the detection solutions with different E. coli concentrations (0, 101, 102 CFU·mL-1) in different food matrices; (b) photograph of the detection solution in a 96-well plate; and (c-e) the relationship between R/B value and E. coli concentration in different food matrices. Error bars represent the standard deviation of three replicates. One asterisk (0.01 < P value < 0.05) indicates difference and two asterisks (P value < 0.01) indicate significant difference. ducted between the control and sample settings, which also confirmed that E. coli concentrations of 102 CFU·mL-1 can be detected using our freeze-dried reagent kit (P value < 0.05). In summary, we have freeze-dried engineered phages to provide rapid and sensitive colorimetric detection of bacteria in resource-limited settings. The freeze-dried phages kept their infectivity after the storage of two months (60 days) at room temperature. Using this detection kit, bacteria concentration as low as 100 CFU·mL-1 in food matrices can be detected without the need for sophisticated instruments or skilled operators, which provides a potential to improve water quality, food safety, and human health in remote parts of Africa, South America, and Southeast Asia.
The authors would like to acknowledge United States Department of Agriculture (2013-02037) and (2016-6701726462) for their support.
ASSOCIATED CONTENT
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Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Bacteria strain, chemical, instruments, and experimental details.
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AUTHOR INFORMATION Corresponding Author
*(S.R.N.) Phone: +1-607-255-9185. Fax: +1-607-2544868. E-mail:
[email protected]. Notes The authors declare no competing financial interests.
ACKNOWLEDGMENT
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