Rapid Detection of Viable Escherichia coli O157 by Coupling Propidium Monoazide with Loop-Mediated Isothermal Amplification

Abstract

Conventional molecular detection methods cannot distinguish between viable and dead Escherichia coli O157 cells. In this study, the loop-mediated isothermal amplification (LAMP) method combined with propidium monoazide (PMA) treatment was developed to selectively detect viable E. coli O157 cells. Four primers, including outer primers and inner primers, were specially designed for the recognition of six distinct sequences on the serogroups (O157) of the specific rfbE gene of the E. coli O157 genome. PMA selectively penetrated through the compromised cell membranes and intercalated into DNA. Amplification of DNA from dead cells was completely inhibited by $3.0{\mu}g/ml$ PMA, whereas the DNA derived from viable cells was amplified remarkably within 1 h by PMA-LAMP. Exhibiting high sensitivity and specificity, PMA-LAMP is a suitable method for evaluating the inactivation efficacy of slightly acidic electrolyzed water in broth. PMA-LAMP can selectively detect viable E. coli O157 cells. This study offers a novel molecular detection method to distinguish between viable and dead E. coli O157 cells.

Introduction

Escherichia coli O157 is a major foodborne pathogen that causes diarrhea, abdominal pain, and more serious complications, such as hemorrhagic colitis and hemolytic-uremic syndrome [24]. According to the latest CDC report, E. coli O157 caused an estimated 63,100 illnesses, 2,100 hospitalizations, and 20 deaths annually [26]. Many outbreaks of disease are closely associated with several kinds of food, such as raw vegetables, fruits, fresh cheese, raw milk, and undercooked beef. Food contamination with E. coli O157 is of increasing concern internationally. The issue has become a great challenge to food safety and human public health. Thus, it is of utmost importance and urgency to develop rapid, low-cost, highly sensitive, and specific methods for the detection of E. coli O157.

Conventional detection methods, such as sorbitol MacConkey agar culture, are too time-consuming for many applications, as enrichment before selective plating and confirmation of positive samples require nearly an entire week. Many molecular methods have been reported to overcome this problem, mainly focused on polymerase chain reaction (PCR) or real-time PCR [4,10,14]. Notomi et al. [23] developed a novel nucleic acid amplification method called loop-mediated isothermal amplification (LAMP) in 2000. This method depends on autocycling strand displacement DNA synthesis performed by the Bst DNA polymerase large fragment [21,23], which differs from PCR and real-time PCR in that four to six primers perform target gene amplification. The amplification is conducted under isothermal conditions of 60-65℃, and amplicons reach numbers as great as 109 within 60 min. The A set of four primers method has been used to detect various foodborne pathogenic microorganisms in recent years, demonstrating that LAMP is a potentially valuable tool for rapidly diagnosing pathogens in food, environmental, or clinical samples [1,9,36].

However, a major obstacle with molecular diagnostic methods is the difficulty in distinguishing between DNA from viable and dead cells, as intact DNA can persist for a considerable time even though the cell is dead, leading to a high incidence of false-positive results [25]. Therefore, current molecular diagnostic methods that are based on this technology tend to overestimate the risk caused by viable cells. A promising way to solve this problem is through the use of nucleic acid intercalating dyes such as ethidium monoazide (EMA) or propidium monoazide (PMA) as a sample pretreatment, so that the dyes can enter cells with damaged membranes and intercalate with DNA bases [22]. PMA has been proven to be more selective owing to the higher charge of the molecule and its selective penetration into membrane-compromised or dead cells, but not into integrated membranes of viable cells [8,22,34]. Slightly acidic electrolyzed water (SAEW) as a novel sanitizer for microbial safety is generated by electrolysis of a diluted hydrochloric acid (HCl) solution and/or sodium chloride (NaCl) solution in a cell with or without a separating membrane. Although widely applied in several fields, evaluating the inactivation effect of SAEW is still performed by the conventional culture method, which lacks high specificity and can be too time-consuming for a number of applications [7]. Hence, the objectives of this study were to develop a method to selectively detect viable cells of E. coli O157 by coupling PMA treatment with subsequent LAMP, and to evaluate the inactivation effect of SAEW in broth.

 

Materials and Methods

Bacterial Strains and Cultivation

A total of 62 strains were used in this study (Table 1). Among them, 12 strains belonged to E. coli O157, 38 strains to non-O157, and 12 strains to non-E .coli bacteria, used for specificity evaluation tests. E. coli O157:H7 reference strains ATCC 43895 and ATCC 43894 were also used for optimization of the reaction conditions. They were routinely grown on Trypticase Soy Broth (TSB, BD Diagnostics Systems, Sparks, MD, USA) and were enumerated by plate count on Trypticase Soy Agar (TSA, BD Diagnostics Systems) at 37℃ for 24 h.

Heat Treatment of Bacterial Cells

The 1.5 ml microcentrifuge tubes containing 0.1 ml of cell suspension (1.6 × 109 CFU/ml) were heated at 100℃ for 5 min in a water bath. The treated tubes were cooled to 4℃ and the absence of viable cells was confirmed by performing the plate count method on TSA.

Preparation of Slightly Acidic Electrolyzed Water and Treatment of Suspension in Broth

Slightly acidic electrolyzed water was produced by electrolysis of a dilute HCl solution (6%) in a chamber without a membrane, using an electrolysis device (BC-360; Cosmic Round Korea Co. Ltd., Seongnam, Korea) at a setting of 2.5 A (current). The pH, oxidation reduction potential (ORP), and available chlorine concentration (ACC) of the treatment solution were measured immediately before treatment with a dual-scale pH meter (Accumet model 15; Fisher Scientific Co., Fair Lawn, NJ, USA) bearing pH and ORP electrodes. The ACC was determined by a colorimetric method, using a digital chlorine test kit (RC-3F; Kasahara Chemical Instruments Corp., Saitama, Japan). The detection range for this measurement was 0-300 mg/l.

The SAEW used in this study had a pH of 5.40, an ORP of 709-764 mV, and an ACC of 21 mg/l. The volume of 9 ml SAEW was transferred to separate sterile screw-capped tubes, and the caps were closed tightly. The volume of 1 ml each bacterial culture was added to each tube under this conditions (dipping time: 3min; pH: 5.4; storage temperature: 25℃), and the tubes were mixed immediately. Following each treatment, 1 ml of each sample was transferred to a tube containing 9 ml of neutralizer (0.85%NaClcontaining0.5%Na2S2O3). Serial 10-fold dilutions were performed in 0.85% saline solution, and the surviving population of bacteria was determined by plating 0.1 ml of each dilution in duplicate on TSA plates. The number of colonies was enumerated on TSA plates after incubation at 37℃ for 24 h. Enrichment was performed to confirm the presence of the lower numbers of survivors that would not be detected by direct plating. For enrichment, 1 ml of each sample solution after treatment was transferred to a 100 ml flask containing 20 ml of sterile TSB and incubated at 37℃ for 24 h. Following enrichment, the culture was plated on TSA plates, and enumerated after incubation at 37℃ for 24 h.

Treatment of Suspensions with Propidium Monoazide

A 1 mg/ml solution of PMA (Biotium, Inc., Hayward, CA, USA) in 20% (v/v) dimethyl sulfoxide (Hayashi Pure Chemical Industries, Ltd., Oakville, Osaka, Japan) was prepared and was stored in the dark at 4℃. A 500 µl portion of each cell suspension in a transparent eppendorf tube was mixed with a series of concentrations of PMA solution and then stored on ice in the dark for 10 min. After dark incubation, the tube was placed horizontally on ice and was exposed to a 650 W halogen lamp (FCW 120V; GE Lighting, Cleveland, OH, USA) at a distance of 15 cm for 2 min with the more transparent side facing upwards [5]. During light exposure, the tube was shaken gently to encourage uniform exposure of the suspension to light.

Table 1.a“+” represents a positive result; “-” represents a negative result.

Preparation of DNA Template

The viable or dead E. coli O157:H7 cells were collected by centrifugation at 10,000 ×g for 5 min. The supernatant was then removed and the cell pellet of the tube was suspended in 200 µl of 1× phosphate-buffered saline Tween 20 buffer solution and was vigorously vortexed for a few seconds. The tube was then placed in boiling water for 5 min (10 min for Gram-positive strains). Thereafter, it was chilled immediately for 5 min on ice. Finally, the supernatant was used as a DNA template after centrifugation at 10,000 ×g for 5 min.

Table 2.aThe positions are numbered based on the coding sequences of the rfbE gene with GenBank ID S83460.

LAMP

A set of four primers were designed from sequence data submitted to GenBank (tyvelose epimerase gene, rfbE, S83460 [2] for LAMP to target six distinct regions using the Primer Explorer V4 software (http://primerexplorer.jp/e/). Forward inner primer (FIP) consisted of the complementary sequence of F1 (F1c) and F2; backward inner primer (BIP) consisted of the complementary sequence of B1 (B1c) and B2. The outer primers F3 and B3 were located outside of the F2 and B2 regions. The sequences of primers used for the LAMP assay are listed in Table 2. LAMP assay was carried out in a total 25 µl reaction mixture containing 1.6 µmol/l (each) of the FIP and BIP primers, 0.2 µmol/l (each) of the F3 and B3 primers, 1.6 mmol/l of deoxynucleoside triphosphates, 6 mmol/ l MgSO4, 1 mol/l betain (Sigma, St. Louis, MO, USA), 1× thermo pol buffer (New England Biolabs, Ipswich, MA, USA), 8 U of Bst DNA polymerase (New England Biolabs), and 2 µl of target DNA. After incubation by a thermal cycler (MyGenie 32; Bioneer, Daejeon, Korea) at 60-65℃ for 30-90 min, the reaction was terminated by heating at 80℃ for 2 min. The experiment was independently repeated twice.

PCR

As a comparison, a PCR assay targeting the E. coli O157:H7 rfbE gene was performed using F3 and B3 primers (Table 2). The 25 µl volume reaction mixture contained PCR buffer, 0.2 mmol/l of each dNTP, 12.5 pmol/l of each PCR primer, 0.25 µl (5 U/µl) of Taq DNA polymerase (TaKaRa Biotech, Dalian, China), and 2 µl of DNA template. After a 5 min denaturation at 94℃, the PCR mixtures were subjected to 30 cycles of amplification at 94℃ for 30 sec, 53℃ for 45 sec, and 72℃ for 45 sec, and a final extension at 72℃ for 10 min, using a thermal cycler (MyGenie 32).

Detection of Amplification Product

The LAMP and PCR amplification products were electrophoresed on a 1.5% agarose gel, and the target bands were visualized by staining with SafeView nucleic acid stains (Applied Biological Materials Inc., Vancouver, Canada). SYBR Green I is a fluorescent dye used widely in detection of nucleic acids. In this study, 25 µl of LAMP reaction products were dyed with 1 µl of SYBR Green I (1000X, Lonza, USA) and determined immediately through both visual observation of the color change by the naked eye and of a fluorescence assay under UV.

 

Results

Optimization of LAMP Assay Conditions and Concentration of PMA Treatment

In order to determine the optimal conditions of LAMP, DNA from E. coli O157:H7 ATCC 43895 was used as a target template. The specific LAMP assay generated many ladder-like pattern bands on 1.5% agarose gel, due to its characteristic structure. The LAMP protocol was carried out as described previously [42]. There were no significant differences under isothermal condition between 60℃ and 65℃; however, the LAMP product amplified at 65℃ showed slightly greater DNA production compared with lower temperature amplifications (data not shown), which was consistent with previous studies [32]. Additionally, the reaction time of 60 min was found to be the best choice based on the LAMP reaction efficiency. In this study, the optimal condition of LAMP was 65℃ for 60 min. As shown in Fig. 1, the viable and dead cell suspensions (1.3 × 108 CFU/ml) were treated with a series of PMA concentrations. As shown in Fig. 1A, there was no obvious difference between the amplification of target DNA derived from viable cells in the PMA-LAMP using different concentrations of PMA. This indicated that PMA had no significant inhibition on DNA amplification derived from viable E. coli O157:H7 cells by the PMA-LAMP method. As can be seen in Fig. 1B, the amplification of DNA derived from dead cells was completely inhibited when PMA at a concentration of 3.0 μg/ml or higher was applied. When the dead cells were treated with 0-2.0 μg/ml PMA, the target DNA was amplified. Therefore, the concentration of 3.0 μg/ml PMA was determined to be suitable for discrimination of target DNA from dead and viable cells by the PMA-LAMP method. Thus, the concentration of 3.0 μg/ml PMA treatment was used in all subsequent trials.

Fig. 1.Concentrations of PMA for inhibiting the amplification of DNA from viable cells (A) and dead cells (B). (A) Lane M, 100 bp marker; lanes 2-9, varying concentrations of PMA (0, 1.0, 2.0, 3.0, 5.0, 10.0, 15.0, and 20.0 μg ml-1). (B) Lane M, 100 bp marker; lanes 2-9, varying concentrations of PMA (0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, and 10.0 μg ml-1).

Sensitivity of PMA-LAMP and PMA-PCR

In order to compare sensitivity, DNA derived from viable E. coli O157:H7 was diluted by 10-fold dilution. The diluted DNA solutions were then subjected to PMA-LAMP and PMA-PCR. The results showed that the limit of detection (LOD) for PMA-LAMP was as low as 1.6 × 102 CFU per reaction tube, whereas the LOD for PMA-PCR was 1.6 × 106 CFU per reaction tube. The comparative sensitivity of PMA-LAMP and PMA-PCR demonstrated that PMA-LAMP was much more sensitive than PMA-PCR.

Fig. 2.Detection of E. coli O157:H7 cells with 1.5% agarose gel electrophoresis by LAMP (A) and PCR (B). Viable or dead cells (1.3× 109 CFU/ml) were prepared from E. coli O157:H7 ATCC 43895 and ATCC 43894. The cells were treated with or without PMA, and DNA was then extracted as a template. Lane 1, ATCC 43895, dead cells with PMA; Lane 2, ATCC 43894, dead cells with PMA; Lane 3, ATCC 43895, dead cells without PMA; Lane 4, ATCC 43894, dead cells without PMA; Lane 5, ATCC 43895, viable cells with PMA; Lane 6, ATCC 43894, viable cells with PMA; Lane 7, ATCC 43895, viable cells without PMA; Lane 8, ATCC 43894, viable cells without PMA.

Evaluating the Specificity of PMA-LAMP and Inactivation Efficacy of SAEW

Among the 72 bacterial strains in a viable state used to determine the specificity of the PMA-LAMP assay, no false-positive or false-negative results were generated (Table 1). As can be seen from Fig. 2, gene rfbE of the viable E. coli O157 was amplified by LAMP and PCR, whether samples were treated or not with PMA (Fig. 2, , lanes 5, 6, 7, and 8 ) The gene from dead cells that were treated with PMA was not amplified by LAMP or PCR (Fig. 2, , lanes 1 and 2); meanwhile, the rfbE gene of PMA-untreated cells was amplified (Fig. 2, lanes 3 and 4). The results illustrated that PMA treatment was a useful tool for the specific detection of viable cells by LAMP and PCR. Although both PMA-LAMP and PMA-PCR revealed amplification of the target gene, the former showed more advantages. PMAPCR needed almost 3 h or more for amplification reaction and product identification. From Fig. 3, it can be seen that the PMA-LAMP products could be visually detected by examining color changes with the naked eye, and with fluorescence under UV light with SYBR Green I. By contrast, PMA-LAMP could finish the DNA amplification reaction and product identification with SYBR Green Ⅰ within 1 h.

Fig. 3.Amplification of LAMP products dyed with SYBR Green I, visually detected by examining color changes with the naked eye (A) and fluorescence under UV light (B). (A) Green indicates a positive result and orange indicates a negative result. (B) Intensely bright cells are determined to be positive and those lacking appreciable fluorescence are deemed negative. Lanes from 1 to 8 are the same samples as those in Fig. 2.

In this study, the SAEW produced in our laboratory, which had a pH of 5.40, an ORP of 709-764 mV, and an ACC of 21 mg/l, was used to inactivate the E. coli O157 cells in broth. Serial 10-fold dilutions of viable E. coli O157 cell suspensions (1.3 × 108 CFU/ml) with 0.1% (w/v) peptone water were prepared to ascertain the inactivation efficacy of SAEW. After treatment with SAEW, there were no surviving populations of E. coli O157 from 1.3 × 106 to 1.3 × 100 CFU/ml by direct plate count, except for the sample of 1.3 × 107 CFU/ml with a mean of 615 isolates. Meanwhile, all the dilution samples after treatment with SAEW were analyzed by PMA-LAMP. There was only one positive result with the sample of 1.3 × 107 CFU/ml and negative results with all other samples. This illustrated that viable cells only remained in the sample of 1.3 × 107 CFU/ml after treatment with SAEW. The comparative results indicated that PMA-LAMP was consistent with the traditional plate count method for the discrimination of viable cells. This study also showed that SAEW had a strong disinfection ability to inactivate microbes in broth, especially in low concentrations of cells suspensions. The PMA-LAMP method presented in this study is an advantageous technique to distinguish between viable and dead E. coli O157 cells. Furthermore, this method can be applied to other bacteria and used in the food industry after optimization.

 

Discussion

Molecular detection methods based mainly on DNA amplification, such as PCR and real-time PCR, cannot distinguish between viable and dead cells, but merely identify viable DNA. Recently, DNA-intercalating dyes that selectively enter compromised cell walls and membranes have been used together with PCR or real-time PCR for the selective detection of viable cells [3,22]. PMA-PCR and PMA-real-time PCR have also been reported to selectively detect viable cells [15,19]. PMA-real-time PCR has many advantages such as high sensitivity, low contamination, and easy standardization. However, owing to the requirement for expensive equipment, application of PMA-real-time PCR in the laboratories is limited [17]. The PMA-LAMP developed in this study could selectively detect viable E. coli O157 without using sophisticated equipment or expensive reagents. Moreover, compared with PMA-PCR, it was much more sensitive. The reaction could be performed in a water bath or heat block in resource-limited sites, and amplification products could be detected by electrophoresis in agarose gel, under UV light, or by SYBR Green I dye. In particular, the use of SYBR Green I visual confirmation was very apparent. However, when applied to large numbers of samples, there is a greater risk of contamination through the use of SYBR Green I due to opening and closing of the LAMP reaction tube. To avoid such problem, preparation of DNA templates and all reaction reagents should be performed aseptically on a clean bench [28].

It is noteworthy that gene rfbE or other genes have been previously used as target genes to design LAMP assays for E. coli O157:H7 [35]. There were also LAMP assays developed recently for STEC by targeting the stx genes [12,13,16,20,30,31]. Recently, viable Salmonellae and Listeria monocytogenes cells were specifically detected by coupling PMA or EMA with LAMP [5,18,29]. However, there is no report available relating to the inhibition effect of PMA concentration on LAMP amplification of DNA from viable and dead E. coli O157 cells. In this study, significant inhibition was not observed for theamplification of the DNA derived from viable E. coli O157 cells with 0-20.0 μg/ml PMA treatment by the PMA-LAMP method. Complete inhibition for the amplification of DNA derived from dead E. coli O157 cells was achieved by 3.0 μg/ml PMA treatment in the PMA-LAMP method. Thus, the 3.0 μg/ml PMA treatment was suitable for discriminating between target DNA from dead or viable cells by the PMA-LAMP method. In comparison with EMA, PMA can inhibit the amplification of DNA derived from dead cells [18,33]. It seems that PMA has an important advantage over EMA of not penetrating live cell membranes. EMA can partly penetrate both dead and live cells though the staining and seemed to be more efficient in the case of dead cells. The main reason for the significantly higher selectivity of PMA is most probably associated with the higher charge of the molecule (EMA has one positive charge, PMA has two) [22].

SAEW is a prominent sanitizer and has been applied to inactivate several foodborne pathogens amidst rising concerns of food safety [6,11]. Until now, evaluating the disinfection ability of sanitizing technologies is mainly based on conventional culture methods, which require much time and lacks high specificity [7]. Unlike the culture technique, PMA-LAMP can specifically detect viable E. coli O157 cells within 1 h, even in viable but non-culturable (VBNC) cells [33]. Therefore, it is a better method to monitor the disinfection efficacy of SAEW in various kinds of samples. A good correlation between PMA-LAMP and the plate count method in broth was observed in this study, but the PMA-LAMP method presented here could not quantitatively detect viable cells. Real-time quantitative loop-mediated isothermal amplification coupling with PMA may solve this problem with a loop-amp real-time turbidimeter [27]. This study may be the first report on the detection of viable E. coli O157 cells by the PMA-LAMP method. Although its application to food or clinical samples still requires further evaluation, this PMA-LAMP method is a potential tool for distinction between the viable and dead cells in various fields, especially in the detection of foodborne pathogens in food safety.