Development of PMA-LAMP for Rapid Detection of Staphylococcus aureus in Viable but Nonculturable State

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1 2016 Joint International Conference on Social Science and Environmental Science (SSES 2016) and International Conference on Food Science and Engineering (ICFSE 2016) ISBN: Development of PMA-LAMP for Rapid Detection of Staphylococcus aureus in Viable but Nonculturable State Qing-Ping ZHONG *, Ying-Qiao CHEN, Li WANG, Xiang FANG, Zhen-Lin LIAO College of Food Science, South China Agricultural University, Guangzhou, , China *Corresponding author Keywords: Staphylococcus aureus, Viable but Non-culturable State, LAMP, Propidium Monoazide. Abstract. Staphylococcus aureus is a major foodborne pathogen. The viable but non-culturable (VBNC) state of S. aureus cannot be detected by traditional culturing methods while remain alive for long time and maintain the potential virulence. The objective of this study was to develop and optimize a method that combines propidium monoazide (PMA) treatment with loop-mediated isothermal amplification (LAMP) to detect VBNC cells of S. aureus. The cell suspension was treated with PMA in the dark for 10 min and was subsequently exposed to a 650 W halogen lamp for 5 min. Then the bacterial cells were harvested and DNA was extracted and amplified by LAMP. The primers targeted six distinct regions in the nuc gene of S. aureus was designed for the PMA-LAMP method. The results indicated that the treatment with 3 μg/ml PMA and a 5min light exposure was suitable for PMA-LAMP to distinguish VBNC cells from dead cells of S. aureus. The optimized assay was specific and sensitive; it could detect as low as 17 CFU/mL VBNC cells in pure culture, 17 CFU/g in spiked milk powder, and 170 CFU/g in spiked dumpling. The assay could detect VBNC state of S. aureus without interference of dead cells and other bacteria. Introduction Staphylococcus aureus is an important foodborne pathogen causing food poisoning due to the ingestion of food containing staphylococcal enterotoxins [1]. This bacterium produce a variety of enterotoxins, and the classical staphylococcal enterotoxins (A, B, C, D and E) are responsible for 95% of cases of food poisoning outbreaks [2]. The traditional detecting method of S. aureus is culture-based assay, which takes about several days and is time consuming. Moreover, viable but non-culturable (VBNC) state of S. aureus cannot be detected by this culture method. VBNC bacteria are those which are viable and maintain metabolic activity but are unable to form colonies on conventional growth media [3]. This state is an adaptive strategy for long-term survival adopted by bacteria when exposed to unfavorable environmental conditions, posing a risk to food safety [4, 5]. So the rapid detection method of S. aureus in VBNC state is an urgent requirement [6]. Loop mediated isothermal amplification (LAMP) is a DNA amplification method which is conducted within 1 h at a single temperature (60-65 ). It has the advantages of high specificity, sensibility, rapidity and simple operation [7]. Propidium monoazide (PMA) is a DNA-intercalating agent which can penetrate membrane-damaged cells and bind to DNA molecule, thus inhibiting DNA amplification of dead cells, allowing amplification of unbound DNA from viable cells [8]. In the present study, we developed a method that combined PMA with LAMP for the rapid detection of the VBNC state of S. aureus. The specificity and sensitivity of the assay were evaluated, and the 451

2 applicability of this assay in detecting the pathogen in food samples was verified. This method could contribute to the rapid detection of the VBNC state of S. aureus. Materials and Methods Bacterial Strains Standard strain of S. aureus (CMCC 26003) was stored on TSA at 4. S. aureus was induced into VBNC state by using 0.5mmol/L potassium sorbate at -20 in oligotrophic condition for 76 days. The plate count method and the LIVE/DEAD BacLight Bacterial Viability Kit method were used to determine whether S. aureus was induced into VBNC state. Vibrio parahaemolyticus (CGMCC1.1614), Salmonella enterica (CGMCC ), Escherichia coli (CGMCC ), Bacillus thuringiensis (CGMCC ), Bacillus subtilis (CGMCC ) were stored in 10% (w/v) glycerol broth at 80. Different Treatments to Obtain Dead Cells of S. aureus The dead cells were obtained as follows: 1) 75 water bath for 10 min, 2) 70% isopropyl alcohol treatment for 10 min. The absence of viable cells was detected by plate count method. PMA Treatment on Cells PMA (Biotium, Inc., Hayward, CA, USA) was dissolved in 20% dimethyl sulfoxide at the concentration of 1 mg/ml. This stock solution was stored in dark at -20. The cells were treated with different concentrations of PMA in Eppendorf tubes in dark at room temperature for 5 min. Then, the tubes were placed into ice bath with lids removed, and exposed to a 650W halogen lamp for 5 min at a distance of 15 cm. During the treatment, the sample tubes were shaken every 30 s to ensure homogeneous exposure to light. After the PMA treatment, the cells were collected and rinsed with a saline solution. Preparation of Bacterial DNA The cell suspensions were collected by centrifugation at 4, 10,000 rpm for 5 min. Genomic DNA was extracted using a DNA kit (Beijing Solarbio Biotech Co., Ltd, Beijing, China), following the manufacturer's instructions. The extracted DNA was stored at -20 or subjected to LAMP. LAMP Primers A set of four primers specific for S. aureus was designed to target six distinct regions on nuc gene. Forward inner primer (FIP) consisted of the sequence (5 -CGTTTACCATTTTTCCATCAGCATA TTTGACAAAGGTCAAAGAACT-3'); backward inner primer (BIP) consisted of the sequence (5'-TCAAGGCTTGGCTAAAGTTGCTTATTCGCTT GTGCTTCACTT-3'). The outer primers F3 (5'-TGCAAAGAAAATTGAAGTCGA-3') and B3(5'-CGTTGTCTTCGCTCCAAAT-3') are located outside of the F2 and B2 regions. The primers were synthesized commercially by Shanghai Jierui Biotechnology Company. Reaction System of LAMP The reaction mixture (Table 1) was incubated at 65 for 60 min. Positive and negative controls were included in each run, and all precautions to prevent cross-contamination were observed. The reaction products were analyzed by electrophoresis in 2.0% agarose gel. In order to facilitate the field application of the assay, the monitoring of amplification products was also carried out through 452

3 naked-eye inspection. Following amplification, 1μl (1:100) of SYBR Green I dye was added into the tube. In case of positive amplification, the original orange color of the dye changes to green. Evaluation of the Specificity of PMA-LAMP The specificity of the assay was assessed by conducting LAMP with the genomic DNA of S. aureus and the strains of non-s. aureus. Table 1. Reaction Mixture of LAMP. Reagent Volume ddh 2O (up to 25 μl) 10 Thermopol reaction buffer mmol/l dntps 3 F3(2.5 μmol/l) 2 B3(2.5 μmol/l) 2 FIP(10 μmol/l) 2 BIP(10 μmol/l) 2 DNA template 1 Bst DNA polymerase (8U) large frament 0.5 Evaluation of the Sensitivity of PMA-LAMP After PMA treatment, the DNA of the treated cells was extracted by kit method and was 10-fold serially diluted in sterile double-distilled water. Aliquots of each 1 ml dilution were amplified, and 1 ml of double-distilled water was used as a negative control. PMA-LAMP Detection in Spiked Food Samples The food samples (milk powder and frozen dumpling) purchased from local markets were determined containing no S. aureus using the standard method (GB/T , China) and the developed PMA-LAMP. Each sample (25 g) was inoculated with 1 ml of VBNC cells of S. aureus, resulting in a spiking level of CFU/g. The uninoculated samples were used as controls. The samples were homogenized with 225 ml of buffered peptone water (BPW; BD Diagnostic Systems, Sparks, MD, USA) for 1 min. The homogenates were centrifuged at 800 rpm for 3 min, the supernatant fractions were 10-fold serially diluted and subjected to PMA treatment, and then heated at 95 for 10 min followed by centrifugation at 10,000 rpm for 2 min. The supernatants were used as DNA templates for PMA-LAMP assays. Results Specificity of LAMP Assay The standard strains of S. aureus and non-s. aureus were used to evaluate the specificity of the assays. The results were showed in Fig. 1 and Fig.2. The specific amplification of nuc gene generated many ladder-like pattern bands on agarose gel, while the negative control and non-s. aureus samples had no amplification (Fig. 1). The positive results were observed in tubes 1-3, and a-c; the negative results in tubes 4 and d (Fig. 2). Figure 1. Specificity of LAMP Reaction. M: 100 bp DNA Ladder, 1: V. parahaemolyticus, 2: S. enterica, 3: E. coli, 4: B. thuringiensis, 5: B. subtilis, 6: Negative Control, 7: S. aureus. 453

4 Figure. 2. LAMP Products Examined with SYBR Green I. (A) Visual Colors Inspected in Natural Light. (B) Visual Colors Inspected under UV. (1) - (3), (a)-(c): Positive Reaction, (4), (d): Negative Reaction. PMA-LAMP of Different States of Cells LAMP results of different states of cells were indicated in Fig. 3. The samples in lanes 1-3 were not treated with PMA, while lanes 4-6 were treated with 3 μg/ml PMA. PMA effectively prevented DNA amplification of dead cells (lane5-6), while did not have effect on VBNC cells (lane 4). Figure 3. LAMP Results of Different States of Cells. M: 100 bp DNA Ladder; Lane 1: Dead Cells Treated with Water Bath; Lane 2: Dead Cells Treated with Isopropyl Alcohol; Lane 3: VBNC Cells; Lane 4: PMA, VBNC Cells; Lane 5: PMA, Dead Cells Treated with Water Bath; Lane 6: PMA, Dead Cells Treated with Isopropyl Alcohol; Lane 7: Positive Control. Optimization of PMA Concentration The results indicated that 3 μg/ml of PMA could effectively suppress DNA amplification of dead cells (Fig. 4). Figure 4. PMA-LAMP Results of Different Cells Treated with Different Concentrations of PMA. (A) VBNC Cells. M: 100 bp DNA Ladder, 1-7: PMA 0μg/mL, 1 μg/ml, 3 μg/ml, 5 μg/ml, 10 μg/ml, 20 μg/ml, 50 μg/ml, 8: Negative Control. (B) Dead cells. M: 100 bp DNA Ladder, 1-6: PMA 1 μg/ml, 3 μg/ml, 5 μg/ml, 10 μg/ml, 20 μg/ml, 50 μg/ml, 7: Negative Control, 8: PMA 0 μg/ml. Development of PMA-LAMP and Its Sensitivity The effective concentration of PMA was 3 μg/ml and the exposure time under halogen lamp was 5 min. The developed PMA-LAMP assay could effectively detect VBNC state of S. aureus. The detection limit was found to be 17 CFU/mL in pure culture (Fig. 5). 454

5 Figure 5. Sensitivity of PMA-LAMP method. M: 100 bp DNA Ladder, 1: Positive Control, 2-9: CFU/mL, CFU/mL, CFU/mL, CFU/mL, CFU/mL, CFU/mL, 17 CFU/mL, 1.7 CFU/mL, 10: Negative Control. PMA-LAMP Detection in Spiked Food Samples As shown in Fig. 6 (A), PMA-LAMP could detect the VBNC state of S. aureus in artificially contaminated milk powder, and its sensitivity was 17 CFU/g. The detection limit was 170 CFU/g in frozen dumpling Fig. 6 (B). Figure 6. Sensitivities of PMA-LAMP in Spiked Food Samples. (A) Spiked Milk Powder. (B) Spiked Frozen Dumpling. M: 100 bp DNA Ladder, 1-5: CFU/g, CFU/g, CFU/g, CFU/g, CFU/g, 6: Negative Control. Conclusions In conclusion, this developed PMA-LAMP assay is specific, sensitive and reliable for rapid detection of VBNC state of S. aureus. It can detect S. aureus as low as 17 CFU/mL VBNC cells in pure culture, 17 CFU/g in spiked milk powder, and 170 CFU/g in spiked dumpling. The assay could detect VBNC state of S. aureus without interference of dead cells and other bacteria. This assay would be a rapid and practical method for the detection of VBNC cells of foodborne pathogens in contaminated food. Acknowledgement This research was financially supported by the National Natural Science Foundation of China ( ). References [1]EFSA, The community summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in the European Union in 2008, EFSA Journal. 8 (2010) [2]Y.H. Al-Tarazi, M.A. Albetar, A.R. Alaboudi, Biotyping and enterotoxigenicity of staphylococci isolated from fresh and frozen meat marketed in Jordan, Food Res. Int. 42 (2009) [3]J. D. Oliver, The viable but nonculturable state in bacteria, The Journal of Microbiology. 43 (2005) [4]J. D. Oliver, Recent findings on the viable but nonculturable state in pathogenic bacteria, FEMS Microbiology Reviews. 34 (2010)

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