Evaluation of a Multiplex Polymerase Chain Reaction for the Simultaneous Detection of Vibrio spp. in Vegetables and Water

Introduction

Due to the increasing tendency toward healthy lifestyles in recent decades, there has been a growing demand for fresh products (Amao, 2018; Elgueta et al., 2019). Vegetables are known to play an essential role in a healthy diet. Vegetables are usually eaten raw and undergo no process, such as cooking or peeling. As a result, they are likely to act as the vehicles of human pathogens (Chen et al., 2019; Balali et al., 2020). In some countries, vegetables are irrigated using wastewater leading to the possibility of being contaminated with enteric pathogenic microorganisms (Steele and Odumeru, 2004; Uyttendaele et al., 2015; Gudda et al., 2020). In recent years, there have been reports of foodborne outbreaks associated with the consumption of vegetables (de Oliveira Elias et al., 2018). Vibrio spp. are among the pathogens which have been noted in raw vegetables (Igbinosa and Odjadjare, 2019).

The most commonly used method for the analysis of Vibrio spp. is a two-step protocol consisting of selective enrichment with Alkaline Peptone Water (APW) medium and then culturing on thiosulfate citrate bile salts (TCBS) agar. The main drawbacks of this conventional method are being labor-intensive and time-consuming (Tunung et al., 2011; Anupama et al., 2019). The sensitivity of the conventional method is low and the detection of the bacteria, when sample concentration is not adequate, is sometimes difficult. Therefore, methods based on polymerase chain reaction (PCR) are suggested to be an effective alternative for the identification of these organisms (di Pinto et al., 2005; Tao et al., 2020).

Several PCR-based methods have been used for identifying Vibrio spp. in recent years, one of which is the multiplex PCR method (Tsai et al., 2019). Multiplex PCR method has been previously reported as a useful and cost-effective tool for the detection of Vibrio species (Awasthi et al., 2019; Han et al., 2019). The objective of this study was to establish a multiplex PCR method to identify V. cholerae, V. parahaemolyticus, V. vulnificus, and V. alginolyticus. Furthermore, we compared the results of the latter technique with the conventional plate culturing method.

Materials and Methods

Sample Collection

The samples used in this study consisted of five groups of green vegetables, namely group A: spinach and onion, group B: cress and radish, group C: tarragon and basil, group D: mint and dill, group E: parsley and coriander, and their surrounding water. The vegetables were collected from the southern parts of Tehran and Varamin, Iran, and were transferred to the laboratory.

Detection of Vibrio spp. and other Bacteria in the Water Surrounding the Vegetables

In order to detect bacteria in the surrounding water of vegetables, two methods of membrane filtration and the most probable number (MPN) were applied.

Membrane Filtration Method

A volume of 1 L of the sample water was filtered onto 0.45 μm filter paper. The filter paper was then put in APW enrichment broth and was incubated at 35°C for 6-8 h. Following incubation, 250 mL of the sample was filtered onto a 0.45 μm cellulose acetate membrane filter which was later placed on MacConkey agar and CHROMagar vibrio plates and was incubated at 37°C for 24 h.

MPN Method

A three- tube MPN procedure was used. The sample was decimally diluted from 1/10 to 1/1000 utilizing sterilized saline and 1 mL of each dilution for 9 mL of culture broth medium in plastic tubes as triplicates for each dilution. Afterwards, the nine tubes were incubated at 35°C for 24 h. An MPN estimate was performed using standard MPN tables.

Detection of Vibrio spp. and Other Bacteria in Vegetables by Plate Culture

The samples were divided into portions of 10 g and were placed in separate stomacher bags. A volume of 90 mL of APW enrichment broth was added to each sample, mixed for 1 min in a stomacher, and the samples were incubated at 35°C for 5-8 h. Following decimal serial dilution in sterilized peptone water, 1 mL of diluted samples was pour plated in TCBS agar and incubated at 35°C for 18-24 h. Because TCBS is not a highly selective media for V. vulnifus, CHROMagar vibrio plates were used as well with the same procedure as TCBS plates. Mannitol salt agar and cetrimide agar were applied to detect Staphylococcus spp. and Pseudomonas spp., respectively.

Detection of Vibrio spp. and Other Bacteria in Vegetables by Multiplex PCR

Genomic DNA was extracted using CinnaGen genomic DNA extraction kit following the procedure provided by the company. The used oligonucleotide primer sets, melting temperatures (T_m), and the size of amplicons used in this study are listed in Table 1. Multiplex PCR was optimized in a 25 μL reaction mixture consisting of 3 μL of DNA template, 3 μL of 10 μM forward primer, 3 μL of 10 μM reverse primer, 12.5 μL of PCR Master Mix, and 3.5 μL of nuclease-free water. A three-step PCR amplification process was carried out in a DNA thermal cycler. Multiplex PCR conditions were optimized using the following temperature-cycling parameters: initial denaturation at 94°C for 180 min followed by 30 cycles of amplification with denaturation at 94°C for 30 sec, primer annealing at 60°C for 30 sec, and primer extension at 72°C for 1 min. Following the amplification cycles, samples were subjected to 72°C for 420 min for the final extension of incompletely synthesized DNA. Next, the PCR products were analyzed using agarose gel electrophoresis 2% (w/v). Agarose gel was stained by erythrogel. Electrophoresis was performed using 1x TBE buffer. All products were visualized and documented with a gel documentation and analysis system.

Results

Detection of Bacteria in Vegetables and Water by Culture

The plates were examined following 24 h incubation. Shiny yellow colonies of 2-3 mm on TCBS were initially considered as V. cholorae and V. alginolyticus. The salt tolerance of the isolates was tested to differentiate V. cholorae and V. alginolyticus as they can grow in 0% and 6%-10% of NaCl, respectively. Green or blue-green colonies on TCBS were considered as V. vulnificus. The results of cultures for different specimens are summarized in Table 2. Vibrio spp. and Staphylococcus aureus were present in all the tested vegetables but not in the surrounding water. Escherichia coli, Pseudomonas aeruginosa, and Streptococcus faecalis were not detected in any of the samples.

Multiplex PCR Results

Representative multiplex PCR gel is shown in Figure 1. The presence of V. alginolyticus and V. vulnificus was confirmed by amplifying the specific regions of 412 bp for V. vulnificus and 144 bp for V. alginolyticus. V. cholerae and V. parahaemolyticus were not detected in multiplex PCR. The annealing temperature, extension time, and primer concentrations used in this multiplex PCR assay were optimized.

Figure 1. Result of the multiplex PCR

Discussion