A High-throughput Platform for the Screening of <em>Salmonella</em> spp./<em>Shigella</em> spp.

Ze Yang; Xin-Bin Chen; Cheng-Ning Tu; Ying Su; Hai-Bo Wang

doi:10.3791/58200

JoVE Journal > Immunology and Infection

Immunologia e infezioni

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Published: November 07, 2018

doi:

10.3791/58200

Ze Yang¹, Xin-Bin Chen¹, Cheng-Ning Tu¹, Ying Su¹, Hai-Bo Wang¹

¹State Key Laboratory of Diarrhea Disease Detection,Zhuhai International Travel Healthcare Center

Summary

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Abstract

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

Introduction

Diarrhea is still a common health issue with a high incidence rate globally¹^,². Though the mortality is relatively low, some patients show various symptoms for weeks (such as loose and watery stools, an urgency to go to the bathroom), which make the socioeconomic impact very high³^,⁴. More seriously, some patients may even develop irritable bowel syndrome if left untreated⁵. There are various kinds of bacteria, viruses and parasites that can cause diarrhea⁶. Salmonella spp./Shigella spp. are among the most common bacteria for the transmission of acute gastroenteritis⁷^,⁸^,⁹^,¹⁰^,¹¹. Therefore, many counties have issued laws or regulations for regular Salmonella spp./Shigella spp. screening among people who would handle food and water. For example, the Chinese government have issued laws for obligatory Salmonella spp./Shigella spp. screening once a year.

The gold standard method for Salmonella spp./Shigella spp. detection is bacteria culture. Through bacteria culture and successive biochemical identification and serological characterization, we can identify the species of bacteria, which could facilitate disease outbreak management and antimicrobial profiling to aid the treatment of patients¹². It could also help trace the source of infection during the Salmonella spp./Shigella spp. outbreak¹³. However, this method is labor-intensive (requiring manual operation) and time-consuming (taking several days), especially for the testing of large numbers of samples⁷. Moreover, viable but non-culturable (VBNC) Salmonella spp./Shigella spp.may exist in some stool samples¹⁴. In view of these drawbacks, many laboratories have tried to develop new techniques for the detection of Salmonella spp./Shigella spp.¹⁵^,¹⁶^,¹⁷^,¹⁸^,¹⁹^,²⁰^,²¹^,²²^,²³^,²⁴^,²⁵. All these methods use the nuclear acid amplification test (NAAT), among which the polymerase chain reaction (PCR) is the most common. One major limitation of these NAAT based methods is that dead bacteria, even bacterial debris containing incomplete genomic DNA, could show positive results²⁶, which could largely influence the accurate diagnosis of disease. Blanco et al. showed that molecular assay is highly sensitive, not only to viable Salmonella in cultures, but also to partial genomes and dead or unviable bacteria from past infections or contamination²⁶. Therefore, new technology should be developed.

Here, we described a novel method that combines the NAAT based method and culturing. As shown in Figure 1, this new method applies real-time PCR screening first and then positive samples are sent for bacteria culture and identification.

Protocol

The protocol follows the guidelines set by the human research ethics committee of Zhuhai International Travel Healthcare Center. Please use standard sterile operation during the experiment.

1. Culture Media Composition and Preparation

Prepare Nutrient Broth: Dissolve 1% peptone, 0.3% beef extract, 0.5% sodium chloride, 0.1% glucose in H₂O, adjust pH to 7.5 and autoclave it in 121 °C for 15 min.
Prepare Selenite Cystine medium: Dissolve 0.5% peptone, 0.4% lactose, 1%Na₂HCO₃, 0.4% sodium hydrogen selenite, 0.001% L-Cystine in H₂O, adjust pH to 7.0 and boil it for 5 min.
Prepare the Xylose, Lysine, Deoxycholate agar (XLD) plate: Dissolve 0.3% yeast extract, 0.5% L-lysine, 0.375% xylose, 0.75% lactose, 0.75% sucrose, 0.5% sodium chloride, 0.008% phenol red, 0.68% sodium thiosulfate, 0.08% ammonium ferric citrate, 0.25% sodium deoxycholate, 1.5% agar in H₂O, and adjust pH to 7.4. Then boil it for 5 min and pour it into 90 mm plates.
Prepare the Salmonella chromogenic agar plate: Dissolve 1.5% agar, 0.7% peptone and yeast extract, 1.29% selective reagent in H₂O. Then boil it for 5 min and pour it into 90 mm plates.
Prepare the Nutrient agar plate: Dissolve 1% peptone, 0.3% beef extract, 0.5% sodium chloride, 1.5% agar in H₂O, and adjust pH to 7.3. Then autoclave it in 121 °C for 15 min and pour it into 90 mm plates.
Prepare the MacConkey agar (MAC) plate: Dissolve 2% peptone, 1% lactose, 0.5% sodium chloride, 0.5% OX Bile Salt, 0.0025% Neutral Red, 1.5% agar, 0.0001% crystal violet in H₂O, and adjust pH to 7.2. Then autoclave it in 115 °C for 20 min and pour it into 90 mm plates.

2. Real-time PCR

Sample collection
1. Insert an anal swab into the patient's anus 3-5 cm deep, and rotate it 360° around.
2. Put the anal swab into a sterile collection tube. Mark sample ID.
3. Send the sample to laboratory as soon as possible.
  NOTE: Samples could be stored at 4 °C for no more than 24 h.
Pre-enrichment
1. Add 3 mL of Nutrient Broth into each sample in the collection tube.
2. Incubate at 36 °C for 6 h in an incubator.
Sample mixing (optional)
1. Collect 100 µL of each pre-enrichment culture and mix 8-10 samples of 1 specimen into a 1.5 mL tube if there are more than 10 samples.
2. Mark properly.
DNA extraction
1. Centrifuge the pre-enrichment culture at 800 x g for 2 min to allow large particles to settle down, and transfer the supernatant to a new tube and centrifuge at 12,000 x g for 5 min. Discard the supernatant by aspiration.
2. Add 100 µL of DNA extraction solution (0.01 M pH 8.0 Tris-EDTA, 0.01% Nonidet P 40 (NP40)) to the pellet. Vortex vigorously for 1 min.
3. Boil at 100 °C for 5 min on a dry bath.
4. Centrifuge at 12,000 x g for 5 min. Collect the supernatant using a new tube, which will be the template for the succeeding real-time PCR analysis.
Real-time PCR
1. Setup the reaction mixture as follow: for each sample, add 12.5 µL of 2x reaction mixture, 0.4 µM of each primer, 0.2 µM of each probe (sequences in Table 1)²⁷, 5 µL of the template as prepared in step 2.4.4, and use ddH₂O to add up to 25 µL total volume.
2. Setup the cycling program as follow: 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s, 55 °C for 30 s, 72 °C for 34 s. Collect fluorescent signals from 6-carboxy-fluorescein (FAM) and Hexachlorofluorescein (HEX) channels at the elongation step (72 °C) automatically by the fluorescent real-time PCR machine.
3. Perform real-time PCR on a fluorescent real-time PCR machine, according to instructions of the instrument.
4. Go to Step 4 directly and issue negative reports if negative results occur on the FAM/HEX channels, which means that samples are negative for Salmonella spp./Shigella spp.
5. Go to step 3.1 and/or 3.2 if positive results occur on the HEX and/or FAM channels, which means that the sample may be positive for Salmonella spp. and/or Shigella spp., respectively.
  NOTE: If sample mixing has been performed in step 2.3, then real-time PCR on individual samples which made up the positive one should be performed to screen out the real positive sample.

3. Guided Culture

Salmonella spp. PCR positive sample
1. Selective culture in medium
  1. Add 100 µL of pre-enrichment culture into 5 mL of Selenite Cystine medium in a test tube. Incubate at 36 °C for 18–24 h in an incubator.
2. Separating culture on plate
  1. Collect one loop of the culture with a micro-loop and spread onto a XLD plate or Salmonella chromogenic agar plate. Incubate at 36 °C for 18-24 h in an incubator.
3. Biochemical identification
  1. Select suspicious colony on XLD plate (pink colony with/without dark heart; dark colony; yellow colony with/without dark heart) or Salmonella chromogenic agar plate (purple or prunosus colony, smooth and round) (Figure 2).
  2. Subject suspicious colony for biochemical identification on automated microbial identification system, according to instructions of the instrument.
4. Serological characterization
  1. O antigen characterization
    1. Add one drop of the O antigen polyvalent sera onto a clean slide.
    2. Collect one loop of the colony with a micro-loop and grind in the sera.
    3. Go to step 3.1.4.1.4 if it looks like flowing sand, which means that the colony is reactive to the sera (Figure 3). Otherwise, go to step 3.1.4.1.5.
    4. Use O antigen monovalent sera to repeat steps 3.1.4.1.1 to 3.1.4.1.3 until the specific O antigen is characterized.
    5. Use Vi sera to repeat steps 3.1.4.1.1 to 3.1.4.1.3. Collect those Vi sera reactive colonies in a tube and boil at 100 °C for 5 min in a dry bath. Centrifuge at 12,000 x g for 5 min. Collect the pellet and repeat steps 3.1.4.1.1 to 3.1.4.1.4 until specific O antigen is characterized.
  2. H antigen characterization
    1. Add one drop of the H antigen polyvalent sera onto a clean slide.
    2. Collect one loop of the colony with a micro-loop and grind in the sera.
    3. Go to step 3.1.4.2.4 if it looks like flowing sand, which means that the colony is reactive to the sera.
    4. Use H antigen monovalent sera to repeat steps 3.1.4.2.1 to 3.1.4.2.3 until specific H antigen is characterized.
      NOTE: Sometimes serum induction may be needed to characterize the second phase H antigen. If so, then the following optional steps should be performed.
    5. (Optional) Add one drop of specific H antigen sera onto Nutrient agar plate. Wait until all the sera are absorbed.
    6. (Optional) Collect one loop of the colony with a micro-loop and spread onto the plate where specific H antigen sera are absorbed. Incubate at 36 °C for 18-24 h in an incubator.
    7. (Optional) Use H antigen monovalent sera to repeat steps 3.1.4.2.1 to 3.1.4.2.3 until second phase H antigen is characterized.
    8. Go to step 4.
Shigella spp. PCR positive sample
1. Separating culture on plate
  1. Collect one loop of the pre-enrichment culture with a micro-loop and spread onto a XLD plate or MAC plate. Incubate at 36 °C for 18–24 h in an incubator.
2. Biochemical identification
  1. Collect suspicious colony on XLD plate (smooth, round, transparent and red colony) or MAC plate (smooth, round, transparent and colorless colony with 2-3 mm in diameter; Shigella sonnei may be bigger and turn to light pink as the elongation of incubation time) (Figure 4).
  2. Subject suspicious colony for biochemical identification on automated microbial identification system, according to instructions of the instrument.
3. Serological characterization
  1. Add one drop of the Shigella spp. polyvalent sera onto a clean slide.
  2. Collect one loop of the colony with a micro-loop and grind in the sera.
  3. Go to step 3.2.3.4 if it looks like flowing sand, which means that the colony is reactive to the sera.
  4. Use Shigella spp. monovalent sera to repeat steps 3.2.3.1 to 3.2.3.3 until specific Shigella spp. is characterized.
  5. Go to step 4.

4. Report

Issue positive or negative reports according to the above results.

Representative Results

The protocol was applied for the screening of Salmonella spp./Shigella spp. in anal stool samples from people who would handle food and water.

In the real-time PCR step, as shown in Figure 5A, there was a successful amplification in HEX channel, which meant that the mixed sample was positive for Salmonella spp. Then a further real-time PCR was conducted on individual samples which made up the positive one. As shown in Figure 5B, sample 2 was positive. Therefore, sample 2 was chosen for the guided culture of Salmonella spp. In Figure 5C, there was a successful amplification in the FAM channel, which meant that the mixed sample was positive for Shigella spp. Then a further real-time PCR was conducted and sample 10 was found to be positive (Figure 5D). Therefore, sample 10 was chosen for the guided culture of Shigella spp.

In the guided culture of Salmonella spp., there were pink colonies and purple colonies on XLD plate and Salmonella chromogenic agar plate, separately, as shown in Figure 2. Then these colonies were subjected to biochemical identification on automated microbial identification system. Results showed that it was Salmonella spp., with species unknown. Therefore, serological characterization was performed (Figure 3) and it was reactive to O4, O12, Hb, H1,2. According to the White-Kauffmann-Le Minor scheme, sample 2 was finally reported as positive for Salmonella paratyphi B. While for the guided culture of Shigella spp., there were pink red and colorless colonies on XLD plate and MAC plate, separately, as shown in Figure 4. Then these colonies were also subjected to biochemical identification on automated microbial identification system. Results showed that it was Shigella sonnei. To confirm its specific serotype, serological characterization was performed (Figure 3) and it was reactive to sonnei phase II. Therefore, sample 10 was finally reported as positive for Shigella sonnei phase II.

Figure 1: The diagram of the protocol. Two major steps were shown and separated by dash line. Please click here to view a larger version of this figure.

Figure 2: Representative culture results of Salmonella spp. on XLD plate and Salmonella chromogenic agar plate. On XLD plate, there were pink colonies with/without dark heart, while on Salmonella chromogenic agar plate, there were purple colonies. Please click here to view a larger version of this figure.

Figure 3: Representative serological characterization result. If the colony was reactive to the sera, it looked like flowing sand (left). Otherwise, it was turbid (right). Please click here to view a larger version of this figure.

Figure 4: Representative culture results of Shigella spp. on XLD plate and MAC plate. On XLD plate, there were red colonies, while on MAC plate, there were transparent and colorless colonies. Please click here to view a larger version of this figure.

Figure 5: Representative real-time PCR results. (A) HEX channel for mix samples. (B) HEX channel for individual samples. (C) FAM channel for mix samples. (D) FAM channel for individual samples. PC: positive control. NC: negative control. Please click here to view a larger version of this figure.

Pathogen	Name	Sequence(5'-3')
Salmonella	Sal-F Sal-R Sal-probe	gctcatattaattccggcatttac caggtcaatagccagaaagg HEX-ataagtaatccaatccgaaatgcctgcgt-Eclipse
Shigella	Shi-F Shi-R Shi-probe	ccgggataaagtcagaactc cagtggagagctgaagtttc FAM-aggccaggtagacttctatctcatccac-Eclipse

Table 1: Primers and probes used. The name and sequences of the primers and probes were provided.

Discussion

Since Salmonella spp./Shigella spp. are often associated with food poisoning and fecal-oral transmission of acute gastroenteritis²⁸^,²⁹ and the routine method is either labor-intensive or time-consuming⁷, we describe a high-throughput platform for the Salmonella spp./Shigella spp. screening, using real-time PCR combined with guided culture.

There are several steps that need consideration to maximize the capability of this platform. The first one is the pre-enrichment step of samples in Nutrient Broth (step 2.2). Though no pre-enrichment step is needed for stool samples collected from those patients with obvious symptoms, such as diarrhea, etc., a general 6 h pre-enrichment step is still needed for anal swab samples, when collected during the pre-employment physical examination for people who would handle food and water, as those people were mainly healthy or at least asymptomatic adults. If the protocol was only applied for the detection of Salmonella spp., then pre-enrichment could be conducted in Selenite Cystine medium in order to increase the sensitivity. The second critical step is the identification of suspicious colonies (step 3.1.3.1 and 3.2.2.1). There are many interfering background flora in the stool samples that may mask the detection and isolation of target pathogens³⁰. Therefore, the characteristic of the suspicious colonies as defined in step 3.1.3.1 and 3.2.2.1 should be kept in mind during the experiment. The third critical step is the serological characterization of Salmonella spp. As a majority of Salmonella spp. contain a second phase for H antigen³¹, serum induction needs to be performed. However, the serum induction is not always successful, and several rounds of induction may be needed to determine the correct second H phase.

The protocol is highly specific and sensitive as verified by our previous study²⁷. The high specificity of the protocol is demonstrated by its ability, in which Salmonella spp./Shigella spp. could be distinguished from other related pathogens²⁷. The lower limit of detection of the protocol is 10⁴CFU/mL and 10³CFU/mL for Salmonella spp. and Shigella spp., respectively²⁷, which are comparable to previous reports⁷^,³²^,³³^,³⁴. As we stated above, the sensitivity of Salmonella spp. detection could be further increased by Selenite Cystine pre-enrichment if the protocol was only applied for the detection of Salmonella spp. Moreover, the protocol could increase the positive rate by two folds and decrease the workload/median turnaround time significantly²⁷.

Similar to other NAAT assays, one major limitation of the protocol, when compared to classic bacteria culture method, is that the protocol could only identify Salmonella spp./Shigella spp., while other common diarrhea-causing bacteria are omitted⁷. In contrast, during classic bacteria culture, those bacteria could be identified in parallel if they existed. Another limitation of the protocol is that some of Salmonella spp./Shigella spp. could not be identified by the real-time PCR due to sequence variations²⁷. However, if negative real-time PCR results appear for those samples from patients with obvious clinical symptoms, laboratory technicians should pay attention and may conduct other experiments to confirm the results. During a large outbreak, we may use a single sample instead of pooled samples for the first round of PCR screening.

In conclusion, the protocol provided here could serve as a valuable platform for the screening of Salmonella spp./Shigella spp.

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

This work was supported by the Science and Technology Program of Zhuhai, China (grant number 20171009E030064), the Science and Technology Program of Guangdong, China (grant number 2015A020211004) and the Science and Technology Program of General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China (grant number 2016IK302, 2017IK224).

Materials

Tris	Sigma	10708976001
EDTA	Sigma	798681
NP40	Sigma	11332473001
ddH₂O	Takara	9012
PrimeSTAR HS (Premix)	Takara	R040Q
Nutrient Broth	LandBridge	CM106
Nutrient agar	LandBridge	CM107
Selenite Cystine medium	LandBridge	CM225
XLD	LandBridge	CM219
MAC	LandBridge	CM908
Salmonella chromogenic agar	CHROMagar	SA130
Salmonella diagnostic serum	Tianrun	SAL60
Shigella diagnostic serum	Tianrun	SHI54
anal swab (collecting tube plus)	Huachenyang
slide	Mingsheng	7102
micro-loop	Weierkang	W511
incubator	Jinghong	DNP-9082
autoclave	AUL	SS-325
dry bath	Jinghong	KB-20
automated microbial identification system	bioMérieux	VITEK2	other equivalent system could be used
fluorescent real-time PCR machine	ThermoFisher	ABI7500	other equivalent machine could be used

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Yang, Z., Chen, X., Tu, C., Su, Y., Wang, H. A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.. J. Vis. Exp. (141), e58200, doi:10.3791/58200 (2018).