Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.
Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4–24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.
Metagenomics sequencing theoretically allows concerted detection and subtyping of foodborne pathogens. However, food samples present challenges to the pathogen analysis by direct sequencing of the food microbiome. First, foodborne pathogens are often present at low levels in food samples. Most of the commercially available rapid detection methods still require 8–48 h culturing to enrich pathogen cells to a detectable level1. Second, many foods contain abundant microflora cells and/or food DNA, making foodborne pathogen DNA a small fraction of food metagenome and an elusive target for detection and subtyping by direct metagenomic sequencing.
Modification of food microbiomes has been reported to allow substantial concentration of foodborne pathogen DNA to facilitate sequencing-based detection of Shiga toxin-producing Escherichia coli2,3 and Salmonella enterica4. Because modified food microbiomes are still mixtures of different microbial species, their sequencing is termed as quasi-metagenomic analysis4. Microbiome modification can be performed by culture enrichment alone2,3 or in combination with immunomagnetic separation (IMS) and multiple displacement amplification (MDA)4,5. IMS can selectively capture pathogen cells from the enrichment culture via antibody-coated magnetic beads. MDA can generate sufficient amounts of genomic DNA for sequencing through the highly efficient ɸ29 DNA polymerase6. IMS-MDA has allowed culture-independent pathogen detection from clinical samples7, and shortening of the culture enrichment for quasi-metagenomic detection and subtyping of Salmonella in food samples4.
The overall goal of this method is to prepare quasi-metagenomic DNA from food samples to allow targeted concentration of Salmonella genomic DNA and subsequent detection and subtyping of the Salmonella contaminant by sequencing. Compared to standard methods for Salmonella detection8,9 and subtyping10, the quasimetagenomic approach can substantially shorten the turnaround time from contaminated food and environmental samples to molecular subtypes of the pathogen by unifying the two typically separated analyses into a single workflow. This method is particularly useful for applications such as foodborne outbreak response and other trace back investigations where robust pathogen subtyping is required in addition to pathogen detection and rapid analytical turnaround is important.
1. Sample Preparation
NOTE: Food samples are prepared for pre-enrichment according to Microbiology Laboratory Guidebook (MLG) of U.S. Department of Agriculture Food Safety and Inspection Service (USDA-FSIS)11 and Bacteriological analytical manual (BAM) of U.S. Food and Drug Administration (FDA)12.
Figure 1: Representative food and environmental samples for quasi-metagenomics detection and subtyping of Salmonella. Samples are placed in sterile filter stomacher bags along with RV broth. Please click here to view a larger version of this figure.
2. Immunomagnetic Separation (IMS) and Multiple Displacement Amplification (MDA)
NOTE: To prevent cross-contamination between samples, work in a biosafety cabinet, change pipette tips for each tube, avoid touching the tube with the pipette, and do not place tubes close together in magnetic stand during the washing step.
3. Real-Time PCR
NOTE: This step is optional for 1) detecting Salmonella without subtyping, and 2) assessing sample quality prior to quasimetagenomics sequencing.
4. Library Preparation for Quasimetagenomic Sequencing
NOTE: The MDA products can be sequenced by both short read and long read (nanopore) sequencing platforms. Use the latest version of DNA library prep kits provided by the manufacturers of the sequencing platforms. Perform DNA library preparation according to manufacturers’ instruction. Use the 2D low-input genomic DNA protocol for library preparation for the long-read sequencing platform5. For library preparation for the short-read sequencing platform, minor modifications to the manufacturer’s protocol are provided below.
Prior to quasimetagenomic sequencing, the overall quantity and purity of IMS-MDA products can be evaluated by fluorospectrometer (Table 1).
Enrichment time (h) | Ct value | Concentration (ng/ul) | Purity (260/280) | |
Brand A | 4 | 24.71 | 812.91 | 1.89 |
8 | 25.80 | 900.36 | 1.87 | |
12 | 15.41 | 753.88 | 1.84 | |
Brand B | 4 | 20.63 | 872.61 | 1.87 |
8 | 22.61 | 993.41 | 1.87 | |
12 | 19.08 | 954.44 | 1.87 |
Table 1: Quality and quantity assessment of DNA samples for quasi-metagenomic sequencing. Salmonella cells were inoculated in 25 g of raw chicken breast of two brands at 1 CFU/g. Inoculated samples were enriched in RV for 4, 8, and 12 h before IMS-MDA.
Targeted quantity assessment of Salmonella DNA can be performed by real-time PCR (Table 1 and Figure 2), which also serves as an alternative for Salmonella detection if subtyping is not needed.
Figure 2: Real-time PCR for Salmonella detection or sample assessment for quasi-metagenomics sequencing. Amplification curves of real-time PCR of Salmonella DNA in IMS-MDA products after 4, 8 and 12 h of enrichment time are shown. A sample after only 2 h enrichment is also included. Salmonella cells (0.27 CFU/g) were inoculated 25 g of raw chicken breast from two brands A and B. Please click here to view a larger version of this figure.
Fluorospectrometer readings allow initial evaluation of sample preparation. Concentration of IMS-MDA products prepared from Salmonella contaminated chicken breast samples ranged from 701.54 to 945.86 ng/µL, suggesting that the sample DNA has been amplified. According to manufacturer's guidance, the minimum DNA sample concentration for short read and long read sequencing is 0.2 ng/µL and 1 µg, respectively. The 260/280 ratio ranged from 1.85 to 1.88 (Table 1), showing high purity of the DNA samples with low levels of protein contamination.
Lower Ct values indicate higher concentrations of Salmonella genomic DNA in the sample. As shown in Table 1 and Figure 2, Ct values of IMS-MDA products decrease as enrichment time increases. In our previous study4, when Ct values were lower than 25, the majority (>50%) of the SE genome was likely to be sequenced, and serotype prediction from raw sequencing reads was successful.
Unsuccessful culture enrichment or IMS can result in a relatively high Ct values indicating low concentration of Salmonella genomic DNA in the final sample as shown in Figure 2. This can happen despite a high DNA concentration and purity of the IMS-MDA products as suggested by fluorospectrometer readings.
Because of the often-low abundance and in-homogenous presence of Salmonella in food and environmental samples, culture enrichment before IMS-MDA is still necessary for Salmonella detection and subtyping; it is therefore a critical step of the protocol. To identify optimal conditions to increase the abundance of Salmonella relative to sample background flora, different enrichment media may be evaluated for specific samples. According to MLG and BAM, both selective medium such as RV broth and non-selective medium such as buffered peptone water and lactose broth can be used for Salmonella enrichment from food samples. The use of RV at 42 °C has been evaluated in our previous study4 for Salmonella enrichment from raw chicken meat, iceberg lettuce and black peppercorns to support quasimetagenomic analysis of the pathogen. According to FDA BAM and AOAC precollaborative8,9and collaborative studies14,15, RV is recommended for the analysis of high microbial and low microbial load foods.
The optimal duration of culture enrichment depends on multiple factors, such as sample type, level of Salmonella contamination, and the physiological state of the Salmonella contaminant. For example, when low levels of Salmonella (e.g., < 1 CFU/g) cells are present in a meat sample and under refrigeration stress, longer enrichment time (e.g., > 12 h) may be needed to revive and enrich Salmonella cells. Sufficient abundance of Salmonella in the modified microbiome leads to adequate sequencing coverage of the Salmonella contaminant genome, which can allow strain-level single nucleotide polymorphism (SNP) typing4. Shorter culture enrichment (e.g., less than 12 h) is possible for rapid detection5 or serotyping of Salmonella4.
Higher levels of Salmonella contamination and/or longer culture enrichment of contaminated food samples can result in higher concentrations of Salmonella genomic DNA in modified food microbiomes, which lead to lower Ct values from the real-time PCR analysis. Ct values display a negative correlation with sequencing coverage of the Salmonella contaminant genome4, and therefore can be used for optimization and modification of the workflow. According to our experience, culture enrichment, especially the length of it, is often the focus of troubleshooting efforts. For example, the Ct value of the IMS-MDA product from a raw chicken breast sample was relatively high at 35.94 (Figure 1). This sample was inoculated with 2.5 CFU/g of SE cells and incubated in BPW at 37 °C for 2 h. Sequencing of this sample did not provide sufficient coverage of the SE genome to allow serotype prediction. In comparison, all the other samples with 4 h or longer enrichment were successfully serotyped from quasimetagenomic sequencing data (data not shown).
After sequencing, we recommend using Kraken16 to identify Salmonella reads for further bioinformatics analyses. CFSAN SNP pipeline17 and SeqSero18 can be used for SNP typing and serotyping prediction from raw sequencing reads, respectively.
This quasimetagenomic technique has several limitations. First, DNA concentration and purity of IMS-MDA products measured by a fluorospectrometer should be used only as a preliminary indicator of proper sample preparation. Due to the high efficiency of MDA, trace amounts of input DNA can be amplified to suffice whole genome sequencing19. Therefore, high quality and quantity DNA samples can still be generated even if culture enrichment or IMS fails to effectively concentrate Salmonella cells. The optional real-time PCR analysis provides a more targeted and informative assessment of the entire sample preparation workflow. Second, difficult-to-culture Salmonella cells, such as viable but nonculturable (VBNC) bacteria20, may not grow during culture enrichment. Therefore, these cells may not be detected by our method.
In summary, compared with traditional methods, our sample preparation method and quasi-metogenomic sequencing approach can allow substantial reduction of the analytical turnaround from Salmonella-contaminated food and environmental samples to robust subtyping of the Salmonella contaminants. In addition to Salmonella, this technique has the potential be applied to rapid and concerted detection and subtyping other foodborne pathogens.
The authors have nothing to disclose.
The authors would like to thank Mark Harrison and Gwen Hirsch of the University of Georgia for kindly providing the bacterial strain and other support to this study.
Laboratory blender bag w/filter | VWR | 10048-886 | |
Buffered peptone water | Oxoid Micorbiology Products | CM0509 | |
Rappaport Vassiliadis broth | Neogen Acumedia | 7730A | |
Polysorbate 20 | Millipore Sigma | P9416 | Tween 20 |
Stomacher blender | Seward | 30010108 | |
Centrifuge | Fisher Scientific | 75005194 | |
50ml Centrifuge tubes | Fisher Scientific | 05-539-6 | |
Thermal Cycler | Techne Prime | EW-93945-13 | |
StepOne Real-Time Thermal cycler | Applied Biosystems | 4.76357 | |
AMPure XP beads | Beckman Coulter | A63881 | PCR purification beads; mix well before use; store at 4C |
Nextera XT library prep kit | Illumina | FC-131-1024 | Store at -80C |
MinIon library prep kit | Oxford Nanopore | SQK-LSK108 | Store at -80C |
NanoDrop | Thermo Scientific | ND-2000 | |
Dynabead Anti-Salmonella beads | Applied Biosystems | 71002 | Vortex well prior to use |
Illustra GenomiPhi V2 DNA amplification kit (MDA kit) -Sample buffer -Reaction buffer -Enzyme mix |
GE Healthcare | 25-6600-30 | Store at -80C |
HulaMixer | Invitrogen | 15920D | |
DynaMag magnetic rack | Invitrogen | 12321D | |
TaqMan Universal PCR mastermix | Applied Biosystems | 4304437 | Mix well before use; store at 4C |
Microfuge | Fisher Scientific | 05-090-100 |