quasi-metagenomic-analysis-salmonella-from-food-environmental

Quasi-metagenomic Analysis of <em>Salmonella</em> 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, ...

Center for Food Safety

A total of 115 desiccated food samples, including agricultural and marine products, were investigated for the presence of Cronobacter. Cronobacter species were characterized with biochemical tests. Antibiotic resistance was assessed with the disk diffusion method, and the molecular subtypes of Cronobacter isolates were identified using an automated repetitive sequence-based PCR (rep-PCR) system. A total of 18 (15.7%) Cronobacter strains were isolated from 115 desiccated food products. Fifteen Cronobacter isolates were C. sakazakii (13%), followed by 2 C. dublinensis (1.7%), and 1 C. universalis (0.9%). The most common antibiotic resistance of Cronobacter observed was against cephalothin (77.8%) followed by ampicillin (5.6%). With exception of 2 strains, all Cronobacter strains isolated from different sources were successfully differentiated by using the automated rep-PCR system, indicating that it can be used for the purpose of contamination or outbreak source tracking of the bacteria.

Isolation and characterization of Cronobacter from desiccated foods in Korea.

Mannitol-yolk-polymyxin B agar (MYPA) was modified by supplementation with trimethoprim. The ability of the supplemented medium to select for and recover Bacillus cereus from pure cultures and food samples with high background microflora was compared with MYPA. For  evaluation of the modified MYPA (mMYPA) in food samples with high background microflora, B. cereus was experimentally spiked into red pepper powder, fermented soybean paste, vegetable salad, and radish sprouts, and then it was recovered on MYPA and mMYPA for comparison. In all food  samples, there was no difference in recoverability (P > 0.05) between mMYPA (red pepper powder, 3.34 ± 0.24 log CFU/g; fermented soybean paste, 3.52 ± 0.47 log CFU/g; vegetable salad, 3.51 ± 0.23 log CFU/g; radish sprouts, 3.32 ± 0.40 log CFU/g) and MYPA  (red pepper powder, 3.18 ± 0.20 log CFU/g; fermented soybean paste, 3.33 ± 0.43 log CFU/g; vegetable salad, 3.36 ± 0.19 log CFU/g; radish sprouts, 3.33 ± 0.31 log CFU/g). However, mMYPA exhibited better selectivity than MYPA, because additional trimethoprim made  the differentiation of suspected colonies easier by inhibiting competing flora. The addition of trimethoprim to conventional media could be a useful option to improve selectivity in foods with high background microflora.

Improvement of mannitol-yolk-polymyxin B agar by supplementing with trimethoprim for quantitative detection of Bacillus cereus in foods.

We presented the first attempt to combine immunomagnetic separation (IMS), whole genome amplification by multiple displacement amplification (MDA) and real-time PCR for detecting a bacterial pathogen in a food sample. This method was effective in enabling real-time PCR detection of low levels of Salmonella enterica Serotype Enteritidis (SE) (∼10 CFU/g) in raw chicken breast without culture enrichment. In addition, it was able to detect refrigeration-stressed SE cells at lower concentrations (∼0.1 CFU/g) in raw chicken breast after a 4-h culture enrichment, shortening the detection process from days to hours and displaying no statistical difference in detection rate in comparison with a culture-based detection method. By substantially improving performance in SE detection over conventional real-time PCR, we demonstrated the potential of IMS-MDA real-time PCR as a rapid, sensitive and affordable method for detecting Salmonella in food.

Rapid detection of Salmonella in raw chicken breast using real-time PCR combined with immunomagnetic separation and whole genome amplification.

Metagenomics analysis of food samples promises isolation-independent detection and subtyping of foodborne bacterial pathogens in a single workflow. Selective concentration of  genomic DNA through immunomagnetic separation (IMS) and multiple displacement amplification (MDA) were shown to shorten culture enrichment of -spiked raw chicken breast samples by over 12 hours while permitting serotyping and high-fidelity single nucleotide polymorphisms (SNP) typing of the pathogen using short shotgun sequencing reads. The herein termed quasi-metagenomics approach was evaluated on -spiked lettuce and black peppercorn samples as well as retail chicken parts naturally contaminated with different serotypes of  Between 8 and 24 h culture enrichment was required for detecting and subtyping naturally occurring  from unspiked chicken parts compared with 4 to 12 h culture enrichment when -spiked food samples were analyzed, indicating the likely need for longer culture enrichment to revive low levels of stressed or injured  cells in food. Further acceleration of the workflow was achieved by real-time nanopore sequencing. After 1.5 hours of analysis on a potable sequencer, sufficient data were generated from sequencing IMS-MDA product of a cultured-enriched lettuce sample to allow serotyping and robust phylogenetic placement of the inoculated isolate. Both culture enrichment and next-generation sequencing remain to be time-consuming processes for food testing where rapid methods for pathogen detection are widely available. Our study demonstrated substantial acceleration of the respective process through IMS-MDA and real-time nanopore sequencing. In one example, the combined use of the two methods delivered a less than 24 h turnaround time from a -contaminated lettuce sample to phylogenetic identification of the pathogen. Improved efficiency like this is important for further expanding the use of whole genome and metagenomics sequencing in microbial analysis of food. Our results suggest the potential of the quasi-metagenomics approach in areas where rapid detection and subtyping of foodborne pathogens is important, such as foodborne outbreak response and precision tracking and monitoring of foodborne pathogens in production environments and supply chains.

Ji-Yeon Hyeon

Center for Food Safety

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples