Evaluation of 16s Long Read Metabarcoding for Characterizing the Microbiome and Salmonella Contamination of Retail Poultry Meat
The traditional gold standard for detection of Salmonella in meat products is bacterial culture with enrichment. While this method is highly sensitive, it is slow and provides an incomplete assessment of isolate taxonomy in positive samples. This study presents a novel PCR-based detection assay whic...
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Elsevier
2025-02-01
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| Series: | Journal of Food Protection |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S0362028X24002187 |
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| author | Zachary Tobar Katie Y. Lee Megan E. Gaa Bryshal P. Moore Xunde Li Maurice E. Pitesky |
| author_facet | Zachary Tobar Katie Y. Lee Megan E. Gaa Bryshal P. Moore Xunde Li Maurice E. Pitesky |
| author_sort | Zachary Tobar |
| collection | DOAJ |
| description | The traditional gold standard for detection of Salmonella in meat products is bacterial culture with enrichment. While this method is highly sensitive, it is slow and provides an incomplete assessment of isolate taxonomy in positive samples. This study presents a novel PCR-based detection assay which amplifies the 16s-ITS-23s region which is an approximately 2,500 base pair region of the larger ribosomal rrn operon. Intra-assay variation was assessed by splitting each biological sample into 3 technical replicates. Limits of detection (LOD) were assessed by utilizing a serial dilution of a pure culture of Salmonella enterica subsp. enterica serovar Heidelberg spiked into either sterile 1 × PBS or 1 × PBS rinsate of a Salmonella culture-negative chicken meat sample. Results indicate the 16s metabarcoding assay evaluated here could not be reliably used for the detection of Salmonella in adulterated retail meat samples as the LOD observed, 4.70 log colony forming units (CFU)/ml, is above the expected concentration of Salmonella in retail poultry meat samples which previous studies have shown range from under 1 to 2 log CFU/ml. However, due to greater taxonomic resolution afforded by using 16s long reads, the assay allowed alpha diversity assessment of the microbiome of raw poultry meat with the ability to assign taxonomy to the species and strain level for some amplicon sequence variants (ASV). This indicates this process may have value characterizing biodiversity and pathogen contamination of poultry samples in earlier steps of the poultry meat production process where bacterial contamination concentrations are likely to be higher. |
| format | Article |
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| institution | Kabale University |
| issn | 0362-028X |
| language | English |
| publishDate | 2025-02-01 |
| publisher | Elsevier |
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| series | Journal of Food Protection |
| spelling | doaj-art-b6335855430d4ee4ad7a8c8d3ce563f62025-02-02T05:26:41ZengElsevierJournal of Food Protection0362-028X2025-02-01882100434Evaluation of 16s Long Read Metabarcoding for Characterizing the Microbiome and Salmonella Contamination of Retail Poultry MeatZachary Tobar0Katie Y. Lee1Megan E. Gaa2Bryshal P. Moore3Xunde Li4Maurice E. Pitesky5Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616, United StatesDepartment of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616, United StatesDepartment of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616, United StatesDepartment of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616, United StatesDepartment of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616, United StatesCorresponding author at: 1089 Veterinary Medicine Dr., Davis, CA 95616, United States.; Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616, United StatesThe traditional gold standard for detection of Salmonella in meat products is bacterial culture with enrichment. While this method is highly sensitive, it is slow and provides an incomplete assessment of isolate taxonomy in positive samples. This study presents a novel PCR-based detection assay which amplifies the 16s-ITS-23s region which is an approximately 2,500 base pair region of the larger ribosomal rrn operon. Intra-assay variation was assessed by splitting each biological sample into 3 technical replicates. Limits of detection (LOD) were assessed by utilizing a serial dilution of a pure culture of Salmonella enterica subsp. enterica serovar Heidelberg spiked into either sterile 1 × PBS or 1 × PBS rinsate of a Salmonella culture-negative chicken meat sample. Results indicate the 16s metabarcoding assay evaluated here could not be reliably used for the detection of Salmonella in adulterated retail meat samples as the LOD observed, 4.70 log colony forming units (CFU)/ml, is above the expected concentration of Salmonella in retail poultry meat samples which previous studies have shown range from under 1 to 2 log CFU/ml. However, due to greater taxonomic resolution afforded by using 16s long reads, the assay allowed alpha diversity assessment of the microbiome of raw poultry meat with the ability to assign taxonomy to the species and strain level for some amplicon sequence variants (ASV). This indicates this process may have value characterizing biodiversity and pathogen contamination of poultry samples in earlier steps of the poultry meat production process where bacterial contamination concentrations are likely to be higher.http://www.sciencedirect.com/science/article/pii/S0362028X2400218716sBioinformaticsMetagenomicsMicrobiomePoultrySalmonella |
| spellingShingle | Zachary Tobar Katie Y. Lee Megan E. Gaa Bryshal P. Moore Xunde Li Maurice E. Pitesky Evaluation of 16s Long Read Metabarcoding for Characterizing the Microbiome and Salmonella Contamination of Retail Poultry Meat Journal of Food Protection 16s Bioinformatics Metagenomics Microbiome Poultry Salmonella |
| title | Evaluation of 16s Long Read Metabarcoding for Characterizing the Microbiome and Salmonella Contamination of Retail Poultry Meat |
| title_full | Evaluation of 16s Long Read Metabarcoding for Characterizing the Microbiome and Salmonella Contamination of Retail Poultry Meat |
| title_fullStr | Evaluation of 16s Long Read Metabarcoding for Characterizing the Microbiome and Salmonella Contamination of Retail Poultry Meat |
| title_full_unstemmed | Evaluation of 16s Long Read Metabarcoding for Characterizing the Microbiome and Salmonella Contamination of Retail Poultry Meat |
| title_short | Evaluation of 16s Long Read Metabarcoding for Characterizing the Microbiome and Salmonella Contamination of Retail Poultry Meat |
| title_sort | evaluation of 16s long read metabarcoding for characterizing the microbiome and salmonella contamination of retail poultry meat |
| topic | 16s Bioinformatics Metagenomics Microbiome Poultry Salmonella |
| url | http://www.sciencedirect.com/science/article/pii/S0362028X24002187 |
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