Prashant Singh

High oxygen‐permeable vacuum skin packaging (10K VSP) is widely used for frozen seafood distribution to mitigate anaerobic pathogen risks, yet post‐thaw microbial dynamics during refrigerated display remain poorly characterized. This study evaluated post‐thaw microbial quality and bacterial community succession across 10 commercially frozen‐then‐thawed seafood species held under simulated retail refrigeration (4°C) on Days 1, 5, and 9. Aerobic plate counts, lactic acid bacteria, and coliform/ Escherichia coli indicator counts were determined, and bacterial communities were characterized using high‐throughput 16S rRNA gene sequencing. Average aerobic plate counts increased from 3.57 ± 1.03 log CFU/g on Day 1 to 6.00 ± 0.84 log CFU/g on Day 5 and 7.30 ± 0.64 log CFU/g on Day 9, although growth patterns differed among seafood products. Several products reached or exceeded microbial acceptability limits by Day 5 or Day 9. Sequencing revealed an ecological transition from initial Photobacterium and Psychrobacter dominance to a shift toward spoilage‐associated genera, particularly Pseudomonas and Brochothrix , during refrigerated storage under the tested oxygen‐permeable VSP condition. These findings provide study‐specific information on post‐thaw microbial changes in 10K VSP seafood products that may help inform future refrigerated handling and display recommendations.

Salmonella enterica is a leading cause of bacterial foodborne illness, frequently associated with raw meat products, including ground beef. Therefore, rapid methods to detect and quantify Salmonella are needed. This study developed and validated a DNA extraction-free digital PCR (dPCR) workflow for the rapid detection and absolute quantification of Salmonella enterica in ground beef. Growth kinetics of 148 S. enterica strains representing 111 serovars were characterized to guide selection of a biologically diverse 12-strain validation panel comprising short- and long-lag phenotypes from six beef-relevant serovars. The assay targeted the invA gene and utilized direct-digital PCR following a 6 h enrichment, eliminating the need for DNA extraction and a standard curve for quantification. The standardized workflow was evaluated using ground beef inoculated with short- and long-lag strains at 1 and 10 CFU/g. Following enrichment, the cell preparations were analyzed using the QIAcuity dPCR platform. The assay consistently detected and quantified Salmonella strains with varying lag phases and background microbiota conditions. Bland–Altman analysis demonstrated strong agreement between dPCR quantification, inoculum counts, and most probable number (MPN) estimates, with variability remaining within acceptable limits. Growth kinetics analysis performed with and without fetal bovine serum (FBS) demonstrated that the presence of blood-derived components can shorten the lag phase of Salmonella strains during enrichment, thereby influencing strain-dependent differences in growth behavior and quantification outcomes. Overall, this workflow enables rapid detection and sensitive quantification of Salmonella in ground beef within nine hours, providing a practical alternative to traditional presence–absence testing for food safety applications.

Rapid detection of Escherichia coli O157:H7 is essential for food safety monitoring, but direct DNA-based assays may detect residual DNA from nonviable cells. This study developed a bacteriophage amplification assay coupled with SYBR Green qPCR (PAA-qPCR) for swift detection of viable E. coli O157:H7. φCAM-21 infection conditions were optimized using microplate growth analysis, and a micro-spin filtration workflow was used to remove unadsorbed phage before progeny-phage amplification. A CAM-21-specific primer pair was designed from a unique genomic region and verified using in silico comparison, gel electrophoresis, melting-curve analysis, and non-target controls. A multiplicity of infection (MOI) of 100 provided the strongest suppression of E. coli O157:H7 C7927 growth and was selected for downstream assays. Progeny phages generated after 1 h of amplification were sufficient for qPCR detection, allowing completion of the workflow in approximately 5 h. Under controlled conditions, the assay detected 10 CFU/mL of E. coli O157:H7. In artificially inoculated foods, consistent detection of 10 CFU/25 g in lettuce and ground beef was achieved after 6 h and 8 h of enrichment, respectively, whereas beef trim and apple juice showed less consistent detection. These results demonstrate the feasibility of CAM-21 PAA-qPCR as a rapid viable-cell detection approach for E. coli O157:H7, although additional validation is required before routine application.

Salmonella quantification in poultry remains a critical gap in routine monitoring because most regulatory and industry workflows still rely on presence/absence results that do not reflect contamination load. This study developed and evaluated an extraction-free digital PCR (dPCR) workflow targeting the invA gene for rapid quantification of Salmonella in chicken breast following short enrichment. Six Salmonella strains representing serotypes Typhimurium, Enteritidis, and serogroup I,4,[5],12,i:- were cold-stressed and inoculated into chicken breast at target levels of 0.1, 1, and 10 CFU/g, followed by 6 h enrichment in modified buffered peptone water (mBPW). Bacterial cells from enrichment were concentrated and used for dPCR without DNA extraction. Quantification performance was benchmarked against a reference most probable number (MPN) (performed from the same inoculated bags), the bioMérieux GENE-UP and Hygiena BAX SalQuant platforms with parallel inoculated bags. Method outputs were log10-transformed and agreement with measured inoculum was assessed using Bland–Altman analysis. Extraction-free dPCR closely tracked inoculum levels across the tested range, with the lowest mean bias (0.195 log10 units) relative to inoculum compared with MPN, GENE-UP, and BAX. MPN showed modest positive deviation (mean bias = 0.35 log10 units), consistent with dilution-based enumeration variability, whereas GENE-UP and BAX estimates were more variable and exhibited a systematic positive deviation (mean bias of 1.16 log10 units and 2.40 log10 units respectively). Overall, the extraction-free dPCR workflow provides a streamlined approach for quantifying Salmonella levels in inhibitor-rich chicken breast matrices, reducing dependence on time-consuming DNA extraction while supporting risk-informed monitoring beyond presence/absence testing.

Vacuum skin packaging (VSP) has emerged as an innovative solution to enhance the safety and freshness of seafood and extend the shelf life of food products. However, its efficacy on seafood products remains completely underexplored. This study aims to evaluate the shelf life and characterize the microbial community dynamics of 10 oxygen permeable skin packed seafood products during refrigeration. Vacuum skin-packed flounder, mahi, perch, salmon, scallop, shrimp, snapper, swai, tilapia, and tuna were collected from a seafood processor in Florida. Samples were defrosted at 4°C and evaluated on days 1, 5, and 9 for aerobic, coliform, and lactic acid bacteria counts. Microbial DNA from samples was extracted and used to characterize bacterial community dynamics during storage. The sequencing data were analyzed using the CLC Genomics Workbench. On day 1, the average aerobic plate count for all 10 samples was 3.5 log CFU/g, which increased to 6 log CFU/g by day 5 and reached 7.5 log CFU/g by day 9. The overall enumeration data indicate that the seafood samples exceeded the spoilage thresholds established by the FDA and FAO by day 5 under refrigeration. Sequencing data revealed Photobacterium, Psychrobacter, Pseudomonas, and Brochothrix as the dominant bacterial genera across most seafood species. Notably, Pseudomonas emerged as the predominant genus by day 5, surpassing all other bacterial genera in abundance. These data provide evidence-based refrigerated handling guidance for oxygen-permeable VSP seafood that processors and retailers can use to manage display time, thereby reducing spoilage-related losses and protecting consumers.

In the United States, testing data indicate that up to 30% of shrimp sold are misrepresented. Among species harvested from U.S. waters, royal red shrimp (Pleoticus robustus) is one of the most valued and is therefore frequently substituted with Argentine red shrimp (Pleoticus muelleri). This study developed and validated an RNase H‑dependent polymerase chain reaction (rhPCR) assay to accurately distinguish P. robustus from P. muelleri. Primer pairs targeting the COI gene were designed, screened for specificity, and subsequently converted into RNase H2‑activated primers labeled with FAM and biotin for lateral‑flow detection. The optimized assay was validated using genomic and crude DNA extracted from 65 barcoded shrimp tissue samples representing seven commercially relevant species: L. setiferus, L. vannamei, P. aztecus, P. duorarum, P. monodon, P. muelleri, and P. robustus. To assess real‑world performance, the assay was further applied to 20 blinded retail samples marketed as royal red shrimp and collected by a state food‑safety division. Results revealed a 60% species‑substitution rate, with 30% of samples identified as P. muelleri, demonstrating the assay’s effectiveness for detecting mislabeling in commercial products. The assay achieved 100% specificity, correctly differentiating royal red and Argentine red shrimp from all non‑target species. Analytical sensitivity reached 0.1 ng/µL for P. robustus and 0.01 ng/µL for P. muelleri crude DNA, with a total turnaround time of 150 minutes for processing 20 samples. These assays provide enforcement agencies with a robust, field‑deployable method for detecting royal red shrimp substitution and supporting evidence‑based regulatory decisions.