Development of PMAxxTM-Based qPCR for the Quantification of Viable and Non-viable Load of Salmonella From Poultry Environment

Abstract

Determining the viable and non-viable load of foodborne pathogens in animal production can be useful in reducing the number of human outbreaks. In this study, we optimized a PMAxx^TM-based qPCR for quantifying viable and non-viable load of Salmonella from soil collected from free range poultry environment. The optimized nucleic acid extraction method resulted in a significantly higher (P < 0.05) yield and quality of DNA from the pure culture and Salmonella inoculated soil samples. The optimized primer for the amplification of the invA gene fragment showed high target specificity and a minimum detection limit of 10² viable Salmonella from soil samples. To test the optimized PMAxx^TM-based qPCR assay, soil obtained from a free range farm was inoculated with Salmonella Enteritidis or Salmonella Typhimurium, incubated at 5, 25, and 37°C over 6 weeks. The survivability of Salmonella Typhimurium was significantly higher than Salmonella Enteritidis. Both the serovars showed moisture level dependent survivability, which was significantly higher at 5°C compared with 25°C and 37°C. The PMAxx^TM-based qPCR was more sensitive in quantifying the viable load compared to the culture method used in the study. Data obtained in the current study demonstrated that the optimized PMAxx^TM-based qPCR is a suitable assay for quantification of a viable and non-viable load of Salmonella from poultry environment. The developed assay has applicability in poultry diagnostics for determining the load of important Salmonella serovars containing invA.

Keywords: PMAxx-based qPCR; Salmonella contamination; Salmonella load; poultry production; viability assay.

FIGURE 1

DNA quantity(ng/μL) obtained through the optimized and the standard kit protocol methods from pure culture of Salmonella. The optimized extraction method resulted in significantly higher yield of DNA. Different superscripts (^A,B) across the bars show significant difference.

FIGURE 2

Amplification efficiency and melting curve analyses of invA determined in 10-fold serial dilutions of DNA. (A) PCR performed on Salmonella DNA extracted from a pure culture with 1 × 10⁹ CFU/mL. (B) Melting curve of invA (605 bp) in DNA obtained from 10-fold serially diluted (10⁸ to 10¹ CFU/g) Salmonella spiked soil samples.

FIGURE 3

Effects of different concentrations (2.5, 5, and 10 mM) of PMAxx^TM on the amplification of target DNA obtained from the viable and non-viable culture of Salmonella. The data showed that PMAxx^TM efficiently inhibited the amplification of the target DNA obtained only from the non-viable culture of Salmonella. This trial was repeated three times and an average of the data was presented. For obtaining non-viable cells, Salmonella in LB broth was heated at 95°C for 5 min, and a 100 μL plated on XLD agar and incubated overnight at 37°C confirmed that the heat treatment was effective in killing 100% of Salmonella in the broth. Asterisks (^∗∗∗) denote significant difference at P < 0.0001, while ns denotes non-significant difference.

FIGURE 4

Overall load of Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) in soil incubated at different temperatures and sampled at weekly intervals. The samples were processed through direct plating for Salmonella load determination. Samples that turned negative through the direct plating were enriched for the qualitative assessment of Salmonella. (A) Mean log₁₀ CFU/g of SE and ST in soil over 6-week of incubation time period. (B) Mean proportion of Salmonella positive samples of SE and ST in soil incubated at different temperatures. (C) Mean moisture level of soil inoculated with SE or ST (D) Mean water activity (a_w) of soil inoculated with SE or ST. Asterisks (^∗∗∗) and (^∗) denote significant difference at P < 0.0001 and P < 0.01, respectively, while ns denotes non-significant difference.

FIGURE 5

Load of Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) in soil incubated at three different temperatures for 6 weeks. (A) Log₁₀ CFU/g of soil of SE and ST affected by storage temperatures (5, 25, and 37°C). (B) Proportion of positive soil samples for SE and ST determined through the enrichment method. Only, the samples that did not show any Salmonella growth after direct plating were enriched. (C) Moisture level of the Salmonella inoculated soil samples incubated at 5, 25, and 37°C. (D) Water activity of the Salmonella inoculated soil samples incubated at 5, 25, and 37°C. The Salmonella inoculated soil samples were processed for Salmonella load through culture, Salmonella detection through enrichment method and moisture and water activity determination at weekly intervals for up to 6 weeks of incubation time period. Asterisk (^∗) denotes significant difference at P < 0.01, while ns denotes non-significant difference. Different superscripts (^a,b,c,d) show significant difference within each treatment group at each sampling timepoint.

FIGURE 6

Regression analysis showing a positive correlation between the moisture level (%) and the load of Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) in soil incubated at 5, 25, and 37°C. (A) Soil inoculated with SE and incubated at 5°C. (B) Soil inoculated with SE and incubated at 25°C. (C) Soil inoculated with SE and incubated at 37°C. (D) Soil inoculated with ST and incubated at 5°C. (E) Soil inoculated with ST and incubated at 25°C. (F) Soil inoculated with ST and incubated at 37°C. Blue lines show moisture level (%), while purple lines show log₁₀ CFU/g of soil.

FIGURE 7

Overall viable and non-viable load quantified by PMAxx^TM -based qPCR from Salmonella inoculated soil affected by temperature. (A) Mean log₁₀ viable and non-viable load of Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) in soil samples incubated at 5, 25, and 37°C and processed at weekly intervals over 6-week of incubation time period. (B) Mean log₁₀ viable and non-viable load of SE and ST affected by temperature. Samples were treated either with PMAxx^TM or left as control before the extraction of DNA. The DNA copy number was calculated through the standard curve and the data were presented as log₁₀ Salmonella load. Asterisks (^∗∗∗) denote significant difference at P < 0.0001.

FIGURE 8

Viable and non-viable load of Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) in soil samples. (A) SE load. (B) ST load. The samples were incubated at 5, 25, and 37°C and processed at weekly intervals for PMAxx^TM -based qPCR. Asterisks (^∗∗∗) show significant difference (P < 0.0001) between the viable and non-viable load of Salmonella affected by each incubation temperature at each sampling timepoint.

FIGURE 9

Correlation between log₁₀ CFU/g and log₁₀ viable load of Salmonella. (A) Soil inoculated with Salmonella Enteritidis (SE) and incubated at 5°C. (B) Soil inoculated with SE and incubated at 25°C. (C) Soil inoculated with SE and incubated at 37°C. (D) Soil inoculated with Salmonella Typhimurium (ST) and incubated at 5°C. (E) Soil inoculated with ST and incubated at 25°C. (F) Soil inoculated with ST and incubated at 37°C. Blue lines represent log₁₀ viable load, while purple lines represent log₁₀ CFU per gram of soil.