Non-stochastic sampling error in quantal analyses for Campylobacter species on poultry products

Abstract

Using primers and fluorescent probes specific for the most common food-borne Campylobacter species (Campylobacter jejuni and Campylobacter coli), we developed a multiplex, most probable number (MPN) assay using quantitative PCR (qPCR) as the determinant for binomial detection: i.e., number of p positive pathogen growth responses out of n = 6 observations each of 4 mL (V) per dilution. Working with media washes of thrice frozen-thawed chicken pieces which had been spiked with known levels of C. jejuni and C. coli, we found that about 20% of the experiments had a significant amount of error in the form of either greater than 25% MPN calculation error (Δε) and/or a low apparent recovery rate (R less than 1 = MPN observed ÷ CFU spiked). Assuming such errors were exacerbated by an excessively small n, we examined computer-generated MPN enumeration data from the standpoint of stochastic sampling error (Δ) and found that such binomial-based assays behaved identically to Poisson-based methods (e.g., counting data) except that fewer technical replicates (n) appeared to be required for the same number of cells per test volume (μ). This result implies that the qPCR detection-based MPN protocol discussed herein should accurately enumerate a test population with a μ ≥ 1 using n = 6 observations per dilution. For our protocol, this equates to ≥ 8 cells per 400-500 g of sampled product. Based on this analysis, the error rate we saw in spiked experiments (where μ >> 1) implied a non-stochastic source. In other experiments we present evidence that this source was, at least in part, related to the cell concentration step (i.e., centrifugation). We also demonstrate that the error rate lessened (from ~38% to ~13%) at lower Campylobacter levels (μ ≤ 40) as would most likely exist in nature. Using this protocol, we were able to quantify 14 to 1,226 MPN per 450 g of naturally contaminated chicken for skinless pieces and 11 to 244 MPN per 450 g for wings, breasts, legs, and thighs (skin on) whereupon about 50% of the 29 samples tested negative for both species. Four of these chicken wash samples did have substantially lower Campylobacter levels (1 to 6 MPN per 450 g) which might be better enumerated using a larger n. However, we established that the limit of quantification of this protocol diminishes for n > 6 because one is ever more diluting the sample, or lessening V, to achieve the requisite n.

Deconvolution of the Error Associated with Random Sampling

“…The counting results alluded to above (P-I, P-II, & MPN) are similar to those observed previously: [5] [15] [16] i.e., stochastic sampling errors associated with microbiological colony counting and MPN data are proportional to the inverse square root of n × μ. Also, the Poisson population-based results compare favorably with those obtained from actual colony counting experiments [14].…”

“…This technique is referred to as the "dilution method" [1] since it involves diluting a test sample's content to extinction ( 0 δ → ). This enumeration protocol is also known as the most probable number (MPN) method and entails sampling from a liquid source, making serial dilutions from this, distributing an aliquot of each of these dilutions into separate receptacles, incubating these under suitable growth conditions, and observing if any growth has occurred based upon some organism-specific detection method [5] [6]. The MPN enumeration procedure is particularly useful when sampling from environmental sources, such as foods, since damaged cells frequently recover in liquid media [7].…”

“…For example, were one to obtain a food sample containing ~14 CFU of a particular organism per 50 g, the cells would typically be washed from the food matrix, concentrated to a few mL (e.g., via centrifugation), and brought up to some appropriate volume (say 40 mL = V sample ) with media [5]. From this, eight 4 mL (V) samples could be randomly selected and distributed into 8 separate receptacles (n = 8 with a dilution factor of 1; i.e., undiluted).…”

“…With the remaining 8 mL from the 0.1 dilution, 4 mL could be diluted again with 36 mL media, mixed and distributed into yet another eight 4 mL replicates (dilution factor = 0.01). After incubation the most likely number (Equation (2), below) of positive occurrences (e.g., presence of a specific gene [5]) observed would be x + = 6, 1, and 0 (out of n = 8 observations per dilution) for dilution factors of 1, 0.1, and 0.01, respectively, and the calculated MPN (±s) per 50 g sample would = 13.8 ± 5.56. Note the relatively large error term.…”

“…as the number of separate n-samplings increases. We also have elaborated on empirical findings developed previously [5] [15] [16] for predicting errors associated with the random sampling of microorganisms as well as comparing the internal variations associated with the three different sampling error data types derived from the Gaussian, binomial (MPN), and Poisson relationships. Thus, new results have been created using the aforementioned probability distributions, Equations (2), (4), and (5), and have been highly replicated since each "experiment", comprising n (= 3, 6, 9, 12, or 24) observations, were repeated 100 times.…”