Method-Dependent Implications in Foodborne Pathogen Quantification: The Case of Campylobacter coli Survival on Meat as Comparatively Assessed by Colony Count and Viability PCR

Abstract

The aim of the present study was to address method-dependent implications during the quantification of viable Campylobacter coli cells on meat over time. Traditional colony counting on selective and non-selective culture media along with an optimized viability real-time PCR utilizing propidium monoazide-quantitative PCR (PMA-qPCR), spheroplast formation and an internal sample process control (ISPC), were comparatively evaluated for monitoring the survival of C. coli on fresh lamb meat during refrigeration storage under normal atmospheric conditions. On day zero of three independent experiments, lamb meat pieces were artificially inoculated with C. coli and then stored under refrigeration for up to 8 days. Three meat samples were tested on different days and the mean counts were determined per quantification method. An overall reduction of the viable C. coli on lamb meat was observed regardless of the applied quantification scheme, but the rate of reduction followed a method-dependent pattern, the highest being observed for colony counting on modified charcoal cefoperazone deoxycholate agar (mCCDA). Univariate ANOVA indicated that the mean counts of viable C. coli using PMA-qPCR were significantly higher compared to Columbia blood agar (CBA) plating (0.32 log10 cell equivalents, p = 0.015) and significantly lower when mCCDA was compared to CBA plating (0.88 log10 CFU, p < 0.001), indicating that selective culture on mCCDA largely underestimated the number of culturable cells during the course of meat storage. PMA-qPCR outperformed the classical colony counting in terms of quantifying both the culturable and viable but non-culturable (VBNC) C. coli cells, which were generated over time on meat and are potentially infectious and equally important from a public health perspective as their culturable counterparts.

Method-Related Impacts on Campylobacter coli Recovery From Sampling Materials And Meat

“…After sampling and during sample analysis in the laboratory, any given quantifi cation method (molecular or classical) will be applied on the same generated homogenized suspension for the enumeration of the targeted microbial species. Evidently, different quantifi cation methods will generate different results due to the inherent capabilities and shortcomings of each method [18]. In the case of meat, all enumeration methods are applied on bacteria that are already detached from the samples and are present in the homogenized suspension (their recovery is already predefi ned at this point).…”

“…The use of qPCR provided a subjective evaluation of the total detachment of C. coli cells from samples, which were quantifi ed as genome copies corresponding to cell equivalents, regardless of the viability of the recovered campylobacters; culturable, viable but nonculturable (VBNC) as well as dead C. coli bacteria were concurrently quantifi ed in this study. Lower counts and recovery rates would be expected if culturebased methods were used instead, since only culturable C. coli bacteria would form colonies on agar plates in this case, in contrast to stressed, injured and/or dead cells [18]. More specifi cally, classical colony counting cannot detect VBNC C. coli cells, which are quickly generated under stressful environmental conditions (e.g.…”

“…More specifi cally, classical colony counting cannot detect VBNC C. coli cells, which are quickly generated under stressful environmental conditions (e.g. low temperatures and atmospheric oxygen) and are potentially infectious and equally important for public health [18,19]. Interestingly, colony-counting on mCCDA (modifi ed Charcoal Cefoperazone Deoxycholate agar), which is the mandatory solid selective medium of choice according to the standard colony-count technique for enumeration of Campylobacter [20], has been reported to underestimate even the culturable C. coli cells due to the selective factors that impose an additional hurdle to the already stressed bacteria present on meat under refrigeration and atmospheric oxygen [18].…”

“…low temperatures and atmospheric oxygen) and are potentially infectious and equally important for public health [18,19]. Interestingly, colony-counting on mCCDA (modifi ed Charcoal Cefoperazone Deoxycholate agar), which is the mandatory solid selective medium of choice according to the standard colony-count technique for enumeration of Campylobacter [20], has been reported to underestimate even the culturable C. coli cells due to the selective factors that impose an additional hurdle to the already stressed bacteria present on meat under refrigeration and atmospheric oxygen [18]. This selective medium should have been used if a standard microbiological method would be included in the present study, since no method of decontamination of the autochthonous microbiota on meat was applied.…”

Using Microbial Responses Viewer and a Regression Approach to Assess the Effect of pH, Activity of Water and Temperature on the Survival of Campylobacter spp.

“…are commensal organisms in bovine, sheep, pigs and poultry alongside various birds and usually do not cause any symptoms in animals. They are Gram-negative, slender, spirally curved rod, non-spore-forming and microaerophilic bacteria [ 1 , 2 ]. It is known that there are 51 species and 16 subspecies belonging to the Campylobacter genus of the Campylobacteraceae family.…”