The effect of different isolation protocols on detection and molecular characterization of Campylobacter from poultry

Abstract

Significance and Impact of Study: The tracing of Campylobacter through the food chain remains important to control campylobacteriosis in humans. Our study points out that the isolation method used affects the genotypes obtained, and this should be considered as a variant when comparing the results of surveillance studies. AbstractWe determined whether different methods to isolate Campylobacter (including the ISO standard 10272:2006-1) affected the genotypes detectable from poultry, at three points during slaughter: caecal content, neck skin and meat. Carcasses from 28 independent flocks were thus sampled (subset A). In addition, ten neck skin samples from four flocks, ten caecal samples from ten different flocks and ten unrelated meat samples obtained from local supermarkets were collected (subset B). Campylobacter was isolated using eight different protocols: with and without enrichment using Bolton broth, Preston broth or Campyfood broth (CFB), followed by culture on either modified Charcoal Cefoperazone Deoxycholate Agar (mCCDA) or Campyfood agar (CFA). All obtained isolates were genotyped for flaA-SVR, and over half of the isolates were also typed by MLST. The strain richness, as a measure of number of detected fla-genotypes, obtained from subset A neck skin and caecal samples was higher than that of meat samples. In half of the cases, within a flock, at least one identical fla-genotype was obtained at all three slaughter stages, suggestive of autologous contamination of carcasses. Enrichment reduced the observed richness of isolates, while CFA plates increased richness compared to mCCDA plates, irrespective of inclusion of an enrichment step. Because the isolation protocol used influences both the yield and the fla-genotype richness obtained from poultry, this variable should be taken into account when different studies are being compared.

“These Aren’t the Strains You’re Looking for”: Recovery Bias of Common Campylobacter jejuni Subtypes in Mixed Cultures

“…This is in contrast to the frequency of recovery observed for the remaining subtypes (Ranks 2, 3 and 4), which collectively decreased from 59.5% of the population recovered by nonenrichment isolation to 37.6% of isolates by enrichment. These results are consistent with studies by and Ugarte-Ruiz et al (2013) in which they assessed the genotype diversity of C. jejuni isolated from poultry samples using a variety of isolation protocols and found method-specific differences in the genotypes and numbers of isolates observed. They concluded that enrichment methods, in particular, limit the "genotypic richness" of the population recovered Ugarte-Ruiz et al, 2013).…”

“…These results are consistent with studies by and Ugarte-Ruiz et al (2013) in which they assessed the genotype diversity of C. jejuni isolated from poultry samples using a variety of isolation protocols and found method-specific differences in the genotypes and numbers of isolates observed. They concluded that enrichment methods, in particular, limit the "genotypic richness" of the population recovered Ugarte-Ruiz et al, 2013). In contrast to using natural samples for experimentation, our controlled experimental set-up allowed us to examine the nature of these findings more directly owing to the removal of potential confounders when dealing with recovery of isolates from samples of unknown strain composition.…”

“…Recovery experiments were performed under controlled laboratory conditions using sterile Bolton's enrichment broth (BB) spiked with normalized quantities of broth culture from four C. jejuni strains of known subtype. While previous studies have documented differences in genotype diversity when comparing isolation procedures (Devane et al, 2013;Ugarte-Ruiz et al, 2013), our experimental design included several features selected to avoid confounding factors related to the analysis of field-collected samples. These included: (a) the use of mixtures composed of known Campylobacter subtypes, which avoided complications related with the analysis of samples comprising unknown subtype composition; (b) the use of controlled culture conditions, which avoided issues related to different sample matrices or background organisms present in samples collected from the field; (c) the use of BB, which was selected for this study because it was used in the original FIGURE 3 | Summary of C. jejuni subtype recovery using enrichment and non-enrichment.…”

“…To date, several studies have noted the potential for biased recovery in the isolation of foodborne pathogens such as Listeria monocytogenes, Salmonella, and Campylobacter (Gorski, 2012;Zilelidou et al, 2016). The use of different isolation protocols and media has been shown to influence the frequency of detection and can affect the diversity of C. jejuni subtypes observed in mixed-strain populations Ugarte-Ruiz et al, 2013). A recovery step in enrichment broth was originally suggested for cases where low numbers of cells may be present (Hutchinson and Bolton, 1984), where cells may be under physiological stress (Bovill and Mackey, 1997), or to help resuscitate cells in the viable but nonculturable state (Jones et al, 1991).…”

“…Several studies have compared non-enrichment and enrichment-based recovery methods and found that sample pre-enrichment could improve Campylobacter recovery over non-enrichment methods (Habib et al, 2011), as has been suggested for isolation from cattle feces (Garcia et al, 1985;Atabay et al, 1998;Stanley et al, 1998;Gharst et al, 2006). However, there is significant evidence suggesting that isolation methods employing a pre-enrichment step may favor certain subtypes and thus bias the subtype representation obtained (Devane et al, 2013;Ugarte-Ruiz et al, 2013). It is not known, however, to what extent different isolation methods affect the distribution of subtypes recovered from animal or environmental samples.…”