Population Analyses Reveal Preenrichment Method and Selective Enrichment Media Affect Salmonella Serovars Detected on Broiler Carcasses

Abstract

Poultry is a major Salmonella reservoir, but conventional culture-based methods typically identify the most abundant serovars while those less abundant remain undetected. Choice of enrichment procedure also introduces bias, and for broiler carcasses, a 1-min rinse before preenrichment is insufficient to release all Salmonella present. The inability to assess serovar diversity means that serovars more often associated with human illness may be masked by more abundant Salmonella. CRISPR-SeroSeq (serotyping by sequencing clustered regularly interspaced short palindromic repeats), an amplicon-based, next-generation sequencing tool, allows detection of multiple serovars and maps the relative serovar frequencies in a sample. To address the preceding limitations, CRISPR-SeroSeq was used on broiler carcasses collected prechilled at a commercial plant. Standard carcass rinse aliquot preenrichments and whole carcass preenrichments that were enriched in Rappaport-Vassiliadis (RV) and tetrathionate (TT) broths were compared. On average, five serovars were observed per carcass, including nine on one carcass. CRISPR-SeroSeq detected serovars comprising as little as 0.005% of the population. CRISPR-SeroSeq data matched (28 of 32) standard culture analysis for abundant serovars. Salmonella serovars Kentucky, Typhimurium, and Schwarzengrund were found on each carcass. Overall, serovar diversity was higher in whole carcass preenrichments that were enriched in RV (P < 0.05). Serovar Schwarzengrund was present at higher frequencies in whole carcass preenrichments compared with rinse aliquot preenrichments (t test, P < 0.05), suggesting it adheres more strongly to the carcass. Salmonella serovar Enteritidis was enriched eightfold more in TT than in RV, and serovars Schwarzengrund and Reading were preferentially enriched in RV. Comparison of preenriched and enriched samples suggests that selective enrichment in RV or TT was inhibitory to some serovars. This article addresses limitations of Salmonella surveillance protocols and provides information related to Salmonella population dynamics.

Characterization of Salmonella Isolates Recovered from Stages of the Processing Lines at Four Broiler Processing Plants in Trinidad and Tobago

“…However, the challenges associated with WCE method, particularly the considerably larger incubator space requirement compared with the use of WCR and NS methods, cannot be disregarded thereby making it an impractical method for routine surveillance testing but applicable as a research tool. It has been reported that the types of samples and the methods of enrichment affect their sensitivities to detect Salmonella in chickens [ 76 , 77 ].…”

Incidence of Salmonella serovars isolated from commercial animal feed mills in the United States and serovar identification using CRISPR analysis

“…PCR products were sequenced at Eton Biosciences, Inc. (San Diego, CA, USA) in both directions using the primers indicated in Table 1. Salmonella CRISPR spacers were identified from the sequence reads using CRISPRFinder (Grissa et al 2007) and were matched to known Salmonella CRISPR loci, including our database that contains CRISPR spacers for 130 different serovars (Shariat et al 2015;Thompson et al 2018;Vosik et al 2018;Cox et al 2019;Richards et al 2020).…”

Determination of Antimicrobial Resistance Patterns in Salmonella from Commercial Poultry as Influenced by Microbiological Culture and Antimicrobial Susceptibility Testing Methods

“…Another important finding in this study was that observed resistance to multiple antimicrobial compounds was associated with culture methods. Salmonella detection and isolation bias imparted by microbiological culture methods and media have been documented and potential implications to the accuracy of surveillance programs were discussed [ 31 , 32 , 33 , 34 ]. Singer et al [ 31 ] reported cultivation media selected preferentially for specific strains of Salmonella in heterogeneous cultures, such as a tetrathionate-RV protocol and a bovine fecal culture.…”

“…A Salmonella Newport strain was more competitive than two Salmonella Typhimurium and one Salmonella Enteritidis strains in both media [ 31 ]. Concentrations of Salmonella Enteritidis at the end of the enrichment period were eight-fold higher in tetrathionate as compared to those in RV, whereas Salmonella Schwarzengrund and Reading preferentially enriched in RV [ 32 ]. Goriski [ 33 ] reported the bias of selective enrichment media (RV and RV nutrient-dense version) on the types of Salmonella isolates from mixed strain culture and cattle fecal cultures.…”