Detection of Salmonella in wheat grains

Abstract

Dry breakfast cereals, breads, pasta, dough, dry mixes for cakes, cookies, batters and coatings contain the cereal grain, wheat. As a raw agricultural product, several studies have shown that wheat grains can be potentially contaminated with foodborne pathogens, such as Escherichia coli, Salmonella, Clostridium, Bacillus, etc., as well as nonpathogenic organisms. Salmonella is of particular interest as a foodborne pathogen because of the involvement in numerous local and national outbreaks. Although the awareness of Salmonella detection in meat, fruits, vegetables and ready to eat foods is well known, our knowledge of the prevalence of Salmonella in cereal grains, such as wheat, is limited. The purpose of this study was to detect and isolate Salmonella from wheat grains that were harvested, transported and stored in different regions of the country. A total of 1,016 wheat grain samples were transported to the laboratory and stored at -80°C until analyzed. Over a thirteen-week period, 625 samples were randomly selected, thawed at 4°C, and tested. Three methods were used for detection and isolation of Salmonella. Method A consisted of 50 g of wheat grains suspended in 450 ml of modified buffered peptone water with pyruvate (mBPWp) and incubated at 37°C for 30 minutes. An aliquot of 10 ml of sample was pipetted into 90 ml of mBPWp with novobiocin (22 [mu]g/ml) and incubated for a total of 24 hours. Ten milliliters of the suspension was added to 90 ml of Rappaport Vassiliadis (RV) broth and incubated for 24 hours at 42°C. Method B was a modified version of method A in which the sample size was decreased to 25 g of wheat grains and suspended in 225 ml of mBPWp. The suspension was incubated at 37°C for 30 minutes and blended in a stomacher before incubation. Method C consisted of the wheat grains incubated in RV broth with novobiocin (22 [mu]g/ml) for 48 hours. In all three methods, DNA was extracted from the RV broth and subjected to quantitative real-time PCR (qPCR) for the detection of invA and pagC genes. Samples positive for both genes were streaked onto Hektoen-Enteric (HE) agar and incubated at 37°C overnight. Presumptive Salmonella colonies were tested for agglutination with Salmonella O antiserum Poly A- I & Vi and retested by qPCR for both genes to confirm the species. Salmonella confirmed isolates were submitted to the National Veterinary Services Laboratory (NVSL) in Ames, IA for serotyping. The isolates were also tested for antimicrobial susceptibility with the National Antimicrobial Resistance Monitoring Systems (NARMS) Sensititre TM CMV3AGNF Gram negative panel. None of the samples were qPCR positive for invA and pagC genes by method A; however, methods B and C identified Salmonella enterica positive samples. Overall, eight samples with a prevalence of 1.3% (8/625) were positive for Salmonella by qPCR and culture methods. Six samples were positive for Salmonella by method B, five samples were positive by method C, and three of the eight samples were identified as positive by both methods. Out of the eight isolates identified five belonged to subsp. enterica and three belonged to subsp. diarizonae. The isolates of subsp. enterica belonged to serotypes Anatum, Hartford, Infantis, Norwich and Oranienburg. The three diarizonae were identified as serotype 61:1,v:1,5(7). Antimicrobial susceptibility testing revealed that five of eight strains were pan-susceptible; however, S. Infantis and one strain of S. diarizonae showed resistance to cefoxitin. Two S. diarizonae strains were resistant to tetracycline and amoxicillin-clavulanic acid. These results showed that harvested wheat grains carry Salmonella and supported other studies showing similar results. Further investigation is needed to determine the source of contamination and pathogenic potential of the isolated strains. In order to assess the virulence potential of the Salmonella strains isolated from these wheat grains, whole genome sequencing of the eight strains was performed and analyzed the for the presence of important virulence genes. In silico analysis of the genomic sequences of the eight strains confirmed the serotyping by NVSL. Multilocus sequence typing (MLST) analysis revealed that all the diarizonae strains belonged to same sequence type (ST-243); however, enterica strains belonged to multiple sequence types. The strains carried virulence genes, including fimbrial genes, typhoid toxin genes, Salmonella pathogenicity island (SPI)-1 and 2 encoded type three secretory system (TTSS) genes and a repertoire of effectors of TTSS. All of the strains carried the aminoglycoside resistance gene, aac(6")-laa. Salmonella phage SEN22 was the only intact phage sequence found in Salmonella strains belonging to subspecies diarizonae, while enterica strains carried diverse phage populations. Based on these studies, analyses of Salmonella strains isolated from wheat grains reveal their virulence potential, suggesting the possibility that these strains could cause foodborne illness in humans.