Effects of different media on the enrichment of low numbers of Shiga toxin-producing Escherichia coli in mung bean sprouts and on the development of the sprout microbiome

Abstract

Sprouted seeds have been implicated in a number of serious outbreaks caused by Salmonella and Shiga toxin-producing Escherichia coli. Sprouts pose a very complex challenge to bacterial pathogen enrichment and detection since they naturally contain high levels of background microflora including members of the Enterobacteriaceae. As such, the currently used method cannot ensure reliable detection of STEC in sprouts. In this study, we compared different media for the enrichment of Enterobacteriaceae in their ability to promote the growth of stressed STEC at 37°C and 42°C. Mung bean sprouts were spiked with low levels of STEC and their growth was recorded over time. In addition, the microbiome of mung bean sprouts was analysed before and after enrichment. Our results indicate that the growth of dry-stressed STEC is comparable in all of the tested enrichment media except for mTSB+Novobiocin and not influenced by the incubation temperature. Low levels of STEC spiked into the sprouts resuspended in media only grew to levels of around 4logcfu/ml during enrichment, which could reduce the probability of detection. Proteobacteria was the dominant phylum detected within the microbiome of non-enriched mung bean sprouts. During enrichment in EE-broth, Proteobacteria remained the most abundant phylum. In contrast, during enrichment in BPW the relative abundance of Proteobacteria decreased whereas Firmicutes increased when compared to the non-enriched mung bean sprout microbiome. The microbiome composition was not significantly influenced by the incubation temperature during enrichment in both BPW and EE-broth. This is the first study to examine the microbiome on sprouted mung bean seeds during BPW and EE enrichment and relates the bacterial community composition changes to the enrichment of pathogens. Abstract (max 400 words) 28This study evaluated the effects of changing the composition of the pre-enrichment medium 29 buffered peptone water (BPW) on the growth of stressed and unstressed Gram-negative 30 foodborne pathogens in a one-broth enrichment strategy. BPW supplemented with an 31 available iron source and sodium pyruvate, along with low levels of 8-hydroxyquinoline and 32 sodium deoxycholate (BPW-S) improved the recovery of desiccated Cronobacter spp. from 33 powdered infant formula. Growth of Salmonella and STEC was comparable in all BPW 34 variants tested for different food matrices. In products with high levels of Gram-negative 35 background flora (e.g. sprouts), the target organisms could not be reliably detected by PCR in 36any of the BPW variants tested unless the initial level exceeded 10 3 cfu/10g of sprouts. 37Based on these results we suggest BPW-S for a one-broth enrichment strategy of stressed 38Gram-negative foodborne pathogens from dry products. However, a one-broth enrichment 39strategy based on BPW variants tested in this evaluation is not recommended for produce 40 with a high level of Gram-negative background flora due to very high detection limits.

How to Warrant Microbiological Food Safety in Food Companies: Problems and a Possible Solution

“…food matrix) can affect bacterial metabolism, and stress conditions such as temperature, pH, saline concentration, or water activity have a notable effect on bacterial growth (Pandey et al 2016). For this reason, protocols often include at least one enrichment step: this not only helps improve sensitivity and detection of even low concentrations of pathogens in a sample (Dwivedi and Jaykus 2011;Gracias and McKillip 2004), but also enhances recovery of stressed (Margot et al 2016;Margot et al 2015) and slow-metabolism bacteria (Vaudaux, Kelley, and Lew 2006) in order to obtain more realistic results.…”

“…dry food). Therefore, food texture and composition, coupled with treatment and storage conditions, can affect bacterial metabolism, with stressing factors altering their growth (Margot et al 2016). For this reason, specific calibration curves are available for specific food matrices based on experimental data considering specific food products categories.…”

Applications of Genomics in Regulatory Food Safety Testing in Canada

“…Metagenomic analysis of enrichment dynamics can be used to inform the development of improved methods for cultural enrichment of pathogens [97][98][99]. A practical approach for this is to selectively amplify hypervariable regions within the 16 s rDNA and to sequence amplicons using WGS technologies.…”

“…You can freely access the chapter at the Web Viewer here. enrichment cultures can be used to evaluate growth of target pathogens relative to background food microbiota over time [97,99]. Such studies can provide valuable insight into species that could potentially interfere with target pathogens that can be applied to the development of improved methodology.…”

Microbial Quality of Raw and Ready-To-Eat Mung Bean Sprouts Produced in Italy

“…Salmonella spp. and Escherichia coli O157:H7 are the most frequently associated causative agents of outbreaks caused by Alfalfa and mung bean sprouts consumption in North America [16][17][18][19][20][21] clearly indicating that mung bean sprouts constitute a significant food safety risk [22]. Furthermore also other shiga toxin producing Escherichia coli (STEC) produced various major outbreak associated with consumption of sprouted-fenugreek seeds in Northern Germany together with 14 EU countries, in USA and Canada [18,23,24].…”