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Abstract
Anaerobic sporeformers, specifically spoilage and pathogenic members of the genus Clostridium, are a concern for producers of dairy products, and of powdered dairy products in particular. As an alternative to testing for individual species, the traditional, and still current, approach to detecting these sporeformers, including non-spoilage/non-pathogenic species, in dairy products has involved testing for a sulphite reducing phenotype [Sulphite reducing Clostridia (SRCs)] under anaerobic conditions. This phenotype is conserved throughout the Order Clostridia. Unfortunately, however, this phenotype is exhibited by other sulphite reducing bacteria (SRBs) also, potentially leading to potential for false positives. Here, this risk was borne out through the identification of several SRBs from industry samples that were identified as Proteus mirabilis and various Bacillus/Paenibacillus sp. Genome wide comparison of a number of representative SRCs and SRBs was employed to determine phylogenetic relationships, especially among SRCs, and to characterize the genes responsible for the sulphite reducing phenotype. This screen identified two associated operons, i.e., asrABC in SRCs, and cysJI in Bacillus/Paenibacillus spp. and P. mirabilis. This screen identified spp. belonging to sensu stricto, Lachnospiraceae and Cluster XIV of the Clostridia all producing the SRC phenotype. This study highlights the inaccuracy of the industry standard SRC test but highlights the potential to generate an equivalent molecular test designed to detect the genes responsible for this phenotype in clostridia.
Highlights
Raw milk is populated by a variety of metabolically and taxonomically diverse bacteria, the majority of which are inactivated by commercial pasteurization (Wells-Bennik et al, 2016)
In order to better understand the prevalence and identity of SRBs in the Irish dairy chain, 101 positive SRB isolates were identified by Sanger sequencing of the corresponding 16S rRNA gene. 77 isolates were identified as clostridia (SRCs), 19 were Bacillus sp., 3 isolates were Proteus mirabilis and 2 Paenibacillus sp. (Table 1)
It was apparent that the SRBs present in the dairy chain were relatively heterogeneous, with the proportion of non-clostridia being notable in light of the purpose of the assay i.e., to detect Sulphite reducing Clostridia (SRCs)
Summary
Raw milk is populated by a variety of metabolically and taxonomically diverse bacteria, the majority of which are inactivated by commercial pasteurization (Wells-Bennik et al, 2016). In the case of C. botulinum, while the presence of the pathogen in PIF has been associated with two incidences of infant botulism previously, one in the United States and one in the United Kingdom in 2001, the links were not conclusively established (Barash et al, 2010) Regardless, this species remains a concern for dairy producers, for those that produce products for infant consumption, as the infectious dose for botulinum spores in infant botulism is thought to be extremely low (ICMSF, 2014) and the reputational damage associated with an outbreak would likely be great. It remains of concern to producers because of its ability to produce a neurotoxin
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