Abstract
Isolates within the Clostridium estertheticum complex (CEC) have routinely been identified through the 16S rRNA sequence, but the high interspecies sequence similarity reduces the resolution necessary for species level identification and often results in ambiguous taxonomic classification. The current study identified CEC isolates from meat juice (MJS) and bovine fecal samples (BFS) and determined the phylogeny of species within the CEC through whole genome sequence (WGS)-based analyses. About 1,054 MJS were screened for CEC using quantitative real-time PCR (qPCR). Strains were isolated from 33 MJS and 34 BFS qPCR-positive samples, respectively. Pan- and core-genome phylogenomics were used to determine the species identity of the isolates. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) were used to validate the species identity. The phylogeny of species within the CEC was determined through a combination of these methods. Twenty-eight clostridia strains were isolated from MJS and BFS samples out of which 13 belonged to CEC. At 95% ANI and 70% dDDH thresholds for speciation, six CEC isolates were identified as genomospecies2 (n=3), Clostridium tagluense (n=2) and genomospecies3 (n=1). Lower thresholds of 94% ANI and 58% dDDH were required for the classification of seven CEC isolates into species C. estertheticum and prevent an overlap between species C. estertheticum and Clostridium frigoriphilum. Combination of the two species and abolishment of current subspecies classification within the species C. estertheticum are proposed. These data demonstrate the suitability of phylogenomics to identify CEC isolates and determine the phylogeny within CEC.
Highlights
Clostridium estertheticum is a strict anaerobic spore forming psychrophile and the major cause of blown pack spoilage (BPS) of vacuum packed chilled meat (Collins, 1992; Kalchayanand et al, 1993)
A surprising result from the phylogenomic trees was the monophyletic clade between species C. estertheticum and C. frigoriphilum (Figures 1, 2), which suggested that they constitute a single species
By leveraging on the genomes of the 13 C. estertheticum complex (CEC) isolates, newly sequenced genomes of five CEC species and 16 publicly available CEC genomes, we identified nine distinct species within CEC through the phylogenomics
Summary
Clostridium estertheticum is a strict anaerobic spore forming psychrophile and the major cause of blown pack spoilage (BPS) of vacuum packed chilled meat (Collins, 1992; Kalchayanand et al, 1993). Based on an early 16S rRNA-based classification of genus Clostridium, C. estertheticum belongs to cluster I named Clostridium sensu stricto (Collins et al, 1994) Within this cluster, C. estertheticum constitute of a group of closely related psychrophilic Clostridium spp. CEC constitutes of seven validly published species based on 16S rRNA classification.. CEC constitutes of seven validly published species based on 16S rRNA classification.1 These include C. algoriphilum, C. bowmanii, C. estertheticum, C. frigoris, C. lacusfryxellense, C. psychrophilum, and C. tagluense, which were isolated from either terrestrial or marine sources (Collins, 1992; Spring, 2003; Shcherbakova et al, 2005; Suetin et al, 2009). The majority of the CEC species have been linked with BPS or other types of meat spoilage (Húngaro et al, 2016)
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