Whole Genome Sequence-Based Identification of Clostridium estertheticum Complex Strains Supports the Need for Taxonomic Reclassification Within the Species Clostridium estertheticum

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.

Targeted Genome Mining Reveals the Psychrophilic Clostridium estertheticum Complex as a Potential Source for Novel Bacteriocins, Including Cesin A and Estercticin A

“…Ten publicly available whole genome sequences of CEC as of December 2020 were downloaded from the NCBI database. Additional 40 genomes from Wambui et al (2021a) and ongoing studies were included in the analysis. The genomes are listed in Supplementary Table 1 .…”

“…Trimming and size end filtering were carried out using Cutadapt v2.5. Illumina sequencing was performed-inhouse as previously described by Wambui et al (2021a) without modification. The genome sequences were assembled de novo and circularized using Unicycler v0.4.8 run with default parameters, using the paired-end Illumina reads and ONT reads larger than 10 kb ( Wick et al, 2017 ).…”

“…The Clostridium estertheticum complex (CEC) constitutes of 11 spore-forming obligate anaerobic and psychrophilic species, most notably, C. estertheticum and C. tagluense ( Collins, 1992 ; Spring, 2003 ; Suetin et al, 2009 ; Palevich et al, 2021 ; Wambui et al, 2021a ). CEC is mainly associated with a distinct meat spoilage phenomenon colloquially referred to as blown pack spoilage (BPS) that occurs in vacuum packed and refrigerated meat ( Wambui and Stephan, 2019 ).…”

“…On the other hand, despite occupying an ecological niche that suggests CEC have adopted a different lifestyle compared to the other Clostridium species, the bacteriocin biosynthetic potential of CEC is currently unknown. The availability of 50 CEC genomes through recent ( Palevich et al, 2021 ; Wambui et al, 2021a ) and other ongoing studies offers a timely opportunity for genome mining aimed at closing this research gap. Therefore, using a targeted approach, we determined bacteriocin production potential of CEC through genome mining.…”

Draft Genome Sequence of Clostridium bowmanii DSM 14206 T , Isolated from an Antarctic Microbial Mat

“…While initial phylogenetic analysis based on 16S rRNA gene sequence data placed DSM 14206 T closest to the clostridial cluster containing Clostridium estertheticum type strain DSM 8809 (ATCC 51377) ( 2 ), subsequent 16S rRNA gene and phylogenomic analyses placed DSM 14206 T closest to Clostridium tagluense DSM 17763 T ( 3 ) and Clostridium sp. strain FP3, which was cultivated from a spoiled chilled vacuum pack of lamb meat ( 4 – 6 ).…”