Comparative Genome Analysis and Phenotypic Characterization of Clostridium gasigenes CGAS001 Isolated From Chilled Vacuum-Packed Lamb Meat

Abstract

Genomic data for psychrophilic bacteria causing blown pack spoilage (BPS) are limited. This study characterizes the genome of a novel Clostridium gasigenes strain CGAS001 isolated from meat juice sample (MJS) of vacuum-packed lamb meat by comparing it with the type strain C. gasigenes DSM 12272 and five strains representing four other BPS-causing Clostridium sensu stricto species. Phenotypic characteristics of the strain, which include biochemical characteristics, antimicrobial resistance and production of putative polyketide, have been determined. The size of its draft genome is 4.1 Mb with 3,845 coding sequences, 28.7% GC content and 95 RNA genes that include 75 tRNAs, 17 rRNAs, and 3 ncRNAs. Average Nucleotide Identity (ANI) and digital DNA-DNA Hybridization (dDDH) predict that C. gasigenes CGAS001 and DSM 12272 constitute a single species (ANI and dDDH = 98.3% for speciation) but two distinct subspecies (dDDH = 73.3% for subspeciation). The genome is characterized by saccharolytic, lipolytic and proteolytic genes as well as hemolysins and phospholipases, which are consistent with its phenotype. The genome also reveals the ability of C. gasigenes to synthesize polyketides which is demonstrated by the antimicrobial activity of a crude polyketide extract against Listeria monocytogenes and Enterococcus devriesei. The strain is resistant to polymyxin B and streptomycin. The genetic and phenotypic analyses suggest that CGAS001 constitutes a novel subspecies of C. gasigenes adapted to a saprophytic lifestyle and can synthesize narrow spectrum antimicrobial compounds.

Keywords: Clostridium gasigenes; antibiotic resistance; blown pack spoilage; chilled meat; genome analysis; polyketides; toxins.

FIGURE 1

Phylogenetic tree of Clostridium gasigenes CGAS001 by 16S rRNA gene sequence. C. gasigenes CGAS001 and DSM 12272 formed a phylogenetic subunit distinct from selected blown pack spoilage causing Clostridium sensu stricto spp. strains. Atopobium parvulum DSM 20469 was used as the outgroup. Number at joint indicated bootstrap values (%) while the bar indicates 0.1 substitutions per nucleotide position. Phylogenetic tree reconstructed by CLC Workbench Genomics based on alignment of the nucleotide sequences.

FIGURE 2

Circular genomic map of C. gasigenes CGAS001 and selected genomes of five Clostridium species causing blown pack spoilage. There was a closer identity of CGAS001 strain CGAS001 to the only other sequenced C. gasigenes strain DSM 12272 than the other selected Clostridium species. The circular map produced using BRIG software.

FIGURE 3

Antimicrobial activity of C. gasigenes CGAS001 crude polyketide extract. (A) The potential of CGAS001 to synthesize antimicrobial compounds was initially identified using the agar streak method whereby the growth of Listeria monocytogenes EGDe (LM) was inhibited by CGAS001 (CG). (B) The crude extract showed narrow spectrum antimicrobial activity against selected Gram-positive and negative bacteria using the disk diffusion method. The extract was active against L. monocytogenes EGDe and Enterococcus devriesei K8-ED (ED), but inactive against Staphylococcus aureus (SA) and Escherichia coli (EC).

FIGURE 4

Biosurfactant activity of C. gasigenes CGAS001’s crude polyketide extract. Addition of the crude extract to distilled water (20 μg/ml) affected the surface tension of the water. Distilled water without the extract was used as a control. Samples were left to stand in atmospheric air for 30 min at room temperature. Crystal violet was added to the mixture for visual effect and had no influence on the surface tension of the distilled water. (A) Front view and (B) top view of 25 μl droplets of distilled water with (H₂O + PK) or without (H₂O) the extract on a non-polar surface (parafilm) after 30 min. (C) Five hundred microliter of distilled water with (H₂O + PK) or without (H₂O + PK) the polyketide extract in 1 ml cuvettes after 30 min.