Detection of Psychrophilic Clostridium spp. in Fecal Samples from Cattle of Different Ages Sampled at the Slaughterhouse Level

Abstract

Clostridium estertheticum and C. estertheticum -like spp. are obligate anaerobic psychrophiles causing “blown pack” spoilage of chilled vacuum-packed meat. The present study aimed at detecting and isolating these spoilage bacteria in fecal samples of cattle of different ages at the slaughterhouse level. One-hundred-and-two swab fecal samples were obtained and enriched anaerobically in pre-reduced peptone-yeast-glucose-starch medium (PYGS) for three weeks at 4°C then screened for C. estertheticum and C. estertheticum -like spp. using a 16S rRNA gene-based real-time PCR assay (RT-PCR). The RT-PCR positive samples were further enriched for 3 weeks in PYGS then subjected to an ethanol (50% v/v) and lysozyme (4 mg/ml) treatment. Isolation was carried out anaerobically on Columbia Agar with 5% defibrinated sheep blood at 4°C for three weeks. Isolated strains were identified morphologically and by their 16S rRNA gene. Thirty-nine percent (n=40/102) of the samples were RT-PCR positive. The frequency of positive samples was 45% (n=9/20), 43% (n=23/54) and 29% (n=8/28) in calves, young cattle and cows/bulls aged ≤ 160, 161-1000 and >1000 days, respectively. Six strains were isolated from six out of the 40 RT-PCR positive samples. Out of these, five were from the calves (n=1) and young cattle (n=4). The six isolates were identified as C. estertheticum (n=1), C. frigoriphilum (n=1) and C. estertheticum -like spp. (n=4). The present findings confirm that feces of cattle are an important source of psychrophilic Clostridium spp. The fecal carriage among livestock animals at slaughter is strongly correlated with the risk of carcass contamination. Therefore, the maintenance of slaughter hygiene is of central importance.

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

“…The prepared samples were serially diluted and plated on CBA and incubated anaerobically for 3–4weeks at 4°C. Finally, 34 qPCR-positive BFS from our previous study ( Wambui et al, 2021 ) were also subjected to the second treatment procedure. The BFS had been incubated anaerobically for 24weeks at 4°C.…”

“…Pan- and core genome-based phylogenomic trees were created from the WGS of the 28 isolates from this study (their accession numbers are given in Supplementary Table 1 ), five newly sequenced genomes each representing an unsequenced CEC species ( Supplementary Table 2 ) and 27 strains either from our previous studies ( Wambui et al, 2020a , b , c , d , 2021 ) or deposited in NCBI database, 3 respectively. Besides CEC, the other representative clostridia groups were C. algidicarnis , C. gasigenes , and C. frigidicarnis that are usually isolated from meat and/or associated with BPS of vacuum-packed chilled meat ( Húngaro et al, 2016 ) as well as C. argentinense and C. peptidovorans that are closely related to CEC ( Lawson and Rainey, 2016 ).…”

“…In the first approach, 14 samples were suspended (1:100) in pre-reduced Peptone Yeast Glucose Starch broth (PYGS; prepared in-house using the previously described formulation; Lund et al, 1993) and incubated anaerobically for 3-4 weeks at 4°C followed by plating of a sample loopful on Columbia agar supplemented with 5% defibrinated sheep blood (CBA; Thermo Fisher Scientific, Waltham, MA). In the second approach, 19 samples were pre-treated in a multistep approach involving elimination of competitive microbiota and spore recovery, as previously described with slight modifications (Wambui et al, 2021). These steps were carried out aerobically.…”

“…Because of the relatedness of 16S rRNA gene sequences, the taxonomy and identification of CEC members has in most cases been debatable ( Spring, 2003 ). This has resulted in ambiguous classification of identified strains as demonstrated in several studies, where strains have been identified as C. estertheticum -like, C. frigoriphilum -like, and C. tagluense -like ( Brightwell and Clemens, 2012 ; Brightwell and Horváth, 2018 ; Dorn-In et al, 2018 ; Wambui et al, 2021 ). This is further exemplified by species C. estertheticum , whereby two strains C. estertheticum subp.…”

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

“…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). Consistent with their involvement in BPS, CEC have primarily been isolated from animal farmyards and meat processing environment (Esteves et al, 2020(Esteves et al, , 2021Wambui et al, 2020dWambui et al, , 2021b. Other sources include permafrost (Shcherbakova et al, 2005;Suetin et al, 2009) and Antarctic microbial mat (Spring, 2003).…”