Detection and isolation of Escherichia coli based on intrinsic acid resistance and identification of a genome-encoded amidase as an antimicrobial against Clostridium perfringens

Abstract

Acid resistance allows Escherichia coli survival in the gastrointestinal tract. The glutamate-dependant acid response (GDAR) is regulated by GadE. The primary goal of this study was to determine the acid resistance of E. coli isolates from ground beef, food products and food animal cecal contents then sequence the gadE open reading frame to assess sequence diversity. Isolates were assigned to acid tolerance groups A (highest tolerance) through D. Of 61 isolates characterized, 24, 21, 10 and 6 isolates were assigned to groups A, B, C, and D, respectively. Of 55 isolates, 92.7% had identical gadE sequences, while 3 isolates had 1 amino acid change and 1 isolate had a predicted truncation. Based on sequence conservation, a gadE-specific real-time PCR assay was developed for confirming E. coli. All bacterial isolates recovered from ground beef enrichments (n=70) and from cecal contents (n=82) had 100% agreement between the PCR assay and biochemical identification. Acid resistance was utilized as a recovery tool for Shiga toxin-producing E. coli (STEC). Ground beef samples were inoculated with STEC and enriched overnight. Target STEC were recovered using immunomagnetic separation (IMS) and plating onto modified Rainbow Agar (mRBA) and Rainbow Agar (RBA). Acid treatment was performed on the post-IMS eluate followed by plating onto mRBA. Using the combination of mRBA and acid treatment, STEC were isolated from 85.8% (103/120) of inoculated samples compared to 56.7% (68/120) using no acid treatment and plating onto RBA, a more selective agar. Clostridium perfringens is a Gram-positive, spore-forming anaerobe implicated in human and animal diseases. Previously discovered C. perfringens bacteriophage lytic enzymes were utilized for in silico identification of prophage lysins or autolysins within C. perfringens genomes. The gene encoding a putative amidase was cloned from C. perfringens genomic DNA and expressed for use as an antimicrobial. By spot assay, lytic zones were observed for the purified amidase. The amidase significantly reduced C. perfringens cultures as shown by turbidity reduction and plate counts. Gene sequence analysis revealed little heterogeneity among multiple C. perfringens strains. The results demonstrate that lytic proteins encoded in bacterial genomes could be used as bacterial control agents.