Evaluation of Virulence Determinants Using Whole-Genome Sequencing and Phenotypic Biofilm Analysis of Outbreak-Linked Staphylococcus aureus Isolates

Abstract

Biofilms are a frequent cause of food contamination of potentially pathogenic bacteria, such as Staphylococcus aureus. Given its vast role in human disease, the possible impact of biofilm-producing S. aureus isolates in a food processing environment is evident. Sixty-nine S. aureus isolates collected from one firm following multiple staphylococcal food poisoning outbreak investigations were utilized for this analysis. Strain evaluations were performed to establish virulence determinants and the evolutionary relationships using data generated by shotgun whole-genome sequencing (WGS), along with end point polymerase chain reaction (PCR) and in vitro phenotypic assessments. S. aureus isolates were grouped into six well-supported clades in the phylogenetic tree, with the relationships within the clades indicating a strong degree of clonal structure. Our analysis identified four major sequence types 47.8% ST1, 31.9% ST45, 7.2% ST5, and 7.2% ST30 and two major spa types 47.8% t127 and 29.0% t3783. Extrapolated staphylococcal enterotoxin (SE) analysis found that all isolates were positive for at least 1 of the 23 SEs and/or SE-like toxin genes. Enterotoxigenic assessments found that 93% of the isolates expressed a classical SE(A–E). SE gene concurrence was observed at 96.2%, based on PCR and WGS results. In total, 46 gene targets were distinguished. This included genes that encode for adhesion and biofilm synthesis such as clfA, clfB, bbp, ebpS, ica, bap and agr. Our evaluation found agr group III to be the most prevalent at 55%, followed by 35% for agr group I. All isolates harbored the complete intercellular adhesion operon that is recognized to contain genes responsible for the adhesion step of biofilm formation by encoding proteins involved in the syntheses of the biofilm matrix. Phenotypic characterization of biofilm formation was evaluated three times, with each test completed in triplicate and accomplished utilizing the microtiter plate method and Congo red agar (CRA). The microtiter plate results indicated moderate to high biofilm formation for 96% of the isolates, with 4% exhibiting weak to no biofilm development. CRA results yielded all positive to intermediate results. The potential to inadvertently transfer pathogenic bacteria from the environment into food products creates challenges to any firm and may result in adulterated food.