Abstract
In a raw-meat-processing environment, members of the Enterobacteriaceae family can coexist with Staphylococcus aureus to form dual-species biofilms, leading to a higher risk of food contamination. However, very little is known about the effect of inter-species interactions on dual-species biofilm formation. The aim of this study was to investigate the interactions between S. aureus and raw-meat-processing environment isolates of Klebsiella oxytoca in dual-species biofilms, by employing an untargeted metabolomics tool. Crystal violet staining assay showed that the biomass of the dual-species biofilm significantly increased and reached its maximum after incubation for 21 h, compared with that of single species grown alone. The number of K. oxytoca in the dual-species biofilm was significantly higher than that of S. aureus. Field emission scanning electron microscopy (FESEM) revealed that both species were evenly distributed, and were tightly wrapped by extracellular polymeric substances in the dual-species biofilms. Ultra-high-pressure liquid chromatography equipped with a quadrupole-time-of-flight mass spectrometer (UHPLC-Q-TOF MS) analysis exhibited a total of 8184 positive ions, and 6294 negative ions were obtained from all test samples. Multivariate data analysis further described altered metabolic profiling between mono- and dual-species biofilms. Further, 18 and 21 different metabolites in the dual-species biofilm were screened as biomarkers by comparing the mono-species biofilms of S. aureus and K. oxytoca, respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were exclusively upregulated in the dual-species biofilm included ABC transporters, amino acid metabolism, and the two-component signal transduction system. Our results contribute to a better understanding of the interactive behavior of inter-species biofilm communities, by discovering altered metabolic profiling.
Highlights
Multiple microbes are important contributors to food spoilage during storage, processing, and distribution
The biomass of the dual-species biofilms (DSBs) formed by S. aureus and K. oxytoca was significantly higher than those of the two monospecies biofilms formed after the 12 h incubation period (p < 0.05)
We used volcano plot analysis to reveal significantly altered metabolite features between mono- and dual-species biofilms formed by S. aureus and K. oxytoca in the positive and negative ion mode, by delineating a log transformation plot of the foldchange difference and the level of statistical significance of each metabolite
Summary
Multiple microbes are important contributors to food spoilage during storage, processing, and distribution. These microbes coexist as communities, compete for resources and nutrients, and are often present as biofilms in the food-processing environment [1,2]. Biofilm formation is generally a dynamic process, and different mechanisms are involved in attachment and biofilm maturation. Staphylococcus aureus, a strain of Grampositive bacteria, is an important commensal opportunistic and life-threatening food-borne pathogen because of its ability to produce staphylococcal enterotoxins in food [4]. The growth of S. aureus cells occurs in two states, the planktonic and biofilm states, with different specifications. Mature S. aureus biofilms are more resistant to environmental stress than their free-living counterparts [5]
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