Ethanol at Subinhibitory Concentrations Enhances Biofilm Formation in Salmonella Enteritidis

Abstract

The survival of Salmonella enteritidis in the food chain is relevant to its biofilm formation capacity, which is influenced by suboptimal environmental conditions. Here, biofilm formation pattern of this bacterium was assessed in the presence of ethanol at sub-minimal inhibitory concentrations (sub-MICs) by microtiter plate assays, cell characteristic analyses, and gene expression tests. It was observed that ethanol at subinhibitory concentrations (1/4 MIC, 2.5%; 1/2 MIC, 5.0%) was able to stimulate biofilm formation in S. enteritidis. The OD595 value (optical density at 595 nm) used to quantify biofilm production was increased from 0.14 in control groups to 0.36 and 0.63 under 2.5% and 5.0% ethanol stresses, respectively. Ethanol was also shown to reduce bacterial swimming motility and enhance cell auto-aggregation ability. However, other cell characteristics such as swarming activity, initial attachment and cell surface hydrophobicity were not remarkedly impacted by ethanol. Reverse transcription quantitative real-time PCR (RT-qPCR) analysis further revealed that the luxS gene belonging to a quorum-sensing system was upregulated by 2.49- and 10.08-fold in the presence of 2.5% and 5.0% ethanol, respectively. The relative expression level of other biofilm-related genes (adrA, csgB, csgD, and sdiA) and sRNAs (ArcZ, CsrB, OxyS, and SroC) did not obviously change. Taken together, these findings suggest that decrease in swimming motility and increase in cell auto-aggregation and quorum sensing may result in the enhancement of biofilm formation by S. enteritidis under sublethal ethanol stress.

Stress Response Mechanisms of Salmonella Enteritidis to Sodium Hypochlorite at the Proteomic Level

“…Enteritidis was observed in the presence of 130 ppm sodium hypochlorite, while complete growth inhibition was achieved when chlorine concentration was increased to 260 or 520 ppm. Chemical agents at sublethal concentrations have been considered to exert an inhibitory but not lethal effect on bacterial pathogens [ 20 ]. Therefore, 130 ppm sodium hypochlorite was selected for sublethal adaptation treatments in the subsequent test.…”

“…In line with the above-mentioned findings, sublethal levels of ethanol were also able to inhibit swimming motility of S . Enteritidis [ 20 ]. Furthermore, flagellar motility of S .…”

“…Flagellar motility of S . Enteritidis in the control (0 ppm sodium hypochlorite) and treated (130 ppm sodium hypochlorite) groups was quantified by a soft agar (0.3%) test as previously described [ 20 ]. LB broth enriched with 0.3% agar was autoclaved and allowed to dry at room temperature.…”

Analysis of the Antimicrobial and Anti-Biofilm Activity of Natural Compounds and Their Analogues against Staphylococcus aureus Isolates

“…The findings were corroborated by the quantitative microtiter plate assay showing OD 630nm of all isolates to be greater than 0.5 ( Figure 1 B). Studies have reported that isolates exhibiting OD > 0.5 are considered strong biofilm formers [ 26 , 27 , 28 ]. The microtiter plate assay is a standard assay used by numerous studies to characterize biofilm-forming pathogens as strong or weak biofilm producers [ 26 , 27 , 28 ].…”

“…Studies have reported that isolates exhibiting OD > 0.5 are considered strong biofilm formers [ 26 , 27 , 28 ]. The microtiter plate assay is a standard assay used by numerous studies to characterize biofilm-forming pathogens as strong or weak biofilm producers [ 26 , 27 , 28 ].…”