Bacillaene Mediates the Inhibitory Effect of Bacillus subtilis on Campylobacter jejuni Biofilms.

Biofilms are the predominant bacterial lifestyle and can protect microorganisms from environmental stresses. Multispecies biofilms can affect the survival of enteric pathogens that contaminate food products, and thus, investigating the underlying mechanisms of multispecies biofilms is essential for food safety and human health. In this study, we investigated the ability of the natural isolate Bacillus subtilis PS-216 to restrain Campylobacter jejuni biofilm formation and adhesion to abiotic surfaces as well as to disrupt preestablished C. jejuni biofilms. Using confocal laser scanning microscopy and colony counts, we demonstrate that the presence of B. subtilis PS-216 prevents C. jejuni biofilm formation, decreases growth of the pathogen by 4.2 log10, and disperses 26-h-old preestablished C. jejuni biofilms. Furthermore, the coinoculation of B. subtilis and C. jejuni interferes with the adhesion of C. jejuni to abiotic surfaces, reducing it by 2.4 log10. We also show that contact-independent mechanisms contribute to the inhibitory effect of B. subtilis PS-216 on C. jejuni biofilm. Using B. subtilis mutants in genes coding for nonribosomal peptides and polyketides revealed that bacillaene significantly contributes to the inhibitory effect of B. subtilis PS-216. In summary, we show a strong potential for the use of B. subtilis PS-216 against C. jejuni biofilm formation and adhesion to abiotic surfaces. Our research could bring forward novel applications of B. subtilis in animal production and thus contribute to food safety. IMPORTANCE Campylobacter jejuni is an intestinal commensal in animals (including broiler chickens) but also the most frequent cause of bacterial foodborne infection in humans. This pathogen forms biofilms which enhance survival of C. jejuni in food processing and thus threaten human health. Probiotic bacteria represent a potential alternative in the prevention and control of foodborne infections. The beneficial bacterium Bacillus subtilis has an excellent probiotic potential to reduce C. jejuni in the animal gastrointestinal tract. However, data on the effect of B. subtilis on C. jejuni biofilms are scarce. Our study shows that the B. subtilis natural isolate PS-216 prevents adhesion to the abiotic surfaces and the development of submerged C. jejuni biofilm during coculture and destroys the preestablished C. jejuni biofilm. These insights are important for development of novel applications of B. subtilis that will reduce the use of antibiotics in human and animal health and increase productivity in animal breeding.

Characterization of Staphylococcus epidermidis strains isolated from industrial cleanrooms under regular routine disinfection.

AIMS: The purpose of this study was the genotypic and phenotypic characterization of 57 strains of Staphylococcus epidermidis isolated from cleanroom environments, based on their biofilm formation and antimicrobial resistance profiles. METHODS AND RESULTS: Biofilm formation was investigated using real-time PCR (icaA, aap, bhp genes), the Congo red agar method and the crystal violet assay. The majority of the strains (59·7%; 34/57) did not form biofilms according to the crystal violet assay, although the biofilm-associated genes were present in 94·7% (54/57) of the strains. Of the biofilm formers (40·4%; 23/57), 39·1% (9/23) have been identified as strong biofilm formers (>4× crystal violet absorbance cut-off). Resistance to a commercial disinfectant and its quaternary ammonium active component, didecyl-dimethyl-ammonium chloride (DDAC), was determined according to minimum inhibitory concentrations (MICs) and the presence of the qac (quaternary ammonium compound) genes. More than 95% (55/57) of the Staph. epidermidis strains had the qacA/B and qacC genes, but not the other qac genes. The MICs for the disinfectant and DDAC varied among the Staph. epidermidis strains, although none were resistant. CONCLUSIONS: Although 59·6% of the Staph. epidermidis strains did not form biofilms and none were resistant to DDAC, more than 94% had the genetic basis for development of resistance to quaternary ammonium compounds, and among them at least 14·0% (8/57) might represent a high risk to cleanroom hygiene as strong biofim formers with qacA/B and qacC genes. SIGNIFICANCE AND IMPACT OF THE STUDY: To assure controlled cleanroom environments, bacterial strains isolated from cleanroom environments need to be characterized regularly using several investigative methods.

Anti-Campylobacter activity of resveratrol and an extract from waste Pinot noir grape skins and seeds, and resistance of Camp. jejuni planktonic and biofilm cells, mediated via the CmeABC efflux pump.

AIMS: To define anti-Campylobacter jejuni activity of an extract from waste skins and seeds of Pinot noir grapes (GSS), resveratrol and possible resistance mechanisms, and the influence of these on Camp. jejuni morphology. METHODS AND RESULTS: Using gene-specific knock-out Camp. jejuni mutants and an efflux pump inhibitor, we showed CmeABC as the most active efflux pump for extrusion across the outer membrane of GSS extract and resveratrol. Using polystyrene surface and pig small intestine epithelial (PSI) and human foetal small intestine (H4) cell lines, GSS extract shows an efficient inhibition of adhesion of Camp. jejuni to these abiotic and biotic surfaces. CONCLUSIONS: Low doses of GSS extract can inhibit Camp. jejuni adhesion to polystyrene surfaces and to PSI and H4 cells, and can thus modulate Camp. jejuni invasion and intracellular survival. SIGNIFICANCE AND IMPACT OF THE STUDY: An understanding of the activities of GSS extract and resveratrol as bacterial growth inhibitors and the specific mechanisms of cell accumulation is crucial for our understanding of Camp. jejuni resistance. GSS extract inhibition of Camp. jejuni adhesion to abiotic and biotic surfaces provides a further step towards the application of new innovative strategies to control Campylobacter contamination and infection via the food chain.

Anti-Campylobacter and resistance-modifying activity of Alpinia katsumadai seed extracts.

AIMS: We tested extracts from Alpinia katsumadai seeds for anti-Campylobacter activity and investigated the roles of the CmeABC and CmeDEF efflux pumps in Campylobacter resistance to these natural phenolics. Additionally, we investigated an A. katsumadai ethanolic extract (AlpE) and other plant extracts as putative efflux pump inhibitors on Campylobacter isolates and mutants in efflux pump genes. METHODS AND RESULTS: AlpE showed antimicrobial activity against sensitive and multidrug-resistant Campylobacter isolates. CmeB inactivation resulted in the greatest reduction in resistance, while cmeF and cmeR mutations produced only moderate effects on minimal inhibitory concentrations (MICs). The chemical efflux pump inhibitors additionally reduced MICs in isolates and mutants, confirming that active efflux is an important mechanism in resistance to AlpE, with additional contributions of other efflux systems. A notable decrease in resistance to tested antimicrobials in the presence of subinhibitory concentrations of AlpE confirms its modifying activity in Campylobacter spp. CONCLUSIONS: AlpE is important anti-Campylobacter source of antimicrobial compounds with resistance-modifying activity. At least two of the efflux systems are involved in the resistance to A. katsumadai antimicrobial seed extracts. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of antimicrobial and resistance-modifying activity of AlpE from A. katsumadai seeds, demonstrating its potential in the control of Campylobacter in the food chain.