Biofilm Formation by Bacillus cereus Is Influenced by PlcR, a Pleiotropic Regulator

Y.-H. Hsueh, E. B. Somers, D. Lereclus, A. C. L. Wong
2006 Applied and Environmental Microbiology  
The ⌬plcR mutant of Bacillus cereus strain ATCC 14579 developed significantly more biofilm than the wild type and produced increased amounts of biosurfactant. Biosurfactant production is required for biofilm formation and may be directly or indirectly repressed by PlcR, a pleiotropic regulator. Coating polystyrene plates with surfactin, a biosurfactant from Bacillus subtilis, rescued the deficiency in biofilm formation by the wild type. Bacillus cereus is a pathogen that causes two distinct
more » ... es two distinct types of food poisoning, the diarrheal and emetic syndromes, as well as a variety of local and systemic infections such as endophthalmitis, endocarditis, meningitis, periodontitis, osteomyelitis, wound infections, and septicemia (12, 32). B. cereus can be readily isolated from food and agricultural products, as well as soil, vegetation, dust, and natural waters. It is regarded as one of the common organisms that impair the quality of dairy products (23, 25, 30, 34) . Its ubiquitous nature, combined with its ability to sporulate and grow at refrigeration temperature, make it difficult to control. B. cereus has been shown to be able to form biofilms on plastic, glass wool, and stainless steel (2, 26, 28), and the biofilm cells were more resistant than planktonic cells to chemical sanitizers (29). Biofilm accumulation in food processing environments can lead to decreased food quality and safety (20, 35, 36) , which impacts public health as well as the economy. In the present study, the role of PlcR in biofilm formation by B. cereus strain ATCC 14579 was investigated. PlcR, a pleiotropic regulator, is activated by a small diffusible peptide (PapR) that acts as a quorum-sensing effector (33). It controls the expression of a variety of genes, many of which encode potential virulence factors, including enterotoxins, hemolysins, phospholipases C, and proteases (1, 7, 15). We found that biofilm formation was enhanced under low nutrient conditions and was dependent on biosurfactant production, which was directly or indirectly repressed by PlcR. Biofilm formation. B. cereus ATCC 14579 and its ⌬plcR mutant (31) were grown in Luria-Bertani (LB) broth (Difco/ Becton Dickinson, Sparks, Md.) at 32°C and 200 rpm overnight to generate inoculum cultures. For the ⌬plcR mutant, kanamycin was added at a final concentration of 150 g/ml. Since nutrient availability is one of the major factors affecting biofilm formation, we compared the ability of the wild-type and mutant strains to develop biofilms in rich and low-nutrient media. Overnight cultures were adjusted to an optical density at 620 nm (OD 620 ) of 0.01 in LB or EPS, a low nutrient medium that contained 7 g of K 2 HPO 4 , 3 g of KH 2 PO 4 , 0.1 g of MgSO 4 · 7H 2 O, 0.01 g of CaCl 2 , 0.001 g of FeSO 4 , 0.1 g of NaCl, 1 g of glucose, and 0.125 g of yeast extract (Difco) per liter (11). Then, 2 ml was added to wells of polystyrene 24-well plates (Falcon/Becton Dickinson, Franklin Lakes, NJ), followed by incubation at 32°C and 50 rpm for 8 h. The total growth (OD 620 ) in each well was measured; planktonic bacteria were removed, and the wells were washed with distilled water and air dried. Biofilm cells were stained with 2 ml of 0.3% crystal violet for 10 min, washed with distilled water, and air dried. The crystal violet in the biofilm cells was solubilized with 2 ml of 70% ethanol, and the optical density at 590 nm (OD 590 ) was measured (13). The total growth of the wild type and the ⌬plcR mutant was similar in EPS and LB; however, biofilm formation in EPS by the ⌬plcR mutant was about four times higher (P Ͻ 0.05) than that by the wild-type strain (Fig. 1) . A dramatic decrease in biofilm development was observed in a rich medium such as LB. Recently, Auger et al. (2) also observed that LB did not support biofilm formation by B. cereus ATCC 14579. We therefore used EPS for subsequent experiments. To monitor biofilm development over time, overnight cultures were adjusted to an OD 620 of 0.01 in EPS, and 10 ml was added to 60-by-15-mm polystyrene petri dishes (Falcon), followed by incubation at 32°C and 50 rpm. At specific times, planktonic cells were removed, and biofilm cells were rinsed and air dried. For enhanced visualization of biofilm cell morphology and structure, cells were fixed with 2 ml of 1% glutaraldehyde, stained with 10 ml of acridine orange (0.025% in 0.026 M citric acid buffer [pH 6.6]; Sigma, St. Louis, Mo.), washed, and air dried. Biofilm cells were observed with an Olympus BH-2 microscope equipped for epifluorescence with an HB0100W mercury burner lamp, a 490-nm excitation filter, and a 515-nm barrier filter. At 6 h, very few cells of the wild type had attached to the bottom of the plate, whereas the ⌬plcR mutant started to develop a biofilm (Fig. 2) . The ⌬plcR mutant biofilm reached maximum density at 12 h, after which the cells started to detach. At 36 h, few cells remained on the plate. A modest increase in attachment was observed for the
doi:10.1128/aem.02182-06 fatcat:53vgwsqirrgavo3biow6gnwp3y