The effect of NaCl, pH, and phosphate on biofilm formation and exopolysaccharide production by high biofilm producers of Bacillus strains.

Biofilm formation is an effective survival strategy of plant-associated microorganisms in hostile environments, so the application of biofilm-forming and exopolysaccharide (EPS)-producing beneficial microbes to plants has received more attention in recent years. This study examined the ability of biofilm and EPS production of Bacillus subtilis and Bacillus thuringiensis strains under different NaCl concentrations (0, 50, 100, 200, and 400 mmol/L), pH values (5.5, 6.5, 7.5, and 8.5), and phosphate levels (0, 25, 50, and 100 mmol/L at 0 and 400 mmol/L NaCl). B. subtilis BS2 and B. thuringiensis BS6/BS7 strains significantly increased biofilm formation in a similar pattern to EPS production under salt stress. B. subtilis BS2/BS3 enhanced biofilm production at slightly acidic pH with a lower EPS production but the other strains formed considerably more amount of biofilm and EPS at alkaline pH. Interestingly, higher levels of phosphate substantially decreased biofilm and EPS production at 0 mmol/L NaCl but increased biofilm formation at 400 mmol/L salt concentration. Overall, contrary to phosphate, salt and pH differently influenced biofilm and EPS production by Bacillus strains. EPS production contributed to biofilm formation to some extent under all the conditions tested. Some Bacillus strains produced more abundant biofilm under salt and pH stress, indicating their potential to form in vivo biofilms in rhizosphere and on plants, particularly under unfavorable conditions.

Bacillus strains exhibit various plant growth promoting traits and their biofilm-forming capability correlates to their salt stress alleviation effect on maize seedlings.

Soil salinity interferes with plant growth and development. Bacillus genus has been used to increase the growth and productivity of a wide variety of crops by alleviating the effects of salt stress. A total of thirty two Bacillus isolates were obtained from maize rhizosphere, and their plant growth-promoting (PGP) traits and biocontrol activities were tested. Bacillus isolates displayed varying degrees of PGP properties-the production of extracellular enzymes, indole acetic acid, hydrogen cyanide, phosphate solubilization, biofilm formation, and antifungal potential against several fungal pathogens. The phosphate-solubilizing isolates belong to B. safensis, B. thuringiensis, B. cereus, and B. megaterium species. Each Bacillus isolate demonstrated different levels of antifungal activity against the fungal pathogens tested. Biofilm production by some salt-tolerant isolates significantly increased at elevated levels of NaCl (p < 0.05). The strains B. safensis B24, B. halotolerans B7/B18, B. subtilis B26, and B. thuringiensis B10 significantly increased the length of root (by 32.7-38.2%) and shoot (by 19.5-29.8%) of maize (p < 0.05). Maize plants treated with some Bacillus strains displayed significantly greater chlorophyll content with an increase of 26.7-32.1% (p < 0.05). Among PGP properties, enhanced biofilm formation played a more important role in maize growth under higher salinity. These salt-tolerant biofilm-forming strains could be efficiently used as bio-inoculant for maize under salinity stress.

The effects of temperature, salt, and phosphate on biofilm and exopolysaccharide production by Azotobacter spp.

Inoculation of agriculturally important biofilms to plants under stress conditions has been of great interest in recent years. Therefore, in this study, biofilm- and exopolysaccharide (EPS)-forming ability of Azotobacter spp. was examined under different temperatures, NaCl concentrations, and phosphate levels. Azotobacter strains formed varying levels of biofilm and EPS depending on the tested factors. The pattern of biofilm formation was similar to that of EPS production under the conditions tested. Biofilm and EPS production at 28 °C was consistently higher than at either 18 or 37 °C. Biofilm production significantly increased in A. chroococcum strains (SBS2, SBS4, and SBS12) and A. vinelandii SBS6 with increasing salinity. Furthermore, a strong negative correlation was observed between biofilm or EPS production and increasing phosphate concentrations. Higher phosphate concentrations decreased biofilm and EPS production. In conclusion, contrary to temperature and phosphate effect, salinity differently affected biofilm and EPS production by Azotobacter strains.

Differential expression of vvhA and CPS operon allele 1 genes in Vibrio vulnificus under biofilm and planktonic conditions.

Examination of genes encoding for the virulence factors, hemolysin/cytolysin (vvhA) and capsular polysaccharide (CPS allele 1), during biofilm formation revealed that their expression was influenced by the maturity of the biofilm as well as by temperature. At 24 °C, expression of vvhA during biofilm formation was low between 4 and 12 h but increased 10-fold by 24 h to (5.1 × 104 ± 6.3 × 103mRNA copies/ml) as the biofilm matured. Compared to planktonic cells, expression of vvhA during biofilm formation at 24 °C was initially up-regulated at 4 h (1.07 ± 0.00-fold) but then was down-regulated almost four-fold during the intermediate and mature stages of biofilm formation. In contrast, vvhA expression at 37 °C was up-regulated almost four-fold in the early stages (4 and 6 h) of biofilm formation and remained two-fold up-regulated by 24 h even as the biofilm was deteriorating. CPS allele 1 expression at 24 °C during biofilm formation was up-regulated (1.50 ± 0.18-fold) during the initial attachment phase of the cells but was strongly down-regulated during the intermediate phases at 8 and 10 h (74.42 ± 42.16-fold and 453.76 ± 193.32-fold, respectively), indicating that capsular polysaccharide (CPS) is not important to intermediate biofilm architecture. Interestingly, as the biofilm matured by 24 h, expression of CPS allele 1 was again up-regulated (1.88 ± 1.07), showing that CPS plays a role in mature biofilm. At 37 °C, CPS allele 1 expression was significantly up-regulated (up to 105) during biofilm formation, indicating that the biofilm form of V. vulnificus may be preferred over the planktonic form in the human host.

The effects of temperature, pH, and iron on biofilm formation by clinical versus environmental strains of Vibrio vulnificus.

Due to the nature of Vibrio vulnificus infections (i.e., gastroenteritis and septicemia), only very few studies of a biofilm-associated form in this pathogen's life cycle have been conducted. We proposed that biofilm production by clinical strains of V. vulnificus would be higher than by environmental strains. Biofilm formation by clinical and environmental reference strains was tested under different temperatures (24, 30, and 37 °C), pH (5.5, 7.5, and 8.5) and iron concentrations (18, 30, 50, 100, and 200 µM). Biofilm production by clinical strains was consistently higher (p < 0.001) at 24 °C than by environmental strains. Higher biofilm production was observed at pH 5.5 by all strains. Growth rates were lowest at pH 5.5 for environmental strains but for clinical strains there were no differences at pH 5.5, 7.5, and 8.5, demonstrating a tolerance to acidic and alkaline conditions. There was a strong, direct correlation between iron concentration in the growth medium and biofilm production by all strains tested. The current study indicates that biofilm formation might be important for the survival of V. vulnificus in vivo as well as in the marine environment. With regard to temperature and pH, higher biofilm production appears to be a trait of clinical strains and could be considered a virulence determinant in V. vulnificus.

Quantitative PCR enumeration of vcgC and 16S rRNA type A and B genes as virulence indicators for environmental and clinical strains of Vibrio vulnificus in Galveston Bay oysters.

Oysters from a reef in Galveston Bay, Texas, USA, were screened for more virulent clinical strains versus less virulent environmental strains of Vibrio vulnificus using a combination of quantitative PCR assays for the virulence correlating gene (clinical variant, vcgC) and 16S rRNA types A and B (type A = environmental, type B = clinical). The combination of vcgC and 16S rRNA type B loci to determine clinical type strains was suitable, as indicated by the strong correlation (R2 = 0.98; p < 0.001) between these gene counts over time and their relative proportion (up to 93.8% and 94.3%, respectively) to vvhA genes used to quantify all strains of V. vulnificus. A strong seasonal shift of V. vulnificus strain types was observed. Environmental strains (16S rRNA type A) predominated from April to mid-June as salinities increased from 22 to 27 PSU (practical salinity unit) and temperatures rose 20 to 28 °C, with peak gene quantities of 16 812 ± 56 CFU/g. As temperatures increased to ≥30 °C from mid-June to September and salinities rose above 27 PSU, clinical strains (16S rRNA type B; vcgC) predominated with peak quantities 31 868 ± 287 and 32 360 ± 178 CFU/g, respectively.