Disinfectant, Soap or Probiotic Cleaning? Surface Microbiome Diversity and Biofilm Competitive Exclusion

This study extends probiotic cleaning research to a built environment. Through an eight-month cleaning trial, we compared the effect of three cleaning products (disinfectant, plain soap, and a probiotic cleaner containing a patented Bacillus spore consortium), and tap water as the control, on the resident microbiome of three common hospital surfaces (linoleum, ceramic, and stainless steel). Pathogens, Escherichia coli and Staphylococcus aureus, were deposited and desiccated, and competitive exclusion was assessed for each microbiome. Cell survival was shown to be an incomplete tool for measuring microbial competitive exclusion. Biofilm competition offered a fuller understanding of competitive dynamics. A test for culturable cell survival showed that both plain soap and probiotic cleaner regimes established a surface microbiome that outcompeted the two pathogens. A different picture emerged when observing biofilms with a deposited and desiccated GFP-labeled pathogen, Pseudomonas aeruginosa. Competitive exclusion was again demonstrated. On surfaces cleaned with disinfectant the pathogen outcompeted the microbiomes. On surfaces cleaned with plain soap, the microbiomes outcompeted the pathogen. However, on surfaces cleaned with probiotic cleaner, despite the exponentially higher surface microbial loads, the microbiome did not completely outcompete the pathogen. Thus, the standard culturable cell test for survival on a surface confirmed the competitive advantage that is typically reported for probiotic cleaners. However, observation of competition in biofilms showed that the more diverse microbiome (according to alpha and beta indices) established on a surface cleaned with plain soap had a better competitive advantage than the monoculture established by the probiotic cleaner. Therefore, microbial diversity appears to be as critical to the competitive exclusion principle as cell numbers. The study showed that both plain soap and probiotic cleaner fostered competitive exclusion far more effectively than disinfectant. Probiotic cleaners with microbial diversity could be worth considering for hospital cleaning.

A Bacillus Spore-Based Display System for Bioremediation of Atrazine

ABSTRACT Owing to human activities, a large number of organic chemicals, including petroleum products, industrial solvents, pesticides, herbicides (including atrazine [ATR]), and pharmaceuticals, contaminate soil and aquatic environments. Remediation of these pollutants by conventional approaches is both technically and economically challenging. Bacillus endospores are highly resistant to most physical assaults and are capable of long-term persistence in soil. Spores can be engineered to express, on their surface, important enzymes for bioremediation purposes. We have developed a Bacillus thuringiensis spore platform system that can display a high density of proteins on the spore surface. The spore surface-tethered enzymes exhibit enhanced activity and stability relative to free enzymes in soil and water environments. In this study, we evaluated a B. thuringiensis spore display platform as a bioremediation tool against ATR. The Pseudomonas sp. strain ADP atzA determinant, an ATR chlorohydrolase important to the detoxification of ATR, was expressed as a fusion protein linked to the attachment domain of the BclA spore surface nap layer protein and expressed in B. thuringiensis. Spores from this strain are decorated with AtzA N-terminally linked on the surface of the spores. The recombinant spores were assayed for ATR detoxification in liquid and soil environments, and enzyme kinetics and stability were assessed. We successfully demonstrated the utility of this spore-based enzyme display system to detoxify ATR in water and laboratory soil samples. IMPORTANCE Atrazine is one of the most widely applied herbicides in the U.S. midwestern states. The long environmental half-life of atrazine has contributed to the contamination of surface water and groundwater by atrazine and its chlorinated metabolites. The toxic properties of ATR have raised public health and ecological concerns. However, remediation of ATR by conventional approaches has proven to be costly and inefficient. We developed a novel B. thuringiensis spore platform system that is capable of long-term persistence in soil and can be engineered to surface express a high density of enzymes useful for bioremediation purposes. The enzymes are stably attached to the surface of the spore exosporium layer. The spore-based system will likely prove useful for remediation of other environmental pollutants as well.

Formulation of Bacillus and Azotobacter Consortia in Liquid Cultures: Preliminary Research on Microbes-Coated Urea

The spore-forming Bacillus and cysts forming Azotobacter are Plant Growth Promoting Rhizobacteria which has been used as biofertilizer in sustainable agriculture since they tolerant to dried soil. Drought resistant microbes will be useful to coat urea in order to reduce the lost of nitrogen. The objectives of this preliminary study were to study the effect of molasse based liquid media on the population of Bacillus spore and Azotobacter vegetative cell and to determine the composition of four bacterial species in liquid formula. In the first experiment The Bacillus subtilis, B. megaterium, A. chroococcum and A. vinelandii were grown separately in 1% cane molasses enriched with 0.1% NH4Cl. As control treatment, The Bacillus and Azotobacter were grown in Nutrient Broth and Ashby’s mannitol broth respectively. In the second experiment, different composition of said Bacillus and Azotobacter were grown in molasses based liquid media prior to count the spore and vegetative cell. The results showed that molasses-based media supported bacterial growth and initial ratio 1:1:1:1 of liquid inoculant was effective to increase bacterial growth. This experiment suggested that the use of organic based media was useful practice of liquid biofertilizer formulation for granule urea coating.