Inactivation of chemical and heat-resistant spores of Bacillus and Geobacillus by nitrogen cold atmospheric plasma evokes distinct changes in morphology and integrity of spores.

Bacterial spores are resistant to severe conditions and form a challenge to eradicate from food or food packaging material. Cold atmospheric plasma (CAP) treatment is receiving more attention as potential sterilization method at relatively mild conditions but the exact mechanism of inactivation is still not fully understood. In this study, the biocidal effect by nitrogen CAP was determined for chemical (hypochlorite and hydrogen peroxide), physical (UV) and heat-resistant spores. The three different sporeformers used are Bacillus cereus a food-borne pathogen, and Bacillus atrophaeus and Geobacillus stearothermophilus that are used as biological indicators for validation of chemical sterilization and thermal processes, respectively. The different spores showed variation in their degree of inactivation by applied heat, hypochlorite, hydrogen peroxide, and UV treatments, whereas similar inactivation results were obtained with the different spores treated with nitrogen CAP. G. stearothermophilus spores displayed high resistance to heat, hypochlorite, hydrogen peroxide, while for UV treatment B. atrophaeus spores are most tolerant. Scanning electron microscopy analysis revealed distinct morphological changes for nitrogen CAP-treated B. cereus spores including etching effects and the appearance of rough spore surfaces, whereas morphology of spores treated with heat or disinfectants showed no such changes. Moreover, microscopy analysis revealed CAP-exposed B. cereus spores to turn phase grey conceivably because of water influx indicating damage of the spores, a phenomenon that was not observed for non-treated spores. In addition, data are supplied that exclude UV radiation as determinant of antimicrobial activity of nitrogen CAP. Overall, this study shows that nitrogen CAP treatment has a biocidal effect on selected Bacillus and Geobacillus spores associated with alterations in spore surface morphology and loss of spore integrity.

Effect of NaOH (caustic wash) on the viability, surface characteristics and adhesion of spores of a Geobacillus sp. isolated from a milk powder production line.

AIM: To investigate the viability, surface characteristics and ability of spores of a Geobacillus sp. isolated from a milk powder production line to adhere to stainless steel surfaces before and after a caustic (NaOH) wash used in clean-in-place regimes. METHODS AND RESULTS: Exposing sessile spores to 1% NaOH at 65°C for 30min decreased spore viability by two orders of magnitude. The zeta potential of the caustic treated spores decreased from -20 to -32 mV and they became more hydrophobic. Transmission electron microscopy revealed that caustic treated spores contained breaks in their spore coat. Under flow conditions, caustic treated spores suspended in 0·1 mol l(-1) KCl were shown to attach to stainless steel in significantly greater numbers (4·6 log(10) CFU cm(-2) ) than untreated spores (3·6 log(10) CFU cm(-2) ). CONCLUSIONS: This research suggests that spores surviving a caustic wash will have a greater propensity to attach to stainless steel surfaces. SIGNIFICANCE OF STUDY: The practice of recycling caustic wash solutions may increase the risk of contaminating dairy processing surfaces with spores.

Characterization of spore surfaces from a Geobacillus sp. isolate by pH dependence of surface charge and infrared spectra.

AIMS: The surfaces of spores from a Geobacillus sp. isolated from a milk powder production line were examined to obtain fundamental information relevant to bacterial spore adhesion to materials. MATERIALS AND RESULTS: The surfaces of spores were characterized using transmission electron microscopy and infrared spectroscopy. Thin sections of spores stained with ruthenium red revealed an exosporium with a hair-like nap around the spores. Attenuated total reflection infrared spectra of the spores exposed to different pH solutions on a ZnSe prism revealed that pH-sensitive carboxyl and phosphodiester groups associated with proteins and polysaccharides contributed to the spore's negative charge which was revealed by our previous zeta potential measurements on the spores. Lowering the pH to the isoelectric point of spores resulted in an increase in intensity of all spectral bands, indicating that the spores moved closer to the zinc selenide (ZnSe) surface as the charged surface groups were neutralized and the spore surface polymers compressed. The attachment of spores to stainless steel was threefold higher at pH 3 compared with pH 7. CONCLUSIONS: This research showed that spore attachment to surfaces is influenced by electrostatic interactions, surface polymer conformation and associated steric interactions. SIGNIFICANCE AND IMPACT OF THE STUDY: The adhesion of thermophilic spores is largely controlled by functional groups of surface polymers and polymer conformation.

Geobacillin 26 - high molecular weight bacteriocin from a thermophilic bacterium.

Bacteriocins are ribosomally synthesized peptides/proteins produced by bacteria. These compounds have antibacterial activity against other bacteria that are usually closely related to the producer strain. Here we describe bacteriocin geobacillin 26 from a thermophilic Gram-positive bacterium Geobacillus stearothermophilus 15. We have purified native bacteriocin, determined its amino acid sequence and heterologously expressed in Gram-negative Escherichia coli. Geobacillin 26 is a heat-labile, high molecular weight antibacterial protein belonging to class III bacteriocins. It has a narrow antibacterial spectrum against other thermophilic bacteria. Our study suggests that this bacteriocin is not a cell wall hydrolyzing enzyme as most of high molecular weight bacteriocins. In addition, geobacillin 26 has no amino acid sequence similarities to other known function proteins. No other class III bacteriocin from a thermophilic bacterium has been reported and well characterized before. Geobacillin 26 as a natural antibacterial agent has a great potential in industry where contamination with other thermophilic bacteria is unwanted. Moreover, this study may prompt to disclose more novel geobacillin 26-like antibacterial proteins, which could find their applications in food industry or medicine.

Characterization of the newly isolated Geobacillus sp. T1, the efficient cellulase-producer on untreated barley and wheat straws.

A thermophile cellulase-producing bacterium was isolated and identified as closely related to Geobacillus subterraneus. The strain, named Geobacillus sp. T1, was able to grow and produce cellulase on cellobiose, microcrystalline cellulose, carboxymethylcellulose (CMC), barley straw, wheat straw and Whatman No. 1 filter paper. However, barley and wheat straws were significantly better substrates for cellulase production. When Geobacillus sp. T1 was cultivated in the presence of 0.5% barley straw, 0.1% Tween 80 and pH 6.5 at 50°C, the maximum level of free cellulase up to 143.50 U/mL was produced after 24h. This cellulase (≈ 54 kDa) was most active at pH 6.5 and 70°C. The enzyme in citrate phosphate buffer (10mM) was stable at 60°C for at least 1h. Geobacillus sp. T1 with efficient growth and cellulase production on straws seems a potential candidate for conversion of agricultural biomass to fuels.