Bacterial metabolites from intra- and inter-species influencing thermotolerance: the case of Bacillus cereus and Geobacillus stearothermophilus.

Bacterial metabolites with communicative functions could provide protection against stress conditions to members of the same species. Yet, information remains limited about protection provided by metabolites in Bacillus cereus and inter-species. This study investigated the effect of extracellular compounds derived from heat shocked (HS) and non-HS cultures of B. cereus and Geobacillus stearothermophilus on the thermotolerance of non-HS vegetative and sporulating B. cereus. Cultures of B. cereus and G. stearothermophilus were subjected to HS (42 or 65 °C respectively for 30 min) or non-HS treatments. Cells and supernatants were separated, mixed in a combined array, and then exposed to 50 °C for 60 min and viable cells determined. For spores, D values (85 and 95 °C) were evaluated after 120 h. In most cases, supernatants from HS B. cereus cultures added to non-HS B. cereus cells caused their thermotolerance to increase (D 50 12.2-51.9) when compared to supernatants from non-HS cultures (D 50 7.4-21.7). While the addition of supernatants from HS and non-HS G. stearothermophilus cultures caused the thermotolerance of non-HS cells from B. cereus to decrease initially (D 50 3.7-7.1), a subsequent increase was detected in most cases (D 50 18-97.7). In most cases, supernatants from sporulating G. stearothermophilus added to sporulating cells of B. cereus caused the thermotolerance of B. cereus 4810 spores to decline, whereas that of B. cereus 14579 increased. This study clearly shows that metabolites in supernatants from either the same or different species (such as G. stearothermophilus) influence the thermotolerance of B. cereus.

A sterilization system using ultraviolet photochemical reactions based on nitrous oxide and oxygen gases.

Active oxygen species (AOS) generated under ultraviolet (UV) lamps can be applied for various industrial processes owing to extremely strong oxidative abilities. We have already reported on an application of the AOS for a sterilization process of microorganisms. Here, a sterilization method using active oxygen generated under ultraviolet (UV) lamps introducing nitrous oxide (N2O) and oxygen gases into a vacuum chamber was investigated. Nitrogen dioxide (NO2) gas was readily produced from N2O by UV photochemical reactions under the low-pressure mercury lamp and then used to sterilize medical devices. We compared the ability of the N2O gas to sterilize Geobacillus stearothermophilus spores with those of conventional methods. Successful sterilization of spores on various biological indicators was achieved within 60 min, not only in sterilization bags but also in a lumen device.

Study of the combined effect of electro-activated solutions and heat treatment on the destruction of spores of Clostridium sporogenes and Geobacillus stearothermophilus in model solution and vegetable puree.

The combined effect of heat treatment and electro-activated solution (EAS) on the heat resistance of spores of Clostridium sporogenes and Geobacillus stearothermophilus was assessed under various heating and exposure time combinations. The acid and neutral EAS showed the highest inhibitory activity, indicating that these solutions may be considered as strong sporicidal disinfectants. These EAS were able to cause a reduction of ≥6 log of spores of C. sporogenes at 60 °C in only 1 min of exposition. For G. stearothermophilus spores, a reduction of 4.5 log was observed at 60 °C in 1 min, while in 5 min, ≥7 log CFU/ml reduction was observed. Inoculated puree of pea and corn were used as a food matrix for the determination of the heat resistance of these spores during the treatments in glass capillaries. The inactivation kinetics of the spores was studied in an oil bath. Combined treatment by EAS and temperature demonstrated a significant decrease in the heat resistance of C. sporogenes. The D100°C in pea puree with NaCl solution was 66.86 min while with acid and neutral EAS it was reduced down to 3.97 and 2.19 min, respectively. The spore of G. stearothermophilus displayed higher heat resistance as confirmed by other similar studies. Its D130°C in pea puree showed a decrease from 1.45 min in NaCl solution down to 1.30 and 0.93 min for acid and neutral EAS, respectively. The differences between the spores of these species are attributable to their different sensitivities with respect to pH, Redox potential and oxygen.

Evaluation of high-intensity ultrasonication for the inactivation of endospores of 3 bacillus species in nonfat milk.

Endospores of Bacillus licheniformis [American Type Culture Collection (ATCC) 6634], Bacillus coagulans (ATCC 12245), and Geobacillus stearothermophilus (ATCC 15952) were spiked in sterile nonfat milk, and subjected to high intensity batch ultrasonication treatment at different amplitudes (80 or 100%) and durations (1 to 10 min). Increasing the amplitude from 80 to 100% did not result in enhanced inactivation of G. stearothermophilus endospores. However, an increase in the duration of ultrasonication from 1 to 10 min significantly increased the inactivation of endospores of all 3 species. About 48.96% of the G. stearothermophilus endospores were inactivated by ultrasonication alone, whereas ultrasonication and pasteurization combined increased the inactivation to 65.74%. Inactivation of endospores could be further enhanced to 75.32% by ultrasonication and higher heat (80 °C/1 min) combination. Endospores of B. licheniformis and B. coagulans were inactivated to a lesser extent compared with G. stearothermophilus spores. Ultrasonicated B. licheniformis endospores germinated in higher numbers when compared with untreated endospores resulting in their greater inactivation during the combined treatment. During microstructure imaging of ultrasonicated endospores, although no structural damage was noticed, they showed irregular shrinkage and wrinkles with surface coarseness. This may also have contributed to their reduced thermal resistance, in addition to sporulation.

Decontamination of sugar syrup by pulsed light.

The pulsed light produced by xenon flash lamps was applied to 65 to 67 °Brix sugar syrups artificially contaminated with suspensions of Saccharomyces cerevisiae and with spores of Bacillus subtilis, Geobacillus stearothermophilus, Alicyclobacillus acidoterrestris, and Aspergillus niger. The emitted pulsed light contained 18.5 % UV radiation. At least 3-log reductions of S. cerevisiae, B. subtilis, G. stearothermophilus, and A. acidoterrestris suspended in 3-mm-deep volumes of sugar syrup were obtained with a fluence of the incident pulsed light equal to or less than 1.8 J/cm(2), and the same results were obtained for B. subtilis and A. acidoterrestris suspended in 10-mm-deep volumes of sugar syrup. A. niger spores would require a more intense treatment; for instance, the maximal log reduction was close to 1 with a fluence of the incident pulsed light of 1.2 J/cm(2). A flowthrough reactor with a flow rate of 320 ml/min and a flow gap of 2.15 mm was designed for pulsed light treatment of sugar syrup. Using this device, a 3-log reduction of A. acidoterrestris spores was obtained with 3 to 4 pulses of incident pulsed light at 0.91 J/cm(2) per sugar syrup volume.

Relevant factors affecting microbial surface decontamination by pulsed light.

Pulsed Light (PL) uses intense flashes of white light rich in ultraviolet (UV) light for decontamination. A log-reduction higher than 5 was obtained in one flash and at fluences lower than 1.8J/cm(2) on spores of a range of spore-forming bacteria, of vegetative cells of non-spore-forming bacteria and on yeasts spread on agar media. Vegetative cells were more sensitive than spores. The inactivation by PL of Bacillus subtilis, B. atrophaeus, B. cereus, Geobacillus stearothermophilus, and Aspergillus niger spores sprayed on polystyrene was similar. The inactivation by PL of B. subtilis and A. niger spores sprayed on glass was slightly lower than on polystyrene. No alteration of the spore structures was detected by scanning electron microscopy for both PL treated B. subtilis and A. niger spores. The inactivation of spores of B. subtilis, B. atrophaeus, B. cereus and B. pumilus by PL or by continuous UV-C at identical fluences was not different, and was much higher by PL for A. niger spores. The increase in the input voltage of the lamps (which also increases the UV-C %) resulted in a higher inactivation. There was no correlation between the resistance to heat and the resistance to PL. The relative effect of UV-C radiations and light thermal energy on PL inactivation was discussed.

Lumen claims of the STERRAD 100NX sterilizer: testing performance limits when processing equipment containing long, narrow lumens.

BACKGROUND: According to manufacturers information, the STERRAD 100NX sterilizer-a low temperature H(2)O(2) gas plasma sterilizer-can adequately process single channel stainless steel lumens with an inside diameter of 0.7 mm or larger and a maximum length of 500 mm using standard cycle sterilizing conditions. The aim of this study was to qualify the performance of this H(2)O(2) gas plasma sterilizer under different experimental settings representing worst case conditions. METHODS: Inoculated carriers were placed at the midpoint position of specified lumens and then submitted to flex scope sterilizing conditions. To simulate insufficient cleaning or crystalline residues, we added organic and inorganic challenges to our inoculated carriers. RESULTS: For experiments done with unchallenged carriers, quantitative analysis reached a log(10) reduction rate of ≥5.71, whereas qualitative results showed no growth in 24 out of 30 biologic indicators tested using flex scope half cycle conditions. Any additional kind of challenge significantly impaired the sterilization outcome. CONCLUSION: The findings of our current study emphasize the importance of a thorough validated cleaning of medical devices as well as timing for cleaning and decontamination before being exposed to the H(2)O(2) sterilization process and, furthermore, the need for strict adherence to manufacturer's recommendations.

Strong and consistently synergistic inactivation of spores of spoilage-associated Bacillus and Geobacillus spp. by high pressure and heat compared with inactivation by heat alone.

The inactivation of spores of four low-acid food spoilage organisms by high pressure thermal (HPT) and thermal-only processing was compared on the basis of equivalent thermal lethality calculated at a reference temperature of 121.1°C (F(z)(121.1)(°)(C, 0.1 MPa or 600 MPa)) and characterized as synergistic, not different or protective. In addition, the relative resistances of spores of the different spoilage microorganisms to HPT processing were compared. Processing was performed and inactivation was compared in both laboratory and pilot scale systems and in model (diluted) and actual food products. Where statistical comparisons could be made, at least 4 times and up to around 190 times more inactivation (log(10) reduction/minute at F(T)(z)(121.1)(°)(C)) of spores of Bacillus amyloliquefaciens, Bacillus sporothermodurans, and Geobacillus stearothermophilus was achieved using HPT, indicating a strong synergistic effect of high pressure and heat. Bacillus coagulans spores were also synergistically inactivated in diluted and undiluted Bolognese sauce but were protected by pressure against thermal inactivation in undiluted cream sauce. Irrespective of the response characterization, B. coagulans and B. sporothermodurans were identified as the most HPT-resistant isolates in the pilot scale and laboratory scale studies, respectively, and G. stearothermophilus as the least in both studies and all products. This is the first study to comprehensively quantitatively characterize the responses of a range of spores of spoilage microorganisms as synergistic (or otherwise) using an integrated thermal-lethality approach (F(T)(z)). The use of the F(T)(z) approach is ultimately important for the translation of commercial minimum microbiologically safe and stable thermal processes to HPT processes.

The dependence of the sporicidal effects on the power and pressure of RF-generated plasma processes.

The sporicidal effect of 20 different radio-frequency plasma processes produced by combining five different gas mixtures [O(2), Ar/H(2) (50/50%), Ar/H(2) (5/95%), O(2)/H(2) (50/50%), O(2)/H(2) (95/5%)] with four power/pressure settings were tested. Sporicidal effects of oxygen-containing plasmas were dependent on power at low pressure settings but not at high pressure settings. In the absence of oxygen no power dependency was observed at either high or low pressure settings. Survivor curves obtained with the use of nonoxygen plasmas typically had a tailing tendency. Only a mixture-optimized Ar/H(2) (15/85%) plasma process was not encumbered by tailing, and produced a decimal reduction time (D value) below 2 min for Bacillus stearothermophilus spores. Scanning electron microscopy showed that a CF(4)/O(2) plasma did more damage to the substrate than the 15/85% Ar/H(2) plasma. The present results indicate that UV irradiation inactivation is swift and power and pressure independent. Additionally, it is produced at low energy. However, it is not complete. Inactivation through etching is highly power and pressure dependent; finally, inactivation by photodesorption is moderately power and pressure dependent. A sterilization process relying on this mechanism is very advantageous because it combines a highly sporicidal effect with low substrate damage.

Optimization of a RF-generated CF4/O2 gas plasma sterilization process.

A sterilization process with the use of RF-generated (13.56 MHz) CF(4)/O(2) gas plasma was optimized in regards to power, flow rate, exposure time, and RF-system type. The dependency of the sporicidal effect on the spore inoculum positioning in the chamber of the RF systems was also investigated. Dried Bacillus stearothermophilus ATCC 7953 endospores were used as test organisms. The treatments were evaluated on the basis of survival curves and corresponding D values. The only parameter found to affect the sterilization process was the power of the RF system. Higher power resulted in higher kill. Finally, when the samples were placed more than 3-8 cm away from a centrally placed electrode in System 2, the sporicidal effect was reduced. The results are discussed and compared to results from the present literature. The RF excitation source is evaluated to be more appropriate for sterilization processes than the MW source.

Assessment of microwave sterilization of foods using intrinsic chemical markers.

The uniformity of microwave processing was investigated by measuring the formation of intrinsic chemical markers in disc-shaped and cylindrically-shaped whey protein gel model systems. These markers are formed as a result of thermally induced reactions of sugar and protein precursors. They were measured in samples placed in a pressurizable Teflon vessel and microwave heated to different peak temperatures using different power levels. Heating uniformity was mapped by sectioning the sample and analyzing for markers. The destruction of B. stearothermophilus spores in alginate beads was correlated with marker formation. The results show that the markers can be used to assess sterility and spatial time-temperature distributions in solid food samples.

Comparative evaluation of the antibacterial effects of intracanal Nd:YAG laser irradiation: an in vitro study.

The purpose of this study was to determine if the Nd:YAG laser was capable of disinfecting contaminated root canals in vitro. Eighty canals of extracted single-rooted teeth were prepared to size 35 K-file. The teeth were sterilized with ethylene oxide gas and then inoculated with Bacillus stearothermophilus. Eight groups were treated as follows: 1, sterility control; 2, positive control, no treatment; 3, hand instrumentation with sterile water filling chamber; 4, hand instrumentation with NaOCl (5.25%) filling chamber; 5, ultrasonic instrumentation with sterile water; 6, ultrasonic instrumentation with NaOCl; 7, laser exposure with sterile water; and 8, laser exposure with NaOCI. All instrumentation times were 1 min. Teeth were then flushed with 1.0 ml of sterile water onto blood agar plates, and the number of colony forming units were determined. Results indicate that: (i) all groups treated with NaOCI effectively inhibited the growth of B. stearothermophilus; (ii) hand and ultrasonic instrumentation with sterile water did not effectively disinfect root canals; and (iii) the Nd:YAG laser was effective in inhibiting the growth of B. stearothermophilus. The effects of the Nd:YAG laser on other bacteria more commonly found in root canal systems should be evaluated.

Evaluation of the antibacterial effects of intracanal Nd:YAG laser irradiation.

Fifty canals of extracted single-rooted teeth were prepared to a size 50 master apical file, sterilized in ethylene oxide, and inoculated with a known quantity of Bacillus stearothermophilus spores. Five groups of 10 canals each were used. The control group received no treatment. The four treatment groups were exposed to pulsed Nd:YAG laser radiation or 0.5% NaOCl alone and in combination. The root canals were flushed with sterile distilled water to recover spores, and serial dilutions were incubated on blood agar and the number of colony-forming units recovered was determined. Analysis of the data indicated a 2-log reduction in colony-forming units among the four treatment groups as compared with the controls; however, no significant differences were observed among the treatment groups. In none of the treatment groups were the root canals sterilized.

Comparative cell targeting in vitro using the CO2 laser.

In keeping with current interest in bactericidal effects of lasers to treat localized infection, we examined the effect of CO2 laser light on three bacterial species. Cell monolayers were placed atop filters minimizing the presence of extracellular water. Samples were exposed to irradiance values between 0 and 520 W/cm2 for 0.1 s. Cell survival was determined by colony counts. There was logarithmic killing of the bacteria with increasing irradiance; 90% kill was effected at 180 W/cm2 for Escherichia coli, 350 W/cm2 for Streptococcus mutans, and 250 W/cm2 for Bacillus stearothermophilus. However, standard deviations suggest no significant difference between cell types. Our results show the bactericidal effect of the CO2 laser with no remarkable difference between Gram types. However, substantial cell kill was achieved at relatively low irradiance. Whereas selectivity of damage may not be afforded by the CO2 laser, it may minimize collateral damage.

[Effect of microwaves on the vegetative and spore forms of Bacillus stearothermophilus]. / Azione delle microonde su forme vegetative e spore di Bacillus stearothermophilus.

Microwave irradiation provides a rapid and effective method for sterilization of stainless steel scalpel blades or cover glasses contaminated by B. stearothermophilus. A study by SEM of vegetative forms showed that microwave irradiation induce a progressive series of alterations and finally the complete destruction of the microorganism. On the contrary there were no significant morphological variations of the spores after lethal irradiation by microwaves.

Dynamic ultraviolet sterilization of different implant types.

This paper investigates the use of the dynamic ultraviolet sterilization process with various dental implants, stainless steel orthopedic cortical bone screws, and polysulfone polymer healing caps. These biomaterials were inoculated with the spores of Bacillus subtilis and Bacillus stearothermophilus. They were then exposed to dynamic ultraviolet radiation in the chamber of a BUD Ultraviolet Device. Samples were incubated in trypticase soy broth at 37 degrees C and 56 degrees C, and they were subcultured onto an enriched agar medium. Results indicate that 16 seconds of dynamic ultraviolet radiation is effective in sterilizing these materials. This is significantly less time than other sterilization techniques presently used.

Effect of mercaptoethylamine on DNA degradation in thermophilic bacteria bac. Stearothermophilus exposed to gamma-, UV-radiation or methylnitrosourea.

The effect of mercaptoethylamine (MEA) on degradation of DNA in thermophilic bacteria Bac. stear. exposed to gamma-, UV-rays or methylnitrosourea (MNU) was studied. Using centrifugation on alkaline and neutral sucrose gradients, it was shown that MEA inhibits the accumulation of breaks in the DNA of Bac. stear. It also lowers the level of DNA degradation in toluene-treated cells of Bac. stear. under the action of the intrinsic nuclease, reduces the activity of the endonuclease specific for apurinic DNA, as well as that of S1-nuclease and DNase-I in vitro. The inhibition in the accumulation of DNA breaks is assumed to be due to a decrease of the endonuclease activity in the cells of thermophilic bacteria.

[Effect of MEA on DNA degradation in permeable irradiated Bac. stearothermophilus cells]. / Vliianie MEA na degradatsiiu DNK v pronitsaemykh obluchennykh kletkakh Bac. stearothermophilus.

It was shown that DNA-degrading activity of permeable, intact and gamma-irradiated cells of Bac. stearothermophilus decreased under the effect of beta-mercaptoethylamine (MEA). MEA decreased also a DNAase activity, in a crude acellular extract of Bac. stearothermophilus, and activities of S1-nuclease and DNAase I. The data obtained prompt an assumption that MEA has an inhibitory action on the activity of endonucleases irrespective of their substrate specificity.

[Effect of MEA on the degradation of thermally-damaged DNA in gamma-irradiated bacterial cells]. / Vliianie MEA na degradatsiiu DNK, soderzhashchuiu termopovrezhdeniia, v gamma-obluchennykh kletkakh bakterii.

It was shown that the radioprotective action of beta-mercaptoethylamine on gamma-irradiated Bacillus stearothermophilus, with regard to survival and diminution of DNA breaks, depended upon the temperature of incubation and the presence of thermal lesions in DNA. beta-Mercaptoethylamine decreased the number of both alkali-labile sites and true single-strand DNA breaks in cells of Bacillus stearothermophilus. beta-Mercaptoethylamine decreased the number of alkali--labile sites and true single-strand breaks in DNA of Escherichia coli cells heated at 52 degrees C.

[Effect of MEA on the accumulation of DNA breaks in Bac. stearothermophilus irradiated with gamma and ultraviolet rays and treated with nitrosomethylurea]. / Vliianie MEA na nakoplenie razryvov v DNK Bac. stearothermophilus, obluchennykh gamma-, UF-radiatsiei i obrabotannykh nitrozometilmochevinoi.

beta-Mercaptoethylamine (MEA) decreased the accumulation of enzymatic single- and double-strand breaks in DNA of thermophilic bacteria exposed to gamma- and UV-radiation and treated with N-nitroso-N-methylurea. The protective effect of MEA, as registered according to accumulation of single-strand and double-strand breaks in DNA of Bac. stearothermophilus immediately after irradiation and after 30 min postirradiation incubation, was similar.