Bactericidal Effects on Geobacillus Stearothermophilus and Bacillus Cereus Microorganisms Angela D

Old Dominion University ODU Digital Commons

Dental Hygiene Faculty Publications Dental Hygiene

2009 Cold Plasma Technology: Bactericidal Effects on Geobacillus Stearothermophilus and Bacillus Cereus Microorganisms Angela D. Morris

Gayle B. McCombs Old Dominion University, [email protected]

Tamer Akan

Wayne L. Hynes Old Dominion University, [email protected]

Mounir Laroussi Old Dominion University, [email protected]

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Repository Citation Morris, Angela D.; McCombs, Gayle B.; Akan, Tamer; Hynes, Wayne L.; Laroussi, Mounir; and Tolle, Susan L., "Cold Plasma Technology: Bactericidal Effects on Geobacillus Stearothermophilus and Bacillus Cereus Microorganisms" (2009). Dental Hygiene Faculty Publications. 4. https://digitalcommons.odu.edu/dentalhygiene_fac_pubs/4 Original Publication Citation Morris, A. D., McCombs, G. B., Akan, T., Hynes, W., Laroussi, M., & Tolle, S. L. (2009). Cold plasma technology: Bactericidal effects on Geobacillus stearothermophilus and Bacillus cereus microorganisms. Journal of Dental Hygiene 83(2), 55-61.

This Article is brought to you for free and open access by the Dental Hygiene at ODU Digital Commons. It has been accepted for inclusion in Dental Hygiene Faculty Publications by an authorized administrator of ODU Digital Commons. For more information, please contact [email protected]. Authors Angela D. Morris, Gayle B. McCombs, Tamer Akan, Wayne L. Hynes, Mounir Laroussi, and Susan L. Tolle

This article is available at ODU Digital Commons: https://digitalcommons.odu.edu/dentalhygiene_fac_pubs/4 ResearchResearch Cold Plasma Technology: Bactericidal Effects on Geobacillus Stearothermophilus and Bacillus Cereus Microorganisms

Angela D. Morris, RDH, MS; Gayle B. McCombs, RDH, MS; Tamer Akan, PhD; Wayne Hynes, PhD; Mounir Laroussi, PhD; and Susan L. Tolle, RDH, MS

Introduction Abstract Low temperature atmospheric Introduction: Cold plasma, also known as Low Temperature Atmo- pressure plasma (LTAPP), also spheric Pressure Plasma (LTAPP) is a novel technology consisting of known as “cold” or nonthermal neutral and charged particles, including free radicals, which can be plasma, is an innovative technology used to destroy or inactivate microorganisms. Research has been con- that has the potential to destroy mi- ducted regarding the effect of cold plasma on gram-positive bacteria; croorganisms.1 Most of the visible however, there is limited research regarding its ability to inactivate the universe is made up of plasma, re- spore-formers Geobacillus stearothermophilus and Bacillus cereus. ferred to as the fourth state of mat- Purpose: The purpose of this study was to determine if cold plasma inac- ter, which is a partly ionized gas tivates G. stearothermophilus and B. cereus vegetative cells and spores. comprised of molecules, atoms, electrons and ions. The remaining Methods: Nine hundred eighty-one samples were included in this 1% of the visible universe consists study (762 experimental and 219 controls). Experimental samples were of three other states of matter: sol- exposed indirectly or directly to cold plasma, before plating and incu- ids, liquids and gases. Most plasmas bating for 16 hours. Control samples were not exposed to cold plasma. are very hot, with temperatures up The percentage-kill and cell number reductions were calculated from to thousands of degrees centigrade; Colony Forming Units (CFU). Data were statistically analyzed at the .05 however, cold plasma denotes tech- level using one-way ANOVA, Kruskal Wallis and Tukey’s tests. nology that operates at or near room Results: There was a statistically significant difference in the inacti- temperature. vation of G. stearothermophilus vegetative cells receiving indirect and Plasma technology can be thought direct exposure (p=0.0001 and p=0.0013, respectively), as well as for of as cold combustion producing B. cereus vegetative cells and spores (p=0.0001 for direct and indirect). highly reactive free radicals via There was no statistically significant difference in the inactivation of G. electron-neutral collisions instead stearothermophilus spores receiving indirect exposure (p=0.7208) or of using heat.2 Researchers have direct exposure (p=0.0835). investigated plasma technology for Conclusion: Results demonstrate that cold plasma exposure effec- a wide spectrum of biomedical and tively kills G. stearothermophilus vegetative cells and B. cereus vegeta- commercial applications including tive cells and spores; however, G. stearothermophilus spores were not decontamination of food and mili- significantly inactivated. tary equipment and sterilization of medical/dental instruments, as well Key Words: Cold plasma, Low Temperature Atmospheric Pressure as the killing of airborne and surface Plasma (LTAPP), bacteria, spores, sterilization pathogens.3 The development of an alternative to traditional sterilization methods that is safer, faster, and impact the health care profession Numerous studies have been con- more cost effective would have far- beyond sterilization purposes; in ducted investigating the effective- reaching implications for the dental particular, inactivating microorgan- ness of cold plasmas to inactivate and medical professions. Moreover, isms associated with oral diseases strains of bacteria, such as Bacillus cold plasma has the potential to and wound infections. atrophaeus (previously called Bacil-

Volume 83 Issue 2 Spring 2009 The Journal of Dental Hygiene 55 Figure 2. Direct Cold Plasma Plume

The purposes of this investiga- positive bacteria following expo- tion were to (1) evaluate the effec- sure, the bacteria remaining were tiveness of cold plasma in destroy- nonviable, suggesting that cold plas- ing G. stearothermophilus and B. ma inactivates the microorganisms cereus, (2) determine which type of without changing their structure. cold plasma exposure (indirect or G. stearothermophilus, a gram- direct) has the greatest kill and, (3) positive, aerobic, spore-forming assess the minimum time needed to microorganism, is extremely resis- achieve a statistically significant re- tant to high heat and pressure.16 This duction in the number of bacterial microorganism is commonly asso- colonies. ciated with spoiling liquid foods in vending machines, such as coffee, Figure 1. Indirect Cold Plasma and is incorporated on biological Chamber Review of the Literature indicator strips as a way to monitor sterilization methods.16 lus subtilis), Escherichia coli, and Sterilization and decontamination B. cereus, a gram-positive, aero- Staphylococcus aureus.4-7 However, are essential components of infection bic, spore-forming, microorganism, there is limited research regard- control within the dental and medi- was chosen because it is an oppor- ing the inactivation of Geobacillus cal communities. Ensuring that ster- tunistic pathogen which commonly stearothermophilus, formerly called ilization techniques are effective is a causes food poisoning and has also Bacillus stearothermophilus, and major concern to health care profes- been associated with periodontal Bacillus cereus. The two microor- sionals. Sterilization occurs when all disease and bacteremias.17-19 Cer- ganisms chosen for this study were microorganisms, spores and viruses tain strains of B. cereus produce selected because of their distinct are destroyed or inactivated.12-14 Cold enterotoxins and emetic toxins, re- differences. G. stearothermophilus plasma technology has the potential sulting in diarrhea and vomiting, is commonly found on biological to become a more cost effective and the classic characteristics of food indicator test strips to verify steam less time-consuming procedure, poisoning.19 According to Marsili or chemical vapor sterilization of re- as well as produce less toxic waste and colleagues, B. cereus microor- sistant microorganisms, whereas B. when compared to the traditional ganisms demonstrate susceptibility cereus is associated with food poi- types of sterilization, such as steam, to plasma treatment by being inac- soning; both are extremely resistant dry heat, ethylene oxide, or chemical tivated within 10 to 30 seconds of to heat at the spore stage.8,9 vapor methods.4,12,14 exposure.20 Moreover, when air was Cold plasma may be utilized to Cold plasma produces greater used as the gas for the plasma, a inactivate microorganisms via in- structurally damaging effects on greater inactivation of B. cereus oc- direct or direct methods. Indirect gram-negative bacteria, such as E. curred after 50 seconds of treatment or “remote” cold plasma exposure coli, than to gram-positive bacte- in comparison to using nitrogen or requires the bacteria to be placed ria.5 Gram-negative bacteria experi- carbon dioxide gas mixtures. Re- away from the plasma discharge; ence structural damage to the outer searchers postulate that this interac- therefore, the samples are placed membrane following exposure to tion may be due to the ozone and in an adjacent chamber (Figure 1). cold plasma, whereas more resistant free radicals that are produced in Conversely, direct cold plasma ex- gram-positive bacteria do not show the breakdown of the air gas, caus- posure occurs when the samples are the same degree of morphological ing inactivation of the B. cereus.20 placed directly under (within inch- effects.15 According to Laroussi and Exposing bacteria inoculated es of) the plasma plume discharge colleagues,5 even though structural on different types of media, such (Figure 2).4,10,11 damage was not observed in gram- as liquid suspension, glass slab, or

56 The Journal of Dental Hygiene Volume 83 Issue 2 Spring 2009 Table 1. Sample Distribution and Cold Plasma Treatment Exposure Times

Dependent Variables Independent Variables Total

Control Bacteria and State Indirect Exposure Direct Exposure Sample Size (No Exposure) G. stearothermophilus 1, 2, 4, 5, 6, 8, 10, 15, 20 and 0 seconds 15, 20, 25 and 30 minutes Vegetative 30 minutes 440 G. stearothermophilus 0 seconds 15, 20, 25 and 30 minutes 10, 20 and 30 minutes Spores Bacillus cereus 1, 2, 3, 4, 5, 10, 15, 20, 25 10, 20, 30, 40 and 50 seconds 0 seconds Vegetative and 30 minutes and 1, 2, 4, 6, 8 and 10 minutes 541 1, 2, 3, 4, 5, 10, 15, 20, 25 30 seconds and 1, 2, 3, 4 and Bacillus cereus Spores 0 seconds and 30 minutes 5 minutes

Total Sample Size 219 344 418 981 polypropylene, may also effect in- as “0 seconds exposure,” utilized G. tative cells and spores were exposed activation by cold plasma.4 Laroussi stearothermophilus and B. cereus to indirect cold plasma for 15, 20, 25 demonstrated that the “survivor vegetative cells and spores that did and 30 minutes, whereas B. cereus curves” of the microorganisms are not receive cold plasma exposure. vegetative cells and spores were ex- related to the culture medium. For Due to the exploratory nature of posed for 1, 2, 3, 4, 5, 10, 15, 20, 25 example, the D-value, or the time this study, various exposure times and 30 minutes (see Table 1). Fol- that was needed to destroy 90% of were evaluated for the indirect and lowing each exposure time, the cold the original concentration of B. at- direct plasma, resulting in a total plasma was turned off and the gases rophaeus on a glass slab, was much sample size of 762 exposed and within the chamber were evacuated shorter than the time required for in- 219 unexposed control samples (N= into a fume hood for 60 seconds. activation of the same bacteria in a 981). The indirect plasma chamber After treatment, the samples were liquid suspension.4 exposed 344 samples and the direct incubated for up to 18 hours, and The inactivation of resistant plasma device exposed 418 samples plates containing between 30 and gram-positive bacteria, such as B. (n=762). The bacteria were pur- 300 CFU were counted. atrophaeus, without causing signifi- chased from the American Type Cul- cant structural damage to the micro- ture Collection (ATCC) (G. stearo- Direct Plasma Exposure. Prior organism, suggests that cold plasma thermophilus ATCC 12980 and B. to direct cold plasma exposure, has the ability to kill without obvious cereus ATCC 14579). Various expo- the microorganisms were cultured morphological changes. Moreover, sure times were selected to evaluate overnight in TSB, diluted before exposing cold plasma to a variety of the time points at which noticeable 10 microliters of the culture was pi- materials used to manufacture dental kill occurred as assessed by counting petted onto a sterile glass slide. The and medical instruments may affect Colony Forming Units (CFU). glass slides were placed directly un- the amount of exposure time needed der the plasma discharge. G. stearo- to effectively destroy resistant mi- thermophilus vegetative cells were croorganisms such as G. stearother- Laboratory Procedures exposed for 1, 2, 4, 5, 6, 8, 10, 15, mophilus and B. cereus. 20, and 30 minutes; G. stearother- Indirect Plasma Exposure. Be- mophilus spores were exposed for fore exposure to cold plasma, the 10, 20, and 30 minutes; B. cereus Methods and Materials microorganisms were cultured in vegetative cells were exposed for trypticase soy broth (TSB), diluted 10, 20, 30, 40, and 50 seconds, as In the present study, the ex- and plated onto trypticase soy agar well as 1, 2, 4, 6, 8, and 10 minutes; perimental group consisted of G. (TSA) (Difco/Becton Dickinson and B. cereus spores were exposed stearothermophilus and B. cereus Laboratories, Sparks, MD 21152) for 30 seconds, 1, 2, 3, 4, and 5 min- vegetative cells (<18 hour culture) or Luria Bertani (LB) (Difco/Bec- utes (see Table 1). After exposure, and spores (48-hour culture) on ton Dickinson Laboratories, Sparks, the glass slide was rinsed with ster- agar or glass slides, then exposed to MD 21152) media for G. stearo- ile saline into a test tube, dilutions either indirect or direct cold plasma. thermophilus and B. cereus, respec- were made and the bacteria plated The control group, also referred to tively. G. stearothermophilus vege- on either TSA or LB media prior to

Volume 83 Issue 2 Spring 2009 The Journal of Dental Hygiene 57 200

180

160 160

140 140

120 120

100 100

80 Mean CFU 80 Mean CFU

60 60

40 40

20 20 0 (control)0 seconds 1 minute 2 minutes 4 minutes 5 minutes 6 minutes 8 minutes 10 minutes 15 minutes 20 minutes 30 minutes (control)0 seconds 10 minutes 20 minutes 30 minutes

(control)0 seconds 15 minutes 20 minutes 25 minutes 30 minutes (control)0 seconds 15 minutes 20 minutes 25 minutes 30 minutes 0

Vegetative Cells Spores Vegetative Cells Spores Figure 3. Mean CFU of G. stearothermo Figure 4. Mean CFU of G. stearothermophilus philus receiving indirect cold plasma receiving direct cold plasma incubation for 12-16 hours. Follow- concentration of cells (CFU/mL) (p-value of 0.7208 for indirect and ing incubation, CFU were counted. was also calculated for each bacteria 0.0835 for direct). and state, exposure type, and time. Data revealed a statistically sig- For data that were roughly nor- nificant kill of B. cereus vegetative Statistical Methods mally distributed, the parametric cells at all time points for indirect ex- test of one-way Analysis of Variance posure and starting at 50 seconds for Data were grouped for statistical (ANOVA) was used to determine direct exposure (p-value of 0.0001 analysis according to bacteria (G. the means and standard deviations, direct and indirect). Statistically sig- stearothermophilus or B. cereus), whereas the Kruskal Wallis test was nificant kill of B. cereus spores oc- bacteria state (vegetative or spores), used to analyze data that were not curred at all time points for indirect cold plasma exposure (direct or in- roughly normally distributed. One- exposure and beginning at 3 minutes direct) and cold plasma exposure way ANOVA and the Kruskal Wallis for direct cold plasma exposure (p- times (variable). Percentage kill test analyzed overall significance, value of 0.0001 for direct and in- was calculated by obtaining the total and the Tukey’s Studentized Range direct) (see Figures 3 - 6 for mean CFU for each bacteria, state, expo- (HSD) test determined which cold CFU and Table 2 for significance). sure type, and time and subtract- plasma treatment times were statis- ing this number from each control tically significant. Discussion group’s CFU, dividing this total by the control CFU and then multiply- Results This study was designed to eval- ing by 100 for the percentage value uate the bactericidal effect of cold [i.e., (control CFU – experimental Results demonstrate there was plasma on G. stearothermophilus CFU / control CFU) x 100% = per- a statistically significant kill of G. and B. cereus vegetative cells and centage kill]. Percentage kill is the stearothermophilus vegetative cells spores. The development of an al- proportion of colonies that were exposed in the indirect chamber ternative to traditional steriliza- killed via cold plasma exposure (ex- at all time points, as well as direct tion methods, such as cold plasma, perimental group) compared to the exposure at 10 minutes (p-value would have a positive impact within number of colonies in the control of 0.0001 for indirect and 0.0013 the medical and dental communities. group. The percentage kill provided for direct); however, there was not Furthermore, vegetative cells and the proportion of the bioburden of a statistically significant kill in G. spores were specifically tested to microorganisms that were effective- stearothermophilus spores exposed determine if differences occurred in ly inactivated by cold plasma.21 The to indirect or direct cold plasma the inactivation rates. Since spores

58 The Journal of Dental Hygiene Volume 83 Issue 2 Spring 2009 would increase its efficacy in killing 140 G. stearothermophilus spores. 120 Exposing bacteria on various types of media, other than agar or 100 glass slides, is recommended. Fu- ture studies are needed to compare 80 the type of media and amount of 60 time required for inactivation of G. Mean CFU stearothermophilus and B. cereus. 40 It has been suggested that the type of media does affect cold plasma 20 exposure times; however, this study

(0) (0) (0) (0) (0) (0) (0.08) (0.92) (0.08) (0.17) (0.17) (0) (0) (0) (0) (0) (0) 4

0 (control)0 seconds 1 minute 2 minutes 3 minutes 4 minutes 5 minutes 10 minutes 15 minutes 20 minutes 25 minutes 30 minutes (control)0 seconds 1 minute 2 minutes 3 minutes 4 minutes 5 minutes 10 minutes 15 minutes 20 minutes 25 minutes 30 minutes did not address this aspect. The indirect chamber exposed 4 samples (Petri dishes) at one time. Vegetative Spores Additional research should evalu- Cells ate variability of sample placement within the chamber. The researchers Figure 5. Mean CFU of B. cereus receiving indirect cold plasma monitored plate location within the chamber (front left, front right, back 250 left or back right); however, the results were not analyzed differentiat- ing between the locations. Addition- ally, a distance of 0.25 inches from 200 the direct plasma output to the glass side was utilized for each exposure. A recommendation for future stud- 150 ies would involve assessing the variability of direct exposure by using different distances between the cold Mean CFU 100 plasma output and the culture. This innovative technology holds commercial promise for a whole host of biomedical and industrial 50 applications. Cold plasma, which could be thought of as room-temperature sterilization, has the poten- (0.06) (0.17) (0.71)

0 (control)0 seconds 10 seconds 20 seconds 30 seconds 40 seconds 50 seconds 1 minute 2 minutes 4 minutes 6 minutes 8 minutes 10 minutes (control)0 seconds 30 seconds 1 minute 2 minutes 3 minutes 4 minutes 5 minutes tial to change the way we currently apply sterilization techniques. Po- tentially, cold plasma offers advan- Vegetative Cells Spores tages over traditional methods, such as being more cost effective and time efficient, and producing less Figure 6. Mean CFU of B. cereus receiving direct cold plasma toxic byproducts than, for instance, ethylene oxide. Plasma technology are more resistant than actively tion.16 These factors may contribute has far-reaching implications for dividing and growing vegetative to the difficulty experienced in kill- the development of an efficient and cells, it was anticipated that vegeta- ing G. stearothermophilus spores safer means of inactivating patho- tive cells would be inactivated at a using cold plasma and may provide genic microorganisms on hard sur- faster rate than spores.11,21,22 These suggestions as to why there were no faces and skin and in the air, as well findings suggest additional research statistically significant reductions in as within the oral cavity. Research- is needed to determine how to best CFU. Since G. stearothermophilus ers envision the implementation of destroy spores. spores were more resistant to cold a cold plasma device that can be G. stearothermophilus spores dem- plasma than B. cereus, future stud- used intraorally to inactivate car- onstrate extreme stability and require ies are required to determine if mod- iogenic and periodontal pathogens, high heat and pressure for inactiva- ifications to the cold plasma device in addition to a device that can be

Volume 83 Issue 2 Spring 2009 The Journal of Dental Hygiene 59 and Dayanand Naik for their assis- Table 2. Statistical Significance of Indirect and Direct tance in conducting the research. Cold Plasma Exposure This research supports “Health Bacteria State Chamber Significance Promotion/Disease Prevention” of the National Dental Hygiene Re- Geobacillus Direct .0013* search Agenda. Vegetative stearothermophilus Indirect .0001* Angela D. Morris, RDH, MS, is a Direct .0835 Spore clinical research associate, John- Indirect .7208 son & Johnson Consumer and Per- Bacillus cereus Direct .0001* sonal Products Worldwide, Division Vegetative Indirect .0001* of Johnson & Johnson Consumer Companies in Morris Plains, New Direct .0001* Spore Jersey. Indirect .0001* Gayle B. McCombs, RDH, MS, is as- * Denotes statistical significance less than or equal to .05 sociate professor and director, Den- tal Hygiene Research Center, School used for surface decontamination. thermophilus and B. cereus vegeta- of Dental Hygiene, Old Dominion The future of plasma technology tive cells and spores. Results dem- University in Norfolk, Virginia. is wide open and far reaching with onstrate that there is a statistically Tamer Akan, PhD, is professor, tremendous potential for state-of- significant reduction in G. stearo- Department of Physics, Eskisehir the-art biomedical and commercial thermophilus vegetative cells and Osmangazi University in Eskisehir, applications. B. cereus vegetative cells and spores Turkey. Despite the limitations of this exposed to cold plasma; however, study, the data support that cold there is no statistically significant Wayne Hynes, PhD, is professor, plasma is effective in killing G. reduction in G. stearothermophilus associate chair, and director, Bio- stearothermophilus vegetative cells, spores. Spores are difficult to inac- logical Sciences, Department of as well as B. cereus vegetative cells tivate; therefore, further analysis is Biological Sciences, Old Dominion and spores, for both direct and indi- needed to determine how to pene- University in Norfolk, Virginia. rect exposure, at various time inter- trate the protective layers by modifi- Mounir Laroussi, PhD, is associ- vals. However, data revealed there cations to the cold plasma devices. ate professor and director of Laser & was not a statistically significant kill Plasma Engineering Institute (LPEI), in G. stearothermophilus spores. Acknowledgements Department of Electrical and Com- puter Engineering, Old Dominion University in Norfolk, Virginia. Conclusion The authors thank the American Dental Hygienists’ Association In- Susan L. Tolle, RDH, MS is professor The present study examined the stitute for Oral Health for funding. and clinic director, School of Dental bactericidal effects of direct and The authors thank Shannon Hens- Hygiene, Old Dominion University indirect cold plasma on G. stearo- ley, Sudhakar Allah, Asma Begum, in Norfolk, Virginia.

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