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ORIGINAL RESEARCH
Inactivation of Escherichia coli and Staphylococcus aureus by Ultrasound Nüzhet Cenk Sesal, PhD, Özge Kekeç
Objectives—The aim of this study was to obtain valuable information about the effect of ultrasonic irradiation with a frequency of 30 kHz frequency and power of 100 W on the inactivation capability of two bacterial groups, namely, Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923, in physiologic water samples. Methods—Ultrasonic irradiation of bacterial samples with different populations of 5 × 103, 1.5 × 104, and 3 × 104 colony-forming units/mL was performed at a constant frequency with various treatment times. The specific energy (γ) values were calculated for these different concentrations of E coli and S aureus. The rate constant for ultrasonic inactivation was estimated in the linear region of a plot representing a survival ratio logarithm versus sonication time. Results—Although a significant death rate for E coli was observed with ultrasound treatment, in contrary to expectations, an increase in S aureus populations was observed. Conclusions—Considering the widespread use of ultrasound for sterilization of tools and equipment used in hospitals, the results obtained in this study indicate that ultrasonic irradiation is not a suitable method for the elimination of the major hospital pathogen S aureus. Key Words—bacterial inhibition; specific energy; ultrasonic irradiation
Received October 28, 2013, from the Department of Biology, Arts and Science Faculty, Marmara University, Istanbul, Turkey. Revision requested December 13, 2013. Revised manuscript accepted for publication December 26, 2013. We thank to Barış Gökalsın for assistance with manuscript preparation. This research was supported by the Marmara University Scientific Research Foundation (project BAPKO Fen-B130612-0222). Address correspondence to Nüzhet Cenk Sesal, PhD, Department of Biology, Arts and Science Faculty, Marmara University, Goztepe Campus, 34722 Istanbul, Turkey. E-mail: [email protected] Abbreviations
cfu, colony-forming units; UV, ultraviolet doi:10.7863/ultra.33.9.1663
I
nactivation of most problem-causing microorganisms is a serious matter for the food industry, the medical field, and the environment.1 Because of microorganism hazards to human health, research has focused on developing alternative inactivation methods. For this purpose, disinfection processes include the use of a wide spectrum of approaches, ranging from conventional strategies such as chemical (chlorination and ozonation) and thermal (pasteurization) treatments to nonconventional strategies such as ultraviolet (UV) light, electrical, mechanical, and ultrasound treatments.1–6 Current disinfection methods such as biocides, UV light, and heat treatment are not effective methods for achieving bacterial inactivation since some microorganisms become resistant to them. Various ultrasound tools have been developed to be used for disinfection as an adjunct to other techniques such as ozonation and UV irradiation.1 Sonication is a powerful disinfection method by itself. Ultrasonic irradiation can be achieved under ambient conditions of pressure and temperature without any chemical compounds, making it one of the most attractive methods. However, high ultrasonic intensities must be used to achieve 100% kill rates when solely using ultrasound.
©2014 by the American Institute of Ultrasound in Medicine | J Ultrasound Med 2014; 33:1663–1668 | 0278-4297 | www.aium.org
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Sesal and Kekeç—Inactivation of E coli and S aureus by Ultrasound
The microbial inactivation mechanism caused by ultrasound is explained in terms of physical and chemical effects generated during the ultrasonic irradiation, which indicates ultrasonic cavitation.1 Piyasena et al1 mentioned that it depends on the species, especially on the structure of the cell wall of the species. Gram-negative bacteria usually have a thin cell wall with an outer membrane, whereas gram-positive bacteria have a thicker cell wall and lack the outer membrane. Likewise, Ananta et al7 also stated that due to this distinguishing feature in the cell structure, ultrasonic biological effects diverge on gram-negative and -positive bacteria. Koda et al8 examined the inactivation of the gram-negative bacterium Escherichia coli and grampositive bacterium Streptococcus mutans by ultrasound at 500 kHz, finding deadly damage to bacteria caused by cavitational bubbles. In this study, E coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were chosen as gram-negative and -positive species, respectively. Escherichia coli is a gramnegative bacterium that can be observed in the lower intestine of warm-blooded organisms. Although most E coli strains are harmless, some cause food poisoning in humans.9 Staphylococcus aureus is a gram-positive bacterium, which can be observed in the human respiratory system and on the skin. It usually causes skin infections, respiratory disease, and food poisoning.10 The objective of this study was to investigate the effect of the ultrasonic irradiation time on the inactivation capability of the selected gram-negative and -positive bacteria in physiologic water samples with various bacterial concentrations.
and a nominal power output up to 100 W was used. Samples of 100 mL were placed in a double-walled jacketed glass container and were subjected to continuous ultrasonic irradiation emitted through a 7-mm-diameter tip at maximum nominal power. During the experiment, the temperature was controlled by a water bath coupled to a circulator and was kept constant at 15°C ± 1°C. The water level inside the container was 4 cm, and the horn (total length, 10 cm) was positioned in the middle of the container with its tip 2 cm above the bottom (Figure 1). The probespecific area of the sonotrode (A), ultrasonic power (σ), ultrasonic density (d), and specific energy (γ) were calculated by the following equations: (1)
2 A= πR ; 4
(2)
σ= P ; A
(3)
d= P ; V
(4)
γ = Pt , BCV
Figure 1. Experimental set up: on, standby indicator (1), cycle (2), amplitude (3), ultrasonic processor (4), power supply (5), horn (6), cold water (7), sample (8), bubble (9), start button (10), sonotrode (11), and ice (12).
Materials and Methods Sample Bacteria and Cultivation Escherichia coli ATCC 25922 and S aureus ATCC 25923 bacterial strains were supplied by the Microbiology Laboratory of the Yeditepe University Faculty of Medicine (Istanbul, Turkey) and were grown in Luria-Bertani medium (Conda, Madrid, Spain) overnight at 37°C. After an overnight incubation, the number of bacteria to be tested in experiments were adjusted to achieve 5 × 103, 1.5 × 104, and 3 × 104 colony-forming units (cfu)/mL via dilutions of McFarland standard compliant optical density of 625 nm using a UV spectrophotometer (DU 700; Beckman Coulter, Inc, Krefeld, Germany). Ultrasonic Irradiation A horn-type sonicator (UP100H; Hielscher GmbH, Teltow, Germany) operating at a fixed frequency of 30 kHz
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where R is the diameter of the sonotrode probe; P is power in watts; BC is the bacterial concentration; V is the sample volume; and t is time. According to Equation 4, the specific energy increases as the bacterial population decreases. Ultrasonic Irradiation on Bacteria To get more insight into the inactivation of E coli and S aureus in culture media, a series of experiments were performed by applying ultrasound at 30 kHz with different sonication times between 5 and 30 minutes. The bacterial suspensions with different specific energies were treated with ultrasound. After transfer of the strains to dishes, they were incubated overnight at 37°C and analyzed by a colony-counting method. Colonies were counted by a colony counter (Funker Gerber, Berlin, Germany). The results were reported as the average survival ratio evaluated from each run (N/N0), where N0 and N represent the numbers of colony-forming units before and after ultrasonic irradiation of the bacteria, respectively. Each result is the average of 3 experiments. For the graphic representation, the readings were normalized to an initial value of 100% cfu. All experimental results were compared with a control group. The rate constant for ultrasonic inactivation was estimated in the linear region of logarithmic bacterial populations against sonication time by the following equation: (5)
The specific energy (γ) values of the measured concentrations of E coli and S aureus are shown in Figure 2. It was observed that between 20 and 30 minutes, specific energy levels of the concentrations were at their maximum. The specific energy of the lowest bacterial concentration (5 × 103 cfu/mL) was approximately 8 times greater than 3 × 104 cfu/mL and nearly 4 times greater than 1.5 × 104 cfu/mL. The survival ratios of the concentrations of E coli and S aureus against irradiation time at the frequency of 30 kHz and power of 100 W are shown in Figures 3–5. For E coli, depending on the application time, a linear increase in the bacterial death rate was observed. The mortality rates were observed to decrease as the concentrations of bacterial samples increased. It can be seen that in the first 5 minutes for the samples at 5 × 103 and 1.5 × 104 cfu/mL, the bacterial population decreased logarithmically. As the Figure 2. Variations in the specific energy with sonication time for the indicated concentrations of bacteria calculated from Equation 4. The specific energy increased as the time increased. The bacterial concentrations were considered constant.
Log(N/N0)av = –2.303kut,
where ku is the ultrasound equation constant; t is time; N0 is the initial bacterial population; and N is the final bacterial population.11 Statistical Analyses Statistical analyses for time-dependent inhibition of bacterial concentrations were performed using groups that had equal variance with a 1-tailed t test in SPSS software for Windows (Microsoft Corporation, Redmond, WA). Three samples were used to average each point, and data with P < .05 were considered significant.
Figure 3. Effect of sonication time on logarithmic inhibition of E coli and S aureus at the concentration of 5 × 103 cfu/mL. Log(N/N0) indicates the logarithmic population change.
Results This study investigated the application of ultrasonic irradiation on different concentrations of bacteria. With an increase in the specific energy (γ), the prohibition of bacteria was expected to happen more effectively. The specific energy increases as the bacterial population decreases (Equation 4). By analyzing the specific energy, the effectiveness depending on time was recorded at concentrations of 5 × 103, 1.5 × 104, and 3 × 104 cfu/mL in descending order (Figure 2).
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treatment time increased, the death rate also increased. At the end of 30 minutes, the changes in the logarithmic values of death depending on time for the samples at 5 × 103, 1.5 × 104, and 3 × 104 cfu/mL were 3.70 to 2.00 log, 4.18 to 2.30 log, and 4.48 to 3.04 log, respectively. These findings represent a decrease in the logarithmic values of the bacterial populations (logN – logN0). All concentration results for E coli were statistically significant at P < .001 (Figures 3–5). No linear increases in death rates depending on application time were observed for S aureus. On the contrary, bacterial populations stayed stable with few significant increases. For the samples containing bacterial concentrations of 1.5 × 104 and 3 × 104 cfu/mL, the values peaked at 10 and 20 minutes. On the other hand, for the sample at 5 × 103 cfu/mL, a decrease at 10 minutes and increases at 5 and 20 minutes were noted. At the end of 30 minutes, all samples completed the study with higher populations, Figure 4. Effect of sonication time on logarithmic inhibition of E coli and S aureus at the concentration of 1.5 × 104 cfu/mL. Log(N/N0) indicates the logarithmic population change.
Figure 5. Effect of sonication time on logarithmic inhibition of E coli and S aureus at the concentration of 3 × 104 cfu/mL. Log(N/N0) indicates the logarithmic population change.
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and the logarithmic values calculated for the populations of 5 × 103, 1.5 × 104, and 3 × 104 cfu/mL were 3.75 log, 4.20 log, and 4.52 log, respectively. These findings also represent a decrease in the logarithmic values of the bacterial populations (logN – logN0). According to the t test, the significance results for the S aureus concentrations of 5 × 103, 1.5 × 104, and 3 × 104 cfu/mL were P < .05, P < .5, and P < .01, respectively (Figures 3–5).
Discussion In this study, we aimed to show the population changes in samples with 3 different concentrations of E coli and S aureus species, which are affected by ultrasound treatment with a fixed frequency of 30 kHz and power of 100 W. In addition, this study shows the effects of sonication time on logarithmic mortality rates. The term ultrasound indicates the application of sound waves with a frequency higher than the human hearing limit (>16 kHz).3 Generally, the frequencies used in ultrasound applications range from 20 to 500 kHz.11 To achieve decontamination of bacteria, usually high-power ultrasound is used at frequencies of around 20 kHz. To substitute or augment the common methods for water disinfection such as chlorination and UV light, ultrasound technology is proposed as an alternative method.3,4 The success rate of this application has led to ultrasound studies as a possible method for water disinfection. Using ultrasound technology for water disinfection is a very valuable application because of its environmentally friendly nature and its capability to disable and decompose clusters of pathogenic microorganisms.12,13 Piyasena et al1 explained that cavitation is the process by which microbubbles are formed and collapsed in a liquid. As a result of bubble collapse, localized heat of around 5500°C and pressure up to 100 MPa are generated, which results in local microbial inactivation. The main reason for microbial cell breakdown is the pressure changes caused by cavitation.14 Balasundaram and Harrison15 stated that “Cavitation is the formation, growth, and collapse of gas and vapor filled bubbles in a liquid. Irradiation of liquids with ultrasound can create acoustic cavitation; turbulent flow of liquids can create hydrodynamic cavitation.” The shock wave plays an important role in the inactivation of bacteria. The specific energy calculated for the bacteria increased proportionally to the sonication time, which indicated a population decrease in the samples. By examining the effect of specific energy on exopolysaccharides, Feng et al16 found that when specific energy increases, the release of exopolysaccharides also increases in the solution.
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Koda et al8 showed a comparison of the survival ratios of E coli and S mutans against irradiation time at a frequency of 500 kHz. Results indicate that increasing the ultrasonic power causes inactivation of bacteria, which decreases logarithmically with sonication time. Observing E coli treated with ultrasound revealed that its cytoplasmic membrane was mostly detached from the cell wall, resulting in a large amount of empty cell envelopes.8 In a study by Jatzwauk et al,17 the temperature of the liquid in the bath was increased from 24°C to 42°C at 60 minutes of sonication treatment. On the contrary, the bath in this study was kept cool with ice to remove the death effect on bacteria due to heat (Figure 1). Their study indicated that sonication at 35 kHz did not lead to increases in death rates for S aureus, Pseudomonas aeruginosa, and Candida albicans. These results show similarities with this study for S aureus. Herceg et al18 mentioned that gram-positive S aureus bacteria are more resistant to ultrasound treatment than gram-negative E coli bacteria at a frequency of 20 kHz. Gram-positive bacteria (S aureus) normally have a wider and more closely attached layer of peptidoglycan than gram-negative (E coli) bacteria, which seems to be a distinctive reason for the number of bacteria killed by ultrasound treatment.18 Unlike their results, this study recorded an increase in the S aureus concentration instead of a decrease. In addition, it is assumed that an increase in bacterial aggregates causes a decrease in the death rate, making the bacteria more difficult to kill. The application of ultrasound is shown to have an effect on declumping the bacteria, which leads to more planktonic bacteria in suspensions. Accordingly, the general effect of ultrasound treatment is a contest between declumping and exterminating the bacteria.13 It can be speculated that the reasons for the higher populations of the gram-positive S aureus samples at the end of the sonication in this study are the structure of the cell walls and the forming of clumps. Staphylococcus species live as grapelike clusters and form greater aggregates under stress conditions. Chu et al19 and Khanal et al20 evaluated the efficiency of ultrasound for bacterial disintegration with increasing sonication time. They found that around the 30th minute of application, breakup of most cell walls arose, and they concluded that an increased sonication time led to complete breakdown of the cell wall. According to our results, the logarithm of the survival ratio of E coli rapidly decreased with irradiation time. In this study, a comparison of the survival curves between E coli and S aureus was performed for the ultrasonic irradiation treatment depending on time and specific energy. The specific energy increases when the bacterial
J Ultrasound Med 2014; 33:1663–1668
population decreases due to inactivation. For the samples at 5 × 103, 1.5 × 104, and 3 × 104 cfu/mL, the specific energy depending on time went from highest to lowest, respectively. Moreover, the effectiveness of ultrasound treatment by the same source of irradiation is determined by the bacterial quantity, on the condition that the volumes of bacterial suspensions such as gram-negative E coli and gram-positive S aureus remain constant. In other words, ultrasound treatment is more effective when the bacterial density is lower (5 × 103 cfu/mL). In conclusion, the particular sonication specifications used in this study are anticipated to be efficient on gramnegative bacterial inactivation, especially if the sonication is applied for longer periods. For the gram-positive bacteria S aureus, the treatment causes the population to increase not only above the initial value but also above a control population that is not treated with ultrasound. Considering the widespread use of ultrasound for sterilization of tools and equipment in hospitals, this method might be ineffective for elimination of the major hospital pathogen S aureus. Nevertheless, extensive future research is required to scrutinize the effect of ultrasonic irradiation on microbial disruption at different specific energy inputs and longer sonication times.
References 1. 2.
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7.
8.
Piyasena P, Mohareb E, Mckellar RC. Inactivation of microbes using ultrasound: a review. Int J Food Microbiol 2003; 87:207–216. Schwartz T, Hoffmann S, Obst U. Formation of natural biofilms during chlorine dioxide and u.v. disinfection in a public drinking water distribution system. J Appl Microbiol 2003; 95:591–601. Gibson JH, Nien Yong DH, Farnood RR, Seto P. A literature review of ultrasound technology and its application in wastewater disinfection. Water Qual Res J Can 2008; 43:23–35. Hulsmans A, Joris K, Lambert N, et al. Evaluation of process parameters of ultrasonic treatment of bacterial suspensions in a pilot scale water disinfection system. Ultrason Sonochem 2010; 17:1004–1009. Madron MS, McClure SR, Griffith RW, Wang C. Absence of bactericidal effect of focused shock waves on an in-vitro biofilm model of an implant. Can J Vet Res 2012; 76:129–135. Jin X, Li Z, Xie L, Zhao Y, Wang T. Synergistic effect of ultrasonic pretreatment combined with UV irradiation for secondary effluent disinfection. Ultrason Sonochem 2013; 20:1384–1389. Ananta E, Voigt D, Zenker M, Heinz V, Knorr D. Cellular injuries upon exposure of Escherichia coli and Lactobacillus rhamnosus to high-intensity ultrasound. J Appl Microbiol 2005; 99:271–278. Koda S, Miyamoto M, Toma M, Matsuoka T, Maebayashi M. Inactivation of Escherichia coli and Streptococcus mutans by ultrasound at 500 kHz. Ultrason Sonochem 2009; 16:655–659.
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9. 10.
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Tarr PI, Gordon CA, Chandler WL. Shiga-toxin–producing Escherichia coli and haemolytic uraemic syndrome. Lancet 2005; 365:1073–1086. Kluytmans J, Van Belkum A, Verbrugh H. Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks. Clin Microbiol Rev 1997; 10:505–520. Thompson LH, Doraiswamy LK. Sonochemistry: science and engineering. Ind Eng Chem Res 1999; 38:1215–1249. Gogate PR, Kabadi AM. A review of applications of cavitation in biochemical engineering/biotechnology. Biochem Eng J 2009; 44:60–72. Joyce E, Phull SS, Lorimer JP, Mason TJ. The development and evaluation of ultrasound for the treatment of bacterial suspensions: a study of frequency, power and sonication time on cultured Bacillus species. Ultrason Sonochem 2003; 10:315–318. Alonzo AG. Microbial inactivation in cloudy apple juice by multi-frequency Dynashock power ultrasound. Ultrason Sonochem 2012; 19:346– 351. Balasundaram B, Harrison STL. Optimising orifice geometry for selective release of periplasmic products using cell disruption by hydrodynamic cavitation. Biochem Eng J 2011; 54:207–209. Feng L, Yan Y, Zhang C, Zhu H, Zhou Q. Effect of ultrasonic specific energy on waste activated sludge solubilization and enzyme activity. Afr J Biotechnol 2010; 9:1776–1782. Jatzwauk L, Schöne H, Pietsch H. How to improve instrument disinfection by ultrasound. J Hosp Infect 2001; 48(suppl A):S80–S83. Herceg Z, Anet Rèek J, Vesna L, Selma Mededovic T. The effect of high intensity ultrasound treatment on the amount of Staphylococcus aureusand Escherichia coli in milk. Food Technol Biotechnol 2012; 50:46–52. Chu CP, Chang BV, Liao GS, Jean DS, Lee DJ. Observations on changes in ultrasonically treated waste-activated sludge. Water Res 2001; 35:1038– 1046. Khanal SK, Isik H, Sung S, Avan Leeuwen J. Ultrasonic conditioning of waste activated sludge for enhanced aerobic digestion. Paper presented at: International Water Association Specialized Conference—Sustainable Sludge Management: State of the Art, Challenges and Perspectives; May 29–31 2006; Moscow, Russia.
J Ultrasound Med 2014; 33:1663–1668
ORIGINAL RESEARCH
Inactivation of Escherichia coli and Staphylococcus aureus by Ultrasound Nüzhet Cenk Sesal, PhD, Özge Kekeç
Objectives—The aim of this study was to obtain valuable information about the effect of ultrasonic irradiation with a frequency of 30 kHz frequency and power of 100 W on the inactivation capability of two bacterial groups, namely, Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923, in physiologic water samples. Methods—Ultrasonic irradiation of bacterial samples with different populations of 5 × 103, 1.5 × 104, and 3 × 104 colony-forming units/mL was performed at a constant frequency with various treatment times. The specific energy (γ) values were calculated for these different concentrations of E coli and S aureus. The rate constant for ultrasonic inactivation was estimated in the linear region of a plot representing a survival ratio logarithm versus sonication time. Results—Although a significant death rate for E coli was observed with ultrasound treatment, in contrary to expectations, an increase in S aureus populations was observed. Conclusions—Considering the widespread use of ultrasound for sterilization of tools and equipment used in hospitals, the results obtained in this study indicate that ultrasonic irradiation is not a suitable method for the elimination of the major hospital pathogen S aureus. Key Words—bacterial inhibition; specific energy; ultrasonic irradiation
Received October 28, 2013, from the Department of Biology, Arts and Science Faculty, Marmara University, Istanbul, Turkey. Revision requested December 13, 2013. Revised manuscript accepted for publication December 26, 2013. We thank to Barış Gökalsın for assistance with manuscript preparation. This research was supported by the Marmara University Scientific Research Foundation (project BAPKO Fen-B130612-0222). Address correspondence to Nüzhet Cenk Sesal, PhD, Department of Biology, Arts and Science Faculty, Marmara University, Goztepe Campus, 34722 Istanbul, Turkey. E-mail: [email protected] Abbreviations
cfu, colony-forming units; UV, ultraviolet doi:10.7863/ultra.33.9.1663
I
nactivation of most problem-causing microorganisms is a serious matter for the food industry, the medical field, and the environment.1 Because of microorganism hazards to human health, research has focused on developing alternative inactivation methods. For this purpose, disinfection processes include the use of a wide spectrum of approaches, ranging from conventional strategies such as chemical (chlorination and ozonation) and thermal (pasteurization) treatments to nonconventional strategies such as ultraviolet (UV) light, electrical, mechanical, and ultrasound treatments.1–6 Current disinfection methods such as biocides, UV light, and heat treatment are not effective methods for achieving bacterial inactivation since some microorganisms become resistant to them. Various ultrasound tools have been developed to be used for disinfection as an adjunct to other techniques such as ozonation and UV irradiation.1 Sonication is a powerful disinfection method by itself. Ultrasonic irradiation can be achieved under ambient conditions of pressure and temperature without any chemical compounds, making it one of the most attractive methods. However, high ultrasonic intensities must be used to achieve 100% kill rates when solely using ultrasound.
©2014 by the American Institute of Ultrasound in Medicine | J Ultrasound Med 2014; 33:1663–1668 | 0278-4297 | www.aium.org
3309jum1531-1668online_Layout 1 8/21/14 8:33 AM Page 1664
Sesal and Kekeç—Inactivation of E coli and S aureus by Ultrasound
The microbial inactivation mechanism caused by ultrasound is explained in terms of physical and chemical effects generated during the ultrasonic irradiation, which indicates ultrasonic cavitation.1 Piyasena et al1 mentioned that it depends on the species, especially on the structure of the cell wall of the species. Gram-negative bacteria usually have a thin cell wall with an outer membrane, whereas gram-positive bacteria have a thicker cell wall and lack the outer membrane. Likewise, Ananta et al7 also stated that due to this distinguishing feature in the cell structure, ultrasonic biological effects diverge on gram-negative and -positive bacteria. Koda et al8 examined the inactivation of the gram-negative bacterium Escherichia coli and grampositive bacterium Streptococcus mutans by ultrasound at 500 kHz, finding deadly damage to bacteria caused by cavitational bubbles. In this study, E coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were chosen as gram-negative and -positive species, respectively. Escherichia coli is a gramnegative bacterium that can be observed in the lower intestine of warm-blooded organisms. Although most E coli strains are harmless, some cause food poisoning in humans.9 Staphylococcus aureus is a gram-positive bacterium, which can be observed in the human respiratory system and on the skin. It usually causes skin infections, respiratory disease, and food poisoning.10 The objective of this study was to investigate the effect of the ultrasonic irradiation time on the inactivation capability of the selected gram-negative and -positive bacteria in physiologic water samples with various bacterial concentrations.
and a nominal power output up to 100 W was used. Samples of 100 mL were placed in a double-walled jacketed glass container and were subjected to continuous ultrasonic irradiation emitted through a 7-mm-diameter tip at maximum nominal power. During the experiment, the temperature was controlled by a water bath coupled to a circulator and was kept constant at 15°C ± 1°C. The water level inside the container was 4 cm, and the horn (total length, 10 cm) was positioned in the middle of the container with its tip 2 cm above the bottom (Figure 1). The probespecific area of the sonotrode (A), ultrasonic power (σ), ultrasonic density (d), and specific energy (γ) were calculated by the following equations: (1)
2 A= πR ; 4
(2)
σ= P ; A
(3)
d= P ; V
(4)
γ = Pt , BCV
Figure 1. Experimental set up: on, standby indicator (1), cycle (2), amplitude (3), ultrasonic processor (4), power supply (5), horn (6), cold water (7), sample (8), bubble (9), start button (10), sonotrode (11), and ice (12).
Materials and Methods Sample Bacteria and Cultivation Escherichia coli ATCC 25922 and S aureus ATCC 25923 bacterial strains were supplied by the Microbiology Laboratory of the Yeditepe University Faculty of Medicine (Istanbul, Turkey) and were grown in Luria-Bertani medium (Conda, Madrid, Spain) overnight at 37°C. After an overnight incubation, the number of bacteria to be tested in experiments were adjusted to achieve 5 × 103, 1.5 × 104, and 3 × 104 colony-forming units (cfu)/mL via dilutions of McFarland standard compliant optical density of 625 nm using a UV spectrophotometer (DU 700; Beckman Coulter, Inc, Krefeld, Germany). Ultrasonic Irradiation A horn-type sonicator (UP100H; Hielscher GmbH, Teltow, Germany) operating at a fixed frequency of 30 kHz
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where R is the diameter of the sonotrode probe; P is power in watts; BC is the bacterial concentration; V is the sample volume; and t is time. According to Equation 4, the specific energy increases as the bacterial population decreases. Ultrasonic Irradiation on Bacteria To get more insight into the inactivation of E coli and S aureus in culture media, a series of experiments were performed by applying ultrasound at 30 kHz with different sonication times between 5 and 30 minutes. The bacterial suspensions with different specific energies were treated with ultrasound. After transfer of the strains to dishes, they were incubated overnight at 37°C and analyzed by a colony-counting method. Colonies were counted by a colony counter (Funker Gerber, Berlin, Germany). The results were reported as the average survival ratio evaluated from each run (N/N0), where N0 and N represent the numbers of colony-forming units before and after ultrasonic irradiation of the bacteria, respectively. Each result is the average of 3 experiments. For the graphic representation, the readings were normalized to an initial value of 100% cfu. All experimental results were compared with a control group. The rate constant for ultrasonic inactivation was estimated in the linear region of logarithmic bacterial populations against sonication time by the following equation: (5)
The specific energy (γ) values of the measured concentrations of E coli and S aureus are shown in Figure 2. It was observed that between 20 and 30 minutes, specific energy levels of the concentrations were at their maximum. The specific energy of the lowest bacterial concentration (5 × 103 cfu/mL) was approximately 8 times greater than 3 × 104 cfu/mL and nearly 4 times greater than 1.5 × 104 cfu/mL. The survival ratios of the concentrations of E coli and S aureus against irradiation time at the frequency of 30 kHz and power of 100 W are shown in Figures 3–5. For E coli, depending on the application time, a linear increase in the bacterial death rate was observed. The mortality rates were observed to decrease as the concentrations of bacterial samples increased. It can be seen that in the first 5 minutes for the samples at 5 × 103 and 1.5 × 104 cfu/mL, the bacterial population decreased logarithmically. As the Figure 2. Variations in the specific energy with sonication time for the indicated concentrations of bacteria calculated from Equation 4. The specific energy increased as the time increased. The bacterial concentrations were considered constant.
Log(N/N0)av = –2.303kut,
where ku is the ultrasound equation constant; t is time; N0 is the initial bacterial population; and N is the final bacterial population.11 Statistical Analyses Statistical analyses for time-dependent inhibition of bacterial concentrations were performed using groups that had equal variance with a 1-tailed t test in SPSS software for Windows (Microsoft Corporation, Redmond, WA). Three samples were used to average each point, and data with P < .05 were considered significant.
Figure 3. Effect of sonication time on logarithmic inhibition of E coli and S aureus at the concentration of 5 × 103 cfu/mL. Log(N/N0) indicates the logarithmic population change.
Results This study investigated the application of ultrasonic irradiation on different concentrations of bacteria. With an increase in the specific energy (γ), the prohibition of bacteria was expected to happen more effectively. The specific energy increases as the bacterial population decreases (Equation 4). By analyzing the specific energy, the effectiveness depending on time was recorded at concentrations of 5 × 103, 1.5 × 104, and 3 × 104 cfu/mL in descending order (Figure 2).
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treatment time increased, the death rate also increased. At the end of 30 minutes, the changes in the logarithmic values of death depending on time for the samples at 5 × 103, 1.5 × 104, and 3 × 104 cfu/mL were 3.70 to 2.00 log, 4.18 to 2.30 log, and 4.48 to 3.04 log, respectively. These findings represent a decrease in the logarithmic values of the bacterial populations (logN – logN0). All concentration results for E coli were statistically significant at P < .001 (Figures 3–5). No linear increases in death rates depending on application time were observed for S aureus. On the contrary, bacterial populations stayed stable with few significant increases. For the samples containing bacterial concentrations of 1.5 × 104 and 3 × 104 cfu/mL, the values peaked at 10 and 20 minutes. On the other hand, for the sample at 5 × 103 cfu/mL, a decrease at 10 minutes and increases at 5 and 20 minutes were noted. At the end of 30 minutes, all samples completed the study with higher populations, Figure 4. Effect of sonication time on logarithmic inhibition of E coli and S aureus at the concentration of 1.5 × 104 cfu/mL. Log(N/N0) indicates the logarithmic population change.
Figure 5. Effect of sonication time on logarithmic inhibition of E coli and S aureus at the concentration of 3 × 104 cfu/mL. Log(N/N0) indicates the logarithmic population change.
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and the logarithmic values calculated for the populations of 5 × 103, 1.5 × 104, and 3 × 104 cfu/mL were 3.75 log, 4.20 log, and 4.52 log, respectively. These findings also represent a decrease in the logarithmic values of the bacterial populations (logN – logN0). According to the t test, the significance results for the S aureus concentrations of 5 × 103, 1.5 × 104, and 3 × 104 cfu/mL were P < .05, P < .5, and P < .01, respectively (Figures 3–5).
Discussion In this study, we aimed to show the population changes in samples with 3 different concentrations of E coli and S aureus species, which are affected by ultrasound treatment with a fixed frequency of 30 kHz and power of 100 W. In addition, this study shows the effects of sonication time on logarithmic mortality rates. The term ultrasound indicates the application of sound waves with a frequency higher than the human hearing limit (>16 kHz).3 Generally, the frequencies used in ultrasound applications range from 20 to 500 kHz.11 To achieve decontamination of bacteria, usually high-power ultrasound is used at frequencies of around 20 kHz. To substitute or augment the common methods for water disinfection such as chlorination and UV light, ultrasound technology is proposed as an alternative method.3,4 The success rate of this application has led to ultrasound studies as a possible method for water disinfection. Using ultrasound technology for water disinfection is a very valuable application because of its environmentally friendly nature and its capability to disable and decompose clusters of pathogenic microorganisms.12,13 Piyasena et al1 explained that cavitation is the process by which microbubbles are formed and collapsed in a liquid. As a result of bubble collapse, localized heat of around 5500°C and pressure up to 100 MPa are generated, which results in local microbial inactivation. The main reason for microbial cell breakdown is the pressure changes caused by cavitation.14 Balasundaram and Harrison15 stated that “Cavitation is the formation, growth, and collapse of gas and vapor filled bubbles in a liquid. Irradiation of liquids with ultrasound can create acoustic cavitation; turbulent flow of liquids can create hydrodynamic cavitation.” The shock wave plays an important role in the inactivation of bacteria. The specific energy calculated for the bacteria increased proportionally to the sonication time, which indicated a population decrease in the samples. By examining the effect of specific energy on exopolysaccharides, Feng et al16 found that when specific energy increases, the release of exopolysaccharides also increases in the solution.
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Koda et al8 showed a comparison of the survival ratios of E coli and S mutans against irradiation time at a frequency of 500 kHz. Results indicate that increasing the ultrasonic power causes inactivation of bacteria, which decreases logarithmically with sonication time. Observing E coli treated with ultrasound revealed that its cytoplasmic membrane was mostly detached from the cell wall, resulting in a large amount of empty cell envelopes.8 In a study by Jatzwauk et al,17 the temperature of the liquid in the bath was increased from 24°C to 42°C at 60 minutes of sonication treatment. On the contrary, the bath in this study was kept cool with ice to remove the death effect on bacteria due to heat (Figure 1). Their study indicated that sonication at 35 kHz did not lead to increases in death rates for S aureus, Pseudomonas aeruginosa, and Candida albicans. These results show similarities with this study for S aureus. Herceg et al18 mentioned that gram-positive S aureus bacteria are more resistant to ultrasound treatment than gram-negative E coli bacteria at a frequency of 20 kHz. Gram-positive bacteria (S aureus) normally have a wider and more closely attached layer of peptidoglycan than gram-negative (E coli) bacteria, which seems to be a distinctive reason for the number of bacteria killed by ultrasound treatment.18 Unlike their results, this study recorded an increase in the S aureus concentration instead of a decrease. In addition, it is assumed that an increase in bacterial aggregates causes a decrease in the death rate, making the bacteria more difficult to kill. The application of ultrasound is shown to have an effect on declumping the bacteria, which leads to more planktonic bacteria in suspensions. Accordingly, the general effect of ultrasound treatment is a contest between declumping and exterminating the bacteria.13 It can be speculated that the reasons for the higher populations of the gram-positive S aureus samples at the end of the sonication in this study are the structure of the cell walls and the forming of clumps. Staphylococcus species live as grapelike clusters and form greater aggregates under stress conditions. Chu et al19 and Khanal et al20 evaluated the efficiency of ultrasound for bacterial disintegration with increasing sonication time. They found that around the 30th minute of application, breakup of most cell walls arose, and they concluded that an increased sonication time led to complete breakdown of the cell wall. According to our results, the logarithm of the survival ratio of E coli rapidly decreased with irradiation time. In this study, a comparison of the survival curves between E coli and S aureus was performed for the ultrasonic irradiation treatment depending on time and specific energy. The specific energy increases when the bacterial
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population decreases due to inactivation. For the samples at 5 × 103, 1.5 × 104, and 3 × 104 cfu/mL, the specific energy depending on time went from highest to lowest, respectively. Moreover, the effectiveness of ultrasound treatment by the same source of irradiation is determined by the bacterial quantity, on the condition that the volumes of bacterial suspensions such as gram-negative E coli and gram-positive S aureus remain constant. In other words, ultrasound treatment is more effective when the bacterial density is lower (5 × 103 cfu/mL). In conclusion, the particular sonication specifications used in this study are anticipated to be efficient on gramnegative bacterial inactivation, especially if the sonication is applied for longer periods. For the gram-positive bacteria S aureus, the treatment causes the population to increase not only above the initial value but also above a control population that is not treated with ultrasound. Considering the widespread use of ultrasound for sterilization of tools and equipment in hospitals, this method might be ineffective for elimination of the major hospital pathogen S aureus. Nevertheless, extensive future research is required to scrutinize the effect of ultrasonic irradiation on microbial disruption at different specific energy inputs and longer sonication times.
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