Listen carefully: Ultrasound and microbial inactivation
Tim Sandle, Ph.D., CBiol, FIScT
Pharmaceutical Microbiologist & Contamination Control Consultant and Expert. Author, journalist, lecturer, editor, and scientist.
Ultrasound processing or sonication is an alternative technology for the inactivation or, sometimes, killing of microorganisms. Ultrasound is defined by the American National Standards Institute as "sound at frequencies greater than 20 kHz". In air at atmospheric pressure, ultrasonic waves have wavelengths of 1.9 cm or less.
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Sonication alone is not very effective in killing bacteria; however, when combined with another physical method, ultrasound can be effective. Examples are:
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Although efficient for certain applications, ultrasound does not meet a demonstrable definition of ‘sterilization’. To date the main applications have been in the food sector and in clinical practice.
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What is ultrasound?
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Ultrasound refers to pressure waves with a frequency of 20 kHz or more. Generally, ultrasound equipment uses frequencies from 20 kHz to 10 MHz. Higher-power ultrasound at lower frequencies (20 to 100 kHz), is sometimes referred to as “power ultrasound”.
During the sonication process (1):
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1.????? Longitudinal waves are created when a sonic wave meets a liquid medium.
2.????? This creates regions of alternating compression and expansion.
3.????? These regions of pressure change cause cavitation to occur.
4.????? Gas bubbles are formed in the medium.
5.????? The bubbles have a larger surface area during the expansion cycle. This increases the diffusion of gas, causing the bubble to expand.
6.????? A point is reached where the ultrasonic energy provided is not sufficient to retain the vapor phase in the bubble; therefore, rapid condensation occurs.
7.????? The condensed molecules collide violently, creating shock waves.
8.????? These shock waves create regions of very high temperature and pressure. The pressure changes resulting from these implosions are the main bactericidal effect in ultrasound.
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In #3 above there is a reference to ‘cavitation’. This is a term used in fluid mechanics relating to when the static pressure of a liquid reduces to below the liquid's vapor pressure. This leads to the formation of small vapor-filled cavities in the liquid (2).
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The cavitation threshold of a medium (the minimum oscillation of pressure that is required to produce cavitation) is determined by:
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The study of ultrasound upon microorganisms -either for growth, inactivation, or even kill, has been undergoing since the 1950s (3).
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How are microorganisms inactivated?
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The mechanism of microbial inactivation or killing is mainly due to thinning of cell membranes, localized heating and production of free radicals. The extent that ultrasound inactivates or kills depends on the process lethality. By inactivation, this means a state creating sublethally injured cells. These are defined as cells capable of forming colonies on nonselective media but not on selective media (4). Reversible damage of cell structures and loss of cell functions can be the result of a sublethal treatment (5).
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An example of microbial kill using ultrasound is:
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20 kHz and 160 W combined with temperatures ranging from 5 to 62°C.
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Other factors that affect the possibility of microbial kill are(6):
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As well as physical effects, ultrasound synergistically enhances the effectiveness of antibacterial agents. The technology can also activate molecules named sonosensitizers, resulting in the formation of compounds toxic to microbial cells.
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Microbial removal
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As well as inactivation, high intensity ultrasound is can be used to remove bacterial cells from surfaces. One example is with the removal of biofilms from the internal pipes of water systems (7). Success within pipes has been achieved using ultrasound frequencies of around 100 kHz and intensities approaching 40 W/cm^2.
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Another study, in what could be an additional potential application for the pharmaceutical sector, used ultrasound to treat reverse osmosis membranes colonized with a monolayer of fast-adhering bacteria. The experimental threshold for detachment was achieved with around 95% of the bacteria were removed (8).
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Growth promotion
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Ultrasound does not always inactivate cells and under certain conditions it can promote bacterial cell growth. Cells can grow in low-intensity insonation due to the ultrasound increasing the transport of small molecules in solution (9).
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Microbial types
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Another variable is the type of microorganism(10). Typically Gram-positive bacteria are more susceptible to ultrasonic treatment than the Gram-negative ones. This notwithstanding, there are other variables that need to be assessed. Taking an example from the food industry and the organism Listeria monocytogenes.
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Ultrasonic treatment (20 kHz and amplitude of 117 μm) at ambient temperature was not very effective giving a decimal reduction time (the time to reduce bacterial activity by 90%) of 4.3 minutes. However, by combining sonication with an increased pressure of 200 kPa (manosonication), the D-value of the ultrasonic treatment can be reduced to 1.5 minutes.
A further increase in pressure to 400 kPa can reduce the D-value to 1.0 minute(11).
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In terms other factors:
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A further variable is the growth phase of the organism. For example, with Escherichia coli susceptibility cells cultivated either on agar or harvested from the stationary phase of liquid culture are significantly more susceptible to ultrasound compared with an equivalent population obtained from the exponential phase of liquid growth (12).
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Endospores
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While ultrasound is effective, under controlled conditions, against vegetative cells, the process also has some effect against endospores. The sonic disruption of spores produces multi-hit kinetics. Studies show how the first hit destroys the exosporium, which protects the spore body from destruction. The second hit destroys the spore body. However, spores stripped of their exosporia are still viable and heat resistant (13).
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Another effect on spores is with breaking aggregates in bacteria spore suspensions, leaving them potentially more open to sterilization methods.
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Process optimization
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Researchers seeking to improve ultrasound methods will often focus on increasing cavitation intensity or by designing combined processes to enhance the lethal effect.
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Clinical application
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An example of a clinical application is the use of ultrasound as a sonobactericide, in the form of an ultrasound-activated microbubble or droplet treatment of bacteria and biofilm for medical devices (14). Success has been reported by combining ultrasound, cavitation nuclei and antibiotic therapy. This is being considered where the three-dimensional structure of a biofilm hinders antibiotic delivery penetration, and effectiveness (15).
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A complication here is with the different morphologies of bacteria. This arises because microbubbles oscillate differently when paired with spherical-shaped Staphylococcus aureus, compared with rod-shaped E. coli. Differences arise in relation to total surface area contact, tension, and rigidity(16).
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Summary
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In this article we have discussed the application of ultrasound, combined with other physical methods or in synergy with an antimicrobial. In some cases ultrasound can be effective in the inactivation microorganisms, although the technology is influenced by the power of ultrasonic waves, exposure time and bacteria type.
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References
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1.????? Berliner, S. (1984). Application of ultrasonic processors. International Biotechnology Laboratory, 2, 42–49
2.????? Riesz, P.; Berdahl, D.; Christman, C.L. (1985) Free radical generation by ultrasound in aqueous and nonaqueous solutions. Environmental Health Perspectives. 64: 233–252
3.????? Kinsloe, H., Ackerman, E., and Reid, J. J. (1954). Exposure of microorganisms to measured sound fields. Journal Bacteriology, 68, 373–380
4.????? Kell D. B., Kaprelyants A. S., Weichart D. H., Harwood C. R., Barer M. R. (1998). Viability and activity in readily culturable bacteria: a review and discussion of the practical issues. Antonie Van Leeuwenhoek Int. J. Gen. Mol. Microbiol. 73 169–187
5.????? Wesche A. M., Gurtler J. B., Marks B. P., Ryser E. T. (2009). Stress, sublethal injury, resuscitation, and virulence of bacterial foodborne pathogens. J. Food Prot. 72 1121–1138
6.????? Alpas, H., Kalchayanand, N., Bozoglu, F., and Ray, B. (2000). Interactions of high hydrostatic pressure, pressurization temperature and pH on death and injury of pressure-resistant and pressure-sensitive strains of foodborne pathogens. International Journal of Food Microbiology, 60, 33–42
7.????? Mott IEC, Stickler DJ, Coakley WT, Bott TR. The removal of bacterial biofilm from water-filled tubes using axially propagated ultrasound. J Appl Microbiol. 1998;84:509–514
8.????? Zips, A., Schaule, G., & Flemming, H. C. (1990). Ultrasound as a means of detaching biofilms. Biofouling, 2(4), 323–333
9.????? Pitt WG, Ross SA. Ultrasound increases the rate of bacterial cell growth. Biotechnol Prog. 2003 May-Jun;19(3):1038-44. doi: 10.1021/bp034068
10.? Allison, D. G., D’Emanuele, A., Egington, P., and Williams, A. R. (1996). The effect of ultrasound on Escherichia coli viability. Journal of Basic Microbiology, 36, 3–11
11.? álvarez, I., Pagán, R., Condón, S., and Raso, J. (2003). The influence of process parameters for the inactivation of Listeria monocytogenes by pulsed electric fields, International Journal of Food Microbiology, 87, 87–95
12.? Dower, W. L., Miller, J. F. and Ragsdale, C. W., 1988. High efficiency transformation of E. coli by high voltage electroporation. Nucl. Ac. Res., 16, 16–27
13.? Berger, J. A., and Marr, G. G. (1960). Sonic disruption of spores of Bacillus cereus. Journal of General Microbiology, 22, 1–64
14.? Pitt WG, McBride MO, Lunceford JK, Roper RJ, Sagers RD. Ultrasonic enhancement of antibiotic action on gram-negative bacteria. Antimicrob Agents Chemother. 1994 Nov;38(11):2577-82
Microbiologist | Antimicrobial resistance | CRISPR | Infectious disease
6 个月Sound or vibration have shown effects on highly pathogenic as well as MDR strain of S.marcescencs collected from patients when we conducted our experiments in the academic lab.