Moist Heat Disinfection and Revisiting the A₀ Concept

Disinfection is defined as a reduction in the number of viable microorganisms on a surface to a level previously specified as appropriate for its intended handling or use.1 This may be achieved using a variety of physical and/or chemical methods.2 Moist heat has been used for many years in the medical device, pharmaceutical, and food processing industries to render products and goods microbiologically safe for use, handling, or consumption. Examples include the pasteurization of foods and beverages,3,4 treatment of products such as bone tissue transplants and vaccines,5–9 sanitization of water distribution systems,10 and the disinfection of medical and dental devices.2,11,12 Moist heat is often used as a terminal disinfection method (following cleaning and rendering them safe for patient use) for a range of devices such as bedpans and anesthesia and respiratory therapy equipment, but is also used as an interim step during the reprocessing of surgical devices to render them safe for handling (prior to inspection, packaging, and terminal sterilization).2 Disinfection processes range from temperatures of 65°C to 100°C (149°F to 212°F),2,11,12 although the use of steam (defined as water in a vapor phase) under various conditions is also an effective disinfection process over time. Considering the use of heat for disinfection (e.g., hot water) and sterilization (steam), our understanding of the microbial resistance profiles to moist heat inactivation (Figure 1) and the impact of heat on the disinfection of microbial populations has been well established.2,13–15 

The lethal effects of temperature on microbial growth and viability are not difficult to comprehend. Moist heat clearly has multiple effects on the structure and function of biomolecules and the coagulated proteins, lipids, and carbohydrates that make up cell and virus structures.2 Most vegetative bacteria and fungi are readily inactivated at temperatures in excess of 55°C (131°F), although some types of bacteria such as Enterococcus and Legionella have been shown to have notable resistance to heat disinfection under certain test conditions compared with other types of bacteria.16,17 Despite these effects, thermal disinfection of vegetative bacteria, fungi, and certain types of protozoa is generally effective above 65°C (149°F). For the purpose of medical device disinfection applications, this would include most bloodborne pathogens such as enveloped viruses (e.g., human immunodeficiency virus and hepatitis B virus), staphylococci, streptococci, Enterobacteriaceae, many nonenveloped viruses (such as poliovirus), and protozoa (e.g., Cryptosporidium and Giardia). Some of the more resistant forms of viruses are the nonenveloped viruses, and a higher level of resistance has been reported with noroviruses and parvoviruses.2 For example, in a study with parvoviruses, moist heat was only effective at temperatures greater than 70°C (158°F) in comparison to model viruses used in disinfection studies (e.g., poliovirus, adenovirus, vaccinia).14 More typical examples of resistance are with bacterial spores requiring temperatures in excess of 100°C, including Clostridium difficile spores (Figure 1).2,15,18,19 

The demonstration of heat disinfection is based on two methodologies, alone or in combination:

• Traditional microbial reduction studies simulating the use of heat disinfection for its intended purpose (e.g., in water, in presence of soil, dried on a device).

• Parametric demonstration by directly measuring the range of temperatures being applied within a test system over time and based on established mathematical modeling.

In this article, we briefly review these methodologies and present some recent data on the thermal disinfection of a range of microorganisms, including when tested in the presence of a developed biofilm. This is particularly considered for disinfection of reusable devices following cleaning to render them safe for staff handling, inspection, and packaging (in preparation for terminal sterilization) or as a terminal disinfection process for noncritical or semicritical devices such as respiratory therapy equipment.

Direct microbiological challenge tests have been used for many years to demonstrate the effectiveness of antimicrobial methods, including thermal disinfection.20 An example are the test requirements for washer-disinfectors defined by the Food and Drug Administration (FDA).21 Testing is recommended with a number of challenge bacteria suspended in an organic soil and dried onto representative surfaces in a washer-disinfector load. These tests are generally performed with a mixed culture of test bacteria. The specific level of disinfection claimed is based on the type of microorganisms and extent of reduction of bacteria demonstrated in the test (Table 1). For example, a high-level disinfection claim is established by demonstrating a 6-log reduction of a mixed population of vegetative bacteria that includes Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and a representative of the Klebsiella-Enterobacter group, in addition to a 6-log reduction in a thermophilic Mycobacterium species, such as Mycobacterium terrae. Similar tests have been recommended in other countries, such as in Germany using Enterococcus.22,23 

Time–temperature relationships for moist heat disinfection are well established in the pasteurization of foods and in industrial and medical applications.15 For medical device applications, the mostly widely described relationship to date is the A₀ concept,23,24 which is essentially based on the principles used for thermal sterilization (e.g., relationships between D values [the time required to achieve inactivation of a 1-log population of a test organism], Z values [the temperature change required to give a log change in the D value], and the F value [the time in minutes to kill a population of bacterial spores when the temperature is at 121°C]).

The A₀ method is based on knowledge of the lethality of a thermal disinfection process at different temperatures to assess the overall lethality of the cycle and to express this as the equivalent exposure time at a specified temperature. This method was first proposed by Dr. David Hurrell and published as a method to demonstrate thermal disinfection in washer-disinfectors. Mathematically, “A” is defined as the equivalent time in seconds at 80°C to produce a given disinfection effect and the “A₀” assumes the Z value (for the target population of microorganisms) of 10°C. So, overall, the A₀ value for moist heat disinfection processes is the equivalent time in seconds at a temperature of 80°C delivered by that process to the product with reference to microorganisms possessing a Z value of 10°C. It can be expressed as follows:

where t is the chosen time interval (in seconds) and T is the temperature in the load (in degrees Celsius).

It is therefore possible to calculate (by integration) the cumulative lethality (or microbial inactivation) during the heat-up, holding, and cooling time of any moist heat disinfection process. In calculating A₀ values, it is also important to note that the lower temperature limit for such integration is set at 65°C (149°F) since, for temperatures below 65°C, the Z value and D value of thermophilic organisms may change dramatically. This cutoff temperature is a matter of some debate, as some pathogens may present with novel thermal resistance even at 65°C and may require a change of this minimum level to 70°C (158°F) or even 75°C (167°F) (McDonnell, personal communication). Practically, for most washer-disinfection applications, the A₀ levels are generally set by considering the holding time for disinfection only, with typical examples shown in Table 2. The acceptable levels of A₀ for disinfection claims in washer-disinfector designs are further defined in parts of the ISO 15883 standard series based on the types of devices being disinfected: for surgical instruments defined in ISO 15883-2 as an A₀ of 600 (i.e., 90°C/194°F for 1 min) and for bedpan washers in ISO 15883-3 at an A₀ of 60 (i.e., 70°C/158°F for 10 min).25,26 The ISO 15883 standard series further define the requirements for testing temperature distribution within the washer and an applicable load in order to validate these minimum requirements.

In order to confirm the applicability of the A₀ concept, testing was performed at A₀ conditions of 60, 600, and 3,000 with various planktonic microorganisms and a mixed culture biofilm model.

Heat inactivation experiments were conducted in thin-walled 0.2-mL polypropylene snap cap PCR tubes and using three 96-well thermal cyclers adjusted to provide A₀ values of 60 (10 min at 70°C ± 0.5°C), 600 (10 min at 80°C ± 0.5°C), and 3,000 (5 min at 90°C ± 0.5°C). Temperature mapping was verified in advance using calibrated thermocouples and a data logger.

Overnight cultures of test organisms were prepared, harvested with sterile saline, centrifuged for 8 to 10 minutes at 3,500 rpm to remove organic debris, and standardized at 490 nm in a spectrophotometer to approximately 1 × 10⁷ CFU/mL to 2 × 10⁸ CFU/mL (CFU = colony-forming unit). Mycobacterium was grown on blood agar for 3 weeks at 30°C to 35°C prior to harvesting and standardization. Aliquots of the standardized suspension were plated in triplicate to confirm the initial inoculum level (N₀). Nine tubes were then inoculated with 0.1 mL of the standardized test organism suspension and three tubes each processed in a thermal cycler under the desired exposure conditions. Following heating, the number of surviving test organisms was determined by plating in triplicate on the indicated medium. Bacteria were incubated for 2 to 4 days at 30°C to 35°C (with the exception of Mycobacterium for 10 days at 30°C to 35°C) and fungi for 4 to 7 days at 20°C to 25°C.

For biofilm testing, a mixed culture biofilm of S. aureus ATCC (American Type Culture Collection) 6538, Escherichia coli ATCC 8739, and Candida albicans ATCC 10231 was prepared on glass microscopic slides as described by McCormick et al.27,28 Following incubation, biofilm formation was verified by staining with SYPRO Ruby and acridine orange (Figure 2). Slides were gently rinsed with sterile water to remove loosely adherent cells and the biofilm harvested by scraping with a sterile scalpel into sterile saline for testing as described above. Preparations were enumerated by ultrasonication for 10 minutes, shaken at 200 rpm for 10 minutes, vortexed, serially diluted, and plated on trypticase soy agar with incubation at 30°C to 35°C for 48 hours.

Thermal mapping in advance of these studies demonstrated that the method used was applicable for evaluating the heat resistance of microorganisms. The heat resistance of a range of planktonic test organisms (microorganisms in a nonaggregated state) was evaluated including Gram-positive bacteria, Gram-negative bacteria, mycobacteria, and fungi (Table 3). Many of the organisms tested are potential pathogens or otherwise associated with nosocomial infections. Test organisms included ATCC cultures and clinical and environmental isolates. None of the test organisms exhibited elevated levels of heat resistance with a ≥6-log reduction of the initial population for all organisms tested at nominal A₀ values of 600 and 3,000. Substantial inactivation was also noted at a nominal A₀ value of 60 with all test organisms exhibiting a ≥6-log reduction, with the exception of an environmental isolate of Micrococcus luteus, which demonstrated a ≥2-log reduction in population. This is not unexpected as M. luteus has been reported to be relatively thermoresistant, and is of concern to the dairy industry.29 Similar results have been reported by others, such as with Enterococcus and Enterobacteriaceae.30,31 The inactivation of methicillin-resistant S. aureus (MRSA) clinical isolates confirms the work of Rutala et al.32 and suggests that heat disinfection is an effective means of limiting the spread of these organisms when reprocessing heat-stable surgical instruments and equipment.

Heat resistance testing was also performed with a mixed culture biofilm of S. aureus, E. coli, and C. albicans. Biofilms are known to form in a wide range of environments, including healthcare applications, and can be highly resistant to antibiotics and chemical disinfection in comparison to the planktonic form of the organism.33 The results of this testing suggest that biofilm formation did not substantially alter the susceptibility of the organisms to heat inactivation (Table 3). Testing performed with the same mixed-culture biofilm model yielded only a 2.1-log reduction in population when exposed to a 2% hydrogen peroxide solution for 60 minutes (results not shown). Although biofilm growth has little impact in heat resistance, chemical disinfection resistance may be a greater concern.2,33 

The use of heat disinfection is clearly appropriate for many healthcare applications, both as an interim reprocessing step to render devices safe for handling and as a terminal step prior to patient use, depending on the criticality of the device. Heat disinfection applications can be verified by direct antimicrobial studies (microorganism inoculation, treatment, and recovery) or by using parametric methods to verify temperature distribution and apply mathematical models such as the A₀ concept.1,24 Given the widespread use of heat disinfection and numerous studies verifying the effectiveness of heat to inactivate microorganisms, it is recommended that the A₀ concept should be more widely applied as a test method to verify the efficacy of moist heat disinfection in washer-disinfectors and other applications. However, it is also recommended that the minimum temperature for application of the A₀ concept should be increased to at least 70°C to 75°C to accommodate the inactivation of thermoresistant bacteria and viruses.