Methods
In vitro testing
Bacterial seed culture preparation
Mycobacterium hassiacum DSM 44199 (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ, Germany) (equivalent to American Type Culture Collection [ATCC] 700660) [15] and Mycobacterium terrae ATCC 15755 (Microbiologics, USA) were grown on Middlebrook 7H11 agar containing 10% oleic acid, dextrose, catalase enrichment (OADC) and incubated at 37 °C for 7 days (M. hassiacum) or 14 days (M. terrae).
Thermal resistance testing
Three independent M. hassiacum and M. terrae suspensions (at least 107 Colony Forming Units per milliliter [CFU/ml] in 0.85% saline + 0.1% Tween 80) were exposed to thermal treatments of various times and temperatures in a heated water bath. Fractional growth data were used to calculate D-values for each Mycobacterium species. A temperature check tube was used to monitor the temperature during treatments.The samples were diluted in peptone water and plated on 7H11 agar supplemented with 10% OADC and incubated at 37 °C for 7 days (M. hassiacum) or 14 days (M. terrae). Following incubation, CFU were counted to determine bacterial survival using the following formula:
$${\text{T}} = { \log }_{ 10} \left( {{\text{N}}*{\text{D}}} \right)$$
where, T = bacterial titer; N = average number of CFU at the valid dilution; D = dilution factor of the valid dilution.
D-value calculation
The D-value is the time required, at a given temperature, to decrease the bacterial population by 1 log10.
Treatment temperatures were determined by calculating the average temperature recorded during the treatment time. For each temperature, the average bacterial count (log10) was plotted on a graph (Y axis: log10 bacterial count; X axis: time of treatment). The D-value for each temperature was calculated using the following formula:
$${\text{D}} = - 1/{\text{slope}}$$
Tests in washer-disinfector
Bacterial culture and test ampoules
Mycobacterium hassiacum ATCC 700660 (Cedarlane, Canada) (equivalent to DSM 44199) [15] was grown at 37 °C in Middlebrook 7H9 broth with glycerol for 7 days. The resulting culture was directly used as test suspension.
Mycobacterium terrae ATCC 15755 (Innovation Diagnostics Inc., Canada) was suspended in 0.85% Saline + 0.1% Tween 80 to an optical density at 620 nm wavelength (OD620) of 0.1 (bacterial titer estimated > 107 CFU/ml).
1.4 ml of test suspensions of M. terrae and M. hassiacum (at least 107 CFU/ml) were sealed in glass ampoules and used to test bacterial thermal resistance during a mock thermal phase in a washer disinfector.
Washer-disinfector test setup
A single-chamber washer disinfector was connected to a preheated water reservoir. Water at the desired temperature was passed through the washer’s chamber for 1 min on a simulated medical instrument load with ampoules placed at the coldest area as determined per thermal profiling (data not shown). The following five treatment temperatures were tested for a 1-min contact time: 65 °C, 70 °C, 75 °C, 80 °C, and 85 °C. Ampoules of M. hassiacum and M. terrae were processed at the same time and thus received identical treatments. Once the 1-min water circulation was achieved, the ampoules were placed in ice cold water to immediately stop the heat treatment. The entire content of each ampoule was plated to assess bacterial survival by counting CFUs following 7-days incubation (M. hassiacum) or 14-days incubation (M. terrae) at 37 °C. The initial titer of each test suspension was also determined to represent the positive control (untreated ampoules).
Results
In vitro testing
The results of the in vitro thermal treatments including bacterial survival and D-values (in seconds) are shown in Fig. 1.
M
. terrae
Treatments for M. terrae were performed at temperatures between 59.5 and 67.2 °C since temperatures higher than 68 °C resulted in complete kill for most of the exposure times tested (data not shown). For all treatment temperatures, bacterial survival of M. terrae decreased as treatment time increased (Fig. 1, upper panel). D-values obtained for M. terrae ranged between 27.8 and 46.6 s. For replicate 1, D-values of 41.1, 38.1, 32.6, and 34.7 s were obtained for treatments at 59.5, 61.0, 66.0, and 66.2 °C, respectively. Replicate 2 resulted in D-values of 32.2, 27.8, and 29.6 s with treatments at 63.7, 64.4, and 65.5 °C, respectively. D-values obtained on replicate 3 were 46.6, 34.4, 34.6, and 27.8 s for treatments at 63.2, 64.8, 64.9, and 67.2 °C, respectively.
M. hassiacum
Treatments on M. hassiacum suspensions were performed at temperatures between 69.2 and 73.6 °C. Bacterial survival decreased when treatment temperature or time increased (Fig. 1, lower panel). D-values obtained for replicate one were 80.5, 40.7, and 21.7 s for treatments at 69.2, 71.5, and 73.6 °C, respectively. On replicate two, treatments at 69.6, 71.4, and 72.7 °C led to D-values of 82.1, 53.2, and 34.2 s, respectively. The third replicate resulted in D-values of 66.3, 75.3, and 37.0 s at 69.3, 71.2, and 73.3 °C.
Comparison of the highest treatment temperatures for M. terrae and the lowest treatment temperatures for M. hassiacum, showed that at 67.2 °C, 1-log10 reduction of M. terrae was achieved after 27.8 s whereas at 69.2 °C, 1-log10 reduction of M. hassiacum was achieved after 80.5 s.
Washer-disinfector testing
When tested in sealed glass ampoules in a washer-disinfector, it was found that M. terrae survival was significantly affected following a 1-min treatment at 65 °C (3.6 log10 reduction from 7.4 log10 CFU/ml to 3.8 ± 1.6 log10 CFU/ml) (Fig. 2). When treatment temperature was increased to 70 °C and above, no surviving CFU were recovered from any ampoules tested for M. terrae.
In contrast, no significant reduction of bacterial survival was observed for M. hassiacum when treated at 65 °C for 1 min. When temperature was raised to 70 and 75 °C, a 1-min treatment lead to 0.2 and 0.6 log10 reduction of the initial titer, respectively. However, the reduction was not significant. When treated for 1 min at 80 and 85 °C, a complete kill of M. hassiacum was observed.
Discussion
Mycobacterium terrae has been the surrogate of choice to safely test tuberculocidal activity of chemical disinfectants in the healthcare industry [6]. Another reliable way to disinfect thermo-resistant medical devices is to use an automated washer-disinfector that can perform thermal disinfection. International standard ISO 15883-2 evaluates thermal disinfection in terms of A0 values [8, 10, 16]. In addition, bacterial thermal resistance can be expressed using the calculated D-values which represent the time needed to decrease a bacterial population of 1 log10 at a given temperature [17].
In vitro testing showed that survival of M. terrae was affected beginning at temperature treatments of 60 °C and higher. M. hassiacum survival was not affected by treatment temperatures below 68 °C (data not shown). Log10 reduction of both M. terrae and M. hassiacum were time and temperature dependent. However, the two strains were not affected at the same temperature ranges. M. terrae depicted a complete kill when submitted to treatments at temperatures higher than 68 °C, which is in accordance with previous observations [11]. Results obtained when testing M. terrae in a washer disinfector were in concordance with in vitro data, i.e. a complete kill at temperatures above 68 °C.
For critical medical devices such as surgical instruments, the ISO 15883 standard series recommends an A0 of 600 for disinfection [1, 7]. This is equivalent to a 1-minute (60 s) treatment at 90 °C. The present study demonstrates that an A0 of 60 (1 min at 80 °C) is enough to kill 7 to 8 log10 of M. terrae or M. hassiacum in a laboratory setup. A sufficient safety margin has to be considered for field application; the A0 600 is therefore reasonable to ensure high-level disinfection of critical devices. More importantly, the data collected during this study demonstrate that M. hassiacum is more suitable than M. terrae as a test organism to validate the tuberculocidal effect of thermal disinfection in an automated washer-disinfector.
To our knowledge, M. terrae thermal resistance has never been studied in detail [11], and there is no evidence of its thermophilic behaviour that justifies its use for thermal disinfection validation tests. M. hassiacum is a strain that was described for its thermophilic properties [13, 15], and showed in the present study a higher resistance to thermal treatment than M. terrae. Therefore, we recommend the use of M. hassiacum as the thermophilic Mycobacterium species surrogate of choice to validate thermal disinfection in automated washer-disinfectors. The choice of model organisms is crucial to keeping a high safety margin to ensure patient safety when reprocessing medical devices in automated washer-disinfectors.