Mycobacterium hassiacum: a thermophilic Mycobacterium species to demonstrate thermal disinfection of medical devices

Objective Reprocessing reusable medical devices is crucial in the healthcare industry. To ensure patient safety, strict standards are dictated to validate thermal disinfection in automated washer-disinfectors. The United States Food and Drug Administration (FDA) has specific recommendations on the vegetative bacterial challenge but comparatively vague guidance on the use of a thermophilic Mycobacterium strain for thermal disinfection studies. This study aims to compare thermal resistance of Mycobacterium hassiacum and Mycobacterium terrae and determine which strain is suitable for medical device thermal disinfection validation testing in automated washer-disinfectors. Results Thermal resistance was demonstrated in vitro by calculating D-values for each strain at different exposure temperatures, and correlated with actual in situ processing conditions. M. terrae was completely killed (> 7 log reduction) at temperatures above 68 °C, with D-values between 46.6 and 27.8 s at temperatures between 59.5 and 67.2 °C. M. hassiacum was completely killed (> 8 log reduction) at temperatures above 75 °C, with D-values between 82.1 and 21.7 s at temperatures ranging between 69.2 and 73.6 °C. In vitro results were correlated in a washer-disinfector performance validation setup.


Introduction
Thousands of reusable medical devices are processed daily at healthcare facilities in order to be re-used on patients. Specially designed washer-disinfectors clean and disinfect reusable medical devices at high throughput following strict standards and guidelines applicable to the field [1,2]. To ensure patient safety, semi-critical medical devices should ideally be sterilized, or at minimum must be subjected to high-level disinfection [3,4]. According to the United States Food and Drug Administration (FDA) guidance, thermal disinfection must be demonstrated by a 6-log 10 reduction of a mixed population of vegetative bacterial strains such as Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and a representative of the Klebsiella/Enterobacteria group (low-level disinfection). In addition, 3-to 6-log 10 reduction of a thermophilic Mycobacterium species must also be achieved for intermediate-and high-level disinfection, respectively [2].
The validation of automated washing and disinfection of reusable medical devices has greatly contributed to the reduction of clinical infections [5]. Chemical disinfection and thermal disinfection are two different processes. Two parameters must be considered to evaluate thermal disinfection: time and temperature of exposure. As per chemical disinfection, the choice of the model organisms is crucial in order to keep a high safety margin. In the late 90s, debates Open Access BMC Research Notes *Correspondence: Bruno_Haas@steris.com 1 STERIS Canada ULC, 490, Boulevard Armand-Paris, Quebec City, QC G1C 8A3, Canada Full list of author information is available at the end of the article in standards committees raised the need for a surrogate strain representative of Mycobacterium tuberculosis and Mycobacterium avium-intracellulare to safely assess mycobactericidal activity in chemical disinfection of medical devices. In 1998, Griffiths et al. suggested the use of Mycobacterium terrae since it possesses similar resistance profile towards disinfectants compared to that of M. tuberculosis and M. avium-intracellulare [6]. However, there are no recommended Mycobacterium strains to validate thermal disinfection. The only guidance, from the U.S. FDA, is to use a thermophilic strain [2].
The International Organization for Strandardization's standard series ISO 15883 addresses thermal disinfection levels using the A 0 concept that represents a time-temperature correlation of a thermal treatment [7]. It is the equivalent time in seconds at 80 °C delivered by the disinfection process with reference to a microorganism with a z value of 10 K [8,9]. In 2016, McCormick et al. detailed the A 0 concept and described Mycobacterium terrae as a model for thermophilic Mycobacterium species [10]. However, no evidence of M. terrae thermophilic properties is available. The only reference to M. terrae susceptibility to thermal treatment was described in 2011 by Pisot et al. [11]. They found a complete inactivation of M. terrae at 65 °C. This behaviour does not correspond with the definition of "thermophilic" [12].
In 1997, Schröder et al. discovered a new Mycobacterium species named Mycobacterium hassiacum [13]. M. hassiacum is characterized as thermophilic as it can show optimal growth at temperatures up to 65 °C [14]. When compared to M. terrae, M. hassiacum could present substantial benefits when used in a thermal disinfection validation setup. First, it meets the FDA guidance that a thermophilic strain of Mycobacterium should be used in thermal disinfection studies. Second, M. hassiacum is a fast-growing species; colonies can generally be obtained within 8 days of incubation at 37 °C whereas M. terrae is a slow-growing species, with colonies appearing after 14 to 21 days of incubation at 37 °C. Finally, M. hassiacum can be easily identified as the test organism, since it has specific phenotypic characteristics including colony shapes and a distinctive yellow color.
In the present study, the thermal resistance of both M. terrae and M. hassiacum was determined by D-value calculation. The resulting D-values were compared in order to determine which strain could be most suitable as a bacterial challenge in thermal disinfection performance validation of medical washer-disinfectors.
Thermal resistance testing Three independent M. hassiacum and M. terrae suspensions (at least 10 7 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: 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 log 10 .
Treatment temperatures were determined by calculating the average temperature recorded during the treatment time. For each temperature, the average bacterial count (log 10 ) was plotted on a graph (Y axis: log 10 bacterial count; X axis: time of treatment). The D-value for each temperature was calculated using the following formula:

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 (OD 620 ) of 0.1 (bacterial titer estimated > 10 7 CFU/ml). 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).

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 (Fig. 1, lower panel)

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 log 10 reduction from 7.4 log 10 CFU/ml to 3.8 ± 1.6 log 10 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 log 10 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 A 0 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 log 10 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). Log 10 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 A 0 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 A 0 of 60 (1 min at 80 °C) is enough to kill 7 to 8 log 10 of M. terrae or M. hassiacum in a laboratory setup. A sufficient safety margin has to be considered for field application; the A 0 600 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 washerdisinfectors. 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.

Limitations
Variability was observed between replicates. Aggregation of the bacteria could have been causing variable results. Mycobacteria are known to aggregate due to their hydrophobicity thus causing difficulties obtaining homogeneous single-cell suspensions. Received: 29 January 2020 Accepted: 25 February 2020