786 infection control & hospital epidemiology july 2017, vol. 38, no. 7
mycobactericidal activity, whether in suspension tests or on carrier disks. No significant loss of bacterial viability was observed in the presence of neutralizing broth in the test solution. The detection limit of the suspension test is 1CFU/mL. All suspension and carrier tests were performed in duplicate using independent bacterial culture batches, and the results of 1 typical experiment are presented in the tables.
Carrier-Disk Tests
For the carrier-disk test, 10 µL of each of the homogeneous bacterial suspensions described above was placed at the center of a 1-cm stainless-steel carrier disk and allowed to air dry for 30 to 40 minutes. Each carrier disk was then sterilely placed, inoculum side up, in a 30-mL straight-side wide-mouth Nalgene jar (ThermoScientific, Rochester, NY). Next, 50 µLof each freshly prepared disinfectant solution preheated to the recommended temperature was applied to the dried inoculum on each carrier disk and held for the desired contact time. Then, 9.95mL sterile neutralizing broth was added to the jar and vortexed for 60 seconds before vacuum filtration was performed as described above. The carrier disk was then vortexed 3 more times with 15mL 0.9% sterile saline/0.1% Tween 80. Vacuum filtration was performed between washes. The MicroFunnel filters were aseptically placed onto 7H11 agar plates supplemented with OADC for CFU counting. The detection limit of the carrier-disk test is 1 CFU. All suspension and carrier-disk tests were performed at least twice.
results Approach
Several of the most commonly used disinfectants were tested against 10 different rapidly growing and slow-growing mycobacteria, including 4 M. chelonae and M. abscessus subsp. massiliense isolates that had been previously reported to display resistance to GTA. Each disinfectant was tested using 2 independent methods, a suspension test and a carrier test. The latter was considered more stringent and more reflective than the former regarding the disinfection of microorganisms on dried surfaces. Tables 1 and 3 show the results of the suspension tests, and Table 2 shows the results of the carrier tests.
Glutaraldehyde-Based Disinfectants
All GTA-based high-level disinfectants displayed the expected mycobactericidal efficacy against the GTA-susceptible control strains, M. bovis BCG (Pasteur strain 1173 P2), M. avium 104, M. terrae ATCC 15755, M. chelonae ATCC 35752, M. abscessus subsp. massiliense CIP 108297, and M. abscessus subsp. massi- liense CRM-0270. During the suspension test, control strains were reduced to no detectable bacterial counts within
liense and M. chelonae isolates,CRM-0019, 9917, Harefield and Epping, showed significant resistance to the GTA-based pro- ducts with <0.5-log10 reduction in CFUs after 10 minutes of exposure to Cidex (1.5% GTA) in suspension tests (<3.2-log10 reduction in CFUs after the recommended time of exposure of 45 minutes) and in the carrier-disk test (<3.6-log10 CFU reduction after 45 minutes) (Tables 1 and 2). For Aldahol (1.8% GTA), the suspension test results for the GTA-resistant strains at the recommended exposure time of 10 minutes at 20° C showed significant resistance to this disinfectant as well (Table 1). The greatest CFU reduction at the 10-minute time point was observed for the GTA-resistant M. abscessus subsp. massiliense strain CRM-0019 (3.33 log10 CFU reduction), while the GTA-resistant M. chelonae strains 9917, Harefield and Epping showed a <1.0-log reduction in CFU counts. When tested at additional time points to 30 minutes, the 3 GTA-resistant M. chelonae strains continued to show greater resistance with a maximum1.0 log10 reduction in CFUs, while M. abscessus subsp. massiliense CRM-0019 was reduced to no detectable CFU by 30 minutes. The carrier-disk testing simi- larly pointed to the significant resistance of M. chelonae 9917, Harefield and Epping with no more than a 4.42-log10 CFU reduction after 20 minutes, while M. abscessus subsp. massi- liense CRM-0019 again showed susceptibility to Aldahol at the extended 20-minute time point, with a>7.0-log10 CFU reduction (Table 2). When the testing temperature for Aldahol was increased from 20°C to 25°C, the suspension testing results showed greater bactericidal efficacy. All GTA-resistant strains except one were reduced to no detectable CFU within
the recommended 10 minutes. However, M. chelonae Hare- field only achieved a 0.68-log10 reduction after 10 minutes and, even at the extended 30-minute time point, CFU counts were only reduced by a meager 1.62-log10 (Table 1). Because the 1.8% GTA Aldahol preparation contains isopropanol,24 15% isopropanol alone was tested for mycobactericidal activity. Neither M. abscessus subsp. massiliense CIP108297 nor the GTA-resistant M. abscessus subsp. massiliense strain CRM-0019 showed any significant reduction in CFU counts with 30 minutes of exposure to this compound (Table 1).
Ortho-phthalaldehyde
Suspension test results for the Cidex OPA (0.3% ortho- phthalaldehyde [OPA]) at 25°C achieved reduction to no
10 minutes of exposure to Cidex (1.5% GTA) at 25°C, except for the slow-growing species M. avium, M. terrae, and M. bovis BCG, which required longer contact times of 15 to 30 minutes (Table 1). Reduction to no detectable bacterial counts was achieved for all control strains within 5 minutes of exposure to Aldahol (1.8% GTA), a GTA-based product containing iso- propanol and potassium acetate,24 at both 20°C and 25°C (Table 1). The carrier-disk test yielded similar results with reduction to no viable bacteria for all GTA-susceptible strains and GTA-based products tested (Table 2). As expected, the GTA-resistant M. abscessus subsp. massi-
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