disinfectant susceptibility of mycobacteria 785
that the selection of aldehyde-resistant strains in clinical settings might select for isolates displaying cross resistance to other disinfectants and/or antibioticsaswellas
increased
pathogenicity.17 Reduced porin expression in the rapidly growing Mycobacterium species, M. smegmatis, has indeed been associated with increased resistance not only to aldehyde-based products but also to quaternary ammonium compounds, isothiazolinones, methylenbisoxazolidine, and lipophilic biocides such as polyhexamethylene biguanine and octenidine dihydrochloride, in addition to multiple antibiotics.17–19 Furthermore, 5 studies have reported that GTA-resistant M. chelonae and M. abscessus strains isolated either experimentally or from endoscope washer disinfectors demonstrated increased resistance to peracetic acid, peroxygen, sodium dichloroisocyanurate, or quaternary ammo- nium compounds.13,14,20–22 Considering the prevalence of aldehyde-resistant rapidly
growing mycobacteria and concerns about the possible development of cross resistance to other biocides, we sought to profile the disinfectant-susceptibility pattern of a panel of aldehyde-susceptible and resistant Mycobacterium isolates to explore alternative high-level disinfection strategies in mycobacterial GTA-resistant outbreaks. A variety of high-level disinfectants commonly used in the reprocessing of endoscopic equipment, including different formulations of aldehyde-based products, oxidizing agents, and alcohol, were tested against these isolates in suspension and carrier tests.
methods Bacterial Strains and Standard Growth Conditions
The 6 control (GTA-susceptible) mycobacterial strains used in this study were 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. massiliense CRM-0270.3 The 4 NTM isolates with known resistance to GTA included M. abscessus subsp. massiliense CRM-0019,3 M. chelonae 9917,17 M. chelonae Harefield and M. chelonae Epping.23 The M. abscessus subsp. massiliense CRM-0270 isolate is genetically closely related to the GTA-resistant outbreak isolate from Brazil but is GTA susceptible.3 All mycobacterial cultures were grown under agitation in Middlebrook 7H9 (Difco Laboratories, Sparks, MD) medium supplemented with 10% OADC (oleic acid, albumin, dextrose, catalase) enrichment (Becton Dickinson, Sparks, MD) and 0.05% Tween 80 (Sigma Aldrich, St Louis, MO) at 30°C to an optical density (OD600) of 1.0.
Disinfectants
Overall, 5 commercial disinfectants labeled for high-level dis- infection were evaluated along with the quaternary ammo- nium compound, cetrimonium bromide (10%; Sigma Aldrich). The 5 products included the GTA-based disinfectants Cidex (2.4% GTA; Johnson & Johnson,
New Brunswick NJ) and Aldahol (3.4% GTA; Alden Medical, West Springfield, MA), the ortho-phthalaldehyde-based disinfectant CidexOPA (0.55%OPA; Johnson&Johnson), the peracetic acid-based products, Reliance DG (Steris, Mentor, OH) and S40 (Steris), and the hydrogen peroxide-based pro- duct Resert XL HLD (Steris). To best reflect the use of the products in clinical settings, minimum recommended con- centrations (MRCs) or minimum effective concentrations (MECs) were chosen for each disinfectant based on the mini- mum concentration of active disinfectant necessary to create a “passing” response on the corresponding manufacturer’s pro- duct testing strips. The passing responses on Cidex and Cidex OPA are based on the MEC, whereas the passing responses for Resert XL HLD and Aldahol test strips are based on the MRC (Tables 1–3). Prior to each series of tests, Cidex and Aldahol were tested for exact concentration of GTA using the hydroxylamine hydrochloride pH titration method and, wherever applicable, diluted to the desired concentration (MEC=1.5% GTA for Cidex; MRC=1.8% GTA for Aldahol) with sterile double-distilled water. Cidex OPA was similarly diluted to 0.3% OPA (MEC value) with sterile double-distilled water. All disinfectants were freshly prepared prior to use. Susceptibility tests were performed at the recommended temperatures.
Suspension Tests
Each mycobacterial strain was prepared for testing by adding 1.0mL of 0.9% sterile saline (Hospira, Lake Forest, IL) con- taining 0.1% Tween 80 to 9.0mL of bacterial suspension (~1010 colony-forming units [CFU]) previously washed 3 times with sterile double-distilled water. The preparation was then dispersed for 1 minute using a sterile 15-mL Wheaton pestle to generate a homogeneous bacterial suspension. Next, 1mL of bacterial suspension was added to 99mL of the disinfectant solution maintained at the recommended temperature under continuous mixing conditions. At predetermined time points, 1.0mL of test solution was removed and placed in either 9.0mL of sterile Dey Engley/1% sodium thiosulfate neutralizing broth (Becton Dickinson; Sigma Aldrich) for oxidizers or 9.0mL of sterile Dey Engley/ 1% sodium bisulfite neutralizing broth (Becton Dickinson; Sigma Aldrich) for GTA and OPA, and immediately vortexed for 60 seconds to neutralize the disinfectant solution. The neutralized test solutions were then vacuum-filtered through a sterile 0.45 µm MicroFunnel filter unit with a Metricel black-gridded membrane (Pall, Ann Arbor, MI). After a final rinse of the filter housing with 30–40mL 0.9% saline/0.1% Tween 80, the filters were aseptically placed onto 7H11 agar plates supplemented with OADC and incubated at 30° for 7 to 21 days, after which CFUs were counted. Control experi- ments performed in parallel on the GTA-susceptible strain M. massiliense CIP 108297 confirmed the neutralizing efficacy of the neutralizing broth with each disinfectant and verified that the neutralizer by itself did not contribute any
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