Abstract
Objective
Activated alkaline glutaraldehyde remains one of the most widely used high-level disinfectant across the world. Yet, several reports have highlighted the potential for nontuberculous mycobacteria to develop high-level resistance to this product. Because aldehyde resistance may lead to cross-resistance to other biocides, we set out to investigate the susceptibility profile of glutaraldehyde-resistant Mycobacterium chelonae and Mycobacterium abscessus isolates to various disinfectant chemistries.
Methods
High-level disinfectants commonly used in the reprocessing of endoscopes and other heat-sensitive, semicritical medical equipment, including different formulations of aldehyde-based products and oxidizing agents, were tested against ten slow- and fast-growing, glutaraldehyde-susceptible and glutaraldehyde-resistant, Mycobacterium isolates in suspension and carrier tests at different temperatures.
Results
While peracetic acid- and hydrogen peroxide-based disinfectants (S40™, Resert™ XL, Reliance™ DG) efficiently killed all of the Mycobacterium isolates, glutaraldehyde- and ortho-phthalaldehyde-based products (Cidex®, Aldahol®, Cidex® OPA) showed variable efficacy against glutaraldehyde-resistant strains despite the ability of some formulations (Aldahol®) to overcome the resistance of some of these isolates, especially when the temperature was increased from 20 to 25°C.
Conclusions
Application permitting, oxidizing chemistries may provide a safe alternative to aldehyde-based products, particularly in the case of GTA-resistant mycobacterial outbreaks.
Introduction
The rapidly growing nontuberculous mycobacteria (NTM), Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium abscessus subsp. abscessus, Mycobacterium abscessus subsp. massiliense and Mycobacterium abscessus subsp. bolletii have been associated with outbreaks and pseudo-outbreaks in health care settings throughout the world.1–4 Infections caused by M. abscessus complex strains are particularly problematic as these species are not only the most pathogenic rapidly-growing mycobacteria, they are also the most antibiotic resistant NTM with treatment failure rates reported to be as high as 60–70% despite years of combination therapy.5
Nosocomial outbreaks of M. abscessus subsp. massiliense infections that reached unprecedented epidemic proportions have occurred in Brazil between 2004 and 2008 following laparoscopic surgeries and cosmetic procedures. Over 2,000 possible cases involving 15 different states and more than 86 hospital sites were reported and shown to have been caused by one single strain of M. abscessus subsp. massiliense, named BRA100.3–4 The most striking feature of BRA100 isolates was their unusually high level of resistance to glutaraldehyde (GTA), the chemical used for disinfection of surgical instruments in all of the hospital sites that had confirmed cases. Survival times of the BRA100 isolates exceeded 10-hour exposure to 2% GTA. This resistance phenotype was advanced as the most likely reason for the selection and dissemination of BRA100 across the country.6 Although by far the largest NTM-associated outbreaks ever reported, sporadic cases of nosocomial infections and pseudo-infections caused by M. chelonae and M. abscessus isolates resistant to aldehyde-based and other high-level disinfectants have been traced to automated endoscope/bronchoscope washer disinfectors and manual dialyzer reprocessing systems in Japan, the Netherlands, the UK and the US.2,7–16
While the ability of rapidly growing mycobacteria (RGM) to survive and persist within treated medical devices and washers may result from their high intrinsic tolerance to biocides, involvement in biofilms and association with free-living amoebae, it is also sometimes due to their ability to develop high level resistance to disinfectants as illustrated by M. abscessus subsp. massiliense BRA100 and a recently characterized GTA-resistant M. chelonae isolate from the UK.3,17 In the latter case, it was shown that defects in porin expression accounted, at least in part, for the resistance phenotype of the isolate, raising concerns that the selection of aldehyde-resistant strains in clinical settings might select for isolates displaying cross-resistances to other disinfectants/antibiotics and increased pathogenicity.17 Reduced porin expression in the rapidly-growing Mycobacterium species, Mycobacterium smegmatis, has indeed been associated with increased resistance not only to aldehyde-based products but also quaternary ammonium compounds, isothiazolinones, methylenbisoxazolidine and lipophilic biocides such as polyhexamethylene biguanine and octenidine dihydrochloride, in addition to multiple antibiotics.17–19 Furthermore, five studies reported that GTA-resistant M. chelonae and M. abscessus strains isolated either experimentally or from endoscope washer disinfectors presented an increased resistance to peracetic acid, peroxygen, sodium dichloroisocyanurate or quaternary ammonium compounds.13–14,20–22
Considering the prevalence of aldehyde-resistant RGM and concerns about the possible development of cross-resistance to other biocides, we undertook to profile the disinfectant-susceptibility pattern of a panel of aldehyde- susceptible and resistant Mycobacterium isolates to explore alternative high-level disinfection strategies in the case of 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, was tested against these isolates in suspension and carrier tests.
Methods
Bacterial Strains and Standard Growth Conditions
The six 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 four 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 Microbiology Systems) and 0.05% Tween 80 (Sigma Aldrich, St. Louis, MO) at 30°C to an optical density (OD600) of 1.0.
Disinfectants
Five commercial disinfectants labeled for high-level disinfection were evaluated along with the quaternary ammonium compound, cetrimonium bromide (10%; Sigma Aldrich). The five products included the GTA-based disinfectants Cidex® (2.4% GTA; Johnson & Johnson) and Aldahol® (3.4% GTA; Alden Medical LLC, West Springfield, MA, USA), the ortho-phthalaldehyde-based disinfectant Cidex® OPA (0.55% OPA; Johnson & Johnson), the peracetic acid-based products, Reliance™ DG (Steris, Mentor, OH, USA) and S40™ (Steris), and the hydrogen peroxide-based product Resert™ XL HLD (Steris). To best reflect the use of the products in clinical settings, Minimum Recommended Concentrations (MRC) or Minimum Effective Concentrations (MEC) were chosen for each disinfectant based on the minimum concentration of active disinfectant necessary to create a pass response on the corresponding manufacturer product testing strips. The pass response on Cidex® and Cidex® OPA is based on the MEC, while the pass response for Resert™ XL HLD and Aldahol® test strips is based on the MRC (see 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.
Table 1.
Mycobactericidal effects of aldehyde-based products against GTA-susceptible and GTA-resistant Mycobacterium isolates in suspension tests.
| Product tested |
Mmas CIP 108297 (GTAS) |
Mmas CRM-0270 (GTAS) |
Mmas CRM-0019 (GTAR) |
Mch ATCC 35752 (GTAS) |
Mch 9917 (GTAR) |
Mch Harefield (GTAR) |
Mch Epping (GTAR) |
M. bovis BCG (Pasteur) (GTAS) |
M. avium 104 (GTAS) |
M. terrae ATCC 15755 (GTAS) |
|---|---|---|---|---|---|---|---|---|---|---|
| Cidex® (1.5% GTA) 25°C | ||||||||||
| 0 min | 8.20 | 8.29 | 7.79 | 7.11 | 7.06 | 7.60 | 7.65 | 6.29 | 8.04 | 7.97 |
| 5 min | 5.27±0.08 | 0 | 7.47±0.04 | 0 | 7.55±0.05 | 7.96±0.00 | 8.02±0.11 | 4.33±0.26 | nd | 4.70±0.18 |
| 10 min | 0 | 0 | 7.48±0.13 | 0 | 7.49 | 7.87±0.06 | 7.95±0.02 | 3.30±2.55 | 5.97±0.03 | 3.81±0.14 |
| 15 min | 0 | 0 | 7.48±0.05 | 0 | 6.95±0.21 | 7.70±0.05 | 7.91±0.04 | 0 | 5.10±0.35 | 3.18±0.21 |
| 30 min | 0 | 0 | 7.23±0.03 | 0 | 7.23±0.03 | 7.50±0.09 | 7.73±0.04 | 0 | 0 | 0 |
| 0 min | nd | nd | 7.39 | nd | 8.83 | 8.33 | 8.38 | nd | nd | nd |
| 45 min | nd | nd | 6.76±0.02 | nd | 5.63±0.38 | 6.85±0.15 | 7.20±0.01 | nd | nd | nd |
| Aldahol® (1.8% GTA) 20°C | ||||||||||
| 0 min | 7.25 | 8.29 | 6.69 | 7.11 | 7.06 | 7.60 | 7.65 | 6.29 | 8.16 | 7.97 |
| 5 min | 0 | 0 | 4.54±0.13 | 0 | 7.39±0.09 | 7.78±0.00 | 7.45±0.04 | 0 | 0 | 0 |
| 10 min | 0 | 0 | 3.36±0.01 | 0 | 7.27 | 7.64±0.01 | 7.15±0.17 | 0 | 0 | 0 |
| 15 min | 0 | 0 | 2.49±0.71 | 0 | 6.97 | 7.44±0.08 | 6.97±0.04 | 0 | 0 | 0 |
| 30 min | 0 | 0 | 0 | 0 | 6.13 | 7.17±0.03 | 6.65±0.00 | 0 | 0 | 0 |
| Aldahol® (1.8% GTA) 25°C | ||||||||||
| 0 min | 7.25 | 8.29 | 6.69 | 7.11 | 8.13 | 7.60 | 7.65 | 6.29 | 8.32 | 7.97 |
| 5 min | 0 | 0 | 0 | 0 | 0 | 7.30±0.02 | 4.70±0 | 0 | 0 | 0 |
| 10 min | 0 | 0 | 0 | 0 | 0 | 6.92±0.25 | 0 | 0 | 0 | 0 |
| 15 min | 0 | 0 | 0 | 0 | 0 | 6.58±0.17 | 0 | 0 | 0 | 0 |
| 30 min | 0 | 0 | 0 | 0 | 0 | 5.98±0.17 | 0 | 0 | 0 | 0 |
| 15% Isopropanol 25°C | ||||||||||
| 0 min | 8.31 | nd | 8.23 | nd | nd | nd | nd | nd | nd | nd |
| 5 min | 8.36±0.07 | nd | 9.02±0.03 | nd | nd | nd | nd | nd | nd | nd |
| 10 min | 8.53±0.15 | nd | 8.86±0.08 | nd | nd | nd | nd | nd | nd | nd |
| 15 min | 8.43±0.03 | nd | 8.87±0.00 | nd | nd | nd | nd | nd | nd | nd |
| 30 min | 8.32±0.05 | nd | 8.88±0.10 | nd | nd | nd | nd | nd | nd | nd |
| Cidex® OPA (0.3% OPA) 25°C | ||||||||||
| 0 min | 7.25 | 8.29 | 7.32 | 7.11 | 8.13 | 7.63 | 7.65 | 6.29 | 8.32 | 7.97 |
| 5 min | 0 | 0 | 0 | 0 | 0 | 3.92±0.21 | 0 | 0 | 0 | 0 |
| 10 min | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 15 min | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 30 min | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Cidex® OPA (0.3% OPA) 20°C | ||||||||||
| 0 min | nd | 5.13 | 7.64 | 7.69 | 8.27 | 7.65 | 7.80 | nd | nd | nd |
| 12 min | nd | 0 | 0 | 0 | 0 | 0 | 0 | nd | nd | nd |
| 30 min | nd | 0 | 0 | 0 | 0 | 0 | 0 | nd | nd | nd |
Shown are the mean log10 CFU counts and standard deviations of duplicate tests for each strain and test product. The 0 min time point (enumeration of CFU in the bacterial suspension prior to disinfectant exposure) is the result of a single serial dilution plating. GTAR, GTA-resistant; GTAS, GTA-susceptible. nd, not determined. Control suspension tests using the GTA-susceptible isolate, M. massiliense CIP 108297, confirmed the neutralizing efficacy of the neutralizing broth with each disinfectant product. The detection limit of the suspension tests is 1 CFU/mL.
Table 3.
Mycobactericidal effects of oxidizing high-level disinfectants and a quaternary ammonium compound against GTA-susceptible and GTA-resistant Mycobacterium isolates in suspension tests.
| Product tested |
Mmas CIP 108297 (GTAS) |
Mmas CRM-0270 (GTAS) |
Mmas CRM-0019 (GTAR) |
Mch ATCC 35752 (GTAS) |
Mch 9917 (GTAR) |
Mch Harefield (GTAR) |
Mch Epping (GTAR) |
M. bovis BCG (Pasteur) (GTAS) |
M. avium 104 (GTAS) |
M. terrae ATCC 15755 (GTAS) |
|---|---|---|---|---|---|---|---|---|---|---|
| Reliance™ DG 50°C | ||||||||||
| 0 min | 8.39 | 8.42 | 9.00 | 8.79 | 7.54 | 8.13 | 7.05 | 6.92 | 9.15 | 7.92 |
| 2 min | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 6 min | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| S40™ 50°C | ||||||||||
| 0 min | 8.63 | 8.73 | 9.11 | 8.58 | 7.75 | 7.92 | 7.91 | 6.92 | 9.15 | 7.92 |
| 2 min | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 6 min | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Resert™ XL 25°C | ||||||||||
| 0 min | 8.63 | 8.73 | 9.11 | 8.58 | 7.89 | 7.92 | 8.40 | nd | 9.15 | nd |
| 4 min | 0 | 3.12±0.18 | 3.51±0.22 | 0 | 0 | 4.17±0.11 | 0 | nd | 0 | nd |
| 8 min | 0 | 0 | 0 | 0 | 0 | 0 | 0 | nd | 0 | nd |
| CTAB 25°C | ||||||||||
| 0 min | 8.39 | 8.42 | 9.00 | 8.79 | 7.89 | 8.13 | 8.40 | nd | nd | nd |
| 60 min | 5.97±0.01 | 6.15±0.02 | nd | 8.33±0.02 | 7.61±0.03 | 7.97±0.04 | nd | nd | nd | nd |
| 1440 min | 6.27 | 5.55±0.12 | 7.16±0.05 | 5.05±0.90 | 6.46±0.16 | 7.43±0.01 | 6.35±0.03 | nd | nd | nd |
| Sterile Water 50°C | ||||||||||
| 0 min | nd | nd | 8.80 | nd | nd | 8.20 | nd | nd | nd | nd |
| 2 min | nd | nd | 8.27±0.11 | nd | nd | 7.96±0.02 | nd | nd | nd | nd |
| 6 min | nd | nd | 8.24±0.13 | nd | nd | 8.00±0.12 | nd | nd | nd | nd |
See Table 1 for legend. Control suspension tests using the GTA-susceptible isolate, M. massiliense CIP 108297, confirmed the neutralizing efficacy of the neutralizing broth with each disinfectant product. The detection limit of the suspension tests is 1 CFU/mL.
Suspension Tests
Each mycobacterial strain was prepared for testing by adding 1.0 mL of 0.9% sterile saline (Hospira, Lake Forest, IL) containing 0.1% Tween 80 to 9.0 mL of bacterial suspension (approximately 1010 Colony Forming Units [CFU]) previously washed three times with sterile double distilled water. The preparation was then dispersed for 1 min using a sterile 15 ml Wheaton pestle to generate a homogeneous bacterial suspension. One mL of bacterial suspension was added to 99 mL of the disinfectant solution maintained at the recommended temperature under continuous mixing conditions. At pre-determined time points, 1.0 mL of test solution was removed and placed in either 9.0 mL of sterile Dey Engley/1% sodium thiosulfate neutralizing broth (BD Difco, Sparks, MD; Sigma Aldrich, St. Louis, MO) for oxidizers or 9.0 mL of sterile Dey Engley/1% sodium bisulfite neutralizing broth (BD Difco, Sparks, MD; Sigma Aldrich, St. Louis, MO) 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 Corporation, Ann Arbor, MI). After a final rinse of the filter housing with 30–40 ml 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 at which point CFU were counted. Control experiments 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 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 1 CFU/mL. All suspension and carrier tests were performed in duplicate using independent bacterial culture batches and the results of one typical experiment are shown.
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 30–40 min. Each carrier disk was then sterilely placed inoculum side up within a 30 ml straight-side wide-mouth Nalgene jar (Thermo Scientific, Rochester, NY). 50 μL of the freshly prepared disinfectant solutions pre-heated to the recommended temperature was applied to the dried inoculum on each carrier disk and held for the desired contact time. 9.95 mL of sterile neutralizing broth was then added to the jar and vortexed for 60 seconds before vacuum filtration as described above. The carrier disk was then vortexed three more times with 15 mL of 0.9% sterile saline/0.1% Tween 80 with vacuum filtration 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 ten different rapidly growing and slowing growing mycobacteria, including four M. chelonae and M. abscessus subsp. massiliense isolates that had been reported previously to display resistance to GTA. Each disinfectant was tested using two independent methods, a suspension test and a carrier test, the latter being considered more stringent and more reflective than the former of the disinfection of microorganisms on dried surfaces. Tables 1 and 3 show the results of the suspension tests, while Table 2 shows the results of the carrier tests.
Table 2.
Mycobactericidal effects of aldehyde-based and oxidizing high-level disinfectants against GTA-susceptible and GTA-resistant Mycobacterium isolates in carrier tests.
| Product tested |
Mmas CIP 108297 (GTAS) |
Mmas CRM-0270 (GTAS) |
Mmas CRM-0019 (GTAR) |
Mch ATCC 35752 (GTAS) |
Mch 9917 (GTAR) |
Mch Harefield (GTAR) |
Mch Epping (GTAR) |
M. bovis BCG (Pasteur) (GTAS) |
M. avium 104 (GTAS) |
M. terrae ATCC 15755 (GTAS) |
|---|---|---|---|---|---|---|---|---|---|---|
| Cidex® (1.5% GTA) 25°C | ||||||||||
| 0 min | 7.55±0.03 | 7.30±0.06 | 7.52±0.00 | 7.59±0.05 | 6.91±0.11 | 7.34±0.01 | 7.44±0.11 | nd | nd | nd |
| 45 min | 0 | 0 | 6.14±0.06 | 0 | 4.59±0.28 | 3.74±0.00 | 5.77±1.3 | nd | nd | nd |
| Aldahol® (1.8% GTA) 20°C | ||||||||||
| 0 min | 7.55±0.03 | 7.30±0.06 | 7.52±0.00 | 7.59±0.05 | 6.91±0.11 | 7.38±0.12 | 7.44±0.11 | nd | nd | nd |
| 20 min | 0 | 0 | 0 | 0 | 4.57±0.17 | 2.96±0.09 | 5.94±0.35 | nd | nd | nd |
| 15% Isopropanol 25°C | ||||||||||
| 0 min | 7.55±0.03 | 7.30±0.06 | nd | 7.59±0.05 | 6.91±0.11 | 7.38±0.12 | 7.44±0.11 | nd | 8.43 | nd |
| 20 min | 7.67±0.34 | 7.77±0.00 | nd | 7.91±0.05 | 6.89±0.02 | 7.45±0.14 | 7.52±0.17 | nd | 6.33±0.00 | nd |
| Cidex® OPA (0.3% OPA) 25°C | ||||||||||
| 0 min | 7.55±0.03 | 7.30±.06 | 7.52±0.00 | 7.59±0.05 | 6.91±0.11 | 7.38±0.12 | 7.44±0.11 | nd | nd | nd |
| 5 min | 0 | 0 | 0 | 0 | 0 | 0 | 0 | nd | nd | nd |
| Reliance™ DG 50°C | ||||||||||
| 0 min | 7.45±0.01 | 7.59±0.06 | 8.31±0.00 | 7.63±0.03 | 6.49±0.00 | 7.63±0.06 | 7.46±0.00 | nd | 8.43±0.03 | 7.00±0.08 |
| 6 min | 0 | 0 | 0 | 0 | 0 | 0 | 0 | nd | 0 | 0 |
| S40™ 50°C | ||||||||||
| 0 min | 7.45±0.01 | 7.59±0.06 | 8.31±0.00 | 7.63±0.03 | 6.49±0.00 | 7.63±0.06 | 7.46±0.00 | nd | 8.43±0.03 | 7.00±0.08 |
| 6 min | 0 | 0 | 0 | 0 | 0 | 0 | 0 | nd | 0 | 0 |
| Resert™ XL 25°C | ||||||||||
| 0 min | 7.55±0.03 | 7.30±0.06 | nd | 7.59±0.05 | 6.91±0.11 | 7.38±0.12 | 7.44±0.11 | nd | nd | nd |
| 8 min | 0 | 0 | nd | 0 | 0 | 0 | 0 | nd | nd | nd |
See Table 1 for legend. Control carrier tests using the GTA-susceptible isolate, M. massiliense CIP 108297, confirmed the neutralizing efficacy of the neutralizing broth with each disinfectant product. The detection limit of the carrier disk tests is 1 CFU.
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. massiliense CRM-0270. Under the suspension test, control strains were reduced to no detectable bacterial counts within 10 min 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 min [Table 1]. Reduction to no detectable bacterial counts was achieved for all control strains within 5 min of exposure to Aldahol® (1.8% GTA), a GTA-based product containing isopropanol 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. massiliense and M. chelonae isolates, CRM-0019, 9917, Harefield and Epping, showed significant resistance to the GTA-based products with less than a half log10 reduction in CFUs after 10 min exposure to Cidex® (1.5% GTA) in suspension tests (less than 3.2 log10 reduction in CFUs after the recommended time of exposure of 45 min), and less than a 3.6 log10 CFU reduction after 45 min in the carrier test [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 min and temperature of 20°C showed significant resistance to this disinfectant as well [Table 1]. The greatest CFU reduction at the 10 min time point was seen 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 less than a 1.0 log reduction in CFU counts. When tested at additional time points out to 30 min, the three GTA-resistant M. chelonae strains continued to show greater resistance with a maximum 1.0 log10 reduction in CFUs while M. abscessus subsp. massiliense CRM-0019 was reduced to no detectable CFU by 30 min. The carrier disk testing similarly pointed to the significant resistance of M. chelonae 9917, Harefield and Epping with no more than a 4.42 log10 CFU reduction after 20 min, while M. abscessus subsp. massiliense CRM-0019 again showed susceptibility to Aldahol® at the extended 20 min time point with a greater than 7.0 log10 CFU reduction [Table 2]. When the testing temperature for Aldahol® was increased from 20 to 25°C, the suspension testing results showed greater bactericidal efficacy. All GTA-resistant strains but one were reduced to no detectable CFU within the recommended 10 min. However, M. chelonae Harefield only achieved a 0.68 log10 reduction after 10 min and, even at the extended 30 min time point, CFU counts were only reduced by a meager 1.62 log10 [Table 1]. Since 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 upon 30 min exposure to this compound [Table 1].
ortho-phthalaldehyde
Suspension test results for the Cidex® OPA (0.3% OPA) at 25°C achieved reduction to no detectable CFU counts at the recommended time point of 5 min for all M. chelonae strains except for M. chelonae Harefield. However, this strain was reduced to no detectable CFU by the 10 min time point at 25°C, and to no detectable CFU by the recommended 12 min time point at 20°C [Table 1]. When 0.3% OPA was tested under the carrier disk method, all test strains were reduced to no viable CFU within 5 min [Table 2].
Oxidizing agents and other disinfectant products
For the two disinfectants requiring usage in automated endoscope washers at 50°C, S40™ and Reliance™ DG (two peracetic acid-based products), CFU counts for all tested strains were reduced below detection levels within 2 min of exposure under the suspension testing method, and by the 6 min time point under the carrier disk method [Tables 2 and 3]. To assess the impact of the 50°C temperature on the viability of mycobacteria, two GTA-resistant strains, M. abscessus subsp. massiliense CRM-0019 and M. chelonae Harefield, were exposed to sterile water at 50°C for up to 6 min; in both cases, no significant loss of viability was detected [Table 3]. For Resert™ XL (a hydrogen peroxide-based product), viable bacterial counts were reduced greater than 7 log10 within 8 min under both the suspension and carrier disk methods [Tables 2 and 3]. Finally, testing of a quaternary ammonium compound found in many disinfectants and antiseptics (10% CTAB) in the suspension test demonstrated, as expected,22 very modest bactericidal activity against rapidly growing mycobacteria, with less than 4 log10 reduction in CFU for all tested strains after 24 hours [Table 3].
Discussion
Whereas OPA is now the leading high-level disinfectant in the USA, activated alkaline GTA at ambient temperature remains one of the most widely used high-level disinfectant across the world for its wide spectrum microbicidal activity, relatively low cost, ease of use and its non-damaging effects on medical equipment and reprocessors. Concerns over the emergence of globally-circulating virulent clones of M. abscessus complex species25 coupled to reports of rapidly growing mycobacteria presenting resistance to aldehyde-based products, including recently, during the largest outbreak ever reported of M. abscessus complex infections in Brazil,3–4,6 and the finding that some of the mechanisms of resistance to aldehydes evolved by NTM may confer cross-resistance to other disinfectants and antibiotics,17,19 led us to systematically investigate the susceptibility profile of a collection of GTA-resistant M. chelonae and M. abscessus subsp. massiliense isolates to various disinfectant chemistries using two different testing methods.
Tests with the GTA-based products Cidex® and Aldahol® confirmed the aldehyde resistance phenotype of the four M. abscessus subsp. massiliense and M. chelonae isolates from the UK and Brazil. Interestingly, increasing the incubation temperature of Aldahol® from 20 to 25°C overcame this resistance in the case of all isolates but one, M. chelonae Harefield, probably reflecting differences in the physiology and mechanisms of aldehyde-resistance evolved by these isolates. Similarly, Cidex® OPA at 25°C showed variable efficacy against the GTA-resistant strains achieving only partial killing of M. chelonae Harefield after the recommended 5 min time point in the suspension test at the minimum effective concentration of 0.3% OPA; however, although CFU counts for all isolates were reduced to 0 upon exposure to 0.3% OPA for 12 min at 20°C. Under the testing conditions used herein that lacked organic load in the suspensions and, therefore, favored disinfectant activity, all of the Mycobacterium isolates tested were fully susceptible to the peracetic acid- and hydrogen peroxide-based disinfectants, independent of their level of resistance to GTA.
In summary, all of the oxidizing disinfectants tested in this study effectively killed GTA-resistant mycobacteria while aldehyde-based products (GTA and OPA) showed variable results despite evidence that changing the products formulation (i.e., Aldahol®) and increasing the temperature might improved their efficacy against some isolates. Although more isolates would need to be tested in the presence and absence of organic load to reach definitive conclusions, it appears that the use of oxidizing chemistry (compatibility with the medical devices permitting) provides a safe alternative to aldehyde-based products, especially when GTA resistance is suspected. Alternatively, increasing the temperature and/or formulation of aldehyde-based products may increase their efficacy but this option should carefully be evaluated on a case-to-case basis.
Acknowledgments
This work received support from the STERIS Foundation, DOW Microbial Control, the College Research Council (College of Veterinary Medicine and Biomedical Sciences) at Colorado State University, and the NIH/NIAID grant AI089718. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The funding sources had no involvement in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the article for publication. All authors report no conflicts of interest relevant to this article. We thank Dr. N. Miner and V. Harris (MicroChem Laboratory Inc.), and Dr. G. McDonnell and A. Fiorello (Steris, Inc.) for their advice with the disinfectant testing methods.
References
- 1.Phillips MS, Fordham von Reyn C. Nosocomial infections due to nontuberculous mycobacteria. Clin Infect Dis. 2001;33:1363–1374. doi: 10.1086/323126. [DOI] [PubMed] [Google Scholar]
- 2.Rutala WA, Weber DJ Healthcare Infection Control Practices Advisory Committee. Guideline for Disinfection and Sterilization in Healthcare Facilities. Centers for Disease Control and Prevention website. 2008 http://www.cdc.gov/ncidod/dhqp/
- 3.Duarte RS, Lourenco MCS, de Souza Fonseca LS, et al. An epidemic of postsurgical infections caused by Mycobacterium massiliense. J Clin Microbiol. 2009;47:2149–2155. doi: 10.1128/JCM.00027-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Leao SC, Viana-Niero C, Matsumoto CK, et al. Epidemic of surgical-site infections by a single clone of rapidly growing mycobacteria in Brazil. Future Microbiol. 2010;5:971–980. doi: 10.2217/fmb.10.49. [DOI] [PubMed] [Google Scholar]
- 5.Jarand J, Levin A, Zhang L, et al. Clinical and microbiologic outcomes in patients receiving treatment for Mycobacterium abscessus pulmonary disease. Clin Infect Dis. 2011;52:565–571. doi: 10.1093/cid/ciq237. [DOI] [PubMed] [Google Scholar]
- 6.Lorena NOS, Duarte RS, Pitombo MB. Rapidly growing mycobacteria infection after videosurgical procedures - The glutaraldehyde hypothesis. Rev Col Bras Cir. 2009;36:266–277. doi: 10.1590/s0100-69912009000300015. [DOI] [PubMed] [Google Scholar]
- 7.Bolan G, Reingold AL, Carson LA, et al. Infections with Mycobacterium chelonei in patients receiving dialysis and using processed hemodialyzers. J Infect Dis. 1985;152:1013–1019. doi: 10.1093/infdis/152.5.1013. [DOI] [PubMed] [Google Scholar]
- 8.Lowry PW, Beck-Sague CM, Bland LA, et al. Mycobacterium chelonae infection among patients receiving high-flux dialysis in a hemodialysis clinic in California. J Infect Dis. 1990;161:85–90. doi: 10.1093/infdis/161.1.85. [DOI] [PubMed] [Google Scholar]
- 9.Centers for Disease Control and Prevention. Nosocomial infection and pseudoinfection from contaminated endoscopes and bronchoscopes - Wisconsin and Missouri. MMWR. 1991;40:675–678. [PubMed] [Google Scholar]
- 10.De Groote MA, Gibbs S, de Moura VC, et al. Analysis of a panel of rapidly growing mycobacteria for resistance to aldehyde-based disinfectants. Am J Infect Control. 2014;42:932–934. doi: 10.1016/j.ajic.2014.05.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Hayes PS, McGiboney DL, Band JD, et al. Resistance of Mycobacterium chelonei-like organisms to formaldehyde. Appl Environ Microbiol. 1982;43:722–724. doi: 10.1128/aem.43.3.722-724.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Centers for Disease Control and Prevention. Epidemiologic notes and reports - Nontuberculous mycobacterial infections in hemodialysis patients - Louisiana, 1982. MMWR. 1983;32:244–246. [PubMed] [Google Scholar]
- 13.van Klingeren B, Pullen W. Glutaraldehyde resistant mycobacteria from bronchoscope washers. J Hosp Infect. 1993;25:147–149. doi: 10.1016/0195-6701(93)90107-b. [DOI] [PubMed] [Google Scholar]
- 14.Griffiths PA, Babb JR, Bradley CR, et al. Glutaraldehyde-resistant Mycobacterium chelonae from endoscope washer disinfectors. J Applied Bacteriol. 1997;82:519–528. doi: 10.1046/j.1365-2672.1997.00171.x. [DOI] [PubMed] [Google Scholar]
- 15.Nomura K, Ogawa M, Miyamoto H, et al. Antibiotic susceptibility of glutaraldehyde-tolerant Mycobacterium chelonae from bronchoscope washing machines. Am J Infect Control. 2004;32:185–188. doi: 10.1016/j.ajic.2003.07.007. [DOI] [PubMed] [Google Scholar]
- 16.Fisher CW, Fiorello A, Shaffer D, et al. Aldehyde-resistant mycobacteria bacteria associated with the use of endoscope reprocessing systems. Am J Infect Control. 2012;40:880–882. doi: 10.1016/j.ajic.2011.11.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Svetlíková Z, Škovierová H, Niederweis M, et al. The role of porins in the susceptibility of Mycobacterium smegmatis and Mycobacterium chelonae to aldehyde-based disinfectants and drugs. Antimicrob Agents Chemother. 2009;53:4015–4018. doi: 10.1128/AAC.00590-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Stephan J, Mailaender C, Etienne G, et al. Multidrug resistance of a porin deletion mutant of Mycobacterium smegmatis. Antimicrob Agents Chemother. 2004;48:4163–4170. doi: 10.1128/AAC.48.11.4163-4170.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Frenzel E, Schmidt S, Niederweis M, et al. Importance of porins for biocide efficacy against Mycobacterium smegmatis. Appl Environ Microbiol. 2011;77:3068–3073. doi: 10.1128/AEM.02492-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Lynam PA, Babb JR, Fraise AP. Comparison of the mycobactericidal activity of 2% alkaline glutaraldehyde and ‘Nu-Cidex’ (0.35% peracetic acid) J Hosp Infect. 1995;30:237–240. doi: 10.1016/s0195-6701(95)90322-4. [DOI] [PubMed] [Google Scholar]
- 21.Stanley PM. Efficacy of peroxygen compounds against glutaraldehyde-resistant mycobacteria. Am J Infect Control. 1999;27:339–343. doi: 10.1016/s0196-6553(99)70054-4. [DOI] [PubMed] [Google Scholar]
- 22.Cortesia C, Lopez GJ, de Waard JH, et al. The use of quaternary ammonium disinfectants selects for persisters at high frequency from some species of non-tuberculous mycobacteria and may be associated with outbreaks of soft tissue infections. J Antimicrob Chemother. 2010;65:2574–2581. doi: 10.1093/jac/dkq366. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Fraud S, Hann AC, Maillard J-Y, et al. Effects of ortho-phthalaldehyde, glutaraldehyde and chlohexidine diacetate on Mycobacterium chelonae and Mycobacterium abscessus strains with modified permeability. J Antimicrob Chemother. 2003;51:575–584. doi: 10.1093/jac/dkg099. [DOI] [PubMed] [Google Scholar]
- 24.Miner N, Harris V, Cao TD, et al. Aldahol high-level disinfectant. Am J Infect Control. 2010;38:205–211. doi: 10.1016/j.ajic.2009.08.009. [DOI] [PubMed] [Google Scholar]
- 25.Bryant JM, Grogono DM, Rodriguez-Rincon D, et al. Emergence and spread of a human-transmissible multidrug-resistant nontuberculous mycobacterium. Science. 2016;354:751–757. doi: 10.1126/science.aaf8156. [DOI] [PMC free article] [PubMed] [Google Scholar]