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. Author manuscript; available in PMC: 2015 Aug 1.
Published in final edited form as: Am J Infect Control. 2014 Aug;42(8):932–934. doi: 10.1016/j.ajic.2014.05.014

Analysis of a Panel of Rapidly Growing Mycobacteria for Resistance to Aldehyde-based Disinfectants

Mary Ann De Groote 1, Sara Gibbs 1, Vinicius Calado Nogueira de Moura 1, Winona Burgess 1, Kris Richardson 2, Shannon Kasperbauer 3, Nancy Madinger 2, Mary Jackson 1,#
PMCID: PMC4121594  NIHMSID: NIHMS600309  PMID: 25087149

Summary

After several accounts across the globe of mycobacterial outbreaks associated with medical procedures and aldehyde disinfectants resistance, we undertook an analysis of mycobacteria isolated from patients seen in a hospital in the United States between 1994 and 2008 to determine prevalence of resistance to aldehyde-based disinfectants. Out of the 117 clinical isolates screened, six isolates belonging to the emerging Mycobacterium abscessus group were found to display significant levels of resistance to glutaraldehyde and ortho-phthalaldehyde.

Infections with rapidly growing, non-tuberculous, mycobacteria (RGM) of the Mycobacterium abscessus group are increasing in prevalence throughout the world.1 A recent outbreak of widespread M. abscessus subsp massiliense infections (more than 2,000 possible cases) following video-assisted surgery in Brazil caused by a single highly virulent clone displaying high-level resistance to glutaraldehyde (GTA) highlights the pathogenic potential of these organisms and the important role that disinfectant failure may play in their spread.2-3 Although by far the largest RGM-associated outbreak ever reported, small outbreaks (9 to 26 cases) of nosocomial infections and pseudo-infections caused by mycobacteria resistant to aldehyde-based disinfectants have also been reported in the United States, and the recovery of RGM capable of surviving more than one to 24 hour exposure to 2% GTA or 2 to 10% formaldehyde from bronchoscope/endoscope-reprocessing machines and processed dialyzers has been reported globally.4-5 GTA and another aldehyde-based disinfectant, ortho-phthalaldehyde (OPA), remain the most widely used chemical disinfectants in hospitals worldwide for the high-level disinfection of semi-critical, temperature-sensitive medical devices. Our previous work has identified reduced porin expression as a possible mechanism through which RGM isolates may develop high-level resistance to aldehydes.6 Given the known impact of porins on both the virulence and resistance of RGM to a variety of antibiotics and biocides6, this finding raises concerns that aldehyde-resistant RGM strains may become a threat to infection control while causing infections that are more difficult to treat. Since RGM clinical isolates are typically not screened for disinfectant resistance unless associated to an outbreak, current data on the prevalence and trends of disinfectant resistance is lacking. The present study was therefore undertaken with the goals of determining the prevalence of GTA and OPA resistance among a panel of randomly selected RGM clinical isolates from the United States and assessing whether GTA or OPA resistance correlated with increased resistance to antibiotics.

One hundred seventeen sequential RGM isolates isolated between 1994 and 2008 from patients seen at a single university teaching hospital (University of Colorado Hospital, Denver) were sub-cultured and their speciation confirmed by standard molecular biology techniques. The panel included isolates of M. abscessus subsp abscessus (n=44), M. fortuitum (n=35), M. chelonae (n=12), M. peregrinum (n=4), M. mucogenicum (n=4), M. abscessus subsp massiliense (n=3), M. abscessus subsp bolletii (n=4), M. phocaicum (n=3), M. porcinum (n=4), M. farcinogenes (n=1), M. goodii (n=1), M. immunogenum (n=1), and M. canariasense (n=1). These isolates were primarily from pulmonary sources but also from skin and soft tissue, sinus, eye, ear and disseminated infections. 64% of patients were from Colorado and others originated from all regions of the United States including Southeastern (32%), Northeastern (16%), West (30%), Midwest (15%) and Hawaii (7%).

GTA was used in the University of Colorado hospital for many years until a switch to OPA was made in 2006. An initial screen for GTA- or OPA-resistant isolates was performed at 25°C in small volume suspension tests in freshly prepared GTA or OPA solutions as described in Fig. 1. The concentrations of GTA and OPA, temperature and incubation times were chosen to meet the processing and minimum effective concentration (MEC) requirements for high-level disinfection recommended by the manufacturer (i.e., minimum of 20 min at 25°C for 2.1% GTA/Cidex Plus®; minimum of 5 min at 25°C for 0.3% OPA/Cidex® OPA). Out of the 117 RGM isolates tested in this primary screen, 13 demonstrated greater than 6 log reduction in CFU counts after the indicated times of exposure to OPA or GTA but yielded a few viable CFUs. They belonged to the M. chelonae (n = 3), M. abscessus (n = 4), M. bolletii (n = 2) and M. fortuitum (n = 4) groups. Three to five subsequent repeat testing on these isolates yielded in most cases no survivors. The presence of a few viable CFUs in some tests may be explained either by bacterial clumping7 or to the existence in some culture batches of a sub-population of, e.g. stationary phase, phenotypically tolerant bacteria. Alternatively, the propensity of these 13 isolates to yield survivors may reflect reduced susceptibility to the disinfectants. To assess the latter hypothesis, the 13 strains were further compared to their reference ATCC strain as well as to other isolates selected at random from our panel for their ability to survive exposure to more diluted solutions of OPA (0.04 to 0.2%) and GTA (0.2 to 0.5%). As shown in Fig. 1, all six M. abscessus subsp abscessus and M. abscessus subsp bolletii isolates (# 34, 43, 53, 78, 91 and 97) consistently showed a significantly higher resistance to 0.5% GTA or 0.2% OPA than the control strains. The relatively high level of resistance to OPA of the M. abscessus subsp abscessus isolates # 91 and 34 in particular is remarkable as Cidex® OPA was only diluted 1.5-fold from the recommended MEC and killing was incomplete even after 15 min. The M. chelonae isolates # 3, 6 and 9 and the M. fortuitum isolates # 7, 41, 42 and 96, in contrast, only yielded survivors at the longest times of exposure when the disinfectant solutions were diluted to 0.2% GTA and 0.04% OPA (7.5 to 10-time dilutions of the recommended MEC) and their susceptibility to the disinfectants was in fact comparable to that of other isolates selected at random from the collection (Fig. 1).

Figure 1. GTA and OPA susceptibility of M. abscessus subsp abscessus, M. abscessus subsp bolletii, M. chelonae and M. fortuitum isolates.

Figure 1

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GTA and OPA susceptibility tests were performed in small volume (1 ml) suspension tests containing ~ 108 to 1010 CFUs in freshly prepared alkaline 2.1% GTA solution under the formulated form of Cidex Plus® (Johnson & Johnson) or 0.3% OPA solution under the form of Cidex® OPA (Johnson & Johnson) essentially as described.10 Results are expressed as Log10 CFU counts upon exposure of the test organisms to the indicated concentrations of disinfectant for 0 to 30 min. The MEC of 2.1% GTA and 0.3% OPA of the concentrated stock solutions were verified prior to each test using test strips specific to each disinfectant (3M Comply™ ColdSterilog™; Cidex OPA Solution Test Strips). Disinfectant neutralization was validated for each disinfectant tested and tests were performed in tri- to pentaplicates on independent culture batches. MAB, M. abscessus subsp abcessus; MBO, M. abscessus subsp bolletii; MCH, M. chelonae; MFO, M. fortuitum. The ATCC reference strains used in these studies (black squares) include M. abscessus subsp abscessus ATCC 19977, M. abscessus subsp bolletii ATCC 14472, M. fortuitum ATCC 6841 and M. chelonae ATCC 35752. The control clinical isolates selected at random from the collection (dashed lines) include the M. abscessus subsp abscessus isolates # 24, 30, 52, 63 and 73, the M. fortuitum isolate # 59 and the M. chelonae isolates # 13, 28 and 49.

While MIC results for the entire collection demonstrated the expected antimicrobial drug susceptibility by species, the MICs measured for isolates # 34, 43, 53, 78, 91 and 97 were comparable to those of other M. abscessus group isolates (data not shown). Thus, no correlation was found between disinfectant susceptibilities and the MIC of the drug classes tested therein.

In conclusion, the recurrent finding of M. abscessus isolates displaying significant levels of resistance to aldehyde-based disinfectants (OPA in particular) in the present study and earlier ones should increase awareness of the existence of such organisms, particularly in clinical settings where the use of disinfectants may promote their emergence, selection and spread in the environment. These organisms are indeed prone to surviving within the tubing of reprocessed heat-sensitive medical devices (e.g., endoscope channels, hemodialysis tubing), washer disinfectors, or any other kind of settings favoring the development of biofilms 8-9.

Acknowledgements

Human subject approval was obtained from the University of Colorado Hospital (HS 10-1197), National Jewish Health (HS-2557CO) and Colorado State University (10-078B). We wish to thank the human subject Institutional review boards of the University Hospital and Colorado State University, and Adrah Levin for assisting with human subject approval at National Jewish Health. We also thank Michelle Barron for advice and information on hospital infection control practices.

Footnotes

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Conflicts of Interests and Financial Disclosure: This work was supported in part by the National Institutes of Health / National Institute of Allergy and Infectious Diseases grant AI089718 and the STERIS Foundation. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

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