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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 9  |  Issue : 2  |  Page : 173-175

Susceptibility of the Mycobacterium abscessus complex to drying: Implications for nebulizer hygiene in patients with cystic fibrosis


Department of Bacteriology, Northern Ireland Public Health Laboratory, Belfast City Hospital, Belfast; School of Biomedical Sciences, Ulster University, Coleraine; School of Medicine, Dentistry and Biomedical Science, The Wellcome.Wolfson Institute for Experimental Medicine, Queenfs University, Belfast, Northern Ireland

Date of Web Publication29-May-2020

Correspondence Address:
John Edmund Moore
Department of Bacteriology, Northern Ireland Public Health Laboratory, Belfast City Hospital, Lisburn Road, Belfast BT9 7AD
Northern Ireland
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmy.ijmy_62_20

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  Abstract 


Background: Nebulizer hygiene and care is important in cystic fibrosis (CF) to minimize device contamination from bacteria, including nontuberculous mycobacteria (NTMs). Most nebulizer manufacturers recommend nebulizer drying, however there is little evidence to understand how nebulizer drying affects NTM survival. Methods: Mycobacterium abscessus subsp. massiliense (n = 2), M. abscessus subsp. bolletii (n = 2), and M. abscessus subsp. abscessus (n = 2) were evaluated for their ability to survive simulated drying conditions associated with routine nebulizer care. Bacterial inocula (circa. 107 colony-forming units) were added to plastic and allowed to dry to completeness for 24 h, employing passive and active drying. Results: NTM isolates of all subspecies could be recovered from all passive and active drying experiments, both in diluent and in sterile sputum, following drying (24 h). There was no combination of drying or physiology that supported NTM cell death, and there was no difference in observed survival with the three species of M. abscessus examined. Conclusion: This study indicates that drying, either passively or actively, for 24 h at room temperature, is unable to eradicate all M. abscessus organisms from dry plastic surfaces, even in the presence of residual sputum contamination. Whilst drying may be advantageous for nebulizer performance, it should not be regarded as an absolute control for the elimination of NTM organisms. With nebulizer hygiene, NTM organisms would be able to survive on a nebulizer following drying for 24 h, which has not undergone any formal disinfection protocol. Therefore, for NTM eradication from washed nebulizers, CF patients should therefore seek an effective alternative control to drying for NTM eradication, i.e., heat disinfection in baby bottle disinfectors. CF patients and health-care professionals should not rely solely on nebulizer drying to achieve NTM eradication.

Keywords: Cystic fibrosis, drying, hygiene, nebuliser, nebulizer


How to cite this article:
Moore JE, Millar BC. Susceptibility of the Mycobacterium abscessus complex to drying: Implications for nebulizer hygiene in patients with cystic fibrosis. Int J Mycobacteriol 2020;9:173-5

How to cite this URL:
Moore JE, Millar BC. Susceptibility of the Mycobacterium abscessus complex to drying: Implications for nebulizer hygiene in patients with cystic fibrosis. Int J Mycobacteriol [serial online] 2020 [cited 2020 Jul 9];9:173-5. Available from: http://www.ijmyco.org/text.asp?2020/9/2/173/285235




  Introduction Top


Cystic fibrosis (CF) is a genetically inherited disease, most commonly associated with European populations. It is a multiorgan disease, associated with mainly the gastrointestinal tract and the lungs, where due to the molecular defect in the Cystic fibrosis transmembrane conductance regulator (CFTR) protein in the respiratory tract, sticky mucus accumulates trapping environmental bacteria in the airways, mainly Pseudomonas aeruginosa and other Gram-negative bacteria, leading to a vicious cycle of infection and inflammation.[1]

The delivery of nebulized therapy is an important component of care in CF patients, where nebulizers are employed to effectively deliver mainly antibiotics and mucolytic agents.[2] Proper maintenance of the nebulizer is important in terms of (i) ensuring that its performance is maintained optimally, as well as (ii) ensuring that nebulizer devices are effectively cleaned and disinfected, therefore becomes an important element of nebulized therapies. In addition, it is important to replace the nebulizer frequently, in accordance with nebulizer manufacturers' guidance.

More recently, the nontuberculous mycobacteria (NTM), particularly the Mycobacterium abscessus complex, consisting of M. abscessus subsp. massiliense, M. abscessus subsp. abscessus, and M. abscessus subsp. bolletii, have emerged as significant clinical pathogens with this disease. For a seminal review of the clinical significance of NTMs in CF, please see studies by Degiacomi et al.[3] and Richards and Olivier.[4] One potential source of NTMs with the CF patient, is the contamination of the patient's nebulizer device, from contaminated water used for nebulizer-cleaning purposes.[5]

As part of CF patients' routine hygienic care and maintenance of their nebulizer, most nebulizer manufacturers recommend drying of the nebulizer, however there is little evidence of the effectiveness of drying on residual contaminating bacteria, especially the NTMs. It was therefore the aim of this study to emulate typical nebulizer-drying procedures and to examine the consequences of these on NTM survival.


  Methods Top


Bacterial strains employed

Isolates (n = 6) from the three members of the M. abscessus complex were employed in this study, as detailed in [Table 1]. These included (i). M. abscessus subsp. massiliense (n = 2),
Table 1: Description of nontuberculous mycobacteria organisms employed and effect of active and passive drying on nontuberculous mycobacteria survival

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(ii). M. abscessus subsp. bolletii (n = 2), and (iii). M. abscessus subsp. abscessus (n = 2). All isolates were clinical reference isolates and were obtained from the Irish National Mycobacterium Reference Laboratory, St. James Hospital, Dublin, Ireland. All isolates were recovered on Columbia Blood agar (Oxoid CM0031, Oxoid Ltd., Basingstoke, UK), supplemented with 5% (v/v) defibrinated horse blood for 5 days at 37°C, under aerobic conditions and passaged a further three times, prior to use. Their identification was confirmed employing the Hain Genotype Mycobacterium assay (NTM-DR VER 1.0) (Hain Lifescience GmbH, Nehren, Germany). With regard to antibiotic resistance, no mutations to MA/AMG were detected in isolates NTM-A and NTM-E. All other NTM isolates harbored erm41 resistance to MA, but no mutations to AMG.

Drying of M. abscessus complex organisms

M. abscessus complex isolates (n = 6), as detailed in [Table 1], were prepared individually by culturing on Columbia Blood agar (Oxoid CM0031, Oxoid Ltd., Basingstoke, UK), supplemented with 5% (v/v) defibrinated horse blood for 5 days at 37°C. Following this, inocula were prepared in (i) sterile 0.1% (w/v) peptone saline diluent (Oxoid CM0733) and (ii) 10% (v/v) sterile CF sputum + 90% (v/v) sterile 0.1% (w/v) peptone saline diluent (Oxoid CM0733). Inocula (100 μL; mean inocula count = 7.38 × 107 colony-forming units) of each M. abscessus complex organism were placed on a plastic screw-cap sterile container (Sterilin, UK) and dried for 24 h, under two conditions, (a) passive drying: inoculated containers (without lids) were stored at room temperature (20°C/no forced air/24 h) in a Class II Biological safety cabinet with the hood closed and (ii) active drying: inoculated containers (without lids) were stored in an incubator (37°C/24 h). After this, 10 ml nutrient broth (Oxoid CM1) was added to each plastic container and incubated for 7 days at 37°C. Broths were visually checked for bacterial growth after 24 h, and each was subcultured (10 μL) onto Columbia Blood agar (Oxoid CM0031, Oxoid Ltd., Basingstoke, UK), supplemented with 5% (v/v) defibrinated horse blood for 5 days at 37°C, under aerobic conditions. Positive growth of M. abscessus complex organisms was confirmed by the presence of cloudy/snowflake growth in broth, along with typical colonial appearance of mycobacterial colonies, when compared to a nondried control culture of original inoculum.


  Results and Discussion Top


All NTM isolates of all subspecies could be recovered from all passive and active drying experiments, both in diluent (0.1% peptone saline) and in sterile sputum [Table 1], following drying for 24 h. There was no combination of drying or physiology that supported NTM cell death. Overall, there was no difference in observed survival with the three species of M. abscessus examined.

In this study, we examined the survival of NTM organisms on plastic surfaces following drying, emulating that of the CF patient and their practices in daily nebulizer care and hygiene. Our choice of three subspecies of M. abscessus was to reflect the important clinical species of NTM, as found in CF patients with NTM-related disease. In these experiments, we set out to emulate the typical manipulations of the nebulizer during nebulizer cleaning. The nebulizer may potentially pick up environmental NTM contamination from several water sources during washing, as well as dilution of contaminating NTM organism on the patients' nebulizer, if they are already NTM sputum positive. We attempted to emulate the drying of residual water from the washing/rinsing stage and during a subsequent 24 h period, prior to the nebulizer being used again. Such a 24 h period would be excessively long, as most patients would require to use their nebulizer after 12 h, but was included to check NTM survival, with an extended period of storage. NTM organisms were suspended in diluent, as well as in a 10% (v/v) diluted sputum concentration, to emulate poor nebulizer-washing practices, resulting in sputum carry over. The importance of including this experimental step was to assess how organisms may gain a survival advantage due to the presence of a matrix, which may protect them from dehydration and subsequent cell death. Postdrying, NTMs were detected qualitatively, as some may have adsorbed onto the plastic surface and thus would have been difficult to recover quantitatively, thereby leading to potentially diminished counts.

Nebulizer hygiene and care in patients with CF are important to minimize risks from microbial contamination from environmental sources, as well as from self-recontamination. Most nebulizer manufacturers recommend drying of the nebulizer, as part of the care and maintenance guidance given by manufacturers to patients. Recent studies have shown that for P. aeruginosa, drying of the nebulizer was lethal to these organisms if performed properly. In addition, any deviation from a thorough drying regimen resulted in the survival and proliferation of P. aeruginosa.[6] Therefore, it is of utmost importance not to extrapolate between CF bacterial pathogens, as these organisms have profoundly different biologies. In the NTMs, the presence of trehalose, a nonreducing disaccharide, is an integral component of glycolipids that are important in cell wall structures and which confer protection against desiccation.[7] Previously, M. abscessus was shown to survive desiccation for up to 2 weeks in the presence of house dust, kaolin, and several chemically defined mineral particulates.[8]


  Conclusions Top


This study indicates that drying, either passively or actively, for 24 h at room temperature, is unable to eradicate all M. abscessus organisms from dry plastic surfaces, even in the presence of residual sputum contamination. Whilst drying may be advantageous for nebulizer performance, it should not be regarded as an absolute control for the elimination of NTM organisms. Within the context of nebulizer hygiene, this means that these NTM organisms would be able to survive on a washed nebulizer following drying for 24 h, which has not undergone any formal disinfection protocol, which can eradicate NTM organisms. Therefore, for NTM eradication from washed nebulizers, CF patients should therefore seek an effective alternative control to drying for NTM eradication, i.e., heat disinfection in baby bottle disinfectors.[5],[9] CF patients and health-care professionals should not rely solely on nebulizer drying to achieve NTM eradication.

Acknowledgments

The authors wish to thank Professor Thomas Rogers and Dr. Margaret Fitzgibbon, Irish National Mycobacterial Reference Laboratory, St James' Hospital, Dublin, Ireland, for provision of the isolates used in this study. Additionally, the authors wish to thanks Mr. Mark Smyth, Northern Ireland Mycobacterial Reference Laboratory, Royal Victoria Hospital, Belfast, Northern Ireland, for preparation of isolates employed in this study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Turcios NL. Cystic fibrosis lung disease: An overview. Respir Care 2020;65:233-51.  Back to cited text no. 1
    
2.
MacFarlane M, Carson L, Crossan A, Bell J, Moore JE, Millar BC. Nebuliser cleaning and disinfection practice in the home among patients with cystic fibrosis. J Infect Prev 2020;21:14-22.  Back to cited text no. 2
    
3.
Degiacomi G, Sammartino JC, Chiarelli LR, Riabova O, Makarov V, Pasca MR. Mycobacterium abscessus, an emerging and worrisome pathogen among cystic fibrosis patients. Int J Mol Sci 2019;20:5868.  Back to cited text no. 3
    
4.
Richards CJ, Olivier KN. Nontuberculous mycobacteria in cystic fibrosis. Semin Respir Crit Care Med 2019;40:737-50.  Back to cited text no. 4
    
5.
Towle D, Callan DA, Lamprea C, Murray TS. Baby bottle steam sterilizers for disinfecting home nebulizers inoculated with non-tuberculous mycobacteria. J Hosp Infect 2016;92:222-5.  Back to cited text no. 5
    
6.
Moore JE, Moore RE, Bell J, Millar BC. Importance of nebulizer drying for patients with cystic fibrosis. Respir Care 2020. [in press] [ahead of print].  Back to cited text no. 6
    
7.
Elbein AD, Pan YT, Pastuszak I, Carroll D. New insights on trehalose: A multifunctional molecule. Glycobiology 2003;13:17R-27R.  Back to cited text no. 7
    
8.
Malcolm KC, Caceres SM, Honda JR, Davidson RM, Epperson LE, Strong M, et al. Mycobacterium abscessus displays fitness for fomite transmission. Appl Environ Microbiol 2017;83:e00562-17.  Back to cited text no. 8
    
9.
Hohenwarter K, Prammer W, Aichinger W, Reychler G. An evaluation of different steam disinfection protocols for cystic fibrosis nebulizers. J Cyst Fibros 2016;15:78-84.  Back to cited text no. 9
    



 
 
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