The International Journal of Mycobacteriology

ORIGINAL ARTICLE
Year
: 2022  |  Volume : 11  |  Issue : 3  |  Page : 256--260

Effect of Ultraviolet-c (UVc) light and ozone on the survival of Mycobacterium abscessus complex organisms associated with cystic fibrosis


John Edmund Moore, Beverley Cherie Millar 
 Laboratory for Disinfection and Pathogen Elimination Studies, Northern Ireland Public Health Laboratory, Belfast City Hospital, Belfast, Northern Ireland, UK

Correspondence Address:
John Edmund Moore
Laboratory for Disinfection and Pathogen Elimination Studies, Northern Ireland Public Health Laboratory, Belfast City Hospital, Belfast, Northern Ireland, BT9 7AD
UK

Abstract

Background: Members of the Mycobacterium abscessus complex have now emerged as clinically significant respiratory pathogens in people with cystic fibrosis (CF), potentially leading to increased disease severity, antibiotic treatment, and persistence dilemmas. Many of these species are resistant to disinfectants and biocides commonly used to clean and disinfect the hospital environment, thus necessitating the need to examine innovative ways to eliminate these organisms from such environments. It was, therefore, the aim of this study to examine the individual effect of ultraviolet-c (UVc) light (λ = 254 nm) and ozone (O3) on the growth of the M. abscessus complex organisms, as well as on seven other clinically significant CF pathogens, including Achromobacter spp., Burkholderia gladioli, Burkholderia cenocepacia, Burkholderia multivorans, Pseudomonas aeruginosa, Staphylococcus aureus, and Stenotrophomonas maltophilia. Methods: Bacterial isolates (n = 46), including M. abscessus complex (n = 6) (M. abscessus subsp abscessus [n = 2], M. abscessus subsp. bolletii [n = 2], M. abscessus subsp. massiliense [n = 2]), and other CF pathogens (n = 40) including Achromobacter spp., B. gladioli, B. cenocepacia, B. multivorans, P. aeruginosa, S. aureus, and S. maltophilia, were exposed for 1 h to UVc light (254 nm), as well as to ozone (O3; 26 ppm). Results: UVc light inactivated all M. abscessus complex organisms (n = 6), as well as the 40 isolates from the other genera and species. No bacterial species tested was able to survive the UVc treatment. O3 was unable to inactivate all isolates of M. abscessus subsp. abscessus (n = 2), M. abscessus subsp. bolletii (n = 2), and one isolate of M. abscessus subsp. massiliense, but killed one strain of M. abscessus subsp. massiliense. Overall, O3 inactivated only 20% of total isolates, allowing the posttreatment growth of the remaining 80% of isolates. There was no difference in the growth dynamic of P. aeruginosa from the environmental waters which had received O3 treatment and the control (untreated with O3). Bacterial growth, while occurring post-O3 treatment, was not as prolific in all remaining organisms, as in the untreated controls, demonstrating some but limited antibacterial effect. Conclusions: From the data presented by this study, UVc light at 254 nm was effective at eliminating all organisms examined, including members of the M. abscessus complex. Given the refractory nature of these organisms against conventional wet chemical disinfection, UVc potentially offers a physical method to control and eliminate the survival of these organisms on health-care surfaces and fomites. For many CF species examined in this study, these data represent the first reports of the organisms susceptibility to UVc light. Further work is now required to establish time/distance parameters incorporated into newly designed innovative devices, to allow disinfection protocols to be optimized, and delivered to exploit this vulnerability with these nontuberculous mycobacterial organisms, as well as with the other bacterial species examined.



How to cite this article:
Moore JE, Millar BC. Effect of Ultraviolet-c (UVc) light and ozone on the survival of Mycobacterium abscessus complex organisms associated with cystic fibrosis.Int J Mycobacteriol 2022;11:256-260


How to cite this URL:
Moore JE, Millar BC. Effect of Ultraviolet-c (UVc) light and ozone on the survival of Mycobacterium abscessus complex organisms associated with cystic fibrosis. Int J Mycobacteriol [serial online] 2022 [cited 2022 Dec 2 ];11:256-260
Available from: https://www.ijmyco.org/text.asp?2022/11/3/256/355931


Full Text



 Introduction



Several recent reports have highlighted the growing clinical concern with the global emergence of nontuberculous mycobacterial (NTM) infections in humans, particularly with Mycobacterium abscessus, in respiratory, skin, and mucosal infections.[1],[2],[3] This emergence has been observed in people with cystic fibrosis (CF) and complicates clinical treatment due to its persistence in the CF lung and difficulty in its treatment with antibiotics, due to its highly resistant nature,[4],[5] as well as its resistance to eradication within the clinical environmental within health care with disinfectants and biocides.[6] In response to such challenges, novel innovations in the discovery of new approaches to tackle such antimicrobial resistance with such NTM organisms have been recently reviewed and discussed.[7],[8]

In a previous study by Caskey et al.,[6] commonly employed hospital biocides, including acetone, propan-2-ol, diethylene glycol, 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one, chlorine dioxide, 4% chlorhexidine, alcohol, and disodium carbonate, compound with hydrogen peroxide, 10% sodium hypochlorite were assayed for their biocidal activity against M. abscessus clinical isolates obtained from CF and non-CF respiratory specimens. Results showed that all isolates survived in alkyldimethylbenzylammonium chloride, 5-chloro-2-methyl-2H-isothiazol-3-one (EC No. 247-500-7) and 2-methyl-2H-isothiazol-3-one, 4% Chlorhexidine™, O-phenylphenol, and Sodium Lauryl Sulfate™ and disodium carbonate, compound with hydrogen peroxide.[6] One out of 13 M. abscessus cultures was killed by Chlorine Dioxide™ and one by sodium dichloroisocyanurate, representing a 5-log kill. Two isolates were killed by alcohol again representing a 5-log kill. Following enrichment, O-phenylphenol and sodium lauryl sulfate showed the greatest biocidal activity with 11/13 isolates, whereas 2/13 cultures were killed by sodium dichloroisocyanurate.[6] All other biocides/culture combinations yielded growth.[6]

The data from this study indicated that M. abscessus may persist after exposure to several common hospital biocides, thereby creating a challenge for control of this NTH organism, both within and outside of health care.

The emergence and clinical mischief that these organisms are now causing can in part be attributed to their environmental origins and the intrinsic biology and strategies that these species have developed to become successful and persistent organisms within the environment. Their spread into the health-care environment and their establishment of chronic infections in susceptible patient populations, including those with CF, has led to urgency in being able to eradicate them from both the physical environment, as well as from anatomical sites of chronic colonization and persistence, especially the lungs.

Previously, there have been numerous reports on a diverse range of environments and fomites, both within and outside of health care, that these organisms are able to colonize and persist on, including musical instruments.[9],[10] Prospective studies testing the survival of an attenuated strain of M. tuberculosis on artificially inoculated reeds from a clarinet showed that this species was able to persist for 13 days postinoculation.[11]

Two technologies, namely ultraviolet (UV) light within the UVc range (200–280 nm; 4.43–12.4 eV; and 0.710–1.987 aJ)[12],[13] and ozone (O3), were examined in this study,[14] as both have previously been reported as having antimicrobial properties, including antimycobacterial properties. As there is a paucity of data on the effect of UVc light and O3 against CF pathogens, including the M. abscessus complex, it was, therefore, the aim of this study to examine the individual effect of UVc light (λ = 254 nm) and O3 on the growth of the M. abscessus complex organisms, as well as on seven other clinically significant CF pathogens, including Achromobacter spp., Burkholderia gladioli, Burkholderia cenocepacia, Burkholderia multivorans, Pseudomonas aeruginosa, Staphylococcus aureus, and Stenotrophomonas maltophilia.

 Methods



Description of bacterial isolates employed

Bacterial isolates (n = 46) were employed in this study, as detailed in [Table 1]. Six strains of M. abscessus complex, including M. abscessus subsp. massiliense (n = 2), M. abscessus subsp. bolletti (n = 2), and M. abscessus subsp. abscessus (n = 2), were employed in this study. All isolates were initially prepared by culturing on CBA supplemented with 5% (v/v) defibrinated horse blood (Oxoid SR0050) and incubated aerobically for 2 weeks at 30°C. In addition, 40 bacterial isolates, from seven species representing clinically significant CF bacterial pathogens, were employed [Table 1]. Isolates were prepared as for the mycobacterial isolates, with the exception of incubation at 37°C for 24–48 h, depending on the individual species' rate of growth.{Table 1}

Inocula of each strain were prepared by emulsifying colonies of each isolate individually into 0.1% (w/v) peptone saline diluent (Oxoid CM0733) (9 ml), equating to a McFarland 1.0 equivalence (approx. 3.0 × 108 colony-forming units (CFUs)/ml). Isolates were inoculated onto fresh CBA medium, as described above, equating to 106 CFUs.

Effect of ultraviolet-c light on Mycobacterium abscessus complex organisms and other cystic fibrosis bacterial pathogens

Inoculated plates (without lids) from (i) above were exposed to UVc light in an enclosed lightbox [Figure 1]. UVc light was generated by 4× PURITEC HNS linear fluorescent tubes (G15T8/OF 25/CS 1/SKU Osram Ltd., UK), positioned at 90° to each other to provide UVc exposure in full 360° rotation. Each lamp operated at 55V with a nominal wattage of 15W (0.33A) producing UVc light with a wavelength of 254 nm, without the production of O3. Inoculated plates were positioned 3–20 cm from at least one light and were exposed to the light for 1 h. Following this, plates were removed from the lightbox and were incubated with lids attached, as above. Duplicate control plates were exposed similarly but in the absence of the UVc light source.{Figure 1}

Effect of ozone on Mycobacterium abscessus complex organisms and other cystic fibrosis bacterial pathogens

Inoculated plates (without lids) from (i) above were exposed to O3 in a Class II PCR cabinet (Microflow Omni PCR Workstation, Bioquell Ltd., UK) of internal volume of 134.52 L. O3 was generated by a commercial O3 generator (Iflyey). The O3 generator was placed in the cabinet and allowed to operate for 1 h to allow the concentration of O3 to equilibrate within the cabinet, before the introduction of the inoculated plates. Inoculated plates were introduced and exposed for 1 h at an O3 concentration of 26 mg O3/l air (26 ppm). Following this, plates were removed from the O3 cabinet and were incubated with lids attached, as above. Duplicate control plates were exposed similarly but in the absence of O3.

Assessment of bacterial growth posttreatment

Following incubation, inoculated plates were examined qualitatively for the presence of bacterial growth. Growth was recorded as +++ (heavy– confluent growth), ++ (heavy growth), + (slight/weak growth), few (few colonies seen), and no growth.

 Results



The individual effects of UVc light and O3 on the growth of M. abscessus complex organisms and the other seven species (including six Gram-negative organisms + 1 Gram-positive organism) are shown in [Table 1]. In summary, UVc light inactivated all 46 isolates from all genera and species. Ozone inactivated 20% of isolates, allowing the posttreatment growth of the remaining 80% of isolates. There was no difference in the growth dynamic of P. aeruginosa from the environmental waters which had received O3 treatment and the control (untreated with O3). Growth was not as prolific in all remaining organisms, as in the untreated controls.

 Discussion



The resistance of M. abscessus complex organisms to several disinfection regimes creates a problem for infection prevention and control strategies within health care. This resistance is in the form of several hospital wet disinfectants to effectively kill NTM organisms.[6] Furthermore, many fomites and electronic devices would not be able to withstand wet or wet immersion disinfection protocols, so it is important to explore physical as well as gaseous interventions that avoid the disinfection target surface from becoming saturated with liquid.

The application of UVc light was successful in eradicating all isolates examined, including the M. abscessus complex organisms. This is an important finding, as this vulnerability of the NTM organisms, as well as the other CF pathogens, to UVc light creates a potential disinfection modality that may be exploited, to aid with cleaning and disinfection regimes both within and outside of health care, where these organisms pose an infection control and prevention risk.

Whilst UVc light may be an effective physical disinfection modality on flat surfaces, care should be taken, as any obstruction in the path of the UV light from its source to its intended target, shadowed area or the presence of blindspots would severely diminish the effectiveness of the disinfection process[15] and thus allow the survival of bacterial cells which have avoided exposure to the UV source. Therefore, such disinfection as UVc, should not be used to disinfect the inner surfaces of fomites such as respiratory nebulizer devices or musical instruments, as the UV light will not be able to penetrate through the fomite nor bend to allow its exposure to viable bacterial cells attached to inner, shadowed, or convulated aspects of the device. In such cases with nebulizer disinfection, this should be achieved through heat using steam through baby bottle disinfector devices, as previously reported.[16],[17]

Limitations of the study

This current study presented its results in qualitative form and hence should be considered a pilot study demonstrating proof of concept. While qualitative data are adequate where we observed complete kill in the case of the UVc studies, it is not possible to determine log reduction of viable numbers of surviving organisms in the O3 arm of the study. Furthermore, the O3 studies examined one concentration of O3 (26 ppm for 1 h). Previous studies employing O3 at a higher concentration of >100 ppm with similar laboratory reference pathogens showed a complete kill of these organisms, after an 8-min O3 infusion followed by 90-min dwell time.[18] This comparator study did not report on O3's ability to kill NTM organisms. In contrast, our study showed the limited effect of O3 to eliminate the same CF pathogens using predominantly wild-type environmental and patient isolates. Further work is, therefore, now required to upscale the O3 concentration and contact time, to anticipate greater kill effects and to potentially define boundaries where O3 may be effective.

 Conclusions



From the data presented by this study, UVc light at 254 nm was effective at eliminating all organisms examined, including members of the M. abscessus complex. Given the refractory nature of these organisms against conventional wet chemical disinfection, UVc potentially offers a physical method to control and eliminate the survival of these organisms on health-care surfaces and fomites. For many CF species examined in this study, these data represent the first reports of the organisms' susceptibility to UVc light. Further work is now required to establish time/distance parameters incorporated into newly designed innovative devices, to allow disinfection protocols to be optimized, and delivered to exploit this vulnerability with these NTM organisms, as well as with the other bacterial species examined.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Data availability statement

All data relevant to the study are included in the article.

Acknowledgments

The authors wish to acknowledge with thanks support from Mr. Alan Murphy, Media Department, Northern Ireland Public Health Laboratory, Belfast City Hospital, for the preparation of the culture media employed in this study. The authors also wish to thank Mr. Benedict Marley and Mr. Darren Canmore, from the Northern Ireland Education Authority's Music Service, for discussions relating to the disinfection of musical instruments.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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