Survival and re-aerosolization in dust of mycobacterium smegmatis - a surrogate for mycobacterium tuberculosis

Tshilombo, Kazadi Valéry (2017-03)

Thesis (MSc)-- Stellenbosch University, 2017.

Thesis

ENGLISH SUMMARY : Background: Mycobacterium tuberculosis (M. tuberculosis) is essentially an airborne pathogen transmitted via aerosols. It remains viable in the soil and outside its hosts for extended periods of time. It has been suggested that M. tuberculosis cannot be re-aerosolized therefore it cannot cause disease once it has landed outside the body. This project aimed answering indirectly several questions relating to M. tuberculosis and the possibility of it being able to cause disease after re-aerosolization: Can M. tuberculosis be re-aerosolized? If it can, does M. tuberculosis remain viable? If it is viable, can it cause infection? Finally, can environmental bio-burden be reduced using copper surfaces? Methodology: This two-phased prospective in vitro study preceded by a pilot study used as surrogate for M. tuberculosis, Mycobacterium smegmatis (M. smegmatis), a fast growing, non-pathogenic mycobacterium. A completely sealed Plexiglas (Polymethyl methacrylate) cabinet was used as aerosol chamber where 125 mg of sterile dust was spread prior to nebulization of 20 ml of 106 CFU/ml of M. smegmatis pCHERRY3. The sampling was performed with a six stage Andersen cascade impactor (ACI) and settle plates before and after the dust lift-up using two small fans. Plates were incubated for three to ten days at 37 degrees Celsius. The numbers of CFU were estimated based on viable plate count. The first phase of the study, the Plexiglas phase, tested the survival of M. smegmatis in dust. The second phase evaluated the impact of copper surface on the survival of M. smegmatis in similar conditions. Results: M. smegmatis survived in dust for more than nineteen days on settle plates after re-aerosolization in the presence of Plexiglas. In copper presence, M. smegmatis survival rate was approximately fifteen days after nebulization in dust, almost 25% less than on Plexiglas. Starting with an inoculum of 106 cells/ml nebulized on Day 0, on Day 1 there were 44.01% lower numbers of M. smegmatis isolated in copper presence compared with Plexiglas surface 24 hours after nebulization. There was a significant decrease in the number of mycobacteria picked up from both copper (100%) and Plexiglas surfaces (35.02%) on Day 2; copper showed significantly lower levels of mycobacteria (p=<0.05). From Day 3 onwards there was no statistically significant difference in M. smegmatis survival between the two surfaces as determined by the ACI level A5 & A6 (0.6-2.1 micrometre) which equates to particle sizes that can reach the alveoli and terminal bronchi. Conclusion: M. smegmatis, the model for M. tuberculosis, survived in dust and remained viable after re-aerosolization more than 19 days on Plexiglas but 15 days on copper. This is particularly relevant in low to middle income countries with high M. tuberculosis burden, where dust is common and sweeping and the use of fans in health care facilities are frequent. It also illustrated that the anti-microbial property of copper surface remains effective in presence of dust. Copper can be used as a touch surface to reduce the bioburden of microbes including mycobacteria that could be re-aerosolized.

AFRIKAANSE OPSOMMING : Agtergrond: Mycobacterium tuberculosis (M. tuberculosis) is in wese ‘n luggedraagde patogeenen word deurmiddel van aerosols oorgedra. Dit bly lewens vatbaar vir lang tyd perke in die grond en buite die gasheer. Daar is al voorgestel dat M. tuberculosis nieweerge-aerosoleerkan word nie en duskandit nie siekte veroorsaak wanneer dit buite die liggaam in die omgewing stof beland het nie. Die doel van hierdieprojek was om ‘n paarvraetevraoor M. tuberculosis en die moontlikheid dat dit weer kan aerosoleer en siekte veroorsaak. Hierdievrae was: Kan M. tuberculosis weeraerosoleer word? Indienwel, bly M. tuberculosis lewens vatbaar? Indien dit lewens vatbaar bly, kandit infeksie veroorsaak? Enlaastens, kan die bio-las in die omgewing deur koper oppervlaktes verminder word? Metodologie: Hierdieprospektieweinvitrostudie was in twee fasesvoltooi, voorafgegaandeur ‘n loodsstudie. Mycobacterium smegmatis (M. smegmatis), ‘n vinnig-groeiende, nie-patogeniese mikobakterie was gebruik as ‘n surrogaatvir M. tuberculosis. ‘n Dig verseelde Plexiglas (polimetielmetakrilaat) kabinet van 90 x 70 x 90 sentimeter was gebruik as aerosolerings kamerwaarin 125mg sterielestofversprei was voor die nebulisering van 20 mL van 106 kolonie vormendeeenhede per milliliterM. Smegmatis pCHERRY3. Die monsters was geneem met behulp van ‘n gekombineerdeses-stadium Andersen lugmonsternemer, asookneerslagplatevoorenna her-aerosolering van die stofdeur twee kleinwaaiers. Die plate was virdrie tot tiendae teen 37 grade Celsius geinkubeer. Die geskatteaantalkolonievormendeeenhede was gebasseer op die lewensvatbareplaattellings. Die eerstefase van die studie (in Plexiglas) het die oorlewing van M. smegmatis in stofgetoets. Die tweedefase het die impak van koperoppervlaktes op die oorlewing van M. smegmatis in soortgelykeomstandighedegetoets. Resultate: M. smegmatis oorleefvirmeer as 19 dae in stof op neerslagplatenadatditweer in die Plexiglas ge-aerosoleer is. In die teenwoordigheid van koper was die oorlewingnanebulisering van M. smegmatis ongeveervyftiendae – bykans 25% minder as op die Plexiglas. Beginnende met ‘n inokulum van 106selle per milliliter op Dag 0, was 44.01% minder M. smegmatis op Dag 1 geisoleer in die teenwoordigheid van koper in vergelyking met Plexiglas oppervlaktes, 24 uurnanebulisering. Daar was ‘n beduidendeafname in die aantalmikobakterieeopgetel van beidekoper (100%) ennie-koper (35.02%) oppervlaktes; die koper het aansienlikelaergetallemikobakterieegetoon (p-waarde<0.05). Vanaf Dag 3 was daargeenstatistiesbeduidendeverskiltussen die twee oppervlaktesnie – soosbepaaldeur die Andersen se kaskadeimpakteerdervlakke A5 en A6 (0.6-2.1 mikrometer), wat gelykstaande is aan partikelgroottes wat in geval van oorlewing van M. smegmatis die alveoli en bronchi soukonbereik. Gevolgtrekking: M. smegmatis, die surrogaatmerker vir M. tuberculosis , het tot 20 dae nadat dit weer aerosoleer was op Plexiglas oorleef en lewensvatbaar gebly in stof, maar vir ‘n korter tydperk in die teenwoordigheid van koper. Hierdie is veral relevant in lae- tot middel-inkomste lande met 'n hoe M. tuberculosis las, waar stof algemeen is en daar gereeld (met ‘n besem) in gesondheidsfasiliteite gevee word en waaiers algemeen gebruik word. Dit het ook aangedui dat die anti-mikrobiese eienskappe van koper in die teenwoordigheid van stof doeltreffend bly. Koper kan as tas-oppervlak gebruik word om die las van mikrobes, insluitend mikobakteriee, te verminder.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/100799
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