Post on 26-Nov-2018
Dishwasher and Car Wash: Man-made
Environments Accommodating Human
Opportunistic Black Yeasts
Emerging Potential of Black Yeasts,
Curitiba, 2011
Nina Gunde-Cimerman and Polona Zalar
Endholites (microscopic cracks in rocks, oligotrophy) Oligotrophes Acidophiles (pH < 3) Alkalophiles (pH > 9) Psychrophiles (T < 15 °C) Hyperthermophiles (T > 80°C) Thermophiles (60–80 °C) Thermoacidophiles (70–80 °C , pH 2 - 3) Halophiles (NaCl > 10% ) Haloalkalophiles (NaCl > 10%; pH > 9)
Osmophiles, xerophiles (npr. glucose > 50%, other carbohydrates;
aridity) Barophiles (high hydrostatic pressure) Radioresistant (ionising radiation)
FUNG I ?
Extremophylic fungi (SSU rDNA)
Hemiascomycetes
Lecanorales
Dothideales
Thelebolales
Pleosporales
Pezizales
Archiascomycetes
Xylariales
Sordariales Ophiostomatales
Microascales
Clavicipitales
Leotiales
Capnodiales
Chaetothyriales
Onygenales
Eurotiales
Hymenomycetes
Heterobasidiomycetes
Ustilaginomycetes
Wallemiomycetes
Teliomycetes
BASIDIOMYCOTA
ASCOMYCOTA
(Trichosporonoides)
(Chrysosporium,
Gymnascella)
(Exophiala) Hortaea,
Phaeotheca,
Trimmatostroma,
Cladosporium
Aureobasidium
Emericella, Eurotium,
Penicillium, Aspergillus
Wallemia
Debariomyces, Pichia,
Yarrowia, Candida
Fusarium, Acremonium
Cryptococcus, Rhodosporidium,
Rhodotorula, Trichosporon
Publication
Household extreme environments?
Conditions in dishwashers
1. Temperature (30 – 80 °C)
2. moisture (0 - cca.20 l of water / washing
cycle)
3. the presence of organic matter (clean –
high presence of organic particles)
4. Changing pH (3-12)
5. Oxidative stress (detergents)
6. Salinity (0-20% NaCl)
Exophiala spp. and Candida
parapsilosis
Dishwashers
• (N= 189): 158strains, 15genera
Paecilomyces
1%
Penicillium
1%
Phaeosphaeria
1%
Acremonium
1%
Fusarium
4%
Magnusiomyces
7%
Pichia
7%Prototheca
1%
Rhodotorula
4%
Sporopachydermia
1% Aspergillus
1%
Aureobasidium
2%
Candida
12%
Exophiala
52%
Cladosporium
5%
Rubber seal
Acremonium Paecilomyces
Aspergillus Penicillium
Aureobasidium Phaeosphaeria
Candida Pichia
Cladosporium Prototheca
Exophiala Rhodotorula
Fusarium Sporopachydermia
Magnusiomyces
List of detected species Aspergillus niger BSL-1
Aureobasidium pullulans BSL-1
Candida inconspicua BSL-1
Candida parapsilosis BSL-1
Cladosporium aff. cladosporioides BSL-1
Cladosporium bruhnei BSL-1
Cladosporium cladosporioides BSL-1
Cladosporium sphaerospermum BSL-1
Exophiala dermatitidis BSL-2
Exophiala phaeomuriformis BSL-2
Magnusiomyces capitatus BSL-2
Geotrichum clavatum BSL-2
Pichia cactophila like BSL-1
Pichia guilliermondii BSL-1
Rhodotorula calyptogenae BSL-1
Rhodotorula mucilaginosa BSL-1
Sporopachydermia cereana BSL-1
Exophiala phaeomuriformis
Sources: Hoog, G.S. de 2000, Atlas of clinical fungi, ed. 2: 1-1126.
Matos et al., 2003, Molecular diversity of oligotropic and neurotropic members of the black yeast
genus Exophiala…
• Original ecological niche: - 1 isolate from underground cave (Czech
Republic)
- 1 isolate from natural hot spring (Slovenia)
HUMAN ENVIRONMENT: bathrooms (tap
water, sinks), steam baths, car surface
• Diseases: - skin infections
- found in sputum
Exophiala dermatitidis
Sources: Hoog, G.S. de 2000, Atlas of clinical fungi, ed. 2: 1-1126
Sudhadham, M. 2009, E. dermatitidis: an opportunistic pathogen emerging from the tropical
rainforest. PHd thesis.
• Original ecological niches: - cactus fruits, berries, feces of
frugivorous birds and bats in tropics
HUMAN ENVIRONMENT: bathing facilties,
steam baths, waste water, railway tiles
(tropics),
Exophiala dermatitidis
Diseases:
- Neurotropic infections
- Oportunistic systemic infections and pseudoepidemics
- Colonization of lungs and cavities
- Intestinal colonization
- Sub-cutaneous infections
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SLOVENIA:
E. dermatitidis, E. phaeomuriformis
Dishwashers – Exophiala dermatitidis + sampling sites
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All
samples Sample no. + -
% %
USA 5 80 20
Canada 7 28,5 71,5
Australia 7 28,6 71,4
Germany 5 60 40
Austria 5 40 60
Spain 5 0 100
UK 2 0 100
Denmark 5 20 80
Belgium 4 25 75
Italy 10 10 90
Israel 5 20 80
China 4 0 100
Japan 5 40 60
South Africa 10 70 30
Slovenia 91 48,3 51,7
ALL 171 41,5 58,5
Directly from a dishwasher
E. dermatitidis: Specialist species with a combination of multiple narrow ecological
amplitudes -
- pH 2,5 - pH 12,5; T 10 - 50C, 0% - 17% NaCl
E. Dermatitidis and E. phaeomuriformis
Temperature tolerance - 4 - 50 C
E. dermatitidis andE. phaeomuriformis
(NaCl)
- 0% - 17% NaCl
―Polyextremophiles―
• ‖ for anaerobic Bacteria and aerobic Archaea from
African alkaline and hypersaline lakes
• Optimal growth at elevated salt concentration (between
• 3.7 and 4.3 M), alkaline pH (above pH 9.5), elevated
temperatures (between 46 and 66 C)
• (Bowers et al. 2009)
Several pathogenic species have recently been
isolated from various domestic habitats.
• Basic growth requirements - similar in both the pathogenic and the
harmless microbial relatives
• Pathogens - additional features
• Growth at elevated temperature - higher potential to colonize warm-
blooded organisms
• Particularly problematic - "polyextremotolerant" fungi
Extreme indoor environments
• Enrichment of potentially pathogenic
species
• Driving of their evolution towards acquiring
the missing virulence factors
• Both factors combined in human
environments - considerable concern.
• Ecological trends
• Increased numbers in less-developed
countries.
Science News
• 'My Dishwasher Is Trying to Kill Me': New
Research Finds Harmful Fungal Pathogens
Living in Dishwasher Seals
• ScienceDaily (June 20, 2011) — A potentially pathogenic fungus
has found a home living in extreme conditions in some of the most
common household appliances, researchers have found. A new
paper published in the British Mycological Society journal, Fungal
Biology, published by Elsevier, shows that these sites make perfect
habitats for extremotolerant fungi (which includes black yeasts).
Some of these are potentially dangerous to human health.
• Your dishwasher wants to kill you!
Summary of responses
• Elsevier newsroom increased in one week, with over 1300 hits to the dishwasher press release
• Traffic to the Fungal Biology page on Elsevier.com tripled
• major media outlets that covered the story:– the combined circulation of these sites (online traffic) per day at a modest estimate is around 100 million – the Telegraph and the Daily Mail (both British) make up nearly 5 million on their own.
• Coverage from the scientific community– Scientific American blog and ScienceBlogs covered the story
• The reach on twitter was in the millions too - a combined reach of over 70,000 users.
• The story went viral!
Newspapers
• News outlet, elespectador.com, Terra, Gandul.info, Sydney Morning Herald, The Telegraph, Daily Mail, The Register, The Times of India, California Watch, the Express, Scientific American (blog), The Mercury, The Star, The Age, Brisbane Times, Western Australia Today, The Independent, Canada.com, Discovery News, Calgary Herald, Agence France-Presse, the Vancouver Sun, The Windsor Star, the Star Phoenix, Ottawa Citizen, Edmonton Joournal, the Province, Montreal Gazette, Times Colonist, MSN News Phillipines, France24, Newstrack India, Gizmodo, UPI Asia, UPI, Infection Control Today, WELT Online, der Standard, ORF.at, Der Westen, RTL, STERN, El Tiempo, Semana, TeleCinco, El Nacional, BBC World (Spanish edition), Son Haber, Ta Nea online, La Stampa, Sued Deutsche, Le Figaro, NU.nl, Компьюлента , Radio Canada, SciencePunk (blog at ScienceBlogs), Microbe.net, RedOrbit, the Straits Times, AD.nl, El Nacional, La Stampa, TG (TeleGiornale), Il quotidiano italiano, 21 Stoleti, Independent, Fox News, MyFox Detroit, MSN Health, U.S. News, Newsday, CNet, Boots WebMD, CNN (blog), Fox News Radio podcast, abc7 (San Fran area)....
Rubber seals
SAMPLING
SEM
-6 years old rubber seal from a dishwasher
- Sterile rubber inoculated with E. dermatitidis
No correlation:
• Type of dishwasher
• Age of dishwasher
• Type of detergent
• Cleanliness of the dishwasher
North of Sweden, July 2011 • 6 months old dishwasher
• Connected to well water
• Exclusively E. dermatitidis!!
Black yeasts can assimilate volatile aromatic hydrocarbons as
the sole source of C and energy
Prenafeta Boldu s sod., 2006
Cars, workshops for car repair,
petrol stations, car washers
-
E. dermatitidis
•Rubber at car petrol tank
•Car repair workshop
•Car washers
Transmission of E. dermatitidis
dishwashers
WATER
Source: Sudhadham, M. 2009,
AIR
WATER AEROSOL
Acknowledgements
• Monika Novak
• Klara Bojanovič
• Jerneja Zupančič
• Špela Koželj
• Exophiala xenobiotica in Exophiala
bergeri,
• in vitro - hlapni aromatski ogljikovodiki.
benzen, toluen in ksilen (Jingjun Zhao in
sod.,2010)
Izolacijska
številka
EXF številka Vzorec odvzema Kraj odvzema Identifikacija po BLAST algoritmu Prekritost
sekvenc (%)
Podobnost
sekvenc (%)
A2-a 6300 avtopralnica Ljubljana Exophiala phaeomuriformis 92 97
A4-a 6302 avtopralnica Maribor Trichoderma sp. *
A7-a 6305 avtopralnica Celje Exophiala phaeomuriformis 92 97
A7-b 6306 avtopralnica Celje Rdeča kvasovka
A10-a 6309 avtopralnica Koper Rhodosporidium diobovatum 100 99
A10-b 6310 avtopralnica Koper Exophiala phaeomuriformis 93 100
A10-c 6311 avtopralnica Koper Candida norvegica 100 99
A12-a 6312 avtopralnica Koper Aspergillus niger *
A12-b 6313 avtopralnica Koper Pichia pastoris 99 99
A14-a 6314 avtopralnica Renkovci Exophiala phaeomuriformis 93 100
A14-b 6315 avtopralnica Renkovci Candida sake 100 99
A15-a 6316 avtopralnica Velika Polana Exophiala phaeomuriformis 93 99
A15-b 6317 avtopralnica Velika Polana Fusarium merismoides var. acetilereum 95 99
A15-c 6318 avtopralnica Velika Polana Exophiala phaeomuriformis 92 100
A17-a 6319 avtopralnica Krško Exophiala phaeomuriformis 100 100
A17-c 6320 avtopralnica Krško Exophiala phaeomuriformis 93 99
A18-a 6321 avtopralnica Krško Galactomyces geotrichum 100 99
A20-a 6322 avtopralnica Jesenice Exophiala xenobiotica 100 99
A20-b 6323 avtopralnica Jesenice Pichia spartinae 100 98
Tabela 6: Rezultati vzorčenja, izolati in identifikacije gliv iz avtopralnic
Metode in potek praktičnega dela
Vzorčenje strojev
Izolacija čistih glivnih kultur
Filtracija vode Shranjevanje v zbirko EX
Ekstrakcija genomske DNA
Verižna reakcija s polimerazo (PCR)
Sekvenciranje
Prstni odtis z M13
Določanje velikosti fragmentov
IDENTIFIKACIJA
Fiziologija
Temperaturna toleranca
pH toleranca
Halotoleranca
Parjenje Vrstična elektronska mikroskopija (SEM)
Rod Exophiala
Vpliv trdote vode
Izolirani in identificirani sevi iz pralnih strojev
• Pralni stroji (N=42): 38 sevov, 12 rodov
Penicillium
3%
Pestalotiopsis
3%
Exophiala
9%
Fusarium
29%
Gibberella
3%
Mucor
6%
Ochroconis
3%
Phoma
6%
Rhodotorula
6%
Aureobasidium
3%Candida
6%
Cladosporium
23%
Predalčki za pralni prašek
Aureobasidium
Cladosporium
Exophiala
Fusarium
Gibberella
Mucor
Ochroconis
Pestalotiopsis
Phoma
Predalčki za mehčalec
Cladosporium
Exophiala
Fusarium
Mucor
Penicillium
Tesnilna guma
Candida
Cladosporium
Rhodotorula
Parjenje in prstni odtis E. dermatitidis:
• genotip A – 7 fragmentov • genotip B – 7 fragmentov • genotip C – 8 fragmentov
pojav mešanega genotipa AxB
Prevalence of E. dermatitidis genotipes
• Dishwashers– Slovenia:
A (A1:A2:A3) : B : C = 30 (0:1:2) : 4 : 2
• Dishwashers - world:
A (A1:A2:A3) : B : C = 23 (0:3:2) : 6 : 0
• ALL dishwashers:
A (A1:A2:A3) : B : C = 53 (0:5:3) : 10 : 2