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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