introduction methods discussion and results€¦ · scan electron microscopy flow cytometry...
TRANSCRIPT
INTRODUCTION
B.R. Oliveira1,2, M. T. Barreto Crespo1,2, V.J. Pereira1,2
1 iBET - Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal2Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal
Limited attention has been given to the presence of fungi in the aquatic environment compared to other microorganisms such as bacteria and virus. Our previous research showed that fungi occur widely in drinking water
sources [1,2] and described many fungi species that have not been previously reported in the aquatic environment. Moreover, many filamentous fungi species present in water were found to be able to grow at high
temperatures and have conidia measurements lower than 5 µm, being therefore considered as potential pathogenic species to humans and animals [2].
Chlorine is the most widely used disinfectant in water treatment. However, Penicillium and Aspergillus species showed a higher resistance to free and combined chlorine disinfection than certain Cladosporium and Phoma
species tested [3,4] and so may resist the conventional treatment. Further research is therefore needed to address the efficiency of different disinfectants for the inactivation of fungi. The use of UV for water treatment has
increased along the years since it is extremely effective to achieve inactivation of protozoans, virus and bacteria and does not require chemical addition. This will also decrease the chlorine dose needed as a final disinfectant
in the distribution system and consequently, decrease the formation of chlorination disinfection by products.
Light-emitting diodes (LED) recently emerged as a promising treatment technology due to their advantages: mercury free lamps, no stabilization time, long lifetimes, and diversity of wavelengths available. LEDs have
already been used for several purposes including the inactivation of several microorganisms in real water sources but, to the best of our knowledge have not been tested in terms of their ability to inactivate filamentous fungi
in water. The aim of this study is therefore to evaluate: (i) the inactivation efficiencies of LEDs with different wavelengths (255 nm and 265 nm) on three Aspergillus’ species (A. fumigatus, A. niger and A. terreus) that were
isolated from drinking water sources and; (ii) the effect of those light sources on their morphology, membrane permeability and enzymatic activity.
METHODS DISCUSSION and RESULTS
Initial After 3 weeks
A. fu
mig
atu
s
A. fumigatus A. niger A. terreus
LE
D 2
55
nm
UV Fluence
Fu
ngal
in
acti
vat
ion
Growth Studies
10 µm10 µmA. fu
mig
atu
s
10 µm10 µm
A. n
iger
10 µm10 µm
A. te
rreu
s
Spores and Mycelium grown for 7 days at 27 °C
1x108 spores/mL spiked into surface water matrix
LED 255 nm and 265 nm
0, 0.5, 1, 5, 10, 15, 30, 45 and 60 min
LE
D 2
55
nm
LE
D 2
65
nm
A. fumigatus A. niger A. terreus
Mem
bra
ne
per
mea
bil
ity a
nd
enzy
mat
ic a
ctiv
ity Size and Granulation
LiveDormant
Dead Damage
FL3 – Propidium Iodide
FL1 - Fluorescein Diacetate
The LED that emits at 265 nm is more efficient to inactivate the fungi species;
A. niger is the most resistant species.
The LED that emits at 265 nm has a higher effect on the fungal spores
morphology, regardless the fungi species
A. fumigatus A. niger A. terreus
No
Ex
po
sure
LE
D 2
55
nm
LE
D 2
65
nm
Fu
ngal
in
acti
vat
ion
Ph
eno
typ
ic e
ffec
t
Karnovsky’s Fixative (overnight):
- Glutaraldehyde (2.5 % v/v)
- Paraformaldehyde (2.0 % v/v)
- Phosphate saline solution (0.1 M)
Osmium tetroxide (1.0 % v/v) for 2 hours
Dehydration with 30, 50, 70, 80, 90, 95 %, 5 min
and 100 %, 10 min, of ethanol
Freeze dried for 30 min
Mounting samples on carbon conductive tape
Cover samples with gold and palladium
Scan Electron Microscopy
Flow Cytometry
CONCLUSIONS
Acknowledgement: Financial support from Fundação para a Ciência e a Tecnologia through the fellowship SFRH/BD/111150/2015 is gratefully acknowledged. iNOVA4Health - UID/Multi/04462/2013, a program financially supported by Fundação para a Ciência e
Tecnologia/Ministério da Educação e Ciência, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement is gratefully acknowledged. The authors also thank Dr. David Bastien from SYSMEX for helping with the flow cytometry
analysis and SYSMEX that has kindly provided the flow cytometer used in these experiments.
References: [1] Pereira, V.J., Basílio, M.C., Fernandes, D., Domingues, M., Paiva, J.M., Benoliel, M.J., Crespo, M.T. and Romão, M.V.S. (2009) Occurrence of filamentous fungi and yeasts in three different drinking water sources. Water Research 43(15), 3813-3819.
[2] Oliveira, B.R., Barreto Crespo, M.T., San Romão, M.V., Benoliel, M.J., Samson, R.A. and Pereira, V.J. (2013) New insights concerning the occurrence of fungi in water sources and their potential pathogenicity. Water Research 47(16), 6338-6347. [3] Pereira, V.J.,
Marques, R., Marques, M., Benoliel, M.J., Barreto Crespo, M.T. (2013) Free chlorine inactivation of fungi in drinking water sources. Water Research, 47, 517-523. [4] Pereira, V.J., Marques, M., Marques, R., Benoliel, M.J., Barreto Crespo, M.T. (2017) Inactivation of
fungi in treated surface water by chloramination. J Am Water Works Assoc, 109, 19-23.
Mem
bra
ne
per
mea
bil
ity a
nd
enzy
mat
ic a
ctiv
ity
The LED that emits at 265 nm has a higher effect on the membrane
permeability and the enzymatic activity of the fungal spores
A. niger is the most resistant species followed by A. fumigatus and A. terreus
The LED that emits at 265 nm is more efficient than the LED that emits
at 255 nm which may be due to the DNA peak absorption at 264 nm
A. niger A. fumigatus A. terreus
Spores’ size: 3.5-4.5 µm Spores’ size: 2.5-3.0 µm Spores’ size: 1.5-2.5 µm
Aspergillus niger was the most resistance species which may also be
due to its higher spores’ size and/or due to the presence of pigments
LiveDormant
Dead Damage
SEM and Flow Cytometry proved to be suitable techniques to evaluate
spores’ morphology, membrane integrity and enzymatic activity
1 mm 1 mm 1 mm
250 µm
250 µm