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Atmosphere 2015, 6, 1290-1306; doi:10.3390/atmos6091290
atmosphere
ISSN 2073-4433
www.mdpi.com/journal/atmosphere
Article
Effects of Stratospheric Conditions on the Viability, Metabolism
and Proteome of Prokaryotic Cells
Dagmar Chudobova
1,2
, Kristyna Cihalova
1,2
, Pavlina Jelinkova
1
, Jan Zitka
1
, Lukas Nejdl
3
,
Roman Guran
1,2
, Martin Klimanek
4
, Vojtech Adam
1,2
and Rene Kizek
1,2,
*
1
Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1,
CZ-613 00 Brno, Czech Republic; E-Mails: dagmar.chudobova@centrum.cz (D.C.);
kriki.cihalova@seznam.cz (K.C.); jelinkova.pav@gmail.com (P.J.); zitka12@gmail.com (J.Z.);
R.Guran@email.cz (R.G.); vojtech.adam@mendelu.cz (V.A.)
2
Central European Institute of Technology, Brno University of Technology, Technicka 3058/10,
CZ-616 00 Brno, Czech Republic
3
Department of Geology and Pedology, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno,
Czech Republic; E-Mail: lukasnejdl@gmail.com
4
Department of Forest Management and Applied Geoinformatics, Mendel University in Brno,
Zemedelska 1, CZ-613 00 Brno, Czech Republic; E-Mail: martin.klimanek@mendelu.cz
* Author to whom correspondence should be addressed; E-Mail: kizek@sci.muni.cz;
Tel.: +420-5-4513-3350; Fax: +420-5-4521-2044.
Academic Editor: Robert W. Talbot
Received: 29 July 2015 / Accepted: 20 August 2015 / Published: 28 August 2015
Abstract: The application of ultraviolet (UV) radiation to inhibit bacterial growth is based
on the principle that the exposure of DNA to UV radiation results in the formation of
cytotoxic lesions, leading to inactivation of microorganisms. Herein, we present the
impacts of UV radiation on bacterial cultures’ properties from the biological, biochemical
and molecular biological perspective. For experiments, commercial bacterial cultures
(Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli
and Salmonella typhimurium) and isolates from patients with bacterial infections
(Proteus mirabilis and Pseudomonas aeruginosa) were employed. The above-mentioned
strains were exposed to UV using a laboratory source and to stratospheric UV using a 3D
printed probe carried by a stratospheric balloon. The length of flight was approximately
two hours, and the probe was enriched by sensors for the external environment
(temperature, pressure and relative humidity). After the landing, bacterial cultures were
OPEN ACCESS
Atmosphere 2015, 6 1291
cultivated immediately. Experimental results showed a significant effect of UV radiation
(both laboratory UV and UV from the stratosphere) on the growth, reproduction, behavior
and structure of bacterial cultures. In all parts of the experiment, UV from the stratosphere
showed stronger effects when compared to the effects of laboratory UV. The growth of
bacteria was inhibited by more than 50% in all cases; moreover, in the case of
P. aeruginosa, the growth was even totally inhibited. Due to the effect of UV radiation, an
increased susceptibility of bacterial strains to environmental influences was also observed.
By using commercial tests for biochemical markers of Gram-positive and Gram-negative
strains, significant disparities in exposed and non-exposed strains were found. Protein
patterns obtained using MALDI-TOF mass spectrometry revealed that UV exposure is able
to affect the proteins’ expression, leading to their downregulation, observed as the
disappearance of their peaks from the mass spectrum.
Keywords: stratosphere; ultraviolet; radiation; prokaryotes; 3D chips; proteomics
1. Introduction
Ultraviolet (UV) radiation is a proven and effective method for the inactivation of microorganisms.
It is not surprising that it has become a widely-used technology in facilities for wastewater treatment.
The advantages of UV radiation are well known throughout the water industry, and technological
advances have stimulated the interest in further research. UV disinfection is based on the ability to
cause DNA damage, leading to the inhibition of vital cellular processes, such as transcription and
replication, and ultimately, this may lead to the death of an organism [1–3]. DNA strongly absorbs
UV-C (220–280 nm) with a maximum at 260 nm, resulting in the formation of lesions between
adjacent nucleobases, primarily pyrimidines [4,5]. Two basic types of lesions may be formed as
cyclobutane pyrimidine dimers (CPDs) and 6–4 photoproducts. CPDs are the majority of lesions
caused by UV-C radiation (approximately 75%), while the remaining 25% consists of
photoproducts [3,6]. Areas in the stratosphere, where bacteria may be exposed to UV-C, are
characterized as regions with a low temperature and a high degree of exposure to UV radiation, in
particular highly biologically-harmful UV-C [7].
The occurrence of live bacteria and fungi in the stratosphere is of interest for researchers [8,9].
Recent studies have shown that bacteria can be isolated from the stratosphere at heights of 20 km [10]
and 41 km [11], which also confirms the previous reports of the presence of stratospheric bacterial
species [12]. Microbes are very abundant in soil, and some of them are adapted for dispersion [13].
While mixing to the border tropopause is limited, a wide range of mechanisms can carry aerosols (or
biological cells) from the troposphere into the stratosphere. These mechanisms include the following: a
volcanic eruption, Brewer–Dobson atmospheric circulation, dust storms, monsoons, electrostatic forces
generated by cells and the starts of rockets [14].
Numerous microbes have evolved mechanisms to repair some UV-induced lesions, including CPDs
and photoproducts [15]. Studies of these mechanisms brought results and conclusion based on these,
and we can say that these are very well understood from the molecular perspective. Briefly, there are
Atmosphere 2015, 6 1292
two main categories of repair of damaged DNA: photoreactivation and excision repair.
Photoreactivation is an enzyme-mediated mechanism stimulated by the exposure of visible and/or near
UV light. Excision is also an enzyme-mediated mechanism, but it is not evoked by the effects of
radiation exposure only [3,15].
It is not known how long microbes may survive in the stratosphere, but many studies have shown
that the period may vary within several months or even years [14,16]. Most of the stratospheric studies
are focused on the characterization of microbes (i.e., the determination of species and the place of
origin), while these do not address the other environmental issues, such as how long they can be viable
in the stratosphere and/or how atmospheric and biological factors control the cell survival. Answers to
these questions can provide a critical framework for understanding the patterns of microbial
biogeography and the evolutionary implications of remote diversion through the routes in the
upper atmosphere [17].
The aim of this study was to reveal the impacts of UV radiation on bacterial cultures from the
biological, biochemical and molecular biological perspective. For experiments, commercial bacterial
cultures (Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli,
Salmonella typhimurium) and isolates from patients with bacterial infections (Proteus mirabilis,
Pseudomonas aeruginosa) were employed.
2. Results and Discussion
2.1. Atmospheric Conditions
Generally, 19 fundamental physical conditions classified into nine categories (a group of
temperatures; precipitations; soils; wind; pressure, vapor and air; relative humidity; sunshine;
cloudiness and group of phenomenon) are measured on climatological stations worldwide. These
values are used for the processing of climatic characteristics and indicators. Measurements and
observation on climatological stations are performed by classical methods through reading the device
or observing the phenomenon daily at 7, 14 and 21 h, local time, measuring of precipitation and snow
cover at 7 o’clock, whereas the occurrence of precipitation and significant weather events are recorded
continuously.
During the flight, there were climatological measurements performed on the Earth’s surface of daily
air temperatures, minimum, maximum and average air pressure and the amount of precipitation. Sunrise
and sunset were also detected. All of these conditions during the transition of stratospheric balloon into
the stratosphere could affect the final values of the experimental flight and are shown in Table 1.
Table 1. Climatic data observed on the Earth’s surface before the start of the stratospheric flight.
Date, Time and
Place of Flight
Climatic Data on the Earth’s Surface
1 May 2015
Spisska Nova Ves
7:45 a.m.–9:45 a.m.
sunshine
daily air
temperature
air pressure (hPa)
atmospheric
precipitations
sunrise sunset /
daily
minimum
air pressure
daily
maximum
air pressure
average
daily air
pressure
/
4:18 18:47 13 °C 980 1015 997 46
Atmosphere 2015, 6 1293
During all of time spent by the stratospheric probe in the stratosphere, the temperature outside the
probe, pressure and relative humidity were monitored (Figure 1). Temperature during the flight ranged
from +10 to −60 °C. The highest temperature was observed at 15,000 meters above sea level. The
pressure was lower with increasing altitude. When observing the relative humidity, a similar trend as in
the case of pressure monitoring was observed.
Figure 1. Conditions observed during the flight of the stratospheric probe in the
stratosphere: (A) temperature outside the stratospheric probe, (B) stratospheric pressure
and (C) relative humidity.
2.2. Effects on Bacteria
The biological part of the experimental stratospheric flight was primarily focused on studying and
the comparison of the effect of laboratory UV radiation (wavelength of 264 nm) and stratospheric UV
radiation [18] on bacterial strains’ growth, their resistance to environmental externalities (antibiotics,
and/or semimetal nanoparticles) and the protein composition of the bacterial cell wall. 3D printing was
used as a technique for producing a probe bearing the bacteria, as this has already been used several
times by us for the production platforms for the detection of nucleic bases, bacteria, viruses
and others [18–21].
2.2.1. Growth Properties
The first part of the biological experimental study looked at the growth characteristics of bacteria.
Lyophilized bacterial cultures were inoculated in cultivated media, which corresponded to the optimal
conditions for bacterial growth by their compositions. Media inoculated with the tested bacterial
cultures (E. coli, S. aureus, MRSA, S. typhimurium, P. mirabilis and P. aeruginosa) were immediately
placed into the microtiter plate, and from the beginning of the inoculation, the absorbance values at
intervals of half an hour were measured. Growth curves showing the progress of the lag phase, the
exponential and stationary growth phase of bacteria are in Figure 2A–F for E. coli, S. aureus, MRSA,
S. typhimurium, P. mirabilis and P. aeruginosa, respectively.
Atmosphere 2015, 6 1294
Figure 2. Growth properties of the bacteria (A) E. coli, (B) S. aureus, (C) MRSA,
(D) S. typhimurium, (E) P. mirabilis and (F) P. aeruginosa) without UV exposure, after
exposure to laboratory UV and after sending the bacteria into the stratosphere on the
surface of the stratospheric probe. Lyophilized bacterial cultures were after the landing of
stratospheric probe immediately cultivated in GTY medium, and absorbance at 37 °C in
half-hour intervals for 24 hours was measured.
In all tested bacterial cultures, the influence of UV radiation on the growth and reproduction of
bacteria was determined, while the laboratory UV of a wavelength of 264 nm always caused lower
growth inhibition than exposure to stratospheric UV. It can be therefore assumed that stratospheric UV
radiation is more intense and harmful (Figure 2).
The influence of UV radiation of specific wavelengths on bacterial growth was observed in the
study done by Poepping et al. [22], which observed the impact of single wavelengths and
sequential wavelengths. Log inactivation values for single-wavelength and sequential wavelength
exposures were representative of the amount of irradiation attributed to the ranges 225–235 nm and
275–285 nm for representative MP UV doses of 50, 100 and 150 mJ/cm
2
. Single-wavelength
exposures at either 280 nm or 228 nm yielded the expected result in the increasing E. coli inactivation
with the increasing dose of the irradiation. Two hundred eighty nanometer single-wavelength
exposures resulted in higher E. coli inactivation levels than 228 nm for similar doses, similar to past
research showing increased inactivation efficiency of 280 nm versus 228 nm [22].
From the perspective of bacterial resistance, it is known that transparent water and high UV
irradiance may maximize the penetration and effect of UV radiation. The study of Escudero et al.
Atmosphere 2015, 6 1295
mainly deals with the identification of the microbial community composition in Aguas Calientes and
with the testing of the UV and antibiotic resistances of some isolates to evaluate co-resistance
mechanisms [23]. Both of the analyzed isolates show further inhibition with larger doses of UV-C
radiation. Significant differences in survival after UV-C irradiation were observed. Higher survival
was observed in Pseudomonas sp. compared to the other gamma Proteobacteria, both isolated from
brine samples. Some recent studies point out that the base of the antibiotic resistance in some isolates
is not mutagenesis, but the possible formation of multi-resistance to UV radiation [23].
2.2.2. Resistance
In terms of biological properties, the effect of UV radiation (laboratory and stratospheric) on the
resistance of bacterial strains to environmental externalities, such as conventional antibiotic drugs or
their alternative as selenium nanoparticles [24,25], was further monitored. It was shown that the
exposure of UV significantly weakened the resistance of bacteria, and the application of antibiotic
drugs or nanoparticles became more effective. When exposed to stratospheric UV, this effect on the
bacteria was even higher in comparison to the laboratory UV (Table 2).
Table 2. Resistance of bacterial cultures to antibiotic drugs or semimetal nanoparticles
(1, erythromycin; 2, penicillin; 3, amoxicillin; 4, tetracycline; 5, lincomycin; and 6,
selenium nanoparticles) without UV exposure, after exposure to laboratory UV and after
exposure to stratospheric UV radiation. Bacterial cultures were exposed to commercial
antibiotic drugs in Petri dishes for 24 hours in an incubator at 37 °C. The sizes of the
resulting inhibition zones in millimeters indicate the level of the bacterial strains’ resistance
to antibiotics. The lower the zone formation, the strain is described as more resistant.
Bacterial
Culture
Type of
Exposure
The Size of The Inhibition Zone (mm)
1 2 3 4 5 6
ERY PNC AMX TTC LNC SeNPs
S. aureus
control 5 1 3 10 10 5
laboratory UV 11 5 10 11 11 7
stratospheric UV 11 6 11 15 12 7
MRSA
control 0 0 1 5 0 0
laboratory UV 0 0 2 6 0 8
stratospheric UV 0 0 2 7 0 9
E. coli
control 0 0 5 5 0 0
laboratory UV 0 0 8 7 0 0
stratospheric UV 0 0 11 10 0 6
S. typhimurium
control 2 0 0 8 0 0
laboratory 4 0 0 9 0 0
stratospheric UV 7 4 7 14 2 0
P. mirabilis
control 0 0 0 3 0 0
laboratory UV 0 0 0 5 0 0
stratospheric UV 2 0 2 7 0 0
P. aeruginosa
control 0 0 0 4 0 0
laboratory UV 0 0 5 5 0 0
stratospheric UV 2 4 7 5 1 3
Atmosphere 2015, 6 1296
2.2.3. Biochemical Markers
Exposure to UV light causes significant biological and biochemical changes. UV radiation
represents the most cytotoxic waveband of solar radiation reaching the Earth’s surface, causing several
structural and physiological effects in organisms. Solar radiation elicits a complex chain of cellular
events in microorganisms, which are not yet fully understood. The data for the role of UV-induced
ROS in biological and biochemical damage to bacteria is rather scarce and mostly obtained indirectly
from transcriptomic and proteomic studies reporting the induction of antioxidant defenses upon
exposure of bacteria to UV-B radiation. The study of Santos et al. showed that the effects of ROS
scavengers on biological and biochemical parameters were variable among the different tested
isolates [26]. In this work, two G
+
(Micrococcus sp. and Staphylococcus sp.) and two
G
−
(Paracoccus sp. and Pseudomonas sp.) phylogenetically-distinct bacterial isolates were used.
Referring to this fact, we confirmed that due to their cell wall characteristics, G
+
bacteria have been
proposed to be more resistant to UV radiation than G
−
strains.
Due to this, the changes in biochemical properties were performed by monitoring of the
biochemical markers using commercial supplied tests. For the purposes of testing of G
+
bacteria
(S. aureus, MRSA), an assay for biochemical markers of staphylococci was used. The results of the
observation of biochemical markers in G
+
bacteria generally points to a very mild effect of UV
radiation on the change in the biochemical properties of tested S. aureus and MRSA. In the case of
bacterial culture S. aureus in all three cases of testing, the same results were observed; therefore, the
influence of UV radiation is assessed as insignificant (Figure 3A). When testing the MRSA
(Figure 3B), a strain, which is in many respects more resistant to environmental influences, exposed to
laboratory UV compared to the control strain, changes in the fermenting of xylose (Figure 3Bb-2-B),
maltose (Figure 3Bb-2-C), mannitol (Figure 3Bb-2-D), trehalose (Figure 3Bb-2-E), sucrose
(Figure 3Bb-2-F), galactose, N-acetyl β-D-glucosamine (Figure 3Bb-2-G), xylitol (Figure 3Bb-3-A),
raffinose (Figure 3Bb-3-B), arabinose (Figure 3Bb-3-C), cellobiose (Figure 3Bb-3-D), fructose
(Figure 3Bb-3-E), ribose (Figure 3Bb-3-F), sorbitol (Figure 3Bb-3-H) and lactose (Figure 3Bb-3-H)
were observed (Figure 3Bb). Stratospheric UV exposure in the case of the MRSA strain caused a
change in ornithine (Figure 3Bc-1-F) and arginine fermentation (Figure 3Bc-1-G) only, which is
shown in Figure 3Bc.
In contrast, testing of the biochemical changes in G
-
bacteria using a test for biochemical markers of
Enterobacteriaceae demonstrated mild changes in laboratory exposure to UV radiation and significant
changes in bacteria exposed to UV light in the stratosphere (Figure 4A–C). In the E. coli bacterial
strain, substantial changes in biochemical parameters can be monitored, where the laboratory UV
exposure to this strain caused the inability to ferment all of the components on the bottom of the wells
in the biochemical assay (Figure 4Ab). A similar trend was observed in the E. coli bacterial strain
exposed to the stratospheric UV, whereas the fermentation occurred in the case of ornithine (Figure
4Ab-1-E), lysine (Figure 4Ab-1-F), hydrogen sulfide (Figure 4Ab-1-G) and indole (Figure 4Ab-1-H).
Atmosphere 2015, 6 1297
Figure 3. Biochemical properties of tested G
+
bacterial strains (A) S. aureus, and/or (B)
MRSA. (a) without UV exposure, (b) after exposure to laboratory UV and
(c) after stratospheric UV radiation. Biochemical changes were observed by biochemical
markers after 24 hours of incubation in a thermostat at 37 °C. Legend: 1A, aesculin; 1B,
phosphatase; 1C, β-glucosidase; 1D, β-glucuronidase; 1E, β galactosidase; 1F, ornithine;
1G, arginine; 1H, urease; 2A, mannose; 2B, xylose; 2C, maltose; 2D, mannite; 2E, trehalose;
2F, saccharose; 2G, galactose; 2H, N-acetyl-β-D-glucosamine; 3A, xylitol; 3B, raffinose;
3C, arabinose; 3D, cellobiose; 3E, fructose; 3F, ribose; 3G, sorbitol; 3H, lactose.
Bacterial culture P. mirabilis showed a completely different trend in terms of the ability to ferment
various substances. While the control culture without UV exposure was able to ferment ornithine
(Figure 4Ba-1-E), lysine (Figure 4Ba-1-F), hydrogen sulfide (Figure 4Ba-1-G) and
indole (Figure 4Ba-1-H), only, using exposure to laboratory UV, changes in the inability to ferment
hydrogen sulfide (Figure 4Bb-1-G) were found. Significant changes in the biochemistry of bacteria
compared to the control by exposure of UV from the stratosphere were observed. P. mirabilis bacteria
after irradiation by stratospheric UV are able to ferment trehalose (Figure 4Bc-2-B), sucrose
(Figure 4Bc-2-C), cellobiose (Figure 4Bc-2-D), adonitol (Figure 4Bc-2-E), inositol (Figure 4Bc-2-F),
β-galactosidase (Figure 4Bc-2-G), phenylalanine (Figure 4Bc-2-H), raffinose (Figure 4Bc-3-C),
melibiose (Figure 4Bc-3-D), rhamnose (Figure 4Bc-3-E), sorbitol (Figure 4Bc-3-F), aesculin
(Figure 4Bc-3-G) and acetoin (Figure 4Bc-3-H). In contrast, this bacteria lose the ability to ferment
ornithine (Figure 4Bc-1-E), lysine (Figure 4Bc-1-F), hydrogen sulfide (Figure 4Bc-1-G) and indole
(Figure 4Bc-1-H), as in the previous cases.
A third tested bacterial culture of G
-
bacteria S. typhimurium exhibited the same biochemical
parameters as E. coli. Changes during the exposure to laboratory UV radiation occurred in more than
half of the components, such as ornithine (Figure 4Cb-1-E), sucrose (Figure 4Cb-2-C), cellobiose
(Figure 4Cb-2-D), adonitol (Figure 4Cb-2-E), inositol (Figure 4Cb-2-F), β-galactosidase
(Figure 4Cb-2-G), phenylalanine (Figure 4Cb-2-H), raffinose (Figure 4Cb-3-C), melibiose
(Figure 4Cb-3-D), rhamnose (Figure 4Cb-3-E), sorbitol (Figure 4Cb-3-F), aesculin (Figure 4Cb-3-G)
and acetoin (Figure 4Cb-3-H). After the exposure to stratospheric UV, S. typhimurium loses the ability
to ferment nearly all components, except ornithine (Figure 4Cc-1-E), lysine (Figure 4Cc-1-F),
hydrogen sulfide (Figure 4Cc-1-G) and indole (Figure 4Cc-1-H). Changes in biochemical properties
were demonstrated in P. aeruginosa bacterial culture (Figure 4D).
Atmosphere 2015, 6 1298
Figure 4. Biochemical properties of tested G
-
bacterial strains (A) E. coli,
(B) P. mirabilis, (C) S. typhimurium and (D) P. aeruginosa. (a) without UV exposure,
(b) after exposure to laboratory UV and (c) after stratospheric UV irradiation. Biochemical
changes were observed by the observation of the biochemical markers of Enterobacteriaceae
after 24 hours of incubation in a thermostat at 37 °C. Legend: 1A, malonate; 1B, Simmons
citrate; 1C, arginine; 1D, urease; 1E, ornithine; 1F, lysine; 1G, hydrogen sulfide; 1H,
indole; 2A, mannitol; 2B, trehalose; 2C, saccharose; 2D, cellobiose; 2E, adonitol; 2F,
inositol; 2G, β galactosidase; 2H, phenylalanine; 3A, glucose; 3B, dulcitol; 3C, raffinose;
3D, melibiose; 3E, rhamnose; 3F, sorbitol; 3G, aesculin; 3H, acetoin.
2.2.4. Proteomic Analyses
Knowledge of the molecular effects of UV radiation on bacteria can contribute to a better
understanding of the environmental consequences of enhanced UV levels associated with global
climate changes and would help to optimize UV-based disinfection strategies. In the study of
Santos et al., it was observed that the exposure to UV radiation caused an increase in methyl groups
associated with lipids, lipid oxidation and also led to alterations in lipid composition, which were
confirmed by gas chromatography [27]. Additionally, mid-infrared spectroscopy revealed the effects of
UV radiation on protein conformation and protein composition, which were confirmed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), oxidative damage to amino acids
and changes in the propionylation, glycosylation and/or phosphorylation status of cell proteins [27].
In the last experiment, the effect of UV radiation on the protein structure in the bacterial cell wall
was evaluated [28]. This experiment revealed the influence of UV radiation on the structure and
composition of bacteria; however, the intensities of the protein peaks of each tested culture were
Atmosphere 2015, 6 1299
evaluated as a pseudo gel view (Figure 5). The mass spectra were also compared in a “stacked” view
(Figure 6).
Figure 5. Pseudo-gel view comparison of MALDI-TOF MS spectra of bacteria without
UV exposure, after exposure to the laboratory and stratospheric UV as (A) S. aureus,
(B) MRSA, (C) E. coli, (D) S. typhimurium, (E) P. mirabilis and (F) P. aeruginosa. Data
were collected in the m/z range 2.000–20.000. For MALDI-TOF analysis, 1 µL of extract
of each bacterial culture was used. The control samples were identified by MALDI
BioTyper
TM
software.
Atmosphere 2015, 6 1300
Figure 6. Comparison of MALDI-TOF MS spectra of bacteria without UV exposure, after
exposure to the laboratory and stratospheric UV: (A) S. aureus; (B) MRSA; (C) E. coli;
(D) S. typhimurium; (E) P. mirabilis; and (F) P. aeruginosa. Data were collected in the
m/z range 2.000–20.000. For MALDI-TOF analysis, 1 µL of extract of each bacterial
culture was used. The control samples were identified by MALDI BioTyper
TM
software.
The differences of protein peak intensities between experimental groups exposed to UV irradiation
in the laboratory and in the stratosphere were significant in all bacterial samples. In a closer view, the
most significant differences were observed in MRSA (Figure 5B and Figure 6B), E. coli (Figure 5C
and Figure 6C) and P. mirabilis (Figure 5E and Figure 6E). In the case of MRSA (Figure 5B), the peak
intensities under stratospheric UV irradiation were lower, and some peaks disappeared within the m/z
range from 10 to 14 m/z (10,487, 10,907, 11,597 and 13,179 m/z). Similar effect was also observed in
the sample of P. mirabilis (Figure 5E), where peaks of 7246, 7690, 8329, 8898 and 9509 m/z
disappeared. On the contrary, in the sample of E. coli (Figure 5C), peaks at 10,141, 10,696 and 11,202
m/z were observed with the lowest intensities in the mass spectrum under laboratory UV irradiation.
For better comparison, a dendrogram from the mass spectra based on the measurement of each
bacterial sample (Figure 7) was also created, which showed that the protein profiles of
P. mirabilis and MRSA were affected the most after stratospheric UV irradiation. It can be concluded
Atmosphere 2015, 6 1301
that UV irradiation has an effect on bacterial protein expression. Stratospheric UV irradiation has a
higher influence on protein expression than laboratory UV irradiation in most cases. This is probably
caused by different conditions in the stratosphere, where other types of radiation with higher intensity
than in the laboratory are also present.
Figure 7. Dendrogram from the mass spectra of bacteria without UV exposure and after
exposure to the laboratory and stratospheric UV. Mass spectra were collected in the
m/z range 2.000–20.000. For MALDI-TOF analysis, 1 µL of extract of each bacterial
culture was used. The dendrogram was created in MALDI BioTyper
TM
.
3. Experimental Section
3.1. Chemicals
The chemicals used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA) in
ACS purity (meets the requirements of American Chemical Society (ACS))
unless noted otherwise.
Deionized water was prepared using reverse osmosis equipment Aqual 25 (Brno, Czech Republic).
Deionized water was further purified by using a Milli-Q Direct QUV apparatus equipped with a UV
lamp. The resistance was 18 MΩ. The pH was measured using pH meter WTW inoLab
(Weilheim, Germany).
3.2. Lyophilization of Bacterial Cultures
Lyophilization was performed using a Lyophilizer Christ Alpha 1-2 (SciQuip Ltd., Shropshire,
United Kingdom). For lyophilization of bacterial cultures, 1 mL of the sample was used in each case.
Atmosphere 2015, 6 1302
3.3. The Experimental Conditions
Bacterial cultures for the full stratospheric flight were always prepared in three groups. The first
group was the control culture without exposure to UV radiation. The second group of the bacterial
culture was exposed to laboratory UV light of a wavelength of 264 nm. The last group of bacterial
cultures was sent into the stratosphere by a stratospheric probe attached to its surface (exposed to UV
from the stratosphere; temperature: −60 °C).
Exposure of Bacterial Cultures to UV Radiation in the Stratosphere
Lyophilized bacterial cultures were transported by stratospheric balloon to a height of 40 km above
sea level. After landing, the samples were immediately transported to the laboratory, where the
bacterial cultures were recultivated in GTY (glucose, tryptone, yeast extract) nutrient medium and
subsequently tested.
3.4. Measurement of Climatic Conditions
Within the project, sunrise and sunset were recorded, and using the climatological measurements,
the daily air temperatures, minimal, maximal and average air pressure and amount of precipitation
were monitored, as well. During the stratospheric flight, temperature outside the probe, pressure and
relative humidity were detected.
3.5. Cultivation of Bacterial Species
Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Salmonella typhimurium
(S. typhimurium) and methicillin-resistant Staphylococcus aureus (MRSA) were obtained from the
Czech Collection of Microorganisms, Faculty of Science, Masaryk University in Brno, Czech
Republic. Proteus mirabilis (P. mirabilis) and Pseudomonas aeruginosa (P. aeruginosa) were
obtained by isolation from smears, which were collected from infected wounds of patients. Isolated
strains were cultivated using selective agars (blood agar; blood agar with 10% NaCl; blood agar with
amikacin; Endo agar) and identified by matrix-assisted laser desorption/ionization mass spectrometry.
The experiments were approved by the Ethics Committee of Trauma hospital in Brno and done with
the agreement of patients. A smear was sampled by rolling motion at the site of skin puncture using a
sterile swab sampler. All tested bacteria were cultivated as pure strains in non-selective broth
(GTY = glucose, tryptone, yeast extract), and these were cultivated for 24 hours on a shaker at 130 rpm
and 37 °C. The bacterial culture was diluted by cultivation medium to OD
600
= 0.1 for the
following experiments.
3.5.1. Determination of Growth Curves
The procedure for the evaluation of bacterial culture growth before and after UV exposure was
carried out in accordance to our previously published study [29].
Atmosphere 2015, 6 1303
3.5.2. Determination of Bacterial Strain Resistance to Antibiotic Drugs
Petri dishes with GTY agar were coated with the tested bacterial cultures diluted to OD
600
= 0.1. On
the surface of the plates with the bacterial cultures, antibiotic commercial discs were placed containing
the following drugs: erythromycin, penicillin, amoxicillin, tetracycline or lincomycin and a disc with
selenium nanoparticles. These dishes were incubated in a thermostat for 24 hours at 37 °C.
3.6. Mass Spectrometry
The following extraction protocol and sample preparation was based on MALDI BioTyper 3.0 User
Manual Revision 2, whereas a similar extraction method was used also in [30]. One colony of bacterial
cultures was re-suspended in 300 µL of deionized water, and 900 µL of ethanol was added. After
centrifugation at 14,000× g for 2 min, the supernatant was discarded, and the obtained pellet was
air-dried. The pellet was then dissolved in 25 µL of 70% formic acid (v/v) and 25 µL of acetonitrile
and mixed. The samples were centrifuged at 14,000× g for 2 min, and 1 µL of the clear supernatant
was spotted in duplicate onto the MALDI target (MTP 384 target polished steel plate; Bruker
Daltonics, Bremen, Germany) and air-dried at room temperature. Then, each spot was overlaid with
1 µL of saturated α-cyano-4-hydroxycinnamic acid (HCCA) matrix solution in organic solvent (50%
acetonitrile and 2.5% trifluoroacetic acid, both v/v) and air-dried completely prior to MALDI-TOF MS
measurement on UltrafleXtreme MS (Bruker). Spectral data were taken in the m/z range of
2.000 Da–20.000 Da, and each was a result of the accumulation of at least 1000 laser shots obtained
from ten different regions of the same sample spot. Spectra with peaks outside the allowed average
were not considered. Modified spectra were loaded into the MALDI BioTyper™ 3.1 Version (Bruker
Daltonics GmbH, Bremen, Germany).
4. Conclusions
The presented study showed the significant effect of UV radiation (laboratory UV and UV from the
stratosphere region) on the growth, behavior and structure of bacterial cultures. Bacteria exposed to
UV radiation from the stratosphere were part of the probe surface, which was sent into the
stratosphere. It was shown that these cultures become more sensitive to adverse environmental
influences with stronger and more intensive action of UV radiation in the stratosphere, whereas their
growth was significantly inhibited and their basic structural characteristics were changed. Even a
short-term effect of UV radiation significantly modulates the protein composition and behavior of
microorganisms, and therefore, it can be assumed that just the UV at a time when the atmosphere was
not as effective as today accelerated the development of the desirable characteristics of organisms and
thus participated in the management of the development of life as we know it today.
Acknowledgments
Financial support from STRATO-NANOBIOLAB CZ/FMP.17A/0436 is highly acknowledged. The
authors wish to express their thanks to Radek Chmela for the perfect technical cooperation.
Atmosphere 2015, 6 1304
Author Contributions
Dagmar Chudobova tested the effect of UV radiation on the biological activity and growth
properties of the tested microorganisms and prepared the manuscript. Kristyna Cihalova tested the
effect of UV radiation on the ability to form resistance to antibiotics of the tested microorganisms and
participated in the preparation of the manuscript. Pavlina Jelinkova tested the effect of UV radiation on
the biochemical properties of the tested microorganisms and participated in the preparation of the
figures. Jan Zitka designed and manufactured the stratospheric probe using 3D printing. Lukas Nejdl
participated in the 3D printing, the preparation of samples for microbiological measurements and
participated in the preparation of the manuscript. Roman Guran tested the effect of UV radiation on the
proteomic profiles of the tested microorganisms. Martin Klimanek cooperated in the coordination of
the flight and the design of the study. Vojtech Adam participated in the design of the study and in the
drafting of the manuscript. Rene Kizek participated in the design and coordination of the study.
Conflicts of Interest
The authors declare that they have no potential conflicts of interests.
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