Bactericidal effects of low temperature oxygen plasma on bacillus stearothermophilus and staphylococcus aureus

Bactericidal effects of low temperature oxygen plasma on bacillus stearothermophilus and staphylococcus aureus

Danijela VU J O ŠE V I Ć 1,3

, Boban M U G O ŠA 2 , Uroš C VE L B A R 3 , Miran M O Z E T I Č 3

, Urška R E PN IK 4 , Tina Z A V A Š N I K - B E R G A N T 4 , Danijela R A J K O V I Ć 1

, Sanja

M ED EN IC A 2

1Centre for Medical Microbiology, Institute of Public Health, Ljubljanska bb, 81000 Podgorica,

Montenegro Email: danijela.vujosevic@ijzcg.me

2 Centre for Prevention and Disease Control, Institute of Public Health, Ljubljanska bb, 81000

Podgorica, Montenegro Email: boban.mugosa@ijzcg.me

3 Plasma Laboratory F4, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia Email:

uros.cvelbar@ijs.si

4 Biochemistry and Molecular Biology Department, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia Email: urska.repnik@ijs.si

Key words:

Oxygen plasma,

sterilization,

bacteria

Synopsis

Plasma of different origins has been shown to possess effective anti-microbial characteristics In this study three complementary techniques: scanning electron microscopy (SEM), fluorescence microscopy and flow cytometry were applied to monitor time-dependent changes in bacterial viability, morphology

and nucleic acids content of bacteria Bacillus stearothermophilus and Staphylococcus aureus

The plasma sterilization capability demonstrated through this study indicated the potential of this low-temperature oxygen plasma as a promising alternative sterilization technique

Klju čne riječi:

Kiseonikova plazma,

sterilizacija,

bakterije

Sinopsis

BAKTERICIDALNI EFEKTI NISKO-TEMPERATURNE KISEONIKOVE PLAZME NA BACILLUS

STEAROTHERMOPHILUS I STAPHYLOCOCCUS AUREUS

antimikrobne osobine U ovoj studiji su korištene tri komplementarne tehnike: elektronska mikroskopija, fluore-scentna

morfologije i sadržaja nukleinskih kiselina bakterija Bacillus stearothermophilus i Staphylococcus aureus, nakon obrade

plazmom

kao obećavajuće alternativne tehnike za sterilizaciju

INTRODUCTION

A novel sterilization method capable of more rapidly killing microorganisms and less damaging material is low-temperature plasma sterilization (CHAU et al., 1996) The plasma sterilization is safer as opposed to classical sterilization of heat sensitive material with ethylene oxide which leaves highly toxic residues absorbed in the sterilized material, whereas no residuals are left on the surface after plasma treatment (PHILIP et al., 2000) Low-temperature plasma is effective against a broad range of bacteria and killing these microorganisms by producing various reactive species like oxygen, hydroxyl free radicals, and other active species, although these killing mechanisms are still being studied (GADRI et al., 2000; LAROUSSI et al., 2004)

Therefore, a relatively simple and inexpensive design and the absence of the toxicity resulting from the treatment itself, give low-temperature plasma the potential

to replace conventional sterilization methods of medical devices, such as implants, dental instruments etc (MOISAN et al., 2002; JACOBS & LIN, 2001; SAMUEL, et al., 1998; HELHEL et al., 2005)

However, the research of low-temperature plasma sterilization has the complex issue, which is further burdened by a large number of experiment variables, is insufficiently definitive about the selected methodologies and experimental conditions Certainly, more tests and in-depth study of low-temperature sterilization are needed to elucidate the mechanism of low-temperature plasma sterilization (CHOI et al., 2006)

There is a need for reliable and accurate monitoring of plasma sterilization during the process and evaluating after In the plasma, a large number of variables influence the whole system between physical and chemical process However, till now no reliable methods have been found to access the sterilization during sample processing in plasma Monitoring during the process can be done by measuring changes of reactive species in plasma Reliable and accurate plasma diagnostic techniques are presently being developed to provide real-time information on plasma during system operation (KANAZAWA, et al., 1989) And more, new, faster and more accurate techniques for evaluation of post treated bacteria need to be developed, apart from standard count plate technique

The purpose of this study was to determine the sterilization effects of low-temperature highly dissociated oxygen plasma on two selected Gram positive

Staphylococcus aureus, commonly involved in infections and food poisoning Three

complementary techniques apart from plate counting; scanning electron microscopy (SEM), fluorescence microscopy and flow cytometry were applied to monitor time-dependent changes in bacterial viability, morphology and nucleic acids content, all in comparison to heat-treated bacteria at 140 °C

MATERIAL AND METHODS

Bacteria Bacillus stearothermophilus (ATCC No 7953) and Staphylococcus aureus (ATCC No 25923) were obtained from the MicroBioLogics CE (MN, USA)

Bacterial cultures were grown overnight on Colombia agar plates (Difco

Staphylococcus aureus Cells were harvested and resuspended in sterile water 100

cells of Bacillus stearothermophilus and

Staphylococcus aureus were evenly distributed on the surface of sterile glass or

aluminum substrate and air dried in a laminar-flow hood Bacteria on carriers were then either exposed to low-temperature oxygen plasma, or vacuum-treated, or

high-temperature dry heated or remained un-treated (control)

Carriers Pyrex glass rectangular substrate and aluminum sheets were used

as carriers for bacteria Glass carriers were used when, later on, fluorescence or flow cytometry were applied, while aluminum sheets were used with SEM, in order to avoid charging effects inside the electron microscope

Carriers’ activation Both carriers were well activated prior to bacteria

deposition to avoid bacteria agglomeration and in order to achieve uniform even distribution of cells across the entire carrier and the formation of thin layer of cells

on the carrier’s surface The activation process was also performed by oxygen plasma treatment for 5 s Plasma with the same characteristic parameters was used for both, the activation of carriers and for the treatment of bacteria

Heat treatment For studying of temperature effects on bacteria degradation

the samples (bacteria on substrate carriers) were treated with high temperature dry heat at 140 ° C for 20 s, 2 min and 10 min

Plasma treatment Inductively coupled radio-frequency generator with the

output power of about 200 W and the frequency of 27.12 MHz was used to create uniform plasma in a glass tube with the inner diameter of 36 mm and the length of

65 cm (Figure 1) The tube was first evacuated to the pressure of 3 Pa and then filled with oxygen to pressure from 30 Pa to 150 Pa Plasma parameters (electron temperature, neutral O-atom density, ion density) were measured with a double Langmuir probe and Fiber Optics Catalytic Probes

Samples were exposed for different treatment time to plasma with the electron

to vacuum conditions but without plasma in order to detect possible changes caused

by evacuation only and not by plasma

F i g u r e 1 S c h e m a t i c o f t h e d i s c h a r g e v e s s e l

Plate counts technique Before and after each treatment the samples (bacteria

on carriers) were placed in sterile containers with 2 ml 0.85% NaCl saline solution The containers were then briefly vortexed in order to wash bacterial cells from carriers The suspension was serially diluted (1/10) in saline to the required concentration range A sample of 100 µl of the diluted suspension was inoculated onto

Colombia agar plate Plates were than incubated at 37 °C (Staphylococcus aureus)

units were finally counted to determine the number of survivors of bacteria

Scanning electron microscopy (SEM) Samples were imaged using

field-emission scanning electron microscopy at a low kinetic energy of primary electrons Low-temperature oxygen plasma-treated bacteria, high temperature dry heat-treated bacteria, vacuum-treated bacteria (control) and un-treated bacteria (control) on aluminum carriers were viewed in the Carl Zeiss Supra 35 VP scanning electron

1000 eV and 600 eV

Fluorescence microscopy Fluorescently labeled low-temperature oxygen

plasma-treated bacteria, high-temperature dry heat-treated bacteria, vacuum-treated bacteria and un-treated bacteria on glass carriers were viewed using wide-field

(Molecular Probes, The Netherlands) was used to stain bacteria on glass carriers according to the manufacturer’s procedure For staining solution two DNA stains SYTO 9 (3.34 mM) and propidium iodide (PI, 20 mM), were mixed together (1.5 µl + 1.5 µl) and diluted with 1 ml of deionised sterile water Bacteria were incubated with

20 µl of staining solution at room temperature in the dark After 15 min, cells were washed with deionised sterile water and viewed under the inverted fluorescence microscope Olympus IX71 with digital camera Olympus DP50 Green fluorescence signal of SYTO 9 and red fluorescence signal of PI were detected using U-M41001 (exc 461 - 500 nm / em 511 - 560 nm) and U-MWIY2 (exc 545 – 580 nm / em > 600 nm) Olympus filter cubes, respectively Oil objectives × 60 (N.A = 1.40) and ×100

(N.A = 1.35) were used

Flow cytometry Fluorescently labeled low-temperature oxygen plasma-treated

bacteria, dry heat-treated bacteria and un-treated bacteria on glass carriers were analyzed on a FACSCalibur flow cytometer using CellQuest software, version 3.3 (Becton Dickinson, CA) After treatment, glass carriers were gently washed with sterile

viability kit L7012 (Molecular Probes, The Netherlands) was used to stain bacteria In

500 µl of a cell suspension, 0.75 µl of 3.34 mM SYTO 9 and 0.75 µl on 30 mM PI were added, and the suspension was then incubated at room temperature for 15 min, protected from the light The analysis gate was set in the untreated sample in dot plots of green (FL1, SYTO 9) and red (FL3, PI) fluorescence versus side scatter

RESULTS WITH DISCUSSION

Plasma characteristics and activation of carriers Prior to experiments

performed on bacteria characteristic parameters in low-temperature oxygen plasma were measured with a double Langmuir probe and a Fibre Optics Catalytic Probes The determined electron temperature was 5 eV, the neutral O-atom density was 8.5 ×

exceeded the ionization fraction by more than 5 orders of magnitude, i.e allowing for almost entire plasma to interact as oxygen radicals with bacteria These plasma parameters were kept constant during all experiments with cells described in continuation

The activation of carriers was done in the period of 5 s Taking into account that

allowed high surface energy by mostly removing impurities of a particular Al and glass carrier, respectively In this way, even distribution of bacteria cells on the entire carrier surface was enabled Furthermore, X-ray photoelectron spectroscopy (XPS) analysis of thus activated Al and glass carriers showed no traces of organic impurities, which could cause a decrease of surface energy and consequently uneven distribution of bacteria later on Therefore, we have concluded that bacteria were deposited on well activated carriers

Samples were exposed to plasma separately for different periods of time The

1 × 1024 m-2

Morphological changes caused by low-temperature oxygen plasma To

better investigate the effects on the cell structure changes during the oxygen plasma sterilization process, scanning electron microscopy (SEM) was used to obtain the cell image on both: un-treated and treated bacteria Representative SEM micrographs of the un-treated bacteria (Figure 2), bacteria treated with dry heat at 140 °C (Figure 3 and 4) and bacteria treated with low-temperature highly dissociated oxygen plasma

(Figure 5) have been taken The un-treated bacteria Bacillus stearothermophilus (Figure 2a) and Staphylococcus aureus (Figure 2b) exhibit regular rod-shaped and

coccoid form, respectively Furthermore, no alterations or lesions of the cell wall were observed for the un-treated bacteria

F i g u r e 2 S E M m i c r o g r a p h s o f u n - t r e a t e d b a c t e r i a o n a l u m i n i u m s u b s t r a t e : ( a ) B a c i l l u s

s t e a r o t h e r m o p h i l u s a n d ( b ) S t a p h y l o c o c c u s a u r e u s

F i g u r e 3 S E M p h o t o g r a p h s o f h e a t - t r e a t e d b a c t e r i a a t 1 4 0 ° C f o r 2 0 s e c o n d s : ( a )

B a c i l l u s s t e a r o t h e r m o p h i l u s a n d ( b ) S t a p h y l o c o c c u s a u r e u s

SEM images of the bacteria treated with dry heat at 140 °C either 20 s (Figs

3a and 3b) or 10 min (Figure 4a and b) show that Bacillus stearothermophilus still retained its typical rod shaped form (Figure 3a and 4a) and Staphylococcus aureus

still its typical coccoid form (Figure 3b and 4b) Both thus appeared quite similar to the un-treated cells in the control experiments (Figure 2a and b)

Nevertheless, some changes in bacterial cell wall morphology were observed after prolonged treatment time at 140 °C indicating certain effect of high temperature

on bacterial cells after 10 min (Figure 4a and b) On the other hand, after 20 s at

140 °C no visible changes in cell wall morphology were observed (Figure 3a and b)

F i g u r e 4 S E M p h o t o g r a p h s o f h e a t - t r e a t e d b a c t e r i a a t 1 4 0 ° C f o r 1 0 m i n u t e s : ( a ) B a c i l l u s

s t e a r o t h e r m o p h i l u s a n d ( b ) S t a p h y l o c o c c u s a u r e u s

F i g u r e 5 S E M p h o t o g r a p h s o f b a c t e r i a t r e a t e d w i t h l o w - t e m p e r a t u r e h i g h l y d i s s o c i a t e d

o x y g e n p l a s m a f o r 2 0 s e c o n d s : ( a ) B a c i l l u s s t e a r o t h e r m o p h i l u s a n d ( b ) S t a p h y l o c o c c u s

a u r e u s

In contrast, treatment with highly dissociated oxygen plasma for 20 s resulted

in a significant reduction of cell size and in modified morphology (Figure 5) compared to the un-treated controls (Figure 2) or thermally treated bacteria (Figure

3 and 4) Bacteria Bacillus stearothermophilus (Figure 5a) and Staphylococcus aureus (Figure 5 b) became badly damaged; their cell membrane and cytoplasm

already after 20 s with cell wall and bacterial cytoplasm becoming barely recognizable (Figure 5)

Even longer than 20 s (up to 240 s) were also tested and, expectedly, bacteria

Bacillus stearothermophilus (Figure 6) and Staphylococcus aureus (Figure 7) were

completely destroyed after the prolonged plasma treatment

F i g u r e 6 S E M p h o t o g r a p h s o f b a c t e r i a B a c i l l u s s t e a r o t h e r m o p h i l u s t r e a t e d w i t h l o w

-t e m p e r a -t u r e h i g h l y d i s s o c i a -t e d o x y g e n p l a s m a : ( a ) f o r 5 5 s e c o n d s ; ( b ) f o r 2 4 0 s e c o n d s

F i g u r e 7 S E M p h o t o g r a p h s o f b a c t e r i a S t a p h y l o c o c c u s a u r e u s t r e a t e d w i t h l o w

-t e m p e r a -t u r e h i g h l y d i s s o c i a -t e d o x y g e n p l a s m a : ( a ) f o r 6 0 s e c o n d s ; ( b ) f o r 2 4 0 s e c o n d s

Another control experiment was performed with Bacillus stearothermophilus and Staphylococcus aureus in such a way that bacteria were exposed to the vacuum

only but without plasma The size and shape of bacteria did not depend on this pre-treatment under vacuum conditions (Figure 8) Furthermore, SEM did not confirm any notable changes on the surface of bacteria which would be caused directly by vacuum, used in a reactor during plasma experiment

We have thus concluded that, in our experiments, evacuation itself did not cause any visible morphological changes of bacteria

These result demonstrated that a strong etching process of the plasma caused the sterilizing effect on the bacteria, when they were exposed to this low-temperature highly dissociated oxygen plasma Bacteria have been heavily damaged, reduced to microscopic debris, ruptured with their cellular contents

released on the substrate surface In addition, for longer exposure times, total cell fragmentation was observed This demonstrates that the plasma has direct physical impact on the cells

F i g u r e 8 S E M p h o t o g r a p h s o f v a c u u m - t r e a t e d b a c t e r i a : ( a ) B a c i l l u s s t e a r o t h e r m o p h i l u s

a n d ( b ) S t a p h y l o c o c c u s a u r e u s

Fluorescence labeling Viability of bacteria was assessed with the

bacteria with intact cell membranes and emits green fluorescence, whereas propidium iodide (PI) penetrates only into bacterial cells with damaged membranes and exhibits red fluorescence When bacterial cells are stained with a mixture of the two dyes, viable cells fluorescence bright green, while dead cells exhibit weaker green fluorescence and also fluoresce red This differential staining was particularly obvious when cells were observed under the fluorescent microscope In Figure 9

(Bacillus stearothermophilus) and Figure 10 (Staphylococcus aureus) binding of

DNA dye SYTO 9 and PI is shown for the un-treated bacteria (a, d), the bacteria treated with high-temperature dry heat at 140 °C for 10 min (b, e) and the bacteria treated with low-temperature highly dissociated oxygen plasma for 20 s (c, f) Binding of DNA dyes to bacteria treated with high-temperature dry heat for 20 s at

observed with SEM after 20 s

In our experiments, the un-treated bacteria Bacillus stearothermophilus

showed strong SYTO 9 staining (green fluorescence, Figure 9a) but very weak PI

staining (red fluorescence, Figure 9d) In high-temperature-treated bacteria, the

number of PI-stained cells stained increased compared to SYTO 9 only-stained cells (Figure 9b and e) Plasma-treated bacteria show neither SYTO 9 (Figure 9c) nor PI (Figure 9f) fluorescence signal SYTO 9 and propidium iodide bind to the nucleic acids, then absence of fluorescence could be explained by denaturation and/or fragmentation bacterial DNA

F i g u r e 9 F l u o r e s c e n c e m i c r o s c o p y i m a g e s o f b a c t e r i a B a c i l l u s s t e a r o t h e r m o p h i l u s

l a b e l l e d w i t h D N A d y e s : S Y T O 9 ( g r e e n f l u o r e s c e n c e i n a , b a n d c ) a n d p r o p i d i u m i o d i d e

( r e d f l u o r e s c e n c e i n d , e a n d f ) ( a , d ) u n - t r e a t e d b a c t e r i a , ( b , e ) b a c t e r i a t r e a t e d w i t h h i g h - t e m p e r a t u r e d r y h e a t f o r 1 0

m i n u t e s a t 1 4 0 ° C a n d ( c, f ) b a c t e ri a tr e a t e d w it h low - te m p e r a tu r e h i g h l y d i s s o c i a t e d

o x y g e n p l a s m a f o r 2 0 s e c o n d s U n - t r e a t e d b a c t e r i a s h o w s t r o n g S Y T O 9 ( a ) s t a i n i n g ( f l u o r e s c e n c e ) b u t w e a k p r o p i d i u m i o d i n e ( P I ) s t a i n i n g ( d ) I n h i g h - t e m p e r a t u r e t r e a t e d

b a c t e r i a P I s t a i n i n g ( r e d f l u o r e s c e n c e ) i n c r e a s e d ( e ) c o m p a r e d t o S Y T O 9 ( b ) P l a s m a

t r e a t e d - b a c t e r i a s h o w n o S Y T O 9 ( c ) o r P I ( f ) f l u o r e s c e n c e s i g n a l

Binding of DNA SYTO 9 and PI in bacteria Staphylococcus aureus exhibited

quite a similar way of staining (Figure 10) The un-treated bacteria showed strong SYTO 9 staining (Figure 10a, green fluorescence) In contrast to the un-treated

bacteria Bacillus stearothermophilus, there were some PI-stained bacteria Staphylococcus aureus cells already in the un-treated sample (Figure 10d) In

high-temperature-treated bacteria, PI-stained bacteria dominated (Figure 10e, red fluorescence) and only a few cells remained SYTO 9 positive (Figure 10b, green

fluorescence) and PI negative (Figure 10e) Plasma treated-bacteria Staphylococcus aureus showed neither SYTO 9 (Figure 10c) nor PI (Figure 10f) fluorescence, the same as Bacillus stearothermophilus

In the case of plasma-treated bacteria, the original DNA is either heavily modified or fully oxidized, so there is practically no DNA left to assure stain binding Degradation of bacterial DNA is achieved by oxygen radicals, especially neutral oxygen atoms The resulted emitted fluorescence is poor: stain is evenly distributed

on the carrier showing no preference to the sites where bacteria reside This