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種々の温度条件下でのバイオフィルム形成とその除 去技術
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九州大学大学院生物資源環境科学府環境農学専攻
https://doi.org/10.15017/26649
出版情報：Kyushu University, 2012, 博士（農学）, 課程博士 バージョン：
権利関係：
BIOFILM FORMATION UNDER VARIOUS TEMPERATURE CONDITIONS AND ITS REMOVAL
TECHNIQUE
MORIMATSU KAZUYA
2013
BIOFILM FORMATION UNDER VARIOUS TEMPERATURE CONDITIONS AND ITS REMOVAL
TECHNIQUE
By
MORIMATSU KAZUYA
A thesis submitted in fulfillment of the requirement for the degree of DOCTOR OF PHILOSOPHY
Postharvest Science Laboratory
Graduate School Bioresource and Bioenvironmental Sciences Kyushu University
Japan
March, 2013
BIOFILM FORMATION UNDER VARIOUS TEMPERATURE CONDITIONS
AND ITS REMOVAL TECHNIQUE
ABSTRACT
Recently, there has been a high demand for food safety because of frequent food-poisoning incidents. In order to provide customers with safe food products, it is necessary to control microorganisms present on a product. However, the attached bacterial cells forming biofilm would come to have excessive tolerance against sterilization. Thus, the presence of bacteria forming biofilm has received much attention as a problem related to food safety. The objectives of this study were to investigate the characteristics of bacterial biofilm formation under various temperature conditions and to explore optimal concentrations and pH values of sodium hypochlorite (NaOCl) against bacteria forming biofilm. The thesis consists of introduction, three main chapters and conclusion.
In chapter 1, the background of the thesis was presented.
In chapter 2, the effect of temperature fluctuation on a bacterial biofilm formation was investigated in a single culture of Salmonella enterica, Staphylococcus aureus and Pseudomonas putida. In the single culture, biofilm formation of Sal. enterica and Sta.
aureus was enhanced even at a low temperature through temperature fluctuation while that of Pseudomonas putida at a low temperature was inhibited by temperature fluctuation.
Moreover, in the result of investigation in a mixed culture of each of Sal. enteica and Sta.
aureus with P. putida, a bacterial interaction between P. putida and each of Sal. enterica and Sta. aureus was induced by a stress of poor nutrient at high temperature, resulting in an acceleration of biofilm formation. This acceleration of biofilm formation was not affected considerably by temperature fluctuation, while it seemed that temperature fluctuation can enhance the attachment of Sal. enterica and Sta. aureus.
In chapter 3, the investigation focused on the effect of temperature on biofilm formation was conducted. In the result, it was shown that a maximum amount of attached biofilm was higher at low temperature than at high temperature in rich nutrient condition. In addition, mature biofilms after it reached the maximum amount were detached at high temperature while the mature biofilms remained on the attachment surface at low temperature. However, these effects of temperature on bacterial biofilm formation could be minimized by the lack of nutrients.
In chapter 4, the purpose of this research was to explore the optimum concentrations and pH values of NaOCl treatment against bacteria forming biofilm. An increase in the available chlorine concentrations and the pH values of NaOCl solution accelerated a
removal of the attached biofilm, especially, the treatment at a strong alkaline state of NaOCl solution gave the quickest removal rate of the biofilm. However, in regardless of conditions of NaOCl treatment, the attached biofilm partly remained attachment after NaOCl treatment. Moreover, the success and failure of sterilization with NaOCl treatment were vague.
In chapter 5, conclusion of this study was proposed.
AKNOWLEDGEMENT
First and foremost, I would like to express my sincere thanks to Prof. Toshitaka Uchino, my supervisor for his valuable guidance and support throughoaut the course of this research work. Indeed, without him, this study would not have been possible. I also extend my grateful thanks to Prof. Eiji Inoue, Laboratory of Bioproduction Engineering and Associate Prof. Fumihiko Tanaka, Laboratory of Postharvest science at Kyushu University, Assistant Prof. Daisuke Hamanaka for their intimate reviews and constructive suggestions for the draft of this dissertations.
I would also like to thank Dr. Takuma Genkawa, Dr. Hung Van Duong, Ms. Vipavee Trivittayasil for their technical advice and laboratory assistance and many thanks to Ayumi Nihara, Hiromi Umezaki, Mami Onishi, Takeshi Watanabe, Masahiro Okubo, Hikaru Imamura, Yudai Eguchi, Fuyuna Tanaka for their useful help in performing experiments during research period. Great thanks also go to Mr. Yoshiaki Hori for his help in installing experimental equipments. Many thanks go to all members of Laboratory of Postharvest Science for their great help during my PhD candidature. Moreover, I want to thank all the members of the Laboratory of Bioproduction Engineering. I would also like to thank JSPS for financial contribution to this research work.
Finally, I am grateful to all of my family members and relatives who have strongly encouraged, support and assisted me.
TABLE OF CONTENTS
ABSTRACT i
ACKNOWLEDGEMENT iv
TABLE OF CONTENTS vi
LIST OF FIGURES ix
LIST OF TABLE xiv
CHAPTER 1: Introduction 1.1. BACKGROUND OF RESEARCH...1
1.2. AIM OF THIS RESEARCH...4
CHAPTER 2: Effect of temperature fluctuation on biofilm formation 2.1. INTRODUCTION...6
2.2. MATERIALS AND METHODS...8
2.2.1. Bacterial strains and growth conditions
2.2.2. Biofilm formation on an inner surface of polystyrene tube in a single culture 2.2.3. Biofilm formation on an inner surface of polystyrene tube in a mixed culture
2.2.4. Quantification assay for an amount of attached biofilm 2.2.5. Bacterial count in biofilm matrix of a mixed culture 2.2.6. Statistical analysis
2.3. RESULTS AND DISCUSSION...15
2.3.1. Biofilm formation in a single culture 2.3.2. Biofilm formation in a mixed culture
2.4. CONCLUSIONS...37
CHAPTER 3:Effect of temperature on biofilm formation
3.1. INTRODUCTION...39
3.2. MATERIALS AND METHODS...40
3.2.1. Bacterial strain and growth condition
3.2.2. Biofilm formation on a coupon of polyvinyl chloride 3.2.3. Quantification assay for an amount of attached biofilm 3.2.4. Bacterial count in biofilm matrix
3.2.5. Statistical analysis
3.3. RESULTS AND DISCUSSION...43
3.4. CONCLUSIONS...51
CHAPTER 4: Effect of sodium hypochlorite treatment on bacteria forming bifoilm 4.1. INTRODUCTION...53
4.2. MATERIALS AND METHODS...55
4.2.1. Bacterial strain and growth condition 4.2.2. Biofilm formation on a coupon of polyvinyl chloride 4.2.3. Conditioning sodium hypochlorite 4.2.4. Sodium hypochlorite treatment 4.2.5. Quantification assay for an amount of attached biofilm after hypochlorite treatment 4.2.6. Bacterial count in biofilm matrix after hypochlorite treatment 4.3. RESULTS AND DISCUSSION...59
4.4. CONCLUSIONS...71
CHAPTER 5: Summary and conclusions...73
References... ...77
LIST OF FIGURES
Fig.1.1 Process of biofilm formation...2
Fig. 2.1 Profile of fluctuating temperature condition...11
Fig.2.2 Outline of experimental method for quantification assay for an amount of attached biofilm and bacterial count in biofilm matrix of a mixed culture...13
Fig. 2.3 Amount of attached biofilm indicated by absorbance in the single cultures of Sal.
enterica, Sta. aureus, and P. putida at 5 C...16
Fig. 2.4 Amount of attached biofilm indicated by absorbance in the single cultures of Sal.
enterica, Sta. aureus, and P. putida at 30 C...17
Fig. 2.5 Amount of attached biofilm indicated by absorbance in the single cultures of Sal.
enterica, Sta. aureus, and P. putida at fluctuating temperature condition of the pattern (i)...19
Fig. 2.6 Amount of attached biofilm indicated by absorbance in the single cultures of Sal.
enterica, Sta. aureus, and P. putida at fluctuating temperature condition of the pattern (ii)...21
Fig. 2.7 Biofilm amount indicated by absorbance and viable bacterial count in biofilm in the mixed culture of P. putida with Sal. enterica at 5 C...23
Fig. 2.8 Biofilm amount indicated by absorbance and viable bacterial count in biofilm in the mixed culture of P. putida with Sta. aureus at 5 C...24
Fig. 2.9 Biofilm amount indicated by absorbance and viable bacterial count in biofilm in the mixed culture of P. putida with Sal. enterica at 30 C...26
Fig. 2.10 Biofilm amount indicated by absorbance and viable bacterial count in biofilm in the mixed culture of P. putida with Sta. aureus at 30 C...27
Fig. 2.11 Biofilm amount indicated by absorbance and viable bacterial count in biofilm in
the mixed culture of P. putida with Sal. enterica at fluctuating temperature condition of the pattern (i)…...30
Fig. 2.12 Biofilm amount indicated by absorbance and viable bacterial count in biofilm in the mixed culture of P. putida with Sta. aureus at fluctuating temperature condition of the pattern (i)...31
Fig. 2.13 Biofilm amount indicated by absorbance and viable bacterial count in biofilm in the mixed culture of P. putida with Sal. enterica at fluctuating temperature condition of the pattern (ii)...34
Fig. 2.14 Biofilm amount indicated by absorbance and viable bacterial count in biofilm in the mixed culture of P. putida with Sta. aureus at fluctuating temperature condition of the pattern (ii)...36
Fig. 3.1 Amounts of biofilm as indicated by absorbance of P. putida cultures incubated at different constant temperatures in 100% TSB...44
Fig. 3.2 Viable bacterial counts as a measure of P. putida biofilm formation in cultures incubated at different constant temperatures in 100% TSB...46
Fig. 3.3 Amounts of biofilm as indicated by absorbance of P. putida cultures incubated at different constant temperatures in 5% diluted TSB...48
Fig. 3.4 Viable bacterial counts as a measure of P. putida biofilm formation in cultures incubated at different constant temperatures in 5% diluted TSB...50
Fig.4.1 Effect of initial amount of biofilm on the residual biofilm amount after NaOCl treatment of 50 ppm in pH 11.7...60
Fig.4.2 Effect of initial amount of biofilm on the residual biofilm amount after NaOCl treatment of 50 ppm in pH 9.8...61
Fig.4.3 Effect of initial amount of biofilm on the residual biofilm amount after NaOCl treatment of 50 ppm in pH 5.7...62
Fig.4.4 Effect of initial amount of biofilm on the residual biofilm amount after NaOCl treatment of 100 ppm in pH 10.1...64
Fig.4.5 Effect of initial amount of biofilm on the residual biofilm amount after NaOCl treatment of 200 ppm in pH 10.4...65
Fig. 4.6 Effect of the initial biofilm amount on the residual biofilm amount after NaOCl treatment...68
Fig.4.7 Viable bacterial count in the residual biofilm after NaOCl treatment for 1 minute...70
LIST OF TABLE
Table 4.1 Culturing condition...57
C H A P T E R 1 I n t r o d u c t i o n
1 . 1 . B A C K G R O U N D O F R E S E A R C H
R e c e n t l y, t h e r e h a s b e e n a h i g h d e m a n d f o r f o o d s a f e t y b e c a u s e o f f r e q u e n t f o o d - p o i s o n i n g i n c i d e n t s . I n o r d e r t o p r o v i d e c u s t o m e r s w i t h s a f e f o o d p r o d u c t s , i t i s n e c e s s a r y t o c o n t r o l m i c r o o r g a n i s m s p r e s e n t o n a p r o d u c t s u r f a c e . T h e r e f o r e , a l o t o f r e s e a r c h e s o n f o o d s a f e t y, f o r e x a m p l e , s t e r i l i z i n g m i c r o b e a n d p r e d i c t i n g m i c r o b i a l g r o w t h , h a s b e e n c o n d u c t e d a c t i v e l y ( B r a t c h e l l e e t a l. , 1 9 9 0 , W i j t z e s e t a l. , 2 0 0 1 , K i m e t a l. , 2 0 0 3 , C h r i s t e n s e n e t a l. , 2 0 0 8 ) . A l t h o u g h m o s t o f r e s e a r c h e s t a r g e t a t p l a n k t o n i c c e l l s , m a n y m i c r o b i a l c e l l s a t t a c h o n t o a m a t e r i a l s u r f a c e i n a r e a l e n v i r o n m e n t o f a f o o d p r o d u c t i o n p r o c e s s , a n d t h e a t t a c h e d c e l l s s o m e t i m e s f o r m b i o f i l m ( J e s s e n e t a l. , 2 0 0 3 ) ( F i g . 1 . 1 ) . I n t h e p r o c e s s o f b i o f i l m f o r m a t i o n , e x t r a c e l l u l a r p o l y s a c c h a r i d e i s p r o d u c e d t o e m b e d t h e a t t a c h e d c e l l s , w h i c h w o u l d c o m e t o h a v e e x c e s s i v e t o l e r a n c e a g a i n s t
a a a a a a a
(1). Bacterial attachment (2). Bacterial growth and biofilm formation Planktonic cells
Fig.1.1 Process of biofilm formation Attachment cells
Extracellular polysaccharide
(3). Biofilm detachment
(1) (2)
(3)
(1). Bacterial attachment (2). Bacterial growth and biofilm formation Planktonic cells
Fig.1.1 Process of biofilm formation Attachment cells
Extracellular polysaccharide
(3). Biofilm detachment (1). Bacterial attachment (2). Bacterial growth and
biofilm formation Planktonic cells
Fig.1.1 Process of biofilm formation Attachment cells
Extracellular polysaccharide
(3). Biofilm detachment
(1) (2)
(3)
s e v e r a l s t r e s s e s s u c h a s h e a t a n d c h e m i c a l t r e a t m e n t ( Z o t t o l a e t a l. , 1 9 9 4 , C h m i e l e w s k i e t a l. , 2 0 0 3 ) . T h e m i c r o b i a l c e l l s i n b i o f i l m s h o w a d i f f e r e n t g r o w t h r a t e f r o m p l a n k t o n i c c e l l s , a s p o i n t e d o u t b y K i n n i m e n t e t a l. ( 1 9 9 2 ) . F r o m t h e s e f a c t s , a t r e a t m e n t o f t h e p r e v i o u s r e s e a r c h u t i l i z i n g t h e p l a n k t o n i c c e l l s s o m e t i m e s c a n n o t c o n t r o l t h e b a c t e r i a f o r m i n g b i o f i l m . T h u s , t h e p r e s e n c e o f b a c t e r i a f o r m i n g b i o f i l m h a s r e c e i v e d m u c h a t t e n t i o n a s a p r o b l e m r e l a t e d t o f o o d s a f e t y, a n d t h e r e s e a r c h e s o n t h e b a c t e r i a f o r m i n g b i o f i l m w e r e p r o m o t e d i n o r d e r t o c o n t r o l t h e p r o b l e m s c a u s e d b y b a c t e r i a l b i o f i l m . I t w a s r e p o r t e d t h a t b a c t e r i a l a t t a c h m e n t a n d b i o f i l m f o r m a t i o n i n f o o d p r o c e s s i n g f a c t o r y c a n b e i n h i b i t e d b y m o d i f y i n g a c h a r a c t e r i s t i c s o f t h e e q u i p m e n t s u r f a c e ( W a n g e t a l. , 2 0 0 3 , N i s h i o k a e t a l. , 2 0 0 4 ) . H o w e v e r , t h i s m e t h o d c a n n o t b e a p p l i e d t o t h e f o o d s u r f a c e t r e a t m e n t . I n a d d i t i o n t o t h e s e s u g g e s t i o n s , C o s t e r t o n e t a l. ( 2 0 0 1 ) s u g g e s t e d s e v e r a l t r e a t m e n t s a g a i n s t b a c t e r i a f o r m i n g b i o f i l m , h o w e v e r , t h e s e t r e a t m e n t s h a v e n o t b e e n d e v e l o p e d s u f f i c i e n t l y f o r e n s u r i n g f o o d s a f e t y. H o u d t e t a l. ( 2 0 1 0 ) a l s o p r o m o t e d t h e n e e d f o r b e t t e r u n d e r s t a n d i n g o f t h e b a c t e r i a l m e c h a n i s m o f b i o f i l m
f o r m a t i o n i n o r d e r t o c o n t r o l t h e p r o b l e m s c a u s e d b y b a c t e r i a l b i o f i l m f o r m a t i o n . T h e r e f o r e , i t i s i m p o r t a n t f o r e n s u r i n g a h i g h s a f e t y o f f o o d t o g a i n m o r e u n d e r s t a n d i n g o f t h e c h a r a c t e r i s t i c s o f b i o f i l m f o r m a t i o n u n d e r e n v i r o n m e n t c o n d i t i o n r e l a t e d w i t h a p r o d u c t i o n p r o c e s s i n f o o d i n d u s t r y a n d t o e x p l o r e t h e t r e a t m e n t a g a i n s t b a c t e r i a f o r m i n g b i o f i l m .
1 . 2 . A I M O F T H I S R E S E A R C H
T h i s r e s e a r c h w a s u n d e r t a k e n t o i n v e s t i g a t e t h e e f f e c t o f e n v i r o n m e n t c o n d i t i o n r e l a t e d w i t h t h e p r o c e s s o f f o o d i n d u s t r y o n b a c t e r i a l b i o f i l m f o r m a t i o n a n d t o e x p l o r e t h e t r e a t m e n t a g a i n s t b a c t e r i a f o r m i n g b i o f i l m . A l t h o u g h u n s t e a d y c o n d i t i o n i s f r e q u e n t l y o b s e r v e d d u r i n g p r o c e s s o f f o o d i n d u s t r y, t h e r e h a v e b e e n n o i n f o r m a t i o n o n a n e f f e c t o f u n s t e a d y c o n d i t i o n o n b a c t e r i a l b i o f i l m f o r m a t i o n . T h e s t u d y i n c h a p t e r 2 i n v e s t i g a t e d b i o f i l m f o r m a t i o n u n d e r f l u c t u a t i n g t e m p e r a t u r e c o n d i t i o n w h i c h i s f r e q u e n t l y o b s e r v e d d u r i n g f o o d d i s t r i b u t i o n . I n t h e r e s u l t , a l t h o u g h a r i s k o f
t e m p e r a t u r e f l u c t u a t i o n w a s r e c o g n i z e d b y t h e r e s u l t o f p r e s e n t s t u d y, a n e f f e c t o f t e m p e r a t u r e o n b a c t e r i a l b i o f i l m f o r m a t i o n w a s u n c l e a r ( M o r i m a t s u e t a l. , 2 0 0 9 , 2 0 1 0 , 2 0 1 2 ) . I n c h a p t e r 3 , t h e i n v e s t i g a t i o n w a s f o c u s e d o n t h e e f f e c t o f t e m p e r a t u r e o n b i o f i l m f o r m a t i o n . A s a r e s u l t , b a c t e r i a f o r m e d a d i f f e r e n t a m o u n t o f b i o f i m w i t h c u l t u r i n g c o n d i t i o n . T h u s , i n c h a p t e r 4 , t o e x p l o r e t h e t r e a t m e n t s t e r i l i z i n g a n y b a c t e r i a f o r m i n g b i o f i l m r e g a r d l e s s o f a m o u n t o f a t t a c h e d b i o f i l m , b a c t e r i a w i t h i n a d i f f e r e n t a m o u n t o f a t t a c h e d b i o f i l m w a s t r e a t e d w i t h h y p o c h l o r i t e s o d i u m ( N a O C l ) , w h i c h i s p o p u l a r l y u t i l i z e d f o r f o o d s a n i t a t i o n . I n c h a p t e r 5 , t h e c h a r a c t e r i s t i c s o f b i o f i l m f o r m a t i o n u n d e r v a r i o u s t e m p e r a t u r e c o n d i t i o n s w e r e s u m m a r i z e d a n d t h e t r e a t m e n t w h i c h c o n t r o l b a c t e r i a f o r m i n g b i o f i l m s u f f i c i e n t l y w a s c o n c l u d e d .
C H A P T E R 2
E f f e c t o f t e m p e r a t u r e f l u c t u a t i o n o n b i o f i l m f o r m a t i o n
2 . 1 . I N T R O D U C T I O N
M o s t o f p r e v i o u s s t u d i e s r e l a t e d w i t h b i o f i l m f o r m a t i o n h a v e f o c u s e d o n a s i n g l e m i c r o b i a l s t r a i n a n d h a v e b e e n c o n d u c t e d u n d e r s t e a d y e n v i r o n m e n t a l c u l t u r e c o n d i t i o n s ( D e w a n t i e t a l. , 1 9 9 5 , R i n a u d i e t a l. , 2 0 0 6 , R o d e e t a l. , 2 0 0 7 , L i a n o u e t a l. , 2 0 1 2 ) . H o w e v e r , t h e r e a r e m a n y k i n d s o f m i c r o b i a l s t r a i n s o n f o o d i n a n a c t u a l s i t u a t i o n , a n d a n y m i c r o b i a l b i o f i l m s a r e g e n e r a l l y d e v e l o p e d u n d e r m u l t i - e x i s t e n t c o n d i t i o n o f v a r i o u s s t r a i n s o f m i c r o o r g a n i s m s ( B a g g e e t a l. , 2 0 0 1 ) . T h e t h i c k n e s s a n d s t a b i l i t y o f b i o f i l m s a r e s o m e t i m e s e n h a n c e d b y a n i n t e r a c t i o n a m o n g v a r i o u s s t r a i n s o f m i c r o o r g a n i s m s , a s J a m e s e t a l. ( 1 9 9 5 ) p o i n t e d o u t . A n i n t e r a c t i o n b e t w e e n d i f f e r e n t b a c t e r i a l s p e c i e s h a s b e e n r e s e a r c h e d a c t i v e l y, a n d i t w a s r e p o r t e d t h a t t h e i n t e r a c t i o n s c a n b e c l a s s i f i e d a s c o o p e r a t i o n , c o m p e t i t i o n , n e u t r a l i s m , a n d m o r e ( H a a c k e t a l. , 1 9 8 2 , S t u r m a n e t a l. , 1 9 9 4 , S i e b e l e t
a l. , 1 9 9 1 ) . A l t h o u g h m o s t o f t h e s e s t u d i e s o n t h e b a c t e r i a l i n t e r a c t i o n h a v e b e e n a l s o c o n d u c t e d u n d e r s t e a d y e n v i r o n m e n t a l c u l t u r e c o n d i t i o n s , t h e s u r r o u n d i n g s a r o u n d t h e m i c r o o r g a n i s m s f r e q u e n t l y f l u c t u a t e . F o r e x a m p l e , a t e m p e r a t u r e f l u c t u a t i o n i s o b s e r v e d d u r i n g d i s t r i b u t i o n o f f o o d a n d a g r i c u l t u r a l p r o d u c e ( J a c x s e n s e t a l. , 2 0 0 2 , U c h i n o e t a l. , 2 0 0 6 ) . H o w e v e r , t h e r e h a v e b e e n n o t m a n y s t u d i e s o f b i o f i l m c o n d u c t e d o n t h e s e u n s t e a d y c o n d i t i o n . T h e r e f o r e , t a k i n g t h e s e e n v i r o n m e n t i n b i o f i l m f o r m a t i o n o n t h e s u r f a c e o f f o o d i n t o c o n s i d e r a t i o n , t h e e f f e c t o f t h e u n s t e a d y t e m p e r a t u r e c o n d i t i o n o n b i o f i l m f o r m a t i o n u n d e r a b a c t e r i a l i n t e r a c t i o n i n a m i x e d c u l t u r e s h o u l d b e i n v e s t i g a t e d .
T h e p r e s e n t s t u d y a i m s f i r s t l y t o c l a r i f y t h e e f f e c t o f t e m p e r a t u r e f l u c t u a t i o n o n b i o f i l m f o r m a t i o n o f S a l m o n e l l a e n t e r i c a, S t a p h y l o c o c c u s a u r e u s a n d P s e u d o m o n a s p u t i d a b y e x a m i n i n g b i o f i l m f o r m a t i o n i n a s i n g l e c u l t u r e o f t h e s e s t r a i n s u n d e r c o n d i t i o n s o f c o n s t a n t t e m p e r a t u r e a n d f l u c t u a t i n g t e m p e r a t u r e , a n d s e c o n d l y t o s t u d y t h e e f f e c t o f t e m p e r a t u r e f l u c t u a t i o n o n t h e b a c t e r i a l i n t e r a c t i o n b y o b s e r v i n g b i o f i l m f o r m a t i o n i n t h e m i x e d c u l t u r e s o f P.
p u t i d a w i t h e a c h o f S a l . e n t e r i c a a n d S t a . a u r e u s.
2.2. MATERIALS AND METHODS
2 . 2 . 1 . B a c t e r i a l s t r a i n s a n d g r o w t h c o n d i t i o n s
T h e b a c t e r i a l s t r a i n s w e r e S t a p h y l o c o c c u s a u r e u s. s u b s p . a u r e u s N B R C 1 0 0 9 1 0 - d e r i v e d s t r a i n f r o m N I T E B i o l o g i c a l R e s o u r c e C e n t e r ( N B R C ) , S a l m o n e l l a e n t e r i c a. s u b s p . e n t e r i c a N B R C 1 3 2 4 5 - d e r i v e d s t r a i n f r o m N B R C a n d P s e u d o m o n a s p u t i d a w h i c h w a s i s o l a t e d f r o m c u c u m b e r f r u i t s a n d i d e n t i f i e d b y a n a l y z i n g b a s e s e q u e n c e o f 1 6 S - r D N A r e g i o n u s i n g P C R m e t h o d . S t a . a u r e u s a n d S a l . e n t e r i c a a r e o n e o f t h e m a i n f o o d p o i s o n i n g b a c t e r i a , P. p u t i d a i s o n e o f t h e g e n u s P s e u d o m o n a s w i t h a g o o d a b i l i t y t o f o r m b i o f i l m . A l l s t r a i n s w e r e c u l t u r e d i n T r y p t i c S o y B r o t h ( T S B ) a t 2 5 C w i t h a g i t a t i o n o f 1 0 5 r p m f o r 7 2 h o u r s .
2 . 2 . 2 . B i o f i l m f o r m a t i o n o n a n i n n e r s u r f a c e o f p o l y s t y r e n e t u b e i n a s i n g l e c u l t u r e
B i o f i l m f o r m a t i o n o n a n i n n e r s u r f a c e o f p o l y s t y r e n e t u b e
i n a s i n g l e c u l t u r e w a s p r e p a r e d a c c o r d i n g t o t h e m e t h o d p r o p o s e d b y P l a n c h o n e t a l. ( 2 0 0 6 ) w i t h s o m e m o d i f i c a t i o n s . E a c h s t a p h y l o c o c c a l , s a l m o n e l l a a n d p s e u d o m o n a l s u b c u l t u r e s w e r e s e p a r a t e l y d i l u t e d w i t h s t e r i l e d i s t i l l e d w a t e r u n t i l o p t i c a l d e n s i t y ( O D ) a t 6 0 0 n m w a s a d j u s t e d t o a v a l u e o f 1 . 0 b y a s p e c t r o p h o t o m e t e r ( V- 5 3 0 , J A S C O C o r p o r a t i o n ) s o t h a t i n i t i a l b a c t e r i a l c o u n t i n t h e s i n g l e c u l t u r e o f S t a . a u r e u s, S a l . e n t e r i c a a n d P. p u t i d a w e r e 5 . 8 × 1 08, 2 . 2 × 1 08 a n d 5 . 0 × 1 08 C F U / m l , r e s p e c t i v e l y. A n a d j u s t e d c u l t u r e o f 0 . 1 m l w a s d i s t r i b u t e d i n a p r e p a r e d 1 5 m l p o l y s t y r e n e t u b e w i t h 5 m l o f T S B a t 1 0 0 % o r 5 % d i l u t e d w i t h s t e r i l e d i s t i l l e d w a t e r , i n o r d e r t o i d e n t i f y t h e e f f e c t o f t h e n u t r i e n t c o n c e n t r a t i o n o n b i o f i l m f o r m a t i o n . F o r b a c t e r i a l a t t a c h m e n t o n t o t h e i n n e r s u r f a c e o f t h e t u b e , t h e s a m p l e s w e r e a l l o w e d t o s t a n d f o r 3 0 m i n u t e s a t r o o m t e m p e r a t u r e . T h e t u b e s w e r e t h e n i n c u b a t e d i n a n i n c u b a t o r f o r 5 d a y s a t c o n s t a n t a n d f l u c t u a t i n g t e m p e r a t u r e c o n d i t i o n s i n o r d e r f o r t h e m i c r o o r g a n i s m s t o f o r m b i o f i l m . T h e c o n s t a n t t e m p e r a t u r e c o n d i t i o n s w e r e s e t a t 5 C a n d 3 0 C , w h i c h r e f l e c t e d a r e f r i g e r a t e d c o n d i t i o n d u r i n g t h e f o o d
d i s t r i b u t i o n a n d a h i g h t e m p e r a t u r e i n s u m m e r . I n a d d i t i o n , t w o p a t t e r n s o f f l u c t u a t i n g t e m p e r a t u r e c o n d i t i o n w e r e a p p l i e d . F o r t h e p a t t e r n ( i ) o f f l u c t u a t i n g t e m p e r a t u r e c o n d i t i o n , t h e t u b e s w e r e i n c u b a t e d a t c a . 5 C f o r 1 d a y, a n d a f t e r t h a t t h e s e w e r e i n c u b a t e d a t c a . 3 0 C f o r 4 d a y s . F o r t h e p a t t e r n ( i i ) , t h e t u b e s w e r e i n c u b a t e d a t c a . 5 C f o r 1 d a y t h e n a t c a . 3 0 C f o r 1 d a y, a f t e r t h a t t h e t u b e s w e r e i n c u b a t e d a t c a . 5 C f o r 3 d a y s . ( F i g . 2 . 1 ) .
2 . 2 . 3 . B i o f i l m f o r m a t i o n o n a n i n n e r s u r f a c e o f p o l y s t y r e n e t u b e i n a m i x e d c u l t u r e
E a c h s t a p h y l o c o c c a l , s a l m o n e l l a a n d p s e u d o m o n a l s u b c u l t u r e s w e r e d i l u t e d w i t h s t e r i l e d i s t i l l e d w a t e r i n t h e s a m e w a y a s s h o w n i n 2 . 2 . 2 . . E a c h s t a p h y l o c o c c a l c u l t u r e a n d s a l m o n e l l a c u l t u r e w e r e m i x e d w i t h p s e u d o m o n a l c u l t u r e s o t h a t i n i t i a l b a c t e r i a l c o u n t i n t h e m i x e d c u l t u r e o f S t a . a u r e u s a n d S a l . e n t e r i c a w i t h P. p u t i d a w e r e 3 . 1 × 1 08 a n d 7 . 4 × 1 08 C F U / m l , r e s p e c t i v e l y. S u b s e q u e n t o p e r a t i o n f o r b i o f i l m f o r m a t i o n o n t h e i n n e r s u r f a c e o f t h e p o l y s t y r e n e t u b e i n t h e m i x e d c u l t u r e w a s p e r f o r m e d a c c o r d i n g t o t h e m e t h o d
a a a a a a a a
Fig. 2.1 Profile of fluctuating temperature condition.
0 10 20 30 40
Incubating temperature(C)
1 2 3 4 5
Culturing time (day) (a) The pattern (i)
0 10 20 30 40
Incubating temperature(C )
1 2 3 4 5
Culturing time (day) (b) The pattern (ii)
0 0 0 10 20 30 40
0 10 20 30 40
Incubating temperature(C)
1 2 3 4 5
1 2 3 4 5
Culturing time (day) (a) The pattern (i)
0 10 20 30 40
0 10 20 30 40
Incubating temperature(C )
1 2 3 4 5
1 2 3 4 5
Culturing time (day) (b) The pattern (ii)
0 0
d e s c r i b e d i n 2 . 2 . 2 . .
2 . 2 . 4 . Q u a n t i f i c a t i o n a s s a y f o r a n a m o u n t o f a t t a c h e d b i o f i l m T h e q u a n t i f i c a t i o n a s s a y f o r a n a m o u n t o f a t t a c h e d b i o f i l m w a s p e r f o r m e d a c c o r d i n g t o t h e m e t h o d p r o p o s e d b y S t e p a n o v i c e t a l. ( 2 0 0 0 ) w i t h s o m e m o d i f i c a t i o n s ( F i g . 2 . 2 ( a ) ) . A f t e r t h e i n c u b a t i o n , s o l u t i o n i n e a c h t u b e w a s d r a i n e d . T h e n , s t e r i l e d i s t i l l e d w a t e r o f 5 m l w a s d i s t r i b u t e d i n e a c h t u b e a n d d r a i n e d t o w a s h . V i a b l e a n d d e a d c e l l s w i t h e x o p o l y s a c c h a r i d e s r e m a i n i n g o n t o t h e p o l y s t y r e n e t u b e a f t e r w a s h i n g w e r e d e f i n e d a s b i o f i l m . S u b s e q u e n t l y , t h e a t t a c h e d c e l l s a n d e x o p o l y s a c c h a r i d e s o n e a c h t u b e w e r e s t a i n e d w i t h 5 m l o f 0 . 1 % c r y s t a l v i o l e t s o l u t i o n f o r 1 0 m i n u t e s . A f t e r d r a i n i n g t h e s o l u t i o n , t h e i n n e r s u r f a c e w a s r i n s e d o f f b y d i s t r i b u t i n g a n d d r a i n i n g 5 m l o f s t e r i l e d i s t i l l e d w a t e r a n d t h e n d r i e d i n a c l e a n b e n c h . T h e b o u n d d y e w a s r e s o l u b i l i z e d w i t h 5 m l o f 9 9 . 5 % e t h a n o l b y u l t r a s o n i c a t i o n a t 1 2 5 W - 4 2 k H z f o r 1 m i n u t e a t r o o m t e m p e r a t u r e . T h e O D o f t h e s o l u t i o n o b t a i n e d w i t h t h e m a n n e r d e s c r i b e d a b o v e w a s m e a s u r e d a t 5 0 0 n m u s i n g a s p e c t r o p h o t o m e t e r a c c o r d i n g t o t h e m e t h o d
a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a
(a) Quantification assay for an amount of attached biofilm
Washing with sterile distilled water
Staining with 0.1% crystal violet solution
Resolubilizing by ultrasonication Distributing
99.5% ethanol
Measuring absorbance at 500nm (b) Bacterial count in biofilm matrix of a mixed culture
Washing with sterile distilled water
Distributing sterile distilled water
Resolubilizing by ultrasonication
Mixing bacterial suspension with agar broth solution
Counting bacterial colonies after incubation
Fig.2.2 Outline of experimental method for quantification assay for an amount of attached biofilm and bacterial count in biofilm matrix of a mixed culture (a) Quantification assay for an amount of attached biofilm
Washing with sterile distilled water
Staining with 0.1% crystal violet solution
Resolubilizing by ultrasonication Distributing
99.5% ethanol
Measuring absorbance at 500nm (a) Quantification assay for an amount of attached biofilm
Washing with sterile distilled water
Staining with 0.1% crystal violet solution
Resolubilizing by ultrasonication Distributing
99.5% ethanol
Measuring absorbance at 500nm (b) Bacterial count in biofilm matrix of a mixed culture
Washing with sterile distilled water
Distributing sterile distilled water
Resolubilizing by ultrasonication
Mixing bacterial suspension with agar broth solution
Counting bacterial colonies after incubation (b) Bacterial count in biofilm matrix of a mixed culture
Washing with sterile distilled water
Distributing sterile distilled water
Resolubilizing by ultrasonication
Mixing bacterial suspension with agar broth solution
Counting bacterial colonies after incubation
Fig.2.2 Outline of experimental method for quantification assay for an amount of attached biofilm and bacterial count in biofilm matrix of a mixed culture
d e s c r i b e d b y H a m a n a k a e t a l. ( 2 0 0 7 ) . T h e q u a n t i f i c a t i o n w a s p e r f o r m e d i n t r i p l i c a t e .
2.2.4. Bacterial count in biofilm matrix of a mixed culture
T o i n v e s t i g a t e t h e c h a n g e i n m i c r o b i a l f l o r a o f t h e b i o f i l m , t h e v i a b l e b a c t e r i a l n u m b e r i n t h e b i o f i l m m a t r i x w a s c o u n t e d a s f o l l o w s ( F i g . 2 . 2 ( b ) ) . A f t e r t h e i n c u b a t i o n , t h e s o l u t i o n i n e a c h t u b e w a s d r a i n e d a n d w a s h e d o n c e w i t h 5 m l o f s t e r i l e d i s t i l l e d w a t e r a n d d r i e d i n t h e c l e a n b e n c h . S u b s e q u e n t l y , 5 m l o f s t e r i l e d i s t i l l e d w a t e r w a s d i s p e n s e d i n t o e a c h t u b e , a n d t h e n t h e b a c t e r i a a t t a c h e d t o t h e i n n e r s u r f a c e o f t h e t u b e w e r e r e s o l u b i l i z e d b y u l t r a s o n i c a t i o n a t 1 2 5 W - 4 2 k H z f o r 1 m i n u t e a t r o o m t e m p e r a t u r e . A f t e r d i l u t i n g t h e b a c t e r i a l s u s p e n s i o n , s t a n d a r d m e t h o d a g a r b r o t h f o r c o u n t i n g b o t h o f P . p u t i d a a n d S a l . e n t e r i c a o r S t a . a u r e u s, X - S A L a g a r f o r S a l . e n t e r i c a a n d m a n n i t o l s a l t a g a r f o r S t a . a u r e u s w a s m i x e d w i t h t h e b a c t e r i a l s u s p e n s i o n i n a p e t r i d i s h . E a c h o f s t a n d a r d m e t h o d a g a r , X - S A L a g a r a n d m a n n i t o l s a l t a g a r p e t r i d i s h e s w e r e i n c u b a t e d a t 2 5 C f o r 4 8 h o u r s , 3 7 C f o r 2 4 h o u r s a n d 3 0 C f o r 4 8 h o u r s , r e s p e c t i v e l y . A f t e r t h e
i n c u b a t i o n , t h e c o l o n i e s o n t h e a g a r w e r e c o u n t e d f o r c a l c u l a t i n g t o t a l b a c t e r i a l c o u n t , s t a p h y l o c o c c a l c o u n t a n d s a l m o n e l l a c o u n t , r e s p e c t i v e l y . T h e e x p e r i m e n t w a s p e r f o r m e d a t l e a s t t h r e e t i m e s .
2 . 2 . 6 . S t a t i s t i c a l a n a l y s i s
T h e m e a n v a l u e o f O D a n d v i a b l e c o u n t f o r e a c h d a y w e r e s t a t i s t i c a l l y e v a l u a t e d u s i n g t h e m o d i f i e d t - t e s t b a s e d o n R y a n ' s m u l t i p l e t e s t . (P< 0 . 0 5 ) .
2.3. RESULTS AND DISCUSSION
2 . 3 . 1 . B i o f i l m f o r m a t i o n i n a s i n g l e c u l t u r e
I n t h e s i n g l e c u l t u r e o f S a l . e n t e r i c a a n d S t a . a u r e u s a t l o w c o n s t a n t t e m p e r a t u r e o f 5 C ( F i g . 2 . 3 ( a ) , ( b ) ) , a n a m o u n t o f a t t a c h e d b i o f i l m r e m a i n e d a l m o s t c o n s t a n t i n a l l c u l t u r i n g d a y s r e g a r d l e s s o f T S B c o n c e n t r a t i o n . W h e r e a s , a t h i g h c o n s t a n t t e m p e r a t u r e o f 3 0 C ( F i g . 2 . 4 ( a ) , ( b ) ) , t h e b i o f i l m a m o u n t i n b o t h o f t h e s i n g l e c u l t u r e s i n c r e a s e d o n t h e 1 s t d a y o f c u l t u r i n g . S i n c e a o p t i m a l t e m p e r a t u r e f o r g r o w t h o f S t a .
a a a a
Fig. 2.3 Amount of attached biofilm indicated by absorbance in the single cultures of Sal. enterica, Sta. aureus, and P. putidaat 5 C.
◆: 100% TSB □: 5% diluted TSB Biofilmamount(OD500 nm)
Culturing time (day)
1 2 3 4 5
0 1 2 3 4 5
0
(a) Sal. enterica
Biofilmamount(OD500 nm)
Culturing time (day)
1 2 3 4 5
0 1 2 3 4 5
0
(b) Sta. aureus
Biofilmamount(OD500 nm) 0.07 0.28
0.14 0.21
0 0.07 0.28
0.14 0.21
0
Culturing time (day)
1 2 3 4 5
0 1 2 3 4 5
0
(c) P. putida 0.05
0.20
0.10 0.15
0 0.05 0.20
0.10 0.15
0
0.03 0.12
0.06 0.09
0 0.03 0.12
0.06 0.09
0
Fig. 2.4 Amount of attached biofilm indicated by absorbance in the single cultures of Sal. enterica, Sta. aureus, and P. putidaat 30 C.
◆: 100% TSB □: 5% diluted TSB Culturing time (day)
1 2 3 4 5
0 1 2 3 4 5
0
(a) Sal. enterica
Culturing time (day)
1 2 3 4 5
0 1 2 3 4 5
0
(b) Sta. aureus
0.07 0.28
0.14 0.21
0 0.07 0.28
0.14 0.21
0
Culturing time (day)
1 2 3 4 5
0 1 2 3 4 5
0
(c) P. putida 0.05
0.20
0.10 0.15
0 0.05 0.20
0.10 0.15
0
0.03 0.12
0.06 0.09
0 0.03 0.12
0.06 0.09
0 Biofilmamount(OD500 nm)Biofilmamount(OD500 nm)Biofilmamount(OD500 nm)
a u r e u s a n d S a l . e n t e r i c a i s 3 5 C a n d 3 7 C , a l o w t e m p e r a t u r e c o u l d p r e v e n t S t a . a u r e u s a n d S a l . e n t e r i c a f r o m n o t o n l y b a c t e r i a l g r o w t h b u t a l s o b i o f i l m f o r m a t i o n w h i l e S t a . a u r e u s a n d S a l . e n t e r i c a a t a h i g h t e m p e r a t u r e c o u l d f o r m b i o f i l m . H o w e v e r , a d i f f e r e n t e f f e c t o n b i o f i l m f o r m a t i o n a t f l u c t u a t i n g t e m p e r a t u r e c o n d i t i o n o f t h e p a t t e r n ( i ) w a s o b s e r v e d a f t e r a r i s e i n t e m p e r a t u r e f r o m 5 C t o 3 0 C ( F i g . 2 . 5 ( a ) ( b ) ) , w h i c h a s i g n i f i c a n t a c c e l e r a t i o n o f b i o f i l m f o r m a t i o n i n c o m p a r i s o n w i t h h i g h c o n s t a n t t e m p e r a t u r e w a s o b s e r v e d w i t h 1 0 0 % T S B i n t h e s i n g l e c u l t u r e o f S a l . e n t e r i c a i n c o n t r a s t t o S t a . a u r e u s (P< 0 . 0 5 ) . T h e r e f o r e , t e m p e r a t u r e f l u c t u a t i o n o f t h e p a t t e r n ( i ) c o u l d a c c e l e r a t e b i o f i l m f o r m a t i o n o f S a l . e n t e r i c a a t h i g h t e m p e r a t u r e u n d e r r i c h n u t r i e n t c o n d i t i o n . I n t h e s i n g l e c u l t u r e o f S a l . e n t e r i c a f r o m t h e 3 r d d a y t o t h e 5 t h d a y o f c u l t u r i n g , a b i o f i l m d e t a c h m e n t a s i n d i c a t e d b y a d e c r e a s e i n t h e b i o f i l m a m o u n t w a s o b s e r v e d a t t h e p a t t e r n ( i ) i n r e g a r d l e s s o f T S B c o n c e n t r a t i o n ( F i g . 2 . 5 ( a ) ) , w h i l e t h e b i o f i l m a m o u n t a t t h e p a t t e r n ( i i ) r e m a i n e d a l m o s t c o n s t a n t i n 1 0 0 % T S B a n d r a t h e r i n c r e a s e d i n 5 % d i l u t e d T S B ( F i g . 2 . 6
a a
Fig. 2.5 Amount of attached biofilm indicated by absorbance in the single cultures of Sal. enterica, Sta. aureus, and P. putidaat fluctuating temperature condition of the pattern (i).
◆: 100% TSB □: 5% diluted TSB Culturing time (day)
1 2 3 4 5
0 1 2 3 4 5
0
(a) Sal. enterica
Culturing time (day)
1 2 3 4 5
0 1 2 3 4 5
0
(b) Sta. aureus
0.07 0.28
0.14 0.21
0 0.07 0.28
0.14 0.21
0
Culturing time (day)
1 2 3 4 5
0 1 2 3 4 5
0
(c) P. putida 0.05
0.20
0.10 0.15
0 0.05 0.20
0.10 0.15
0
0.03 0.12
0.06 0.09
0 0.03 0.12
0.06 0.09
0 Biofilmamount(OD500 nm)Biofilmamount(OD500 nm)Biofilmamount(OD500 nm)
( a ) ) . I n a d d i t i o n , a l s o i n t h e s i n g l e c u l t u r e o f S t a . a u r e u s r e g a r d l e s s o f T S B c o n c e n t r a t i o n ( F i g . 2 . 6 ( b ) ) , b i o f i l m f o r m a t i o n w a s s h o w n a f t e r a d e c l i n i n g f r o m 3 0 C t o 5 C a t t h e p a t t e r n ( i i ) d e s p i t e n o b i o f i l m f o r m a t i o n a t 5 C o f c o n s t a n t t e m p e r a t u r e c o n d i t i o n . T h u s , t e m p e r a t u r e f l u c t u a t i o n o f t h e p a t t e r n ( i i ) c o u l d a c c e l e r a t e t h e b i o f i l m f o r m a t i o n o f S a l . e n t e r i c a a n d S t a . a u r e u s e v e n a t l o w t e m p e r a t u r e . M e a n w h i l e , i n t h e s i n g l e c u l t u r e o f P. p u t i d a i n c o n t r a s t t o t h e o t h e r s i n g l e c u l t u r e s w i t h b o t h o f T S B c o n c e n t r a t i o n s , a n a c t i v e b i o f i l m f o r m a t i o n w a s o b s e r v e d a t l o w c o n s t a n t t e m p e r a t u r e w h i l e n o c o n s i d e r a b l e b i o f i l m f o r m a t i o n w a s o b s e r v e d a t h i g h c o n s t a n t t e m p e r a t u r e i n a l l c u l t u r i n g d a y s ( F i g . 2 . 3 ( c ) , F i g . 2 . 4 ( c ) ) . A n d , c o m p a r i n g b e t w e e n b i o f i l m f o r m a t i o n a t l o w c o n s t a n t t e m p e r a t u r e a n d t h a t a t f l u c t u a t i n g t e m p e r a t u r e c o n d i t i o n o f t h e p a t t e r n ( i i ) ( F i g . 2 . 3 ( c ) , F i g . 2 . 6 ( c ) ) , i t w a s s e e m e d t h a t t e m p e r a t u r e f l u c t u a t i o n o f t h e p a t t e r n ( i i ) c o u l d p r e v e n t b i o f i l m f o r m a t i o n o f P. p u t i d a.
Fig. 2.6 Amount of attached biofilm indicated by absorbance in the single cultures of Sal. enterica, Sta. aureus, and P. putidaat fluctuating temperature condition of the pattern (ii).
◆: 100% TSB □: 5% diluted TSB Culturing time (day)
1 2 3 4 5
0 1 2 3 4 5
0
(a) Sal. enterica
Culturing time (day)
1 2 3 4 5
0 1 2 3 4 5
0
(b) Sta. aureus
0.07 0.28
0.14 0.21
0 0.07 0.28
0.14 0.21
0
Culturing time (day)
1 2 3 4 5
0 1 2 3 4 5
0
(c) P. putida 0.05
0.20
0.10 0.15
0 0.05 0.20
0.10 0.15
0
0.03 0.12
0.06 0.09
0 0.03 0.12
0.06 0.09
0 Biofilmamount(OD500 nm)Biofilmamount(OD500 nm)Biofilmamount(OD500 nm)
2 . 3 . 2 . B i o f i l m f o r m a t i o n i n a m i x e d c u l t u r e
I n b o t h o f t h e m i x e d c u l t u r e s , a n i n c r e a s e i n t h e a m o u n t o f b i o f i l m w i t h c u l t i v a t i o n t i m e s w a s o b s e r v e d a t l o w c o n s t a n t t e m p e r a t u r e w i t h b o t h o f 1 0 0 % a n d 5 % d i l u t e d T S B t o b e s i m i l a r t o t h a t i n t h e s i n g l e c u l t u r e o f P. p u t i d a ( F i g . 2 . 7 , F i g . 2 . 8 ) . I n a d d i t i o n , r e g a r d l e s s o f T S B c o n c e n t r a t i o n , t o t a l b a c t e r i a l c o u n t i n c r e a s e d w i t h c u l t i v a t i o n t i m e i n b o t h o f t h e m i x e d c u l t u r e s w h i l e s t a p h y l o c o c c a l c o u n t r e m a i n e d a l m o s t c o n s t a n t a n d s a l m o n e l l a c o u n t s l i g h t l y d e c r e a s e d i n e a c h o f t h e m i x e d c u l t u r e . T h e r e f o r e , i n b o t h o f t h e m i x e d c u l t u r e s , P.
p u t i d a p r i n c i p a l l y f o r m e d b i o f i l m a t 5 C w h i l e S t a . a u r e u s a n d S a l . e n t e r i c a c o u l d n e i t h e r f o r m b i o f i l m n o r g r o w w e l l i n e a c h o f t h e m i x e d c u l t u r e s , r e s p e c t i v e l y. A b a c t e r i a l i n t e r a c t i o n c o u l d n o t b e r e c o g n i z e d i n b o t h o f t h e m i x e d c u l t u r e s a t t h e l o w c o n s t a n t t e m p e r a t u r e b e c a u s e t h e b i o f i l m a m o u n t i n b o t h o f t h e m i x e d c u l t u r e s d i d n o t m a r k e d l y i n c r e a s e i n c o m p a r i s o n w i t h t h e s i n g l e c u l t u r e o f P. p u t i d a.
A t t h e h i g h c o n s t a n t t e m p e r a t u r e i n t h e m i x e d c u l t u r e o f S a l . e n t e r i c a, t h e a m o u n t o f a t t a c h e d b i o f i l m i n 1 0 0 % T S B d e c r e a s e d f r o m t h e 2 n d d a y t o t h e 5 t h d a y a f t e r a i n c r e a s e i n
a a a a
Fig. 2.7 Biofilm amount indicated by absorbance and viable bacterial count in biofilm in the mixed culture of P. putidawith Sal. entericaat 5 C.
■: biofilm amount in mixed culture of P. putidawith Sal. enterica
◆: total bacterial count ◇: salmonella count Culturing time (day)
（a）100% TSB
Biofilmamount (OD500 nm) 0.06 0.30
0.12 0.18
0 0.24
0 2 4 6 8 10
0 2 4 6 8 10
Viable bacterial count (log CFU/ml)
1 2 3 4 5
0
Culturing time (day)
（b）5% diluted TSB
Biofilmamount (OD500 nm) 0.06 0.30
0.12 0.18
0 0.24
0 2 4 6 8 10
0 2 4 6 8 10
Viable bacterial count (log CFU/ml)
1 2 3 4 5
0
Fig. 2.8 Biofilm amount indicated by absorbance and viable bacterial count in biofilm in the mixed culture of P. putidawith Sta. aureusat 5 C.
■: biofilm amount in mixed culture of P. putidawith Sta. aureus
◆: total bacterial count ◇: staphylococcal count Culturing time (day)
（a）100% TSB
Biofilmamount (OD500 nm) 0.06 0.30
0.12 0.18
0 0.24
0 2 4 6 8 10
0 2 4 6 8 10
Viable bacterial count (log CFU/ml)
1 2 3 4 5
0
Culturing time (day)
（b）5% diluted TSB
Biofilmamount (OD500 nm) 0.06 0.30
0.12 0.18
0 0.24
0 2 4 6 8 10
0 2 4 6 8 10
Viable bacterial count (log CFU/ml)
1 2 3 4 5
0
t h e b i o f i l m a m o u n t o n t h e 1 s t d a y w h i l e a m a i n t e n a n c e o f t h e b i o f i l m a m o u n t w a s o b s e r v e d i n 5 % d i l u t e d T S B a f t e r a i n c r e a s e i n t h e b i o f i l m a m o u n t u n t i l t h e 3 r d d a y ( F i g . 2 . 9 ) . I n t h e m i x e d c u l t u r e o f S t a . a u r e u s, t h e b i o f i l m a m o u n t i n 1 0 0 % T S B k e p t a l m o s t c o n s t a n t o v e r t h e c u l t u r i n g p e r i o d a f t e r a i n c r e a s e i n t h e b i o f i l m a m o u n t o n t h e 1 s t d a y w h i l e a i n c r e a s e i n t h e b i o f i l m a m o u n t w a s o b s e r v e d f r o m t h e b e g i n n i n g t o t h e e n d o f c u l t u r i n g i n 5 % d i l u t e d T S B ( F i g . 2 . 1 0 ) . I n b o t h o f t h e m i x e d c u l t u r e s , a l t h o u g h a d e l i c a t e d i f f e r e n t b i o f i l m f o r m a t i o n f r o m a l l o f t h e s i n g l e c u l t u r e s w a s o b s e r v e d u n d e r h i g h c o n c e n t r a t i o n T S B c o n d i t i o n , a s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e b i o f i l m a m o u n t i n e a c h o f t h e m i x e d c u l t u r e s a n d t h a t i n a l l o f t h e s i n g l e c u l t u r e s w a s n o t o b s e r v e d . W h i l e i n 5 % d i l u t e d T S B , a s i g n i f i c a n t i n c r e a s e i n t h e a m o u n t o f a t t a c h e d b i o f i l m i n c o m p a r i s o n w i t h e a c h o f t h e s i n g l e c u l t u r e s w a s o b s e r v e d i n b o t h o f t h e m i x e d c u l t u r e (P< 0 . 0 5 ) . T h e r e f o r e , b a c t e r i a f o r m i n g b i o f i l m c o u l d s u r v i v e u n d e r r i c h n u t r i e n t c o n d i t i o n e v e n w i t h o u t a b a c t e r i a l i n t e r a c t i o n r e l a t e d w i t h b i o f i l m f o r m a t i o n , w h i l e a s t r e s s o f p o o r n u t r i e n t c o n d i t i o n c o u l d i n d u c e t h e b a c t e r i a l i n t e r a c t i o n a n d
a a a a a
Fig. 2.9 Biofilm amount indicated by absorbance and viable bacterial count in biofilm in the mixed culture of P. putidawith Sal. entericaat 30 C.
■: biofilm amount in mixed culture of P. putidawith Sal. enterica
◆: total bacterial count ◇: salmonella count Culturing time (day)
（a）100% TSB
Biofilmamount (OD500 nm) 0.06 0.30
0.12 0.18
0 0.24
0 2 4 6 8 10
0 2 4 6 8 10
Viable bacterial count (log CFU/ml)
1 2 3 4 5
0
Culturing time (day)
（b）5% diluted TSB
Biofilmamount (OD500 nm) 0.06 0.30
0.12 0.18
0 0.24
0 2 4 6 8 10
0 2 4 6 8 10
Viable bacterial count (log CFU/ml)
1 2 3 4 5
0
Fig. 2.10 Biofilm amount indicated by absorbance and viable bacterial count in biofilm in the mixed culture of P. putidawith Sta. aureusat 30 C.
■: biofilm amount in mixed culture of P. putidawith Sta. aureus
◆: total bacterial count ◇: staphylococcal count Culturing time (day)
（a）100% TSB
Biofilmamount (OD500 nm) 0.06 0.30
0.12 0.18
0 0.24
0 2 4 6 8 10
0 2 4 6 8 10
Viable bacterial count (log CFU/ml)
1 2 3 4 5
0
Culturing time (day)
（b）5% diluted TSB
Biofilmamount (OD500 nm) 0.06 0.30
0.12 0.18
0 0.24
0 2 4 6 8 10
0 2 4 6 8 10
Viable bacterial count (log CFU/ml)
1 2 3 4 5
0
a c c e l e r a t e b a c t e r i a l b i o f i l m f o r m a t i o n . I n t h e m i x e d c u l t u r e o f S a l . e n t e r i c a u s i n g 5 % d i l u t e d T S B , a n a c c e l e r a t i o n o f b i o f i l m f o r m a t i o n f r o m t h e b e g i n n i n g t o t h e 3 r d d a y o f t h e c u l t u r i n g m i g h t b e a b a c t e r i a l i n t e r a c t i o n r e l a t e d w i t h Q u o r u m - s e n s i n g s y s t e m , i n w h i c h b a c t e r i a p r o d u c e a s i g n a l m a t e r i a l f o r r e c o g n i z i n g a d e n s i t y o f b a c t e r i a . G r a m - n e g a t i v e b a c t e r i a , s u c h a s S a l . e n t e r i c a a n d P. p u t i d a, p r o d u c e N - a c y l - h o m o s e r i n e l a c t o n e s ( A H L s ) a s a s i g n a l m a t e r i a l t o e n h a n c e b i o f i l m f o r m a t i o n , a n d A H L s w o r k s b e t w e e n d i f f e r e n t b a c t e r i a l s t r a i n s , e v e n s a l m o n e l l a b a c t e r i a a n d p s e u d o m o n a l b a c t e r i a ( K j e l l e b e r g e t a l. , 2 0 0 2 ) . T h e r e f o r e , S a l . e n t e r i c a a n d P. p u t i d a c o u l d c o o p e r a t e t h r o u g h Q u o r u m - s e n s i n g s y s t e m t o f o r m b i o f i l m , i n t h e r e s u l t s , t h e s a m e t e n d e n c y o f a c h a n g e o f t o t a l b a c t e r i a l c o u n t a n d s a l m o n e l l a c o u n t w a s o b s e r v e d i n a l l c u l t u r i n g d a y s . W h e r e a s , i n t h e m i x e d c u l t u r e o f S t a . a u r e u s w i t h 5 % d i l u t e d T S B t h r o u g h o u t a n a c c e l e r a t i o n o f b i o f i l m f o r m a t i o n f r o m t h e 3 r d d a y t o t h e 5 t h d a y, t o t a l b a c t e r i a l c o u n t i n c r e a s e d w h i l e s t a p h y l o c o c c a l c o u n t d e c r e a s e d . O n e o f t h e s t u d i e s f o r t h e i n t e r a c t i o n w a s r e p o r t e d b y Q i n e t a l. ( 2 0 0 9 ) t h a t P.
a e r u g i n o s a e x o p o l y s a c c h a r i d e s d i s r u p t e d t h e e s t a b l i s h m e n t o f S t a . e p i d e r m i d i s b i o f i l m i n o r d e r t o c o m p e t e m o r e s u c c e s s f u l l y f o r n u t r i e n t s . T h u s , i n t h i s s t u d y, P. p u t i d a m i g h t a l s o f o r m e x o p o l y s a c c h a r i d e s t o d i s r u p t s t a p h y l o c o c c a l b i o f i l m s a n d d e c r e a s e s t a p h y l o c o c c a l c o u n t i n o r d e r t o c o m p e t e m o r e s u c c e s s f u l l y w i t h S t a . a u r e u s f o r n u t r i e n t s .
A t f l u c t u a t i n g t e m p e r a t u r e c o n d i t i o n o f t h e p a t t e r n ( i ) i n t h e m i x e d c u l t u r e o f S a l . e n t e r i c a u n d e r l o w T S B c o n c e n t r a t i o n ( F i g . 2 . 1 1 ) , a n i n c r e a s e i n t h e b i o f i l m a m o u n t s i m i l a r t o t h e h i g h c o n s t a n t t e m p e r a t u r e w a s o b s e r v e d f r o m t h e 1 s t d a y t o t h e 3 r d d a y a f t e r t h e t e m p e r a t u r e r i s e . I n a d d i t i o n , a l s o i n t h e m i x e d c u l t u r e o f S t a . a u r e u s u n d e r l o w T S B c o n c e n t r a t i o n ( F i g . 2 . 1 2 ) , a n a c c e l e r a t i o n o f b i o f i l m f o r m a t i o n i n d u c e d b y t h e c o m p e t i t i v e i n t e r a c t i o n w a s o b s e r v e d . H o w e v e r , a s i g n i f i c a n t d i f f e r e n c e b e t w e e n b i o f i l m f o r m a t i o n a t h i g h c o n s t a n t t e m p e r a t u r e a n d t h e p a t t e r n ( i ) w a s n o t o b s e r v e d . T h e r e f o r e , t h e t e m p e r a t u r e r i s e c o u l d h a v e n o e f f e c t o n b a c t e r i a l i n t e r a c t i o n r e l a t e d w i t h b i o f i l m f o r m a t i o n . H o w e v e r , i n t h e m i x e d c u l t u r e w i t h S t a . a u r e u s i n 1 0 0 % T S B , a n e x p e r i m e n t a l r e s u l t r e l a t e d w i t h e f f e c t o f t e m p e r a t u r e r i s e o n
a a a a a
Fig. 2.11 Biofilm amount indicated by absorbance and viable bacterial count in biofilm in the mixed culture of P. putidawith Sal. entericaat fluctuating temperature condition of the pattern (i).
■: biofilm amount in mixed culture of P. putidawith Sal. enterica
◆: total bacterial count ◇: salmonella count Culturing time (day)
（a）100% TSB
Biofilmamount (OD500 nm) 0.06 0.30
0.12 0.18
0 0.24
0 2 4 6 8 10
0 2 4 6 8 10
Viable bacterial count (log CFU/ml)
1 2 3 4 5
0
Culturing time (day)
（b）5% diluted TSB
Biofilmamount (OD500 nm) 0.06 0.30
0.12 0.18
0 0.24
0 2 4 6 8 10
0 2 4 6 8 10
Viable bacterial count (log CFU/ml)
1 2 3 4 5
0
