FEMS Microbiology Letters Advance Access published July 17, 2015
1
Escherichia coli O8-antigen enhances biofilm formation under
2
agitated conditions
3 4
Akash Kumar1, Dhriti Mallik1, Shilpa Pal1, Sathi Mallick1, Sujoy Sarkar1, Ajoy
5
Chanda2, Anindya S. Ghosh1 *
6 7
Author details
8
1
9
Bengal, India, PIN-721302. 2Department of Chemical Engineering, Indian Institute of
10
Department of Biotechnology, Indian Institute of Technology, Kharagpur, West
Technology, Kharagpur, West Bengal, India, PIN-721302
11 12 13
E-mail addresses:[email protected] (Akash Kumar), [email protected]
14
(Dhriti Mallik), [email protected] (Shilpa Pal), [email protected]
15
(Sathi Mallick), [email protected] (Sujoy K. Sarkar), [email protected]
16
(Ajoy Chanda) [email protected] (Anindya S. Ghosh)*Correspondence and
17
reprints.
18 19
*Correspondence:Anindya S. Ghosh
20
1
21
Bengal, India, PIN-721302.Tel.: +91-3222-283798, Fax: +91-3222-278707
Department of Biotechnology, Indian Institute of Technology, Kharagpur, West
22
1
23
Abstract
24
Bacterial surface components have a major role in the development of biofilms. In the
25
present study, the effect of Escherichia coli O8-antigen on biofilms was investigated
26
using two E. coli K-12 derived strains that differed only in the O8-antigen
27
biosynthesis. In presence of O8-antigen both bacterial adhesion and biofilm formation
28
slightly decreased under static conditions whereas a substantial increase in adhesion
29
and biofilm formation was observed under agitated conditions. It was noted that
30
irrespective of the O8-antigen status, the hydrophobic interactions played an
31
important role in bacterial adhesion under both static and agitated conditions.
32
However, under agitated conditions, the extent of bacterial adhesion in the O8-antigen
33
bearing strain was predominately determined by the electrostatic interactions. Results
34
showed that the presence of O8-antigen decreases the surface hydrophobicity and
35
surface charge. Moreover, O8-antigen facilitates adhesion on hydrophilic and
36
hydrophobic surfaces as revealed through the tests with modified substrata. Our
37
results indicate that O8-antigen, which appears dispensable for biofilm formation
38
under static conditions, actually enhances E. coli biofilm formation under agitated
39
conditions.
40 41
Keywords
42
Escherichia coli; Adhesion; Biofilm; O-antigen; Hydrophobic; Lipopolysaccharide.
43 44 45 46 47
2
48
Introduction
49
Biofilms are the communities of microorganisms encased within extracellular
50
polymeric substances, adhered to biotic or abiotic surfaces and often the cause of
51
persistent infections (Stewart & William Costerton, 2001). For example, the biofilms
52
of Gram-negative bacteria Escherichia coli formed on catheters and in the bladder
53
epithelium are commonly associated with the urinary tract infections (Nakao et al.,
54
2012). The Lipopolysaccharide (LPS), a macromolecule present on the outer
55
membrane (OM) of Gram-negative bacteria, has been discerned as one of the surface
56
structures that influences the bacterial adhesion (Walker et al., 2005). LPS consists of
57
three components: lipid A, core oligosaccharide and O-antigen, and provides
58
resistance against bile salts, hydrophobic antibiotics, complement system and
59
macrophages (Kong et al., 2012).
60
The O-antigens, attached to the core oligosaccharide, are responsible for
61
antigenic specificity and O-serogroup determination (DebRoy et al., 2011). The
62
mutants lacking O-antigens are defective either in the O-antigen (rfb) or in the LPS
63
core (rfa) gene cluster (Liu & Reeves, 1994). The OM of several E. coli K-12
64
laboratory strains have defective LPS core. This affects the O-antigen polysaccharide
65
side chain attachment to the core, resulting in the inability of the K-12 strains to
66
produce O-antigen (Liu & Reeves, 1994). However, O-antigen is usually present in
67
the pathogenic E. coli strains and plays a major role in pathogenesis (DebRoy et al.,
68
2011).
69
Previous studies have addressed the role of LPS on biofilm formation or
70
bacterial adhesion, the first step of biofilm formation. However these studies present
71
conflicting evidence with respect to O-antigen where its presence is associated with
3
72
either increased or decreased adhesion and/or biofilm formation (Cockerill et al.,
73
1996, Kierek & Watnick, 2003).
74
The E. coli O8-antigen is a linear homopolymer of α-mannose terminating in
75
3-O-methylmannose (Ghosh et al., 2006). The O8-antigen lowers the deformities that
76
appear in the penicillin-binding protein (PBP) deletion mutants and increases the
77
susceptibility to beta-lactam antibiotics (Ghosh et al., 2006, Sarkar & Ghosh, 2008).
78
In this work, attempts are made to determine the role of O-antigen in the process of
79
biofilm formation using two E. coli K-12 derived strains, differing only with respect
80
to the O8-antigen biosynthesis (Rick et al., 1994). To the best of our knowledge so far
81
no study has simultaneously compared the effect of O-antigen on biofilm formation
82
under both static and agitated conditions. Most of the previous studies are performed
83
under static conditions, though the conditions in the human host or in the environment
84
are rarely static. Therefore, the goal of this study was to demarcate the differences in
85
the biofilm-forming abilities of these strains under both static and agitated conditions.
86 87
Materials and Methods
88
Bacterial strains and growth conditions
89
Strains derived from E. coli K-12 were used in this study. E. coli AB1133 is
90
an O-antigen-deficient strain; and E. coli 2443 strain possess intact rfbO8 gene cluster
91
and synthesizes O8-antigen (Rick et al., 1994). The strains were grown in Luria
92
Bertani (LB) broth and/or agar (Hi-Media). Unless otherwise specified, chemicals and
93
reagents were purchased from Sigma Chemical Co., (St. Louis, MO, USA).
94
4
95 96
Statistical analysis The mean value of the experimental data was represented in bar graphs with
97
error bars showing standard deviation in the results section. The statistical
98
significance (P value) of the data was determined by performing an unpaired, two-
99
tailed t test using GraphPad Prism (GraphPad Software, San Diego, CA). For
100
additional confirmation of the level of significance, a one-way analysis of variance
101
(ANOVA) was performed. To compare the change in different parameters, fold
102
difference was calculated which is defined as the ratio of values between the strains,
103
provided the ratio ≥ 1 or, its reciprocal if the ratio is < 1.
104
Biofilm formation assay
105
Biofilm formation was assessed in 24-well polystyrene tissue culture plates as
106
described previously (Kumar et al., 2012), with little modifications. In brief, each
107
well having 900 µL of LB was inoculated with 100 µL of culture (OD600 nm ~ 0.2) and
108
the plates were incubated for 24 h at 37 °C under static or agitated conditions
109
(agitated continuously at 90 rpm in an orbital shaker). For microscopy of biofilms,
110
polystyrene coupons were placed along with the culture in the wells of the microtiter
111
plate. The biofilms formed were stained with 0.1% crystal violet (CV). Excess stain
112
was washed and the absorbance of the retained CV extracted with 33% acetic acid
113
was measured at 600 nm. Biofilm Formation Index (BFI) for each well was calculated
114
using the formula, BFI = (B-C)/G, where, B is the stain released from the test
115
wells, C is the stained control wells and G is the absorbance of cell density in broth.
116
The experiments were repeated for at least six times.
117
5
118 119
Microscopic analysis of biofilms Biofilms grown on polystyrene coupons were rinsed with PBS (pH 7.0), fixed
120
with 2% glutaraldehyde, dehydrated in ethanol and observed through scanning
121
electron microscopy (SEM) at 1000× magnification (JSM5800 JEOL, Japan). For
122
fluorescence microscopy, the biofilms stained with BacLight Live/Dead dye
123
(Invitrogen Inc., CA, USA) were visualized at 200× magnification using OLYMPUS
124
IX 81 fluorescence microscope (Olympus Inc., Japan).
125 126 127
COMSTAT analysis For quantitative evaluation, biofilms were stained with 0.01% (wt/vol)
128
acridine orange for 15 min and washed to remove the excess stain. Images were
129
acquired using a confocal laser scanning microscope (CLSM) FV1000 (Olympus Inc.,
130
Japan) through 40× objective lens. Five image stacks, each containing five optically
131
sectioned images (512- by 512-pixel tagged image file format) per strain were
132
collected at random. COMSTAT software was used to analyze the acquired Z-stacks
133
images to determine the biovolume and mean thickness of the biofilms (Heydorn et
134
al., 2000).
135 136
Adhesion assay
137
To check bacterial adhesion, 1 mL of bacterial suspensions in LB (OD600 nm ~
138
0.5) with or without Tween 80 (0.1%) or 100 mM NaCl, were incubated at 37 °C for
139
1 h in either static or agitated conditions. Unattached cells were removed and the
140
adherent bacteria were stained with CV for 15 min. After removing the excess dye,
141
retained CV was solubilized in 33% acetic acid and Adhesion Index (AI) was 6
142
calculated in a manner similar to BFI. Experiments were repeated for at least six
143
times.
144 145
Determination of the cell surface hydrophobicity
146
Bacterial cell surface hydrophobicity was assessed using Microbial Adhesion
147
To Hydrocarbons (MATH) and by measuring the contact angles (θw) (Van der Mei et
148
al., 2003, Saxena et al., 2009). MATH was measured using overnight grown cells
149
washed and resuspended (OD600 nm ~ 0.5) in PUM buffer (100 mM K2HPO4, 50 mM
150
KH2PO4, 30 mM Urea, 1 mM MgSO4). Cell suspensions were vigorously mixed with
151
n-hexadecane and the mixture was incubated at room temperature to allow
152
partitioning of the phases. The absorbance of the lower aqueous phase was measured
153
at OD600 nm and Hydrophobicity index (HPBI) was calculated as the change in
154
percentage from the original absorbance (Ao) to the final absorbance (Af) upon
155
hexadecane exposure [HPBI = (Ao−Af)/Ao×100]. Each experiment was repeated at
156
least for three times.
157
Water contact angles (θw) was measured on the lawns of bacterial strains using
158
the sessile drop technique (Van der Mei et al., 2003). In brief, centrifuged cell
159
suspension (OD600 ~ 1.0) was deposited and air dried on polystyrene surface. The
160
stable ‘plateau contact angles’ of the water droplets were measured with a Goniometer
161
(Rame-Hart 190-F2, Rame-Hart Instrument Co., Netcong, NJ, USA) in triplicate for
162
each strain.
163 164 165 166
Surface charge measurements Bacterial surface charge was estimated by measuring the zeta potential (Van der Mei et al., 2003). Washed cells suspended in 10 mM potassium phosphate buffer 7
167
(pH 7.2) were dispensed into zeta cells and the zeta potential of each sample was
168
determined using a zeta-sizer Nano ZS (Malvern Instruments, UK). The experiments
169
were performed at least three times for each culture.
170 171
Silane coating and Plasma treatment
172
To increase the hydrophobicity of the polystyrene, an organosilane
173
(Dimethyldichlorosilane) was coated on the polystyrene plate and air-dried. Each
174
plate was later rinsed with 10 mM potassium phosphate buffer (pH 7.2) just before
175
use (Cunliffe et al., 1999). To increase the hydrophilicity, polystyrene plates were
176
treated with atmospheric pressure plasma by employing a plasma generator, as
177
described elsewhere (Atmospheric Pressure Plasma ULS series, Acxys Technologies,
178
France) (Saxena et al., 2009).
179 180
Results and discussion
181
O8-antigen enhances biofilm formation under agitated conditions
182
Biofilm forming ability of E. coli 2443 (O8-antigen present) and AB1133 (O8-
183
antigen deficient) (Supplementary Figure 1 and Supplementary Figure 2) was
184
analyzed semi-quantitatively, under both static and agitated conditions (Figure 1a).
185
Under static conditions 2443 (2443-S) strain produced 22% (P< 0.05) less biofilm
186
than AB1133 (AB1133-S). However, under agitated conditions 2443(2443-A)
187
produced 96% (P< 0.001) more biofilm as compared to AB1133 (AB1133-A). The
188
formed biofilms, visualized through SEM (Figure 2a) and fluorescence microscopy
189
(Figure 2b) showed enhanced level of biofilm formation in both the cultures grown
190
under agitated conditions as compared to the static conditions. LIVE/DEAD staining
191
did not show any significant difference in the cellular viability of biofilms, regardless
8
192
of the strain or condition. COMSTAT analysis of the image stacks of biofilms showed
193
2443 under agitated conditions had higher biovolume (~1.3 fold; P< 0.05) and mean
194
thickness (~1.3 fold; P< 0.05) than AB1133 biofilms (Table 1).
195 196
O8-antigen enhances bacterial adhesion under agitated conditions
197
Bacterial adhesion was assessed to check whether variation in biofilm
198
formation was linked to it. E. coli 2443 under static condition was 21% less adherent
199
(P< 0.05) than AB1133 (Figure 1b), but under agitated conditions was 82% more
200
adherent (P < 0.001) than AB1133. The adhesion results corresponded closely with
201
the biofilm formation values (correlation coefficient r2 > 0.95), which affirmed that
202
the bacterial adhesion is the main factor that accounts for the observed variation in
203
biofilm formation under both conditions. Similar results were reported in an another
204
study with adherent-invasive Escherichia coli (AIEC) strains where the higher
205
adhesion indices of both AIEC and non-AIEC strains correlated with higher specific
206
biofilm formation indices (Martinez-Medina et al., 2009). In another report, the
207
attachment of E. coli K-12 strain to surfaces, mimicking cellular interfaces, presented
208
significant differences between stagnant and flowing conditions (Barth et al., 2008).
209
Such observations illustrate that cell surface structure and composition affects
210
adhesion in different ways under different conditions.
211 212 213
Hydrophobic and electrostatic interactions influence bacterial adhesion To ascertain the role of hydrophobic and/or electrostatic interactions in
214
bacterial adhesion, adhesion assays were performed in the presence of NaCl and
215
Tween 80. Tween 80 (a surfactant) reduces hydrophobic interactions while NaCl
216
supplies counter-ions for blocking electrostatic interactions (Crittenden et al., 2001).
9
217
The addition of Tween 80 decreased adhesion under both the conditions in both the
218
strains (Figure 3). In static conditions, the level of adhesion decreased by 63 and 56%
219
in AB1133 and 2443, respectively (P < 0.001 in both cases), while under agitated
220
conditions, AB1133 and 2443 depicted a decrease in adhesion by 50 and 51%,
221
respectively (P < 0.001 in both cases). This suggests that hydrophobic interactions are
222
important for adhesion, irrespective of the O8-antigen status of the strains.
223
The presence of NaCl, which interferes with electrostatic interactions, did not
224
markedly affect the adhesion under static conditions in both the strains. This shows
225
under static conditions, presence or absence of O8-antigen had little effect on
226
electrostatic interactions and consequently on bacterial adhesion. Similarly, presence
227
of NaCl did not affect AB1133 adhesion under agitated conditions; however, it
228
decreased the adhesion of 2443 by ~30% (P < 0.05). As the electrostatic interactions
229
among the molecules reduces in the presence of high concentration of salts like NaCl
230
(Volle et al., 2008), the significant decrease in the adhesion of 2443 strain under
231
agitated conditions implies the involvement of an electrostatic component in the
232
adhesion events that diminishes when the participating charged groups of O8-antigen
233
are shielded by additional salt molecules.
234
Taken as a whole, adhesion assay indicated that hydrophobic interactions play
235
an important role under both conditions irrespective of the O8-antigen status.
236
However, in the presence of O8-antigen and under agitated conditions, electrostatic
237
interactions have a crucial role in adhesion.
238 239
10
239 240
O8-antigen decreases both surface hydrophobicity and surface charge
241
Cell surface hydrophobicity is an important factor associated with the
242
attachment, and increased cell hydrophobicity is associated with increased adhesion
243
(McEldowney & Fletcher, 1986). Bacterial cell surface hydrophobicity, as determined
244
by the bacterial adhesion to hydrocarbons and the contact angle measurement,
245
revealed that growth conditions, i.e., static or agitated, do not affect the surface
246
hydrophobicity of the cell (Figure 4). More importantly, AB1133 was significantly
247
more hydrophobic than 2443 (~1.6 fold; P< 0.05 for both tests, under both conditions)
248
suggesting that O8-antigen decreases surface hydrophobicity in bacterial cells. This
249
might lead to diminution in the hydrophobic interactions and ultimately resulting in
250
decreased adhesion, as in the case of 2443 under static conditions. It is proposed that
251
the hydrophobic lipid A embedded in the outer membrane imparts cell surface
252
hydrophobicity (Boyer et al., 2011) and O-antigen, a predominantly hydrophilic
253
polymer, masks the hydrophobic properties of lipid A. Therefore, its absence
254
enhances hydrophobicity of the cells (Boyer et al., 2011). It is also demonstrated that
255
bacteria with long O-antigen show decreased adhesion as compared to those lacking
256
an O-antigen layer (Salerno et al., 2004).
257
Earlier studies have revealed that surface charge plays a significant role in
258
bacterial aggregation and adhesion (Kłodzińska et al., 2010). When measured, zeta
259
potential of AB1133 was significantly more negative than that of 2443 (~3 fold; P
1
Escherichia coli O8-antigen enhances biofilm formation under
2
agitated conditions
3 4
Akash Kumar1, Dhriti Mallik1, Shilpa Pal1, Sathi Mallick1, Sujoy Sarkar1, Ajoy
5
Chanda2, Anindya S. Ghosh1 *
6 7
Author details
8
1
9
Bengal, India, PIN-721302. 2Department of Chemical Engineering, Indian Institute of
10
Department of Biotechnology, Indian Institute of Technology, Kharagpur, West
Technology, Kharagpur, West Bengal, India, PIN-721302
11 12 13
E-mail addresses:[email protected] (Akash Kumar), [email protected]
14
(Dhriti Mallik), [email protected] (Shilpa Pal), [email protected]
15
(Sathi Mallick), [email protected] (Sujoy K. Sarkar), [email protected]
16
(Ajoy Chanda) [email protected] (Anindya S. Ghosh)*Correspondence and
17
reprints.
18 19
*Correspondence:Anindya S. Ghosh
20
1
21
Bengal, India, PIN-721302.Tel.: +91-3222-283798, Fax: +91-3222-278707
Department of Biotechnology, Indian Institute of Technology, Kharagpur, West
22
1
23
Abstract
24
Bacterial surface components have a major role in the development of biofilms. In the
25
present study, the effect of Escherichia coli O8-antigen on biofilms was investigated
26
using two E. coli K-12 derived strains that differed only in the O8-antigen
27
biosynthesis. In presence of O8-antigen both bacterial adhesion and biofilm formation
28
slightly decreased under static conditions whereas a substantial increase in adhesion
29
and biofilm formation was observed under agitated conditions. It was noted that
30
irrespective of the O8-antigen status, the hydrophobic interactions played an
31
important role in bacterial adhesion under both static and agitated conditions.
32
However, under agitated conditions, the extent of bacterial adhesion in the O8-antigen
33
bearing strain was predominately determined by the electrostatic interactions. Results
34
showed that the presence of O8-antigen decreases the surface hydrophobicity and
35
surface charge. Moreover, O8-antigen facilitates adhesion on hydrophilic and
36
hydrophobic surfaces as revealed through the tests with modified substrata. Our
37
results indicate that O8-antigen, which appears dispensable for biofilm formation
38
under static conditions, actually enhances E. coli biofilm formation under agitated
39
conditions.
40 41
Keywords
42
Escherichia coli; Adhesion; Biofilm; O-antigen; Hydrophobic; Lipopolysaccharide.
43 44 45 46 47
2
48
Introduction
49
Biofilms are the communities of microorganisms encased within extracellular
50
polymeric substances, adhered to biotic or abiotic surfaces and often the cause of
51
persistent infections (Stewart & William Costerton, 2001). For example, the biofilms
52
of Gram-negative bacteria Escherichia coli formed on catheters and in the bladder
53
epithelium are commonly associated with the urinary tract infections (Nakao et al.,
54
2012). The Lipopolysaccharide (LPS), a macromolecule present on the outer
55
membrane (OM) of Gram-negative bacteria, has been discerned as one of the surface
56
structures that influences the bacterial adhesion (Walker et al., 2005). LPS consists of
57
three components: lipid A, core oligosaccharide and O-antigen, and provides
58
resistance against bile salts, hydrophobic antibiotics, complement system and
59
macrophages (Kong et al., 2012).
60
The O-antigens, attached to the core oligosaccharide, are responsible for
61
antigenic specificity and O-serogroup determination (DebRoy et al., 2011). The
62
mutants lacking O-antigens are defective either in the O-antigen (rfb) or in the LPS
63
core (rfa) gene cluster (Liu & Reeves, 1994). The OM of several E. coli K-12
64
laboratory strains have defective LPS core. This affects the O-antigen polysaccharide
65
side chain attachment to the core, resulting in the inability of the K-12 strains to
66
produce O-antigen (Liu & Reeves, 1994). However, O-antigen is usually present in
67
the pathogenic E. coli strains and plays a major role in pathogenesis (DebRoy et al.,
68
2011).
69
Previous studies have addressed the role of LPS on biofilm formation or
70
bacterial adhesion, the first step of biofilm formation. However these studies present
71
conflicting evidence with respect to O-antigen where its presence is associated with
3
72
either increased or decreased adhesion and/or biofilm formation (Cockerill et al.,
73
1996, Kierek & Watnick, 2003).
74
The E. coli O8-antigen is a linear homopolymer of α-mannose terminating in
75
3-O-methylmannose (Ghosh et al., 2006). The O8-antigen lowers the deformities that
76
appear in the penicillin-binding protein (PBP) deletion mutants and increases the
77
susceptibility to beta-lactam antibiotics (Ghosh et al., 2006, Sarkar & Ghosh, 2008).
78
In this work, attempts are made to determine the role of O-antigen in the process of
79
biofilm formation using two E. coli K-12 derived strains, differing only with respect
80
to the O8-antigen biosynthesis (Rick et al., 1994). To the best of our knowledge so far
81
no study has simultaneously compared the effect of O-antigen on biofilm formation
82
under both static and agitated conditions. Most of the previous studies are performed
83
under static conditions, though the conditions in the human host or in the environment
84
are rarely static. Therefore, the goal of this study was to demarcate the differences in
85
the biofilm-forming abilities of these strains under both static and agitated conditions.
86 87
Materials and Methods
88
Bacterial strains and growth conditions
89
Strains derived from E. coli K-12 were used in this study. E. coli AB1133 is
90
an O-antigen-deficient strain; and E. coli 2443 strain possess intact rfbO8 gene cluster
91
and synthesizes O8-antigen (Rick et al., 1994). The strains were grown in Luria
92
Bertani (LB) broth and/or agar (Hi-Media). Unless otherwise specified, chemicals and
93
reagents were purchased from Sigma Chemical Co., (St. Louis, MO, USA).
94
4
95 96
Statistical analysis The mean value of the experimental data was represented in bar graphs with
97
error bars showing standard deviation in the results section. The statistical
98
significance (P value) of the data was determined by performing an unpaired, two-
99
tailed t test using GraphPad Prism (GraphPad Software, San Diego, CA). For
100
additional confirmation of the level of significance, a one-way analysis of variance
101
(ANOVA) was performed. To compare the change in different parameters, fold
102
difference was calculated which is defined as the ratio of values between the strains,
103
provided the ratio ≥ 1 or, its reciprocal if the ratio is < 1.
104
Biofilm formation assay
105
Biofilm formation was assessed in 24-well polystyrene tissue culture plates as
106
described previously (Kumar et al., 2012), with little modifications. In brief, each
107
well having 900 µL of LB was inoculated with 100 µL of culture (OD600 nm ~ 0.2) and
108
the plates were incubated for 24 h at 37 °C under static or agitated conditions
109
(agitated continuously at 90 rpm in an orbital shaker). For microscopy of biofilms,
110
polystyrene coupons were placed along with the culture in the wells of the microtiter
111
plate. The biofilms formed were stained with 0.1% crystal violet (CV). Excess stain
112
was washed and the absorbance of the retained CV extracted with 33% acetic acid
113
was measured at 600 nm. Biofilm Formation Index (BFI) for each well was calculated
114
using the formula, BFI = (B-C)/G, where, B is the stain released from the test
115
wells, C is the stained control wells and G is the absorbance of cell density in broth.
116
The experiments were repeated for at least six times.
117
5
118 119
Microscopic analysis of biofilms Biofilms grown on polystyrene coupons were rinsed with PBS (pH 7.0), fixed
120
with 2% glutaraldehyde, dehydrated in ethanol and observed through scanning
121
electron microscopy (SEM) at 1000× magnification (JSM5800 JEOL, Japan). For
122
fluorescence microscopy, the biofilms stained with BacLight Live/Dead dye
123
(Invitrogen Inc., CA, USA) were visualized at 200× magnification using OLYMPUS
124
IX 81 fluorescence microscope (Olympus Inc., Japan).
125 126 127
COMSTAT analysis For quantitative evaluation, biofilms were stained with 0.01% (wt/vol)
128
acridine orange for 15 min and washed to remove the excess stain. Images were
129
acquired using a confocal laser scanning microscope (CLSM) FV1000 (Olympus Inc.,
130
Japan) through 40× objective lens. Five image stacks, each containing five optically
131
sectioned images (512- by 512-pixel tagged image file format) per strain were
132
collected at random. COMSTAT software was used to analyze the acquired Z-stacks
133
images to determine the biovolume and mean thickness of the biofilms (Heydorn et
134
al., 2000).
135 136
Adhesion assay
137
To check bacterial adhesion, 1 mL of bacterial suspensions in LB (OD600 nm ~
138
0.5) with or without Tween 80 (0.1%) or 100 mM NaCl, were incubated at 37 °C for
139
1 h in either static or agitated conditions. Unattached cells were removed and the
140
adherent bacteria were stained with CV for 15 min. After removing the excess dye,
141
retained CV was solubilized in 33% acetic acid and Adhesion Index (AI) was 6
142
calculated in a manner similar to BFI. Experiments were repeated for at least six
143
times.
144 145
Determination of the cell surface hydrophobicity
146
Bacterial cell surface hydrophobicity was assessed using Microbial Adhesion
147
To Hydrocarbons (MATH) and by measuring the contact angles (θw) (Van der Mei et
148
al., 2003, Saxena et al., 2009). MATH was measured using overnight grown cells
149
washed and resuspended (OD600 nm ~ 0.5) in PUM buffer (100 mM K2HPO4, 50 mM
150
KH2PO4, 30 mM Urea, 1 mM MgSO4). Cell suspensions were vigorously mixed with
151
n-hexadecane and the mixture was incubated at room temperature to allow
152
partitioning of the phases. The absorbance of the lower aqueous phase was measured
153
at OD600 nm and Hydrophobicity index (HPBI) was calculated as the change in
154
percentage from the original absorbance (Ao) to the final absorbance (Af) upon
155
hexadecane exposure [HPBI = (Ao−Af)/Ao×100]. Each experiment was repeated at
156
least for three times.
157
Water contact angles (θw) was measured on the lawns of bacterial strains using
158
the sessile drop technique (Van der Mei et al., 2003). In brief, centrifuged cell
159
suspension (OD600 ~ 1.0) was deposited and air dried on polystyrene surface. The
160
stable ‘plateau contact angles’ of the water droplets were measured with a Goniometer
161
(Rame-Hart 190-F2, Rame-Hart Instrument Co., Netcong, NJ, USA) in triplicate for
162
each strain.
163 164 165 166
Surface charge measurements Bacterial surface charge was estimated by measuring the zeta potential (Van der Mei et al., 2003). Washed cells suspended in 10 mM potassium phosphate buffer 7
167
(pH 7.2) were dispensed into zeta cells and the zeta potential of each sample was
168
determined using a zeta-sizer Nano ZS (Malvern Instruments, UK). The experiments
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were performed at least three times for each culture.
170 171
Silane coating and Plasma treatment
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To increase the hydrophobicity of the polystyrene, an organosilane
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(Dimethyldichlorosilane) was coated on the polystyrene plate and air-dried. Each
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plate was later rinsed with 10 mM potassium phosphate buffer (pH 7.2) just before
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use (Cunliffe et al., 1999). To increase the hydrophilicity, polystyrene plates were
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treated with atmospheric pressure plasma by employing a plasma generator, as
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described elsewhere (Atmospheric Pressure Plasma ULS series, Acxys Technologies,
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France) (Saxena et al., 2009).
179 180
Results and discussion
181
O8-antigen enhances biofilm formation under agitated conditions
182
Biofilm forming ability of E. coli 2443 (O8-antigen present) and AB1133 (O8-
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antigen deficient) (Supplementary Figure 1 and Supplementary Figure 2) was
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analyzed semi-quantitatively, under both static and agitated conditions (Figure 1a).
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Under static conditions 2443 (2443-S) strain produced 22% (P< 0.05) less biofilm
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than AB1133 (AB1133-S). However, under agitated conditions 2443(2443-A)
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produced 96% (P< 0.001) more biofilm as compared to AB1133 (AB1133-A). The
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formed biofilms, visualized through SEM (Figure 2a) and fluorescence microscopy
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(Figure 2b) showed enhanced level of biofilm formation in both the cultures grown
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under agitated conditions as compared to the static conditions. LIVE/DEAD staining
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did not show any significant difference in the cellular viability of biofilms, regardless
8
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of the strain or condition. COMSTAT analysis of the image stacks of biofilms showed
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2443 under agitated conditions had higher biovolume (~1.3 fold; P< 0.05) and mean
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thickness (~1.3 fold; P< 0.05) than AB1133 biofilms (Table 1).
195 196
O8-antigen enhances bacterial adhesion under agitated conditions
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Bacterial adhesion was assessed to check whether variation in biofilm
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formation was linked to it. E. coli 2443 under static condition was 21% less adherent
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(P< 0.05) than AB1133 (Figure 1b), but under agitated conditions was 82% more
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adherent (P < 0.001) than AB1133. The adhesion results corresponded closely with
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the biofilm formation values (correlation coefficient r2 > 0.95), which affirmed that
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the bacterial adhesion is the main factor that accounts for the observed variation in
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biofilm formation under both conditions. Similar results were reported in an another
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study with adherent-invasive Escherichia coli (AIEC) strains where the higher
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adhesion indices of both AIEC and non-AIEC strains correlated with higher specific
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biofilm formation indices (Martinez-Medina et al., 2009). In another report, the
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attachment of E. coli K-12 strain to surfaces, mimicking cellular interfaces, presented
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significant differences between stagnant and flowing conditions (Barth et al., 2008).
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Such observations illustrate that cell surface structure and composition affects
210
adhesion in different ways under different conditions.
211 212 213
Hydrophobic and electrostatic interactions influence bacterial adhesion To ascertain the role of hydrophobic and/or electrostatic interactions in
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bacterial adhesion, adhesion assays were performed in the presence of NaCl and
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Tween 80. Tween 80 (a surfactant) reduces hydrophobic interactions while NaCl
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supplies counter-ions for blocking electrostatic interactions (Crittenden et al., 2001).
9
217
The addition of Tween 80 decreased adhesion under both the conditions in both the
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strains (Figure 3). In static conditions, the level of adhesion decreased by 63 and 56%
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in AB1133 and 2443, respectively (P < 0.001 in both cases), while under agitated
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conditions, AB1133 and 2443 depicted a decrease in adhesion by 50 and 51%,
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respectively (P < 0.001 in both cases). This suggests that hydrophobic interactions are
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important for adhesion, irrespective of the O8-antigen status of the strains.
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The presence of NaCl, which interferes with electrostatic interactions, did not
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markedly affect the adhesion under static conditions in both the strains. This shows
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under static conditions, presence or absence of O8-antigen had little effect on
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electrostatic interactions and consequently on bacterial adhesion. Similarly, presence
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of NaCl did not affect AB1133 adhesion under agitated conditions; however, it
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decreased the adhesion of 2443 by ~30% (P < 0.05). As the electrostatic interactions
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among the molecules reduces in the presence of high concentration of salts like NaCl
230
(Volle et al., 2008), the significant decrease in the adhesion of 2443 strain under
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agitated conditions implies the involvement of an electrostatic component in the
232
adhesion events that diminishes when the participating charged groups of O8-antigen
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are shielded by additional salt molecules.
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Taken as a whole, adhesion assay indicated that hydrophobic interactions play
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an important role under both conditions irrespective of the O8-antigen status.
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However, in the presence of O8-antigen and under agitated conditions, electrostatic
237
interactions have a crucial role in adhesion.
238 239
10
239 240
O8-antigen decreases both surface hydrophobicity and surface charge
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Cell surface hydrophobicity is an important factor associated with the
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attachment, and increased cell hydrophobicity is associated with increased adhesion
243
(McEldowney & Fletcher, 1986). Bacterial cell surface hydrophobicity, as determined
244
by the bacterial adhesion to hydrocarbons and the contact angle measurement,
245
revealed that growth conditions, i.e., static or agitated, do not affect the surface
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hydrophobicity of the cell (Figure 4). More importantly, AB1133 was significantly
247
more hydrophobic than 2443 (~1.6 fold; P< 0.05 for both tests, under both conditions)
248
suggesting that O8-antigen decreases surface hydrophobicity in bacterial cells. This
249
might lead to diminution in the hydrophobic interactions and ultimately resulting in
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decreased adhesion, as in the case of 2443 under static conditions. It is proposed that
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the hydrophobic lipid A embedded in the outer membrane imparts cell surface
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hydrophobicity (Boyer et al., 2011) and O-antigen, a predominantly hydrophilic
253
polymer, masks the hydrophobic properties of lipid A. Therefore, its absence
254
enhances hydrophobicity of the cells (Boyer et al., 2011). It is also demonstrated that
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bacteria with long O-antigen show decreased adhesion as compared to those lacking
256
an O-antigen layer (Salerno et al., 2004).
257
Earlier studies have revealed that surface charge plays a significant role in
258
bacterial aggregation and adhesion (Kłodzińska et al., 2010). When measured, zeta
259
potential of AB1133 was significantly more negative than that of 2443 (~3 fold; P
