Accepted Manuscript Title: Characterization of Mannheimia haemolytica biofilm formation in vitro Author: Ismail Boukahil Charles J. Czuprynski PII: DOI: Reference:
S0378-1135(14)00524-0 http://dx.doi.org/doi:10.1016/j.vetmic.2014.11.012 VETMIC 6816
To appear in:
VETMIC
Received date: Revised date: Accepted date:
17-2-2014 4-11-2014 12-11-2014
Please cite this article as: Boukahil, I., Czuprynski, C.J.,Characterization of Mannheimia haemolytica biofilm formation in vitro, Veterinary Microbiology (2014), http://dx.doi.org/10.1016/j.vetmic.2014.11.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Highlights Mannheimia haemolytica forms a biofilm in vitro.
3
Biofilm formation is greater in tissue culture medium than bacteriological broth.
4
The biofilm is largely protein with lesser amounts of carbohydrate and nucleic acid.
5
Biofilm cells are more resistant than planktonic cells to some antibiotics.
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Characterization of Mannheimia haemolytica biofilm formation in vitro
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Ismail Boukahila and Charles J. Czuprynskia*
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Department of Pathobiological Sciences, University of Wisconsin-Madison, School of Veterinary Medicine, 2015 Linden Drive, Madison, WI 53706, USA
*Corresponding Author:
Department of Pathobiological Sciences University of Wisconsin-Madison
12
4174 Veterinary Medicine Building
13
2015 Linden Drive
14
Madison, WI 53706
15
608-262-8102 (phone)
16
608-263-0438 (fax)
17
Email: [email protected]
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IB email: [email protected]
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Abstract Mannheimia haemolytica is the primary bacterial agent in the bovine respiratory disease complex. It is thought that M. haemolytica colonizes the tonsillar crypts of cattle as a commensal and
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subsequently descends into the lungs to cause disease. Many bacterial species persist in the host as
24
biofilms. There is limited information about the ability of M. haemolytica to form biofilms. The aim of
25
this study was to develop an in vitro model for M. haemolytica biofilm formation. We found that M.
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haemolytica required at least 36 hours to form robust biofilms on plastic in vitro when incubated in
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RPMI-1640 tissue culture medium at 37°C, with maximal biofilm formation being evident at 48 hours.
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Biofilm formation was inhibited by adding the monosaccharides D (+) galactose and D (+) mannose to
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the growth medium. Addition of antibodies to the M. haemolytica surface protein OmpA also reduced
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biofilm formation. Upon evaluating the macromolecules within the biofilm extracellular polymeric
31
substance we found it contained 9.7 µg/cm2 of protein, 0.81 µg/cm2 of total carbohydrate, and 0.47
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µg/cm2 of extracellular DNA. Furthermore, proteinase K treatment significantly decreased biofilms
33
(P
S0378-1135(14)00524-0 http://dx.doi.org/doi:10.1016/j.vetmic.2014.11.012 VETMIC 6816
To appear in:
VETMIC
Received date: Revised date: Accepted date:
17-2-2014 4-11-2014 12-11-2014
Please cite this article as: Boukahil, I., Czuprynski, C.J.,Characterization of Mannheimia haemolytica biofilm formation in vitro, Veterinary Microbiology (2014), http://dx.doi.org/10.1016/j.vetmic.2014.11.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Highlights Mannheimia haemolytica forms a biofilm in vitro.
3
Biofilm formation is greater in tissue culture medium than bacteriological broth.
4
The biofilm is largely protein with lesser amounts of carbohydrate and nucleic acid.
5
Biofilm cells are more resistant than planktonic cells to some antibiotics.
cr
ip t
2
Ac ce p
te
d
M
an
us
6
1 Page 1 of 33
6
Characterization of Mannheimia haemolytica biofilm formation in vitro
7
Ismail Boukahila and Charles J. Czuprynskia*
10
Department of Pathobiological Sciences, University of Wisconsin-Madison, School of Veterinary Medicine, 2015 Linden Drive, Madison, WI 53706, USA
*Corresponding Author:
Department of Pathobiological Sciences University of Wisconsin-Madison
12
4174 Veterinary Medicine Building
13
2015 Linden Drive
14
Madison, WI 53706
15
608-262-8102 (phone)
16
608-263-0438 (fax)
17
Email: [email protected]
19
an
M
d
te
Ac ce p
18
us
11
ip t
9
a
cr
8
IB email: [email protected]
2 Page 2 of 33
19 20 21
Abstract Mannheimia haemolytica is the primary bacterial agent in the bovine respiratory disease complex. It is thought that M. haemolytica colonizes the tonsillar crypts of cattle as a commensal and
23
subsequently descends into the lungs to cause disease. Many bacterial species persist in the host as
24
biofilms. There is limited information about the ability of M. haemolytica to form biofilms. The aim of
25
this study was to develop an in vitro model for M. haemolytica biofilm formation. We found that M.
26
haemolytica required at least 36 hours to form robust biofilms on plastic in vitro when incubated in
27
RPMI-1640 tissue culture medium at 37°C, with maximal biofilm formation being evident at 48 hours.
28
Biofilm formation was inhibited by adding the monosaccharides D (+) galactose and D (+) mannose to
29
the growth medium. Addition of antibodies to the M. haemolytica surface protein OmpA also reduced
30
biofilm formation. Upon evaluating the macromolecules within the biofilm extracellular polymeric
31
substance we found it contained 9.7 µg/cm2 of protein, 0.81 µg/cm2 of total carbohydrate, and 0.47
32
µg/cm2 of extracellular DNA. Furthermore, proteinase K treatment significantly decreased biofilms
33
(P
