Biofilm formation by Escherichia coli O157:H7 under reducing conditions.

ANGEL VILLEGAS N, BECERRA MC, BARONETTI JL, ALBESA I, PARAJE MG.

Córdoba

Congreso; 1° Reunión Internacional de Ciencias Farmacéuticas RICIFA.; 2010

Dto. de Farmacia, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba y el Dto. de Farmacia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario.

Introduction Escherichia coli O157:H7 was first identified as being responsible for causing outbreaks of enteric infection in 1982. Symptoms include diarrhoea, haemorrhagic colitis, and haemolytic uraemic syndrome (1). The ability of E.coli O157:H7 to form biofilm has been described. A biofilm can be defined as a sessile bacterial community of cells that live attached to each other and to surfaces (2).   Objective This study evaluated the capability to form biofilms of  E. coli O157 strains under reduction conditions and their relationship to bacterial stress.   Materials and methods In this study, the biofilm formation of the reference strain of  E.coli EDL 933 and eight isolates were tested using thioglycollate as a culture medium with and without 0.5% glucose. The biofilm-forming ability was measured by adhesion to biofilms on polystyrene microtiter plates and stained with crystal violet (3). The Biofilm Biomass Unit (BBU) was arbitrarily defined with OD595 0.1 being equal to 1 BBU. The extracellular production of Reactive Oxygen Species (ROS) was detected in 48 h cultures by the reduction of nitro blue tetrazolium to nitroblue diformazan. The reaction was proportional to the ROS generated in biofilm and was measured by OD at 540 nm. The Nitric oxide (NO·) was evaluated as nitrite by a microplate assay method using the Griess reagent. 50 ml aliquots were mixed with 100 ml of Griess reagent. Absorbance was measured at 540 nm in a microplate reader and results were expressed as mM.   Results There was a significant increase in biofilm formation and a significant decrease in ROS and nitrite in all strains tested using thioglycollate as a culture medium supplemented with 0.5% glucose.   Conclusions In conclusion, we suggest that biofilm formations in reduction conditions are influenced by cellular stress. However, improved knowledge of ROS and RNI regulation may help in clarifying the relevance of biofilm formation in the pathogenesis of infections, and furthermore could also be of great value in the development of better preventive and therapeutic measures.   Acknowledgments NAV is a research fellow of CONICET. This work was supported by the following Grants: FONCyT, CONICET and SECyT.     References 1)     Ryu JH, Kim H, Frank JF, Beuchat LR. Attachment and biofilm formation on stainless steel by Escherichia coli O157:H7 as affected by curli production. Applied Microbiology. 2004. 39:359–362 2)     Ryu Jh, Beuchat LR. Biofilm Formation by Escherichia coli O157:H7 on Stainless Steel: Effect of Exopolysaccharide and Curli Production on Its Resistance to Chlorine. Appl Environ Microbiol. 2005. 71(1):247–254. 3)     O'Toole GA, Kolter R. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signaling pathways: a genetic analysis. Mol.Microbiol. 1998.28:449–461.             Introduction Escherichia coli O157:H7 was first identified as being responsible for causing outbreaks of enteric infection in 1982. Symptoms include diarrhoea, haemorrhagic colitis, and haemolytic uraemic syndrome (1). The ability of E.coli O157:H7 to form biofilm has been described. A biofilm can be defined as a sessile bacterial community of cells that live attached to each other and to surfaces (2).   Objective This study evaluated the capability to form biofilms of  E. coli O157 strains under reduction conditions and their relationship to bacterial stress.   Materials and methods In this study, the biofilm formation of the reference strain of  E.coli EDL 933 and eight isolates were tested using thioglycollate as a culture medium with and without 0.5% glucose. The biofilm-forming ability was measured by adhesion to biofilms on polystyrene microtiter plates and stained with crystal violet (3). The Biofilm Biomass Unit (BBU) was arbitrarily defined with OD595 0.1 being equal to 1 BBU. The extracellular production of Reactive Oxygen Species (ROS) was detected in 48 h cultures by the reduction of nitro blue tetrazolium to nitroblue diformazan. The reaction was proportional to the ROS generated in biofilm and was measured by OD at 540 nm. The Nitric oxide (NO·) was evaluated as nitrite by a microplate assay method using the Griess reagent. 50 ml aliquots were mixed with 100 ml of Griess reagent. Absorbance was measured at 540 nm in a microplate reader and results were expressed as mM.   Results There was a significant increase in biofilm formation and a significant decrease in ROS and nitrite in all strains tested using thioglycollate as a culture medium supplemented with 0.5% glucose.   Conclusions In conclusion, we suggest that biofilm formations in reduction conditions are influenced by cellular stress. However, improved knowledge of ROS and RNI regulation may help in clarifying the relevance of biofilm formation in the pathogenesis of infections, and furthermore could also be of great value in the development of better preventive and therapeutic measures.   Acknowledgments NAV is a research fellow of CONICET. This work was supported by the following Grants: FONCyT, CONICET and SECyT.     References 1)     Ryu JH, Kim H, Frank JF, Beuchat LR. Attachment and biofilm formation on stainless steel by Escherichia coli O157:H7 as affected by curli production. Applied Microbiology. 2004. 39:359–362 2)     Ryu Jh, Beuchat LR. Biofilm Formation by Escherichia coli O157:H7 on Stainless Steel: Effect of Exopolysaccharide and Curli Production on Its Resistance to Chlorine. Appl Environ Microbiol. 2005. 71(1):247–254. 3)     O'Toole GA, Kolter R. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signaling pathways: a genetic analysis. Mol.Microbiol. 1998.28:449–461.                           Introduction Escherichia coli O157:H7 was first identified as being responsible for causing outbreaks of enteric infection in 1982. Symptoms include diarrhoea, haemorrhagic colitis, and haemolytic uraemic syndrome (1). The ability of E.coli O157:H7 to form biofilm has been described. A biofilm can be defined as a sessile bacterial community of cells that live attached to each other and to surfaces (2).   Objective This study evaluated the capability to form biofilms of  E. coli O157 strains under reduction conditions and their relationship to bacterial stress.   Materials and methods In this study, the biofilm formation of the reference strain of  E.coli EDL 933 and eight isolates were tested using thioglycollate as a culture medium with and without 0.5% glucose. The biofilm-forming ability was measured by adhesion to biofilms on polystyrene microtiter plates and stained with crystal violet (3). The Biofilm Biomass Unit (BBU) was arbitrarily defined with OD595 0.1 being equal to 1 BBU. The extracellular production of Reactive Oxygen Species (ROS) was detected in 48 h cultures by the reduction of nitro blue tetrazolium to nitroblue diformazan. The reaction was proportional to the ROS generated in biofilm and was measured by OD at 540 nm. The Nitric oxide (NO·) was evaluated as nitrite by a microplate assay method using the Griess reagent. 50 ml aliquots were mixed with 100 ml of Griess reagent. Absorbance was measured at 540 nm in a microplate reader and results were expressed as mM.   Results There was a significant increase in biofilm formation and a significant decrease in ROS and nitrite in all strains tested using thioglycollate as a culture medium supplemented with 0.5% glucose.   Conclusions In conclusion, we suggest that biofilm formations in reduction conditions are influenced by cellular stress. However, improved knowledge of ROS and RNI regulation may help in clarifying the relevance of biofilm formation in the pathogenesis of infections, and furthermore could also be of great value in the development of better preventive and therapeutic measures.   Acknowledgments NAV is a research fellow of CONICET. This work was supported by the following Grants: FONCyT, CONICET and SECyT.     References 1)     Ryu JH, Kim H, Frank JF, Beuchat LR. Attachment and biofilm formation on stainless steel by Escherichia coli O157:H7 as affected by curli production. Applied Microbiology. 2004. 39:359–362 2)     Ryu Jh, Beuchat LR. Biofilm Formation by Escherichia coli O157:H7 on Stainless Steel: Effect of Exopolysaccharide and Curli Production on Its Resistance to Chlorine. Appl Environ Microbiol. 2005. 71(1):247–254. 3)     O'Toole GA, Kolter R. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signaling pathways: a genetic analysis. Mol.Microbiol. 1998.28:449–461.