Method for producing enhanced antigenic enteric bacteria

A method of growing an enteric bacterium having an enhanced antigenic property, wherein the enteric bacterium is selected from the group consisting of Yersinia spp., Bacteroides spp., Klebsiella spp., Enterobacter spp., Gastrospirillum spp., Salmonella spp., Aeromonas spp., Vibrio spp., Clostridium spp., Enterococcus spp. and Escherichia coli, which method comprises growing a culture of the enteric bacterium in vitro in a culture medium with a combination of conditions comprising:

• a) a divalent cation chelator selected from the group consisting of about 1.0 to about 25 .mu.M of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester, 0.5 to about 10 mM of ethylene-bis(oxyethylenenitrilo)-tetraacetic acid, about 1.0 to about 100 .mu.M of ethylene-bis(oxyethylenenitrilo)-tetraacetic acid/acetoxymethyl ester;
• b) at a temperature between about 30.degree. C. and about 42.degree. C.;
• and
• c) in air or an microaerophilic condition, wherein the microaerophilic condition comprises i) about 5% to about 20% CO.sub.2 with about 80% to about 95% air;
• ii) about 5% to about 20% CO.sub.2 with about 80% to about 95% N.sub.2 ;
• or iii) about 5% to about 10% O.sub.2 with about 10% to about 20% CO.sub.2 with about 70% to about 85% N.sub.2 ;
• for a sufficient time so that the culture is in a growth phase at about early log phase, between early log phase and stationary phase, or at about stationary phase, wherein the enhanced antigenic property is a higher level of an immunogenic antigen or a new immunogenic antigen when compared to the antigenic property of bacteria from a culture of the enteric bacterium grown in brain heart infusion broth.

An enteric bacterium having an enhanced antigenic property, wherein the enteric bacterium is selected from the group consisting of Yersinia spp., Bacteroides spp., Klebsiella spp., Enterobacter spp., Gastrospirillum spp., Salmonella spp., Aeromonas spp., Vibrio spp., Clostridium spp., Enterococcus spp. and Escherichia coli, which enteric bacterium is harvested from a culture of the enteric bacterium grown in vitro in a culture medium with a combination of conditions comprising:

• a) about 0.05% to about 3% bile or about 0.025% to about 0.6% of one or more bile acids or salts thereof;
• b) at a temperature between about 30.degree. C. and about 42.degree. C.;
• c) in air or an microaerophilic condition, wherein the microaerophilic condition comprises i) about 5% to about 20% CO.sub.2 with about 80% to about 95% air;
• ii) about 5% to about 20% CO.sub.2 with about 80% to about 95% N.sub.2 ;
• or iii) about 5% to about 10% O.sub.2 with about 10% to about 20% CO.sub.2 with about 70% to about 85% N.sub.2 ;
• and
• d) a divalent cation chelator selected from the group consisting of 0 to about 100 .mu.M of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester, 0 to about 10 mM of ethylene-bis(oxyethylenenitrilo)-tetraacetic acid, 0 to about 100 .mu.M of ethylene-bis(oxyethylenenitrilo)-tetraacetic acid/acetoxymethyl ester,
• wherein said enteric bacterium culture is at about early log phase, between early log phase and stationary phase, or at about stationary phase, wherein the enhanced antigenic property is a higher level of an immunogenic antigen or a new immunogenic antigen when compared to the antigenic property of bacteria from a culture of the enteric bacterium grown in brain heart infusion broth.

An enteric bacterium having an enhanced antigenic property, wherein the enteric bacterium is selected from the group consisting of Yersinia spp., Bacteroides spp., Klebsiella spp., Gastrospirillum spp., Enterobacter spp., Salmonella spp., Shigella spp., Aeromonas spp., Vibrio spp., Clostridium spp., Enterococcus spp. and Escherichia coli, which enteric bacterium is harvested from which a culture of the enteric bacterium grown in vitro in a culture medium with a combination of conditions comprising:

• a) a divalent cation chelator selected from the group consisting of about 1.0 to about 25 .mu.M of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester, 0.5 to about 10 mM of ethylene-bis(oxyethylenenitrilo)-tetraacetic acid, about 1.0 to about 100 .mu.M of ethylene-bis(oxyethylenenitrilo)-tetraacetic acid/acetoxymethyl ester;
• b) at a temperature between about 30.degree. C. and about 42.degree. C.;
• and
• c) in air or an microaerophilic condition, wherein the microaerophilic condition comprises i) about 5% to about 20% CO.sub.2 with about 80% to about 95% air;
• ii) about 5% to about 20% CO.sub.2 with about 80% to about 95% N.sub.2 ;
• or iii) about 5% to about 10% O.sub.2 with about 10% to about 20% CO.sub.2 with about 70% to about 85% N.sub.2,
• wherein said enteric bacterium culture is at about or between early log phase to stationary phase, wherein the enhanced antigenic property is a higher level of an immunogenic antigen or a new immunogenic antigen when compared to the antigenic property of bacteria from a culture of the enteric bacterium grown in brain heart infusion broth.