Resistance nodulation cell division (RND)-type efflux transporters play the main 
role in intrinsic resistance to various antimicrobial agents in many 
gram-negative bacteria. Here, we estimated 12 RND-type efflux transporter genes 
in Vibrio parahaemolyticus. Because VmeAB has already been characterized, we 
cloned the other 11 RND-type efflux transporter genes and characterized them in 
Escherichia coli KAM33 cells, a drug hypersusceptible strain. KAM33 expressing 
either VmeCD, VmeEF, or VmeYZ showed increased minimum inhibitory concentrations 
(MICs) for several antimicrobial agents. Additional four RND-type transporters 
were functional as efflux pumps only when co-expressed with VpoC, an outer 
membrane component in V. parahaemolyticus. Furthermore, VmeCD, VmeEF, and VmeYZ 
co-expressed with VpoC exhibited a broader substrate specificity and conferred 
higher resistance than that with TolC of E. coli. Deletion mutants of these 
transporter genes were constructed in V. parahaemolyticus. TM32 (ΔvmeAB and 
ΔvmeCD) had significantly decreased MICs for many antimicrobial agents and the 
number of viable cells after exposure to deoxycholate were markedly reduced. 
Strains in which 12 operons were all disrupted had very low MICs and much lower 
fluid accumulation in rabbit ileal loops. These results indicate that resistance 
nodulation cell division-type efflux transporters contribute not only to 
intrinsic resistance but also to exerting the virulence of V. parahaemolyticus.