TWO DIFFERENT MECHANISMS OF AMPICILLIN RESISTANCE OBSERVED IN STRAINS OF VIBRIO CHOLERAE O1

 

Masahiko Ehara1, Nguyen Binh Minh2, Tran Huy Hoang2, Nguyen Dong Tu2,

Nguyen Thi Phuong Lan3, Tran Thi My Trinh3, and Masaaki Iwanaga4

1 Department of Bacteriology, Institute of Tropical Medicine, Nagasaki University, Japan

2 Department of Bacteriology, National Institute of Hygiene and Epidemiology, Hanoi, Vietnam

3 Department of Microbiology and Immunology, Pasteur Institute of Ho Chi Minh City

4 Department of Bacteriology, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan

 

Background: It is well known clinically that ampicillin is not a first choice for treatment of cholera. But why ampicillin is not effective for treatment of cholera is not clearly understood except for the presence of beta-lactamases. Even in the absence of beta-lactamases, ampicillin is not effective for cholera.

Objective: Ampicillin resistant strains of Vibrio cholerae O1 both of fimbriate and non-fimbriate phase were analyzed to elucidate which proteins play important role in the resistancemechanism.

Methodology: Six rough strains isolated in Vietnam in 1995, one rough strain isolated in Laos in 1998 and one fimbriate strain (Bgd17) were used in this study. Isolation of resistant strains; Rough strains and fimbriate strains form biofilm when cultured in liquid media. These biofilms were inoculated in LB broth containing 50 microgram/ml of ampicillin and cultured overnight at 37° with vigorous shaking, then spread over BTB agar plates containing 100 microgram/ml of ampicillin. Thus obtained resistant strains were analyzed by SDS-PAGE together with their parent strains sensitive to ampicillin. Several overproduced proteins from the resistant Bgd17 strain were purified by combination of ion-exchange column chromatography and gel filtration chromatography. Antibodies against these purified proteins were developed in rabbits. The N-terminal aminoacid sequence was also determined for several purified proteins.

Results: Non-fimbriate strains, when changed into resistant to ampicillin, markedly reduced the production of OmpU protein (an outer membrane protein, porin). On the other hand, the fimbriate Bgd17 strain overproduced CpxP (a stress-combative member of the CpX regulon) depending on the concentration of ampicillin. It is a noteworthy finding that all wild strains tested do not express CpxP protein when analyzed by Western blot.

Conclusion: Two different machineries were observed operating in non-fimbriate- and fimbriate phases of Vibrio cholerae O1 strains. One is passively operating in non-fimbriate strains by reducing the expression of OmpU protein, the other is operating more actively in the fimbriate strain by overproducing CpxP protein in parallel with the concentration of ampicillin.

Acknowledgment: The financial support of the Japan Society for the Promotion of Science (JSPS) is acknowledged.

Keywords: fimbriate, non-fimbriate, ampicillin resistance, OmpU, CpxP,