Detection and Inhibition of Bacterial Cell–Cell Communication - Bacterial Pathogenesis: Methods and Protocols - page 54

non-cholera Vibrio species are Vibrio parahaemolyticus , which is responsible
for gastroenteritis and wound infections, and Vibrio alginolyticus , which causes
wound infections. The most commonly isolated species is V. vulnificus , which
is of great medical importance due to the high mortality rate associated with
infections (12) . Because of the introduction of antibiotics for the treatment of
cholera, the occurrence of drug-resistant V. cholerae strains is being reported
with increasing frequency. Ninety-eight percent of clinical O1 isolates showed
resistance to one or more antibiotics, whereas 50% of the environmental isolates
showed resistance to one to three drugs, and one clinical and one environmental
isolate showed resistant to seven antibiotics (13) .
The trend of acquisition of resistance to antibiotics has increased for
many pathogenic bacterial species. Yet, the number of therapeutically useful
compounds that emerge is continuously decreasing, and only one new class
of antibiotic has been introduced in the last 40 years. Only the development
of new classes of antimicrobials with novel mechanisms of action can address
the drug resistance problem we face today. Schmidt (14) states “a prioritized
goal in the search for new targets is to identify and inactivate disease-causing
functions instead of focusing on killing. Since these factors have no vital
functions for microbes, there is less pressure to develop resistance, which would
ultimately reduce global selective pressure and prevent the acceleration and
dissemination of resistance. ” Thus, quorum-sensing systems such as the AHL
and AI-2 regulatory systems represent novel targets for the control of biofilm
formation and virulence factor production by bacteria.
2. Materials
2.1. Detection and Inhibition of N-Acylated Homoserine Lactone
Quorum Sensing
2.1.1. ABt Medium (15)
1. Solution A—in 100 mL of distilled water (dH 2 O),4gof(NH 4 ) 2 SO 4 ,6gof
Na 2 HPO 4 ·2H 2 O,3gofKH 2 PO 4 , 3 g of NaCl. Autoclave and store at room
temperature.
2. 1 M MgCl 2 .
3. 0.1 M CaCl 2 .
4. 0.01 M FeCl 2 (make fresh as Fe oxidizes rapidly into insoluble hydroxides).
5. Thiamine, 1 mg/mL.
6. Solution B—to 1 L of sterile dH 2 O,add1mLof1 M MgCl 2 , 1 mL of 0.1 M
CaCl 2 , 1 mL of 0.01 M FeCl 2 , and 2.5 mL of thiamine at 1 mg/mL. Store at room
temperature.
7. 20% glucose, filter sterilized. Store at room temperature.
8. 20% casamino acids, filter sterilized. Store at room temperature.