Group 6 Copy 173
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C-30 promotes swarming of nalD mutant


Finally after some long delay we started the project, incorporating a short stay undergraduate Peruvian student from Buffalo University miss Carolina Millan Leon.

So far we have the following strains:

Strain

Characteristic

PA14 wild-type

 

PA14 mexR

Transposon mutant with a disrupted repressor of the efflux pump MexAB-OprM

PA14 nalD

Transposon mutant with a disrupted repressor of the efflux pump MexAB-OprM

PA14 mexA

Transposon mutant with a disrupted structural gene of the efflux pump MexAB-OprM

INP 49R

Clinical isolate from Cystic Fibrosis patient with dysfunctional mexA and mexE genes

 

First swarming motility of each strain was evaluated; however pre-cultures of mexR had notorious lysis so this strain may have something wrong hence we will try to obtain another copy.

Next we evaluated the effect of the QS inhibitor C-30 which is efflux by MexAB-OprM which also efflux the QS signal 3-Oxo-C12-HSL and several antibiotics.

As expected C-30 at 50 uM inhibited swarming of the PA14 wt strain and of the mexA strain (that lacks a functional MexAB-OprM pump) at 12.5-25 uM, in contrast at 25 uM it promoted the swarming of nalD!

Our explanation is that the nalD strain which over express the pump efflux the signal so the intracellular concentration is low and then QS controlled phenotypes like swarming are low, however adding C-30 will keep the pump busy, hence the QS signal will accumulate and QS dependent phenotypes will increase.

Now we are testing swarming of the other strains in the presence and absence of C-30, and antibiotics like ceftazidime and carbenicillin which are efflux by MexAB-OprM, and also growth curves and we will begin to measure other QS controlled phenotypes such as pyocyanin and exoproteases.

 

NOTE that the second plate is mexA with C30 at 12.5 uM and not 25 uM

Tolerance against the antibiotics Ceftazidime and Carbenicillin is higher in the nalD mutant than in the wild type and lower in mexA, as expected, since nalD have higher expression of the efflux pump MexAB-OprM and mexA have no active pump.


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About This Project

Each year in the U.S. 23,000 people die as a direct result of antibiotic resistant infections. I hypothesize that by combining classical antibiotics, quorum sensing, and virulence inhibitors we can increase the virulence of multidrug resistant Pseudomonas aeriginosa. The results of this study simulate the evolution of multidrug resistant bacteria predicting the worst case scenarios, so we can be better prepared to fight these infections in the future.

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