Cockroach survival assay with Stenotrophomonas maltophilia and vancomycin

Today I am repeating the cockroach survival assay with S.malt. Instead of strains G and K, I will be using 13270 and the E.coli strain DH5alpha along with BAA84 and BAA2423 from liquid cultures I prepared yesterday.  I updated the protocol accordingly. Also, I placed the roaches in the 37C incubator 24 hours before injections so the roached will be better accumulated to incubation post injection. The purpose of this repeat is to see if we obtain similar results to what was observed in the first trial. In addition to the previous method, we added a titer plate method for calculating concentration of bacteria given in each injection to better understand results.

Protocol for Cockroach Survival Assay 

Materials 

• Bacterial cultures  
• Stenotrophomonas maltophilia isolates BAA 2423, BAA 84, and 13270 
• Escherichia coli DH5alpha  
• LB broth media  
• LB + Vancomycin100 broth media 
• 90 Cockroaches ~1 inch  
• Insulin Syringe with 31G needle 
• Repeat micropipetter  
• 2 mL Eppendorf tubes 
• p1000 Pipettes and tips
• 37°C incubator (shaking and stationary)

Procedure 

1. From fresh plates of bacteria, make an overnight culture using 3 ml of LB broth in a sterile 15mL centrifuge tube.  Suspend a colony of the bacteria in the media and place in a 37°C shaking incubator for ~16 hours. 
2. Transfer 1mL of the overnight liquid culture into a 2mL eppendorf tube.  
• Make 2 replicates of each isolate for +/- vancomycin 
3. Spin down cultures (2 min at 10,000g) into a pellet 
4. Discard supernatant and resuspend one pellet with 1mL LB broth and the other with 1mL LB + Vancomycin100.  Repeat for each isolate.
5. Load an insulin syringe with resuspended bacterial culture and inject 25μL into each roach in a set of 10 using a repeat micropipetter (set at 2.5 where 1 Div= .01 mL). Repeat with each bacterial culture +/-vancomycin. 
• Make sure roaches have been incubated at 37°C for at least 24 hours before injection. 
6. Create control group by injecting 10 roaches with 25μL LB+Vancomycin100. 
7. Place 3-4 roaches of the same group into petri plates and store in 37°C incubator. Observe for survival for one week.
8. Create a titer for each strain by serial diluting  cultures 1:10 to the -8 dilution.
• This can be done by filling 8 1.5mL eppendorf tubes with 900μL sterile PBS and transferring 100μL to the subsequent tube (change pipette tips in between transfers and mix each suspension thoroughly). Repeat for each isolate used.
• From the -5 to -8 dilutions, spread 100μL onto an LB plate and place in 37°C incubator overnight. (Final dilutions with therefore be -6 to -9)
• Count colonies and determine CFU/mL for injection cultures. 

RESULTS (number of roaches alive out of 10)

Group                         Day1   Day2    Day3    Day4    Day5    Day6    Day7  %survival

Control (LB+Vanco)    10         9           9          9           9          9           9           90%

BAA2423                      9          8           8          7          7           7           7           70%

BAA2423+Vanco          3          3          2           2          2           1          1           10%

BAA84                          8          8           8          7          7           7           7           70%

BAA 84+Vanco            2           2           3          2          2           2          1           10%

13270                            2          1           1          1           1          1           1           10%

13270+Vanco                2          2           2          2          2           2           2           20%

DH5 alpha**                8          5           5           4          2          2           2            20%
DH5 alpha**+Vanco   10         5           2          1           1          1           1           10%

CFU/25μL (dosage per roach):
BAA 2423    3.7x10^7
BAA 84        1.6x10^7
13270          2.4x10^7
DH5 alpha   7.4x10^6

*During time of injections, the roaches seemed much healthier than the previous trial--may result in different immune response. 
**We are not confident in the identity of this strain of E.coli. Through various results we believe this strain is not DH5alpha, and therefore the study will be repeated using a different strain of either E.coli B/r or Klebsiella aerogenes. Furthermore, the study will be repeated with a lower concentraion of the same S.malt strians in order to observe results at a 1:10 dilution. 

//BEE