Cloning and Characterization of E. meningoseptica Beta Lactamase

Elizabethkingia meningoseptica is a rod-shaped bacterium that has been known to cause outbreaks of meningitis, and occasionally sepsis in infants and adults with weakened immune systems. This bacteria has been shown to be resistance to most antibiotics, or antimicrobials, that are used to treat Gram-negative bacterial infections. The purpose of our experiment is to target the predicted genes associated with antibiotic resistance in E. meningoseptica , clone the specific gene in this pathogenic bacterium, and characterize the gene to further understand the function. With the published genomic sequence of E. meningoseptica ATCC 13253 type strain, we will design primers for PCR amplification of multi-drug RND-efflux pumps specifically for beta-lactamase enzymes, and clone the PCR products into E. coli strain DH5a using the plasmid vector pBBR1MCS-2.n In this experiment, we inserted our gene into DH5a cells, which are a modified E. coli used for cloning to look at a specific gene. The gene is inserted into the strain in order to further look at the function of the targeted gene. (E. Coli) We will isolate the recombinant plasmids using a DNA mini-prep method and then confirm the DNA sequence cloned into these plasmids is as expected.  We will then screen these clone strains for acquired antimicrobial resistance and degree of acquired resistance. We made antibiotic plates to test the resistance to several different clinically relevant antibiotics.