Lab 2: Genetic Analysis to Investigate Outbreaks

what is a plasmid

extrachromosomal double stranded DNA molecules, typically circular DNA although some are linear

how do plasmids spread from one bacterium to another?

genes carried on the plasmid ensure replication of the plasmid.

transferred by either a conjugation or transformation (liberation from dead cells and uptake of naked plasmid DNA by competent bacteria

plasmid genes may integrate into the chromosome and undergo replication in the normal way

which kinds of genes are commonly found in plasmids?

genes which are helpful but not essential to the bacterium. R plasmids confer antimicrobial resistance; other plasmids encode genes for toxin production, enzymes or other virulence characteristics

Listeria monocytogenes

In healthy people it is mild and self-limiting or asymptomatic

In immunocompromised serious/life threatening

pregnant women can cause intrauterine infection via crossing the placenta resulting in still births, miscarriages or babies with systemic listeriosis (meningitis, encephalitis, or sepsis)

transmission is through contaminated foods and may survive refrigeration temperatures

heating foods (when appropriate) above 72 degrees will kill the bacteria

What is the main difference in colony morphology between the cephexin sensitive and resistant strains

cephexin resistant colonies are mucoid (mucus)

this makes the colonies wet, shiny, slimy, and sticky

this difference could be attributed to the presence of a capsule (consisting of mainly hydrated sugar polymers)

why are capsulated bacteria generally more virulent than non-capsulated bacteria?

resist phagocytosis

poorly antigenic and masks underlying antigens preventing complement activation by classical (antibody-dependent) and alternate pathways (anti-body independent)

Agglutination Test for Capsular Vi Antigen

clumping: positive for agglutination (resistant E. coli)

no clumping: negative for agglutination (sensitive E. coli)

this antigen is not found in E. coli so if it has acquired the Vi antigen than it is resistant to normal antibiotics

Could genes for cephexin resistance and Vi-containing capsule be encoded on the same plasmid?

When the plasmid is lost, the E. coli reverts to cephexin sensitivity. The change in morphology as well as mucoid appearance indicates loss of the capsule (suggesting it is also encoded on a plasmid and probably the genes for the capsule and drug resistance are on the same plasmid).

it is possible the factors are on separate plasmids and both phenotypes are lost at the same time (seems unlikely given both phenotypes are lost reliably) but it is possible

DNA fingerprinting

we used it to find out if all cephexin resistant strains have the same plasmid digesting and extracted DNA with restriction enzyme. The enzyme cuts the plasmid DNA into different sized fragments which give a specific banding pattern when subjected to gel electrophoresis. (Restriction Length Polymorphism/ RFLP)

How many bands were present in plasmid digest from cephexin-resistant E. coli

6 bands

Was there any plasmid DNA present in the cured E. coli strain

no

was your digest the same or different compared to the other digests

the same (suggesting all the disease associated E. coli have the same plasmid and that these are all the same E. coli strain

Conclusion from the results of the RFLP

the E. coli strain obtained a plasmid from Salmonella Typhi that encoded for cephexin resistant capsule production (both of these seem to be on the same plasmid)