microbiology and molecular biology

[pradeep]

hello everyone.... this is pradeep here i have a planned project which deals with isolating a specific microorganism isolating plasmid and transforming another susceptible organism with the previous one and check wether the antibiotic resistance and the typical charecteristic is plasmid controlled. my problem is to isolate the typical bacteria i m unable to decide which one. so my question is which bacteria should i isolate and how,keeping in mind that it should have a charecteristic property and should be antibiotic resistant

[shijun]

Hi Pradeep,

I've contacted one of our Microbiology experts about your questions and this is what she has to say:

I would be happy to offer some suggestions, but there are some open
questions/considerations that I have.

>From what I understand, the investigator would like to start with a
resistant strain of bacteria, do a plasmid preparation/isolation, and
attempt a transfer experiment to a known strain of bacteria that does not
have antibiotic resistance (by bacterial transformation), in order to
determine if the antibiotic resistance is due to a resistance locus on a
plasmid. Please let me know if this is not the case.

1. There are several mechanisms of antibiotic resistance that bacteria
employ and multiple ways that bacteria can maintain resistance. Please keep
in mind that plasmid-borne resistance contrasts with strains that have
resistance encoded on the bacterial chromosome. Strains that have
plasmid-borne resistance will easily lose the plasmid should the antibiotic
no longer be in use. For example, it is a common lab technique to put an
ampicillin-resistance encoding plasmid in E. coli strains to carry a gene.
Should ampicillin be omitted from the growth media, the bacteria will lose
the plasmid because there is no selective pressure to retain the plasmid.

Some good sites to look at that cover the mechanisms of resistance include: http://www.foodsafetynetwork.ca/animal/ ... pr-wjp.htm
http://www.antibioresistance.be/

2. Where does the investigator intend on isolating the resistant strain
from? Quite often, it is difficult to isolate a known resistant strain that
has not been engineered or is not a clinical sample. Clinical samples can be
obtained through a clinic or hospital, but I imagine that there would be
some biological hazards and issues with that. In converse, if the
investigator wanted to isolate a resistant strain from the environment,
there would be some antibiotic resistance/susceptibility testing required to
determine if strains are resistant to any antibiotics.

For instance, it would be feasible grow bacteria from an environmental swab
on an agar plate supplemented with a common antibiotic, for example,
ampicillin/carbenicillin. Any bacteria that grows on this plate would be
resistant to that antibiotic. Then the investigator could transform common
strains of E. coli (most commonly used in research) with the plasmid that
was isolated from the resistant bacteria and check for bacterial survival on
antibiotic-agar plates in comparison to untransformed bacteria. Such common
experimental tools can be found commercially, for example through Invitrogen
(http://www.invitrogen.com).

I will give this some more thought and forward any other comments.