Saturday, January 31, 2009

Qui gladio ferit, gladio perit

Bacterial conjugation-based antimicrobial agents.
Marcin Filutowicz, Richard Burgess, Richard L. Gamelli, Jack A. Heinemann, Brigitta Kurenbach, Sheryl A. Rakowski, Ravi Shankar
Plasmid 60 (2008) 38–44

Horizontal gene transfer is an essential mechanism in the adaptive evolution of bacteria. In genomic era it is more evident that the antibiotic resistance genes are spread across microbial population. It is also known that these genes are often located on mobile genetic elements, of which conjugative plasmids represent a major group and are found in nearly all Prokaryotes. Broad host range plasmids and conjugation are the main ways of spreading antibiotic resistance through bacterial population. But “He who lives by the sword shall die by the sword”, so could we take advantage of conjugation and use it to kill pathogenic bacteria.
In this short review paper authors summarize the work on so called bacterial conjugation-based technologies (BCBT). These technologies exploit plasmid biology for combating the rising tide of antibiotic-resistant bacteria. Specifically, the concept utilizes conjugationally delivered plasmids as antimicrobial agents, and it builds on the accumulated work of many scientists dating back to the discoveries of conjugation and plasmids themselves.
It is easy to imagine how it works. Genetic information carried by plasmid DNA is expressed in the recipient cells upon conjugation. So if plasmids carry instruction for destruction of the host cells it is executed. Authors present 3different ways to kill the host cell by plasmid.

The first approach is very simple. It uses so called runaway plasmid which can replicate without any control in a host cell. Replication of plasmid DNA acts like a trap to capture all of the cell’s available replication machinery to the exclusion of chromosomal replication.

The second is production of plasmid- or chromosome-encoded bacteriocins. In almost all instances, cells producing a bacteriocin also produce a bacteriocin-specific antidote, typically a peptide or RNA. For BCBT purpose, an anti-kill antidote, which can neutralize the expression of a plasmid-encoded antimicrobial agent, can be integrated into the chromosome of the donor bacteria. Susceptible recipients are killed after plasmid transfer from the protected donor cells.

In the third approach, a donor might be rendered insensitive to a killer plasmid by using a tightly regulatable promoter-operator system in which the expression of a lethal bacteriocin gene is prevented by a repressor made only in the donor cell. An engineered example of a plasmid with multiple toxins that are independently regulated has been built and employed in the proof-of-concept experiments which are described in the paper.

The BCBT has been successfully used by ConjuGon Inc. (Madison, WI), and the Loyola University Medical Center’s Burn and Shock Trauma Institute (Maywood, IL), in eradicating Acinetobacter baumannii in vitro and in an in vivo murine burn sepsis model. A. baumannii is a Gram-negative opportunistic human pathogen that is found in soil and water and is easily transmitted in health care settings. Wounds such as burns are routinely treated with topical antibiotics at high enough doses to achieve therapeutic concentration; however, such antibiotic treatment is compromised if the wound is infected with multidrug- resistant bacterial strains. Many clinically-isolated strains of A. baumannii are pan resistant (resistant to all antibiotics) and the incidence of nosocomial infections caused by such strains is increasing in critically injured and immunocompromised patients who are hospitalized for prolonged periods. So BCBT could be extremely useful in such cases.
The other target for BCBT is to kill pathogenic bacteria which are living inside the host cells. These bacteria establish themselves in the intracellular milieu of their host, thereby evading administered antibiotics as well as the host’s immune system. To target those pathogens the intra- or inter-species conjugation can be used. In that case a non pathogenic strain (such as Salmonella) acts a donor of killer-plasmid. In the case of Mycobaterium tuberculosis and M. avium infections bacteriophages were used instead of plasmid to reduce number of pathogens.

There are also some disadvantages of this technology. The efficiency of killing pathogenic bacteria depends first on plasmid transfer efficiency, and second, on plasmid killing properties itself. Thus it is important to increase the ability of plasmid to be transfer to the specific target host. It is also good to find highly efficient killing system. But even with high efficient conjugation and killing system it seems to be very unlikely to eliminate all susceptible bacteria in the environment, because they still have some “defense” systems like restriction systems to protect. On the other hand the commercial antibiotics also do not kill all susceptible bacteria. But the decreased number of pathogenic cells allows immunological system to finish the job.

Another thing is the use of bacterial strains containing modified genetic information and “releasing” them to the “environment”. Authors present an assortment of applications of live bacteria approved by U.S. government agencies for use or further study.

To summarize, in some cases the BCBT could be an alternative method of dealing with bacterial infections and as a new technology can be developed in all possible ways...

dr Jaroslaw E. Krol

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