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Rendering environmental pathogens sensitive to
antibiotics prior to infection

Final Report Summary - RESTORING SENSITIVIT (Rendering environmental pathogens sensitive to <br/>antibiotics prior to infection)

Our initial goal was to provide proof of concept for the ability to render hospital-residing pathogens sensitive to antibiotics by the use of phages as genetic delivery vehicles.

Aim I. Construct lambda phages harboring genes that confer antibiotic sensitivity in a dominant fashion thus, restoring antibiotic sensitivity to their hosts.
The genetic elements we introduced into the pathogens, using lysogenic phages, are dominant sensitive genes of antibiotic resistance. One is the rpsL gene, an essential highly conserved protein. Most of the acquired resistance to streptomycin (sm) is due to mutations in this rpsL gene. Fortunately, introduction of the non-mutant (wt) copy of the gene rpsL into these resistant strains render them sensitive to sm. The other gene is the gyrA, conferring sensitivity in a dominant fashion to quinolones as nalidixic acid, was also used in the transferred DNA cassette conferring multiple drug sensitivity (MDS) to the pathogens.
Achievement #1:
We have constructed a lambda phages encoding two genes conferring antibiotic sensitivity, rpsL or gyrA.
Achievement #2
We demonstrated the ability of the engineered phages to restore sensitivity to streptomycin and nalidixic acid to antibiotic resistance bacterial strains, mutated in rpsL or gyrA respectively.

Thus, this aim has been mostly established, as the proof of principle for sensitization of resistant bacteria has been delivered for some of the proposed constructs.

The results of this study were published in:
Edgar R., Friedman N., Molshanski-Mor S., and Qimron U. Reversing Bacterial Resistance to Antibiotics by Phage-Mediated Delivery of Dominant Sensitive Genes. 2012. Appl. Environ. Microbiol. 78(3):744-51.
Research highlight on this paper was published in Nat Rev Microbiol. 2011 Dec 28;10(2):83 by Jermy A. News Feature on this paper was published in Nat. Med. 2012 Sep 19(19):1318 by Lauren Gravitz. News Feature on this paper was published also in The wall street journal on 2012 Jan by Ann Lukits ".

Aim 2. Construct lambda phages encoding sRNAs that counteract drug-resistance genes thus, restoring antibiotic sensitivity to their hosts
As a complementary method, which will further enhance the system, we planned to construct phages harboring small RNAs (sRNA) aimed against antibiotic resistance cassettes. The plan was to clone DNA encoding sRNAs against various resistance markers and test their efficiency to counteract resistance.
While working on this aim, a new way to target genes by RNA was described in the literature. As this system was more affective then most systems based on sRNA, we have proceeded in this direction. We are now close to publication. As this field is highly competitive, we rather not elaborate.
Achieving the above two aims allow us to target two major ways by which antibiotic resistance is acquired, namely, mutations in target genes and acquisition of resistance elements.
Achievement #3
An RNA based system that counteract drug-resistance genes was established

A Complete summary of these results is now in preparation.

Aim 3. Construct a safe selection marker that makes the introduction of the sensitizing genes favorable for host uptake
One of the unique features in our approach is applying an ex vivo selective pressure so that pathogens that do not harbor the phage conferring the drug sensitivities will be killed. We used the E. coli encoded tehAB operon, which increased the minimal inhibitory concentration (MIC) of tellurite against E. coli 50-100 fold upon expression from an active promoter. Transducing the tellurite resistance gene along with the MDS cassette, followed by application of tellurite in the cleansing solutions will thus result in enrichment of antibiotic-sensitive pathogens in nosocomial infections. The tellurite treatment combined with the constructed phage will enrich for antibiotic treatable pathogens, creating a new situation in which the “escaping” pathogens will be treatable with antibiotics.
Achievement #4:
We constructed a lambda phage harboring the tellurite resistance genes.
Achievement #5
We demonstrates that tellurite can be used as a selective agent, so that only bacteria harboring the phage survive.
Achievement #6
In the new RNA system we have developed we demonstrates that RNA can be used as a selective agent, so that only bacteria harboring the phage survive.

Thus, this aim has been partially completed.

A summary of part these results can be found in:
Edgar R., Friedman N., Molshanski-Mor S., and Qimron U. Reversing Bacterial Resistance to Antibiotics by Phage-Mediated Delivery of Dominant Sensitive Genes. 2012. Appl. Environ. Microbiol. 78(3):744-51.
Research highlight on this paper was published in Nat Rev Microbiol. 2011 Dec 28;10(2):83 by Jermy A. News Feature on this paper was published in Nat. Med. 2012 Sep 19(19):1318 by Lauren Gravitz. News Feature on this paper was published also in The wall street journal on 2012 Jan by Ann Lukits ".

The other results are now close to publication.

Aim 4. Construct a maintenance system for the sensitizing genes
Although the lysogen is stable by its nature, the antibiotic treatment might create selective pressure against the introduced construct. Therefore, we introduced an alternative maintenance genes to counteract the antibiotic pressure. We planned to take advantage of a naturally evolved in bacteria for maintaining extrachromosomal elements, the toxin-antitoxin system. Introduction of such a system to the DNA construct should maintain the DNA construct despite the antibiotic treatment. Pathogens that lose the DNA construct are killed by the toxin because they lose the antitoxin encoding DNA.
Achievement #6:
We used the hok/sok plasmid maintenance system demonstrating it can enhance lysogenized phage stability.
Achievement #7
Also, we constructed the toxin-antitoxin system linked to the sensitizing genes and showed that it increased maintenance 100 fold as compared to the control construct.

Aim 5. Combine the constructs into a single delivery vehicle and test its efficiency in conferring antibiotic sensitivity to resistant pathogens
Upon successful completion of the above aims, a large DNA fragment, multi-drug sensitization
(MDS) cassette were constructed. The final should contain (i) sensitizing DNA elements against sm and other resistance genes (Aims I+II) (ii) ex vivo selection markers for tellurite resistance (Aim III) (iii) the hok/sok plasmid maintenance system (Aim IV).
Achievement #8:
We have combined the tellurite resistance genes and the sensitizing gene rpsL or gyrA. We were able to show that tellurite can be used as a selective pressure so that only bacteria harboring the sensitizing construct survive.
Achievement #9:
Lambda phage harboring this primary sensitizing cassette was constructed.
We were able to show that tellurite can be used as a selective pressure so that only bacteria harboring the sensitizing phage survive.

Thus, this aim has been partially completed

Addendum article on this work was also published:
Yosef I, Kiro R, Molshanski-Mor S, Edgar R, Qimron U. Different approaches for using bacteriophages against antibiotic-resistant bacteria. 2014. Bacteriophage. 4(1):e28491.

Additionally, a patent (application number 13/477213) was submitted.