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Modified lactic acid bacteria detect and inhibit multiresistant enterococci.

Borrero J, Chen Y, Dunny GM, Kaznessis YN - ACS Synth Biol (2014)

Bottom Line: The peptides inhibit enterococcal growth and reduce viability of enterococci in the vicinity of L. lactis.Sensitive detection and specific inhibition occur both in agar and liquid media.The engineered L. lactis also inhibited growth of multidrug-resistant E. faecium strains, when induced by cCF10.

View Article: PubMed Central - PubMed

Affiliation: †Department of Chemical Engineering and Materials Science, ‡Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455, United States.

ABSTRACT
We designed Lactococcus lactis to detect Enterococcus faecalis. Upon detection, L. lactis produce and secrete antienterococcal peptides. The peptides inhibit enterococcal growth and reduce viability of enterococci in the vicinity of L. lactis. The enterococcal sex pheromone cCF10 serves as the signal for detection. Expression vectors derived from pCF10, a cCF10-responsive E. faecalis sex-pheromone conjugative plasmid, were engineered in L. lactis for the detection system. Recombinant host strains were engineered to express genes for three bacteriocins, enterocin A, hiracin JM79 and enterocin P, each with potent antimicrobial activity against E. faecalis. Sensitive detection and specific inhibition occur both in agar and liquid media. The engineered L. lactis also inhibited growth of multidrug-resistant E. faecium strains, when induced by cCF10. The presented vectors and strains can be components of a toolbox for the development of alternative antibiotic technologies targeting enterococci at the site of infection.

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β-gal activity(Miller Units) from recombinant L.lactis strains carrying engineered plasmids (pBK1, pBK2,pBK2iDT, pBK2Z, pBK2idTZ). Exponential cultures of each strain wereinduced with 100 ng/mL cCF10 90 min prior to harvesting cells forβ-gal assay. The results are shown as averaged results fromtwo independent experiments, each done in triplicate. The error barsrepresent the standard deviation.
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fig1: β-gal activity(Miller Units) from recombinant L.lactis strains carrying engineered plasmids (pBK1, pBK2,pBK2iDT, pBK2Z, pBK2idTZ). Exponential cultures of each strain wereinduced with 100 ng/mL cCF10 90 min prior to harvesting cells forβ-gal assay. The results are shown as averaged results fromtwo independent experiments, each done in triplicate. The error barsrepresent the standard deviation.

Mentions: As expected, highest levels of β-gal productionwere observed for L. lactis-pBK1, where the absenceof prgX prevents the repression of PQ byPrgX, and therefore PQ works as a constitutive promoter,with the expression of lacZ always on (Figure 1). Strains carrying either pBK2 or pBK2idT bothproduced β-gal upon induction with the pheromone cCF10, withthe latter strain showing higher levels of induced expression (Figure 1). Although we presented induction results using50 ng/mL of cCF10, we found that lower levels of pheromone (25 ng/mLand 10 ng/mL) resulted in comparable expression.


Modified lactic acid bacteria detect and inhibit multiresistant enterococci.

Borrero J, Chen Y, Dunny GM, Kaznessis YN - ACS Synth Biol (2014)

β-gal activity(Miller Units) from recombinant L.lactis strains carrying engineered plasmids (pBK1, pBK2,pBK2iDT, pBK2Z, pBK2idTZ). Exponential cultures of each strain wereinduced with 100 ng/mL cCF10 90 min prior to harvesting cells forβ-gal assay. The results are shown as averaged results fromtwo independent experiments, each done in triplicate. The error barsrepresent the standard deviation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4384838&req=5

fig1: β-gal activity(Miller Units) from recombinant L.lactis strains carrying engineered plasmids (pBK1, pBK2,pBK2iDT, pBK2Z, pBK2idTZ). Exponential cultures of each strain wereinduced with 100 ng/mL cCF10 90 min prior to harvesting cells forβ-gal assay. The results are shown as averaged results fromtwo independent experiments, each done in triplicate. The error barsrepresent the standard deviation.
Mentions: As expected, highest levels of β-gal productionwere observed for L. lactis-pBK1, where the absenceof prgX prevents the repression of PQ byPrgX, and therefore PQ works as a constitutive promoter,with the expression of lacZ always on (Figure 1). Strains carrying either pBK2 or pBK2idT bothproduced β-gal upon induction with the pheromone cCF10, withthe latter strain showing higher levels of induced expression (Figure 1). Although we presented induction results using50 ng/mL of cCF10, we found that lower levels of pheromone (25 ng/mLand 10 ng/mL) resulted in comparable expression.

Bottom Line: The peptides inhibit enterococcal growth and reduce viability of enterococci in the vicinity of L. lactis.Sensitive detection and specific inhibition occur both in agar and liquid media.The engineered L. lactis also inhibited growth of multidrug-resistant E. faecium strains, when induced by cCF10.

View Article: PubMed Central - PubMed

Affiliation: †Department of Chemical Engineering and Materials Science, ‡Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455, United States.

ABSTRACT
We designed Lactococcus lactis to detect Enterococcus faecalis. Upon detection, L. lactis produce and secrete antienterococcal peptides. The peptides inhibit enterococcal growth and reduce viability of enterococci in the vicinity of L. lactis. The enterococcal sex pheromone cCF10 serves as the signal for detection. Expression vectors derived from pCF10, a cCF10-responsive E. faecalis sex-pheromone conjugative plasmid, were engineered in L. lactis for the detection system. Recombinant host strains were engineered to express genes for three bacteriocins, enterocin A, hiracin JM79 and enterocin P, each with potent antimicrobial activity against E. faecalis. Sensitive detection and specific inhibition occur both in agar and liquid media. The engineered L. lactis also inhibited growth of multidrug-resistant E. faecium strains, when induced by cCF10. The presented vectors and strains can be components of a toolbox for the development of alternative antibiotic technologies targeting enterococci at the site of infection.

Show MeSH
Related in: MedlinePlus