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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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lacZ expression of recombinant L. lactis in coculture with E. faecalis. (A) Plate experiments:10 μL dot of E. faecalis OG1RF and 5 μLdot of recombinant L. lactis (pBK1, pBK2Z and pBK2idTZ)grown in BHI plates supplemented with X-gal. The blue staining correspondsto the expression of lacZ by recombinant L. lactis strains. These are representative images of anexperiment that was repeated several times. (B) Absorbance at OD650 and image of E. faecalis OG1RF and L. lactis-pBK2idTZ grown in cocultures at different ratios(v/v) in M9X media. The results shown are averaged from two independentexperiments, each done in triplicate. The error bars represent thestandard deviation.
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fig2: lacZ expression of recombinant L. lactis in coculture with E. faecalis. (A) Plate experiments:10 μL dot of E. faecalis OG1RF and 5 μLdot of recombinant L. lactis (pBK1, pBK2Z and pBK2idTZ)grown in BHI plates supplemented with X-gal. The blue staining correspondsto the expression of lacZ by recombinant L. lactis strains. These are representative images of anexperiment that was repeated several times. (B) Absorbance at OD650 and image of E. faecalis OG1RF and L. lactis-pBK2idTZ grown in cocultures at different ratios(v/v) in M9X media. The results shown are averaged from two independentexperiments, each done in triplicate. The error bars represent thestandard deviation.

Mentions: When E. faecalis and recombinant L. lactis were grown together on agar plates, we observed three differentlevels of β-gal production: high, medium, and low. As observedin Figure 2A, L. lactis-pBK1exhibited high levels of β-gal production on the plate. Theinteraction between recombinant L. lactis-pBK2Z or L. lactis-pBK2idTZ with E. faecalis OG1RFtriggered the expression of lacZ by these two strains.β-gal production was higher when using L. lactis-pBK2idTZ. No β-gal production was detected in those colonieslocated outside the area of interaction with E. faecalis OG1RF.


Modified lactic acid bacteria detect and inhibit multiresistant enterococci.

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

lacZ expression of recombinant L. lactis in coculture with E. faecalis. (A) Plate experiments:10 μL dot of E. faecalis OG1RF and 5 μLdot of recombinant L. lactis (pBK1, pBK2Z and pBK2idTZ)grown in BHI plates supplemented with X-gal. The blue staining correspondsto the expression of lacZ by recombinant L. lactis strains. These are representative images of anexperiment that was repeated several times. (B) Absorbance at OD650 and image of E. faecalis OG1RF and L. lactis-pBK2idTZ grown in cocultures at different ratios(v/v) in M9X media. The results shown are averaged from two independentexperiments, each done in triplicate. The error bars represent thestandard deviation.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: lacZ expression of recombinant L. lactis in coculture with E. faecalis. (A) Plate experiments:10 μL dot of E. faecalis OG1RF and 5 μLdot of recombinant L. lactis (pBK1, pBK2Z and pBK2idTZ)grown in BHI plates supplemented with X-gal. The blue staining correspondsto the expression of lacZ by recombinant L. lactis strains. These are representative images of anexperiment that was repeated several times. (B) Absorbance at OD650 and image of E. faecalis OG1RF and L. lactis-pBK2idTZ grown in cocultures at different ratios(v/v) in M9X media. The results shown are averaged from two independentexperiments, each done in triplicate. The error bars represent thestandard deviation.
Mentions: When E. faecalis and recombinant L. lactis were grown together on agar plates, we observed three differentlevels of β-gal production: high, medium, and low. As observedin Figure 2A, L. lactis-pBK1exhibited high levels of β-gal production on the plate. Theinteraction between recombinant L. lactis-pBK2Z or L. lactis-pBK2idTZ with E. faecalis OG1RFtriggered the expression of lacZ by these two strains.β-gal production was higher when using L. lactis-pBK2idTZ. No β-gal production was detected in those colonieslocated outside the area of interaction with E. faecalis OG1RF.

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