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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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E. faecalis viability when grown in coculturewith recombinant L. lactis strains. (A) E.faecalis OG1RF viability at different time points when coculturedwith L. lactis-pBK2idTZ (blue diamonds), L. lactis-pBK1:Bac (purple x’s) or L. lactis-pBK2idTZ:Bac induced with 100 ng/mL cCF10 at time 0 (yellow circles).The viability is expressed as the log10 of the total E. faecalis OG1RF colony forming units per ml (CFU/mL).(B) E. faecalis OG1RF viability at different timepoints when cocultured with L. lactis-pBK2idTZ:Bac(red squares) and, shown again for cross-reference to Figure 4A, L. lactis-pBK2idTZ (blue diamonds). Also shown is E. faecalis JRC101 (a cCF10 deficient strain) viabilitywhen cocultured with L. lactis-pBK2idTZ:Bac (greentriangles). The viability is expressed as the total number of E. faecalis CFU/mL. Cell ratios are as described in thetext. All results are shown as averaged results from two independentexperiments, each done in triplicate. The error bars represent thestandard deviation.
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fig4: E. faecalis viability when grown in coculturewith recombinant L. lactis strains. (A) E.faecalis OG1RF viability at different time points when coculturedwith L. lactis-pBK2idTZ (blue diamonds), L. lactis-pBK1:Bac (purple x’s) or L. lactis-pBK2idTZ:Bac induced with 100 ng/mL cCF10 at time 0 (yellow circles).The viability is expressed as the log10 of the total E. faecalis OG1RF colony forming units per ml (CFU/mL).(B) E. faecalis OG1RF viability at different timepoints when cocultured with L. lactis-pBK2idTZ:Bac(red squares) and, shown again for cross-reference to Figure 4A, L. lactis-pBK2idTZ (blue diamonds). Also shown is E. faecalis JRC101 (a cCF10 deficient strain) viabilitywhen cocultured with L. lactis-pBK2idTZ:Bac (greentriangles). The viability is expressed as the total number of E. faecalis CFU/mL. Cell ratios are as described in thetext. All results are shown as averaged results from two independentexperiments, each done in triplicate. The error bars represent thestandard deviation.

Mentions: The ultimate goalof this work was to engineer L. lactis strains todetect E. faecalis in the environment and then producea set of antimicrobial peptides. Once we established that both detectionof E. faecalis and production of active bacteriocinsby recombinant L. lactis were separately functional,we tested whether L. lactis could perform both tasksconcurrently. The lactococcal strain selected for this set of experimentswas L. lactis pBK2idTZ:Bac, the one with the highestsensitivity to cCF10 as well as the highest antimicrobial activityagainst E. faecalis. We grew this strain in coculturewith E. faecalis OG1RF, under two different conditions,with or without an external addition of cCF10, and measured the enterococcalviability over time. We also cocultured L. lactis pBK1:Bac with E. faecalis OG1RF, in order to determinethe inhibition caused by the constitutive system. To further verifythat the inhibition of E. faecalis is caused by theexpression and production of the three bacteriocins and discard anyantimicrobial activity from the native lactococcal strain, we culturedthe control strain L. lactis-pBK2idTZ together with E. faecalis OG1RF. To show that the induction of the AMPsis specifically triggered by cCF10, we cultured L. lactis pBK2idTZ:Bac together with E. faecalis JRC101,an E. faecalis OG1RF-derivative with a knockout inthe gene coding for cCF10. As observed in Figure 4, the highest reduction of E. faecalis OG1RFcounts was achieved by L. lactis-pBK1:Bac and L. lactis-pBK2idTZ:Bac induced with cCF10. Under these conditionswe observed reductions of the enterococcal strain of up to 6 and 4logarithmic units, respectively, in the first 2 h postinoculation.However, when using uninduced L. lactis-pBK2idtZ:Bacwe observed a reduction of up to 2-fold in the total number of enterococcalCFU in the coculture in comparison with the control (Figure 4). These differences were not significant duringthe first 3 h, and reached the highest levels after 5–9 h ofcoculture.


Modified lactic acid bacteria detect and inhibit multiresistant enterococci.

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

E. faecalis viability when grown in coculturewith recombinant L. lactis strains. (A) E.faecalis OG1RF viability at different time points when coculturedwith L. lactis-pBK2idTZ (blue diamonds), L. lactis-pBK1:Bac (purple x’s) or L. lactis-pBK2idTZ:Bac induced with 100 ng/mL cCF10 at time 0 (yellow circles).The viability is expressed as the log10 of the total E. faecalis OG1RF colony forming units per ml (CFU/mL).(B) E. faecalis OG1RF viability at different timepoints when cocultured with L. lactis-pBK2idTZ:Bac(red squares) and, shown again for cross-reference to Figure 4A, L. lactis-pBK2idTZ (blue diamonds). Also shown is E. faecalis JRC101 (a cCF10 deficient strain) viabilitywhen cocultured with L. lactis-pBK2idTZ:Bac (greentriangles). The viability is expressed as the total number of E. faecalis CFU/mL. Cell ratios are as described in thetext. All results are shown as averaged results from two independentexperiments, each done in triplicate. The error bars represent thestandard deviation.
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Related In: Results  -  Collection

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fig4: E. faecalis viability when grown in coculturewith recombinant L. lactis strains. (A) E.faecalis OG1RF viability at different time points when coculturedwith L. lactis-pBK2idTZ (blue diamonds), L. lactis-pBK1:Bac (purple x’s) or L. lactis-pBK2idTZ:Bac induced with 100 ng/mL cCF10 at time 0 (yellow circles).The viability is expressed as the log10 of the total E. faecalis OG1RF colony forming units per ml (CFU/mL).(B) E. faecalis OG1RF viability at different timepoints when cocultured with L. lactis-pBK2idTZ:Bac(red squares) and, shown again for cross-reference to Figure 4A, L. lactis-pBK2idTZ (blue diamonds). Also shown is E. faecalis JRC101 (a cCF10 deficient strain) viabilitywhen cocultured with L. lactis-pBK2idTZ:Bac (greentriangles). The viability is expressed as the total number of E. faecalis CFU/mL. Cell ratios are as described in thetext. All results are shown as averaged results from two independentexperiments, each done in triplicate. The error bars represent thestandard deviation.
Mentions: The ultimate goalof this work was to engineer L. lactis strains todetect E. faecalis in the environment and then producea set of antimicrobial peptides. Once we established that both detectionof E. faecalis and production of active bacteriocinsby recombinant L. lactis were separately functional,we tested whether L. lactis could perform both tasksconcurrently. The lactococcal strain selected for this set of experimentswas L. lactis pBK2idTZ:Bac, the one with the highestsensitivity to cCF10 as well as the highest antimicrobial activityagainst E. faecalis. We grew this strain in coculturewith E. faecalis OG1RF, under two different conditions,with or without an external addition of cCF10, and measured the enterococcalviability over time. We also cocultured L. lactis pBK1:Bac with E. faecalis OG1RF, in order to determinethe inhibition caused by the constitutive system. To further verifythat the inhibition of E. faecalis is caused by theexpression and production of the three bacteriocins and discard anyantimicrobial activity from the native lactococcal strain, we culturedthe control strain L. lactis-pBK2idTZ together with E. faecalis OG1RF. To show that the induction of the AMPsis specifically triggered by cCF10, we cultured L. lactis pBK2idTZ:Bac together with E. faecalis JRC101,an E. faecalis OG1RF-derivative with a knockout inthe gene coding for cCF10. As observed in Figure 4, the highest reduction of E. faecalis OG1RFcounts was achieved by L. lactis-pBK1:Bac and L. lactis-pBK2idTZ:Bac induced with cCF10. Under these conditionswe observed reductions of the enterococcal strain of up to 6 and 4logarithmic units, respectively, in the first 2 h postinoculation.However, when using uninduced L. lactis-pBK2idtZ:Bacwe observed a reduction of up to 2-fold in the total number of enterococcalCFU in the coculture in comparison with the control (Figure 4). These differences were not significant duringthe first 3 h, and reached the highest levels after 5–9 h ofcoculture.

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