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Efflux-mediated resistance to a benzothiadiazol derivative effective against Burkholderia cenocepacia.

Scoffone VC, Ryabova O, Makarov V, Iadarola P, Fumagalli M, Fondi M, Fani R, De Rossi E, Riccardi G, Buroni S - Front Microbiol (2015)

Bottom Line: Here a new compound, belonging to the 2,1,3-benzothiadiazol-5-yl family (10126109), with a bactericidal effect and a minimal inhibitory concentration (MIC) of 8 μg/ml against B. cenocepacia, is described.Indeed, rnd-9 overexpression was confirmed by quantitative reverse transcription PCR, and RND-9 was identified in the membrane fractions of the mutant strains.Moreover, the increase in the MIC values of different drugs in the mutant strains, together with complementation experiments, suggested the involvement of RND-9 in the efflux of 10126109, thus indicating again the central role of efflux transporters in B. cenocepacia drug resistance.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Microbiology, Dipartimento di Biologia e Biotecnologie "Lazzaro Spallanzani," Università degli Studi di Pavia Pavia, Italy.

ABSTRACT
Burkholderia cenocepacia is a major concern for people suffering from cystic fibrosis as it contributes to serious respiratory tract infections. The lack of drugs effective against this opportunistic pathogen, along with the high level of resistance to multiple antibiotics, render the treatment of these infections particularly difficult. Here a new compound, belonging to the 2,1,3-benzothiadiazol-5-yl family (10126109), with a bactericidal effect and a minimal inhibitory concentration (MIC) of 8 μg/ml against B. cenocepacia, is described. The compound is not cytotoxic and effective against B. cenocepacia clinical isolates and members of all the known B. cepacia complex species. Spontaneous mutants resistant to 10126109 were isolated and mutations in the MerR transcriptional regulator BCAM1948 were identified. In this way, a mechanism of resistance to this new molecule was described, which relies on the overexpression of the RND-9 efflux pump. Indeed, rnd-9 overexpression was confirmed by quantitative reverse transcription PCR, and RND-9 was identified in the membrane fractions of the mutant strains. Moreover, the increase in the MIC values of different drugs in the mutant strains, together with complementation experiments, suggested the involvement of RND-9 in the efflux of 10126109, thus indicating again the central role of efflux transporters in B. cenocepacia drug resistance.

No MeSH data available.


Related in: MedlinePlus

Time-kill curve of Burkholderia cenocepacia J2315 exposed to 10126109. A B. cenocepacia J2315 culture in exponential phase of growth (∙) was split and concentrations of 10126109 corresponding to 0.5 (○), 1 (▴), 2 (■), and 4-fold (□) the MIC value were added. The viable counts were determined at 37°C during 32 h. The dotted line indicates a reduction of 3 log10.
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Figure 2: Time-kill curve of Burkholderia cenocepacia J2315 exposed to 10126109. A B. cenocepacia J2315 culture in exponential phase of growth (∙) was split and concentrations of 10126109 corresponding to 0.5 (○), 1 (▴), 2 (■), and 4-fold (□) the MIC value were added. The viable counts were determined at 37°C during 32 h. The dotted line indicates a reduction of 3 log10.

Mentions: In order to assess if 10126109 exerts a bacteriostatic or a bactericidal effect on Burkholderia cells, a time-kill curve was constructed using concentrations ranging from 0.5 to 4 times the MIC value (Figure 2). A bactericidal effect was observed using two and fourfold the MIC of 10126109 (i.e., a reduction of 99.9% (≥3 log10) of the total number of CFU ml-1 in the original inoculum was observed), while the number of B. cenocepacia cells remained constant when an amount of compound equal to the MIC was added to the culture. These data suggest that bacterial killing is concentration dependent, as already reported for ciprofloxacin (Silva et al., 2011).


Efflux-mediated resistance to a benzothiadiazol derivative effective against Burkholderia cenocepacia.

Scoffone VC, Ryabova O, Makarov V, Iadarola P, Fumagalli M, Fondi M, Fani R, De Rossi E, Riccardi G, Buroni S - Front Microbiol (2015)

Time-kill curve of Burkholderia cenocepacia J2315 exposed to 10126109. A B. cenocepacia J2315 culture in exponential phase of growth (∙) was split and concentrations of 10126109 corresponding to 0.5 (○), 1 (▴), 2 (■), and 4-fold (□) the MIC value were added. The viable counts were determined at 37°C during 32 h. The dotted line indicates a reduction of 3 log10.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525489&req=5

Figure 2: Time-kill curve of Burkholderia cenocepacia J2315 exposed to 10126109. A B. cenocepacia J2315 culture in exponential phase of growth (∙) was split and concentrations of 10126109 corresponding to 0.5 (○), 1 (▴), 2 (■), and 4-fold (□) the MIC value were added. The viable counts were determined at 37°C during 32 h. The dotted line indicates a reduction of 3 log10.
Mentions: In order to assess if 10126109 exerts a bacteriostatic or a bactericidal effect on Burkholderia cells, a time-kill curve was constructed using concentrations ranging from 0.5 to 4 times the MIC value (Figure 2). A bactericidal effect was observed using two and fourfold the MIC of 10126109 (i.e., a reduction of 99.9% (≥3 log10) of the total number of CFU ml-1 in the original inoculum was observed), while the number of B. cenocepacia cells remained constant when an amount of compound equal to the MIC was added to the culture. These data suggest that bacterial killing is concentration dependent, as already reported for ciprofloxacin (Silva et al., 2011).

Bottom Line: Here a new compound, belonging to the 2,1,3-benzothiadiazol-5-yl family (10126109), with a bactericidal effect and a minimal inhibitory concentration (MIC) of 8 μg/ml against B. cenocepacia, is described.Indeed, rnd-9 overexpression was confirmed by quantitative reverse transcription PCR, and RND-9 was identified in the membrane fractions of the mutant strains.Moreover, the increase in the MIC values of different drugs in the mutant strains, together with complementation experiments, suggested the involvement of RND-9 in the efflux of 10126109, thus indicating again the central role of efflux transporters in B. cenocepacia drug resistance.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Microbiology, Dipartimento di Biologia e Biotecnologie "Lazzaro Spallanzani," Università degli Studi di Pavia Pavia, Italy.

ABSTRACT
Burkholderia cenocepacia is a major concern for people suffering from cystic fibrosis as it contributes to serious respiratory tract infections. The lack of drugs effective against this opportunistic pathogen, along with the high level of resistance to multiple antibiotics, render the treatment of these infections particularly difficult. Here a new compound, belonging to the 2,1,3-benzothiadiazol-5-yl family (10126109), with a bactericidal effect and a minimal inhibitory concentration (MIC) of 8 μg/ml against B. cenocepacia, is described. The compound is not cytotoxic and effective against B. cenocepacia clinical isolates and members of all the known B. cepacia complex species. Spontaneous mutants resistant to 10126109 were isolated and mutations in the MerR transcriptional regulator BCAM1948 were identified. In this way, a mechanism of resistance to this new molecule was described, which relies on the overexpression of the RND-9 efflux pump. Indeed, rnd-9 overexpression was confirmed by quantitative reverse transcription PCR, and RND-9 was identified in the membrane fractions of the mutant strains. Moreover, the increase in the MIC values of different drugs in the mutant strains, together with complementation experiments, suggested the involvement of RND-9 in the efflux of 10126109, thus indicating again the central role of efflux transporters in B. cenocepacia drug resistance.

No MeSH data available.


Related in: MedlinePlus