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Genetic Basis and Functional Consequences of Differential Expression of the CmeABC Efflux Pump in Campylobacter jejuni Isolates.

Grinnage-Pulley T, Zhang Q - PLoS ONE (2015)

Bottom Line: Several examined amino acid substitutions in CmeR did not affect its binding to the cmeABC promoter, but a mutation that led to C-terminal truncation of CmeR abolished its DNA-binding activity.Overexpression of cmeABC did not affect the susceptibility of C. jejuni to most tested antimicrobials except for chloramphenicol, but promoted the emergence of ciprofloxacin-resistant mutants under antibiotic selection.These results link CmeABC overexpression in natural C. jejuni isolates to various mutations and indicate that this phenotypic change promotes the emergence of antibiotic-resistant mutants under selection pressure.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa, United States of America.

ABSTRACT
The CmeABC multidrug efflux transporter of Campylobacter jejuni plays a key role in antimicrobial resistance and is suppressed by CmeR, a transcriptional regulator of the TetR family. Overexpression of CmeABC has been observed in laboratory-generated mutants, but it is unknown if this phenotype occurs naturally in C. jejuni isolates and if it has any functional consequences. To answer these questions, expression of cmeABC in natural isolates obtained from broiler chickens, turkeys and humans was examined, and the genetic mechanisms and role of cmeABC differential expression in antimicrobial resistance was determined. Among the 64 C. jejuni isolates examined in this study, 43 and 21 were phenotypically identified as overexpression (OEL) and wild-type expression (WEL) levels. Representative mutations of the cmeABC promoter and/or CmeR-coding sequence were analyzed using electrophoretic mobility shift assays and transcriptional fusion assays. Reduced CmeR binding to the mutated cmeABC promoter sequences or decreased CmeR levels increased cmeABC expression. Several examined amino acid substitutions in CmeR did not affect its binding to the cmeABC promoter, but a mutation that led to C-terminal truncation of CmeR abolished its DNA-binding activity. Interestingly, some OEL isolates harbored no mutations in known regulatory elements, suggesting that cmeABC is also regulated by unidentified mechanisms. Overexpression of cmeABC did not affect the susceptibility of C. jejuni to most tested antimicrobials except for chloramphenicol, but promoted the emergence of ciprofloxacin-resistant mutants under antibiotic selection. These results link CmeABC overexpression in natural C. jejuni isolates to various mutations and indicate that this phenotypic change promotes the emergence of antibiotic-resistant mutants under selection pressure. Thus, differential expression of CmeABC may facilitate Campylobacter adaptation to antibiotic treatments.

No MeSH data available.


Emergence of ciprofloxacin-resistant mutants from WEL (circle) and OEL (triangle) isolates during treatment with ciprofloxacin.In panel A, the experiment was performed with an initial inoculum of 107 CFU/ml of each isolate, while in panel B, the inoculum was 106 CFU/mL for each isolate. The culture medium was MH broth containing 4 μg/mL of ciprofloxacin. Three WEL and OEL isolates were used in each experiment with cultures prepared in triplicate. Each point represents the number of ciprofloxacin-resistant mutants from a single culture. Bars represent mean log10 CFU/mL for each group. Means for each phenotypic group were compared for each day with multiple unpaired Student’ t-tests and Holm-Šídák method for multiple comparisons. The significance level was set at 0.05.
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pone.0131534.g006: Emergence of ciprofloxacin-resistant mutants from WEL (circle) and OEL (triangle) isolates during treatment with ciprofloxacin.In panel A, the experiment was performed with an initial inoculum of 107 CFU/ml of each isolate, while in panel B, the inoculum was 106 CFU/mL for each isolate. The culture medium was MH broth containing 4 μg/mL of ciprofloxacin. Three WEL and OEL isolates were used in each experiment with cultures prepared in triplicate. Each point represents the number of ciprofloxacin-resistant mutants from a single culture. Bars represent mean log10 CFU/mL for each group. Means for each phenotypic group were compared for each day with multiple unpaired Student’ t-tests and Holm-Šídák method for multiple comparisons. The significance level was set at 0.05.

Mentions: The spontaneous mutation rate to ciprofloxacin was examined for selected isolates using the fluctuation assay. Although there was a trend that the mutation rate was higher in OEL than in WEL, the difference was not statistically significant (p> 0.05) (S3 Fig). However, OEL isolates showed increased emergence of ciprofloxacin-resistant (CipR) mutants during in vitro treatment (Fig 6). Two experiments were performed with the initial inoculum levels of 107 and 106 CFU/mL, respectively. For the inoculums at 107 CFU/mL, there were no significant difference in the mean numbers of pre-existing CipR mutants between the WEL and OEL cultures on day 0 (Fig 6A). CipR populations from both WEL and OEL cultures expanded over days 1 to 3. The mean CipR mutant populations were 0.9 logs higher for OEL on day 1, 1.5 logs higher on day 2, and 2 logs higher than WEL on day 3. However, the means were not significantly different between the OEL and WEL groups (p > 0.05).


Genetic Basis and Functional Consequences of Differential Expression of the CmeABC Efflux Pump in Campylobacter jejuni Isolates.

Grinnage-Pulley T, Zhang Q - PLoS ONE (2015)

Emergence of ciprofloxacin-resistant mutants from WEL (circle) and OEL (triangle) isolates during treatment with ciprofloxacin.In panel A, the experiment was performed with an initial inoculum of 107 CFU/ml of each isolate, while in panel B, the inoculum was 106 CFU/mL for each isolate. The culture medium was MH broth containing 4 μg/mL of ciprofloxacin. Three WEL and OEL isolates were used in each experiment with cultures prepared in triplicate. Each point represents the number of ciprofloxacin-resistant mutants from a single culture. Bars represent mean log10 CFU/mL for each group. Means for each phenotypic group were compared for each day with multiple unpaired Student’ t-tests and Holm-Šídák method for multiple comparisons. The significance level was set at 0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4488513&req=5

pone.0131534.g006: Emergence of ciprofloxacin-resistant mutants from WEL (circle) and OEL (triangle) isolates during treatment with ciprofloxacin.In panel A, the experiment was performed with an initial inoculum of 107 CFU/ml of each isolate, while in panel B, the inoculum was 106 CFU/mL for each isolate. The culture medium was MH broth containing 4 μg/mL of ciprofloxacin. Three WEL and OEL isolates were used in each experiment with cultures prepared in triplicate. Each point represents the number of ciprofloxacin-resistant mutants from a single culture. Bars represent mean log10 CFU/mL for each group. Means for each phenotypic group were compared for each day with multiple unpaired Student’ t-tests and Holm-Šídák method for multiple comparisons. The significance level was set at 0.05.
Mentions: The spontaneous mutation rate to ciprofloxacin was examined for selected isolates using the fluctuation assay. Although there was a trend that the mutation rate was higher in OEL than in WEL, the difference was not statistically significant (p> 0.05) (S3 Fig). However, OEL isolates showed increased emergence of ciprofloxacin-resistant (CipR) mutants during in vitro treatment (Fig 6). Two experiments were performed with the initial inoculum levels of 107 and 106 CFU/mL, respectively. For the inoculums at 107 CFU/mL, there were no significant difference in the mean numbers of pre-existing CipR mutants between the WEL and OEL cultures on day 0 (Fig 6A). CipR populations from both WEL and OEL cultures expanded over days 1 to 3. The mean CipR mutant populations were 0.9 logs higher for OEL on day 1, 1.5 logs higher on day 2, and 2 logs higher than WEL on day 3. However, the means were not significantly different between the OEL and WEL groups (p > 0.05).

Bottom Line: Several examined amino acid substitutions in CmeR did not affect its binding to the cmeABC promoter, but a mutation that led to C-terminal truncation of CmeR abolished its DNA-binding activity.Overexpression of cmeABC did not affect the susceptibility of C. jejuni to most tested antimicrobials except for chloramphenicol, but promoted the emergence of ciprofloxacin-resistant mutants under antibiotic selection.These results link CmeABC overexpression in natural C. jejuni isolates to various mutations and indicate that this phenotypic change promotes the emergence of antibiotic-resistant mutants under selection pressure.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa, United States of America.

ABSTRACT
The CmeABC multidrug efflux transporter of Campylobacter jejuni plays a key role in antimicrobial resistance and is suppressed by CmeR, a transcriptional regulator of the TetR family. Overexpression of CmeABC has been observed in laboratory-generated mutants, but it is unknown if this phenotype occurs naturally in C. jejuni isolates and if it has any functional consequences. To answer these questions, expression of cmeABC in natural isolates obtained from broiler chickens, turkeys and humans was examined, and the genetic mechanisms and role of cmeABC differential expression in antimicrobial resistance was determined. Among the 64 C. jejuni isolates examined in this study, 43 and 21 were phenotypically identified as overexpression (OEL) and wild-type expression (WEL) levels. Representative mutations of the cmeABC promoter and/or CmeR-coding sequence were analyzed using electrophoretic mobility shift assays and transcriptional fusion assays. Reduced CmeR binding to the mutated cmeABC promoter sequences or decreased CmeR levels increased cmeABC expression. Several examined amino acid substitutions in CmeR did not affect its binding to the cmeABC promoter, but a mutation that led to C-terminal truncation of CmeR abolished its DNA-binding activity. Interestingly, some OEL isolates harbored no mutations in known regulatory elements, suggesting that cmeABC is also regulated by unidentified mechanisms. Overexpression of cmeABC did not affect the susceptibility of C. jejuni to most tested antimicrobials except for chloramphenicol, but promoted the emergence of ciprofloxacin-resistant mutants under antibiotic selection. These results link CmeABC overexpression in natural C. jejuni isolates to various mutations and indicate that this phenotypic change promotes the emergence of antibiotic-resistant mutants under selection pressure. Thus, differential expression of CmeABC may facilitate Campylobacter adaptation to antibiotic treatments.

No MeSH data available.