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Beyond Antimicrobial Resistance: Evidence for a Distinct Role of the AcrD Efflux Pump in Salmonella Biology

View Article: PubMed Central - PubMed

ABSTRACT

For over 20 years, bacterial multidrug resistance (MDR) efflux pumps have been studied because of their impact on resistance to antimicrobials. However, critical questions remain, including why produce efflux pumps under non-antimicrobial treatment conditions, and why have multiple pumps if their only purpose is antimicrobial efflux? Salmonella spp. possess five efflux pump families, including the resistance-nodulation-division (RND) efflux pumps. Notably, the RND efflux pump AcrD has a unique substrate profile, distinct from other Salmonella efflux pumps. Here we show that inactivation of acrD results in a profoundly altered transcriptome and modulation of pathways integral to Salmonella biology. The most significant transcriptome changes were central metabolism related, with additional changes observed in pathogenicity, environmental sensing, and stress response pathway expression. The extent of tricarboxylic acid cycle and fumarate metabolism expression changes led us to hypothesize that acrD inactivation may result in motility defects due to perturbation of metabolite concentrations, such as fumarate, for which a role in motility has been established. Despite minimal detectable changes in flagellar gene expression, we found that an acrD mutant Salmonella enterica serovar Typhimurium isolate was significantly impaired for swarming motility, which was restored by addition of fumarate. The acrD mutant outcompeted the wild type in fitness experiments. The results of these diverse experiments provide strong evidence that the AcrD efflux pump is not simply a redundant system providing response resilience, but also has distinct physiological functions. Together, these data indicate that the AcrD efflux pump has a significant and previously underappreciated impact on bacterial biology, despite only minor perturbations of antibiotic resistance profiles.

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Impact of acrD inactivation on bacterial fitness. (a) Fitness under standard laboratory conditions in vitro. LB broth was inoculated with a 50:50 mixture of wild-type and acrD mutant cells and grown at 37°C with aeration. Samples were taken at the indicated time points, and fresh cultures were inoculated every 24 h. Data presented are the means of three independent experiments with three biological replicates each, ± standard deviations. (b) Competitive infection of polarized Caco-2 epithelial cells. After infection with a 50:50 mixture of wild-type and acrD mutant cells, association was determined at 2 h postinfection prior, to gentamicin treatment. A gentamicin protection assay was used to calculate invasion and persistence at later time points. Replica plating was used to determine the proportion of wild-type versus acrD::aph bacteria. Data presented are the means of three independent experiments performed, with 4 replicates each, ± standard deviations. *, P < 0.05; **, P < 0.001.
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fig4: Impact of acrD inactivation on bacterial fitness. (a) Fitness under standard laboratory conditions in vitro. LB broth was inoculated with a 50:50 mixture of wild-type and acrD mutant cells and grown at 37°C with aeration. Samples were taken at the indicated time points, and fresh cultures were inoculated every 24 h. Data presented are the means of three independent experiments with three biological replicates each, ± standard deviations. (b) Competitive infection of polarized Caco-2 epithelial cells. After infection with a 50:50 mixture of wild-type and acrD mutant cells, association was determined at 2 h postinfection prior, to gentamicin treatment. A gentamicin protection assay was used to calculate invasion and persistence at later time points. Replica plating was used to determine the proportion of wild-type versus acrD::aph bacteria. Data presented are the means of three independent experiments performed, with 4 replicates each, ± standard deviations. *, P < 0.05; **, P < 0.001.

Mentions: Since no differences were seen in growth when evaluated under a variety of conditions, yet expression levels of 403 genes were altered, we hypothesized that these numerous gene expression changes would alter the fitness of the acrD mutant in a competitive environment. Bacterial fitness can be determined in a number of different ways; initially we tested fitness under standard laboratory conditions in vitro (LB broth, 37°C with aeration, inoculation with a 50:50 ratio of wild-type Salmonella to acrD mutant). Under these conditions, the acrD mutant outcompeted the wild type as early as 4 h postinoculation (Fig. 4a). After 2 h of growth, the acrD mutant composed 57% of the population, and by 4 h 92% of the bacteria were acrD mutants (P < 0.001). This high proportion rose to 97% at 6 h (P < 0.001) and then varied between 96% and 99% (P < 0.001) for the remainder of the 72 h, which included passaging into fresh medium at 12, 24, and 48 h.


Beyond Antimicrobial Resistance: Evidence for a Distinct Role of the AcrD Efflux Pump in Salmonella Biology
Impact of acrD inactivation on bacterial fitness. (a) Fitness under standard laboratory conditions in vitro. LB broth was inoculated with a 50:50 mixture of wild-type and acrD mutant cells and grown at 37°C with aeration. Samples were taken at the indicated time points, and fresh cultures were inoculated every 24 h. Data presented are the means of three independent experiments with three biological replicates each, ± standard deviations. (b) Competitive infection of polarized Caco-2 epithelial cells. After infection with a 50:50 mixture of wild-type and acrD mutant cells, association was determined at 2 h postinfection prior, to gentamicin treatment. A gentamicin protection assay was used to calculate invasion and persistence at later time points. Replica plating was used to determine the proportion of wild-type versus acrD::aph bacteria. Data presented are the means of three independent experiments performed, with 4 replicates each, ± standard deviations. *, P < 0.05; **, P < 0.001.
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fig4: Impact of acrD inactivation on bacterial fitness. (a) Fitness under standard laboratory conditions in vitro. LB broth was inoculated with a 50:50 mixture of wild-type and acrD mutant cells and grown at 37°C with aeration. Samples were taken at the indicated time points, and fresh cultures were inoculated every 24 h. Data presented are the means of three independent experiments with three biological replicates each, ± standard deviations. (b) Competitive infection of polarized Caco-2 epithelial cells. After infection with a 50:50 mixture of wild-type and acrD mutant cells, association was determined at 2 h postinfection prior, to gentamicin treatment. A gentamicin protection assay was used to calculate invasion and persistence at later time points. Replica plating was used to determine the proportion of wild-type versus acrD::aph bacteria. Data presented are the means of three independent experiments performed, with 4 replicates each, ± standard deviations. *, P < 0.05; **, P < 0.001.
Mentions: Since no differences were seen in growth when evaluated under a variety of conditions, yet expression levels of 403 genes were altered, we hypothesized that these numerous gene expression changes would alter the fitness of the acrD mutant in a competitive environment. Bacterial fitness can be determined in a number of different ways; initially we tested fitness under standard laboratory conditions in vitro (LB broth, 37°C with aeration, inoculation with a 50:50 ratio of wild-type Salmonella to acrD mutant). Under these conditions, the acrD mutant outcompeted the wild type as early as 4 h postinoculation (Fig. 4a). After 2 h of growth, the acrD mutant composed 57% of the population, and by 4 h 92% of the bacteria were acrD mutants (P < 0.001). This high proportion rose to 97% at 6 h (P < 0.001) and then varied between 96% and 99% (P < 0.001) for the remainder of the 72 h, which included passaging into fresh medium at 12, 24, and 48 h.

View Article: PubMed Central - PubMed

ABSTRACT

For over 20&nbsp;years, bacterial multidrug resistance (MDR) efflux pumps have been studied because of their impact on resistance to antimicrobials. However, critical questions remain, including why produce efflux pumps under non-antimicrobial treatment conditions, and why have multiple pumps if their only purpose is antimicrobial efflux? Salmonella spp. possess five efflux pump families, including the resistance-nodulation-division (RND) efflux pumps. Notably, the RND efflux pump AcrD has a unique substrate profile, distinct from other Salmonella efflux pumps. Here we show that inactivation of acrD results in a profoundly altered transcriptome and modulation of pathways integral to Salmonella biology. The most significant transcriptome changes were central metabolism related, with additional changes observed in pathogenicity, environmental sensing, and stress response pathway expression. The extent of tricarboxylic acid cycle and fumarate metabolism expression changes led us to hypothesize that acrD inactivation may result in motility defects due to perturbation of metabolite concentrations, such as fumarate, for which a role in motility has been established. Despite minimal detectable changes in flagellar gene expression, we found that an acrD mutant Salmonella enterica serovar Typhimurium isolate was significantly impaired for swarming motility, which was restored by addition of fumarate. The acrD mutant outcompeted the wild type in fitness experiments. The results of these diverse experiments provide strong evidence that the AcrD efflux pump is not simply a redundant system providing response resilience, but also has distinct physiological functions. Together, these data indicate that the AcrD efflux pump has a significant and previously underappreciated impact on bacterial biology, despite only minor perturbations of antibiotic resistance profiles.

No MeSH data available.


Related in: MedlinePlus