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1. Fitness trade-offs of multidrug efflux pumps in Escherichia coli K-12 in acid or base, and with aromatic phytochemicals

   https://doi.org/10.1128/aem.02096-23  

   Liu, Yangyang; Van_Horn, Andrew M; Pham, Minh_T N; Dinh, Bao_Ngoc N; Chen, Rachel; Raphael, Slaybrina_D R; Paulino, Alejandro; Thaker, Kavya; Somadder, Aaryan; Frost, Dominick J; et al (February 2024, Applied and Environmental Microbiology)

   Zhou, Ning-Yi (Ed.)

   ABSTRACT Multidrug efflux pumps are the frontline defense mechanisms of Gram-negative bacteria, yet little is known of their relative fitness trade-offs under gut conditions such as low pH and the presence of antimicrobial food molecules. Low pH contributes to the proton-motive force (PMF) that drives most efflux pumps. We show how the PMF-dependent pumps AcrAB-TolC, MdtEF-TolC, and EmrAB-TolC undergo selection at low pH and in the presence of membrane-permeant phytochemicals. Competition assays were performed by flow cytometry of co-culturedEscherichia coliK-12 strains possessing or lacking a given pump complex. All three pumps showed negative selection under conditions that deplete PMF (pH 5.5 with carbonyl cyanide 3-chlorophenylhydrazone or at pH 8.0). At pH 5.5, selection against AcrAB-TolC was increased by aromatic acids, alcohols, and related phytochemicals such as methyl salicylate. The degree of fitness cost for AcrA was correlated with the phytochemical’s lipophilicity (logP). Methyl salicylate and salicylamide selected strongly against AcrA, without genetic induction of drug resistance regulons. MdtEF-TolC and EmrAB-TolC each had a fitness cost at pH 5.5, but salicylate or benzoate made the fitness contribution positive. Pump fitness effects were not explained by gene expression (measured by digital PCR). Between pH 5.5 and 8.0,acrAandemrAwere upregulated in the log phase, whereasmdtEexpression was upregulated in the transition-to-stationary phase and at pH 5.5 in the log phase. Methyl salicylate did not affect pump gene expression. Our results suggest that lipophilic non-acidic molecules select against a major efflux pump without inducing antibiotic resistance regulons.IMPORTANCEFor drugs that are administered orally, we need to understand how ingested phytochemicals modulate drug resistance in our gut microbiome. Bacteria maintain low-level resistance by proton-motive force (PMF)-driven pumps that efflux many different antibiotics and cell waste products. These pumps play a key role in bacterial defense by conferring resistance to antimicrobial agents at first exposure while providing time for a pathogen to evolve resistance to higher levels of the antibiotic exposed. Nevertheless, efflux pumps confer energetic costs due to gene expression and pump energy expense. The bacterial PMF includes the transmembrane pH difference (ΔpH), which may be depleted by permeant acids and membrane disruptors. Understanding the fitness costs of efflux pumps may enable us to develop resistance breakers, that is, molecules that work together with antibiotics to potentiate their effect. Non-acidic aromatic molecules have the advantage that they avoid the Mar-dependent induction of regulons conferring other forms of drug resistance. We show that different pumps have distinct selection criteria, and we identified non-acidic aromatic molecules as promising candidates for drug resistance breakers. 

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2. Resensitization of Resistant Bacteria to Antimicrobials

   Patel, Mohit B.; Meisel, Joseph W.; Negin, Saeedeh; Gokel, Michael R.; Garrad, Evan; Gokel, George W. (April 2017, Annals of pharmacology and pharmaceutics)

   null (Ed.)

   Hydraphiles are synthetic amphiphiles that form pores in bilayer membranes. A study was undertaken to determine if the formation of pores could assist the penetration of antibiotics into bacteria. The disruption of ion homeostasis by the pore-formers leads to microbial toxicity. Co-administration of hydraphiles at concentration ≤ ½ MIC and antimicrobials to E. coli or P. aeruginosa showed potency enhancements of up to 30-fold. A possible mechanism is the enhancement of antibiotic influx owing to membrane disruption and/or altering the ion balance within the bacterial cells. 

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3. Horizontal Gene Transfer Is the Main Driver of Antimicrobial Resistance in Broiler Chicks Infected with Salmonella enterica Serovar Heidelberg

   https://doi.org/10.1128/mSystems.00729-21  

   Oladeinde, Adelumola; Abdo, Zaid; Press, Maximilian O.; Cook, Kimberly; Cox, Nelson A.; Zwirzitz, Benjamin; Woyda, Reed; Lakin, Steven M.; Thomas, Jesse C.; Looft, Torey; et al (August 2021, mSystems)

   Garrido, Daniel (Ed.)

   ABSTRACT The overuse and misuse of antibiotics in clinical settings and in food production have been linked to the increased prevalence and spread of antimicrobial resistance (AR). Consequently, public health and consumer concerns have resulted in a remarkable reduction in antibiotics used for food animal production. However, there are no data on the effectiveness of antibiotic removal in reducing AR shared through horizontal gene transfer (HGT). In this study, we used neonatal broiler chicks and Salmonella enterica serovar Heidelberg, a model food pathogen, to test if chicks raised antibiotic free harbor transferable AR. We challenged chicks with an antibiotic-susceptible S . Heidelberg strain using various routes of inoculation and determined if S . Heidelberg isolates recovered carried plasmids conferring AR. We used antimicrobial susceptibility testing and whole-genome sequencing (WGS) to show that chicks grown without antibiotics harbored an antimicrobial resistant S . Heidelberg population at 14 days after challenge and chicks challenged orally acquired AR at a higher rate than chicks inoculated via the cloaca. Using 16S rRNA gene sequencing, we found that S . Heidelberg infection perturbed the microbiota of broiler chicks, and we used metagenomics and WGS to confirm that a commensal Escherichia coli population was the main reservoir of an IncI1 plasmid acquired by S . Heidelberg. The carriage of this IncI1 plasmid posed no fitness cost to S . Heidelberg but increased its fitness when exposed to acidic pH in vitro . These results suggest that HGT of plasmids carrying AR shaped the evolution of S . Heidelberg and that antibiotic use reduction alone is insufficient to limit antibiotic resistance transfer from commensal bacteria to Salmonella enterica . IMPORTANCE The reported increase in antibiotic-resistant bacteria in humans has resulted in a major shift away from antibiotic use in food animal production. This shift has been driven by the assumption that removing antibiotics will select for antibiotic susceptible bacterial taxa, which in turn will allow the currently available antibiotic arsenal to be more effective. This change in practice has highlighted new questions that need to be answered to assess the effectiveness of antibiotic removal in reducing the spread of antibiotic resistance bacteria. This research demonstrates that antibiotic-susceptible Salmonella enterica serovar Heidelberg strains can acquire multidrug resistance from commensal bacteria present in the gut of neonatal broiler chicks, even in the absence of antibiotic selection. We demonstrate that exposure to acidic pH drove the horizontal transfer of antimicrobial resistance plasmids and suggest that simply removing antibiotics from food animal production might not be sufficient to limit the spread of antimicrobial resistance. 

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4. Extreme Acid Modulates Fitness Tradeoffs of Multidrug Efflux Pumps MdtEF-TolC and AcrAB-TolC in Escherichia coli K-12

   https://doi.org/https://doi.org/10.1128/AEM.00724-21  

   Schaffner, SH; Lee, AV; Pham, MT; Kassaye, BB; Li, H; Tallada, S; Lis, C; Lang, M; Liu, Y; Ahmed, N; et al (January 2021, Applied and environmental microbiology)

   null (Ed.)

   Bacterial genomes encode various multidrug efflux pumps (MDR) whose specific conditions for fitness advantage are unknown. We show that the efflux pump MdtEF-TolC, in Escherichia coli, confers a fitness advantage during exposure to extreme acid (pH 2). Our flow cytometry method revealed pH-dependent fitness tradeoffs between bile acids (a major pump substrate) and salicylic acid, a membrane-permeant aromatic acid that induces a drug-resistance regulon but depletes proton motive force (PMF). The PMF drives MdtEF-TolC and related pumps such as AcrAB-TolC. Deletion of mdtE (with loss of pump MdtEF-TolC) increased the strain’s relative fitness during growth with or without salicylate or bile acids. However, when the growth cycle included a 2-h incubation at pH 2 (below the pH growth range), MdtEF-TolC conferred a fitness advantage. The fitness advantage required bile salts but was decreased by the presence of salicylate, whose uptake is amplified by acid. For comparison, AcrAB-TolC, the primary efflux pump for bile acids, conferred a PMF-dependent fitness advantage with or without acid exposure in the growth cycle. A different MDR pump, EmrAB-TolC, confered no selective benefit during growth in the presence of bile acids. Without bile acids, all three MDR pumps incurred a large fitness cost with salicylate when exposed at pH 2. These results are consistent with the increased uptake of salicylate at low pH. Overall, we showed that MdtEF-TolC is an MDR pump adapted for transient extreme-acid exposure; and that low pH amplifies the salicylate-dependent fitness cost for drug pumps. IMPORTANCE Antibiotics and other drugs that reach the gut must pass through stomach acid. Yet little is known of how extreme acid modulates the effect of drugs on gut bacteria. We find that extreme-acid exposure leads to a fitness advantage for a multidrug pump that otherwise incurs a fitness cost. At the same time, extreme acid amplifies the effect of salicylate selection against multidrug pumps. Thus, organic acids and stomach acid could play important roles in regulating multidrug resistance in the gut microbiome. Our flow cytometry assay provides a way to measure the fitness effects of extreme-acid exposure to various membrane-soluble organic acids including plant-derived nutrients and pharmaceutical agents. Therapeutic acids might be devised to control the prevalence of multidrug pumps in environmental and host-associated habitats. 

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5. Constitutive Activation of RpoH and the Addition of L-arabinose Influence Antibiotic Sensitivity of PHL628 E. coli

   https://doi.org/10.3390/antibiotics13020143  

   Frizzell, Jenna K; Taylor, Ryan L; Ryno, Lisa M (February 2024, Antibiotics)

   Antibiotics are used to combat the ever-present threat of infectious diseases, but bacteria are continually evolving an assortment of defenses that enable their survival against even the most potent treatments. While the demand for novel antibiotic agents is high, the discovery of a new agent is exceedingly rare. We chose to focus on understanding how different signal transduction pathways in the gram-negative bacterium Escherichia coli (E. coli) influence the sensitivity of the organism to antibiotics from three different classes: tetracycline, chloramphenicol, and levofloxacin. Using the PHL628 strain of E. coli, we exogenously overexpressed two transcription factors, FliA and RpoH.I54N (a constitutively active mutant), to determine their influence on the minimum inhibitory concentration (MIC) and minimum duration of killing (MDK) concentration for each of the studied antibiotics. We hypothesized that activating these pathways, which upregulate genes that respond to specific stressors, could mitigate bacterial response to antibiotic treatment. We also compared the exogenous overexpression of the constitutively active RpoH mutant to thermal heat shock that has feedback loops maintained. While FliA overexpression had no impact on MIC or antibiotic tolerance, RpoH.I54N overexpression reduced the MIC for tetracycline and chloramphenicol but had no independent impact on antibiotic tolerance. Thermal heat shock alone also did not affect MIC or antibiotic tolerance. L-arabinose, the small molecule used to induce expression in our system, unexpectedly independently increased the MICs for tetracycline (>2-fold) and levofloxacin (3-fold). Additionally, the combination of thermal heat shock and arabinose provided a synergistic, 5-fold increase in MIC for chloramphenicol. Arabinose increased the tolerance, as assessed by MDK99, for chloramphenicol (2-fold) and levofloxacin (4-fold). These experiments highlight the potential of the RpoH pathway to modulate antibiotic sensitivity and the emerging implication of arabinose in enhanced MIC and antibiotic tolerance. 

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