Differential development of antibiotic resistance and virulence between Acinetobacter species.

The two species that account for most cases of Acinetobacter-associated bacteremia in the United Kingdom are Acinetobacter lwoffii, often a commensal but also an emerging pathogen, and Acinetobacter baumannii, a well-known antibiotic-resistant species. While these species both cause similar types of human infection and occupy the same niche, A. lwoffii (unlike A. baumannii) has thus far remained susceptible to antibiotics. Comparatively little is known about the biology of A. lwoffii, and this is the largest study on it conducted to date, providing valuable insights into its behaviour and potential threat to human health. This study aimed to explain the antibiotic susceptibility, virulence, and fundamental biological differences between these two species. The relative susceptibility of A. lwoffii was explained as it encoded fewer antibiotic resistance and efflux pump genes than A. baumannii (9 and 30, respectively). While both species had markers of horizontal gene transfer, A. lwoffii encoded more DNA defense systems and harbored a far more restricted range of plasmids. Furthermore, A. lwoffii displayed a reduced ability to select for antibiotic resistance mutations, form biofilm, and infect both in vivo and in in vitro models of infection. This study suggests that the emerging pathogen A. lwoffii has remained susceptible to antibiotics because mechanisms exist to make it highly selective about the DNA it acquires, and we hypothesize that the fact that it only harbors a single RND system restricts the ability to select for resistance mutations. This provides valuable insights into how development of resistance can be constrained in Gram-negative bacteria. IMPORTANCE: Acinetobacter lwoffii is often a harmless commensal but is also an emerging pathogen and is the most common cause of Acinetobacter-derived bloodstream infections in England and Wales. In contrast to the well-studied and often highly drug-resistant A. baumannii, A. lwoffii has remained susceptible to antibiotics. This study explains why this organism has not evolved resistance to antibiotics. These new insights are important to understand why and how some species develop antibiotic resistance, while others do not, and could inform future novel treatment strategies.

Microbial Primer: Multidrug efflux pumps.

Multidrug efflux pumps are molecular machines that sit in the bacterial cell membrane and pump molecules out from either the periplasm or cytoplasm to outside the cell. While involved in a variety of biological roles, they are primarily known for their contribution to antibiotic resistance by limiting the intracellular accumulation of antimicrobial compounds within bacteria. These transporters are often overexpressed in clinical isolates, leading to multidrug-resistant phenotypes. Efflux pumps are classified into several families based on their structure and understanding the characteristics of each family is important for the development of novel therapies to restore antibiotic potency.

Pseudomonas aeruginosa increases the susceptibility of Candida albicans to amphotericin B in dual-species biofilms.

BACKGROUND: Biofilms are the leading cause of nosocomial infections and are hard to eradicate due to their inherent antimicrobial resistance. Candida albicans is the leading cause of nosocomial fungal infections and is frequently co-isolated with the bacterium Pseudomonas aeruginosa from biofilms in the cystic fibrosis lung and severe burn wounds. The presence of C. albicans in multispecies biofilms is associated with enhanced antibacterial resistance, which is largely mediated through fungal extracellular carbohydrates sequestering the antibiotics. However, significantly less is known regarding the impact of polymicrobial biofilms on antifungal resistance. RESULTS: Here we show that, in dual-species biofilms, P. aeruginosa enhances the susceptibility of C. albicans to amphotericin B, an effect that was biofilm specific. Transcriptional analysis combined with gene ontology enrichment analysis identified several C. albicans processes associated with oxidative stress to be differentially regulated in dual-species biofilms, suggesting that P. aeruginosa exerts oxidative stress on C. albicans, likely through the secretion of phenazines. However, the mitochondrial superoxide dismutase SOD2 was significantly down-regulated in the presence of P. aeruginosa. Monospecies biofilms of the sod2Δ mutant were more susceptible to amphotericin B, and the susceptibility of these biofilms was further enhanced by exogenous phenazines. CONCLUSIONS: We propose that in dual-species biofilms, P. aeruginosa simultaneously induces mitochondrial oxidative stress, while down-regulating key detoxification enzymes, which prevents C. albicans mounting an appropriate oxidative stress response to amphotericin B, leading to fungal cell death. This work highlights the importance of understanding the impact of polymicrobial interactions on antimicrobial susceptibility.

Chelating silica nanoparticles for efficient antibiotic delivery and particle imaging in Gram-negative bacteria.

The inefficacy of antibiotics against Gram-negative bacteria is a major challenge for treatment of many clinically important bacterial infections. The complex structure of the double cell membrane of Gram-negative bacteria makes it inaccessible to many key antibiotics such as vancomycin and also presents a major challenge for drug development. In this study we design of a novel hybrid silica nanoparticle system bearing membrane targeting groups with the antibiotic encapsulated together with a ruthenium luminescent tracking agent, for optical detection of the nanoparticle delivery in the bacterial cell. The hybrid system shows delivery of vancomycin and efficacy against a library of Gram negative bacterial species. Evidence of penetration of nanoparticles in bacteria cells is achieved via luminescence of the ruthenium signal. Our studies show that nanoparticles modified with aminopolycarboxylate chelating groups are an effective delivery system in bacterial growth inhibition in species whereas the molecular antibiotic is ineffective. This design provides a new platform for delivery of antibiotics that cannot alone penetrate the bacterial membrane.

E. coli ST11 (O157:H7) does not encode a functional AcrF efflux pump.

Escherichia coli is a facultative anaerobe found in a wide range of environments. Commonly described as the laboratory workhorse, E. coli is one of the best characterized bacterial species to date, however much of our understanding comes from studies involving the laboratory strain E. coli K-12. Resistance-nodulation-division efflux pumps are found in Gram-negative bacteria and can export a diverse range of substrates, including antibiotics. E. coli K-12 has six RND pumps; AcrB, AcrD, AcrF, CusA, MdtBC and MdtF, and it is frequently reported that all E. coli strains possess these six pumps. However, this is not true of E. coli ST11, a lineage of E. coli, which is primarily composed of the highly virulent important human pathogen, E. coli O157:H7. Here we show that acrF is absent from the pangenome of ST11 and that this lineage of E. coli has a highly conserved insertion within the acrF gene, which when translated encodes 13 amino acids and two stop codons. This insertion was found to be present in 97.59 % of 1787 ST11 genome assemblies. Non-function of AcrF in ST11 was confirmed in the laboratory as complementation with acrF from ST11 was unable to restore AcrF function in E. coli K-12 substr. MG1655 ΔacrB ΔacrF. This shows that the complement of RND efflux pumps present in laboratory bacterial strains may not reflect the situation in virulent strains of bacterial pathogens.

Characteristics of antimicrobial peptide OaBac5mini and its bactericidal mechanism against Escherichia coli.

Introduction: Antimicrobial peptides (AMPs) play an important role in defending against the attack of pathogenic microorganisms. Among them, the proline-rich antibacterial peptides (PrAMPs) have been attracting close attention due to their simple structure, strong antibacterial activity, and low cell toxicity. OaBac5mini is an active fragment of the sheep-derived OaBac5 belonging to the PrAMPs family. Methods: In this study, the antibacterial activity of OaBac5mini was investigated by testing the MICs against different stains of E. coli and S. aureus as well as the time-kill curve. The bactericidal mechanism was explored by determining the effect of OaBac5mini on the cell membrane. The stability and biosafety were also evaluated. Results: The susceptibility test demonstrated that OaBac5mini showed potent antibacterial activity against the multidrug-resistant (MDR) E. coli isolates. It is noticeable that the absence of inner membrane protein SbmA in E. coli ATCC 25922 caused the MIC of OaBac5mini to increase 4-fold, implying OaBac5mini can enter into the cytoplasm via SbmA and plays its antibacterial activity. Moreover, the antibacterial activity of OaBac5mini against E. coli ATCC 25922 was not remarkably affected by the serum salts except for CaCl2 at a physiological concentration, pH, temperature, repeated freeze-thawing and proteases (trypsin < 20 µg/mL, pepsin or proteinase K). Time-kill curve analysis showed OaBac5mini at the concentration of 200 µg/mL (8 × MICs) could effectively kill E. coli ATCC 25922 after co-incubation for 12 h. In addition, OaBac5mini was not hemolytic against rabbit red blood cells and also was not cytotoxic to porcine small intestinal epithelial cells (IPEC-J2). Bioinformatic analysis indicated that OaBac5mini is a linear peptide with 8 net positive charges. Furthermore, OaBac5mini significantly increased the outer membrane permeability and impaired the inner membrane integrity and ultrastructure of E. coli ATCC25922. Conclusion: OaBac5mini is a stable and potent PrAMP that kills E. coli by two different modes of action - inhibiting intracellular target(s) and damaging cell membrane.

RND pumps across the genus Acinetobacter: AdeIJK is the universal efflux pump.

Acinetobacter are generally soil-dwelling organisms that can also cause serious human infections. A. baumannii is one of the most common causative agents of Acinetobacter infections and is often multidrug resistant. However, an additional 25 species within the genus have also been associated with infection. A. baumannii encodes six resistance nodulation division (RND) efflux pumps, the most clinically relevant class of efflux pumps for antibiotic export, but the distribution and types of RND efflux pumps across the genus is currently unknown. Sixty-four species making up the genus Acinetobacter were searched for RND systems within their genomes. We also developed a novel method using conserved RND residues to predict the total number of RND proteins including currently undescribed RND pump proteins. The total number of RND proteins differed both within a species and across the genus. Species associated with infection tended to encode more pumps. AdeIJK/AdeXYZ was found in all searched species of Acinetobacter, and through genomic, structural and phenotypic work we show that these genes are actually homologues of the same system. This interpretation is further supported by structural analysis of the potential drug-binding determinants of the associated RND-transporters, which reveal their close similarity to each other, and distinctiveness from other RND-pumps in Acinetobacter, such as AdeB. Therefore, we conclude that AdeIJK is the fundamental RND system for species in the genus Acinetobacter. AdeIJK can export a broad range of antibiotics and provides crucial functions within the cell, for example lipid modulation of the cell membrane, and therefore it is likely that all Acinetobacter require AdeIJK for survival and homeostasis. In contrast, additional RND systems, such as AdeABC and AdeFGH, were only found in a subset of Acinetobacter that are associated with infection. By understanding the roles and mechanisms of RND efflux systems in Acinetobacter, treatments for infections can avoid efflux-mediated resistance and improve patient outcomes.

Transcriptional profiling of Pseudomonas aeruginosa mature single- and dual-species biofilms in response to meropenem.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen frequently isolated from chronic infections of the cystic fibrosis lung and burn wounds, and is a major cause of antimicrobial-resistant nosocomial infections. P. aeruginosa is frequently co-isolated with the opportunistic fungal pathogen Candida albicans, with the presence of C. albicans in dual-species biofilms promoting tolerance to meropenem. Here, transcription profiling of mature P. aeruginosa single- or dual-species biofilms was carried out to understand the molecular mechanism(s) by which C. albicans enhances meropenem tolerance. C. albicans appeared to have a mild impact on the transcriptome of P. aeruginosa mature biofilms, with most differentially regulated genes being involved in interkingdom interactions (i.e. quorum sensing and phenazine biosynthesis). The addition of meropenem to mature single- or dual-species biofilms resulted in a significant bacterial transcriptional response, including the induction of the beta-lactamase, ampC, genes involved in biofilm formation. P. aeruginosa elicited a similar transcriptional response to meropenem in the presence of C. albicans, but C. albicans promoted the expression of additional efflux pumps, which could play roles in increasing the tolerance of P. aeruginosa to meropenem.

Molecular mechanisms of antibiotic resistance revisited.

Antibiotic resistance is a global health emergency, with resistance detected to all antibiotics currently in clinical use and only a few novel drugs in the pipeline. Understanding the molecular mechanisms that bacteria use to resist the action of antimicrobials is critical to recognize global patterns of resistance and to improve the use of current drugs, as well as for the design of new drugs less susceptible to resistance development and novel strategies to combat resistance. In this Review, we explore recent advances in understanding how resistance genes contribute to the biology of the host, new structural details of relevant molecular events underpinning resistance, the identification of new resistance gene families and the interactions between different resistance mechanisms. Finally, we discuss how we can use this information to develop the next generation of antimicrobial therapies.

EnvR is a potent repressor of acrAB transcription in Salmonella.

BACKGROUND: Resistance nodulation division (RND) family efflux pumps, including the major pump AcrAB-TolC, are important mediators of intrinsic and evolved antibiotic resistance. Expression of these pumps is carefully controlled by a network of regulators that respond to different environmental cues. EnvR is a TetR family transcriptional regulator encoded upstream of the RND efflux pump acrEF. METHODS: Binding of EnvR protein upstream of acrAB was determined by electrophoretic mobility shift assays and the phenotypic consequence of envR overexpression on antimicrobial susceptibility, biofilm motility and invasion of eukaryotic cells in vitro was measured. Additionally, the global transcriptome of clinical Salmonella isolates overexpressing envR was determined by RNA-Seq. RESULTS: EnvR bound to the promoter region upstream of the genes coding for the major efflux pump AcrAB in Salmonella, inhibiting transcription and preventing production of AcrAB protein. The phenotype conferred by overexpression of envR mimicked deletion of acrB as it conferred multidrug susceptibility, decreased motility and decreased invasion into intestinal cells in vitro. Importantly, we demonstrate the clinical relevance of this regulatory mechanism because RNA-Seq revealed that a drug-susceptible clinical isolate of Salmonella had low acrB expression even though expression of its major regulator RamA was very high; this was caused by very high EnvR expression. CONCLUSIONS: In summary, we show that EnvR is a potent repressor of acrAB transcription in Salmonella, and can override binding by RamA so preventing MDR to clinically useful drugs. Finding novel tools to increase EnvR expression may form the basis of a new way to prevent or treat MDR infections.

The role of bacterial transport systems in the removal of host antimicrobial peptides in Gram-negative bacteria.

Antibiotic resistance is a global issue that threatens our progress in healthcare and life expectancy. In recent years, antimicrobial peptides (AMPs) have been considered as promising alternatives to the classic antibiotics. AMPs are potentially superior due to their lower rate of resistance development, since they primarily target the bacterial membrane ('Achilles' heel' of the bacteria). However, bacteria have developed mechanisms of AMP resistance, including the removal of AMPs to the extracellular space by efflux pumps such as the MtrCDE or AcrAB-TolC systems, and the internalization of AMPs to the cytoplasm by the Sap transporter, followed by proteolytic digestion. In this review, we focus on AMP transport as a resistance mechanism compiling all the experimental evidence for the involvement of efflux in AMP resistance in Gram-negative bacteria and combine this information with the analysis of the structures of the efflux systems involved. Finally, we expose some open questions with the aim of arousing the interest of the scientific community towards the AMPs-efflux pumps interactions. All the collected information broadens our understanding of AMP removal by efflux pumps and gives some clues to assist the rational design of AMP-derivatives as inhibitors of the efflux pumps.

Seeking patterns of antibiotic resistance in ATLAS, an open, raw MIC database with patient metadata.

Antibiotic resistance represents a growing medical concern where raw, clinical datasets are under-exploited as a means to track the scale of the problem. We therefore sought patterns of antibiotic resistance in the Antimicrobial Testing Leadership and Surveillance (ATLAS) database. ATLAS holds 6.5M minimal inhibitory concentrations (MICs) for 3,919 pathogen-antibiotic pairs isolated from 633k patients in 70 countries between 2004 and 2017. We show most pairs form coherent, although not stationary, timeseries whose frequencies of resistance are higher than other databases, although we identified no systematic bias towards including more resistant strains in ATLAS. We sought data anomalies whereby MICs could shift for methodological and not clinical or microbiological reasons and found artefacts in over 100 pathogen-antibiotic pairs. Using an information-optimal clustering methodology to classify pathogens into low and high antibiotic susceptibilities, we used ATLAS to predict changes in resistance. Dynamics of the latter exhibit complex patterns with MIC increases, and some decreases, whereby subpopulations' MICs can diverge. We also identify pathogens at risk of developing clinical resistance in the near future.

Mathematical Modelling Highlights the Potential for Genetic Manipulation as an Adjuvant to Counter Efflux-Mediated MDR in Salmonella.

Bacteria have developed resistance to antibiotics by various mechanisms, notable amongst these is the use of permeation barriers and the expulsion of antibiotics via efflux pumps. The resistance-nodulation-division (RND) family of efflux pumps is found in Gram-negative bacteria and a major contributor to multidrug resistance (MDR). In particular, Salmonella encodes five RND efflux pump systems: AcrAB, AcrAD, AcrEF, MdsAB and MdtAB which have different substrate ranges including many antibiotics. We produce a spatial partial differential equation (PDE) model governing the diffusion and efflux of antibiotic in Salmonella, via these RND efflux pumps. Using parameter fitting techniques on experimental data, we are able to establish the behaviour of multiple wild-type and efflux mutant Salmonella strains, which enables us to produce efflux profiles for each individual efflux pump system. By combining the model with a gene regulatory network (GRN) model of efflux regulation, we simulate how the bacteria respond to their environment. Finally, performing a parameter sensitivity analysis, we look into various different targets to inhibit the efflux pumps. The model provides an in silico framework with which to test these potential adjuvants to counter MDR.

Contribution of the efflux pump AcrAB-TolC to the tolerance of chlorhexidine and other biocides in Klebsiella spp.

Introduction. We are becoming increasingly reliant on the effectiveness of biocides to combat the spread of Gram-negative multi-drug-resistant (MDR) pathogens, including Klebsiella pneumoniae. It has been shown that chlorhexidine exposure can lead to mutations in the efflux pump repressor regulators SmvR and RamR, but the contribution of each individual efflux pump to biocide tolerance is unknown.Hypothesis. Multiple efflux pumps, including SmvA and AcrAB-TolC, are involved in increased tolerance to biocides. However, strains with upregulated AcrAB-TolC caused by biocide exposure are more problematic due to their increased MDR phenotype.Aim. To investigate the role of AcrAB-TolC in the tolerance to several biocides, including chlorhexidine, and the potential threat of cross-resistance to antibiotics through increased expression of this efflux pump.Methodology. Antimicrobial susceptibility testing was performed on K. pneumoniae isolates with ramR mutations selected for after exposure to chlorhexidine, as well as transposon mutants in components and regulators of AcrAB-TolC. RTPCR was used to detect the expression levels of this pump after biocide exposure. Strains from the globally important ST258 clade were compared for genetic differences in acrAB-TolC and its regulators and for phenotypic differences in antimicrobial susceptibility.Results. Cross-resistance to antimicrobials was observed following mutations in ramR. Exposure to chlorhexidine led to increased expression of acrA and its activator ramA, and transposon mutants in AcrAB-TolC have increased susceptibility to several biocides, including chlorhexidine. Variations in ramR within the ST258 clade led to an increase in tolerance to certain biocides, although this was strain dependent. One strain, MKP103, that had increased levels of biocide tolerance showed a unique mutation in ramR that was reflected in enhanced expression of acrA and ramA. MKP103 transposon variants were able to further enhance their tolerance to specific biocides with mutations affecting SmvA.Conclusions. Biocide tolerance in K. pneumoniae is dependent upon several components, with increased efflux through AcrAB-TolC being an important one.

A role for the periplasmic adaptor protein AcrA in vetting substrate access to the RND efflux transporter AcrB.

Tripartite resistance-nodulation-division (RND) efflux pumps, such as AcrAB-TolC of Salmonella Typhimurium, contribute to antibiotic resistance and comprise an inner membrane RND-transporter, an outer membrane factor, and a periplasmic adaptor protein (PAP). The role of the PAP in the assembly and active transport process remains poorly understood. Here, we identify the functionally critical residues involved in PAP-RND-transporter binding between AcrA and AcrB and show that the corresponding RND-binding residues in the closely related PAP AcrE, are also important for its interaction with AcrB. We also report a residue in the membrane-proximal domain of AcrA, that when mutated, differentially affects the transport of substrates utilising different AcrB efflux channels, namely channels 1 and 2. This supports a potential role for the PAP in sensing the substrate-occupied state of the proximal binding pocket of the transporter and substrate vetting. Understanding the PAP's role in the assembly and function of tripartite RND pumps can guide novel ways to inhibit their function to combat antibiotic resistance.

Breaking antimicrobial resistance by disrupting extracytoplasmic protein folding.

Antimicrobial resistance in Gram-negative bacteria is one of the greatest threats to global health. New antibacterial strategies are urgently needed, and the development of antibiotic adjuvants that either neutralize resistance proteins or compromise the integrity of the cell envelope is of ever-growing interest. Most available adjuvants are only effective against specific resistance proteins. Here, we demonstrate that disruption of cell envelope protein homeostasis simultaneously compromises several classes of resistance determinants. In particular, we find that impairing DsbA-mediated disulfide bond formation incapacitates diverse ß-lactamases and destabilizes mobile colistin resistance enzymes. Furthermore, we show that chemical inhibition of DsbA sensitizes multidrug-resistant clinical isolates to existing antibiotics and that the absence of DsbA, in combination with antibiotic treatment, substantially increases the survival of Galleria mellonella larvae infected with multidrug-resistant Pseudomonas aeruginosa. This work lays the foundation for the development of novel antibiotic adjuvants that function as broad-acting resistance breakers.

Antibiotics, like penicillin, are the foundation of modern medicine, but bacteria are evolving to resist their effects. Some of the most harmful pathogens belong to a group called the 'Gram-negative bacteria', which have an outer layer ­ called the cell envelope ­ that acts as a drug barrier. This envelope contains antibiotic resistance proteins that can deactivate or repel antibiotics or even pump them out of the cell once they get in. One way to tackle antibiotic resistance could be to stop these proteins from working. Proteins are long chains of building blocks called amino acids that fold into specific shapes. In order for a protein to perform its role correctly, it must fold in the right way. In bacteria, a protein called DsbA helps other proteins fold correctly by holding them in place and inserting links called disulfide bonds. It was unclear whether DsbA plays a role in the folding of antibiotic resistance proteins, but if it did, it might open up new ways to treat antibiotic resistant infections. To find out more, Furniss, Kaderabkova et al. collected the genes that code for several antibiotic resistance proteins and put them into Escherichia coli bacteria, which made the bacteria resistant to antibiotics. Furniss, Kaderabkova et al. then stopped the modified E. coli from making DsbA, which led to the antibiotic resistance proteins becoming unstable and breaking down because they could not fold correctly. Further experiments showed that blocking DsbA with a chemical inhibitor in other pathogenic species of Gram-negative bacteria made these bacteria more sensitive to antibiotics that they would normally resist. To demonstrate that using this approach could work to stop infections by these bacteria, Furniss, Kaderabkova et al. used Gram-negative bacteria that produced antibiotic resistance proteins but could not make DsbA to infect insect larvae. The larvae were then treated with antibiotics, which increased their survival rate, indicating that blocking DsbA may be a good approach to tackling antibiotic resistant bacteria. According to the World Health Organization, developing new treatments against Gram-negative bacteria is of critical importance, but the discovery of new drugs has ground to a halt. One way around this is to develop ways to make existing drugs work better. Making drugs that block DsbA could offer a way to treat resistant infections using existing antibiotics in the future.

Dynamic Boolean modelling reveals the influence of energy supply on bacterial efflux pump expression.

Antimicrobial resistance (AMR) is a global health issue. One key factor contributing to AMR is the ability of bacteria to export drugs through efflux pumps, which relies on the ATP-dependent expression and interaction of several controlling genes. Recent studies have shown that significant cell-to-cell ATP variability exists within clonal bacterial populations, but the contribution of intrinsic cell-to-cell ATP heterogeneity is generally overlooked in understanding efflux pumps. Here, we consider how ATP variability influences gene regulatory networks controlling expression of efflux pump genes in two bacterial species. We develop and apply a generalizable Boolean modelling framework, developed to incorporate the dependence of gene expression dynamics on available cellular energy supply. Theoretical results show that differences in energy availability can cause pronounced downstream heterogeneity in efflux gene expression. Cells with higher energy availability have a superior response to stressors. Furthermore, in the absence of stress, model bacteria develop heterogeneous pulses of efflux pump gene expression which contribute to a sustained sub-population of cells with increased efflux expression activity, potentially conferring a continuous pool of intrinsically resistant bacteria. This modelling approach thus reveals an important source of heterogeneity in cell responses to antimicrobials and sheds light on potentially targetable aspects of efflux pump-related antimicrobial resistance.

Efflux Impacts Intracellular Accumulation Only in Actively Growing Bacterial Cells.

For antibiotics with intracellular targets, effective treatment of bacterial infections requires the drug to accumulate to a high concentration inside cells. Bacteria produce a complex cell envelope and possess drug export efflux pumps to limit drug accumulation inside cells. Decreasing cell envelope permeability and increasing efflux pump activity can reduce intracellular accumulation of antibiotics and are commonly seen in antibiotic-resistant strains. Here, we show that the balance between influx and efflux differs depending on bacterial growth phase in Gram-negative bacteria. Accumulation of the fluorescent compound ethidium bromide (EtBr) was measured in Salmonella enterica serovar Typhimurium SL1344 (wild type) and efflux deficient (ΔacrB) strains during growth. In SL1344, EtBr accumulation remained low, regardless of growth phase, and did not correlate with acrAB transcription. EtBr accumulation in the ΔacrB strains was high in exponential phase but dropped sharply later in growth, with no significant difference from that in SL1344 in stationary phase. Low EtBr accumulation in stationary phase was not due to the upregulation of other efflux pumps but instead was due to decreased permeability of the envelope in stationary phase. Transcriptome sequencing (RNA-seq) identified changes in expression of several pathways that remodel the envelope in stationary phase, leading to lower permeability. IMPORTANCE This study shows that efflux is important for maintaining low intracellular accumulation only in actively growing cells and that envelope permeability is the predominant factor in stationary-phase cells. This conclusion means that (i) antibiotics with intracellular targets may be less effective in complex infections with nongrowing or slow-growing bacteria, where intracellular accumulation may be low; (ii) efflux inhibitors may be successful in potentiating the activity of existing antibiotics, but potentially only for bacterial infections where cells are actively growing; and (iii) the remodeling of the cell envelope prior to stationary phase could provide novel drug targets.

Interchangeability of periplasmic adaptor proteins AcrA and AcrE in forming functional efflux pumps with AcrD in Salmonella enterica serovar Typhimurium.

BACKGROUND: Resistance-nodulation-division (RND) efflux pumps are important mediators of antibiotic resistance. RND pumps, including the principal multidrug efflux pump AcrAB-TolC in Salmonella, are tripartite systems with an inner membrane RND transporter, a periplasmic adaptor protein (PAP) and an outer membrane factor (OMF). We previously identified the residues required for binding between the PAP AcrA and the RND transporter AcrB and have demonstrated that PAPs can function with non-cognate transporters. AcrE and AcrD/AcrF are homologues of AcrA and AcrB, respectively. Here, we show that AcrE can interact with AcrD, which does not possess its own PAP, and establish that the residues previously identified in AcrB binding are also involved in AcrD binding. METHODS: The acrD and acrE genes were expressed in a strain lacking acrABDEF (Δ3RND). PAP residues involved in promiscuous interactions were predicted based on previously defined PAP-RND interactions and corresponding mutations generated in acrA and acrE. Antimicrobial susceptibility of the mutant strains was determined. RESULTS: Co-expression of acrD and acrE significantly decreased susceptibility of the Δ3RND strain to AcrD substrates, showing that AcrE can form a functional complex with AcrD. The substrate profile of Salmonella AcrD differed from that of Escherichia coli AcrD. Mutations targeting the previously defined PAP-RND interaction sites in AcrA/AcrE impaired efflux of AcrD-dependent substrates. CONCLUSIONS: These data indicate that AcrE forms an efflux-competent pump with AcrD and thus presents an alternative PAP for this pump. Mutagenesis of the conserved RND binding sites validates the interchangeability of AcrA and AcrE, highlighting them as potential drug targets for efflux inhibition.