AcrAB-TolC Multidrug Efflux Pump Research Articles

Mg2+-dependent mechanism of environmental versatility in a multidrug efflux pump.

Tripartite resistance nodulation and cell division multidrug efflux pumps span the periplasm and are a major driver of multidrug resistance among Gram-negative bacteria. The periplasm provides a distinct environment between the inner and outer membranes of Gram-negative bacteria. Cations, such as Mg2+, become concentrated within the periplasm and, in contrast to the cytoplasm, its pH is sensitive to conditions outside the cell. Here, we reveal an interplay between Mg2+ and pH in modulating the dynamics of the periplasmic adaptor protein, AcrA, and its function within the prototypical AcrAB-TolC multidrug efflux pump from Escherichia coli. In the absence of Mg2+, AcrA becomes increasingly plastic within acidic conditions, but when Mg2+ is bound this is ameliorated, resulting in domain specific organisation in neutral to weakly acidic regimes. We establish a unique histidine residue directs these structural dynamics and is essential for sustaining pump efflux activity across acidic, neutral, and alkaline conditions. Overall, we propose Mg2+ conserves the structural mobility of AcrA to ensure optimal AcrAB-TolC function within rapid changing environments commonly faced by the periplasm during bacterial infection and colonization. This work highlights that Mg2+ is an important mechanistic component in this pump class and possibly across other periplasmic lipoproteins.

Identification of Potential Therapeutics of Mentha Essential Oil Content as Antibacterial MDR Agents against AcrAB-TolC Multidrug Efflux Pump from Escherichia coli: An In Silico Exploration.

Multidrug-resistant bacterial pathogens, such as E. coli, represent a major human health threat. Due to the critical need to overcome this dilemma, since the drug efflux pump has a vital function in the evolution of antimicrobial resistance in bacteria, we have investigated the potential of Mentha essential oil major constituents (1-19) as antimicrobial agents via their ability to inhibit pathogenic DNA gyrase and, in addition, their potential inhibition of the E. coli AcrB-TolC efflux pump, a potential target to inhibit MDR pathogens. The ligand docking approach was conducted to analyze the binding interactions of Mentha EO constituents with the target receptors. The obtained results proved their antimicrobial activity through the inhibition of DNA gyrase (1kzn) with binding affinity ΔG values between -4.94 and -6.49 kcal/mol. Moreover, Mentha EO constituents demonstrated their activity against MDR E. coli by their ability to inhibit AcrB-TolC (4dx7) with ΔG values ranging between -4.69 and -6.39 kcal/mol. The antimicrobial and MDR activity of Mentha EOs was supported via hydrogen bonding and hydrophobic interactions with the key amino acid residues at the binding site of the active pocket of the targeted receptors.

High prevalence of intermediate resistance to ciprofloxacin in Salmonella enterica isolated from a Brazilian poultry production chain, located in Minas Gerais state

We characterized the distribution and diversity of antimicrobial-resistance Salmonella enterica isolated from a poultry production chain in Minas Gerais, Brazil, with special attention to ciprofloxacin and multidrug resistance (MDR). S. enterica (n = 96) of different serotypes and from different processing steps were subjected to broth dilution assay to estimate the minimum inhibitory concentration (MIC) for 12 antibiotics (8 classes) and screened using PCR for the presence of 17 antimicrobial-resistance genes. Isolates presented mainly resistance to ampicillin (11/96), and most presented intermediate resistance to ciprofloxacin (92/96). Roughly one-third (33/96) were resistant to streptomycin based on our interpretive criteria. Most strains resistant to streptomycin and ciprofloxacin were PCR-positive for aphA (51/96) and qnrB (94/96), respectively. Ciprofloxacin resistance was further investigated through high-resolution melting qPCR (HRM-qPCR) and sequencing of quinolone resistance-determining region (QRDR: gyrA, gyrB, parC, and parE). Minor differences were identified in melting temperatures (Tm), and a Thr57Sr mutation was observed in parC. MDR isolates harboring acrA and capable of expressing the AcrAB-TolC multidrug efflux pump were resistant to ethidium bromide at 0.4 mg/mL. The intermediate resistance to ciprofloxacin may be associated with qnrB, and the potential role of Thr57Ser mutation warrants further investigation. The high prevalence of antibiotic related genes and its association with the observed intermediary resistance to ciprofloxacin indicates the widespread of this hazard in the studied poultry production chain.

Insights into the effects of pulsed antimicrobials on the chicken resistome and microbiota from fecal metagenomes

Antimicrobial resistance has become a global problem that poses great threats to human health. Antimicrobials are widely used in broiler chicken production and consequently affect their gut microbiota and resistome. To better understand how continuous antimicrobial use in farm animals alters their microbial ecology, we used a metagenomic approach to investigate the effects of pulsed antimicrobial administration on the bacterial community, antibiotic resistance genes (ARGs) and ARG bacterial hosts in the feces of broiler chickens. Chickens received three 5-day courses of individual or combined antimicrobials, including amoxicillin, chlortetracycline and florfenicol. The florfenicol administration significantly increased the abundance of mcr-1 gene accompanied by floR gene, while amoxicillin significantly increased the abundance of genes encoding the AcrAB-tolC multidrug efflux pump (marA, soxS, sdiA, rob, evgS and phoP). These three antimicrobials all led to an increase in Proteobacteria. The increase in ARG host, Escherichia, was mainly attributed to the β-lactam, chloramphenicol and tetracycline resistance genes harbored by Escherichia under the pulsed antimicrobial treatments. These results indicated that pulsed antimicrobial administration with amoxicillin, chlortetracycline, florfenicol or their combinations significantly increased the abundance of Proteobacteria and enhanced the abundance of particular ARGs. The ARG types were occupied by the multidrug resistance genes and had significant correlations with the total ARGs in the antimicrobial-treated groups. The results of this study provide comprehensive insight into pulsed antimicrobial-mediated alteration of chicken fecal microbiota and resistome.

Drug Efflux Pump Inhibitors: A Promising Approach to Counter Multidrug Resistance in Gram-Negative Pathogens by Targeting AcrB Protein from AcrAB-TolC Multidrug Efflux Pump from Escherichia coli

Simple SummaryMultidrug-resistant bacterial infections, especially that caused by Gram-negative bacteria, have posed serious health issues worldwide. Bacteria have different mechanisms that can confer multidrug resistance to bacteria, among these mechanisms are drug efflux pumps that play the main role in conferring multidrug resistance by recognizing then expelling a wide range of compounds, especially antibiotics, and reducing their concentration to sub-toxic levels. Small molecule inhibitors that target drug efflux pumps especially the AcrAB-TolC multidrug efflux pump, from E. coli, appear as a new promising and attractive approach that could increase the required accumulation of antimicrobials to eliminate bacteria as well as leading to reverse antibiotic resistance and prevent the development of resistance in clinically relevant bacterial pathogens and enhances the activity of antibiotics or prolong their effectiveness.Infections caused by multidrug resistance (MDR) of Gram-negative bacteria have become one of the most severe public health problems worldwide. The main mechanism that confers MDR to bacteria is drug efflux pumps, as they expel a wide range of compounds, especially antibiotics. Among the different types of drug efflux pumps, the resistance nodulation division (RND) superfamily confers MDR to various Gram-negative bacteria species. The AcrAB-TolC multidrug efflux pump, from E. coli, a member of RND, is the best-characterized example and an excellent model for understanding MDR because of an abundance of functional and structural data. Small molecule inhibitors that target the AcrAB-TolC drug efflux pump represent a new solution to reversing MDR in Gram-negative bacteria and restoring the efficacy of various used drugs that are clinically relevant to these pathogens, especially in the high shortage of drugs for multidrug-resistant Gram-negative bacteria. This review will investigate solutions of MDR in Gram-negative bacteria by studying the inhibition of the AcrAB-TolC multidrug efflux pump.

Improving E. coli organic solvent tolerance by 1,4-dihydroxy-2-naphthoic acid.

Improving the organic solvent tolerance of bacteria is beneficial for the bioproduction of various valuable chemicals. In this study, we found that 1,4-dihydroxy-2-naphthoic acid (DHNA), known as a prebiotic, increased organic solvent tolerance in Escherichia coli. The AcrAB-TolC multidrug efflux pump contributes to the intrinsic organic solvent tolerance of E. coli. The addition of DHNA increased this pump's expression level. Transcriptional activators MarA, SoxS, and Rob proteins are known to control the expression of marA/soxS/rob regulon genes, including acrAB and tolC. Evaluation of the organic solvent tolerances of ΔmarA mutant, ΔsoxS mutant, and Δrob mutant showed that ΔmarA mutant and ΔsoxS mutant did not improve organic solvent tolerance by the addition of DHNA. In addition, DHNA increased the promoter activities of both marA and soxS. These results indicated that DHNA induces the AcrAB-TolC pump through both the marRAB system and the soxRS system.

Copper-based fungicide copper hydroxide accelerates the evolution of antibiotic resistance via gene mutations in Escherichia coli

The extensive use of copper-based fungicides in orchards, especially in vineyards, leads to the accumulation of copper, which has caused growing concern. However, data on the acquisition of antibiotic resistance in opportunistic pathogens under copper-based fungicides are scarce. In this study, we investigated the potential development of antibiotic resistance in Escherichia coli K12 under selective copper hydroxide pressure. The results indicated that copper hydroxide at concentrations of 100 mg/L and 200 mg/L evolved resistance against chloramphenicol and tolerance against tetracycline to 4–8 and 2.00–2.67 times than the initial minimal inhibitory concentrations (MICs), respectively. Whole-genome sequencing analysis showed that the obtained resistant strains carried gene mutations including AcrAB-TolC multidrug efflux pump (acrB and marR), outer membrane porin (evZ), and another indirect pathways. Furthermore, the expression of multidrug efflux pump genes and oxidative stress-related genes were significantly upregulated, whereas outer membrane porin genes were downregulated. Thus, our results could well explain the emergence of antibiotic resistance and resistance mechanisms selected by copper-based fungicide, and provide a basis for the management of copper-based fungicide in agriculture to avoid the spread of antibiotic resistance.

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

BackgroundResistance-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. MethodsThe 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.ResultsCo-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.ConclusionsThese 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.

Molecular Insights into an Antibiotic Enhancer Action of New Morpholine-Containing 5-Arylideneimidazolones in the Fight against MDR Bacteria.

In the search for an effective strategy to overcome antimicrobial resistance, a series of new morpholine-containing 5-arylideneimidazolones differing within either the amine moiety or at position five of imidazolones was explored as potential antibiotic adjuvants against Gram-positive and Gram-negative bacteria. Compounds (7–23) were tested for oxacillin adjuvant properties in the Methicillin-susceptible S. aureus (MSSA) strain ATCC 25923 and Methicillin-resistant S. aureus MRSA 19449. Compounds 14–16 were tested additionally in combination with various antibiotics. Molecular modelling was performed to assess potential mechanism of action. Microdilution and real-time efflux (RTE) assays were carried out in strains of K. aerogenes to determine the potential of compounds 7–23 to block the multidrug efflux pump AcrAB-TolC. Drug-like properties were determined experimentally. Two compounds (10, 15) containing non-condensed aromatic rings, significantly reduced oxacillin MICs in MRSA 19449, while 15 additionally enhanced the effectiveness of ampicillin. Results of molecular modelling confirmed the interaction with the allosteric site of PBP2a as a probable MDR-reversing mechanism. In RTE, the compounds inhibited AcrAB-TolC even to 90% (19). The 4-phenylbenzylidene derivative (15) demonstrated significant MDR-reversal “dual action” for β-lactam antibiotics in MRSA and inhibited AcrAB-TolC in K. aerogenes. 15 displayed also satisfied solubility and safety towards CYP3A4 in vitro.

Molecular characterization of carbapenem-resistant and virulent plasmids in Klebsiella pneumoniae from patients with bloodstream infections in China

ABSTRACT Bloodstream infections (BSIs) caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) are potentially life-threatening and an urgent threat to public health. The present study aims to clarify the characteristics of carbapenemase-encoding and virulent plasmids, and their interactions with the host bacterium. A total of 425 Kp isolates were collected from the blood of BSI patients from nine Chinese hospitals, between 2005 and 2019. Integrated epidemiological and genomic data showed that ST11 and ST307 Kp isolates were associated with nosocomial outbreak and transmission. Comparative analysis of 147 Kp genomes and 39 completely assembled chromosomes revealed extensive interruption of acrR by ISKpn26 in all Kp carbapenemase-2 (KPC-2)-producing ST11 Kp isolates, leading to activation of the AcrAB-Tolc multidrug efflux pump and a subsequent reduction in susceptibility to the last-resort antibiotic tigecycline and six other antibiotics. We described 29 KPC-2 plasmids showing diverse structures, two virulence plasmids in two KPC-2-producing Kp, and two novel multidrug-resistant (MDR)-virulent plasmids. This study revealed a multifactorial impact of KPC-2 plasmid on Kp, which may be associated with nosocomial dissemination of MDR isolates.

Novel mechanisms of TolC-independent decreased bile-salt susceptibility in Escherichia coli.

Bile salts, including sodium deoxycholate (DOC), are secreted into the intestine to aid fat digestion and contribute to antimicrobial protection. Gram-negative pathogens such as Escherichia coli, however, are highly resistant to DOC, using multiple mechanisms of which the multidrug efflux pump AcrAB-TolC is the dominant one. Given that TolC-mediated efflux masks the interaction of DOC with potential targets, we sought to identify those targets by identifying genes whose mutations cause an increase in the MIC to DOC relative to the ∆tolC parental strain, that lacks TolC-associated functional efflux pumps. Using a mutant screen, we isolated twenty independent spontaneous mutants that had a higher MICDOC than the E. coli parental ∆tolC strain. Whole genome sequencing of these mutants mapped most mutations to the ptsI or cyaA gene. Analysis of knock-out mutants and complementation showed that elimination of PtsI, a component of the carbohydrate phosphotransferase system, or one of the two key proteins involved in cAMP synthesis and signaling, adenylate cyclase (CyaA) or cAMP receptor protein (Crp) causes low-level increased resistance of a ∆tolC E. coli strain to DOC.

Loss of the Periplasmic Chaperone Skp and Mutations in the Efflux Pump AcrAB-TolC Play a Role in Acquired Resistance to Antimicrobial Peptides in Salmonella typhimurium.

Bacterial resistance to antibiotics is a major concern worldwide, leading to an extensive search for alternative drugs. Promising candidates are antimicrobial peptides (AMPs), innate immunity molecules, shown to be highly efficient against multidrug resistant bacteria. Therefore, it is essential to study bacterial resistance mechanisms against them. For that purpose, we used experimental evolution, and isolated a Salmonella enterica serovar typhimurium-resistant line to the AMP 4DK5L7. This AMP displayed promising features including widespread activity against Gram-negative bacteria and protection from proteolytic degradation. However, the resistance that evolved in the isolated strain was particularly high. Whole genome sequencing revealed that five spontaneous mutations had evolved. Of these, three are novel in the context of acquired AMP resistance. Two mutations are related to the AcrAB-TolC multidrug efflux pump. One occurred in AcrB, the substrate-binding domain of the system, and the second in RamR, a transcriptional regulator of the system. Together, the mutations increased the minimal inhibitory concentration (MIC) by twofold toward this AMP. Moreover, the mutation in AcrB induced hypersusceptibility toward ampicillin and colistin. The last mutation occurred in Skp, a periplasmic chaperone that participates in the biogenesis of outer membrane proteins (OMPs). This mutation increased the MIC by twofold to 4DK5L7 and by fourfold to another AMP, seg5D. Proteomic analysis revealed that the mutation abolished Skp expression, reduced OMP abundance, and increased DegP levels. DegP, a protease that was reported to have an additional chaperone activity, escorts OMPs through the periplasm along with Skp, but is also associated with AMP resistance. In conclusion, our data demonstrate that both loss of Skp and manipulation of the AcrAB-TolC system are alternative strategies of AMP acquired resistance in Salmonella typhimurium and might represent a common mechanism in other Gram-negative bacteria.

In situ structure and assembly of the multidrug efflux pump AcrAB-TolC

Multidrug efflux pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug efflux pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug efflux pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled efflux pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with efflux pump inhibitors.

Role of AcrAB-TolC multidrug efflux pump in drug-resistance acquisition by plasmid transfer.

Drug-resistance dissemination by horizontal gene transfer remains poorly understood at the cellular scale. Using live-cell microscopy, we reveal the dynamics of resistance acquisition by transfer of the Escherichia coli fertility factor-conjugation plasmid encoding the tetracycline-efflux pump TetA. The entry of the single-stranded DNA plasmid into the recipient cell is rapidly followed by complementary-strand synthesis, plasmid-gene expression, and production of TetA. In the presence of translation-inhibiting antibiotics, resistance acquisition depends on the AcrAB-TolC multidrug efflux pump, because it reduces tetracycline concentrations in the cell. Protein synthesis can thus persist and TetA expression can be initiated immediately after plasmid acquisition. AcrAB-TolC efflux activity can also preserve resistance acquisition by plasmid transfer in the presence of antibiotics with other modes of action.

Genetic Variability of the AcrAB-TolC Multidrug Efflux Pump Underlies SkQ1 Resistance in Gram-Negative Bacteria.

SkQ1, a novel antibiotic targeting bacterial bioenergetics, is highly effective against both gram-positive and gram-negative bacteria. However, some gram-negative bacteria, such as Escherichia coli and Klebsiella pneumoniae, are highly resistant to it. In different gram-negative bacteria, this resistance is associated with the identity of their AcrB transporter protein sequence with the sequence of the AcrB protein from E. coli. SkQ1 is expelled from E. coli cells by the AcrAB-TolC multidrug efflux pump. In this study, we demonstrate that SkQ1 resistance in E. coli, in contrast to chloramphenicol resistance, does not depend on the presence of the multidrug efflux pump accessory protein AcrZ.

Fitness cost constrains the spectrum of marR mutations in ciprofloxacin-resistant Escherichia coli.

ObjectivesTo determine whether the spectrum of mutations in marR in ciprofloxacin-resistant clinical isolates of Escherichia coli shows evidence of selection bias, either to reduce fitness costs, or to increase drug resistance. MarR is a repressor protein that regulates, via MarA, expression of the Mar regulon, including the multidrug efflux pump AcrAB-TolC.MethodsIsogenic strains carrying 36 different marR alleles identified in resistant clinical isolates, or selected for resistance in vitro, were constructed. Drug susceptibility and relative fitness in growth competition assays were measured for all strains. The expression level of marA, and of various efflux pump components, as a function of specific mutations in marR, was measured by qPCR.ResultsThe spectrum of genetic alterations in marR in clinical isolates is strongly biased against inactivating mutations. In general, the alleles found in clinical isolates conferred a lower level of resistance and imposed a lower growth fitness cost than mutations selected in vitro. The level of expression of MarA correlated well with the MIC of ciprofloxacin. This supports the functional connection between mutations in marR and reduced susceptibility to ciprofloxacin.ConclusionsMutations in marR selected in ciprofloxacin-resistant clinical isolates are strongly biased against inactivating mutations. Selection favours mutant alleles that have the lowest fitness costs, even though these cause only modest reductions in drug susceptibility. This suggests that selection for high relative fitness is more important than selection for increased resistance in determining which alleles of marR will be selected in resistant clinical isolates.

Biased partitioning of the multidrug efflux pump AcrAB-TolC underlies long-lived phenotypic heterogeneity.

The molecular mechanisms underlying phenotypic variation in isogenic bacterial populations remain poorly understood. We report that AcrAB-TolC, the main multidrug efflux pump of Escherichia coli, exhibits a strong partitioning bias for old cell poles by a segregation mechanism that is mediated by ternary AcrAB-TolC complex formation. Mother cells inheriting old poles are phenotypically distinct and display increased drug efflux activity relative to daughters. Consequently, we find systematic and long-lived growth differences between mother and daughter cells in the presence of subinhibitory drug concentrations. A simple model for biased partitioning predicts a population structure of long-lived and highly heterogeneous phenotypes. This straightforward mechanism of generating sustained growth rate differences at subinhibitory antibiotic concentrations has implications for understanding the emergence of multidrug resistance in bacteria.

An allosteric transport mechanism for the AcrAB-TolC multidrug efflux pump.

Bacterial efflux pumps confer multidrug resistance by transporting diverse antibiotics from the cell. In Gram-negative bacteria, some of these pumps form multi-protein assemblies that span the cell envelope. Here, we report the near-atomic resolution cryoEM structures of the Escherichia coli AcrAB-TolC multidrug efflux pump in resting and drug transport states, revealing a quaternary structural switch that allosterically couples and synchronizes initial ligand binding with channel opening. Within the transport-activated state, the channel remains open even though the pump cycles through three distinct conformations. Collectively, our data provide a dynamic mechanism for the assembly and operation of the AcrAB-TolC pump.

Detection of potential AcrAB-TolC multidrug efflux pump inhibitor in calyces extract of Hibiscus sabdariffa

Aim: The aim of this study is to investigate occurrence of potential efflux pump inhibitor (EPI) against AcrAB-TolC efflux pump in the methanol extract of H. sabdariffa. Materials and Methods: Calyces of H. sabdariffa were purchased from the local market in April 2014, methanol extract of H. sabdariffa was subjected to agar plate diffusion against Escherichia coli TG1 and its ∆acrB-∆tolC and thin layer chromatography (TLC) bioassay. The corresponding EPI fraction was eluted by methanol. The synergistic effect of antimicrobials and EPI fraction was measured by minimum inhibitory concentration (MIC) determination for E. coli and Erwinia amylovora strains, and the ability of EPI fraction to enhance EtBr accumulation was conducted. Results: E. coli TG1 was more sensitive to the methanol extracts of H. sabdariffa than E. coli ∆acrB-∆tolC, and inhibition zone corresponding to flavones on TLC bioassay plate has been formed which might be related to the fraction of potential EPI. The MIC values revealed that EPI fraction enhanced the activity of the used antimicrobials by 4 to 8 folds in E. coli TG1 and by 4 to 10 folds in E. amylovora 1189. Addition of EPI fraction in a dose-dependent manner increased the intercellular accumulation of Ethidium Bromide (EtBr) in the wild type stains of E. coli TG1 and E. amylovora 1189. Conclusion: EPI fraction behaves like a multidrug efflux pump inhibitor and further investigation should be conducted for determination of the chemical structure of EPI fraction.

In vitro characterization and inhibition of the interaction between ciprofloxacin and berberine against multidrug-resistant Klebsiella pneumoniae.

Ciprofloxacin is a quinolone antibiotic used to treat Klebsiella pneumoniae infections in the clinic. Previous studies have demonstrated that berberine exhibits antibacterial activity and less acquired resistance related to efflux pumps. The multidrug efflux pump acrAB-tolC can be stimulated to expel as much toxic material as possible from the cells, but a detrimental effect can be produced owing to an overcrowded periplasm with excess expression products, which inhibits bacterial growth. In this study, the in vitro antibacterial activities of ciprofloxacin in combination with berberine were evaluated and compared with those of ciprofloxacin and berberine alone by evaluating the MIC, MBC and summation fractional IC against 20 clinical multidrug-resistant K. pneumoniae isolates, 1 quality control bacterium and 1 induced-resistance bacterium. Susceptibility tests showed that the MIC for the combination of berberine and ciprofloxacin was 1/2 that of the individual agents or less. Antimicrobial activities of 18.18% synergy and 77.27% additivity were found. Furthermore, synergism was verified through a time-kill assay, which suggested that the synergistic antibacterial effect of the two-drug combination may, to some extent, be related to the high expression of the acrAB-tolC and acrR multidrug efflux pumps. Indeed, the expression of these genes was increased >14-fold in the isolates affected by ciprofloxacin–berberine combination synergism.