Berberine Is a Novel Type Efflux Inhibitor Which Attenuates the MexXY-Mediated Aminoglycoside Resistance in Pseudomonas aeruginosa

The emergence and spread of multidrug-resistant P. aeruginosa infections is of great concern, as very few agents are effective against strains of this species. Methanolic extracts from the Coptidis Rhizoma (the rhizomes of Coptis japonica var. major Satake) or Phellodendri Cortex (the bark of Phellodendron chinense Schneider) markedly reduced resistance to anti-pseudomonal aminoglycosides (e.g., amikacin) in multidrug-resistant P. aeruginosa strains. Berberine, the most abundant benzylisoquinoline alkaloid in the two extracts, reduced aminoglycoside resistance of P. aeruginosa via a mechanism that required the MexXY multidrug efflux system; berberine also reduced aminoglycoside MICs in Achromobacter xylosoxidans and Burkholderia cepacia, two species that harbor intrinsic multidrug efflux systems very similar to the MexXY. Furthermore this compound inhibited MexXY-dependent antibiotic resistance of other classes including cephalosporins (cefepime), macrolides (erythromycin), and lincosamides (lincomycin) demonstrated using a pseudomonad lacking the four other major Mex pumps. Although phenylalanine-arginine beta-naphthylamide (PAβN), a well-known efflux inhibitor, antagonized aminoglycoside in a MexXY-dependent manner, a lower concentration of berberine was sufficient to reduce amikacin resistance of P. aeruginosa in the presence of PAβN. Moreover, berberine enhanced the synergistic effects of amikacin and piperacillin (and vice versa) in multidrug-resistant P. aeruginosa strains. Thus, berberine appears to be a novel type inhibitor of the MexXY-dependent aminoglycoside efflux in P. aeruginosa. As aminoglycosides are molecules of choice to treat severe infections the clinical impact is potentially important.

The emergence and spread of multidrug-resistant P. aeruginosa infections is of great concern, as very few agents are effective against strains of this species. Methanolic extracts from the Coptidis Rhizoma (the rhizomes of Coptis japonica var. major Satake) or Phellodendri Cortex (the bark of Phellodendron chinense Schneider) markedly reduced resistance to anti-pseudomonal aminoglycosides (e.g., amikacin) in multidrug-resistant P. aeruginosa strains. Berberine, the most abundant benzylisoquinoline alkaloid in the two extracts, reduced aminoglycoside resistance of P. aeruginosa via a mechanism that required the MexXY multidrug efflux system; berberine also reduced aminoglycoside MICs in Achromobacter xylosoxidans and Burkholderia cepacia, two species that harbor intrinsic multidrug efflux systems very similar to the MexXY. Furthermore this compound inhibited MexXY-dependent antibiotic resistance of other classes including cephalosporins (cefepime), macrolides (erythromycin), and lincosamides (lincomycin) demonstrated using a pseudomonad lacking the four other major Mex pumps. Although phenylalanine-arginine beta-naphthylamide (PAβN), a well-known efflux inhibitor, antagonized aminoglycoside in a MexXY-dependent manner, a lower concentration of berberine was sufficient to reduce amikacin resistance of P. aeruginosa in the presence of PAβN. Moreover, berberine enhanced the synergistic effects of amikacin and piperacillin (and vice versa) in multidrug-resistant P. aeruginosa strains.
Thus, berberine appears to be a novel type inhibitor of the MexXY-dependent aminoglycoside efflux in P. aeruginosa. As aminoglycosides are molecules of choice to treat severe infections the clinical impact is potentially important.
Keywords: Pseudomonas aeruginosa, efflux, mexXY, aminoglycoside resistance, berberine INTRODUCTION Pseudomonas aeruginosa is a metabolically versatile bacterium that can cause a wide range of severe opportunistic infections in patients with serious underlying medical conditions (Gellatly and Hancock, 2013). Infections caused by P. aeruginosa often are hard to treat; inappropriate chemotherapy readily selects multidrugresistant P. aeruginosa against which very few agents are effective (Poole, 2011;Morita et al., 2014). This so-called "antibiotic resistance crisis" has been compounded by the lag in antibiotic discovery and development programs in recent years, and is jeopardizing the essential role played by antibiotics in current medical practices (Rossolini et al., 2014). Moreover, P. aeruginosa possesses an intrinsic resistance to many antimicrobials because of the bacterium's outer-membrane barrier, the presence of multidrug efflux transporters, and endogenous antimicrobial inactivation (Poole, 2011;Morita et al., 2015b). Although, antipseudomonal agents (e.g., carbapenems) have been discovered and developed, P. aeruginosa readily acquires resistance to individual agents via chromosomal mutations and lateral gene transfer (Poole, 2011;Morita et al., 2015b).
The phenyl-arginine-β-naphthylamide (PAβN, MC-207,110) is well-known as a non-specific inhibitor against the RND-type multidrug efflux pumps in P. aeruginosa . However it antagonized the anti-pseudomonas activity of aminoglycosides (amikacin and netilmicin) in a MexXYdependent manner even though it inhibited MexXY-dependent fluoroquinolone (levofloxacin) resistance of P. aeruginosa (Mao et al., 2001). MP 601384, the only inhibitor of the MexXYmediated aminoglycoside resistance has been reported to date (Jassem et al., 2011) although its chemical structure was not revealed.
In the current study we identify berberine, a natural isoquinoline alkaloid produced by a variety of plant species (Tillhon et al., 2012), potentiates aminoglycoside activity against P. aeruginosa including multidrug resistant isolates through screening from traditional Japanese herbal preparations, frequently prescribed as Kampo prescriptions (Watanabe et al., 2011). This compound has been reported as a potent efflux inhibitor against Gram-positive bacteria such as Staphylococcus aureus (Tillhon et al., 2012) but it has a weak activity against Gram-negative bacteria such as P. aeruginosa in part due to a substrate of multidrug efflux pumps (Tegos et al., 2002).

Bacterial Strains, Plasmids, and Growth Conditions
The bacterial strains and plasmids used in this study are listed in Table 1. Of note various P. aeruginosa strains in which contribution of the MexXY efflux system to aminoglycoside resistance were assessed before (Morita et al., 2012a) are used in the main (PAO1; a reference strain, PAGU 1498 and PAGU 1606; multidrug resistant clinical isolates, PAGU 1569; panaminoglycoside clinical isolates) ( Table 1). Among the four strains PAGU 1498 is the agrZ-type MexXY-overproducing mutant (Morita et al., 2012a).
Bacterial cells were grown (unless otherwise indicated) in Luria (L) broth and on L agar (1.5%) under aerobic conditions at 37 • C, as previously described, with antibiotics as specified (Morita et al., 2015b). Bacterial growth was quantified by measuring the optical density at 600 nm on an Ultrospec 2100 Pro Spectrophotometer (GE Healthcare Corp., Tokyo, Japan), unless otherwise indicated. Cells harboring the plasmid pEX18Tc (Hoang et al., 1998) or derivatives thereof were maintained on medium supplemented with 2.5-10 µg/ml tetracycline for E. coli or and selected on medium supplemented with 20-150 µg/ml tetracycline for P. aeruginosa. Cells harboring the plasmid pFLP2 (Hoang et al., 1998) were maintained and selected on medium supplemented with 100 µg/ml ampicillin for E. coli or 50-200 µg/ml carbenicillin for P. aeruginosa.

Construction of P. aeruginosa Mutants
In-frame deletion mutants and/or aacC1-gfp insertion mutants of mexXY, mexZ, and mexVW from P. aeruginosa PAO1 derivatives were constructed using the previously described sacB-based strategy (Morita et al., 2006(Morita et al., , 2010(Morita et al., , 2015a. The plasmids and resulting P. aeruginosa mutants are listed in Table 1, while the primer pairs are listed in Table 2. To introduce gene deletions into strains of P. aeruginosa, deletion constructs were first prepared in plasmid pEX18Tc by cloning PCR-amplified 0.75kb DNA fragments corresponding to the regions upstream and downstream of the gene sequences to be deleted. The selection concentrations of tetracycline and gentamicin for the recombination events were adjusted to reflect the endogenous tetracycline and gentamicin MICs of the P. aeruginosa strains. These constructs were confirmed by colony PCR.

Preparation of Crude Drug Extracts
A total of 96 crude drugs (listed in Table S1) that are used in Kampo prescriptions in Japan (Tanabe et al., 2014) were prepared as follows. Ten grams of each crude drug was placed into 100 ml of methanol and extracted for 1 day at room temperature, and then the solution was filtered. The extraction process was performed three times. Each filtrate was mixed and concentrated in vacuo at 40 • C by using a rotary evaporator N-1000 (EYELA, Tokyo, Japan) equipped with a coolant system CCA-1110 (EYELA, Tokyo, Japan). Each final extract was dissolved in dimethyl sulfoxide (DMSO) and adjusted to the concentration of 100 mg/ml. Each of the crude drugs was resuspended at 1 mg/ml in DMSO, and evaluated on the condition of the broth microdilution method described above at the Section of Preparation of Crude Drug Extracts for the ability to restore effectiveness of either 16 µg/ml imipenem, 4 µg/ml ciprofloxacin, or 32 µg/ml amikacin against highly multidrug-resistant P. aeruginosa PAGU 1606.

High Performance Liquid Chromatographic Method for Quantification of Berberine and Coptisine
The stock solutions of berberine and coptisine were both prepared in methanol. The extracts also were dissolved in methanol to a concentration of 1 mg/ml. In order to construct calibration curves, a series of standard solution of each compound was prepared by appropriate dilution of the stock solutions. Calibration curves were constructed by plotting the peak area ratio vs. the concentration.

Molecular Biology Techniques
Plasmid DNA isolation from E. coli, DNA purification, measurement of DNA concentration, DNA digestion with restriction enzymes, DNA dephosphorylation, DNA ligation, isolation of chromosomal DNA from P. aeruginosa, PCR conditions, nucleotide sequencing, competent cell preparation from E. coli, transformation of E. coli, and transfer of plasmids into P. aeruginosa via conjugation were performed as described previously (Morita et al., 2015b), unless otherwise indicated. DNA sequences and amino acid sequences were analyzed using the Pseudomonas Genome Database (Winsor et al., 2011), Basic Local Alignment Search Tool (BLAST), and the software DNASIS Pro (Ver. 2.1; Hitachi, Japan).

Isolation of a Novel Inhibitor of the MexXY Multidrug Efflux System from the Rhizomes of Coptis Japonica and the Bark of Phellodendron Amurense
Extracts from the Coptidis Rhizoma (the rhizomes of Coptis japonica var. major Satake) or Phellodendri Cortex (the bark of Phellodendron chinense Schneider) restored the effectiveness of 32 µg/ml amikacin against the highly multidrug-resistant P. aeruginosa PAGU 1606. None of the tested extracts restored susceptibility of the strain to 16 µg/ml imipenem or 4 µg/ml ciprofloxacin (data not shown). The extracts of both Coptis rhizome and Phellodendron bark reduced MICs of amikacin and gentamicin (but not those of imipenem or ciprofloxacin) by eight-fold or more in the two multidrug resistant P. aeruginosa strains (PAGU 1606 and PAGU 1498) ( Table 3).

Berberine Is a Novel Inhibitor of Aminoglycoside Resistance Dependent on the MexXY-Efflux System in P. aeruginosa
Berberine is reported the most abundant benzylisoquinoline alkaloid in the two active extracts (Tillhon et al., 2012). Therefore, we next tested the activity of berberine along with coptisine, a similar benzylisoquinoline alkaloid. Berberine constituted 34.4% of the extract from the rhizomes of C. japonica and 28.3% of extract from the bark of Phellodendron amurense ( Figure S1). Coptisine was the second-most abundant benzylisoquinoline alkaloid from the Coptis extract, constituting 4.1% of the extract from the rhizomes of C. japonica ( Figure S1). Although berberine was ineffective as a solo antibacterial (with an MIC >512 µg/ml MIC against P. aeruginosa; data not shown), berberine inhibited aminoglycoside resistance by two-to >eight-fold against four P. aeruginosa strains ( Table 4). Such a reduction was not observed in isogenic mutants deficient in the RND-type multidrug efflux system MexXY, which is considered one of the major aminoglycoside resistance determinants in P. aeruginosa (Morita et al., 2012b) (Table 4). Coptisine showed activity similar  to that of berberine (data not shown). These results indicate that the inhibitory activity of the two extracts reflected, at least in part, the activity of berberine and/or coptisine. Of note berberine also inhibited aminoglycoside resistance of A. xylosoxidans PAGU 0002 T and B. cepacia PAGU 0013 T but not that of A. baumannii PAGU 1567 T ( To test the effect of berberine on non-aminoglycoside susceptibility, we, first of all, evaluated MICs against the wild-type strain (PAO1) and then, in order to remove any effects of the RND pumps, evaluated MICs in a multidrug-sensitive P. aeruginosa strain mutated in the loci encoding four RND multidrug efflux systems (mexXY, mexAB, mexCD-oprJ, and mexEF-oprN) (Morita et al., 2001b;Li et al., 2003) (Table 6). Like the parent strain, the quadruple efflux mutant (PAGU g 0850) also exhibited an eight-fold decrease in MIC of erythromycin in the presence of berberine ( Table 6). However mutation of one of the remaining RND multidrug efflux systems (mexVW) attenuated the effect of berberine, with the quintuple mutant (PAGU g 1933) exhibiting only a two-fold decrease in erythromycin MIC upon exposure to berberine ( Table 6). Similar effects were seen for lincomycin resistance ( Table 6). These results suggest that berberine attenuation of resistance to the macrolides and lincomycin depends on the MexVW multidrug efflux system. The molecular mechanism underlying the berberine effects on the MexVW system will be the subject of further research. We sought to better understand the molecular mechanism of berberine attenuation of the MexXY-mediated efflux. Therefore, adjuvant effects of berberine on resistance to various antimicrobial substrates of the MexXY efflux pump were compared in two RND mutant strains differing only by the absence or presence of mexXY (PAGU g 1929 vs PAGU g 1933, respectively; Table 6). Specifically, the mexXY genes were overexpressed in PAGU g 1933 due to deletion of the mexZ repressor gene. The effects of mexXY overexpression were being determined in the absence of other major RND multidrug efflux systems, since the two strains shared deletions in the mexAB, mexCD-oprJ, mexEF-oprN, and mexVW loci. As seen in the earlier experiments (e.g., Table 6), resistance to erythromycin, lincomycin, cefpirome, and aminoglycosides was attenuated eight-fold in the presence of berberine, and these effects were observed only in the presence of the MexXY system ( Table 6). While resistance to ciprofloxacin and tetracycline also was attenuated eight-fold in the presence of berberine, a partial berberine effect (four-fold decrease in MIC) was observed for ciprofloxacin and tetracycline even in the absence of mexXY overexpression (compare PAGU g 1929 and PAGU g 1933; Table 6). It might suggest that berberine possibly has another target sites other than 5 RND pumps which are involved in resistance to ciprofloxacin and tetracycline. In control experiments (data not shown), we confirmed that berberine did not display antimicrobial activity (at concentrations up to 512 µg/ml) in the PAGU g 1929 strain (that is, despite deletion of mexAB, mexCD-oprJ, mexEF-oprN, and mexVW). We additionally confirmed that the attenuation activity of berberine in the PAGU g 1929 background was still concentrationdependent; the aminoglycoside adjuvant activity of berberine in this strain was not detected at a berberine concentration of 8 µg/ml (data not shown).

Comparison of Berberine with the Known Efflux Inhibitor Phenylalanine-Arginine β-Naphthylamide (PAβN)
We compared the effects of berberine with those of the previously reported efflux inhibitor PAβN. Consistent with earlier reports (Mao et al., 2001), PAβN did not reduce MexXY-dependent gentamicin resistance (as seen in PAGU g 1929), but PAβN did inhibit ciprofloxacin resistance in the quadruple efflux mutant (mexAB, mexCD-oprJ, mexEF-oprN, and mexVW; PAGU g 1929) ( Table 7). This distinction suggests that berberine inhibits the MexXY system via a mechanism distinct from that of PAβN. Given that berberine is known to be substrates of many multidrug efflux pumps, including RND-type multidrug efflux pumps (Tegos et al., 2002), PAβN is expected to facilitate accumulation of berberine in P. aeruginosa cells. Indeed, exposure to PAβN provided inhibition of MexXY-dependent aminoglycoside resistance at a lower concentration of berberine, as shown for the multidrug-resistant P. aeruginosa strain PAGU 1606 (Table S2).

Berberine Synergistically Inhibits
MexXY-Mediated Aminoglycoside Resistance in P. aeruginosa FIC indices were determined in multiple strains for the combination of gentamicin and berberine ( Table 8). The combination exhibited synergy only in MexXY-positive strains (PAGU g 1927 andPAGU g 1929). This result suggests that berberine and gentamicin act synergistically on the MexXY system. Puzzlingly, berberine MICs in the presence of gentamicin are significantly higher in the MexXY-deficient P. aeruginosa mutant cells than in the MexXY-overproducing P. aeruginosa mutant cells (Table 8). This observation implies that berberine accumulates more effectively in the presence of the MexXY system, although classically the pump facilitates the efflux of substrates.
Berberine Promotes Combined Effects of Amikacin with Piperacillin (and Vice Versa) in Multidrug-Resistant P. aeruginosa Strains Aminoglycoside uptake is known to be facilitated by inhibitors of bacterial cell wall synthesis such as β-lactams (Taber et al., 1987). Anti-pseudomonal β-lactams and polymyxins thus are expected to promote the adjuvant effects of berberine observed above. We tested several such agents (data not shown) and found that piperacillin, an anti-pseudomonal β-lactam, was among the most potent. The effects of this cell wall inhibitor were seen in all of the tested multidrug-resistant P. aeruginosa strains (Table 9).
Notably, exposure to the combination of piperacillin (32 µg/ml) and berberine (512 µg/ml) reduced the amikacin MIC of PAGU 1606 to a level below the amikacin breakpoint of 32 µg/ml ( Table 9). In contrast, exposure to piperacillin or berberine alone did not have as large an effect in reducing amikacin resistance. We additionally noted a reduction in the MIC of piperacillin in PAGU 1606 when grown in the presence of both of berberine (512 µg/ml) and sub-breakpoint amikacin (16 µg/ml), with the piperacillin MIC reduced to a level similar to that seen when grown in the presence of 128 µg/ml of amikacin alone (Table 9). Indeed, the FIC index of amikacin in combination with piperacillin of PAGU 1606 was calculated as follows: amikacin MIC in the presence of piperacillin/amikacin MIC) + (piperacillin MIC in the presence of amikacin/piperacillin MIC) = 64/256+16/256<0.5. It suggested synergy of these two compounds in PAGU 1606 (Table 9).

DISCUSSION
The MexXY multidrug efflux system is a significant determinant of resistance to aminoglycosides in P. aeruginosa, although aminoglycosides are not standard RND pump substrates (Morita et al., 2012b). The MexXY pump typically is encoded by a two-gene operon and comprises a periplasmic membrane fusion protein (MexX) and an inner-membrane (IM) drug/H + antiporter (MexY; the RND component); the pump is functional only when combined with an outer-membrane (OM) channel encoded by a separate locus (e.g., OprM, which is encoded by the third gene of another multidrug efflux operon, mexAB-oprM Morita et al., 2012b). In some P. aeruginosa strains, such as the taxonomic outlier strain PA7, the mexXY genes are adjacent to a third gene (oprA in PA7) encoding an outer membrane channel; the resulting three-gene operon is similar to axyXY-oprZ of A. xylosoxidans and amrAB-oprA of various Burkholderia species (e.g., B. pseudomallei and B. cepacia complexes) (Morita et al., 2012a,b). In the PA7-related strains, MexXY seem to cooperate with both OprM and OprA, as either of these Opr proteins can compensate for the genetically engineered suppression of the other (Morita et al., 2012b;Li et al., 2015). In P. aeruginosa, the MexXY system mediates resistance to aminoglycosides as well as various clinically relevant antimicrobials such as fluoroquinolones, some β-lactams (e.g., cefepime), tetracyclines  (Hoang et al., 1998).
Frontiers in Microbiology | www.frontiersin.org (including tigecycline), and macrolides such as azithromycin (Morita et al., 2012b). In wild-type cells, the mexXY operon is induced by agents that target ribosomes (Jeannot et al., 2005;Morita et al., 2006); the operon is overexpressed in mutant cells (e.g., Morita et al., 2012b;Guénard et al., 2014). Potent inhibitors of RND-type efflux pumps could be used as adjunctive therapies that would increase the potency of existing antibiotics and decrease the emergence of multidrug resistance in Gramnegative pathogens such as P. aeruginosa and Enterobacteriaceae (Gill et al., 2015;Opperman and Nguyen, 2015;Venter et al., 2015). Several potent inhibitors of RND-type efflux pump (e.g., PAβN , D13-9001 (Nakashima et al., 2013), and MBX2319 Vargiu et al., 2014 have been reported. However, only a single inhibitor of MexXY (MP 601384) has been reported to date (Jassem et al., 2011); the chemical structure of that agent was not revealed.
In the present study, berberine was shown to synergistically inhibit aminoglycoside resistance in P. aeruginosa in a MexXY-dependent manner. We observed a similar effect in A. xylosoxidans and B. cepacia, distinct species that possess MexXY orthologues (Morita et al., 2012b). On the other hand, we did not observe berberine attenuation of aminoglycoside resistance in A. baumannii. In Acinetobacter, the AdeAB RND-type pump mediates aminoglycoside resistance (Magnet et al., 2001), and the AdeAB system exhibits more sequence similarity to P. aeruginosa MexCD than to P. aeruginosa MexXY (Morita et al., 2012b). Furthermore, berberine attenuated MexXY-dependent resistance to aminoglycosides in a P. aeruginosa harboring MexXY but lacking another four RND multidrug efflux systems. We observed the same effect in this background for antimicrobials of other classes (including erythromycin, cefepime, and lincomycin) that are considered substrates of the MexXY pump (Morita et al., 2012b). Berberine is a natural isoquinoline alkaloid produced by a variety of plant species; this compound has been reported to possess a number of biological activities, including antimicrobial effects (Tillhon et al., 2012). Recently, berberine's antibacterial properties were shown to be due primarily to inhibition of the cell division protein FtsZ (Domadia et al., 2008;Boberek et al., 2010). However, this compound's antibacterial activity is not strong against Gram-negative bacteria such as P. aeruginosa at least in part because many bacterial multidrug efflux pumps recognize berberine as a substrate (e.g., Morita et al., 1998;Tegos et al., 2002). Berberine is more potent against Gram-positive bacteria such as Mycobacterium tuberculosis and S. aureus by inhibiting MF-type multidrug efflux pumps such as NorA (Tegos et al., 2002). The MexXY system confers berberine resistance in Escherichia coli lacking the major RND-type mulidrug efflux system AcrAB (data not shown) as well as confers PAβN resistance in P. aeruginosa lacking the major RND pumps ( Table 6), suggesting that each of the two agents acts as a competitive inhibitor of substrate binding and/or extrusion. However each spectrum of antimicrobial potentiation is not the same. Unfortunately, the adjuvant activity of berberine requires (in Pseudomonas) a relatively high (>100 µg/ml) concentration of the compound, consistent with a limited ability to inhibit the MexXY efflux. Clinical development would require optimization of the structure of berberine to provide better synergistic activity with aminoglycosides against P. aeruginosa. Recently a region of MexY (the substrate-specificity-determining RND component of this pump) that corresponds to a proximal binding pocket of AcrB was shown to be involved in aminoglycoside recognition and efflux (Lau et al., 2014;Li et al., 2015). This protein domain would be a good starting place for understanding details of aminoglycoside recognition and export by MexY. Although the substrate specificity of E. coli AcrAB-TolC (the best studied RND-type multidrug efflux pump) is extraordinarily broad, the complex does not recognize aminoglycosides as substrates (Li et al., 2015). The design and development of better MexXY inhibitors will require improved understanding of the molecular mechanisms of MexXY-mediated aminoglycoside resistance.
In conclusion berberine is the first efflux inhibitor that restores aminoglycosides activity in multidrug resistant P. aeruginosa. As aminoglycosides are molecules of choice to treat severe infections the clinical impact is potentially important.

AUTHOR CONTRIBUTIONS
YM conceived and designed the experiment. YM, KeN, and KK performed the experiments. YM, KeN, KuN, KK, JT, MI, and YK analyzed the data. YM wrote the paper.