Natural and synthetic compounds such as trimethoprim behave as inhibitors of efflux in Gram-negative bacteria

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1 Journal of Antimicrobial Chemotherapy Advance Access published March 19, 2010 J Antimicrob Chemother doi: /jac/dkq079 atural and synthetic compounds such as trimethoprim behave as inhibitors of efflux in Gram-negative bacteria Laura J. V. Piddock 1 *, Mark I. Garvey 1, M. Mukhlesur Rahman 2 and Simon Gibbons 2 1 Antimicrobial Agents Research Group, School of Immunity and Infection, College of dical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK; 2 Department of Pharmaceutical and Biological Chemistry, The School of Pharmacy, University of London, Brunswick Square, London WC1 1AX, UK *Corresponding author. Tel: ; Fax: ; l.j.v.piddock@bham.ac.uk Received 9 December 2009; returned 10 January 2010; revised 17 February 2010; accepted 18 February 2010 bjectives: We hypothesized that small heterocyclic or nitrogen-containing compounds could act as RD efflux pump inhibitors (EPIs). To ascertain possible EPIs, we sought to identify compounds that synergized with substrates of RD efflux pumps for wild-type bacteria and those that overexpress an efflux pump, but had no synergistic activity against strains in which a gene encoding a component of the AcrAB-TolC efflux pump had been inactivated. thods: Twenty-six compounds plus L-phenylalanyl-L-arginyl-b-naphthylamide (PAb) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were screened by bioassay to identify compounds that synergized with ciprofloxacin for a range of Enterobacteriaceae and Pseudomonas aeruginosa. The MICs of ciprofloxacin, tetracycline, chloramphenicol, erythromycin and ethidium bromide+synergizing compounds were determined, and the ability to inhibit the efflux of Hoechst was measured. Results: Two compounds, trimethoprim and epinephrine, consistently showed synergy with antibiotics for most strains. The combinations did not show synergy for Salmonella enterica serovar Typhimurium in which the AcrAB-TolC efflux pump was inactive. Both compounds inhibited the efflux of Hoechst Conclusions: Two compounds, trimethoprim and epinephrine, which are already licensed for use in man, may warrant further analysis as EPIs. The combination of trimethoprim with another antibiotic is a well-used combination in anti-infective chemotherapy, and so combination with another agent, such as a quinolone, may be a viable option and further studies are now required. Keywords: AcrAB-TolC, antibiotic resistance, EPIs Introduction The numbers of antibiotic-resistant bacteria have increased in recent years and such resistance can compromise the efficacy of antimicrobial therapy. 1 Antibiotic-resistant bacteria can be associated with infections with higher mortality than those caused by antibiotic-susceptible strains and fluoroquinolone resistance has been shown to be a particular risk factor for mortality associated with infections by some bacterial species. 2 Fluoroquinolones are also the agents most commonly associated with the selection of constitutive chromosomally mediated multidrug resistance (MDR), frequently presumed to be conferred by the overproduction of chromosomally encoded MDR efflux pumps. 3 The RD efflux pump AcrAB-TolC in Enterobacteriaceae and homologues thereof in other Gram-negative bacteria is the system most commonly associated with innate and acquired chromosomally mediated MDR. 3 Clinical isolates that overexpress efflux pumps usually overproduce this pump and there is an association of MDR mediated by efflux with prior use of fluoroquinolones. 4 In the laboratory, in vitro mutants selected after fluoroquinolone exposure comprise various phenotypes, of which 30% are MDR due to enhanced efflux. 5 Due to the lack of new antibacterial agents, there is considerable interest in restoring the activity of older antimicrobials. ne way to do this is to inhibit the action of MDR efflux pumps that confer innate resistance to these older agents; this is an area of active drug development by pharmaceutical companies. 6 Efflux pump proteins make attractive targets for drug discovery programmes as: (i) inhibition confers multidrug susceptibility; and (ii) a functional RD efflux pump is necessary for mutants resistant to substrates of the pump to be selected, 7 10 and for pathogens to colonize, persist and cause infection in their # The Author Published by xford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org 1of9

2 Piddock et al. 3,7 9,11 hosts. Agents that inhibit efflux (whether innate or enhanced) are termed efflux pump inhibitors (EPIs). However, this is a broad definition that includes agents that: (i) interact with the efflux pump protein(s); (ii) dissipate the proton-motive force required for pump activity, e.g. carbonyl cyanide m-chlorophenylhydrazone (CCCP), as in the case of RD family efflux pumps; or (iii) inhibit expression of the efflux pump gene, as has been shown for chlorpromazine and acrb. 12 The majority of studies searching for efflux pump inhibitors do so by in vitro determination of antimicrobial activity in the presence of the test compound, and by measuring efflux activity in the presence and absence of the test compound. Rarely do studies examine the precise mechanism of action of the presumed EPI. To date, there is only one agent that has been developed as an EPI for Gram-negative bacteria: L-phenylalanyl-L-arginyl-b-naphthylamide (PAb). 10 This agent is routinely used in the laboratory as a screen to indicate efflux-mediated antibiotic resistance in Gram-negative bacteria. However, PAb is not used in the clinical setting due to toxicity and bioavailability issues. 7 Several teams worldwide are searching for molecules whose actions are suggestive of EPIs. Publications predominantly focus on the identification of molecules with activity against Gram-positive bacteria, in particular Staphylococcus aureus. 13 More recently, as there is a desperate need for new agents to treat infections by Gram-negative bacteria, the search for inhibitors of efflux has been widened. However, for Gram-negative bacteria such as Salmonella enterica serovar Typhimurium, there are no clinically useful EPIs. Building upon the studies by Lomovskaya et al., 10 where PAb was identified as a Pseudomonas aeruginosa xb inhibitor, other molecules have subsequently been identified to have in vitro activity, although as yet none have been licensed for use. Many of the molecules, such as D-ornithinyl-D-homophenylalanyl-3-aminoquinoline, were modified to decrease their in vivo toxicity. 14 Data for other molecules suggest that they are also EPIs, including Table 1. Bacterial strains 1-(1-naphthylmethyl)-piperazine, which has been shown to be active against Acinetobacter baumannii and Enterobacteriaceae thers have shown that drugs that are currently licensed for uses other than as anti-infective agents, have EPI-like properties; these include selective serotonin reuptake inhibitors 18 and phenothiazines. 12 We hypothesized that simple heterocyclic nitrogen-containing compounds could also act as inhibitors of efflux in Gramnegative bacteria. Therefore, in a proof-of-principle study, we screened 26 compounds for EPI-like activity by identifying molecules that had synergistic activity with ciprofloxacin for wildtype Salmonella Typhimurium and strains that overexpressed the AcrAB-TolC efflux pump, but had no activity against mutants in which AcrA, AcrB or TolC had been inactivated. For those compounds that synergized with ciprofloxacin, the MICs of a range of agents+the test compound were determined for several different Gram-negative bacterial species. Growth kinetics and, as a measure of efflux activity, accumulation of Hoechst were determined for the most active compounds. Materials and methods Bacterial strains, storage and growth All bacteria used in this study are listed in Table 1. Construction of mutants derived from S. enterica serovar Typhimurium (hereafter referred to as Salmonella Typhimurium) strains SL1344 and 14028s with the acra, acrb and tolc genes disrupted has been described previously. Salmonella Typhimurium L3 is a human pre-therapy isolate and L10 is a human post-therapy clinical isolate that overexpresses acrab, both of which have been previously described. 4,22 All bacteria were stored on Protect TM beads at 2808C until required. The identification of each species was confirmed by Gram stain and analytical profile index (API 20E; biomérieux, Marcy l Étoile, France). Strain Species Description Reference/source L354 Salmonella Typhimurium SL L109 Salmonella Typhimurium SL1344DtolC 21 L785 Salmonella Typhimurium ATCC (LT2) 31 L828 Salmonella Typhimurium ATCC 14028s ATCC a L829 Salmonella Typhimurium ATCC 14028s DtolC 32 L830 Salmonella Typhimurium ATCC 14028s DacrB 32 L831 Salmonella Typhimurium ATCC 14028s DacrAB 32 L3 Salmonella Typhimurium human pre-therapy clinical isolate 21 L10 Salmonella Typhimurium human post-therapy MDR clinical isolate, acrab A1 Enterobacter cloacae CTC CTC b B14 Serratia marcescens CTC 2847 CTC b G1 Pseudomonas aeruginosa CTC CTC b H42 Klebsiella pneumoniae CTC CTC b H43 Klebsiella pneumoniae CTC 9633 CTC b I114 Escherichia coli CTC CTC b J29 Morganella morganii CTC 235 CTC b a ATCC, American Type Culture Collection. b CTC, ational Collection of Type Cultures (HPA, Colindale, London, UK). 2of9

3 Inhibitors of efflux JAC dia and chemicals Bacteria were routinely grown on LB agar plates (xoid, Basingstoke, UK) and in LB broth (xoid). All antibiotics and compounds were obtained from Sigma (Poole, UK). Bioassay screening Screening with ciprofloxacin+26 test compounds (trimethoprim, cathinone, theobromine, norepinephrine, epinephrine, theophylline, caffeine, quinine, arecoline, atropine, ephedrine, norephedrine, morphine, ergometrine, ergotamine, harmine, harmaline, strychnine, amphetamine, nicotine, papaverine, emetine, quinidine, pilocarpine, salbutamol and ajmaline) for synergistic activity was carried out by bioassay, essentially as described by Lund et al. 23 In brief, this consisted of large sterile square dishes (24 24 cm; Fisher Scientific, Loughborough, Leicestershire, UK) containing 250 ml of Iso-Sensitest agar (xoid) solidified for 2 h at 48C. The plates were then air-dried at 508C for 15 min. An overnight 25 ml culture of Salmonella Typhimurium was diluted 1:10000 to obtain cfu/ml (determined by 10 successive replicate plate counts) and then 10 ml of the diluted culture was poured onto the surface of the agar plate. The excess liquid was removed and the plates left to dry at room temperature for 15 min. Six rows of 6 wells (total 36 wells), each of 8 mm diameter, were cut out of each agar plate using an agar cutter (Fisher Scientific) and 200 ml of sterile Iso-Sensitest broth containing ciprofloxacin (0.5, 1 and 2 mg/l) was added to three sets of wells respectively. This volume and concentrations gave a zone of inhibition of 15 cm diameter. To determine whether the test compound synergized with ciprofloxacin, 200 ml of a solution containing both ciprofloxacin (0.5, 1 and 2 mg/l respectively) and the test compound (100 mg/l) or epinephrine and trimethoprim (10 mg/l) in broth was added to three separate wells. The plates were incubated overnight at 378C and the diameters of the zones of growth inhibition were measured. Each experiment contained three technical repeats and was repeated on three separate days. This allowed the inherent variability of the method to be established and statistical analyses of data. A zone of inhibition of the antibiotic plus test compound larger than that of the antibiotic alone was taken to suggest synergy. The positive control was ciprofloxacin plus PAb (40 mg/l) or CCCP (100 mg/l) and the negative control was Iso- Sensitest broth alone. Compound alone was measured to determine whether the test agent had inherent antimicrobial activity. Determination of MICs of antibiotics +synergizing compounds The MICs of ciprofloxacin, tetracycline, chloramphenicol, erythromycin and ethidium bromide+test compounds (10 mg/l) or trimethoprim and epinephrine (10 mg/l) were determined by the standardized agar doubling dilution method and by the microbroth dilution method following the BSAC recommended protocol. 24,25 All MIC determinations were repeated at least three times in independent experiments. All antibiotics, compounds and efflux pump inhibitors were made up and used according to the manufacturer s instructions. Accumulation of Hoechst candidate EPI-like compounds by Salmonella Typhimurium The efflux activity of five strains [L3, L10, SL1344, ATCC (LT2) and ATCC 14028s] was determined by measuring the accumulation of the fluorescent dye Hoechst (bis-benzimide; 2.5 mm)+known EPIs (100 mg/l CCCP and 40 mg/l PAb) and+the putative EPIs (epinephrine and trimethoprim, both at 10 mg/l, and theobromine and norepinephrine, both at 100 mg/l). asurements were taken at excitation and emission wavelengths of 350 and 460 nm, respectively, over 30 min using a FLUstar PTIMA (BMG Labtech, Aylesbury, UK), as previously described. 8 Differences in the accumulation seen+putative EPI were analysed for statistical significance using the two-tailed Student s t-test. P values 0.05 were taken as significant. Growth kinetics + putative EPIs The growth kinetics of L3, L10, SL1344, ATCC (LT2) and ATCC 14028s+putative EPIs were determined by monitoring the optical density at 600 nm every 10 min at 378C for 24 h using a FLUstar PTIMA (BMG Labtech). The putative EPIs (epinephrine and trimethoprim, both at 10 mg/l, and theobromine and norepinephrine, both at 100 mg/l) were added to the bacterial cultures at mid-logarithmic growth phase (2 h). Samples were also removed and visualized microscopically in order to detect any gross changes to cell morphology, i.e. filamentation. A two-tailed Student s t-test was used to compare the generation times. P values 0.05 were taken as significant. Results Choice of compounds to investigate for EPI properties Many EPIs are either nitrogen-containing heterocyclic compounds 15,26 or possess amino acid residues, such as in the case of PAb. 10 Therefore, we selected a range of natural and synthetic compounds possessing either heterocyclic functional groups or straight chain amino groups. Some of these compounds, e.g. epinephrine, norepinephrine and cathinone, possessed a phenylethylamine moiety, which appears to be a common structural motif in some EPIs. Identification of compounds that synergized with ciprofloxacin Twenty-six compounds (listed in the Materials and methods section) plus PAb and CCCP were screened by bioassay to identify compounds that synergized with ciprofloxacin. In addition to PAb and CCCP, 13 compounds showed synergy with ciprofloxacin. f these, trimethoprim, cathinone, theobromine, norepinephrine and epinephrine had the greatest synergy (Figure 1 and Table 2). The MICs of trimethoprim for all strains were mg/l and of epinephrine were mg/l. The MICs of theobromine and norepinephrine were.256 mg/l for all strains. Salmonella Typhimurium ATCC 14028s and SL1344 in which acra, acrb, acrab or tolc were inactivated were hypersusceptible to ciprofloxacin, tetracycline, erythromycin, ethidium bromide and chloramphenicol (Table 3 and data not shown). However, the MICs of epinephrine, norepinephrine, trimethoprim and theobromine were the same for the parental strain and the mutants in which the efflux pump gene had been inactivated (data not shown). When the MICs of ciprofloxacin were determined in combination with the test compounds (at one-quarter to one-half MIC or lower), the synergy observed in the bioassays was not always replicated for all strains (Table 3). onetheless, all compounds identified in the bioassay synergized with all antibiotics and ethidium bromide for at least six strains, including CTC type strains of different species (Tables 3 5). The combination that showed synergy most often was that of trimethoprim with ciprofloxacin. 3of9

4 Piddock et al. H 2 H H H 2 H H PAβ H H H Epinephrine H Theobromine orepinephrine H Theophylline Figure 1. Structures of five test compounds and theophylline compared with that of PAb. Table 2. Compounds from the bioassay screen having the greatest synergy with ciprofloxacin H H H H 2 H H 2 H 2 Cathinone H 2 Trimethoprim Ciprofloxacin plus Strain Description PAb TMP Cat The re Ene Thp Caf L354 SL1344 S S S S S S S S L785 ATCC (LT2 wild) S S S S S S S E L828 ATCC 14028s S S S S S S E S L3 human pre-therapy clinical isolate S S S S S E S E L10 human post-therapy MDR clinical isolate, acrab +++ S S S S S S E E S, synergy; E, no effect; TMP, trimethoprim; Cat, cathinone; The, theobromine; re, norepinephrine; Ene, epinephrine; Thp, theophylline; Caf, caffeine. The MIC values of ciprofloxacin were reduced by 2 4-fold in the presence of trimethoprim for the wild-type strains of Salmonella Typhimurium and CTC type strains of Enterobacter cloacae, Serratia marcescens, P. aeruginosa, Klebsiella pneumoniae and Escherichia coli (Table 3). However, no synergy was seen for trimethoprim with ciprofloxacin for the strains of Salmonella Typhimurium in which acra, acrb, acrab or tolc were inactivated. Theobromine with ciprofloxacin also showed synergy for eight other bacterial strains, including wild-type strains of Salmonella Typhimurium and CTC type strains of E. cloacae, P. aeruginosa, K. pneumoniae and E. coli. Except for the mutant in which tolc had been inactivated, no synergy was seen with this combination, or with mutants in which acra, acrb or acrab were inactivated. Ciprofloxacin and epinephrine was also a synergistic combination for some strains/species, but increased activity of the combination versus ciprofloxacin alone was also seen for mutants in which a gene encoding a component of the AcrAB-TolC pump was inactivated. However, the combination had no activity against the parental strains (e.g. L828, Table 3). The combination of ciprofloxacin and norepinephrine was only synergistic for two 4of9

5 Inhibitors of efflux JAC strains (one Salmonella Typhimurium and the CTC type strain of K. pneumoniae). Table 3. MIC (mg/l) of ciprofloxacin+the four most active compounds against Gram-negative bacteria CIP plus Strain CIP PAb a Ene b re c TMP b The c L L L L L L L A B G H H I J CIP, ciprofloxacin; Ene, epinephrine; re, norepinephrine; TMP, trimethoprim; The, theobromine. Bold text indicates synergistic combinations. a PAb at 40 mg/l. b TMP and Ene at 10 mg/l. c re and The at 100 mg/l. Epinephrine, norepinephrine, trimethoprim and theobromine synergize with other antibiotics PAb showed synergy with ciprofloxacin for 10 strains (Table 3). PAb also synergized with tetracycline, chloramphenicol and ethidium bromide for seven to nine strains/species (Tables 4 and 5). However, only for S. marcescens was synergy seen between PAb and erythromycin. When the MICs of the four test compounds in combination with other antibiotics were determined, it was revealed that the synergy observed with ciprofloxacin was not always reproduced with the other agents. For instance, norepinephrine and chloramphenicol, norepinephrine and ethidium bromide, and theobromine with erythromycin were not synergistic combinations. However, as seen with ciprofloxacin, trimethoprim in combination with any of the other four agents was synergistic for six to eight strains/species (Tables 4 and 5). Epinephrine in combination with tetracycline, chloramphenicol and ethidium bromide was also synergistic for five to seven strains/species. Theobromine formed a synergistic combination with tetracycline, chloramphenicol and ethidium bromide for three to five strains. orepinephrine combined with tetracycline or erythromycin showed synergy for three to four strains. Where synergism was observed, the activity for a particular antibiotic was similar, irrespective of the synergizing compound. Efflux activity of test compounds Due to the synergistic activity seen for the compounds with some antibiotics and strains in which AcrAB-TolC was produced, and the lack of synergy seen for mutants in which components of this pump were not produced, it was hypothesized that Table 4. MIC (mg/l) of tetracycline and erythromycin+the four most active compounds against Gram-negative bacteria TET plus ERY plus Strain TET PAb a Ene b re c TMP b The c ERY PAb a Ene b re c TMP b The c L L L L L L L A B G H H I J TET, tetracycline; Ene, epinephrine; re, norepinephrine; TMP, trimethoprim; The, theobromine; ERY, erythromycin. Bold text indicates synergistic combinations. a PAb at 40 mg/l. b TMP and Ene at 10 mg/l. c re and The at 100 mg/l. 5of9

6 Piddock et al. Table 5. MIC (mg/l) of chloramphenicol and and ethidium bromide+the four most active compounds against Gram-negative bacteria CHL plus EtBr plus Strain CHL PAb a Ene b re c TMP b The c EtBr PAb a Ene b re c TMP b The c L L L L L L L A B G H H I J CHL, chloramphenicol; Ene, epinephrine; re, norepinephrine; TMP, trimethoprim; The, theobromine; EtBr, ethidium bromide. Bold text indicates synergistic combinations. a PAb at 40 mg/l. b TMP and Ene at 10 mg/l. c re and The at 100 mg/l. some compounds could be EPIs that target AcrAB-TolC. Therefore, the effect of trimethoprim, epinephrine and theobromine upon the accumulation of Hoechst was measured for five strains of Salmonella Typhimurium (SL1344, LT2, ATCC 14028s, L3 and L10). The growth kinetics of the bacteria were unaffected at the concentrations of compound tested (10 mg/l trimethoprim and epinephrine; 100 mg/l theobromine) (data not shown). A compound with EPI-like properties, such as PAb, inhibits efflux and so the bacterium is unable to export Hoechst 33342; therefore, the concentration of Hoechst accumulated by the bacterium increases. Trimethoprim and epinephrine both significantly increased the concentration of Hoechst accumulated (1.5- and fold, respectively) (Figures 2 and 3). However, accumulation of Hoechst by Salmonella Typhimurium appeared unaffected by theobromine (data not shown). Discussion This proof-of-principle study revealed that combinations of several agents (including trimethoprim) had synergistic antibacterial activity. The compounds were chosen on the basis of being heterocyclic nitrogen-containing drug-like molecules, e.g. the phenylethylamines epinephrine, norepinephrine and cathinone. As ciprofloxacin is a substrate of many efflux pumps in different bacterial species, 3 initial experiments focused upon identifying compounds that displayed synergistic activity with ciprofloxacin in a simple bioassay. Furthermore, to identify possible EPIs, we sought to identify compounds that synergized with substrates of RD efflux pumps for wild-type bacteria and those that overexpress an efflux pump, but had no synergistic activity against strains in which a gene encoding a component of the AcrAB-TolC efflux pump had been inactivated. Three of the agents identified in the bioassay that synergized with ciprofloxacin, namely epinephrine, norepinephrine and cathinone, are members of the phenylethylamine class of natural product, and all possess this structural motif, as does PAb. Many of the EPIs identified to date appear to possess either monocyclic or bicyclic (e.g. naphthyl) aromatic rings with side chains possessing nitrogen atoms, for example D-ornithinyl-D-homophenylalanyl- 3-aminoquinoline 14 and 1-(1-naphthylmethyl)-piperazine. 15,16 Where synergy was confirmed in the MIC tests and was seen with a combination of compound plus an antibiotic or ethidium bromide, it was typically seen for CTC strains of different species. This suggests broad synergistic activity rather than species-specific activity. However, these data require confirmation in studies with a larger number of strains of each species. Two compounds, trimethoprim and epinephrine, consistently showed synergy with antibiotics for most strains. The combinations did not show synergy for strains in which the AcrAB-TolC efflux pump was inactive. These data suggest that trimethoprim and epinephrine either inhibit expression of the genes encoding AcrAB-TolC, as found for acrb and chlorpromazine, 12 or interact with a component of the pump directly, as for PAb and xb. These hypotheses are supported by data showing that both compounds inhibited the efflux of Hoechst Whilst trimethoprim and epinephrine appear to be structurally unrelated, they both possess an aromatic ring linked through a chain to a basic nitrogen-containing moiety: a pyrimidine ring in the case of trimethoprim and a primary amine in the case of epinephrine (Figure 1). These appear to be common features of many EPIs, including PAb, D-ornithinyl-D-homophenylalanyl- 3-aminoquinoline and 1-(1-naphthylmethyl)-piperazine. The pyrimidine ring of trimethoprim also bears striking structural and electronic similarity to the A-rings of theobromine and 6of9

7 Inhibitors of efflux JAC Hoechst Ene 6000 Fold difference Fluorescence units Time (s) Hoechst TMP Hoechst alone Figure 2. Accumulation of Hoechst (bis-benzimide)+epinephrine (Ene; 10 mg/l) or trimethoprim (TMP; 10 mg/l) over 25 min in Salmonella Typhimurium SL1344. All compounds were added at time zero L3 L3 + Ene L3 + TMP L10 L10 L10 + Ene + TMP L354 L354 L354 + Ene + TMP L785 L785 L785 L828 L828 L828 + Ene + TMP + Ene + TMP Figure 3. Fold change in accumulation of Hoechst (bis-benzimide)+epinephrine (Ene) or trimethoprim (TMP) after 10 min of exposure of Salmonella Typhimurium strains L3, L10, SL1344, LT2 and ATCC 14028s (Table 1). All compounds were added at time zero. Hoechst (bis-benzimide) was added after 2 min. theophylline. A recent study by Bohnert et al. 27 has shown that the AcrB multidrug efflux pump protein contains a hydrophobic phenylalanine-rich binding site. It is possible that aromatic nitrogen-containing EPIs interact and bind at this site via p p interactions between their aromatic rings and those of the phenylalanine residues. 7of9

8 Piddock et al. Two compounds, epinephrine and norepinephrine, had little or no effect upon the activity of ciprofloxacin for Salmonella Typhimurium ATCC 14028s. However, this combination was synergistic against mutants lacking acrab or tolc. These data suggest that these compounds are substrates of the AcrAB-TolC efflux pump. In the absence of a functional pump, these agents accumulate within the bacterial cell and have an additive antibacterial effect with ciprofloxacin. Furthermore, this suggests that these two compounds have intrinsic antibacterial activity and interact with an intracellular target. Epinephrine is licensed for the treatment of anaphylactic shock (type 1 hypersensitivity allergic reactions) and is a component of the Epi-pen (British ational Formulary; BF). It is a catecholamine, which is a sympathomimetic monoamine derived from the amino acids phenylalanine and tyrosine. The concentration administered intramuscularly is 1 mg/l, but in a medical emergency 100 mg/l is administered by slow intravenous injection (BF). Therefore, it is unlikely that a combination of epinephrine plus an antibiotic would be licensed for use as an anti-infective. Trimethoprim is an inhibitor of dihydrofolate reductase. It is usually administered as a combination antibiotic with another inhibitor of folic acid biosynthesis, sulfamethoxazole (BF). Therefore, the synergistic and EPI-like behaviour of trimethoprim with other antibiotics is interesting. Synergy of a quinolone with trimethoprim has been reported previously for Gram-negative and Gram-positive bacteria, although the mechanism has not been explored. 28,29 The combination of trimethoprim with another antibiotic, e.g. sulfamethoxazole to give co-trimoxazole, is a well-used combination in anti-infective chemotherapy and so combination with another agent, such as a quinolone, may be a viable option. Although the use of trimethoprim alone has been associated with the selection of trimethoprim-resistant bacteria, this was reduced when in combination with sulfamethoxazole. In conclusion, we have shown that much simpler aromatic nitrogen-containing compounds than PAb and D-ornithinyl-D-homophenylalanyl-3-aminoquinoline, which are already licensed for use in man, may warrant further analysis as EPIs. The simplicity of the phenylethylamine and pyrimidine moieties will make these compounds an attractive synthetic target as EPIs. Funding This work was supported by The Leverhulme Trust (project F/00 /094/AR). Transparency declarations one to declare. References 1 Taubes G. The bacteria fight back. Science 2008; 321: Helms M, Ethelberg S, Mølbak K et al. International Salmonella Typhimurium DT104 infections, Emerg Infect Dis 2005; 11: Piddock LJ. Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria. Clin Microbiol Rev 2006; 19: Piddock LJV, Griggs DJ, Hall MC et al. Ciprofloxacin-resistance in clinical isolates of Salmonella typhimurium obtained from two patients. Antimicrob Agents Chemother 1993; 37: Ricci V, Piddock LJV. Ciprofloxacin selects for multi-drug resistance in Salmonella enterica serovar Typhimurium mediated by at least two different pathways. J Antimicrob Chemother 2009; 63: Lomovskaya, Bostian KA. Practical applications and feasibility of efflux pump inhibitors in the clinic a vision for applied use. Biochem Pharmacol 2006; 71: Ricci V, Tzakas P, Buckley A et al. Ciprofloxacin-resistant Salmonella enterica serovar Typhimurium strains are difficult to select in the absence of AcrB and TolC. Antimicrob Agents Chemother 2006; 50: Webber MA, Randall LP, Cooles S et al. Triclosan resistance in Salmonella enterica serovar Typhimurium. J Antimicrob Chemother 2008; 62: Ricci V, Piddock LJV. nly for substrate antibiotics is a functional AcrAB-TolC efflux pump and RamA required to select multidrug resistant Salmonella Typhimurium. J Antimicrob Chemother : Lomovskaya, Warren MS, Lee A et al. Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy. Antimicrob Agents Chemother 2001; 45: Piddock LJ. Multidrug-resistance efflux pumps not just for resistance. at Rev Microbiol 2006; 4: Bailey AM, Paulsen IT, Piddock LJV. RamA confers multidrug resistance in Salmonella enterica via increased expression of acrb, which is inhibited by chlorpromazine. Antimicrob Agents Chemother 2008; 52: argotra A, Koul S, Sharma S et al. Quantitative structure activity relationship (QSAR) of aryl alkenyl amides/imines for bacterial efflux pump inhibitors. Eur J d Chem 2009; 44: Renau TE, Léger R, Filonova L et al. 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