Potential role of non-antibiotics (helper compounds) in the treatment of multidrug-resistant Gram-negative infections: mechanisms for their direct and indirect activities

A

uaao©

K

1

siafal

eT

0d

International Journal of Antimicrobial Agents 31 (2008) 198–208

Review

Potential role of non-antibiotics (helper compounds) in the treatmentof multidrug-resistant Gram-negative infections: mechanisms

for their direct and indirect activities

Marta Martins a,b, Sujata G. Dastidar c, Seamus Fanning d, Jette E. Kristiansen e,Joseph Molnar f, Jean-Marie Pages g, Zsuzsanna Schelz f, Gabriella Spengler f,

Miguel Viveiros a, Leonard Amaral a,b,∗a Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 96, 1349-008 Lisboa, Portugal

b UPMM, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 96, 1349-008 Lisboa, Portugalc Division of Microbiology, Department of Pharmaceutical Technology, Jadavpur University, Calcutta 700032, India

d Centre for Food Safety, School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Irelande Department of Clinical Microbiology, Sønderborg, Southern Danish University, Sydvang 1, 6400 Sønderborg, Denmark

f Institute of Medical Microbiology and Immunology, University of Szeged, H-6720 Szeged, Dom ter 10, Hungaryg UMR-MD-1, IFR48, Facultes de Medecine et de Pharmacie, Universite de la Mediterranee, Marseille, France

Received 25 October 2007; accepted 25 October 2007

We dedicate this article to our friend and colleague J David Williams whom we miss dearly.

bstract

Multidrug resistance in Gram-negative bacteria is now known to be primarily caused by overexpression of efflux pumps that extrudenrelated antibiotics from the periplasm or cytoplasm of the bacterium prior to their reaching their intended target. This review focuses on
variety of agents that have been shown to be efflux pump inhibitors (EPIs) and which, if used as ‘helper compounds’ in combination with
ntibiotics to which the organism is initially resistant, may produce the required cure. Although not all of the EPIs may serve a helper rolewing to their toxicity, they may nevertheless serve as lead compounds.

2007 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

eywords: Multidrug resistance; Gram-negative bacteria; Efflux pumps; Efflux pump inhibitors; Non-antibiotics; Helper compounds

t[Tenu

. Introduction

An antibiotic as defined by Webster’s dictionary is aubstance produced by a microorganism that destroys ornhibits the growth of another microorganism. In contrast ton antibiotic, there are medicinal compounds that are used
or the therapy of non-infectious pathology and that haventimicrobial properties [1]. For lack of a better name, theseatter compounds are termed ‘non-antibiotics’ [2] and, given
∗ Corresponding author at: Unit of Mycobacteriology, Instituto de HigieneMedicina Tropical, Rua da Junqueira, 96, 1349-008 Lisboa, Portugal.el.: +351 21 365 2600; fax: +351 21 363 2105.

E-mail address: [email protected] (L. Amaral).

st[stbrt

924-8579/$ – see front matter © 2007 Elsevier B.V. and the International Societyoi:10.1016/j.ijantimicag.2007.10.025

heir potential for the therapy of some problematic infections3,4], they may eventually achieve antimicrobial status.he group of ‘non-antibiotics’ consists of two subgroups,ach with activities that are distinctly different. Thoseon-antibiotics that have direct antimicrobial activity makep the first group (antimicrobial non-antibiotics) [3]. Theecond group consists of two subclasses, one of which altershe permeability of the microorganism to a given antibiotic5–7], termed ‘helper compounds’, whilst the secondubgroup enhances the killing activity of macrophages
hat have phagocytosed the microorganism [8] and maye termed ‘macrophage modulators’. The purpose of thiseview of ‘non-antibiotics as helper compounds’ is to discusshe mechanisms by which some non-antibiotics have direct
of Chemotherapy. All rights reserved.

mailto:[email protected]

dx.doi.org/10.1016/j.ijantimicag.2007.10.025

M. Martins et al. / International Journal of Antimicrobial Agents 31 (2008) 198–208 199

F t Mycoc gs are j

aotnioh

1

dacattiodofaupatet

rt(apbhtpi

1

sta(oblaapl

ig. 1. Structure of phenothiazine and its derivatives with activity againsompounds characterised by a three-ring structure in which two benzene rin

ntibacterial activity, some assist the penetration of antibi-tics by their ability to inhibit efflux of the antibiotic prioro it reaching its intended target, others directly transformon-killer cells into effective killers of bacteria which causenfections that are problematic to manage, and lastly somef which have the ability to cure bacteria of plasmids andence may have potential in the area of animal husbandry.

.1. History of ‘non-antibiotics’

Prior to end of the 19th century, the work of Paul Ehrlichemonstrated the antimicrobial activity of methylene blue,dye that is the ‘poster child’ for a group of heterocyclic

ompounds, the phenothiazines (Fig. 1). Methylene blue waslso shown to obviate the mobility of microorganisms andhis finding quickly spurred studies by others to see whetherhe dye would also affect the mobility of a mammal andndeed it did, inasmuch as intravenous (i.v.) administrationf this dye produced lethargy in humans [9]. Because theye turned the patient blue, it could not be used for therapyf neurological disease that required restraining the patientrom overactive activity. Nevertheless, interest in the dye aspotential neuroleptic was maintained for the next 50 years,ltimately resulting in the first neuroleptic non-colouredhenothiazine, chlorpromazine (CPZ) [10]. Interest in the
ntimicrobial properties of methylene blue was not sufficiento motivate its exploitation as an antimicrobial agent. How-ver, with global use of CPZ, anecdotal reports demonstratedhat patients receiving CPZ therapy could be cured of bacte-
[b[m

bacterium tuberculosis (TB). Phenothiazines are a group of heterocyclicoined by a sulphur and nitrogen atom at non-adjacent positions.

ial infections [11]. Nevertheless, because the appearance ofhese reports took place during the ‘golden age of antibiotics’1950s to 1970s), there was little need for the use of CPZ asnother antibiotic, especially since the serious side effectsroduced by this agent were rather frequent [12]. However,ecause antibiotic resistance, especially multidrug resistance,as become common [13], a number of investigators turnedheir interest to CPZ and other derived phenothiazines asotential agents against multidrug-resistant (MDR) organ-sms.

.2. Antimicrobial activity of non-antibiotics

The in vitro antimicrobial activities of non-antibiotics areummarised in part by Tables 1 and 2. These demonstratehe wide gamut of antibacterial activities of phenothiazinesnd their derivatives (Table 1) as well as cardiovascular drugsTable 2). However, with few exceptions the concentrationsf these agents needed to inhibit in vitro growth are welleyond those that can be achieved clinically [11]. Neverthe-ess, phenothiazines such as trimethoprim and trimeprazine,t concentrations that are tolerated by the mouse, protect thenimal from developing Salmonella infections [77]. Similarrotection of the mouse against infection by highly viru-ent Salmonella strains is afforded by diclofenac sodium
7]. Non-antibiotic phenothiazines and their derivatives haveeen shown to protect mice against Escherichia coli infection78]. However, although the above non-antibiotics protect theouse against a Gram-negative infection, they have not yet

200 M. Martins et al. / International Journal of Antimicrobial Agents 31 (2008) 198–208

Table 1In vitro antimicrobial activity of the non-antibiotics phenothiazines and related compounds

Bacteria MIC (mg/L)

Md, Fz CPZ, TZ, Ft, Tf, Tz, Tm, MD, Pr, Pz, Pc Dc, Dm Bd, Dp

Mycobacterium tuberculosisa N.D. 20–40 N.D. N.D.Staphylococcus aureusb 5–25 10–50 5–25 50–100Bacillus spp.c 10–50 25–100 50–100 100–200Streptococcus spp.d 10–50 25–100 25–50 50–100Escherichia colie 50–200 50–400 25–200 50–200Salmonella spp.f 25–100 25–100 10–100 150Shigella spp.g 25–100 25–100 10–100 150Klebsiella spp.h 200–400 300–400 400–500 500Proteus spp.i 100–300 100–400 100–200 200–300Pseudomonas spp.j 200–400 400–500 500 500Vibrio choleraek 5–25 10–50 5–25 50–100Vibrio parahaemolyticusl 5–25 10–50 5–25 50–100

MIC, minimum inhibitory concentration; Md, methdilazine; Fz, fluphenazine; CPZ, chlorpromazine; TZ, thioridazine; Ft, flupenthixol; Tf, trifluoperazine;Tz, trimeprazine; Tm, triflupromazine; MD, methyldopa; Pr, promazine; Pz, promethazine; Pc, prochlorperazine; Dc, diclofenac; Dm, dicyclomine; Bd,bromodiphenhydramine; Dp, diphenhydramine; N.D., no data. a–lReferences for data: aCPZ [14], TZ [15], Ft [16], Tf [17,18], Tm [19,20], MD [19,21], Pr[20,22], Pz [6,21]; bMd [23], CPZ [24–26], TZ [12,27], Ft [28], Tf [29,30], Tz [25], Tm [31], Pr [25,32], Pz [25], Pc [32,33], Dc [34,35], Dm [36], Dp [37];cFz [38], CPZ [39,40], Tz [41], Pr [40,42], Pz [43], Pc [33], Dp [37]; dFz [44], TZ [45], Dp [37]; eCPZ [46,47,48], TZ [27,49], Tf [50], Pr [51], Pz [42,52,53],P Tf [30][ , Dm [3[ ]; kMd

bltt

ncttrh[ncca

TA

B

SBESSKPV

MNa

d

[

miskptwaattaa

c [33], Dc [34,54], Bd [55], Dp [55]; fMd [55], Fz [38], CPZ [56], Ft [28],36,62], Dp [53,63]; gMd [55], CPZ [64], TZ [27], Tf [30], Tz [41], Pc [33]41], Pz [65]; jCPZ [66], TZ [29], Tf [29,30], Tz [41], Pz [43,53,66], Dc [34

een shown to cure a Salmonella-infected mouse. Neverthe-ess, children who present with recurrent pyelonephritis dueo E. coli can be cured with CPZ and gentamicin, whereasherapy with the antibiotic alone results in failure [79].

Although the use of phenothiazines for therapy of Gram-egative infections is still very far from being seriouslyonsidered, phenothiazines appear to have great potential forhe therapy of MDR tuberculosis (TB) [3,4,6,8,11]. As ishe case for in vitro activity against Gram-negative bacte-ia, a large number of phenothiazines have been shown toave in vitro activity against Mycobacterium tuberculosis14,17–20,22,80–85]. However, the concentrations of phe-othiazines that inhibit in vitro growth cannot be achieved
linically [12]. Nevertheless, because phenothiazines areoncentrated as much as 100-fold by macrophages [86,87]nd M. tuberculosis is an intracellular infection of the human
able 2ntibacterial activities of cardiovascular drugs in vitro

acteria MIC (mg/L)

Ox, Am Nf, Lc, Db

taphylococcus aureusa 5–25 –acillus spp.b 5–25 –scherichia colic 25–100 100–400almonella spp.d 50–100 100–200higella spp.e 5–100 –lebsiella spp.f 100–300 –seudomonas spp.g 200–400 –ibrio choleraeh 5–25 –

IC, minimum inhibitory concentration; Ox, oxyfedrine; Am, amlodipine;f, nifedipine; Lc, lacidipine; Db, dobutamine; –, no references available.

–hReferences for data: aAm [69,70]; bAm [70,71]; cAm [69], Nf [72,73];Ox [74,75], Am [70,76], Db [75]; eAm [70]; fAm [70]; gAm [70]; hAm70].

pitoaosesoft

1e

1

pc

, Tz [41,57], Tm [31], Pr [57,58], Pc [33,59], Dc [7,60,61], Dm [36,62], Bd6,62], Bd [55], Dp [55]; hTZ [29], Tf [29], Tz [41], Pc [33]; iCPZ [65], Tz[67], CPZ [67,68], Ft [28], Bd [55], Dp [37,55]; lTZ [27], Tf [30], Tz [41].

acrophage [88], phenothiazines have been studied for activ-ty against intracellular mycobacteria. These studies havehown that killing of phagocytosed M. tuberculosis by non-illing macrophages is enhanced by concentrations of thehenothiazine that are well below those employed for theherapy of psychoses [15,17,86,89]. It is important to note thathereas many compounds have been shown to have in vitro

ctivity, few of these penetrate the macrophage and retainctivity against intracellular mycobacteria. With respect tohe phenothiazines, because these compounds are concen-rated many-fold by the macrophage, it is probable thatny phenothiazine will have intracellular antimycobacterialctivity. In line with the demonstration that phenothiazinesromote the killing of intracellular mycobacteria, micenfected with M. tuberculosis have been cured of this infec-ion when treated with thioridazine [90] and with analoguesf CPZ [91]. Because a given phenothiazine has the samectivity against M. tuberculosis regardless of its resistance tone, two or more antibiotics [14], phenothiazines should beeriously considered for the therapy of MDR-TB [4,8,11] andxtensively drug-resistant (XDR)-TB [92]. However, becauseome phenothiazines such as CPZ produce a plethora of seri-us side effects, only those phenothiazines that are relativelyree of serious effects are to be considered as candidates forhe therapy of MDR-TB [3,4,8,11,14].

.3. Mechanism of action by which phenothiazinesxpress in vitro and ex vivo activity

.3.1. In vitroPhenothiazines inhibit transport of calcium (Ca2+) by

reventing its binding to Ca2+-binding proteins such asalmodulin [93]. This means that enzyme systems which

l of An

aa[paribpptnpsttMedirttIptIe[rthpftvbsrai

1

khtv[tColtKi

biin

1

tipttdsnmTcbaitba

ca[citsiiwssurcrceauwoiamd

M. Martins et al. / International Journa

re dependent upon Ca2+, such as those involved in gener-ting cellular energy from hydrolysis of ATP, are inhibited94]. Among these phenothiazine-sensitive systems are trans-orters that extrude from the cell a variety of noxious agentsnd hence protect the cell from these agents. All bacte-ia studied to date contain a variety of transporters thatntrinsically recognise noxious agents, such as antimicro-ial agents and detergents, and extrude these agents from theeriplasmic space of the cell envelope [95]. When these trans-orters are overexpressed, the bacterium becomes resistanto a variety of unrelated antibiotics, hence their MDR phe-otypes. Phenothiazines have been shown to reverse MDRhenotypes of bacteria and therefore render these bacteriausceptible to antibiotics to which they were initially resis-ant [24,45,56,96–98]. Because multidrug resistance is dueo the overexpression of efflux pumps that are the cause of

DR bacteria [99] and these efflux systems are driven bynergy provided by the proton-motive force which is depen-ent upon Ca2+-dependent enzyme systems [100,101], thenhibition of Ca2+ binding to Ca2+-dependent enzymes willender the bacterium susceptible to that which they were ini-ially resistant [102]. Therefore, it is not surprising to notehat phenothiazines reverse MDR phenotypes of bacteria.nhibition of intrinsic efflux of a noxious agent such as ahenothiazine is believed to result in greater permeabilityo other noxious agents, including the phenothiazine itself.ncreased permeability ensures that phenothiazine moleculesventually reach the DNA, intercalate between the bases103], inhibit all DNA-based processes [81] and hence inhibiteplication. The in vitro concentrations of the phenothiazinehat inhibit replication of the bacterium are generally manyundred-fold greater than that which can be achieved in theatient. Why then can a phenothiazine protect the mouserom a Salmonella infection (extracellular) at a concentra-ion that is many hundred-fold lower than that achieved initro? As administration of phenothiazines to the mouse haseen shown to induce the secretion of cytokines by T-cell sub-ets and these cytokines are involved in T-cell responses thateduce the infective bacterial process [104], the protectivectivity of the phenothiazine is not related to that producedn vitro.

.3.2. Ex vivoIn contrast to the in vitro activity, the enhancement of

illing of intracellular bacteria promoted by a phenothiazineas generally been considered to be due to concentration ofhe phenothiazine to a level that is compatible with the initro concentration that inhibits replication of the bacterium8,86,89]. However, because phenothiazines can inhibit anyransport process, including those for potassium (K+) anda2+ [49,105], and these ions are essential for acidificationf the phagolysosome and subsequent activation of its hydro-
ases [106], the possibility that enhanced killing is not dueo a concentration effect but is related to the inhibition of
+ and Ca2+ transport processes has been considered andnvestigated [107]. Although the concentrating effect cannot

waaF

timicrobial Agents 31 (2008) 198–208 201

e ruled out, recent experiments have shown that commonnhibitors of K+ and Ca2+ transport enhance the killing ofntracellular bacteria and support the contention that phe-othiazines enhance killing by the same mechanism [108].

.4. Efflux pump inhibitors (EPIs) of MDR bacteria

Antibiotic-sensitive bacteria that are gradually exposedo increasing concentrations of a given antibiotic developncreasing resistance to that antibiotic [109,110]. Accom-anying this induced resistance are increases in resistanceo other unrelated antibiotics [46,110]. This induced resis-ance can be reversed with the transfer of the bacterium to arug-free medium or by exposure of the bacterium to an EPIuch as phenylalanine arginyl �-naphthylamide (PA�N), phe-othiazines such as thioridazine and CPZ, carbonyl cyanide-chlorophenylhydrazone (CCCP) and reserpine [24,111].hese studies suggest that exposure to an antibiotic at con-entrations that do not completely inhibit replication of theacterium serves to induce the bacterium to withstand thection of the antibiotic even though its concentration has beenncreased. Development of a MDR phenotype suggests thathe development of multidrug resistance in Gram-negativeacteria in patients treated with subinhibitory doses of thentibiotic occurs via the same mechanism.

Nevertheless, the above studies only demonstrate an asso-iation of induced MDR phenotypes with the development ofn overexpressed putative efflux pump system. Recent studies46] have demonstrated that exposure of E. coli to increasingoncentrations of tetracycline (TET) results in significantlyncreased activity of genes that regulate genes coding forransporters of the resistance-nodulation-cell division (RND)uperfamily efflux pumps. The stress genes soxS and rob arencreased when the organism is first exposed to TET. Thiss followed by increased activity of marA, marB and marR,hich remain increased in activity during prolonged expo-

ure to increasing concentrations of TET, whereas those ofoxS and rob decrease to levels that approximate to that ofnexposed controls. Together with the increased activity ofegulator genes, there are increases in the activity of genes thatode for the two main transporters, AcrB and YhiV, whichemain at their highest level whilst the organism remainsultured in the presence of high concentrations of TET. Inter-stingly, the response to an antibiotic is not limited to thectivation of efflux pump genes. micF, a gene that downreg-lates the post-transcription of porins [111], is increased, asell as ompX, a gene that is considered to be a downregulatorf porins by interfering with their assembly [46]. Althoughnducement of TET resistance does not significantly alter thectivity of ompF and ompC genes, the amounts of the outerembrane proteins OmpF and OmpC are decreased, possibly

ue to the increased activity of genes that code for proteases
hich degrade these outer membrane proteins prior to their
ssembly into the tri-barrel porin [109]. However, we cannott this time rule out the possibility that downregulation of typeporin is due to the increased activity of the micF gene as

2 l of An

nbtrMroebtabtb

1

anbbsoeclt

maocapaeEstToo2aubnboleariip

pcatiMro[

ccistcatifmfimoplqctstOiimbcj[idopirbE

1ei

02 M. Martins et al. / International Journa

oted [46]. This gene produces a short anti-sense mRNA thatinds to the tail end of the OmpF mRNA, thereby preventingranslation of OmpF mRNA. Hence, increase of micF activityesults in less OmpF protein [112]. It may be concluded that

DR phenotypic resistance of a Gram-negative bacteriumesults from treatment of a patient with subinhibitory dosesf an antibiotic and that this MDR phenotype involves over-xpression of efflux pumps and downregulation of porins,oth systems working together to reduce the permeability ofhe organism to that antibiotic as well as to other unrelatedntibiotics. Because the MDR efflux pump can be inhibitedy a variety of agents, the use of EPIs as helper compoundso antibiotics to which the bacterium is initially resistant hasecome a focus of investigation.

.4.1. The search for EPIsEnterobacter aerogenes and Klebsiella pneumoniae are

mong the commonest Gram-negative bacteria involved inosocomial respiratory and urinary tract infections. Theseacteria exhibit a marked decrease in antibiotic suscepti-ility. In various clinical isolates, the MDR phenotype istrongly associated with a marked decrease in the synthesisf non-specific porins and the overproduction of active drugfflux systems. These modifications of envelope permeabilityontribute to a high level of resistance for structurally unre-ated molecules such as �-lactams, quinolones, macrolides,etracyclines and chloramphenicol [99,113].

Today, one challenge is to synthesise and characterise newolecules that are capable of circumventing efflux activity

nd restoring the internal concentration of common antibi-tics that are substrates of efflux pumps. In addition, theseompounds must be devoid of any intrinsic antibacterialctivity at the concentration used [46]. E. aerogenes and K.neumoniae clinical isolates that exhibit a MDR phenotypend active efflux mechanisms have been used to test sev-ral quinoline derivative molecules as chemosensitisers orPIs. This group of molecules has been selected due to theirtructural homology with the quinolones and with attentiono previous results obtained with resistant microbes [114].he respective minimum inhibitory concentrations (MICs)f these quinoline compounds are quite similar to thosebtained with the commercially available EPI PA�N (MC-07,110), which is the first inhibitor previously developedgainst Pseudomonas aeruginosa [115]. PA�N is currentlysed to detect efflux pump activities in various Gram-negativeacteria, including resistant E. aerogenes and K. pneumo-iae strains [115,116]. Of the quinoline derivatives that haveeen tested for their capability to decrease resistance, certainf them (belonging to alkoxyquinoline, alkylaminoquino-ine, thioalkoxyquinoline and chloroquinoline subclasses) arefficient chemosensitisers of chloramphenicol activity, withn 8- and 32-fold decrease in the chloramphenicol MIC in
esistant strains. In addition, they significantly increase thentracellular accumulation of chloramphenicol or norfloxacinn resistant bacteria that exhibit an overproduction of effluxumps, especially the AcrB pump in E. aerogenes and K.
hta

timicrobial Agents 31 (2008) 198–208

neumoniae. The subsequent increase of intracellular drugoncentration reached a level similar to that obtained withn energy poison which disrupts the inner membrane pro-on gradient required for the activity of the efflux pump,ndicating a clear effect on the efflux mechanism [116].

oreover, some of these compounds were also able toestore partially norfloxacin and tetracycline susceptibilityf resistant clinical isolates and of a resistant variant strain114,117].

The variation obtained in the level of restoration of sus-eptibility conferred by the quinoline derivatives to antibioticlasses could be attributed to the differences in the pump sitesnvolved in drug transport. It is possible that EPIs may havepecific effects on drug transport mechanisms depending onheir own affinity for specific residues located inside the pumpavity (affinity site). There are two possibilities that generatecollapse in drug transport. First, interactions resulting in

he pump–inhibitor complex may saturate all the drug affin-ty sites if the antibiotic and the inhibitor have equal affinityor the same sites. Alternatively, inhibitor–pump interactionsay induce steric hindrance if the respective binding sites

or antibiotic and inhibitor on the pump are in close proxim-ty [117]. It has been hypothesised previously that quinoline

olecules may act as competitive inhibitors of the antibi-tic flux that takes place inside the transporter, e.g. AcrBump [116]. This proposal has been supported by some pre-iminary results showing a relationship between the dose ofuinoline compound and the level of intracellular drug con-entration reached during the incubation time. However, athe moment no direct and clear assay is available to mea-ure and characterise the extrusion of intracellular antibiotichrough transporter channels present in resistant isolates.wing to the presence of active efflux mechanisms in var-

ous bacteria and the increase of resistant phenotypes, its necessary to develop an efficient assay to decipher the

olecular parameters of transporters [116]. This assay wille very useful for the determination of specificity, kineticonstants for various drugs and efficiency of EPIs. In con-unction with molecular modelling using crystallisation data118,119], these kinetic data will yield great improvementn the design of future efflux inhibitors. With the recentetermination of several three-dimensional (3D) structuresf efflux transporters [118,120,121], various models pro-osed for efflux activity and results obtained with currentnhibitors, it seems reasonable to propose that this approachegarding ‘pump–antibiotic–inhibitor’ interactions [117] wille used to improve the design of the new generation ofPIs.

.4.2. Could modulation of the tripartite CmeABC RNDfflux system in Campylobacter reduce the risk of humannfection?

Widespread use of antimicrobials in veterinary medicineas resulted in the emergence of strains of Campylobac-er displaying a MDR phenotype [122,123]. These strainsre being transmitted to humans, usually through the

l of An

cpratircnatino

C1dtexhqrri[ieTanMapntTi

drusrmR

sidiioei

frIpadsut

roeitetbtbeatt

1

rebifplacbBattm

dotaticm


onsumption of undercooked contaminated food, particularlyoultry. Of concern to public health is the increase in strainsesistant to fluoroquinolone and macrolide drugs, importantntibiotics used in the front-line treatment of campylobac-eriosis [124]. The tripartite RND efflux pump, CmeABC,s known to contribute both to the acquired and intrinsicesistance to these antimicrobials. Moreover, upregulation ofmeABC increases the extrusion rate of bile salts, probably theatural substrate for these pumps, giving rise to an improveddaptation of the organism in the avian gut [125]. An abilityo modulate chemotherapeutically the activity of RND pumpsn Campylobacter may provide a means of effectively elimi-ating these organisms from the avian gut, thereby reducingnward transmission to humans.

Multiple mechanisms of antibiotic resistance exist inampylobacter [126]. Efflux was recognised in the mid990s, and in 2002 the first RND efflux system wasescribed in Campylobacter jejuni and later in Campylobac-er coli [127–130]. The cmeABC system is constitutivelyxpressed in wild-type Campylobacter strains and has aenobiotic substrate profile. Although target gene mutationsave been recognised in these bacteria both for fluoro-uinolones (including the quinolone resistance-determiningegion (QRDR) of gyrA) and macrolides (domain V of the 23SRNA gene), there is a body of evidence demonstrating themportance of CmeABC in acquired and intrinsic resistance131,132]. The CmeABC efflux system in Campylobacters composed of three proteins, a CmeB energy-dependentfflux pump, the CmeC outer membrane channel (similar toolC in other Gram-negative strains including Salmonella)nd CmeA, the adaptor protein. All are regulated by a CmeRegative repressor, located proximal to these three genes.utations in cmeR give rise to overexpression of cmeABC

nd the associated MDR phenotype. Other putative effluxumps have been located in Campylobacter, but these doot appear to contribute to the acquired or intrinsic resis-ance to fluoroquinolones, macrolide, chloramphenicol orET [133,134]. Nevertheless, there may be a synergistic

nteraction that needs to be further studied.The availability of EPIs could be useful as a means of

irectly modulating the activity of these pumps, therebyestoring a susceptible phenotype [95]. PA�N is the onlyseful EPI identified to date and has been shown to increaseusceptibility to fluoroquinolones in one study. Others haveefuted this observation [130]. Nevertheless, site-directedutation of cmeB unequivocally established a role for thisND pump in resistance to these drugs.

Birds are the main reservoir of Campylobacter; as coloni-ation results in a harmless commensal relationship, unliken humans where infection occurs. The ability to colonise isependent on an organism’s ability to resist multiple stressesn the gastrointestinal tract, including the antibacterial activ-
ty of bile acids. Membrane-bound porins facilitate exchangesf small molecules between bacteria and their immediatenvironment. In C. jejuni, the major outer membrane porins expressed at higher levels at temperatures similar to those
csdt


ound in avians [135], suggesting that temperature-dependentegulation may be at least one important step in colonisation.n Gram-negative bacteria, efflux of bile salts from the cyto-lasm is the best characterised mechanism of bile resistance,nd in Campylobacter this is the only mechanism defined toate. Inactivation of cmeABC increases the susceptibility oftrains to bile acids. Furthermore, bile salts can also dereg-late the expression of cmeABC by reducing CmeR bindingo the promoter region.

Recognising the importance of efflux pumps and theirole in antibiotic/xenobiotic resistance in Campylobacter andther bacteria is important given the diminishing arsenal offfective drugs [99]. In the past, systematic screening hasdentified a number of possible EPI compounds. Althoughhese have been useful to define further the functioning offflux pumps, none are suitable for clinical application inheir current form. Nevertheless, rational design approachesased on better understanding of the 3D structure provideshe possibility of improved EPI designs in the future. It cane anticipated that not only would specific EPIs restore thefficacy of existing drugs, they may also be useful as feeddditives designed to decrease colonisation of the animal gas-rointestinal tract and thus reduce transmission to humans viahe food chain [124].

.5. Anti-plasmid activities of non-antibiotics

Antibiotic resistance is based on a complex of multifacto-ial processes in which the transfer of mobile genetic elementsncoding resistance further aggravates the spread of resistantacterial strains. Moreover, the use of antimicrobial drugsn clinical and veterinary medicine is a recognised drivingorce for the selection of resistant bacteria. This selectiveressure is a major contributor to the emergence and evo-ution of MDR phenotypic bacteria. Bacterial plasmids playmajor role in these processes with their ability to be repli-

ated independently from the chromosome and be transferredy conjugation from a host cell to a recipient bacterium.acterial resistance constitutes a considerable therapeuticnd economic burden, requiring new therapeutic approacheso overcome. Plasmid-mediated bacterial resistance may beackled by curing of these resistance-carrying genetic ele-

ents.For plasmid curing, replication should be inhibited at three

ifferent levels simultaneously: the intracellular replicationf plasmid DNA; partition; and intercellular transconjugalransfer. Various methods involving chemical and physicalgents have been tested for this purpose and have revealedhat super-optimal temperature, DNA intercalators (ethid-um bromide, acridine orange, acriflavine or surface-activeompounds, e.g. sodium dodecyl sulphate) are potent plas-id eliminators [136]. Based on these findings, Molnar and

olleagues carried out a systematic assay to determine thetructure–activity relationship among tricyclic antipsychoticrugs that interfere with the genetic elements encoding resis-ance [136–138]. Several members of this drug group and

2 l of An

tdseicntilpbdmaioi

csa1isaahtwatcrnTrtmavrRbtpftfiptinndc

rlm[

2

pMcp‘aNitb

EFFMP

R


heir derivatives, synthesised according to computer-aidedrug design, exerted anti-plasmid activity. The antidepres-ants possessing a secondary amine side chain are moreffective in inhibiting bacterial growth and in eliminat-ng plasmids than are the drugs with tertiary amine sidehains, whereas the plasmid-eliminating potency of quater-ary amines is weaker as they are probably unable to penetratehe cell membrane. Inhibition of plasmid replication resultedn a single nick outside the replication origin of the superhe-ical structure. The process leads to further relaxation of thelasmid DNA. Intercalation of the compounds was provedy the increase in the melting point of DNA and by circularichroism measurements [139,140]. When the native plas-id DNA and its promethazine complex were analysed by

garose gel electrophoresis, the superhelical form was miss-ng from the promethazine-treated plasmid DNA. The ratiof open circular and linear forms of plasmid DNA increasedn promethazine-treated samples.

It has been proposed that the HOMO orbital energy, theonjugated �-electron system of the tricyclic skeleton, theymmetric �-electron distribution to the L-molecular region,nd the superdelocalisability of the �-electron system on 10,2, 13 atoms have special importance in anti-plasmid activ-ty. In a computer-aided structure–activity relationship study,ome correlations were found between anti-plasmid effectnd the symmetry of the HOMO orbitals of phenothiazinesnd related compounds. The correlation coefficient was asigh as 0.97 when the electrophilic superdelocalisability ofhe heavy atoms of the tricyclics on atoms C8, C9b and N10ere taken into consideration. In this way, the anti-plasmid

nd carcinogenic molecular orbitals were clearly differen-iated [12,141–145]. A possible mechanism of action is aomplex formation of tricyclics with the guanine–cytosine-ich regions of the plasmid DNA, which are necessary forormal plasmid replication in an uncomplexed form [146].he use of phenothiazine drugs as resistance modifiers is

estricted owing to their toxicity, because the concentra-ions required for anti-plasmid effects are beyond that which

ay be clinically achievable; therefore, only limited datare available regarding their resistance-modifying activity inivo. Promethazine was studied in children with frequentlyecurring pyelonephritis, in combination with gentamicin.esults were positive; the combination reduced the num-er of recurrences of urinary tract infections compared withhe control group [79]. In another in vivo study, someatients recovered from urogenital infections despite theact that plasmid elimination in the urine did not occur andhe causative agents were resistant to gentamicin. Thesendings suggest that promethazine may affect the specificili-mediated and plasmid-encoded adhesion or multiplica-ion of bacteria on epithelial cells [147]. Promethazine andmipramine were investigated in the inhibition of adhesion of
ephropathogenic E. coli strains in tissue culture using scan-ing electron microscopy. It was found that in addition to theirect antibacterial effect, it can be presumed that a low con-entration of promethazine and imipramine can inhibit the

eversible and irreversible attachment of bacteria to epithe-ial cells, since both drugs interfere with the function of the

icrofilaments of cells and bacteria via membrane effects144,148,149].

. Concluding remarks

The group of compounds termed ‘non-antibiotics’ exhibitsroperties that render them important for the therapy ofDR infections. However, with the exception of infections

aused by MDR-TB and XDR-TB where some of these haveromise, non-antibiotics are at this time best considered ashelper compounds’ to be co-administered with conventionalntibiotics to which the MDR organism was initially resistant.evertheless, regardless of how certain we may feel regard-

ng their use, it still remains for clinical trials to demonstrateheir importance in the therapy of some of the most seriousacterial infections.

Funding: This work was partially supported by grantsU-FSE/FEDER-POCI/SAU-MMO/59370/2004 and EU-SE/FEDER-PTDC/BIA-MIC/71280/2006 provided by theundacao para a Ciencia e a Tecnologia (FCT) of Portugal.M was supported by grant SFRH/BD/14319/2003 (FCT,

ortugal).Competing interests: None declared.Ethical approval: Not required.

eferences

[1] Haug BE, Strom MB, Svendsen JS. The medicinal chemistry ofshort lactoferricin-based antibacterial peptides. Curr Med Chem2007;14:1–18.

[2] Nishimura T. Application and problems of combination drugtherapy—the combination of antibiotics and non-antibiotics. NipponRinsho 1986;44:936–41.

[3] Kristiansen JE, Amaral L. The potential management of resis-tant infections with non-antibiotics. J Antimicrob Chemother1997;40:319–27.

[4] Amaral L, Viveiros M, Kristiansen JE. ‘Non-antibiotics’: alter-native therapy for the management of MDRTB and MRSA ineconomically disadvantaged countries. Curr Drug Targets 2006;7:887–91.

[5] Amaral L, Lorian V. Effects of chlorpromazine on the cell enve-lope proteins of Escherichia coli. Antimicrob Agents Chemother1991;35:1923–4.

[6] Viveiros M, Amaral L. Enhancement of antibiotic activity againstpoly-drug resistant Mycobacterium tuberculosis by phenothiazines.Int J Antimicrob Agents 2001;17:225–8.

[7] Dutta NK, Annadurai S, Mazumdar K, Dastidar SG, Kristiansen JE,Molnar J, et al. Potential management of resistant microbial infectionswith a novel non-antibiotic: the anti-inflammatory drug diclofenacsodium. Int J Antimicrob Agents 2007;30:242–9.

[8] Amaral L, Martins M, Viveiros M. Enhanced killing of intracellu-lar multidrug-resistant Mycobacterium tuberculosis by compounds
that affect the activity of efflux pumps. J Antimicrob Chemother2007;59:1237–46.
[9] Amaral L, Kristiansen JE. Phenothiazines: potential management ofCreutzfeldt–Jacob disease and its variants. Int J Antimicrob Agents2001;18:411–17.

l of An
[10] Lopez-Munoz F, Alamo C, Cuenca E, Shen WW, Clervoy P, Rubio G.History of the discovery and clinical introduction of chlorpromazine.Ann Clin Psychiatry 2005;17:113–35.

[11] Amaral L, Kristiansen JE. Phenothiazines: an alternative to conven-tional therapy for the initial management of suspected multidrugresistant tuberculosis. A call for studies. Int J Antimicrob Agents2000;14:173–6.

[12] Amaral L, Viveiros M, Molnar J. Antimicrobial activity of phenoth-iazines. In Vivo 2004;18:725–31.

[13] World Health Organization/International Union Against Tuberculosisand Lung Disease Global Project on Anti-Tuberculosis Drug Resis-tance Surveillance. Anti-tuberculosis drug resistance in the world:report no. 3. Geneva, Switzerland: WHO; 2004.

[14] Amaral L, Kristiansen JE, Abebe LS, Millett W. Inhibition of the res-piration of multi-drug resistant clinical isolates of Mycobacteriumtuberculosis by thioridazine: potential use for initial therapy offreshly diagnosed tuberculosis. J Antimicrob Chemother 1996;38:1049–53.

[15] Martins M, Schelz Z, Martins A, Molnar J, Hajos G, Riedl Z, et al. Invitro and ex vivo activity of thioridazine derivatives against Mycobac-terium tuberculosis. Int J Antimicrob Agents 2007;29:338–40.

[16] Dutta NK, Mazumdar K, Dastidar SG, Chakrabarty AN, Shirataki Y,Motohashi N. In vitro and in vivo antimycobacterial activity of anantihypertensive agent methyl-L-DOPA. In Vivo 2005;19:539–45.

[17] Reddy MV, Nadadhur G, Gangadharam PR. In-vitro intracellularantimycobacterial activity of trifluoperazine. J Antimicrob Chemother1996;37:196–7.

[18] Gadre DV, Talwar V. In vitro susceptibility testing of Mycobacteriumtuberculosis strains to trifluoperazine. J Chemother 1999;11:203–6.

[19] Ratnakar P, Rao SP, Sriramarao P, Murthy PS.Structure–antitubercular activity relationship of phenothiazine-typecalmodulin antagonists. Int Clin Psychopharmacol 1995;10:39–43.

[20] Bate AB, Kalin JH, Fooksman EM, Amorose EL, Price CM, WilliamsHM, et al. Synthesis and antitubercular activity of quaternizedpromazine and promethazine derivatives. Bioorg Med Chem Lett2007;17:1346–8.

[21] Molnar J, Beladi I, Foldes I. Studies on antituberculotic action ofsome phenothiazine derivatives in vitro. Zentralbl Bakteriol [Orig A]1977;239:521–6.

[22] Bettencourt MV, Bosne-David S, Amaral L. Comparative in vitroactivity of phenothiazines against multidrug-resistant Mycobacteriumtuberculosis. Int J Antimicrob Agents 2000;16:69–71.

[23] Chattopadhyay D, Mukherjee T, Pal P, Saha B, Bhadra R. Alteredmembrane permeability as the basis of bactericidal action of methdi-lazine. J Antimicrob Chemother 1998;42:83–6.

[24] Kristiansen MM, Leandro C, Ordway D, Martins M, Viveiros M,Pacheco T, et al. Thioridazine reduces resistance of methicillin-resistant Staphylococcus aureus by inhibiting a reserpine-sensitiveefflux pump. In Vivo 2006;20:361–6.

[25] Shibl AM, Hammouda Y, Al-Sowaygh I. Comparative effects ofselected phenothiazine tranquilizers and antihistaminics on bacte-rial cells and possible interactions with antibiotics. J Pharm Sci1984;73:841–3.

[26] Kristiansen JE, Blom J. Effect of chlorpromazine on the ultrastruc-ture of Staphylococcus aureus. Acta Pathol Microbiol Scand [B]1981;89:399–405.

[27] Radhakrishnan V, Ganguly K, Ganguly M, Dastidar SG, ChakrabartyAN. Potentiality of tricyclic compound thioridazine as an effectiveantibacterial and antiplasmid agent. Indian J Exp Biol 1999;37:671–5.

[28] Jeyaseeli L, Gupta AD, Asok Kumar K, Mazumdar K, Dutta NK,Dastidar SG. Antimicrobial potentiality of the thioxanthene flu-penthixol through extensive in vitro and in vivo experiments. Int J
Antimicrob Agents 2006;27:58–62.
[29] Hendricks O, Butterworth TS, Kristiansen JE. The in-vitro antimicro-bial effect of non-antibiotics and putative inhibitors of efflux pumps onPseudomonas aeruginosa and Staphylococcus aureus. Int J Antimi-crob Agents 2003;22:262–4.


[30] Mazumder R, Ganguly K, Dastidar SG, Chakrabarty AN. Trifluoper-azine: a broad spectrum bactericide especially active on staphylococciand vibrios. Int J Antimicrob Agents 2001;18:403–6.

[31] Dastidar SG, Debnath S, Mazumdar K, Ganguly K, ChakrabartyAN. Triflupromazine: a microbicide non-antibiotic compound. ActaMicrobiol Immunol Hung 2004;51:75–83.

[32] Kaatz GW, Moudgal VV, Seo SM, Kristiansen JE. Phenothiazines andthioxanthenes inhibit multidrug efflux pump activity in Staphylococ-cus aureus. Antimicrob Agents Chemother 2003;47:719–26.

[33] Mazumder R, Chaudhuri SR, Mazumder A. Antimicrobial potential-ity of a phenothiazine group of antipsychotic drug—prochlorperazine.Indian J Exp Biol 2002;40:828–30.

[34] Kruszewska H, Zareba T, Tyski S. Search of antimicrobial activity ofselected non-antibiotic drugs. Acta Pol Pharm 2002;59:436–9.

[35] Dastidar SG, Ganguly K, Chaudhuri K, Chakrabarty AN. The anti-bacterial action of diclofenac shown by inhibition of DNA synthesis.Int J Antimicrob Agents 2000;14:249–51.

[36] Karaka P, Kumar KA, Basu LR, Dasgupta A, Ray R, DastidarSG. Experimental analysis of antimicrobial action of dicyclominehydrochloride. Biol Pharm Bull 2004;27:2010–13.

[37] Dastidar SG, Saha PK, Sanyamat B, Chakrabarty AN. Antibacterialactivity of ambodryl and benadryl. J Appl Bacteriol 1976;41:209–14.

[38] Dastidar SG, Chaudhury A, Annadurai S, Roy S, Mookerjee M,Chakrabarty AN. In vitro and in vivo antimicrobial action offluphenazine. J Chemother 1995;7:201–6.

[39] Orlowski M, Goldman M. Action of chlorpromazine on spore-forming Bacillus species. Can J Microbiol 1974;20:1689–93.

[40] Chan YY, Ong YM, Chua KL. Synergistic interaction betweenphenothiazines and antimicrobial agents against Burkholderia pseu-domallei. Antimicrob Agents Chemother 2007;51:623–30.

[41] Dastidar SG, Jairaj J, Mookerjee M, Chakrabarty AN. Studies onantimicrobial effect of the antihistaminic phenothiazine trimeprazinetartrate. Acta Microbiol Immunol Hung 1997;44:241–7.

[42] Klubes P, Fay PJ, Cerna I. Effect of chlorpromazine on cell wallbiosynthesis and incorporation of orotic acid into nucleic acids inBacillus megaterium. Biochem Pharmacol 1971;20:265–77.

[43] Kawase M, Motohashi N, Sakagami H, Kanamoto T, Nakashima H,Ferenczy L, et al. Antimicrobial activity of trifluoromethyl ketonesand their synergism with promethazine. Int J Antimicrob Agents2001;18:161–5.

[44] Mortensen I, Kristiansen JE, Christensen AV, Hvidberg EF. Theantibacterial effect of some neuroleptics on strains isolated frompatients with meningitis. Pharmacol Toxicol 1992;71:449–51.

[45] Kristiansen JE, Hendricks O, Delvin T, Butterworth TS, Aagaard L,Christensen JB, et al. Reversal of resistance in microorganisms byhelp of non-antibiotics. J Antimicrob Chemother 2007;59:1271–9.

[46] Viveiros M, Dupont M, Rodrigues L, Couto I, Davin-Regli A, MartinsM, et al. Antibiotic stress, genetic response and altered permeabilityof E. coli. PLoS ONE 2007;2:e365.

[47] Labedan B. Increase in permeability of Escherichia coli outer mem-brane by local anesthetics and penetration of antibiotics. AntimicrobAgents Chemother 1988;32:153–5.

[48] Molnar J, Schneider B. Plasmid curing and antibacterial effects ofsome chlorpromazine derivatives in relation to their molecule orbitals.Acta Microbiol Acad Sci Hung 1978;25:291–8.

[49] Chukhlova EA, Sadykov IuKh, Kholmukhamedov EL, Grenader AK.Effect of trimecaine, ajmaline, stenopril and chloracizine on fluc-tuations in K+ currents in rat liver mitochondria. Ukr Biokhim Zh1984;56:207–10 [in Russian].

[50] Spengler G, Miczak A, Hajdu E, Kawase M, Amaral L, MolnarJ. Enhancement of plasmid curing by 9-aminoacridine and twophenothiazines in the presence of proton pump inhibitor 1-(2-
benzoxazolyl)-3,3,3-trifluoro-2-propanone. Int J Antimicrob Agents2003;22:223–7.
[51] Heller CS, Sevag MG. Prevention of the emergence of drug resistancein bacteria by acridines, phenothiazines, and dibenzocycloheptenes.Appl Microbiol 1966;14:879–85.

2 l of An
[52] Lehtinen J, Lilius EM. Promethazine renders Escherichia coli suscep-tible to penicillin G: real-time measurement of bacterial susceptibilityby fluoro-luminometry. Int J Antimicrob Agents 2007;30:44–51.

[53] Gunics G, Motohashi N, Amaral L, Farkas S, Molnar J. Interactionbetween antibiotics and non-conventional antibiotics on bacteria. IntJ Antimicrob Agents 2000;14:239–42.

[54] Mazumdar K, Dutta NK, Dastidar SG, Motohashi N, Shirataki Y.Diclofenac in the management of E. coli urinary tract infections. InVivo 2006;20:613–9.

[55] Chattopadhyay D, Dastidar SG, Chakrabarty AN. Antimicrobialproperties of methdilazine and its synergism with antibiotics andsome chemotherapeutic agents. Arzneimittelforschung 1988;38:869–72.

[56] Amaral L, Kristiansen JE, Frolund Thomsen V, Markovich B. Theeffects of chlorpromazine on the outer cell wall of Salmonellatyphimurium in ensuring resistance to the drug. Int J AntimicrobAgents 2000;14:225–9.

[57] Dash SK, Dastidar SG, Chakrabarty AN. Antibacterial property ofpromazine hydrochloride. Indian J Exp Biol 1977;15:324–6.

[58] Dastidar SG, Chakraborty P, Mookerjee M, Ganguly M, ChakrabartyAN. Studies on the existence of synergism between differentantibiotics and a phenothiazine tranquilizer, promazine, possess-ing antimicrobial property. Acta Microbiol Immunol Hung 1994;41:41–9.

[59] McGuigan JE, Berry WC, Carlquist PR. Comparison of chlo-ramphenicol, oxytetracycline, and prochlorperazine in Salmonellagastroenteritis. U S Armed Forces Med J 1960;11:1288–93.

[60] Annadurai S, Guha-Thakurta A, Sa B, Dastidar SG, Ray R,Chakrabarty AN. Experimental studies on synergism between amino-glycosides and the antimicrobial antiinflammatory agent diclofenacsodium. J Chemother 2002;14:47–53.

[61] Annadurai S, Basu S, Ray S, Dastidar SG, Chakrabarty AN. Antibac-terial activity of the antiinflammatory agent diclofenac sodium. IndianJ Exp Biol 1998;36:86–90.

[62] Karak P, Kumar KA, Mazumdar K, Mookerjee M, DastidarSG. Antibacterial potential of an antispasmodic drug dicyclominehydrochloride. Indian J Med Res 2003;118:192–6.

[63] Colombo CI. Antibacterial activity of diphenhydramine HCI. BollChim Farm 1962;101:715–20.

[64] Sack RB. Diarrhoea management: drug treatment. Dialogue Diar-rhoea 1986;25:5.

[65] Molnar J, Ren J, Kristiansen JE, Nakamura MJ. Effects ofsome tricyclic psychopharmacons and structurally related com-pounds on motility of Proteus vulgaris. Antonie Van Leeuwenhoek1992;62:319–20.

[66] Molnar J, Mandi Y, Kiraly J. Antibacterial effect of some phenoth-iazine compounds and R-factor elimination by chlorpromazine. ActaMicrobiol Acad Sci Hung 1976;23:45–54.

[67] Rabbani GH. Mechanism and treatment of diarrhoea due to Vibriocholerae and Escherichia coli: roles of drugs and prostaglandins. DanMed Bull 1996;43:173–85.

[68] Kristiansen JE, Gaarslev K. The antibacterial effect of selected neu-roleptics on Vibrio cholerae. Acta Pathol Microbiol Immunol Scand[B] 1985;93:49–51.

[69] Kruszewska H, Zareba T, Tyski S. Examination of antimicro-bial activity of selected non-antibiotic drugs. Acta Pol Pharm2004;61(Suppl.):18–21.

[70] Kumar KA, Ganguly K, Mazumdar K, Dutta NK, Dastidar SG,Chakrabarty AN. Amlodipine: a cardiovascular drug with powerfulantimicrobial property. Acta Microbiol Pol 2003;52:285–92.

[71] Bayes M, Rabasseda X, Prous JR. Gateways to clinical trials. MethodsFind Exp Clin Pharmacol 2002;24:431–55.

[72] Gunics G, Motohashi N, Molnar J, Farkas S, Kawase M, Saito S, et al.Enhanced antibacterial effect of erythromycin in the presence of 3,5-dibenzoyl-1,4-dihydropyridines. Anticancer Res 2001;21:269–73.

[73] Gunics G, Farkas S, Motohashi N, Shah A, Harsukh G, Kawase M, etal. Interaction between 3,5-diacetyl-1,4-dihydropyridines and ampi-


cillin, and erythromycin on different E. coli strains. Int J AntimicrobAgents 2002;20:227–9.

[74] Mazumdar K, Dutta NK, Kumar KA, Dastidar SG. In vitro and invivo synergism between tetracycline and the cardiovascular agentoxyfedrine HCl against common bacterial strains. Biol Pharm Bull2005;28:713–17.

[75] Mazumdar K, Ganguly K, Kumar KA, Dutta NK, Chakrabarty AN,Dastidar SG. Antimicrobial potentiality of a new non-antibiotic:the cardiovascular drug oxyfedrine hydrochloride. Microbiol Res2003;158:259–64.

[76] Asok Kumar K, Mazumdar K, Dutta NK, Karak P, Dastidar SG, RayR. Evaluation of synergism between the aminoglycoside antibioticstreptomycin and the cardiovascular agent amlodipine. Biol PharmBull 2004;27:1116–20.

[77] Guha Thakurta AG, Mandal SK, Ganguly K, Dastidar SG,Chakrabarty AN. A new powerful antibacterial synergistic combi-nation of trimethoprim and trimeprazine. Acta Microbiol ImmunolHung 2000;47:21–8.

[78] Komatsu N, Motohashi N, Fujimaki M, Molnar J. Inductionof a protective immunity in mice against Escherichia coli byphenothiazines,10-[n-(phthalimido)alkyl]-2-substituted-10H-pheno-thiazines and 1-(2-chloroethyl)-3-(2-substituted-10H-phenothia-zines-10-yl)alkyl-1-ureas. In Vivo 1997;11:13–16.

[79] Molnar J, Haszon I, Bodrogi T, Martonyi E, Turi S. Synergistic effectof promethazine with gentamycin in frequently recurring pyelonephri-tis. Int Urol Nephrol 1990;22:405–11.

[80] Gadre DV, Talwar V, Gupta HC, Murthy PS. Effect of trifluoperazine, apotential drug for tuberculosis with psychotic disorders, on the growthof clinical isolates of drug resistant Mycobacterium tuberculosis. IntClin Psychopharmacol 1998;13:129–31.

[81] Ratnakar P, Murthy PS. Trifluoperazine inhibits the incorporation oflabelled precursors into lipids, proteins and DNA of Mycobacteriumtuberculosis H37Rv. FEMS Microbiol Lett 1993;110:291–4.

[82] Chakrabarty AN, Bhattacharya CP, Dastidar SG. Antimycobacterialactivity of methdilazine (Md), an antimicrobic phenothiazine. APMIS1993;101:449–54.

[83] Ratnakar P, Murthy PS. Antitubercular activity of trifluoper-azine, a calmodulin antagonist. FEMS Microbiol Lett 1992;76:73–6.

[84] Madrid PB, Polgar WE, Toll L, Tanga MJ. Synthesis and anti-tubercular activity of phenothiazines with reduced binding todopamine and serotonin receptors. Bioorg Med Chem Lett 2007;17:3014–17.

[85] Yano T, Li LS, Weinstein E, Teh JS, Rubin H. Steady-state kinetics andinhibitory action of antitubercular phenothiazines on Mycobacteriumtuberculosis type-II NADH-menaquinone oxidoreductase (NDH-2).J Biol Chem 2006;281:11456–63.

[86] Ordway D, Viveiros M, Leandro C, Bettencourt R, Almeida J, Mar-tins M, et al. Clinical concentrations of thioridazine kill intracellularmultidrug-resistant Mycobacterium tuberculosis. Antimicrob AgentsChemother 2003;47:917–22.

[87] Daniel WA, Wojcikowski J. The role of lysosomes in the cellulardistribution of thioridazine and potential drug interactions. ToxicolAppl Pharmacol 1999;158:115–24.

[88] Saunders BM, Britton WJ. Life and death in the granu-loma: immunopathology of tuberculosis. Immunol Cell Biol2007;85:103–11.

[89] Crowle AJ, Douvas GS, May MH. Chlorpromazine: a drug poten-tially useful for treating mycobacterial infections. Chemotherapy1992;38:410–19.

[90] Martins M, Viveiros M, Kristiansen JE, Molnar J, Amaral L. Thecurative activity of thioridazine on mice infected with Mycobacterium
tuberculosis. In vivo 2007;21:771–6.
[91] Weinstein EA, Yano T, Li LS, Avarbock D, Avarbock A, HelmD, et al. Inhibitors of type II NADH:menaquinone oxidoreductaserepresent a class of antitubercular drugs. Proc Natl Acad Sci USA2005;102:4548–53.

l of An
[92] Amaral L, Martins M, Viveiros M. Phenothiazines as anti-multi-drug resistant tubercular agents. Infect Disord Drug Targets2007;7:257–65.

[93] Weiss B, Prozialeck W, Cimino M, Barnette MS, Wallace TL.Pharmacological regulation of calmodulin. Ann N Y Acad Sci1980;356:319–45.

[94] Garcia JJ, Tuena de Gomez-Puyou M, Gomez-Puyou A. Inhibitionby trifluoperazine of ATP synthesis and hydrolysis by particulateand soluble mitochondrial F1: competition with H2PO4

−. J BioenergBiomembr 1995;27:127–36.

[95] Piddock LJV. Clinically relevant chromosomally encoded mul-tidrug resistance efflux pumps in bacteria. Clin Microbiol Rev2006;19:382–402.

[96] Kristiansen MM, Leandro C, Ordway D, Martins M, Viveiros M,Pacheco T, et al. Phenothiazines alter resistance of methicillin-resistant strains of Staphylococcus aureus (MRSA) to oxacillin invitro. Int J Antimicrob Agents 2003;22:250–3.

[97] Michalak K, Wesolowska O, Motohashi N, Molnar J, HendrichAB. Interactions of phenothiazines with lipid bilayer and theirrole in multidrug resistance reversal. Curr Drug Targets 2006;7:1095–105.

[98] Kawase M, Motohashi N. New multidrug resistance reversal agents.Curr Drug Targets 2003;4:31–43.

[99] Piddock LJ. Multidrug-resistance efflux pumps—not just for resis-tance. Nat Rev Microbiol 2006;4:629–36.

[100] Plishker GA. Phenothiazine inhibition of calmodulin stimulatescalcium-dependent potassium efflux in human red blood cells. CellCalcium 1984;5:177–85.

[101] Molnar J, Hever A, Fakla I, Fischer J, Ocsovski I, Aszalos A.Inhibition of the transport function of membrane proteins by somesubstituted phenothiazines in E. coli and multidrug resistant tumorcells. Anticancer Res 1997;17:481–6.

[102] Bhatnagar K, Singh VP. Ca2+-dependence and inhibition of transfor-mation by trifluoperazine and chlorpromazine in Thermoactinomycesvulgaris. Curr Microbiol 2003;46:265–9.

[103] Rohs R, Sklenar H. Methylene blue binding to DNA with alternatingAT base sequence: minor groove binding is favored over intercalation.J Biomol Struct Dyn 2004;21:699–711.

[104] Mengozzi M, Fantuzzi G, Faggioni R, Marchant A, Goldman M,Orencole S, et al. Chlorpromazine specifically inhibits peripheral andbrain TNF production, and up-regulates IL-10 production, in mice.Immunology 1994;82:207–10.

[105] Kim KS, Kim EJ. The phenothiazine drugs inhibit hERG potassiumchannels. Drug Chem Toxicol 2005;28:303–13.

[106] Pillay CS, Elliott E, Dennison C. Endolysosomal proteolysis and itsregulation. Biochem J 2002;363:417–29.

[107] Wittekindt OH, Schmitz A, Lehmann-Horn F, Hansel W, Griss-mer S. The human Ca2+-activated K+ channel, IK, can be blockedby the tricyclic antihistamine promethazine. Neuropharmacology2006;50:458–67.

[108] Ahluwalia J, Tinker A, Clapp LH, Duchen MR, Abramov AY, PopeS, et al. The large-conductance Ca2+-activated K+ channel is essentialfor innate immunity. Nature 2004;427:853–8.

[109] Viveiros M, Jesus A, Brito M, Leandro C, Martins M, Ordway D, et al.Inducement and reversal of tetracycline resistance in Escherichia coliK-12 and expression of proton gradient-dependent multidrug effluxpump genes. Antimicrob Agents Chemother 2005;49:3578–82.

[110] Martins A, Couto I, Aagaard L, Martins M, Viveiros M, KristiansenJE, et al. Prolonged exposure of methicillin-resistant Staphylococcusaureus (MRSA) COL strain to increasing concentrations of oxacillinresults in a multidrug-resistant phenotype. Int J Antimicrob Agents2007;29:302–5.

[111] Guillier M, Gottesman S, Storz G. Modulating the outer membranewith small RNAs. Genes Dev 2006;20:2338–48.

[112] Chen S, Zhang A, Blyn LB, Storz G. MicC, a second small-RNA reg-ulator of Omp protein expression in Escherichia coli. J Bacteriology2004;186:6689–97.


[113] Poole K. Efflux-mediated antimicrobial resistance. J AntimicrobChemother 2005;56:20–51.

[114] Mahamoud A, Chevalier J, Davin-Regli A, Barbe J, Pages JM. Quino-line derivatives as promising inhibitors of antibiotic efflux pump inmultidrug resistant Enterobacter aerogenes isolates. Curr Drug Tar-gets 2006;7:843–7.

[115] Lomovskaya O, Bostian KA. Practical applications and feasibility ofefflux pump inhibitors in the clinic—a vision for applied use. BiochemPharmacol 2006;71:910–18.

[116] Pages JM, Masi M, Barbe J. Inhibitors of efflux pumps in Gram-negative bacteria. Trends Mol Med 2005;11:382–9.

[117] Mahamoud A, Chevalier J, Alibert-Franco S, Kern WV, Pages JM.Antibiotic efflux pumps in Gram-negative bacteria: the inhibitorresponse strategy. J Antimicrob Chemother 2007;59:1223–9.

[118] Higgins CF. Multiple molecular mechanisms for multidrug resistancetransporters. Nature 2007;446:749–57.

[119] Lomovskaya O, Zgurskaya HI, Totrov M, Watkins WJ. Waltzingtransporters and ‘the dance macabre’ between humans and bacteria.Nat Rev Drug Discov 2007;6:56–65.

[120] Murakami S, Nakashima R, Yamashita E, Matsumoto T, YamaguchiA. Crystal structures of a multidrug transporter reveal a functionallyrotating mechanism. Nature 2006;443:173–9.

[121] Seeger MA, Schiefner A, Eicher T, Verrey F, Diederichs K, Pos KM.Structural asymmetry of AcrB trimer suggests a peristaltic pumpmechanism. Science 2006;313:1295–8.

[122] Aarestrup FM, Engberg J. Antimicrobial resistance of thermophilicCampylobacter. Vet Res 2001;32:311–21.

[123] Angulo FJ, Nargund VN, Chiller TC. Evidence of an associationbetween use of anti-microbial agents in food animals and anti-microbial resistance among bacteria isolated from humans and thehuman health consequences of such resistance. J Vet Med B InfectDis Vet Public Health 2004;51:374–9.

[124] Quinn T, Bolla J-M, Pages J-M, Fanning S. Antibiotic-resistantCampylobacter: could efflux pump inhibitors control infection? JAntimicrob Chemother 2007;59:1230–6.

[125] Lin J, Cagliero C, Guo B, Barton YW, Maurel MC, Payot S, et al. Bilesalts modulate expression of the CmeABC multidrug efflux pump inCampylobacter jejuni. J Bacteriol 2005;187:7417–24.

[126] Taylor DE, Courvalin P. Mechanisms of antibiotic resistancein Campylobacter species. Antimicrob Agents Chemother1988;32:1107–12.

[127] Pumbwe L, Piddock LJ. Identification and molecular characterisa-tion of CmeB, a Campylobacter jejuni multidrug efflux pump. FEMSMicrobiol Lett 2002;206:185–9.

[128] Lin J, Michel LO, Zhang Q. CmeABC functions as a multidrug effluxsystem in Campylobacter jejuni. Antimicrob Agents Chemother2002;46:2124–31.

[129] Corcoran D, Quinn T, Cotter L, O’Halloran F, Fanning S.Characterization of a cmeABC operon in a quinolone-resistant Campy-lobacter coli isolate of Irish origin. Microb Drug Resist 2005;11:303–8.

[130] Corcoran D, Quinn T, Cotter L, Fanning S. Relative contributionof target gene mutation and efflux to varying quinolone resistancein Irish Campylobacter isolates. FEMS Microbiol Lett 2005;253:39–46.

[131] Corcoran D, Quinn T, Cotter L, Fanning S. An investigation of themolecular mechanisms contributing to high-level erythromycin resis-tance in Campylobacter. Int J Antimicrob Agents 2006;27:40–5.

[132] Gibreel A, Taylor DE. Macrolide resistance in Campylobacterjejuni and Campylobacter coli. J Antimicrob Chemother 2006;58:243–55.

[133] Ge B, McDermott PF, White DG, Meng J. Role of efflux pumps
and topoisomerase mutations in fluoroquinolone resistance inCampylobacter jejuni and Campylobacter coli. Antimicrob AgentsChemother 2005;49:3347–54.
[134] Pumbwe L, Randall LP, Woodward MJ, Piddock LJ. Evidence formultiple-antibiotic resistance in Campylobacter jejuni not medi-

2 l of An
ated by CmeB or CmeF. Antimicrob Agents Chemother 2005;49:1289–93.

[135] Dedieu L, Pages J-M, Bolla J-M. Environmental regulation ofCampylobacter jejuni outer membrane protein porin expression inEscherichia coli monitored by green fluorescent protein. Appl EnvironMicrobiol 2002;68:4209–15.

[136] Mandi Y, Molnar J, Holland B, Beladi I. Efficient curing of anEscherichia coli F-prime plasmid by phenothiazines. Genet Res1976;26:109–11.

[137] Molnar J, Kiraly J, Mandi Y. The antibacterial action and R-factorinhibiting activity by chlorpromazine. Experientia 1975;31:444–5.

[138] Molnar J, Domonkos K, Mandi Y, Foldeak S, Holland IB. The possi-ble mechanism of plasmid elimination by phenothiazines and relateddrugs. In: Usdin E, Eckert H, Forrest IS, editors. Phenothiazines andstructurally related drugs: basic and clinical studies. Amsterdam, TheNetherlands: Elsevier-North Holland; 1980. p. 115–18.

[139] Nikaido H. Preventing drug access to targets: cell surface perme-ability barriers and active efflux in bacteria. Semin Cell Dev Biol2001;12:215–23.

[140] Barabas K, Molnar J. Lack of correlation between intercalation and
plasmid curing ability of some tricyclic compounds. Acta MicrobiolAcad Sci Hung 1980;27:55–61.
[141] Motohashi N, Sakagami H, Kurihara T, Csuri K, Molnar J.Antiplasmid activity of phenothiazines, benzo[a]phenothiazines andbenz[c]acridines. Anticancer Res 1992;12:135–40.


[142] Tanaka M, Wayda K, Molnar J, Parkanyi C, Aaron JJ, Motohashi N.Antimutagenicity of benzo[a]phenothiazines in chemically inducedmutagenesis. Anticancer Res 1997;17:839–42.

[143] Motohashi N, Kawase M, Saito S, Miskolci CS, Berek L, MolnarJ. Plasmid elimination and immunomodulation by 3-benzazepines invitro. Anticancer Res 1999;19:5075–8.

[144] Molnar J, Foldeak S, Nakamura MJ, Rausch H, Domonkos K, SzaboM. Antiplasmid activity: loss of bacterial resistance to antibiotics.APMIS Suppl 1992;30:24–31.

[145] Spengler G, Molnar A, Schelz Z, Amaral L, Sharples D, Molnar J.The mechanism of plasmid curing in bacteria. Curr Drug Targets2006;7:823–41.

[146] Miskolci C, Labadi I, Kurihara T, Motohashi N, Molnar J.Guanine–cytosine rich regions of plasmid DNA can be the targetin anti-plasmid effect of phenothiazines. Int J Antimicrob Agents2000;14:243–7.

[147] Kasler M, Poczik M, Molnar J, Agoston E. A Pipolphen plazmidtorlohatasanak vizsgalata urogenitalis fertozesek eseten. Urol NephrolSzemle 1982;9:130–3.

[148] Molnar J, Mucsi I, Kasa P. Inhibition of the adhesion of E. coli
on cultured human epithelial cells in the presence of promet-hazine or imipramine. Zentralbl Bakteriol Mikrobiol Hyg [A]1983;254:388–96.
[149] Hirota Y. The effect of acridine dyes on mating type factors inEscherichia coli. Proc Natl Acad Sci USA 1960;46:57–64.

Documents

Potential role of non-antibiotics (helper compounds) in the treatment of multidrug-resistant Gram-negative infections: mechanisms for their direct and indirect activities