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Polymyxin B, in combination with fluconazole, exerts a potentfungicidal effect

Bing Zhai 1, Henry Zhou2, Liangpeng Yang3, Jun Zhang 2, Kathy Jung4, Chou-Zen Giam3, Xin Xiang2

and Xiaorong Lin1*

1Department of Biology, Texas A&M University, College Station, TX, USA; 2Department of Biochemistry and Molecular Biology, UniformedServices University of the Health Sciences, Bethesda, MD, USA; 3Department of Microbiology and Immunology, Uniformed Services

University of the Health Sciences, Bethesda, MD, USA; 4National Institute on Alcohol Abuse and Alcoholism, Rockville, MD, USA

*Corresponding author. Department of Biology, Texas A&M University, 3258 TAMU, College Station, TX 77843-3258, USA. Tel: 1-979-845-7274;Fax: 1-979-845-2891; E-mail: [email protected]

These authors contributed equally to this study.

Received 4 November 2009; returned 18 December 2009; revised 27 January 2010; accepted 28 January 2010

Objectives: The objective of this study was to identify existing clinical compounds that either possess a fungi-cidal activity alone or can act synergistically with fungistatic antifungals.

Methods: We screened a clinical compound library for drugs that exhibited anti-Aspergillus activity. Amongselected compounds, the cationic peptide antibiotic polymyxin B was chosen for further characterizationbecause it can be used parenterally and topically. The fungicidal effect of polymyxin B and its synergistic inter-actions with azole antifungals were tested against a variety of fungal species. The toxicity of the drug combi-nation of polymyxin B and fluconazole was compared with that of each drug alone in mammalian cell cultures.

Results: We found that polymyxin B possesses a broad-spectrum antifungal activity at relatively high concen-trations. However, because of its synergistic interactions with azole antifungals, polymyxin B at much lowerconcentrations exerts a potent fungicidal effect against Cryptococcus neoformans, Candida albicans andnon-albicans Candida species and moulds when combined with azoles. The combination of polymyxin B andfluconazole at concentrations within susceptible breakpoints is particularly potent against C. neoformans iso-

lates, including fluconazole-resistant strains. The drug combination displayed no additional toxicity comparedwith polymyxin B alone when tested in cell culture.

Conclusions: The combination of polymyxin B and fluconazole has the potential to be used in the clinic to treatsystemic cryptococcosis. Our findings suggest that combining cationic peptide antibiotics with azole antifungalscould provide a new direction for developing novel antifungal therapies.

Keywords: antifungal, combination therapy, antibiotic, fungal membrane

Introduction

Systemic fungal infections have drastically increased over thepast three decades due to the rising immunocompromised

population as a result of transplantation, cancer chemotherapy,steroid therapy and, in particular, HIV infection (AIDS).15 Unfor-tunately, the outcome of current antifungal therapy is far fromsatisfactory. For example, the three major global invasivemycoses, aspergillosis, candidiasis and cryptococcosis causedby fungal species of Aspergillus, Candida and Cryptococcus,respectively, typically have mortality rates ranging from 10% to90%.613 This poor outcome is in part due to the limitednumber of clinically available antifungals and the fact that manyantifungals either lack potency or are toxic to the host.1416

The emergence of resistant fungal strains to current antifungals,which is exacerbated by the necessity for long-term usage ofantifungals in immunocompromised individuals, causesadditional difficulty in treatment.15,1724 For example, a recent

global survey of close to 3000 Cryptococcus neoformans isolatesindicated that .11% of the isolates are resistant to flucona-zole.25 Therefore, there is an urgent need for new therapies.

Through a screen of a clinical compound library, we havefound that polymyxin B, an antibiotic used to treat bacterialinfections, possesses fungicidal activity. Similar to what hasbeen observed previously,2628 polymyxin B is fungicidal at rela-tively high concentrations. Interestingly, in combination with flu-conazole or itraconazole, polymyxin B at low concentrationsdemonstrates a killing effect against Aspergillus fumigatus,

# The Author 2010. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.For Permissions, please e-mail: [email protected]

J Antimicrob Chemother 2010; 65: 931938doi:10.1093/jac/dkq046 Advance publication 18 February 2010

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Rhizopus oryzae, Candida albicans and non-albicans Candidaspecies. The combination at clinically relevant low concen-trations is particularly potent against C. neoformans, includingstrains of varied resistance to fluconazole.

Materials and methodsStrains and media

For the initial drug screening, the Aspergillus nidulans strain R2129 wasgrown on YAG medium (0.5% yeast extract, 2% agar and 2% glucose).For microscopic analyses of the effects of drugs, R21 and the A. fumigatusstrain B523330 were grown on YG liquid medium (0.5% yeast extract and2% glucose) supplemented with 0.12% uridine and uracil (YUU). Allyeast strains were maintained on yeast peptone dextrose (YPD)medium. Drug disc diffusion and microdilution assays were performedusing RPMI 1640 medium buffered with MOPS.

Clinical compound library screening

The Johns Hopkins Clinical Compound Library (JHCCL version 1.0), a col-

lection of 1514 FDA-approved (1082) and foreign approved (432) drugs(FAD),31 was screened for drugs with an inhibitory effect on A. nidulanscolony growth. The library was assembled in a 96-well plate formatwith 25 mL aliquots of 10 mM stocks of each compound in either wateror DMSO. An aliquot of 2 mL from each well was spotted onto a YAGplate and 1105 spores ofA. nidulans strain R21 were point-inoculatedonto the spots of the original drug application. The cells were incubatedat 378C for 2 days. Drugs that either significantly or completely inhibitedA. nidulans colony formation were selected. Selected drugs at 2 mM weretested again for an inhibitory effect on A. nidulans in liquid medium.A. nidulans spores (105) were inoculated into 500 mL of drug-containingliquid medium and incubated at 378C overnight in the 8-chamberedLab-Tek Borosilicate Coverglass System. Twenty-three compounds thatsignificantly inhibited colony growth of A. nidulans strain R21 at this con-centration were identified. Inhibition of spore germination or germ tube

extension of the A. nidulans and A. fumigatus isolates was examinedmicroscopically in the presence of polymyxin B at the indicated concen-trations for the time period indicated in the text. Viability of those inhib-ited cells was examined microscopically after they had been cultured indrug-free medium for 24 h.

Disc diffusion halo assay for antifungal activity

Briefly, yeast cells at a cell density of5106 were spread onto RPMI1640 agar medium with L-glutamine and without sodium bicarbonate.The plates were allowed to solidify and dry. Whatman paper discs(7 mm) containing water, fluconazole, polymyxin B and their combi-nation at various concentrations were dried and placed on the solidifiedagar surface. The cells were incubated for 2448 h at 378C.

Microdilution assay for antifungal activity

The microdilution assay was performed according to the CLSI (formerlyNCCLS) standard32 except that the cells were incubated at 378C. Briefly,yeast cells at a final concentration of 1103 cells/mL (or Aspergillusor Rhizopus spores at a final concentration of5104 cells/mL) wereinoculated in the RPMI 1640 liquid medium with serial (2) dilutions ofeach drug being tested. The concentrations used for polymyxin B and flu-conazole are indicated in the text. Wells that contained no drugs or noyeast inoculation were included as positive and negative controls. TheMIC100 of polymyxin B or the combination was defined as the lowestdrug concentration that resulted in a 100% decrease in absorbancecompared with that of the control in drug-free medium. The MIC 90 of

fluconazole was defined as the lowest drug concentration that resultedin a 90% decrease in absorbance compared with that of the control indrug-free medium. MICs were read after incubation without agitationfor 24 h for Candida strains and 48 h for the Aspergillus, Rhizopus orCryptococcus isolates. Fungicidal effect was examined by counting thecfu of the suspension from each well after plating it onto the YPD

medium and incubating for 24 (for Candida strains) to 48 h (forCryptococcus, Aspergillus and Rhizopus strains). The minimal fungicidalconcentration (MFC) was defined as the minimal drug concentrationthat causes at least 99% of cells to be killed compared with the originalinoculum. A synergistic fungicidal effect between fluconazole and poly-myxin B for each strain was calculated based on the fractional fungicidalconcentration index (FFCI), FFCI[F]/MFCF[P]/MFCP, where MFCF andMFCP are the MFCs of fluconazole and polymyxin B, respectively, and [F]and [P] are the concentrations at which fluconazole and polymyxin B,in combination, are fungicidal. FFCIs 0.5 indicate synergistic inter-actions, FFCIs .0.5 4.0 indicate no interaction and FFCIs.4.0 indicateantagonistic interactions.33

Generation of fluconazole-resistant H99FR

C. neoformans H99 reference strain was cultured in yeast nitrogen base(YNB) liquid medium with fluconazole at an inhibitory but sublethal con-centration of 2.4 mg/L. The culture was maintained at 378C with shaking.An aliquot of the culture was transferred to fresh YNB medium with flu-conazole every other day and continued for 16 weeks.

Proliferation of HeLa and human monocytic THP-1 cellsin the presence of the drugs

HeLa cells were grown in Dulbeccos modified Eagles medium (DMEM)supplemented with 10% fetal bovine serum, 2 mM L-glutamine and100 U/L each of penicillin and streptomycin. THP-1 cells were grown inRPMI 1640 medium with the same supplements. HeLa cells (1103)were seeded in a well of a 96-well culture plate with 50 mL of DMEMand were grown overnight in a 378C incubator with 5% CO2. Polymyxin

B and/or fluconazole were added to the medium to reach final concen-trations of 40 and 100 mg/L, respectively. At 0, 24, 48 and 72 h afterdrug addition, cell growth was examined microscopically and quantified.One hundred thousand THP-1 cells were grown in 1 mL of medium andtreated similarly. Triplicate samples were taken for each treatment ateach timepoint. The experiment was repeated twice.

Results

The antibiotic polymyxin B showed anti-A. nidulansactivity

The Johns Hopkins Clinical Compound Library,31 consisting of1514 FADs and FDA-approved drugs was screened for anti-A.nidulans activity. Twenty-three drugs were found to either signifi-

cantly or completely inhibit the colony growth of the A. nidulansstrain R21 when 2 mL aliquots of the drugs from 10 mM stockwere directly spotted onto each point inoculum containing1105 spores. Eleven compounds significantly inhibited sporegermination in liquid medium at a concentration of 2 mM. Themajority of them were known antifungals belonging to the azolefamily. One antibiotic (polymyxin B sulphate) and three antisepticcompounds were also identified. Because polymyxin B has thepotential to treat systemic infections, it was chosen for furtherstudies. The effect of polymyxin B on A. nidulans and its patho-genic relative A. fumigatus is fungicidal. Both spores andpre-germinated germ tubes inhibited by polymyxin B failed to

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re-grow after transfer to fresh drug-free medium (Figure 1adand data not shown).

Polymyxin B alone is fungicidal at relativelyhigh concentrations

Because A. nidulans is rarely pathogenic to humans, the suscep-tibility to polymyxin B of the pathogenic filamentous fungi

A. fumigatus and R. oryzae and pathogenic yeasts includingC. neoformans, C. albicans, Candida glabrata, Candida krusei andCandida parapsilosis was examined using the standard microdilu-tion assay. Microscopic examination of polymyxin-treated

A. fumigatus (B5233) revealed that polymyxin B at 28 or

56 mg/L significantly inhibited germination of A. fumigatusspores compared with the untreated control (Figure 1e). Thosespores failed to grow even after removal of the drug, consistentwith the fungicidal effect of polymyxin B (Figure 1c and data notshown). Pre-germinated germ tubes also failed to grow in thepresence of polymyxin B as evidenced by lack of long hyphae(Figure 1d). However, some spores present in the populationwere resistant and were able to form hyphae in the presenceof the drug after an overnight incubation. Thus, as expected,both B5233 and Af293 isolates showed strong resistance topolymyxin B in the disc diffusion assay (data not shown) andthe microdilution assay (MIC100.1000 mg/L) based on visual

examination. In contrast, the R. oryzae strain 99-880 is more sus-ceptible, with a MIC100 of 32 mg/L. The pathogenic yeast strainstested also showed varied susceptibility to polymyxin B, with theMIC100 ranging from 8 to 256 mg/L (Table 1, columns 1 and 2).This range is consistent with MICs reported in the litera-ture.27,28,34 Given that bacterial strains with MICs .8 mg/L areconsidered resistant to polymyxin B,35 the relatively high MICsobserved in these fungal species preclude the clinical use of poly-myxin B as a monotherapy in treating fungal infections.

In combination with fluconazole, polymyxin B at lowerconcentrations is fungicidal for all yeast strains tested

Polymyxins act against Gram-negative bacteria by binding lipo-polysaccharide (LPS) and anionic phospholipids in the bacterialmembrane, disrupting membrane integrity.3640 It is possiblethat polymyxin B binds the fungal membrane in an analogousmanner, but with reduced efficiency because eukaryotic mem-branes have low membrane potentials, high levels of sterolsand higher contents of neutral lipid.4143 The azole antifungals

target the lanosterol 14a-demethylase Erg11 in the fungalergosterol biosynthesis pathway.44 This results in a reducedergosterol level and altered membrane property.14,4547 Wereason that polymyxin B and azole antifungals may have syner-gistic interactions against fungi. To test this hypothesis, the sus-ceptibility ofC. neoformans, C. albicans, C. glabrata, C. krusei andC. parapsilosis strains to fluconazole, polymyxin B and a combi-nation of these two compounds was analysed by disc diffusionhalo assay. As shown in Figure 2, the strains demonstratedvaried susceptibility to fluconazole, with the C. krusei strainDUMC132.91 and the C. parapsilosis strain MMRL1594 beinghighly resistant. Polymyxin B alone at 20 mg per disc had no orminimal fungicidal activity against the strains tested(Figure 2b). Interestingly, when fluconazole was combined with

polymyxin B, the halo surrounding the disc was significantlymore clear (Figure 2a and b), an indication of potential fungicidalactivity.

Because the disc diffusion method was not quantitative indetermining the susceptibility to polymyxin B due to the rela-tively large size of this molecule (mol. wt .1300 Da),48 thesynergy of the drug combination was further examined usingthe microdilution assay. Cells from the microdilution assayafter incubation with polymyxin B alone, fluconazole alone orthe combination at various concentrations were also plated ondrug-free medium to count the cfu for determination of theMFC. As shown in Table 1, the strains showed varied susceptibilityto polymyxin B and fluconazole, and there is no apparent corre-lation between the susceptibility towards polymyxin B and the

susceptibility towards fluconazole. Consistent with results fromthe disc diffusion assays, the C. krusei strain DUMC132.91 andthe C. parapsilosis strain MMRL1594 are highly resistant to fluco-nazole (the susceptibility breakpoint for fluconazole is 8 mg/Laccording to the CLSI standard). The MFC of polymyxin B foreach strain tested is similar to the MIC100 (columns 2 and 3),supporting its fungicidal property. Conversely, the MFC of fluco-nazole can be much higher than the MIC90 (columns 4 and 5),and complete cell killing is often not achievable. A synergisticfungicidal interaction between polymyxin B and fluconazolewas observed against all fungal strains tested (last twocolumns). Similar synergistic interactions against the

80

Sporegermina

tionrate(%)

60

40

20

0

56 mg/L

100e(a)

(b) (c) (d)

Control 28 mg/L

Figure 1. Polymyxin B is fungicidal against Aspergillus fumigatus.(a) A. fumigatus spores germinated and formed long hyphae afterovernight growth in drug-free medium. (b) Polymyxin B at 20 mM(28 mg/L) final concentration in YUU liquid medium inhibitedgermination of the majority of A. fumigatus spores. (c) A. fumigatusconidia transferred to drug-free medium after an overnight treatment

with 20m

M polymyxin B did not germinate after 24 h of incubation at378C. (d) Pre-germinated germ tubes of A. fumigatus did not grow inthe presence of 20 mM polymyxin B. In this experiment, germ tubeswere formed after a 7 h incubation of spores in a drug-free medium,and then subjected to an overnight treatment with 20 mM polymyxin B,and subsequently followed by an incubation in drug-free medium for24 h. Note that the germ tubes did not become longer hyphae, incontrast to the non-treated cells shown in (a). (e) Quantitative analysisof the effectiveness of polymyxin B at 20 mM (28 mg/L) or 40mM(56 mg/L) in inhibiting the germination of A. fumigatus spores. Sporesof A. fumigatus strain B5233 were incubated at 378C for 9.5 h. Onehundred and fifty cells were counted for each treatment. The averagesand standard deviations based on three independent experiments areshown in the graph.

Polymyxin B with fluconazole is fungicidal

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C. neoformans H99 strain were also observed for polymyxin B anditraconazole (another azole antifungal), and for polymyxin B and

amphotericin B combinations (data not shown).The susceptibility of the filamentous fungal (mould) patho-gens A. fumigatus strain Af293 and R. oryzae strain 99-880towards polymyxin B alone, itraconazole alone and the drugcombination was also examined using the microdilution assay.As shown at the bottom of Table 1, synergistic interactionsbetween itraconazole and polymyxin B were also observed.

The combination of polymyxin B and fluconazole atclinically relevant concentrations is effective againstCryptococcus strains with varied fluconazole resistance

Because the C. neoformans strain H99 is more susceptible topolymyxin B than the Candida strains tested (Table 1), we

decided to further examine whether the susceptibility to poly-myxin B and to the drug combination is specific to the H99strain or is a general trait of Cryptococcus. C. neoformans hasthree serotypes (A, D and the less common AD hybrids). SerotypeA causes .95% of cryptococcosis cases, and cryptococcosiscaused by serotype A is also more severe.49 Serotype A isolatesare in general more resistant to antifungals compared withserotype D.50 Thus, additional clinical and environmentalC. neoformans serotype A isolates that are genetically distinctwere examined (Table 1). These strains belong to three majorC. neoformans molecular types of serotype A (VNI, VNII orVNB) and they are of either a or a mating type (Table 2).5153

Despite the variations in susceptibility towards polymyxin or flu-conazole, the combination of polymyxin B at 2 mg/L and fluco-

nazole at 8 mg/L was able to achieve.

99% cell killing for allstrains tested except for two strains, Bt81 and A2-102-5,where 9396% killing was achieved. We also obtained a rela-tively fluconazole-resistant strain H99FR by culturing susceptibleH99 in the presence of sublethal fluconazole for 16 weeks. Thestrain H99FR showed a 16-fold increase in MIC90 of fluconazole(Table 2) and significantly increased resistance to itraconazole(data not shown). Again, a synergistic interaction againstH99FR was observed between polymyxin B and fluconazole,and the combination of polymyxin B at 2 mg/L and fluconazoleat 8 mg/L was again effective (Table 2).

The combination of polymyxin B and fluconazole displaysno adverse effect on the growth and proliferation of

HeLa and THP-1 cells when used at a concentration >10times higher than that required for anti-Cryptococcusactivity

Although both polymyxin B and fluconazole have been used inhuman populations to treat infectious diseases for years andtheir toxicity profiles are known, the potential side effectscaused by the combination of these two drugs in animals orhumans remain unknown. As polymyxin B is accumulated to amuch higher level in the kidney (nephrotoxicity), we decided totest the toxicity of the drug combination at higher doses thanthose required for the anti-Cryptococcus activity in tissue

Table 1. Polymyxin B and fluconazole exhibit a synergistic fungicidal effect (mg/L)

Yeast strains MIC100 PMB MFC PMB MIC90 FLC MFC FLC MFC PMB/FLC FFCI

Cryptococcus neoformans H99a76 8 8 1 8 1 2 0.266

Candida albicans SC5314a 128 .256 0.2 64 6 8 0.125

Candida glabrata PAT2ISO3a 256 256 8 .64 25 15 0.236

Candida krusei DUMC132.91a 32 32 64 64 8 10 0.160

Candida parapsilosis MMRL1594a 128 256 .64 .64 10 10 0.078

Saccharomyces cerevisiae BY474177 32 64 4 16 2 4 0.252

Filamentous fungal strains MIC100 PMB MFC PMB MIC90 ITC MFC ITC MFC PMB/ITC FFCI

Aspergillus fumigatus Af29378 .1000 .1000 0.4 12.8 12 1.6 0.137

Rhizopus oryzae 9988079 32 32 0.4 1.6 4 0.4 0.375

aStrains were obtained from Duke University Medical Center.MFC is defined as the lowest drug concentration at which at least 99% of cells were killed compared with the original inocula. Suspensions fromthe microdilution assay after 24 or 48 h (for Cryptococcus) of incubation were plated on drug-free medium to obtain the numbers of cfu to determine

the MFC. Successive 2 serial dilutions of the drugs were used. When fluconazole was used alone, sometimes the MFC could not be achieved in theconcentration range tested and is indicated by ..PMB stands for polymyxin B (susceptible breakpoint for bacteria is 2 mg/L according to the BSAC or 4 mg/L according to the CLSI); FLC, fluconazole(susceptible breakpoint for yeasts is 8 mg/L); ITC, itraconazole (susceptible breakpoint for moulds is 1.0 mg/L). The highest concentrations testedfor PMB, FLC and ITC were 256 (except for A. fumigatus, where 1000 mg/L was the highest concentration tested), 64 and 6.4 mg/L, respectively.The fractional fungicidal concentration index (FFCI)[FLC]/MFCFLC[PMB]/MFCPMB, where MFCFLC and MFCPMB are the concentrations of fluconazole andpolymyxin B, respectively, and [FLC] and [PMP] are the concentrations at which fluconazole and polymyxin B, in combination, killed at least 99% ofcells compared with the original inocula. When the MFC was not achieved (. is used in these cases), the concentration 2 that of the highest con-centration tested is used for FFCI calculation. FFCIs 0.5 indicate synergistic interactions, FFCIs between 0.5 and 4.0 suggest no interaction and FFCIs.4.0 indicate antagonistic interactions.33 The same formulation was used to calculate the FFCI for the interaction between polymyxin B anditraconazole.

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culture. Here, we examined the effect of polymyxin B aloneat 40 mg/L (!20-fold anti-Cryptococcus concentrations), fluco-nazole alone at 100 mg/L (.12-fold anti-Cryptococcus

concentrations) and the combination of both drugs at theabove concentrations on the growth of HeLa cells and humanmonocytic THP-1 cells. None of the treatments displayed any

H2Ocontrol

PMB + FLC20mg + 4mg

FLC4mg

PMB20mg

C.krusei

C.glabrata

C.albicans

Control(a) (b) PMB20mg

FLC4mg

PMB + FLC20mg + 4mg

C.parapsilosis

C. neoformans

Figure 2. Synergistic interaction between fluconazole and polymyxin B against Candida and Cryptococcus. Discs containing water, polymyxin B,fluconazole and the drug combination were dried and overlaid on a lawn of yeast cells derived from the strains indicated. Cells were incubated for24 h (Candida species) or 48 h (Cryptococcus). Inhibition of fungal growth in regions surrounding the disc produces a halo. A completely clear haloindicates fungicidal activity. (a) Disc diffusion halo assays of the C. albicans strain SC5314 produced a clear halo when fluconazole and polymyxinB were used in combination. (b) Microscopic observations reveal significant clearing of the zone of inhibition (halo) when fluconazole andpolymyxin B were used in combination. The left-hand side of each image shows the edge of the disc. PMB, polymyxin B; FLC, fluconazole.

Table 2. In combination with fluconazole, polymyxin B at low concentrations is effective against different Cryptococcus neoformans isolates

Strains Genotypea

Sourcea

Virulencea

MIC100 PMB MFC PMB MIC90 FLC MFC FLC MFC PMB/FLC

H99 (a)76 VNI (A1) clinical high 8 8 1 4 1 2

C45 (a)51 VNII clinical high 20 24 2 6 2 8

A7-35-23 (a)51 VNII environmental low 16 20 4 .64 2 8

C23 (a)51 VNI (A1) clinical high 8 20 8 64 2 8

Bt65 (a)52,53 VNB (A15) clinical 12 12 8 12 2 8

Bt31 (a)52,53 VNB (A4) clinical 8 8 12 32 2 8

Bt81 (a)52,53 VNB (A15) clinical 24 32 12 .64 2 8b

Bt85 (a)52,53 VNB (A21) clinical 8 8 12 .64 2 8

A2-102-5 (a)51 VNI (A2) environmental low 10 10 16 32 2 8b


H99FR VNI (A1) laboratory 5 6 16 48 2 8


PMB, polymyxin B; FLC, fluconazole.The highest concentration of fluconazole tested was 64 mg/L.MICs/MFCs are given in terms of mg/L.aInformation was obtained from previous reports.5153 VNI, VNII and VNB indicate the three molecular types of C. neoformans. An upper case Afollowed by a number indicates further genotypic classification based on the amplified fragment length polymorphism (AFLP) genetic typingpattern. The symbol indicates that no information is available.bThe combination at the indicated concentrations achieved 93%96% rather than .99% killing.


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adverse effect on the growth or the morphology of HeLa cells orTHP-1 cells (Figure 3 and data not shown).

Discussion

Here we showed that the combination of polymyxin B and fluco-nazole (or itraconazole) at low concentrations is fungicidalagainst a variety of pathogenic fungal species and also relativelyfluconazole-resistant strains, indicating the potential of thiscombination in treating systemic and localized fungal infections.

Because polymyxin B (1000 mg/L) is already used in eye drop/ointment and in creams to treat bacterial infections, it could becombined with azole antifungals to treat non-systemic fungalinfections, such as fungal keratitis and mucocutaneous candidia-sis (oral thrush, vaginal candidiasis) that affect the general aswell as the immunocompromised populations.

This drug combination could also potentially be an effectivetreatment for cryptococcosis that involves the brain. Polymyxin Bcan penetrate the brain tissue with a low but appreciableefficiency,54,55 and has been used to treat bacterial meningitiswhen administered intravenously, intrathecally or intraventricu-larly.35,54,5662 Thus, it is conceivable that the combination ofpolymyxin B and fluconazole could be efficacious against crypto-coccal meningitis. Even if polymyxin B could not reach a level at

which it can act synergistically with fluconazole in the braintissue when it is administered intravenously, more effectiveclearance/reduction of the fungal burden in other tissues/organs may exert a sink effect that could help further reducebrain fungal burden. Assessing the efficacy of the drug combi-nation against systemic cryptococcosis with different routes ofadministration in animal models in the future will provide valu-able information regarding the potential efficacy of this drugcombination in the clinic.

Based on the well-known mechanism by which polymyxin Bkills Gram-negative bacteria, we hypothesize that polymyxin Bkills fungi through binding anionic lipids on fungal membrane

and disruption of membrane integrity. This cationic heptapeptidewith a hydrophobic tail derives its bactericidal activity from itselectrostatic interaction with negatively charged lipids (LPS oranionic phospholipids) and its hydrophobic interaction withmembrane lipids. These interactions allow polymyxin B to formchannels to destroy the integrity of the cytoplasmic mem-brane.41,6367 The observation that magainin 2, another anti-biotic that is structurally different from polymyxin B but sharesa similar mode of action against bacteria,63,68,69 is also fungicidaland acts synergistically with fluconazole against H99 (data notshown) indicates a conserved mechanism of cationic peptideantibiotics against fungi.

The lower efficiency of polymyxin B alone (or other cationicpeptide antibiotics such as magainin 2) against eukaryotes com-pared with bacteria28,42,70,71 could be partly due to the presenceof sterols in eukaryotic membrane, as sterols have been shownto reduce the insertion of cationic peptides into anionic mixedmembranes to form pores.72 The fact that azole treatmentlowers fungal ergosterol levels and renders fungi more suscep-tible to polymyxin B supports this hypothesis.

Although our toxicity study in cell culture suggests that thespecificity of azoles against fungal ergosterol biosynthesis(without affecting mammalian cholesterol biosynthesis) mayprevent increased toxicity of the drug combination towardshosts compared with polymyxin B alone, the safety concern ofthe current formulation of polymyxin B might limit its use inthe clinic to treat systemic fungal infections. However, the resur-ging interest in using polymyxin B to treat bacterial infectionscaused by multidrug-resistant isolates may drive the develop-ment of safer and more effective new formulations. Regardlessof the clinical potential of polymyxin B per se in treating fungalinfections, given the recent discoveries of cationic peptides aspotential antifungals,7375 investigation into the fungicidalactivity of polymyxin B and the synergy between polymyxin B

and azoles will probably have a far-reaching impact on the devel-opment of novel, effective and safer antifungal therapies.

AcknowledgementsWe thank Drs David Sullivan and Jun Liu for providing the clinicalcompound library, Drs Joseph Heitman, Alexander Idnurm and YunChang for providing strains, and Drs Ron Morris, Joseph Heitman,Alexander Idnurm, David Sullivan, Gudrun Ihrke and Yun Chang fordiscussions.

FundingThis work was supported by: the Department of Biology of Texas A&MUniversity (the startup fund to X. L.); CNP Department of Defense(grant G171IM to X. X.); and National Institutes of Health (grantnumbers GM069527 to X. X. and CA115884 to C.-Z. G.).

Transparency declarationsNone to declare.

50

25

Cellnumbe

r(104)

00 1 2

Days after treatment

3

Control

Fluconazole

Polymyxin

Polymyxin + Fluconazole

Figure 3. The combination does not produce any additional adverseeffect on host cells. The growth of human monocytic THP-1 cells wasnot affected by the combination of polymyxin B sulphate andfluconazole at final concentrations of 40 and 100 mg/L, respectively.For all three timepoints, there are no statistical differences based on

Students t-tests between the control and treatments, with P values nogreater than 0.01.

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References1 Anaissie EJ. Diagnosis and therapy of fungal infection in patients withleukemianew drugs and immunotherapy. Best Pract Res Clin Haematol2008; 21: 68390.

2 Meunier F, Lukan C. The First European Conference on Infections inLeukaemiaECIL1: a current perspective. Eur J Cancer2008; 44: 21127.

3 Black KE, Baden LR. Fungal infections of the CNS: treatment strategiesfor the immunocompromised patient. CNS Drugs 2007; 21: 293 318.

4 Bohme A, Karthaus M. Systemic fungal infections in patients withhematologic malignancies: indications and limitations of the antifungalarmamentarium. Chemotherapy1999; 45: 31524.

5 Mitchell TG, Perfect JR. Cryptococcosis in the era of AIDS100 yearsafter the discovery of Cryptococcus neoformans. Clin Microbiol Rev 1995;8: 51548.

6 Steinbach WJ, Stevens DA. Review of newer antifungal andimmunomodulatory strategies for invasive aspergillosis. Clin Infect Dis2003; 37 Suppl 3: S15787.

7 van der Horst CM, Saag MS, Cloud GA et al. Treatment of cryptococcal

meningitis associated with the acquired immunodeficiency syndrome.National Institute of Allergy and Infectious Diseases Mycoses StudyGroup and AIDS Clinical Trials Group. N Engl J Med 1997; 337: 1521.

8 Pitisuttithum P, Tansuphasawadikul S, Simpson AJ et al. A prospectivestudy of AIDS-associated cryptococcal meningitis in Thailand treatedwith high-dose amphotericin B. J Infect 2001; 43: 22633.

9 Filioti J, Spiroglou K, Panteliadis CP et al. Invasive candidiasis inpediatric intensive care patients: epidemiology, risk factors,management, and outcome. Intensive Care Med 2007; 33: 127283.

10 Powderly WG. Recent advances in the management of cryptococcalmeningitis in patients with AIDS. Clin Infect Dis 1996; 22 Suppl 2: S11923.

11 Latge J-P, Steinbach WJ. Aspergillus fumigatus and Aspergillosis.Washington, DC: ASM Press, 2009.

12 Longley N, Muzoora C, Taseera K et al. Dose response effect of

high-dose fluconazole for HIV-associated cryptococcal meningitis insouthwestern Uganda. Clin Infect Dis 2008; 47: 155661.

13 Pukkila-Worley R, Mylonakis E. Epidemiology and management ofcryptococcal meningitis: developments and challenges. Expert OpinPharmacother2008; 9: 110.

14 Odds FC, Brown AJ, Gow NA. Antifungal agents: mechanisms ofaction. Trends Microbiol 2003; 11: 2729.

15 Georgopapadakou NH. Antifungals: mechanism of action andresistance, established and novel drugs. Curr Opin Microbiol 1998; 1:54757.

16 Kontoyiannis DP, Mantadakis E, Samonis G. Systemic mycoses in theimmunocompromised host: an update in antifungal therapy. J HospInfect 2003; 53: 24358.

17 Lamb D, Kelly D, Kelly S. Molecular aspects of azole antifungal action

and resistance. Drug Resist Updat 1999; 2: 390 402.18 Perlin DS. Resistance to echinocandin-class antifungal drugs. DrugResist Updat 2007; 10: 12130.

19 Sar B, Monchy D, Vann M et al. Increasing in vitro resistance tofluconazole in Cryptococcus neoformans Cambodian isolates: April 2000to March 2002. J Antimicrob Chemother 2004; 54: 5635.

20 Berg J, Clancy CJ, Nguyen MH. The hidden danger of primaryfluconazole prophylaxis for patients with AIDS. Clin Infect Dis 1998; 26:1867.

21 Armengou A, Porcar C, Mascaro J et al. Possible development ofresistance to fluconazole during suppressive therapy for AIDS-associated cryptococcal meningitis. Clin Infect Dis 1996; 23: 13378.

22 Paugam A, Dupouy-Camet J, Blanche P et al. Increased fluconazoleresistance of Cryptococcus neoformans isolated from a patient withAIDS and recurrent meningitis. Clin Infect Dis 1994; 19: 9756.

23 Bicanic T, Harrison T, Niepieklo A et al. Symptomatic relapse ofHIV-associated cryptococcal meningitis after initial fluconazolemonotherapy: the role of fluconazole resistance and immune

reconstitution. Clin Infect Dis 2006; 43: 106973.

24 Friese G, Discher T, Fussle R et al. Development of azole resistanceduring fluconazole maintenance therapy for AIDS-associatedcryptococcal disease. AIDS 2001; 15: 23445.

25 Pfaller MA, Diekema DJ, Gibbs DL et al. Results from the ARTEMIS DISKGlobal Antifungal Surveillance Study, 1997 to 2007: 10.5-year analysis ofsusceptibilities of noncandidal yeast species to fluconazole andvoriconazole determined by CLSI standardized disk diffusion testing.J Clin Microbiol 2009; 47: 11723.

26 Nicholls MW. Polymyxin sensitivity of Candida tropicalis. J MedMicrobiol 1970; 3: 52938.

27 Schwartz SN, Medoff G, Kobayashi GS et al. Antifungal properties ofpolymyxin B and its potentiation of tetracycline as an antifungal agent.Antimicrob Agents Chemother1972; 2: 3640.

28 Falla TJ, Karunaratne DN, Hancock RE. Mode of action of theantimicrobial peptide indolicidin. J Biol Chem 1996; 271: 19298303.

29 Xiang X, Zuo W, Efimov VP et al. Isolation of a new set of Aspergillusnidulans mutants defective in nuclear migration. Curr Genet 1999; 35:62630.

30 Tsai HF, Wheeler MH, Chang YC et al. A developmentally regulatedgene cluster involved in conidial pigment biosynthesis in Aspergillusfumigatus. J Bacteriol 1999; 181: 646977.

31 Chong CR, Chen X, Shi L et al. A clinical drug library screen identifiesastemizole as an antimalarial agent. Nat Chem Biol 2006; 2: 4156.

32 National Committee for Clinical Laboratory Standards. ReferenceMethod for Broth Dilution Antifungal Susceptibility Testing of YeastsSecond Edition: Approved Standard M27-A2. NCCLS, Wayne, PA, USA, 2002.

33 Odds FC. Synergy, antagonism, and what the chequerboard putsbetween them. J Antimicrob Chemother 2003; 52: 1.

34 Moneib NA. In-vitro activity of commonly used antifungal agents inthe presence of rifampin, polymyxin B and norfloxacin against Candidaalbicans. J Chemother 1995; 7: 5259.

35 Kwa A, Kasiakou SK, Tam VH et al. Polymyxin B: similarities to anddifferences from colistin (polymyxin E). Expert Rev Anti Infect Ther2007; 5: 81121.

36 Srimal S, Surolia N, Balasubramanian S et al. Titration calorimetricstudies to elucidate the specificity of the interactions of polymyxin Bwith lipopolysaccharides and lipid A. Biochem J 1996; 315: 67986.

37 Vaara M. Agents that increase the permeability of the outermembrane. Microbiol Rev 1992; 56: 395 411.

38 Hancock RE, Chapple DS. Peptide antibiotics. Antimicrob AgentsChemother1999; 43: 131723.

39 Storm DR, Rosenthal KS, Swanson PE. Polymyxin and related peptideantibiotics. Annu Rev Biochem 1977; 46: 72363.

40 Teuber M, Bader J. Action of polymyxin B on bacterial membranes.Binding capacities for polymyxin B of inner and outer membranesisolated from Salmonella typhimurium G30. Arch Microbiol 1976; 109:518.

41 Hancock RE. Peptide antibiotics. Lancet 1997; 349: 41822.

42 HsuChen CC, Feingold DS. The mechanism of polymyxin B action andselectivity toward biologic membranes. Biochemistry1973; 12: 210511.

43 Teuber M, Miller IR. Selective binding of polymyxin B to negativelycharged lipid monolayers. Biochim Biophys Acta 1977; 467: 2809.


937

JA


8/8

44 Jandrositz A, Turnowsky F, Hogenauer G. The gene encoding squaleneepoxidase from Saccharomyces cerevisiae: cloning and characterization.Gene 1991; 107: 15560.

45 Anderson JB. Evolution of antifungal-drug resistance: mechanismsand pathogen fitness. Nat Rev Microbiol 2005; 3: 54756.

46 Cowen LE. The evolution of fungal drug resistance: modulating thetrajectory from genotype to phenotype. Nat Rev Microbiol 2008; 6:18798.

47 Lupetti A, Danesi R, Campa M et al. Molecular basis of resistance toazole antifungals. Trends Mol Med 2002; 8: 7681.

48 Gales AC, Reis AO, Jones RN. Contemporary assessment ofantimicrobial susceptibility testing methods for polymyxin B andcolistin: review of available interpretative criteria and quality controlguidelines. J Clin Microbiol 2001; 39: 18390.

49 Casadevall A, Perfect JR. Cryptococcus neoformans. Washington, DC:ASM Press, 1998.

50 Dannaoui E, Abdul M, Arpin M et al. Results obtained with variousantifungal susceptibility testing methods do not predict early clinicaloutcome in patients with cryptococcosis. Antimicrob Agents Chemother2006; 50: 246470.

51 Litvintseva AP, Mitchell TG. Most environmental isolates ofCryptococcus neoformans var. grubii (serotype A) are not lethal formice. Infect Immun 2009; 77: 318895.

52 Litvintseva AP, Marra RE, Nielsen K et al. Evidence of sexualrecombination among Cryptococcus neoformans serotype A isolates insub-Saharan Africa. Eukaryot Cell 2003; 2: 11628.

53 Litvintseva AP, Thakur R, Vilgalys R et al. Multilocus sequence typingreveals three genetic subpopulations of Cryptococcus neoformans var.grubii (serotype A), including a unique population in Botswana. Genetics2006; 172: 222338.

54 Jimenez-Mejias ME, Pichardo-Guerrero C, Marquez-Rivas FJ et al.Cerebrospinal fluid penetration and pharmacokinetic/pharmacodynamicparameters of intravenously administered colistin in a case ofmultidrug-resistant Acinetobacter baumannii meningitis. Eur J Clin

Microbiol Infect Dis 2002; 21: 2124.55 Jimenez-Mejias ME, Becerril B, Marquez-Rivas FJ et al. Successfultreatment of multidrug-resistant Acinetobacter baumannii meningitiswith intravenous colistin sulfomethate sodium. Eur J Clin MicrobiolInfect Dis 2000; 19: 9701.

56 Segal-Maurer S, Mariano N, Qavi A et al. Successful treatment ofceftazidime-resistant Klebsiella pneumoniae ventriculitis withintravenous meropenem and intraventricular polymyxin B: case reportand review. Clin Infect Dis 1999; 28: 11348.

57 Benifla M, Zucker G, Cohen A et al. Successful treatment ofAcinetobacter meningitis with intrathecal polymyxin E. J AntimicrobChemother2004; 54: 2902.

58 Michalopoulos A, Falagas ME. Colistin and polymyxin B in critical care.Crit Care Clin 2008; 24: 37791, x.

59 Evans ME, Feola DJ, Rapp RP. Polymyxin B sulfate and colistin: oldantibiotics for emerging multiresistant gram-negative bacteria. AnnPharmacother1999; 33: 9607.

60 Levin AS, Barone AA, Penco J et al. Intravenous colistin as therapy fornosocomial infections caused by multidrug-resistant Pseudomonasaeruginosa andAcinetobacter baumannii. ClinInfect Dis 1999; 28: 100811.

61 Garrod LP, OGrady F, Barber M. Antibiotic and Chemotherapy.Baltimore: Williams and Wilkins, 1971.

62 Stein A, Raoult D. Colistin: an antimicrobial for the 21st century? ClinInfect Dis 2002; 35: 9012.

63 Savage PB, Li C, Taotafa U et al. Antibacterial properties of cationicsteroid antibiotics. FEMS Microbiol Lett 2002; 217: 17.

64 Vaara M, Vaara T. Sensitization of Gram-negative bacteria toantibiotics and complement by a nontoxic oligopeptide. Nature 1983;303: 5268.

65 Sanders WE Jr, Sanders CC. Toxicity of antibacterial agents:mechanism of action on mammalian cells. Annu Rev Pharmacol Toxicol1979; 19: 5383.

66 Stansly PG, Schlosser ME. Studies on polymyxin: isolation andidentification of Bacillus polymyxa and differentiation of polymyxin fromcertain known antibiotics. J Bacteriol 1947; 54: 54956.

67 Li J, Nation RL, Milne RW et al. Evaluation of colistin as an agentagainst multi-resistant Gram-negative bacteria. Int J Antimicrob Agents2005; 25: 1125.

68 Matsuzaki K, Nakamura A, Murase O et al. Modulation of magainin

2-lipid bilayer interactions by peptide charge. Biochemistry 1997; 36:210411.

69 Matsuzaki K, Sugishita K, Harada M et al. Interactions of anantimicrobial peptide, magainin 2, with outer and inner membranes ofGram-negative bacteria. Biochim Biophys Acta 1997; 1327: 11930.

70 Bearer EL, Friend DS. Anionic lipid domains: correlation with functionaltopography in a mammalian cell membrane. Proc Natl Acad Sci USA1980; 77: 66015.

71 Ogita A, Nagao Y, Fujita K et al. Amplification of vacuole-targetingfungicidal activity of antibacterial antibiotic polymyxin B by allicin,an allyl sulfur compound from garlic. J Antibiot (Tokyo) 2007; 60:5118.

72 Mason AJ, Marquette A, Bechinger B. Zwitterionic phospholipidsand sterols modulate antimicrobial peptide-induced membranedestabilization. Biophys J 2007; 93: 428999.

73 Ahmad I, Perkins WR, Lupan DM et al. Liposomal entrapment of theneutrophil-derived peptide indolicidin endows it with in vivo antifungalactivity. Biochim Biophys Acta 1995; 1237: 10914.

74 Falla TJ, Hancock RE. Improved activity of a synthetic indolicidinanalog. Antimicrob Agents Chemother 1997; 41: 7715.

75 Garibotto FM, Garro AD, Masman MF et al. New small-sizepeptides possessing antifungal activity. Bioorg Med Chem 2010; 18:15867.

76 Perfect JR, Lang SD, Durack DT. Chronic cryptococcal meningitis: anew experimental model in rabbits. Am J Pathol 1980; 101: 17794.

77 Winzeler EA, Shoemaker DD, Astromoff A et al. Functionalcharacterization of the S. cerevisiae genome by gene deletion andparallel analysis. Science 1999; 285: 9016.

78 Nierman WC, Pain A, Anderson MJ et al. Genomic sequence of thepathogenic and allergenic filamentous fungus Aspergillus fumigatus.Nature 2005; 438: 11516.

79 Ma LJ, Ibrahim AS, Skory C et al. Genomic analysis of the basal lineagefungus Rhizopus oryzae reveals a whole-genome duplication. PLoS Genet2009; 5: e1000549.

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