CFAD V. Anacor, |PR20’|5-01776 ANACOR EX. 2104 - “I/“I2
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`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2104 - 1/12
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`_.ANTll\/IICROBIAL AGENTS AND
`
`CHEMOTHERAPY
`VOLUME .12 s OCTOBER 1998 o NUMBER 10
`
`."»'.>.11r.r..i1'1,-,3
`
`George A. Jacoby. Editor in C/lief
`(2111111)
`‘
`La/Icr C/iltir
`B11!‘/I/igfotl, 1l’[(I.\'.S'.‘
`Karen Bush, Editor (1999)
`R,
`ll.’/(I/1111.80}! ‘P,/I((lI7I(1(L’l111((I(
`R(’S(’{Il(/1 ]II.$11f!(1(’
`R‘"mm' NJ‘
`_
`p
`I
`William A. Craig, 1;tlrt0r (2002)
`Iii"//i(mI S. 11/Iizlt/lcioti 1)’I(3II1()l'1(1/
`lv'r*tc1'rr/1.5‘ Hospiml
`t)[t(t(1.YOIl,
`1-Viv.
`
`I
`
`._
`
`_
`
`V
`
`V
`
`.
`
`,, ]
`)
`(“H”)
`)‘t.[(;mm,m [,’,‘cw_ C((]1:/:
`George M. Eliopuulosq Ednor
`(7000)
`Bet/i I.s't'itc/ Deucu/rem Meclicul Center
`B0510”,
`)\«[(,35,
`
`_
`
`Barbara E. Murray, E(11I()I' (211112)
`Utzi't'et:\'ity of 7‘e_\m' Ht’(l/1/I S('1'cIice
`Center
`Ilou.s'i‘01i. T(’.\‘.
`,Spruance, E(([tr)i' (211110)
`Spotswood
`UIII1(,'1.S.I(_\. of Lm/I School of
`1)/It’!/I(!I1(’
`Sal’ I1!/W Cm‘ Um/I _
`Lowell S. Young, E11110!‘ (211111)
`Kitzell Institute for Ar’!/rt'iti.s‘ & I/1_/‘ectic.
`Disc(t.s'e.s'
`
`Suit Fm/tci.vc0, Crtlifi
`
`Peter C. Appelbaum (211110)
`(,‘nrdon L. Archer (1998)
`Michel Arthur (1999)
`Masanori Baba (1998)
`Frank Balis (311111))
`John F. Barrett (1999)
`Arthur L. Barry (1998)
`John (,1. Bartlett (1999)
`Larry A. Batter (1998)
`Arnold S. Bayer (20110)
`Jellrey M. Becker (1999)
`Michel G. Bergeron (1998)
`_Ionatltan D. Berrnan (2111111)
`Joseph S. Bertino (1999)
`John Anthony Bilello (2111111)
`Karen K. Biron (1999)
`Jurg Blaser (20111))
`F. Douglas Boudinot (2000)
`l’atricia Bradford (3000)
`Claude Carbon (1993)
`Henry F. Chambers (211110)
`Sarah H. Cheesernan (1998)
`l)unal(l M. Coen (1999)
`l’atrice Conrvalin (2001))
`William L. Current (1999)
`Micltael H. Cynainon (1998)
`Gar) V. Doern (1999)
`A. Kirk Field (1999)
`1.. Mark Fisher (1999)
`Robert K. Flarnin (201111)
`Jean-Marie Frere (211011)
`
`EDITORIAL BOARD
`Steven .\'leshnick (Z1100)
`John N. Galgiani (1999)
`r\'1icltael H. Miller (1999)
`f\'. H. Georgopapadakou (2001))
`Johan W. Mouton (2000)
`Dale N. Gerding (301111)
`Thomas D. Gootz (1999)
`Henry W. Murray (1999)
`Shenvoud L. Gorbach (I998)
`P. K. Narang (1999)
`Fl'ankliri A. Neva (I998)
`Regine Hakenbeek (1998)
`Thalia I. Nicas (1999)
`Scott M. Hammer (1998)
`Robert E. W. Hancock (1998)
`Wright Nichols (1998)
`H. Hunter Handsfield (1998)
`Charles H. Nightingale (1999)
`Hiroshi Nikaitlo (1998)
`David Hecht (3000)
`Frank C. Odds (20011)
`David C. Hooper (1999)
`James E. Pennington (1998)
`Clark B. lnderlied (1998)
`John R. Perfect (1999)
`Susan E. Jensen (21100)
`Brent G. l’e|t_\' (1998)
`James 11. Jorgensen (1999)
`Michael A. Plaller (211111))
`A. W. Karclinter (20011)
`Robert E. Kessler (1999)
`Ronald Polk (1999)
`R. Keith Poole (1999)
`Michael E. Klepscr (2111111)
`Steven J. Projan (21101))
`V Roger Labia (2111111)
`Robert 1\l. Rakita (1999)
`Marie Thérese Labro (1999)
`Mieltael D. Reed (2000)
`Brendan A. Larder (1998)
`Catherine P. Reese (1998)
`Philip Todd l.a\'in (1998)
`Richard C. Rciclnnan (1998)
`Jaiues E. Leggett (1999)
`John Rex (1999)
`Stephen A. Lerner (1998)
`Louis Rice (1998)
`Stuart B. Levy (1998)
`David Livermore (1998)
`Douglas D. Riclnnan (1999)
`Keith A. Rotlvold (1999)
`J. Joseph Marr (1998)
`Allan Ronald (1999)
`Henry Masur (1999)
`John C. Rotschafer (1998)
`Douglas Mayers (2111111)
`Robert H. Rtibin (1999)
`Antone A. r\'Iedeiros (1998)
`Michael J. R_\'l)ak (1998)
`Jolni Mellors (1999)
`
`Daniel F. Sahrn (1998)
`Dominique Sanglard (21100)
`W. Michael Scheld (1998)
`Raymond F. Schinazi (1998)
`Robert T. Schooley (1999)
`Richard S. S(‘l1\\‘flll)t! (211011)
`David M. Shlaes (2111111)
`Tltontas R. Shryock (1998)
`Robert W. Sidwell (1999)
`Lynn L. Silver (1999)
`Jean-Pierre Sonunadossi (191.
`James M. Steckelberg (1999)
`Daniel S. Stein (1998)
`Stephen E. Straus (1999)
`Andrew H. Strayer (1999)
`Janice R. Sufrin (2111111)
`Alan r\l. Sugar (1998)
`Francis C. Szoka. Jr. (1998)
`Diane E. 'l‘a_\'lor (1999)
`Fred C. Tenover (2001))
`Kenneth S.
`'l'honrson (1999)
`Clyde Tl1ornslJerr_v (1998)
`Patrick Triett-Crtot (2111111)
`Hugo Vanden Bossche (1998)
`.los ran (ler Meet‘ ( 1999)
`C. (3. Wang (2000)
`Bernard \\"eisblnm (20110)
`Richard J. \Vl1itle_V (1999)
`Richard Wise (1998)
`Gcolflrey J. Yuen (1998)
`
`Barbara H. Iglewslti. ('Imr't'tm1tr. I’rrbIicuti0ti.s‘ Board
`Arthur Gelmis, Pmrluction Eilitor
`
`Linda M. Illig. Diiectot; .IUHI'tl(I/X
`Robert D. Loper. Assi.s't(rtri Proilrtcriwi Editor
`
`ISSN (1116(r—'—1S114). an interdisciplinary pttblicatiori of the American Society for 1\/lierobiolog_\' (ASP
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`CFAD V. Anacor, |PR2015-01776 ANACOR EX. 2104 - 2/12
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`CODEN: AMACCQ
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`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2104 - 2/12
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`

`
`MECHANISMS OF ACTION: PHYSIOLOGICAL
`EFFECTS
`
`Accumulation ol'Ampl1otericin B in Human Macrophages
`Enhances Activity against Aspetgillusflmzigutus Conidia:
`Quantification of Conidial Kill at the Single-Cell Level
`
`Targeted Antimicrobial Photoehemotherapy
`
`Antiparasitic Elfects of the Intra-Golgi Transport Inhibitor
`Megalomicin
`
`Inhibitory Activities of Gatifloxacin (AM-1155), a Newly
`Developed Fluoroquinolone, against Bacterial and
`Mammalian T_vpe II Topoisomerases
`
`Translation Elongation Factor 2 is Part of the Target for a
`New Family of Antifungals
`
`Proteasolne Inhibitors Block Development of Plasmodium
`spp.
`
`Enhancement of Antimicrobial Activity of Neuropeptide Y by
`N-Terminal Truncation
`
`MECHANISMS OF RESISTANCE
`
`Sequencing of Gyrase and Topoisomerase IV Quinolone-
`Resistanee-Determining Regions of Chlamyditl trac/10n1m‘is
`and Characterization of Quinolone-Resistant Mutants
`Obtained In Vitro
`
`Characterization of 11140 of Enrerolmcrer aeragenes BM2688,
`a Class I Integron with Two New Gene Cassettes, cmlA2 and
`qucl’
`
`Roles of Amino Acids 161 to 179 in the PSE--I 9 Loop in
`Substrate Specificity and in Resistance to Ceftazidime
`
`Rapid, Transient Fluconazole Resistance in Crmdirla (llbicans
`Is Associated with Increased mRNA Levels of CDR
`
`Characterization of Mutations in the 17208 Gene That
`Confer Rifampin Resistance in Slap/IyI0c0ccu.s' aureus
`
`Characterization of a Glyeosyl Transferase Inactivating
`Macro|i(les. Encoded by gim/I from Srreptonrvces aml20facieII.s'
`
`Bernhard Jahn, Albert Rampp.
`Christian Dick, Andreas Jahn.
`Michael Palmer. and Sucharit
`Bhakdi
`
`Nikolaos S. Soukos. Laurie Ann
`Ximenez-Fyvie. Michael R.
`Hamblin. Sigmund S. Socransky.
`and Tayyaba Hasan
`
`Pedro Bonay. Isabel Dur2in—Chica.
`Manuel Fresno. Balbino Alareon.
`and Antonio Aleina
`
`Masaya Tal<ei. Hideyuki Fukuda.
`Tokularo Yasue. Masaki Hosaka.
`and Yasuo Oomori
`
`Laura Capa. Alfonso Mendoza. Jose
`L. Lavandera. Federico Gomez de
`las Heras. and Jose F. Garcia-Bustos
`
`Soren M. Gantl. Joon Mo Myung.
`Marcelo R. S. Briones. Wei Dong
`Li. E. J. Corey. Satoshi Omura.
`Victor Nussenzweig, and Photini
`Sinnis
`
`Mayumi Shimizu. Yasushi Shigeri,
`Yoshiro Tatsu. Susumu Yoshikawa.
`and Noboru Yumoto
`
`Sophie Dessus—Babus, Céeile M.
`Bébéar. Alain Charron, Christiane
`Bébéar. and Bertille de Barbeyrac
`
`Marie-Céeile Ploy, Patrice
`Courvalin. and Thierry Lambert
`
`Christian Therrien. Francois
`Sansehagrin. Timothy Palzkill. and
`Roger C. Levesque
`
`Kieren A. Marr. Christopher N.
`Lyons. Tiger Rustad. Raleigh A.
`Bowden. and Theodore C. White
`
`Helene Auhry—Damon. Claude-
`James Soussy. and Patrice Courvalin
`Anne Gourmelen. Marie—Helene
`Blondelet-Rouault. and Jean—Lue
`Pernodet
`
`Continued on fol/rm'i/lg /Juge
`
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`

`
`
`
`C0I1!iI1ue(Ifi'0m pmccrling page
`
`Mechanism of Fluconazole Resistance in Camlida krusei
`
`Alison S. Orozco, Lindsey M.
`Higginbotham. Christopher A.
`Hitchcock. Tanya Parkinson, Derek
`Falconer, Ashraf S. Ibrahim,
`Mahmoud A. Ghannoum. and Scott
`G. Filler
`
`gyrA Mutations Associated with Fluoroquinolone Resistance
`in Eight Species of Enterobacreriaceae
`
`Linda M. Weigel. Christine D.
`Steward. and Fred C. Tenovcr
`
`Explaining the Bias in the 23S rRNA Gene Mutations
`Associated with Clarithromycin Resistance in Clinical
`Isolates of Helicolmcter pylori
`
`Y. J. Debets—Ossenkopp, A. B.
`Brinkman, E. J. Kuipers, C. M. J. E.
`Vandcnbroucke-Grauls. and J. G.
`Kustcrs
`
`2645-2649
`
`2661-2667
`
`2749-2751
`
`Characterization of ISI8, an Element Capable of Activating
`the Silent (mc(6')-Ij Gene ofAcinetaImcter sp. 13 Strain
`BM27l6 by Transposition
`
`Description of Two New Isolates of Streptococcus pneumoniac
`in Spain That Are Highly Resistant to Cefotaxime
`
`Eric Rudant. Patrice Courvalin, and
`Thierry Lambert
`
`2759-2761
`
`Joaquin Ruiz. Marco Sempcre.
`Encarnacion Simarro, and Asuncion
`Fcnoll
`
`2768-2769
`
`SUSCEPTIBILITY
`
`Structure-In Vitro Activity Relationships of Pentamidine
`Analogues and Dication-Substituted Bis-Benzimidazoles as
`New Antifungal Agents
`
`In Vitro Antifungal Activities of a Series of I)ication-
`Substituted Carbazoles, Furans, and Benzimidazoles
`
`Intracellular Delivery and Antibacterial Activity of
`Gentamicin Encapsulated in pH-Sensitive Liposomes
`
`Identification and Characterization of Novel Antimicrobial
`Decapeptides Generated by Combinatorial Chemistry
`
`Comparison of Inhibitory and Bactericidal Activities and
`Postantibiotic Effects of LY333328 and Ampicillin Used
`Singly and in Combination against Vancomycin-Resistant
`En!er0coccu.s' faecium
`
`Antibiotic Susceptibility Patterns of Streptococcus pnetunoniac
`in China and Comparison of MICS by Agar Dilution and
`E-Test Methods
`
`Haemophilus ducreyi Is Susceptible to Protegrin
`
`In Vitro Activity of the Echinocandin Antifungal Agent
`LY303,366 i11 Comparison with Itraconazole and
`Alnphotericin B against AspeI'giI1u.s' spp.
`
`Maurizio Del Poeta, Wiley A.
`Schell, Christine C. Dykstra, Susan
`Jones. Richard R. Tidwell,
`Agnicszka Czarny, Miroslav Bajic,
`Marina Bajic, Arvind Kumar. David
`Boykin, and John R. Perfect
`
`Maurizio Dcl Poeta, Wiley A.
`Schell, Christine C. Dykstra, Susan
`K. Jones, Richard R. Tidwell,
`Arvind Kumar, David W. Boykin,
`and John R. Perfect
`
`Peter Lutwychc, Carol Cordeiro,
`David J. Wiscman. Marysc S1—Louis,
`Mitchell Uh. Michael J. Hope,
`Murray S. Webb, and B. Brett
`Finlay
`
`Sung Yu Hong. Jong Eun Oh,
`Mi yun Kwon. Myeong Jun Choi.
`Ji Hye Lee. B0l\’ Luel Lee. Hong
`Mo Moon. and Kenn Hyeung Lee
`
`Aldona L. Baltch. Raymond P.
`Smith, William J. Ritz, and
`Lawrence H. Bopp
`
`2495-2502
`
`2503-2510
`
`2511-2520
`
`2534-2541
`
`2564-2568
`
`Hui Wang. Robin Huebncr. Minjun
`Chen. and Keith Klugman
`
`2633-2636
`
`Kate Fortncy, Patricia A. Tolten,
`Robert 1. Lehrcr. and Stanley M.
`Spinola
`
`Karen L. Oakley. Caroline B.
`1\/Ioore. and David W. Dcnning
`
`2690-2693
`
`2726-2730
`
`In Vitro Studies of Pharmacodynamic Properties of
`Vancomycin against Staphylococcus (lllI'(’ll.X' and Stuplrvlacoccus
`
`E. L(')\vdin.
`0. Cars
`
`l. Odcnholt. and
`
`2739-2744
`
`»
`
`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2104 - 4/12
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`

`
`Ciprofloxacin, Otloxacin, Doxycycline, and Clarithromycin
`In Vitro Activities of 15 Antimicrobial Agents against
`Clinical Isolates of South African Enterococci
`
`In Vitro Susceptibilities ofCl1l(Imydiapneunzuniae Strains
`Recovered from Atherosclerotic Coronary Arteries
`PMX-622 (Polymy.\'in B-Dextran 70) Does Not Alter In Vitro
`Activities of II Antimicrobial Agents
`
`ANALYTICAL PROCEDURES
`
`M. C. Struwig, P. L. Botha. and
`L. J. Chalklcy
`Jens Gielfers, Werner Solbaeh, and
`Matthias Maass
`
`Peter C. Fuchs, Arthur L. Barry,
`and Steven D. Brown
`
`An Esclzericliia coli System Expressing Human
`Deoxyribonucleoside Salvage Enzymes for Evaluation of
`Potential Antiproliferative Nucleosidc Analogs
`
`Jianghai Wang, Jan Neuhard, and
`Stalfan Eriksson
`
`ANTIVIRAL AGENTS
`
`Restoration of Innmme Response by a Cationic Amphiphilic
`Drug (AY 9944) In Vitro: A New Approach to Chemotherapy
`against Human lnnmmodeficiency Virus Type 1
`
`Genotypic and Phenotypic Characterization of Human
`Immunodeficiency Virus Type I Variants Isolated from
`Patients Treated with the Protease Inhibitor Nelfinavir
`
`EXPERIMENTAL THERAPEUTICS
`
`Granulocyte Colony-Stimulating Factor and Azole
`Antifungal Therapy in Murine Aspergillosis: Role of
`Immune Suppression
`
`Mucoadhesive Microspheres Containing Amo\'icillin for
`Clearance of Helicobacter pylori
`
`Interaction of Streptococcus pnemnoniae and Moraxelltt
`camrrlmlisz Investigation of the Indirect Pathogenic Role of
`B-Lactamase-Producing Moraxellae by Use of a Continuous-
`Culture Biofilm System
`
`Reduction by Cefodizime of the Pulmonary Inflammatory
`Response Induced by Heat-Killed Streptococcus ptzezunorziue
`in Mice
`
`Etficacies of KY62 against Leislnnania rmzazonensis and
`Leislznmnia rlonowmi in Experimental Murine Cutaneous
`Leishmaniasis and Visceral Leishmaniasis
`
`Interleukin-12 Enhances In Vivo Parasiticidal Elfeet of
`Benznidazole during Acute Experimental Infection with a
`Naturally Drug-Resistant Strain of Ttypanosoma cruzi
`
`In Vitro and In Vivo Ellicacy of the Triazole TAK-187
`against Ciyptococcus neofor-mans
`
`Ammar Achour, Jean-Charles
`Landureau, Rosangela Salerno-
`Concalves, Jean-Claude Mazicre,
`and Daniel Zaguiy
`
`A. K. Patick, M. Duran, Y. Cao, D.
`Shugarts, M. R. Keller, E. Mazabel,
`M. Knowles, S. Chapman, D. R.
`Kuritzkes, and M. Markowitz
`
`John R. Graybill, Rosie Bocanegra,
`Laura K. Najvar, David Loebenberg,
`and Mike F. Luther
`
`Naoki Nagahara, Yohko Akiyama.
`Masafumi Nakao, Mayumi Tada,
`Megumi Kitano. and Yasuyuki
`Ogawa
`R. K. Budhani and J. K. Struthers
`
`Yves Bergeron, Nathalie Ouellel,
`Anne—Marie Deslauriers, Marie
`Simard, Martin Olivier, and Michel
`G. Bergeron
`
`Hail M. Al-Abdely, John R.
`Graybill, Rosie Bocanegra, Laura
`Najvar, Eleanor Montalbo, Steven
`L. Regen, and Peter C. Melby
`
`Vladimir Michailowsky, Silvane
`M. F. Murta, Leonardo Caivalho—
`Oliveira, Maria E. S. Pcreira,
`Ludmila R. P. Fcrreira, Zigman
`Brener, Alvaro J. Romanha, and
`Ricardo T. Gazzinelli
`
`Wiley A. Sehell, Gisele Madeira
`Duboe De Almeida, Richard K.
`Dodge, Kcnji Okonogi, and John R.
`Perfect
`
`Co/ninuezl on following page
`
`CFAD V. Anacor, IPR2015-01776 ANACOR EX. 2104 - 5/12
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`

`
`
`
`Cori/imted _/ivm /Jrwct/ilzg page
`
`Pharmacodynamics of Gatifloxaciu in Cerebrospinal Fluid
`in Experimental Cephalosporin-Resistant Pneumocoecal
`
`In Vivo Etficacy of ABT-255 against Drug-Sensitive
`and -Resistant M_vc0bacteI'iuni tuberculosis Strains
`
`Use of Mierosphere Technology for Targeted Delivery of
`Rifampin to Mycolmcterimn tuberculosis-Inlected
`
`Treatment of Vaucomyein-Resistant Enterococcus faecimn
`with RP 59500 (Quimipristin-Dalfopristin) Administered by
`Intermittent or Continuous Infusion, Alone or in
`Coinl)ination with Doxyeycline. in an In Vitro
`Pharmacodynamic Infection Model with Simulated
`Endocardial Vegetations
`
`Antitiypanosomal Activity ot' a New Triazine I)erivative,
`SIPI 1029, In Vitro and in Model Infections
`
`Visceral Leislunaniasis in the BALB/c Mouse: A
`Comparison of the Efficacy of a Nouionic Surfactant
`Formulation of Sodium Stibogluconate with Those of Three
`Proprietar_v Formulations ot' Amphotericin B
`
`PHARMACOLOGY
`
`Stability of Cephalosporin Prodrug Esters in Human
`Intestinal Juice: Implications for Oral Bioavailability
`
`Secretion of Sparflo.\acin from the Human Intestinal Caco-2
`Cell Line Is Altered by P-Glyeoprotein Inhibitors
`
`Rifampin Concentrations in Various Compartments of the
`Human Brain: A Novel Method for Determining Drug
`Levels in the Cerebral Extracellular Space
`
`Development of a New Cartridge Radioimnumoassay for
`Determination of Intracellular Levels of Lamivudine
`Triphosphate in the Peripheral Blood Mononuclear Cells of
`Human Immunodeficiency Virus-Infected Patients
`
`Compartmental Pharmacokineties and Tissue Drug
`Distribution of the Pradimiein Derivative BMS 181184 in
`
`Single-I)ose Pharmacokinetics of a Pleconaril (VP63843)
`Oral Solution and Elfect of Food
`
`A New Approach for Early Assessment of the Epileptogenic
`Potential ot'Quii1olones
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`lrja Lutsan. Ian R. Friedland.
`Loretta Wubbel. Cynthia C.
`McCoig. Hasan S. Jafri. Winston
`Ng. Faryal Ghatfar. and George H.
`McCracken. Jr.
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`Andy Oleksijew. Jon Meulbroek.
`Patty Ewing. Ken Jarvis. Mike
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`Cyrus J. Bacchi. Marcus Vargas,
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`and Weicheng Zhou
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`Jean Paul Laneury, Patrick
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`Frangois Huneau, Agnes Mordrelle.
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`Brian L. Robbins, Thu T. Tran.
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`Susan M. Abdel—Rahman and
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`Gregory L. Kearns
`Annie Delon. Claudine Pariat.
`Philippe Courtois. Serge Bouquet.
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`
`Apparent Weak Efficacy of Ivcrmectin for Treatment of
`Human To.\'ocariasis
`
`.lcan»Franc0is l\/lagnaval
`
`Iuhibitor-Resistant TEM B-Lactamases: Revision of the
`TEM-41 Sequence
`
`ERRATUA/I
`
`I£i1e1'g_v-Dcpemlent Accumulation ot' Fluoroquinoloues in
`Quinolone-Resistant Klebsiellu pneunzoniae Strains
`
`AUTHOR ’S CORRECTION
`
`Roger Labia. Danielle Sirot.
`Catlierinc Clianal-Claris. Paul
`Stapleton. Kcvin Shannon. and Gan’
`French
`
`Luis Martincz-Martinez. lsabcl
`Garcia. Sofia Ballcsta. Vicente
`Javier Bcnccli. Santiago Hci‘nzin(lcz—
`Allés. and Alvaro Pascual
`
`Incidence and Mechanisms of Resistance to the
`Combination of Amoxicillin and Clavulanic Acid in
`Esclzericlzia coli
`
`Paul Stapleton. Pei-Jun Wu. Anna
`King. Kevin Shannon. Gary French.
`and Ian Phillips
`
`E01’ your convenience, the full text of the 1998 AAC issues is available online. Access will be free
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`
`Mechanism of Fluconazole Resistance in Candida krusei
`
`ALISON S. OROZCO,l LINDSEY M. HIGGINBOTHAM.‘ CHRISTOPHER A. HITCHCOCK,3
`TANYA PARKINSON} DEREK FALCONER,3 ASHRAF S. lBRAHIM,l
`MAHMOUD A. GHANNOUMWKT AND SCOTT G. FlLLERl'3*
`
`St. John '3 Cardiovascular Research Center, Division of Infectious Diseases. Harbor-UCLA Research and Education
`Institute, Torrance, California 90502‘; Pfizer Central Research, Sandwic/1, Kent, United Kingdomz; and the UCLA
`School of Medicine, Los Angeles, California 900243
`
`Received 2 April 19‘)8/Returned for modification 30 May 1998/Accepted 9 July 1998
`
`The mechanisms of fluconazole resistance in three clinical isolates of Candida krasei were investigated.
`Analysis of sterols of organisms grown in the absence and presence of fluconazole demonstrated that the
`predominant sterol of C. krusei is ergosterol and that fluconazole inhibits 140:-(lemethylase in this organism.
`The 140:-demethylase activity in cell extracts of C. krusei was 16- to 46-fold more resistant to inhibition by
`fluconazole than was 1-lot-demethylase activity in cell extracts of two fluconazole-susceptible strains of Candida
`albicans. Comparing the carbon monoxide difference spectra of microsomes from C. krusei with those of
`microsomes from C. albicans indicated that the total cytochrome P-450 content of C. krusei is similar to that
`of C. albicans. The Soret absorption maximum in these spectra was located at 448 nm for C. krasei and at 450
`nm for C. albicans. Finally, the fluconazole accumulation of two of the C. krusei isolates was similar to if not
`greater than that of C. albicans. Thus, there are significant qualitative differences between the 1401-demethylase
`of C. albicans and C. krusei. In addition, fluconazole resistance in these strains of C. krusei appears to be
`mediated predominantly by a reduced susceptibility of 14oz-demethylase to inhibition by this drug.
`
`As the use of azole antifungal agents has risen dramatically,
`there has been 2111 increase in the number of reports of azole-
`resistant isolates of Candida. especially in patients with ad-
`vanced AIDS. In addition to the development of resistance by
`Candida albicans and Candida glabrata,
`it has been reported
`that the frequent use of fluconazole can select for the emer-
`gence of Candida kmsei as a commonly isolated opportunistic
`pathogen in some medical centers (1, 33). This finding is clin-
`ically significant because C. knisei can cause serious infections
`in susceptible patients (8, 19). Furthermore, this organism is
`usually intrinsically resistant to fluconazole, botl1 in vitro (3)
`and in vivo (4).
`Three general mechanisms of azole resistance have been
`described for species of Candida. The first is an alteration in
`the target enzyme, 14ot—demethylase. Inhibition of this enzyme
`by azoles causes an accumulation of CH methylated sterols
`which likely disrupt membrane structure (9). In some resistant
`organisms,
`there is overexpression of the 140:-demethylase
`gene and/or the enzyme is less susceptible to azole inhibition
`(15, 24, 32). The second mechanism is decreased drug accu-
`mulation, mediated by either diminished uptake or increased
`etllux of the drug (22, 26). The third mechanism of resistance
`is the presence of a deficiency in C5(,,) desaturase. Organisms
`deficient in this enzyme produce 14-methylfecosterol and re-
`main viable when 14oz-demethylase activity is inhibited (5, 14).
`To determine if fluconazole resistance in C. krusei is medi-
`ated by one or more of these mechanisms, we analyzed the
`eltects of this antifungal agent on sterol synthesis by three
`strains of C. krusei. In addition, the fluconazole uptake and
`cytochrome P-450 content of these organisms were measured.
`Our results indicate that the predominant mechanism of flu-
`
`Corresponding author. Mailing address: Division of Infectious
`Diseases, Harbor-UCLA Research and Education Institute, 1124 West
`Carson St., RB—2, Torrance, CA 90502. Phone: (310) 222-6426. Fax:
`(310) 782-2016. E-mail: Filler@HUMC.EDU.
`T Present address: Department of Dermatology, Center for Medical
`Mycology, Case Western Reserve University, Cleveland, OH 44106.
`
`conazole resistance in these organisms is a 14ot-demethylase
`with reduced susceptibility to the inhibitory etfects of flucon-
`azole.
`
`MATERIALS AND METHODS
`Fungal strains and susceptibility testing. Three clinical isolates of C. Iomei,
`strains 91-1158. 91-1159, and 91-1161. were generously provided by Michael
`Rinaldi (San Antonio, Tex.). A fourth isolate of C. krusei, ATCC 6258, was
`obtained from the American Type Culture Collection (Rockville, Md.). Two
`clinical isolates of C. albicans, Y01.345 and SC5314, were supplied by Christo-
`pher Hitchcock and William Fonzi (Georgetown University School of Medicine,
`\’»/ashington, D.C.), respectively. The susceptibilities of the organisms to flucon-
`azole and itraconazole were determined at 24 and 48 h by the National Com-
`mittee for Clinical Laboratory Standards M27-A broth microdilution method at
`an inoculum of 103 organisms per \vcll (20). The medium was RPMI 1640 (Irvine
`Scientific. Santa Ana, Calif.) buffered to pH 7.0 with 0.165 M morpl1olinepro-
`panesulfonie acid (MOPS). The MICs were defined as the concentrations of drug
`that reduced growth by St)”/r compared to that of organisms grown in the absence
`of drug.
`Sterol analysis. For sterol extraction, each strain of C. krurei was grown for
`24 h at 37°C on a rotary shaker. The medium was Sabouraud dextrose broth
`(Difeo, Detroit, Mich.), with and without fluconazole. The concentration of
`tluconazole was 16 p_g/ml, the highest concentration at which these organisms
`would grow in this medium. The organisms were harvested by centrifugation and
`washed twice in 0.85% saline, and their total sterols were extracted by ethanolic
`K01-I, as described previously (6. 7). The resultant suterols were further purified
`by thin-layer chromatography on PK6F silica gel 60—A plates (Whatman, Clifton,
`NJ.) with a solvent system of petroleum ether-diethyl ether (321, vol/vol) (27).
`The sterols were eluted from the silica in chloroform-diethyl ether-ethanol (1:
`1:1). After being derivatized with hexamethyldisilazane and trimethylchlorosi—
`lane (28). the sterols were redissolved in hexane and analyzed by gas chroma-
`tography-mass spectrometry. The sterols were identified by comparison to known
`standards and published data (16, 17, 23).
`Fluconazole accumulation. A filter-based assay was used to measure the ac-
`cumulation of fluconazole by the organisms (22). These organisms were grown to
`exponential phase in Sabouraud dextrose broth and suspended in phosphate-
`buifered saline (pH 7.5) containing 5% glucose (wt/vol) at 108 organisms per ml.
`Next. a mixture of [3H]tluconazole (specific activity, 715 GBq/mmol) and unla-
`beled fluconazole was added to the cells so that
`the final concentration of
`fluconazole was 100 nM (0.2 nCi/ml). At selected intervals, aliquots were re-
`moved and the organisms were collected by filtration. Next, the organisms were
`washed four times in pltospliate-bullered saline containing 100 nM unlabeled
`fluconazole. The amount of cell—associated radioactivity was determined by scin-
`tillation counting. All experiments were performed in triplicate.
`Carbon monoxide difference spectra of microsomes. The cytochrome P-450
`content of the organisms was analyzed by measuring their carbon monoxide
`dilterence spectra. Organisms were grown to exponential phase in Sabouraud
`
`2645
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`
`OROZCO ET AL.
`
`ANTIMICROB. AGENTS CHEMOTHER.
`
`TABLE 1. Antifungal susceptibilities of C. ulbicmis and C. /(rusei
`
`RESULTS
`
`MIC (t.Lg/I'I'll)
`
`Fluconazole
`24 h
`48 h
`
`ltraconazole
`24 h
`~ 48 11
`
`0.25
`0.5
`32
`16
`16
`
`<0.25
`1
`64
`32
`32
`
`0.125
`0.5
`0.5
`0.5
`0.5
`
`0.125
`0.5
`0.5
`0.5
`0.5
`
`dextrose broth at 37°C and harvested by centrifugation. They \vere spheroplasted
`with lyticase (Sigma, St. Louis, Mo.) in sorbitol buffer (1.5 M sorbitol in 10 mM
`Tris butler. pH 7.4) and resuspended in 10 mM Tris buffer, pH 7.4. containing
`0.65 M sorbitol, 0.1 n1M EDTA, 0.1 mM glutathione. and protease inhibitors
`(Boehringer Mannheim, Indianapolis. Ind.) (10). All subsequent steps were
`carried out at 4°C. The spheroplasts \vere broken into microsomes by sonication.
`after which debris and unbroken cells were removed b_v centrifugation at 1.500 X
`g for 10 min followed by 25,000 X g for 25 min. The microsomes \vere harvested
`with calcium chloride by the method of Kappeli et al. (13), \vashed once in 10
`n1M Tris buffer, pH 7.4, containing 150 mM potassium chloride. and resuspended
`in 100 mM Tris containing 0.65 M glycerol, 0.1 mM EDTA, and 0.1 ml\’l gluta-
`thione. The carbon monoxide difference spectra of the microsomes were mea-
`sured. and their cytochrome P-450 content was calculated by using an extinction
`coefficient of 91 liters/nunol/cm (21). The protein concentration of each micro-
`some suspension was determined by the Bio-Rad protein assay (Bio-Rad. Her-
`cules. Calif). For each organism, the cytochrome P-450 contents of at least three
`different preparations of microsomes were measured.
`Sterol biosynthesis by cell extracts. To determine the effects of fluconazole on
`the synthesis of ergosterol from [”C]meva1onic acid, the organisms were grown
`in Sabouraud dextrose broth until late exponential phase. The organisms were
`broken by vortexing with glass beads in 0.1 M potassium phosphate, pH 7.5.
`Debris and unbroken cells were removed b_v centrifugation at 2,000 X g for 5 min
`followed b_v 10.000 x g for 10 min. To measure sterol biosynthesis, 925 l.Ll of the
`resultant supernatant was added to 75 pl of cofactor hul1'er to achieve the
`following final concentrations: 1 pM NADP.
`1
`|J.l\/1 NADPH, 1 pM NAD. 7 uM
`glucose-6-phosphate. 5 pM ATP. 3 p,l\'l reduced glutathione. 2 i.t1\/1 MnCl3. and
`0.25 |.|.Ci ["C]mevalonic acid (10. 12). Selected concentrations of lluconazole
`were added to the reaction mixtures before the addition ofthe cell extracts. After
`incubation at 37°C for 2 h. the reaction was stopped with ethanolic KOH. The
`samples were saponified at 80°C for -15 min. after which the nonsaponiliable
`lipids were extracted with petroleum ether (bp 40 to 60”C). The samples were
`dried under nitrogen and redissolved in chloroform. The sterols were separated
`by thin-|a_ver chromatography on silica gel LK6D (Whatman) with a solvent
`system of petroleum ether-diethyl ether (3:1. vol/vol) (27). The sterols \vcre
`visualized by iodine staining and identified by comparison with commerciall_v
`available standards which were run in parallel. Next. the sterol-containing bands
`were scraped from the plates and “C incorporation was determined by liquid
`scintillation counting. All experiments were repeated at least three times with
`
`Growth in fluconazole reduced the ergosterol content of C.
`krusei. Fluconazole MICs for C. krusei 91-1158, 91-1159, and
`91-1161 were all high, whereas both strains of C. albicans were
`susceptible to this drug (Table 1). C. a/bicrms Y01.345 was
`slightly more susceptible to itraconazolc than C. albicmzs
`SC5314 or the three strains of C. kmsei. Analysis of the sterols
`of the organisms by gas chromatography-mass spectrometry
`revealed that the predominant sterol in C. kmsei was ergosterol
`(Table 2). Growing all three strains of C. kmsei in the presence
`of fluconazole caused a decrease in their ergosterol content
`and a marked increase in lanosterol. Other 14-methyl sterols
`that were increased in organisms exposed to fluconazole were
`14-methylfecosterol and eburicol. These results suggest
`that
`fiuconazole inhibits 1401-demethylase in C. kmsei. No 14-meth-
`