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Fluoroquinolones, Broad- Spectrum Β-Lactams

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Fluoroquinolones, Broad- Spectrum Β-Lactams Practical cases of interpretative reading and clinical impact of the antibiogram-quinolones Dóra Szabó Institute of Medical Microbiology Semmelweis University, Budapest, Hungary ESCMID POSTGRADUATE© by author EDUCATION COURSE Quinolones: from Bench to Bedside. October 5-7. 2011 ESCMID Online Lecture Library Mechanisms of quinolone resistance Mutations in the bacterial enzymes: DNA gyrase - gyrA DNA topoisomerase IV – parC Efflux pumps multidrug transporters of the resistance- nodulation-cell division (RND) family AcrAB efflux pump© by author Porin changes Plasmid-mediated quinolone resistance (PMQR)ESCMID Online Lecture Library Plasmid-mediated quinolone resistance (PMQR) Qnr proteins - protecting DNA gyrase from quinolones qnrA, qnrS, qnrB, qnrC, qnrD AAC(6’)-Ib-cr – a variant aminoglycoside acetyl-transferase © by author Plasmid-mediated quinolone efflux OqxAB QepAESCMID Online Lecture Library Problem in detection fluoroquinolone resistance Low-level fluoroquinolone resistance First-step mutation in the bacterial enzymes: DNA gyrase - gyrA DNA topoisomerase IV – parC Plasmid-mediated quinolone© by author resistance (PMQR) ESCMID Online Lecture Library In vitro activity of quinolones against wild- type E. coli carrying gyrA PMQR deteminants MIC (µg/ml) MIC (µg/ml) MIC (µg/ml) Agent E. coli J53 CLSI EUCAST E. coli (gyrA mutant) Ciprofloxacin ≤1 ≤0.5 0.008 0.25 Levofloxacin ≤2 ≤1 0.0015 0.5 Nalidixic acid ≤16 © by4 author≥256 Premafloxacin 2 0.25 ESCMID Online LectureMartinez- MartinezLibrary et al. JAC 2003, 51,1037 The effect of qnrA genes on MIC values Increasing the MIC values of quinolone Increased from 0.008 mg/L to 0.25 mg/L ciprofloxacin MIC in E. coli J54 (range 0.125 mg/L to 2 mg/L) (Wang et al. AAC 2003 47,2242) Least pronounced for nalidixic acid (twofold to eightfold increaes in MIC) (Wang et al. AAC 2003 47,2242) © by author For example non-typhi Salmonella reduced susceptibiltiy to ciprofloxacin but susceptible to nalidixic acid (Hopkins et al. EID 2008 14,340; Hopkins et al. JAC 2007 ESCMID59,1071) Online Lecture Library The effect of qnrA genes on MIC values qnrA harboring plasmids yielded very similar quinolone susceptibilities in transconjugants (Wang et al. AAC 2003 47, 2242) Ciprofloxacin susceptibiliy varied 16-fold Presence of additional plasmid mediated quinolone determinants © by author Copy number Transcriptional level (RodrigezESCMID-Martinez JM Online et al. CMI 2006 Lecture 12,440; Wu XS etLibrary al. AAC 2007 5, 4105) In vitro activity of quinolones against wild- type E. coli carrying qnr PMQR deteminants MIC (µg/ml) MIC (µg/ml) MIC (µg/ml) Agent CLSI EUCAST E. Coli E. coli E. coli E. coli E. coli E. coli 1 2 3 4 5 J53 qnrA qnrS qnrB qnrC qnrD Ciprofloxacin ≤1 ≤0.5 0.008 0.25 0.25 0.25 0.25 0.06 Levofloxacin ≤2 ≤1 0.0015 0.5 0.38 0.5 0.25 Nalidixic acid ≤16 4 16 16 16 16 Gatifloxacin 0.25 Ofloxacin ≤0.5 © 2by author 0.125 1 Wang et al. AAC 2004 48,1400 2 Hata et al. AAC, 2005 49,801 3 Jacoby et al AAC, 2006 50, 1178 4 Wang et al. AAC, 2009 53, 1892 ESCMID Online Lecture5 Cavaco Library et al. 2009 53,603 The effect of qnrS and qnrB genes on MIC values Similar to qnrA1 Increased MIC of nalidixic acid, ciprofloxacin and ofloxacin 8-, 83-, and 24 fold (Cattoir V et al. CMI 2007 13, 940) Nalidixic acid can be resistant by CLSI breakpoint More studies overall© bynalidixic author acid 2- 8 fold and ciprofloxacin 8- to 32 fold increase in MIC (JacobyESCMID GA et al. AAC 2006 Online 50,1178; Gay Lecture KA et al. CID 2006 Library 43, 297) Interaction between Qnr and other resistance mechanisms qnrA is often with other resistance mechanisms alteration in DNA gyrase Topoisomerase IV Efflux Outer porin chanels A few studies more than one qnr gene Two genes product may© competeby author for binding gyrase (Cattoir VL et al. AAC 2007 51,2652; Hu FP et al.Acta Phar Sin 2008 29,320) ESCMID Online Lecture Library The effect of aac(6’)-Ib-cr gene on MIC values Selective for ciprofloxacin and norfloxacin Both have piperazinyl amine side chain Increase in MICs is modest, smaller than conferred by Qnr proteins © by author ESCMID Online Lecture Library The effect of qepA gene on MIC values Multiresistance profile Aminoglycosides, fluoroquinolones, broad- spectrum β-lactams Nalidixic acid 2- fold Ciprofloxacin 32-fold Norfloxacin 64-fold© by author (Yamane KJ et al. AAC 2007 51,3354) TwofoldESCMID increase for Onlineerythromycin, Lecture acriflavine, Library ethidium-bromide In vitro activity of quinolones against wild- type E. coli carrying other PMQR determinants MIC (µg/ml) MIC (µg/ml) MIC (µg/ml) Agent CLSI EUCAST E. coli E. coli E. coli J53 qepA21 aac(6’)-Ib-cr2 Ciprofloxacin ≤1 ≤0.5 0.008 0.12 0.004-0.008 Gatifloxacin ≤2 0.008 0.12 Levofloxacin ≤2 ≤1 0.015 0.015 0.015 Nalidixic acid ≤16 4 4 Moxifloxacin ≤0.5© by author0.03 0.12 Norfloxacin ≤1 ≤0.5 0.06 1 0.64 ESCMID Online Lecture1 Cattoir Library et al. AAC 2008 52, 3801 2 Robicsek et al. Nat med 2006 12,83 Quinolone© by author susceptibility in Enterobacteriaceae ESCMID Online Lecture Library EUCAST Clinical breakpoints for Enterobacteriaceae 2011 Fluoroquinolnes MIC breakpoint Disk Zone diameter (mg/L) content breakpoint (mm) (µg) S≤ R> S≥ R< Ciprofloxacin 0.5 1 5 22 19 Levofloxacin 1 2 5 22 19 Moxifloxacin 0.5 1 5 20 17 Nalidixic acid NA NA© by authorNA NA NA (screen) Norfloxacin 0.5 1 10 22 19 Ofloxacin ESCMID0.5 Online1 Lecture5 Library22 19 Changes Version 1.3, January 2011 Revised comments on ampicillin, ampicillin-sulbactam, piperacillin- tazobactam (typo error) cefoxitin and tigecycline. Revised or new zone diameter breakpoints for ampicillin- sulbactam, amoxicillin-clavulanate, cefalexin, cefoxitin (screen), ceftazidime and tobramycin. Nalidixic acid zone diameter breakpoints previously recommended for detection of fluoroquinolone resistance removed. Nalidixic acid does© by not detectauthor qnr-mediated resistance and low-level resistance in Enterobacteriaceae (exception Salmonella spp) is no longer of major interest since high-level resistance is now common in most EnterobacteriaceaeESCMID Online species. Lecture Library CLSI Clinical breakpoints for Enterobacteriaceae 2011 © by author ESCMID Online Lecture Library Differences between EUCAST and CLSI quinolone breakpoint recommendation Ciprofloxacin EUCAST S≤ 0.5, R> 1 CLSI S≤ 1; R> 4 Levofloxacin EUCAST S≤ 1; R> 2 CLSI S≤ 2; R>2 Norfloxacin EUCAST S≤ 0.5; R> 1 CLSI S≤ 4, R>16 © by author Ofloxacin EUCAST S≤ 0.5; R> 1 CLSI S≤ 2; R> 8 ESCMID Online Lecture Library Differences between EUCAST and CLSI quinolone breakpoint recommendation Recommendation only in EUCAST for Moxifloxacin Recommendation only in CLSI for Nalidixic acid Lomefloxacin Enoxacin Gatifloxacin Gemifloxacin Grepafloxacin © by author Fleroxacin ESCMID Online Lecture Library 69 Escherichia coli isolates 62 Salmonella isolates susceptible isolates well-characterized nalidixic-acid resistant strains with one or two mutations in the QRDRs of the topoisomerase genes low-level-resistant strains with either qnrA, qnrB, or qnrS and aac(6’)Ib-cr © by author measured the MICs and inhibition zone diameters for a panel of quinolones ESCMID Online Lecture Library EUCAST breakpoints for © by author susceptibility CLSI breakpoints for susceptibility ESCMID Online Lecture Library aac(6’)Ib-cr positive isolate © by author ESCMID Online Lecture Library EUCAST susceptibility CLSI susceptibility aac(6’)Ib-cr positive isolate EUCAST breakpoints for © by author susceptibility CLSI breakpoints for susceptibility ESCMID Online Lecture Library aac(6’)Ib-cr positive isolate © by author ESCMID Online Lecture Libraryaac(6 ’)Ib-cr positive isolate EUCAST susceptibility CLSI susceptibility Differencies in drug potencies Specificities toward substrate Carrying aac(6’)Ib-cr In qnr-positive isolates the expression level can be different Structural differences© by between author quinolones tested ESCMID Online Lecture Library Quinolones Nalidixic acid Very good for detection of mutants NOT GOOD for qnr- or aac(6’)Ib-cr positive E. coli or Salmonella isolates Oxolinic acid or flumequine Better results for detection© by author of qnr genes ESCMID Online Lecture Library Fluoroquinolones Very good for detection of qnr genes For the detection of aac(6’)Ib-cr gene only ciprofloxacin or norfloxacin are useful But they still might be undetected when low breakpoints are used because of the low-level mutations © by author ESCMID Online Lecture Library Screening with nalidixic acid is efficient for the detection of mutants, but it is not so efficient for the detection of qnr and aac(6)Ib-cr. Detection would be maximized by screening with either ciprofloxacin or norfloxacin by both MIC determination and disk diffusion assays. Furthermore, a low ©concentration by author of ciprofloxacin (1 g) in the disks seemed to increase the sensitivity of the disk diffusion assay. ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Case reports © by author ESCMID Online Lecture Library Minimal inhibitory concentration (µg/ml) used in this study Antimicrobial agent MIC(µg/ml) E. coli K-12 K. pneumoniae E. coli U8699 E. coli m4353 E. coli m4353 RX85 U8699 transconjugant transconjugant recipient Nalidixic acid 4 16 32 16 32 Norfloxacin ≤2 4 ≤2 ≤2 ≤2 Ciprofloxacin 0.16 0.38 0.19 0.25 0.19 Imipenem 0.19 >32 1 4 4 Meropenem 0.094 6 © by author0.19 0.25 0.19 Aztreonam 0.032 4 0.2 0.094 0.032 PCR/sequencing none blaCMY-13, blaSHV- blaCMY-13, blaVIM-1, bla TEM-1 blaVIM-1, qnrB1 11, blaVIM-1 qnrA1 blaVIM-1 qnrA1 like1, qnrB1 ESCMID Online Lecture Library Galani et al IJAA 2010 36, 252 First report of presence qnrA1 and qnrB2 on the same transferable plasmid with bla VIM-1 and blaCMY-13 in Klebsiella pneumoniae and with blaVIM-1 in E.
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