ACCEPTED ANTIPNEUMOCOCCAL ACTIVITY OF LBM415 COMPARED TO OTHER AGENTS. KLAUDIA KOSOWSKA-SHICK KIM L. CREDITO GLENN A. PANKUCH BONIFACIO DEWASSE

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1 AAC Accepts, published online ahead of print on 20 November 2006 Antimicrob. Agents Chemother. doi: /aac Copyright 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. MULTISTEP RESISTANCE SELECTION AND PAE STUDIES ON ANTIPNEUMOCOCCAL ACTIVITY OF LBM415 COMPARED TO OTHER AGENTS. KLAUDIA KOSOWSKA-SHICK KIM L. CREDITO GLENN A. PANKUCH BONIFACIO DEWASSE PAMELA MCGHEE PETER C. APPELBAUM Department of Pathology, Hershey Medical Center, Hershey, PA Running title: LBM415 antipneumococcal activity Correspondence to: Dr. Peter C. Appelbaum Department of Pathology Hershey Medical Center P.O. Box 850 Hershey, PA Telephone: (717) Telefax: (717)

2 ABSTRACT LBM415 is a peptide deformylase inhibitor active against Gram-positive and some Gramnegative species. In multiselection studies, LBM415 had low MICs against all S.pneumoniae strains tested regardless of genotype and selected resistant clones after days. MIC increases correlated with changes mostly in 70 GXGXAAXQ 77 motif in peptide deformylase (PDF). Post-antibiotic effect (PAE) of LBM415 ranged between 0.3 and 1.4 h. 2

3 LBM415 is a peptide deformylase with predominant activity against Gram-positive bacteria and good potency against pneumococci (4-6,9,10,19). The current study extends the antipneumococcal activity of LBM415 by i) testing capability of LBM415 compared to different classes of antibiotics which show clinical significance in treatment of community-required pneumococcal respiratory tract infections: amoxicillin/clavulanate, cefuroxime, cefdinir, ceftriaxone, imipenem, azithromycin, clarithromycin, levofloxacin, gatifloxacin and moxifloxacin to select for resistance in 12 strains by multistep methodology; ii) testing resistance mechanism of selected resistant clones; iii) examining the post-antibiotic effect (PAE) of the above compounds against 9 pneumococci. For multistep resistance selection, 4 erythromycin susceptible, 3 erm(b) positive, 2 mef(a) and 1 strain each with erm(b) + mef(a), L4 and 23S rrna mutations were studied: of these, 3 were penicillin-susceptible, 4 penicillin-intermediate and 5 penicillin-resistant; 3 strains were quinolone-resistant and 9 quinolone-susceptible. For PAE studies, strains comprised 3 erythromycin-susceptible ( 0.06 µg/ml), 3 erm(b) positive and 3 with mef(a): of these, 3 were penicillin-susceptible, 2 penicillin-intermediate and 4 penicillin-resistant, and all were quinolone-susceptible. LBM415 laboratory-grade powder was obtained from Novartis Laboratories, Hanover, NJ. Other antimicrobials were obtained from their respective manufacturers. Prior MIC testing of all strains was by CLSI macrodilution (15). Multipassage resistance selection was done as described previously (11). Daily passages were continued until >4-fold increase in MIC was found (minimum 14, maximum 50 passages). Stability of acquired resistance was determined by MIC after 10 passages on drug-free agar. Identities of all resistant strains and parent isolates were tested by pulse-field gel electrophoresis (PFGE)(11). All LBM415-resistant mutants and their parents were analyzed for presence of mutations in defb 3

4 gene encoding peptide deformylase (PDF). Gene defb was amplified using the following primers: defb6up 5 -GAGAAAATGTCTGCAATAGAACGT 3 and defb6dn 5 - CCCCGTCTTGCAACGGGA-3. These primers were used for sequencing analysis as well. Transformation of S.pneumoniae was performed as described previously (7,18). S.pneumoniae R6 strain was used as DNA recipient and strain A9 served as a control in transformation experiments. Purified DNA (final concentration 1 µg/ml) from isolates with defined defb mutations was used (Table 1). Selection was done on BHI agar plates with 5% sheep blood and appropriate antibiotics (LBM415: 1 or 2 µg/ml; streptomycin: 100 µg/ml). All quinolone-resistant clones and their parental strains were analyzed for presence of alterations in quinolone resistance determinant regions (QRDR) encoding by parc, pare, gyra and gyrb genes using primers and cycling conditions described previously (14,16,17). Genes encoding ribosomal proteins L4, L22 and domains II and V of 23S rrna were amplified and sequenced for macrolide-resistant parents and multistep selected macrolide-resistant clones (18). PAE was performed as described previously (3,11) with exposure to 10 x MIC for 1 h. Results of multistep resistance selection tests are presented in Table 1 and 2. LBM415 yielded resistant clones in 11/12 strains after days, with MIC (µg/ml) increases from (parents) to 2.0->16.0 (mutants); moxifloxacin MICs rose from (parents) to (mutants) in days for 7/12 strains. Gatifloxacin yielded MICs which rose from 1-2 µg/ml to 16 µg/ml in 3/12 strains after days; levofloxacin MICs increased in 2/12 strains after days from 1 (parents) to 16 (mutants); azithromycin MICs rose from to 0.25->64 in 3/6 strains after days and clarithromycin MICs rose from to >128 in 4/8 strains after days. Amoxicillin/clavulanate, cefuroxime, cefdinir, ceftriaxone, and imipenem did not yield resistant clones after 50-days. 4

5 Among LBM415 selected clones none showed cross-resistance (defined as > 4-fold increase in MIC). Cross-reactivity was observed only within the same group of antibiotics: quinolones (8 clones) and macrolide (4 clones)(table 2). All quinolone-resistant selected clones (except clones 3243 and 3009 with raised moxifloxacin and gatifloxacin MICs, respectively) had changes in QRDR region (Table 2). Among macrolide-resistant selected clones, three had changes in 23S rrna or L22 protein (Table 2). Six out of eleven LBM415 selected clones had change in PDF protein (Table 1). Results of PAE studies are presented in Table 3. As can be seen, LBM415 had PAEs ranging between 0.3 and 1.4 h. LBM415 PAEs were not affected by the strains susceptibility to β-lactams or macrolides. β-lactam PAEs ranged between h. Macrolide and quinolone PAEs ranged between 1.8 and 6 h and h, respectively. Macrolides and quinolones gave longer PAEs compared to LBM415 and ß-lactams. LBM415 yielded resistant clones, with defined and stepwise mutations in the PDF protein, in most strains tested after days. Among eleven selected LBM415 clones with raised MICs, only two clones (Table 1) retained raised MIC after 10 days drug-free subculture, suggesting that this selected resistance may be unstable in the absence of the drug. LBM415 MICs (µg/ml) of 16.0 and 4 were associated with substitutions in 69 GGVGLAAPQ 77 (G69V/R/C substitutions) motif (Table 1). The GXGXAAXQ motif is highly conserved among many bacterial species (12,13) and is involved in the structural stability and catalytic mechanism of PDFs. Transfer of the defb gene with substitutions G69V/R/C and/or Q172H and/or P76R from 3009, 3243 and 37 strains to susceptible S. pneumoniae R6 resulted in increase of LBM415 MICs from 0.25 of R6 strain to 16, 8 and 4 µg/ml, respectively. A previous study has described that S. pneumoniae R6 strain expressing mutated defb gene encoding single substitution Q172H displayed reduced susceptibility to 5

6 actinonin, member of the PDF inhibitor drug class (12). In our study, change within zinc binding motif 173 HEIDH 177, resulted in MICs of 8 µg/ml, but transformation of defb with substitution D176N (donor strains: 1076 and 1077) failed and no R6 mutant was selected, even on selection plates with 1 µg/ml of LBM415. Alterations G139F and R135C, near 128EGCLS 132 motif, which is essential for enzyme activity (12,13) resulted in MICs of 2-4 µg/ml, but only mutation G139F (donor strain 24) was essential to select an R6 mutant with MIC 4 µg/ml. Changes in PDF close or within 70 GXGXAAXQ 77 conserved motif were crucial to increase of LBM415 resistance levels in mutated clones. Also, frequency of introducing a mutated gene (defined as number of transformants grown on selective medium to number of clones grown on medium without selective agent) with two substitutions: G69R and P76R was times higher than in other mutated forms of defb gene tested. Mutations near or within 173 HEIDH 177 and 128 EGCLS 132 motifs appear not to have influenced low-level of resistance in mutants tested. The LBM415 resistance mechanisms in the other three mutants with no alteration in PDF protein remain unknown and require further investigation. Mechanisms of resistance for quinolones and macrolides (Table 2) have been described before (1,2,8,11). However, substitutions S478I and P413S in GyrB have not, to our knowledge, been reported before. LBM415 had short, but detectable PAEs against all nine strains tested. PAEs did not depend on the strain s susceptibility to other unrelated agents. PAEs for macrolides and quinolones were usually higher than those of LBM415 and all ß-lactams tested. Our results suggest that LBM415 might be of potential use for therapy (including potentially short-course regimens) of pneumococcal infections. This hypothesis will need 6

7 to be clarified by pharmacokinetic/pharmacodynamic and animal experimentation studies. This study was supported by a grant from Novartis Laboratories, Hanover, NJ. We thank Dr. Joyce Sutcliffe for providing strain A9 and advice on transformation experiments. 7

8 REFERENCES 1. Browne, F.A., C. Clark, B. Bozdogan, B.E. Dewasse, M.R. Jacobs, and P.C. Appelbaum Single and multi-step resistance selection study in Streptococcus pneumoniae comparing ceftriaxone with levofloxacin, gatifloxacin and moxifloxacin. Int. J. Antimicrob. Agents. 20: Canu, A., B. Malbruny, M. Coquemont, T.A. Davies, P.C. Appelbaum, and R. Leclercq Diversity of ribosomal mutations conferring resistance to macrolides, clindamycin, streptogramin, and telithromycin in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 46: Craig, W.A., and S, Gudmondsson Postantibiotic effect, p In V. Lorian (ed.), Antibiotics in laboratory medicine. The Williams and Wilkins Co., Baltimore, MD. 4. Ednie, L.M., G. Pankuch, and P.C. Appelbaum Antipneumococcal activity of LBM415, a new peptide deformylase inhibitor compared to other agents. Antimicrob. Agents Chemother. 48: Fritsche, T.R., H.S. Sader, R. Cleeland, and R.N. Jones Comparative antimicrobial characteristics of LBM415 (NVP PDF-713), a new peptide deformylase inhibitor of clinical importance. Antmicrob. Agents Chemother. 49: Fritsche, T.R., and H.S. Sader Antimicrobial spectrum and activity of NVP-PDF 713, a novel peptide deformylase inhibitor, tested against 1,837 recent gram-positive clinical isolates. Diagn. Microbiol. Infect. Dis. 49: Havarstein, L.S., G. Coomaraswamy, and D.A. Morrison An unmodified heptadecapeptide pheromone induces competence for genetic transformation in Streptococcus pneumoniae. Proc. Natl. Acad. Sci. USA 92: Janoir, C., V. Zeller, M.-D. Kitzis, N.J. Moreau, and L. Gutmann High-level 8

9 fluoroquinolone resistance in Streptococcus pneumoniae requires mutations in parc and gyra. Antimicrob. Agents Chemother. 40: Jones, R.N., T.R. Fritsche, and H.S. Sader Antimicrobial spectrum and activity of NVP-PDF 713, a novel peptide deformylase inhibitor, tested against 1,837 recent grampositive clinical isolates. Diagn. Microbiol. Infect. Dis. 49: Jones, R.N., G.J. Moet, H.S. Sader, and T.R. Fritsche Potential utility of a peptide deformylase inhibitor (NVP-PDF 713) against oxazolidinone-resistant or streptograminresistant Gram-positive organism isolates. J. Antimicrob. Chemother. 53: Matic, V., K. Kosowska, B. Bozdogan, L.M. Kelly, K. Smith, L.M. Ednie, G. Lin, K.L. Credito, C.L Clark, P. McGhee, G.A. Pankuch, M.R. Jacobs, and P. C. Appelbaum Antipneumococcal Activities of Two Novel Macrolides, GW and GW , Compared with Those of Erythromycin, Azithromycin, Clarithromycin, Clindamycin, and Telithromycin 48: Margolis, P., C. Hackbarth, S. Lopez, M. Maniar, W. Wang, Z. Yuan, R. White, and J. Trias Resistance of Streptococcus pneumoniae to deformylase inhibitors is due to mutations in defb. Antimicrob. Agents Chemother. 45: Meinnel, T., C. Lazennec, S. Villoing, and S. Blanquet Structure function relationships within the peptide deformylase family. Evidence for a conserved architecture of the active site involving three conserved motifs and a metal ion. J. Mol. Biol. 267: Nagai, K., T.A. Davies, G.A. Pankuch, B.E. DeWasse, M.R. Jacobs, and P.C. Appelbaum In vitro selection of resistance to clinafloxacin, ciprofloxacin, and trovafloxacin in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 44:

10 15. National Committee for Clinical Laboratory Standards Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically - fifth edition; approved standard. NCCLS publication no. M7-A7. National Committee for Clinical Laboratory Standards, Wayne, PA. 16. Pan, X.S., J. Ambler, S. Mehtar, and L.M. Fisher Involvement of topoisomerase IV and DNA gyrase as ciprofloxacin targets in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 40: Pankuch, G.A., S.A. Juenemann, T.A. Davies, M.R. Jacobs, and P.C. Appelbaum In vitro selection of resistance to four ß-lactams and azithromycin in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 42: Tait-Kamradt, A, T. Davies, P.C. Appelbaum, F. Depardieu, P. Courvalin, J. Petitpas, L. Wondrack, A. Walker, M.R. Jacobs, and J. Sutcliffe Two new mechanisms of macrolide resistance in clinical strains of Streptococcus pneumoniae from Eastern Europe and North America. Antimicrob. Agents Chemother. 44: Watters, A.A., R.N. Jones, J.A. Leeds, G. Denys, H.S. Sader, and T.R. Fritsche Antimicrobial activity of a novel peptide deformylase inhibitor, LBM415, tested against respiratory tract and cutaneous infection pathogens: a global surveillance report ( ). J. Antimicrob. Chemother. 57:

11 Table 1. S. pneumoniae multistep resistance selection, mechanism of resistance detection and transformation results for LBM415. Selected Resistance Retest MIC of LBM415 Strain Initial MIC (µg/ml) after 10 Antibiotic-free Mutations in PDF Transformation/MIC (macrodilution) MIC (µg/ml) # Passages Subcultures >16 14 >16 G69V + a /16 µg/ml D176N - a D176N - a c NT b G69C, Q172H + a /8 µg/ml R135C - a c NT b G139F + a /4 µg/ml c NT b G69R, P76R + a /4 µg/ml c NT b Abbreviation: PDF, peptide deformylase. a transformation experiment: + positive selection on LBM415 plates (LBM415-2 µg/ml), - no mutants obtained on LBM415 plates (1 and 2 µg/ml LBM415) b NT; not tested c mechanism not detected 11

12 Table 2. S. pneumoniae multistep resistance selection, and mechanism of resistance detection for macrolides and quinolones used in the study. Selected Initial Retest MIC after 10 Antibiotic-free Subcultures Resistance Strain Antibiotic MIC (µg/ml) MIC # (µg/ml) Passages LBM415 AMC IMP CXM CDR CRO AZM CLR LVX GAT MXF 3009 LVX <0.008 <0.004 < a 4 Detected resistance mechanism Mutations: GyrA (S81Y) & ParC (S79Y) 3009 GAT > b 3009 MXF > Mutations: GyrA (S81F) & ParC (D83N) 3009 CLR 16 > <0.008 < b 1076 MXF >128 > ParC mutation: S79Y 1076 GAT >128 > ParC mutation: S79Y 1077 AZM 0.03 > >64 > S rrna substitution: A2058G c 3665 MXF GyrA mutation: S81Y 3665 CLR b 3676 MXF Mutations: GyrA (E85G), GyrB (S478I) & ParC (S79Y) 3676 CLR b 3243 MXF < b 3243 AZM L22 insertion: 93SFRPRA CLR S rrna substitution: A2059T c 19 MXF >128 > GyrA mutation: S81F 19 LVX >128 > Mutations: GyrA (S81Y) & ParC (D83Y) 24 MXF >128 > Mutations: GyrA (S81Y), GyrB (P413S) & ParC (S79F) 2527 AZM b 37 GAT >128 > Mutations: GyrA (S81Y), GyrB (E474K) & ParC (S79Y) Abbreviations: AMC, amoxicillin-clavulanic acid; CRO, ceftriaxone; CXM, cefuroxime; CDR, cefdinir; IPM, imipenem; AZM, azithromycin; CLR, clarithromycin; LVX, levofloxacin; GAT, gatifloxacin; MXF, moxifloxacin. a cross-resistant strains are depicted in bold b mechanism not detected c Escherichia coli numbering 12

13 Table3. MICs and PAEs of nine S. pneumoniae strains. Drug MIC Range (µg/ml) PAE (h) a LBM ( ) Amoxicillin/clavulanate ( ) Cefuroxime ( ) Cefdinir ( ) Ceftriaxone ( ) Imipenem ( ) Azithromycin b ( ) Clarithromycin b ( ) Levofloxacin ( ) Gatifloxacin ( ) Moxifloxacin ( ) a Mean (range of two experiments). PAE induced by 1 hour exposure at 10 x MIC. b Three erythromycin-sensitive and three erythromycin-resistant mef(a) strains were tested. 13