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Serum bactericidal activities of moxifloxacin and levofloxacin against aerobic and anaerobic intra-abdominal pathogens
ARTICLE IN PRESS
Anaerobe 14 (2008) 8–12 www.elsevier.com/locate/anaerobe
Antimicrobial susceptibility
Serum bactericidal activities of moxifloxacin and levofloxacin against aerobic and anaerobic intra-abdominal pathogens Gary E. Steina,, Sharon Schooleya, Kerin L. Tyrrellb, Diane M. Citronb, David P. Nicolauc, Ellie J.C. Goldsteinb,d a
Department of Medicine, Michigan State University, B320 Life Sciences, East Lansing, MI 48824, USA b R. M. Alden Research Laboratory, Santa Monica, CA 90404, USA c Hartford Hospital, Hartford, CT 06102, USA d David Geffen School of Medicine at UCLA, Los Angeles, CA 90073, USA Received 23 April 2007; received in revised form 24 August 2007; accepted 21 September 2007 Available online 7 October 2007
Abstract We studied the serum bactericidal activity (SBA) of moxifloxacin and levofloxacin against common pathogens associated with complicated intra-abdominal infections. Ten healthy volunteers received a single dose of moxifloxacin (400 mg) and levofloxacin (750 mg) and serum samples were collected at 2, 4, 8, 12, and 24 h after the dose of each drug. Bactericidal titers in serum over time were determined for aerobic gram-negative bacilli (Escherichia coli, Klebseilla pneumoniae, and Enterobacter cloacae) and anaerobic bacteria (Bacteroides fragilis, Bacteroides thetaiotaomicron, Prevotella bivia, and Finegoldia magna). Both fluoroquinolones provided rapid (2 h) attainment and prolonged (24 h) SBA (titers X1:8) against each of the aerobic bacilli studied. SBA was observed for at least 12 h against B. fragilis strains with MICsp2 mg/ml to moxifloxacin and p4 mg/ml to levofloxacin. Prolonged (12 h) SBA (titers X1:2) was also observed against isolates of B. thetaiotaomicron, P. bivia, and F. magna with moxifloxacin MICsp2 mg/ml. r 2007 Elsevier Ltd. All rights reserved. Keywords: Moxifloxacin; Levofloxacin; Anaerobes; Intra-abdominal; Bacteroides
1. Introduction Various therapeutic regimens are recommended for the treatment of community-acquired complicated intraabdominal infections (cIAI). The current recommendation for fluoroquinolone-based therapy, from the Infectious Diseases Society of America, is to use these agents in combination with metronidazole for improved coverage against anaerobic bacteria [1]. This recommendation is primarily based upon data from clinical trials of ciprofloxacin plus metronidazole [2]. In contrast to ciprofloxacin, moxifloxacin has good in vitro activity against anaerobes which enhances its ability to treat mixed aerobic/anaerobic infections [3]. Based upon newer clinical trials, moxifloxacin has received US Food & Drug Administration (FDA) approval for the treatment of cIAI Corresponding author. Tel.: +1 517 353 5126; fax: +1 517 353 1922.
E-mail address: [email protected] (G.E. Stein). 1075-9964/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2007.09.005
[4]. The list of FDA-approved anaerobic pathogens includes Bacteroides fragilis, Bacteroides thetaiotaomicron, Peptostreptococcus species, and Clostridium perfringens. Bacterial eradication rates for antibiotics utilized in the treatment of patients with cIAI are substantially determined from clinical responses, since repeat clinical specimens are often not available. Furthermore, a significant variety of anaerobes can be cultured from these infections that have an extensive range of minimum inhibitory concentrations (MICs) [5,6]. These factors make it difficult to designate a single susceptibility breakpoint concentration for anaerobic pathogens. In response to these concerns, pharmacodynamic investigations of different bacteria can be employed to help determine an appropriate susceptibility breakpoint concentration [7]. In this investigation, we tested the serum concentrations of moxifloxacin against pathogens commonly associated with cIAI. Moreover, selected anaerobes were chosen to further analyze the susceptibility breakpoint concentration
ARTICLE IN PRESS G.E. Stein et al. / Anaerobe 14 (2008) 8–12
for moxifloxacin. We also included levofloxacin as a comparative agent in this study, since this fluoroquinolone, with and without metronidazole, has become the most commonly prescribed antibiotic in the United States for the treatment of intra-abdominal infections [8]. 2. Patients and methods 2.1. Subjects Ten healthy volunteers (five males and five females) participated in this investigation. Each gave written informed consent that was approved by the University Committee on Research in Human Subjects at Michigan State University. None of these subjects had a history of chronic disease, were pregnant, or were receiving medications. Female volunteers had a negative screening pregnancy test and were directed to either abstain from sexual activity or protect themselves during intercourse during the study. The male volunteers had a mean age of 35 years (range 20–45 years) and a mean weight of 77 kg (range 55–91 kg). The female volunteers had a mean age of 37 years (range 21–46 years) and a mean weight of 72 kg (range 55–105 kg). 2.2. Drug administration Each subject received a single 400 mg oral dose of moxifloxacin (Shering-Plough, Kenilworth, NJ) and a single 750 mg oral dose of levofloxacin (Ortho-McNeil, Raritan, NJ). There was a 1-week washout period between doses. The study drugs were taken on an empty stomach following a 12-h fast. Food intake was allowed 2 h after receiving the dose of antibiotic. 2.3. Serum samples Venous blood samples were obtained immediately before (time zero, control) and at 2, 4, 8, 12, and 24 h after the dose of each drug. Following centrifugation, serum samples were aliquotted and stored at 70 1C until the time of analysis. The concentration in serum samples of levofloxacin and moxifloxacin were measured for each time period by a validated high-performance liquid chromatographic method [9]. Pharmacokinetic parameters for these antibiotics were determined by the use of non-compartmental analysis using Win Non Lin (version 3.3, Pharsight Corporation, Mountain View, CA) [10]. 2.4. Bacterial strains The inhibitory activities of levofloxacin and moxifloxacin were determined by broth microdilution for aerobes and agar dilution for anaerobes as recommended by Clinical and Laboratory Standards Institute (CLSI) [11,12]. Broth microdilution was performed using cation-
9
adjusted Mueller–Hinton broth. Isolates were suspended in Mueller–Hinton broth to a density equal to 0.5 McFarland standard and diluted to give a final inoculum of 104 CFU/well; trays were incubated in ambient air at 37 1C for 24 h. Susceptibility testing of anaerobic organisms was performed using Brucella agar supplemented with hemin, vitamin K, and 5% laked sheep blood. Isolates were suspended in Brucella broth to a density equal to a 0.5 McFarland standard. The suspensions were applied to the antibiotic plates at 105 CFU/spot; plates were incubated in an anaerobic chamber at 36 1C for 44–48 h. The MIC was defined as the lowest concentration of antibiotic that prevented a change in growth. Three commonly isolated aerobic gram-negative bacilli (Escherichia coli, Klebseilla pneumoniae, and Enterobacter cloacae) associated with intra-abdominal infections were studied. An S. aureus (ATCC 29213) was used as a control strain. The intra-abdominal anaerobes tested included recent clinical isolates of B. fragilis, B. thetaiotaomicron, P. bivia, and Finegoldia (Peptostreptococcus magnus) magna [6]. A range of B. fragilis isolates were selected to determine a MIC breakpoint for serum bactericidal activity (SBA). Selected strains of other anaerobic pathogens with specific MICs to moxifloxacin where chosen to verify the breakpoint concentration obtained for B. fragilis isolates. The control strains tested included B. fragilis strain ATCC 25285 and B. thetaiotaomicron strain ATCC 29741. 2.5. Bactericidal titers in serum Inhibitory and bactericidal titers in serum were determined according to CLSI standards [13]. Each determination was performed in duplicate. Wells with no visible growth and the first growth well were sub-cultured to supplemented Mueller–Hinton agar (aerobes) or Brucella agar (anaerobes) plates that were incubated for 2 (aerobes) or 3 (anaerobes) days prior to counting colonies. Both aerobic and anaerobic isolates were tested against serum collected at each time period for all subjects. The bactericidal titer (up to 1:32)-in-serum endpoint was determined as the highest dilution of serum yielding 99.9% killing. The median and geometric mean bactericidal titers at each time period were calculated and used to determine SBA. 3. Results Each of the 10 subjects received the study antibiotics according to the protocol, and no adverse experiences were observed or reported. The calculated pharmacokinetic parameters for moxifloxacin and levofloxacin in these subjects were found to be similar to other single-dose studies in healthy volunteers (Table 1) [14,15]. Of note, the AUC for levofloxacin (750 mg) was approximately twice that of moxifloxacin (400 mg).
ARTICLE IN PRESS G.E. Stein et al. / Anaerobe 14 (2008) 8–12
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Table 1 Pharmacokinetic parameters of single-dose moxifloxacin (400 mg) and levofloxacin (750 mg) in the study subjects Drug
Tmax (h)
Cmax (mg/ml)
T1/2 (h)
AUC024 (mg h/ml)
Levofloxacin Moxifloxacin
1.370.4 2.271.2
9.272.0 3.170.5
7.671.6 9.971.8
94713 45715
Tmax, time to maximal serum concentration; Cmax, maximal serum concentration; T1/2, elimination half-life; AUC0–24, area-under-the-serum-concentration curve.
Table 2 Median serum bactericidal titers over time of moxifloxacin and levofloxacin against aerobic bacteria Organisms and time point (h)
Moxifloxacin
Levofloxacin
MIC (mg/ml)
SBT (range)
MIC (mg/ml)
SBT (range)
0.06
1:32 (1:32) 1:32 (1:32) 1:16 (1:8–1:32)
0.06
1:32 (1:32) 1:32 (1:32) 1:16 (1:16–1:32)
K. pneumoniae 117 2 0.12 12 24
1:32 (1:32) 1:32 (1:16–1:32) 1:8 (1:8–1:32)
0.03
E. cloacae 015 2 12 24
1:32 (1:32) 1:32 (1:16–1:32) 1:16 (1:8–1:32)
0.03
E. coli 082 2 12 24
0.06
1:32 (1:8–1:32) 1:32 (1:16–1:32) 1:16 (1:8–1:16) 1:32 (1:32) 1:32 (1:16–1:32) 1:32 (1:8–1:32)
SBT, serum bactericidal titer.
The MICs of moxifloxacin and levofloxacin for the study isolates are presented in Tables 2 and 3. The potency of these two fluoroquinolones was similar for the three Enterobacteriaceae studied. Moxifloxacin was found to be two to eight times more potent against the anaerobic isolates selected for the study. This was especially true for strains of F. magna. Both fluoroquinolones provided rapid (2 h) attainment and prolonged (24 h) SBA against each of the aerobic bacilli studied. Serum bactericidal titers of 1:32 were maintained for a minimum of 12 h by each drug (Table 2). Against anaerobic isolates, both these fluoroquinolones were able to attain rapid and prolonged SBA. SBA (titers X1:2) was observed for at least 12 h against B. fragilis strains with MICsp2 mg/ml to moxifloxacin and p4 mg/ml to levofloxacin. No SBA was observed against the B fragilis isolate with a moxifloxacin MIC ¼ 4 mg/ml. These findings were further supported from testing selected isolates of B. thetaiotaomicron, P. bivia, and F. magna (Table 3). Prolonged (12 h) SBA (titers X1:2) was observed against each of these anaerobes with moxifloxicin MICsp2 mg/ml. Similar findings were observed with levofloxacin against isolates with MICsp4 mg/ml. No SBA was observed with
Table 3 Median serum bactericidal titers over time of moxifloxacin and levofloxacin against anaerobic bacteria Organisms and time point (h)
Moxifloxacin
Levofloxacin
MIC (mg/ml)
MIC (mg/ml)
SBT (range)
SBT (range)
B. fragilis 13778 2 0.5 12 24
1:4 (1:4–1:8) 1:4 (1:2–1:4) 1:2 (o1:2–1:2)
1.0
1:4 (1:2–1:4) 1:4 (1:2–1:4) o1:2 (o1:2)
B. fragilis 15077 2 1.0 12 24
1:4 (1:4–1:8) 1:4 (1:2–1:4) 1:2 (o1:2–1:2)
1.0
1:4 (1:2–1:4) 1:2 (o1:2–1:4) o1:2 (o1:2–1:2)
B. fragilis 16330 2 2.0 12 24
1:4 (1:2–1:4) 1:2 (o1:2–1:4) 1:2 (o1:2–1:4)
4.0
1:4 (o1:2–1:4) 1:2 (o1:2–1:4) 1:2 (o1:2–1:4)
B. fragilis 13832 2 4.0 12 24
o1:2 (o1:2–1:2) o1:2 (o1:2) o1:2 (o1:2)
16.0
B. thetaiotaomicron 13592 2 1.0 12 24
1:4 (1:2–1:4) 1:2 (1:2–1:4) 1:2 (o1:2–1:2)
4.0
1:4 (1:2–1:8) 1:2 (o1:2–1:2) o1:2 (o1:2–1:4)
B. thetaiotaomicron 13286 2 2.0 12 24
1:2 (1:2) 1:2 (1:2) o1:2 (o1:2–1:2)
4.0
1:2 (1:2–1:4) 1:2 (o1:2–1:2) o1:2 (o1:2–1:2)
F. magna 13504 2 1.0 12 24
1:2 (o1:2–1:2) 1:2 (1:2) o1:2 (o1:2)
8.0
o1:2 (o1:2) o1:2 (o1:2) o1:2 (o1:2)
F. magna 13272 2 2.0 12 24
1:2 (o1:2–1:2) 1:2 (1:2) o1:2 (o1:2)
16.0
o1:2 (o1:2) o1:2 (o1:2) o1:2 (o1:2)
P. bivia 13104 2 1.0 12 24
1:2 (1:2–1:4) 1:2 (o1:2–1:2) o1:2 (o1:2)
2.0
1:4 (1:2–1:4) 1:2 (o1:2–1:2) o1:2 (o1:2)
P. bivia 13543 2 2.0 12 24
1:2 (1:2) 1:2 (o1:2–1:2) o1:2 (o1:2)
4.0
1:4 (1:2–1:4) 1:2 (o1:2–1:2) o1:2 (o1:2)
o1:2 (o1:2) o1:2 (o1:2) o1:2 (o1:2)
SBT, serum bactericidal titer.
levofloxacin against strains of F. magna due to the high MICs (X4 mg/ml) of these isolates. 4. Discussion In this study, single doses of both moxifloxacin and levofloxacin provided bactericidal titers (X1:2) in serum for a prolonged period of time against common aerobic and anaerobic pathogens associated with cIAI. We believe that this observation has clinical relevance for several
ARTICLE IN PRESS G.E. Stein et al. / Anaerobe 14 (2008) 8–12
moxifloxacin against B. fragilis, C. perfringens, and grampositive anaerobic cocci in an in vitro pharmacokinetic model [19]. Moxifloxacin produced a 2-to-3 log decrease in counts at 24 h against these organisms, with AUC/MIC ratios X18. These ratios are lower than observed with aerobic bacteria and support our findings that prolonged bactericidal activity against anaerobic bacteria can be obtained with relatively low, extended serum concentrations compared to the MIC [22]. This is in contrast to our studies with Staphylococcus species, where no SBA was observed with moxifloxacin against isolates with MICs X1.0 mg/ml [7]. In our initial experiments with B. fragilis isolates, we observed prolonged bactericidal activity with moxifloxacin at MICsp2 mg/ml (levofloxacin, p4 mg/ml). No SBA was observed with either drug for the strain of B. fragilis with a MIC ¼ 4 mg/ml to moxifloxacin (Fig. 1). Additional studies with isolates of B. thetaiotaomicron, P. bivia, and F. magna confirmed this serum susceptibility breakpoint of concentration of 2 mg/ml for moxifloxacin. Prolonged SBA was observed for each of these bacteria at MICp2 mg/ml. These findings lend further support to the FDA susceptibility breakpoint concentration of 2 mg/ml for moxifloxacin against anaerobes, which was derived from phase 3 clinical trials of patients with cIAI (4). A susceptibility breakpoint concentration for levofloxacin against anaerobic bacteria has not been established by the FDA.
8
8
6
6
Median 1/SBT
Median 1/SBT
reasons. This ex vivo pharmacodynamic model integrates antimicrobial activity with pharmacokinetic parameters in human subjects. Furthermore, serum bactericidal titers allow for an evaluation of antimicrobial acitivity in the presence of factors such as antibodies, complement, and protein binding, as well as actual clinically relevant drug concentrations. Moreover, the use of serum from normal volunteers provides bactericidal values similar to those from patients when conducting serum bactericidal tests [16]. In modeling techniques, the duration of killing has been shown to be a good predictor of the antimicrobial effects of fluoroquinolones (time-kill and regrowth) and useful in determining the most appropriate dosing regimen [17,18]. However, the serum bactericidal test cannot predict either bacteriologic failure or clinical outcome. In contrast to studies with aerobic bacteria, where concentration-dependent killing is observed, concentration-independent killing was discovered against anaerobes with newer fluoroquinolones [19,20]. This finding is supported from time-kill assays, where maximal bactericidal activity against various anaerobic bacteria has been observed at concentrations twice the MIC with these agents [3]. In time-kill experiments of newer fluoroquinolones against a strain of B. thetaiotaomicron, Ross et al. found that AUC/MIC ratios X11 produced a 3-log kill by 14 h [21]. Noel et al. studied the antibacterial effects of
4 2
11
0
4 2 0
0
2
4
8
12
24
0
2
4
8
12
24
12
24
Time (h)
8
8
6
6
Median 1/SBT
Median 1/SBT
Time (h)
4 2 0
4 2 0
0
2
4
8 Time (h)
12
24
0
2
4
8 Time (h)
Fig. 1. Serum bactericidal activities of moxifloxacin (m) and levofloxacin (}) against B. fragilis isolates: (A) Moxi MIC ¼ 0.5 mg/ml, Levo MIC ¼ 1.0 mg/ml; (B) Moxi MIC ¼ 1.0 mg/ml, Levo MIC ¼ 1.0 mg/ml; (C) Moxi MIC ¼ 2.0 mg/ml, Levo MIC ¼ 4.0 mg/ml; and (D) Moxi MIC ¼ 4.0 mg/ml, Levo MIC ¼ 16 mg/ml. SBT, serum bactericidal titer.
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G.E. Stein et al. / Anaerobe 14 (2008) 8–12
It is important to note that moxifloxacin has FDA approval only for the treatment of common anaerobic pathogens associated with cIAI. Most clinical isolates of anaerobic gram-positive cocci and C. perfringens have MICsp1.0 mg/ml to moxifloxacin, whereas many B. fragilis group species often require higher concentrations for inhibition [6]. For example, the MIC90 (MIC for 90% of strains) for moxifloxacin against B. fragilis and B. thetiotaomicron isolates is 4–8 mg/ml [6]. Overall, Goldstein et al. found that 83% (763/923) of anaerobes isolated from patients with cIAI were susceptible to moxifloxacin at a MICp2 mg/ml [6]. This level of susceptibility at 2 mg/ml is similar to the overall bacteriologic eradication rate (82%) for moxifloxacin against anaerobes isolated from patients with cIAI [4]. In summary, this pharmacodynamic analysis along with microbiologic and clinical outcome studies support a susceptibility breakpoint concentration of 2 mg/ml for moxifloxacin when used in the treatment of anaerobic bacteria associated with cIAI.
[8]
[9]
[10]
[11]
[12]
[13]
[14]
Acknowledgment This study was supported in part by a research grant from Schering-Plough, Kenilworth, NJ.
[15]
References [16] [1] Solomkin JS, Mazuski JE, Baron EJ, et al. Guidelines for the selection of antiinfective agents for complicated intra-abdominal infections. Clin Infect Dis 2003;37:997–1005. [2] Matthaiou DK, Peppas G, Bliziotis IA, Falagas ME. Ciprofloxacin/ metronidazole versus b-lactam-based treatment of intra-abdominal infections: a meta-analysis of comparative trials. Int J Antimicrob Agents 2006;28:159–65. [3] Stein GE, Goldstein EJC. Fluoroquinolones and anaerobes. Clin Infect Dis 2006;42:1598–607. [4] Malangoni MA, Song J, Herrington J, et al. Randomized controlled trial of moxifloxacin compared with piperacillin-tazobactam and amoxicillin-clavulanate for the treatment of complicated intraabdominal infections. Ann Surg 2006;244:204–11. [5] Edminston CE, Krepel CJ, Seabrook GR, et al. In vitro activities of moxifloxacin against 900 aerobic and anaerobic surgical isolates from patients with intra-abdominal and diabetic foot infections. Antimicrob Agents Chemother 2004;48:1012–6. [6] Goldstein EJC, Citron DM, Warren YA, et al. In vitro activity of moxifloxacin against 923 anaerobes isolated from human intraabdominal infections. Antimicrob Agents Chemother 2006;50:148–55. [7] Stein GE, Schooley S, Kaatz GW. Serum bactericidal activity of the methoxyfluoroquinolones gatifloxacin and moxifloxacin against
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[22]
clinical isolates of Staphylcoccus species: are the susceptibility breakpoints too high? Clin Infect Dis 2003;37:1392–5. Linder JA, Huang ES, Steinman MA, et al. Fluoroquinolone prescribing in the United States: 1995–2002. Am J Med 2005;118: 259–68. Xuan D, Zhong M, Mattoes H, et al. Streptococcus pneumoniae response to repeated moxifloxacin or levofloxacin exposure in a rabbit tissue cage model. Antimicrob Agents Chemother 2001;45: 794–9. Florea NR, Tessier PR, Zhang C, et al. Pharmacodynamics of moxifloxacin and levofloxacin at simulated epithelial lining fluid drug concentrations against Streptococcus pneumoniae. Antimicrob Agents Chemother 2004;48:1215–21. Clinical and Laboratory Standards Institute. Methods for dilution of antimicrobial susceptibility test for bacteria that grow aerobically. Approved standard, 6th ed. CLSI document M7-A6. Wayne, PA: CLSI; 2003. Clinical and Laboratory Standards Institute. Methods for antimicrobial susceptibility testing of anaerobic bacteria. Approved standard, 6th ed. CLSI document M11-A6. Wayne, PA: CLSI; 2004. Clinical and Laboratory Standards Institute. Methodology for the serum bactericidal test. Approved guideline M21-A. Wayne, PA: CLSI; 1999. Chien S-C, Wong FA, Fowler CL, et al. Double-blind evaluation of the safety and pharmacokinetics of multiple oral once-daily 750milligram and 1-gram doses of levofloxacin in healthy volunteers. Antimicrob Agents Chemother 1998;42:885–8. Stass H, Dalhoff A, Kubitza D, Schuhly U. Pharmacokinetics, safety, and tolerability of ascending single doses of moxifloxacin, a new 8-methoxyquinolone administered to healthy subjects. Antimicrob Agents Chemother 1998;42:2060–5. Kays MB, White RL, Friedrich LV. Effect of serum from different patient populations on the serum bactericidal test. J Antimicrob Chemother 2001;48:417–20. Firsov AA, Zinner SH. Use of modeling techniques to aid in antibiotic selection. Curr Infect Dis Rep 2001;3:35–43. MacGowan AP, Rogers CA, Holt HA, et al. Pharmacodynamics of gemifloxacin against Streptococcus pneumoniae in an in vitro pharmacokinetic model of infection. Antimicrob Agents Chemother 2001;45:2916–21. Noel AR, Bowker KE, MacGowan AP. Pharmacodynamics of moxifloxacin against anaerobes studied in an in vitro pharmacokinetic model. Antimicrob Agents Chemother 2005;49:4234–9. Peterson ML, Hovde LB, Wright DH, et al. Pharmacodynamics of trovafloxacin and levofloxacin against Bacteroides fragilis in an in vitro pharmacodynamic model. Antimicrob Agents Chemother 2002; 46:203–10. Ross GH, Wright DH, Hovde DH, et al. Fluoroquinolone resistance in anaerobic bacteria following exposure to levofloxacin, trovafloxacin, and sparfloxacin in an in vitro pharmacodynamic model. Antimicrob Agents Chemother 2001;45:2136–40. Wright DH, Brown GH, Peterson ML, Rotschafer JC. Application of fluoroquinolone pharmacodynamics. J Antimicrob Chemother 2000; 46:669–83.
Anaerobe 14 (2008) 8–12 www.elsevier.com/locate/anaerobe
Antimicrobial susceptibility
Serum bactericidal activities of moxifloxacin and levofloxacin against aerobic and anaerobic intra-abdominal pathogens Gary E. Steina,, Sharon Schooleya, Kerin L. Tyrrellb, Diane M. Citronb, David P. Nicolauc, Ellie J.C. Goldsteinb,d a
Department of Medicine, Michigan State University, B320 Life Sciences, East Lansing, MI 48824, USA b R. M. Alden Research Laboratory, Santa Monica, CA 90404, USA c Hartford Hospital, Hartford, CT 06102, USA d David Geffen School of Medicine at UCLA, Los Angeles, CA 90073, USA Received 23 April 2007; received in revised form 24 August 2007; accepted 21 September 2007 Available online 7 October 2007
Abstract We studied the serum bactericidal activity (SBA) of moxifloxacin and levofloxacin against common pathogens associated with complicated intra-abdominal infections. Ten healthy volunteers received a single dose of moxifloxacin (400 mg) and levofloxacin (750 mg) and serum samples were collected at 2, 4, 8, 12, and 24 h after the dose of each drug. Bactericidal titers in serum over time were determined for aerobic gram-negative bacilli (Escherichia coli, Klebseilla pneumoniae, and Enterobacter cloacae) and anaerobic bacteria (Bacteroides fragilis, Bacteroides thetaiotaomicron, Prevotella bivia, and Finegoldia magna). Both fluoroquinolones provided rapid (2 h) attainment and prolonged (24 h) SBA (titers X1:8) against each of the aerobic bacilli studied. SBA was observed for at least 12 h against B. fragilis strains with MICsp2 mg/ml to moxifloxacin and p4 mg/ml to levofloxacin. Prolonged (12 h) SBA (titers X1:2) was also observed against isolates of B. thetaiotaomicron, P. bivia, and F. magna with moxifloxacin MICsp2 mg/ml. r 2007 Elsevier Ltd. All rights reserved. Keywords: Moxifloxacin; Levofloxacin; Anaerobes; Intra-abdominal; Bacteroides
1. Introduction Various therapeutic regimens are recommended for the treatment of community-acquired complicated intraabdominal infections (cIAI). The current recommendation for fluoroquinolone-based therapy, from the Infectious Diseases Society of America, is to use these agents in combination with metronidazole for improved coverage against anaerobic bacteria [1]. This recommendation is primarily based upon data from clinical trials of ciprofloxacin plus metronidazole [2]. In contrast to ciprofloxacin, moxifloxacin has good in vitro activity against anaerobes which enhances its ability to treat mixed aerobic/anaerobic infections [3]. Based upon newer clinical trials, moxifloxacin has received US Food & Drug Administration (FDA) approval for the treatment of cIAI Corresponding author. Tel.: +1 517 353 5126; fax: +1 517 353 1922.
E-mail address: [email protected] (G.E. Stein). 1075-9964/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2007.09.005
[4]. The list of FDA-approved anaerobic pathogens includes Bacteroides fragilis, Bacteroides thetaiotaomicron, Peptostreptococcus species, and Clostridium perfringens. Bacterial eradication rates for antibiotics utilized in the treatment of patients with cIAI are substantially determined from clinical responses, since repeat clinical specimens are often not available. Furthermore, a significant variety of anaerobes can be cultured from these infections that have an extensive range of minimum inhibitory concentrations (MICs) [5,6]. These factors make it difficult to designate a single susceptibility breakpoint concentration for anaerobic pathogens. In response to these concerns, pharmacodynamic investigations of different bacteria can be employed to help determine an appropriate susceptibility breakpoint concentration [7]. In this investigation, we tested the serum concentrations of moxifloxacin against pathogens commonly associated with cIAI. Moreover, selected anaerobes were chosen to further analyze the susceptibility breakpoint concentration
ARTICLE IN PRESS G.E. Stein et al. / Anaerobe 14 (2008) 8–12
for moxifloxacin. We also included levofloxacin as a comparative agent in this study, since this fluoroquinolone, with and without metronidazole, has become the most commonly prescribed antibiotic in the United States for the treatment of intra-abdominal infections [8]. 2. Patients and methods 2.1. Subjects Ten healthy volunteers (five males and five females) participated in this investigation. Each gave written informed consent that was approved by the University Committee on Research in Human Subjects at Michigan State University. None of these subjects had a history of chronic disease, were pregnant, or were receiving medications. Female volunteers had a negative screening pregnancy test and were directed to either abstain from sexual activity or protect themselves during intercourse during the study. The male volunteers had a mean age of 35 years (range 20–45 years) and a mean weight of 77 kg (range 55–91 kg). The female volunteers had a mean age of 37 years (range 21–46 years) and a mean weight of 72 kg (range 55–105 kg). 2.2. Drug administration Each subject received a single 400 mg oral dose of moxifloxacin (Shering-Plough, Kenilworth, NJ) and a single 750 mg oral dose of levofloxacin (Ortho-McNeil, Raritan, NJ). There was a 1-week washout period between doses. The study drugs were taken on an empty stomach following a 12-h fast. Food intake was allowed 2 h after receiving the dose of antibiotic. 2.3. Serum samples Venous blood samples were obtained immediately before (time zero, control) and at 2, 4, 8, 12, and 24 h after the dose of each drug. Following centrifugation, serum samples were aliquotted and stored at 70 1C until the time of analysis. The concentration in serum samples of levofloxacin and moxifloxacin were measured for each time period by a validated high-performance liquid chromatographic method [9]. Pharmacokinetic parameters for these antibiotics were determined by the use of non-compartmental analysis using Win Non Lin (version 3.3, Pharsight Corporation, Mountain View, CA) [10]. 2.4. Bacterial strains The inhibitory activities of levofloxacin and moxifloxacin were determined by broth microdilution for aerobes and agar dilution for anaerobes as recommended by Clinical and Laboratory Standards Institute (CLSI) [11,12]. Broth microdilution was performed using cation-
9
adjusted Mueller–Hinton broth. Isolates were suspended in Mueller–Hinton broth to a density equal to 0.5 McFarland standard and diluted to give a final inoculum of 104 CFU/well; trays were incubated in ambient air at 37 1C for 24 h. Susceptibility testing of anaerobic organisms was performed using Brucella agar supplemented with hemin, vitamin K, and 5% laked sheep blood. Isolates were suspended in Brucella broth to a density equal to a 0.5 McFarland standard. The suspensions were applied to the antibiotic plates at 105 CFU/spot; plates were incubated in an anaerobic chamber at 36 1C for 44–48 h. The MIC was defined as the lowest concentration of antibiotic that prevented a change in growth. Three commonly isolated aerobic gram-negative bacilli (Escherichia coli, Klebseilla pneumoniae, and Enterobacter cloacae) associated with intra-abdominal infections were studied. An S. aureus (ATCC 29213) was used as a control strain. The intra-abdominal anaerobes tested included recent clinical isolates of B. fragilis, B. thetaiotaomicron, P. bivia, and Finegoldia (Peptostreptococcus magnus) magna [6]. A range of B. fragilis isolates were selected to determine a MIC breakpoint for serum bactericidal activity (SBA). Selected strains of other anaerobic pathogens with specific MICs to moxifloxacin where chosen to verify the breakpoint concentration obtained for B. fragilis isolates. The control strains tested included B. fragilis strain ATCC 25285 and B. thetaiotaomicron strain ATCC 29741. 2.5. Bactericidal titers in serum Inhibitory and bactericidal titers in serum were determined according to CLSI standards [13]. Each determination was performed in duplicate. Wells with no visible growth and the first growth well were sub-cultured to supplemented Mueller–Hinton agar (aerobes) or Brucella agar (anaerobes) plates that were incubated for 2 (aerobes) or 3 (anaerobes) days prior to counting colonies. Both aerobic and anaerobic isolates were tested against serum collected at each time period for all subjects. The bactericidal titer (up to 1:32)-in-serum endpoint was determined as the highest dilution of serum yielding 99.9% killing. The median and geometric mean bactericidal titers at each time period were calculated and used to determine SBA. 3. Results Each of the 10 subjects received the study antibiotics according to the protocol, and no adverse experiences were observed or reported. The calculated pharmacokinetic parameters for moxifloxacin and levofloxacin in these subjects were found to be similar to other single-dose studies in healthy volunteers (Table 1) [14,15]. Of note, the AUC for levofloxacin (750 mg) was approximately twice that of moxifloxacin (400 mg).
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Table 1 Pharmacokinetic parameters of single-dose moxifloxacin (400 mg) and levofloxacin (750 mg) in the study subjects Drug
Tmax (h)
Cmax (mg/ml)
T1/2 (h)
AUC024 (mg h/ml)
Levofloxacin Moxifloxacin
1.370.4 2.271.2
9.272.0 3.170.5
7.671.6 9.971.8
94713 45715
Tmax, time to maximal serum concentration; Cmax, maximal serum concentration; T1/2, elimination half-life; AUC0–24, area-under-the-serum-concentration curve.
Table 2 Median serum bactericidal titers over time of moxifloxacin and levofloxacin against aerobic bacteria Organisms and time point (h)
Moxifloxacin
Levofloxacin
MIC (mg/ml)
SBT (range)
MIC (mg/ml)
SBT (range)
0.06
1:32 (1:32) 1:32 (1:32) 1:16 (1:8–1:32)
0.06
1:32 (1:32) 1:32 (1:32) 1:16 (1:16–1:32)
K. pneumoniae 117 2 0.12 12 24
1:32 (1:32) 1:32 (1:16–1:32) 1:8 (1:8–1:32)
0.03
E. cloacae 015 2 12 24
1:32 (1:32) 1:32 (1:16–1:32) 1:16 (1:8–1:32)
0.03
E. coli 082 2 12 24
0.06
1:32 (1:8–1:32) 1:32 (1:16–1:32) 1:16 (1:8–1:16) 1:32 (1:32) 1:32 (1:16–1:32) 1:32 (1:8–1:32)
SBT, serum bactericidal titer.
The MICs of moxifloxacin and levofloxacin for the study isolates are presented in Tables 2 and 3. The potency of these two fluoroquinolones was similar for the three Enterobacteriaceae studied. Moxifloxacin was found to be two to eight times more potent against the anaerobic isolates selected for the study. This was especially true for strains of F. magna. Both fluoroquinolones provided rapid (2 h) attainment and prolonged (24 h) SBA against each of the aerobic bacilli studied. Serum bactericidal titers of 1:32 were maintained for a minimum of 12 h by each drug (Table 2). Against anaerobic isolates, both these fluoroquinolones were able to attain rapid and prolonged SBA. SBA (titers X1:2) was observed for at least 12 h against B. fragilis strains with MICsp2 mg/ml to moxifloxacin and p4 mg/ml to levofloxacin. No SBA was observed against the B fragilis isolate with a moxifloxacin MIC ¼ 4 mg/ml. These findings were further supported from testing selected isolates of B. thetaiotaomicron, P. bivia, and F. magna (Table 3). Prolonged (12 h) SBA (titers X1:2) was observed against each of these anaerobes with moxifloxicin MICsp2 mg/ml. Similar findings were observed with levofloxacin against isolates with MICsp4 mg/ml. No SBA was observed with
Table 3 Median serum bactericidal titers over time of moxifloxacin and levofloxacin against anaerobic bacteria Organisms and time point (h)
Moxifloxacin
Levofloxacin
MIC (mg/ml)
MIC (mg/ml)
SBT (range)
SBT (range)
B. fragilis 13778 2 0.5 12 24
1:4 (1:4–1:8) 1:4 (1:2–1:4) 1:2 (o1:2–1:2)
1.0
1:4 (1:2–1:4) 1:4 (1:2–1:4) o1:2 (o1:2)
B. fragilis 15077 2 1.0 12 24
1:4 (1:4–1:8) 1:4 (1:2–1:4) 1:2 (o1:2–1:2)
1.0
1:4 (1:2–1:4) 1:2 (o1:2–1:4) o1:2 (o1:2–1:2)
B. fragilis 16330 2 2.0 12 24
1:4 (1:2–1:4) 1:2 (o1:2–1:4) 1:2 (o1:2–1:4)
4.0
1:4 (o1:2–1:4) 1:2 (o1:2–1:4) 1:2 (o1:2–1:4)
B. fragilis 13832 2 4.0 12 24
o1:2 (o1:2–1:2) o1:2 (o1:2) o1:2 (o1:2)
16.0
B. thetaiotaomicron 13592 2 1.0 12 24
1:4 (1:2–1:4) 1:2 (1:2–1:4) 1:2 (o1:2–1:2)
4.0
1:4 (1:2–1:8) 1:2 (o1:2–1:2) o1:2 (o1:2–1:4)
B. thetaiotaomicron 13286 2 2.0 12 24
1:2 (1:2) 1:2 (1:2) o1:2 (o1:2–1:2)
4.0
1:2 (1:2–1:4) 1:2 (o1:2–1:2) o1:2 (o1:2–1:2)
F. magna 13504 2 1.0 12 24
1:2 (o1:2–1:2) 1:2 (1:2) o1:2 (o1:2)
8.0
o1:2 (o1:2) o1:2 (o1:2) o1:2 (o1:2)
F. magna 13272 2 2.0 12 24
1:2 (o1:2–1:2) 1:2 (1:2) o1:2 (o1:2)
16.0
o1:2 (o1:2) o1:2 (o1:2) o1:2 (o1:2)
P. bivia 13104 2 1.0 12 24
1:2 (1:2–1:4) 1:2 (o1:2–1:2) o1:2 (o1:2)
2.0
1:4 (1:2–1:4) 1:2 (o1:2–1:2) o1:2 (o1:2)
P. bivia 13543 2 2.0 12 24
1:2 (1:2) 1:2 (o1:2–1:2) o1:2 (o1:2)
4.0
1:4 (1:2–1:4) 1:2 (o1:2–1:2) o1:2 (o1:2)
o1:2 (o1:2) o1:2 (o1:2) o1:2 (o1:2)
SBT, serum bactericidal titer.
levofloxacin against strains of F. magna due to the high MICs (X4 mg/ml) of these isolates. 4. Discussion In this study, single doses of both moxifloxacin and levofloxacin provided bactericidal titers (X1:2) in serum for a prolonged period of time against common aerobic and anaerobic pathogens associated with cIAI. We believe that this observation has clinical relevance for several
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moxifloxacin against B. fragilis, C. perfringens, and grampositive anaerobic cocci in an in vitro pharmacokinetic model [19]. Moxifloxacin produced a 2-to-3 log decrease in counts at 24 h against these organisms, with AUC/MIC ratios X18. These ratios are lower than observed with aerobic bacteria and support our findings that prolonged bactericidal activity against anaerobic bacteria can be obtained with relatively low, extended serum concentrations compared to the MIC [22]. This is in contrast to our studies with Staphylococcus species, where no SBA was observed with moxifloxacin against isolates with MICs X1.0 mg/ml [7]. In our initial experiments with B. fragilis isolates, we observed prolonged bactericidal activity with moxifloxacin at MICsp2 mg/ml (levofloxacin, p4 mg/ml). No SBA was observed with either drug for the strain of B. fragilis with a MIC ¼ 4 mg/ml to moxifloxacin (Fig. 1). Additional studies with isolates of B. thetaiotaomicron, P. bivia, and F. magna confirmed this serum susceptibility breakpoint of concentration of 2 mg/ml for moxifloxacin. Prolonged SBA was observed for each of these bacteria at MICp2 mg/ml. These findings lend further support to the FDA susceptibility breakpoint concentration of 2 mg/ml for moxifloxacin against anaerobes, which was derived from phase 3 clinical trials of patients with cIAI (4). A susceptibility breakpoint concentration for levofloxacin against anaerobic bacteria has not been established by the FDA.
8
8
6
6
Median 1/SBT
Median 1/SBT
reasons. This ex vivo pharmacodynamic model integrates antimicrobial activity with pharmacokinetic parameters in human subjects. Furthermore, serum bactericidal titers allow for an evaluation of antimicrobial acitivity in the presence of factors such as antibodies, complement, and protein binding, as well as actual clinically relevant drug concentrations. Moreover, the use of serum from normal volunteers provides bactericidal values similar to those from patients when conducting serum bactericidal tests [16]. In modeling techniques, the duration of killing has been shown to be a good predictor of the antimicrobial effects of fluoroquinolones (time-kill and regrowth) and useful in determining the most appropriate dosing regimen [17,18]. However, the serum bactericidal test cannot predict either bacteriologic failure or clinical outcome. In contrast to studies with aerobic bacteria, where concentration-dependent killing is observed, concentration-independent killing was discovered against anaerobes with newer fluoroquinolones [19,20]. This finding is supported from time-kill assays, where maximal bactericidal activity against various anaerobic bacteria has been observed at concentrations twice the MIC with these agents [3]. In time-kill experiments of newer fluoroquinolones against a strain of B. thetaiotaomicron, Ross et al. found that AUC/MIC ratios X11 produced a 3-log kill by 14 h [21]. Noel et al. studied the antibacterial effects of
4 2
11
0
4 2 0
0
2
4
8
12
24
0
2
4
8
12
24
12
24
Time (h)
8
8
6
6
Median 1/SBT
Median 1/SBT
Time (h)
4 2 0
4 2 0
0
2
4
8 Time (h)
12
24
0
2
4
8 Time (h)
Fig. 1. Serum bactericidal activities of moxifloxacin (m) and levofloxacin (}) against B. fragilis isolates: (A) Moxi MIC ¼ 0.5 mg/ml, Levo MIC ¼ 1.0 mg/ml; (B) Moxi MIC ¼ 1.0 mg/ml, Levo MIC ¼ 1.0 mg/ml; (C) Moxi MIC ¼ 2.0 mg/ml, Levo MIC ¼ 4.0 mg/ml; and (D) Moxi MIC ¼ 4.0 mg/ml, Levo MIC ¼ 16 mg/ml. SBT, serum bactericidal titer.
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It is important to note that moxifloxacin has FDA approval only for the treatment of common anaerobic pathogens associated with cIAI. Most clinical isolates of anaerobic gram-positive cocci and C. perfringens have MICsp1.0 mg/ml to moxifloxacin, whereas many B. fragilis group species often require higher concentrations for inhibition [6]. For example, the MIC90 (MIC for 90% of strains) for moxifloxacin against B. fragilis and B. thetiotaomicron isolates is 4–8 mg/ml [6]. Overall, Goldstein et al. found that 83% (763/923) of anaerobes isolated from patients with cIAI were susceptible to moxifloxacin at a MICp2 mg/ml [6]. This level of susceptibility at 2 mg/ml is similar to the overall bacteriologic eradication rate (82%) for moxifloxacin against anaerobes isolated from patients with cIAI [4]. In summary, this pharmacodynamic analysis along with microbiologic and clinical outcome studies support a susceptibility breakpoint concentration of 2 mg/ml for moxifloxacin when used in the treatment of anaerobic bacteria associated with cIAI.
[8]
[9]
[10]
[11]
[12]
[13]
[14]
Acknowledgment This study was supported in part by a research grant from Schering-Plough, Kenilworth, NJ.
[15]
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