Journal of Antimicrobial Chemotherapy Advance Access published May 14, 2014
J Antimicrob Chemother doi:10.1093/jac/dku152
Humanized tissue pharmacodynamics of cefazolin against commonly isolated pathogens in skin and skin structure infections Jami G. Jain, Seth T. Housman and David P. Nicolau* Center for Anti-Infective Research and Development, Hartford Hospital, Hartford, CT, USA *Corresponding author. Tel: +1-860-545-3941; Fax: +1-860-545-3992; E-mail: [email protected]
Received 18 December 2013; returned 2 March 2014; revised 17 March 2014; accepted 8 April 2014
Methods: Nine clinical isolates [five MSSA (cefazolin MIC range 0.5 –2.0 mg/L), two Escherichia coli (cefazolin MICs 1.0 and 2.0 mg/L) and two Klebsiella pneumoniae (cefazolin MICs of 1.0 and 2.0 mg/L)] were evaluated with a starting inoculum (0 h) of 106 cfu/mL. Time – kill curves were built and the area under the bacterial killing and regrowth curve (AUBC) was calculated. Results: The starting inoculum had a mean+SD of 6.3+0.28 log10 cfu/mL. Cefazolin human simulated targets for peak, trough and half-life were 13.0 mg/L, 2.6 mg/L and 2.6 h, respectively. Control isolates grew to 8.5+ 0.2 log10 cfu/mL. Against MSSA, cefazolin achieved a reduction from 0 h of 21.18+0.67 and 23.58+1.24 log10 cfu/mL, at 4 and 24 h, respectively. Cefazolin achieved a reduction in bacterial density of 23.45+0.35 and 22.68+0.99 log10 cfu/mL at 4 and 24 h, respectively, when tested against Enterobacteriaceae. No significant difference was observed when comparing AUBC based on MIC values. The rate of initial bacterial reduction of Enterobacteriaceae was rapid, with a decrease of .3 log10 cfu/mL by 4 h, while MSSA exhibited a gradual reduction in bacterial density over one dosing interval. Conclusions: The observed antibacterial effects of cefazolin support its continued utility against susceptible S. aureus, E. coli and K. pneumoniae in skin and skin structure infections. Keywords: Staphylococcus aureus, in vitro, Enterobacteriaceae, antibiotic exposures, lower limb infections
Introduction Comorbidities such as diabetes mellitus and peripheral vascular disease commonly result in patients having reduced blood flow to the lower extremities and increased risk of infection. 1 This poses the possibility that patients with chronic lower limb infections may not receive adequate antimicrobial exposures at the site of infection. Consequently, antimicrobial concentrations in the blood may not always correlate with concentrations at the site of infection. These patients often have reduced immune function, which further reduces their ability to fight off infection. Therefore, it is paramount that clinicians take into account any differences in pharmacokinetics when treating lower limb infections. While Staphylococcus aureus is the most prevalent pathogen implicated in acute bacterial skin and skin structure infections (ABSSSIs), polymicrobial infections, including Escherichia coli and Klebsiella pneumoniae, are also a well-recognized clinical
burden.2 – 6 As a result of its antibacterial spectrum, cefazolin is used for both empirical and directed therapy of ABSSSIs.7 In the context of altered perfusion and tissue concentrations, a reassessment of cefazolin’s pharmacodynamic profile is necessary. A recent study by Bhalodi et al.8 measured cefazolin tissue concentrations in patients with chronic lower limb infections. Using an in vivo microdialysis technique previously described,9,10 they concluded that tissue exposures of cefazolin were adequate to reach clinical pharmacodynamic targets [percentage of free time above the MIC (%fT.MIC)]. While these data provide the pharmacokinetic profile of cefazolin in the infected patient, additional pharmacodynamic studies are required to assess the regimen’s potential efficacy against target pathogens. We set out to assess the efficacy of human simulated tissue concentrations following 1 g of cefazolin every 8 h against methicillin-susceptible Staphylococcus aureus (MSSA) and Enterobacteriaceae in a onecompartment in vitro pharmacodynamic (IVPD) model.
# The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: [email protected]
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Objectives: The objective of this study was to assess the efficacy of humanized cefazolin tissue concentrations against methicillin-susceptible Staphylococcus aureus (MSSA) and Enterobacteriaceae in an in vitro pharmacodynamic model.
Jain et al.
Materials and methods Bacterial strains Nine clinical isolates (five MSSA isolates, two E. coli isolates and two K. pneumoniae isolates) were selected for use in this study. The MIC for all organisms was determined a minimum of two times by the broth microdilution method in accordance with the CLSI.11 In addition to cefazolin, MICs of ceftaroline, ceftriaxone, daptomycin, oxacillin, tigecycline and vancomycin for MSSA were also determined. MICs of cefazolin, cefepime, ceftaroline, ceftriaxone, ciprofloxacin, piperacillin/tazobactam and tigecycline for Enterobacteriaceae were determined.
Antibiotics
Simulated drug exposures Tissue concentrations for cefazolin were based on patient pharmacokinetic data generated by Bhalodi et al.8 In brief, an in vivo microdialysis technique was used to measure interstitial concentrations of cefazolin in seven patients, each with a chronic lower limb infection. This technique provided continuous measurements over a dosing interval, describing the antimicrobial exposures in these patients with compromised lower limb vasculature. These human data were simulated in an IVPD model with a peak tissue concentration of 13 mg/L and a half-life of 2.6 h, resulting in a tissue trough concentration of 2.6 mg/L.
In vitro pharmacodynamic model A one-compartment in vitro model was used for all experiments. Each experiment consisted of three independent models (two experimental treatment models and one growth control model), which ran concurrently for each isolate. To achieve uniform temperatures of 378C, the models were stationed in a temperature-modulated circulating water bath. All experiments were performed with a starting (0 h) inoculum of 106 cfu/mL. Inoculum preparation included making a bacterial suspension of 108 cfu/mL from subcultures of the test isolate (incubated at 378C overnight) with 0.9% normal saline for injection. Three millilitres of inoculum was injected into each 300 mL model to achieve the starting inoculum of 106 cfu/mL. Cation-adjusted Mueller – Hinton broth (CAMHB) (Becton, Dickinson and Company, Sparks, MD, USA) was used as the bacterial growth medium for all experiments. Once inoculated, the bacteria were allowed to enter the log phase of growth over 30 min. Cefazolin was then administered as a bolus into the model (0 h) to simulate a steady-state trough of free drug concentration achieved in human lower limb tissue after intravenous doses of 1 g of cefazolin every 8 h. Following this initial bolus, cefazolin was infused at the beginning of each dosing interval, using a peristaltic pump (Masterflex L/S Model 7524-10; Cole-Palmer, USA), into the model at a pre-determined rate. Each experiment was conducted over 24 h and performed in duplicate to assure reproducibility. Bacterial density over time was determined by sampling from each model at pre-determined timepoints (0, 1, 2, 4, 6, 8, 10, 12, 16, 18, 20 and 24 h) and serial dilution in normal saline. Aliquots of diluted sample were plated for quantitative culture utilizing trypticase soy agar plates (100 mm diameter) with 5% sheep blood. Colony counts were read after 16 – 20 h of incubation at 378C. The lower limit of detection for bacterial density was 1.7 log10 cfu/mL. To evaluate the antibiotic activity of the regimen of 1 g of cefazolin every 8 h, bacterial density was measured by the change in log10 cfu/mL from 0 h at 4 and 24 h. Bactericidal activity was
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Antibiotic concentration and exposure determinations Samples of CAMHB broth taken from each of the treatment models were assayed for cefazolin at 0, 2, 4, 6, 8, 10, 16, 18 and 24 h. All samples were immediately stored at 2808C until analysis. Cefazolin was analysed by a validated HPLC method at the Center for Anti-Infective Research and Development, as described previously.12 For the assay, the lower and upper detection limits were 0.5 and 30 mg/L, respectively. The interday coefficient of variation (CV) for the cefazolin check samples of 0.8 and 20 mg/mL was 5.9% and 5.1%, respectively (n ¼ 22), while the intraday CV was 2.1% and 3.1% (n¼10), respectively. The cefazolin assay was linear over a concentration range of 0.5 – 30 mg/L. Based on achieved concentrations in each model, the peak and trough concentrations were reported as those observed by analysis and the half-life was calculated as 0.693/k, where k is the estimated elimination rate constant calculated as the natural log (ln) of the measured peak/trough divided by its change in time. The AUBC over each 24 h period (log10 cfu.h/mL) was calculated using a model-specific growth control for comparison.
Statistical analysis Differences in AUBC at 24 h within MSSA and Enterobacteriaceae, based on MIC values, were assessed by one-way ANOVA or by the t-test where applicable. In addition, mean bacterial reductions at 4 and 24 h were compared between MSSA and Enterobacteriaceae using the t-test. An a priori P value ,0.05 was considered statistically significant.
Results MIC determination The MICs of cefazolin for MSSA F 2-13, MSSA F 10-29, MSSA F 44-14, MSSA F 19-13 and MSSA F 18-8 were 0.5, 0.5, 1, 1 and 2 mg/L, respectively. The MICs of cefazolin for the Enterobacteriaceae isolates E. coli AZ 18-25, E. coli AZ 6-17, K. pneumoniae JJ 7-10 and K. pneumoniae JJ 7-33 were 1, 2, 1 and 2 mg/L, respectively. Phenotypic profiles of all isolates used are shown in Table 1.
Pharmacokinetic analysis Pharmacokinetic parameters (peak, trough and half-life) observed in the models were 13.68+2.66 mg/L, 3.34+1.03 mg/L and 3.02+0.08 h, respectively. A comparison between observed patient pharmacokinetics and observed model pharmacokinetics can be seen in Figure 1. These resulted in a mean cefazolin %fT.MIC of 100% for all MSSA and Enterobacteriaceae isolates, as would be expected in the clinical setting when 1 g of cefazolin every 8 h is administered.8
Antibacterial activity MSSA isolates The average bacterial density of the starting inoculum was 6.42+0.25 log 10 cfu/mL. Control isolates grew to 8.44+ 0.19 log10 cfu/mL over 24 h. Figure 2 summarizes the time – kill curves for cefazolin against all MSSA isolates. The mean changes in bacterial density at 4 and 24 h against the five MSSA isolates
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Cefazolin (Apotex Corp., Weston, FL, USA; lot 101D026; expiry date, March 2015) was obtained from the Department of Pharmacy at Hartford Hospital.
defined as a reduction in bacterial density of at least 3 log10 cfu/mL. Time– kill curves were constructed and the area under the bacterial killing and regrowth curve (AUBC) was calculated to assess antibiotic efficacy.
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Humanized tissue pharmacodynamics of cefazolin
Table 1. Phenotypic profiles of MSSA and Enterobacteriaceae isolates Isolate
CFZ
OXA
TZP
FEP
CRO
CPT
VAN
CIP
DAP
TGC
MSSA F 2-13 MSSA F 10-29 MSSA F 44-14 MSSA F 19-13 MSSA F 18-8 E. coli AZ 18-25 K. pneumoniae JJ 7-10 E. coli AZ 6-17 K. pneumoniae JJ 7-33
0.5 0.5 1 1 2 1 1 2 2
0.25 0.5 0.5 0.5 2 ND ND ND ND
ND ND ND ND ND 2 2 2 1
ND ND ND ND ND ,0.06 ,0.06 ,0.06 ,0.06
2 4 4 8 8 ,0.25 ,0.25 ,0.25 ,0.25
0.25 0.25 0.25 0.25 0.5 ,0.06 ,0.06 ,0.06 ,0.06
1 1 1 1 1 ND ND ND ND
ND ND ND ND ND ,0.015 0.03 ,0.015 0.03
0.25 0.5 0.25 0.5 0.5 ND ND ND ND
0.064 0.125 0.125 0.25 0.125 0.125 0.5a 0.125 0.5a
Mean bacterial density (log10 cfu/mL)
Cefazolin concentration (mg/L)
10
10
2 mg/L 1 mg/L
1
0.5 mg/L 0
2
4
6
8
10
12
14
16
18
20
22
24
8
6
4
2
0 0
2
Time (h) Human tissue mean concentrations Model simulated mean concentrations Figure 1. Human versus simulated cefazolin pharmacokinetic profiles and corresponding MICs. Data are plotted as the means of the multiple models and the means of observed human pharmacokinetic profiles.
are provided in Table 2. MSSA isolates exhibited a significantly slower progression of bacterial reduction by 4 h (P, 0.0001) when compared with the rapid reductions observed with Enterobacteriaceae. No significant differences in AUBC reductions were seen, regardless of MIC (P . 0.05). The AUBCs over 24 h for cefazolin are also summarized in Table 2.
Enterobacteriaceae isolates The average bacterial density of the starting inoculum was 6.08+ 0.21 log10 cfu/mL. Control isolates grew to 8.74+0.07 log10 cfu/ mL over 24 h in the models. Figure 3 summarizes the time – kill curves for cefazolin against all Enterobacteriaceae isolates. The mean changes in bacterial density at 4 and 24 h against the four Enterobacteriaceae isolates are provided in Table 2. When comparing 24 h bacterial reductions observed with MSSA and
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10 12 14 16 18 Time (h) Mean control MSSA F2-13/MIC = 0.5 MSSA F10-29/MIC = 0.5 MSSA F44-14/MIC = 1 MSSA F19-13/MIC = 1 MSSA F18-8/MIC = 2 Limit of detection 1.7 log10 cfu/mL 6
8
20
22
24
Figure 2. Mean bacterial densities over 24 h for MSSA isolates. Data are plotted as the means of the multiple models for the treatments and the mean of all corresponding growth control isolates. The lower limit of detection (broken line) was 1.7 log10 cfu/mL.
Enterobacteriaceae, no statistically significant difference was found (P .0.05). As with MSSA, Enterobacteriaceae showed no significant difference in AUBC, regardless of MIC (P .0.05). The AUBCs over 24 h for cefazolin are also summarized in Table 2.
Discussion With today’s focus on emerging pathogens with various resistances to many mainstay regimens, it is imperative that clinicians optimize antimicrobials pharmacodynamically whenever
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CFZ, cefazolin; OXA, oxacillin; TZP, piperacillin/tazobactam; FEP, cefepime; CRO, ceftriaxone; CPT, ceftaroline; VAN, vancomycin; CIP, ciprofloxacin; DAP, daptomycin; TGC, tigecycline; ND, not determined. This table shows the reported modal MIC (mg/L) unless otherwise noted. a Median.
Jain et al.
Table 2. Mean change in bacterial density at 4 and 24 h and AUBC from 0 to 24 h for cefazolin against all isolates Change in bacterial density (log10 cfu/mL) from 0 ha Isolate
AUBC (log10 cfu.h/mL)a
4h
24 h
1 g of cefazolin every 8 h
control
MSSA F 2-13 MSSA F 10-29 MSSA F 44-14 MSSA F 19-13 MSSA F 18-8 Mean+SDb
0.5 0.5 1 1 2
22.07 20.71 20.95 20.01 21.51 21.18+0.67
24.24 24.28 23.03 24.66 22.64 23.58+1.24
77.58 88.70 92.81 82.42 88.30 86.46+9.15
196.40 194.52 195.77 198.07 192.76 195.5+2.00
E. coli AZ 18-25 K. pneumoniae JJ 7-10 E. coli AZ 6-17 K. pneumoniae JJ 7-33 Mean+SDb
1 1 2 2
23.30 23.17 23.97 23.36 23.45+0.35
22.25 22.84 23.85 21.78 22.68+0.99
92.66 59.28 61.80 86.85 75.15+17.55
204.12 201.05 197.56 202.42 201.29+2.78
a
Values for each isolate are the means of two to five independent time– kill experiments. Data are presented as mean+SD of all time–kill curves (two to five per isolate). Negative numbers indicate reductions in cfu from 0 h.
b
Mean bacterial density (log10 cfu/mL)
10
8
6
4
2
0 0
2
4
6
8
10 12 14 Time (h)
16
18
20
22
24
Mean control E. coli AZ 18-25/MIC = 1 E. coli AZ 6-17/MIC = 2 K. pneumoniae JJ 7-10/MIC = 1 K. pneumoniae JJ 7-33/MIC = 2 Limit of detection 1.7 log10 cfu/mL Figure 3. Mean bacterial densities over 24 h for Enterobacteriaceae isolates. Data are plotted as the means of the multiple models for the treatments and the mean of all corresponding growth control isolates. The lower limit of detection (broken line) was 1.7 log10 cfu/mL.
possible. Cefazolin is commonly used to treat ABSSSIs, caused by MSSA. Antibiotics can vary in their ability to achieve effective concentrations in foot lesions due to specific drug pharmacokinetics and patients’ vascular permeabilities. As new tissue concentration data are generated, a correlation between concentrations at the site of infection and efficacy against clinical isolates should be assessed.8 Here we set out to assess the antibacterial effects of human simulated tissue concentrations following 1 g of cefazolin every 8 h against both MSSA and Enterobacteriaceae.
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In this study, five MSSA clinical isolates with cefazolin MICs in the range of 0.5 –2 mg/L were used. It is important to note that these isolates are on the upper end of the MIC distribution, with MIC50 and MIC 90 being 0.5 and 1 mg/L, respectively. 13 The %fT.MIC of 100 was achieved for all isolates, exceeding the required pharmacodynamic target of 30% – 40% of the dosing interval.14 As expected, on average cefazolin exhibited bactericidal activity against MSSA. The changes in bacterial density at 4 and 24 h were observed in order to depict the rate of kill for both MSSA and Enterobacteriaceae. A slow progression of killing was observed across all MSSA models regardless of MIC, indicated by less than half of its overall bacterial density reductions occurring at 4 h. However, after 24 h of simulating a regimen consisting of 1 g of cefazolin every 8 h, the mean bacterial density was reduced by .3 log10 cfu/mL. As the only MSSA isolate with an MIC of 2 mg/L, F 18-8 exhibited a slight rebound in growth, resulting in a 24 h bacterial reduction of 1 log10 cfu/mL less than the mean stated above. These simulated cefazolin tissue concentrations seem to provide more than adequate antimicrobial exposures for all the tested MSSA isolates. It would be expected that patients being treated with monotherapy of 1 g of cefazolin every 8 h, under these pharmacokinetic and vasculature conditions, would reach pharmacodynamic targets against MSSA. Additional isolates were tested to represent commonly involved Gram-negative pathogens in ABSSSIs. Four clinical isolates of Enterobacteriaceae (two E. coli isolates and two K. pneumoniae isolates) with cefazolin MICs of 1 mg/L and at the susceptible breakpoint of 2 mg/L were tested. As with MSSA, all Enterobacteriaceae isolates had a %fT.MIC that exceeded current pharmacodynamic targets. It is important to note that, when treating Gram-negative pathogens with cefazolin, the %fT.MIC is required to be higher (60%–70%) than when treating Gram-positive pathogens (30% – 40%).14 Bacterial reductions were observed with all isolates. No differences in bacterial reductions were seen between isolates when comparing MICs. All Gram-negative isolates exhibited a rapid rate of killing that was characterized by reductions of .3 log10 cfu/mL in bacterial
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MIC (mg/L)
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Humanized tissue pharmacodynamics of cefazolin
References 1 Suaya JA, Eisenberg DF, Fang C et al. Skin and soft tissue infections and associated complications among commercially insured patients aged 0–64 years with and without diabetes in the U.S. PLoS One 2013; 8: e60057. 2 Ray GT, Suaya JA, Baxter R. Incidence, microbiology, and patient characteristics of skin and soft-tissue infections in a U.S. population: a retrospective population-based study. BMC Infect Dis 2013; 13: 252. 3 Ki V, Rotstein C. Bacterial skin and soft tissue infections in adults: a review of their epidemiology, pathogenesis, diagnosis, treatment and site of care. Can J Infect Dis 2008; 19: 173–84. 4 Edelsberg J, Taneja C, Zervos M et al. Trends in US hospital admissions for skin and soft tissue infections. Emerg Infect Dis 2009; 15: 1516– 8. 5 Pallin DJ, Egan DJ, Pelletier AJ et al. Increased US emergency department visits for skin and soft tissue infections, and changes in antibiotic choices, during the emergence of community-associated methicillin-resistant Staphylococcus aureus. Ann Emerg Med 2008; 51: 291–8. 6 McCaig LF, McDonald LC, Mandal S et al. Staphylococcus aureus-associated skin and soft tissue infections in ambulatory care. Emerg Infect Dis 2006; 12: 1715– 23. 7 Stevens DL, Bisno AL, Chambers HF et al. Practice guidelines for the diagnosis and management of skin and soft-tissue infections. Clin Infect Dis 2005; 41: 1373– 406. 8 Bhalodi AA, Housman ST, Shepard A et al. Tissue pharmacokinetics of cefazolin in patients with lower limb infections. Antimicrob Agents Chemother 2013; 57: 5679– 83. 9 de la Pena A, Liu P, Derendorf H. Microdialysis in peripheral tissues. Adv Drug Deliv Rev 2000; 45: 189– 216. 10 de Lange EC, de Boer AG, Breimer DD. Methodological issues in microdialysis sampling for pharmacokinetic studies. Adv Drug Deliv Rev 2000; 45: 125–48. 11 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty-third Informational Supplement M100-S23. CLSI, Wayne, PA, USA, 2013.
Acknowledgements
12 Dudley MN, Nightingale CH, Drezner AD et al. Comparative penetration of cefonicid and cefazolin into the atrial appendage and pericardial fluid of patients undergoing open-heart surgery. Antimicrob Agents Chemother 1984; 26: 347–50.
We thank Christina Sutherland for conducting cefazolin HPLC analysis. Additionally we thank Pam Tessier and Henry Christensen for their help with the in vitro experimentation.
13 Zhanel GG, Adam HJ, Baxter MR et al. Antimicrobial susceptibility of 22746 pathogens from Canadian hospitals: results of the CANWARD 2007– 11 study. J Antimicrob Chemother 2013; 68 Suppl 1: i7– 22.
Funding
14 Craig WA. Pharmacokinetic/pharmacodynamic parameters: rationale for antibacterial dosing of mice and men. Clin Infect Dis 1998; 26: 1 –10; quiz 11–2.
This work was funded internally by the Center for Anti-Infective Research and Development at Hartford Hospital.
15 Clouse FL, Hovde LB, Rotschafer JC. In vitro evaluation of the activities of telavancin, cefazolin, and vancomycin against methicillin-susceptible and methicillin-resistant Staphylococcus aureus in peritoneal dialysate. Antimicrob Agents Chemother 2007; 51: 4521– 4.
Transparency declarations
16 Brill MJ, Houwink AP, Schmidt S et al. Reduced subcutaneous tissue distribution of cefazolin in morbidly obese versus non-obese patients determined using clinical microdialysis. J Antimicrob Chemother 2014; 69: 715– 23.
None to declare.
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density 4 h after initiation of cefazolin. This was in contrast with the observations made with MSSA isolates, but the exact reason for this difference is unknown. Some concentration-dependent killing was observed in the models, characterized by repeated increasing and decreasing bacterial densities around the simulated troughs and peaks, respectively. Overall, this observation did not seem to grossly affect the sustained bacterial reductions at 24 h. Historically, bacterial reductions observed at 24 h have been predictive of efficacy.15 That being said, additional studies may be warranted to assess the activity of cefazolin over an extended period of time. Nonetheless, these data confirm that, in a patient with intact immunity, it can be expected that 1 g of cefazolin every 8 h would be sufficient to treat a lower limb infection when exhibiting the described tissue concentrations. A study by Brill et al.16 evaluated 2 g of cefazolin intravenously in obese and non-obese patients undergoing laparoscopic Toupet fundoplication surgery. The authors observed decreased tissue concentrations following the administration of a one-time dose of 2 g of cefazolin. While it is hard to apply the observations in that study to our observations, given the stark differences in study design, it appears that higher doses may be needed in morbidly obese patients (BMI .40) in the setting of organisms with MICs approaching the breakpoint. In this study, we evaluated the pharmacodynamic tissue concentration profile resulting from the administration of 1 g of cefazolin every 8 h against susceptible isolates of S. aureus, E. coli and K. pneumoniae. When simulating the tissue concentrations observed in infected patients, sustained bacterial reductions were observed over 24 h, with rapid bactericidal activity observed against Gram-negative isolates and progressive, sustained kill of S. aureus. These data support the clinical utility of the regimen of 1 g of cefazolin every 8 h in patients with lower limb infections due to susceptible staphylococci, E. coli and K. pneumoniae.
J Antimicrob Chemother doi:10.1093/jac/dku152
Humanized tissue pharmacodynamics of cefazolin against commonly isolated pathogens in skin and skin structure infections Jami G. Jain, Seth T. Housman and David P. Nicolau* Center for Anti-Infective Research and Development, Hartford Hospital, Hartford, CT, USA *Corresponding author. Tel: +1-860-545-3941; Fax: +1-860-545-3992; E-mail: [email protected]
Received 18 December 2013; returned 2 March 2014; revised 17 March 2014; accepted 8 April 2014
Methods: Nine clinical isolates [five MSSA (cefazolin MIC range 0.5 –2.0 mg/L), two Escherichia coli (cefazolin MICs 1.0 and 2.0 mg/L) and two Klebsiella pneumoniae (cefazolin MICs of 1.0 and 2.0 mg/L)] were evaluated with a starting inoculum (0 h) of 106 cfu/mL. Time – kill curves were built and the area under the bacterial killing and regrowth curve (AUBC) was calculated. Results: The starting inoculum had a mean+SD of 6.3+0.28 log10 cfu/mL. Cefazolin human simulated targets for peak, trough and half-life were 13.0 mg/L, 2.6 mg/L and 2.6 h, respectively. Control isolates grew to 8.5+ 0.2 log10 cfu/mL. Against MSSA, cefazolin achieved a reduction from 0 h of 21.18+0.67 and 23.58+1.24 log10 cfu/mL, at 4 and 24 h, respectively. Cefazolin achieved a reduction in bacterial density of 23.45+0.35 and 22.68+0.99 log10 cfu/mL at 4 and 24 h, respectively, when tested against Enterobacteriaceae. No significant difference was observed when comparing AUBC based on MIC values. The rate of initial bacterial reduction of Enterobacteriaceae was rapid, with a decrease of .3 log10 cfu/mL by 4 h, while MSSA exhibited a gradual reduction in bacterial density over one dosing interval. Conclusions: The observed antibacterial effects of cefazolin support its continued utility against susceptible S. aureus, E. coli and K. pneumoniae in skin and skin structure infections. Keywords: Staphylococcus aureus, in vitro, Enterobacteriaceae, antibiotic exposures, lower limb infections
Introduction Comorbidities such as diabetes mellitus and peripheral vascular disease commonly result in patients having reduced blood flow to the lower extremities and increased risk of infection. 1 This poses the possibility that patients with chronic lower limb infections may not receive adequate antimicrobial exposures at the site of infection. Consequently, antimicrobial concentrations in the blood may not always correlate with concentrations at the site of infection. These patients often have reduced immune function, which further reduces their ability to fight off infection. Therefore, it is paramount that clinicians take into account any differences in pharmacokinetics when treating lower limb infections. While Staphylococcus aureus is the most prevalent pathogen implicated in acute bacterial skin and skin structure infections (ABSSSIs), polymicrobial infections, including Escherichia coli and Klebsiella pneumoniae, are also a well-recognized clinical
burden.2 – 6 As a result of its antibacterial spectrum, cefazolin is used for both empirical and directed therapy of ABSSSIs.7 In the context of altered perfusion and tissue concentrations, a reassessment of cefazolin’s pharmacodynamic profile is necessary. A recent study by Bhalodi et al.8 measured cefazolin tissue concentrations in patients with chronic lower limb infections. Using an in vivo microdialysis technique previously described,9,10 they concluded that tissue exposures of cefazolin were adequate to reach clinical pharmacodynamic targets [percentage of free time above the MIC (%fT.MIC)]. While these data provide the pharmacokinetic profile of cefazolin in the infected patient, additional pharmacodynamic studies are required to assess the regimen’s potential efficacy against target pathogens. We set out to assess the efficacy of human simulated tissue concentrations following 1 g of cefazolin every 8 h against methicillin-susceptible Staphylococcus aureus (MSSA) and Enterobacteriaceae in a onecompartment in vitro pharmacodynamic (IVPD) model.
# The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: [email protected]
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Downloaded from http://jac.oxfordjournals.org/ at Ondokuz Mayis University on May 21, 2014
Objectives: The objective of this study was to assess the efficacy of humanized cefazolin tissue concentrations against methicillin-susceptible Staphylococcus aureus (MSSA) and Enterobacteriaceae in an in vitro pharmacodynamic model.
Jain et al.
Materials and methods Bacterial strains Nine clinical isolates (five MSSA isolates, two E. coli isolates and two K. pneumoniae isolates) were selected for use in this study. The MIC for all organisms was determined a minimum of two times by the broth microdilution method in accordance with the CLSI.11 In addition to cefazolin, MICs of ceftaroline, ceftriaxone, daptomycin, oxacillin, tigecycline and vancomycin for MSSA were also determined. MICs of cefazolin, cefepime, ceftaroline, ceftriaxone, ciprofloxacin, piperacillin/tazobactam and tigecycline for Enterobacteriaceae were determined.
Antibiotics
Simulated drug exposures Tissue concentrations for cefazolin were based on patient pharmacokinetic data generated by Bhalodi et al.8 In brief, an in vivo microdialysis technique was used to measure interstitial concentrations of cefazolin in seven patients, each with a chronic lower limb infection. This technique provided continuous measurements over a dosing interval, describing the antimicrobial exposures in these patients with compromised lower limb vasculature. These human data were simulated in an IVPD model with a peak tissue concentration of 13 mg/L and a half-life of 2.6 h, resulting in a tissue trough concentration of 2.6 mg/L.
In vitro pharmacodynamic model A one-compartment in vitro model was used for all experiments. Each experiment consisted of three independent models (two experimental treatment models and one growth control model), which ran concurrently for each isolate. To achieve uniform temperatures of 378C, the models were stationed in a temperature-modulated circulating water bath. All experiments were performed with a starting (0 h) inoculum of 106 cfu/mL. Inoculum preparation included making a bacterial suspension of 108 cfu/mL from subcultures of the test isolate (incubated at 378C overnight) with 0.9% normal saline for injection. Three millilitres of inoculum was injected into each 300 mL model to achieve the starting inoculum of 106 cfu/mL. Cation-adjusted Mueller – Hinton broth (CAMHB) (Becton, Dickinson and Company, Sparks, MD, USA) was used as the bacterial growth medium for all experiments. Once inoculated, the bacteria were allowed to enter the log phase of growth over 30 min. Cefazolin was then administered as a bolus into the model (0 h) to simulate a steady-state trough of free drug concentration achieved in human lower limb tissue after intravenous doses of 1 g of cefazolin every 8 h. Following this initial bolus, cefazolin was infused at the beginning of each dosing interval, using a peristaltic pump (Masterflex L/S Model 7524-10; Cole-Palmer, USA), into the model at a pre-determined rate. Each experiment was conducted over 24 h and performed in duplicate to assure reproducibility. Bacterial density over time was determined by sampling from each model at pre-determined timepoints (0, 1, 2, 4, 6, 8, 10, 12, 16, 18, 20 and 24 h) and serial dilution in normal saline. Aliquots of diluted sample were plated for quantitative culture utilizing trypticase soy agar plates (100 mm diameter) with 5% sheep blood. Colony counts were read after 16 – 20 h of incubation at 378C. The lower limit of detection for bacterial density was 1.7 log10 cfu/mL. To evaluate the antibiotic activity of the regimen of 1 g of cefazolin every 8 h, bacterial density was measured by the change in log10 cfu/mL from 0 h at 4 and 24 h. Bactericidal activity was
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Antibiotic concentration and exposure determinations Samples of CAMHB broth taken from each of the treatment models were assayed for cefazolin at 0, 2, 4, 6, 8, 10, 16, 18 and 24 h. All samples were immediately stored at 2808C until analysis. Cefazolin was analysed by a validated HPLC method at the Center for Anti-Infective Research and Development, as described previously.12 For the assay, the lower and upper detection limits were 0.5 and 30 mg/L, respectively. The interday coefficient of variation (CV) for the cefazolin check samples of 0.8 and 20 mg/mL was 5.9% and 5.1%, respectively (n ¼ 22), while the intraday CV was 2.1% and 3.1% (n¼10), respectively. The cefazolin assay was linear over a concentration range of 0.5 – 30 mg/L. Based on achieved concentrations in each model, the peak and trough concentrations were reported as those observed by analysis and the half-life was calculated as 0.693/k, where k is the estimated elimination rate constant calculated as the natural log (ln) of the measured peak/trough divided by its change in time. The AUBC over each 24 h period (log10 cfu.h/mL) was calculated using a model-specific growth control for comparison.
Statistical analysis Differences in AUBC at 24 h within MSSA and Enterobacteriaceae, based on MIC values, were assessed by one-way ANOVA or by the t-test where applicable. In addition, mean bacterial reductions at 4 and 24 h were compared between MSSA and Enterobacteriaceae using the t-test. An a priori P value ,0.05 was considered statistically significant.
Results MIC determination The MICs of cefazolin for MSSA F 2-13, MSSA F 10-29, MSSA F 44-14, MSSA F 19-13 and MSSA F 18-8 were 0.5, 0.5, 1, 1 and 2 mg/L, respectively. The MICs of cefazolin for the Enterobacteriaceae isolates E. coli AZ 18-25, E. coli AZ 6-17, K. pneumoniae JJ 7-10 and K. pneumoniae JJ 7-33 were 1, 2, 1 and 2 mg/L, respectively. Phenotypic profiles of all isolates used are shown in Table 1.
Pharmacokinetic analysis Pharmacokinetic parameters (peak, trough and half-life) observed in the models were 13.68+2.66 mg/L, 3.34+1.03 mg/L and 3.02+0.08 h, respectively. A comparison between observed patient pharmacokinetics and observed model pharmacokinetics can be seen in Figure 1. These resulted in a mean cefazolin %fT.MIC of 100% for all MSSA and Enterobacteriaceae isolates, as would be expected in the clinical setting when 1 g of cefazolin every 8 h is administered.8
Antibacterial activity MSSA isolates The average bacterial density of the starting inoculum was 6.42+0.25 log 10 cfu/mL. Control isolates grew to 8.44+ 0.19 log10 cfu/mL over 24 h. Figure 2 summarizes the time – kill curves for cefazolin against all MSSA isolates. The mean changes in bacterial density at 4 and 24 h against the five MSSA isolates
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Cefazolin (Apotex Corp., Weston, FL, USA; lot 101D026; expiry date, March 2015) was obtained from the Department of Pharmacy at Hartford Hospital.
defined as a reduction in bacterial density of at least 3 log10 cfu/mL. Time– kill curves were constructed and the area under the bacterial killing and regrowth curve (AUBC) was calculated to assess antibiotic efficacy.
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Humanized tissue pharmacodynamics of cefazolin
Table 1. Phenotypic profiles of MSSA and Enterobacteriaceae isolates Isolate
CFZ
OXA
TZP
FEP
CRO
CPT
VAN
CIP
DAP
TGC
MSSA F 2-13 MSSA F 10-29 MSSA F 44-14 MSSA F 19-13 MSSA F 18-8 E. coli AZ 18-25 K. pneumoniae JJ 7-10 E. coli AZ 6-17 K. pneumoniae JJ 7-33
0.5 0.5 1 1 2 1 1 2 2
0.25 0.5 0.5 0.5 2 ND ND ND ND
ND ND ND ND ND 2 2 2 1
ND ND ND ND ND ,0.06 ,0.06 ,0.06 ,0.06
2 4 4 8 8 ,0.25 ,0.25 ,0.25 ,0.25
0.25 0.25 0.25 0.25 0.5 ,0.06 ,0.06 ,0.06 ,0.06
1 1 1 1 1 ND ND ND ND
ND ND ND ND ND ,0.015 0.03 ,0.015 0.03
0.25 0.5 0.25 0.5 0.5 ND ND ND ND
0.064 0.125 0.125 0.25 0.125 0.125 0.5a 0.125 0.5a
Mean bacterial density (log10 cfu/mL)
Cefazolin concentration (mg/L)
10
10
2 mg/L 1 mg/L
1
0.5 mg/L 0
2
4
6
8
10
12
14
16
18
20
22
24
8
6
4
2
0 0
2
Time (h) Human tissue mean concentrations Model simulated mean concentrations Figure 1. Human versus simulated cefazolin pharmacokinetic profiles and corresponding MICs. Data are plotted as the means of the multiple models and the means of observed human pharmacokinetic profiles.
are provided in Table 2. MSSA isolates exhibited a significantly slower progression of bacterial reduction by 4 h (P, 0.0001) when compared with the rapid reductions observed with Enterobacteriaceae. No significant differences in AUBC reductions were seen, regardless of MIC (P . 0.05). The AUBCs over 24 h for cefazolin are also summarized in Table 2.
Enterobacteriaceae isolates The average bacterial density of the starting inoculum was 6.08+ 0.21 log10 cfu/mL. Control isolates grew to 8.74+0.07 log10 cfu/ mL over 24 h in the models. Figure 3 summarizes the time – kill curves for cefazolin against all Enterobacteriaceae isolates. The mean changes in bacterial density at 4 and 24 h against the four Enterobacteriaceae isolates are provided in Table 2. When comparing 24 h bacterial reductions observed with MSSA and
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10 12 14 16 18 Time (h) Mean control MSSA F2-13/MIC = 0.5 MSSA F10-29/MIC = 0.5 MSSA F44-14/MIC = 1 MSSA F19-13/MIC = 1 MSSA F18-8/MIC = 2 Limit of detection 1.7 log10 cfu/mL 6
8
20
22
24
Figure 2. Mean bacterial densities over 24 h for MSSA isolates. Data are plotted as the means of the multiple models for the treatments and the mean of all corresponding growth control isolates. The lower limit of detection (broken line) was 1.7 log10 cfu/mL.
Enterobacteriaceae, no statistically significant difference was found (P .0.05). As with MSSA, Enterobacteriaceae showed no significant difference in AUBC, regardless of MIC (P .0.05). The AUBCs over 24 h for cefazolin are also summarized in Table 2.
Discussion With today’s focus on emerging pathogens with various resistances to many mainstay regimens, it is imperative that clinicians optimize antimicrobials pharmacodynamically whenever
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CFZ, cefazolin; OXA, oxacillin; TZP, piperacillin/tazobactam; FEP, cefepime; CRO, ceftriaxone; CPT, ceftaroline; VAN, vancomycin; CIP, ciprofloxacin; DAP, daptomycin; TGC, tigecycline; ND, not determined. This table shows the reported modal MIC (mg/L) unless otherwise noted. a Median.
Jain et al.
Table 2. Mean change in bacterial density at 4 and 24 h and AUBC from 0 to 24 h for cefazolin against all isolates Change in bacterial density (log10 cfu/mL) from 0 ha Isolate
AUBC (log10 cfu.h/mL)a
4h
24 h
1 g of cefazolin every 8 h
control
MSSA F 2-13 MSSA F 10-29 MSSA F 44-14 MSSA F 19-13 MSSA F 18-8 Mean+SDb
0.5 0.5 1 1 2
22.07 20.71 20.95 20.01 21.51 21.18+0.67
24.24 24.28 23.03 24.66 22.64 23.58+1.24
77.58 88.70 92.81 82.42 88.30 86.46+9.15
196.40 194.52 195.77 198.07 192.76 195.5+2.00
E. coli AZ 18-25 K. pneumoniae JJ 7-10 E. coli AZ 6-17 K. pneumoniae JJ 7-33 Mean+SDb
1 1 2 2
23.30 23.17 23.97 23.36 23.45+0.35
22.25 22.84 23.85 21.78 22.68+0.99
92.66 59.28 61.80 86.85 75.15+17.55
204.12 201.05 197.56 202.42 201.29+2.78
a
Values for each isolate are the means of two to five independent time– kill experiments. Data are presented as mean+SD of all time–kill curves (two to five per isolate). Negative numbers indicate reductions in cfu from 0 h.
b
Mean bacterial density (log10 cfu/mL)
10
8
6
4
2
0 0
2
4
6
8
10 12 14 Time (h)
16
18
20
22
24
Mean control E. coli AZ 18-25/MIC = 1 E. coli AZ 6-17/MIC = 2 K. pneumoniae JJ 7-10/MIC = 1 K. pneumoniae JJ 7-33/MIC = 2 Limit of detection 1.7 log10 cfu/mL Figure 3. Mean bacterial densities over 24 h for Enterobacteriaceae isolates. Data are plotted as the means of the multiple models for the treatments and the mean of all corresponding growth control isolates. The lower limit of detection (broken line) was 1.7 log10 cfu/mL.
possible. Cefazolin is commonly used to treat ABSSSIs, caused by MSSA. Antibiotics can vary in their ability to achieve effective concentrations in foot lesions due to specific drug pharmacokinetics and patients’ vascular permeabilities. As new tissue concentration data are generated, a correlation between concentrations at the site of infection and efficacy against clinical isolates should be assessed.8 Here we set out to assess the antibacterial effects of human simulated tissue concentrations following 1 g of cefazolin every 8 h against both MSSA and Enterobacteriaceae.
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In this study, five MSSA clinical isolates with cefazolin MICs in the range of 0.5 –2 mg/L were used. It is important to note that these isolates are on the upper end of the MIC distribution, with MIC50 and MIC 90 being 0.5 and 1 mg/L, respectively. 13 The %fT.MIC of 100 was achieved for all isolates, exceeding the required pharmacodynamic target of 30% – 40% of the dosing interval.14 As expected, on average cefazolin exhibited bactericidal activity against MSSA. The changes in bacterial density at 4 and 24 h were observed in order to depict the rate of kill for both MSSA and Enterobacteriaceae. A slow progression of killing was observed across all MSSA models regardless of MIC, indicated by less than half of its overall bacterial density reductions occurring at 4 h. However, after 24 h of simulating a regimen consisting of 1 g of cefazolin every 8 h, the mean bacterial density was reduced by .3 log10 cfu/mL. As the only MSSA isolate with an MIC of 2 mg/L, F 18-8 exhibited a slight rebound in growth, resulting in a 24 h bacterial reduction of 1 log10 cfu/mL less than the mean stated above. These simulated cefazolin tissue concentrations seem to provide more than adequate antimicrobial exposures for all the tested MSSA isolates. It would be expected that patients being treated with monotherapy of 1 g of cefazolin every 8 h, under these pharmacokinetic and vasculature conditions, would reach pharmacodynamic targets against MSSA. Additional isolates were tested to represent commonly involved Gram-negative pathogens in ABSSSIs. Four clinical isolates of Enterobacteriaceae (two E. coli isolates and two K. pneumoniae isolates) with cefazolin MICs of 1 mg/L and at the susceptible breakpoint of 2 mg/L were tested. As with MSSA, all Enterobacteriaceae isolates had a %fT.MIC that exceeded current pharmacodynamic targets. It is important to note that, when treating Gram-negative pathogens with cefazolin, the %fT.MIC is required to be higher (60%–70%) than when treating Gram-positive pathogens (30% – 40%).14 Bacterial reductions were observed with all isolates. No differences in bacterial reductions were seen between isolates when comparing MICs. All Gram-negative isolates exhibited a rapid rate of killing that was characterized by reductions of .3 log10 cfu/mL in bacterial
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MIC (mg/L)
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Humanized tissue pharmacodynamics of cefazolin
References 1 Suaya JA, Eisenberg DF, Fang C et al. Skin and soft tissue infections and associated complications among commercially insured patients aged 0–64 years with and without diabetes in the U.S. PLoS One 2013; 8: e60057. 2 Ray GT, Suaya JA, Baxter R. Incidence, microbiology, and patient characteristics of skin and soft-tissue infections in a U.S. population: a retrospective population-based study. BMC Infect Dis 2013; 13: 252. 3 Ki V, Rotstein C. Bacterial skin and soft tissue infections in adults: a review of their epidemiology, pathogenesis, diagnosis, treatment and site of care. Can J Infect Dis 2008; 19: 173–84. 4 Edelsberg J, Taneja C, Zervos M et al. Trends in US hospital admissions for skin and soft tissue infections. Emerg Infect Dis 2009; 15: 1516– 8. 5 Pallin DJ, Egan DJ, Pelletier AJ et al. Increased US emergency department visits for skin and soft tissue infections, and changes in antibiotic choices, during the emergence of community-associated methicillin-resistant Staphylococcus aureus. Ann Emerg Med 2008; 51: 291–8. 6 McCaig LF, McDonald LC, Mandal S et al. Staphylococcus aureus-associated skin and soft tissue infections in ambulatory care. Emerg Infect Dis 2006; 12: 1715– 23. 7 Stevens DL, Bisno AL, Chambers HF et al. Practice guidelines for the diagnosis and management of skin and soft-tissue infections. Clin Infect Dis 2005; 41: 1373– 406. 8 Bhalodi AA, Housman ST, Shepard A et al. Tissue pharmacokinetics of cefazolin in patients with lower limb infections. Antimicrob Agents Chemother 2013; 57: 5679– 83. 9 de la Pena A, Liu P, Derendorf H. Microdialysis in peripheral tissues. Adv Drug Deliv Rev 2000; 45: 189– 216. 10 de Lange EC, de Boer AG, Breimer DD. Methodological issues in microdialysis sampling for pharmacokinetic studies. Adv Drug Deliv Rev 2000; 45: 125–48. 11 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty-third Informational Supplement M100-S23. CLSI, Wayne, PA, USA, 2013.
Acknowledgements
12 Dudley MN, Nightingale CH, Drezner AD et al. Comparative penetration of cefonicid and cefazolin into the atrial appendage and pericardial fluid of patients undergoing open-heart surgery. Antimicrob Agents Chemother 1984; 26: 347–50.
We thank Christina Sutherland for conducting cefazolin HPLC analysis. Additionally we thank Pam Tessier and Henry Christensen for their help with the in vitro experimentation.
13 Zhanel GG, Adam HJ, Baxter MR et al. Antimicrobial susceptibility of 22746 pathogens from Canadian hospitals: results of the CANWARD 2007– 11 study. J Antimicrob Chemother 2013; 68 Suppl 1: i7– 22.
Funding
14 Craig WA. Pharmacokinetic/pharmacodynamic parameters: rationale for antibacterial dosing of mice and men. Clin Infect Dis 1998; 26: 1 –10; quiz 11–2.
This work was funded internally by the Center for Anti-Infective Research and Development at Hartford Hospital.
15 Clouse FL, Hovde LB, Rotschafer JC. In vitro evaluation of the activities of telavancin, cefazolin, and vancomycin against methicillin-susceptible and methicillin-resistant Staphylococcus aureus in peritoneal dialysate. Antimicrob Agents Chemother 2007; 51: 4521– 4.
Transparency declarations
16 Brill MJ, Houwink AP, Schmidt S et al. Reduced subcutaneous tissue distribution of cefazolin in morbidly obese versus non-obese patients determined using clinical microdialysis. J Antimicrob Chemother 2014; 69: 715– 23.
None to declare.
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density 4 h after initiation of cefazolin. This was in contrast with the observations made with MSSA isolates, but the exact reason for this difference is unknown. Some concentration-dependent killing was observed in the models, characterized by repeated increasing and decreasing bacterial densities around the simulated troughs and peaks, respectively. Overall, this observation did not seem to grossly affect the sustained bacterial reductions at 24 h. Historically, bacterial reductions observed at 24 h have been predictive of efficacy.15 That being said, additional studies may be warranted to assess the activity of cefazolin over an extended period of time. Nonetheless, these data confirm that, in a patient with intact immunity, it can be expected that 1 g of cefazolin every 8 h would be sufficient to treat a lower limb infection when exhibiting the described tissue concentrations. A study by Brill et al.16 evaluated 2 g of cefazolin intravenously in obese and non-obese patients undergoing laparoscopic Toupet fundoplication surgery. The authors observed decreased tissue concentrations following the administration of a one-time dose of 2 g of cefazolin. While it is hard to apply the observations in that study to our observations, given the stark differences in study design, it appears that higher doses may be needed in morbidly obese patients (BMI .40) in the setting of organisms with MICs approaching the breakpoint. In this study, we evaluated the pharmacodynamic tissue concentration profile resulting from the administration of 1 g of cefazolin every 8 h against susceptible isolates of S. aureus, E. coli and K. pneumoniae. When simulating the tissue concentrations observed in infected patients, sustained bacterial reductions were observed over 24 h, with rapid bactericidal activity observed against Gram-negative isolates and progressive, sustained kill of S. aureus. These data support the clinical utility of the regimen of 1 g of cefazolin every 8 h in patients with lower limb infections due to susceptible staphylococci, E. coli and K. pneumoniae.
