Effects of Urine Matrix and pH on the Potency of Delafloxacin and Ciprofloxacin against Urogenic Escherichia coli and Klebsiella pneumoniae Wonhee So, Jared L. Crandon and David P. Nicolau*,† From the Center for Anti-Infective Research and Development (WS, JLC, DPN) and Division of Infectious Diseases, Hartford Hospital (DPN), Hartford, Connecticut
Purpose: We assessed the effects of the urine matrix and its varying pH on the potency of the novel broad-spectrum fluoroquinolone delafloxacin and of ciprofloxacin against 16 urogenic Enterobacteriaceae in the urine of patients with suspected urinary tract infection. Materials and Methods: We determined minimum inhibitory concentrations in broth and urine using microdilution in 9 Escherichia coli and 7 Klebsiella pneumoniae specimens. The change in potency between broth and urine was calculated. Results: Against 16 highly ciprofloxacin resistant Enterobacteriaceae with a broth minimum inhibitory concentration of 32 mg/l or greater the minimum inhibitory concentration in delafloxacin in broth was 2 mg/l (1 and 0 isolates of E. coli and K. pneumoniae, respectively), 4 mg/l (3 and 0), 8 mg/l (3 and 1), 16 mg/l (2 and 4) and 32 mg/l (0 and 2). Across the 143 collected urines pH ranged from 4.7 to 9.0 with 71% at pH 6.5 or less. The delafloxacin minimum inhibitory concentration measured in 80% urine from 100 unique patient samples (pH 5.0 to 8.3) was 2 mg/l or less (18% and 0.8% for E. coli and K. pneumoniae, respectively), 4 mg/l (23% and 6%), 8 mg/l (21% and 18%), 16 mg/l (23% and 33%) and 32 mg/l or greater (15% and 42%). For E. coli and K. pneumoniae combined the median changes in the delafloxacin minimum inhibitory concentration were a 1 doubling dilution decrease at pH 6.0 or less, no change at pH 6.1 to 7.0 and a 1 doubling dilution increase at pH 7.1 or greater. Unlike delafloxacin, ciprofloxacin showed a 1 doubling dilution increase for E. coli and no change for K. pneumoniae at pH 7.0 or less with no change observed at pH 7.1 or greater. Conclusions: Most urines collected from patients with urinary tract infection had a pH of 6.5 or less. Delafloxacin broth minimum inhibitory concentrations were twofold to fivefold doubling dilutions lower than those of ciprofloxacin. In contrast to ciprofloxacin, the potency of delafloxacin was further enhanced in the acidic environment commonly observed in the setting of urinary tract infection.
Abbreviations and Acronyms CA-MHB ¼ cation-adjusted MHB MHB ¼ Mueller-Hinton broth MIC ¼ minimum inhibitory concentration UTI ¼ urinary tract infection Accepted for publication January 19, 2015. Study received Hartford Hospital institutional review board approval. Supported by Melinta Therapeutics, New Haven, Connecticut, which reviewed the final manuscript before submission. * Correspondence: Center for Anti-Infective Research and Development, Hartford Hospital, 80 Seymour St., Hartford, Connecticut 06102 (telephone: 860-972-3941; FAX: 860-545-3992; e-mail: [email protected]). † Financial interest and/or other relationship with Melinta Therapeutics.
Key Words: urinary tract infections, Enterobacteriaceae, fluoroquinolones, ciprofloxacin, hydrogen-ion concentration
URINARY tract infections are the most common bacterial infections with clinical and economic burdens that impact community and hospital settings.1,2 Although various pathogens
are responsible for UTIs, Enterobacteriaceae, including Escherichia coli and Klebsiella pneumoniae, are most commonly isolated in uncomplicated and complicated UTIs.3,4
0022-5347/15/1942-0563/0 THE JOURNAL OF UROLOGY® © 2015 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH, INC.
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EFFECTS OF URINE MATRIX AND pH ON POTENCY OF DELAFLOXACIN AND CIPROFLOXACIN
Delafloxacin is an investigational fluoroquinolone with potent broad-spectrum activity against aerobic and anaerobic gram-positive and gram-negative organisms, including these commonly pathogenic Enterobacteriaceae.5e7 Like other fluoroquinolones, delafloxacin inhibits DNA synthesis by interacting with DNA gyrase and topoisomerase IV.5 However, in contrast to other fluoroquinolones, delafloxacin shows atypically equivalent functional activity at each enzyme,5,8 which results in activity against fluoroquinolone resistant bacteria since mutations in the 2 are rare.9 Along with inherent activity against fluoroquinolone resistant bacteria at neutral pH a further enhancement of delafloxacin potency in acidic environment was noted during its development.10 That is, delafloxacin showed tenfold higher potency at pH 5.5 compared to pH 7.4 against Staphylococcus aureus while the opposite was observed with moxifloxacin. Unlike other currently marketed zwitterionic fluoroquinolones10 the absence of a protonatable substituent of delafloxacin confers a weak acid character to the molecule, which leads to enhanced potency in acidic environments.11,12 Given that urine pH can vary among patients with UTI, understanding the impact of this observation on delafloxacin activity may provide additional insight into the ongoing development program to evaluate the preclinical and clinical efficacy of delafloxacin to treat UTIs, including those of resistant strains. Therefore, we examined the effect of the urine matrix and its varying pH on the potency of delafloxacin and ciprofloxacin against urogenic isolates of E. coli and K. pneumoniae.
MATERIALS AND METHODS The study was approved by the Hartford Hospital institutional review board. Informed consent was waived since urine samples were collected as part of standard clinical practice. No other intervention was done for study purposes.
Study Population All adult patients with a urine sample collected for urinalysis and sent to the chemistry department as part of clinical care from March to May 2014 were considered for study. From this cohort we selected patients whose urinalyses were sent to the microbiology department for suspected infection, ie positive nitrite, 5 or greater white blood cells, or small or greater (100 or greater) leukocyte esterase, for consecutive measurement of urine pH. After processing urine samples as described samples with insufficient volume or antibacterial effects were further excluded from MIC testing.
Antimicrobial Agents We used analytical grade delafloxacin meglumine (lot 71263AA002, ScinoPharm Taiwan, Tainan, Taiwan,
Republic of China) and analytical grade ciprofloxacin (lot P500044, Sigma-AldrichÒ). Analytical powders were weighed in a quantity sufficient to achieve the desired concentrations and reconstituted immediately before making MIC trays.
Bacterial Isolates We studied 16 urogenic Enterobacteriaceae, including 9 E. coli and 7 K. pneumoniae. All isolates were clinical isolates collected from a urine source. Four of these isolates were collected during the study period from the microbiology department at our institution. The remaining isolates were obtained from the isolate library at the Center for Anti-infective Research and Development at our institution.
Susceptibility Testing in Broth Broth MICs of delafloxacin and ciprofloxacin were determined using the broth microdilution methodology outlined by CLSI (Clinical and Laboratory Standards Institute).13 Modal MICs of all experiments were reported.
Urine pH Measurement We determined pH with a Model 320 Orion PerpHecTÒ LogRÒ pH meter and a Model 911600 gel filled pH electrode (Thermo Fisher ScientificÒ) according to manufacturer instructions. All samples were stored at e80C in a freezer.
Procedure Development Studies Freeze-Thaw and pH. To measure the freeze-thaw effect on pH we measured the pH of urine (range 4.9 to 7.8) from 10 patients at the time of acquisition from the clinical laboratory. Samples were frozen at e80C. A sample was thawed for each patient after 1 to 5 days in the freezer and pH was reassessed. Time and pH. We assessed the effect of time on pH between clinical sample collection and receipt at our laboratory. We first measured the pH (range 5.2 to 6.1) of the original sample of 5 patients at the time of processing. We then remeasured pH 3, 8, 12 and 24 hours after processing at room temperature (25C) and under refrigeration (2C to 8C). Broth and pH. To evaluate the magnitude of pH changes after adding CA-MHB (BDÒ) (pH 7.2) to a urine sample 12 urines with various pH values (range 5.0 to 7.5) were selected. We measured pH after adding broth at 10%, 20%, 30% or 50% of total volume. Growth Study of Urine and Broth Combination. To examine the ability of various urine-to-broth ratios to support the growth of organisms 8 Enterobacteriaceae (4 E. coli and 4 K. pneumoniae) were tested for growth in 90%, 80% and 50% urine. Three patient urines, including 1 with pH 4.8 and 2 with pH 5.3, were tested for each combination. Notably 100% urine was not tested since inocula had to be made in CA-MHB during the MIC study. Effect on MIC of 0.5 pH Changes. To measure the effect of small pH changes (ie 0.5) on delafloxacin MICs we measured the urine MICs of delafloxacin against 1 E. coli
EFFECTS OF URINE MATRIX AND pH ON POTENCY OF DELAFLOXACIN AND CIPROFLOXACIN
and 3 K. pneumoniae at pH 5, 5.5 and 6. Urine sample pH was adjusted with 0.1 N hydrochloric acid or 0.1 N sodium bicarbonate.
Urine Sample Processing At our laboratory clinical samples were filter sterilized and reassessed for sterility via culture, antibacterial effects and pH as described. Filtering. Samples were centrifuged at 1,000 gravity for 10 minutes at 25C. Urine supernatant was filtered with a 0.45 mm MillexÒ polyvinylidene fluoride syringe filter, followed by sterile filtration with a 0.22 mm Millex GV polyvinylidene fluoride syringe filter. Sterility Assessment. The sterile filtered sample (10 ml) was plated on trypticase soy agar with 5% sheep blood plate (BD). Plates were incubated at 37C for 16 to 20 hours to assure sterility. Antibacterial Effect Assessment. Given the difficulty of interpreting the urological exposure of administered antibiotics (ie timing relative to sample collection, variability in renal clearance, etc) directly from the medical record, we took further steps to physically determine the antibacterial activity of each urine sample and exclude those that showed antibacterial effects. This was done by applying 10 ml of undiluted urine sample on an agar plate previously inoculated with a susceptible quality control strain of E. coli (ATCCÒ 25922). The plates were incubated at 37C for 16 to 20 hours. If the organism grew without inhibition, urine was considered to be antibacterial free.
Data Collection Patient demographics, including age, gender and location, and urinalysis results were collected from the medical record. On the day of urine MIC testing the pH of 80% urine (8:2 urine-to-broth ratio) was recorded for data analysis.
Susceptibility Testing in Urine Urine MICs of delafloxacin and ciprofloxacin were determined using the microdilution methodology according to CLSI.13 Trays were made of 80 ml patient specific urine and inocula were made in 20 ml broth. For each urine MIC study 4 to 7 unique patient urines served as a set. Broth MICs were repeated in each set to establish baseline MICs for comparison.
Data Analysis The MIC change between broth and urine for delafloxacin and ciprofloxacin was calculated as (log2[MIC in urine] e log2[MIC in broth]). Linear regression analysis was performed using SigmaPlotÔ, version 12 to characterize the relationship between pH and the MIC change of all E. coli and all K. pneumoniae.
RESULTS Study Population Across the 143 urines collected the measured pH ranged from 4.7 to 9.0 with 71% at pH 6.5 or less, 13% at pH 6.6 to 7.0 and 16% at pH 7.1 or greater
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(figs. 1 and 2). Of the pH values measured at our laboratory 77% were within 0.5 of the pH reported according to the urinalysis. Of these samples 15 and 24 were excluded from study due to insufficient volume and a positive antibacterial effect, respectively, while 4 were excluded due to other operational errors. Table 1 lists demographic data on the 100 patients in the urine MIC studies. Median age was 58 years (range 18 to greater than 89). Age greater than 89 years was aggregated into 1 category of 8 patients according to the Safe Harbor Method [x164.514(b)(2)] in Guidance on DeIdentification of Protected Health Information in Accordance with the HIPAA (Health Insurance Portability and Accountability Act) Privacy Rule. We tested the samples of 92 patients with a urine volume of 40 ml or greater for 9 E. coli and 7 K. pneumoniae. The samples of 8 patients with a urine volume of less than 40 ml were tested for 5 E. coli and 3 K. pneumoniae (table 2). Susceptibility Testing in Broth Table 2 lists the broth MICs of the 16 urogenic Enterobacteriaceae, including 9 E. coli and 7 K. pneumoniae. Ciprofloxacin MICs were 32 mg/l or greater against all 16 isolates but delafloxacin MICs were 2 to 16 mg/l for E. coli and 8 to 32 mg/l for K. pneumoniae. Procedure Development Studies Freeze-Thaw pH. Two of 10 patient samples showed a 0.2 change in pH. The remaining patient samples showed no greater than a 0.1 change in pH, suggesting that freeze-thaw had little effect on pH. Time pH. All measured pH values remained at 0.1
of that of the original sample pH whether at room temperature or refrigerated during 24 hours. Therefore, clinical samples were processed within 24 hours of collection and refrigerated (temperature 2C to 8C) until processed. Broth pH. Across 12 patient urines tested the
changes in pH of the urine-to-broth combination was 0.2 or less at a ratio of 9:1, 0.3 or less at 8:2, 0.5 or less at 7:3 and 0.8 or less at 5:5. Therefore, only the 9:1 and 8:2 combinations were further evaluated in growth studies to minimize pH changes from the original samples. Growth Study of Urine and Broth Combination. Based
on the broth pH study as noted, growth experiments were performed in 90% and 80% urine as well as in 50% urine for comparison. In 50% urine all 8 Enterobacteriaceae tested grew in all 3 urines. However, in 90% and 80% urine the positive growth rate decreased to 66% and 79%, respectively. Based on the results of broth pH and growth studies urine MIC studies were performed in 80% urine.
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Figure 1. Influence of urine matrix on delafloxacin (A) and ciprofloxacin (B) MICs for E. coli. MIC change was e8.67 þ 1.39 pH (r2 ¼ 0.554) (A) and 2.56 0.32 pH (r2 ¼ 0.085) (B). Bars represent frequency of pH in urine of 143 patients. Numbers indicate observations of 9 E. coli tested in 100 patient urines. Diagonal line indicates linear regression.
Effect on MIC of 0.5 pH Change. Across the 4
Enterobacteriaceae tested 87.5% of samples showed no shift in MICs over the change in pH by 0.5. The remaining 12.5% of urines showed 1 doubling dilution shift. Therefore, during MIC testing in 80% urine pH was not readjusted back to the original 100% urine pH. Susceptibility Testing in Urine Table 3 lists the frequency of observed delafloxacin urine MICs measured in 100 unique patient samples at pH 5.0 to 8.3. Figures 1 and 2 show MIC
shifts from broth to urine for E. coli and K. pneumoniae with delafloxacin and ciprofloxacin. Figures 1 and 2 also show the results of linear regression analysis. The magnitude of the MIC change for delafloxacin was similar for E. coli and K. pneumoniae with a median decrease of 1 doubling dilution at pH 6.0 or less, no change at pH 6.1 to 7.0 and an increase of 2 (E. coli) and 1 (K. pneumoniae) doubling dilution at pH 7.1 or greater. On the other hand, ciprofloxacin MICs remained relatively unchanged at 47% for E. coli and 82% for K. pneumoniae across
EFFECTS OF URINE MATRIX AND pH ON POTENCY OF DELAFLOXACIN AND CIPROFLOXACIN
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Figure 2. Influence of urine matrix on delafloxacin (A) and ciprofloxacin (B) MICs for K. pneumoniae. MIC change was e7.56 þ 1.18 pH (r2 ¼ 0.495) (A) and 1.91 0.27 pH (r2 ¼ 0.066) (B). Bars represent frequency of pH in urine of 143 patients. Numbers indicate observations of 9 K. pneumoniae tested in 100 patient urines. Diagonal line indicates linear regression.
the pH ranges. There was a median MIC change of 1 doubling dilution increase for E. coli, no change for K. pneumoniae at pH 7.0 or less and no change at pH 7.1 or greater.
DISCUSSION The novel fluoroquinolone delafloxacin is distinguished from other fluoroquinolones by its improved activity against certain increasing populations of fluoroquinolone resistant gram-negative organisms such as E. coli and K. pneumoniae.14e16 In addition, further enhancement was noted of delafloxacin potency in an acidic environment.10e12 In this
study we observed a change in delafloxacin activity between broth and patient specific urines of varying pH against highly ciprofloxacin resistant urogenic Enterobacteriaceae. In contrast to ciprofloxacin, which had poor activity against these pathogens with relatively unchanged MICs, delafloxacin showed enhanced activity in the acidic environment commonly observed in our patients with suspected UTI. The normal range of urinary pH was previously reported to be as wide as 4.5 to 8.6.17,18 Likewise our 143 consecutively collected urine samples from patients with signs of infection showed a wide range of 4.7 to 9.0. Although we did not explicitly explore
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EFFECTS OF URINE MATRIX AND pH ON POTENCY OF DELAFLOXACIN AND CIPROFLOXACIN
Table 1. Characteristics of 100 patients No. Pts No. female No. male No. location: Intensive care unit inpt Non intensive care unit inpt Emergency department Urinalysis criteria for culture: Pos nitrite 5 or Greater white blood cells on microscopic examination 100 or Greater (small or greater) leukocyte esterase
83 17 9 57 34 26 90 88
patient characteristics and their effect on urinary pH, data in the literature suggest that urinary pH can be affected by multiple factors.17,18 For example, the presence of urea-splitting organisms such as Proteus mirabilis and some strains of Klebsiella species, Pseudomonas species, Providencia species and Staphylococcus species are associated with increased urinary pH. Alkaline urine is also observed in systemic alkalosis and renal tubular acidosis, and with the use of carbonic anhydrase inhibitors.17 On the other hand, the presence of uric acid, and the administration of cranberry juice, ascorbic acid and ammonium chloride reportedly decrease urinary pH.19,20 In addition, groups suggested that there is lower urinary pH with bacterial infection (pH 3.5 to 6.0) compared to healthy urine (pH 6.5 to 7.5).21,22 Similarly despite the multifactorial nature of urinary pH and the broad range of pH in our 143 clinical samples with signs of infection, 71% had pH no greater than 6.5. As expected from previous reports,5,6 delafloxacin broth MICs against these 16 highly ciprofloxacin resistant Enterobacteriaceae were 2 to 5 doubling dilutions lower than the ciprofloxacin broth MICs. Nilius et al reported delafloxacin MICs of 4 to 32 mg/l against 10 E. coli (ciprofloxacin MIC 32 to
greater than 128 mg/l) and 2 to 8 mg/l against 10 K. pneumoniae (ciprofloxacin MIC 64 to greater than 128 mg/l).5 Similarly Almer et al reported delafloxacin MICs of 1 to 16 mg/l against 27 ciprofloxacin resistant E. coli (MIC 4 to greater than 128 mg/l) and 1 to 4 mg/l against 22 ciprofloxacin resistant K. pneumoniae (MIC 4 to 64 mg/l).6 From 100 patient urines (pH 5.0 to 8.3) in which urine MIC testing was performed delafloxacin MICs decreased by 1 to 3 doubling dilutions in 53% of observations for E. coli and 68% for K. pneumoniae with a median of 1 doubling dilution decrease at pH 6.0 or less. While 48% of the urinary pH distribution was for pH 6.0 or less, after excluding samples with insufficient volume or antibacterial effect only 37% of our total observations were made at pH 6.0 or less (table 3). Similar reductions in frequency were seen between the urinary pH distribution and the number of observations at pH 6.5 or less (ie 71% vs 59%). As such the median decrease in MIC could have been greater if we had had more urine samples with increased acidity in our observations. Enhanced activity of a fluoroquinolone in an acidic environment was also observed for finafloxacin, another investigational fluoroquinolone.21,23 Finafloxacin showed up to a 5 doubling dilution decrease of MICs at pH 5.8 compared to pH 7.2 against Acinetobacter baumannii isolates while ciprofloxacin MICs increased up to greater than 4 doubling dilutions at pH 5.8.21 Wagenlehner et al determined MICs of finafloxacin and ciprofloxacin in CA-MHB at a pH of 5.8, 7.2 and 8.0 as well as in synthetic urine adjusted to pH 5.8.23 Similar to our observations, compared to the MICs in CA-MHB at pH 7.2 the finafloxacin MICs were 1 to 2 doubling dilutions higher at pH 8.0 and 2 to 3 doubling dilutions lower at pH 5.8 against E. coli. Interestingly at pH 5.8 finafloxacin MICs in synthetic urine were higher than in
Table 2. Broth MICs of 16 Enterobacteriaceae Isolate No. E. coli: 443* 348 429* 435* 310 350* 432* 130 133 K. pneumoniae: 368* 331A 350B* 369* 378 320 390
Delafloxacin (mg/l)
Ciprofloxacin (mg/l)
2 4 4 4 8 8 8 16 16
64 Greater than 64 64 64 Greater than 64 Greater than 64 64 64 64
8 16 16 16 16 32 32
Greater Greater Greater Greater Greater Greater
* Isolates used when urine volume was less than 40 ml.
32 than than than than than than
64 64 64 64 64 64
Known Resistance Mechanism Extended spectrum Not determined Extended spectrum Not determined Extended spectrum Not determined Extended spectrum Extended spectrum Extended spectrum
b-lactamases b-lactamases b-lactamases b-lactamases b-lactamases b-lactamases
K. pneumoniae carbapenemases K. pneumoniae carbapenemases K. pneumoniae carbapenemases K. pneumoniae carbapenemases K. pneumoniae carbapenemases Extended spectrum b-lactamases K. pneumoniae carbapenemases
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Table 3. Urine MIC in 80% urine from 100 unique patient samples and 20% broth % E. coli MIC (pH) 5.0e6.0 No. observations* Delafloxacin MIC (mg/l): 2 or Less 4 8 16 32 or Greater Ciprofloxacin MIC (mg/l): 16 32 or Greater
253 35.2 24.5 20.2 17.4 2.8 0 100
5.0e6.5 414 28.7 28.5 20.5 18.4 3.9 0 100
5.0e7.0 570 21.6 27.5 22.6 20.2 8.1 0 100
% K. pneumoniae MIC (pH) 5.0e8.3 695 17.8 23.2 21.4 22.7 14.8 0.7 99.3
5.0e6.0 216 2.3 13.4 34.3 38.0 12.0 0 100
5.0e6.5 342 1.5 9.4 26.6 40.1 22.5 0 100
5.0e7.0 484 1.0 6.8 21.1 36.6 34.5 0 100
5.0e8.3 589 0.8 5.6 17.8 33.4 42.3 0.8 99.2
* For example, number at pH 6.1 to 6.5 for E. coli was 161 (414 minus 253).
CA-MHB, in contrast to our observations using delafloxacin in patient urine. The mechanism of the enhanced activity of delafloxacin or finafloxacin in an acidic environment, as distinguished from other zwitterionic fluoroquinolones such as moxifloxacin, levofloxacin and ciprofloxacin, is partly explained by the different physiochemical properties of the agents and the resultant change in lipophilicity and transmembrane diffusion depending on the surrounding pH.10,24,25 In addition, a possible influence of pH on bacterial gene expression was suggested, leading to modifications in influx or efflux transporters.21,26 On the other hand, consistent with previous reports of zwitterionic fluoroquinolones,10,20 ciprofloxacin MICs showed a 1 doubling dilution increase for E. coli and no change for K. pneumoniae under an acidic pH. Our study uniquely reveals the effect of the host related factor (ie urinary pH) on ciprofloxacin MIC, which to our knowledge was reported only once previously by Zhanel et al.27 In that study MICs of ciprofloxacin in urine at pH 5.5 were compared to MICs in MHB at pH 7.3. They observed 6 doubling dilution increases in the ciprofloxacin urine MIC at pH 5.5 compared to MHB at pH 7.3. While the magnitude of the ciprofloxacin MIC increase in that series (6 doubling dilutions) was greater than in ours (1 doubling dilution), all 3 isolates in the study by Zhanel et al were highly susceptible to ciprofloxacin with a broth MIC of 0.016 mg/l. In addition, ciprofloxacin MICs greater than 64 mg/l were not further defined in our series, which may have limited our ability to identify a correlation between a MIC greater than 64 mg/l
and pH. Also of note is that while methodologically we could not perform these studies in pure urine, we anticipate similar findings for these pH dependent observations.
CONCLUSIONS Delafloxacin broth MICs were twofold to fivefold doubling dilutions lower than those of ciprofloxacin against these 16 urogenic Enterobacteriaceae. While urinary pH can fluctuate depending on multiple factors, this study provides the approximate distribution of urinary pH in patients with suspected UTI, most highlighting the distribution in those with acidic urine (pH 6.5 or less). Although ciprofloxacin MICs remained relatively unchanged or increased across the distribution, delafloxacin showed enhanced activity against E. coli and K. pneumoniae in the acidic urinary environment (pH 5.0 to 6.0). Moreover, this improved potency resulted in MIC values that were predominantly 8 mg/l or less in the biological urine matrix. Future studies are required to fully understand the clinical implications of delafloxacin enhanced potency at the infection site.
ACKNOWLEDGMENTS Mary Anne Banevicius, Henry Christensen, Jennifer Hull, Lucinda Lamb, Debora Santini, Christina Sutherland, Sara Robinson and Kim Greenwood assisted with the study. David O’Sullivan, Hartford Hospital, assisted with statistics. Delafloxacin meglumine was provided by Melinta Therapeutics.
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14. Aypak C, Altunsoy A and Duzgun N: Empiric antibiotic therapy in acute uncomplicated urinary tract infections and fluoroquinolone resistance: a prospective observational study. Ann Clin Microbiol Antimicrob 2009; 8: 27. 15. Goettsch W, van Pelt W, Nagelkerke N et al: Increasing resistance to fluoroquinolones in Escherichia coli from urinary tract infections in the Netherlands. J Antimicrob Chemother 2000; 46: 223. 16. Talan DA, Krishnadasan A, Abrahamian FM et al: Prevalence and risk factor analysis of trimethoprimsulfamethoxazole- and fluoroquinolone-resistant Escherichia coli infection among emergency department patients with pyelonephritis. Clin Infect Dis 2008; 47: 1150. 17. Meng MV, Walsh TJ, Stoller ML et al: Urologic disorders. In: Current Medical Diagnosis and Treatment 2014. Edited by MA Papadakis and SJ McPhee. New York: McGraw-Hill 2014; chapt 23. Available at http://accessmedicine. mhmedical.com/content.aspx?bookid¼330&Sec tionid¼44291025. Accessed August 23, 2014. 18. Wagenlehner FME, Kinzig-Schippers M, Tischmeyer U et al: Pharmacokinetics of ciprofloxacin XR (1000mg) versus levofloxacin (500mg) in plasma and urine of male and female healthy volunteers receiving a single oral dose. Int J Antimicrob Agents 2006; 27: 7.
21. Higgins PG, Stubbings W, Wisplinghoff et al: Activity of the investigational fluoroquinolone finafloxacin against ciprofloxacin-sensitive and -resistant Acinetobacter baumannii isolates. Antimicrob Agents Chemother 2010; 54: 1613. 22. Byerly TC: Handbook of biological data. Science 1953; 118: 3. 23. Wagenlehner FME, Wagenlehner CM, Blenk B et al: Urinary pharmacokinetics and bactericidal activity of finafloxacin (200 and 800 mg) in healthy volunteers receiving a single oral dose. Chemotherapy 2011; 57: 97. 24. Nikaido H and Thanassi DG: Penetration of lipophilic agents with multiple protonation sites into bacterial cells: tetracyclines and fluoroquinolones as examples. Antimicrob Agents Chemother 1993; 37: 1393. 25. Park HR, Kim TH and Bark KM: Physicochemical properties of quinolone antibiotics in various environments. Eur J Med Chem 2002; 37: 443. 26. Begic S and Worobec EA: Fluoroquinolone resistance of Serratia marcescens: sucrose, salicylate, temperature, and pH induction of phenotypic resistance. Can J Microbiol 2007; 53: 1239. 27. Zhanel GG, Karlowsky JA, Davidson RJ et al: Influence of human urine on the in vitro activity and postantibiotic effect of ciprofloxacin against Escherichia coli. Chemotherapy 1991; 37: 218.
Purpose: We assessed the effects of the urine matrix and its varying pH on the potency of the novel broad-spectrum fluoroquinolone delafloxacin and of ciprofloxacin against 16 urogenic Enterobacteriaceae in the urine of patients with suspected urinary tract infection. Materials and Methods: We determined minimum inhibitory concentrations in broth and urine using microdilution in 9 Escherichia coli and 7 Klebsiella pneumoniae specimens. The change in potency between broth and urine was calculated. Results: Against 16 highly ciprofloxacin resistant Enterobacteriaceae with a broth minimum inhibitory concentration of 32 mg/l or greater the minimum inhibitory concentration in delafloxacin in broth was 2 mg/l (1 and 0 isolates of E. coli and K. pneumoniae, respectively), 4 mg/l (3 and 0), 8 mg/l (3 and 1), 16 mg/l (2 and 4) and 32 mg/l (0 and 2). Across the 143 collected urines pH ranged from 4.7 to 9.0 with 71% at pH 6.5 or less. The delafloxacin minimum inhibitory concentration measured in 80% urine from 100 unique patient samples (pH 5.0 to 8.3) was 2 mg/l or less (18% and 0.8% for E. coli and K. pneumoniae, respectively), 4 mg/l (23% and 6%), 8 mg/l (21% and 18%), 16 mg/l (23% and 33%) and 32 mg/l or greater (15% and 42%). For E. coli and K. pneumoniae combined the median changes in the delafloxacin minimum inhibitory concentration were a 1 doubling dilution decrease at pH 6.0 or less, no change at pH 6.1 to 7.0 and a 1 doubling dilution increase at pH 7.1 or greater. Unlike delafloxacin, ciprofloxacin showed a 1 doubling dilution increase for E. coli and no change for K. pneumoniae at pH 7.0 or less with no change observed at pH 7.1 or greater. Conclusions: Most urines collected from patients with urinary tract infection had a pH of 6.5 or less. Delafloxacin broth minimum inhibitory concentrations were twofold to fivefold doubling dilutions lower than those of ciprofloxacin. In contrast to ciprofloxacin, the potency of delafloxacin was further enhanced in the acidic environment commonly observed in the setting of urinary tract infection.
Abbreviations and Acronyms CA-MHB ¼ cation-adjusted MHB MHB ¼ Mueller-Hinton broth MIC ¼ minimum inhibitory concentration UTI ¼ urinary tract infection Accepted for publication January 19, 2015. Study received Hartford Hospital institutional review board approval. Supported by Melinta Therapeutics, New Haven, Connecticut, which reviewed the final manuscript before submission. * Correspondence: Center for Anti-Infective Research and Development, Hartford Hospital, 80 Seymour St., Hartford, Connecticut 06102 (telephone: 860-972-3941; FAX: 860-545-3992; e-mail: [email protected]). † Financial interest and/or other relationship with Melinta Therapeutics.
Key Words: urinary tract infections, Enterobacteriaceae, fluoroquinolones, ciprofloxacin, hydrogen-ion concentration
URINARY tract infections are the most common bacterial infections with clinical and economic burdens that impact community and hospital settings.1,2 Although various pathogens
are responsible for UTIs, Enterobacteriaceae, including Escherichia coli and Klebsiella pneumoniae, are most commonly isolated in uncomplicated and complicated UTIs.3,4
0022-5347/15/1942-0563/0 THE JOURNAL OF UROLOGY® © 2015 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH, INC.
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EFFECTS OF URINE MATRIX AND pH ON POTENCY OF DELAFLOXACIN AND CIPROFLOXACIN
Delafloxacin is an investigational fluoroquinolone with potent broad-spectrum activity against aerobic and anaerobic gram-positive and gram-negative organisms, including these commonly pathogenic Enterobacteriaceae.5e7 Like other fluoroquinolones, delafloxacin inhibits DNA synthesis by interacting with DNA gyrase and topoisomerase IV.5 However, in contrast to other fluoroquinolones, delafloxacin shows atypically equivalent functional activity at each enzyme,5,8 which results in activity against fluoroquinolone resistant bacteria since mutations in the 2 are rare.9 Along with inherent activity against fluoroquinolone resistant bacteria at neutral pH a further enhancement of delafloxacin potency in acidic environment was noted during its development.10 That is, delafloxacin showed tenfold higher potency at pH 5.5 compared to pH 7.4 against Staphylococcus aureus while the opposite was observed with moxifloxacin. Unlike other currently marketed zwitterionic fluoroquinolones10 the absence of a protonatable substituent of delafloxacin confers a weak acid character to the molecule, which leads to enhanced potency in acidic environments.11,12 Given that urine pH can vary among patients with UTI, understanding the impact of this observation on delafloxacin activity may provide additional insight into the ongoing development program to evaluate the preclinical and clinical efficacy of delafloxacin to treat UTIs, including those of resistant strains. Therefore, we examined the effect of the urine matrix and its varying pH on the potency of delafloxacin and ciprofloxacin against urogenic isolates of E. coli and K. pneumoniae.
MATERIALS AND METHODS The study was approved by the Hartford Hospital institutional review board. Informed consent was waived since urine samples were collected as part of standard clinical practice. No other intervention was done for study purposes.
Study Population All adult patients with a urine sample collected for urinalysis and sent to the chemistry department as part of clinical care from March to May 2014 were considered for study. From this cohort we selected patients whose urinalyses were sent to the microbiology department for suspected infection, ie positive nitrite, 5 or greater white blood cells, or small or greater (100 or greater) leukocyte esterase, for consecutive measurement of urine pH. After processing urine samples as described samples with insufficient volume or antibacterial effects were further excluded from MIC testing.
Antimicrobial Agents We used analytical grade delafloxacin meglumine (lot 71263AA002, ScinoPharm Taiwan, Tainan, Taiwan,
Republic of China) and analytical grade ciprofloxacin (lot P500044, Sigma-AldrichÒ). Analytical powders were weighed in a quantity sufficient to achieve the desired concentrations and reconstituted immediately before making MIC trays.
Bacterial Isolates We studied 16 urogenic Enterobacteriaceae, including 9 E. coli and 7 K. pneumoniae. All isolates were clinical isolates collected from a urine source. Four of these isolates were collected during the study period from the microbiology department at our institution. The remaining isolates were obtained from the isolate library at the Center for Anti-infective Research and Development at our institution.
Susceptibility Testing in Broth Broth MICs of delafloxacin and ciprofloxacin were determined using the broth microdilution methodology outlined by CLSI (Clinical and Laboratory Standards Institute).13 Modal MICs of all experiments were reported.
Urine pH Measurement We determined pH with a Model 320 Orion PerpHecTÒ LogRÒ pH meter and a Model 911600 gel filled pH electrode (Thermo Fisher ScientificÒ) according to manufacturer instructions. All samples were stored at e80C in a freezer.
Procedure Development Studies Freeze-Thaw and pH. To measure the freeze-thaw effect on pH we measured the pH of urine (range 4.9 to 7.8) from 10 patients at the time of acquisition from the clinical laboratory. Samples were frozen at e80C. A sample was thawed for each patient after 1 to 5 days in the freezer and pH was reassessed. Time and pH. We assessed the effect of time on pH between clinical sample collection and receipt at our laboratory. We first measured the pH (range 5.2 to 6.1) of the original sample of 5 patients at the time of processing. We then remeasured pH 3, 8, 12 and 24 hours after processing at room temperature (25C) and under refrigeration (2C to 8C). Broth and pH. To evaluate the magnitude of pH changes after adding CA-MHB (BDÒ) (pH 7.2) to a urine sample 12 urines with various pH values (range 5.0 to 7.5) were selected. We measured pH after adding broth at 10%, 20%, 30% or 50% of total volume. Growth Study of Urine and Broth Combination. To examine the ability of various urine-to-broth ratios to support the growth of organisms 8 Enterobacteriaceae (4 E. coli and 4 K. pneumoniae) were tested for growth in 90%, 80% and 50% urine. Three patient urines, including 1 with pH 4.8 and 2 with pH 5.3, were tested for each combination. Notably 100% urine was not tested since inocula had to be made in CA-MHB during the MIC study. Effect on MIC of 0.5 pH Changes. To measure the effect of small pH changes (ie 0.5) on delafloxacin MICs we measured the urine MICs of delafloxacin against 1 E. coli
EFFECTS OF URINE MATRIX AND pH ON POTENCY OF DELAFLOXACIN AND CIPROFLOXACIN
and 3 K. pneumoniae at pH 5, 5.5 and 6. Urine sample pH was adjusted with 0.1 N hydrochloric acid or 0.1 N sodium bicarbonate.
Urine Sample Processing At our laboratory clinical samples were filter sterilized and reassessed for sterility via culture, antibacterial effects and pH as described. Filtering. Samples were centrifuged at 1,000 gravity for 10 minutes at 25C. Urine supernatant was filtered with a 0.45 mm MillexÒ polyvinylidene fluoride syringe filter, followed by sterile filtration with a 0.22 mm Millex GV polyvinylidene fluoride syringe filter. Sterility Assessment. The sterile filtered sample (10 ml) was plated on trypticase soy agar with 5% sheep blood plate (BD). Plates were incubated at 37C for 16 to 20 hours to assure sterility. Antibacterial Effect Assessment. Given the difficulty of interpreting the urological exposure of administered antibiotics (ie timing relative to sample collection, variability in renal clearance, etc) directly from the medical record, we took further steps to physically determine the antibacterial activity of each urine sample and exclude those that showed antibacterial effects. This was done by applying 10 ml of undiluted urine sample on an agar plate previously inoculated with a susceptible quality control strain of E. coli (ATCCÒ 25922). The plates were incubated at 37C for 16 to 20 hours. If the organism grew without inhibition, urine was considered to be antibacterial free.
Data Collection Patient demographics, including age, gender and location, and urinalysis results were collected from the medical record. On the day of urine MIC testing the pH of 80% urine (8:2 urine-to-broth ratio) was recorded for data analysis.
Susceptibility Testing in Urine Urine MICs of delafloxacin and ciprofloxacin were determined using the microdilution methodology according to CLSI.13 Trays were made of 80 ml patient specific urine and inocula were made in 20 ml broth. For each urine MIC study 4 to 7 unique patient urines served as a set. Broth MICs were repeated in each set to establish baseline MICs for comparison.
Data Analysis The MIC change between broth and urine for delafloxacin and ciprofloxacin was calculated as (log2[MIC in urine] e log2[MIC in broth]). Linear regression analysis was performed using SigmaPlotÔ, version 12 to characterize the relationship between pH and the MIC change of all E. coli and all K. pneumoniae.
RESULTS Study Population Across the 143 urines collected the measured pH ranged from 4.7 to 9.0 with 71% at pH 6.5 or less, 13% at pH 6.6 to 7.0 and 16% at pH 7.1 or greater
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(figs. 1 and 2). Of the pH values measured at our laboratory 77% were within 0.5 of the pH reported according to the urinalysis. Of these samples 15 and 24 were excluded from study due to insufficient volume and a positive antibacterial effect, respectively, while 4 were excluded due to other operational errors. Table 1 lists demographic data on the 100 patients in the urine MIC studies. Median age was 58 years (range 18 to greater than 89). Age greater than 89 years was aggregated into 1 category of 8 patients according to the Safe Harbor Method [x164.514(b)(2)] in Guidance on DeIdentification of Protected Health Information in Accordance with the HIPAA (Health Insurance Portability and Accountability Act) Privacy Rule. We tested the samples of 92 patients with a urine volume of 40 ml or greater for 9 E. coli and 7 K. pneumoniae. The samples of 8 patients with a urine volume of less than 40 ml were tested for 5 E. coli and 3 K. pneumoniae (table 2). Susceptibility Testing in Broth Table 2 lists the broth MICs of the 16 urogenic Enterobacteriaceae, including 9 E. coli and 7 K. pneumoniae. Ciprofloxacin MICs were 32 mg/l or greater against all 16 isolates but delafloxacin MICs were 2 to 16 mg/l for E. coli and 8 to 32 mg/l for K. pneumoniae. Procedure Development Studies Freeze-Thaw pH. Two of 10 patient samples showed a 0.2 change in pH. The remaining patient samples showed no greater than a 0.1 change in pH, suggesting that freeze-thaw had little effect on pH. Time pH. All measured pH values remained at 0.1
of that of the original sample pH whether at room temperature or refrigerated during 24 hours. Therefore, clinical samples were processed within 24 hours of collection and refrigerated (temperature 2C to 8C) until processed. Broth pH. Across 12 patient urines tested the
changes in pH of the urine-to-broth combination was 0.2 or less at a ratio of 9:1, 0.3 or less at 8:2, 0.5 or less at 7:3 and 0.8 or less at 5:5. Therefore, only the 9:1 and 8:2 combinations were further evaluated in growth studies to minimize pH changes from the original samples. Growth Study of Urine and Broth Combination. Based
on the broth pH study as noted, growth experiments were performed in 90% and 80% urine as well as in 50% urine for comparison. In 50% urine all 8 Enterobacteriaceae tested grew in all 3 urines. However, in 90% and 80% urine the positive growth rate decreased to 66% and 79%, respectively. Based on the results of broth pH and growth studies urine MIC studies were performed in 80% urine.
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EFFECTS OF URINE MATRIX AND pH ON POTENCY OF DELAFLOXACIN AND CIPROFLOXACIN
Figure 1. Influence of urine matrix on delafloxacin (A) and ciprofloxacin (B) MICs for E. coli. MIC change was e8.67 þ 1.39 pH (r2 ¼ 0.554) (A) and 2.56 0.32 pH (r2 ¼ 0.085) (B). Bars represent frequency of pH in urine of 143 patients. Numbers indicate observations of 9 E. coli tested in 100 patient urines. Diagonal line indicates linear regression.
Effect on MIC of 0.5 pH Change. Across the 4
Enterobacteriaceae tested 87.5% of samples showed no shift in MICs over the change in pH by 0.5. The remaining 12.5% of urines showed 1 doubling dilution shift. Therefore, during MIC testing in 80% urine pH was not readjusted back to the original 100% urine pH. Susceptibility Testing in Urine Table 3 lists the frequency of observed delafloxacin urine MICs measured in 100 unique patient samples at pH 5.0 to 8.3. Figures 1 and 2 show MIC
shifts from broth to urine for E. coli and K. pneumoniae with delafloxacin and ciprofloxacin. Figures 1 and 2 also show the results of linear regression analysis. The magnitude of the MIC change for delafloxacin was similar for E. coli and K. pneumoniae with a median decrease of 1 doubling dilution at pH 6.0 or less, no change at pH 6.1 to 7.0 and an increase of 2 (E. coli) and 1 (K. pneumoniae) doubling dilution at pH 7.1 or greater. On the other hand, ciprofloxacin MICs remained relatively unchanged at 47% for E. coli and 82% for K. pneumoniae across
EFFECTS OF URINE MATRIX AND pH ON POTENCY OF DELAFLOXACIN AND CIPROFLOXACIN
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Figure 2. Influence of urine matrix on delafloxacin (A) and ciprofloxacin (B) MICs for K. pneumoniae. MIC change was e7.56 þ 1.18 pH (r2 ¼ 0.495) (A) and 1.91 0.27 pH (r2 ¼ 0.066) (B). Bars represent frequency of pH in urine of 143 patients. Numbers indicate observations of 9 K. pneumoniae tested in 100 patient urines. Diagonal line indicates linear regression.
the pH ranges. There was a median MIC change of 1 doubling dilution increase for E. coli, no change for K. pneumoniae at pH 7.0 or less and no change at pH 7.1 or greater.
DISCUSSION The novel fluoroquinolone delafloxacin is distinguished from other fluoroquinolones by its improved activity against certain increasing populations of fluoroquinolone resistant gram-negative organisms such as E. coli and K. pneumoniae.14e16 In addition, further enhancement was noted of delafloxacin potency in an acidic environment.10e12 In this
study we observed a change in delafloxacin activity between broth and patient specific urines of varying pH against highly ciprofloxacin resistant urogenic Enterobacteriaceae. In contrast to ciprofloxacin, which had poor activity against these pathogens with relatively unchanged MICs, delafloxacin showed enhanced activity in the acidic environment commonly observed in our patients with suspected UTI. The normal range of urinary pH was previously reported to be as wide as 4.5 to 8.6.17,18 Likewise our 143 consecutively collected urine samples from patients with signs of infection showed a wide range of 4.7 to 9.0. Although we did not explicitly explore
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EFFECTS OF URINE MATRIX AND pH ON POTENCY OF DELAFLOXACIN AND CIPROFLOXACIN
Table 1. Characteristics of 100 patients No. Pts No. female No. male No. location: Intensive care unit inpt Non intensive care unit inpt Emergency department Urinalysis criteria for culture: Pos nitrite 5 or Greater white blood cells on microscopic examination 100 or Greater (small or greater) leukocyte esterase
83 17 9 57 34 26 90 88
patient characteristics and their effect on urinary pH, data in the literature suggest that urinary pH can be affected by multiple factors.17,18 For example, the presence of urea-splitting organisms such as Proteus mirabilis and some strains of Klebsiella species, Pseudomonas species, Providencia species and Staphylococcus species are associated with increased urinary pH. Alkaline urine is also observed in systemic alkalosis and renal tubular acidosis, and with the use of carbonic anhydrase inhibitors.17 On the other hand, the presence of uric acid, and the administration of cranberry juice, ascorbic acid and ammonium chloride reportedly decrease urinary pH.19,20 In addition, groups suggested that there is lower urinary pH with bacterial infection (pH 3.5 to 6.0) compared to healthy urine (pH 6.5 to 7.5).21,22 Similarly despite the multifactorial nature of urinary pH and the broad range of pH in our 143 clinical samples with signs of infection, 71% had pH no greater than 6.5. As expected from previous reports,5,6 delafloxacin broth MICs against these 16 highly ciprofloxacin resistant Enterobacteriaceae were 2 to 5 doubling dilutions lower than the ciprofloxacin broth MICs. Nilius et al reported delafloxacin MICs of 4 to 32 mg/l against 10 E. coli (ciprofloxacin MIC 32 to
greater than 128 mg/l) and 2 to 8 mg/l against 10 K. pneumoniae (ciprofloxacin MIC 64 to greater than 128 mg/l).5 Similarly Almer et al reported delafloxacin MICs of 1 to 16 mg/l against 27 ciprofloxacin resistant E. coli (MIC 4 to greater than 128 mg/l) and 1 to 4 mg/l against 22 ciprofloxacin resistant K. pneumoniae (MIC 4 to 64 mg/l).6 From 100 patient urines (pH 5.0 to 8.3) in which urine MIC testing was performed delafloxacin MICs decreased by 1 to 3 doubling dilutions in 53% of observations for E. coli and 68% for K. pneumoniae with a median of 1 doubling dilution decrease at pH 6.0 or less. While 48% of the urinary pH distribution was for pH 6.0 or less, after excluding samples with insufficient volume or antibacterial effect only 37% of our total observations were made at pH 6.0 or less (table 3). Similar reductions in frequency were seen between the urinary pH distribution and the number of observations at pH 6.5 or less (ie 71% vs 59%). As such the median decrease in MIC could have been greater if we had had more urine samples with increased acidity in our observations. Enhanced activity of a fluoroquinolone in an acidic environment was also observed for finafloxacin, another investigational fluoroquinolone.21,23 Finafloxacin showed up to a 5 doubling dilution decrease of MICs at pH 5.8 compared to pH 7.2 against Acinetobacter baumannii isolates while ciprofloxacin MICs increased up to greater than 4 doubling dilutions at pH 5.8.21 Wagenlehner et al determined MICs of finafloxacin and ciprofloxacin in CA-MHB at a pH of 5.8, 7.2 and 8.0 as well as in synthetic urine adjusted to pH 5.8.23 Similar to our observations, compared to the MICs in CA-MHB at pH 7.2 the finafloxacin MICs were 1 to 2 doubling dilutions higher at pH 8.0 and 2 to 3 doubling dilutions lower at pH 5.8 against E. coli. Interestingly at pH 5.8 finafloxacin MICs in synthetic urine were higher than in
Table 2. Broth MICs of 16 Enterobacteriaceae Isolate No. E. coli: 443* 348 429* 435* 310 350* 432* 130 133 K. pneumoniae: 368* 331A 350B* 369* 378 320 390
Delafloxacin (mg/l)
Ciprofloxacin (mg/l)
2 4 4 4 8 8 8 16 16
64 Greater than 64 64 64 Greater than 64 Greater than 64 64 64 64
8 16 16 16 16 32 32
Greater Greater Greater Greater Greater Greater
* Isolates used when urine volume was less than 40 ml.
32 than than than than than than
64 64 64 64 64 64
Known Resistance Mechanism Extended spectrum Not determined Extended spectrum Not determined Extended spectrum Not determined Extended spectrum Extended spectrum Extended spectrum
b-lactamases b-lactamases b-lactamases b-lactamases b-lactamases b-lactamases
K. pneumoniae carbapenemases K. pneumoniae carbapenemases K. pneumoniae carbapenemases K. pneumoniae carbapenemases K. pneumoniae carbapenemases Extended spectrum b-lactamases K. pneumoniae carbapenemases
EFFECTS OF URINE MATRIX AND pH ON POTENCY OF DELAFLOXACIN AND CIPROFLOXACIN
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Table 3. Urine MIC in 80% urine from 100 unique patient samples and 20% broth % E. coli MIC (pH) 5.0e6.0 No. observations* Delafloxacin MIC (mg/l): 2 or Less 4 8 16 32 or Greater Ciprofloxacin MIC (mg/l): 16 32 or Greater
253 35.2 24.5 20.2 17.4 2.8 0 100
5.0e6.5 414 28.7 28.5 20.5 18.4 3.9 0 100
5.0e7.0 570 21.6 27.5 22.6 20.2 8.1 0 100
% K. pneumoniae MIC (pH) 5.0e8.3 695 17.8 23.2 21.4 22.7 14.8 0.7 99.3
5.0e6.0 216 2.3 13.4 34.3 38.0 12.0 0 100
5.0e6.5 342 1.5 9.4 26.6 40.1 22.5 0 100
5.0e7.0 484 1.0 6.8 21.1 36.6 34.5 0 100
5.0e8.3 589 0.8 5.6 17.8 33.4 42.3 0.8 99.2
* For example, number at pH 6.1 to 6.5 for E. coli was 161 (414 minus 253).
CA-MHB, in contrast to our observations using delafloxacin in patient urine. The mechanism of the enhanced activity of delafloxacin or finafloxacin in an acidic environment, as distinguished from other zwitterionic fluoroquinolones such as moxifloxacin, levofloxacin and ciprofloxacin, is partly explained by the different physiochemical properties of the agents and the resultant change in lipophilicity and transmembrane diffusion depending on the surrounding pH.10,24,25 In addition, a possible influence of pH on bacterial gene expression was suggested, leading to modifications in influx or efflux transporters.21,26 On the other hand, consistent with previous reports of zwitterionic fluoroquinolones,10,20 ciprofloxacin MICs showed a 1 doubling dilution increase for E. coli and no change for K. pneumoniae under an acidic pH. Our study uniquely reveals the effect of the host related factor (ie urinary pH) on ciprofloxacin MIC, which to our knowledge was reported only once previously by Zhanel et al.27 In that study MICs of ciprofloxacin in urine at pH 5.5 were compared to MICs in MHB at pH 7.3. They observed 6 doubling dilution increases in the ciprofloxacin urine MIC at pH 5.5 compared to MHB at pH 7.3. While the magnitude of the ciprofloxacin MIC increase in that series (6 doubling dilutions) was greater than in ours (1 doubling dilution), all 3 isolates in the study by Zhanel et al were highly susceptible to ciprofloxacin with a broth MIC of 0.016 mg/l. In addition, ciprofloxacin MICs greater than 64 mg/l were not further defined in our series, which may have limited our ability to identify a correlation between a MIC greater than 64 mg/l
and pH. Also of note is that while methodologically we could not perform these studies in pure urine, we anticipate similar findings for these pH dependent observations.
CONCLUSIONS Delafloxacin broth MICs were twofold to fivefold doubling dilutions lower than those of ciprofloxacin against these 16 urogenic Enterobacteriaceae. While urinary pH can fluctuate depending on multiple factors, this study provides the approximate distribution of urinary pH in patients with suspected UTI, most highlighting the distribution in those with acidic urine (pH 6.5 or less). Although ciprofloxacin MICs remained relatively unchanged or increased across the distribution, delafloxacin showed enhanced activity against E. coli and K. pneumoniae in the acidic urinary environment (pH 5.0 to 6.0). Moreover, this improved potency resulted in MIC values that were predominantly 8 mg/l or less in the biological urine matrix. Future studies are required to fully understand the clinical implications of delafloxacin enhanced potency at the infection site.
ACKNOWLEDGMENTS Mary Anne Banevicius, Henry Christensen, Jennifer Hull, Lucinda Lamb, Debora Santini, Christina Sutherland, Sara Robinson and Kim Greenwood assisted with the study. David O’Sullivan, Hartford Hospital, assisted with statistics. Delafloxacin meglumine was provided by Melinta Therapeutics.
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