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Ceftaroline is a new broad-spectrum cephalosporin being developed for the treatment of serious bacterial infections, including those caused by aerobic Gram-positive and Gram-negative bacteria. The purpose of the present study was to investigate the effect of administration of ceftaroline on the intestinal flora of healthy subjects. Twelve healthy subjects (6 males and 6 females), 20 to 41 years of age, received ceftaroline (600 mg) by intravenous infusion every 12 h (q12h) for 7 days. Plasma and feces were collected for determination of ceftaroline concentration and analysis of fecal flora. Fecal specimens were cultured on nonselective and selective media. Different colony types were counted, isolated in pure culture, and identified to the genus level. All new strains of colonizing bacteria were tested for susceptibility to ceftaroline. The concentrations of ceftaroline in plasma were as follows: on day 2, 17.5 to 34.8 mg/liter; on day 5, 19.7 to 33.2 mg/liter; and on day 7, 18.0 to 29.8 mg/liter. No ceftaroline concentrations were found on day −1, 9, 14, or 21. No measurable concentrations in feces were found on day −1, 2, 5, 7, 9, 14, or 21. There was a minor impact on the numbers of Escherichia coli strains, while the numbers of enterococci and Candida albicans strains were not affected. There were moderate decreases in the numbers of bifidobacteria and lactobacilli during the first 7 days, while the numbers of clostridia increased during the same period. No impact on the numbers of Bacteroides bacteria was noticed. No new colonizing aerobic or anaerobic bacteria resistant to ceftaroline (MIC ≥ 4 mg/liter) were found. Ceftaroline had no significant ecological impact on the human intestinal microflora.
Administration of antimicrobial agents, including those that are administered parenterally and excreted in the bile, can cause several adverse effects on the intestinal microflora. Emergence of resistance among bacteria in the normal flora and distribution of resistant genes by transfer of DNA in the microbial community can contribute to an increased load of resistant, potentially pathogenic microorganisms (8). The extent of disturbances is influenced by an antimicrobial agent's spectrum, dose, route of administration, pharmacokinetic and pharmacodynamic properties, and in vivo inactivation (8). The disturbances may result in clinical conditions that can be serious, such as systemic infections in immunocompromised patients and antibiotic-associated diarrhea (9).
Ceftaroline is a new semisynthetic parenteral cephalosporin with broad-spectrum activity (11). In contrast to other cephalosporins, ceftaroline retains activity against methicillin-resistant Staphylococcus aureus (MRSA) and penicillin-resistant pneumococci. Furthermore, ceftaroline maintains good activity against Gram-negative pathogens (3). Ceftaroline has been evaluated for healthy subjects, patients with mild or moderate renal impairment, and patients with complicated skin and skin structure infections and bacterial pneumonia (10, 11). The clinical responses have been favorable, and ceftaroline has been well tolerated. The purpose of the present study was to investigate the effect of ceftaroline on the human intestinal microflora during and after 7 days of administration.
Twelve healthy subjects (6 males and 6 females), 20 to 41 years of age, were included in the study. They were recruited through information about the study on the Clinical Pharmacology Trial Unit website on the Internet. Physical examination was carried out on each subject before the start of the study. The examination included measurements of blood pressure and heart rate, electrocardiogram (ECG) tests, clinical laboratory safety tests, and an interview on medical and surgical history. The female subjects were tested for pregnancy. The inclusion criteria were as follows: age of between 18 and 45 years, normal findings in the medical history and physical examination, body mass index (BMI) within the range of 18 to 30 kg/m2, and nonsmoker status. The exclusion criteria were as follows: regular use of medication, except contraceptive tablets; treatment with antimicrobial agents within 3 months preceding the study; participation in a trial with another investigational drug within 1 month preceding the study; present or residual gastrointestinal, liver, or kidney disease or other conditions known to interfere with the absorption, distribution, metabolism, or excretion of drugs; known history of Clostridium difficile infection; history of hypersensitivity to beta-lactam antibiotics; pregnancy or breast feeding; estimated creatinine clearance of less than 80 ml/min; positive screen for hepatitis B or C or HIV; history of alcohol abuse or drug abuse; and donation of blood or blood products within 1 month preceding the study. Before being admitted to the study, the subject gave consent to participate after the nature, scope, and possible consequences of the study had been explained in a form understandable to him or her. The subject was also given written information and a copy of the signed consent form.
The study protocol submitted to the Ethics Committee of Karolinska Institute, Stockholm, Sweden, and to the Medical Products Agency, Uppsala, Sweden, was approved before the trial was started.
The subjects each received ceftaroline (600 mg) by intravenous (i.v.) infusion over 60 (±10) min every 12 h (q12h) for 6 days and once on study day 7, 12 h after the second infusion given on study day 6, for a total of 13 doses.
Plasma samples were obtained for bioassay analyses of ceftaroline on study day −1, at 60 (±5) min after the start (within 5 min before the end) of the first ceftaroline infusions on study days 2 and 5, on study day 7 at 60 (±5) min after the start (within 5 min before the end) of the ceftaroline infusion, and on study days 9, 14, and 21.
Samples were obtained for bioassay analyses of ceftaroline from fecal specimens passed on study days −1, 2, 5, 7, 9, 14, and 21 unless no specimen was passed on a given day, in which instance the first subsequent specimen passed after that day and before the next collection time point was collected. If more than 1 specimen was produced on a specified day, only the first specimen of that day was collected.
The plasma and fecal samples for the analyses were collected in sterile tubes and containers and frozen at −70°C until processed.
The concentrations of ceftaroline in plasma were determined by suspension in antibiotic medium 1 (Difco, Sparks, MD), with Micrococcus luteus ATCC 9341 used as the test organism, based on a lower limit of quantification of 0.25 mg/liter in plasma. The plates were incubated for 18 h at 37°C. Best-fit standard curves were obtained by linear regression analysis. Intra-assay and interassay precision levels were determined, and results were considered to be acceptable when both intra- and interassay differences were less than 10%.
Ceftaroline concentrations in feces were determined by suspension in antibiotic medium 1 (Difco, Sparks, MD), with M. luteus ATCC 9341 used as the indicator strain. Two validation reports were provided by the investigator, providing information on linearity ranges, lower limit of detection, and intra-assay and interassay coefficients of variation.
Standards were prepared in pooled plasma for blood samples and in feces for fecal samples.
The samples were obtained for culture and ceftaroline susceptibility tests from fecal specimens passed on study days −1, 2, 5, 7, 9, 14, and 21, unless no specimen was passed on a given day, in which instance the first subsequent specimen passed was collected. If more than one specimen was produced on a specified day, only the first specimen of that day was collected. The samples were suspended in prereduced peptone yeast extract medium, diluted 10-fold, and inoculated on nonselective and selective agars as described by Nord et al. (6). The aerobic agar plates were incubated for 24 h at 37°C and the anaerobic plates for 48 h at 37°C in anaerobic jars (GasPak; BBL, Cockeysville, MD). After incubation, different colony types were counted, isolated in pure culture, and identified to the genus level. All isolates were analyzed according to Gram reaction and colony morphology, followed by biochemical tests (5). Anaerobic microorganisms were identified by gas chromatographic analysis of metabolites from glucose (5). The lower limit of detection for feces was 102 CFU/g. Clostridium difficile strains were typed using a multiplex real-time PCR-based assay (Xpert C. difficile assay; Cepheid, San Francisco, CA) (4).
The MICs for ceftaroline were determined for new colonizing strains from the fecal samples by the agar dilution method (1, 2). The final inoculum for aerobic bacteria was 104 CFU per spot, and that for anaerobic bacteria was 105 CFU per spot. The inoculated plates were incubated for 24 h (aerobic bacteria) and 48 h (anaerobic bacteria). The reference strains were Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Bacteroides fragilis ATCC 25285, and Clostridium difficile ATCC 700057. The strains were considered resistant according to the break-off points used in the CLSI recommendations. A provisional breakpoint for ceftaroline of 4 mg/liter was used. The MIC was defined as the lowest concentration of the drug that inhibited growth completely. MIC50/90 corresponded to the concentrations that inhibited the growth of 50% and 90% of the strains tested, respectively.
The following safety assessments were performed: physical examination on study days −1, 2, 5, 7, and 9, with a follow-up visit and an end-of-study visit; assessment of vital signs; ECG testing (days −1, 7, and 9); and adverse-event monitoring and monitoring of clinical laboratory parameters.
Statistics for the values estimated for the fecal specimens (as log numbers of microorganisms per gram feces and as concentrations of ceftaroline in feces) and statistics for the pharmacokinetic analysis were calculated by use of Wilcoxon's signed-rank test.
The concentrations in plasma are shown in Table Table1.1. The concentrations were as follows: on day 2, 17.5 to 34.8 mg/liter (mean value, 25.9 mg/liter); on day 5, 19.7 to 33.2 mg/liter (mean value, 24.5 mg/liter); and on day 7, 18.0 to 29.8 mg/liter (mean value, 23.8 mg/liter). No ceftaroline concentrations were found on day −1, 9, 14, or 21.
No measurable concentrations in feces were found on day −1, 2, 5, 7, 9, 14, or 21.
The effect of ceftaroline on the aerobic intestinal microflora is shown in Fig. Fig.1.1. The numbers of enterococci and Candida albicans strains were within the normal variation. Median E. coli counts decreased by approximately 2.0 log CFU/g of feces from study day −1 to study day 7 and by 1.5 log CFU/g feces from study day −1 to study day 9, with recovery to baseline counts on study day 14. This decrease was not significant (P > 0.05). The median values for other Enterobacteriaceae did not change significantly from study day −1 through study day 14. On study day 21, there were increased numbers of Klebsiella pneumoniae bacteria in 1 subject and of Citrobacter species (C. braaki, C. freundii, C. koseri, and C. youngae) bacteria in 5 subjects.
Figure Figure22 presents the effect of ceftaroline on the anaerobic microflora. From study day −1 to study day 7, there were moderate decreases of approximately 2.1 log CFU/g feces in numbers of bifidobacteria and of approximately 1.7 log CFU/g feces in numbers of lactobacilli. From study day −1 to study day 7, there was a moderate increase of approximately 2.0 log CFU/g feces in numbers of Clostridium species bacteria. No impact on the numbers of bacteria of Bacteroides spp. was noticed. C. difficile strains were isolated from two subjects on study days 5, 7, and 9. All isolates were toxin B positive by a cytotoxin assay and positive for the ToxA and ToxB genes. No strains were positive for the binary toxin gene. No isolates belonged to any known international PCR ribotype. No clinical symptoms were observed in these subjects. Therefore, these findings are likely to have no clinical relevance.
No new colonizing aerobic and anaerobic bacteria resistant to ceftaroline (MIC ≥ 4 mg/liter) were found. All B. fragilis group strains (n = 123) isolated during the observation period were beta-lactamase producers and resistant to ceftaroline (MIC ≥ 64 mg/liter). The MIC values for the six C. difficile strains were 4 to 8 mg/liter.
There were 5 subjects with 9 adverse events, all mild in severity. One event, nasopharyngitis (common cold), was deemed to be unrelated. Other adverse events were deemed to be possibly related to ceftaroline and included a rash (small and irregular on the forehead), nausea and abdominal pain (both in 1 subject), vomiting and nausea (both in 1 subject), tendon pain, and diarrhea plus headache (both in 1 subject). None of these subjects were positive for C. difficile growth, toxin A, toxin B, or binary toxin. No subjects had potentially clinically significant postbaseline vital-sign values. There were no subjects who had potentially clinically significant results for postbaseline hematology, chemistry, or urine analysis, based on both normal ranges and percent change from the baseline. There were no significant changes in ECG parameters from baseline to postbaseline levels in any subjects.
Ceftaroline has good in vitro activity against aerobic Gram-positive bacteria, including MRSA and multidrug-resistant Streptococcus pneumoniae (MDRSP) (3). The broad-spectrum activity of ceftaroline includes many Gram-negative bacterial species but not extended-spectrum-beta-lactamase-producing or AmpC-depressed enterobacteria or Pseudomonas aeruginosa. Among anaerobic bacteria, ceftaroline is active against Gram-positive species such as Propionibacterium spp., Peptostreptococcus spp., Clostridium perfringens, and Clostridium septicum but marginally active against C. difficile (MIC90, 4 mg/liter). B. fragilis and Prevotella species are resistant to ceftaroline (MIC90 > 32 mg/liter) (7).
Ceftaroline is a promising antibacterial agent for treatment of complicated skin and skin structure infections and community-acquired bacterial pneumonia (11). Ceftaroline is well tolerated, with good safety and tolerability, which was also observed in the present trial.
The administration of antibacterial agents can cause disturbances in the ecological balance between the host and the microorganisms. These changes are dependent on the spectrum of activity, the dose, the route of administration, the pharmacokinetic and pharmacodynamic properties, and the in vitro inactivation of the agent. Secretion of an agent by intestinal mucosa or bile may also have an impact on the intestinal microflora, causing antibiotic resistance. The impact of ceftaroline on the normal intestinal microflora has not been studied before. In the present investigation, no measurable concentration of ceftaroline in feces was detected, possibly due to the fact that ceftaroline and its metabolites are eliminated primarily through renal excretion. This may also explain the findings which noted only a minor effect on intestinal microflora, with no new strains of colonizing aerobic or anaerobic bacteria resistant to ceftaroline (MIC ≥ 4 mg/liter). Broad-spectrum antibiotics such as cephalosporins, fluoroquinolones, and amoxicillin are most often involved in causing C. difficile infection (CDI). The absence of concentration in feces and the minimal level of disruption of intestinal microflora observed in the present study suggest a low risk for development of CDI associated with the use of ceftaroline. On the basis of these findings, ceftaroline appears to have no significant impact on the human intestinal microflora.
The study was supported by a grant from Cerexa, Inc., a wholly owned subsidiary of Forest Laboratories, Inc.
Published ahead of print on 15 March 2010.