Plasmid-mediated complementation of gyrA and gyrB in fluoroquinolone-resistant Bacteroides fragilis.

OBJECTIVES: To identify whether mutations in gyrA and gyrB confer fluoroquinolone resistance in Bacteroides fragilis. METHODS: Eight fluoroquinolone-resistant (FQR) strains were complemented with plasmid-mediated B. fragilis wild-type gyrA (pMP1) and gyrB (pMP2), and MICs determined. Sequence analysis of the gyrA and gyrB quinolone resistance determining region (QRDR) was performed for all strains. RESULTS: MICs of fluoroquinolones were two- to 32-fold higher than wild-type for all mutants. Five mutants had a substitution in GyrA (Ser-82-->Phe), one mutant had a substitution in GyrA (Asp-81-->Gly), one mutant had a substitution in GyrB (Glu-478-->Lys), and one resistant strain did not contain mutations in the QRDR of gyrA or gyrB. Following complementation with pMP1 or pMP2, the MICs of fluoroquinolones were reduced two- to 32-fold for the mutants. CONCLUSION: These studies verify that substitutions in GyrA and GyrB confer resistance in B. fragilis. Other mechanisms are also responsible for resistance since not all resistant strains fully complemented to the wild-type phenotype.

Pharmacodynamics of trovafloxacin and levofloxacin against Bacteroides fragilis in an in vitro pharmacodynamic model.

An in vitro pharmacodynamic investigation was conducted to explore whether the area under the concentration time curve from 0 to 24 h (AUC(0-24))/MIC ratio could predict fluoroquinolone performance against Bacteroides fragilis. An in vitro model was used to generate kill curves for trovafloxacin (TVA) and levofloxacin (LVX) at AUC(0-24)/MIC ratios of 1 to 406 against three strains of B. fragilis (ATCC 25285, ATCC 23745, and clinical isolate M97-117). TVA and LVX were bolused prior to the start of experiments to achieve the corresponding AUC(0-24)/MIC ratio. Experiments were performed in duplicate over 24 h and in an anaerobic environment. Analyses of antimicrobial performance were conducted by comparing the rates of bacterial kill (K) using nonlinear regression analysis with 95% confidence intervals. Statistical significance was defined as a lack of overlap in the 95% confidence limits generated from the slope of each kill curve. For both TVA and LVX, K was maximized once an AUC(0-24)/MIC ratio of > or =40 was achieved and was not further increased despite a 10-fold increase in AUC(0-24)/MIC from approximately 40 to 400 against all three strains of B. fragilis. No significant differences were found in K between AUC(0-24)/MIC ratios of approximately 40 to 200. In experiments where AUC(0-24)/MIC ratios that were > or = 5 and < or = 44 were conducted, 64% demonstrated regrowth at 24 h. Resistant strains were selected in 50% of those experiments, demonstrating regrowth, which resulted in increased MICs of two- to 16-fold for both TVA and LVX. Regrowth did not occur, nor were resistant strains selected in any studies with an AUC/MIC that was > 44. Our findings suggest that fluoroquinolones provide antibacterial effects against B. fragilis in a concentration-independent manner associated with an AUC(0-24)/MIC ratio of > or =40. Also, the potential for the selection of resistant strains of B. fragilis may increase with an AUC(0-24)/MIC ratio of < or =44.

What do we really know about antibiotic pharmacodynamics?

Antibiotic pharmacodynamics is an evolving science that focuses on the relationship between drug concentration and pharmacologic effect, which is an antibiotic-induced bacterial death that also can manifest as an adverse drug reaction. The pharmacologic action of antibiotics usually can be described as concentration dependent or independent, although such classifications are highly reliant on the specific antibiotic and bacterial pathogen being studied. Quantitative pharmacodynamic parameters, such as ratio of the area under the concentration-time curve during a 24-hour dosing period to minimum inhibitory concentration (AUC0-24:MIC), ratio of maximum serum antibiotic concentration to MIC (Cmax:MIC), and duration of time that antibiotic concentrations exceed MIC (T>MIC), have been proposed as likely predictors of clinical and microbiologic success or failure for different pairings of antibiotic and bacteria. Thus far, most pharmacodynamic data reported have focused on fluoroquinolones, but work has been conducted on vancomycin, beta-lactams, macrolides, aminoglycosides, and other antibiotics. Despite the development of a number of different pharmacodynamic modeling systems, remarkable agreement exists between in vitro, animal, and limited human data. Although still somewhat premature and requiring additional clinical validation, antibiotic pharmacodynamics will likely advance on four fronts: the science should prove to be extremely useful and represent a cost-effective and efficient method to help develop new antibiotics; formulary committees will likely use pharmacodynamic parameters to assist in differentiating antibiotics of the same chemical class in making antibiotic formulary selections; pharmacodynamic principles will likely be used to design optimal antibiotic strategies for patients with severe infections; and limited data to date suggest that the application of pharmacodynamic concepts may limit or prevent the development of antibiotic resistance. The study of antibiotic pharmacodynamics appears to hold great promise and will likely become a routine part of our daily clinical practices.

Comparison of once-daily versus twice-daily administration of cefdinir against typical bacterial respiratory tract pathogens.

In an in vitro pharmacodynamic model, a twice-daily cefdinir dosing regimen was more effective than a once-daily regimen against common bacterial respiratory pathogens in producing 3-log(10) killing and preventing the occurrence of regrowth at 24 h. Twice-daily administration is likely the more appropriate cefdinir dosing strategy for the treatment of community-acquired pneumonia.

Fluoroquinolone resistance in anaerobic bacteria following exposure to levofloxacin, trovafloxacin, and sparfloxacin in an in vitro pharmacodynamic model.

This investigation explored pharmacodynamic characteristics of fluoroquinolones against Bacteroides thetaiotamicron and the potential for development of resistance. An in vitro model was used to generate kill curves with three fluoroquinolones at various area under the concentration-time curve (AUC)/MIC ratios. Concentration-independent killing was observed. Increases in MICs were noted following exposure to fluoroquinolones at AUC/MIC ratios of 6 to 14.

Intensive care unit antimicrobial resistance and the role of the pharmacist.

Over the past 20 yrs, pharmacists have successfully integrated their services and expertise to gain acceptance as full members of pediatric, surgical, medical, and intensive care unit (ICU) patient care teams. The pharmacists' training in pharmacology, pharmacokinetics, pharmacodynamics, and pharmacoeconomics complements the expertise of other members of the patient care team. Generally, a strong background in infectious diseases and critical care also provides a focal point for clinical pharmacy service intervention. Although practitioners often focus on issues exclusively related to their specific hospital or ICU, the issues surrounding antibiotic resistance are more global and societal in nature. Medical, surgical, and pharmaceutical practices inside the hospital and ICU extend their influence into the community. Customs and practices of daily living in our society coupled with use of agents capable of altering microbial flora impact our hospital and ICU when patients from the community are admitted. The misuse of antibiotics and the lack of effective infection control programs are often identified as key components in the perpetuation of these phenomena. The focus for the pharmacist and the ICU team must be on the optimization of antibiotic use and infection control guidelines. This review will address the many issues that surround the appropriate use of antibiotics and what role the pharmacist can play in ensuring the optimal use of infection control measures in the ICU and hospital.

In vitro pharmacodynamic analysis of single daily dosing versus conventional dosing of gentamicin administered with penicillin against Enterococcus faecalis.

STUDY OBJECTIVE: To compare the effectiveness of single daily dosing (SDD) versus conventional dosing of gentamicin when administered with penicillin to treat enterococcal infections. DESIGN: In vitro pharmacodynamic model. SETTING: Hospital laboratory. MEASUREMENTS AND MAIN RESULTS: A 24-hour in vitro pharmacodynamic model was employed to simulate SDD and 3 times/day dosing of gentamicin, in conjunction with continuously infused penicillin, against Enterococcus faecalis. Duplicate 24-hour kill curves were generated with varying concentrations of penicillin and gentamicin alone and in combination. No difference in the rate of kill was seen between any combination of penicillin and gentamicin. Regrowth occurred only with drug combinations in which penicillin was administered continuously at the minimum inhibitory concentration. Variations in the gentamicin dosing regimen did not affect regrowth. CONCLUSION: In the treatment of enterococcal infections, an SDD regimen for gentamicin shows no efficacy benefit compared with conventional dosing.

Application of fluoroquinolone pharmacodynamics.

Pharmacodynamics provides a rational basis for optimizing dosing regimens by describing the relationship between drug, host and antimicrobial effect. The successful identification of meaningful pharmacodynamic outcome parameters can, therefore, greatly assist clinicians in making objective prescribing decisions rather than relying on static in vitro MIC data. While pharmacodynamic outcome parameters have been proposed for select antimicrobial agents, their clinical application remains to be defined fully. Quinolone antibiotics are generally considered to have concentration-dependent bactericidal activity and peak/MIC and AUC/MIC ratios have been identified as possible pharmacodynamic predictors of clinical and microbiological outcome as well as the development of bacterial resistance. Investigators have suggested that AUC/MIC ratios of 100-125 or peak/MIC ratios of >10 are required to predict clinical and microbiological success and to limit the development of bacterial resistance. These conclusions are derived primarily from studies of Gram-negative bacteria, and recent data suggest that these ratios may not be applicable for Streptococcus pneumoniae, where an AUC/MIC ratio of <40 appears to be a more accurate predictor. There is considerable variation in pharmacodynamic calculations and outcome parameters appear to be quinolone- and pathogen-specific. Additional prospective clinical research is needed to characterize quinolone pharmacodynamic parameters and answer unresolved questions regarding optimal pharmacodynamic outcome predictors for Gram-positive bacteria, anaerobes and atypical respiratory pathogens.

Decreased in vitro fluoroquinolone concentrations after admixture with an enteral feeding formulation.

BACKGROUND: The purpose of this study was to determine if mixing of fluoroquinolones with a common enteral feeding formulation, Ensure (Ross Products Division, Abbott Laboratories, Columbus, OH), would alter the measured in vitro quinolone concentrations over a 24-hour period. METHODS: Tablets of ciprofloxacin (500 mg), levofloxacin (500 mg), and ofloxacin (300 mg) were crushed and mixed with 240 mL of Ensure, water and calcium chloride (500 mg/L), water and magnesium chloride (200 mg/L), water and calcium chloride and magnesium chloride, and water alone. Fluoroquinolone concentrations of the mixtures were measured, via high-performance liquid chromatography, at baseline and serially over 24 hours. Experiments were performed in duplicate, at three temperatures (5 degrees C, 25 degrees C, and 37 degrees C). RESULTS: Average decreases of 82.5% +/- 1.5% for ciprofloxacin, 61.3% +/- 5.2% for levofloxacin, and 45.7% +/- 10.1% for ofloxacin (mean +/- 95% CI) were observed in vitro for Ensure over the two experimental sets at baseline. Serial analysis revealed no further significant change in any of the quinolone concentrations over the remaining 24-hour period. No significant decrease was noted with the quinolones when mixed in water and calcium, water and magnesium, water and calcium and magnesium, or water alone. This phenomenon appears to be unaffected by time and temperature. CONCLUSIONS: These data suggest there is an immediate and significant loss of fluoroquinolone when mixed with Ensure. An explanation for the loss of fluoroquinolone remains unclear.

Fluoroquinolone resistance in Bacteroides fragilis following sparfloxacin exposure.

In vitro pharmacodynamic studies investigating the antimicrobial properties of five fluoroquinolones, (trovafloxacin, sparfloxacin, clinafloxacin, levofloxacin, and ciprofloxacin) against Bacteroides fragilis ATCC 23745 were conducted. The times required to reduce the viable counts by 3 log units were as follows: clinafloxacin, 2.9 h; levofloxacin, 4.6 h; trovafloxacin, 6 h; and sparfloxacin, 10 h. Exposure to ciprofloxacin did not achieve a 3-log decrease in viable counts. The susceptibility of B. fragilis was determined both prior to exposure and following 24 h of exposure to each of the five fluoroquinolones tested. The MICs of clinafloxacin, levofloxacin, trovafloxacin, sparfloxacin, ciprofloxacin, metronidazole, cefoxitin, chloramphenicol, and clindamycin were determined by the broth microdilution method. The MICs for B. fragilis preexposure were as follows: clinafloxacin, 0.25 microg/ml; trovafloxacin, 0.5 microg/ml; sparfloxacin, 2 microg /ml; levofloxacin, 2 microg/ml; and ciprofloxacin, 8 microg/ml. Similar pre- and postexposure MICs were obtained for cultures exposed to trovafloxacin, clinafloxacin, levofloxacin, and ciprofloxacin. However, following 24 h of exposure to sparfloxacin, a fluoroquinolone-resistant strain emerged. The MICs for this strain were as follows: clinafloxacin, 1 microg/ml; trovafloxacin, 4 microg/ml; sparfloxacin, 16 microg/ml; levofloxacin, 16 microg/ml; and ciprofloxacin, 32 microg/ml. No changes in the susceptibility of B. fragilis pre- and postexposure to sparfloxacin were noted for metronidazole (MIC, 1 microg/ml), cefoxitin (MIC, 4 microg /ml), chloramphenicol (MIC, 4 microg/ml), and clindamycin (MIC, 0.06 microg/ml). Resistance remained stable as the organism was passaged on antibiotic-free agar for 10 consecutive days. Mutant B. fragilis strains with decreased susceptibility to clinafloxacin, trovafloxacin, sparfloxacin, levofloxacin, and ciprofloxacin were selected on brucella blood agar containing 8x the MIC of levofloxacin at a frequencies of 6.4 x 10(-9), 4x the MICs of trovafloxacin and sparfloxacin at frequencies of 2.2 x 10(-9) and 3. 3 x 10(-10), respectively, and 2x the MIC of clinafloxacin at a frequency of 5.5 x 10(-11); no mutants were selected with ciprofloxacin. The susceptibilities of strains to trovafloxacin, levofloxacin, clinafloxacin, sparfloxacin, and ciprofloxacin before and after exposure to sparfloxacin were modestly affected by the presence of reserpine (20 microg/ml), an inhibitor of antibiotic efflux. The mechanism of fluoroquinolone resistance is being explored, but it is unlikely to be efflux due to a lack of cross-resistance to unrelated antimicrobial agents and to the fact that the MICs for strains before and after exposure to sparfloxacin are minimally affected by reserpine.

Bacillus anthracis: medical issues of biologic warfare.

Recent world events refocused attention on the possibility of nations engaging in biologic warfare, including an attack with Bacillus anthracis. The single available anthrax vaccine in the United States for human use, formerly known as MDPH-PA, has decreased ability to protect laboratory animals against virulent B. anthracis strains, especially compared with new vaccines being developed. Studies with these vaccines, however, have several shortcomings. The pathogenesis, diagnosis, treatment, and prophylaxis of anthrax are discussed, as well as the implications that an attack with B. anthracis would place on the health care system.

Implications of vancomycin degradation products on therapeutic drug monitoring in patients with end-stage renal disease.

In renally impaired patients, vancomycin concentrations typically are maintained at body temperature for extended periods of time due to the drug's prolonged half-life. Both time and increased temperature potentiate production of vancomycin crystalline degradation products (CDP-1). Commercially available vancomycin assays, such as fluorescence polarization immunoassay (FPI) and radioimmunoassay, cross-react with CDP-1 isomers. Overestimation of vancomycin concentrations by 40-53% due to cross-reactivity of CDP-1 with active factor B vancomycin occurs with FPI. As FPI is the most common method of analyzing serum vancomycin, clinicians must be aware of its potential shortcomings and be prepared to alter vancomycin dosages in renally impaired patients. The possibility of adverse affects due to elevated concentrations of CDP-1 or therapeutic failures due to subtherapeutic levels of factor B vancomycin cannot be excluded.

Gram-positive infections: pharmacy issues and strategy for quinupristin/dalfopristin.

The development of the first streptogramin antibiotic, quinupristin/dalfopristin represents an attempt to bring new antimicrobial strategies on line to combat the menacing problem of Gram-positive-resistant bacteria. With introduction to the medical center formulary, the pharmacy will need to be aware of several practical issues surrounding the use of quinupristin/dalfopristin. Cost and unit size will be important issues. Initially, this drug will only be available in 500-mg vials which may not always accommodate the suggested dose of 7.5 mg/kg of actual body weight. In addition, the drug can only be reconstituted with D5W or sterile water, and it can not be mixed with normal saline, heparin, or other drugs. Institutions adding this drug to their formularies must address the expected logistical concerns with their medical, nursing, and pharmacy staffs prior to patient usage.

Cyclospora: review of an emerging parasite.

Cyclospora is a parasite traditionally associated with diarrhea in travelers to endemic countries. Recently, several cases of cyclosporiasis were reported in nontravelers in the United States and Canada, implicating various fruits and vegetables as vehicles of infection. The life cycle of cyclospora is not fully known, but is believed to involve both asexual and sexual stages of proliferation. Food- and water-borne transmission of infection have been implicated. Patients infected with Cyclospora cayetanensis have protracted watery diarrhea. Various generalized symptoms are also present, making cyclosporiasis indistinguishable from infectious diarrhea caused by other microorganisms. Diagnosis depends on identifying the organism by microbiologic examination of stool samples. Treatment consists of supportive care, maintenance of fluid and electrolyte status, symptomatic relief, and antibiotic therapy. Trimethoprim-sulfamethoxazole is the only antibiotic available that is effective in eradicating the organism.

Determination of quinolone antibiotics in growth media by reversed-phase high-performance liquid chromatography.

A simple, accurate, precise, and versatile high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of three quinolone antibodies in Mueller-Hinton broth. The fluoroquinolone agents studied were ciprofloxacin, ofloxacin, and sparfloxacin; other quinolone agents have been identified using this method but not validated in this matrix (levofloxacin, clinafloxacin, temafloxacin, and trovafloxacin). In addition, several other biological growth mediums have been investigated (human serum, human urine, Todd-Hewitt growth media, Ensure enteral feeding solution, and Haemophilus growth media). This method uses UV detection (280 nm), a simple, one-step protein precipitation extraction, and separation using a C18 column with an isocratic, ion-pairing mobile phase. An appropriate internal standard was obtained by using another quinolone antibiotic of differing retention time. The calibration curves were linear (r2> or =0.999) over a concentration range of 0.0625-20.0 microg/ml with a lower limit of quantification of 0.1 microg/ml. The intra-day and inter-day coefficients of variation were less than 15%.

Current management of human bites.

Human bites should be considered dangerous injuries with potentially serious complications. Their characteristics vary from an uninfected abrasion to a serious infection such as cellulitis or osteomyelitis. An estimated 10% of the injuries become infected; suspected pathogens include oral and skin flora. Management consists of history and examination, wound care, surgical intervention if necessary, assessment of risk of disease transmission, and appropriate antibiotic prophylaxis or treatment. The best choice for oral or intravenous antibiotic therapy remains the combination of a beta-lactam antibiotic with a beta-lactamase inhibitor. Among the most serious human bites are clenched fist injuries, which often require surgical intervention and intravenous antibiotic therapy.

Wide variation in single, daily-dose aminoglycoside pharmacokinetics in patients with burn injuries.

Five to seven mg/kg single, daily-dose aminoglycoside regimens have been recently advocated as effective alternatives to traditional aminoglycoside regimens. The rationale for single, daily-dose aminoglycoside therapy is to produce an optimal ratio between aminoglycoside peak concentrations (Cmax) and pathogen minimal inhibitory concentration to maximize bacterial killing and to produce an aminoglycoside-free period during the 24-hour dosing interval. Single, daily-dose aminoglycoside therapy has not been recommended to date for use in the population of patients with burn injuries. The purpose of this study was to determine the magnitude and variability of aminoglycoside Cmax and the duration of the aminoglycoside-free period after simulated single, daily-dose regimens in patients with burn injuries. Fifty-two patients receiving gentamicin or tobramycin in the burn unit were studied retrospectively to determine the individualized pharmacokinetic parameters and the simulated Cmax and 24-hour after the dose trough minimum concentrations for 5 and 7 mg/kg single, daily-dose aminoglycoside regimens. Patients were only included in the final analysis if they had been treated for burn wound infections and exhibited a calculated creatinine clearance exceeding 60 ml/min (N = 40). Mean [percentage coefficient of variation] Cmax/minimum concentrations were 15.4[30.5]/0.03[200.0] and 21.6[30.6]/0.04[200.0] mg/L for 5 and 7 mg/kg daily doses, respectively. The mean coefficient of variation time to reach an extrapolated concentration of 0.1 mg/L was 15.9[30.8] hours and 17.0[30.6] hours for the 5 and 7 mg/kg daily doses, respectively. Substantial variability in aminoglycoside Cmax and duration of the aminoglycoside-free period was observed. These data suggest that many patients with burn injuries are not candidates for single, daily-dose aminoglycoside therapy because of restrictive creatinine clearance criteria and pronounced variability in length of the aminoglycoside-free interval. If single, daily-dose aminoglycoside therapy is to be used in this patient population, therapeutic drug monitoring is recommended to screen for appropriate candidates and to optimize Cmax and minimal inhibitory concentration ratios and duration of the aminoglycoside-free interval.