Application of pharmacokinetic/pharmacodynamic concepts to the development of treatment regimens for sporadic canine urinary tract infections: Challenges and paths forward.

Antimicrobial efficacy can be predicted based on infection site exposure to the antimicrobial agent relative to the in vitro susceptibility of the pathogen to that agent. When infections occur in soft tissues (e.g., muscle, blood, and ligaments), exposure at the infection site is generally assumed to reflect an equilibrium between the unbound concentrations in plasma and that in the interstitial fluids. In contrast, for sporadic urinary tract infections (UTIs) in dogs and uncomplicated UTIs in humans, the primary site of infection is the bladder wall. Infection develops when bacteria invade the host bladder urothelium (specifically, the umbrella cells that form the urine-contacting layer of the stratified uroepithelium) within which these bacteria can avoid exposure to host defenses and antimicrobial agents. Traditionally, pathogen susceptibility has been estimated using standardized in vitro tests that measure the minimal concentration that will inhibit pathogen growth (MIC). When using exposure-response relationships during drug development to explore dose optimization, these relationships can either be based upon an assessment of a correlation between clinical outcome, drug exposure at the infection site, and pathogen MIC, or upon benchmark exposure-response relationships (i.e., pharmacokinetic/pharmacodynamic indices) typically used for the various drug classes. When using the latter approach, it is essential that the unbound concentrations at the infection site be considered relative to the MIC within the biological matrix to which the pathogen will be exposed. For soft tissue infections, this typically is the unbound plasma concentrations versus MICs determined in standardized media such as cation-adjusted Mueller Hinton broth, which is how many indices were originally established. However, for UTIs, it is the unbound drug concentrations within the urine versus the MICs in the actual urine biophase that needs to be considered. The importance of these relationships and how they are influenced by drug resistance, resilience, and inoculum are discussed in this review using fluoroquinolones and beta-lactams as examples.

Approaches to developing judicious uses of veterinary antibacterial drugs.

Development of new veterinary antibacterials is an important and challenging endeavor. Global recognition of antimicrobial resistance as a threat across human, animal, plant, food, and environmental sectors has increased the level of scrutiny on veterinary antibacterial use. Rigorous scientific evaluation of these products has and continues to be the underpinning of effectiveness evaluations and how hazards are identified, characterized, and ultimately used to make evidence-based and risk-based safety decisions. Some scientific factors commonly considered in the development of veterinary antibacterials include the pathogenesis and sequelae of the indicated disease, clinical and bacteriological improvement, dosage regimen (dose amount, route, duration, frequency), and antimicrobial-resistance qualitative risk assessments. Key discussion areas covered are how culture and susceptibility testing help determine if antibacterial effects are primarily responsible for clinical improvement and how pharmacokinetic and pharmacodynamic data can help predict success, aid in defining an adequate dosage regimen, and help minimize resistance emergence and spread.

Proposed Epidemiological Cutoff Values for Ceftriaxone, Cefepime, and Colistin in Salmonella.

We tested a diverse set of 500 isolates of nontyphoidal Salmonella enterica subsp. enterica from various animal, food, and human clinical sources for susceptibility to antimicrobials currently lacking epidemiological cutoff values (ECOFFs) set by the European Committee on Antimicrobial Susceptibility Testing. A consortium of five different laboratories each tested 100 isolates, using broth microdilution panels containing twofold dilutions of ceftriaxone, cefepime, and colistin to determine the minimum inhibitory concentrations of each drug when tested against the Salmonella isolates. Based on the resulting data, new ECOFFs of 0.25 µg/mL for ceftriaxone, 0.12 µg/mL for cefepime, and 2 µg/mL for colistin have been proposed. These thresholds will aid in the identification of Salmonella that have phenotypically detectable resistance mechanisms to these important antimicrobials.

Establishing Genotypic Cutoff Values To Measure Antimicrobial Resistance in Salmonella.

Whole-genome sequencing (WGS) has transformed our understanding of antimicrobial resistance, helping us to better identify and track the genetic mechanisms underlying phenotypic resistance. Previous studies have demonstrated high correlations between phenotypic resistance and the presence of known resistance determinants. However, there has never been a large-scale assessment of how well resistance genotypes correspond to specific MICs. We performed antimicrobial susceptibility testing and WGS of 1,738 nontyphoidal Salmonella strains to correlate over 20,000 MICs with resistance determinants. Using these data, we established what we term genotypic cutoff values (GCVs) for 13 antimicrobials against Salmonella For the drugs we tested, we define a GCV as the highest MIC of isolates in a population devoid of known acquired resistance mechanisms. This definition of GCV is distinct from epidemiological cutoff values (ECVs or ECOFFs), which currently differentiate wild-type from non-wild-type strains based on MIC distributions alone without regard to genetic information. Due to the large number of isolates involved, we observed distinct MIC distributions for isolates with different resistance gene alleles, including for ciprofloxacin and tetracycline, suggesting the potential to predict MICs based on WGS data alone.

Evaluation of the renal effects of experimental feeding of melamine and cyanuric acid to fish and pigs.

OBJECTIVE: To determine whether renal crystals can be experimentally induced in animals fed melamine or the related triazine compound cyanuric acid, separately or in combination, and to compare experimentally induced crystals with those from a cat with triazine-related renal failure. ANIMALS: 75 fish (21 tilapia, 24 rainbow trout, 15 channel catfish, and 15 Atlantic salmon), 4 pigs, and 1 cat that was euthanatized because of renal failure. PROCEDURES: Fish and pigs were fed a target dosage of melamine (400 mg/kg), cyanuric acid (400 mg/kg), or melamine and cyanuric acid (400 mg of each compound/kg) daily for 3 days and were euthanatized 1, 3, 6, 10, or 14 days after administration ceased. Fresh, frozen, and formalin-fixed kidneys were examined for crystals. Edible tissues were collected for residue analysis. Crystals were examined for composition via Raman spectroscopy and hydrophilic-interaction liquid chromatography-tandem mass spectrometry. RESULTS: All animals fed the combination of melamine and cyanuric acid developed goldbrown renal crystals arranged in radial spheres (spherulites), similar to those detected in the cat. Spectral analyses of crystals from the cat, pigs, and fish were consistent with melamine-cyanurate complex crystals. Melamine and cyanuric acid residues were identified in edible tissues of fish. CONCLUSIONS AND CLINICAL RELEVANCE: Although melamine and cyanuric acid appeared to have low toxicity when administered separately, they induced extensive renal crystal formation when administered together. The subsequent renal failure may be similar to acute uric acid nephropathy in humans, in which crystal spherulites obstruct renal tubules.

Antimicrobial Drug Resistance in Fish Pathogens.

Major concerns surround the use of antimicrobial agents in farm-raised fish, including the potential impacts these uses may have on the development of antimicrobial-resistant pathogens in fish and the aquatic environment. Currently, some antimicrobial agents commonly used in aquaculture are only partially effective against select fish pathogens due to the emergence of resistant bacteria. Although reports of ineffectiveness in aquaculture due to resistant pathogens are scarce in the literature, some have reported mass mortalities in Penaeus monodon larvae caused by Vibrio harveyi resistant to trimethoprim-sulfamethoxazole, chloramphenicol, erythromycin, and streptomycin. Genetic determinants of antimicrobial resistance have been described in aquaculture environments and are commonly found on mobile genetic elements which are recognized as the primary source of antimicrobial resistance for important fish pathogens. Indeed, resistance genes have been found on transferable plasmids and integrons in pathogenic bacterial species in the genera Aeromonas, Yersinia, Photobacterium, Edwardsiella, and Vibrio. Class 1 integrons and IncA/C plasmids have been widely identified in important fish pathogens (Aeromonas spp., Yersinia spp., Photobacterium spp., Edwardsiella spp., and Vibrio spp.) and are thought to play a major role in the transmission of antimicrobial resistance determinants in the aquatic environment. The identification of plasmids in terrestrial pathogens (Salmonella enterica serotypes, Escherichia coli, and others) which have considerable homology to plasmid backbone DNA from aquatic pathogens suggests that the plasmid profiles of fish pathogens are extremely plastic and mobile and constitute a considerable reservoir for antimicrobial resistance genes for pathogens in diverse environments.

Determination of oxytetracycline levels in rainbow trout serum on a biphenyl column using high-performance liquid chromatography.

We developed a simple and sensitive high-performance liquid chromatography method on a biphenyl column to determine oxytetracycline (OTC) levels in rainbow trout serum. The assay used deproteination, filtration, and subsequent separation on a reverse-phase biphenyl column, with UV detection at 355 nm. OTC (7.8-7.9 min) was completely resolved from structurally similar riboflavin (10.4-10.5 min), a common feed supplement. Estimated limits of detection and quantitation of OTC were 0.01 and 0.04 microg/mL, respectively. The average recovery for OTC was 102% with a R.S.D. of 8.34%. Calibration standards were linear from 0.01 to 10 microg/mL.

Epidemiologic cutoff values for antimicrobial agents against Aeromonas salmonicida isolates determined by frequency distributions of minimal inhibitory concentration and diameter of zone of inhibition data.

OBJECTIVE: To develop epidemiologic cutoff values by use of frequency distributions for susceptibility to 4 antimicrobial agents when tested against a representative population of a major aquaculture pathogen, Aeromonas salmonicida. SAMPLE POPULATION: 217 typical and atypical A salmonicida isolates obtained from 20 states and 12 countries. PROCEDURES: Species identification of A salmonicida isolates was confirmed by detection of specific nucleotide sequences by use of a PCR assay. Minimal inhibitory concentration (MIC) and diameter of the zone of inhibition for oxytetracycline, ormetoprim-sulfadimethoxine, oxolinic acid, and florfenicol were determined for each isolate in accordance with standardized antimicrobial susceptibility testing methods that have been approved by the Clinical and Laboratory Standards Institute for bacterial isolates from aquatic animals. Susceptibility data were tabulated in a scattergram and analyzed by use of error rate bounding. RESULTS: Susceptibility tests for oxytetracycline, ormetoprim-sulfadimethoxine, and oxolinic acid revealed 2 distinct populations of bacteria. Isolates tested against florfenicol clustered into a single population. Oxolinic acid susceptibility data revealed higher MICs in the non-United States A salmonicida isolates. Slow-growing (atypical) A salmonicida isolates were generally more susceptible than typical isolates for all antimicrobials, except oxolinic acid. CONCLUSIONS AND CLINICAL RELEVANCE: Use of frequency distributions of susceptibility results to develop epidemiologic cutoff values appears to be applicable to aquatic isolates. Frequency distributions of susceptibility results for A salmonicida revealed clear divisions between isolate susceptibilities. This type of data, considered in conjunction with pharmacokinetic and efficacy data, may be useful for developing clinical breakpoints for use in aquaculture.

Oxytetracycline pharmacokinetics in rainbow trout during and after an orally administered medicated feed regimen.

The pharmacokinetic-pharmacodynamic predictor of antimicrobial activity for tetracyclines is reported to be the area under the concentration-time curve at steady state (AUC(ss)) divided by the minimal inhibitory concentration of the targeted pathogen. Here, we estimate AUC(ss) values for oxytetracycline (OTC) in serum of rainbow trout Oncorhynchus mykiss by using a destructive sampling study design. Seventy-two rainbow trout were fed OTC-medicated feed at 74.7 +/- 1.5 mg/kg (mean +/- SD) body weight (BW) by oral gavage for 10 consecutive days. Serum was collected from nine fish at 1, 3, 6, 8, 10, 12, 15, and 22 d after dosing began. Serum OTC concentrations were measured by high-performance liquid chromatography with a 0.01-microg/mL limit of detection. The average OTC AUC(ss) was 29.2 microg x h/mL and was estimated using nonlinear mixed-effects modeling and bootstrap resampling techniques. The elimination half-life was estimated as 85.0 h, and the fraction of steady state achieved was estimated as 0.85. The calculated AUC(ss) (24.8 microg x h/mL) following 10 d of oral dosing with 75 mg OTC/kg BW was less than the estimated AUC(ss). Results suggest that the pharmacokinetics of OTC exposure, including the AUC(ss), is better evaluated by using multiday dosimetry than by using a standard single-dose protocol.

Determination of cyanuric acid residues in catfish, trout, tilapia, salmon and shrimp by liquid chromatography-tandem mass spectrometry.

In May 2007, investigators discovered that waste material from the pet food manufacturing process contaminated with melamine (MEL) and/or cyanuric acid (CYA) had been added to hog and chicken feeds. At this time, investigators also learned that adulterated wheat gluten had been used in the manufacture of aquaculture feeds. Concern that the contaminated feed had been used in aquaculture and could enter the human food supply prompted the development of a method for the determination of CYA residues in the edible tissues of fish and shrimp. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed as a sensitive technique for the analysis of CYA in catfish, tilapia, salmon, trout and shrimp tissue. CYA was extracted from ground fish or shrimp with an acetic acid solution, defatted with hexane, and isolated with a graphitic carbon black solid-phase extraction column. Residues were separated from matrix components using a porous graphitic carbon LC column, and then analyzed with electrospray ionization in negative ion mode on a triple quadrupole mass spectrometer. Selective reaction monitoring was performed on the [M-H](-)m/z 128 ion resulting in the product ions m/z 85 and 42. Recoveries from catfish, tilapia and trout fortified with 10-100 microgkg(-1) of CYA averaged 67% with a relative standard deviation (R.S.D.) of 18% (n=107). The average method detection limit (MDL) for catfish, tilapia and trout is 3.5 microgkg(-1). An internal standard, (13)C(3)-labeled CYA, was used in the salmon and shrimp extractions. Average recovery of CYA from salmon was 91% (R.S.D.=15%, n=18) with an MDL of 7.4 microgkg(-1). Average recovery of CYA from shrimp was 85% (R.S.D.=10%, n=13) with an MDL of 3.5 microgkg(-1).

Determination and confirmation of melamine residues in catfish, trout, tilapia, salmon, and shrimp by liquid chromatography with tandem mass spectrometry.

Pet and food animal (hogs, chicken, and fish) feeds were recently found to be contaminated with melamine (MEL). A quantitative and confirmatory method is presented to determine MEL residues in edible tissues from fish fed this contaminant. Edible tissues were extracted with acidic acetonitrile, defatted with dichloromethane, and cleaned up using mixed-mode cation exchange solid-phase extraction cartridges. Extracts were analyzed by liquid chromatography with tandem mass spectrometry with hydrophilic interaction chromatography and electrospray ionization in positive ion mode. Fish and shrimp tissues were fortified with 10-500 microg/kg (ppb) of MEL with an average recovery of 63.8% (21.5% relative standard deviation, n = 121). Incurred fish tissues were generated by feeding fish up to 400 mg/kg of MEL or a combination of MEL and the related triazine cyanuric acid (CYA). MEL and CYA are known to form an insoluble complex in the kidneys, which may lead to renal failure. Fifty-five treated catfish, trout, tilapia, and salmon were analyzed after withdrawal times of 1-14 days. MEL residues were found in edible tissues from all of the fish with concentrations ranging from 0.011 to 210 mg/kg (ppm). Incurred shrimp and a survey of market seafood products were also analyzed as part of this study.