In vitro elution studies of amikacin and cefazolin from polymethylmethacrylate

OBJECTIVE: To compare the in vitro elution characteristics of amikacin and cefazolin from polymethylmethacrylate (PMMA) alone and in combination. STUDY DESIGN: A prospective, controlled, experimental study. METHODS: Three aliquots of 6 g sterile PMMA were measured and to them added (1) 750 mg amikacin; (2) 1050 mg cefazolin; and (3) 750 mg amikacin and 1050 mg cefazolin. Ten beads of each antimicrobial/PMMA combination were placed in 5 mL phosphate-buffered saline (PBS) at pH 7.4 and room temperature with constant agitation. PBS was sampled at 15 time points between 1 hour and 30 days. Amikacin concentrations were determined by fluorescence polarization immunoassay and cefazolin concentrations by high-performance liquid chromatography. RESULTS: Amikacin and cefazolin eluted at concentrations greater than 8 and 4 times, respectively, above the minimum inhibitory concentration (MIC) for susceptible bacteria over 30 days. Co-elution of the antibiotics resulted in a greater rate and proportion of antibiotic eluted. Concentrations of amikacin and cefazolin in the co-eluted fluid were not maintained sufficiently above the MIC for selected bacteria over 30 days. CONCLUSIONS: PMMA beads of only amikacin or cefazolin-eluted concentrations greater than the MIC for selected bacteria for 30 days. Co-elution of the antibiotics at the selected doses resulted in a significantly shorter duration of elution and may not be effective for treatment of wound infection. CLINICAL RELEVANCE: Co-elution of amikacin and cefazolin from PMMA at the selected doses cannot be recommended for sustained treatment of infection.     

Elution of metronidazole and gentamicin from polymethylmethacrylate beads

OBJECTIVE: To characterize the elution and bioactivity of metronidazole and gentamicin sulfate polymerized, individually and in combination, with polymethylmethacrylate (PMMA). STUDY DESIGN: In vitro experimental study. METHODS: PMMA beads containing metronidazole (3 concentrations), gentamicin sulfate, or metronidazole and gentamicin sulfate were immersed in 5 mL of phosphate-buffered saline in triplicate. Eluent was replaced at specified time intervals for 1 or 21 days, and antibiotic concentrations were measured by high-performance liquid chromatography. Changes in antibiotic bioactivity attributable to polymerization or copolymerization of the antibiotics with PMMA, ethylene oxide sterilization, and storage of AIPMMA beads containing metronidazole were evaluated. RESULTS: Antibiotic elution patterns were similar for all groups. Day 1 elution for groups containing metronidazole or gentamicin individually represented a mean 63%-66% and 79%, respectively, of the 21-day total. Approximately 50% of the day 1 elution occurred during the first hour. The elution of metronidazole was dose dependent. The elution of metronidazole (day 3-21) and gentamicin (all days) was significantly greater when metronidazole and gentamicin were combined (P <.05). The addition of metronidazole delayed polymerization of PMMA. Neither polymerization nor copolymerization of metronidazole and gentamicin with PMMA, gas sterilization, or 2-month storage of beads containing metronidazole significantly affected antimicrobial bioactivity. CONCLUSIONS: Metronidazole elution from PMMA was dose dependent. Copolymerization of metronidazole and gentamicin sulfate in PMMA resulted in increased rates of elution. Intraoperative preparation of metronidazole-impregnated PMMA beads is not practical, but sterilization and storage for 2 months should not affect efficacy. CLINICAL RELEVANCE: The local delivery of biologically active metronidazole and gentamicin by elution from PMMA is feasible.     

In vitro evaluation of antibiotic elution from polymethylmethacrylate (PMMA) and mechanical assessment of antibiotic-PMMA composites

OBJECTIVE: To determine whether different methods of sterilization of antibiotic vials or the heat of polymerization altered the antimicrobial activity or mechanical properties of antibiotic/polymethylmethacrylate (PMMA) composites when compared to antibiotic-free PMMA. STUDY DESIGN: In vitro study. METHODS: Steam-sterilized, gas-sterilized, and non-sterilized 1 gram vials of cefazolin and injectable gentamicin sulfate (high and low doses) were mixed with PMMA to prepare composites for antibiotic elution evaluation, compression, and elongation testing. Blocks of PMMA that contained antibiotic were assayed for antibacterial activity using an agar gel diffusion method or were placed in phosphate buffered saline (PBS) to assess elution of antibiotic. Phosphate buffered saline samples from steam-sterilized cefazolin and high-dose gentamicin groups were assayed on days 1, 2, 5, and 9 for cefazolin or gentamicin concentration by high-pressure liquid chromatography or fluorescent polarization immunoassay, respectively. RESULTS: PMMA blocks containing antibiotic inhibited bacterial growth of Staphylococcus aureus 25923 for an average of 9 days. Cefazolin and gentamicin concentration in PBS decreased dramatically after the first 24 hours, but remained above minimum inhibitory concentration (MIC) throughout the experiment for all groups except low-dose gentamicin. Compressive strength of plugs made from plain cement and plugs made from PMMA mixed with untreated and steam-sterilized cefazolin was similar, but was significantly different from the other groups. There appeared to be an inverse relationship between compressive strength and elongation. CONCLUSION: PMMA/antibiotic composites inhibited bacterial growth for 7 to 10 days. Compressive strength was affected by different additions of antibiotic. CLINICAL RELEVANCE: Bacteria introduced during a surgical procedure may be inhibited by elution of antibiotic from PMMA at the time of contamination.     

In Vitro Elution and Antibacterial Activity of Clindamycin,Amikacin, and Vancomycin from R‐gel Polymer

Objective: To characterize the in vitro elution and bioactivity of 2 formulations of antibiotics in a novel, dissolvable, cross‐linked dextran polymer matrix: Formulation 1—amikacin and clindamycin (AC); Formulation 2—amikacin, clindamycin, and vancomycin (ACV). Study Design: Prospective, in vitro, experimental study. Methods: Aliquots of the antibiotic impregnated polymer were incubated in PBS buffer for 10 days. PBS was changed every 24 hours and concentrations of the antibiotics eluted into saline were quantified. Antimicrobial activity of the eluent from each sampling period was tested for growth inhibition of Staphylococcus aureus. Results: Both formulations of R‐gel^? had a rapid initial release of antibiotics within the first 24 hours and then the concentrations decreased gradually over 10 days. The concentration of amikacin, clindamycin, and vancomycin remained above the breakpoint minimum inhibitory concentration of each drug for a minimum of 9 days. No significant difference (P=.9938, P=.9843) was present in the elution pattern or total amount of antibiotic eluted from clindamycin or amikacin, respectively. Eluent from both groups demonstrated bioactivity against S. aureus for the entire 10‐day study period. Conclusions: Amikacin and clindamycin together, or in combination with vancomycin, elute from R‐gel^? effectively and at gradually decreasing concentrations for at least 10 days. The antibiotics maintained their bioactivity following polymerization and elution from the R‐gel^?.     

In vitro elution of gentamicin from Plaster of Paris beads

OBJECTIVE: To determine the in vitro elution characteristics of gentamicin from Plaster of Paris-gentamicin (POP-gent) beads. STUDY DESIGN: In vitro, controlled, experimental study. METHODS: The POP-gent beads were made using a bead mold from 20 g calcium sulfate hemihydrate, 5 mL (500 mg) gentamicin solution, and 3 mL of phosphate buffered saline (PBS). Control beads were made similarly, using 30 g of dried powder and 8 mL of PBS. Beads were left in the mold overnight, gas-sterilized with ethylene oxide, and stored at room temperature for 5 months before testing. Bead chains were placed in sterile tubes containing porcine serum, and tubes were placed in a 37 degrees C incubator on a rocker. Serum was removed at intervals over 14 days and the concentration of gentamicin determined by fluorescent polarization immunoassay. Serum antibacterial activity was determined against an equine origin Escherichia coli. RESULTS: POP-gent beads released gentamicin for the 14-day sampling period. Eighty percent of the gentamicin incorporated in the beads was released over the first 48 hours. Eluent from POP-gent beads inhibited the growth of E coli at all time periods. No gentamicin was eluted from control beads and control eluent did not inhibit growth of E coli. CONCLUSIONS: In this experimental model, POP-gent beads released bactericidal drug for 14 days. Eighty percent of the gentamicin incorporated into the beads was released during the first 48 hours. The drug retains its bactericidal activity after ethylene oxide sterilization and storage at room temperature for up to 5 months. CLINICAL RELEVANCE: Pop-gent beads may be a useful repository device to deliver gentamicin locally in tissues.     

In vitro elution of amikacin and ticarcillin from a resorbable, self-setting, fiber reinforced calcium phosphate cement

Objective: To determine in vitro elution characteristics of amikacin and ticarcillin from fiber reinforced calcium phosphate beads (FRCP). Sample Population: Experimental. Methods: FRCP beads with water (A), amikacin (B), ticarcillin/clavulanate (C), or both amikacin and ticarcillin/clavulanate (D) were bathed in mL phosphate‐buffered saline (PBS) at 37°C, 5% CO₂ and 95% room air. PBS was sampled (eluent) and beads were placed in fresh PBS at time points 1 and 8 hours and 1, 2, 3, 4, 5, 6, 7, 10, 12, 14, 18, 21, 25, 28, 35, 42, 49, and 56 days. Antibiotic concentration and antimicrobial activity of eluent against Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae were determined. Results: Both antibiotics eluted in a bimodal pattern. Beads with a single antibiotic eluted 20.8 ± 2.5% of amikacin and 29.5 ± 0.8% of ticarcillin over 56 days. Coelution of the antibiotics resulted in a lower proportion (AUC_0–∞) of antibiotics eluted for both amikacin (9.5 ± 0.2%) and ticarcillin (21.7 ± 0.09%). Bioassay of antimicrobial activity of the eluent (t=1, 8, and 24 hours) established reduced antimicrobial activity of amikacin from combination beads (D). Conclusions: FRCP beads with amikacin or ticarcillin/clavulanate, but not the combination, are suitable carriers for wound implantation. Clinical Relevance: Duration before complete resorption of FRCP beads in vivo should be determined before clinical use as a resorbable depot. The results of this study underscore the importance of testing drug combinations, despite success of the combination systemically, before their use in local applications.     

In vitro Elution of Amikacin and Vancomycin from Impregnated Plaster of Paris Beads

Objective: To describe in vitro elution characteristics of amikacin and vancomycin from calcium sulfate hemihydrate 98% (plaster of Paris, POP) beads and characterize eluent inhibition of Staphylococcus spp. Study Design: Experimental study. Methods: POP beads were impregnated with amikacin or vancomycin alone or in combination and then incubated alone or in combination for 84 days at 37°C in plastic tubes containing sterile phosphate‐buffered saline (PBS). Beads containing no antimicrobial served as negative control. Beads were intermittently moved to a new tube containing drug‐free PBS. Antimicrobial was measured in the eluent using a polarized fluorescent immunoassay. Eluent inhibition of Staphylococcus spp. was determined at each time point. Results: Antimicrobial release from beads was characterized by an initial rapid phase then a slower phase. Although antimicrobial release from beads occurred throughout the 84 days, most was in the first 24 hours, except for vancomycin alone. Duration of eluent inhibition of Staphylococcus spp. growth ranged from 0.5 (amikacin alone) to 56 days (vancomycin alone). Control eluent did not inhibit bacterial growth. Conclusions: Amikacin elution from POP beads was rapid, inhibiting growth for <24 hours with or without vancomycin. Vancomycin elution was slower and inhibited growth for 56 days alone or for 5 days with amikacin. Clinical Relevance: Vancomycin‐impregnated beads appear to be reasonable as a therapeutic option whereas amikacin‐impregnated POP beads and amikacin and vancomycin combinations may require further study before considering as a therapeutic option.     

The effect of implanting gentamicin-impregnated polymethylmethacrylate beads in the tarsocrural joint of the horse

OBJECTIVE: To determine the effect of intra-articular gentamicin-impregnated polymethylmethacrylate (PMMA) beads inserted in the equine tarsocrural joint on the synovial fluid, synovial lining, and cartilage, and to determine the peak and sustainable gentamicin concentrations in synovial fluid and plasma. STUDY DESIGN: Pharmacokinetic, cytologic, and histologic study of the effect of gentamicin-impregnated PMMA on normal equine tarsocrural joints. ANIMALS: Five healthy adult horses. METHODS: Gentamicin-impregnated PMMA bead strands (3 strands each of 40 beads, with each strand containing 100 mg gentamicin) were surgically inserted into one radiographically normal tarsocrural joint in 5 horses. Each horse had both joints flushed with 1 L of lactated Ringer's solution before bead administration. Synovial fluid total protein concentration, white blood cell (WBC) count, gentamicin concentration, synovial histology, cartilage integrity, and cartilage glycosaminoglycan (GAG) concentrations were determined. RESULTS: Gentamicin concentration (mean +/- SEM peak concentration, 27.9 +/- 2.27 microg/mL) occurred in the first 24 hours and remained above 2 microg/mL for 9 days. Gentamicin concentrations in control joints and the plasma remained below detectable levels. The synovial fluid WBC count for treated joints was increased compared with control joints for 72 hours, but was similar at day 6. The synovial protein concentration in gentamicin-treated joints remained increased for 21 days. Synovium in treated joints had diffuse synovitis, whereas control joints had less fibrovascular proliferation. Superficial cartilage erosion was present in all treated joints. There was no difference in the GAG content of treated and control joint cartilage. CONCLUSIONS: Short-term implantation of gentamicin (300 mg)-impregnated PMMA beads can provide therapeutic levels of gentamicin (>2 microg/mL) in the normal tarsocrural joint for 9 days; however, gentamicin-impregnated PMMA beads induce synovitis and superficial cartilage erosion. CLINICAL RELEVANCE: Temporary intra-articular administration of antibiotic-impregnated PMMA may be an effective way to treat septic joints that require constant high concentrations of antibiotics.     

Elution profiles of cefazolin from PMMA and calcium sulfate beads prepared from commercial cefazolin formulations

Antibiotic beads have become popular for the treatment of local bacterial infections. The preparation of antibiotic beads from commercial pharmaceutical antibiotics is a convenient method in clinic. The elution characteristics of cefazolin from polymethylmethacrylate (PMMA) (SmartSet HV, Depuy I and Cemfix 3) beads and calcium sulfate beads were studied. Commercial cefazolin formulation was incorporated in PMMA or calcium sulfate at 1 g cefazolin/10 g of matrix substances to form beads. The concentrations of eluted cefazolin during 15 days were greater than MIC for Staphylococcus aureus (ATCC 25923). The eluted cefazolin concentrations were in the range of 3.6 ± 1.2 to 4.6 ± 0.4 mg for PMMA beads and 15.4 ± 1.7 mg for calcium sulfate beads. The accumulated eluted cefazolin from PMMA beads and calcium sulfate beads for 15 days were 34.41 ± 3.93 to 38.67 ± 3.04% and 95.94 ± 3.93%, respectively. The various storage conditions; at room temperature or 4°C, with or without light-protection, for 6 months had little effects on the amounts of eluted cefazolin. The results showed both in-housed cefazolin-PMMA beads and cefazolin-calcium sulfate beads could be the effective tools for the treatment of local bacterial infections.     

Effects of porcine small intestinal submucosa on elution characteristics of gentamicin-impregnated plaster of Paris

OBJECTIVE: To evaluate effects of small intestinal submucosa (SIS) on elution properties of plaster of Paris (POP). SAMPLE POPULATION: 27 POP cylinders, 27 POP spheres, and 9 polymethylmethacrylate (PMMA) spheres. PROCEDURES: Pellets were loaded with gentamicin (50 mg/g) and divided into 7 groups of 9 beads each: PMMA spheres; POP cylinders coated with 0, 4, or 8 layers of SIS; and POP spheres coated with 0, 4, or 8 layers of SIS. Gentamicin concentration was measured 6, 12, 18, 24, 32, 40, and 48 hours and 3, 4, 5, 7, 14, 21, 28, 35, and 42 days after wrapping. Porosity was evaluated via scanning electron microscopy. Curvature factor of elution curves, total amount of drug released (TDR), time required to reach 50% of total release (TDR(t50)), and number of days with concentrations > or = 1 microg/mL were compared among groups. RESULTS: SIS decreased the curvature factor and increased the TDR(t50) and TDR of POP spheres and cylinders. Curvature factor of the PMMA-release curve remained lower than that for any POP group, but all POP groups wrapped in SIS released more gentamicin than PMMA spheres. Gentamicin concentrations remained > or = 1 microg/mL in SIS-wrapped POP and PMMA groups throughout the study. Wrapping POP in SIS minimized the increase in porosity of pellets. CONCLUSIONS AND CLINICAL RELEVANCE: Wrapping POP with SIS slows the release and increases the amount of gentamicin leaching from spheres and cylinders. All groups wrapped in SIS maintained antimicrobial concentrations greater than the minimum inhibitory concentration of most pathogens.     

Synovial fluid and plasma concentrations of ceftiofur after regional intravenous perfusion in the horse

OBJECTIVE: To determine radiocarpal (RC) joint synovial fluid and plasma ceftiofur concentrations after regional intravenous perfusion (RIP) and systemic intravenous (IV) administration. STUDY DESIGN: Experimental cross-over study. ANIMALS: Five normal adult horses. METHODS: One RC joint was randomly selected for RIP and the contralateral RC joint was sampled to determine intrasynovial ceftiofur concentrations after IV administration. Wash-out between IV and RIP was > or = 14 days. After surgical introduction of an intraarticular catheter, ceftiofur (2 g) was administered under general anesthesia either IV or by RIP after tourniquet application. Plasma and synovial fluid were collected over 24 hours. Samples were analyzed using high-performance liquid chromatography with ultraviolet detection and the results were statistically analyzed using a linear mixed effect model. RESULTS: Mean synovial fluid ceftiofur concentrations were consistently higher after RIP than after IV administration and were > 1 mug/mL (minimal inhibitory concentration [MIC] for common pathogens) for >24 hours. Mean synovial fluid peak concentration of ceftiofur after RIP and IV administration was 392.7+/-103.29 microg/mL at 0.5 hours postinjection (HPI) and 2.72+/-0.31 mug/mL at 1 HPI, respectively. Large variations in synovial fluid and plasma ceftiofur concentrations were observed between horses regardless of administration technique. RIP did not cause adverse effects. CONCLUSIONS: Under the present experimental conditions RIP with ceftiofur (2 g) induced significantly higher intraarticular antibiotic concentrations in the RC joint in comparison with IV administration. Moreover, after RIP, synovial fluid ceftiofur concentrations remain above the MIC for common pathogens (1 microg/mL) for > 24 hours. No adverse effects from the technique or the antibiotic were observed. CLINICAL RELEVANCE: RIP with high doses of ceftiofur may be a beneficial adjunctive therapy when treating equine synovial infections which are caused by cephalosporin susceptible microorganisms.     

Quantitative susceptibility of Streptococcus suis strains isolated from diseased pigs in seven European countries to antimicrobial agents licensed in veterinary medicine

The susceptibility of Streptococcus suis strains (n=384) isolated from diseased pigs in seven European countries to 10 antimicrobial agents was determined. For that purpose a microbroth dilution method was used according to CLSI recommendations. The following antimicrobial agents were tested: ceftiofur, cefquinome, enrofloxacin, florfenicol, gentamicin, penicillin, spectinomycin, tetracycline, tilmicosin and trimethoprim/sulphamethoxazole. Using breakpoints established by CLSI for veterinary pathogens, all strains were susceptible to ceftiofur, florfenicol, enrofloxacin and penicillin. MIC-90 values of these antibiotics were < or = 0.03, 0.5, 2 and < or = 0.13 microg/mL, respectively. A low degree of resistance was observed for gentamicin (1.3%), spectinomycin (3.6%) and trimethoprim/sulphamethoxazole (6.0%). MIC-90 values of these antibiotics were 8, 16 and 2 microg/mL, respectively. A high level of resistance was observed for tetracycline (75.1%). A MIC-90 value of 64 microg/mL was found for this antibiotic. Serotype-associated differences in MIC-90 values were observed for tetracycline, tilmicosin and trimethoprim/suphamethoxazole.     

Antimicrobial susceptibility of Riemerella anatipestifer isolated from ducks and the efficacy of ceftiofur treatment.

The in vitro susceptibilities of 50 field isolates of Riemerella anatipestifer from ducks to ceftiofur and 16 other commonly used antimicrobials were determined. The MIC90 values (MIC refers to minimum inhibitory concentrations) for the antimicrobials used in this study are as follows: penicillin was 16 microg/ml; ceftiofur was 32 microg/ml; cephalothin, chloramphenicol, flumequine, and kanamycin were 64 microg/ml; nalidixic acid, nitrofurantoin, and sulfamethoxazole were 128 microg/ml; amikacin, ampicillin, gentamicin, lincomycin, spectinomycin, streptomycin, tetracycline, and trimethoprim were > or = 256 microg/ml. The therapeutic efficacy of ceftiofur against a highly lethal experimental R. anatipestifer infection in ducks was also evaluated. All experimental ducks were infected through the infraorbital sinus with 1 ml of 9 x 10(9) CFU of R. anatipestifer. Ceftiofur (0, 0.25, 0.5, 1, and 2 mg/kg) was injected subcutaneously 5 hours after infection. A single dose of 2 mg/kg resulted in 73% survival as compared with 10% survival in the infected, but untreated controls.     

Conception in mares following intrauterine therapy with amikacin

The semisynthetic aminoglycoside amikacin is a potentially valuable antibiotic in broodmare practice due to its efficiency against Gram negative bacteria which have developed resistance to gentamicin. Each of 4 mares conceived when amikacin at 200 ug/ml in buffered extender was used in artificial insemination or as a prebreeding infusion. After intrauterine infusion of 2 g amikacin in 200 ml saline, 4 mares conceived to natural service or insemination with untreated semen after seven hours. In unbuffered saline solutions, amikacin (1 g/100 ml) and gentamicin (1 mg/ml) were spermicidal in vitro.     

鸡白痢沙门菌的分离鉴定与耐药性分析

本研究采集某鸡场疑似感染鸡白痢的病死鸡组织,进行了沙门菌的分离、鉴定,以及对10种常见抗生素的药物敏感性试验。结果显示:分离株均为鸡白痢沙门菌,对阿米卡星(0%)、庆大霉素(0%)、头孢噻肟(3.70%)的耐药率较低,对链霉素(88.89%)、四环素(62.96%)、阿莫西林(51.85%)的耐药率较高;分离株至少可对1种药物产生耐药性,最多可对5种药物产生耐药性,70.37%的菌株可耐3种以上药物,其中耐3种药物的菌株数量最多(48.15%)。该研究结果不仅为临床合理用药提供指导,也为该地区鸡白痢的耐药性监测提供依据。     

Biodegradable Drug Delivery Systems for Gentamicin Release and Treatment of Synovial Membrane Infection

OBJECTIVE: This study investigated two biodegradable drug delivery systems (BDDS) for elution of gentamicin and elimination of synovial membrane infection. STUDY DESIGN: The effect of BDDS on control and infected synovial explants was determined. ANIMALS OR SAMPLE POPULATION: Synovial explants from four adult equine cadavers. METHODS: First, BDDS were placed in phosphate buffered saline for 14 days. Eluent was tested for gentamicin concentration (G) and bioactivity. Second, synovial explants were divided into four groups (n = 14/group): Group 1 (control); Group 2 (infected control) 405 cfu Staphylococcus aureus added at 6 hours; Group 3 (antibiotic BDDS [Ab-BDDS]) Ab-BDDS added at 24 hours; Group 4 (infected Ab-BDDS) 405 cfu S. aureus added at 6 hours, Ab-BDDS added at 24 hours. Both types of Ab-BDDS were used (n = 7/type/group). Explants were incubated in standard medium for 4 days. Medium was cultured and analyzed for (G) and hyaluronic acid concentration (HA). Explants were analyzed for viability and morphologic changes. RESULTS: The Ab-BDDS released >500 microg/mL of active gentamicin for 10 days. In Group 3, infection was eliminated within 24 hours, but histologic scores did not return to normal. Viability was significantly reduced by infection, but if eliminated, viability tended to return to normal. In Group 3, the Ab-BDDS had no significant effect on viability or (HA). Histopathologic scores were significantly higher for infected synovium. Infection, even if treated, significantly reduced (HA). CONCLUSIONS: Both Ab-BDDS eliminated infection within 24 hours. However, synovial morphology, viability and function did not return to normal. CLINICAL RELEVANCE: The Ab-BDDS may be useful for treatment of synovial membrane infection.     

Biofilm bacteria: formation and comparative susceptibility to antibiotics

The Calgary Biofilm Device (CBD) was used to form bacterial biofilms of selected veterinary gram-negative and gram-positive pathogenic bacteria from cattle, sheep, pigs, chicken, and turkeys. The minimum inhibitory concentration (MIC) and minimum biofilm eradication concentration (MBEC) of ampicillin, ceftiofur, cloxacillin, oxytetracycline, penicillin G, streptomycin, tetracycline, enrofloxacin, erythromycin, gentamicin, tilmicosin, and trimethoprim-sulfadoxine for gram-positive and -negative bacteria were determined. Bacterial biofilms were readily formed on the CBD under selected conditions. The biofilms consisted of microcolonies encased in extracellular polysaccharide material. Biofilms composed of Arcanobacterium (Actinomyces) pyogenes, Staphylococcus aureus, Staphylococcus hyicus, Streptococcus agalactiae, Corynebacterium renale, or Corynebacterium pseudotuberculosis were not killed by the antibiotics tested but as planktonic bacteria they were sensitive at low concentrations. Biofilm and planktonic Streptococcus dysgalactiae and Streptococcus suis were sensitive to penicillin, ceftiofur, cloxacillin, ampicillin, and oxytetracycline. Planktonic Escherichia coli were sensitive to enrofloxacin, gentamicin, oxytetracycline and trimethoprim/ sulfadoxine. Enrofloxacin and gentamicin were the most effective antibiotics against E. coli growing as a biofilm. Salmonella spp. and Pseudomonas aeruginosa isolates growing as planktonic populations were sensitive to enrofloxacin, gentamicin, ampicillin, oxytetracycline, and trimethoprim/sulfadoxine, but as a biofilm, these bacteria were only sensitive to enrofloxacin. Planktonic and biofilm Pasteurella multocida and Mannheimia haemolytica had similar antibiotic sensitivity profiles and were sensitive to most of the antibiotics tested. The CBD provides a valuable new technology that can be used to select antibiotics that are able to kill bacteria growing as biofilms.     

Comparison of the use of regulatory assays and high-performance liquid chromatography for detection of residues of ceftiofur sodium metabolites in tissue specimens of culled dairy cattle

OBJECTIVE: To compare the results of regulatory screening and confirmation assays with those of high-performance liquid chromatography (HPLC) in the detection of ceftiofur metabolites in the tissues of culled dairy cattle. ANIMALS: 17 lactating Holstein dairy cows. PROCEDURE: Daily IM injections of ceftiofur sodium were administered at a dose of 2.2 mg of ceftiofur equivalents/kg (n = 6) or 1.0 mg of ceftiofur equivalents/kg (10) for 5 days. Following withdrawal times of 12 hours (high-dose ceftiofur) and either 5 or 10 days (low-dose ceftiofur), cows were slaughtered and liver, kidney, and diaphragmatic muscle specimens were harvested and analyzed by HPLC and standard regulatory methods that included the following assays: the swab test on premises, the fast antimicrobial screen test, the calf antibiotic and sulfa test, and the 7-plate bioassay confirmation test. RESULTS: In all tissue specimens, residues of ceftiofur and desfuroylceftiofur-related metabolites, as measured by HPLC, were less than regulatory tolerance, as defined by the FDA. False-positive screening assay results were more likely for tissue specimens that had been frozen for shipment to a federal laboratory, compared with fresh tissue specimens that were assayed at the slaughter establishment (23% vs 3% false-positive results, respectively). CONCLUSIONS AND CLINICAL RELEVANCE: The observation that fresh tissues had negative results on screening assays, whereas subsets of the same tissue specimens had false-positive results on screening assays following freezing, suggests that freezing and thawing interferes with microbial inhibition-based regulatory screening assays.     

Effect of antibiotics on in vitro and in vivo avian cartilage degradation

Antibiotics are used in the livestock industry not only to treat disease but also to promote growth and increase feed efficiency in less than ideal sanitary conditions. However, certain antibiotic families utilized in the poultry industry have recently been found to adversely affect bone formation and cartilage metabolism in dogs, rats, and humans. Therefore, the first objective of this study was to determine if certain antibiotics used in the poultry industry would inhibit in vitro cartilage degradation. The second objective was to determine if the antibiotics found to inhibit in vitro cartilage degradation also induced tibial dyschondroplasia in growing broilers. Ten antibiotics were studied by an avian explant culture system that is designed to completely degrade tibiae over 16 days. Lincomycin, tylosin tartrate, gentamicin, erythromycin, and neomycin sulfate did not inhibit degradation at any concentration tested. Doxycycline (200 microg/ml), oxytetracycline (200 microg/ml), enrofloxacin (200 and 400 microg/ml), ceftiofur (400 microg/ml), and salinomycin (10 microg/ml) prevented complete cartilage degradation for up to 30 days in culture. Thus, some of the antibiotics did inhibit cartilage degradation in developing bone. Day-old chicks were then administered the five antibiotics at 25%, 100%, or 400% above their recommended dose levels and raised until 21 days of age. Thiram, a fungicide known to induce experimental tibial dyschondroplasia (TD), was given at 20 ppm. Birds were then killed by cervical dislocation, and each proximal tibiotarsus was visually examined for TD lesions. The results showed that none of these antibiotics significantly induced TD in growing boilers at any concentration tested, whereas birds given 20 ppm thiram had a 92% incidence rate.