In vitro antiseptic susceptibilities for Staphylococcus pseudintermedius isolated from canine superficial pyoderma in Japan

Background –  Topical therapy, particularly with chlorhexidine, is becoming increasingly common as a treatment option for canine pyoderma; however, there are limited studies on the susceptibility of Staphylococcus pseudintermedius to chlorhexidine compounds. Objectives –  To determine the in vitro susceptibility of both meticillin‐resistant and meticillin‐susceptible S. pseudintermedius isolates to chlorhexidine and other antiseptic agents and the presence of multidrug efflux pump genes. Samples –  One hundred S. pseudintermedius isolates from 23 initial and 77 recurrent cases of canine pyoderma. Methods –  After bacterial identification and mecA testing, minimal inhibitory concentrations (MICs) of antiseptic agents were determined. Multidrug efflux pump genes, including qacA, qacB and smr, were identified. Results –  Of the 100 isolates, 57 were identified as meticillin‐resistant S. pseudintermedius. The MIC₉₀ of chlorhexidine acetate, chlorhexidine gluconate, acriflavine, ethidium bromide and benzalkonium chloride were 1, 1, 2, 0.5 and 2 μg/mL, respectively. Multidrug efflux pump genes qacA, qacB and smr were not detected in any of the isolates. Conclusions and clinical importance –  The MICs for chlorhexidine and other antiseptics remain low, and multidrug efflux pump genes were not found in the tested isolates.     

Susceptibility of canine and feline bacterial pathogens to pradofloxacin and comparison with other fluoroquinolones approved for companion animals

In this study, 908 bacterial pathogens from defined infections of dogs and cats were tested for their susceptibility to the novel fluoroquinolone pradofloxacin, which was approved in 2011 for use in cats and dogs. Most of the bacteria tested (Staphylococcus aureus, Staphylococcus pseudintermedius, Escherichia coli, β-haemolytic streptococci, Pasteurella multocida and Bordetella bronchiseptica) exhibited low pradofloxacin MIC₉₀ values of ≤0.25 μg/ml. Solely Proteus spp. and Pseudomonas aeruginosa had higher MIC₉₀ values of ≥4 μg/ml. Only six (3.4%) of 177 S. pseudintermedius and 12 (5.3%) of 227 E. coli isolates showed pradofloxacin MICs of ≥2 μg/ml. Analysis of the quinolone resistance determining regions of the target genes identified double mutations in GyrA that resulted in amino acid exchanges S83L + D87N or S83L + D87Y and single or double mutations in ParC that resulted in amino acid exchanges S80I or S80I + E84G in all 12 E. coli isolates. The six S. pseudintermedius isolates exhibited amino acid exchanges S84L or E88K in GyrA and S80I in GrlA. Comparative analysis of the MICs of pradofloxacin and the MICs determined for enrofloxacin and its main metabolite ciprofloxacin, but also marbofloxacin, orbifloxacin, difloxacin and ibafloxacin was conducted for the target pathogens S. pseudintermedius, E. coli and P. multocida. This comparison confirmed that pradofloxacin MICs were significantly lower than those of the other tested fluoroquinolones.     

Resistant cutoff values and optimal scheme establishments for florfenicol against Escherichia coli with PK‐PD modeling analysis in pigs

Florfenicol, a structural analog of thiamphenicol, has broad‐spectrum antibacterial activity against gram‐negative and gram‐positive bacteria. This study was conducted to investigate the epidemiological, pharmacokinetic–pharmacodynamic cutoff, and the optimal scheme of florfenicol against Escherichia coli (E. coli) with PK‐PD integrated model in the target infectious tissue. 220 E. coli strains were selected to detect the susceptibility to florfenicol, and a virulent strain P190, whose minimum inhibitory concentration (MIC) was similar to the MIC₅₀ (8 μg/ml), was analyzed for PD study in LB and ileum fluid. The MIC of P190 in the ileum fluid was 0.25 times lower than LB. The ratios of MBC/MIC were four both in the ileum and LB. The characteristics of time‐killing curves also coincided with the MBC determination. The recommended dosages (30 mg/kg·body weight) were orally administrated in healthy pigs, and both plasma and ileum fluid were collected for PK study. The main pharmacokinetics (PK) parameters including AUC_24 hr, AUC_0–∞, T_max, T_1/2, C_max, CL_b, and K_e were 49.83, 52.33 μg*h/ml, 1.32, 10.58 hr, 9.12 μg/ml, 0.50 L/hr*kg, 0.24 hr^?1 and 134.45, 138.71 μg*hr/ml, 2.05, 13.01 hr, 16.57 μg/ml, 0.18 L/hr*kg, 0.14 hr^?1 in the serum and ileum fluid, respectively. The optimum doses for bacteriostatic, bactericidal, and elimination activities were 29.81, 34.88, and 36.52 mg/kg for 50% target and 33.95, 39.79, and 42.55 mg/kg for 90% target, respectively. The final sensitive breakpoint was defined as 16 μg/ml. The current data presented provide the optimal regimens (39.79 mg/kg) and susceptible breakpoint (16 μg/ml) for clinical use, but these predicted data should be validated in the clinical practice.     

Prevalence of and Resistance to Anti‐Microbial Drugs in Selected Microbial Species Isolated from Bulk Milk Samples

The prevalence of strains of Staphylococcus aureus, coagulase‐negative (CN) staphylococci, Listeria monocytogenes, Escherichia coli, Enterococcus faecalis, E. faecium and Bacillus cereus, was investigated in 111 bulk milk samples. Staphylococcus aureus was isolated from 38 samples, CN staphylococci from 63 samples, E. coli from 49 samples, E. faecalis or E. faecium from 107 samples, and L. monocytogenes from two samples. Bacillus cereus was not found in any of the samples and three samples were free of any of the selected species. Sensitivity to the anti‐microbial drugs amikacin, ampicillin, ampicillin + sulbactam, cephalothin (CLT), cephotaxime, clindamycin, chloramphenicol (CMP), co‐trimoxazole, erythromycin (ERY), gentamicin, neomycin, norfloxacin, oxacillin, penicillin, streptomycin (STR), tetracycline (TTC) and vancomycin was tested using the standard dilution technique. Minimum inhibitory concentration (MIC) characteristics (MIC₅₀, MIC₉₀, MIC range) were determined for each microbial species. Resistance against one or more anti‐microbial drugs was found in 93% of S. aureus, 40% of CN staphylococci, 73% of E. coli, 88% of E. faecalis, 55% of E. faecium, and one L. monocytogenes strain. Most of the strains, particularly enterococci, were resistant to STR, TTC, and ERY (MIC₅₀ 4 μg/ml). A high percentage of staphylococci were resistant to β‐lactam antibiotics. High resistance to CLT was found in 11 strains of E. coli (MIC 256 μg/ml) and strains resistant to CMP (MIC₉₀ 16 μg/ml) were detected. The highest numbers of resistance phenotypes were found in E. coli (16) and CN staphylococci (12). Eighteen identical resistance phenotypes were demonstrated in indicator bacteria (E. coli, E. faecalis, E. faecium) and pathogens (S. aureus, CN staphylococci) isolated from the same bulk milk sample. The obtained resistance data were matched against the herd owners' information on therapeutic use of the drugs. This confrontation could not explain the findings of strains resistant to ERY or CMP. Our findings are evidence of selection of resistant strains among not only pathogenic agents, but also among indicator bacteria which can become significant carriers of transmissible resistance genes.     

Pharmacokinetic/pharmacodynamic integration and modelling of oxytetracycline for the porcine pneumonia pathogens Actinobacillus pleuropneumoniae and Pasteurella multocida

Pharmacokinetic–pharmacodynamic (PK/PD) integration and modelling were used to predict dosage schedules of oxytetracycline for two pig pneumonia pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida. Minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) were determined in broth and porcine serum. PK/PD integration established ratios of average concentration over 48 h (C_av0–48 h)/MIC of 5.87 and 0.27 μg/mL (P. multocida) and 0.70 and 0.85 μg/mL (A. pleuropneumoniae) for broth and serum MICs, respectively. PK/PD modelling of in vitro time–kill curves established broth and serum breakpoint values for area under curve (AUC_0–24 h)/MIC for three levels of inhibition of growth, bacteriostasis and 3 and 4 log₁₀ reductions in bacterial count. Doses were then predicted for each pathogen, based on Monte Carlo simulations, for: (i) bacteriostatic and bactericidal levels of kill; (ii) 50% and 90% target attainment rates (TAR); and (iii) single dosing and daily dosing at steady‐state. For 90% TAR, predicted daily doses at steady‐state for bactericidal actions were 1123 mg/kg (P. multocida) and 43 mg/kg (A. pleuropneumoniae) based on serum MICs. Lower TARs were predicted from broth MIC data; corresponding dose estimates were 95 mg/kg (P. multocida) and 34 mg/kg (A. pleuropneumoniae).     

In vitro evaluation of topical biocide and antimicrobial susceptibility of Staphylococcus pseudintermedius from dogs

Background – Staphylococcus pseudintermedius is an important canine pathogen, and the emergence and widespread dissemination of meticillin‐resistant strains (MRSP) is of significant concern. Multidrug‐resistant infections may require alternative approaches, such as the use of topical therapy. There is minimal information about the in vitro susceptibility of meticillin‐susceptible S. pseudintermedius (MSSP) and MRSP to biocides and topical antimicrobials. Hypothesis/Objectives – The hypothesis was that clinical isolates of MSSP and MRSP would not have universal susceptibility to topical biocides and antimicrobials. The goal of this study was to assess the susceptibility of a collection of S. pseudintermedius isolates to selected antimicrobials and biocides. Animals – The study was performed on clinical isolates of MSSP and MRSP from dogs with skin and soft tissue infections collected throughout North America between 2006 and 2008. Methods – The minimal inhibitory concentrations (MICs) of chlorhexidine digluconate, benzalkonium chloride, triclosan, accelerated hydrogen peroxide, geranium oil, tea tree oil and grapefruit seed extract were tested for 25 MRSP and 25 MSSP isolates from dogs using the agar dilution method. The MICs of fusidic acid, bacitracin and mupirocin were determined using Etests. Results – Triclosan demonstrated excellent activity against all bacterial isolates, with no growth at the lowest concentration evaluated (MIC ≤ 0.5 μg/mL). Conversely, grapefruit seed extract did not inhibit growth at the highest concentration tested (MIC > 3.84 μg/mL). All isolates were susceptible to mupirocin, fusidic acid and bacitracin. There were no significant differences noted in the range, MIC₅₀ or MIC₉₀ between MSSP and MRSP isolates. Conclusions and clinical importance – While isolates were susceptible to most of the tested compounds, universal susceptibility to all compounds with potential antimicrobial activity cannot be assumed, and specific testing is required.     

In vitro miconazole susceptibility of meticillin-resistant Staphylococcus pseudintermedius and Staphylococcus aureus

Background – The emergence and dissemination of meticillin‐resistant staphylococci has created significant treatment challenges in veterinary medicine and increased interest in topical therapy for superficial infections. Concern has been expressed regarding the use of some topical antimicrobials in animals because of the potential for emergence of resistance, and additional options are required. Miconazole has limited antibacterial properties that include antistaphylococcal activity. Hypothesis/Objectives – The objective of this study was to assess the in vitro susceptibility of Staphylococcus pseudintermedius and Staphylococcus aureus to miconazole. Methods – In vitro susceptibility of 112 meticillin‐resistant S. pseudintermedius (MRSP), 53 meticillin‐resistant S. aureus (MRSA) and 37 meticillin‐susceptible S. pseudintermedius (MSSP) to miconazole was assessed using agar dilution. Results – The minimal inhibitory concentration (MIC) range, MIC₅₀ and MIC₉₀ for MRSP were 1–8, 2 and 4 μg/mL, respectively. Corresponding results for MRSA were 1–8, 2 and 6 μg/mL, and for MSSP 1–4, 2 and 2 μg/mL. The MIC for MSSP was a significantly lower MIC than that for both MRSP (P = 0.006) and MRSA (P < 0.001), while the MIC for MRSP was significantly lower than that for MRSA (P = 0.001). Conclusions and clinical importance – These in vitro data suggest that miconazole could be a useful therapeutic option for superficial infections caused by meticillin‐susceptible and meticillin‐resistant staphylococci, but proper clinical investigation is required.     

Pharmacokinetics of enrofloxacin HCl‐2H2O (ENRO‐C), PK/PD,and Monte Carlo modeling vs. Leptospira spp. in cows

The pharmacokinetics, PK/PD ratios, and Monte Carlo modeling of enrofloxacin HCl‐2H₂O (Enro‐C) and its reference preparation (Enro‐R) were determined in cows. Fifty‐four Jersey cows were randomly assigned to six groups receiving a single IM dose of 10, 15, or 20 mg/kg of Enro‐C (Enro‐C₁₀, Enro‐C₁₅, Enro‐C₂₀) or Enro‐R. Serial serum samples were collected and enrofloxacin concentrations quantified. A composite set of minimum inhibitory concentrations (MIC) of Leptospira spp. was utilized to calculate PK/PD ratios: maximum serum concentration/MIC (C_max/MIC₉₀) and area under the serum vs. time concentration of enrofloxacin/MIC (AUC_0‐24/MIC₉₀). Monte Carlo simulations targeted C_max/MIC = 10 and AUC_0‐24/MIC = 125. Mean C_max obtained were 6.17 and 2.46 μg/ml; 8.75 and 3.54 μg/ml; and 13.89 and 4.25 μg/ml, respectively for Enro‐C and Enro‐R. C_max/MIC₉₀ ratios were 6.17 and 2.46, 8.75 and 3.54, and 13.89 and 4.25 for Enro‐C and Enro‐R, respectively. Monte Carlo simulations based on C_max/MIC₉₀ = 10 indicate that only Enro‐C₁₅ and Enro‐C₂₀ may be useful to treat leptospirosis in cows, predicting a success rate ≥95% when MIC₅₀ = 0.5 μg/ml, and ≥80% when MIC₉₀ = 1.0 μg/ml. Although Enro‐C₁₅ and Enro‐C₂₀ may be useful to treat leptospirosis in cattle, clinical trials are necessary to confirm this proposal.     

Pharmacokinetics,bioavailability and PK/PD relationship of cefquinome for Escherichia coli in Beagle dogs

The pharmacokinetics and bioavailability of cefquinome in Beagle dogs were determined by intravenous (IV), intramuscular (IM) or subcutaneous (SC) injection at a single dose of 2 mg/kg body weight (BW). The minimum inhibitory concentrations (MIC) of cefquinome against 217 Escherichia coli isolated from dogs were also investigated. After IV injection, the plasma concentration‐time curve of cefquinome was analyzed using a two‐compartmental model, and the mean values of t_1/2α (h), t_1/2β (h), V_ss (L/kg), Cl_B (L/kg/h) and AUC (μg·h/mL) were 0.12, 0.98, 0.30, 0.24 and 8.51, respectively. After IM and SC administration, the PK data were best described by a one‐compartmental model with first‐order absorption. The mean values of t_1/2Kel, t_1/2Ka, t_max (h), C_max (μg/mL) and AUC (μg·h/mL) were corresponding 0.85, 0.14, 0.43, 4.83 and 8.24 for IM administration, 0.99, 0.29, 0.72, 3.88 and 9.13 for SC injection. The duration of time that drug levels exceed the MIC (%T > MIC) were calculated using the determined MIC₉₀ (0.125 μg/mL) and the PK data obtained in this study. The results indicated that the dosage regimen of cefquinome at 2 mg/kg BW with 12‐h intervals could achieve %T > MIC above 50% that generally produced a satisfactory bactericidal effect against E. coli isolated from dogs in this study.     

Physicochemical characterization and pharmacokinetics in broiler chickens of a new recrystallized enrofloxacin hydrochloride dihydrate

Enrofloxacin, a key antimicrobial agent in commercial avian medicine, has limited bioavailability (60%). This prompted its chemical manipulation to yield a new solvate‐recrystallized enrofloxacin hydrochloride dihydrate entity (enro_C). Its chemical structure was characterized by means of mass spectroscopy, Fourier transformed infrared spectroscopy, X‐ray powder diffraction, and thermal analysis. Comparative oral pharmacokinetics (PK) of reference enrofloxacin (enro_R) and enro_C in broiler chickens after oral administration revealed noticeable improvements in key parameters and PK/PD ratios. Maximum serum concentration values were 2.61 ± 0.21 and 5.9 ± 0.42 μg/mL for enro_R and enro_C, respectively; mean residence time was increased from 5.50 ± 0.26 h to 6.20 ± 0.71 h and the relative bioavailability of enro_C was 336%. Considering C_max/MIC and AUC/MIC ratios and the MIC values for a wild‐type Escherichia coli O78/H12 (0.25 μg/mL), optimal ratios will only be achieved by enro_C (C_max/MIC = 23.6 and AUC/MIC = 197.7 for enro_C; vs. C_max/MIC = 10.4 and AUC/MIC = 78.1 for enro_R). Furthermore, enro_C may provide in most cases mutant prevention concentrations (C_max/MIC ≥ 16). Ready solubility of powder enro_C in drinking water at concentrations regularly used (0.01%) to provide an additional advantage of enro_C in the field. Further development of enro_C is warranted before it can be recommended for clinical use in veterinary medicine.     

Multidose pharmacokinetics of orally administered florfenicol in the channel catfish (Ictalurus punctatus)

Plasma disposition of florfenicol in channel catfish was investigated after an oral multidose (10 mg/kg for 10 days) administration in freshwater at water temperatures ranging from 24.7 to 25.9 °C. Florfenicol concentrations in plasma were analyzed by means of liquid chromatography with MS/MS detection. After the administration of florfenicol, the mean terminal half‐life (t_1/2), maximum concentration at steady‐state (C_ss(max)), time of C_ss(max) (T_max), minimal concentration at steady‐state (C_ss(min)), and V_c/F were 9.0 h, 9.72 μg/mL, 8 h, 2.53 μg/mL, and 0.653 L/kg, respectively. These results suggest that florfenicol administered orally at 10 mg/kg body weight for 10 days could be expected to control catfish bacterial pathogens inhibited in vitro by a minimal inhibitory concentration value of <2.5 μg/mL.     

Nanoemulsion formulation of florfenicol improves bioavailability in pigs

Nanotechnology applications in medicine have seen a tremendous growth in the past decade and are being employed to enhance the stability and bioavailability of lipophilic substances, such as florfenicol. This study aimed to examine the pharmacokinetic properties of the formulated oil‐in‐water florfenicol‐loaded nanoemulsion (FF‐NE). FF‐NE and florfenicol control (Nuflor^®) were administered to the pigs at a dose of 20 mg/kg. Nanoemulsion formulation of florfenicol was highly influenced in vivo plasma profile. The in vivo absorption study in pigs indicated that C_max (14.54 μg/mL) was significantly higher in FF‐NE, 3.42 times higher than the marketed formulation. In comparison with the control group, the relative bioavailability of formulated nanoemulsion was up to 134.5%. Assessment of bioequivalence using log‐transformed data showed that the 90% confidence intervals (90% CI) of C_max and AUC_0–∞ were 2.48–4.60 and 1.21–1.72, respectively.     

Some pharmacokinetic indices of oral fluconazole administration to koalas (Phascolarctos cinereus) infected with cryptococcosis

Three asymptomatic koalas serologically positive for cryptococcosis and two symptomatic koalas were treated with 10 mg/kg fluconazole orally, twice daily for at least 2 weeks. The median plasma C_max and AUC_0‐8 h for asymptomatic animals were 0.9 μg/mL and 4.9 μg/mL·h, respectively; and for symptomatic animals 3.2 μg/mL and 17.3 μg/mL·h, respectively. An additional symptomatic koala was treated with fluconazole (10 mg/kg twice daily) and a subcutaneous amphotericin B infusion twice weekly. After 2 weeks the fluconazole C_max was 3.7 μg/mL and the AUC_0‐8 h was 25.8 μg/mL*h. An additional three koalas were treated with fluconazole 15 mg/kg twice daily for at least 2 weeks, with the same subcutaneous amphotericin protocol co‐administered to two of these koalas (C_max: 5.0 μg/mL; mean AUC_0‐8 h: 18.1 μg/mL*h). For all koalas, the fluconazole plasma C_max failed to reach the MIC₉₀ (16 μg/mL) to inhibit C. gattii. Fluconazole administered orally at either 10 or 15 mg/kg twice daily in conjunction with amphotericin is unlikely to attain therapeutic plasma concentrations. Suggestions to improve treatment of systemic cryptococcosis include testing pathogen susceptibility to fluconazole, monitoring plasma fluconazole concentrations, and administration of 20–25 mg/kg fluconazole orally, twice daily, with an amphotericin subcutaneous infusion twice weekly.     

A microbiological assay to estimate the antimicrobial activity of parenteral tildipirosin against foodborne pathogens and commensals in the colon of beef cattle and pigs

Tildipirosin (TIP) is a novel 16‐membered‐ring macrolide authorized for the treatment of bovine and swine respiratory disease. The pH dependency of macrolide antimicrobial activity is well known. Considering that the pH in the colon contents of growing beef cattle and pigs is usually below pH 7.0, the minimum inhibitory concentrations (MIC) of TIP against foodborne bacterial pathogens such as Campylobacter (C.) coli, C. jejuni and Salmonella enterica and commensal species including Enterococcus (E.) faecalis, E. faecium and Escherichia coli were determined under standard (pH 7.3 ± 1) or neutral as well as slightly acidic conditions. A decrease in pH from 7.3 to 6.7 resulted in an increase in MICs of TIP. Except for the MICs > 256 μg/mL observed in the resistant subpopulation of the C. coli and the Enterococcus species, the MIC ranges increased from 2–8 μg/mL to 64–> 256 μg/mL for Salmonella enterica and E. coli, from 8–16 μg/mL to 32–128 μg/mL for the two Campylobacter species, and from 4–32 μg/mL to 128–> 256 μg/mL for both Enterococcus species. To estimate the antimicrobial activity of TIP in the colon contents of livestock during recommended usage of the parenterally administered TIP (Zuprevo^®), and to compare this with the increased MICs at the slightly acidic colonic pH, we developed and validated a microbiological assay for TIP and used this to test incurred faecal samples collected from cattle and pigs. Microbiological activity of luminal TIP was determined in aqueous supernatants from diluted faeces, using standard curves produced from TIP‐spiked faecal supernatants. The limit of quantification (LOQ) for TIP was 1 μg/mL (ppm). In a cattle study (n = 14), 3 of 28 faecal samples collected 24 and 48 h post‐treatment were found to contain TIP above the LOQ (concentrations of 1.3–1.8 ppm). In another cattle study (n = 12) with faecal samples collected at 8, 24 and 48 h post‐treatment, TIP concentrations were above the LOQ in 4 of the 8 h samples (1.2–2.6 ppm) and one of the 24‐h samples (1.3 ppm). In a pig study (n = 12) with faecal samples collected 24, 48 and 72 h post‐treatment, only one sample contained TIP above the LOQ (concentration 1.5 ppm). In another pig study (n = 12), with samples collected at 8, 24 48 and 96 h post‐treatment, TIP concentrations were above the LOQ in one 8‐h sample (1.1 ppm) and two 24‐h samples (2.3 and 2.5 ppm). None of the 48‐h and 96‐h samples from these 4 studies contained measurable TIP concentrations. Thus, in cattle and pigs, only a small fraction of faecal samples collected up to 24 h postdosing contained measurable microbiologically active TIP, with its maximum limited to 2.6 μg/mL. This is several log₂ dilution steps below the MICs of TIP against foodborne pathogens and commensals collected under acidic conditions comparable with those in the colonic contents and may explain a lack of intestinal dysbacteriosis with parenteral tildipirosin in livestock.     

Biofilm production by pathogens associated with canine otitis externa,and the antibiofilm activity of ionophores and antimicrobial adjuvants

Otitis externa (OE) is a frequently reported disorder in dogs associated with secondary infections by Staphylococcus, Pseudomonas and yeast pathogens. The presence of biofilms may play an important role in the resistance of otic pathogens to antimicrobial agents. Biofilm production of twenty Staphylococcus pseudintermedius and twenty Pseudomonas aeruginosa canine otic isolates was determined quantitatively using a microtiter plate assay, and each isolate was classified as a strong, moderate, weak or nonbiofilm producer. Minimum biofilm eradication concentration (MBEC) of two ionophores (narasin and monensin) and three adjuvants (N‐acetylcysteine (NAC), Tris‐EDTA and disodium EDTA) were investigated spectrophotometrically (OD_570nm) and quantitatively (CFU/ml) against selected Staphylococcus and Pseudomonas biofilm cultures. Concurrently, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of planktonic cultures were assessed. 16/20 of the S. pseudintermedius clinical isolates were weak biofilm producers. 19/20 P. aeruginosa clinical isolates produced biofilms and were distributed almost equally as weak, moderate and strong biofilm producers. While significant antibiofilm activity was observed, no MBEC was achieved with narasin or monensin. The MBEC for NAC ranged from 5,000–10,000 µg/ml and from 20,000–80,000 µg/ml against S. pseudintermedius and P. aeruginosa, respectively. Tris‐EDTA eradicated P. aeruginosa biofilms at concentrations ranging from 6,000/1,900 to 12,000/3,800 µg/ml. The MBEC was up to 16‐fold and eightfold higher than the MIC/MBC of NAC and Tris‐EDTA, respectively. Disodium EDTA reduced biofilm growth of both strains at concentrations of 470 µg/ml and higher. It can be concluded that biofilm production is common in pathogens associated with canine OE. NAC and Tris‐EDTA are effective antibiofilm agents in vitro that could be considered for the treatment of biofilm‐associated OE in dogs.     

Pharmacokinetics of florfenicol after intravenous administration in Escherichia coli lipopolysaccharide‐induced endotoxaemic sheep

Experiments in different animal species have shown that febrile conditions, induced by Escherichia coli lipopolysaccharide (LPS), may alter the pharmacokinetic properties of drugs. The objective was to study the effects of a LPS‐induced acute‐phase response (APR) model on plasma pharmacokinetics of florfenicol (FFC) after its intravenous administration in sheep. Six adult clinically healthy Suffolk Down sheep, 8 months old and 35.5 ± 2.2 kg in body weight (bw), were distributed through a crossover factorial 2 × 2 design, with 4 weeks of washout. Pairs of sheep similar in body weight were assigned to experimental groups: Group 1 (LPS) was treated with three intravenous doses of 1 μg/kg bw of E. coli LPS before FFC treatment. Group 2 (control) was treated with an equivalent volume of saline solution (SS) at similar intervals as LPS. At 24 h after the first injection of LPS or SS, an intravenous bolus of 20 mg/kg bw of FFC was administered. Blood samples (5 mL) were collected before drug administration and at different times between 0.05 and 48.0 h after treatment. FFC plasma concentrations were determined by liquid chromatography. A noncompartmental pharmacokinetic model was used for data analysis, and data were compared using a Mann–Whitney U‐test. The mean values of AUC_0–∞ in the endotoxaemic sheep (105.9 ± 14.3 μg·h/mL) were significantly higher (P < 0.05) than values observed in healthy sheep (78.4 ± 5.2 μg·h/mL). The total mean plasma clearance (CL_T) decreased from 257.7 ± 16.9 mL·h/kg in the control group to 198.2 ± 24.1 mL·h/kg in LPS‐treated sheep. A significant increase (P < 0.05) in the terminal half‐life was observed in the endotoxaemic sheep (16.9 ± 3.8 h) compared to the values observed in healthy sheep (10.4 ± 3.2 h). In conclusion, the APR induced by the intravenous administration of E. coli LPS in sheep produces higher plasma concentrations of FFC due to a decrease in the total body clearance of the drug.     

Pharmacokinetic and pharmacodynamic characterization of ceftiofur crystalline‐free acid following subcutaneous administration in domestic goats

Pharmacokinetic (PK)–pharmacodynamic (PD) integration of crystalline ceftiofur‐free acid (CCFA) was established in six healthy female goats administered subcutaneously (s.c.) on the left side of the neck at a dosage of 6.6 mg/kg body weight. Serum concentrations of ceftiofur and desfuroylceftiofur (DFC) were determined using high‐performance liquid chromatography. Mutant prevention concentration (MPC), minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ceftiofur were determined for Pasteurella (P.) multocida. Mean terminal half‐life and mean residence time of ceftiofur + DFC were 48.6 h and 104 h, respectively. In vitro plasma protein binding of ceftiofur was 46.6% in goats. The MIC and MBC values of ceftiofur were similar in serum and MHB and a very small difference between these values confirmed bactericidal activity of drug against P. multocida. In vitro and ex vivo time–kill curves for P. multocida demonstrated a time‐dependent killing action of drug. Considering target serum concentration of 0.20 μg/mL, PK‐PD values for AUC_24 h/MIC₉₀ and T > MIC₉₀, respectively, were 302 h and 192 h against P. multocida. A MPC/MIC ratio of 10–14 indicated that selective pressure for proliferation of resistant mutants of P. multocida is minimal after CCFA single‐dose administration. Based on MPC = 1.40 μg/mL for P. multocida, the PK‐PD indices, viz. T > MPC and AUC₂₄/MPC, were 48 h and 43 h, respectively. The data suggested the use of single dose (6.6 mg/kg, s.c.) of CCFA in goats to obtain clinical and bacteriological cure of pneumonia due to P. multocida.     

The acute phase response induced by Escherichia coli lipopolysaccharide modifies the pharmacokinetics and metabolism of florfenicol in rabbits

The aim of this study was to determine the effect of Escherichia coli lipopolysaccharide (LPS)‐induced acute phase response (APR) on the pharmaco‐kinetics and biotransformation of florfenicol (FFC) in rabbits. Six rabbits (3.0 ± 0.08 kg body weight (bw)) were distributed through a crossover design with 4 weeks of washout period. Pairs of rabbits similar in bw and sex were assigned to experimental groups: Group 1 (LPS) was treated with three intravenous doses of 1 μg/kg bw of E. coli LPS at intervals of 6 h, and Group 2 (control) was treated with an equivalent volume of saline solution (SS) at the same intervals and frequency of Group 1. At 24 h after the first injection of LPS or SS, an intravenous bolus of 20 mg/kg bw of FFC was administered. Blood samples were collected from the auricular vein before drug administration and at different times between 0.05 and 24.0 h after treatment. FFC and florfenicol‐amine (FFC‐a) were extracted from the plasma, and their concentrations were determined by high‐performance liquid chromatography. A noncompartmental pharmacokinetic model was used for data analysis, and data were compared using the paired Student t‐test. The mean values of AUC_0–∞ in the endotoxaemic rabbits (26.3 ± 2.7 μg·h/mL) were significantly higher (P < 0.05) than values observed in healthy rabbits (17.2 ± 0.97 μg·h/mL). The total mean plasma clearance (CL_T) decreased from 1228 ± 107.5 mL·h/kg in the control group to 806.4 ± 91.4 mL·h/kg in the LPS‐treated rabbits. A significant increase (P < 0.05) in the half‐life of elimination was observed in the endotoxaemic rabbits (5.59 ± 1.14 h) compared to the values observed in healthy animals (3.44 ± 0.57 h). In conclusion, the administration of repeated doses of 1 μg/kg E. coli LPS induced an APR in rabbits, producing significant modifications in plasma concentrations of FFC leading to increases in the AUC, terminal half‐life and mean residence time (MRT), but a significant decrease in CL_T of the drug. As a consequence of the APR induced by LPS, there was a reduction in the metabolic conversion of FFC to their metabolite FFC‐a in the liver, suggesting that the mediators released during the APR induced significant inhibitory effects on the hepatic drug‐metabolizing enzymes.     

Anti‐microbial Susceptibility for east1+Escherichia coli Isolated from Diarrheic Pigs in Korea

The in vitro susceptibilities of 128 isolates of east1⁺Escherichia coli from pre‐weaned and post‐weaned pigs with diarrhoea were tested with nine commonly used anti‐microbial agents by an agar dilution minimal inhibitory concentration (MIC) procedure according to National Committee for Clinical Laboratory Standards guidelines. For the isolates from pre‐weaned and post‐weaned pigs, most of them were susceptible to low concentrations (MIC₉₀) of tetracycline (4 and 2 μg/ml), ceftiofur (2 and 2 μg/ml), and colistin (4 and 2 μg/ml). Marked resistance was found in others.     

Pharmacokinetics of chloramphenicol following administration of intravenous and subcutaneous chloramphenicol sodium succinate,and subcutaneous chloramphenicol,to koalas (Phascolarctos cinereus)

Clinically normal koalas (n = 19) received a single dose of intravenous (i.v.) chloramphenicol sodium succinate (SS) (25 mg/kg; n = 6), subcutaneous (s.c.) chloramphenicol SS (60 mg/kg; n = 7) or s.c. chloramphenicol base (60 mg/kg; n = 6). Serial plasma samples were collected over 24–48 h, and chloramphenicol concentrations were determined using a validated high‐performance liquid chromatography assay. The median (range) apparent clearance (CL/F) and elimination half‐life (t_1/2) of chloramphenicol after i.v. chloramphenicol SS administration were 0.52 (0.35–0.99) L/h/kg and 1.13 (0.76–1.40) h, respectively. Although the area under the concentration–time curve was comparable for the two s.c. formulations, the absorption rate‐limited disposition of chloramphenicol base resulted in a lower median C_max (2.52; range 0.75–6.80 μg/mL) and longer median t_max (8.00; range 4.00–12.00 h) than chloramphenicol SS (C_max 20.37, range 13.88–25.15 μg/mL; t_max 1.25, range 1.00–2.00 h). When these results were compared with susceptibility data for human Chlamydia isolates, the expected efficacy of the current chloramphenicol dosing regimen used in koalas to treat chlamydiosis remains uncertain and at odds with clinical observations.