P‐74 Evaluation of the in vivo effects of Tris‐EDTA and chlorhexidine digluconate 0.15% solution in chronic bacterial otitis externa: 11 cases

The objectives of this study were to evaluate in vivo tolerance, and antimicrobial and clinical activities of a topical otic preparation containing EDTA tromethamine (Tris) and chlorhexidine digluconate 0.15% solution (Otodine^®) in dogs with chronic bacterial otitis externa. Eleven dogs were included. The affected ears were filled with the solution once daily during a 2‐week period. Dogs were evaluated on days 0, 14 and 28. Three clinical parameters (exudate, erythema, pain) and three cytologic parameters (Malassezia, cocci, rods) were scored (0–4 scale) by otoscopic and cytological examinations of otic exudate. Bacterial cultures were performed at each time point. If there were bacteria on cytological examination on day 14, the dogs were treated with the original product, with the addition of enrofloxacin (5%) applied 10 min after the original product, for a further 2 weeks. All 11 cases yielded isolates of resistant gram‐negative bacteria; gram‐positive bacteria were also isolated from six of 11 dogs. On day 14, six of 11 dogs were negative on culture examination; on day 28, 10 of 11 were negative and only one case had a positive culture. On day 14, clinical and microbial scores (cytology) were reduced by 54.6 and 71.1%, respectively, and by 85.7 and 94% on day 28. All cases reported good tolerance of the treatment. The results show that this ear solution was helpful in the management of chronic bacterial otitis externa in dogs and was well tolerated. There seems to be a synergistic effect of the combination of Tris‐EDTA/chlorhexidine digluconate 0.15% solution, and an antimicrobial agent (enrofloxacin) against resistant gram‐positive and gram‐negative bacteria. Funding: Self‐funded.     

Pharmacokinetics of an intravenous and oral dose of enrofloxacin in white rhinoceros (Ceratotherium simum)

South Africa currently loses over 1000 white rhinoceros (Ceratotherium simum) each year to poaching incidents, and numbers of severely injured victims found alive have increased dramatically. However, little is known about the antimicrobial treatment of wounds in rhinoceros. This study explores the applicability of enrofloxacin for rhinoceros through the use of pharmacokinetic‐pharmacodynamic modelling. The pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin were evaluated in five white rhinoceros after intravenous (i.v.) and after successive i.v. and oral administration of 12.5 mg/kg enrofloxacin. After i.v. administration, the half‐life, area under the curve (AUC_tot), clearance and the volume of distribution were 12.41 ± 2.62 hr, 64.5 ± 14.44 μg ml^?1 hr^?1, 0.19 ± 0.04 L h^?1 kg^?1, and 2.09 ± 0.48 L/kg, respectively. Ciprofloxacin reached 26.42 ± 0.05% of the enrofloxacin plasma concentration. After combined i.v. and oral enrofloxacin administration oral bioavailability was 33.30 ± 38.33%. After i.v. enrofloxacin administration, the efficacy marker AUC₂₄: MIC exceeded the recommended ratio of 125 against bacteria with an MIC of 0.5 μg/mL. Subsequent intravenous and oral enrofloxacin administration resulted in a low Cmax: MIC ratio of 3.1. The results suggest that intravenous administration of injectable enrofloxacin could be a useful drug with bactericidal properties in rhinoceros. However, the maintenance of the drug plasma concentration at a bactericidal level through additional per os administration of 10% oral solution of enrofloxacin indicated for the use in chickens, turkeys and rabbits does not seem feasible.     

Pharmacokinetics of enrofloxacin and ceftiofur in plasma,interstitial fluid,and gastrointestinal tract of calves after subcutaneous injection,and bactericidal impacts on representative enteric bacteria

This study's objectives were to determine intestinal antimicrobial concentrations in calves administered enrofloxacin or ceftiofur sodium subcutaneously, and their impact on representative enteric bacteria. Ultrafiltration devices were implanted in the ileum and colon of 12 steers, which received either enrofloxacin or ceftiofur sodium. Samples were collected over 48 h after drug administration for pharmacokinetic/pharmacodynamic analysis. Enterococcus faecalis or Salmonella enterica (5 × 10⁵ CFU/mL of each) were exposed in vitro to peak and tail (48 h postadministration) concentrations of both drugs at each location for 24 h to determine inhibition of growth and change in MIC. Enrofloxacin had tissue penetration factors of 1.6 and 2.5 in the ileum and colon, while ciprofloxacin, an active metabolite of enrofloxacin, was less able to cross into the intestine (tissue penetration factors of 0.7 and 1.7). Ceftiofur was rapidly eliminated leading to tissue penetration factors of 0.39 and 0.25. All concentrations of enrofloxacin were bactericidal for S. enterica and significantly reduced E. faecalis. Peak ceftiofur concentration was bactericidal for S. enterica, and tail concentrations significantly reduced growth. E. faecalis experienced growth at all ceftiofur concentrations. The MICs for both organisms exposed to peak and tail concentrations of antimicrobials were unchanged at the end of the study. Enrofloxacin and ceftiofur achieved intestinal concentrations capable of reducing intestinal bacteria, yet the short exposure of ceftiofur in the intestine may select for resistant organisms.     

Antimicrobial activity of enrofloxacin against Staphylococcus intermedius strains isolated from canine pyodermas

This study examined and compared the minimal inhibition concentrations (MICs) of enrofloxacin against 393 Staphylococcus intermedius strains isolated in France from canine pyodermas during three different years, 1995 (174 isolates), 1997 (101 isolates) and 1999 (118 isolates). The MICs of enrofloxacin against these strains ranged from 0.063 to 64 mg L^?1, with MIC₅₀ and MIC₉₀ equal to 0.125 and 0.25 mg L^?1, respectively. Two resistant strains were found, but only among isolates collected in 1999. The data show that resistance to enrofloxacin among S. intermedius strains is still rare in dogs, but the selection in vitro of variants in which the MICs were increased 4–16‐fold after 10 serial passages in subinhibitory concentrations of enrofloxacin suggests that inappropriate use might favour the development of resistant strains in vivo.     

Prospective evaluation of the safety of compounded bulk material L‐asparaginase in dogs with lymphoma

Use of compounded L‐asparaginase became routine in veterinary oncology when manufacturing of Elspar^® was discontinued in 2012. The objective of this study was to evaluate the safety of compounded L‐asparaginase (CLASP, KRS Global Biotechnology, Boca Raton, FL, USA) in comparison with Elspar^® (Lundbeck LLC, Deerfield, IL, USA). In addition, we documented the response to CLASP in combination with a corticosteroid in this population of dogs with lymphoma. Dogs were prospectively treated with 10 000 IU/m² CLASP or Elspar^® subcutaneously. Corticosteroids were administered concurrently. Adverse events (AE) were assessed according to the Veterinary Cooperative Oncology Group Common Terminology Criteria for Adverse Events v1.1 (VCOG‐CTCAE). Response was recorded. Seventy‐three dogs received 75 treatments (CLASP, n = 47; Elspar^®, n = 28). No AE were attributed to CLASP. Grade I and II AE probably or possibly related to treatment were observed following two Elspar^® treatments. The overall response rate to the combination of CLASP and a corticosteroid was 80% (24% CR and 56% PR). In combination with a steroid, the compounded L‐asparaginase evaluated in this study is safe and demonstrates activity against canine lymphoma. In the face of the discontinuation of Elspar^®, veterinarians should seek compounded LASP products that have been tested for activity, purity, and sterility.     

Effects of endotoxin-induced fever and probenecid on disposition of enrofloxacin and its metabolite ciprofloxacin after intravascular administration of enrofloxacin in goats

Pharmacokinetics of enrofloxacin and its active metabolite ciprofloxacin were investigated in normal, febrile and probenecid‐treated adult goats after single intravenous (i.v.) administration of enrofloxacin (5 mg/kg). Pharmacokinetic evaluation of the plasma concentration–time data of enrofloxacin and ciprofloxacin was performed using two‐ and one‐compartment open models, respectively. Plasma enrofloxacin concentrations were significantly higher in febrile (0.75–7 h) and probenecid‐treated (5–7 h) goats than in normal goats. The sum of enrofloxacin and ciprofloxacin concentrations in plasma ≥0.1 μg/mL was maintained up to 7 and 8 h in normal and febrile or probenecid‐treated goats, respectively. The t1/2β, AUC, MRT and ClB of enrofloxacin in normal animals were determined to be 1.14 h, 6.71 μg.h/mL, 1.5 h and 807 mL/h/kg, respectively. The fraction of enrofloxacin metabolized to ciprofloxacin was 28.8%. The Cmax., t1/2β, AUC and MRT of ciprofloxacin in normal goats were 0.45 μg/mL, 1.79 h, 1.84 μg.h/mL and 3.34 h, respectively. As compared with normal goats, the values of t1/2β (1.83 h), AUC (11.68 μg ? h/mL) and MRT (2.13 h) of enrofloxacin were significantly higher, whereas its ClB (430 mL/h/kg) and metabolite conversion to ciprofloxacin (8.5%) were lower in febrile goats. The Cmax. (0.18 μg/mL) and AUC (0.99 μg.h/mL) of ciprofloxacin were significantly decreased, whereas its t1/2β (2.75 h) and MRT (4.58 h) were prolonged in febrile than in normal goats. Concomitant administration of probenecid (40 mg/kg, i.v.) with enrofloxacin did not significantly alter any of the pharmacokinetic variables of either enrofloxacin or ciprofloxacin in goats.     

The addition of calcium ion chelator,EGTA to thawing solution improves fertilizing ability in frozen–thawed boar sperm

In our previous study, seminal plasma effectively suppressed the induction of sperm to capacitation‐like status and acrosome loss during the thawing process. However, because boar seminal plasma is contaminated with various kinds of bacteria and/or viruses, it is necessary to develop a thawing solution without animal‐derived materials. In this study, we focused on the increase of intracellular Ca²⁺ ([Ca²⁺]_i) in sperm after thawing and the negative effects of sperm qualities. After thawing, the fluorescent intensity of [Ca²⁺]_i indicator, Fluo‐3/AM, and the level of phosphorylated tyrosine residue of protein were increased in the sperm. Next, we investigated whether the addition of Ca²⁺ chelators (ethylenediaminetetraacetic acid (EDTA) and ethyleneglycoltetraacetic acid (EGTA)) improved post‐thawed sperm motility. When the frozen–thawed sperm were treated with 6 mmol/L EDTA + 6 mmol/L EGTA, sperm motility was significantly increased as compared with control (6 mmol/L EDTA alone) at all incubation periods (P < 0.05). The combinational treatment significantly suppressed the elevation of [Ca²⁺]_i and the tyrosine phosphorylation, which improved the acrosomal status and fertilizing ability in vitro. Furthermore, when the thawing method was applied for artificial insemination, the fertilization rate was significantly higher than control (P < 0.05, 33% vs. 82%). Thus, we conclude that the addition of EDTA + EGTA to thawing solution is a beneficial tool for artificial insemination using frozen–thawed boar sperm.     

In vitro antimicrobial activity of a commercial ear antiseptic containing chlorhexidine and Tris–EDTA

Minimum bactericidal concentrations (MBCs) of a commercial ear antiseptic containing chlorhexidine 0.15% and Tris–EDTA (Otodine^®) were determined by broth microdilution for 150 isolates representing the most common pathogens associated with canine otitis. The microorganisms were classified into three groups according to their levels of susceptibility. The most susceptible group included Staphylococcus pseudintermedius, Malassezia pachydermatis, Streptococcus canis and Corynebacterium auriscanis, which were generally killed by 1 : 64 dilution of the antiseptic product (MBC = 23/0.8 μg/mL of chlorhexidine/Tris–EDTA). The most resistant organism was Proteus mirabilis, which survived up to 1 : 8 dilution of the product (MBC = 375/12 μg/mL). Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus displayed intermediate MBCs ranging between 188/6 and 47/1.5 μg/mL. Interestingly, S. pseudintermedius was more susceptible than S. aureus, and no significant difference was observed between meticillin‐resistant and meticillin‐susceptible isolates within each species, indicating that antiseptic use is unlikely to co‐select for meticillin resistance. Although the concentrations required for killing (MBCs) varied considerably with microorganism type, the combination of chlorhexidine 0.15% and Tris–EDTA was active against all the pathogens most commonly involved in canine otitis.     

Effects of essential oil supplementation of a low‐energy diet on performance,intestinal morphology and microflora,immune properties and antioxidant activities in weaned pigs

A total of 144 weaned piglets were used to evaluate the effects of essential oil (EO) supplementation of a low‐energy diet on performance, apparent nutrient digestibility, small intestinal morphology, intestinal microflora, immune properties and antioxidant activities in weaned pigs. Pigs received a low‐energy diet (negative control, NC, digestible energy = 3250 kcal/kg), NC plus 0.025% EO or a positive control diet (PC, digestible energy = 3400 kcal/kg) for 28 days. Growth performance was similar between the EO group and PC group. However, EO supplementation increased (P < 0.05) average daily gain and the apparent digestibility of dry matter, crude protein and energy compared with pigs fed the NC diet. Greater (P < 0.05) villus height and lower (P < 0.05) counts of Escherichia coli and total anaerobes in the rectum in the EO group were observed compared with NC or PC groups. Pigs fed EO diet had higher (P < 0.05) concentrations of albumin, immunoglobulin A (IgA), IgG and total antioxidant capacity and lower fecal score than pigs fed the PC and NC diets. Above all, this study indicates that supplementation of EO to a low‐energy pig diet has beneficial results and obtains similar performance compared with normal energy (PC) diet.     

Thymus satureioides and Origanum majorana essential oils improve the quality of Beni Arouss buck semen during storage at 4°C

This study aims to investigate the effects of essential oils (EOs), extracted from Thymus satureioides (TS) and Origanum majorana (OM), on Beni Arouss buck semen quality stored in skimmed milk at 4°C. EOs were extracted by hydro-distillation, and the chemical compounds were determined. Ejaculates were collected from six Beni Arouss bucks, once a week for 10 weeks, and they were pooled, divided into five equal aliquots and diluted to 400 × 10⁶ sperm/ml with skimmed milk supplemented with 0.01% of OM EO, 0.01% of TS EO, 0.05% of OM EO and 0.05% of TS EO. Non-supplemented skimmed milk was considered as a control. Semen motility, kinematic parameters, viability, abnormality, membrane integrity and lipid peroxidation were evaluated at 0, 4, 8, 24, 28, 32 and 48 hr of liquid storage at 4°C. The main EO components were carvacrol (31.7%), thymol (28.0%) and borneol (14.4%) for TS, and terpinene-4-ol (31.2%), γ-terpinene (17.4%) and α-terpinene (12.7%) for OM. The results highlighted a dose-dependent effect of TS and OM EOs on all semen quality parameters. 0.01% of both EOs had a beneficial effect on the sperm preservation stored at 4°C compared with control (p < .05) excepted for the straight-line velocity. The 0.05% EO addition had harmful effects during storage particularly for TS EO. In conclusion, 0.01% of TS and OM EOs are recommended to improve the Beni Arouss buck semen preservation at 4°C.     

Mutant prevention concentration,pharmacokinetic–pharmacodynamic integration,and modeling of enrofloxacin data established in diseased buffalo calves

The pharmacokinetic–pharmacodynamic (PK/PD) modeling of enrofloxacin data using mutant prevention concentration (MPC) of enrofloxacin was conducted in febrile buffalo calves to optimize dosage regimen and to prevent the emergence of antimicrobial resistance. The serum peak concentration (C_max), terminal half‐life (t_1/2K₁₀₎, apparent volume of distribution (Vd_(area)/F), and mean residence time (MRT) of enrofloxacin were 1.40 ± 0.27 μg/mL, 7.96 ± 0.86 h, 7.74 ± 1.26 L/kg, and 11.57 ± 1.01 h, respectively, following drug administration at dosage 12 mg/kg by intramuscular route. The minimum inhibitory concentration (MIC), minimum bactericidal concentration, and MPC of enrofloxacin against Pasteurella multocida were 0.055, 0.060, and 1.45 μg/mL, respectively. Modeling of ex vivo growth inhibition data to the sigmoid E_max equation provided AUC_24 h/MIC values to produce effects of bacteriostatic (33 h), bactericidal (39 h), and bacterial eradication (41 h). The estimated daily dosage of enrofloxacin in febrile buffalo calves was 3.5 and 8.4 mg/kg against P. multocida/pathogens having MIC₉₀ ≤0.125 and 0.30 μg/mL, respectively, based on the determined AUC_24 h / MIC values by modeling PK/PD data. The lipopolysaccharide‐induced fever had no direct effect on the antibacterial activity of the enrofloxacin and alterations in PK of the drug, and its metabolite will be beneficial for its use to treat infectious diseases caused by sensitive pathogens in buffalo species. In addition, in vitro MPC data in conjunction with in vivo PK data indicated that clinically it would be easier to eradicate less susceptible strains of P. multocida in diseased calves.     

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.     

Evaluation of enrofloxacin use in koalas (Phascolarctos cinereus) via population pharmacokinetics and Monte Carlo simulation

Clinically normal koalas (n = 6) received a single dose of intravenous enrofloxacin (10 mg/kg). Serial plasma samples were collected over 24 h, and enrofloxacin concentrations were determined via high‐performance liquid chromatography. Population pharmacokinetic modeling was performed in S‐ADAPT. The probability of target attainment (PTA) was predicted via Monte Carlo simulations (MCS) using relevant target values (30–300) based on the unbound area under the curve over 24 h divided by the minimum inhibitory concentration (MIC) (fAUC_0–24/MIC), and published subcutaneous data were incorporated (Griffith et al., 2010). A two‐compartment disposition model with allometrically scaled clearances (exponent: 0.75) and volumes of distribution (exponent: 1.0) adequately described the disposition of enrofloxacin. For 5.4 kg koalas (average weight), point estimates for total clearance (SE%) were 2.58 L/h (15%), central volume of distribution 0.249 L (14%), and peripheral volume 2.77 L (20%). MCS using a target fAUC_0–24/MIC of 40 predicted highest treatable MICs of 0.0625 mg/L for intravenous dosing and 0.0313 mg/L for subcutaneous dosing of 10 mg/kg enrofloxacin every 24 h. Thus, the frequently used dosage of 10 mg/kg enrofloxacin every 24 h subcutaneously may be appropriate against gram‐positive bacteria with MICs ≤ 0.03 mg/L (PTA > 90%), but appears inadequate against gram‐negative bacteria and Chlamydiae in koalas.     

Determination of Intracellular Concentrations of Free and Two Types of Liposome‐Encapsulated Enrofloxacin in Anatolian Shepherd Dog Monocytes

In this study, it was evaluated the accumulation of free and two types of liposome‐encapsulated enrofloxacin (LEE) at the doses of 0.25, 0.5 and 1 μg/ml, which were clinically relevant concentrations into monocytes of healthy Anatolian shepherd dogs. Enrofloxacin was encapsulated with two different types of liposome in multilamellar large vesicles (MLV). Type A MLV composed of 15 mg egg phosphatidylcholine and 35 mg cholesterol, Type B MLV composed of phosphatidylcholine (PC), cholesterol and enrofloxacin, in a molar ratio of 1 : 1 : 1. The mean sizes of Type A and Type B liposome were found to be 7.65 and 4.27 μm, respectively. However, the mean encapsulation rate determined of Type A (13 ± 2%) was found lower than Type B liposome (44 ± 3%). The amounts of intracellular enrofloxacin concentrations were determined by high performance liquid chromatography. Type B LEE accumulated significantly higher level into monocytes when compared to free drug or Type A liposome. This study showed that Type B LEE markedly concentrated within monocytes and may improve the antibacterial efficacy of the antibiotic.     

Comparative Pharmacokinetics of Enrofloxacin and Tissue Concentrations of Parent Drug and Ciprofloxacin after Intramuscular Administrations of Free and Liposome‐Encapsulated Enrofloxacin in Rabbits

Pharmacokinetic properties and tissue concentrations of enrofloxacin and ciprofloxacin were compared after intramuscular (i.m.) administrations of free and liposome‐encapsulated enrofloxacin at the dose of 5 mg/kg body weight (bw). Twelve healthy adult New Zealand white rabbits were used in the experiment. Blood samples were obtained at 10, 20, 40, 60 and 90 min and 2, 4, 6, 8 and 12 h and tissue samples were collected 24 h after injection. Concentrations of drugs in serum were determined by high‐performance liquid chromatography. Pharmacokinetics were best described by a two‐compartment open model. Results indicated that absorption rate was slow, peak concentration was higher (P < 0.05), and the time to peak concentration (t_max ? 1.5 h) was significantly longer (P < 0.05) for liposome‐encapsulated enrofloxacin (LEE) when compared with free enrofloxacin. Values of elimination half‐life (t_1/2β = 12.9 h) and mean residence time (MRT = 17.6 h) of liposome‐encapsulated enrofloxacin were longer (P < 0.05) and total clearance (Cl = 0.43 l/h/kg) was lower than those of free form. Moreover, the distribution volume at steady‐state (V_d(ss) = 14.4 l/kg) of enrofloxacin administered encapsulated into liposomes was significantly higher (P < 0.05) than that of free enrofloxacin (FE). The tissue levels of enrofloxacin and ciprofloxacin after LEE injection were not different (P > 0.05) from FE. In conclusion, the result of present study suggest that LEE may be a beneficial and valuable formulation in the treatment of infectious diseases caused by sensitive pathogens in animals, providing sustained drug release from injection side and prolonged therapeutic serum concentrations after i.m. administration.     

Strategic administration of enrofloxacin in poultry to achieve higher maximal serum concentrations

To achieve a higher maximal serum concentration (Cs(max)) of enrofloxacin after oral administration of 10mg/kg/day of three commercial preparations of enrofloxacin to chickens, two concentrations of the drug were tested (0.1 and 0.2%), under controlled laboratory conditions. A single oral bolus dose was delivered directly into the proventriculus of each of 240 chickens, which were equally divided into six groups: three received the customary concentration (0.1%), and three received the higher concentration. A quantitative/qualitative microbiological analytical method to determine serum concentrations of enrofloxacin and a software program to obtain pharmacokinetic variables, revealed that time vs. concentration relationships best fitted double peak shape curves, Cs(max1) and Cs(max2). Statistically significant (P>0.01) increments were obtained when 0.2% enrofloxacin oral solutions from the three different commercial preparations were administered. The increments ranged from 175% to 338% for Cs(max1) and 69% to 342% for Cs(max2). Optimal bactericidal concentrations of enrofloxacin are usually twice the value of their minimal inhibitory concentration. Although clinical trials are now required, it would appear that increments in the serum concentration of enrofloxacin may reduce to the rate at which bacterial resistance occurs and so increase clinical efficacy without affecting the cost per treatment.     

Comparative mutant prevention concentration and mechanism of resistance to veterinary fluoroquinolones in Staphylococcus pseudintermedius

Background – The problem of antibacterial drug resistance is increasing worldwide, in part due to the therapeutic concentrations currently used based on the minimal inhibitory concentration (MIC) as a measure of potency are often the very concentrations required to selectively enrich the resistant mutant portion of the population. A mutant prevention concentration (MPC)‐based dosing strategy is suggested to improve the therapeutic outcome based on the MIC. Objective – Our aim was to investigate the MPC and mechanism of resistance to various fluoroquinolones using recent Staphylococcus pseudintermedius isolates from canine pyoderma. Methods – The broth microdilution method for MIC and a series of agar plates containing different concentrations of fluoroquinolones were inoculated with ～10¹⁰ colony‐forming units of the bacterial culture for MPC were used. PCR was used to identify mutation in the resistant isolates. Results – The rank order of potency based on MIC and MPC was ciprofloxacin = enrofloxacin ≥ marbofloxacin > difloxacin ≥ orbifloxacin. Integrating our data with reported pharmacokinetic data at the recommended dose ranges revealed that only high doses of ciprofloxacin, enrofloxacin and marbofloxacin could achieve a maximal plasma concentration (C_max) greater than the MPC of 90% of isolates (C_max/MPC₉₀). The overall rank of potency against S. pseudintermedius, based on C_max/MIC, C_max/MPC, the area under concentration–time curve (AUC)/MIC and AUC/MPC values, was in decreasing order: enrofloxacin > ciprofloxacin ≥ marbofloxacin ≥ orbifloxacin = difloxacin. Sequencing of the quinolone resistant determining region of gyrA, gyrB, grlA and grlB of resistant strains showed a base‐pair substitution in both gyrA and gyrB that resulted in Ser‐84 to Leu and Ser‐80 to Arg amino acid changes, respectively. Conclusions and clinical importance – High doses of ciprofloxacin, enrofloxacin and marbofloxacin could minimize the selection of resistant mutants, whereas the possibility of selecting mutants with the conventional doses of difloxacin and orbifloxacin, and low clinical doses of all fluoroquinolones, seems high.     

Comparative in vitro antimicrobial efficacy of commercial ear cleaners

The aim of this study was to compare the antimicrobial efficacy of ear cleaners against Staphylococcus intermedius, Pseudomonas aeruginosa and Malassezia pachydermatis. Single isolates of each organism were incubated in duplicate at 38 °C for 30 min with each ear cleaner diluted 1/2 to1/256 in phosphate‐buffered saline. Positive and negative controls were included. Aliquots were then incubated for 16–18 h on sheep blood agar (bacteria) or for 3 days on Sabouraud's dextrose agar (Malassezia) at 38 °C. The lowest dilutions exhibiting 100% antimicrobial efficacy for S. intermedius were: Cleanaural^® Dog 1/32; Sancerum^® 1/16; Otoclean^® 1/4; EpiOtic^® 1/2; MalAcetic^® 1/2; and Triz Plus^® 1/2. The results for P. aeruginosa were Sancerum^® and Triz Plus^® 1/16; Cleanaural^® Dog and EpiOtic^® 1/8; Otoclean^® 1/4; and MalAcetic^® 1/2. Results for M. pachydermatis were: Cleanaural^® Dog 1/32; Sancerum^®, Otoclean^®, EpiOtic^® and Triz Plus^® 1/8; and MalAcetic^® 1/4. Cleanaural^® Cat, MalAcetic HC^® and Triz EDTA^® did not display any antimicrobial activity at any dilution. Antimicrobial activity appeared to be associated with the presence of isopropyl alcohol, parachlorometaxylenol and a low pH. The results of this study may help clinicians make evidence‐based decisions when selecting ear cleaners for use in individual cases.     

The Content of β-endorphin-like Immunoreactivity in Porcine Corpus Luteum and the Potential Roles of Progesterone, Oxytocin and Prolactin in the Regulation of β-endorphin Release from Luteal Cells in vitro

The amount of β‐endorphin‐like immunoreactivity (β‐END‐LI) in porcine corpora lutea from several stages of the oestrous cycle and the effects of progesterone, oxytocin, and prolactin on β‐END‐LI secretion in vitro by luteal cells were studied. Porcine corpora lutea obtained on days 1–5, 6–10, 11–13, 14–18, and 19–21 of the cycle were used to prepare extracts for β‐END‐LI determination. Additionally, corpora lutea from days 11–13 and 14–18 were enzymatically dissociated and isolated luteal cells were used for further study of β‐endorphin secretion in vitro. Cells were cultured in serum‐free defined M 199 medium (10⁶ cells/ml) at 37°C under 5% CO₂ in air, for 12 h. The influences of the following factors on β‐END‐LI secretion by luteal cells were tested: progesterone (10^–9, 10^–7 and 10^–5M ), oxytocin (0.01, 0.1, 1 and 10 ng/ml), and prolactin (0.1, 1, 10 and 100 ng/ml). The β‐END‐LI contents in extracts and media were measured by radioimmunoassay. The tissue concentration of β‐END‐LI was lowest on days 1–5 of the cycle (0.35 ± 0.03 ng/g wet tissue). Subsequently, it constantly increased to the highest value on days 14–18 (16.58 ± 0.52 ng/g wet tissue) and on days 19–21 it declined (11.10 ± 0.52 ng/g wet tissue). Progesterone at a low dose (10^–9 M ) resulted in significant (p < 0.05) increases and decreases in β‐END‐LI secretion by luteal cells from days 11–13 and 14–18, respectively. Higher doses of progesterone (10^–7 and 10^–5 M ) had no effect on β‐END‐LI release, compared with the control group. All dose‐levels of oxytocin used decreased β‐END‐LI secretion by luteal cells on days 11–13 and 14–18 of the cycle. Prolactin at doses of 0.1 and 1 ng/ml on days 11–13, and all doses tested on days 14–18 resulted in decreases in β‐END‐LI release from luteal cells. These results document evident changes in β‐END‐LI content in the pig corpus luteum during its development and indicate the potential roles of progesterone, oxytocin, and prolactin in luteal cell secretion of β‐END‐LI.