Vet Dermatol 2013; 24: 519e122

DOI: 10.1111/vde.12071

TrisEDTA signicantly enhances antibiotic efcacy against multidrug-resistant Pseudomonas aeruginosa in vitro
Laura M. Buckley*, Neil A. McEwan* and Tim Nuttall
*School of Veterinary Science, The University of Liverpool, Leahurst Campus, Neston, Cheshire, CH64 7TE, UK The Royal (Dick) School of Veterinary Studies, Easter Bush Veterinary Centre, The University of Edinburgh, Roslin, Midlothian, EH25 9RG, UK Correspondence: Tim Nuttall, The Royal (Dick) School of Veterinary Studies, Easter Bush Veterinary Centre, The University of Edinburgh, Roslin EH25 9RG, UK. E-mail: tim.nuttall@ed.ac.uk.

Background Multidrug-resistant Pseudomonas aeruginosa commonly complicates chronic bacterial otitis in dogs. Hypothesis/Objectives The aim of this in vitro study was to determine the effect of ethylenediaminetetraacetic acidtromethamine (TrisEDTA) on the minimal bactericidal concentrations (MBCs) and minimal inhibitory concentrations (MICs) of marbooxacin and gentamicin for multidrug-resistant P. aeruginosa isolates from cases of canine otitis. Methods Eleven isolates were identied as multidrug resistant on disc diffusion; 10 were resistant to marbooxacin and two were resistant to gentamicin. Isolates were incubated for 90 min with each antibiotic alone and in combination with TrisEDTA at concentrations of 0.075 lg/mL to 5 mg/mL for marbooxacin, 0.001 lg/mL to 10 mg/mL for gentamicin and 17.8:4.7 to 0.14:0.04 mg/mL for TrisEDTA. Positive and negative controls were included. Aliquots of each antibiotic and/or TrisEDTA concentration were subsequently transferred to sheep blood agar to determine the MBCs, and tryptone soy broth was added to the remaining suspensions to determine the MICs. Results TrisEDTA alone was bacteriostatic but not bactericidal at any concentration. The addition of Tris EDTA signicantly reduced the median MBC (from 625 to 468.8 lg/mL; P < 0.001) and MIC (from 29.3 to 2.4 lg/mL; P = 0.008) of marbooxacin, and the median MBC (from 625 to 39.1 lg/mL) and MIC (from 19.5 to 1.2 lg/mL) of gentamicin (both P < 0.001). Conclusions and clinical importance TrisEDTA signicantly reduced the MBCs and MICs of marbooxacin and gentamicin for multidrug-resistant P. aeruginosa in vitro. This may be of use to clinicians managing these infections in dogs.

Introduction
Pseudomonas spp. are ubiquitous environmental, Gramnegative bacteria. They are transient organisms of canine skin and opportunistic invaders in pathological processes such as otitis.1 Pseudomonas aeruginosa is the most common Gram-negative isolate in cases of canine otitis.26 The severity of these infections is a considerable therapeutic challenge and welfare concern in veterinary dermatology. The pathogenesis and management of chronic otitis is complex due to the need to manage primary, predisposing and perpetuating factors.7,8 Further difculty lies in selecting antibiotics in cases complicated with multidrug-resistant P. aeruginosa.9

Accepted 13 April 2012 Sources of Funding: Virbac SA and The University of Liverpool School of Veterinary Science. Conict of Interest: Tim Nuttall and Neil McEwan have received research and other funding from Virbac Animal Health, Novartis Animal Health and Dechra Veterinary Products. These companies have products relevant to otitis. 2013 ESVD and ACVD, Veterinary Dermatology, 24, 519e122.

Antibiotic resistance is seen commonly in P. aeruginosa. Intrinsic resistance to a number of antimicrobials exists due the low permeability of the outer membrane and chromosomally encoded antibiotic efux pumps.1012 Resistance to cefalexin, clindamycin, fusidic acid and amoxicillinclavulanic acid has been reported in 94100% of P. aeruginosa isolates obtained from canine and feline otitis.5 Acquired resistance can also develop via the induction of b-lactamase and aminoglycoside-modifying enzymes, mutations in the genes encoding topoisomerase II (gyrA and gyrB) and IV (parC and parE), diminished expression of outer membrane proteins and upregulation of the genes encoding efux pumps.1315 Resistance to enrooxacin is seen in 2638% of isolates.5,6 The accumulation of multiple resistance mechanisms leads to the development of multidrug-resistant strains. Tissue concentrations of enrooxacin capable of killing resistant organisms cannot be achieved within the ear canal using systemic therapy.16 Topical antibiotic solutions are often preferred in cases of chronic otitis because much higher antibiotic concentrations can be achieved.7,8,17 The use of home-made topical solutions containing injectable antibiotics, such as marbooxacin
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and gentamicin, is common practice in cases of canine Pseudomonas otitis. However, little information is available on the most appropriate antibiotic concentrations for topical use, particularly for multidrug-resistant isolates. Ethylenediaminetetraacetic acidtromethamine (Tris EDTA) has been shown to inhibit the growth of P. aeruginosa in vitro18 and in vivo19 and to potentiate the action of some antibiotics against P. aeruginosa in vitro2027 and in vivo.28 TrisEDTA has also been shown to reduce the minimal inhibitory concentration (MIC) of enrooxacin against ciprooxacin-resistant P. aeruginosa.29 It may therefore be of benet in managing cases of P. aeruginosa otitis, particularly those with multidrug resistance. It is, however, unknown whether TrisEDTA exerts similar effects for other antibiotics and whether TrisEDTA reduces the minimal bactericidal concentration (MBC). The aim of this in vitro study was to determine the impact of TrisEDTA on MBCs and MICs of two commonly used injectable antibiotic solutions, marbooxacin and gentamicin, for multidrug-resistant P. aeruginosa isolates from cases of otitis.

Isolate preparation
For each assay, the isolates were incubated overnight at 37C on sheep blood agar. Bacterial colonies were then placed in 4 mL sterile phosphate-buffered saline (PBS) and washed by vortexing for 30 s. The suspension was centrifuged for 3 min at 75 g and the supernatant discarded. The cell pellets were then resuspended by vortexing for 30 s in 4 mL PBS. The dilution of each suspension was adjusted by the addition of PBS to give an optical density (OD) of 0.3 at 570 nm (Cecil instruments spectrophotometer, Cambridge, UK). Preliminary studies showed that these solutions contained approximately 2 9 106 colony-forming units/mL.

Antibiotic/TrisEDTA preparation
Antibiotic concentrations of 0.15 lg/mL to 10 mg/mL for marbotoquinol UK Ltd, Buckingham, oxacin (Marbocyl SA 200 mg; Ve UK) and 0.002 lg/mL to 20 mg/mL for gentamicin (Genticin 40 mg/mL injection; Hospira UK Ltd, Royal Leamington Spa, UK) were achieved by performing doubling dilutions of the stock antibiotic solution with PBS. One hundred microlitres of each antibiotic concentration was placed into the wells of a sterile 96-well culture plate (NuncTM NunclonTM Delta 96-Well MicroWell; Fisher Scientic UK, Loughborough, UK). Fifty microlitres of PBS plus 50 lL of each microbial suspension (1 9 105 colony-forming units) were added to each well to achieve nal antibiotic concentrations of 0.075 lg/mL to 5 mg/mL for marbooxacin and 0.001 lg/mL to 10 mg/mL for gentamicin. The process was repeated with the addition of 50 lL of 4.5:1.2 mg/mL TrisEDTA to each antibiotic solution in the place of PBS. To determine the effect of TrisEDTA alone, the process was repeated using 100 lL of TrisEDTA (TrizAuralTM; Dechra Veterinary Products, Shrewsbury, UK) at concentrations ranging from 35.5:9.4 to 0.3:0.07 mg/mL. Following the addition of the microbial suspension and PBS, this gave nal TrisEDTA concentrations of 17.8:4.7 to 0.14:0.04 mg/mL. Positive control wells using 100 lL of each microbial suspension and 100 lL of PBS and three negative control wells with 200 lL of PBS, 200 lL of TrisEDTA and 200 lL of each antibiotic only were included. All the wells were run in duplicate, and the plates were incubated at 37C for 90 min.

Materials and methods

The methods were adapted from a previous study by Swinney et al.30

Isolates
The P. aeruginosa isolates were obtained from and tested by two commercial bacteriology laboratories. The 11 isolates selected for use in the study were from clinical cases of canine otitis externa and/ or media and had been identied as multidrug resistant by disc diffusion using the Clinical and Laboratory Standards Institutes methodology (Table 1).31

Table 1. The results of antimicrobial disc diffusion susceptibility testing* for the 11 multidrug-resistant Pseudomonas aeruginosa isolates used in the study Isolate 1 2 3 4 5 6 7 8 9 10 11 bps bpr ca r r r r r r r r r r r 18 13 c r r r r r r r r r r nt

cf r r r r r r r r r r nt 24 20

ot r r r r r r r r r r nt

ts r r r r r r r r r r s 16 10

e r r r r r r r r r r r 23 16

m r r r r r r r r r s r 20 14

g s s s s s s s r s r s 15 12

n r r s r r r nt r r r nt 17 15

t s s s s s s s r r s s 15 14

cp r r r r s r nt s s s s 21 15

a s s s s s s s r s r s 17 14

tb s s s s s s s r s r s 15 12

p s s s s s s nt s s s s 12 11

d nt nt nt nt nt nt r nt nt nt r 19 14

ap nt nt nt nt nt nt r nt nt nt r 17 13

cl nt nt nt nt nt nt nt nt nt nt r 21 14

co nt nt nt nt nt nt nt nt nt nt r 18 14

Abbreviations: a, amikacin; ap, ampicillin; bpr, zone diameter breakpoint to the nearest whole millimetre compatible with resistance to antibiotic; bps, zone diameter breakpoint to the nearest whole millimetre compatible with sensitivity to antibiotic; c, cefalexin; ca, amoxicillinclavulanic acid; cf, cefovecin; cl, clindamycin; co, cephalothin; cp, ciprooxacin; d, doxycycline; e, enrooxacin; g, gentamicin; m, marbooxacin; n, neomycin; nt, not tested; ot, oxytetracycline; p, polymixin B; r, resistant; s, sensitive; t, ticarcillin; tb, tobramycin; and ts, trimethoprimsulphonamide. *Testing was carried out by commercial bacteriology laboratories according to CLSI performance standards for antimicrobial disc and dilution susceptibility tests for bacteria isolated from animals document M31-A3 2008. Inferred from cephalothin according to performance standards for antimicrobial susceptibility testing, CLSI document M100-S21 2011. Manufacturers guidelines (Zoetis, UK). Inferred from tetracycline (r 1 mm, s 15 mm) according to performance standards for antimicrobial susceptibility testing, CLSI document M100-S21, 2011.

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Determination of minimal bactericidal concentrations

The MBCs were determined by plating 10 lL of each suspension onto sheep blood agar and incubating at 37C for 18 h. The results were recorded as the mean total number of colony-forming units or as conuent growth. The purity of each culture was determined by gross colony appearance and cytological examination of cell morphology stained with Rapi-Diff II (Bios-Europe, Skelmersdale, UK). The MBC was considered to be the lowest concentration of antibiotic that prevented bacterial growth.

Table 3. Minimal bactericidal concentrations and MICs for marbooxacin alone and in combination with TrisEDTA for the 11 multidrug-resistant P. aeruginosa isolates used in this study MBC (lg/mL) Isolate 1 2 3 4 5 6 7 8 9 10 11 Median m 625 625 625 625 78.1 625 1250 625 1250 156.3 2500 625 mt 625 468.8 312.5 312.5 3.7 625 937.5 312.5 625 156.3 1250 468.8 MIC (lg/mL) m 39.1 625 14.7 29.3 9.8 625 625 7.3 3.7 1.8 312.5 29.3 mt 14.65 1.5 2.4 7.4 0.6 78.1 156.3 0.61 1.2 0.2 78.1 2.4

Determination of minimal inhibitory concentrations

The MICs were determined following the addition of 100 lL of tryptone soy broth to each well and incubation at 37C for 18 h. The OD at 570 nm was recorded using a Multiskan microtitre plate reader (Thermo Scientic, Loughborough, UK). The results were expressed as the mean OD corrected for background, and the MIC was considered to be the lowest concentration of antibiotic that prevented bacterial growth.

Statistical analysis
Graphpad Prism version 4 (Graphpad Inc., San Diego, CA, USA) was used to compare the MBCs and MICs for each antibiotic and each antibiotic plus TrisEDTA using the Wilcoxon signed rank test. Values of P < 0.05 were considered signicant.

Abbreviations: m, marbooxacin alone; MBC, minimal bacteriocidal concentration; and mt, marbooxacin and TrisEDTA.

Results
Controls In all cases, the microbial isolates incubated in PBS resulted in conuent growth on sheep blood agar. No bacterial growth was seen following culture of samples taken from the antibiotic-, PBS- or TrisEDTA-only wells. TrisEDTA When used alone, TrisEDTA was not bactericidal at any concentration. The MICs for TrisEDTA ranged from 2.2:0.6 mg/mL to 8.9:2.4 mg/mL (Table 2). Marbooxacin The results for marbooxacin are shown in Table 3. Bactericidal concentrations of marbooxacin for all the P. aeruginosa isolates were achieved with the concentration ranges used in the study. The median MBC for marbooxacin for the 11 isolates was 625 lg/mL (range 78.12500 lg/mL). When marbooxacin was combined

with TrisEDTA, there was a signicant (P < 0.001) reduction in the median MBC to 468.8 lg/mL (range 3.7 1250 lg/mL). The median MIC was 29.3 lg/mL (range 1.8625 lg/mL). When marbooxacin was combined with TrisEDTA, there was a signicant (P = 0.008) reduction in the median MIC to 2.4 lg/mL (range 0.2 156 lg/mL). Gentamicin The results for gentamicin are shown in Table 4. Bactericidal concentrations of gentamicin for all the P. aeruginosa isolates were achieved with the concentration ranges used in the study. The median MBC for gentamicin was 625 lg/mL (range 156.35000 lg/mL). When gentamicin was combined with TrisEDTA, the median MBC was signicantly (P < 0.001) reduced to 39.1 lg/mL (range 19.5 234.4 lg/mL). The median MIC was 19.5 lg/mL (range 2.478.1 lg/mL). When gentamicin was combined with TrisEDTA, the median MIC was signicantly (P < 0.001) reduced to 1.2 lg/mL (range 0.56.1 lg/mL).

Table 2. Minimal inhibitory concentrations (MICs) for TrisEDTA for the 11 multidrug-resistant P. aeruginosa isolates used in this study Isolate 1 2 3 4 5 6 7 8 9 10 11 TrisEDTA MIC (mg/mL) 8.9:2.4 4.4:1.2 4.4:1.2 2.2:0.6 4.4:1.2 2.2:0.6 4.4:1.2 4.4:1.2 8.9:2.4 4.4:1.2 4.4:1.2

Table 4. Minimal bactericidal concentrations and MICs for gentamicin alone and in combination with TrisEDTA for the 11 multidrugresistant P. aeruginosa isolates used in this study MBC (lg/mL) Isolate 1 2 3 4 5 6 7 8 9 10 11 Median g 625 468.8 156.3 312.5 468.8 1875 937.5 1250 625 5000 1250 625 gt 19.5 19.5 24.4 39.1 39.1 78.1 58.6 39.1 58.6 78.1 234.4 39.1 MIC (lg/mL) g 19.5 9.8 2.4 9.8 19.5 19.5 14.7 29.3 19.5 78.1 14.7 19.5 gt 6.1 0.5 1.2 0.6 0.6 1.2 1.2 0.6 1.8 4.9 1.8 1.2

The pairs of values are the MIC of Tris and the MIC of EDTA, respectively.

Abbreviations: g, gentamicin; and gt, gentamicin and TrisEDTA.

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Discussion
This is the rst study to demonstrate that TrisEDTA is able to enhance signicantly both the bactericidal and the inhibitory effects of marbooxacin and gentamicin against multidrug-resistant P. aeruginosa isolates. The small variations in the MICs of the TrisEDTA solution between the different isolates did not appear to inuence the results when the TrisEDTA was combined with the antibiotics. EDTA damages the outer cell wall membrane of Gramnegative bacteria by chelating divalent cations.32,33 This causes the release of lipopolysaccharides and renders the bacteria more permeable to other agents.3436 Tris is an alkaline buffer that potentiates the chelating action of EDTA.33,37 Marbooxacin is a third-generation uoroquinolone that kills Gram-negative bacteria by inhibiting DNA synthesis.38,39 It induces conformational changes in two enzymes, topoisomerase II (DNA gyrase) and topoisomerase IV, that inhibit normal enzyme activity.39,40 Gentamicin kills Gram-negative bacteria by irreversibly binding to receptors on the 30S subunit of bacterial ribosomes and inhibiting protein synthesis.4144 As marbooxacin and gentamicin act intracellularly and in a concentrationdependent manner, disruption of the bacterial cell wall by TrisEDTA should enhance their action. This could explain the reduction in MBCs and MICs seen in this study. The ability of TrisEDTA to damage the Gram-negative bacterial cell wall and potentiate the action of antimicrobial agents has been widely investigated and supports the ndings of this study.1829,45 For the 11 isolates used in this study, TrisEDTA appears to have a greater effect on the action of gentamicin than on that of marbooxacin. This may be due to differences in the mechanisms of action of the two antibiotics. Aminoglycosides have a number of actions in addition to the inhibition of protein synthesis. These actions include interference with the bacterial cellular electron transport system, misreadings in translation and damage to the bacterial cell membrane.41,46,47 Damage to the cell membrane involves the binding of gentamicin to outer membrane sites. This competitively displaces divalent cations that are required to cross-bridge lipopolysaccharide molecules.48 This process promotes further gentamicin uptake into the cell and can cause the formation of holes in the cell membrane, leading to cell lysis.42 As TrisEDTA works by a similar mechanism, it is likely that it is able to potentiate the activity of gentamicin to a greater extent. Another in vitro study using amikacin and neomycin combined with TrisEDTA against P. aeruginosa came to similar conclusions.27 The reason for the lack of reduction in MBCs for three of the P. aeruginosa isolates (1, 6 and 10) tested with marbooxacin and TrisEDTA is unclear. The results might be explained by the presence of mutations conferring high-level resistance to marbooxacin. Resistance to uoroquinolones can be acquired via mutation of the genes regulating efux pumps and those encoding topoisomerase II or IV.49 Topoisomerase II is the primary uoroquinolone target in the inhibition of DNA synthesis, and topoisomerase IV is a secondary target.13,40,50 Chromosomal resistance (step522

wise point mutations in target enzyme genes) is the major contributing factor to high uoroquinolone MICs, although outer membrane permeability and/or efux pumps also contribute. Consequently, TrisEDTA may help to overcome resistance induced by upregulation of efux pumps, but in the presence of mutations leading to high-level resistance in the primary and secondary targets, increased permeability of the bacterial cell wall may be insufcient to bring about a bactericidal effect. However, isolate 10 was sensitive to marbooxacin according to the disc diffusion breakpoints,31 so high-level resistance is unlikely to explain the lack of reduction in MBC in this case. Sequencing of the gyrA and gyrB, parC and parE genes would help to clarify why some isolates were unaffected by TrisEDTA. In addition, MIC testing with the presence of an efux inhibitor could assist in dening the resistance phenotype. This study could be criticised for not including a standard strain of P. aeruginosa as a control to validate the MIC testing and for using a method that differs from the published recommendations.31 Our aim was to design a study that would be comparable to the management of clinical cases of P. aeruginosa to determine whether TrisEDTA is able to enhance the action of commonly used injectable antibiotic solutions. The isolates were chosen to be difcult from the point of view of clinical management. The method used avoided the numerous confounding factors that would be encountered in an in vivo study but limited the ability to compare results with published standards. The antibiotic concentrations required to kill P. aeruginosa in this in vitro study can easily be achieved in the clinical setting and, although the results cannot be converted directly to the clinical setting, it is possible that the topical use of similar concentrations could kill bacterial isolates identied as resistant on disc diffusion and MIC testing. This has been suggested in another in vitro study, where MICs for an enrooxacinsilver sulfadiazine combination were determined for resistant P. aeruginosa isolates.51 In conclusion, this in vitro study demonstrates the ability of TrisEDTA signicantly to reduce the MBCs and MICs of marbooxacin and gentamicin against P. aeruginosa. Using TrisEDTA in combination with these topical antimicrobials may assist the management of dogs with otitis associated with multidrug-resistant P. aeruginosa. Further investigation is needed to determine how best to apply these ndings clinically. Further work to determine the contact time of the antibiotic with P. aeruginosa within the ear canal, the optimal concentration of antibiotic and TrisEDTA against P. aeruginosa and whether pretreatment or concurrent treatment with TrisEDTA is most effective would also be benecial.

Acknowledgements
The authors are grateful to Peter Graham of NationWide Laboratories and to Dorina Timofte and Andy Wattret of The University of Liverpool School of Veterinary Science for providing the bacterial isolates.
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sume  Re riennes chroniques canines sont fre quemment complique es par des PseudoContexte Les otites bacte sistantes. monas aeruginosa multi-re ses/Objectifs Le but de cette e tude in vitro e tait de de terminer leffet du Tris-EDTA (ethylenediHypothe ricides (MBCs) et les concenaminetetraacetic acidtromethamine) sur les concentrations minimales bacte trations minimales inhibitrices (MICs) de la marbooxacine et de la gentamicine pour les souches de sistantes issues de cas dotite canine. P. aeruginosa multi-re thodes Onze souches ont e  te  identie es comme multi-re sistantes sur disque de diffusion ; 10 e taient Me sistantes  taient re sistantes   te  incube es re a la marbooxacine et deux e a la gentamicine. Les souches ont e pendant 90 minutes avec chaque antibiotique seul et en combinaison avec le Tris-EDTA avec des concentrations de marbooxacine de 0.075 g/mL  a 5 mg/mL, de gentamicine de 0.001 g/mL  a 10 mg/mL et de ^les positifs et ne gatifs ont e  te  inclus. Une certaine Tris-EDTA de 17.8:4.7 to 0.14:0.04 mg mL. Les contro  de chaque antibiotique et/ou de Tris-EDTA a e  te  ensuite transfe  re e sur ge lose au sang de mouton quantite terminer les MBCs, et du bouillon tryptone soja a e  te  ajoute  aux suspensions restantes pour an de de terminer les MICs. de sultats Le Tris-EDTA seul a e  te  bacte riostatique mais pas bacte ricide quelque soit la concentration. Re duit signicativement la MBC me diane (de 625  Lajout de Tris-EDTA a re a 468.8 g/mL; P < 0.001) et la diane (de 625  MIC (de 29.3  a 2.4 g/mL; P = 0.008) de la marbooxacine, et la MBC me a 39.1 g/mL) et la MIC (de 19.5  a 1.2 g/mL) de la gentamicine (P < 0.001). Conclusions et importance clinique Le Tris-EDTA diminue signicativement les MBCs et les MICs de sistantes. Ceci pourrait e ^tre la marbooxacine et de la gentamicine pour les P. aeruginosa in vitro multi-re rer ces infections chez le chien. utile aux cliniciens an de ge Resumen  n Pseudomonas aeruginosa resistente a mu ltiples antibacterianos complica con frecuencia Introduccio nica bacteriana en perros. casos de otitis cro  tesis/Objetivos el propo sito de este estudio in vitro fue determinar el efecto de  Hipo acido etilendiaminotetraacetico-trometamina (Tris-EDTA) en las concentraciones m nimas bactericidas (MBCs) y concentraciones inhibitorias m nimas (MICs) de marbooxacina y gentamicina para aislados de P. aeruginosa multiresistente de casos de otitis canina. todos once aislados se identicaron como multiresistentes mediante difusio n en disco; 10 fueron Me resistentes a marbooxacina y dos resistentes a gentamicina. Los aislados se incubaron durante 90 minutico por si solo o en combinacio n con Tris-EDTA a concentraciones de 0,075 lg/mL a tos con cada antibio 5 mg/mL para marbooxacina; 0,001 lg/mL a 10 mg/mL para gentamicina y 17,8: 4,7 a 0,14: 0,04 mg/mL n de antibio tico de Tris-EDTA. Se incluyeron controles positivos y negativos. Al cuotas de cada concentracio s transferidos a placas de agar con sangre de oveja para determinar la MBCs, y/o Tris-EDTA fueron despue ~adido a las suspensiones restantes para determinar las MICs. y caldo de triptona de soja fue an lo fue bacteriost n. La adicio n Resultados Tris-EDTA so atico pero no bactericida a ninguna concentracio de Tris-EDTA redujo signicativamente la media MBC (de 625 a 468,8 lg/mL; P < 0,001) y MIC (de 29,3 a 2,4 lg/mL; P = 0,008 de marbooxacina y la media de MBC (625 a 39,1 lg/mL) y MIC (de 19,5 a 1,2 lg/ mL) de gentamicina (en ambos P < 0,001). Conclusiones e importancia cl nica Tris-EDTA redujo signicativamente las MBCs y MICs de marbooxacina y gentamicina para P. aeruginosa multiresistente in vitro. Esto puede ser de utilidad para los cl nicos en el manejo de estas infecciones en perros. Zusammenfassung Hintergrund Der auf viele Wirkstoffe resistente Pseudomonas aeruginosa verkompliziert h aug chronisch bakterielle Otitiden von Hunden. Hypothese/Ziele Das Ziel dieser in vitro Studie war es, die Wirksamkeit von ethylendiamintetraessigsaurem Tromethamin (Tris-EDTA) auf die minimalen bakteriziden Konzentrationen (MBCs) und die minimalen
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TrisEDTA and otic Pseudomonas isolates

ber multiresistenten inhibitorischen Konzentrationen (MICs) von Marbooxacin und Gentamicin gegenu P. aeruginosa Isolaten bei Otitisf allen von Hunden zu bestimmen. Methoden Elf Isolate wurden mittels Diskdiffusion als multiresistent identiziert; 10 waren resistent ber Marbooxacin und zwei waren resistent gegenu ber Gentamicin. Die Isolate wurden 90 Mingegenu uten lang mit jedem einzelnen Antibiotikum und in Kombination mit Tris-EDTA bei Konzentrationen von r Marbooxacin, 0,001 lg/mL bis 10 mg/mL fu r Gentamicin und 17,8:4,7 bis 0,075 lg/mL bis 5 mg/mL fu r Tris-EDTA inkubiert. Es wurden positive und negative Kontrollen inkludiert. In der 0,14:0,04 mg/mL fu r jedes Antibiotikum und/oder jede Tris-EDTA Konzentration auf Schafsblutagar Folge wurden Aliquote fu he wurde den u brigen Suspensionen beigefu gt transferiert, um die MBCs zu bestimmen, Trypton Sojabru um die MICs zu bestimmen. Ergebnisse Tris-EDTA alleine wirkte bakteriostatisch aber bei keiner Konzentration bakterizid. Der Zusatz von Tris-EDTA reduzierte den Medianwert der MBC (von 625 bis 468,8 lg/mL; P < 0,001) und die MIC r Marbooxacin, und den Medianwert der MBC (von 625 auf (von 29,3 bis 2,4 lg/mL; P = 0,008) fu r Gentamicin (beide P < 0,001) signikant. 39,1 lg/mL) und die MIC (von 19,5 bis 1,2 lg/mL) fu r MarSchlufolgerung und klinische Bedeutung Tris-EDTA reduzierte in vitro die MBCs und MICs fu ber einem multiresistenten P. aeruginosa signikant. Dieses Ergebnis booxacin und Gentamicin gegenu nnte fu r Kliniker beim Management dieser Infektionen von Hunden wichtig sein. ko

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