In recent years, detection of fluoroquinolone (FQ) resistance determinants in _Escherichia coli_ (_E. coli_) isolated from animals indicated that this is an important public health issue and

can create a high risk for the treatment of infectious diseases at the recommended available dosage regimens.1 In Gram-negative bacteria, resistance to FQs primarily occurs from gene

mutations in the quinolone resistance-determining region (QRDR) of the genes encoding the drug target enzymes (DNA gyrase and topoisomerase IV).2 In addition, the pentapeptide repeat

proteins (QnrA, QnrB and QnrS) increase MIC of FQ for _E. coli_ (0.125–16 μg ml−1) by protecting DNA gyrase from inhibition.3, 4 FQs have been approved for use in the treatment of infectious

treatment of various infections, including urinary, skin and otitis infections. There are a few reported drug interactions among FQs that have veterinary significance.5, 6, 7 Interactions

between main compounds (enrofloxacin and ibafloxacin) and their metabolites (ciprofloxacin and 8-hydroxy-ibafloxacin) against _E. coli_, _Staphylococcus_ spp. and _Pseudomonas aeruginosa_

(_P. aeruginosa_) were investigated in these studies. In addition, Pankey and Ashcraft8 showed that there was a synergistic interaction between ciprofloxacin and gatifloxacin against _P.

aeruginosa_. The objective of this work was to identify if a synergistic interaction between danofloxacin and orbifloxacin against FQ-resistant _E. coli_ isolates from animals occurs. The

drug combination studies were carried out using the checkerboard and time-kill methods. Seven _E. coli_ isolates carrying _gyrA_ mutations or _qnr_ genes from the Laboratory of Molecular

Pharmacology were selected for the checkerboard and time-kill studies. The _gyrA_ mutant _E. coli_ isolates were obtained from three healthy fowl (E224, E245, E246), the _qnr-_containing _E.

coli_ isolates were from one healthy cow (E101), one cow (E103) and one sheep (E248) with gastroenteritis, and one healthy dog (E300). Broth microdilution testing was performed to determine

the MICs of the compounds according to the guidelines of the Clinical Laboratory Standards Institute.9 _E. coli_ ATCC25922 was used as control for antimicrobial susceptibility testing. QRDR

and plasmid-mediated quinolone resistance (PMQR) genes were amplified using specific primers10, 11 and PCR products of _gyrA_ were sequenced by Macrogen Inc. (Korea). The DNA sequences of

_gyrA_ were analyzed using the BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Presence of the _qnrA_ and _qnrS_ genes was determined by PCR amplification, as described previously

by Robicsek _et al._12 and Cengiz _et al._1 The primers used are as follows: _gyrA_, 5′-ACGTACTAGGCAATGACTGG-3′ (forward) and 5′-AGAAGTCGCCGTCGATAGAAC-3′ (reverse); _qnrA_,

5′-ATTTCTCACGCCAGGATTTG-3′ (forward) and 5′-GATCGGCAAAGGTTAGGTCA-3′ (reverse); and _qnrS_, 5′-ACGACATTCGTCAACTGCAA-3′ (forward) and 5′-TAAATTGGCACCCTGTAGGC-3′ (reverse). Fractional

inhibitory concentration index/indices (FIC index/indices) of danofloxacin and orbifloxacin were determined using checkerboard method.13 Danofloxacin concentrations ranged from 0.064 to 256 

μg ml−1 and orbifloxacin concentrations ranged from 0.128 to 512 μg ml−1. FIC index/indices were calculated as follows: FICA=MIC drug A in combination/MIC drug A alone FICB=MIC drug B in

combination/MIC drug B alone FIC index/ΣFIC=FICA+FICB The FIC index was interpreted as follows: synergy=FIC index⩽0.5; indifference=0.5⩽4; antagonism=FIC index>4. Time-kill experiments

were slightly modified from the method described by Petersen _et al._14 A liquid overnight bacterial culture of the _gyrA_ mutant and _qnr-_containing _E. coli_ isolates was diluted with

Mueller–Hinton Broth (Becton, Dickinson and Company, Sparks, MD, USA) and drug stock solutions to achieve an initial inoculum of ∼106 c.f.u. ml−1. Each 10 ml culture was incubated at 37 °C,

and samples were withdrawn for the determination of bacterial counts at 0, 6 and 24 h. Colony counts were determined by plating 100 μl of each diluted sample onto Plate Count Agar (Becton,

Dickinson and Company) with an automated spiral plater (WASP; Don Whitley Scientific Ltd., Shipley, UK) and then counting using an colony counter (UVITEC Cambridge, Cambridge, UK). Synergy

was defined as a ⩾2 log10 decrease in colony count at 6 or 24 h with the combination compared with the initial inoculum. The drug combination was considered to be antagonist if there was a

⩾2 log10 increase in c.f.u. ml−1 and indifference was the interpretation of a <2 log10 change in c.f.u. ml−1. The MICs of danofloxacin and orbifloxacin for _E. coli_ ATCC25922 were 0.032

and 1 μg ml−1, respectively. Microbiological activity (MIC90) of danofloxacin and orbifloxacin to _E. coli_ isolated from animals was reported as 0.015–0.25 and 0.5 μg ml−1, respectively.15

_E. coli_ isolates presented an alteration in _gyrA_ (E224, E245, E246: Ser-83→Leu; E224: Asp-87→Asn, E245: Asp-87→Glu) and the _qnr_ genes detected were _qnrA1_ (E101) and _qnrS1_ (E103,

E248, E300). The amino-acid substitutions in _gyrA_ were at the most frequently identified site (codon 83).2 The MICs of the compounds and FIC values of the combination for _gyrA_ mutant and

_qnr_-containing _E. coli_ isolates are shown in Table 1. FIC index of the combination for resistant _E. coli_ isolates ranged from 0.09 to 1. The incidence of synergy and

additivity/indifference was 71% and 29%, respectively. Antagonism was not detected for any of _E. coli_ isolates by checkerboard method. By using the time-kill method, the _in vitro_

activity of the combination against _gyrA_ mutant and _qnr_-containing _E. coli_ isolates are shown in Table 1. At 6-h incubation, the combination resulted ⩾2 log10 reduction in viable

counts against all _E. coli_ isolates and it showed synergic activity. At 24-h incubation, this was also achieved for E103, E224 and E245 isolates. However, regrowth was observed for four of

seven _E. coli_ isolates after 24 h incubation. There are increasing numbers of antibiotic-resistant infections, especially by Gram-negative bacteria, which are innately multi-drug

resistant.16 In addition, Gram-negative bacteria such as _E. coli_ are increasingly resistant to the few effective agents available for treatment via the acquisition of transmissible

elements, and when isolated from animals have multiple and different mechanisms of antibiotic resistance.1, 17 Therefore, to restore the efficacy of licensed veterinary FQs against resistant

Gram-negative bacteria has become important. The two methods used most commonly to assess antimicrobial interactions _in vitro_ are the checkerboard and time-kill assays.7 In this study,

these methods were used to assess synergy of danofloxacin+orbifloxacin combination against seven clinical isolates of _E. coli_. Synergy using the checkerboard method was detected for five

of seven _E. coli_ isolates with 0.09–0.5 ΣFIC. Pankey and Ashcraft8 used Etest synergy method and found a similar interaction between ciprofloxacin and gatifloxacin against _P. aeruginosa_

with 19% incidence. Enrofloxacin is unique in that it is partially metabolized to ciprofloxacin and both active drugs circulate in treated animals.5 Lautzenhiser _et al._7 showed that for

staphylococcal and _E. coli_ isolates, FIC indices of enrofloxacin+ciprofloxacin combination were between 0.5 and 4.0, indicating that the combination acted additively _in vitro_. The

combination of ibafloxacin plus its major active metabolite 8-hydroxy-ibafloxacin had synergistic action in two _E. coli_ isolates and additive effects in _E. coli_ ATCC25922.6 The

synergistic activity of the active metabolite contributes additionally to the antimicrobial activity of the parent compound.5, 6, 7 In this study, by the time-kill method, synergy was mainly

shown after 6 h of exposure for all isolates accompanied by regrowth after 24 h for four of them. These results showed that for _E. coli_ synergy incidence detected by the time-kill method

is higher than checkerboard method. Elipoulos and Moellering13 indicated that in contrast to the checkerboard technique, which typically provides only inhibitory data, the killing-curve

technique measures the microbicidal activity of the combination being tested. For this reason, it is presumably more relevant for clinical situations in which bactericidal effect.13 Cengiz

_et al._18 showed that the genetic mechanisms of FQ resistance were determinative for the bactericidal activity of enrofloxacin alone against _E. coli_. The results of this study clearly

indicated that danofloxacin (second-generation FQs) and orbifloxacin (third-generation FQs) can exert synergistic activity against some strains FQ-resistant _E. coli_ isolates, and this

combination could be considered for augmenting of their efficacy _in vivo_. This study was financed by the Scientific and Technological Research Council of Turkey-TUBITAK (TOVAG-110O478) and

supported by COST Action BM0701 ‘ATENS’. REFERENCES * Cengiz, M. _et al_. Molecular characterization of quinolone resistance in _Escherichia coli_ from animals in Turkey. _Vet. Rec._ 171,

155 (2012). Article  CAS  PubMed  Google Scholar  * Hopkins, K. L., Davies, R. H. & Threlfall, E. J. Mechanisms of quinolone resistance in _Escherichia coli_ and _Salmonella_: recent

developments. _Int. J. Antimicrob. Ag_ 25, 358–373 (2005). Article  CAS  Google Scholar  * Poirel, L. _et al_. Expanded-spectrum β-lactamase and plasmid-mediated quinolone resistance.

_Emerg. Infect. Dis._ 13, 803–805 (2007). Article  PubMed  PubMed Central  Google Scholar  * Rodriguez-Martinez, J. M., Briales, A., Velasco, C., Martinez-Martinez, L. & Pascual, A.

Discrepancies in fluoroquinolone clinical categoris between the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and CLSI for _Escherichia coli_ harbouring qnr genes and

mutations in _gyrA_ and _parC_. _J. Antimicrob. Chemoth._ 66, 1406–1453 (2011). Google Scholar  * Blondeau, J. M., Borsos, S., Blondeau, L. D. & Blondeau, B. J. _In vitro_ killing of

_Escherichia coli_, _Staphylococcus pseudintermedius and Pseudomonas aeruginosa_ by enrofloxacin in combination with its active metabolite ciprofloxacin using clinically relevant drug

concentrations in the dog and cat. _Vet. Microbiol_ 155, 284–290 (2012). Article  CAS  PubMed  Google Scholar  * Coulet, M., Van Borssum Waalkes, M., Cox, P. & Lohuis, J. _In vitro_ and

_in vivo_ pharmacodynamic properties of the fluoroquinolone Ibafloxacin. _J. Vet. Pharmacol. Therap._ 25, 401–411 (2002). Article  CAS  Google Scholar  * Lautzenhiser, S. J., Fialkowski, J.

P., Bjorling, D. & Rosin, E. _In vitro_ antibacterial activity of enrofloxacin and ciprofloxacin in combination against _Escherichia coli_ and staphylococcal clinical isolates from dogs.

_Res. Vet. Sci._ 70, 239–241 (2001). Article  CAS  PubMed  Google Scholar  * Pankey, G. A. & Ashcraft, D. S. _In vitro_ synergy of ciprofloxacin and gatifloxacin against

ciprofloxacin-resistant _Pseudomonas aeruginosa_. _Antimicrob. Agents Ch._ 49, 2959 (2005). Article  CAS  Google Scholar  * Clinical Laboratory Standards Institute.. _Performance Standards

for Antimicrobial Susceptibility Testing; Nineteenth Informational Supplement M100-S19_, C. S.: Wayne, PA, USA, (2009). * Everett, M. J., Jin, Y. F., Ricci, V. & Piddock, L. J. V.

Contributions of individual mechanisms to fluoroquinolone resistance in 36 _Escherichia coli_ strains isolated from humans and animals. _Antimicrob. Agents Ch._ 40, 2380–2386 (1996). Article

  CAS  Google Scholar  * Wang, M. _et al_. New plasmid-mediated quinolone resistance gene, _qnrC_, found in a clinical isolate of _Proteus mirabilis_. _Antimicrob. Agents Ch._ 53, 1892–1897

(2009). Article  CAS  Google Scholar  * Robicsek, A., Strahilevitz, J., Sahm, D. F., Jocoby, G. A. & Hooper., D. C. _qnr_ prevalence in ceftazidime-resistant _Enterobacteriaceae_

isolates from United States. _Antimicrob. Agents Ch._ 50, 2872–2874 (2006). Article  CAS  Google Scholar  * Elipoulos, G. M. & Moellering, R. C. Antimicrobial combinations in _Antibiotic

in Laboratory Medicine_ 4th edn, ed.Lorian V, 330–396 William and Wilkins: Baltimore, MD, (1996). Google Scholar  * Petersen, P. J., Labthavikul, P., Jones, H. C. & Bradford, P. A. _In

vitro_ antibacterial avtivities of tigecycline in combination with other antimicrobial agents determined by chequerboard and time-kill kinetic analysis. _J. Antimicrob. Chemoth._ 57, 573–576

(2006). Article  CAS  Google Scholar  * Prescott, J. F., Baggot, J. D. & Walker, R. D. _Antimicrobial Theraphy in Veterinary Medicine_ 3rd edn. Iowa State University Press: Ames,

320–321 (2000). Google Scholar  * Livermore, D. M. Has the era of untreatable infections arrived? _J. Antimicrob. Chemoth._ 64 (Suppl. 1), 29–36 (2009). Article  Google Scholar  *

Karczmarczyk, M., Martins, M., Quinn, T., Leonard, N. & Fanning, S. Mechanisms of fluoroquinolone resistance in _Escherichia coli_ isolates from food-producing animals. _Appl Environ

Microbiol._ 77, 7113–7120 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Cengiz, M. _et al_. _In vitro_ bactericidal activity of enrofloxacin against _gyrA_ mutant and

_qnr-_containing _Escherichia coli_ isolates from animals. _Vet. Rec._ 172, 474 (2013). Article  CAS  PubMed  Google Scholar  Download references AUTHOR INFORMATION AUTHORS AND AFFILIATIONS

* Department of Pharmacology and Toxicology, Laboratory of Molecular Pharmacology, Faculty of Veterinary Medicine, Uludag University, Nilufer, Turkey Murat Cengiz & Pinar Sahinturk

Authors * Murat Cengiz View author publications You can also search for this author inPubMed Google Scholar * Pinar Sahinturk View author publications You can also search for this author

inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Murat Cengiz. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Cengiz, M., Sahinturk, P.

Assessment of synergistic interactions of danofloxacin and orbifloxacin against quinolone-resistant _Escherichia coli_ isolated from animals by the checkerboard and time-kill methods. _J

Antibiot_ 66, 629–631 (2013). https://doi.org/10.1038/ja.2013.62 Download citation * Received: 27 November 2012 * Revised: 07 May 2013 * Accepted: 20 May 2013 * Published: 19 June 2013 *

Issue Date: October 2013 * DOI: https://doi.org/10.1038/ja.2013.62 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a

shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * danofloxacin * _Escherichia coli_

* orbifloxacin * synergism