clinical histories and condition of host dogs : in total , 173 cotton rectal 
 swabs were collected from 93 dogs treated at rakuno gakuen university ( rgu ) veterinary 
 teaching hospital ( ebetsu , japan ; university hospital ) and from 80 dogs treated at 8 
 companion animal clinics ( 10 samples per clinic , from different dogs ) in the community of 
 ebetsu ( community clinics ) from june to december 2005 ( regardless of the clinical condition 
 seen for the animal ) .
all dogs admitted to the university hospital had also visited the 
 community clinics previously .
university hospital cases ( 15 male and 20 female dogs ) included those with tumor , cataract , 
 glaucoma , keratitis , hip dysplasia , cushing syndrome and herniated intervertebral discs . 

community clinic cases ( 27 male and 24 female dogs ) included those undergoing castration , 
 panovario - hysterectomy or treatment for urinary tract infections , cystitis , chronic 
 diarrhea , dermatitis , otitis externa , gingivitis , pharyngitis and keratitis .
dogs were aged 
 016 years ( university dogs : 8.2  3.7 y [ mean  sd ] ; community dogs : 5.5  4.2 y ) .
the 
 6-month history of antimicrobial use prior to sampling was also compared for the 54 dogs 
 admitted to the university hospital and the 56 dogs admitted to the community clinics .
samples were streaked on deoxycholate  hydrogen sulfide  lactose ( dhl ) 
 agar ( nissui , tokyo , japan ) and incubated for 24 hr at 37c .
colonies of suspected 
 e. coli growing on these dhl agar plates were picked and subcultured on 
 nutrient agar ( nissui ) .
after incubation , the biochemical properties of these colonies were 
 assessed using triple sugar iron agar ( nissui ) , lysine indole motility medium ( nissui ) and 
 cytochrome oxidase tests .
final identification of e. coli was performed 
 using api20e codes ( biomrieux , tokyo , japan ) .
the 173 canine samples were also assessed on 
 dhl agar supplemented with 4 g / ml of enrofloxacin ( enr ; 
 bayer , osaka , japan ) .
susceptibility testing : susceptibilities to a panel of antimicrobials were 
 examined using the agar dilution method , according to the guidelines of the clinical and 
 laboratory standards institute ( clsi ) . 

mueller  hinton ( mh ) agar was obtained from oxoid ( basingstoke , u.k . ) .
ampicillin ( amp ) , 
 amoxicillin ( amx ) , cefazolin ( cfz ) , cephalexin ( lex ) , gentamicin ( gen ) , kanamycin ( kan ) , 
 dihydrostreptomycin ( dsm ) , oxytetracycline ( otc ) and chloramphenicol ( chl ) were obtained 
 from sigma - aldrich ( st . louis , mo , u.s.a . ) , and cefpodoxime ( cpd ) was purchased from daiichi 
 sankyo co. , ltd .
staphylococcus aureus atcc29213 , 
 enterococcus faecalis atcc29212 , e. coli atcc25922 and 
 pseudomonas aeruginosa atcc27853 were used as controls .
resistance to dsm ( 32 
 g / ml ) and otc ( 16 
 g / ml ) was microbiologically defined as described in the 
 japanese veterinary antimicrobial - resistance monitoring system .
intermediate interpretations for dsm and otc were defined as having 
 two - fold lower minimum inhibitory concentration ( mic ) than those of the resistance category . 

phe - arg--naphthylamide ( pan ; sigma - aldrich ; final concentration : 20 
 g / ml ) was used as an efflux - pump inhibitor . organic solvent tolerance :
organic solvent tolerance ( ost ) was 
 investigated as previously described   with slight 
 modifications .
an overnight culture of e. coli was diluted with 0.9% nacl 
 ( approximately 1  10 cells / ml ) .
a drop of cell suspension ( 5 
 l ) was spotted onto mh agar medium to form a circle with a diameter of 8 
 mm .
the surface of the agar was overlaid with a mixture ( 3:1 , 1:1 , or 1:3 [ vol / vol ] ) of 
 n - hexane ( 96.0% pure ; kishida chemical co. , ltd .
, osaka , japan ) and 
 cyclohexane ( > 99% pure ; merck kgaa , darmstadt , germany ) to a depth of 3 mm .
cyclohexane 
 is an organic solvent known to be a more effective agent against e. coli 
 than n - hexane .
bacterial growth 
 was assessed after the plates were incubated at 37c for 1618 hr in a sealed vessel . 

confluent growth of the cells ( confluent ) was considered to be indicative of tolerance to 
 the solvent tested .
when only a few colonies ( < 10 ) grew on the plate or when no growth 
 was observed , the cells were considered to be sensitive to the solvent tested 
 ( non - confluent ) .
determination of qrdr mutations , pmqrs , -lactamases and chl - resistance 
 genes : mutations in qrdrs of gyra , parc , 
 pare and gyrb were examined by direct dna sequencing of 
 pcr products , as described by everett et al . .
pmqr genes ( qnra , qnrb , 
 qnrs , aac ( 6 ) ib - cr and qepa ) were 
 detected by pcr using specific primers ( table 
 1table 1.sequences of oligonucleotides and fluorescence - labeled oligonucleotides used for 
 pcr , direct sequencing and real - time rt - pcr in this studygeneforward primer ( 53)reverse primer ( 53)fluorescent probe ( 53)purposereferencegyraacgtactaggcaatgactggagaagtcgc cgtcgatagaacpcr and sequencinggyrbtgtatgcgatgtctgaactgctcaatagcagctcggaatapcr and sequencingparctgtatgcgatgtc tgaactgctcaatagcagctcggaatapcr and sequencingparetaccgag ctgttccttgtggggcaatgtgcagaccat cagpcr and sequencingqnraagaggatttctcacgccaggtgccaggcacagatcttgacpcrqnrbggmathgaaattcgccactgtttgcygyycgccagtcgaapcrqnrsgcaagttcattgaacagggttctaaaccgtcgagttcggcgpcraac ( 6)-ibttgcgatgctctatgagtggctactcgaatgcctggcgtgtttpcr and sequencingqepaaactgcttgagcccgtagatgtctacgccatggacctcacpcrblatematgagtattcaacattttcgttaccaatgcttaatcagtgpcr and sequencingblashvatgcgttatattcgcctgtgttagcgttgccagtgctcgapcrcata1agttgctcaatgtacctataaccttgtaattcattaagcattctgccpcrcata2acactttgccctttatcgtctgaaagccatcacatactgcpcrcata3ttcgccgtgagcattttgtcggatgagtatgggcaacpcrflorcgccgtcattcctcaccttcgatcacgggccacgctgtgtcpcrcmlattgcaacagtacgtgacatacacaacgtgtacaaccagpcracractatcaccctacgctctatcttcgcgcgcacgaacatacccgaacccggatcacactctrt - pcracrbgcggtcgtgtgaagaaagtttaactcccaacgagaagaggagaatgaccatcagcagcacgaacataccagtrt - pcrthis studytolcggtacgttgaacgagcaggatcccatcagcaatagcattctgttccctggcactgaacaatgcgctgagcaart - pcrthis studygapaaaaggcgctaacttcgacaagaacggtggtcatcagacctcaacgataacttcggcatcart - pcrthis studya ) m , a , or c ; h , a , or c or t ; y , c , or t. ) and direct dna sequencing [ 4 , 15 , 21 ] . to 
 identify the amp - resistance mechanism , -lactamase genes , viz .
, 
 blatem and blashv , were detected 
 by pcr and direct dna sequencing .
, cata1 , cata2 , cata3 , 
 flor and cmla , were detected by pcr as described in 
 previous studies [ 17 , 27 ] .
nucleotide sequences were determined using a bigdye terminator v3.1 cycle 
 sequencing kit with a 3130 genetic analyzer ( applied biosystems , foster city , ca , 
 u.s.a . ) .
a ) m , a , or c ; h , a , or c or t ; y , c , or t. real - time reverse transcription - pcr : overnight cultures of e. 
 coli isolates were diluted 1:100 in lb broth and grown to the mid - logarithmic 
 phase .
rna was isolated using an rneasy mini kit ( qiagen , hilden , germany ) according to the 
 manufacturer s instructions and stored at 80c until used .
the concentration of rna was 
 determined spectrophotometrically ( biospectrometer , eppendorf , hamburg , germany ) .
gene 
 expression ( acra , acrb and tolc ) was 
 estimated by quantitative reverse transcription ( rt ) taqman - pcr .
the respective primer pairs 
 and probes ( table 1 ) used for 
 acrb , tolc and gapa in this study were 
 designed according to the sequence of e. coli strain k12 substrain mg1655 , 
 which is deposited in genbank ( accession number u00096 ) .
the probes were labeled by the 
 manufacturer ( sigma - aldrich ) with the reporter dye 6-carboxyfluorescein ( 6-fam ) at the 
 5-end and with the quencher dye 6-carboxytetramethylrhodamine ( tamra ) at the 3-end . 

purified rna ( 2.5 ng ) was used in one - step rt and real - time pcr 
 amplification .
rt - pcr amplification mixtures ( 20 l ) contained purified 
 rna , 2 quantitect probe rt - pcr master mix , 0.2 l of quantitect rt mix 
 ( quantitect probe rt - pcr kit , qiagen ) , 0.2 m of probe and 0.5 
 m forward and reverse primers .
the cycle conditions comprised 20-min 
 reverse transcription at 50c ; a 15-min initial activation step at 95c ; and 45 cycles each 
 of 55c for 1 min and at 60c for 30 sec in a lightcycler 480 ( roche , mannheim , germany ) . 

e. 
 coli strain ag100 ( k-12 arge3 thi-1 rpsl xyl mtl d 
 ( gal - uvrb)supe44 ) was kindly donated by dr helen i. 
 zgurskaya ( university of oklahoma , norman , ok , u.s.a . ) and used as a reference strain .
statistical analysis : statistical significance of differences between the 
 isolates obtained from dogs admitted to the 2 types of treatment facilities was determined 
 by student s t - test and fisher s exact test .
antimicrobial - resistance profile of canine e. coli isolates : there was a 
 significant difference in the ages ( p<0.05 ) , but not in the gender 
 distribution of the dogs admitted to the university hospital or the community clinics .
seventy - four e. coli isolates were obtained from 93 rectal samples from 
 dogs admitted to the university hospital ( 79.6% ) and 66 isolates from 80 rectal samples 
 obtained from dogs admitted to the community clinics ( 82.5% ) , after culture on dhl agar 
 plates that had not been supplemented with enr .
there was no significant difference in the 
 frequency of e. coli isolation between dogs admitted to the 2 types of 
 treatment facilities ( p>0.05 ) .
of all the canine e. coli isolates , 44.3% ( 62 of 140 isolates ) were 
 resistant to at least 1 antimicrobial agent tested with aminopenicillin resistance being the 
 most frequent ( approximately 30% ) ; approximately 50% of aminopenicillin - resistant isolates 
 were also resistant to cephalosporins ( cfz and cpd ) .
although there was no significant 
 difference in the rate of resistance to amp or amx between isolates derived from university 
 hospital cases and isolates derived from community clinics cases , when considering isolates 
 with resistance as well as those with an intermediate interpretation to amp and amx , this 
 rate was significantly more prevalent in the university hospital than in the community 
 clinics samples ( p<0.05 , table 
 2table 2.antimicrobial susceptibility of e. coli strains derived from 
 dogs attending rakuno gakuen university veterinary teaching hospital ( rgu ; university ) 
 and animal clinics in the community ( community)antimicrobial(break point , 
 g / ml)groupsrange(g / ml)mic50(g / ml)mic90(g / ml)number of strains ( % ) siri + ramp(32)university2>1284>12844 ( 59.5)*4 ( 5.4)26 ( 35.1)30 ( 40.5)*community0.5>1284>12850 ( 75.8)016 ( 24.2)16 ( 24.2)amx(32)university1>12816>12832 ( 43.2)**15 ( 20.3)**27 ( 36.5)42 ( 56.8)**community4>1284>12850 ( 75.8)016 ( 24.2)16 ( 24.2)cfz(32)university1>1282>12861 ( 82.4)1 ( 1.4)12 ( 16.2)13 ( 17.6)community1>1282>12857 ( 86.3)09 ( 13.6)9 ( 13.6)lex(32)university8>1288>12854 ( 73.0)5 ( 6.8)15 ( 20.3)20 ( 27.0)community4>1288>12847 ( 71.2)9 ( 13.6)10 ( 15.1)19 ( 28.8)cpd(8)university<0.125>1280.512862 ( 83.8)012 ( 16.2)12 ( 16.2)community0.25>1280.512857 ( 86.4)09 ( 13.6)9 ( 13.6)kan(64)university1>12823266 ( 89.2)1 ( 1.4)7 ( 9.5)8 ( 10.8)community2>1282>12859 ( 89.4)07 ( 10.6)7 ( 10.6)gen(16)university0.5>1281867 ( 90.5)07 ( 9.5)7 ( 9.5)community0.5641262 ( 93.9)04 ( 6.1)4 ( 6.1)dsm(32)university2>1284>12855 ( 74.3)1 ( 1.4)18 ( 24.3)19 ( 25.7)community2>1284>12848 ( 72.7)1 ( 1.5)17 ( 25.8)18 ( 27.3)otc(16)university2>1282>12856 ( 75.7)3 ( 4.1)15 ( 20.3)18 ( 24.3)community1>1282>12850 ( 75.8)016 ( 24.2)16 ( 24.2)chl(32)university4>12886454 ( 73.0)*9 ( 12.2)11 ( 14.9)*20 ( 27.0)*community4>1288861 ( 92.4)3 ( 4.5)2 ( 3.0)5 ( 7.6)enr(4)university0.011280.036459 ( 79.7)*015 ( 20.3)*15 ( 20.3)*community0.01640.031661 ( 92.4)05 ( 7.5)5 ( 7.6)amp : ampicillin , amx : amoxicillin , cfz : cefazolin , chl : chloramphenicol , cpd : 
 cefpodoxime , dsm : dihydrostreptomycin , enr : enrofloxacin , gen : gentamicin , kan : 
 kanamycin , lex : cephalexin , otc : oxytetracycline .
the prevalence of chl - resistant and enr - resistant isolates was also 
 significantly higher in the university hospital than in the community clinics samples ( table 2 ) .
amp : ampicillin , amx : amoxicillin , cfz : cefazolin , chl : chloramphenicol , cpd : 
 cefpodoxime , dsm : dihydrostreptomycin , enr : enrofloxacin , gen : gentamicin , kan : 
 kanamycin , lex : cephalexin , otc : oxytetracycline .
p<0.05 , * * p<0.01 ; difference 
 versus community . in terms of susceptibilities to aminopenicillin and chl among the 15 enr - resistant isolates 
 derived from the university hospital samples , 7 isolates showed resistance and/or 
 intermediate interpretation to aminopenicillin , as well as resistance to chl .
six of these 
 isolates showed resistance and/or intermediate interpretation to aminopenicillin , but 
 susceptibility to chl , while the remaining 2 isolates showed susceptibility to both 
 aminopenicillin and chl ( data not shown ) . among the 5
enr - resistant isolates derived from 
 the community clinics samples , 1 isolate showed both resistance to aminopenicillin and chl , 
 and 4 isolates showed resistance and/or an intermediate interpretation to aminopenicillin , 
 but susceptibility to chl ( data not shown ) .
the prevalence of resistance to aminoglycosides 
 ( kan , gen and dsm ) and otc was not significantly different between isolates derived from the 
 university hospital cases and from the community clinics cases .
enr - resistant isolates 
 frequently demonstrated concomitant resistance to aminopenicillins , cephalosporins , gen , dsm 
 and chl ( table 3table 3.prevalence of concomitant antimicrobial resistance on enr - resistant e. 
 coli isolates derived from non - supplemented agarisolatesprevalence of concomitant resistance ( % ) ampamxcezlexcpdkangendsmotcchlenr - resistant ( 20)90.0**90.0**75.0**80.0**70.0**15.030.0**65.0**35.040.0**enr - susceptible ( 120)20.020.85.07.55.89.24.218.320.02.5amp : ampicillin , amx : amoxicillin , cfz : cefazolin , chl : chloramphenicol , cpd : 
 cefpodoxime , dsm : dihydrostreptomycin , enr : enrofloxacin , gen : gentamicin , 
 kan,:kanamycin , lex : cephalexin , otc : oxytetracycline . 
 *
prevalence of ost was significantly higher in isolates from the university 
 hospital cases than from the community clinics cases ( table 4table 4.organic solvent tolerance ( ost ) of e. coli strains derived from 
 dogs in attending rakuno gakuen university veterinary teaching hospital ( rgu ; 
 university ) and animal clinics in the community ( community)ost(n - hexane : cyclohexane)groupsnon - confluentconfluent3:1university32 ( 43.2)**42 ( 56.8)**community55 ( 83.3)11 ( 16.7)1:1university60 ( 81.1)**14 ( 18.9)**community63 ( 95.5)3 ( 4.5)1:3university67 ( 90.5)7 ( 9.5)*community65 ( 98.5)1 ( 1.5)values indicate the number of e. coli isolates and ( percentage of 
 the total ) . *
amp : ampicillin , amx : amoxicillin , cfz : cefazolin , chl : chloramphenicol , cpd : 
 cefpodoxime , dsm : dihydrostreptomycin , enr : enrofloxacin , gen : gentamicin , 
 kan,:kanamycin , lex : cephalexin , otc : oxytetracycline . 
 *
values indicate the number of e. coli isolates and ( percentage of 
 the total ) .
the average number of antimicrobials used for each dog was significantly higher in the 
 university hospital than in the community clinics cases ( table 5table 5.status of antimicrobial use in dogs attending rakuno gakuen university veterinary 
 teaching hospital ( rgu ; university ) and animal clinics in the community 
 ( community)antimicrobial useuniversitycommunityaverage number of antimicrobialsused for each dog1.4**0.8frequency of dogs treatedby fluoroquinolone24.1%**14.3%frequency of dogs treatedby all antimicrobials74.1%**50.0%we could obtain antimicrobial use history for 6 months prior to sampling from 54 dogs 
 in the university and 56 dogs in the community .
the frequencies of dogs treated with any antimicrobials and with 
 fluoroquinolones were also significantly higher in the university hospital than in the 
 community clinics cases ( table 5 ) .
in addition , 
 prevalence of fluoroquinolone - resistant isolates was significantly higher in dogs that had 
 been treated with fluoroquinolones compared with that in dogs that had not been treated with 
 this agent ( p<0.05 ; data not shown ) .
we could obtain antimicrobial use history for 6 months prior to sampling from 54 dogs 
 in the university and 56 dogs in the community . *
isolation of fluoroquinolone - resistant e. coli using enr - supplemented dhl agar 
 plates : to investigate fluoroquinolone - resistance mechanisms and the occurrence 
 of multidrug resistance involving fluoroquinolone , we selected enr - resistant e. 
 coli on enr - supplemented dhl agar plates ( fig . 
 1fig .
1.influence of enrofloxacin selection on isolation frequencies of e. 
 coli isolated from canine rectal samples .
amp , ampicillin ; amx , 
 amoxicillin ; cfz , cefazolin ; chl , chloramphenicol ; cpd , cefpodoxime ; dsm , 
 dihydrostreptomycin ; enr , enrofloxacin ; gen , gentamicin ; kan , kanamycin ; lex , 
 cephalexin ; otc , oxytetracycline . * statistical difference for isolation with 
 deoxycholate hydrogen sulfide lactose ( dhl ) medium without antimicrobials ; 
 p<0.05 . ) .
rate of resistance to aminopenicillins , cephalosporins , gen , dsm , otc and chl was 
 significantly higher in isolates obtained from enr - supplemented dhl agar plates than those 
 obtained from dhl agar plates that had not been supplemented with enr .
isolates obtained 
 from enr - supplemented dhl agar plates were most frequently amp resistant .
influence of enrofloxacin selection on isolation frequencies of e. 
 coli isolated from canine rectal samples .
amp , ampicillin ; amx , 
 amoxicillin ; cfz , cefazolin ; chl , chloramphenicol ; cpd , cefpodoxime ; dsm , 
 dihydrostreptomycin ; enr , enrofloxacin ; gen , gentamicin ; kan , kanamycin ; lex , 
 cephalexin ; otc , oxytetracycline . * statistical difference for isolation with 
 deoxycholate hydrogen sulfide lactose ( dhl ) medium without antimicrobials ; 
 p<0.05 .
we further characterized the 31 e. coli isolates derived from 
 enr - supplemented dhl agar plates ( table 
 6table 6.characterization of antimicrobial and organic solvent susceptibility , qrdr 
 mutations , existence of resistant genes and expression levels of acrab in e. 
 coli isolates derived by enr - supplemented agarstraincanine case historyantimicrobial usefor 6 monthsbefore samplingqrdr mutationsmic ( g / ml)-lactamasegenepmqrcp - resistancegeneexpression levelgyraparcpareampcpdenrchlacraacrbtolcs83d87s80e84a108university groupre18mastocytomacfz , lexlnig-->128>128128 ( 16)32 ( 16)n.d.n.d.n.d.2.623.763.27re21abdominal tumoramp , cfz , lex , enrlnig-->128>128128 ( 32)>128n.d.n.d.cata12.313.774.65re28rhabdomyosarcomacfz , lexlnig-->128>128128 ( 32)64 ( 16)blatem-1n.d.n.d2.177.133.46re33mastocytomacfz , lex , ofxlnig-->128>128128 ( 32)>128blatem-1n.d.cata12.8114.295.88re61herniated intervertebral discslexlnig-->1280.564 ( 8)16 ( 8)blatem-1n.d.n.d.1.432.343.21re63unknownamplnig--16 ( 8)264 ( 8)16 ( 8)n.d.n.d.n.d.0.752.681.87re20lung tumoramc , cfz , enrlniv-->128>12832 ( 4)16 ( 8)blatem-1n.d.n.d.2.162.93.95re2tumor of the breastgen , frmlni - t->128>128128 ( 16)32 ( 16)n.d.n.d.n.d.2.82.874.07re72glaucomacfz , ofx , orblni - t->128164 ( 8)16 ( 8)blatem-1n.d.n.d.0.890.961.77re80oral tumornonelni - t->128164 ( 8)16 ( 8)blatem-1n.d.n.d.1.021.011.72re4osteosarcomaamp , amx , frmlni -- e460a>128>12864 ( 16)>128blatem-1n.d.cata11.361.52re64oral tumorcfzlni -- e460a>1280.564 ( 16)>128blatem-1n.d.cata10.761.081.7re65unknownnonelni -- e460a>1280.564 ( 16)>128blatem-1n.d.cata10.81.041.29re22multiple myelomaenr , minlni -- s458a>1284128 ( 16)16 ( 8)blatem-1n.d.n.d.1.941.672.55re26unknowncfz , lexlni---4 ( 4)0.2516 ( 8)8 ( 4)n.d.n.d.n.d.1.11.582.36re50keratitisofx , orblni--->128>128128 ( 16)16 ( 8)blatem-1n.d.n.d.1.321.652.74re54biopsy of vertebral bodycfzlni--->128232 ( 16)>128blatem-1n.d.cata11.542.674.09re17cushing syndromecfzlwig--16 ( 8)164 ( 8)16 ( 8)n.d.n.d.n.d.1.362.763.87re39herniated intervertebral discsamplwig-->128>128256 ( 16)>128blatem-1n.d.cata11.183.283.74total ( % ) r84.263.110052.6 * 1.83 * 3.48**3.06**i + r94.752.610094.7**(13.9)(8.6*)community groupce7otitis externalex , genlnia-->128>128128 ( 16)>128n.d.n.d.cata10.881.42.01ce5unknownnonelnig-->1282128 ( 16)16 ( 8)blatem-1n.d.n.d.1.441.631.22ce6unknownlex , cfz , genlnig-->128>12864 ( 4)8 ( 4)n.d.n.d.n.d.0.250.370.43ce10diarrheanonelniv--4 ( 4)0.564 ( 8)4 ( 2)n.d.n.d.n.d.0.632.122.02ce14unknownnonelniv-->1280.5128 ( 16)4 ( 2)blatem-1n.d.n.d.0.841.261.6ce1unknownnonelni - t->128>12864 ( 8)16 ( 8)blatem-1n.d.n.d.0.91.271.32ce9otitis externalex , gen , lvxlni -- e460a>128>12832 ( 8)16 ( 8)blatem-1n.d.n.d.1.71.891.72ce12gingivitisclilni -- s458t>1280.532 ( 4)4 ( 4)blatem-1n.d.n.d.1.341.711.52ce13diarrheasxtlni -- s458a>1282128 ( 8)8 ( 4)n.d.aac ( 6)-ib - crn.d.0.240.460.56ce3pharyngitisamp , lex , ofxlgr--->1283232 ( 8)8 ( 4)blatem-1n.d.n.d.0.911.61.82ce4unknownnonelgr--->1283232 ( 4)8 ( 8)blatem-1n.d.n.d.0.50.91.25ce8keratitislex , lvxlgi--->128>12832 ( 8)8 ( 4)n.d.n.d.n.d.0.951.511.7total ( % ) r91.758.31008.30.971.341.43i + r91.766.710033.3(8.0)(4.6)amc : amoxicillin - clavulanic acid , cli : clindamycin , frm : fradiomycin , lvx : 
 levofloxacin , min : minocycline , ofx : ofloxacin , orb : orbifloxacin , sxt : 
 trimethoprim - sulfamethoxazole .
b ) 
 wild - type c ) fold - reduction of mic by pan .
e ) mrna expression 
 levels derived from real - time rt - pcr ( relative amount of ag100 ) . 

* p<0.05 , * * p<0.01 ; statistical difference 
 versus community group . ) .
all enr - resistant isolates had nucleotide substitutions in qrdrs accompanied 
 by changes in 3 or 4 amino acids in qrdrs .
the aac ( 6 ) ib - cr , one of the 
 genes encoding pmqrs , was detected in only 1 strain . in total , more than 70% of the 
 enr - resistant isolates had resistance or intermediate interpretation to amp and/or chl , and 
 74% of isolates with an amp mic of>128 g / ml possessed 
 blatem-1 , and 100% of isolates with a chl mic of>128 
 g / ml possessed cata1(table 6 ) .
expression levels of 
 acra , acrb and tolc and the effect of 
 pan were higher in chl  resistance and chl  intermediate interpretable isolates than in 
 chl - susceptible isolates ( table 6 ) . isolates 
 exhibiting ost had high acrb expression , while isolates with an 
 intermediate interpretation to amp also exhibited ost and had higher acrb 
 expression than did isolates that were amp - susceptible ( data not shown ) .
amc : amoxicillin - clavulanic acid , cli : clindamycin , frm : fradiomycin , lvx : 
 levofloxacin , min : minocycline , ofx : ofloxacin , orb : orbifloxacin , sxt : 
 trimethoprim - sulfamethoxazole .
b ) 
 wild - type c ) fold - reduction of mic by pan .
e ) mrna expression 
 levels derived from real - time rt - pcr ( relative amount of ag100 ) . 

* p<0.05 , * * p<0.01 ; statistical difference 
 versus community group . among dogs from which we isolated e. coli on enr - supplemented dhl agar 
 plates ,
the frequency of dogs treated with any antimicrobials was significantly higher in 
 the university hospital ( 89.5% ) than in the community clinics ( 58.3% ) cases ( table 6 ) .
in contrast , the frequency of dogs treated 
 with fluoroquinolones was not significantly different between the university hospital 
 ( 31.6% ) and community clinics ( 25.0% ) cases .
twenty - seven of 31 e. coli 
 isolates obtained on enr - supplemented dhl agar plates showed resistance or an intermediate 
 interpretation to amp and/or chl . among the 27 dogs from which we isolated e. 
 coli with resistant or an intermediate interpretation to amp and/or chl on 
 enr - supplemented dhl agar plates ,
18 dogs had been treated with fluoroquinolone and/or 
 -lactam antimicrobials ( table 6 ) .
in this study , e. coli isolates with resistant or an intermediate 
 interpretation to aminopenicillins , chl or fluoroquinolone were more frequently obtained 
 from dogs admitted to the universal hospital than from those admitted to the community 
 clinics . remarkably , isolates with resistance to fluoroquinolones
more frequently showed 
 resistance to aminopenicillins , cephalosporins , gen , dsm and chl , as compared with 
 fluoroquinolone - susceptible isolates .
this result suggested that the difficulty of providing 
 effective antimicrobial treatment increases in secondary medical care .
it indicated a need 
 to investigate the mechanism underlying the emergence of this multidrug - resistance 
 phenotype . to characterize in detail the fluoroquinolone - resistant isolates obtained from the 
 university hospital and community clinics studied here , we investigated 
 antimicrobial - resistance mechanisms of e. coli isolates derived from dogs 
 using enr - supplemented dhl agar plates .
all enr - resistant isolates obtained from 
 enr - supplemented dhl agar plates possessed 3 or 4 mutations in qrdrs .
a previous study 
 showed that in vitro exposure to fluoroquinolone caused mutations in qrdrs 
 and acrab 
this may indicate 
 that in vivo fluoroquinolone exposure can also cause an increase in 
 fluoroquinolone - resistant e. coli possessing multiple mutations in qrdrs 
 and acrab  tolc overexpression .
indeed , prevalence of fluoroquinolone - resistant isolates was 
 significantly higher in dogs that had been treated with fluoroquinolones compared with that 
 in dogs that had not been treated with this agent , as determined using on dhl agar plates 
 that had not been supplemented with enr .
moreover , fluoroquinolone - resistant isolates 
 derived from the university hospital had higher levels of acra , 
 acrb and tolc expression than did such isolates obtained 
 from the community clinics , as determined using enr - supplemented dhl agar plates .
these 
 findings suggested that the high prevalence of fluoroquinolone - resistant e. 
 coli isolates derived from the university hospital may have been caused by 
 frequent fluoroquinolone use in the university hospital and/or continuous fluoroquinolone 
 use in the community clinics and the university hospital .
this may have resulted in 
 development of a mechanism that decreased fluoroquinolone susceptibility , viz . , 
 overexpression of acrab  tolc . in this study ,
chl , in addition to enr was another agent to which isolates derived from the 
 university hospital showed a significantly higher prevalence of resistance than did those 
 derived from the community clinics .
all enr - resistant isolates with a chl mic of > 128 
 g / ml that had been derived from enr - supplemented dhl 
 agar plates possessed cata1 .
however , other resistant isolates with a chl 
 mic of 32 and 64 g / ml and an intermediate interpretation 
 isolates with a chl mic of 16 g / ml did not possess any 
 specific chl - resistance gene . among all antimicrobial agents that we tested , isolates with 
 enr resistance were most frequently co - resistant to aminopenicillins , and all the isolates 
 showing resistance to amp , but not to cepharosporins , possessed 
 blatem-1 .
however , isolates with intermediate interpretation 
 to amp did not possess any of the -lactamase genes for which we tested .
these results 
 indicated that the main resistance mechanisms for fluoroquinolones , amp and chl involved by 
 acquisition of mutations in qrdrs and a resistance - associated gene , e.g. , 
 blatem-1 or cata1 , although there may also be 
 other mechanisms that decreased their susceptibilities and conferred co - resistance to these 
 agents . to evaluate the mechanism underlying decreased susceptibilities and co - resistance to 
 fluoroquinolone , aminopenicillins and chl , we investigated acrab  tolc function , because 
 acrab  tolc is a major resistance  nodulation  division ( rnd ) family - type efflux pump that 
 excretes several antimicrobials [ 14 , 19 , 20 ] .
acrab 
 overexpression increases the mics of aminopenicillins and chl to an intermediate 
 interpretation ( 16 g / ml ) or resistance ( 32 or 64 
 g / ml ) level , and its effect is not limited to 
 fluoroquinolone resistance [ 13 , 21 ] .
tolc is also known to cause the efflux of several organic 
 solvents , which cause cell death by breaking down microbial membranes ; therefore , investigation of ost is useful for identifying e. 
 coli isolates that have active acrab  tolc .
we observed that ost isolates with higher acrb expression 
 and isolates with an intermediate interpretation to aminopenicillins and chl , as well as 
 isolates resistant to aminopenicillins and/or with chl mics of 32 and 64 
 g / ml , also exhibited ost and higher 
 acrb expression than did susceptible isolates , as seen by analysis using 
 enr - supplemented dhl agar plates
. a higher prevalence of isolates with ost , decreased 
 aminopenicillin susceptibility and decreased chl susceptibility , was observed in isolates 
 obtained from university hospital compared to those from community clinics cases , as seen on 
 agar plates without enr supplementation .
tolc function contributes to a decrease in susceptibility to aminopenicillins and chl 
 mics in some e. coli isolates obtained from dogs .
our study revealed that the frequency of total antimicrobial treatment as well as 
 fluoroquinolone use was significantly higher in the university hospital than in the 
 community clinics .
this evidence suggested that the frequent use of antimicrobials in dogs 
 admitted to the university hospital and/or their continuous use in dogs moving from the 
 community clinics to the university hospital facilitate selection of antimicrobial resistant 
 e. coli strains with qrdr mutations , beta - lactamase gene and 
 cata1 .
in addition , our study also revealed that dogs admitted to the 
 university hospital tend to be treated with multiple antimicrobials .
indeed , our 
 results showed that enr - resistant e. coli isolates had higher rates of 
 resistance to several antimicrobials compared with enr - susceptible e. coli 
 isolates , and enr - resistant isolates derived from the university hospital cases on 
 enr - supplemented dhl agar showed a stronger development of the acrab 
tolc than did 
 enr - resistant e. coli isolates derived from the community clinics cases .
we 
 considered that these findings substantially reflect the situation in japanese companion 
 animal medicine , because the samples in this study were successively .
in addition , a 
 previous study also showed that amp or enr treatment led to the emergence of 
 aminopenicillin  enr  chl - resistant e. coli isolates in dogs in the united 
 states [ 2 , 8 ] . 

moreover , fluoroquinolone  aminopenicillin  chl - resistant e. coli isolates 
 with overexpression of acrab  tolc were frequently isolated from humans in university 
 hospitals .
these findings indicate that the 
 emergence of this multidrug - resistant phenotype may mirror the same phenomenon in human and 
 companion animal clinical fields in several countries in some cases . in these cases , a 
 clearer strategy for choice and use of antimicrobials suitable to treatments is required in 
 order to prevent the emergence and spread of these fluoroquinolone - resistant e. 
 coli with decreased susceptibilities to several other antimicrobials . in 
 particular
, we suggest that it may be important to share the history of antimicrobials usage 
 across the first and secondary medical care settings of companion animals to avoid treatment 
 with several antimicrobials in the same period and to avoid extensive , continuous treatment 
 with the same class antimicrobial . in conclusion , this study revealed that the higher prevalence of concomitant resistant and 
 intermediate interpretations to fluoroquinolones , aminopenicillins and chl in isolates from 
 the university hospital than in isolates from the community clinics was due not only to the 
 acquisition of specific resistance mechanisms , such as -lactamases , cata1 
 and qrdr mutations , but also to overexpression of the acrab  tolc efflux pump in canine 
 e. coli .
 abstractunderstanding the prevalence 
 of antimicrobial - resistance and the relationship between emergence of resistant bacteria 
 and clinical treatment can facilitate design of effective treatment strategies . 
 we here 
 examined antimicrobial susceptibilities of escherichia coli isolated from 
 dogs admitted to a university hospital ( university hospital ) and companion animal clinics 
 ( community clinics ) in the same city and investigated underlying multidrug - resistance 
 mechanisms . 
 the prevalence of e. coli with intermediate and resistant 
 interpretations to ampicillin ( amp ) , enrofloxacin ( enr ) and chloramphenicol ( chl ) was 
 higher in the university hospital than in the community clinics cases . 
 use of 
 antimicrobials , including fluoroquinolone , was also significantly higher in the university 
 hospital than in the community clinics cases . upon isolation using enr - supplemented agar 
 plates , all enr - resistant isolates had 34 nucleotide mutations that accompanied by amino 
 acid substitutions in the quinolone - resistance - determining regions of 
 gyra , parc and pare , and 94.7% of all 
 isolates derived from the university hospital showed amp and/or chl resistance and 
 possessed blatem and/or cata1 . 
 the average 
 mrna expression levels of acra , acrb and 
 tolc and the prevalence of organic solvent tolerance , in isolates 
 derived from enr - supplemented agar plates were significantly higher in the university 
 hospital than in the community clinics isolates . 
 thus , e. coli derived 
 from the university hospital cases more often showed concomitant decreased 
 susceptibilities to aminopenicillins , fluoroquinolones and chl than did those derived from 
 the community clinics ; this was related to an active acrab  tolc efflux pump , in addition 
 to acquisition of specific resistance genes and genetic mutations . 