study design and enrolled cats : nine domestic short - haired cats living at 
 a research facility were enrolled ; mean age was 6.11  1.71 years , and body weight was 3.51 
  0.42 kg .
all cats were selected from a research colony maintained under standard 
 laboratory conditions at the tottori university .
none of the cats had received 
 antimicrobials for at least six months prior to the study , and all cats were deemed healthy 
 based on a physical examination and a hematological examination .
the cats were randomized 
 into three groups ( groups a , b and c ) of three cats each .
the cats in groups a and b 
 received a daily oral dose of 5 and 2.5 mg orb / kg ( ds pharma animal health , osaka , japan ) , 
 respectively , for 7 consecutive days , as approved dosages in japan .
further , the cats were fed commercial dry cat food and received no 
 medication other than orb during the study .
all cats were housed in separate cages located 
 in one room , and direct contact among the cats was prevented during the study .
only when 
 administered with orb , the cats were separately led out of each cage .
this study was approved by tottori 
 university animal use committee ( approval number , 13-t-29 ) . isolation and identification of fecal coliforms :
stool samples were 
 collected from each of the 9 cats on days 1 , 3 , 5 , 7 , 9 , 11 , 13 , 15 and 17 . on day 1 ,
serial 10-fold dilutions were then prepared 
 from 1 g of each stool sample in 0.1% peptone water .
once the appropriate dilution was 
 prepared , 0.1 ml was plated onto eosin methylene blue ( emb ) agar ( nissui 
 pharmaceutical co. , ltd . ,
tokyo , japan ) containing either no drugs or orb at the 
 concentration of 2 g / ml , which was defined as the 
 concentration of intermediate susceptibility in to the clinical and laboratory standards 
 institute guidelines .
on emb agar , coliform 
 colonies develop a metallic luster , other gram - negative bacteria appear colorless , and 
 gram - positive bacteria can not grow .
the inoculated plates were incubated at 35c for 24 hr , 
 and the number of coliform bacteria was enumerated as colony - forming units ( cfu)/g of feces . 

a maximum of 10 coliform colonies per cat per sampling were picked up and subjected to 
 identification of bacterial species .
isolates were confirmed to be e. coli 
 by gram staining , the typical colony shape on deoxycholate hydrogen sulphide lactose agar 
 ( nissui pharmaceutical co. , ltd . ) and detection of the uid gene by pcr 
 .
when isolates were not identified as e. 
 coli , they were identified using the api 20e kit ( sysmex biomrieux co. , ltd .
the isolates were stored in 10% skim milk at 
 80c for antimicrobial susceptibility testing and genetic analysis .
the remaining stool 
 samples were frozen at 80c until high performance liquid chromatography ( hplc ) 
 analysis .
antimicrobial susceptibility testing : susceptibility testing against orb 
 was conducted using the agar dilution method , according to the clinical and laboratory 
 standards institute guidelines . in addition , 
 susceptibilities to ampicillin ( amp ) , cefazolin ( cez ) , tetracycline ( tet ) , chloramphenicol 
 ( chl ) , kanamycin ( kan ) and trimethoprim / sulfamethoxazole ( sxt ) were determined using the 
 disk diffusion method .
cez - resistant isolates , ampc -lactamase and extended - spectrum -lactamase ( esbl ) were 
 phenotypically screened using cefoxitin disks ( 30 g / disk ) and cefotaxime 
 ( 3 g ) , respectively ; the results were considered positive , if the 
 inhibition zone diameters were 14 and 27 mm , respectively [ 10 , 28 ] .
further , ampc - positive isolates 
 were defined as derepressed ampc mutants or inducible ampc producers , as previously 
 described .
analysis of the mechanism of fq resistance : isolates with an orb mic of 1 
 g / ml were assessed for the presence of mutations of the 
 quinolone resistance - determining region ( qrdr ) and pmqr determinants .
the qrdr of the gyra gene was amplified by pcr with previously described 
 primers .
the qrdr of the parc gene 
 was also amplified and sequenced using previously described primers , when qrdr mutations were detected in gyra .
pcr 
 products were then randomly selected and bidirectionally sequenced with the same primers for 
 confirmation .
enterobacterial repetitive intergenic consensus sequence - based pcr : 
 enterobacterial repetitive intergenic consensus sequence - based pcr ( eric - pcr ) was carried 
 out to investigate epidemiologic relationship among the pmqr - positive isolates .
the 
 procedure was slightly modified from the previous studies [ 27 , 31 ] .
briefly , the pcr reaction was set 
 up in a 20 l reaction volume containing 2 l of a 10  
 extaq buffer , 1 u of extaq dna polymerase ( takara bio inc . ,
otsu , japan ) , 0.25 mm each of 
 the dntps , 1030 ng of bacterial dna and 20 pmol of each primer ( i.e. 
 eric1r and eric2 ) .
dna amplifications were performed with an initial denaturation ( 7 min at 
 94c ) followed by 30 cycles of denaturation ( 1 min at 94c ) , annealing ( 1min at 52c ) and 
 extension ( 8 min at 65c ) with a final extension ( 15 min at 65c ) .
measurement of orb concentration in feces : stool samples were obtained 
 from cats in groups a and b on days 1 , 3 , 5 , 7 and 9 and from cats in group c on days 1 and 
 9 .
the concentration of orb in feces was determined at the research institute for animal 
 science in biochemistry and toxicology ( sagamihara , japan ) . in brief ,
orb in stool samples 
 was extracted with acetonitrile containing 1% formic acid and purified via liquid  liquid 
 partition , salting - out and a mini - column ( oasis max , nihon waters k.k . ,
orb was analyzed using an hplc system ( shimadzu corporation , kyoto , japan ) equipped 
 with a binary pump , an autosampler , a column heater and a fluorescence detector . for 
 separation ,
supelcosil abzplus ( sigma  aldrich co. , llc , tokyo , japan ) was concurrently used 
 with lichrocart 4 - 4 lichrosher 100 rp-18 guard column ( merck , tokyo , japan ) .
the quality control was carried 
 out by analyzing stool samples together with samples of known concentrations ( i.e. 0.2 and 2 
 g / g ) every time .
kramer multiple comparison test was used to compare the mean age and body weight of 
 the enrolled cats , and mean cfu / g of stool samples among groups a , b and c. the mann 
whitney 
 u test was used to compare mean concentrations of orb in feces between 
 groups a and b. fisher s exact test was used to compare rates of antimicrobial resistance 
 between groups and periods ( i.e. before and after treatment ) .
a p value of 
 < 0.05 was considered as statistically significant in all analyses .
enrolled cats : there were no differences in age or body weight among the 
 three groups ( p>0.05 ) .
total number of coliforms in cats : the total number of coliforms during 
 the test schedule is shown in fig . 1fig .
1.the means of the total number of coliforms and orb concentration in the feces of cats 
 in groups a , b and c during the test schedule . in groups a and b , orb was administered 
 orally daily at a dose of 5.0 and 2.5 mg / kg , respectively , on days 17 
 ( ) .
see supplemental figs . 1 and 2 for the total number of coliforms and 
 orb concentration , respectively
significantly lower number 
 of coliforms in groups a and b , respectively , compared with group c 
 ( p<0.05 ) .
* there were significant differences between groups a 
 and b ( p<0.05 ) . and supplemental fig .
1 . according to the one - way analysis of variance and the 
 tukey 
kramer multiple comparison test , pre - treatment coliform counts in groups a , b and c 
 were not significantly different ( 7.08  0.53 , 5.39  0.72 and 6.55  1.11 , 
 respectively ) .
the means of the total number of coliforms and orb concentration in the feces of cats 
 in groups a , b and c during the test schedule . in groups
a and b , orb was administered 
 orally daily at a dose of 5.0 and 2.5 mg / kg , respectively , on days 17 
 ( ) .
see supplemental figs . 1 and 2 for the total number of coliforms and 
 orb concentration , respectively , in each cat .
significantly lower number 
 of coliforms in groups a and b , respectively , compared with group c 
 ( p<0.05 ) .
* there were significant differences between groups a 
 and b ( p<0.05 ) . in groups
a and b , within 3 days of orb administration , the number of fecal coliforms 
 decreased rapidly and continued to be significantly lower than that of group c 
 ( p<0.05 ) , until day 9 or 7 , respectively .
after cessation of orb 
 treatment , coliform counts in the cats of groups a and b mostly reached detectable levels by 
 days 9 ( cat 3 ) and 11 ( cats 1 and 2 ) and by days 7 ( cat 5 ) and 9 ( cat 6 ) , respectively . 

there were no significant differences in coliform counts between groups a and c from day 11 
 ( p>0.05 ) .
on the other hand , significant differences were found in 
 coliform counts between groups b and c ( p<0.05 ) on days 13 , 15 and 17 , 
 because of extremely low counts of coliform in cats 4 and 6 .
significant differences between 
 groups a and b were not seen during the study period ( p>0.05 ) .
no 
 remarkable fluctuations were observed in coliform counts in the cats of group c during the 
 study period .
rates of antimicrobial resistance of coliforms in cats : by antimicrobial 
 susceptibility testing , orb resistance was not detected in groups a and b before the 
 treatment . after the treatment , orb resistance was found in group b ( 6.0% ) ; however , there 
 was no significant difference in the resistance rates before and after the treatment 
 ( p>0.05 ) . on the other hand ,
compared to before treatment , rates of amp resistance were significantly high in groups a 
 and b after the treatment ( 0% vs. 26.2% and 0% vs. 76.2% , respectively , 
 p<0.01 ) ; rates of cez resistance were also significantly high in these 
 groups after the treatment ( 0% vs. 26.2% and 0% vs. 76.2% , respectively , 
 p<0.01 ) .
there were significant differences in rates of resistance to 
 the two drugs between groups a and b after the treatment ( p<0.01 ) .
in group c , resistance to any antimicrobials tested was not detected during test 
 period .
changes in the composition of the bacterial population of fecal coliforms : 
 coliform species are shown in table 1table 1.bacterial species of coliforms from stool samples obtained from cats during the 
 test schedulegroupcatdate of sampling ( no . of isolates ) 1357911131517a1eccfececec2ececec / cfcfec3ececececec / cfec / cfb4eccf5eccfcfec / cfececcf6eccfcfcfecc7ececececececececec8ececececececececec9ecececececececececa ) groups a and b were treated with 5 and 2.5 mg orb / kg , respectively .
c ) the isolates were also detected using emb agar containing orb 
 ( 2 g / ml ) and exhibited low ( mic : 1 
 g / ml ) or intermediate susceptibility to orb ( mic : 
 24 g / ml ) .. on day 1 , all isolates from cats in groups a , b and c were identified as 
 e. coli .
a ) groups a and b were treated with 5 and 2.5 mg orb / kg , respectively .
c ) the isolates were also detected using emb agar containing orb 
 ( 2 g / ml ) and exhibited low ( mic : 1 
 g / ml ) or intermediate susceptibility to orb ( mic : 
 24 g / ml ) . after orb treatment , the isolates of citrobacter freundii were detected in 
 all cats in groups a and b. in group a , c. freundii isolates were detected 
 on days 11 , 13 , 15 and/or 17 , whereas in group b , the bacteria were detected on days 7 , 9 , 
 11 , 13 and/or 17 .
furthermore , e. coli isolates were detected alone or 
 along with c. freundii isolates in all cats except cat 4 ( from group b ) , in 
 whom coliforms were not detected until day 15 after treatment initiation , and only 
 c. freundii isolates were detected on day 17 .
using emb agar containing orb , coliforms were detected with 10 cfu / g in cat 
 3 ( from group a ) on day 15 and with 10 cfu / g in cat 4 ( from group b ) on day 
 17 after treatment initiation ; these isolates were identified as e. coli 
 and c. freundii ( cat 3 ) and c. freundii alone ( cat 4 ) .
no 
 growth of coliforms was detected in the other cats by using orb - containing emb agar .
coliform bacteria other than e. coli or c. freundii were 
 not identified during the test schedule .
resistance mechanisms in e. coli and c. freundii : as for orb resistance , 
 e. coli isolates were highly susceptible ( mic , 0.0310.25 
 g / ml ) in cats 1 and 2 ( from group a ) , and 4 , 5 and 6 
 ( from group b ) not only before treatment but also after treatment ( table 2table 2.antimicrobial susceptibility and fq resistance mechanisms of coliforms from cats 
 before and after orb administrationgroupcatperiodisolatesmic range of 
 orbifloxacin(g / ml)qrdr mutationpmqrsusceptibility to other antimicrobialsspeciesngyraparca1pre - treatmentec100.063susceptiblepost - treatmentec300.0630.125susceptiblecf104noneqnrbamp cez2pre - treatmentec100.0630.125susceptiblepost - treatmentec220.0310.125susceptiblecf184noneqnrbamp cez3pre - treatmentec100.063susceptiblepost - treatmentec50.0310.063susceptible391s83lnonenonesusceptiblecf64noneqnrbamp cezec412s83lnonenonesusceptiblecf64noneqnrbamp cezb4pre - treatmentec100.0630.125susceptiblepost - treatmentcf104noneqnrbamp cezcf104noneqnrbamp cez5pre - treatmentec100.063susceptiblepost - treatmentec90.0630.25susceptiblecf254noneqnrbamp cez6pre - treatmentec100.063susceptiblepost - treatmentec100.0630.125susceptiblecf254noneqnrbamp cez58noneqnrbamp cezc7allec890.0630.125susceptible8allec900.0630.125susceptible9allec870.0630.125susceptiblea ) groups a and b were treated with 5 and 2.5 mg orb / kg , respectively .
c ) the isolates were detected using emb agar containing orb ( 2 
 g / ml ) .
d ) s83l : codon position 83 , 
 serine - to - leucine mutation . ) . on the other hand , isolates with low or intermediate susceptibility ( mic , 
 12 g / ml ) were predominantly detected in cat 3 ( from 
 group 1 ) after treatment , and these isolates had a single qrdr mutation in 
 gyra at codon position 83 ( serine to leucine : s83l ) . in group c , all 
 e. coli isolates were highly susceptible to orb ( mic , 0.0630.125 
 g / ml ) during the test schedule . all c. 
 freundii isolates from cats 15 demonstrated intermediate susceptibility ( mic , 4 
 g / ml ) , whereas the resistant isolates ( mic , 8 
 g / ml ) were barely detectable only in cat 6 on days 9 , 
 11 and 13 . according to pcr and sequencing results , none of the c. freundii 
 isolates had qrdr mutations in gyra , but carried qnrb , one 
 of the pmqr genes .
a ) groups a and b were treated with 5 and 2.5 mg orb / kg , respectively .
c ) the isolates were detected using emb agar containing orb ( 2 
 g / ml ) .
d ) s83l : codon position 83 , 
 serine - to - leucine mutation .
all e. coli isolates were susceptible to the 5 tested antimicrobials , 
 whereas all c. freundii isolates were resistant to amp and cez .
all 
 c. freundii isolates were phenotypically confirmed to be inducible ampc 
 producers , but not esbl producers .
genetic relationship between c. freundii isolates from cats : four 
 c. freundii isolates each from cats 16 ( from groups a and b ) were 
 selected and subjected to eric - pcr . as the result , all of these isolates had the identical 
 banding pattern ( data not shown ) .
orb concentration in feces of orb - treated cats : the changes in orb 
 concentration in feces are shown in fig . 1 and 
 supplemental fig .
orb was detected in cats of groups a and b between days 3 and 9 after 
 treatment initiation .
highest concentration of orb was determined in cats 2 and 6 of groups 
 a and b , respectively , between days 3 and 7 .
the peaks of mean concentration in groups a and 
 b were observed on day 3 ( 141.0 and 61.3 g / g , respectively ) . on days 5 and 
 7 , significantly higher concentrations of orb were detected in group a than in group b 
 ( p<0.05 ) .
on the other hand , orb was not detected in all of cats in 
 group c on days 1 and 9 .
to the best of our knowledge , this is the first study to evaluate the effects of oral fq on 
 the fecal flora of cats .
we have demonstrated that the counts of coliform bacteria 
 significantly decreased during and after the period for administration of orb .
a similar 
 reduction in the number of coliforms was reported in dogs treated with enrofloxacin .
thus , these data indicate that fq administration 
 reduces the number of coliforms in the fecal flora of cats and dogs . as for the composition 
 of the bacterial population of the fecal coliform bacteria ,
only e. coli 
 was detected in all cats on day 1 , whereas isolates of c. freundii and 
 e. coli were detected as a dominant species of all cats in groups a and b 
 after cessation of orb treatment .
few reports have addressed the effect of antimicrobial 
 treatment on the composition of the bacterial population of fecal or gut flora in animals . 

johnson et al .   detected the 
 emergence of streptococcus spp . and corynebacterium spp . 
 in cats after the administration of metronidazole .
confirmed the increase in the number of enterococcal 
 cells in dogs after the administration of cefovecin .
although there were several differences 
 in test condition between studies , our and their results suggest that administration of 
 antimicrobials can cause increase , in addition to decrease , of specific bacterial species of 
 fecal or gut flora , which may be explained by microbial substitution .
pharmacokinetics of orb has not yet been evaluated in animals treated with different doses 
 of this antibiotic . in this study , we found clear differences in orb concentrations in feces 
 between the two doses ( 5.0 and 2.5 mg / kg ) .
this dose - dependent kinetics of orb should be 
 considered when administering this drug . moreover , our results reveal remarkably high 
 concentrations of orb in feces of cats receiving either the high - dose or low - dose treatment . 
 thus , orb is likely to be mostly excreted into the feces after oral administration : a notion 
 supported by a previous study , revealing that a 
 higher concentration of orb is present in bile acid than in serum of cats after orb 
 treatment .
these pharmacokinetic properties of the drug may be responsible for the 
 significant reduction of fecal coliforms in cats after treatment .
our study showed some variations in the numbers of coliforms and orb concentrations in 
 feces between cats administered with the drug , suggesting that effect of orb treatment on 
 fecal coliforms and pharmacokinetics of the drug may vary by individual .
such 
 interindividual variations should be taken into account when administering the drug for 
 cats .
susceptibility testing revealed that no orb - resistant e. coli appeared 
 because all isolates failed to develop more than one qrdr mutation : the necessary condition 
 for the acquisition of fq resistance .
, wherein all fecal e. coli isolates exhibit high - level 
 resistance to enrofloxacin after treatment of dogs with the drug . in general
, the concept of 
 a mutant selection window has considerable implications for the acquisition of fq 
 resistance , and antimicrobials at concentrations beyond the mutant prevention concentration 
 ( mpc ) can prevent the development of fq - resistant mutants . in our study , the fecal orb concentration in cats treated with the doses of 
 2.5 and 5.0 mg / kg far exceeds the mpc value of e. coli ( 0.532 
 g / ml ) ; this result has been confirmed by another study 
 .
these findings suggest that oral orb poses a 
 low risk of selection of highly fq - resistant mutants among fecal e. coli 
 isolates in cats because of high gut levels .
compared with e. coli isolates , c. freundii isolates show 
 higher orb mic values .
such low susceptibilities to fqs may give a competitive advantage to 
 c. freundii when administered with orb .
most strains of c. 
 freundii maintained intermediate sensitivity to orb . among the tested pmqr genes , 

this 
 finding can be explained by a study by jacoby et al . , revealing that chromosomal qnrb is prevalent in 
 c. freundii isolates . on the other hand ,
no qrdr mutations of 
 gyra , the basis of the fq resistance mechanism , were identified in c. freundii isolates .
cesaro 
 et al .   reported that qrdr 
 mutations can be more effectively suppressed in qnr - positive e. 
 coil strains than in qnr - negative e. coli 
 strains ; this property of qnr may elucidate our present results , namely 
 that qnrb - positive c. freundii isolates fail to acquire 
 strong orb resistance .
it should be considered that gut flora of cats can act as a reservoir 
 of qnr - positive bacteria , which are possibly selected as a result of fq 
 use . in coliform - positive samples after orb treatment ,
qnr - positive 
 c. freundii isolates were detected more frequently in group b ( 8 of 11 
 samples ) than in group a ( 5 of 13 samples ) .
this finding implies that low - dose treatment of 
 orb might facilitate the selection of qnr - positive bacteria , compared with 
 high - dose treatment .
however , to clarify this point , further large - scale studies would be 
 needed . as for susceptibilities to the antimicrobials other than fqs ,
all e. coli 
 isolates exhibited susceptibility to all the tested antimicrobials both before and after orb 
 treatment . as a result
, multidrug - resistant e. coli isolates , which were 
 reported in dogs after enrofloxacin treatment , 
 were not detected in the present study .
on the other hand , all c. freundii 
 isolates exhibited resistance to amp and cez by the production of ampc , but not esbls .
this 
 finding resulted in the significant increase of resistance rates to amp and cez in isolates 
 of e. coli and c. freundii after orb treatment .
similarly , 
 the high prevalence of ampc in this bacterial species was previously found in human isolates 
 in a study by kanamori et al . . 

our study suggests that fq use poses a risk of coselection of ampc - producing c. 
 freundii isolates in feces of cats .
eric - pcr revealed that c. freundii isolates , which were 
 qnrb - positive and ampc - producing , from cats 16 ( from groups a and b ) 
 were clonal or genetically identical .
this result indicates that c. 
 freundii isolates spread clonally in the cats ; however , the cause of the clonal 
 spread could not be identified .
one hypothesis is cross - contamination between the cats 
 during the study , despite deliberate efforts to prevent this .
another is that orb 
 treatment might select c. freundii isolates that had spread horizontally 
 among enrolled cats before the study . in either case ,
more aggressive and effective measures 
 to prevent any transfer of bacteria would be needed for future studies .
firstly , this study was carried out as a pilot study 
 by using a small number of cats , and thus , the present results might be somewhat biased . 
 secondly , the effect of orb treatment remains to be clarified in cats with clinical signs or 
 household cats , because we used healthy experimental cats in research settings .
fecal or gut flora in cats is composed of a 
 variety of aerobic and anaerobic bacteria , in 
 addition to coliform bacteria . therefore , this study could not entirely clarify an effect of 
 orb treatment on fecal flora of cats .
nevertheless , we have described the effects of orb administration on fecal coliforms in 
 healthy cats .
we revealed alterations in bacterial composition , e.g. selection of 
 qnrb- and ampc - positive c. freundii isolates , in 
 addition to a decrease in the total number of coliforms .
moreover , we could not detect 
 strongly fq - resistant mutants among isolates of e. coli and c. 
 freundii .
further studies using household cats with and without clinical signs 
 are required to assess clinical and public health implications of the effects of fq use on 
 the fecal flora of cats .
 the study objective was to determine the effect of oral orbifloxacin ( orb ) on 
 antimicrobial susceptibility and composition of fecal coliforms in cats . 
 nine cats were 
 randomized to two groups administered a daily oral dose of 2.5 and 5.0 mg orb / kg for 7 
 days and a control group ( three cats per group ) . 
 coliforms were isolated from stool 
 samples and were tested for susceptibilities to orb and 5 other drugs . 
 orb concentration 
 in feces was measured using high - performance liquid chromatography ( hplc ) . 
 the coliforms 
 were undetectable after 2 days of orb administration , and their number increased in most 
 cats after termination of the administration . 
 furthermore , only isolates of 
 escherichia coli were detected in all cats before administration , and 
 those of citrobacter freundii were detected after termination of the 
 administration . 
 e. coli isolates exhibited high orb susceptibility 
 [ minimum inhibitory concentration ( mic ) , 0.125 g / ml ] 
 or relatively low susceptibility ( mic , 12 g / ml ) with a 
 single gyra mutation . 
 c. freundii isolates largely 
 exhibited intermediate orb susceptibility ( mic , 4 
 g / ml ) , in addition to resistance to ampicillin and 
 cefazolin , and harbored qnrb , but not a gyra mutation . 
 
 hplc revealed that the peaks of mean concentration were 61.3 and 141.0 
 g / g in groups receiving 2.5 and 5.0 mg / kg , respectively . 
 our findings 
 suggest that oral orb may alter the total counts and composition of fecal coliform , but is 
 unlikely to yield highly fluoroquinolone - resistant mutants of e. coli and 
 c. freundii in cats , possibly because of the high drug concentration in 
 feces . 