sample collection : chicken fecal samples were collected from vietnam , 
 indonesia and thailand by local collaborators . the countries chosen in this study are areas of major chicken production in 
 the region and are considered representative of chicken farming in other southeast asian 
 countries . one hundred fecal samples were collected from a poultry slaughterhouse ( suburbs 
 of hanoi ) in vietnam during september 2007 . fecal samples were obtained from chickens that 
 had been brought from 23 farms located in 19 provinces ( bac giang , bac ninh , cao bang , ha 
 nam , ha noi , ha tay , hai duong , hai phong , hung yen , long son , nam dinh , ninh binh , phu tho , 
 quang ninh , thai binh , tahi nguyen , thanh hoa , tuyen quang and vinh phuc ) ( 5 
 samples / province ; 10 samples were obtained from ha noi ) . from indonesia , a total of 144 
 fecal samples were collected at seven farms from seven different provinces ( north sumatra , 
 west sumatra , lampung , yogyakarta , south kalimantan , bali and south sulawesi ) ( 20 to 22 
 samples / province ) during january and february 2007 . from thailand , a total of 100 fecal 
 samples were collected from three farms located in three provinces ( bangkok , burirum and 
 surin ) ( 43 samples / bangkok , 37 samples / burirum and 20 samples / surin ) during january 2006 . all samples were placed in sterile plastic tubes and stored at 20c or 80c until use . bacterial isolation : to isolate e. coli , chicken fecal 
 samples were spread onto desoxycholate - hydrogen sulfate - lactose agar ( nissui pharmaceutical , 
 tokyo , japan ) and incubated at 37c overnight . for individual samples , a maximum of 2 
 colonies were identified as e. coli on the basis of colony morphology and 
 then selected for further analysis . to confirm that these colonies were e. 
 coli , the selected colonies were then processed for api 20e testing ( sysmex , 
 kobe , japan ) . samples from thailand were not used for e. coli 
 isolation . to isolate enterococci , chicken fecal samples were spread onto enterococcosel ( ecs ) agar 
 ( becton dickinson , franklin lakes , nj , u.s.a . ) , incubated at 37c for 4872 hr and enriched 
 using ecs broth . after incubation at 37c for 16 hr , enriched cultures were streaked onto 
 ecs agar . for individual samples , we selected a maximum of two colonies identified as 
 enterococci on the basis of colony morphology . the colonies were applied to the api 20 strep 
 system ( sysmex ) for bacterial species identification . dna was extracted from the colonies 
 using a commercial kit ( instagenematrix , biorad , tokyo , japan ) and following the 
 manufacturer s instructions . to distinguish between e. faecalis and 
 e. faecium , we performed multiplex pcr . antimicrobial susceptibility tests : we determined minimal inhibitory 
 concentrations ( mics ) using the agar dilution method according to guidelines set by the 
 clinical laboratory standards institute ( clsi ) . we 
 tested the following antimicrobials on e. coli : ampicillin , cefazolin , 
 kanamycin , gentamicin , dihydrostreptomycin , oxytetracycline , chloramphenicol , nalidixic 
 acid , enrofloxacin ( all obtained from sigma - aldrich , st . louis , mo , u.s.a . ) and cefpodoxime 
 ( daiichi - sankyo , tokyo , japan ) . on e. faecalis and e. 
 faecium , we tested the following antimicrobial agents : ampicillin , kanamycin , 
 gentamicin , dihydrostreptomycin , erythromycin , lincomycin , oxytetracycline , vancomycin , 
 chloramphenicol and enrofloxacin ( all obtained from sigma - aldrich ) . the breakpoints 
 for kanamycin , gentamicin and enrofloxacin for e. faecalis and gentamicin 
 for e. faecium have not been defined by the clsi , and therefore , we used 
 the guidelines set by kojima et al . . in this study , we defined the breakpoint of kanamycin and enrofloxacin for 
 e. faecium by taking into consideration the midpoint between the peaks of 
 each mic distribution . e. coli atcc25922 , staphylococcus 
 aureus atcc29213 , e. faecalis atcc29212 and pseudomonas 
 aeruginosa atcc27853 were used as quality control strains . enterococcus isolates that exhibited low susceptibility to vancomycin 
 ( mic=8 mg / l ) were selected for further tests . detection of vancomycin - resistance genes : the vancomycin - resistance genes , 
 vana , vanb , vanc1 , vanc2/3 , vand and vane , were detected 
 in the isolates that exhibited low susceptibility to vancomycin by performing pcr as 
 previously described [ 12 , 17 ] . statistical analysis : e. coli resistance to the selected 
 antimicrobial agents was compared between isolates from vietnam and indonesia using the 
  test . when a two - by - two table contained at least 1 expected value below 5 , 
 the fisher s exact test was performed . e. faecalis and e. faecium isolates were obtained from 
 vietnam , indonesia and thailand . the prevalence of antimicrobial resistant bacterial strains 
 across each country was compared using multiple comparisons . a chi - square test was first 
 performed for each antimicrobial agent to detect significant differences within a country . when the result was significant , a test for multiple comparisons of proportions   was performed using r ( version 2.14.2 ) statistical 
 software . if we did not identify any resistant isolates in 2 of the 3 countries , we 
 performed a pairwise comparison between the country with the resistant isolate and the 
 countries that did not have a resistant isolate using either the  test or 
 fisher s exact test . the criteria used to select between these tests are mentioned above ( as 
 described for e. coli ) . e. coli : in total , we identified 125 e. coli isolates and 
 described any antimicrobial resistance for all agents tested ( table 1table 1.antimicrobial susceptibility of e. coli isolates from chickens 
 obtained from southeast asian countriesantimicrobialbreak point(mg / l)mic range(mg / l)mic50 ( mg / l)mic90 ( mg / l)% of antimicrobial resistancevietnam(n=47)indonesia(n=78)total(n=125)ampicillin321>512256>51283.043.658.4cefazolin80.25512284.35.14.8cefpodoxime8<0.1251280.2514.35.14.8kanamycin642>5122>51248.916.728.8gentamicin160.3>51213244.7016.8dihydrostreptomycin321>512851282.924.446.4oxytetracycline160.5>51212851293.661.573.6chloramphenicol320.25>512825651.125.635.2nalidixic acid322>512128>51280.142.356.8enrofloxacin2<0.1256416470.235.948.8mic : minimal inhibitory concentration . a ) the value was the clsi break point , b ) the 
 percentage of antimicrobial resistance in vietnam was significantly higher than that 
 in indonesia ( p<0.05 ) . ) . the majority of e. coli isolates from vietnam and indonesia 
 were resistant to oxytetracycline ( 73.6% ) , nalidixic acid ( 56.8% ) and ampicillin ( 58.4% ) . the prevalence of e. coli isolates from vietnam that were resistant to 
 almost all antimicrobials , except cefazolin and cefpodoxime , was significantly higher than 
 of those from indonesia ( p<0.05 ) . the diversity of multidrug resistant 
 phenotypes among the e. coli isolates is shown in table 2table 2.multidrug resistant phenotypes of e. coli isolates from chickens 
 obtained from southeast asian countriesno . exhibitingantimicrobialresistancemdr patternsno . of isolatesvietnam(n=47)indonesia(n=78)total(n=125)10accdkgdocpne2028akgdocpne6067akgdone505akdocpne325agdocpne202akgdocpn101ackgdon101accdkdne0116adocpne224akdone303agdocpn202akdocpn101akocpne011kgdone0115aocpne347accdkd033akone022adone112kdone112adocpn011aocpne0114aone224adon303gdocp101done101ocpne0113one055don134doc033ado022aocp022akn011ako0112ao033ne033do022ad101ag101ocp011mdr : multidrug resistance , a : ampicillin , c : cefazolin , cd : cefpodoxime , k : 
 kanamycin , g : gentamicin , d : dihydrostreptomycin , o : oxytetracycline , cp : 
 chloramphenicol , n : nalidixic acid , e : enrofloxacin .. forty - three ( 91.5% ) of the 47 isolates obtained from vietnam exhibited 
 multidrug resistance , and the isolates were resistant to 210 different antimicrobials . forty - nine ( 62.8% ) out of 78 isolates obtained from indonesia exhibited multidrug 
 resistance , and each isolate was resistant to 27 antimicrobial agents . a ) the value was the clsi break point , b ) the 
 percentage of antimicrobial resistance in vietnam was significantly higher than that 
 in indonesia ( p<0.05 ) . mdr : multidrug resistance , a : ampicillin , c : cefazolin , cd : cefpodoxime , k : 
 kanamycin , g : gentamicin , d : dihydrostreptomycin , o : oxytetracycline , cp : 
 chloramphenicol , n : nalidixic acid , e : enrofloxacin . the isolates exhibited antimicrobial resistance for 8 of the 10 agents tested 
 ( table 3table 3.antimicrobial susceptibility of e. faecalis isolates from 
 chickens obtained from southeast asian countriesantimicrobialbreak point(mg / l)mic range(mg / l)mic50(mg / l)mic90(mg / l)% of antimicrobial resistancevietnam(n=22)indonesia(n=58)thailand(n=37)total(n=117)ampicillin16<0.1258120000kanamycin6432>51264>51277.329.32737.6gentamicin5124>5121651240.96.9011.1dihydrostreptomycin25616512128>51295.441.440.551.3erythromycin8<0.125>512>512>51290.977.648.670.9lincomycin12832>512>512>51290.979.354.173.5oxytetracycline161512256>51210065.556.869.2vancomycin320.58240000chloramphenicol3241281612886.38.621.627.4enrofloxacin160.2512816436.4195.417.9mic : minimal inhibitory concentration . a ) the value was the clsi break point , b ) the 
 value was the jvarm breakpoint , c)e ) 
 superscripts indicate significantly higher percentage of antimicrobial resistance in 
 vietnam than in indonesia ( c ) , vietnam than in thailand ( d ) , indonesia than in 
 thailand ( e ) ( p<0.05 ) . ) . e. faecalis resistance against lincomycin ( 73.5% ) , 
 erythromycin ( 70.9% ) and oxytetracycline ( 69.2% ) was common in all 3 countries . mg / l ) , but the isolates 
 did not have vancomycin - resistance genes . the prevalence of e. faecalis 
 isolates from vietnam that were resistant to almost all antimicrobials tested was 
 significantly higher than of those collected from other countries 
 ( p<0.05 ) . the prevalence of e. faecalis isolates from 
 indonesia that were resistant to erythromycin and lincomycin was significantly higher than 
 the prevalence of those from thailand ( p<0.05 ) . the diversity of 
 multidrug resistant phenotypes among e. faecalis isolates is shown in table 4table 4.multidrug resistant phenotypes of e. faecalis isolates from 
 chickens obtained from southeast asian countriesno . : multidrug resistance , a : ampicillin , k : kanamycin , g : gentamicin , d : 
 dihydrostreptomycin , em : erythromycin , l : lincomycin , o : oxytetracycline , cp : 
 chloramphenicol , e : enrofloxacin .. twenty - two ( 100% ) out of 22 isolates obtained from vietnam exhibited 
 multidrug resistance and were found to be resistant to 28 different antimicrobials . forty - nine ( 84.5% ) out of 58 isolates obtained from indonesia exhibited multidrug resistance 
 and were found to be resistant to 26 different antimicrobials . twenty - seven ( 73.0% ) out of 
 37 isolates obtained from thailand exhibited multidrug resistance and were determined to be 
 resistant to 27 different antimicrobials . a ) the value was the clsi break point , b ) the 
 value was the jvarm breakpoint , c)e ) 
 superscripts indicate significantly higher percentage of antimicrobial resistance in 
 vietnam than in indonesia ( c ) , vietnam than in thailand ( d ) , indonesia than in 
 thailand ( e ) ( p<0.05 ) . mdr : multidrug resistance , a : ampicillin , k : kanamycin , g : gentamicin , d : 
 dihydrostreptomycin , em : erythromycin , l : lincomycin , o : oxytetracycline , cp : 
 chloramphenicol , e : enrofloxacin . antimicrobial resistance was found for nine of the 10 antimicrobials tested 
 ( table 5table 5.antimicrobial susceptibility of e. faecium isolates from 
 chickens obtained from southeast asian countriesantimicrobialbreak point(mg / l)mic range(mg / l)mic50(mg / l)mic90(mg / l)% of antimicrobial resistancevietnam(n=89)indonesia(n=58)thailand(n=33)total(n=180)ampicillin16<0.125644819.1009.4kanamycin648>512128>51250.669.081.862.2gentamicin25615128>51227.00313.9dihydrostreptomycin25616>51264>51261.829.315.242.8erythromycin8<0.125>512512>51291.051.797.079.4lincomycin1288>512512>51288.86997.0 83.9oxytetracycline160.551225651297.88197.0 92.2vancomycin320.58120000chloramphenicol32212881610.1005.0enrofloxacin20.5128812886.56997.0 82.8mic : minimal inhibitory concentration . a ) the value was the clsi break point , b ) the 
 value was set as the midpoint between the peaks of each mic distribution , c ) the value 
 was the jvarm breakpoint , d)i ) 
 superscripts indicate a significantly higher percentage of antimicrobial resistance in 
 vietnam than in indonesia ( d ) , vietnam than in thailand ( e ) , indonesia than in vietnam 
 ( f ) , indonesia than in thailand ( g ) , thailand than in vietnam ( h ) , thailand than in 
 vietnam ( i ) ( p<0.05 ) . ) . e. faecium isolate resistance to oxytetracycline ( 92.2% ) , 
 lincomycin ( 83.9% ) , enrofloxacin ( 82.8% ) , erythromycin ( 79.4% ) and kanamycin ( 62.2% ) was 
 common in all three countries . mg / l ) , but the isolate did not contain 
 vancomycin - resistance genes . the prevalence of e. faecium isolates from 
 vietnam that were resistant to almost all the antimicrobials tested was significantly higher 
 than of those from indonesia ( p<0.05 ) , except for kanamycin and 
 vancomycin . the prevalence of e. faecium isolates from vietnam that were 
 resistant to ampicillin , gentamicin , dihydrostreptomycin and chloramphenicol was 
 significantly higher than of those from indonesia ( p<0.05 ) . the 
 prevalence of e. faecium isolates from thailand that were resistant to 
 erythromycin , lincomycin , oxytetracycline and enrofloxacin was significantly higher than of 
 those from indonesia ( p<0.05 ) . the diversity of multidrug resistant 
 phenotypes among the e. faecium isolates is shown in table 6table 6.multidrug resistant phenotypes of e. faecium isolates from 
 chickens obtained from southeast asian countriesno . exhibitingantimicrobial resistancemdr patternsno . of isolatesvietnam(n=89)indonesia(n=58)thailand(n=33)total(n=180)9akgdemlocpe10018akgdemloe6006kgdemlocpe2002akdemlocpe10017kgdemloe130013agdemloe3003kgdemlocp1012kdemlocpe10016kdemloe710320kgemloe7119ademloe5005kdemlocp10015kemloe242026demloe8008kdemlo3115kdloe0202kdemle01014emloe154625kemlo1506kloe0404kemoe2002demlo0202kdlo0101kdle0101demle01013loe1405klo0303koe0303aoe1001kdo1001emle1001deml01012oe1304kem0011ke0101do1001lo0101mdr : multidrug resistance , a : ampicillin , k : kanamycin , g : gentamicin , d : 
 dihydrostreptomycin , em : erythromycin , l : lincomycin , o : oxytetracycline , cp : 
 chloramphenicol , e : enrofloxacin .. eighty - five ( 95.5% ) out of 89 isolates obtained from vietnam exhibited 
 multidrug resistance and were resistant to 29 different antimicrobials . fifty - three ( 91.4% ) 
 out of the 58 isolates obtained from indonesia exhibited multidrug resistance and were 
 resistant to 26 different antimicrobials . thirty - three ( 100% ) out of the 33 isolates 
 obtained from thailand exhibited multidrug resistance and were resistant to 27 different 
 antimicrobials . a ) the value was the clsi break point , b ) the 
 value was set as the midpoint between the peaks of each mic distribution , c ) the value 
 was the jvarm breakpoint , d)i ) 
 superscripts indicate a significantly higher percentage of antimicrobial resistance in 
 vietnam than in indonesia ( d ) , vietnam than in thailand ( e ) , indonesia than in vietnam 
 ( f ) , indonesia than in thailand ( g ) , thailand than in vietnam ( h ) , thailand than in 
 vietnam ( i ) ( p<0.05 ) . mdr : multidrug resistance , a : ampicillin , k : kanamycin , g : gentamicin , d : 
 dihydrostreptomycin , em : erythromycin , l : lincomycin , o : oxytetracycline , cp : 
 chloramphenicol , e : enrofloxacin . in this study , e. faecium was more frequently isolated from chicken feces 
 than e. faecalis , which is consistent with previous reports [ 3 , 10 ] that 
 suggested that e. faecium is the most predominant enterococcus in chicken 
 feces . resistance against enrofloxacin and ampicillin was more commonly observed in 
 e. faecium than e. faecalis . in contrast , resistance to 
 chloramphenicol was more commonly observed in e. faecalis than e. 
 faecium . the same trend has been observed in several previous studies [ 4 , 19 ] . these 
 results suggest that the frequency of acquiring antimicrobial resistance differs according 
 to the species . the prevalence of e. coli and enterococci isolates resistant to 
 antimicrobials , especially fluoroquinolone , was higher than in developed countries [ 5 , 16 , 18 ] . in this study , most e. coli and 
 enterococci isolates exhibited multidrug resistance . in all three countries , previous work 
 has shown that a resistance of indicator bacteria against each class of antimicrobial is 
 proportional to the total amount of the respective antimicrobial used to treat animals 
 . a high frequency of oxytetracycline resistance 
 and multidrug resistance may be a result of the large amount of oxytetracycline and multiple 
 other antimicrobials being used as feed additives to promote chicken growth as well as the 
 excessive use of these drugs in the veterinary setting . a comparison of the results from the three countries revealed that e. coli 
 isolates and enterococci from vietnam exhibited a significantly higher prevalence for 
 resistance to the majority of the antimicrobials tested . in addition , multidrug resistance 
 was more frequently observed in isolates from vietnam as compared with those from indonesia 
 and thailand . in vietnam , several studies have suggested that the high percentage of 
 antimicrobial resistance in retail meats is caused by the unregulated and/or inappropriate 
 use of antimicrobials in animal farming [ 11 , 22 , 25 ] . our 
 results suggest that the usage of antimicrobials in chickens in southeast asian countries , 
 especially in vietnam , is higher than that in developed countries [ 11 , 22 , 25 ] . in this study , the prevalence of bacteria resistant to fluoroquinolones was extremely high 
 compared to that in developed countries [ 5 , 16 , 18 ] . the who 
 considers both fluoroquinolones and third - generation cephalosporins to be critically 
 important medicines for both humans and animals . however , in developing countries , enrofloxacin ( a fluoroquinolone ) is often used not only as 
 a medicine , but also as a feed additive to promote animal growth ( personal communication 
 with local veterinary officer ) . in the united states , the use of fluoroquinolone in poultry 
 has been banned since 2004 . in japan , 
 fluoroquinolones have been recommended for therapeutic use as second - line drugs in the 
 veterinary field , since enrofloxacin was approved as a first - line veterinary fluoroquinolone 
 in 1991 . with increased globalization , the increased prevalence of fluoroquinolone - resistant 
 bacteria in southeast asian countries places many other countries at high risk . our current 
 data suggest that the governments of southeast asian countries should strictly regulate the 
 use of antimicrobials , particularly fluoroquinolones , in the treatment of food - producing 
 animals . in this study , three e. faecalis isolates and one e. 
 faecium isolate exhibited low susceptibility to vancomycin ( 8 
 mg vancomycin - resistant enterococci ( vres ) have previously been 
 isolated from chicken meat that was imported into japan from thailand [ 15 , 23 ] . while we did attempt to 
 identify the genes responsible for vancomycin - resistance , such as vana or 
 vanb , we were unable to detect them . in japan , vres were not detected in 
 food - producing animals in 1999 due to the ban on avoparcin ( a vancomycin analogue ) as a 
 treatment for food - producing animals , which began in 1997 . however , in denmark during 2006 , vres were detected in 3% of e. 
 faecalis samples obtained from pigs despite a ban restricting avoparcin use as a 
 treatment for food - producing animals that began in 1995 . notably , avoparcin was still given to chickens in the three countries during 
 this period ( personal communication with local veterinary officer ) . therefore , although vres 
 were not detected in this study , we advocate that continual surveillance for vres in 
 food - producing animals is important to accurately evaluate the future risk of infection for 
 both humans and food - producing animals . in this study , we determined the antimicrobial resistance of indicator bacteria obtained 
 from chickens in three southeast asian countries . our results show that monitoring the 
 development and prevalence of antimicrobial resistance in developing countries is required . ongoing surveys of antimicrobial resistance in developing countries are important , as the 
 information obtained will help establish guidelines for the prudent use of 
 antimicrobials .
abstractto determine the prevalence of indicator bacteria resistant to antimicrobials 
 among poultry in three southeast asian countries ( vietnam , indonesia and thailand ) , we 
 examined the antimicrobial susceptibilities of commensal bacteria isolated from chickens . 
 in total , 125 , 
 117 and 180 isolates of escherichia coli , 
 enterococcus faecalis and enterococcus faecium , 
 respectively , were used to test for antimicrobial susceptibility . bacterial resistance to 
 antimicrobial treatment was most frequently observed with oxytetracycline with a 
 prevalence of 73.6% ( e. coli ) , 69.2% ( e. faecalis ) and 
 92.2% ( e. faecium ) . 
 resistance to fluoroquinolones , which are critically 
 important medicines , was also frequently observed in e. coli ( 48.8% ) , 
 e. faecalis ( 17.9% ) and e. faecium ( 82.8% ) . 
 the 
 prevalence of indicator bacteria resistant to most of the antimicrobials tested in these 
 countries was higher than those for developed countries . 
 the factors underlying 
 antimicrobial resistance may include inappropriate and/or excessive use of antimicrobials . 
 these results highlight the need for monitoring the emergence and prevalence of 
 antimicrobial resistance in developing countries .