aeromonas strains : one hundred and nineteen strains were isolated from eel 
 ( n=70 ) , pet fish ( n=36 ) and koi carp ( n=13 ) . the eel strains were identified at the species 
 level in our previous study by phylogenetic analysis using housekeeping genes ( gyrb and 
 rpod ) [ 11 , 22 ] ; they consisted of a. dhakensis ( n=22 ) , a. caviae 
 ( n=16 ) , a. hydrophila ( n=12 ) , a. veronii ( n=13 ) , 
 a. jandaei ( n=4 ) , a. media ( n=2 ) and a. trota 
 ( n=1 ) . the pet fish strains were identified as 5 aeromonas 
 spp.a . veronii ( n=31 ) , a. allosaccharophila 
 ( n=2 ) , a. dhakensis ( n=1 ) , a. hydrophila ( n=1 ) 
 and a. jandaei ( n=1)by phylogenetic analysis using gyrb 
 gene sequences . the same identification method was applied for the koi carp strains , which 
 consisted of a. hydrophila ( n=8 ) and a. veronii ( n=5 ) . all 
 strains were stored at 70c using cryocare bacteria preservers ( key scientific products , 
 stamford , tx , u.s.a . ) until used for antimicrobial susceptibility tests and the detection of 
 -lactamase - encoding genes ( blatem , 
 blaoxa - b and blactx - m ) . antimicrobial susceptibility test : the antimicrobial susceptibility test 
 ( ast ) was implemented using the vitek 2 system with a veterinary susceptibility test card 
 for gram - negative bacteria ( ast - gn38 ) ( biomrieux , lyon , france ) , according to the 
 manufacturer s instructions . the ast - gn38 card contains the following -lactam antimicrobial 
 agents as dehydrated substances at the indicated concentrations : ceftiofur ( tio)1 and 2 
 g / ml ; amoxicillin and clavulanic acid ( amc)4/2 , 16/8 
 and 32/16 g / ml ; ampicillin ( am)4 , 8 and 32 
 g / ml ; cefpodoxime ( pod)0.5 , 1 and 4 
 g/l ; and piperacillin ( pip)4 , 16 , 32 and 64 
 g / ml . the cutoff for resistance to each antimicrobial 
 agent was designated as a resistance of greater than  intermediate  as defined by the 
 criteria of the vitek 2 system ( version 04.01 ) based on the clsi m100-s18 . detection of -lactam resistance genes : the genomic dna from strains was 
 purified using the accuprep genomic extraction kit ( bioneer , seoul , korea ) . the 
 -lactamase - encoding genes , blatem , 
 blaoxa - b and blactx - m , were 
 subjected to pcr assays using the same primers and conditions as used in previous studies 
 [ 8 , 18 ] . the 
 specificity for each pcr assay was demonstrated by direct sequencing analysis using the same 
 primer sets for amplification on randomly selected pcr products . however , all pcr products 
 for the blactx - m gene were sequenced at the macrogen service 
 center ( seoul , korea ) . the generated sequences ( 277 to 704 , based on the e. 
 coli numbering of the blactx - m-3 gene ; accession 
 number ab231615 ) were aligned with those of well - known blactx - m 
 genes ( genbank ) according to ctx - m-1 , 2 , 8/25 and 9 groups . genetic distances were 
 determined using the kimura two - parameter models , and neighbor - joining phylogenetic trees 
 were constructed using the mega5 program , according to the previously reported method . table 1table 1.resistance to five different -lactams according to aeromonas 
 spp . used in the present studyspeciesno . of resistant strain againstamamcpippodtioa . jandaei ( n=5)5 ( 0)2 ( 2)5 ( 1)02 ( 1)a . media ( n=2)2 ( 0)2 ( 1)2 ( 0)1 ( 0)2 ( 0)a . allosaccharophila ( n=2)2 ( 0)02 ( 0)00a . trota ( n=1)1 ( 1)01 ( 0)1 ( 0)1 ( 0)total ( n=119)118 ( 2)73 ( 59)101 ( 8)33 ( 15)63 ( 10 ) * parenthesized numbers indicate the number of strains with intermedate 
 resistance am and pip showed resistance in 99.2% and 84.9% of all 
 aeromonas strains , respectively . major species ( a. 
 veronii , a. hydrophila , a. caviae and 
 a. dhakensis ) revealed variable resistance levels to the other -lactams . 
 of these -lactams , the most resistant species to pod was a. dhakensis 
 ( 78.3% ) , followed by a. caviae ( 56.3% ) , a. hydrophila 
 ( 9.5% ) and a. veronii ( 4.1% ) . in addition , pod resistant strains ( 27.7% of 
 119 strains ) were simultaneously resistant to tio . tio resistant strains were observed in 
 52.9% of all strains including 21 strains of a. dhakensis ( 91.3% ) , 15 
 strains of a. caviae ( 93.8% ) , 10 strains of a. hydrophila 
 ( 47.6% ) and 12 strains of a. veronii ( 24.5% ) . in the case of amc 
 resistance , however , 89.0% ( n=105 ) of am - resistant strains ( n=118 ) 
 showed below intermediate resistance to amc . when compared with aeromonas 
 spp . , the decreased resistance was frequently observed in aeromonas spp . , 
 except for a. caviae strains ( table 
 1 ) . eleven different resistance patterns for -lactams strains could be classified among all 
 aeromonas strains ( table 
 2table 2.prevalence of blactx - m-1 group in aeromonas spp . 
 according to resistance patterns of -lactamsresistance patterndetection of blactx - m-1 
 group in aeromonas spp . mediatotalnone0000 ( 1)00000 ( 1)am001 ( 1)0 ( 8)00001 ( 9)am+amc000 ( 3)0 ( 2)00000 ( 5)am+pip1 ( 1)*1 ( 1)0 ( 2)0 ( 20)0 ( 3)00 ( 2)02 ( 29)am+pip+tio000 ( 2)0 ( 3)00000 ( 5)am+amc+tio1 ( 1)0 ( 1)0000001 ( 2)am+amc+pip1 ( 1)00 ( 5)0 ( 6)00001 ( 12)am+amc+pip+tio2 ( 2)0 ( 5)0 ( 6)0 ( 7)0 ( 2)000 ( 1)2 ( 23)am+pip+pod+tio0000 ( 1)00 ( 1)000 ( 2)am+amc+pod+tio00 ( 1)0000000 ( 1)am+amc+pip+pod+tio15 ( 18)1 ( 8)2 ( 2)0 ( 1)0000 ( 1)18 ( 30)total20 ( 23)2 ( 16)3 ( 21)0 ( 49)0 ( 5)0 ( 1)0 ( 2)0 ( 2)25 ( 119)*nonparenthesized and parenthesized numbers indicate the number of strains carrying 
 blactx - m-1 group genes and showing each grouped 
 phenotypic antimicrobial resistance , respectively . 3 out of 15 strains 
 harbored blactx - m-33 gene . ) . the predominant pattern was simultaneous resistance to all -lactams ( 30 
 strains ) , followed by am+pip ( 29 strains ) , am+amc+pip+tio ( 23 strains ) , am+amc+pip ( 12 
 strains ) and only am ( 9 strains ) . of these patterns , simultaneous resistance to all 
 -lactams was the most frequently observed patterns for a. dhakensis . * nonparenthesized and parenthesized numbers indicate the number of strains carrying 
 blactx - m-1 group genes and showing each grouped 
 phenotypic antimicrobial resistance , respectively . 3 out of 15 strains 
 harbored blactx - m-33 gene . in investigation of genetic background to -lactam resistances , all of the 119 strains 
 harbored the blatem , and sequences of random pcr products had 
 100% identity to gene encoding tem-1-type -lactamase ( genbank no . the 
 blaoxa gene was encountered in three strains : one a. 
 allosaccharophila strain from pet fish and two a. caviae strains 
 from eel . in the case of blactx - m gene , prevalence of the gene 
 showed differences according to aeromonas spp . and -lactam resistance 
 patterns ( table 2 ) . the 
 blactx - m genes were detected in 25 strains including 20 of 
 a. dhakensis ( 19 eel strains and 1 pet fish strain ) , 2 eel strains of 
 a. caviae and 3 eel strains of a. hydrophila . the pcr 
 products were subjected to phylogenetic analysis by direct sequencing analysis using the 
 primer set for blactx - m consensus . the constructed phylogenetic tree showed that 25 
 blactx - m - positive strains were grouped with genes encoding 
 ctx - m-1-group -lactamases ( fig . 1.phylogenetic identification of blactx - m-1-group gene 
 sequences resulting from blactx - m - positive strains . the 
 identification was performed by alignments with major sequences belonging to 
 blactx - m-1-group genes in genbank ( accession numbers 
 are within parentheses ) . the branch numbers refer to the percentage confidence as 
 estimated by a bootstrap analysis with 1,000 replications . ) . on the other hand , 3 eel strains of a. dhakensis were clustered 
 with the gene encoding ctx - m-33 belonging to ctx - m-1 group . in addition , the genes were 
 detected more among strains with resistance to all -lactams ( 18/30 ) than among strains with 
 other resistance patterns ( table 2 and fig . phylogenetic identification of blactx - m-1-group gene 
 sequences resulting from blactx - m - positive strains . the 
 identification was performed by alignments with major sequences belonging to 
 blactx - m-1-group genes in genbank ( accession numbers 
 are within parentheses ) . the branch numbers refer to the percentage confidence as 
 estimated by a bootstrap analysis with 1,000 replications . dhakensis could be a reservoir for genes encoding ctx - m-1-group -lactamases in 
 aquatic farming , and ( ii ) this is the first time that strains harboring 
 blactx - m-33 gene  which encodes a variant of ctx - m-15 
 -lactamases  have been isolated in worldwide veterinary aquatic medicine and the korean 
 microbiology community . in general , most of 
 aeromonas strains were resistant to aminopenicillins ( e.g. , amoxicillin and ampicillin ) , 
 regardless of the isolation sources [ 9 , 17 ] . therefore , the results were agreed with the previous hypothesis , 
 suggesting the production of naturally occurring aminopenicillinase in 
 aeromonas spp . . in addition , 
 all of the present strains harbored the blatem gene encoding 
 tem - type -lactamase . when compared with the previous studies [ 1 , 8 , 17 ] , a particularly interesting finding of the present study was the 
 frequent occurrence of strains with resistance to ceftiofur and cefpodoxime . neither of 
 these antibiotics is approved for aquaculture use , but they are both approved for veterinary 
 use . although its occurrence is rare , a previous study found resistance to both antibiotics 
 in aeromonas spp . and pseudomonas spp . from rainbow trout 
 ( oncorhynchus mykiss ) . it is 
 possible that the resistance to both antibiotics is a consequence of their off - label use in 
 aquaculture and/or anthropogenic contamination from other farmed areas . cefpodoxime - resistant strains were found to be simultaneously resistant to ceftiofur . the 
 prevalence of resistance to both antibiotics differed among the aeromonas 
 spp . : ceftiofur resistance was more prevalent in a. caviae and a. 
 dhakensis than in other aeromonas spp . , and a. 
 dhakensis was the predominant species for cefpodoxime resistance . these results 
 could be related to differences in genetic determinants related to antibiotic resistance 
 among aeromonas spp . previous studies have also frequently detected genes 
 encoding ctx - m - type cephalosporinase in ceftiofur - resistant 
 enterobacteriaceae isolates from domestic animals [ 4 , 10 ] . on the other hand , most of 
 present strains were highly resistant to ampicillin , but they showed decreased resistance to 
 combination of aminopenicillin and -lactamase inhibitor . the behavior was observed in all 
 aeromonas spp . except a. caviae and was similar to 
 extended - spectrum -lactamase - producing strain [ 4 , 
 5 , 7 , 10 ] . in these aspects , the present study subjected all 
 strains to pcr assays in order to detect blactx - m genes , 
 followed by sequencing analysis of the amplicons . the ctx - m-1 group - encoding genes were 
 frequently detected in strains with resistance to all -lactams , with the pattern commonly 
 observed in a. dhakensis strains of eel . this result supports our 
 expectation of different genetic backgrounds to -lactams antibiotic resistance in 
 aeromonas spp . a previous study found no 
 blactx - m genes , but frequently detected 
 blamox genes in 47 clinical strains of a. 
 dhakensis with high prevalence of resistances to amoxicillin and 
 amoxicillin - clavulanic acid . therefore , it is 
 unclear whether the distribution of blactx - m-1-group genes is 
 species specific , as for previous reports on cpha - related genes in 
 aeromonas spp . . based on 
 differences in the isolation sources between the present and previous studies , a. dhakensis is regarded as having 
 different genetic backgrounds under selective pressure according to antibiotic use . in 
 addition , ampc producer might be a. caviae strains with high resistance to 
 ampicillin and ampicillin - clavulanic acid in the present study . to the best of our knowledge , no previous study has investigated the prevalence of 
 blactx - m genes in aquatic cultured fish species . in 
 addition , this study is the first to isolate the strains harboring the 
 blactx - m-33a gene encoding a variant ctx - m-15 
 -lactamases  from aquatic medicine after its first description in clinical e. 
 coli isolates in greece . this study 
 indicates that a. dhakensis could be the main carrier for spreading 
 blactx - m-1-group genes in aquatic farming , hence , its risk 
 to public health needs to be determined and to be monitored seriously .
the prevalence of resistant 
 genes against -lactams in 119 aeromonas strains was determined . a large 
 number ( 99.2% ) of the present fish strains were resistant to one or more - lactams 
 including ceftiofur , amoxicillin - clavulanic acid , ampicillin , piperacillin and 
 cefpodoxime . among antibiotic resistance phenotypes , 
 the simultaneous resistance to all 
 -lactams occurred in 25.2% ( n=30 ) of all strains , which consisted of 18 strains of 
 a. dhakensis , 8 strains of a. caviae , 2 strains of 
 a. hydrophila and only one strain of a. veronii . for 
 exploring genetic background of the antibiotic resistances , 
 multiple pcr assays were 
 subjected to detect -lactamase - encoding genes , blatem , 
 blaoxa - b and blactx - m . in the 
 results , 
 the blatem-1 gene was harbored in all strains , 
 whereas only 3 strains harbored blaoxa gene . in the case of 
 blactx - m gene , 
 the gene was detected in 21.0% ( 25 out of 
 119 ) of all strains , which countered with 80% ( 20 out of 25 ) of a. 
 dhakensis , 8% ( 2 out of 25 ) of a. caviae and 12% ( 3 out of 25 ) 
 of a. hydrophila . 
 in addition , most of the 
 blactx - m positive strains showed simultaneous resistance to 
 all -lactams ( 18 out of 30 strains ) . in sequence analysis for 
 blactx - m genes detected , they were ctx - m group 1-encoding 
 genes including blactx - m-33 from 3 eel strains of a. 
 dhakensis . therefore , a. dhakensis obtained from cultured fish 
 could represent a reservoir for spreading genes encoding ctx - m group 1 enzymes and hence 
 should be carefully monitored , especially for its potential risk to public health .