samples from 96 birds ( 41 wild ducks , 29 chickens , 23 ducks , and 3 geese ) were collected from the padmachar area of rajshahi district during january 2009 . in this area ,
a lake hosts several thousand wintering wild birds ; that lake also is frequented by poultry from surrounding households .
each fecal sample , collected by swirling a cotton swab in a bird s cloaca or droppings , was submerged in a bacterial freeze medium and handled as described ( 5 ) .
each sample was placed on an uriselect 4 agar plate ( bio - rad laboratories , marnes - la - coquette , france ) , and assessed for e. coli by biochemical testing and api 20e biochemical strips ( biomrieux sa , marcy - l'etoile , france ) .
one e. coli isolate per positive bird sample was tested by disk diffusion against 15 antimicrobial drugs ( table 1 ) according to the recommendations of the european committee on antimicrobial susceptibility testing ( www.eucast.org ) .
each sample was also enriched in brain - heart infusion broth ( becton dickinson , franklin lakes , nj , usa ) supplemented with vancomycin 16 g / ml ( icn biomedicals inc .
aurora , oh , usa ) for 18 h at 37c . for detection of esbl - producing bacteria and genes ( blactx - m , blashv , and blatem ) in putative esbl isolates ,
carbapenem - resistant isolates were screened on mueller - hinton agar plates supplemented with 2 g / ml or 8 g / ml meropenem and incubated overnight at 37c .
the genetic profiles of the esbl - producing e. coli isolates were determined by using repetitive element pcr .
the reaction mixture contained 1 taq pcr buffer , 0.625 mol / l primer eric1r ( 5-atgtaagctcctggggattcac-3 ) , 1.9 mmol / l mgcl2 , 50 mol / l dntps , 0.6 u taq polymerase , and template in a total volume of 20 l .
cycling parameters were 1 min at 94c ; 1 min at 36c and 2 min at 72c for 45 cycles , and a final extension for 5 min at at 72c .
isolates that had identical strong band patterns but an addition or a loss of a weak band were assigned subtype numbers .
one representative for each repetitive element pcr genotype and subtype ( n = 18 ) was characterized by multilocus sequence typing ( mlst ) ( 10 ) . after sequencing ,
allele profiles and sequence types were determined by using the e. coli mlst database ( http://mlst.ucc.ie/mlst/dbs/ecoli/ # ) .
one representative sample for each genotype was tested for transferability of the esbl plasmid by conjugation to recipient e. coli da11782 ( mcra , mrr - hsdrms - mcrbc , lacx74 , deor , reca1 , arad139 [ ara - leu ] 7697 , galk , rpsl , enda1 , nupg , rif ) . equal amounts of donor and recipient overnight cultures in luria - bertani broth were mixed and incubated , without shaking , overnight at 37c .
approximately 10 cfu of conjugation mixture was placed on selective plates containing 10 g / ml cefotaxime , 100 g / ml rifampin , and 50 g / ml nalidixic acid and incubated overnight at 37c .
e. coli was isolated from 66 samples , yielding an isolation rate of 73.3% regardless of bird species .
thirty - five ( 53% ) of the 66 isolates were resistant to 1 antimicrobial compounds .
the 3 next most common resistances were to ampicillin , trimethoprim / sulfamethoxazole , and nalidixic acid ( table 1 ) .
multidrug resistance was found in 22.7% ( 15/66 ) of the isolates , and 13.6% ( 9/66 ) of the isolates were resistant to 4 or 5 classes of antimicrobial drugs .
the overall prevalence of esbl carriage among birds was 30% ( 27/90 ) ; 36 e. coli isolates produced esbl .
thirty - four of them belonged to the ctx - m-1 group ( 2 blactx - m-1 and 32 blactx - m-15 ) and 2 to the ctx - m-9 group , the latter of which were ctx - m-14like .
combinations of blactx - m-15 or blactx - m-1 and blatem-1 were detected in 50% of the isolates , whereas none harbored shv - genes .
the genetic fingerprints of the esbl - producing e. coli isolates identified 15 genotypes , of which 19 ( 53% ) of 36 were type a ( table 2).this genotype was found in wild and domestic birds .
mlst analysis revealed 15 different sequence types ( sts ) and 1 nontypeable isolate ( table 2 ) .
four isolates had new allele types or a new combination of allele types and were given novel sts ( st2690st2693 ) .
conjugation was successful for 9/18 isolates , indicating the transferability of plasmids carrying esbl genes .
the carriage rate of esbls was high and the predominating antimicrobial - resistant phenotypes of wild birds and poultry appeared to correlate with antimicrobial prescription patterns in bangladesh ( 6 ) .
most esbl - positive samples originated from poultry , and household poultry was the predominant carrier of the blactx - m-15 genotype and the ctx - m-14like enzymes .
the ctx - m-15 gene shows a global distribution in clinical settings but has been reported from poultry in the united kingdom ( 11 ) and from wild birds in sweden ( 5 ) , which indicates that this esbl type also is widely disseminated in the environment .
the pcr - based genotyping showed the diversity of the esbl - producing e. coli isolates .
wild birds and domestic poultry harbored the same strains , and some of the ducks had the same strains as chickens .
this commonality of strains might be caused by a common use of natural water resources , and shows with what ease e. coli can travel between species .
mlst analysis identified several human - associated genotypes , including st448 , st405 , st744 , st648 , and st131 .
the epidemic e. coli strain o25bst131 did not carry the more common ctx - m-15 gene but a ctx - m-14like gene , a frequent finding in hospitals in taiwan ( 12 ) .
metallo--lactamases of the new delhi metallo--lactamase type have not been found in the environment of bangladesh ( 13 ) , but st405 and st648 are associated with new delhi metallo--lactamase-1producing organisms on the subcontinent of india ( 14 ) .
esbl - producing e. coli st744 has been reported previously in humans in laos ( 15 ) .
we showed that e. coli that produces ctx - m-15 is endemic to birds in bangladesh .
our findings suggest that wild birds and free - range poultry might be crucial environmental indicators of antimicrobial drug resistance .
they also might take a more active part than previously thought as spreaders and as long - term reservoirs of medically threatening pathogens and resistance genes .
several factors are likely to contribute to the widespread dispersal of esbls in bangladesh , including dense population , poor sanitation , and close contact with livestock combined with a high selective pressure created by unrestricted use of antimicrobial drugs in human medicine , veterinary medicine , and aquaculture .
development of a countrywide antimicrobial resistance surveillance system in livestock , wildlife species , and humans in bangladesh , as well as other measures , are needed immediately to control the situation .
 multidrug resistance was found in 22.7% of escherichia coli isolates from bird samples in bangladesh ; 30% produced extended - spectrum -lactamases , including clones of ctx - m genes among wild and domestic birds . 
 unrestricted use of antimicrobial drugs in feed for domestic birds and the spread of resistance genes to the large bird reservoir in bangladesh are growing problems . 
The Study
Conclusions