a 2-year - old child was admitted to the department of pediatrics at the jawaharlal institute of postgraduate medical education and research ( jipmer ) , puducherry , india , with a provisional diagnosis of cholera . before the child received antimicrobial therapy , a fecal specimen
meanwhile , a strain of v. cholerae o1 el tor ogawa was isolated from the fecal specimen .
this strain was biochemically identified ( 3 ) and confirmed by agglutination with specific antiserum ( bd difco , becton dickinson , sparks , md , usa ) .
antimicrobial drug susceptibility testing was conducted by kirby  bauer method in accordance with the clinical and laboratory standards institute ( 4 ) against ampicillin ( 10 g ) , ceftriaxone ( 30 g ) , ciprofloxacin ( 5 g ) , furoxone ( 300 g ) , cotrimoxazole ( 25 g ) , and tetracycline ( 30 g ) . the mic for ceftriaxone was determined by the agar dilution method and etest for the el tor ogawa strain . for the agar dilution method ,
we doubled dilutions of ceftriaxone sodium ( himedia , mumbai , india ) from 0.5 g / ml to 128 g / ml , which included the recommended break points ( 5 ) .
american type culture collection escherichia coli 25922 was spread on each plate as growth control .
the etest was performed according to the manufacturers instructions ( ab biomrieux , solna , sweden ) .
we performed the combination disk test using cefotaxime and ceftazidime , alone and in combination with clavulanic acid , to detect extended - spectrum -lactamase ( esbl ) in accordance with clinical and laboratory standards institute guidelines ( 4 ) .
the modified hodge test and imipenem - edta disk synergy test for the phenotypic detection of carbapenemase were performed as described ( 4,7 ) .
pcr was performed to detect esbl genes , i.e. , blactx ( 8) , blashv ( 8) , and blatem ( 9 ) ; a multiplex pcr was used to detect the ampc group of genes , i.e. , blamox , blacit , bladha , and blaacc ( 10 ) . a multiplex pcr to detect carbapenemase genes , i.e. , blakpc , blaimp , blavim , and blandm , was performed ( 11 ) .
the strain was resistant to ampicillin , ceftriaxone , cotrimoxazole , and furoxone and sensitive only to ciprofloxacin and tetracycline .
because the strain was negative for the phenotypic and the genotypic tests for esbl production , in our attempt to determine the reason for the increased mic , we further tested for the ampc type of -lactamase production .
the strain , an ampc -lactamase producer , was positive for the bladha gene ( 405 bp ) by multiplex pcr . on sequencing ,
the bladha gene detected had 99% identity with klebsiella pneumoniae -lactamase bladha-1 gene ( genbank accession no .
ay635140.1 ) . because carbapenems are considered the treatment of choice for ampc - producing organisms , we tested the strain for the production of carbapenemase and found it to be a carbapenemase producer by a positive modified hodge test and by the edta disk synergy test .
the multiplex pcr for detection of carbapenemase genes yielded an amplicon of 660 bp , which was confirmed by sequencing to be an blandm-1 gene and had 100% identity with e. coli strain hk-01 plasmid pndm - hk ( genbank accession no .
to demonstrate the presence of a plasmid bearing the blandm-1 gene , we isolated a plasmid using the alkaline lysis method ( 12 ) from the v. cholerae strain .
this plasmid was subjected to the multiplex pcr to detect the carbapenemase genes mentioned earlier ( 11 ) , which yielded an amplicon of 660 bp matching that of the blandm-1 gene amplicon .
( 13 ) and used e. coli j53 as the recipient , which was isolated on meropenem ( 4 mg / l ) containing luria - bertani agar and macconkey agar , meropenem ( 4
we also subjected the v. cholerae plasmid to the multiplex pcr to detect the ampc gene , as mentioned earlier , but none of the genes were amplified .
we failed to transfer the ampc gene ( bladha-1 ) by using the plasmid transfer experiment .
( 15 ) . of the many ampc genes known are 2 types of bladha genes : bladha-1 and bladha-2
; both are inducible plasmid - mediated genes . because the strain reported here was already resistant to a third - generation cephalosporin , i.e. , ceftriaxone
, we did not perform the inducible ampc test and proceeded to perform the ampc disk test .
plasmid - mediated inducible -lactamases are extremely rare and are most often constitutive ( 15 ) .
the ampc disk test does not differentiate between chromosomal and plasmid - mediated ampc -lactamases .
carbapenems are the treatment of choice for ampc - producing strains ( 6 ) and are hydrolyzed by carbapenemases , which have many families , including the metallo--lactamases ( 7 ) .
the most recently described blandm-1 ( new delhi metallo--lactamase ) is plasmid - mediated metallo--lactamase and has been isolated from the environment and from hospitals ( 14 ) .
we isolated a clinical strain of v. cholerae producing an ampc -lactamase and a carbapenemase . our findings ,
the critical role of all bacteriology laboratories needs to be emphasized for determining not only resistance patterns but also the mechanisms of resistance .
health care  associated networks need to be strengthened to ensure justified and appropriate use of antimicrobial agents that will result in safe drinking water and improved sanitation ; these can have remarkable effect in reducing the spread of many communicable diseases , such as cholera , and can go a long way in controlling the growing menace of antimicrobial drug resistance . in light of the above findings , the antimicrobial drug profile of organisms , such as v. cholerae , needs to be under constant surveillance in the community .
 vibrio cholerae resistance to third - generation cephalosporins is rarely reported . 
 we detected a strain that was negative for extended - spectrum -lactamase and positive for the ampc disk test , modified hodge test , and edta disk synergy test and harbored the bladha-1 and blandm-1 genes . 
 the antimicrobial drug susceptibility profile of v. cholerae should be monitored . 
The Study
Conclusions