a total of 130 consecutive p. aeruginosa isolates recovered from different clinical specimens at sanjay gandhi postgraduate institute of medical sciences in lucknow , uttar pradesh , india , during november 2013april 2014 were included in the study ; all specimens were collected from within the state ( figure 1 ) .
aeruginosa isolates were identified by standard microbiological techniques ( 6 ) and further confirmed by phoenix automated identification and sensitivity systems ( bd biosciences , san jose , ca , usa ) .
the drug susceptibility profile was interpreted by using clinical and laboratory standards institute breakpoints ( 7 ) .
these isolates were subjected to pcr by using blandmspecific primers ( 8) followed by amplicon sequencing .
the isolates were further screened for high - level aminoglycoside resistance by their ability to grow on muller hinton agar containing amikacin and gentamicin 256 mg / l each as a marker for 16s rmtase ( 3 ) .
a total of 33 ( 86.84% ) isolates were positive for high - level aminoglycoside resistance .
each of these isolates were further subjected to pcr for detection of 16s rmtases ( arma and rmta  rmth ) by using primers and conditions described previously ( 25 ) ; 17 ( 51% ) isolates were positive for 16s rmtases .
their distributions were as follows : arma in 6 ( 18% ) ; rmtb in 4 ( 12% ) ; arma + rmtb in 4 ( 12% ) ; rmtc in 2 ( knpa1a and knpa1c ) ( 6% ) ; and rmtc + rmtf in 1 ( knpa1b ) ( 3% ) .
knpa1a , knpa1b , and knpa1c were further characterized ; sequence analysis of amplicons confirmed rmtc with 100% nucleotide identity originally described in proteus mirabilis strain ars68 from japan ( 9 ) and assigned embl / genbank nucleotide accession nos .
mics of the 3 isolates for different aminoglycosides , -lactams , -lactam/-lactamase inhibitor combinations , carbapenems , and colistin are provided in the table .
location of uttar pradesh state , india , showing geographic location of patients infected with 16s rrna methyl transferase  positive pseudomonas aeruginosa ( gray shading ) and rmtc - positive isolates knpa1a , knpa1b , and knpa1c ( black dots ) ; knpa1b was also positive for rmtf .
knpa1a was isolated from surgical drainage from a woman , 59 years of age , who had hypertension and underwent an abdominal hysterectomy for cervical carcinoma , followed by external beam radiotherapy .
her condition stabilized , and she was discharged with advice for repair of the fistula , but she did not return for further treatment . during hospitalization , she received multiple antimicrobial drugs .
knpa1b was isolated from endoscopic nasobiliary drainage ( enbd ) collected from a man , 57 years of age , who had extrahepatic biliary obstruction as a complication of hilar cholangiocarcinoma .
stent block and fever occurred , necessitating a repeat of the procedure and drainage of fluid .
he was discharged with the drainage tube in situ and was advised to return for surgery .
knpa1c was isolated from a man , 68 years of age , who had diabetes mellitus and hypertension .
he had stricture of the urethra and meatal narrowing after having a transurethral prostate resection .
a urinary tract infection was diagnosed , and p. aeruginosa ( knpa1c ) was recovered from urine .
the patient received piperacillin / tazobactam and colistin combination therapy ; urine culture was sterile on day 3 posttreatment .
resistance genes such as metallo--lactamases ( e.g. , imp , vim , sim , gim , spm ) and extended - spectrum -lactamases such as tem , shv , ctx - m , and ampc were detected by using pcr ( 8) ( table ) . to study genetic relatedness among the 3 isolates , genomic dna in agarose blocks
was separated on 1.0% agarose gels in 0.5  tris - borate - edta buffer with the chef ii d - mapper xa pulsed - field gel electrophoresis system ( bio - rad , hercules , ca , usa ) following standard conditions ( 10 ) .
multilocus sequence typing ( mlst ) was done according to protocols described in the pseudomonas 
 aeruginosa mlst database ( http://pubmlst.org/paeruginosa ) .
seven chromosomal genes were pcr amplified and sequenced ; the sequences were compared with those on the mlst database to determine allele numbers and sequence types ( sts ) .
knpa1a , knpa1b , and knpa1c belonged to st764 , st902 , and st880 , respectively .
* caz , ceftazidime ; ctx , cefotaxime ; fep , cefepime ; atm , aztreonam ; cps , cefoperazone / sulbactam ; tzp , piperacillin / tazobactam ; ipm , imipenem ; mer , meropenem ; col , colistin ; ak , amikacin ; g , gentamicin .
lane 1 ,  ladder ; 2 , knpa1a ; 3 , knpa1b ; 4 , knpa1c .
b ) chromosomal location of rmtc , rmtf , and blandm-1 genes by i - ceui - digested genomic dna of p. aeruginosa isolates .
lane 5 , knpa1a ; 6 , knpa1b ; 7 , knpa1c ; smears show southern blot analysis of genomic dna with probes specific to 16s rrna , rmtc , rmtf , and ndm-1 genes .
isecp1 was previously shown to promote both expression and transposition of rmtc ( 11 ) ; hence , to assess association of isecp1 with rmtc , pcr was performed on the 3 isolates with primer pairs isecpir - f and rmtc - down and isecp15 and rmtc - r , as described ( 12 ) .
sequence analysis of amplicons revealed association of an intact isecp1 element with rmtc in knpa1a ; however , complete isecp1 could not be amplified in knpa1b and knpa1c , corroborating earlier observations of either partial deletion of this element or role of a different isecp1-like element in the spread of rmtc in gram - negative bacteria ( 13 ) .
attempts to transfer rmtc from all 3 isolates to rifampin - resistant escherichia coli 20r764 and ciprofloxacin - resistant p.
repeated attempts to obtain amikacin - resistant ( mic 16 g / ml ) transformants of e. coli dh5 and p. aeruginosa pa01 by electroporation with plasmid preparation by using the kado and liu method ( 14 ) were also unsuccessful , despite successful transfer of control plasmids . to determine the location of rmtc , rmtf , and blandm-1 , genomic dna from the 3 isolates
was digested separately with restriction enzyme i - ceu-1 ( new england biolabs , beverly , ma , usa ) , separated by pfge , and subsequently assayed with 16s rrna , rmtc , rmtf , and blandm-1 probes ( 13 ) .
all these probes were hybridized with chromosomal dna ( figure 2 ) and not with plasmid extract .
this result shows that rmtc , rmtf , and blandm-1 were located and stabilized on the chromosome of p. aeruginosa .
we describe an occurrence of 16s rmtases rmtc and rmtf in clinical isolates of p. aeruginosa co - producing blandm-1 . the rmtc and rmtf genes might have been acquired from plasmids as part of mobile genetic elements and finally integrated and stabilized on the chromosome , but the underlying mechanism of transmission needs to be elucidated .
further , spread of multidrug - resistant p. aeruginosa strains expressing rmtc with and without an intact isecp1 element and ndm-1 is of major clinical concern and calls for further studies to limit the spread of such strains .
 we investigated 16s rrna methyltransferases in 38 blandm-1positive pseudomonas aeruginosa isolates and found rmtc in 3 isolates , 1 of which also harbored rmtf . 
 the isolates were clonally unrelated ; rmtc and rmtf genes were located on a chromosome with the blandm-1 gene . 
 strategies are needed to limit the spread of such isolates . 