
 k. pneumoniae belonging to the family of enterobacteriaceae is an important opportunist human pathogen associated with hospital - acquired infections such as pneumonia , urinary tract infections , or bacteraemias [ 13 ] .
this species naturally produces a broad - spectrum -lactamase of the shv-1 type or penicillinase len that confers resistance to penicillins and to first - generation cephalosporins .
the widespread use of expanded - spectrum antibiotics for treatment of serious infections due to gram negative bacteria has led to the appearance of extended - spectrum -lactamases ( esbls ) which are able to hydrolyze these compounds [ 48 ] . since their first identification in germany and
the united states of america , extended - spectrum -lactamases ( esbls ) have spread worldwide [ 6 , 7 ] .
these enzymes are mostly plasmid - encoded variants of tem-1 , tem-2 , and shv-1 by one or more amino acids or are from a rapidly evolving class called ctx - m .
they confer resistance to broad - spectrum penicillins , monobactams , and narrow - spectrum and third - generation cephalosporins , respectively [ 4 , 8 ] .
the prevalence of esbl producing k. pneumoniae isolates in hospitals ranges from 5 to 25% in several parts of the world . in september 2012 ,
more than 100 shv variants have been identified ( http://www.lahey.org/ ; j. a. jacoby and k. bush ) . in tunisia , shv-2a and shv-12 were the predominant shv - type esbl produced by k. pneumoniae . in this work ,
we searched for the presence of potential new shv 's variants produced by k. pneumoniae isolates in tunisia clinical settings . from this survey
, we isolated a k. pneumoniae strain ( ml2011 ) resistant to third - generation cephalosporin and aztreonam .
our goal was to study the contribution of shv-104 in the multiresistant phenotype of the strain .

 k. pneumoniae ml2011 was collected in july 2004 , from intensive care unit of the military hospital of tunis ( tunisia ) .
identification of strains was performed by using both the vitek automated system ( biomrieux , marcy l'etoile , france ) and api 20 e system ( biomrieux , marcy l'etoile , france ) .
biochemical and serological confirmation of the identity of this strain was done at the laboratory of bacteriology , military hospital , tunisia .
e. coli dh5 and e. coli hb101 were used , respectively , for the transformation and conjugation experiments .
e. coli bl21 and bl21 ( de3 ) ( plyss ) were used for the resistance profile of the different strains and for the overexpression of shv-104 , respectively .
disc susceptibility testing was performed according to clinical and laboratory standards institute ( clsi ) guidelines   on mueller - hinton agar ( bio rad , france ) .
detection of esbl activity was achieved with a double - disk synergy test , by screening isolate for synergism between amoxicillin - clavulanate and cefotaxime , ceftriaxone , ceftazidime , and aztreonam , respectively
. minimal inhibitory concentrations ( mics ) of ampicillin , ticarcillin , ceftriaxone , cefotaxime , ceftazidime , cefoxitin , imipenem , streptomycin , and chloramphenicol were determined by etest strips , according to the manufacturer 's instructions ( ab biodisk , solna , sweden ) , and interpreted according to clinical breakpoints from the clsi and eucast .
pi of the -lactamases produced by k. pneumoniae ml2011 , the hb101 transconjugants , and the bl21 transformants were determined as described previously .
plasmid dna of the clinical isolate was extracted with a plasmid extraction kit gfx micro plasmid prep ( amersham biosciences , uk ) , according to the manufacturer 's instructions .
plasmid dna electrophoresis was performed in 1% agarose gel and visualized with ethidium bromide under uv light .
the plasmid dna of e. coli rp4 ( 50  kb ) was used as a molecular size marker [ 13 , 14 ] .
transformants were selected on luria bertani ( lb ) medium agar plates supplemented with ampicillin ( 100  g / ml ) .
transfer of resistance phenotypes was performed by a liquid mating method   on lb broth medium .
culture mixtures were incubated overnight at 37c with transformants e. coli dh5/pml2011 as donors and e. coli hb101 as recipient strain .
after conjugation , bacterial suspensions were plated onto agar containing ampicillin ( 100  g / ml ) and streptomycin ( 100  g / ml ) .
amplifications of the blatem , blashv , and blactx - m genes by pcr were carried out using the plasmid pml2011 and the chromosomal dna of k. pneumoniae ml2011 as the template .
the pcr products ( corresponding to shv and ctx - m genes ) were visualized by agarose gel electrophoresis , purified with a gfx pcr dna and gel band purification kit ( amersham biosciences , uk ) . prior to their cloning into ptz57r
/ t vector ( fermentas , usa ) , the sequence of the pcr fragment was verified by giga genomics facility ( ulg , belgium ) . the ligation mix was transformed into e. coli dh5 competent cells .
positive colonies were selected on lb broth medium supplemented with ampicillin ( 100  g / ml ) .
the insert sequences were performed by giga genomics facility in belgium , using forward and reverse m13 universal primers : puc / m13f ( 5-cgc cag ggt ttt ccc agt cac gac-3 ) and puc / m13 r ( 5-tca cac agg aaa cag cta tga c-3 ) . 

ndei and bamhi restriction sites were inserted at the 5 and 3 extremities of blashv , respectively , by pcr .
the nucleotides sequences of the pcr - generated fragments were firstly verified by sequencing and secondly cloned into the pet26b ( + ) vector ( novagen inc . ,
/ shv-104 , was used to transform e. coli bl21 ( de3 ) ( plyss ) competent cells ( novagen inc . ,
the shv-104 enzyme was produced in terrific broth ( tb ) medium containing kanamycin ( 50  g / ml ) and chloramphenicol ( 30  g / ml ) as selecting agents during growth of the bacteria at 37c under orbital shaking . 40  ml of an overnight preculture in tb was inoculated to 1  l of fresh tb supplemented with kanamycin and chloramphenicol . at an absorbance value of 0.7 at 600  nm ,
iptg ( final concentration 0.5  mm ) was added and the culture was incubated for 5 additional hours .
cells were harvested by centrifugation ( 5,000  g for 10  min at 4c ) ; the pellet was resuspended in 50  ml of 20  mm tris / hcl buffer ph 7.5 ( buffer a ) .
bacteria were disrupted with the help of cell disrupter equipment ( emulsiflex c3 , germany ) .
cell debris were removed by centrifugation ( 30  000  g for 45  min at 4c ) and the supernatant was dialysed overnight against buffer a at 4c .
the crude extract was loaded onto a q - sepharose ff column ( 2.6 by 34  cm , pharmacia , sweden ) equilibrated in buffer a. the -lactamase shv-104 was eluted in the flow through .
the solution was dialysed overnight against 20  mm tris / hcl buffer ph 8.5 and loaded in a hitrap q - sepharose hp ( 5  ml ; ge healtcare ) .
the enzyme was eluted by a linear nacl gradient ( 00.7  m ) in ten column volumes .
the active fractions were collected and concentrated on ym-10 membrane ( amicon , beverly , mass . ) to a final volume of 3  ml . the sample was loaded in a molecular sieve sephacryl-100 ( 1.5  56  cm ) column equilibrated in 25  mm sodium phosphate buffer ph 7.0 containing 0.2  m nacl .
the fractions exhibiting -lactamase activity were collected and their specific activity was followed by measuring the rate of nitrocefin hydrolysis . the fractions exhibiting a constant specific activity were pooled and concentrated to a final concentration of 0.5  mg / ml . the enzyme preparation was stored at 20c in 25  mm sodium phosphate buffer ph 7.0 .
the n - terminal sequence was determined with the help of a gas - phase sequencer ( prosite 492 ; applied biosystems , calif ) .
all the measurements were done on a spectrophotometer specord 50 analytik jena ( analis , belgium ) connected to a personal computer via an rs232c interface .
bovine serum albumin ( 20  g / ml ) was added to diluted solutions of -lactamase in order to prevent enzyme denaturation . the steady state kinetic parameters ( km and kcat ) were determined using the hanes ' linearization of the michaelis - menten equation .
the kcat and km values were determined for a representative set of -lactam antibiotics . a model of the shv-104 structure
mics for k. pneumoniae ml2011 showed that this strain was resistant to all -lactams tested except imipenem ( table 2 ) .
k. pneumoniae ml20011 exhibited a high level of resistance to oxyimino - cephalosporins ( cefotaxime , ceftriaxone , and cefpirome : mic 512  mg / l ; ceftazidime : mic 256  mg / l ) .
the strain was also resistant to chloramphenicol , ciprofloxacin , nalidixic acid , and tetracycline .
the disk diffusion method performed according to the clsi guidelines   showed synergistic effect between ceftazidime , cefotaxime , aztreonam , ceftriaxone , and amoxicillin - clavulanic acid suggesting the presence of an esbl enzyme .
plasmid analysis revealed the presence of a 50  kb transferable plasmid by transformation and conjugation experiments .
the frequency of conjugational transfer performed with k. pneumoniae ml2011 as donors and e. coli hb101 as the recipient was 10/donor .
the e. coli transformants and transconjugants were resistant to penicillins and expanded - spectrum cephalosporins but were susceptible to cefoxitin , aminoglycosides , quinolones , chloramphenicol , and imipenem ( table 2 ) .
the examination of the crude extract of k. pneumoniae ml2011 by isoelectric focusing showed the presence of four -lactamase bands with apparent pis of 5.5 , 7.3 , 7.6 , and 8.6 . only the -lactamases with pi 8.6 and
pcr analyses confirmed the presence of blatem , blactx - m-1 group , and blashv in k. pneumoniae ml2011 .
blashv can be present on both the chromosomal dna of k. pneumoniae ml2011 and the plasmid pml2011 .
the complete nucleotide sequences were performed and compared for shv gene with strain k. pneumoniae kp297 ( ddbj / embl / genebank accession number ef035567 ) and ctx - m gene with ctx - m-1 genebank accession number x92506 .
these comparisons revealed the presence of ctx - m-28 previously described in   and 2 shv : blashv-1 on the chromosomal dna and an open reading frame that was similar to blashv-1 with one mutation on the plasmid pml2011 .
the mutation consisted of the replacement of the ( r ) residue at position 202 ( codon cgt ) to s ( codon agt ) .
as this substitution was not present in all known shv -lactamases , the sequence of blashv was named blashv-104 and was deposited in genbank ( accession number eu274581 ) .
the analysis of the shv sequences coding mutation at amino acid 202 suggests that shv-104 has evolved directly from shv-1 .
moreover , the modification r202s may result in a change of the pi to 7.3 .
the best results , in terms of production of shv-104 , were found in growing e. coli bl21(de3 ) ( plyss ) in tb , added 0.5  mm iptg after 5-hour incubation .
the shv-104 was extracted from the periplasmic space and purified by three chromatographic steps ( q - sepharose ff , hitrap q - sepharose hp , and molecular sieve sephacryl-100 ) to obtain at the end ~10  mg ( 90% protein pure ) for liter .
kinetic parameters values , kcat and km , of shv-104 -lactamase for representative -lactam antibiotics were shown in table 3 .
kcat values of shv-104 against ticarcillin , nitrocefin , cephalothin , and cefuroxime were higher than those of shv-1 , and km values were higher ( 43 to 540  m versus 21 to 100  m ) except for ticarcillin .
overall , the catalytic efficiency against penicillins was higher for shv-104 ( kcat / km , 0.001 to 19  m 
s ) than for shv-1 ( kcat / km , 0.3 to 14  m  s ) , except for cefuroxime ( kcat / km , 0.001  m  s versus 0.005  m  s ) . in contrast
, shv-104 had a low catalytic efficiency against benzylpenicillin ( 30-fold ) than shv-1 and exhibited catalytic activity against cefotaxime ( an oxyimino - cephalosporin ) .
kinetic analysis showed that shv-104 was more active against nitrocefin and cephalothin , but hydrolysis remained inefficient , owing to a high km .
, only cefotaxime was slowly hydrolysed by shv-104 . on the basis of the fact that a group 2be enzyme should hydrolyze one or more oxyimino--lactams such as cefotaxime , ceftazidime , or aztreonam at 10%
the rate at which benzylpenicillin is hydrolyzed [ 17 , 18 ] , the activity of the novel enzyme was insufficient for the enzyme to count as an extended - spectrum -lactamase ( esbl ) .
finally , a model of the shv-104 structure was built with the help of easypred program using shv-1 as template .
the r202s mutation may affect a salt bridge present in shv-1 between r202 and e92 via a water molecule ( figure 1 ) .
analysis of shv-104 structure model showed that it is reasonable to think that this mutation can not have a direct effect on the broadening of the activity profile of the shv enzyme .
a plausible hypothesis relies on the fact that the absence of a salt bridge in shv-104 compared to shv-1 could increase the overall flexibility of the protein and indirectly the flexibility of the -lactamase active site .
the conversion of a non - esbl to an esbl for the shv family can be achieved by different mutations .
unlike the earliest described esbls ( shv-4 , -5 , -7 , -9 , -10 , -12 , -15 , -22 , -45 , -46 , -55 , -64 , and -66 ) , shv-104 does not possess these mutations , g238s and e240k , thought initially to be necessary for the hydrolysis of cefotaxime .
mutation at position 179 was responsible for the esbl phenotype in shv-6 , -8 , and -24 .
random mutagenesis of 179 in tem-1 produced three e. coli transformants ( 179n , g , and y ) with increased levels of resistance to ceftazidime .
shv-7 variant was found with a serine at position 43 and is resistant to cefotaxime , ceftazidime , and aztreonam .
the hydrogen bonding from r43 to the segment at positions 64 to 69 running behind the -sheet may be important in maintaining the position of s70 . in conclusion
, we could demonstrate that the production of shv-104 is not the major factor for the resistance pattern of k. pneumonia ml2011 .
our study indicates that shv-104 can be an intermediate in the process of selection of a new shv boras spectrum -lactamase .
we could also conclude that the coexpression of ctxm-28 by this strain will increase the resistance profile of the strain toward beta - lactam antibiotics .
 
 klebsiella pneumoniae ml2011 , a multiresistant isolate , was isolated from the military hospital of tunis ( tunisia ) . 
 the determination of the minimal inhibitory concentrations exhibited by k. pneumoniae ml2011 was performed by etest . 
 the crude extract of the isolates contains four different -lactamases with pi 5.5 , 7.3 , 7.6 , and 8.6 . only the -lactamases with pi 7.3 and 
 pi 8.6 were transferred by transformation and conjugation experiment . 
 molecular characterization of these genes was performed by pcr and sequencing . 
 the chromosomal -lactamases are tem ( pi 5.5 ) and shv-1 ( 7.6 ) . 
 ctx - m-28 ( pi 8.6 ) and the novel variant of shv named shv-104 ( pi 7.3 ) were encoded by bla gene located on a 50  kb highly conjugative plasmid . 
 the shv-104 -lactamase was produced in e. coli and purified . 
 its profile of activity was determined . 
 compared to shv-1 , shv-104 contains one mutation , r202s . 
 their kinetic parameters were similar except for cefotaxime . 
 the analysis of the predicted structure of shv-104 indicated that the r202s mutation suppresses a salt bridge present in shv-1 . 
 therefore , the overall flexibility of the protein increased and might improve the hydrolysis of cefotaxime . 
 we can conclude that the multiresistant phenotype of k. pneumoniae ml2011 strain is mainly linked to the production of ctx - m-28 since shv-104 possesses a narrow spectrum of activity . 
1. Introduction
2. Materials and Methods
3. Results and Discussion