bacterial resistance to -lactams and -lactamase inhibitors is an ever - increasing problem that compromises their clinical utility . among gram - negative bacteria ,
of particular concern are enzymes able to target the expanded - spectrum -lactams , including the ampc enzymes ( class c cephalosporinases ) , the so - called extended - spectrum--lactamases ( esbl ; classes a and d ) , and the carbapenemases , which hydrolyze most -lactams , including the carbapenems ( classes a , b , and d).1 in order to restore their antibacterial activity against gram - negative pathogens , -lactams have been paired with inhibitors of -lactamases . those currently used in the clinical setting ( clavulanate , tazobactam , and sulbactam ) have a spectrum of inhibition essentially covering class a enzymes .
all three marketed inhibitors contain a -lactam core and share a similar mechanism of inhibition .
they react with serine enzymes to form a covalent acyl - enzyme intermediate ; opening of the four - member -lactam ring is followed by considerable molecular rearrangement before hydrolysis to regenerate the active enzyme.2 avibactam ( nxl104 ) is a non--lactam -lactamase inhibitor that displays a broad - spectrum inhibition profile , with potent inhibition of class a , class c , and some class d enzymes .
the inhibitor is characterized by high carbamylation efficiency and slow decarbamylation , resulting in a long half - life of the inactive covalent adduct.3 in addition , the decarbamylation step results in regeneration of intact avibactam , and not hydrolysis.4 avibactam has little intrinsic antibacterial activity , but efficiently protects -lactams from hydrolysis in a variety of class a , class c , and some class d - producing strains , including esbl , klebsiella pneumoniae carbapenemase ( kpc ) , and oxa-48 producers.57 many strains of the enterobacteriaceae family , as well as some nonfermenters , such as pseudomonas aeruginosa and acinetobacter baumannii , encode a chromosomal ampc -lactamase , although the regulation of enzyme production is different among the various enzyme - producing strains . in escherichia
coli , enzyme expression is usually constitutive and low - level , whereas in other species , such as enterobacter spp .
, it can be transiently induced to higher levels by several -lactam compounds , with carbapenems and cephamycins generally having the highest induction potential.8 derepression can also occur by mutations favoring constitutive production of very high levels of -lactamase .
the mechanism of induction is complex , via a system involving ampd , ampr , ampg , and intermediates in peptidoglycan recycling.9 induction of ampc does not necessarily correlate with a risk of clinical failure , particularly when the rate of bactericidality is high .
however , the potential for ampc induction has to be carefully examined when considering administration of a -lactamase inhibitor , because it can antagonize the antibacterial activity of its partner -lactam.1012 indeed , the antibacterial activity of a given -lactam with limited stability to ampc is preserved , provided that its potential for ampc induction is low .
in contrast , its activity would be compromised if combined with a -lactamase inhibitor that induces significant ampc production .
the aim of this study was to investigate the ability of avibactam to induce ampc expression in e. cloacae strains in vitro . because -lactamase / avibactam complexes are known to have a long half - life , it was not possible to evaluate ampc induction by measuring directly the -lactamase activity produced by avibactam - treated cells .
thus , it was measured by quantitation of cellular ampc messenger ribonucleic acid ( mrna ) .
e. cloacae isolates used in this study were obtained from the novexel culture collection , originally collected from a variety of clinical or laboratory sources .
minimal inhibitory concentration ( mic ) determinations were performed according to the clinical and laboratory standards institute broth - microdilution methods using cation - adjusted mueller 
hinton broth.13 mic values were measured for cefoxitin ( sigma - aldrich , st louis , mo , usa ) and ceftazidime ( novartis , basel , switzerland ) ; the latter was tested alone or in association with clavulanate ( us pharmacopeial convention , rockville , md , usa ) or avibactam , at a constant inhibitor concentration of 4 mg / l .
bertani broth ( interchim , montlugon , france ) , then diluted to an optical density ( od600 nm ) value of 0.1 and incubated with shaking for 24 hours to reach midlog - growth phase . at this point
, the test inducer ( cefoxitin , clavulanate , or avibactam ) was added at the appropriate concentration ( 8 , 16 , 32 , or 64 g / ml ) , whereas control cultures were grown in the absence of inducer .
approximately 5  10 cells were sampled for rna extraction just before addition of inducer , and at timed intervals thereafter up to 6 hours .
each induction experiment was performed at least three times for all three e. cloacae strains .
total cellular rna was extracted with an rneasy rna protect mini kit ( qiagen , venlo , the netherlands ) , and residual deoxyribonucleic acid ( dna ) was eliminated by treatment with an rnase - free dnase set ( qiagen ) .
analysis of rna integrity and total rna quantification was performed using the agilent 2100 rna bioanalyzer and the nano 6000 kit ( agilent technologies , santa clara , ca , usa ) . polymerase chain reaction ( pcr ) primers were designed with primer express software for ampc ( forward 5-tggcgtatcgggtcaatgt-3 ; probe 5-tcagggtctgggctgggagatgc-3 ; reverse 5-cctccacgggccagttg-3 ) and for rpls ( forward 5-caggtgacaccgtggaagtg- ; probe 5-aagtatgggttgttgaaggttccaa-3 ; reverse 5-cgaatgcctgcagacgttt-3 ) .
the probe primers were modified by addition of 6-fam ( 6-carboxy - fluorescein ) at the 5 end and tamra ( 6-carboxy - tetramethyl - rhodamine ) at the 3 end .
real - time pcr ( rt - pcr ) reactions were carried out in the abi prism 7000 sequence - detection system ( life technologies , carlsbad , ca , usa ) using a quantitect probe rt - pcr kit ( qiagen ) .
individual reactions were set up in triplicate for either ampc or rpsl genes , according to the manufacturer s recommendations .
briefly , complementary dna was syn - thesized from 0.5 ng of rna using moloney murine leuke - mia virus reverse transcriptase and 0.7 m of each primer ; reverse transcription was carried out at 50c for 30 minutes .
pcr conditions were as follows : initial activation of dna polymerase at 95c for 15 minutes , and pcr for 40 cycles at 95c for 15 seconds , 60c for 60 seconds .
absence of genomic dna contamination was verified for each rna preparation by running the assay in the absence of reverse transcriptase .
data were analyzed using sequence detection 2.0 software ( life technologies ) . to correct for differences in the amount of starting material ,
the ribosomal e. cloacae rpls gene ( encoding ribosomal protein 19 ) was chosen as a housekeeping reference gene .
values obtained were then normalized to that of ampc from e. cloacae strain p99 for measurement of basal expression , or to that of ampc in the test strain before induction .
relative quantitation was carried out by using the 2 t method , as recommended by the manufacturer.14 normalized ampc expression in culture 2 relative to that in culture 1 was calculated as follows : crude bacterial extracts were prepared by vortexing bacterial cells with glass beads in 100 mm phosphate buffer ph 7 containing 0.1 mg / ml of bovine serum albumin and 2% v / v glycerol ( about 10 bacterial cells / ml ) .
-lactamase activity was measured using a spectrophotometer at 485 nm for 15 minutes using 180 l of crude cell lysate at appropriate dilution , and 20 l of 1 mm nitrocefin ( oxoid sr112c ) .
results were expressed as initial reaction rates ( a485 nm / minute ) per 10 cells or per milligram of protein .
e. cloacae isolates used in this study were obtained from the novexel culture collection , originally collected from a variety of clinical or laboratory sources .
minimal inhibitory concentration ( mic ) determinations were performed according to the clinical and laboratory standards institute broth - microdilution methods using cation - adjusted mueller 
hinton broth.13 mic values were measured for cefoxitin ( sigma - aldrich , st louis , mo , usa ) and ceftazidime ( novartis , basel , switzerland ) ; the latter was tested alone or in association with clavulanate ( us pharmacopeial convention , rockville , md , usa ) or avibactam , at a constant inhibitor concentration of 4 mg / l .
bacterial strains were grown overnight at 37c in luria  bertani broth ( interchim , montlugon , france ) , then diluted to an optical density ( od600 nm ) value of 0.1 and incubated with shaking for 24 hours to reach midlog - growth phase . at this point , the test inducer ( cefoxitin , clavulanate , or avibactam ) was added at the appropriate concentration ( 8 , 16 , 32 , or 64 g / ml ) , whereas control cultures were grown in the absence of inducer .
approximately 5  10 cells were sampled for rna extraction just before addition of inducer , and at timed intervals thereafter up to 6 hours .
each induction experiment was performed at least three times for all three e. cloacae strains .
total cellular rna was extracted with an rneasy rna protect mini kit ( qiagen , venlo , the netherlands ) , and residual deoxyribonucleic acid ( dna ) was eliminated by treatment with an rnase - free dnase set ( qiagen ) .
analysis of rna integrity and total rna quantification was performed using the agilent 2100 rna bioanalyzer and the nano 6000 kit ( agilent technologies , santa clara , ca , usa ) .
polymerase chain reaction ( pcr ) primers were designed with primer express software for ampc ( forward 5-tggcgtatcgggtcaatgt-3 ; probe 5-tcagggtctgggctgggagatgc-3 ; reverse 5-cctccacgggccagttg-3 ) and for rpls ( forward 5-caggtgacaccgtggaagtg- ; probe 5-aagtatgggttgttgaaggttccaa-3 ; reverse 5-cgaatgcctgcagacgttt-3 ) .
the probe primers were modified by addition of 6-fam ( 6-carboxy - fluorescein ) at the 5 end and tamra ( 6-carboxy - tetramethyl - rhodamine ) at the 3 end .
real - time pcr ( rt - pcr ) reactions were carried out in the abi prism 7000 sequence - detection system ( life technologies , carlsbad , ca , usa ) using a quantitect probe rt - pcr kit ( qiagen ) .
individual reactions were set up in triplicate for either ampc or rpsl genes , according to the manufacturer s recommendations .
briefly , complementary dna was syn - thesized from 0.5 ng of rna using moloney murine leuke - mia virus reverse transcriptase and 0.7 m of each primer ; reverse transcription was carried out at 50c for 30 minutes .
pcr conditions were as follows : initial activation of dna polymerase at 95c for 15 minutes , and pcr for 40 cycles at 95c for 15 seconds , 60c for 60 seconds .
absence of genomic dna contamination was verified for each rna preparation by running the assay in the absence of reverse transcriptase .
data were analyzed using sequence detection 2.0 software ( life technologies ) . to correct for differences in the amount of starting material ,
the ribosomal e. cloacae rpls gene ( encoding ribosomal protein 19 ) was chosen as a housekeeping reference gene .
values obtained were then normalized to that of ampc from e. cloacae strain p99 for measurement of basal expression , or to that of ampc in the test strain before induction .
relative quantitation was carried out by using the 2 t method , as recommended by the manufacturer.14 normalized ampc expression in culture 2 relative to that in culture 1 was calculated as follows :
crude bacterial extracts were prepared by vortexing bacterial cells with glass beads in 100 mm phosphate buffer ph 7 containing 0.1 mg / ml of bovine serum albumin and 2% v / v glycerol ( about 10 bacterial cells / ml ) .
-lactamase activity was measured using a spectrophotometer at 485 nm for 15 minutes using 180 l of crude cell lysate at appropriate dilution , and 20 l of 1 mm nitrocefin ( oxoid sr112c ) .
results were expressed as initial reaction rates ( a485 nm / minute ) per 10 cells or per milligram of protein .
both cefoxitin and ceftazidime are good ampc substrates ; cefoxitin is also a good ampc inducer , whereas ceftazidime has limited potential for induction .
the differential activity of cefoxitin and ceftazidime can therefore be used to infer the presence of an inducible ampc gene , as strains without significant levels of ampc enzyme remain susceptible to cefoxitin.9,15 in order to select potentially inducible strains for this study , three e. cloacae strains ( 293la2 , 293ht107 , and 293uc1 ) were chosen on the basis of resistance to cefoxitin and susceptibility to ceftazidime .
the e. cloacae p99 strain was included in this study as a reference , having a cefoxitin - resistant ( cefoxitin - r ) and ceftazidime - r phenotype ( stably derepressed high - level ampc producer ) .
the mic values obtained for these four strains are shown in table 1 . as expected , owing to its spectrum of coverage limited to class a enzymes , clavulanate had no effect on the high ceftazidime mic value for the p99 strain ; in contrast , avibactam reduced ceftazidime mic to a susceptible level in this strain .
induction of -lactamases is most frequently assessed by assaying -lactamase activity using spectrophotometric assays of nitrocefin hydrolysis , in the presence or absence of an inducer .
however , this is not technically possible when testing the induction potential of a compound that can form highly stable complexes with ampc enzymes ( half - life around 7 days for avibactam / p99 ampc complex).5 therefore , induction was measured by quantitation of ampc transcripts using rt - pcr .
however , -lactamase enzymatic activity was measured using nitrocefin in parallel with rt - pcr in the experiments that did not involve exposure to -lactamase inhibitors . basal expression of ampc mrna in those three e. cloacae strains was compared to that of derepressed ampc p99 and found to be 150- to 300-fold lower ( table 1 ) . concomitantly , basal -lactamase activity in crude cell extracts reported using the nitrocefin substrate was also higher ( 1,000- to 1,800-fold ) in p99 than in the three selected e. cloacae strains .
basal levels of ampc transcripts and -lactamase activity were therefore fully consistent with the susceptibility / resistance phenotype of the strains .
the inducibility of -lactamase activity was studied in the presence of 132 mg / l cefoxitin .
the three e. cloacae strains tested showed similar induction profiles : ampc mrna peaked at 12 hours following induction , then slowly declined to reach basal levels at 46 hours .
figure 1 shows the kinetics of one representative experiment with the strain 293ht107 treated with cefoxitin at 1 or 2 mg / l . increased mrna concentrations were detectable soon after incubation start ( as soon as 10 minutes ; data not shown ) and peaked after 12 hours of incubation .
-lactamase activity was delayed slightly when compared to ampc mrna and continuously increased throughout the 4 hours of incubation with cefoxitin . when treated with 1632 mg /
l of cefoxi - tin , the maximal ampc transcriptional level after 2 hours of culture was between 100 and 200 times the basal level of both 293ht107 ( figure 2 g ) and 293la2 ( figure 2d ) strains , and around 50 times the basal level of the 293uc1 strain ( figure 2a ) .
the potential of avibactam and clavulanate for induction of ampc expression was evaluated on the three e. cloacae strains at 1664 mg / l .
clavulanate had no significant effects on 293uc1 and 293la2 strains during the 6-hour incubation period ( figure 2b and e ) , whereas it was a moderate ampc inducer for 293ht107 with about a 40-fold increase of ampc mrna at 64 mg / l after 2 hours of incubation ( figure 2h ) .
in contrast , avibactam had no detectable effect on ampc mrna levels in the three strains tested ( figure 2 , c , f , and i ) . at the concentrations used for induction studies , avibactam had no effect on the growth of the bacterial strains tested , as testified by the od values measured at each time point .
enterobacter spp . are recognized to be among the most common nosocomial pathogens , with current resistance rates presenting a serious therapeutic dilemma .
resistance through overexpression of ampc can occur in the vast majority of strains possessing a chromosomally encoded cephalosporinase , and ampc induction is recognized as a widespread resistance mechanism . in a study examining 200 clinical isolates of p. aeruginosa , enterobacter spp .
, it was shown that 85% of the collected strains showed inducible ampc production , of which 11% were stably derepressed and only 3% were not induced by either cefoxitin or imipenem.16 approximately 12% of hospital strains of the european meropenem yearly susceptibility test information collection ( mystic ) program in the years 19972000 were due to potential ampc - producing strains of enterobacter spp .
s. marcescens , in which 28% represented stably derepressed ampc - producing phenotypes.17 much of what is known about ampc regulation is from studies in e. coli , c. freundii , and e. cloacae ; the induction mechanism in response to exposure to certain -lactams is complex and closely linked to the peptidoglycan - recycling pathway.9 different effector proteins and regulation mechanisms have been recently evidenced for p. aeruginosa ampc induction , suggesting that the process is more complex in that species and distinct from the current paradigm established following studies of enterobacteriaceae species.18 -lactams differ in their inducing abilities , with carbapen - ems and cephamycins having the highest potential.8 the clavu - lanate -lactamase inhibitor is also an ampc inducer , and was shown in vitro to antagonize the antibacterial activity of various -lactams.10,11 in this context , the potential for induction of the new -lactamase inhibitor avibactam was evaluated . at sub - mic concentrations ,
cefoxitin induced a major dose - dependent synthesis of ampc in all three e. cloacae strains tested here , whereas clavulanate triggered synthesis of ampc mrna in two out of the three strains , at the highest concentration tested ( 64 mg / l ) .
in contrast , in the same range of concentrations , avibactam had no effect on cellular ampc mrna concentration in any of the three e. cloacae strains during the 6-hour incubation period .
from these initial findings , it is concluded that there is little likelihood of antagonism between -lactam antibiotics and the novel -lactamase inhibitor avibactam in enterobacter spp .
questions remain about other bacterial species producing inducible chromosomal ampc enzymes , and will be the focus of future studies .
in contrast with the inhibitors currently available ( clavulanate , tazobactam , and sulbactam ) , which all have relatively limited activity against the class c enzymes , avibactam is a potent inhibitor of ampc -lactamases.3,5 it is the first non--lactam -lactamase inhibitor to advance to clinical development , currently undergoing phase ii 
iii clinical trials in combination with ceftazidime and with cef - taroline ( http://www.clinicaltrials.gov ) .
ceftaroline , like some third - generation cephalosporins , is a weak inducer of ampc enzymes at sub - mic concentrations , resulting in a propensity to select ampc - derepressed or ampc - hyperinducible mutants.19 pairing ceftaroline with avibactam should thus be an effective strategy to limit the risk of selection of mutants , and to restore ceftaroline activity against ampc - hyperproducing strains , as well as to other -lactamase producers .
 induction of ampc -lactamase expression can often compromise antibiotic treatment and is triggered by several -lactams ( such as cefoxitin and imipenem ) and by the -lactamase inhibitor clavulanic acid . 
 the novel -lactamase inhibitor avibactam ( nxl104 ) is a potent inhibitor of both class a and class c enzymes . 
 the potential of avibactam for induction of ampc expression in enterobacter cloacae was investigated by ampc messenger ribonucleic acid quantitation . 
 cefoxitin and clavulanic acid were confirmed as ampc inducers , whereas avibactam was found to exert no effect on ampc expression . 
 thus , avibactam is unlikely to diminish the activity of any partner -lactam antibiotic against ampc - producing organisms . 
Introduction
Materials and methods
Bacterial strains and susceptibility testing
Induction experiments
Reverse-transcription polymerase chain reaction
-Lactamase activity assays
Results
Discussion