avibactam is a non--lactam--lactamase inhibitor with activity against class c enzymes , most class a enzymes ( including kpcs ) , and some class d ( oxa ) carbapenemases . when ceftazidime is combined with avibactam , the spectrum of activity is expanded to include organisms producing these -lactamases [ 24 ] . this combination is currently in phase 3 clinical trials for the treatment of gram - negative infections . it is important to know if small variations from standard clinical laboratory standards institute ( clsi ) testing guidelines cause any significant changes in testing outcome . these variations can have effects on the mic when testing -lactams , including cephalosporins and -lactam--lactamase inhibitor combinations [ 57 ] . the effect of varying the broth microdilution in vitro testing parameters on the ceftazidime - avibactam mic of -lactamase positive and negative enterobacteriaceae and pseudomonas aeruginosa isolates was determined and compared with the effects on meropenem and piperacillin - tazobactam mics . isolates were obtained from atcc ( manassas , va ) , jmi laboratories ( north liberty , ia ) , or the cerexa , inc . culture collection . mics were determined for ceftazidime - avibactam ( ceftazidime : usp , avibactam : forest laboratories , inc . ) , meropenem ( usp ) , and piperacillin - tazobactam ( piperacillin : sigma , tazobactam : usp ) by broth microdilution assay according to clsi guidelines [ 8 , 9 ] . the effects of the following parameters on the mic were determined : medium ph 5 , 6 , and 8 ( standard ph 7.27.4 ) ; incubation with 5% co2 ( standard incubation ambient air ) ; the addition of 2.5% laked horse blood ( lhb ) to cation - adjusted mueller hinton broth ( camhb ) and testing in haemophilus test medium ( htm ) ( standard medium camhb ) ; testing in non - cation - adjusted media ( mhb without added cations containing 2.95.9  mg / l calcium and 3.25.2  mg / l magnesium ) and testing in 50  mg / l calcium in mhb ( standard cation concentration 2025  mg / l calcium and 1012.5  mg / l magnesium ) ; and inoculum densities of 5  10  cfu / ml and 5  10  cfu / ml ( standard density 5  10  cfu / ml ) . human serum and human serum albumin were previously shown to have no effect on ceftazidime - avibactam mics . in medium with acidic ph ( ph 5 or 6 ) , mics for ceftazidime - avibactam were increased by 4- to 16-fold for 6 of 9 enterobacteriaceae isolates and were decreased by 4-fold for 2 of 4 p. aeruginosa isolates ( table 1 ) . acidic media caused increased piperacillin - tazobactam mics against 4 isolates and decreased mics against 3 isolates by 4-fold ( table 2 ) . acidic media caused increased meropenem mics against 4 isolates and decreased meropenem mics against 3 isolates by 4- to 8-fold ( table 3 ) . mic increases were observed at ph 5 against the reference strain e. coli atcc 25922 ( -lactamase negative ) . thus , the ph effect on mic appears to be related to the intrinsic antibacterial activity of the -lactams , rather than having an effect on the inhibition of -lactamases . the ceftazidime - avibactam mic for one e. coli isolate was increased by 4-fold when incubated in 5% co2 ( table 1 ) . incubation in 5% co2 caused the piperacillin - tazobactam mics against 2 p. aeruginosa isolates to change by 4-fold ( table 2 ) . testing in htm had no effect on mics with any drug except for one isolate that had 4-fold increased ceftazidime - avibactam mic ( table 1 ) . addition of 2.5% lhb had no effect on ceftazidime - avibactam mics ( table 1 ) , caused a 4-fold decreased piperacillin - tazobactam mic against one isolate ( table 2 ) , and caused 8-fold and > 32-fold increased meropenem mics for 2 isolates ( table 3 ) . ceftazidime - avibactam and piperacillin - tazobactam mics were unaffected by changes in calcium concentration of media ( tables 1 and 2 ) . in medium with trace calcium ( mhb ) , mics of meropenem decreased by 4-fold against one isolate and increased by > 4-fold against another ( table 3 ) . three isolates had ceftazidime - avibactam mics that were increased by 4- to 8-fold when the inoculum was increased 10-fold ( 5  10  cfu / ml ) ( table 1 ) . increased inoculum raised the mics 4- to 16-fold for piperacillin - tazobactam against 3 isolates , and lower inoculum ( 5  10  cfu / ml ) resulted in a 4-fold decreased mic for one isolate ( table 2 ) . high inoculum density caused meropenem mics to increase by 4- to 64-fold for 7 isolates , while low inoculum density caused meropenem mics to decrease by 4- to 8-fold for 2 isolates ( table 3 ) . in summary , most of the variations in broth microdilution parameters had little effect on ceftazidime - avibactam mics . the two parameters that did have an effect were acidic media ( 6 isolates ) and high inoculum density ( 3 isolates ) . to testing parameters only affected ceftazidime - avibactam mics for a single isolate by only 4-fold in each instance . it is possible that these individual cases are due to random variation rather than reproducible effects on mic . piperacillin - tazobactam and meropenem mics were also affected by changes in ph and inoculum density indicating that these effects are not unique to the ceftazidime - avibactam combination . the magnitude of the changes in piperacillin - tazobactam mics was more difficult to assess due to the high mics of most isolates under standard testing conditions . the inoculum effects noted in this study with ceftazidime - avibactam mics are not surprising given that inoculum effects have been previously documented with ceftazidime mic testing , particularly against -lactamase producing strains [ 57 ] . an inoculum effect on other cephalosporin and carbapenem mics has also been previously documented ; several studies have shown that mics for piperacillin - tazobactam , and to a lesser extent meropenem , are increased with higher inocula of -lactamase producing and nonproducing isolates [ 6 , 7 , 1113 ] . these findings indicate that slight variations in testing parameters during routine mic testing will likely have no significant effect on ceftazidime - avibactam mic values ; however , special attention should be paid to closely follow clsi guidelines with respect to the ph of the media and the inoculum used . since most laboratories use commercially prepared media , ph should not be an issue due to the buffering capacity of these media . additionally , clsi recommends validating inoculum density on a weekly basis as part of standard quality control so any variations in inoculum should be easily identified . therefore , if clsi guidelines and quality control recommendations are followed , there should not be any issues with routine ceftazidime - avibactam mic testing .
effects of varying in vitro susceptibility testing parameters of the broth microdilution assay on ceftazidime - avibactam mics were determined and compared to meropenem and piperacillin - tazobactam for 9 enterobacteriaceae and 4 pseudomonas aeruginosa isolates . 
 the effect of varying incubation conditions ( ambient air or 5% co2 ) , ph of medium , medium composition ( cation - adjusted mueller hinton broth with and without laked horse blood and haemophilus test medium ) , cation content of the medium , and inoculum density were tested . 
 most variations had no effect on ceftazidime - avibactam mic values ( no more than a 2-fold change ) . however , acidic ph or high inoculum resulted in 4- to 16-fold changes in mic , which was similar to those observed for meropenem and piperacillin - tazobactam under these conditions . 
 overall , this study shows that slight variations in testing parameters during routine mic testing will likely have no significant effect on ceftazidime - avibactam mic values .