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at present , enterobacteriaceae producing broad - spectrum beta - lactamases belong to the most feared group of bacterial pathogens .
of particular clinical importance are esbl and ampc enzymes capable of hydrolyzing broad - spectrum penicillins , monobactams and cephalosporins .
the prevalence of broad - spectrum beta - lactamases in pathogenic bacteria is clinically very important .
it increases the risk of failure of antibiotic therapy , resulting in higher morbidity , mortality and , last but not least , treatment costs .
for example , kang et al reported initial antibiotic therapy fails significantly more often ( 73% ) in patients with spontaneous bacterial peritonitis due to esbl - positive strains as compared with peritonitis of esbl - negative etiology ( 17% ) .
moreover , they showed 30-day mortality to be higher in patients with esbl - positive pathogens ( 60% ) than in those with esbl - negative enterobacteriaceae ( 23% ) .
pai et al documented high rates of failure of cefotaxime or ceftazidime therapy in patients with bloodstream infections caused by klebsiella pneumoniae strains producing ampc beta - lactamases .
tumbarello et al also reported mortality associated with bloodstream infections due to esbl - positive enterobacteriaceae of as much as 60% in cases of inadequate antibiotic therapy as compared with 19% when therapy was adequately targeted to the above multiresistant bacteria .
schwaber et al documented a 30% mortality rate in patients with bacteremia due to esbl - producing strains of escherichia coli , klebsiella spp . and proteus spp .
as compared with 16% mortality in patients with non - esbl - associated bacteremia . the frequency and number of different types of esbl and ampc enzymes continue to increase .
the earss data on the resistance of invasive isolates of escherichia coli and klebsiella pneumoniae suggest that the frequency of third - generation cephalosporin resistance has increased in the recent years .
this negative trend is assumed to be determined by the spread of strains producing broad - spectrum beta - lactamases , in particular esbl . in their multicentre study ,
paterson et al found that 31% of klebsiella pneumoniae strains isolated from blood produced esbl , with esbl production being detected in 44% of isolates from intensive care units ( icus ) . in their 2004 multicentre study comprising 16 health care facilities ,
kolar et al detected esbl production in 26% of klebsiella pneumoniae strains , with the prevalence reaching 39% in icus .
european icus showed that the collaborating laboratories incorrectly identified 29% of esbl - positive isolates as susceptible to ceftriaxone and 37% as susceptible to cefotaxime . according to tenover et al , the proportion of laboratories that did not detect production of esbl and ampc enzymes in enterobacteriaceae ranged from 24% to 32% , depending on the types of enzymes present .
a 2006 italian study showed that all tested esbl producers were correctly detected by only 42% of laboratories , while a 2008 spanish study reported that all tested esbl producers were correctly detected by 91% of laboratories , but only 47% of laboratories correctly identified all ampc strains [ 10 , 11 ] . for detection ,
phenotypic and genotypic methods may be used , with phenotyping being preferred in routine microbiology practice . in the case of esbl ,
the double - disk synergy test ( ddst ) , etest or clsi test are used , which are based on inhibiting the activity of these enzymes by their inhibitors ( e.g. , clavulanic acid ) and decreasing the minimum inhibitory concentration ( mic ) value of a particular cephalosporin , enlarging the zone of growth inhibition around the disk or its extension and formation of a characteristic pattern between the substrate disk and the disk containing the inhibitor [ 1214 ] .
ampc beta - lactamases may be detected using a modified ampc disk test with disks containing ceftazidime , ceftazidime with clavulanic acid and their combination with 3-aminophenylboronic acid , an inhibitor of ampc beta - lactamases .
our study aimed to compare the sensitivity of phenotypic methods for detecting esbl and ampc enzymes in a group of strains with their production detected by molecular biology methods .
the strains were identified by standard microbiology methods and the phoenix automated system ( becton dickinson ) .
esbl detection was based on pcr amplification of blatem , blashv and blactx - m genes encoding the most frequent types of these beta - lactamases , with subsequent determination of a particular variation by direct sequencing [ 1618 ] .
ampc beta - lactamases were determined by multiplex pcr capable of differentiation of various types of these enzymes . for the isolates ,
the results of the disk diffusion and microdilution methods and the accuracy of the phoenix automated system were assessed . in esbl - positive strains ,
the sensitivity of a modified microdilution method with determination of cefoxitin mic and the ratio of mic of cefoperazone with the cefoperazone / sulbactam combination ( chart 1 ) , as well as of the esbl etest ( ab biodisk ) and a modified ddst ( chart 2 ) were assessed .
ampc strains were tested by a modified ampc disk test using 3-aminophenylboronic acid ( chart 3 ) and the modified microdilution method with the determination of cefoxitin mic and the ratio of mic of cefotaxime and ceftazidime with the cefotaxime/3-aminophenylboronic acid and ceftazidime/3-aminophenylboronic acid combinations ( chart 1 ) .
the strains were identified by standard microbiology methods and the phoenix automated system ( becton dickinson ) .
esbl detection was based on pcr amplification of blatem , blashv and blactx - m genes encoding the most frequent types of these beta - lactamases , with subsequent determination of a particular variation by direct sequencing [ 1618 ] .
ampc beta - lactamases were determined by multiplex pcr capable of differentiation of various types of these enzymes .
for the isolates , the results of the disk diffusion and microdilution methods and the accuracy of the phoenix automated system were assessed . in esbl - positive strains ,
the sensitivity of a modified microdilution method with determination of cefoxitin mic and the ratio of mic of cefoperazone with the cefoperazone / sulbactam combination ( chart 1 ) , as well as of the esbl etest ( ab biodisk ) and a modified ddst ( chart 2 ) were assessed .
ampc strains were tested by a modified ampc disk test using 3-aminophenylboronic acid ( chart 3 ) and the modified microdilution method with the determination of cefoxitin mic and the ratio of mic of cefotaxime and ceftazidime with the cefotaxime/3-aminophenylboronic acid and ceftazidime/3-aminophenylboronic acid combinations ( chart 1 ) .
pcr and direct sequencing were used to detect esbl production in 85 strains and ampc production in 21 strains ( table 2 ) . when using the microdilution method with the clsi criteria ,
the tested strains were shown to be highly false - susceptible to cefoperazone ( 21% for esbl and 76% for ampc ) , cefotaxime ( 29% and 67% ) , ceftazidime ( 32% and 29% ) and cefepime ( 60% for esbl ) . also with the disk method , a large percentage of strains were identified as false - susceptible to third- and fourth - generation cephalosporins according to the clsi criteria ( cefoperazone 9% for esbl and 43% for ampc ; cefotaxime 8% and 14% ; ceftazidime 45% and 14% ; cefepime 52% ) .
after the breakpoints for cefepime , cefotaxime and ceftazidime were decreased to 1 mg / l , as recommended by the eucast , the percentage of false susceptibility in the tested strains of enterobacteriaceae producing esbl and ampc enzymes decreased significantly .
although the sensitivity of esbl detection by the phoenix automated system was high ( 99% ) , it only reached 38% in the group of ampc - positive strains .
high sensitivity was achieved with tests specific for a given type of a broad - spectrum beta - lactamase .
the modified ddst showed 100% sensitivity for esbl producers , whereas the modified ampc test with 3-aminophenylboronic acid proved correct in detecting 95% of ampc - positive strains . in the group of esbl - positive strains ,
mg / l or less and a ratio of mic of cefoperazone and cefoperazone / sulbactam > 2:1 .
the sensitivity of the modified microdilution method for ampc beta - lactamases was 95% ( table 5 ) .
from a microbiological and clinical point of view , early and correct detection of esbl and ampc beta - lactamase production is essential . in routine microbiology practice , this remains complicated by false - negative results , especially if traditional methods for determination of bacterial resistance to antimicrobial agents are used . in severe bacterial infections , particularly in intensive care patients , antibiotic therapy should be initiated as soon as possible , preferably within an hour of diagnosis .
however , microbiological results of phenotypic tests ( including correct determination of susceptibility / resistance of bacterial pathogens to antimicrobial agents ) remain an important primary source for selecting adequate antibiotic therapy and , if needed , de - escalation antibiotic therapy . results of our study show a high percentage of false susceptibility in the standard microdilution method ( 2176% ) and disk diffusion method ( 852% ) when using the 2009 clsi criteria . when decreasing the breakpoints for cefepime , ceftazidime and cefotaxime to 1
mg / l , in accordance with the eucast clinical breakpoints , false susceptibility drops to 543% . the use of the eucast criteria for the disk diffusion method results in a clear decrease of false susceptibility to 1038% .
detection of broad - spectrum beta - lactamases should be based on specific phenotypic methods , especially the modified ddst and ampc disk method .
sensitivity of the mddst used for esbl detection was 100% , and 95% in the mampc test .
although very good results were also obtained with the esbl etest ( a sensitivity of 95% ) , this might not be the most suitable method for common use in microbiology due to higher operating costs associated with this test .
the standard microdilution method with a modified set of antibiotics seems to be an effective method since it meets the criteria of high sensitivity ( 87% for esbl and 95% for ampc ) and simultaneous detection of the 2 enzyme types .
wiegand et al tested 3 semi - automated and 3 manual phenotypic methods for esbl detection . in the semi - automated systems ,
the sensitivity rates were 99% for phoenix , 86% for vitek 2 and 84% for microscan .
the compared manual methods included agar diffusion assays , the ddst , esbl etest and clsi disk method .
thus , the manual diffuse tests were more precise than the automated systems , which is in accordance with our results documenting 100% sensitivity of the mddst .
linscott and brown compared 4 phenotypic methods for esbl detection ; however , their production was not confirmed by pcr . unlike the study by wiegand et al .
, they reported higher sensitivity of semi - automated methods ( microscan esbl plus esbl confirmation panel 100% ; vitek 1 99% ) as compared with agar diffusion assays ( esbl etest 97% ; clsi esbl disk method 96% ) .
it is obvious that 100% sensitivity in the detection of esbl production is difficult to achieve .
our results , especially those of the modified ddst , suggest that the best solution seems to be a combination of 2 phenotypic methods . in case of a high prevalence of ampc - positive enterobacteriaceae
, routine testing should include a phenotypic method to detect potential ampc production . as a suitable highly sensitive method , the ampc disk test with 3-aminophenylboronic acid could be recommended . for basic screening , it would be advisable to use the microdilution method with a modified set of antibiotics able to detect both esbl and ampc enzyme production in a single assay .
summarybackgroundenterobacteriaceae producing esbl and ampc enzymes can be associated with failure of antibiotic therapy and related morbidity and mortality . 
 their routine detection in microbiology laboratories is still a problem . 
 the aim of this study was to compare the sensitivity of selected phenotypic methods.material/methodsa total of 106 strains of the enterobacteriaceae family were tested , in which molecular biology methods confirmed the presence of genes encoding esbl or ampc . in esbl - positive strains , 
 the sensitivity of the esbl etest ( ab biodisk ) and a modified double - disk synergy test ( ddst ) were evaluated . 
 ampc strains were tested by a modified ampc disk method using 3-aminophenylboronic acid . for simultaneous detection of esbl and ampc , the microdilution method with 
 a modified set of antimicrobial agents was used.resultsthe sensitivity of the esbl etest was 95% ; the modified ddst yielded 100% sensitivity for esbl producers and the ampc test correctly detected 95% of ampc - positive strains . 
 the sensitivity of the modified microdilution method was 87% and 95% for esbl and ampc beta lactamases , respectively.conclusionsthe detection of esbl and ampc beta lactamases should be based on specific phenotypic methods such as the modified ddst , esbl etest , ampc disk test and the modified microdilution method .