A New Phenotypic Method for Detection of Extended Spectrum β-lactamases in AmpC β-lactamase -producing Klebsiella Species

Abstract

SUMMARY E SBL-producing Enterobacteriaceae are recognized worldwide as nosocomial pathogens of major importance. Extended–spectrum ß-lactamases are enzymes that mediate resistance to oxyimino-cephalosporins and aztreonam. AmpC ß-lactamases, in contrast to ESBLs, hydrolyze broad and extended cephalosporins (cephamycins as well as to oxymino-ß-lactams) but are not inhibited by ß-lactamase inhibitors such as clavulanic acid and hence the presence of an ESBL can be masked by the expression of an AmpC. So the exact detection of ESBLs in isolates that produce both ESBLs and AmpCs is important for both treatment and epidemiology, as it can decrease the spread of resistance in hospital strains. Therefore, there is a need for alternative simple method that detects ESBL in Enterobacteriaceae isolates with a high sensitivity even though the isolates harbor AmpC. The aim of our study was focused on the modification of DDST to detect ESBL production in isolates which harbor AmpC this was done by using of cefepime (4th generation cephalosporin) as an indicator drug which is known to be a poor substrate for AmpC B-lactamases instead of other 3rd generation cephalosporins, and the using of tazopactam instead of clavulanic acid as the later may act as an inducer of high level AmpC producing a false negative result in the ESBL elective tests and adjusting distances between the two discs centre to centre at 20 mm. This study was conducted on a total number of 90 klebsiella isolates collected from different clinical specimens referred to the Central Microbiology Laboratory of Ain Shams University Hospitals for routine culture and sensitivity from September to December 2013. All isolates have been subjected to the following: Screening for ESBLs production by disc diffusion method (DDM), Double disc synergy test (DDST) using (amoxicillin/clavulanate) (AMC) (20/10µg) and 3rd generation cephalosporins; CAZ, CTX, CPD and FEP. Detection of ESBLs production by modified double disc synergy (MDDST) using FEP (30µg) disc and (piperacillin/tazobactam) (TZP) (100/10µg) disc, Confirmation of ESBL production by confirmatory CLSI method using ceftazidime/ceftazidime-clavulanate (CAZ-CLA) E-test ESBL strips. Screening for AmpC production by resistance to cefoxitin disc (FOX) (30µg) and Confirmation of AmpC by modified three dimensional test (M3D). Fifty seven / 90 isolates were ESBL positive by E-test (golden standard test). Fourty seven out of 57 were positive by DDST, while MDDST detected all the 57 ESBL positive isolates. Sixty out of 90 isolates were AmpC producers by M3D (golden standard test). Fifty seven out of 60 isolates that were positive by M3D test were resistant to FOX with a sensitivity of 95% and specificity of 60% for detection of AmpC production with positive predictive value and negative predictive value of (83% and 86%) respectively. Sixty out of 90 isolates were positive AmpC by M3D test. Thirty two out of 60 isolates were positive ESBL by MDDST. On the other hand 24 /60 were positive by DDST. The sensitivity of DDST was 75% and specificity of 100% for detection of ESBL in presence of AmpC. Thirty out of 90 isolates were negative AmpC by M3D test. Twenty five out of 30 isolates were positive ESBL by MDDST. Twenty three out of 30 were positive by double disc synergy test. The sensitivity of DDST was 92% and specificity of 100% for detection of ESBL in absence of AmpC in the isolates. This means that the sensitivity of the DDST when M3D was negative is better from its sensitivity when the latter was positive , where MDDST is positive in eight isolates more than double disc test in positive M3D isolates and two in negative M3D isolates.