الفهرس 
Beta-LactamasesOnly 14 pages are availabe for public view
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Abstract
AmpC β-lactamases have gained importance since the late 1970s as one of the mediators of antimicrobial resistance in Gram negative bacilli. These enzymes are typically produced by isolates of E. coli, Klebsiella spp., Proteus, and Salmonella spp. and are associated with multiple antibiotic resistance that leaves few therapeutic options.
Enterobacteriaceae producing AmpC β-lactamases (AmpC) have become a major therapeutic challenge. The detection of AmpC-producing Klebsiella spp. is of significant clinical relevance since AmpC producers may appear susceptible to expanded-spectrum cephalosporins when initially tested. This may lead to inappropriate antimicrobial regimens and therapeutic failure. Thus, a simple and reliable detection for AmpC producers is needed.
Cefoxtin screening test is reported as useful in screening for AmpC. Modified Three Dimensional Test (M3D) is reported as simple and sensitive phenotypic detection method. Many studies have validated the use of cloxacillin (as AmpC inhibitor) to detect AmpC β-lactamases among Gram-negative bacteria. Also, many studies have validated the use of imipenem and cefoxitin as inducing agents to detect the presence of inducible AmpC enzymes among enterobacterial strains.
Even though multiplex-PCR is a gold standard method to detect AmpC β-lactamase genes (differentiating between chromosomal and plasmid-mediated AmpC β-lactamases and the different types or families of PMABLs), but the test is still costly and time consuming and equipment availability is limited to few laboratories, hence looking for specific and sensitive phenotypic tests have always been a challenge.
Many clinical laboratories show interests in performing phenotypic tests as these are cost effective, simple to perform, easy to implement in the diagnostic laboratory to avoid inappropriate therapy and for better infection control, distinguishing between the cefoxitin-resistant AmpC producers and cefoxitin-resistant non AmpC producers which may be due to reduced membrane permeability and also, differentiation of organisms expressing ESBLs from organisms expressing plasmid-mediated AmpC β-lactamases in order to address surveillance and epidemiology as well as hospital infection control issues associated with these resistance mechanisms.
The aim of this work is to detect of the occurrence of PMABLs in Klebsiella spp. isolates by phenotypic tests and detect its gene types by multiplex-PCR.
This study was conducted on a total number of one hundred clinical isolates of Klebsiella spp. collected from different clinical specimens referred to the Central Microbiological Laboratory of Ain Shams University Hospitals from September 2012 to February 2013. Isolates were screened by Cefoxtin Disk resistance using Disk Diffusion Method (DD). All isolates were subjected to seven phenotypic methods: Cloxacillin Combined Disk Diffusion Test (CC-DD), Cefoxitin-Cloxacillin Double-Disk Synergy Test (CC-DDS), Ceftazidime-Imipenem Antagonism Test (CIAT), Cefoxitin-Cefotaxime (FOX-CTX) Antagonism Test and Modified Three Dimensional Test (M3D) for detection of AmpC β-lactamases. Detection of ESBLs production was done by using Double Disk Synergy Test (DDS) and detection of Plasmid-mediated AmpC genes (ACC, FOX, MOX, DHA, CIT and EBC) by Multiplex-PCR.
AmpC production was detected in 57 (57%) out of the 100 isolates by M3D test, 44 (77.2%,) out of 57 M3D-positive isolates were cefoxtin resistant with a sensitivity of 77.2%, a specificity of 95.3% and an excellent agreement between the results of both tests (kappa=0.719). Thirteen (22.8%,) out of 57 M3D-positive isolates were positive by CC-DD test with more specificity (95.3%) than sensitivity (22.8%) and a poor agreement between the results of both tests (kappa=0.162) while seven (12.3%) out of 57 M3D-positive isolates were positive by CC-DDS test with lower sensitivity (12.3%), a specificity of 41.9% and fair negative agreement between the results of both tests (kappa= - 0.428). Fourty-three (75.4%) out of 57 M3D-positive isolates were only AmpC producers and 14 (24.6%) out of 57 M3D-positive isolates were ESBL/AmpC co-producers.
Plasmid-mediated AmpC β-lactamases were detected in 13 (32.5%) out of 40 isolates by PCR. Only eight (61.5%) out of 13 PCR-positive isolates were cefoxitin resistant with a sensitivity of 61.5%, a specificity of 66.7% and week agreement between the results of both tests (kappa=0.261). Eight (61.5%) out of 13 PCR-positive isolates were positive by CC-DD test with more specificity (81.5%) than sensitivity (61.5%) and fair agreement between the results of both tests (kappa=0.430), while five (38.5%) out of 13 PCR-positive isolates were positive by CC-DDS test with less sensitivity (38.5%) than specificity of 77.8% and poor agreement between the results of both tests (kappa=0.169). One (7.7%) out of 13 PCR-positive isolates were positive by CIAT with lower sensitivity (7.7%), higher specificity (96.3%) and poor agreement between results of both tests (kappa=0.051). Two (15.4%) out of 13 PCR-positive isolates were positive by FOX-CTX antagonism test with lower sensitivity (15.4%), higher specificity (88.9%) and poor agreement between the results of both tests (kappa=0.051). Eight (61.5%) out of 13 PCR-positive isolates were only AmpC producers and five (38.5%) out of 13 PCR-positive isolates were ESBL/AmpC co-producers.
Plasmid-mediated AmpC genes were detected in 13 (32.5%) out of 40 isolates by Multiplex-PCR using family-specific primers. Eight (61.5%) out of 13 PCR-positive isolates have the blaMOX gene, nine (69.2%) out of 13 PCR-positive isolates have the blaCIT gene, five (38.5%) out of 13 PCR-positive isolates have the blaFOX gene, five (38.5%) out of 13 PCR-positive isolates have the blaACC gene, one (7.7%) out of 13 PCR-positive isolates has the blaEBC gene and one (7.7%) out of 13 PCR-positive isolates has the blaDHA gene.