الفهرس 
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Abstract
Antimicrobial resistance has become a serious global problem and affects almost every bacterial species for which treatment with antibiotics is available. Resistant bacteria are emerging worldwide as a threat to favor¬able outcomes of treatment of common infections in com¬munity and hospital settings. One of the most important resistant mechanisms in gram-negative bacteria against beta-lactam antibiotics is induced by production of beta-lactamase enzymes. The increased prevalence of Enterobacteriaceae producing ESBLs creates a great need for identifying these organisms for infection control and epidemiological surveillance.
The aim of the present study was to (i) Determine the incidence of ESBL producing E. coli and K. pneumoniae in different clinical samples by phenotypic and molecular methods. (ii) Evaluate the diagnostic performance of the selective chromogenic culture media, chromID ESBL, as a rapid identification method for phenotypic detection of ESBL producing strains of E. coli and K. pneumoniae. (iii) Determine the prevalence of the genes blaTEM, blaSHV, and blaCTX-M, which are responsible for ESBL production in clinical isolates of E. coli and K. pneumoniae. (iv) Identify the sequence types of the most prevalent ESBL genes among E. coli and K. pneumoniae in Alexandria.
One hundred isolates of ESBL producing E. coli and K. pneumoniae were isolated from 173 different clinical specimens. All isolates were subjected to antimicrobial susceptibility testing by the Bauer-Kirby disk diffusion technique, CLSI Phenotypic confirmatory test (combined disk method), double disk synergy test, modified double disk synergy test (MDDST), and culture on chromID ESBL agar. Conventional PCR was performed for detection of the most prevalent genes responsible for ESBL production. Twelve randomly selected TEM, SHV and CTX-M genes were subjected to nucleic acid sequencing.
An ESBL phenotype was confirmed in 100/173 (57.8%) isolates according to the CLSI guideline by combined disk test using both cefotaxime and ceftazidime alone and in combination with clavulanic acid.
Out of 100 strains phenotypically confirmed as ESBL producers by using both disks, two Klebsiella strains failed to be detected by cefotaxime combined disk (CTX/CCT), on the other hand 2 E. coli and 2 K. pneumoniae strains failed to be detected by ceftazidime combined disk (CAZ/CCA). Using this technique, cefotaxime combined disk achieved the highest sensitivity of 98%. Among the phenotypic detection methods, MDDST using cefepime with piperacillin-tazobactam was the most sensitive (86%), followed by MDDST using cefepime with amoxicillin-clavulanate (70%), followed by DDST with 20 mm distance between augmentin and β-lactam antibiotics (44%), and lastly the DDST with 30 mm distance between augmentin and β-lactam antibiotics (8%). As regard chromID ESBL, the sensitivity was 97% for E. coli strains as two strains failed to be detected because they did not produce the expected-colored colony (non-colored colonies), while the sensitivity was 100% for K. pneumoniae with overall sensitivity of 98%.
In isolates with ESBL/AmpC co-producer, MDDST using cefepime with piperacillin-tazobactam was able to detect all isolates. Using either CTX/CCT or CAZ/CCA combined disks as a phenotypic confirmatory test failed to detect 2 (5.9%) and 4 (11.8%) ESBL/AmpC co-producer isolates respectively. chromID ESBL was able to detect 33 (97.1%) isolates of ESBL/AmpC co-producer.
Out of 100 phenotypically ESBL positive isolates, 80 (80%) were positive for at least one of the three studied genes. More positive strains were found among K. pneumoniae 81.8% compared to E. coli 79.1%.
Among ESBL-producing isolates, blaTEM was the most prevalent β-lactamase-encoding gene. It was detected in 76.2% of the ESBL-producing isolates, whereas blaSHV and blaCTX-M were present in 16.2% and 36.2% of isolates respectively.
All phenotypic methods showed 100% specificity except chromID ESBL which gave 95.9% specificity in detecting ESBL producing isolates. For genotypic methods, it gave 100% specificity.
Sequence analysis of PCR product of selected isolates showed that blaTEM were identical to TEM-1, the SHV types were SHV-11, and that of CTX-M were characterized as CTX-M-15.