الفهرس 
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Abstract
Carbapenemases production by extended-spectrum β-lactamases (ESβLs)-
producing Escherichia coli (E. coli) has been increasingly found and may be considered
as a major cause of morbidity and mortality in hospital-acquired infection.
The aim of this work was to determine resistance pattern and frequency of
carbapenem resistance and presence of class D carbapenemases among ESβLsproducing
E. coli isolated from patients in Menoufia University Hospitals.
This study was conducted at Medical Microbiology and Immunology
Department, Faculty of Medicine, Menoufia University during the period from April
2018 to September 2019. Clinical samples were collected (270) from patients (1 month-
74 years old) admitted to different departments of MUHs. The study protocol was
approved by local ethics committee of Menoufia University. An informed consent was
obtained from each patient or the guardians of unaware patients.
Clinical samples (125 urine, 70 sputum and bronchial aspirate, 30 pus swabs, 22
blood samples, 15 burn swabs and 8 surgical drains) were collected, processed, and
cultured on different bacteriological media. E. coli isolates were identified by the
standard microbiological methods. E. coli isolates were preserved on tryptic soy broth
with 16% glycerol and frozen at -80°C.
Antimicrobial susceptibility testing was performed by disk diffusion method
against different antimicrobial agents (Oxoid, Basingstoke, UK) as recommended by
clinical laboratory standard institute (CLSI) including: piperacillin (PRL) (100 μg),
amoxacillin/clavulanic acid (AMC) (20/10 μg), piperacillin/ tazobactam (TZP) (100/10
μg), ceftazidime (CAZ) (30 μg), ceftriaxone (CRO) (30 μg), cefepime (CPM).
Cefoxitin( FOX) (30 μg) (30 μg), meropenem (MEM) (10 μg), imipenem (IPM) (10
μg), ertapenem (ETP)(10 μg), Aztreonam (ATM) (30 μg), amikacin (AK) (30 μg),
gentamicin (GM) (10μg), tobramycin (TOB) (10 μg), tetracycline (TET) (30 μg),
doxycycline (DOX) (30μg),ciprofloxacin (CIP)(5 μg), levofloxacin (LEV) (5 μg),
Gatifloxacin (GAT) (5 μg)and tigecycline (TGC) (30μg). Minimal inhibitory
concentration (MIC) of imipenem (Sigma, St. Louis, Missouri, USA) was determined
by agar dilution method according to CLSI guidelines.
E. coli isolates showing zone of inhibition ≤ 21 mm for ceftazidime, ≤ 27 mm
for cefotaxime, and ≤ 25 mm for ceftriaxone were considered potential ESβL-producers
and confirmed by cephalosporins/clavulanate combination test and the ESβL-NDP test.
Carbapenems (imipenem, meropenem and ertapenem) - resistant ESβLsproducing
E. coli isolates by disk diffusion were further confirmed by imipenem MIC
agar dilution method and carbaNP test. Different classes of carbapenemases (class A, B
and D) production were detected by inhibitor-based methods (boronic acid combined
disk, imipenem/EDTA combined disk and imipenem MIC with addition of sodium
chloride tests respectively).
Molecular detection of bla oxa23 and bla oxa48 (class D carbapenemases genes)
was done by multiplex PCR while bla shv gene (ESβLs gene) was detected by
conventional PCR