الفهرس 
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Abstract
Urinary tract infection is one of the most common nosocomial infections. Uropathogens resistant to β-lactam agents due to production of various enzymes have increased in recent years. The dissemination in hospitals and the location of these enzymes on highly mobile genetic elements have contributed to their rapid spread and the frequent co-transfer of multiple other antibiotic resistance factors. This results in multidrug resistance in uropathogens. The aim of this work was to determine the incidence of multi-drug resistant Enterobacteriacaeae isolated from urine of patients admitted to different departments at Menofia University Hospitals (MUHs). To detect the presence of ESβLs, AmpC β-lactamases and carbapenemases among the isolated Enterobacteriaceae using phenotypic methods and to detect the presence of bla KPC and bla NDM resistance genes among the enterobacterial isolates using real- time PCR. The study included 260 patients admitted to different departments and ICUs at MUHs and developed UTIs. Urine samples were collected from all patients. The specimens were processed according to standard microbiological methods. One hundred and twenty Enterobacteriaceae isolates were isolated and identified by conventional methods and bacterial isolates subjected to molecular diagnosis were identified using API system (Microbact 12ATM, Oxoid). Antimicrobial susceptibility test was done for Enterobacteriaceae isolates by disk diffusion method and results were interpreted according to the Clinical and Laboratory Standard Institute guidelines.
Summary
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Extended-spectrum β-lactamases (ESβLs) production was detected by disk diffusion screening test (resistance to 3rd generation cephalosporins and aztreonam) and by combined disk confirmatory test. Detection of AmpC production was done by cefoxitin disk screening test and by AmpC disk confirmatory test. The obtained isolates were tested against imipenem, meropenem and ertapenem by disk diffusion method as a screening method for suspected carbapenemase production. This was followed by performing the phenotypic confirmatory tests for detection of carbapenemase production. The modified Hodge test (MHT) was evaluated as a phenotypic confirmatory test to detect both class A and class B carbapenemases production. Novel boronic acid-based method (a combined-disk synergy test) using imipenem in combination with phenylboronic acid as an inhibitor was used as a confirmatory tool for class A (KPC) detection. A combined disk test using imipenem in combination with EDTA was also applied to confirm class B (MβLs) production. Being the gold standard for detection of carbapenemase production, a real time PCR assay was performed (as the molecular confirmatory test) using specific primers for blaKPC and blaNDM genes. Results of real- time PCR were compared with those of MHT and combined disk synergy tests to evaluate the sensitivity and specificity of each.
The results obtained in this study revealed that out of 260 urine samples obtained from the patients, only 149/260 (57%) revealed positive culture results with 154 isolates. The most frequent nosocomial uropathogens were
Summary
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Enterobacteriaceae spp. (120/154, 78%). Among isolated Enterobacteriaceae, E. coli was the most common (58/120, 48.4%), followed by K. pneumoniae (22/120, 18.3%) followed by Enterobacter spp. (16/120, 13.3%), K. oxytoca (9/120, 7.5%), Proteus spp. (6/120, 5%), Citrobacter spp. (4/120, 3.3%), Serratia spp. ( 3/120, 2.5%) and lastly Morganella morganii (2/120, 1.7%). Studying the risk factors associated with acquiring NUTI revealed that UTI was more common among old patients (>50 years old; 35.8%, P<0.001). Females (36.5%) were more affected than males (P<0.001).Married patients had UTI more (46.9%, P<0.001) than single patients. Higher rates of UTI were found in patients with duration of hospital stay more than 14 days (40.8%, P<0.001). UTIs were found more in patients with history of associated co-morbidities (47.3%) compared to those with no history of associated co-morbidities (P<0.001). Diabetic patients had statistically (P<0.001) higher rate of UTI than non diabetics. Regarding catheterization, higher rates of UTI were found in patients with urinary catheter (43.5%) compared to those having no urinary catheter (P<0.05). UTI were found in patients with duration of catheterization more than 7 days with a high statistically significant difference (P<0.001). Patients whose catheter is inserted in general ward had higher rate of UTI (71.7%) than ICU (22.1%) and operation room (6.2%) with (P<0.001).
Antimicrobial susceptibility tests using disk diffusion method revealed that all isolates were resistant to piperacillin and amoxicillin/clavulanic acid. They were highly resistant to co trimexazole(95%), ciprofloxacin (94.2%), cefoxitin (90%), aztreonam (85%), cefotaxime (84.2%), ceftriaxone (83.3%) and ceftazidime (82.5%).Susceptibility to amikacin, levofloxacin, piperacillin/tazobactam, ertapenem, imipenem, meropenem and tigecycline was observed in 51.7%, 37.5%, 49.2%,57.5%, 55%,59.5%, and 80.8% of isolates, respectively. In this study, 56%
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(67/120) of Enterobactericeae isolates were multidrug resistance (MDR), 33 % (40/120) were extreme drug resistance (XDR) and 11% (13/120) of Enterobactericeae isolates were pandrug resistance (PDR). Regarding the detection of ESβL, the disk diffusion screening method revealed that 85% (102/120) of Enterobacteriaceae isolates were ESβL producers by disc diffusion screening method, while combined disk confirmatory method detected only 55.8% of Enterobacteriaceae as ESβL producers (P<0.001). E. coli was the predominant ESβL-producer (31/67, 46.3%) followed by K. pneumoniae (17.9%), Enterobacter spp. (16.4%), K. oxytoca (7.4%), Citrobacter spp. (4.5%), Proteus spp. (3%) and Serratia spp. (1.5%). Regarding the detection of AmpC beta-lactamase, the cefoxitin disk screening method revealed that 90% (108/120) of Enterobacteriaceae isolates were detected as AmpC producers by cefoxitin disk screening method. While, AmpC disk confirmatory test detected only 30.8% (37/120) of Enterobacteriaceae as AmpC producers. Statistically there is a high significant difference between two methods (p<0.001). Enterobacter spp. (12/37, 32.5%) were the predominant AmpC β-lactamase producers followed by K. pneumoniae (21.6%), K. oxytoca (16.2%), E. coli (16.2%), Citrobacter spp. (5.4%), Serratia spp. (5.4%) and Morganella morganii (2.7%).