الفهرس 
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Abstract
Integron system is a dynamic force in the evolution of multi - drug resistance and it helps bacteria to acquire novel combinations of resistance genes. Class 1 integrons play a role in the emergence of multi-resistant bacteria by facilitating the recruitement of gene cassettes encoding antibiotic resistance.
Class 1 integrons are widely prevalent among clinical isolates of resistant E. coli, especially in ESβl-producers and are probably a reservoir for producing multi-drug resistance and nosocomial infections in hospitals.
The purposes of this work were to detect the occurence of class 1 integrons among clinical as well as commensal E. coli isolates. To investigate the distribution of class 1 integrons among E. coli isolates from different types of infection and E. coli isolates with different antibiotic susceptibility patterns, and to evaluate the contribution of class 1 integrons to the dissimination of multi-drug resistance in E. coli particularly ESβL producers.
The present study was carried out between September 2012 and February 2014. It involved 210 patients admitted to Menoufia University Hospital having infections that became evident 48 hours or more after hospital admission. The study included two groups; group I, 80 patients (34 males and 46 females), located in different wards and intensive care units (ICUs) of Menoufia University Hospitals having E. coli nosocomial infections. Their ages ranged from 1 month-87 years old (mean 43.60±18.35). Group II were 20 healthy subjects (volunteers; 9 males and 11 females), matched by age and
sex, they were not exposed to antibiotics or hospital environment in the three months prior to sample collection. Their ages ranged from 2–72 years old (mean 33.3±22.47).
After sampling, culture and isolation of bacteria were done using appropriate media. Identification of isolates was according to the standard microbiological methods. E. coli isolates were identified by colony morphology, culture characteristics, microscopic examination and conventional biochemical reactions.
Antimicrobial susceptibility testing for isolates by disk diffusion method was interpreted according to the Clinical and Laboratory Standards Institute guidelines. Screening by disk diffusion test for ESβL producing organisms was done according to the Clinical and Laboratory Standards Institute guidelines and phenotypic confirmatory methods, double disk synergy test (DDST) and cephalosporin/clavulanate combination disks test were used.
Detection of class 1 integrons was carried out using PCR for all studied E. coli clinical and commensal isolates using specific intI1 gene primers.
Thirty two clinical E. coli isolates and 10 commensals (including intI1 positive and negative isolates) were selected for detection of ESβL genes SHV,TEM and CTX-M by PCR.
In our study, E. coli was represented by 34.7 % of total isolates (n=230). forty five percent of E. coli was isolated from urine, 38.8 % from pus samples, 31.8 % from wound swabs, 35.6 % from sputum and 11.1 % from blood.
High significant difference (p<0.001) was found between E. coli C.Is. and E. coli commensals regarding their susceptibility to different antibiotics. All E. coli C.Is. were resistant to 3 or more
antibiotic classes (i.e. multi-drug resistant, MDR). Fourteen E. coli commensals were resistant to 3 or more antibiotics.
Regarding class 1 integron distribution, there was statistical significant difference (p<0.05) between studied E. coli C.Is. and commensals where 63.8 % of E. coli C.Is. and 30 % of E. coli commensals had class 1 integron.
There was high statistical significant difference (p< 0.001) between subjects having intI1 gene and those had not the gene according to the age group, where 73 % of the age group between 25–50 years old had intI1gene and 92.9% of the age group more than 50 years old had intI1 gene, while none of age group < 5 years old had intI1 gene.
For presence of intI1 gene in relation to clinical history among Group I, there was statistical significant difference (p>0.05) between patients admitted and not admitted to ICU in having intI1 gene. Eighty percentages of admitted patients to ICU had intI1 gene. Eighty percentages of patients stayed more than 4 weeks in the hospital had intI1 gene. Class 1 integron gene increased with subjects exposed to invasive procedures, administrated antibiotics, corticosteroids and/or cytotoxic drugs.
There was statistical significant difference (P<0.05) between the presence of IntI1 gene in relation to the type of specimen where, the presence of intI1 was significantly higher in urinary C.Is. (33/45: 73.3%) followed by isolates from sputum (11/16:68.75%) and pus (4/7: 57.1%).
There was significant difference (p<0.05) between clinical isolates and commensal isolates having intI1 gene in relation to resistance to most antibiotics e.g. ciprofloxcacin, norfloxacin , cefamandole, cefoxitin, trimethoprim sulfamethoxazole ( 1.25
μg/23.75 μg) and tetracycline with highly significant difference (p<0.001) between them in having intI1 gene in relation to resistance to ampicillin, piperacine, amoxicillin /clavulinic acid, amikacin, gentamycin, tobramycin, streptomycin, , chloramphenicol and nitrofurantoin and non significant difference between them in having intI1 gene in relation to resistance to cefotaxime, ceftraixone, ceftazidime, cefepime, azteronam and imipenem.
The more the resistance against different antibiotics the more the chance of presence of intI1 gene. All E. coli C.Is. that had integrons were MDR.
There were 17 isolates which were resistant to all 21 antibiotics tested in this study (pan-resistance) including extended spectrum β-lactams, quinolones, imipenem, amoxicillin clavulanic acid and cefepime. Also another 14 isolates were resistant to all above mentioned drugs except cefepime.
Regarding phenotypic methods for ESβL detection among studied E. coli isolates, by using screening disc diffusion method for ESβL detection, there was high significant difference (p<0.001) between clinical isolates (45 isolates were ESβL positive and 35 were ESβL negative) and commensals (2 isolates were ESβL positive and 18 were ESβL negative) while no significant difference (p>0.05) between them was observed by using DDST for ESβL detection (12 clinical isolates were ESβL positive and 68 clinical isolates were ESβL negative while all commensals were ESβL negative). By using cephalosporin/clavulanate combination disks test, significant difference (p<0.05) was observed between clinical isolates and commensals (25 clinical isolates were ESβL positive and 55 were ESβL negative and among commensals 1 isolate was ESβL positive and 19 isolates were ESβL negative).
155There were different risk factors among patients in relation to positive ESβL E. coli C.Is. detected by cephalosporins/clavulinate combination test with significant difference (P<0.05) between patients regarding the cause and the duration of hospital stay and highly significant difference (P<0.001) regarding the admission to ICU (positive ESβL E. coli C.Is. were presented more in patients admitted to ICU: 76 %), corticosteroids and/or cytotoxic drugs administration and associated co-morbidities, Non significant difference (p>0.05) between patients regarding invasive procedures in having ESβL E. coli C.Is.
In our results, thirty two E. coli C.Is and ten E. coli commensals were studied by PCR for ESβL genes detection where there was significant difference between cephalosporin/clavulanate combination disks test and molecular method in detecting positive ESβL E. coli C.Is. where 24 were positive by two tests and one isolate was positive phenotypically but negative by PCR. Similar results were conducted by both methods for E. coli commensals.
Regarding PCR results, the sensitivity of cephalosporin/clavulanate combination disks test was 89.3 % and its specificty was 92.8 % with 90.4 % accuracy for detection of ESβL producing isolates.
In our study, the total SHV genes were 19 (70.4 %) in E. coli C.Is. and 1 in E. coli commensales. Total TEM were 10 ( 37.0 %) in E. coli C.Is. and total CTX-M were 8 (29.6%) in E. coli C.Is. with significant difference between E. coli C.Is. and commensals results (p< 0.05).
There were 28 E. coli isolates positive for ESβL gene from which twenty seven were E. coli C.Is., with twenty one (77.7 %) had intI 1
gene (10 isolates carried SHV gene , 4 isolates carried TEM gene , 1 isolate carried CTX-M gene, 4 isolates carried SHV, TEM, CTX-M genes and 2 isolates carried SHV, CTX-M genes) and six clinical isolates had not intI1 gene (22.3 %) (3 isolates carried SHV gene, 2 isolates carried TEM gene and 1 isolate carried CTX-M gene). The only E. coli commensal +ve for ESβL gene had SHV type.
Our results reveals that there was fair agreement (K= 0.21) between the presence of class 1 integron and the presence of ESβL in clinical E. coli isolates and slight agreement ( K=0.14) between the presence of class 1 integron gene and the presence of ESβL in commensal E. coli isolates. There was non statistical significant difference (p>0.05) between E. coli C.Is. and E. coli commensals regarding the relation between the presence of class 1 integron and ESβL production.