الفهرس 
?-Lactam antibioticsOnly 14 pages are availabe for public view
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Abstract
Extended-spectrum-beta-lactamases have spread widely worldwide among the Enterobacteriaceae, predominantly in Klebsiella spp. and E.coli. Organisms producing ESBL have traditionally been responsible for serious nosocomial infections that are often occur in outbreaks and are usually exhibiting an extremely resistant MDR phenotype with a very limited therapeutic choices and therefore they usually associated with increased morbidity, mortality, and health-care associated costs.
Continuous microbiological and molecular screening for ESBLs is extremely important for implementing appropriate measures for their prevention and control. Although, several studies have pointed to a high prevalence of ESBLs in Egypt but data regarding the detailed molecular types are scarce.
In the current study, we screened for ESBL production among all specimens of nosocomial infections caused by E.coli and klebsiella isolates (n=1508) received at Ain Shams University microbiology laboratory. All isolates of E.coli and klebsiella spp. were subjected to routine culture on blood agar and MacConkey agar media for 24 to 48hrs incubation at 35C. Identification of the isolates was done using Gram stain morphology and conventional biochemical tests. Antimicrobial susceptibility testing and phenotypic detection of ESBL production were carried on Muller-Hinton agar by using the standard disk-diffusion method and interpreted as recommended by CLSI, (2008) guidelines.
We found that the overall prevelance of ESBL among all the isolates of E.coli and Klebsiella spp. during the study period of one year was 42% (638/1508); with a prevelance of 41% among E.coli isolates (225/638) and 43% among Klebsiella spp. isolates (413/638).
A random selection of a total of 118 of phenotypically confirmed ESBL producing E.coli and klebsiella spp., that were obtained from 116 patients, were subjects for assessment of risk factors predisposing for ESBL infections and for further molecular analysis using; PCR, DNA sequence analysis, and PFGE. ESBL Cases were compared to 148 patients with non-ESBL-EK positive bacterial culture.
We found a significant association between infection with ESBL-EK and two hospital wards; the surgical wards and the geriatric ICU. We also found that the majority (47%) of the randomly collected ESBL-EK isolates were obtained from wound cultures.
The independent risk factors for nosocomial infections with ESBL-EK after multivariate analysis was; old age, long hospital stay prior to infection, having mechanical ventilation, diabetes mellitus, and renal disease, prior antibiotics intake; with the most common antibiotics associated with ESBL infections were extended spectrum cephalosporins (especially cefotaxime), ciprofloxacin, and vancomycin.
On the other hands, we noted that a significantly greater number of non-ESBL positive patients had prior -lactam/-lactamase inhibitors (augmentin or unasyn) exposure than ESBL positive patients. Thus, we recommend these agents to be used as a save alternative to the previously mentioned agents during the propohylactic or empirical therapies, as it has shown to be useful for reducing the resistance among E.coli and Klebsiella spp.
The studied ESBL-EK isolates were characterized by MDR phenotypes with the only agent that remained effective against all the tested isolates was the carbapenems (imipenem and meropenem).
PCR analysis revealed that blaTEM was the most prevalent ESBL-type among our ESBL-EK isolates (79%), followed by blaCTX-M (78%), then blaOXA (75%). However, neither of these genes could be detected in 3% (4/118) of the studied isolates. Searching for bla SHV in these four negative samples revealed the presence of SHV in two of them.
We also found that the majority of tested ESBL-EK strains harboured more than one type of ESBL genes, with the most prevalent pattern was the production of a combination of three enzymes (bla TEM/ blaCTX-M/ blaOXA) which is detected in 48% of ESBL-EK, the second pattern was the production of any two of ESBL enzymes (detected in 1%-15% of the isolates). On the other hand, few isolates were expressing only one ESBL gene (detected in 1%-3% of the isolates). Sequencing of the amplified PCR products indicated that all the representative isolates harboured the blaTEM-1, blaCTX-M-15, blaOXA-1, and blaSHV-2a.
In the present study, we also tried to predict the presence of the highly virulent epidemic clone ST131 among our CTX-M-producing E.coli isolates that exhibited ciprofloxacin resistance using a triplex PCR-based phylogenetic typing mechanism. It showed that 44% of tested isolates were derived from the phylogenetic group D, 39% were from group A, 12% were belonging to group B2, and only 5% were from the group B1.
Our results suggesting that those isolates that were belonging to group B2 could be the clone ST131 as they were fulfilling most of the criteria characterizing this clone as they were; CTX-M 15-producing E.coli, showing ciprofloxacin resistance, and belonging to the phylogenetic group B2. Howevere, unfortunately, we did not perform MLST analysis which is required for definitive typing of clone ST131.
Finally, we examined the different patterns of chromosomal DNA from 12 ESBL-K isolates, recovered from two suspected outbreaks, using PFGE after total chromosomal DNA digestion with XbaI. Analyses of the different patterns revealed extensive diversity among the tested isolates, except for two of them obtained six months apart.
The absence of the clonal link among most of these strains suggesting that they did not represent an outbreak strains. However, ESBL-K isolates seem to be endemic in our hospital and their spread was due to the dissemination of plasmids rather than the spread of one single clone. However, we did not analye the plasmids.