الفهرس 
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Abstract
Carbapenems are widely used in the treatment of ESBL producing K.pneumoniae. However, as carbapenems are more frequently utilized, an increasing number of CR-KP has been observed worldwide. CR in K.pneumoniae has been attributed not only to enzymatic degradation but also to permeability barriers due to outer membrane proteins disruption.Two major porins, namely OmpK35 and OmpK36 have been described in K.pneumoniae. The disruption of these porins plays a role in CR acting as a permeability barrier, as a result, carbapenems reach low concentrations in the periplasmic space and their activity may be then compromised by enzymes with carbapenemase activity, leading to CR.The aim of this study was to phenotypically detect CR among ESBL producing K. pneumoniae, followed by the evaluation of the role of ompK35 and ompK36 gene expression among CR-KP isolates.One hundred ESBL producing K. pneumoniae isolates were included in this study. The MIC of imipenem was performed for all isolates by broth microdilution method. For CR-KP isolates, the phenotypic detection of KPC, MBL and AmpC enzymes was performed followed by Realtime qRT-PCR to detect and quantify ompK35 and ompK36 gene expression.Statistical analysis of the data was carried out and the results were discussed.CR was detected in 42% of the 100 ESBL producing K. pneumoniae isolates where 12 isolates (12%) exhibited LLR (≥4-32 µg/ml) and 30 isolates (30%) exhibited HLR (>32-512 µg/ml).
Out of the 42 CR-KP strains, 20 (47.62%) were isolated from ICUs, 12 (28.57%) from surgical wards and 10 isolates (23.81%) from internal medicine wards. The highest isolation was observed among sputum and BAL specimens.By the phenotypic detection methods, all 42 isolates were KPC producers whether singly in 9/42 isolates or in combination with other enzymes: [KPC/AmpC] and [KPC/AmpC/MBL] in 13/42 isolates for each, and [KPC/MBL] in 7/42 isolates.
The analysis of expression levels of ompK35 and ompK36 genes by real time qRT-PCR showed that among the 42 isolates, only 5 isolates (11.91%) showed normal expression of both porins (OmpK35 and OmpK36), one isolate (2.38%) showed reduced expression of ompK36 only, 14 (33.33%) showed reduced expression of ompK35 only while reduced expression of both ompK35 and ompK36 occurred in a significant number of isolates (22/42, 52.38%).
When comparing the imipenem MIC results with the expression levels of porins, among the 30 HLR isolates, 20 isolates exhibited reduced expression of both ompK35 and ompK36 and 9 isolates exhibited reduced expression of ompK35 only and this was statistically significant (p<0.05)Meanwhile, among the 12 LLR isolates, 5 isolates showed normal expression of both porins, 5 isolates showed reduced expression of ompK35 alone while 2 isolates only showed reduced expression of both ompK35 and ompK36.To study the contribution of reduced expression of these porins to the increase of imipenem MIC values, the correlation between ompK35/ompK36 genes expression and MIC values was analyzed.
Our data demonstrated a significant correlation between reduced expression of ompK36 and increased MIC values, while this correlation could not be established between reduced expression of ompK35 and high MIC values.This study also demonstrated that the combined effect of MBL production together with reduced expression of ompK35 and ompK36 resulted in significant increase in imipenem MIC. Furthermore, the combined effect of AmpC production together with reduced expression of ompK35 also resulted in significant increase in imipenem MIC.Therefore, the CR among our isolates was attributed to the combined effect of enzymes production together with downregulation of ompK35 and/or ompK36.