الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Due to the severity of the disease, resistance to a number of antibiotics, and the difficulties of treatment, K. pneumoniae has recently captured the interest of researchers all over the world. Increased MDR K. pneumoniae strains in recent years may be due to overuse and uncontrolled use of antimicrobial agents to treat diseases and infections caused by this bacterium.
K. pneumoniae has established several mechanisms for resistance to different antimicrobials. One of the important mechanisms for developing the MDR is efflux pump systems. Efflux pumps are protein-based structures that are capable to extrude the different toxic substances out of cells. The aim of this study was to determine the expression of efflux pump genes in MDR K. pneumoniae isolates obtained from different clinical cases, and their relation to antibiotic susceptibility. A total of 50 K. pneumoniae isolates were obtained from different clinical specimens including; blood, sputum, urine, ETT aspirate, CSF, and BAL from pediatric and adult patients. All samples were selected for subsequent testing based on antimicrobial susceptibility results, 40 MDR and 10 non-MDR isolates (the control group). All these samples were referred to Microbiology department of the Medical Research Institute, Alexandria University over a period of three months from February 2022 to April 2022. Isolation and identification of K. pneumoniae was done by conventional methods, including culturing on blood and MacConkey agar, Gram stain, and biochemical identification. Susceptibility test of K. pneumoniae to different antibiotics was carried out by the Kirby-Bauer disc diffusion method, except for colistin was performed by MIC method. Phenotypic screening of extended spectrum β-lactamase enzyme (ESBL) was done by double-disk synergy test (DDST) for all K. pneumoniae isolates. Phenotypic detection of efflux pumps activity was conducted using ethidium bromide agar-based method (cartwheel-method), Genotypic detection of K. pneumoniae efflux pump encoding genes (acrA, oqxA, kexE, kdeA, and kpnE) was done for all isolates using SYBR green quantitative real-time PCR by using Thermo Scientific Maxima SYBR Green qPCR master Mix (2X)(Thermo Fisher Scientific, Vilnius, Lithuania). Finally, Relative quantification was calculated by Comparative CT method (2 ˗ΔΔCT method). In this study the majority of K. pneumoniae isolates were obtained from blood (32%), urine (30%), sputum (26%), ETT (8%), finally (2%) from CSF and BAL. Most of the isolates were from NICU (40%), and PICU (26%). Our isolates were mostly resistant to β-lactam group of antibiotics, ranged from 48% to 86%, followed by resistance to aminoglycosides group ranged from 38% to 68%, to fluoroquinolones group were from 54% to 60%, to polymyxins group; colistin 18%, and folate pathway inhibitors; trimethoprim-sulfamethoxazole 46%.
Summary, Conclusions, and Recommendations
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Regarding phenotypic ESBL detection by DDST, only (10/40, 25%) of the MDR tested positive. Most of the MDR K. pneumoniae were obtained from sputum and blood 30% each, followed by urine samples (25%). Phenotypic detection of efflux pump revealed that all the 50 (100%) of K. pneumoniae isolates (40 MDR+10Non MDR) produced pink fluorescence under UV light at concentration 1.25-2.5 mg/L of EtBr and indicated the presence of active efflux pump. Genotypically, kexE efflux pump gene was the most upregulated in 70%, 60% of MDR and non-MDR isolates respectively, the median gene expression level of MDR was 2.2(0.9 to 4.4), and 1.1 (1.0 to 2.0) for non-MDR. The acrA efflux pump gene expression was slightly upregulated (nearly at a baseline level of expression) among 47.5% of MDR and 50% of non-MDR groups, withe median expression level value of 1.0 (0.5 to 1.6) and 1.1 (0.8 to 1.5) respectively. The expression level of the other three tested efflux pumps oqxA, kdeA, kpnE, was down regulated in both groups MDR and non-MDR. Among MDR isolates was 95%,87.5%,87.5% with median value 0.1 (0.0 to 0.2), 0.3 (0.2 to 0.5), 0.2 (0.1 to 0.5) respectively. While, among non-MDR was 80% for each, with median value 0.0 (0.0 to 0.8), 0.3 (0.2 to 0.9), 0.3 (0.0 to 0.9) respectively. No statistically significant difference was detected between MDR and non-MDR regarding upregulation versus downregulation.
The gene expression between 5 efflux pump genes among MDR isolates (n= 40) showed a statistically significantly different (p-value ≤0.05), except for kpnE & kdeA (p= 0.4) and kpnE & oqxA (p= 0.06). While, among non-MDR isolates (n= 10) the gene expression between 5 efflux pumps reveled that there was a statistically significantly difference (p-value ≤0.05) for oqxA and kexE (p= 0.037) only. Among the 40 MDR K. pneumoniae isolates there was a statistically significant difference between ESBLs and non-ESBLs producers in fold change of gene expression of acrA, and kdeA (p =0.022, p =0.004) respectively, and no significant difference was found in the other efflux pump genes. Several low positive significant correlations between the following pairs of efflux pumps relative gene expression: acrA & kpnE (p= 0.025), oqxA & kpnE (p= 0.006), kexE & oqxA (p=0.013), kexE & kdeA (p=0.0022), and kpnE & kdeA (p= 0.008) were detected. And a high positive significant correlation between oqxA & kdeA (p<0.000001) was also detected. Correlation of overexpression of efflux pump genes with antimicrobial resistance results of each antibiotic revealed a significant association between acrA expression and high resistance to Amikacin (p= 0.046), and the two carbapenems; Imipenem (p= 0.004) and Meropenem (p= 0.010).
Also, we marked a significant correlation between the expression of kexE, and the high resistance to colistin (p= 0.016), doxycycline (p= 0.048) and cefadroxil (p= 0.042).