Expression of mexAB-oprM Operon of Septicemic Pseudomonas aeruginosa in Relation to Antibiotic Resistance

number: 
2959
إنجليزية
Degree: 
Imprint: 
Medicine
Author: 
Ibtesam Ghadban Auda Al-Grawi
Supervisor: 
Dr. Sabah Abdul-Latif Bilal
Dr. Amera khdier Al-Absali
year: 
2011
Abstract:

This study was designed as a try to clarify the role of an important efflux pump, MexAB-OprM, in the resistance of Pseudomonas aeruginosa isolated from cases of septicemia (SPA) to some antibiotics. The study recruited 53 SPA isolates obtained from four sources, 18 of the isolates taken from Dr. Amir Hani, Microbiology department\College of Medicine –Al-Nahrain University, 13 from Mrs. Marwa Al-Kabbi, College of Science- Al-Mustansyria University, 19 from Dr. Mohammed Ali, pharmacology department\ Al-Kindey College of Medicine- Baghdad University and 3 isolates from Al-Kadhymia teaching hospital\Baghdad. The SPA isolates were isolated from cases of leukemia (22), lymphoma (5), cases of urogenital (9), gastrointestinal (5), and breast (3) cancers, or suffering from burn infections (6) and from neonatal patients (3). All of these isolates were collected in a period from February to December 2010. Minimum inhibitory concentration (MIC) of some antibiotics of 53 SPA isolates were determined, antibiotics tested were ciprofloxacin(Cip), gentamicin (NG), doxycycline(Do), and five β-lactams, imipenem(I), cefixime(Fox) ,carbenicillin(Py), cefotaxime(CTX) and ceftazidime(CAZ). The percentage of resistance of each was determined and was as follows: 75.5%, 66%, 98.1%, 0%, 96.2%, 90.6%, 47.2% and 51%, respectively. The numbers and percentages of high and low resistant isolates of each antibiotic were determined, except imipenem; hence all of SPA isolates were susceptible to this antibiotic. There were 6(15%), 21(60%), 37(71%), 13(52%), 21(45.6%), 50(94.3%) and 20(74%) isolates were highly resistant to Cip, GN, Do, CTX, Py, Fox, and CAZ respectively. While there were 34(85%), 14(40%), 15(29%), 12(48%), 27(54.4%), 1(5.7%) and 7(26%) were showed low level resistant to these antibiotics. All of SPA isolates were tested for metallo-β lactamase production. P. aeruginosa producers were 68%, of these 70.6% of Fox resistant and 75% of Py resistant was found MBL producers. There is significant association between Fox and Py resistance and metallo-β-lactamase production (p<0.05 and p<0.001, respectively). No significant association between MBL production and high level Fox and Py resistance was found. Significant association was found between no MBL production and low level of Py resistance (p < 0.05). A second mechanism of β-lactam antibiotic resistance, cefotaximases, were investigated by amplification of common sequence of blaCTX gene, 72% of CTX resistant and 69.2% of CAZ resistant SPA isolates were positive. There is a significant association between occurrence of blaCTX and CTX resistance and CAZ resistance (p < 0.05 each). On the other hand, a significant association between CTX and CAZ susceptibility and absence of blaCTX was found (p < 0.001 each). No significant association between level of CTX and CAZ resistance and occurrence of blaCTX gene Plasmid profile of SPA isolates reveals occurrence of megaplasmids in most isolates, as well as relatively small plasmids. Multiplex PCR was applied on the chromosomal DNA and on eluted plasmids of SPA isolates to amplify two of mexAB-oprM efflux pump operon and regulatory gene mexR. All of SPA isolates were positive to all tested genes (mexA, mexR and mexB) on the chromosomal DNA and were negative on eluted plasmids of SPA isolates. The expression of mexAB-oprM was analyzed by single step reverse transcriptase PCR (SS-RT-PCR), mexB gene expression was used as indicator to the expression of all operon. Only 28.3% of SPA isolates were mexB expressers. The association between mexB gene expression of SPA isolates and antibiotic resistance was investigated. Non mexB expression was significantly associated with Cip susceptible isolates (p<0.05) No significant relationship between Cip, GN, Do, Py and Fox resistance and expression of mexB gene was found. Cefotaxime and CAZ resistance associated with expression of mexB gene (p<0.01 each) Cefotaxime and CAZ susceptibility associated with no mexB expression (p<0.001, each). The relationship between expression of mexB gene and level of antibiotic resistant of SPA isolates was determined. There is a significant association between expression of mexB gene and high level of Cip (p < 0.01) Do (p < 0.01), Py (p <0.001), CTX (p<0.05) and CAZ resistance (p<0.05) was found. No significant association between mexB expression and high Fox and GN level resistance On the other hand, there is a significant association between no mexB gene expression and low level of Cip (p<0.01), Py (p <0.001), CAZ (p<0.05) and CTX resistance (p <0.05). In conclusion, antibiotic resistance of SPA is a complicated events, SPA isolates may express more than one resistance mechanism for each antibiotic, as proved in this study in case of β-lactams resistance. When more than one resistance mechanism occurs in the same isolates high level of resistance will be expected. Expression of predominant MexAB–OprM efflux pump associated with high level of resistance, but not with the resistance itself, hence its expression is low and there is many other resistance mechanisms may express in the same isolates.