Research J. Pharm. and Tech. 10(12): December 2017
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ISSN 0974-3618 (Print) www.rjptonline.org
0974-360X (Online)
RESEARCH ARTICLE
Detection of Efflux Pumps Genes in Clinical Isolates of Acinetobacter baumannii
Raad Abdulabass AL-Harmoosh1*, Eman M. Jarallah2, Anmar M. AL-Shamari3,
Hussein M. AL-Khafaji4
1University of Kufa, Faculty of Science, Najaf, IRAQ
2University of Babylon, Faculty of Science, Babylon, IRAQ
3Ministry of Health and Environment, Babylon Health and Environment Office, Babylon, IRAQ.
4Ministry of Health and Environment, Babylon Health Office, Babylon, IRAQ.
*Corresponding Author E-mail: raada.almusawi@uokufa.edu.iq
ABSTRACT:
One of the most interesting features of Acinetobacter baumannii is the ease by which it can acquire resistance to various antibiotics. Mutations, new gene acquisitions, up-regulating expression of existing genes (should those be genes of drug-destroying enzymes or efflux pumps), or losing them, like in the case of porins, all may contribute to the broad spectrum resistance of these strains.This study was directed to decide the event of efflux pumpsgenes harboring A. baumannii isolates recovered from Babylon hospitals. Isolates were recognized by biochemical tests and more affirmed utilizing API 20E system. Antibioticssusceptibility was measured by utilizing disks diffusion test. Ten (0.76%) A. baumannii isolates were recovered from clinical specimens. One (10%) isolate was observed to be imipenem and meropenem resistant (MIC > 512 ?g/ml). There are indicative appearance of efflux pump genes ; (50%) for Ade-A and Ade-B each other , (40%) for Ade-C, (80%) for all of Ade-R and Ade-S , (100%) for all of Ade-I and Ade-J , and (50%) for Ade-Y. All isolates of A. baumannii appear as MDR , while only one appear to be as PDR.
KEYWORDS: Efflux pump, Ade-ABC, Acinetobacter, Carbapenem, Ade-RS, Ade-IJ).
INTRODUCTION:
Resistance of Acinetobacter baumannii can affect practically any drugs used in clinical practice. As a result of the rapid acquisition of resistance genes to different and multiple classes of antibiotics, several drugs have already been eliminated from treatment options for A. baumannii infections such as penicillins, cephalosporins, aminoglycosides, quinolones and tetracyclines1.
Efflux systems pump several compounds, which including antibiotics, out of the bacterial cell and can play a major role in bacterial resistance to different classes of antibiotics2.
Received on 08.06.2017 Modified on 21.08.2017
Accepted on 11.09.2017 © RJPT All right reserved
Research J. Pharm. and Tech 2017; 10(12): 4231-4236.
DOI: 10.5958/0974-360X.2017.00775.2
pumps have a relatively broad spectrum of compound-specificity hence they may lower the intracellular concentration of various classes of drugs. This may lead, in case of some drugs, to significant resistance on its own; while in case of others it may contribute to significant resistance if other mechanisms (e.g. ?-lactamases) are already functional. The latter situation is commonly seen among carbapenem resistant non-fermenters. An up-regulated efflux is often combined with overproduction of enzymes (e.g. cephalosporinases, AmpC beta lactamases) which, in synergism with the increased efflux, add up to resistance3. The efflux pump systems are grouped into different families. The resistance nodulation- division (RND) family is the best studied among A. baumannii especially the Ade-ABC group 4. This group has been found to pump a wide range of antibiotics out of the cells including the ?-lactams. Another two groups in the RND family were described in A. baumannii named Ade-IJK and Ade-
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FGH groups. The first group has the ability to expel Carbapenems, while the second group was found to play a role in chloramphenicol, clindamycin and fluoroquinolone resistance 5,6,7. Taken together, although the role of limited uptake and increased efflux ?-lactam resistance of A. baumannii is undeniable, the major modes by which this bacterium counteracts these drugs are the production of a broad array of enzymes, i.e., various ?-lactamases3.
MATERIALS AND METHODS
Isolation and Identification of A. baumannii :
A total of 1300 clinical specimens (included 588 burn swabs, 136 wound swabs, 50 from throat, 204 urine, 110 stool, 20 sputum, 162 blood, 15 ears and 15 eyes) were gathered from patients in Babylon Province hospitals more than one year time span beginning from March, 2014 to March, 2015. Isolates were recovered from clinical specimens after culturing onMacConkey agar and incubated for overnight at 37°C, lactose non fermenting bacteria were sub-cultured and incubated for extra overnight. Suspected bacterial isolates which their cells are Gram negative coccobacillary or diplobacillus and negative to oxidase which further distinguished utilizing API20 E system.
Antimicrobial Susceptibility Testing:
Isolates were cultured on Mueller-Hinton agar and their susceptibilities to various antibiotic agents were tested by disk diffusion method as indicated by the Clinical and Laboratory Standard Institute s rules 8.
MIC Determination:
Depending upon producer s guidelines the antibiotic stripes (E-test) were applied to the agar surface , the antibiotics promptly diffuses into the encompassing medium in high to low concentration from one end of the strip to the next. The gradient stays stable after dispersion , and the zone of inhibition made takes the form of oval (Liofilchem manufacture). Furthermore microbroth dilution method was done.
Genotypic Recognition of Efflux Pumps genes:
DNA was extricated from the isolates by utilizing genomic extraction mini kit according to the manufacture instructions (Bioneer Company, Korea). Primers used in this study were chosen depending different references as shown in table (1).
Amplification was performed in a 20 ?l volume as recommended by PromegaMaster mix instruction. PCR amplifications were carried out on a thermal cycler (Prime, England). The cycling conditions for amplification were showed in table (2). Amplified products were distinguished by agarose gel electrophoresis in 1% Tris-borate-EDTA (TBE) agarose (Promega, USA) and staining with ethidium bromide. The electrophoresis result was identified by utilizing gel documentation system (Claver, England).
Table (1): Primers used in this study
Gene
Primer Sequence
Product size (bp)
References
Ade-A
F
5?-CTC TAG CCG ATG TCG CTC AA-3
510
9
R
5?-ATA CCT GAG GCT CGC CAC TG-3
Ade-B
F
5?-ACC GCG CTA ACT TAG GTA TGC-3
931
R
5?-TCT GAA CTT GCA CCT GAG GCA-3
Ade-C
F
5?-TGC GGC AGG TTA GCC CAT CG-3
435
R
5?-GCG GAA CAG GAT GAC CTG CT-3
Ade-R
F
5?-GAC TAC GAT ATT GGC GAC AT-3
560
R
5?-ACG GTT CGC TCT AGT GCA TC-3
Ade-S
F
5?-GAT TGG CAT GCG CCT CGC AA-3
580
R
5?-GCC TGC ATG TGA ATA GCG TA-3
Ade-I
F
5?-ATC GCG CTT GTT GGT TGT AG -3
541
10
R
5?-AAG CAC CAG CCG TTA CTG AA-3
Ade-J
F
5?-ATT GCA CCA CCA ACC GTA AC -3
453
R
5?-TAG CTG GAT CAA GCC AGA TA -3
Ade-Y
F
5?- CAA TCT GCA ACT GCG CTT -3
587
R
5?- TCA ACA GCT TCT GCG GTA -3
Table (2): PCR Thermo-cycling conditions
Gene
Temperature (°C) / Time
Number of cycles
Initial
denaturation
Cycling condition
Finalextension
Denaturation
Annealing
Extension
Ade-A
95/2min
95/30 sec
56/1min
72/1min
72/5min
30
Ade-B
95/2min
95/30 sec
59/30sec
72/100sec
72/5min
35
Ade-C
95/2min
95/30 sec
56/1min
72/50sec
72/5min
25
Ade-R
94/3min
94/30 sec
56/30sec
72/1min
72/3min
25
Ade-S
94/3min
94/30 sec
56/1min
72/1min
72/3min
25
Ade-I
95/2min
95/30 sec
56/1min
72/1min
72/5min
25
Ade-J
95/2min
95/30 sec
56/30sec
72/50sec
72/5min
30
Ade-Y
95/2min
95/30 sec
54/30sec
72/1min
72/5min
30
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RESULTS
Isolation and Identification of A. baumannii :
Depending on the biochemical tests and API20E system it has been able to isolate and identify of 10 (0.76%) isolates as A. baumannii from the 1300 clinical samples.
Antimicrobial susceptibility testing :
As determined by disk-diffusion method, every one of the A. baumannii isolates showed distinctive pattern of resistance to various antibiotic agents (Fig.1), exhibiting highest resistance to penicillins (carbenicillin and ampicillin) with resistance rate of (100%), while 3(30%) of resistance were resistant to piperacillin.
High resistance rates were watched for each of amoxicillin/clavulanic acid and aztreonam (80%), (70%) for cefepime, (60%) for each of ceftazidime and cefotaxime. The results likewise revealed that were high resistance rates for each of tobramycine, and gentamicin (70%) and moderate to amikacin (50 %). The isolates demonstrated low resistance rates for the carbapenem antibiotic agents, imipenem , meropenem and ertapenem (10%). The percentage of resistance rate of the remaining antibiotic agents were as the following: (80%) for chloramphenicol, followed by colistin sulfate with (70%), polymyxin B (50%), trimethoprim-sulfamethoxazole (50%), (40%) for quinolones, (ciprofloxacin) and (20%) to each of tetracycline and doxycycline. Results revealed that all tested isolates were resistant at least of three classes of antibiotics, so that these isolates were considered to be multidrug resistant.
Figure (1) : Antibiotics susceptibility profile of A. baumannii isolates by disk diffusion method (n=10)
(IMP,Imipenem ; MEM,Meropenem ; ETP,Ertapenem ; FEP,Cefepime ; CAZ,Ceftazidime ; CTX,Cefotaxime ; AM,Ampicillin ; PY,Carbenicillin ; PRL,Piperacillin ; AMC,Amoxi-clav; ATM,Aztreonam ; AK,Amikacin ; TOB,Tobramycine ; CN,Gantamicin ; TE,Tetracycline ; DO,Doxycycline ; CIP,Ciprofloxacin ; SXT,Trimethoprim-Sulfamethoxazole ; C,Chloramphenicol ; PB,Polymyxin B ; CT,Colistinsulphate).
MIC determination:
The results revealed that A. baumannii isolates were resistant to imipenem, meropenem, and ertapenem with concentrations beyond values: 0.032 ?g/ml - >512 ?g/ml.
Genotypic detection of efflux pumps genes :
The efflux pumps genes distribution have different pattern among A. baumannii isolates as shown in table(3) .
Table (3): Distribution of efflux pumps genes among A. baumannii isolates.
Genes
Isolates No.
Total (%)
1
2
3
4
5
6
7
8
9
10
Ade-A
-
+
-
+
-
+
+
-
+
-
5 (50%)
Ade-B
-
+
-
+
+
+
+
-
-
-
5 (50%)
Ade-C
-
+
-
+
+
+
-
-
-
-
4 (40%)
Ade-R
+
+
-
+
+
+
+
-
+
+
8 (80%)
Ade-S
+
+
-
+
+
+
+
-
+
+
8 (80%)
Ade-I
+
+
+
+
+
+
+
+
+
+
10 (100%)
Ade-J
+
+
+
+
+
+
+
+
+
+
10 (100%)
Ade-Y
-
+
-
+
+
+
+
-
-
-
5 (50%)
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Figure (2):Agarose gel electrophoresis (1.5% agarose,70 volt for 1-2 hrs) for Ade-A gene product (amplified size 510 bp). Lane (M), DNA molecular size marker (100- bp Ladder). Lanes (2 , 4, 6, 7 and 9) show positive results . Lanes (1, 3, 5,8 and 10) show negative results.
Figure (3):Agarose gel electrophoresis (1.5% agarose,70 volt for 1-2 hrs) for Ade-B gene product (amplified size 931 bp). Lane (M), DNA molecular size marker (100- bp Ladder). Lanes (2 , 4, 5, 6 and 7) show positive results. Lanes (1, 3, 8, 9 and 10) show negative results.
Figure (4):Agarose gel electrophoresis (1.5% agarose,70 volt for 1-2 hrs) for Ade-C gene product (amplified size 435 bp). Lane (M), DNA molecular size marker (100- bp Ladder). Lanes (2 , 4 , 5 and 6) show positive results. lanes (1, 3, 7, 8, 9 and 10) show negative results.
Figure (5):Agarose gel electrophoresis (1.5% agarose,70 volt for 1-2 hrs) for Ade-R gene product (amplified size 560 bp). Lane (M), DNA molecular size marker (100- bp Ladder). Lanes (1 , 2 , 4, 5, 6, 7 , 9 and 10) show positive results. Lanes (3 and 8) shows negative result.
Figure (6):Agarose gel electrophoresis (1.5% agarose,70 volt for 1-2 hrs) for Ade-S gene product (amplified size 580 bp). Lane (M), DNA molecular size marker (100- bp Ladder). Lanes (1 , 2 , 4, 5, 6, 7 , 9 and 10) show positive results. Lanes (3 and 8) shows negative result.
Figure (7):Agarose gel electrophoresis (1.5% agarose,70 volt for 1-2 hrs) for Ade-I gene product (amplified size 541 bp). Lane (M), DNA molecular size marker (100- bp Ladder). All lanes (from 1 to 10) show positive results.
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Figure (8):Agarose gel electrophoresis (1.5% agarose,70 volt for 1-2 hrs) for Ade-J gene product (amplified size 453 bp). Lane (M), DNA molecular size marker (100- bp Ladder). All lanes (from 1 to 10) show positive results.
Figure (9):Agarose gel electrophoresis (1.5% agarose,70 volt for 1-2 hrs) for Ade-Y gene product (amplified size 587 bp). Lane (M), DNA molecular size marker (100- bp Ladder). Lanes (2 , 4 , 5 , 6 and 7) show positive results. Lanes (1, 3, 8, 9 and 10) show negative results.
DISCUSSION:
The isolation rate of Acinetobacter baumannii appeared with low percentage (0.76%) accordingly to the biochemical tests and API20E.This result agreed with several local study11.
High resistance rates were observed for most of the antimicrobials agents studied, including, penicillin, amoxicillin/ clavulanic acid, aztreonam, and chloramphenicol. Antimicrobial resistance considerably restricts the available treatment options, especially resistance to carbapenem, which is considered to be the first option to treat severe infections due to Acinetobacter spp.12. El-Astal, mentioned that inappropriate and incorrect administration of antimicrobial agents and lack of appropriate infection control strategies may be the possible reasons behind increasing resistant rate of A. baumannii to common used antimicrobial drugs 13.
Results from table (3) revealed different distribution of these efflux pumps genes. In the present study Ade-A and Ade-B appeared in 5(50%) of A. baumannii isolates (Fig. 2,3). These results had an agreement with Beheshti, et al., (2014)14 in tetracycline-resistant A. baumannii. Lina, et al., (2008) documented that the majority of A. baumannii isolates (75%) have a positive amplification of Ade-ABC cluster. Kor, and his colleagues 10also who recorded a closely similar result with (67.4%) have positive result in Ade-A genes . Ade-R and Ade-S as a regulatory genes was detected in 8(80%) of tested A. baumannii in the present study (Figures 5,6) . These results appeared more closely with Lina, et al., (2008)15 who documented that the majority of A. baumannii isolates (75%) have a positive amplification of Ade-RS.
However, the present study showed Ade-I and Ade-J genes in all isolates (Fig.7,8). In a parallel study by Kor10 that showed Ade-I and Ade-J genes have been appeared in (67.4%) of isolates. In another study Yoon, et al., (2013)16 who documented that all of examined isolates have Ade-I and Ade-J genes.
Whereas, Ade-Y results in present study showed a 5(50%) positive amplification (Fig.9) which are disagreement with Kor, et al., (2014)10 who hadn t detected Ade-Y genes in all A. baumannii isolates.
Results showed some isolates have no efflux pumps gene but it appeared with high antibiotics resistance! the reason for this situation may be due to presence of enzymatic mechanisms of resistance 17,18.
CONCLUSION:
Our study has demonstrated low spreading rate of multidrug resistant and efflux pumps genesharbored A. baumannii isolates among patients with various infections. Sadly, numerous antibiotics endorsed to individuals are superfluous. As well as the overuse and misuse of antibiotics helps to produce drug-resistant bacteria.So that efflux pumps have critical role in resistance mechanism.
ACKNOWLEDGEMENTS :
The authors are exceptionally thankful to department of biology in faculty of science. The authors thank the unknown reviewers for their significant remarks and accommodating correction recommendations.
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