Molecular characterization of multidrug resistant pseudomonas aeruginosa / Abdelkodose Mohammed Hussen Abdulla
Pseudomonas aeruginosa is one of the main causes of healthcare-associated infections among hospitalized patients. Healthcare-associated infections predominantly lead to pneumonia, urinary tract infections, as well as skin and soft-tissue infections. This organism is commonly multiresistant and le...
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| Format: | Thesis |
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2012
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| Online Access: | http://studentsrepo.um.edu.my/7127/ http://studentsrepo.um.edu.my/7127/1/MOLCULAR_CHARACTERIZATION_OF_MDR_P._AERUGINOSA.pdf |
| Summary: | Pseudomonas aeruginosa is one of the main causes of healthcare-associated
infections among hospitalized patients. Healthcare-associated infections predominantly lead
to pneumonia, urinary tract infections, as well as skin and soft-tissue infections. This
organism is commonly multiresistant and leads to morbidity and mortality. In this study,
the resistance mechanisms of 88 clinical P. aeruginosa isolates from the Department of
Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur,
Malaysia were evaluated. The antibiotics resistance profiles of these isolates were
determined and this was followed by evaluating the expression levels of AmpC
cephalosporinase, the multidrug efflux pumps, the OprD outer membrane porin and the
penicillin binding protein (PBP2, PBP3). Selected clinical isolates that were resistant to
imipenem and meropenem were evaluated for metallo--lactamase (MBL) and extended
spectrum β-lactamases (ESBL) production. The antimicrobial agents tested in this work
were piperacillin/tazobactam, ceftazidime, aztreonam, amikacin, gentamicin, ciprofloxacin,
imipenem, meropenem and colistin. These agents were selected as representatives of the
primary antibiotic classes used to treat P. aeruginosa infections.
The clinical specimens of P. aeruginosa isolates were isolated from urine (53.4%),
wound (21.6 %), sputum (5.7%), blood (5.7%) and in dwelling medical devices (13.6 %) as
shown in Table 3.1a. Samples were collected from patients hospitalized in the surgical (31),
medical (20), orthopedic (13), paediatric (7), neurosurgery (7), intensive care unit (ICU)
(5), otorhinolaryngology (ENT) (3) and gynaecology wards (2). Of these isolates, 47 were
from urine, 19 from wounds, 12 from in dwelling medical devices, 5 from blood, and 5
from sputum.
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In the isolates tested. The highest resistance was observed for gentamicin (94.0%),
ciprofloxacin and ceftazidime (92%), imipenem (74.0%), meropenem (78%), amikacin
(66.0%), piperacillin/tazobactam (58.0%), aztreonam (56.0%) and colistin (7.0%), with the
surgical department having the highest of numbers of isolates resistant for all antibiotics
except colistin with urine and wound specimens being most resistant to the antibiotics
except for colistin.
The gene expression analysis of 88 P. aeruginosa isolates showed overexpression of efflux
pump genes for MexY (82.0%, from 2.0 to 1731.0 fold), MexB (73.0%, from 2.0 to 50.0
fold), MexEF (68.0%, from 2.9 to 371), MexZ (66.0%) and MexCD (48.0%, from 2.0 to
522), while for AmpC overexpression it was 65.0% (from 10.4 to 1806.0 fold). Down
regulation were noted for OprD (97.0%, from 0.2 to 0.7 fold), PBP2 (77.0%, from 0.1 to
0.7 fold), PBP3 (84.0%, from 0.01 to 0.7 fold) and OprM (65%, from 0.2 to 0.7) as
compared to those of P. aeruginosa ATCC 27853.
Among the resistant isolates overexpressed for the MexB and MexY efflux gene, the lowest
mRNA expression was noted for isolates resistant to colistin, whereas the highest mRNA
expression was noted for isolates resistant to ciprofloxacin and amikacin respectively. For
the overexpression of MexCD and MexEF genes, the lowest mRNA expression was seen
with isolates resistant to piperacillin/tazobactam. However, the highest overexpression for
MexCD was seen with isolates resistant to meropenem and aztreonam while the highest
overexpression for MexEF gene was noted with isolates resistant to colistin. For the
overexpression of AmpC gene, the lowest mRNA expression was seen with isolates
resistant to colistin and the highest mRNA expression was noted with isolates resistant to
piperacillin/tazobactam. All isolates resistant to meropenem, imipenem and colistin
demonstrated lower mRNA expression for OprD gene while as for OprM gene, the lowest
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mRNA expression was observed in isolates resistant to amikacin. For the PBP gene,
isolates resistant to colistin had lower mRNA expression for PBP2, PBP3 while those
resistant to imipenem had higher mRNA expression. With regard to the significance of the
above results, the overexpressions of MexY gene were significantly different in isolates
resistant to amikacin, gentamicin and ciprofloxacin as compared to other antibiotics (p <
0.05), while, for the AmpC gene, mRNA expression in P. aeruginosa isolates demonstrated
high significant differences towards piperacillin/tazobactam antibiotics as compared to
other antibiotics (p < 0.05).
Sixty-five of the clinical P. aeruginosa isolates that were resistant to imipenem and
meropenem were then evaluated for detection of 6 different metallo-beta-lactamase (MBL)
genes (blaIMP, blaVIM, blaGIM, blaSIM, blaSPM and blaNDM). In addition, these isolates were
tested for the extended spectrum beta-lactamase (ESBL) production genes (blaVEB, blaTEM,
blaCTX-M, blaSHV and blaPER) using PCR. Among the 65 imipenem and meropenem resistant,
41 isolates were metallo-β-lactamase (MBL) producers and these genes blaIMP, blaVIM,
blaGIM, blaNDM and blaSIM were detected in 20, 14, 4, 2, and in 1 isolates respectively.
Thirty-three of these 65 isolates tested positive for 3 ESBL genes out of which PCR
positive isolates were present in 25 isolates for the blaVEB gene, 5 isolates for the blaTEM
gene and 3 isolates for the blaCTX-M gene respectively.
Most of the P. aeruginosa clinical isolates had a high level of resistance to
examined antibiotics except colistin. Multidrug resistance phenotype in these clinical
isolates was caused by the interaction of several different resistance mechanisms occurring
within the same strain such as overexpression of efflux, AmpC overproduction or decreased
outer membrane porin OprD, alteration of penicillin binding protein. Additionally, these
strains highlight the ability of P. aeruginosa to develop dual resistance to different classes
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of antimicrobial agents through independent mechanisms of resistance and highlight the
need for the judicious use of therapy when dealing with P. aeruginosa to prevent multidrug
resistance. The clinical P. aeruginosa isolates that were resistant to imipenem and
meropenem demonstrated high efflux overproduction, MBL and ESBL production that
confirm these resistance genes has the ability to increase the resistance to imipenem and
meropenem among P. aeruginosa. |
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