Hasnur, Nur Nadhra Adila
(2018)
Demonstration of antigenic and specific surface association heat shock protein(s) of acinetobacter baumannii.
Masters thesis, Universiti Sains Malaysia.
Abstract
Acinetobacter baumannii is an important nosocomial pathogen associated with
high mortality. It is a major concern of hospital acquired infection (HAI) worldwide.
Until recently, the resistance of Acinetobacter baumannii has increased steadily against
all first-line antibiotics. Due to this situation, the choice of antimicrobial agents to treat
Acinetobacter baumannii infections is limited. Currently, the gold standard method for
identification of Acinetobacter species is the DNA-DNA hybridization while the two
recommended methods that are widely accepted for identification of Acinetobacter
species are amplified rDNA restriction analysis (ARDRA) and amplified fragment
length polymorphism (ALFP). However, these methods are too laborious for everyday
diagnostic use. Thus, there is a need to develop an early detection of Acinetobacter
baumannii to reduce the time consuming in detecting the infection in patients.
Development of a specific and sensitive diagnostic test requires discovery of
biomarker(s) which does not cross react with other bacteria and specific only to
Acinetobacter baumannii. Heat shock proteins (HSP) are proteins that expressed in
bacteria during stress environment and these proteins have potential as biomarker in
diagnostic field. Thus, the aim of this study is to detect the presence of HSPs and
biomarker(s) in the surface associated proteins (SAPs) of Acinetobacter baumannii. The
SAPs profile from the ATCC 19606 strain and clinical isolate of Acinetobacter
baumannii were demonstrated using Sodium Dodecyl Sulfate-Polyacrylamide Gel
Electrophoresis (SDS-PAGE). This study demonstrated that the expression level ofSAPs of Acinetobacter baumannii varies with increasing temperature. The SAPs
profiles expressed at 37°C was compared with 38.5°C and 41°C to assess the effect of
temperatures on the expression of the SAPs. The protein was subjected to Western blot
using serum from patients infected with Acinetobacter baumannii as well other
bacteremia infections. Result of this study demonstrated various antigenic bands
detected when probed with sera from patients with Acinetobacter baumannii infections
(incubated at 37°C and 41°C) against IgA, IgM and IgG isotypes. All the antigenic
bands were checked for cross reaction using sera from patients infected with
Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella
spp, Staphylococcus aureus, Enterococcus faecalis and methicillin-resistant
Staphylococcus aureus (MRSA). Five specific and antigenic proteins were selected for
further identification by MALDI-ToF analysis. The protein 96.7kDa, 62.0kDa, 37.9kDa,
25.0kDa and 16.0kDa from Acinetobacter baumannii were identified as catalase HPII,
Hsp60 chaperonin, phosphate ABC transporter substrate binding protein, Ycei-like
protein, and Lipopolysaccharide export system protein LptA, respectively. The
increased expression of antigenicity level of these proteins probably is a survival
mechanism of the bacteria at higher temperature in the host body and could be potential
diagnostic biomarkers for diagnostic and vaccine development.
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