Mechanisms of resistance to Carbapenems in Acinetobacter species

Wong, Eng Hwa (2008) Mechanisms of resistance to Carbapenems in Acinetobacter species. PhD thesis, University of Malaya.

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Carbapenem-resistant Acinetobacter spp. have gained increasing significance as opportunistic pathogens in hospitalized patients. Reports of multidrug-resistant isolates have increased considerably over the last decade, partly due to the extensive use of broad-spectrum antibiotics. Carbapenem resistance is often associated with the loss and or decrease in outer membrane proteins and overexpression of multidrug efflux systems. However, carbapenem-hydrolyzing β-lactamases of Ambler Class B (metallo-enzymes) and Ambler Class D (oxacillinases) have also been detected in Acinetobacter spp. In this study, we describe a study on carbapenem resistance mechanisms involved in Acinetobacter spp. isolated from University Malaya Medical Centre (UMMC). A total of 39 carbapenem-resistant clinical isolates of Acinetobacter spp. were obtained from inpatients of UMMC from August 2003 until March 2004. All 39 strains were identified as Acinetobacter baumannii-calcoaceticus complex using the API20NE system and Fluorescent in-situ hybridization (FISH). Growth at 44°C distinguished the 36 A. baumannii strains from the 3 A. calcoaceticus. The antibiotic susceptibility profiles to β-lactams were determined by the minimum inhibitory concentration (MIC) using the agar dilution method as described by the Clinical and Laboratory Standards Institute, CLSI. ATCC strains of Escherichia coli 25922 and Pseudomonas aeruginosa 27853 were used as controls. Out of the 39 imipenem-resistant strains screened by the IMP-EDTA-double disk synergy test and Modified Hodge test, only two strains, S26 and S90 were positive for the presence of metallo-β-lactamases, whereby these enzymes were inhibited by EDTA or 2-mercaptopropanoic acid. Both these strains had similar MIC values for imipenem, and cefotaxime at 32 and 512 μg/ml, respectively. IEF analysis showed that both strains had a band of pI 8.0 which corresponded to that of blaIMP-4, while an additional band of pI 7.0 was present in strain S90. The two strains, S26 and S90, were PCR positive for blaIMP. Amplification and subsequent nucleotide sequencing of the entire coding region of blaIMP confirmed the identity of the blaIMP amplicon to be blaIMP-4. However, none of the 39 isolates harbored blaVIM. Besides that, 37 out of the 39 strains harboured blaOXA-23. Plasmid analysis revealed that the two imipenem-metallo-β-lactamase-producing strains (MBLs), S26 and S90, carried plasmids: 147 kb, 50 kb, 36 kb in both strains with an additional 7 kb plasmid in S26. Southern blot hybridization showed that the blaIMP-4 gene was located on the 50 kb plasmid. Screening for Class 1 integrons revealed two bands of 2.5 kb and 3 kb in 31 out of the 39 carbapenem-resistant strains of Acinetobacter spp. Restriction enzyme analysis with AluI revealed 3 fingerprint profiles. Sequencing of the entire gene cassette representatives of each profile revealed that strains with profile 1 (2.5kb) contained the aacC1 gene, whereas the larger (3kb) integron cassette (profile 2) was identical to that of profile 1 with the exception of an additional copy of the orfX gene. Both the blaIMP-4 producing A. calcoaceticus strains, S26 and S90, had a unique profile 3. Nucleotide sequencing of the inserted gene cassettes revealed identical genes: blaIMP4, qacG, aacA4, and catB3. Although identical cassette arrays were found in integrons of the IMP-4 producing strains, PFGE analyses showed that S26 and S90 had different genotypes. Southern blot hybridization using blaIMP-4 probe showed positive hybridization of the Class 1 integron. Screening of efflux pump genes among the 39 strains showed that 36 strains carried all the 3 genes (adeA, adeB, and adeC), while the remainder 3 strains had one or two of the genes detected. Inactivation of adeB gene has led to decreased MIC levels to meropenem indicating its contribution towards the development of carbapenem resistance. Besides that, all the strains showed loss of a 29-kDa OMP. The polyclonal antibodies produced showed bactericidal effect against the organism tested and it specifically killed the bacteria grown in iron deficit medium, suggesting a potential target for antimicrobial therapy. Overall, in this study, the clinical isolates of A. baumannii and A. calcoaceticus posses several characteristics that make it well equipped to emerge as significant nosocomial pathogen in the hospital settings of UMMC. Majority strains of about 36 that were identified as A. baumannii harboured the blaOXA-23 (Class D enzyme). This enzyme belongs to the three unrelated groups of clavulanic acid-resistant β-lactamases, represented by OXA-23, OXA-24, and OXA-58, those posses as intrinsic carbapenem-hydrolyzing oxacillinases and may play a role in carbapenem resistance. However, blaOXA-23 gene was not detected in the two A. calcoaceticus, S26 and S90 which harboured the blaIMP-4 gene. The blaIMP-4 gene that harboured by these strains was found to be carried on the Class 1 integron which is located in the plasmid and this may assist in development of resistance. Besides the enzymatic based mechanisms, broad-spectrum antimicrobial resistance can also be due to a variety of intrinsic and acquired mechanisms. One such important feature is loss or decreased permeability of the outer cell membrane. In this study, a 29-kDa OMP has been identified to be the major outer membrane protein in A. baumannii / A. calcoaceticus and loss of this porin has contributed to carbapenem resistance. Finally, efflux pumps belonging to the Resistance Nodulation Cell Division (RND) family has also been determined in these strains and was found to be associated with resistance to aminoglycosides, fluoroquinolones, cefotaxime and also to meropenem. These suggest that, multiple mechanisms are involved for carbapenem resistance in Acinetobacter spp. and therefore, understanding carbapenem resistance mechanisms might be crucial for the development of novel therapeutic strategies. However, it will be an important approach in the near future if one attempts to determine possible targets for new agents to control antimicrobial resistance in nosocomial pathogens such as Acinetobacter spp.

Item Type: Thesis (PhD)
Subjects: R Medicine > R Medicine (General)
Divisions: Faculty of Medicine, Health and Life Sciences > School of Medicine
Date Deposited: 23 Jul 2013 06:26
Last Modified: 23 Jul 2013 06:26

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