Evaluation of active efflux in multi-drug resistant isolates of

Acinetobacter baumanii
A Abdi-ali, P. Nikasa, Z. Falahi, A. Rahmani-badi
Department of Biology, Faculty of Science, Alzahra Universty, Tehran, I.R.Iran

ABSTRACT
65 clinical isolates were obtained throughout a one year period from different clinical samples (urine,
blood, bone marrow and burns) from 3 hospitals. Isolates tested were all A.baumanii. Using Kirbybauer drug susceptibility test for 12 antibiotics, 22 multidrug resistant strains which showed resistance
to more than 10 of these antibiotics were screened for high resistance to ciprofloxacin, gentamicin and
ceftazidime by determining MICs. The MICs of antibiotics were measured by the microdilution method.
Subsequently, to show that the high resistance in these strains depend on proton gradient-dependent
efflux pump, MICs were also measured in presence and absence of carbonyle cyanide m-chlorophenyl
hydrazone (CCCP), using microdilution method. CCCP was added to a concentration of 30 M.
Results of MIC for ciprofloxacin, gentamicin and ceftazidime were more than 16, 128 and 1024 g/ml,
respectively. CCCP decreased the level of MICs at least in 1 dilution. Consequently the MICs in the
presence of CCCP were lower than the MICs in the absence of the reagent.
This study indicates the MICs and accumulation of EtBr in presence and absence of CCCP were
different. These results demonstrate the presence of proton gradient-dependent efflux pumps in
clinical isolates of A. baumannii.

INTRODUCTION
Acinetobacter spp.,particularly A.baumannii, are important opportunistic pathogens that can cause
variety of nosocomial infections, including bacteremia, urinary tract infection, and secondary
meningitis, but their predominant role is as agent of nosocomial pneumonia, particularly ventilatorassociated pneumonia in patients confined to hospital intensive care units (ICUs). Such infections are
often extremely difficult for the clinician to treat because of the widespread resistance of these bacteria
to the major groups of antibiotics[1,2].
A.baumannii is noted for its intrinsic resistance to antibiotics and for its ability to acquire genes
encoding resistance determinants [3]. Intrinsic resistance can be generated by the interplay of
decreased permeability and constitutive expression of active efflux systems and it can affect unrelated
antimicrobial agents [4].
Foremost among the mechanisms of resistance in this pathogen is the production of B-lactamases
and aminoglycoside-modifying enzymes. Additionally, diminished expression of outer membrane
proteins, mutations in topoisomerases and up-regulation of efflux pumps play an important part in
antibiotic resistance [3]. Two resistance-nodulation-cell division (RND) type active efflux systems,
AdeABC and AdeDE, have been described in Acinetobacter [5].
Active efflux gives rise to level of MICs of antimicrobial agents. Therefore, the level of MICs for
ciprofloxacin, gentamicin and ceftazidime decreased in presence of CCCP compared with the level of
MICs in absence of the reagent [6].

MATHERIALS & METHODS

Bacterial strains. Sixty five clinical isolates of Acinetobacter spp. were collected from three hospitals
in Tehran. The identification of A.baumannii was performed by standard biochemical reactions.
Susceptibility testing. Antibiotic susceptibility was tested by disk diffusion on Muller-Hinton agar.
Subsequently susceptibility of A.baumannii strains to ciprofloxacin, gentamicin and ceftazidime was
determined in presence and absence of CCCP, by using microbroth dilution method. CCCP was
added to final conentration of 30M. Results were reported as MIC, the concentration of antibiotic in
presence and absence of CCCP that inhibited visible growth determined by absence of turbidity in the
broth after 18 h of incubation at 37 C. Experiments were performed in triplicate [1,7,8].
Accumulation of ethidium bromide. The kinetics of ethidium bromide accumulation were monitored
by a fluorimetric assay for 22 multidrug resistant strains [1].

Fig.1. Distribution of antibiotic susceptibilities showed by A.baumannii.

Fig.2. The level of MIC in presence and absence of CCCP.

Gentamicin (G), Ceftazidime (ca), Ciprofloxacin (Cf)

REFRENCES:

[1].

RESULTS & DISCUSSION

Antimicrobial resistance was as follows: amikacin (81.5%), ampicillin-sulbactam (33.8%), ceftazidime
(92.3%), ciprofloxacin (79.7%), colistin (1.6%), cotrimoxazole (92.2%), gentamicin (85.9%), imipenem
(42.2%), kanamycin (93.7%), ofloxacin (78.5%), piperacillin (96.9%), and tobramycin (73.8%) [Fig.1].
Results of MIC for ciprofloxacin, gentamicin and ceftazidime were more than 16, 128 and 1024 g/ml,
respectively. CCCP decreased the level of MICs at least in 1 dilution. Consequently the MICs in the
presence of CCCP were lower than the MICs in the absence of the reagent [Fig.2].
In three strains of 22 multidrug resistants the rate of accumulation of EtBr was increased in the presence of
CCCP, whilst the rest accumulated EtBr at a similar rate[Fig.3].
This study indicates that A.baumannii exhibits high resistance to a wide range of antimicrobial agents and
the MICs of these antibiotics in presence and absence of CCCP, a compound that disrupts the proton
gradient-dependent pumps, were different.
Accumulation of EtBr confirms that contrary to the decrease of MICs in the presence of CCCP, only three
multidrug resistant strains show active efflux mechanism.

Fig.3. Ethidium bromide accumulation in A.baumannii.

At 420 s CCCP was added to the bacterial suspensions
at a final concentration of 100 M. Active efflux (AE).

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