Asian Journal of Medical Sciences 4(5): 179-183, 2012
ISSN: 2040-8773
© Maxwell Scientific Organization, 2012
Submitted: August 09, 2012
Accepted: September 17, 2012
Published: October 25, 2012
Identification of Multidrug-Resistant Genes in Acinetobacter baumannii in
Sulaimani City-Kurdistan Regional Government of Iraq
Aras A.K. Shali
Department of Biology, School of Science, Faculty of Science and Educational Sciences,
University of Sulaimani, Kurdistan Regional Government, Iraq
Abstract: Acinetobacter baumannii is an opportunistic pathogen responsible for hospital-acquired infections.
A. baumannii epidemics described world-wide were caused by few genotypic clusters of strains. The occurrence of
epidemics caused by multi-drug resistant strains assigned to novel genotypes have been reported over the last few
years. Multiple drug-resistant strains of Acinetobacter have created therapeutic problems worldwide. This study was
conducted to determine the antimicrobial susceptibility patterns of blaOXA-type carbapenemases among isolates of
Acinetobacter spp obtained from clinical specimens. Twenty one Acinetobacter isolates were identified at the
species level and their susceptibilities to different antibiotics were determined using Vitek® 2 system. Resistant
percentages for all isolates were recorded; highest resistant rate was against ampicillin (100%) while lowest rate was
against imipenem (57.1%). The MICs of imipenem for the resistant isolates were ≥16. All isolates show multi drug
resistance to different antibiotics used. The Isolates were then subjected to multiplex-PCR targeting blaOXA genes.
All strains of A. baumannii possess a blaOXA-51-like gene. The co-existence of blaOXA-51-like and blaOXA-23-like were
detected in 17 strains while in 4 strains blaOXA-51-like was the only presented gene. Detection of blaOXA-51-like can be
used as a simple and reliable method to differentiate A. baumannii strains from other species.
Keywords: BlaOXA-51-like Acinetobacter baumannii, Kurdistan Acinetobacter baumannii, multidrug resistant
A. baumannii
leaving carbapenems as the only effective drug to treat
severe infections (Pournaras et al., 2006). Resistance to
carbapenems in the population of Acinetobacter strains
is high with the majority of isolates showing multidrug
resistance. On the other hand, carbapenem resistance
has been observed on every continent; examples
include the UK, Greece, North America, Asia-Pacific
region and Iran (Pournaras et al., 2006; Turton et al.,
2006a; Morgan et al., 2009; Mendes et al., 2009;
Feizabadi et al., 2008). However, MDR (carbapenem
resistant) Acinetobacter baumannii originating from
injured Canadian military personnel returning from
Afghanistan and Iraq have also been described (Tien
et al., 2007).
In 1993, the first of a novel group of narrowspectrum OXA-type β-lactamases was discovered in an
imipenem-resistant A. baumannii strain from a patient
in the Royal Infirmary of Edinburgh that was found to
possess carbapenem hydrolyzing activity (Paton et al.,
1993). Moreover, blaOXA-58 was found to be the likely
cause of carbapenem resistance while another isolate
had an insertion sequence element upstream of its
intrinsic blaOXA-51 (McCracken et al., 2009).
Polymerase Chain Reaction (PCR) testing of the
synergy-positive isolates for carbapenemase genes was
done by using consensus primers for blaIMP (Senda
et al., 1996), blaVIM (Tsakris et al., 2000), blaSPM
(Toleman et al., 2002), blaOXA-23-like, blaOXA-24-like,
INTRODUCTION
Acinetobacter baumannii (AB) is a Gram-negative
coccobacillus that is ubiquitous in fresh water and soil
and is also found frequently as a skin and throat
commensal in humans (Houang et al., 2001).
Acinetobacter spp has become major pathogens in
hospital-associated infections, especially in critical care
settings such as Intensive Care Units (ICUs) and units
for patients with severe burns (Murray and Hospenthal,
2008). They can survive in the hospital environment for
long periods and have a remarkable propensity to
develop resistance to multiple classes of antimicrobial
agents (Boo et al., 2009). During the past decade,
nosocomial outbreaks of Acinetobacter baumannii have
been described with increasing frequency, occurring
mostly in intensive care units, burn units and surgical
wards (Mandell et al., 2000; Bergogne-Bérézin and
Towner, 1996). Epidemic strains of Acinetobacter
baumannii are often resistant to several antimicrobial
drugs, which reduces treatment effectiveness.
Nosocomial transmission is from patient to patient and
associated with environmental reservoirs (BergogneBérézin and Towner, 1996). In a Canadian study,
Acinetobacter baumannii has been ranked as the 20th
most common organism identified from ICUs (Zhanel
et al., 2008). Antimicrobial resistance is increasingly
being reported in Acinetobacter baumannii, often
179
Asian J. Med. Sci., 4(5): 179-183, 2012
blaOXA-58-like (Poirel et al., 2005) and blaOXA-51-like
(Héritier et al., 2005). In two Greek hospitals, 5
Metallo-Β-Lactamase (MBL) -positive Acinetobacter
baumannii isolates were found. The isolates were
unrelated and carried blaVIM-1 in a class 1 integron;
blaOXA-51- and blaOXA-58-like carbapenemase genes were
also detected (Athanassios et al., 2006). Two classes of
molecular carbapenemase, classes B and D have been
found among strains of Acinetobacter. The enzymes in
class D (OXA enzymes) have emerged as the major
carbapenemases in the world, although metallo
enzymes are mainly prevalent in East Asia (Livermore,
2002). OXA enzymes (encoded by blaOXA genes) can
be sub classified into eight distinct subgroups: OXA23-like, OXA-24-like, OXA-51-like and OXA-58-have
been identified in Acinetobacter spp. Reports from
different countries have shown that blaOXA-51- type
genes are intrinsically harbored by A. baumannii
isolates and they support the presence of a direct
reservoir of β-lactam-resistance genes within the
nosocomial environment (Livermore, 2002; Brown and
Amyes, 2006). Detection of blaOXA-58-like can be used as
a simple and reliable method to differentiate
A. baumannii strains from other species (Feizabadi
et al., 2008; Turton et al., 2006b). Furthermore,
Acinetobacter spp isolates that gave a band for blaOXA51-like identified as A. baumannii (Turton et al., 2006a).
Hence this study was designed to identify
Acinetobacter spp using molecular methods to species
level.
MATERIALS AND METHODS
Microbiological investigations: This study was carried
out at Biotechnology Research Laboratory in the
Department of Biology, School of Science, Faculty of
Science and Educational Sciences-University of
Sulaimani. A total of 21 bacterial isolates were
recovered from clinical specimens (burn and urine) in
Central Health Laboratory and Burn and Plastic
Surgery Hospital/Emergency in Sulaimani city during
the period from February, 2010 to March, 2012.
Presumptive
identification
based
on
culture
characteristics and gram stain. Standard identification,
confirmation and complete methods were conducted
including using the commercial identification systems;
API® 20E (bioMérieux, France) and Vitek® 2 system
(bioMérieux, France). Non Acinetobacter baumannii
isolates were excluded from the study. Confirmatory
identification and speciation was carried out by PCR for
the detection of the blaOXA-51- and blaOXA-58-like
carbapenemase genes, as described by Turton et al.
(2006b) and Feizabadi et al. (2008).
Antimicrobial susceptibility testing and MIC:
Antimicrobial susceptibility test was performed and
MICs were determined using Vitek® 2 susceptibility
test
system
(bioMe´rieux) for the following
antimicrobial
agents:
Ampicillin,
Amoxicillin/
Clavulanic Acid, Pipracillin/Tazobactam, Cefazolin,
Cefepime,
Imipenem,
Amikacin,
Gentamicin,
Ciprofloxacin,
Nitrofurantoin,
Trimethoprime,
Cefoxitin, Ceftazidim and Cefotaxim.
PCR amplification of blaOXA genes: A single bacterial
colony cultured previously on nutrient agar was
suspended in 50 µL ddH2O. The cells then disrupted by
heating for 10 min at 99°C in PCR machine. The
samples were centrifuged at 13000 rpm for 10 min
(Person et al., 2007) and 5 µL of the supernatant was
used as template in the PCR reactions. To amplify the
genes encoding carbapenemases, a multiplex-PCR
assay was run using the primers blaOXA-51-like (353 bp:
5´-TAA TGC TTT GAT CGG CCT TG-3´ and 5´-TGG
ATT GCA CTT CAT CTT GG-3´), blaOXA -23- like (501
bp: 5´-GAT CGG ATT GGA GAA CCA GA-3´ and 5´ATT TCT GAC CGC ATT TCC AT-3´), blaOXA -24- like
(246 bp: 5´-GGT TAG TTG GCC CCC TTA AA-3´
and 5´-AGT TGA GCG AAA AGG GGA TT-3´) and
blaOXA-58-like (599 bp: 5´-AAG TAT TGG GGC TTG
TGC TG-3´ and 5´-CCC CTC TGC GCT CTA CAT
AC-3´) as described by Turton et al. (2006a) and Niel
et al. (2006). Amplification was performed in a final
volume of 20 µL according to instruction manual
(Cinnagen, Iran), containing Master max (4 µL).
Forward and Reverse primer (each 0.6 µL) mixed with
5 µL DNA sample and completed to 20 µL by ddH2O.
The thermocycler run was programmed at 94°C for 5
min followed by 30 cycles of 25s at 94°C, 40s at 53°C,
50s at 72°C and a final cycle of 6 min at 72°C
(Feizabadi et al., 2008).
RESULTS
Results obtained from standard identification
methods show that all isolates were Acinetobacter
baumannii. The result of antimicrobial susceptibility
test show that all strains (n = 21) were resistant
Table1: Percentage of antimicrobial resistance and MICs of A.
baumannii isolates to different antibiotics
Antimicrobial agent
Resistance (%)
MIC range
(µg/mL)
Ampicillin
100
16-32
Amoxicillin/lavulanic acid
85.7
4-32
Pipracillin/tazbactam
90.4
≥128
Cefazolin
100
≥64
Cefeime
90.4
≥64
Imipenem
57.1
≥16
Amikacin
66.6
≥64
Gentamicin
85.7
≥16
Ciprofloxacin
95.2
64-320
Nitrofurantion
95.2
≥512
Trimethopreime
95.2
≥4
Cefoxitin
100
≥64
Ceftazidim
85.7
≥64
Cefotaxim
85.7
≥64
180 Asian J. Med. Sci., 4(5): 179-183, 2012
Fig. 1: Detection of genes encoding OXA carbapenemase in Acinetobacter baumannii by multiple PCR
A-Lane 1: Ladder (1500bp); Lane -ve: (ddH2O); Lane 3-13: Acinetobacter baumannii; B-Lane L: Ladder (1500bp); Lane
11-21: Acinetobacter baumannii; Lane -ve: Negative control- Pseudomonas aeruginosa.
Table 2: Distribution of two blaOXA alleles and determination of MICs for imipenem, piperacillin/tazobactam and cefotaxime among carbapenem
resistance A. baumannii showing resistance to these antimicrobial agents by Vitek® 2 system
Acinetobacter spp
BlaOXA allele
No. of strain
No. of resistant strains
Antibiotic
MIC (μg/mL)
A. baumannii
blaOXA-51/OXA-23
17
10, 15, 14
Imipeneme, Piperacillin/
≥16≥128
tazobactam/cefotaxime
16-≥64
A. baumannii
blaOXA-51
4
2, 3, 3
Imipeneme, Piperacillin/
8≥128
tazobactam/cefotaxime
16-≥64
to ampicillin (MIC≥32), while 57.1% (n = 12) of the
strains showed resistant to imipenem (MIC≥16), these
strains considered as carbapenem resistant A.
baumannii, about 90% (n = 19) strains were resistant to
Pipracillin/Tazobactam (MIC≥128) whereas 85.71%
(n = 18) of the strains showed resistance to cefotaxim
(MIC 16-≥64) (Table 1).
Multiplex PCR results revealed that all strains
possess a blaOXA-51-like gene (amp icon size: 353 bp).
The co-existence of blaOXA-51-like and blaOXA-23-like (amp
icon size: 501 bp) were detected in 17 strains, in which
58.8% of them (n = 10) were resistant to imipenem,
while in 4 strains blaOXA-51-like gene was only present
(Fig. 1 and Table 2). Pseudomonas aeruginosa used as
negative control (Feizabadi et al., 2008).
DISCUSSION
Accurate identification and typing of bacterial
isolates are essential, particularly when determining
strains involved in hospital outbreaks. Inappropriate
infection control measures and inaccurate antibiotic
usage are highly potential factors that might increase
the prevalence and spread of antibiotic resistant
A. Baumannii isolates (Dhabaan et al., 2011). Class D
β-lactamase-mediated resistance to β-lactams has been
increasingly reported during the last decade. Those
enzymes also known as oxacillinases or OXAs are
widely distributed among Gram negatives. Genes
encoding class D β-lactamases are known to be intrinsic
in many Gram-negative rods, including Acinetobacter
baumannii and Pseudomonas aeruginosa, but play a
minor role in natural resistance phenotypes (Niel et al.,
2006). The OXAs are characterized by an important
genetic diversity and a great heterogeneity in terms of
β-lactam hydrolysis spectrum. The acquired OXAs
possess either a narrow spectrum or an expanded
spectrum of hydrolysis, including carbapenems in
several instances. Acquired class D β-lactamase genes
are mostly associated to class 1 integron or to insertion
sequences (Poirel et al., 2010). However, blaOXA-51-like
was sought in clinical strains of Acinetobacter
baumannii in a multiplex PCR, which also detects
blaOXA-23-like. All isolates that gave a band for blaOXA-51like identified as A. baumannii. This gene was detected
in all of 21 strains of A. baumannii but not in the
181 Asian J. Med. Sci., 4(5): 179-183, 2012
negative control (Fig. 1). This result agrees with that of
Turton et al. (2006b). Although it is clear that blaOXA-51like genes are present in the vast majority of isolates of
A. baumannii, there has been some debate as to whether
they are present in all isolates of this species (Brown
and Amyes, 2006). Furthermore, strains that considered
as carbapenem resistant (MIC≥16) possess both blaOXA51-like and blaOXA-23-like genes were highly resistant to
imipenem than the strains show intermediate resistant
to imipenem (n = 2, MIC = 8) that have only blaOXA-51like which describe the lower MIC range (Table 2).
However, intermediate ranges of susceptibility are
considered as resistant (Raffaele et al., 2004).
These results provide evidences that detection of
blaOXA-51-like can be used as a simple and reliable way
for identifying A. baumannii. It has been found that
blaOXA-51-like exists in all isolates of A. baumannii and
those strains that show carbapenem resistance are
almost possess blaOXA-51-like and blaOXA-23-like genes.
ACKNOWLEDGMENT
Special thanks to Mrs. Paywast Jamal Jalal for her
assistance as well as Central Health Laboratory and
Burn and Plastic Surgery Hospital/Emergency in
Sulaimani city for collaboration.
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