First Detection Of The blaOXA-23 Genes In A Clinical Isolates Of
Acinetobacter baumannii IN Hillah Hospitals-IRAQ

Raad Abdulabass AL-Harmoosh* And Eman M. Jarallah**
* University of Kufa / College of Science, Najaf, IRAQ.
** University of Babylon / college of Science, Babylon, IRAQ
Abstract: Carbapenemase producing Acinetobacter baumannii is frequently associated with
nosocomial infections. Increasing resistance to carbapenems, may significantly reduce the choice of
effective antibiotics. This study was conducted to determine the occurrence of carbapenmase
producing A. baumannii isolates obtained from Hillah hospitals. Isolates were identified according
to biochemical tests and more confirmed using API 20E system. Carbapenem susceptibility was
assayed by using disks diffusion test. Phenotypic detection of carbapenemase was performed
using the imipenem-EDTA disk and modified Hodg tests. Then isolates were subjected to monoplex PCR
targeting blaOXA-23 genes. 10 (0.76%) A. baumannii isolates were recovered from clinical samples.
One (10%) of isolate was found to be imipenem and meropenem resistant (MIC > 512 ??gg//mmll))..
66((6600%%)) gavgavee posposiittiivvee rreessuulltt wwiitthh tthehe iimmiipepenneemm--EEDDTTAA
ddiisskk aandnd mmododiiffiieedd HHodgodg tteesstt.. PPCCRR experiments showed only four (40%)
isolates were harbored blaOXA-23genes. The present findings suggest that emergence of blaOXA-23
carbapenemase producing A. baumannii clinical isolates in Hillah hospitals. Keywords: blaOXA-23,
Acinetobacter, Oxacillinase, Carbapenem, MDR















I. Introduction
Acinetobacter baumannii have been implicated in recent years as important nosocomial pathogen
especially in intensive care unit (8). A. baumannii is responsible for 3-5% of nosocomial
pneumonia, and one of the most common presentations in mechanically ventilated patients in
intensive care units (14). Carbapenems are the drugs of choice for this pathogen and
carbapenem-resistant A. baumannii has been frequently hospital encountered (13). Meropenem and
imipenem are carbapenems that remain active against organisms carrying most Ambler classes of
?-lactamases which include many Gram-negative bacilli, including Acinetobacter spp. However,
carbapenem resistance is increasingly encountered in Acinetobacter isolates worldwide (1). One of
the major mechanisms of carbapenem resistance in this pathogen is the production of carbapenem
hydrolyzing ?-lactamases. These specific groups of ?-lactamases are categorized into class B
metallo ?-lactamases (MBLs) including IMP and VIM, NDM and class D (Oxacillinases) including
OXA-23-like, OXA-24/40-like and OXA- 58 (4).





























In A. baumannii, the OXA-type class D ?-lactamases are arranged into five families: the
OXA-23-like, OXA-40-like, OXA-51-like, OXA-58-like and OXA-143-like groups, respectively. It is
noteworthy that the genes encoding for the OXA-51 like enzymes are intrinsic to the species, while
for other families the genes have been acquired from other sources (35).
The first oxacillinase gene was identified in A. baumannii on a plasmid, named ARI-1 but after its
detailed characterization it was renamed OXA-23 (32).
Epidemics with A. baumannii strains harboring the blaOXA23 have been reported worldwide (9 ,17, 20,
45). In a recent survey, it was found that the isolates carrying the blaOXA23 are found globally
and
consistent with the clonal spread (21).
In Iraq, little attention has been paid to the carbapenmase producing isolates. However, in the
Hillah city, no information are regarding the molecular studies of the occurrence of OXA-23-like A.
baumannii producing clinical isolates. Hence, the proposed aim of this study is to identify and
detect OXA-23-like producing A. baumannii isolates recovered from hospital settings in the Hillah
city.

II. Material And Methods
Isolation and Identification of Isolates:
A total of 1300 clinical specimens (included 588 burn swabs, 136 wound swabs, 50 from throat, 204
urine, 110 stool, 20 sputum, 162 blood, 15 ears and 15 eyes) were collected from patients in Hillah
hospitals over one year period starting from March, 2014 to March, 2015. Isolates were recovered
from clinical samples after culturing on MacConkey agar and incubated for overnight at 37°C, non
lactose fermenting bacteria were sub-cultured and incubated for additional overnights. Suspected
bacterial isolates which their cells are Gram negative coccobacillary or diplobacillus and negative
to oxidase which further identified using API20 E system.


First Detection Of The Blaoxa-23 Genes In A Clinical Isolates Of Acinetobacter baumannii in ….



Antimicrobial susceptibility testing:
Isolates were cultured on Mueller-Hinton agar and their susceptibilities to different antibiotics
were
tested by disk diffusion method according to the Clinical and Laboratory Standard Institute’s
guidelines (12).

MIC determination:
Depending on manufacturer s instructions the antibiotic stripes were applied to the agar surface ,
the
antibiotic instantaneously diffuses into the surrounding medium in high to low concentration from
one end of the strip to the other. The gradient remains stable after diffusion , and the zone of
inhibition created takes the form of ellipse (Liofilchem manufacture).

Imipenem-EDTA double disks method:
Screening for metallo ?-lactamases (MBL) was performed using disks containing 1900 ?g of EDTA
plus 10 ?g of imipenem disk were placed on the inoculated plates containing Muller Hinton agar. An
increase of
? 17 mm in zone diameter in the presence 1900 ?g of EDTA compared to imipenem alone indicated the
presence of an MBL (25)

Modified Hodg test:
Imipenem was used for carbapenemase detection as described by Lee and his colleagues (24). Positive
test has a clover leaf-like indentation of E. coli Top-10 growing along the test organism growth
streak within the imipenem disk diffusion zone.

PCR amplification:
DNA was extracted from the isolates by using genomic extraction mini kit according to
the
manufacture instructions (Bioneer company, Korea) . To amplify the genes encoding carbapenemases, a
monoplex-PCR was run using the primers of blaOXA-23 (501bp: F/ 5-GAT CGG ATT GGA GAA CCA GA- 3 and
R/ 5-ATT TCT GAC CGC ATT TCC AT-3 ) were described by Turton and his colleagues (43).
Amplification was performed in a 20 ?l volume as recommended by Promega Master mix instruction.
PCR amplifications were carried out on a thermal cycler (Prime, England). The cycling conditions
for amplification were as follows: initial denaturation of 94°C for 3 min and 30 cycles of 1 min at
94° C, 1 min at 57° C, and 1 min at 72°C, followed by 3 min at 72°C. Amplified products were
detected by agarose gel electrophoresis in 1% Tris-borate-EDTA (TBE) agarose (Promega, USA) and
staining with ethidium bromide. The electrophoresis result was detected by using gel documentation
system (Claver, England).

III. Results
Based on the conventional biochemical tests and API20E system enabled to isolation and
identification of 10 (0.76%) isolates as A. baumannii from the 1300 clinical samples (Table 1). As
determined by disk-
diffusion method, all A. baumannii isolates exhibited different pattern of resistance to different
antibiotic agents (Fig.1), demonstrating highest resistance to penicillins (carbenicillin and
ampicillin) with rate of resistance of 10(100%) isolates, whereas 3(30%) of isolates were
resistance to piperacillin.
Resistance to other drug classes varied among the isolates. For cephalosporin antibiotics, a higher
resistance was also detected with 6(60%) of isolates being resistant to ceftazidime and cefotaxime
each other , and 7(70%) to cefepime. The results also revealed that were high resistant rates
8(80%) isolates for each amoxi- clav and aztreonam of monobactam s antibiotics.
For the carbapenem antibiotics, imipenem , meropenem and ertapenem displayed the lowest resistant
rate 1(10%) isolates. Aminoglycosides resistance was variable , 7(70%) to each of tobramycine,
andgentamicin and 5 (50 %) to amikacin.
The resistance to quinolones, (ciprofloxacin) was detected as 4(40%) isolates.
Percentages of
resistance of isolates to the remaining antibiotics were as follows : 2(20%) to each of
tetracycline and doxycycline , trimethoprim-sulfamethoxazole 5(50%), chloramphenicol 8(80%) ,
Polymyxin B 5(50%) and Colstin sulphate 7(70%) . Results revealed that all tested isolates were
resistant to at minimum of 3 classes of antibiotics, hence these isolates were considered to be
multidrug resistant.








































































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First Detection Of The Blaoxa-23 Genes In A Clinical Isolates Of Acinetobacter baumannii in ….



Table (1): Distribution of bacterial isolates recovered from clinical samples among different
hospitals in Hillah city.







Hospital s name No. of samples






No. (%) of Acinetobacter baumannii isolates






No. (%) of other bacterial spp. isolates






No. (%) of no growth cultures










Al- Hillah Teaching Hospital 885 7 (0.8%)
710 (80%) 168 (18%)












Babylon Teaching Hospital for
Maternity and Pediatric 415 3 (0.7%)
235 (56.6%) 177 (42.6%)















Total 1300 10 (0.76%)
945 (72.69%) 345 (26.5%)






















100
90
80
70
60
50
40
30
20
10
0



















Type of Antibiotic











IMMP EMETPFEPCAZCTXAM PY PRLAMACTMAKTOBCN TE DOCIPSXT C PB CT


































Sensitive 90 90 90 10 0 0 0 0 60 20 20 30 10 20 70 80 40 30 20 30 20
Intermediate 0 0 0 20 40 40 0 0 10 0 0 20 20 10 10 0 20 20 0 40 10
Resistant 10 10 10 70 60 60 10 10 30 80 80 50 70 70 20 20 40 50 80 30 70







































Figure (1) : Antibiotics susceptibility profile of A. baumannii isolates by disk diffusion method
(n=10) (IMP,Imipenem ; MEM,Meropenem ; ETP,Ertapenem ; FEP,Cefepime ; CAZ,Ceftazidime ;
CTX,Cefotaxime ; AM,Ampicillin ; PY,Carbenicillin ; PRL,Piperacillin ; AMC,Amoxi-clav;
ATM,Aztreonam ; AK,Amikacin ; TOB,Tobramycine ; CN,Gantamicin ; TE,Tetracycline ;
DO,Doxycycline ; CIP,Ciprofloxacin ; SXT,Trimethoprim-Sulfamethoxazole ; C,Chloramphenicol
; PB,Polymyxin B ; CT,Colstin sulphate).

MIC determination:
Minimum inhibitory concentrations of A. baumannii isolates against carbapeneme antibiotics
(imipenem, meropenem, ertapenem) were determined by using E-test and microbroth dilution methods
according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Table (2) revealed
that A. baumannii isolates were resistant to imipenem, meropenem, and ertapenem with concentrations
beyond values:
0.032 ?g /ml - >512 ?g /ml.
























































Isolates No.






















































Table (2) : MIC of carbapenem antibiotics
MIC (?g/ml)
IMP MER ERT

























































1 0.75 0.032
0.032

2 0.25 0.38
2

3 1 0.047
0.023

4 0.19 0.38
1.5

5 0.38 1.5
6

































































6 > 512 > 512
> 512

7 0.19 0.75
3

8 1 0.5
2
9 1.9 0.047
0.032
10 1 0.064
0.047








































































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First Detection Of The Blaoxa-23 Genes In A Clinical Isolates Of Acinetobacter baumannii in ….



Phenotypic detection of carbapenem production:
1(10%) isolate demonstrated enhancement of inhibition zone, with the imipenem-EDTA test whereas
six isolates showed positive results with modified Hodg test.

Genotypic detection of blaOXA-23 genes :
blaOXA-23genes were appeared in (40%) of A. baumannii isolates PCR products using specific primers
gene. (Fig. 2). Consequently, table (3) shows the isolates that harbored blaOXA-23 genes appeared
as extensive drug resistant (XDR), which exhibited resistance to at least 5 classes of antibiotics
were used in this study.













Figure (2): Agarose gel electrophoresis (1.5% agarose,70 volt for 1-2 hrs) for blaOXA-23 gene
product (amplified size 501bp) using DNA template of Acinetobacter baumannii isolates extracted by
using genomic extracting Mini Kit. Lane (M), DNA molecular size marker (100- bp Ladder). Lanes (2 ,
4, 6, and 7) of A. baumannii isolates show positive results with blaOXA-23 gene . lanes (1, 3, 5,
8, 9 and 10) show negative results with blaOXA-23 gene.

Table (3) : Antibiotics resistant pattern of carbapenemase genes positive A. baumannii isolates.
Type of resistance No. (%) of A. baumannii isolates (N=10)
No. of antibiotics classes
resisted
MDR 10 (100%)
3 or more
2 (20%) 9








































XDR







































3 (30%) 7
4 (40%) 6
5 (50%) 5










































PDR 1 (10%)
11












































IV. Discussion
The current report provided an evidence that the A. baumannii may be silently spread with low
proportion 0.76% (Table 1) in a hospital settings and highlighted the threat of undetected
reservoirs. However, The source of infection may include health care device or the environment can
involve with transfer of microorganisms between staff and patients (22).
Results from Figure (1) revealed that higher resistant rate was found for ampicillin and
carbenicillin were (100%) for each other, while for piperacillin was (30 %) . This result in
agreement with a pervious study in Hillah by Al-Warid (3), who found that all 11 (100%) ?-lactam
resistant A. baumannii isolates were resistant to ampicillin , carbencillin and piperacillin .
Alsehlawi and his colleagues (2) stated that all A. baumannii isolates were resistant to ampicillin
, carbenicillin (100%) for each other and (91.6%) to ticarcillin . High resistance to this class of
antibiotics may be due to widespread use of these antibiotics in Hilla hospitals. The present study
showed a high level of resistance to cephalosporins : ceftazidime and cefotaxime (60%) each other,
and (70%) for cefepime. There is also a wide range of resistance to aztreonam (80%). Alsehlawi, and
his colleagues (2)
reported that resistance rate of A. baumannii isolates to ceftazidime , cefotaxime and cefepime
were (100%). A report by Chaiwarith and his colleagues (10) documented that susceptibility to
ceftazidim and cifpirome were equal , (70%) in A. baumannii isolates collected from Thailand
hospitals. High level of resistance to third generation cephalosporins could be attributed to the
production of ESBLs, since it mediates resistance to broad spectrum cephalosporins (e.g.
,ceftazidime ,ceftriaxone and cefotaxime) and aztreonam (37). In the present
study there was a high level of resistance (80%) to ?-lactam / ?-lactamase inhibitor combination
(amoxicillin / clavulanic acid). This is likely due to the heavy selection pressure from overuse of
this antibiotic and seem to be losing the battle. A similar result was recorded by Alsehlawi and
his colleagues (2) who noted that (75%) of A. baumannii were resistant to amoxi-clav. In spite of
the restricted use of cefotaxime in treatment of bacterial infection in Iraq , results of this
study revealed a higher resistance (70%) to cefotaxime among clinical isolates








































































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of A. baumannii. This result is in accordance with the findings recorded by other researchers,
Al-Warid,(100%) in Hillah(3) and AlSehlawi and his colleagues,(100%) in Najaf(2). The resistance to
cephalosporins may be as a result of the development of porin-deficient mutants (28). In addition,
increasing numbers of bacterial strains express different types of ?-lactamases including inducible
and / or plasmid mediated AmpC type of enzyme may also increase the chance for resistance to
cephalosporins (38).
Results from (Fig.1) showed that resistance rates to imipenem, meropenem and ertapenem were (10%).
This result is lower than that reported by other studies contacted in Turkey which reported that
the resistance rate of A. baumannii isolates collected from clinical samples to imipenem was
(46.7%) and to meropenem was (53.3%) (31). While in a local study in Najaf , AlSehlawi and his
colleagues (2) found that only four isolates
(41.6 %) of A. baumannii were resistant to imipenem and meropenem antibiotics . Leepethacharat and
Oberdorfer demonstrated (65%) and (50%) resistance to imipenem and meropenem , respectively by A.
baumannii in a surveillance study at Chiang Mai University Hospital in Thailand (27). While in USA
Trottier and his colleagues who found that (87%) of A. baumannii resist to imepenem(43).
Meropenem is well tolerated and offers several potential advantages, including greater in vitro
activity against Gram negative pathogens and the option of bolus administration (46) . Beside these
, problem of renal metabolism of imipenem , and risk of seizures (36), and availability of
meropenem only in Hillah hospitals might be the reasons behind possible greater use of meropenem
over imipenem and hence the high prevalence of resistance.
Ertapenem is the least active carbapenem against most strains producing carbapenemase and therefore
the first marker that indicates the likelihood of carbapenemase occurrence (30,41). Specificity is
questioned because bacteria with ESBL and porin mutations are also resistant to ertapenem (19)
Reasons behind resistance may be due to inappropriate duration of antibiotic therapy and sub-
therapeutic concentrations of the drug (6,34) .
Results of the present study revealed that amikacin was more effective (80%) than other
aminoglycosides, gentamicin and Tobramycine (60%) for each other. This result was parallel with
other studies worldwide , as with Leepethacharat and Oberdorfer in Thailand (27) and ?zdemir and
his colleagues In Turkey(31). In another study in Najaf Alsehlawi and his colleagues who found that
resistance against aminoglycosides were (58.3%) to amikacin , whereas gentamicin and Tobramycine
(83.3%),(2).
High efficiency of amikacin may be due to its less vulnerability to bacterial enzymes than other
aminoglycosides . In both A. baumannii, gentamicin and Tobramycine resistance is often due to the
expression of a variety of modifying enzymes including aminoglycoside modifying enzymes (AME),
acetylases , phosphorlyases and adenylases which can impair the effectiveness of antibiotics. Other
resistance mechanisms include changes in bacterial membrane permeability and altered ribosomal
proteins (7) .
As shown in figure (1) resistance to quinolones antibiotics , (ciprofloxacin) was (40%). Quinolone
resistance is typically encoded chromosomally .In this study, resistance against fluroquinolones
may reflect antibiotic pressure in Hilla hospitals. In China , Zhou and his colleagues reported
high resistance to quinolones (> 95%) among clinical isolates of A. baumannii (48). In another
study in Najaf Alsehlawi and his colleagues who found that resistance against ciprofloxacin was
(91.6 %),(2).
Quniolone resistance results from mutations in the chromosomally encoded type II topoisomerases,
and via the up regulation of efflux pumps, or point- related genes (15,42). The plasmid qnr genes
play an emerging role in the dissemination of fluoroquinolone resistance (18).
Results of present study show highly resistant level to colstin sulphate (80%) with moderate
resistance to Polymyxin B (50%). This result had been convergent with study at Chiang Mai
University Hospital in Thailand (27).
Percentages of resistance of isolates to the remaining antibiotics were as follows : tetracycline
(20%), doxycycline (20 %) , trimethoprim-sulfamethoxazole (50%) and chloramphenicol (80%). These
results were parallel with previous studies in Brasil (41) and with Patwardhan, and his colleagues
at India (33).This may be
due to multiresistance plasmid harboring A. baumannii (33).
The high levels of resistance to antibiotics in the present study may be as a result of both
intrinsic and acquired mechanisms .The resistance is widespread and constitutes serious clinical
threat (29). In addition, the selection pressure of antibiotics in hospital environment lead to
multiple resistance to these drugs. El-Astal, mentioned that inappropriate and incorrect
administration of antimicrobial agents and lack of appropriate infection control strategies may be
the possible reasons behind increasing resistant rate of A. baumannii to common used antimicrobial
drugs (16).
All A. baumannii isolates were screened by two phenotypic tests for carbapenemase production. The
present study showed that 6 (60%) of the isolates gave positive results by imipenem- EDTA disk test
. Different studies which have used the IMP-EDTA to detect MBLs production in A. baumannii reported
that (33%) of isolates have enhancement of inhibition zone, with the IMP-EDTA test (2) . However,
there are four isolates which gave negative results with EDTA disk synergy test.








































































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The most easily performed test for Carbapenemase detection is the modified Hodge s test, which has
been found to be 100% sensitive for the detection of the carbapenemase (24). Out of the 10 A.
baumannii isolates which were enrolled in this study, 6 (60%) isolates were found to produce the
carbapenemase enzyme by MHT and all the remaining isolates were found to be carbapenemase negative
. In a previous local study, Alsehlawi and his colleagues reported that 4 (33.3%) of A. baumannii
isolates recovered from Najaf hospitals were confirmed as carbapenemase producer using modified
Hodge s test, whereas the same isolate gave negative result with imipenem-EDTA synergy test (2).
Another study from Croatia the Hodge test showed that 74% (72/97) of the A. baumannii isolates were
positive for carbapenemase production. (18) , whereas in a study from Pakistan has shown that 17 %
of A. baumannii were positive for carbapenemase production by MHT (5).
Oxacillinases are only weakly active against carbapenems and are largely confined to Pseudomonas
and Acinetobacter species and only rarely in Enterobacteriaceae (47). blaOXA-23 represented a new
subset of the OXA family. It has been identified in outbreaks of carbapenem-resistant Acinetobacter
in Brazil , Korea and United Kingdom (13, 22, 39, 44). Result from present study show that 4(40%)
A. baumannii isolates had blaOXA-23 genes (Fig. 2) . In another study in Iraq , Alsehlawi, and his
colleagues who found 2(40%) A. baumannii isolates had blaOXA-23 genes positive in Najaf hospitals
(2) . In contrast in Taiwan study , Lee, and his colleagues documented only one isolate (4%) of
A. baumannii was harbored blaOXA-23 gene (26).
As in the present study, such isolates (blaOXA-23 positive A. baumannii) exhibit resistance to most
antimicrobials that recommended by CLSI (2012) and appeared to be extensive antibiotic resistance
(XDR) (Table 3), this may creating a serious problem for choice of therapy, this results was more
identical with the report of emergence XDR in A. baumannii isolates from patients in ICUs of
Samsung Medical Center in Seoul, South Korea (23). Hence, The occurrence of isolates contain
blaOXA-23 in Hillah hospitals may resulted from transfer of plasmid among resistant isolates rather
than, several isolate may produce identical restriction pattern suggest the dissemination of
blaOXA-23 due to a clonal spread of resistant A. baumannii isolates (11).

V. Conclusion
Our study has shown the spreading of multidrug resistant and blaOXA-23 harbored A. baumannii
isolates among patients with different infections. Hence, it is suggested that, such isolates,
which consequently poses an increased threat to hospitalized patients in Hillah hospitals and more
importantly, avoiding misuse and overuse of antibiotics may reverse the undesired effects of
multidrug resistant and OXA-23producing bacteria.

Acknowledgment
The authors are very grateful to department of biology in faculty of science. The authors thank the
anonymous reviewers for their valuable comments and helpful revision suggestions.

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