Emergence of Multidrug resistance(MDR)
Acinetobacter baumannii Isolated from Iraqi hospitals .
Sawsan S. AL-Jubori1 , Israa Mohamed Safi Abd Ali AL-Kadmy1 , Zuhar I. Jassim2
1:Biotechnology branch \Department of Biology –College of Science .Al-Mustansiriyah Uni .Baghdad. Iraq.
2: central public health lab, microbiology, Baghdad/Iraq
E.mail for corresponding author: sawsan_sajid_ma2000@yahoo.com , israaalkadmy@gmail.com
Abstract : Twenty isolates primarily diagnosed as A.baumannii were isolated from wounds infections
from patients submitted to Baghdad hospitals\Iraq. Bacterial diagnosis was performed depending on rapid
morphological tests followed by identification using complementary Vitek system. recA gene(a house
keeping gene ) was used for genotypic diagnosis ,amplified size was 425bp. Antimicrobial susceptibility
testing was performed by disk diffusion and 13 antibiotics related to different groups were used .The result
revealed that most isolates displayed multidrug resistanse (MDR) pattern ,and all isolates were resisted to
gentamicin ,piperacillin and ceftazidim while ticracillin and Tigecycline were most effective when only 1
and 2 isolates were susceptible respectively .The results of rapid ESβLs production via ESβL strips
revealed that 20\20(100%) gave positive results in the preliminary screening of β-lactamases, 18\20 (90%)
were ESβLs producers ,for MβL, the rate was reduced to 12\20 (60%), finally 6 \20(30%) of the isolates
were AmpC producers .In Hodeg test , up to 80%(16\20) gave positive results.Genotypic Screening test
for antibiotic resistance genes showed that all the isolates covered MDR pattern and the most provenance
gene was blaTEM (100%) followed by aminoglycoside modifying enzymes genes type aac(3)-I (65%) ,
aac(6)-Ib (55%), and aph(3′)VI (55%) .The percentage of Efflux pump genes were 50% and 60% for mexX
and mexY ,respectively . The rate of carbapenemase genes IMP-1 and VIM were 50% and 25%
respectively whiles gyrA gene(quinolone resistance ) and bla-CMY (Cefipem resistance ) were 45% and
20%, respectively .The lowest percentages of resistance were for 23s ribosomal methylation enzymes
rmtC ( 10|%) , rmtB (0%) and rmtD (5%) . Up to 14 genes were detected in A.baummani no.1 ,a sign for
MDR emergence or a step for XDR among Iraqi isolates .
Key words : Multidrug resistance A.baummani , ESBLs ,Hodeg test , Aminoglycoside modifying enzymes
,Carbapenemase genes. ribosomal methylation enzymes .
Introduction :
1
Acinetobacter is a gram-negative bacterium commonly found in soil and water. There are many types
or “species” of Acinetobacter that cause human disease Ranged from pneumonia to serious blood and
wound infections(1) .The species A. baumannii causes about 80% of reported infections(2). Its ability
to colonize and spread among hospitalized patients ,producing different virulence factors and their
antibiotics resistance rendering them a successful nosocomial infected agent(3) .
In last three decades, clinicians are increasingly faced with infections of A. baumannii isolates resistant
to almost all clinically applicable antibiotics(4). The ability of A. baumannii to persist in the hospital
environment and to colonize patients in addition to its multidrug-resistant (MDR) phenotype makes
control of outbreaks caused by this organism very difficult(5) .Multidrug-resistant A.
baumannii(MDRAB) often defined as resistant to three or more antimicrobials groups (e.g.
aminoglycoside, ampicillin-sulbactam, antipseudomonal carbapenem, antipseudomonal cephalosporin
, fluoroquinolone)(6). MDRAB infections occurs most often in Health care settings (hospital
colonization and human reservoirs) housing very ill patients (5,7).They rarely occur outside of health
care settings; however, community-acquired A. baumannii has been reported in China and tropical
Australia, and has been mostly identified in patients with co-morbidity(8). Acinetobacter appears to be
particularly effective at acquiring genetic material from other organisms and thus rapidly developing
extreme drug resistance(XDR) caused by A. baumannii that lacked susceptibility to all antimicrobial
drugs tested except colistin (Multidrug-resistant A. baumannii has emerged as a substantial problem
worldwide Numerous outbreaks (9). XDR are increasly common and been mentioned as reports of
injured USA soldiers returning from Iraq and Afghanistan infected with A. baumannii (10) .Iraqibacter
isolates are extremely difficult to treat and pose considerable infection control issues(11). Aims of this
study involved Isolation and identification of A.baumannii from different clinical infections ,
Performing antibiotic susceptibility test against different antibiotics using simple agar diffusions then
molecular detection of different antibiotics resistant genes in Iraqi clinical isolates of A.baumannii
Collection and diagnosis of Bacterial isolates
Twenty isolates primarily diagnosed as A.baumannii were isolated from wounds infections from patients
submitted to
Baghdad
hospitals\Iraq .Bacterial diagnosis
was performed depending on rapid
morphological tests followed by identification using complementary Vitek system.
Genotyping detection for isolates
recA gene(a house keeping gene ) was used for genotypic diagnosis. Specific primers listed in table(1)
were employed and the amplified size was 425bp .Template DNA was prepared by boiling method by
Ruppé et al.(12). Briefly, few isolated colonies of overnight growth bacteria were suspended thoroughly
2
in 1 mL distilled water and boiled in a water bath for 10 min. After centrifugation, supernatant was used
as template DNA.PCR mixture of composed from 12.5 of GoTaq®Green Master Mix (2x) , 5 µl template
DNA , 1.5 µl primers (for each) final concentration (0.6pmol/µl),and nuclease free water up to 25 µl
(4.5 µl).
Antimicrobial susceptibility testing: It was performed by the disk diffusion method according to the
CLSI guidelines( 13) .The following discs(μg/disc),
were used TGC:Tigecycline(15μg), GN:
gentamicin(30 μg), AK: amikacin(30μg),TIC: ticarcillin(75μg), CP: ciprofloxacin(5μg),TCC: ticarcillinclavunic acid(75/10μg), CT: cefotaxime(30μg), IP: imipenem(10μg),PI: pipracillin(100μg), TZ:
ceftazidime(30μg),
T: tetracycline(30μg), TS: trimethoprim-sulfamethoxazole(1.25/23.75μg ), NT:
netilmicin(30μg).
Rapid Detection of ESβL Production Via ESβL Strips.
The detection of β-lactamases production was performed using Rapid ESβL detection kit ( Mast group.
UK). This kit includes four tests: Preliminary screening kit, Metallo β lactamases, ESβLs confirmation
and AmpC detection. The test was performed according to the procedure suggested by the manufacturing
company. Brifly,one drop of test substrate (approximately 20μl) was dispensed on the filter pad of the
strip. The test substrate was added immediately to the strip before testing.Using a loop, several identical
colonies were picked up and spread on the filter pad of the test strip .Any change in color (from yellow
to red) observed around the streaked line was considered a positive result. The tested strips were
observed after 2 to 15 minutes at room temperature, and the result was read after 15 minutes.
Modified Hodge test:
Modified Hodge test (MHT) was performed according to Amjad et al.(14) for detection of
carbapenemase production in Gram negative rods. Briefly, 5 ml of brain heart infusion broth culture
for Escherichia coli ATCC 25922 equal to 0.5 McFarland dilution was prepared. 1:10 dilution was
streaked as lawn on to a Mueller Hinton agar plate. Ten μg /disc meropenem susceptibility disk was
placed in the center of the test area. Test organism was streaked in a straight line from the edge of the
disk to the edge of the plate. The plate was incubated overnight at 37 cº for 24 hours. After 24 hrs,
MHT Positive test (carbapenemase producer) showed a clover leaf-like indentation of the Escherichia
coli 25922 growing along the test organism growth streak within the disk diffusion zone. MHT negative
test represent by normal growth for the standard strain or without the formation of clover leaf-like shape
PCR Amplification for Antibiotics resistance genes: All the isolates were subjected to molecular
screening study using PCR amplification technique in to detect MDR profile .Different primers ( table
1 )were used. PCR mixture was composed from 5μl Template DNA prepared by boiling method as
3
described above ,12.5 μl of GoTaq®Green Master Mix (2x) , 1.5 μl from forward and reverse primers
(final concentration 0.6pmol\ μl) for all genes except for blaAmpC, IMP-1, rmtC, rmtD, rmtB when 1µl
primers ( final concentration 1pmol\ μl ) were used .The volume was complete to 25 μl with nuclease free
water. PCR was run under the following conditions: primary denaturation step 95°C for 5 min; 30 40 repeated cycles (according to gene listed in table 1)of 94°C for 30sec , 43- 62 °C(table1) annealing
temperature(table-1) for 60 sec and 72°C for 1 min then final extension step at 72 °C for 6 min. PCR
products were electrophoresed in 1% agarose gel and visualized under UV light according to Sambrook
and Russell (15)
Table (1): primers used in the current study
4
Results and discussion:
In the current study, 20 isolates of A.bummani from wounds infections . Peleg et al.(8) considered
Acinetobacter as one of the most troublesome pathogens for health care institutions and hospitals worldPrimer
RecA
mexX
MexY
aac(3)-I
aac(6)-Ibant(4’)IIbaph(3′)VI
blaTEM
blaCTX-M
blaSHV
blaAmpC
IMP-1
VIM
blaCMY
RmtC
RmtD
RmtB
GyrA
Sequences (5'_3')
Size
product
425
Tm
54
No.
cycle
35
326
62
30
250
59
30
227
43
35
482
56
35
F-GACGACGACAAGGATATGGAATTGCCCAATATTATT
R- GGAACAAGACCCGTTCAATTCAATTCATCAAGTTT
364
57
35
F-TAT CTC GGC GGC GGT CGA GT
R- CAC GCG GGG AAA CGC GAG AA
F- GAGTATTCAACATTTCCGTGTC
R- TAATCAGAGGCACCTATCTC
R-GTTGGGGTAGTTGCGATTGG
F-CGCTTTGCGATGTGAAG
F-AAGATCCACTATCGCCAGCAG
R-ATTCAGTTCCGTTTCCCAGCGG
F-ATGCAACAACGACAATCCATC
R-GTTGGGGTAGTTGCGATTGG
F-CTACGCCAGCAGAGTCTTTG
R-AACCAGTTTTGCCTTACCAT
F-GTTTGGTCGCATATCGCAAC
R-AATGCGCAGCACCAGGATAG
F-GACAGCCTCTTTCTCCACA
R-TGGAACGAAGGCTACGTA
F-CGA AGA AGT AAC AGC CAA AG
R-ATC CCA ACA TCT CTC CCA CT
F-TCAAAAAGGAAAAGGACGTG
R-CGATGCGACGATCCATTC
F-CCC AAA CAG ACC GTA GAG GC
R-CTC AAA CTC GGC GGG CAA GC
F- GGCATTTATTCGTCACCGCC
R-TTCCGGGAACTCGGTTAACG
800
55
35
Bartual,
et
al.,
2005)16)
Llanes et al. 2004)
)17)
Xavier, et al. 2010)
)18
Ndegwa et al.
(2012)19
Haldorsenet
al.
(2011)20
Haldorsen et al.
(2011)20
)Vaziri et al . 2011)21
800
48
30
Oliveira
et
al.,
)2011)22
550
48
30
Oliveira
et
al.,
)2011)22
200
59
30
Oliveira
et
al.,
)2011)22
1150
58
40
Oliveira
500
55
35
382
57
35
1014
50
30
711
53
40
500
52
40
584
59
40
886
60
30
F- CCTGAATCTTCYGGTAAAAC
R- GTTTCTGGGCTGCCAAACATTAC
F- TGA AGG CGG CCC TGG ACA TCA GC
R- GAT CTG CTC GAC GCG GGT CAG CG
F- CCGCTACAACGGCTATCCCT
R- AGCGGGATCGACCAGCTTTC
F-AGCCCGCATGGATTTGA
R- GGCATACGGGAAGAAGT
F- TTG CGA TGC TCT ATG AGT GGC TA
R- CTC GAA TGC CTG GCG TGT TT
Reference
et
al.,
)2011)22
Ramazanzadeh
)2010( ,23
Mendes
et al.,
)2007)24
Zhao et al.
(2003)25
Doiet al.
(2007)26
Tijet et al .,
( 2011)27
Lee et al.(2006)28
According
study
to
this
wide and undergone significant taxonomic modification over the last 30 years. Beside its remarkable
ability to up regulate or acquire antibiotic resistance genetic elements , making it one of the most
threatening pathogen(10,11). Unfortunately, the quick upgrade for multiple drug resistant (MDR) isolates
including newer extended-spectrum -lactamases and different carbapenemases are emerging fast, leading
to pan-resistant strains of A. baumannii. (6).Figure (1) represent the percentage of resistance towards
antibiotics used in the current study. The percentage differ from completely resist (100%) towards
gentamicin, pipracillin and ceftazidime to 5% and 10% for tetracycline and Tigecycline respectively
.Very high resistance appeared to cefotaxime and ticarcillin(95% for each ) , ticarcillin-clavunic acid(90%)
5
followed by netilmicin(75%) . Mild activity appeared with imipenem (60%), ciprofloxacin(55%) and
amikacin(45%) as compared with good activity of trimethoprim-sulfamethoxazole when only 20% were
resisted to this combination .
TS, 4(20%) NT, 15(75%) TGC, 2(10%)
GN, 20(100%)
T, 1(5%)
AK, 9(45%)
TZ, 20(100%)
Tic, 19(95%)
PI, 20(100%)
CP, 11(55%)
IP, 12(60%)
TCC, 18(90%)
CT, 19(95%)
Figure (1): Percentage of resistance towards antibiotics used in the current study . TGC:Tigecycline/ GN:
gentamicin/ AK: amikacin/ TIC: ticarcillin/ CP: ciprofloxacin/ TCC: ticarcillin-clavunic acid/ CT:
cefotaxime/ IP: imipenem/ PI: pipracillin/ TZ: ceftazidime/ T: tetracycline/ TS: trimethoprimsulfamethoxazole/ NT: netilmicin
Figure (2) represents number of antibiotics that each isolate showed its ability to resist .As clear from
the figure ,some isolates like A.baummani no.7,11, 9, 10 and 20 were able to resist at least 10 to 12
antibiotics related to different groups .other isolates were able to resist in between 8 to 4 antibiotics .
Maragkis and Perl(5) illustrated that
therapeutic options are limited for multidrug-resistant
Acinetobacter infection and the development or discovery of new therapies, well-controlled clinical trials
of existing antimicrobial regimens and combinations, and greater emphasis on the prevention of health
care–associated transmission of multidrug-resistant Acinetobacter infection are essential. Such highly
resisted isolates that lacking susceptibility to all commercially available antimicrobial drugs also is referred
as XDR because no therapeutic options are available (3).In our local isolates , A.baummani no.7 and 20
might considered as XDR for their ability to resist almost all commercially used antibiotics .Karaiskos and
Giamarellou (29)mentioned in their reports that Colistin is active in vitro and effective in vivo against
XDR carbapenemase-producing microorganisms in the critically ill host, whereas tigecycline, with the
exception of P. aeruginosa, has a similar spectrum of activity. . Evidence to date suggests that colistin
constitutes the backbone of therapy, but the unique pharmacokinetic properties of colistin have led many
to suggest the use of combination antimicrobial therapy(30).
6
14
12
12
12
11
10
11
10
8
9
9
8
6
9
8
8
8
9
9
8
7
6
6
6
4
4
2
0
Figure (2): Number of resisted antibiotics by A. baumanni isolates
Table 2 shows the results of β- Lactemase and Hodeg tscreening test. Out of 20 isolates, 20 (100%) gave
positive results in the preliminary screening of β-lactamases, 18 (90%) were ESβLs producer isolates . For
MβL, the rate was reduced to 12 (60%), finally 6 (30%) of the isolates were AmpC producers Al-jubori
et al.(31) used the same screening test for P.aeruginosa. For Hodeg test , up to 80%(16\20) gave positive
results and Figure (3) shows some of these results .In the current study four isolates( A.baumannii 1,7, 11
and 20) were positive in the five screening test. Amjad et al.(14) illustrated that Modified Hodge test is
a simple test which can be performed in the routine lab for phenotypic detection of carbapenemases in
isolates showing intermediate or sensitive zone diameter on disc diffusion but in his study 12% of
Acinetobacter gave positive results .The production of carbapenemases is an important mechanism
responsible for the carbapenem resistance(32). A simple and inexpensive testing method for screening
metallo-β-lactamases (MBLs) and AmpC β-lactamases in non fermentative Gram negative bacteria is
required (14,32).In Noyal et al.(32) study 46\100 (59.0%) A. baumannii gave a positive Modified Hodge
test and were resistant to meropenem.In a study carried by Amudhan et al. (33) the MHT was positive in
94.4% (n =169) of their isolates . Purohit et al.(34) demonstrated that neither any standard guidelines are
available nor any method has been found to be perfect for carbapenamases like metallo-β-lactamases
(MBLs) detection and various methods have shown discordant results, depending upon the employed
methodology, β-lactamase substrate and MBL inhibitor used.
.
7
Table -2: Percentage of β-lactamase enzymes and Hodeg positive test
Isolate no
No.of
β-lactamase types
AmpC 6\20(30%)
Hodeg test
16\20
)80%)
+test
Preliminary
screening
test 20\20(100%)
ESβLs
(90%)
18\20
MβL 12\20 (60%)
A.b1
5
+
+
+
+
+
A.b 2
3
+
+
------
-----
+
A.b 3
4
+
+
+
-----
+
A.b 4
3
+
+
+
-----
+
A.b 5
3
+
+
----
-----
----
A.b 6
1
+
----
----
-----
+
A.b 7
5
+
+
+
+
+
A.b 8
4
+
+
+
-----
+
A.b 9
4
+
+
+
----
+
A.b 10
4
+
+
+
+
---
+
+
+
+
+
A.b 11
5
A.b 12
2
+
+
------
------
-----
A.b 13
3
+
+
------
-----
+
A.b 14
3
+
+
-----
-----
+
A.b 15
4
+
+
+
-----
+
A.b 16
4
+
----
+
+
+
A.b 17
3
+
+
-----
-----
+
A.b 18
3
+
+
-------
------
+
A.b 19
3
+
+
+
----
---
A.b 20
5
+
+
+
+
+
8
Figure 3: Hoges test for some A.baumanni isolates .positive results appear in no. 1,3, 7,8 and negative
results in 5 and 12
Molecular detection for antibiotic resistance genes revealed that all the isolates harbored more than one
type of resistant mechanisms . Figure (4) illustrates agarose gel electrophorese for some amplified
virulence genes :A Multiplex PCR for CTX and TEM genes responsible for cefotaxime and ampilcillin
resistance , respectively. B: MexX and C : MexY genes which conferring resistance for Efflux pump system
while E: rmtC and F: rmtD genes were used for detecting ribosomal methylation resistance . PCR was
performed for the detection of MBL ,blaIMP and blaVIM genes as clear in G and H , respectively ,beside
detecting aminoglycoside modyfing enzymes (G: aph(3)-VI, I : acc(3)-I, K: aac(6)-Ib and ant(4’)IIb ). Most of
these isolates harbored more than one resistance genes, some isolates were conferring unusual resistance
pattern (figures: 5 and 6) as with A.banmannii no.1 (14 genes), no.7( 12 genes )and no.20(13 genes )making
them MDR type. The most prevalence genes among our local isolates was blaTEM and it was detected in
all the isolates (100%) followed by the percentage of Aminoglycoside genes aac(3)-1 (65%) , aac(3)-1
and aph(3)VI (55% for each) . The rate of Efflux pump genes mexX and mexY were 50% and 60%
,respectively.For the carbapenamase genes, the results showed that IMP-1was more predominate than VIM
when their percentage were 25% and 50% , respectively, and two isolates (A.banmannii no.1 and 20 )
harbored both of them while in Amudhan et al. (33) study , only one isolate showed positive result for
both of them . gyrA gene(Quinolone resistance ) and bla-CMY (Cefipem resistance ) were 45% and 20% ,
respectively .The lowest percentages were those related to 23s ribosomal methylation enzymes rmtC (
10|%) , rmtB (0%) and rmtD (5%). Prashanth et al . (6) demonstrated that A. baumannii, like Pseudomonas
aeruginosa, produces a naturally occurring AmpC βlactamase, together with a naturally occurring oxacillinase with carbapenemase properties. ESBLs are
plasmid-mediated β-lactamases of predominant class A. ESBLs are capable of efficiently hydrolyzing
penicillin, cephalosporin, the oxyimino group containing cephalosporins (cefotaxime, ceftazidime) and
monobactams (aztreonam) and wide range of class A ESBLs have been reported in Acinetobacter sp. such
as TEM, SHV, CTX-M and VEB. In a research done in Tehran by Asadollahi et al. (35) they mentioned the
diversity of aminoglycoside modifying enzyme(AME) genes among multidrug resistant A. baumannii
genotypes isolated from nosocomial infections ,and their results focused on AME types were
9
A
B
C
E
D
F
G
I
H
J
K
L
Figure 4: Agarose gel electrophoreses(1% agarose, 7 V/cm for 90 min) for some amplified virulence genes :A :
Multiplex PCR for CTX and TEM (550 and 800 bp, respectively ), B: MexX (326 bp) ,C : MexY (250bp) ,D: recA (425
bp) , E: rmtC ( 711bp) ,F: rmtD(500bp) , G:Multiplex PCR for aph(3)-VI and IMP( 800 and 500 bp, respectively ) , H:
VIM(382 bp) ,I : acc(3)-I( 227bp), J:SHV (200 bp) ,K: aac(6)-Ib (482bp) and ant(4’)IIb (364 bp) .
10
no.of Antibiotic resisted genes
16
14
12
10
8
6
4
2
0
A.b A.b A.b A.b A.b A.b A.b A.b A.b A.b A.b A.b A.b A.b A.b A.b A.b A.b A.b A.b
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Series 1 14
7
7
6
6
6
12
7
10
8
7
4
4
4
3
4
1
1
3
13
Figure (5): The number of resisted genes within twenty A.baumannii isolates
25
20(100%)
20
15
12(60%) 13(65%)
11(55%)
10 (50%)
11(55%)
10(50%)
10
5
6(30%)
4(20%)
4(20%)
5(25%)
9(45%)
5(20%)
4(20%)
2(10%)
1(5%) (0%)
0
Figure (6): Prevalence and percentage of Antibiotics resistance genes within A.baumannii isolates
aph-6 (90% ), aad A1(82.5%) , aacC1 (65% )and aad B( 20%) among aminoglycosides resistant A.
baumannii isolates. The amikacin resistance gene aph(3)-VI also called (aphA6 ) was first detected in the
nosocomial pathogen Acinetobacter baumannii and analysis of 133 whole-genome sequences covering the
taxonomic diversity of Acinetobacter spp. Revealed that this gene was detected in all Acinetobacter
spp.(36). With an increase in the use of colistin methansulfonate (CMS) to treat carbapenem-resistant A.
baumannii infections, colistin resistance is emerging,in Zubair et al.(37) study twenty patients with
colistin-resistant A.baumannii were identified and usually Colistin-resistant A. baumannii occurred
almost exclusively among patients who had received CMS for treatment of carbapenem-resistant, colistinsusceptible A. baumannii infection. Such isolates will return to killer pathogen, incurable and highly
problematic.
11
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