Comparison between Phenotypic and Genotypic Detection of Metallo Beta Lactamase Enzyme among Gram Negative Bacteria Isolated from Burn Patient

Abstract

Background: Acquired metallo β-lactamases (MBLs) are emerging worldwide as powerful resistance determinants in Gram negative

Full text

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Journal of Kirkuk Medical College
Vol. 7, No. 1, 2019
Comparison between Phenotypic and Genotypic Detection of Metallo Beta
Lactamase Enzyme among Gram Negative Bacteria Isolated from Burn Patient
*Fattma A. Ali, **Khanda Abdulateef Anoar, **Sherko Ali Omer
Department of Medical Microbiology/ College of Health Sciences/ Hawler Medical
University/ Iraq
Department of Microbiology/ School of Medicine/ University of Sulaimani/ Iraq
Abstract:
Background: Acquired metallo β-lactamases (MBLs) are emerging worldwide as powerful resistance
determinants in Gram negative bacteria Rapid dissemination and spread between different bacterial species
by large gene transfer favor by globalization and travel represent a high risk of worldwide pandemic among
Enterobacteriaceae
Objective:This study design to screen for phenotypic detection of MBL and genes coding for metalloβ-
lactamase(MBL) such as, blaVIM, blaIMP and bla NDM among infected burn wound patients in Sulaimani
city /Iraq.
Materials and Methods: Out of 230 burn wound samples, 201 wound swabs and 29 tissue biopsy were
collected from hospitalized burn patients with second and third degrees burnfrom Burn and Plastic Surgery
Hospital in Sulaimani city from the period of April to October 2011. According to direct gram stain ,
bacterial cultural , biochemical tests, analytic profile identification(API) system and vitek 2 Compact system
, antimicrobial susceptibility Testing by using Kirby-Bauer disk diffusion method and vitek compact system,
phenotypic tests for Metalloβ-lactamase by Double disk synergy test and Combined EDTA disk test and
Modified Hodge test and also Molecular detection of Metallo β-lactamase enzyme by using polymerase
chain reaction.
Results: Out of 230 burn wound samples177 samples gram negative bacteria were isolated; inflammatory
cells showed significantly associated with positive bacterial culture. The most frequent bacteria isolated
were Pseudomonas species 48(27.12%), in which Pseudomonas aeruginosa account for a higher percentage
46(25.98%) and one species (0.56%) of each P. stutzeri and P.florescence wasisolated followed by
Acinetobacterspecies 44 (24.86%) in which Acinetobacterbaumanniiwas the commonest and accounts for a
higher percentage 41 (23.16%) and one species(0.56%) of each of A. ursingi, A.
hemolyticasandA.complexwere identified. On the other hand in the family of Enterobacteriaceae,the most
common bacteria were Klebsiellapneumoniae44(24.86%), Enterobacter cloacae 18 (10.17%) and
Escherichia species 11(6.21%). The susceptibility of bacterial isolates against 18 antibiotics from different
classes of antibiotics was tested and it was found that most of the isolated species of non fermenter bacteria
such as Acinetobacterand Pseudomonas species show multidrug resistant pattern and high resistance against
most of the antibiotics commonly used. The most resistant antibiotics against non fermenter bacteria were
Ticarcillin (81.82%) against Pseudomonas species and Ticarcillin, Tazobactam- Pipracillin and Cefoxitin
(93.18%) against Acinetobacterspecies .The most resistant antibiotic against K. pneumonia
andE.cloacaewere Cefoxitin(84.9%)and Amoxicillin –Clavulanic acid (84.9%).The most resistant antibiotics
against Escherichia specieswere Cefotaxime, Trimethoprim and Cefipime (90.91%) but the most effective
antibiotic with a highsensitivity rate was Imipenem (90.91).Phenotypic tests were carried out for the
detection of MBL enzyme for all studied isolates and it was found that Combined disk test was the most
sensitive test giving the highest percentage (31.07%) followed by Double disk synergy test(28.8%),
Modified Hodge test (20.9%) .Polymerase chain reaction assay was used for genotypic detection of MBL
genes (blaIMP,blaVIM, ,blaNDM ) in all isolates and the results revealed that the gene blaIMPwas locatedin
33(18.64%),blaVIM,in19 (10.73%), 2(1.12%)forblaNDM Also these genes were detected in 25 stock
cultures of Gram negative bacteria preserved in 25 °C since 2008 to 2010. blaIMP gene was detected
in11(44%) , blaVIM in 12(48%) and 2(8%) for blaNDM, all these genes were detected in K. pneumonae,P.
aeruginosa and A. baumanniiwith length amplified genes (230) bp for IMP and (390)bp for VIM and(
621)bp for NDM.
Conclusions:These results indicate that most of the Meropenem resistant strains from infected burn wound
strains in this study were producing MBL enzymes The presence of MBL genes among Meropenem
sensitive strains indicates that there might be a hidden MBL gene among isolated strains which cannot be
diagnosed by phenotypic tests leading to the dissemination of these genes in the hospital silently among
patients even within normal health workers who act as carriers for MBL genes in future
Keywords: Metallo beta lactamase, Gram negative bacteria, Genes.

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Introduction:
Mortality rates from burn injury vary
across regions of the world. Low and
middle income countries suffer higher
mortality and morbidty rates from burns
as these countries require improved
surveillance of burn injury via
epidemiology studies, which will
determine the incidence and prevalence
of burn injury among sub populations (1).
One of the significant public health
problems in many area of the world is
burn injury and despite advances in the
use of topical and parenteral
antimicrobial therapy, bacterial infection
remains a major problem in the
management of burn victims today (2). A
variety of organisms have been isolated
from burn wound colonization such as
aerobic bacterial isolates ranging from
gram-positive organisms like
Staphylococcus aureus, and
Enterococcus spp. to gram negative
organisms such Acinetobacter spp.
which are apparently the only group of
gram-negative bacteria that may be
natural residents of human skin (3). Non-
fermenter Gram negative bacilli are
ubiquitous in nature with a wide
geographic distribution and they are
found in most of the environments; soil,
water, plants, food stuff and decay
vegetation, therefore they prefer moist
environment (4). They are common in
hospital setting, and may be found on
the surface of humidifiers, ventilator
machines, dialysis machines and other
equipment, as well as from the skin of
hospital personnel especially immune
compromised. A report by the Infectious
Diseases Society of America specifically
addressed three categories of multidrug
resistance of Gram negative bacilli,
namely extended-spectrum
cephalosporin resistance E.coli,
Klebsiella spp., Pseudomonas spp. and
Carbapenem resistance Acinetobacter
spp. Although Acinetobacter spp. are
frequently resistant to Fluoroquinolones,
Aminoglycosides, and all -lactams
with the exception to Carbapenems (5),
which were considered to be the last
drug of choice for the treatment of
infections due to Acinetobacter spp. (6).
However, resistance to Carbapenems is
emerging and represents a significant
threat to the management of MDR
isolates in many areas of the world (7),
which were mediated by mechanisms
such as hyper-production of a β-
lactamase with weak Carbapenems
hydrolyzing activity such as AmpC-type
Cephalosporinase or an extended-
spectrum β-lactamase respectively (8).
Metallo beta lactamase are metallo
enzymes of Ambler class B which are
Clavulanic acid resistance enzymes.
They require divalent cations of zinc as
co-factors for enzymatic activity and are
universally inhibited by EDTA as well
as other chelating agents of divalent
cation. There are two dominant types of
transferable MBL genes among clinical
isolates, blaIMPand blaVIM, which are
frequently present on gene cassettes
inserted into integrons located on the
chromosome or on plasmids (9). New
Dehlimetallo β-lactamase has recently
emerged in India, Pakistan and the
United Kingdom and it represents a
serious threat of rapid dissemination of
multiple antibiotic resistances since the
majority of blaNDM producing
Enterobacteriaceae have been reported
to remain susceptible only to Colistin
and Tigecycline (10). Some of the
blaOXA type β-lactamase variants also
have the ability to hydrolyze ESBL,
whereas other variants such as blaOXA
type, Carbapenemase are able to
hydrolyze Carbapenems. The

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occurrence of blaOXA type
carbapenemase genes in Acinetobacter
spp. represents vertical inheritance in
specific species but horizontal
acquisition in other species (11). Class D
carbapenemase belong to blaOXA
family and are commonly present in
Acinetobacter spp. and occasionally in
Enterobacteriaceae and Pseudomonas
spp. These enzymes hydrolyze
Carbapenems weakly and are poorly
inhibited by Clavulanate (12) which are
able significantly to hydrolyze
Aminopenicillins such as Ampicillin,
Aamoxicillin and Carboxypenicillins
such as Carbencillin and Ticarcillin (13).
Methods:
Study population and specimens
Study was carried out in the Burn and
Plastic Surgery Hospital in Sulaimani
city at a period from April - October
2011. Two hundred and thirty (230)
burned patients with suspected burn
wound infection of any total body
surface area or burn degree were
planned to participate in this study. The
swabs samples were obtained from deep
areas of the burn wound, the samples
were taken from the areas which was
appear deep with discharge, and thick
eschar while for dry wound, the swab
was moistened with sterile saline before
swabbing.
Sample Prosessing
Out of 230 burn wound samples, 201
wound swabs and 29 tissue biopsy were
collected from hospitalized burn patients
with second and third degrees burn from
Burn and Plastic Surgery Hospital in
Sulaimani city from the period of April
to October 2011. Out of 230 samples,
According to direct gram stain, bacterial
cultural, biochemical tests, analytic
profile identification (API) system and
vitek 2 Compact system. After taking
the samples, four quadrant method was
used to strike the surface of blood agar,
MacConkey agar and Nutrient agar and
the growth was inspected after 24 hours
of aerobic incubation at 37°C of all the
samples (14).
Antibiotics disks
Two different sets of antimicrobial disks
were used according to standards
institute for antimicrobial susceptibility
Testing. One set (14 antimicrobials) for
Enterobacteriaceae The second set (14
antimicrobials) for non-fermenter
(Kirby-Bauer disk diffusion method) (15),
standard bacterial strains of K.
pneumoniae ATCC 700603, E. coli
ATCC 25922 and P. aeruginosa ATCC
25853 were used as quality control
strains.
Phenotypic tests for Metalloβ-lactamase
Double disk synergy test (DDST)
This test is performed by inoculating the
tested organism onto muller hinton agar
plate. A 10 μgMeropenem disk and a
blank filter paper disk 6 mm in diameter
were placed 10 mm apart from edge to
edge, then, 10 μl of 0.5 M EDTA
solution was applied to the blank disk,
after 18hours of incubation at 37ºC, the
presence of extension of zone towards
the impregnated EDTA disk was
interpreted as EDTA synergy test
positive (16).
Combined EDTA disk test
An overnight broth culture of the test
strain with an opacity adjusted to 0.5
McFarland standards was used to
inoculate a plate of Mueller-Hinton
agar. 4 µml of the sterilized EDTA
solution was added to 10 µg Meropenem
disk, then the EDTA impregnated
antibiotic disks were dried immediately
in an incubator and stored at -20 ºC in
airtight vials without any desiccants
until used after drying of MHA plate, a
10 μgMeropenem disk and meropenem
disk combined with EDTA was placed

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Journal of Kirkuk Medical College
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20 mm apart. After 24 hours incubation
at 37 ºC an increase in the zone size of
at least 7 mm around the Meropenem
combined EDTA impregnated disk
compared to Meropenem disks alone
was recorded as MBL producing
strains(17).
Modified Hodge test
All tested gram negative bacteria were
subjected to MHT test according to what
was described by (18). An overnight
culture suspension of pan sensitive
strain of E.coli was prepared by adding
two to three isolated colonies of E.coli
strain to 5 ml of normal saline, and the
suspension was further diluted by
adding 1 ml of suspension to 4 ml of
(0.85%) NaCl and the mixture were
adjusted to 0.5 McFarland‘s standard
and this suspension was streaked across
the entire plate of MHA plate. After
drying 10 µg of Meropenem disk was
placed at the center of the plate and up
to 4 different isolates of gram negative
bacteria were streaked linearly from the
periphery of the plate into the direction
of Meropenem disk at the center and the
test plate was incubated for 18 hours at
37ºC. The presence of a ‗cloverleaf
shaped‘ zone of inhibition around each
tested strain is interpreted as
Carbapenemases producing strain or a
positive result.
Molecular detection of Metallo β-
lactamase enzyme.
All gram negative bacteria isolated from
hospitalized burn patient and 25 clone
stocks randomly selected from
Bacteriology Department reservoir
culture of Burn and Plastic Surgery
Hospital in Sulaimani in which they
were isolated from MDR strains of
severely burned patients since 2008 and
cultured on clone stock culture and
preserved at 25ºC were screened for
presence of MBL gene (blaIPM,
blaVIM and blaNDM) by multiplex
PCR reaction.
Reactivation of bacterial stock culture
For reactivation of bacterial strain, the
frozen surface of the a stock culture was
scraped with a sterile inoculating needle
or tip, and immediately immersed in 2
ml of Tryptic Soya broth and incubated
for 24 hours in a shaking incubator with
200 rpm at 37 ºC (19).
Plasmid extraction
Plasmid extraction was performed as
recommended by the manufacturer of
Gene JET plasmid Miniprep Kit. The
column was discarded and purified
plasmid DNA concentration was read by
Nanodrop®spectrophotmetery via the
program of Nanodrp 2000/2000c
program. The extracted DNA samples
were stored at -20 ºC. All bacterial
plasmids were run on (1%) agaros gel
for the detection of their pattern. 15 µl
of extracted plasmid was mixed with 3
µl of loading dye (6×) and the mixture
was loaded into prepared agaros gel and
the gel was passed in to the gel tank for
30 minute at a voltage of 1 to 5 volts/
cm2 of the gel size. The results were
read using ultra violet light in Gel
Documentation System (20).
Polymerase Chain Reaction (PCR)
Polymerase chain reaction mixtures
were prepared in duplicates and negative
controls were included in each run to
validate the reaction. Each reaction
mixture was prepared to a volume of
25l in a sterile PCR tube table (1).
Lyophilized primers provided by
Cinagen were processed in order to
produce a stock concentration of 100
m by mixing the concentrated
lyophilized primer with specific
volumes of nuclease free deionized

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water according to the primer
manufacture (Cinagen). From this stock
concentration a working primer
concentration of 10m was prepared by
mixing 90l of deionized H2O with
10l stock primer.
Table (1): Recommended volumes used to prepare the PCR mixture.
Mixture component
Volume
Final concentration
Green master mix 2 X
12.5 µl
1X
Forward primer 10 µm
1µl
0.1-1 µg/ml
Reveres primer 10 µm
1µl
0.1-1 µg/ml
DNA template
3µl
< 250 ng
Nuclease free Deionized water
To reach 25 µl
In Multiplex PCR, the reaction was
prepared to contain three sets of primers
which were first prepared by mixing the
revers and the forward of each primer
then all the primers were mixed together
in a specified volume and these were
added to other components of the PCR
reaction. Polymerase chain reaction
amplification conditions were modified
according to the annealing temperature
of the primer sets table (2).
Table (2): Primers used in the study.
Primer name
Sequence of primer
Ann. Temp.
Target gene
Amplicon in bp
VIM-R
TGGTGTTTGGTCGCAAT
48 °C
bla VIM
390bp
VIM-F
CGAATGCGCACCAG
IMP-R
GGAATAGAGTGGCTTAACTCTC
bla IMP
232bp
IMP-F
GTTTAACAAAACAACCACC
NDM-R
CGGAATGGCTCATCACGATC
bla NDM
621 bp
NDM-F
GGTTTGGCGATCTGGTTTTC
Agaros gel preparation and gel
purification of DNA products
The agaros gel was prepared according
to what was performed by Ozeret al.,
(21), PCR products from agaros gel were
recovered by using GeneJETrM Gel
Extraction Kit (Fermentas). The content
of the kit was prepared prior to use as
recommended by the manufacturers.
Sequencing of PCR products
Sequencing of PCR products was done
in University of Koya/ Erbil/ Iraq,
Genome Centre, and Sequencing
Department. The purified PCR product
was sent after its purification from
agaros in an 1.5 ml Eppendorf tube and
15 µl of both the revers and forward
primers was also sent in a separate tube.
The sequencing reactions were
performed using BigDye® Terminator
V3.1 Cycle Sequencing Kit (Applied
Biosystem), and High-performance 4-
capillary 3130 Genetic Analyzer pop7
polymer was used for separation. Data
collection software V3.0 was used for
sequence analysis in the center and
DNAMAN program (Lynnon
Corporation, version 4.13) was used for
the analysis of sequenced data.
Statistical analysis
Statistical analysis was done by using
SPSS program (Statistical Package for
Social Science) version 18 Inc.,
Evanston. Type of work was a
descriptive study in which the data were
analyzed between factors as categorical

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using 2-sided Pearsonchi-square test to
see significant differences between them
at p value of < 0.05, correlation was
performed at (99%) confidence interval.
Result:
Primary cultivation was done for all 230
burn wound specimens in which
201specimens were wound swabs and
29 were tissue biopsies and the growth
was detected in 211(91.74%) samples
and 19(8.26%) samples were found to
be negative table (3) and both types of
specimens showed positive cultures that
is statistically significant (p<0.05).
Out of 211 samples which give positive
cultures, 177 isolates of Gram negative
bacteria were isolated from both wound
swab and tissue biopsies. The most
frequently isolated bacteria among burn
patients was the group of non glucose
fermenting Gram negative bacilli
followed by family of
Enterobacteriaceae in which
Pseudomonas species 48(27.12%) was
the most frequently isolated out of all
Gram negative isolates. The most
frequently isolated species was P.
aeruginosa 46 followed by one isolate of
each of P. florescence and P. stutzeri.
Among the 46 species of P.aeruginosa, 6
isolates showed blue green pigment
(pyocyanin) and 2 isolates showed red
pigmentation (pyoverdin) with
appearance of β-hemolysis over blood
agar plate in 32 species. The second
common isolated bacteria from burn
wound was Acinetobacterspecies
account for 44(24.86%) From the family
of Enterobacteriaceae, K.pneumoniae
was the most frequently isolated
bacteria (24.86%) followed by
Enterobacter cloacae (10.17) asin
figure(1).
Antimicrobial susceptibility tests
All Gram negative isolates were tested
for antimicrobial susceptibility testing
using disk diffusion methods and the
results were interpreted according to
standard values provided by Clinical
and Laboratory Standard Institute of
antimicrobial susceptibility testing (15).
Tables (4, 5) illustrate the susceptibility
profile for all Gram negative isolates by
using 14 antimicrobial agents. Most of
Gram negative isolates in this study
showed resistance to at least eight of
antimicrobial agentswere considered to
be multidrug resistant (MDR) isolates.
Pseudomonas spp. shows resistance to at
least 3 classes and most of resistance
were observed against Ticarcillin (81.2
%), followed by Pipracillin –
Tazobactam (72.9%), Gentamicin and
Tobramycin (70.83%), Cefoxtin
(68.75%), Acinetobacter species which
revealed high resistant pattern against
Cefoxitin (93.18%), Ticarcillin (93.1%)
Pipracillin–Tazobactam (93.1%),
Trimethoprim (90.9%), Amikacin (90.9
%), and Cefipime (88.4 %). From the
family of Enterobacteriaceae, the
susceptibility pattern was different.
Klebsiella pneumonia showed resistance
to Amoxicillin–Clavulanic acid and
Cefoxitin (84%), followed by Ticarcillin
and Cefazolin (81.8%) and Ceftazidime
(79.5).
Out of 177 gram negative isolates that
were screened for susceptibility to
carbapenem by disk diffusion method,
70(39.5%) bacterial species were
carbapenem resistant and from these
66(37.2 %) were Meropenem resistant
strains table (6).
IMP: Imipenem, MEM: Meropenem
Double disk synergy test (DDST) were
performed by observing synergism
between Meropenem disk and EDTA
disk figure (2), and this test showed that
51(28.81%) bacteria from all isolated

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Journal of Kirkuk Medical College
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bacteria isolated from burn patients
were positive by this test, and
Acinetobacterspecies (15.82%) were the
most frequently isolated bacteria
observed to be positive by this test
among all gram negative bacteria
followed by pseudomonas species
(8.47%) as in table (7).
Phenotypic detection of MBL enzyme
by using combined EDTA disk test
CMDT.
This test was performed to detect MBL
production. If the zone of inhibition
around Meropenem with EDTA was
more than 7 mm compared to
Meropenem disk alone, the test is
considered to be positive figure (3).
Among 177 isolates, 55(31.07%) were
found to be positive by this test, and
(95%) of positive bacteria by this test
showed a high rate of zone of inhibition
> 20 mm and Acinetobacter species
were the most common bacteria
observed to be positive by this test
(14.69%), followed by Pseudomonas
species (14.12), as in table (7). Out of
the 177 isolated bacteria tested by this
method, only 2 isolates were found to be
positive in which both of the strains
were Meropenem resistant, while 175
samples were negative, but 51 isolates
showed entire growth along the strip and
this will be regarded as undermined
results.
Phenotypic detection of MBL enzyme
by using Modified Hodge test
Modified Hodge test was performed as a
screening test for all Gram negative
bacilli isolated from burn patients
including both meropenem resistant and
sensitive strains. Positive test was
recorded as indentation of the zone of
inhibition along the tested bacteria
towards the Meropenem disk as seen in
figure (4). MHT test also was detected
in 37(20.9%) out of all 177 Gram
negative bacteria, from which only 6
isolates were from Meropenem resistant
strains and the reaming 31 samples were
Meropenem sensitive strains table (7).
Molecular detection of MBL genes by
PCR
Plasmid extractions were performed for
all 177 isolated Gram negative bacteria
from infected wounds of burn patients
and 25 bacterial stocks. Genes
responsible for metallo β-lactamase
(MBL) can be detected by using
polymerase chain reaction (PCR).
Several possible genes in this study such
as blaVIM, blaIMP and blaNDMwere
targeted to be screened by multiplex
PCR mixture figure (5).
Polymerase chain reaction products
were resolved using (1%) agarose gel
electrophoresis and suspected bands
were gel purified and sent for
sequencing. Three blaVIMfromP.
aeruginosawere subjected to sequencing
using their amplification primer sets.
The results of forward and reverse
primers sequencing data were subjected
to BLAST service available at National
Center for Biotechnology Information.
The amplified PCR of blaVIM product
belongs to one of several genes of MBL
integrated into P. aeruginosa, Elizabeth
kingia meningo septica, A. bereziniaer
or other organisms. When the reverse
primers sequence data were uploaded
into the BLAST query, the same results
of MBL were obtained. When sequence
data from three blaVIM gene products
were aligned together using DNAMAN
program using both forward sequence
and reverse sequence data the following
results were obtained showing
homology ranging from (96.77%) for
forward sequences to (98.14%) for
reverse sequences, figures (6, 7).
Concering sequencing blaIMP a nd

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Journal of Kirkuk Medical College
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NDM gene products, sequencing failed
to show any results and this will need
further work to confirm these products.
Detection of blaIMP gene was observed
in 33(18.6%) Gram negative isolates
with implified gene (230) bp from
which 29 isolates were Meropenem
resistant and 4 were Meropenem
susceptible while blaVIM gene was
detected from 19(10.7%) Gram negative
bacteria with implified genes (390) bp
from which 14 isolates were
Meropenem resistant and 5 were
Meropenem susceptible strains as
presented in table 8. Concerning
blaNDM with implified genes (621) bp.
Two possible products were amplified
and one of them was isolated from
multidrug resistant Klebsiella
pneumonia and the other one from
Meropenem sensitive strain of
Klebsiella pneumonia figure (8). PCR
assay screening for MBL gene was
performed for all 177 isolated Gram
negative bacteria and 25 Gram negative
stock samples which were stored at 25
°C since 2008-2010 in laboratory of
Emergency and Plastic Surgery Hospital
in Sulaimani. Out of 177 Gram negative
bacteria isolated from burn patients,
46(25.9%) isolates were positive for
different MBL genes by PCR , from
which 39(22%) isolates were from
Meropenem resistant strains and
7(3.9%) from Meropenem sensitive
strains as in table (9). Statistically
significant correlations were found
between PCR results and Meropenem
susceptibility (p< 0.05).
Genotypic detection of MBL gene in
Gram negative bacteria isolated from
stocks culture.
Polymerase chain reaction was performed
for the detection of different MBL genes
(blaIMP, VIM, NDM) in 25 stock samples
since 2008, and all these stocks were from
multidrug resistant strains of severely
burned patients from ICU and ABU in
Emergency and Plastic Surgery Hospital in
Sulaimani. MBL genes were isolated form
17 Gram negative out of 25 Gram negative
stock cultures. Out of 25 stock samples
from different Gram negative bacteria,
17(68%) strains were MBL producers by
PCR from which blaIMP, account for
11(44%), bla VIM12 (48%) and 2(8%) for
blaNDMas clarified in table (9) with length
amplified genes (230) bp for IMP and (390)
bp for VIM and ( 621) bp for NDM. The
times of isolation of five strains date back
into 2008, three strains from 2009, and nine
strains from 2010. MBL genes were
detected among 6(24%) isolates of P.
aeruginosa, 5(16) of A.baumannii and
14(32%) were K. pneumoniae as figure (9)
and table (9).
Comparing the phenotypic and gene
detection methods, it was found that each
test showed different results for MBL. The
highest percentage of phenotypic detection
of MBL (20.9%) was observed by using
CMDT, and the highest percentage of MBL
gene such as blaIMP 30(16.9) was mostly
observed among A.baumannii 19(10.7) as
seen in table 10. while bla VIM genes were
detected in 19(10.73%) among all Gram
negative isolates specifically P. aeruginosa
(5%). Statistically, no significant relations
were observed between each gene with
phenotypic test (p>0.05).
Table (3): Relation of bacterial culture with types of specimens.
Specimens
Positive culture
Negative culture*
Total
No.(%)
No.(%)
No. (%)
Wound swab
192 (83.48)
9 (3.91)
201 (87.39)
Tissue biopsy
19 (8.26)
10 (4.35)
29 (12.61)
Total
211 (91.74)
19 (8.26)
230 (100)

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Journal of Kirkuk Medical College
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Figure (1): Number of Gram negative bacteria isolated from hospitalized burn patients.
Table (4): Antibiotics Susceptibility of Enterobacteriaceae.
Antibiotic
names
Klebsiella
E.coli
Enterobacter
Morganella
Providencia
Proteus
R
I
S
R
I
S
R
I
S
R
I
S
R
I
S
R
I
S
AN
No.
21
4
19
5
0
6
6
1
11
0
2
2
1
0
2
0
0
2
%
47.73
9.09
43.18
45.45
0
54.55
33.33
5.56
61.11
0
50
50
33.33
0
66.67
0
0
100
ATM
No.
34
1
9
9
1
1
12
0
6
4
0
0
1
0
2
2
0
0
%
77.27
2.27
20.45
81.82
9.09
9.09
66.67
0
33.33
100
0
0
33.33
0
66.67
100
0
0
CZ
No.
36
1
7
9
0
2
15
0
3
4
0
0
1
0
2
2
0
0
%
81.82
2.27
15.91
81.82
0
18.18
83.33
0
16.67
100
0
0
33.33
0
66.67
100
0
0
CTX
No.
34
2
8
10
0
1
13
0
5
4
0
0
1
0
2
2
0
0
%
77.27
4.55
18.18
90.91
0
9.09
72.22
0
27.78
100
0
0
33.33
0
66.67
100
0
0
IMP
No.
5
1
38
1
0
10
0
1
17
1
0
3
0
0
3
1
1
0
%
11.36
2.27
86.36
9.09
0
90.91
0
5.56
94.44
25
0
75
0
0
100
50
50
0
MEM
No.
6
1
37
2
0
9
1
0
17
0
0
4
0
0
3
0
0
2
%
13.64
2.27
84.09
18.18
0
81.82
5.56
0
94.44
0
0
100
0
0
100
0
0
100
CAZ
No.
35
1
8
8
1
2
11
0
7
4
0
0
1
0
2
1
1
0
%
79.55
2.27
18.18
72.73
9.09
18.18
61.11
0
38.89
100
0
0
33.33
0
66.67
50
50
0
TMP
No.
31
4
9
10
0
1
8
3
7
1
0
3
1
0
2
1
1
0
%
70.45
9.09
20.45
90.91
0
9.09
44.44
16.67
38.89
25
0
75
33.33
0
66.67
50
50
0
TZP
No.
31
0
13
6
1
4
14
0
4
0
3
1
1
0
2
2
0
0
%
70.45
0
29.55
54.55
9.09
36.36
77.78
0
22.22
0
75
25
33.33
0
66.67
100
0
0
AMC
No.
37
0
7
9
1
1
16
0
2
4
0
0
1
0
2
2
0
0
%
84.09
0
15.91
81.82
9.09
9.09
88.89
0
11.11
100
0
0
33.33
0
66.67
100
0
0
CIP
No.
9
5
30
5
1
5
6
5
7
0
1
3
1
0
2
1
0
1
%
20.45
11.36
68.18
45.45
9.09
45.45
33.33
27.78
38.89
0
25
75
33.33
0
66.67
50
0
50
FEP
No.
34
0
10
10
0
1
12
1
5
1
2
1
1
0
2
2
0
0
%
77.27
0
22.73
90.91
0
9.09
66.67
5.56
27.78
25
50
25
33.33
0
66.67
100
0
0
FOX
No.
37
0
7
7
1
3
13
2
3
0
4
0
1
0
2
2
0
0
%
84.09
0
15.91
63.64
9.09
27.27
72.22
11.11
16.67
0
100
0
33.33
0
66.67
100
0
0
CM
No.
34
0
10
7
0
4
13
1
4
1
0
3
1
0
2
2
0
0
%
77.27
0
22.73
63.64
0
36.36
72.22
5.56
22.22
25
0
75
33.33
0
66.67
100
0
0
TC
No.
36
0
8
7
0
4
12
1
5
4
0
0
0
0
3
0
0
2
%
81.82
0
18.18
63.64
0
36.36
66.67
5.56
27.78
100
0
0
0
0
100
0
0
100
48
44
44
18
11
4
3
2
1
1
1
Peudomonas species
Acinetobacter species
Klebsiella pneumonia
Enterobacter cloacae
Escherichia species
Morganella morganii
Providencia rettegeri
proteus mirabilis

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Journal of Kirkuk Medical College
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Table (5): Antibiotics Susceptibility of non fermenter gram negative bacteria.
Antibiotic
Name
Pseudomonas
Acinetobacter
Barkholderia
Achromobacter
Ralastonia
R
I
S
R
I
S
R
I
S
R
I
S
R
S
AN
No
29
2
17
40
0
4
1
0
0
1
0
0
1
0
%
60.42
4.17
35.4
90.91
0
9.09
100
0
0
100
0
0
FEP
No
32
1
15
39
2
3
0
0
1
1
0
0
0
0
%
66.67
2.08
31.3
88.64
4.55
6.82
0
0
100
100
0
0
COL
No
3
0
45
5
1
38
0
0
1
0
0
1
0
1
%
6.25
0
93.8
11.36
2.27
86.4
0
0
100
0
0
100
0
100
IMP
No
11
5
32
21
5
18
0
0
1
0
0
1
0
0
%
22.92
10.4
66.7
47.73
11.36
40.9
0
0
100
0
0
100
0
0
MEM
No
25
3
20
30
4
10
0
0
1
0
0
1
0
1
%
52.08
6.25
41.7
68.18
9.09
22.7
0
0
100
0
0
100
0
100
TZP
No
35
1
12
41
0
3
1
0
0
1
0
0
1
0
%
72.92
2.08
25
93.18
0
6.82
100
0
0
100
0
0
100
0
TOB
No
34
0
14
36
0
8
1
0
0
1
0
0
1
0
%
70.83
0
29.2
81.82
0
18.2
100
0
0
100
0
0
100
0
CIP
No
24
5
19
36
5
3
0
1
0
0
1
0
1
0
%
50
10.4
39.6
81.82
11.36
6.82
0
100
0
0
100
0
100
0
CAZ
No
31
0
17
36
5
3
1
0
0
0
1
0
1
0
%
64.58
0
35.4
81.82
11.36
6.82
100
0
0
0
100
0
100
0
CM
No
34
3
11
35
1
8
1
0
0
1
0
0
1
0
%
70.83
6.25
22.9
79.55
2.27
18.2
100
0
0
100
0
0
100
0
TC
No
39
0
9
41
0
3
1
0
0
1
0
0
1
0
%
81.25
0
18.8
93.18
0
6.82
100
0
0
100
0
0
100
0
CTX
No
33
0
15
43
0
1
1
0
0
1
0
0
1
0
%
68.75
0
31.3
97.73
0
2.27
100
0
0
100
0
0
100
0
AMS
No
25
4
19
11
4
29
1
0
0
1
0
0
0
1
%
52.08
8.33
39.6
25
9.09
65.9
100
0
0
100
0
0
0
100
TMP
No
26
5
17
40
2
2
0
1
0
1
0
0
1
0
Table (6): Numbers and percentages of Carbapenem resistance among Gram negative bacteria.
Bacterial species
IMP
No. (%)
MEM
No.(%)
Both
No.(%)
Carbapenem
resistance
No. (%)
Total
Pseudomonas species
1(0.56)
17(9.6)
10(5.65)
28(15.82)
48 (27.12)
Acinetobacter species
1(0.56)
10 (5.65)
20(11.3)
31(17.51)
44 (24.86)
Klebsiellapneumonia
1(0.56)
2(1.12)
4(2.25)
7 (3.95)
44 (24.86)
Enterobacter cloacae
-
1(0.56)
-
1 (0.56)
18 (10.17)
Escherichia coli
-
1(0.56)
1(0.56)
2 (1.11)
11 (6.21)
Morganellamorganii
1(0.56)
-
-
1 (0.56)
4 (2.25)
Providenciarettegeri
-
-
-
-
3 (1.69)
Proteus mirabilis
-
-
-
-
2 (1.12)
Barkholderiapseudomallei
-
-
-
-
1 (0.56)
Achromobacterxylosoxidans
-
-
-
-
1 (0.56)
Ralastoniapauculae
-
-
-
-
1 (0.56)
Total
4 (2.26%)
31 (17.51%)
35 (19.77%)
70 (39.55)
177 (100)

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Vol. 7, No. 1, 2019
Figure (2): Double disk synergy test for MBL.positive MBL test indicated by synergy
between Meropenem disk and EDTA disk (black arrow).
Figure (3): Combined EDTA disk testA: EDTA mixed with Meropenem disk, B: Meropenem
disk, positive MBL test indicated by zone of inhibition around combined disk is > 7mm to
that of meropenem disk alone.
Figure (4): Photograph of Modified Hodge test.
A: Klebsiellapneumoniae (positive), B: Acinetobacterbaumannii (negative), C: Pseudomonas
aeruginosa (negative), D: E. coli (negative). Positive MBL: indentation of the zone of
inhibition along the tested bacteria (A) designated by black arrow towards Meropenem disk.
B
A
D
A
B
C
Meropenem disk

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Journal of Kirkuk Medical College
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Table (7): Phenotypic test for MBL detection.
Figure (5): PCR products for MBL genes.
A: 1. DNA ladder (1 Kbp), 2: Klebsiellapneumoniea negative, 3. K. pneumoniae
positive blaIMP, VIM, NDM (red arrow), 4. Pseudomonas aeruginosanegative, 5.
AcinetobacterbaumanniiblaIMP, bla VIM positive, 6. AcinetobacterbaumanniiblaIMP
positive, 7 and 8: Acinetobacterbaumanniinegative, 9. Pseudomonas
aeruginosapositive blaVIM.
Meropenem resistance Bacterial species
Positive phenotypic test
Hodge test
DDST
CMD
pseudomonas aeruginosa
8
12
25
Acinetobacterbaumannii
7
22
26
Barkholderiapseudomallei
0
0
0
Klebsiella pneumonia
4
3
3
Enterobacterclocca
0
0
0
Escherichia coli
1
1
1
Total
20
38
55
500 bp
621 bpblaNDM
1
2
3
4
6
9
390 bpblaVIM
232bpblaIMP
5
7
8
1500 bp
400 bp
300 bp
200 bp
100 bp
500 bp
600 bp

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Journal of Kirkuk Medical College
Vol. 7, No. 1, 2019
Table (8): Different MBL genes amplified using PCR .
MBL
genes
Bacterial species
blaIMP,
No. (%)
bla VIM
No.(%)
blaNDM
No.(%)
Total gene
isolated
Total No. of
bacteria
Pseudomonas species
7 (3.95)
9 (5.08)
-
16 (9.04)
48 (27.12)
Acinetobacter species
19 (10.73)
5 (2.82)
-
24 (13.56)
44 (24.86)
Klebsiella pneumonia
7 (3.95)
5 (2.82)
2 (1.1)
14 (7.91)
44 (24.86)
Enterobacter cloacae
-
-
-
-
18 (10.17)
Escherichia species
-
-
-
-
11 (6.21)
Morganella morganii
-
-
-
-
4 (2.25)
Providencia rettegeri
-
-
-
-
3 (1.69)
proteus mirabilis
-
-
-
-
2 (1.12)
Barkholderia pseudomallei
-
-
-
-
1 (0.56)
Achromobacter xylosoxidans
-
-
-
-
1 (0.56)
Ralastonia paucula
-
-
-
-
1 (0.56)
Total
33 (18.64)
19 (10.73)
2 (1.12)
54 (30.51)
177 (100)
bla :β- lactamase gene , VIM: Verona integron metallo β- lactamase, IMP: Imipenemase,
NDM: new delhi metallo β- lactamase.
Figuers (6): Sequence alingment of three sequence data from forward primers of blaVIM .
Using DNAMAN sequence alingment, A homology of (96.77%) was obtined.

177
Journal of Kirkuk Medical College
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Figuers (7): Sequence alingment of three sequnece data from revers Primers of blaVIM gene.
DNAMAN sequence alingment. A homology of (98.14%) was obtined.
Figure (8): PCR product on Gel electrophoresis for MBL genes among burn patients.
lane 7: DNA ladder , lane 6 : blaNDM positive K.pneumoniae, lane2, 3, 5, 6 blaIMP positive P.
aeruginosaand A. baumannii, lane 4,1 :390 bpbla VIM positive (red arrow ) P. aeruginosa.
500bp
400bp
300bp
200bp
600bp
700bp
1500bp
390 bpblaVIM
232 bpblaIMP
1
5
6
7
3
4
2
621 bpblaNDM

178
Journal of Kirkuk Medical College
Vol. 7, No. 1, 2019
Figure (9): PCR product among stock cultures.
Lane 1: DNA ladder, 2, 3, 4, 5, blaIMP positive A.baumanii and P.aeruginosa , lane 6: blaVIM
positive K.pneumonia,
Table (9): MBL genes detection by PCR among stock culture.
MBL
genes
Bacterial species
blaIMP,
No. (%)
bla
VIMNo.(%)
blaNDM
No.(%)
Total gene
isolated
Total
bacterial No.
Pseudomonas aeruginosa
3 (12)
3 (12)
-
6 (20)
6 (24)
Acinetobacterbaumannii
2 (8)
3 (12)
-
5 (16)
5 (20)
Klebsiellapneumonae
6 (24)
6 (24)
2 (8)
14 (32)
8 (32)
Enterobacter cloacae
-
-
-
-
3 (12)
Escherichia coli
-
-
-
-
3 (12)
Total
11 (44)
12 (48)
2 (8)
25 (100)
25 (100)
Relation of PCR with different phenotypic tests used for the detection of MBL genes.
Table (10): Prevalence rate of MBL enzyme by phenotypic and PCR assay among burn patients.
Gram negative bacteria
Positive MBL genes
positive phenotypic test of MBL
Total No. of
Gram
negative
No. (%)
No. (%)
blaVIM
blaIMP
blaNDM
DDST
CMDT
MHT
Pseudomonas species
9 (5.08)
7 (3.95)
-
15(8.47)
25(14.12)
8(4.51)
48 (27.12)
Acinetobacter species
5 (2.82)
19 10.73)
-
28(15.82)
26( 14.69)
9 (5.08)
44 (24.86)
Klebsiella pneumonia
5 (2.82)
7 (3.95)
2 1.13)
7 (3.95)
3 (1.69)
18 (10.17)
44 (24.86)
Enterobacter cloacae
-
-
-
-
1 (0.56)
18 (10.17)
Escherichia species
-
-
-
1 0.56)
1 (0.56)
1 (0.56)
11 (6.21)
Morganellamorganii
-
-
-
-
-
-
4 (2.26)
Providenciarettegeri
-
-
-
-
-
-
3 (1.69)
Proteus mirabilis
-
-
-
-
-
-
2 (1.12)
Burkholderiapseudomallei
-
-
-
-
-
-
1 (0.56)
Achromobacterxylosoxidans
-
-
-
-
-
-
1 (0.56)
Ralastoniapaucula
-
-
-
-
-
-
1 (0.56)
Total
19(10.73)
33(18.64)
2 1.12)
51 (28.8)
55 (31.07)
37 (20.9)
177 (100)
DDST: Double disk synergy test, CMDT: combined disk test, MHT: Modified hodge test, E-
test: Episilometer test, bla :β- lactamase gene , VIM: Verona integrin metallo β- lactamase,
IMP: Imipenemase, NDM: new delhimetallo β- lactamase. p >0.05
390 bpblaVIM
232 bpblaIMP
1500 bp
500 bp
100 bp
200 bp
300 bp
400 bp
600 bp
1
1
2
1
6
1
5
1
3
1
7
1
8
1
4
1

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Journal of Kirkuk Medical College
Vol. 7, No. 1, 2019
Discussion:
One of the risk factors for bacterial
infection of burn is the duration of
hospitalization, as the mean of hospital
stay in this study was 9 days which was
a too long period for the acquisition of
bacteria from different sources such as
the environment or patients in burn
units. This was in agreement with study
done in Tunisia by Fekih et al. (22). Other
risk factors are acquisition of infection
from relatives of patients or health care
workers, inadequate sterilization of the
burn surface which provides a suitable
site for bacterial colonization and
multiplication that is rich in protein as it
was opened into environment and is
easily contaminated (23). The risk factors
for nosocomial infection among burn
patients are unlike to what were
concluded in Turkey by Alp et al., (24) in
which advanced age, and underlying
disease were found to be risk factors for
nosocomially infection among burn
patients. The relative frequency of
bacterial isolation in burn patients varies
among different studies and different
countries. In this study, Gram negative
bacteria (76.9%) were isolated from
burn wound infection but it was higher
than what was recorded in Erbil (43%)
by Hussen (25), in Karbalae (67.44%) by
Kawkab, (26), in which Gram negative
bacteria were regarded as a common
microorganism isolated from burn
patients but with different prevalence
rate that may be due to differences in
samples size.
In the present study, P. aeruginosawas
the most frequently isolated gram
negativebacteria (27.1%) over all gram
negative bacteria from burn patients,
which was in agreement with studies
done in Hilla from Iraq by Nktel et al.,
(27) and in India (28) in which
Pseudomonas was the most commonly
isolated microorganism but with high
prevalence rate in India (55%) and in
Iraq (32%),The second bacteria isolated
in this study wasAcinetobacter spp.
(24.86%) as A. baumanniiwas the
commonest isolates and mostly occur
among seriously ill patients indicating
that the group of non- glucose
fermenting gram negative bacteria was
the most frequently isolated among
hospitalized burn patients. This is
similar to what was concluded by
Vitkauskienėet al. (29) in which
prevalence rate of (34%) among burn
patients, This results disagree entirely to
what was identified in United Kingdom
regarding types of microorganisms
isolated among burn patients as a cause
of burn wound infection such as
Stenotrophomonasmaltophilia, Vibrio
species Chryseobacteriumspecies,
Alcaligenesxylosoxidans (30) which
might be due to changing
microorganisms in this country
regarding microbial flora or
environmental distribution of Gram
negative bacteria.
Colonization of skin also remains one of
the risk factors for infection by
Acinetobacterspecies as recorded in the
studies done by Balkhyetal., (31) in Sudia
Arabia in which colonization of the skin
prior to the burn injury was regarded as
one of the risk factors for burn wound
infection. The family of
Enterobacteriaceae comes after the non
fermenter group in which K. pneumonia
was the most frequent bacteria (24.8%)
followed by E. cloaca (10.2%), E. coli,
M. morganiiand Proteus species. Most
of the bacterial isolates in this study
were similar in pattern to bacteria
isolated from other studies in Iraq such
as Baghdad and Mosel and Saudi Arabia
(32) in that Enterobacteriaceae comes

180
Journal of Kirkuk Medical College
Vol. 7, No. 1, 2019
after the group of non fermenter apart
from absence of Acinetobacterspecies
while there are other studies done in
neighboring countries such as Turkey by
Oncul et al., (33) which recorded the
pattern of gram negative bacteria among
burn patients with P. aeruginosaasthe
commonest isolates followed by K.
pneumoniae, A.
baumanniiandproteusspp. But in Iran the
same patterns of Gram negative bacteria
were recorded with addition of E.coli
(34). The reason for this high prevalence
of gram negative microorganisms may
be related to their virulence factors, and
prevalence in hospital environment, to
the source of infection that may arise
from overcrowding in the hospital
wards, frequent visitors to the burn
patients, colonization by normal flora
such Acinetobacter species.
The result of antimicrobial sensitivity
showed that P. aeruginosa and all other
non-fermenter gram negative bacilli
were highly resistant to Pipracillin,
Tazobactum and Ticarcillin in the range
of (72-81%) and for third generation
cephalosporin from (63-69%). This is in
agreement with a study done in India in
which resistance against third generation
Cephalosporin such as Ceftazidim
(68%) was recorded (28) while
Pseudomonas species were
sensitiveagainst Colistin and Imipenem.
Our result in this study that in
agreement with what was done by
Branskiet al., (35) in Texas in which
Colistin was the sensitive drug (100%)
against all MBL producing
Pseudomonasspecies. Resistance was
highly detected against most of the
currently used antimicrobial drugs such
as Cefotaxim, Cefoxintin,
Ciprofloxacillin making the outcome
serious regarding treating the patients in
burn unit. These results were in
agreement with those done in USA, Iran
and Nigeria in which most of the
bacteria isolated from burn unite were
MDR and posses resistance to
floroquinolon and third generation
cephalosporin drugs while Imipenem
drugs give sensitivity (66.67%) for
Pseudomonas specieswhich was higher
to a study done in Tunisia by
Zoghlamiet al,. (36) as they were
recording (31%) sensitivity to Imipenem
drug.
Carbapenems still remain a major issue
in the management of hospitalized
patients and are increasingly used
because of the emergence of ESBL in
Enterobacteriaceae, particularly among
E. coli and K. pneumonia (37) also as the
last resort for treating infections caused
by organisms producing ESBL such as
P. aeruginosa and antibiotic resistant
Acinetobacterspp. (38). This jeopardizes
the effective use of Carbapenem and
may lead to the generation of a new
class of Gram negative ―superbugs‖ of
particular concern which are often
commensals. So, the risk of spread from
asymptomatic carriers outside of the
hospital setting is high (39). In this study,
Carbapenem resistance was detected in
70(30.4%) among Gram negative
bacteria isolated from burn patients and
66(28.6%) of these isolates were
Meropenem resistant strains. These
results unlike the study done in Turkey
in which Carbapenem still remains the
therapeutic choice in the burn hospital
(40). Out of 70 Carbapenem resistant
strains, 30 isolates of Acinetobacter spp.
and 28 of pseudomonas spp. were
meropenem resistant strains. This
indicates that among Gram negative
bacteria isolated from burn patients,
58(82.8%) were non fermenter Gram
negative bacilli with Meropenem
resistant strains which disagrees with

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what was recorded in India by Goel et
al., (41) in which Meropenem resistant
was only observed among
Acinetobacterspecies (18%) while all
Pseudomonas specieswere sensitive to
it. Another study done in China (24)
revealed that all A. baumannii isolated
from burn patients were (100%)
sensitive to Meropenem. It was clear
that most of the Meropenem resistance
isolates in this study showed resistance
to more than 6 drugs from different
classes of antibiotics that were
designated as MDR strain. This finding
was the same as other studies done in
Tehran, Israel, Sweden and Sanandaj
from Iran (4) and the reasons for the
increase in prevalence of MDR are multi
factorial and it will differ according to
the geographical area such as
endogenous colonization or exogenous
spread from hospital environment
having a high burden of MDR patients,
ground water or other water sources
contaminated because of inadequate
waste management or sewage disposal
system and miss uses of antibiotic (43).
The importance of surveillance is
underscored by the fact that the
appearance of acquired carbapenemase
in different countries has been
associated with imported cases, mainly
due to the transfer of patients from
geographical areas where this problem
is widely established. Demographic
change, environmental changes,
improved medical technology, bacterial
evolution, and the breakdown of public
health systems, urban migration.
Double disk synergy test detect MBL in
51(28.8%) out of all gram negative
bacterial strains in which (95%) of
Meropenem resistant isolates showed a
significant zone of enhancement toward
EDTA disk which was in agreement
with a study done in Ahwaz and
Teheran (44) which detected (26.5%),
Beside using DDST, other tests, such as
CMDT, were used which show high
positive results in (98%) of Meropenem
resistance strains with zone of inhibition
> 7 mm. Interpretation of the CMDT
assay results is more objective than that
of the DDST results according to what
was concluded previously (45), because
the DDST depends upon the technician's
expertise in discriminating true
synergism from intersection of
inhibition zone whereas CMDT is easy
to be interpreted as it needs no
synergisms just an increase zone of
inhibition around the combined
Meropenem disk by EDTA. The
prevalence rate of CMDT in the current
study was 55(31.07%) among all Gram
negative bacteria which was in
agreement with that done by Eser et al.,
(46) which found (76%) positive CMDT
among Gram negative bacteria but it
disagrees with what was recorded by
Guptaet al., (47) in which the sensitivity
of CMDT was equal to that of E-test.
MHT was detected in 37(20.9%)
isolated Gram negative bacteria from
which 20(30.3%) isolates were from
Meropenem resistance strains and
19(17.1%) from Meropenem sensitive
strains that were isolated from different
units in burn hospital. These results
indicate that the positive MHT test
among Meropenem sensitive strains was
indicative of MBL positive strains or
presence of other types of β-lactamase
enzymes such as class A
Carbapenemase KPC which is in
agreement to studies done in India and
Iran (48) which concluded that MHT test
is a good tool for the diagnosis of MBL
even in small percentages (12% and
16%). Out of all Gram negative isolates,
K. pneumonia was found to be positive
for MHT test at 17(9.6%) out of all

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Meropenem sensitive and resistant
strains. This result was similar to study
done in Pakistan but with a higher MHT
positive (27%) among Gram negative
bacteria isolated from burn patients (18)
while lower percentage of MHT positive
test was observed among non fermenter
group such as Acinetobacter and
Pseudomonas species which were lower
than K. pneumonia in the current study.
PCR detection of three MBL genes
yielded many positive results. Obtaining
the expected size of amplicon was
considered as an indicator for the gene
presence. These amplicon sizes were
consistent and the same results were
obtained when PCR was repeated on the
same samples. For blaVIM gene, three
different amplicons from different
isolates were sequenced and the data
showed homology to genes documented
to be for MBL resistance gram negative
bacteria and all three amplicons, when
aligned together, show homology of
more than (96%) indicating to be for the
same gene in different isolates. In this
study, the prevalence of MBL
production by using PCR was different
according to each gram negative
bacteria isolated among burn patients
but in general it was demonstrated in
46(25.9 %) isolated among all gram
negative isolates in which 39(22 %)
isolated were from Meropenem resistant
strains and 7(3.9%) from Meropenem
sensitive strains. These results indicate
that most of the Meropenem resistant
strains from infected burn wound strains
in this study were producing MBL
enzymes which were distributed in all
the burn units of the hospital especially
in the ICU (41.3%), ABU (39.1%), and
PBU (13%) but plastic units harbor the
least number of MBL producers that
indicates most of the MBL produce
strains distributed among severely
burned patients with weak immune
system and they were at the risk of
acquiring these resistance genes. Similar
studies were done in other countries
with different prevalence rates such as
(16%) in Italy, (17.8%) in UK and
(30%) in India (28). The presence of
MBL genes among Meropenem
sensitive strains indicates that there
might be a hidden MBL gene among
isolated strains which cannot be
diagnosed by phenotypic tests leading to
the dissemination of these genes in the
hospital silently among patients even
within normal health workers who act as
carriers for MBL genes in future. Other
causes are due to the fact that in this
study Meropenem and Imipenem were
selected to be tested against isolated
bacteria as an example of Carbapenem
agent, but there were other generations
such as Etrapenem and Doripenem
which were not used and resistance
might be detected in these 7 cases of
MBL positive strains. Our results
revealed that there was a difference in
the prevalence of MBL production
among each Gram negative bacteria
isolated. (56.8%) of A.baumannii were
MBL producer by PCR reaction which
was higher in comparison to other
bacteria in the hospital although the
main mechanism of Carbapenem
resistance in Acinetobacter species is
class D blaOXAcarbapenemase
specifically blaOXA51 like which is
intrinsic to the most species of
Acinetobacterbaumannii (49). MBL
producers were (33.3%) for
P.aeruginosa while (31.8%) of
K.pneumoniae were recorded among all
isolated Klebsiella species. These
findings disagree with what were found
by other workers in Iran (50) in which
Pseudomonas species was the most
frequent MBL producer among Gram

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negative bacteria and even within its
own species at the rate of (68%).
Regarding MBL gene, three types of
MBL genes blaIMP, bla VIM, and bla
NDM were isolated from the current
study. The most frequently detected
gene was blaIMP gene 33(18.6%)
isolate among all Gram negative
bacteria isolated from burn patients.
Similar results were recorded in Turkey
and Spain (51) in which the detection of
MBL genes (blaIMP, bla VIM) were
observed but in lower percentages to
that of our study being (2.5%) and
(11%) respectively. it was commonly
detected in 19(75%) of A. baumannii
isolates while 7 isolates from each P.
aeruginosa and K. pneumoniae were
found to be positive for blaIMP gene
which is consistent with a study done in
Turkey at different periods observing
(39%) which are much lower than our
results (46). On the other hand, bla VIM
gene was recorded in 19(10.7%) among
all Gram negative bacteria isolated from
burn patients. In this context, it was
clear that the gene was present in all
hospital wards especially in the ICU and
ABU, as most of the Meropenem
resistant cases were from these two
units. This is consistent with the study in
Tehran by Bahar and
Samadikuchaksaraei, (52) and the first
report published from newly hospital in
Iraq from Baghdad by Huang et al., (53)
in which they recorded bla VIM to be
(11%) and (12.3%) respectively.
Though bla VIM is distributed all over
the world, different areas in the world
were recorded to carry this gene blaVIM
especially in neighboring countries at
various prevalence rates lower than our
study which were (2.3%) for bla VIM-2
in Teheran (54), and (6.7%) in India (55).
The cause of lower prevalence rate of
bla VIM in those studies in spite of
larger sample size is due to restriction of
work in P. aeruginosa only from burn
unit. There were 2 cases of newest
superbug gene blaNDM (NDM)
depending on the amplicon size of the
PCR products (621bp). All of the two
isolates were from Klebsiella
pneumonia thatneed further work, due to
the fact that the PCR reaction for MBL
detection in this study was multiplex
reaction which faced difficulty during
sequencing process. This superbug gene
disseminates rapidly in all the parts of
the world as it was identified in thirteen
countries in Europe (56) over the last
three years, and recorded by researchers
in the world, majority of cases were in
the UK (57). Apart from European
countries, blaNDM gene was
disseminated in all other neighboring
countries such as one case report in Iran
by Gaibani et al., (58), Turkey by Poirel
et al., (59) and there was a single report
from each of Israel, Oman, United Arab
Emirates and Iran, but they were few in
numbers in comparison to blaVIM and
blaIMPgenes. Although blaNDM gene
is a new type of MBL gene but it is
present and it is likely that the number
of cases reported is underestimated in
most countries in which they do not
have an idea about the gene and
confirmation tests that are routinely
performed for MBL or there may be
diagnosed cases but they were not
notified by Reference Laboratories. Till
now there are no published cases in Iraq
but there are two cases reports published
in France and Lebanon from Iraqi
patients travelling via medical tourists to
these countries. They were diagnosed as
MDR strains with blaNDM gene
positive. This result indicates that this
new superbug gene was present in Iraq
and is regarded as a source of
international disseminations and may be

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this MDR gene is transmitted to our
locality from tourists of other
neighboring countries such as Iran,
Turkey and South and Middle of Iraq
and transmission mostly occurs by
plasmid caring genes encoding for MBL
enzyme. Regarding those Meropenem
resistant bacterial strains that give
negative results for PCR reaction for
MBL genes, the causes might be due to
the presence of other types of MBL
gene that are responsible for β-
lactamase production such as class A
Klebsiella pneumonia carbapenemase
(KPC) enzyme or the presence of
blaoxagenes which are specific to
A.baumanniiespecially blaoxa 23, and
blaoxa 48, and blaoxa 51(60). Apart
from being Carbapenem-resistant, all
MBL positive strains were resistant to
important groups of tested
antimicrobials, including third
generation Cephalosporins,
Aminoglycosides and which are a
characteristic feature of MBL-producers
which supports the results recorded in
Iran by Peshattiwar and Peerapur (61) as
they recorded resistant isolates to
multiple classes of antimicrobial agents
in spite of MBL positive. All positive
MBL in this study had hospital stay for
more than 9 days and with an invasive
device such as IV line, urinary catheter
which was regarded as a major risk
factor in combination of TSAB of the
MBL patients.
MBL genes were also isolated from
different Gram negative bacterial stock
culture from previous years since 2008
in Emergency and Plastic Surgery
Hospital in Sulaimani indicating that
MBL genes were present in this hospital
and were circulating in environment of
the hospital. MBL genes were isolated
form 17 samples out of 25 stock cultures
selected randomly among Gram
negative bacteria, dating back to 2008,
2009 and 2010 indicating that these
genes were present since these years and
continuously circulate in the hospital
environment that acts as source of MBL
genes in this hospital. The
microorganisms that were positive for
MBL gene from stock culture were the
same of the current study.
In this study, the evaluation of simple
phenotypic tests for MBL detection such
as DDST, CMDT, MHT was done for
the comparison of the efficacy and
sensitivity of PCR for MBL gene
detection. It was found that (94.8%) of
all Gram negative bacteria with MBL
positive by PCR were positive by
CMDT and (64.1%) were recorded for
DDST, but only (28.2%) of MBL
positive by PCR were positive by MHT
test which is in agreement with two
studies performed in India. One of them
observed the strong relation between the
phenotypic tests and PCR results but
with restriction of the study to the
Pseudomonas species only, and (100%)
sensitivity of DDST observed among
PCR positive and (92%) for E-test (62),
The rate of sensitivity was higher than
this study which may be due to the fact
that all the tested strains were MDR non
fermenter group in contrast to this study
in which all isolated Gram negative
bacteria (Meropenem resistant and
Meropenem sensitive strains ) were
screened by PCR.
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