Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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DETECTION OF bla-VIM1GENE IN CARBAPENEMRESISTANT
Pseudomonas aeruginosa ISOLATED FROM
CLINICAL SAMPLES IN WASIT PROVINCE HOSPITALS.
Zeyad Khalaf Hussein ,Israa Jabbar Shamkhi
Department of Bacteriology, Al–Zahraa Teaching Hospital ,Wasit ,Iraq
Keywords; P. aeruginosa; Carbapenems; Metallo-beta-lactamases.
Corresponding Author: [email protected]
ABSTRACT
The present study investigated the presence of bla-VIM1in clinical isolates of
Pseudomonas aeruginosa. During the period from November 2017 to February 2018,
a total of two hundred patients admitted to (Al-Zahraa Teaching Hospital, Al-Karama
Teaching Hospital and Al-Kut Hospital for Gynecology, Obstetrics and Pediatrics) in
Wasit province. One hundred and three of isolates were diagnosed as P. aeruginosa.
High prevalence of P. aeruginosa isolates were detected in burn swab samples 70
(35%) followed by sputum 12 (6%) and ear 11 (5.5%). All 103 P. aeruginosa isolates
were primarily screened for carbapenems - resistance, 36 (34.95%) were resistant to
carbapenems. Carbapenems resistant isolates were underwent antimicrobial
susceptibility to 14 antibiotics using Kirby-Bauer disk diffusion method. The
phenotypic and molecular methods of carbapenem resistance were investigated.
Carbapenems resistant isolates were identified by the Double disc synergy test, which
detects the probability of isolates able to produce Metallo-beta- lactamases (MBL).
Out of the isolates 32 (88.89%)were positive. Imipenem-EDTA combined disk
method showed 30 (83.33%) of isolates possessed ability to produce the Metallobeta-
lactamases. In addition , the Modified Hodge test (MHT) showed the ability of
isolates to produce Carbapenemases enzyme. 16 (44.44%) of the isolates were
positive to Modified Hodge test. Carbapenemase gene were detected by PCR
technique. The results demonstrated that out of 36 carbapenems resistant
Pseudomonas aeruginosa (CRPA) isolates 34 (94.44%) were positive to bla-VIM1
gene.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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INTRODUCTION
Pseudomonas aeruginosa is an aerobic Gram-negative rod-shaped. It is
widely distributed in nature and can adapt to many environments, it can be isolated
from nearly any conceivable source within hospitals (1). It is an important cause of
both community and hospital-acquired infections. Infections with these bacteria have
been associated with high mortality and morbidity when compared with other
bacterial pathogens (2) P. aeruginosa infections are clinical problem, it’s a difficult to
treat because of high resistant to many antibiotics (Multi-drug resistant) and a high
risk of emergence of resistance during therapy (3).
Beta-lactam as antibacterial agent are broadly used to treat diseases caused by
Gram-negative Pathogens However, the adequacy of these medications is lessened
impressively because of the presence of extended-spectrum beta lactamases (ESBLs)
and the consequent emergence of multi-drug resistant (MDRs) strains (3).
Carbapenems are a group of β-lactam antibiotics with a broad spectrum of
antibacterial activity. Their structure makes them highly resistant for most β-
lactamases (4) They include meropenem and imipenem, which are among the few
therapeutic options still available for treating infections caused by P. aeruginosa (5).
Carbapenems are considered to be antimicrobial agents of choice and are frequently
used for the treatment of hard-to-manage P. aeruginosa infections. However,
carbapenem resistance in P. aeruginosa has been reported to increase steadily over
the years across the world, but the relative contribution of different carbapenems
resistance mechanisms is not well established (6,7). The goal of this research was to
detect the presence of bla-VIM1 producers in isolates of P. aeruginosa isolates
producer between clinical P. aeruginosa isolates in Wasit hospitals. Also, study the
relationships between the presence of resistance genes bla-VIM1and the sensitivity to
ten antibiotics.
MATERIALS AND METHODS
Collection of samples
Over four months from November 2017 to February 2018, different samples
including (burn swab, ear swab, urine, sputum and wound swab) from two hundred
patients admitted to (Al-Zahraa Teaching Hospital, Al-Karama Teaching Hospital and
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
32
Al-Kut Hospital for Gynecology, Obstetrics and Pediatrics) in Wasit province were
enrolled in this study.
Identification of P. aeruginosa
In the case of swab samples, two swabs were taken from each patient, while
sputum and urine were divided directly into two parts, the first swab was prepared for
stained smear preparation (Gram stain), and the other was used for culturing on
different culture media for further isolation and characterization of the causative
agents. The isolated bacteria were identified by standard laboratory methods and
API20E system (BioMerieux), P. aeruginosa were isolates in Brain-Heart Infusion
(BHI) broth containing 15% glycerol, and the tubes were stored in freezing at -20 C̊
(8).
Antimicrobial susceptibility testing
Resistance patterns of the P. aeruginosa isolates to different antibiotics was
determined using disk diffusion test (Kirby-Bauer) on Muller Hinton agar media (9),
the antibiotic discs used in this study were Levofloxacin (5 μg), Meropenem (10 μg),
Imipenem(10 μg), Aztreonam (30 μg), Ceftazidime (30 μg), Amikacin (30 μg),
Gentamicin (30 μg), Ciprofloxacin (10 μg), Piperacillin (10 μg), Colistin sulphate (25
μg). The standard isolate from central public health laboratory E. Coli ATCC25922
was used as a negative control. The resulting zones of inhibition were measured and
compared with the break points standard value of Clinical Laboratory Standards
Institute CLSI (9). The minimum inhibitory was determined by Vitik2 - compact
system AST card, and standard agar dilution method (10) according to the CLSI (9).
Phenotypic detection of Metallo-β-lactamases (MBL)
All imipenem and meropenem-resistant isolates were examined for MBL
production using the IMP-EDTA double disk synergy test as described by (11),
furthermore, Modified Hodge test (MHT) was used for detection of Carbapenemases
production P. aeruginosa isolates according to CLSI guidelines using 10 μg
meropenem susceptibility disk, which was placed in the center of the test area. P.
aeruginosa 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 C̊ in ambient air for 16-24 hours. After
24 hours, MHT positive test showed a clover leaf-like (12).
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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DNA Extraction and polymerase chain reaction (PCR) amplification
In this study, both plasmid DNA and chromosomal DNA were extracted,
plasmid DNA was extracted according to (13), while chromosomal DNA was
extracted by Genomic DNA Mini Kit (Genaid) according to company instructions.
All carbapenem-resistant isolates were screened by standard PCR conventional using
specific primers for blaNDM-1 gene as shown in table (1). PCR reaction tubes were
transferred into thermal cycler (eppendroff,Germany) that was programmed as
following: initial denaturation for 4 mints at 95 C̊, 30 cycles were performed (the
conditions for each cycle were: 30 sec. at 94 C̊, 1mints. at 56 C̊ and 90 sec. at 72 C̊ ),
and final extension at 72 C̊ for 5 mints. Amplified products were electrophoresed on
1.5% agarose for 90 mints at 5 V/cm.
Table 1. Sequences of primer that used in the detection blaNDM-1 gene
Gene Nucleotide sequences
(5' 3')
Products
size bp References
bla-VIM1 F
R
AGT GGT GAG TAT CCG ACA G
ATG AAA GTG CGT GGA GAC 261 (14)
Statistical Analysis
Statistical analysis was performed with Graph Pad Prism version 6 software,
percentages were used for the comparison between samples of the study. Data
analysis was done using Chi-square for the comparison of categorical data.
RESULTS
A total of two hundred samples were recorded in this study which include,
burn swabs (n=105, 52.50%), ear swabs (n=19, 9.50%), wound swab (n=36, 18.00%),
sputum from patients with lower respiratory tract infection (n=7, 3.50%) The patient's
ages ranged from one to greater than 61 years table (2).
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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Table 2. Distribution of Pseudomonas aeruginosa isolates according to age
groups
Age groups P. aeruginosa Others bacteria Negative Total
1-10 yr. 11 10 2 23
11-20 yr. 10 9 2 21
21-30 yr. 20 12 4 36
31-40 yr. 27 13 2 42
41-50 yr. 11 14 3 28
51-60 yr. 9 7 4 20
≥61 yr. 15 13 2 30
Total 103 78 19 200
One hundred and eighty one bacterial species were isolated from these samples with
the rate of P. aeruginosa (n=103, 51.50%) followed by E. coli (n=28, 14.00%) and
the lowest percentage were K. pneumoniae (n=2, 1.00%). There is non-significant
association between Pseudomonas infections and age groups.
Antimicrobial susceptibility test
The results of antibiotic susceptibility test for isolated P. aeruginosa indicated
different antibiotic profiles as shown in table (4). In total, 55.5% (n=103) resistance
to the third generation ceftazidime 57.28% of the isolates exhibited resistance to the
fourth generation cefepime. While the resistance to monobactams, aztreonam was
51.46%. The highest resistance percentage was found against gentamicin (91.26%).
According to the results of the fluoroquinolones susceptibility testing, 83.50% and
60.19% of the isolates were resistant to ciprofloxacin and levofloxacin, respectively
table (3).
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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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Table 3. Susceptibility patterns of Pseudomonas aeruginosa to different antibiotics
Antibiotic
Sensitive (S) Intermediate (IR) Resistant (R)
No. of
isolates % No. of
isolates % No. of
isolates %
Imipenem 67 65.05 0 0 36 34.95
Meropenem 67 65.05 0 0 36 34.95
Ciprofloxacin 12 11.65 5 4.85 86 83.50
Levofloxacin 38 36.89 3 2.91 62 60.19
Amikacin 9 8.74 6 5.83 88 85.44
Gentamicin 5 4.85 4 3.88 94 91.26
Cefepime 42 40.77 6 5.83 55 53.40
Ceftazidime 39 37.86 5 4.85 59 57.28
Aztreonam 40 38.83 10 9.71 53 51.46
Piperacillin 17 16.50 16 15.53 70 67.96
Piperacillin/Tazobactam 50 48.54 8 7.77 45 43.69
Colistin 102 98.03 0 0 1 0.97
Ticarcillin/Clavulanic acid 47 45.63 5 4.85 51 49.51
Ticarcillin 40 38.83 7 6.80 56 54.37
Phenotypic detection of MBLs
From 36 of P. aeruginosa carbapenem-resistant isolates, MHT revealed 16
(44.44%) were positive showing their ability to produce carbapenemases figure (1),
moreover, double disc synergy indicate that in 32 (88.89%) isolates figure ( 2), and 30
(83.33%) isolates were positive to Imipenem-EDTA combined disk test figure (3).
Those isolates, which were found MBL positive by Double disc synergy test ,
Imipenem-EDTA combined disk test and were also found to be MBL positive MHT
table (4).
Figure 1. The Modified Hodge test (MHT) of carbapenemase production
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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-ve =Negative carbapenemase production.
+ve = Positive carbapenemase production.
Figure 2. Combined disk test for P. aeruginosa (CRPA) showed positive test for
MBLs production using imipenem and imipenem with EDTA. 1- Imipenem without
EDTA. 2- Imipenem with EDTA.
Figure 3. Double disc synergy test for P. aeruginosa (CRPA) showed positive test for
MBLs production using imipenem disk and EDTA disk.
Table 4 . Phenotypic test for carbapenemase production
Test Positive Negative
Double disc synergy test 32 (88.89%) 4 (11.11%)
Imipenem-EDTA combined disk method 30 (83.33%) 6 (16.67%)
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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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modified Hodge test 16 (44.44%) 20 (55.56%)
PCR screening for NDM-1 encoding gene
PCR was carried out by using specific primers for VIM-1 and performed on
all the Carbapenems-resistant isolates for generation of specific amplification band
with certain molecular weight that were 261 bp fragment which represented bla-
VIM1gene. The results showed MBL gene bla-VIM1 (261 bp) was detected in 34
(94.44%) of the carbapenem-resistant isolates on plasmid DNA, while MBL gene
bla-VIM1gene not found on chromosomal DNA figure (1).
Figure 1. Gel electrophoresis of amplified plasmid DNA for detection of MBL bla-
NDM1 gene (261bp) using PCR with specific primers; 1,5% Agarose for 90 minutes
at 70 V\cm. Lane M: Marker DNA ladder Size (100bp), Lane C: Negative control and
Lanes (1-14) positive for bla-VIM1 (٢٦١ bp) except ( ٩,١٠)
DISCUSSION
The current emergence of P. aeruginosa carbapenem-resistant represents a
major threat to the clinical approach because it exhibits intrinsically decreased
susceptibility to a range of antimicrobials and possesses a great ability to develop
resistance to multiple classes of agents (15). Among two hundred samples were
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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recorded in this study, the mean age of the patients were 36.61 years. Results of
current study revealed that, there is non-significant (P ˃ 0.05) association between
Pseudomonas infections and age. It is noteworthy to mention that result was disagreed
with a study conducted by Magliano et al. (16) who was reported the high rate of P.
aeruginosa infection among age group ( ≥ 60 years). In the present study, P.
aeruginosa has been the predominant bacterial isolated among study group followed
by E. coli (14%) and the lowest percentage were K. pneumoniae (1%). These findings
are compatible with study conducted in Egypt (17), the present study is incompatible
with a study in Baghdad (18) who found that Acinetobacter baumannii (31% ) is
even more common than P. aeruginosa (12%), and study conducted in Baghdad
(19), who reported that S. aureus (30%) was the most common agents, then P.
aeruginosa (14.6%) this difference in results can be attributed to sample difference
and kind of test used in isolation and diagnosis of different bacterial species.
Carbapenems are a class of β-lactam antibiotics with good antimicrobial
activity against P. aeruginosa but the arises and spread of acquired carbapenemresistance
in this species have challenged the success of therapeutic and control
efforts (20). Result in current study showed there was no difference found in activity
of imipenem and meropenem to P. aeruginosa (both of them have the same
percentage 34.95% resistant, respectively), which disagree with Gupta et al. 2006
who found that the imipenem had a better activity than meropenem (21). Furthermore,
current finding indicated that higher resistance against imipenem and meropenem
have compared with study in Najaf (22), who reported that the resistance rate was
7.4% and 14.8%, respectively. The percentage of fluoroquinolone-resistant isolates
was 83.50% and 60.19% of isolates resistant to ciprofloxacin and Levofloxacin,
respectively identified in this study is considerably higher than that reported in study
conducted in Najaf Hospitals, in which resistance were 73.4 % for ciprofloxacin and
55.5 % for Levofloxacin (22) also it is in harmony with previous study in Najaf (23).
Fluoroquinolone resistance among P. aeruginosa isolates looks to be increasing in the
Wassit hospitals, perhaps because of high increasing fluoroquinolone use, and the
lack of adherence to approved infection control practices by hospitals. The P.
aeruginosa isolates were most resistant to amikacin (85.44%) and gentamicin
(91.26%), the resistance rate was higher when compared with other study reported
(22) in Najaf, who revealed that only 64.8% of the P. aeruginosa isolates resistant to
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
39
this antibiotic. However, the findings of P. aeruginosa antibiogram in the present
study disagree with a study done in United States of America (24). Results in the
current research, showed that 51.46% of the P. aeruginosa isolates were resistant to
aztreonam, Present findings are higher than previous study done by Abdullah, who
showed low rate of aztreonam resistance among P. aeruginosa clinical isolates (25).
Colistin resistance is not dependent upon bacterial metabolic activity and acquired
resistance is rare (26). In present study, the resistance of the isolates to Colistin was
2.78%, this result disagreed with the study in Turkey, who mentioned that all
multidrug-resistant strains were 100% susceptible to Colistin (26). The present
investigation showed that Colistin was only antibiotic that may remain highly active
against carbapenem resistance P. aeruginosa (CRPA) isolates, these results accepted
with study in Iran (27). This might be explained by the high cost of Colistin and
limited use out of the hospitals. The high rate of resistance observed in P. aeruginosa
isolates in this study, may be explained by incorrectly prescribed antibiotics,
extensive of antibiotics in animal food which in turn transfers to humans by meat and
egg consumption, and availability of few new antibiotics.
The production of MBLs is the most common mechanism for carbapenem
resistance in Enterobacteriaceae and P. aeruginosa isolates (28,29).The resistant
isolates were tested by MHT revealed 16 (44.44%) were positive isolates, in addition
double disc synergy test showed that 32 (88.89%) isolates out of 36 P. aeruginosa
(CRPA) were positive. Present study revealed that these two tests may be useful in
screening for MBL, but these tests cannot be routinely performed in all national
laboratories. The current results showed that 94.44% percentage of P. aeruginosa
(CRPA) isolates have bla-VIM1 gene in plasmid DNA, the percentage of bla-
VIM1gene in the current study was higher than previous study in Saudi Arabia was
noted VIM appeared
in 29.4% of our isolates (30). In another study in Turkey, 100 P. aeruginosa isolates
were collected from patients in a Turkish university hospital 1% isolate was found to
carry bla-VIM gene (31).
When compared to previous studies in Iran, current finding indicated that the
rate of blaVIM-1 was higher in this study compared with previously reported by
Mahmood (32), who found the rate was 18%. The bla VIM genes are located in class
1 integrons as a gene cassettes and have been identified on plasmid with different
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
40
replicon types (33), increasing the possibility of dissemination and linkage to other
antibiotic resistance genes. However, this constitutes the first report on prevalence
and detection of bla-VIM1 gene in Wasit province.
CONCLUSION
We concluded Rate of occurrence of bla-VIM1producers was highest among
carbapenem- resistant Pseudomonas aeruginosa isolated from clinical samples in
Wassit Province hospitals. Therefore, the detection of bla-VIM1 positive P.
aeruginosa isolates in this study indicates importance of strengthening surveillance to
prevent the nosocomial infection and dissemination of VIM1 in Wassit.
الکشف الجزیئی عن جین المیتالوبیتالاکتامیز
المقاومة لمضادات Pseudomonas aeruginosa فی جرثومة (blaVIM-1)
الکاربابینیم والمعزولة من مرضى مستشفیات محافظة واسط
زیاد خلف حسین ، اسراء جبار شمخی
الخلاصھ
بین عزلات بکتریا blaVIM- تھدف الدراسة الحالیة الى التحری عن موروث المیتالوبیتالاکتامیز 1
Pseudomonas تم الحصول على ١٠٣ عزلة بکتیریة تابعة لبکتریا Pseudomonas aeruginosa
من مجموع ٢٠٠ عینة سریریة ومن ثلاث مستشفیات رئیسیة فی محافظة واسط للفترة من aeruginosa
تم عزلھا من عینات Pseudomonas aeruginosa تشرین الثانی ٢٠١٧ الى شباط ٢٠١٨ . ان اکثر عزلات
.11 ( 12 والاذن (% 5.5 ( 70 تلھتا عینات الجروح (% 6 ( الحروق (% 35
مقاومة للمضادات الحیویة من Pseudomonas aeruginosa وقد اظھر المسح الاولی ل ١٠٣ عزلة
عائلة الکاربابنیم وعددھا ٣٦ عزلة. اختبرت حساسیة العزلات المقاومة لمضادات الکاربابنیم للمضادات الحیویة
بطریقة انتشار القرص.
تم دراسة الطرق المظھریة للعزلات المقاومة للکاربابنیم .بالأضافة الى انھُ تم تحدید العزلات المقاومة
فی الکشف عن قابلیة العزلات على انتاج انزیمات (Double disc synergy test) للکاربابنیم بواسطة اختبار
٨٨.٨٩ %) موجبة لھذا ) وکان من بین العزلات ٢٣ ، Metallo-beta-lactamases (MBL) البیتا لاکتامیز
٨٣.٣٣ %) من ) اظھرت ٣٠ . Imipenem-EDTA combined disk الفحص. وکذلک طریقة فحص
Modified Hodge العزلات تمتلک القدرة على انتاج انزیمات البیتالاکتامیز .بالاضافة الى ذلک اظھر اختبار
٤٤.٤٤ %) من العزلات ایجابیة لھذا ) قدرة العزلات على انتاج انزیم الکاربابنیمیز حیث اظھرت ١٦ test
الفحص.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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حیث اظھرت ( PCR) بواسطة تقنیة سلسلة تفاعل انزیم البلمرة bla-VIM تم الکشف عن جین 1
٣٤ عزلة (%٩٤.٤٤) ، ( CRPA) مقاومة للکاربابنیم Pseudomonas aeruginosa النتائج لل ٣٦ عزلة
عزلة موجبة لجین bla-VIM کانت موجبة لجین 1
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