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MECHANISMS
Emergence of Imipenem-Resistant Pseudomonas
aeruginosa Clinical Isolates from Egypt Coharboring
VIM and IMP Carbapenemases
Ramadan Ahmed El-Domany,
1
Mohamed Emara,
2
Mohammed A. El-Magd,
3
Walaa H. Moustafa,
2
and Nesma M. Abdeltwab
1
Pseudomonas aeruginosa is an important human pathogen and the leading cause of nosocomial infections.
P. aeruginosa is characterized by massive intrinsic resistance to a multiple classes of antibiotics with carba-
penems being the most potent inhibitor of P. aeruginosa and considered the first choice for its treatment.
Therefore, it is crucial to investigate novel mechanisms of resistance of P. aeruginosa to carbapenems for
achieving successful therapy. A total of 114 P. aeruginosa isolates from two university hospitals in Egypt were
recruited in this study. Antimicrobial susceptibility testing revealed that 50 isolates (43.8%) exhibited
multidrug-resistant (MDR) phenotype, of them 14 isolates (12.2%) were imipenem (IPM)-resistant. Of these 14
isolates, 13 isolates (11.4%) exhibited the metallo-b-lactamase (MBL) phenotype. MBLs encoding genes, VIM
and IMP, were identified by PCR. PCR results revealed that four isolates harbored the VIM gene alone, one
isolate harbored IMP gene alone, and four isolates harbored both genes. The correct size of PCR products of
VIM and IMP genes (390 and 188 bp, respectively) were sequenced to confirm results of PCR and to look for
any possible polymorphism among MBL genes of tested isolates. Data analysis of these sequences showed
100% identity of nucleotide sequences of MBL genes among tested Egyptian patients. To our knowledge, this
is the first report of IMP carbapenemase-encoding gene in Africa and the first detection of the emergence of
P. aeruginosa coproducing VIM and IMP genes in Egypt.
Keywords: metallo-b-lactamases, sequencing, carbapenems, imipenem, VIM, IMP, Pseudomonas aeruginosa,
PCR, MDR
Introduction
Pseudomonas aeruginosa is a dangerous human
pathogen that is commonly associated with nosocomial
infections. P. aeruginosa has a remarkable ability to
acquire resistance mechanisms to various classes of anti-
microbial agents and, therefore, it is termed as multidrug-
resistant ( MDR) pathogen.
1
Extended-spectrum blactamases (ESBL) and metallo-b-
lactamases ( MBLs)-mediated resistance are the most impor-
tant emerging mechanisms of resistance in P. aeruginosa.
2,3
Class B b-lactamases, also known as MBLs, have sub-
stantial ability to efficiently hydrolyze carbapenems and other
b-lactams and are not inhibited by b-lactamase inhibitors.
4
MBLs are categorized into three structural classes, namely
B1, B2, and B3, based on the structure of their active site.
5
Many acquired MBLs have been identified so far, in-
cluding the IMP and VIM which were first detected
6,7
in
addition to SPM-, GIM-, SIM-, KHM-, NDM-, AIM-, DIM-,
SMB-, TMB-, and FIM-type enzymes, which have subse-
quently been reported.
8,9
The majority of these MBLs are typically encoded by
plasmid or transposon-borne genes, often on integron.
10,11
The worldwide emergence of carbapenem-resistant strains
with similar mobile genetic elements indicates the dissemi-
nation of genes encoding carbapenemases through horizontal
gene transfer. For example, the IMP and VIM genes, re-
sponsible for MBLs production are horizontally transferable
through plasmids and can rapidly spread to other bacteria.
12,13
A considerable increase in prevalence of MBLs-mediated
carbapenem resistance has been reported in several coun-
tries, however, little or no information about the prevalence
of VIM and IMP carbapenemase-encoding genes was re-
corded among imipenem (IPM)-resistant P. aeruginosa in
Egypt.
4
Therefore, the ultimate goal of the current study was
to determine the prevalence of the MBLs, VIM and IMP
1
Department of Microbiology and Immunology, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh, Egypt.
2
Department of Microbiology and Immunology, Faculty of Pharmacy, Helwan University, Cairo, Egypt.
3
Department of Anatomy and Embryology, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrelsheikh, Egypt.
MICROBIAL DRUG RESISTANCE
Volume 00, Number 00, 2017
ªMary Ann Liebert, Inc.
DOI: 10.1089/mdr.2016.0234
1
carbapenemase-encoding genes, among IPM-resistant
Egyptian P. aeruginosa clinical isolates.
Materials and Methods
Bacterial strains and antibiotic susceptibility testing
Bacterial isolates were collected from in-patients at-
tending Tanta and Mansoura University Hospitals. A total
of 114 P. aeruginosa clinical samples from various sour-
ces, including urine (n=38), wound swab (n=14), sputum
(n=20), and blood (n=42), were collected using proper
sampling techniques
14,15
and were inoculated onto Pseu-
domonas P agar (Tech agar or king A agar). All 114
P. aeruginosa isolates were tested for their susceptibil-
ity to different classes of antimicrobial agents, includ-
ing ampicillin, amoxicillin, amoxicillin/clavulanic acid,
ciprofloxacin, ofloxacin, gentamicin, amikacin, ceftazi-
dime, cefoperazone, cefuroxime, imipenem, meropenem,
ticarcillin, ticarcillin/clavulanic acid, piperacillin, piper-
acillin/tazobactam, and colistin using VITEK 2 Compact
System.
16
Molecular identification of P. aeruginosa
by PCR and sequencing
The genomic DNA was extracted using the GeneJET
Genomic DNA Extraction Kit following the manufacturer’s
protocol (#K0721; Fermentas). Primers that have been used
to detect IMP and VIM genes in IPM-resistant P. aeruginosa
were designed as previously described by Ellington et al.
17
with the following sequences; IMP gene forward primer
5¢GGAATAGAGTGGCTTAAYTCTC3¢, reverse primer
5¢CCAAACYACTASGTTATCT3¢and VIM gene forward
primer 5¢GATGGTGTTTGGTCGCATA3¢, reverse primer
5¢GATGGTGTTTGGTCGCATA3¢.
The PCR mixture was carried out in a final volume of
50 ml consisting of 5 ml of DNA template (5–20 ng/ml); 25 ml
of PCR master mix; 2.5 ml of forward primer (0.1–0.5 mM);
2.5 ml of reverse primer (0.1–0.5 mM), and 15 ml nuclease-
free water. The thermal cycler (Verti; Applied Biosystem)
was programmed with the following conditions; initial de-
naturation at 95C for 5 min, followed by 35 cycles of de-
naturation at 94C for 30 sec, annealing at 52C for 40 sec,
extension at 72C for 50 sec, and final cycle of amplification
at 72C for 5 min. A positive and a negative control for each
PCR were included. After agarose gel electrophoresis with
ethidium bromide, the PCR products were analyzed under
UV light.
Samples were purified using the PCR Purification Kit
(#PP-201S; Jena Bioscience, Germany) according to the
manufacturer’s protocol. The purified PCR products were
sent to Macrogen Company (South Korea) to be sequenced
in both directions using ABI 3730XL DNA sequencer
(Applied Biosystem). The Sequences were analyzed using
the Chromas Lite 2.1 program (http://technelysium.com
.au/?page_id=13) and the identity of the sequenced PCR
products were examined using Blast search against Gen-
Bank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The
alignments, annotations, and assembly of the sequences were
performed using Geneious 4.8.4 software www.geneious
.com/web/geneious/home.
Results
Identification of clinical isolates and antimicrobial
susceptibility testing
A total of 114 P. aeruginosa clinical isolates, collected
from urine, wound, sputum, and blood, were identified by
Gram staining and culturing methods. Pseudomonas P agar
(Tech agar) was used as a simple, fast, effective, and reliable
method for identification of P. aeruginosa based on the
accurate detection of the characteristic pigment Pyocyanin,
which is produced by P. aeruginosa. Additional confirma-
tory biochemical tests were carried to further identify the
isolates and identification was further scrutinized by VITEK
2 compact system. The susceptibility of all P. aeruginosa
clinical isolates to tested antibiotics is listed in (Table 1).
Antimicrobial susceptibility to various antibiotics (ampicil-
lin, amoxicillin, amoxicillin/clavulanic acid, ciprofloxacin,
ofloxacin, gentamicin, amikacin, ceftazidime, cefoperazone,
cefuroxime, and imipenem) has been tested. This revealed
that 50 isolates (43.8%) exhibited MDR phenotype (resis-
tance to more than three antimicrobial agents from different
classes). IPM was the most effective antimicrobial agent as
only 14 isolates (12.2%) were resistant to it. Moreover, the
susceptibility of the 14 IPM-resistant isolates to another
group of antibiotics (ticarcillin, ticarcillin/clavulanic, piper-
acillin, piperacillin/tazobactam, meropenem, and colistin)
Table 1. Antibiotic Susceptibility Pattern
of Tested Pseudomonas aeruginosa Isolates
Antimicrobial agent
No. of
resistant
isolates
Percentage
of resistant
isolates, %
Ampicillin 114 100
Amoxicillin 114 100
Amoxicillin/clavulanic acid 114 100
Ciprofloxacin 51 44.7
Imipenem 14 12.2
Gentamicin 63 55.3
Ceftazidime 57 50
Amikacin 29 25.5
Ofloxacin 46 40.4
Cefoperazone 57 50
Cefuroxime 78 68.5
FIG. 1. Ethidium bromide-stained gel shows PCR prod-
ucts of VIM genes with size of 390 bp (lanes 2–10); lane 1,
positive control; lane 11, negative control; M, 100 bp marker
(ladder).
2 EL-DOMANY ET AL.
was further investigated. This showed that nine isolates
(64%) were resistant to ticarcillin and to ticarcillin/clavulanic
acid, eight isolates (57%) were resistant to piperacillin, nine
isolates (64%) were resistant to piperacillin/tazobactam, and
nine isolates (64%) were resistant to meropenem. Interest-
ingly, all IPM-resistant isolates were susceptible to colistin.
Molecular detection of MBLs genes by PCR
Isolation of bacterial DNA. Bacterial DNA was extracted
from P. aeruginosa isolates as revealed by gel electropho-
resis, which showed intact bands indicating pure genomic
DNA. The DNA concentration and purity were determined
by NanoDrop (UV-VIS spectrophotometer Q5000; Qua-
well) by measuring the absorption at 260 and 280 nm.
Detection of MBLs genes by PCR. The MBL genes de-
tected in this study were consistent with those of positive
control, with PCR products matching the predicted sizes,
VIM =390 bp and IMP =188 bp (Figs. 1 and 2, respectively).
Out of 114 tested P. aeruginosa isolates, 14 P. aeruginosa
clinical isolates exhibited MBLs genotype; of them four
isolates harbored the VIM gene alone, one isolate harbored
IMP gene alone, and four isolates harbored both genes.
Isolates harboring either VIM or IMP exhibited either ex-
tensive drug resistance (XDR) or MDR phenotype (Table 2).
VIM and IMP genes have been detected in isolates col-
lected from blood or wound, whereas isolates collected from
urine only harbored the VIM gene (Table 3).
DNA sequencing and alignments of MBL genes. The se-
quences of VIM and IMP genes were submitted to the
GenBank databases and assigned the following accession
numbers KX 452684 and KX 452681, respectively. Data
analysis of these sequences showed 100% identity of nu-
cleotide sequences of MBL genes among the tested isolates.
This was the impetus to check the identity between these
sequences and other published sequences. Only one single-
nucleotide polymorphism (SNP) was detected at the level of
the third nucleotide of IMP, where A (our sequence) re-
placed by T in LC103182 (Iran). This SNP is synonymous
as there was no change in amino acid sequence.
Discussion
The emergence of MBLs-mediated carbapenem resis-
tance in P. aeruginosa is substantially alarming because of
the ongoing spread and its intrinsic and acquired resistance
mechanisms, which limit treatment options of this trouble-
some pathogen.
4,18
There are few studies that have deter-
mined the resistance of P. aeruginosa to carbapenems in
Egypt.
19,20
Therefore, we thought to determine the preva-
lence of MBLs among IPM-resistant P. aeruginosa clinical
isolates collected from two university hospitals in Egypt.
In this study, the susceptibility pattern of 114 clinical
isolates of P. aeruginosa was determined and subsequently
the IPM-resistant isolates were assessed for MBLs pro-
duction by molecular analysis targeting VIM and IMP
genes. IPM-resistant P. aeruginosa clinical isolates ex-
hibited high level of resistance to tested antibiotics. By
contrast, all IPM-resistant P. aeruginosa clinical isolates
were sensitive to colistin, which is in accordance with
previously published work recommending colistin as a
FIG. 2. Ethidium bromide-stained gel shows PCR prod-
ucts of IMP gene with size of 188 bp (lanes 3, 4, 7, and 8);
lane 1, positive control; lanes 2, 5, and 6, negative samples;
lane 9, negative control; M 100 bp marker (ladder).
Table 2. Prevalence of VIM and IMP Genes Among Isolates and Their Related
Antimicrobial Resistance Profile
MBL
gene
Prevalence (%)
among all
isolates
Prevalence (%)
among
IPM-resistant
isolates
Prevalence (%)
among
XDR-isolates
Prevalence (%)
among
IPM-resistant
XDR-isolates
Prevalence (%)
among
MDR-isolates
Prevalence (%)
among
IPM-resistant
MDR-isolates
VIM 8/114 (7%) 8/14 (57%) 3/64 (4.7%) 3/8 (25%) 5/50 (10%) 5/8 (63.5%)
IMP 5/114 (4%) 5/14 (35%) 1/64 (1.6%) 1/8 (12.5%) 4/50 (8%) 4/8 (50%)
IPM, imipenem; MBL, metallo-b-lactamase; MDR, multidrug resistant; XDR, extensive drug resistance.
Table 3. Distribution of VIM and IMP Genes
Among P. aeruginosa Clinical
Isolates from Various Sources
Isolate number MBL gene Clinical source
MUH4 VIM Blood
MUH9 VIM Blood
MUH10 VIM Urine
MUH12 VIM Urine
MUH7 IMP Blood
MUH8 VIM and IMP Blood
MUH18 VIM and IMP Wound
TUH48 VIM and IMP Blood
TUH56 VIM and IMP Blood
MUH, Mansoura University Hospital; TUH, Tanta University
Hospital.
DETECTION OF VIM- AND IMP-PRODUCING PSEUDOMONAS AERUGINOSA 3
potential alternative to carbapenems in the treatment of
infections caused by P. aeruginosa.
21
PCR data obtained revealed the presence of VIM gene in
8 isolates (57%) out of the 14 IPM-resistant P. aeruginosa
clinical isolates, which is lower than the results of a previ-
ously published study from Egypt, where Zafer et al. re-
ported a high prevalence (85%) of VIM-2 in MBL-positive
isolates from Egypt.
20
In two studies from Iran, Moosavian
and Rahimzadeh
22
and Neyestanaki et al.
23
reported prev-
alence rates of VIM gene of 1.6% and 55%, respectively,
compared with 32.89% in a study from Canada,
24
which are
less than what we found in the current study. On the con-
trary, a higher prevalence rate of P. aeruginosa carrying
VIM gene was reported from Poland (85.3%).
25
Sequence
analysis showed 100% identity with the VIM gene of
P. aeruginosa (accession number KX 452684). The se-
quencing results were further confirmed; there is less variance
among VIM genes in the current study when compared with
global genotypes.
PCR results also revealed the production of IMP by 5
isolates (35%) out of 14 IPM-resistant P. aeruginosa clinical
isolates. Lower prevalence rates have been reported by other
studies, where prevalence rates of 1.75% (IMP-7) and 3%
(IMP-1) were reported in Canada
24
and Iran,
23
respectively.
On the contrary, higher prevalence (55%) of P. aeruginosa
carrying IMP gene was also reported from Iran.
22
Sequence
analysis showed 100% identity with the IMP gene of
P. aeruginosa (accession number KX 452681). Results re-
vealed identification of four IPM-resistant P. aeruginosa
isolates that coproduce both VIM and IMP and to the best of
our knowledge, the copresence of VIM and IMP has not
previously been reported in Egypt.
Furthermore, sequence analysis of IMP amplicon from
our isolates showed variation in the sequence when com-
pared with IMP isolated from Iranian patients (LC103182)
using BLAST analysis. In this study, a transversion type of
mutation at the third nucleotide position was detected,
wherein substitution of ‘‘A’’ with ‘‘T’’ was noticed. This
SNP is synonymous as there was no change in amino acid
sequence. This observation appears to be ‘‘Novel and Un-
ique’’ and it might be very significant, but needs further
investigations.
Taken together, this study reports the emergence of
P. aeruginosa clinical isolates that harbor VIM and IMP
carbapenemase-encoding genes either alone or in combi-
nation. To our knowledge, this is the first report of detection
of IMP gene in P. aeruginosa in Africa. Moreover, this is
also the first report of the emergence of P. aeruginosa co-
harboring both VIM and IMP carbapenemase-encoding
genes in Egypt.
Authors’ Contributions
Conception and design: R.A.E.; Acquisition of data/
isolates: N.M.A.; Data analysis and interpretation: M.E.,
R.A.E., M.A.A., and N.M.A.; drafting the article: M.E.,
N.M.A., and W.H.M.: Critical revision of the article:
R.A.E., M.A.A., and M.E.; and all authors read and ap-
proved the final article.
Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Ramadan Ahmed El-Domany, PhD
Department of Microbiology and Immunology
Faculty of Pharmacy
Kafrelsheikh University
Post Box 3356
Kafrelsheikh 3356
Egypt
E-mail: ramadaneldomany@yahoo.com
DETECTION OF VIM- AND IMP-PRODUCING PSEUDOMONAS AERUGINOSA 5
