Department of Microbiology, Dhaka Medical College, Dhaka, Bangladesh
Background and objectives: Ventilator-associated respiratory tract infection (VARTI)
is a major cause of morbidity and mortality among the critically ill patients
of intensive care units (ICU). Acinetobacter
baumannii, an important offending pathogen in VARTI, has been found to be
resistant to several antibiotics including carbapenems. The present study was
conducted to determine the rate of New Delhi metallo-β-lactamase 1 (NDM-1) producing A. baumannii causing VARTI among the patients admitted in an ICU of
a large tertiary care hospital.
Methods: The study was conducted from July 2013
to June 2014. Endotracheal aspirates (ETA) were collected from patients with
clinically suspected VARTI. Samples were collected from patients who were on
mechanical ventilation for more than 48 hours. ETA samples were cultured aerobically and isolated A. baumannii were tested for susceptibility to carbapenem. Presence
of NDM-1 encoded by the blaNDM-1 gene was detected by polymerase
chain reaction (PCR).
Results: A total of 138 VARTI cases were included in the study. Total
107 (77.5%) bacteria were isolated from 138 ETA samples of which 38 were A. baumannii. Out of 38 isolated A. baumannii, 35 (92.1%) were resistant
to imipenem/meropenem and 33 (86.8%) were positive for blaNDM-1 gene
Conclusion: The present study demonstrated that high proportion of A. baumannii isolated from VARTI cases
in ICU were carbapenem resistant and blaNDM-1 positive. Careful infection
control program should be considered to contain the spread of this
multi-resistant organism to other hospital and community.
IMC J Med Sci 2018; 12(1): 37-41
Correspondence: Dr. Shahida Akhter, Assistant
Professor, Department of Microbiology, Ibrahim Medical College, 122 Kazi Nazrul
Islam Avenue, Dhaka 1000, Bangladesh; Email: email@example.com
respiratory tract infections (VARTI) in ICU patients include
ventilator-associated pneumonia (VAP) and tracheobronchitis (VAT). The
incidence of VAP and VAT in ICU patients ranges from 7% to 70% and 3% to 10%
respectively [1-6]. Most cases of VAP are caused by bacterial pathogens that
normally colonize upper respiratory tract and gastrointestinal tract of the
patient. External sources like transmission from caregivers, environmental
surfaces or other patients have been implicated. Detection of causative organisms and their antibiotic
susceptibility is crucialfor diagnosis and effective treatment of VAP . Several Gram positive and negative organisms namely
methicillin-resistant Staphylococcus aureus
(MRSA), Pseudomonas aeruginosa, ESBL producing Enterobacteriaceae
and multi-resistant A. baumannii have
been isolated from cases of VARTI [8,9,4]. Besides in Klebsiella pneumoniae and Escherichia
coli, metallo-b-lactamase (MBL) producing blaNDM-1 gene conferring
resistance to carbapenem has recently been identified in A. baumannii in different countries of the world [10-14]. In view
of the above, the present study was conducted to determine the presence of blaNDM-1 gene in A. baumannii isolated from ICU patients
with VARTI of a tertiary care hospital in Dhaka city.
Materials and methods
The study was carried out at the ICU of Dhaka Medical
College Hospital, Dhaka from July, 2013 to June 2014. All patients suspected to
have either VAP or VAT were included in the study. The study was approved by
the Institutional Review Board of Dhaka Medical College.
Study population and
sample collection: Patients at ICU on mechanical ventilator
for more than 48hours with suspected VAP and VAT were enrolled in the study.
Criteria for suspected VAP include a presence of new and persistent (>48
hours) or progressive radiographic pulmonary infiltrate plus two of the
following: temperature of >380C or <360C, blood
leukocyte count of >10,000 cells/µl
or <5,000 cells/µl, purulent tracheal secretions, and gas exchange
degradation .VAT was suspected in intubated patients with clinical signs of
lower respiratory tract infection (such as fever, leukocytosis and purulent
sputum), presence of bacteria within neutrophils
in tracheal aspirate by Gram stain and growth of significant bacteria by semi-quantitative
culture method in the absence of a new or progressive infiltrate on chest radiography
Endotracheal tube aspirates (ETA) were collected from
clinically suspected VAP and VAT cases by gently introducing a 50cm/14Fr
suction catheter through the endotracheal tube for a distance of approximately
25-26 cm. The ETA was obtained by suction, without instilling saline. Two milliliters
of sterile phosphate buffered saline (PBS) was injected into the lumen of the
catheter with a sterile syringe to flush the exudates. The exudates were
collected into a sterile 50 ml Falcon tube and transported immediately to the
laboratory for further processing. Only one ETA sample was collected from each
of A. baumannii and antibiotic susceptibility test: ETA was mechanically liquefied and homogenized by vortexing
for one minute with glass beads (1-2 glass beads). After vortexing sample was centrifuged at 2000 rpm for 10 minutes.
Supernatant was discarded using a sterile pipette and the deposit was further
mixed by vortexing. The processed specimen was used for Gram staining and culture
in recommended media. A. baumannii
was identified by standard biochemical tests . All isolated A. baumannii were tested for
susceptibility to imipenem (10 µg), meropenem (10
µg), piperacillin-tazobactam (30 µg), ceftazidime (30 µg), ceftriaxone (30
µg), cefepime (30 µg), amoxicillin-clavulanic acid (20/10 µg), TMP-SMX
(1.25/23.75 µg), ciprofloxacin (5 µg), gentamicin (10 µg), amikacin (30 µg) and
colistin (10 µg) by disc diffusion technique [18,19]. The zone of inhibition
around the antibiotic disc was measured after 18 hours of incubation of plates
at 370C.The zone of inhibition was interpreted as sensitive and resistant according to CLSI guideline
. Potency of the disks and antimicrobial agents were standardized using the
reference strain E. coli ATCC 25922.
of blaNDM-1 gene by PCR: The isolates were screened for the presence of blaNDM-1 MBL gene by PCR with
the primers reported previously . The sequence of the primers is shown in
Table-1. In brief, PCR was performed in a final reaction volume of 25μl in a PCR tube, containing 10μl
of master mix (mixture of dNTP, taq polymerase, MgCl2 and PCR
buffer), 4μl primers (Promega corporation, USA), 3 μl extracted DNA and 8μl of
nuclease free water. PCR assay was performed in Eppendorf AG thermal cycler.
After initial denaturation at 940C for 10 minutes, the reaction was
subjected to 36 cycles. Each cycle consisted of denaturation at 940C
for one minute, annealing at 600C for one minute and elongation at
720C for 90 seconds followed by final extension at 720C
for 10 minutes. The product was analyzed by electrophoresis in 1.5% agarose gel
containing ethidium bromide (0.5 μg/ml) in
TBE buffer (0.04 M Tris acetate, 0.001 M EDTA; pH 8.6) and photographed under
UV illumination. DNA of known imipenem sensitive K. pneumonia was used as
The sequence of primers
used for detection of blaNDM-1
gene in A. baumannii by PCR 
Total 138 VARTI cases were enrolled in
the study of which 65 (47.1%) and 73 (52.9%) were VAP and VAT cases
respectively. A total of 107 (77.5%) bacteria were isolated from ETA samples of
which 38 were A. baumannii. Of the 38
isolates, 17 (26.2%) were isolated from VAP cases while 21(28.8%) were from VAT
cases. Antimicrobial susceptibility of A. baumannii to different antibiotics
is shown in Table-2. The resistance to imipenem/ meropenem, aminoglycosides, quinolones
and third generation cephalosporins ranged from 92.1% to 100%. However, only
13.2% A. baumannii were resistant to
colistin. PCR revealed presence of MBL blaNDM-1
gene in 33 (86.9%) out of 38 isolated A.
baumannii (Table-3 and Fig-1). All of them were resistant to carbapenem.
Table-2: Resistance pattern of A. baumannii to different antibiotics
Table-3: Distribution of blaNDM-1 gene in A.
Fig.1: PCR analysis of A. baumannii isolates from VARTI cases
showing presence of 155 bp blaNDM-1 gene (Lane 2, 3, 5, 6, 7); Negative control
(L1 and 8); L4: 100 bp DNA ladder.
Infection by MBL producing organism containing
blaNDM-1 gene are
increasing in the last few years in Bangladesh [12,21,22]. In 2011, about 3.5% blaNDM-1 positive Escherichia coli, K. pneumoniae, A. baumannii,
Providencia rettgeri and Citrobacter freundii
were reported from Bangladesh . In 2013, another study from Bangladesh,
reported the presence of blaNDM-1
gene in 22% of the imipenem resistant A.
baumannii . However, the present study has revealed that over 86% of A. baumannii isolated from high risk ICU
patients were positive for blaNDM-1
gene and were resistant to several groups of antibiotics apart from carbapenem.
MBL containing organisms are usually sensitive to polymyxins and tigecycline .
In the present study, though majority (>90%) of our blaNDM-1 positive A.
baumannii were resistant to several classes of antibiotics, but 86.9% of them
were sensitive to colistin.
Therefore, the results of present study
emphasize the necessity of strong infection control program and continuous monitoring
of antibiotic susceptibility of offending organisms to contain the spread of
multi-drug resistant blaNDM-1
positive A. baumannii in high risk
areas of the hospitals. Also, strict and judicious use of effective antibiotic
like colistin is necessary.
designed the study, performed the experiments and wrote the manuscript. SMS
conceived, designed and supervised the study.
declare no conflict of interest.
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