Co-existence of bla OXA-23 and bla NDM-1 genes of Acinetobacter baumannii isolated from Nepal: antimicrobial resistance and clinical significance
Joshi et al. Antimicrobial Resistance and Infection Control
Co-existence of bla and bla OXA-23 NDM-1 genes of Acinetobacter baumannii isolated from Nepal: antimicrobial resistance and clinical significance
Prabhu Raj Joshi 3
Mahesh Acharya 3
Trishna Kakshapati 2
Udomluk Leungtongkam 0
Rapee Thummeepak 0
Sutthirat Sitthisak 0 1
0 Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University , Phitsanulok , Thailand
1 Centre of Excellence in Medical Biotechnology, Faculty of Medical Science, Naresuan University , Phitsanulok , Thailand
2 Annapurna Neurological Institute and Allied Sciences , Maitighar, Kathmandu , Nepal
3 Central Department of Microbiology, Tribhuvan University , Kirtipur, Kathmandu , Nepal
Background: Molecular analysis of carbapenem-resistant genes in Acinetobacter baumannii, an emerging pathogen, is less commonly reported from Nepal. In this study we determined the antibiotic susceptibility profile and genetic mechanism of carbapenem resistance in clinical isolates of A. baumannii. Methods: A. baumannii were isolated from various clinical specimens and identified based on Gram staining, biochemical tests, and PCR amplification of organism specific 16S rRNA and blaOXA-51 genes. The antibiotic susceptibility testing was performed using disc diffusion and E-test method. Multiplex PCR assays were used to detect the following β-lactamase genes: four class D carbapenem hydrolyzing oxacillinases (blaOXA-51, blaOXA-23, blaOXA-24 and blaOXA-58). Uniplex PCRs were used to detect three class B metallo-β-lactamases genes (blaIMP, blaVIM and blaNDM-1), class C cephalosporin resistance genes (blaADC), aminoglycoside resistance gene (aphA6), and ISAba1 of all isolates. Insertion sequence ISAba125 among NDM-1 positive strains was detected. Clonal relatedness of all isolates were analyzed using repetitive sequence-based PCR (rep-PCR). Results: Of total 44 analyzed isolates, 97.7% (n = 43) were carbapenem-resistant A. baumannii (CR-AB) and 97.7% (n = 43) were multidrug resistant A. baumannii (MDR-AB). One isolate was detected to be extremely drug resistant A. baumannii (XDR-AB). All the isolates were fully susceptible to colistin (MICs < 2 μg/ml). The blaOXA-23 gene was detected in all isolates, while blaNDM-1 was detected in 6 isolates (13.6%). Insertion sequence, ISAba1 was detected in all of blaOXA-23 positive isolates. ISAba125 was detected in all blaNDM-1 positive strains. The blaADC and aphA6 genes were detected in 90.1 and 40. 1%, respectively. The rep-PCR of all isolates represented 7 different genotypes. Conclusion: We found high prevalence of CR-AB and MDR-AB with blaOXA-23 gene in a tertiary care hospital in Nepal. Systemic network surveillance should be established for monitoring and controlling the spread of these resistant strains.
Acinetobacter baumannii; Carbapenem resistance; blaOXA-23 and blaNDM-1 carbapenemase genes
Acinetobacter baumannii, an emerging pathogen of
healthcare centers, shows intrinsic as well as acquired
drug-resistance mechanisms . Multidrug-resistant A.
baumannii can be resistant to all of the currently
available antibiotics, and in its deadliest form these are only
susceptible to potentially toxic polymyxins and colistins,
leaving limited options for treatment . Infections with
carbapenem- and colistin-resistant A. baumannii are
emerging globally .
Carbapenem resistance in A. baumannii encompasses
production of class B, C and class D carbapenemase,
decreased membrane permeability, altered
penicillinbinding proteins, and overexpression of efflux pumps [4,
5]. Most commonly, Acinetobacter spp. develop
carbapenem resistance by production of OXA-type
carbapenemase and metallo-β-lactamases (MBLs) [6, 7];
blaOXA23-like, blaOXA-40-like, blaOXA-58-like and blaOXA-51-like
carbapenemases are broadly reported, where
blaOXA-51like β-lactamases, intrinsic to A. baumannii, is used for
species identification [8–10]. Among multiple MBL
genes, blaIMP and blaVIM types (chromosomal or plasmid
encoded) encode carbapenemase in A. baumannii . A.
baumannii harboring plasmid encoded New Delhi
metallo-β-lactamase-1 (NDM-1), a novel carbapenemase
gene, is reported from many countries [11, 12]. In
addition, detection of class C β-lactamase genes (blaADC)
which mediated cephalosporin resistances and
aminoglycoside resistant genes (aphA6) has increased in recent
years in A. baumannii clinical isolates [13, 14].
A. baumannii remains a critical problem in many
healthcare settings throughout the world despite the
implementation of infection control practices. There are limited
data on carbapenem-resistant A. baumannii in Nepal. The
objective of this study was to determine antibiotic
susceptibility profile, antibiotic resistance genes and genetic
mechanism of carbapenem resistance of A. baumannii in clinical
isolates at a tertiary care hospital, Nepal.
Bacterial isolation and identification
A. baumannii isolates were collected from inpatient
units of a tertiary hospital, Nepal. Forty-four
nonduplicate isolates were collected (24 male and 20 female;
age range between 24 to 80 years) over 9 months periods
(October 2014 to June 2015). All isolates were identified
by classical biochemical methods and confirmed by PCR
method for detecting 16S rRNA gene and blaOXA-51
gene [15, 16]. Isolates were identified as A. baumannii
by PCR result of positive for both PCRs.
Antibiotic susceptibility testing
The antibiotic susceptibility of amikacin (30),
cefotaxime (30), ceftazidime (30), ceftriaxone (30), cefepime
(30), ciprofloxacin (5), gentamicin (10), imipenem
(10), meropenem (10), trimethoprim/sulfamethoxazole
(1.25/23.75), tetracycline (30), and
piperacillin/tazobactam (100/10) (Oxoid) was determined on Mueller
Hinton Agar (High Media, India) according to the
antibiotic disk diffusion method . The plates were
incubated at 37 °C for 24 h. The zones of inhibition
were determined whether the microorganism was
susceptible, intermediately resistant, or resistant to each
antibiotic according to Clinical and Laboratory
Standards Institute (CLSI) guidelines. E-test was
performed to determine the Minimum inhibitory
concentration (MIC) of ceftazidime, imipenem, tigecycline
and colistin (High Media, India) according to
manufacturer instructions and interpreted as per CLSI
guidelines except for tigecycline. Multidrung-resistant
A. baumannii (MDR-AB) was defined when A.
baumannii resistant to multiple antibiotics, often defined
as three or more antibiotic classes. Extensively drug
resistant A. baumannii (XDR-AB) was defined when
A. baumannii was resistant to all antimicrobial agents
except polymyxins (colistin) .
PCR amplification of antibiotic resistance genes
PCR assays to detect blaOXA-23, blaOXA-24, blaOXA-51,
blaOXA-58, blaIMP, blaVIM, blaNDM, blaADC and ahpA6
genes were performed using primers as describe
previously (Table 1). The amplification reaction was
performed using A. baumannii cell lysate as DNA
template. Each PCR was performed in triplicate in a
thermocycler with a PCR condition as described
previously [14, 16, 19–21]. All PCR assays used 16S rRNA
or blaOXA-51 genes as the internal control. The ISAba1
of blaOXA-23 gene was detected using combination of
primers ISAba1-F/ISAba1-R and
ISAba1-F/blaOXA-23R (Table 1) . The ISAba125 of blaNDM-1 gene were
determined in all blaNDM-1 positive strains using
combination of primers ISA125-F/ISA125-R and
ISA125F/blaNDM-R (Table 1). PCR products of the blaNDM-1
genes were purified and sequenced. BLAST was used
to compare the sequences of blaNDM-1 genes against
the GenBank Database. PCR products were analyzed
by electrophoresis in 1% agarose gel containing
0.5 μg/ml ethidium bromides.
IPM-EDTA combined disk test
All blaNDM-1 positive strains were tested for MBL
production by IPM-EDTA combined disk test. The
test was performed as previously described . After
24 h incubation, the difference of inhibition zone
diameter between IPM-EDTA disk and IPM disk
alone (≥7 mm) was considered the positive criteria
for the presence of MBL.
Table 1 List of primer for detection of genes used in this study
Size/ Annealing temp.
Repetitive element PCR-mediated DNA fingerprinting
Genomic DNA of each isolates was extracted from the
overnight cultures using GF-1 bacterial DNA extraction
kit (Vivantis, Malaysia). Rep-PCR was performed by
using genomic DNA as a template for PCR amplification
with the ERIC-2 primer (Table 1) using condition as
describe previously [24, 25]. PCR-banding patterns and
rep-PCR types were analyzed and interpreted as
previously described .
Demographic characteristic of patients
Demographic characteristics of the inpatients with A.
baumannii infection were analyzed; 24 (54.5%) were
male and 20 (45.5%) were female. Most of the specimens
were from ICU wards (n = 27, 61.4%) (Fig. 1). Isolates
were collected from sputum (n = 26, 59.1%), tracheal
aspirates (n = 9, 20.4%), catheter tip, (n = 4, 9.1%), pus (n =
4, 9.1%) and urine (n = 1, 2.3%) (Fig. 1).
Of the 44 isolates, resistance was found against
ciprofloxacin (n = 43, 97.7%), cefotaxime (n = 43, 97.7%),
ceftazidime (n = 42, 95.4%), ceftriaxone (n = 41, 93.2%),
cefepime (n = 39, 88.6%), amikacin (n = 19, 43.2%),
gentamicin (n = 23, 52.3%), trimethoprim/sulfamethoxazole
(n = 41, 93.2%), tetracycline (n = 21, 47.7%) and
piperacillin/tazobactam (n = 43, 97.7%). Only one isolate of
A.baumannii was susceptible to all tested antibiotics.
Most isolates (97.7%, n = 43) were carbapenem resistant
A. baumannii (CR-AB); all CR-AB were MDR-AB. One
isolate was detected to be XDR-AB. All the isolates were
fully susceptible to colistin (MICs < 2 μg/ml) and MIC of
tigecycline was determined to be <2.5 μg/ml (Table 2).
Antibiotic resistance genes and IS element in A.
Aminoglycoside resistance gene, aphA6 and
cephalosporin resistance genes, blaADC were detected in 40.1%
(18/44) and 90.1% (40/44), respectively. The blaOXA-23
was present in all isolates. Other class D β-lactamase
Fig. 1 Distribution of A. baumannii carrying carbapenemase genes in different specimen types a and wards b
genes, including blaOXA-24 and blaOXA-58, markers of
carbapenem resistance in A. baumannii, were not
detected in analyzed isolates. ISAba1 was detected in all of
blaOXA-23 positive isolates (100%). Of total analyzed
isolates, 6 (13.6%) also harbored blaNDM-1 gene in addition
to blaOXA-23 and blaOXA-51. All NDM-1 positive strains
exhibited insertion sequence ISAba125 detecting with
primers ISA125-F/ISA125-R. All isolates also detected a
band of 1.6 kb in a PCR using ISA125-F/blaNDM-R
primers. Metallo-β-lactamase (MBL) genes, including
blaVIM and blaIMP, were not detected in all isolates. The
sequences of the blaNDM-1 gene yielded 99-100%
sequence identity to the blaNDM-1 gene from Acinetobacter
lwoffii strain WJ10621 plasmid pNDM-BJ01 (Accession:
JQ001791) obtained from the GenBank Database.
Six A. baumannii isolates harbored blaNDM-1 gene were
detected for MBL production. All of blaNDM-1 positive
strains were positive for MBL production. MBL positive
strains showed resistance to fluoroquinolones and
Clonal relationship among isolates were studied using
rep-PCR typing. The fingerprinting represented 7
different DNA patterns consisting of 2 to 5 DNA fragment
sizes. The amplicons size for ERIC-2 PCR was 500–
4000 bp. The genotype was named A-G as shown in
Fig. 2. The high prevalence genotype was type C (n = 14;
31.8%) and D (n = 12; 27.3%). Genotype A, B, C and D
were disseminated in all isolated ward (ICU, general
ward and post-operative ward). Among 44 isolates, one
isolate of type F (2.3%) and G (2.3%) was found. Type F
was obtained from a catheter tip specimen from the ICU
ward. Type G was obtained from sputum of a patient
from a general ward. All NDM-1 positive strains
exhibited genotype A (n = 1), B (n = 1), C (n = 3) and D
(n = 1).
A. baumannii harboring blaOXA-51-like gene has been
identified as a marker for species identification. An
intrinsic blaOXA-51-like gene detected in all isolates in this
study supports the use of this gene as a surrogate
marker of A. baumannii identification [8–10]. High
prevalence of cephalosporin resistance genes, blaADC
(90.1%) was found in this study. In addition, we found a
high rate of cepharosporin resistant antibiotics
(cefotaxime, ceftazidime, ceftriaxone) using the disk diffusion
method. These data indicated that cephalosporins no
longer work to treat A. baumannii isolated from Nepal.
Carbapenem resistance in A. baumannii is a major
concern and is most often associated with class D
Table 2 The carbapenemases gene patterns, rep-PCR types and MIC determination of A. baumannii isolated from difference wards
blaOXA-51/ OXA-23/ NDM-1 4
Abbreviations: CAZ ceftazidime, IPM imipenem, TG tigecycline, CL colistin
A (2), B (6), C (8), D (6), E (2), F (1)
A (1), B (1), C (1), D (5), G (1)
A (1), B (1), C (2)
A (1), C (2) D (1)
Fig. 2 Rep-PCR-based DNA fingerprint patterns of A. baumannii isolates. The lanes marked M contain molecular markers. Each lane represents
genotype patterns of A–G
β-lactamases and MBLs. The full susceptibility of all
CRAB to colistin in this study indicates that colistin is still
an option of drug for the treatment of infections caused
by A. baumannii in Nepal hospital.
OXA-type carbapenemases are predominant in A.
baumannii [6, 7]. In agreement with this finding, high
prevalence of blaOXA-23 carrying A. baumannii strains
has been reported in Nepalese patients . The
acquired blaOXA-23 is the dominant genetic determinant in
Asia. The blaOXA-23 gene located on plasmid can be
transferred between A. baumannii through conjugation.
Thus, antibiotic resistant bacteria have been rapidly
increasing worldwide . The blaOXA-24 and blaOXA-58
were not detected in any isolates from this study. The
blaOXA-24/40 and blaOXA-58 genes were common in A.
baumannii isolated from Europe [2, 28]. Recently,
blaOXA-143 and blaOXA-235, which are novel class D
βlactamase genes in A. baumannii have been identified.
To date, these determinants were detected only in Brazil,
Mexico and the USA [29, 30]. ISAba1 was detected in
widespread clones of A. baumannii worldwide. Our
study found ISAba1 upstream of blaOXA-23 in all A.
baumannii isolates. A correlation between A. baumannii
clusters carrying the ISAba1/blaOXA-23 gene and
increased minimal inhibitory concentrations for
carbapenems was reported . One isolate (AB-13) that was
recovered from catheter tips of long-stay hospital
patients showed an extreme drug resistance pattern
(Additional file 1: Table S1). This isolate represented
blaOXA23, blaADC and aphA6 genes. Further molecular study to
detect other antibiotic resistance genes is needed to
explain what factors correlated with extreme drug
resistance. We also found one isolate (AB-25) harboring
blaOXA-23, blaADC and aphA6 genes was sensitive to all
tested drugs (Additional file 1: Table S1). This may be due
to the lack of promoter or mutation of ISAba1 or blaOXA-23
gene. Further study is needed to warrant the conclusion.
The blaNDM-1 carrying A. baumannii has recently been
emerged in many countries, including Germany, Spain,
Israel, Egypt, Switzerland, Libya, India, Pakistan and
Nepal [11, 26, 32, 33]. The blaNDM-1 gene has been
identified as a chimeric gene constructed by the fusion of the
aminoglycoside-resistance gene aphA6 with a
mannosebinding lectin gene. This event most likely occurs in
Acinetobacter spp., indicating that these bacteria are likely
the origin of this gene . In this study, we identified
13.6% of A. baumannii carrying blaNDM-1 gene. Previous
study has identified high prevalence (24.6%) of the A.
baumannii harbored the blaNDM-1 gene in Nepal in 2013–
2014 . Taking into consideration the relationship
between India, China and Nepal, the spread of blaNDM-1 is
likely to occur rapidly, mostly through A. baumannii
rather than Enterobacteriaceae. A. baumannii able to
transfer the blaNDM-1 gene via conjugation to the recipients and
Tn125 appears to be the main vehicle for dissemination of
the blaNDM-1 genes in A. baumannii . Poirel et al.
reported that the blaNDM-1 gene was located within the
composite transposon Tn125 bracketed by two copies of a
strong promoter of blaNDM-1 gene called ISAba125 .
This report was correlated with our finding that found
ISAba125 in 100% of NDM-1 producing A. baumannii.
The previous study reported that the most of A.
baumannii isolates harboring blaNDM-1 belonged to ST85
and ST25 [35–37]. In Libyan hospital, Libya, the main
clone of imipenem-resistant NDM-1-producing A.
baumannii belonged to ST2 . We used rep-PCR typing
to determine the clonal relationship in NDM-1
producing A. baumannii. Our study highlighted that most of
NDM-1-producing A. baumannii isolates belonged to 4
genotypes using rep-PCR. Rep-PCR is a method that
generates DNA fingerprints to discriminate between
bacterial strains, and has been used to characterize A.
baumannii isolates from hospitalized patients . Our
rep-PCR typing represented a high genetic diversity
(A-G) among A. baumannii isolates from Nepal. Some
clonally related groups (A, B, C and D) were observed in
the all wards represented the disseminated of these
clones in the hospital. Four genotypes (A, B, C, and D)
of co-existence of blaOXA-23 and blaNDM-1 A. baumannii
isolates were found. In addition, dissemination of these
four genotypes into different wards also confirms as a
major epidemic. Since rep-PCR is less discriminatory for
molecular typing of bacterial strains, further study using
multi-locus sequence typing could be useful for
Antibiotic resistance in A. baumannii is considered to
be a major future challenge in Nepal. Beyond OXA-type
carbapenemase, there is no doubt the emergence and
spreads of NDM-1 encoding A. baumannii–a superbug–
will further limit chemotherapeutic options and threaten
the public health of Nepal. The mechanism of hospital
adaptiveness beyond antibiotic resistance will be more
demanded in order to fully understand and combat
MDR and XDR A. baumannii.
Additional file 1: Table S1. Type of clinical specimen, ward, antibiotic
susceptibility patterns, rep-PCR types, resistance genes and MIC of 44 A.
baumannii isolates. (DOCX 23 kb)
CR-AB: Carbapenem-resistant Acinetobacter baumannii; ICU: Intensive care
unit; IPM-EDTA: Imipenem-ethylenediaminetetraacetic acid; MBL:
Metallobeta-lactamase; MDR-AB: Multidrug-resistant Acinetobacter baumannii;
MIC: Minimum inhibitory concentration; NDM: New Delhi
metallo-betalactamase; PCR: Polymerase chain reaction; XDR-AB: Extremely drug resistant
We thank Dr. Basant Pant, Neurosurgeon, Annapurna Neurological Institute
and Allied Sciences for providing laboratory facility and moral support. We
also acknowledge Nepal Health Research Council for approving this study.
Availability of data and materials
Please contact author for data requests.
PRJ and MA designed the study, collected data, analyzed the data and
prepared the manuscript, TK supervised the study, UL and RT collected data,
SS, analyzed the data, supervised the study and prepared the manuscript. All
authors read and approved the manuscript.
Consent for publication
Ethics approval and consent to participate
Ethical approval was obtained from the Ethical Review Board of Nepal Health
Research Council (NHRC) (Reg. 27/2015). Informed consent was taken from
all the patients or patients’ guardians. The research was in compliance with
the Helsinki Declaration.
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