Introduction
Acinetobacter baumannii is a leading cause of nosocomial infections with increased morbidity and mortality [
1].
A. baumannii is intrinsically resistant to most antibiotics and has the ability to acquire resistance genetic determinants from the environment as well [
2]. Antimicrobial resistance to β-lactams in
A. baumannii includes: (1) enzymatic mechanisms or production of β-lactamases and (2) non-enzymatic mechanisms such as modification of membrane permeability by either loss of or decrease in expression of outer membrane porins or an increased expression of efflux pumps [
2,
3].
Carbapenems are the drug of choice to treat infections caused by multidrug-resistant
A. baumannii. However, resistance to carbapenems is increasing and has been reported worldwide. Carbapenem resistance in
A. baumannii can be either carbapenemase mediated or non-carbapenemase mediated. Carbapenemase-mediated resistance is mainly due to the production of class A (serine proteases), class B (metallo-beta-lactamases) and class D (oxacillinases) carbapenemases, whereas non-carbapenemase-mediated resistance involves upregulation of the efflux pumps and/or loss of outer membrane porins [
4].
Resistance to carbapenem in
A. baumannii is most frequently due to oxacillinases, which can be intrinsic or acquired. The intrinsic
blaOXA-51 gene is considered to be specific for
A. baumannii, since the said gene is carried in its chromosome. Acquired OXA enzymes, which are encoded by the
blaOXA-23,
blaOXA-40 and
blaOXA-58 genes, are more predominant in
A. baumannii, whereas MBLs encoded by the
blaIMP,
blaVIM,
blaNDM and
blaSIM genes are more predominant in non-baumannii Acinetobacter isolates [
5].
The insertion sequence has a major role in the overproduction and dissemination of OXA genes in
A. baumannii. Overexpression of OXA genes is driven by insertion sequences, which provide promoter sequences, thereby contributing to high levels of carbapenem resistance. The insertion sequence
ISAba1 belongs to the IS4 family and has been associated with several resistance genes in
A. baumannii, such as
blaOXA-23,
blaOXA-51 and
blaOXA-58 [
6].
Limited data are available on the resistance pattern and molecular determinants responsible for carbapenem resistance in A. baumannii from India. The objectives of this study were (1) to determine the trend in the antibiotic resistance profile for invasive Acinetobacter species and (2) to detect the prevalence of the resistance genes responsible for beta lactamases such as extended spectrum and carbapenamases in Acinetobacter species.
Results
A total of 103 invasive clinical isolates of Acinetobacter species were included in this study. Thirty isolates were from blood specimens, and 73 isolates were from ETA.
All 103 isolates (100%) were confirmed as Acinetobacter baumannii by the presence of the blaOXA-51 gene.
Antimicrobial Susceptibility
Antimicrobial susceptibility testing revealed that all the isolates were resistant to ceftazidime, cefepime, piperacillin/tazobactam, cefoperazone/sulbactam, aztreonam, imipenem, meropenem, amikacin, netilmycin, tetracycline, tobramycin, levofloxacin and co-trimoxazole. Almost 102 (98%) of the study isolates were susceptible to polymyxin B.
Phenotypic Detection
Among the 103 clinical isolates, the CarbAcineto NP test was positive in 94 (91.2%) isolates and negative in 9 (8.7%) (Table
1).
Table 1
CarbAcineto NP test and distribution of different types of β-lactamase genes among invasive isolates of Acinetobacter baumannii
Blood (n = 30) | 30 (32) | 0 (0) | 6 (14) | 2 (25) | 0 (0) | 1 (100) | 0 (0) | 6 (30) | 30 (30) | 30 (30) | 0 (0) | 29 (33) | 24 (29) |
ETA (n = 73) | 64 (68) | 9 (100) | 36 (86) | 6 (75) | 1 (100) | 0 (0) | 6 (100) | 14 (70) | 73 (70) | 73 (70) | 2 (100) | 60 (67) | 58 (70) |
Total (n = 103) | 94 | 9 | 42 | 8 | 1 | 1 | 6 | 20 | 103 | 103 | 2 | 89 | 82 |
PCR for ESBL, MBL and OXA Genes
Among the 103 isolates tested for ESBLs, the PER gene was present in 42 isolates (41.5%) followed by the TEM gene in 8 (7.9%), SHV gene in 1 (0.9%) and VEB gene in 1 (0.9%) isolate. PER and TEM genes coexisted in seven (6.9%) isolates followed by PER and SHV coexisting in one isolate (2%). None of the isolates had the GES gene (Table
1).
Twenty isolates were positive for the NDM gene (19.2%), and six isolates were positive for the VIM gene (5.7%). One isolate (0.9%) had both VIM and NDM genes. None of the isolates had KPC, IMP, SPM and SIM genes (Table
1).
The acquired OXA carbapenemase,
blaOXA-23 gene was present in 101 isolates (98%). Two isolates (2%) had both
blaOXA-23 and
blaOXA-24 genes. None of the isolates had the
blaOXA-58 gene (Table
1).
ISAba1 and Mapping PCR
ISAba1 and mapping PCR was performed for all the clinical isolates (
n = 103). The
ISAba1 element was found upstream of the corresponding
blaOXA-23 gene in 82 isolates (79.6%). Seven isolates (6.7%) were positive for the
ISAba1 element but not found upstream of the
blaOXA-23 gene. Fourteen isolates (14%) were negative for the
ISAba1 element (Table
1).
Discussion
Multidrug-resistant
A. baumannii is increasingly reported in healthcare-associated infections worldwide. This scenario has left carbapenems as the drug of choice to treat severe infections caused by MDR
A. baumannii. However, increased resistance to carbapenems due to diverse intrinsic and acquired resistance mechanisms is emerging. Production of class D OXA carbapenemases and class B metallo-β-lactamases plays a predominant role in contributing to carbapenem resistance to
A. baumannii worldwide [
17].
The SENTRY study reports that the susceptibility rate of imipenem to
A. baumannii has declined from 73.7% in 2001–2004 to 37.4 in 2009 in the Asia-Pacific region [
18]. The Tigecycline Evaluation and Surveillance Trial (TEST) study between 2005 and 2009 reported the overall susceptibility rate for imipenem and meropenem was 54.1% and 51.8%, respectively [
1]. A recent study from South India has reported 34–93.6% carbapenem susceptibility in
A. baumannii isolates [
19].
Due to the intrinsic low permeability of carbapenems, phenotypic detection of carbapenemase is difficult in
Acinetobacter sp. A number of phenotypic tests such as the modified Hodge test and inhibition-based tests have been proposed for the rapid detection of carbapenemases in
Acinetobacter sp. These tests were efficient in detecting IMP and VIM producers, leaving the most predominant carbapenemases such as OXA and NDM producers undetected [
10]. Although CLSI 2015 recommends the use of the carba NP test, it has not been well validated for the detection of carbapenemase-producing
Acinetobacter sp. The rate of imipenem hydrolysis achieved with OXA enzymes is too low to be detected with the carba NP test [
10]. Therefore, a modified version of the Carba NP known as the CarbAcineto NP was proposed for the phenotypic detection of carbapenemase production in
Acinetobacter sp. The sensitivity and specificity of the CarbAcineto NP test are reported to be 100% and 94.7%, respectively, in comparison to the carba NP test where the sensitivity is 11.9% only [
10]. However, the CLSI is non-committal about this modification.
In this study, 94 out of 103 clinical isolates were positive for the CarbAcineto NP test, and all were positive for the blaOXA-23 gene. Nine isolates were negative for the CarbAcineto NP.
Among the study isolates, OXA carbapenemases were detected in 101 (98%) isolates of carbapenem-resistant
A. baumannii. The
blaOXA-23-like oxacillinases were the most common type. A study from East India also showed the OXA-23 genes as the prevalent type of oxacillinase contributing to carbapenem resistance [
17]. Another study from South India showed the prevalence of the OXA-23 gene to be 56.8% [
19]. Among the metallo β-lactamases (MBLs), 20 isolates were positive for the NDM gene while 6 were positive for the VIM gene. This study showed NDM as the predominant MBL gene. However, studies by Saranathan et al. and Amudhan et al. showed IMP-like and VIM-like as the prevalent MBL genes [
19,
20]. Of the 103 isolates tested, 52 (50%) were positive for 1 of the ESBL genes such as PER, TEM and SHV. In addition, we found coexistence of PER, TEM and OXA-23 in six isolates, PER, TEM, VIM and OXA-23 in one isolate, and PER, SHV and OXA-23 in one isolate.
Class D OXA enzymes possess weak carbapenemase activity. However, the presence of an insertion sequence upstream of the OXA gene promotes their expression and can modulate the mobility of OXA genes. Evans et al. reported that a promoter for the
blaOXA-23-like gene is provided by
ISAba1 when it is inserted 25-bp upstream of the gene [
21]. Also studies have shown that transposons such as Tn2006 can be formed by two
ISAba1 elements bracketing the
blaOXA-23 gene, which in turn involves in the dissemination of resistance determinants [
22]. In this study, we found that 79.6% of the isolates had
ISAba1 upstream of the OXA-23-like gene, which could provide a promoter for OXA-23 gene overproduction.
Even though the
blaOXA-51-like gene is chromosomally encoded and has weak carbapenemase activity, studies have reported that the presence of
ISAba1 upstream to this gene can provide a promoter that allows the hyper-production of carbapenemase, resulting in carbapenem resistance [
6]. In this study, 6% of the carbapenem-resistant
A. baumannii isolates had an
ISAba1 element, which was not found upstream but seen elsewhere than the
blaOXA-23 gene. Carbapenem resistance in these isolates might be due to overexpression of the
blaOXA-51 gene. Future studies are required to confirm the role of insertion sequence-induced overexpression of the
blaOXA-51 gene. Studies have reported that carbapenem resistance in
A. baumannii is mainly due to carbapenemase mediated [
4]. However, non-carbapenemase-mediated resistance mechanisms such as reduced membrane permeability due to porin changes and overexpression of efflux pumps make a trivial contribution toward carbapenem resistance in
A. baumannii [
23]. Heritier et al. reported that the synergism between acquired oxacillinases and the RND efflux pump, AdeABC, was implicated in the increased levels of resistance toward carbapenems [
24]. Therefore, future studies on the role of non-carbapenemase-mediated mechanisms in
A. baumannii are required.
Acknowledgments
The authors gratefully acknowledge Dr. Kamini Walia, Scientist E, Division of Epidemiology and Communicable Diseases, Indian Council of Medical Research (ICMR), New Delhi, and the ICMR Council itself for providing the grant for this research and the Institutional Review Board of the Christian Medical College, Vellore (83-i/11/13). All named authors meet the International Committee of Medical Journal Editors (ICMJE) criteria for authorship for this manuscript, take responsibility for the integrity of the work as a whole and have given final approval for the version to be published. The study has been funded by Indian Council of Medical Research, New Delhi, India (ref. no. AMR/TF/54/13ECDHII dated 23/10/2013).