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  Table of Contents  
Year : 2013  |  Volume : 31  |  Issue : 3  |  Page : 237-241

Multiple carbapenem hydrolyzing genes in clinical isolates of Acinetobacter baumannii

1 Department of Microbiology, University College of Medical Sciences and Guru Teg Bahadur Hospital, Dilshad Garden, New Delhi, India
2 Department of Microbiology and Infectious Diseases, Chacha Nehru Bal Chikitsalaya, New Delhi, India

Date of Submission16-Aug-2013
Date of Acceptance04-Apr-2013
Date of Web Publication25-Jul-2013

Correspondence Address:
S Rai
Department of Microbiology, University College of Medical Sciences and Guru Teg Bahadur Hospital, Dilshad Garden, New Delhi
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0255-0857.115626

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 ~ Abstract 

Purpose: Carbapenem resistance in Acinetobacter baumannii has become highly rampant, which has been ascribed to the presence of multiple carbapenemases. The objective of the present study was to prospectively investigate the presence of multiple carbapenemase encoding genes in clinical isolates of A. baumannii. Materials and Methods: A total of 30 imipenem resistant, consecutive non-repeat clinical isolates A. baumannii from a Tertiary Care Centre of Delhi were subjected to antimicrobial susceptibility testing (AST), screening for carbapenemase production by modified Hodge test (MHT) and determination of minimum inhibitory concentration for imipenem by E-Test® . These were subjected to Real time PCR for blaIMP-1 and 2 , blaVIM-1 and 2 , blaOXA23, 24, 51 and 58 using SYBR green-I. These were grouped together on the basis of their genotype as each isolate harboured multiple carbapenemases and correlated with their AST profile. Detection of the novel carbapenemase blaNDM-1 was performed by real time PCR using TaqMan probes on 14 isolates. Results: Colistin appeared to be the most effective drug in vitro, followed by tetracycline and beta lactam/beta lactamase inhibitor combinations. All, but one isolate were positive for the MHT. All 30 isolates were positive for blaOXA-51 like gene as well as blaIMP-1 and blaVIM-1 genes. blaOXA 24 and 58 were not detected in any of the isolates. blaIMP-2 , blaVIM-2 , blaOXA-23 were present in 15, 6 and 14 isolates respectively. Grouping based on the genotypic profile did not correlate with susceptibility pattern. Nine among the 14 isolates also harboured the novel blaNDM-1 gene. Conclusions: This is the first study from North India, which comprehensively detected the presence of multiple carbapenemases as well the blaNDM-1 gene. The presence of the novel gene blaNDM-1 indicated ability of A. baumannii to acquire new carbapenemase genes despite the existence of multiple carbapenemase genes. The present study confirmed the presence of multiple genetic mechanisms for carbapenemases production among the clinical isolates of A. baumannii in north India.

Keywords: Acinetobacter baumannii, multiple carbapanemases, real time PCR

How to cite this article:
Niranjan D K, Singh N P, Manchanda V, Rai S, Kaur I R. Multiple carbapenem hydrolyzing genes in clinical isolates of Acinetobacter baumannii. Indian J Med Microbiol 2013;31:237-41

How to cite this URL:
Niranjan D K, Singh N P, Manchanda V, Rai S, Kaur I R. Multiple carbapenem hydrolyzing genes in clinical isolates of Acinetobacter baumannii. Indian J Med Microbiol [serial online] 2013 [cited 2021 Mar 3];31:237-41. Available from:

 ~ Introduction Top

Acinetobacter baumannii has emerged as an extensively-drug-resistant pathogen implicated in healthcare associated infections (HCAIs) such as ventilator associated pneumonia, urinary tract infection, bacteremia, septicemia, secondary meningitis, wound infection etc. [1] Community acquired infections due to Acinetobacter spp. are also on the rise with community acquired pneumonia being the most common followed by meningitis and cellulitis. [2],[3]

One of the most striking features of A. baumannii is its extraordinary ability to develop resistance against major antibiotic classes. [1],[3] In the recent past carbapenems had been drugs of choice for serious infections with A. baumannii, but carbapenem resistant strains are rapidly emerging. There are several factors leading to carbapenem resistance in A. baumannii, most important being the acquisition of carbapenem hydrolysing β-lactamases. Other mechanisms include the presence of mobile genetic elements, reduced expression of outer membrane proteins, altered affinity or expression of penicillin-binding proteins and multidrug efflux pumps. [4]

On the basis of molecular studies, carbapenem hydrolysing β-lactamases can be either metallo-β-lactamases (MBLs) or serine carbapenemases. MBLs may either be located on chromosomes or encoded by transferable genes. Genes encoding different MBLs identified in A. baumannii includes blaIMP , blaVIM, and recently the blaNDM gene. Serine carbapenemases enzymes are either of Ambler Class A or Class D. Class A carbapenemases (KPC, GES, SME, NMC, and IMI) have not been described for A. baumannii. Class D or, the oxacillinases have been characterized from A. baumannii and include blaOXA 23-27 , blaOXA-40 , blaOXA-48 , blaOXA-51 , and blaOXA-58 . Despite having a weak carbapenemase activity, these enzymes are able to confer resistance to imipenem and meropenem and are only partially inhibited by clavulanic acid. [5]

There is a paucity of studies highlighting presence of multiple genes encoding carbapenem hydrolyzing enzymes in A. baumannii including the novel MBL blaNDM-1 . The present study was carried out to identify the presence of multiple carbapenem hydrolyzing enzymes among the clinical isolates of A. baumannii. The present study is first of its kind from India on the genotypic identification of MBLs and oxacillinases in A. baumannii that attempted to identify blaIMP-1 , blaIMP-2 , blaVIM-1 , blaVIM-2 and blaNDM-1 using real time PCR.

 ~ Materials and Methods Top

The present study is a prospective observational study conducted at a Tertiary Care Hospital at Delhi, India.

Bacterial strains

A total of 30 non-repeat, consecutive imipenem resistant A. baumannii isolates obtained from tracheal aspirates (13), pus (13) and blood samples (4) received during October 2008 to September 2009 were included. The isolates were identified as Acinetobacter calcoaceticus-baumannii complex by phenotypic methods using standard methods including Gram's stain followed by the biochemical reactions including, positive catalase and negative oxidase tests, oxidative utilization of glucose, citrate utilization as the sole source of carbon; and negative indole, methyl red, Voges Proskauer, and urease tests. [6] They were differentiated up to genospecies 2 (A. baumannii) or 13TU level based on malonate and lactate utilization and growth at different temperatures. [7] The genotypic identification of A. baumannii was confirmed by the presence of blaOXA-51 like carbapenemase gene in all isolates using the primers for the 353 bp fragment by real time PCR [Table 1]. [8]
Table 1: Primers used for carbapenamase detection in the present study

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Antimicrobial susceptibility testing (AST)

AST was performed by disc diffusion method as per CLSI guidelines for 16 antibiotics including piperacillin (100 μg), ceftazidime (30 μg), cefepime (30 μg), imipenem (10 μg), meropenem (10 μg), gentamicin (10 μg), amikacin (30 μg), tobramycin (10 μg), netilmicin (30 μg), trimethoprim/sulpha-methoxazole (1.25/23.75 μg), ciprofloxacin (5 μg), tetracycline (30 μg), colistin (30 μg), and beta lactam/beta lactamase inhibitor (BL/BLI) combinations like ticarcillin/clavulanic acid (75/10 μg), ampicillin/sulbactam (10/10 μg), piperacillin/tazobactam (100/10 μg), (HiMedia, India). Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and E. coli ATCC 35218 were used as quality control strains. [9] Minimum inhibitory concentration (MIC) for imipenem was determined by agar gradient technique using E-test® (BioMerieux, France). MBL producing strains were identified using the Modified Hodge test (MHT) and E-test MBL strips as per manufacturer's instructions. [9],[10]

Genotypic identification of carbapenemases

DNA preparation

For real time PCR, bacterial DNA was extracted from overnight growth of A. baumannii on sheep blood agar using MagNA Pure Compact® (Roche Applied Sciences, USA) automated system as per manufacturer's instructions.

Real time PCR for bla IMP , bla VIM and bla oxa using SYBR green chemistry

The 20 μl reaction mixture comprised of five μl extracted DNA, one μl of forward and reverse primer each (conc. 10 pmol/l), LightCycler® DNA Master SYBR Green-I 10 μl and three μl nuclease free water in one well of 96 well reaction plate. The reaction plate was loaded, and real time PCR was performed on Roche Light Cycler® 480 (Roche Applied Sciences, India). Primers used for detection of carbapenamases by real time PCR (other than blaNDM ) with the amplicon size and their PCR conditions are summarized in the [Table 1] and [Table 2] respectively. [11]
Table 2: Real time PCR cycles used in the present study

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Real-time PCR for bla NDM-1 using TaqMan primer probe

Real-time PCR assay was performed on 14 isolates using the primer and probe sequences derived from the Genbank sequence AB571289. Due to limited consumables for probe based RT-PCR, alternate strains were chosen out of the 30 isolates. Primer sequence F-(5'- GCT GGC GGT GGT GAC TC-3'), R-(5'- GGC AAG CTG GTT CGA CAA C-3') and TaqMan probe sequence 'FAM-TGG CAT AAG TCGCAA TCC CCG C - BBQ' as published previously. [12] E. coli ATCC 25922, K. pneumoniae ATCC 700603, A. baumannii ATCC 19609 and P. aeruginosa ATCC 27853 were included in the test panel as negative control strains. In house strains of E. coli, K. pneumoniae and A.baumannii harbouring blaNDM-1 gene, identified by PCR and gene sequencing were included as positive controls. [12]

 ~ Results Top

All A. baumannii isolates were susceptible to colistin (100%). Susceptibilities for other antimicrobials included tetracycline (80%), BL/BLI combinations (ticarcillin/clavulanic acid, ampicillin/sulbactam and piperacillin/tazobactam) (40%), netilmicin (23%), and tobramycin (16%). For imipenem, the MIC 50 and MIC 90 values were 16 and 32 μg/mL respectively. Twenty nine strains were identified as MBL producers by E-test (MBL) while one strain was indeterminate. All the strains were found to produce carbapenemases by MHT.

The presence of the blaoxa-51 -like gene in all 30 isolates confirmed the identification. In addition, all isolates harboured blaIMP-1 and blaVIM-1 genes. blaIMP-2 , blaVIM-2 and blaOXA-23 were found positive in 15, 6 and 14 isolates respectively [Figure 1]a-f. Based on the real time PCR results and antibiogram pattern the isolates were grouped together in an attempt to correlate the phenotypic antibiogram pattern with the genotype [Table 3]. Four strains found to harbour blaIMP1 and 2 and blaVIM1 and 2 positive were placed in group 1. Another set of 11 isolates found positive for blaIMP-1 and 2 and blaVIM-1 were placed in group 2. Thirteen isolates were found positive from blaIMP-1 and blaVIM-1 . These three groups were subdivided into subgroups a and b on the basis of presence of blaOXA-23 . The fourth group consisted of blaIMP-1 , blaVIM-1 and 2 positive isolates. The blaNDM-1 was found to be present in nine out of 14 isolates tested [Figure 2]. Among these nine isolates, two were susceptible to colistin only, four were susceptible to colistin and tetracycline, and three were susceptible to colistin, tetracycline and BL/BLI combinations.
Figure 1: a, b, c, d, e and f demonstrates the real time PCR (SYBR green based) amplification and melting curves for blaIMP-1, blaIMP-2, blaVIM-1, blaVIM-2, blaOXA-23 and blaOXA-51 respectively. All 30 Acinetobacter baumannii isolates were negative for blaOXA-24 and blaOXA-58

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Figure 2: Demonstrates real time PCR (TaqMan Probe Based) amplification curves for blaNDM-1

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Table 3: Grouping of isolates based on presence of different sets of carbapenemase genes and their susceptibility profile. blaOXA-51 being the genetic marker was present in all 30 isolates while bla OXA-24 and 58 were absent

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 ~ Discussion Top

Carbapenem resistant A. baumannii (CRAB) is rapidly emerging as a common cause of HCAI's. Such strains have been implicated in many recent outbreaks occurring mostly in intensive care units where extensive use of antibiotics has contributed to the selection of highly resistant strains. These isolates have been found to harbour multiple resistance genes against carbapenems. [1]

All isolates in the present study were multi drug resistant (MDR) as defined previously. [13],[1] Colistin (100% sensitive) was found as the only drug for management of infections caused by such isolates. MDR in A. baumannii may be explained by the fact that MBL genes in A. baumannii found on highly mobile genetic element within Class 1 integrons containing an array of resistance gene cassettes that have inserted themselves in the bacterial chromosome or plasmid between the 5'-conserved segment (5'- CS) and the 3'- CS together with other antibiotic resistance genes, mostly encoding aminoglygoside-modifying enzymes. [1] Imipenem MIC for strains in the present study ranged between 8 and 32 μg/ml which was similar to other reported studies. [14],[15],[16],[17] There was no correlation between the genotypic profile and susceptibility pattern. This may have been because of other mechanisms present, but their detection was out of the scope of this study. [4]

The IMP and VIM gene variants confer carbapenem resistance in A. baumannii isolates but they do not lead to very high MIC values. The co-presence of a masked extended spectrum beta lactamases or AmpC beta lactamases may render carbapenems ineffective against such isolates. [1] The isolates of the study were shown to harbour multiple carbapenem hydrolysing enzyme genes. All 30 isolates harboured blaIMP-1 , and blaVIM-1 genes besides blaOXA-51 which are usually found embedded in Class 1 integrons. [1] There has been a recent report from south India on the occurrence of oxacillinase production, which also demonstrated predominant presence of blaVIM along with blaOXA-23 among clinical isolates of CRAB.[17] Another report form south India has investigated co-presence of blaOXA-23 with blaNDM-1, [18] but this is the first comprehensive study from India which has probed for multiple genetic mechanisms, blaIMP-1 and 2 , blaVIM-1 and 2 , different blaOXA genes and the novel blaNDM-1 gene in A. baumannii isolates. Results of the present study are in agreement with reports about the presence of carbapenemase genes in the A. baumannii isolates. [8] The chromosomal blaOXA-51 -like gene, intrinsic to A. baumannii species, can confer carbapenem resistance when an insertion sequence ISAb/a1 element is inserted upstream of the gene; however, the present work did not investigate the source of carbapenemases, class-1 integron and ISAba1.The presence of the novel gene blaNDM-1 emphasizes the instant reception of A. baumannii to carbapenemase genes despite existence of multiple carbapenemase genes, a process previously described as "genetic capitalism." [19] The present study confirms multiple genetic mechanisms of carbapenemases production among the clinical isolates of A. baumannii in our setting.

 ~ References Top

1.Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii: Emergence of a successful pathogen. Clin Microbiol Rev 2008;21:538-82.  Back to cited text no. 1
2.Song JY, Kee SY, Hwang IS, Seo YB, Jeong HW, Kim WJ, et al. In vitro activities of carbapenem/sulbactam combination, colistin, colistin/rifampicin combination and tigecycline against carbapenem-resistant Acinetobacter baumannii. J Antimicrob Chemother 2007;60:317-22.  Back to cited text no. 2
3.Héritier C, Poirel L, Lambert T, Nordmann P. Contribution of acquired carbapenem-hydrolyzing oxacillinases to carbapenem resistance in Acinetobacter baumannii. Antimicrob Agents Chemother 2005;49:3198-202.  Back to cited text no. 3
4.Yu YS, Yang Q, Xu XW, Kong HS, Xu GY, Zhong BY. Typing and characterization of carbapenem-resistant Acinetobacter calcoaceticus-baumannii complex in a Chinese hospital. J Med Microbiol 2004;53:653-6.  Back to cited text no. 4
5.Queenan AM, Bush K. Carbapenemases: The versatile beta-lactamases. Clin Microbiol Rev 2007;20:440-58, table of contents.  Back to cited text no. 5
6.Winn W, Allen S, Janda W, Koneman E, Procop G, Schreckenberger P, et al., editors. Koneman's Color Atlas and Textbook of Diagnostic Microbiology. 6 th ed. Philadelphia: Lippincott Williams and Wilkins; 2006.  Back to cited text no. 6
7.Gerner-Smidt P, Tjernberg I, Ursing J. Reliability of phenotypic tests for identification of Acinetobacter species. J Clin Microbiol 1991;29:277-82.  Back to cited text no. 7
8.Turton JF, Woodford N, Glover J, Yarde S, Kaufmann ME, Pitt TL. Identification of Acinetobacter baumannii by detection of the blaOXA-51-like carbapenemase gene intrinsic to this species. J Clin Microbiol 2006;44:2974-6.  Back to cited text no. 8
9.Wayne PA. Clinical Laboratory Standards Institute, Performance standards for antimicrobial susceptibility testing, 19 th informational supplement. (M100-S19) Clinical Laboratory Standards Institute; 2009.  Back to cited text no. 9
10.Rai S, Manchanda V, Singh NP, Kaur IR. Zinc-dependent carbapenemases in clinical isolates of family Enterobacteriaceae. Indian J Med Microbiol 2011;29:275-9.  Back to cited text no. 10
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12.Manchanda V, Rai S, Gupta S, Rautela RS, Chopra R, Rawat DS, et al. Development of TaqMan real-time polymerase chain reaction for the detection of the newly emerging form of carbapenem resistance gene in clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii. Indian J Med Microbiol 2011;29:249-53.  Back to cited text no. 12
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13.Manchanda V, Sanchaita S, Singh N. Multidrug resistant acinetobacter. J Glob Infect Dis 2010;2:291-304.  Back to cited text no. 13
14.Takahashi A, Yomoda S, Kobayashi I, Okubo T, Tsunoda M, Iyobe S. Detection of carbapenemase-producing Acinetobacter baumannii in a hospital. J Clin Microbiol 2000;38:526-9.  Back to cited text no. 14
15.Jeon BC, Jeong SH, Bae IK, Kwon SB, Lee K, Young D, et al. Investigation of a nosocomial outbreak of imipenem-resistant Acinetobacter baumannii producing the OXA-23 beta-lactamase in korea. J Clin Microbiol 2005;43:2241-5.  Back to cited text no. 15
16.Merkier AK, Catalano M, Ramírez MS, Quiroga C, Orman B, Ratier L, et al. Polyclonal spread of bla (OXA-23) and bla (OXA-58) in Acinetobacter baumannii isolates from Argentina. J Infect Dev Ctries 2008;2:235-40.  Back to cited text no. 16
17.Amudhan SM, Sekar U, Arunagiri K, Sekar B. OXA beta-lactamase-mediated carbapenem resistance in Acinetobacter baumannii. Indian J Med Microbiol 2011;29:269-74.  Back to cited text no. 17
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18.Karthikeyan K, Thirunarayan MA, Krishnan P. Coexistence of blaOXA-23 with blaNDM-1 and armA in clinical isolates of Acinetobacter baumannii from India. J Antimicrob Chemother 2010;65:2253-4.  Back to cited text no. 18
19.Baquero F. From pieces to patterns: Evolutionary engineering in bacterial pathogens. Nat Rev Microbiol 2004;2:510-8.  Back to cited text no. 19


  [Figure 1], [Figure 2]

  [Table 1], [Table 2], [Table 3]

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