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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 23  |  Issue : 2  |  Page : 63-68

Detection of Metallo-β-lactamase production amongst Acinetobacter species from a tertiary care hospital


Department of Microbiology, Bharati Vidyapeeth Deemed to be University Medical College, Pune, Maharashtra, India

Date of Submission06-Oct-2021
Date of Decision08-Oct-2021
Date of Acceptance24-Nov-2021
Date of Web Publication27-Jan-2022

Correspondence Address:
Dr. Vrushali Harsh Thakar
Department of Microbiology, Bharati Vidyapeeth Deemed to be University Medical College, Dhankawadi, Pune - 411 043, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jacm.jacm_64_21

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  Abstract 


INTRODUCTION: In the past few years, resistance to antimicrobial drugs has been increasing in Acinetobacter spp., which will likely become a substantial treatment challenge in the future. Carbapenems have potent activity against Acinetobacter spp. and are usually the drugs of choice against multidrug-resistant Acinetobacter baumannii. Acinetobacter spp. may develop resistance to carbapenems by producing Metallo-β-lactamases (MBLs). The emergence of MBL-encoding genes is worrisome, since they are usually carried by mobile genetic structures with great ability to spread.
AIMS AND OBJECTIVES: The aim of this study was undertaken to find out the prevalence of MBL producing Acinetobacter species from a tertiary care hospital.
MATERIALS AND METHODS: Acinetobacter species were identified by conventional methods and antimicrobial susceptibility tests were done by the Kirby Bauer's technique. The presence of MBL was detected by the combination disc method and by VITEK 2. Verona integrin-encoded MBL (VIM) gene was detected in 15 MBL positive strains by polymerase chain reaction (PCR). Five PCR positive strains were sent for sequence analysis.
RESULTS: A total of 72 Acinetobacter strains were processed. Thirty-two strains were MBL positive phenotypically. All MBL positive strains were susceptible to colistin. Of 32 MBL positive strains, PCR was done on 15 strains. VIM gene was detected in all 15 strains.
CONCLUSION: This study highlights the emergence of MBL strains. Hence, proper hospital infection control practices and rapid MBL detection by laboratory is important to avoid treatment failures.

Keywords: Acinetobacter, bla-VIM, Metallo-β-lactamase


How to cite this article:
Thakar VH, Chakraborthy A, Modak M, Krunal L. Detection of Metallo-β-lactamase production amongst Acinetobacter species from a tertiary care hospital. J Acad Clin Microbiol 2021;23:63-8

How to cite this URL:
Thakar VH, Chakraborthy A, Modak M, Krunal L. Detection of Metallo-β-lactamase production amongst Acinetobacter species from a tertiary care hospital. J Acad Clin Microbiol [serial online] 2021 [cited 2022 Jul 1];23:63-8. Available from: https://www.jacmjournal.org/text.asp?2021/23/2/63/336589




  Introduction Top


In the past few years, resistance to antimicrobial drugs has been increasing in Acinetobacter spp., which will likely become a substantial treatment challenge in the future. Carbapenems have potent activity against Acinetobacter spp. and are usually the drugs of choice against multidrug-resistant Acinetobacter baumannii isolates.[1].Acinetobacter spp. may develop resistance to carbapenems through various mechanisms including class B and D carbapenemase production, decreased permeability or altered penicillin-binding proteins.[2]

Predominant carbapenemase elaborated by Acinetobacter is class D oxacillinases. However, there are increasing reports of metallo-β-lactamase (MBL) production by Acinetobacter.[3]

Two types of carbapenemases are recognised: serine β-lactamases and MBLs. Based on amino acid sequence homologies, 5 MBL types have been recognised: IMP (imipenase), VIM (Verona integrin-encoded MBL), SPM (Sao Paulo MBL), GIM (German imipenase) and SIM (Seoul imipenase).[4]

The emergence of MBL-encoding genes is worrisome, since they are usually carried by mobile genetic structures with great ability to spread.[5].Moreover, increased mortality rates have been documented for patients infected with MBL-producing organisms, especially due to inadequate empirical therapy. Therefore, early detection of MBL-producing organisms is crucial to establish appropriate antimicrobial therapy and to prevent their inter- and intra-hospital dissemination.[6]

The study was undertaken to find out the prevalence of MBL-producing Acinetobacter species from a tertiary care hospital.


  Materials and Methods Top


Total 72 non-duplicate strains of Acinetobacter spp. isolated from various clinical specimens during 1 year (November 2016–December 2017) were included in the study. Isolates were identified by routine conventional methods. Antimicrobial susceptibility testing was done by the Kirby–Bauer's disc diffusion method and by VITEK 2.

All isolates were tested for MBL production by doing combined disc method as described by Yong et al.[7]

Combination disc method

Test organism was inoculated on Muller-Hinton Agar plates according to the The Clinical and laboratory Standards Institute CLSI guidelines[8] for antimicrobial susceptibility testing. An imipenem (10ug) disc was placed centre-to-centre from imipenem-ethylenediaminetetraacetic acid (EDTA) disc (750ug) [procured from Himedia laboratories]. The inhibition zones of imipenem and imipenem-EDTA discs were compared after 16–18 h of incubation at 37°c.

Metallo-β-lactamase positive

Strains showing >7 mm increase in zone size of imipenem-EDTA disc as compared to plain imipenem disc.

Control strain used-ATCC P. aeruginosa 27853 (negative control).

Known MBL-producing Acinetobacter baumanii strain isolated in laboratory was used as positive control.

Metallo-β-lactamase negative

No increase in zone size of combination disc.

All phenotypically MBL positive strains were preserved in Trypticase Soy broth at- 20°c for further studies.

Polymerase chain reaction

A total of 15 MBL positive strains were selected for genotypic confirmation. Deoxyribonucleic acid (DNA) extraction was done using QIAGEN DNA extraction mini kit for Gram-negative bacilli.

Primers used for the detection of VIM gene were:

Bla-VIM primers: Bla-VIM-F (5′-GTTTGGTCG CATATCGCAAC-3′).

Bla-VIM-R (5′-AATGCGCAGCACCAGGATAG-3′).[9]

Polymerase chain reaction mixture (25ul each) was as follows:

12.5 ul of master mix (Genetix Biotech Asia Pvt., Ltd) containing (400uMdATP, 400uMdGTP, 400uMdCTP, 400uMdTTP, 4 mMMg2+ and Gene Max TM Hot Start DNA polymerase), 1ul forward primer, 1ul reverse primer, 2ul DNA and 8.5ul nuclease-free water.

Polymerase chain reaction conditions included

Polymerase chain reaction (PCR) conditions included: initial denaturation at 94°C for 5 min, followed by 33 cycles each of 94°C for 25 s, 53°C for 40 s and 72°C for 50 s followed by a single final elongation step at 72°C for 6 min. The PCR product of 382 bp for bla-VIM was visualised by 2% agarose gel electrophoresis containing ethidium bromide 0.5 μg/ml (VWR life sciences). VWR is largest supplier of laboratory chemicals and apparatus. VWR was acquired by Avantor in 2017. Indian distributor is in Bengaluru. Long form I couldn't find.

Sequence analysis

Five VIM-positive PCR products were sent for sequencing to Eurofins Genomics, and VIM gene sequence was found out.


  Results Top


Specimen wise distribution of Acinetobacter strains is given in [Table 1].
Table 1: Specimen wise distribution of Acinetobacter strains

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Of 72 Acinetobacter strains, 32 (44.44%) strains were MBL positive by combination disc method [Figure 1] and by VITEK 2. Specimen wise distribution of MBL positive strains is shown in [Table 2]. The maximum number of MBL (12) positive strains were isolated from respiratory samples (37.5%).
Figure 1: MBL positive by combination disc method

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Table 2: Specimen wise distribution of MBL positive strains

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All MBL positive strains were isolated from indoor patients except one which was from the outdoor patient.

Antimicrobial susceptibility pattern of MBL positive strains is shown in [Table 3].
Table 3: Antimicrobial susceptibility pattern of MBL positive Acinetobacter spp. (percentage resistance)

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All MBL positive strains were susceptible to colistin.

PCR was done for VIM gene detection on 15 MBL positive strains. VIM gene was detected in all 15 strains [Figure 2].
Figure 2: Gel showing PCR products

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Five PCR products were sent for sequence analysis.

Follow-up of patients

All indoor patients infected with MBL positive strains were critical and were admitted to the intensive care unit. Hence, they were treated with injection colistin. The Outpatient department (OPD) patient was from urology OPD. The patient was catheterised post-surgery. Acinetobacter baumanii isolated was considered catheter coloniser. The change of urinary catheter was advised. The patient was treated with fluoroquinolone.


  Discussion Top


MBLs have been identified from clinical isolates worldwide, with an increasing frequency over the past few years and strains producing these enzymes have been responsible for prolonged nosocomial outbreaks that were accompanied by serious infections.[10]

The prevalence rate of MBL-producing Acinetobacter spp in our study was 44.44%. Similar rates (49%) were observed by Peymani et al.[11] at a tertiary hospital in Iran and by Goel et al. (48%) in Belgum, Karnataka.[12].

About 37.5% of MBL positive strains were isolated from respiratory samples of patients admitted to the intensive care unit. Similar findings have been observed by Hare Krishna Nath in Assam.[13]

The majority of MBL positive strains were resistant to commonly used antibiotics including cephalosporins, aminoglycosides, fluoroquinolones, carbapenems, etc., except colistin, a characteristic feature of MBL producers. Kumar et al.[14] and Irfan et al.[15] have reported such a high level of resistance amongst MBL positive Acinetobacter strains.

Recently, the Clinical and Laboratory Standards Institute has included the Carba NP test for testing carbapenemase production amongst imipenem-resistant Acinetobacter spp. However, we did combination disc method as it was easier to perform routinely. Results of the combination disc method were also confirmed by VITEK 2 and PCR.

We did PCR for VIM gene detection only on 15 strains. However, all the strains were positive for VIM gene. Purohit et al.[9] have also detected VIM gene amongst MBL-producing Acinetobacter spp using similar primers.

Sequence analysis-amplified sequences were identified with the help of BLAST program.[16] All 10 (forward and reverse) sequences have shown 90%–99% identity with Acinetobacter baumannii strains. [Table 4] shows our sequences.
Table 4: blast hit results with our amplified Polymer chain reaction sequences

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We have compared all these sequences with reported VIM genes of A. baumanii. Total 19 sequences were compared with above 10 sequences for identity, but none of the reported VIM genes shown any type of identity with our amplified VIM genes. The reported VIM genes of A. baumanii are given in [Table 5].
Table 5: Reported verona integrin-encoded MBL genes of Acinetobacter baumanii in NCBI nucleotide database

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We have submitted the sequences to the Genbank of National Center for Biotechnology Information (NCBI) database. The temporary accession number is 'BankIt 2144757'. Further studies are needed to find out whether our VIM gene sequence is a new one or not.


  Conclusion Top


MBL production in Acinetobacter spp is alarming as it makes these organisms resistant to all commonly used antibiotics. Acinetobacter strains were also commonly isolated from respiratory samples of indoor patients prolonging their morbidity. Restricting the use of carbapenems and proper infection control policies to reduce the spread of these multi-drug organisms is the need of the hour.

Combination disc method is simple to perform, so it can be used as a screening test for early detection of MBL production. Bla-VIM was detected in our study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Tsakris A, Ikonomidis A, Pournaras S, Tzouvelekis LS, Sofianou D, Legakis NJ, et al. VIM-1 Metallo-beta-lactamase in Acinetobacter baumannii. Emerg Infect Dis 2006;12:981-3.  Back to cited text no. 1
    
2.
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. 2
    
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Zarrilli R, Giannouli M, Tomasone F, Triassi M, Tsakris A. Carbapenem resistance in Acinetobacter baumannii: The molecular epidemic features of an emerging problem in health care facilities. J Infect Dev Ctries 2009;3:335-41.  Back to cited text no. 3
    
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Mohmed N, Raafat D. Phenotypic and genotypic detection of metallo-beta-lactamase in imipenem resistant Acinetobacter baumanii isolated from tertiary hospital in Alexandria, Egypt. Res J Microbiol 2011;6:750-60.  Back to cited text no. 4
    
5.
Picão RC, Andrade SS, Nicoletti AG, Campana EH, Moraes GC, Mendes RE, et al. Metallo-beta-lactamase detection: Comparative evaluation of double-disk synergy versus combined disk tests for IMP-, GIM-, SIM-, SPM-, or VIM-producing isolates. J Clin Microbiol 2008;46:2028-37.  Back to cited text no. 5
    
6.
Cornaglia G, Akova M, Amicosante G, Cantón R, Cauda R, Docquier JD, et al. Metallo-beta-lactamases as emerging resistance determinants in Gram-negative pathogens: Open issues. Int J Antimicrob Agents 2007;29:380-8.  Back to cited text no. 6
    
7.
Yong D, Lee K, Yum JH, Shin HB, Rossolini GM, Chong Y. Imipenem-EDTA disk method for differentiation of metallo-beta-lactamase-producing clinical isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol 2002;40:3798-801.  Back to cited text no. 7
    
8.
Clinical Laboratory Standards Institute. Performance Standards for Antimicrobial Disk Susceptibility Testing, M02-A11. 11th ed. Wayne, PA: CLSI; 2012. p. 68.  Back to cited text no. 8
    
9.
Purohit M, Mendiratta DK, Deotale VS, Madhan M, Manoharan A, Narang P. Detection of metallo-β-lactamases producing Acinetobacter baumannii using microbiological assay, disc synergy test and PCR. Indian J Med Microbiol 2012;30:456-61.  Back to cited text no. 9
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Senda K, Arakawa Y, Nakashima K, Ito H, Ichiyama S, Shimokata K, et al. Multifocal outbreaks of metallo-beta-lactamase-producing Pseudomonas aeruginosa resistant to broad-spectrum beta-lactams, including carbapenems. Antimicrob Agents Chemother 1996;40:349-53.  Back to cited text no. 10
    
11.
Peymani A, Nahaei MR, Farajnia S, Hasani A, Mirsalehian A, Sohrabi N, et al. High prevalence of metallo-beta-lactamase-producing Acinetobacter baumannii in a teaching hospital in Tabriz, Iran. Jpn J Infect Dis 2011;64:69-71.  Back to cited text no. 11
    
12.
Goel V, Hogadel SA, Karadesai SG. Prevalence of extended spectrum beta lactamases, Amp C beta lactamases and metallobeta lactamases producing Pseudomonas aeruginosa, Acinetobacter baumanii in an ICU in a tertiary care hospital. J Sci Soc 2013;40:28-30.  Back to cited text no. 12
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Nath H, Barkataki D. Study of Acinetobacter isolates from clinical specimens in tertiary care hospital and their antimicrobial susceptibility pattern. Int J Curr Microbiol Appl Sci 2016;5:842-8.  Back to cited text no. 13
    
14.
Kumar SH, De AS, Baveja SM, Gore MA. Prevalence and risk factors of Metallo β-lactamase producing Pseudomonas aeruginosa and Acinetobacter species in burns and surgical wards in a tertiary care hospital. J Lab Physicians 2012;4:39-42.  Back to cited text no. 14
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Irfan S, Zafar A, Guhar D, Ahsan T, Hasan R. Metallo-beta-lactamase-producing clinical isolates of Acinetobacter species and Pseudomonas aeruginosa from Intensive Care Unit patients of a tertiary care hospital. Indian J Med Microbiol 2008;26:243-5.  Back to cited text no. 15
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Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990;215:403-10.  Back to cited text no. 16
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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