|Year : 2021 | Volume
| Issue : 2 | Page : 63-68
Detection of Metallo-β-lactamase production amongst Acinetobacter species from a tertiary care hospital
Vrushali Harsh Thakar, Ankana Chakraborthy, M Modak, Lahiri Krunal
Department of Microbiology, Bharati Vidyapeeth Deemed to be University Medical College, Pune, Maharashtra, India
|Date of Submission||06-Oct-2021|
|Date of Decision||08-Oct-2021|
|Date of Acceptance||24-Nov-2021|
|Date of Web Publication||27-Jan-2022|
Dr. Vrushali Harsh Thakar
Department of Microbiology, Bharati Vidyapeeth Deemed to be University Medical College, Dhankawadi, Pune - 411 043, Maharashtra
Source of Support: None, Conflict of Interest: None
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|| |
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..Acinetobacter spp. may develop resistance to carbapenems through various mechanisms including class B and D carbapenemase production, decreased permeability or altered penicillin-binding proteins.
Predominant carbapenemase elaborated by Acinetobacter is class D oxacillinases. However, there are increasing reports of metallo-β-lactamase (MBL) production by Acinetobacter.
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).
The emergence of MBL-encoding genes is worrisome, since they are usually carried by mobile genetic structures with great ability to spread..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.
The study was undertaken to find out the prevalence of MBL-producing Acinetobacter species from a tertiary care hospital.
| Materials and Methods|| |
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.
Combination disc method
Test organism was inoculated on Muller-Hinton Agar plates according to the The Clinical and laboratory Standards Institute CLSI guidelines 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.
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.
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′).
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.
Five VIM-positive PCR products were sent for sequencing to Eurofins Genomics, and VIM gene sequence was found out.
| Results|| |
Specimen wise distribution of Acinetobacter strains is given in [Table 1].
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%).
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)|
Click here to view
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].
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|| |
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.
The prevalence rate of MBL-producing Acinetobacter spp in our study was 44.44%. Similar rates (49%) were observed by Peymani et al. at a tertiary hospital in Iran and by Goel et al. (48%) in Belgum, Karnataka..
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.
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. and Irfan et al. 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. 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. 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|
Click here to view
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|
Click here to view
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|| |
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
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]