Comparison of loop-mediated isothermal amplification, multiplex PCR, and REP- PCR techniques for identification of carbapenem-resistant Acinetobacter baumannii clinical isolates
,
Abstract
Background and Objectives: Acinetobacter baumannii, an opportunistic pathogen, is related to hospital-acquired infections and increased mortality. This study aimed to develop the loop-mediated isothermal amplification (LAMP) test for the fast-detecting of A. baumannii isolates as well as determining genetic relatedness for these isolates via the REP-PCR technique.
Materials and Methods: LAMP primers and multiplex PCR primers were designed for recognizing A. baumannii isolates harboring the blaSHV-1, blaPER-1, blaTEM-1, AMPC, qnr, and aac (6)-1 genes, were collected (October 2020 to February 2021) from Shahid Motahari Hospital, Tehran, Iran. Combination disc test (CDT) results were used to assess the phenotypic identification of isolates from ESBL producers. The sensitivity of the LAMP method was evaluated using a range of serial dilutions of genomic DNA. Results were compared between the LAMP technique, and multiplex PCR. The genetic diversity of clinical isolates was determined by REP-PCR.
Results: Among one hundred A. baumannii samples and based on the combined disc test, 56% of isolates were ESBL producers. The sensitivity of the LAMP technique for the identification of A. baumannii was 4.06 ng/μl whilst the multiplex PCR was (16.2 ng/μl). Regarding multiplex PCR, (68%) of the isolates were blaSHV-1 positive, (40%) blaPER-1, (85%) aac (6́)-1, AMPC (67%), blaTEM-1 (63%), and (15%) qnr respectively. While in LAMP, (69%) of isolates were blaSHV-1 positive, (86%) aac (6')-1, and (20%) qnr. The results of AMPC, blaTEM-1, and blaPER-1 genes showed 100% compatibility between multiplex PCR and LAMP assays. The results of REP-PCR indicated there were 17 clones, clone A at 14% was the most prevalent of the isolates.
Conclusion: Wherever equipment and financial constraints are crucial, the LAMP test offers a better and more potent detection rate for the identification of A. baumannii isolates than multiplex PCR. Furthermore, the genetic diversity of A. baumannii in these clinical isolates showed frequent commonality of genotypes.
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2. Lima WG, Silva Alves GC, Sanches C, Antunes Fernandes SO, de Paiva MC. Carbapenem-resistant Acinetobacter baumannii in patients with burn injury: A systematic review and meta-analysis. Burns 2019; 45: 1495-1508.
3. Abdi SN, Ghotaslou R, Asgharzadeh M, Mehramouz B, Hasani A, Baghi HB, et al. AdeB efflux pump gene knockdown by mRNA mediated peptide nucleic acid in multidrug resistance Acinetobacter baumannii. Microb Pathog 2020; 139: 103825.
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6. Al-Sheboul SA, Al-Moghrabi SZ, Shboul Y, Atawneh F, Sharie AH, Nimri LF. Molecular characterization of carbapenem-resistant Acinetobacter baumannii isolated from intensive care unit patients in Jordanian Hospitals. Antibiotics (Basel) 2022; 11: 835.
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8. Bansal G, Allen-McFarlane R, Eribo B. Antibiotic susceptibility, clonality, and molecular characterization of carbapenem-resistant clinical isolates of Acinetobacter baumannii from Washington DC. Int J Microbiol 2020; 2020: 2120159.
9. Wong MH, Chan BK, Chan EW, Chen S. Over-expression of ISAba1-linked intrinsic and exogenously acquired OXA type carbapenem-hydrolyzing-class D-ß-Lactamase-encoding genes is key mechanism underlying carbapenem resistance in Acinetobacter baumannii. Front Microbiol 2019; 10: 2809.
10. Castanheira M, Simner PJ, Bradford PA. Extended-spectrum β-lactamases: an update on their characteristics, epidemiology and detection. JAC Antimicrob Resist 2021; 3: dlab092.
11. Krause KM, Serio AW, Kane TR, Connolly LE. Aminoglycosides: an overview. Cold Spring Harb Perspect Med 2016; 6: a027029.
12. Kishk R, Soliman N, Nemr N, Eldesouki R, Mahrous N, Gobouri A, et al. Prevalence of aminoglycoside resistance and aminoglycoside modifying enzymes in Acinetobacter baumannii among intensive care unit patients, Ismailia, Egypt. Infect Drug Resist 2021; 14: 143-150.
13. Zárate SG, De la Cruz Claure ML, Benito-Arenas R, Revuelta J, Santana AG, Bastida A. Overcoming aminoglycoside enzymatic resistance: design of novel antibiotics and inhibitors. Molecules 2018; 23: 284.
14. Venkataramana GP, Lalitha AKV, Mariappan S, Sekar U. Plasmid-mediated fluoroquinolone resistance in Pseudomonas aeruginosa and Acinetobacter baumannii. J Lab Physicians 2022; 14: 271-277.
15. Havenga B, Reyneke B, Ndlovu T, Khan W. Genotypic and phenotypic comparison of clinical and environmental Acinetobacter baumannii strains. Microb Pathog 2022; 172: 105749.
16. Sigmund IK, Renz N, Feihl S, Morgenstern C, Cabric S, Trampuz A. Value of multiplex PCR for detection of antimicrobial resistance in samples retrieved from patients with orthopaedic infections. BMC Microbiol 2020; 20: 88.
17. Sharma A, Gaind R. Development of loop-mediated isothermal amplification assay for detection of clinically significant members of Acinetobacter calcoaceticus–baumannii complex and associated carbapenem resistance. Front Mol Biosci 2021; 8: 659256.
18. Mancuso G, Midiri A, Gerace E, Biondo C. Bacterial antibiotic resistance: the most critical pathogens. Pathogens 2021; 10: 1310.
19. Tarafdar F, Jafari B, Azimi T. Evaluating the antimicrobial resistance patterns and molecular frequency of bla (oxa-48) and bla (GES-2) genes in Pseudomonas aeruginosa and Acinetobacter baumannii strains isolated from burn wound infection in Tehran, Iran. New Microbes New Infect 2020; 37: 100686.
20. Nogbou N-D, Nkawane GM, Ntshane K, Wairuri CK, Phofa DT, Mokgokong KK, et al. Efflux pump activity and mutations driving multidrug resistance in Acinetobacter baumannii at a tertiary Hospital in Pretoria, South Africa. Int J Microbiol 2021; 2021: 9923816.
21. Abdar MH, Taheri-Kalani M, Taheri K, Emadi B, Hasanzadeh A, Sedighi A, et al. Prevalence of extended-spectrum beta-lactamase genes in Acinetobacter baumannii strains isolated from nosocomial infections in Tehran, Iran. GMS Hyg Infect Control 2019; 14: Doc02.
22. Ranjbar R, Tolon SS, Zayeri S, Sami M. The frequency of antibiotic resistance and ESBLs among clinically Acinetobacter baumannii strains isolated from patients in a major hospital in Tehran, Iran. Open Microbiol J 2018; 12: 254-260.
23. Pournaja A, Rajabnia R, Razavi S, Solgi S, Ardebili A, Yaghoubi S, et al. Molecular characterization of carbapenem-resistant Acinetobacter baumannii isolated from pediatric burns patients in an Iranian hospital. Trop J Pharm Res 2018; 17: 135-141.
24. Goyal A, Mani NK, Chahar R, Soni A, Goyal S. Comparison of various phenotypic methods in detection of carbapenemases and metallo-beta-lactamases in carbapenem resistant clinical isolates of Acinetobacter species at a tertiary care centre in North India. Int J Curr Microbiol App Sci 2018; 7: 3023-3030.
25. Wolff N, Hendling M, Schroeder F, Schönthaler S, Geiss AF, Bedenic B, et al. Full pathogen characterisation: species identification including the detection of virulence factors and antibiotic resistance genes via multiplex DNA-assays. Sci Rep 2021; 11: 6001.
26. Tahbaz SV, Azimi L, Lari AR. Characterization of aminoglycoside resistance mechanisms in Acinetobacter baumannii isolates from burn wound colonization. Ann Burns Fire Disasters 2019; 32: 115-121.
27. Ranjbar R, Farahani A. Study of genetic diversity, biofilm formation, and detection of Carbapenemase, MBL, ESBL, and tetracycline resistance genes in multidrug-resistant Acinetobacter baumannii isolated from burn wound infections in Iran. Antimicrob Resist Infect Control 2019; 8: 172.
28. Liu Y, Liu X. Detection of AmpC β-lactamases in Acinetobacter baumannii in the Xuzhou region and analysis of drug resistance. Exp Ther Med 2015; 10: 933-936.
29. Chaudhary M, Payasi A. Molecular characterization and antimicrobial susceptibility study of Acinetobacter baumannii clinical isolates from Middle East, African and Indian patients. J Proteomics Bioinform 2012; 5: 265-269.
30. Mirnejad R, Heidary M, Bahramian A, Goudarzi M, Pournajaf A. Evaluation of polymyxin B susceptibility profile and detection of drug resistance genes among Acinetobacter baumannii clinical isolates in Tehran, Iran during 2015-2016. Mediterr J Hematol Infect Dis 2018; 10(1): e2018044.
31. Poirier AC, Kuang D, Siedler BS, Borah K, Mehat JW, Liu J, et al. Development of loop-mediated isothermal amplification rapid diagnostic assays for the detection of Klebsiella pneumoniae and carbapenemase genes in clinical samples. Front Mol Biosci 2022; 8: 794961.
32. Chu J, Shin J, Kang S, Shin S, Chung Y-J. Rapid and sensitive detection of Salmonella species targeting the hilA gene using a loop-mediated isothermal amplification assay. Genomics Inform 2021; 19(3): e30.
33. Garciglia Mercado C, Gaxiola Robles R, Ascencio F, Silva-Sanchez J, Estrada-Garcia MT, Gomez-Anduro G. Development of a LAMP method for detection of carbapenem-resistant Acinetobacter baumannii during a hospital outbreak. J Infect Dev Ctries 2020; 14: 494-501.
34. Nogbou N-D, Phofa DT, Nchabeleng M, Musyoki AM. Investigating multi-drug resistant Acinetobacter baumannii isolates at a tertiary hospital in Pretoria, South Africa. Indian J Med Microbiol 2021; 39: 218-223.
35. Kian B, Mirnejad R, Moradli G, Mirkalantari S, Golmohammadi R. Molecular Genotyping of Acinetobacter baumannii species isolated from patients in Tehran, Iran, by repetitive element PCR fingerprinting. Iran J Pathol 2018; 13: 144-150.
2. Lima WG, Silva Alves GC, Sanches C, Antunes Fernandes SO, de Paiva MC. Carbapenem-resistant Acinetobacter baumannii in patients with burn injury: A systematic review and meta-analysis. Burns 2019; 45: 1495-1508.
3. Abdi SN, Ghotaslou R, Asgharzadeh M, Mehramouz B, Hasani A, Baghi HB, et al. AdeB efflux pump gene knockdown by mRNA mediated peptide nucleic acid in multidrug resistance Acinetobacter baumannii. Microb Pathog 2020; 139: 103825.
4. Vrancianu CO, Gheorghe I, Czobor IB, Chifiriuc MC. Antibiotic resistance profiles, molecular mechanisms and innovative treatment strategies of Acinetobacter baumannii. Microorganisms 2020; 8: 935.
5. Gedefie A, Demsis W, Ashagrie M, Kassa Y, Tesfaye M, Tilahun M, et al. Acinetobacter baumannii biofilm Formation and its role in disease pathogenesis: A review. Infect Drug Resist 2021; 14: 3711-3719.
6. Al-Sheboul SA, Al-Moghrabi SZ, Shboul Y, Atawneh F, Sharie AH, Nimri LF. Molecular characterization of carbapenem-resistant Acinetobacter baumannii isolated from intensive care unit patients in Jordanian Hospitals. Antibiotics (Basel) 2022; 11: 835.
7. Vijayakumar S, Jacob JJ, Vasudevan K, Mathur P, Ray P, Neeravi A, et al. Genomic characterization of mobile genetic elements associated with carbapenem resistance of Acinetobacter baumannii from India. Front Microbiol 2022; 13: 869653.
8. Bansal G, Allen-McFarlane R, Eribo B. Antibiotic susceptibility, clonality, and molecular characterization of carbapenem-resistant clinical isolates of Acinetobacter baumannii from Washington DC. Int J Microbiol 2020; 2020: 2120159.
9. Wong MH, Chan BK, Chan EW, Chen S. Over-expression of ISAba1-linked intrinsic and exogenously acquired OXA type carbapenem-hydrolyzing-class D-ß-Lactamase-encoding genes is key mechanism underlying carbapenem resistance in Acinetobacter baumannii. Front Microbiol 2019; 10: 2809.
10. Castanheira M, Simner PJ, Bradford PA. Extended-spectrum β-lactamases: an update on their characteristics, epidemiology and detection. JAC Antimicrob Resist 2021; 3: dlab092.
11. Krause KM, Serio AW, Kane TR, Connolly LE. Aminoglycosides: an overview. Cold Spring Harb Perspect Med 2016; 6: a027029.
12. Kishk R, Soliman N, Nemr N, Eldesouki R, Mahrous N, Gobouri A, et al. Prevalence of aminoglycoside resistance and aminoglycoside modifying enzymes in Acinetobacter baumannii among intensive care unit patients, Ismailia, Egypt. Infect Drug Resist 2021; 14: 143-150.
13. Zárate SG, De la Cruz Claure ML, Benito-Arenas R, Revuelta J, Santana AG, Bastida A. Overcoming aminoglycoside enzymatic resistance: design of novel antibiotics and inhibitors. Molecules 2018; 23: 284.
14. Venkataramana GP, Lalitha AKV, Mariappan S, Sekar U. Plasmid-mediated fluoroquinolone resistance in Pseudomonas aeruginosa and Acinetobacter baumannii. J Lab Physicians 2022; 14: 271-277.
15. Havenga B, Reyneke B, Ndlovu T, Khan W. Genotypic and phenotypic comparison of clinical and environmental Acinetobacter baumannii strains. Microb Pathog 2022; 172: 105749.
16. Sigmund IK, Renz N, Feihl S, Morgenstern C, Cabric S, Trampuz A. Value of multiplex PCR for detection of antimicrobial resistance in samples retrieved from patients with orthopaedic infections. BMC Microbiol 2020; 20: 88.
17. Sharma A, Gaind R. Development of loop-mediated isothermal amplification assay for detection of clinically significant members of Acinetobacter calcoaceticus–baumannii complex and associated carbapenem resistance. Front Mol Biosci 2021; 8: 659256.
18. Mancuso G, Midiri A, Gerace E, Biondo C. Bacterial antibiotic resistance: the most critical pathogens. Pathogens 2021; 10: 1310.
19. Tarafdar F, Jafari B, Azimi T. Evaluating the antimicrobial resistance patterns and molecular frequency of bla (oxa-48) and bla (GES-2) genes in Pseudomonas aeruginosa and Acinetobacter baumannii strains isolated from burn wound infection in Tehran, Iran. New Microbes New Infect 2020; 37: 100686.
20. Nogbou N-D, Nkawane GM, Ntshane K, Wairuri CK, Phofa DT, Mokgokong KK, et al. Efflux pump activity and mutations driving multidrug resistance in Acinetobacter baumannii at a tertiary Hospital in Pretoria, South Africa. Int J Microbiol 2021; 2021: 9923816.
21. Abdar MH, Taheri-Kalani M, Taheri K, Emadi B, Hasanzadeh A, Sedighi A, et al. Prevalence of extended-spectrum beta-lactamase genes in Acinetobacter baumannii strains isolated from nosocomial infections in Tehran, Iran. GMS Hyg Infect Control 2019; 14: Doc02.
22. Ranjbar R, Tolon SS, Zayeri S, Sami M. The frequency of antibiotic resistance and ESBLs among clinically Acinetobacter baumannii strains isolated from patients in a major hospital in Tehran, Iran. Open Microbiol J 2018; 12: 254-260.
23. Pournaja A, Rajabnia R, Razavi S, Solgi S, Ardebili A, Yaghoubi S, et al. Molecular characterization of carbapenem-resistant Acinetobacter baumannii isolated from pediatric burns patients in an Iranian hospital. Trop J Pharm Res 2018; 17: 135-141.
24. Goyal A, Mani NK, Chahar R, Soni A, Goyal S. Comparison of various phenotypic methods in detection of carbapenemases and metallo-beta-lactamases in carbapenem resistant clinical isolates of Acinetobacter species at a tertiary care centre in North India. Int J Curr Microbiol App Sci 2018; 7: 3023-3030.
25. Wolff N, Hendling M, Schroeder F, Schönthaler S, Geiss AF, Bedenic B, et al. Full pathogen characterisation: species identification including the detection of virulence factors and antibiotic resistance genes via multiplex DNA-assays. Sci Rep 2021; 11: 6001.
26. Tahbaz SV, Azimi L, Lari AR. Characterization of aminoglycoside resistance mechanisms in Acinetobacter baumannii isolates from burn wound colonization. Ann Burns Fire Disasters 2019; 32: 115-121.
27. Ranjbar R, Farahani A. Study of genetic diversity, biofilm formation, and detection of Carbapenemase, MBL, ESBL, and tetracycline resistance genes in multidrug-resistant Acinetobacter baumannii isolated from burn wound infections in Iran. Antimicrob Resist Infect Control 2019; 8: 172.
28. Liu Y, Liu X. Detection of AmpC β-lactamases in Acinetobacter baumannii in the Xuzhou region and analysis of drug resistance. Exp Ther Med 2015; 10: 933-936.
29. Chaudhary M, Payasi A. Molecular characterization and antimicrobial susceptibility study of Acinetobacter baumannii clinical isolates from Middle East, African and Indian patients. J Proteomics Bioinform 2012; 5: 265-269.
30. Mirnejad R, Heidary M, Bahramian A, Goudarzi M, Pournajaf A. Evaluation of polymyxin B susceptibility profile and detection of drug resistance genes among Acinetobacter baumannii clinical isolates in Tehran, Iran during 2015-2016. Mediterr J Hematol Infect Dis 2018; 10(1): e2018044.
31. Poirier AC, Kuang D, Siedler BS, Borah K, Mehat JW, Liu J, et al. Development of loop-mediated isothermal amplification rapid diagnostic assays for the detection of Klebsiella pneumoniae and carbapenemase genes in clinical samples. Front Mol Biosci 2022; 8: 794961.
32. Chu J, Shin J, Kang S, Shin S, Chung Y-J. Rapid and sensitive detection of Salmonella species targeting the hilA gene using a loop-mediated isothermal amplification assay. Genomics Inform 2021; 19(3): e30.
33. Garciglia Mercado C, Gaxiola Robles R, Ascencio F, Silva-Sanchez J, Estrada-Garcia MT, Gomez-Anduro G. Development of a LAMP method for detection of carbapenem-resistant Acinetobacter baumannii during a hospital outbreak. J Infect Dev Ctries 2020; 14: 494-501.
34. Nogbou N-D, Phofa DT, Nchabeleng M, Musyoki AM. Investigating multi-drug resistant Acinetobacter baumannii isolates at a tertiary hospital in Pretoria, South Africa. Indian J Med Microbiol 2021; 39: 218-223.
35. Kian B, Mirnejad R, Moradli G, Mirkalantari S, Golmohammadi R. Molecular Genotyping of Acinetobacter baumannii species isolated from patients in Tehran, Iran, by repetitive element PCR fingerprinting. Iran J Pathol 2018; 13: 144-150.
| Files | ||
| Issue | Vol 15 No 5 (2023) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v15i5.13871 | |
| Keywords | ||
| Acinetobacter baumannii; Antibiotic resistance; Loop-mediated isothermal amplification (LAMP); Multiplex polymerase chain reaction; Repetitive extragenic palindromic polymerase chain reaction | ||
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How to Cite
1.
Abbood Al jebur A, Soleimani N, Hosseini SM. Comparison of loop-mediated isothermal amplification, multiplex PCR, and REP- PCR techniques for identification of carbapenem-resistant Acinetobacter baumannii clinical isolates. Iran J Microbiol. 2023;15(5):654-664.
