The Expression of VanA Gene in Enterococcus faecium and Enterococcus faecalis Under Influence of Vancomycin

Zolfaghar, Ghazal; Rahnamaye Farzami, Marjan; Hosseini, Farzaneh; Akhavan Sepahi, Abbas

year 18, Issue 6 (November - December 2024) Iran J Med Microbiol 2024, 18(6): 411-422 | Back to browse issues page

Mendeley

Zotero

RefWorks

Zolfaghar G, Rahnamaye Farzami M, Hosseini F, Akhavan Sepahi A. The Expression of VanA Gene in Enterococcus faecium and Enterococcus faecalis Under Influence of Vancomycin. Iran J Med Microbiol 2024; 18 (6) :411-422
URL: http://ijmm.ir/article-1-2502-en.html

The Expression of VanA Gene in Enterococcus faecium and Enterococcus faecalis Under Influence of Vancomycin

Ghazal Zolfaghar¹

, Marjan Rahnamaye Farzami²

, Farzaneh Hosseini¹

, Abbas Akhavan Sepahi³

1- Department of Microbiology, School of Biological Sciences, North Tehran Branch, Islamic Azad University, Tehran, Iran
2- Department of Microbiology, Research Center of Health Reference Laboratory, Ministry of Health and Medical Education, Tehran, Iran
3- Department of Microbiology, School of Biological Sciences, North Tehran Branch, Islamic Azad University, Tehran, Iran , sepahiakh@gmail.com

Abstract: (231 Views)

Background & Objective: Enterococci are increasingly recognized as important pathogens in clinical settings due to their intrinsic and acquired antibiotic resistance. The aim of this study was to investigate the prevalence of Enterococcus (E.) faecalis and E. faecium in clinical samples, their antibiotic resistance patterns, and the correlation between vancomycin resistance and the presence of the vanA gene in both species.
Methods: A total of 120 isolates were identified from clinical samples. Phenotypic specification was confirmed by targeting D-alanine-D-alanine ligases specific for E. faecalis and E. faecium using PCR analysis. Antibiotic resistance was determined by antimicrobial susceptibility testing using disk diffusion method. The presence of van genes was investigated by multiplex PCR analysis. Real-time RT-PCR was used to determine the expression of vanA gene among vancomycin-resistant isolates.
Results: In this study, 82 E. faecalis (68.3%) and 38 E. faecium (31.6%) isolates from a total of 120 clinical samples were identified. Antimicrobial susceptibility testing revealed increased resistance rates among E. faecium, with resistance to ampicillin at 89.5% and penicillin at 84.2%, while E. faecalis showed lower rates of resistance at 5.8% and 7.2%, respectively. Multiplex PCR analysis confirmed that 54.1% of E. faecalis and 69.2% of E. faecium resistant isolates contained vanA gene. Notably, vancomycin-treated isolates exhibited 8.6-fold increase in vanA gene expression compared to untreated E. faecalis, while E. faecium showed 2.6-fold increase.
Conclusion: This study found a high prevalence of vancomycin-resistant Enterococcus (VRE) isolates, with majority carrying vanA gene. Resistance rates to multiple antibiotics were significantly higher in E. faecium compared to E. faecalis. Furthermore, vanA gene expression was notably increased in VRE isolates treated with vancomycin. The research highlights the genetic and environmental factors driving VRE emergence and proposes potential treatment strategies to combat this public health concern.

Keywords: Enterococcus, Gene expression, Resistance, Vancomycin, VRE

Full-Text [PDF 640 kb] (73 Downloads)

Type of Study: Original Research Article | Subject: Medical Bacteriology
Received: 2024/10/10 | Accepted: 2025/01/9 | ePublished: 2025/01/29

References

1. Rosenberg Goldstein RE, Micallef SA, Gibbs SG, George A, Claye E, Sapkota A, et al. Detection of vancomycin-resistant enterococci (VRE) at four U.S. wastewater treatment plants that provide effluent for reuse. Sci Total Environ. 2014;466-467:404-11. [DOI:10.1016/j.scitotenv.2013.07.039] [PMID] [PMCID]

2. Mubarak AG, El-Zamkan MA, Younis W, Saleh SO, Abd-Elhafeez HH, Yoseef AG. Phenotypic and genotypic characterization of Enterococcus faecalis and Enterococcus faecium isolated from fish, vegetables, and humans. Sci Rep. 2024;14(1):21741. [DOI:10.1038/s41598-024-71610-0] [PMID] [PMCID]

3. Krawczyk B, Wityk P, Gałęcka M, Michalik M. The Many Faces of Enterococcus spp.-Commensal, Probiotic and Opportunistic Pathogen. Microorganisms. 2021;9(9):1900. [DOI:10.3390/microorganisms9091900] [PMID] [PMCID]

4. Bortolaia V, Espinosa-Gongora C, Guardabassi L. Human health risks associated with antimicrobial-resistant enterococci and Staphylococcus aureus on poultry meat. Clin Microbiol Infect. 2016;22(2):130-40. [DOI:10.1016/j.cmi.2015.12.003] [PMID]

5. Arabestani MR, Nasaj M, Mousavi SM. Correlation between Infective Factors and Antibiotic Resistance in Enterococci Clinical Isolates in West of Iran. Chonnam Med J. 2017;53(1):56-63. [DOI:10.4068/cmj.2017.53.1.56] [PMID] [PMCID]

6. Osman KM, Ali MN, Radwan I, ElHofy F, Abed AH, Orabi A, et al. Dispersion of the Vancomycin Resistance Genes vanA and vanC of Enterococcus Isolated from Nile Tilapia on Retail Sale: A Public Health Hazard. Front Microbiol. 2016;7:1354. [DOI:10.3389/fmicb.2016.01354]

7. Cho S, Barrett JB, Frye JG, Jackson CR. Antimicrobial Resistance Gene Detection and Plasmid Typing Among Multidrug Resistant Enterococci Isolated from Freshwater Environment. Microorganisms. 2020;8(9):1338. [DOI:10.3390/microorganisms8091338] [PMID] [PMCID]

8. Ramos S, Silva V, Dapkevicius MLE, Igrejas G, Poeta P. Enterococci, from Harmless Bacteria to a Pathogen. Microorganisms. 2020;8(8):1118. [DOI:10.3390/microorganisms8081118] [PMID] [PMCID]

9. Hashimoto Y, Taniguchi M, Uesaka K, Nomura T, Hirakawa H, Tanimoto K, et al. Novel Multidrug-Resistant Enterococcal Mobile Linear Plasmid pELF1 Encoding vanA and vanM Gene Clusters From a Japanese Vancomycin-Resistant Enterococci Isolate. Front Microbiol. 2019;10:2568 [DOI:10.3389/fmicb.2019.02568] [PMID] [PMCID]

10. Sivertsen A, Pedersen T, Larssen Kjersti W, Bergh K, Rønning Torunn G, Radtke A, et al. A Silenced vanA Gene Cluster on a Transferable Plasmid Caused an Outbreak of Vancomycin-Variable Enterococci. Antimicrob Agents Chemother. 2016;60(7):4119-27. [DOI:10.1128/AAC.00286-16] [PMID] [PMCID]

11. Zarrilli R, Tripodi MF, Di Popolo A, Fortunato R, Bagattini M, Crispino M, et al. Molecular epidemiology of high-level aminoglycoside-resistant enterococci isolated from patients in a university hospital in southern Italy. J Antimicrob Chemother. 2005;56(5):827-35. [DOI:10.1093/jac/dki347] [PMID]

12. Monteiro da Silva BN, Faria AR, Souza SdSR, Colodette SS, Morais JM, Teixeira LM, et al. Expression of VanA-type vancomycin resistance in a clinical isolate of Enterococcus faecium showing insertion of IS19 in the vanS gene. Int J Antimicrob Agents. 2020;55(4):105897. [DOI:10.1016/j.ijantimicag.2020.105897] [PMID]

13. Brtkova A, Filipova M, Drahovska H, Bujdakova H. Characterization of enterococci of animal and environmental origin using phenotypic methods and comparison with PCR based methods. Veterinární medicína. 2010;55(3):97-105. [DOI:10.17221/159/2009-VETMED]

14. James S. Lewis II, PharmD, FIDSA. Performance Standards for Antimicrobial Susceptibility Testing. 35th ed. Order Code PDF: CLSI M100Ed35E. 2025. Malvern, PA, USA: Clinical and Laboratory Standards Institute (CLSI) Press. Pages:428.

15. Shen H, Liu Y, Qu J, Cao B. Comparison of vanA gene mRNA levels between vancomycin-resistant Enterococci presenting the VanA or VanB phenotype with identical Tn1546-like elements. J Microbiol Immunol Infect. 2016;49(6):866-71. [DOI:10.1016/j.jmii.2014.09.003] [PMID]

16. Georges M, Odoyo E, Matano D, Tiria F, Kyany'a C, Mbwika D, et al. Determination of Enterococcus faecalis and Enterococcus faecium Antimicrobial Resistance and Virulence Factors and Their Association with Clinical and Demographic Factors in Kenya. J Pathog. 2022;2022:3129439. [DOI:10.1155/2022/3129439] [PMID] [PMCID]

17. Boccella M, Santella B, Pagliano P, De Filippis A, Casolaro V, Galdiero M, et al. Prevalence and Antimicrobial Resistance of Enterococcus Species: A Retrospective Cohort Study in Italy. Antibiotics (Basel). 2021;10(12):1552. [DOI:10.3390/antibiotics10121552] [PMID] [PMCID]

18. Sattari-Maraji A, Jabalameli F, Node Farahani N, Beigverdi R, Emaneini M. Antimicrobial resistance pattern, virulence determinants and molecular analysis of Enterococcus faecium isolated from children infections in Iran. BMC Microbiol. 2019;19(1):156. [DOI:10.1186/s12866-019-1539-y] [PMID] [PMCID]

19. Sumangala B, Sharlee R, Sahana Shetty NS. Identification of Enterococcus.faecalis and E.faecium among Enterococci isolated from clinical samples in a teaching hospital Mandya Institute of Medical Sciences, Mandya. Indian J Microbiol Res. 2020;7(3):284-7. [DOI:10.18231/j.ijmr.2020.051]

20. Nasiri M, Hanifian S. Enterococcus faecalis and Enterococcus faecium in pasteurized milk: Prevalence, genotyping, and characterization of virulence traits. LWT. 2022;153:112452. [DOI:10.1016/j.lwt.2021.112452]

21. Ismail Hakki EKİN SA, Gülşah TOLLU, Ozgul GULAYDIN, Kemal GURTURK, Ziya ILHAN, Cihat OZTURK. The Presence and Prevalence of Enterococcus faecalis and Enterococcus faecium Strains in Urine and Stool Samples. Turk J Vet Res. 2018;2(1):14-8.

22. Yamin D, Uskoković V, Wakil AM, Goni MD, Shamsuddin SH, Mustafa FH, et al. Current and Future Technologies for the Detection of Antibiotic-Resistant Bacteria. Diagnostics. 2023;13(20):3246. [DOI:10.3390/diagnostics13203246] [PMID] [PMCID]

23. Angeletti S, Lorino G, Gherardi G, Battistoni F, De Cesaris M, Dicuonzo G. Routine molecular identification of enterococci by gene-specific PCR and 16S ribosomal DNA sequencing. J Clin Microbiol. 2001;39(2):794-7. [DOI:10.1128/JCM.39.2.794-797.2001] [PMID] [PMCID]

24. Gholizadeh Y, Prevost M, Van Bambeke F, Casadewall B, Tulkens PM, Courvalin P. Sequencing of the ddl gene and modeling of the mutated D-alanine:D-alanine ligase in glycopeptide-dependent strains of Enterococcus faecium. Protein Sci. 2001;10(4):836-44. [DOI:10.1110/ps.39101] [PMID] [PMCID]

25. Ozawa Y, Courvalin P, Gaiimand M. Identification of enterococci at the species level by sequencing of the genes for D-alanine:D-alanine ligases. Syst Appl Microbiol. 2000;23(2):230-7. [DOI:10.1016/S0723-2020(00)80009-0] [PMID]

26. Moghimbeigi A MM, Dousti M, Kiani F, Sayehmiri F, Sadeghifard N, Nazari A. Prevalence of vancomycin resistance among isolates of enterococci in Iran: a systematic review and meta-analysis. Adolesc Health Med Ther. 2018;15(9):177-88. [DOI:10.2147/AHMT.S180489] [PMID] [PMCID]

27. Adeyemi FM, Yusuf N-A, Adeboye RR, Oluwajide OO, Ako-Nai AK. Comparative Analysis of Prevalence and Antibiotic Resistance in Vancomycin-Resistant Enterococcus from Clinical Samples - Demographics and Phenotypes. Avicenna J Clin Microbiol Infect. 2021;8(2):57-65. [DOI:10.34172/ajcmi.2021.11]

28. Freitas AR, Tedim AP, Francia MV, Jensen LB, Novais C, Peixe L, et al. Multilevel population genetic analysis of vanA and vanB Enterococcus faecium causing nosocomial outbreaks in 27 countries (1986-2012). J Antimicrob Chemother. 2016;71(12):3351-66. [DOI:10.1093/jac/dkw312] [PMID]

29. Bhardwaj P, Ziegler E, Palmer Kelli L. Chlorhexidine Induces VanA-Type Vancomycin Resistance Genes in Enterococci. Antimicrob Agents Chemother. 2016;60(4):2209-21. [DOI:10.1128/AAC.02595-15] [PMID] [PMCID]

30. Resende M, Caierão J, Prates JG, Narvaez GA, Dias CA, d'Azevedo PA. Emergence of vanA vancomycin-resistant Enterococcus faecium in a hospital in Porto Alegre, South Brazil. J Infect Dev Ctries. 2014;8(2):160-7. [DOI:10.3855/jidc.4126] [PMID]

31. Bahman Mirzaei TNF, Puran Juhari, Masoome Aslani Mehr, Reyhane Babaei. Investigation of the Prevalence of vanA and vanB genes in vancomycin resistant enterococcus (VRE) by Taq Man real time PCR Assay. J Microbiol Infect Dis. 2013;3(4):192-8. [DOI:10.5799/ahinjs.02.2013.04.0107]

32. Moosavian M, Ghadri H, Samli Z. Molecular detection of vanA and vanB genes among vancomycin-resistant enterococci in ICU-hospitalized patients in Ahvaz in southwest of Iran. Infect Drug Resist. 2018;11:2269-75. [DOI:10.2147/IDR.S177886] [PMID] [PMCID]

33. Werner G, Klare I, Witte W. Arrangement of the vanA gene cluster in enterococci of different ecological origin. FEMS Microbiology Letters. 1997;155(1):55-61. [DOI:10.1111/j.1574-6968.1997.tb12685.x] [PMID]

34. Abdullah HM, Marbjerg LH, Andersen L, Hoegh SV, Kemp M. A Simple and Rapid Low-Cost Procedure for Detection of Vancomycin-Resistance Genes in Enterococci Reveals an Outbreak of Vancomycin-Variable Enterococcus faecium. Diagnostics. 2022;12(9):2120. [DOI:10.3390/diagnostics12092120] [PMID] [PMCID]

35. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402-8. [DOI:10.1006/meth.2001.1262] [PMID]

36. Ahn K, Hwang GY, Uh Y, Yoon KJ, Hyun S. Vancomycin Resistance due to vanA Gene Expression in an Aerococcus viridans Isolate: First Case in Korea. Ann Lab Med. 2017;37(3):288-9. [DOI:10.3343/alm.2017.37.3.288] [PMID] [PMCID]

37. Kariyama R, Mitsuhata R, Chow JW, Clewell DB, Kumon H. Simple and reliable multiplex PCR assay for surveillance isolates of vancomycin-resistant enterococci. J Clin Microbiol. 2000;38(8):3092-5. [DOI:10.1128/JCM.38.8.3092-3095.2000] [PMID] [PMCID]

38. Patel R, Uhl JR, Kohner P, Hopkins MK, Cockerill FR, 3rd. Multiplex PCR detection of vanA, vanB, vanC-1, and vanC-2/3 genes in enterococci. J Clin Microbiol. 1997;35(3):703-7. [DOI:10.1128/jcm.35.3.703-707.1997] [PMID] [PMCID]