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Survey on phenotypic resistance in Enterococcus faecalis: comparison between the expression of biofilm-associated genes in Enterococcus faecalis persister and non-persister cells

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Abstract

Background

Phenotypic resistance is considered as a serious therapeutic challenge for which a definitive remedy has not been discovered yet. Biofilm and persister cell formation are two well-studied phenotypic resistance phenomena, leading to the recalcitrance and relapse of different types of chronic infections. The presence of persister cells in biofilm structures seems to be one of the main factors contributing to the relapse of infections and treatment failure. Given the dormant and inert nature of persister cells, they can be easy targets for the immune system factors. Biofilm formation can be a survival strategy for the defenseless persister cells. Thus, this study was aimed to evaluate the expression of biofilm-associated genes in Enterococcus faecalis persister and non-persister cells.

Methods

Vancomycin susceptibility and biofilm formation ability were investigated among 95 E. faecalis clinical isolates using microtiter broth dilution and microtiter plate assays, respectively. PCR was used to determine the presence of biofilm-related genes (gelE, esp, and agg) among the vancomycin-susceptible, biofilm producer E. faecalis isolates (91 isolates). Minimum bactericidal concentration for biofilms (MBCB) were determined for vancomycin using the MTP assay. Bacterial persister assay was performed using an enzymatic lysis assay. Finally, the expression of biofilm-related genes was compared between the persister and non-persister isolates of E. faecalis using real-time qPCR.

Results

E. faecalis isolates showed a high level of susceptibility (95.8%) to vancomycin (MIC < 1 µg/mL). The gelE, esp, and agg genes were found in 91 (100%), 72 (79.12), and 74 (81.32) of the isolates, respectively. All the E. faecalis isolates were tolerant to vancomycin in the biofilm condition, showing a MBCB of > 2500 µg/mL. Based on the enzymatic lysis assay, only 3 isolates, out of the 91, had the ability to form persister cells. The expression of biofilm-associated genes was higher among the persister compared to non-persister E. faecalis isolates.

Conclusions

Biofilm-associated persister cells indicated a high vancomycin tolerance compared to non-persister cells. Moreover, persister isolates showed a higher tendency for biofilm formation and a higher expression level of the biofilm-associated genes, compared to non-persister isolates.

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Funding

This research was supported by Ilam University of Medical Sciences, Ilam, Iran. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Authors and Affiliations

  1. Department of Medical Microbiology, Faculty of Medicine, Ilam University of Medical Science, Ilam, Iran

    Vahab Hassan Kaviar, Parisa Asadollahi, Behrooz Sadeghi Kalani & Iraj Pakzad

  2. Clinical Microbiology Research Center, Ilam University of Medical Sciences, Ilam, Iran

    Vahab Hassan Kaviar, Saeed Khoshnood, Parisa Asadollahi, Behrooz Sadeghi Kalani, Abbas Maleki & Iraj Pakzad

  3. Department of Microbiology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

    Sanaz Yarahmadi

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  1. Vahab Hassan Kaviar
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Contributions

IP and VHK contributed to the conception, design of the work, acquisition, analysis and interpretation of data. AM and SY contributed in administrative, technical, and material support PA, BSK, SK and VHK contributed in drafting of the manuscript, revising and final approval of the version to be published.

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Correspondence to Iraj Pakzad.

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This investigation was approved by the Medical Research and Ethics Committee of Ilam University of Medical Science (IR.MEDILAM.REC.1397.046) (Ethics Approval Number: 971013/38).

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Kaviar, V.H., Khoshnood, S., Asadollahi, P. et al. Survey on phenotypic resistance in Enterococcus faecalis: comparison between the expression of biofilm-associated genes in Enterococcus faecalis persister and non-persister cells. Mol Biol Rep 49, 971–979 (2022). https://doi.org/10.1007/s11033-021-06915-8

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