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Evaluation of the antimicrobial activities of ultrasonicated spinach leaf extracts using RAPD markers and electron microscopy

Abstract

Spinach (Spinacia oleracea L.) leaves represent an important dietary source of nutrients, antioxidants and antimicrobials. As such, spinach leaves play an important role in health and have been used in the treatment of human diseases since ancient times. Here, the aims were to optimize the extraction methods for recovering antimicrobial substances of spinach leaves, determine the minimum inhibitory concentrations (MICs) of the antimicrobial substances against Escherichia coli and Staphylococcus aureus and, finally, evaluate the effects of spinach leaves’ antimicrobials on bacterial DNA using central composite face-centered methods. The effect of the extracts on both Gram-positive and Gram-negative bacterial models was examined by scanning electron microscopy (SEM) and random amplification of polymorphic (bacterial) DNA (RAPD). The optimal extraction conditions were at 45 °C, ultrasound power of 44% and an extraction time of 23 min. The spinach extracts exhibited antimicrobial activities against both bacteria with MICs in the 60–100 mg/ml range. Interestingly, SEM showed that the treated bacterial cells appear damaged with a reduction in cell number. RAPD analysis of genomic DNA showed that the number and sizes of amplicons were decreased by treatments. Based on these results, it was inferred that spinach leaf extracts exert bactericidal activities by both inducing mutations in DNA and causing cell wall disruptions.

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Acknowledgements

The authors would like to thank the Higher Committee for Education Development in Iraq (HCED) for the financial support to this work. Also, the authors are thankful to Dr. Alan Walters for providing spinach samples and Dr. Vjollca Konjufca for providing the bacterial strains and research facilities for the study.

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Authors

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AA designed, carried out and wrote the manuscript. DAL supervised, helped design the research and proofread the article. NL designed, analyzed and carried out the RAPD experiments and edited drafts of the manuscript. AGA helped edit drafts of the manuscript.

Corresponding author

Correspondence to Naoufal Lakhssassi.

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The authors declare that they have no competing interests.

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This study did not involve humans, human data or animals; no ethics approval or consent is required to publish the results.

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Communicated by Erko Stackebrandt.

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Altemimi, A., Lakhssassi, N., Abu-Ghazaleh, A. et al. Evaluation of the antimicrobial activities of ultrasonicated spinach leaf extracts using RAPD markers and electron microscopy. Arch Microbiol 199, 1417–1429 (2017). https://doi.org/10.1007/s00203-017-1418-6

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Keywords

  • Spinach
  • Antimicrobial activity
  • SEM
  • RAPD
  • Ultrasonic extraction
  • Escherichia coli
  • Bacterial pathogens
  • Staphylococcus aureus