Isolation and Molecular Identification of Vahlkampfiidae and Vermamoeba Vermiformis from Fresh Vegetables: A Neglected Source of Infections
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Abstract
Background: Naegleria spp., Tetramitus spp., and Vermamoeba vermiformis are potential pathogenic free-living amoebae (FLA) causing diseases such as keratitis, meningoencephalitis, and lung infections. We aimed to investigate the presence of Vahlkampfiidae and V. vermiformis in raw vegetables commonly consumed in Iran.
Methods: Totally, 70 samples of vegetables samples including watercress (22), leeks (12), parsley (10), basil (13) and mint (13) were collected from municipal markets of Tehran, the capital of Iran during June to October 2021. After washing vegetables, samples were cultivated onto 2% non-nutrient agar (NNA) medium. After morphological confirmations, DNA was extracted and identical fragments of the FLA were amplified and sequenced.
Results: Out of 70 cultured samples, 11 samples (15.71 %) were morphologically positive, of which four and seven were V. vermiformis and Vahlkampfiidae isolates, respectively. According to the PCR/sequencing results two, one, one, and one strains belonged to N. australiensis, N. americana, Vahlkampfia sp., V. inornata, and T. aberdonicus, respectively. All Vermamoeba genus were characterized as V. vermiformis.
Conclusion: The results of current study revealed the contamination of fresh raw vegetables with Vahlkampfiidae and V. vermiformis. In addition, to our knowledge this is the first report of T. aberdonicus in raw vegetables. Our findings highlight the public health importance of vegetables in transmission of FLA, as well as the potential role of FLA in transmission of potential pathogenic microorganisms via consuming of fresh raw vegetables.
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2. Scheid P: Free-living amoebae as human parasites and hosts for pathogenic microorganisms. Proceedings. 2018; 2(11):692.
3. Andalib S, Mohammad Rahimi H, Niyyati M, et al. Free-living amoebae in an oil refinery wastewater treatment facility. Sci Total Environ. 2022;839:156301.
4. Saberi R, Seifi Z, Dodangeh S, et al. A systematic literature review and meta-analysis on the global prevalence of Naegleria spp. in water sources. Transbound Emerg Dis. 2020; 67(6):2389-2402.
5. Pazoki H, Niyyati M, Javanmard E, Lasjerdi Z, Spotin A, Mirjalali H, Behravan MR. isolation and phylogenetic analysis of free-living amoebae (Acanthamoeba, Naegleria, and Vermamoeba) in the farmland soils and recreational places in Iran. Acta Parasitol. 2020; 65(1):36-43.
6. Javanmard E, Niyyati M, Lorenzo-Morales J, et al. Molecular identification of waterborne free living amoebae (Acanthamoeba, Naegleria and Vermamoeba) isolated from municipal drinking water and environmental sources, Semnan province, north half of Iran. Exp Parasitol. 2017; 183:240-244.
7. Movahedi Z, Shokrollahi MR, Aghaali M, Heydari H. Primary amoebic meningoencephalitis in an Iranian infant. Case Rep Med. 2012;2012:782854.
8. Niyyati M, Lorenzo-Morales J, Motevalli Haghi A, Valladares B. Isolation of Vahlkampfiids (Willaertia magna) and Thecamoeba from soil samples in Iran. Iran J Parasitol. 2009;4(1): 48-52
9. Sousa-Ramos D, Reyes-Batlle M, Bellini NK, Rodríguez-Expósito RL, Piñero JE, Lorenzo-Morales J. Free-living amoebae in soil samples from Santiago island, Cape Verde. Microorganisms. 2021; 9(7):1460.
10. Berrilli F, Di Cave D, Novelletto A, Montalbano Di Filippo M. PCR-based identification of thermotolerant free-living amoebae in Italian hot springs. Eur J Protistol. 2021; 80:125812.
11. Bradbury RS. Free-living amoebae recovered from human stool samples in Strongyloides agar culture. J Clin Microbiol. 2014; 52(2):699-700.
12. Cabello-Vílchez AM, Mena R, Zuñiga J, Cermeño P, et al. Endosymbiotic Mycobacterium chelonae in a Vermamoeba vermiformis strain isolated from the nasal mucosa of an HIV patient in Lima, Peru. Exp Parasitol. 2014; 145 Suppl:S127-130.
13. Fatemi M, Niyyati M, Rouhani S, Karamati SA, Mirjalali H, Karanis P. Contamination of fresh vegetables in municipal stores with pathogenic Acanthamoeba genotypes; a public health concern. Int J Environ Health Res. 2023; 33(10):1010-1021.
14. Moran S, Mooney R, Henriquez FL. Diagnostic Considerations for Non-Acanthamoeba Amoebic Keratitis and Clinical Outcomes. Pathogens. 2022;11(2):219.
15. Niyyati M, Lorenzo-Morales J, Rezaie S, et al. First report of a mixed infection due to Acanthamoeba genotype T3 and Vahlkampfia in a cosmetic soft contact lens wearer in Iran. Exp Parasitol. 2010;126(1):89-90.
16. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X. molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018; 35(6):1547-1549.
17. Soler L, Moreno Y, Moreno-Mesonero L, Amorós I, Alonso JL, Ferrús MA. Microbiome of free-living amoebae (FLA) isolated from fresh organic produce: potential risk to consumers? Foods. 2023; 12(16): 3102.
18. Moreno-Mesonero L, Hortelano I, Moreno Y, Ferrús MA. Evidence of viable Helicobacter pylori and other bacteria of public health interest associated with free-living amoebae in lettuce samples by next generation sequencing and other molecular techniques. Int J Food Microbiol. 2020; 318:108477.
19. Vaerewijck MJ, Sabbe K, Van Hende J, Baré J, Houf K. Sampling strategy, occurrence and diversity of free-living protozoa in domestic refrigerators. J Appl Microbiol. 2010; 109(5):1566-1578.
20. Reyes-Batlle M, Díaz FJ, Sifaoui I, et al. Free living amoebae isolation in irrigation waters and soils of an insular arid agroecosystem. Sci Total Environ. 2021; 753:141833.
21. Reyes-Batlle M, Rizo-Liendo A, Viera-Santana RA, et al. Isolation and molecular identification of Naegleria australiensis in irrigation water of Fuerteventura island, Spain. Acta Parasitol. 2019; 64(2):331-335.
22. Pazoki H, Niyyati M, Javanmard E, Lasjerdi Z, Spotin A, Mirjalali H, Karanis P. Isolation of N. philippinensis and N. americana strains from irrigation waters of farmland soils in Iran. Environ Sci Pollut Res Int. 2020; 27(19):24568-24573.
23. Huang SW, Hsu BM. Survey of Naegleria and its resisting bacteria-Legionella in hot spring water of Taiwan using molecular method. Parasitol Res. 2010;106(6):1395-1402.
24. Denet E, Coupat-Goutaland B, Nazaret S, Pélandakis M, Favre-Bonté S. Diversity of free-living amoebae in soils and their associated human opportunistic bacteria. Parasitol Res. 2017; 116(11):3151-3162.
25. De Jonckheere JF, Brown S. The identification of vahlkampfiid amoebae by ITS sequencing. Protist. 2005; 156(1):89-96.
26. Robinson BS, De Jonckheere JF, Dobson PJ. Two new Tetramitus species (Heterolobosea, Vahlkampfiidae) from cold aquatic environments. Eur J Protistol. 2007; 43(1):1-7.
27. Murtagh GJ, Dyer PS, Rogerson A, Nash GV, Laybourn-Parry J. A new species of Tetramitus in the benthos of a saline antarctic lake. Eur J Protistol. 2002; 37(4):437-443.
28. Baumgartner M, Eberhardt S, De Jonckheere JF, Stetter KO. Tetramitus thermacidophilus n. sp., an amoeboflagellate from acidic hot springs. J Eukaryot Microbiol. 2009; 56(2):201-206.
29. Schönborn W. FC Page, A new key to freshwater and soil gymnamoebae, with instructions for culture, freshwater biological association, ambleside (1988), 122 S., 55 Abb., zT als Tafeln. Brosch. In.: Urban & Fischer; 1989.
30. Sadaka HA, el-Nassery SF, abou Samra LM, Awadalla HN. Isolation and identification of free-living amoebae from some water sources in Alexandria. J Egypt Soc Parasitol. 1994; 24(2):247-257.
31. Rivera F, Ramírez P, Vilaclara G, Robles E, Medina F. A survey of pathogenic and free-living amoebae inhabiting swimming pool water in Mexico City. Environ Res. 1983; 32(1):205-211.
32. Siddiqui R, Makhlouf Z, Khan NA. The increasing importance of Vermamoeba vermiformis. J Eukaryot Microbiol. 2021; 68(5):e12857.
33. Delafont V, Perraud E, Brunet K, Maisonneuve E, Kaaki S, Rodier MH. Vermamoeba vermiformis in hospital network: a benefit for Aeromonas hydrophila. Parasitol Res. 2019; 118(11):3191-3194.
34. Pagnier I, Valles C, Raoult D, La Scola B: Isolation of Vermamoeba vermiformis and associated bacteria in hospital water. Microb Pathog. 2015; 80:14-20.
35. Reyes-Batlle M, Wagner C, Zamora-Herrera J, et al. Isolation and Molecular Identification of Vermamoeba vermiformis strains from soil sources in El Hierro island, Canary islands, Spain. Curr Microbiol. 2016; 73(1):104-107.
36. Nisar MA, Ross KE, Brown MH, Bentham R, Hinds J, Whiley H. Molecular screening and characterization of Legionella pneumophila associated free-living amoebae in domestic and hospital water systems. Water Res. 2022; 226:119238.
37. Moreno-Mesonero L, Soler L, Amorós I, Moreno Y, Ferrús MA, Alonso JL. Protozoan parasites and free-living amoebae contamination in organic leafy green vegetables and strawberries from Spain. Food Waterborne Parasitol. 2023; 32:e00200.
2. Scheid P: Free-living amoebae as human parasites and hosts for pathogenic microorganisms. Proceedings. 2018; 2(11):692.
3. Andalib S, Mohammad Rahimi H, Niyyati M, et al. Free-living amoebae in an oil refinery wastewater treatment facility. Sci Total Environ. 2022;839:156301.
4. Saberi R, Seifi Z, Dodangeh S, et al. A systematic literature review and meta-analysis on the global prevalence of Naegleria spp. in water sources. Transbound Emerg Dis. 2020; 67(6):2389-2402.
5. Pazoki H, Niyyati M, Javanmard E, Lasjerdi Z, Spotin A, Mirjalali H, Behravan MR. isolation and phylogenetic analysis of free-living amoebae (Acanthamoeba, Naegleria, and Vermamoeba) in the farmland soils and recreational places in Iran. Acta Parasitol. 2020; 65(1):36-43.
6. Javanmard E, Niyyati M, Lorenzo-Morales J, et al. Molecular identification of waterborne free living amoebae (Acanthamoeba, Naegleria and Vermamoeba) isolated from municipal drinking water and environmental sources, Semnan province, north half of Iran. Exp Parasitol. 2017; 183:240-244.
7. Movahedi Z, Shokrollahi MR, Aghaali M, Heydari H. Primary amoebic meningoencephalitis in an Iranian infant. Case Rep Med. 2012;2012:782854.
8. Niyyati M, Lorenzo-Morales J, Motevalli Haghi A, Valladares B. Isolation of Vahlkampfiids (Willaertia magna) and Thecamoeba from soil samples in Iran. Iran J Parasitol. 2009;4(1): 48-52
9. Sousa-Ramos D, Reyes-Batlle M, Bellini NK, Rodríguez-Expósito RL, Piñero JE, Lorenzo-Morales J. Free-living amoebae in soil samples from Santiago island, Cape Verde. Microorganisms. 2021; 9(7):1460.
10. Berrilli F, Di Cave D, Novelletto A, Montalbano Di Filippo M. PCR-based identification of thermotolerant free-living amoebae in Italian hot springs. Eur J Protistol. 2021; 80:125812.
11. Bradbury RS. Free-living amoebae recovered from human stool samples in Strongyloides agar culture. J Clin Microbiol. 2014; 52(2):699-700.
12. Cabello-Vílchez AM, Mena R, Zuñiga J, Cermeño P, et al. Endosymbiotic Mycobacterium chelonae in a Vermamoeba vermiformis strain isolated from the nasal mucosa of an HIV patient in Lima, Peru. Exp Parasitol. 2014; 145 Suppl:S127-130.
13. Fatemi M, Niyyati M, Rouhani S, Karamati SA, Mirjalali H, Karanis P. Contamination of fresh vegetables in municipal stores with pathogenic Acanthamoeba genotypes; a public health concern. Int J Environ Health Res. 2023; 33(10):1010-1021.
14. Moran S, Mooney R, Henriquez FL. Diagnostic Considerations for Non-Acanthamoeba Amoebic Keratitis and Clinical Outcomes. Pathogens. 2022;11(2):219.
15. Niyyati M, Lorenzo-Morales J, Rezaie S, et al. First report of a mixed infection due to Acanthamoeba genotype T3 and Vahlkampfia in a cosmetic soft contact lens wearer in Iran. Exp Parasitol. 2010;126(1):89-90.
16. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X. molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018; 35(6):1547-1549.
17. Soler L, Moreno Y, Moreno-Mesonero L, Amorós I, Alonso JL, Ferrús MA. Microbiome of free-living amoebae (FLA) isolated from fresh organic produce: potential risk to consumers? Foods. 2023; 12(16): 3102.
18. Moreno-Mesonero L, Hortelano I, Moreno Y, Ferrús MA. Evidence of viable Helicobacter pylori and other bacteria of public health interest associated with free-living amoebae in lettuce samples by next generation sequencing and other molecular techniques. Int J Food Microbiol. 2020; 318:108477.
19. Vaerewijck MJ, Sabbe K, Van Hende J, Baré J, Houf K. Sampling strategy, occurrence and diversity of free-living protozoa in domestic refrigerators. J Appl Microbiol. 2010; 109(5):1566-1578.
20. Reyes-Batlle M, Díaz FJ, Sifaoui I, et al. Free living amoebae isolation in irrigation waters and soils of an insular arid agroecosystem. Sci Total Environ. 2021; 753:141833.
21. Reyes-Batlle M, Rizo-Liendo A, Viera-Santana RA, et al. Isolation and molecular identification of Naegleria australiensis in irrigation water of Fuerteventura island, Spain. Acta Parasitol. 2019; 64(2):331-335.
22. Pazoki H, Niyyati M, Javanmard E, Lasjerdi Z, Spotin A, Mirjalali H, Karanis P. Isolation of N. philippinensis and N. americana strains from irrigation waters of farmland soils in Iran. Environ Sci Pollut Res Int. 2020; 27(19):24568-24573.
23. Huang SW, Hsu BM. Survey of Naegleria and its resisting bacteria-Legionella in hot spring water of Taiwan using molecular method. Parasitol Res. 2010;106(6):1395-1402.
24. Denet E, Coupat-Goutaland B, Nazaret S, Pélandakis M, Favre-Bonté S. Diversity of free-living amoebae in soils and their associated human opportunistic bacteria. Parasitol Res. 2017; 116(11):3151-3162.
25. De Jonckheere JF, Brown S. The identification of vahlkampfiid amoebae by ITS sequencing. Protist. 2005; 156(1):89-96.
26. Robinson BS, De Jonckheere JF, Dobson PJ. Two new Tetramitus species (Heterolobosea, Vahlkampfiidae) from cold aquatic environments. Eur J Protistol. 2007; 43(1):1-7.
27. Murtagh GJ, Dyer PS, Rogerson A, Nash GV, Laybourn-Parry J. A new species of Tetramitus in the benthos of a saline antarctic lake. Eur J Protistol. 2002; 37(4):437-443.
28. Baumgartner M, Eberhardt S, De Jonckheere JF, Stetter KO. Tetramitus thermacidophilus n. sp., an amoeboflagellate from acidic hot springs. J Eukaryot Microbiol. 2009; 56(2):201-206.
29. Schönborn W. FC Page, A new key to freshwater and soil gymnamoebae, with instructions for culture, freshwater biological association, ambleside (1988), 122 S., 55 Abb., zT als Tafeln. Brosch. In.: Urban & Fischer; 1989.
30. Sadaka HA, el-Nassery SF, abou Samra LM, Awadalla HN. Isolation and identification of free-living amoebae from some water sources in Alexandria. J Egypt Soc Parasitol. 1994; 24(2):247-257.
31. Rivera F, Ramírez P, Vilaclara G, Robles E, Medina F. A survey of pathogenic and free-living amoebae inhabiting swimming pool water in Mexico City. Environ Res. 1983; 32(1):205-211.
32. Siddiqui R, Makhlouf Z, Khan NA. The increasing importance of Vermamoeba vermiformis. J Eukaryot Microbiol. 2021; 68(5):e12857.
33. Delafont V, Perraud E, Brunet K, Maisonneuve E, Kaaki S, Rodier MH. Vermamoeba vermiformis in hospital network: a benefit for Aeromonas hydrophila. Parasitol Res. 2019; 118(11):3191-3194.
34. Pagnier I, Valles C, Raoult D, La Scola B: Isolation of Vermamoeba vermiformis and associated bacteria in hospital water. Microb Pathog. 2015; 80:14-20.
35. Reyes-Batlle M, Wagner C, Zamora-Herrera J, et al. Isolation and Molecular Identification of Vermamoeba vermiformis strains from soil sources in El Hierro island, Canary islands, Spain. Curr Microbiol. 2016; 73(1):104-107.
36. Nisar MA, Ross KE, Brown MH, Bentham R, Hinds J, Whiley H. Molecular screening and characterization of Legionella pneumophila associated free-living amoebae in domestic and hospital water systems. Water Res. 2022; 226:119238.
37. Moreno-Mesonero L, Soler L, Amorós I, Moreno Y, Ferrús MA, Alonso JL. Protozoan parasites and free-living amoebae contamination in organic leafy green vegetables and strawberries from Spain. Food Waterborne Parasitol. 2023; 32:e00200.
| Files | ||
| Issue | Vol 19 No 2 (2024) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijpa.v19i2.15854 | |
| Keywords | ||
| Vegetables Free living amoeba Vahlkampfiidae Vermamoeba vermiformis | ||
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How to Cite
1.
Fatemi M, Niyyati M, Karamati SA, Mirjalali H. Isolation and Molecular Identification of Vahlkampfiidae and Vermamoeba Vermiformis from Fresh Vegetables: A Neglected Source of Infections. Iran J Parasitol. 2024;19(2):183-191.
