Research Article
Volume 2 Issue 2 - 2017
Investigation of the Existence of Gram Negative Endophytic Bacteria Potentially Pathogenic For Man in Vegetables from Organic Cultures
María Beatriz Riverón Acosta* and Luiz Gilherme Coimbra Duarte
Center for Biological and Health Sciences. Microbiology Laboratory. Universidade Presbiteriana Mackenzie, São Paulo, Brazil
*Corresponding Author: María Beatriz Riverón Acosta, Center for Biological and Health Sciences. Microbiology Laboratory. Mackenzie Presbiterian University, São Paulo, Brazil.
Received: November 21, 2017; Published: November 27, 2017
Endophytic bacteria live in vegetable tissues colonizing them actively and exerting beneficial functions for the host. The objective of this study was to investigate the presence of Gram negative endophytic bacteria in vegetables from organic cultures (lettuce, chard, spinach, basil, cauliflower, coriander) purchased at food markets. After disinfecting leaves with a healthy aspect, fragments of approximately 1 cm2 were placed on LB agar. After the development of bacteria around the fragments, they were isolated in McConkey agar. Several lactose fermenting and non-fermenting colonies were chosen and identified by biochemical assays. Of 46 identified colonies, 27 (58.6%) corresponded to enterobacteria, 14 (30.4%) to the Acinetobacter genus and 5 (10.9%) to the Pseudomonas genus. A relevant number of endophytic pathogen bacteria, including Salmonella, Yersinia and Shigela, among others, was verified. The risk of using animal manure as compost for organic cultures is well known. Generally, the hygiene of vegetables and fruits is given importance in relation with contaminant epiphytic microbiota, but the endophytic microbial population pathogenic for humans is rarely considered, fact which can explain outbreaks where it is not possible to detect the origin of the contamination.
Keywords: Endophytic Gram negative bacteria; Pathogenic bacteria; Organic cultures
The importance of the existence of microorganisms is indisputable, without them, higher forms of life could not have arisen, even if maintained. They have fundamental roles in the planet we inhabit, because there are important interactions between microorganisms and animals and microorganisms and plants, and with man, both in the recycling of key nutrients and in the degradation of organic matter, which may be very beneficial or extremely harmful to their hosts [1].
A portion of microorganisms, mainly bacteria and fungi, inhabit the interior of plants. They are the endophytes, colonizing the healthy tissues of the aerial parts of the plant, at some stage of its life cycle, without causing any apparent damage. This concept is also extended to root microorganisms. Endophytes differ from epiphytes that live on the surface of plants, and phytopathogens, which cause disease. Among the endophytic microorganisms, the fungi and bacteria that form the nodules in the roots of the plants to which they are associated, are well studied because of their importance in agriculture, particularly for their participation in the fixation of atmospheric nitrogen (diazotic mycorrhizal rhizosphere). On the contrary, the endophytic of the aerial parts of the plants have only recently aroused the interest of the scientific community, especially for its potentials in the production of biotechnological interest metabolites [2-5].
Although the existence of the endophytic microbiota is well understood, much research has yet to be done on the ecological, genetic and physiological aspects of this interaction. In addition, it is interesting to know the diversity of these organisms, their presence, frequency and functions. There are a number of reasons to deepen the studies of endophytes such as the lack of information to elucidate the biological basis of these interactions and the advantages to the plants already attributed to their presence [6].
Although endophytic-plant interactions are still not well understood, they are known to be symbiotic, neutral, or antagonistic. In symbiotic interactions, microorganisms produce or induce the production of primary and secondary metabolites that may confer several plant advantages such as herbivore and insect attack decrease, abiotic stress tolerance increase, and control of other microorganisms. Examples of metabolites that can be induced by endophytes are phytoalexins, low molecular weight substances with antimicrobial activities produced by plants against the action of microorganisms or stressing agents, mycotoxins and other secondary metabolites produced by fungi that can cause diseases in humans and other animals, and/or, present antimicrobial, antioxidant and antihypertensive properties. New antibiotics, antimycotics such as cryptocardine, immunosuppressants and antineoplastics such as taxol, powerful antitumor, are some examples of secondary metabolites produced by endophytic microorganisms of extreme relevance in the pharmaceutical industry [7-17].
In addition to the importance described for the pharmaceutical industry, endophytic microorganisms are valuable tools as vectors for introducing genes of interest in plants for the production of pest and pathogen inhibitors [18-21].
Organic farming or organic farming is the term often used to designate the production of vegetables and other plant products without the use of synthetic chemicals such as inorganic fertilizers (nitrates, phosphates, etc.) and pesticides, or genetically modified organisms. This production system, which excludes the use of fertilizers, agrochemicals and growth regulators, is based on the use of animal manures, composting and biological control of pests and diseases. But it is precisely in fertilization with manure that if not properly treated before employment (and it is known that even pig feces and human faeces are used in soil fertilization) which results in a high source of microbial contamination.
The objective of this work was to investigate the presence of potentially pathogenic Gram negative bacteria in the feet of vegetables grown from organic crops (lettuce and curd, chard, basil, spinach, coriander and cabbage) purchased from supermarkets in the Higienópolis region of the city of São Paulo.
Material and Methods
Sterilization of the collected material: The collected material was processed within 24 hours, after collection, washing it abundantly with running water and neutral detergent. Then, in an aseptic chamber, the material was immersed in 70% (v/v) ethyl alcohol for 1 minute, then 3% (v/v) sodium hypochlorite for 4 minutes and again in 70% ethyl alcohol per 30 seconds to remove excess hypochlorite, and finally rinsed with sterile distilled water 3 times to remove any residue.
Endophytic isolation: After several colonies isolated from bacteria developed around the fragments of leaves and stems were randomly selected and transferred (culture by depletion) to plates containing McConkey selective medium. After incubation for 24-48 hours, isolated fermentative colonies of lactose (red) and non-fermenting colonies of lactose (whitish colonies) were transferred for biochemical tests (reactions provided by the Enterokit ™ Probac® Brazil) and further testing complementary, as proof of the presence of the enzyme cytochrome oxidase for those bacteria suspected to be Pseudomonas and/or Acinetobacter.
Different vegetables derived from organic crops purchased from a supermarket in the Higienópolis region, São Paulo city: Lettuce (Lactuca sativa, family Astaraceae) were qualitatively analyzed; basil (Ocimum gratissimum L., family Lamiaceae); Chard (Beta vulgaris var. cicla (L) K, Koch, family Amaranthaceae); spinach (Spinacia oleraceae, Amaranthaceae family); cabbage (Brassica oleraceae, Brassicaceae family) and coriander (Coriandrum sativum L., family Apiaceae).
From the lettuce, two feet of crisp lettuce, a flat lettuce foot and a crisp purple lettuce foot were analyzed. Of the other vegetables, only one foot. Of all, three leaves were taken at random to make the analyses. A total of 46 Gram negative bacterial colonies were identified, of which 27 (58.7%) were enterobacteria, 14 (30.4%) to the genus Acinetobacter and 5 (10.9%) to the genus Pseudomonas (Table 1).
Lineages found Classification
A1 Acinetobacter sp.
A2 Acinetobacter sp.
A3 Acinetobacter sp.
A4 Acinetobacter sp.
A5 Pseudomonas aeruginosa
A6 Escherichia coli
A7 Acinetobacter sp.
A8 Acinetobacter sp.
A9 Acinetobacter sp.
A10 Acinetobacter sp.
A11 Acinetobacter sp.
A12 Klebsiella pneumoniae
A13 Serratia marcescens
A14 Hafnia alvei
A15 Yersinia enterocolitica
A16 Shigella spp. or Yersinia pestis
A17 Pseudomonas sp.
A18 Klebsiella pneumoniae
M1 Proteus vulgaris
M2 Escherichia coli
M3 Acinetobacter sp.
M4 Acinetobacter sp.
M5 Pseudomonas aeruginosa
M6 Acinetobacter sp.
C1 Salmonella sp.
C2 Proteus mirabilis
C3 Proteus vulgaris
C4 Salmonella sp.
C5 Pseudomonas putrefasciens
C6 Klebsiella oxytoca
ARC1 Acinetobacter sp.
ARC2 Proteus vulgaris
ARC3 Edwarsiella parda
ARC4 Salmonella typhi
AC1 Klebsiella oxytoca
AC2 Proteus mirabilis
AC3 Klebsiella pneumoniae
AC4 Serratia marcescens
E1 Serratia marcescens
E2 Escherichia coli
E3 Acinetobacter sp.
Co1 Escherichia coli
Co2 Pseudomonas putrefasciens
Co3 Proteus vulgaris
Co4 Salmonella spp.
Co5 Salmonella typhi
Table 1: Classification of Gram-negative endophytic bacteria found in vegetables from organic crops based on biochemical tests.
Note: The genera Pseudomonas do not belong to Enterobaceriaceae and Acinetobacter. Subtitles: M1 to M6 lineages isolated from basil. Sections C1 to C6 isolated from cabbage. ARC1 to ARC4 strains isolated from purple curly lettuce. Lines AC1 to AC4 isolated from Swiss chard. E1 to E3 lineages isolated from spinach. Co1 to Co5 strains isolated from coriander.
Endophytic microorganisms (bacteria and fungi) are those that live in at least one period of their life cycle. Healthy plant tissues can remain dormant or actively colonize tissues in a localized or systemic way, exerting several beneficial activities for the host plant as well as nitrogen fixation, production of biologically active metabolites such as antibacterial and antifungals, growth factors, etc. However, many endophytic bacterial species are pathogenic or potentially pathogenic to humans [2].
Organic agriculture or organic farming excludes the use of synthetic chemicals such as inorganic fertilizers (nitrates, phosphates, etc.) pesticides, and genetically modified organisms, but is based on the use of animal manures, composting and biological control of pests and diseases. But, it is precisely in fertilizing with manures that if not properly treated before employment that results in a high source of microbial contamination.
The use of excreta is motivated by the recognition of its valuable nutrient content for plants. It is known that pig manures and human faeces are used as vegetable fertilizers. Human excreta may also contain pathogenic microorganisms, which directly threaten human health. Diarrhea and parasitic diseases are important factors contributing to the Global Burden of Disease, where the major causes are the environmental transmission through contaminated water and food crops, or through direct contact with fecal contaminated sources. Excreta usually does not contain industrialized chemical contaminants, but must be treated to reduce levels of pathogens at safety levels. Human metabolites such as hormones may also exist, but reuse on farmland would decrease the negative impact as well as on water sources [24].
A system for the control and management of microbial exposure with respect to wastewater and excreta use was drafted and published by the World Health Organization (WHO) in the 1980s [25] and revised thereafter [26]. This report focused on the management and management of feces and urine, taking into account current information on risk management and limiting a strategy of separation at source.
The presence of disease-causing organisms in human excreta is the result of infection of the individuals. This type of infection does not necessarily manifest itself with clinical symptoms (asymptomatic carriers) but may lead to excretion of the pathogens in question. For organisms that infect the gastrointestinal tract, this contamination occurs primarily through feces. In this work, several bacterial genera of clinical relevance such as Klebsiella, Serratia, Hafnia, Yersinia, Shigella, Salmonella, Edwarsiella, Proteus, Escherichia, and the non-fermenters Acinetobacter and Pseudomonas were identified.
All the microorganisms identified in the present study occupy the same ecological niche, such as water, soil, plants, intestinal tract of warm-blooded animals, etc. and can trigger diseases such as urinary tract infections and gastroenteritis, and if not treated quickly and effectively can lead to severe conditions and even death.
Even after undergoing a thorough disinfection process, the organic vegetables analyzed presented pathogenic potential for consumers, since if the individual has the impaired immune system, when eating this type of vegetable, it can develop some of the diseases mentioned above.
People generally buy organic products because they are labelled healthier because they do not have pesticides and pesticides. But the use of natural manure, composed of animal waste and animal waste is susceptible to bacterial contamination. All plants have endophytic microorganisms that may be symbiotic or commensal; however, few studies have been conducted regarding the interaction of these endophytic organisms with the direct consumers of host plants. Therefore, it is necessary to carry out studies to establish the relationship between consumption of this type of food (organic vegetables) and outbreaks, once which demonstrated in this work the existence of bacteria potentially pathogenic to humans.
  1. Madigan MT., et al. Brock Biology of Microorganisms. 8th edition. New Jersey, Prentice Hall (1997).
  2. Melo IS de and Azevedo JL. “Ecologia microbiana”. Jaguariúna Embrapa-CNPMA (1998): 488.
  3. Souza A., et al. “Atividade antimicrobiana de fungos endofíticos isolados de plantas tóxicas da Amazônia: Palicourea longiflora (aubl) rich e Strychnos cogens bentham”. Acta Amazônica 34.2 (2004):185-195.
  4. Cao l., et al. “Isolation and characterization on endophytic Streptomyces strains from surface sterilized tomato (Lycopersicon esculentum) roots”. Letters in Applied Microbiology 39.5 (2004): 425-430. 
  5. Ryan RP., et al. “Bacterial endophytes: recent developments and applications”. FEMS Microbiology Letters 278.1 (2008): 1-9.
  6. Bhagat J., et al. “Molecular and functional characterization of endophytic fungi from traditional medicinal plants”. World Journal of Microbiology and Biotechnology 28.3 (2012): 963-971. 
  7. Cordeiro NF and DietrIich SMC. “Phytoalexin induction by leafsurface fungi of tropical Rubiaceae”. Ciência e Cultura 44.5 (1992): 342-344.
  8. Araújo WL. “Isolamento, identificação e caracterização genética de bactérias endofíticas de porta-enxertos de citros”. Dissertação de Mestrado, ESALQ, Piracicaba: São Paulo (1996):
  9. D´Mello JPF and Macdonald AM. C. “Mucotoxins”. Animal Feed Science and Technology 69. 1-3 (1997): 155-166.
  10. Wang J., et al. “Taxol from Tubercularia sp. strain TF5, an endophytic fungus of Taxus mairei”. FEMS Microbiology Letters 193.2 (2000): 249-253. 
  11. Strobel G., et al. “Natural products from endophytic microorganisms”. Journal of Natural Products 67.2 (2004): 257- 268. 
  12. Rothballer M., et al. “Endophytic root colonization of gramineous plants by Herbaspirillum frisingense”. FEMS Microbiology Ecology 66.1 (2008): 85-95.
  13. Zhang HW., et al. “Cephalosol: an antimicrobial metabolite whitle an unprecedent skelton from endophytic Cephalosporium acremonium IFB-E007”. Chemistry 14 (2008):10670-10674. 
  14. Tejesvi MV., et al. “Antioxidant, antiheipertensive, and antibacterial properties of endophytic Pestalotiopsis spp. from medical plants”. Canadian Journal of Microbiology 54 (2008): 769-780.
  15. Artani N., et al. “Screening of endophytic fungi having ability for antioxidative and alphaglucosidase inibitor isolated form Taxus sumatrana”. Pakistan Journal of Biological Sciences 14.22 (2011):1019-1023.
  16. Zhang Y., et al. “Alkaloids produced by endophytic fungi: a review”. Natural Product Communications 7.7 (2012): 963-968. 
  17. Ding L., et al. “Kandenols A-E, eudemenes from an endophytic Streptomyces sp. of the mangrove tree Kandelia candel”. Journal of Natural Products 75.12 (2012): 2223-2227.
  18. Hallmann J and Sikora RA. “Toxicity of fungal endophytic secondary metabolites to plant parasitic nematodes and soil-born plant pathogeny fungi”. European Journal of Plant Pathology 102.2 (1996): 155-162. 
  19. Wagner BL and Lewis LC. “Colonization of corn Zea mays, by the entomopathogenic fungus Beauveria bassiana”. Applied and Environmental Microbiology 66.8 (2000): 3468-3473.
  20. Ownley BH., et al. “Bauveria bassiana: endophytic colonization and plant disease control”. Journal of Invertebrate Pathology 98.3 (2008): 267-270.
  21. Ikeda AC., et al. “Morphological and genetic characterization of endophytic bacteria isolated from roots of different maize genotypes”. Microbial Ecology 65.1 (2013): 54-60.
  22. Koneman E. Diagnóstico microbiológico. 6ª edition. Rio de Janeiro: Guanabara Koogan (2006).
  23. Bergey´s Manual of Systematic Bacteriology. Volume 2. 2nd edition, USA: Williams & Wilkins (2005).
  24. Schönning C., et al. “Diretrizes para o uso de urina e fezes nos sistemas de saneamento ecológico”. Estocolmo Suecia: Instituto Ambiental de Estocolmo (2004).
  25. WHO. Guidelines for the safe use of wastewater and excreta in agriculture and aquaculture. Geneva, Switzerland: World Health Organization (1989).
  26. WHO. Diet, nutrition and the prevention of chronic diseases. Technical report series 916. (2003).
Citation: María Beatriz Riverón Acosta and Luiz Gilherme Coimbra Duarte. “Investigation of the Existence of Gram Negative Endophytic Bacteria Potentially Pathogenic For Man in Vegetables from Organic Cultures”. Nutrition and Food Toxicology 2.2 (2017): 311-316.
Copyright: © 2017 María Beatriz Riverón Acosta and Luiz Gilherme Coimbra Duarte. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.