Prevalence of plant beneficial and human pathogenic bacteria isolated from salad vegetables in India

Nithya and Babu BMC Microbiology (2017) 17:64 DOI 10.1186/s12866-017-0974-x RESEARCH ARTICLE Open Access Prevalence of plant beneficial and human pathogenic bacteria isolated from salad vegetables in India Angamuthu Nithya and Subramanian Babu* Abstract Background: The study aimed at enumerating, identifying and categorizing the endophytic cultivable bacterial community in selected salad vegetables (carrot, cucumber, tomato and onion). Vegetable samples were collected from markets of two vegetable hot spot growing areas, during two different crop harvest seasons. Crude and diluted vegetable extracts were plated and the population of endophytic bacteria was assessed based on morphologically distinguishable colonies. The bacterial isolates were identified by growth in selective media, biochemical tests and 16S rRNA gene sequencing. Results: The endophytic population was found to be comparably higher in cucumber and tomato in both of the sampling locations, whereas lower in carrot and onion. Bacterial isolates belonged to 5 classes covering 46 distinct species belonging to 19 genera. Human opportunistic pathogens were predominant in carrot and onion, whereas plant beneficial bacteria dominated in cucumber and tomato. Out of the 104 isolates, 16.25% are human pathogens and 26.5% are human opportunistic pathogens. Conclusions: Existence of a high population of plant beneficial bacteria was found to have suppressed the population of plant and human pathogens. There is a greater potential to study the native endophytic plant beneficial bacteria for developing them as biocontrol agents against human pathogens that are harboured by plants. Keywords: Bacteriome, Endophyte, Human pathogenic, Plant beneficial, Salad, Vegetables Background Fresh vegetables are considered as the essential components of healthy diet of people and the consumption of vegetables in the form of salads has increased in many parts of the world, including India. In contrast to the potential health benefits of fresh vegetables, a concern about the safety and the quality of vegetables has also raised due to outbreaks of infectious diseases reported from by Center for Disease Control and Prevention (CDC), US Food and Drug Administration (FDA), World Health Organization (WHO) and Center for Science in the Public Interest (CSPI). These changes are mainly due to change in the ecology of human pathogens to persist in non-host environments. * Correspondence: School of Bio Sciences and Technology, VIT University, Vellore 632014, India Since the contamination of salad vegetables with human pathogenic bacteria has reached concerning proportions in recent years, which has been evidenced by reports of various public health agencies through enhanced epidemiological and surveillance techniques, the raw vegetables are undoubtedly the portable source of infectious microorganisms, which has been revealed by numerous outbreaks associated with the consumption of salad vegetables [1, 2]. In general, fresh vegetables are known to harbour large bacterial populations [3], which may be of plant endophytes, plant pathogenic and human pathogenic in nature. The most important features of plant host colonization is by the adaptation of pathogens to the host defence response, physiology, immunity, native microflora, physical barriers, mobility and temperature. The pathogenic or non-pathogenic bacteria have several points of opportunities to contaminate fresh vegetables © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Nithya and Babu BMC Microbiology (2017) 17:64 Page 2 of 16 from the field through the time of consumption [4]. However, the route cause for the contamination of these vegetables, survival rate of endophytic bacterial communities, survival rate of pathogens which may be for plant or/and human, their interaction strategies, survival mechanisms are still under exploration. Since the fresh vegetables in the form of salads are consumed raw, the pathogens present in it lead to widespread disease outbreaks. The non-pathogenic microbes associated with plants as a commensal or pathogen may leads to allergies which is still undeterminable due to change in the interaction strategies of microbes with the endophytic bacterial community and the plant host [5]. There are several reported outbreaks related to salad vegetables from the past decades to the present. Recently, Listeriosis outbreak was reported by CDC on January 28, 2016 from the consumption of mixed salad vegetables; E. coli 0157 outbreak was reported by USA Today newsletter related to salad vegetables in costco chicken; multistate Salmonella outbreak was linked to cucumbers in 2015 and thus the outbreaks are expanding. In order to step on to the control of these outbreaks, detail reports of the endophytic bacterial community of vegetables used for salads, survival rate of pathogens in non-host environment etc. have to be identified. The present study was undertaken to find the endophytic bacterial community of the most commonly used South Indian salad vegetables like carrot, cucumber onion and tomato sold in vegetable markets. The study was undertaken by collecting the vegetable samples from two different vegetable growing hot spots of Tamil Nadu, India. Further, the endophytes were classified based on the evolutionary relationship to identify the predominant endophytic taxonomical group in salad vegetables and further classified based on their specific known functions such as human pathogens, human commensals, plant pathogens, plant commensals and environmentally beneficial bacteria. solution of iodine (2%), and allowed to air dry inside a laminar airflow cabinet. The outer layer of onion was peeled and washed with deionized water and the external surface was scrubbed with an alcoholic solution of iodine (2%). Sterilization efficacy was evaluated by cutting the scrubbed external surface with sterile scalpel blade and placing directly on the surface of nutrient agar medium. The method chosen for sterilization, treatment with alcoholic solution of 2% iodine and drying under UV light on each side of vegetables, proved to be effective in killing the surface associated bacteria. For every sample batch, two samples of each vegetable were randomly chosen following surface sterilization and placing the external surface on nutrient agar plate. After 24 h of incubation at 37 °C, none of the tested vegetables showed bacte (...truncated)