Disinfection of waterborne coliform bacteria by Neem oil

Robert L. Matthews1, Michael R. Templeton2*, Sabitri K. Tripathi3, Kiran Bhattarai4

1 Water and Health Research Centre, Department of Civil Engineering, University of Bristol, Bristol, United Kingdom, BS8 1UB. Email: Tel: +44(0)1173315292.

2* Corresponding Author: Department of Civil and Environmental Engineering, Imperial College London, London, United Kingdom, SW7 2AZ. Email: . Tel: +44(0)2075946099, Fax: +44(0)2075946124.

3 Department of Science and Humanities, Nepal Engineering College, Bhaktapur, Kathmandu, Nepal, GPO Box 10210. Email: .

4 Department of Civil Engineering, Nepal Engineering College, Bhaktapur, Kathmandu, Nepal, GPO Box 10210. Email: .

Submitted for publication in Environmental Engineering Science

Submitted 10 February 2009

Revised and re-submitted 26 May 2009


Abstract

This study assessed the ability of Neem oil to disinfect an Escherichia coli isolate in pure laboratory-grade water and total and faecal coliform bacteria in two unfiltered surface waters. Neem oil doses as low as 2.13 g∙l-1 with five minutes of mixing time achieved high levels of inactivation of the E. coli in pure laboratory-grade water (>99% inactivation). However, the disinfection of total and faecal coliforms in the unfiltered surface waters was variable and limited to <70% inactivation typically. Increasing the Neem oil dose and mixing time generally resulted in an increase in the mean inactivation of total coliform bacteria but not always of faecal coliform bacteria. The inactivation of total coliforms was consistently greater than the inactivation of faecal coliforms, suggesting a potential range of sensitivities to Neem oil among coliform bacteria. The reduced effectiveness of the Neem oil in the unfiltered surface waters compared to the pure laboratory-grade water suggests a possible interference by natural water constituents (e.g. suspended particles, dissolved organic matter) which may inhibit the anti-bacterial potential of Neem oil, or that naturally occurring bacteria are more resistant to the anti-microbial effects of Neem oil. Overall, Neem oil was found to be insufficient on its own for use as a disinfectant of potable water, however further investigation is recommended into the performance of Neem oil disinfection when combined with pre-treatment steps (e.g. sand or cloth filtration) and into methods for concentrating the active anti-microbial ingredients of Neem oil to form more potent disinfectant solutions.

Key Words: Disinfection, coliform; Neem; essential oils; drinking water; bacteria


Introduction

The Moringa oleifera seed is an example of a naturally occurring material that is effective as a means for treating water and has been successfully adopted for use in developing countries (e.g. Sudan) (Sutherland et al., 1989). The seed contains substances which act as a coagulant and thereby assist to settle out particles and pathogens from water. In India, lentils and seeds from the Tamarind tree and several other plants have also been proven as effective coagulants for waters with high turbidity (Schulz and Okun, 1984).

Comparatively little is known about the potential existence of natural disinfectants (i.e. substances with the ability to kill/inactivate pathogenic microorganisms), even though many herbs and plant extracts are used in traditional medicine and as pesticides in developing countries (Ketkar et al., 1995; Ross, 1999). One such plant, the Neem tree (Azadirachta indica), was selected as the focus of this study, however there are many other natural essential oils that have been reported to have anti-microbial properties (e.g. tea tree oil, eucalyptus, thyme) (Hammer et al., 1999; Lambert et al., 2001).

The Neem tree grows widely in arid tropical and subtropical areas around the world (Schmutterer, 1995). The medicinal properties of components of the Neem tree have been known in India for thousands of years; it is referred to in Sanskrit texts as “Arishtha”, the reliever of sickness (Ketkar et al., 1995). Traditional medical uses of Neem products have covered a vast range of illnesses, from leprosy to intestinal worms. Neem leaf tea is also often used as a treatment for the symptoms of malaria and for diarrhoea (Shultz et al, 1992). Extracts from dried leaves have been reported to be effective in the treatment of skin infections such as ringworm and scabies (Biswas et al, 2002).

A substantial number of anti-bacterial active compounds have been isolated from various parts of the Neem tree. The most active compound with regard to pest and insect control is thought to be azadirachtin (Schmutterer, 1990; Eppler et al., 1995) while nimbidin extracted from seed oil has been reported to exhibit strong anti-bacterial properties (Biswas et al., 2002). The anti-microbial components of essential oils generally consist of a large group of mainly terpenoid compounds, such as limonene, carvone and pinene (Sikkema et al., 1995; Cotton, 1996). Among the most commonly investigated with regard to anti-microbial activity are phenolic terpenes such as eugenol, carvacrol and thymol (Juven et al., 1994; Tassou et al., 2000; Lambert et al., 2001; Arfa et al., 2006; Marwah et al., 2007). The most important active component of Neem in terms of bactericidal activity is believed to be the nimbin group of tri-terpenoids (Kraus and Ermel, 1995; Biswas et al., 2002). The mechanism by which these compounds affect bacteria is primarily impairment of the cell membrane; the lypophillic molecules dissolve in the cell membrane, disrupting membrane permeability and resulting in the leakage of ions, adenosine triphosphate (ATP), nucleic acids and amino acids (Sikkema et al., 1995; Griffin et al., 1999; Utlee et al., 1999; Tassou et al., 2000; Lambert et al., 2001).

There have been only a small number of experimental attempts to assess the anti-bacterial activity of Neem products. One study using the disc diffusion method demonstrated the anti-bacterial activity of Neem seed oil against various Gram-negative bacteria including Escherichia coli and some Gram-positive bacteria such as Staphylococcus pyogenes (Rao et al., 1986). Reports of the effectiveness Neem leaf extract are somewhat conflicting, with some studies reporting anti-bacterial activity towards certain bacterial species and others reporting no effect on any bacterial species (Eppler et al., 1995). A recent study tested Neem leaf extracts against a range of foodborne pathogens and found that ethanolic Neem extracts exhibited anti-bacterial activity against several Gram-positive bacteria but no activity against any of the Gram-negative bacteria tested, including six strains of E. coli (Hoque et al., 2007). In contrast, another recent study tested the performance of methanolic Neem leaf extract against various multi-drug resistant strains of Vibrio cholerae and found significant anti-bacterial activity (Thakurta et al., 2007). The latter study also included a toxicology assay and found that the leaf extract showed no signs of toxicity in mice following an oral dose of 1800 mg/kg.

It is difficult to extract conclusions on the anti-bacterial properties of Neem components from the published studies to-date, since the studies were performed using vastly different experimental methods, different parts of the Neem tree, and different types of bacteria. In addition, the majority of the studies employed agar diffusion techniques which may be unreliable for the testing of oils (Mann and Markham, 1997). To-date there has been no research on Neem extracts specifically as a potential disinfectant of waterborne pathogens. Therefore, the specific objectives of this study were:

·  To assess the anti-bacterial activity of Neem oil against a sludge-isolated culture of E. coli in pure laboratory-grade water.

·  To assess the anti-bacterial activity of Neem oil against total and faecal coliform bacteria in unfiltered surface waters.

·  To comment on the practicality of using Neem oil as a viable water disinfectant based on the required dose levels observed in this study.


Materials and Methods

Microbiological methods

A culture of E. coli isolated from sewage sludge was maintained on a plate of Lauria Bertani (LB) agar consisting of LB broth (Fisher Scientific) and Agar No.1 (Lab M International Diagnostics Group). To prepare the inocula, a loop of culture was transferred to LB broth and incubated for 18 to 24 hours on a shaker plate. After incubation, the bacteria were washed in phosphate-buffered saline (PBS) solution (Oxoid) following which the bacteria were refrigerated in PBS and used in experiments within 24 hours.

Total and faecal coliform bacteria were used as indicator organisms to evaluate the disinfection potential of Neem oil when added to unfiltered surface waters. These groups are standard indicator organisms for the microbiological quality of drinking water (WHO, 1993). The membrane filter procedure was chosen as the primary method for all the surface water testing, according to Standard Methods 9222B and 9222D (APHA, 2005).

It was beyond the aims and scope of this research to examine the disinfection performance of Neem oil against a range of other strains of E. coli or other waterborne pathogens (e.g. viruses, protozoa), however this is recommended for further investigation.


Water sources

Surface water samples were characterised upon collection in terms of pH, temperature, ultraviolet absorbance at 254 nm, and turbidity (Table 1). Samples were transported in an insulated cooler, stored at 4oC, and were used in experiments on the same day as collection. Sample bottles were disinfected beforehand with Virkon© solution and rinsed with reverse-osmosis treated water. All the water required for a particular test run was taken from a single sample bottle. Each bottle was agitated to redistribute any settled matter before the sample volume was dispensed.

Surface waters from England and Nepal (the study countries) were collected. The surface water from England was a pond with faecal and organic pollution from a large resident waterfowl population. The surface water from Nepal was a river near Kathmandu with high levels of faecal pollution from the discharge of untreated wastewater and human faeces upstream. The river water is used for bathing and is known to contaminate local wells that are used for drinking water. Neither surface water is used directly as a source of drinking water, however they were selected in order to ensure high enough bacteria counts to be able to meaningfully quantify the level of disinfection achieved by the Neem oil. They may also be considered to represent a worst-case, contaminated drinking water source in a developing country context.

Neem oil

For the tests in England, Neem oil was obtained from a commercial source (The Neemteam, Newport, UK). The Neem oil was produced by cold pressing and was well defined by the manufacturer in terms of concentration of key active ingredients (azadirachtin: 1375 ppm, nimbin: 1500 ppm, salanin: 1500 ppm). The same batch was used throughout testing.

Neem oil is readily available in Nepal although it is usually imported from India. The Neem oil used in this study was manufactured by Shree Baidyanath, Patna, India. It was darker in colour and possessed a stronger bitter smell, suggesting that it was produced by steam or solvent extraction rather than cold pressing (Conrick, 2001). The same batch was used for all tests.

Disinfection potential of Neem oil against E. coli in lab-grade water

The inocula were prepared at concentrations of 2 x 108 to 6 x 108 CFU∙ml-1. The doses of Neem oil trialled were 2.13 g∙l-1, 4.27 g∙l-1, 8.53 g∙l-1 and 17.1 g∙l-1. Inocula (3 ml) were added to 27 ml of autoclaved PBS in a 125 ml conical flask to create a 10-1 dilution. The Neem oil dose was applied to this dilution which was then sealed and mixed for five minutes on a shaker plate at 170 rpm. From the disinfected sample 10-2, 10-3 and 10-4 dilutions were generated. Samples (0.1 ml) of each of these dilutions were spread-plated on LB agar and incubated for 24 hours at 35oC in order to obtain plates with between 20 and 200 countable colonies. Samples for plating were extracted from the water column below the oil layer; the oil layer was allowed to separate for one minute before sample collection. It is possible that some fraction of the targeted coliform bacteria were separated into the oil phase and hence not enumerated in the samples collected from the water column below. This hypothesis could not be tested since samples taken from the oil layer could not be analysed for coliform counts; the oil confounded the membrane filtration technique and would have exerted a continued anti-microbial effect during enumeration (i.e. there was no way to ‘neutralise’ the oil). As such, this was accepted as a limitation of the experimental method of this study.

Disinfection potential of Neem oil against coliform bacteria in surface water samples

Surface water samples were mixed with a range of Neem oil doses over several contact times, to investigate dose- and time-dependence of the disinfection performance. As in the trials with lab-grade water, each sample was left to stand for one minute to allow the oil and water to separate; a sample of water containing a minimal amount of oil could then be decanted and filtered. This careful separation was necessary, as otherwise the oil accumulated on the surface of the membrane filter, either causing it to clog or interfering with colony growth. The total coliform and faecal coliform filter trials for a given Neem oil dose were performed using the same sample. A positive control consisted of following an identical method but without the addition of Neem oil, and a negative control consisted of Neem oil added to deionised water with no bacteria.

To test varying mixing times at a fixed concentration, the disinfected sample was prepared in batches of 250 ml with the application of 17.1 g∙l-1 Neem oil. The samples were then mixed on a shaker plate at 180 rpm for one minute, four minutes, 12 minutes or one hour. The samples were then analysed as described above. The positive control consisted of surface water with no Neem oil addition placed on the shaker plate for one hour.

The methodology was kept as similar as possible for the trials in Nepal. The same membrane filter unit and supplies of membrane filters used for the England-based investigation were taken to Nepal for the testing.


Statistical analyses

All tests were replicated a minimum of three times. The results are displayed as bar and whisker plots, with the bars indicating the mean values and the whiskers showing the minimum and maximum values in each data set. The surface water coliform inactivation results are reported in terms of percent inactivation, while the cultured E. coli inactivation results are reported as log10 inactivation.

Results and Discussion