ORIGINAL RESEARCH ARTICLE

Comparative Analysis of Oligodynamic Virtue of Various Metals on Bacterial Population

Khushboo Lilaria, Nisha Gupta, Jai Shankar Paul*, Shailesh Kumar Jadhav

School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India.

khushboolilaria@gmail.com, nishagupta1210@gmail.com, jaishankar_paul@yahoo.com, Jadhav9862@gmail.com

^*Corresponding Author Email- jaishankar_paul@yahoo.com

Introduction

Water is the most crucial resource on Earth which is a vital substance for sustaining the entire ecosystem and regulating and maintaining the different physiological functions in a body. Even a single life cannot be imagined without water, as it is vital for the survival and existence of every living organism. According to World Health Organization (WHO) and the United Nations Children Fund (UNICEF), there are around 2.5 billion people who do not have proper sanitation (Bhuiyan et al. 2013). As stated by UNICEF, >3000 children died each day globally due to the consumption of contaminated drinking water (Villapún et al. 2016). Following data obtained from the Central Bureau of Health Intelligence (CBHI) and the Ministry of Health and Family Welfare, waterborne diseases are mainly responsible for most of the infections in children (Barakat 2011; Yatigammana et al. 2011).

There has been an increased risk to human health since the day of the population explosion on Earth and an enhancement in the rate of pollution of water sources. The water gets polluted by several chemicals such as fertilizer and pesticides from agricultural run-off, sewage and food processing waste, heavy metals, chemical waste from industries, urbanization and oil spills.

The ions like mercury, silver, copper, iron, zinc, bismuth, gold and aluminium exert an anti-microbial effect (Alexander et al. 2009; Villapún et al. 2016). However, the utilization of copper as an anti-microbial substance is not a new motive. Copper-based preparations have been used since ancient times to treat wounds, burns, infections, rashes and inflammation. Various mechanisms have been suggested via which copper facilitate antimicrobial activity. The exact mechanism is still not known but some findings suggest that copper damage proteins either by altering the protein conformation or by binding to its active site. Copper may damage nucleic acids by crosslinking within or between the strands of DNA or can damage proteins or can cause the formation of radical ions which can ultimately lead to the destruction of the bacterial cell (Borkow et al. 2009; Villapun et al. 2016). The present study was to undertaken to investigate the oligodynamic potential of various metals including copper, steel, aluminum, brass, and bell metal on bacterial population in different time intervals.

Materials and Methods

All the chemicals used in the current study were of analytical grade and were purchased from HiMedia Laboratories Pvt. Ltd. Mumbai, India, Sigma-Aldrich, USA or Merck India Limited, Mumbai, India.

Preparation of medium

Nutrient Agar Medium (NAM) was used in the current study for the isolation and bacterial growth with a composition (g/L); peptone–5, beef–3, sodium chloride–5 and agar–15g in 1000 mL distilled water. The pH of the medium was kept 7.0±0.2 followed by its sterilization at 15 psi pressure at 121°C for 15–20 minutes. Subsequently, sterile NAM plates were prepared for further use in the study.

Investigating the oligodynamic potential of metals

The metal pots made up of copper, steel, aluminum, brass, and bell metal were selected for the study. The soil pot was also used along with these metals. The above pots were sterilized properly using 70% (v/v) ethanol and were left for drying for about 10-15 min in hot air oven. The metal pots were then filled with 100 mL of tap water and kept at room temperature. After regular interval of time of 12, 24, 36, 48 and 60 h, spread plate method was applied for assessing the bacterial growth and for the analysis of oligodynamic actions of various metal pots via serial dilution.

Isolation of bacteria by spread plate method

For analysis of oligodynamic effect on bacterial growth, stored water samples were taken individually after definite interval of time for isolating the bacteria. Bacterial colony was isolated through spread plate method in sterile NAM plates in triplicates. After the preparation of dilution series from the water sample, 100µL of each water sample of suitable dilution was spread uniformly with the help of a spreader on a sterile NAM plate. The inoculated plates were kept in incubator for 24 h at 37° C.

Morphological characterization of the bacterial isolates

The total numbers of bacterial colonies were counted and the reduction of bacterial density in water sample with increasing holding time interval in the metal pots was recorded. Then the isolated colonies were characterized morphologically.

Results and discussion

Microbial analysis of water stored in different storage containers like copper, steel, aluminium, brass, bell metal, and silver was assessed for their oligodynamic potential after a regular interval of time (0-60 hours of incubation). There was a very significant decrease in the bacteria population after 24 hours of incubation in the water sample kept in copper metal, silver and soil pots (Table 1 and 2). However, the water stored for about the same time in brass, aluminium, and steel didn’t show any decrease in the bacterial colonies and hence indicating negligible bacterial population inhibition. Water cultures were tested up to 60 hours of storage and copper metal showed maximum inhibition (about 70.44 %) of bacterial growth (Table 1, 2 and Fig. 1).

The morphological characterization of all the bacterial population of water sample kept in various metal pots was carried out. Majorly, the bacterial population found in the water sample of various metal pots has raised elevation, punctiform with an entire margin and is creamish in colour (Table 3).

The current findings are in line with Shrestha et al. (2010) who reported the oligodynamic virtue of copper metal against the gram-negative bacteria (Salmonella paratyphi). He observed that the bacterial colonies reduced completely within 4 hours of holding time in the copper metal. Packiyam et al. (2016) reported a significant reduction in the bacterial colonies from the water samples kept in mud pots, silver, copper, and brass. Moreover, there was no reduction in the coliform bacteria in the water sample stored in the glass, plastic, ceramics, aluminium, coconut shell and stainless steel. The effect of metals on water-borne and skin-borne pathogens was studied by Dhar (2019) using copper, silver, gold, zinc, and brass metals and reported that silver could eliminate Escherichia coli, Salmonella typhi and Vibrio cholerae by >98% within 24 hours of holding time.

From the current study, it can be concluded that copper metal is the most promising metal in inhibiting the bacterial population within 48–60 hours of holding time. Our results are in corroboration with previous findings. Copper and silver have been shown to have fast reaction rates against the bacterial population according to Varkey (2010) and Praveen (2018).

Figure 1: Bacterial colonies isolated from water sample kept in different pots (a–f) after 60 hours