‘Honeytrapping’ Pseudomonas From Water: A Sustainable Prototype For Water Disinfection

This paper introduces a novel prototype for the removal of Pseudomonas from water samples. Bacterial cells have the tendency to get attracted towards specic chemicals (chemotaxis); a ‘honeytrap’ strip was conceptualized by integrating a combination of serine, pseudomonas specic chemo-attractant and honey to attract and inhibit the bacteria in situ. Honey, a natural antimicrobial agent, has garnered attention in its effective inhibitory role in Pseudomonal biolms and wound infections. Dipping serine side of the strip attracted bacteria towards honeytrap, wherebythe porous nature of the strip facilitated the ‘trapping’ and subsequent diffusion of the bacterial cells towards honey-adsorbed end of the strip. This ‘honeytrap’ reportedly leads to the targeted elimination of Pseudomonas, hence facilitating its removal. The percentage ecacy of this ‘honeytrap’ device is 96% with a log reduction equivalent to 1.6 within a time frame of 2 hours. Pseudomonas aeruginosa, although, not a natural contaminant of potable water, enters circulation due to improperly maintained plumbing xtures and storage facilities. Honeytrap strip is an easy to use, biodegradable and cost effective sustainable solution, and thus a scaled up version ofthis device may enablesubstantial improvement in quality of potable water.


Introduction
Consumption of contaminated water affects the health of approximately 1.8 billion people all over the world. Bioaccumulation of non-degradable contaminants has aggravated the intensity and propensity of fatal diseases (Jamshaid et al., 2018). Potability of water is decided by the physicochemical and biological parameters according to World Health Organization (WHO) (Galal-Gorchev, 1993;Van Leeuwen, 2000), Environment Protection Agency (EPA) (Oberdiek, 2007), Environment Quality Standard (EQS) (Gobelius et al., 2018). Although comprehensive water testing mechanisms ensure that international standards for water quality are stringently met, yet local contaminations during storage and distribution have been observed resulting in increase in bacterial load (Brick et al., 2004;Han et al., 1989).One of the major concern is that water acts as a reservoir and aids easy transmission of pathogens like Pseudomonas aeruginosa (P. aeruginosa), Flavobacteriummeningosepticum, Aeromonashydrophilla (Rutala and Weber, 1997).Hence, there is an urgent need for eld-applicable devices for testing and sanitization of potable water especially at household levels.
P. aeruginosa, an opportunistic, gram negative bacteria is the prominent cause of both community and hospital based infections (Pachori et al., 2019). Currently, sensitive detection of P. aeruginosa and its metabolites is based on genotyping techniques such as Polymerase Chain Reaction (PCR)(Héry-Arnaud  (Fitzgerald and DerVartanian, 1969) established that about 0.5 mg/L of chlorine is capable of removing 99.9% P. aeruginosa within an hour; but with decreasing chlorine concentration the effectiveness of this treatment diminishes (Price and Ahearn, 1988). Pseudomonas has been detected in the range of 10-2300 CFU/mL in drinking water and tap water (Mena and Gerba, 2009),with infective load varying upto 10 9 CFU/ml (George, 1989),which is contrary to the permitted levels set by European Union.
As of now, no technique is capable of detecting and disinfecting P. aeruginosa simultaneously from solutions. In this work, we have proposed and tested a device for sanitizing the potable water against Pseudomonas. Chemo-attractants like sugar and amino acids have been found to pull bacteria (Sourjik and Wingreen, 2012),especially serine which attracts P.aeruginosa (Moulton and Montie, 1979). Several naturally occurring non-toxic materials such as coffee(Martínez-Tomé et al., 2011),turmeric oil (Negi et al., 1999),capsicum extract (Careagaet al., 2003),moringa leaf extract (Peixoto et al., 2011),honey (Cooper et al., 2002;Shenoy et al., 2012;Wilkinson and Cavanagh, 2005) have been established as inhibitors ofP. aeruginosa.
Our approach of device fabrication uses a combination of serine as chemoattractant and honey as an inhibitor for removal of P. aeruginosa,hence the name, honeytrap.The attractant lures bacteria instantly followed by diffusion of the cells through the contact surface, exposing it to the inhibitor, thus reducing the microbial load.
This inexpensive and eld-applicable 'honeytrap' has been successfully validated using a combinatorial approach. In the future, honeytrap may nd its application in households as a personal pocket water sterilant Experimental Section Materials P. aeruginosastrain was obtained from IMTech, Chandigarh. Whatman lter papers were obtained from Whatman International Ltd Maidstone England. LB Broth, LB Agar and serine was procured from HiMedia Laboratories Pvt. Ltd. Honey (Dabur Ltd, India ) was purchased locally. Safranin dye was procured from CDH. The water used in this study was double distilled water.

Preparation of Serine and Honey strips
The Whatman lter paper was cut into strips (60 mm X 10 mm). Serine solution (1mM) was prepared with autoclaved distilled water and sterilized using a syringe lter (0.22µm nylon membrane). The solution was subsequently stored in at bottom vials. The autoclaved strip was loaded with 2ml of serine solution. The strip, after an incubation of 1 hour, was removed and air dried in sterile conditions. Serine strips (SS) thus prepared, were used in further experiments. Sterilized strips were similarly used for the preparation of honey strips (HS). About 2 ml of sterilized stock solution (25% honey V/V) was prepared and paper strips were likewise incubated for an hour to generate HS. Control strips (C) were also designed containing SS on one end and water imbibed on another end.
HS and SS prepared were annexed to each other via biologically friendly glue to attain a serine-honey strip (SHS) containing an attractant on one end and an inhibitor on another end (Dasgupta et al., 2016).
Preparation of arti cially spiked water Pseudomonas was grown in nutrient broth overnight at 37ºC with proper aeration in the rotating incubator. The overnight culture was subsequently inoculated in 25 ml fresh broth and OD 600 was monitored till 0.123 corresponding to 3 x 10 8 CFU/ml according to the McFarland scale. The culture was centrifuged at 8000g for 15 min and the cell pellet was resuspended in 25 ml distilled ltered water (spiked water). This spiked water was hereafter used for testing the e cacy of honeytrap for removal of Pseudomonal cells.

Chemoattractant potential of SS
To assess the trapping mechanism ofP. aeruginosacells by SHS, glass slides loaded with 200 µl of 'spiked' water were horizontally exposed to the SHS (Serine end) for different incubation periods (15min, 30 min, 45min, 1hr, 1.5hr, 2hr). Bacterial chemotaxis was monitored intermittently via microscope (Magnus Opto Systems India Pvt. Ltd.) at various time durations. Subsequently, strips were placed on nutrient agar to assess the possible adsorption of P. aeruginosaby honeytrap and incubated overnight at 37 ºC.
Trapping e cacy of honeytrap 'Spiked' water sample (2ml) containing P. aeruginosawas taken and approximately 1 cm 2 of honeytrap was dipped into the vials at the serine side. Vials were exposed to different incubation times (15min, 30 min, 45min, 1hr, 1.5hr, 2hr) with SHS and absorbance at 600nm was monitored. The change in OD was attributed to the fact that there was a change in the bacterial concentration. Spectroscopic monitoring for where B 2hr = number of viable microorganisms after treatment Electrical conductivity has been considered as a criterion to measure the concentration of bacteria in suspension (HARRIS and PATTY, 1949).Hence, serine as a trapping constituent of P. aeruginosawas assessed by measuring the change in electrical conductivity of the suspension post incubation periods.
Brie y, 2 ml 'spiked' water samples were incubated with honeytrap and change in electrical conductivity of the culture was monitored using a digital 3-in-1 TDS/EC/Temp meter (Labcare,India) post incubation periods. The alteration in the electrical conductivity of suspension was plotted against the incubation time and correlation studied with the change in percentage of bacterial cells.

Effect of HS on adsorbed Pseudomonas
Two sets of 2 ml 'spiked' water samples were taken and exposed to control strips and honeytrap respectively and incubated for different time periods. Subsequently, strips were incubated overnight at 37ºC on nutrient agar to assess the possible adsorption and inhibition of P. aeruginosa by control and SHS strips.

Imaging and characterization
The functionality of serine as an attractant was con rmed using an optical microscope (40X magni cation). 200µl of overnight bacteria culture was placed on the glass slide with a honeytrap placed at one end of the glass slide. Bacterial adsorption on the device was observed via Field emission scanning electron microscopy (Fe-SEM) (Nova Nano FE-SEM 450) after exposing the strip (1cm 2 ) to vacuum gold sputter coater and the images were observed with 5.00 kV accelerating voltage of electrons and at a magni cation of 10,000X.

Result And Discussion
Design of the SHS bait A chemotactic trap with serine as an attractant, speci c for P.aeruginosa(Moulton and Montie, 1979)was a xed in a contiguous manner with an inhibitory strip. This trap takes the advantage that it can autonomously attract, trap and destroy Pseudomonas in a co-ordinated mechanism as depicted in Scheme 1

Functioning of SHS
A three pronged approach was used to verify the attraction, trapping, and destruction machinery of the SHS strip and each mechanism was monitored sequentially.

Attraction of cell by SHS
The SHS doped with 1 mM serine was tested for its bacterial 'chemotactic' potential. Conventional mechanism of monitoring chemotaxis was modi ed using SHS strip on glass slide whereby serine of SHS induced active migration of cells (Fig. 1). With an increase in the incubation time, there was a clear demarcation and movement of theells towards SS owing to its chemoattractant property. The cell suspensions were exposed to different incubation periods and residual numbers of cells were enumerated.There was a perceptive increase in the cell concentration towards the serine end of the device within 15 min as compared to the control strip containing water. Negligible cells were observed near the CS. The structure of the paper enabled the movement of bacteria within the SHS strip thus contributing to real time removal of bacterial cells.
The other method used to con rm the effectiveness of SHS was by measuring the OD of the spiked water with a speci c cell density. According to the Mcfarland standard, a cell density of 3X10 8 CFU corresponds to 0.123 at OD 600 . The strip was dipped approx 1cm from the end of the sample and was kept for the designated incubation time (15 min, 45 min, 60 min, 90 min). After each incubation, the strip was removed and the OD of the sample was taken at 600 nm. Results con rmed that despite abundant load of cells, the strip removed about 96% of the total load after 2 hours of incubation (Fig. 2). The decrease in the residual cells was additionally con rmed by measuring the electrical conductivity. There was a consistent increase observed in the electrical conductivity with a smooth transition from 3788 mS/cm to 4052 mS/cm within 2 hours (Fig. 2). It has been earlier observed that an increase in conductivity is proportional to the corresponding decrease in cells in a suspension thus con rming the decrease in suspended bacterial cells (Harris and Patty, 1949).Furthermore, honeytrap indicated that the strip has a percent reduction value of 96% and a log reduction equivalent to 1.6.

Trapping and removal by SHS
Reduction in suspended bacterial cells by the honeytrap was further con rmed using SEM. Approximately 1cm 2 cutouts were made 2 cm away from the SS (SSc) and HS (HSc) ends and analyzed (Fig. 3).
Bacterial cells were observed in the SSc cellulose bers within 30 min of incubation (Fig. 3a) whereas no bacterial cells were observed in HSc (Fig. 3d). The pores of the strip allow capillary action and enabled the bacteria to diffuse towards the HS end. After 60 min, the elevated cell numbers was observed at the SSc strips (Fig. 3b) and comparatively fewer bacterial cells could be observed in HSc (Fig. 3e), thus indicative of honey susceptibility A lesser number of cells in cutout from the honey end, HSc may either indicate a slower diffusion rate or the inhibitory activity of honey. Bacteria have been found to alter their size and shape in response to stress factors and this phenomenon could be correlated with the morphological changes and clumping observed in Pseudomonassignifying plausible cell death (Brudzynski and Sjaarda, 2014; Combarros-Fuertes et al., 2020) (Fig. 3a).

Qualitative analysis of Chemo-inhibitory effect of SHS on Pseudomonas
The medicinal use of pure honey has been known since ancient times but its application in therapeutics and as an antimicrobial has been recently employed (Albaridi 2019, Husain et al., 2019). Studies indicated a concentration range of 20 % − 25 % has 100 % antimicrobial e cacy with 24 hrs incubation (Shenoy et al., 2012). In this case, the chemo-inhibitory effect of the SHS strip was con rmed by a simple plate incubation method. The incubation time varied from 15-90 min. The effect of honey was markedly evident as signi cantly lesser growth was perceived with the time, associated with the progressive disappearance of growth from the serine side of the strip.
The Road Ahead P. aeruginosa is widely distributed in the environment. By selectively using the unique combination of organism speci c chemo-attractant and inhibitor, the honeytrap ensures passive targeting of Pseudomonas cells facilitating their e cient removal from water. The experimental setup for honeytrap indicated that the strip has a percent reduction value of 96% and a log reduction equivalent to 1.6 thus, indicating a water treatment potency of more or less 90%-92%.
Currently, many point of care device are available with a range of e cacy and target contaminants. Previously a micro uidic device had been designed based on the chemotactic behavior of E.coli for subsequent disinfection of contaminated water (Kim et al., 2015). Other household water disinfection devices are available but may present various disadvantages like higher cost and use of chemicals ( Table 1), Some of the instruments use UV light as a sterilant, however it has been observed that UV light may lead to mutation of the microbial species already present in the source (Shibai et al., 2017). Hence, honeytrap presents itself with a unique green technology designed with already proven antimicrobials that do not have any deleterious effect on humans require a power source and sometimes leave traces of chemicals, which may be harmful over time. The prototype proposed in this research is based on green technology, cost effective, biodegradable and tends to destruct the targeted bacteria in situ. Although further research and testing is required to attain the nesse suitable for acting as a household water treatment device, the honeytrap device envisions an exciting arena towards the development of a new generation of inexpensive and sustainable. The prototype may be extended to attract and destroy other motile related species. As an extension to current technology, In addition to honey, other natural inhibitors may be incorporated to make a device effective against a greater microbial diversity.   Supplementary Files This is a list of supplementary les associated with this preprint. Click to download.