Solvation of Ampicillin in Nanoparticle doped water and the

affect on its stability and bactericidal effect.

Barel. 1,2
R , Mor.A 2
1- The Department of biotechnology engineering Ben-Gurion University
2- Do-Coop technologies Ltd.
Abstract:
Water is one of the most common and ordinary substances we encounter on a day to day basis. It is the most important molecule for all organisms. Most of water features
are attributed to its unique physical and chemical properties. Nanoparticle doped water - Neowater® (NW) is a novel technology developed at “Do-Coop Technologies", in
which a heated crystal material is dropped into a basin containing ultra pure water (UPW) cooled to a point slightly below the anomaly and which is also radiated with RF
waves. This patented and unique process leads to a stable composition of water, nano-particle and gas which modifies the physical properties of water. The technology
enables wide and various applications in the fields of chemistry and biology such as enhanced solubility of organic molecules and increasing bioavailability and stability of
various molecules. These properties together with the fact that water serves as the close environment of bacteria as well as the vehicle for antibiotics delivery raised a
hypothesis that antibiotic dissolved in NW will affect bacteria differently comparing to one dissolved in water and yelled different efficacy levels throw enhanced delivery or
stability. Ampicillin (Amp) was dissolved in NW and in UPW. Its effect on both non resistant and Amp resistant (by pUC19 plasmid insertion) DH5α E.coli's bacterial
growth was assessed using 3 different parameters: decreasing and increasing Amp concentrations, different incubation time at 37°C (7-24 hours) and storage conditions of
the dissolved Amp (Temperatures from -20°C to 50°C) Amp dissolved in NW and stored at 40°C or 50°C showed a greater inhibition effect on non resistant E. coli growth
than Amp dissolved in UPW and stored under the same condition, showing the preservative effect of NW.

Objectives:
Studying the activity and stability of Ampicillin when dissolved in Neowater® as compared to Ampicillin dissolved in Ultra Pure Water.
Methods:
Experiment A
Amp was dissolved in NW and in UPW (C=0.1g/mL). Every 0.1g/mL 0.8 0.8
Amp NW 031207-3 Amp NW 031207-3
stock was split into two tubes, one stored in 50°C and the other stored 0.7 Amp UPW 0.7 Amp UPW
at -20°C. In order to test Amp bactericidal activity 5µL of each stock 0.6
negative control
0.6
negative control

(either in NW or in UPW stored in both temperatures) were added to 0.5 0.5

O.D 595 nm

O.D 595 nm
10mL of liquid LB containing 100µL of DH5α E. coli + pKYB1
0.4 0.4
(kanamycin resistant) bacteria starter. As a negative control 3 additional
0.3 0.3
tubes were set with 5µL of NW without Amp. The bacteria were
0.2 0.2
incubated for 7h in an incubator shaker set on 37°C and 220rpm. The
0.1 0.1
bacterial growth curves were monitored by OD595 readings in 1h
0.0 0.0
intervals. 0 2 4 6 8
0 2 4 6 8
Experiment B time(hours) time(hours)
The experiment was identical to experiment A only the Amp was stored Figure 2: Growth curves of E. coli (DH5α +pKYB1- kanamycin Figure 3: Growth curve of E. coli (DH5α +pKYB1) with 50µg/mL
in 40°C instead of 50°C. resistant) with 50µg/mL Amp dissolved in Neowater® or in UPW Amp in Neowater® or in UPW stored at 50°C Conducted 4 days
Experiment C stored at -20°C Conducted 4 days after Amp storage at -20°C. The after Amp storage at 50°C. Amp dissolved in UPW group is
negative control contains bacteria and Neowater® without Amp to statistically significant compared to Amp dissolved in Neowater®
Amp was dissolved in NW and in UPW (C=0.1g/mL). The stocks were eliminate NW toxicity. as jugged by student’s Ttest (P=0.0381)
stored at -20°C.To different 10mL liquid LB tubes contacting 100µL of
0.8 0.8
Amp NW 010707-1
an overnight starter of DH5α E. coli + pUC19 (Amp resistant). 30µL of Amp UPW
Amp NW 010707-1
0.7 0.7 Amp UPW
the stock solution was added (C=300µg/mL). As a negative control 3 negative control negative control
0.6 0.6
additional tubes were set with 30µL of NW each without Amp. The
0.5
O.D 595 nm

O.D 595 nm

bacteria were incubated for 7h in an incubator shaker set on 37°C and 0.5
0.4
220rpm.The bacterial growth curves were monitored by OD595 readings 0.4

in 1h intervals. (Only end point results are shown). 0.3 0.3

0.2 0.2

0.1
Results: 0.0
0.1

0.0
0.70 0 2 4 6 8 0 2 4 6 8
Amp neowater 031207-3 time(hours)
time(hours)
0.68 Amp UPW
control Figure 4: Growth curve of E. coli (DH5α+pKYB1) with 50µg/mL Figure 5: Growth curve of E. coli (DH5α +pKYB1) with
0.66 Amp in Neowater® or in UPW stored at -20°C Conducted 9 days 50µg/mL Amp in Neowater or in UPW stored at 40°C Conducted
0.64
after Amp storage at -20°C . The negative control contains bacteria 9 days after Amp storage at 40°C. Amp dissolved in UPW group
and Neowater® without Amp to eliminate Neowater® toxicity. is statistically significant compared to Amp dissolved in
O.D 595 nm

0.62 Neowater® as jugged by student’s Ttest (P=0.0342)

0.60
Results summary:
0.58

0.56
1.For non Amp resistant bacteria - Amp dissolved in Neowater® shows better
0.54 bacterial inhibition than Amp dissolved UPW after 4 days of storage in 50°C
0.52 and after 9 days of storage in 40°C
Figure 1: Turbidity of E. Coli (DH5α +pUC19) with 300µg/mL
Amp in Neowater® and UPW 7 hours after amp addition both 2.For Amp resistant bacteria - When increasing Amp concentrations, lower
groups are statistically significant compared to Amp dissolved in
Neowater® as jugged by students Ttest (P≤0.034)
concentrations of Amp dissolved in Neowater® are needed to achieve decrease
in bacterial growth in comparison to Amp dissolved in UPW.
Conclusions:
NW stabilize Amp when stored at high temperatures .This effect can be implanted on other heat sensitive molecules and will
allow using them in the industry with fewer limitations.
Lower concentrations of Amp are need for the inhibition of growth of Amp resistant bacteria when Amp is dissolved in NW in
comparison to Amp dissolved in UPW. This effect can assist treating antibiotic resistant bacteria without the need of
developing new antibiotics.