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Vol. 3. Núm. S1.
1st International WOSQUAL-2019 Conference (selected medicine proceedings)
(junio 2020)
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Vol. 3. Núm. S1.
1st International WOSQUAL-2019 Conference (selected medicine proceedings)
(junio 2020)
Original article
Open Access
Evaluation of different type of electrolyzed water against bacterial colonization of diabetic foot ulcers: Study in vitro
Visitas
1903
Edy Supardia, Saldy Yusufb,
Autor para correspondencia
saldy_yusuf@yahoo.com

Corresponding author.
, Muh Nasrum Massic, Hasniati Haeruddind
a Nani Hasanuddin College of Health Sciences Makassar, Indonesia
b Faculty of Nursing, Hasanuddin University, Indonesia
c Medicine Departement, Faculty of Medicine, Hasanuddin University, Indonesia
d Gema Insan akademik College of Health Sciences Makassar, Indonesia
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Tablas (5)
Table 1. Frequency distribution of participants based on demographic data.
Table 2. Frequency distribution based on the participants’ diabetes mellitus status.
Table 3. Frequency distribution of participants based on a history of diabetic foot ulcers and diabetic foot status.
Table 4. Frequency distribution of bacterial types in participants’ diabetic foot ulcer based on the wound classification of University of Texas Classification system.
Table 5. Analysis of the effect of electrolyzed water on bacterial colonization.
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Abstract
Objective

This study aims to evaluate the effect of electrolyzed water effect on bacterial colonization on diabetic foot ulcer (DFU) patients.

Method

This is an in vitro study. Eight bacteria are incubated into 5 types of electrolyzed water (ESAW), electrolyzed weak acid water (EWAW), electrolyzed neutral water (ENAW), electrolyzed weak alkaline water (EWAlW), and electrolyzed strong alkaline water (ESAlW). Evaluations were performed 2, 24, 48 and 72h after incubation. Data were analyzed using repeated ANOVA test used to compare the difference of electrolyzed water effect on bacterial colonization of DFU patients.

Results

ESAW (pH 2.5) significantly (p=0.001) had a better bactericidal effect than EWAW, ENAW, EWAlW and ESAlW.

Conclusion

Current study confirmed the positive effect ESAW on bacterial colonization in DFU patients.

Keywords:
Electrolyzed water
pH water
Diabetic foot ulcer
Texto completo
Introduction

Diabetic foot ulcers (DFUs) associated with various devastating complications, such as; long term care, high cost,1,2 infection,3 amputation4,5 and mortality.6 Therefore prompt and aggressive treatment are needed to accelerate wound healing progress, which will be prevent worsening of ulcers, maintain extremity intact and quality of life.4

One of essential modality in management of DFUs is wound cleansing. Wound Cleansing is one of wound care procedure.7,8 At least normal saline and tap water are reported as main wound cleansing agent wound cleansing,9 meanwhile the advantages of acid cleansing has less evaluated.

Previous study revealed that Hypochlorus acid improved wound healing since it has antimicrobial effect including in DFUs.10 The benefits of pH neutral in a study of Neutralized electrolyzed water (NEW) effects that were preceded by an in vitro study showed that NEW pH 7.7 significantly reduced the number of Aspergillus flavus spores and Penicillium expansum spores.11 Although electrolyzed alkaline water has many health benefits including improving abnormal intestinal fermentation, chronic diarrhea, gastric hyperacidity, dyspepsia, improving constipation, suppressing body fat accumulation, expelling early melamine, reducing skin damage due to ultraviolet light, modulation of immune responses and repair diabetes.12 Despite many studies has reported the benefit of pH water, the effectiveness as wound cleansing remain unknown. Thus, the aim of current study to evaluate the effect of electrolyzed water effect on bacterial colonization on diabetic foot ulcer (DFU) patients.

MethodsSetting

This was an experiment in vitro study. The swabbing were taken from DFUs patients both inpatient-outpatient clinic (Hospital) and three home care setting (private wound care practices) by an accidental sampling technique. HbA1C>6% and Texas university classification system (grade B), as inclusion criteria.

Swabbing were taken using Levine technique. Culture of wound specimens through inoculation on the medium, incubated for 24h to identify bacteria and its gram properties.

Preparation

The electrolysis process of water uses the electrolysis machine (Leveluk SD 501, Japan), to prepare five different types of electrolyzed water (EW). Status of EW were measured by pH meter (Hanna pHep Tester HI98107, Romania), and validated at the Makassar Environmental Health and Disease Control Laboratory.

In vitro

The identified bacteria were made suspended in 0.9% NaCl solution using Mcfarlan 0.5 standard, then insert 5 types of electrolyzed water (EW) into the 9ml screw tube, sterilize. Suspension of 8 types of bacteria 1ml were added into a tube which each contains 5 types of EW, then mixed until homogeneous. Incubation running for 2h then put into sterile petri dish and adding medium plate count agar (PCA) to the petri dish, mix until homogeneous. After PCA solidified, incubate it into an incubator at 37°C for 2h. After 2h incubation, the number of bacterial colonies that grow on the medium were calculated. The procedure were repeated until the second day (48h) and third (72h).

Analysis

Demography data presented in frequency table (n, %), while evaluation of electrolyzied watered against bacterial colonization were analyzed using repeated one way anova (SPSS, ver 22, Chicago Inc). Ethical clearance was approved from Ethical Committee, Faculty of Medicine, Hasanuddin University. No. 437/H4.8.4.5.31/PP36-KOMETIK/2017.

ResultsCharacteristics of participants

Half of participants on early elderly on age group (46–55 years) (n: 10, 50.0%), the mean and standard deviation of the participants’ age (53.1, ±6.7), dominantly male gender (n: 14, 70.0%), Islam (n: 19, 95.0%), Bugis ethnic (n: 12, 60.0%), the last education for Senior High School (n: 8, 40.0%), government officer and self-employed (n: 8, 40.0%) (Table 1).

Table 1.

Frequency distribution of participants based on demographic data.

Variabel  Total
  n: 20 
Age (mean, +SD)  53.1  6.7 
Late adults (36–45 years old)  15.0 
Early elderly (46–55 years old)  10  50.0 
Late elderly (56–65 years)  35.0 
Gender
Male  14  70.0 
Female  30.0 
Religion
Islam  19  95.0 
Christian Protestant  5.0 
Ethnic
Bugis  12  60.0 
Makassar  30.0 
Toraja  5.0 
Java  5.0 
Last education
Primary School  15.0 
Junior High School  5.0 
Senior High School  40.0 
Diploma  10.0 
Bachelor/Masters/Doctorate  30.0 
Occupation
Government officer/Army/Police/Lecture  40.0 
Honorary  5.0 
Entrepreneur  35.0 
Farmer  5.0 
Housewife  15.0 

DM status based on diabetes duration <5 years (n: 8, 40.0%), mean and standard deviation of HbA1C (10.3, ±2.6%), Glycemic Control by oral (n: 8, 40.0%), history of never smoking (n: 8, 40.0%), the mean and standard deviation of the participants’ height (165.9cm, ±7.4), weight (62.9kg, ±10.1), Body Mass Index (BMI) (22.7kg/m2, ±2.4), the normal BMI category (18.50–24.99) (n: 16, 80.0%), systolic blood pressure (130.1mmHg, ±19.5), and diastole blood pressure (84.5mmHg, ±12.8) (Table 2).

Table 2.

Frequency distribution based on the participants’ diabetes mellitus status.

Variabel  Total
  n: 20 
Duration of diabetes
<5 years  40.0 
5–10 years  25.0 
>10 years  35.0 
Glycemic Control
There is no  15.0 
Oral  40.0 
Insulin  25.0 
Oral and insulin  20.0 
Smoking history
Never  40.0 
Ever  40.0 
Active  20.0 
     
HbA1C (mean, ±SD)  10.3  2.6 
Blood pressure
Sistole (mmHg) (mean, ±SD)  130.1  19.5 
Diastole (mmHg) (mean, ±SD)  84.5  12.8 
Height (cm) (mean, +SD)  165.9  7.4 
Weight (Kg) (mean, +SD)  62.9  10.1 
Body Mass Index (BMI) (kg/m2) (mean, +SD)  22.7  2.4 
BMI category
Underweight (<18.49)  5.0 
Normal (18.50–24.99)  16  80.0 
Overweight (25.00–29.99)  15.0 

DFU history based on wound duration 1–2 months (n: 11, 55.0%), causes of DFU injuries due to trauma (n: 10, 50.0%), there was a history of previous injuries (n: 15, 75.0%), no amputation history (n: 15, 75.0%), Neuropathy (n: 11, 55.0%), and angiopathy (n: 12, 60.0%) (Table 3).

Table 3.

Frequency distribution of participants based on a history of diabetic foot ulcers and diabetic foot status.

Variables  Total
  n: 20 
Duration of ulcer DFU
<1 month  35.0 
1–2 months  11  55.0 
>2 months  10.0 
Causes of ulcers
Unknown  40.0 
Trauma  10  50.0 
Non trauma  10.0 
History of ulcer
None  25.0 
Yes  15  75.0 
History of amputation
None  15  75.0 
Yes  25.0 
Neuropathy status
Non neuropathy  45.0 
Neuropathy  11  55.0 
Angiopathy status
Non Angiopathy  12  60.0 
Angiopathy  40.0 

The dominant aerobic bacterial colonies found in all participants were Proteus mirabilis (n: 6, 30.0%). Based on The classification of University of Texas Classification system, the dominant type of proteus mirabilis bacteria was found in stage B grade 1 (n: 5, 45.5%), while Eschericia coli bacteria were dominantly found in stage B grade 3 (n: 4, 50%) (Table 4).

Table 4.

Frequency distribution of bacterial types in participants’ diabetic foot ulcer based on the wound classification of University of Texas Classification system.

The type of bacteria  University of Texas Classification system
  Stage BGrade 1Stage BGrade 2Stage BGrade 3Total
  n=11  n=n=n=20 
Alkaligenes faecalis  9.1  5.0 
Eschericia coli  50.0  20.0 
Enterobacter agglumerans  9.1  5.0 
Klebsiella Sp  9.1  100  10.0 
Proteus mirabilis  45.5  12.5  30.0 
Proteus vulgaris  9.1  25.0  15.0 
Providencia alkalifaesen  9.1  5.0 
Providencia stuarti  9.1  12.5  10.0 

The effect of electrolyzed water in reducing the number of bacterial colonies based on the evaluation time of 2h, 24h, 48h to 72h. This indicates bactericidal effects have occurred at 2h after incubation (Fig. 1).

Fig. 1.

Estimated effect of electrolyzed water on time bacterial colonization from the evaluation time of at 2, 24, 48, and 72h (Factor1*: 1, 2, 3, 4).

(0.23MB).

There are significant differences in the effect of electrolyzed water on bacterial colonization, where ESAW (pH 2.5) significantly (p = 0.001) which confirmed had a better bactericidal effect compared to EWAW, ENAW, EWAlW and ESAlW. However, between ENW pH 7.0 and EWAlW pH 8.5 there seemed no significant difference, where the p value=0.951 (Table 5).

Table 5.

Analysis of the effect of electrolyzed water on bacterial colonization.

Electrolyzed water  Mean difference  95% CI  p value 
Electrolyzed strong acid water (ESAW) (pH 2.5)
ESAW (pH 2.5) v.s EWAW (pH 6.0)  −177.4  (−)204.9–(−)149.8  0.001 
ESAW (pH 2.5) v.s ENW (pH 7.0)  −253.7  (−)281.2–(−)226.1  0.001 
ESAW (pH 2.5) v.s EWAlW (pH 8.5)  −246.8  (−)274.3–(−)219.2  0.001 
ESAW (pH 2.5) v.s ESAlW (pH 11.5)  −48.8  (−)76.3–(−)21.2  0.001 
Electrolyzed weak acid water (EWAW) (pH 6.0)
EWAW (pH 6.0) v.s ENW (pH 7.0)  −76.3  (−)103.8–(−)48.7  0.001 
EWAW (pH 6.0) v.s EWAlW (pH 8.5)  −69.3  (−)96.9–(−)41.8  0.001 
EWAW (pH 6.0) v.s ESAlW (pH 11.5)  128.7  101.1–156.2  0.001 
Electrolyzed neutral water (ENW) (pH 7.0)
ENW (pH 7.0) v.s EWAlW (pH 8.5)  6.9  (−)20.7–34.5  0.951 
ENW (pH 7.0) v.s ESAlW (pH 11.5)  204.9  177.3–232.6  0.001 
Electrolyzed weak alkaline water (EWAlW) (pH 8.5)
EWAlW (pH 8.5) v.s ESAW (pH 11.5)  198.0  170.4–225.6  0.001 
Electrolyzed weak acid water (ESAlW) (pH 11.5)
ESAlW (pH 11.5) v.s ESAW (pH 2.5)  48.8  21.2–76.3  0.001 
ESAlW (pH 11.5) v.s EWAW (pH 6.0)  −128.7  (−)156.2–(−)101.1  0.001 
ESAlW (pH 11.5) v.s ENW (pH 7.0)  −204.9  (−)232.5–(−)177.3  0.001 
ESAlW (pH 11.5) v.s EWAlW (pH 8.5)  −198.0  (−)225.6–(−)170.4  0.001 
Discussion

Electrolyze water produces strong and weak acid and base solutions.13 PH levels Electrolyzed neutral water is located at 7 to 8. Electrolyzed strong acid water has a pH value of 3 to 2, while electrolyzed strong alkaline water has a pH value of 10 to 13. pH values between 5.0 to 6.5 and 8.0 to 10 each is slightly acidic and slightly alkaline Electrolyzed water has strong bactericidal, fungicidal and virucidal effects in various fields such as medicine.14 Determination of in vitro microbiology whether antibacterial agents are bactericidal or bacteriostatic is influenced by growth conditions, bacterial density, duration of test, and degree of decrease in bacterial count. antibacterial is better described as potentially bactericidal and bacteriostatic.15 In this study ESAW (pH 2.5) had a better bactericidal effect from the first 2h to 72h after the incubation of proteus mirabilis, Eschericia coli, proteus vulgaris, alkaligenes faecalis and enterobacter compared to EWAW (pH 6.0), ENW (pH 7.0), EWAlW (pH 8.5), and ESAlW (pH 11.5). The bactericidal potential possessed by ESAW (pH 2.5) in this study was due to the presence of low pH levels, ORP and residual chlorine. Although EW's bactericidal activity and its mechanism of action are still not fully understood,14 some scientists consider the presence of chlorine in EW as the main factor responsible for bactericidal activity, while others consider ORP to be a major factor.16 Chlorine concentration (Cl2), ORP and pH can affect the bactericidal effectiveness of EW.17 Low pH is known to be responsible for decreasing bacterial production and making bacterial cells more susceptible to dynamic chlorine.19 Bacterial cell inactivation occurs because of a high ORP (1150mV) in acid EW, causing oxidation on the cell surface, damaging various cell layers, and disrupting the metabolic pathway in the cell. In principle, low pH and high ORP in EW Acid acts synergistically with chlorine in reactivating microorganisms.20

The results of this study are in line with previous study which confirmed electrolyzed water acid (pH 2.6) has a disinfecting effect in reducing bacteria,21 thus electrolyzed water acid is effective in reducing bacteria,22 and has a higher bactericidal effect than EW pH 5.6–5.7 and pH 8.23 In addition, the bacterial colonies in EWAW (pH 6.0) is lower than ESAW (pH 2.5) and ESAlW (pH 11.5). Nevertheless, the bactericidal effect possessed by EWAW (pH 6.0) from the first 2h to 72h after the bacteria was incubated is still better than ENW (pH 7.0) and EWAlW (pH 8.5). Active chlorine in the form of hypochlorous acid (HOCl), which dominates when the pH of the solution is 5.0–6.5. HOCl is dissociated with hypochlorite ions (OCl) at high pH or chlorine gas (Cl2) at low ph.18 This study are in line with previous study that slightly electrolyzed water acid (pH 6.0–6.5) was effective in deactivating Eschericia coli and Staphylococcus aureus.24 One study also reported that slightly acidic electrolyzed water pH range 5.6–5.7 significantly has a bactericidal effect in reducing Eschericia coli.23 Using a slightly acidic electrolyzed water (pH 6.3) has a bactericidal effect in deactivating bacteria25

Electrolyzed neutral water and electrolyzed weak alkaline water in this study have the same lower bactericidal effect on the amount of bacterial colonization compared to other EW types. This is because the decrease in the average number of colonies in these two types of EW is not significant. The optimal pH level for bacterial growth is 4–9.18,26 The ability to deactivate all organisms decreases at pH 9.14

Electrolyzed strong alkaline water (pH 11.5) also has a better bactericidal effect than EWAW (pH 6.0), ENW (pH 7.0), EWAlW (pH 8.5), but is no better than the bactericidal effect of ESAW (pH 2.5) because its bactericidal activity begins at 24h evaluation. The results of this study are consistent with previous finding, that alkaline EW (pH 11.3) has bacterial reduction ability.27

However, with a small sample size and study design, caution must be applied, as the findings might not be different in clinical study, particularly potential negative effect on wound tissue which need more investigation.

Conclusion

Electrolyzed strong acid water (pH 2.5) has a better bactericidal effect compared to other types of electrolyzed water. Electrolyzed weak acid water (pH 6.0) has a better bactericidal effect than EW pH 7 and EW pH 8.5 There is no difference in the effect of electrolyzed neutral water (pH 7.0) with electrolyzed weak alkaline water (pH 8.5) on the reduction of bacterial colonization and both have an effect bactericidal which is worse than other EW. Electrolyzed strong alkaline water (pH 11.5) has a better bactericidal effect than EW pH 6.0, EW pH 7.0 and EW pH 8.5.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

Current study granted from Ministry of Research and Technology of the Republic of Indonesia as the magister scholarship.

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Peer-review under responsibility of the scientific committee of the International Conference on Women and Societal Perspective on Quality of Life (WOSQUAL-2019). Full-text and the content of it is under responsibility of authors of the article.

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