Bacteriophage ɸPT1b-Based Hand Sanitizer Gel for Reducing Pathogenic Escherichia coli Infection

Abstract

Foodborne disease is caused by consuming pathogenic microorganism-contaminated food that generates poisoning. Escherichia coli is a bacterium that causes foodborne disease, which is neutralized using gel hand sanitizer containing a bacteriophage with hydroxypropyl methylcellulose (HPMC) and active glycerin ingredients. Phages are viruses that infect bacteria naturally. This study aims to examine the effect of HPMC and glycerin on the physical properties and activity of bacteriophage ɸPT1b-based hand sanitizer gel, as well as determining the optimum composition of the combination of HPMC and glycerin in the same. The results of the study shows that the HPMC and glycerin factors show a positive value for inhibitory response, with the HPMC factor showing the best results. The optimum formula results using Design Expert 12.0 software were 0.75% for HPMC and 7.5% for glycerin, while the values for viscosity, dispersal power, and inhibitory power were 32,500 dPas, 7,737 cm, and 1.300 cm, respectively. In conclusion, an increase in HPMC concentration affects the increment of the viscosity score and decreases spread response. However, the glycerin concentration increment reduces the viscosity score but raises the spread value.

Introduction

Consuming food contaminated by pathogenic microorganisms causes foodborne diseases, one of which being diarrhea. WHO data states that approximately 2.2 million people, many of them children in developing countries, are dying due to unhygienic food, with recent studies showing that the incidence and prevalence of diarrhea in Indonesia are at 3.5% and 7%, respectively. The areas reporting the highest incidence and prevalence of diarrhea in Indonesia are Papua (6.3%, 14.7%), South Sulawesi (5.2%, 10.2%), Aceh (5.0%, 9.3%), West Sulawesi (4.7%, 10.1%), and Central Sulawesi (4.4%, 8.8%). The first isolation of E. coli was performed by Theodor Escherich in 1885, using the stool of a small child [1]. E. coli is gram-negative, clinically meaningful, and most frequently found in coliform bacteria. Wild type E. coli can be terminated using antibiotics such as ampicillin and chloramphenicol; however, E. coli has a complex antigen structure of O, K, and H antigens [2].

Phages can kill the bacteria within hours and reduce bacterial infection within minutes, and recently, the application of phages used in biotechnology and medical domains has included rapid bacterial detection, disease diagnosis, bacteriophage vaccines, therapy, and biocontrol. In the lytic cycle, phages release their genetic material directly without integration with the host cell's genetics. It replicates independently and kills the host cell quickly—the penetration process aided by lysozyme in the bacteriophage’s tail [3].

Furthermore, phage application as a preharvest strategy, such as a barrier for animal diseases, minimizes pathogens in the gastrointestinal tract, thereby preventing the entry of pathogens into food supplies. Using phages is also an effective method for bacterial biocontrol through polysaccharide degradation, as phages meet the requirements because they are non-toxic, specific, and effective [4].

There are two kinds of hand sanitizer, alcohol-based and active-ingredients-based, such as chlorhexidine, chloroxylenol, hexachlorophene iodine quaternary, ammonium compounds, and triclosan. Alcohol, as an antimicrobial, is only short-acting. However, phages are viruses that infect bacteria naturally and can enhance antibiotic activity through phage-antibiotic synergy (PAS) [5]. Phages can survive in a free environment and infect pathogenic bacteria, and phages based on HPMC and glycerin can be easily used in the form of gel hand sanitizer. HPMC has the clear and neutral physical properties of a gel and can maintain its viscosity, while glycerin is a humectant material that effectively increases the ability to absorb water and maintain skin moisture [6, 7].

Materials and Methods

Rejuvenation of Escherichia coli

Pure isolates of E. coli O157:H7 was grown at the GeMBio Laboratory of Universitas Jember, where it was rejuvenated in nutrient agar in petri dishes and slant tubes. The streak method was used for both media, which were incubated at room temperature for 24 h [8].

Propagation of Phage ɸPT1b

The bacteriophages were collected from Narulita et al. [9], and the spot test method was used to perform the particle propagation. Meanwhile, the bacteria were isolated from the logarithmic phase (± 4 h incubation) with 300 µL of bacteria mixed with the top medium agar (0.8%). Using solidified agar, 5 µL of bacteriophage particles were tested by spot test and incubated for 24 h at 36^o C. Spot test results were watered with sodium chloride, magnesium sulfate, and a gelatin buffer (SM buffer) and re-incubated at 4^o C for 10 min. Finally, the supernatant was filtered through a 0.20 µm membrane.

Determination of Phage Titer

The bacteriophage titer was determined using quantification expressed in plaque-forming units (PFU/mL), and the method used was a plaque assay. Bacteriophage filtrate was diluted with a serial 10^–1 to 10^–6, then the phage titer was measured by calculating the number of clear zones [10]. The phage titer result was 1.25 × 10⁴ plaque-forming units (PFU/mL) (Fig. 1).

Fig. 1.

Determination of Phage Titer on Luria Bertani. A Spot Test; B Plaque Assay

Gel Formulation of Phage-Based Hand Sanitizer

The best formulation of gel recipes with the active bacteriophage ɸPT1b was obtained by using HPMC (gelling agent) and glycerin (humectant) in various concentrations (Table 1). Propyl and methylparaben were dissolved into glycerin and mixed with raised HPMC by adding 20 mL aquadest at ± 85ºC. Next, the remaining aquadest (80 mL) was gradually added at room temperature, before the final step, when phage ɸPT1b was added to the cold solution.

Table 1.

Ingredients of bacteriophage ɸPT1b hand sanitizer formula

Formula	HPMC	Glycerin	ɸPT1b	Methyl Paraben	Propyl Paraben	Aquadest
	(g)	(g)	(g)	(g)	(g)	(ml)
F1	0.75	5	0.02	.18	0.02	Ad 100
FA	1.75	5	0.02	.18	0.02	Ad 100
FB	0.75	7.5	0.02	.18	0.02	Ad 100
FAB	1.75	7.5	0.02	.18	0.02	Ad 100

Characterization of Bacteriophage ɸPT1b-Based Hand Sanitizer Gel

Bacteriophage ɸPT1b-based hand sanitizer gel was observed in physical appearance, especially in terms of its odor, color, and dosage form [11]. The acceptable pH range is 6–8 [12] based on normal skin pH (4.5–6.5) [13] or the suggested pH for skin of 4.8–8.0 (SNI 16–43,399-1996). Viscosity was measured by viscometer with an initial speed of 50 rpm [14], and the good viscosity gels were found to have a range of 2000–4000 cps [15].

Inhibition Test

The inhibition test aimed to determine the inhibition of bacteriophage ɸPT1b-based hand sanitizer gel against E. coli O157:H7 by employing a paper disc diffusion method using a LB (Luria–Bertani) medium. The inhibitory test results appeared after 24 h of incubation with the formation of a clear zone around the disc. The positive control contained bacteriophage ɸPT1b only, while the negative control was hand sanitizer gel with no bacteriophage [9].

Dispersal Test

This test aimed to ensure gel distribution during application. One-gram gel was weighed and placed in the middle of a scaled-round glass before another round glass or other transparent material was set on the gel with ballast poundage of up to 150 g for one minute. The diameter distribution to the end of the steps was examined. Sufficient gel spread was in 5–7 cm [14] range.

Determination of Optimum Formula

The optimum formula was determined through analyzing data using a factorial design, and the response factors for submitting were the viscosity, dispersion, and inhibition values. The two-factor mixture formula with factorial design was:

$$\text{Y}={\text{b}}_0+{\text{b}}_1\left({\text{X}}_1\right)+{\text{b}}_2\left({\text{X}}_2\right)+{\text{b}}_{12}\left({\text{X}}_1\right)\left({\text{X}}_2\right)$$ 1

with Y: Viscosity, dispersion, and inhibitory responses, X₁, X₂: Level factors (HPMC and glycerin), b₀, b₁, b₂, b₁₂: Coefficient calculated from the results of the experiment, b₀: Average results of all experiments, b₁, b₂, b₁₂: ∑XY/2ⁿ.

Data Analysis

The data analysis used ANOVA (analysis of variants) with specific LSD (least significant difference) tests by comparing the bacterial number on a single phage test with combination formulas. The significant difference revealed a p value of < 0.05 with a 95% confidence level [16].

Result

Figure 2 shows the results of the bacteriophage ɸPT1b-based gel formula with pH conditions of all formulas ranging from 6.5 to 7.5.

Fig. 2.

Gel Formula of Bacteriophage ɸPT1b F1; FA; FB, and FAB. F1 (formula with an HPMC factor of 0.75% and glycerin factor of 5%); FA (formula with a 1.75% HPMC factor and 5% glycerin); FB (formula with 0.75% HPMC factor and 7.5% glycerin); FAB (Formula with a 1.75% HPMC factor and 7.5% glycerin)

The results of the gel's viscosity test for skin application showed that F1 and FB best suit the viscosity range [Table 2] with a significant effect on the HPMC factor (p < 0.0001). However, the glycerin and interaction between the two factors present an insignificant effect (p = 0.9614). While the inhibition test result [Table 3] and (Fig. 3) determined that the data correlates with the better inhibition capability, HPMC and glycerin factors significantly affect the inhibitory response (p-value 0.0358) while the interaction of both factors has an insignificant effect (p-value of 0.0590).

Table 2.

Results of viscosity test of bacteriophage ɸPT1b Gel preparation

Replication	Viscosity (dPas)
	(1)	(A)	(B)	(AB)
1	34	220	32.5	230
2	33	250	32.5	230
3	32	220	32.5	230
Average	33 ± 0.81	230 ± 14.14	32.5 ± 0	230 ± 0

F1 (formula with an HPMC factor of 0.75% and glycerin factor of 5%); FA (formula with HPMC factor of 1.75% and glycerin of 5%), FB (formula with HPMC factor of 0.75% and glycerin of 7.5%), FAB (formula with HPMC factor 1.75% and glycerin 7.5%)

Table 3.

Results of inhibition test of bacteriophage ɸPT1b Gel

Replication	Inhibition Zone (cm)
	F1	FA	FB	FAB	N1	NA	NB	NAV	K+
1	0.77	0.98	1.36	1.38	0.50	0.6	0.47	0.73	0.4
2	0.75	1.00	1.24	1.2445	0.54	0.7	0.14	0.62	0.41
3	0.755	0.86	1.30	1.37	0.86	0.76	0.54	0.87	1.36
Average	0.76 ± 0.01	0.95 ± 0.06	1.30 ± 0.05	1.33 ± 0.06	0.63 ± 0.16	0.69 ± 0.07	0.38 ± 0.17	0.74 ± 0.10	0.72 ± 0.45

F1 (formula with an HPMC factor of 0.75% and glycerin factor of 5%); FA (formula with a 1.75% HPMC factor and 5% glycerin); FB (formula with 0.75% HPMC factor and 7.5% glycerin); FAB (formula with a 1.75% HPMC factor and 7.5% glycerin); N1 (F1 without bacteriophages); NA (FA without bacteriophages); NB (FB without bacteriophages); NAB (FAB without bacteriophages); and K + (PT1b bacteriophage in the sodium chloride, magnesium sulfate, and gelatin buffer (SM buffer)

Fig. 3.

Test Results of Inhibitory Test of Bacteriophage ɸPT1b Gel. F1 (low level); FA (dominant HPMC); FB (dominant glycerin); FAB (interaction of dominant A and B factors); N1 (F1 without bacteriophage); NA (FA without bacteriophage); NB (FB without bacteriophage); NAB (FAB without bacteriophages); and K + (PT1b bacteriophages in SM buffer)

The dispersion of gel bacteriophage ɸPT1b hand sanitizer results [Table 4] indicates that the combination factors of HPMC and glycerin show insignificant effects on the scatter efficacy response (p-value of 0.2936).

Table 4.

Results for bacteriophage ɸPT1b Gel spreads

Replication	Spread of bacteriophage gel (cm)
	F1	FA	FB	FAB
1	7,355	4,845	7.51	5,16
2	7,61	4.73	7.52	5,22
3	7.48	4.72	8.18	5.01
Average	7.48 ± 0.10	4.77 ± 0.06	7.74 ± 0.31	5.13 ± 0.09

F1 (formula with an HPMC factor of 0.75% and glycerin factor of 5%); FA (formula with HPMC factor of 1.75% and glycerin of 5%); FB (formula with HPMC factor of 0.75% and glycerin 7.5%); and FAB (formula with HPMC factor 1.75% and glycerin 7.5%)

HPMC or glycerin alone has a significant effect with a p-value of 0.0178. An overlay graph was used to determine the optimum area of the phage ɸPT1b gel formula (Fig. 4).

Fig. 4.

Overlay plot of optimum formula

The yellow area indicates the optimum area, which means the HPMC and glycerin factors meet the criteria. The three best formulas are presented in Table 5 with the highest desirability value of 0.940 [17] as the optimum formula.

Table 5.

Solutions offered by factorial designs

No.	(HPMC)	Glycerin	Viscosity	Scattering	Inhibition	Desirability
1	0750	7,500	32,500	7,737	1,300	0.940	Selected
2	0.756	7,500	33,708	7,722	1,300	0.938
3	0828	7,500	47,850	7,550	1,302	0.923

Discussion

Hand sanitizer is an antimicrobial product that is popular in the community. Bacteriophages can be used as active ingredients when making hand sanitizers and are viruses that infect bacteria. They can also help reduce the resistance of pathogenic bacteria to existing antibiotics and can survive in a free environment for a long time. Optimization of hand sanitizer gel formulations with active bacteriophage ingredients with an HPMC base and glycerin humectants is very important because its application on the skin is only useful for a certain period.

The organoleptic describes the physical properties of the bacteriophage ɸPT1b hand sanitizer gel. As a gelling agent, the HPMC factor mainly affected the gel form and color intensity [18]. The optimized result shows that a greater concentration of the HPMC factor causes higher viscosity and a slight color difference in preparation. The pH test aimed to ensure the safety of the bacteriophage ɸPT1b gel with an expected pH range of 4.5–6.5, the normal pH of the skin [13]. SNI 16–43,399-1996 specified a pH range that can exist on the skin, which ranges from 4.8 to 8.0. The pH test results of gel preparations F1, FA, FB, and FAB were within the 6.5–7.05 range [19], and therefore safe for skin application. Meanwhile, phages can exist in neutral pH 6–8 in either wet or dry conditions. Thus, four different formulas of gel hand sanitizer preparation suit the storage of phages for an extended period [20].

Viscosity is another essential physical property of gel and is measured by the Brookfield viscometer. The preparations F1 and FA used spindle number 1, while FB and FAB use spindle number 2 because they are physically different. The expected viscosity range was 2000–4000 cps [16] with the viscosity value increasing following the HPMC increment. However, the increased glycerin concentration decreased the viscosity of the gel preparation. Nevertheless, the interaction of both factors produced a proper viscosity. The HPMC had a dominant effect on the viscosity shown [Table 3] at 1.75% HPMC, and 5% glycerin showed the highest average viscosity value of 230 dPas.

The viscosity results are vital as viscosity affects the dispersal response of the gel's active substances, as an optimum viscosity can hold the active substance and facilitate its application [21]. The HPMC as a gelling agent enriches the polymer fibers to bind more liquid, causing the viscosity to increment. Glycerin concentration also contributed to water binding and increased molecular units. As this molecular unit increases, it increases resistance to flow and spread. In other words, a higher viscosity value will reduce the spreadability value.

The material contents (HPMC and glycerin) only affect the physical properties in terms of the convenience of application. The transparent zone formation in the medium is due to the active ingredient bacteriophage ɸPT1b [Fig. 4]. Methylparaben and propylparaben have an antibacterial property that inhibits growth in the negative control [22], and this paraben is pharmaceutically used as a preservative to avoid microorganism contamination. In this study, the concentrations of methylparaben and propylparaben at the lowest possible levels were 0.015% and 0.01%, respectively [23], and combining the two parabens increased the preservative effectiveness by inhibiting the growth of mold and fungus so that the gel preparation was available for long-lasting storage.

All formulas of F (A, B, AB) and N (A, B, AB) demonstrated an inhibition effect due to the addition of the phage's active ingredients. The measurement of preparation dispersion used two round glasses with 150-g ballast, and the best spread range was 5–7 cm. The positive values showed an increasing effect, while negative values demonstrated a lowering effect. The increase of HPMC factors caused the reduction of the dispersion of the gel phage ɸPT1b, and the glycerin factor showed a positive effect by increasing the spread response. The interaction value of the two factors showed a positive value, which means the gel spreadability was increased. The dispersion test determined the adequate preparations that spread well and were safely applied, and greater spreading power ensures the active substance spreads broadly on the skin surface [14].

The yellow section indicates the optimum area where HPMC and glycerin factors meet the definitive viscosity, inhibition, and dispersal power. The most optimal formula offered was the FB formula with 0.75% of HPMC and 7.5% of glycerin [Table 5], which produced viscosity response, inhibition, and dispersal values of 32,500, 1300, and 7.737, respectively. The highest desirability value (close to 1) of 0.940 indicates that FB meets 94.0% of the predetermined response criteria [17].

A physical stability test was applied to ensure the physical quality of the preparation during storage. The characteristics of physical instability in the gel preparation include organoleptic changes, i.e., changes in color, odor, phase separation, consistency, formation of gases, and other physical changes [24]. The stability test used the freeze and thaw methods over five cycles, which determined whether the preparation underwent physical changes after being stored at different temperatures (4 °C and 35 °C). There were no changes observed in the physical appearance of the shape, color, and smell of the phage ɸPt1b-based hand sanitizer gel over the five cycles, demonstrating that the gel was organoleptically stable [25]. The freeze–thaw test also affected the viscosity responses, which initially increased in cycle two, decreased in cycles three and four, and then increased again in cycle five; these changes were due to the storage temperature. The viscosity changes indicated the instability of the preparation during storage. Viscosity theory explains that tilapia has a significant viscosity at low temperatures, and a small viscosity value at high temperatures [26], showing that Viscosity is inversely proportional to temperature. The higher the temperature, the faster the fluid particles move, and therefore the viscosity decreases. Meanwhile, extreme temperature also affects the dispersal responses, as the higher the viscosity value, the lower the dispersibility of the gel preparation. Based on the freeze–thaw results, the dispersal value changes dynamically over the five cycles, with the viscosity changes caused by the gel’s unstable dispersibility.

The pH measurement aimed to determine whether the pH of the preparation was under the skin's pH range as an unsuitable pH would cause skin irritation. Based on the observation results, the pH was categorized as unstable yet within the normal range. The correlation between temperature and pH showed a positive relationship but almost no effect, while the data showed that pH increases according to the temperature increases [27].

Conclusion

Increasing the HPMC concentration factor will increase the value of the viscosity response and decrease the value of the spreadability response, while increasing the concentration of the glycerin factor decreases the value of the viscosity response and increases the value of the spreadability response. The interaction of the two showed a positive effect on the viscosity response and spreadability, but the value was relatively small and therefore the effect was also relatively small. An increase in the HPMC factor produced a positive value, which means it increased the response to inhibition, while an increase in the glycerin factor also produced a positive value to the response to inhibition, although the value was relatively small compared to the that of the HPMC factor. The interaction of the two produced a negative value to the inhibition response, which means that the value of the inhibition response decreases. The optimal formula was formula (B) which had an HPMC concentration of 0.75% and glycerin of 7.5%, producing a viscosity response value of 32,500 dPas, a dispersal power value of 7,737 cm, and inhibitory power of 1.300 cm. The optimum formula chosen was the one that has the highest desirability value of 0.940, indicating that FB meets 94.0% of the predetermined response criteria.

Declarations

Conflict of interest

The authors report no conflicts of interest.

Ethical Approval

This article does not contain any studies performed by any of the authors involving animals or human participants.