Highly Efficiently Inactivation of Microbial Pathogensusing Advanced Ozone Generator Unit as An Eco- Friendly Promising Strategy

Research Article | DOI: https://doi.org/10.31579/2688-7517/208

Highly Efficiently Inactivation of Microbial Pathogensusing Advanced Ozone Generator Unit as An Eco- Friendly Promising Strategy

  • Khalifa Ali 1*
  • Mohammed G. Barghoth 1
  • Said E. Desouky 1
  • Hanaa H. Elsayed 2
  • Mohamed Roushdy 1

1 Department of Botany and Microbiology, Faculty of Science (Boys), Al-Azhar University, 11884 Nasr, Cairo, Egypt.

2 Department of Food Chemistry and Metabolism, National Nutrition Institute, 16 Elkaser Alainy St. Cairo, Egypt.

*Corresponding Author: Khalifa Ali, Department of Botany and Microbiology, Faculty of Science (Boys), Al-Azhar University, 11884 Nasr, Cairo, Egypt.

Citation: Khalifa Ali, Mohammed G. Barghoth, Said E. Desouky, Hanaa H. Elsayed, Gleisy Kelly N. Gonçalves, (2024), Highly Efficiently Inactivation of Microbial Pathogensusing Advanced Ozone Generator Unit as An Eco- Friendly Promising Strategy, J. Pharmaceutics and Pharmacology Research, 7(9); DOI:10.31579/2688-7517/208

Copyright: © 2024, Khalifa Ali. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: 17 May 2024 | Accepted: 04 June 2024 | Published: 02 September 2024

Keywords: ozone treatment; microbial elimination; s. aureus; b. subtilis; b.spizizenii; e. coli; p.aeroginosa; c. albicans

Abstract

Searching for an alternative disinfection and sanitization strategy to control and prevent the contamination and diseases caused by microbial pathogens represents one of the critical challenges for all world governments. So that the antimicrobial efficiency of ozone gas as a terminal disinfectant was estimated at a relatively small level (1.2 mg/l/h) using a unit that was generated as a local unit assembled at the faculty of science against six reference strains including Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 9372), Bacillus spizizenii (ATCC 6633), Escherichia coli (ATCC 8739), Pseudomonas aeroginosa (ATCC 9027), and Candida albicans (ATCC 10231) for 10, 20, 30 and 40 minutes under laboratory conditions. After 10 min of ozone treatment, the log reduction of cell viability was 97.15%, 59.25%, 24.20%, 24.09%, 14.50 %, 13.47%, and 0.46% for P. aeroginosa, strains combination, E. coli, C. albicans, B. spizizenii, B. subtilis, and S. aureus, respectively. The twenty-minute exposure to ozone resulted in a reduction in microbial viability percent 98.17%, 82.88%, 69.63%, 62.79%, 49.43%, 29.57%, and 28.08%, for P. aeroginosa, strains combination, B. subtilis, and E. coli, C. albicans, S. aureus, and B. spizizenii, respectively. The efficacy of ozone for P. aeroginosa, E. coli, and strains combination increased by more than 98% after 30 min of ozone treatment followed by 90.41%, 86.76%, 52.63%, and 36.64% for B. subtilis, C. albicans, B. spizizenii, and S. aureus, respectively. The maximum ozone efficacy reached 100% for all reference stains except B. spizizenii (62.10%) after 40 min of ozone treatment making this strategy a candidate tool recommended for the management and control of the pathogenic microorganisms.

Introduction

Effective sanitation and cleaning of surgical materialsand wastes as well as food manufacturing equipment are major challenges in the medical and industrial fields. Due to the inappropriate cleaning method, the contamination by pathogenic microorganisms increased dramatically during the last decades raisingsubstantial public healthconcerns Silva et al., (2021).Disinfection used in the medical and industrial fields has typically been accomplished using heat, such as hot wateror steam, or traditional chemicals, such as chlorinethat are completely dissolved in water and easy to use at low cost. Unfortunately, not only are there growing environmental implications about the presenceof chemical by-products such as halo-organics created when liquidchemicals are employedas disinfectants but there are also developing public health problemssuch as respiratory tract and skin irritation (Alvaro et al., 2009; Selma et al., 2008; Shen et al., 2012).

The kind of disinfecting agent for using is determined by the nature of microorganisms to be destroyed as well as the quality of the cleaningequipment. If the sanitizing agent is ineffective, the microorganisms multiply and accumulate again on the equipment surfaces (Dosti et al., 2005). Among the new disinfectant strategies, ozone (O3) appearsto be a viable methodfor preventing microbiological contamination by spoilageor microbial pathogens. Ozone, in fact,has a significant antimicrobial activity and is considered an environmentally beneficial technology because of its low environmental impact (Bigi et al., 2021).

Ozone is widely applied in several applications as a disinfectant including; treatment of food plant wastes, reuse of waste water, sterilization of food plant equipment’s, food surface hygiene and dairy industry (Graham, 1997; Panebianco et al., 2022). Ozone is the second strongest oxidizing agent after fluorine (Miller et al., 1978). Because of its high oxidation capability, ozone is particularly effective at killing microorganisms. Ozone exhibited biocidal effects on a wide range of species, including Gram- positive and Gram-negative bacteria, spores, and vegetative cells (Guzel-Seydim et al., 2004; Lezcanoetal., 1999). Ozone has also been shown to kill a variety of viruses, including the hepatitis A, influenza A, and others (Dosti et al., 2005).

For practicaluses of ozone, different generation techniques are available, with the electrically andphotocatalytic (UV) techniques being the most widespread for instance, in the electric corona discharge process, in which the oxygen molecules pass through an electrical field between two electrodes and split (Brodowska et al., 2018). Ozone can also be produced by ultraviolet radiation, which involves the passing of oxygen gas molecules through a high-energy short-wave UV light (Baggio et al., 2020).

Chemical, thermal,chemo-nuclear, and electrolytic processes are among the othermethods of ozone generating (Varga& Szigeti2016).

Based on the Environmental Protection Agency, ozone is more effective than chemicals in the disinfection process because it has a faster reaction time and does not allow bacteria to regrowth again. The residual ozone breaks down rapidly into oxygen and therefore does not pose a real threat (EPA, 1999). Alsono toxic by- productsare found during the ozone treatment process, in comparison to the generation of disinfection by-products (DBP) during using chemicals such as chlorination, which may be mutagenic and carcinogenic. Moreover, the resistances of some organisms to disinfectants, as well as the demandfor considerably increased doses for microbial inactivation, are two of the most critical problemsin disinfection (Gomes et al., 2019; Greene et al., 1993).

Thus, the purpose of this investigation is to evaluate the action of direct application of ozone gas generated through advanced units against several bacterial species and yeast actingas pathogenic and spoilage organismsincluding; S. aureus,

B. subtilis, B. spizizenii, E. coli, P. aeroginosa, and C. albicans. Also, to evaluatethe optimal time and the efficacyof ozone gas required to attenuate or control the growth of these pathogens.

  1. Materials and methods Ozone generation unit

Ozone was generated during this study based on plasma technology through a new design reactor called Dielectric Barrier Discharge plasma reactor (DBD plasma reactor). In this unit, plasma is produced over the dielectric barrier's surfaceand along the perimeter of the top electrode that is exposedto the surrounding air as represented in Figure. [1].

This device was designed and manufactured by Prof. Dr. Safwat Hasaballah at the Faculty of Science, Al-Azhar University. As follows, the two copper electrodes were placed around the outer plastic tube and the inner electrode of the dielectric material (composed of hydrocarbon/ceramic RO4350B™).The thickness of the outer Cu+2 wire

electrode is 0.2 mm and the length of the inner metal electrode is 60 cm. An alternating potentialdifference between the two electrodes is used to create DBD plasmaaround the outer Cu+2 wire electrode that is connected to the higher potential (0–10 kv, 50Hz) and exposed to air. Plasma does not form on the bottom side of the reactor because the inner electrode is grounded and covered by a layer of Kapton tape (Portugal et al., 2017; Choudhury et al., 2018).

1.2 Culture preparation Test organisms

Reference strainsthat were used during this study includedGram- positive bacteriasuchas Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 9372,and Bacillus spizizenii ATCC6633 as well as Gram-negative bacteria represented in Escherichia coli ATCC 8739, and Pseudomonas aeroginosa ATCC 9027 while unicellular yeast represented in Candida albicans ATCC 10231. Bacterial strains were seeded on nutrient agar plates and incubated for 24 h at 37 °C while C. albicans was cultured onSabouraud dextrose agar at 30 °C for 24 h.

Separate colony of each referencestrains was transferred from the agar medium to 300 mL of the Muller Hinton broth and allowed to multiply at 37°C for 24 hours. Cells cultured in Muller Hinton brothmedium were collected at room temperature using centrifugation at 3000 ×g for 10 min. The supernatantwas removed and the cells were harvested and re-suspended in the phosphate-buffered saline (PBS; 0.07M, pH 7.0) until the bacterial inoculum density was equal to 2 McFarland (2MF) approximately 6 x 108 cells/ml when measured spectrophotometrically at absorbance 620 nm (CLSI, 2018; Sowhini et al.,2020). TwoMcFarland standard was prepared by mixing 0.2 mL of

1.175 percent barium chloridedihydrate (BaCl2.2H2O) with 9.8 mL of 1 percent sulfuricacid (H2SO4) (Zapata& Ramirez-Arcos,2015).

1.3 Experimental set-up

Ozone createdby the plasma reactor was transferred to a beakercontaining sterile broth media and inoculated with 2 McFarland (6 x 108 CFU/ mL) of the reference test organisms. At pH 6–8 and room temperature 20–25 oC, 50 ml of bacterial suspension was exposed to 1.2 mg/l/h of ozone dosage for interval times ranging from 10 to 40 minutes. The experiments were conducted for each strain alone and in combination for all testorganisms with each other. After each test was measured, 180 µL of ozonated bacterial suspension was kept in sterile Eppendorf tubes until the end of the experiments César et al.,(2012).

The collected Eppendorf tubes were incubated for 30 min at 37 oC to make sure that the ozone was completely consumed before adding the indicator dye (resazurin). Then 20 µL of resazurinsolution was added to each Eppendorf tube and incubated for 4 h at 37oC. All experiments were conducted in triplicate for each test organism Morganet al., (2009).

1.4 Bio-indicator dye preparation

Resazurin solution (7-Hydroxy-3H-phe-noxazin-3-one 10-oxide) was prepared by dissolving 337.5 mg of this dye in 50 mL of sterile distilled water and homogeneity obtained by mixing for 1 hour in a sterile vortex mixer. The preparation processes werecarried out in the dark, and the resazurin solutionwas maintained in a dark bottle to avoid light exposure (Teh et al., 2013). The color of resazurin dye was converted from blue topink or red fluorescent material (resorufin) by oxidoreductase within live bacterial cells that were used to determine the antibacterial impact of ozone. The change in resazurin color was measured at 570 nm and 600 nm (Pettit et al., 2005; Morgan et al., 2009).

1.5 Statistical analysis

Data analysiswas performed using ANOVA and the Tukey test after converting the CFU/ml countsto a logarithmic. A statistically significant difference was defined as a P value of less than 0.05. Bycomparing the groups that were sterilized with ozonated broth media for 10, 20, 30, and 40 minutes to the control group, the hundredpercent reductions in CFU/mL for each microorganism investigated was calculated (César et al., 2012). Two control groups were used during this study firstly negative group: contained 180 µL of sterile nutrient broth media and 20 µL indicator, without a test organism in which the color of dye remained blue even at the end of the experiment. Secondlypositive group: contained180 µL of inoculated nutrient broth media and a 20 µL indicator of 180 µL without exposure to ozone in which the color of the dye was changed to pink at the end of the experiment.

1.6 Results and Discussion

Managing and controlling microbialcontamination are represented as one of the main challenges in all life aspects, especially in the medicalfield. Maintaining a suitablehygienic environment is critical to prevent the dispersion and contamination formed by microbial pathogens. Also searching for advanced strategies to preventmicrobial contamination or pathogenic bacteriarepresents one of the largestchallenges facing all industrial and medical branches in the following upcoming years. Therefore, newly ozone designed unit was used to generate ozone gas with a low dosage of 1.2 mg/l/h in an aqueous solution.

The antimicrobial action of generated ozone was studied on six microbial reference strains including Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 9372), Bacillus spizizenii (ATCC 6633), Escherichia coli (ATCC 8739), Pseudomonas aeroginosa (ATCC 9027), and Candida albicans (ATCC 10231) for 10, 20, 30 and 40 minutesas represented in Table 1. The initial microbialconcentration was adjustedat 2 MF approximately equal to 6 x 108 CFU/ ml or 8.76 ±0.02 log number of cells.

For E. coli (ATCC 8739), there are statistically significant differences observed after ozone exposurefor 10 min up to 40 min if comparedwith the controlgroup. The obtained results showed that the viability of E. coli cells gradually decreased after 20 min until disappeared of bacterial cells, this indicated that the activityof ozone is based actuallyon the exposure time duration.A significant effect

of ozone at a concentration of 2 mg/l/h was used to remove E. coli cells in biofilms on lettucesurfaces as reportedby Ölmez & Temur (2010). Moreover, the effective inactivation of E. coli O157:H7 by ozonated water dose 35 and 45 mg L-1 for 0, 5, 15, and 25 min was studied by Souza et al. (2019). Also, Mohammadet al. (2019) demonstrated that the mean log reductions of Shiga toxin-producing E. coli when treated with 5 mg/l/h of aqueous ozone after 10, 15, and 20 min, were found to be 1.5 ± 0.4, 1.6 ± 0.4, and 2.1 ± 0.5, respectively.

The same results were obtained for Pseudomonas aeroginosa (ATCC 9027) except that the log- transformed CFU/ml was rapidly full down during the first 10 min of ozone exposure from log 8.76±0.02 to 0.25±0.02 and these results indicated that the highest effectiveness of ozone on P. aeroginosa occurred at short exposure time [Table 1]. These results are full consistence with data reported by Dosti et al. (2005) they found that the largest log reduction of P. fragi (ATCC 4973), P. putida (ATCC 795), and P. fluorescens (ATCC 948) was produced after the 10 min of ozonation process. Our observation is greater than the results observed by Marino et al. (2018) who studied the effectiveness of ozonated water instatic and dynamic conditions to demonstrate the sensitivity of P. fluorescens to ozone treatments. After 60 minutesof treatment, the authors found that the gaseous ozone at 20 ppm reducedthe number of P. fluorescens by 5.51 log CFU/cm2 (Marino et al., 2018).

On the other hand, no statistically significant differences were observed by

S. aureus (ATCC 6538) after 10 min of exposure to gaseous ozone this may be due to the bacterial resistance or the duration of ozone dose not being enough to reduce the number of bacterial cells. The higher reduction of S. aureus cells was obtained after 30 min of ozone exposureand this confirmedthat the reduction of bacterial cell numbers was dependent on the ozone exposure duration and this indicated that the greater log reduction was obtained after 30 min of treatment than 10 and 20 min [Table 1]

Table 1 Effect of ozone on reference strains suspended in broth culture at different experimental times.
Reference StrainsGroupsMean ± SD.MinimumMedianMaximum(p)
E. coli (ATCC 8739)Control8.76a ±0.028.748.768.78(<0>
10 min6.64b ±0.096.536.676.71 
20 min3.26c ±0.752.403.593.78 
30 min0.06d ±0.010.050.060.07 
40 min0.0d ±0.010.00.00.01 
S. aureus (ATCC 6538)Control8.76a ±0.028.748.768.78(<0>
10 min8.72a ±0.018.718.728.73 
20 min6.17b ±0.106.076.176.27 
30 min5.55c ±0.045.525.535.60 
40 min0.03d ±0.010.020.030.04 
P. aeroginosa (ATCC 9027)Control8.76a ±0.028.748.768.78(<0>
10 min0.25b ±0.020.230.250.27 
20 min0.16c ±0.030.130.170.19 
30 min0.03d ±0.030.010.020.07 
40 min0.0d ±0.00.00.00.0 
C. albicans (ATCC 10231)Control8.76a ±0.018.758.768.77(<0>
10 min6.65b ±0.036.636.656.68 
20 min4.43c ±0.024.414.434.45 
30 min1.16d ±0.051.111.151.21 
40 min0.21e ±0.020.190.200.23 
B. spizizenii (ATCC 6633)Control8.76a ±0.018.758.768.77(<0>
10 min7.49b ±0.017.487.497.50 
20 min6.30c ±0.016.296.306.31 
30 min4.15d ±0.034.124.154.17 
40 min3.32e ±0.423.013.143.80 
B. subtilis (ATCC 9372)Control8.76a ±0.028.748.758.78(<0>
10 min7.58b ±0.027.567.597.60 
20 min2.66c ±0.052.622.642.71 
30 min0.84d ±0.050.790.840.89 
40 min0.0e ±0.00.00.00.0 
Combination of all referencestrainsControl8.76a ±0.028.748.758.78(<0>
10 min3.57b ±0.053.513.593.60 
20 min1.50c ±0.011.491.501.51 
30 min0.11d ±0.020.090.110.13 
40 min0.0e ±0.00.00.00.0 
SD: Standarddeviation, means in the same column with small commonletters are not significant at p ≤ 0.05 (i.e., Means with Different letters are significant) 

S. aureus was highly sensitive to ozonation treatments under dynamic conditions, according to Marino et al. (2018), while contact with gaseous ozone at high doses (20 ppm) resulted in a lowering of 4.72 Log CFU/cm2 of S. aureus biofilm.

Genus Bacillus is one of the most significant spore-forming bacteria, with considerable variety and adaptation to many environmental conditions, as well as the capacity to attach and survive on a variety of surfaces and the ability to producesome biofilm forms(Shemesh & Ostrov, 2020).They are easilycapable of formingresistant spores that can surviveafter the conventional cleaning methods that increased the additional disinfectant compounds and raised the cost during the lastfew yearsto manage the microbial contamination formed by spoilage or pathogenic microorganisms (Panebianco etal., 2022).

The bactericidal action of gaseous ozone on Bacillus subtilis (ATCC 9372) and Bacillus spizizenii (ATCC6633) demonstrated that lowering reductionin log- transformed CFU/ml 7.58 ±0.02 and 7.49 ±0.01, respectively after the first 10 min of ozone exposure comparing in Contrast to the control group 8.76 ±0.02. Both strains showed some resistance toward ozone gas for the first 30 min of contact with ozone followed by a quick fall down in the bacterial cell numbers and this was revealed primarily by B. subtilis thenB. spizizenii and this may be due to the presenceof sporulation character of Bacillus genus and hence it required for prolongation of ozone gas exposure also the results indicated that the 30 min of ozone treatment is effective sanitization method to destroy these pathogens

All of the treatments after 10 min of Bacillus exposure differ substantially from the controls. The presence of spores may decrease ozone's efficiency. Ozone penetration is likely prevented by the multilayer structure of spores' outer coat. Bacillus cereus and Bacillus megaterium spores were found to be 10–15 times more resistant to ozone than their vegetative cells as determined by Broadwater et al. (1973). Ozone did not considerably reduce the number of Bacillus subtilis spores, according to the data recorded by Herbold et al. (1989). The same results were obtained by Dosti et al. (2005) who noted that the response of B. licheniformis, was found after 10-min of ozonation treatment. Also, the effect of ozonated water on B. cereus biofilms was evaluated by Babu et al. (2016) on dairy processing membranes and they found that theaverage reduction in treat membranesis 1.0 log CFU/cm2.

The antimicrobial nature of ozone on Candida albicans (ATCC 10231) showed a statistically significant difference relative to the controlgroup after 10 min of exposure (6.65±0.03) and the largest reduction of Candida cells obtained after passing 20 min of ozone exposure. The effect of ozonated solution against the combination of all reference strains showed the reduction of microbial quantitiesfrom 8.76± 0.02 to 3.57±0.05 during the first ten minutes. The complete microbial reduction obtained after 30 min of exposure indicated the higher effectiveness of ozone against the collection of several strains of microbial pathogens.

The efficacy of ozone againstthe reference strains based on the mean logarithmic reduction concerning the control group after 10 min of exposure to 1.2 mg/l/h of ozone dose was found to be 97.15%, 59.25%, 24.20%, 24.09%, 14.50 %, 13.47%, and 0.46% for P. aeroginosa, strains combination, E. coli, C. albicans, B. spizizenii, B. subtilis, and S. aureus, respectively and this indicated that the largest reduction percent was observed by P. aeroginosa while the smallest was observed by S. aureus[Fig. 2]. 

Figure 2: the efficacy of ozone treatment against the reference strains after 10 min of exposure.

Whereas the highest reductionpercent achieve after 20 min of ozone treatment was 98.17%, 82.88%, 69.63%, 62.79%, for P. aeroginosa, strains combination, B. subtilis, and E. coli, respectively, and the smallest was found at 49.43%,29.57%, and 28.08%,for C. albicansS. aureus,and B. spizizenii, respectively [Figure. 3].

Figure. 3 the efficacy of ozone treatment against the reference strains after 20 min of exposure.

After passing 30 min of ozone treatment the efficacy of ozone toward reference strainsincreased by more than 99% for P. aeroginosa and E. coli andmore than 98% for strainscombination followed by 90.41%, 86.76%,52.63%, and 36.64% for B. subtilis, C. albicans, B. spizizenii, and S. aureus, respectively [Figure. 4].

Figure 4: the efficacy of ozone treatment against the reference strains after 30 min of exposure.

 Furthermore, the efficacy of ozone reachedthe maximum reductionyield (100%) for all reference stainsexcept B. spizizenii (62.10%) after exposure to ozone for 40 min [Figure. 5].

As a result, the net log reduction in the cell viability of the reference strains was found to be largest after 30min for P. aeroginosa and E. coli and 40 min for the other strains.

Recently ozone has an expansiveantimicrobial range, and each microorganism speciesis responsive to the gas in a different manner.Control of fungi,Gram-negative and Gram-positive bacteria, and viruses have all been investigated using the ozonationprocess (Alwi & Ali 2014; Khadreet al., 2001). Microorganisms are inactivated by ozone throughthe rupture of the cellularmembrane and subsequent dispersion of internal cytoplasmic components due to the higher oxidative potential of thisgas. Ozone interacted with the cellular component such as unsaturated fatty acids, nucleic acids, cell wall constituent’s enzymes, and other proteins (Sanchez et al., 2016). Based on Manousaridis et al.'s observation, the antimicrobial effect of ozone in aqueous media is attributed to their dissociation into molecular ozone, superoxide, and hydroxyl, as well as hydroperoxyl radicals (Manousaridis et al., 2005). However, in the presenceof moisture, the harmful effectsof ozone are hypothesized to be enhanceddue to the increase of these radicals’production

(Kowalski et al., 1998).

Our findings indicate that Gram-negative bacteria are more susceptible to gaseous ozone than Gram-positive bacteria similar to the observation reported by Moore et al. (2000) who concluded that the Gram-negative bacteria were significantly moresensitive than the Gram-positive bacteria than the yeast strain after being exposed to 

1.7 Conclusion

Throughout this study, comparatively small concentrations of gaseous ozone exhibited good antimicrobial capabilities when applied to pathogenic microorganisms. Exposure of six reference strains included Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 9372), Bacillus spizizenii (ATCC 6633), Escherichiacoli (ATCC 8739), Pseudomonasaeroginosa (ATCC 9027), and Candida albicans (ATCC 10231) to ozone gas at the level of 1.2 mg/l/h for 40 min resulted in at least 62 to 100% reductionof the viability of the test organisms. The higher efficiency of the ozone strategy used in this study appeared well among the combination of the strains with 98% and 100% reduction of cell viability after 30 and 40 min, respectively. Gaseous ozone could be utilized as a successful disinfectant agent in different fields, according to the findings of this study, if applied after regular cleaning. Although it is recommended ozone as an effective tool used for traditional sanitation approaches replacement of chemical disinfectants.

References

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Mina Sherif Soliman Georgy

We would like to thank the Journal of Thoracic Disease and Cardiothoracic Surgery because of the services they provided us for our articles. The peer-review process was done in a very excellent time manner, and the opinions of the reviewers helped us to improve our manuscript further. The editorial office had an outstanding correspondence with us and guided us in many ways. During a hard time of the pandemic that is affecting every one of us tremendously, the editorial office helped us make everything easier for publishing scientific work. Hope for a more scientific relationship with your Journal.

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Layla Shojaie

The peer-review process which consisted high quality queries on the paper. I did answer six reviewers’ questions and comments before the paper was accepted. The support from the editorial office is excellent.

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Sing-yung Wu

Journal of Neuroscience and Neurological Surgery. I had the experience of publishing a research article recently. The whole process was simple from submission to publication. The reviewers made specific and valuable recommendations and corrections that improved the quality of my publication. I strongly recommend this Journal.

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Orlando Villarreal

Dr. Katarzyna Byczkowska My testimonial covering: "The peer review process is quick and effective. The support from the editorial office is very professional and friendly. Quality of the Clinical Cardiology and Cardiovascular Interventions is scientific and publishes ground-breaking research on cardiology that is useful for other professionals in the field.

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Katarzyna Byczkowska

Thank you most sincerely, with regard to the support you have given in relation to the reviewing process and the processing of my article entitled "Large Cell Neuroendocrine Carcinoma of The Prostate Gland: A Review and Update" for publication in your esteemed Journal, Journal of Cancer Research and Cellular Therapeutics". The editorial team has been very supportive.

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Anthony Kodzo-Grey Venyo

Testimony of Journal of Clinical Otorhinolaryngology: work with your Reviews has been a educational and constructive experience. The editorial office were very helpful and supportive. It was a pleasure to contribute to your Journal.

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Pedro Marques Gomes

Dr. Bernard Terkimbi Utoo, I am happy to publish my scientific work in Journal of Women Health Care and Issues (JWHCI). The manuscript submission was seamless and peer review process was top notch. I was amazed that 4 reviewers worked on the manuscript which made it a highly technical, standard and excellent quality paper. I appreciate the format and consideration for the APC as well as the speed of publication. It is my pleasure to continue with this scientific relationship with the esteem JWHCI.

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Bernard Terkimbi Utoo

This is an acknowledgment for peer reviewers, editorial board of Journal of Clinical Research and Reports. They show a lot of consideration for us as publishers for our research article “Evaluation of the different factors associated with side effects of COVID-19 vaccination on medical students, Mutah university, Al-Karak, Jordan”, in a very professional and easy way. This journal is one of outstanding medical journal.

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Prof Sherif W Mansour

Dear Hao Jiang, to Journal of Nutrition and Food Processing We greatly appreciate the efficient, professional and rapid processing of our paper by your team. If there is anything else we should do, please do not hesitate to let us know. On behalf of my co-authors, we would like to express our great appreciation to editor and reviewers.

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Hao Jiang

As an author who has recently published in the journal "Brain and Neurological Disorders". I am delighted to provide a testimonial on the peer review process, editorial office support, and the overall quality of the journal. The peer review process at Brain and Neurological Disorders is rigorous and meticulous, ensuring that only high-quality, evidence-based research is published. The reviewers are experts in their fields, and their comments and suggestions were constructive and helped improve the quality of my manuscript. The review process was timely and efficient, with clear communication from the editorial office at each stage. The support from the editorial office was exceptional throughout the entire process. The editorial staff was responsive, professional, and always willing to help. They provided valuable guidance on formatting, structure, and ethical considerations, making the submission process seamless. Moreover, they kept me informed about the status of my manuscript and provided timely updates, which made the process less stressful. The journal Brain and Neurological Disorders is of the highest quality, with a strong focus on publishing cutting-edge research in the field of neurology. The articles published in this journal are well-researched, rigorously peer-reviewed, and written by experts in the field. The journal maintains high standards, ensuring that readers are provided with the most up-to-date and reliable information on brain and neurological disorders. In conclusion, I had a wonderful experience publishing in Brain and Neurological Disorders. The peer review process was thorough, the editorial office provided exceptional support, and the journal's quality is second to none. I would highly recommend this journal to any researcher working in the field of neurology and brain disorders.

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Dr Shiming Tang

Dear Agrippa Hilda, Journal of Neuroscience and Neurological Surgery, Editorial Coordinator, I trust this message finds you well. I want to extend my appreciation for considering my article for publication in your esteemed journal. I am pleased to provide a testimonial regarding the peer review process and the support received from your editorial office. The peer review process for my paper was carried out in a highly professional and thorough manner. The feedback and comments provided by the authors were constructive and very useful in improving the quality of the manuscript. This rigorous assessment process undoubtedly contributes to the high standards maintained by your journal.

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Raed Mualem

International Journal of Clinical Case Reports and Reviews. I strongly recommend to consider submitting your work to this high-quality journal. The support and availability of the Editorial staff is outstanding and the review process was both efficient and rigorous.

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Andreas Filippaios

Thank you very much for publishing my Research Article titled “Comparing Treatment Outcome Of Allergic Rhinitis Patients After Using Fluticasone Nasal Spray And Nasal Douching" in the Journal of Clinical Otorhinolaryngology. As Medical Professionals we are immensely benefited from study of various informative Articles and Papers published in this high quality Journal. I look forward to enriching my knowledge by regular study of the Journal and contribute my future work in the field of ENT through the Journal for use by the medical fraternity. The support from the Editorial office was excellent and very prompt. I also welcome the comments received from the readers of my Research Article.

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Dr Suramya Dhamija

Dear Erica Kelsey, Editorial Coordinator of Cancer Research and Cellular Therapeutics Our team is very satisfied with the processing of our paper by your journal. That was fast, efficient, rigorous, but without unnecessary complications. We appreciated the very short time between the submission of the paper and its publication on line on your site.

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Bruno Chauffert

I am very glad to say that the peer review process is very successful and fast and support from the Editorial Office. Therefore, I would like to continue our scientific relationship for a long time. And I especially thank you for your kindly attention towards my article. Have a good day!

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Baheci Selen

"We recently published an article entitled “Influence of beta-Cyclodextrins upon the Degradation of Carbofuran Derivatives under Alkaline Conditions" in the Journal of “Pesticides and Biofertilizers” to show that the cyclodextrins protect the carbamates increasing their half-life time in the presence of basic conditions This will be very helpful to understand carbofuran behaviour in the analytical, agro-environmental and food areas. We greatly appreciated the interaction with the editor and the editorial team; we were particularly well accompanied during the course of the revision process, since all various steps towards publication were short and without delay".

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Jesus Simal-Gandara

I would like to express my gratitude towards you process of article review and submission. I found this to be very fair and expedient. Your follow up has been excellent. I have many publications in national and international journal and your process has been one of the best so far. Keep up the great work.

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Douglas Miyazaki

We are grateful for this opportunity to provide a glowing recommendation to the Journal of Psychiatry and Psychotherapy. We found that the editorial team were very supportive, helpful, kept us abreast of timelines and over all very professional in nature. The peer review process was rigorous, efficient and constructive that really enhanced our article submission. The experience with this journal remains one of our best ever and we look forward to providing future submissions in the near future.

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Dr Griffith

I am very pleased to serve as EBM of the journal, I hope many years of my experience in stem cells can help the journal from one way or another. As we know, stem cells hold great potential for regenerative medicine, which are mostly used to promote the repair response of diseased, dysfunctional or injured tissue using stem cells or their derivatives. I think Stem Cell Research and Therapeutics International is a great platform to publish and share the understanding towards the biology and translational or clinical application of stem cells.

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Dr Tong Ming Liu

I would like to give my testimony in the support I have got by the peer review process and to support the editorial office where they were of asset to support young author like me to be encouraged to publish their work in your respected journal and globalize and share knowledge across the globe. I really give my great gratitude to your journal and the peer review including the editorial office.

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Husain Taha Radhi

I am delighted to publish our manuscript entitled "A Perspective on Cocaine Induced Stroke - Its Mechanisms and Management" in the Journal of Neuroscience and Neurological Surgery. The peer review process, support from the editorial office, and quality of the journal are excellent. The manuscripts published are of high quality and of excellent scientific value. I recommend this journal very much to colleagues.

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S Munshi

Dr.Tania Muñoz, My experience as researcher and author of a review article in The Journal Clinical Cardiology and Interventions has been very enriching and stimulating. The editorial team is excellent, performs its work with absolute responsibility and delivery. They are proactive, dynamic and receptive to all proposals. Supporting at all times the vast universe of authors who choose them as an option for publication. The team of review specialists, members of the editorial board, are brilliant professionals, with remarkable performance in medical research and scientific methodology. Together they form a frontline team that consolidates the JCCI as a magnificent option for the publication and review of high-level medical articles and broad collective interest. I am honored to be able to share my review article and open to receive all your comments.

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Tania Munoz

“The peer review process of JPMHC is quick and effective. Authors are benefited by good and professional reviewers with huge experience in the field of psychology and mental health. The support from the editorial office is very professional. People to contact to are friendly and happy to help and assist any query authors might have. Quality of the Journal is scientific and publishes ground-breaking research on mental health that is useful for other professionals in the field”.

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George Varvatsoulias

Dear editorial department: On behalf of our team, I hereby certify the reliability and superiority of the International Journal of Clinical Case Reports and Reviews in the peer review process, editorial support, and journal quality. Firstly, the peer review process of the International Journal of Clinical Case Reports and Reviews is rigorous, fair, transparent, fast, and of high quality. The editorial department invites experts from relevant fields as anonymous reviewers to review all submitted manuscripts. These experts have rich academic backgrounds and experience, and can accurately evaluate the academic quality, originality, and suitability of manuscripts. The editorial department is committed to ensuring the rigor of the peer review process, while also making every effort to ensure a fast review cycle to meet the needs of authors and the academic community. Secondly, the editorial team of the International Journal of Clinical Case Reports and Reviews is composed of a group of senior scholars and professionals with rich experience and professional knowledge in related fields. The editorial department is committed to assisting authors in improving their manuscripts, ensuring their academic accuracy, clarity, and completeness. Editors actively collaborate with authors, providing useful suggestions and feedback to promote the improvement and development of the manuscript. We believe that the support of the editorial department is one of the key factors in ensuring the quality of the journal. Finally, the International Journal of Clinical Case Reports and Reviews is renowned for its high- quality articles and strict academic standards. The editorial department is committed to publishing innovative and academically valuable research results to promote the development and progress of related fields. The International Journal of Clinical Case Reports and Reviews is reasonably priced and ensures excellent service and quality ratio, allowing authors to obtain high-level academic publishing opportunities in an affordable manner. I hereby solemnly declare that the International Journal of Clinical Case Reports and Reviews has a high level of credibility and superiority in terms of peer review process, editorial support, reasonable fees, and journal quality. Sincerely, Rui Tao.

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Rui Tao

Clinical Cardiology and Cardiovascular Interventions I testity the covering of the peer review process, support from the editorial office, and quality of the journal.

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Khurram Arshad

Clinical Cardiology and Cardiovascular Interventions, we deeply appreciate the interest shown in our work and its publication. It has been a true pleasure to collaborate with you. The peer review process, as well as the support provided by the editorial office, have been exceptional, and the quality of the journal is very high, which was a determining factor in our decision to publish with you.

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Gomez Barriga Maria Dolores

The peer reviewers process is quick and effective, the supports from editorial office is excellent, the quality of journal is high. I would like to collabroate with Internatioanl journal of Clinical Case Reports and Reviews journal clinically in the future time.

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Lin Shaw Chin

Clinical Cardiology and Cardiovascular Interventions, I would like to express my sincerest gratitude for the trust placed in our team for the publication in your journal. It has been a true pleasure to collaborate with you on this project. I am pleased to inform you that both the peer review process and the attention from the editorial coordination have been excellent. Your team has worked with dedication and professionalism to ensure that your publication meets the highest standards of quality. We are confident that this collaboration will result in mutual success, and we are eager to see the fruits of this shared effort.

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Maria Dolores Gomez Barriga

Dear Dr. Jessica Magne, Editorial Coordinator 0f Clinical Cardiology and Cardiovascular Interventions, I hope this message finds you well. I want to express my utmost gratitude for your excellent work and for the dedication and speed in the publication process of my article titled "Navigating Innovation: Qualitative Insights on Using Technology for Health Education in Acute Coronary Syndrome Patients." I am very satisfied with the peer review process, the support from the editorial office, and the quality of the journal. I hope we can maintain our scientific relationship in the long term.

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Dr Maria Dolores Gomez Barriga

Dear Monica Gissare, - Editorial Coordinator of Nutrition and Food Processing. ¨My testimony with you is truly professional, with a positive response regarding the follow-up of the article and its review, you took into account my qualities and the importance of the topic¨.

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Dr Maria Regina Penchyna Nieto

Dear Dr. Jessica Magne, Editorial Coordinator 0f Clinical Cardiology and Cardiovascular Interventions, The review process for the article “The Handling of Anti-aggregants and Anticoagulants in the Oncologic Heart Patient Submitted to Surgery” was extremely rigorous and detailed. From the initial submission to the final acceptance, the editorial team at the “Journal of Clinical Cardiology and Cardiovascular Interventions” demonstrated a high level of professionalism and dedication. The reviewers provided constructive and detailed feedback, which was essential for improving the quality of our work. Communication was always clear and efficient, ensuring that all our questions were promptly addressed. The quality of the “Journal of Clinical Cardiology and Cardiovascular Interventions” is undeniable. It is a peer-reviewed, open-access publication dedicated exclusively to disseminating high-quality research in the field of clinical cardiology and cardiovascular interventions. The journal's impact factor is currently under evaluation, and it is indexed in reputable databases, which further reinforces its credibility and relevance in the scientific field. I highly recommend this journal to researchers looking for a reputable platform to publish their studies.

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Dr Marcelo Flavio Gomes Jardim Filho

Dear Editorial Coordinator of the Journal of Nutrition and Food Processing! "I would like to thank the Journal of Nutrition and Food Processing for including and publishing my article. The peer review process was very quick, movement and precise. The Editorial Board has done an extremely conscientious job with much help, valuable comments and advices. I find the journal very valuable from a professional point of view, thank you very much for allowing me to be part of it and I would like to participate in the future!”

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Zsuzsanna Bene