Available online on 15.04.2024 at http://jddtonline.info

Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

Copyright  © 2024 The   Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

Open Access  Full Text Article                                                                                                                                                  Research Article

The Effect of Honey on Bacteria Isolated from Urinary Tract Infections among Patients Attending Ruhengeri Referral Hospital 

Ishimwe Alain Prudence ^1,2,3*, Munezero Pierre Celestin ^1,2, Migabo Hiberte ¹, Itangimbabazi Josue ¹, Saramba Eric ²

¹ Ines-Ruhengeri, Faculty of Applied Fundamental Sciences, Department of Biomedical Laboratory Sciences, Rwanda  

² University of Rwanda, College of Medicine and Health Sciences, Department of Biomedical Laboratory Sciences, Rwanda 

³ Kibogora Polytechnic, Department of Biomedical Laboratory Sciences, Rwanda   

INTRODUCTION 

Urinary tract infections (UTIs) are common bacterial infections affecting millions of individuals worldwide, with women being particularly susceptible (Bhargava et al., 2022). In most cases, UTIs are caused by coliform bacteria, such as Escherichia coli, leading to symptoms including dysuria, frequency, and lower abdominal pain (Mandracchia et al., 2016). Although antibiotics represent the standard UTI treatment, the rising prevalence of antibiotic-resistant bacteria necessitates the exploration of alternative therapies, including natural products like honey ¹.

Honey has been employed for medicinal purposes for millennia and is recognized for its antimicrobial, anti-inflammatory, and antioxidant properties ². Recent research has demonstrated the ability of honey to effectively inhibit the growth of various pathogenic bacteria, including those implicated in UTIs ³. The antimicrobial mechanism of action is of honey is believed to be multifactorial, with elements such as high sugar content, acidity, and hydrogen peroxide production contributing to its antibacterial properties ⁴. Despite honey's potential therapeutic benefits, data on its effectiveness in treating UTIs remains scarce, particularly in resource-limited settings. In Rwanda, UTIs are a prevalent especially among women, and are associated with poor hygiene practices and limited access to clean water ⁵. A review of UTIs in Rwanda identified E. coli as the most common pathogen and reported high resistance rates to commonly used antibiotics ⁶. This underlines the urgent need for alternative therapies to treat UTIs in vulnerable populations. 

This study aimed to investigate honey's effect on bacteria isolated from urine obtained from patients suffering from UTIs at the Ruhengeri Referral Hospital in Rwanda. 

MATERIALS AND METHODS

Study Setting and Design

This cross-sectional study was conducted at Ruhengeri Referral Hospital in the Northern Province of Rwanda. Upon enrolment, each participant donated a random urine sample that was transported to the microbiology laboratory at INES Ruhengeri for bacterial culture, identification and sensitivity testing.

Data Collection

The study enrolled consenting patients diagnosed with urinary tract infections who attended Ruhengeri Referral Hospital. A sample size of 30 eligible and consecutive patients who visited the hospital between August 2022 and October 2022 was selected. Inclusion criteria were a UTI diagnosis, while patients with infections at other anatomical sites were excluded. Urine samples were collected in sterile containers and transported to the microbiology laboratory at INES Ruhengeri for isolation and identification of bacteria and susceptibility testing.

Bacterial isolation, identification and susceptibility testing

Each sample was inoculated on different culture media such as Blood Agar, MacConkey and Mannitol Salt Agar using streaking method. After gram staining of each distinct single isolated colony of bacteria, the latter was confirmed using biochemical tests, including catalase, coagulase, citrate test, sugar fermentation and hydrogen sulphide production, motility test, urease test, and indole production.   

Antibiotic Susceptibility Test

Antimicrobial susceptibility testing was conducted using the Kirby-Bauer disk diffusion method in accordance with the guidelines provided by the Clinical Laboratory Standards Institute (CLSI) on Muller-Hinton Agar (CLSI, 2020). The test organisms were prepared by creating a suspension of 3-5 freshly grown colonies, equivalent to a 0.5 McFarland standard. The swab containing the suspension was swirled to cover the surface of the Muller-Hinton agar. The plates were allowed to dry for 3-5 minutes at room temperature before a sterile dropper was used to apply a drop of honey to the inoculated plates' tip hole. Subsequently, the plates were incubated aerobically at 37°C for 24 hours. The diameter of the clear zone surrounding the drop of honey was measured using a ruler, and the results were reported as the diameter of the zone of inhibition. 

Ethical Considerations

The authorization to carry out this study was obtained from both Ruhengeri Referral Hospital Ruhengeri Institute of Higher Education. Each patient has signed the consent form prior to be enrolled into the study. All patients a unique study identifier.

RESULTS

Frequency of bacterial isolates

The most frequently isolated bacteria were S. aureus (33.3%), followed by P. aeruginosa (13.8%) and Coagulase negative Staphylococcus (CoNS) (13.8%), E. coli, K.  pneumonia, and Streptococcus spp. each accounted for 11.2%, and the least isolated was   E. aerogenes at 5.5% (Fig. 1).

Figure 1: The distribution of bacterial species isolated from study participants

Antimicrobial Effect of Honey

Two types of honey, processed honey and raw honey with no additives or dilution, were evaluated for antimicrobial activity against the bacteria isolated from the study participants. The processed honey did not exhibit significant activity against the isolated bacteria, as evidenced by the absence of any inhibition zone and bacteria growth on the Mueller-Hinton agar (MHA). On the other hand, raw honey demonstrated antimicrobial activity against the bacterial isolates, with varying degrees of inhibition zones depending on the type of bacteria. E. coli and Streptococcus species were the most susceptible to the honey, demonstrated by mean diameter of inhibition zone (mIZ) (mIZ = 36 ±2.83; ±4.24 mm, respectively), followed by S. aureus and CoNS (mIZ = 36 ±4.24; ±5.66 mm, respectively), K. pneumonia (mIZ = 28 ±5.66 mm), P. aeruginosa (mIZ = 26 ±1.41 mm), and E. aerogenes (mIZ =24 ±2.83 mm) (Table 1)

Table 1: Mean zone of bacterial growth inhibition by raw honey on different isolated bacteria.

DISCUSSION

Urinary tract infections are caused by a wide range of microorganisms, including bacteria (gram-positive and –negative) and fungi, which are often isolated from patients' urine samples ⁷(Bhargava et al., 2022). Bacteria commonly associated with UTIs include E. coli, S. aureus, K. pneumoniae, P. aeruginosa, E. aerogenes, CoNS, and Streptococcus spp (Hodson & Craig, 2015). Previous studies reported E. coli as the most prevalent bacteria causing UTIs ⁸, typically gaining entry into the urinary tract through stool containing the bacteria [9]. Women are more susceptible to UTIs than men due to their shorter urethra, which is also situated closer to the anus, a source of E. coli ⁹. K. pneumoniae infections are primarily nosocomial. Individuals with weakened immune systems, such as  those who are sick or injured, and those undergoing various medical procedures are at a higher risk of contracting K. pneumoniae infection than the general population ¹⁰. Infections can occur through the use of contaminated medical equipment, such as ventilators and intravenous catheters ¹¹. 

In this study Staphylococcus species (S. aureus and CoNS) were most prevalent isolated bacteria and S. aureus was more frequent than CoNS. Gessese et al. reported them at the same proportion ¹². Contrary, Flores-Mireles et al. mentioned that CoNS are more prevalent than S. aureus ⁷. Staphylococcus species are often the predominant bacterial species isolated from patients' urine samples, as it is primarily spread by contaminated hands ¹². Although skin and mucous membranes generally serve as effective barriers against infection, damage to these barriers allows bacteria to penetrate and cause infection ¹³. Individuals with weakened immune systems or invasive medical devices, like catheters, are especially vulnerable to S. aureus infections ¹⁴. Current results differ from those reported by Loubet et al, that S. aureus is an uncommon isolate in urine cultures contributing to only 0.5–6% of positive urine cultures, except in patients with risk factors for urinary tract colonization ¹. This discrepancy might possibly be explained by poor collection of urine specimens leading to contamination ¹⁵. 

E. aerogenes was  the least frequently isolated bacteria from urine samples in the present study, as it is primarily associated with healthcare-related infections and is responsible for numerous nosocomial infections, including UTIs ¹⁶. Enterobacter species can be part of the mammalian gastrointestinal tract's microflora and may be present on human skin surfaces, in water, certain foods, soil, and sewage ¹⁷.

The antimicrobial susceptibility test was performed using honey. The properties of honey include; low pH, high osmotic effect, high sugar concentration, hydrogen peroxide generation and a high content of antioxidants, lysozymes, polyphenols, phenolic acids, flavonoids, methylglyoxal, and bee peptides, which together possibly contribute to the antimicrobial properties against some bacteria that cause UTIs ¹⁸. The unadulterated quality and composition of honey are essential for it to function as an antimicrobial agent ¹⁹.

Artificial or processed honey, prepared using a mixture of mono- and disaccharides at concentrations similar to those found in natural honey, often did not demonstrate effective bacterial inhibition ²⁰. The osmotic pressure exerted by honey's high sugar content is a crucial aspect of its antibacterial activity, and the level of inhibition depends on honey's concentration ²¹.

Studies have demonstrated that natural honey can inhibit growth and kill isolated bacteria from UTIs, with varying zones of inhibition ²². Studies have emphasized on role of hydrogen peroxidase produced by honey in antimicrobial activity ². However their mode of action differ depending on the type of the honey used. For instance, manuka honey was used as characteristic of honey with peroxidase activity while pasture honey with non-peroxidase activity ²³.  The same authors demonstrated the antimicrobial activity of both honey against CoNS. While in this study the processed honey did not show any effect on isolated bacteria, French et al. demonstrated a weak effect of simulated honey in inhibiting isolates ²³. 

This study has shown the antimicrobial activity of natural honey to both gram-negative and positive bacteria. In addition to the above-mentioned components of the honey, many other component were mentioned to involve in its capacity to inhibit bacterial growth and these include high viscosity which is mostly due to the high concentration of sugar, and the low content of water. To these, it adds the mild acidity which is an adverse environment for bacterial growth ²⁴. Hydrogen peroxidase was also described as an important component in antibacterial activity of the honey. However, its activity requires the dilution of the honey to allow the honey-containing glucose oxidase to act on endogenous glucose which in turn produces hydrogen peroxidase ²⁵. On the other hand, the non-peroxidase antibacterial activity of honey has been documented. One of the components that come in action is the phenolic compound ²⁶. That is why, manuka honey possesses higher content of phenolic acid than pasture honey ²⁷.

CONCLUSION

With different bacteria that are currently showing resistance to different classical antibiotics, the natural showed to be a remedy to controlling bacteria isolated from the urinary tract. Further studies are of needed to compare the inhibition capacity of natural honey with classical antibiotics. Additionally, in vivo studies will also shed light on the efficacy of natural honey as nutritional regimen. That is why the practical way to use honey to get targeted area is also an interested point that need to be investigated.

ACKNOWLEDGMENTS

We extend our gratitude to Ruhengeri Referral Hospital administration for facilitating this study at their health facilities.

Conflict of interest

Authors declare no conflict of interest

Availability of raw data and material

Raw data and information on material should be obtained from the corresponding author upon request.

REFERENCES 

1. Loubet, P., et al., Alternative Therapeutic Options to Antibiotics for the Treatment of Urinary Tract Infections. Front Microbiol, 2020. 11: p. 1509. https://doi.org/10.3389/fmicb.2020.01509 PMid:32719668 PMCid:PMC7350282

2. Mandal, M.D. and S. Mandal, Honey: its medicinal property and antibacterial activity. Asian Pac J Trop Biomed, 2011. 1(2): p. 154-60. https://doi.org/10.1016/S2221-1691(11)60016-6 PMid:23569748

3. Vallianou, N., Honey and its Anti-Inflammatory, Anti-Bacterial and Anti-Oxidant Properties. General Medicine: Open Access, 2014. 02. https://doi.org/10.4172/2327-5146.1000132

4. Yupanqui Mieles, J., et al., Honey: An Advanced Antimicrobial and Wound Healing Biomaterial for Tissue Engineering Applications. Pharmaceutics, 2022. 14(8). https://doi.org/10.3390/pharmaceutics14081663 PMid:36015289 PMCid:PMC9414000

5. Amiri, F.N., et al., Hygiene practices and sexual activity associated with urinary tract infection in pregnant women. East Mediterr Health J, 2009. 15(1): p. 104-10. https://doi.org/10.26719/2009.15.1.104 PMid:19469432

6. Muvunyi, C.M., et al., Decreased susceptibility to commonly used antimicrobial agents in bacterial pathogens isolated from urinary tract infections in Rwanda: need for new antimicrobial guidelines. Am J Trop Med Hyg, 2011. 84(6): p. 923-8. https://doi.org/10.4269/ajtmh.2011.11-0057 PMid:21633029 PMCid:PMC3110351

7. Flores-Mireles, A.L., et al., Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nat Rev Microbiol, 2015. 13(5): p. 269-84. https://doi.org/10.1038/nrmicro3432 PMid:25853778 PMCid:PMC4457377

8. Lee, D.S., S.J. Lee, and H.S. Choe, Community-Acquired Urinary Tract Infection by Escherichia coli in the Era of Antibiotic Resistance. Biomed Res Int, 2018. 2018: p. 7656752. https://doi.org/10.1155/2018/7656752 PMid:30356438 PMCid:PMC6178185

9. Bhargava, K., et al., Bacterial profile and antibiotic susceptibility pattern of uropathogens causing urinary tract infection in the eastern part of Northern India. Front Microbiol, 2022. 13: p. 965053. https://doi.org/10.3389/fmicb.2022.965053 PMid:36016776 PMCid:PMC9396120

10. Chang, D., et al., Clinical Epidemiology, Risk Factors, and Control Strategies of Klebsiella pneumoniae Infection. Front Microbiol, 2021. 12: p. 750662. https://doi.org/10.3389/fmicb.2021.750662 PMid:34992583 PMCid:PMC8724557

11. Rawashdeh, A.Z.A., Mohammed, E. Y., Arab, A. R. Al, Alara, M., & Al-Rawashdeh, B. , Advantages and disadvantages of using E-learning in university education: Analyzing students' perspectives. Electronic Journal of E-Learning, 2021. 19(2): p. 107-117. https://doi.org/10.34190/ejel.19.3.2168

12. Gessese, Y.A., et al., Urinary pathogenic bacterial profile, antibiogram of isolates and associated risk factors among pregnant women in Ambo town, Central Ethiopia: a cross-sectional study. Antimicrob Resist Infect Control, 2017. 6: p. 132. https://doi.org/10.1186/s13756-017-0289-6 PMid:29299306 PMCid:PMC5747040

13. Zachary, J.F., Mechanisms of Microbial Infections. Pathologic Basis of Veterinary Disease, 2017: p. 132-241.e1. https://doi.org/10.1016/B978-0-323-35775-3.00004-7 PMCid:PMC7158295

14. Pietrocola, G., et al., Colonization and Infection of Indwelling Medical Devices by Staphylococcus aureus with an Emphasis on Orthopedic Implants. Int J Mol Sci, 2022. 23(11). https://doi.org/10.3390/ijms23115958 PMid:35682632 PMCid:PMC9180976

15. Mason, C.Y., A. Sobti, and A.L. Goodman, Staphylococcus aureus bacteriuria: implications and management. JAC Antimicrob Resist, 2023. 5(1): p. dlac123. https://doi.org/10.1093/jacamr/dlac123 PMid:36644414 PMCid:PMC9833284

16. Canada, G.o., Final Screening Assessment of Enterobacter aerogenes strain ATCC 13048. 2018. 1-54.

17. Ramirez, D., Giron, M. , Enterobacter Infections. [Updated 2022 Jun 27]. In: StatPearls [Internet]. . 2023, StatPearls: Treasure Island (FL).

18. Almasaudi, S., The antibacterial activities of honey. Saudi J Biol Sci, 2021. 28(4): p. 2188-2196. https://doi.org/10.1016/j.sjbs.2020.10.017 PMid:33911935 PMCid:PMC8071826

19. Nolan, V.C., J. Harrison, and J.A.G. Cox, Dissecting the Antimicrobial Composition of Honey. Antibiotics (Basel), 2019. 8(4). https://doi.org/10.3390/antibiotics8040251 PMid:31817375 PMCid:PMC6963415

20. Hossain, M.L., et al., A Review of Commonly Used Methodologies for Assessing the Antibacterial Activity of Honey and Honey Products. Antibiotics (Basel), 2022. 11(7). https://doi.org/10.3390/antibiotics11070975 PMid:35884229 PMCid:PMC9312033

21. Piotr, S., Antimicrobial Activity of Honey, in Honey Analysis, T. Vagner de Alencar Arnaut de, Editor. 2017, IntechOpen: Rijeka. p. Ch. 10.

22. Johnston, M., et al., Antibacterial activity of Manuka honey and its components: An overview. AIMS Microbiol, 2018. 4(4): p. 655-664. https://doi.org/10.3934/microbiol.2018.4.655 PMid:31294240 PMCid:PMC6613335

23. French, V.M., R.A. Cooper, and P.C. Molan, The antibacterial activity of honey against coagulase-negative staphylococci. J Antimicrob Chemother, 2005. 56(1): p. 228-31. https://doi.org/10.1093/jac/dki193 PMid:15941774

24. Albaridi, N.A., Antibacterial Potency of Honey. Int J Microbiol, 2019. 2019: p. 2464507. https://doi.org/10.1155/2019/2464507 PMid:31281362 PMCid:PMC6589292

25. Bang, L.M., C. Buntting, and P. Molan, The effect of dilution on the rate of hydrogen peroxide production in honey and its implications for wound healing. J Altern Complement Med, 2003. 9(2): p. 267-73. https://doi.org/10.1089/10755530360623383 PMid:12804080

26. Wahdan, H.A., Causes of the antimicrobial activity of honey. Infection, 1998. 26(1): p. 26-31. https://doi.org/10.1007/BF02768748 PMid:9505176

27. Lachman, J., et al., Evaluation of antioxidant activity and total phenolics of selected Czech honeys. LWT - Food Science and Technology, 2010. 43(1): p. 52-58. https://doi.org/10.1016/j.lwt.2009.06.008