Effect of Fluconazole, An Anti-Fungal Drug, On Human Flora Bacteria

Research Article

J Bacteriol Mycol. 2020; 7(2): 1130.

Effect of Fluconazole, An Anti-Fungal Drug, On Human Flora Bacteria

Zarf S and Arman I*

Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Zonguldak Bulent Ecevit University, Zonguldak, Turkey

*Corresponding author: Ibrahim Arman, Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Bulent Ecevit University, 67100 Zonguldak, Turkey

Received: May 13, 2020; Accepted: May 26, 2020; Published: June 02, 2020

Abstract

Fluconazole is an antifungal drug, which is in the class of azole derivatives and it is widely used worldwide. To date, there have been some studies published in the literature on the genotoxicity and cytotoxicity of fluconazole, indicating that the drug has a serious toxic effect. According to our research, there is no study on the antibacterial effect of fluconazole. Therefore, this research aimed to investigate the effect of fluconazole on the growth of some flora bacterial strains. The Mueller Hinton liquid medium containing different concentrations of fluconazole (24, 12, 6, and 3 mM) was prepared and inoculated with bacterial suspensions to adjust to Macfarland 0.5. After the culture was incubated for 24 hours at 150 rpm at 37°C, the growth of the strains was examined using the UV-visible spectrophotometer and CFU (colony forming unit) count. In addition, the growth of strains was repeated using sterile discs containing concentrations of fluconazole (24, 12, 6, and 3 mM) on the Mueller Hinton solid medium. After treated with the 24mM concentration of fluconazole, the growth rate of Enterococcus faecalis, Enterobacter cloacae, Escherichia coli and Proteus mirabilis strains, were reduced about 35%, 20%, 20% and 22%, respectively. Any of the drug concentration did not showed inhibitory effect on the bacterial strains on the Mueller Hinton solid medium.

Keywords: Fluconazole; Antifungal; Flora; Bacteria; Colony

Introduction

Fluconazole, a bis-triazole derivative, was synthesized in 1981 by replacing its imidazole nucleus [1]. The drug is a relatively small and water-soluble molecule compared to ketoconazole and itraconazole [2]. Fluconazole is widely used worldwide as an antifungal drug, especially in our country Turkey [3].

Oral dosage of the drug is in the form of tablets of 50, 100, 150 and 200 mg and its daily dose intake is 50-400 mg. In the treatment of candidemia, the maximum dose for Candida species is 400 mg daily, but for Candida glabrata is 800 mg. Increasing doses could be tolerated by patients, but increased hepatic and other side effects as the dose increased [4]. Side effects reported about the fluconazole are often associated with their interaction with the gastrointestinal tract. Serious side effects such as abdominal pain, diarrhea, flatulence, nausea, vomiting, leukopenia, thrombocytopenia and hyperlipidemia have been reported [5]. Somchit et al. [6] demonstrated the dose and time-dependent toxic effect of the drug on hepatocyte cells (Somchit et al. 2002). Balance of population is as important as the variety of bacterial strains in human flora [7]. The balanced condition between the host and intestinal microbiota is called symbiosis [8]. Microbiota can undergo changes by factors such as age, diet habits, lifestyle and genetic predisposition [9]. The amount and frequency of use of drugs can permanently change the microbiome [10]. Today, it is accepted that dysbiosis can cause many diseases in human [11]. Therefore, the flora bacteria are important. The number of pathogens increases in the impaired intestine; toxic substances accumulate as a result of the lack of useful flora to detoxify. Microorganisms have important functions in maintaining a healthy person’s life, and sometimes disorders in the variety and balance of bacteria in the intestine can lead to the dominance of other pathogenic bacteria [12]. The family Enterobacteriaceae consists of many species and strains colonized in the small and large intestine, and also contains pathogenic members of the non-pathogenic commensal microbiota [13]. From Enterobacteriaceae, Escherichia coli, Enterobacter cloacae, Proteus mirabilis, Proteus vulgaris are the most frequently isolated strains from human intestins [14]. Most enterococcus species are normal flora of the gastrointestinal tract of humans. Enterococcus fecalis is one of enterococcus species. Therefore, this study aimed to analyze the effect of fluconazole on the Escherichia coli, Enterococcus fecalis, Proteus mirabilis and Enterobacter cloacae.

Materials and Methods

Reagents

The lab grade of fluconazole was purchased from Sigma-Aldrich (PHR1160-1G) and used as a test substance. The molecular weight of fluconazole (FCZ) was 306.27 g/ml and its chemical structure was as shown below.

Effect of fluconazole on bacterial strains by broth dilution method

Mueller Hinton broth medium containing different concentrations of fluconazole (serial dilutions made by creating a two-fold dilution with a starting concentration of 24 mM) were prepared and inoculated with an overnight culture of Escherichia coli (ATCC-25922), Enterococcus fecalis (ATCC-29212), Proteus mirabilis (ATCC7002) and Enterobacter cloacae (ATCC 13047) followed by adjusting to standard turbidity of 0.5 McFarland. After that, the cultures were incubated at 37°C at 150 rpm for overnight to determine MIC (minimum inhibitory concentration) using UVVisible spectrophotometry (Optizen 2120 UV) and CFU (colony forming units) counting.

Effect of fluconazole on bacterial strains by disc diffusion method

The bacterial strains of Escherichia coli (ATCC-25922), Enterococcus fecalis (ATCC-29212), Proteus mirabilis (ATCC7002) and Enterobacter cloacae (ATCC 13047) were cultured on Mueller Hinton agar plats by spread plate technique. After that sterile blank discs containing 24, 12, 6, and 3 mM of fluconazole were placed on the culture before incubation. Then the cultures were incubated at 37°C for an overnight and the antibacterial effect of the fluconazole tried to assay by the zone diameter formed around the colonies [15].

Result

The maximum concentration of fluconazole (24 mM) had an inhibitory effect on the bacterial strains. After treated with the 24 mM concentration of fluconazole, the growth rate of Enterococcus faecalis, Enterobacter cloacae, Escherichia coli and Proteus mirabilis strains, were reduced about 35%, 20%, 20% and 22%, respectively. As it was shown in the (Figure 1), the inhibitory effect on the growth of bacteria is concentration dependent manner. In addition, as shown in Figure 2, not any zone was observed around the sterile disks containing fluconazole concentrations (24, 12, 6 and 3 mM) on the Mueller Hinton solid medium.

Figure 1: Relative growth of the bacterial strains in broth medium containing various concentrations of fluconazole. Control samples of Escherichia coli, Enterococcus faecalis, Enterobacter cloacae and Proteus mirabilis that were considered as 100% contained 7.6735E+14 CFU/ml, 5.5745E+21 CFU/ml, 2.72E+21 CFU/ml and 2.037E+22 CFU/ml respectively. All of the tests were conducted as three repeats.

    

    
    

    

    Figure 1: Relative growth of the bacterial strains in broth medium containing
various concentrations of fluconazole. Control samples of Escherichia coli,
Enterococcus faecalis, Enterobacter cloacae and Proteus mirabilis that were
considered as 100% contained 7.6735E+14 CFU/ml, 5.5745E+21 CFU/ml,
2.72E+21 CFU/ml and 2.037E+22 CFU/ml respectively. All of the tests were
conducted as three repeats.

Figure 2: Growth of Escherichia coli (a), Enterobacter cloacae (b), Enterococcus faecalis (c) and Proteus mirabilis (d) on Mueller Hinton agar using sterile blank discs. Concentrations of fluconazole of the discs present in the plate-halves labeled by 1, 2, 3 and 4 contained 24, 12, 6 and 3 mM of fluconazole, respectively. Plate-halves labeled by zero did not contain fluconazole and it was control sample.

    

    
    

    

    Figure 2: Growth of Escherichia coli (a), Enterobacter cloacae (b),
Enterococcus faecalis (c) and Proteus mirabilis (d) on Mueller Hinton agar
using sterile blank discs. Concentrations of fluconazole of the discs present
in the plate-halves labeled by 1, 2, 3 and 4 contained 24, 12, 6 and 3 mM
of fluconazole, respectively. Plate-halves labeled by zero did not contain
fluconazole and it was control sample.

Discussion

Since human intestinal microbiota affects a lot of vital physiological processes, any change in microbial composition (dysbiosis) has a direct impact on human health [16]. In this research, drug concentration was calculated considering the concentration of fuconazole in the small intestine (105 ± 72 mL and 54 ± 41 mL) after maximum oral dose intake [17]. When fuconazole is given to patients orally (50, 100, 200 and 400 mg), it is directly contact the microorganism population in the gastrointestinal tract for a long time. After treated with the maximum concentration of fuconazole (24 mM) the growth rate of Enterococcus faecalis, Enterobacter cloacae, Escherichia coli and Proteus mirabilis strains, were reduced about 35%, 20%, 20% and 22%, respectively. The inhibitory effect on the growth of the bacteria is less common as we move from high concentration to low concentrations. Before, a study on fish showed that a diet supplemented with E. faecalis significantly increased protease and lipase activities compared to a control feed. E. faecalis supplement significantly increased the production of propionic and butyric acid in the intestine [18]. In addition, decrease of Escherichia coli strain in the intestinal of the patients with ulcerative colitis cause to increase in the amount of non-normal flora bacteria even pathogenic strains in the intestinal tract [19]. Because colicin proteins expressed by E. coli inhibit bacterial growth of other species [20]. In a study on patients with ulcerative colitis, a reduced amount of E. coli bacteria was observed in the intestinal microflora, which was associated with joint involvement in patients with ulcerative colitis [21]. Patients with ulcerative colitis using fluconazole should consider this adverse effect, since culture concentrations of E. coli treated with fluconazole are reduced. The increase in the growth of E. cloacae in humans has been associated with obesity [22, 23]. In vitro studies have shown that in adipocytes induced by flagellin, they can increase hepatic fat accumulation by increasing lipolysis and glycerol synthesis from adipocytes [24, 25].

Acknowledgment

This research was a master thesis that was conducted under the supervision of assistant professor Ibrahim ARMAN (Ebrahim Valipour) in Zonguldak Bulent Ecevit University-TURKEY. We wish to thank the Zonguldak Bulent Ecevit University Scientific Research Commission for supporting our study through project grants no. 2017-50737594-01.

References

  1. Sugar AM, Lyman CA. A practical guide to medically important fungi and the diseases they cause: Lippincott-Raven. 1997.
  2. Dismukes WE. Introduction to antifungal drugs. Clin Infect Dis. 2000; 653- 657.
  3. Ayub AC, Gomes AD, Lima MV, Vianna-Soares CD, Ferreira LA. Topical delivery of fluconazole: in vitro skin penetration and permeation using emulsions as dosage forms. Drug Dev Ind Pharm. 2007; 33: 273-280.
  4. Sanguineti A, Carmichael JK, Campbell K. Fluconazole-resistant Candida albicans after long-term suppressive therapy. Arch Intern Med. 1993; 153: 1122-1124.
  5. Sweetman SC. Martindale: the complete drug reference: Pharmaceutical press London. 2009.
  6. Somchit N, Hassim S, Samsudin S. Itraconazole and fluconazole-induced toxicity in rat hepatocytes: a comparative in vitro study. Hum Exp Toxicol. 2002; 21: 43-48.
  7. Holzapfel WH, Haberer P, Snel J, Schillinger U, in’t Veld JHH. Overview of gut flora and probiotics. Int J Food Microbiol. 1998; 41: 85-101.
  8. Chow J, Lee SM, Shen Y, Khosravi A, Mazmanian SK. Host-bacterial symbiosis in health and disease. Adv Immunol. 2010; 107: 243-274.
  9. Altuntas Y, Batman A. Mikrobiyota ve metabolik sendrom. Turk Kardiyol Dern Ars. 2017: 286-296.
  10. Langdon A, Crook N, Dantas G. The effects of antibiotics on the microbiome throughout development and alternative approaches for therapeutic modulation. Genome Med. 2016; 8: 39.
  11. Petersen C, Round JL. Defining dysbiosis and its influence on host immunity and disease. Cell Microbiol. 2014; 16: 1024-1033.
  12. Ma N, Guo P, Zhang J, He T, Kim SW, Zhang G, et al. Nutrients mediate intestinal bacteria–mucosal immune crosstalk. Front Immunol. 2018; 9: 5.
  13. Schierack P, Walk N, Reiter K, Weyrauch KD, Wieler LH. Composition of intestinal Enterobacteriaceae populations of healthy domestic pigs. Microbiology. 2007; 153: 3830-3837.
  14. Søgaard P. Population analysis of susceptibility to cefotaxime in Enterobacteriaceae. Acta Pathologica Microbiologica Scandinavica Series B: Microbiology. 1985; 93: 365-369.
  15. Valipour R, Yilmaz MB, Valipour E. Study of DNA-Binding Activity and Antibacterial Effect of Escitalopram Oxalate, an Extensively Prescribed Antidepressant. Drug research. 2019.
  16. Barko P, McMichael M, Swanson KS, Williams DA. The gastrointestinal microbiome: a review. J Vet Intern Med. 2018; 32: 9-25.
  17. Schiller C, Fröhlich CP, Giessmann T, Siegmund W, Mönnikes H, Hosten N, et al. Intestinal fluid volumes and transit of dosage forms as assessed by magnetic resonance imaging. Aliment Pharmacol Ther. 2005; 22: 971-979.
  18. Allameh S, Ringø E, Yusoff F, Daud H, Ideris A. Dietary supplement of Enterococcus faecalis on digestive enzyme activities, short-chain fatty acid production, immune system response and disease resistance of Javanese carp (Puntius gonionotus, Bleeker 1850). Aquac Nutr. 2017; 23: 331-338.
  19. Walujkar SA, Dhotre DP, Marathe NP, Lawate PS, Bharadwaj RS, Shouche YS. Characterization of bacterial community shift in human Ulcerative Colitis patients revealed by Illumina based 16S rRNA gene amplicon sequencing. Gut Pathog. 2014; 6: 22.
  20. Omerovic M, Müstak HK, Kaya IB. Escherichia coli Patotiplerinin Virulens Faktörleri. Etlik Vet Mikrobiyol Derg. 2017; 28: 1-6.
  21. Koca T. Bağirsak mikroflorasinin inflamatuvar hastalik patogenezindeki yeri. Arsiv kaynak tarama dergisi. 2015; 24: 78-91.
  22. Fei N, Zhao L. An opportunistic pathogen isolated from the gut of an obese human causes obesity in germfree mice. ISME J. 2013; 7: 880-884.
  23. Keskitalo A, Munukka E, Toivonen R, Hollmen M, Kainulainen H, Huovinen P, et al. Enterobacter cloacae administration induces hepatic damage and subcutaneous fat accumulation in high-fat diet fed mice. PLoS One. 2018; 13: e0198262.
  24. Munukka E, Wiklund P, Partanen T, Välimäki S, Laakkonen EK, Lehti M, et al. Adipocytes as a link between gut microbiota-derived flagellin and hepatocyte fat accumulation. PLoS One. 2016; 11: e0152786.
  25. Karimi-Zarchi M, Peighmbari F, Karimi N, Rohi M, Chiti Z. A Comparison of 3 Ways of Conventional Pap Smear, Liquid-Based Cytology and Colposcopy vs Cervical Biopsy for Early Diagnosis of Premalignant Lesions or Cervical Cancer in Women with Abnormal Conventional Pap Test. Int J Biomed Sci. 2013; 9: 205-210.

Citation: Zarf S and Arman I. Effect of Fluconazole, An Anti-Fungal Drug, On Human Flora Bacteria. J Bacteriol Mycol. 2020; 7(2): 1130.