Efficacy of Quercetin against Polymicrobial Biofilm on Catheters

 

Hasyrul Hamzah1,2, Triana Hertiani3*, Sylvia Utami Tunjung Pratiwi3, Titik Nuryastuti4

1Program Doctoral Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, 55281 Indonesia.

2Faculty of Health and Pharmacy, Universitas Muhammadiyah Kalimantan Timur, Samarinda,

Kalimantan Timur 75124, Indonesia.

3Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Gadjah Mada,

Yogyakarta, 55281 Indonesia.

4Department of Microbiology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta–55281, Indonesia.

*Corresponding Author E-mail: hertiani@ugm.ac.id

 

ABSTRACT:

Every year, the catheter-associated urinary tract infections (CAUTIs) experience a very significant number of increases. Urinary tract infections constitute about 30% of nosocomial infections, and about 75% of all bacterial species show biofilm production, which provides survival benefits to offering protection from environmental stresses and causing decreased susceptibility to antimicrobial agents. Until now the discovery of catheter antibiofilm compounds is still minimal, therefore the development of a new candidate antibiofilm for polymicrobial biofilms in catheters is a challenge that must be overcome in preventing catheter-associated urinary tract infections (CAUTIs). This study aimed to determine the effectiveness of quercetin in inhibited and decreased polymicrobial biofilm formation in catheters: Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Candida albicans. The test for inhibition of the degradation of polymicrobial biofilms on catheters was determined using the microtiter broth method. The use of quercetin on polymicrobial biofilms was analyzed by calculating the minimum biofilm inhibitory concentration (MBIC50) and the minimum value of biofilm eradication (MBEC50). The relationship of quercetin work against S. aureus, P. aeruginosa, E. coli, and C. albicans polymicrobial biofilms was tested by using scanning electron microscopy (SEM). Quercetin 1% gave 50% inhibitory activity to the formation of polymicrobial catheter biofilms at 24 h and 48 h at 53.55±0.01 and 50.38 ± 0.01 in comparison to control (nystatin and chloramphenicol). The results also proved evidence of quercetin activity that can degrade polymicrobial catheter biofilms by 46.48%±0.01 and damage the polymicrobial biofilm matrix extracellular polymeric substance (EPS) on the catheter. Quercetin seems to inhibitory activity against the formation of polymicrobial biofilms in catheters and is very potential to be developed as a candidate for new antibiofilm drugs against urinary tract infections.

 

KEYWORDS: Biofilm, catheter, urinary tract infections, quercetin.

 

 


INTRODUCTION:

Catheter-associated urinary tract infections (CAUTIs or CAUTI) is one type of infection associated with health care (healthcare-associated infection or HAI) the most commonly found hospital. Catheter urine is the leading cause of CAUTI, with 70-80 % scene any infection caused by biofilms1.

 

The frequency of CA-UTI in recent reports is 4.32/1000 device days for acute care hospitals2 (Weber dkk., 2011). In 2007, CA-UTI occurred in 2 to 3/1000 urine catheter days in US adult intensive care units (ICUs) participating in the CDC National Healthcare Safety Network3. At a Veterans hospital, 0.3% of catheter days involved asymptomatic UTI4. From 1990 to 2007, the incidence of CA-UTI in US critical care units declined, with a proportional decrease varying from 18.6% in cardiothoracic units to 67% in medical/surgical units. The CA-UTI infection rate in 398 intensive care units in Shanghai over five years was 6.4/1000 catheter days and varied from 0 in burn ICUs to 12.8/1000 in coronary care units4.

The high number of CAUTIs is associated with biofilm mode of growth of microbes. The biofilm mode of growth is advantageous for several reasons. The artificial surface of the implants facilitates adhesion of bacteria, which can, therefore, form a biofilm. The bacteria in biofilm are protected against drying, mechanical damage, and other influences of the outerenvironment. In the human body, the bacteria in biofilm are protected against the immunity system and antibiotic treatment5.

 

The organisms most frequently associated with CA-UTI in acute care facilities are Escherichia coli, Candida species, Enterococcus species, Pseudomonas aeruginosa, and Klebsiella species. In long-term care facility residents with chronic catheters, the most common bacteria are E. coli, Klebsiella pneumonia, Proteus mirabilis, P. aeruginosa. For bacteremic CA-UTI in these residents, E. coli, P. mirabilis, and Enterococcus species are most frequently isolated6.

 

Biofilms formed on indwelling urinary catheters and other urinary devices differ from
biofilms on non-urinary devices (indwelling vascular lines or endotracheal tubes)
because of the incorporation of urine components7.

 

Biofilm is a collection of microbial cells attached to irreversible on a surface and encased in a matrix Extracellular Polymer Substance (EPS) produced by themselves which are also accompanied by an increase in the number of phenotypes such as climate change and gene transcription from planktonic cells or free cells 8,9,10,11.

 

Until now there has not been much discovery of polymicrobial antibiofilm candidate combinations on catheters. Therefore we are competing to see the quercetin compensation for polymicrobial biofilms in catheters to look for new strategies to fight infections produced by biofilms on catheters.

 

MATERIALS AND METHODS:

Materials:

Quercetin is collections from Pharmacy-Biology Laboratory, Faculty of Pharmacy, UGM, Yogyakarta, Indonesia. Other materials include the following: crystal violet (Merck, Germany), ethyl acetate (Merck, Germany), Brain heart infusion (Oxoid) (Merck, Germany), RPMI 1640 (Sigma-Aldrich), catheter, ethanol 95% (Merck, Germany), nystatin, chloramphenicol (Sigma-Aldrich, Germany).

 

Equipment:

Laminar Air Flow, incubator (IF-2B) (Sakura, Japan), micropipette pipetman (Gilson, France), multichannel micropipette (Socorex, Swiss), microplate flat-bottom polystyrene 24 well (Iwaki, Japan), microtiter plate reader (Optic Ivymen System 2100-C, Spain), spektrofotometri (Genesys 10 UV Scanning, 335903) (Thermo Scientific Spectronic, USA), autoclave (Sakura, Japan), incubator with orbital shaker S1500 (Stuart, UK), analytical scales (AB204 -5, Switzerland).

 

Bacterial Strains:

A standard strain of Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), was cultured in tryptic soy broth (TSB) medium and incubated at 37⁰C for 72 h. Candida albicans (ATCC 10231) was cultured in Sabouraud Dextrose Broth (SDB) medium and incubated at 37⁰C with agitation at (120rpm) for 24 h. The optical densities (OD600) of microbial cultures will be adjusted to 0.1 (equal of the 0.5 McFarland standard ~1,5 x 108 CFU/ml), and subsequently diluted in fresh medium to OD600 0.01 for each microbial species.

 

Catheter biofilm inhibition activity:

Method of using12,13,14 with a little modification. The catheter is cut by one centimeter and then sterilized in 70% ethanol and allowed to dry. A total of 200µL of media inserted in each microtiter plate well are then incubated at ± 37ºC for 24 and 48 hours. After the incubation phase, the plate was washed with PBS. There was 200uL media that contained pure isolate with concentration series (1% b/v - 0125% b/v), was added to all washed-pits. Media that contained ethanol 1% was used as solvent control, and microbe suspension was used as negative control. A microbe suspension that was used an antifungal and anti-bacterial (chloramphenicol and nystatin 1% b/v) previously was used as positive control, while a media with no microbial growth was used as media control. The plate was then incubated at 37°C for 24 hours for mid-phase biofilm forming and 48 hours for maturing phase. Then, the plate was washed with PBS. Next, 125uL crystal violet 1% solution was added to each pit, next they were incubated at room temperature for 15 minutes. After the incubation, the microplate was washed with PBS and added with 200uL of ethanol 96% in each pit to dilute the formed biofilm. An Optical Density (OD) examination was performed with a microplate reader at 595nm wavelength.

 

Catheter biofilm degradation activity:

This method is similar to the method of catheter biofilm inhibition activity, but the difference is the laying time of the testing, in the degradation of the catheter biofilm for 6 days following the method9,14 with a little modification. Biofilm was inoculated inside a microtiter plate with the same manner as explained above. After incubated at 370C for 48 hours, the cultures from each pit were decantated, and planktonic cells were diminished by washing it with PBS. The biofilm cells were exposed by quercetin at several concentrations, started from 1% b/v and up to 0.125% b/v, later on they were incubated at 37oC for 48 hours. chloramphenicol and nystatin 1% b/v was used as positive control. After incubated, plates were washed three times with 200mL of sterile PBS in order to diminish any attached cells. Biofilm degradation was quantified by 125uL of crystal violet 1% solution in each pit; then incubated at room temperature for 15 minutes. After incubation, microplates were washed with PBS and ethanol 96% was added inside each pit to dilute the biofilm formed. An Optical Density (OD) examination was performed with microplate reader at 595 nm wavelength.

 

Scanning electron microscopy:

The catheter was inserted inside the microtiter plate round bottom polystyrene 24 well that contained testing suspension that had been given a similar treatment with biofilm inhibition assay. The catheter then incubated on 37 ºC for 24-48 hours, continued to the careful washing of the catheter for three-time with sterile aquadest, then fixated with 2,5 % (v/v) glutaraldehyde inside cacodylate buffer for ± 24 hours with the aim of cell's death without changing the cell's structure that will be observed. Next, a dehydration process using methanol was done for 30 minutes to minimize the water amount so that the observing process could not be interrupted. The sample then observed under Scanning Electron Microscopy (SEM) with a voltage of 10 Kv16.

 

RESULT:

Quercetin Effect on Mid-phase (24 h) Polymicrobial biofilm on catheter:

In this study, we evaluated the potential of quercetin antibiofilm on inhibition of polymicrobial biofilms on catheters. Our results indicate that quercetin 1% provides an activity equal to 53,55 % ± 0,01*, while the drug control was chloramphenicol and nystatin as large as 66.05± 0,01*, 57,58± 0,01* (Fig. 1)

 

Figure 1. Quercetin Effect on Mid-phase (24 h) Polymicrobial biofilm on catheter.

 

These results indicate that quercetin comparison can inhibit 50% growth of polymicrobial biofilms on catheters and not much different from the drug control of nystatin and lower than the drug control of chloramphenicol. This is evidence that quercetin inhibits the growth of biofilm C. albicans compared to biofilms made by bacteria on catheters.

 

Quercetin Effect on Maturation-phase (48 h) Polymicrobial biofilm on catheter:

In the 48-hour phase the quercetin compound gave inhibition of 50.58 ± 0.02 *, control of the drug nystatin and control of chloramphenicol as much as 55,95 % ± 0,01, 64,20 % % ± 0,03 (Fig 2).

 

Figure 2. Quercetin Effect on Maturation-phase (48 h) Polymicrobial biofilm on catheter

 

These data show that quercetin can still inhibit biofilm growth and almost has an inhibition practically equivalent to the drug control of nystatin, although biofilm growth in this phase is more stable and well organized.

 

Quercetin Effect on degradation- Polymicrobial biofilm on catheter:

The degradation phase is the most extended phase in biofilm formation, where the quercetin compound is only able to provide inhibition of 46,48 % ±0,02* whereas nystatin and chloramphenicol give the inhibitory activity of 52,85 % ± 0,01, 60,88 % ± 0,01 (Fig 3).

 

Figure 3. Quercetin Effect on degradation- Polymicrobial biofilm on catheter

 

In this phase quercetin compounds are more difficult to penetrate polymicrobial biofilm defenses on catheters because the matrix structure of biofilms formed in this phase is more numerous, complex and very strong, as well as drug control of nystatin and chloramphenicol, drug control has decreased inhibitory activity compared to phase 24 and 48 hours, because the biofilm structure in this phase forms a community and cell communication called quorum sensing.

 

The Result of Scanning Electron Microscopy Polymicrobial Biofilm on catheter With No Treatment

Polymicrobial biofilms on catheters that are not treated show a very dense and accumulated cell density and show very thick EPS production (Fig 4).

 

In this picture, microbes can form highly structured and complex biofilms on the catheter duct; they attach and change the surface of the urinary catheter by providing or blocking the receptor area for uropathogens. This causes the compound to experience difficulty in providing maximum inhibition due to the thickness of the EPS matrix, which protects the biofilm on the catheter.

 

According to Beiko (2004) the structure of the biofilm is very complex and consists of 3 layers: the innermost part or connecting layer that attaches to the surface of the tissue or catheter material, the base layer in which there are microorganisms attached, and the outer layer called the surface layer can be used as an entrance for planktonic organisms25.

 

Also presence of microbes in different stages and forms plays an important role in the mixed-species biofilm formation, for example the Candida species form pseudohyphae in their biofilm mode of growth; the strains of the genus Proteus may profit from close contact with each other, because in the formation of parallel cells they are capable of faster movement on the catheter surface and they produce higher amount of extracellular polysaccharides26.

 

The Result of Scanning Electron Microscopy Polymicrobial Biofilm on catheter with administration of quercetin compounds 1% b/v:

Giving quercetin compounds 1% b/v (Fig. 4b) in polymicrobial biofilms P. aeruginosa - E. coli - C. albicans - S. aureus on the catheter causes a decrease in the amount of cell density indicated by cells becoming ruptured, deformed and cell leakage. This is because quercetin compounds damage EPS, which is a protective biofilm.

 

Another mechanism of quercetin in inhibiting and killing polymicrobial biofilms in catheters based on changes in biofilm layers is lack of nutrients and decreased oxygen levels possessed by biofilms due to the entry of quercetin compounds, this condition causes cells to become abnormal and experience dead. according to Hamzah (2019), Cell's leakage was caused by the hydrophobic bond that consisted of the membrane-forming components such as protein and phospholipid were broken27.

 

DISCUSSION:

Biofilm formation, in some cases, is the first step in the creation of crust in the catheter. The urea-producing bacteria cause an increase in the pH of the urine and once tangent to the biofilm, causing precipitation and magnesium and calcium to form the struvite crust along the catheter surface17,28,29,30

 

This process occurs due to bacteria forming biofilms on the catheter providing a relatively strong defense, which causes the bacteria S. aureus, E. coli and P. aeruginosa to synergize to create an involved community and cause quercetin compounds not easily penetrate the case of bacterial biofilms. The structure of biofilms and bacteria that are embedded in them is like tissue from a high-level organism. It is shown that bacteria in biofilms can communicate with each other and initiate the release and attachment of cells.

 

Organisms associated with growth biofilms are firmly attached to the surface and biofilm matrix. Biofilm matrix consists of microorganisms and extracellular polymeric tissue produced by microbes involved. One of the best distinguishing characteristics of this specific biofilm is the development of antimicrobial resistance that can reach 1,000 times18. Based on the literature it is explained that bacteria can synergistically form biofilms with other bacterial species, and physically and physiologically the thicker and stronger biofilm structure19,20,31,32,33

 

Urinary tract infections in patients who have catheters installed can arise in various ways. Urinary tract infections can be extensively or intraluminal. Our results show that in the 48-hour phase, there was a decrease in quercetin inhibitory activity against biofilm formation in the catheter. This is because, in this phase, the growth of the biofilm in the catheter is longer than the 24-hour phase. This situation causes the biofilm formed on the catheter to be thicker and more complex than the 24-hour phase. Consequently, quercetin compounds and drug control in this phase are more difficult to penetrate the target cell. Lewis (2002) suggested that the effectiveness of beta-lactam antibiotics decreased when antibiotics inhibited bacterial biofilm cells.

 

According to Reid (1996), the initial step of biofilm formation on a catheter is a buildup of materials contained in urine (protein, electrolytes, and organic molecules) which is called the conditioning layer22. This process takes place within a matter of minutes after the device is installed and changes the surface of the urine catheter material by providing or blocking the receptor area for urupatogens.

 

Interaction between multi-species plays a role in colonization and causes of infection. Infections that occur because biofilms are resistant to antibiotics, causing disease to worsen and high mortality23,35,36,37

 

This caused by the more extended the biofilm growth time, the joint venture among the bacteria community was stronger, so the antibiotic was hard to penetrate 9. In this phase, the bacteria form a very thick biofilm along the catheter surface and make the catheter duct become blocked. As a result, the drug compound cannot reach the target cell.

 

Studies of catheter-related Candida infection have shown that retention of vascular catheters colonized with Candida species is associated with prolonged fungemia, high antifungal therapy failure rates, an increased risk of metastatic complications, and death 24

 

The degradation phase causes the biofilm to form crystals, and these biofilms can build on the outer surface of the catheter around the balloon and the tip of the catheter, and result in trauma to the bladder and epithelial urethra. At the time of the bladder and initiate stone formation, the main complication is blocking the flow of urine through the catheter which results in the buildup of crystalline material in the lumen. As a result of the urine sometimes exits the catheter.

 

CONCLUSION:

Quercetin compounds have activity as polymicrobial antibiofilm on catheters by inhibiting the middle phase and maturation of biofilms at concentration levels 1% b/v. Based on the results of Scanning electron microscopy (SEM), quercetin was able to damage the EPS matrix of polymicrobial biofilms on the catheter. Because quercetin is very potential to be developed as a candidate for polymicrobial antibiofilm drugs in catheters.

 

ACKNOWLEDGEMENT:

Authors want to extend their gratitude to the Microbiology Laboratory in the Faculty of Pharmacy Universitas Gadjah Mada.

 

REFERENCES:

1.          Nicolle, L.E. Catheter-associated urinary tract infections. Antimicrobial Resistance and Infection Control, 2014. 3: 23.

2.          Weber, D.J., Sickbert-Bennett, E.E., Gould, C.V., Brown, V.M., Huslage, K., dan Rutala, W.A. Incidence of catheter-associated and non-catheter-associated urinary tract infections in a healthcare system. Infection Control and Hospital Epidemiology, 2011. 32: 822–823.

3.          Burton, D.C., Edwards, J.R., Srinivasan, A., Fridkin, S.K., dan Gould, C.V. Trends in catheter-associated urinary tract infections in adult intensive care units-United States, 1990-2007. Infection Control and Hospital Epidemiology, 2011. 32: 748–756.

4.          Tao, L., Hu, B., Rosenthal, V.D., Gao, X., dan He, L. Device-associated infection rates in 398 intensive care units in Shanghai, China: International Nosocomial Infection Control Consortium (INICC) findings. International Journal of infectious diseases: IJID: official publication of the International Society for Infectious Diseases, 2011. 15: e774-780.

5.          Stepanovic S, Djukic N, Opavski N, and Djukic S. Significance of inoculum size in biofilm formation by staphylococci. New Microbiol. 2003. 26:129–32.

6.          Mylotte, J.M. Nursing home-acquired bloodstream infection. Infection Control and Hospital Epidemiology, 26: 833–837 (2005).

7.          Tenke, P., Riedl, C.R., Jones, G.L., Williams, G.J., Stickler, D., dan Nagy, E. Bacterial biofilm formation on urologic devices and heparin coating as a preventive strategy. International Journal of Antimicrobial Agents, 2004. 23 Suppl 1: S67-74.

8.          Keshvardoust, P. et al. Biofilm formation inhibition and dispersal of multi-species communities containing ammonia-oxidising bacteria. npj Biofilms and Microbiomes. 2019. Vol. 5: 22.

9.          Hamzah, H., Pratiwi, S.U.T., and, Hertiani, T. Efficacy of Thymol and Eugenol Against Polymicrobial Biofilm. E. coli, 2018. 29: 8.

10.        Pierce, C.G., Vila, T., Romo, J.A., Montelongo-Jauregui, D., Wall, G., Ramasubramanian, A. The Candida albicans Biofilm Matrix: Composition, Structure, and Function. Journal of Fungi (Basel, Switzerland). 2017

11.        Pratiwi SUT., Lagendijk EL., de Weert S., Hertiani T., Idroes R., Van Den Hondel CAMJJ. Effect of Cinnamomumburmannii Nees ex Bl. and Massoiaaromatica Becc. Essential oils on planktonic growth and biofilm formation of Pseudomonas aeruginosa and Staphylococcus aureus in vitro. International Journal of Applied Research in Natural Product. 2015. 8, 1-13.

12.        Hasyrul Hamzah, Triana Hertiani, Sylvia Utami Tunjung Pratiwi and Titik Nuryastuti. Inhibitory activity and degradation of curcumin as Anti-Biofilm Polymicrobial on Catheters. Int. J. Res. Pharm. Sci. 2002. 11, 830–835.

13.        Stepanovic S, Djukic N, Opavski N, and Djukic S. Significance of inoculum size in biofilm formation by staphylococci. New Microbiol; 2003. 26:129–32.

14.        Holá V., Růžička F., Votava M.: Impact of surface coating on the adherence of slime producing and nonproducing Staphylococcus epidermidis; Microbiologica; 2004. vol. 27; 3, p. 305-308.

15.        Hess, D.J., Henry-Stanley, M.J., Barnes, A.M.T., Dunny, G.M., Wells, C.L. Ultrastructure of a Novel Bacterial Form Located in Staphylococcus aureus In Vitro and In Vivo Catheter-Associated Biofilms. J. Histochem. Cytochem.2012. 60, 770–776.

16.        Sofer, M. dan Denstedt, J.D. Encrustation of biomaterials in the urinary tract. Current Opinion in Urology, 2000. 10: 563–569.

17.        Andes, D., Nett, J., Oschel, P., Albrecht, R., Marchillo, K., dan Pitula, A. Development and Characterization of an In Vivo Central Venous Catheter Candida albicans Biofilm Model. Infection and Immunity, 2004. 72: 6023–6031.

18.        Andersson, S., Kuttuva Rajarao, G., Land, C.J., dan Dalhammar, G. Biofilm formation and interactions of bacterial strains found in wastewater treatment systems: Biofilm formation and interactions of bacterial strains. FEMS Microbiology Letters, 2008. 283: 83–90.

19.        Cowan, S.E., Gilbert, E., Liepmann, D., dan Keasling, J.D., n.d. 2015. 'Commensal Interactions in a Dual-Species Biofilm Exposed to Mixed Organic Compounds

20.        Leriche, V., Briandet, R., dan Carpentier, B. Ecology of mixed biofilms subjected daily to a chlorinated alkaline solution: spatial distribution of bacterial species suggests a protective effect of one species to another. Environmental Microbiology, 2003. 5: 64–71.

21.        Reid, G., H. C. van der Mei, C. Tieszer, and H. J. Busscher. Uropathogenic Escherichia coli adhere to urinary catheters without using fimbriae. FEMS Immunol. Med. Microbiol. 1996. 16:159–162.

22.        Harriott, M.M. dan Noverr, M.C. Importance of Candida–bacterial polymicrobial biofilms in disease. Trends in Microbiology, 2011. 19: 557–563.

23.        Lewis, R.E., Kontoyiannis, D.P., Darouiche, R.O., Raad, I.I., dan Prince, R.A. Antifungal activity of amphotericin B, fluconazole, and voriconazole in an in vitro model of Candida catheter-related bloodstream infection. Antimicrobial Agents and Chemotherapy, 2002. 46: 3499–3505.

24.        Luzzati, R., Amalfitano, G., Lazzarini, L., Soldani, F., Bellino, S., Solbiati, M. Nosocomial candidemia in non-neutropenic patients at an Italian tertiary care hospital. European Journal of Clinical Microbiology and Infectious Diseases: Official Publication of the European Society of Clinical Microbiology, 2000. 19: 602–607.

25.        Beiko, D. T., Knudsen, B. E., Watterson, J. D. Urinary tract biomaterials.J Urol ;171(6 Pt 1):2438–44(2004)

26.        Stickler, D., Hughes, G.: Ability of Proteus mirabilis to swarm over urethral catheters; Eur.J.Clin.Microbiol.Infect.Dis. 1999. 18, 3, 206-208.

27.        Hamzah, H., Pratiwi, S.U.T., Hertiani and Nuryastuti.T. The activity of Tannin on the Formation of Mono-Species and Polymicrobial Biofilm Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. TradMedJ. 2019. Vol 24, No 2.

28.        Hamzah, H., Hertiani, T., Pratiwi, S. U. T., Murti, Y. B. and Nuryastuti, T. The Inhibition and Degradation Activity of Demethoxycurcumin as Antibiofilm on C. albicans ATCC 1023. Research J. Pharm. and Tech. 2020, (13), 1.

29.        T, T. D. and P, G. Biofilm Formation Among Enterococci Species. Res. J. Pharm. Technol. 9, 1877–1879 (2016).

30.        Abbas, H. A., Serry, F. M. and EL-Masry, E. M. N-acetylcysteine and Ambroxol: can mucolytics dissolve the resistance of biofilms to antibiotics. Res. J. Pharm. Technol. 5, 912–917 (2012).

31.        Abbas, H. A., Serry, F. M. and EL-Masry, E. M. Non-steroidal anti-inflammatory drugs and sodium ascorbate potentiate the antibiotic activity against Pseudomonas aeruginosa biofilms. Res. J. Pharm. Technol. 5, 1124–1129 (2012).

32.        Abbas, H. A., Serry, F. M. and EL-Masry, E. M. Biofilms: The Microbial Castle of Resistance. Res. J. Pharm. Technol. 6, 01–03 (2013).

33.        Abbas, H. A., Abdo, I. M. and Moustafa, M. Z. In vitro Antibacterial and Antibiofilm Activities of Hibiscus sabdariffa L. Extract and Apple Vinegar against Bacteria Isolated from Diabetic Foot Infections. Res. J. Pharm. Technol. 7, 131–136 (2014).

34.        Abbas, H. A., El-Sayed, M. A., Kamel, M. M. and Gamil, L. Allium kurrat and Eruca sativa are Natural agents for Inhibition and Eradication of Enterohemorrhagic Escherichia coli O157:H7 Biofilm. Res. J. Pharm. Technol. 7, 425–428 (2014).

35.        Pushpam, A. C., Chelvan, R. K. Y., Ramalingam, K. and Vanitha, M. C. Evaluation of the Antibiofilm Properties of Arthrobacter defluvii AMET1677 Strain Isolated from Shrimp Pond Sediment against Marine Biofilm Forming Bacteria. Res. J. Pharm. Technol. 9, 373–380 (2016).

36.        Kareem, M. H. and Hasan, A. Y. Inhibition of Biofilm formation of Imipenem-resistant Acinetobacter baumannii using Curcuma longa extracts, silver nanoparticles and Azithromycin. Res. J. Pharm. Technol. 12, 4463–4470 (2019).

37.        Yazigi, H., Khamees, A. and Wakil, H. Acquired Urinary Tract Infection in the Public Hospital. Res. J. Pharm. Technol. 12, 1255–1258 (2019).

 

 

 

 

Received on 31.01.2020           Modified on 18.03.2020

Accepted on 03.05.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2020; 13(11):5277-5282.

DOI: 10.5958/0974-360X.2020.00923.3