FIELD OF THE INVENTION

The present disclosure is generally related to nanocomposites. Particularly, the present disclosure is related to a nanocomposite for treating urinary tract infections. More particularly, the present disclosure is related to: a nanocomposite for treating urinary tract infections; and method of synthesis thereof.

BACKGROUND OF THE INVENTION

Recurrences of urinary tract infections are a leading cause of antibiotic failure. Uropathogenic E.coli strains cause more than 80% of urinary tract infections worldwide. They can survive inside a gut, in addition to colonising, in extraintestinal sites, thereby, causing infections. Further, they are resistant, to all classes of antibacterial drugs available (including the most recent classes of drugs).

Though type-A procyanidins have been shown to reduce bacterial adherence, to bladder cells, they suffer from poor bioavailability; hence, their usage is limited.

Recently, metal-based nanoparticles have been explored, as an option, for urinary tract infections. But they come with a cost of damage, to host cells and tissues. Entry of zinc oxide nanoparticles, into human organs and organelles, has adverse effects. These effects include: increased macrophage aggregation; inflammation; platelet aggregation; cardiopulmonary toxicity; and/or the like.

There is, therefore, a need in the art, for: a nanocomposite for treating urinary tract infections; and method of synthesis thereof, which overcome the aforementioned drawbacks and shortcomings.

SUMMARY OF THE INVENTION

A nanocomposite for treating bacterial urinary tract infections is disclosed. Said nanocomposite broadly comprises: an about 10 mg/mL of zinc oxide nanoparticles; an about 2 mg/mL of type-A procyanidin; and methanol. Said zinc oxide nanoparticles are of sizes that range between about 20 nm and about 80 nm. A method of synthesising said nanocomposite broadly comprises following steps:

An about 10 mg/mL of zinc oxide nanoparticles are dispersed, in methanol, to obtain dispersed zinc oxide nanoparticles. Said zinc oxide nanoparticles are of sizes that range between about 20 nm and about 80 nm.

An about 2 mg/mL of type-A procyanidin (dissolved in methanol) is mixed, with said dispersed zinc oxide nanoparticles, to obtain a mixture. Said mixture is kept, under constant stirring, at about 150 RPM, for about 24 hours.

Unbound type-A procyanidin is washed several times, by centrifuging, and kept for drying, to obtain said nanocomposite.

Said nanocomposite was determined to be non-toxic, to macrophages, at all tested concentrations. In vero kidney cell lines and T24 bladder cell lines, said nanocomposite was determined to be toxic, only at high concentrations.

The disclosed nanocomposite and method offer at least the following synergistic advantages and effects: low MIC50 values; good bacterial biofilm inhibiting properties; eradicate pre-formed bacterial biofilms and dispersed bacterial cells; eradicate adhered bacterial cells; offer superior safety profiles.

The disclosed nanocomposite may be directly coated, onto catheters, for treating bacterial urinary tract infections. Alternatively, or in addition, it may be part of a kit, for treating bacterial urinary tract infections.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1(a) and Figure 1(b) illustrate colour change, during synthesis of TZnO nanoparticles, in accordance with an embodiment of the present disclosure;

Figure 1(c) illustrates ultraviolet-visible spectra, of ZnO nanoparticles, TZnO nanoparticles, and TAP, in accordance with various embodiments of the present disclosure;

Figure 1(d) illustrates X-ray diffraction spectra, of ZnO nanoparticles and TZnO nanoparticles, in accordance with various embodiments of the present disclosure; Figure 2(a) illustrates a field emission scanning electron micrograph of ZnO nanoparticles, in accordance with an embodiment of the present disclosure;

Figure 2(b) illustrates a field emission scanning electron micrograph of TZnO nanoparticles, in accordance with an embodiment of the present disclosure;

Figure 2(c) illustrates comparative Fourier-transform infrared spectra, of ZnO nanoparticles, TZnO nanoparticles, and TAP, in accordance with various embodiments of the present disclosure;

Figure 2(d) illustrates results thermogravimetric analyses, of ZnO nanoparticles, TZnO nanoparticles, and TAP, in accordance with various embodiments of the present disclosure;

Figure 3(a) illustrates results of dose-response studies, of ZnO nanoparticles and TZnO nanoparticles, against UTI89 E.coli strain, in accordance with various embodiments of the present disclosure;

Figure 3(b) illustrates results of dose-response studies, of ZnO nanoparticles and TZnO nanoparticles, against QSLUPEC5 E.coli strain, in accordance with various embodiments of the present disclosure;

Figure 3(c) illustrates results of dose-response studies, of ZnO nanoparticles and TZnO nanoparticles, against QSLUPEC6 E.coli strain, in accordance with various embodiments of the present disclosure;

Figure 3(d) illustrates results of dose-response studies, of ZnO nanoparticles and TZnO nanoparticles, against QSLUPEC7 E.coli strain, in accordance with various embodiments of the present disclosure;

Figure 4(a) illustrates reductions, in metabolically active cells of UTI89 E.coli strain, upon treatments, with ZnO nanoparticles and TZnO nanoparticles, at MIC50 and MIC values, in accordance with various embodiments of the present disclosure;

Figure 4(b) illustrates reductions, in metabolically active cells of QSLUPEC5 E.coli strain, upon treatments, with ZnO nanoparticles and TZnO nanoparticles, at MIC50 and MIC values, in accordance with various embodiments of the present disclosure; Figure 4(c) illustrates reductions, in metabolically active cells of QSLUPEC6 E.coli strain, upon treatments, with ZnO nanoparticles and TZnO nanoparticles, at MIC50 and MIC values, in accordance with various embodiments of the present disclosure;

Figure 4(d) illustrates reductions, in metabolically active cells of QSLUPEC7 E.coli strain, upon treatments, with ZnO nanoparticles and TZnO nanoparticles, at MIC50 and MIC values, in accordance with various embodiments of the present disclosure;

Figure 5(a) illustrates a scanning electron micrograph of untreated controls, in accordance with an embodiment of the present disclosure;

Figure 5(b) illustrates a scanning electron micrograph, upon treatments of UTI89 E.coli strain with TZnO nanoparticles, in accordance with an embodiment of the present disclosure;

Figure 5(c) illustrates a scanning electron micrograph, upon treatments of UTI89 E.coli strain with ZnO nanoparticles, in accordance with an embodiment of the present disclosure;

Figure 6(a) illustrates results of dose-response studies (biofilm inhibitory activities), upon treatments, with ZnO nanoparticles and TZnO nanoparticles, at sub-MIC values, against UTI89 E.coli strain, QSLUPEC5 E.coli strain, QSLUPEC6 E.coli strain, and QSLUPEC7 E.coli strain, in accordance with various embodiments of the present disclosure;

Figure 6(b) illustrates reductions, in metabolically active UTI89 biofilms, QSLUPEC5 biofilms, QSLUPEC6 biofilms, and QSLUPEC7 biofilms, upon treatments, with ZnO nanoparticles and TZnO nanoparticles, at MBIC50 and MBIC values, in accordance with various embodiments of the present disclosure;

Figure 7(a) illustrates a confocal laser scanning microscope image of untreated controls, in accordance with an embodiment of the present disclosure;

Figure 7(b) illustrates a confocal laser scanning microscope image, upon treatments of UTI89 biofilms with TZnO nanoparticles, in accordance with an embodiment of the present disclosure; Figure 7(c) illustrates a confocal laser scanning microscope image, upon treatments of UTI89 biofilms with ZnO nanoparticles, in accordance with an embodiment of the present disclosure;

Figure 7(d) illustrates reductions, in average thickness, upon treatments of UTI89 biofilms, with ZnO nanoparticles and TZnO nanoparticles, in accordance with various embodiments of the present disclosure;

Figure 7(e) illustrates reductions, in average biomass, upon treatments of UTI89 biofilms, with ZnO nanoparticles and TZnO nanoparticles, in accordance with various embodiments of the present disclosure;

Figure 8(a) illustrates a scanning electron micrograph of untreated controls, in accordance with an embodiment of the present disclosure;

Figure 8(b) illustrates a scanning electron micrograph, upon treatments of UTI89 E.coli strain with TZnO nanoparticles, in accordance with an embodiment of the present disclosure;

Figure 8(c) illustrates a scanning electron micrograph, upon treatments of UTI89 E.coli strain with ZnO nanoparticles, in accordance with an embodiment of the present disclosure;

Figure 9(a) illustrates results of gene expression studies, of adhesion and invasion-related genes, upon treatments of UTI89 E.coli strain, with ZnO nanoparticles and TZnO nanoparticles, at MBIC values, in accordance with various embodiments of the present disclosure;

Figure 9(b) illustrates results of gene expression studies, of adhesion and invasion-related genes, upon treatments of QSLUPEC5 E.coli strain, with ZnO nanoparticles and TZnO nanoparticles, at MBIC values, in accordance with various embodiments of the present disclosure;

Figure 9(c) illustrates results of gene expression studies, of adhesion and invasion-related genes, upon treatments of QSLUPEC6 E.coli strain, with ZnO nanoparticles and TZnO nanoparticles, at MBIC values, in accordance with various embodiments of the present disclosure; Figure 9(d) illustrates results of gene expression studies, of adhesion and invasion-related genes, upon treatments of QSLUPEC7 E.coli strain, with ZnO nanoparticles and TZnO nanoparticles, at MBIC values, in accordance with various embodiments of the present disclosure;

Figure 10(a) illustrates reductions, in metabolically active dispersed cells and biofilms, of UTI89 E.coli strain, upon treatments, with ZnO nanoparticles and TZnO nanoparticles, in accordance with various embodiments of the present disclosure;

Figure 10(b) illustrates reductions, in metabolically active dispersed cells and biofilms, of QSLUPEC5 E.coli strain, upon treatments, with ZnO nanoparticles and TZnO nanoparticles, in accordance with various embodiments of the present disclosure;

Figure 10(c) illustrates reductions, in metabolically active dispersed cells and biofilms, of QSLUPEC6 E.coli strain, upon treatments, with ZnO nanoparticles and TZnO nanoparticles, in accordance with various embodiments of the present disclosure;

Figure 10(d) illustrates reductions, in metabolically active dispersed cells and biofilms, of QSLUPEC7 E.coli strain, upon treatments, with ZnO nanoparticles and TZnO nanoparticles, in accordance with various embodiments of the present disclosure;

Figure 11(a) illustrates a confocal laser scanning microscope image of untreated controls, in accordance with an embodiment of the present disclosure;

Figure 11(b) illustrates a confocal laser scanning microscope image, upon treatments of UTI89 biofilms with TZnO nanoparticles, in accordance with an embodiment of the present disclosure;

Figure 11(c) illustrates a confocal laser scanning microscope image, upon treatments of UTI89 biofilms with ZnO nanoparticles, in accordance with an embodiment of the present disclosure;

Figure 11(d) illustrates reductions, in average biomass, upon treatments of UTI89 biofilms, with ZnO nanoparticles and TZnO nanoparticles, in accordance with various embodiments of the present disclosure; Figure 11(e) illustrates reductions, in average thickness, upon treatments of UTI89 biofilms, with ZnO nanoparticles and TZnO nanoparticles, in accordance with various embodiments of the present disclosure;

Figure 12(a) illustrates a scanning electron micrograph of untreated controls, in accordance with an embodiment of the present disclosure;

Figure 12(b) illustrates a scanning electron micrograph, upon treatments of UTI89 E.coli strain with TZnO nanoparticles, in accordance with an embodiment of the present disclosure;

Figure 12(c) illustrates a scanning electron micrograph, upon treatments of UTI89 E.coli strain with ZnO nanoparticles, in accordance with an embodiment of the present disclosure;

Figure 13 illustrates results of toxicity studies, of TZnO nanoparticles, compared with ZnO nanoparticles, against RAW macrophages, in accordance with various embodiments of the present disclosure;

Figure 14(a) illustrates release of reactive oxygen, upon treatments with TZnO nanoparticles, in accordance with various embodiments of the present disclosure;

Figure 14(b) illustrates release of reactive oxygen, upon treatments with ZnO nanoparticles, in accordance with various embodiments of the present disclosure;

Figure 14(c) illustrates release of nitrates, upon treatments with TZnO nanoparticles, in accordance with various embodiments of the present disclosure;

Figure 14(d) illustrates release of nitrates, upon treatments with ZnO nanoparticles, in accordance with various embodiments of the present disclosure;

Figure 15(a) illustrates results of toxicity studies, of TZnO nanoparticles, compared with ZnO nanoparticles, against vero kidney epithelial cells, in accordance with various embodiments of the present disclosure;

Figure 15(b) illustrates results of toxicity studies, of TZnO nanoparticles, compared with ZnO nanoparticles, against T24 urinary bladder epithelial cells, in accordance with various embodiments of the present disclosure; Figure 16(a) illustrates surfaces of T24 bladder cells colonised, by cells of UTI89 E.coli strain, in untreated controls, in accordance with an embodiment of the present disclosure;

Figure 16(b) illustrates surfaces of T24 bladder cells colonised, by cells of UTI89 E.coli strain, upon treatments with TZnO nanoparticles, in accordance with an embodiment of the present disclosure;

Figure 16(c) illustrates surfaces of T24 bladder cells colonised, by cells of UTI89 E.coli strain, upon treatments with ZnO nanoparticles, in accordance with an embodiment of the present disclosure;

Figure 16(d) illustrates reductions, in adhered bacteria, upon treatments with TZnO nanoparticles, in accordance with various embodiments of the present disclosure;

Figure 17(a) illustrates T24 bladder monolayer cells invaded, by cells of UTI89 E.coli strain, in untreated controls, in accordance with an embodiment of the present disclosure;

Figure 17(b) illustrates T24 bladder monolayer cells invaded, by cells of UTI89 E.coli strain, upon treatments with TZnO nanoparticles, in accordance with an embodiment of the present disclosure;

Figure 17(c) illustrates T24 bladder monolayer cells invaded, by cells of UTI89 E.coli strain, upon treatments with ZnO nanoparticles, in accordance with an embodiment of the present disclosure;

Figure 17(d) illustrates reductions, in invaded cells, upon treatments with TZnO nanoparticles, in accordance with various embodiments of the present disclosure;

Figure 18(a) illustrates T24 bladder cells infected and adhered, with cells of UTI89 E.coli strain, in untreated controls, in accordance with an embodiment of the present disclosure;

Figure 18(b) illustrates T24 bladder cells infected and adhered, with cells of UTI89 E.coli strain, upon treatments with TZnO nanoparticles, in accordance with an embodiment of the present disclosure;

Figure 18(c) illustrates T24 bladder cells infected and adhered, with cells of UTI89 E.coli strain, upon treatments with ZnO nanoparticles, in accordance with an embodiment of the present disclosure; Figure 18(d) illustrates reductions, in invaded cells, upon treatments with TZnO nanoparticles, in accordance with various embodiments of the present disclosure;

Figure 19(a) illustrates a microscopic image of T24 bladder cells infected and adhered, with cells of UTI89 E.coli strain, in untreated controls, in accordance with an embodiment of the present disclosure;

Figure 19(b) illustrates a microscopic image of T24 bladder cells infected and adhered, with cells of UTI89 E.coli strain, upon treatments with TZnO nanoparticles, in accordance with an embodiment of the present disclosure;

Figure 19(c) illustrates a microscopic image of T24 bladder cells infected and adhered, with cells of UTI89 E.coli strain, upon treatments with ZnO nanoparticles, in accordance with an embodiment of the present disclosure;

Figure 20(a)(i) illustrates a topological view of an uncoated catheter, in accordance with an embodiment of the present disclosure;

Figure 20(a)(ii) illustrates a topological view of a catheter coated with TZnO nanoparticles, in accordance with an embodiment of the present disclosure;

Figure 20(a)(iii) illustrates a topological view of a catheter coated with ZnO nanoparticles, in accordance with an embodiment of the present disclosure;

Figure 20(b)(iv) illustrates a cross-sectional view of an uncoated catheter, in accordance with an embodiment of the present disclosure;

Figure 20(b)(v) illustrates a cross-sectional view of a catheter coated with TZnO nanoparticles, in accordance with an embodiment of the present disclosure;

Figure 20(b)(vi) illustrates a cross-sectional view of a catheter coated with ZnO nanoparticles, in accordance with an embodiment of the present disclosure;

Figure 20(c)(vii) illustrates an extraluminal side of an uncoated catheter, in accordance with an embodiment of the present disclosure;

Figure 20(c)(viii) illustrates an extraluminal side of a catheter coated with TZnO nanoparticles, in accordance with an embodiment of the present disclosure; Figure 20(c)(ix) illustrates an extraluminal side of a catheter coated with ZnO nanoparticles, in accordance with an embodiment of the present disclosure;

Figure 21(a) illustrates a scanning electron micrograph of bacterial accumulations, in an uncoated catheter, in accordance with an embodiment of the present disclosure;

Figure 21(b) illustrates a scanning electron micrograph of bacterial accumulations, in a catheter coated with TZnO nanoparticles, in accordance with an embodiment of the present disclosure;

Figure 21(c) illustrates a scanning electron micrograph of bacterial accumulations, in a catheter coated with ZnO nanoparticles, in accordance with an embodiment of the present disclosure; and

Figure 21(d) illustrates reductions, in biofilms, in catheters coated with ZnO nanoparticles and TZnO nanoparticles, in accordance with various embodiments of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this specification, the use of the words “comprise” and “include”, and variations such as “comprises”, “comprising”, “includes”, and “including”, may imply the inclusion of an element (or elements) not specifically recited. Further, the disclosed embodiments may be embodied, in various other forms, as well.

Throughout this specification, the use of the acronym “ZnO” is to be construed as: “Zinc Oxide”.

Throughout this specification, the use of the acronym “TAP” is to be construed as: “Type- A Procyanidin”.

Throughout this specification, the use of the phrase “TZnO nanoparticles” is to be construed as: “synthesised nanocomposite” (or synthesised nanocomposites).

Throughout this specification, the use of the phrase “treatments with TZnO nanoparticles” is to be construed as: “treatments with synthesised nanocomposite” (or treatments with synthesised nanocomposites). Throughout this specification, the use of the acronym “MIC” is to be construed as: “Minimum Inhibitory Concentration”.

Throughout this specification, the use of the acronym “MIC50” is to be construed as: “MIC value, at which, 50% of isolates, in a test population, are inhibited”.

Throughout this specification, the use of the acronym “MBIC” is to be construed as: “Minimum Biofilm Inhibitory Concentration”.

Throughout this specification, the use of the acronym “MBIC50” is to be construed as: “MBIC value, at which, 50% of biofilms, in a test population, are inhibited”.

Throughout this specification, the use of the word “bacteria” is to be construed as being inclusive of: uropathogenic E.coli.

Throughout this specification, the disclosure of a range is to be construed as being inclusive of: the lower limit of the range; and the upper limit of the range.

Throughout this specification, the words “the” and “said” are used interchangeably.

Also, it is to be noted that embodiments may be described as a method. Although the operations in a method are described as a sequential process, many of the operations may be performed in parallel, concurrently, or simultaneously. In addition, the order of the operations may be re-arranged. A method may be terminated, when its operations are completed, but may also have additional steps.

A nanocomposite is disclosed. The disclosed nanocomposite was synthesised and tested, as follows:

Synthesis and Functionalisation of Zinc Oxide Nanoparticles

An about 10 mg/mL of ZnO nanoparticles (of sizes, between about 20 nm and about 80 nm) were dispersed, in methanol, to obtain dispersed ZnO nanoparticles. An about 2 mg/mL TAP (dissolved in methanol) was mixed, with the dispersed ZnO nanoparticles, to obtain a mixture. The mixture was kept, under constant stirring, at about 150 RPM, for about 24 hours. Unbound TAP was washed several times, by centrifuging, and kept for drying, to obtain TZnO nanoparticles. The TAP was obtained, from Indus Biotech Private Limited, Pune, Maharashtra, India. Briefly, the method of synthesising the TAP broadly comprises following steps (as mentioned, in US 8,835,415 B2 and/or Indian Patent 270492, the contents of which, are incorporated herein, by reference): extracting pulverized plant mass using organic solvent, to remove toxic substances; drying the mass, to remove the organic solvent; re-extracting the dried mass using aqueous solvent, to obtain extract; and purifying the extract, through chromatographic column. followed by concentrating, purifying, standardizing and drying to obtain the composition.

Characterisations

As illustrated, in Figure 1(a) and Figure 1(b), a colour change, from white to brown, was observed, upon synthesis of the TZnO nanoparticles. As illustrated, in Figure 1(c), ultraviolet-visible spectroscopy confirmed presence of the ZnO nanoparticles (Xmax = about 370 nm), in the TZnO nanoparticles, indicating a band gap of about 3.3. eV.

As illustrated, in Figure 1(d), Bragg angular peaks, with 29, at about 31.8 degrees, about 34.46 degrees, about 36.3 degrees, about 47.6 degrees, about 56.62 degrees, about 62.9 degrees, about 67.98 degrees, about 69.12 degrees, and about 76.92 degrees, confirmed that the ZnO nanoparticles had pure hexagonal wurtzite, as per JCPDS - 75-076.

Average sizes of the ZnO nanoparticles were determined to be about 58.35 ± 5.79 nm (as illustrated, in Figure 2(a)), whereas, those for the TZnO nanoparticles, was determined to be about 96.33 ± 5.76 nm (as illustrated, in Figure 2(b)). Functional group analyses of the TAP, the TZnO nanoparticles, and the ZnO nanoparticles further confirmed the functionalisation of the ZnO nanoparticles (synthesis of the TZnO nanoparticles).

Figure 2(c) illustrates comparative Fourier-transform infrared spectra, of the ZnO nanoparticles, the TZnO nanoparticles, and the TAP. As illustrated, in Figure 2(d), thermogravimetric analyses were carried out, to further confirm the TAP loading, onto the ZnO nanoparticles. Primary weight loss of water, from the TAP, was determined to occur, at about 500 degrees Centigrade.

In case of the TZnO nanoparticles, after an initial weight loss, at about 150 degrees Centigrade, secondary weight loss was determined to occur, at the same temperature of about 500 degrees Centigrade. Loading of the TAP, onto the ZnO nanoparticles, was determined to be about 5.37%, by roughly calculating differences, between mass losses of the TZnO nanoparticles and the ZnO nanoparticles.

Bacteriostatic Effects Against Multiple Drug Resistant Strains of Uropathogenic E.coli

Antibacterial effects of treatments, with the TZnO nanoparticle and the ZnO nanoparticles, were evaluated, against uropathogenic E.coli UTI89, and three other clinical multiple drug resistant strains. As illustrated, in Figure 3(a), Figure 3(b), Figure 3(c), and Figure 3(d), MIC50 values of the TZnO nanoparticle treatments were determined to be lower, in all cases, when compared with those of the ZnO nanoparticle treatments.

At the MIC50 and MIC values, there were significant reductions, in metabolically active cells, as illustrated, in Figure 4(a), Figure 4(b), Figure 4(c), and Figure 4(d) (one-way ANOVA; n=3; ***p=0.009; ****p<0.0001). Upon treatments with the TZnO nanoparticles, the reductions, in the metabolically active cells, were determined to be substantially higher, when compared with treatments with the ZnO nanoparticles. As illustrated, in Figure 5(a), Figure 5(b), and Figure 5(c), scanning electron micrographs depicted absence of cell membrane disruptions and complete eliminations of the bacterial cells.

Inhibitions of Biofilm Formation at Sub -MIC Levels

50% biofilm inhibitory concentration value was determined to be about 1 pg/mL, which increased, in a concentration-dependent manner, with a maximum inhibition of about 70% (at about 5 pg/mL), as illustrated, in Figure 6(a). At about 70% and about 50% inhibitory concentrations, viable biofilms were significantly reduced, when compared with the untreated controls (as illustrated, in Figure 6(b)). Upon the treatments with the TZnO nanoparticles, the reductions, in the viable biofilms, were determined to be substantially higher, when compared with the treatments with the ZnO nanoparticles.

In Figure 6(a) and Figure 6(b), statistical analyses were performed, through one-way ANOVA (n=3; *p=0.037; **p=0.067; ***p=0.002; ****p<0.0001).

Biofilm inhibitions were further confirmed, through confocal laser scanning microscopic techniques, as illustrated, in Figure 7(a), Figure 7(b), and Figure 7(c). As illustrated, in Figure 7(d) and Figure 7(e), significant reductions, in average thickness and biofilm biomass of the UTI89 strain, were determined (n=3; unpaired student t-test; ns=0.75; **p=0.001; ***p=0.0003).

Scanning electron micrographs showed thick biofilms of untreated biofilm controls (the UTI89 strain) being encased, in an exopolysaccharide matrix, as illustrated, in Figure 8(a). Reductions in biofilms were determined, in case of the treatments, with the TZnO nanoparticles and the ZnO nanoparticles, as illustrated, in Figure 8(b) and Figure 8(c).

In subsequent gene expression analyses, genes that control bacterial adherence were considered. For all four multiple drug-resistant strains, the expression of adhesins and fimbriae were determined to be downregulated (by about 1.5 times to about 2 times log 10-fold). As illustrated, in Figure 9(a), Figure 9(b), Figure 9(c), and Figure 9(d), striking differences, between the TZnO nanoparticle treatments and the ZnO nanoparticle treatments, were determined, in type I fimbriae adhesion system, fimA andfimH (one-way ANOVA; n=3; ns=0.42; *p=0.032; **p=0.0021; ***p=0.0003).

Eradications of Pre-Formed Biofilms and Dispersed Cells

From a therapeutic perspective, biofilm eradication is the need of the hour. On this front, studies were conducted, to evaluate biofilm eradication activities, at the MIC values. The concentration range was strictly restricted, to within the MIC range.

The treatments, with the TZnO nanoparticles and the ZnO nanoparticles, rendered metabolically inactive biofilms and dispersed cells. As illustrated, in Figure 10(a) and Figure 10(b), upon the treatments with the TZnO nanoparticles, the reductions, in the metabolically active biofilms and dispersed cells, were determined to be substantially higher, when compared with the treatments with the ZnO nanoparticles. In Figure 10(a), Figure 10(b), Figure 10(c), and Figure 10(d), statistical analyses were performed, through one-way ANOVA (n=3; *p=0.013; **p=0.001; ***p= 0.0003).

Biofilm eradications were further confirmed, through confocal laser scanning microscopic techniques, as illustrated, in Figure 11(a), Figure 11(b), and Figure 11(c). As illustrated, in Figure 11(d) and Figure 11(e), significant reductions, in average thickness and biofilm biomass of the UTI89 strain, were determined (n=3; unpaired student t-test; ns=0.0612; *p=0.0201; **p=0.0091).

Scanning electron micrographs showed thick biofilms of untreated biofilm controls (the UTI89 strain) being encased, in the exopolysaccharide matrix, as illustrated, in Figure 12(a). Reductions in biofilms were determined, in case of the treatments, with the TZnO nanoparticles and the ZnO nanoparticles, as illustrated, in Figure 12(b) and Figure 12(c).

Non-Toxicity to RAW Macrophages, Vero Kidney Cell Lines, and T24 Urinary Bladder Cell Lines

Figure 13 illustrates comparative toxicity profiles, of the TZnO nanoparticle treatments and the ZnO nanoparticle treatments, at concentrations about the MIC, MBIC, and MB EC (Minimum Biofilm Eradication Concentration) values (one-way ANOVA; n=6; ns=0.13; *p=0.0195; **p=0.008; ***p=0.0007). The TZnO nanoparticle treatments were determined to be non-toxic, to macrophages, at all tested concentrations. On the other hand, the ZnO nanoparticle treatments were determined to be significantly toxic, to the macrophages.

Correspondingly, release of reactive oxygen and nitrates was determined to be significantly increased, upon the treatments with the ZnO nanoparticles, as illustrated, in Figure 14(a), Figure 14(b), Figure 14(c), and Figure 14(d) (one-way ANOVA; n=6; ns=0.13; *p=0.0195; **p=0.006; ***p=0.0006). “LPS” denotes bacterial lipopolysaccharide (positive controls).

Figure 15(a) and Figure 15(b) illustrate toxicity profiles, of the TZnO nanoparticle treatments and the ZnO nanoparticle treatments, against vero kidney cell lines and T24 bladder cell lines (one-way ANOVA; n=6; ns=0.1349; *p=0.13; **p=0.0037; ***p=0.0002). In both the cell lines, the TZnO nanoparticle treatments were determined to be toxic, only at high concentrations. The ZnO nanoparticle treatments, on the other hand, were determined to be harmful, to the vero kidney cell lines, even at low concentrations.

Adhesion and Invasion to T24 Bladder Cell Lines

Figure 16(a) illustrates the T24 bladder epithelial cells infected with the UTI89 strain. As illustrated, in Figure 16(b) and Figure 16(c), the TZnO nanoparticle treatments and the ZnO nanoparticle treatments were determined to significantly reduce bacterial colonisations. Figure 16(d) illustrates significant reductions (by about two times log-10 fold), in adhered bacteria, upon treatments with the TZnO nanoparticles, when compared with the controls (unpaired student t-test; n=6; ns=0.1; **p=0.004).

Figure 17(a) illustrates the T24 bladder epithelial cells invaded (by the UTI89 strain). As illustrated, in Figure 17(b) and Figure 17(c), the TZnO nanoparticle treatments and the ZnO nanoparticle treatments were determined to reduce invasion of cells, thereby, protecting host cells. However, host cell damages were determined, in case of the ZnO nanoparticle treatments. Figure 17(d) illustrates significant reductions, upon treatments with the TZnO nanoparticles, when compared with the controls (unpaired student t-test; n=6; ns=0.0848; *p=0.024; **p=0.0071).

Eradications of Adhered Bacterial Cells

Figure 18(a) illustrates the T24 bladder epithelial cells infected with the UTI89 strain, and the bacterial cells colonised to the urothelial cells. The TZnO nanoparticle treatments and the ZnO nanoparticle treatments were determined to significantly reduce bacterial colonisations, as illustrated, in Figure 18(b) and Figure 18(c). Figure 18(d) illustrates significant reductions (by about three times log- 10 fold), in the adhered bacteria, upon treatments with the TZnO nanoparticles, when compared with the controls (unpaired student t-test; n=6; ns = 0.1059; *p=0.015; **p=0.0023).

As illustrated, in Figure 19(a), the UTI89 cells invaded the urothelial cells, leading to host cell death. As illustrated, in Figure 19(b) and Figure 19(c), the TZnO nanoparticle treatments and the ZnO nanoparticle treatments were determined to reduce the invasion of cells, thereby, protecting the host cells. However, host cell damages were determined, in case of the ZnO nanoparticle treatments. Coating of Nanocomposite to Silicone Catheters

Figure 20(a)(i), Figure 20(b)(iv), and Figure 20(c)(vii) illustrate topological views, of: uncoated catheters; catheters coated with the TZnO nanoparticles; and catheters coated with the ZnO nanoparticles, respectively.

Figure 20(a)(ii), Figure 20(b)(v), and Figure 20(c)(viii) illustrate cross-sectional views, of: the uncoated catheters; the catheters coated with the TZnO nanoparticles; and the catheters coated with the ZnO nanoparticles, respectively. Red arrow, in Figure 20(b)(v) shows edges of the catheters.

Figure 20(a)(iii), Figure 20(b)(vi), and Figure 20(c)(ix) illustrate extraluminal sides, of: the uncoated catheters; the catheters coated with the TZnO nanoparticles; and the catheters coated with the ZnO nanoparticles, respectively. Red arrow, in Figure 20(b)(viii) shows edges of the catheters.

The coating of catheters was carried out, according to Yassin et al; 2019 (the contents of which, is incorporated herein, by reference), with slight modifications.

Sterile coated and uncoated catheters were placed, in artificial urine media, and Uropathogenic E.coli cells were inoculated. The catheters were incubated, for about seven days, and the media was changed after about every 24 hours. On about the seventh day, the catheters were vortexed and sonicated, to remove adhered bacteria.

As illustrated, in Figure 21(a), Figure 21(b), and Figure 21(c), through scanning electron microscopy analyses, it was determined that there were aggregations of live/dead cells, in the catheters coated with the ZnO nanoparticles. Such cell aggregates can act as a base, for further accumulations of bacteria, leading to failures of the catheters.

Figure 21(d) illustrates reductions, in biofilms, upon treatments with the TZnO nanoparticles and the ZnO nanoparticles, when compared with the controls (unpaired student t-test; n=6; ns=0.52; **p=0.0029).

The disclosed nanocomposite and method offer at least the following synergistic advantages and effects: low MIC50 values; good bacterial biofilm inhibiting properties; eradicate pre-formed bacterial biofilms and dispersed bacterial cells; eradicate adhered bacterial cells; and superior safety profiles. The disclosed nanocomposite may be directly coated, onto catheters, for treating bacterial urinary tract infections. Alternatively, or in addition, it may be part of a kit, for treating bacterial urinary tract infections.

It will be apparent to a person skilled in the art that the above description is for illustrative purposes only and should not be considered as limiting. Various modifications, additions, alterations, and improvements, without deviating from the spirit and the scope of the disclosure, may be made, by a person skilled in the art. Such modifications, additions, alterations, and improvements should be construed as being within the scope of this disclosure.