Estimation of Some Plant Extract Activity against Bacterial Cystitis Isolated from Urinary Tract Infection

Abstract

In this study, 60 urine samples were collected from patients with urinary tract infections who were admitted to Al-Hussein Teaching Hospital between December and February of 2018–2019. A urine sample was collected for culture and crystal formation. Only 57 (95 percent) of the 60 samples on culture were isolated from urinary tract infections caused by various causes. According to the results of the isolation and laboratory diagnosis, as well as biochemical tests, Staphylococcus saprophyticus, Streptococcus agalactiae, Escherichia coli, Klebsiella pneumonia, proteus spp., Morganella morgani, and Pseudomonas aueroginosa were identified in this study. S. saprophyticus is the ore predominant in UTIs infection While Morganella morganii is the least common result, 8% of the total The isolates are varied in their ability to produce urease enzyme and stone (cast) they were varied in their hemolytic activity. Isolates that able to produce urease in different level which provided as main step in pathogenesis in urinary tract infections and cast formation, Zea mays, curcumine and canberry were shown very high effectively to inhibit stone in the percent of (11–13), respectively coffee and Ziziphus gave results varied in their activity.

1. Introduction

Urease production is regarded as an important virulence factor in bacterial pathogenicity because the ammonia produced by this enzyme raises the pH, which has important medical implications. Urease is a virulence factor found in pathogenic bacteria that cause gastric ulcers, urinary stone formation, pyelonephritis, and other human health issues [1].

Although symptoms can aid in the diagnosis of a UTI, they may not accurately localize the infection within the urinary tract. However, in many cases, urinary tract colonization is asymptomatic. Cystitis (bladder infection) is the most common type of UTI, characterized by irritative symptoms such as urinary urgency, frequency, dysuria, hematuria, foul-smelling urine, and suprapubic pain. In addition to cystitis, these symptoms are also common for urethritis and prostatitis. An associated epididymitis, diagnosed reliably by physical examination in men, is an easily localizable variation of UTI. Symptoms of “upper urinary tract” infections, such as pyelonephritis, may include those associated with cystitis, as well as fever, rigors, flank or abdominal pain, and nausea and vomiting [2]. The aims of our study is evaluation of some plant extract on bladder stone and well done by this objectives:-

1. Isolation the pathogenic bacterial SPP. From urinary tract infections

2. Investigateion bacterial isolates on urease production abbility.

3. In vitro stone foramtion

4. Estimation the activity of some plants on this stone

As a result, the organism has an easily assimilated nitrogen source. Urease promotes virulence in uropathogenic bacteria. As a result, the urinary tract becomes alkaline. Increased urine pH can cause the formation of struvite stones, which contain the infecting organism, increased attachment of bacteria to the renal epithelium, direct renal tissue damage, and the activation of certain antibiotics [3].

Because urinary stones are a mixture of compounds de-posited as a result of metabolic disorders and infections, their chemical composition is not uniform. The fact that urolithiasis promotes the development of infections, and bacteria easily colonize porous stone surfaces, may explain the diverse composition of the stones [4].

Struvite stones are also referred to as ‘infection stones’ and ‘triple phosphate stones’. Struvite stone formation can only be sustained if ammonia production is increased and the urine pH is raised to reduce phosphate solubility. Only when urine is infected with a urease-producing organism, such as Proteus, can both of these requirements be met. Urease degrades urea to produce ammonia and carbon dioxide: 2NH3 + CO2 = Urea With a pK of 9.0, the ammonia/ammonium buffer pair produces highly alkaline urine rich in ammonia. Urease is an enzyme that breaks down urea into ammonia and carbonic acid.

alkaline urine rich in ammonia. Urease splits urea into ammonia and carbonic acid:

$$\mathrm{NH}2-\mathrm{CO}-\text{NH2}\to H2\text{CO3}+2\text{NH3}$$E1

Ammonia then mixes with water to produce ammonium hydroxide and under these alkaline conditions, carbonic acid moves toward bicarbonate and carbonate ions.

$${\mathrm{NH}}_3+2H_{2}O\to 2{{\mathrm{NH}}_4}^{+}+2{\mathrm{oh}}^{-}$$E2

Thus, the alkalinisation of urine by the urease reaction causes the formation of NH₄⁺, which favors the formation of carbonate ions (CO₃²⁻) and trivalent phosphate ions (PO₄³⁻). This in turn causes struvite and carbonate apatite formation (Sun et al., 2010).

$${{\mathrm{CO}}_3}^{2-}+10{\mathrm{Ca}}^{2+}+6{{\mathrm{PO}}_4}^{3-}\to {\mathrm{Ca}}_{10}{\left({\mathrm{PO}}_4\right)}_6{\mathrm{CO}}_3$$E3

$$6H_{2}O+{\mathrm{Mg}}^{2+}+{{\mathrm{NH}}_4}^{+}+{{\mathrm{PO}}_4}^{3-}\to {\text{MgNH}}_4{\mathrm{PO}}_4{\text{6H}}_{2}O$$E4

These components all play different roles in the plant, resulting in a variety of potential health benefits from their consumption. Microorganisms have developed resistance to various antibiotics that are currently in use, as is well known in the medical field. This phenomenon has caused enormous clinical issues in the treatment of diseases caused by such microorganisms.

It appears to be a urease inhibitor with a mechanism. Plants have a natural polyphenol structure that consists of two o-methoxy phenols attached symmetrically through a, −unsaturated -diketone linker that also induces keto-enol tautomerism. It is possible that this compound inhibits urease activity via a chelate interaction that binds to the urease active site; as a result, some plant extracts form a stable complex with urease and, as a result, the enzyme is inhibited.

Proteus is a Gram-negative, rod-shaped bacteria, non-spore forming and facultative anaerobic, catalase positive, oxidase negative and non- lactose fermenter. It belongs to the family Enterobacteriaceae [5].

The first description of Proteus bacteria was made in 1885 by Hauser, who named them after the character in Homer’s Odyssey who ‘has the power of assuming different shapes to escape being questioned’ (Wang and Pan, 2014). There are currently four recognized species of Proteus: P. mirabilis, Proteus penneri, P. vulgaris, and Proteus myxofaciens [6].

This bacterium has to measure (1–3) μm in length and (0.4–0.8) μm in diameter, motile by peritrichous flagella, chemo-organotrophic, made a respiratory and a fermentative type of metabolism not require oxygen, so it called facultative anaerobic [7], made pale colonies shape when growing on MacConkey and when grown on blood agar (that causes β hemolysis on blood agar) with distinct fishy odor. Also, it made rings of swarming motility in an agar media [8].

Morganella morganii:- Morganii and M. sibonii are Gram-negative bacilli that belong to the tribe Proteae of the family Enterobacteriaceae. It is naturally present in the environment and in the intestinal tracts of humans, mammals, and reptiles. Despite its widespread distribution, it is an uncommon cause of human infections [9].

M. morganii was first identified as a cause of urinary tract infections in the late 1930s, and only a few reports of infections due to this pathogen have been published since then. It is most common in postoperative patients and is mostly associated with urinary tract infections. Bacteremia/sepsis in both children and adults Skin and soft tissue infections, meningitis, ecthyma, and endophthalmitis [10].

The genus Zizyphus belongs to the Rhamnaceae family. It is a genus of about 100 species of deciduous or evergreen trees and shrubs found in tropical and subtropical regions. PACs have recently received a lot of attention due to the health benefits that they have been linked to [11].

Cranberries are composed of 88% water and a mixture of organic acids, vitamin C, flavonoids, anthocyanidins, proanthocyanidins (PACs), catechins, and triterpinoids [12].

Curcumin (diferuloylmethane, chemical formula: C₂₁H₂₀O₆ is a yellow-orange pigment extracted from the roots of turmeric (Curcuma longa). It usually exists in two tautomeric forms: keto and enol, The enol form has a higher energy stability. Curcumin has gained popularity due to its diverse biological activities. Turmeric gained popularity in the 1970s after it was discovered to have anti-inflammatory properties [12].

The significance of turmeric in medicine has changed since the antioxidant properties of other plants were discovered, therefore, it is found thatplants possesses antitumour, antibacterial, antifungal and antiviral properties additionally, these plant does not exhibit toxicity to either animal or humans even at high doses, the case of struvite crystallization induced by bacteria in relation to urinary stone formation. Urease inhibitors bind to it and inhibit its activity by preventing the hydrolysis of urea to ammonia and carbon dioxide. There are two types of urease inhibitors: I mechanism-based directed mode; (ii) active-site directed mode The active-site directed inhibitors resemble urea, the enzyme’s substrate, in structure. Mechanism-based directed inhibitors interfere with the catalysis mechanism of the enzyme, causing it to be inhibited or inactivated [13].

It appears to be a mechanism-based urease inhibitor. Plants have a chemical structure that consists of two o-methoxy phenols attached symmetrically via a, −unsaturated -diketone linker, which also induces keto-enol tautomerism, making it a natural polyphenol. It is possible that this compound inhibits urease activity via a chelate interaction, which binds to the urease active site; as a result, some plant extracts form a stable complex with urease, inhibiting urease activity. The diketone moiety of plants has chelating properties toward transition metals, including nickel. Curcumin chelation toward transition metals such as iron and copper has been found to be beneficial in the treatment of Alzheimer’s disease [14].

2. Materials

2.1 Specimens

A total of 60 urine samples were collected from patients admitted to Al-Hussein teaching Hospital, during a period extending from September 2018 to December 2018.

Urine samples were collected in sterile cups, as all patients had signs and symptoms of UTI and they were diagnosed as having UTI by the Urologists.

3. Methods

3.1 Preparation of reagents and solutions

3.1.1 Reagents

3.1.1.1 Catalase reagent

This reagent was used with a concentration of 3%. It was prepared by adding H2O2 to D.W. (v/v). It was used for identification of catalase producing bacteria by looking for appearance of air bubbles which indicate positivity.

3.1.1.2 Oxidase reagent

This reagent was prepared freshly, by dissolving 1gm of (tetramethyl-paraphenylene-diamine-dihydrochloride) in 100 ml of D.W. and kept in a dark bottle. It immediately used for the identification of bacteria positive for oxidase production by the appearance of dark purple color as a positive result.

3.1.1.3 Methyl red reagent

Methyl red reagent is prepared by dissolving 0.1 gm of methyl red in 300 ml of (95%) ethanol and then the volume is completed to 500 ml by D.W. It is used to identify the complete glucose hydrolysis.

3.1.1.4 Coagulase test

It is an important method for detection of ability to produce coagulase and differentiation between coagulase-producing and non-producing staphylococci. Bacterial broth was added to fresh plasma, and incubated at 37°C and examined after 1,2,3 and 4 hours. The test was read by tilting the tube and observing for clot formation in the plasma.

4. Preparation of culture media

A group of culture media were prepared according to the instructions of the manufactures company and sterilized by autoclaving at 121°C for 15 minutes.

4.1 Blood agar medium

Blood agar medium was prepared by dissolving blood agar base in distal water. It was autoclaved cooled to 50°C. 5% fresh human blood was added and mixed well. This medium was used to cultivate bacterial isolates and to determine the ability of bacteria to hemolyse blood cell.

4.2 Mannitol-salt agar medium

Mannitol salt agar was prepared according to manufacturer. It was used to detect ability to ferment mannitol.

4.3 Nutrient agar medium

Nutrient agar medium was prepared according to the manufacturing company. It was used for general experiments, cultivation and activation of bacterial isolates when it is necessary.

4.4 Nutrient broth

This medium was used to grow and preserve the bacterial isolates. Nutrient broth medium was prepared according to the method suggested by the manufacturing company.

4.5 Urea agar medium

Urea agar medium was prepared by adding 10 ml of urea solution (20% sterilized by Millipore filter paper) in volume of autoclaved urea agar base and completed up to 100 ml distilled water and cooled to 50°C, the pH was adjusted to 7.1. then medium was put into test tubes and allowed to solidify in a slant form. It was used to test ability of bacteria to produce urease enzyme.

4.6 Brain heart infusion broth

Brain-heart infusion broth was prepared according to the manufacturing company. It was used for detection of different biochemical tests.

4.7 MacConkey agar medium

MacConkey agar medium was prepared according to the method recommended by the manufacturing company. It was used for the primary isolation of most Gram-negative bacteria and to differentiate lactose fermenters from non-lactose fermenters.

4.8 Brain heart infusion (BHI) broth: glycerol medium

This medium was prepared by mixing 5 ml of glycerol with 95 ml of BHI broth (sterilized by autoclave). It was used for preservation of bacterial isolates as stock for long time.

4.9 Simmon-citrate medium

Simmon-citrate medium is prepared according to the manufacturing company. It has been used for determining the ability of bacteria to utilize citrate as the sole source of carbon.

4.10 Kligler iron agar medium

Kligler-Iron agar was used for determining glucose and lactose fermentation and possible hydrogen sulfide (H2S) production as a first step in the identification of Gram-ve bacilli.

4.11 Motility medium

It was prepared by dissolving 0.5 gm of agar-agar in 100 ml of brain-heart infusion broth and autoclaved, then the contents were dispensed into test tube.

5. Collection of specimens

Urine specimens were collected under aseptic procedure, to avoid any possible contamination. 10 ml of mid-stream urine samples are collected from patients suffering from UTIs, urine samples are collected in sterile screw-cap containers. Each specimen is immediately inoculated onto the blood agar plates and MacConkey’s plates. All plates are incubated at 37°C for 24 hrs.

6. Laboratory diagnosis

According to the diagnostic procedures recommended, The isolation and identification of Staphylococci species in specimens are performed as follows:

6.1 Colonial morphology and microscopic examination

6.1.1 Colonial morphology

All specimens were cultivated on blood agar, Nutrient and MacConkey agar media by swabbing and incubated at 37°C for 18-24 hrs.Each primary positive culture is identified depending on the phenotypic properties such as (colony size, shape, color and nature of pigments, translucency, edge, elevation, and texture).

6.1.2 Microscopic examination

The morphology of bacterial cells is investigated by Gram-stain to observe shape, arrangement of cells and type of reaction by using Gram-stain. Then, specific biochemical tests are done for each isolates for the final identification.

6.1.3 Motility test

After preparation of semisolid media, the bacterial colonies were inoculated by stabbing-down to the center of the tube to about half the depth of the medium. The cultured tubes were incubated at 37°C and were examined after 6 hours, 1 and 2 days. Non-motile bacteria had generally restricted to the stab line and given sharply defined margins with leaving the surrounding medium clearly transparent, while motile bacteria give diffuse hazy growth that spread throughout the medium rendering it slightly opaque.

6.2 Biochemical tests

The following biochemical tests are performed for the identification of Staphylococcus species isolates:

6.2.1 Coagulase test

Method of Benson (2001), include several colonies of bacterial growth were transferred with a loop to a tube containing 5 ml of Nutrient broth. The tube was covered to prevent evaporation and incubated at 37°C in the incubator over-night. After incubation, tube mixed and centrifuged, 0.5 ml of the supernatant withdrawn and mixed with 0.5 ml of fresh human plasma, then incubated in the water bath at 37°C for several hours.

If the plasma is coagulated, the organism is coagulase-positive. Some coagulations occurred in 30 minutes or several hours later. Any degree of coagulation, from a loose clot suspended in plasma to a solid clot, was considered to be a positive result, even if it takes 24 hours to occur.

6.2.2 Catalase test

Transferring the bacterial growth by wood stick and put it on the surface of a clean slide and add a drop of (3% H2O2), positive result when the gas bubbles appear.

6.2.3 Oxidase test

Filter paper is soaked with a freshly made reagent, and the colony to be tested is taken up with a sterile wooden stick and put over the filter paper. A positive result is indicated by a deep purple color which appeared within 5–10 seconds.

6.2.4 Citrate utilization test

The surface of simmons citrate slant medium is inoculated with the colony of tested bacteria and incubated at 37°C 18-24 hrs. Conversion of the indicator’s color from green to blue indicates that the organism was able to utilize citrate as a sole source of carbon.

6.2.5 Methyl red test

Tubes of MR-VP broth are seeded with the selected bacterial colonies and incubated at 37°C for 48 hrs. Then 5 drops of methyl red reagent are added to it, the appearance and observation of red color means a positive result and a complete analysis of glucose.

6.2.6 Mannitol fermentation test

Mannitol Salt Agar was inoculated with bacterial colonies then incubated at 37C° for 24 hours. The color changed from pink to bright yellow as the bacteria was lactose fermenter, or unchanging color of the medium was a negative result.

6.3 Virulence factors test

6.3.1 Urease test

This test was done by inoculating the prepared urea medium with bacterial growth. The tubes were incubated for 24–48 hours at 37°C. The change of color medium into pink indicated a positive result.

6.3.2 Haemolysin production on blood agar

Detection of hemolysin production was carried out by inoculating a blood agar with bacterial isolates, then incubated at 37°C for 24 hrs. The appearance of a clear zone around the colonies indicated a complete hemolysis (ß- hemolysis) while greenish zone around the colonies referred to partial hemolysis (α- hemolysis), no change in the medium referred to no hemolysis (γ- hemolysis).

6.4 In vitro stone formation

A fresh urine sample was obtained from a healthy control with no history of urinary stones or urogenital infectious diseases. It was sterilized by filtration. Ability to forma stone was detected by growing bacterial species aerobically at 37°C for 24 hours in brain heart infusion broth which was enriched with 1% Tween 80 and 10% serum. 1 ml of 1: 10 dilution in human urine of an overnight culture of bacteria was inoculated in to 9 ml of the sterile urine.

E. coli (known to be non-urease-producer) was inoculated in the same mentioned above.

All inoculums gave a final count of about 107 CFU/ml.

A control of 10 ml of urine (from the same person) was also studied.

All tubes were incubated at the same incubation conditions (at 37°Cfor overnight).

Ammonium concentration (indophenol method; appendix), turbidity and pH were determined at the beginning of experiment and after 4, 8 and 24 hours of incubation.

Sediment was examined at the same intervals and crystals, if any, were identified both macro- and microscopically.

The inhibition factor were added.

6.5 Plant extract preparation

50 gm of the powdered of each samples (curcumine, Zea mays, Ziziphus, coffee) dissolved in 500 ml of distilled water the final concentration of each plant extract is 50 gm/ml while the cranberry were used as a template with its exact concentration.

7. Results and discussion

7.1 Isolation and identification bacterial urinary tract infections

7.1.1 Isolation of species

A total of 60 urine samples were obtained from patients suffering from urinary tract infection who are admitted to Al-Hussein Teaching Hospital, at the period from December to February 2018–2019. Among 60 clinical samples, only 57 showed positive results, as shown in Table 1.

No. of urine samples	No. of culture
	No. of negative culture	No. of positive culture
60 samples	3(5%)	57(95%)

Table 1.

Number and percentage of bacteria isolated from urine samples of patients with urinary tract infections.

In this study, Staphylococcus saprophyticus, Streptococcus agalactiae, Escherichia coli, Klebsiella pneumonia, proteus spp., Morganella morgani and Pseudomonas aueroginosa that were isolated and identified in this study, as shown in Table 2 and the Figure 1 Show the distribution rate of isolation.

Bacterial spp.	No. of isolation			%
	Male	Female	Total
Klebsiella	2	5	7	12%
Staphylococcus saprophyticus	—	12	12	21%
Proteus	6	4	10	19%
Morganella; morgani	2	3	5	8%
Pseudomonas aueroginosa	5	3	8	14%
E.coli	4	5	9	16%
Streptococcus agalactiae	—	6	6	10%
Total	19 (34%)	38(66%)	57	100%

Table 2.

Prevalence and distribution of bacterial pathogens according to gender of patients.

Figure 1.

Distribution of bacterial Spp. isolation rate.

It was observed that Klebsiella pneumoniae is rods, catalase positive, oxidase negative and hemolysin producer, non-motile, lactose fermenter on MacConkey agar, the colonies appear pink to red in color, with mucoid phenotype.

Besides, the isolates were urease and capsule producers, having positive results for vogues proskaur and negative for methyl red and H₂S production test. All the isolates of klebsiella pneumoniae were fermenter for glucose, lactose sucrose, L-arabinose, D-mannitol and maltos.

Regarding, Staphylococcus saprophyticus, this bacteria is isolated only from women patients with UTI at a rate 21%. This bacteria is highly prevalence among young women, and also is considered pathogen in UTI. Many studies indicate that S. saprophyticus can cause UTI in young females and considered as the second causes of UTI among this age group (below 15 years old) after E. coli [15, 16, 17].

Streptococcus agalactiae (group B Streptococcus) is a major cause of neonatal infectious disease in humans in many countries and is carried asymptomatically by a large proportion of adults. It is also recognized as an emerging pathogen in human adults [18].

GBS is also an important cause of morbidity and mortality in the elderly and in immuno-compromised adults. Primary manifestations of adult GBS disease include bacteremia, skin and soft tissue infections, pneumonia, osteomyelitis and urinar y tract infections [19, 20].

According to [21] only (16.7%) of urine samples were obtained from patients with suspected UTI were positive from coagulase negative staphylococcus, and this result is lesser than that obtained in this study, Isolation rate of Proteus spp. Similar to [22] who found that percentage 7(7%) of Proteus. The variation in bacterial isolation between studies may be attributed to many factors such as sanitary practices in hospitals and staff, environmental conditions, isolation and identification techniques, social and cultural level of patients, and use multidrug (antibiotics) that may lead to developing in bacterial resistance ability, or may be due to differences in the size of samples; all these factors may employ together and play an important role in inhibit or stimulate the growth and distribution of pathogenic bacteria in hospitals.

7.2 Identification of bacterial spp

The identification of any bacteria depends mainly on the cultural, biochemical characteristics and microscopic patterns. These organisms varying from cocci to (bacilli) rods microscopically searching on is motile or not, spore forming, coagulase and catalase.

Most Staphylococcus; on blood agar, the colonies tend to be non-pigmented, smooth, entire, glistening, and opaque colonies, However, the isolates were shown to be catalase positive, oxidase negative, coagulase negative, and mannitol fermentation negative. Also, all isolates were non-motile and negative for each starch hydrolysis, gelatin hydrolysis, methyl red and citrate utilization test.

The identification of Proteus is Gram- negative bacilli, catalase positive,have white colony with fushy oder and showe swarming motality. M. morganii can produce the enzyme catalase, so is able to convert hydrogen peroxide to water and oxygen. This is a common enzyme found in most living organisms. In addition, it is indole test-positive representing this organism can split tryptophan to indole, pyruvate, and ammonia. Methyl redtests positive in Morganella morganii, an indicator dye that turns red in acidic solutions. is facultatively anaerobic and oxidase-negative. Its colonies appear off-white and opaque in color, when grown on blood agar plates. It is straight rods, about 0.6–0.7 μm in diameter and 1.0–1.7 μm in length.

8. Complicated and simple urinary tract infections

A total (57) isolates of bacteria 33 (18.66%) isolated obtained from patient with complicated urinary tract infection and a 24 (39.2%) isolated obtained from patients with simple urinary tract infection from both sex as shown in Table 3, While the Figure 2 shows the comparison between them.

No. of bacterial Isolated	Complicated UTIs	simple UTIs
57	33(57.8%)	24(42.1%)

Table 3.

Bacterial isolated from complicated and simple urinary tract infections.

Figure 2.

Show the comparison between complicated and simple UTIs.

In the case of complicated UTI which includes several cases shown in Table 4.

Cause of UTI	No. of patients
Renal stone	20
Nephrostomy	1
JJ stent	1
Bladder stone	1
Chronic pyelonephritis	1
Ca. of bladder	2
Urethral tumor	1
BPH	3
Immune compromised, leukemia	2
Urethral stricture	1

Table 4.

Patient with complicated urinary tract infection.

Complicated urinary infection occurs in both women and men, and in any age group. Because uncomplicated urinary infection is rare in men, any male urinary infection is usually considered complicated (Figure 3) [23].

Figure 3.

Distribution of complicated UTI cases.

Recurrent urinary infection in postmenopausal women is associated with genetic and behavioral risk factors similar to those seen in younger women with acute uncomplicated urinary infection, such as a higher likelihood of being a no secretor and a history of prior urinary infection [24].

However, postmenopausal women with recurrent urinary infection are also more likely to have increased residual urine volume, cystoceles and prior genitourinary surgery than are women without infection, and these associations are consistent with complicated infection. Thus, as a population, postmenopausal women with recurrent urinary infection encompass elements consistent with both uncomplicated and complicated urinary infection.

In case of complicated UTI, the result of this study was agreement with results obtained by [25] who found that these bacteria found in complicated UTI with percentage (60.8%). This result was dis- agreement with the result obtained by [26] as they found that the percentage of isolated percentage.

Also, the result in this study is closed to that obtained by [27] who found that percentage 44% from complicated UTI, and the results obtained by [28] that they were isolated bacterial spp. from renal calculi in percentage 21% was not agreement with results in this study.

On the other hand, the case of simple UTI with includes seven isolated from woman, including four pregnant women, two males from the other nine samples.

9. In vitro struvite stone formation by bacterial spp.

Struvite stones are thought to develop in urinary tract infected with urea splitting bacteria; the bacterial urease hydrolyzes urea, leading to hyperammonuria and alkalinization of urine with consequent crystallization of struvite.

In the present study, the stone forming ability of urease positive isolates investigated through an experiment testing the pH and crystals of human urine inoculated with, E. coli as a control and controlled urine with no bacteria at 0, 4, 8, and 24 hours of incubation.

When human urine was inoculated with bacteria a gradual increase in the cell density was noticed until the 4th hour when the rate of proliferation became faster, increasing the microbial population to the maximum when examined after an overnight incubation, during the experimental bacterial growth in human urine, the primary urine pH, and crystals were measure, and re-measured again at 4, 8 and 24 hours of incubation. At the onset of the experiment, the urine pH was about (5.5). When isolates grew in human urine, there was a slight elevation in the urine pH at the first 4 hours, reaching about (6.2), at 8 hours reach to (7.5) and maximized after 24 hours up to (9). However, this is not the case when E. coli grew at the same experimental conditions, as it was expected, there was a very minor elevation in the pH, whereas, the final reading was 6.0. On the other hand there were no changes in the pH of control urine which was free of bacteria during the period of study. The results were shown in Figure 4.

Figure 4.

Stone formation by positive urease, bacteria (40×).

Struvite stones formation associated with urinary infection by urease production isolates are thought to be as a consequence of hyper ammonuria and alkalinization of urine associated with this bacterium growth [29].

The numbers of crystals seen microscopically increased gradually parallel with that of both the pH, reaching a maximum number after 24 hours of incubation when white sediment appeared at the bottom of the tube at that time (Table 5).

Bacterium	No. of crystals/Hpf microscope at hour
	zero	4	8	24
Strep. agalactiae	0–4	4–8	8–12	12–20
Staph. saprophyticus	0–6	6–8	8–11	12–25
Merothrips morgani	0–6	6–10	10–15	15–20
Klebsiella	0–7	7–10	11–17	12–28
Proteus	0–3	3–8	5–10	20–30
E. coli	0–3	0–3	0–3	0–3
P. aueroginosa	0–2	0–2	0–2	0–2

Table 5.

Number of crystals according to the time in bacterial isolated from complicated and simple urinary tract infection.

However, as it was expected E. coli growth resulted in no increase in the number of crystals seen microscopically as in the case of other non-urease producer organisms, and urine inoculated with no bacteria did not alter the number of crystals at all (Figure 5). There was no sediment observed in case of both E coli pseudomonas the control urine after an overnight incubation (Figure 6).

Figure 5.

Shows stone formation according to the time.

Figure 6.

Stone formation by bacteria with correlation to the time.

P. merabilis is a relatively fastidious microorganism and is probably less virulent than other gram negative bacilli usually involved in urinary tract infection, urease enzyme produced by this bacterium plays a major role in the pathogenesis of struvite stone formation, and it is agreed to be the direct cause of hyperammonuria and alkalinization of urine seen in UTI caused by this bacterium.

Struvite is one of the main components of infectious urinary stones, which are caused by the activity of microorganisms that produce urease, primarily Proteus species. One of the primary causes of urinary stone formation is the aggregation of precipitating particles and bacteria.

10. Effect of some plants extracts on struvite stone formation

The effect of some plant extract on stone formation was investigated; it was found that the activity of curcumin were performed an in vitro experiment of struvite growth from human urine. The results demonstrate that curcumin exhibits the effect against isolates inhibiting the activity of urease—an enzyme produced by these microorganisms. Addition of curcumin decreases the efficiency of growth of struvite compared with the absence of curcumin.

The results show that the urine pH was about (4.5). When bacteria grew in human urine and give a very high rate of stone formation followed by, and added curcumin and crambery, there was a slight elevation in the urine pH at the first 4 hours, reduced increase pH to reaching about (5.5), at 8 hours the pH was reach to (6) and after 24 hours the pH was (7.5) (Table 6).

No. of cast Bacterial Isolates	Free without inhibition	Plant extract
		Zea mays	Curcumin	Cranberry	Ziziphus	Coffee
Proteus	30	19	17	19	22	25
Klebsiella	28	9	11	8	10	12
Morganella morganii	20	8	10	10	11	11
Staphylocuccus	25	9	11	11	18	18
Streptococcus	20	10	10	17	19	20

Table 6.

Effect of plant extract against struvite stone.

This result was agrrement with result obtained by [30] who found that the experiment of added curcumin has demonstrated that the curcumin a lower concentration has inhibitor urease activity (Figure 7).

Figure 7.

Shows the plant extract activity against stone.

In the case of addition of curcumin our experiment runs differently. First, the solubility of curcumin in the solution of human urine is relatively low. Therefore, we have initially observed unsolvable particles of curcumin, resulting in “stellar” aggregates. Furthermore, individual struvite crystals appear later in the absence of curcumin. Furthermore, the addition of curcumin reduces the size and number of struvite crystals.

Struvite crystals form much later when curcumin is present than when it is not. Two factors could contribute to a slower pH increase. For starters, curcumin has the potential to act as a bactericide. Second, while curcumin may inhibit urease activity, it has no effect on the viability of the bacterium. In contrast, curcumin inhibits urease activity.

Curcumin as well as Zea mays and Ziziphus leave has attracted attention because of its various biological activities. Modern interest in turmeric began in 1970s when it was found that turmeric possesses anti-inflammatory properties. Therefore, plant extracts are widely studied and it is found that curcumin possesses also antitumor, antibacterial, antifungal and antiviral properties, Curcumin is a powerful agent that can be used in a variety of pharmacological applications. Furthermore, curcumin is not toxic to either animals or humans at high doses. Even at doses of 8–10 g/day, curcumin is pharmacologically safe [31]. Tween 80 in culture media promotes the formation of struvite stone by stabilizing urease activity. The current study has confirmed that urease is important in stone formation.

Cranberries are made up of 88 percent water and a variety of organic acids, vitamin C, flavonoids, anthocyanidins, catechins, and triterpinoids. These components all play different roles in the plant, which results in a variety of potential health benefits from their consumption. PACs have recently received a lot of attention due to the health benefits that have been linked to them.

Certain cranberry components have anti-adhesive effects on specific uropathogens. Cranberries contain three types of Mavonoids (Mavonols, anthocyanins, and as well as catechins, hydroxyl cinnamic acid, and other phenolic acids and triterpenoids. Anthocyanins are absorbed and transported through the human circulatory system without causing any chemical changes in the urine.

Therefore, PACs can reduce the bacterial attachment to host tissues and prevent biofilm synthesis. It has been suggested by that disruption of quorum sensing by PACs might be the other reason of decrease in biofilm production. Cranberry can be an effective preventive measure for UTIs as it inhibits adhesion and biofilm formation of uropathogenic bacteria.

11. Conclusion

In this chapter, it is included that.

The prevalence of pathogens in complicated UTI is more than in simple UTI because its virulence as well as its ability to causes diseases.

Staphylococcus saprophyticus are the more predominant pathogen in UTI infection in female at the reproductive age.

Urease enzyme are sole of strutative cystitis.

All urease-producing bacteria are able to cause cystitis.

Successful uses of curcumine, zea mays, chrampry, ziziphus leave and coffee to eradicated the Bacterial cystitis.

Acknowledgments

We want to thanks and appreciation all staff in Al-Husseiny teaching hospital emergency for their cooperation during our research.