Influence of various uropathogens on crystallization of urine mineral components caused by Proteus mirabilis

Accepted Manuscript Influence of various uropathogens on crystallization of urine mineral components caused by Proteus mirabilis Agnieszka Torzewska, Katarzyna Bednarska, Antoni Różalski PII:

S0923-2508(18)30179-7

DOI:

https://doi.org/10.1016/j.resmic.2018.11.005

Reference:

RESMIC 3702

To appear in:

Research in Microbiology

Received Date: 27 July 2018 Revised Date:

19 November 2018

Accepted Date: 26 November 2018

Please cite this article as: A. Torzewska, K. Bednarska, A. Różalski, Influence of various uropathogens on crystallization of urine mineral components caused by Proteus mirabilis, Research in Microbiologoy, https://doi.org/10.1016/j.resmic.2018.11.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Title:

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Influence of various uropathogens on crystallization of urine mineral components caused by

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Proteus mirabilis Authors: Agnieszka Torzewskaa*, Katarzyna Bednarskaa, Antoni Różalskia

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University of Łódź, Banacha 12/16, 90-237 Łódź, Poland

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Department of Biology of Bacteria, Faculty of Biology and Environmental Protection ,

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E-mail:

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Correspondence and reprints - [email protected] ( Agnieszka Torzewska)

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[email protected] (Katarzyna Bednarska)

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[email protected] (Antoni Różalski)

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Abstract

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Infectious urolithiasis is a consequence of long-standing urinary tract infections with urease-

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positive bacteria, especially Proteus spp. However, because of the often mixed nature of

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urinary tract infections, in the case of urinary stones formation, several species of bacteria

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may be involved in the process. The purpose of the study was to determine the impact of the

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bacterial species: Escherichia coli, Klebsiella pneumoniae, Providencia stuartii,

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Pseudomonas aeruginosa and Staphylococcus aureus on the crystallization caused by

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Proteus mirabilis. The studies were conducted in synthetic urine with the addition of P.

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mirabilis and a representative of another species. During the experiments the viability of

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bacteria, pH, presence and morphology of crystals, and the intensity of crystallization were

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assessed. Crystallization of calcium and magnesium phosphates occurred in all investigated

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configurations. However, there were differences observed in the course and intensity of

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crystallization between the mixed culture and the P. mirabilis culture. Although most intense

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crystallization took place in the pure culture of P. mirabilis it was also demonstrated that the

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presence of other uropathogens increased the survival of P. mirabilis. This synergistic effect

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could be responsible for the persistence and recurrence of urolithiasis in the urinary tract.

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Key words: uropathogen, Proteus mirabilis, multibacterial infection, urinary tract infection,

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urolithiasis

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1. Introduction Urolithiasis is a multifactorial disease resulting from metabolic disorders, poor diet, obesity,

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incorrect concentration of crystallization inhibitors and promoters in urine or bacterial

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infection. Almost 10% of the human population suffers from this disease [1]. Urinary stones

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formed as a result of urinary tract infection caused by urease-producing bacteria account for

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about 10-15 % of all urinary calculi [2]. This urolithiasis is distinguished by rapid growth of a

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stone (4–6 weeks are enough to form a stone), persistence and high rate, up to 50% of

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recurrence, even with proper treatment [3]. Proteus mirabilis is the species that most

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commonly causes the development of infectious urolithiasis. Precipitation and crystallization

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of the urine components are caused by urease of these bacteria. An increase in urinary pH and

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the reactions occurring as a result of urea hydrolysis cause a rise in the concentrations of ions:

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NH4+, PO43-, CO32-, which, in the presence of calcium and magnesium even in normal

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concentrations, leads to the precipitation of phosphate salt and promotes crystallization of

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struvite (MgNH4PO4 x 6 H2O) and carbonate apatite (Ca10(PO4)6CO3) [4, 5]. In the further

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steps of urinary stones formation the crystals aggregate and are retained in the urinary tract. In

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these stages bacterial extracellular polysaccharides and microorganism macromolecules such

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as glycosaminoglycans are involved [2, 6]. Moreover, P. mirabilis bacteria, which are able to

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survive in the cells of the urinary tract and to avoid antimicrobial factors and mechanisms of

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the host immune system [7], cause the formation of microcrystals inside the urothelial cells

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[7, 8]. Intracellular crystals grow in isolated conditions, can reach the size that allows further

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mineral salt deposition, attachment of other crystals and macromolecules derived from the

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host and bacteria, and form a stone. In addition, intracellular bacteria are a source of recurrent

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infections. These two phenomena are likely to be responsible for the recurrence and

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persistence of infectious urolithiasis caused by Proteus bacilli [8].

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Pathogenesis of urinary stones formation as a result of P.mirabilis infection is well understood but there is still little information about the participation of other microorganisms

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in the development of P.mirabilis- induced urolithiasis. Clinical studies have shown that it is

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rarely the case that only one species of microorganisms is isolated from urinary calculi.

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Besides P.mirabilis the bacteria most commonly isolated from urinary stones are Escherichia

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coli, Klebsiella pneumoniae and Pseudomonas aeruginosa [9, 10, 11]. P. mirabilis is most

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frequently isolated from struvite stones, while E. coli and K. pneumoniae are more often

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found in calculi built of calcium oxalate and calcium phosphate [ 9,11]. How urease-negative

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bacteria contribute to urinary stone formation has still not been explained. It is also worth

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emphasizing that urinary tract infections can be polymicrobial infections, which are

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frequently caused by several microorganisms simultaneously. This occurs particularly in

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people with a weakened immune system or in long-term catheterized patients [12]. In these

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cases, P. mirabilis is a common cause of both complicated UTI (12%) and catheter-associated

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bacteriuria in long-term catheterized patients (15%) [13, 14]. In such situations it can be

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assumed that both infection and urinary stones formation may be accompanied by the

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occurrence of synergistic interactions. Therefore, the purpose of this work was to determine

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the impact of the most common uropathogens on crystallization caused by P. mirabilis.

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2. Material and Methods

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2.1.

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Bacterial strains

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Uropathogenic strains of Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis,

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Providencia stuartii, Pseudomonas aeruginosa, and Staphylococcus aureus were used in the

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study. All bacteria were obtained from encrusted biofilm formed on Foley’s urinary catheters

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of long-term catheterized patients (from several months to several years) and were deposited

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in the bacterial strains collection at the Department of Biology of Bacteria, the University of

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ACCEPTED MANUSCRIPT Lodz. Method of isolation of bacteria and their identification has been already described

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earlier by Moryl et al. [15]. Strains selected for this research came from catheters with surface

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visibly encrusted with mineral salts. Before the experiment, the bacteria were cultured on

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tryptic soy broth (TSB, BTL, Łódź, Poland) for 18 h at 37 °C. These cultures were mixed

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with synthetic urine to achieve a bacterial suspension with a density 1x 106 CFU mL-1 (colony

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forming units per milliliter). In practice, this meant at least a 1000-fold dilution of the culture

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in synthetic urine. The number of bacteria was checked spectrophotometrically at 550 nm

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(Ultrospec2000, Pharmacia Biotech, Vienna , Austria) using a standard curve of a directly

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proportional relationship between the absorbance (A 550) and the number of bacteria in the

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suspension.

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2.2.

Synthetic urine

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Synthetic urine was prepared using the modified method previously described by Griffith et

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al. [16] which is now widely used in in vitro studies of uropathogens [17]. The solution

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consisted of the following components (g L-1): urea, 25.0; NaCl, 4.6; KH2PO4, 2.8; Na2SO4,

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2.3; KCl, 1.6; NH4Cl, 1.0; creatinine, 1.1; CaCl2 × 2H2O, 0.651; MgCl2 × 6H2O, 0.651;

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sodium citrate, 0.65; sodium oxalate, 0.02; and tryptic soy broth, 10.0 (Sigma-Aldrich,

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Poznan, Poland). Synthetic urine composition corresponds to the mean concentration of the

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mineral components found during a 24-hour period in normal human urine. Prior to the

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experiment, pH was adjusted to 5.8 and the synthetic urine was sterilized by passing through a

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0.2 µm pore-size filter.

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2.3.

Analysis of urease activity

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Urease activity was determined using the phenol hypochlorite ammonia determination assay

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[18]. One unit of urease activity was defined as µg NH3 produced per 1 min, and expressed as

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ACCEPTED MANUSCRIPT units per mg of total bacterial protein (U/mg). First, 1 ml of bacterial culture (TSB medium,

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37°C, 24 h) was centrifuged (8000g/5min), suspended in 1 ml of synthetic urine and

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incubated for 15 min at 37°C. Then 10 µl of bacterial suspension in synthetic urine was mixed

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with 150 µl phenol sodium nitroprusside solution (1 % w/v phenol, 0.05 % w/v sodium

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nitroprusside) and 150 µl sodium hypochlorite solution (0.5 % w/v sodium hydroxide, 0.5 %

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w/v sodium hypochlorite) and incubated for 30 min at 37°C. The absorbance value which was

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measured at 625 nm with a Multiskan Ex (Labsystems, Helsinki, Finland), using all reagents

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without bacteria as a negative control (blank) was compared to that given by standard

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solutions of ammonium sulphate. Bacterial suspension remaining in synthetic urine was

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centrifuged (8000g/5min), after that 2M NaOH was added to the pellet and incubated

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overnight at 37°C to lyse bacterial cells. The amount of protein was measured by the Lowry

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method [19]. In addition, the urease activity of all bacterial strains was detected on the

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Christensen’s urea medium.

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2.4.

Crystallization experiment

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2.4.1. Determination of the effect of other bacteria on crystallization caused by P.

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mirabilis

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The differences in crystallization intensity were determined in mixed bacterial cultures in 20

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ml of synthetic urine in a 50 ml tube with conical bottom after 24 h of incubation at 37°C

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without stirring. Suspensions of bacteria were prepared as described above. In each

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experiment another bacterial species (E.coli, K. pneumoniae, P. stuartii, P. aeruginosa, S.

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aureus) were added to the P. mirabilis suspension, each at a concentration of 1x106 CFU mL-

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number of bacteria were assessed. Crystallization intensity was determined by chemical

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analysis and direct phase-contrast microscopy. A pure culture of P. mirabilis was used as a

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. After 5 h , and 24 h incubation periods, the pH, the intensity of crystallization and the

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ACCEPTED MANUSCRIPT reference system. Crystals observation was performed using an inverted microscope Nikon

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Eclipse S2000 with software enabling image analysis. For chemical analyses, 1 ml of each

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sample containing urine with bacteria and crystals was collected, centrifuged (8000g/5min)

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and treated for mineralization with HNO3 (65%, POCh, Gliwice, Poland) for 60 min at 100

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°C. The phosphate concentration was measured by the colorimetric method based on the

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reduction of phosphomolybdic acid [20] and atomic absorption spectroscopy (SpectrAA-300

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Varian, Palo Alto, California ) was used to determine calcium and magnesium concentrations.

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To determine the number of bacteria in pure and mixed cultures, the suspensions were serially

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diluted in saline solution. In the case of mixed cultures of E.coli, K.pneumoniae, P.mirabilis

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and P.stuartii aliquots of 100 µl were spread on McConkey medium. For mixed cultures of

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P.mirabilis with S.aureus diluted suspensions were spread both on McConkey (P. mirabilis

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growth) and TSB agar (P.mirabilis and S.aureus growth) and for P.mirabilis with

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P.aeruginosa McConkey (P.mirabilis growth) and Cetrimide agar (P. aeruginosa growth)

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were used. After overnight incubation at 37°C, colonies on the plates were counted to

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determine the number of CFU mL-1 (colony forming units per milliliter). In mixed cultures,

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the ability to grow, breakdown of lactose and different colony morphology were taken into

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account to determine the number of bacteria of individual species.

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Statistics

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The data are presented as mean ± standard deviation (SD) of five to seven independent

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experiments. Statistical analyses were based on the Mann-Whitney U test the Anova Kruskal-

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Wallis test and performed using Statistica software version 12 pl (StatSoft ,Poland). The

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results were considered to be statistically significant at p < 0.05. Statistical differences

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between groups are indicated in the text.

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3. Results

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3.1.

Viability of bacteria in pure and mixed cultures

At the beginning of each experiment, the suspension in the synthetic urine had a density of 0.8

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to 1.2 x 106 CFU mL-1 for each bacteria. As shown in Fig. 1, the number of all tested bacteria

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during incubation increased significantly at 5 h reaching a value of 107 CFU mL-1 with the

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exception of Pseudomonas aeruginosa and Staphylococcus aureus of which growth was 10-

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times weaker at that time. After 24 h, probably due to the high pH of the synthetic urine, the

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growth of the bacteria was inhibited. In the case of Proteus mirabilis, the number of bacteria

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reached a value 39.5 ± 19.8 x 106 CFU mL-1, after 5 h of incubation in synthetic urine, but at

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24 h their viability drastically decreased, even 100-fold. When comparing the number of

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P.mirabilis cells in the pure culture to that of the number of bacteria in the mixed cultures, it

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can be seen that during the shorter incubation (5 h) the presence of other bacteria did not

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affect P. mirabilis growth (p > 0.05), whereas after 24 h, in all mixed cultures, the number of

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P. mirabilis bacilli was much higher than in the pure culture of this microorganism (p ≤

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0.001). The highest number of P.mirabilis cells was observed in co-cultured infection with

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Escherichia coli. The numbers of cells of the other species that were co-cultured with P.

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mirabilis ranged between 45.2 – 2.9 x 106 CFU mL-1 after 5 h of incubation, whereas at 24h

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the presence of live bacteria was detected only in the case of Providencia stuartii, P.

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aeruginosa and S. aureus (0.2 – 12 x 106 CFU mL-1). In the presence of E. coli and K.

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pneumoniae no growth was observed after this time, which might have been caused by the

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higher sensitivity of these bacteria at high pH.

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Changes in urine pH due to the activity of bacterial urease

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ACCEPTED MANUSCRIPT During the growth of urease-positive bacteria in urine the pH increases over time. As shown

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in Table 1, apart from P.mirabilis, two of tested strains exhibited urease activity i.e.

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Klebsiella pneumoniae and Providencia stuartii. The activity of this enzyme was highest in P.

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mirabilis (189 U/mg). While analyzing pH changes in the prepared cultures, it was found that

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in all tested samples of both mixed and pure P. mirabilis cultures the pH was increased (Table

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2). After 5h the pH values varied from 7.29 to 7.90. The highest pH was found when P.

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mirabilis was present together with P. stuartii. After 24 h of culture in all tests the pH values

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were very similar (9.48 - 9.57).

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Bacterial effect on the crystallization in synthetic urine

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As mentioned above, crystallization of mineral compounds in urine is provoked by an

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increase in the pH caused by bacterial urease. In the experiments, crystallization was induced

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by P. mirabilis bacteria. Chemically, the resulting crystals were found to be calcium and

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magnesium phosphates, and therefore in the performed experiments, the degree of

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crystallization was measured by the presence and amount of the Mg, Ca and phosphate ions.

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The reference point for these results was the calcium (1.86 ± 0.23 µg mL-1) and magnesium

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(1.31 ± 0.26 µg mL-1) content in a urine sample containing bacteria measured at 0 h. As

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shown in Figure. 2, for almost all tested samples, both after 5 and 24 hours of incubation, the

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amounts of ions were statistically significantly different from the reference (p ≤ 0.001), which

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indicated that crystallization had occurred. The initial crystallization (after 5h) is

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characterized by a higher content of calcium phosphate (apatite) than magnesium phosphate

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(struvite). In the sample containing P. mirabilis and S. aureus, at that point struvite (p=0.77,

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compared with reference point – 0 h) had not been crystallized yet. After 24h the differences

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between the amounts of calcium and magnesium were much smaller. At the beginning of the

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experiment, after 5 hours, crystallization was most intense when P. mirabilis alone was

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ACCEPTED MANUSCRIPT present in a culture. In the case of mixed cultures, only in the presence of P. stuartii and E.

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coli together with P. mirabilis the amounts of tested ions were clearly higher than in the

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control, while in the other samples the levels of ions were slightly different from the amount

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present in the control. In the presence of S. aureus, crystallization occurred particularly late.

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Similarly to the situation observed at 5h, after 24 h the amount of crystallized minerals in the

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P. mirabilis monoculture was higher than in the case of co-culture with other bacteria but this

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difference in each case, as indicated in Fig. 2, was not statistically significant. In particular,

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when P. mirabilis was accompanied by E. coli, the level of calcium and magnesium

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phosphate precipitates was equal to their amount in the pure culture (p > 0.05).

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During the whole experiment crystallization was also evaluated by observing the

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suspension under the phase contrast microscope. The time of the appearance of the first

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crystals, their size and morphology provide important information about the crystallization

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rate. It is known that precipitates of apatite (for example, carbonate apatite) appear as the first

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when the pH rises above 6.5 and that crystals of struvite are formed later when the pH rises

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above 7.2 [2]. These minerals have a distinctive appearance. Fig. 3 shows amorphous apatite

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and “coffin-like” crystals of struvite. After 5h only apatite was visible in all samples, with the

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largest amounts of the precipitates in the pure culture of P. mirabilis and co-culture of P.

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mirabilis with P. aeruginosa. At the end of the experiment (after 24h) the pH increased above

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9.0 and, apart from apatite, struvite crystals were also visible. The size and morphology of

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these crystals were varied. The smallest crystals of struvite were found in the culture of P.

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mirabilis with P. aeruginosa (17-24 µm), while the largest ones, exceeding 80 µm, were

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present in the urine infected with P. mirabilis alone. In most of the samples, the crystals were

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present in both coffin-like and dendritic form, with the exception of the culture of P. mirabilis

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with P. aeruginosa containing no dendrites, which may indicate decelerated crystallization.

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4. Discussion The formation of urinary stones as a result of infection by bacteria of the genus Proteus is a

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well-recognized phenomenon [5, 3]. However, most of the studies have been conducted on

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pure cultures of P. mirabilis, which does not fully reflect conditions that occur during urinary

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tract infections and complications such as urinary stone formation. Urinary tract infections are

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often polymicrobial and in such conditions interactions between microorganisms may

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influence the process of urinary stones formation. Polymicrobial urinary tract infections are

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most commonly associated with long-term urinary catheterization. Moryl et al [15] studying

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the distribution of Proteus mirabilis in multi-species biofilms on 88 urinary catheters showed

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that P. mirabilis accounted for about 18% of the bacteria isolated from the catheters and was

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often accompanied by such bacteria as: Pseudomonas aeruginosa, Providencia stuartii,

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Klebsiella pneumoniae, Escherichia coli, Morganella morganii and Enterococcus faecalis.

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Such a composition of mixed biofilms on urological catheters has also been confirmed by

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other studies [21, 22, 23]. Due to the fact that the same microorganisms are simultaneously

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isolated from urinary stones, strains isolated from encrusted catheters were used in the present

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study. In mixed culture or polymicrobial infection, microorganisms can compete for nutrients

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or provide them to each other, promote or inhibit colonization (adhesion, invasion), acquire

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new features by transfer of genetic information, or eliminate each other by secretion of

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antibacterial components [4]. These are dynamically changing interactions depending on the

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composition of mixed culture of microorganisms. In mixed cultures both positive and

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negative interactions between microorganisms have been observed. There are only a few

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studies which show the interactions between microorganisms in the formation of urinary

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stones [24,25,26]. On the basis of the results of the present study, it can be concluded that the

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ACCEPTED MANUSCRIPT highest intensity of mineral salts crystallization is noted when the urine is infected with P.

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mirabilis alone. The presence of other microorganisms seems to be delaying the process of

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crystallization and to inhibit its intensity. Previously, Armbruster et al [24] demonstrated in

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studies on mice that a simultaneous colonization with P. mirabilis and P. stuartii enhances

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the process of urinary stones formation. The effect of this interaction is explained by the

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synergistic intensification of the urease activity of both uropathogens. In further studies using

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the CAUTI mouse model these authors showed positive effects of other uropathogens such as

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Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa on

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the urease activity of P. mirabilis [25]. The activation mechanism is not yet known but seems

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very important in the pathogenicity of Proteus mirabilis in polymicrobial infection.

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Previously mentioned interactions between P. mirabilis and P. stuartii were not observed in

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these studies. Although the pH was the highest at 5h of incubation in the presence of P.

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mirabilis and P. stuartii , the level of crystallization, compared with the sample where P.

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mirabilis was present alone, was lower and the crystals were smaller. The differences in the

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results of these two studies may be due to different experimental models as well as the

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properties of the tested strains, which may indicate strain-specific dependency. Besides P.

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stuartii, another urease - positive uropathogen – K. pneumoniae was used in this study and in

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this case, the presence of P. mirabilis together with another microorganism producing urease

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did not intensify crystallization. It indicates that urease activity is required to induce the

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crystallization process but the level of crystallized salts depends on other bacterial factors and

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the environment of the macroorganism. Many authors have emphasized the role of

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polysaccharides of bacteria in modulating the crystallization intensity during the formation of

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infectious urinary stones [3, 27]. In accordance with their results, bacterial polysaccharides

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that bind cations of calcium and magnesium can accelerate crystallization of struvite and

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apatite. In the case of in vivo studies ,the presence of inhibitors and promoters of

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ACCEPTED MANUSCRIPT crystallization (e.g. glycosaminoglycans, ) as well as various concentrations of ions (e.g.

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magnesium, calcium) in the urine which are involved in crystallization are also significant [6,

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28].

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The effect of mixed cultures on the formation of infectious urinary stones may not be directly

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related to the crystallization process but it can also be associated with different degree of

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adaptation of bacteria to unfavorable growth environment such as urine. Urine has a low pH

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and high concentration of urea which inhibits the growth of most bacteria. Bacteria can

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hydrolyze urea (urease - positive e.g. P. mirabilis) and endure its adverse effects [3] or have

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osmoadaptative systems, like accumulation of betaine inside cells, e.g. E. coli [29]. Bacteria

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can also use urine components for a growth which is allowed by the metabolic pathways

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characteristic for this environment [29]. The advantage is also given to microorganisms with

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siderophores which allow them to obtain iron ions from the environment. In the case of

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polymicrobial bacteriuria bacteria can mutually favor the growth of other bacteria or inhibit it.

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In the presented results As shown in Fig. 1, both in pure and in mixed cultures the number of

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bacteria decreased at 24 h of incubation due to the high pH. However in all cases, the number

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of P. mirabilis bacteria was higher in mixed cultures, which indicates that in presence of

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other uropathogens infections P. mirabilis has better conditions for growth and colonization

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of the organism. It also seems probable that the ureolytic activity and the changing pH of the

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growth environment can affect the number and type of species that are co-found in long-term

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infections. In our research, in the case of P. mirabilis and E. coli co-culture infection, the

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number of E. coli bacteria dropped to almost zero but the viability of P. mirabilis was not

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altered compared to 5h. Previously, co-infections were studied in mouse experimental model

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in the context of polymicrobial infections by Alteri et al. [26]. These authors have shown that

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the co-infection of E. coli and P. mirabilis results in greater colonization of both

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ACCEPTED MANUSCRIPT microorganisms in the urinary tract of mice. This effect is due to the metabolic properties of

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both microorganisms consisting in the complementary use of carbon sources [30]. Contrary to

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these findings, in our studies co-culture infection did not have a positive effect on E. coli .

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This is probably due to the large accumulation of bacteria in the environment with elevated

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pH, a high concentration of ammonia and an increasingly low carbon content. In vivo, such a

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situation can occur in the biofilm on the urothelium or on the urinary catheters as well as in

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the external or internal structure of the urinary stone.

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Research is preliminary and requires continuation, especially for in vivo studies in

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which the contribution of macroorganism’s factors will be taken into account. From a point of

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view of the urinary stones formation it is particularly interesting to assess both the individual

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differences in the composition of normal human urine, which cannot be demonstrated by

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using synthetic urine and the involvement of immune system factors. However, the results

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obtained clearly indicate that in the presence of other microorganisms the crystallization

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caused by P. mirabilis is delayed and that P. mirabilis in such co-infections have a better

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viability, which may affect the persistence of infectious urolithiasis and increase the duration

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of treatment. It is not without importance to further analyze cell components of urease-

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negative bacteria which influence the crystallization process during the formation of urinary

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stones.

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Declaration of interest

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The authors report no conflicts of interest

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Acknowledgments

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This work was supported by the Ministry of Science and Higher Education, Poland (core

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funding for statutory research activity, Grant 1132 - Department of Biology of Bacteria,

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University of Lodz).

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References

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[1] Knoll T. Epidemiology, pathogenesis, and pathophysiology of urolithiasis. Eur Urol Suppl

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2010;9 :802-806.

332

[2] Flannigan R, Choy WH, Chew B, Lange D. Renal struvite stones—pathogenesis,

333

microbiology, and management strategies. Nat Rev Urol 2014;11:331-341.

334

[3] McLean R J C, Nickel J C, Cheng K-J, Costerton JW. The ecology and pathogenicity of

335

urease-producing bacteria in the urinary tract. CRC Crit Rev Microbiol 1988;16:37-79.

336

[4] Prywer J, Torzewska A. Bacterially induced struvite growth from synthetic urine:

337

experimental and theoretical characterization of crystal morphology. Cryst Growth Des 2009;

338

9: 3538–3543.

339

[5] Bichler KH, Eipper E, Naber K, Braun V, Zimmermann R, Lahme S. Urinary infection

340

stones. Int. J. Antimicrob. Agents 2002; 19:488–498.

341

[6] Torzewska A, Różalski A. In vitro studies on the role of glycosaminoglycans in

342

crystallization intensity during infectious urinary stones formation. APMIS 2014;122(6):505-

343

11. doi: 10.1111/apm.12191.

344

[7] Mathoera RB, Kok DJ,1 Verduin CM, Nijman RJM. Pathological and therapeutic

345

significance of cellular invasion by Proteus mirabilis in an enterocystoplasty infection stone

346

model. Infect Immun 2002; 70: 7022–7032.

AC C

EP

TE D

M AN U

SC

329

16

ACCEPTED MANUSCRIPT [8] Torzewska A, Budzyńska A, Białczak-Kokot M, Rozalski A. In vitro studies of

348

epithelium associated crystallization caused by uropathogens during urinary calculi

349

development. Microb Pathogenesis 2014; 71-72: 25-31.

350

[9] Tavichakorntrakool R, Prasongwattana V, Sungkeeree S, Saisud P, Sribenjalux P,

351

Pimratana C, et al. Extensive characterizations of bacteria isolated from catheterized urine and

352

stones matrices in patients with nephrolithiasis. Nephrol Dial Transplant 2012; 27: 4125-

353

4130.

354

[10] Lewi H J E., White A, Hutchinson G, Scott R. The bacteriology of the urine and renal

355

calculi. Urol Res 1984; 12: 107-109.

356

[11] Mufarrij PW, Lange JN, Assimos DG, Mirzazadeh M, Holmes RP. Multibacterial

357

growth from a surgical renal stone culture: a case report and literature review. Rev Urol

358

2012;14:108-114.

359

[12] Short F L, Murdoch S L, Ryan R P. Polybacterial human diseases: the ills of social

360

networking. Trends Microbiol 2014; 22:508-516.

361

[13] Jacobsen SM, Stickler DJ, Mobley HLT, Shirtliff ME. Complicated catheter-associated

362

urinary tract infections due to Escherichia coli and Proteus mirabilis. Clin Microbiol Rev

363

2008;21:26–59.

364

[14] Tay W H, Chong K K L, Kline K A. Polymicrobial-host interactions during infection. J

365

Mol Biol 2016;428:3355-3371.

366

[15] Moryl M, Torzewska A, Jalmuzna P, Rozalski A. Analysis of Proteus mirabilis

367

distribution in multi-species biofilms on urinary catheters and determination of bacteria

368

resistance to antimicrobial agents. Pol J Microbiol 2013;62:377-384.

369

[16] Griffith D P, Musher D M, Itin C. Urease, the primary cause of infection-induced urinary

370

stones. Invest Urol 1976;13:346-350.

AC C

EP

TE D

M AN U

SC

RI PT

347

17

ACCEPTED MANUSCRIPT [17] Ipe DS, Ulett GC. Evaluation of the in vitro growth of urinary tract infection-causing

372

gram-negative and gram-positive bacteria in a proposed synthetic human urine (SHU)

373

medium. J Microbiol Methods 2016;127:164-171.

374

[18] Weatherburn M W. Phenol-hypochlorite reaction for determination of ammonia. Anal

375

Chem 1967; 39: 971-3.

376

[19] Lowry JOH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with the

377

Folin phenol reagent. J Biol Chem 1951; 193: 260-5.

378

[20] Ames BH, Dubin DT. The role of polyamines in the neutralization of bacteriophage

379

deoxiribonucleic acid. J Biol Chem 1960;235:769-775.

380

[21] MacLeod S M, Stickler D J. Species interactions in mixed-community crystalline

381

biofilms on urinary catheters. J Med Microbiol 2007;56:1549-1557.

382

[22] Ganderton L, Chawla J, Winters C, Wimpenny J, Stickler D. Scanning electron

383

microscopy of bacterial biofilms on indwelling bladder catheters. Eur J Clin Microbiol Infect

384

Dis 1992;11:789-796.

385

[23] Armbruster C, Prenovost K, Mobley HLT, Mody L. How often do clinically diagnosed

386

catheter-associated urinary tract infections in nursing homes meet standardized criteria? J Am

387

Geriatr Soc 2017b;65:395–401.

388

[24] Armbruster CE,. Smith SN, Yep A, Mobley HTL. Increased incidence of urolithiasis and

389

bacteremia during Proteus mirabilis and Providencia stuartii coinfection due to synergistic

390

induction of urease activity. J Infect Dis 2013;26:1-9.

391

[25] Armbruster ChE, Smith SN, Johnson AO, DeOrnellas V, Eaton KA, Yep A, et al. The

392

pathogenic potential of Proteus mirabilis is enhanced by other uropathogens during

393

polymicrobial urinary tract infection. Infect Immun 2017a, 85, e:00808-16.

AC C

EP

TE D

M AN U

SC

RI PT

371

18

ACCEPTED MANUSCRIPT [26] Alteri ChJ, Himpsl SD, Mobley HLT. Preferential use of central metabolism in vivo

395

reveals a nutritional basis for polymicrobial infection. PLoS Pathog 2015;11(1): e1004601.

396

doi: 10.1371/journal.ppat.1004601.

397

[27] Dumanski A J, Hedelin H, Edin-Liljegren A, Beauchemin D, McLean RJ. Unique ability

398

of the Proteus mirabilis capsule to enhance mineral growth in infectious urinary calculi.

399

Infect Immun 1994;62: 2998-3003.

400

[28] Ebisuno S, Komura T, Yamagiwa K. Urease-induced crystallizations of calcium

401

phosphate and magnesium ammonium phosphate in synthetic urine and human urine. Urol

402

Res 1997;25(4):263-7.

403

[29] Ipe DS, Horton E, Ulett . GC. The basic of bacteriuria: strategies of microbes for

404

persistence in urine. Front Cell Infect Microbiol 2016;6: 14

405

[30] Norsworthy AN, Pearson MM. From catheter to kidney stone: the uropathogenic

406

lifestyle of Proteus mirabilis. Trends Microbiol 2017;25:304–315.

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Legends to figures

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Fig.1 Bacterial viability in mixed and pure cultures during incubation in synthetic urine after:

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A -5h, B-24h. The values represent means ± SD of 5-8 experiments. **- p < 0.05, *- p < 0.001

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for P. mirabilis viability in mixed culture vs pure culture, U Mann- Whitney test Anova

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Kruskal-Wallis test.

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Fig.2 Intensity of crystallization induced by P.mirabilis after A – 5h, B- 24h of incubation.

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The values represent means ± SD of 5-8 experiments. **- p < 0.05, *- p < 0.001 for comparison

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of each ion concentration to control culture of P. mirabilis, U Mann- Whitney test Anova

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Kruskal-Wallis test.

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Fig.3 Amorphous apatite (A) and struvite crystals (S) in synthetic urine infected with: 1-

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P.mirabilis, 2 – P.mirabilis + E.coli, 3- P.mirabilis + K.pneumoniae, 4 – P.mirabilis +

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5h, b-after 24h of incubation. The scale bar represents 20 µm.

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Table 1. Differences in the ureolytic activity of the tested strains. Strain

Activity of urease Culture method* Colorimetric method [U/mg]** positive 189.9 ± 9.8 positive 39.9 ± 2.3 positive (after 48h) 24.9 ± 4.1 negative 5.0 ± 1.2 negative 4.1 ± 2.9 negative 3.6 ± 1.9

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Proteus mirabilis Providencia stuartii Klebsiella pneumoniae Pseudomonas aeruginosa Escherichia coli Staphylococcus aureus

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* Urease activity was detected on Christensen’s medium, **- urease activity was expressed as µg NH3 produced -1 -1 x 1 min x (mg total bacterial protein) by colorimetric methods see Materials and methods section, results are presented as means ± standard deviation (SD) of five experiments

ACCEPTED MANUSCRIPT Table 2.Changes in urinary pH induced by bacteria.

Sample

Time of incubation 5h 24h 7.57 ± 0.73 7.29 ± 0.29 7.43 ± 0.32 7.90 ± 0.23 7.29 ± 0.09 7.34 ± 0.14

9.48 ± 0.10 9.50 ± 0.03 9.56 ± 0.04 9.57 ± 0.04 9.49 ± 0.04 9.47 ± 0.06

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P. mirabilis P. mirabilis+ E. coli P. mirabilis + K. pneumoniae P. mirabilis +P. stuartii P. mirabilis + P. aeruginosa P. mirabilis + S. aureus

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Results are presented as means ± standard deviation (SD) of five experiments

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