Contents lists available at ScienceDirect Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath Allicin prevents the formation of Proteus-induced urinary crystals and the blockage of catheter in a bladder model in vitro Hamed Imani Rad a , Hadi Peeri a , Mojtaba Amani b,c , Alireza Mohammadnia d , Abiodun David Ogunniyi e , Manouchehr Khazandi e , Henrietta Venter f , Mohsen Arzanlou a,* a Department of Microbiology, School of Medicine, Ardabil University of Medical Sciences, Ardabil, Iran b Department of Clinical Chemistry, School of Pharmacy, Ardabil University of Medical Sciences, Ardabil, Iran c Department of Biochemistry, School of Medicine, Ardabil University of Medical Sciences, Ardabil, Iran d Department of Information Technologies, School of Medicine, Ardabil University of Medical Sciences, Ardabil, Iran e Australian Centre for Antimicrobial Resistance Ecology, School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy, South Australia, Australia f School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, 5000, Australia ARTICLE INFO Keywords: Allicin Proteus mirabilis Synthetic urine Crystallization Urinary catheter Blockage Bladder model ABSTRACT Stone formation and catheter blockage are major complications of Proteus UTIs. In this study, we investigated the ability of allicin to inhibit P. mirabilis-induced struvite crystallization and catheter blockage using a synthetic bladder model. Struvite crystallization inhibition study was carried out using P. mirabilis lysate as urease enzyme source in synthetic urine (SU). Struvite productions were monitored by phase contrast light microscopy and measurements of pH, Mg 2+ and Ca 2+ precipitation and turbidity. A catheter blockage study was performed in a synthetic bladder model mimicking natural UTI in the presence of allicin at sub-MIC concentrations (MIC = 64 μg/ml). The results of crystallization study showed that allicin inhibited pH rise and consequently turbidity and precipitation of ions in a dose-dependent manner. The results of catheter blockage study showed that allicin at sub-MIC concentrations (2, 4, 8 μg/ml) significantly increased the time for catheter blockage to occur to 61, 74 and 92 h respectively compared to allicin-free control (48 h). In a similar way, the results showed that allicin delayed the increase of SU pH level in bladder model in a dose-dependent manner compared to allicin-free control. The results also showed that following the increase of allicin concentration, Mg 2+ and Ca 2+ deposition in catheters were much lower compared to allicin-free control, further confirmed by direct ob- servation of the catheters’ eyehole and cross sections. We conclude that allicin prevents the formation of Proteus- induced urinary crystals and the blockage of catheters by delaying pH increase and lowering Mg 2+ and Ca 2+ deposition in a dose-dependent manner. 1. Introduction Proteus mirabilis is the third common cause of complicated urinary tract infections (UTIs) especially in those with structural or functional urinary tract abnormalities [1]. Among the Gram-negative bacteria, P. mirabilis shows the greatest adherence to catheters [2]. Accordingly, it is the second most common cause of catheter-associated UTIs (CAUTI) in patients with long-term indwelling catheters [3]. In spite of decreases in the incidence of most hospital acquired infections (HAIs), the risks of CAUTI are still high [1] and CAUTIs accounting for about 40% of hospital acquired infections. The duration of catheterization is one of the important risk factors associated with CAUTI development. Up to 50% of patients catheterized for short term, develop bacteriuria in a week and 90% of patients undergoing long-term catheterization will develop CAUTI within 4 weeks [4]. CAUTIs with urease positive or- ganisms are problematic since they can cause urinary stones and crys- talline biofilm on catheters [1]. Infected stones account for about 15% of all urinary stones [5]. Stone formation and catheter blockage are one of the most important complications in Proteus UTIs. P. mirabilis pro- duces many virulence factors that are involved in the establishment of these complications such as fimbriae that mediate attachment to ur- inary tract cells and the catheter surface, as well as urease enzyme which hydrolyses urea to the carbon dioxide and ammonia [4]. An increased amount of ammonia elevates the pH of the urine and biofilm. Under alkaline conditions struvite (MgNH 4 PO 4 × 6H 2 O) and carbonate apatite (Ca 10 (PO 4 ) 6 × CO 3 ) crystals are formed and precipitate in the urinary tract and on the catheter surface. The crystals form structures in the catheter, eventually blocking the catheter eyehole or drainage https://doi.org/10.1016/j.micpath.2019.05.016 Received 12 February 2019; Received in revised form 8 May 2019; Accepted 9 May 2019 * Corresponding author. E-mail address: m.arzanlou@arums.ac.ir (M. Arzanlou). Microbial Pathogenesis 132 (2019) 293–301 Available online 10 May 2019 0882-4010/ © 2019 Elsevier Ltd. All rights reserved. T