Chỉ số độ bám dính giảm tới 75% đối với cả chủng UPEC và chủng P. mirabilis. Kết quả nghiên cứu cho thấy rằng, ngoài công dụng chống bám dính với các chủng E. coli gây bệnh đường tiết niệu , Proanthocyanidin loại A2 còn hoạt động trên các chủng P. mirabilis , làm giảm khả năng bám dính vào các tế bào biểu mô của bàng quang.
Anti-Adhesion Activity of A2-type Proanthocyanidins (a Cranberry Major Component) on Uropathogenic E. coli and P. mirabilis Strains
Abstract
Urinary tract infections (UTIs) are relatively common in women and may be classified as uncomplicated or complicated, depending upon the urinary tract anatomy and physiology. Acute uncomplicated cystitis (AUC) occurs when urinary pathogens from the bowel or vagina colonize the periurethral mucosa and reach the bladder. The vast majority of episodes in healthy women involving the same bacterial strain that caused the initial infection are thought to be reinfections. About 90% of AUC are caused by uropathogenic Escherichia coli (UPEC), but Proteus mirabilis also plays an important role. Several studies support the importance of cranberry (Vaccinium macrocarpon) proanthocyanidins in preventing adhesion of P-fimbriated UPEC to uroepithelial cells. In this study, we evaluated the in vitro anti-adhesion activity of A2-linked proanthocyanidins from cranberry on a UPEC and Proteus mirabilis strains and their possible influence on urease activity of the latter. A significant reduction of UPEC adhesion (up to 75%) on the HT1376 cell line was observed vs. control. For the strains of P. mirabilis there was also a reduction of adhesion (up to 75%) compared to controls, as well as a reduction in motility and urease activity. These results suggest that A2-type cranberry proanthocyanidins could aid in maintaining urinary tract health.
1. Results and Discussion
1.1. Adhesion Assay
The assay of adhesion carried out on the strains treated with PACs showed a reduction of up to 75% of the adhesion index both for the UPEC strains and for the strains of P. mirabilis, with respect to the same strains cultured without PACs. Such a large reduction was obtained only at the concentration of 50 µg/mL. The lower concentrations had a variable efficacy in reducing adhesion of the bacteria in the study, as well as providing discrepant results. At the concentration of 15 µg/mL, E. coli shows a decrease between 13.8% and 24.1%, while P. mirabilis presents a high variability in its values, with percentages ranging from 3.3% up to 49.6%. With 25 µg/mL, a decrease between 36.9% and 53.6% for E. coli and between 44.9% and 68.4% for P. mirabilis occurs. With 50 µg/mL, values appear more consistent, with reduction percentages varying from 67.1% to 75.4% for E. coli and from 61.4% to 75.4% for P. mirabilis. With 75 µg/mL, the observed decrease is between 74.3% to 79.9% for E. coli and from 66.7% to 77.4% for per P. mirabilis. At the concentration of 100 µg/mL, the reduction goes from 78% up to 84.8% for E. coli and from 77.6% up to 81.7% for P. mirabilis. These results are shown in Figure 1 and Figure 2 demonstrating the constant decrease of adhesion by all the strains examined. The count of adhering bacteria was carried out manually, both for controls (Figure 3) and treated strains (Figure 4).
Adhesion indexes for the strains of E. coli.
Adhesion indexes for the strains of P.mirabilis. In both figures the reduction of the adhesion indexes is clear.
(a)(b) P. mirabilis adherent to a cell on a control slide.
(a–c) Strains of P. mirabilis adherent to cells after treatment with PACs at the concentration of 50 µg/mL. The lower number of adherent bacteria can be clearly seen.
1.2. Scanning Electron Microscopy
From the images obtained from scanning electron microscopy, the cells are integral and the adherent bacteria are fewer than those in the controls (Figure 5).
(a,b) A cell with few adhering bacteria at the concentration of 50 µg/mL. In the outer part, which is in contact with the environment, the cells have cilia, flagella and microvilli; (c) In the controls the cells are also integral but with a higher number of adherent bacteria.
1.3. Motility Test
Only for P. mirabilis 2 and P. mirabilis 4 we observed a reduction of swarming capacity with respect to the control (Figure 6). For the other P. mirabilis strains, substantial differences in the swarming were not observed. The cause of this different behavior is unknown at present but will be the subject of further in-depth analysis.
(a, b) Reduction of swarming capacity observed for two P. mirabilis strains. The lower size of growth halo can be seen in both right plates.
1.4. Production of Urease
The capacity to hydrolyze urea, thanks to an enzyme called urease, producing ammonia and carbon dioxide, is considered one of the principal characteristics of the species of Proteus. This was thus used as an identifying criterion to distinguish them from other non-fermenting bacteria belonging to the family of the Enterobacteriaceae.
In Christensen’s medium, Proteus was able to use the urea as the only source of nitrogen, thus producing a dose of ammonia sufficient to change the color of the medium from yellow to fuchsia. The change of color was due to the presence of red phenol, contained in the medium, which is yellow up to pH 6.8 and then becomes a bright red at pH 8.2.
The absorbance mean value for P. mirabilis 3 was 0.075 OD with respect to 0.203 OD of the control. The pH mean value was 7 vs. 8.5 of the control. P. mirabilis 4 showed the same behavior, with 0.105 as average OD (versus 0.417 of the control) and 7.5 as average pH (versus 9 of the control) respectively. On the contrary no substantial differences were observed for the remaining two strains examined, neither in absorbance nor in pH. The change of media color, actually, was not evident for both strains, showing a limited urease activity. The causes for the discrepancy in the results observed for the examined strains is still unknown and will be the subject of further analyses.
1.5. Discussion
Cranberries have been used for decades for the prevention and treatment of UTIs. Numerous studies have been carried out supporting their efficacy in reducing the number of relapses in women subject to recurrent infections [13,14,15,16,17]. It is well known that their efficacy depends on the anti-adhesive effect of proanthocynidins that they contain on the fimbriae of the uropathogenic strains of E. coli. In this study we wanted to demonstrate a similar effect also on the fimbriae expressed by P. mirabilis, as well as a reduction of motility and urease activity, two other important virulence factors.
P. mirabilis can exist in two different morphotypes: in the first it has 6/10 peritrichous flagella that allow individual movement; in the second it has thousands of flagella that allow a particular type of movement, defined swarming. The two different types of movement alternate during the growth of Proteus. When the bacterium is of the second morphotype, the cells move a lot then undergo a temporary halt when the bacterium changes to the first morphotype. The result of this alternation is the characteristic growth in concentric circles that can be seen on the agar dishes. Both these types of movement are made by means of the flagella, the filaments composed principally of a protein called flagellin. The protein FlaA is codified by the flaA gene [20] and by flhD. It seems that the activity of this gene, over expressed during the phase of swarming, is notably reduced by exposure to PACs [21].
Also, the urease activity is slightly inhibited. By means of hydrolysis of the urea, the bacteria obtains a source of nitrogen but, contemporarily, this leads to the formation of crystals that can block the catheter or damage kidneys and bladder [22,23]. For this reason, infection from Proteus causes severe damage to tissues with respect to that from E. coli. The activity of cranberries, and of proanthocyanidins in particular, on the species of Proteus has to date been poorly investigated [21]. From the results of this study it seems that they could also play an important role in the prevention of infections caused by this microorganism.
2. Experimental
2.1. Bacterial Strains
For our experiments we used 5 strains of P. mirabilis and 5 of E. coli isolated from the urine of women with a history of recurrent cystitis, a standard strain of P. mirabilis (DSM4479) and a standard strain of E. coli (ATCC11775).
2.2. Adhesion Assay
The adhesion assay was carried out using the method of Di Martino et al. [24] slightly modified. The HT1376 cells, from human bladder carcinoma, were grown on a sterile cover glass in 24-well plates at 37 °C in Minimal Essential Medium (MEM) with fetal bovine serum at 10%, 2 mM of glutamine and 100 µg/mL of streptomycin. Before infection the cells were washed with PBS to remove any antibiotic present in the culture medium.
The bacteria were grown for 36 h at 37 °C in Luria-Bertani broth with the addition of type A proanthocyanidins, at the most efficacious concentration of 50 µg/mL [18] and then at the concentrations of 100, 75, 25 and 15 µg /mL. They were then centrifuged and re-suspended in MEM at a concentration of 0.5 McFarland, which corresponds to 108 CFU/mL. Then the prepared bacterial suspensions were placed in contact with the cells (1 mL of suspension per well) and incubated at 37 °C for 3 h. Finally they were washed three times with PBS to remove any bacteria that had not adhered, the cells were then fixed with methanol, colored with Giemsa at 10% and observed at the microscope. The adhesion index obtained is the average number of adhering bacteria per cell, from an examination of 100 cells. Each test was performed in triplicate.
2.3. Scanning Electron Microscopy
Sterile cover glasses similar to those used for adhesion assay were fixed with 3% glutaraldehyde in 0.12 M phosphate buffer solution (pH 7.2) at 4 °C for 1 h. The samples were post-fixed in 1% osmium tetroxide in the same buffer, dehydrated in ethanol, critical point dried and sputter coated with a 5 nm gold layer using an Emscope SM 300. A Hitachi S-4000 field emission scanning electron microscope was used for the observation [25].
2.4. Motility Test
To evaluate the activity of type A PACs on the motility of P. mirabilis, a drop was removed from the 6 suspensions used for the experiment described above and inoculated in the same number of plates containing Mueller-Hinton agar. As control, a further 6 plates were inoculated with a suspension of bacteria grown in Luria-Bertani broth for 36 h at 37 °C without the addition of PACs. The plates were then incubated for 24 h at 37 °C and the swarming was then observed [26].
2.5. Production of Urease
To evaluate the inhibition activity of PACs on the production of urease Christensen’s medium was used in the liquid form, with the addition of a solution of urea at 40%. For the inoculum we used the suspensions already prepared for the adhesion assay in which the 5 wild strains of P. mirabilis and the collection strain were grown both in the presence and absence of PACs. A drop of each suspension was inoculated in a test tube containing 9.5 mL of medium and 0.5 mL of urea and incubated for 3 h at 37 °C. Obviously in all the test tubes the change in color from yellow to fuchsia was observed. At this point the suspensions were read at the spectrophotometer at 570 nm to show the difference between the strains used as control and those grown in contact with PACs. The test was repeated three times. Moreover, PH of all solutions was examined through a pH Meter
3. Conclusions
This work evaluated the efficacy of type A2 PACs, contained in cranberries, in the prevention of UTIs [28]. Our results show that, other than the well-known activity on uropathogenic strains of E. coli, they are active also on strains of P. mirabilis, reducing the ability to adhere to the epithelial cells of the bladder. These results indicate that PACs also partly inhibit the activity of the flagella, resulting in reduced motility and urease production. Further studies are needed to better characterize the latter features.
Nguồn: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4790394/
