Study of the influence of Dr fimbriae on cell adherence to the surface of calcium oxalate and tribasic calcium phosphate crystals in various buffers - test tube test

Recent findings indicate that frequent urinary tract infections (UTIs) can lead to both infection and metabolic stones. However, the exact processes by which UTIs cause metabolic stones are still unclear . Due to the fact that Dr fimbriae are closely associated with uropathogenic strains of E. coli causing chronic urinary tract infections, it is interesting to investigate its impact on the bacteria adherence to the calcium oxalate and tribasic calcium phosphate – model elements of kidney stones. For this experiment, recombinant AAEC191A strains were used. Initially, all strains were suspended in following buffers: PBS pH=7.4, PBS pH=5.7, PBS+40 mM sodium oxalate pH=5.7, PBS+20 mM sodium oxalate pH=5.7, PBS+5 mM sodium oxalate pH=5, 7 and citrate buffer (21.75 mM sodium citrate, 8.25 mM citric acid, 120 mM sodium chloride., and brought to the same OD600. To examine the percentage adherence of cells to selected calcium salts, 5 ml of the aqueous phase was prepared, containing 0,5 ml, 1 ml, or 2 ml of culture suspended in the appropriate buffer, to which 1 ml of 20% suspension of tribasic calcium phosphate (hydroxyapatite) or calcium oxalate monohydrate was added at the end. Then, all samples were mixed for 10 minutes, allowing the bacteria to atach to the crystal suspension, which resulted in a loss of bacteria in the aqueous phase. No calcium salt suspension was added to the control samples; however, they were subjected to the same experimental conditions as the test samples. In the case of samples containing hydroxyapatite or calcium oxalate monohydrate suspension, the transition of bacteria to the suspension phase resulted in a decrease in the optical density, OD600, of the aqueous layer compared to the optical density of the corresponding control sample. The OD600 of each sample was measured in three technical replicates.
The catalog contains a Word document with graphs of the percentage of cell adherence of AAEC191A/pACYC177 and AAEC191A/pCC90 strains to monohydrate calcium oxalate crystals and to tribasic calcium phosphate in various buffers.

近期研究表明，反复发作的尿路感染（Urinary Tract Infections, UTIs）可同时诱发感染性结石与代谢性结石。然而，UTI引发代谢性结石的具体机制仍未明确。鉴于Dr菌毛（Dr fimbriae）与引发慢性尿路感染的尿路致病性大肠埃希菌菌株（uropathogenic strains of E. coli）密切相关，探究其对细菌黏附于草酸钙（calcium oxalate）与磷酸三钙（tribasic calcium phosphate）——肾结石（kidney stones）的典型组成成分——的影响具备重要研究意义。本实验采用重组AAEC191A菌株（recombinant AAEC191A strains）。首先，将所有菌株重悬于以下缓冲液体系：pH 7.4的磷酸盐缓冲液（Phosphate Buffered Saline, PBS）、pH 5.7的PBS、含40 mM草酸钠（sodium oxalate）的pH 5.7 PBS、含20 mM草酸钠的pH 5.7 PBS、含5 mM草酸钠的pH 5 PBS、含5 mM草酸钠的pH 7 PBS，以及柠檬酸盐缓冲液（citrate buffer，组成为21.75 mM柠檬酸钠、8.25 mM柠檬酸、120 mM氯化钠），并将所有菌液调整至相同的600 nm光密度（OD600）。为检测细胞对选定钙盐（calcium salts）的黏附率，配制5 mL水相体系：分别取0.5 mL、1 mL或2 mL重悬于对应缓冲液的菌液，随后终浓度加入1 mL的20%磷酸三钙（羟基磷灰石，hydroxyapatite）或一水合草酸钙（calcium oxalate monohydrate）悬浮液。将所有样品混匀10分钟，使细菌黏附于晶体悬浮液，此过程将导致水相中的细菌数量减少。对照组样品不添加钙盐悬浮液，但与实验组样品接受完全一致的实验处理。对于添加了羟基磷灰石或一水合草酸钙悬浮液的样品，细菌转移至悬浮相后，其水相层的600 nm光密度（OD600）相较于对应对照组样品会出现下降。每个样品的OD600均设置3次技术重复（technical replicates）进行测定。本数据集包含一份Word文档，其中收录了AAEC191A/pACYC177与AAEC191A/pCC90菌株在不同缓冲液中对一水合草酸钙晶体及磷酸三钙的细胞黏附率图表。