| Urinary tract infection (UTI) is one of the most common clinical infectious diseases. The infections that occur2times in six months or3times a year are defined as recurrent UTI (RUTI). The replapse of this disease and the resistance of the pathogens are great difficulties of clinical treatment. An alternative to the antibiotics treatment is of great values for clinical treatment. Traditional Chinese Medicine (TCM) treatment of UTI, especially RUTI, has special advantages over Western medicine. The present study is aimed to carry out clinical and experimental study on TCM treatment of RUTI, and systematic analysis of the pathogen UPEC in terms of genotype, pathogenesis and resistance. The present study include:(1) Clinical treatment of RUTI by Syndrome differentiation treatment with Fuzhengqingrelishi.(2) Etiological analysis of the pathogen:Genotype, pathogenesis, and resistance analysis of UPEC isolates.(3) Treatment of UPEC UTI mouse model by TCM Fuzhengqingrelishi.Part I:Clinical treatment of recurrent urinary tract infection by FuzhengqingrelishiObjective:Though parallel, randomized, controlled, blinded prospective clinical study design, the rates of symptom alleviation, bacterial clearance, relapse and recurrence were observed to evaluate the treatment efficacy.Methods:Though parallel, randomized, controlled, blinded prospective clinical study design, according to TCM differentiation standards, the RUTI patients were divided into two layers of Yinxushire and Yangxujiashi, each of the layers were grouped into treatment and control groups. A total of90cases were included in the study. The Yangxu group included40cases of TCM treatment, and20cases of controls. The Yangxu Group included20TCM treatment cases and10control cases. The Yangxu TCM group was treated with Ziyintonglin and the Yangxu TCM group was treated with Wenyangtonglin for4weeks. The control cases were treated with sensitive antibiotics together with placebo for1week. All the cases were followed for six months to evaluate the symptom alleviation, urine WBC negative conversion, bacterial clearance and recurrences.Results:1, Analysis of the age, onset times, pathogen distribution showed that all the cases were female, with average ages of56.5. The most isolated pathogens were Gram negative bacteria, of which E. coli is mostly represented, accounting for63.8%. No difference in the percent was observed for the Yinxu group (63%) and Yangxu group (64%). Cases which were isolated E. coli, Klebsiella pneumonia and Enterococcus were attacked over5times in six months.2, Symptom curation and effect percent of TCM was higher than control group, while the total efficacy is comparable. Alleviation of frequent urination in Yinxu treatment group was greatly better than control group (P<0.05), no significant differences in other symptoms. Yangxu TCM group sowed better alleviation of belly bulge, facial swelling, limb edema symptom than the control group (P<0.05), greatly improvement of fatigue and feces were observed (P<0.001). Symptom evaluation showed that efficacy cure rate of Yangxu TCM was higher than control, but no statistically differences observed.3, Combining Yangxu and Yinxu, TCM group efficacy was higher than control in terms of urine WBC (p<0.001).4, Regardless of the layers of Yinxu and Yangxu, bacterial clearance rate of TCM group was better than control at2weeks (P <0.05), but as the monitoring time extended, the differences decreased, and at the end of the treatment, the treatment and control group showed identical clearance rates. At14days post the first treatment, about half of the cases showed urine culture negative conversion in TCM group, while only one thirds of cases in control group. At the end of the treatment, similar clearance rates were observed for both TCM and control groups. For cases caused by ESBL positive E. coli, methicillin-resistant coagulase-negative staphylococci (MRSCON), methicillin-resistant Staphylococcus epidermidis (MRSE), the clearance rate of TCM was75%, while no clearance was observed in control group.5, Recurrence rate of TCM was lower than that of control group, and Yinxu group was statistically lower than control group (P<0.001).Conclusion:1, The older postmenopausal women were susceptible to RUTI caused by E. coli, the most represented pathogen of RUTI.2, Treatment efficacy of Fuzhengqingrelishi was higher for some cases in Yangxu group than control group, while the efficacy for Yinxu cases was similar to that of control group.3, Fuzhengqingrelishi was helpful for alleviation of inflammation and organ destructions.4, Bacterial clearance, especially the ESBL positive pathogen in TCM group was higher than control group.5, Fuzhengqingrelishi treatment efficacy was higher than western medicine for RUTIs.Part Ⅱ:Etiological analysis of RUTIs, Genotype, virulence gene and resistance of UPEC isolatesObjective:Clinical isolates of the most represented species UPEC were selected for genotype, pathogenesis and resistance analysis. The possible correlation of genotype, virulence and resistance were putatively investigated.Methods:By systematically optimization of the template, amplification condition, electrophoresis, rep-PCR for UPEC isolates were developed and205clinical isolates were analyzed. Primers for24virulence genes were synthesized and used to screen for virulence gene distribution. Disk diffusion method was used to analyze resistance of UPEC isolates against17antibiotics. Taken together these data, the genotype, virulence and resistance of UPEC isolates were analyzed and their correlations were defined.Results:1, After condition optimization, rep-PCR for UPEC was developed.2, A total of205UPEC isolates were genotyped by rep-PCR, which generated158genotypes and clustered into8groups.3, Virulence gene screening showed that, for the24genes,15were observed in part of the strains. The most distributed virulence genes included fimH, traT, aerJ, papGⅢ, PAI, fyuA and papG, which were greatly differentially distributed.4, Resistance profile analysis showed that the highest resistance rate peaked by PIP,78.54%, followed by GEN, LVS, CZO, whose resistance rate is66.34%,65.37%and50.24%respectively. Resistance of the UPEC isolates differed among different department.5, Main genotypes of the isolates contained similar virulence genes.6, Virulence genes were positively correlated with ESBL, resistance. TraT and PAI were positively correlated with resistance.Conclusion:1, UPEC isolates were greatly diversified and different genotypes are circulated in different departments.2, Most of the the isolates are multi-resistant and even pan-drug resistant.3, Virulence gene is positively correlated with resistance, and the more virulence gene, the higher resistance.4, No correlation between genotype and resistance was observed, implied the possible horizontal transfer of resistance.Part III:Development of UPEC UTI model and treatment of UTI by Fuzhengqingrelishi TCMObjective:Based on virulence and resistance distributions, a highly pathogenic and resistant strain was selected to construct mouse model. The mouse model was treated with Fuzhengqingrelishi, and the mouse death rate, status of survival mice, bacterial clearance rate in urine and organs were tested to evaluate the efficacy. In vitro inhibition assay was used to test the bacteria killing or inhibition role of the TCM.Methods:Based on the virulence gene and resistance profile, UPEC isolate was used to infect Balb/c mice with dose of1×109,1×108and1×107cfu through urinary tract. The death rate, status and bacterial clearance in urine were tested. Then, the mouse model were divided into high dose, low dose, model and antibiotics groups which were treated or untreated accordingly. The negative controls are normal untreated mice. The treatment efficacies were evaluated by comparison of mouse death rate, status of survival mice, discharge of bacteria from urine, bacteria in organs of spleen, kidney and bladder, and organ indexes. Through in vitro inhibition assay, the MIC and MPC of UPEC201were defined. TCM sera were prepared and used to test the inhibition roles on UPEC.Results:1, UPEC201was selected as the stain to develop mouse model. Mouse model of UPEC201was developed by infection with high dose. Bacteria in urine but remained for21days for the high dose group.2, Compared with the untreated group, the death rate was decreased. The cure rrate for high dose, low dose and antibiotics group were80%,40%and80%, respectively. After the treatment, the status was significantly improved, and at the end of the treatment, the survival mice returned to normal level.3, The bacteria in urine decreased after the treatment, and at the7th day post the treatment, the bacteria were completely cleared in group of high dose of TCM and antibiotics group. Bacteria analysis in organs showed that bacteria could not be completely cleared. The organ indexes decreased in all treatment groups, but the high dose and antibiotics groups showed the highest recovery of organ indexes.4, Bacterial strains isolated from treated mouse showed higher sensitivity to several antibiotics and have lost part of the plasmids.5, The MIC of Fuzhengqingrelishi for UPEC201was250mg/ml. TCM sera generated from mouse did not inhibit growth of UPEC201. MIC of sera containing TCM for UPEC201WAS125mg/ml.Conclusion:1, A mouse model was developed with UPEC201. Fuzhengqingrelishi could treat UTI mouse models with similar efficacy to that of antibiotics.2, After treatment, bacteria in urine were cleared, but the bacteria still exist in organs, which might be the reason for RUTI.3, Bacterial load in blader and spleen in TCM group was lower than that in model and antibiotics groups. But the bacterial load in urine and kidney remained silimar level, implying that TCM have higher inhibitition activity on UPEC in organs.4, TCM treatment showed identical alleviation of organ index to antibiotics;5, TCM treatment could clear plasmid in UPEC and dcrease the resistance.4, TCM could inhibit UPEC, but could not kill the bacteria.Innovations1, By using parallel, parallel, randomized, controlled, blinded prospective clinical study design, the rates of symptom alleviation, relapse and recurrence of RUTI after treatment by fuzhengqingrelishi were systematically evaluated.2, Rep-PCR for UPEC isolates were developed and the genotypes of205clinical isolates were analyzed. For UPEC, the most common pathogen of RUTIs, the virulence, and resistance were analysed. Correlations were observed between virulence pathogenesis, and resistance, but not between genotypes and resistance, implying that resistance is possibly acquired by horizontal gene transfer. 3, A RUTI mouse model were developed by using UPEC201strain and treated with the method of Chinese medicine of fuzhengqingrelishi. This is the first report of this type of TCM for RUTIs.4, Treatment of RUTI model by fuzhengqingrelishi could not completely clear the bacteria in mouse, but could clear part of the resistant plasmids. |
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