| Clostridium difficile (C. difficile) is a Gram-positive, spore forming anaerobic bacillus. It can cause antibiotic-associated diarrhea and pseudomembranous colitis (PMC), which are called C. difficile associated diarrhea (CDAD). Over the past decade, the morbidity and mortality of CD AD has significant increased in North America and Europe. This is primarily due to the emergence of the epidemic hypervirulent C. difficile NAP1/027 and 078 clones. Now difficile is the major pathogen of hospital-acquired diarrhea. Although the nationalwide surveillance data is still paucity, some data from local study showed that CDAD in China is not rare. Huang et al reported the incidence of CD AD was 15-17 cases per 10000 inpatients. This number was similar to the reports of other countries when there no outbreak occurs.The standard antimicrobial therapy for CD AD, even recurrence has changed little since the disease was first described. In Europe and North America, metronidazole and vancomycin have been still the first-line agents in treating CD AD. However, the decreased response and increased recurrence have been reported in recent. Furthermore, presumed potential for selection of vancomycin-resistant enterococci (VRE) has limited the usage of vancomycin. Fidaxomicin, a new drug approved by FDA in 2011, has showed good antibacterial activity against C.diffcile in vitro and in vivo. However, fidaxomicin is so expensive and not available in China up to now. Other investigational treatments including the use of adjunct probiotics or fecal microbiota transplant to reconstruct the intestinal normal flora, toxin-binding resin, and immunotherapy such as monoclonal antibodies and vaccines are all still in clinical trial or pre-clinical trial stage. Therefore, developing new treatment method has become an urgent matter.Berberine, a well-known alkaloid, can be extracted from herbs such as Coptis chinensis. Berberine has been used for the treatment of diarrhea in China for thousands of years. It is reported that berberine has bacteriostatic or bactericidal effect against variety Gram-positive and Gram-negative bacteria in vitro, and also, it can reduce the release of bacterial toxins. Berberine protects intestinal injury in mice. The synergism of anti-MRSA effect of berberine combined oxacillin is reported as well.This is a comprehensive study using a variety of research methods to investigate the antibacterial activity of berberine with or without vancomycin against C. difficile in vitro and in vivo. The results will provide a new treatment option for CDAD.There are two parts in this study:Part one. In vitro antibacterial activity of berberine alone or in combination with vancomycin against C. difficile.Part two. Protective effect of berberine alone or in combination with vancomycin in a hamster model of CDAD.Part One. In vitro antibacterial activity of berberine alone or in combination with vancomycin against CdifficileFifty C. difficile isolates were collected from Fudan University Hospital Huashan between 2008 and 2009. The minimum inhibitory concentration (MIC) of berberine and vancomycin against the C. difficile clinical isolates were determined by agar dilution method. To determine the activity of berberine in combination with vancomycin, checkerboard method was used. The antimicrobial activity against selected strains was retested by E-test method. Four isolates:SH9 which was resistant to vancomycin; SH178 which was decreased sensitivity to vancomycin; 027, a Sweden clinical isolate which belonged to ribotype 027, and ATCC 43596c were selected for the time-kill assay. The concentration of vancomycin was 1/2 MIC-2 MIC whereas that of berberine was 64 mg/L and 128 mg/L. The sporulation rates of Cdifficile were detected by phase contrast microscope.The MIC50, MIC90 against Cdifficile isolates were 512 mg/L,>512 mg/L for berberine and 0.5 mg/L,1 mg/L for vancomycin, respectively. Three strains were resistant to vancomycin (MIC= 4 mg/L). The checkerboard method results showed that synergistic, additive and unrelated interaction of berberine in combination with vancomycin against 50 Cdifficile strains were 12%,80%,8%, respectively. No antagonism effects were found. The results of E-test agreed with that of the checkerboard. In time-killing studies, berberine with vancomycin showed additive interaction against clinical isolates 027, SH9 and SHI78 in certain concentration combinations; however, unrelated interaction occurred against standard strain ATCC 43596c. Compared to the single drug vancomycin, berberine in combination with vancomycin decreased the sporulation rate of Cdifficile strains.Part Two. Protective effect of berberine alone or in combination with vancomycin in a hamster model of CD AD.The purpose of this part was to establish the clindamycin induced CD AD model of golden hamster and evaluate the protective efficacy of berberine alone or in combination with vancomycin against C.difficile in vivo. The hypervirulent strain 027 was selected as experimental strain. The animals were golden hamsters, female, weight 80g approximately. The hamsters randomly divided into 5 groups, berberine group (n=7); vancomycin group (n=7); berberine and vancomycin combination group (n=7); the no treatment control group (n=5) and the health control group (n=5). Acute infection observation period was from gavages to seven days, the end point was 27days after administration. The evaluating indicators included the stool character, weight changing, and survival rates in different groups. According to the results, the Kaplan-Miere survival cure was made.During the whole period of experiment, the health control group had no diarrhea, weight lose and death. The no treatment control group animals all developed severe diarrhea after infection with C. difficile and died on day 3 with weight loss 19.9%± 8.6% of their initial weight. The same were observed in the berberine-treated animals with weight loss 14.1% ±6.4%. Some of vancomycin-treated animals developed mild-to-moderate diarrhea after infection with C. difficile. After 4-5 days of vancomycin-treated, the surviving animals’diarrhea symptom improved. The mean weight loss among the vancomycin-treated animals was 2.4% ±5.8% of their initial weight. The survival rate at the end of acute stage was 57.1%, during the follow up period the recurrent rate was 75%, the final survival rate was 14.3%. Some of berberine in combination with vancomycin-treated animals developed mild-to-moderate diarrhea after infection with C. difficile as well and recovered after 4-5 days of treatment. The mean weight loss among the berberine and vancomycin-treated animals was 1.1%±5.3% of their initial weight. The survival rate at the end of acute stage was 71.4%, during the follow up period the recurrent rate was 20%, the final survival rate was 57.1%. Taking the animal’s colon contents to culture, detect toxins and PCR ribotyping,027 experiment strains were recovered. There were no significant differences of the colon histopathological damage among each group.In conclusion, the results of this study showed that:1. Berberine alone demonstrated limited antimicrobial activity against C.difficile in vitro and in vivo;2. Berberine in combination with vancomycin showed additive interaction against C.difficile.3. Berberine in combination with vancomycin decreased the sporulation rate of C.difficile than vancomycin alone.4. Berberine in combination with vancomycin reduced the recurrence rate and improved the survival rate in a hamster model of CDAD than vancomycin alone.This is the first study on the antimicrobial effect of berberine used alone or in combination with vancomycin against C.difficile in vitro and in vivo. The results provide a new option for the treatment of CD AD. If the efficacy and safety of berberine in combination with vancomycin for the treatment of CDAD can be confirmed by clinical trials, it is possible to put berberine into overseas markets as artemisinin. The limitation of this study is the relatively fewer of C.difficile isolates tested in the hamster CD AD model, more studies are needed to fatherly evaluate the protective effect of berberine in combination with vancomycin in vivo. |
PDF Full Text Request