Enteric Dysbacteriosis Caused By Antibiotic And Modulation Of The Intestinal Microflora By Lactobacillus Spp.

The normal microflora acts as a barrier against colonization of potential pathogenic microorganisms and against overgrowth of already present opportunistic microorganisms. Administration of antimicrobial agents therapeutically and intake passively by the food chain may result in disturbance of microflora and sometimes in severe intestinal complications. It is therefore important to investigate the variation of these antibiotic-related modifications.In order to explore the relationship between the dysbacteriosis and disease, further to find the efficient therapeutic strategy, it's very necessary to get a standard animal model which can simulate the complex relationship between the host and the intestinal microflora. So, two kinds of animal models which exposed in different doses of ceftriaxone were produced and a strain of lactobacillus was screened to regulate the disturbance caused by ceftriaxone. The efficacy and the safety of the modulation were evaluated respectively in vivo and in vitro.PART ONEObjective: To produce the mouse models of intestinal microflora dysbiosis which induced by different dosed of ceftriaxone.Methods:(1)50mg/d ceftriaxone was given to the SPF BALB/c mice intragastrically for 5 days and next the Candida albicans was inoculated in order to simulate the over-growth of fungi;(2)Clean mice were treated with 300μg/ml,30μg/ml and 3μg/ml ceftriaxone for 45 days in drinking water respectively so that to imitate the antimicrobial agent residues;(3)Changes of mice fecal flora were observed by optical microscope and SEM directly. Enumerations of intestinal microflora and the elimination of Candida albicans were detected by vital cell counting. Alterations of diversity of bacterial community in murine feces were monitored by PCR-DGGE analysis and the variation of colonization resistance of murine fecal microbiota were tested by simulated intestinal conditions.Results:(1)Treatment with ceftriaxone at dosage of 50mg/d would result in a severe damage of murine intestinal microflora. The numbers of anaerobic bacterium,lactobacilli and enterococci decreased conspicuously. during the treatment (P<0.01). Neither bifidobacteria nor enterobacteriaceae could be detected. DGGE profiles indicated that the richness and diversity of total bacteria,lactobacilli and enterococci descent remarkably(P<0.05);Bacillus sp. lose the predominance and a strain of Lactobacillus reuteri success to dominant bacteria in the gastrointestine. At the same time, the membrane flora adhered to the intestinal mucosa disappeared and the colonization resistance of the microbiota was broken seriously(.2)Balance of microecology in murine feces was slightly broken in both 300μg/ml and 30μg/ml ceftriaxone treated groups. Except the enterococci ,numbers of anaerobe,lactobacilli and enteric bacterium reduced first but increased in the end. In contrast to the overgrowth of enteric bacterium, numbers of bifidobacterium decreased marked (P<0.01) and irreversibly.(P>0.05).No obvious changes of fecal microbiota were observed in 3μg/ml ceftriaxone treated group. DGGE analysis showed an obvious decrease of diversities both in total bacteria as well as lactobacilli, but the variety of enterococci was independent of the ceftriaxone. From the DGGE profiles we also found a temporary resident of two strains of Ruminococcus torques while the band corresponding with enterococcus faecium remained throughout.Moreover, two strains of dominant lactobacilli in murine intestine were proved to be Lactobacillus reuteri and Lactobacillus salivarius subsp. Salivarius by sequencing. As to the colonization resistance, the inhibition to the Candida albicans was weakened partly, but the defense still worked.PART TWOObjective:To access the inhibitory effect of eight strains of lactobacillus spp. to Candia albicans in vitro and screen the best one to modulate the abnormal intestine microflora in vivo.Methods:(1)Crystal violet-based germ tube assay was used to detect the germination of Candida albicans and the inhibition efficiency of strains and SCFAs were tested by germination rate. The control of lactobacilli to the growth of Candida albicans was confirmed by co-incubation and agar diffusion assy.(2)At the different time points, 108CFU LGG were gavaged to the mice pre-treated by ceftraxone with therapeutic dose and 109CFU LGG were added synchronously to the drinking water contained low doses of ceftraxone(.3)Optical microscopic examination was used to observed the changes of mice fecal flora and vital counts was used to enumerate the numbers of intestinal microflora .PCR-DGGE analysis was proceeded to detect the diversities of bacterial community in the murine microbiota and simulated intestine system was used to evaluate the colonization resistance.Results:(1)Lactobacillus rhamnous GG (LGG) showed the best ability to inhibit the germination and growth of Candida albicans in vitro and among the SCFAs only butyric acid could inhibit the germ tube formation availably.(2)A noticeable reconstitution of fecal microflora was found when LGG was gavaged to the mice and obvious results had been achieved by increases of anaerobe,latobacilli and bifidobacteria. Besides the quantitative changes of bacterial numbers, richness and diversity index of lactobacilli were back to normal (P>0.05) as well. We also found that the rebuild intestinal microbiota supplied with LGG could exclude Candida albicans dramatically which indicated that the colonization resistance of the flora recovered. Interestingly, all of the efficiency was impressed in the LGG pre-treated group.(3)In the low dosage ceftriaxone treated group, the growth of bifidobacteria and the restore of lactobacillus diversity demonstrated a positive effect of LGG to modulate the murine fecal microbiota, but at the same time, the outgrowth of enteric bacteria as well as a low diversity index of total bacteria indicated the modulation of LGG on the maladjustment was limited. However, compared with ceftriaxone treated group, supply of LGG could strengthen the colonization resistance of the fecal bacteria significantly(P<0.01).PART THREEObjective:To evaluate the risk of transmission of antimicrobial resistance mediated by lactobacillus spp..Methods:(1)The disc-diffusion assay (Kirby-Bauer assay ) was performed to detect the antibiotic susceptibility of 8 strains of lactobacillus spp..(2)Double disc diffusion assay was used to test the ESBLs(Extended-Spectrumβ-lactamase).(3)Plasmid was extracted from LGG to observed the mobile genetic elements which was potential to mediate the transit of antibiotic resistance genes . Results:(1)The lactobacilli tested in our experiment were multi-drug resistant and LGG appeared the resistance to 5 antimicrobial agents.(2)Double-disc diffusion assay showed a negative results production of ESBLs of LGG and no plasmids were detected in LGG.Conclusion:1. Clinical dosage of ceftriaxone could disturb the balance of murine intestinal microflora dramatically and the mouse model reproduced the symptom of fungi overgrowth occurred frequently in clinical patients. There were significant decreases both in numbers and diversities of fecal microorganism and the innate colonization resistance of normal flora was seriously destroyed so that resulted in an abnormal overgrowth of Candida albicans. So, this mouse model demonstrated that a cause-effect relationship potentially exist between enteric dysbacteriosis and infectious intestinal diseases.2. Long-term exposed in low dosage of ceftriaxone could resulted in a slightly disturbance of murine intestinal microbiota and the mouse model successfully simulated the process and the consequence of a passive intake of antibiotic residues from food chain.3. LGG, a select strain of lactobacillus spp. in vitro, could promote the reconstruction of the fecal microbiota in both animal models characterized above. With the supply of LGG, the numbers,bacterial population and colonization resistance of fecal microflora tended to return to normal condition. Otherwise, the risk of LGG to mediate transission of antimicrobial resistance was under control.