| In recent years, there have been questions concerning the effects of consumption of trace levels of antimicrobial residues in foods on the indigenous human gut microflora, such as selection of resistant bacteria and disruption of the colonization barrier of the resident gut flora. Currently, European, Australian and United States guidelines for veterinary drug registration require a safety assessment of microbiologic hazards from consumption of antimicrobial residues taking into account the potentially adverse effects on human intestinal micro?ora. In our country, we also have made efforts in assessing the microbiogical safety of ingested antimicrobial residues to the consumer.Human ?ora-associated rodent model was relevant to humans, since the inoculant is fecal flora from healthy adult. It is therefore been recommended to determine the effects of antimicrobial residues on human microflora by national agency. However, in comparison with adult, the infant gut microflora is more variable in its composition and less stable over time. The results obtained from HFA rodent model could not reflect the impact of antimicrobial residues on the infant gut microflora. So, it is necessary to develop a more susceptive animal model to assess the effect of antimicrobial residues on the infant gut microflora. Infant mice model can meet the need.In order to facilitate the evaluation of the microbiological safety of antimicrobial residues in food in our country, two kinds of animal models, HFA mice model and infant mice model, will be developed in this paper. HFA mice model will be applied in assessing the effect of enrofloxacin on the gut microflora and infant mice model will be applied in assessing long-term impact of amoxicillin on the development of gut microflora.PART ONEObjective: to develop HFA mice model for assessing the effect of enrofloxacin on the gut microflora Methods: (1) The germ-free KM mice were bred by artificially rearing the cesarean delivered mice pups in the isolators; (2) The germ-free KM mice were inoculated intragastrically with the fecal flora from healthy adult; (3) The resemblance of gut microflora in HFA-KM mice model to the human inoculant was analyzed by PCR-DGGE; (4) HFA-KM mice models were treated with 30mg/L, 3mg/L and 300μg/L enrofloxacin for 35 days in drinking water respectively; (5) Enumerations of intestinal microflora and the ciprofloxacin-resistant bacteria in HFA-KM mice feces were detected by vital cell counting. Alterations of diversity of bacterial community in murine feces were monitored by PCR-DGGE analysis.Results: (1) The germ-free status of germ-free KM mice had been proved by national institute for the control for the pharmaceutical and biological products. (2) The DGGE profiles confirmed that the major components of microflora in inoculated human feces could be successfully transferred into the HFA-KM mice model, although the dominant bacterial group in HFA mice might change from those present in inoculated human feces. Meanwhile, two strains, belonged to Family Lachnospiraceae, and one strain, relative to Bacteriodes vulgates can't be colonized in the HFA-KM mice. (3) The levels of total aerobe and enterobacteria in HFA-KM mice feces were decreased significantly in 30mg/L and 3mg/L enrofloxacin-treated groups (P<0.05). No obvious changes of fecal microflora were observed in 300μg/L enrofloxacin-treated group. (4) The levels of total resistant aerobe and anaerobe in HFA-KM mice feces were increased significantly in 30mg/L and 3mg/L enrofloxacin-treated groups (P<0.05). No obvious changes of fecal microflora were observed in 300μg/L enrofloxacin-treated group. (5) The richness and Shannon index were decreased significantly in DGGE profile from the HFA-KM fecal microflora in all the three enrofloxacin-treated groups (P<0.05).PART TWOObjective: to apply the infant mice model in the assessing the long-term impact of amoxicillin on the development of gut microflora, especially the genus Lactobacillus and BifidobacteriumMethods: (1) From postnatal day 7 (PND7) to PND21 the suckling BALB/C mice received a daily dose of 100 mg/kg body weight of either amoxicillin (amoxicillin-treated group) or the equivalent volume of saline (control group) by intragastric gavage. At PND 21 and 56, five mice from each dam were sacrificed by cervical dislocation. Colons were recovered immediately after the mice were killed. The digesta of each intact colon was collected by manual extrusion. (2) The Lactobacillus-specific and Bifidobacterium-specific 16S rRNA gene clone library were constructed. (3) Alterations of diversity of bacterial community in murine feces were monitored by PCR-DGGE analysis. (4) The changes in the levels of the genus Lactobacillus and Bifidobacterium in the colons of BALB/C mice were detected by real-time PCR.Results: (1) The dominant Lactobacillus species were L. murinus (61.2%) and L. johnsonii (29.5%). The dominant Bifidobacterium species were B. pseudolongum (88.5%). (2) Five weeks after the treatment, the richness and Shannon index in the V3-DGGE profile from the mice in amoxicillin-treated group were still lower than those in control group (P<0.05). In detail, the numbers of lactobacilli in amoxicillin-treated mice colon digesta had no difference from those in control, but the richness and Shannon index in the Lactobacillus-specific DGGE profile were decreased significantly in comparison with control (P<0.05). The numbers of Bifidobacterium in amoxicillin-treated mice colon digesta were still lower than those in control group (P<0.05), but no differences were observed in the Bifidobacterium-specific DGGE profile between the amoxicillin-treated group and the control group.Conclusions:1. HFA-KM mice model was developed by inoculating the germ-free KM mice intragastrically with the fecal flora from healthy adult. The major components of microflora in inoculated human feces could be successfully transferred into the HFA-KM mice model. So, this mouse model could be used in assessing the microbiogical safety of ingested antimicrobial residues to the consumer.2. The lowest doses of enrofloxacin, equivalent to the ADI for enrofloxacin, could reduce the biodiversity of fecal microflora in HFA-KM mice model significantly, although no effect on either the bacteria counts or the resistant bacteria counts was observed. Concerning the biodiversity of gut microflora, the safety of ADI for enrofloxacin is deserved to be checked again.3. Neonatal amoxicillin treatment led to a long-term impact on the biodiversity of the gut microflora in infant mice model, especially the genus Lactobacillus and Bifidobacterium. Thus, in view of its susceptibility, infant mice model could be used as a supplement to HFA mice model. |
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