Establishment Of An IHFA Mouse Model For Evaluating The Effect Of Amoxicillin Treatment On Infant Gut Microbiota

Antibiotic use in early life is considered as one of the most important environmentalfactors affecting the infant gut microbiota colonization. The administration of broad spectrumantibiotics, usually applied in pediatric clinical treatment, has been shown to decrease thebiodiversity of gut microbiota. To avoid the ethical problems associated with utilisingantibiotics in health infant human volunteers, the infant human-flora associated (IHFA)animals provide an attractive model for determining the effect of residual and therapeuticdoses of antibiotics on the infant gut microbiota. There several studies focus on constructionand application of IHFA model. However, there still lack of data on the colonization patternof infant gut microbiota in germ free animals. Further more, it is not clear whether IHFArodents can be used to study the effect of neonatal antibiotics treatment on microbiota.Actually, there's lack of germ free animal for study related problems in our country. In thisstudy, an automatic method for artificial rearing of germ free mice pups were developed forefficiently obtaining germ free mice; An IHFA mice model were established by infected germfree mice with infant feces, and the effects of age and strain on the microbiota colonization ingerm free mice were detected; And then the ecological impact of neonatal amoxicillintreatment was investigated in the IHFA mouse model.Methods1. To establish an automatic artificial rearing method for mice, an intragastric cannulawas placed into3-d-old-C57BL/6J mice (n=48), and the mice were artificially reared (AR)with mouse milk substitute (MMS). Littermate pups (n=20) were maternally reared (MR) ascontrols, mice pups growth was observed. The feces of13-d pups were analyzed byPCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting technique. The automaticartificial rearing method was applied to grow germ free mice pups in the isolator, germ freemice pups growth was observed. Reproductive capacity and biochemical indicator of adultmice obtained by artificial rearing were measured. 2. To study the colonization pattern of infant gut microbiota in germ free animals, anIHFA model was developed by infected postnatal day (PND)1germ-free mice (Kunming,n=10; Balb/c, n=10) with feces from a breast-fed infant. Microbiota in the feces of Balb/c andKuming mice (at PND7,14, and21) was analyzed by PCR-DGGE. Bifidobacteria andLactobacillus levels in the feces of Balb/c and Kunming mice (at PND7,14, and21) weredetected by quantitative real-time PCR (qRT-PCR).3. To determine whether IHFA mice can be used to study the effect of neonatalantibiotics treatment on gut microbiota, IHFA mice received a daily intragastric gavage ofamoxicillin (100mg/kg, n=10) or saline (n=10) from PND7to PND21. Microbiota patterns inthe feces of amocillin treated and control IHFA mice (at PND21and53) were compared byPCR-DGGE and Bifidobacteria/Lactobacillus-specific qRT-PCR.Results1. Automatic artificial rearing method had been applied to grow germ free mice pups inthe isolator. After11days of artificial rearing,37of48C57BL/6J pups had survived. Therewere no significant changes in the number of DGGE bands, Shannon index between the twogroups. However, several bands in the AR group were obviously different from that of theMR group in the DGGE profile. After21days of artificial rearing, survival rate of germ freemice pups was62.5%. Litter Sizes of AR acquired female mice were smaller than those ofMR acquired female mice (P <0.05). These is no different changes in biochemical indicatorsbetween AR and MR acquired mice (P>0.05).2. The microbiota patterns of the IHFA mice model vary with age and mouse strain. TheDice similarity coefficient (Cs) for the fecal microbiota of Balb/c mice in comparison with theHD donor sample was higher for Balb/c mice than for Kunming mice (P <0.05). In addition,The Cs of Kuming mice at PND14and PND7were significantly lower than that of PND21(P <0.05). The Bifidobacteria and Lactobacillus species colonizing the Balb/c mice weresimilar to those in the Kunming mice (at PND7/14/21). The Bifidobacteria counts increasedwith age in both mouse strains, whereas the Lactobacillus counts decreased with age in bothstrains.3. Amoxicillin treatment lead to a significant impact on the IHFA gut microbiota patternand the adult microbiota is not completely recovered to normal state after neonatal treatment.The Shannon indexes other than number of DGGE bands were significantly increased by amoxicillin treatment at PND21(P <0.05). Further more, the fecal Lactobacillus andBifidobacteria counts were decreased by amoxicillin treatment at PND21(P <0.05).Although the Evenness and levels of Lactobacillus/Bifidobacteria in the amoxicillin treatedgroup were similar to those in the control group at PND53(P>0.05). However, the Shannonindexes, numbers of DGGE bands seem still different between the two groups at PND53(P <0.05).Conclusion1. Automatic artificial rearing method had been applied to grow germ free mice pups inthe isolator, which is beneficial to obtain germ free mice efficiently. It is possible to implantintragastric cannulas into both common and germ free mice pups. Both common and germfree mice pups grow well by artificial rearing. The microbiota of common mice pups wouldbe affected by artificial rearing. Adult germ free mice obtained by artificial rearing seemhealth, and importantly, are able to breed offspring. These results indicate that the novelautomatic artificial rearing method is suitable for efficiently obtaining germ free mice.2. The DGGE results suggested that the microbiota patterns of the IHFA mouse modelvary with age and mouse strain. However, qRT-PCR suggested that mouse age not strainsignificantly influence Bifidobacteria and Lactobacillus numbers in the IHFA mice model. Inaddition, the composition of human infant gut microbiota appeared to be modified by themice.3. Amoxicillin treatment lead to a significant impact on the IHFA gut microbiota patternand the adult microbiota is not completely recovered to normal state after neonatal treatment.This study demonstrates the high value IHFA mice may have as an animal model for neonatalantibiotic treatment. To our knowledge, this is the first report of neonatal antibiotic treatmenton the gut microbiota in an IHFA model.