Federici Sara, Miragoli Francesco, Pisacane Vincenza, Rebecchi Annalisa, Morelli Lorenzo, Callegari Maria Luisa
Centro Ricerche Biotecnologiche, Università Cattolica del Sacro Cuore, Via Milano 24, 26100 Cremona, Italy.
Istituto di Microbiologia, Università Cattolica del Sacro Cuore, Via E. Parmense 84, 29122 Piacenza, Italy.
FEMS Microbiol Lett. 2015 May;362(10). doi: 10.1093/femsle/fnv064. Epub 2015 Apr 22.
Methanogens commonly inhabit swine intestine. We analyzed the gut archaeal population by extracting DNA from the feces of nine piglets. We performed PCR to target the V6-V8 region of the 16S rRNA gene. Subsequent denaturing gradient gel electrophoresis (DGGE) revealed the presence of Methanobrevibacter boviskoreani, which has not previously been identified in pigs. We confirmed these data with a PCR-DGGE analysis of the mcrA gene, and subsequent sequencing. At 63 days old, the only band in fecal samples corresponded to M. boviskoreani. The DGGE analysis also showed that Methanobrevibacter smithii, which was abundant at 28 days, was dramatically reduced at 42 days, and it completely disappeared at 63 days. To confirm these data, we quantified M. smithii and the total archaeal population by quantitative PCR (qPCR); moreover, we designed a new set of species-specific primers based on the 16S rRNA gene of M. boviskoreani. The qPCR results confirmed the reduction in M. smithii over time and a simultaneous increase in M. boviskoreani. At 63 days, the total numbers of archaea and M. boviskoreani genomes were comparable, which suggested that M. boviskoreani represented the dominant archaea. This work showed that the archaeal population shifted during weaning, and M. boviskoreani replaced M. smithii.
产甲烷菌通常栖息于猪的肠道中。我们通过从9只仔猪的粪便中提取DNA来分析肠道古菌群落。我们进行PCR以靶向16S rRNA基因的V6 - V8区域。随后的变性梯度凝胶电泳(DGGE)显示存在牛科短柄产甲烷菌,此前在猪中尚未鉴定出该菌。我们通过对mcrA基因进行PCR - DGGE分析及后续测序来确认这些数据。在63日龄时,粪便样本中的唯一条带对应于牛科短柄产甲烷菌。DGGE分析还表明,在28日龄时丰富的史氏短柄产甲烷菌在42日龄时显著减少,并在63日龄时完全消失。为了确认这些数据,我们通过定量PCR(qPCR)对史氏短柄产甲烷菌和古菌总数进行定量;此外,我们基于牛科短柄产甲烷菌的16S rRNA基因设计了一组新的种特异性引物。qPCR结果证实了史氏短柄产甲烷菌随时间减少以及牛科短柄产甲烷菌同时增加。在63日龄时,古菌和牛科短柄产甲烷菌基因组的总数相当,这表明牛科短柄产甲烷菌是主要的古菌。这项工作表明在断奶期间古菌群落发生了变化,牛科短柄产甲烷菌取代了史氏短柄产甲烷菌。