Culture of Methanogenic Archaea from Human Colostrum and Milk

Abstract

Archaeal sequences have been detected in human colostrum and milk, but no studies have determined whether living archaea are present in either of these fluids. Methanogenic archaea are neglected since they are not detected by usual molecular and culture methods. By using improved DNA detection protocols and microbial culture techniques associated with antioxidants previously developed in our center, we investigated the presence of methanogenic archaea using culture and specific Methanobrevibacter smithii and Methanobrevibacter oralis real-time PCR in human colostrum and milk. M. smithii was isolated from 3 colostrum and 5 milk (day 10) samples. M. oralis was isolated from 1 milk sample. For 2 strains, the genome was sequenced, and the rhizome was similar to that of strains previously isolated from the human mouth and gut. M. smithii was detected in the colostrum or milk of 5/13 (38%) and 37/127 (29%) mothers by culture and qPCR, respectively. The different distribution of maternal body mass index according to the detection of M. smithii suggested an association with maternal metabolic phenotype. M. oralis was not detected by molecular methods. Our results suggest that breastfeeding may contribute to the vertical transmission of these microorganisms and may be essential to seed the infant's microbiota with these neglected critical commensals from the first hour of life.

Figure 1

Meta-analysis comparing the frequency of Methanobrevibacter species in human feces from obese and lean individuals. The study of Million et al. included individuals from previous studies from the same center^,. The study of Ignacio et al. was performed in Brazil and confirmed the decreased frequency of M. smithii in obesity. No substantial heterogeneity was observed between the 2 studies (I² = 0%), with a highly significant association (p = 0.004). These results were consistent with those reported by Scwhiertz et al. focusing on Methanobrevibacter species with very low heterogeneity (I² = 12%) suggesting a consistent and significant (p = 0.0003) effect at the genus level. Studies with a lower taxonomical resolution did not show any consistent result. For instance, studies at the Methanobacteriales order level included M. stadtmanae associated with proinflammatory properties in vitro and in clinical studies. These results suggest a genus-specific effect of human methanogenic Archaea on weight regulation and obesity and support that M. smithii is a neglected critical commensal for human health.

Figure 2

Molecular phylogenetic analysis by maximum likelihood method of the new isolates and their closest species. Bootstrap values ≥90% indicated at nodes. The evolutionary history was inferred by using the maximum likelihood method based on the Kimura 2-parameter model. The tree with the highest log likelihood (−3941.91) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The initial tree for the heuristic search was obtained automatically by applying the maximum parsimony method. A discrete gamma distribution was used to model the evolutionary rate differences among sites (5 categories (+G, parameter = 0.3801)). The tree is drawn to scale, with branch lengths measured according to the number of substitutions per site. The analysis involved 18 nucleotide sequences. There were a total of 1490 positions in the final dataset. Evolutionary analyses were conducted in MEGA7.

Figure 3

Comparison of rhizomes of Methanobrevibacter smithii and Methanobrevibacter oralis isolated from human milk with archaea previously described in the digestive tract (M. smithii) and mouth (M. oralis). M. smithii strain C2 CSUR P5816 was isolated from the colostrum of mother_2 (Table 1). Its genome (GenBank number: SAMEA104570327) was compared to the genome of a strain isolated from human feces (=WWM1085, GenBank Number: NQLD000000000000). M. oralis strain M2 CSUR was isolated from the milk (day 10 after delivery) of mother_11. Its genome P5920 (GenBank Number: SAMEA10457076) was compared to the genome of the type strain of M. oralis strain ZR (Genome Number: NZ_LWMU00000000.1) isolated from the human oral cavity (=DSM7256, =JCM 30027).

Figure 4

Distribution of maternal BMI values according to the detection of M. smithii in colostrum and/or milk. Density histograms of pre-pregnancy maternal BMI values for mothers with (left) or without (right) detection of M. smithii in colostrum and/or milk.