The microbiota of a newborn develops rapidly after birth and is dependent on the mother's microbiota, mode of delivery, gestational age, and early feeding practices. It has also been suggested that sanitation, hygiene, and climate may guide the succession of microbes (1–3). The normal microbiota contributes to the gut barrier function with a decisive role in the development of the immune system. Indeed, aberrant phenomena in the compositional development of the microbiota have been documented to increase the risk of immunological and inflammatory diseases (4,5). Moreover, it is increasingly evident that gut microbes may influence energy harvest and storage and shape the host metabolic network, thereby affecting the growth pattern and risk of obesity and related disorders (6).
Malnutrition in the low-income world and infectious diseases in high-income countries are a major health problem for infants and children worldwide (1), but they are especially devastating in poorer regions. Different challenges beset the high-income countries, where allergies and autoimmune and chronic inflammatory diseases are reaching epidemic proportions. To meet the respective nutritional challenges in the low- and high-income worlds, malnutrition and overnutrition or unbalanced diet, specific dietary approaches, have been devised. Nonetheless, breast-feeding remains the basic foundation in infants. The World Health Organization recommends that infants be exclusively breast-fed for the first 6 months of life and this should continue for at least 2 years with solid foods added at 6 months of age (7). The diet in low-income countries is often based on locally produced foods and is naturally high in fiber and complex carbohydrates, which have the natural ability to modulate intestinal microbiota (8). Such foods are often introduced to infants early along with breast-feeding. In high-income countries, infant formula and some complementary foods are added when breast-feeding is not sufficient.
The aim of the present study was to characterize the gut microbiota of 6-month-old infants living in Malawi and Finland, all being breast-fed and living in an environment typical for each area, thus representing infants in a low-income country and infants in a high-income country.
The Malawian study population comprised 44 healthy 6-month-old rural infants, who were enrolled for an epidemiological clinical trial assessing the effect of selected dietary interventions on early childhood growth. The trial adhered to the principles of the Declaration of Helsinki and regulatory guidelines in Malawi. Written informed consent was obtained from all of the participants’ mothers and the trial protocol was reviewed and approved by the College of Medicine research and ethics committee (University of Malawi) and the ethical committee of the Pirkanmaa Hospital District, Finland. The online-only trial profile of the Malawian and Finnish study cohorts can be reviewed at http://links.lww.com/MPG/A90.
The inclusion criteria included the availability of fecal samples and signed informed consent from at least 1 guardian. Subjects included were permanent residents of the Lungwena Health Center or the Malindi Hospital catchment area. The exclusion criteria comprised severe illness, history of allergy toward peanut, history of anaphylaxis or serious allergic reaction to any substance, requiring emergency medical care, and concurrent participation in any other clinical trial. Therefore, of the total 840 participants in the main trial, included in the present study, 3 sets of 16 consecutive Malawian participants were selected in order of recruitment, these representing 3 different seasons, which reflect variation of food availability during the year. Among these 48 infants, a stool sample was not available in 4 cases, yielding a sample size of 44. The stool samples were collected at enrollment for the clinical trial, that is, before the participants had undergone any intervention. The clinical characteristics and patterns of present dietary intakes of the Malawian infants participating in the study are presented in Table 1. The 44 infants represented a population typical of that age in Malawi. The data were collected after the infants were 6 months old. After that, the infants were randomized to different nutritional treatments. Upon enrollment, the mothers answered a questionnaire on whether the infants had received breast milk or foods other than breast milk the previous day or ever.
There is no information as to the modes of delivery for the Malawian participants, but because cesarean sections are in general rare in rural Malawi, the great majority were presumably born vaginally. Exclusive breast-feeding is also rare in rural Malawi and infant diets are typically complemented soon after birth with fluids such as tea, water, rice water, and solid foods such as thin maize porridge and bean and fish meat soups.
The Finnish study population comprised 31 healthy 6-month-old infants (9,10) participating in an ongoing prospective randomized study in the city of Turku and neighboring areas in southwestern Finland, with a total of 256 mother–infant pairs. The infants included in the present study did not receive probiotics or prebiotics and their mothers, if breast-feeding, did not have probiotic or prebiotic products in their diet. The mothers were interviewed during the first 6 months for the feeding status of the infant, infants’ food diaries were collected, and infants’ weight and height were measured. The mothers’ diets reflected the typical Finnish diet, as previously reported (11). The clinical characteristics of the infants are presented in Table 1. The 31 infants represented a population typical of that age in Finland, the majority being born vaginally and their mean growth reflecting the population reference. The infants were breast-fed and received additional infant formula along with complementary foods. The latter included pureed vegetables (eg, potato, carrot), fruits (eg, peach, banana, blueberry), chicken or fish meat, and cereals (eg, oats, rice). The study complied with the Declaration of Helsinki as revised in 2000. Written informed consent was obtained from all of the participant mothers, and the study protocol was approved by the ethics committee of the Hospital District of Southwest Finland.
From both Malawian and Finnish study groups, fecal samples were collected. These were stored frozen and transported to the laboratory in the Functional Foods Forum, University of Turku in Turku, Finland, and stored at −70°C until analyzed. The samples were examined to identify the microbiota composition and characterize differences between the low-income and high-income area infant microbiota. The microbiota composition represents that of the colonic lumen.
Sampling Preparation and DNA Extraction
The fecal samples (0.5 g) were weighed, diluted 1:10 (wt/vol) in phosphate-buffered saline (PBS) (pH 7.4), and homogenized by thorough agitation in a vortex. Aliquots of these dilutions were used for DNA extraction. DNA from both feces and pure cultures of the different bacterial strains used as reference were extracted using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions.
Real-time Polymerase Chain Reaction Analysis
Quantitative real-time polymerase chain reaction (PCR) was used to characterize the fecal microbiota using group- and species-specific primers as previously described. These oligonucleotides were purchased from the Thermo Electron Corporation (Thermo Biosciences, Ulm, Germany).
Briefly, PCR amplification and detection were performed with an ABI PRISM 7300-real-time PCR system (Applied Biosystems, Foster City, CA). Each reaction mixture of 25 μL was composed of Power SYBR Green PCR Master Mix (Applied Biosystems), 1 μL of each of the specific primers at a concentration of 0.2 mol/L, and 1 μL of template DNA. The fluorescent products were detected in the last step of each cycle. A melting curve analysis was made after amplification to distinguish the targeted from the nontargeted PCR product.
The bacterial concentration in each sample was calculated by comparing the Ct values obtained from standard curves. A standard curve was made from serial dilutions of DNA isolated from each pure culture of the different reference strains. A linear relation was observed between cell numbers and Ct values (r2 = 0.99–0.97).
The following reference strains were used to construct the corresponding standard curves: Bifidobacterium longum (DSM 20219) (this strain was also used as the standard strain for quantification of the Bifidobacterium genus), B catenulatum (JCM 7130), B bifidum (DSM 20456), B adolescentis (DSM 20083), B breve (DSM 20213), Clostridium coccoides (DSM 935T), C leptum (DSM 753T), C difficile (DSM 1296T), C perfringens (DSM 756), Akkermansia muciniphila (ATTC BAA-835), and Staphylococcus aureus (DSM 20231). The primer sequences of the reference strains and the annealing temperatures of the primers have been published elsewhere (9,12–14).
Flow Cytometry-Fluorescent In Situ Hybridization Analysis
Homogenized fecal samples were fixed overnight in 4% paraformaldehyde and stored in PBS-ethanol at −20°C until analyzed. Fluorescent in situ hybridization (FISH) with flow cytometer was performed as previously described (5,9). In brief, samples were hybridized at specific temperatures in hybridization buffer with specific probes at a concentration of 5 ng/μL. After overnight hybridization, they were washed with buffer without sodium dodecyl sulfate, pelleted, and resuspended in PBS. A Eubacterium (EUB) 338 probe was covalently linked at their 5′-end with fluorescein isothiocyanate (FITC) and other probes with carbocyanine 3 (Thermo Biosciences). Probes included EUB 338 for the total bacteria (15), Bif164 for the Bifidobacterium group, Bac303 for the Bacteroides-Prevotella group, and Chis150 for the C histolyticum group, with the references to the probe sequences (9), respectively. Data acquisition was performed with an LSR II flow cytometer equipped with an high-throughput screening 96-well plate reader (Becton Dickinson, San Jose, CA); 40 μL of each sample were collected in duplicate. Data were analyzed with BD FACSDiva software (Becton Dickinson).
Total amounts of bacteria were determined with an EUB 338-FITC probe. Determination of specific bacteria was made by combining each of the group-specific carbocyanine 3 probes with the EUB 338-FITC probe and counting double-positive cells (16).
Continuous variables were compared between Malawian and Finnish infants using the Kruskal-Wallis test. Continuous variables are presented using medians with interquartile ranges (IQR). The Fisher exact test was used to test the percentages of positive values for quantitative PCR. Statistical analysis was performed using SAS for Windows (version 9.2, SAS Institute Inc, Cary, NC).
Differences in Gut Microbiota Composition Between Malawian and Finnish Infants
Differences in the gut microbiota composition of Malawian and Finnish infants were observed in most bacteria assessed. Percentages of the Bifidobacterium group, Bacteroides-Prevotella group, and C histolyticum group detected by FCM-FISH were significantly higher in Malawian than in Finnish infants (70.8% vs 46.8%, P < 0.001; 17.2% vs 4.7%, P < 0.001; 4.4% vs 2.8%, P = 0.01, respectively) (Fig. 1).
Likewise, the counts of Bifidobacterium, Bacteroides-Prevotella, and C histolyticum groups and the total number of bacteria detected by FCM-FISH were significantly higher in the Malawian infants (9.2 log cells/g, IQR 8.4–9.6 vs 8.4 log cells/g, IQR 8.0–8.7, P < 0.001; 8.6 log cells/g, IQR 8.1–8.9 vs 7.5 log cells/g, IQR 6.9–8.0, P < 0.001; 8.0 log cells/g, IQR 7.6–8.4 vs 7.3 log cells/g, IQR 6.2–7.6, P < 0.001; 9.4 log cells/g, IQR 8.8–9.8 vs 8.8 log cells/g, IQR 8.4–9.0, P = 0.003, respectively).
At the same time, using quantitative PCR higher counts of the Bifidobacterium genus group and the species B longum and B bifidum were detected in higher amounts in Malawian compared to Finnish infants (10.4 vs 10.0 log cells/g, 10.3 vs 9.1 log cells/g, 10.1 vs 5.9 log cells/g, respectively). The B catenulatum, C difficile, and A muciniphila species were rare in Malawian as compared with Finnish infants. Bacteria belonging to the B adolescentis, C perfringens, and S aureus species, present in Finnish infants, were not detectable in Malawian (Table 2) infants.
Our study is the first to compare the microbiota of southeastern African and northern European infants. The Malawian infants’ microbiota showed an abundance of Bifidobacterium, Bacteroides-Prevotella, and C histolyticum as compared with the levels of these bacteria in Finnish infants. The most likely explanation here is the diet of the African children, this being composed of plant polysaccharides, which are introduced together with breast-feeding. Among Malawian infants, both were more frequent than they were among Finnish children. In addition, it has been reported that the availability of food in Malawi varies significantly among the seasons (17). Thus, our cohort has been chosen to reflect all of the seasons and availability of food in Malawi. In recent studies from Ley et al (6,18), a high Firmicutes/Bacteroidetes ratio has been reported in obese individuals, whereas an increase in Bacteroidetes accompanies weight loss. These previous results are in accord with the difference observed in the numbers of Bacteroidetes coinciding with the weight differences between Malawian and infants from southwestern Finland, whether related or unrelated to the diet.
In recent studies from Finland, a high concentration of Bacteroidetes is seen in obese individuals (5,19), and at the same time Bacteroidetes levels were significantly higher in infants from obese mothers during the first 6 months of life (19), demonstrating that maternal health status and the diet have an effect on microbiota composition in infants (8).
Bifidobacteria have been seen to typify the gut microbiota of healthy breast-fed infants. This appears to be the case in both populations studied here. It has been reported that numerous specific bifidobacterial species preferentially consume small-mass oligosaccharides, which are abundant early in the lactation cycle (20). A more detailed breakdown of species composition reveals species differences among subpopulations of infants (21,22). These findings could be associated with the different immunomodulatory properties of specific bifidobacterial species present in the gut. B adolescentis has been associated with inflammatory effects, whereas B longum and B bifidum have been shown to possess immunomodulatory properties (9,23). Interestingly, B adolescentis was detectable only in Finnish infants and B catenulatum in both groups. In Malawian infants, the prominent strains were B longum, B bifidum, and B breve. Thus, the potential role of Bifidobacterium species composition and quantity of total microbiota in infants in high-income countries may be more marked, favoring the allergy risk. In low-income countries such as Malawi, the situation may be the opposite, providing clues for measures preventive of the trend in high-income areas.
It was noted that Malawian infants were less often colonized with clostridial species, staphylococci, and Akkermansia-like bacteria, which were abundant in the Finnish infants. The preponderance of these bacterial species could also reflect disease risk because allergic infants are more often colonized by C difficile and S aureus and less by bifidobacteria (19). In addition, higher levels of clostridial species and staphylococci have been reported in obese and overweight infants and adults (5,18,19,24). A muciniphila has been shown to be a common member of the infant gut microbiota in Europe, typically beginning to colonize the intestine early in infancy and increasing in numbers again toward adulthood (25), and further shown to degrade the intestinal mucus because this bacterium contains numerous candidate mucinase-encoding genes (26). The mucus degradation caused by Akkermansia may affect the innate and adaptive immune responses in that it disturbs the first defense protection of the host mucosal surfaces, suggesting that it may play a role in the pathogenesis of inflammatory diseases (27) and even obesity (5,9,19). The absence of Akkermansia-like bacteria in Malawian infants could thus constitute a protective health factor against Westernized diseases.
In the same fashion, it has been debated whether the specific microbiota composition in Malawian infants could be protective against potentially pathogenic microbiota. Such a conception is supported by a study from the Ivory Coast (28), which reported that anemic African children carry an unfavorably high ratio of fecal enterobacteria to bifidobacteria and lactobacilli. This ratio is further increased by iron fortification. The specific microbiota composition found in Malawian infants could be a barrier against some potentially pathogenic intestinal microbes constantly challenging infants in low-income areas with poor hygiene.
Taken together, our findings indicate that the gut microbiota in 6-month-old Malawian and Finnish infants is dominated mainly by bifidobacteria. They further suggest that specific Bifidobacterium species may be necessary for their health-promoting role in different areas of the world. Conversely, microbiota compositions including members of the Bacteroidetes phylum and Akkermansia-like species may have been guided by the initial environmental exposures, this including diet and hygiene. The role of microbiota, diet, and hygiene may thus be more prominent than expected in providing protection against aberrant phenomena and inflammatory challenges. To unravel the interrelations between diet composition and the activity of the gut microbiota as well as their relation to human genotype and environmental factors clearly calls for further research.
We thank Jaakko Matomaki for statistical consultation during the data analysis and Robert MacGilleon for language review of the manuscript.
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