What Is Known
- Lactobacilli are believed to be associated with a healthy gut, and therefore commonly used as probiotics.
- Early colonization by specific lactobacilli has been associated with a lower risk of developing atopic and allergic diseases.
- A low prevalence of lactobacilli has also been linked with excessive crying during infancy.
What Is New
- Malawian infants had a more abundant, richer, and more diverse Lactobacillus microbiota with a distinct composition compared with Finnish infants.
- Lactobacillus rhamnosus GG, a widely consumed probiotic in Finland, was detected in a third of the Finnish infants, although neither they nor their mothers received probiotics.
The microbial colonization of the intestine is a step-wise process, which is affected by the mother's microbiota, mode of delivery, gestational age, early feeding practices, and the environment (1). It is widely recognized that low microbial exposure in early life may enhance the risk of inflammatory and immune-mediated disorders, including atopic diseases (2). Such diseases are reaching epidemic proportions in the high-income countries, while remaining rare in low-income countries, where in contrast malnutrition and infectious diseases are a significant health concern. Interestingly, gut microbiota may also have an impact on energy harvest and storage and thus affect child growth and malnutrition (3), and differences in fecal microbiota composition have been linked to the development of overweight and obesity (4,5)(4,5).
Among the first microbes to colonize the human body are the lactobacilli, possibly already in utero (6,7)(6,7), and a majority of the microbes in the birth canal (8) and breast milk (9) belong to this group (including the genera Lactobacillus, Leuconostoc, Pediococcus, and Weissella). Lactobacilli are believed to be associated with a healthy gut and therefore members of this group are also commonly used as probiotics. Early colonization with some lactobacilli, L casei group to be more specific, has been associated with a decreased risk of developing allergy (10) and atopic dermatitis (11). In addition, a low prevalence of Lactobacillus spp has been reported as a possible risk factor for excessive crying and fussing during infancy (12) and some Lactobacillus sp may also have a role in weight regulation (13). Nevertheless, little is known as to the Lactobacillus microbiota composition of infants living in low-income countries and how it compares with that in infants from high-income nations.
In this study, we characterized the Lactobacillus group composition in the feces of healthy 6-month-old infants living in rural Malawi and Southwestern Finland, representing typical infants in these areas. For this purpose, we used quantitative polymerase chain reaction method (qPCR) for the enumeration of the whole Lactobacillus group and PCR-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprinting to give a detailed view of the Lactobacillus microbiota composition at species level in the infant fecal samples.
Subjects and Samples
The study populations comprised healthy 6-month-old infants living in rural Malawi (n = 44) and Southwestern Finland (n = 31). Malawi is a landlocked country in Southeast Africa, where infant mortality is high and life expectancy is low, whereas Finland is the opposite (14). The infants from Malawi were enrolled in an epidemiological clinical trial assessing the impact of selected dietary interventions on early childhood growth (identifier: NCT00524446). This study was approved by the College of Medicine research and ethics committee (University of Malawi) and the ethics committee of the Pirkanmaa Hospital District, Finland. For the purpose of the present study, samples from the Malawian infants were acquired at baseline before dietary interventions. The Finnish infants were participants in a prospective randomized study in Turku and neighboring areas in Southwestern Finland (identifier: NCT00167700). The study protocol was approved by the ethics committee of the Hospital District of Southwest Finland. The study populations and characteristics have been described in detail elsewhere (15,16)(15,16).
The clinical characteristics of Malawian and Finnish infants are presented in Table 1. The majority of the Finnish and presumably all the Malawian infants were born vaginally. In both locations, the infants had an age-appropriate diet typical for each area. None of the infants in Malawi was exclusively breast-fed in Malawi. Liquids, including tea, water, rice water, and solid foods such as thin maize porridge and bean and fish meat soups were introduced shortly after birth. In Finland, infants were mainly breast-fed, but some received additional infant formula and some complementary foods, including pureed vegetables (eg, potato, carrot), fruits (eg, peach, banana, blueberry), chicken or fish meat, and cereals (eg, oats, rice). None of the infants or their mothers received any probiotics or prebiotics. Further clinical characteristics of the cohorts have been supplied by groups under Grzeskowiak et al (15) and Mangani et al (16).
From both study groups, fecal samples were collected and stored frozen and transported to the laboratory in the Functional Foods Forum, University of Turku, Finland, and stored at −70°C until analyzed (15).
Bacterial Strains and Growth Conditions
The following strains obtained from the DSMZ culture collection (Braunschweig, Germany) were used as references: L johnsonii DSM 10533T, L reuteri DSM 20016T, L sakei DSM 20017T, L rhamnosus DSM 20021T, L delbreuckii subsp delbrueckii DSM 20074T, L acidophilus DSM 20079T, L plantarum DSM 20174T, Weissella confusa DSM 20196T, L gasseri DSM 20243T, and Leuconostoc mesenteroides DSM 20343T. The strains were routinely grown in MRS broth (Oxoid Ltd, Basingstoke, Hampshire, England) at 37°C overnight.
The DNA was extracted from the reference strains using the method described by Endo et al (17). The DNA was diluted to 10 ng/μL before PCR amplification. From the fecal samples, the DNA was extracted using QIAmp DNA Mini Kit (Qiagen, Venlo, The Netherlands) as described by Grzeskowiak et al (15).
PCR-DGGE and Numerical Analysis
The primer set Lac1 and Lac2-GC (Table 1) designed by Walter et al (18), which amplifies a fragment of the V3 region of the 16S rRNA gene of the genera Lactobacillus, Leuconostoc, Pediococcus, and Weissella, was used to analyze the Lactobacillus group community in the fecal samples. The reaction mixture was prepared as described by Endo et al (19) and the thermal cycling was performed as indicated by Walter et al (18). Amplification was confirmed by gel electrophoresis in 1.0% agarose.
The DGGE analysis was conducted using the DCode system (Bio-Rad Laboratories, Hercules, CA.) according to the method by Endo et al (20). In this study, we used a denaturing gradient of 35% to 50% and the electrophoresis was performed at a constant voltage of 55 V for 16 hours. Gel staining, excision of unknown bands, and their sequencing were done as described by Endo et al (20). The sequencing was carried out by the Finnish Microarray and Sequencing Center. The accession numbers of the sequences are KJ809074-KJ809085.
Species- and Strain-Specific PCRs
Because it is not possible to differentiate L casei group species (L casei, L paracasei, and L rhamnosus) by the DGGE technique used in the present study (18), the samples which produced a band identified as L casei group in the PCR-DGGE analysis were further analyzed by species-specific PCR with L rhamnosus- (21), L casei- (22), and L paracasei- (21) specific primer sets (Table 2) (18,21–24)(18,21–24)(18,21–24)(18,21–24)(18,21–24). The PCR reaction mixture (25 μL) contained 0.2 μmol/L of each primer, 2 mmol/L of MgCl2, 0.2 mmol/L of each deoxyribonucleotide phosphate, 1 unit of AmpliTaq Gold DNA polymerase, reaction buffer and 1 μL of template DNA, and the following thermal cycling program was used: 10 minutes at 95°C; 45 cycles of denaturation for 15 seconds at 95°C, annealing at a primer-specific temperature for 40 seconds, and extension for 30 s at 72°C; and a final extension for 5 minutes at 72°C. The primer-specific temperatures for L rhamnosus, L casei, and L paracasei were 61°C, 58°C, and 60°C, respectively.
Samples that produced an amplicon of an expected size in L rhamnosus species-specific PCR were further analyzed using a L rhamnosus GG strain-specific PCR method developed by Brandt et al (23).
Quantitative PCR Analysis of the Lactobacillus Group in the Fecal Samples
Quantitative PCR with the primer set Lac1 and S-G-Lab-0677-a-A-17 described by Rinttilä et al (24) was used to enumerate the number of Lactobacillus group cells in each sample. PCR amplification and detection were performed with an ABI PRISM 7300-PCR sequence detection system (Applied Biosystems, Foster City, CA) using the following amplification program: 95°C for 10 minutes and 40 cycles of 95°C for 20 seconds, 58°C for 20 seconds, and 72°C for 30 seconds. The reaction mixture was composed of Power SYBR Green PCR Master Mix (Applied Biosystems), 1 μL of each of the primers at a concentration of 0.2 mol L−1 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 the amplification to distinguish the targeted PCR product from the nontargeted. The bacterial concentration in each sample was determined by comparing the Ct values obtained from standard curves. The standard curves were formed from serial dilutions of DNA extracted from a known amount of L rhamnosus DSM 20021T cells.
The Lactobacillus group bacterial counts (log cells/g) assessed by qPCR are presented as means with 95% confidence intervals. The bacterial counts in Malawian and Finnish infants were compared using independent samples t test.
The DGGE images were imported to Bionumerics software version 6.6 (Applied Maths, St-Martens-Latem, Belgium) for normalization and band detection and were numerically analyzed. Band searching and matching using 1% band tolerance was performed as implemented in the Bionumerics software. Moreover, bands and band matching were manually corrected when deemed necessary, and cluster analysis of the DGGE patterns was performed by the unweighted pair-group method using arithmetic averages based on the Pearson correlation similarity coefficient. The band presence/absence matrix was exported from Bionumerics and imported to R version 3.1.2 (25) for principal component analysis and permutational multivariate analysis of variance (PERMANOVA). The principal component analysis based on the Euclidean distances was performed using the R function prcomp, and the first 2 principal components were used to generate a 2-dimensional scatter plot. The PERMANOVA analysis was made using the adonis function of the R software package vegan (26). A Euclidean distance matrix was calculated with 999 permutations.
Furthermore, matrices based on the presence/absence and intensities of bands were exported from Bionumerics and imported to Microsoft Excel for the calculation of richness and Shannon diversity indices. The number of bands present in the DGGE profile was used as a measure of richness for the samples, and the Shannon diversity index, H’, was calculated using the equation H’ = −Σ Pi ln (Pi). Pi is the importance probability of the bands in a gel lane and is calculated as Pi = ni/N, in which ni is the intensity of band i and N is the sum of intensities of all bands in the densitometric profile.
Richness in the samples was not normally distributed and is thus presented as medians with interquartile range (IQR) and analyzed using the Mann-Whitney U test. The Shannon diversity indices were normally distributed and are expressed as means with 95% confidence intervals (CI) and analyzed by independent samples t test. Fisher exact test was used to determine statistical difference in the prevalence of each band class between the Malawian and Finnish infants. Band classes identified as the same species were combined.
The aforementioned methods were also used to assess whether breast-feeding or vaginal delivery was associated with bacterial counts, richness, diversity, or the composition of the Lactobacillus microbiota in the infants. Similarly, we assessed whether animal milk consumption or domestic animals (cows, goats, sheep, or chickens) in the family was associated with colonization by L ruminis in the Malawian infants.
The statistical analysis was conducted with SPSS version 21.0 (IBM SPSS Statistics, Armonk, NY).
The qPCR analysis indicated that the Malawian infants had higher counts of Lactobacillus-group bacterial cells than the Finnish infants (7.45 log cells/g [95% CI 7.32–7.57] vs 6.86 log cells/g [95% CI 6.57–7.16]; P < 0.001, respectively) (Table 3).
For the PCR-DGGE, an amplicon of an expected size was obtained from all samples. The number of different Lactobacillus group DNA fragments per sample, the richness, was higher in the Malawian compared with the Finnish infants according to PCR-DGGE (5 bands [IQR 3.25–6.00] vs 4 bands [IQR 2.00–4.00]; P = 0.001, respectively) (Table 3). Bacterial diversity as measured by the Shannon diversity index, H’, based on the DGGE band intensity values, showed a higher diversity in the Malawian compared with Finnish infants (1.31 [95% CI 1.21–1.41] vs 0.98 [95% CI 0.83–1.13]; P < 0.001, respectively) (Table 3).
Differences were also observed in the composition of Lactobacillus microbiota between the Malawian and the Finnish infants based on the PCR-DGGE analysis. Leuconostoc citreum, W confusa, L ruminis, L gasseri, L acidophilus, and L mucosae were detected more often in the Malawian than in the Finnish infants (100.0% vs 74.2%, P < 0.001; 95.5% vs 41.9%, P < 0.001; 59.1% vs 0.0%, P < 0.001; 38.6% vs 9.7%, P = 0.005; and 29.5% vs 0%, P < 0.001; 22.7% vs 3.2%, P = 0.017, respectively) (Table 4). Bands identified as L casei group, L delbrueckii, and L parabuchneri were detected less often in the Malawian than in the Finnish infants (0.0% vs 58.1%, P < 0.001; 0.0% vs 19.4%, P = 0.004; 0% vs 12.9%, P = 0.026, respectively) (Table 4).
A total of 18 Finnish samples (58.1% in total), which produced L casei group bands in DGGE profiles, were further analyzed by species-specific PCR with L rhamnosus-, L paracasei-, and L casei- specific primer sets and an L rhamnosus GG strain-specific primer set. All of the 18 samples presented L rhamnosus, 8 of them presented L paracasei, but none had L casei. Furthermore, the strain L rhamnosus GG was detected in 10 of the Finnish samples.
A comparison of formula-fed (n = 10) and breast-fed (n = 24) (included exclusive and nonexclusive breast-feeding) Finnish infants revealed that breast-feeding was not associated with the Lactobacillus group cell counts or the richness and diversity of the Lactobacillus microbiota in the present study (P > 0.05). The L casei group, however, was detected more often in the breast-fed than in the formula-fed infants (76.2% vs 20.0%, P = 0.005). The species L rhamnosus of the L casei group was more common in breast-fed compared with formula-fed infants (76.2% vs 20.0%, P = 0.005). Furthermore, mode of delivery was not associated with the bacterial counts, richness, diversity, or the composition of the Lactobacillus microbiota in the Finnish infants.
Unweighted pair-group method using arithmetic averages analysis of the DGGE fingerprints based on the Pearson correlation similarity coefficient revealed 2 main clusters (Fig. 1). Cluster 1 was composed of infants from both countries and was characterized by bright bands recovered from L citreum and W confusa. Furthermore, this cluster included a Malawian subcluster, which showed a strong intensity band of L ruminis in addition to the L citreum and W confusa bands. Cluster 2 comprised primarily Finnish samples, which produced a bright L casei group band and a weak or no Leuconostoc or Weissella band. Moreover, the principal components analysis (Fig. 2) also supported the cluster seperation seen in Figure 1. PERMANOVA confirmed these results indicating a significant difference in species composition between the Malawian and the Finnish infants (P < 0.001; F = 9.96).
The 2 countries studied involve different challenges related to the gut, that is, allergies and gastrointestinal disorders (IBS, IBD, etc) in Finland, which are absent in Malawi, and bacterial (or viral) diarrhea in Malawi. The microbiota is closely linked to these challenges. Earlier, Bifidobacterium, Bacteroides-Prevotella, and Clostridium histolyticum groups have been characterized from the same infants by Grzeskowiak et al (15), using quantitative polymerase chain reaction (qPCR) and flow cytometry-fluorescent in situ hybridization (FCM-FISH). The authors found significant differences in the composition of the gut microbiota between the study groups: the Malawian infants yielded greater proportions of bifidobacteria, Bacteroides-Prevotella, and Clostridium histolyticum, whereas the species Bifidobacterium adolescentis, Clostridium perfringens, and Staphylococcus aureus were absent. Here we report that the Malawian infants had a more abundant, richer, and diverse Lactobacillus microbiota than the Finnish infants, which shed further light on the differences in the gut microbiota composition of infants from low-income and high-income countries. This information may be useful in understanding why some gut-related diseases are more common in high-income or low-income countries.
The Malawian infants here had higher counts of lactobacilli than their Finnish counterparts. This concurs with earlier findings: a higher prevalence of lactobacilli in the feces of infants has been reported in areas where allergic and immune-mediated diseases are less common compared with urbanized areas with a high prevalence of these disorders. For instance, higher levels of lactobacilli have been detected in rural Thai infants versus Singaporean infants (27) and also in Estonian versus Swedish infants (28). It has been hypothesized that colonization with specific lactobacilli species during infancy may have an impact on the development of the immune system and thus affect health later in life (10).
Leuconostoc citreum and Weissella confusa were the most common lactobacilli in both the Finnish and the Malawian samples, but were more prevalent in the Malawian infants. This was also reflected in the clustering analysis, where the infants colonized by these species formed the largest cluster (Fig. 1). The predominance of these species in infant stools has never been reported, but these findings are not surprising in that Leuconostoc spp and Weissella spp have been identified as the predominant microorganisms in breast milk (9). Although both genera have been described as opportunistic pathogens associated with neonatal bacteremia (29), our results and those of earlier studies suggest that these organisms are common inhabitants of the infant gut and seem to form a fundamental part of the Lactobacillus microbiota of infants irrespective of geographical location.
A species identified as L ruminis was detected only in the Malawian infants. This organism has been described as one of the predominant Lactobacillus species in adults (30), but only few studies report it part of the microbiota of infants. Interestingly, L ruminis has been detected in infants from Malawi as well (31), suggesting that this species might be a characteristic of infants from this area. It is moreover possible that this species originates from animal sources because it has been identified as an indigenous species in several animals, including pigs (32), cows (33), and chickens (34). Animal milk consumption or the domestic animals in the family, however, were not associated with colonization by L ruminis in the present study (data not shown), suggesting that the species could also originate from wild animals in the rural area.
The Malawian infants were also characterized by the presence of L gasseri, L acidophilus, and L mucosae. L gasseri is often encountered in breast milk (35) and has been described as one of the predominant Lactobacillus species in infants from Sweden during the first 2 months of life (36). The other 2 species, L acidophilus and L mucosae, have also been regarded as normal inhabitants of the infant gut (36). Interestingly, the presence of L acidophilus has been reported to be inversely related to colic in infants (37).
L delbrueckii and L parabuchneri were more common in the Finnish infants, although their presence remained generally low. L delbrueckii might originate from dairy products because it is used in the production of for example yogurt. L parabuchneri colonization in the gut has not as yet been well characterized and to our knowledge the species has not hitherto been isolated from fecal samples from infants or adults. L parabuchneri was originally isolated from human saliva (38) and strains of this species may also be present in some cheeses as well (39,40)(39,40).
The L casei group, containing the species L casei, L paracasei, and L rhamnosus, was only detected in the Finnish infants and thus was also a discriminating factor in the cluster analysis (Fig. 1). Interestingly, infants colonized by the L casei group were less often colonized by Leuconostoc spp or Weissella spp. It is possible that these species share adhesion sites and may also compete for the same nutrients.
Further analysis of the L casei group-positive samples by species-specific PCR revealed L rhamnosus to be the most abundant species of this group in the Finnish infants, and we also noted that this species was more common in breast-fed than in formula-fed infants. L rhamnosus has also been reported as a predominant species in Sweden (36) and Greece (41), and the findings in Sweden were similar to those in our study: breast-fed infants were more commonly colonized by L rhamnosus than the weaned infants at 6 months of age. Moreover, early colonization by L rhamnosus has been associated with a decreased risk of developing an allergy later in life (10). In addition to L rhamnosus, some of the L casei group-positive samples from Finnish infants contained L paracasei. Similar to L rhamnosus, early colonization by L paracasei has been linked to a decreased risk of allergy development later in life (10) and to a decreased risk of atopic dermatitis in children (11). Interestingly, absence of the whole L casei group has been associated with celiac disease in children (42). All in all, it seems that the L casei group species, especially L rhamnosus, may be regarded as healthy parameters in Western infants.
Surprisingly, more than half of the L rhamnosus-positive samples contained the L rhamnosus GG strain even though neither the infants nor the mothers received any probiotic products. A similar observation of L rhamnosus GG appearing in almost half of the fecal samples in a placebo group has been reported in Finnish infants (43). L rhamnosus GG has long been provided in different food matrices in Finland and hence it has been hypothesized that L rhamnosus GG might colonize and produce derivative strains in the human gut (44). Nonetheless, the source of L rhamnosus GG in the gut of Finnish infants remains to be elucidated.
Differences in diet practices and sanitation levels in Malawi and Finland offer 1 possible explanation for the differences in the Lactobacillus group microbiota composition and abundance. In addition to breast-feeding, local foods naturally rich in fiber and complex carbohydrates capable of modulating the microbiota are introduced to infants in Malawi relatively early, whereas in Finland, infant formula and complementary foods are in contrast introduced when breast-feeding was not sufficient (15). Plant polysaccharides have been reported as an important factor modulating the microbiota in infants from another African country, Burkina Faso (45). It must also be borne in mind that the composition of breast milk might differ between the Malawian and Finnish mothers. Breast milk is a continuous source of microbes and other microbiota-modulating factors to infants, and for instance, Cabrera-Rubio et al (9) recently reported that the breast-milk microbiome is affected by the weight of the mothers and by the mode of delivery.
Hygiene and the presence of animals could be another important factor in discriminating the gut microbiota between the 2 countries, as in Malawi, hygiene standards are generally lower compared with Finland, and people live in close contact with animals. For instance, perinatal pet exposure has been reported to impact on the composition of the gut microbiota and wheezy bronchitis in infants (46), and a recent study has shown that humans actually share microbiota with their companion animals (47). Direct contact with animals, however, might not even be needed. The dust from a house, which has pets, has as such been reported to increase the amount of lactobacilli and protect against airway allergens in mice (48).
Lactobacilli are often used as probiotics in infant intervention studies, including cases of atopic dermatitis and the results have been promising for specific probiotic strains (49). Moreover, studies on probiotic use in 1 Norwegian birth cohort also suggest that consumption of specific Lactobacillus probiotics (consisting of 3 main probiotic products available at the time of study) during the perinatal period may promote a reduction in the risk of atopic eczema in infants (50). The present findings clearly indicate that the composition of Lactobacillus microbiota may differ between countries, and thus the question arises: could such differences in microbiota composition affect the efficacy of a probiotic? For instance, L casei-group species have been widely used as probiotics for the same purposes in several countries around the globe, regardless of whether the species are indigenous to the population or not.
Taken together, the Malawian infants in our study had higher amounts of lactobacilli and the community was richer and more diverse than that in the Finnish infants, and we believe that the environment, including diet and hygiene, may be among the factors associated with these differences. Although the lactobacilli constitute only a small part in adult microbiota, they seem to play an important role in the development of the gut microbiota, the health status of an infant, and also in protecting against various diseases later in life, as has been indicated by earlier reports. It is not clear how persistent the Lactobacillus species are, however, in the intestines of the infants in this study, because we have now assessed only 1 time-point at 6 months of age. Further characterization of the developing microbiota needs to be assessed to see how the gut microbiota of these infants develops in general, and what role early lactobacillus colonization has in the perspective of health later in life. It seems that a continuous microbial exposure in early stages of life is needed for the development of a normal and healthy gut microbiota.
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Keywords:© 2015 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology,
Finland; infant; Lactobacillus; Malawi; microbiota