|Year : 2017 | Volume
| Issue : 4 | Page : 1022-1029
Paediatric health significance of prevalent stratified indigenous Lactobacillus spp. species in diet-dependent infantile faecal specimens
Adenike A O. Ogunshe
Applied Microbiology and Infectious Diseases, Department of Microbiology, Faculty of Science, University of Ibadan, Ibadan, Nigeria
|Date of Submission||27-Mar-2016|
|Date of Acceptance||29-Jul-2016|
|Date of Web Publication||04-Apr-2018|
Adenike A O. Ogunshe
Applied Microbiology and Infectious Diseases Unit, Department of Microbiology, Faculty of Science, University of Ibadan, Ibadan
Source of Support: None, Conflict of Interest: None
Composition of early gut microbiome changes in breast-fed and formula-fed infants as solids foods are introduced into their diet.
This study aims to investigate the stratified of easily culturable indigenous Lactobacillus spp. with probiotic potentials in infant faecal specimens.
Participants and methods
Questionnaires were administered on 41 healthy and well-nourished neonates and infants between 1.5 and 11 months of age through their mothers. Isolated bacterial florae were identified using standard phenotypic cultural and taxonomic procedures and tools.
Most of the infants were fed on breast milk and industrial baby cereal foods; however, more male babies (seven) were exclusively breast-fed than female babies (two). The pH range of the infantile faecal specimens was 4.8–5.9, but the enumerated viable counts as colony-forming units were between 1.1 × 103 and 7.2 × 103 cfu g-1. No lactobacilli were isolated from 39% faecal specimens of 4–16 days old infants, but there were higher recovery rates of Lactobacillus strains from faecal specimens of older (5–11 months) infants. Diversity of isolated 151 Lactobacillus strains included one or more different phenotypes characterized as Lactobacillus acidophilus, Lactobacillus bifidus, Lactobacillus brevis, Lactobacillus casei, Lactobacillus plantarum and Lactobacillus reuteri. The overall most-prevalent Lactobacillus strains were L. reuteri (46.4%), L. casei (23.2%) and L. acidophilus (19.9%), whereas L. bifidus (7.28%), L. brevis (1.99%) although L. plantarum (1.32%) were isolated from faecal specimens of healthy infants above 5 months of age. L. acidophilus (32.5%), L. casei (26.0%), L. reuteri (31.2%) vs. L. casei (20.0%) and L. reuteri (62.7%) were the most-prevalent phenotypes from female and male babies, respectively.
Diet-dependent Lactobacillus spp., which reflected rich indigenous infantile bacterial consortium, were recoverable from faeces of less than 12-month-old breast milk-fed and industrial cereal-fed, healthy Nigerian infants without antibiotherapy.
Keywords: infant feeding, informed consents, intestinal microbial colonisation, Lactobacillus diversity, paediatric diets
|How to cite this article:|
O. Ogunshe AA. Paediatric health significance of prevalent stratified indigenous Lactobacillus spp. species in diet-dependent infantile faecal specimens. Menoufia Med J 2017;30:1022-9
|How to cite this URL:|
O. Ogunshe AA. Paediatric health significance of prevalent stratified indigenous Lactobacillus spp. species in diet-dependent infantile faecal specimens. Menoufia Med J [serial online] 2017 [cited 2020 May 27];30:1022-9. Available from: http://www.mmj.eg.net/text.asp?2017/30/4/1022/229207
| Introduction|| |
Microbiota of the human gastrointestinal tract plays a key role in nutrition and health, such as gastrointestinal problems,, due to normal human microflora being a complex ecosystem which in part is dependent on enteric nutrients for establishing colonization. There has, therefore, been increase in the use of dietary components that help to maintain or even improve the gut microbial balance,. Several studies have shown that lactobacilli constitute one of the dominant groups of human intestinal and faecal microorganisms, of human infants. Moreover, members of the genus Lactobacillus make an integral part of the healthy human intestinal flora, such that much attention has been paid to their health-promoting properties, as it has been claimed that when administered in adequate amounts, they confer a health benefit on the host,. However, a delicate balance exists between the gut microbial flora and its host, and a number of different environmental factors such as age and diets may affect the gut microbial flora,.
Infant feeding practices, particularly breast-feeding and formula-feeding, have been shown to influence the structure and function of developing infantile gut microbiome,. In spite of the fact that bacterial colonization of a previously germ-free human gut begins at birth, diet and environmental conditions can influence this ecosystem. A delayed bacterial colonization of the gut with a limited number of bacterial species tends to be virulent, whereas bacterial overgrowth is one of the major factors that promote bacterial translocation,. Moreover, the development of the infant intestinal microbiome in response to dietary and other exposures may shape long-term metabolic and immune functions, as infant feeding practices, particularly breast-feeding and formula-feeding, have been shown to influence the structure and function of the developing infantile microbiome.
Ingestion of Lactobacillus has been found to improve the overall resistance ability of the intestinal mucosa and markedly decrease the chance of translocation. Also, the microbial communities that colonize different regions of the human gut usually influence many aspects of health by contributing nutrients and energy to the host through fermentation of nondigestible dietary components in the large intestine, and a balance is thus maintained with the host's metabolism and immune system [14, 15]. Similarly, presence of Lactobacillus spp. has been found to be able to sustain and control the human intestinal microflora of infants,, and because of these benefits, considerable research is being directed at promoting the growth of lactobacilli in the large intestine.
Early colonization of the infant gastrointestinal tract is crucial for the overall health of infants, and, in addition, establishment and maintenance of nonpathogenic intestinal microbiota may reduce several neonatal inflammatory conditions. Much effort has, therefore, been devoted to the manipulation of the composition of the microbiota through the role of early infant nutrition,. Meanwhile, as reported earlier, information about human flora has mostly been gathered from faeces and many of the validly described gut Lactobacillus spp. have been isolated from human faeces [13,18–20]. The major available data on microbiota composition of neonates have been obtained using faecal samples; however, knowledge on postnatal microbial development is far from complete. The aim of this study, therefore, was to identify the diversity of major Lactobacillus spp. in diet-dependent or diet-specific infantile gut flora that are of clinical importance.
| Participants and Methods|| |
Babies brought for immunization by their mothers were the participants for the current study. Short structured questionnaires on age, sex, tribe and type of feeding were administered on the babies through their mothers. Questions about age, general educational and employment status of the mothers were asked as well. Brief status of the infants in the current study is as described in [Table 1] and [Table 2].
|Table 1: Diversity and frequency of occurrence of Lactobacillus spp. isolated from infantile faecal specimens of female babies|
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|Table 2: Diversity and frequency of occurrence of Lactobacillus spp. isolated from infantile faecal specimens of male babies|
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Healthy and well-nourished neonates and infants between 1.5 and 11 months of age were eligible for inclusion in this study.
- Age more than 1 year.
- Babies presented with diarrhoea and/or vomiting.
- Babies on antimicrobial therapy within 6 months before collection of faecal specimens.
Prior to the commencement of the study, research approval for the current study was obtained from Department of Paediatrics and Institute of Child Health, University College Hospital, University of Ibadan, Ibadan, Oyo State, Nigeria, and informed consent was obtained from mothers for questionnaire administration and collection of freshly egested faecal specimens of babies.
Collection of specimens
Nursing mothers, aged 19–68 years (the 68-year-old was a grandmother), who brought their babies for immunization were briefed on the concept of the investigation, and specific verbal informed consents were obtained from the participating mothers. Freshly egested infantile faecal samples (n = 41) that were microbially analysed in the present study were randomly collected from 18 males and 23 females between the ages of 1.5 and 11 months. All participating infants, except one, were born after 37 gestational weeks with birth weights of more than 2500 g. Infants collection of faecal samples used in the current study spanned between 1 and 5 month(s).
At the commencement of infantile faecal sample collections, the Senior Matron in charge of the Institute of Child Health explained the concepts of the research study to nursing mothers that brought their babies for immunization, followed by detailed information on the benefits of the study by the research scientist.
Sterile cotton swabs were used in collecting freshly egested faeces of the babies during defecation and were immediately transferred into 5 ml sterile de Man, Rogosa and Sharpe (MRS) broth (pH 6.5) in tightly closed sterile McCartney bottles, followed by incubation at 32–35°C for 24 h. One millilitre volume of each overnight broth cultures of infantile faecal samples were then separately cultured on sterile MRS agar plates by the pour-plate method, incubated aerobically and anaerobically in 5% CO2(Gas Pak Anaerobic System; Oxoid) Limited, Basingstoke, Hampshire, UK, 32–35°C for 24–48 h. Faecal samples were then quantitatively analysed by bacterial enumeration based on colony-forming units/g cfu/g of the faecal specimens.
Isolated Lactobacillus strains from infantile faecal samples were subcultured to obtain pure cultures, which were examined microscopically. Initial identification and grouping of the lactobacilli were based on Grams reaction, catalase reaction with hydrogen peroxide, growth at 15 and 45°C in MRS medium, gas and acid production from glucose, and fermentation of lactose, sucrose, arabinose, fructose and mannitol. Isolates that met preliminary identification criteria were grown in replicates overnight (18–24 h) in 10 ml Rogosa broth at 35°C until the weight of the cell mass was 0.05–0.10 g, whereas additional taxonomic studies were carried out according to standard related methods [20–24]. Purified cells were centrifuged, washed twice in sterile 0.9% NaCl solution, and then stored at 4°C in Hogness freezing buffer (3.6 mmol/l K2 HPO4, 1.3 mmol/l KH2 PO4, 2.0 mmol/l Na-citrate, 1.0 mmol/l MgSO4, 12% glycerol) as stock cultures.
| Results|| |
Strong suspicion and peer influence, which cut across age, educational status, religion and tribe, was observed among the nursing mothers during collection of faecal specimens of babies. Personal one-on-one interactions with the nursing mothers yielded only little cooperation with regard to collection of faecal specimens from their babies. Exclusive breast-feeding beyond 3 months was difficult among working-class nursing mothers due to lack of time and adequate cold-storage facilities. More babies of nonworking mothers were exclusively breast-fed beyond 4 months.
Faecal specimens of 41 healthy and well-nourished neonates and infants between 1.5 and 11 months were sampled in this study. Most of the female babies were 8 and 10 months old,, although most of the male babies were 9 and 10 months old. The infants were mostly of Yoruba origin, and all the infants were fed on breast milk and/or industrial baby cereal foods, although more of the male babies (seven) were exclusively breast-fed than female babies (two). None of the infants was fed on solid diet alone [Table 1] and [Table 2] and [Figure 1] and [Figure 2].
The pH of the infantile faecal specimens were between 4.5 and 5.8, and enumerated viable counts between 1.1 × 103 and 7.2 × 103 cfu g -1. No microbial growth was recorded for faecal specimen CH001, and no lactobacilli growths were observed in 16 infantile faecal specimens (CH002, CH003, CH006, CH007, CH008, CH010, CH011, CH014, CH022, CH023, CH029, CH038, CH045, CH046, CH056 and CH057) of participants that were 4–16 days old. One or more different phenotypes of Lactobacillus spp. were isolated from each infant above 16 days old [Table 1] and [Table 2] and [Figure 3],[Figure 4],[Figure 5].
|Figure 3: Frequency of overall occurrence of Lactobacillus species isolated from infantile faecal specimens of female babies.|
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|Figure 4: Frequency of overall occurrence of Lactobacillus species isolated from infantile faecal specimens of male babies.|
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|Figure 5: Comparative frequency of occurrence of Lactobacillus species isolated from infantile faecal specimens of female and male babies.|
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A total of 151 (n = 77 females and n = 74 males) isolated Lactobacillus strains from infantile faecal specimens were characterized in this study as Lactobacillus acidophilus (19.9%), Lactobacillus bifidus (7.28%), Lactobacillus brevis (1.99%), Lactobacillus casei (23.2%), Lactobacillus plantarum (1.32%)and Lactobacillus reuteri (46.4%). L. reuteri, L. casei and L. acidophilus were the overall most-prevalent Lactobacillus species from faecal specimens of female and male subjects, but L. bifidus, L. brevis and L. plantarum were obtained from infantile faecal specimens of healthy infants and children above 5 months of age. L. acidophilus (16.6%), L. casei (13.2%) and L. reuteri (15.89%) were the most-prevalent Lactobacillus spp. isolated from faecal specimens of female babies, whereas L. casei (9.93%) and L. reuteri (30.6%) were the most-prevalent Lactobacillus spp. isolated from faecal specimens of male babies [Table 1] and [Table 2] and [Figure 5]. There were relatively higher recovery rates of Lactobacillus strains from faecal specimens of older healthy infants and children (5–11 months) [Table 1] and [Table 2] and [Figure 1] and [Figure 2].
More of L. acidophilus, L. casei and L. reuteri were recovered from faecal specimens of infants fed on breast milk+baby cereal food (g), whereas L. acidophilus, L. bifidus, L. brevis, L. casei and L. reuteri were prevalent in faecal specimens of infants exclusively fed on breast milk. L. brevis was recovered only from infantile faecal specimens of healthy infants and children above 5 months of age [Table 1] and [Table 2].
Using correlation and regression analyses of one-way analysis of variance, the significance level value (0.024) indicates that there were no significant differences between pH of the infantile faecal specimens, diet and tribes of the babies on the total viable counts of the Lactobacillus spp. isolated from faecal specimens of the babies.
| Discussion|| |
Ethical permit is quite necessary for scientific, bio (medical) research activities prior to research, and is, therefore, a must. Whereas based on the findings of the current study, more than ethical permits would be needed to conduct human infantile-related biomedical research studies in some localities, such as the south-western parts of Nigeria, as there is very strong suspicion and peer influence among nursing mothers about collection of clinical specimens from their children. In spite of formal briefings and sociocultural factors, as soon as the first nursing mother refuses to allow collection of faecal specimen from her baby, other mothers would also claimed that their babies had not defecated (pooh pooh), even, including the nursing mothers cleaning up their defecating babies.
Few mothers who readily allowed their children's faecal specimens to be collected provided the reason for noncooperation of other mothers as being based on fear of fetish (occult/voodoo) practices that may later harm their babies. Each mother was then subsequently personally interacted with after formal briefings, and although more mothers were readily responsive, majority still did not cooperate with regard to collection of faecal specimens from their babies. Thus, these indicates that in certain communities, in spite of ethical approvals, parental (mother's) believes can strongly influence infantile scientific research studies that involve specimens' collections from babies, if it does not directly involve curing ill children.
Breast milk is the main source of nourishment for children in their first months of life,, but complementary foods are usually introduced between 4 and 6 months of age,. In the current study, according to the infants' mothers, none of the infants was fed on solid diet alone, although almost all of them were fed on breast milk and baby cereals (industrial weaning foods), except few who were exclusively fed on breast milk. In spite of series of initiatives and advertisements on breast feeding, including activities of baby-friendly clinics, most of the average Nigerian nursing mothers breast feed their babies without knowing certain major biotherapeutic benefits of breast feeding,. The two common beliefs are that, breast milk is nutritionally beneficial to babies, and breast feeding serves as natural means of contraception, although most of times breast feeding had been found to fail as a standard natural contraceptive measure.
During one-on-one interaction sessions with the nursing mothers, it was confirmed that working-class nursing mothers found it challenging to practice exclusive breast-feeding beyond 3-month maternity leave policy for nursing mothers, which is even only commonly practised in government agencies. This was in addition to the lack of paternity leave for working-class fathers and the lack of adequate cold-storage facility such as refrigeration due to erratic electric supply. Meanwhile, more babies whose mothers were not in the working-class group were exclusively breast-fed well above 4 months of age. More male infants were also exclusively breast-fed compared with female infants. Even, a 7 months (CHF012) and a 10 months (CHF054) old male babies were exclusively breast-fed, which could be due to preference placed on male children in Nigeria or due to more incessant crying request to be fed by male babies. Although recovery of clinically important Lactobacillus spp. from faecal specimens of infants was not tribe dependent, it was, however, age and sex dependent. Considering that all the infants had breast milk inclusive in their diets, which is related to recovery of Lactobacillus spp., breast milk seems to constitute a good functional source for lactobacilli infant gut colonization.
Lactic acid bacteria thrive best at low pH; thereby, providing bacteriostatic or bactericidal potentials, and can, thus, protect infant guts against infectious microbes. Microbially analysed neonatal and children faecal samples in the present study were in the acidic pH (4.5–5.8) range, which is similar to earlier reported infantile faecal specimens' pH, mostly within 4.5 and 5.5. Easier recovery of Lactobacillus spp. between low and high acidic pH in the present study infers their ability to survive passage through the stomach's relatively low pH, small intestine's relatively high pH and also survival in the presence of bile acids, which are all first-line properties screened for when selecting probiotic strains. That no lactobacilli were isolated from faecal samples of breast-fed infants between the ages of 4 and 16 days is, however, in accordance with trends of previous reports, including those of Flint et al., Mitsuoka, Hill et al., where they stated that about Furthermore, the pattern of bacterial colonization in premature neonatal guts is different from those of healthy, full-term infant guts, because establishment of beneficial microflora is quite slow in premature infants. Therefore, absence of growth on culture plates of sample CH001, obtained from a 4-day-old, low-birth weight, female neonate, might be a reflection of low milk intake as well as immaturity of digestive organs of the baby [32–35].
Identification methods for Lactobacillus strains in this study combined cultural, microscopic, biochemical and physiological techniques to establish correlations of characteristics, which were methods that have been previously proved to be highly successful in characterizing lactobacilli from a variety of sources,. Apart from L. acidophilus, L. bifidus, L. brevis, L. casei, L. plantarum and L. reuteri, which were isolated from infantile faecal specimens of healthy infants in the current study, other Lactobacillus spp. that can be isolated from infantile faecal specimens include Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus leichmanii, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus coryniformis, Lactobacillus salivarius, etc.,. Identified Lactobacillus strains in this study were thus similar to those previously isolated from same or related sources,, although observed slight variation and increase in the intestinal Lactobacillus microbiome of older human infantis may be due to differences in human milk and nonhuman milk (solid food) diets fed to the infants. Also, the slight observed interspecies variation in the most-prevalent lactobacilli flora of infantile faecal samples obtained in the current study, and some previous studies, may be accounted for by differences in geographical-based diets and certain lifestyle characteristics, such as those associated with anthroposophy ,,.
Infant feeding practices, particularly breast milk-feeding and formula-feeding, have been shown to influence the structure and function of the developing human infant gut microbiome, as human milk provides infants with a rich microbial consortium and a variety of oligosaccharides, which are prebiotics that ensure gut colonization by microbes that are beneficial for metabolism and immune development,. Since the gut microbiota of breast-fed infants has been characterized as having less diverse colonization, whereas the microbiomes in mixed-fed infants have been assumed to be intermediate between those of breast-fed and formula-fed infants. In the current study, there was no significant difference in diversity of Lactobacillus flora due to tribe and and pH of the infantile faecal specimens. According to Alles et al., poor feeding practices of some pregnant women, infants and young children have been reported to contribute to the burden of malnutrition as well as childhood morbidity and mortality in sub-Saharan Africa. Thus, appropriate nutrition for healthy pre-term and term infants, and young children as well, is a paediatric issue of special importance. Microbiota of the human intestinal tracts, however, play an important role in health, particularly in mediating many of the effects of diet upon gut health. As stated earlier, control and manipulation of bacterial colonization in neonatal guts may be a new approach for prevention and treatment of intestinal infectious diseases of various aetiologies.
Considering that all the infants in the current study had breast milk inclusive in their diets, breast milk seems to constitute a good functional (prebiotic) source for colonization of infant gut by lactobacilli. Potential probiotic Lactobacillus candidates can thus enhance nutritional health status of infants through Nigerian indigenous Lactobacillus fermented foods and beverages, which can serve as fortified animal or plant milk, dairy or carbohydrate food supplements/additives and dietary prebiotics and probiotics combinations (pro-prebiotics) for infant feeding. Such potential pro-prebiotics Lactobacillus strains can also demonstrate other likely beneficial effects on intestinal environment, such as modulation of gut microbiota in clinical paediatric cases, like infantile diarrhoea, malnutrition and even as weaning foods, especially for infants born to mothers living with HIV, instead of the present national policy of breast feeding of babies by mothers living with HIV.
Diet-dependent Lactobacillus spp., which reflected rich indigenous infantile bacterial consortium, were recoverable from faeces of less than 12-month-old breast milk-fed and industrial cereal-fed, healthy Nigerian infants without antibiotherapy. These not only reflected geographic dependent indigenous, infantile gut microbiota but also provided useful information that can assist in selection of indigenous probiotic candidates of paediatric nutrition/health importance.
The author acknowledges Matron Giwa of Institute of Child Health, University College Hospital (UCH), Ibadan, for assistance in obtaining informed consent for collection of infantile faecal samples as well as Professor Nottidge of Department of Paediatrics and Professor Akinkugbe of Institute of Child Health, University College Hospital (UCH), Ibadan, Nigeria, for ethical approval.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]