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Analysis of the gut microbiota using MALDI-TOF and cultural techniques in breastfed infants delivered vaginally and through caesarean section
*Corresponding author: Sanjeev B. Rai, Chief of Research, Father Muller Research Center, Mangaluru, Karnataka, India. raibs11@gmail.com
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Received: ,
Accepted: ,
How to cite this article: Bagambila S, Antony B, Dsouza JG, Devadiga S, Praseena K, Rai SB. Analysis of the gut microbiota using MALDI-TOF and cultural techniques in breastfed infants delivered vaginally and through caesarean section. Karnataka Paediatr J 2023;38:117-22. doi: 10.25259/KPJ_43_2023
Abstract
Objectives:
The gastrointestinal tract of newborns is colonised immediately after birth with microbes from the environment, mainly from the mother. According to studies, the early neonatal microbiota is crucial for developing the postnatal immune system. The objective of this study was to investigate the relationship between neonatal gut flora and the mode of delivery during gut microbiota colonisation.
Material and Methods:
A total of 30 neonates – 16 born vaginally and 14 by caesarean section – participated in this study of the intestinal bacterial composition at 3 days of age. Stool cultures and matrix-assisted laser desorption ionisation time-of-flight analyses determine aerobic and anaerobic bacterial species.
Results:
Neonates delivered by caesarean section appeared to have a less diverse gut microbiota regarding bacterial species than vaginally delivered neonates. Bifidobacterium species are absent from the gut microbiota after caesarean delivery. Even though every newborn vaginally born had a unique microbial profile, the most prevalent bacterial species were Streptococcus spp., Veillonella atypica, Bacteroides vulgatus and Bifidobacterium spp.
Conclusion:
Our results suggest that the mode of birth significantly influences the gut microbiota composition in the 1st year of human life. This study opens the path to further investigations to confirm the link between microbiota composition and enterotypes of the gut microbiome of breastfed neonates. In addition, we underline the importance of MALDI-TOF for species-level identification of organisms within a fraction of a second.
Keywords
Neonates
Vaginal delivery
Caesarean delivery
Gut microbiota
INTRODUCTION
The largest microbiota in the human body is found in the gastrointestinal tract (GIT), with approximately 1013–1014 microorganisms. The foetal intestine is sterile and surrounded by amniotic fluid. The gut microbiome is essential for human health and disease.[1] In contrast to the adult gut microbiome, premature infants have a relatively dynamic and unstable microbiome.[1,2] As a source of nutrient influx, GIT is an ideal environment for microbial colonisation.[3] The diversity of the intestinal microbiome could significantly affect human body functions.[4]
Exclusive breastfeeding is essential throughout the first 6 months of life, according to the World Health Organisation (WHO), which makes this claim in its global strategy. The WHO recommends early breastfeeding initiation within the 1st hours after birth.[5] Researchers using neonatal stool culture in the late 1950s were able to study the development of the gut microbiota after birth.[6] Several studies have isolated probiotic bacteria from human breast milk.[7] Infant formula lacks the diverse bacterial communities found in human breast milk.[8] Breastfed infants have a gut microbiota composed predominantly of Bifidobacteria, whereas formula-fed infants have a more heterogeneous composition with a lower proportion of Bifidobacteria.[9] Traditionally, Lactobacillus and Bifidobacterium species are most commonly used as probiotics in humans and are present in breast milk.[10]
Breastfeeding reduces the risk of ear infections, respiratory infections, diarrhoea and sudden infant death syndrome in newborns. Lower risks of diabetes, food allergies and asthma are other benefits.[11] Colonisation of the neonatal microbiota begins in utero.[12] The delivery method and the end of breastfeeding are essential for developing a gut microbiota similar to that of an adult. During the 1st year of life, the microbial composition changes abruptly. In younger and older adults, the gut microbiota changes gradually over time.[12] The gut microbiota influences the growth and differentiation of intestinal epithelial cells and plays a critical role in nutrition, metabolism, immunology and protection.[2]
Using matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry, it is possible to characterise and identify microorganisms quickly, precisely and affordably. The current study, focusing on culture and MALDI-TOF analyses, investigates the effects of the mode of delivery on the gut microbiota of breastfed infants. During vaginal delivery, the newborn is exposed to various maternal microorganisms, some of which colonise the infant’s gut. Caesarean sections prevent vertical transmission of intestinal microbes by not exposing the newborn to the mother’s microbes.[13]
Healthy individuals can be classified by the differential enrichment of microbial taxa in their gut. These enriched taxa, termed enterotypes, are independent of age, sex, body mass index and region.[14] For Firmicutes/Bacteroidetes and Proteobacteria/Actinobacteria, the ratio of taxa abundance at the strain level was calculated using the relative abundances of taxa at the country level.[14]
The enterotypes ‘P-type’, ‘A-type’, ‘F-type’ and ‘B-type’, which are relatively abundant in Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes, respectively, were found in the overall comparison. Regional variations exist in the makeup of the four enterotypes.[14,15]
There is compelling evidence that the early neonatal microbiota’s composition has a significant impact on postnatal immune development. Immediately after birth, microorganisms from the environment, primarily the mother, colonise the neonatal GIT.
The present study investigated the relationship between the neonatal gut microbiota and delivery technique. 16 infants born vaginally and 14 by caesarean section participated in studying intestinal bacterial composition on the 3rd day of life. Aerobic and anaerobic bacteria were differentiated using species-level MALDI-TOF analyses and anaerobic and aerobic cultures. By identifying and assessing the gut microflora of infants, researchers can determine how maternal-infant contact, feeding style and birth affect early microbiota development. This will allow a better understanding of the relationship between the gut microbiota and the type of feeding and delivery. Further analysis will be possible if probiotics and prebiotics influence microbiota development and treat or avert disease.
MATERIAL AND METHODS
A prospective study was conducted in the microbiology research centre laboratory. Samples were collected from hospitalised newborns at Father Muller Medical College Hospital in Kankanady, Mangalore, India. Medical records of infants admitted to the neonatal intensive care unit, paediatric intensive care unit and paediatric wards were reviewed.
The Ethics Committee of FMIEC, Mangalore, approved the study (Reg. No. FMIEC/CCM/755/2022), and the participants were recruited after obtaining written informed consent from the mothers.
The participants were newborns admitted to the neonatal unit of a hospital in Mangalore, India. Newborns who were exclusively breastfed and delivered both vaginally and by caesarean section were included in the study. Critically ill patients, mixed-breastfed infants, neonates who died or were discharged within 24 hours and neonates who received prebiotics and probiotics were excluded.
Data collection and analysis
A structured data collection form was used between December 2022 and February 2023 to collect information from hospitalised neonates and their mothers on their demographic data, clinical and laboratory data, antibiotic treatment and discharge records. For calculating the ratio of taxa abundance at the phylum level (Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria), the country-level relative abundances of the taxa were used.
Sample collection
A fresh stool sample was collected in a sterile, clean container, taken to the laboratory without delay and processed for aerobes and anaerobes.
Sample preparations
The media used to process aerobes were Blood agar (BA) and MacConkey agar (MAC). Samples were inoculated at BA and MAC and then incubated at 37°C for 24–48 h. Isolates were subjected to MALDI-TOF analysis.
For anaerobes, the faeces were inoculated onto anaerobic media such as Robertson’s Cooked Meat Medium (RCM), Laked BA, Neomycin BA and Bacteroides Bile Esculin agar plates. A metronidazole disc (5 μg) was placed in the plates for the presumptive identification of anaerobes and incubated in an anaerobic jar (BD GasPak jar EZ). An anaerobe pack was used for achieving anaerobiosis, which is a paper sachet, preserved in a foil bag that absorbs O2 and generates carbon dioxide, immediately in contact with air. The jar was incubated for 48–72 h at 37°C. The jar was opened after incubation, and the colonies that showed a definite zone of inhibition to metronidazole were presumptively identified as anaerobes. The Anaerobic Laboratory Manuals were used to further characterise the isolates based on aerotolerance, Gram stain, colony morphology and biochemical reactions.[16-18] As mentioned, RCM, a supplemental medium, was incubated for 24 hours and subcultured on the anaerobic media. Identification was carried out using MALDI-TOF analysis at the species and genus levels.
MALDI-TOF extended direct transfer method
After colonies appear on the plate, MALDI-TOF will be performed for species-level identification. An isolated colony was smeared as a thin film directly onto an empty position on the MALDI target plate, overlaid the material with 1.0 μL of 70% formic acid and dried at room temperature within 30 min. After drying at room temperature, the smear was overlayed with 1 µL of matrix solution (α- Cyano-4-hydroxycinnamic acid - HCCA) and dried at room temperature,[18-21] and the sample plate was put into the MALDI-TOF system, the patient’s demographic information was entered and the acquisition process began.
Statistical analysis
Statistical data were analysed using a Microsoft Excel spreadsheet. The Chi-square tests and Fisher tests were also performed using the Statistical Package For The Social Sciences (SPSS). Data were collected using a self-designed form that included information on feeding habits, feeding type, frequency, demographics, anthropometric status, diagnosis, drug therapy and other relevant information.
RESULTS
Infants delivered vaginally have a more diversified gut flora than neonates delivered by caesarean. A lack of Bifidobacterium species in the gut microbiota following caesarean delivery is remarkable. Even though they displayed individual microbial profiles, vaginally delivered neonates were characterised by prominent species such as Streptococcus spp., Veillonella atypica, Bacteroides vulgatus, Bifidobacterium spp. and Escherichia coli. The caesarean-delivered neonates show predominant group organisms such as Streptococcus spp. and Clostridium spp., Pseudomonas aeruginosa and Klebsiella pneumoniae. The intestinal microorganisms of breastfed infants born vaginally and by caesarean section are compared in [Table 1]. Significant differences were observed in Streptococcus spp., Enterococcus spp. and Clostridium spp., but no significant difference in Staphylococcus spp., Gram-negative bacilli, Bifidobacterium spp., B. vulgatus and V. atypica.
| Categories | Organisms | Vaginal delivery (n=16) | Caesarean delivery (n=14) | P-value |
|---|---|---|---|---|
| Gram-positive cocci | Streptococcus spp. (parasanguinis, anginosus and salivarius) | 4 | 0 | 0.04 |
| Staphylococcus spp. (aureus, haemolyticus, epidermidis, hominis and caprae) | 7 | 6 | 0.96 | |
| Enterococcus spp. (faecium and faecalis) | 0 | 7 | 0.002 | |
| Gram-negative bacilli | Escherichia coli | 9 | 5 | 0.26 |
| Klebsiella pneumoniae | 6 | 7 | 0.49 | |
| Pseudomonas aeruginosa | 1 | 2 | 0.46 | |
| Anaerobes | Bifidobacterium spp. | 3 | 0 | 0.08 |
| Veillonella atypica | 1 | 0 | 0.34 | |
| Clostridium spp. | 0 | 3 | 0.05 | |
| Bacteroides vulgatus | 1 | 0 | 0.34 |
Bold values represent statistically significant values.
The gut enterotypes from vaginally and caesarean-born, breastfed infants are shown in [Table 2]. Our study revealed that infants in India predominantly belong to P-type and F-type enterotypes. There is no significant difference between the intestinal enterotypes of vaginally and caesarean-born neonates.
| Groups | Organisms | Mode of delivery | |||
|---|---|---|---|---|---|
| Vaginal delivery | Enterotypes | Caesarean delivery | Enterotypes | ||
| Firmicutes | Streptococcus spp. (parasanguinis, anginosus and salivarius) | 4 | FFFF | 0 | - |
| Firmicutes |
Staphylococcus spp. (aureus, caprae, hominis haemolyticus and epidermidis) |
7 | FFFFFFF | 6 | FFFFFF |
| Firmicutes |
Enterococcus spp. (faecium and faecalis) |
0 | - | 7 | FFFFFFF |
| Proteobacteria | Escherichia coli | 9 | PPPPPPPPP | 5 | PPPPP |
| Proteobacteria | Klebsiella pneumoniae | 6 | PPPPPP | 7 | PPPPPPP |
| Proteobacteria | Pseudomonas aeruginosa | 1 | P | 2 | PP |
| Actinobacteria | Bifidobacterium spp. | 3 | AAA | 0 | - |
| Firmicutes | Veillonella atypica | 1 | F | 0 | - |
| Firmicutes | Clostridium spp. | 0 | - | 3 | FFF |
| Bacteroidetes | Bacteroides vulgatus | 1 | B | 0 | - |
F: Firmicutes, B: Bacteroidetes, P: Proteobacteria, A: Actinobacteria, Bold values represent significant enterotypes and highest culture positive results. Numeric values indicate the difference in colonisation in vaginally born and caesarean born babies. Bifidobacterium spp., Streptococcus spp., primarily colonised in vaginally born neonates but not in delivered by caesarean section.
However, Bifidobacterium spp., Streptococcus spp., Staphylococcus spp., E. coli and Klebsiella species were primarily colonised in vaginally born neonates. E. coli, Enterococcus spp., K. pneumoniae, Staphylococcus spp. and Clostridium spp. were the most prevalent bacteria found in the colons of neonates delivered by caesarean section.
Table 3 shows the characteristics of the study population by birth type and maternal demographics. Compared to female infants (42.8%), most male infants (56.2%) were delivered vaginally. The mean value of the mother undergone vaginal delivery in the study population was 27.6 and caesarean-delivered mothers 26.
| Variables | Vaginal delivery | Caesarean delivery |
|---|---|---|
| Gender, n (%) | ||
| Female | 7 (43.75) | 8 (57.14) |
| Male | 9 (56.25) | 6 (42.86) |
| Mother’s age (Mean±SD) | 27.6±4.71 | 26±4.47 |
| Gestational age/week (Mean±SD) | 38.2±2.04 | 37.5±1.84 |
| Baby’s birth weight (Mean±SD) | 3026±543 | 2703±736 |
| Primigravida, n (%) | 7 (43.75) | 6 (42.86) |
| Multigravida, n (%) | 9 (56.25) | 8 (57.14) |
| Mothers haemoglobin (Before delivery) (Mean±SD) |
11.36±0.72 | 11.55±0.90 |
| Aerobes, n (%) | 15/16 (93.75) | 14/14 (100) |
| Anaerobes, n (%) | 4/16 (25) | 3/14 (21.43) |
| No growth, n (%) | 1/16 (6.25) | 0/14 (00) |
| Single isolates, n (%) | 4/16 (25) | 3/14 (21.43) |
| Mixed population, n (%) | 11/16 (68.75) | 11/14 (78.57) |
SD: Standard deviation
DISCUSSION
Mode of delivery and postnatal diet are two of the most significant factors affecting the gut microbiota, according to Adamek et al.’s study on the development of the gut microbiota during pregnancy and the early childhood years.[4] The composition of the gut flora is influenced by pre- and probiotic supplementation, antibiotic therapy, gestational age at birth and other environmental factors. The complexity of the flora ensures that its composition is unique to each individual.[4]
The gut microbiome influences immunologic, endocrine and neuronal pathways and plays a vital role in child development. Several factors influence the colonisation of the infant gut microbiome. Different microbial colonisation patterns are related to vaginal and caesarean delivery, antibiotic exposure and infant feeding habits. Infant microbial colonisation patterns can potentially affect long-term physical and neurocognitive development and disease risk due to the broad physiological impact.
Understanding these influences will impact neonatal care and parenting. Long-term gut microbiota dysbiosis can have long-lasting functional effects and lead to various diseases. Changes in the microbiota significantly increase the risk of obesity, diabetes, asthma and food allergies. Infants born vaginally had higher concentrations of Bacteroides, Bifidobacteria and Lactobacillus in the first few days of life and showed more significant microbial variability in subsequent weeks. Staphylococcus, Streptococcus and Clostridium predominate in the less diverse and similar microbiome of infants born through caesarean section to that of the mother’s skin and hospital environment.
Newborns receive a wider variety of colonising microorganisms that help build and prime the immune system from the maternal vaginal microbiota. Vaginal birth is the ideal birth route, and a caesarean section should be performed if there are medical indications. In our study, we compared the faecal microbiota of 30 breastfed infants depending on the mode of delivery. Enterococcus faecium, Enterococcus faecalis and Clostridium spp. were higher in infants born through caesarean section. Infants born vaginally had higher levels of Streptococcus parasanguinis, Streptococcus anginosus, Streptococcus salivarius and Bifidobacterium bifidum.
Our results suggest that the delivery mode strongly influences newborns’ gut bacteria during the first 3 days of life. Infants delivered vaginally and by caesarean section appear to have different gut flora.
According to Layuk et al.,[5] breast milk contains various prebiotics passed from mother to child through breastfeeding. The most prevalent prebiotics vary depending on the type of breast milk and include different amounts of Lactobacillus spp., Bifidobacterium spp. and Staphylococcus spp.[5] Bifidobacterium spp. is also a component of the breast milk microbiota but was found only in the faecal microbiota of vaginally delivered infants in the present study. Human milk, in addition to the birth canal, is a significant source of Bifidobacterium and contributes to the establishment of this genus in the gut microbiota of infants.
By global comparison, mainly four different enterotypes, referred to as ‘P-type’, ‘A-type’, ‘F-type’ and ‘B-type’, which were relatively abundant in Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes, respectively. The compositions of the four enterotypes differ geographically. Some studies have reported that there were no significant differences in the faecal relative abundance of Bifidobacterium between caesarean-delivered and vaginally delivered infants; however, the prevalence of the species Bifidobacterium longum was lower in caesarean. Akkermansia and Kluyvera were more abundant in the faeces of caesarean newborns, whereas Bacteroides was similarly less abundant.[20-24] However, in our study, we noticed a significant difference in the abundance of Bifidobacterium between caesarean-delivered and vaginally delivered infants. All infants delivered by both routes had E. coli, K. pneumonia and Staphylococcus spp. in their faeces.
A study by Shao et al. reported the disrupted transmission of maternal Bacteroides strains and high-level colonisation by opportunistic pathogens associated with the hospital environment, including Enterococcus, Enterobacter and Klebsiella species in babies delivered by caesarean section.[24] In our study, beneficial organisms such as Bifidobacterium, B. vulgatus, Streptococcus and Veillonella species are found in infants delivered by the vaginal route, and pathogenic bacteria such as Klebsiella, Enterococcus and Clostridium species were colonised in babies delivered by caesarean section.
CONCLUSION
In summary, these studies show that delivery technique significantly affects the gut microbiota composition during the neonatal period, which may affect the gut biota of early childhood. The beneficial effects of the gut microbiota have long been known; it also plays an essential role in immunity against pathogenic microbes. Our findings indicate that the manner of delivery significantly impacts the gut microbiota’s composition during the neonatal period, which may impact the gut microbiota of young children. In addition, our findings demonstrate that probiotic producers such as Bifidobacterium spp. and Veillonella spp. are more prevalent in the gut microbiota of infants born vaginally than in the gut microbiota of infants born through caesarean section. Healthcare providers could use this information to develop strategies to promote healthy gut microbiota in newborns, such as promoting vaginal birth and breastfeeding.
The study could be improved by including a larger sample and a more diverse population to provide more comprehensive conclusions about the association between mode of delivery, breastfeeding and gut microbiota.
Ethical approval
Ethics approval Reg No: FMIEC/CCM/755/2022.
Declaration of patient consent
Patient’s consent not required as the patients’ identity is not disclosed or compromised.
Conflicts of interest
Sanjeev B. Rai is the member of the editorial board of the journal.
Use of artificial intelligence (AI)-assisted technology for manuscript preparation
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript, and no images were manipulated using AI.
Financial support and sponsorship
FMRC Grant.
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