The relevance of probiotics in Caesarean-born neonates

‘An ounce of prevention is worth a pound of cure’ is an axiom by Benjamin Franklin, one that is relevant especially in the current COVID-19 pandemic. In Australia, rates of delivery by Caesarean-section (CS) have increased and reached 35% in 2017¹. Antibiotics are used regularly for both prophylaxis and treatment of infections in mothers who deliver babies through CS². This excess use is important for its potential role in driving antimicrobial resistance worldwide³ and also has an impact on the establishment of the neonatal gut microbiome.

CS is associated with significant difficulties in initiating breastfeeding when compared with vaginal birth⁴. The microbiome of breast milk contains bacteria, including lactic-acid bacteria (LAB), and is important in establishing the gut microbiome of neonates⁵. Breastfeeding helps to establish healthy gut microbiome. LAB were first described by Pasteur as part of fermentation to prevent spoilage approximately 70 years before the discovery of penicillin in 1928⁶ (Figure 1).

Figure 1.  Timeline of probiotic development in comparison to the antibiotic development.

CS-born infants generally lack LAB, i.e. Lactobacillus spp. and Bifidobacterium spp., which appear important in establishing the neonatal immune system⁷. Recent data support the theory that probiotic administration to CS-born infants may prevent allergy in children and young people⁸. Certain species of Bifidobacterium spp. may only be isolated from human breast milk within a few days after birth⁹. Early intervention through probiotic administration in neonates, especially in neonates born via CS may improve general health, given their susceptibility to various chronic diseases⁷ as well as potential prevention of chronic inflammatory diseases, such as inflammatory bowel disease, rheumatoid arthritis, coeliac disease and diabetes mellitus later in life¹⁰.

Probiotics, in particular Lactobacillus spp. and Bifidobacterium spp., are considered normal flora and part of human gut microbiota⁷. Lactobacillus spp. and Bifidobacterium spp. are considered generally regarded as safe, especially for oral administration¹¹. In international guidelines such as the FAO/WHO guideline, probiotics are recognised as having a role in maintaining gut health and may modulate host immunity¹¹. In this article, the genomes of Lactobacillus spp. and Bifidobacterium spp. are described along with the mechanisms of action of LAB in interfering against pathogenic bacteria.

Very recently the taxonomy within genus Lactobacillus spp. was re-classified into 25 genera¹². As the changes were very recent, and these new genera have not been adopted to the WHO/FAO guideline for probiotics, genus Lactobacillus will be used for this article. It is proposed that genus Lactobacillus of L. casei, L. paracasei and L. rhamnosus as genus Lacticaseibacillus¹². L. salivarius and L. fermentum have been named as Ligilactobacillus salivarius and Limosilactobacillus fermentum, respectively¹². Genus Lactobacillus of L. acidophilus and L. gasseri have not changed¹².

There have been extensive studies of the use of probiotics in neonates including preterm infants^13–16. In particular, these studies have focussed on the role of probiotics in reducing the incidence of necrotising enterocolitis (NEC) and sepsis. Most significantly, a randomised controlled trial of a symbiotic preparation including L. plantarum in 4500 term neonates in the community resulted in a 42% reduction in neonatal sepsis¹⁷.

In addition to its impact on neonatal sepsis, probiotics may reduce gastrointestinal complications in neonates though the evidence is mixed¹⁸. The large Probiotics in Preterm Infants Study (PiPS) Trial randomised 1310 pre-term babies to treatment with Bifidobacterium breve BBG-001 or placebo and showed no reduction in rates of sepsis, NEC or death¹⁶. In contrast, the ProPrems trial, a randomised-controlled trial that included 1099 preterm infants from Australia and New Zealand demonstrated a reduction of NEC of approximately 50%¹⁹. The strains being used in the ProPrems trial were B. infantis, S. thermophilus and B. lactis. A metagenomic approach to characterise the gut microbiota was also used in a sub-study of ProPrems trial, which showed abundance of Bifidobacterium spp. in the infants administered with probiotic¹⁵. In neonates, while considered generally safe, cases of Lactobacillus bacteraemia have been reported including in a <1000 g weight pre-term infant following a laparotomy²⁰.

L. plantarum, L. gasseri and L. salivarius have been isolated from infants’ oral and faecal samples^21–23. Therefore, these three genera are considered normal infants’ microbiota and warrant further research. Thus far, there is no published research study of using L. gasseri and L. salivarius in neonates. Further, these strains are not commercially available for infants or neonates yet. Research is required to include species not typically in the current formulation of probiotics for neonates. As L. plantarum may reduce atopic dermatitis²⁴, and L. gasseri and L. salivarius may have immunomodulatory effects^21,22, these should be considered for inclusion in probiotic formula for neonates. In the potential formulation of the probiotics, Bifidobacterium spp. have been reported as predominant genus in breastmilk microbiome⁷. Therefore, to add another strain to the formula would need to consider the species proportion in the breastmilk, i.e. with lower CFU than Bifidobacterium spp. Of note, L. plantarum, L. gasseri and L. salivarius are in the commercial formula available for adults.

Probiotic administration has also significantly reduced the length of stay in pre-term infants²³. A cost-saving analysis in pre-term infants supplemented with probiotics showed a saving of €2000 per infant²⁵. The clinical impact and cost effectiveness of probiotic administration require further well designed laboratory, clinical and cost analysis research.

Probiotics interfere with pathogens through acid production, hydrogen peroxide production and bacteriocin activity²⁶. Production of bacteriocins, small peptides with anti-bacterial activity of Lactobacillus spp. has been reported to inhibit pathogen growth²⁶. Specific short-chain fatty acids (SCFA) have been studied to understand their beneficial properties, such as butyrate for the antagonistic activity against cancer cells and anti-inflammatory property²⁷. SCFA production by Bifidobacterium spp. in the gastrointestinal tract results in a lower pH and inhibition of potentially pathogenic bacteria²⁸.

Bacterial exopolysaccharide has been known to possess immunostimulatory properties^29,30. Extracellular vesicles (EV) in Gram-negative bacteria have been studied for their pathogenicity, host-pathogen interaction and potential targets in vaccine development³¹. Very recently, studies on EV were performed in probiotic strains and revealed potential delivery of bacteriocins and other beneficial properties through the EV^32,33. Advancing research on EV of probiotics is highly recommended as it will provide further understanding on molecular mechanisms of probiotic bactericidal properties against pathogens and immune modulation. Evidence of Bifidobacterium spp. in boosting immune systems has been demonstrated mostly in the mouse model, marked by the stimulation of IL-6 and IL-10 in the ileal Peyer’s patches and in weaned pig model, marked by the increase of IgA against the parasite and IgG^34,35.

Genome data provides comprehensive data that might also help to determine the beneficial properties and the potential virulence determinants in the strains. Genome data enable the comparison of the strains with publicly available genome data. We limit the discussion of the genomes to the strains being used commercially in humans. The recent genus Lactobacillus name changes have not impacted the species and genomes, as we abbreviate the genera.

L. rhamnosus GG (LGG) has been the most commercially popular probiotic strain. More than 1100 studies on L. rhamnosus GG were found in NCBI (accessed 22 April 2020). L. plantarum 299v has shown beneficial properties such as effectiveness to treat irritable bowel syndrome³⁶; regardless, only 112 studies on L. plantarum 299v versus 204 studies on L. plantarum WCFS1 were in NCBI. Very few studies were on L. salivarius with 39 studies of L. salivarius UCC118 found from NCBI. As previously described, the L. plantarum WCFS1 genome was first sequenced in the early 2000s and has been well described with its genome of 3 308 273 bp (GenBank accession number NC_004567.2) and a total of nearly 1200 identified proteins. The beneficial properties of L. plantarum WCFS1 include the ability of this strain to survive in a wide range of environments with temperature and pH changes³⁷. The parental strain of L. plantarum WCFS1 is L. plantarum NCIMB 8826, which was isolated from human saliva³⁸. L. plantarum NCIMB 8826 colonises the oral cavity well but not the human intestine, although it has been demonstrated to survive in the gastrointestinal tract, including faeces³⁹. L. gasseri ATCC33323 (Accession Number of NC_008530.1) was the complete reference L. gasseri genome in the NCBI database with its genome of 1 894 360 bp. L. gasseri ATCC33323 is an autochthonous microbe in the gastrointestinal system⁴⁰. Therefore, oral application of the L. gasseri ATCC33323 for intestinal colonisation may be well tolerated. For a comprehensive genome description of L. salivarius UCC118, the reference strain being used here is available through the study by Claesson and colleagues⁴¹. The size of the chromosomal genome of L. salivarius UCC118 was 1 827 111 bp (GenBank accession number: NC_007929.1). General probiotic properties of L. salivarius were the ability to eliminate pathogens and the adaptation to the gastrointestinal niche⁴². L. salivarius UCC118 has broad spectrum activity versus Gram-positive bacteria⁴³. Therefore, L. salivarius UCC118 has very strong probiotic properties and is autochthonous to the gastrointestinal tract.

Genomes of Bifidobacterium spp. have also been described, i.e. B. longum (n = 349), B. breve (n = 109), B. bifidum (n = 104) and B. animalis (n = 83) (from NCBI, accessed 2 March 2020). B. longum NCC2705, B. breve DSM 20213, B. bifidum PRL2010 and B. animalis subsp. lactis DSM 10140 are the reference genomes in NCBI with genome sizes of 2.257, 2.257, 2.215 and 1.938 Mb, respectively (GenBank Accession Numbers: NC_004307.2, NZ_JDUD000000000.1, NC_014638.1 and CP001606.1, respectively).

Probiotic supplementation in neonates has been frequently studied. In an era of interventional birth leading to high rates of CS, probiotics may have a role in establishing a healthy gut microbiome. The impact of probiotics in this setting may include a reduction in important acute complications such as neonatal sepsis, and NEC and longer-term impacts relating to the development of mucosal immunity and atopy. The heterogeneity of trial results may relate to the differing strains used. Genomic and metagenomics approaches to analysing the gut microbiome may improve understanding of gut dysbiosis and its role in these complications.

L. rhamnosus, L. casei, L. acidophilus, L. plantarum, L. gasseri and L. salivarius are listed in the three main regulatory bodies in European Food Safety Authority (EFSA), Canada and China as strains can be added in food⁴⁴, which may broaden the use of these strains as human food supplement in countries outside Europe, e.g. China and Canada. Europe has been the epicentre for probiotic development and generation so far. EFSA allowed 37 different Lactobacillus spp. for consumption through food⁴⁴. Therefore, supplementation of Lactobacillus spp. and Bifidobacterium spp. to infants and neonates can be categorised as natural administration of beneficial microbes or probiotics to maintain gut microbiota and immune systems⁴⁵.

Infloran containing Bifidobacterium bifidum and Lactobacillus acidophilus is a commercial probiotic widely used in neonatal units in Australia. Other probiotics available in pharmacies are listed in Table 1. Many commercial preparations are not included in the table due to a lack of published data on the strain identity and CFU counts. Guidelines in choosing the right probiotics are available from International Scientific Association for Probiotics and Prebiotics website (https://isappscience.org/). Industry-related probiotic information can be found from the International Probiotic Association website (http://internationalprobiotics.org/). As probiotic administration is now becoming broader than oral administration, the use of the food-medicine interface guidance tool within Therapeutic Goods Australia (https://www.tga.gov.au/) is highly recommended in translating probiotic research to industry.

Table 1.  Commercially available probiotics for infants including neonates.

In summary, with the increasing evidence of CS births in Australia and worldwide, and antibiotic prophylaxis administration in CS births, as well as the potential delay of the breastfeeding initiation, it would be highly recommended to provide probiotics those commonly isolated from breastmilk, to CS born neonates. Probiotic administration mimicking the LAB of breastmilk will be likely a better option than inoculation of swabs originated from vagina, often called seeding. Future studies that include microbiome analysis, neurocognitive development as well as economic analysis of probiotic administrations are highly recommended.

The authors declare no conflicts of interest.