The role of Lactic Acid Bacteria Microbiota in Determining the pH Value of Newborn Faeces

The low pH is considered a protective mechanism of the body against infections by providing unfavourable conditions for their causers. Analysis of fecal pH of newborns have been performed around the world during the last century, and a disturbing tendency has been surveilled due to industrialization and lifestyle change of mothers and their babies. Considering the lack of information of that kind in Bulgaria, we aimed at analysing the pH values of solely breastfed and mix-fed infants, and its relation to the quantity of the found lactic acid bacteria, their taxa and respective metabolic characteristics. A total of fifteen faecal samples were collected from neonates aged 0 to 6 months. The pH values and presence of the Lactobacillaceae family, along with significant representatives of the genus Bifidobacterium were measured and evaluated by using multiplex semi-quantitative PCR. The gathered information about infants’ supplementation intake and feeding was considered. The solely breastfed neonates showed the lowest pH values, and the highest bifidobacterial content in comparison to the mixed-fed. The average pH value is around 5.1, which we interpret as relatively low, compared to the data in the literature. No relation was observed between the probiotic supplementation and the respective stool samples. These results suggest that breastfeeding plays the main role in manifesting gut health, inducing the growth of beneficial bacteria and immunity development. More participants and additional analyses are needed.


1.
Introduction Neonate gut microbiome formation is a complicated process, depending on too many diverse factors, which influences the gastrointestinal health later in life.Therefore, the interest in infant gut colonization has increased tremendously during the last century.The history of infant gut microbiota studies starts with Henry Tissier [1], the first to remark the presence of Gram-positive Y-shaped bacteria, later known as bifidobacteria in infant faecal samples, continues with Logan [2], who in 1913 described them as dominant in infant stool and along with others underline the key role of these actinobacteria in the overall neonate well-being.Common surveillances describe the infant gut throughout the last century.However, analogous results are nowadays rarely reported after the rise of industrialization and the tremendous lifestyle change, and are only distinctive to concrete rural areas.
As being considered a significant symbiont of the neonate gut, genus Bifidobacterium contains species known for their ability to ferment the human milk oligosaccharides (HMOs), secrete acidic metabolic products and lead to decrease in faecal pH values.HMOs are indigestible for the infant host, which leads to their excretion in the stool if not metabolized.Major protective function of human HMOs is their ability to bind to infection-inducing toxins, viruses and bacteria, and restricting their 1305 (2024) 012015 IOP Publishing doi:10.1088/1755-1315/1305/1/012015 2 access to the gut mucus.The profuse structural range of secreted HMOs suggests additional defensive implications.Equally important is their assimilation by certain bacterial species, reportedly Bacteroides spp.and Bifidobacterium spp.[2].However, only bifidobacteria produce acidic endproducts of HMOs consummation, and a specific species, Bifidobacterium longum subsp.infantis, (B.infantis) evidently has the ability to utilize all known structures of HMOs.Consequently, their presence benefits the bifidobacterial population and attenuates the growth of other bacteria, including pathogenic and potentially pathogenic [3].Another infection-preventive strategy, characteristic of lactic acid bacteria is the decline in pH that result from their dominance.In fact, the more dominating the bifidobacterial representatives in the gut ecosystem are, the more decreased the pH appears to be [4].The presence of these bacteria is extensively linked to birth mode and supplementation.Newborns delivered by Caesarean Section (CS) are claimed to be inoculated with skin-related microbiota and lack the beneficial lactic acid microbiota characteristic for the vaginally born [5].Supplementation of any kind also alters the presence of crucial members of LAB.
The purpose of the current study was to investigate the relation between the pH of faecal samples collected from infants at the age of under six months, and the presence of specific lactic acid bacteria.A comparison of LAB quantity between neonates born vaginally and by CS was made.Any kind of probiotic supplementation and formula feeding was taken into account when evaluating the results.Faecal microbiota is a complicated analysis material, owing to the compounds found in faecal samples, for instance, bile salts, but at the same time extremely microbiologically abundant.The conventional approaches to studying the faecal microbiota includes bacterial culturing, a process highly predisposed to incorrect end results, considering the existence of non-cultivable bacteria, along with the need of special sample handling that would assure the viability of microorganisms.Presently, the most appropriate method for an accurate, fast and informative analysis of microbiota is the real-time PCR.It uses species-specific and group-specific primers, necessary for the amplification of genomic regions that belong to the target microorganism.The obtained results are transformed by the software into information about the presence of the aimed species or group based on the number of DNA copies produced during amplification.Some variations in the PCR methodology allow combination of identification of a number of taxa, using the multi-tube and multiplex assay.

Samples
Fecal samples were collected from and donated by 15 healthy Bulgarian infants which did not take antibacterial medications at least 3 weeks before sample collection as mothers and their newborns.Main characteristics of the participants are shown in table 1.
Therefore, the timing of collection is an important factor to take into consideration when interpretating the results.In addition, subjects with a history of antibiotic use or probiotic intake were noticed in this study also.Written informed consent was obtained from all mothers according to the institutional guidelines, and the protocol was approved by the Ethics commission of Sofia University "St.Kliment Ohridski.Test kits containing a sterile plastic tube (Zymo Research, USA) with a screw cap to collect the stool samples were provided to the participants.The samples were collected by the participants themselves and immediately stored in home freezers until delivery to the experimental laboratory.In the lab the samples were stored at -80 o C until DNA was extracted.

DNA Extraction from Collected Fecal Samples
Approximately 200 mg of each stool sample was used for DNA extraction.Fecal DNA was subsequently extracted using commercial extraction kits PREP-NA-PLUS ("DNA-Technology", LLC) according to the manufacturer's instructions.The concentration and quality of the extracted genomic DNA was determined by spectrophotometric analysis (NanoDrop One c , Thermo Fisher Scientific, USA).All extracted DNA samples were stored at -20 o C until use in further experiments.

Performing the RT-PCR
All samples were analyzed using Enteroflor Kiddy real-time PCR reagent kits (DNA-Technology TS, Russia) and the instrument DT-96 PCR detection system (R&P DNA Technology, Russia).The kit was used for evaluation of the microbiota of the specific fecal microbiota by RT-PCR.The method has been certified and is successfully in routine use for the last years.

Interpretation of Test Results
Results of PCR analysis are analyzed and interpreted automatically by the software designed for Real time PCR thermocyclers produced by DNA-Technology, LLC.Calculation of the total bacterial load (TBL) of the fecal microbiota, as well as absolute quantities of microorganisms in genome equivalents (GE), are carried out by the software based on a mathematical formula that takes into account the number of the threshold cycle during RT-PCR.The software calculates the relative amounts of a microorganism or a group of microorganisms as the difference between the absolute values of Lg10 of the microorganism/group of microorganisms and the TBL.This study was performed in accordance with the Nurenberg Code.This human study was approved by Ethics commission of Sofia University -approval: № 93-И-8≠1/24.01.2023.All parents, guardians or next of kin provided written informed consent for the minors to participate in this study.All adult participants provided written informed consent to participate in this study.

Lactic acid bacteria content depends on the birth mode
The majority of infants participating in this research were delivered via Caesarean Section (CS group) (table 2, figure 1, figure 2), and six of them were born vaginally (VD).The total amount of Bifidobacteria was higher in naturally-delivered compared to CS-born, in the range of 8.9 GE/g to 9.8 GE/g, while the CS group showed the lowest amount at 6.9 GE/g and the highest at 9.5 GE/g.The average Bifidobacterial content for the VD group was 9.26 GE/g, and for Caesarean infants, it was 8.72 GE/g.The proportion of metabolically active bifidobacteria showed results unrelated to the birth mode, with three samples lower than 10%, two of which belonging to the group of vaginally delivered neonates, and one to the CS group.The diversity score of metabolically active "child" bifidobacteria, presented in PCS, ranged from 2 to 4, VD group had a midpoint of 2.83, while the CS group had one of 3.22, which suggests a slightly higher diversity of bifidobacteria in Caesarean neonates.Almost all of VD group had a 100% proportion of metabolically active "child" bifidobacteria, with one exception at sample 12 that exhibited a significantly lower proportion of 7.1%.Four out of the nine CS samples showed a 100% proportion of metabolically active "child" bifidobacteria, while the other five had from 71.6% to 99.90%.
The total Bifidobacteria content is divided into two groups of 1) Metabolically active "infant" bifidobacteria (bifidobacteria common and of metabolical importance to newborn) and 2) Metabolically active "adult" bifidobacteria (typical for and of metabolical significance to adults), represented in Lg (GE/g faeces), depending on the host age they belong to.Unsurprisingly, the "infant" bifidobacteria were widely distributed among the samples, but at the same time, B. infantis 4 being equally non-present in around half of each group of neonates.B. longum subsp.longum (B.longum) and B. bifidum were found in all samples, with one or two exclusions, and B. breve was the third relatively well-represented bifidobacteria, with no relation between any on the results and mode of birth.In like manner, although sporadically occurring among samples, the metabolically active "adult" bifidobacteria presence was unrelated to the mode of birth.The lactic acid bacteria from the Firmicutes philum recognized as beneficial gut inhabitants, belong to the Lactobacillaceae family.The CS group exhibited a larger population of lactobacilli, with the average of 4.68 GE/g, whereas the VD group showed 2.95 GE/g, even though the vaginal delivery is extensively related to transfer of Lactobacillaceae representatives from the mother's vaginal microbiota to the infant's microbiome.Notwithstanding, all of the analyzed samples belonged to infants at the age of 3 weeks to 6 months, a major factor regarding the microbiota transferring during birth.Many types of modifications, following delivery might have potentially occurred, and therefore altered the primordial gut colonization.

Lactic acid bacteria content depends on the type of feeding and supplementation
Breastfeeding is considered the golden standard for neonate feeding for at least the first six months of life.Twelve out of fifteen participants in the current study were solely breastfed, whereas the other three were mix-fed.Interestingly, the sample of the only 4-month-old that appears to be formula-fed at each feeding shows the lowest total amount of bifidobacteria at 6.9 GE/g, significantly lower than the results of the other counterparts.The proportion of metabolically active "infant" bifidobacteria, although not the lowest result, showed a relatively decreased values, which confirms the irreplaceability of breast milk.On the contrary, one of the other two probes of mix-fed new-borns showcased the highest bifidobacteria quantity, proportion and diversity among samples, which suggests a more profound consideration of the used formula in these cases.All of the probiotic-supplemented samples, three in total, had been prescribed the same probiotic formula, containing Limosilactobacillus reuteri, and even though the amount of Lactobacillaceae representatives is supposed to be increased, in fact, samples of non-supplemented neonates showed more elevated genome copies of lactobacilli, for instance, samples 1, 7 and 10.However, the VD group lacked total presence of Lactobacillaceae, except for the ones that had been supplemented with the mentioned above formula.

Faecal pH is influenced by the specific lactic acid bacteria taxa
The results for faecal pH values were in the range of 4.69 to 6.50, with average pH of 5.10.The two lowest pH values were observed at samples 15 and 9 that were also characterized by the highest amounts of total bifidobacterial content, and some of the highest metabolically active "infant" bifidobacteria.B. infantis is presented in only seven out of fifteen samples, and is at its largest quantities in samples 10, 15, and 2. The same probes were three of the samples with the most acidic faecal pH.Moreover, the elevated diversity of metabolically active "child" bifidobacteria that includes the presence of B. longum, B. bifidum and B. breve was surveilled in the samples with the most decreased pH.Conversely, the exclusively increased pH of 6.50 at sample 6 showed the exceptionally low total amount of bifidobacteria at 6.9 GE/g, and metabolically active "infant" bifidobacteria at 5.7.The rest of the probes showed faecal pH values between 4.85 and 5.4 and bifidobacteria presence around the average amounts, with the exception of sample 12.Where the amounts of metabolically active "adult" bifidobacteria surpass the "infant" bifidobacteria, and totally lack LAB representatives of philum Firmicutes, which indicates a possible dysbiosis.No relation between Lactobacillaceae amounts and pH values was detected in the tested samples.More research is required to confirm the statements.

Lactic acid bacteria content depends on the birth mode
Birth mode is thought to affect the neonate intestinal microbiota by transferring bacteria dominating the mother's skin or the vaginal ecosystems in Caesarian Section and vaginal delivery, respectively.Dominguez-Bello et al., [5] research the influence of delivery mode on the new-borns' intestinal habitats and suggest that naturally born infants acquire microbiota of their mothers' vaginal microbiome, which manifests individuality, but nonetheless is broadly represented by the Lactobacillaceae family.The importance of the dominance of these LAB, not only plays pivotal role in the woman's vaginal health [6], but by being introduced to the neonate's gut early-on provides protection against pathogenic bacteria.On the contrary, the succession of skin-related microbiome in the CS-delivered new-borns leads to not only higher susceptibility to infection-inducing bacteria, but also the need of probiotic administration due to their increased vulnerability to asthma [7] and allergies [8].In spite of the expected lactobacilli dominance in VD infants, the only probes from the VD group that contained certain amounts of the Lactobacillaceae family representatives were the ones supplemented with a probiotic product containing L. reuteri.The lack of lactobacilli genomic equivalents in the other samples alludes either the possibility of non-lactobacillus dominating vaginal microbiome of some mothers or the contribution of other factors that had possibly let to the depletion of the Lactobacillaceae population in VD infants.In fact, sample 8, of a breastfed CS neonate held the highest amount of 7.6 GE/g, which signifies the likelihood of other possible roads of transmission of lactic acid bacteria between mother and child.
Conversely, the bifidobacterial total presence in GE/g is increased in the VD group, however, the diversity of "child" bifidobacteria showed to be higher on average in the CS group.Regardless of historical linkages between the excistance and variety of the Bifidobacterium genus and natural delivery [3] [9], the minor differences in these parameters we surveilled in the two groups give us no opportunity to draw a solid conclusion.Additional analysis including more participants and sampling points are needed.

Lactic acid bacteria content depends on the type of feeding and supplementation
All of the participating neonates in the current study were sampled on the sixth month after their birth or prior, suggesting that breastfeeding was the main nutritional source.The individuality of mothers, their genetic and lifestyle differences are a predisposition for diversity in the microbiological consistence of their breast milk, which proceeds to demonstrate microbiological distinctions in the stool samples of their respective infants.All solely breastfed neonates exhibited relatively high total amount of bifidobacteria between 8.5 GE/g and 9.5 GE/g.The other three samples of mix-fed infants form two completely different perspectives of the role of formula-feeding.In the case of sample 6, the newborn was supplemented with an adaptive formula intended for infants with increased risk of allergies to cow milk's protein.The determinative composition of this breast milk substitute, along with its designation, suppose certain gut microbiota alterations that had presumably let to total bifidobacterial decrease, and almost equal quantities of "infant" and "adult" bifidobacterial species.In addition, the new-born was delivered via Caesarean Section, a birth mode associated with IgEmediated food allergies [10].However, considering the lack of similar instances in our study, we suggest that additional research including more participants are necessary to confirm the linkage between CS and food allergies.In contrast, the other two mix-fed neonate, probe 4 and 9, exhibited some of the largest total amount of bifidobacterail content, almost fully represented by the "infant" Bifidobacterial species.The formula used in these two cases, containing galacto-and fructooligosaccharides, that would presumably enhance the growth of bifidobacteria.According to Sims and Tannock [11] the GOS and FOS usually added to ruminant milk are utilized by many bifidobacterial species, including B. breve, B. bifidum, B. longum, and could potentially overgrow B. infantis.We hypothesize that similar event had occurred in this case that would explain the largest amounts of B. longum and B. bifidum, compared to B. infantis, which on the other hand, is extensively associated with utilization of all variations of HMOs [2].Additionally, the neonate providing sample 4 was supplemented this formula five to seven times a day, which is around three times more than the infant that supplied sample 9. Similar dynamics were observed with B. infantis in low (sample 9) or nonexistent (sample 4) quantities, and medium to increased presence of B. longum, B. bifidum and B. breve, with the exception of B. longum in sample 4 that was shown as relatively decreased.Moreover, the only "adult" bifidobacteria found in both samples was B. dentium, occasionally isolated from infant stool, sharing some characteristic with B. longum, B. bifidum and B. breve, of metabolizing specific structures of oligosaccharides [12].
Only three out of fifteen infants were supplemented with probiotic, containing Limosilactobacillus reuteri DSM 17938, a strain related to overall improvement of gastrointestinal (GI) functions [13].Among the beneficial properties of this strains are the increase of bacterial diversity [14], alleviated stool passing and colic decrease [15].Unlike Marti et al., [14], we did not surveil acceleration in the Lactobacillaceae presence in the supplemented samples.However, our results imply a general representation of the whole family that eliminates more specific information of different species and strains proximity.Further analysis are required for more detailed assessment of the results.

Faecal pH is influenced by the specific lactic acid bacteria taxa
Bifidobacterium is proven to be the genus with the most significant properties for newborns due to the unique metabolism of the representatives that not only plays key role in the developments of the immune system [16], but also exerts defensive features.Frese et al., [4] conclude that even though there are bacteria capable of utilizing HMOs (Bacteroidacese), only bifidobacteria produce SCFAs (short chain fatty acids) (acetate and lactate) as a result of digesting them, which makes their presence fundamental for achieving optimal faecal pH values.Another specific of bifidobacteria-rich infant stool is the significantly lower amounts of HMOs, a phenomenon Frese et al., [4] observed after supplementing neonates B. infantis EVC001.The average faecal pH of B. infantis EVC001-colonized infants was 5.15, while those lacking this strain showcased a relative pH of 5.97 [4].We surveilled B. infantis in various quantities in seven out of fifteen samples and a pH midpoint value of 5.04, slightly lower than the one Frese et al., report.However, the average faecal pH of neonates lacking B. infantis was 5.15.These result could be explained with the higher amounts of other members of genus Bifidobacterium and their species and strain specifications.Two of the three probes of mix-fed newborns showed rather decreased presence of B. infantis, whereas seven out of twelve non-supplemented infants lacked B. infantis.Potential explanations of this peculiar population dynamics are the different maternal HMOs secretion types [17] that might have altered the bifidobacterial-inducing effect of these oligosaccharides, antibiotic use [18] or probable fall to undetectable amounts of bifidobacterial levels that vary day-to-day [3].Additional analyses that evaluate the faecal quantity of SCFAs and B. infantis presence including more sampling points during a prolonged period of time are needed to support these statements.

Conclusion
The present study was a pilot study to test the Enteroflor Kiddy real-time PCR Detection Kit which is designed for detection of DNA of colon-associated microorganisms.There were no published data in the literature worldwide generated using this kit.The reliability of the results we confirmed with duplicates and using negative and positive control as described by the manufacturer.Our research tested the protocol, data collection and sustainability, sample preparation and techniques in preparation for a larger study in the future in Bulgaria.

Figure 1 .
Figure 1.Percentage distribution of the identified species of Bifidobacterium and family

Figure 2 .
Figure 2. Quantity distribution in GE/g of the identified species of Bifidobacterium and family Lactobacilaceae.

Table 1 .
Characteristics of the Study Subjects Infant

Table 2 .
Main results after performing real time PCR reactions.
BM -Breast milk CS -delivered via Caesarean Section VD -Vaginal delivery