Assessment of the Safety, Growth, and Gut Microbiota Modulation of Neonates Consuming Infant Formula with Bifidobacterium animalis ssp. lactis CP-9 or Lactobacillus salivarius AP-32: Insights from a Four-Month Clinical Study in Newborn Infants Under Two Months Old

doi:10.21203/rs.3.rs-3718044/v1

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Assessment of the Safety, Growth, and Gut Microbiota Modulation of Neonates Consuming Infant Formula with Bifidobacterium animalis ssp. lactis CP-9 or Lactobacillus salivarius AP-32: Insights from a Four-Month Clinical Study in Newborn Infants Under Two Months Old

https://doi.org/10.21203/rs.3.rs-3718044/v1

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Background

Breast milk is a natural treasure for newborns, and its microbiota contains a rich array of bacterial species. When breastfeeding is not possible, infant formula with probiotics can be used as a sole source or as a supplement to breast milk. The aim of this study was to investigate the safety, tolerance, growth, and gut microbiota modulation resulting from the consumption of an infant formula with Bifidobacterium animalis ssp. lactis CP-9 (B. animalis CP-9) or Lactobacillus salivarius AP-32 (L. salivarius AP-32), which were isolated from breast milk and the healthy human gut of newborn infants.

Methods

One hundred and fifty-nine healthy infants were enrolled in this study and separated into three groups: the control group, L. salivarius AP-32 group, and B. animalis CP-9 group. The clinical study evaluated adverse events, growth effects, the incidence of allergies, and gastrointestinal disorders in infants consuming infant formula containing B. animalis CP-9 or L. salivarius AP-32. Finally, the impact of the probiotic infant formula on the gut microbiota was elucidated by next-generation sequencing (NGS) analysis.

Results

A 4-month intervention revealed that body weight, recumbent length, and head circumference were similar among the three groups. No adverse events related to the intervention were observed. The microbiota composition was more diverse on day 0 and became more uniform by month 4. B. animalis CP-9 and L. salivarius AP-32 were found to be antibiotic susceptible and displayed a low risk of horizontal antibiotic gene transfer.

Conclusions

The use of B. animalis CP-9 and L. salivarius AP-32 in infant formula was considered safe and well tolerated.

Trial registration

The trial was registered in the US Library of Medicine (clinicaltrials.gov) with the number NCT03993301 on 20/06/2019.

Probiotics

Infant formula

Gastrointestinal disorders

Baby growth

Over the first year of life, nutrition is the principal regulator of growth, with a minimal contribution from growth hormone. Breastfeeding is a natural and preferred method of feeding [1]. When breastfeeding is not possible, formula can be used as the sole source or as a supplement to breast milk. Infant formula supplemented with probiotics is commonly used worldwide. A major need when addressing the issue of using infant formula in healthy infants is its safety and tolerance when administered for extended periods. Concerns related to the early administration of infant formula supplemented with probiotics include the timing, duration, and delivery in the form of a specific matrix (infant formula), which could be the only source of feeding for an infant over a prolonged period [2]. Some cases of human infection caused by lactic acid bacteria have been reported, raising public concerns about food safety[3]. Antibiotic resistance testing of probiotics is advised to ensure that antibiotic resistance determinants are not introduced into a context where these genes are at risk of being transferred to pathogenic organisms [4].

In 2010, the Committee of Nutrition of the European Society for Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) commented on infant formula supplemented with probiotics (and/or prebiotics) [5]. Probiotics were defined as ‘‘live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” [6]. As one of the earliest identified probiotic strain, Lactobacillus rhamnosus GG ATCC 53103 (LGG) has been shown to have a positive impact on the immune system and gastrointestinal health in children [7–9]. Even no adverse effects have been reported thus far, a longitudinal study remained necessary to evaluate the effect of probiotic-enriched formula on growth and the gut microflora in healthy term infants [10]. Growth studies should include at least measurements of weight, length, velocity and head circumference[11]. Despite their growth, allergic diseases and gastrointestinal illnesses are common health problems in newborn infants. Gastrointestinal illnesses are a major cause of morbidity and mortality in children in developing countries [12, 13]. Allergic diseases are becoming increasingly common worldwide and result from complex interactions between genetic susceptibility and environmental factors [14, 15]. Because allergic sensitization may occur as early as gestation through the first 6 months of life, intervention strategies, such as probiotic administration, are requested for this critical period [16].

Bifidobacterium animalis ssp. lactis CP-9 was separated from breast milk, and Lactobacillus salivarius AP-32 was separated from the healthy human gut. Both strains were analyzed via whole-genome sequencing, and several studies were performed to examine their beneficial effects on human health. The safety of B. animalis CP-9 was assessed in rodents [17], and the combination of B. animalis CP-9 with other probiotic strains was shown to exhibit antioxidative effects in middle-aged mice [18]. L. salivarius AP-32 exhibited antibacterial activity against Helicobacter pylori and reduced lymphocyte infiltration and inflammatory chemokine expression in H. pylori-infected rats[19]. In addition, L. salivarius AP-32 was able to ameliorate mitochondrial function and reshape the gut microbiota in Parkinson’s disease rats [20, 21]. Both B. animalis CP-9 and L. salivarius AP-32 exhibited antibacterial activity against oral pathogens and increased IgA concentrations in the oral mucosa [22, 23]. The multi-strain probiotic supplementations containing B. animalis CP-9 and L. salivarius AP-32 reduced inflammation and attenuated glycemic levels in type 2 diabetic rats and type 1 diabetic patients[24, 25]. Moreover, the combination of B. animalis CP-9, L. salivarius AP-32, and L. rhamnosus bv-77 improved the blood lipid profile in obese rats[26] and reformed obesity-related gut dysbiosis in obese children[27]. Although B. animalis CP-9 and L. salivarius AP-32 were manufactured for human use for more than one decade, their safety use in infant formula still needs to be particularly evaluated.

This study primarily aimed to evaluate the growth outcome of infants consuming infant formula containing B. animalis CP-9 or L. salivarius AP-32. The safety of B. animalis CP-9 and L. salivarius usage was also investigated in the aspect of antibiotic resistance.

Clinical study in infants

2.1 Inclusion and exclusion criteria

This double-blind randomized trial was performed at China Medical University Children's Hospital. All the babies were divided into three groups: (I) the probiotic A formula (L. salivarius AP-32, 10⁷ cfu/g) group and (II) the probiotic B formula (B. animalis CP-9, 10⁷ cfu/g). The control group was treated with (III) regular formula (Youluck infant formula). The probiotic lyophilized powder was sponsored by Glac Biotech Co., Ltd. (Tainan, Taiwan), and the infant formula was manufactured by Youluck International, Inc., Taiwan. This study was approved by the China Medical University & Hospital Research Ethics Committee (IRB No. CMUH108-REC2-005) and registering in the US Library of Medicine (www.clinicaltrial.gov) with the number NCT03993301. This study assessed the growth and incidence of allergies and gastrointestinal disorders in infants. All newborns whose parents provided written informed consent and satisfied the inclusion and exclusion criteria mentioned below were included in the study.

The inclusion criteria for patients were as follows: (1) had NSD full-term health infants (≥ 37 weeks) and birth weights greater than 2,500 grams and (2) were expected to breastfeed. Patients were eligible for the trial after informed consent was signed by the parents. The exclusion criteria for infants were as follows: chronic diarrhea, hemangioma, cerebral hemorrhage, severe asphyxia (stage III), fetal chromosomal abnormalities, cyanotic congenital heart disease, intestinal hypoplasia or abnormal immune function after birth, liver failure, breastfeeding within two months after birth, taking other probiotic products within two weeks after birth, treatment with antibiotics during acute infection and diagnosis of allergies, gastroenteritis, or respiratory infections.

2.2 Study populations

The purpose of this trial was to evaluate the effect of infant formula containing probiotics on neonatal growth. The main endpoint was weight gain after four months of intervention. To examine whether the weight gain of the experimental group would be different from that of the control group (placebo), the sample size was calculated according to the research of Puccio et al. in 2007[28]. In that study, the average weight gain rate increased by 3.9 ± 6.1 g/day in babies fed infant formula containing probiotics compared to babies fed infant formula without probiotics. Forty-five evaluable infants per group were needed to have 85% power of the test at the significance level of α = 0.05. To accommodate the expected 25% dropout rate, a total of 60 infants per group were enrolled in the study. The physiological values were evaluated in the per-protocol population (per-protocol analysis).

2.3 Intervention

The infant formula with probiotics (10⁷ cfu/g) or the regular formula were prepared by nurses who were blinded to the groupings. The team members followed the orders from a sealed envelope. Therefore, the only personnel who knew of the infants’ group assignments were the investigators. The study was conducted for 4 months, and the participants were requested to consume at least 2 scoops (17.2 g) of infant formula per day. When fed, the formula was dissolved in warm water (≤ 37°C). All babies were monitored for adverse conditions such as vomiting, diarrhea or bloating.

Subsection safety assessment in vitro

2.4 The minimum inhibitory concentration (MIC)

Single colonies of B. animalis CP-9 and L. salivarius AP-32 were prepared using the quadrant streaking method from their stock cultures. The determination of the MICs of various antibiotics against B. animalis CP-9 and L. salivarius AP-32 was conducted by the Food Industry Research and Development Institute (FIRDI) in Hsinchu City, 30062, Taiwan. This determination followed the microdilution method as outlined in the ISO 10932 guidelines (ISO 10932: 2012, 'Milk and milk products - Determination of the minimal inhibitory concentration of antibiotics applicable to Bifidobacteria and no enterococcal lactic acid bacteria'). The data were aligned with FIRDI Report No. 110SN00745 and 110SN01622.

2.5 Genome annotation

The whole-genome sequences of B. animalis CP-9 and L. salivarius AP-32 were obtained by sample submission to the BGI Genomics Sequencing Service (Guangdong, China). After the genome sequence assembly, the drug resistance-related genes and mobile element information were annotated using the Rapid Annotation Subsystem Technology server (http://RAST.nmpdr.org/, version 2.0) [29]. Moreover, promoter analysis was performed using the online tool SAPPHIRE (https://sapphire.biw.kuleuven.be/) to predict whether there was a promoter sequence structure upstream of the drug resistance-related genes. The similarity of the sequences is indicated by the E value.

2.6 Fecal DNA extraction and NGS analysis

Fecal samples were collected from all the recruited newborn babies 2 times (day 0 and month 4 of the study). Bacterial DNA was extracted from fecal samples using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Germany) with some modifications. Briefly, the storage buffer was removed from the stool sample by centrifuging at 13,200 rpm for 10 mins, after which the cells were lysed by using InhibitEX buffer. After homogenization, the processed supernatant was obtained by adding proteinase K and ethanol. Finally, a QIAamp spin column was used to obtain the DNA from the supernatant, and the DNA was eluted with elution buffer. The DNA concentration and purity were monitored by a NanoDrop™ 2000 spectrophotometer (Thermo Scientific), and a 10× dilution was then performed with elution buffer. The standard V3–V4 region of the 16S rRNA gene was used to construct the gut microbiome library. DNA amplification was carried out with KAPA HiFi hotstart readymix (Roche, United States), and purification was performed with AMPure XP magnetic beads (Beckman Coulter, United States). The quality and quantity of the library were assessed by a Fragment Analyzer (Advanced Analytical, United States) and a Qubit 3.0 Fluorometer, respectively. Afterwards, the library sequence was analyzed via MiSeq (Illumina, United States) with paired-end reads (2 × 301 nt) and at least 100,000 reads per sample.

The quality control and feature table construction of the resulting sequence data were performed via QIIME 2 version 2020.11 (https://qiime2.org/, accessed on 23 Aug 2023) in conjunction with the DADA2 pipeline. Quality filtering, denoising, and chimera removal were performed to create amplicon sequence variants (ASVs) for subsequent in-depth analyses. The Shannon index was used to measure alpha diversity. The examination of beta diversity was conducted utilizing unweighted UniFrac, a metric calculated via MicrobiomeAnalyst (https://www.microbiomeanalyst.ca/, accessed on 29 Aug 2023). Permutational multivariate analysis of variance (PERMANOVA) was applied to examine and validate the statistical differences in beta diversity. The Greengenes (v13.8) 99% operational taxonomic units were used as reference sequences to assign ASVs. The data are presented as the means ± SDs, and significant differences were determined using Student’s t test; a P value less than 0.05 was considered to indicate statistical significance.

2.7 Statistical analysis

All the data are presented as the mean ± standard deviation (mean ± SD). Pearson's chi-square test was used for categorical variables, and the Kruskal‒Wallis test was used for continuous variables. All the statistical data were analyzed using SPSS 19.0, and the significance level was set at p < 0.05.

3.1 Disposition of Subjects

The planned enrollment was 180 evaluable participants; however, the study ended in August 2022 due to the shelf life of the infant formula. From February 2020 through August 2022, one hundred and fifty-nine (159) healthy, full-term, spontaneous vaginally delivered infants were enrolled and randomized to the CP-9 (n = 60), AP-32 (n = 39), or placebo (n = 60) groups. All randomized subjects received allocated treatment. The study was conducted for 4 months, and the participants were requested to consume at least 2 scoops (17.2 g) of infant formula per day (Fig. 1A). One hundred eighteen (118) patients completed the 4-month treatment, 8 (5.0%) withdrew consent, and 7 (4.4%) were lost to follow-up before completing the treatment (Fig. 1B). The dropout rate in the CP-9 group was 30% for reasons including low consumption perception, infant colic, lactose intolerance, galactosemia, diarrhea, heart problems, and surgery. The dropout rate in the AP-32 group was 23.1% for reasons including poor compliance, low consumption perception, lactose intolerance, and constipation. The dropout rate in the placebo group was 23.3% for reasons including poor compliance, infant colic, personal concern, low consumption perception, lactose intolerance, and constipation.

3.2 Demographics and characteristics

Subject demographics and baseline characteristics are summarized in Table 1. The percentage of females was slightly greater in the AP-32 group (63.3%), and the percentage of males was slightly greater in the placebo group (56.5%). The difference was not significant between the probiotic group and the placebo group (P = 0.237). The gestational age ranged from 37 to 40 weeks in all the groups (P = 0.291). The baseline age ranged from 0 to 47 days (P = 0.410). The treatment duration ranged from 119 to 162 days (P = 0.696). The age ranged from 120 to 183 days when the subject finished the treatment at month 4 (P = 0.826). The data showed that the demographic and baseline characteristics were similar among the treatment groups.

Table 1

Subjects Demographics and Characteristics.
	Placebo	CP-9	AP-32	P value
Gender(M/F)	26/20(56.5%/43.5%)	20/22(47.6%/52.4%)	11/19(36.7%/63.3%)	0.237
Gestational age(weeks)	38.59 ± 0.91	38.67 ± 0.87	38.33 ± 0.88	0.291
Baseline age (days)	2.63 ± 4.52	4.10 ± 9.20	3.07 ± 6.06	0.410
Duration(days)	132.80 ± 10.59	131.07 ± 9.96	131.70 ± 8.81	0.696
Age at month 4 (days)	134.43 ± 11.86	134.17 ± 14.21	133.77 ± 10.07	0.826

The data are presented as the number and percentage of subjects (N, %) or mean ± standard deviation (SD).

3.3 The probiotic formula did not affect the growth of infants

One of the endpoints was the weight gain rate (in grams/day) of the individual infants during the 4 months of the study. The baseline weights recorded on day 0 were similar among all groups before the intervention started (placebo: 3.14 ± 0.43 kg; CP-9: 3.10 ± 0.29 kg; AP-32: 3.02 ± 0.28 kg; P = 0.415; Fig. 2A), and the body weights measured at month 4 of the intervention were not significantly different (placebo: 7.30 ± 0.83 kg; CP-9: 7.05 ± 0.68 kg; AP-32: 6.95 ± 0.58 kg; P = 0.167; Fig. 2A). The weight gain rate was calculated by subtracting the baseline weight from the weight at the end of the study (month 4), which was then divided by the treatment duration. The mean weight gain was similar between the two probiotic groups and the placebo group (placebo: 31.47 ± 5.35 g/day; CP-9: 30.24 ± 5.30 g/day; AP-32: 30.03 ± 4.23 g/day; P = 0.379; Fig. 2B).

The growth rates (in millimeters/day) of recumbent length and head circumference were two other endpoints of the assessment. The baseline length and circumference recorded on day 0 were similar among all groups before the intervention started (placebo: 49.62 ± 2.26 cm; CP-9: 49.68 ± 1.68 cm; AP-32: 49.04 ± 1.51 cm; P = 0.388; placebo: 33.45 ± 1.48 cm; CP-9: 33.11 ± 1.06 cm; AP-32: 33.03 ± 1.13 cm; P = 0.540; Fig. 2C and E); the length and circumference measured on month 4 of the intervention exhibited no difference (placebo: 63.33 ± 2.17 cm; CP-9: 63.67 ± 2.91 cm; AP-32: 63.01 ± 2.19 cm; P = 0.462; and placebo: 41.21 ± 1.36 cm; CP-9: 41.13 ± 1.63 cm; AP-32: 40.7 ± 1.30 cm; P = 0.255; Fig. 2C and E). The mean growth rates in terms of length and circumference were similar between the two probiotic groups and the placebo group (P = 0.925 and P = 0.628; Fig. 2D and F).

3.4 No intervention-related adverse events were observed by the pediatricians.

Forty-six (39.0%) of the 118 infants had one adverse event (AE) during the study: 16 (38.1%) in the CP-9 group, 16 (53.3%) in the AP-32 group, and 14 (30.4%) in the placebo group (Table 2). Gastrointestinal disorders were the most common organ system involved (n = 24, 21.8%), with infantile colic being the most common manifestation (n = 15, 13.6%). A few cases of abdominal bloating (n = 5, 4.5%) were recorded, while cases of constipation, enteritis, anal fissures, and inguinal hernia were all single cases (n = 1, 0.9%). There was no apparent difference between the probiotic groups and the placebo group in terms of the number of infants with these AEs (n = 8/9/7, P = 0.284). Skin disorders were the second most common organ system involved, with 22 (20.0%) infants experiencing AEs within this category. Atopic dermatitis (n = 11, 10.0%) was the most common, followed by seborrheic dermatitis (n = 8, 7.3%), rash (n = 2, 1.8%), and infantile eczema (n = 1, 0.9%). The number of patients with these AEs was similar between the two probiotic groups and the placebo group (n = 8/7/7, P = 0.672). A total of 46 AEs were reported during the study, and none of them were diagnosed as possibly related to the study product by pediatricians.

Table 2

Summary of Gastrointestinal Disorders and Allergic Diseases.
	Total N = 118	Placebo N = 46	CP-9 N = 42	AP-32 N = 30	P value
Total	46 (39.0%)	14 (30.4%)	16 (38.1%)	16 (53.3%)	0.134
Gastrointestinal disorders	24 (21.8%)	7 (15.2%)	8 (19%)	9 (30%)	0.284
Constipation	1 (0.9%)	--	1 (2.4%)	--	0.402
Infantile colic	15 (13.6%)	5 (10.9%)	5 (11.9%)	5 (16.7%)	0.745
Abdominal bloating	5 (4.5%)	2 (4.4%)	1 (2.4%)	2(6.7%)	0.672
Enteritis	1 (0.9%)	--	--	1 (3.3%)	0.228
Anal Fissures	1 (0.9%)	--	--	1 (3.3%)	0.228
Inguinal hernia	1 (0.9%)	--	1 (2.4%)	--	0.402
Allergic diseases	22 (20.0%)	7 (15.2%)	8 (19.0%)	7 (23.3%)	0.672
Seborrheic dermatitis	8 (7.3%)	1 (2.2%)	4 (9.5%)	3 (10.0%)	0.281
Atopic dermatitis	11 (10.0%)	5 (10.9%)	3 (7.1%)	3 (10.0%)	0.826
Rash	2 (1.8%)	1 (2.2%)	--	1 (.3.3%)	0.530
Eczema infantile	1 (0.9%)	--	1 (2.4%)	--	0.402

3.5 B. animalis CP-9 and L. salivarius AP-32 were susceptible to most of the listed antibiotics of the European Food Safety Agency (EFSA).

The antimicrobial resistance patterns of B. animalis CP-9 and L. salivarius AP-32 were assessed by comparing the observed MICs with the most recent EFSA breakpoint values (Table 3). B. animalis CP-9 exhibited susceptibility to vancomycin, streptomycin, tetracycline, erythromycin, clindamycin, chloramphenicol, and ampicillin, with MIC values below the cutoff established by the EFSA. However, it displayed an MIC of 128 µg/mL for gentamicin. On the other hand, L. salivarius AP-32 was susceptible to gentamicin, streptomycin, tetracycline, erythromycin, clindamycin, chloramphenicol, and ampicillin, with MIC values less than the EFSA cutoff. Nevertheless, it displayed an MIC of 256 µg/mL for kanamycin. B. animalis CP-9 and L. salivarius AP-32 might be intrinsically resistant to gentamicin and kanamycin, respectively.

To understand the nature of gentamicin and kanamycin resistance in B. animalis CP-9 and L. salivarius AP-32, respectively, gene annotation of the genome sequences was performed. Gentamicin belongs to the antibiotic class of aminoglycoside. The genome of B. animalis CP-9 contained only one circular chromosome (1,944,145 bp) and no plasmid (Fig. 3A). These results suggested that a potential gene structure may contribute to the higher gentamicin resistance of B. animalis CP-9. (Table S1). Considering that a potential gene structure was not located near any mobile elements or transposases, this resistance is likely intrinsic. Kanamycin is also an aminoglycoside. The genome of L. salivarius AP-32 consists of one circular chromosome (1,727,032 bp) and two plasmids (269,712 bp and 17,451 bp; Fig. 3B). The gene annotation indicated a potential gene structure that was not located near any mobile elements or transposases, suggesting that this resistance could be intrinsic (Table S2).

Table 3

Antibiotic susceptibility profiles are reported in MIC (µg/mL) using the microdilution method in accordance with ISO 10932 guidelines^a.
Antibiotic	CP-9	AP-32
Gentamicin	128/64	8/16
Vancomycin	1/2	n.r.
Streptomycin	64/128	64/64
Tetracycline	4/8	4/8
Erythromycin	0.25/1	0.5/1
Clindamycin	0.25/1	0.5/1
Chloramphenicol	4/4	4/4
Ampicillin	2/2	0.5/4
Kanamycin	n.r.	256/64

^a The results are presented as the measured value/cutoff value in EFSA Journal 2012; 10(6):2740.

^b n.r.: not needed.

3.6 The diversity of the gut microbiota changes with age

In all the groups, the alpha diversity increased, and the beta diversity shifted at month 4 (P < 0.001; Fig. 4A and B). There were no significant differences in either alpha or beta diversity among the three groups on day 0 or at month 4. The four most abundant phyla were Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes in all the groups. The proportions of Firmicutes and Bacteroidetes remained similar between day 0 and month 4. Interestingly, at month 4, the percentage of Proteobacteria decreased, while that of Actinobacteria increased (Fig. 4C and 5A). The ten most abundant genera were Bifidobacterium, Bacteroides, Streptococcus, Enterococcus, Veillonella, Clostridium, Rothia, Lactobacillus, Parabacteroides, and others. The proportions of the top 10 genera varied among the three groups on day 0 and became more uniform by month 4 (Fig. 4D).

The abundance of the Bifidobacterium genus increased in correlation with the increase in the Actinobacteria phylum in all three groups (Fig. 5A and B). Although the content of the Lactobacillus genus remained unaffected by the intervention, the proportions of the Streptococcus, Enterococcus, and Veillonella genera within the Firmicutes phylum were altered (Figure S1). Remarkably, the abundance of Bifidobacterium longum species significantly increased in all groups, while supplementation with B. animalis CP-9 did not lead to a greater proportion of B. animalis species (Fig. 5C). Conversely, supplementation with L. salivarius AP-32 successfully elevated the proportion of L. salivarius species (Fig. 5C). No significant alterations were identified in either Klebsiella (Fig. 5B) or other genera (Figure S2), despite the concurrent reduction in the abundance of the Proteobacteria phylum (Fig. 5A).

According to our clinical observations, the subjects were 2.63–4.10 days old at the beginning of the intervention and 133.77–134.43 days old at the end of the intervention. The growth of body weight, recumbent length, and head circumference was similar among the groups. According to the WHO child growth standards [30], the 50th percentile for 0–4-month body weight increase is 3636 g for boys and 3210 g for girls, corresponding to a growth rate of 26.31–29.80 g/day. Our subjects displayed a growth rate of 30.03–31.47 g/day, indicating that infant formula containing B. animalis CP-9 or L. salivarius AP-32 did not affect normal body weight. For 0–4-month body length increments, the 50th percentile is 14 cm for boys and 13 cm for girls, with a growth rate of 0.107–0.115 cm/day. Our subjects showed a normal growth rate of 0.104–0.107 cm/day, suggesting that the intervention did not affect body length growth. The 50th percentile for the 0–4-month head circumference increase was 7.1 cm for boys and 6.7 cm for girls, corresponding to a growth rate of 0.055–0.058 cm/day. Our subjects exhibited a normal growth rate of 0.059–0.062 cm/day, indicating that the growth of head circumference was not influenced by the infant formula containing B. animalis CP-9 or L. salivarius AP-32.

Unexpectedly, the allocation in the L. salivarius AP-32 group was lower, resulting in a sample size smaller than that originally estimated. This reduction in sample size affected the statistical power of the study, and the results should be interpreted with caution[31]. Nevertheless, the intervention involving infant formula containing B. animalis CP-9 or L. salivarius AP-32 had no adverse effects on baby weight, height, or head circumference. This result was similar to that of our recent study in which these two strains were supplied at a higher dosage to 1-month-old infants[32]. A systematic review revealed that supplementation with certain strains may reduce the number of colic episodes, days with fever, and antibiotic use, but no significant effects were observed on baby weight or height, with a mean duration of intervention of 5.6 ± 2.8 months [33]. The present 4-month study suggested that the use of B. animalis CP-9 or L. salivarius AP-32 are generally safe to use as formula additives. Further studies are needed to determine whether these two strains can exert significant effects on newborn growth.

The antibiotic resistance of our probiotic strains was investigated to ensure that the material was suitable for intervention in newborn babies. Surprisingly, B. animalis CP-9 did not exhibit tetracycline resistance even though it harbored four tetracycline resistance-related genes, including one tet(W) gene [34]. On the other hand, B. animalis CP-9 exhibited greater gentamicin resistance than the EFSA breakpoint for the Bifidobacterium genus. In general, Bifidobacteria are inherently resistant to aminoglycosides due to the absence of cytochrome-mediated drug transport [35, 36]. Our gene annotation suggested that this resistance might be intrinsic, and this in silico-based screen provided a potential target for further laboratory-based investigations, such as insertional inactivation [36]. Similarly, L. salivarius AP-32 did not exhibit tetracycline resistance even though it harbored two drug resistance-related genes, one related to tetracycline resistance, on the larger plasmid[37, 38]. On the other hand, L. salivarius AP-32 displayed greater kanamycin resistance than the EFSA breakpoint value for facultative heterofermentative Lactobacillus. However, this resistance is not unique to the L. salivarius AP-32 strain [39]. Despite our gene annotation, kanamycin resistance might be intrinsic to L. salivarius AP-32, but the underlying mechanism requires further laboratory-based investigations [40].

The dynamic evolution of the gut microbiota composition progresses rapidly during the first 6 months after birth. The relative abundances of some genera that are abundant at birth decrease, while the abundances of other genera begin to increase at detectable levels as infants age[41]. Participants in this study were healthy, full-term, spontaneous vaginally delivered infants, which minimized the influence of delivery mode on the initial microbiota [42]. Notably, a more diverse microbiota was observed among the three groups before the intervention, and the microbiota composition became more uniform by the end of the intervention. For instance, the proportion of Proteobacteria was slightly greater in the AP-32 group than in the other two groups on day 0, and it was significantly lower by month 4. Proteobacteria include a wide variety of pathogenic genera, and an increase in the abundance of Proteobacteria in the gut microbiota can indicate a potential disruption or dysbiosis in the microbial balance of the gut[43]. Various implications were associated with an overgrowth or higher abundance of this phylum, such as inflammation, metabolic disorders, gastrointestinal conditions, altered gut barrier function, risk of infections and childhood asthma[44–46]. Therefore, the higher proportion of Proteobacteria and lower proportion of Bifidobacterium may indicate the intestinal microflora dysbiosis in the AP-32 group before the intervention. Dysbiosis is possibly related to the slightly higher incidence of gastrointestinal and allergic events recorded in that group. Our results suggest that probiotic supplementation is capable of promoting the restoration of healthy microbiota in newborns. Further investigations, including longer interventions and additional follow-up periods, are needed to determine whether the modulation of the microbiota can lead to improvements in gastrointestinal and allergic conditions.

Infant formula serves as a human milk substitute, offering nutritional solutions for infants who cannot receive sufficient breast milk from their mothers. Although supplementing with one or a few probiotic strains cannot replicate the complexity of human milk, which contains numerous bioactive compounds, including oligosaccharides and immune cells, as well as varying levels of bacteria and their metabolites[47], infant formula can become more comprehensive as additional safe probiotic strains are considered for use in infants.

The present study demonstrated that the bacterial strains B. animalis CP-9 and L. salivarius AP-32 had low resistance to antimicrobials. The supplementation of B. animalis CP-9 or L. salivarius AP-32 did not affect normal growth or support the normal evolution of the gut microbiota in healthy infants. Therefore, these two strains are considered safe and suitable for use in infants.

Ethics approval and consent to participate:This trial was conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from all patients prior to commencing the study. The trial was registered in the US Library of Medicine (clinicaltrials.gov) with the number NCT03993301 on June 20, 2019.

Consent for publication: Not applicable.

Availability of data and materials: The data supporting the findings of this study have been deposited in the BioProject with the primary ID PRJNA1051835, [http://www.ncbi.nlm.nih.gov/bioproject/1051835].

Competing interests: Glac Biotech Co., Ltd. provided financial support in the form of salaries for [J. F. Chen, H.S. Wang, Y.Y. Huang, K.C. Hsia, J.H. Lin, Y.W. Kuo, C.M. Li, Y.C. Hsu, S.Y. Tsai, H.H. Ho] but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. S.P. Shen and H.C. Lin declared that they have no competing interests.

Funding: This study was supported by a grant from Glac Biotech Co., Ltd., Tainan, Taiwan.

Authors' contributions: S.P. Shen: Project administration. H.C. Lin: Project administration. J. F. Chen: Original draft preparation and data analysis. H.S. Wang: Reviewing, Data analysis. Y.Y. Huang: Methodology, Data visualization. K.C. Hsia: Data analysis. J.H. Lin: Methodology, Data analysis. Y.W. Kuo: Project design. C.M. Li: Resources. Y.C. Hsu: Review, Editing. S.Y. Tsai: Review, Resources. H.H. Ho: Supervision, Project design.

Acknowledgments: We sincerely thank Professor Yao-Tsung Yeh at the Aging and Disease Prevention Research Center, Fooyin University, Kaohsiung City 831, Taiwan, for performing the NGS and gut microbiota analysis.

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Assessment of the Safety, Growth, and Gut Microbiota Modulation of Neonates Consuming Infant Formula with Bifidobacterium animalis ssp. lactis CP-9 or Lactobacillus salivarius AP-32: Insights from a Four-Month Clinical Study in Newborn Infants Under Two Months Old

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

Trial registration

Figures

1. Introduction

2. Materials and Methods

2.1 Inclusion and exclusion criteria

2.2 Study populations

2.3 Intervention

2.4 The minimum inhibitory concentration (MIC)

2.5 Genome annotation

2.6 Fecal DNA extraction and NGS analysis

2.7 Statistical analysis

3. Results

3.1 Disposition of Subjects

3.2 Demographics and characteristics

3.3 The probiotic formula did not affect the growth of infants

3.4 No intervention-related adverse events were observed by the pediatricians.

3.5 B. animalis CP-9 and L. salivarius AP-32 were susceptible to most of the listed antibiotics of the European Food Safety Agency (EFSA).

3.6 The diversity of the gut microbiota changes with age

4. Discussion

5. Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

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