Lactic acid bacteria isolated from women’ breast milk and infants’ faeces have appreciable immunogenic and probiotic potentials against diarrheagenic E. coli strains

Diarrheal diseases remain the leading cause of high mortality among the infants, particularly in the developing countries; Probiotic intervention for diarrhea has been an ongoing novel approach to diarrheal prevention and treatment. This study aims to characterize immunogenic and probiotic properties of lactic acid bacteria (LAB) isolated from human breast milk and neonates’ faeces. The LAB isolates from 16 mothers’ breast milk and 13 infants’ faeces were screened and identified by 16 S rRNA gene partial sequencing. Their antimicrobial activities against 5 strains of diarrheagenic Escherichia coli were tested. Organic acids production was quantified by HPLC, and antibiotic resistance pattern were determined by VITEK®. Autoaggregation, co-aggregation and hydrophobicity properties were assessed by UV spectrophotometry and immunomodulatory effect was determined in mouse model. Ninety-three LAB of five genera were identified. The most abundant species was Lactiplantibacillus plantarum with inhibition zones ranged from 8.0 to 25.0 ± 1 mm. Lacticaseibacillus rhamnosus A012 had 76.8 mg/mL lactic acid, (the highest concentration), was susceptible to all antibiotics tested. L. plantarum A011 and L. rhamnosus A012 were highly resistance to gastrointestinal conditions. L. rhamnosus A012 produced hydrophobicity of 25.01% (n-hexadecane), 15.4% (xylene) and its autoaggregation was 32.52%. L. rhamnosus A012 and L. plantarum A011 exert immunomodulatory effects on the cyclophosphamide-treated mice by upregulating anti-inflammatory cytokine and downregulating proinflammatory cytokines. Lactobacillus sp. demonstrated good probiotic and immunomodulatory properties. Further works are ongoing on the practical use of the strains. Supplementary Information The online version contains supplementary material available at 10.1186/s12866-024-03502-2.


Introduction
Diarrheal disease is mostly caused by viral, bacterial, and parasitic microorganisms [1] and often refers to as 'leading killer' of children under the age of five worldwide [2].According to UNICEF and WHO report, about 1.7 billion cases yearly was attributed to childhood diarrheal disease worldwide [3] with around 525,000 deaths of children under the age of five.In 2015, 300,000 children death was recorded globally while over 1300 death was recorded each day representing 484,000 deaths of young children in 2019, however, most of these deaths take place majorly in Africa [2][3][4][5][6].Despite the awareness and management of diarrhea globally, in 2021, UNICEF still recorded high percentage of death due to diarrhea and reported this disease as a leading killer with approximately 9 per cent of all deaths among children below age 5 globally [4].The updated UNICEF data report in January 2024 recoded 1,200 young children dying each day, amounting to 443,832 children per year, The occurrence of diarrheal disease is normally high in developing countries, India has the highest numbers of diarrhea cases among children and Nigeria follows with the second largest percentage [7,8].Moreover, current UNICEF data showed that for every 1000 birth in Nigeria, 107.2 deaths are recorded annually [4], in which high proportion of these deaths are recorded from Northern Nigeria [9].
Most often, antibiotics and Oral Rehydration Therapy (ORT) are employed as a direct approach to combat acute and severe Diarrheagenic E. coli (DEC) diseases in children [14][15][16].However, these interventions are not sufficient considering the inconsistence in pathogenicity of diarrhea and frequent use of antibiotics disrupts gastrointestinal microbiota [17].Therefore, a non-antibiotic intervention such as probiotics and fermented foods are encouraged in the management of infantile diarrhea caused by DEC.
Probiotics are beneficial living microorganisms with significant health benefits.They positively affect gut microflora by contributing to intestinal microbial balance [18], general antimicrobial properties and decreasing the harmful microbial toxic activity [19,20].They also restore and maintain the gut microbial niche [21].Probiotic strains possess the potential to reverse the infectious effect due to diarrheagenic microorganisms.The mechanisms of interaction between gut microbial populations and the host immune system reveal that these beneficial microorganisms stimulate and regulate the nerve, endocrine, and immune cells [22].They also modulate series of activities like digestion, metabolism, competitive removal of pathogens, and neural plasticity [23].
Breastmilk is safe, clean, and best food with necessary nutrients given to infants in their first months of life to promote a sound health and rapid growth [24].It contains antibody that protects the infants against common illness and infections.According to Moles et al. [24] and Timmerman et al. [25], breastmilk is one of the factors that contributes to the colonization of baby's gut microbiota as it contains some beneficial microbes such as Lactobacillus, Streptococcus, Bifidobacterium, Enterobacterium, Enterococcus, Staphylococcus among others.Breastmilk microbiota on the other hand influence the colonization and proliferation of the neonate's gut microbiota and the maturation of immune system.These gut bacteria display their mechanism of actions against gastroenteritis through bidirectional interactions such as direct microbiota-microorganism interactions or indirectly through modulating the host nerve cells and immune cells [22,26].
A stable microbial community is highly needed to encourage host-microbial and microbial-microbial interaction to improve good health condition among children [27,28].Beneficial microbes e.g.lactic acid bacteria (LAB) have been shown to have antibacterial activities against pathogens implicated in gastroenteritis [29][30][31].Lactobacillus and Bifidobacterium are added to baby formula to promote a balanced gut microbiota and prevents the growth of harmful bacteria that could cause infections and inflammation [32].Currently, there is scarcity of information on LAB from healthy infants' gut and humans' breast milk as potential probiotic strains against DEC strains in a diarrhea endemic region.Therefore, this study was designed to characterize and evaluate probiotic potentials of LAB isolated from human mothers' breastmilk and their infants' faeces in the management of infantile DEC.

Samples collection
The study was ethically reviewed and approved by the ethics committee of the Ekiti State University Teaching Hospital Ethics and Research Committee and was registered as EKSUTH/A67/2015/03/008.Prior to the collection of samples, informed consent of the participants (mother and child) was obtained from the mothers.Fresh mothers' breastmilk and infants' faecal samples were collected from the participants (nursing mothers and children) at the Department of Community Medicine, Ekiti State University Teaching Hospital, Ado Ekiti, Ekiti State, Nigeria.A total of 16 breastmilk samples and 13 fecal samples were collected from 16 mothers and 13 infants.The age of the mothers ranged between 23 years to 33 years while their neonates age ranged between 1 day to 9 months old.The collection of samples was done by health personnel from the Community Medicine Unit of the hospital in an aseptic environment.Nipples and areola of the mothers' breast were cleaned properly with soap and sterile water before manually expressing the breast milk, the first three drops of breastmilk were discarded.Five mL of breastmilk samples was collected from mothers into sterile collection tubes.Approximately 6-10 g of fresh fecal samples were also collected from the neonates into sterile collection tubes.The samples collected were stored on ice until delivery to the laboratory for immediate microbiological analysis.The test pathogenic microorganisms used for the study were 5 diarrheagenic E. coli strains; EPEC H62E, ETEC H40B, STEC H77E, EIEC H68D, and EAEC H40C obtained from Molecular and Biotechnology laboratory, Pharmaceutical Microbiology Department, University of Ibadan, Nigeria.

Isolation of lactic acid bacteria
The isolation of LAB from the samples was carried out using modified method of Medjaoui et al. [33].In summary, 1 mL of breastmilk sample/1 g of faecal sample was homogenized with 9 mL of de Man-Rogosa and Sharpe broth (Oxoid, U.K) supplemented with 0.05 mg/100 mL of L-cysteine hydrochloride (MERCK, Darmstadt, Germany), (MRS-cys) to make a 10 − 1 dilution.The mixture was incubated at 37 o C under anaerobic condition (AnaerogenGen TM Oxoid, UK) for 24 h.The resulting cultures were homogenized for 1 min, ten-fold serial dilutions were done and 100 µL of last 3 dilutions was plated out on MRS-cys agar for 48 h.Morphologically distinct colonies were sub-cultured onto MRS-cys agar plates until pure colony was achieved.Gram positive and catalase negative LAB isolates were preserved and stored as glycerol/MRS-cys stocks at − 80 °C.

Identification of lactic acid bacterial strains
The Genomic DNA of LAB was extracted from overnight broth culture using AccuPrep® Genomic DNA Extraction kit (Bioneer, South Korea), according to the manufacturer`s instructions.The extracted DNA was used in a PCR reaction with 27 F (AGAGTTT-

GATCMTGGCTCAG) and 1389R ( A C G G G C G G T G T G T A C
A A G) primers.The PCR reaction was an initial denaturation at 95 °C for 4 min followed by 25 cycles of 95 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min 30 s and a final 7 min extension at 72 °C [34].The amplicons obtained were stained using Gel-Red, analyzed by agarose gels electrophoresis (1.5% w/v agarose), and their sizes were visualized with Gel documentation system (E-Box CX5.TS.26MXTrans-illuminator System, VIL-BER, France).The nucleotides sequences obtained were analyzed using The Basic Local Alignment Search Tool (BLAST) at http://www.ncbi.nlm.nih.gov/BLAST.and the identified sequences were placed in the European Molecular Biology Laboratory (EMBL) with accession number PRJNA628165 (https://www.ncbi.nlm.nih.gov/sra/PRJNA628165).

Determination of antimicrobial activities of isolated strains
To determine the anti-diarrheagenic E. coli activities of isolated LAB, agar well diffusion method was used [35].Briefly, fresh LAB isolates were cultured for 24 h at 37 °C, centrifuged for 10 min at 12,000 rpm, and sterilized by filtration through a 0.45 μm filter (Millipore, Billerica, MA, United States).A 0.1 mL of 24 h fresh cultured of each diarrheagenic E. coli strain (10 6 cfu/mL) was seeded onto a sterile petri dish containing pre-solidified Mueller Hinton agar (Becton, Dickson and Co, Spark, MD, USA) and allowed to dry.The equidistant 6 mm diameter wells were made with a sterile borer.An aliquot of 100 µL of the cell free supernatant (CFS) was dispensed into each well bored and was allowed to diffuse at room temperature for 60 min.The plates were incubated for 24 h at 37 o C. The antimicrobial activity of the LAB indicated by zones of inhibitions surrounding each well containing CFS were measured and recorded.All the tests were performed in triplicates.
The agar overlay method described by Ayeni et al. [19] was employed and modified to determine the activity of viable LAB cells against the 5 diarrheagenic E. coli strains.Briefly, a fresh cultured LAB isolate was prepared overnight in MRS broth culture, 50 µL of LAB was added on MRS-cys agar plates as a straight line and incubated anaerobically for 48 h at 37 o C. Thereafter, the agar plates were overlaid with a fresh broth culture of each diarrheagenic E. coli (10 6 cfu/mL) vehiculated in 10 mL Mueller Hinton soft agar (0.7%).The overlay agar was left to set and incubated aerobically for 24 h at 37 o C. The zone of inhibition around the straight LAB lines were measured.All the assays were conducted in triplicate.

Growth inhibition of pathogenic strains by LAB using co-culture assay
The growth inhibition of diarrheagenic E. coli strains by selected LAB isolates (they were previously selected due to good antimicrobial activities results) were carried out in a kinetic study.Ten (10) ml MRS-MH broth that contained 5 ml of MRS-cys (double strength) and 5 ml of Mueller Hinton broths (double strength) were prepared to allow the respective growth of LAB and E. coli.The coculture broth was inoculated with 10 8 cfu/mL of each of 15 Lactobacillus isolates, and 10 6 cfu/mL of each of the 5 diarrheagenic E. coli strains and incubated at 37 o C for 24 h.Each Lactobacillus isolate and diarrheagenic E. coli strain were mono-cultured and used as the control.Viable counts of E. coli and LAB strains in co-culture tubes were done at 0, 8, 16, 24 h by plating them onto sterile Eosin Methylene Blue agar plates (Sigma-Aldrich, USA) and sterile MRS-cys agar plates for the determination of viable cell.The plates were incubated for 24 h at 37 o C [36,37].
One of the major mechanisms of antimicrobial activity of LAB is production of organic acid, therefore, LAB isolates were analyzed for organic acid productions.The organic acids, which include propionic, acetic, lactic, and butyric acids were measured and quantified by HPLC (Adept CECIL CE 4200).The HPLC procedure was carried out using 20 µL of the supernatant LAB isolates.The supernatant was pipette directly into the HPLC system fitted to the UV absorbance detector with 210 nm wavelength.H 2 SO 4 at 55 o C was the mobile phase and the HPLC standard curves were plotted using the retention time data and response factor of the organic acids.Then, the area (mAs), height (mA) and the quantity of organic acids (mg/mL) produced by the isolates were measured with linear coefficients (R 2 ) greater than 0.99 [37].

Resistance to low pH
The acid tolerance of the LAB isolates at different pH level was assessed using the modified method of Hassanzadazar et al. [38].Overnight cultures of LAB were centrifuged at 12,000 rpm for 10 min.The supernatants were discarded, and the pellets were carefully rinsed twice with phosphate buffer saline (PBS; 7.2).The cell pellets were re-suspended in 10 mL of adjusted pH 2.0 and 3.0 MRS-cys broth and incubated for 3 h at 37 o C. The survived bacterial cells at 0 and 3 h were determined by plating out 1 mL of the broth and incubating at 37 o C for 24 h, the colonies were counted using colony counter.The LAB isolates cultured without any adjustment of pH were used as control.Each assay was conducted in triplicate.

Tolerance of LAB to bile salt
The tolerance of LAB to bile salt was determined by a modified method of Hassanzadazar et al. [38].Overnight cultures of LAB isolates were freshly prepared in MRScys broth at 37 °C, the prepared cultures were centrifuged at 12,000 rpm for 10 min.The supernatants were discarded, and the cell pellets were gently rinsed twice in 7.2 pH phosphate buffer saline, re-suspended in a 10 ml sterile MRS-cys broth supplemented in 0.3% (w/v) bile salt and incubated at 37 °C for 4 h under anaerobic condition (AnaeroGen™ 3.5 L).The survived bacterial cells at 0 and 4 h were determined by plating out 1 mL of the broth and incubating at 37 o C for 24 h, the colonies were counted using colony counter.The LAB isolates cultured without any adjustment of pH was used as control.Each assay was conducted in triplicate. .

Consecutive acid and bile tolerance test
The ability of LAB isolates to survive gastrointestinal transit of consecutive low pH and bile supplementation was evaluated for the selected 20 LAB isolates with reference to their antibacterial potential and tolerance to mono bile and acid environment.Freshly prepared LAB broth cultures were centrifuged at 12,000 rpm for 10 min.The bacterial supernatants were discarded, and cells pellets were gently rinsed twice in 7.2 pH phosphate buffer saline and re-suspended in a 10 mL sterile MRS-cys broth.An aliquot of 0.1 mL of the suspension was reintroduced into 10 mL MRS broth adjusted to pH 3 with 1 M HCl (the initial viable counts were determined by plating out and incubating 1 mL of the mixture) and then the tubes were incubated for 3 h at 37 o C under anaerobic condition, thereafter, 0.1 ml of the mixture was further inoculated into 10 ml of MRS-cys broth of 0.3% bile salt (w/v), and incubated at 37 o C for 4 h under anaerobic condition.The final survived bacterial cells at 4 h were determined by plating out 1 mL of the broth and incubating at 37 o C for 24 h, The LAB isolates cultured without any adjustment, nor any supplementation were used as control.The log reduction of final count in comparison with the initial count was determined.Each experiment was conducted in triplicate.

Cell surface hydrophobicity
Cell surface hydrophobicity is an important factor of Microbial Adhesion to Hydrocarbon (MATH), it is employed to determine the contact of bacteria with their host cells [39].Based on this, ninety-three strains of LAB were evaluated for cell surface hydrophobicity using a previously described method [36] with modification.A fresh overnight LAB broth culture was prepared and centrifuged (5,000 rpm for 15 min).to obtain the cell pellet.LAB cells were washed two times with sterile PBS.The re-suspended sterile PBS (3mL) and the optical densities was adjusted to 600 nm absorbance for initial reading with the use of UV spectrophotometer (Unico, Flinn Scientific, Canada).Then, 1 mL each of two hydrocarbons; xylene (Sigma, USA) and n-hexadecane (Sigma, USA) was pipetted and added to the suspension.The tubes were vortexed vigorously for 30 s, incubated for 1 h at 37 °C.The aqueous phase was carefully collected and its absorbance at 600 nm was measured.The result was expressed in percentage.Cell surface hydrophobicity was calculated using the equation below: Optical Density initial absorbance (600nm) × 100

Autoaggregation and Co-aggregation assay
Auto-aggregation is used most often as a pre-test to evaluate the adhesion property of the LAB to mucosal and epithelial surfaces [39].This assay is used to evaluate aggregation of bacteria cell, based on the ability of LAB isolates to survive the gastric-intestinal environment and their adherence to hydrocarbon, the auto-aggregation of 15 potential probiotic LAB isolates (they were previously selected due to good gastrointestinal tolerance results) was evaluated using previously described method [40] with some modification.A fresh overnight LAB broth culture was prepared and centrifuged (5,000 rpm for 15 min) to obtain the cell pellet.LAB cells were washed two times with sterile PBS.The re-suspended sterile PBS (3 mL) was adjusted to 600 nm absorbance for initial reading with the use of UV spectrophotometer (Unico, Flinn Scientific, Canada).Thereafter, the remaining suspension was also vortexed and left to incubate for 5 h at 37 o C. At an hour interval up to 5 h (t1, t2, t3, t4 and t5), an aliquot (1 mL) from the top of the suspension was carefully removed and its absorbance at 600 nm was measured and recorded.Autoaggregation result was determined using the following equation: Note A 0 indicates the absorbance at time 0 h.A t indicates the absorbance every hour, up to 5 h.To determine the co-aggregation potential of LAB, the modified method of Pessoa et al. [40] was used.Briefly, cell suspensions of selected LAB and 5 diarrheagenic E. coli strains were prepared.Then, 2 mL of mixed suspensions (1 ml of LAB cell suspension and 1 mL diarrheagenic E. coli) were vortex and incubated for 5 h at 37 o C co-cultured.Also, 2 ml of each (LAB cell suspension and diarrheagenic E. coli strains cell suspensions) were incubated at the same conditions (control).The absorbance at 600 nm wavelength of co-cultured and mono-cultured suspensions were measured using UV spectrophotometer (Chongqing Gold, China).The co-aggregation in % was calculated as written below: Ax and Ay represents absorbance of LAB and diarrheagenic E coli strains separately.A(x + y) represents the absorbance of mixed culture of LAB suspensions and diarrheagenic E coli suspensions.

Evaluation of antibiofilm potential of LAB
Due to the protective properties of LAB against pathogens and their associated biofilms, fifteen selected LAB that previously produced efficient inhibition against EAEC H40C was analysed to produce antibiofilm agents against a characterized EAEC 042 strain [41] using modified method of Jadhav et al. [42].Three dilutions of CSF of LAB cells (1:1, 1:10 and 1:100) were prepared.180 µl of high glucose Dulbecco′s Modified Eagle′s Medium (DMEM) broth (Sigma Aldrich, USA), was placed into each 96-well flat bottom polystyrene plates together with 15 µl of CFS of LAB and 5 µl of EAEC 042 culture to make a total of 200 µl suspensions.A high glucose DMEM broth with no EAEC 042 was used as control.Absorption of 595 nm OD was measured for initial growth inhibition; the sealed plates were aerobically incubated for 18 h at 37 0 C. The plates were washed thoroughly using 200 µL of sterile PBS in a microplate washer (Global diagnostics Micro wash 1100) thrice, the air-dried plate was fixed for 10 min using 75% ethanol.The plate was stained for 5 min with 0.5% crystal violet, then, rinsed to remove the unbound dye.The plate was dried and eluted using 200 µL of 95% ethanol for 20 min.
The biofilm produced was quantified using microplate spectrophotometer (BioRad, Richmond, CA, USA), the optical density of the eluted crystal violet was measured at 570 nm.The amount of the stain absorbed determined the quantity of the biofilm formed.
The biofilm inhibition was expressed in percentage and was calculated using the following formula:

Evaluation of antibiotics susceptibility pattern of selected lactic acid bacteria
The minimum inhibitory concentration (MIC) of overnight cultures of 15 selected potential probiotic LAB isolates was determined using AST-GP75 test cards with Vitek 2 (Biomerieux diagnostics, France) equipment according to the manufacturers` instructions.The MIC results were interpreted as resistant or susceptible according to European Food Safety Authority (EFSA) [43,44] breakpoint guidelines.

Haemolytic pattern of lactic acid bacteria
The haemolytic potential of all LAB isolates was observed using a previously described method [45].Fresh LAB were streaked on blood agar supplemented with 5% human blood and incubated at 37 o C for 24 h.Haemolytic properties such as β-, α-and γ-haemolysis production were observed.LAB isolates with γ-haemolysis were recorded as non-haemolytic.

In-vivo stimulation of immune markers by selected lactic acid bacteria Experimental animals
The use of animals for experimental purpose was approved by Afe Babalola University Ethical Committee with the approved reference number AB/EC/19/06/047.The procedures involving the careful use and proper handling of animals was judiciously followed.Male Swiss mice (5 wk) of 22 ± 4 g weight was obtained from Animal Breeding Experimental Center, Ekiti State University Nigeria and housed in clean ventilated cages with appropriate animal house condition (12/12 light, temperature, humidity).The mice were allowed to acclimatize to their new environment for 1 week and fed with right proportions of laboratory mice pellets and water ad libitum.During the study, the weight of the mice were monitored every 4 days with a table top weighing balance, this was done till the end of the experiment.

Preparation of bacterial strains
Freshly prepared L. plantarum A011 and L. rhamnosus A012 were cultured using MRS-cys at 37 o C for 20 h in anaerobic condition.1.0 × 10 8 cfu/mL of the cells were harvested, centrifuged for 10 min at 4,000 rpm using compact centrifuge (Biocompare, USA).The cell pellets were rinsed two times, re-suspended in 10 mL of sterile PBS.The suspensions were prepared daily for oral use.

Experimental design
After a week of acclimatization, the mice were initially divided into seven groups of six mice per group.The first four groups were administered with an immunosuppressive agent, cyclophosphamide (CTX), and were classified as immunosuppressed group, the next two groups were given L. rhamnosus A012 and L. plantarum A011 respectively, they were classified as immunocompetent group, and the last group was given PBS and was classified as normal group.The detailed of the treatment in each group is as follows: group 1 (CTX + PBS) received cyclophosphamide 80 mg/kg BW/d via intraperitoneal injection (i.p) for three days, thereafter, received PBS (vehicle) for 15 days.Group 2 (CTX + Lev.) received cyclophosphamide 80 mg/kg BW/d via i.p. for three days and treated orally with a standard drug levamisole hydrochloride 40 mg/kg for 15 days.Group 3 (CTX + L. rham) received cyclophosphamide 80 mg/kg BW/d via i.p. for three days, thereafter, treated orally with 0.2 ml of L. rhamnosus A012 (1.0 × 10 8 cfu/mL) for 15 days.Group 4 (CTX + L. pla) received cyclophosphamide 80 mg/kg BW/d via i.p. for three days and treated orally with 0.2 ml of L. plantarum A011 (1.0 × 10 8 cfu/mL) for 15 days, Group 5 (L.rham alone) received 0.2 ml of L. rhamnosus A012 (1.0 × 10 8 cfu/mL) orally for 18 days.Goup 6 (L.pla alone) received 0.2 ml of L. plantarum A011 (1.0 × 10 8 cfu/mL) orally for 18 days and group 7 (PBS alone) received 0.2 ml of PBS orally for 18 days.

Analysis of immune organ index and white blood cells quantification
As described above, the study was carried out for 18 days.The experimental mice were euthanized by inhalation of phentermine hydrochloride at 0.1-0.2ml depending on the body weight of the animal to induce loss of consciousness.The blood of each mice was collected from abdominal aorta and dispensed immediately in EDTA bottle for whole blood analysis and sterile sample bottle to prepare serum.Cervical dislocation was carried out, the colon and the spleen were harvested and placed immediately in PBS, then refrigerated for ELISA analysis.The spleen index was calculated as follows: Spleen or thymus indices (mg/g) = spleen or thymus weight (mg) Bodyweight (g) The white blood cells in the whole blood previously collected were microscopically counted with haemocytometer.and recorded as number of cells/ µL.

Cytokine quantitation
The serum and the spleen concentrations of TNF-α, IL-6, and IL-10 were determined using ELISA kit (Leg-endMax™, BioLegend, U.K) at room temperature according to the manufacturers` instructions.The quantity of pro-and anti-inflammatory cytokines produced were measured using microplate reader at 450 nm.The concentration of each cytokine was extrapolated using line of regression from the standard curves of TNF-α, IL-6, and IL-10 and expressed as pg /mL.

Statistical analysis
Graph pad prism 5 and 8.0 statistical software program was used to analyze the results.The statistical significance of the data was determined with one-way ANOVA and the p values less than 0.05.

Diversity of lactic acid bacteria in human breast milk and infant faeces
Ninety-three LAB strains were identified from the 2 samples (i.e.breast milk and neonates' faeces  1).
The viable cells of LAB induced large zones of inhibition against the tested pathogens (Table 1).Fourteen LAB isolates produced antimicrobial activities against all tested diarrheagenic E. coli strains.Eighty-one (87.10%) strains of LAB produced antimicrobial activity against EPEC H62E, E. faecalis A077 produced the largest zone that is designated as ++++ (> 20 mm), 91.40% LAB produced zones of inhibition against ETEC H40B with E. durans A004 having the highest zone that is > 20 mm.76.34% LAB produced zones of inhibition against EAEC H40C in which E. durans A098 produced the highest zone that is > 20 mm, 73.12% LAB produced antimicrobial activity against EIEC H68D, L. pseudomesenteroides A082 had the highest zone that is > 20 mm.93.55% LAB produced antagonistic effect against STEC H77E, L. pseudomesenteroides A019 produced the highest inhibition that is > 20 mm.
The inhibition of growth of 17 LAB against diarrheagenic E. coli strains in co-culture experiment was observed (Fig. 2a-e).L. plantarum A011, L. rhamnosus A012 and L. rhamnosus A072 inhibited the growth of all the tested pathotypes of diarrheagenic E. coli at 16 h, however, P. pentosaceus A074, L. pseudomesenteroides A044, W. cibaria B3a and L. pentosus B1b inhibited the growth of all pathogens at 16 h and 24 h (Fig. 2a-e).
All the tested LAB isolates (100%) were non haemolytic (γ-haemolysis).However, the screened LAB isolates exhibited varying resistance pattern to tested antibiotics.Larger percentage of LAB showed resistance to ampicillin (80%), 26.7% were resistant to tetracycline, while 33.3% showed resistance to erythromycin.However, L. rhamnosus A012 was susceptible to all the antibiotics, while L. plantarum A011 and L. pentosus A4c were resistant to only ampicillin while L. pentosus A028 was resistant to only clindamycin (Table 2).

Lactic acid bacterial isolates survived simulated gastrointestinal conditions
Among the 93 LAB isolates screened for their tolerance to low acidic condition, 92.47% of the isolates demonstrated high rate of survival after exposure to pH 3 for 3 h (83.36-99.84%survival rate) out of which 54.54% of LAB had 1 log 10 reduction.However, 1.08% LAB isolates had complete growth inhibition (completely killed) while, 6.45% of the isolates produced higher cfu/mL in acidic condition (Supplementary Table 2).Meanwhile, at pH 2, the growth rate was 55.91%, in which 4.3% had 1 log 10 reduction of cfu/mL, 19.35% had 2 log 10 reductions, 26.88% had 3 log 10 reductions and 6.45% had 4 log 10 reductions after 3 h of incubation.On the other hand, 44.09% isolates had no growth after exposing them to pH 2 for 3 h (Supplementary Table 2).

Immunomodulatory activity of lactic acid bacteria
General observation following cyclophosphamide intraperitoneal injection.
The mice in group 1, 2, 3 and 4 showed lassitude, lackluster, fur piloerection, and lack of enthusiasm to environmental activity on day 1 after the intraperitoneal administration of CTX, they had loose-watery bowel movements, with reduction in their food and water intake especially on day 2-3 after the intraperitoneal administration of CTX.There was no weight loss at the onset of the experiment (Fig. 6a).However, following administration of cyclophosphamide (i.p), the immunosuppressed groups (CTX + PBS, CTX + Lev, CTX + Lev and L. rh + CTX) experienced a drastic weight loss when compared with immunocompetent groups (L .rhalone L.pl alone and PBS alone) as shown in Fig. 6a.Nonetheless, after the intervention with lactobacilli and levamisole hydrochloride, there was a significant increase (p˂0.05) in body weight of mice in these treatment groups compared to mice in the untreated group (CTX + PBS group).This finding infers the ability of Lactobacillus sp in restoring the weight loss due to administration of cyclophosphamide.Groups 5, 6 and 7 only had gradual body weight increase (Fig. 6a).
The spleen index value in CTX + PBS group was significantly reduced compared to CTX + Lev, L. rh + CTX and PBS alone groups with p˂0.0001.However, a reduction was also observed in L. plant + CTX group compared to CTX + Lev, L. rh + CTX and PBS groups but with no significance difference (Fig. 6b).There was also a significant reduction in immunocompetent groups (L.rh alone and L. plant alone) as compared to CTX group with p value ˂0.001 (Fig. 6b).The white blood cell count of the experimental mice was increased significantly in CTX + Lev, L. rh + CTX compared to CTX + PBS and PBS groups with significant difference of p < 0.0001 (Fig. 6c).
The effect of treatment with L. rhamnosus A012 and L. plantarum A011 on both serum and spleen level of TNFα, IL-6 and IL-10 in mice is presented in Fig. 7a and c, & Fig. 8a and c.Administration of CTX in mice caused significant increase in TNF-α level (p < 0.001), however, L. rhamnosus A012 and L. plantarum A011 treatment significantly decreased (p < 0.001) the spleen and serum level of TNF-α showing ameliorative effect of Lactobacillus sp.(Figures 7a and 8a).In like manner, intervention treatment with L. rhamnosus A012 and L. plantarum A011 also significantly reduced the level of IL-6 in both serum and spleen of the mice as shown in Figs.7b and 8b.
However, the administration of CTX significantly decreased (p < 0.05) the serum and spleen level of IL-10, but this was averted by the intervention of L. rhamnosus A012 and L. plantarum A011 (Figs. 7c and 8c).Administration of Lactobacillus species significantly (p < 0.001) reduced the concentrations of both TNF-α and IL-6 in the serum and tissue of mice treated with L. rhamnosus A012 and L. plantarum A011 in comparison with the CTX + PBS treated mice, ameliorating the effect of cyclophosphamide in the treated groups (Fig. 7a and b).In addition, there was a significant reduction of IL-10  in the serum and spleen of animals in CTX + PBS group compared to PBS group (control).However, after treating the animals with L. rhamnosus A012 and L. plantarum A011, the quantity of IL-10 produced in the serum and spleen of animals in these groups significantly increased (p < 0.01) showing anti-inflammatory ability of L. rhamnosus A012 and L. plantarum A011 and the ameliorating effect of LAB on cyclophosphamide induced immunosuppression mice.

Discussion
Breast milk was thought to be sterile until new facts emerge about the presence of beneficial bacteria, which protects infants from gastroenteritis infections, asthma and allergy [46].Likewise, the presence of LAB in the GIT determines a healthy gut and can protect a child from infection [47].In the current study, the diversity of LAB cultured from human breast milk and child faeces shows 93 lactic acid bacteria of 15 different species belonging to 4 genera viz.: Lactobacillus, Enterococcus, Weisella, Leuconostoc, and Pediococcus.We observed that Lactobacillus was the most frequent genera identified and L. plantarum was the most abundant specie in human breast milk (36.36%) and neonates' faeces (18.42%), respectively.This collaborates other studies in which Lactobacillus species were abundantly identified from breast milk [48][49][50] and faeces of breastfed infants [51].This is however contrary to a report where S. epidermidis and E. faecalis were predominantly identified from breast milk and neonates' faeces [33].Lactic acid bacteria have also been isolated from cattle, pigs, pickles, and sausage [37,52], showing that LAB can also be found in other sources.In this study, L. xiangfangensis was isolated from breast milk; this is novel specie that was previously isolated from Chinese pickle [53].In addition, E. thailadicus was identified from breast milk, supporting the claim that human milk-faecal microbiome consists of thousands of species of microorganisms that are yet to be identified [40].L. plantarum, L. rhamnosus and some other strains were found in both human breast milk and neonate's faeces, this supports the claim that composition of LAB in faeces of exclusive breastfeeding neonates usually reflect mothers breast milk [54].
An essential criterion for the selection of probiotic strains is the ability of LAB strains to exert antimicrobial effect against pathogenic microorganisms, through the production of some organic acid metabolites such as lactic, acetic, butyric, propionic acid.Hence, this study reports that L. rhamnosus A012, P. pentosaceus A074, L. plantarum A023, L. pseudomesenteroides A044, L. pentosus B1b, L. pentosus A028 L. rhamnosus A072, L. plantarum A011, L. pentosus A4c, L. plantarum A051, L. plantarum A051 and L. plantarum A014 produce significant inhibition against all diarrheagenic E. coli strains tested through various methods such as agar well diffusion, agar overlay, and time-kinetic study.Similarly, antimicrobial activity of LAB isolated from breast milk and neonates' faeces against E. coli, Shigella spp., Pseudomonas spp., Salmonella spp., and different pathotypes of diarrheagenic E. coli strains have been reported [51,55].In like manner, LAB isolates from different countries were also found to produce antimicrobial activity against pathogenic fungi such as Candida albicans and Grampositive bacteria like Bacillus cereus, Staphylococcus albus, Streptococcus faecalis, Streptococcus thermophilus.Similarly, LAB from fermented food produce antimicrobial activity against diarrheagenic E. coli strains [31].Furthermore, co-culturing LAB strains with diarrheagenic E. coli strains also showed its probiotic potential by completely inactivating the organisms within 24 h of contact.Interestingly, L. rhamnosus A072, L. plantarum A011, L. pentosus A028, L. plantarum A084, L. rhamnosus A012, L. pentosus B1b, and P. pentosaceus A074 inhibited all the diarrheagenic E. coli strains between 8 h and 16 h of contact.This study correlates the report of [29,33,36,37] and [30].
The lactic acid bacteria strains in this study exert their antimicrobial activity against the pathogenic strains by releasing inhibitory substances such as lactic acid, acetic acid, and other compounds against diarrheagenic E. coli strains.This claim is supported by high quantity of lactic and acetic acid released by some LAB strains, such as L. rhamnosus A012, thereby corroborating the report of [37] and [56].
The production of biofilm by many pathogenic strains such as S. aureus, S. viridans, K. pneumonia, P. mirabilis, P. aeruginosa and E. coli enhances antimicrobial resistance.Our study shows that lower dilution of CFS possessed antibacterial activity against EAEC strain 042, while at the high dilution of 1:100, L. plantarum A011 produced outstanding anti-biofilm potential showing the interference of LAB with the biofilm integrity of EAEC strain 042 to completely prevent biofilm formation.The antibiofilm mechanisms could be their ability to produce inhibitory substance or ability to form auto-and coaggregation.Hence, the antibiofilm activity exhibited by L. plantarum A011 and other LAB validates the reports that CFS of Lactobacillus strains could produce antibiofilm activities against pathogens [57]; [58,59].
The survival of LAB in gastrointestinal condition is an essential feature that must be possessed by potential oral probiotics for the strains to survive the gastrointestinal effect and get to the colon.In line with this, the current study reports the survival of LAB strains in both pH 2.0 and 3.0, although smaller percentage of the LAB strains survived pH 2.0, larger percentage of the strains survived pH 3.0 with the viable cells range of 8.05 and 8.16 log.However, few LAB strains were able to grow in the bile salt as their survival rate was over 100%.At the initial exposure to 0.3% bile salt, their viable cells reduced but were found improved and survived after 4 h of exposure to the stress condition indicating that these LAB strains may contain certain functional proteins that protect and allow better survival to the bile stress condition [60]; [61]; [62]; [63].The identified potential probiotics strains through survival in gastrointestinal conditions in this study include L. plantarum A011, P. pentosaceus A074, L. rhamnosus A072, L. rhamnosus A012 and L. pentosus A4c among others, this is in line with the characteristics of the potential probiotics that thrive in gastrointestinal conditions [64]; [65]; [66].In addition, these LAB isolates survived the adverse environment of consecutive gastrointestinal transit with survived viable cells range of 6.78 to 8.42 log.The survival ability of LAB isolates through GI tract condition may be due to the production of organic acid and/or production of resistant cell wall material [67], the production of bile resistance gene (bsh-1 and bsh-2) in LAB cells [68] or their evolutionary stress-sensing system defense mechanism which allow them to migrate across the intestine [69,70], the following strains P. pentosaceus A074, L. rhamnosus A012, L. pentosus A4c, L. plantarum A011 and L. rhamnosus A072 maintain their viability and falls within the range of 1.0 × 10 6 and 1.0 × 10 10 in simulated gastrointestinal environment [71].
Another essential criterion in the selection of probiotic strain is its ability to adhere to mucus and/or human epithelial cells.The adherence of LAB strains to mucus layer makes use of specific and non-specific mechanisms to exclude the pathogenic organism [72,73].However, according to van Zyl et al. [74] the activities of LAB are strain specific, therefore, the mechanisms of action used by one bacterial species to exclude other bacteria from the intestinal tract differ and may comprise microbemicrobe interactions mediated by binding to the mucosal interface of the host at specific sites of attachment [74].Cell surface hydrophobicity, a biophysical measurement uses cell-surface interaction mechanism.Therefore, LAB strains with high cell surface hydrophobicity showed affinity to non-polar interface with high adhesion to cell-surface interphase, thereby use non-specific mechanism to exclude the pathogens and prevent them from colonizing mucus cell lines.The current study demonstrated that the screened LAB isolates have the potential to colonize and adhere to epithelial layer of the intestine with the isolates displaying both high and low affinity to n-Hexadecane and Xylene, thereby, producing a nonspecific adherence through cell surface hydrophobicity.It has been reported that LAB isolates from faeces possess higher affinity to surface hydrophobicity [75], this is in accordance with the current study as LAB isolates from fecal samples demonstrated high percentage of cell surface hydrophobicity more than the isolates from breast milk.We therefore report LAB isolates to possess a protective ability on the host epithelia layer supporting [50] that Lactobacillus and Bifidobacterium species protect the epithelia layer of intestine from mechanical damage and bacterial infection.
The LAB strains in this study demonstrated both autoaggregation and co-aggregation quality as it was observed in L. rhamnosus A012, L. rhamnosus A072, L. pentosus B1b and P. pentosaceus A074.This shows that the LAB cell form biofilm which protect them from environmental stress or any irrational from response host cells [57][58][59].LAB also form aggregate around pathogens of the same species or different species, these are destroyed by their production of organic acid [75].The selected LAB strains excluded the diarrheagenic E. coli in the co-aggregation assay, and competitively displayed EPEC and EIEC, this indicates the ability of LAB to protect the GIT corroborating earlier researchers' reports that LAB possess the ability to produce adhesion property in-vitro forming a protective barrier through autoaggregation and co-aggregation [75][76][77].
According to WHO guidelines on the use of probiotics, probiotic strain should not possess any antibiotic resistance gene that is transferrable as this can pose a potential risk on human health [648].Consistent with early research [72,78], this study found that nearly all screened LAB isolates were susceptible to gentamicin, vancomycin, and kanamycin, in addition, the LAB strains were susceptible to tetracycline except P. pentosaceus A074, L. pseudomesenteroides A064, and W. cibaria B3a.The high resistance of LAB strains to ampicillin support [79].The LAB strains were also susceptible to erythromycin and clindamycin [72,80] contrary to other report [81] and [82].It is noteworthy that L. rhamnosus A012 was susceptible to all the 21 antibiotics used, correlating [83] report on L. rhamnosus GG being safe.However, L. rhamnosus A072, was resistant to clindamycin, ampicillin, streptomycin and erythromycin establishing the notion of strain specificity of probiotic.Therefore, we report that L. rhamnosus A012 could be a potential probiotic but cannot be co-administered with these antibiotics.However, L. pentosus B1b, L. pentosus A4c, and L. plantarum A011 strains could be co-administered with antibiotics as there has not been a report of their gene carry resistance gene.Potential probiotics must not haemolyse blood [84].This requirement was followed in this study as all the LAB strains possess γ-haemolysis (nonhaemolytic) [85,86].
Ability of probiotic strain to modulate immune responses is a desirable criterion of probiotics.Bifidobacteria and lactic acid bacteria play essential role using defense mechanism to protect host cell, they stimulate, modulate, and regulate immune system [87,88].Cyclophosphamide, as a chemotherapy in cancer treatment has also been clinically confirmed to be used in immune suppressor for nephrotic syndrome, granulomatosis with polyangiitis, and organ transplant among other conditions [89][90][91], hence, the reason for its selection in modelling immunosuppression.In this study, the administration of CTX resulted in weight loss, loss of appetite, inaction, uncontrollable turning of neck and diarrhea in immunosuppressed mice correlating the report of [91] where administration of CTX resulted in immunological damage.However, the treatment with levamisole hydrochloride and Lactobacillus species alleviated the immunological damage previously observed by significantly accelerating the weight increase, gastrointestinal stability and recovering of white blood cells in the treated mice, this indicates protective features of Lactobacillus strains [87].The spleens accommodate the immune cells, which protect the host and play a major role in regulating the immune responses [92,93].From our report, Lactobacillus alleviated the spleen injury caused by CTX to the immune organ thereby, protecting the spleen from further damage, correlating the previously reported work [89,90].
TNF-α triggers the immune system molecules to induce neutrophil activation [88].In this study, administration of CTX triggered immunosuppression thereby elevating the pro-inflammatory cytokines in mice.
However, oral administration of Lactobacillus species reversed the effect and prevented the excessive rise in the concentration of TNF-α and IL-6.Excessive production of pro-inflammatory cytokines can also result in apoptosis of epithelial cell, however, the intervention of Lactobacillus sp has been reported to ameliorate this effect as it down-regulated the production of TNF-α and IL-6 in the spleen, this has also been reported by [94] and [88], where Lactobacillus strains decreased the expression of TNF-α in contrast to [89] report in which Lactobacillus sp upregulated TNF-α.Meanwhile, healthy, and immunocompetent mice showed normal expression of TNF-α supporting the claim that TNF-α might be very low in a healthy individual [94].
Interleukin-6 response triggers the pro-inflammatory and an anti-inflammatory profile [95].The data presented in this study showed that IL-6 was upregulated with administration of CTX but downregulated with the intervention of Lactobacillus species and levamisole hydrochloride (p < 0.001).This supports the report of [94] and [89], in which Lactobacillus strains modulate the production of IL-6 cytokines.
IL-10, an anti-inflammatory cytokine, regulates the production of pro-inflammatory cytokines to maintains the immune system balance.IL-10 binds to IL-10R1 to initiate the signal transduction activity using Jak-STAT pathway [88].Through this, intestinal epithelial barrier is strengthened and serve as the central regulator of the inflammatory response [88,95].In the current study, the level of IL-10 was significantly elevated upon Lactobacillus treatment.Treatment with Lactobacillus species upregulated the production of IL-10, thereby strengthening and protecting the cells from injuries.In this study, L. plantarum A011 and L. rhamnosus A012 possessed anti-inflammatory effect, as these strains elevated the level of IL-10 in both serum and tissue than the standard drug (Levamisole hydrochloride).This indicates the ameliorating effect of Lactobacillus strains to restore gut microbes and damaged intestinal epithelial barrier.Our results, therefore, agrees with [89,90,96] and [88] claim, that Lactobacillus strains modulate IL-10 response in immunosuppressed mice.Thus, L. plantarum A011 and L. rhamnosus A012 have good potential to maintain the intestinal immune balanced through stimulation and regulation of cytokines (TNF-α, IL-6 and IL-10).
This study demonstrated the anti-diarrheagenic E. coli activities of lactic acid bacteria isolated from human breast milk and infant's faeces in vitro and their potentials to function as probiotic candidate in ameliorating the damaged effect of immune deficiency.The two selected Lactobacillus strains, L. plantarum A011 and L. rhamnosus A012 demonstrates promising probiotic properties both in vitro and in vivo.

Fig. 4
Fig. 4 Concentration (mg/mL) of organic acid produced by LAB

Fig. 5 a
Fig. 5 a and b: Survival of LAB in successive low pH and bile salt supplement

Table 1
Antibacterial activity of lactic acid bacteria against diarrheagenic Escherichia coli strains

Table 3
Autoaggregation of LAB and co-aggregation of LAB with diarrhoeagenic E. Coli strains