Phenotypic and Genotypic Identification of Bacteria from Women Breast-Milk and the Feces of their Childs in the Western Region of Algeria

1Laboratoire de Génétique moléculaire, Département de Génétique Moléculaire Appliquée. Université des Sciences et de la Technologie d’Oran Mohamed Boudiaf (USTO-MB), Algérie. 2Institut de Recherches Microbiologiques JM Wiame, Av. E. Gryson 1, B-1070 Bruxelles, Belgique. 3Laboratoire de Physiologie de la Nutrition et Sécurité Alimentaire, Département de Biologie, Faculté des Sciences, Université d'Oran 1, Ahmed Ben Bella, Algérie. 4Service de Bactériologie, Etablissement Hospitalier Universitaire d’Oran (EHUO), Algérie. 5Département de Biotechnologie, Faculté des Sciences, Université d’Oran 2, Mohamed Ben Ahmed, Algérie. 6Laboratoire de biologie du développement et de la différenciation, Université d’Oran 1, Ahmed Ben Bella, Algérie.

found in the infant's feces and their mother's milk 7 8 9 10 11 .The microbiome of human milk is very diverse.Sequencing of the 16S rRNA gene has shown the complexity of the bacterial community in breast-milk 12 13 14 , its variability among women and its relative stability over time 15 12 .Culture-independent methods consistently show Staphylococcus, Pseudomonas, and Streptococcus having the most relative abundance 16 12 17 .
On the other hand, culture-dependent methods have shown the prevalence of the facultative anaerobic genera Staphylococcus, Streptococcus and Propionibacterium.Other genera isolated comprise Bifidobacterium, Enterococcus, Lactobacillus, Veilonella, and Rothia 9 18 19 17 20 .The presence of lactic acid bacteria (LAB) in human milk is well documented 7 21 19 .
Therefore, milk is a source of lactic bacteria for the infant gut 7 19 .LAB form a large group of anaerobic and aero-tolerant, non-sporulating, Gram-positive, catalase and oxidase-negative rods and cocci, generally with complex nutritional requirements.They produce antimicrobial substances, such as bacteriocins and organic acids, which can inhibit the growth of gastro intestinal pathogens in the infant's gut 22 23 24 25 .Bacteriocins have important potential applications in food and pharmaceutical industries 26 27 The transmission of LAB with antibacterial activity through breast-feeding could play an important role in the prevention of intestinal infections and in the establishment of a healthy gut microbiome in the infant.The aim of our research was to identify the bacteria from mother-infant couples in Algeria.The second aim was to assess the antibacterial properties of LAB from breast milk and infants' feces.Few publications have addressed the question of the antimicrobial activity of LAB from breast-milk or infants, despite its potential contribution to their probiotic potential 28 29 30 .
Moreover, previous studies on breast milk and infant gut microbiota have been conducted mostly in western countries or China.Yet there are geographical differences in infant's gut microbiota and environmental factors, such as diet and familial environment, are known to influence its composition 31 6 .In our work, we have screened mother-infant couples in Algeria, a country where few studies have been conducted in the field 32 .

Sampling and isolation of LAB
The 32 couples mothers-infant were recruited from the hospital "Etablissement Hospitalier Universitaire d'Oran, EHUO".The collected breast milk samples and feces were analyzed within the Service de Bactériologie, Etablissement Hospitalier Universitaire d'Oran (EHUO).
All participants were selected according to the following criteria: healthy women without current or past infection, full-term pregnancy and absence of infant or maternal perinatal problems.The infants were full-term vaginally delivered and breastfed.The mothers were informed of the procedures and the aims of the experiment, and gave their consent to the taking of samples.The milk samples were collected in sterile tubes, by manual expression using sterile gloves and after wiping the nipples and areola with a swab soaked in sterile water.The first drops of milk were not collected.Feces samples of the newborns (1g) were collected with sterile swab immersed, in 9 mL of sterile saline to allow the dispersion of samples.
Samples were collected from 32 mothers and their infants aged from 1 to 6 months.The samples were processed immediately after collection.Serial dilutions of the samples were plated in triplicate on specific culture media: de Man, Rogosa and Sharpe agar (MRS, Oxoid) supplemented with 0.5g/L of L-cysteine (MRScys) and Bifidobacteria Selective Medium agar (BSM) 33 .The plates were incubated at 37°C under anaerobic conditions (AnaeroGen, Oxoid) for 48 h to 72 h.Colonies with different morphologies were streaked on the appropriate medium.Purified isolates were tested for catalase and oxidase, and submitted to Gram staining.All isolates which were catalase-negative, oxidase negative and Grampositive were kept for further analysis and stored at -80°C as frozen cultures in MRS broth with 50% glycerol.

Identification of the isolates
An identification and biochemical characterization of some isolates was obtained with the VITEK® 2 Advanced Colorimetry™ (BioMérieux).Cell suspensions and subsequent analyses were made according to the manufacturer's recommendations.
For the molecular identifications, an almost complete 16S rRNA gene fragment was amplified with the primers pH (AAGAGGTGATCCAGCCGCA) and 10-27Fd (a slightly shorter pA primer AGTTTGATCCTGGGTCAG) 34 or BifiFd (TYMTGGCTCAGGATGAAC).The latter primer corresponds to position 17 to 34, which is conserved in lactic acid bacteria and bifidobacteria, and was used when the initial amplification with the 10-27Fd primer failed.We used a standard PCR program of: 1 min.at 95°C followed by 40 cycles of 30 s at 95°C, 30 s at 55°C and 2 min at 72°C, and ending by 10 min final elongation at 72°C in a T100 Thermocycler (BioRad).The amplification products were sequenced (Beckman Coulter Genomics, UK) with the primers used for the amplification.
A preliminary Blast analysis 35 of the sequences provided the genus to which each isolate belonged.The MEGA 5.2 software 36 was used to build phylogenetic trees from the multiple alignments with the type strains of the genus, using both the neighbour-joining method 37 and maximum likelihood.A taxonomic allocation was inferred from the closest reference strains the isolates clustered with.All sequences were deposited in GenBank under the access numbers KY552930 to KY552956.

Assay of antibacterial activity
Six commonly used indicator strains: Escherichia coli ATCC 25 923, Pseudomonas aeruginosa ATCC 43 300, Staphylococcus aureus ATCC 25 923, Listeria innocua ATCC 29 523, Listeria ivanovii ATCC 29 723 and Micrococcus luteus ATCC4698, were used for the detection of antimicrobial activity.They were cultivated in TSB (Tryptic Soy Broth, Difco) and 200 µl of the indicator culture were spread on to the surface of TSA (Tryptic Soy Agar, Difco) plates, to make indicator plates.Cell-free supernatants of the isolates to be tested were obtained by centrifugation of fully grown cultures at 13000 rpm for 5 min and filtration of the supernatant through a 0.45µm filter.The pH of all supernatants was adjusted to pH 6.5 with NaOH 1M.
Antibacterial activity was tested by the paper disc diffusion assay 38 and by the welldiffusion assay.In the first case, sterile paper discs (9 mm antibiotic assay paper discs) were placed on the surface of the indicator plate immediately after spreading and 100 µl of the cell-free supernatant of the isolates to be tested were deposited on the paper discs.The plates were incubated for 18 h to 24 h at 37°C.In the second case, wells were aseptically punctured into the indicator plates and filled with 100 µl of the cell-free supernatants.The plates were left at 4°C for 30 min and then incubated at 37°C for 24 h.The antimicrobial activity of the isolates was estimated from the size of the inhibition zone around the wells or the paper disc 39 .

Proteolytic treatment
A 20-µL volume of proteolytic enzymes (proteinase K, Thermofisher or trypsin) in their recommended buffers, and at a final concentration of 0.1mg/mL, was added to 200µL of supernatans (filtered and neutralized as stated before).The tubes were incubated at 37°C for 2 hours, and then puted at 100°C for 5 min to stop proteolysis.

Acid and bile tolerance test
Resistance to bile was tested as stated in 40 41 .In short, cells pretreated in PBS pH2.0 for 3 hours, were used to inoculate MRS medium with 0.2% and 3% bile (bovine bile, Sigma).Growth was assessed after 24h anaerobic incubation at 37° by plating serial dilution of the cultures.

Statistical analysis
The statistical analysis was performed with SPSS 20 software.The Pearson's tests were applied on different species of bacteria from breast milk and infant feces; urban and rural lifestyles of mothers.In the descriptive analysis, correlation coefficient was used for statistical analysis of this study.A P-value of <0.05 was considered to be statistically significant.

Diversity of lactic bacteria in breast milk and infant's feces
Suitable dilutions of the milk and feces samples from the 32 mother-infant couples were spread on MRS-Cys and BSM agar plates.For each sample, colonies with different phenotypes were picked up and purified.A total of 155 isolates We noted that Enterococcus feacium was isolated in the majority of the milk and fecal samples.
A positive correlation is observed for 6 groups and negative for Enterococcus feacalis.For Leuconostoc mesenteroides and Lactococcus lactis, the correlation is significant at the level of 0.01 with (P-value= 0).
According to the mother's lifestyle, we noticed that the genus Enterococcus was the most frequently isolated from rural mother's milk as well as urban mother's milk, respectively : E. faecium  1 and Table 3).

Antimicrobial activity
All LAB isolates were tested for their antimicrobial activity against 6 indicator strains (E.coli, L. innocua, L.ivanovii, M. luteus, P. aeruginosa, and S. aureus).Thirty isolates (21% of all LAB isolates) had a marked inhibitory effect against at least one of the indicator strains in a plate assay.Isolates with antimicrobial activity were not distributed evenly between the different species (Table 4).None of the 40 E. durans isolates (group 5 in Vitek2-identified Enterococcus isolates) had any antagonist activity against the indicator strains, and neither did the Leuconostoc isolates.Most of isolates with antibacterial activity belonged to the species E. faecium.Nine isolates were antagonist against more than one indicator strain (Table 5).Multi-antagonist isolates were found among E. faecalis (M4) and E. faecium (D21, D26 and D27).
Interestingly, none of the isolates had any effect on E. coli or S. aureus, but a high proportion (20 of the 30: 67%) of the antagonist isolates had an inhibitory effect on Pseudomonas.Isolates D8 (E.faecium) and M29 (E.faecalis) and the Lactococcus isolate had the highest antibacterial activity against P. aeruginosa.Large inhibition zones (of more than 30 mm) were also produced by four E. faecium isolates against M. luteus (Table 5).
The active supernatant retained their antibacterial activities after heat treatment (30 min at 100°).A treatment with proteinase K or trypsin results in the loss of the antimicrobial activity of all the active supernatants.This suggests that the antimicrobial activity is due to a heat stable, peptidic, bacteriocin-like product.
Most antagonist strains grew in the presence of bile salt (Table 5), an interesting capacity when looking for potential probiotic strains.

DISCUSSION
Our study was done on a sampling of the Algerian population, 32 mothers-child couples resident in rural regions (n=16) and others in urban regions (n=16).Mother's maternal milk and feces of their babies aged from 1 and 6 months old.
Most of the LAB isolated during this study belonged to the genus Enterococcus.This confirms what has been related in other studies were Enterococcus was predominant in breast-milk and infants feces 17 .
Studies have indicated that Enterococci are the most commonly isolated species during the first two months of infancy, with Lactobacilli being the second most common early colonizer 19 .Our results are also in accordance with previous studies of human milk based on a culturedependent approach where S. epidermidis was one of the predominant species 19 17 .Staphylococcus appears also as a dominant genus through the 16S rRNA gene abundance in culture-independent approaches 16 17 .
No significant difference (P-value > 0.05) was observed for four genera : Lactobacillus fermentum, Enterococcus durans, Enterococcus feacalis, Staphylococcus epidermidis isolated from the babie's feces and the maternal milk.This means that there is a homogeneous distribution of these genera in these two environments.Our results are in agreement with the previous studies which showed that the bacterial composition of the fecal microbiote of the breast-fed babies reflects that of the maternal milk 42 Our data show the presence of lactobacilli (with 8.88 %) only in the maternal milk of resident mothers in rural areas 43 noticed that lactobacilli tends to have more important percentages in the milk of the resident women in rural areas.Our results are also in agreement with previous studies realized in South Africa, in Japan and South Korea 44 .Another Japanese study showed that rural women seem to have a higher prevalence of L. reuteri colonization than women from the other countries.This may be related to the wide use of functional foods, probiotics and various fermented foods as an important part of the Japanese diet 45 .
In this study, the diet of lactating mothers was using a questionnaire that in rural maternal local bread, local fermented cheeses, raw goats' milk and druid fruit consumption was higher suggesting that the incorporation of probiotics in the mother's diet before delivery and in the infant diet during breastfeeding may positively influence the maturation process of gut immunity.
In our study, bifidobacteria was not identified as noticed in our previous works 46 .On the other hand, the majority of the papers published TALHI-MEKHICI et al.: STUDY OF BACTERIA FROM WOMEN BREAST-MILK previously show the presence of Bifidobacterium.This could reflect a diminished importance of members of this genus in the Algerian-motherinfant couples, due to the differences in nutrition, the way of life and genetics.All this factors influenced microbial composition of human milk 47 .
We have identified in this study, the species Enterococcus faecium, E. faecalis, Lactobacillus fermentum and Lactococcus lactis as the most promising in terms of antagonism.Some recognized probiotic strains belonged to those species 48 .As we tested the antimicrobial activity on a limited range of commonly used indicator strains, it can be expected that a more extended survey, on a higher number of strains, would reveal a higher proportion of antagonists among the milk or feces isolates.
Our results suggest that the production of bacteriocins could explain the antimicrobial activity of the isolates.A conventional bacteriocin would not act on Gram-negative species.Yet, we have observed a high proportion of isolates with an antibacterial effect on the Gram-negative P. aeruginosa, a less frequent property of antagonistic LAB, although observed in some studies 49 .If the peptidic nature of the antagonist molecule was to be confirmed, its mechanism of action would be worth investigating.
Eight isolates (D21, D26, M7, M10, M24, M29, M4, M25) cumulate an antagonist effect against P. aeruginosa with an effect on the Grampositif species of Micrococcus and Listeria.This could reflect the possibility that some LAB possess several antimicrobial mechanisms.
In this study we performed a screening of 30 antagonist strains.The advantages of these thirty particular isolates deserve investigation of a new in vivo study to determine their produced antimicrobial substances.Some of these strains could be exploited in the development of dairy formulas for babies or new food products.Bacteriocin-producing strains can also play an important role in food fermentation and preservation.

CONCLUSION
This present research is the first study related to the microbiome of women breast-milk from different regions, done in Algeria.This work indicates that lifestyle of mothers (rural and urban) influences the presence of bacteria in their breast milk, which we have noted for the 32 milk samples of resident mothers from different regions.A significant difference is observed for two species, Lactobacillus fermentum and Staphylococcus epidermidis.
Our study confirms that human milk and infant feces analyzed in pairs share the same species, indicating that breastfeeding could contribute to the bacterial transfer from mother to child and thus colonize the intestinal flora of the baby.
To compare the flora of breast milk according to the lifestyle we suggest increasing the number of samples to confirm this result and collect accurate data on lifestyle such as diet, social conditions and economical life.

Fig. 1 .
Fig. 1.Distribution of different isolates (species) of the milk mothers from the rural and the urban regions

Table 1 .
TALHI-MEKHICI et al.: STUDY OF BACTERIA FROM WOMEN BREAST-MILK Phenotypic identification of Enterococci by VITEK® 2 Advanced Colorimetry™

Table 2 .
Comparison of frequencies of bacterial genera detected in breast milk and infant feces

Table 4 .
Distribution per species of isolates with antibacterial activity

Table 5 .
Probiotic and antagonism properties of the isolates with antimicrobial activity