Isolation and Identification of Dominant Bacteria from Raw Donkey Milk Produced in a Region of Morocco by QIIME 2 and Evaluation of Their Antibacterial Activity

Recently, the interest in donkey milk has increased considerably because it proved high nutritive and functional values of their ingredients. Its chemical composition is widely studied, but its microbiota, especially lactic acid bacteria, remains less studied. This study focuses on analyzing, isolating, and identifying lactic acid bacteria and evaluating their capacity to produce biomolecules with antibacterial activity. Among 44 strains identified, 43 are Gram-positive, and most are catalase-negative and cocci-shaped. Five strains were selected to evaluate their antibacterial activity against Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli. Different induction methods allowed to amplify the antibacterial effects against these pathogenic strains.


Introduction
In recent decades, overuse and misuse of antibiotics has led to the emergence and spread of high bacterial resistance, making difficult to treat infections involving tremendous research efforts by the medical and scientific community [1,2]. is concern led the international authorities to adopt concerted plans to optimize the use of antibiotics and to promote the research for alternative solutions [3]. Since, research is focused in the searching for a new bioactive molecule with antibiotic capabilities.
LAB are microorganisms forming a group composed of bacilli and cocci [7]. e common characteristic of these bacteria is the production of lactic acid, which is the end product of the fermentation process of several sugars [8]. In addition, these germs represent a potential source of several metabolites with antimicrobial and antifungal activities like organic acids [9], reuterin, hydrogen peroxide, diacetyl [5] carbon dioxide, and bacteriocins [10][11][12][13]. ey are present in different microbial biotopes such as soil, plants, the digestive system of human, and dairy products [14,15].
Among these dairy products, we chose donkey milk (DM), which has attracted scientists' attention because of its convincing nutritive and functional elements [16,17]. Because of its chemical composition similar to human milk [18,19], it has therefore been used as an adequate alternative for infants with multiple food intolerances and allergies.
Whereas chemical composition of donkey milk is very much studied, its microbiota remains less studied; therefore, this study focused (i) to analyze basic DM parameters, (ii) to isolate and identify LAB, (iii) and finally evaluate their antibacterial effect.

Measurement of Some Physicochemical Parameters.
e milk pH was determined using a pH meter [21], the acidity was determined by using a methanolic solution of phenolphthalein as a colored indicator [21], and the density was determined according to Afnor [22]. e dry matter content was estimated by evaporation in a waterbath at 70°C and then oven drying for 3 hours [21].

Isolation of Bacteria.
e isolation was carried out by the successive depletion method on MRS medium agar and M17 medium agar (Biokar diagnostics). Seven subcultures were made until isolation of pure colonies.

Phenotypic and Biochemical Identification.
Gram stain was performed using the standard bacteriological procedure [23].
(1) Catalase Test. A colony was removed and then emulsified on a slide containing a drop of 3% v/v hydrogen peroxide.
(2) Kligler Test. Using a sterile needle, a colony was sown by simple pitting at the bottom of the agar tube (Kligler medium, Biokar diagnostics) and by tight streaks on the inclined surface of the tube, before being incubating at 32°C for 24 h. is double inoculation permits bacterial growth on the aerobic surface and in the largely anaerobic part of the tube.
(3) Mobility Mannitol Test. Using a sterile needle, a colony was removed and introduced into the medium up to 1 cm from the bottom of the tube, keeping the loop in the same trajectory at the entrance and at the outlet of the agar medium. Incubation was performed at 32°C for 24 h.

Molecular Identification
2.4.1. DNA Extraction. DNA extraction was performed by a chloroform phenol method described elsewhere [24]. Quantitation of the DNA was performed using NanoDrop Lite Spectrophotometer ( ermo Fisher Scientific).
Sequencing products were separated and detected in a 3730xl Genetic Analyzer (Applied Biosystems). Chromatogram revision and trimming was performed with the sequence scanner (Applied Biosystems), and alignment was obtained from ClustalX implemented in BioEdit [26].

Taxonomic Inference.
e 16SrDNA sequences were analyzed with QIIME 2 2019.10 [27]. e Sanger sequences in FASTA format were imported into a QIIME2 artefact, aligned with MAFFT [28], and a phylogenetic tree was inferred using fasttree2 [29]. e assignment to diverse taxonomic levels was performed with the q2-feature-classifier [30] and classify-sklearn Naive Bayes taxonomy classifier against the Green genes 13_8 99% OTUs reference sequences [31]. In addition, each sequence was analyzed using the alignment tool BLASTN [32].

Diffusion of Agar.
e well diffusion method was carried out following a modification of the Schillinger and Lücke [33] method. Petri dishes containing 20 mL of the LB medium supplemented with agar 0.6% (w/v) are inoculated aseptically a 10 6 CFU/mL bacteria suspension. After solidification, 5 mm wells were created. en, 50 μL of each supernatant was deposited in each well. All dishes were put at 4°C for 4 hours and then incubated at 37°C overnight. Two negative controls were used; One containing only the indicator strain, and the other, the modified MRS medium (Table 1), to test the effect on the indicator strains. e presence of an inhibition zone (Zi) around the wells is synonymous with the production of antimicrobial substances (one or more). Zi is determined following formula: Zi � diameter of the Zi obtained − diameter of the well(5 mm). (1)

Milk Analysis.
Milk stability tests have shown that analyzed milk is stable and does not contain any chemical contamination, such as formalin and alcohol like found by other studies [34].
Physicochemical parameters are given in Table 2. e titratable acidity of the milk is 16.3°D, which explains that the milk is not altered and that the pH of the DM is neutral; this is due to the low content in casein and phosphate [35].
is result is similar to those found by Guo et al. and Salimei [36,37]. Comparing with other milks, we find that the pH of DM is similar to human milk (7-7, 3) [19], but more basic than camel milk, cow milk, and goat milk, which is around 6 [38].

Microbiological Identification.
e phenotypic and biochemical characteristics were evaluated for the 44 isolated bacterial strains. e morphological results showed that all these bacteria are Gram-positive, except for one strain, and with dominance for cocci shapes. Most strains are catalase negative, whereas only 17 strains showed a positive result (Table S1, supplementary material). Our results are consistent with those found by de Garnica et al. [39]. e strains were tested for their ability to use glucose and lactose in the Kligler medium [24]. All strains fermented glucose without gas production, as expected from homofermentary bacteria. None of the tested strains were able to produce H 2 S, the majority ferment lactose and mannitol, and they are immobile [24,39]. e high level of lysozyme in DM selects for the presence of Gram-positive bacteria, as observed from the characterization of analyzed samples [40].

Molecular Identification.
e molecular identification of LAB from 16S rDNA sequences showed the existence of 9 groups of bacteria (Figure 1), resolved at different taxonomic levels.
A first group is characterized by the genus Bacillus with 15 isolates, with a dominance of the species Bacillus amyloliquefaciens, as inferred from the BLASTN search. A second group was characterized by the genus Enterococcus with 9 isolates. A third group included 7 isolates from the genus Staphylococcus. ree isolated from the identified Aerococcus viridans were included in a fourth group. A fifth group was characterized by the genus Leuconostoc with a frequency of 2 isolates. e other groups included a single isolate, Rothia, Acinetobacter, and Streptococcus. e taxonomic inference combining a BLASTN search and the importation of moderate Sanger sequence data into the standard NGS metagenomic pipeline resulted in congruent and reliable taxa assignation.
All these species have been previously isolated and studied from dairy products, such as cow's milk, goat's milk, and sheep's milk [39,[41][42][43][44][45]. Another study aimed at characterizing the bacterial community of donkey milk and showed that the basic microbiota was composed of genera including LAB and species typically present in plants, soil, and water [46].

Antibacterial Activity.
e LABs were put under different culture conditions to optimize the production of the antibacterial substances; the inhibitory spectra of these bacteria are given in Table 3.
ese results reveal that the supernatants of the 5 studied strains show an inhibition against the pathogenic indicator strains, with the exception of Aerococcus viridans and Enterococcus viikkiensis. ese species showed a weak inhibition under different culture conditions against Staphylococcus aureus and Escherichia coli, respectively. All strains exhibited significant inhibition against Listeria monocytogenes. e maximum activity was observed with Tween 20, UV, and in coculture with Listeria with diameters of 28, 20, and 21 mm respectively. Subsequent studies have demonstrated the ability of Tween 20 to increase bacteriocin production and as a result demonstrated inhibitory activity in its presence [47][48][49]. Furthermore, another study has shown that the regulation of bacteriocin expression depends on an external stimulus such as cocultivation with other strains [50]. Referring to the controls (Table S2), the inhibitory activity observed in the presence of SDS appears to be caused by SDS itself and not by lactic acid strains. is hypothesis has been confirmed by Chen and Yanagida [51]. e Scientific World Journal

Conclusion
e present study has shown that the five lactic strains tested are capable of producing active biomolecules with inhibitory activity on the pathogenic strains: Listeria monocytogenes, Escherichia coli, and Staphylococcus aureus.
is antibacterial activity could be due to the production of bacteriocins or other biomolecules. Studies are underway to characterize and identify these antimicrobial compounds.
Data Availability e microbiological and molecular data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Supplementary Materials
e supplementary material of the research article "Isolation and identification of bacterial population of donkey milk from a region of Morocco by QIIME 2 and evaluation of their biomolecules" by Derdak et al. is composed by two tables. Table S1: phenotypic and biochemical characteristics of isolated lactic bacteria.