Unveiling the Antibiotic Susceptibility and Antimicrobial Potential of Bacteria from Human Breast Milk of Pakistani Women: An Exploratory Study

Abstract

Background. Human life quality and expectancy have increased dramatically over the past 5 decades because of improvements in nutrition and antibiotic’s usage fighting against infectious diseases. Yet, it was soon revealed that the microbes adapted to develop resistance to any of the drugs that were used. Recently, there is great concern that commensal bacteria from food and the gastrointestinal tract of humans and animals could act as a reservoir for antibiotic resistance genes. Methodology. This study was intended for evaluating the phenotypic antibiotic resistance/sensitivity profiles of probiotic bacteria from human breast milk and evaluating the inhibitory effect of the probiotic bacteria against both Gram-negative and Gram-positive bacteria. Results. The results point out that some of the isolated bacteria were resistant to diverse antibiotics including gentamycin, imipenem, trimethoprim sulfamethoxazole, and nalidixic acid. Susceptibility profile to certain antibiotics like vancomycin, tetracycline, ofloxacin, chloramphenicol, streptomycin, rifampicin, and bacitracin was also observed. The antimicrobial qualities of cell-free supernatants of some probiotic bacteria inhibited the growth of indicator bacteria. Also, antimicrobial properties of the probiotic bacteria from the present study attributed to the production of organic acid, bacterial adhesion to hydrocarbons (BATH), salt aggregation, coaggregation with pathogens, and bacteriocin production. Some isolated bacteria from human milk displayed higher hydrophobicity in addition to intrinsic probiotic properties like Gram-positive classification, catalase-negative activity, resistance to gastric juice (pH 2), and bile salt (0.3%) concentration. Conclusion. This study has added to the data of the antibiotic and antimicrobial activity of some probiotic bacteria from some samples of Pakistani women breast milk. Probiotic bacteria are usually considered to decrease gastrointestinal tract diseases by adhering to the gut epithelial and reducing population of pathogens and in the case of Streptococcus lactarius MB622 and Streptococcus salivarius MB620 in terms of hydrophobicity and exclusion of indicator pathogenic strains.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

Resistance to antibiotics is a worldwide health concern that is essential to be addressed from diverse perspectives. The capability of microbes to endure and flourish when exposed to antibiotics that they were initially vulnerable to is called antimicrobial resistance. Antibiotics are extensively misused and overused in humans, animals producing dairy and meat, aquaculture, and agriculture, and this unquestionably contributes to the occurrence of antimicrobial resistance [1, 2]; resistance to antibiotics can be inherent or attained [3–5]. Inheritance of intrinsic resistance is a natural antimicrobial-resistant trait presented by some probiotic bacteria. Intrinsic resistance can be explained as the nonsusceptibility of a bacterium to a known lethal concentration of antibiotic at the appropriate dose. This type of resistance typically does not compromise the safety of bacteria and is not transferable [6]. Probiotic bacteria with inherent resistance can persist very high dose of antibiotics rendering the bacteria less vulnerable to antibiotics [7]. A bacterium can attain antibiotic resistance to antimicrobial substances by getting new traits by mutations in intrinsic genes or receiving resistance genes by horizontal transfer [8]. Horizontal transfer of antimicrobial resistance genes is typically facilitated by mobile genetic components like transposons and plasmids. Antimicrobial resistance genes containing probiotic bacteria are naturally occurring that can be found in living organisms, in breast milk, and in other fermented foods [2, 6, 7, 9]. Additionally, the significant routes for the spread of antibiotic-resistant probiotic bacteria are food chain and gastrointestinal tract (GIT) [2, 10]. So these microbes can function as vectors for the transfer of antimicrobial resistance genes from foodstuff to living organisms [3, 11, 12]. Probiotics are considered as beneficial bacterial category of lactic acid bacteria, and safety of probiotics may be compromised if they function as a vector for transmittance of antimicrobial genes to potential pathogenic bacteria [6, 12]. Frequency of misusing antibiotics among women of childbearing age in developing world including Pakistan is much higher because of ineffectiveness of satisfactory guidelines on antibiotics [13, 14]. Antimicrobial resistance genes possessing probiotic bacteria could transfer those genes vertically from mother to infant during delivery or breast feeding [2, 6].

The increase in antibiotic resistance among microbes and subsequent increase in antibiotic failure to treat microbial diseases have encouraged more investigation into substitute antimicrobial compounds. Effective and favorable antimicrobial compounds are now being examined. Many studies exist on antimicrobial substances produced by lactic acid bacteria [15, 16]. The antimicrobial activity of probiotic lactic acid bacteria has been linked with production of effective metabolites including organic acids (lactic and acetic acids), hydrogen peroxide, carbon dioxide, ethanol, and bacteriocins [17, 18]. The powerful antimicrobial compounds are bactericidal against pathogenic microorganisms, thus also vital in food preservation [19, 20].

Organic acids are of great significance and effective antimicrobial compound produced by potential probiotics [19, 21]. Probiotic preparations usually comprise of probiotic bacterial strains that are acid-tolerant bacteria producing lactic acid (an organic acid) after carbohydrate fermentation as a main metabolic end-product. The antimicrobial effects caused by organic acids have been well acknowledged [22–24]. The low pH will decrease the pathogenic bacteria while assisting the propagation of the probiotic and other advantageous, organic acid-tolerant microbes in the gastrointestinal tract [25].

The main selection criterion for probiotics is their capability of adhering to the mucus produced by intestinal epithelium. This ability may raise their survival capabilities in the gut and consequently let bacteria exert their health-benefiting effects [26–29]. Though adhesion capability of probiotics does not essentially certify health benefits, their adhesion to intestinal epithelial wall can have a defensive role contrary to harmful bacteria through competition for binding sites to the host cells [30]. Commonly, adhesion is a complicated procedure comprising specific and nonspecific (hydrophobic connections between cell surfaces) ligand receptor interactions [31]. Along with that, exopolysaccharides or lipoteichoic acid manufactured by bacteria can take part in its adhesion to host epithelial cells [30, 32]. Communications between all these components are significant for adhesion in turn providing the benefits of intestine colonization of probiotic bacteria. Cell surface hydrophobicity is considered as an important functionality in general adhesion capacity. Most commonly, it is calculated by assessing the affinity of the tested strain to a hydrocarbon solvent such as the bacterial adhesion to hydrocarbon (BATH) method that measures membrane hydrophobicity of bacteria or hydrophilic nature of the cell surface. In a recent study by [33], microorganisms with elevated hydrophobicity can adhere better to epithelial cells in turn influencing the adhesion capacity. Falah et al. [34] stated that the hydrophobicity investigation can be done as a prerequisite for the adhesion capability to epithelial cells by probiotic bacteria. Hydrophobicity is considered as one of the essential properties improving the first interaction among bacteria and host cells. Aggregation (auto as well as co) and surface hydrophobicity are features that offer potential benefits for microorganisms in colonizing the gastrointestinal tract [35]. Produced by probiotic bacteria, high molecular mass antimicrobial components are strong bacteriocins and other antibacterial proteinaceous substances with narrow and broad range activity against pathogenic microbes. Among the entire techniques, agar well diffusion assay is the most used method for bacteriocin production [36].

So, we planned to evaluate the antibiotic resistance/sensitivity profile of probiotic bacteria isolated from human breast milk. The study also explores the antimicrobial activity of some of the identified probiotic bacteria to produce antimicrobial metabolites that will prevent the proliferation of pathogenic bacteria which is among the conditions for the selection of probiotic bacteria.

2. Materials and Methods

2.1. Materials

The materials used for the experimental work of this study include deMan, Rogosa, and Sharpe (MRS) agar (Merck Millipore, cat # 110660), BHI agar (BD Difco, cat # 241830), sodium hydroxide pellets (Merck Millipore, cat # 106482), ammonium sulfate (Merck Millipore, cat # 101216), and antibiotic discs (Oxoid, cat # HP0053A). Glass wear and plastic wear used in the study were purchased from Thermo Scientific.

2.2. Isolation and Identification

Sampling was done, by manual expression after disinfecting the skin with chlorhexidine, from healthy mothers after getting their informed consent from the local hospitals in Rawalpindi, Pakistan. Milk samples were kept on ice while transferring to the Microbiology and Biotechnology Laboratory, Fatima Jinnah Women University, Rawalpindi, for immediate isolation. The milk isolation was done on minimal media, deMan, Rogosa, and Sharpe (MRS) agar, and the distinct colonies were sent for 16S rRNA sequencing to Macrogen, Korea. Retrieved sequences were run through basic local alignment sequence tool (BLAST), and sequence of the identified strain was then submitted to the National Center for Biotechnology Information (NCBI).

2.3. Antibiotic Resistance/Sensitivity Profile

Antibiotic resistance of isolates was affirmed by 15 commonly used antibiotics by disc diffusion method [37]. In the management, screening, and killing of bacteria, critical role is played by antibiotics during different biotechnological processes. The vulnerability of bacteria to antibiotics is termed as antibiotic sensitivity of that specific bacterium. Clear zone which appeared around the commercially available “antibiotic disc” if bacteria are sensitive to antibiotic is called zone of inhibition. Bacteria that are resistant to antibiotic still grow around the disc.

For antibiotic sensitivity assay, MRS agar medium was prepared, autoclaved, and poured in sterile Petri plates under aseptic conditions. After solidification, plates were inverted and kept at room temperature. Three MRS agar plates were taken for each strain. Inoculum was prepared by mixing loopful of bacterial culture in 1 ml distilled water, and 50 μl was spread on the surface of agar. Then, antibiotic discs of various antibiotics were placed with the help of forceps on the plates. Five discs of different antibiotics were placed on each plate, and every disc was softly pressed with the tip of forceps. The plates were incubated at 37°C. After 24 hrs, the diameter of zones of inhibition around the discs was measured in millimeters [38]. The presence or absence of bacterial growth revealed the resistance/sensitivity of bacteria against that antibiotic.

2.4. Antimicrobial Activity

Antimicrobial compounds (like bacteriocin, lactic acid, and hydrogen peroxide) are produced by probiotic bacteria to compete with pathogens that lead to increase the immune response of the host [39]. In vitro antagonistic assay was performed using the agar double-layer diffusion method. Bacterial isolates were spotted onto MRS agar’s surface in a Petri dish and incubated at 37°C for 24 to 48 hrs. After incubation, cells were killed by exposure to chloroform for 30 min, and the residual chloroform was allowed to evaporate for another 30 min. The plates were overlaid with 3.5 ml of BHI (Difco) soft agar (0.75%) which were inoculated with 10⁶ CFU/ml of the indicator bacteria and incubated at 37°C for 24 to 48 hrs. The plates were then checked for the presence or absence of an inhibitory halo around the spot [40]. The indicator ATCC strains were obtained from Microbiology and Biotechnology Lab, Fatima Jinnah Women University, including Bacillus subtilis MB405, Bacillus pumilus MB407, Bacillus cereus MB401, Alcaligenes faecalis MB090, Microbacterium oxydans MB325, Pseudomonas geniculata MB321, Streptomyces laurentii MB319, Enterococcus faecium, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae MB081, and Staphylococcus aureus.

2.5. Organic Acid Production Assay

Lactic acid bacteria are known for the production of organic acids specifically lactic acids. Isolated lactic acid bacteria were considered to have the same property, and in order to determine this ability, acid production assay was conducted [38].

Powdered skimmed milk was purchased from the local market. Autoclaved distilled water was taken and mixed with 10% of powdered skimmed milk to make sterile skimmed milk with pH 6.68. Five ml of skimmed milk was inoculated with 24 hrs fresh bacterial culture and incubated at 37°C for 24, 48, and 72 hrs. After incubation, coagulated skimmed milk was filtered, and pH of each filtrate was measured with digital electrode pH meter for lactic acid production. The filtrate was also titrated against 0.1 N NaOH, and organic acid production was quantified in terms of percentage strength [41].

2.6. Hydrophobicity Assay

Bacterial adherence to hydrocarbons (BATH) was performed to assess bacterial surface hydrophobicity of isolated strains. Bacterial cells from an overnight culture were harvested by centrifugation ($\mathrm{5,000}\times \text{g}$, 20 min, 4°C), washed twice with phosphate-buffered saline PBS, and suspended in the same buffer. Absorbance (A600 nm) was adjusted to $0.70\pm 0.02$ in order to standardize the number of bacteria (200–250 CFU/ml). The optical density (OD₆₀₀ nm) of a homogenized bacterial suspension was recorded; then, the same suspension repeated and left to rest for 24 hrs at 37°C without vortexing. The aggregation percentage was expressed as $$\begin{array}{}\text{(1)}& \text{BATH}\%=1-\frac{A\text{Time}}{A0}\times 100,\end{array}$$where $A\text{Time}$ represents the absorbance of the mixture at 24 hrs and $A0$ is the absorbance at time 0 [42].

2.7. Salt Aggregation Test

Salt aggregation test (SAT) was performed to assess cell surface hydrophobicity of isolated strains in complement with BATH [43]. Overnight cultures of the lactobacilli and pathogens were harvested by centrifugation at $5000\times \text{g}$ at room temperature. The pellet was washed twice with PBS (0.002 M, pH 6.7) and then resuspended in this buffer to a final concentration of about $1\times {10}^8$ CFU/ml. Then, 25 μl of the bacterial suspensions was mixed with equal volumes of ammonium sulfate at various molarities (0.2 M to 4.5 M in 0.002 M PBS with pH 6.7) on glass slide. After gentle mixing for 1 min, the lowest ammonium sulfate concentration to cause visual bacterial cell clumping was recorded as the SAT value. The SAT value is inversely proportional to the hydrophobic nature [44].

2.8. Coaggregation with Pathogens

Coaggregation assay was performed using the method by Collado et al. [45] with minor modifications. Overnight cultures of isolates and pathogen strains (same as used in antimicrobial assay) were washed twice with PBS (pH 6.7) and resuspended in PBS to a final concentration of $1\times {10}^8$ CFU/ml. Equal volumes (1.5 ml) of isolated strains and pathogen strains were mixed, by vortexing for 10 s, and incubated at 37°C for 2 hrs without agitation. The supernatant liquids were then measured at 600 nm (A600). All experiments were performed in triplicate. Coaggregation was calculated according to the following equation: $$\begin{array}{}\text{(2)}& \text{Coaggregation}\%=1-A_{\text{mix}}{A}_{\text{isolate}}+\frac{A_{\text{pathogen}}}{2}\times 100,\end{array}$$where $A_{\text{isolate}}$, $A_{\text{pathogen}}$, and $A_{\text{mix}}$ represent the test strains, pathogenic strains, and their mixture after incubation for 2 hrs, respectively.

2.9. Statistical Analysis

All the experiments were done in triplicate, and the static significance was measured using Excel statistics. The correlation among the isolates was also done using formulas from Excel software. Heatmap was constructed using R programming using ggplot and pheatmap packages.

3. Results

3.1. Isolation and Identification

From the breast milk of eleven healthy mothers, seventeen distinct colonies were sent for 16S rRNA sequence identification. Retrieved sequence was submitted to NCBI GenBank nucleotide database under the accession numbers starting from MG751364 to MG751380. Identified bacterial strains from human breast milk belong mainly from Staphylococcus and Streptococcus genera. Mainly nine identified strains were used in this study including Staphylococcus hominis MB606, Staphylococcus hominis MB613, Staphylococcus hominis MB614, Staphylococcus hominis MB615, Bacillus sp. MB618, Staphylococcus hominis MB619, Streptococcus salivarius MB620, Staphylococcus epidermidis MB621, and Streptococcus lactarius MB622.

3.2. Antibiotic Resistance/Sensitivity Profiling

The bacterial strains isolated from human milk were exposed to multiple antibiotics like gentamicin (CN 10 μg), amoxicillin (AMC 10 μg), erythromycin (E 15 μg), streptomycin (S 10 μg), imipenem (IMI 10 μg), tetracycline (TE 30 μg), kanamycin (K 30 μg), bacitracin (BA 10 μg), nalidixic acid (NA 30 μg), vancomycin (VA 30 μg), ofloxacin (OFX 5 μg), rifampicin (RD 5 μg), clindamycin (CD 2 μg), chloramphenicol (C 30 μg), and trimethoprim sulfamethoxazole (SXT 25 μg) to study their antibiotic resistance in order to generate antibiograms for the isolates. Multiple antibiotic resistance (MAR) index was also calculated for these isolates.

Isolated probiotics showed sensitivity to antibiotics, and zones of inhibition of various diameters were observed against different antibiotics on Mueller-Hinton (MH) agar medium (Table 1). These zones were compared with standard zones for specific antibiotic standards mentioned in CLSC (clinical laboratory standard charts, 2007). All the probiotics from human milk were susceptible to chloramphenicol, while variation was observed for vancomycin (89%), rifampicin (89%), ofloxacin, streptomycin and bacitracin (78%), and amoxicillin (67%). All probiotics showed resistance against imipenem, trimethoprim, and nalidixic acid (Table 2). MAR index of the isolated bacteria revealed that almost all the isolates had the $\text{values}\ge 0.2$ showing high resistance towards antibiotics, and the index was calculated using the formula $\text{MAR}=a/b$ where “$a$” is the number of antibiotics to which tested isolates were resistant and “$b$” is the total number of antibiotics used in the assay (Table 3). The correlation of isolated bacteria was calculated using Excel statistics to determine the correlation coefficient among isolates against their antibiotic resistance sensitivity mechanism. The values of correlation coefficient more than 0.7 showed significance to the isolates which might be sharing the same mechanism of antibiotic resistance. The values between 0.5 and 0.7 showed moderate correlation, and values below revealed very little correlation among the stains. The correlation matrix showed that MB606 and MB613 were very closely related in terms of their antibiotic resistance/sensitivity profile and rest of the matrix showed variable results (Table 4). Heatmap summarizes the resistance and sensitivity of probiotic bacteria from human milk and grouped the strains on the basis of similarity and difference in the resistance/sensitivity profile (Figure 1).

Table 1

Antibiotic resistance and sensitivity profiling of various probiotics isolated from human milk against selected antibiotics.

Strains

AMC

CN

VA

TE

OFX

E

C

IMI

S

RD

SXT

CD

BA

NA

MB606

11.5 (R)

20.75 (S)

20.75 (S)

26 (S)

24.5 (S)

23.3 (S)

30.5 (S)

0 (R)

25.5 (S)

29.3 (S)

9 (R)

20.5 (S)

19.5 (S)

0 (R)

MB613

10 (R)

21.75 (S)

19.5 (S)

19.8 (S)

23.5 (S)

24.5 (S)

32.8 (S)

0 (R)

26.5 (S)

33 (S)

9 (R)

16.5 (I)

21.8 (S)

0 (R)

MB614

13.25 (R)

10.5 (R)

20.25 (S)

15.8 (I)

0 (R)

8 (R)

29.3 (S)

0 (R)

14.25 (I)

32 (S)

0 (R)

16.5 (S)

20.8 (S)

0 (R)

MB615

10 (R)

16 (S)

13.25 (R)

17.3 (I)

23.5 (S)

18.3 (I)

25.8 (S)

0 (R)

30 (S)

24.5 (S)

9 (R)

16.8 (I)

17.8 (I)

0 (R)

MB618

17.25 (I)

16.5 (R)

19.5 (S)

23.8 (S)

32 (S)

23 (S)

27.3 (S)

0 (R)

26.5 (S)

9.5 (R)

0 (R)

18.5 (I)

8.5 (S)

8.25 (R)

MB619

27.5 (S)

19.5 (S)

20.5 (S)

31.3 (S)

22 (S)

22.3 (I)

31.5 (S)

0 (R)

26.5 (S)

35.8 (S)

0 (R)

10.5 (R)

23.5 (S)

0 (R)

MB620

0 (R)

11.75 (R)

21.25 (S)

16.8 (I)

9.5 (R)

0 (R)

20.5 (S)

0 (R)

22.25 (S)

27.5 (S)

0 (R)

9 (R)

22.3 (S)

0 (R)

MB621

15.25 (R)

18.5 (R)

15.75 (S)

28 (S)

21.5 (S)

23.3 (S)

28.5 (S)

0 (R)

22.5 (S)

29.8 (S)

8 (R)

14.3 (I)

18.3 (S)

0 (R)

MB622

19 (S)

19.75 (R)

28 (S)

27.8 (S)

22 (S)

26.3 (S)

35.8 (S)

0 (R)

11.75 (I)

22.8 (S)

0 (R)

18.8 (I)

34.3 (S)

0 (R)

AMC = amoxicillin; CN = gentamicin; VA = vancomycin; TE = tetracycline; OFX = ofloxacin; E = erythromycin; C = chloramphenicol; IMP = imipenem; S = streptomycin; RD = rifampicin; SXT = trimethoprim sulfamethoxazole; CD = clindamycin; BA = bacitracin; NA = nalidixic acid; S = sensitive; R = resistant.

Table 2

Antibiogram for probiotic isolates from human milk.

Antibiotics	Concentration (μg)	Susceptible (%)	Intermediate (%)	Resistant (%)
Amoxicillin	10	22	11	67
Gentamicin	10	44	0	56
Vancomycin	30	89	0	11
Tetracycline	30	67	33	0
Ofloxacin	5	78	0	22
Erythromycin	15	56	22	22
Chloramphenicol	30	100	0	0
Imipenem	10	0	0	100
Streptomycin	10	78	22	0
Rifampicin	5	89	0	11
Trimethoprim sulfamethoxazole	25	0	0	100
Clindamycin	2	22	56	22
Bacitracin	10	78	11	11
Nalidixic acid	30	0	0	100

Table 3

Multiple antibiotic resistance (MAR) index of probiotic bacteria isolated from human milk.

Strains	MAR index
Staphylococcus hominis MB606	0.2857143
Staphylococcus hominis MB613	0.321428
Staphylococcus hominis MB614	0.571428
Staphylococcus hominis MB615	0.5
Bacillus sp. MB618	0.5
Staphylococcus hominis MB619	0.321428
Streptococcus salivarius MB620	0.60714
Staphylococcus epidermidis MB621	0.392857
Streptococcus lactarius MB622	0.3571428

Table 4

Correlation matrix of probiotics from human milk in terms of their antibiotic resistance mechanism.

	MB606	MB613	MB614	MB615	MB618	MB619	MB620	MB621	MB622
MB606	1
MB613	0.959033	1
MB614	0.592638	0.498561	1
MB615	0.748331	0.755447	0.277181	1
MB618	0.512348	0.517219	0.168687	0.273861	1
MB619	0.605705	0.732484	0.324065	0.566585	0.431016	1
MB620	0.528932	0.600706	0.795536	0.359833	0.246361	0.600706	1
MB621	0.81744	0.843416	0.617431	0.539749	0.656962	0.588571	0.699422	1
MB622	0.564288	0.595547	0.494027	0.287914	0.613267	0.686174	0.505672	0.789342	1

Bold, positive/strong correlation; italic, moderate correlation; underline, low correlation.

[figure(s) omitted; refer to PDF]

3.3. Organic Acid Production Assay

Organic acid production ability of probiotic bacteria isolated from human milk was assayed where all the isolated probiotics were able to coagulate skimmed milk (Table 5) and produced organic acid along with a gradual decrease in medium pH (Figure 2). Bacterial strains Staphylococcus hominis MB606; Staphylococcus epidermidis MB607; Staphylococcus epidermidis MB608; Staphylococcus hominis MB609, MB610, and MB611; Staphylococcus epidermidis MB612; Staphylococcus hominis MB613, MB614, MB615, MB616, and MB617; Bacillus sp. MB618; Staphylococcus hominis MB619; Streptococcus salivarius MB620; Staphylococcus epidermidis MB621 (Figure 3); and Streptococcus lactarius MB622 showed decreased organic acid molarity (when titrated against 0.1 M NaOH) with the increase in time of incubation (Figure 4).

Table 5

Milk coagulation, pH measurement, and organic acid production by probiotic bacteria isolated from human milk.

Bacterial strains	Milk coagulation	Incubation time
		24 hrs		48 hrs		72 hrs
		pH	Organic acid molarity	pH	Organic acid molarity	pH	Organic acid molarity
MB606	+	5.925	0.128205	5.885	0.119048	5.655	0.116279
MB613	+	6.28	0.212766	5.7	0.128205	5.385	0.114943
MB614	+	5.85	0.185185	5.27	0.134943	5.03	0.125
MB615	+	6.45	0.222222	5.705	0.158205	5.56	0.138889
MB618	+	6.66	0.3125	6.795	0.199254	5.99	0.172414
MB619	+	6.255	0.175439	5.385	0.166667	5.075	0.12987
MB620	+	5.4	0.172414	5.275	0.140845	5.19	0.120482
MB621	+	6.145	0.263158	5.58	0.136986	5.135	0.10989
MB622	+	6.32	0.181818	5.625	0.178571	5.545	0.133333

[figure(s) omitted; refer to PDF]

3.4. Hydrophobicity Assay

To assess bacterial surface hydrophobicity, bacterial adherence to hydrocarbons (BATH) was performed. Staphylococcus hominis MB606, MB613, MB614, MB615, and MB619 showed 51, 74, 86, 78, and 79% hydrophobicity values, respectively. Hydrophobicity recorded for different strains was 5% (Bacillus sp. MB618), 59% (Streptococcus salivarius MB620), 75% (Staphylococcus epidermidis MB621), and 78% (Streptococcus lactarius MB622) as shown in Figure 5.

[figure(s) omitted; refer to PDF]

3.5. Antimicrobial Activity of Lactic Acid Bacteria

Antimicrobial resistance genes containing probiotic bacteria are naturally occurring that can be found in living organisms thus in their products such as in human breast milk. In order to assess the probiotic potential of some of the identified probiotic isolates from human milk, the study on antimicrobial activity of 9 selected strains was carried out. The antimicrobial activity of probiotics against 12 pathogenic bacteria was investigated, and selected strains showed variable results for inhibition of growth of all the pathogens.

3.6. Agar Well Diffusion Assay

Bacteriocin production activity of isolated probiotic bacteria was assayed against different available pathogenic bacteria (indicator strains) by well diffusion method on MH agar medium. Bacterial strain Staphylococcus epidermidis MB621 produced bacteriocin against Bacillus subtilis MB405, Microbacterium oxydans MB325, Streptomyces laurentii MB319, Bacillus cereus MB401, E. coli, and Staphylococcus aureus. Staphylococcus hominis MB614, Streptococcus salivarius MB620, and Streptococcus lactarius MB622 gave inhibitory zone against Bacillus subtilis MB405, Streptomyces laurentii MB319, Bacillus cereus MB401, and Staphylococcus aureus (Table 6).

Table 6

Agar well diffusion assay against indicator strains.

Indicator strains	Control	MB614	MB620	MB621	MB622
Bacillus subtilis MB405	0	24	14	25.5	18
Bacillus pumilus MB407	0	0	0	0	0
Alcaligenes faecalis MB090	0	0	0	0	0
Microbacterium oxydans MB325	0	0	0	11	0
Pseudomonas geniculata MB321	0	0	0	0	0
Streptomyces laurentii MB319	0	8	10	14.5	11
Enterococcus faecium	0	0	0	0	0
Bacillus cereus MB401	0	9	9	11	8
Enterococcus faecalis	0	0	0	0	0
E. coli	0	0	0	24	0
Klebsiella pneumoniae MB081	0	0	0	0	0
Staphylococcus aureus	0	8	9	9	9

Zones were measured in millimeter around the well.

3.7. Salt Aggregation Test

Salt aggregation test (SAT) proved to be a screening test for detecting bacteria with high surface hydrophobicity (Figure 6) due to surface protein of fimbrial (occurrence of fimbria protein suggestively associated with pathogenicity) and nonfimbrial nature. The isolated bacteria from human milk and the indicator strains (used previously in antimicrobial assay) aggregated in the ammonium sulfate salt solution of various molarities (0.5-4.5 M). Streptococcus salivarius MB620 and Streptococcus lactarius MB622 aggregated at 1 M and 0.5 M salt concentration, respectively (Table 7).

[figure(s) omitted; refer to PDF]

Table 7

SAT for probiotic isolates and indicator strains.

Probiotic isolates	Salt aggregation molarity	Indicator strains	Salt aggregation molarity
Staphylococcus hominis MB606	0.5	Bacillus subtilis MB405	0.5
Staphylococcus hominis MB613	0	Bacillus pumilus MB407	2.5
Staphylococcus hominis MB614	0	Alcaligenes faecalis MB090	0
Staphylococcus hominis MB615	0.5	Microbacterium oxydans MB325	3
Bacillus sp. MB618	0	Pseudomonas geniculata MB321	4
Staphylococcus hominis MB619	0.5	Streptomyces laurentii MB319	3
Streptococcus salivarius MB620	1	Enterococcus faecium	3
Staphylococcus epidermidis MB621	0.5	Bacillus cereus MB401	0.5
Streptococcus lactarius MB622	0.5	Enterococcus faecalis	2
		E. coli	1.5
		Klebsiella pneumoniae MB081	1
		Staphylococcus aureus	2.5

3.8. Coaggregation with Pathogens

Representative isolated probiotic bacteria (Figures 7 and 8) showed coaggregation with B. subtilis (33-45%), B. cereus (33-44%), B. pumilus (35-36%), and M. oxydans (33-37%). Among the strains tested, Streptococcus salivarius MB620 exhibited maximum coaggregation ability with B. subtilis (45%) and B. cereus (44%). Streptococcus lactarius MB622 showed the maximum coaggregation abilities with B. pumilus (35%). Both Streptococcus salivarius MB620 and Streptococcus lactarius MB622 showed good coaggregation activity against E. coli, i.e., 35% and 33%, respectively.

[figure(s) omitted; refer to PDF]

4. Discussion

In order to consider probiotic bacteria safe to be utilized in food products or as supplement, the Joint FAO/WHO Expert Committee on Food Additives Meeting and World Health Organization [46] guidelines are for the screening of antibiotic resistance/sensitivity profile. Hence, the phenotypic antibiotic resistance/sensitivity profile of probiotic bacteria isolated from human breast milk was studied. Most of the tested probiotic bacteria were sensitive to quinolones (ofloxacin) demonstrating inhibition of bacterial DNA synthesis. Also, 33% probiotic bacteria were susceptible to beta-lactams (amoxicillin), and 67% showed resistance to amoxicillin. The beta-lactams are recognized for their disruption of bacterial cell wall synthesis [47]. Furthermore, comparable to the current study, probiotic bacteria from human breast milk were susceptible to beta-lactams [6]. The probiotic bacteria were resistant to the carbapenems (imipenem), chloramphenicol, and quinolones (nalidixic acid). Similar trend was reported in the study with a total of 140 probiotics isolated from 35 kinds of Korean commercially available kimchi, where disc diffusion assay showed a resistance incidence of 98.6% for nalidixic acid [48]. Also, 78% of the isolated strains were sensitive to clindamycin, quinolones, aminoglycosides (streptomycin), and polypeptides (bacitracin), and a study with human breast milk isolates showed susceptibility to clindamycin [49]. In another study with lactococci isolated from dairy origin, the highest resistance frequency was observed against streptomycin, trimethoprim, nalidixic acid, and rifampicin, whereas intermediate level of resistance was seen against antibiotics like gentamycin, tetracycline, and clindamycin; susceptibility was detected against amoxicillin, erythromycin, vancomycin, ofloxacin, bacitracin, and chloramphenicol [50]. The results are mostly similar to the findings in the present study which may be because of similar origin with some difference which may be because of variation in the strains. All the probiotic bacterium isolates presented susceptibility against chloramphenicol and vancomycin (except Streptococcus hominis MB615) similar to the study by Sharma et al. in 2017 showing all the probiotic bacterial strains sensitive to ampicillin and vancomycin. Former reports propose inherent resistance of lactococcal strains to trimethoprim and cefoxitin and to the aminoglycosides—gentamicin and kanamycin [19, 51], which may somewhat clarify the resistance seen here in case of trimethoprim and other aminoglycoside drugs. Different levels of resistance to chloramphenicol, tetracycline, clindamycin, rifampicin, and erythromycin have also been reported before in Lactococcus lactis [19, 52]. Resistance against trimethoprim, gentamicin, erythromycin, and cephalothin for L. lactis strains isolated from broiler chicken feces has also been reported [53]. The variance with the current study may be because of numerous factors such as the origin of the isolates and the screening methods used. Sharma et al. [54] and Kozak et al. [49] described vancomycin resistance in Lactobacillus plantarum and Lactobacillus pentosus from human breast milk. This was also witnessed for Leuconostoc and Weissella species from fermented dairy milk [8]. In the current study, 89% of the isolates were susceptible to vancomycin. This study validates with the research finding of Jiménez et al. [55] which also stated susceptibility of Enterococcus faecium from human breast milk to vancomycin. The results of the current study and that of Jiménez et al. [55] suggest that the inherent resistance to vancomycin claimed for probiotic bacteria is not applicable to all species. Multidrug resistance was observed in the probiotic bacteria investigated with the highest seen in Streptococcus salivarius MB620, Staphylococcus hominis MB614, and Bacillus sp. MB618 which exhibited resistance to 7, 6, and 5 of the antibiotics used in this study, respectively. This trend was observed by Kıvanç et al. [56] and Reis et al. [57] who reported multiple drug resistance in Enterococcus faecium isolated from human breast milk to ciprofloxacin, ampicillin, gentamicin, penicillin, and vancomycin. Antimicrobial resistance is a complex problem that could be accredited to numerous kinds of mode of transmission and selection pressures [58]. Many reports have proposed that some favorable bacteria like probiotic bacteria and Bifidobacterium originate from the gut of a mother [59–62]. Supporting this concept, the existence of antimicrobial resistance in the probiotic bacteria tested could be linked to translocation of probiotic bacteria with these features from maternal gut to mammary glands. Maternal gastrointestinal tract and skin microflora are possible reservoirs of antimicrobial-resistant bacteria which could be transmitted to newborns and infants [49]. All of the maternal nipples were cleansed with disinfected swabs former to the collection of breast milk samples. But cleaning of breast prior to breast feeding between lactating mothers is seldom practiced in both developed and developing worlds. In probiotic bacteria, high MAR index for antibiotics as shown by the present study may perhaps be attributed to misuse of antibiotic also to the extent of antibiotic exposure to the childbearing age women and lactating mothers. Additionally, this is combined with the food chain, since once an antibiotic-resistant bacterium was ingested, it could be passed to the gut to be transferred to infants through breast milk. Native gut lactic acid bacteria have varied AMR pattern that could be transferred to other bacteria inside the gut [63]. Isolation of bacteria that are resistant to antibiotic in ready-to-eat food products specifies likelihood of spreading the bacteria from food to humans [64]. Antibiotic residues often exist in edible animal flesh and dairy products [65]. Antibiotics like tetracycline, streptomycin, penicillin, gentamicin, and erythromycin are improperly used for protection and therapeutic purpose in egg-laying poultry [66, 67]. In developing countries including Pakistan, practice of self-medication with different antibiotics is common, and antibiotics are effortlessly obtained over the counter without prescription. Lack of proper regulation and legislation add considerably to absurd antibiotic prescription and self-medication is unavoidably raising the antibiotic resistance in bacteria [68, 69].

Certain probiotic bacteria are regarded as safe by the United States Food and Drug Administration [70] due to their prehistoric use of unfermented foods and dairy. If probiotics have probability of transferring antimicrobial resistance (AMR) genes, the safety of those probiotics could be at stake [54]. Resistance to antibiotics by some probiotics in food chain has been qualified to unselective application of antibiotics [64, 71]. Antibiotic misuse by child-bearing women is also connected with global AMR [55, 72]. Furthermore, vertical transfer of AMR genes among probiotics from lactating mother to the child is likely to occur [73].

Prevailing study evaluated the resistance profile of the nine breast milk isolates against multiple antibiotics. Sensitivity of all the isolates to chloramphenicol, almost 90% to vancomycin and rifampicin and almost 80% to ofloxacin, streptomycin, and bacitracin was observed. Also, all isolates were resistant to imipenem, trimethoprim sulfamethoxazole, and nalidixic acid. However, some of the isolates were resistant to amoxicillin and gentamycin. Sensitivity of probiotic bacterial species to next-generation antibiotics from ampicillin and penicillin groups, witnessed in the present study, is consistent with other finding from human milk [54]. Resistance to vancomycin is quite common among probiotic bacteria [8, 74]. Furthermore, Kozak et al. [49] and Sharma et al. [54] described vancomycin resistance in isolated L. plantarum and L pentosus from human milk. Resistance to clindamycin, tetracycline, levofloxacin, gentamicin, and erythromycin of some probiotics as stated in the present study is consistent to the studies of other researchers [49, 54]. Constant misuse of tetracycline and erythromycin in human and in crops occurs worldwide [55, 64]. Consequently, it contributes to greater prevalence of resistance to these antibiotics and in some probiotics. According to FDA, in children with ages less than eight years, tetracycline is not suggested [75]. Therefore, the presence of some tetracycline sensitivity in almost all the isolates stated in the study is considered as less probable to impart safety risks to infants. Several investigations on human alpha-lactalbumin made lethal to tumor cells (HAMLET), a constituent of human milk harvesting favorable results on its antibiotic-potentiating influence on various bacterial species including Staphylococci and Streptococci with multiple drug resistance [76–79].

The antimicrobial activity of bacteria is important in the selection of potential probiotic bacteria. There are numerous mechanisms of action by probiotics associated with antibacterial, antifungal, antiparasitic, antiviral, anticancerous, antiallergic, and antidiabetic and enhancement of the reproductive, cardiovascular, and central nervous systems [80–82]. Antagonistic substance production by probiotic bacteria like lactic acid, bacteriocins, hydrogen peroxide, and other antimicrobial substances is linked with potential benefits of probiotic [83, 84]. Antimicrobial substances secreted by probiotic bacteria in fact have bactericidal and bacteriostatic properties on pathogenic bacteria [85, 86]. The study also surveyed antimicrobial action of probiotic bacteria against particular indicator strains. The results from agar diffusion assay using cell-free supernatant were comparatively poor in inhibition of pathogenic indicator bacteria. But Staphylococcus hominis MB614, Streptococcus salivarius MB620, Staphylococcus epidermidis MB621, and Streptococcus lactarius MB622 inhibited growth of Bacillus subtilis MB405, Bacillus cereus MB401, Streptomyces laurentii MB319, and Staphylococcus aureus. Staphylococcus epidermidis MB621 inhibited Microbacterium oxydans MB325 and Escherichia coli. By agar diffusion assay, the limited inhibition of pathogens was witnessed in this study which could be qualified for little concentration of antimicrobial substances along with reduced diffusion of supernatants. In [87, 88], Al-Otaibi et al. also stated reduced antimicrobial properties of probiotics from camel milk and yoghurt, in agar well diffusion assay.

Organic acid particularly lactic acid might be the utmost incompatible substance that inhibited some pathogens [89]. This study designated the proliferation of organic acid production with the time course of incubation, with a reduction in pH. Highest acid molarity of 0.3 M was observed for Bacillus sp. MB618; after that, 0.2 M acid production was observed in Staphylococcus hominis MB613 and MB615 and Staphylococcus epidermidis MB621; after 24 hrs of incubation at 37°C, organic acid molarity decreases during the course of incubation considerably because of other basic metabolite production during stationary phase of bacterial growth. The capability of coagulating milk when inoculated by all of the isolated strains was observed.

The antimicrobial characteristics have been accredited to the probiotic potential of isolated bacteria [90]. The study was conducted in order to evaluate the preventive properties of the isolated bacteria against both Gram-positive and Gram-negative microorganisms. Some of the probiotic bacterial strains from the study avert growth of particular indicator (pathogenic) strains by the agar well diffusion assay. The cell-free supernatants of some bacterial isolates under consideration prevented the indicator bacterial growth, showing antimicrobial abilities. Furthermore, antimicrobial properties of the isolated probiotic bacteria probably might be qualified for the organic acid (lactic acid) production, BATH, salt aggregation, coaggregation with pathogens, and bacteriocin production. This research has consequently added to the data of the antimicrobial properties of particular isolated probiotics from certain samples of Pakistani women’s milk.

5. Conclusion

The probiotic strains were resistant to multiple antibiotics (gentamycin, imipenem, trimethoprim sulfamethoxazole, and nalidixic acid) while showing sensitivity to vancomycin, tetracycline, ofloxacin, chloramphenicol, streptomycin, rifampicin, and bacitracin. Bacterial antimicrobial behavior showed that selected probiotics were able to inhibit the growth of certain pathogens. The antimicrobial properties of probiotic bacteria may also be related to the production of lactic acid, bacterial adhesion to hydrocarbons (BATH), salt aggregation, coaggregation with pathogens, and bacteriocin production. Streptococcus lactarius MB622 and Streptococcus salivarius MB620 displayed higher hydrophobicity (78% and 59%, respectively) in addition to intrinsic probiotic properties. Probiotic bacteria are considered to safeguard gastrointestinal track (GIT) by adhering to the gut epithelial cells and reducing population of pathogens. This research has consequently added to the data of the antimicrobial activity of some isolated probiotic bacteria from some samples of Pakistani women’s breast milk.

6. Future Applications

It would be advantageous to conduct a detailed study on the immunomodulatory properties of these bacteria from human milk. The use of starter culture with antimicrobial properties is of considerable interest due to the fact that the production of such compounds is a significant factor that helps to promote the safety and quality of fermented food. This study has financial and time constrains of investigating the potential probiotic ability to transfer antibiotic resistance gene in order to ensure their safety to be used as food supplements.

Ethical Approval

The strains used in this study were isolated from human breast milk collected by manual expression.

Consent

Informed consent was obtained from the mother to use the bacterial strains from the milk in our research works.

Acknowledgments

The study was funded by the Higher Education Commission Pakistan under HEC indigenous scholarship Batch II Phase II. This study was supported by Researchers Supporting Project Number (PNURSP2023R30), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

References

[1] S. Andleeb, M. Jamal, S. Bukhari, S. Sardar, M. Majid, "Trends in antimicrobial use in food animals, aquaculture, and hospital waste," Antibiotics and Antimicrobial Resistance Genes, pp. 95-138, DOI: 10.1007/978-3-030-40422-2_5, 2020.

[2] J. Azevedo, E. S. Dos Anjos, S. M. Cordeiro, M. S. Dos Santos, E. C. Escobar, P. R. Lobo, M. da Gloria Carvalho, M. G. Reis, J. N. Reis, L. C. Campos, "Genetic profiles and antimicrobial resistance of Streptococcus pneumoniae non-PCV10 serotype isolates recovered from meningitis cases in Salvador, Brazil," Journal of Medical Microbiology, vol. 65 no. 10, pp. 1164-1170, DOI: 10.1099/jmm.0.000346, 2016.

[3] N. Karapetkov, R. Georgieva, N. Rumyan, E. Karaivanova, "Antibiotic susceptibility of different lactic acid bacteria strains," Beneficial Microbes, vol. 2 no. 4, pp. 335-339, DOI: 10.3920/BM2011.0016, 2011.

[4] S. Nayyar, S. Kadyan, H. Parveen, R. H. Mallappa, D. Pradhan, "Antimicrobial resistance in food grade lactic acid bacteria," Biological and Chemical Hazards in Food and Food Products, pp. 91-120, DOI: 10.1201/9781003189183-5, 2022.

[5] A. Saeed, A. Yasmin, M. Baig, M. A. Ahmed, Z. U. R. Farooqi, "Streptococcus lactarius MB622 and Streptococcus salivarius MB620 isolated from human milk reduce chemokine IL-8 production in response to TNF- α in Caco-2 cell line, an exploratory study," Cytokine, vol. 168, article 156232,DOI: 10.1016/j.cyto.2023.156232, 2023.

[6] M. Sharma, A. Wasan, R. K. Sharma, "Recent developments in probiotics: an emphasis on Bifidobacterium," Food Bioscience, vol. 41, article 100993,DOI: 10.1016/j.fbio.2021.100993, 2021.

[7] T. M. Uddin, A. J. Chakraborty, A. Khusro, B. R. M. Zidan, S. Mitra, T. B. Emran, K. Dhama, M. K. H. Ripon, M. Gajdács, M. U. K. Sahibzada, M. J. Hossain, N. Koirala, "Antibiotic resistance in microbes: history, mechanisms, therapeutic strategies and future prospects," Journal of Infection and Public Health, vol. 14 no. 12, pp. 1750-1766, DOI: 10.1016/j.jiph.2021.10.020, 2021.

[8] A. B. Flórez, I. Campedelli, S. Delgado, Á. Alegría, E. Salvetti, G. E. Felis, B. Mayo, S. Torriani, "Antibiotic susceptibility profiles of dairy Leuconostoc, analysis of the genetic basis of atypical resistances and transfer of genes in vitro and in a food matrix," PLoS One, vol. 11 no. 1, article e0145203,DOI: 10.1371/journal.pone.0145203, 2016.

[9] A. Saeed, A. Yasmin, M. Baig, K. Khan, M. B. B. Heyat, F. Akhtar, Z. Batool, A. Kazmi, A. Wahab, M. Shahid, M. A. Ahmed, I. Ahmad, "Isolation and Characterization of Lactobacillus crispatus , Lactococcus lactis , and Carnobacterium divergens as Potential Probiotic Bacteria from Fermented Black and Green Olives ( Olea europaea ): An Exploratory Study," BioMed Research International, vol. 2023,DOI: 10.1155/2023/8726320, 2023.

[10] S. B. Kumar, S. R. Arnipalli, O. Ziouzenkova, "Antibiotics in food chain: the consequences for antibiotic resistance," Antibiotics, vol. 9 no. 10,DOI: 10.3390/antibiotics9100688, 2020.

[11] European Food Safety Authority, European Centre for Disease Prevention and Control, "The European Union summary report on antimicrobial resistance in zoonotic and indicator bacteria from humans, animals and food in 2018/2019," EFSA Journal, vol. 19 no. 4, article e06490,DOI: 10.2903/j.efsa.2021.6490, 2021.

[12] M. Fatahi-Bafghi, S. Naseri, A. Alizehi, "Genome analysis of probiotic bacteria for antibiotic resistance genes," Antonie Van Leeuwenhoek, vol. 115 no. 3, pp. 375-389, DOI: 10.1007/s10482-021-01703-7, 2022.

[13] O. J. Olayemi, B. O. Olayinka, A. I. Musa, "Evaluation of antibiotic self-medication pattern amongst undergraduate students of Ahmadu Bello University (Main Campus) Zaria," Research Journal of Applied Sciences, Engineering and Technology, vol. 2 no. 1, pp. 35-38, 2010.

[14] E. V. Yeika, B. Ingelbeen, B. L. Kemah, F. S. Wirsiy, J. N. Fomengia, M. A. Van der Sande, "Comparative assessment of the prevalence, practices and factors associated with self-medication with antibiotics in Africa," Tropical Medicine & International Health, vol. 26 no. 8, pp. 862-881, DOI: 10.1111/tmi.13600, 2021.

[15] O. Ajao, K. Banwo, O. Ogunremi, A. Sanni, "Antimicrobial properties and probiotic potentials of lactic acid bacteria isolated from raw beef in Ibadan, Nigeria," Journal of Microbiology, Biotechnology and Food Sciences, vol. 8 no. 2, pp. 770-773, DOI: 10.15414/jmbfs.2018.8.2.770-773, 2018.

[16] M. P. Arena, V. Capozzi, P. Russo, D. Drider, G. Spano, D. Fiocco, "Immunobiosis and probiosis: antimicrobial activity of lactic acid bacteria with a focus on their antiviral and antifungal properties," Applied Microbiology and Biotechnology, vol. 102 no. 23, pp. 9949-9958, DOI: 10.1007/s00253-018-9403-9, 2018.

[17] K. Monika, T. Malik, R. Gehlot, K. Rekha, A. Kumari, R. Sindhu, P. Rohilla, "Antimicrobial property of probiotics," Environment Conservation Journal, vol. 22 no. SE, pp. 33-48, DOI: 10.36953/ECJ.2021.SE.2204, 2021.

[18] M. A. D. Sousa, G. R. Rama, C. F. Volken de Souza, C. E. Granada, "Acid lactic lactobacilli as a biotechnological toll to improve food quality and human health," Biotechnology Progress, vol. 36 no. 2, article e2937,DOI: 10.1002/btpr.2937, 2020.

[19] M. S. Ammor, A. B. Flórez, B. Mayo, "Antibiotic resistance in non-enterococcal lactic acid bacteria and bifidobacteria," Food Microbiology, vol. 24 no. 6, pp. 559-570, DOI: 10.1016/j.fm.2006.11.001, 2007.

[20] L. Nunziata, M. Brasca, S. Morandi, T. Silvetti, "Antibiotic resistance in wild and commercial non-enterococcal lactic acid bacteria and bifidobacteria strains of dairy origin: an update," Food Microbiology, vol. 104, article 103999,DOI: 10.1016/j.fm.2022.103999, 2022.

[21] E. Likotrafiti, P. Valavani, A. Argiriou, J. Rhoades, "In vitro evaluation of potential antimicrobial synbiotics using Lactobacillus kefiri isolated from kefir grains," International Dairy Journal, vol. 45, pp. 23-30, DOI: 10.1016/j.idairyj.2015.01.013, 2015.

[22] G. M. Jadhao, S. M. Wankhede, D. H. Rekhate, A. P. Dhok, P. S. Bankar, H. N. Rewatkar, "Effect of supplementation of acidifiers with probiotic on performance of broiler chicken," Indian Journal of Animal Nutrition, vol. 36 no. 3, pp. 325-328, DOI: 10.5958/2231-6744.2019.00053.7, 2019.

[23] P. Rodjan, K. Soisuwan, K. Thongprajukaew, Y. Theapparat, S. Khongthong, J. Jeenkeawpieam, T. Salaeharae, "Effect of organic acids or probiotics alone or in combination on growth performance, nutrient digestibility, enzyme activities, intestinal morphology and gut microflora in broiler chickens," Journal of Animal Physiology and Animal Nutrition, vol. 102 no. 2, pp. e931-e940, DOI: 10.1111/jpn.12858, 2018.

[24] I. M. Youssef, A. S. Mostafa, M. A. Abdel-Wahab, "Effects of dietary inclusion of probiotics and organic acids on performance, intestinal microbiology, serum biochemistry and carcass traits of broiler chickens," Journal of World's Poultry Research, vol. 7 no. 2, pp. 57-71, 2017.

[25] P. Markowiak, K. Śliżewska, "Effects of probiotics, prebiotics, and synbiotics on human health," Nutrients, vol. 9 no. 9,DOI: 10.3390/nu9091021, 2017.

[26] B. A. Behbahani, M. Noshad, F. Falah, "Inhibition of Escherichia coli adhesion to human intestinal Caco-2 cells by probiotic candidate Lactobacillus plantarum strain L15," Microbial Pathogenesis, vol. 136, article 103677,DOI: 10.1016/j.micpath.2019.103677, 2019.

[27] A. Fariq, A. Saeed, "Production and biomedical applications of probiotic biosurfactants," Current Microbiology, vol. 72 no. 4, pp. 489-495, DOI: 10.1007/s00284-015-0978-4, 2016.

[28] G. Krausova, I. Hyrslova, I. Hynstova, "In vitro evaluation of adhesion capacity, hydrophobicity, and auto-aggregation of newly isolated potential probiotic strains," Fermentation, vol. 5 no. 4,DOI: 10.3390/fermentation5040100, 2019.

[29] S. Lebeer, I. J. Claes, T. L. Verhoeven, J. Vanderleyden, S. C. De Keersmaecker, "Exopolysaccharides of Lactobacillus rhamnosus GG form a protective shield against innate immune factors in the intestine," Microbial Biotechnology, vol. 4 no. 3, pp. 368-374, DOI: 10.1111/j.1751-7915.2010.00199.x, 2011.

[30] A. Monteagudo-Mera, R. A. Rastall, G. R. Gibson, D. Charalampopoulos, A. Chatzifragkou, "Adhesion mechanisms mediated by probiotics and prebiotics and their potential impact on human health," Applied Microbiology and Biotechnology, vol. 103 no. 16, pp. 6463-6472, DOI: 10.1007/s00253-019-09978-7, 2019.

[31] G. Liu, M. Chu, S. Nie, X. Xu, J. Ren, "Effects of Ilisha elongata protein, soy protein and whey protein on growth characteristics and adhesion of probiotics," Current Research in Food Science, vol. 5, pp. 2125-2134, DOI: 10.1016/j.crfs.2022.10.024, 2022.

[32] F. Chaffanel, F. Charron-Bourgoin, C. Soligot, M. Kebouchi, S. Bertin, S. Payot, Y. Le Roux, N. Leblond-Bourget, "Surface proteins involved in the adhesion of Streptococcus salivarius to human intestinal epithelial cells," Applied Microbiology and Biotechnology, vol. 102 no. 6, pp. 2851-2865, DOI: 10.1007/s00253-018-8794-y, 2018.

[33] B. M. S. de Souza, T. F. Borgonovi, S. N. Casarotti, S. D. Todorov, A. L. B. Penna, "Lactobacillus casei and Lactobacillus fermentum strains isolated from mozzarella cheese: probiotic potential, safety, acidifying kinetic parameters and viability under gastrointestinal tract conditions," Probiotics and Antimicrobial Proteins, vol. 11 no. 2, pp. 382-396, DOI: 10.1007/s12602-018-9406-y, 2019.

[34] F. Falah, A. Vasiee, B. A. Behbahani, F. T. Yazdi, S. Moradi, S. A. Mortazavi, S. Roshanak, "Evaluation of adherence and anti-infective properties of probiotic Lactobacillus fermentum strain 4-17 against Escherichia coli causing urinary tract infection in humans," Microbial Pathogenesis, vol. 131, pp. 246-253, DOI: 10.1016/j.micpath.2019.04.006, 2019.

[35] M. S. R. Rajoka, H. M. Mehwish, M. Siddiq, Z. Haobin, J. Zhu, L. Yan, D. Shao, X. Xu, J. Shi, "Identification, characterization, and probiotic potential of Lactobacillus rhamnosus isolated from human milk," LWT, vol. 84, pp. 271-280, DOI: 10.1016/j.lwt.2017.05.055, 2017.

[36] R. H. Kadi, A. M. Baghdadi, "Screening and estimation of antibacterial activity of bacteriocins produced by Enterococcus faecium LR isolated from raw horse milk samples," Pakistan Journal of Pharmaceutical Sciences, vol. 35 no. 4, 2022.

[37] J. Hudzicki, "Kirby-Bauer disk diffusion susceptibility test protocol," American Society for Microbiology, vol. 15, pp. 55-63, 2009.

[38] M. Z. Hoque, F. Akter, K. M. Hossain, M. S. M. Rahman, M. M. Billah, K. M. D. Islam, "Isolation, identification and analysis of probiotic properties of Lactobacillus spp. from selective regional yoghurts," World Journal of Dairy & Food Sciences, vol. 5 no. 1, pp. 39-46, 2010.

[39] S. Karimi, F. Azizi, M. Nayeb-Aghaee, L. Mahmoodnia, "The antimicrobial activity of probiotic bacteria Escherichia coli isolated from different natural sources against hemorrhagic E. coli O157: H7," Electronic Physician, vol. 10 no. 3, pp. 6548-6553, DOI: 10.19082/6548, 2018.

[40] Q. S. Damaceno, J. P. Souza, J. R. Nicoli, R. L. Paula, G. B. Assis, H. C. Figueiredo, V. Azevedo, F. S. Martins, "Evaluation of potential probiotics isolated from human milk and colostrum," Probiotics and Antimicrobial Proteins, vol. 9 no. 4, pp. 371-379, DOI: 10.1007/s12602-017-9270-1, 2017.

[41] Z. Zalán, J. Hudáček, J. Štětina, J. Chumchalová, A. Halász, "Production of organic acids by Lactobacillus strains in three different media," European Food Research and Technology, vol. 230 no. 3, pp. 395-404, DOI: 10.1007/s00217-009-1179-9, 2010.

[42] C. Kotzamanidis, A. Kourelis, E. Litopoulou-Tzanetaki, N. Tzanetakis, M. Yiangou, "Evaluation of adhesion capacity, cell surface traits and immunomodulatory activity of presumptive probiotic Lactobacillus strains," International Journal of Food Microbiology, vol. 140 no. 2-3, pp. 154-163, DOI: 10.1016/j.ijfoodmicro.2010.04.004, 2010.

[43] G. I. Geertsema-Doornbusch, H. C. Van der Mei, H. J. Busscher, "Microbial cell surface hydrophobicity the involvement of electrostatic interactions in microbial adhesion to hydrocarbons (MATH)," Journal of Microbiological Methods, vol. 18 no. 1, pp. 61-68, DOI: 10.1016/0167-7012(93)90072-p, 1993.

[44] D. Y. Ren, C. Li, Y. Q. Qin, R. L. Yin, S. W. Du, F. Ye, H. F. Liu, M. P. Wang, Y. Sun, X. Li, M. Y. Tian, N. Y. Jin, "Lactobacilli Reduce Chemokine IL-8 Production in Response to TNF- α and Salmonella Challenge of Caco-2 Cells," BioMed Research international, vol. 2013,DOI: 10.1155/2013/925219, 2013.

[45] M. C. Collado, J. Meriluoto, S. Salminen, "Measurement of aggregation properties between probiotics and pathogens: in vitro evaluation of different methods," Journal of Microbiological Methods, vol. 71 no. 1, pp. 71-74, DOI: 10.1016/j.mimet.2007.07.005, 2007.

[46] Joint FAO/WHO Expert Committee on Food Additives Meeting, World Health Organization, "Safety Evaluation of Certain Contaminants in Food," vol. 82, 2006.

[47] H. Cho, T. Uehara, T. G. Bernhardt, "Beta-lactam antibiotics induce a lethal malfunctioning of the bacterial cell wall synthesis machinery," Cell, vol. 159 no. 6, pp. 1300-1311, DOI: 10.1016/j.cell.2014.11.017, 2014.

[48] Y. R. Yun, J. J. Lee, H. J. Lee, Y. J. Choi, J. H. Lee, S. J. Park, S. H. Park, H. Y. Seo, S. G. Min, "Comparison of quality characteristics of commercial kimchi manufactured in Korea, China, and the United States," Foods, vol. 10 no. 10,DOI: 10.3390/foods10102488, 2021.

[49] K. Kozak, D. Charbonneau, R. Sanozky-Dawes, T. Klaenhammer, "Characterization of bacterial isolates from the microbiota of mothers' breast milk and their infants," Gut Microbes, vol. 6 no. 6, pp. 341-351, DOI: 10.1080/19490976.2015.1103425, 2015.

[50] S. B. Singh, K. Young, L. L. Silver, "What is an "ideal" antibiotic? Discovery challenges and path forward," Biochemical Pharmacology, vol. 133, pp. 63-73, DOI: 10.1016/j.bcp.2017.01.003, 2017.

[51] A. Sirichoat, A. B. Flórez, L. Vázquez, P. Buppasiri, M. Panya, V. Lulitanond, B. Mayo, "Antibiotic susceptibility profiles of lactic acid bacteria from the human vagina and genetic basis of acquired resistances," International Journal of Molecular Sciences, vol. 21 no. 7,DOI: 10.3390/ijms21072594, 2020.

[52] E. Kazancıgil, T. Demirci, H. İ. Öztürk-Negiş, N. Akın, "Isolation, technological characterization and in vitro probiotic evaluation of Lactococcus strains from traditional Turkish skin bag Tulum cheeses," Annals of Microbiology, vol. 69 no. 12, pp. 1275-1287, DOI: 10.1007/s13213-019-01512-4, 2019.

[53] M. Wang, J. Tang, G. Wang, Y. Guo, L. Guo, E. Li, "Isolation and identification of an acid-tolerant Lactobacillus species from chicken intestine and its application," Journal of Biotech Research, vol. 13, pp. 142-151, 2022.

[54] C. Sharma, S. Gulati, N. Thakur, B. P. Singh, S. Gupta, S. Kaur, S. K. Mishra, A. K. Puniya, J. P. S. Gill, H. Panwar, "Antibiotic sensitivity pattern of indigenous lactobacilli isolated from curd and human milk samples," Biotech, vol. 7 no. 1,DOI: 10.1007/s13205-017-0682-0, 2017.

[55] E. Jiménez, V. Ladero, I. Chico, A. Maldonado-Barragán, M. López, V. Martín, L. Fernández, M. Fernández, M. A. Álvarez, C. Torres, J. M. Rodríguez, "Antibiotic resistance, virulence determinants and production of biogenic amines among enterococci from ovine, feline, canine, porcine and human milk," BMC Microbiology, vol. 13 no. 1,DOI: 10.1186/1471-2180-13-288, 2013.

[56] S. A. Kıvanç, M. Kıvanç, T. Yiğit, "Antibiotic susceptibility, antibacterial activity and characterisation of Enterococcus faecium strains isolated from breast milk," Experimental and Therapeutic Medicine, vol. 12 no. 3, pp. 1732-1740, DOI: 10.3892/etm.2016.3545, 2016.

[57] N. A. Reis, M. A. F. Saraiva, E. A. A. Duarte, E. A. de Carvalho, B. B. Vieira, N. S. Evangelista-Barreto, "Probiotic properties of lactic acid bacteria isolated from human milk," Journal of Applied Microbiology, vol. 121 no. 3, pp. 811-820, DOI: 10.1111/jam.13173, 2016.

[58] L. I. I. Ouoba, V. Lei, L. B. Jensen, "Resistance of potential probiotic lactic acid bacteria and bifidobacteria of African and European origin to antimicrobials: determination and transferability of the resistance genes to other bacteria," International Journal of Food Microbiology, vol. 121 no. 2, pp. 217-224, DOI: 10.1016/j.ijfoodmicro.2007.11.018, 2008.

[59] R. Albesharat, M. A. Ehrmann, M. Korakli, S. Yazaji, R. F. Vogel, "Phenotypic and genotypic analyses of lactic acid bacteria in local fermented food, breast milk and faeces of mothers and their babies," Systematic and Applied Microbiology, vol. 34 no. 2, pp. 148-155, DOI: 10.1016/j.syapm.2010.12.001, 2011.

[60] L. Fernández, S. Langa, V. Martín, A. Maldonado, E. Jiménez, R. Martín, J. M. Rodríguez, "The human milk microbiota: origin and potential roles in health and disease," Pharmacological Research, vol. 69 no. 1,DOI: 10.1016/j.phrs.2012.09.001, 2013.

[61] T. Jost, C. Lacroix, C. Braegger, C. Chassard, "Impact of human milk bacteria and oligosaccharides on neonatal gut microbiota establishment and gut health," Nutrition Reviews, vol. 73 no. 7, pp. 426-437, DOI: 10.1093/nutrit/nuu016, 2015.

[62] S. Selvamani, D. J. Dailin, V. K. Gupta, M. Wahid, H. C. Keat, K. H. Natasya, R. A. Malek, S. Haque, R. Z. Sayyed, B. Abomoelak, D. Sukmawati, H. A. El Enshasy, "An insight into probiotics bio-route: translocation from the mother’s gut to the mammary gland," Applied Sciences, vol. 11 no. 16,DOI: 10.3390/app11167247, 2021.

[63] J. M. Rolain, "Food and human gut as reservoirs of transferable antibiotic resistance encoding genes," Frontiers in Microbiology, vol. 4,DOI: 10.3389/fmicb.2013.00173, 2013.

[64] Q. Zhang, G. Lambert, D. Liao, H. Kim, K. Robin, C. K. Tung, N. Pourmand, R. H. Austin, "Acceleration of emergence of bacterial antibiotic resistance in connected microenvironments," Science, vol. 333 no. 6050, pp. 1764-1767, DOI: 10.1126/science.1208747, 2011.

[65] F. M. Treiber, H. Beranek-Knauer, "Antimicrobial residues in food from animal origin—a review of the literature focusing on products collected in stores and markets worldwide," Antibiotics, vol. 10 no. 5,DOI: 10.3390/antibiotics10050534, 2021.

[66] O. O. Adebowale, O. K. Adeyemo, O. Awoyomi, R. Dada, O. Adebowale, "Antibiotic use and practices in commercial poultry laying hens in Ogun State Nigeria," Revue d’élevage et de médecine vétérinaire des pays tropicaux, vol. 69 no. 1, pp. 41-45, DOI: 10.19182/remvt.31170, 2016.

[67] M. D. Mund, U. H. Khan, U. Tahir, B. E. Mustafa, A. Fayyaz, "Antimicrobial drug residues in poultry products and implications on public health: a review," International Journal of Food Properties, vol. 20 no. 7, pp. 1433-1446, DOI: 10.1080/10942912.2016.1212874, 2017.

[68] P. Chaturvedi, P. Shukla, B. S. Giri, P. Chowdhary, R. Chandra, P. Gupta, A. Pandey, "Prevalence and hazardous impact of pharmaceutical and personal care products and antibiotics in environment: a review on emerging contaminants," Environmental Research, vol. 194, article 110664,DOI: 10.1016/j.envres.2020.110664, 2021.

[69] J. Tanwar, S. Das, Z. Fatima, S. Hameed, "Multidrug resistance: an emerging crisis," Interdisciplinary perspectives on infectious diseases, vol. 2014,DOI: 10.1155/2014/541340, 2014.

[70] A. B. Smith, "The regulation of probiotics in the United States," Lactic Acid Bacteria, pp. 693-710, 2019.

[71] A. Li, Y. Wang, S. Suolang, K. Mehmood, X. Jiang, L. Zhang, Z. Li, M. Waqas, M. Iqbal, W. Basang, J. Li, "Isolation and identification of potential Bacillus probiotics from free ranging yaks of Tibetan Plateau, China," Pakistan Veterinary Journal, vol. 39 no. 3, pp. 377-382, DOI: 10.29261/pakvetj/2019.032, 2019.

[72] M. J. Gosalbes, Y. Vallès, N. Jiménez-Hernández, C. Balle, P. Riva, S. Miravet-Verde, L. E. de Vries, S. Llop, Y. Agersø, S. J. Sørensen, F. Ballester, M. P. Francino, "High frequencies of antibiotic resistance genes in infants’ meconium and early fecal samples," Journal of Developmental Origins of Health and Disease, vol. 7 no. 1, pp. 35-44, DOI: 10.1017/S2040174415001506, 2016.

[73] D. V. Patangia, C. A. Ryan, E. Dempsey, C. Stanton, R. P. Ross, "Vertical transfer of antibiotics and antibiotic resistant strains across the mother/baby axis," Trends in Microbiology, vol. 30 no. 1, pp. 47-56, DOI: 10.1016/j.tim.2021.05.006, 2022.

[74] M. Gueimonde, B. Sánchez, C. G. de los Reyes-Gavilán, A. Margolles, "Antibiotic resistance in probiotic bacteria," Frontiers in Microbiology, vol. 4,DOI: 10.3389/fmicb.2013.00202, 2013.

[75] M. E. Sanders, L. M. Akkermans, D. Haller, C. Hammerman, J. T. Heimbach, G. Hörmannsperger, G. Huys, D. D. Levy, F. Lutgendorff, D. Mack, P. Phothirath, G. Solano-Aguilar, E. Vaughan, "Safety assessment of probiotics for human use," Gut Microbes, vol. 1 no. 3, pp. 164-185, DOI: 10.4161/gmic.1.3.12127, 2010.

[76] F. Alamiri, K. Riesbeck, A. P. Hakansson, "HAMLET, a protein complex from human milk, has bactericidal activity and enhances the activity of antibiotics against pathogenic Streptococci," Antimicrobial Agents and Chemotherapy, vol. 63 no. 12,DOI: 10.1128/AAC.01193-19, 2019.

[77] A. P. Hakansson, H. Roche-Hakansson, A. K. Mossberg, C. Svanborg, "Apoptosis-like death in bacteria induced by HAMLET, a human milk lipid-protein complex," PLoS One, vol. 6 no. 3, article e17717,DOI: 10.1371/journal.pone.0017717, 2011.

[78] L. R. Marks, E. A. Clementi, A. P. Hakansson, "The Human Milk Protein-Lipid Complex HAMLET Sensitizes Bacterial Pathogens to Traditional Antimicrobial Agents," PLoS ONE, vol. 7 no. 8, article e43514,DOI: 10.1371/journal.pone.0043514, 2012.

[79] L. R. Marks, E. A. Clementi, A. P. Hakansson, "Sensitization of Staphylococcus aureus to methicillin and other antibiotics in vitro and in vivo in the presence of HAMLET," PLoS One, vol. 8 no. 5, article e63158,DOI: 10.1371/journal.pone.0063158, 2013.

[80] S. Kadaikunnan, T. S. Rejiniemon, J. M. Khaled, N. S. Alharbi, R. Mothana, "In-vitro antibacterial, antifungal, antioxidant and functional properties of Bacillus amyloliquefaciens," Annals of Clinical Microbiology and Antimicrobials, vol. 14 no. 1,DOI: 10.1186/s12941-015-0069-1, 2015.

[81] S. S. Malik, A. Saeed, M. Baig, N. Asif, N. Masood, A. Yasmin, "Anticarcinogenecity of microbiota and probiotics in breast cancer," International Journal of Food Properties, vol. 21 no. 1, pp. 655-666, DOI: 10.1080/10942912.2018.1448994, 2018.

[82] A. Raheem, L. Liang, G. Zhang, S. Cui, "Modulatory effects of probiotics during pathogenic infections with emphasis on immune regulation," Frontiers in Immunology, vol. 12, article 616713,DOI: 10.3389/fimmu.2021.616713, 2021.

[83] A. M. Leite, M. A. L. Miguel, R. S. Peixoto, P. Ruas-Madiedo, V. M. F. Paschoalin, B. Mayo, S. Delgado, "Probiotic potential of selected lactic acid bacteria strains isolated from Brazilian kefir grains," Journal of Dairy Science, vol. 98 no. 6, pp. 3622-3632, DOI: 10.3168/jds.2014-9265, 2015.

[84] J. Šušković, B. Kos, J. Beganović, A. Leboš Pavunc, K. Habjanič, S. Matošić, "Antimicrobial activity–the most important property of probiotic and starter lactic acid bacteria," Food Technology and Biotechnology, vol. 48 no. 3, pp. 296-307, 2010.

[85] S. Jara, M. Sánchez, R. Vera, J. Cofré, E. Castro, "The inhibitory activity of Lactobacillus spp. isolated from breast milk on gastrointestinal pathogenic bacteria of nosocomial origin," Anaerobe, vol. 17 no. 6, pp. 474-477, DOI: 10.1016/j.anaerobe.2011.07.008, 2011.

[86] X. Pan, F. Chen, T. Wu, H. Tang, Z. Zhao, "The acid, bile tolerance and antimicrobial property of Lactobacillus acidophilus NIT," Food Control, vol. 20 no. 6, pp. 598-602, DOI: 10.1016/j.foodcont.2008.08.019, 2009.

[87] M. M. Al-Otaibi, "Probiotics yoghurt and their effect on growth of the pathogenic microorganism," Alexandria Science Exchange Journal, vol. 34 no. 1,DOI: 10.21608/asejaiqjsae.2013.158971, 2013.

[88] M. M. Al-Otaibi, N. S. Al-Zoreky, H. A. El-Dermerdash, "Camel's milk as a natural source for probiotics," Research Journal of Microbiology, vol. 8 no. 2, pp. 70-80, DOI: 10.3923/jm.2013.70.80, 2013.

[89] G. Tellez-Isaias, C. N. Vuong, B. D. Graham, C. M. Selby, L. E. Graham, R. Senas-Cuesta, T. L. Barros, L. C. Beer, M. E. Coles, A. J. Forga, J. Ruff, B. M. Hargis, "Developing probiotics, prebiotics, and organic acids to control Salmonella spp. in commercial turkeys at the University of Arkansas, USA," Turkey Diseases, Production and Management, vol. 1 no. 3,DOI: 10.51585/gjvr.2021.3.0014, 2021.

[90] D. R. Silva, J. D. C. O. Sardi, N. de Souza Pitangui, S. M. Roque, A. C. B. da Silva, P. L. Rosalen, "Probiotics as an alternative antimicrobial therapy: current reality and future directions," Journal of Functional Foods, vol. 73, article 104080,DOI: 10.1016/j.jff.2020.104080, 2020.