Characterization and evaluation of anti-Salmonella enteritidis activity of indigenous probiotic lactobacilli in mice

Amina Mustafa; Muhammad Nawaz; Masood Rabbani; Muhammad Tayyab; Madiha Khan

doi:10.1515/biol-2022-0100

Artikel Open Access

Characterization and evaluation of anti-Salmonella enteritidis activity of indigenous probiotic lactobacilli in mice

Amina Mustafa , Muhammad Nawaz , Masood Rabbani , Muhammad Tayyab und Madiha Khan

Veröffentlicht/Copyright: 17. August 2022

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Abstract

Lactobacilli (n = 24), isolated from human infants and yogurt, showed variable in vitro activity against Salmonella enteritidis (8.0 ± 1.0 to 16.6 ± 0.5 mm) and other gut pathogens (9.0 ± 1.0 to 15.3 ± 0.5 mm), as determined by a well diffusion assay. The isolates were identified as Limosilactobacillus fermentum (FY1, FY3, FY4, IL2, and IL5), Lactobacillus delbrueckii (FY6 and FY7), Lactobacillus sp. (IL7), and Lactobacillus gasseri (IL12). All isolates showed variable in vitro tolerance to acidic pH for 3 h and visible growth at pH 4 and in the presence of 0.3% ox-bile. The antibiotic susceptibility profile of Lactobacillus isolates indicated resistance against vancomycin, ciprofloxacin, streptomycin, and lincomycin. Isolates had variable auto-aggregation and showed variable capabilities to co-aggregate with S. enteritidis. Based on all tested parameters, L. fermentum IL2, L. fermentum IL5, and L. gasseri IL12 were selected for co-culture experiments, followed by in vivo evaluation in Balb/c mice. All the selected isolates resulted in a 100% reduction in S. enteritidis in broth. Lactobacillus isolates efficiently colonized mouse guts and inhibited S. enteritidis colonization. Overall, there was ≥99.06% and ≤4.32 Mean log₁₀ reduction in Salmonella counts in mice feces within 7 days. The study, thus, provided characterized lactobacilli that could be considered as potential ingredients for probiotic formulations intended to prevent S. enteritidis infection in humans.

Keywords: lactobacilli; Salmonella enteritidis ; mice; yogurt; human infants

1 Introduction

Salmonella enterica is a leading bacterial cause of foodborne illnesses, causing a considerable burden globally [1]. Out of 2,600 salmonellae, Salmonella enterica serovar Enteritidis is a significant pathogen of public health importance [2]. Salmonella enteritidis causes gastrointestinal inflammation, with diarrhea as the most common symptom. Diarrhea may cause excessive water loss while removing bacteria from the body. Thus, the innovation of effective control strategies against Salmonella enteritidis infections has been a moving target [3]. In Pakistan, it is an endemic pathogen causing regular outbreaks associated with poultry products [4]. Moreover, it can be transmitted both vertically and horizontally in poultry and eventually to humans [5]. In the 20th century, Salmonella enteritidis emerged as a major egg-associated pathogen [6]. Although numerous antibiotics are available for treating Salmonella infections, their excessive use has mounted anti-microbial resistance, treatment failures, and a negative impact on human health [7]. Isolation of multi-drug resistant Salmonella enteritidis from different regions of Pakistan has disclosed a potential threat not only to humans but also to the poultry industry of Pakistan [8]. Therefore, Pakistan has restricted the preventive and medical use of antibiotics in poultry and livestock sectors [9]. However, managing drug-resistant Salmonella enteritidis infections by non-antibiotic prophylactic regimens (including probiotics) is an acceptable alternative [10].

Probiotics or direct-fed microbes are currently receiving considerable interest as an alternative to antibiotics. According to World Health Organization and Food and Agriculture Organization, probiotics are the living microorganisms which when administered in adequate amount confer a health benefit to the host [11]. These have been classified into two broad categories, including classical probiotics (Bifidobacterium, Lactobacillus, and Saccharomyces boulardii) and next-generation probiotics (Faecalibacterium prausnitzii and Akkermancia muciniphila) [12]. The different strains of probiotics are being used in numerous supplements, drugs, food products, milk powder, cheese, yogurt, ice cream, and fruit juices [13].

Probiotics are distinguished for providing health benefits, including immune boosting, protection against gut pathogens, binding of mycotoxins, strengthening of gut function and microbiota, and increased absorption of nutrients [14]. It has been hypothesized that probiotic lactobacilli have antibacterial potential and, thus, can prevent the growth of pathogenic bacteria like Salmonella, Escherichia coli, and Campylobacter [15,16]. The anti-Salmonella activity of lactic acid bacteria and their potential to act as a probiotic feed additive, especially against salmonellosis, have been demonstrated previously [17]. Currently, the mechanistic action of probiotics is still under consideration. The mechanisms of action of probiotics have not been fully revealed. However, the most favored mechanisms include the production of antibacterial compounds like bacteriocins, organic acids, and H₂O₂, a decline in gut pH, competition for nutrients, immune modulation, modulation of cytokine pattern and toxin action, reduction in bacterial enzyme activity, neutralization of enterotoxins, mycotoxin removal by physical binding, competitive exclusion of pathogen, and antagonism of the pathogen, followed by strengthening of normal flora and enhanced digestive activity [18,19].

The anti-microbial effect of probiotics is species and strain-specific, which is more prominent against pathogens from the same origin. Therefore, extensive screening is required to select probiotic strains [20]. Due to the beneficial attributes and lesser side effects, there has been increased interest in searching out novel probiotic strains. Thus, the present work is aimed to isolate anti-S. enteritidis lactobacilli, determine their in vitro probiotic properties, and evaluate the resultant probiotics for the prevention of Salmonella enteritidis colonization in mice.

2 Methods

2.1 Isolation and identification of lactobacilli

Yogurt samples (n = 15), including both homemade (n = 7) and commercial (n = 8), were collected from different areas of Lahore. Fecal swab samples (n = 15) were obtained from healthy humans (breastfed infants) after taking informed consent from their parents/guardians. All samples were transported to the Institute of Microbiology, the University of Veterinary and Animal Sciences (UVAS), Lahore, where these were serially diluted (ten-fold) and plated on de Man, Rogosa, and Sharpe (MRS) agar (Merck, Germany), as described in a previous study [21]. After 24–48 h incubation at 37°C (equivalent to human body temperature) in an anaerobic jar (Oxoid Anaerobic indicator BR0055), individual colonies were purified and stored in MRS broth supplemented with 20% glycerol at −20°C. Lactobacilli were initially identified by Gram’s staining and a catalase test, as described by Bergey’s Manual of Determinative Bacteriology [22]. DNAs of lactobacilli were isolated using a DNA extraction kit (Geneall, South Korea), according to the manufacturer’s instructions. Lactobacillus genus was confirmed using specific primers XB5-F (5′-GCCTTGTACACACCGCCCGT-3′) and LbLMA1-R (5′-CTCAAAACTAAACAAAGTTTC-3′) targeting 16S–23S inter-spacer regions, as used earlier [23]. Whereas, 16S rDNAs were amplified using universal primers 8FLP-F (5′-AGTTTGATCNCTGGCTCAG-3′) and XB4-R (5′-GTGTGTACAAGGCCCGGGAAC-3′) [24]. The fragments were amplified in a Bio-Rad T100^TM Thermo-cycler using the following program: initial denaturation at 94°C for 10 min, followed by 35 cycles of final denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and initial extension at 72°C for 1 min. In the end, the final extension was performed at 72°C for 10 min. The sequences of 16S rDNA amplicons (∼1400 bp) or 16S–23S rDNA inter-spacer (∼250 bp) were submitted to GenBank, and accession numbers were obtained.

Informed consent: Informed consent has been obtained from the guardians of all individuals included in this study.
Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance with the tenets of the Helsinki Declaration, and has been approved by the authors’ institutional review board or equivalent committee.

2.2 Screening of anti-Salmonella enteritidis lactobacilli

Lactobacilli were screened for their activity against Salmonella enteritidis (D) (ATCC13076^TM) and antibiotic-resistant salmonellae (Salmonella enteritidis 54, Salmonella enteritidis 56, Salmonella enteritidis 58, and Salmonella enteritidis 91) of poultry origin, that were isolated previously by Yasmin [9]. The antibiotic susceptibility patterns of salmonellae are mentioned in Table 1.

Table 1

Antibiotic susceptibility patterns of salmonellae

Strain	Antibiotic resistance profile
S.E 54	AMP^R, AMX^R, CHL^R, CIP^R, NAL^R, TET^R, CRO^R, and CFM^R
S.E 56	AMP^R, AMX^R, CHL^R, CIP^R, NAL^R, TET^R, CRO^R, CFM^R, and CAZ^R
S.E 58	AMP^R, AMX^R, CHL^R, CIP^R, NAL^R, TET^R, CAZ^R, GEN ^R, and SXT ^R
S.E 91	AMP^R, AMX^R, CHL^R, CIP^R, NAL^R, and GEN ^R

Note: S.E: Salmonella enteritidis; R: resistant; AMP: ampicillin; AMX: amoxicillin; CHL: chloramphenicol; CIP: ciprofloxacin; NAL: nalidixic acid; TET: tetracycline; CRO: ceftriaxone; CFM: cefixime; CAZ: ceftazidime; GEN: gentamicin; SXT: sulfamethoxazole.

An agar diffusion assay was performed following the previous method with slight modifications [1,15,16,25]. In brief, ∼0.5 McFarland inoculum of Salmonella enteritidis was swabbed on Mueller–Hinton (MH) agar. Wells were created, sealed with molten agar, and filled with cell-free supernatants (CFSs; 80–100 µL) of lactobacilli. CFSs were also tested after neutralization using NaOH (1 mol L⁻¹) and inactivation of H₂O₂ using catalase. MRS broth was used as a negative control in the test. After 24 h incubation at 37°C, the diameter of inhibition zones was measured in millimeters.

2.3 In vitro probiotic properties of lactobacilli

All the tests essential for the assessment of probiotic potential were performed in accordance with the previous related studies [10,21,26].

2.3.1 Tolerance to acidic pH and ox-bile

Acid tolerance of lactobacilli was determined as described by Asghar et al. [26], with some modifications. Briefly, lactobacilli (3 × 10⁷ CFU) were exposed to normal saline having pH 2, 3, 4, and 7, adjusted with HCl (0.1 mol L⁻¹)/NaOH (1 mol L⁻¹). After 3 h incubation at 37°C, 20 µL of pH-exposed lactobacilli (6 × 10⁶ CFU) were re-cultured in 200 µL of MRS broth (pH = 7) supplemented with L-cysteine (0.5 g L⁻¹), using a 96-well microtiter plate. The plate was then incubated at 37°C. The pH tolerance was estimated by calculating the difference in optical density (OD) values at 24 h and 0 min, recorded at 630 nm using a Rayto Microtiter plate reader (RT-2100C). To monitor the ability of lactobacilli to survive acidic conditions, their growth was observed in MRS broth with pH adjusted to 3, 4, and 7. The bacterial growth was indicated by an increase in OD values after 24 h incubation.

To check tolerance to ox-bile, lactobacilli (3 × 10⁷ CFU) were inoculated in 200 µL of MRS broth supplemented with 0.3, 1.0, and 1.8% purified ox-bile (Hi-Media) and l-cysteine (0.5 g L⁻¹) [27,28]. After 24 h incubation at 37°C, tolerance was assessed by calculating the difference in OD values at 0 min and 24 h.

2.3.2 Activity against other gut pathogens

The activity of isolates against Campylobacter jejuni (ATCC33291^TM), E. coli ZQN9, and Salmonella Gallinarum G1 was determined by the agar well diffusion method as described earlier.

2.3.3 Antibiotic susceptibility pattern

Minimum inhibitory concentrations (MICs) of ampicillin, amoxicillin, chloramphenicol, vancomycin, erythromycin, tetracycline, penicillin, ciprofloxacin, lincomycin, streptomycin, doxycycline, and levofloxacin against lactobacilli (n = 24) were determined by the broth microdilution method in 96-well microtiter plates as demonstrated by Saleem [29]. Two-fold dilutions of antibiotics (0.25–128 μg mL⁻¹) were prepared in the LAB susceptibility test medium using microtiter plate wells. Inoculums of lactobacilli (∼1 McFarland) were prepared by suspending their fresh growth in normal saline, followed by dilution (1/10³) in MRS broth. Then, the doubly diluted antibiotics (50 μL) were inoculated with prepared inoculum (100 μL, ∼3 × 10⁴ CFU). Microtiter plates were then incubated at 37°C for 24 h in anaerobic conditions, followed by observation of visible growth or turbidity. Minimum concentrations of antibiotics inhibiting the visible growth of isolates were considered as MICs. Isolates were designated as resistant, intermediate, and sensitive to the tested antibiotics following the breakpoints given by the European Food Safety Authority (EFSA) [30], except the breakpoints for ciprofloxacin and levofloxacin were adopted from the Clinical and Laboratory Standards Institute.

2.3.4 Auto-aggregation and Co-aggregation assays

Auto-aggregation and co-aggregation abilities of lactobacilli were determined according to the procedure of Angelov [31]. Briefly, ∼1 McFarland suspension of freshly grown Lactobacillus and Salmonella enteritidis were incubated alone and in combination (1:1) at 37°C. Afterward, OD of 0.2 mL supernatant was recorded at 0 min, 1, 2, and 16 h. Percentage auto-aggregation and co-aggregation were calculated using the same formulae that were used by Angelov [31].

2.4 Salmonella enteritidis inhibition in broth cultures

A broth inhibition test was performed by adapting the procedure used by Khan et al. [21]. Suspensions (100 µL) of Salmonella enteritidis (0.5 McFarland) and Lactobacillus (1 McFarland) were mixed in skim milk broth (10 mL) and incubated at 37°C. Salmonella enteritidis and lactobacilli were enumerated from this mixture at different intervals, using Salmonella Shigella and MRS agar, respectively.

2.5 In vivo evaluation of probiotic lactobacilli for prevention of Salmonella enteritidis in mice

The probiotic potential of selected lactobacilli against Salmonella enteritidis was evaluated in mice using the previous protocol with some modifications in experimental design [32]. In vivo experiments were conducted in compliance with the institutional ethical guidelines. Ethical approval was obtained from the institutional ethical review committee of UVAS, Lahore (approval No. 408; dated: 22/04/2019). Healthy conventional Balb/c mice were purchased from the University of Health Sciences, Lahore, and moved to the cages placed in controlled mouse rooms of the Institute of Microbiology, UVAS, at 22 ± 2°C temperature, with 65% humidity and alternate 12 h periods of light and dark. All the males mice at 8 weeks of age were divided into 5 groups, each with 5 mice. They were supplied with potable water and commercial standard fat diet ad libitum and housed in cages (filled with wood shavings). Negative control mice were administered with sterilized normal saline. The positive control group received only Salmonella enteritidis. The experimental groups were orally administered with a probiotic dose (0.6 × 10⁸ CFU), using orogastric gavage, for 17 days. Experimental and positive control groups were additionally challenged (twice) with Salmonella enteritidis (D) ATCC13076^TM (∼10⁷ CFU mL⁻¹) on the 7th and 15th day of the experiment. Fresh fecal samples were collected using sterile forceps and diluted (ten-fold) using sterile normal saline. The diluted feces (0.2 mL) were spread on both the Salmonella Shigella agar and MRS agar supplemented with vancomycin (100 µg mL⁻¹) and fluconazole (100 µg mL⁻¹), separately. The plates were then incubated at 37°C for 24–48 h. Lactobacillus counts and Salmonella counts were then performed.

Ethical approval: The research related to animal use has been complied with all the relevant national regulations and institutional policies for the care and use of animals.

2.6 Statistical analysis

All analyses were carried out using one-way ANOVA followed by Tukey’s test, using GraphPad Prism 5.0 software. A p-value < 0.05 was considered significant.

3 Results

3.1 Isolates identified as Lactobacillus spp.

Lactobacillus isolates (n = 24) were purified from 30 samples, 9 from yogurt and 15 from feces of healthy human infants who were fed on breast milk and did not receive any previous treatment with probiotics and antibiotics. All isolates were Gram-positive rods and catalase-negative. All 24 isolates were confirmed as lactobacilli using Lactobacillus genus-specific polymerase chain reaction. Isolates were confirmed as L. fermentum (FY1 [MN153531], FY3 [MN153532], FY4 [MN153533], IL2 [MN153536 – 16S rDNA; MN161530 – 16S–23S inter-spacer], and IL5 [MN161531]), L. delbrueckii (FY6 [MN153534] and FY7 [MN153535]), Lactobacillus sp. (IL7), L. gasseri (IL12 [MN153537 – 16S rDNA; MN161532 – 16S–23S inter-spacer]), and other species of Lactobacillus.

3.2 Anti-microbial effect of lactobacilli against S. enteritidis

The activity of lactobacilli against Salmonella enteritidis was indicated by ≤16.6 ± 0.5 mm inhibition zones against salmonellae, determined by a well diffusion assay (Figure 1).

Figure 1

Anti-Salmonella enteritidis activity (mean value ± SD) of CFSs of lactobacilli. No zones of inhibition were detected in neutralized CFSs except a few with ≤8.6 ± 0.5 mm zone diameter. Inactivation of H₂O₂ did not affect the activity of supernatants. S.E: Salmonella enteritidis; SD: Standard Deviation. a, b, c, and d indicate statistically significant difference (p < 0.05) among different bars of the same group.

3.3 In vitro probiotic properties of lactobacilli

None of the isolates showed growth at pH 3, except FY9, IL4, IL9, and IL17 which showed an increase in OD (≤0.148 ± 0.015). Most lactobacilli showed better growth at pH 4 (increase in OD ≤ 1.076 ± 0.006) compared to pH 3, except FY3, FY5, FY7, FY9, and IL1. The IL2 and IL13 showed the highest growth at pH 4 (Figure 2).

Figure 2

Growth of lactobacilli at different pH values within 24 h. SD: standard deviation; OD: optical density. a, b, and c indicate statistically significant difference (p < 0.05) among different bars of the same group.

The isolates also showed varying degrees of tolerance to pH 4 (increase in OD ≤ 1.291 ± 0.068) for 3 h, except FY1, FY5, and IL6. Only 9 isolates tolerated pH 3 (increase in OD ≤ 1.359 ± 0.4) for 3 h, as shown in Table 2. Isolates FY3 and IL15 additionally tolerated pH 2 (increase in OD ≤ 1.067 ± 0.05) for 3 h. In short, lactobacilli were more tolerant to pH 4 compared to lower pH values.

Table 2

Tolerance of lactobacilli to different pH values for 3 h

Sr No	Isolate	Tolerance for 3 h (increase in OD)
Sr No	Isolate	pH 7	pH 4	pH 3	pH 2
1	FY1	0.716 ± 0.5^a	0.015 ± 0.01^b	1.017 ± 0.005^a	0.034 ± 0.002^b
2	FY2	1.063 ± 0.04^a	1.061 ± 0.08^a	0.982 ± 0.01^a	0.076 ± 0.003^b
3	FY3	0.916 ± 0.04^a	1.028 ± 0.05^a	0.07 ± 0.008^b	0.609 ± 0.5^b
4	FY4	1.008 ± 0.07^a	0.942 ± 0.06^a	0.068 ± 0.008^b	0.062 ± 0.008^b
5	FY5	1.140 ± 0.09^a	0.006 ± 0.01^b	0.624 ± 0.02^b	0.202 ± 0.1^c
6	FY6	1.190 ± 0.16^a	0.466 ± 0.65^b	0.058 ± 0.003^b	0.046 ± 0.01^b
7	FY7	1.234 ± 0.13^a	0.043 ± 0.021^b	0.059 ± 0.005^b	0.238 ± 0.313^b
8	FY8	0.72 ± 0.52^a	0.65 ± 0.52^a	0.051 ± 0.003^a	0.049 ± 0.006^a
9	FY9	0.909 ± 0.38^a	1.29 ± 0.12^a	0.047 ± 0.005^b	0.046 ± 0.001^b
10	IL1	0.047 ± 0.06^a	1.073 ± 0.02^b	1.359 ± 0.4^b	0.021 ± 0.002^a
11	IL2	0.812 ± 0.66^a	1.024 ± 0.07^a	0.98 ± 0.03^a	0.027 ± 0.003^b
12	IL3	1.136 ± 0.04^a	1.07 ± 0.05^a	0.075 ± 0.05^b	0.029 ± 0.001^b
13	IL4	1.045 ± 0.02^a	1.078 ± 0.11^a	0.025 ± 0.02^b	0.02 ± 0.01^b
14	IL5	1.120 ± 0.07^a	1.065 ± 0.06^a	1.045 ± 0.04^a	0.201 ± 0.1^b
15	IL6	1.124 ± 0.07^a	1.159 ± 0.22^a	0.053 ± 0.01^b	0.013 ± 0.013^b
16	IL7	1.215 ± 0.21^a	0.069 ± 0.006^b	0.543 ± 0.03^c	0.043 ± 0.04^b
17	IL8	1.077 ± 0.21^a	1.148 ± 0.12^a	1.096 ± 0.07^a	0.024 ± 0.001^b
18	IL9	1.147 ± 0.01^a	1.291 ± 0.068^b	0.992 ± 0.01^c	0.023 ± 0.03^d
19	IL10	0.405 ± 0.54^a	1.136 ± 0.09^b	0.090 ± 0.008^a	0.016 ± 0.01^a
20	IL12	1.021 ± 0.02^a	1.19 ± 0.18^a	1.057 ± 0.003^a	0.013 ± 0.02^b
21	IL13	1.166 ± 0.01^a	0.482 ± 0.7^b	0.078 ± 0.002^b	0.012 ± 0.01^b
22	IL14	1.115 ± 0.06^a	1.083 ± 0.09^a	0.304 ± 0.385^b	0.004 ± 0.03^b
23	IL15	1.177 ± 0.18^a	1.277 ± 0.24^a	1.121 ± 0.01^a	1.067 ± 0.05^a
24	IL17	0.893 ± 0.1^a	0.934 ± 0.04^a	0.059 ± 0.007^b	0.02 ± 0.01^b

Note: a, b, c, and d indicate statistically significant difference (p < 0.05) among different columns of the same row. SD: standard deviation; OD: optical density. Increase in OD = OD after 24 h – OD at 0 min.

Lactobacillus isolates (n = 18) showed good growth rates in the presence of 0.3% ox-bile (increase in OD ≤ 0.603 ± 0.4), and 1% ox-bile (increase in OD ≤ 0.339 ± 0.02) as indicated in Figure 3.

Figure 3

Growth of lactobacilli in MRS broth supplemented with different concentrations of ox-bile within 24 h. a, b, c, and d indicate statistically significant difference (p < 0.05) among different bars of same group; SD: dtandard deviation.

All the isolates had variable auto-aggregation and co-aggregation (with Salmonella enteritidis) capabilities, ranging between 0.8 ± 0.2 and 55.6 ± 0.1% and 0.8 ± 0.1 and 32.7 ± 0.2%, respectively (Figures 4 and 5).

Figure 4

Auto-aggregation of lactobacilli within different time intervals.

Figure 5

Co-aggregation of lactobacilli with Salmonella enteritidis within different time intervals.

The safety profile of lactobacilli indicated sensitivity to all the tested antibiotics except vancomycin, ciprofloxacin, lincomycin, and streptomycin (Table 3). Lactobacilli also had variable activity against other gut pathogens, including Campylobacter jejuni ATCC (≤15.3 ± 0.5 mm), E. coli ZQN9 (≤11.3 ± 0.5 mm), and Salmonella Gallinarum G1 (≤12 ± 0 mm).

Table 3

Antibiotic resistance pattern of lactobacilli

Sr No.	Isolates	Resistance profile
1	FY1	VAN^R, CIP^R, and STR^R
2	FY2	VAN^R, CIP^R, and STR^R
3	FY3	VAN^R, CIP^R, and STR^R
4	FY4	VAN^R, CIP^R, and STR^R
5	FY5	VAN^R, CIP^R, and STR^R
6	FY6	CIP^R
7	FY7	CIP^R and STR^R
8	FY8	VAN^R, CIP^R, and STR^R
9	FY9	VAN^R, CIP^R, and STR^R
10	IL1	VAN^R, CIP^R, LCM^R, and STR^R
11	IL2	VAN^R, CIP^R, and LCM^R
12	IL3	VAN^R, CIP^R, LCM^R, and STR^R
13	IL4	VAN^R, CIP^R, LCM^R, and STR^R
14	IL5	VAN^R, LCM^R, and STR^R
15	IL6	VAN^R, CIP^R, LCM^R, and STR^R
16	IL7	VAN^R, CIP^R, LCM^R, and STR^R
17	IL8	VAN^R, CIP^R, LCM^R, and STR^R
18	IL9	VAN^R, CIP^R, LCM^R, and STR^R
19	IL10	CIP^R, LCM^R, and STR^R
20	IL12	VAN^R, CIP^R, and LCM^R
21	IL13	VAN^R, CIP^R, and STR^R
22	IL14	VAN^R, CIP^R, and STR^R
23	IL15	VAN^R, CIP^R, and STR^R
24	IL17	VAN^R, CIP^R, and STR^R

Note: R: resistant; VAN: vancomycin; CIP: ciprofloxacin; STR: streptomycin; LCM: lincomycin.

3.4 In vitro and in vivo activities of probiotic lactobacilli against S. enteritidis

All the selected isolates (L. fermentum IL5, L. fermentum IL2, and L. gasseri IL12) resulted in a 100% reduction in S. enteritidis in broth (Table 4), followed by efficient colonization in mouse guts, as indicated by increased Lactobacillus counts (8.77 ± 0.02 log₁₀ CFU mL⁻¹) in mice feces (Table 5). No Salmonella was detected in mice feces of IL5 and IL12 groups at the end of the trial. On the other hand, there was a 2 log reduction in Salmonella counts in the case of IL2 group. There was no significant reduction in Salmonella counts in mice feces of positive and negative control groups. Within a week, there was 2.02, 3.37, and 4.32 mean log₁₀ reduction in Salmonella counts in mice feces by IL2, IL5, and IL12, respectively. In other words, the tested isolates (IL2, IL5, and IL12) showed 99.06, >99.9, and >99.99% reduction in Salmonella counts, respectively. Fortunately, no mortality was observed during the in vivo experiment.

Table 4

Reduction in Salmonella enteritidis in co-culture with selected probiotics

Lactobacilli	Log₁₀ CFU mL⁻¹ (mean value ± SD) of Salmonella enteritidis				Salmonella enteritidis reduction at 48 h (%)
Lactobacilli	0 min	4 h	24 h	48 h	Salmonella enteritidis reduction at 48 h (%)
IL2	6.35 ± 0.03^a	8.92 ± 0.02^c	7.65 ± 0.03^b	ND	100
IL5	5.92 ± 0.02^a	7.23 ± 0.02^c	6.55 ± 0.05^b	ND	100
IL12	4.91 ± 0.03^a	8.82 ± 0.02^c	6.68 ± 0.02^b	ND	100

Note: a, b, and c indicate statistically significant difference (p < 0.05) among different columns of the same row; ND: not detected; SD: standard deviation; CFU: colony-forming unit.

Table 5

Effect of probiotic lactobacilli on Salmonella enteritidis colonization in mice

Group	log₁₀ CFU mL⁻¹ (mean value ± SD)
Group	Day 1		Day 8		Day 10		Day 16		Day 17
	S.E	Lactobacillus	S.E	Lactobacillus	S.E	Lactobacillus	S.E	Lactobacillus	S.E
NC	—	—	2.68 ± 0.02^a	7.33 ± 0.02^a	2.57 ± 0.02^a	8.16 ± 0.03^a	2.68 ± 0.02^a	7.57 ± 0.02^a	2.31 ± 0.03^a
PC	—	—	3.35 ± 0.05^b	8.21 ± 0.01^b	3.47 ± 0.02^b	7.57 ± 0.02^b	4.82 ± 0.02^b	3.6 ± 0.07^b	4.32 ± 0.02^b
IL2	ND	3.16 ± 0.01^a	4.94 ± 0.02^c	7.78 ± 0.02^c	2.74 ± 0.02^c	7.91 ± 0.01^c	2.95 ± 0.02^c	7.56 ± 0.02^a	2.30 ± 0.02^a
IL5	ND	3.44 ± 0.04^b	ND	8.77 ± 0.02^d	ND	8.12 ± 0.02^a	ND	8.05 ± 0.05^c	ND
IL12	2.83 ± 0.02	4.31 ± 0.07^c	2.62 ± 0.02^a	7.82 ± 0.02^c	5.61 ± 0.02^d	8.67 ± 0.02^d	2.48 ± 0.01^d	8.54 ± 0.04^d	ND

Note: a, b, c, and d indicate statistically significant difference (p < 0.05) among different rows of the same column; CFU: colony-forming units; SD: standard deviation; S.E: Salmonella enteritidis; NC: negative control; PC: positive control.

4 Discussion

The frequent use of antibiotics against Salmonella enteritidis has resulted in antibiotic-resistant salmonellae [9,23]. One of the recently proposed sustainable alternatives to antibiotics for the prevention of Salmonella enteritidis is the use of Lactobacillus as probiotics [33]. Various dietary probiotic microbes, including L. rhamnosus, L. reuteri, L. casei, L. acidophilus, E. coli, Bifidobacterium, Enterococci faecium, and Saccharomyces boulardii have been successfully investigated for medicinal use, either as single strain or as mixed cultures [34]. Lactobacillus is a natural resident of human, animal, and avian gastrointestinal tracts (GIT) and has been isolated from all parts of intestines, plants, and fermented foods [21,23,26,35]. Other studies isolated probiotic L. fermentum, L. reutri, L. gallinarum, L. plantarum, and L. brevis from homemade yogurt, Brazilian food products, and indigenous poultry [36,37]. Likewise, the current research isolated the local lactobacilli not only from yogurt but also from breastfed babies. As these lactobacilli are of human origin and were already a part of microflora, they may exert better probiotic effects on human GIT. Likewise, lactobacilli have also been isolated from the stool of breastfed healthy babies in China using the same strategy [24]. Ligilactobacillus salivarius CECT 5713, isolated from a breastfed 1-month-old infant, proved to be a potential probiotic strain [38]. In a recent study, Dobreva et al. [39] isolated Lactiplantibacillus plantarum strains, having activity against S. Typhimurium, from breast milk and tested their efficiency in simulated real conditions in the food chain. L. gasseri has also been reported as an important component of vaginal microflora [40]; thus, L. gasseri IL12 isolated from the stool of breastfed infants in the present study may be a representative of the mother’s reproductive tract flora.

Many studies have investigated the effects of probiotics against Salmonella [21,26,41,42]. Usually, in vitro investigations are carried out before in vivo studies for probiotic screening. However, the results of all the in vitro assays are not always consistent with the outcomes of in vivo experiments. In order to screen anti-Salmonella enteritidis lactobacilli, a well diffusion assay was used in the present study, while Kowalska et al. [43] used the agar slab method for the same purpose. Moreover, Zhou et al. [44] showed the anti-Salmonella mode of action of natural L-phenyl lactic acid purified from L. plantarum. Another study showed the successful inhibition of Salmonella invasion to intestinal epithelium HT29 cells by probiotic lactobacilli (L. salivarius and L. agilis) of porcine origin [45]. Recently, anti-fungal and anti-aflatoxigenic lactobacilli have also been screened [46].

To reach the intestine, microorganisms must survive acidity stress (pH 1.5–2) in the stomach. Lactobacillus is capable of using a proton antiport system for pH maintenance in a wide range of environments [47]. The ability of an organism to grow in MRS broth at acidic pH or to tolerate acid stress is often used for probiotic selection [24]. Current research showed visible growth of Lactobacillus isolates at both pH 4 and pH 7. In addition, tolerance to acid was also monitored by checking survival after exposure to acid stress without any nutrition. For the said purpose, normal saline (0.89% NaCl) was preferentially used because it provides osmotic protection to bacterial cells. Normal saline normally has a pH of around 5.5. To expose isolates to acid stress without any nutrition, the pH of saline was adjusted to 3, 4, and 7 using HCl (0.1 mol L⁻¹)/NaOH (1 mol L⁻¹). The similar tolerant behavior of lactobacilli has also been previously identified by Khan et al. [21] and Asghar et al. [26], employing the same strategy. Mostly, researchers used to record log counts of survivors after exposure to acid or bile. In contrast, the present study measured the OD, with increased OD values confirming the capability of isolates to survive in/after stressed conditions. According to the previous report, lactobacilli showed 41% survival after 90 min of exposure to pH 2 and 3 [26]. Similarly, Aazami et al. [41] reported high growth rates of Limosilactobacillus reuteri and Ligilactobacillus salivarius at pH 2.5. G-Alegría et al. [48] also showed that Lactiplantibacillus plantarum isolates could grow well at pH 3.2. To establish in the gut, lactobacilli should possess the ability to grow in the presence of at least 0.3% bile salts [26,49]. Lactobacilli isolated in present study showed growth in the presence of 0.3% ox-bile (increase in OD ≤ 0.603 ± 0.4), and 1% ox-bile (increase in OD ≤ 0.339 ± 0.02). Previously reported probiotics also showed a similar degree of resistance [26]. The greater resistance of isolates against 0.3% ox-bile compared to 1% and 1.8% agrees with previous findings [21]. A high degree of bile tolerance in L. fermentum, L. plantarum, and L. brevis has also been examined in a previous study [37].

The auto-aggregation capability of a probiotic strain is necessary for attachment to the gut epithelium. Whereas co-aggregation provides a barrier effect against pathogen colonization. The present study indicated variable auto-aggregation and co-aggregation (with Salmonella enteritidis) capabilities of lactobacilli, ranging between 0.8 ± 0.2 and 55.6 ± 0.1% and 0.8 ± 0.1 and 32.7 ± 0.2%, respectively. Several prior research works also reported similar effects of lactobacilli against Salmonella [21,26,41].

The EFSA has declared lactobacilli as generally recognized as safe microorganisms [49]. However, antibiotic susceptibility of food origin probiotics should be determined to prevent the transfer of potential anti-microbial resistance traits to commensal flora of animals, humans, and pathogens [50]. Lactobacillus sp. is generally sensitive to protein synthesis and cell wall synthesis inhibitors, except aminoglycoside and vancomycin, respectively. Whereas lactobacilli are mostly resistant to DNA synthesis inhibitors [19]. All lactobacilli isolated in the present study were sensitive to most of the tested antibiotics. This study also revealed vancomycin, ciprofloxacin, and streptomycin-resistant lactobacilli (Table 2) as reported previously [19]. Criteria for selecting probiotic strain also involve the activity against gut pathogens. Anti-Salmonella lactobacilli also showed activity against Campylobacter jejuni ATCC, E. coli ZQN9, and Salmonella Gallinarum G1. Recent reports are also available, indicating 6.5–12 mm inhibition zone radii [51,52,53]. In this way, probiotic bacteria suppress the growth of undesirable bacteria in the gut, resulting in the stabilization of the digestive system [54].

Surface layer proteins of Lactobacillus and adhesins (lectins) found in the bacterial cell wall and intestinal epithelium favor the colonization of Lactobacillus in the gut and the competitive exclusion of pathogen [55]. Due to this character, oral administration of probiotic potential lactobacilli can reduce Salmonella enteritidis counts in the intestine, resulting in reduced infections. The results of co-culture experiments indicated the ability of lactobacilli to compete for limiting nutrients, while the growth of Salmonella enteritidis was completely inhibited after 48 h. A previous study also obtained similar results from a co-culture experiment just after 10 h [56]. Overall, ≥99.06 percent reduction and ≤4.32 Mean log₁₀ reduction in Salmonella counts from mice feces indicated the inability of Salmonella to adhere to the gut in the presence of Lactobacillus isolates. This may result from lactic acid production in the gut or competitive exclusion of Salmonella enteritidis. Another previous research showed the anti-microbial effect of Lactobacillus against Salmonella in a mouse model [42]. Recently, yeasts have also been subjected to co-aggregation with Salmonella, antagonism to salmonellosis, and in vivo survival, which presented high co-aggregation and satisfactorily survived the passage through the GIT of mice [57]. These Lactobacillus isolates can be proposed as potential probiotics to prevent Salmonella enteritidis infections in humans but only after confirmation using further experiments. For example, a recent study indicated the probiotic effect of Artemisia argyi broth fermented with Lactiplantibacillus plantarum against Salmonella Typhimurium after showing in vitro growth inhibition, reduction in adherence, and invasion of HT-29 cell line, decreased bacterial load not only in GIT but also in internal organs (spleen, liver, and mesenteric lymph nodes) of mice, decreased level of pro-inflammatory cytokines in serum, and improved epithelial barrier integrity [58]. In the current work, lactobacilli also showed significant in vitro activity against salmonellae of poultry origin; thus, these can also be evaluated in poultry birds like a previous study which indicated probiotic Bacillus licheniformis and Bacillus subtilis as tools for reducing Salmonella load in broiler GIT [59]. A significant reduction in mean Salmonella counts in feces has also previously been observed in probiotic-treated animals [60]. Various in vitro models suggested the blockage of Salmonella binding sites by lactobacilli. For example, L. fermentum was found to cause an effective reduction in the colonization of Salmonella Pullorum by 77%, whereas Ligilactobacillus animalis inhibited the adhesion of Salmonella Pullorum, Salmonella enteritidis, and Salmonella Gallinarum to host epithelial fragments by 90, 88, and 78%, respectively [61]. An antagonistic effect of Lactobacillus acidophilus LA10 against Salmonella enteritidis SE86 was also demonstrated in conventional mice upon the oral administration of 10⁸ CFU of each of SE86 and LA10 [32]. Recently, Liu et al. [42] also found the reduction in lethal effects of Salmonella by pretreatment of mixed Lactobacillus strains in a mouse model, especially L. plantarum exhibited remarkable performance with better preventive effects. During in vivo evaluations, the effect of lactobacilli on other parameters like body weight gain, feed conversion ratio, phytate solubilization, enhanced immune response, and the effects on gut morphology and intestinal absorption capacity have also been examined previously [14,26,36,52]. Further research may include studying the factors affecting the activity of lactobacilli, such as the latest research showing that xylooligosacharides promote the anti-Salmonella activity of L. plantarum [62].

The present study is the first step of a multistep project to develop indigenous probiotics against Salmonella enteritidis to prevent and mitigate Salmonella enteritidis-associated foodborne infections. A lot of probiotic products are imported in Pakistan but none of these use the beneficial strains of the local environment. It is imperative to explore the local strains for added benefits. Thus, current work provided characterized L. fermentum IL2, L. fermentum IL5, and L. gasseri IL12, resulting in the availability of local probiotic lactobacilli in Pakistan, which can be considered potential ingredients for the mass production and further development of probiotic products for humans and poultry, after further evaluations. Thus, it will help to cope with the insufficiency of local probiotic products in Pakistan, and consumers’ health will be improved. In this way, by using probiotics as an alternative, the emergence of antibiotic-resistant strains could also be controlled. In the next phase, we will explore other benefits of these strains, i.e., immunomodulatory effects and suitable foods as the delivery vehicle.

tel: +923337773240; fax: +92-9211449-178

Acknowledgments

We thank Merryn Fraser for revising the language of the manuscript.

Funding information: Authors state that the work was funded by the Higher Education Commission (HEC); project # 7069/Punjab/NRPU/R&D/HEC/2017.
Author contributions: M.N. and M.R. designed the study. A.M. and M.K. executed the research work. M.N. and A.M. performed data analysis. A.M., M.N., and M.T. played role in manuscript preparation. All authors approved the final version of the manuscript after revising critically.
Conflict of interest: Authors state no conflict of interest.
Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. The gene sequences obtained during the current study are available in the NCBI repository, https://www.ncbi.nlm.nih.gov/.

References

[1] Abou Elez RMM, Elsohaby I, El-Gazzar N, Tolba HMN, Abdelfatah EN, Abdellatif SS, et al. Antimicrobial resistance of Salmonella enteritidis and Salmonella typhimurium isolated from laying hens, table eggs, and humans with respect to antimicrobial activity of biosynthesized silver nanoparticles. Animals. 2021;11(12):3554.10.3390/ani11123554Suche in Google Scholar PubMed PubMed Central

[2] Saleh S, Van Puyvelde S, Staes A, Timmerman E, Barbé B, Jacobs J, et al. Salmonella Typhi, Paratyphi A, Enteritidis and Typhimurium core proteomes reveal differentially expressed proteins linked to the cell surface and pathogenicity. PLoS Negl Trop Dis. 2019;13(5):e0007416.10.1371/journal.pntd.0007416Suche in Google Scholar PubMed PubMed Central

[3] Chai SJ, White PL, Lathrop SL, Solghan SM, Medus C, McGlinchey BM, et al. Salmonella enterica serotype Enteritidis: increasing incidence of domestically acquired infections. Clin Infect Dis. 2012;54(Suppl_5):S488–S97.10.1093/cid/cis231Suche in Google Scholar PubMed

[4] Yaqoob E, Hussain I, Rahman S. Molecular characterization by using random amplified polymorphic DNA (RAPD) analysis of Salmonella enteritidis isolates recovered from avian and human sources. Pak Vet J. 2007;27(2):102.Suche in Google Scholar

[5] Gole VC, Roberts JR, Sexton M, May D, Kiermeier A, Chousalkar KK. Effect of egg washing and correlation between cuticle and egg penetration by various Salmonella strains. Int J Food Microbiol. 2014;182:18–25.10.1016/j.ijfoodmicro.2014.04.030Suche in Google Scholar PubMed

[6] Rabsch W, Hargis BM, Tsolis RM, Kingsley RA, Hinz K-H, Tschäpe H, et al. Competitive exclusion of Salmonella enteritidis by Salmonella Gallinarum in poultry. Emerg Infect Dis. 2000;6(5):443–8.10.3201/eid0605.000501Suche in Google Scholar PubMed PubMed Central

[7] Sandel DC, Wang C, Kessler S. Urinary tract infections and a multidrug-resistant Escherichia coli clonal group. N Engl J Med. 2002;346(7):535–6.10.1056/NEJM200202143460717Suche in Google Scholar PubMed

[8] Asif M, Rahman H, Qasim M, Khan TA, Ullah W, Jie Y. Molecular detection and antimicrobial resistance profile of zoonotic Salmonella enteritidis isolated from broiler chickens in Kohat, Pakistan. J Chin Med Assoc. 2017;80(5):303–6.10.1016/j.jcma.2016.11.007Suche in Google Scholar PubMed

[9] Yasmin S. Antibiotic susceptibility pattern of salmonellae isolated from poultry from different districts of Punjab, Pakistan. Pak Vet J. 2019;40:98–102.10.29261/pakvetj/2019.080Suche in Google Scholar

[10] Kamollerd C, Surachon P, Maunglai P, Siripornadulsil W, Sukon P. Assessment of probiotic potential of Lactobacillus reuteri MD5-2 isolated from ceca of Muscovy ducks. Korean J Vet Res. 2016;56(1):1–7.10.14405/kjvr.2016.56.1.1Suche in Google Scholar

[11] FAO/WHO. Guidelines for the evaluation of probiotics in food. Food and Agricultural Organization of the United Nations and World Health Organization Working Group Report; 2002.Suche in Google Scholar

[12] Sehrawat N, Yadav M, Singh M, Kumar V, Sharma VR, Sharma AK. Probiotics in microbiome ecological balance providing a therapeutic window against cancer. Sem Cancer Biol. 2021;70:24–36.10.1016/j.semcancer.2020.06.009Suche in Google Scholar PubMed

[13] Nagpal R, Kumar A, Kumar M, Behare PV, Jain S, Yadav H. Probiotics, their health benefits and applications for developing healthier foods: a review. FEMS Microbiol Lett. 2012;334(1):1–15.10.1111/j.1574-6968.2012.02593.xSuche in Google Scholar PubMed

[14] Khan I, Nawaz M, Anjum AA, Ahmad MD, Mehmood A, Rabbani M, et al. Effect of indigenous probiotics on gut morphology and intestinal absorption capacity in broiler chicken challenged with Salmonella enteritidis. Pak J Zool. 2020;52(5):1825.10.17582/journal.pjz/20190518090547Suche in Google Scholar

[15] Divyashree S, Anjali PG, Somashekaraiah R, Sreenivasa MY. Probiotic properties of Lactobacillus casei – MYSRD 108 and Lactobacillus plantarum-MYSRD 71 with potential antimicrobial activity against Salmonella Paratyphi. Biotechnol Rep. 2021;32:e00672.10.1016/j.btre.2021.e00672Suche in Google Scholar PubMed PubMed Central

[16] Pratiwi ST, Siagian A, Raja ENL, editors. The effect of pH on the activity of bacteriocin from Lactobacillus acidophilus on the growth of Salmonella Typhi. 12th Annual Scientific Meeting, Medical Faculty, Universitas Jenderal Achmad Yani, International Symposium on” Emergency Preparedness and Disaster Response during COVID 19 Pandemic”(ASMC 2021)). Atlantis Press; 2021.10.2991/ahsr.k.210723.001Suche in Google Scholar

[17] Adetoye A, Pinloche E, Adeniyi BA, Ayeni FA. Characterization and anti-salmonella activities of lactic acid bacteria isolated from cattle faeces. BMC Microbiol. 2018;18(1):96.10.1186/s12866-018-1248-ySuche in Google Scholar PubMed PubMed Central

[18] Plaza-Diaz J, Ruiz-Ojeda FJ, Gil-Campos M, Gil A. Mechanisms of action of probiotics. Adv Nutr. 2019;10(Suppl_1):S49–66.10.1093/advances/nmy063Suche in Google Scholar PubMed PubMed Central

[19] Pessoa WFB, Melgaço ACC, de Almeida ME, Ramos LP, Rezende RP, Romano CC. In vitro activity of lactobacilli with probiotic potential isolated from cocoa fermentation against Gardnerella vaginalis. Bio Med Res Int. 2017;2017, Article ID 3264194, 10 pages. 10.1155/2017/3264194.Suche in Google Scholar PubMed PubMed Central

[20] Shokryazdan P, Sieo CC, Kalavathy R, Liang JB, Alitheen NB, Faseleh Jahromi M, et al. Probiotic potential of Lactobacillus strains with antimicrobial activity against some human pathogenic strains. BioMed Res Int. 2014;2014:927268.10.1155/2014/927268Suche in Google Scholar PubMed PubMed Central

[21] Khan I, Nawaz M, Anjum AA, Ahmad M-u-D. Isolation and in vitro Characterization of Anti-Salmonella enteritidis Probiotic Potential of Indigenous Lactobacilli from Poultry. Pak Vet J. 2019;39:563–7.10.29261/pakvetj/2019.022Suche in Google Scholar

[22] Holt JG, Krieg NR, Sneath PH, Staley JT, Williams ST. Bergey’s manual of determinative bacteriology. 9th edn. Baltimore: A Waverly Company Williams and Wilkins; 1994.Suche in Google Scholar

[23] Saleem N, Nawaz M, Ghafoor A, Javeed A, Mustafa A, Yousuf MR, et al. Phenotypic and molecular analysis of antibiotic resistance in lactobacilli of poultry origin from Lahore, Pakistan. Pak Vet J. 2018;38:4.Suche in Google Scholar

[24] Nawaz M, Wang J, Zhou A, Ma C, Wu X, Xu J. Screening and characterization of new potentially probiotic lactobacilli from breast-fed healthy babies in Pakistan. Afr J Microbiol Res. 2011;5(12):1428–36.10.5897/AJMR10.737Suche in Google Scholar

[25] Chin SF, Romainor ANB, Pang SC, Lihan S. Antimicrobial starch-citrate hydrogel for potential applications as drug delivery carriers. J Drug Delivery Sci Technol. 2019;54:101239.10.1016/j.jddst.2019.101239Suche in Google Scholar

[26] Asghar S, Arif M, Nawaz M, Muhammad K, Ali MA, Ahmad MD, et al. Selection, characterisation and evaluation of potential probiotic Lactobacillus spp. isolated from poultry droppings. Benef Microbes. 2016;7(1):35–44.10.3920/BM2015.0020Suche in Google Scholar PubMed

[27] Riaz Rajoka MS, Mehwish HM, Siddiq M, Haobin Z, Zhu J, Yan L, et al. Identification, characterization, and probiotic potential of Lactobacillus rhamnosus isolated from human milk. LWT. 2017;84:271–80.10.1016/j.lwt.2017.05.055Suche in Google Scholar

[28] Rajoka MSR, Hayat HF, Sarwar S, Mehwish HM, Ahmad F, Hussain N, et al. Isolation and evaluation of probiotic potential of lactic acid bacteria isolated from poultry intestine. Microbiology. 2018;87(1):116–26.10.1134/S0026261718010150Suche in Google Scholar

[29] Saleem N. Phenotypic and molecular analysis of antibiotic resistance in lactobacilli of poultry origin from Lahore, Pakistan. Pak Vet J. 2018;38:4–413.10.29261/pakvetj/2018.084Suche in Google Scholar

[30] Additives EPo, Feed PoSuiA. Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance. EFSA J. 2012;10(6):2740.10.2903/j.efsa.2012.2740Suche in Google Scholar

[31] Angelov A. Aggregation and co-aggregation abilities of potentially probiotic amylolytic lactic acid bacteria strains. Germany: Proceedings of the ENGIHR Conference; 2014.Suche in Google Scholar

[32] Scapin D, Grando WF, Rossi EM, Perez KJ, Malheiros PdS, Tondo EC. Antagonistic activity of Lactobacillus acidophilus LA10 against Salmonella enterica serovar Enteritidis SE86 in mice. Brazilian J Microbiol. 2013;44(1):57–61.10.1590/S1517-83822013005000024Suche in Google Scholar PubMed PubMed Central

[33] Tellez G, Pixley C, Wolfenden RE, Layton SL, Hargis BM. Probiotics/direct fed microbials for Salmonella control in poultry. Food Res Int. 2012;45(2):628–33.10.1016/j.foodres.2011.03.047Suche in Google Scholar

[34] Gupta V, Garg R. Probiotics. Indian J Med Microbiol. 2009;27(3):202–9.10.1017/CBO9781139855952.235Suche in Google Scholar

[35] Melo TA, dos Santos TF, Pereira LR, Passos HM, Rezende RP, Romano CC. Functional profile evaluation of Lactobacillus fermentum TCUESC01: a new potential probiotic strain isolated during cocoa fermentation. BioMed Res Int. 2017;2017:5165916.10.1155/2017/5165916Suche in Google Scholar PubMed PubMed Central

[36] Arif A. Screening, characterization and physicochemical optimization of phosphorus solubilization activity of potential Probiotic Lactobacillus spp. Pak Vet J. 2018;38:316–20.10.29261/pakvetj/2018.061Suche in Google Scholar

[37] Ramos CL, Thorsen L, Schwan RF, Jespersen L. Strain-specific probiotics properties of Lactobacillus fermentum, Lactobacillus plantarum and Lactobacillus brevis isolate from Brazilian food products. Food Microbiol. 2013;36(1):22–9.10.1016/j.fm.2013.03.010Suche in Google Scholar PubMed

[38] Martín R, Jiménez E, Olivares M, Marín ML, Fernández L, Xaus J, et al. Lactobacillus salivarius CECT 5713, a potential probiotic strain isolated from infant feces and breast milk of a mother–child pair. Int J Food Microbiol. 2006;112(1):35–43.10.1016/j.ijfoodmicro.2006.06.011Suche in Google Scholar PubMed

[39] Dobreva L, Danova S, Georgieva V. Anti-Salmonella activity of lactobacilli from different habitats. Bulg J Vet Med. 2022;25(1).10.15547/bjvm.2395Suche in Google Scholar

[40] Verstraelen H, Verhelst R, Claeys G, De Backer E, Temmerman M, Vaneechoutte M. Longitudinal analysis of the vaginal microflora in pregnancy suggests that L. crispatus promotes the stability of the normal vaginal microflora and that L. gasseri and/or L. iners are more conducive to the occurrence of abnormal vaginal microflora. BMC Microbiol. 2009;9(1):116.10.1186/1471-2180-9-116Suche in Google Scholar

[41] Aazami N, Salehi Jouzani G, Khodaei Z, Meimandipour A, Safari M, Goudarzvand M. Characterization of some potentially probiotic Lactobacillus strains isolated from Iranian native chickens. J Gen Appl Microbiol. 2014;60(6):215–21.10.2323/jgam.60.215Suche in Google Scholar

[42] Liu J, Hu D, Chen Y, Huang H, Zhang H, Zhao J, et al. Strain-specific properties of Lactobacillus plantarum for prevention of Salmonella infection. Food Funct. 2018;9(7):3673–82.10.1039/C8FO00365CSuche in Google Scholar

[43] Kowalska JD, Nowak A, Śliżewska K, Stańczyk M, Łukasiak M, Dastych J. Anti-Salmonella potential of new Lactobacillus strains with the application in the poultry industry. Pol J Microbiol. 2020;69(1):5–18.10.33073/pjm-2020-001Suche in Google Scholar

[44] Zhou Q, Gu R, Li P, Lu Y, Chen L, Gu Q. Anti-Salmonella mode of action of natural l-phenyl lactic acid purified from Lactobacillus plantarum ZJ316. Appl Microbiol Biotechnol. 2020;104(12):5283–92.10.1007/s00253-020-10503-4Suche in Google Scholar

[45] Casey PG, Casey GD, Gardiner GE, Tangney M, Stanton C, Ross RP, et al. Isolation and characterization of anti-Salmonella lactic acid bacteria from the porcine gastrointestinal tract. Lett Appl Microbiol. 2004;39(5):431–8.10.1111/j.1472-765X.2004.01603.xSuche in Google Scholar

[46] Azeem N, Nawaz M, Anjum AA, Saeed S, Sana S, Mustafa A, et al. Activity and anti-aflatoxigenic effect of indigenously characterized probiotic lactobacilli against aspergillus flavus – a common poultry feed contaminant. Animals. 2019;9(4):166.10.3390/ani9040166Suche in Google Scholar

[47] Lorca GL, de Valdez GF. A low-pH-inducible, stationary-phase acid tolerance response in Lactobacillus acidophilus CRL 639. Curr Microbiol. 2001;42(1):21–5.10.1007/s002840010172Suche in Google Scholar

[48] G-Alegría E, López I, Ruiz JI, Sáenz J, Fernández E, Zarazaga M, et al. High tolerance of wild Lactobacillus plantarum and Oenococcus oeni strains to lyophilisation and stress environmental conditions of acid pH and ethanol. FEMS Microbiol Lett. 2004;230(1):53–61.10.1016/S0378-1097(03)00854-1Suche in Google Scholar

[49] Marteau P, Minekus M, Havenaar R, Huis In’t Veld JHJ. Survival of lactic acid bacteria in a dynamic model of the stomach and small intestine: validation and the effects of bile. J Dairy Sci. 1997;80(6):1031–7.10.3168/jds.S0022-0302(97)76027-2Suche in Google Scholar

[50] Armas F, Camperio C, Marianelli C. In vitro assessment of the probiotic potential of Lactococcus lactis LMG 7930 against ruminant mastitis-causing pathogens. PLoS One. 2017;12(1):e0169543.10.1371/journal.pone.0169543Suche in Google Scholar PubMed PubMed Central

[51] Li A, Wang Y, Suolang S, Mehmood K, Jiang X, Zhang L, et al. Isolation and identification of potential bacillus probiotics from free ranging yaks of Tibetan Plateau, China. Pak Vet J. 2019;39(3):377–82.10.29261/pakvetj/2019.032Suche in Google Scholar

[52] Khan M, Anjum AA, Nawaz M, Awan AR, Ali MA. Effect of newly characterized probiotic lactobacilli on weight gain, immunomodulation and gut microbiota of Campylobacter jejuni challenged broiler chicken. Pak Vet J. 2019;39(4):473–8.10.29261/pakvetj/2019.051Suche in Google Scholar

[53] Halder D, Mandal S. Antibacterial potentiality of commercially available probiotic Lactobacilli and curd Lactobacilli strains, alone and in combination, against human pathogenic bacteria. Transl Biomed. 2016;7:1–7.10.21767/2172-0479.100061Suche in Google Scholar

[54] Guo Z, Wang J, Yan L, Chen W, Liu X-M, Zhang H-P. In vitro comparison of probiotic properties of Lactobacillus casei Zhang, a potential new probiotic, with selected probiotic strains. LWT-Food Sci Technol. 2009;42(10):1640–6.10.1016/j.lwt.2009.05.025Suche in Google Scholar

[55] Chen X, Xu J, Shuai J, Chen J, Zhang Z, Fang W. The S-layer proteins of Lactobacillus crispatus strain ZJ001 is responsible for competitive exclusion against Escherichia coli O157:H7 and Salmonella typhimurium. Int J Food Microbiol. 2007;115(3):307–12.10.1016/j.ijfoodmicro.2006.11.007Suche in Google Scholar PubMed

[56] Abhisingha M, Dumnil J, Pitaksutheepong C. Selection of potential probiotic Lactobacillus with inhibitory activity against Salmonella and fecal coliform bacteria. Probiotics Antimicrob Proteins. 2018;10(2):218–7.10.1007/s12602-017-9304-8Suche in Google Scholar PubMed

[57] Andrade GC, Andrade RP, Oliveira DR, Quintanilha MF, Martins FS, Duarte WF. Kluyveromyces lactis and Torulaspora delbrueckii: Probiotic characterization, anti-Salmonella effect, and impact on cheese quality. LWT. 2021;151:112240.10.1016/j.lwt.2021.112240Suche in Google Scholar

[58] Tao X, Tian L, Zhan H, He Y, Zhong C, Wei H. In vitro and in vivo assessments of Artemisia argyi fermented with Lactobacillus plantarum WLPL01 as an alternative anti-Salmonella agent. Food Control. 2021;126:108079.10.1016/j.foodcont.2021.108079Suche in Google Scholar

[59] Shanmugasundaram R, Applegate T, Selvaraj R. Effect of Bacillus subtilis and Bacillus licheniformis probiotic supplementation on cecal Salmonella load in broilers challenged with salmonella. J Appl Poult Res. 2020;29(4):808–16.10.1016/j.japr.2020.07.003Suche in Google Scholar

[60] Casey PG, Gardiner GE, Casey G, Bradshaw B, Lawlor PG, Lynch PB, et al. A five-strain probiotic combination reduces pathogen shedding and alleviates disease signs in pigs challenged with Salmonella enterica Serovar Typhimurium. Appl Env Microbiol. 2007;73(6):1858–63.10.1128/AEM.01840-06Suche in Google Scholar PubMed PubMed Central

[61] Gusils C, González SN, Oliver G. Some probiotic properties of chicken lactobacilli. Can J Microbiol. 1999;45(12):981–7.10.1139/w99-102Suche in Google Scholar PubMed

[62] Liu J, Li X, Song F, Cui S, Lu W, Zhao J, et al. Dietary supplementation with low-dose xylooligosaccharide promotes the anti-Salmonella activity of probiotic Lactiplantibacillus plantarum ZS2058 in a murine model. Food Res Int. 2022;151:110858.10.1016/j.foodres.2021.110858Suche in Google Scholar PubMed

Received: 2021-12-11

Revised: 2022-05-09

Accepted: 2022-05-23

Published Online: 2022-08-17

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Artikel in diesem Heft

https://doi.org/10.1515/biol-2022-0100

Schlagwörter für diesen Artikel

lactobacilli; Salmonella enteritidis; mice; yogurt; human infants

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BY 4.0