Antimicrobial Effects of Different Synbiotic Compounds against Pathogenic Bacteria Isolated from Beef, Mutton, and Chicken

Introduction

Meat is an essential source of nutrients such as proteins, vitamins, lipids, and minerals. As it has high nutrient content, it is a suitable culture media for the growth of many species of microorganisms (e.g., bacteria and fungi) ( 1 ). Food-borne diseases remain a significant concern in developing countries caused by Escherichia coli, Salmonella, Shigella, Listeria monocytogenes, Staphylococcus aureus, Clostridium perfringens, and Campylobacter jejuni. In addition, it has been reported that antibiotic resistance is prevalent among food-borne pathogens ( 2 ). The application of naturally produced antimicrobials without any side effects on human health to stop the bacterial spread in food is an ideal way to solve the problems related to food contamination ( 3 ). Probiotics like Lactic acid bacteria are usually recognized as live microorganisms or their end products, which, when used in sufficient amounts, beneficially affect the host by modulating the intestinal immune system ( 4 ). They contain bacteria belonging to the genera Lactobacillus, Bifidobacteriums, Leuconostoc, Pediococcus, Propioni bacterium, and Bacillus. Streptococcus Salivarius subsp thermophiles is also a probiotic microorganism ( 5 ).The antimicrobial properties of probiotics are related to producing organic acids, such as lactic and acetic acid, hydrogen peroxide, antimicrobial enzymes, inhibitory compounds, and reducing the availability of the essential nutrients for pathogens in their living environment ( 6 ). Prebiotics are non-digestible organic food component fibers that positively affect the host by promoting intestinal health and providing substrates for confident intestinal bacteria like lactobacilli and bifidobacteria, which have positive effects on the host’s health to ferment. Coupling the application of probiotics with prebiotics is known as synbiotic which is believed to be more beneficial in terms of gut health and function ( 7 ). The present study aimed to investigate in vitro effects of synbiotic combinations of L. acidophilus, B. cereus, and S. salivarius with inulin, trehalose, and raffinose upon bacterial species isolated from beef, mutton, and chicken meet.

2. Materials and Methods

2.1 Sample Collection

Between December 2018 till April 2019, 60 retail raw meat samples were collected from Urmia city of Iran, including beef (n=20), mutton (n=20), and chicken (n=20). All samples were collected within 8 h post-slaughter and were kept below 4 °C during transportation to the lab ( 2 ).

2.2. Sample Preparation

Twenty-five grams of each sample was weighed and transferred to sterile flasks with 100 ml of phosphate buffer saline. After homogenizing by the meat grinder, the samples were stored for further analysis ( 2 ).

2.3. Isolation and Identification of Pathogens

Homogenized meat samples were transferred to nutrient agar, mannitol salt agar, MacConkey agar, Eosin- Methylene blue agar, selenite F broth, and listeria selective agar (Merck, Germany). Standards biochemical methods such as gram staining, oxidase, catalase, citrate, sulfide indole motility, triple sugar iron, DNase tests, and urea hydrolysis were used for bacterial identification ( 1 ). Then, pathogenic isolates were stored at -80 oC in 15% (w/w) glycerol ( 8 ). Escherichia coli ATTC 15224, Staphylococcus aureus ATTC 12600, Listeria monocytogenes ATTC 2374, and Salmonella enterica ATTC 14028 were used as indicator organisms.

2.4. Probiotics, prebiotics, and their synbiotic combinations

The L. acidophilus ATCC 4356 were inoculated in 5 ml Man Rogosa Sharpe (MRS) broth (Sigma-Aldrich, USA) containing the carbohydrate substrate of inulin, trehalose, or raffinose (1%w/w; Sigma-Aldrich, USA) and incubated for 24 h at 37 °C under microaerobic conditions with turbidity equivalent to 0.5 McFarland (1.5×10⁸ CFU/ml) ( 9 ). Bacillus cereus var. toyoi (Toyocerin^®, Asahi Vet. S.A., Barcelona, Spain, 200 mg/kg, containing 2×10⁵/g Bacillus cereus var. toyoi spore) was grown in 5 ml nutrient broth with 1% w/w of inulin, trehalose, or raffinose. Then, incubated for 24 h at 37 °C under aerobic conditions until reaching 1.5 × 10⁸ CFU/ ml ( 10 ). Finally, S. salivarius k12 (OralBiotic^® Lozenges, Canada) was inoculated into mitis-salivarius broth (Sigma-Aldrich, USA) supplemented with inulin, trehalose, or raffinose and incubated at 37 °C under anaerobic conditions to give approximately 1.5 × 10⁸ cfu/ml ( 11 ).

2.5. Preparing Supernatant of Probiotic Bacteria

Bacterial cells were removed by centrifugation at 2000 g for 10 min, and pH was adjusted for each culture supernatant to 7.0 using (1M NaOH) and sterilized by filtration using 0.22 µm Millipore filters (Sigma-Aldrich, USA).

2.6. Well Diffusion Agar

In this method, culture was done on Muller Hinton agar (MHA) plates with a sterile swab from a suspension of E. coli, Salmonella, S. aureus, and L. monocytogenes in TSB broth medium (0.5 McFarland). Wells of 10 mm in diameter were cut into agar plates with a sterile Pasteur pipette, and 100µl of supernatants of probiotic bacteria (L. acidophilus ATCC 4356 growing on MRS, Bacillus cereus var. toyoi growing on nutrient broth, and S. salivarius k12 growing on mitis-salivarius broth each supplemented with 1% w/w of prebiotics) was placed into each well. All the plates were incubated for 24 h at 37 °C, and antimicrobial activity was measured as growth-free inhibition zones around the wells (mm) ( 12 ).

2.7. Radial Streak Method

Nutrient agar, MRS agar, and MSA agar plates were cultured with 0.5 McFarland of B. cereus, L. acidophilus, and S. salivarius suspensions by covering a circular area in the center of the plate. After incubation of 48 h at 37 °C, the plates were seeded with E. coli, Salmonella, S. aureus, and L. monocytogenes (0.5 McFarland) by radial lines of inoculum from the border to the center of the petri dish. After 24 h of incubation at 37 °C, the growth inhibitory activity was measured by subtracting the circle diameter (cm) of the probiotic spreading zone from the inhibition zone diameter detected ( 8 ).

2.8. Assessment of Minimum Inhibitory Concentration (MIC)

The MIC of formulations was determined using the broth microdilution method in 96 well plates. After adding 100µl Muller Hinton broth to each well, 100µl of each probiotic culture supernatant was added to the first well, and then serial dilutions were made (100, 50, 25, 12.5, and 6.25µg/ml). Then, 100µl of each indicator strain (1.5×10⁸ CFU/ml) in Muller Hinton broth was added to each well. After overnight incubation at 37 °C, the plates were read using a microplate reader at the wavelength of 600 nm, and the highest dilution in which no growths was determined as the MIC ( 11 ).

2.9. Assessment of Minimum Bactericidal Concentration (MBC)

For MBC testing, aliquots (20µl) of broth from wells containing no growth were plated onto MHA plates and again incubated overnight at 37 °C. The MBCs were detected as the lowest concentration of the cell-free supernatant at which bacterial growth was not seen ( 11 ).

Statistical analysis was performed using the SPSS statistical program (version 25). Data were presented as mean ± SE, analysis of variance (ANOVA) was used to compare the data, and (P≤0.05) was considered statistically significant.

3. Results

Figure 1 presented data for the microbial analysis of different meat samples. All 60 meat samples (beef =20, mutton =20, and chicken =20) had microbial contaminations. Seventy percent of mutton samples were contaminated with S. aureus and L. monocytogenes. The contamination percentage with E. coli and Salmonella among mutton samples was 20% and 30%, respectively. When chicken samples were analyzed for microbial quality, they were contaminated with different kinds of bacteria, namely, L. monocytogenes (20%), S. aureus (50%), E. coli (20%), and Salmonella (40%). Among beef samples, L. monocytogenes was detected in 90% of samples, which was followed by S. aureus (50%), E. coli (50%), and Salmonella (10%).

Figure 1. Frequency of food-borne bacteria isolated from beef (n=20), mutton (n=20), and chicken (n=20)

3.1. Antimicrobial Activity by Agar Well Diffusion and Radial Streak Methods

The antimicrobial effect of the supernatant of tested probiotics against L. monocytogenes and S. aureus isolated from beef, mutton, and chicken samples using well diffusion assay is shown in tables 1, 2, and 3. The diameter of inhibition zones was different among tested probiotics. The widest diameter of inhibition zones was related to S. salivarius+raffinose, S. salivarius+inulin, and S. salivarius+trehalose, respectively. The results of the study on antimicrobial activity of indicated probiotics supernatants against L. monocytogenes and S. aureus using the radial streak method on different meat samples were the same as well diffusion method (tables 4, 5, and 6). In Salmonella and E. coli, well diffusion and radial streak methods results indicated that the highest inhibitory effects were for B. cereus+inulin, B. cereus+raffinose, and B. cereus+trehalose, respectively (tables 1-6).

3.2. Determination of MIC and MBC

Tables 7-9 show MICs and MBCs of different synbiotic formulations against bacterial strains isolated from different meat samples. In L. monocytogenes and S. aureus of sheep origin, the highest inhibitory efficacies were for S. salivarius+raffinose (12.5), S. salivarius+inulin (12.5), and S. salivarius+trehalose (25), respectively. In sheep originating Salmonella isolates, the highest inhibitory effects were for B. cereus+inulin (12.5), B. cereus+raffinose (12.5), and B. cereus+trehalose (25), and in E. coli of the same origin were for B. cereus+inulin (6.25), B. cereus+raffinose (12.5), and B. cereus+trehalose (25). Moreover, the highest inhibitory efficacies of studied formulations against L. monocytogenes and S. aureus with the chicken origin, were related to S. salivarius+raffinose (12.5), S. salivarius+inulin (12.5), and S. salivarius+trehalose (25) and in chicken originated Salmonella were for B. cereus+inulin (12.5), B. cereus+raffinose (25), and B. cereus+trehalose (25). Finally, in E. coli of chicken origin, were for B. cereus+inulin (6.25), B. cereus+raffinose (6.25), and B. cereus+trehalose (12.5). About L. monocytogenes and S. aureus with cow origin, the highest inhibitory effects were belonging to S. salivarius+raffinose (12.5), S. salivarius+Inulin (12.5), and S. salivarius+trehalose (25) and in cow originated Salmonella, were for B. cereus+inulin (6.25), B. cereus+raffinose (12.25), and B. cereus+trehalose (25). In E. coli of the same origin, the highest inhibitory properties were for B. cereus+inulin (12.5), B. cereus+raffinose (12.25) , and B. cereus+trehalose (25).

The MBC values obtained for extracts against L. monocytogenes and S. aureus of sheep origin resulted in the highest bactericidal efficacies for S. salivarius+raffinose (12.5), S. salivarius+inulin (12.5), and S. salivarius+trehalose (25). In sheep originated Salmonella were for B. cereus+inulin (25), B. cereus+raffinose (25), and B. cereus+trehalose (25) and in E. coli of the same origin were related to B. cereus+inulin (6.25), B. cereus+raffinose (25), and B. cereus+trehalose (25). Among chicken L. monocytogenes and S. aureus isolates, the highest bactericidal activity was observed in S. salivarius+raffinose (25), S. salivarius+inulin (25), and S. salivarius+trehalose (25), in chicken Salmonella strains, were for B. cereus+inulin (25), B. cereus+raffinose (25), and B. cereus+trehalose (25), and in chicken E. coli isolates were for B. cereus+Inulin (12.5), B. cereus+raffinose (12.5), and B. cereus+trehalose (25). Among L. monocytogenes and S. aureus of cow origin, the highest bactericidal properties were related to S. salivarius+raffinose (12.5), S. salivarius+inulin (25), and S. salivarius+trehalose (25), in cow originate Salmonella were for B. cereus+inulin (6.25), B. cereus+raffinose (25), and B. cereus+trehalose (25). Finally, in cow originated E. coli were for B. cereus+inulin (12.5), B. cereus+raffinose (12.5), and B. cereus+trehalose (25).

4. Discussion

Enteric infections are the fifth leading cause of death worldwide. About 70% of these infections are food-borne. Antibiotics are usually the choice drug for preventing and treating such infections, and contaminated raw meat is an essential source of food-borne diseases. Additionally, the extent of meat contamination is highly related to the standard of hygiene ( 1 , 2 ). Nowadays, the application of probiotics, prebiotics, and synbiotics has been increased as an alternative strategy in fighting against food-borne pathogens ( 13 ). The present study aimed to access the antibacterial properties of synbiotic compounds containing L. acidophilus, B. cereus, and S. salivarius as probiotics and inulin, trehalose, and raffinose as prebiotics against pathogenic bacteria (e.g., Salmonella, E. coli, L. monocytogenes, and S. aureus) isolated from beef, mutton, and chicken meat. IrohaI., UgboE. ( 2 ) investigated the bacterial contamination of raw meat, including beef, chicken, and chevron. Among isolated bacteria, the most frequent isolates were E. coli, K. pneumoniae, S. typhi, S. dysenteriae, P. aeruginosa, and S. aureus, respectively ( 2 ). In another study by Acharya, Poudel ( 1 ), pathogenic bacteria were isolated from meat processing units. The isolated pathogenic microorganisms were Vibrio spp, Salmonella spp, Shigella spp, Proteus spp, and Staphylococcus. As in our study, the highest prevalence was found in L. monocytogenes and S. aureus, respectively. A similar result was reported by Acharya, Poudel ( 1 ), ( 2 ). However, Salmonella and E. coli had the lowest occurrence in our studied samples, which is different from Acharya and Iroha,s findings. Lactobacillus is one of the critical groups of probiotic microorganisms. L. acidophilus, L. casei, L. reuteri, L. rhamnosus, and L. plantarum are commonly used probiotics in functional foods ( 14 ). Tharmaraj and Shah ( 3 ) studied the antimicrobial properties of some probiotic formulations against selected pathogenic and spoilage bacteria in cheese-based dips. The highest antimicrobial effects were for L. rhamnosus, L. acidophilus, L. casei, and L. paracasei, respectively. The most potent impact of all probiotics was against B. cereus, and the weakest was against E. coli. Our isolates showed nearly similar antagonistic activity against B. cereus and E. coli ( 3 ). In 2013, the antibacterial activity of L. acidophilus strains was characterized against E. coli and S. aureus. The metabolites of L. acidophilus showed antimicrobial properties against two tested pathogens ( 15 ). The results of all these studies are in accordance with our findings on mutton and chicken isolates. Although in our research, synbiotic compounds containing L. acidophilus exhibited antimicrobial activity against studied bacteria of mutton and chicken origins, they have no antibacterial effects on L. monocytogenes and S. aureus of cow origin. However, their inhibitory properties were weak compared to other formulations.

Bacillus spp have been used for food production and preservation for many years. Their ability to produce secretory proteins, enzymes, antimicrobial compounds, and carotenoids, tolerate a hostile environment of the gastrointestinal tract, and stability during food processing and storage make them suitable candidates for health elevating formulations ( 16 ). Jadamus, Vahjen ( 17 ) evaluated the growth behavior of B. cereus var. toyoi in the gastrointestinal tract of chickens and piglets. This bacteria germinated rapidly in both animal species, which is essential for its probiotic effects. Here we also used B. cereus var. toyoi against pathogenic bacteria isolated from different meat types. In addition, in the study of Altmeyer, Kroger ( 18 ), B. cereus var. toyoi, was investigated for its positive effect on pigs. The present assessment reveals that synbiotics formulations containing B. cereus showed the highest antagonistic activity against E. coli and Salmonella strains of beef, mutton, and chicken origin.

S. salivarius is an oral streptococcal species with no disease relationships in healthy humans ( 19 ). According to a study by Wescombe, Upton ( 20 ), although S. salivarius TOVE-R could decrease dental caries in animal models, it showed weak bacteriocin activity in vitro. In 2013, Burton, Drummond ( 19 ) investigated the effects of probiotic S. salivarius strain M18 on indices of dental health. The M18-treated children exhibited reduced cariogenic bacteria indicating the antibacterial properties of the M18 probiotic. In addition, S. salivarius K12 can prevent different upper respiratory tract infections, including streptococcal sore throat, otitis media, and halitosis ( 4 , 21 - 23 ). Furthermore, Fantinato, Camargo ( 24 ) studied S. salivarius strains for potential application as a probiotic for producing bacteriocin against S. pyogenes. The bacteriocin test showed that 133 strains could prevent S. pyogenes ( 24 ). In this study, synbiotic compounds containing S. salivarius were the most effective mixtures against L. monocytogenes and S. aureus with beef, mutton, and chicken origin. In conclusion, synbiotic formulations exhibit promise as alternatives for antibiotics as pressure to omit growth-promoting antibiotic use increases.

Authors' Contribution

The idea of the project: S. N. G. and P. H.

Interpretation of data: S. N. G., P. H. and S. M.

Preparing the manuscript: S. N. G. and P. H.

Critical revision of the manuscript for the development of the protocol and abstracting the data: S. N. G. and P. H.

Ethics

All procedures were approved by the ethics committees of the University of Tabriz, Tabriz, Iran.

Conflict of Interest

The authors declare that they have no conflict of interest.

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