Abstract

Infant meals are approved as a primary source of nutrition for babies, thus they should be nutrient-dense and come from a variety of sources. The current study seeks to assess the hygiene properties of infant foods in the cities of Taif and Makkah in order to improve their quality to satisfy industry standards. In Makkah and Taif, Saudi Arabia, 300 infant food samples were obtained at random from supermarkets and pharmacies. Microbiological examinations were done on the obtained samples. Also, molecular techniques were applied to confirm B. cereus pathogenicity. Probiotic bacteria were tested of their anti-microbial activity, and then L. rhamnosus as anti-B. cereus was added to infant diets and evaluated its activity as detoxifier using mice model. In almost all of the samples analyzed, total bacterial counts were found, with mean complete viable counts ranging from 1.586 to 3.543 log10 cfu/g. In addition, 196 Bacillus colonies were noted, with 27% of them including B. cereus. Despite being a rare species, B. cereus is the most deadly strain due to its pathogenicity and synthesis of severe toxins. As a result, the high effect of L. rhamnosus as anti-B. cereus led to its usage for detoxification and de-pathogenicity of the target pathogen, and their linked genes to enterotoxins were genetically detected. To validate that, L. rhamnosus was mixed with B. cereus and/or its emetic toxins and fed to mice as a food sample. The findings of this study demonstrated that L. rhamnosus reduced the toxicity of B. cereus and its enterotoxins, paving the way for this probiotic strain to be used as an edible and safe agent to protect infant's meals from hazardous microbial elements.

1. Introduction

As breast milk feeding is highly recommended in the 1^st six months of a baby's life, it is significant to support it by perfecting hygienic foods from the 2^nd six months due to their benefits and nutritious value ^{1, 2}. Infant foods serve as the source of the primary nutrients for children (6-24 months), which are obtained from different origins, hence exposure to the food-borne pathogen. Children and infants are classified as part of the high-risk group due to their limited food. Thus, contamination of their foods with any microbes can result in infection because their immune systems are not completely developed ³. So, the hygienic value of such foods is very important. Food borne diseases are global causes of health problems which include a wide range of infections related to bacterial, viral, fungal spores, parasitic, or food chemicals. In this regard, the WHO reported that B. cereus is the most significant common pathogen in pasteurized foods ^{4, 5, 6}. Zhang et al. ⁶ stated that B. cereus might have a different ecological niche, high levels of enterotoxin in baby foods, mainly in those appropriate for infants over six months old. Therefore, there is a need to draft suitable safety principles and hygienic control trials for infant foods to prevent B. cereus infection.

B. cereus is a human and animal psychrotrophic and facultative anaerobic spore-forming pathogen ⁷. It has been associated with outbreaks of food-related infection and the capability for producing toxins during growth temperatures ranging between 6-10°C ^{7, 8}. Also, it can grow at refrigeration temperatures and produce enterotoxins within seven days of incubation ⁹. Two types of diseases, emesis and diarrhea, are produced by B. cereus toxins which cause emetic syndrome by toxins performed in food, and diarrheal by one or more enterotoxins ⁷. The vegetative stage of B. cereus cells produces enterotoxins and causes diarrheal ¹⁰ due to the consumption of higher spores/cells numbers of more than 10⁵ cfu/g ¹¹.

Lactobacillus species have intense hostile activity against many pathogens (bacteria or fungi) ¹². Ahmad et al. ¹³ noticed that lactic acid bacteria bacteriocins are active against a broad spectrum of pathogens and stable under a wide range of pH and temperature values. Also, Rohmatussolihat et al. ¹⁴ reported that lactic acid bacteria could generate antimicrobial substances that help prevent food spoilage and the growth of bacteria in foods. For this purpose, this study was designed to evaluate the hygiene of infant foods in Taif and Makkah cities to improve these foods' quality. So, many infant food samples were collected randomly from pharmacies and supermarkets from Taif and Makkah cities. Also, isolation of Bacillus spp., identification and production of B. cereus toxins in infant foods was performed. Also, the evaluation of detoxification and de-pathogenicity activity of L. rhamnosus as anti-B. cereus was investigated.

2. Materials and Methods

A total of 300 babies milk based powder and ready to use were sampled on behalf of 75 samples with honey and milk based, infant food with wheat and milk-based, infant food with rice and milk-based, ready to use infant food with vegetables, ready to use infant food with fruit, (15 samples of each), 25 samples of infant food with fruit and milk-based, infant milk powder (35 samples) and infant food with vegetables and milk based (15 samples). All samples were obtained randomly from diverse supermarkets and pharmacies in Taif and Makkah cities, Saudi Arabia. During sampling, sterile techniques for packaging and transferring were applied, and the samples were delivered to the laboratory for analysis, according to American Public Health Association ¹⁵. Each sample was analyzed for total bacteria, total Bacillus cereus counts, and total psychrotrophic spore formers, as reported by ¹⁵.

Four probiotic bacterial strains (LAB) were applied in the current work. Lactobacillus rhamnosus B-445, Lactobacillus dulbrueckii subsp. bulgaricus Lb-12 DRI-VAC strains were delivered from the Northern Regional Research Laboratory (NRRL), Illinois, USA. Leuconostoc mesenteroides B-118 was delivered from Chr. Hansen's Lab, Denmark. Lactobacillus cremoris was obtained from the Department of Dairy, National Research Centre, Egypt. They were proved to be probiotics ¹⁶ by the possession of these characteristics; acid tolerance, susceptibility to an antibiotic, survivability in variable concentrations of bile salt, and antimicrobial actions against definite pathogenic microbes as designated ¹⁷. Prior to strains application, each strain was sub-cultured two times in MRS broth medium and then preserved in % (w/v) sterile reconstructed skim milk powder at 37°C for 24 h, except Leuconostoc mesenteroides which were incubated at 30°C for 18 h.

For all collected infant food samples, about 25 g of each sample in 225 ml of sterile peptone water was done and then mixed well using a sterile homogenizer for 2 min. The serial dilution technique was applied to prepare an appropriate number of bacterial counts. Total bacterial counts were counted using tryptic soya agar and plate count agar media ¹⁸. In case of a psychrotrophic spore-forming bacterial count, the plate count agar medium was applied with the incubation temperature at 7°C for 10 days ¹⁹.

Three to five isolates from typical colonies of the total psychrotrophic spore-forming plates from each plate were picked randomly. Each isolated colony was purified and characterized as psychrotrophic Bacillus spp., and then inoculated into 10 ml of tryptic soya broth. Finally, the inoculated cultures were incubated at 37°C for 24 h and then subjected to several biochemical examinations and API 50 CHB (Biomerieux, France) for identification.

Extraction of bacterial DNA of the 22 B. cereus isolates was obtained by lysozyme (20 mg/ml) and proteinase K (1 mg/ml) method, which was detailed previously ²⁰. Three virulence genes, hbla, nhec and cytk were amplified as the enterotoxin genes using extracted DNA and confidential primers as outlined in Table 1. PCR amplification was carried out, which started with denaturation at 95°C for 5 min, followed by 30 cycles of 95°C for 1 min, 58°C for 1 min, 72°C for 1 min, and a final elongation step at 72°C for 10 min, with a final hold at 4°C ²¹. PCR products were analyzed in 1% (w/v) TAE agarose gel. All PCR experiments for each strain were carried out twice ²².

One isolate of B. cereus was had the three tested virulence genes. Thus, it was identified using molecular biology techniques. Amplification reaction using the PCR of 16 S rRNA genes was performed using extracted DNA and the two primers ^{23, 24, 25}. The PCR amplification started with an initial denaturation at 95 °C for 5 min, followed by 35 cycles of 95°C for 30 s, 55 °C for 30 s, and 72 °C for 45 s for a complete extension. PCR product was purified by QIAquick Gel Extraction Kit (QIAGEN, USA) and sequenced. Identification was achieved using the BLAST program (National Centre for Biotechnology Information). Sequences alignment was done using Jukes Cantor Model. The phylogenetic reconstruction was carried out using the neighbor-joining (NJ) algorithm; with bootstrap values and submitted to Gene Bank.

The antimicrobial activity of the 4 probiotic strains against B. cereus growth and its production of toxins was evaluated. The cell-free supernatant containing metabolites of probiotic strains were obtained through centrifugation of the cultures (overnight growth) of the tested probiotic bacteria (37°C in MRS broth) at 4000 rpm for 15 min at 4°C. The supernatant was passed through 0.22 μm Millex-GV filter (Millipore) and pH was adjusted to 6 for excluding the organic acid effects. The activity of the produced metabolites was tried against the B. cereus strain by the use of an agar well diffusion test as detailed by Elshahawyet al. ²⁶. Testing of metabolite trials was done in triplicate and the plates were incubated overnight at 37°C. Inhibition of B. cereus growth was evaluated by measurement of the diameter (mm) of the clear inhibition zone formed around wells ^{27, 28}.

The antagonism activity of L. rhamnosus B-445 strain against B. cereus strain was evaluated into broth culture media system. Briefly, B. cereus strain was inoculated into tryptic soya broth medium, and the tested probiotic strain was cultured in MRS broth and incubated for 24 h at 37°C. The cultures dilution was separately done in saline solutions to 10⁵ and 10⁷ cfu/ml for B. cereus and LAB, respectively, and then mixed into 100 ml tryptic soya broth. The counts of B. cereus and L. rhamnosus in the co-cultures were measured after incubation at different time points (0, 6, 24, 48 h) on the separated medium. B. cereus was counted by spreading 0.1 ml of the appropriate dilution onto PEMBA medium ²⁹. L. rhamnosus were counted by De Man, Rogosa, and Sharpe agar MRS, and the plates were incubated anaerobically at 30°C for 3 days.

Evaluation of pathogenicity and production of toxins by B. cereus isolate was carried out using the mice toxicity technique. Also, the effect of L. rhamnosus on this pathogenicity was evaluated. B. cereus was cultured in 100 ml of 10% skim milk medium and incubated at 37°C for 18 h under shaking conditions (150 rpm), then centrifuged (4500 rpm for 30 min). The supernatant was autoclaved (121°C for 15 min) to destroy heat-labile toxins like diarrheal enterotoxin (the emetic enterotoxin is heat stable) and kill B. cereus vegetative cells and spores. Partial purification of the emetic toxin available in the culture supernatant was done using ammonium sulphate precipitation protocol by adding an equal volume of 100% (NH₄)₂SO₄ solution to give a final concentration of 50% saturation. The mixture was kept at 4°C for 1 h, then centrifuged undercooling (5000 rpm for 30 min), and the supernatant was discarded. The pellets were re-suspended in absolute methanol (HPLC grade) and shacked at 30°C until homogenized. The final mixture was centrifuged to remove non-toxic materials (methanol-insoluble), because the enterotoxin is soluble in absolute methanol ³⁰.

For the preparation of B. cereus cells, it was grown in a brain heart infusion broth medium at 37°C for 24 h. After that, the culture was centrifugated at 5000 rpm for 10 min. The pellet was re-suspended in a suitable quantity of phosphate buffer to attain a bacterial suspension equivalent to 0.5 McFarland standard ³¹. In another case, L. rhamnosus strain was cultured in 1% (w/v) reconstituted sterile skim milk powder (RSM) at 37°C for 24 h. After bacterial cultures and B. cereus crude toxins were prepared, an equal volume of L. rhamnosus culture was mixed with B. cereus culture or its emetic toxin and incubated at 37°C for 6 h.

To evaluate the pathogenicity of B. cereus, and study the role of L. rhamnosus in attenuating the toxicity of B. cereus, 30 adult males mice (Mus musculus domesticus) with an average weight of 100 g were obtained from the animal house at Taif University. The animals were placed in polycarbonate boxes in a room atmosphere in the laboratory and acclimatized for one week before starting the experiments. The mice were divided into six groups containing five mice each for the control and treatment groups. The animals were fed a basal diet containing 50% corn starch, 20% casein, 10% sugar cane, 10% corn oil, 5% cellulose, 4% salt mixture, and 1% vitamin mixture ³². Half ml of each treatment was given orally to the mice by intragastric gavages for three days. The animals were caged in groups and fed with normal food and water in an animal house under the supervision of officials. The trials (6 groups) were as follows; the control group was fed with phosphate-buffer saline and their regular food for each animal, 2^nd group was fed with B. cereus, the emetic toxin, 3^rd group was fed with B. cereus culture, 4^th group was fed with a mixture of B. cereus the emetic toxin and L. rhamnosus culture, 5^th group was fed with a mixture of B. cereus and L. rhamnosus cultures, and the 6^th group was fed with L. rhamnosus culture. The result was recorded after follow-up clinical signs of mice after three days, and the animals were thoroughly observed for any toxic signs through this period. Convulsions, motor activity, tremors, sedation, aggressiveness, relaxation of muscle, analgesia, hypnosis, ptosis, paralysis, lacrimation, skin color, and diarrhea were noted during the experiment. The observed death was also recorded in each group. At the end of the experiment, harvesting of the liver, heart, spleen, and kidney was done, and their weight per total body was measured. The organs were observed for major changes in an appearance before considering.

The experiments were performed in triplicate and the results are expressed as the mean ± standard deviation (SD). Statistical significance was evaluated using analysis of variance (ANOVA, SAS software) followed by the determination of the least significant difference (LSD) at 0.05.

All experimental procedures were performed according to the principles of the Ethics Committee of Taif University (Approval No. HAO-02-T-105) (Taif, Saudi Arabia).

3. Results

Three hundred infant food samples were collected randomly from pharmacies and supermarkets in Makkah and Taif cities. The collected samples were subjected to microbiological analyses. The results exposed that the total bacterial counts (TBC) were detected in almost all examined samples, with mean total viable counts ranging from 1.586 to 3.543 log10 cfu/g. The results revealed that the total psychrotrophic spore bacteria were noticed in 40 and 60% of the whole samples of milk based infant food with vegetables and fruits, respectively.

The samples were exposed to examine their content of Bacillus spp. by isolation of different morphological aerobic spore-forming colonies, and then characterized (morphologically, biochemically and API CHB50 tests) using acceptable protocols of Bacillus spp. Identification. The occurrence of diverse isolated Bacillus was illustrated in Figure 1. The results exposed that B. subtilis was the most habitually occurred compared with other Bacillus spp. with an incidence of 36%, followed by B. cereus (27%), B. circulans (20%), B. licheniformis (12%), and B. coagulans (5%). Vegetables and fruit milk based infant foods were had the highest numbers of Bacillus spp. count. From the obtained Bacillus isolates, B. subtilis and B. cereus, due to their hydrolytic activities, are essential for food hygiene.

It is plain the whole B. cereus group calculated on PEMBA medium was noticed in common of tested samples with the highest percentages with milk based infant food with fruit and vegetable and reached to 62.2 and 26.6%, respectively (Figure 2). On conflicting, B. cereus count was not noticed in the ready to use infant food with fruit and vegetables. Generally, 40 out of 300 samples (26.67%) of the examined infant foods are positive for B. cereus counts.

Due to Bacillus cereus isolate No. 8 pathogenicity, it was identified using molecular biology techniques to confirm the biochemical and API CHB50 tests. B. cereus isolate 8 was selected as a potent strain based on its pathogenicity and production of 3 virulent harmful toxins relating genes. The total genomic DNA of this isolate was extracted and purified using lysozyme/isopropyl technique, and the 16S rRNA gene was amplified. The obtained sequence was compared with available sequences in the GenBank database, and the similarity percentage of this isolate accounted for 98% with Bacillus cereus strains. Also, the phylogenetic tree was performed, and it showed that this strain was very close to the type strains of B. cereus deposited in the culture collection center of the National Centre for Biotechnology Information (Figure 3).

Three virulent genes, hemolytic BL complex hbla gene, non-hemolytic enterotoxin complex nhec gene and cytotoxic cytk gene were detected by PCR protocol using related primers. Twenty-five B. cereus isolates were checked for their having the three virulent harmful genes, and the images resulting from PCR amplification of these genes were illustrated in Figure 4. From the data represented in Figure 4, 2 isolates (8 and 9) appeared to have the three tested genes. The results revealed that B. cereus isolate 8 was had hbla gene, which isolated from infant food with fruit milk. The PCR product of this gene amplification was 1200 pb amplicon size (Figure 4A); it was detected in five out of 25 isolates. The same tendency of hbla gene distribution with diverse ratio was achieved from B. cereus isolates obtained from infant food with rice milk based, wheat milk based and honey milk based with percentages of 19.35, 12, and 5.55%, respectively. In the case of nhec virulence gene, it looks to be widely spread among B. cereus isolates like infant food with fruit milk based, infant food with vegetables milk based, infant food with honey milk based and infant food with wheat milk based, as well as it wasn't detected in infant food with rice milk based (Figure 4C). Also, it was clear that from Figure (4B) the cytk gene was observed in higher percent compared to nhec and hbla genes, it was detected by 92% of B. cereus isolates in this research. Also, it was detected in isolates obtained from milk based infant food with fruit, honey, vegetables, wheat and rice.

Anti-Bacillus cereus activity of probiotic strains, L. rhamnosus B-445, L. dulbrueckii subsp. bulgaricus Lb-12 DRI-VAC, Leuconostoc mesenteroides B-118 and L. cremoris strain against B. cereus growth was investigated in this experiment. The cell-free supernatant containing bioactive metabolites of probiotic strains was obtained and employed against B. cereus strain using an agar well diffusion assay. The data noticed from pictures illustrated in Figure 5 stated that the strain of L. rhamnosus B-445 was produced highly antibacterial activity against B. cereus strain since recorded 21 mm of inhibition zone diameter, followed by L. dulbrueckii and L. cremoris, but Leuconostoc mesenteroides appeared no activity.

The antagonism activity of L. rhamnosus against B. cereus was assessed in the broth culture media system, and the results are illustrated in Figure 6. From that, we can clearly show the antagonism between the two tested strains. The growth of B. cereus strain was inhibited under mixed culture with L. rhamnosus strain started from 6 h of incubation. L. rhamnosus exposed a high inhibitory activity against B. cereus strain resulting in decreasing the total counts from 5.3 to 3.3 Log10 cfu/ml after 24 h with reduction percent of 37.7% and reached to 2.0 Log10 cfu/ml with reduction percent of 62.2% after 48 h of incubation.

The probiotic strain L. rhamnosus was mixed with B. cereus and/or its emetic toxins and added as diets samples to mice. After three days of the experiment, some clinical signs were observed in groups two and three, which were treated with (B. cereus cells and its emetic toxin only) like dying and decreasing weight in comparison with the other groups. The control group didn't show any abnormal observations with weights (ranging between 100 and 103 g) after three days. While mice fed with bacterial cells of B. cereus showed loss of their body weight and slow motion, their weights ranged from 72 to 75 g, and two of five mice died. Also, four out of five mice in the group fed with the emetic toxins have died, and the rest mice had a clear decrease of their weight to 60 g and slow motion. However, the group fed with a mixture of B. cereus cells and L. rhamnosus exhibited normal appearance and movement with consequences reaching 93-94 g. Moreover, the mice group provided with a mix of crude toxins and L. rhamnosus culture displayed normal appearance, motion, and weight, with weights ranging between 98 and 101 g. For a group fed with a diet containing L. rhamnosus culture, it appeared to be normal observations, and their weight reached 100-105 g. The data in Figure 7 illustrated swollen intestinal tract due to the accumulation of fluid in the lumen of the intestine and inflammation of the intestines and stomach, especially when mice's fed with the bacterial cells of B. cereus. In contrast, no significant changes showed in the stomach and intestines of mice treated with crude emetic enterotoxin. Ulcers and dark red were seen in the liver of groups fed with bacterial cells and emetic enterotoxin of B. cereus, which reflects severe vascular effects of the toxin, especially in mice fed with an emetic toxin (Figure 7). In contrast, group fed with mixture of bacterial cells of B. cereus and L. rhamnosus showed normal stomach, intestine and liver in comparison with the control group. Also, the group fed with mixture of B. cereus emetic enterotoxin and L. rhamnosus B-445 showed normal stomach, intestine and liver. Moreover, the group fed with diet involves L. rhamnosus B-445 appeared normal observations in all body contents.

4. Discussion

Infant foods are certified as a primary nutrition source for babies from the 2^nd six months to 2 years old. Thus, it should represent a rich source of nutrients from several origins. However, any germs in babies' food can cause severe infection due to their immune systems not being developed completely ³. So, the hygienic quality of their foods is essential. This study evaluated the hygiene of infant foods at Taif and Makkah cities and tried to improve these foods' quality. To achieve this objective, three hundred infant food samples were collected randomly from pharmacies and supermarkets in Makkah and Taif cities, Saudi Arabia. The collected samples were subjected to microbiological analyses. The results exposed that the total bacterial counts (TBC) were detected in almost samples, with mean complete viable counts ranging from 1.586 to 3.543 log10 cfu/g. A similar observation was reported by other researchers ^{33, 34}. TBC may be in the range of standard regulations. Thus, it is not essential like spore-forming and pathogenic bacteria. Therefore, total psychrotrophic spore-forming counts were also detected. The high TBC indicated poor hygienic practices such as handling, packing during processing, and low-quality milk or ingredients ³⁵. The authority of psychrotrophic bacteria in the TBC is even more marked when producing milk under poor hygienic conditions and/or increasing somatic cells numbers. The current parameter was tested as a device for infant foods quality. The results revealed that the total psychrotrophic spore bacteria were noticed in 40 and 60% of the total samples of milk based infant food with vegetables and fruits. At this point, Ahmed et al. ³⁶ cited that the count of psychrotrophic spore bacteria was 2.9 х102 cfu/g in baby's milk foods. Also, Sadek et al. ³³ reported that 40% of based infant food with vegetables was positive in psychrotrophic spore bacteria.

The samples were exposed to examine their content of Bacillus spp. by isolation of different morphological aerobic spore-forming colonies and then characterized (morphologically, biochemically and API CHB50 tests) using acceptable protocols of Bacillus spp. Identification as reported by ³⁷. The results exposed that B. subtilis was the most habitually occurred compared with other Bacillus spp. Vegetables and fruit milk based infant foods had the highest numbers of Bacillus spp. count. From the obtained isolates, B. subtilis and B. cereus, due to their hydrolytic activities, are essential for food hygiene for food constituents and their ability of them to produce toxins and/or grow at refrigerator temperature. These results are in agreement with the observation of ³⁸, who stated that the public bacillus species noticed in the marketable baby formulas were B. subtilis (28%) followed by B. lichenifomis (20%) and B. cereus (14%). Although B. cereus wasn't the most common species, it is the most harmful strain due to its pathogenicity and production of some dangerous toxins. Therefore, it is necessary to study the distribution of B. cereus through the obtained samples and its pathogenicity as well as detection of its relating toxins. B. cereus spores are globally distributed. Consequently, it is necessary to examine the presence of B. cereus in infant food and its pathogenicity. According to ³⁹ regulations, the acceptable limit for B. cereus in infant food must be less than and not exceed 100 cfu/g. It is plain the whole B. cereus group calculated on PEMBA medium was noticed in common of tested samples with the highest percentages with milk based infant food with fruit and vegetable. On contradictory, B. cereus count was not noticed in the ready to use infant food with fruit and vegetables. The study published by Ioana and Alexandra ⁴⁰ described that B. cereus was noticed in 6 out of 30 powder milk samples for newborns. Furthermore, Kim et al. ⁴¹ presented that B. cereus was detected in infant foods, including 40.0% of biscuits, 23.0% of cereal-based infant foods and 7.4% of liquid infant foods. The high differences in percentage and range of B. cereus may be due to the types of samples and tests and diverse of formulas. To solve this problem, new nontoxic and high stable bioactive constituents were developed in previous studies for controlling the pathogens in different food forms ⁴² and can apply to this target to decrease the number of B. cereus count. In addition, food-grade natural materials may be applied also as the best chance to control the pathogens ⁴³.

The pathogenicity of B. cereus was evaluated based on detecting toxicogenic relating genes using molecular biology techniques. Hemolytic BL complex hbla gene, non-hemolytic enterotoxin complex nhec gene, and cytotoxic cytk gene were detected by PCR protocol using related primers. Two isolates (8 and 9) appeared to have the three tested genes from the obtained data. Toxigenic B. cereus strains have been reported in diverse starchy foods like vegetables, sauces, puddings, cereals, dairy products, and infant cereal formulations fried and cooked rice ^{44, 45}. Therefore, it is essential to evaluate the food safety of different baby food products widely manufactured, distributed, and sold in Saudi Arabia and other countries. PCR technique was recently applied to detect enterotoxins-related genes in B. cereus ⁴⁶. The results revealed that B. cereus isolate eight was had hbla gene which isolated from infant food with fruit milk. The same tendency of hbla gene distribution with diverse ratio were achieved from B. cereus isolates obtained from infant food with rice milk based, wheat milk based and honey milk based. Furthermore, Dhuha and Habeeb ⁴⁷ stated that the gene of hbla was noticed in B. cereus strains isolated from milk, dairy products, and child food. These variations are recognized based on several factors like season, collection condition, and sample type. For nhec virulence gene, it looks to be widely spread among B. cereus isolates like infant food with fruit milk based, infant food with vegetables milk based, infant food with honey milk based and infant food with wheat milk based, as well as it wasn't detected in infant food with rice milk based. These results were similar that those published by Dhuha and Habeeb ⁴⁷, who stated that nhec gene was found in 80% of B. cereus isolated from infant food. Also, it was clear that the cytk gene was observed in a higher percentage compared to nhec and hbla genes. Also, it was detected in isolates obtained from milk based infant food with fruit, honey, vegetables, wheat, and rice. The cytotoxic gene, cytk of B. cereus (as clinical isolate), was the only cause of severe food poisoning outbreak that killed people and had necrotic and hemolytic action ^{48, 49}. Due to Bacillus cereus isolate No. 8, pathogenicity was identified using molecular biology techniques to confirm the biochemical and API CHB50 tests. One B. cereus isolates eight was selected as a potent strain based on its pathogenicity and production of 3 virulent harmful toxins relating genes. This isolate's total genomic DNA was extracted and purified using lysozyme/isopropyl technique, and the 16S rRNA gene was amplified. The obtained sequence was compared with available sequences in the GenBank database, and the similarity percentage of this isolate accounted for 98% with Bacillus cereus strains. Also, the phylogenetic tree was performed, and it showed that this strain is very close to the type strains of B. cereus deposited in the culture collection center of the National Centre for Biotechnology Information.

Anti-Bacillus cereus activity of probiotic strains, L. rhamnosus B-445, L. dulbrueckii subsp. bulgaricus Lb-12 DRI-VAC, Leuconostoc mesenteroides B-118 and L. cremoris strain against B. cereus growth were investigated in this experiment. The cell-free supernatant containing bioactive metabolites of probiotic strains was obtained and employed against B. cereus strain using an agar well diffusion assay. The L. rhamnosus B-445 produced highly antibacterial activity against B. cereus strain at a 21 mm inhibition zone diameter, followed by L. dulbrueckii and L. cremoris and Leuconostoc mesenteroides. The recorded variations in anti-Bacillus cereus activity of the examined probiotic strains may be due to their ability to produce various bioactive substances like enzymes, phenolic compounds, organic and/or amino acids, polysaccharides, bacteriocins and/or biochemical derivatives. In this target, many researchers published many papers about producing bioactive materials from microbial cells ^{27, 50, 51, 52}. Also, several reports showed that LAB strains had antimicrobial activities against pathogenic bacteria like Bacillus strains such as L. rhamnosus B-445 and L. dulbrueckii subsp. Bulgaricus ⁵³. The high activity of L. rhamnosus against Bacillus cereus led to the use of this strain as a safe agent for detoxification and de-pathogenicity of the target pathogen in this study. The antagonism activity of L. rhamnosus against B. cereus was assessed in the broth culture media system. From that, we can clearly show the antagonism between the two tested strains. The growth of B. cereus strain was inhibited under mixed culture with L. rhamnosus strain started from 6 h of incubation. L. rhamnosus exposed a high inhibitory activity against B. cereus strain, decreasing the total counts from 5.3 to 3.3 Log10 cfu/ml after 24 h with a reduction percent of 37.7% and reached 2.0 Log10 cfu/ml with a reduction percent of 62.2% after 48 h of incubation. This antagonism may be due to the antibacterial metabolites as bacteriocin, and similar inhibitory substances that are produced by L. rhamnosus and showed potential applications in food bio-preservation ^{54, 55, 56} reported that the L. bulgaricus Z55 produce bacteriocin that inhibited many food-borne pathogens.

Since infant foods are one of the primary young child and baby diets, it is essential that the infant foods have high levels of hygiene quality ⁵⁷. B. cereus strain is harmful, and pathogen bacteria distribute in various environments. It is dangerous to find clinical or pathogenic B. cereus in baby-related foods. Its spore-forming (it will be resistant to thermal treatments) and its toxins are stable toward hygienic controls ⁸. It is an excellent chance to find an environmental-safe, non-toxic and safe agent for detoxification and de-pathogenicity of harmful B. cereus. To investigate this objective, the probiotic strain L. rhamnosus was mixed with B. cereus and/or its emetic toxins and added as diets samples to mice. After three days of experiment, some clinical signs were observed in groups number 2 and 3, which were treated with (B. cereus cells and its emetic toxin) like dying and decreasing in weight compared to the other groups. The control group didn't show any abnormal observations after three days. While mice fed with bacterial cells of B. cereus showed loss of their body weight and slow motion, their weights ranged from 72 to 75 g. Also, four out of five mice in the group fed with the emetic toxins have died, and the rest of the mice had an apparent decrease of their weight to 60 g and slow motion. However, the group fed with a mixture of B. cereus cells and L. rhamnosus exhibited a normal appearance and movement with weights reached to 93-94 g. Moreover, the mice group fed with a mixture of crude toxins and L. rhamnosus culture displayed normal appearance, motion, and weight, ranging between 98 and 101 g. The group fed with a diet containing L. rhamnosus culture appeared to have normal observations, and their weight reached 100-105 g. The swollen intestinal tract due to accumulation of fluid in the lumen of the intestine and inflammation of the intestines and stomach are specially recorded when mice's fed with the bacterial cells of B. cereus. In contrast, no significant changes showed in the stomach and intestines of mice treated with crude emetic enterotoxin. Ulcer and dark red color showed in the liver of the groups were fed with bacterial cells and emetic enterotoxin of B. cereus, which reflects severe vascular effects of the toxin, especially in mice fed with the emetic toxin. In contrast, the group fed with a mixture of bacterial cells of B. cereus and L. rhamnosus showed normal stomach, intestine, and liver compared to the control group. Also, the group fed with a mixture of B. cereus emetic enterotoxin and L. rhamnosus B-445 showed normal stomach, intestine, and liver. Moreover, the group fed with a diet involving L. rhamnosus B-445 appeared to have normal observations in all body contents. Thus, applying L. rhamnosus B-445 as an environmentally safe anti-B. cereus agent in infant foods is essential.

5. Conclusions

This study concluded that psychrotrophic B. cereus in infant foods could be a potential hazard for the babies' health due to its ability to grow and produce enterotoxins in infant foods products. Also, the study concluded that incorporating L. rhamnosus with infant foods led to attenuating the toxic effects of the B. cereus strain.

Acknowledgment and Funding

This work was supported by Taif University Researchers Supporting Program (project number: TURSP-2020/151), Taif University, Saudi Arabia.

References