الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
FBDs are a global public health issue that not only has major impacts on human health and healthcare systems, but also affects international trade. It is estimated that 600 million people, almost one in 10 people in the world, fall ill annually from consuming contaminated food, with diarrheal diseases being the most common form of these illnesses. FBDs were defined as any disease resulting from food contamination by pathogenic microorganisms during the food supply chain, pre, and post-harvest procedures; production, distribution and even before consumption.
Salmonella spp., L.monocytogenes and E. coli O157:H7 are a group of the major foodborne pathogens. Collectively, these agents account for an estimated public health burden of 1.1 million annual cases of illness, nearly 23,000 hospitalizations and 653 deaths. Not only do these three pathogens have detrimental public health impacts in terms of morbidity, mortality and economics, but they also represent significant economic burden for food producers.
Most bacteria undergo a transition from planktonic mode to biofilm mode depending on bacterial community and environmental conditions. Bacterial biofilms are a natural complex of microorganisms embedded in a protective slimy matrix composed of various types of polysaccharides, proteins, nucleic acids and lipids. Unlike the planktonic cells, biofilm cells have distinctive characteristics such as antibiotic resistance, preservative tolerance and enhanced virulence, leading to chronic infections. Therefore, more attention has been paid to the prevention and control of biofilm formation of bacterial pathogens in food and clinical applications. In this study, probiotic strains were used as an alternative strategy for controlling biofilm formation of three selected foodborne pathogens using three different techniques (competition, exclusion and displacement) and applying both
in-vitro and in-vivo (laboratory animal model) studies.
The present study aimed to:
Evaluate probiotic LAB as a strategy for controlling biofilm formation by foodborne pathogens. This included the following:
1 To isolate and identify the different LAB species from different food samples.
2 To evaluate the probiotic properties of the identified LAB.
3 To examine the selected LAB isolates for their ability to prevent the colonization and biofilm formation of the three chosen foodborne pathogens both in-vitro and in-vivo.
The present study was conducted on a total of 100 food samples of most commonly consumed types of yoghurt, cheese, buttermilk, pickles and honey that were randomly purchased from various markets in Alexandria. All the collected samples were transported within 2 hours in an ice box to the HIPH Microbiology Laboratory for processing. The collected samples were subjected to all microbiological procedures for the isolation and identification of probiotic LAB.
Seven LAB isolates were identified (L.acidophilus, L.delbrukii, L.lactis, L.pentosus, L.paracasei, L.plantarum & B.animalis) and subjected to the probiotic properties testing and examined for their ability to produce biofilms. The ability of LAB isolates to inhibit pathogenic biofilms of L.monocytogenes, S.Typhimurium and E.coli O157:H7 was then evaluated using three different assays: (competition, exclusion and displacement assays).
This study evaluated the ability of the chosen LAB isolates (L.acidophilus, L.plantarum and B.animalis), that revealed the best probiotic properties in-vitro, to have an in-vivo inhibitory effect against the selected foodborne pathogens (S. Typhimurium, E. coli 157:H7 and L. monocytogenes) biofilms. The ability of LAB isolates to inhibit pathogenic biofilms in-vitro was evaluated by three different assays (competition, exclusion and displacement) but only the best assay (exclusion) that showed the highest inhibitory effect was applied in-vivo in the laboratory animal study.
The results of the present study revealed that:
1- Of the 100 examined food samples, 58 (58%) probiotic LAB were isolated.
2- Pickles yielded the highest percentage of LAB isolates (95.0%). This was followed by yoghurt samples (70.0%), which represented the highest percentage among the tested dairy products. Cheese samples were the least to yield LAB isolates (35.0%). No LAB isolates were recovered from any of the examined honey samples.
3- Of the 58 isolated LAB L.pentosus and L.plantarum accounted for 48.3 % of all identified LAB isolates with the percentages of 25.9% and 22.4%, respectively. B.animalis and L.paracasei were the least encountered isolates (5.2% and 3.4 %, respectively). L.pentosus was the main LAB isolate from pickles (79.0%)
4- The majority of yoghurt LAB isolates were identified as L.delbrukii with a percentage of 38.1%. While L.plantarum was the main LAB isolate from both cheese and buttermilk samples with percentages of 71.0% and 72.7%, respectively.
5- All LAB isolates had auto-aggregation abilities but with different percentages. L.delbrukii and L.lactis isolates had the lowest mean auto- aggregation percentages (22.0% and 36.0%, respectively), while L.animalis, L.acidophilus and L.plantarum isolates had the highest mean auto-aggregation activity (62.0%, 61.0% and 65.0%, respectively). L.paracasei and L.pentosus showed auto-aggregation activity means of 50.0% and 55.0%, respectively.
6- The tested strains of L.monocytogenes, S.Typhimurium and E. coli O157:H7 had auto-aggregation capabilities with mean percentages of 71.67%, 53.33% and 57.17%, respectively.
7- All LAB isolates were able to co-aggregate with the tested pathogenic reference strains of L.monocytogenes, S.Typhimurium and E.coli O157:H7 with mean percentages ranging from 31.0% to 77.0%.
8- A statistically significant difference was found between the 7 tested probiotic LAB and between the 3 tested foodborne pathogens regarding their auto-aggregation activity, with a p value of <0.001.
9- All LAB isolates revealed high co-aggregation activity with L.monocytogenes, where L.acidophilus and L.plantarum had the highest mean percentages of 73.0% and 77.0%, respectively. Co-aggregation with E.coli O157:H7 was the best by B.animalis and L.plantarum with a mean of 75.0% each. While co-aggregation activity mean of LAB isolates with S.Typhimurium ranged from 36.0% in case of L.delbrukii and 68.0% with L.plantarum. L.lactis isolates had the least co-aggregation activity with all examined pathogenic reference strains.
10- All of the 7 tested probiotic LAB isolates co-aggregated significantly with each of the 3 examined foodborne pathogens with p values of < 0.001 except for L.paracasei (p= 0.003).
11- All the examined LAB isolates had an increase in their count reduction with the increase in acidity from pH 2.5 to pH 1.5.
12- All LAB isolates revealed resistance to acidity of pH 2.5 after 3 hours of incubation at 37◦C with only one log count reduction. LAB isolates showed moderate to poor resistance to acidity of pH 2. Moderate resistance was encountered with B.animalis, L.lactis, L.acidophilus and L.plantarum with final log count of 3 to 4 and poor with a final of 2 log count with the rest of LAB isolates.
13- All LAB isolates revealed no resistance to acidity of pH 1.5 (count= zero log10 CFU/ml) after 3 hours of treatment except for L.acidophilus and B.animalis that showed very poor resistance.
14- No significant reduction was recorded in the count of B.animalis, L.acidophilus and L.plantarum after 3 hours of incubation at pH 2 and 2.5. There was
a statistically significant reduction in the count of all of the 7 tested probiotic LAB isolates at pH1.5. While only L.lactis, L.delbrukii, L.paracasei and L.pentosus showed a significant count reduction at pH 2.
15- After one hour of 0.5% bile salt treatment, LAB isolates log counts were reduced by one log regarding L.acidophilus, B.animalis and L.plantarum and 4log in L.delbrukii. The count reduction continued after the second hour till the fourth hour for all LAB isolates except for L.acidophilus, L.plantarum and B.animalis that ended with only two log reduction from the starting count.
16- L.lactis and L.delbrukii had statistically significant log counts reduction after 2 hours and 4 hours of bile salts treatment (0.5 %). p:(0.026, 0.001) and (0.001,0.001), respectively. On the other hand, L.paracasei and L.pentosus showed a significant reduction after 4 hours of 0.05% bile salt treatment, p:0.026 and 0.007, respectively.
17- This study reveals that there was no statistically significant reduction in the count of B.animalis, L.acidophilus and L.plantarum after 1, 2 and 4 hours of incubation, in presence of 0.5% bile salts concentration.
18- All the examined LAB isolates CFCs except L.delbrukii, had antimicrobial effects against L.monocytogenes with zones of inhibition that exceeded that of positive control.
19- All LAB isolates developed zones of inhibition against S.Typhimurium that ranged from 7 to 19 mm in diameter, except L.delbrukii that revealed no inhibition effect on S.Typhimurium. L.pentosus, L.plantarum, Lacidophilus and B.animalis demonstrated an antimicrobial activity against S.Typhimurium with zones of inhibition equal to or more than 15 mm (control inhibition zone diameter).
20- L.plantarum, Lacidophilus and B.animalis also proved their ability to develop zones of inhibition against E.coli O157:H7 with diameters equal to kanamycin positive control, while L.paracasei revealed moderate antimicrobial action against the same pathogen. Also L.pentosus showed excellent antimicrobial activity against all studied foodborne pathogens. There was a statistically significant difference between all of these figures. P= 0.003.
21- All LAB isolates were susceptible to ciprofloxacin, clindamycin and kanamycin. Except for L.lactis, all LAB isolates were susceptible to ampicillin and erythromycin. All LAB isolates were susceptible for gentamycin excluding L.lactis and L.delbrukii.
22- All LAB isolates revealed resistance to chloramphenicol except for L.delbrukii and L.pentosus. Tetracyclin was an effective antibiotic against all tested LAB isolates except for L.acidophilus and L.paracasei. All LAB isolates were resistant to vancomycin.
23- All of the seven tested LAB isolates in this study showed a non haemolytic activity after their culture on sheep blood agar and anaerobic incubation for 24 hours at 37◦C. That demonstrated their non haemolysin producing character which is considered as positive property in their safety assessment.
24- All of the 3 examined foodborne pathogens were strong biofilm producers.
25- All probiotic LAB isolates had biofilm forming ability but with different degrees, where L.delbrukii was weak biofilm producer and L.lactis had moderate biofilm forming ability however, the rest of LAB isolates were strong biofilm producers.
26- With the exception of L.delbrukii, displacement assay effect of all LAB isolates reduced the log count of L.monocytogenes in a range of one log reduction (in case of L.lactis) to 4log reduction (in case of B.animalis and L.acidophilus). While S.Typhimurium log count was not affected after displacement by L.lactis, L.delbrukii and L.pentosus, the effect of L.acidophilus, B.animalis and L.plantarum reached 37.5% reduction of log count.
27- Limited displacement effect of LAB isolates on E.coli O157:H7 with maximum of 3 log reduction (37.5%) was noted with L.acidophilus and B.animalis.
28- Using competition assay L.monocytogenes count was reduced with about 62.5% by L.acidophilus and by 50.0% regarding B.animalis and L.plantarum isolates, while the rest of LAB isolates had reduced competition effects.
29- S.Typhimurium count was decreased by 50.0% as a result of L.acidophilus competition and 37.5% by each of B.animalis and L.plantarum. E.coli O157:H7 was the least affected by LAB isolates with a maximum of two log reduction or no effect in presence of L.lactis and L.delbrukii.
30- The seven LAB isolates had an exclusion effect against the selected three foodborne pathogens with variation in the percentage of reduction.
31- L.monocytogenes count was significantly reduced by exclusion effect especially for B.animalis (87.5%), L.acidophilus (87.5%) and L.plantarum (75.0%), while the minimum reduction was with 3 log (37.5%) concerning L.lactis, L.delbrukii and L.pentosus. S.Typhimurium count was reduced by 62.5 % with B.animalis and 50.0% by L.acidophilus and L.plantarum, in addition to two log reduction with the rest of LAB isolates.
32- Exclusion effect on E.coli O157:H7 was the highest with L.acidophilus (62.5%) while L.plantarum and B.animalis were reduced by 50%. L.lactis, L.delbrukii, L.paracasei and L.pentosus were excluded by E.coli O157:H7 with percentages ranging from 12.5 % to 25.0%.
33- There was a statistically significant count reduction percentage of L.monocytogenes using the exclusion assay by B.animalis, L.acidophilus and L.plantarum p=0.001, 0.001 and 0.007, respectively. On the other hand, B.animalis caused a significant count reduction of S.Typhimurium (p=0.026) and L.acidophilus showed
a significant count reduction for E.coli O157:H7 (p=0.026).
34- Using exclusion assay, the selected three LAB isolates produced a complete in-vivo inhibition of L.monocytogenes, S.Typhimurium and E.coli O157:H7 biofilm formation. While L.plantarum caused a log count reduction of S.Typhimurium and E.coli O157:H7 from 5 to 1 log10 CFU/ml (80%).
from the present study it could be concluded that:
1- Pickles and yoghurt samples yielded the highest percentages of LAB isolates.
2- Dairy products (youghurt, cheese and buttermilk) are considred a good source of probiotic bacteria that have the best properties.
3- All LAB isolates have auto-aggregation abilities and were able to co-aggregate significantly with the tested pathogenic reference strains of L.monocytogenes, S.Typhimurium and E.coli O157:H7.
4- B.animalis, L.acidophilus and L.plantarum had a significant resistance to bile salts of 0.5% after 4 hours of incubation, and acidity after 3 hours of incubation at pH 2 and 2.5.
5- All the examined LAB isolates CFCs had statistically significant antimicrobial effects.
6- B.animalis, L.acidophilus and L.plantarum were the best in probiotic properties.
7- B.animalis, L.acidophilus and L.plantarum showed the best antibiofilm activities with each of the three tested biofilm inhibition assays.
8- Among the 3 tested biofilm inhibition assays, exclusion method was found to be the best assay and it was significantly effective in both in-vitro and laboratory animal in-vivo experiments.
9- Probiotic LAB are consisdered an effective strategy for controlling biofilm formation ability of foodborne pathogens (L.monocytogenes, S.Typhimurium and E.coli O157:H7) and could be used as an alternative to antibiotics against MDR foodborne pathogens.
from the results of the present study, the following recommendations are suggested:
1- B.animalis, L.acidophilus or L.plantarum are suggested to be used in the processing of dairy products due to their effective probiotic properties.
2- As the tested probiotic LAB (B.animalis, L.acidophilus or L.plantarum) showed their best biofilm inhibition activities using the exclusion method, thus their prior consumption could be a beneficial strategy to control the BFA of the most commonly encountered foodborne pathogens (L.monocytogenes, S.Typhimurium and E.coli O157:H7).
3- Further in-vivo studies are needed to confirm the inhibitory biofilm capabilities of B.animalis, L.acidophilus and L.plantarum probiotics against foodborne pathogens in human GIT.