THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY TRINH THI MY DUYEN Topic title: STUDY ON THE EVALUATION OF PROBIOTICS AS ENVIRONMENTAL CLEANING AGENTS... UNIVERSITY OF A
INTRODUCTION
Background
Multi-drug resistant bacteria pose significant clinical challenges worldwide, driven largely by the widespread use of antibiotics, leading to increased resistance among pathogens causing both hospital- and community-acquired infections Resistant microorganisms contribute to higher healthcare costs, increased morbidity, and mortality, particularly in developing countries Pseudomonas aeruginosa, an opportunistic Gram-negative bacterium, is a major cause of nosocomial infections—accounting for nearly 10% of hospital-acquired infections in surgical sites, respiratory, and urinary tracts—and is associated with serious underlying diseases It exhibits high genotypic diversity and inherent resistance to most antibiotics, including aminoglycosides, anti-pseudomonal penicillins, newer cephalosporins, imipenem, and fluoroquinolones, complicating its treatment Similarly, Staphylococcus aureus, a Gram-positive cocci commonly found in human anterior nares, is a prevalent pathogen associated with various infections, emphasizing the need for effective strategies to combat antibiotic resistance in these bacteria.
S.aureus has a characteristic of biofilm formation When S aureus enters into the circulatory system, it avoids the detection by the immune system, binds to a specific surface including infection area and forms a biofilm to survive in the host (Lowy et al., 1998) Moreover, its biofilm can be created on both biotic and abiotic surface; so, S aureus shows resistance to antibiotics that becomes a problem in treatment (Fedtke et al., 2004; Greenber et al., 1989; Herrmann, 2002; D Joh et al., 1999; PW Park et al., 1996; Patti et al., 1994) Some infectious diseases related to the S aureus’ biofilm formation were arthritis, endocarditis, and cystic fibrosis (Costa et al., 1999; Lancet, 1998; Rajan, 2002) Bacteria could have characteristics to form a biofilm The extracellular polymeric matrix is made from the combination of exopolysaccharides, proteins, teichoic acids, enzymes, and extracellular DNA (Melchior et al., 2006; Parra-Ruiz et al., 2012) Regarding to previous studies, the matrix’s structure is changed from strains to strains due to the environment and the conditions (Rohde et al., 2001; Landini, 2009) By living in a community, biofilms have various benefits and advantages from their parts and one of them is resistant to the immune system and antibiotics Recent reports have documented the role of exogenous Lactobacilli in the prevention and treatment of some infections Lactobacillus acidophilus is gram-positive bacteria naturally living in the human and animal digestive system Lactobacillus is also used in dairy products including milk, yogurt… in combination with other microbes Lactobacillus has the potential to be used as an antibiotic medicine and a drug deliver ( TTV Doan et al., 2013) Recent reports have documented the role of exogenous Lactobacilli in the prevention and treatment of some infections
Lactobacillus strains are natural inhabitants of the human body and have proven beneficial in combating various bacterial infections Oral administration of Lactobacillus has been shown to inhibit the growth of harmful pathogens, primarily through the secretion of antibacterial substances like lactic acid and hydrogen peroxide The primary goal of this study was to investigate whether Lactobacillus can effectively inhibit the growth of Staphylococcus aureus and Pseudomonas aeruginosa, two common pathogenic bacteria.
Objectives
This in vitro experiment aimed to identify bacterial strains to analyze their antibacterial activity and evaluate the effectiveness of probiotic bacteria under various biological and abiotic conditions The findings lay the groundwork for developing natural disinfectant agents that could replace chemical-based products by harnessing the antibacterial properties of these strains The primary goal of the study is to promote environmental protection by proposing probiotic-based cleaning agents that not only eliminate pathogenic bacteria but are also eco-friendly.
Scope of study
- The provided bacteria cultured in MRS broth environment
- The bacteria DNA extraction The amplification genes by PCR reaction using 16S rRNA primers
- Ligation and transformation reactions to joining of two nucleic acid fragments through the action of an enzyme performed using T4 DNA ligase
- Sequencing of bacterial gene and compare to with gene bank in the NCBI
- Use DNA star software to identify selected strains of bacteria
- Test and evaluate antibacterial activity by the pouch hold method with identified bacteria strains.
LITERATURE REVIEW
Definition of Probiotics
The term "probiotic" is derived from Greek, meaning "for life," reflecting its longstanding association with beneficial microorganisms Originally, it described substances produced by microorganisms that stimulated the growth of others, and later expanded to include tissue extracts and animal feed supplements that promote intestinal health (Fuller, 1999) Fuller’s earlier definition characterized probiotics as live microbial feed supplements that enhance the host's microbial balance, contributing to health (Fuller, 1989) The current definition by the FAO/WHO describes probiotics as "live microorganisms which, when administered in adequate amounts, confer a health benefit on the host," emphasizing their role in promoting health through consumption in food (FAO/WHO, 2001) This evolution highlights the growing understanding of probiotics' mechanisms and their importance in supporting human and animal health.
Recent studies highlight the growing interest in the potential applications of probiotics in unrecognized food and agricultural products The selection of new probiotic strains and the development of innovative applications are gaining significant importance Agricultural uses of probiotics for enhancing animal, fish, and plant production have seen notable growth However, uncertainties still exist regarding the technological, microbiological, and regulatory aspects of incorporating probiotics into these applications (Krửckel L, 2006).
Biofertilizers
Bio-fertilizers naturally stimulate plant growth by encouraging the production of growth-promoting compounds, eliminating the need for harmful chemical fertilizers that can endanger user health They enhance soil fertility and productivity through the accumulation of humus produced by beneficial microorganisms decomposing organic residues Application of bio-fertilizers promotes a balanced nutrient cycle and creates a "biological buffer" that helps plants withstand extreme cultivation conditions and stress The beneficial microorganisms introduced via bio-fertilizers boost the plant's immune system, providing natural protection against pests and diseases, which can significantly reduce the reliance on chemical pesticides.
Biofertilizer technology is an eco-friendly and renewable resource-based alternative to chemical fertilizers, reducing environmental pollution and offering a low-cost solution especially suitable for developing nations with inexpensive labor It involves micro-organisms that enhance nutrient availability, including nitrogen fixation and phosphate solubilization, forming symbiotic relationships with plant roots that improve soil health and plant growth Phosphorus plays a crucial role alongside nitrogen in supporting plant metabolism, with micro-organisms acting as natural agents to mobilize these nutrients from the soil Future advancements in biofertilizer research will likely include multi-functional microbial blends and innovative biotechnologies, such as transferring nitrogen-fixing genes into plants, to decrease reliance on chemical fertilizers and promote sustainable agriculture (Rao, N S S., 1982).
MATERIAL AND METHOD
Equipment and materials
No Name of device Type Country
6 Dry bath incubator MD-02N Taiwan
9 Orbital shaker incubator LM-570RD Singapore
14 Thermo scientific MY SPIN 6 HSF 51902 Taiwan
16 Laminar airflow cabinet JW.4N Taiwan
Two groups of beneficial bacteria, supporting overall health and joint support, were provided by my supervisor, Mr Douglas JH Shyu Initially focused on using probiotics to improve the health of animals and plants, this study evolved into a broader project dedicated to environmental protection.
Figure 2.1 The powder of bacteria in this study
In this study, MRS broth and American Bacteriological Agar served as nutrient media for cultivating plant growth bacteria, with concentrations of 52.25 g/l and 16 g/l respectively, and a pH of 7.3 The media were sterilized through autoclaving at 121ºC for one hour and stored at 4ºC to ensure contamination-free conditions for optimal bacterial growth.
Figure 2.2 MRS broth medium and American Bacteriological Agar
Lysozyme buffer, prepared for DNA extraction from gram-positive bacteria, contains 20 µg/ml lysozyme, 20 nM Tris-HCl, 2 nM EDTA, 1% Triton X-100, and is adjusted to pH 8.0 The buffer is sterilized by autoclaving at 121°C for 20 minutes and stored at 4ºC to ensure stability and sterility for effective DNA extraction.
Figure 2.3 Lysozyme Buffer for DNA extraction
Escherichia coli strain DH5α, as shown in Figure 2.4, was utilized for cloning purposes in this study This bacterial strain was provided by the Functional Genomics Laboratory at the Department of Biology, National Pingtung University of Science and Technology, ensuring reliable and efficient genetic manipulation for our research.
Figure 2.4 The cell of Escherichia coli (DH5α) in this study.
Method
In order to culture well-grown bacteria, a specific type of medium was required In this study, I used MRS Broth contains the best nutrition for bacteria to grow well
Table 2.2 Formular in g/l (Medium to facilitate de growth of lactobacilli)
1 Firstly, prepared plate agar and liquid medium to culturing bacteria
15.675 MRS Broth + 300ml of H2O (mix well) and add 3ml liquid on tubes Add 3ml tubes
15.675 MRS Broth + 6g Agar American + 300ml H2O (mix well)
Sterilize them all in an autoclave for 1 hour And then spill liquid to the plate Store both liquid and the solid medium in 4° cabinets
2 The bacteria were removed from the original storage tube and then diluted it to cultured spread on the surface of the medium plate according to the ‘‘Z’’ shape by the culture rods that heated on the fire of alcohol
3 Then it was incubated in the incubator at 37ºC for 48 hours
4 The result would be the appearance of single colonies
5 Picked up each one single colony in a plate put in one liquid culture tube, then the sample shook and incubated in the incubator for 37ºC overnight to facilitate DNA extraction in the next process
1 Add about 1 drop of crystal violet stain over the fixed culture Let stand for 60 seconds
2 Pour off the stain and gently rinse the excess stain with a stream of H2O
3 Add about 1 drop of the iodine solution on the smear, enough to cover the fixed culture Let stand for 30 seconds
4 Pour off the iodine solution and rinse the slides with running water Shake off excess water from the surface
5 Add a few drops of alcohol so the solution trickles down the slide Rinse it off with water after 5 seconds Stop when the solvent is no longer colored as it flows over the slide
6 Counterstain with 5 drops of the Safranin solution for 20 seconds
7 Wash off the red Safranin solution with water Blot with bibulous paper to remove any excess water Alternatively, the slide may be shaken to remove most
8 Examine the finished slide under a microscope
Genomic DNA extraction kit which was used for DNA extraction in my study The process includes the following basic steps (Figure 2.5)
Figure 2.5 The process of DNA extraction bacteria
1 Add 1ml sample to microtube, centrifuge at 13000rpm/1min then remove supernatant
2 Add 200àl Lysozyme Buffer and vortex ( keep in room temperature 10 min and shake it by every 3 min)
3 Add 200àl GB Buffer ( vortex 10 times to mix sample)
4 Incubate 70°C for 10 minutes until the sample lysate is clear, invert the tube every 3 minutes At this time, preheat required Elution Buffer at 70°C DNA binding
5 Add 200àl of Ethanol 95% to the sample lysate and vortex immediately for 10 seconds to mix samples If precipitate appears, break up by pipetting
6 Place a GD Column on a 2 ml Collection Tube
7 Apply all the mixture (including any precipitate) from the previous step to the GD Column
8 Close the cap and centrifuged at 13,000rpm for 2 minutes Discard the flow-through and return the GD column to the 2ml collection tube
9 Add 400àl W1 Buffer to the GD Column Centrifuge at 13,000rpm for 1min then discard the flow-through and return the GD Column to the 2ml Collection Tube
10 Add 600àl of Wash Buffer to the GD Column Centrifuge at 13000rpm for 1min
11 Discard the flow-through and return the GD Column the 2ml Collection Tube Centrifuge for an additional 3 minutes to dry the column
12 Transfer dried GD Column into a clean 1.5ml microcentrifuge tube
13 Add 50àl of preheated Elution Buffer to the center of the column matrix
14 Allow standing for 2 minutes until the Elution Buffer is absorbed by the matrix
15 Centrifuge at 13000rpm for 1min to elute purified DNA
DNA electrophoresis was performed using a 1.2% agarose gel prepared with approximately 0.3 grams of agarose dissolved in 30 ml of 0.5X TAE buffer, following proper heating and cooling procedures for complete dissolution The gel was cast and solidified for about 30 minutes before being submerged in 1X TAE buffer within the electrophoresis tank DNA samples mixed with 6X loading dye were loaded into the wells, with the first well containing a 1 kb DNA marker to enable size comparison of the samples Electrophoresis was carried out at 100V for 30 minutes, and the gel was stained with Vison DNA prior to running The process was stopped once the DNA marker reached the penultimate mark line, confirming successful separation of the genetic material.
* DNA electrophoresis was conducted in 0,5X TAE buffer
PCR reactions were performed in a 10 µl final volume using the Takara PCR Thermal Cycler Dice® Gradient (Code TP600) The amplification process included an initial denaturation at 94ºC for 5 minutes, followed by 25 cycles of 94ºC for 30 seconds and 53ºC for 60 seconds, with a final extension at 72ºC for 10 minutes These reactions were conducted at the Biotechnology Laboratory, National Pingtung University of Science and Technology.
Table 2.5 The component of PCR reaction amplification gen 16S rRNA
Table 2.6 The sequences of primer used for PCR reaction to identify
Characterization of rhizobacteria in sesame
16S-F3R3 TAC GGG AGG CAG CAG
TAC CTT GTT ACG ACT TCA
The temperature cycles for PCR reaction
3.2.6 DNA purification from Agarose gel
PCR products generated through gene amplification often contain impurities such as primers, PCR buffers, nucleotides, and potential by-products that can interfere with downstream separation processes, making purification essential After confirming the PCR product via DNA electrophoresis, the target fragment is carefully excised under UV light using a clean scalpel and transferred into a microcentrifuge tube The purified DNA is then obtained using the FavorPrepTM Gel/PCR purification kit (Favorgen Biotech Corp., Taiwan), following the standard purification steps to ensure high-quality DNA for subsequent applications.
Figure 2.6 The process of DNA purification from Agarose gel
1 Excise the agarose gel with a clean scalpel
• Remove the extra agarose gel to minimize the size of the gel slice
2 Transfer up to 200 mg of the gel slice into a microcentrifuge tube
3 Add 500 àl of FADF Buffer to the sample and mix by vortexing
4 Incubate at 60 °C for 10 minutes and vortex the tube every 2 ~ 3 minutes until the gel slice dissolved completely
5 During incubation, interval vortexing can accelerate the gel dissolved
6 Make sure that the gel slice has been dissolved completely before proceed the next step
7 Cooldown the sample mixture to room temperature And place a FADF Column into a Collection Tube
8 Transfer all of the sample mixtures to the FADF Column Centrifuge at 13,000 x g for 1 minute, then discard the flow-through
9 Add 750 àl of Wash Buffer (ethanol added) to the FADF Column Centrifuge at 13,000 x g for 30 1 minute, then discard the flow-through
• Make sure that ethanol (96-100 %) has been added into Wash Buffer when first use
10 Centrifuge again at full speed (~ 18,000 x g) for an additional 3 minutes to dry the column matrix
• Important step! The residual liquid should be removed thoroughly on this step
11 Place the FADF Column to a new microcentrifuge tube
12 Add 40àl of Elution Buffer to the membrane center of the FADF Column Stand the FADF Column for 1 min
• Important step! For effective elution, make sure that the elution solution is dispensed onto the membrane center and is absorbed completely
• Important: Do not elute the DNA using less than the suggested volume (10àl) It will lower the final yield
13 Centrifuge at 13,000 x g for 1 min to elute DNA Keep the sample at -20 o C
3.2.7 Cloning of Screened Gene into yT&A-Vector following Transformed into DH5α 3.2.7.1 Ligation
The PCR product was successfully cloned into the yT&A Vector using the RBC Rapid Ligation Kit protocol, ensuring efficient preparation for further experiments The ligation mixture was prepared on ice, containing 1.5 µl of PCR product, 1X reaction buffer, 0.5 µl of yT&A cloning vector, and 0.5 µl of T4 DNA ligase, with distilled water added to reach a total volume of 10 µl Gentle pipetting was used to mix the reaction, which was then incubated at 4°C for 18 hours to facilitate optimal ligation efficiency.
Table 2.7 The component of the ligation reaction
The mixture of reaction ligation was transformed into competent cell E coli
DH5 α by heat shock (Foger and Hall, 2007) The process includes the following basic steps (Figure 2.8)
Figure 2.7 The process of transformation reaction
1 Take 20ul DH5a to a centrifuge tube
2 Add 200ul CaCl2(0.1M) into centrifuge tube
3 Add 10ul ligation products keep 40min of the mixture in the ice and shake it every 10 min
4 Move centrifuge tube to an incubator for 42oC in the only 90secound
5 Then keep centrifuge tube 5mins more in ice
6 Add to mixture 600ul of LB
7 Culture bacteria in 1hour at 37oC
8 Centrifuge 8,000rpm for 1min and discard 600ul of the liquid and mix well
9 Spread 100ul of a mixture to LB plate(add AP) and culture at 37oC for 12 hours
Culture bacteria at 37 °C over 12 hours
Bacteria were cultured by inoculating obtained cultures into 3 ml of liquid LB medium containing 100 μg/ml of Ampicillin, and incubated at 37°C with shaking for 6 to 12 hours to ensure optimal growth The plasmid DNA was then extracted using the FavorPrep Plasmid Extraction Mini Kit (Favorgen) following the manufacturer's standard protocol.
1 Transfer 1 ml of well-grown bacterial culture into a centrifuge tube
2 Centrifuge the tube at 13,000 x g for 1 minute to pellet the cells and discard the supernatant completely
3 Add 200 àl of FAPD1 Buffer (RNase A added) to the cell pellet and resuspend the cells completely by pipetting
• No cell pellet should be visible after resuspension of the cells
4 Add 200 àl of FAPD2 Buffer and gently invert the tube 5 ~ 10 times Incubate the sample mixture at room temperature for 2 ~ 5 minutes to lyse the cells
• Do not vortex, a vortex may shear genomic DNA If necessary, continue inverting the tube until the lysate becomes clear
• Do not proceed with the incubation over 5 minutes
5 Add 300 àl of FAPD3 Buffer and invert the tube 5 ~ 10 times immediately to neutralize the lysate
• Invert immediately after adding FAPD3 Buffer will avoid asymmetric precipitation
6 Centrifuge at 13,000 x g for 10 min to clarify the lysate During centrifugation, place a FAPD Column in a Collection Tube
7 Transfer the supernatant carefully to the FAPD Column and centrifuge at 13,000 x g for 5 minutes Discard the flow-through and place the column back to the Collection Tube
• Do not transfer any white pellets into the column
8 Add 400 àl of W1 Buffer to the FAPD Column and centrifuge at 13,000 x g for 1 minute Discard the flow-through and place the column back to the Collection Tube
9 Add 600 àl of Wash Buffer to the FAPD Column and centrifuge at 13,000 x g for 1 minute Discard the flow-through and place the column back to the Collection Tube
• Make sure that ethanol (96-100 %) has been added into Wash Buffer when first use
10 Centrifuge at full speed (~ 18,000 x g) for an additional 3 minutes to dry the FAPD Column
• Important step! The residual liquid should be removed thoroughly on this step
11 Place the FAPD Column to a new 1.5 ml microcentrifuge tube
12 Add 35àl of Elution Buffer to the membrane center of the FAPD Column Stand the column for 1 minute
• Important step! For effective elution, make sure that the elution solution is dispensed on the membrane center and is absorbed completely
13 Centrifuge at 13,000 x g for 5 minutes to elute plasmid DNA and store the DNA at -20 °C
Figure 2.8 The process of Plasmid DNA extraction
The screened gene was confirmed through restriction enzyme digestion using EcoRI and HindIII The eluted plasmid DNA was prepared by transferring 10 µl into a reaction mixture with CytSmart and the restriction enzymes, then incubated at 37°C for 2 hours A 3 µl sample of the digested DNA, mixed with dye, was loaded onto a 1.2% agarose gel for electrophoresis The digestion was verified by running the gel at 100V for approximately 30 minutes, followed by staining with Ethidium Bromide and imaging to confirm successful restriction enzyme activity.
Table 2.8 The component of restriction enzyme digestion reaction
Pouch hold method (A formula has been shown by my professor to test antibacterial activity)
Each strain of bacteria will be tested antibacterial activity assay with 2 pathogenic bacteria are staphylococcus aureus and pseudomonas aeruginosa about
To evaluate the effectiveness of probiotics, gradually increase the concentration of biological bacteria from 100 µL to 200 µL and then to 300 µL over a period of 10 to 12 hours This approach helps determine whether higher probiotic concentrations produce a stronger biological response Monitoring these incremental changes allows for an accurate assessment of probiotic potency and effectiveness.
Figure 2.9 The image illustrates how to prepare work on the MRS agar plate
To evaluate the antibacterial activity, randomly select various strains and combine them in a 1:1 ratio to form a total volume of 200ml Each mixture will then undergo an antibacterial activity assay against two pathogenic bacteria, with incubation periods ranging from 10 to 12 hours This approach helps determine the efficacy of the bacterial combinations in inhibiting pathogenic growth, providing valuable insights into their potential therapeutic applications.
RESULTS
The result of the culture of bacteria
The bacteria strains were grown in MRS Broth medium at 37ºC in 48 hours in the incubator After two days, the bacteria culture appeared singles colony (Figure 3.1)
Figure 3.1 The two groups of bacteria isolated growth on media
The result of gram staining
Gram staining is an experimental method that distinguishes bacterial species into two groups (Gram-positive and Gram-negative) based on the physical and chemical properties of the cell wall
Observe the slide under a microscope
Gram-positive: dark blue or purple
Gram-negative: yellow red or burgundy
Figure 3.2: The shape and arrangements of bacteria observed under the microscope
The image displays bacteria characterized by their color and shape after Gram staining, revealing them as purple, indicating they are Gram-positive bacteria These bacteria are rod-shaped and typically arrange themselves in straight chains of varying lengths.
The results of the DNA extraction of six strains in the study
Bacteria samples were collected and their DNA was extracted using a DNA extraction kit in the laboratory The extracted DNA was then analyzed by running the samples on 1.2% agarose gels and staining with Clear Vision DNA stain The results of the total DNA extraction are presented in Figure 3.2, demonstrating successful isolation of bacterial DNA for further analysis.
Figure 3.3 The results of DNA extraction
DNA was successfully extracted from the cell envelope, providing high-quality genetic material suitable for molecular biology research and applications The extraction process yielded intact DNA with bright, clear bands, indicating minimal degradation These results confirm that the DNA is suitable for use in subsequent experiments, ensuring reliable and efficient downstream analyses.
The results of PCR amplification
Figure 3.4 The results of PCR amplification with primer pair 16S-F3R3
Optimizing the specific priming temperature was essential for successful DNA cloning via PCR The PCR amplification with research primers produced a single, clear, and intact band approximately 1100 bp in size, as confirmed by electrophoresis on a 1.2% agarose gel for 30 minutes Using the primer pair 16S-F3R3 yielded this distinct bright band, indicating successful amplification The resulting PCR products are suitable for subsequent cloning into a splitting vector, facilitating further molecular analysis.
The results of gene cloning
4.5.1 Results of transforming plasmid DNA into variable cells of E coli DH5α
Colony Ht1 Colony Ht4 Colony Ht6
Figure 3.5 The result of transforming the recombinant vector into competent cells
Figure 3.5 demonstrates successful transformation of the recombinant vector into host cells, indicated by the appearance of numerous white colonies on the culture plate The colonies' density and quantity facilitated an efficient selection process Selected colonies were subsequently cultured in 3 ml LB liquid medium supplemented with 1 ml/l ampicillin, shaken at 200 rpm overnight, to enable plasmid DNA extraction for downstream analysis.
4.5.2 The results of DNA Plasmid Extraction by Restriction Enzyme digestion
To verify whether the extracted plasmids carry the gene of interest, they should be digested with EcoRI and Hind III restriction enzymes This enzymatic digestion typically results in two DNA fragments: the lower fragment represents the inserted gene segment, while the upper fragment is the remaining plasmid backbone after the gene has been excised Confirming these fragments through gel electrophoresis ensures successful cloning and proper gene incorporation within the plasmid.
Healthy colony group Figure 3.6 The results of electrophoresis of enzyme-cut products by EcoRI and
HindIII enzyme of the 16S-F3R3 primer
The plasmid separation results are divided into two parts, including size DNA bands of 600 bp and 1100 bp respectively that exactly as originally planned.
Identify and analyze the nucleotide sequence of the DNA markers
Once the DNA segments are successfully cloned, they are sent to Kiron Meek Genomics, Taiwan's leading sequencing company The sequencing process is performed using an automated sequencer based on Sanger's method, ensuring accurate and reliable results After sequencing, the data is processed with BioEdit software and compared with existing sequences in the GenBank (NCBI) database for detailed analysis This approach facilitates precise identification and validation of the genetic information, supporting comprehensive research and further studies.
Figure 3.7 The software to compare sequences on gene banks on the NCBI website
Results of identification and sequence analysis of 16S-F3R3 gene
The J2-5; J3-1; J6-7; Ht1-6; Ht4-4 and Ht6-10 strains were sequenced with 16S-F3R3 gene sequences with the following results:
The analysis of the gene sequence revealed it is approximately 1100 bp, consistent with estimated sizes, with notable differences valuable for classification purposes The J2-5 strain exhibits a 99.3% genetic homology with Lactobacillus pentosus or Lactobacillus plantarum, while the J6-7 strain shows a 98.6% homologous rate with Lactobacillus plantarum The Ht4-4 strain demonstrates a 99.7% similarity to Lactobacillus rhamnosus, and Ht6-10 shares a 97.9% homology with Lactobacillus paracasei Conversely, J3-1 and Ht1-6 yielded results identified as "vector" during sequencing, indicating unsuccessful identification Overall, these four strains are confirmed beneficial bacteria that are documented in the NCBI database across various countries.
Figure 3.8 Phylogenetic tree gene and homologous rate of J2-16S-F3R3 strain
Figure 3.9 Phylogenetic tree gene and homologous rate of J6-16S-F3R3 strain
Figure 3.10 Phylogenetic tree gene and homologous rate of Ht4-16S-F3R3 strain
Figure 3.11 Phylogenetic tree gene and homologous rate of Ht6-16S-F3R3 strain
Table 3.1 Identify 6 strains in the study on gene bank
No Strains Primer Size (bp) Similar
J2: Lactobacillus pentosus or Lactobacillus plantarum subsp plantarum
The results of the antibacterial activity assay
4.7.1 Antibacterial of mix strains of bacterial
Table 3.2 The antibacterial level of mix strains is measured in units of millimeters
Figure3.12 The level of anti-Staphylococcus aureus
J2-J6 and J6-Ht4 demonstrate the most effective antibacterial activity against Staphylococcus aureus, each forming a 3 mm bactericidal zone Following closely, J6-Ht6 exhibits a 2.5 mm antibacterial zone, indicating significant efficacy The other groups show smaller antibacterial zones, measuring no more than 2 mm in diameter Notably, J2-Ht4 displays the least antibacterial activity, with a zone of only 0.5 mm, highlighting its limited effectiveness against bacterial resistance.
Figure3.13 The level of anti- Pseudomonas aeruginosa
Experiments with Pseudomonas aeruginosa demonstrated that the J6-Ht4 and J6-Ht6 groups achieved the best results at a 3mm distance, indicating superior bacterial efficacy In contrast, the J2-Ht6 group showed the lowest performance, likely due to insufficient bacterial concentration to effectively combat pathogenic bacteria.
In short, all bacterial groups have the ability to fight off pathogenic bacteria Among them, the three groups J2-J6, J6-Ht4 and J6-Ht6 are the best about the antibacterial ability
Table 3.3 The anti-Staphylococcus aureus level
Figure3.3 The anti- Staphylococcus aureus level of J2, J6, HT4, and HT6 strain
According to Table 3.5, bacteria strains J2 and J6 demonstrate comparable anti-Staphylococcus aureus activity, forming significant inhibition zones In contrast, Ht4 and Ht6 exhibit bacterial invasion across the entire agar surface, indicating less effective antibacterial properties Therefore, J2 and J6 possess superior antibacterial efficacy against Staphylococcus aureus compared to Ht4 and Ht6.
Table 3.4 The anti-Pseudomonas aeruginosa
Figure 3.15 The anti-Pseudomonas aeruginosa level of J2, J6, HT4, and HT6 strain
Table 3.4 clearly demonstrates that bacterial inhibition depends on concentration, with higher concentrations producing stronger antibacterial effects All four bacterial strains exhibit anti-Pseudomonas aeruginosa properties, indicating broad-spectrum antibacterial activity The consistent size of the inhibition zones at each concentration suggests that these strains have similar antibacterial efficacy against Pseudomonas aeruginosa Overall, the data highlights the concentration-dependent antibacterial potency of these strains, emphasizing their potential as effective antimicrobial agents.
CONCLUSIONS
Conclusion
Both the Joint and Health bacteria groups contain a total of six bacterial strains After conducting experiments and sending samples for sequencing, four specific strains were identified among the tested samples.
J2: Lactobacillus pentosus or Lactobacillus Plantarum subsp plantarum J6: Lactobacillus Plantarum
This experiment primarily focused on evaluating antibacterial activities, showcasing that strains J2 and J6 exhibit superior antibacterial efficacy compared to Ht4 and Ht6 against pathogenic bacteria Notably, higher bacterial concentrations resulted in increased inhibition, indicating a dose-dependent antibacterial effect Additionally, combining different bacterial strains demonstrated a synergistic effect, enhancing their ability to inhibit pathogenic bacteria effectively.
The table highlights the bacterial inhibition levels of both a random mix of strains and four specific Lactobacillus strains, including Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus pentosus, and Lactobacillus plantarum subsp plantarum These findings demonstrate the varying effectiveness of these probiotic strains in inhibiting bacterial growth, revealing their potential applications in food preservation and gut health The study emphasizes the significance of selecting specific Lactobacillus strains for targeted bacterial inhibition, which can enhance probiotic efficacy Incorporating these strains into functional foods may improve their antioxidant and antimicrobial properties, supporting overall health benefits.
This experiment demonstrates that all tested bacteria possess the ability to suppress pathogenic bacteria effectively The size of the antibacterial zone surrounding the opening is influenced by the bacterial concentration, indicating that higher concentrations may enhance antibacterial activity These findings highlight the potential of these bacteria as natural agents in controlling harmful pathogens, which is valuable for developing eco-friendly antimicrobial strategies.
Table 3.4 presents the antibacterial cycle sizes, highlighting that the J6 and Ht4 mixture exhibited the most effective bacterial elimination, with a cycle width of approximately 3mm against Staphylococcus aureus and Pseudomonas aeruginosa The J2 and Ht4 groups demonstrated the shortest anti-Staphylococcus aureus cycle of around 0.5mm, indicating limited effectiveness Notably, the J2-Ht6 group did not show any activity against Pseudomonas aeruginosa, and the antibacterial cycle sizes for the remaining groups did not exceed 3mm, suggesting varying degrees of antibacterial efficacy across different treatments.
In conclusion, four lactobacillus strains have ability resistant to the two types of pathogenic bacteria, which is actually potential bacteria for producing friendly cleaning agents.
Recommendations
- Based on the antibacterial properties of probiotic bacteria, which are beneficial bacteria, it is necessary to continue research and development
- Testing of this product to purify the environment from this probiotic bacterium in the future
- Experimental survey and compare the superiority of probiotics compared with conventional detergents
- It is therefore suggested the idea of the bacterial strain or a group of bacteria strains that have the best cleaning as an environmental cleaner
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