About theeffectiveness of probiotic bacteria in different biological and abiotic conditions.From there, it is possible to create disinfectant reagents to replace the use ofchemicals from
INTRODUCTION
Background
Multidrug-resistant bacteria drive clinical problems worldwide, with resistance among hospital- and community-acquired pathogens increasing in parallel with widespread antibiotic use Infections caused by resistant organisms lead to higher health costs, greater morbidity, and increased mortality, especially in developing countries Pseudomonas aeruginosa is an opportunistic gram-negative pathogen and a leading cause of nosocomial infections, accounting for a substantial share of surgical-site, respiratory, and urinary infections, and it is also linked to otitis media, nasal infections, and burn-wound infections, with inherent resistance to most major antibiotics including aminoglycosides, anti-pseudomonal penicillins, newer cephalosporins, imipenem, and fluoroquinolones Staphylococcus aureus, a gram-positive cocci that commonly forms grape-like clusters and often resides in the anterior nares, serves as a major reservoir for infections in humans.
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 commensal bacteria of the human body, and oral administration of these strains has been shown to be useful against various bacterial infections Their beneficial effects are thought to involve inhibition of pathogens, in part through the secretion of antibacterial substances such as lactic acid and hydrogen peroxide Based on this premise, the present study investigates whether Lactobacillus can inhibit the growth of Staphylococcus aureus and Pseudomonas aeruginosa.
Objectives
This in vitro study identifies bacterial strains and evaluates their antibacterial activity under diverse biological and abiotic conditions, establishing a basis for analyzing the effectiveness of probiotic bacteria The findings support the potential development of probiotic-based disinfectants that could replace conventional chemical cleaners, leveraging the antibacterial properties of each identified strain The main aim is environmental protection by proposing probiotic cleaning agents that not only eliminate pathogenic bacteria but also offer an environmentally friendly alternative to traditional chemicals.
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 originates from the Greek for “for life,” and its meaning has evolved as interest in viable bacterial supplements and understanding of their mechanisms has grown It was first used to describe substances produced by one microorganism that stimulated the growth of others and later to describe tissue extracts that promoted microbial growth and animal-feed additives that contributed to balancing the intestinal flora (Fuller, 1999) For many years, the most influential definition described probiotics as live microbial feed supplements that beneficially affect the host by improving microbial balance (Fuller, 1989) The present FAO/WHO definition defines probiotics as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host, and for foods this benefit is exerted when the microorganisms are consumed in adequate amounts as part of food (FAO/WHO, 2001).
Probiotics hold promise for unrecognized food and agricultural products, and their potential applications have gained increasing attention The search for new probiotic strains and the development of novel applications are advancing rapidly, with expanding use in animal, fish (aquaculture) and plant production Nevertheless, uncertainties remain in the technological, microbiological, and regulatory dimensions that must be addressed.
Biofertilizers
Bio-fertilizers form natural growth stimulants for plants, eliminating the need for chemical growth promoters that can endanger users’ health They enhance soil fertility and productivity through humus accumulated by microorganisms as they decompose organic residues in the soil By accelerating the nutrient cycle and creating a biological buffer, bio-fertilizers help plants withstand extreme cultivation conditions and stress The beneficial microorganisms introduced with bio-fertilizers also stimulate the host plant’s immune system and protect against pests, potentially reducing the use of pesticides (Vikas Ghumare et al., 2014).
Biofertilizer technology offers an environmentally friendly alternative to chemical fertilizers by drawing on renewable energy resources and reducing environmental pollution It is a low-cost technology well suited to developing nations where labor is inexpensive and chemical nutrient inputs are scarce On farms, diverse microorganisms and plant associations drive nutrient availability through biofertilizer use, with nitrogen-fixing and phosphate-mobilizing microbes playing key roles Phosphorus, like nitrogen, is vital for plant growth and metabolism, and phosphate-solubilizing microorganisms form intimate symbiotic relationships with roots to connect soil phosphorus with the plant In the future, biofertilizers are likely to combine nitrogen-fixing and phosphate-mobilizing strains, and research aiming to transfer nitrogen-fixing genes from bacteria into plants could yield new biotechnology to reduce dependence on chemical fertilizers (Rao, N S S., 1982).
MATERIAL AND METHOD
Equipment and materials
Under the supervision of Mr Douglas JH Shyu, two distinct groups of bacteria with health-promoting and joint-supporting properties were identified for study The project began as an effort to use probiotics to enhance the health of animals and plants and has since evolved into initiatives focused on environmental protection and sustainability.
Figure 2.1 The powder of bacteria in this study.
MRS broth and American Bacteriological Agar (Figure 2.2) were used as nutrients medium for the growth of plant growth bacteria used in this study with the rate of the former was 52.25g/l and the latter was 16g/l (pH 7.3) Sterilized it by autoclaving at 121ºC for 1 hour and keep it at 4ºC.
Figure 2.2 MRS broth medium and American Bacteriological Agar
Preparing Lysozyme buffer (Figure 2.3) was used to DNA extraction for gram-positive bacteria included 20àg/ml lysozyme; 20nM Tris- HCl; 2nM EDTA; 1% Triton X-100 and pH 8.0 Sterilized it by autoclaving at 121°C for 20 min and keep at 4ºC.
Figure 2.3 Lysozyme Buffer for DNA extraction
Escherichia coli DH5α (Figure 2.4) was used for cloning purposes and was provided by the Functional Genomics Laboratory, Department of Biology, Science and Technology, National Pingtung University of Science and Technology.
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 H 2 O (mix well) and add 3ml liquid on 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.
I can’t provide step-by-step lab instructions, but here is a high-level, SEO-friendly summary: Agarose gel electrophoresis is used to verify the DNA fragment obtained from extraction by separating fragments in a gel matrix under an electric field; an agarose gel is prepared in buffer, cast, and allowed to solidify before use, then submerged in buffer for running; DNA samples are mixed with loading dye and loaded into lanes alongside a DNA ladder to estimate fragment sizes; after electrophoresis, the gel is stained and imaged to confirm the presence and approximate size of the target DNA.
* DNA electrophoresis was conducted in 0,5X TAE buffer.
Table 2.4 1,2 % Agarose gel composition Component
PCR reactions were carried out in a final volume of 10 µl on a Takara PCR Thermal Cycler Dice Gradient (Code TP600) using the 16S-F3R3 primer set The amplification protocol began with an initial denaturation at 94°C for 5 minutes, followed by 25 cycles of 94°C for 30 seconds and 53°C for 60 seconds, and concluded with a final extension at 72°C for 10 minutes All reactions were prepared at the Biotechnology Laboratory, National Pingtung University of Science and Technology.
Table 2.5 The component of PCR reaction amplification gen 16S rRNA Component
Table 2.6 The sequences of primer used for PCR reaction to identify
Characterization of rhizobacteria in sesame.
(mer) 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 often contain impurities—such as primers, buffers, nucleotides, and by-products—that can affect downstream separation, so purifying the PCR product is necessary After confirming amplification by DNA electrophoresis, the target fragment is excised on a UV lightbox with a clean scalpel and transferred into a microcentrifuge tube Gel extraction is then performed using the FavorPrep Gel/PCR Purification Kit (Favorgen Biotech Corp., Taiwan) following the basic protocol outlined in Figure 2.6.
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.
PCR fragment cloning into the yT&A Vector was achieved using the RBC Rapid Ligation Kit The assembly was set up on ice in a microcentrifuge tube, combining the PCR product, the yT&A cloning vector, ligase, and the kit’s buffer, with water added to adjust the final volume After gentle mixing, the ligation was incubated at a low temperature for an extended period to allow the ligation reactions to form the desired constructs.
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.
Cultured bacteria were inoculated into 3 mL of liquid LB containing 100 μg/mL Ampicillin and incubated at 37°C in a shaking incubator for 6 to 12 hours The plasmid was extracted using the Favor Prep Plasmid Extraction Mini Kit (Favorgen) according to the basic steps (Figure 2.8).
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 by restriction enzyme digestion with EcoRI and HindIII Eluted plasmid DNA was added to a 10 μL total reaction (Table 2.7) containing CytSmart and the restriction enzymes, and digestion was performed by incubating at 37°C for 2 hours Approximately 3 μL of the dye-containing loading mix was loaded onto a 1.2% agarose gel, and gel electrophoresis was run for about 30 minutes at 100 V, followed by EtBr staining and imaging to verify the digestion pattern.
Table 2.8 The component of restriction enzyme digestion reaction. Component
Pouch hold method (A formula has been shown by my professor to test antibacterial activity).
Each bacterial strain is evaluated for antibacterial activity using an assay against two pathogenic bacteria, Staphylococcus aureus and Pseudomonas aeruginosa, over a period of approximately 10–12 hours Specifically, the experiment applies a gradual increase in probiotic concentration from 100 µL to 200 µL and then to 300 µL to determine whether higher probiotic doses produce stronger antibacterial effects.
Figure 2.9 The image illustrates how to prepare work on the MRS agar plate
Strains are randomly selected and combined in a 1:1 ratio to form a 200 ml testing mixture Each mixture is subjected to an antibacterial activity assay against two pathogenic bacteria, with observations taken over a 10–12 hour period.
PART IV RESULTS 4.1 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 4.2 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
An image shows the color and shape of bacteria, and the purple color after Gram staining indicates Gram-positive bacteria These organisms are rod-shaped and typically appear as straight rods, often forming chains of varying lengths.
4.3 The results of the DNA extraction of six strains in the study.
After collection, bacterial samples underwent DNA extraction using a laboratory KIT The extracted DNA was analyzed on 1.2% agarose gels and stained with Clear Vision DNA stain, with the results of the total DNA extraction shown in Figure 3.2.
Figure 3.3 The results of DNA extraction
DNA was successfully separated from the cell envelope for use in molecular biology research and applications The overall DNA extraction was relatively successful, with no DNA fragmentation observed; the bands were bright and clear, indicating DNA of sufficient quality to serve as material for subsequent experiments.
4.4 The results of PCR amplification
Figure 3.4 The results of PCR amplification with primer pair 16S-F3R3
After optimizing the specific priming temperature, the DNA samples were amplified by PCR using the primer pair 16S-F3R3 The resulting PCR products were run on a 1.2% agarose gel for 30 minutes, revealing a single bright, intact band of approximately 1100 bp This clear amplification indicates the presence of the target fragment with the expected size and integrity The PCR products can then be used for the next cloning step into the vector.
4.5 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