OPTIMIZATION OF COLONY POLYMERASE CHAIN REACTION FOR THE 16SRRNA OF DIFFERENT STRAINS OF ESCHERICHIA COLI

Objective: This work aimed to enhance colony polymerase chain reaction (PCR) for the 16S rRNA of several Escherichia coli strains. Methods: The isolation of E. coli is done from the gut of the chicken and soil. Then, we optimized the condition for colony PCR for the amplification of 16s ribosomal RNA. We successfully designed primer 3 for 16s ribosomal RNA and made the dilution solution with PCR grade water that is 1:10. Moreover, finally, we made a 20 μ L solution that contains the master mix of our isolated colony and forward and reverse base primer for amplification. After the conventional PCR, the amplified 16s ribosomal RNA was then run on Gel to obtain the desired bands. And finally saw the bands in the Gel Doc picture. Results: Our result shows that the technique of colony PCR is an efficient and quick method than other existing methods that are too costly, tedious, and time-consuming procedures that deter their exploitation in various experimentations and for the identification of


INTRODUCTION
Escherichia coli, belonging to the Enterobacteriaceae family, encompass commensal and pathogenic strains [1]. Virulence traits are encoded on mobile genetic elements, leading to distinct strain variations [2]. The emergence of new E. coli strains necessitates the development of innovative diagnostic methods. A novel technique utilizing rRNA primers enables the amplification of the 16SrRNA gene for mutation evaluation, strain detection, and antibiotic resistance analysis. Gel Electrophoresis aids in the visualization of the amplified gene, facilitating rapid identification [3]. E. coli is categorized into intestinal pathogenic, extra-intestinal pathogenic and commensal classes [3]. Enteropathogenic E. coli (EPEC) is responsible for infant diarrhea in developing countries [4]. EPEC attaches to epithelial cells through the LEE gene, resulting in cytoskeletal changes [5].
Enteroaggregative E. coli (EAEC) is a notable contributor to prolonged diarrhea in individuals of all ages, spanning across global populations [9,10]. It colonizes the intestinal mucosa, secretes enterotoxin and cytotoxin, and induces mild but notable mucosal damage, primarily in the colon. Attachment is facilitated by aggregative adherence fimbriae. EAEC synthesizes toxins, including the autotransporter protease referred to as pic, moreover, Shigella flexneri encompasses diverse strains that harbor the oligomeric enterotoxin, recognized as shigella enterotoxin (SHET1) [9,10].
Enteroinvasive E. coli (EIEC) shares genetic and biochemical similarities with Shigella species and can cause invasive inflammatory colitis, occasionally resulting in dysentery. EIEC and Shigella, both responsible for dysentery, exhibit similar symptoms including fever, abdominal cramps, and bloody and mucus-containing diarrhea. In many instances, the diarrhea caused by EIEC presents as a watery form, making it challenging to differentiate from diarrhea induced by other pathogenic strains of E. coli [11].
ExPEC is a major causative agent of UTIs, accounting for 75-95% of cystitis and pyelonephritis cases in the United States [13,14]. UTIs are categorized as lower (cystitis) or upper (pyelonephritis) infections [15]. Women are more susceptible to UTIs due to anatomical differences [16]. Commensal E. coli strains that colonize the intestinal tract have a symbiotic relationship with the host and confer health benefits [17][18][19].
preventing pathogen colonization in catheters and establishing growth in the urinary tract without triggering an immune response [26].
In conclusion, E. coli encompasses a range of commensal and pathogenic strains that contribute to severe diseases such as intestinal disorders and watery diarrhea. Particular strains, such as E. coli O157:H7, are capable of producing verotoxins or Shiga-like toxins, which are cytotoxins encoded by bacteriophages [37]. These toxins can cause sporadic cases of diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome [37]. Verotoxin-producing E. coli/Shiga toxin-producing E. coli strains, characterized by the absence of the enterotoxin gene, carry a distinct fimbrial adhesion that is encoded by 60-megadalton plasmids. Effective detection methods, including hybridization probes and polymerase chain reaction (PCR)-based techniques, have been developed for their identification [38]. The utilization of nucleic acid-based tests, including PCR, has revolutionized bacterial pathogen detection in clinical laboratories, ensuring high sensitivity and specificity. Proper procedures and primer selection are crucial to achieve accurate results [38][39][40][41][42]. Colony PCR offers a rapid method for selecting specific DNA sequences without the need for DNA purification, making it useful for various applications in bacterial research [43][44][45][46].

METHODS
The study was conducted at the Centre of Biotechnology and Microbiology, University, and ethical permission was granted by the department's ethical committee.

Sample collection
Soil samples were collected from different regions in Peshawar University to isolate E. coli strains. The soil samples were wrapped in sterile Aluminum foil to prevent contamination.

Serial dilution
The collected soil samples were transferred to a Laminar Flow Hood (LFH) where sterile conditions were maintained to prevent contamination. Serial dilution of the samples was performed up to 10-5 in LFH.

Preparation of media
Selective Eosin Methylene Blue Agar (EMB) media was used to culture the samples to grow E. coli strains. Media was prepared according to the manufacturer's instructions and autoclaved at 121°C and 15psi pressure. The media was poured into sterile petri plates and incubated at 37°C for 24 h to check for sterility. LFH was sterilized by washing it with 70% ethanol followed by turning on the ultraviolet (UV) light for 15 min. A sterile environment was maintained throughout the research to prevent contamination.

Sample inoculation
The collected samples were inoculated on EMB media to obtain the growth of E. coli on selective media. After streaking the inoculums, the plates were kept in an incubator for 24 h at 37°C to observe growth.

Biochemical tests for identification of E. coli
Biochemical tests were performed to identify E. coli strains. E. coli strains as shown in Table 1. E. coli strains are Gram-negative and give pink-colored colonies that are arranged either singly or in pairs [47]. They are lactose fermenters and catalase positive. For quick identification, "IMViC" tests were performed. These bacteria are indole and methyl red positive and Voges Proskauer and citrate utilization negative [48]. They do not produce H2S gas while other species of this genus produce gas using indole, glucose, mannitol, lysine, arabinose, xylose, and trihalose [49]. Thomas in 1988 carried out biochemical tests of E. coli species and found that most of the E. coli species ferment lactose; however, 10% of the species are late lactose fermenters while some of the species are non-lactose fermenters [50].
Detailed procedure PCR Amplification of Bacteria 16s rRNA Genes Directly from a Colony:

Materials required
PCR-grade water, Master mix, Forward and Reverse base primers, Template (E. coli colony), Falcons tubes, PCR tube rack.

Primer designing and dilution
Specific primer 3 was designed for 16s ribosomal RNA amplification. Primer dilution was necessary because primers are highly concentrated. To dilute, 10 μL of forward and reverse primers were mixed with 90 μL of PCR-grade water at a 1:10 dilution.

PCR mixture
To make the absolute mixture up to 20 μL, 13 μL of PCR-grade water was taken, and 4 μL of the master mix was added to it. Then, 1 μL of the forward base primers and 1ul of the reverse base primers were added to the PCR tubes. A colony (1 μL) from the EMB plate containing the E. coli culture was picked, and the 20 μL mixture was made. The PCR tubes were placed in a thermal cycler and run on the specific desired conditions required for denaturation, annealing, and amplification. After completion of the PCR, the PCR tubes were transferred to a freezer and kept at 4°C.

Materials required for gel electrophoresis
Agarose powder, TAE buffer, Ethidium bromide, Loading dye, DNA ladder, Gel casting tray and comb, Gel electrophoresis apparatus, UV transilluminator.

Gel preparation
We prepared a 1% of agarose gel by adding 1 g of agarose powder to 100 mL of 1X TAE buffer in a flask. The mixture was heated in a microwave until the agarose dissolved completely and then allowed to cool to 60°C. We added 1 μL of ethidium bromide to the gel mixture and poured the mixture into the gel casting tray with the comb inserted. The gel was allowed to solidify for 30 min at room temperature.

Sample loading
We mixed 5 μL of loading dye with 10 μL of the PCR product and loaded the mixture into the wells of the gel using a micropipette. We also loaded 5 μL of DNA ladder into one of the wells as a size reference.

Electrophoresis
The gel was run in the electrophoresis apparatus for 45 min at 100 V. After the run was complete, the gel was removed from the apparatus and placed on a UV transilluminator. The DNA bands were visualized by UV light and photographed using a gel documentation system.

E. coli culture
Sample of E. coli is cultured on MacConkey agar media which are obtained from soil (Fig. 1) and from the gut of chicken (Fig. 2) and stored at 4°C in the freezer. The cultured plates of E. coli are then processed for 16s r RNA extraction through colony PCR. Moreover, after the completion of PCR, we run it on agarose gel and then finally saw the bands on the Gel Doc picture (Fig. 3).

PCR results
After the completion of conventional PCR, the sample was then run on Gel to obtain the desired bands, and then finally see the bands on the Gel Doc picture.

Gel doc picture
The sample was loaded to wells on the gel, in well no 1 st 100bp DNA ladder was loaded to find the number of nucleotides as a positive control. After the completion of Gel electrophoresis, the Gel is kept in Gel Doc to see the bands on the Gel Doc picture (Fig. 3).

DISCUSSION
E. coli, a Gram-negative, facultative anaerobe, belongs to the Enterobacteriaceae family and is known to colonize a human infant's gastrointestinal tract a few hours after birth. While E. coli strains are less likely to cause intestinal illness, certain strains such as EPEC and enterohemorrhagic E. coli can cause diarrhea and hemolytic uremic syndrome.
Several techniques are used to detect pathogenic E. coli, including biochemical tests, PCR-based diagnosis, and 16s ribosomal RNA [40]. The most effective method for identifying specific DNA sequences is colony PCR, which involves picking a bacterial colony and using PCR to identify the desired sequence. The 16s ribosomal RNA sequence is frequently used as a marker for taxonomy categorization and phylogenetic study of microorganisms due to its highly conserved primer design and hypervariable region [39].
Shigella and EIEC share many characteristics and cause dysentery and invasive inflammatory colitis, respectively. The most prevalent pathogen, ExPEC, is responsible for 75-95% of instances of cystitis and pyelonephritis in the US [14].
In conclusion, identifying specific strains of E. coli is essential for treating and preventing the conditions caused by them. Colony PCR and 16s ribosomal RNA sequencing is effective techniques for identifying pathogenic E. coli and studying their taxonomy and phylogenetic characteristics.

CONCLUSION
This study demonstrates that 16s ribosomal RNA can be amplified directly from bacterial colonies using colony PCR without the need for extraction and purification of total genomic DNA. Our findings suggest that this technique can potentially be used for the direct identification of bacterial strains without studying the genetic protocol of DNA extraction and purification. We have successfully designed rRNA primers for E. coli species that can be amplified to evaluate various types of mutations, strain detection, and antibiotic resistance. The three-step quick and novel method for the amplification of E. coli 16SrRNA gene sequences can be a convenient technique for biomedical personnel to rapidly identify the E. coli variants. This study contributes to the development of a faster and simpler technique for bacterial identification and characterization.

ACKNOWLEDGMENT
We would like to thank Centre of Biotechnology and Microbiology, University of Peshawar for generous help in research.

CONFLICTS OF INTEREST STATEMENT
None.