Molecular characterization and phylogenetic analysis tree of Shiga toxin-producing Escherichia coli (STEC) from local Basturma in Mosul city, Iraq

1. Isolation and identification of Escherichia coli from Basturma using Classical methods.
2. Detection some of genes encoding virulence factors in Escherichia coli.
3. Find the relationship among Escherichia coli isolates using phylogenic tree.

Introduction

Escherichia coli (E. coli) stands out as the predominant microorganism transmitted through food, causing various diseases that can result in human fatalities (1). E. coli contains a range of virulence factors, including genes like uidA, Shiga toxin 1 and 2 (Stx1, Stx2) and Rfb (2). The uidA gene in E. coli encodes β-glucuronidase enzyme which plays an important role for hydrolyzes glucuronides into glucuronic acid, while Shiga toxin-producing Escherichia coli (STEC), is known as a minor global problem worry among foodborne pathogens associated with meat, this is due to reported outbreaks and isolated instances of STEC infections occurring worldwide (3). STEC has the potential to induce various health issues, including difference types of diarrheas, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, and renal failure (4). E. coli O157:H7, which produces Shiga toxin, is a significant strain of E. coli that can cause a range of diseases in both humans being and animals (5). E. coli O157:H7 has the specific-species Rfb gene and E. coli O157:H7 can synthesis the Stx1 and Stx2 toxins, which are responsible for causing the food poisoning in consumers (6). Beef meat products have been recognized as the primary sources in the popular of reported foodborne disease outbreaks associated with E. coli O157:H7 (7). The meat contained the pathogenic bacteria can exist at any stage of the food chain, spanning from the slaughtering process to the consumer (8). While healthy animals and its products are generally regarded as sterile under typical conditions, certain procedures conducted during the processing in the slaughterhouse that have the potential to introduce microbial contamination, these processes may lead to contaminate meats and instruments with pathogenic bacteria (9). In addition, Beef meat products may be exposed to contamination of STEC during the procedure of synthesis and becomes the reservoir of STEC which causes the risks to human (10). A common technique for attributing the human disease burden caused by food-borne illness to particular origins is the "microbiological approach" this method entails isolating the pathogen from both the food and infected humans (11). The safety of raw cured fermented sausages relies on several factors, including the incorporation of salt and nitrite, the process of drying that effect on the amplification of bacteria on the meat, and stored at a cold or moderately low temperature for a specified period, allowing for the desired curing effect (12). The acknowledgment of dry fermented sausages as a probable hazard to food care has prompted certain lands to implement regulations aimed at reducing associated risks (13). Many studies emphasize the importance of fermented meats as a notable source of disease occurrences (14). Basturma, an Iraqi meat product, is extensively produced in various regions of the country, with notable production in Mosul city, located in the Nineveh Governorate. This product shares similarities with Turkish dry-fermented sausage the heavily smoked Spanish dry fermented sausage. These products, whether partially dried or smoked, are recommended for consumption only after undergoing heat processing, such as frying or grilling (15). Numerous researchers have redirected their focus towards the utilization of additives, such as acidulants and other substances, in the processing of meat trimmings intended for ground meat. This approach serves as a method to prevent contamination of the final ground meat product (16).

The goals of this study included isolating and identifying E. coli from Basturma, to emphasize the specific risks linked to raw cured fermented meats and other primary product kinds., and to detect the presence of the Stx1 and Stx2 genes, which causing food poisoning using the molecular biology technique.

Materials and methods

Ethical approval

The study was conducted in accordance with the ethical standards set by the Institutional Animal Care and Use Committee at the University of Mosul, College of Veterinary Medicine. All samples were collected with the owners' consent under the approved ID: UM.Vet.2024.066.

Samples collection

The primary focus of our research centered on the city of Mosul in Iraq, encompassing various neighborhoods in Mosul city. Over the period spanning from April 2023 to June 2023. A total of 45 samples of locally produced Basturma collected from a range of local stores, including 8 samples from each region (First Qadsia, Ras Al-Jada, Bab Al-Tob, and Al-Zahra), and 5 samples from Al-Tahrer area). These Basturma were meticulously gathered in sterile containers and subsequently placed in containers containing peptone water. Following this, a pre-enrichment process was carried out at 37°C for 18-24 h to prepare samples for analysis. Subsequently, these samples were carefully transported in an icebox to the Research Center and Laboratories at Mosul University.

Isolation and identification E. coli

The Basturma samples underwent a rigorous analysis for isolating and identifying of E. coli. All samples were cultured for 24 hours at 37°C in nutritional broth that was purchased from LAB (United Kingdom). A tiny amount of nutrient broth was then placed over EMB agar and MacConkey agar, which were also obtained from LAB (UK), in accordance with the conventional laboratory methods. These agar plates were incubated for 24 h at 37°C to facilitate growth and identification. Additionally, Oxoid's (UK)Brilliance E. coli/coliform Agar was used to distinguish between coliform and generic E. coli bacteria. Numerous biochemical studies provided additional confirmation of the probable E. coli isolates. In order to preserve the E. coli isolates, they were stored at -80ºC in Nutrient broth that contained 15% glycerol until they were required for additional analysis.

DNA Isolation

The following careful procedures were carried out in order to extract and analyze the potentially hazardous E. coli isolates. The samples were first grown on Brilliance E. coli/coliform medium and incubated at 37°C for 24 hours. The purpose of this specific agar medium is to encourage the growth and differentiation of E. coli and coliform bacteria. Performing the extraction process as directed, we isolated DNA from the E. coli isolates using the Qiagen (Germany) DNeasy Blood and Tissue Kit. The Nanodrop (Jenway, UK) instrument was subsequently employed for measuring the isolated DNA, providing an accurate measurement of the DNA content. The E. coli DNA was measured, isolated, and then delicately stored at -20°C in order to maintain its stability and cleanliness for later research. The DNA is protected in this condition of storage and is readily accessible for additional study and experimentation.

Reaction of PCR

Table 1 displays how particular sequences of the E. coli genes uidA, Stx1, Stx2, and rfb have been produced employing the polymerase chain reaction (PCR) technique. A PCR reaction totaling 25 μl was prepared. The reaction mixture comprised 12.5 μl of Promega Corporation's (USA) 2× GoTaq Green Mix Master, 1 μl each of primers 1 and 2, 6.5 μl of Promega Corporation's (USA) DNeasy-free water, and 4 μl of the E. coli DNA template. A 1.5% agarose gel from Peqlab (Erlangen, Germany) was prepared for gel electrophoresis, and the amplicons were placed into the wells together with a 100 bp ladder DNA marker. The amplified DNA fragments have been divided and seen using electrophoresis, and their sizes determined via comparing them to the DNA ladder. Under suitable heat cycling conditions, the PCR amplification was carried out. These parameters, which include denaturation, annealing, and extension temperatures and durations, were tuned for the primer set and DNA template under investigation and were customized to the PCR method being employed.

DNA sequencing

To purify and sequence six PCR amplicons derived from isolates of Basturma, each previously identified as E. coli-positive through classical PCR, the samples were outsourced to Macrogen, a commercial sequencing company based in South Korea. The target gene for sequencing was uidA. Subsequently, the obtained the uidA gene sequences were subjected to a comparative analysis against previously published E. coli sequences available on GenBank, utilizing the NCBI BLASTn tool accessible at http://www.ncbi.nlm.nih.gov. The alignment and comparison of these sequences were further examined through online multiple sequence alignment using CLUSTALW from GenomeNet [available at].

For the construction of phylogenetic trees, the same CLUSTALW tool from GenomeNet, in conjunction with the Neighbor-Joining (NJ) program, was employed. The uidA gene sequences from E. coli served as an outgroup in the phylogenetic tree construction, involving 500 replicates to enhance robustness. This comprehensive approach aimed to elucidate the genetic relationships among the E. coli isolates from Basturma by leveraging purification, sequencing, and subsequent bioinformatics analyses, ultimately contributing to a better understanding of the phylogenetic context of these isolates.

Table 1: The sequence Primers and PCR program used for detecting of the uidA, Stx1, Stx2 and rfb gene

PCR program: I=35 times (94°C – 30s, 57°C – 30s, 72°C – 30s), II=35 times (94°C – 60s, 55°C – 60s, 72°C – 60s), III=35 times (94°C – 60s, 62°C – 60s, 72°C – 60s), IV=35 times (94°C – 60s, 52°C – 60s, 72°C – 60s)

Results

The classical microbiology methods for local Basturma found that 6/45 (13.4%) of isolates was E. coli. As indicated by the table 2, there was no E. coli isolated from First Qadsia and Al-Noor. The percentage rate of E. coli recovered from Ras Al-Jada and Bab Al-Tob was 25% (2/8), from Al-Zahra it was 12.5% (1/8), and from Al-Tahrer it was 20% (1/5) (Table 2). All E. coli isolate was confirmed by using the PCR assay which appeared that all E. coli isolates have the uidA gene (100%) which have the molecular weight 623 bp (Figure 1 and Table 3). The PCR technique revealed that 1/6 (16.7%) of isolates possessed the Stx1 gene (Figure 2 and Table 3). Not one of the isolates of E. coli carried the Stx2 and rfb genes (Figure 3).

Table 2: The number of samples and percentage of positive E. coli isolates

Table 3: The percentage of the uid A, Stx1, Stx2, and rfb genes in E. coli isolates

The sequencing results, individual sequencing analysis (BLASTn) was performed on five novel uidA gene sequences, all of which were obtained from Basturma. The sequences of E. coli listed in the NCBI Genbank are indexed below the subsequent accession numbers (PP332803, PP332803, PP332805, PP332806, and PP332807) as shown as in the table 4. In addition, this study revealed that the individual sequencing analysis (BLASTn) for five sequences of the uidA gene comprises: every item taken from Basturma. Using the maximum likelihood approach in MEGA11 software, the phylogenetic tree analysis showed that the local sequences of E. coli isolated from Ras Al-Jada (PP332803) and Bab Al-Tob areas (PP332806) was closely related (100% identity), while, the local sequences of E. coli isolated from Ras Al-Jada and Bab Al-Tob areas (PP332805) was closely related (99% identity), the local sequences of E. coli isolated from Al-Tahrer areas was closely related with Ras Al-Jada and Bab Al-Tob areas (PP332805) (74% identity) (Figure 5).

Additionally, local uidA gene sequences showed a distinct relationship from those obtained from the GenBank database that were previously reported, based on a phylogenetic tree analysis utilizing the maximum likelihood technique in MegAlign software. The close relationship between our sequence types Seq E. coli-1, PP332804.1, PP332806, and PP332807 and the sequence type AP023205. 1 China, CP040269.1 Norway, and CP054564.1 USA was 99%. In addition, the sequence types PP332803 and PP332805 showed similarity with CP019961. 1 Hong Kong, and CP057580. 1 UK was 96.3% (Figures 6 and 7).

Figure 1: The molecular weight of the uidA gene with molecular weight 623 bp based on Agarose Gel Electrophoresis (2%). The amplification of DNA appears as a ladder-like pattern. Lanes M are DNA Marker 100 bp ladder (Biozym Diagnostic), Lane 1 is the positive control, and Lanes 2 – 6 represent positive isolates, and Lane 7 is the negative control.

Figure 2: The molecular weight of the Stx1 gene with molecular weight 347 bp based on Agarose Gel Electrophoresis (2%). The amplification of DNA appears as a ladder-like pattern. Lanes M are DNA Marker 100 bp ladder (Biozym Diagnostic), Lane 1 is the positive control, and Lanes 2 – 6 represent positive isolates, and Lane 7 is the negative control.

Figure 3: The molecular weight of the Stx2 gene with molecular weight 779 bp based on Agarose Gel Electrophoresis (2%). The amplification of DNA appears as a ladder-like pattern. Lanes M are DNA Marker 100 bp ladder (Biozym Diagnostic), Lane 1 is the positive control, and Lanes 2 – 6 represent negative isolates, and Lane 7 is the negative control.

Figure 4: The perfect amplicon of the rfb gene with molecular weight 259 bp. The molecular weight of the Rfb gene with molecular weight 259 bp based on Agarose Gel Electrophoresis (2%). The amplification of DNA appears as a ladder-like pattern. Lanes M are DNA Marker 100 bp ladder (Biozym Diagnostic), Lane 1 is the positive control, and Lanes 2 - 6 represent negative isolates, and Lane 7 is the negative control

Table 4: The NCBI GenBank accession numbers for the E. coli sequences in Basturma

Figure 5: Phylogenetic tree of the partial sequence of the uidA gene of the local sequences of the E. coli reported in the NCBI GenBank, The numbers at the branches indicate bootstrap supports (500 replications)

Figure 6: Clustering analysis of the uidA gene sequence of E. coli and other uidA sequences of E. coli isolates retrived from NCBI GenBank.

Table 7: uidA gene sequence similarity and divergencea^a of each pair for E. coli calculated by DNASTAR. a percentage divergence is computed by contrasting sequence pairs with the tree that MegAlign DNASTAR has generated. Sequences are directly compared using percent similarity, which ignores evolutionary relationships.

Discussion

Food is a medium via which many pathogenic bacteria can spread; E. coli is a major foodborne microorganism that cause food poisoning in the globe (21). Numerous investigations have found that meat and its products had greater levels of E. coli contamination (22). Butcher shops are a hub for E. coli contamination of meat and its products via worker hands and utensils, according to a prior study conducted in Iraq (23). Basturma, an Iraqi meat product is regarded one of most famous food which ate in Winter season in various areas of Iraq, especially in Mosul city (Nineveh governorate). In this investigation, none of isolates was E. coli O:157, H7, the percentage of E. coli in Basturma was 13.4% due to exposed the meat to several stage of contamination started from farm passed through the slaughterhouse ended in the supermarket (24). Different cultures or geographical features of various systems for feeding, variances when preparing meat, and changes in the techniques employed in the microbiological testing could all contribute to varying contamination rates of E. coli in meats and its products (25). When preparing Basturma, added spicy, garlic, and salt to meat that lead to lowers the pH of the flesh which causes inhibiting the formation and proliferation of bacteria in meat (26). During storage, the moisture content of Basturma dropped, which also caused an E. coli fall (27). The majority of people in underdeveloped nations choose to purchase inexpensive meats from unofficial marketplaces, which do not follow safety regulations or hygienic guidelines for storing meat (28, 29).

Furthermore, this study showed that only one strain of E. coli has the Stx1 gene (16.7%) and lacks the Stx2 gene. Previous studies' findings showed that the Stx1 gene was more commonly detected in E. coli than the Stx2 gene (30). Numerous investigations found that the Stx1 gene produced findings that were greater than ours results; in Spain, the E. coli possessed the Stx1 gene were 29% (31) and in Namibia, 40.68% (30). Simultaneously, the Stx1 and Stx2 genes failed to identify any E. coli isolated from Vietnamese meat and seafood (32). Furthermore, a close relationship between E. coli isolated from Ras Al-Jada and Bab Al-Tob areas was observed in the phylogenetic tree of the uidA gene sequences in E. coli isolated from Mosul city. This relationship could be explained by the animals being raised on the same farm and exposed to E. coli infection, or by the animals contracting the infection during the processing of carcasses in the abattoir such as cutting equipment and tools, an unclean atmosphere, and a decline in worker hygiene (33). Genomic sequencing analyses were carried out to verify the isolates' identities even more. The findings demonstrated a high degree of sequence similarity range from 96.3% to 99% between the sequence of this study with the sequence of E. coli isolated in world. It has been established that these molecular targets are effective tools for E. coli genotyping (34-38). The study's high similarity findings supported the identification of the samples as E. coli species. This thorough genetic research supported the finding that PCR was an effective method for characterizing E. coli molecules at the molecular level.

Conclusion

The primary food-borne pathogen responsible for producing the Shiga toxins is E. coli that cause several problems in digestion system food poisoning in humans. Meat products can contaminate via E. coli. The tools that butchers utilize (knives, hooks, tables, machinery, and employee hands) are crucial in the contamination and spread of E. coli to meats. Basturma is a one types of meat products which may be contaminate with E. coli or other foodborne pathogens. Furthermore, this work provides a valuable molecular diagnostic tool to identify and character of E. coli isolates, providing insight into the possible implications of this species in companion animals and facilitating larger-scale study.

Acknowledgment

I want to express my profound appreciation to the University of Mosul's College of Veterinary Medicine for all of their help and support during this research. This research was completed successfully thanks in large part to the college's facilities, resources, and advice.

Conflict of interest

The authors of this manuscript have declared that no conflicts of interest arose during the writing or data analysis phases