Molecular confirmation of some virulence genes of E. coli isolated from lambs' colibacillosis

1. The objective of the study was to molecularly diagnose E. coli causing colibacillosis and investigate some of its virulence genes responsible for disease development in lambs within the region.
2. All isolates examined were confirmed as E. coli based on the amplification results of the uidA gene.
3. Colibacillosis is the most common and significant cause of diarrhea or dysentery in lambs in Mosul city.
4. EHEC (STEC) strains predominated in lambs in Mosul city, with Shiga toxin genes (stx1/stx2) as key virulence factors, while the uidA gene was effective for species confirmation.

Introduction

Neonatal diarrhea and enteritis are the most common gastrointestinal disorders in newborn lambs, especially in lambs within the first month of age. Weakness, dehydration, and frequent watery stools are their hallmarks. In severe and advanced cases, high mortality is more common (1). Colibacillosis is the most significant disease in the first few weeks of lambs' age, caused by pathogenic Escherichia coli; this leads to neonatal diarrhea and/or septicemia and, finally, a high mortality rate, particularly in conventional farms. It deforms growth, health disorders, and immunity, resulting in great economic losses due to cost and less effective treatment (2). Enteropathogenic E. coli (EPEC) and Enterohemorrhagic (EHEC) are the main types of E. coli that cause colibacillosis in lambs, kids, and other ruminants (3). Enterohemorrhagic strains produce highly potent Shiga toxins, so these types of E. coli are named Shiga toxin-producing E. coli (STEC). Enterohemorrhagic E. coli produces at least one kind of Shiga toxin, differentiated biochemically and molecularly and called Sxt1 and Sxt2 (3,4). STEC is the most prevalent bacterial population that can infect humans. Humans can contract STEC through fecal-oral routes by consuming tainted food or water, as ruminant animals are the pathogen's natural reservoirs (5). Small ruminants are a significant carrier of STEC for many hosts, including humans (6), as such, recognition of STEC in sheep is crucial due to their role in human infections. EHEC and EPEC strains can also both harbor the intimin gene (eaeA), which is one of the virulence factors implicated in attachment and biofilm production (7).

Because there has been little molecular research on colibacillosis and the molecular diagnosis of its causative agent in lambs in Iraq in general, and particularly in Mosul city, the aim of this study was the molecularly diagnose E. coli causing colibacillosis and investigate some of its virulence genes responsible for the production of disease in lambs within the region.

Materials and methods

Ethical approve

The Institutional Animal Care and Use Committee issued numbered UM.VET.2024.041 dated 9/7/2024 licensed this study.

Conventional diagnosis of isolates

In a prior investigation conducted by the same researchers (8), 65 E. coli isolates were derived from 67 rectal swabs of lambs from diverse localities that were obtained and analyzed for bacterial isolation. The isolated bacteria were identified by another study through their growth on some selective culture media, specifically MacConkey's broth, MacConkey's agar, and Eosin Methylene Blue. Then, the detection of E. coli was done depending on different biochemical tests, including Indole, Methyl Red, Voges-Proskauer, and Citrate (IMVC) (9). The diagnosed E. coli isolates were cultivated on the chromogenic agar (Hicrom^TM E. coli agar-Himedia, India) to differentiate Shiga toxin-producing from non-producing E. coli colonies by making greenish-blue colonies (according to manufacturing instructions).

DNA extraction

All isolates were cultured on EMB agar for 24 hours at 37°C, and then the bacterial DNA was extracted depending on the manufacturer's protocol of the extraction kit (AddBio/Korea). The extracted DNA was kept at -80°C, and used for subsequent steps.

Determining the DNA purity and concentration:

The concentration and purity of DNA of the isolates were analyzed by Implen NanoPhotometer (model N50, Germany). The purity of the DNA was evaluated at A260/A280 and A260/A230 (10-12).

Primers used in PCR

Four pairs of gene-specific primers (uidA, stx1, stx2, and eaeA) produced by Macrogen- Korea were used in the present study (13). The uidA primer pair was used for the molecular confirmation of E. coli isolates (14) with an amplicon size of 623bp. The three other primer pairs were used to differentiate between EPEC and EHEC (15,16) (Table 1).

Table 1: Sequences of primers used in the study

Amplification program

The amplification mixture consisted of master mix (12.5 µL Promega, USA), F-primer (10 pmol) (2.5 µL), R-primer (10 pmol) (2.5 µL), DNA template (5 µL), and PCR water (up to 25 µL). Amplification processes were done according to the specific program for each gene (Tables 2-4).

Table 2: Amplification program of the uidA gene

Table 3: Amplification program of the stx1 and stx2 genes

Table 4: Amplification program of the eaeA gene

Agarose gel electrophoresis

The agarose gel was prepared as described by Green & Sambrook (20). Briefly, 100 ml of 1X TBE (Promega, USA) was transferred into a beaker, and 2g of the agarose powder was mixed with the buffer and heated in a microwave oven for complete dissolution. The mixture was allowed to cool to 50-60°C, and 5 microliters of safe dye (AddBio) was added. The mixture was poured into the casting tray and the comb was placed at its place to make wells. The agarose solution was allowed to solidify at room temperature. Then, the comb was removed carefully, and the gel tray was put into an electrophoresis chamber, filled with 1X TBE buffer until the buffer level reached up to 3-5 mm above the surface of the gel. Afterward, a mixture of 3μl of amplified DNA (PCR product) and 9μl of Promega loading dye was loaded into all wells except one of them, which was loaded with a 100bp DNA marker (Promega, USA). Then, the agarose gel electrophoresis was run for 1 hour at 80V until the DNA bands were migrated, and finally, the bands were visualized and photographed by using a UV transilluminator.

Results

Bacterial isolates

Of the 65 pure E. coli isolates cultivated on HiCrome™ E. coli agar, 52 isolates (80%) exhibited a greenish-blue coloration, while 13 isolates (20%) displayed a white coloration (Figure 1).

Figure 1: Isolates of E. coli grown on chromogenic agar.

Concentration and purity of DNA samples

The concentrations of DNA measured for 65 samples ranged from 23.85 ng/µl to 241.60 ng/µl. The samples had a 260/280 absorbance ratio between 1.89- 2.087. Whereas at A260/A230, the samples ratio ranged from 1.92-2.086 (Figure 2).

Figure 2: Determination of DNA samples concentration and purity.

Confirmation of diarrheagenic E. coli in lambs using PCR

All 65 isolates were confirmed as E. coli by detection of the uidA gene using the PCR technique. The size of products was 623bp (Figure 3).

Figure 3: Confirmation of E. coli isolates by amplification of the uidA gene. Agarose gel electrophoresis (2%) confirms the E. coli isolates and shows bands of approximately 623 bp as the expected size of the uidA gene partially amplified by the PCR. M: 100 bp DNA marker, lanes 1-18 positive PCR products, and lane NC: negative control.

Detection of virulence genes

All E. coli isolates that were confirmed molecularly were tested to detect the presence of virulence genes in this study, which are stx1, stx2, and eaeA genes, using molecular techniques. Sixty E. coli isolates 92.3% bore the stx1 gene with a size of PCR products of 366 bp (Figure 4), and 47 (72.3%) isolates had the stx2 gene with a size of products of 282 bp (Figure 5). At the same time, 17 isolates (26.15%) showed the existence of the eaeA gene with a size of products of 629 bp (Figure 6). According to the above results, 64 out of 65 isolates of E. coli were EHEC 98.5% due to the presence of stx1 or stx2, or both of them, with or without the eaeA gene. In contrast, one isolate 1.5% lacked the three virulence factor genes (Table 5). According to the results, EPEC isolates did not exist in the current study (Table 5).

Figure 4: Confirmation of the presence of the stx1 gene. Agarose gel electrophoresis 2% confirms the E. coli isolates and shows bands of approximately 366bp as the expected size of the stx1 gene partially amplified by the PCR. M: 100 bp DNA marker, lanes 1-7, 9-10, 12-18 positive PCR products; 8, 11 negative PCR products, and lane NC: negative control.

Figure 5: Confirmation of the presence of the stx2 gene. Agarose gel electrophoresis 2% confirms the E. coli isolates and shows bands of approximately 282bp as the expected size of the stx1 gene partially amplified by the PCR. M: 100 bp DNA marker, lanes 4-7, 11-18 positive PCR products; 1-3, 8-10 negative PCR products, and lane NC: negative control.

Figure 6: Confirmation of the presence of the eaeA gene. Agarose gel electrophoresis 2% confirms the E. coli isolates and shows bands of approximately 629bp as the expected size of the stx1 gene partially amplified by the PCR. M: 100 bp DNA marker, lanes 6-7, 12, 17-18 positive PCR products; 1-5, 8-11, 13-16 negative PCR products, and lane NC: negative control.

Table 5: Classification of EHEC isolates according to the presence of the stx1, stx2, and eaeA genes

 X letter meaning the isolate bearing the gene.

Linking between molecular diagnosis and phenotypic diagnosis of EHEC (STEC) and EPEC isolates

The phenotypic results based on the growth on HiCrome™ E. coli agar did not relate to the molecular results, except in diagnosing the isolates as E. coli. The second clear benefit was the identification of isolates carrying the gene Stx2 alone or with other genes (Table 6).

Table 6: Linking between molecular diagnosis and phenotypic diagnosis of EHEC

Discussion

Diarrhea in lambs is caused by several diseases, which lead to great economic losses. Colibacillosis is the most common and important cause of dysentery or diarrhea, and it is a bacterial disease caused by pathogenic E. coli in newborn animals, mainly lambs, within 1-3 days after birth, and may also occur in older ages and around the time of weaning (20,21). The results of the existing study revealed that the extraction process was fantastic since concentrations of measured DNA samples were from 23.85 ng/µl to 241.60 ng/µl, with a range of 87.36 ng/µl, and consistent with the scientific articles, the concentration up to 20ng/μl is very satisfied for amplification (22). Kapa Biosystems (23) indicated that isolated DNA quantities ranging between 10 and 100 ng/μl are necessary for PCR investigation. The absorbance ratio A260/A280 for purity indicated that the samples had a ratio ranging from 1.89 to 2.087, classifying them as pure samples. Whereas the A260/A230 ratio of the samples varied from 1.92 to 2.086, indicating exceptional purity. In general, the results of DNA extraction are considered satisfactory if the purity value falls between 1.8 and 2.0, and the concentration exceeds 20 ng/μl (24,25). One of the first steps in DNA testing that is crucial to the effectiveness of PCR testing is extraction. This supports the assertion made by Fulton et al. (26) and Joshi et al. (27) that the success of PCR analysis depends on DNA extraction. Ibrahim (28) states that in order to move on to the amplification stage of PCR analysis, high-quality and high-quantity DNA extraction findings are required, because in the PCR amplification process, the enzyme functions slowly when contaminating substances like polysaccharides and polyphenols are present in the extracted DNA (29).

All isolates under investigation were confirmed as E. coli according to the amplification results of the uidA gene, which is a typical species-specific gene sequence and unique to E. coli, so it is the perfect gene for the diagnosis of E. coli (30,31). Numerous studies, local and universal, have relied on the uidA gene for molecular confirmation of E. coli isolates associated with various infections in animals, humans, and foodborne illnesses (30-32).

This work indicates that the distribution of virulence factors among E. coli isolates is intricate, with a notably high incidence of Shiga toxins, highlighting significant public health risks and potential economic losses. Depending on the current results, 92.3% of the isolates contained the stx1 gene, and 72.3% of the isolates exhibited the stx2 gene. At the same time, 26.15% of the isolates showed the existence of the eaeA gene. In contrast, 1.5% of isolates were negative to the three virulence genes. The presence of stx1, stx2 alone or together, and with or without the presence of eaeA, is the leading marker for virulence in STEC, which plays a crucial role in the occurrence of colibacillosis in lambs, so that 98.5% of the isolated E. coli in the present study were classified as EHEC isolates because any E. coli strain that is positive for stx1 and/or stx2 and for eaeA is referred to as EHEC (33,34). EHEC isolates are the main causes of colibacillosis in lambs (30-35). Concerning EPEC isolates, they did not exist in the current study.

Shiga toxins affirm the significant pathogenicity of E. coli, resulting in intestinal damage that causes diarrhea and enteritis in infected lambs. This injury mainly damages the endothelium of the blood vessels, leading to thrombosis and pathological activation of intravascular coagulation by preventing endothelial and other cells from synthesizing proteins (36-38). The two forms of Shiga toxins have different levels of toxicity and clinical effects. For example, in animals, Shiga toxin 2 was found to be about 100 times more toxic than Stx1. For instance, studies revealed that Stx2 is 40-400 times more effective than Stx1 in infected mice. This high potency is linked to Stx2's A1 subunit, which exhibits more ribosome attraction and catalysis than Stx1 (39,40). Thus, the existence of both virulence factors makes the strain more virulent.

 The eaeA gene encodes Intimin, which promotes attachment to enterocytes and produces attaching and effacing (A/E) lesions in the intestinal mucosa (7,41). Therefore, even though not all isolates harbored both Stx1 and Stx2 genes and the majority of them lost the eaeA gene, the data point to the high frequency of Shiga toxin-producing E. coli in lambs in Mosul city. Consequently, excessive attention should be paid to these high rates and their prevalence among lambs, which represent one of the most important food sources for humans in this city, in particular, and across the country in general. For instance, but not exclusively, to illustrate the significance of these STEC isolates' transmission through food sources, 4,769 people were impacted by 466 documented STEC outbreaks between 2010 and 2017, of which 3,353 cases were deemed foodborne in the US (42).

Compared to a previous study conducted in Nineveh Governorate (43), where they found that the stx1 and stx2 genes were present in all isolates, the present study showed that a high percentage 40% of the isolates carried both the Shiga toxin genes (stx1 and stx2), a large percentage of the isolates 26.15% expressed stx1 only, with a low prevalence of isolates 6.2% expressed stx2 gene alone. There are also differences between the results of the existing study and another local study that was carried out in Kirkuk province, where the stx1, stx2, and eaeA genes were identified in 64.28%, 67.85%, and 100% of the isolates, respectively (44). The findings of the current study revealed the prevalence of stx1 in the isolates, followed by stx2 and eaeA. The observed discrepancies are ascribed to sample variability; the current study focused on colibacillosis cases in lambs, whereas the previously mentioned local studies utilized human feces and food samples, and other research encompassed general diarrhea cases, as well as samples from sheep pens, workers' hands, and animal products.

 Numerous global studies (45-47) recorded the Shiga toxin and Intimin separately or together as virulence factors in the isolated E. coli strains. A study conducted in southern Brazil identified the eae gene and categorized the isolates as EPEC strains in 19.2% of the sheep investigated, with positivity observed exclusively in lambs (45). Another article in Argentina (46), after determining the presence of stx and eaeA, the isolated E. coli was classified as STEC and EPEC strains. EPEC carriers were detected in 50% of the farms evaluated, while STEC carriers were found in all of the farms. The risk associated with the farm-to-table food supply should not be ruled out. In Iran, according to a study, 4.48% of the isolates were EPEC, while 40.34% of the isolates belonged to STEC. The high incidence of STEC suggested that diarrheal lambs are a significant human reservoir (47). Furthermore, in a Turkish study 48.7% of the E. coli isolates from diarrheal lambs and goat kids were identified as Shiga toxigenic, 30.8% as enterotoxigenic, and 20.5% as enteropathogenic. These three pathotypes were found to be highly prevalent in the western region of Turkey (48). The cause for the discrepancy in the findings of the aforementioned research papers could be the age of the animal, the sample size and source, geographic and environmental conditions, and the local pathogen load (49-51).

The correlation between phenotypic identification on Chromo agar and molecular identification for differentiating STEC from EPEC isolates in the present study was inconclusive, as stx1 isolates exhibited both greenish-blue and white colonies. Consequently, the cultivation of E. coli isolates on Chromo agar has not definitively distinguished between Shiga toxin-producing and non-producing isolates. The only notable outcome was with stx2 isolates that appeared greenish blue colonies. The cultivation on chromo agar is unreliable for differentiating E. coli isolates from other Enterobacteriaceae, as 20% of isolates confirmed molecularly as E. coli, based on the presence of the uidA gene, manifested as white colonies. This phenomenon may be due to the lack of the β-D-glucuronidase (GUD) enzyme, which imparts a greenish-blue hue to colonies and is produced by 94-96% of E. coli strains (52).

Conclusions

In conclusion, colibacillosis is the most common and important cause of diarrhea or dysentery in lambs in Mosul city. The isolated bacteria are highly virulent, possessing virulence factors represented by the occurrence of the Shiga toxin genes in addition to the presence of Intimin as an adhesion factor in some isolates. This raises the likelihood that lambs serve as a reservoir for these zoonotic germs, which cause many syndromic disorders in people, particularly in youngsters. This study confirms colibacillosis as a major disease in lambs, primarily caused by pathogenic E. coli isolates carrying virulence genes. EHEC (STEC) strains dominated in lambs in Mosul city, with Shiga toxin genes (stx1/stx2) as key virulence factors, while the uidA gene proved ideal for species confirmation. The eaeA (intimin) gene contributed significantly to pathogenicity, though its absence in some isolates suggests alternative mechanisms.

Acknowledgment

The authors extend their sincere gratitude to the College of Veterinary Medicine, University of Mosul, for their invaluable support and resources that facilitated this study.

Conflicts of interest

Regarding the research data and instruments utilized in this work, the authors declare that there is no conflict of interest.