Molecular detection of some virulence genes, Shiga toxins and enterotoxin of E. coli and S. aureus isolated from dairy cows, workers and shared farm environments in Karbala Governorate-Iraq
Saleh Abed ALWahed1, Mahdi Kamil M AL-Jobori 2 ,Wafaa S. M. Mohsen3
1 Institute of Genetic Engineering and Biotechnology for Postgraduate Studies- University of Baghdad
2 College of Science, University of Karbala
*Corresponding author: [email protected]
Available from. http://dx.doi.org/10.21931/RB/2023.08.03.117
Staphylococcus aureus and E. coli may contain one or more genes that encode toxins, including the staphylococcal enterotoxins, Shiga toxins and shock syndrome toxins. These can interact with several cellular targets to produce diseases, such as food poisoning and toxic shock syndrome. This study was designed to characterize the prevalence of Shiga toxins-producing E. coli (STEC) and enterotoxins-producing S. aureus among cow farms, worker farms and shared environments. Five dairy farms were recruited in the Karbala governorate. After gaining the owner's permission to visit the farms, A total of 400 samples were collected from cows (1 nose and 1 teat sample from each cow and 10 cows from each farm), ( 1 nose and 1 hand sample from each worker and ten workers from each farm), (one sample of milk from each cow and ten cows from each farm) and 30 environment samples per farm (10 swabs from milking tools, 10 swabs from the feeding place and 10 swabs from the cows' shelter). Sampling took place between June 2021 to October 2021. Farm workers were invited to volunteer to participate in the survey and sampling. The molecular profiling of E. coli isolates showed negative results for the presence of eaeA, stx1 and stx2 genes.10% (4 out of 40) of S. aureus isolates exhibited favorable PCR products for femA gene and the remaining 36 as femA negative. However, none of the S. aureus isolates were positive for SE production (sea and seb). In conclusion, the risk for severe human infections is low due to the loss of virulence, Shiga and enterotoxins-associated genes in E. coli and S. aureus adapted to livestock.
Keyword: Shiga toxins, Enterotoxins, eaeA, femA, Cows, Human, Environment.
Staphylococcus aureus and E. coli are major causative pathogens that threaten farmers; they are commonly found in environmental settings, such as bedding, clothes, farmers' hands, and water used on farms. It ubiquitous bacterial organisms that are found in a wide variety of places, including the human intestine, where they can lead to diarrheal disease and a range of extraintestinal infections 1 . Developing countries are affected mainly by foodborne infections, numerous epidemiological reports have implicated raw milk is usually colonized by a variety of zoonotic foodborne pathogens.These pathogens have originated from the environment on the farm, mixing clean milk with mastitis milk, manure, soil, and contaminated water. Therefore, food-producing animals are primary sources of most foodborne pathogens 2,3
The significant virulence genes of STEC are the stx genes encoding the Shiga toxins and the eaeA gene encoding the intimin protein, which is responsible for adhesion. This protein is essential for the tight binding of bacteria to target cells and is encoded on a chromosomal pathogenicity island termed the locus for enterocyte effacement (LEE). The LEE has an attaching and effacing (A/E) lesion, which allows the binding of the toxins and, hence, results in an infection. The vast majority of virulence factors are encoded in mobile elements of the DNA: pathogenicity islands, transposons, plasmids and phages (Martin, 2017). The mechanism of disease cause, E. coli, is divided into six groups of pathotypes. Enteropathogenic E. coli (EPEC), Attaching and effacing E. coli (A/EEC), Enterotoxigenic E. coli (ETEC), Enteroinvasive E. coli (EIEC), EHEC and Enteroaggregative E. coli (EAEC). E. coli strains that produce the Stx toxins have been referred to as Vero Toxin-producing E. coli (VTEC), Shiga-toxigenic E. coli (STEC) and enterohaemorrhagic E. coli (EHEC) 4. The events of pathogenesis can be summarized as follows: 5 (i) the colonization of the gut,( ii) the effect of the virulence factors on the host and (iii) disease caused by the virulence factors. STEC contamination is not limited to cattle products, but there has been a higher frequency of cases involving beef or veal 5. Ruminants, especially cattle and sheep, unspecified meat and sheep meat, young cattle and unpasteurized milk from cattle have constituted a vast range of STEC. It is not surprising that humans most frequently become infected with STEC by ingesting contaminated food or water or by direct contact with animals, resulting in sporadic disease or outbreaks involving up to several thousand individuals. The polymerase chain reaction assay should aid in quickly detecting this virulent serotype and help curb the severe epidemic of human diseases associated with STEC infections 6.
Various virulence factors work together in the pathogenic process of S. aureus. The broad range of infections by S. aureus is related to several virulence factors that allow it to adhere to the surface, invade or avoid the immune system, and cause harmful toxic effects to the host 7. The femA and femB genes encode proteins that influence the methicillin resistance level. Both are involved in the formation of the cell wall. The femA gene is involved in the glycine content of peptidoglycan and peptidoglycan biosynthesis of S. aureus. It mediates the effect on drug sensitivity, and thus, it is involved in methicillin resistance 8. There are nine major antigenic types of staphylococcal enterotoxins (SEs) have been reported (SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI, and SEJ), while only one serotype of TSST (comprised of TSST-1 and TSSTovine) has been characterized 9. This study aimed to detect the Shigathe toxin of E. coli and enterotoxin of S. aureus on cows, humans and shared environments using PCR
MATERIALS AND METHODS
Five dairy farms were recruited in the Karbala governorate. After gaining the owner's permission to visit the farms, samples were collected from cows (1 nose and 1 teat sample from each cow and ten cows from each farm), ( 1 nose and 1 hand sample from each worker and ten workers from each farm), (one sample of milk from each cow and ten cows from each farm) and 30 environment samples per farm (10 swabs from milking tools, 10 swabs from the feeding place and 10 swabs from the cows' shelter). Sampling took place between June 2021 to October 2021. Farm workers were invited to volunteer to participate in the survey and sampling.
In other research, we isolated and identified E. coli and S. aureus isolates based on cultural and biochemical criteria and confirmed by Vitek-2-system and molecular detection of the 16SrRNA gene for each bacteria. The obtained results were 33 E. coli and 40 S. aureus from 400 samples collected.
Polymerase chain reaction (PCR) was performed to amplify target genes using primers for the following genes: eaeA, Stx1 and Stx2 for E. coli, and femA, Sea and Seb for S. aureus. The sequences of these primers are listed in Table 1.
DNA extraction of bacterial genome
Chromosomal DNA of the overnight broth culture of E. coli and S. aureus isolates was obtained according to the protocol of ABIOpure Total DNA Kit (ABIOpure, USA); the procedure was explained in detail in the user's manual. The extracted DNA was validated by Nanodrop and stored at −20°C in the refrigerator until further usage. After PCR amplification, agarose gel electrophoresis was adopted to confirm the presence of amplification. PCR was utterly dependable on the extracted DNA criteria 10.
Table 1. Sequences of primers used in the conventional PCR.
Molecular detection of Shiga toxin and eaeA gene in E. coli isolates
The PCR technique was used for molecular profiling of E. coli isolates through amplifying Shiga toxin-encoding genes stx1, stx2, and the intimin-encoding gene (even). The PCR reaction was performed using primers listed in (Table 1) in a Thermal Cycler (Thermo Fisher Scientific, USA). Approximately 2 ng of bacterial DNA was added to 10 μl Master Mix (10X), 1 μl of each primer and the final volume was adjusted to 20 μl by 6 µl adding Nuclease Free Water. The amplification conditions started with initial denaturation for 3 min at 95°C followed by 30 cycles of 95°C for 3 min, 63.4, 62.5 and 58°C for 40 s, and 72°C for 2 min. The final cycle was followed by a 72°C final extension for 5 min. The amplified DNA fragments were separated by 1.5% agarose gel electrophoresis (Optima, Japan) in 1x TBE buffer and captured and visualized on a UV transilluminator. A 100 bp plus DNA Ladder was used to determine each amplicon size.
Molecular detection of enterotoxin and femA gene in S. aureus
The presence of enterotoxins (sea and seb ) and femA gene amplification were detected using PCR. The PCR mixture reactions were performed for E. coli. The amplification conditions started with initial denaturation for 5 min at 95°C followed by 30 cycles of 95°C for 3 min, 58, 58 and 55°C for 3 min, and 72°C for 3 min. The final cycle was followed by a 72°C final extension for 7 min. Sequences of the used primers are listed in (Table 1). The amplified DNA fragments were separated by 1.5% agarose gel electrophoresis (Optima, Japan) in 1x TBE buffer, captured, and visualized on a UV transilluminator. A 100 bp plus DNA Ladder was used to determine each amplicon size.
Detection of eaeA and Shiga toxin (stx1, stx2) genes
The polymerase chain reaction assay should aid quick detection of the virulent serotype and help suppress the severe epidemic of human diseases associated with STEC infections. It is well known that STEC is a zoonotic food and water-borne pathogen associated with diarrhea and renal failure, particularly in children, and can cause attaching and effacing properties in diarrhea cases 11. Also, previous reports revealed that virulence genes were detected in only a few E. coli strains, which may be due to the fact that there are occasional strains that have the genes but do not express the toxins 15. On the other hand, many studies reveal that cattle have been recognized as the main reservoir of STEC strains 16
Figure 1.The amplification of eaeA, stx1 and stx2 genes of E. coli were fractionated on 1.5% agarose gel electrophoresis stained with Eth. Br. M: 100bp ladder marker.
Table 2. Detection of 16SrRNA and some virulence genes in E. coli isolated from five areas in Karbala.
Detection femA and enterotoxins (Sea, Seb) genes
The femA gene, which encodes a protein precursor, is involved in peptidoglycan biosynthesis and has been used as a molecular marker for identifying S. aureus (Jukes et al., 2010). The amplified DNA (98-bp) region of femA gene could be identified through Agarose gel electrophoresis with a suitable 100bp ladder DNA marker (Figure 2). 10% (4 out of 40) of S. aureus isolates exhibited favorable PCR products for femA gene and the remaining 36 as femA negative (Table 3), which disagreed with the results of 19 who found that the femA (147) and femB (138)genes were the most frequent in 148 samples of S. spp samples.
Figure 2. The amplification of femA gene were fractionated on 1.5% agarose gel electrophoresis stained with Eth. Br. M: 100bp ladder marker.
Table 3. Detection of 16SrRNA and some virulence genes in S. aureus isolated from five areas in Karbala.
Staphylococcus aureus isolates can produce enterotoxins, posing a public health threat. This means that the detection of Enterotoxins is very crucial 21. In the current study, the molecular detection of sea and seb genes was beneficial for properly characterizing Enterotoxins-producing S. aureus. PCR was used to screen the existence of the enterotoxins (sea and seb) genes in 40 S. aureus isolates (Figure 3). None of the 40 S. aureus isolates were positive for SE production (sea and seb) (Table 3).
Figure 3. The amplification of sea and seb genes was fractionated on 1.5% agarose gel electrophoresis stained with Eth. Br. M: 100bp ladder marker.
Several primers that amplify these genes have previously been reported, including the ones used in this study 11,12. The molecular profiling of E. coli isolates showed negative results for the presence of eaeA, stx1 and stx2 genes (Figure 1). These results were correlated with previous studies, which showed that eaeA-positive E. coli was not found in clinical mastitis cases 13, and with the results obtained by 14 none of the isolates had stx2 shown by PCR. Numerous investigators have underlined the strong association between the carriage of eaeA gene and the capacity of STEC strains to cause severe human illnesses.
This study demonstrated the absence of pathogenic E. coli in cows, workers and environmental sources by detecting the virulence genes associated with the pathogenic (Table 2). These results imply that the presence of drug-resistant strains of non-pathogenic E. coli isolates from the environment is possible. This can threaten management programs for farm 17 since one study also reported that non-pathogenic E. coli can serve as a reservoir of antibiotic resistance genes and could transfer the genes to other pathogenic E. coli if conditions are suitable 18.
The absence of femA and femB is related to glycine reduction in the peptidoglycan, thus making the cell wall more susceptible to beta-lactams 20.
The risk for acute human infections is low due to the loss of virulence-associated genes and adaptation of S. aureus to livestock 21. These results agree with the previous result of other authors 22, who showed that sea and seb genes were not detected in all isolates. However, disagree with the results of 23 who reported that Of all 77 staphylococcus enterotoxin (SE)-positive isolates, more than 90% could produce enterotoxins, and 24 when used commercial test kits (SET-RPLA ) showed that 21.4% (3 out of 14 isolates) of S. aureus isolates produced classic enterotoxins (sea, sec, sed). The findings of this study indicate that all isolates isolated from the five study areas do not produce enterotoxins.
The present study uses molecular tools to provide new information on the genotypic traits of E. coli and S. aureus isolates from cow farms, workers and shared environments. The risk for severe human infections is low due to the loss of virulence, Shiga and enterotoxins-associated genes in E. coli and S. aureus adapted to livestock. In the future, surveillance studies using large sample size should be conducted to make the findings robust.
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Received: 25 June 2023/ Accepted: 26 August 2023 / Published:15 September 2023
Citation: ALWahed, S.A.; AL-Jobori, M.K.M.; Mohsen, WSM Molecular detection of some virulence genes, Shiga toxins and enterotoxin of E. coli and S. aureus isolated from dairy cows, workers and shared farm environments in Karbala Governorate-Ira. Revis Bionatura 2023;8 (3) 117 http://dx.doi.org/10.21931/RB/2023.08.03.117