Virulent shiga toxin-producing Escherichia coli (STEC) O157:H7 ST11 isolated from ground beef in Brazil

Abstract

In this study, a total of 248 ground beef samples were analyzed for the presence of Shiga toxin-producing Escherichia coli (STEC). Out of these samples, only one (0.4%) tested positive for STEC. Further analysis using PCR confirmed the presence of all tested genes associated with STEC, including stx1, stx2, eae, ehx, uid, rfbO157, and fliCH7 in this isolate. Interestingly, no STEC strains were detected in the remaining 100 beef cut samples or the 100 chicken cut samples, indicating the absence of detectable STEC contamination in those specific samples. The isolated strain exhibited significant cytotoxic activity in Vero cells, indicating its ability to produce cytotoxic Shiga toxins. To further investigate the strain, whole-genome sequencing (WGS) analyses were performed. The resistome analysis revealed the absence of acquired antimicrobial resistance genes, indicating a pan-susceptible phenotype. However, this strain presented chromosomal mutations in gyrA, gyrB, parC, parE, pmrA, pmrB, and folP. Plasmid analysis identified the presence of two plasmids, namely IncFIB(AP001918) and IncFII. The multi-locus sequence typing (MLST) identified the strain as belonging to sequence type (ST) 11, which is associated with E. coli O157:H7 strains. The virulome analysis confirmed the presence of several canonical virulence markers, including stx1, stx2, eae-g01-gamma, ehxA, stx1a-O157, and stx2a-O157. Overall, this study identified for the first time a rare occurrence of STEC contamination in ground beef, with the isolated strain belonging to the highly virulent O157:H7 serotype. These findings contribute to our understanding of STEC prevalence and characteristics in food samples, highlighting the importance of effective food safety measures to prevent potential health risks associated with STEC contamination.

Introduction

Shiga toxin-producing Escherichia coli (STEC) is responsible for causing a spectrum of gastrointestinal illnesses. Initial symptoms may include mild to severe diarrhea, which can progress to hemorrhagic colitis characterized by bloody stools. In some cases, the infection can further escalate and lead to a severe complication known as hemolytic uremic syndrome (HUS), which is a life-threatening condition [1]. HUS is characterized by the destruction of red blood cells, kidney damage, and potentially other organ complications. It is important to seek medical attention promptly if symptoms of STEC infection, such as diarrhea and bloody stools, are observed, particularly in cases where HUS is suspected [2, 3]. HUS is largely a pediatric illness but occasionally can affect adults at any age as observed during a German outbreak [4].

Shiga toxins encoded by stx₁and stx₂ genes are the main virulence factors, responsible for cytotoxins that inhibit host cells protein synthesis [2, 5]. Other factors such as intimin, mediated by the eae gene [2, 5] located in the locus of enterocyte effacement– LEE [6], and enterohemolysin (ehxA), encoded by a gene in a 60 Mda plasmid, are of great importance. Ruminants, especially cattle, are recognized as the major reservoir of STEC serogroups [7]. Indeed, cases or outbreaks of STEC infections are commonly associated with the consumption of meat and meat products that have been contaminated by animal feces. However, it is important to note that other types of food have also been implicated in STEC outbreaks, especially vegetables, as highlighted by the FAO/WHO [8]. Additionally, incidents involving the consumption of soy nut butter contaminated with STEC were reported [9]. These examples highlight the importance of practicing food safety measures and proper hygiene when handling and preparing various food items to minimize the risk of STEC infections.

Proper hygiene measures during slaughter and subsequent processing steps are crucial to prevent the contamination of bovine and poultry products by STEC. While there are several reports available on the occurrence of STEC in raw bovine meat, such as the study conducted by White et al. [10], relatively little is known about its occurrence in poultry meat. However, a few studies have shed some light on the subject. For instance, Chinen et al. [11] and Doregiraee et al. [12] have explored the presence of STEC in poultry meat. These studies contribute to our understanding of the potential contamination sources and emphasize the importance of implementing stringent hygiene practices throughout the entire production chain to minimize the risk of STEC transmission through meat products.

Brazil holds the distinction of being the largest meat exporter in the world [13]. However, the available records regarding the occurrence of STEC in meat and meat products produced in the country are not extensive. Some studies, such as those conducted by Bergamini et al. [14], Cerqueira et al. [15], and Rodolpho and Marin [16], have provided insights into this matter. However, the information remains limited. It is worth noting that to the best of our knowledge, there are no specific reports on the occurrence of STEC in chicken cuts in Brazil. In contrast, Argentina has experienced the highest incidence of hemolytic uremic syndrome (HUS) worldwide due to STEC infections [17]. While a few cases of HUS associated with STEC have been reported in Brazil [18, 19], the prevalence appears to be relatively low compared to Argentina. These observations emphasize the need for further research and surveillance to better understand the occurrence and epidemiology of STEC in meat products, particularly in Brazil.

Given the limited availability of data regarding the presence of STEC in Brazilian meat and meat products, the objective of this study was to examine the prevalence of STEC in ground beef, beef cuts, and chicken cuts sold at retail establishments in São Paulo, the largest market in Brazil. The research aimed to fill the knowledge gap regarding the occurrence of STEC in these specific meat products within the country.

Materials and methods

Bacterial strains

E. coli O157:H7 strain EDL-933, generously provided by R. M. F. Piazza from Instituto Butantan, Sao Paulo, SP, and STEC strains O103, O111, and O26, kindly provided by B. E. C. Guth from Federal University of Sao Paulo, SP, were utilized as positive controls in this study. E. coli K12 was used as the negative control. All strains were stored at -70 °C in trypticase soy broth (TSB) supplemented with 20% glycerol, sourced from Oxoid (Basingstoke, UK) and Synth (Diadema, SP, Brazil), respectively.

Samples and sampling

The study involved a total of 448 samples, consisting of 248 samples of ground beef, 100 samples of beef cuts, and 100 samples of chicken cuts. These samples were collected from various supermarkets and butcher shops located in different districts of Sao Paulo city, Brazil. To ensure proper preservation, the samples were transported to the laboratory using an insulated box with reusable ice, maintaining appropriate temperature conditions during transit.

Tests for Shiga toxin-producing Escherichia coli (ISO 16,654 and surveillance group for diseases and infections of animals NRM 006)

For each sample, 25 g were combined with 225 mL of modified tryptone soy broth (mTSB + N) from Oxoid (Basingstoke, UK), supplemented with 20 mg/L novobiocin obtained from Sigma (St. Louis, MO, USA). The mixture was subjected to stomaching to ensure thorough homogenization. Subsequently, the samples were incubated for 24 h at 41.5 °C. Five aliquots of 1 mL were removed and subjected to immunomagnetic separation (IMS), using Dynabeads against STEC O157, O26, O103, O111 and O145, following the manufacturer´s instructions (Dynal, Oslo, Norway).

To detect serogroup O157, a bead complex consisting of 1 µL, 49.5 µL, and 49.5 µL of the sample was streaked onto three different agar plates. These included MacConkey sorbitol agar (Oxoid), MacConkey sorbitol agar (CT-SMAC) containing cefixime (0.05 mg/L) and potassium tellurite (2.5 mg/L) (Oxoid), and CHROMagar^® (Invitrogen, Carlsbad, USA). The plates were then incubated at 37 °C for 24 h. Suspected colonies that appeared on the plates were subjected to biochemical tests following the methods outlined by Toledo et al. [20, 21] and Ewing et al. [22]. These biochemical tests were used to further confirm the presence of serogroup O157.

To isolate serogroups O26, O103, O111, and O145, 50 µL of the bead complex was streaked onto MacConkey agar plates. The plates were then incubated at 37 °C for 24 h. Following incubation, three to five colonies displaying typical and atypical characteristics were selected. These selected colonies were transferred to trypticase soy broth (TSB) supplemented with 20% glycerol from Synth (v/v). The isolates were preserved at -70 °C until they were subjected to PCR analysis for further identification and characterization.

Detection of virulence genes by PCR

Two multiplex PCR were performed: one for genes stx₁, stx₂ and eae (23) and another for genes rfb_O157 [23] and fliC_H7 [24]. Two other simplex PCR were also done for genes ehx and uid according to Feng and Monday [25]. DNA extraction was performed using the DNeasy Blood and Tissue kit from Qiagen (Hilden, Germany). All PCR reactions were carried out using the GoTaq Green Master Mix kit (Promega Corporation, Madison, USA). The reaction mixture consisted of 12.5 µL of the GoTaq Green Master Mix, 300 nM of each primer (Table 1), 3 µL of the extracted DNA, and sterile deionized water to achieve a final volume of 25 µL.

Table 1.

Primers used in this study

Primer	Sequence	Fragment size (bp)	Reference
stx 1	F: cag tta atg tgg tgg cga agg R: cac cag aça atg taa ccg ctg	348	Feng and Monday [25]
stx 2	F: atc cta ttc ccg gga gtt tac g R: gcg tca tcg tat aca cag gag c	584	Feng and Monday [25]
eae	F: att acc atc cac aca gac ggt R: aca gcg tgg ttg gat caa cct	397	Feng and Monday [25]
ehx	F: gtt tat tct ggg gca ggc tc R: ctt cac gtc acc ata cat at	158	Feng and Monday [25]
uid	F: gcg aaa act gtg gaa ttg gg R: tga tgc tcc atc act tcc tg	252	Feng and Monday [25]
rfb O157	F: cgg aca tcc atg tga tat gg R: ttg cct atg TAC agc taa tcc	259	Paton and Paton [26]
fliC H7	F: gcg ctg tcg agt tct atc gagc R: caa cgg tga ctt tat cgc cat tcc	625	Gannon et al., [24]

*F: forward

**R: reverse

The amplification products were stored at 4ºC until further analysis. E. coli O157:H7 (EDL933) was used as the positive control in the PCR reactions, while ultrapure water served as the negative control. The amplified PCR products were analyzed by performing 1.5% agarose gel electrophoresis supplemented with ethidium bromide. In cases where a positive result was obtained for a pool, the individual colonies from that pool were subsequently subjected to separate PCR analysis. If the pool tested positive for the presence of stx1 and/or stx2 and eae genes, further testing was conducted on individual colonies obtained from that pool.

Biochemical and serological tests

Isolates that tested positive for stx1 and/or stx2 were subjected to biochemical identification following the methods previously described [20–22]. The hemolysis phenotype of these isolates was assessed based on the protocol outlined by Beutin et al. [27].

For the isolates that displayed positive results for both stx1 and/or stx2 and hemolysis, further characterization of their O: H serotype was performed at the Instituto Adolfo Lutz, located in Sao Paulo, Brazil. The O: H serotyping process is a method used to identify the specific serotype of the isolates based on their O antigen (lipopolysaccharide) and H antigen (flagellar) characteristics.

Shiga toxin production using Vero cell

To assess the production of Shiga toxins, a Vero cell assay was conducted following the protocol outlined by Gentry and Dalrymple [28] and Karmali [29]. Briefly, positive samples for stx1 and stx2 were grown in Luria broth for 18–24 h at 37 °C and the sterile supernatants were inoculated into Vero cells monolayers. The Shiga cytotoxic activity was observed after 24 and 48 h of incubation at 37 °C.

Whole-genome sequencing (WGS)

DNA extraction was carried out using the PureLink Quick Gel Extraction Kit from Life Technologies (Carlsbad, CA). Following extraction, the quality and quantity of the DNA were assessed using a NanoDrop spectrophotometer from Thermo Scientific. The DNA concentration was further determined using a Qubit 2.0 fluorometer from Life Technologies.

To prepare the genomic library, the Nextera XT DNA Library Preparation Kit from Illumina (San Diego, CA) was employed. The library was subsequently sequenced using the Illumina NextSeq 550 platform, generating paired-end reads with a length of 2 × 75 base pairs. The sequencing process aimed to achieve a genome coverage of 290.0×, ensuring comprehensive and accurate sequencing of the sample.

The FastQ data obtained from sequencing were imported into the CLC Genomic Workbench 10 software from Qiagen for further analysis. The raw reads underwent a quality evaluation process to assess their overall sequencing quality. To ensure the reliability of the assembly, a trimming and cleanup step was performed to remove any potential contaminants such as barcodes, adapter sequences, and excessive N bases.

To achieve an accurate assembly, the software enforced specific criteria on the read length, aiming for a size of approximately 300 base pairs, and a G + C content of around 50%. These criteria were set to optimize the assembly process and improve the accuracy of the final results.

Following the quality control and trimming steps, the de novo assembly was conducted using the default settings provided by the CLC Genomic Workbench. These default settings include parameters such as automatic word size (set to 20) and bubble size (set to 50), as well as a minimum contig length of 200 nucleotides. The software also automatically detected the paired distances between reads and performed read mapping back to the assembled contigs. By employing these settings and procedures, the CLC Genomic Workbench facilitated the de novo assembly process, generating contigs from the reads and providing valuable information about the genomic composition of the sample.

We further characterized this strain by performing a phylogenomic analysis. In this regard, genome assemblies of 20 E. coli strains belonging to serotype O157:H7 from different sources and countries were retrieved from the Escherichia/Shigella Enterobase database (https://enterobase.warwick.ac.uk). A maximum likelihood phylogenetic tree of the A1 3104 strain was reconstructed alongside the 20-genome assemblies retrieved from Enterobase using the default settings of CSI Phylogeny 1.4 (https://cge.cbs.dtu.dk/services/CSIPhylogeny). The genome of Escherichia coli O157:H7 str. Sakai was used as reference (RefSeq assembly accession: GCF_000008865.2). The resulting phylogeny was visualized and annotated using iTol version 6.

Results

Out of the 248 ground beef samples tested, only one sample (0.4%) was found to be positive for STEC (Shiga toxin-producing Escherichia coli). PCR analysis confirmed the presence of all tested genes associated with STEC (stx1, stx2, eae, ehx, uid, rfbO157, and fliCH7) in this isolate, and biochemical tests confirmed its identification as Escherichia coli. No STEC strains were isolated from the remaining 100 beef cut samples or the 100 chicken cut samples, indicating the absence of detectable STEC contamination in those specific samples.

The isolated strain exhibited more than 50% cytotoxic activity in Vero cells within 24 h, indicating its ability to produce cytotoxic Shiga toxins. However, it did not express the entero-hemolysin gene. Serological testing revealed that the isolate belonged to the O157:H7 serotype, a known pathogenic strain of E. coli associated with severe illnesses.

Whole-genome sequencing (WGS) analyses further supported the earlier findings. The resistome, plasmid replicons, multilocus sequence type (MLST), serotype, and virulome were identified using ResFinder 4.1, PlasmidFinder 2.1, MLST 2.0, SerotypeFinder 2.0, and VirulenceFinder 2.0 databases (95% of identity and 60% of minimum length), respectively (http://genomicepidemiology.org/).

Indeed, the resistome revealed that there is no presence of antimicrobial resistance (AMR) genes, indicating a pan-susceptible phenotype. However, this strain presented chromosomal mutations in gyrA, gyrB, parC, parE, pmrA, pmrB, and folP. The lack of AMR genes is typically find in highly virulent strains, such as E. coli O157:H7 ST11. On the other hand, plasmidome encompassed two plasmids, IncFIB(AP001918) and IncFII. Genomic analysis revealed that the IncFIB(AP001918) plasmid carried the virulence genes ehxA and etpD, which are known to contribute to the pathogenicity of the strain. Additionally, the IncFII plasmid was found to harbor two other virulence genes (anr and katP). These findings indicate the presence of multiple plasmids carrying virulence determinants within the strain of interest.

This strain assigned to a virulent multi locus sequence type (ST) 11 and E. coli serotype O157:H7 confirming serological results. In addition, this strain was classified as belonging to phylogroup D based on the presence of chuA and absence of yjaA, as determined by Clermont typing.

The virulome revealed the presence of several canonical virulence markers such as, stx1, stx2, eae-g01-gamma, ehxA, stx1a-O157, stx2a-O157, espA, espB, espJ, espP, espY2, etpD, fdeC, fimH, gad, hlyE, iha, iss, katP, nleA, nleB, nleC, nlpl, ompT, AsIA, anr, astA, chuA, csgA, terC, tir, toxB, and traT. In addition, the genes terC, espA, espB, tir, and eae were found to be associated with insertion sequence IS630, while the presence of ISEc26, another insertion sequence element, was also identified. The genome sequence of E. coli strain A1 3104 have been deposited at DDBJ/ENA/GenBank with accession number JAUCBH000000000.

The phylogenomic results revealed that our strain clustered together with an E. coli strain (ESC MB4782AA) isolated from Human in Uruguay in 2011. Interestingly, other strains from Brazil, Argentina, and Paraguay did not cluster with the E. coli A1 3104 strain suggesting that there are more than one lineage circulating in Latin America (Fig. 1).

Fig. 1.

Maximum-likelihood phylogenetic tree of 20 E. coli strains from different sources, countries, and years

Discussion

The isolated STEC strain was obtained from a ground beef sample acquired at a butcher shop. It is worth noting that the butcher shop maintained an acceptable hygiene condition. Despite the adherence to acceptable hygiene practices, the presence of STEC in the ground beef sample indicates that contamination may occur even under seemingly satisfactory conditions. This highlights the importance of implementing rigorous food safety measures throughout the entire production and handling process to minimize the risk of STEC contamination and associated health hazards.

Indeed, various foods have been identified as potential vehicles for STEC, but ground beef has been particularly associated with foodborne illnesses caused by E. coli O157:H7. Research studies, such as the one conducted by White et al. [10], have highlighted the significant role of ground beef in transmitting STEC infections.

Despite Brazil’s position as one of the leading beef-producing and exporting countries [13], there has been a scarcity of research focusing on the detection and occurrence of STEC in both bovine meat and chicken within the retail market of Sao Paulo city. This information gap emphasizes the need for more comprehensive studies to assess the prevalence and potential risks associated with STEC contamination in these food products. Such research can contribute to the development of effective food safety measures and regulations to mitigate the transmission of STEC infections and protect public health.

Although studies have reported a low occurrence of STEC in Brazilian meat and meat products [14–16], as well as on carcasses [30] and animal feces [31–33], these results should not be disregarded. Even though the occurrence may be relatively low, the presence of STEC in meat and animal feces is still a concern due to the potential risks it poses to public health.

It is important to recognize that even a low occurrence of STEC can lead to foodborne illness outbreaks, especially considering the virulence and severity of certain STEC strains, such as E. coli O157:H7 ST11 lineage [1]. The consequences of infection can range from mild gastrointestinal symptoms to severe complications like bloody diarrhea and hemolytic uremic syndrome (HUS), which can be life-threatening.

Therefore, despite the relatively low occurrence, it is crucial to continue monitoring and implementing strict food safety measures throughout the entire food production chain to minimize the risk of STEC contamination and ensure the safety of meat and meat products for consumers.

Indeed, studies have demonstrated the ability of STEC O157:H7 to form biofilms on various surfaces, including equipment and utensils [34–36]. The formation of biofilms by STEC O157:H7 on equipment and utensils, such as grinders and knives, poses a challenge in the control and prevention of STEC contamination during grinding processes. If proper cleaning and sanitation protocols are not followed, residual biofilms can serve as a source of contamination and contribute to the spread of STEC during food processing.

To effectively control STEC during grinding, it is essential to implement rigorous cleaning practices that target the removal and prevention of biofilm formation. This includes thorough cleaning of equipment surfaces, regular inspection and maintenance of equipment to ensure its cleanliness, and the use of appropriate sanitizers or disinfectants to eliminate any remaining microbial contamination. By adopting proper cleaning procedures and maintaining high hygiene standards, the risk of STEC contamination can be minimized, contributing to the overall safety and quality of meat products.

The occurrence of STEC in beef, and particularly in ground beef, varies among countries. The difference on prevalence of STEC in Argentina and Brazil can be explained by differences in animal breeding or feeding during winter that could influence the colonization of the rumen.

The results of the present investigation are similar to the ones reported in Turkey by Sarimehmetoglu et al. [37]. These authors isolated STEC in 0.79% of the ground beef samples acquired at retail level. The isolates presented the genes stx1, stx2 and eae, as well as hly. Also in Turkey, Cadirci et al. [38] reported that STEC was present in 2.5% of samples of meat products, such as meatballs, where two isolates were positive for genes stx1, stx2 and eae, and four were positive for stx2 and eae. On the other hand, Ju et al. [39], in the United States of America, reported that 8.5% of the samples of ground beef were contaminated with non-O157 STEC presenting the gene stx_2, but not eae, some of them expressing cytotoxicity on the Vero cell assay. Sallam et al. [40], in Egypt, detected much higher levels (26,7%) of contamination of STEC O157 in ground beef, whereas Hoang Minh et al. [41], in Japan, isolated STEC in 4.4% of the tested meat samples, observing that 20% of the isolates harbored eae gene but none of them reacted with O157 serum. On the other hand, 92% of these strains produced Shiga toxin.

Bai et al. [42], in China, reported the presence of STEC in raw beef (11.0%), chicken (0.5%), pork (4.4%) mutton (20.6%), and duck (7.7%) samples. Sixty-three isolates were confirmed as STEC being 20 positive for stx₁, 32 stx₂-positive and 11 stx₁ + stx₂.

Cattle are recognized as a significant source of STEC, making it crucial to exercise caution during the slaughtering process. The presence of STEC in meat can be attributed to cross-contamination at various stages of the production chain, with skinning and evisceration being critical points of concern. Therefore, strict adherence to Good Manufacturing Practices (GMP) and Good Hygiene Practices (GHP) becomes essential to minimize the risk of contamination. GMP and GHP play pivotal roles in the meat industry, ensuring food safety and preventing STEC contamination. GMP entails following rigorous protocols and procedures to maintain a hygienic production environment, while GHP focuses on personal hygiene, sanitation, and proper handling of equipment and surfaces.

The implementation of immunomagnetic separation (IMS) during the enrichment step in this study may have enhanced the isolation of serogroup O157, allowing for its successful detection. However, similar results were not observed for serogroups O26, O103, O111, and O145.

WGS analyses provided additional insights into the characteristics of the isolated STEC strain. The resistome analysis indicated the absence of antimicrobial resistance genes, suggesting that this particular strain is not resistant to commonly used antibiotics. This is promising in terms of treatment options, as the strain would likely respond well to common antimicrobial agents used for E. coli infections. However, it is important to note that the absence of detected AMR genes does not guarantee long-term susceptibility. The emergence and acquisition of AMR genes can occur through various mechanisms, including horizontal gene transfer (HGT) and mutational events. Therefore, continuous monitoring of antimicrobial susceptibility patterns and continuous surveillance for the emergence of resistance in the population remains crucial.

The plasmidome analysis revealed the presence of two plasmids, IncFIB(AP001918) and IncFII, which may contribute to the strain’s virulence and potential for HGT. The presence of ehxA and etpD on the IncFIB plasmid suggests that this plasmid may play a significant role in the strain’s pathogenicity by enabling the expression of cytotoxic and adherence-related factors. On the other hand, the presence of anr and katP on the IncFII plasmid suggests the involvement of this plasmid in additional virulence mechanisms, potentially contributing to the strain’s ability to evade host immune responses and establish infection.

The identification of these virulence genes on plasmids highlights the potential for HGT, a process that allows the transfer of genetic material between bacteria. This mechanism can facilitate the spread of virulence factors among bacterial populations, leading to the emergence of more pathogenic strains.

Understanding the genetic composition and distribution of virulence genes, particularly those carried on plasmids, is crucial for comprehending the pathogenic potential of the strain and informing strategies for infection control and treatment. Further investigations are needed to explore the genetic context of these virulence genes, including their association with other genetic elements and their potential influence on the strain’s virulence phenotype. Furthermore, the strain was assigned to a virulent sequence type (ST11), further supporting its pathogenic potential.

The virulome analysis revealed the presence of several canonical virulence markers associated with STEC, indicating the strain’s ability to cause severe infections. The presence of IS630 and ISEc26 in proximity to the virulence genes indicates their involvement in the genetic evolution and adaptation of the strain. The association of these virulence genes with insertion sequences suggests a potential role in the mobility and rearrangement of genetic elements within the genome of the strain and can provide valuable insights into the strain’s pathogenic potential and evolutionary dynamics.

Overall, this study identified a rare occurrence of STEC contamination in ground beef in Brazil, with the isolated strain belonging to the highly virulent E. coli O157:H7 ST11 lineage [1]. These findings contribute to our understanding of STEC prevalence and characteristics in food samples, highlighting the importance of effective food safety measures to prevent potential health risks associated with STEC contamination.

Conclusion

In order to support the current knowledge regarding the epidemiological distribution of pathogenic strains in foods, our results provide valuable information related to the occurrence of highly virulent STEC O157:H7 ST11 lineage in ground beef. Although our results shown low occurrence of STEC in the tested meat samples, the presence of this highly virulent strain in ground beef raises a particular concern, since the strain exhibited cytotoxic activity in Vero cells within 24 h and carried a range of virulence markers, indicating its potential to cause severe illnesses. The findings emphasize the importance of continued monitoring and implementation of rigorous hygiene practices throughout the production and retail chain to mitigate the risk of STEC contamination and ensure food safety.

Data availability

Upon request.

Code availability

NA.

Declarations

Conflict of interest

The authors have no conflict of interest to declare.