Epidemiological Characteristics of Shiga Toxin-Producing Escherichia coli Responsible for Infections in the Polish Pediatric Population

Shiga toxin-producing Escherichia coli (STEC), also known as verocytotoxin-producing E. coli (VTEC) strains are responsible for serious diseases such as hemorrhagic colitis and hemolytic uremic syndrome (HUS), especially among children and elderly. STEC are zoonotic pathogens, with ruminants considered the main reservoir. Enterohaemorrhagic E. coli (EHEC) is a subgroup of STEC serotypes strongly associated with bloody diarrhea and HUS in industrialized countries. HUS is defined as a combination of acute kidney injury, low platelet counts, and hemolytic anemia. After HUS,some children never recover kidney function and require long-term replacement therapy, while those who recover are at risk of developing chronic kidney disease and arterial hypertension. Some have residual non-renal problems, including neurological defects, insulin-dependent diabetes, pancreatic failure, or gastrointestinal complications. STEC associated with diarrhea may resolve without any long-term consequences (WHO 2018).

STEC serotypes produce high levels of various toxins in the large intestine which are closely related to the potent cytotoxins produced by Shigella dysenteriae type 1. They appear to directly damage mucosal and vascular endothelial cells in the gut wall. If absorbed, they exert toxic effects on another vascular endothelium, e.g., renal. The mainstay of treatment for STEC infection is supportive. Although E. coli is susceptible to most used antibiotics, they have not been shown to alleviate symptoms, reduce carriage of the organism, or prevent HUS.

The knowledge of how to reduce the incidence of STEC on cattle farms still needs to be elucidated. However, tests can be performed to determine whether a given animal carries the bacterium. If necessary, meat can be given bactericidal treatment, which involves heating or irradiation. Although these techniques are helpful, they do not guarantee the absence of STEC in food. Effective prevention requires the application of strict hygiene practices throughout the entire food chain, from producer to consumer. To prevent infection, children under the age of 5 should not come into contact with farm animals, especially cattle and sheep, and their environment as well as should avoid eating unpasteurized cheese or not washed fruits, vegetables, and herbs if they are eaten raw; nor drink water that has not undergone microbiological testing, e.g., from a well or spring (The Institut Pasteur 2023).

The aim of the study was to evaluate epidemiological characteristics of Shiga toxin-producing E. coli causing infection in the pediatric population in Poland.

Outbreaks and sporadic cases of STEC have been reported in several countries. Typical HUS caused by STEC presents with acute illness with symptoms of bloody diarrhea. About 25% of typical HUS do not show diarrhea. Almost 90% of individuals recover without sequelae, either spontaneously or after plasma infusion or replacement plasma (Noris et al. 2021). In our study, the most common outcome was complete recovery in 83.3% (15/18) of patients. However, we reported some complications of HUS, such as neurological complications and insulin-dependent diabetes, in 11.1% and 5.6%, respectively.

Our study showed that among the analyzed STEC patients, the stx2 gene predominated with a frequency of 84.4%, and equally, 6.7% were identified with only the stx1 gene and with both stx1 and stx2 genes. The stx2 gene was reported as most frequently detected by others 49.1% – 83.3% (Jenssen et al. 2014). Januszkiewicz and Rastawicki (2016) described strains isolated in Poland from humans, cattle, and food between 1996 and 2010. The authors confirmed the presence of the stx2 gene in 11.8% and the simultaneous occurrence of stx1 and stx2 in 88,2% of isolates but did not detect any stx1 in human isolates. Our results confirm the circulation of E. coli producing verotoxin1 in the Polish pediatric population. Other researchers reported that the stx1 gene is dominant in 57.7–83%, but the stx2 gene in 11.7–17% of isolates (Bouzari et al. 2018).

Shiga toxins (ST) are a group of bacterial toxins involved in human and animal diseases. STs are produced by E. coli isolates. Other species reported as ST producers besides S. dysenteriae type 1 are occasionally C. freundii, Enterobacter cloacae, and Shigella flexneri (Herold et al. 2004). In our study, in one case, C. freundii was detected in the bloody stool of a patient suffering from diarrhoea having the stx1 gene.

The fact that our study mainly includes high-risk STEC might support the serotyping results, as 40 (97.6%) of the strains tested had serotype O157 and encoded the Shiga toxin 2 gene. Reports of non-O157 STEC serotypes associated with severe disease vary among countries. According to the ECDC report, until 2021, the most frequently reported serogroup in human STEC cases was O157, followed by O26, but in recent years, there has been an increasing trend in number of non-O157 STEC infections. The last ECDC report on STEC infections (ECDC 2022) shows serogroup O26 as the most common (34%) among HUS cases, followed by O157 (19.8%). The results of our study concern the period 2018–2022, and sporadic cases of HUS in pediatric population. Detection of the O157 serogroup was not influenced by diagnosis as PCR amplification of Shiga toxin coding genes was applied for detection. Our results agree with some other studies suggesting that E. coli O157: H7 serotype causes in children with HUS more severe diseases than non-O157. In the USA, serotype O157:H7 was the most commonly associated with STEC and most often associated with HUS (Hermos et al. 2011). Similar results were described in a recent study from Sweden (Hua et al. 2021) and China (Zhu et al. 2023). These results are inconsistent with the currently described infections and HUS associated with non-O157 E. coli serotypes, even from Poland (Rastawicki et al. 2020). This phenomenon requires further, more in-depth research.

ST production is necessary but not enough for STEC virulence. Shiga toxin production by O157:H7 is mediated by sensing quorum (Mühlen and Dersch 2020). Serotype O157:H7 harbors the locus of the pathogenic enterocyte efflux island, which encodes genes involved in removing intestinal epithelial cell microvilli and the tight adherence of bacteria to the epithelial cell membrane. The molecular mechanism underlying the pathogenicity of different STEC strains requires further elucidation. Stx2d has been described as a Shiga toxin whose cytotoxicity is activated 10- to 1,000-fold by elastase present in the intestinal mucus of mice or man (Matussek et al. 2023). Clinical STEC strains may have the following genes: stx2a, stx2b, stx2c, stx2d, stx2e, stx2f, stx2g, stx2h, stx2i, stx2j, stx2k, stx2l, stx2m (Bai et al. 2021). Bacterial genetic factors identified as molecular predictors of the development of HUS included stx subtype stx2a, stx2a, and stx2c and genes encoding intimin, toxins, secretory system proteins, and transcription regulators (Sallée et al. 2013). Rapid diagnostic targeting of these genes directly in stool samples could lead to improved care and infection control measures, reducingthe number of patients suffering from severe STEC disease (Matussek et al. 2023).

The likelihood that a patient infected with E. coli O157:H7 will develop HUS varies with age. Fifteen to twenty percent of children less than 10 years of age with culture-confirmed E. coli O157:H7 infection develop HUS. Our study confirmed this because 77% of children with confirmed STEC infection and HUS were under 5 years of age.

Fatima and Aziz (2023) reported a seasonal pattern of STEC infections, with increased incidence during the summer months. Similarly, in the 2018–2022 analyzed period, we observed that infections most frequently occurred in June, July, August, and September, with a peak in July.

Because antibiotics are strong inducers of the bacterial SOS response, which initiates the production and release of phages from bacteria, antibiotic treatment is not recommended. It was mentioned that bacterial DNA damage due to antibiotic therapy may increase the expression of genes encoding verotoxins and increase the risk of HUS (Noris et al. 2021). It is believed that disease worsening after administration of antibiotics also occurs due to changes in the commensal intestinal flora, which allows STEC to attach to the intestinal wall (Harkins et al. 2020). There is no therapy against STEC infections, and current treatment is only supportive and includes hydration therapy and, if necessary, dialysis (Koyanagi et al. 2019). An in vivo mouse model using E. coli O86 demonstrated reduced Shiga toxin production and mortality following azithromycin administration and reduced duration of STEC excretion in feces with a lower frequency of long-term STEC O104:H4 transfer (Nitschke et al. 2012). Cefotaxime, meropenem, ceftazidime, gentamicin, and kanamycin did not affect the stx gene expression or had little induction operation. Antibiotics affecting protein biosynthesis, such as polycyclic, do not affect the expression of the stx2 gene (Chen et al. 2013). Patients treated with intravenous ciprofloxacin and meropenem significantly reduced the duration of ST excretion in stool, with a concomitant reduction in the incidence of seizures and death compared with patients who did not receive antibiotics (Prado et al. 1995). According to Prado et al. (1995) use of β-lactams during the first 3 days of illness was associated with an increased risk of STECHUS. Intravenous meropenem treatment promoted ST release by E. coli O157:H7 but not the O104:H subtype. Ciprofloxacin and norfloxacin increased stx2 expression thousands of times compared to controls. Oral consumption of quinolones by E. coli patients infected with O157:H7 did not increase the possibility of progression to HUS (Chen et al. 2013). The use of trimethoprim did not show significant progression to STEC-HUS, symptomatic improvement or change in Shiga toxin excretion (Tarr et al. 2018). In a prospective study of 259 children below 10 years of age infected with E. coli O157:H7 (Wong et al. 2012) HUS was more common in children who received antibiotics, which included trimethoprim-sulfamethoxazole, β-lactams, metronidazole, and azithromycin (36 versus 12 percent for children who did not receive antibiotics). After controlling for other variables, the absolute increase in the risk of HUS associated with antibiotic therapy was 25 percent, equivalent to one case of HUS in every four children treated with antibiotics. Data in adults are sparser. In three adult studies that were included in the meta-analysis, antibiotics administered early in STEC infection were associated with the subsequent development of HUS (Wong et al. 2012). A multicenter study observing the application of fosfomycin in STEC infection showed a reduction/progression of STEC-HUS after administration within the first five days (Kurabayashi et al. 2015). Combination treatment with neutralizing antibodies against Stx1 and Stx2 and tigecycline reduced the toxicity levels of STEC isolates, E. coli cell counts, and the likelihood of transmission (Skinner et al. 2015). Bacterial DNA-interacting antibiotics, such as fluoroquinolones, trimethoprim/sulfamethoxazole, and β-lactams, induce bacteriophage lysogeny, leading to increased ST expression in vitro and increased transfer of toxin-encoding prophages to previously nontoxic strains in vivo (Bitzan 2009). Harkins et al. (2020) recommend that antibiotic therapy should be considered safe if the following criteria are met: i) confirmed absence of STEC with a virulence profile associated with HUS in the stool sample; ii) absence of acute or chronic kidney disease; iii) in long-term carriers of STEC associated with HUS, antibiotic therapy should not be started earlier than 2 weeks after the first isolation of the pathogen, and identical types of STEC should be isolated in two separate samples. Although the usefulness of antibiotic treatment in patients with STEC infections is a subject of ongoing debate, knowledge of STEC antimicrobial susceptibility patterns in different geographic areas is important for therapeutic and strain characterization purposes. Prado et al. (1995) found that the strains were sensitive to furazolidone, ciprofloxacin, gentamicin, and amikacin. Resistance was detected to tetracycline at 4%, chloramphenicol – 5%, trimethoprim/sulfamethoxazole – 24%, and ampicillin – 25% (Kurabayashi et al. 2015). All our isolates were susceptible to cefepime, imipenem, meropenem, and amikacin. All strains were ESBL negative. Bielaszewska et al. (2011) reported a 2011 outbreak in Germany caused by E. coli O104:H4 stx2-positive with expanded CTX-M-1 β-lactamase. STEC serotype O121 ESBL-positive isolates were described in 2019 by Kikuchi et al. (2019). Our study showed that in justified cases, all permitted antibiotics can be used in the empirical therapy of STEC infections.

In our study, PFGE analysis of 23 selected E. coli and one C. freundii strain showed 18 PFGE types; however, types A, B, and C with two subtypes showed genetic relatedness. The remaining 15 PFGE types were derived from single patients and were not related to each other. All strains with genetic relatedness have been found in Mazovian voivodeships. However, strains with type B were also found in Podlasie and type C in Warmian-Masurian voivodeships. The STEC strains with A, B, and C PFGE types derived from different persons, times, and areas reflect no epidemic transmission of these strains and show their probable persistence in the environment. On the other hand, the occurrence of the stx2 gene in the remaining sporadic STEC strains indicates other than clonal distribution of this Shiga toxinproducing gene. To evaluate the character of this distribution, further analysis should be performed. Contreras et al. (2011) described nineteen types of pulsed fields in 20 STEC isolates. Japanese scientists recovered STEC O157:H7 isolates from experimentally infected cattle and observed twelve new PFGE profiles. Changes in the PFGE profiles of STEC O157:H7 isolates occurred after passing through the gastrointestinal tract of livestock (Yoshii et al. 2009). Strain circulation is observed due to the persistence of dominant strains or repeated exposure of cattle to different strains, which can be demonstrated by PFGE analysis. No significant differences have been described between the fecal microflora of excretors and non-excretors (Bibbal et al. 2022). Matussek et al. (2023) performed whole-genome multilocus sequence typing and genome-wide phylogeny analysis to assess the phylogenetic relatedness of STEC isolates from HUS and non-HUS patients and suggested genetic differences of pathogenic STEC strains in different geographic regions and populations. Human immunity and/or bacteria-host interaction may affect STEC-related disease progression. Accessory genes were differentially presented in HUS-STEC and non-HUS-STEC strains in a pangenome-wide association study. There was no significant difference in virulence genes between O157 strains from patients with and without HUS. These data suggest that the infection dose and differences in host innate and adaptive immunity, e.g., genetic variation in patients’ complement genes, are predisposing factors to atypical HUS (Sallée et al. 2013) and may play a vital role in the pathogenesis and development of STEC-HUS. Susceptibility to STEC infection varies among people. Personalized medicine and sequencing of patient’s DNA in the STEC-HUS diagnostic process may answer the question of what genetic variability in human genes is a critical factor and why STEC diarrhea in a patient resolves spontaneously while in others it leads to HUS. Whole-genome phylogeny and multiple pangenome correspondence analysis did not separate HUS-STEC strains from non-HUS-STEC strains, suggesting that STEC strains of different genetic origins may independently acquire genes determining their pathogenicity and that other non-bacterial factors may play a key role in the pathogenicity of HUS development (Matussek et al. 2023). Recent data suggest the involvement of the complement system in the pathogenesis of STEC-HUS (Mele et al. 2014). The gut microbiota plays a pivotal role in host defense against STEC. Precolonization of Clostridium ramosum in STEC-infected mice reduced the level of secreted STX2 and prevented kidney histopathological changes. C. ramosum inhibits growth and ST production by STEC more strongly than C. perfringens (Koyanagi et al. 2019).

STEC has been shown to be an important pathogen causing severe post-infectious complications and deaths among the youngest children in Poland. In our study, the genetic diversity of STEC strains was seen. Almost 86.4% of isolates carried the stx2 gene, and 97.6% belonged to the serotype O157. The isolates were susceptible to antibiotics. The STEC infections were observed in the summer months, mainly in children 1–5 years (77.8%). Contact with animals was the most frequent HUS risk factor. Epidemiological analysis of STEC isolates is crucial for understanding the disease and tracking outbreaks. Collaboration between physicians and microbiologists when diagnosing patients with diarrhea, especially in cases of contact with animals, is vital. Due to the occurrence of these infections as independent epidemic outbreaks in human-animal relationships, epidemiological surveillance should be conducted considering the One Health concept.