A natural approach to combating antibiotic-resistant pathogens in livestock: Hibiscus sabdariffa-derived hibiscus acid as a promising solution.

We examined the antibacterial efficacy of streptomycin, hibiscus acid, and their combination against multidrug-resistant Shiga-toxin-producing Escherichia coli (STEC) and Salmonella Typhimurium in mice. We determined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for streptomycin, hibiscus acid, and their combination against STEC and Salmonella. Fifteen sets of six mice in each set were utilised: six groups were orally exposed to 4 log10 colony forming units (CFUs) of S. Typhimurium and another six to STEC, and three acted as the controls. Six hours post-inoculation, specific groups of mice received either oral solutions containing hibiscus acid at 5 and 7 mg/ml; streptomycin at 50 and 450 µg/ml; hibiscus acid/streptomycin (5 mg/ml hibiscus acid and 50 µg/ml streptomycin); or isotonic saline. The study determined the MIC and MBC of 7 mg/ml of hibiscus acid; 300 and 450 µg/ml of streptomycin; and two concentrations of hibiscus/streptomycin (3 mg/ml / 20 µg/ml and 5 mg/ml / 50 µg/ml). Interestingly, the mice that were infected and subsequently treated with hibiscus acid at 7 mg/ml alone or in conjunction with streptomycin did not have either STEC or Salmonella in their faecal samples, and none of the mice died. In contrast, the untreated mice and those exclusively treated with streptomycin had the pathogens present in their stool, leading to the mortality of all the subjects.

O80:H2-Associated Hemolytic Uremic Syndrome without Hemorrhagic Colitis: A Case Report.

Introduction: Hemolytic uremic syndrome (HUS) is characterized by progressive kidney injury accompanied by thrombotic microangiopathy, which is clinically defined as microangiopathic hemolytic anemia with thrombocytopenia and organ injury. Shiga toxin-producing Escherichia coli (STEC)-HUS is caused by infection with pathogenic E. coli strains, typically O157, O26, and O111. However, the prevalence of other types of pathogenic E. coli has been increasing, and these pathogens sometimes cause atypical clinical manifestations of STEC-HUS. Case Presentation: We report the case of a 3-year-old girl diagnosed with STEC-HUS associated with a rare O80:H2 stx2 serotype, characterized by an atypical clinical course. She presented with severe hemolytic anemia and mild renal dysfunction but did not have enterohemorrhagic diarrhea. The first culture test of her stool sample collected using a swab upon admission yielded no signs of STEC, leading to an initial diagnosis of atypical HUS; thus, eculizumab was administered adding to red blood cell transfusion and recombinant thrombomodulin alfa and haptoglobin. However, a subsequent culture test of her second stool sample revealed the presence of O80:H2 stx2, confirming the diagnosis of STEC-HUS. Subsequently, the patient's condition improved, and her serum creatinine level gradually normalized over the course of 3 months. Conclusion: Diligently diagnosis is crucial in cases lacking typical STEC-HUS symptoms. We advocate for repeated stool culture testing to ensure accurate identification and timely management of such cases.

Prevalence, Molecular Characterization, and Antimicrobial Resistance Profiles of Shiga Toxin-Producing Escherichia coli Isolated from Raw Beef, Pork, and Chicken Meat in Vietnam.

Shiga toxin-producing Escherichia coli (STEC) is one of the most important foodborne pathogens, and the rise of antibiotic resistance to it is a significant threat to global public health. The purpose of this study is to investigate the prevalence, molecular characterization, and antibiotic resistance of STEC isolated from raw meat in Vietnam. The findings in this study showed that the prevalence of STEC in raw beef, pork, and chicken meat was 9.72% (7/72), 5.56% (4/72), and 1.39% (1/72), respectively. The STEC isolates were highly resistant to ampicillin (91.67%) and tetracycline (91.67%), followed by trimethoprim/sulfamethoxazole (83.33%), streptomycin (75%), and florfenicol (66.67%). The incidence of STEC virulence-associated genes, including stx1, stx2, eae, and ehxA, was 8.33% (1/12), 91.67% (11/12), 33.33% (4/12), and 58.33% (7/12), respectively. STEC serogroups O157, O26, and O111 were detected in 3 out of 12 STEC isolates. Two isolates were found to be ESBL producers carrying the blaCTX-M-55 gene, and three isolates were colistin-resistant strains harboring the mcr-1 gene. Notably, a STEC O111 isolate from chicken meat harbored both the blaCTX-M-55 and mcr-1 genes.

Enterohemorrhagic Escherichia coli: trigger for small vessel vasculitis.

We describe the case of a 19-year-old woman who presented with abdominal pain, vomiting, and a palpable purpuric rash. The patient subsequently developed dysentery and was found to have an infection from Shiga toxin-producing Escherichia coli. The patient also met diagnostic criteria for IgA vasculitis (also known as Henoch Schönlein purpura) but had negative immunofluorescence biopsies of the rash. The patient was treated with steroids and achieved recovery. To our knowledge, this is the first documented case of IgA vasculitis in the setting of an enterohemorrhagic E. coli infection. This case highlights an atypical presentation of IgA vasculitis and the need to include small vessel vasculitis as a differential diagnosis when treating patients of all ages.

Eculizumab for Shiga-toxin-induced hemolytic uremic syndrome in adults with neurological involvement.

The role of eculizumab in treating Shiga-toxin-producing Escherichia coli (STEC) hemolytic uremic syndrome (HUS) patients with neurological involvement remains unclear. We describe two distinctly different STEC-HUS patients with neurologic involvement successfully managed with eculizumab, and perform a literature review of all published cases. Both patients had complete resolution of neurological symptoms after initiation of eculizumab. Eighty patients with STEC-HUS treated with eculizumab were identified in the literature, 68.7% had complete resolution of neurological symptoms. Based on our experience and literature review, three prevailing themes were noted: 1) Early eculizumab administration optimized neurological outcomes, 2) Symptom resolution may not be immediate, neurological symptoms may initially worsen before improvement, and 3) Plasma exchange yielded no benefit. Early administration of eculizumab may reverse neurotoxicity in patients with STEC-HUS.

Lung Ultrasound to Evaluate Fluid Status and Optimize Early Volume-Expansion Therapy in Children with Shiga Toxin-Producing Escherichia Coli-Haemolytic Uremic Syndrome: A Pilot Study.

Background: Shiga toxin-producing Escherichia coli-haemolytic uremic syndrome (STEC-HUS) can result in kidney and neurological complications. Early volume-expansion therapy has been shown to improve outcomes, but caution is required to avoid fluid overload. Lung ultrasound scanning (LUS) can be used to detect fluid overload and may be useful in monitoring hydration therapy. Methods: This prospective observational pilot study involved children with STEC-HUS who were recruited from a regional paediatric nephrology centre. B-line quantification by LUS was used to assess fluid status at the emergency department (ED) admission and correlated with the decrease in patient weight from the target weight. A control group of children on chronic dialysis therapy with episodes of symptomatic fluid overload was also enrolled in order to establish a B-line threshold indicative of severe lung congestion. Another cohort of "healthy" children, without renal or lung-related diseases, and without clinical signs of fluid overload was also enrolled in order to establish a B-line threshold indicative of euvolemia. Results: LUS assessment was performed in 10 children with STEC-HUS at ED admission, showing an average of three B-lines (range 0-10). LUS was also performed in 53 euvolemic children admitted to the ED not showing kidney and lung disease (healthy controls), showing a median value of two B-lines (range 0-7), not significantly different from children with STEC-HUS at admission (p = 0.92). Children with STEC-HUS with neurological involvement during the acute phase and those requiring dialysis presented a significantly lower number of B-lines at admission compared to patients with a good clinical course (p < 0.001). Patients with long-term renal impairment also presented a lower number of B-lines at disease onset (p = 0.03). Conclusions: LUS is a useful technique for monitoring intravenous hydration therapy in paediatric patients with STEC-HUS. A low number of B-lines at ED admission (<5 B-lines) was associated with worse short-term and long-term outcomes. Further studies are needed to determine the efficacy and safety of an LUS-guided strategy for reducing complications in children with STEC-HUS.

Increased resistance against tellurite is conferred by a mutation in the promoter region of uncommon tellurite resistance gene tehB in the ter-negative Shiga toxin-producing Escherichia coli O157:H7.

Resistance to potassium tellurite (PT) is an important indicator in isolating Shiga toxin-producing Escherichia coli (STEC) O157:H7 and other major STEC serogroups. Common resistance determinant genes are encoded in the ter gene cluster. We found an O157:H7 isolate that does not harbor ter but is resistant to PT. One nonsynonymous mutation was found in another PT resistance gene, tehA, through whole-genome sequence analyses. To elucidate the contribution of this mutation to PT resistance, complementation of tehA and the related gene tehB in isogenic strains and quantitative RT‒PCR were performed. The results indicated that the point mutation not only changed an amino acid of tehA, but also was positioned on a putative internal promoter of tehB and increased PT resistance by elevating tehB mRNA expression. Meanwhile, the amino acid change in tehA had negligible impact on the PT resistance. Comprehensive screening revealed that 2.3% of O157:H7 isolates in Japan did not harbor the ter gene cluster, but the same mutation in tehA was not found. These results suggested that PT resistance in E. coli can be enhanced through one mutational event even in ter-negative strains. IMPORTANCE: Selective agents are important for isolating Shiga toxin-producing Escherichia coli (STEC) because the undesirable growth of microflora should be inhibited. Potassium tellurite (PT) is a common selective agent for major STEC serotypes. In this study, we found a novel variant of PT resistance genes, tehAB, in STEC O157:H7. Molecular experiments clearly showed that one point mutation in a predicted internal promoter region of tehB upregulated the expression of the gene and consequently led to increased resistance to PT. Because tehAB genes are ubiquitous across E. coli, these results provide universal insight into PT resistance in this species.

A challenging STEC strain isolation from patients' stools: an O166:H15 STEC strain with the stx2 gene.

Two patients with acute gastroenteritis tested positive for Shiga toxin-producing Escherichia coli (STEC) by polymerase chain reaction (PCR), and both strains carried the Shiga toxin 2 encoding gene. Since routine culture using CHROMagar STEC failed to recover these isolates, immunomagnetic separation (IMS) targeting the top six non-O157:H7 serotypes was used for isolate recovery. After two subsequent IMS runs, the STEC strains were isolated from trypticase soy broth with and without overnight enrichment for runs 1 and 2, respectively. Serotyping based on whole-genome sequencing revealed that both patients carried the strain O166:H15 STEC with the stx2 gene. Hence, the magnetic beads used in IMS appeared to have cross-reactivity with other E. coli serotypes. When the STEC isolates from both stools were cultured on CHROMagar STEC and sheep blood agar (BAP), two distinct colony sizes were apparent after overnight incubation. The small and large colonies were picked and separately cultured on both media, and colony growth was observed for 2 weeks at room temperature after an initial overnight incubation at 37°C. After 1 week, the colonies showed concentric ring structures with a darker center and a lighter surrounding on CHROMagar STEC and a "fried egg"-resembling structure with a raised circular center and a flat surrounding on BAP. Both colony types remained morphologically different on CHROMagar STEC throughout the 15 days. However, on BAP, their appearance was comparable by day 7. IMPORTANCE: Shiga toxin-producing E. coli (STEC) infections can lead to severe complications such as bloody diarrhea and hemolytic uremic syndrome (HUS), especially in young children and the elderly. Strains that carry the shiga toxin 2 gene (stx2), such as O157:H7, have been mostly linked with severe disease outcomes. In recent years, outbreaks caused by non-O157:H7 strains have increased. E. coli O166:H15 has been previously reported causing a gastroenteritis outbreak in 1996 as a non-STEC strain, however the O166:H15 serotype we recovered carried the stx2 gene. It was particularly challenging to isolate this strain from stools by culture. Consequently, we tested immunomagnetic separation for the STEC recovery, which was a novel approach on clinical stools. Virulence genes were included for the characterization of these isolates.

Characterization of Shiga Toxin-producing Escherichia coli Isolated from Cattle Around Ulaanbaatar City, Mongolia.

Shiga toxin-producing Escherichia coli (STEC) are associated with severe infections including hemorrhagic colitis and hemolytic uremic syndrome in humans. Ruminants are known as reservoirs of STEC; however, no data are available on STEC in ruminants in Mongolia, where more than 5 million cattle and 25 million sheep are raised. To disclose the existence and characteristics of STEC in Mongolia, in this study, we isolated and characterized STEC from cattle in Mongolia. We collected 350 rectal swabs of cattle from 30 farms near Ulaanbaatar city and isolated 45 STEC from 21 farms. Rectal swabs were precultured with modified Escherichia coli broth and then inoculated to Cefixime-Tellurite Sorbitol MacConkey agar plate and/or CHROMagar STEC agar plate for the isolation of STEC. The isolation ratios in each farm were from 0% to 40%. Multiplex PCR for the estimation of O- and H-serotypes identified 12 O-genotypes (Og-types) and 11 H-genotypes (Hg-types) from 45 isolates; however, Og-types of 19 isolates could not be determined. Stx gene subtyping by PCR identified 2 stx1 subtypes (1a and 1c) and 4 stx2 subtypes (2a, 2c, 2d, and 2g). Forty-five isolates were divided into 21 different groups based on the Og- and Hg-types, stx gene subtypes and the existence of virulence factors, ehxA, eae, and saa, which includes several major serotypes associated with human illness such as O26:H11 and O157:H7. The most dominant isolate, OgUT:H19 [stx1a (+), stx2a (+), ehxA (+) and saa (+)], was isolated from eight farms. This is the first report on the characterization of STEC in cattle in Mongolia, and the results suggest the importance of further monitoring of STEC contamination in the food chains as well as STEC infection in humans.

Genomic analysis of an outbreak of Shiga toxin-producing Escherichia coli O183:H18 in the United Kingdom, 2023.

In June 2023, UKHSA surveillance systems detected an outbreak of severe gastrointestinal symptoms caused by a rare serotype of Shiga toxin-producing Escherichia coli, STEC O183:H18. There were 26 cases aged 6 months to 74 years (42 % cases were aged 0-9 years), distributed across the UK with onset dates range between 22 May 2023 and 4 July 2023. The epidemiological and food chain investigations were inconclusive, although meat products made from beef mince were implicated as a potential vehicle. The outbreak strain belonged to sequence type (ST) 657 and harboured a Shiga toxin (stx) subtype stx2a located on a prophage that was unique in the UKHSA stx-encoding bacteriophage database. Plasmid encoded, putative virulence genes subA, ehxA, saa, iha, lpfA and iss were detected, however, the established STEC virulence genes involved in attachment to the gut mucosa (eae and aggR) were absent. The acquisition of stx across the global population structure of ST657 appeared to correspond with the presence of subA, ehxA, saa, iha, lpfA and iss. During the outbreak investigation, we used long read sequencing to characterise the plasmid and prophage content of this atypical STEC, to look for evidence to explain its recent emergence. Although we were unable to determine source and transmission route of the outbreak strain, the genomic analysis revealed potential clues as to how novel strains for STEC evolve. With the implementation of PCR capable of detecting all STEC, and genome sequencing for typing and virulence profiling, we have the tools to enable us to monitor the changing landscape of STEC. Improvements in the standardised collection of epidemiological data and trace-back strategies within the food industry, will ensure we have a surveillance system capable of alerting us to emerging threats to public health.

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

Shiga toxin-producing Escherichia coli (STEC) are zoonotic pathogens causing hemorrhagic colitis and hemolytic uremic syndrome (HUS) in children and the elderly. Stool samples were collected from 180 children hospitalized in five pediatric centers in Poland in 2018-2022. Direct stx1/stx2 gene detection by PCR in feces and E. coli isolates was performed. Antibiotic susceptibility was tested according to EUCAST v.12. Randomly selected isolates were serotyped with O157 antiserum and genotyped by pulsed-field gel electrophoresis (PFGE). A total of 44 E. coli isolates were confirmed as STEC by PCR. Among them, 84.4% were positive for stx2, and equally 6,8% for only stx1 and both stx1 and stx2 genes. The stx1 gene was also found in one Citrobacter freundii isolate. E. coli serotype O157 was present in 97.6% of the isolates. STEC infections most often occurred between June-October with a peak in July and August (51%). The highest, 77.8% of STEC isolates were found in the 1-5 years old group. No extended-spectrum ß-lactamases (ESBL) were found. Resistance only to amoxicillin/clavulanic acid (24.4%), piperacillin/tazobactam (3%), cefotaxime (6%), gentamicin (6%), ciprofloxacin (3%), azithromycin (3%), trimethoprim/sulfamethoxazole (24,2%) was detected. PFGE analysis showed 18 PFGE types with no clonal distribution. Eight isolates with A, B, and C PFGE types showed genetic relatedness in the type with no detection of transmission way of distribution. STEC strains pose a serious threat to human health, therefore demographic and epidemiological characteristics are crucial for their surveillance.

First Isolation of the Heteropathotype Shiga Toxin-Producing and Extra-Intestinal Pathogenic (STEC-ExPEC) E. coli O80:H2 in French Healthy Cattle: Genomic Characterization and Phylogenetic Position.

The emerging heteropathotype shigatoxigenic (STEC) and extra-intestinal pathogenic Escherichia coli (ExPEC) O80:H2 has been the second leading cause of pediatric HUS in France since the mid-2010s. In contrast with other highly pathogenic STEC serotypes, for which ruminants have clearly been identified as the main human infection source, this heteropathotype's reservoir remains unknown. In this context, we describe for the first time the isolation of seven STEC O80:H2 strains from healthy cattle on a single cattle farm in France. This study aimed at (i) characterizing the genome and (ii) investigating the phylogenetic positions of these O80:H2 STEC strains. The virulomes, resistomes, and phylogenetic positions of the seven bovine isolates were investigated using in silico typing tools, antimicrobial susceptibility testing and cgMLST analysis after short-read whole genome sequencing (WGS). One representative isolate (A13P112V1) was also subjected to long-read sequencing. The seven isolates possessed ExPEC-related virulence genes on a pR444_A-like mosaic plasmid, previously described in strain RDEx444 and known to confer multi-drug resistance. All isolates were clonally related and clustered with human clinical strains from France and Switzerland with a range of locus differences of only one to five. In conclusion, our findings suggest that healthy cattle in France could potentially act as a reservoir of the STEC-ExPEC O80:H2 pathotype.

Survival of O157 and non-O157 shiga toxin-producing Escherichia coli in Korean style kimchi.

Korean style kimchi contaminated with Shiga toxin-producing Escherichia coli (STEC) O157:H7 was the cause of an outbreak in Canada from December 2021 to January 2022. To determine if this STEC O157:H7 has greater potential for survival in kimchi than other STEC, the outbreak strain and six other STEC strains (O26:H11, O91:H21, O103:H2, O121:H19, and two O157:H7) were inoculated individually at 6 to 6.5 log CFU/g into commercially sourced kimchi and incubation at 4 °C. At intervals of seven days inoculated and control kimchi was plated onto MacConkey agar to enumerate lactose utilising bacteria. The colony counts were interpreted as enumerating the inoculated STEC, since no colonies were observed on MacConkey agar plated with uninoculated kimchi. Over eight weeks of incubation the pH was stable at 4.10 to 4.05 and the STEC strains declined by 0.7-1.0 log, with a median reduction of 0.9 log. The linear rate of reduction of kimchi outbreak STEC O157:H7 was -0.4 log per 30 days (Slope Uncertainty 0.05), which was not significantly different from the other O157 and nonO157 STEC strains (P = 0.091). These results indicate that the outbreak was not due to the presence of strain better adapted to survival in kimchi than other STEC, and that STEC can persist in refrigerated Korean style kimchi with a minimal decline over the shelf-life of the product.

Evaluating disinfectant efficacy on mixed biofilms comprising Shiga toxigenic Escherichia coli, lactic acid bacteria, and spoilage microorganisms.

This study aimed to investigate the impact of temperature and the presence of other microorganisms on the susceptibility of STEC to biocides. Mature biofilms were formed at both 10°C and 25°C. An inoculum of planktonic bacteria comprising 106 CFU/mL of spoilage bacteria and 103 CFU/mL of a single E. coli strain (O157, O111, O103, and O12) was used to form mixed biofilms. The following bacterial combinations were tested: T1: Carnobacterium piscicola + Lactobacillus bulgaricus + STEC, T2: Comamonas koreensis + Raoultella terrigena + STEC, and T3: Pseudomonas aeruginosa + C. koreensis + STEC. Tested biocides included quaternary ammonium compounds (Quats), sodium hypochlorite (Shypo), sodium hydroxide (SHyd), hydrogen peroxide (HyP), and BioDestroy®-organic peroxyacetic acid (PAA). Biocides were applied to 6-day-old biofilms. Minimum Bactericidal Concentrations (MBC) and Biofilm Eradication Concentrations (BEC) were determined. Planktonic cells and single-species biofilms exhibited greater susceptibility to sanitizers (p < 0.0001). Lactobacillus and Carnobacterium were more susceptible than the rest of the tested bacteria (p < 0.0001). Single species biofilms formed by E. coli O111, O121, O157, and O45 showed resistance (100%) to Shypo sanitizer (200 ppm) at 25°C. From the most effective to the least effective, sanitizer performance on single-species biofilms was PAA > Quats > HyP > SHyd > Shypo. In multi-species biofilms, spoilage bacteria within T1, T2, and T3 biofilms showed elevated resistance to SHyd (30%), followed by quats (23.25%), HyP (15.41%), SHypo (9.70%), and BioDestroy® (3.42%; p < 0.0001). Within T1, T2, and T3, the combined STEC strains exhibited superior survival to Quats (23.91%), followed by HyP (19.57%), SHypo (18.12%), SHyd (16.67%), and BioDestroy® (4.35%; p < 0.0001). O157:H7-R508 strains were less tolerant to Quats and Shypo when combined with T2 and T3 (p < 0.0001). O157:H7 and O103:H2 strains in mixed biofilms T1, T2, and T3 exhibited higher biocide resistance than the weak biofilm former, O145:H2 (p < 0.0001). The study shows that STEC within multi-species biofilms' are more tolerant to disinfectants.

Using CHROMagar™ STEC medium exclusively does not recover all clinically relevant Shiga toxin-producing Escherichia coli in Aotearoa, New Zealand.

Diagnostic laboratories in Aotearoa, New Zealand (NZ) refer cultures from faecal samples positive for Shiga toxin genes to the national Enteric Reference Laboratory for isolation of Shiga toxin-producing Escherichia coli (STEC) for epidemiological typing. As there was variation in the culture media being referred, a panel of 75 clinical isolates of STEC, representing 28 different serotypes, was used to assess six commercially available media and provide guidance to clinical laboratories. Recommendations were subsequently tested for a 3-month period, where STEC isolations and confirmations were assessed by whole genome sequencing analysis against the culture media referred. CHROMagar™ STEC (CH-STEC; CHROMagar Microbiology, Paris, France) or CH-STEC plus cefixime-tellurite sorbitol MacConkey agar was confirmed inferior to CH-STEC plus blood agar with vancomycin, cefsulodin, and cefixime (BVCC). The former resulted in fewer STEC types (n = 18) being confirmed compared to those from a combination of CH-STEC and BVCC (n = 42). A significant (P < .05) association with an STEC's ability to grow on CH-STEC and the presence of the ter gene cluster, and eae was observed. Culturing screen positive STEC samples onto both CH-STEC and BVCC ensures a consistently higher recovery of STEC from all clinical samples in NZ than CH-STEC alone.

Management of pediatric hemolytic uremic syndrome.

Classical clinical triad of hemolytic uremic syndrome (HUS) is microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury associated with endothelial cell injury. Several situations, including infections, medications, malignancies, and transplantation can trigger endothelial damage. On the HUS spectrum, atypical hemolytic uremic syndrome (aHUS) deserves special attention in pediatric patients, as it can cause endstage kidney disease and mortality. A dysfunction in the alternative complement pathway, either acquired or genetic, has been shown to be the main underlying cause. In the last decades, breathtaking advances have been made in understanding the pathophysiology of this rare disease, which has led to more efficient treatment. Recent studies have implicated genes in pathways beyond the alternative complement system, such as DGKE, TSEN2, and INF2 highlighting the importance of personalized management. Eculizumab has brought about dramatic improvements in the treatment of aHUS. Beyond eculizumab, there are many alternative therapeutics in the pipeline that target the complement system. Because of the rarity of aHUS, data from multiple patient registries are very important. The present report aimed to summarize the most important aspects of diagnosing and treating aHUS based on the Turkish national registry and the literature so as to improve clinical practice.

Multiple-Drug Resistant Shiga Toxin-Producing E. coli in Raw Milk of Dairy Bovine.

INTRODUCTION: Raw milk may contain pathogenic microorganisms harmful to humans, e.g., multidrug-resistant Escherichia coli non-O157:H7, which can cause severe colitis, hemolytic uremia, and meningitis in children. No studies are available on the prevalence of Shiga toxin-producing E. coli (STEC O157:H7) in sick or healthy dairy animals in the Khyber Pakhtunkhwa Province of Pakistan. AIM: This study aimed to isolate, characterize, and detect antibiotic resistance in STEC non-O157:H7 from unpasteurized milk of dairy bovines in this province. MATERIALS AND METHODS: We collected raw milk samples (n = 800) from dairy farms, street vendors, and milk shops from different parts of the Khyber Pakhtunkhwa Province. E. coli was isolated from these samples followed by latex agglutination tests for serotyping. The detection of STEC was conducted phenotypically and confirmed by the detection of virulence genes genotypically. An antibiogram of STEC isolates was performed against 12 antibiotics using the disc diffusion method. RESULTS: A total of 321 (40.12%) samples were found to be positive for E. coli in this study. These samples were processed for the presence of four virulence genes (Stx1, Stx2, ehxA, eae). Forty samples (5.0%) were STEC-positive. Of these, 38%, 25%, 19%, and 18% were positive for Stx1, Stx2, ehxA, and eae, respectively. Genotypically, we found that 1.37% of STEC isolates produced extended-spectrum beta-lactamase (ESBL) and contained the blaCTX M gene. Resistance to various antibiotics ranged from 18% to 77%. CONCLUSION: This study highlights the risk of virulent and multidrug-resistant STEC non-O157:H7 in raw milk and the need for proper quality surveillance and assurance plans to mitigate the potential public health threat.

Isolation and characterization of a relatively broad-spectrum phage against Escherichia coli.

Multiple pathogenic types or serotypes restrict treatment for colibacillosis. In addition, rising antibiotic resistance has heightened public awareness to prevent and control pathogenic Escherichia coli. The bacteriophage is a viable technique to treat colibacillosis as an alternative to antibiotics. In this study, PH444, a relatively broad-spectrum obligate lytic phage, was screened from 48 Shiga toxin-producing Escherichia coli (STEC) phages isolated from farm manure samples and sewage samples in order to conduct genome-wide analysis, biological characterization, and a bacterial challenge experiment in milk. The results demonstrated that PH444 was a T7-like phage with a double-stranded DNA of 115,111 bp that belongs to the Kuravirus and was stable at temperatures between 4 and 50 °C and a pH range of 3 to 11. After adding PH444, the bacterial load in milk could be reduced from 3 × 103 PFU/ mL to zero within 1 h. In consideration of the biological properties of phage PH444, it was, therefore, demonstrated that PH444 has the potential to be used in phage biocontrol.

Prevalence and Characteristics of Plasmid-Encoded Serine Protease EspP in Clinical Shiga Toxin-Producing Escherichia coli Strains from Patients in Sweden.

Shiga toxin-producing Escherichia coli (STEC) infection can cause a broad spectrum of symptoms spanning from asymptomatic shedding to mild and bloody diarrhea (BD) and even life-threatening hemolytic-uremic syndrome (HUS). As a member of the serine protease autotransporters of Enterobacteriaceae (SPATE) family, EspP has the ability to degrade human coagulation factor V, leading to mucosal bleeding, and also plays a role in bacteria adhesion to the surface of host cells. Here, we investigated the prevalence and genetic diversity of espP among clinical STEC isolates from patients with mild diarrhea, BD, and HUS, as well as from asymptomatic individuals, and assessed the presence of espP and its subtypes in correlation to disease severity. We found that 130 out of 239 (54.4%) clinical STEC strains were espP positive, and the presence of espP was significantly associated with BD, HUS, and O157:H7 serotype. Eighteen unique espP genotypes (GTs) were identified and categorized into four espP subtypes, i.e., espPα (119, 91.5%), espPγ (5, 3.8%), espPδ (4, 3.1%), and espPε (2, 1.5%). espPα was widely distributed, especially in strains from patients with BD and HUS, and correlated with serotype O157:H7. Serogroup O26, O145, O121, and O103 strains carried espPα only. Ten GTs were identified in espPα, and espPα/GT2 was significantly associated with severe disease, i.e., BD and HUS. Additionally, espP was strongly linked to the presence of eae gene, and the coexistence of espPα and stx2/stx2a + stx2c was closely related to HUS status. To sum up, our data demonstrated a high prevalence and genetic diversity of the espP gene in clinical STEC strains in Sweden and revealed an association between the presence of espP, espP subtypes, and disease severity. espP, particularly the espPα subtype, was prone to be present in more virulent STEC strains, e.g., "top-six" serotypes strains.

Antimicrobial resistance, ß-lactamase genotypes, and plasmid replicon types of Shiga toxin-producing Escherichia coli isolated from different animal hosts.

AIMS: The primary objective of this study was to analyze antimicrobial resistance (AMR), with a particular focus on ß-lactamase genotypes and plasmid replicon types of Shiga toxin-producing Escherichia coli (STEC) strains originating from various animal hosts. METHODS AND RESULTS: A total of 84 STEC strains were isolated from cattle (n = 32), sheep/goats (n = 26), pigeons (n = 20), and wild animals (n = 6) between 2010 and 2018 in various regions of Iran. The Kirby-Bauer susceptibility test and multiple polymerase chain reaction (PCR) panels were employed to elucidate the correlation between AMR and plasmid replicon types in STEC isolates. The predominant replicon types were IncFIC and IncFIB in cattle (46.8%), IncFIC in sheep/goats (46.1%), IncA/C in pigeons (90%), and IncP in wild animals (50%). STEC of serogroups O113, O26, and O111 harbored the IncFIB (100%), IncI1 (80%), and IncFIC + IncA/C (100%) plasmids, respectively. A remarkable AMR association was found between ciprofloxacin (100%), neomycin (68.7%), and tetracycline (61.7%) resistance with IncFIC; amoxicillin + clavulanic acid (88.8%) and tetracycline (61.7%) with IncA/C; ciprofloxacin (100%) with IncFIB; fosfomycin (85.7%) and sulfamethoxazole + trimethoprim (80%) with IncI1. IncI1 appeared in 83.3%, 50%, and 100% of the isolates harboring blaCTX-M, blaTEM, and blaOXA ß-lactamase genes, respectively. CONCLUSIONS: The emergence of O26/IncI1/blaCTX-M STEC in cattle farms poses a potential risk to public health.