Synthetic phage-based approach for sensitive and specific detection of Escherichia coli O157.

Escherichia coli O157 can cause foodborne outbreaks, with infection leading to severe disease such as hemolytic-uremic syndrome. Although phage-based detection methods for E. coli O157 are being explored, research on their specificity with clinical isolates is lacking. Here, we describe an in vitro assembly-based synthesis of vB_Eco4M-7, an O157 antigen-specific phage with a 68-kb genome, and its use as a proof of concept for E. coli O157 detection. Linking the detection tag to the C-terminus of the tail fiber protein, gp27 produces the greatest detection sensitivity of the 20 insertions sites tested. The constructed phage detects all 53 diverse clinical isolates of E. coli O157, clearly distinguishing them from 35 clinical isolates of non-O157 Shiga toxin-producing E. coli. Our efficient phage synthesis methods can be applied to other pathogenic bacteria for a variety of applications, including phage-based detection and phage therapy.

Occurrence, molecular characterization, and antimicrobial susceptibility of sorbitol non-fermenting Escherichia coli in lake water, fish and humans in central Oromia, Ethiopia.

Contaminated lake water and fish can be sources of bacterial pathogens of public health concern, including pathogenic E. coli. Within Ethiopia, specifically, Central Oromia, raw fish consumption is a common practice. Although there are few reports on occurrence of E. coli O157 in fish destined for human consumption and children under five years, information on the transmission pathways of E. coli O157 and other sorbitol non-fermenting (SN-F) E. coli from water-to-fish-to-human, and their virulence factors and antimicrobial resistant determinants along the fish supply chain is lacking. The study aimed to investigate the occurrence, molecular characteristics, and antimicrobial susceptibility of E. coli O157 and other SN-F E. coli strains in fish, lake water and humans in central Oromia, Ethiopia. A total of 750 samples (450 fish samples, 150 water samples, 150 human stool samples) were collected from five lakes and three health facilities. The samples were processed following the standard protocol recommended by European Food Safety Authority and Kirby-Bauer disc diffusion method for detection of the bacteria, and antimicrobial susceptibility tests, respectively. Molecular characterization of presumptive isolates was performed using Whole-Genome Sequencing (WGS) for serotyping, determination of virulence factors, antimicrobial resistance traits, and genetic linkage of the isolates. Overall, 3.9% (29/750) of the samples had SN-F E. coli; of which 6.7% (n = 10), 1.8% (n = 8) and 7.3% (n = 11) were retrieved from water, fish, and diarrheic human patients, respectively. The WGS confirmed that all the isolates were SN-F non-O157: H7 E. coli strains. We reported two new E. coli strains with unknown O-antigen from fish and human samples. All the strains have multiple virulence factors and one or more genes encoding for them. Genetic relatedness was observed among strains from the same sources (water, fish, and humans). Most isolates were resistant to ampicillin (100%), tetracycline (100%), cefotaxime (100%), ceftazidime (100%), meropenem (100%), nalidixic acid (93.1%) and sulfamethoxazole/trimethoprim (79.3%). Majority of the strains were resistant to chloramphenicol (58.6%) and ciprofloxacin (48.3%), while small fraction showed resistance to azithromycin (3.45%). Isolates had an overall MDR profile of 87.5%. Majority, (62.1%; n = 18) of the strains had acquired MDR traits. Genes encoding for mutational resistance and Extended-spectrum beta-lactamases (ESBL) were also detected. In conclusion, our study revealed the occurrence of virulent and MDR SN-F E. coli strains in water, fish, and humans. Although no genetic relatedness was observed among strains from various sources, the genomic clustering among strains from the same sources strongly suggests the potential risk of transmission along the supply chain at the human-fish-environment interface if strict hygienic fish production is not in place. Further robust genetic study of the new strains with unknown O-antigens, and the epidemiology of SN-F E. coli is required to elucidate the molecular profile and public health implications of the pathogens.

Detection of the chuA gene encoding the invasive enterohemorrhagic species Escherichia coli 0157:H7 using qPCR in horse feces samples on Sumbawa Island, Indonesia.

Background: Bacterial identification can be done using various testing techniques. Molecular techniques are often used to research dangerous diseases, an approach using genetic information on the pathogenic agent. The enterohemorrhagic invasive species Escherichia coli 0157:H7 was identified from the feces of working horses on the island of Sumbawa. Another advance in molecular technology is genome amplification with qPCR which is the gold standard for detecting E. coli. Aim: This study aims to detect and identify the invasive species E. coli 0157:H7 using the gene encoding chuA with the qPCR method sourced from horse feces. Methods: Fresh fecal samples from horses on Sumbawa Island were isolated and identified, then continued with molecular examination using the gene encoding chuA using the qPCR method. Results: qPCR testing in this study showed that six sample isolates that were positive for E. coli 0157:H7 were detected for the presence of the chuA gene, which is a gene coding for an invasive species of E. coli bacteria. The highest to lowest Cq values and Tm from the qPCR results of the sample isolates were 15.98 (4KJ), 14.90 (19KG), 14.6 (3KJ), 13.77 (20KG), 12.56 (5KGB), and 12.20 (6KJ). Tm values are 86.7 (4KJ), 86.69 (3KJ), 86.56 (5KGB), 85.88 (20KGB), 85.81 (19KG), and 85.74 (6KJ). Conclusion: Validation, standardization of the development, and modification of qPCR technology must be carried out to harmonize testing throughout to avoid wrong interpretation of the test results so that the determination of actions to eradicate and control diseases originating from animals in the field does not occur.

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.

The heme binding protein ChuX is a regulator of heme degradation by the ChuS protein in Escherichia coli O157:H7.

Escherichia coli O157:H7 possesses an 8-gene cluster (chu genes) that contains genes involved in heme transport and processing from the human host. Among the chu genes, four encode cytoplasmic proteins (ChuS, ChuX, ChuY and ChuW). ChuX was previously shown to be a heme binding protein and to assist ChuW in heme degradation under anaerobic conditions. The purpose of this work was to investigate if ChuX works in concert with ChuS, which is a protein able to degrade heme by a non-canonical mechanism and release the iron from the porphyrin under aerobic conditions using hydrogen peroxide as the oxidant. We showed that when the heme-bound ChuX and apo-ChuS protein are mixed, heme is efficiently transferred from ChuX to ChuS. Heme-bound ChuX displayed a peroxidase activity with ABTS and H2O2 but not heme-bound ChuS, which is an efficient test to determine the protein to which heme is bound in the ChuS-ChuX complex. We found that ChuX protects heme from chemical oxidation and that it has no heme degradation activity by itself. Unexpectedly, we found that ChuX inhibits heme degradation by ChuS and stops the reaction at an early intermediate. We determined using surface plasmon resonance that ChuX interacts with ChuS and that it forms a relatively stable complex. These results indicate that ChuX in addition to its heme transfer activity is a regulator of ChuS activity, a function that was not described before for any of the heme carrier protein that delivers heme to heme degradation enzymes.

Ferrous gluconate triggers ferroptosis in Escherichia coli: Implications of lipid peroxidation and DNA damage.

Microbial ferroptosis has been proved to combat drug-resistant pathogens, but whether this pattern can be applied to the prevention and control of Escherichia coli remains to be further explored. In this study, ferrous gluconate (FeGlu) showed remarkable efficacy in killing E. coli MG1655 with a mortality rate exceeding 99.9%, as well as enterotoxigenic E. coli H10407 (ETEC H10407) and enterohemorrhagic E. coli O157:H7 (EHEC O157:H7). Bacteria death was instigated by the infiltration of Fe2+, accompanied by a burst of intracellular reactive oxygen species (ROS) and lipid peroxidation. Notably, mitigating lipid peroxidation failed to alleviate death of E. coli. Further findings confirmed that FeGlu induced DNA damage, and ΔrecA mutant showed more sensitive, implicating that DNA damage was involved in the death of E. coli. The direct interaction of Fe2+ with DNA was demonstrated by fluorescent staining, gel electrophoresis, and circular dichroism (CD). Moreover, proteomic analysis unveiled 50 differentially expressed proteins (DEPs), including 18 significantly down-regulated proteins and 32 significantly up-regulated proteins. Among them, the down-regulation of SOS-responsive transcriptional suppressor LexA indicated DNA damage induced severely by FeGlu. Furthermore, FeGlu influenced pathways such as fatty acid metabolism (FadB, FadE), iron-sulfur cluster assembly (IscA, IscU, YadR), iron binding, and DNA-binding transcription, along with α-linolenic acid metabolism, fatty acid degradation, and pyruvate metabolism. These pathways were related to FeGlu stress, including lipid peroxidation and DNA damage. In summary, FeGlu facilitated ferroptosis in E. coli through mechanisms involving lipid peroxidation and DNA damage, which presents a new strategy for the development of innovative antimicrobial strategies targeting E. coli infections.

Decoding the Structure-Function Relationship of the Muramidase Domain in E. coli O157.H7 Bacteriophage Endolysin: A Potential Building Block for Chimeric Enzybiotics.

Bacteriophage endolysins are potential alternatives to conventional antibiotics for treating multidrug-resistant gram-negative bacterial infections. However, their structure-function relationships are poorly understood, hindering their optimization and application. In this study, we focused on the individual functionality of the C-terminal muramidase domain of Gp127, a modular endolysin from E. coli O157:H7 bacteriophage PhaxI. This domain is responsible for the enzymatic activity, whereas the N-terminal domain binds to the bacterial cell wall. Through protein modeling, docking experiments, and molecular dynamics simulations, we investigated the activity, stability, and interactions of the isolated C-terminal domain with its ligand. We also assessed its expression, solubility, toxicity, and lytic activity using the experimental data. Our results revealed that the C-terminal domain exhibits high activity and toxicity when tested individually, and its expression is regulated in different hosts to prevent self-destruction. Furthermore, we validated the muralytic activity of the purified refolded protein by zymography and standardized assays. These findings challenge the need for the N-terminal binding domain to arrange the active site and adjust the gap between crucial residues for peptidoglycan cleavage. Our study shed light on the three-dimensional structure and functionality of muramidase endolysins, thereby enriching the existing knowledge pool and laying a foundation for accurate in silico modeling and the informed design of next-generation enzybiotic treatments.

Differential microbiota shift on whole romaine lettuce subjected to source or forward processing and on fresh-cut products during cold storage.

Romaine lettuce in the U.S. is primarily grown in California or Arizona and either processed near the growing regions (source processing) or transported long distance for processing in facilities serving distant markets (forward processing). Recurring outbreaks of Escherichia coli O157:H7 implicating romaine lettuce in recent years, which sometimes exhibited patterns of case clustering in Northeast and Midwest, have raised industry concerns over the potential impact of forward processing on romaine lettuce food safety and quality. In this study, freshly harvested romaine lettuce from a commercial field destined for both forward and source processing channels was tracked from farm to processing facility in two separate trials. Whole-head romaine lettuce and packaged fresh-cut products were collected from both forward and source facilities for microbiological and product quality analyses. High-throughput amplicon sequencing targeting16S rRNA gene was performed to describe shifts in lettuce microbiota. Total aerobic bacteria and coliform counts on whole-head lettuce and on fresh-cut lettuce at different storage times were significantly (p < 0.05) higher for those from the forward processing facility than those from the source processing facility. Microbiota on whole-head lettuce and on fresh-cut lettuce showed differential shifting after lettuce being subjected to source or forward processing, and after product storage. Consistent with the length of pre-processing delays between harvest and processing, the lettuce quality scores of source-processed romaine lettuce, especially at late stages of 2-week storage, was significantly higher than of forward-processed product (p < 0.05).

CRISPR/Cas12a powered air-displacement enhanced evanescent wave fluorescence fiber-embedded microfluidic biochip for nucleic acid amplification-free detection of Escherichia coli O157:H7.

Simple yet ultrasensitive and contamination-free quantification of environmental pathogenic bacteria is in high demand. In this study, we present a portable clustered regularly interspaced short palindromic repeats-associated protein 12a (CRISPR/Cas12a) powered Air-displacement enhanced Evanescent wave fluorescence Fiber-embedded microfluidic Biochip (AEFB) for the high-frequency and nucleic acid amplification-free ultrasensitive detection of Escherichia coli O157:H7. The performance of AEFB was dramatically enhanced upon employing a simple air-solution displacement process. Theoretical assays demonstrated that air-solution displacement significantly enhances evanescent wave field intensity on the fiber biosensor surface and increases the V-number in tapered fiber biosensors. Consequently, light-matter interaction is strengthened, and fluorescence coupling and collection efficiency are improved, considerably enhancing sensitivity. By integrating the CRISPR biosensing mechanism, AEFB facilitated rapid, accurate, nucleic acid amplification-free detection of E.coli O157:H7 with polymerase chain reaction (PCR)-level sensitivity (176 cfu/mL). To validate its practicality, AEFB was used to detect E.coli O157:H7 in surface water and wastewater. Comparison with RT-PCR showed a strong linear relationship (R2 = 0.9871), indicating the excellent accuracy and reliability of this technology in real applications. AEFB is highly versatile and can be easily extended to detect other pathogenic bacteria, which will significantly promote the high-frequency assessment and early-warning of bacterial contamination in aquatic environments.

Genomic island-encoded regulatory proteins in enterohemorrhagic Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) is an important zoonotic pathogen that is a major cause of foodborne diseases in most developed and developing countries and can cause uncomplicated diarrhoea, haemorrhagic colitis, and haemolytic uraemic syndrome. O islands (OIs), which are unique genomic islands in EHEC O157:H7, are composed of 177 isolated genomic features and harbour 26% of the total genes that are absent in the non-pathogenic E. coli K-12 genome. In the last twenty years, many OI-encoded proteins have been characterized, including proteins regulating virulence, motility, and acid resistance. Given the critical role of regulatory proteins in the systematic and hierarchical regulation of bacterial biological processes, this review summarizes the OI-encoded regulatory proteins in EHEC O157:H7 characterized to date, emphasizing OI-encoded regulatory proteins for bacterial virulence, motility, and acid resistance. This summary will be significant for further exploration and understanding of the virulence and pathogenesis of EHEC O157:H7.

Highly sensitive SERS platform for pathogen analysis by cyclic DNA nanostructure@AuNP tags and cascade primer exchange reaction.

The capacity to identify small amounts of pathogens in real samples is extremely useful. Herein, we proposed a sensitive platform for detecting pathogens using cyclic DNA nanostructure@AuNP tags (CDNA) and a cascade primer exchange reaction (cPER). This platform employs wheat germ agglutinin-modified Fe3O4@Au magnetic nanoparticles (WMRs) to bind the E. coli O157:H7, and then triggers the cPER to generate branched DNA products for CDNA tag hybridization with high stability and amplified SERS signals. It can identify target pathogens as low as 1.91 CFU/mL and discriminate E. coli O157:H7 in complex samples such as water, milk, and serum, demonstrating comparable or greater sensitivity and accuracy than traditional qPCR. Moreover, the developed platform can detect low levels of E. coli O157:H7 in mouse serum, allowing the discrimination of mice with early-stage infection. Thus, this platform holds promise for food analysis and early infection diagnosis.

Oral phages prophylaxis against mixed Escherichia coli O157:H7 and Salmonella Typhimurium infections in weaned piglets.

Escherichia coli and Salmonella Typhimurium are the main pathogens of diarrhea in weaned piglets. The prevention of bacterial diarrhea in weaned piglets by phage is rarely reported. We conducted this study to evaluate the preventive effect of phages on mixed Escherichia coli and Salmonella Typhimurium infections in weaned piglets. A novel phage named NJ12 was isolated by using Salmonella Typhimurium SM022 as host bacteria and characterized by electron microscopy, genomic analysis and in vitro bacteriostatic activity. Phage NJ12 and a previously reported phage EP01 were microencapsulated with sodium alginate to make phage cocktail. Microencapsulated phage cocktail and PBS (Phosphate buffer solution) were used to piglets the phage and phage-free group through oral administration before bacterial infection 2 h, respectively. Piglets of the phage and phage-free group were consumed with feed contaminated with 6 mL (108CFU/mL) Escherichia coli O157:H7 GN07 (GXEC-N07) and 6 mL (108CFU/mL) SM022 every day for seven consecutive days. The results showed that piglets in the phage-free group had more severe diarrhea, larger decreased average weight gain and higher levels of neutrophils compared with piglets in phage group. Meanwhile, piglets in the phage-free group had higher load of SM022 and GN07 in jejunal tissue and more severe intestinal damage compared with piglets in group phage in vivo. In addition, oral administration phage can significant decreased the relative abundance of Enterobacteriaceae but hardly repaired the changes of diversity and composition of gut microbiota caused by the mixed infection of SM022 and GN07. This implies that phage used as a feed additive have a marvelous preventive effect on bacterial diarrhea during weaning of piglets.

G-triplex/hemin DNAzyme mediated colorimetric aptasensor for Escherichia coli O157:H7 detection based on exonuclease III-assisted amplification and aptamers-functionalized magnetic beads.

Escherichia coli O157: H7 (E. coli O157: H7) is one of the most common foodborne pathogens and is widespread in food and the environment. Thus, it is significant for rapidly detecting E. coli O157: H7. In this study, a colorimetric aptasensor based on aptamer-functionalized magnetic beads, exonuclease III (Exo III), and G-triplex/hemin was proposed for the detection of E. coli O157: H7. The functional hairpin HP was designed in the system, which includes two parts of a stem containing the G-triplex sequence and a tail complementary to cDNA. E. coli O157: H7 competed to bind the aptamer (Apt) in the Apt-cDNA complex to obtain cDNA. The cDNA then bound to the tail of HP to trigger Exo III digestion and release the single-stranded DNA containing the G-triplex sequence. G-triplex/hemin DNAzyme could catalyze TMB to produce visible color changes and detectable absorbance signals in the presence of H2O2. Based on the optimal conditions, E. coli O157: H7 could be detected down to 1.3 × 103 CFU/mL, with a wide linear range from 1.3 × 103 to 1.3 × 107 CFU/mL. This method had a distinguished ability to non-target bacteria, which showed good specificity. In addition, the system was successfully applied to detect E. coli O157: H7 in milk samples.

Engineering of a Chimeric Template Triggers RNase H-Based Isothermal Amplification Approach for Sensitive Detection of Pathogen RNA.

RNA-based detection of pathogenic organisms is an emerging field of research that is crucial for disease diagnosis and environmental and food safety. By rationally engineering an RNA-DNA tandem (RDT) structural template, we proposed a novel RNase H-based isothermal exponential amplification (RH-IEA) reaction to rapidly identify long-stranded RNA. In this strategy, the rigid and compact RDT template selectively recognized the target RNA and formed a stable hybrid with it. Upon site-specific cleavage of RNase H, the 3' overhang of the target RNA was cut off, and a free hydroxyl end at the hydrolysis site was generated to trigger an exponential amplification reaction (EXPAR). This method maintained the high efficiency and rapid amplification kinetics of EXPAR. As a result, the RH-IEA strategy was able to sensitively and specifically detect the characteristic sequence of Escherichia coli O157:H7 RNA, with a detection sensitivity of 1 fg/µL. Besides, the RDT template can be used as an RNA protector to prevent specific segments of the target RNA from being degraded by RNase enzymes, allowing the sample to be stored at room temperature for a long time. With this advantage, the practicality of RH-IEA will be more flexible than the reverse transcription polymerase chain reaction. It was successfully applied in the identification of E. coli O157:H7 in milk with a minimum detection concentration of 1.0 × 102 CFU/mL. Therefore, the RH-IEA method will serve as a powerful tool for detecting long-stranded RNA and will also shed light on the pathogen detection in food safety and molecular diagnosis.

Genetic Characterization of Intimin Gene (eae) in Clinical Shiga Toxin-Producing Escherichia coli Strains from Pediatric Patients in Finland.

Shiga toxin (Stx)-producing Escherichia coli (STEC) infections cause outbreaks of severe disease in children ranging from bloody diarrhea to hemolytic uremic syndrome (HUS). The adherent factor intimin, encoded by eae, can facilitate the colonization process of strains and is frequently associated with severe disease. The purpose of this study was to examine and analyze the prevalence and polymorphisms of eae in clinical STEC strains from pediatric patients under 17 years old with and without HUS, and to assess the pathogenic risk of different eae subtypes. We studied 240 STEC strains isolated from pediatric patients in Finland with whole genome sequencing. The gene eae was present in 209 (87.1%) strains, among which 49 (23.4%) were from patients with HUS, and 160 (76.6%) were from patients without HUS. O157:H7 (126, 60.3%) was the most predominant serotype among eae-positive STEC strains. Twenty-three different eae genotypes were identified, which were categorized into five eae subtypes, i.e., γ1, ß3, ε1, θ and ζ3. The subtype eae-γ1 was significantly overrepresented in strains from patients aged 5-17 years, while ß3 and ε1 were more commonly found in strains from patients under 5 years. All O157:H7 strains carried eae-γ1; among non-O157 strains, strains of each serotype harbored one eae subtype. No association was observed between the presence of eae/its subtypes and HUS. However, the combination of eae-γ1+stx2a was significantly associated with HUS. In conclusion, this study demonstrated a high occurrence and genetic variety of eae in clinical STEC from pediatric patients under 17 years old in Finland, and that eae is not essential for STEC-associated HUS. However, the combination of certain eae subtypes with stx subtypes, i.e., eae-γ1+stx2a, may be used as risk predictors for the development of severe disease in children.

Comparative genomics analysis and characterization of Shiga toxin-producing Escherichia coli O157:H7 strains reveal virulence genes, resistance genes, prophages and plasmids.

Escherichia coli O157:H7 is a foodborne pathogen that has been linked to global disease outbreaks. These diseases include hemorrhagic colitis and hemolytic uremic syndrome. It is vital to know the features that make this strain pathogenic to understand the development of disease outbreaks. In the current study, a comparative genomic analysis was carried out to determine the presence of structural and functional features of O157:H7 strains obtained from 115 National Center for Biotechnology Information database. These strains of interest were analysed in the following programs: BLAST Ring Image Generator, PlasmidFinder, ResFinder, VirulenceFinder, IslandViewer 4 and PHASTER. Five strains (ECP19-198, ECP19-798, F7508, F8952, H2495) demonstrated a great homology with Sakai because of a few regions missing. Five resistant genes were identified, however, Macrolide-associated resistance gene mdf(A) was commonly found in all genomes. Majority of the strains (97%) were positive for 15 of the virulent genes (espA, espB, espF, espJ, gad, chuA, eae, iss, nleA, nleB, nleC, ompT, tccP, terC and tir). The plasmid analysis demonstrated that the IncF group was the most prevalent in the strains analysed. The prophage and genomic island analysis showed a distribution of bacteriophages and genomic islands respectively. The results indicated that structural and functional features of the many O157:H7 strains differ and may be a result of obtaining mobile genetic elements via horizontal gene transfer. Understanding the evolution of O157:H7 strains pathogenicity in terms of their structural and functional features will enable the development of detection and control of transmission strategies.

Enterohaemorrhagic E. coli utilizes host- and microbiota-derived L-malate as a signaling molecule for intestinal colonization.

The mammalian gastrointestinal tract is a complex environment that hosts a diverse microbial community. To establish infection, bacterial pathogens must be able to compete with the indigenous microbiota for nutrients, as well as sense the host environment and modulate the expression of genes essential for colonization and virulence. Here, we found that enterohemorrhagic Escherichia coli (EHEC) O157:H7 imports host- and microbiota-derived L-malate using the DcuABC transporters and converts these substrates into fumarate to fuel anaerobic fumarate respiration during infection, thereby promoting its colonization of the host intestine. Moreover, L-malate is important not only for nutrient metabolism but also as a signaling molecule that activates virulence gene expression in EHEC O157:H7. The complete virulence-regulating pathway was elucidated; the DcuS/DcuR two-component system senses high L-malate levels and transduces the signal to the master virulence regulator Ler, which in turn activates locus of enterocyte effacement (LEE) genes to promote EHEC O157:H7 adherence to epithelial cells of the large intestine. Disruption of this virulence-regulating pathway by deleting either dcuS or dcuR significantly reduced colonization by EHEC O157:H7 in the infant rabbit intestinal tract; therefore, targeting these genes and altering physiological aspects of the intestinal environment may offer alternatives for EHEC infection treatment.

Lcn2 deficiency accelerates the infection of Escherichia coli O157:H7 by disrupting the intestinal barrier function.

Bacterial infections result in intestinal inflammation and injury, which affects gut health and nutrient absorption. Lipocalin 2 (Lcn2) is a protein that reacts to microbial invasion, inflammatory responses, and tissue damage. However, it remains unclear whether Lcn2 has a protective effect against bacterial induced intestinal inflammation. Therefore, this study endeavors to investigate the involvement of Lcn2 in the intestinal inflammation of mice infected with Enterohemorrhagic Escherichia coli O157:H7 (E. coli O157:H7). Lcn2 knockout (Lcn2-/-) mice were used to evaluate the changes of inflammatory responses. Lcn2 deficiency significantly exacerbated clinical symptoms of E. coli O157:H7 infection by reducing body weight and encouraging bacterial colonization of. Compared to infected wild type mice, infected Lcn2-/- mice had significantly elevated levels of pro-inflammatory cytokines in serum and ileum, including interleukin (IL)-6, IL-1ß, and tumor necrosis factor-α (TNF-α), as well as severe villi destruction in the jejunum. Furthermore, Lcn2 deficiency aggravated intestinal barrier degradation by significantly reducing the expression of tight junction proteins occludin and claudin 1, the content of myeloperoxidase (MPO) in the ileum, and the number of goblet cells in the colon. Our findings indicated that Lcn2 could alleviate inflammatory damage caused by E. coli O157:H7 infection in mice by enhancing intestinal barrier function.

A paper-embedded thermoplastic microdevice integrating additive-enhanced allele-specific amplification and silver nanoparticle-based colorimetric detection for point-of-care testing.

This study introduces a thermoplastic microdevice integrated with additive-enhanced allele-specific amplification and hydrazine-induced silver nanoparticle-based detection of single nucleotide polymorphism (SNP) and opportunistic pathogens. For point-of-care testing of SNP, an allele-specific loop-mediated isothermal amplification reaction using nucleotide-mismatched primers and molecular additives was evaluated to discriminate single-nucleotide differences in the samples. The microdevice consists of purification and reaction units that enable DNA purification, amplification, and detection in a sequential manner. The purification unit enables the silica-based preparation of samples using an embedded glass fiber membrane. Hydrazine-induced silver nanoparticle formation was employed for endpoint colorimetric detection of amplicons within three min at room temperature. The versatile applicability of the microdevice was demonstrated by the successful identification of SNPs related to sickle cell anemia, genetically-induced hair loss, and Enterococcus faecium. The microdevice exhibited a detection limit of 103 copies per µL of SNP targets in serum and 102 CFU mL-1 of Enterococcus faecium in tap water within 70 min. The proposed microdevice is a promising and versatile platform for point-of-care nucleic acid testing of different samples in low-resource settings.

Establishment of real-time fluorescence and visual LAMP for rapid detection of Escherichia coli O157:H7 and kits construction.

Escherichia coli O157:H7 is a common pathogenic bacterium in food and water that can pose a threat to human health. The aim of this study was to develop loop-mediated isothermal amplification (LAMP) method for the detection of E. coli O157:H7 in food based on the specific gene Ecs_2840 and to construct rapid detection kits based on the established methods. Specifically, we established two methods of real-time fluorescent LAMP (RT-LAMP) and visual LAMP with calcein as an indicator. In pure bacterial culture, the cell sensitivity and genomic sensitivity of the RT-LAMP kit were 8.8 × 100 CFU ml-1 and 4.61 fg µl-1, respectively. The sensitivity of the visual LAMP kit was 2.35 × 100 CFU ml-1 and 4.61 fg µl-1. Both kits had excellent specificity and anti-interference performance. In addition, milk inoculated with 2.26 × 100 CFU ml-1E. coli O157:H7 could be detected within the reaction time after enrichment for 3 h. The results showed that the LAMP kits were rapid, sensitive, and specific for the detection of E. coli O157:H7 in food and had good application prospects in food safety surveillance.