First molecular characterization of Escherichia coli O157:H7 isolates from clinical samples in Paraguay using whole-genome sequencing Molecular characterization of Escherichia coli O157 isolates from Paraguay / Primera caracterización molecular de aislamientos de Escherichia coli O157:H7 provenientes de muestras clínicas en Paraguay utilizando secuenciación de genoma completo

Abstract Escherichia coli O157:H7 is a foodborne pathogen implicated in numerous outbreaks worldwide that has the ability to cause extra-intestinal complications in humans. The Enteropathogens Division of the Central Public Health Laboratory (CPHL) in Paraguay is working to improve the genomic characterization of Shiga toxin-producing E. coli (STEC) to enhance laboratory-based surveillance and investigation of foodborne disease outbreaks. Whole genome sequencing (WGS) is proposed worldwide to be used in the routine laboratory as a high-resolution tool that allows to have all the results in a single workflow. This study aimed to carry out for the first time, the genomic characterization by WGS of nine STEC O157:H7 strains isolated from human samples in Paraguay. We were able to identify virulence and resistance mechanisms, MLST subtype, and even establish the phylogenetic relationships between isolates. Furthermore, we detected the presence of strains belonging to hypervirulent clade 8 in most of the isolates studied.

Resumen Escherichia coli O157:H7 es un patógeno transmitido por alimentos implicado en numerosos brotes en todo el mundo y es capaz de causar complicaciones extraintestinales en humanos. La sección de «Enteropatógenos¼ del Laboratorio Central de Salud Pública trabaja en mejorar la caracterización genómica de STEC, de modo de potenciar la vigilancia laboratorial y la investigación de brotes de enfermedades transmitidas por alimentos. La secuenciación de genoma completo (WGS, por sus siglas en inglés) se propone a nivel mundial como una herramienta de alta resolución para ser utilizada en el laboratorio de rutina, ya que permite obtener todos los resultados en un único proceso. El objetivo de este trabajo fue llevar a cabo, por primera vez, la caracterización genómica por WGS de nueve cepas STEC O157:H7 aisladas en Paraguay a partir de muestras de origen humano. Pudimos identificar los factores de virulencia, los mecanismos de resistencia, el subtipo MLST, e incluso pudimos establecer la relación filogenética entre los aislamientos. Además, detectamos que la mayoría de las cepas pertenecían al clado hipervirulento 8.

Multi-enzyme isothermal rapid amplification assay for the detection of Escherichia coli O157∶H7 / 上海预防医学

ObjectiveA rapid enrichment and detection method for Escherichia coli O157∶H7 was developed by using multienzyme isothermal rapid amplification （MIRA） fluorescence method combined with metal organic frameworks immunomagnetic beads. MethodsUsing rfbE gene as the target， the primers， probes and reaction system were screened， and the specificity， sensitivity and practical application of this method were investigated. ResultsThe detection limit of Escherichia coli O157∶H7 was 1.18×105 CFU‧mL-1， and the detection limit of DNA concentration was 9 pg‧μL-1. The detection process was completed in 20 minutes. The test results of 47 strains （24 target strains and 23 non-target strains） were consistent with real-time PCR （RT-PCR）. ConclusionA method based on metal-organic framework immunomagnetic beads enrichment combined with MIRA assay is developed in this study. The method is simple， rapid and suitable for rapid enrichment and detection of Escherichia coli O157∶H7 in food.

Cambios en el perfil protéico de E. coli O157:H7 frente al tratamiento con Ib-M1 e IONP@Ib-M1 / Changes in the protein profile of E. coli O157: H7 against treatment with Ib-M1 and IONP@Ib-M1 / Identificação de alterações no perfil protetor de E. coli O157: H7 contra o tratamento com Ib-M1 e IONP@Ib-M1

Resumen Escherichia coli 0157:H7 es una bacteria patógena reconocida por su capacidad de resistencia a diversos antibióticos; razón por la cual, se generan complicaciones en el tratamiento de infecciones producidas por esta bacteria. El péptido Ib-M1 y el bioconjugado I0NP@Ib-M1 han surgido como una nueva alternativa antimicrobiana contra E. coli 0157:H7. El mecanismo de acción de Ib-Mi e I0NP@Ib-M1 contra esta bacteria aún es desconocido; por lo tanto, el objetivo de esta investigación fue identificar el cambio en el perfil de proteínas de E. coli 0157:H7 luego del tratamiento con Ib-M1 e I0NP@ Ib-M1 como primer paso para determinar su mecanismo de acción. Para esto, se llevó a cabo la obtención de proteínas, posteriormente se realizó una electroforesis bidimensional para finalmente realizar la determinación de la variabilidad de los perfiles proteicos. Una vez obtenidos estos perfiles, se llevó a cabo un análisis de varianza (AN0VA). Se identificaron 72 proteínas expresadas diferencialmente, las cuales pueden relacionarse con el efecto sobre el crecimiento de la bacteria en presencia de Ib-M1 e I0NP@Ib-M. Estas proteínas se encuentran involucradas en procesos de transferencia de grupos acilo (proteína Yhbs), translocación de lipoproteínas (proteína LolA) y transporte de aminoácidos (proteína GpmA), entre otros.

Abstract Escherichia coli 0157: H7 is a pathogenic bacterium which is recognized for the ability to resist multiple antibiotics; accordingly, complications occur in the treatment of infections caused by this bacterium. The Ib-M1 peptide and the I0NP @ Ib-M1 bioconjugate have emerged as a new antimicrobial alternatives against E. coli 0157: H7. The mechanism of action of Ib-M1 and I0NP @ Ib-M1 against this bacterium is still unknown; therefore, the goal of this research was to identify the change in the proteins profile of E. coli 0157: H7 after treatment with Ib-M1 and I0NP @ Ib-M1 as a first step to determine its mechanism of action. For this, the proteins were obtained first, and then a two-dimensional electrophoresis was performed to finally determine the variability of the protein profiles. 0nce the protein profiles were obtained, an analysis of variance (AN0VA) was carried out. 72 differentially expressed proteins were identified, which can be connected to the effect on the bacterium's growth in the presence of Ib-M1 and I0NP @ Ib-M. These proteins are involved in acyl groups transfer processes (Yhbs protein), lipoprotein translocation (LolA protein) and amino acid transport (GpmA protein), among others.

Resumo Escherichia coli O157: H7 é uma bactéria patogênica reconhecida por sua capacidade de resistir a vários antibióticos; razão pela qual, complicações são geradas no tratamento de infecções produzidas por essa bactéria. O peptídeo Ib-M1 livre e imobilizado em nanopartículas magnéticas de óxido de ferro (IONP @ Ib-M1) surgiu como uma nova alternativa antimicrobiana contra E. coli O157: H7 e isolados clínicos desta bactéria. O mecanismo de ação de Ib-M1 e IONP @ Ib-M1 contra E. coli O157: H7 ainda é desconhecido; Portanto, o objetivo desta pesquisa foi identificar a alteração no perfil proteico de E. coli O157: H7 após o tratamento com Ib-M1 e IONP @ Ib-M1 como um primeiro passo para determinar seu mecanismo de ação. Para isso, foi realizada a obtenção das proteínas, posteriormente foi realizada uma eletroforese bidimensional para finalmente determinar a variabilidade dos perfis protéicos. Uma vez obtidos os perfis de proteínas, foi realizada uma análise de variância (ANOVA). Os resultados mostram a identificação de proteínas expressas diferencialmente e que estão envolvidas em processos de transferência de grupos acila (proteína Yhbs), translocação de lipoproteínas (proteína LolA) e transporte de aminoácidos (proteína GpmA), entre outros.

Research progress on antibiotic resistance of Escherichia coli O157:H7 / 预防医学

@#Contamination of foodborne pathogens is the main cause of related diseases. Escherichia coli O157:H7 (E.coli O157:H7), as a representative of pathogenic E.coli, is one of the most severe and commonly reported E.coli in the world, but there is still no effective clinical treatment against the infection. Antibiotics show effective in the early infection of E.coli O157:H7. However, their extensive use has led to drug-resistant bacteria and genes in recent years, which becomes a great threat to public health. This article reviews the research progress of E.coli O157:H7 from the prevalence of antibiotic-resistant bacteria, the resistance mechanism, and the prevention and control methods, in order to provide a reference for its prevention, early clinical treatment and related research.

First detection of Shiga toxin producing Escherichia coli O157:H7 (HlyA gene) and enumeration of most probable number (MPN) of fecal coliforms and Escherichia coli in cage cultured oysters (Crassostrea iredalei) and water from southern Malaysia

Aims@#Escherichia coli O157:H7 is known to be transmitted via fecal-oral route, where water plays a role in the transmission process. Oysters as bivalves, bio accumulate pathogens from the water through filter feeding and are suspected to play a role as disease transmission vector. In Malaysia, the data on oyster’s microbiological quality are limited. Hence, it was vital to conduct oyster related studies in Malaysia. The main objectives of this study include the enumeration of most probable number (MPN) of fecal coliforms and E. coli and isolation of E. coli from oyster (Crassostrea iredalei) and water sample for the detection of 16S rRNA and HlyA (Hemolysin A) genes of E. coli O157:H7. @*Methodology and results@#A total of 120 oysters and water samples (n=6) were collected from a fisherman village located in southern Malaysia. Total fecal coliforms and E. coli were determined using the MPN procedure. Colonies of E. coli were identified based on Gram staining, biochemical test, and PCR detection for the presence of 16S rRNA and HlyA gene of E. coli O157:H7. The enumeration results showed that the MPN of the fecal coliforms and E. coli found in the collected oyster samples do not meet the standard to be directed for human consumption (0.72 ± 0.19 × 104 MPN/100 g and 0.13 ± 0.03 × 10 4 MPN/100 g, respectively). The PCR assays showed that 16 out of the 104 (15.38%) of E. coli isolated from water and oysters showed the presence of HlyA gene. The phylogenetic tree analysis showed there were genetic relationships between the HlyA gene of the E. coli isolated in this study with the ones isolated from calf and human faeces.@*Conclusion, significance and impact of study@#The detection of Shiga toxin producing E. coli O157:H7 (HlyA gene) in cage cultured oysters (C. iredalei) and water from southern Malaysia was first time reported here. In the future, more study can be conducted to study the expression of the HlyA gene and confirm of its identity as E. coli O157:H7 using different target genes such as eaeA (encodes a 94 kD outer membrane protein called intimin) and Stx1 (Shiga toxin, Shigella dysenteriae type 1).

Evaluación de Lactobacillus plantarum microencapsulado y su viabilidad bajo condiciones gastrointestinales simuladas e inhibición frente a Escherichia coli O157:H7 / Evaluation of microencapsulated Lactobacillus plantarum and its viability under simulated gastrointestinal conditions and inhibition against Escherichia coli O157: H7

RESUMEN Se evaluó la viabilidad de Lactobacillus plantarum microencapsulado, su efecto de inhibición sobre Escherichia coli O157:H7 y su crecimiento en condiciones gastrointestinales simuladas. Se utilizaron L. plantarum ATCC 8014® y E. coli ATCC 43888®. Se realizaron pruebas de inhibición de L. plantarum sobre E. coli y test de susceptibilidad a CTX (30 µg), P (10 IU), GN (10 µg), DCX (1 µg), CIP (5 µg) y KF (30 µg) para ambas cepas. En la bacteria láctica se determinó la cinética de fermentación y la presencia de péptidos y aminoácidos por HPLC en ambas cepas. Se evaluó el crecimiento a 37 y 45°C de L. plantarum y se valoró la viabilidad de su microencapsulación mediante condiciones gastrointestinales (bilis, sales biliares y pH ácido), al igual que la supervivencia y estabilidad de preparado y sus características físicas y morfológicas. Los resultados indicaron que L. plantarum inhibió a E. coli y el microencapsulado, resultados positivos con una viabilidad del 83,3%; eficiencia de 88,4%; humedad de 7,79%; actividad de agua 0,4; humectabilidad de 1 min, 56 s; solubilidad del 96%; morfología esférica y tamaño entre 15,18 a 35,68 pm. Finalmente, se observó un alto potencial de L. plantarum como agente inhibidor para E. coli O157:H7.

ABSTRACT The viability of microencapsulated Lactobacillus plantarum, its inhibition effect on Escherichia coli O157: H7 and growth in simulated gastrointestinal conditions was evaluated. L. plantarum ATCC 8014® and E. coli ATCC 43888® were used. Inhibition tests of L. plantarum on E. coli and susceptibility test to CTX (30 µg), P (10 IU), GN (10 µg) DCX (1 µg), CIP (5 µg) and KF (30) were performed µg) for both strains. In the lactic bacterium the fermentation kinetics were determined, and the presence of peptides and amino acids by HPLC in both strains. The growth at 37 ° C and 45 ° C of L. plantarum was evaluated and the viability of its microencapsulation was assessed by gastrointestinal conditions (Bile, Bile salts and acidic pH), as well as the survival and stability of the preparation and its physical characteristics and morphological. The results indicated that L. plantarum inhibited E. coli and the microencapsulated positive results with a viability of 83.3%, efficiency 88.4%, Humidity 7.79%, water activity 0.4, wettability of 1 min, 56 s, 96% solubility, spherical morphology and size between 15.18 to 35.68 pm. Finally, a high potential of L. plantarum was observed as an inhibitory agent for E. coli O157: H7.

Síndrome hemolítico urémico asociado a Shigatoxina: ¿Cómo prevenirlo? / Shiga-toxin associated hemolytic uremic syndrome: how to prevent it?

Resumen: El síndrome hemolítico urémico (SHU) asociado a infección intestinal por bacterias productoras de Shigatoxina, que afecta principalmente a población infantil, puede causar morbilidad aguda grave, secuelas crónicas en varios órganos, y la muerte prematura en algunos de ellos. Dado su carácter zoonótico, adecuadas medidas de manejo agropecuario y correcta higiene de lo que consumimos es indispensable a la hora de prevenir la infección. Actualmente, una vez gatillado el SHU el manejo es médico y, principalmente, de soporte. En los últimos años diversas estrategias terapéuticas se han ido desarrollando para evitar que esta enfermedad ocurra, o, al menos, que pueda ser atenuada en sus consecuencias de morbi-mortalidad. El presente artículo describe acciones específicas a diferentes niveles de prevención de esta patología.

Abstract Hemolytic uremic syndrome (HUS) associated with intestinal infection by Shiga toxin-producing bacteria, which mainly affects children, can cause severe acute morbidity, chronic sequelae in seve ral organs, and premature death in some of them. Given its zoonotic nature, adequate measures of agricultural management and proper hygiene of what we consume are essential to prevent infection. Once the HUS is triggered, medical management is currently mainly supportive. In recent years, va rious therapeutic strategies have been developed to prevent this disease from occurring or, at least, to mitigate its morbidity and mortality consequences. This article describes specific actions at different levels of prevention of this pathology.

A fluorometric method for direct detection of inorganic polyphosphate in enterohemorrhagic O157:H7 / 南方医科大学学报

OBJECTIVE@#To establish a quantitative fluorescent detection method using DAPI for detecting inorganic polyphosphate (polyP) in enterohemorrhagic Escherichia coli (EHEC) O157:H7.@*METHODS@#The DNA of wild-type strain of EHEC O157:H7 was extracted and purified. DAPI was combined with the extracted DNA and polyP45 standards for measurement of the emission spectra at 360 nm and 415 nm fluorescence spectrophotometry. The fluorescence of DAPI-DNA and DAPI-polyP complexes was detected by fluorescence confocal microscopy to verify the feasibility of DAPI for detecting polyP. To determine the optimal pretreatment protocol for improving the cell membrane permeability, the effects of 6 pretreatments of the cells (namely snap-freezing in liquid nitrogen, freezing at -80 ℃, and freezing at -20 ℃, all followed by thawing at room temperature; heating at 60 ℃ for 10 min; treatment with Triton x-100; and placement at room temperature) were tested on the survival of EHEC O157:H7. The fluorescence values of the treated bacteria were then measured after DAPI staining. A standard calibration curve of polyP standard was established for calculation of the content of polyP in the live cells of wildtype EHEC strain and two mutant strains.@*RESULTS@#At the excitation wavelength of 360 nm, the maximum emission wavelength of DAPI-DNA was 460 nm, and the maximum emission wavelength of DAPI-polyP was 550 nm at the excitation wavelength of 415 nm. The results of confocal microscopy showed that 405 nm excitation elicited blue fluorescence from DAPIDNA complex with the emission wavelength of 425-475 nm; excitation at 488 nm elicited green fluorescence from the DAPIpolyP complex with the emission wavelength of 500-560 nm of. Snap-freezing of cells at -80 ℃ followed by thawing at room temperature was the optimal pretreatment to promote DAPI penetration into the live cells. The standard calibration curve was =1849+127.5 (R=0.991) was used for determining polyP content in the EHEC strains. The experimental results showed that wild-type strain had significantly higher polyP content than the mutant strains with deletion.@*CONCLUSIONS@#We established a convenient quantitative method for direct and reliable detection polyP content to facilitate further study of polyP and its catalytic enzymes in EHEC O157:H7.

Growth kinetics of Escherichia coli O157: H7 on the epicarp of fresh vegetables and fruits

ABSTRACT Despite the increasing reports on the incidence of fresh vegetables and fruits as a possible vehicle for human pathogens, there is currently limited knowledge on the growth potential of Escherichia coli O157:H7 on different plant substrates. This study analyzed the selective adhesion and growth of E. coli O157:H7 on chili habanero (Capsicum chinense L.), cucumber (Cucumis sativus), radish (Raphanus sativus), tomato (Lycopersicon esculentum), beet (Beta vulgaris subsp. vulgaris), and onion (Allium cepa L.) under laboratory conditions. The Gompertz parameters were used to determine the growth kinetics. Scanning electron microscopy was used to visualize the adhesion of E. coli O157:H7 on the epicarp of the samples. Predictive models were constructed to compare the growth of E. coli O157:H7 on the samples with different intrinsic factors and to demonstrate the low selectivity of the pathogen. No significant difference was observed in the lag-phase duration (LPD), generation time (GT), and exponential growth rate (EGR) of the pathogen adhered to the samples. The interaction between the microorganism and the substrate was less supportive to the growth of E. coli O157:H7 for onion, whereas for tomato and cucumber, the time for the microorganism to attain the maximum growth rate (M) was significantly longer than that recorded for other samples.

Immunogenicity of the nanovaccine containing intimin recombinant protein in the BALB/c mice

PURPOSE: Escherichia coli O157:H7 is one of the most important pathogens which create hemorrhagic colitis and hemolytic uremic syndrome in human. It is one of the most prevalent causes of diarrhea leading to death of many people every year. The first diagnosed gene in the locus of enterocyte effacement pathogenicity island is eae gene. The product of this gene is a binding protein called intimin belonging to the group of external membrane proteins regarded as a good stimulants of the immune system. Chitosan with its lipophilic property is an environmentally friendly agent able to return to the environment. MATERIALS AND METHODS: Intimin recombinant protein was expressed in pET28a vector with eae gene and purification was performed using Ni-NTA and finally the recombinant protein was approved through western blotting. This protein was encapsulated using chitosan nanoparticles and the size of nanoparticles was measured by Zetasizer. Intimin encapsulated was prescribed for three sessions among three groups of oral, injection, and oral-injection using Chitosan nanoparticles. Challenge was performed for all three groups with 108 E. coli O157:H7 bacteria. RESULTS: Intimin produced by chitosan nanoparticles improves immunological responses through the adjuvant nature of chitosan nanoparticles. Chitosan may be used as a carrier for transportation of the prescribed vaccine. Among the mice, encapsulated intimin could be able to provide suitable titers of IgG and IgA by the aid of chitosan nanoparticles. Results of mice challenge showed that decreased the bacterial shedding significantly. CONCLUSION: Results showed that the chitosan nanovaccine with intimin protein may be used as a suitable candidate vaccine against E. coli O157:H7.

Preparation and evaluation of fluorescent carbon dots (CDs) and CDs based immunofluorescence probe / 医疗卫生装备

Objective To fabricate an immunofluorescence probe system of carbon dots conjugated antibody based on antigen-antibody reaction principles.Methods A green one-step microwave assisted pyrolysis method was applied to preparing fluorescent carbon dots (CDs) using aminoglucose as carbon source and the obtained CDs were conjugated with antibody via EDC/NHS reactions to build CDs based fluorescent probe.Furthermore,the properties of CDs and CDs based probe system were evaluated by Fourier transform infrared (FTIR) spectra,transmission electron microscopy (TEM),UV-vis absorption and so on.Results The as-prepared CDs showed excellent fluorescence and hydrophilicity and CDs based immunofluorescence probe exhibited the capability of quick detection of E.coli O157:H7.Cinclusion Fluorescent CDs as one new emerging environment-friendly nanomaterial has great potential in biosensors.

Inhibiting effect of Lactobacillus acidophilus on intestinal colonization of enterohaemorrhagic E.coli O157:H7 in vitro and vivo / 实用医学杂志

Objective To test the inhabiting effect of Lactobacillus acidophilus on E.coli O157: H7 in intestinal colonization and explore its mechanism. Methods The suppressive effects of Lactobacillus acidophilus against E.coli O157:H7 adhering to Ht29 cells were carried out by competition , exclusion and replacement as-says. Furthermore, we evaluated the cytokine levels of IL-4, IL-12, and INF-γ in serum of mice. In addition, E.coli O157:H7 fecal shedding was monitored and the pathological changes of intestines were observed in mice. Results The competition, exclusion and replacement assays showed Lactobacillus acidophilus inhibited E.coli O157:H7 adhering to Ht29 cells. In vivo, the mice of treatment group were induced significantly higher level of IL-4, IL-12, and INF-γ, though prevention group induced IL-12 only. Fifteen days after E.coli O157:H7 infec-tion, there were 8 mice (80%) in prevention group and 5 mice (50%) in treatment group stopped shedding. Moreover, the pathological changes of intestines of both prevention group and treatment group appeared normal , but control groups showed seriously damaged in intestinal villus. Conclusion Lactobacillus acidophilus inhibits E.coli O157:H7 in intestinal colonization and the preventative effect was better than treatment effect. Thus , Lac-tobacillus acidophilus can be used for E.coli O157:H7 in prevention and treatment infection as probiotics.

Immunofluorescence probe based on fluorescent carbon dots and its application to the special recognition of Escherichia coli / 军事医学

Objective Carbon dots (CDs) are an emerging carbon nano-material which is environmentally-friendly, economical , efficient and stable .Their fluorescence properties can match the semiconductor quantum dot .Moreover , CDs have more excellent biocompatibilities .The purpose of this experiment is to apply CDs to the fluorescent immune probe to make them a new label , which can replace the traditional fluorescent dyes .Methods Using microwave heating method , the high strength fluorescent carbon dots were prepared .Wtih the EDC coupling method , the high strength fluorescent car-bon dots could bond with Escherichia coli antibodies to form a complex immune fluorescent probe .Specific recognition exper-iments were carried out in the model of E.coli O157∶H7.Results CDs were successfully applied to immune recognition of E.coli O157∶H7 and multicolor fluorescence was observed .Conclusion CDs can serve as a label of the fluorescent im-mune probe , and are expected to become a new type of low toxicity biosensor with independent intellectual property rights .

Prevalence of Escherichia Coli O157:H7 and Enterobacteriaceae on Hands of Workers in Halal Cattle Abattoirs in Peninsular Malaysia

Background: Several occupational diseases of multiple origins are encountered among abattoir workers. Presence of indicator microorganisms (coliforms) on hands of workers can be used a gauge for hygienic practices. Methods: A cross-sectional study was performed to assess the prevalence of E.coli and enterobacteriaceae among Halal abattoir workers in some government halal abattoirs of Malaysia. A total of one hundred and sixty-five hand swab samples were collected from workers of Halal abattoirs in Malaysia. The samples were subjected to microbiological analysis for characterisation and serotyping. Results: The results have shown that no Escherichia coli O157:H7 was isolated on the hands of abattoir workers before and after work. However, a total prevalence of 9.7% was recorded for all samples during work. For non-O157:H7, total prevalence of 33.3% during work and 13% after work were obtained. High prevalence was recorded in sample taken during work from Tampin, Jasin and Kemaman (100% each) while low prevalence where observed in Shah Alam, Banting and Ipoh (20% each). Conclusions: Based on the findings the hygienic practices of hand washing among the workers in few locations was found to be low especially after work.

Detection of E. coli O157:H7 and Shigella dysenteriae toxins in clinical samples by PCR-ELISA

Shiga toxin producing bacteria are potential causes of serious human disease such as hemorrhagic colitis, severe inflammations of ileocolonic regions of gastrointestinal tract, thrombocytopenia, septicemia, malignant disorders in urinary ducts, hemolytic uremic syndrome (HUS) Shiga toxin 1 (stx1), shiga toxin 2 (stx2), or a combination of both are responsible for most clinical symptoms of these diseases. A lot of methods have been developed so far to detect shiga toxins such as cell culture, ELISA, and RFPLA, but due to high costs and labor time in addition to low sensitivity, they have not received much attention. In this study, PCR-ELISA method was used to detect genes encoding shiga toxins 1 and 2 (stx1 and stx2). To detect stx1 and stx2 genes, two primer pairs were designed for Multiplex-PCR then PCR-ELISA. PCR products (490 and 275, respectively) were subsequently verified by sequencing. Sensitivity and specificity of PCR-ELISA method were determined by using genome serial dilution and Enterobacteria strains. PCR-ELISA method used in this study proved to be a rapid and precise approach to detect different types of shiga toxins and can be used to detect bacterial genes encoding shiga toxins.

Effect of Lactobacillus sp. isolates supernatant on Escherichia coli O157: H7 enhances the role of organic acids production as a factor for pathogen control / Efeito do sobrenadante de isolados de Lactobacillus sp. sobre Escherichia coli O157: H7 reforça o papel da produção de ácidos orgânicos como fator de controle patogênico

Many attempts have been made to establish the control of foodborne pathogens through Lactobacillus isolates and their metabolism products with success being obtained in several situations. The aim of this study was to investigate the antagonistic effect of eight Lactobacillus isolates, including L. casei subsp. pseudoplantarum, L. plantarum, L. reuteri and L. delbrueckii subsp. delbrueckii, on the pathogenic Escherichia colistrain O157:H7. The inhibitory effect of pure cultures and two pooled cultures supernatants of Lactobacillus on the growth of pathogenic bacteria was evaluated by the spot agar method and by monitoring turbidity. Antimicrobial activity was confirmed for L. reuteri and L. delbrueckii subsp. delbrueckii and for a pool of lactic acid bacteria. The neutralized supernatant of the pool exerted a higher antimicrobial activity than that of the individual strains. Furthermore, D-lactic acid and acetic acid were produced during growth of the Lactobacillus isolates studied.(AU)

Muitas tentativas têm sido feitas para se estabelecer o controle de patógenos de origem alimentar através do uso de estirpes de Lactobacillus e dos seus produtos de metabolismo, com sucesso sendo sucedido em várias situações. O objetivo deste trabalho foi investigar o efeito antagônico do sobrenadante de culturas de oito isolados de Lactobacillus, incluindo L. casei subsp. pseudoplantarum, L. plantarum L. reuteri e L. delbrueckii subsp. delbrueckii, sobre Escherichia coli amostra O157:H7. Os efeitos inibidores de culturas puras e de dois "pools" de cultura de Lactobacillus sobre o crescimento da bactéria foram avaliados através do método de inibição em ágar e através do monitoramento da turbidez da cultura bacteriana. A atividade antimicrobiana foi confirmada para Lactobacillus reuteri e Lactobacillus delbrueckii subsp. delbrueckii e para o "pool" de bactérias acido-láctica. O sobrenadante neutralizado do "pool" de Lactobacillus exerceu uma atividade antimicrobiana mais elevada do que aquela das estirpes individuais. Além disso, ácido D-láctico e ácido acético foram produzidos durante o crescimento dos Lactobacillus estudados(AU)

Screening of the novel colicinogenic gram-negative rods against pathogenic Escherichia coli O157:H7.

Purpose: Escherichia coli (E. coli) O157:H7 is gram‑negative enteric pathogen producing different types of Shiga toxin. This bacterium is the most corporate cause of haemorrhagic colitis in human. Administration of antibiotics (particularly sulfa drugs) against this pathogen is a debatable topic as this may increase the risk of uremic syndrome; especially in children and aged people. Around the world, microbiologists are in search of alternative therapeutic methods specially probiotics against this pathogen. In the present study, we have focused on the investigation of alternate bio‑therapeutics (probiotics) for the treatment of patients infected with E. coli O157:H7. This study is based on the identification of colicin‑producing gram‑negative bacteria (particularly enterobacteriaceae) which can competently exclude E. coli O157:H7 from the gut of the infected individual. Materials and Methods: Hundred samples from human, animal faeces and septic tank water were analysed for nonpathogenic gram‑negative rods (GNRs). Results: Out of these samples, 175 isolates of GNRs were checked for their activity against E. coli O157:H7. Only 47 isolates inhibited the growth of E. coli O157:H7, among which majority were identified as E. coli. These E. coli strains were found to be the efficient producers of colicin. Some of the closely related species i. e., Citrobacter sp, Pantoea sp. and Kluyvera sp. also showed considerable colicinogenic activity. Moreover, colicinogenic species were found to be nonhaemolytic, tolerant to acidic environment (pH 3) and sensitive to commonly used antibiotics. Conclusion: Nonhaemolytic, acid tolerant and sensitive to antibiotics suggests the possible use of these circulating endothelial cells (CEC) as inexpensive and inoffensive therapeutic agent (probiotics) in E. coli O157:H7 infections.

Development of Droplet Digital Polymerase Chain Reaction for Quantifying Escherichia Coli O157:H7 / 分析化学

A droplet digital polymerase chain reaction ( ddPCR) method for quantifying E. coli O157:H7 by targeting rfbE gene was developed. The probe concentration in ddPCR was optimized and the linearity range, precision, limit of detection ( LOD) and limit of quantification ( LOQ) were also evaluated. The optimized probe concentration was 300 nmol/L. The ddPCR response was linear over the E. coli O157:H7 genome DNA concentration range from 4 to 1. 25×105 copies in 20 μL ddPCR system and the linear correlation coefficient (R2) was 0. 999. The ddPCR precision (RSD) was less than 5% over the DNA concentration range from 760 to 88400 copies/20 μL. The LOD and LOQ was 3 copies in 20 μL and 4 copies in 20 μL, respectively. Specificity test showed that the ddPCR was specific for detecting E. coli O157:H7. Both ddPCR and standard real time quantitative PCR showed the same results for 16 real samples of chicken meat, pork and beef, which indicated that ddPCR method was suitable for detection of E. coli O157:H7 in food.

Development of up-converting phosphor technology based lateral flow assay for quan-titative detection of foodborne pathogens / 军事医学

Objective To develop an up-converting phosphor technology based lateral flow (UPT-LF) assay for rapid detection of Salmonella paratyphi A, S.paratyphi B, Escherichia coli O157 ∶H7 and Vibrio parahaemolyticus. Methods With up-converting phosphor nano-particles ( UCP-NPs ) as the bio-marker, four double-antibody-sandwich mode based UPT-LF strips for detecting the above mentioned four pathogens were prepared respectively and their sensitivi-ty, accuracy, linearity and specificity were evaluated .Furthermore, the feasibility of detecting bacteria in food samples was evaluated by different food samples artificially contaminated with less than 10 CFU target pathogens .Results The sensitivi-ty of UPT-LF assays for four pathogens was 105 ~106 CFU/ml with excellent specificity .The four strips had a good linear response with the linear fitting coefficient of determination (r) for each target pathogen ranging from 0.985 to 0.996.The positive rate of detecting pathogens from samples was acceptable .Conclusion The four developed UPT-LF strips provide a new choice for rapid , specific and sensitive and quantitative detection of S.paratyphi A , S.paratyphi B, E.coli O157∶H7 and V.parahemolyticus.

Construction and preliminary study of biological functions of O157∶H7 typeⅢsecre-tion system effector NleF gene knockout mutant / 军事医学

Objective To construct Escherichia coli O157∶H7 T3SS effector NleF gene knockout mutant and its com-plementary strain, and probe its effects on bacterial growth and cell death .Methods T3SS Effector NleF gene knockout mutant ΔnleF was constructed with λ-Red homologous recombination .Complementary strain ΔnleF/NleF was constructed by transferring pET-24a(+)-NleF into ΔnleF competent cells.Wild type,ΔnleF and ΔnleF/NleF were cultured in LB and DMEM(10%FBS) respectively,D600 was measured every hour , and the growth curve was drawn .HeLa cells were infected with three kinds of strains , the supernatant of LDH release was detected with cytotoxicity detection kit ,and the cytotoxicity was calculated .Results ΔnleF and ΔnleF/NleF were constructed .The growth rates of wild type , ΔnleF and ΔnleF/NleF was not significantly different .Wild type O157 infection induced cell death .Cytotoxicity was increased as much in ΔnleF in-fected cells as in ΔnleF/NleF infected cells.Conclusion EHEC O157∶H7 T3SS Effector NleF has no significant effect on bacterial growth ,but might inhibit host cell death caused by bacterial infection .