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.

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.

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.

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.

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.

Evaluation of zoonotic potency of Escherichia coli O157: H7 through arbitrarily primed PCR methods

Objective: To evaluate the zoonotic potency of Escherichia coli O157:H7 through arbitrarily primed-PCR (AP-PCR) methods as one of the DNA fingerprinting methods. Methods: A total of 14 isolates consisted of 11 isolates originated from human feces with renal failure symptoms, 2 isolates originated from cattle feces, and 1 control isolate were used in this study. DNA of each isolate was extracted, and their profiles were studied by using AP-PCR method with M13 F and M13 R arbitrary primers. Results: The results founded that all of 14 isolates had similarity range from 54.6% to 88.5%. Isolates KL-106(3) and KL-55(6) originated from humans showed the degree of similarity with isolates SM-25(1) and SM-7(1) originated from cattle as high as 85% and 77%, respectively. Conclusions: The high degree of similarity between isolates originated from cattle and human indicated the high potency of zoonoses. The results also concluded AP-PCR method as a briefly fingerprinting method in order to trace the epidemiological of E. coli O157:H7.

Regulatory effects of Stx phage on the T3SS in E.coli O157 ∶H7 / 中华微生物学和免疫学杂志

Objective To investigate the effects of shiga toxin ( Stx ) phage on the expression of type Ⅲsecretion system (T3SS) in E.coli O157 ∶H7.Methods A standard E.coli O157 ∶H7 strain, EDL933 and a natural Stx phage defective mutant of EDL 933 strain, TUV93-0 were used for this study .The expression of T3SS proteins was compared between EDL 933 and TUV93-0 strains.The expression of five operons ( LEE1-LEE5 ) was evaluated by measuring the green fluorescent protein ( GFP ) in five different plasmids with LEE1-LEE5 promoters, respectively.Results The expression of T3SS proteins in TUV93-0 mutant were significantly increased than those in EDL 933 strain.Moreover, the expression of LEE1, LEE2 and LEE5 were also increased in TUV93-0 mutant.Conclusion The deletion of Stx phage might enhance T3SS expression through the regulation of LEE 1.

Isolation and identification of Escherichia coli O157∶H7 from cow and its biological characteristics / 中国人兽共患病学报

To understand the Escherichia coli (E .coli) O157∶ H7 isolated from cow in Zhengzhou ,Henan Province ,a total of 146 samples of cow fecal and milk were collected in the different farms ,and E .coli O157∶ H7 was detected with mul-tiplex polymerase chain reaction (PCR) in our laboratory .Then the biochemical characteristics ,growth dynamic ,the biofilm formation ,and the toxin genes of the E .coli O157∶ H7 isolates were analyzed .The results showed that 2 strains of E .coli O157∶H7 were found ,with the detection rate of 1 .4% ,and the isolates were named as L1 and L2 in current study ,respec-tively .The E .coli O157∶H7 clinical isolates had the same biochemical characteristics with that of the typical E .coli .The L1 and L2 isolates presented similar growth curve ,which entered into the log phase earlier than that of the standard strain .L1 strain formed thick ,confluent ,complete biofilm after 48 hours post-inoculation ,and the biofilm of L2 strain was formed com-pletely in 36 hours .The two E .coli O157∶ H7 isolates were positive with eaeA and hlyA genes ,and the L1 strain also carried the Stx2 virulence gene .Our results reinforce the epidemiological data of E .coli O157∶H7 ,and underscore the need for more effective surveillance of animal-derived E .coli O157∶H7 isolates in Zhengzhou City ,China .

Survival of Escherichia coli o157: h7 co-cultured with different levels of Pseudomonas fluorescens and Lactobacillus plantarum on fresh beef

The purpose of this study was to investigate the effect of different levels of Pseudomonas fluorescens (10² and 10(6)log10 cfu/ml)and Lactobacillus plantarum (10² and 10(4)log10 cfu/ml)on the growth of Escherichia coli O157:H7 on beef loins. Beef loins inoculated with E. coli O157:H7 and P. fluorescens were aerobically stored for 7 days at 4 ºC, while those inoculated with E. coli O157:H7 and L. plantarum were vacuum packaged and stored for 8 weeks at 4 ºC. Aerobic Plate Counts (APC), E. coli O157:H7 and either P. fluorescens or L. plantarum counts were determined at different storage intervals. For the aerobically packaged beef loins, E. coli O157:H7 was detected throughout the 7 day storage period regardless of the P. fluorescens level in the inoculum. For the vacuum packaged beef loins, similar inoculum levels of E. coli O157:H7 and L. plantarum allowed E. coli O157:H7 to survive until week 5 of storage, while a higher inoculum level of L. plantarum inhibited E. coli O157:H7 from week 3. Once fresh beef has been contaminated with E. coli O157:H7, the level of P. fluorescens in the background flora does not inhibit its survival and growth. However, under vacuum storage, the application of L. plantarum as a biopreservative inhibits the survival of E. coli O157:H7 on beef. The higher the level of L. plantarum in the system, the earlier the onset of the inhibition. Farmers and abattoirs have to strengthen preventive strategies to eliminate contamination of beef carcasses with E. coli O157:H7.

Respiration system FNR protein effect on type Ⅲ secretion system in E.coli O157 ∶ H7 / 中华微生物学和免疫学杂志

Objective To investigate the effect of FNR on type Ⅲ secretion system (T3SS) in E.coli O157 ∶ H7.Methods fnr mutant was constructed by λRed recombineering technology promoted by Bet and Exo proteins using PCR products.Results Through bacterial infection assays and immunofluorescence microscopy,it was found that the adhesion ability was decreased insignificantly infnr mutant compared to the wild type ZAP198.However,the secreted proteins were reduced significantly in the mutant from the secretion profile.Conclusion The reason might be that high ClpXP protein caused by the deletion of fnr degraded GrlA resulting in the inhibition of LEE(locus of enterocyte effacement) and T3SS.

Antimicrobial activity of essential oils against Vancomycin-Resistant enterococci (VRE) and Escherichia coli O157: H7 in feta soft cheese and minced beef meat

Eleven essential oils (EOs) were evaluated for their antibacterial properties, against Vancomycin-Resistant Enterococci (VRE) and E. coli O157:H7. EOs were introduced into Brain Heart Infusion agar (BHI) (15ml) at a concentration of 0.25 to 2 percent (vol/vol) to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for each pathogen evaluated. Results showed that the most active essential oils against bacteria tested were thyme oil, with MIC90 and MBC90 for the VRA strains of 0.25 percent and 0.5 percent, respectively. Eucalyptus, juniper and clove oils were the least potent agent, with MIC90 and MBC90 of 2 percent. Furthermore, the inhibitory effect of these EO were evaluated against VRE and E. coli O157:H7, experimentally inoculated (10³ cfu/g) in Feta soft cheese and minced beef meat, which was mixed with different concentrations (0.1 percent, 0.5 percent and 1 percent) of the EO and stored at 7 ºC for 14 days. Out of eucalyptus, juniper, mint, rosemary, sage, clove and thyme oils tested against target bacteria sage and thyme showed the best results. Clove and mint did not show any effect on VRE and E. coli O157:H7 in both kinds of studied foods. The addition of thyme oil at concentrations of 0.5 and 1 percent caused best significant reduction in the growth rate of VRE and E. coli O157:H7 in cheese and meat at 7 ºC. It is concluded that selected plant EOs can act as potent inhibitors of both microorganisms in a food product. The results revealed the potential of thyme oil as a natural preservative in feta soft cheese and minced beef meat against VRE and E. coli O157:H7 contamination.

Detection of Escherichia coli O157 and Escherichia coli O157:H7 by the immunomagnetic separation technique and stx1 and stx2 genes by multiplex PCR in slaughtered cattle in Samsun Province, Turkey

This study was conducted to investigate the presence of Escherichia (E.) coli O157 and E. coli O157:H7 and stx1 and stx2 genes on cattle carcasses and in rectal samples collected from Samsun Province of Turkey. A total of 200 samples collected from cattle carcasses and the rectal contents of 100 slaughtered cattle from two commercial abattoirs were tested using the immunomagnetic separation technique and multiplex PCR methods. E. coli O157 and E. coli O157:H7 were detected in 52 of the 200 samples (26%) tested. Of the positive samples, 49 were E. coli O157 and three were E. coli O157:H7. The E. coli O157 strain was isolated from 24 carcasses and 25 rectal samples, while E. coli O157:H7 was isolated from two carcasses and one rectal sample. Of the 49 samples positive for E. coli O157, 32 were from the rectal and carcass samples of the same animal, while two E. coli O157:H7 isolates were obtained from rectal swabs and carcasses of the same animal. The stx1 and stx2 genes were both detected in 35 E. coli O157 isolates and one E. coli O157:H7 isolate, but the stx2 gene was only detected alone in two E. coli O157 isolates. Overall, 16 carcasses tested positive for E. coli O157 and one carcass tested positive for E. coli O157:H7 based on both carcass and rectal samples. Overall, the results of this study indicate that cattle carcasses pose a potential risk to human health due to contamination by E. coli O157 and E. coli O157:H7 in the feces.

Caracterización de aislados de Escherichia coli O157: H7 en canales de bovinos y porcinos mediante PCR / Charactertization of Escherichia ciki O157: H7 isolates obtained from bovine and porcine carcasses

Muchos de los brotes causados por Escherichia coli O157:H7 se han asociado al consumo de carne bovina mal cocida, pero también se ha reportado su presencia en la carne de otros animales domésticos. En México existe poca información sobre la presencia de este patógeno en canales de res y de cerdo. El objetivo de este estudio fue determinar la presencia de E. coli O157:H7 en canales de res y cerdo y su caracterización mediante PCR. De un total de 18 aislados, 12 fueron positivas por PCR para los genes rfbE y fliC que determinan el serotipo O157:H7. De estos 12, uno de canal de res y tres de canales de cerdo fueron positivos por PCR para los genes stx1, stx2 y eaeA, por lo que fueron considerados como enterohemorrágicos. Las diferencias encontradas en el número de canales positivas para los genes caracterizados no fueron estadísticamente significativas, y los resultados señalan que E. coli O157:H7 puede ser encontrada en ambos tipos de canal, representando un riesgo para la salud, por lo que se deben tomar medidas más estrictas de higiene y manejo para evitar que canales que no cumplan con el carácter de inocuidad lleguen a los consumidores finales.

Many Escherichia coli O157:H7 outbreaks have been associated to consumption of undercooked beef, but the presence has also been reported in the meat of other domestic animals. In Mexico, little information exists on the presence of this pathogen in bovine and pork carcasses. The objective of this study was to determine the presence and/or absence of E. coli serotype O157:H7 in bovine and pork carcasses and their characterization by means of PCR. Of 18 isolates, 12 were positive by PCR to rfbE and fliC genes, which determine O157:H7 serotype. Of these 12, one from bovine carcass and three of pork carcasses were positive by PCR to stx1, stx2 and eaeA genes, therefore, they were considered enterohemorrhagic strains. The differences found in the positive carcass number to any of the genes were not statistically significant. The results show that E. coli O157:H7 could be found in both carcasses types, representing a risk for the health, so strict hygienic and handling measures should be taken in order to avoid that carcasses which do not fulfill the food safety aspect might arrive to the final consumers.

Detection of Escherichia coli O157:H7 by multiplex PCR-DHPLC / 中华微生物学和免疫学杂志

Objective To develop the PCR-denaturing high performance liquid chromatography (DHPLC) for detection of E. Coli O157: H7. Methods The virulence genes of Shiga-like toxin(SLT) and rfbE were specifically amplified by 2 sets of primers. The target gene fragments of the PCR assay were 224 bp and 499 bp, respectively. Results Analysis of 37 strains demonstrated that this PCR system was specific. The detection limit of the PCR was 4 CFU/ml. Conclusion These results indicated that the multiplex PCR-DHPLC assay can be used for specific and sensitive detection of E. Coli O157:H7.

Detecção de Escherichia coli O157: H7 inoculada experimentalmente em amostras de leite cru por método convencional e PCR multiplex / Detection of E. coli O157: H7 experimentally inoculated in raw milk samples by conventional method and multiplex PCR

Padronizou-se um método de reação em cadeia da polimerase (PCR) multiplex para detecção de Escherichia coli O157:H7 e avaliou-se a eficiência da PCR e de um método de cultivo convencional em placas na detecção desse patógeno experimentalmente adicionado em leite estéril e em leite cru com baixa contagem bacteriana total (média de 4,01 x 10³ UFC/ml) e com alta contagem bacteriana (média de 2,10 x 10(6) UFC/ml). Foram padronizadas duas reações de PCR com o uso dos primers: "A" (RfbF; RfbR e FLICh7F/FLICh7R) e "B" (SLT-IF/SLTIR e SLT-IIF/SLT-IIR). A detecção de E. coli O157:H7 (1UFC/ml) a partir do leite estéril e do leite cru com baixa contaminação bacteriana foi possível quando se utilizou o método de contagem em placas e a PCR. A sensibilidade dos dois métodos foi menor quando se testou o leite cru com alta contaminação microbiana, sendo o método convencional mais sensível. Os resultados indicam que a presença de outros microrganismos, em alta quantidade no leite, dificulta a detecção de E. coli O157:H7 pelos métodos utilizados.

This experiment was carried out in order to evaluate the effect of the raw milk bacterial count on the efficiency of a multiplex polymerase chain reaction and a conventional plate count method for detection of Escherichia coli O157:H7. This pathogen was experimentally inoculated into sterile milk, raw milk with low bacterial count (count mean of 4.01 x 10³ cfu/ml) and, raw milk with high bacterial count (mean 2.10 x 10(6) cfu/ml). Two protocols of PCR were standardized using primers "A" (Rfbf and Rfbr and FLICh7F/FLICh7R) and "B" (SLT-IF/SLTIR and SLT-IIF/SLT-IIR). Both conventional plate count and PCR methods were able to detect the presence of E. coli O157:H7 in either sterile milk or raw milk with low bacterial count initially inoculated with 1cfu of E. coli O157:H7 per ml. The sensibility of both methods for high-contaminated raw milk samples was lower, being the conventional approach more sensitive. These results indicate that high bacterial count in raw milk can affect E. coli O157:H7 detection.

Survival and replication of Escherichia coli O157:H7 inside the mice peritoneal macrophages

The replication of Escherichia coli O157:H7 on the resident peritoneal macrophages of four mice strains (BALB/c, CD1, C57BL, and Swiss) has been investigated. Macrophagial bactericidal killing activity was estimated via studying their ability to internalize (gentamicin-protected) E. coli during 2, 4, 24, and 48 h assays. Host genetic background has been found to show no significant effect on the ability of resident peritoneal macrophages to kill E. coli O157:H7.

A multiplicação de Escherichia coli O157:H7 em macrófagos peritoniais residentes de quatro linhagens de camundongos (BALB/C, CD1, C57BL e Swiss) foi investigada. A capacidade bactericida dos macrófagos foi estimada através da avaliação de sua capacidade de internalizar E.coli (protegidos pela gentamicina) em ensaios de 2, 4, 24 e 48h. Observou-se que as características genéticas do hospedeiro não têm efeito significativo na capacidade dos macrófagos peritoniais eliminarem E. coli O157:H7.

Escherichia coli O157:H7 adherence to HEp-2 cells is implicated with curli expression and outer membrane integrity

Escherichia coli (E. coli) has ability to express thin aggregative fimbriae, known as curli, on the cell surface. Previously, a few example of curli expression in serogroup O157:H7 of enterohemorrhagic E. coli (EHEC) were reported, compared to other E. coli groups. However, significance of curliation in the EHEC pathobiology has not been described well in the literature. A highly curliated O157:H7 strain was used in this study in order to elucidate role of curliation in EHEC adherence to cultured HEp-2 cells. The expression of curli in the EHEC isolate was consistent with strong positive indication of Congo-red (CR) binding and formation of clumps in the bottom of the tube containing Luria-Bertani (LB) broth when cultured overnight at 37 degress C. A few CR-binding negative (CR-) colonies occurred spontaneously within the population of CR+ isolate. The CR+ EHEC showed massive aggregative adhesion pattern, whereas the spontaneous CR- strain showed typical localized adherence on HEp-2 cells. Electron microscopy confirmed highly curliated bacteria in the CR+ EHEC sample. Interestingly, the curliation disappeared in a msbB1 and msbB2 double mutant derived from the CR+ EHEC. These results suggest that the compromised outer membrane integrity caused by msbB mutations may abrogate curli production in the CR+ EHEC harbouring penta-acylated lipid A structure in their outer membrane.

Homologous analysis of E.coli O_(157)∶H_7 by pulsed-field gel electrophoresis and random amplified polimorphic DNA / 中华传染病杂志

Objective To analyze the molecular epidemiological characteristics of E. coli O 157∶H 7 of Xuzhou, Jiangsu. Methods The virulence gene spectrum of E. coli O 157∶H 7 strains were analyzed by PCR and the homology of E. coli O 157∶H 7 strains were detected by PFGE and RAPD. Results In all E. coli O 157∶H 7 strains isolated from epidemic area, 100% possess Hly and eaeA gene, 95.35% possess SLT 2 gene, and 11.63% possess SLT 1 gene. The PFGE spectrum showed that the strains isolated from epidemic area were distinctively different from the strains isolated from Japan, and similar to but not identical with the standard strain 882364. The PFGE spectrum of strains isolated from epidemic area patients were identical with those of strains isolated from excrements of poultries, domestic animals and insect intestine.Conclusions Poultries and domestic animals which carry E.coli O 157∶H 7 could be the source of infection. PFGE could be used to analyze E.coli O 157∶H 7 and played an important role in epidemiology study. The results showed that the method of analysis of E. coli O 157∶H 7 by RAPD was convenient and time saving.