Engineered Bacillus subtilis alleviates intestinal oxidative injury through Nrf2-Keap1 pathway in enterotoxigenic Escherichia coli (ETEC) K88-infected piglet / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Engineered probiotics can serve as therapeutics based on their ability of produce recombinant immune-stimulating properties. In this study, we built the recombinant Bacillus subtilis WB800 expressing antimicrobial peptide KR32 (WB800-KR32) using genetic engineering methods and investigated its protective effects of nuclear factor-E2-related factor 2 (Nrf2)‍-Kelch-like ECH-associated protein 1 (Keap1) pathway activation in intestinal oxidative disturbance induced by enterotoxigenic Escherichia coli (ETEC) K88 in weaned piglets. Twenty-eight weaned piglets were randomly distributed into four treatment groups with seven replicates fed with a basal diet. The feed of the control group (CON) was infused with normal sterilized saline; meanwhile, the ETEC, ETEC+WB800, and ETEC+WB800-KR32 groups were orally administered normal sterilized saline, 5×1010 CFU (CFU: colony forming units) WB800, and 5×1010 CFU WB800-KR32, respectively, on Days 1‍‒‍14 and all infused with ETEC K88 1×1010 CFU on Days 15‍‒‍17. The results showed that pretreatment with WB800-KR32 attenuated ETEC-induced intestinal disturbance, improved the mucosal activity of antioxidant enzyme (catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx)) and decreased the content of malondialdehyde (MDA). More importantly, WB800-KR32 downregulated genes involved in antioxidant defense (GPx and SOD1). Interestingly, WB800-KR32 upregulated the protein expression of Nrf2 and downregulated the protein expression of Keap1 in the ileum. WB800-KR32 markedly changed the richness estimators (Ace and Chao) of gut microbiota and increased the abundance of Eubacterium_rectale_ATCC_33656 in the feces. The results suggested that WB800-KR32 may alleviate ETEC-induced intestinal oxidative injury through the Nrf2-Keap1 pathway, providing a new perspective for WB800-KR32 as potential therapeutics to regulate intestinal oxidative disturbance in ETEC K88 infection.

Factores asociados a la diarrea del viajero en Quito, Provincia de Pichincha, Ecuador / Factors associated with traveler's diarrhea in Quito, Pichincha Province, Ecuador

La diarrea del viajero (DV) es la complejidad más común y su repercusión varía en función de muchas variables: edad, lugar de destino, estación del año, tipo de residencia, origen del viajero y, por supuesto, la dieta durante el viaje, en definición es la emisión de heces sueltas tres o más veces al día, se adquiere mayormente al consumir alimentos y bebidas contaminados con patógenos que ocasionen diarrea. A nivel mundial, las causas más comunes de DV son dos patotipos de Escherichia coli (enterotoxigénica y enteroagregativa) y Campylobacter, aunque existen variaciones significativas según el área geográfica visitada. Algunos autores han detectado la presencia de diarrea en más de la mitad de los viajeros a países en vías de desarrollo. Se realizó un estudio descriptivo de corte transversal en 15 hospitales centinelas de la zona 9 del Sistema de Vigilancia de la resistencia antimicrobiana, en la provincia de Pichincha Quito, Ecuador entre enero 2019 a enero 2020, con el objeto de caracterizar los eventos de DV. 517 registros, solo en 298 se asento la procedencia, pudiendo atribuirse a ese 42,34% la procedencia por nacionalidad, se estimó que 108 casos presentaron infección mixta de al menos una bacteria y otro tipo de agresor, lo que representa 36,24%, siendo el agente causal principalmente involucrado ETEC, seguida de E. coli H7:O157 (N=60)(AU)

Traveler's diarrhea (DV) is the most common complexity and its impact varies depending on many variables: age, place of destination, season of the year, type of residence, origin of the traveler and, of course, the diet during the trip, In definition, it is the emission of loose stools three or more times a day, it is acquired mostly by consuming food and beverages contaminated with pathogens that cause diarrhea. Worldwide, the most common causes of DV are two Escherichia coli pathotypes (enterotoxigenic and enteroaggregative) and Campylobacter, although there are significant variations depending on the geographic area visited. Some authors have detected the presence of diarrhea in more than half of the travelers to developing countries. A descriptive cross-sectional study was carried out in 15 sentinel hospitals in zone 9 of the Antimicrobial Resistance Surveillance System, in the province of Pichincha Quito, Ecuador between January 2019 and January 2020, in order to characterize VD events. 517 records, only in 298 the origin was established, and that 42.34% can be attributed the origin by nationality, it was estimated that 108 cases presented mixed infection of at least one bacterium and another type of aggressor, which represents 36.24% , the causative agent being mainly involved ETEC, followed by E. coli H7: O157 (N = 60). Likewise, ETEC is responsible for the greatest severity in clinical pictures, being responsible for 50.00% of the cases in this category(AU)

Caracterización de Escherichia coli D7111 productora de ß-LACTAMASA TEM-176 / Characterization of Escherichia coli D7111 producing the ß-LACTAMASA TEM-176

RESUMEN El presente reporte es la descripción original de bla TEM-176. Se caracterizaron los mecanismos de resistencia a antimicrobianos de un aislamiento de Escherichia coli enterotoxigénica, determinándose la resistencia a 22 antimicrobianos categorizados en 15 grupos diferentes mediante difusión en agar, estableciéndose grupo filogenético, mecanismos de resistencia y presencia de integrones de Clase 1 y 2 mediante PCR. Integrones y genes de resistencia a β-lactámicos fueron secuenciados. El aislamiento del grupo filogenético A, mostró resistencia o sensibilidad disminuida a ampicilina, amoxicilina más ácido clavulánico, ácido nalidíxico, ciprofloxacino, estreptomicina, kanamicina, tetraciclina, trimetoprim, sulfisoxazol, cotrimoxazol, azitromicina y nitrofurantoina, detectándose la presencia de bla TEM, aadA1/2, aphA1, sul3, tet(A) y un integron de Clase 2 conteniendo un gen dfrA1. La resistencia a quinolonas se relacionó con la substitución Ser83Ala. La secuencia de TEM mostró la substitución Ala222Val, la cual a la fecha no había sido descrita, reportándose como una nueva β-lactamasa, con el nombre de bla TEM-176.

ABSTRACT The present report is the original description of bla TEM-176. The mechanisms of resistance to antimicrobial agents were determined in an enterotoxigenic Escherichia coli, determining the susceptibility to 22 antimicrobials classified in 15 different groups by agar diffusion and establishing the phylogenetic group, mechanisms of resistance and presence of Class 1 and 2 integrons. Integrons and β-lactam resistance genes were sequenced. The isolate, belonging to phylogenetic group A, showed the presence of resistance or diminished susceptibility to a ampicillin, amoxicillin plus clavulanic acid, nalidíxic acid, ciprofloxacin, streptomycin, kanamycin, tetracycline, trimethoprim, sulfisoxazole, cotrimoxazole, azithromycin and nitrofurantoin, carrying bla TEM, aadA1/2, aphA1, sul3, tet(A) and a Class 2 integron containing a dfrA1 gene. Quinolone resistance was related to the substitution Ser83Ala. The TEM sequencing showed the presence of the new substitution Ala222Val, which led to the description of the new β-lactamase bla TEM-176.

Caracterización de Escherichia coli D7111 productora de ß-LACTAMASA TEM-176 / Characterization of Escherichia coli D7111 producing the ß-LACTAMASE TEM-176

RESUMEN El presente reporte es la descripción original de bla TEM-176. Se caracterizaron los mecanismos de resistencia a antimicrobianos de un aislamiento de Escherichia coli enterotoxigénica, determinándose la resistencia a 22 antimicrobianos categorizados en 15 grupos diferentes mediante difusión en agar, estableciéndose grupo filogenético, mecanismos de resistencia y presencia de integrones de Clase 1 y 2 mediante PCR. Integrones y genes de resistencia a β-lactámicos fueron secuenciados. El aislamiento del grupo filogenético A, mostró resistencia o sensibilidad disminuida a ampicilina, amoxicilina más ácido clavulánico, ácido nalidíxico, ciprofloxacino, estreptomicina, kanamicina, tetraciclina, trimetoprim, sulfisoxazol, cotrimoxazol, azitromicina y nitrofurantoina, detectándose la presencia de bla TEM, aadA1/2, aphA1, sul3, tet(A) y un integron de Clase 2 conteniendo un gen dfrA1. La resistencia a quinolonas se relacionó con la substitución Ser83Ala. La secuencia de TEM mostró la substitución Ala222Val, la cual a la fecha no había sido descrita, reportándose como una nueva β-lactamasa, con el nombre de bla TEM-176.

ABSTRACT The present report is the original description of bla TEM-176. The mechanisms of resistance to antimicrobial agents were determined in an enterotoxigenic Escherichia coli, determining the susceptibility to 22 antimicrobials classified in 15 different groups by agar diffusion and establishing the phylogenetic group, mechanisms of resistance and presence of Class 1 and 2 integrons. Integrons and β-lactam resistance genes were sequenced. The isolate, belonging to phylogenetic group A, showed the presence of resistance or diminished susceptibility to a ampicillin, amoxicillin plus clavulanic acid, nalidíxic acid, ciprofloxacin, streptomycin, kanamycin, tetracycline, trimethoprim, sulfisoxazole, cotrimoxazole, azithromycin and nitrofurantoin, carrying bla TEM, aadA1/2, aphA1, sul3, tet(A) and a Class 2 integron containing a dfrA1 gene. Quinolone resistance was related to the substitution Ser83Ala. The TEM sequencing showed the presence of the new substitution Ala222Val, which led to the description of the new β-lactamase bla TEM-176.

Lowland and highland Varieties of Dioscorea esculenta tubers stimulate growth of Lactobacillus sp. over enterotoxigenic E. coli in vitro

@#<p style="text-align: justify;"><strong>Objectives:</strong> Probiotic supplementation often only leads to transient improvement in the gut microbiome. Potential prebiotics, such as the oligosaccharide-rich varieties of Dioscorea esculenta tubers, can potentially bridge the gap between supplementation and persistent colonization. Thus, this study aimed to assess the ability of D. esculenta tubers to promote the growth of probiotic Lactobacillus sp. in vitro selectively.</p><p style="text-align: justify;"><strong>Methods:</strong> The prebiotic activity of the selected varieties of Dioscorea esculenta tubers was evaluated via compe titive growth assay, wherein the ratios of probiotic Lactobacillus sp. over enterotoxigenic Escherichia coli (ETEC) or "prebiotic ratios" were compared following treatment.</p><p style="text-align: justify;"><strong>Results:</strong> Negative control (0.9% NaCl solution) produced a ratio of 0.88, Lowland and Highland varieties produced ratios of 1.26 and 1.29, respectively, and positive control (inulin) produced 1.54. The two varieties had comparable ratios to one another (p > 0.05), and significantly higher ratios than the negative control (p < 0.05). Both varieties have significant prebiotic activity. Compared to inulin, the two varieties' prebiotic activity was 84% as effective.</p><p style="text-align: justify;"><strong>Conclusion:</strong> Overall, the tubers promoted the growth of Lactobacillus sp. over ETEC. The crude tuber samples, given their availability and affordability, can be easily integrated into the local diet to contribute to the improvement of the general population's health.</p>

Caracterização de Escherichia coli diarreiogênica isolada de água subterrânea para consumo humano em um assentamento rural / Characterization of diarrheagenic Escherichia coli isolated from underground water for human consumption in a rural settlement

Escherichia coli diarreiogênica (DEC) é um importante agente de infecções gastrointestinais transmitidas pela água. O trabalho teve como objetivo avaliar a qualidade microbiológica e físicoquímica de 58 amostras de água subterrânea in natura para consumo de um assentamento rural e caracterizar os isolados de E. coli, genotipicamente dentro dos patotipos de DEC, pela técnica da PCR, e fenotipicamente. Todas as amostras apresentaram contaminação por coliformes totais e 36 (62,1%) por E. coli. Em células HEp-2, dos 170 isolados de E. coli, 106 (62,36%) apresentaram AA, 15 (8,82%) AD, 17 (10%) adesão não caracterizado e 32 (18,82%) foram não aderentes. Quanto à formação de biofilme, 126 (74,12%) cepas são formadoras, e 44 (25,88%) não formaram biofilme. Foram identificadas DEC em 6,89% das amostras de água: três (5,17%) ETEC e uma (1,72%) EAEC. EAEC apresentou AA e as três cepas de ETEC apresentaram AD, AA e não definida. Todas as DEC foram formadoras de biofilme. Dois isolados de ETEC apresentaram resistência à ampicilina e tetraciclina, uma ETEC à aminoglicosídeos, e EAEC foi sensível a todos os antibióticos testados. As ETEC foram classificadas no filogrupo B1 e a EAEC no E. Os sorotipos encontrados foram: O24:H21, OR:H21, O17:H46 e O6:H12. Todas as amostras estavam dentro dos parâmetros normais de flúor e 13 (22,4%) apresentaram resultados acima do padrão permitido de turbidez. A presença de E. coli e DEC nas amostras de água indica a necessidade de adoção de medidas que evitem a contaminação da água fornecida à população, evitando, assim, a transmissão de doenças.(AU)

Diarrheogenic Escherichia coli (DEC) is an important agent of waterborne gastrointestinal infections. The objective of this work was to evaluate the microbiological and physico-chemical quality of 58 freshwater groundwater samples for the consumption of a rural settlement and to characterize the E. coli isolates, genotyped within the DEC pathophyses, by the PCR technique, and phenotypically. All water samples presented contamination with total coliforms, and 36 (62.1%) with E. coli. In HEp-2 cells, of 170 E. coli isolates, 106 (62.36%) showed AA, 15 (8.82%) AD, 17 (10%) noncharacterized adhesion and 32 (18.82%) were non-adherent. As for biofilm formation, 126 (74.12%) strains are forming, and 44 (25.88%) did not form biofilm. DEC was identified in 6.89% of the water samples with E. coli: three (5.17%) with enterotoxigenic E. coli (ETEC), and one (1.72%) with enteroaggregative E. coli (EAEC). EAEC showed aggregative adherence, whereas various ETEC strains presented diffuse, aggregative, and non-defined adherence. All DEC strains were biofilm forming. Two isolates of ETEC showed resistance to ampicillin and tetracycline, and one isolate showed resistance to aminoglycosides. The EAEC isolate was sensitive to all antibiotics tested. All ETECs were classified into phylogenetic B1 and EAEC in E. The serotypes identified in our study were: O24:H21, ONT:H21, O17:H46, and O6:H12. All water samples were within normal fluorine parameters; however, measured turbidity was above the permitted standard in 13 (22.4%) samples. The presence of E. coli and DEC in water samples indicates the need to take action to prevent contamination of water resources accessed by people to prevent the transmission of diseases.(AU)

Escherichia coli enterotoxigénica y enteroagregativa: prevalencia, patogénesis y modelos múridos / Enterotoxigenic and enteroaggregative Escherichia coli: prevalence, pathogenesis and murine models

Resumen Los patotipos de Escherichia coli enterotoxigénica (ETEC) y enteroagregativa (EAEC) son importantes agentes etiológicos causantes de diarrea en niños menores de cinco años de México y países en desarrollo, en quienes causan numerosas muertes. Ambos se han asociado con retraso en el crecimiento infantil y son los principales agentes causales de la "diarrea del viajero". La patogénesis de ambas bacterias se inicia cuando estas se adhieren al epitelio intestinal mediante fimbrias, denominadas factores de colonización en las cepas ETEC aisladas de humano y fimbrias de adherencia agregativa en las cepas de EAEC. Una vez que ETEC se adhiere al enterocito produce una o ambas de sus toxinas e induce la secreción de iones de cloruro, sodio y agua al lumen intestinal, produciendo su característica diarrea acusa. EAEC se une al epitelio intestinal formando una biopelícula, induce la producción de moco, libera sus toxinas y promueve inflamación. Modelos de infección de EAEC y ETEC con ratones C57BL/6 silvestres y deficientes del ligando de CD40 (con microbiotas intactas), respectivamente, revelaron que la desnutrición y la dieta baja en cinc incrementan la infección de EAEC causando retraso en el crecimiento y que ETEC coloniza, persiste e induce respuesta inmune humoral local y sistémica.

Abstract Enterotoxigenic (ETEC) and enteroaggregative Escherichia coli (EAEC) pathotypes are important etiological agents causative of diarrhea in children younger than 5 years of age in Mexico and in developing countries, where they cause numerous deaths. Both have been associated with delayed growth in children and are the main causative agents of traveler's diarrhea. The pathogenesis of both bacteria starts by adhering to the intestinal epithelium by means of fimbriae, called colonization factors in human ETEC isolates and aggregative adherence fimbriae in EAEC isolates. Once ETEC adheres to the enterocyte, it produces one or both of its toxins and induces the secretion of chloride and sodium ions and water into the intestinal lumen, producing its characteristic watery diarrhea. EAEC binds to the intestinal epithelium forming a biofilm, induces the production of mucus, releases its toxins and promotes inflammation. EAEC and ETEC infection models with wild-type C57BL/6 and CD40 ligand-deficient mice (with intact microbiota), respectively, revealed that undernutrition and low-zinc diet increases EAEC infection, causing growth retardation, and that ETEC colonizes, persists and induces local and systemic humoral immune response.

Adaptação de Escherichia coli enterotoxigênica ao eugenol / Adaptation of enterotoxigenic Escherichia coli to eugenol

O objetivo do trabalho foi verificar a capacidade de adaptação de ETEC- ATCC 35401 ao antimicrobiano eugenol. Para isso, a Concentração Mínima Bactericida (CMB) de eugenol foi determinada e em seguida,as células de ETEC foram expostas a concentrações subletais de eugenol (CMB/4, CMB/8 e CMB/16). Posteriormente testadas frente a diferentes concentrações do composto (CMB/2; CMB; 1,2CMB; 1,4CMB; 1,6CMB; 1,8CMB e 2CMB), estas foram incubadas e plaqueadas em TSA (Ágar Triptona de Soja) empregando a técnica de microgotas. As células de ETEC foram classificadas como capazes de se adaptarem quando essas cresceram em placas após cultivo em presença do componente em concentração igual ou maior que a CMB do eugenol (1,0% (v/v)). As células de ETEC apresentaram a capacidade de adaptação por crescerem na CMB, após exposta a CMB/16. Os resultados demonstram a necessidade de obtenção da concentração de uso adequado de eugenol a fim de evitar a adaptação.

Prevalence of O-serogroups, virulence genes, and F18 antigenic variants in Escherichia coli isolated from weaned piglets with diarrhea in Korea during 2008–2016

To diagnose colibacillosis, detection of O-serogroups and virulence genes has been recommended worldwide. The prevalence of virulence factors can fluctuate over time. The objectives of this study were to determine the prevalence of O-serogroups, virulence genes, and F18 subtypes among pathogenic Escherichia coli isolated from weaned piglets with diarrhea in Korea. Between 2008 and 2016, 362 E. coli were isolated from weaned piglets with diarrhea. Hemolysis was determined in blood agar, and O-serogroups were identified using the slide agglutination technique. The genes for the toxins and fimbriae were amplified by polymerase chain reaction (PCR). Real-time PCR was conducted to discriminate between F18 subtypes. Although the most prevalent serogroup was O149 (11.3%) in the last 9 years, O139 (19.1%) became the most prevalent in recent years (2015–2016). The most predominant pathotype was enterotoxigenic E. coli (61.3%). The frequencies of Shiga-like toxin-producing E. coli (STEC) (23.4%), O139 (19.1%), Stx2e (35.1%), and F18ab (48.7%) increased over the most recent years. Although enterotoxigenic E. coli was the most predominant pathotype, the frequencies of O139, Stx2e, STEC, and F18ab have increased in recent years. These results demonstrate that there have been temporal changes in the predominant O-serogroups and virulence genes over the last decade in Korea. These findings can be practicable for use in epidemiology and control measures for enteric colibacillosis in Korean piggeries.

Molecular Characterization of Enterotoxigenic Escherichia coli in Foodborne Outbreak

Diarrheagenic Escherichia coli (E. coli) is a main cause of diarrhea worldwide. This study reports the investigation on the occurrence of enterotoxigenic E. coli (ETEC) serotype O27:H7-associated foodborne gastrointestinal disease that occurred at two schools, one middle school and one high school, in Seoul, Korea in June 2015. The immediate government investigation in 1,216 students and 19 food handlers in these two schools revealed that 116 students, 32 students in the middle school and 84 students in the high school, and 2 food handlers, one from middle school and the other from high school, developed gastrointestinal illness symptoms including diarrhea. Following lab investigation identified 29 ETEC serotype O27:H7 strains, 27 from 116 students and 2 from 19 food handlers. Pattern of pulsed-field gel electrophoresis analysis of ETEC isolates suggested that ETEC serotype O27:H7 caused the diarrheal outbreak in June 2015 in Seoul, Korea was a specific clone. In addition, these ETEC serotype O27:H7 isolates were highly resistance to the several antibiotics. The results from the present study provide the evidence that ETEC serotype O27:H7 can be an important cause of domestic foodborne outbreak in Korea.

Piglet colibacillosis diagnosis based on multiplex polymerase chain reaction and immunohistochemistry of paraffin-embedded tissues

Enterotoxigenic Escherichia coli (ETEC) causes diarrhea in pigs, referred to as colibacillosis. The aim of this study was to optimize multiplex polymerase chain reaction (PCR) and immunohistochemistry (IHC) analyses of paraffin-embedded material to detect pathogenic E. coli strains causing colibacillosis in pigs. Multiplex PCR was optimized for fimbriae (F18, F4, F6, F5, and F41) and toxins (types A and B heat-stable toxins [STaP and STb], heat-labile toxin [LT], and type 2 Shiga toxin [ST(x2e)]), and IHC was optimized for an anti-E. coli polyclonal antibody. Samples (132) from pigs received between 2006 and 2014 with clinical and histopathological diagnoses of colibacillosis were analyzed. E. coli was detected by IHC in 78.7%, and at least one virulence factor gene was detected in 71.2%. Pathogenic strains of ETEC with at least one fimbria and one toxin were detected in 40% of the samples in multiplex PCR. The most frequent virulence types were F18-STaP (7.5%), F18-STaP-STb (5.7%), and F4-STaP (3.8%). A statistically significant association was noted between virulence factors F4, F18, STaP, and STb and positive immunostaining results. Colibacillosis diagnosis through multiplex PCR and IHC of paraffin-embedded tissues is a practical approach, as samples can be fixed and stored for long periods before analysis.

High Frequency of Enteric Protozoan, Viral, and Bacterial Potential Pathogens in Community-Acquired Acute Diarrheal Episodes: Evidence Based on Results of Luminex Gastrointestinal Pathogen Panel Assay

Infectious diarrhea is endemic in most developing countries. We aimed to investigate the protozoan, viral, and bacterial causes of acute diarrhea in Taif, Saudi Arabia. A cross-sectional prospective 1-year study was conducted on 163 diarrheal patients of various ages. Stool samples were collected, 1 per patient, and tested for 3 protozoa, 3 viruses, and 9 bacteria with the Luminex Gastrointestinal Pathogen Panel. Overall, 53.4% (87/163) of samples were positives (20.8% protozoa, 19.6% viruses, 2.8% bacteria, and 9.8% mixed). Rotavirus (19.6%), Giardia duodenalis (16.5%), and Cryptosporidium spp. (8.5%) were the mostly detected pathogens. Adenovirus 40/41 (4.2%), Salmonella (3%), Shiga toxin-producing Escherichia coli (3%), and Entamoeba histolytica (2.4%) were also detected. Norovirus GI/II, Vibrio cholerae, Yersinia enterocolitica, and Clostridium difficile toxin A/B were not detected in any patients. All pathogens were involved in coinfections except E. histolytica. Giardia (5.5%) and rotavirus (3%) were the most commonly detected in co-infections. Enterotoxigenic E. coli (2.4%), Campylobacter spp. (2.4%), E. coli 0157 (1.8%), and Shigella spp. (1.2%) were detected in patients only as co-infections. Infections were more in children 0–4 years, less in adults 40 years, with statistically significant differences in risk across age groups observed with rotavirus (P < 0.001), Giardia (P=0.006), and Cryptosporidium (P=0.036) infections. Lastly, infections were not significantly more in the spring. This report demonstrates the high burden of various enteropathogens in the setting. Further studies are needed to define the impact of these findings on the clinical course of the disease.

Construction of a live attenuated Salmonella strain expressing FanC protein to prevent bovine enterotoxigenic Escherichia coli and evaluation of its immunogenicity in mice

To construct a novel vaccine candidate against bovine enterotoxigenic Escherichia coli (ETEC), FanC, the major subunit of K99 fimbriae adhesion, was inserted into secretion plasmid pYA3560 containing a β-lactamase secretion system. This was then transformed into Δasd Δcrp Salmonella (S.) Typhimurium and designated as JOL950. Secretion of recombinant fanC fimbrial antigens was confirmed by immunoblot analysis. Groups of mice were inoculated with single or double doses of JOL950. Another group was used as a negative control. Compared to control mice, all immunized mice had significantly higher levels (p < 0.05) of serum immunoglobulin (Ig)G, and secretory IgA against FanC. The IgG2a and IgG1 titer assays revealed that immunization highly induced IgG2a compared to that of IgG1, indicating that T helper-1- related cell-mediated immune responses may be elicited by JOL950. The results show that both systemic and mucosal immunities against selected fimbrial antigens of bovine ETEC expressed by a live attenuated S. Typhimurium strain are prominently produced in mice immunized with JOL950 via an oral route.

Virulence profiles of enterotoxigenic Escherichia coli isolated from piglets with post-weaning diarrhea and classification according to fecal consistency / Perfis de virulência de Escherichia coli enterotoxigênica isoladas de leitões desmamados com diarreia do Sul do Brasil e classificação das amostras de acordo com a consistência fecal

The aim of this study was to assess the frequency and association of virulence factors of Escherichia (E.) coli isolated from weaned piglets with diarrhea and to correlate it with fecal consistency. A total of 152 rectal swabs were collected from 25-40 day-old piglets with diarrhea, in farms of Southern Brazil. Phenotypical and molecular techniques were used for bacterial isolation, characterization and classification of enterotoxigenic E. coli (ETEC) pathotypes. Statistical analysis was carried out to determine the frequency of virulence factors and virotypes, of fimbriae F4, F5, F6, F18, F41 and toxins LT, STa, STb and STx2e. Out of 456 E. coli isolates, 287 (62.9%) samples showed significant growth of E. coli. Among them, 194 (67.6%) samples showed at least one virulence factor, indicating that ETEC is an important etiological agent of diarrhea in weaned piglets. Higher frequencies were found of fimbria F4 and F18 and enterotoxins LT, STa and STb. Significant association was found to F4, LT, STa and STb; between F18 and STa and STx2e; between F5 and LT, STa and STb. The most frequent virotypes were F18-STa, F4-LT-STa-STb, F4-STa, F4-LT-STb and F18-STa-STx2e. Beta-hemolysis was observed in 47.4% of samples and there was significant association between hemolytic samples and virulence factors F4, F18, STa and STx2e. Regarding fecal consistency, there was significant association of liquid feces and F4 fimbria, STa toxin and virotypes F4-STa and F4-F5-LT-STa-STb. Since there was significant association of ETEC and liquid feces in nursery piglets, it is important to prioritize the sampling of liquid feces for the diagnosis etiologic cause of diarrhea.

O objetivo deste estudo foi avaliar a frequência e associação de fatores de virulência de Escherichia (E.) coli isoladas de leitões desmamados com diarreia e correlacioná-la com consistência fecal. Suabes retais foram coletados em leitões com 25-40 dias de idade com sinal clínico de diarreia, em granjas do Sul do Brasil, totalizando 456 amostras. Foram utilizadas técnicas fenotípicas e moleculares para isolamento bacteriano, caracterização e classificação de patotipos de E. coli enterotoxigênica (ETEC). A análise estatística foi realizada para determinar a frequência de fatores de virulência e virotipos, de fímbrias F4, F5, F6, F18, F41 e toxinas LT, STa, STB e STx2e. Duzentas e oitenta e sete (62,9%) amostras apresentaram crescimento significativo de E. coli. Entre os quais, 194 (67,6%) amostras apresentaram pelo menos um fator de virulência, indicando que ETEC é um importante agente etiológico de diarreia em leitões desmamados. As frequências mais elevadas foram encontradas para as fímbrias F4 e F18 e enterotoxinas LT, STa e STb. Associação significativa foi encontrada para F4, LT, STa e STb; entre F18 e STa e STx2e; entre F5 e LT, STa e STb. Os virotipos mais frequentes foram F18-STa, F4-LT-STa-STb, F4-STa, F4-LT-STb e F18-STa-STx2e. Beta-hemólise foi observada em 47,4% das amostras e houve associação significativa entre amostras hemolíticas e fatores de virulência F4, F18, STa e STx2e. Quanto consistência fecal, houve associação significativa de fezes líquidas e fímbria F4, toxina STa e virotipos F4-STa e F4-F5-LT-STa-STb. A associação significativa da ETEC e fezes líquidas em leitões de creche, é importante para priorizar a amostragem de fezes com essa consistência para no diagnóstico etiológico da diarreia.

Epidemiologic and etiologic characteristics of diarrheagenic Escherichia coli infection in population in Shenzhen / 中华流行病学杂志

<p><b>OBJECTIVE</b>To understand the epidemiologic and etiologic characteristics of diarrheagenic Escherichia (E.) coli infections in Shenzhen.</p><p><b>METHODS</b>Stool samples were collected from acute diarrheal patients in four sentinel hospitals in Shenzhen and diarrheagenic E. coli strains were isolated and identified with multiplex real-time PCR. Serotyping and pulsed-field gel electrophoresis (PFGE) typing were conducted for the diarrheagenic E. coli isolates.</p><p><b>RESULTS</b>A total of 74 diarrheagenic E. coli strains were isolated from 1 823 stool samples (4.06%). The patients were mainly young children aged <3 years and adults aged 20-39 years, and the infections mainly occurred during May-September of a year. Enterotoxigenic E. coli (ETEC) and enteropathognic E. coli (EPEC) were predominant (45.9% and 31.1%). Serogroups and PFGE patterns varied among the diarrheagenic E. coli isolates. However, serogroup O159 were predominant in ETEC and there were 5 clusters with ≥2 strains sharing same PFGE patterns.</p><p><b>CONCLUSIONS</b>ETEC and EPEC were predominant in diarrheagenic E. coli strains isolated from diarrheal patients in Shenzhen. Age and season specific characteristics of diarrheagenic E. coli infections were observed. The serotypes and PFGE patterns of diarrheagenic E. coli strains varied. Close attention should be paid to the possible ETEC outbreak.</p>

Composição química e concentração mínima bactericida de dezesseis óleos essenciais sobre Escherichia coli enterotoxigênica / Determination of minimum bactericidal concentration of sixteen essential oils on enterotoxigenic Escherichia coli

RESUMO Este trabalho teve por objetivo avaliar o efeito bactericida in vitro de dezesseis óleos essenciais sobre Escherichia coli enterotoxigênica (ETEC). Dentre os óleos essenciais estudados, três foram extraídos in situ por arraste a vapor e treze foram adquiridos comercialmente. Todos os óleos foram analisados por CG-EM e CG-DIC. A atividade bactericida foi avaliada pelo método de microdiluição utilizando-se caldo triptona de soja e microplacas de poliestireno de 96 poços, com posterior plaqueamento das culturas em ágar triptona de soja. Os óleos essenciais de Cinnamomum cassia e de Thymus vulgaris apresentaram concentração mínima bactericida (CMB) de 0,12% e 0,25%, respectivamente. Já os óleos comerciais de Syzygium aromaticum e Origanum vulgare apresentaram ambos CMB de 0,50% e os óleos extraídos in situ de Cymbopogon citratus e Origanum vulgare apresentaram ambos CMB de 1,00%. Os dezesseis óleos essenciais apresentaram composição química qualitativa e quantitativa distintas. As análises químicas dos óleos essenciais de Cinnamomum cassia e de Thymus vulgaris tiveram a presença majoritária de E-cinamaldeído (84,52%) e timol (50,89%). Conclui-se que os óleos de C. cassia e T. vulgaris foram os mais eficazes na inibição do crescimento in vitro dessa bactéria, a qual possui diferentes níveis de sensibilidade dependendo da composição química do óleo.

ABSTRACT This study aimed to evaluate the bactericidal effect in vitro of sixteen essential oils on enterotoxigenic Escherichia coli (ETEC). Among the essential oils, three were extracted in situ by steam distillation and thirteen were purchased commercially. All oils were analyzed by GC-MS and GC-FID. The bactericidal activity was evaluated by the microdilution method using tryptone soy broth, and 96-well polystyrene microplates with subsequent plating of the cultures in tryptone soy agar. Cinnamomum cassia and Thymus vulgaris essential oils showed minimal bactericidal concentration (MBC) 0.12% and 0.25%, respectively. Both commercial oils of Syzygium aromaticum and Origanum vulgare showed MBC of 0.50% and the oils extracted in situ Origanum vulgare and Cymbopogon citratus showed both MBC of 1.00%. The sixteen essential oils pointed out distinct qualitative and quantitative chemical composition. Chemical analysis of Cinnamomum cassia and Thymus vulgaris oils had the predominant presence of E-cinnamaldehyde (84.52% ± 0.07%) and thymol (50.89% ± 0.31%). In conclusion, T. vulgaris and C. cassia oils were the most effective in inhibiting in vitro growth of this bacterium, which has different sensitivity levels depending on the chemical composition of the oil.

Eficiencia de la desingección con aceites esenciales y ultrasonido sobre Escherichia coli inoculada en frutos de tomate y el impacto sobre la actividad antioxidante / Efficacy of disinfection treatments using essential oils and ultrasound on tomato fruits inoculated with escherichia coli and impact on antioxidant activity

La creciente demanda de frutos frescos puede constituir un riesgo para la salud de los consumidores, teniendo en cuenta la gran variedad de microorganismos que estos suelen albergar. El objetivo del presente estudio fue evaluar la eficacia de varios procedimientos de desinfección sobre Escherichia coli enterotoxigénica (enterotoxigenic E. coli [ETEC]) inoculada en tomate y la conservación de las propiedades antioxidantes de los frutos desinfectados. Los frutos fueron sumergidos durante 5 o 10min en dispersiones de aceites esenciales de orégano o tomillo (5 o 10ppm), combinados o no con la aplicación de ultrasonido. La actividad antioxidante se determinó por la neutralización del radical 2,2-difenil-1-pricrilhidrazil (DPPH) y se reportó como porcentaje de inhibición (%I). Los tratamientos de desinfección más eficaces para una significativa reducción log10 UFG/g (S) de ETEC fueron con 10ppm de aceite de orégano durante 10min, con reducciones S=3,05 en tratamientos individuales y S=4,03 en mixtos. Los %I más altos se lograron con tratamientos individuales con sonicación (69,52 y 72,48) y en tratamientos combinados con aceite de tomillo 5ppm y ultrasonido durante 5 y 10min, con valores de 51,27 y 53,31%, respectivamente

Fresh produce often harbors a great number of microorganisms; hence, its growing demand may constitute a risk for consumers. The aim of this study was to evaluate the efficacy of several disinfection procedures against enterotoxigenic Escherichia coli (ETEC) inoculated on tomato fruits and the conservation of the antioxidant properties of these disinfected fruits. Fruits were immersed for 5 or 10min in oregano or thyme essential oil dispersions (5, 10ppm), with or without ultrasound treatment. Antioxidant activity of disinfected fruits was determined as the ability to scavenge 2,2-diphenyl-1-pricrylhydrazyl (DPPH) radicals and was reported as percentage of inhibition (%I). The most efficient disinfectant treatments showing significant differences (p≤.05) between the reductions log10 CFU/g (S) of ETEC were those using 10ppm oregano for 10min, with S=3.05 in individual treatments and S=4.03 in mixed treatments. The highest %I was obtained with individual sonication treatments (69.52 and 72.48), while in combined treatments the %I values increased with thyme oil 5ppm and ultrasound for 5min (51.27%) and 10min (53.31%)

Surveillance for diarrheagenic Escherichia coli in Shanghai, 2012-2013 / 中华流行病学杂志

<p><b>OBJECTIVE</b>To understand the distribution of diarrheagenic Escherichia (E.) coli in population in Shanghai and discuss the practice model of cooperation in enteric infectious disease prevention and control between public health institution and hospital.</p><p><b>METHODS</b>Sentinel hospitals were assigned, standard detection and identification of diarrheagenic E. coli were conducted, incidence curve of diarrheagenic E. coli infection was drawn and epidemiologic survey and laboratory detection were conducted for suspect diarrheagenic E. coli infection outbreaks.</p><p><b>RESULTS</b>A total of 7 204 stool specimens were collected from diarrhea patients in 4 hospitals during 2012-2013, in which 712 (9.9% ) were diarrheagenic E. coli positive, including 351 enteropathogenic E. coli (EPEC) strains, 292 enterotoxigenic E. coli (ETEC) strains, 32 enteroinvasive E. coli(EIEC) strains and 6 Shiga toxin-producing E. coli (STEC/EHEC) strains, as well as 31 mixed strains. EPEC infection mainly occurred in children aged 1-5 years; and all of these infections were caused by aEPEC. The incidence peak of ETEC infection was during August, the positive rate was >20%. The ETEC infection mainly occurred in infants aged 1-28 days in 2012 and in people aged 20-60 years in 2013 (P<0.05). ST was the major type (59.6%), followed by LT (27.8%) and ST/LT (12.6%). EIEC infection increased in children obviously in 2013 (P<0.01). No EHEC O157:H7 case was detected, but two EHEC O26:H11 (eae-hlyA-stx1a) cases in children were reported for the first time in Shanghai. The survey result indicated that the multidrug-resistant ETEC (STh-CS21-CFA/I-ClyA-EatA-ST2332-SHNL0005) strain causing outbreak in 15 newborns in Shanghai in 2012 was in the same clone as the strain detected in Zigong in Sichuan province.</p><p><b>CONCLUSION</b>Significant change has occurred in diarrheagenic E. coli distribution in Shanghai in recent years, ETEC has potential risk to cause outbreak of hospital acquired infection in neonates and food borne infection. The active surveillance on ETEC and other enteric pathogens by both public health institutions and hospitals need to be improved.</p>

Epidemiological Investigation on an Outbreak of Enterotoxigenic E. coli among the Baseball Club Students of High School in Ulsan City, 2014 / 農村醫學 地域保健

OBJECTIVES: An outbreak of food poisoning occurred among the baseball club students at a high school in Ulsan city in 2014. An epidemiological investigation was carried out to examine the infection source and the transmission route of pathogen, and to prevent a recurrence. METHODS: A questionnaire survey was conducted for 26 male students and 2 food handlers. Rectal swabs were examined in 7 students and the 2 food handlers, and an environmental investigation was performed. A retrospective cohort study was used to evaluate the association between risk factors and disease. RESULTS: The attack rate was 35.7% (10 persons/28 persons) from June 9 to 14, and Enterotoxigenic E. coli ST/LT was isolated from 7 among 28 persons. The study revealed that no food was a significant risk factor for the outbreak. There were no connection between environmental factors and the outbreak. CONCLUSIONS: The major risk factors for this outbreak were presumed to be the contaminated ice cube and ice making machines and eating ice cube from the machines. More strict personal and environmental hygiene need to be enforced to prevent such outbreaks.

A CssA, CssB and LTB chimeric protein induces protection against Enterotoxigenic Escherichia coli

OBJECTIVES: Enterotoxigenic Escherichia coli (ETEC), a major cause of diarrhea in children under 5, is an important agent for traveler's diarrhea. Heat-labile enterotoxin (LT) and colonization factors (CFs) are two main virulence mechanisms in ETEC. CS6 is one of the most prevalent CFs consisting of two structural subunits viz., CssA, CssB, necessary for attachment to the intestinal cells. METHODS: In the present research, a chimeric trivalent protein composed of CssB, CssA and LTB was constructed. The chimeric gene was synthesized with codon bias of E. coli for enhanced expression of the protein. Recombinant proteins were expressed and purified. Mice were immunized with the recombinant protein. The antibody titer and specificity of the immune sera were analyzed by ELISA and Western blotting. Efficiency of the immune sera against ETEC was evaluated. RESULTS: Antibody induction was followed by immunization of mice with the chimeric protein. Pretreatment of the ETEC cells with immunized animal antisera remarkably decreased their adhesion to Caco-2 cells. DISCUSSION: The results indicate efficacy of the recombinant chimeric protein as an effective immunogen, which induces strong humoral response as well as protection against ETEC adherence and toxicity. .