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.

Toxigenic Profiling of Enterotoxin-Producing Bacillus cereus Isolated from Marketed Raw Chicken Meat and Human Subjects by Triplex and Multiplex PCR

Bacillus cereus incorporates the most important group of endospore-forming micro organism and can cause emetic and diarrheal food poisoning. A total of 42 B. cereus strains isolated from marketed raw chicken meat and human subjects swab samples were assessed by a triplex and multiplex PCR for the presence of enterotoxin genes. The detection rate of nheB, hblA, hblD, cytK, nheA,CER, hblC and entFM enterotoxin genes among all B. cereus strains was 83.33%, 80.95%, 69.04%, 21.42%, 47.61%, 0%, 61.90%, and 92.85% respectively. Enterotoxigenic profiles were determined in enterotoxin-producing strains showed 19 different patterns. The results offer essential information on toxin genes prevalence and toxigenic profiles of B. cereus from sources of origin. The present study was taken into consideration about extreme fitness danger for public health and insuring extra ability in difficulty to food safety amongst all B. cereus group members. Also, there may be need for extensive and continuous tracking of food products embracing both emetic toxin and enterotoxin genes.

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.

Surveillance for enterotoxigenic & enteropathogenic Escherichia coli isolates from animal source foods in Northwest Iran

Background & objectives: Diarrhoeagenic Escherichia coli strains are common agents of diarrhoea particularly in developing countries. Food products of animal origin are considered as common carriers of E. coli. This study was undertaken to identify enterotoxigenic Escherichia coli (ETEC) and enteropathogenic E. coli (EPEC) pathotypes in animal-source foods (ASF). Methods: A total of 222 ASF samples were investigated. Based on the culture and biochemical tests, 109 E. coli isolates were identified. Duplex-polymerase chain reaction assay was used to detect ETEC and EPEC. The target genes selected for each category were the lt and st for the ETEC, and eae and bfp for the EPEC isolates. Results: The occurrence of E. coli in dairy and meat products was 45 and 52.5 per cent, respectively. Among the E. coli isolates, two ETEC, one typical EPEC and three atypical EPEC were detected in meat samples, whereas only one typical EPEC and one atypical EPEC were detected in dairy samples. Interpretation & conclusions: Our results showed presence of ETEC and EPEC strains in ASFs. The milk without pasteurization and traditional dairy products produced in unhygienic conditions are most likely the main sources of E. coli pathotypes and other zoonotic pathogens and thus can be considered a potential hazard to the health of the community.

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.

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.

Antimicrobial susceptibility profile of enterotoxigenic and enteropathogenic Escherichia coli isolates obtained from fecal specimens of children with acute diarrhea / Perfil de suscetibilidade a drogas antimicrobianas de amostras de Escherichia coli enterotoxigênica e enteropatogênica isoladas de espécimes fecais de crianças com doença diarreica aguda

ABSTRACT Among the diseases which etiopathogenesis is associated with Escherichia coli, acute diarrhea stands out. Studies on the characterization of the antimicrobial susceptibility profile contribute to the selection of appropriate empirical antimicrobial therapy. In this study, the antimicrobial susceptibility profile of 98 enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC) strains isolated from fecal specimens of children with acute diarrhea was evaluated. The resistance rates to ampicillin, sulfamethoxazole/trimethoprim, amoxicillin/clavulanate, and nalidixic acid were high, ranging from 34.7% to 10.2%. The result of this research recommends the use of cefotaxime and ceftriaxone for the empirical treatment of children with acute diarrhea which the etiology suggested is ETEC or EPEC.

RESUMO Entre as doenças cuja etiopatogenia está associada à Escherichia coli, destaca-se a doença diarreica aguda. Estudos que visam à caracterização do perfil de suscetibilidade antimicrobiana contribuem para o delineamento de antibioticoterapia empírica eficaz. Neste estudo, foi avaliado o perfil de suscetibilidade a antimicrobianos de 98 amostras de E. coli enterotoxigênica (ETEC) e E. coli enteropatogênica (EPEC) isoladas de crianças com doença diarreica. As frequências de resistência a ampicilina, sulfametoxazol-trimetoprima, amoxicilina-clavulanato e ácido nalidíxico foram elevadas, variando entre 34,7% e 10,2%. Esta pesquisa recomenda o emprego de cefotaxima e ceftriaxona para o tratamento empírico de crianças com quadro de diarreia cuja etiologia sugerida seja ETEC ou EPEC.

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.

Enterotoxigenic Bacillus cereus from cooked chicken meat: A potential public health hazard

Aims: This study was conducted to isolate Bacillus cereus from raw and cooked chicken meat from selected retail shops and wet markets in Kota Bharu and to determine the antimicrobial resistance patterns of B. cereus. Methodology and results: A total of sixty samples (30 from raw and 30 from cooked chicken meat) were tested for presence of B. cereus. Isolation and identification of B. cereus was done by using routine bacterial culture and Polymerase Chain Reaction (PCR). Bacillus cereus was detected in 16.67% (10/60) of the samples tested. All isolates were negative for the enterotoxigenic gene, nhe genes, however, six of the isolates were found to be positive for hbla genes. B. cereus isolates showed 100% resistance towards beta lactam antibiotics. Conclusion, significance and impact study: Although only 60 samples are analysed in the current study, the fact that toxigenic strains of B. cereus were isolated in cooked chicken meat intended for human consumption implies the potential public health risk it might pose. Further study with increased sample size, screening other toxigenic strains of B. cereus and molecular typing is recommended to have a more detailed understanding of the occurrence of the bacteria in chicken meat in Kota Bharu. It is necessary to educate the public on the risks of food contamination by bacteria that may cause food borne illnesses. Some precautions such as routine checking of the freshness of food before consumption, hygienic preparation and proper cooking of food can be implemented to reduce the risks of food borne illnesses related B. cereus and other potentially dangerous bacteria.

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.

The study of using siRNA technology to silence expression of LT gene of Enterotoxigenic Escherichia coli / 国际检验医学杂志

The distinctive LT siRNAs were designed according to the LT sequence .During the process of cultivation ,siRNA targeting the LT gene ,non‐specific control siRNA ,negative control siRNA and culture medium were added into siRNA group (siRNA‐LT1 group , siRNA‐LT2 group) ,siRNA‐coa3 group ,siRNA‐NC group and blank control group ,respectively ,and three times in each group (1 nmol each time) .After siRNA added at the first time ,bacteria was collected in 45 min (A) ,90 min (B) and 135 min (C) time points .The expression of mRNA in three time points (A ,B and C) were detected by real‐time fluorescence quantitative PCR .The protein level of LT in siRNA‐LT1 group ,siRNA‐LT2 group and blank control group were detected by Western blot in three time points .Results The results of real‐time fluorescence quantitative PCR showed that inhibition of siRNA‐LT1 on the expression of LT mRNA at the three time points(A ,B and C)were 70 .9% ,70 .1% ,72 .5% respectively ,and inhibition of siRNA‐LT2 on the ex‐pression of LT mRNA at the three time points(A ,B and C)were 70 .1% ,69 .2% and 70 .5% respectively .In the three time points (A ,B and C)the inhibition rate of the expression of LT mRNA in siRNA‐LT1 group and siRNA‐LT2 group were statistically lower than that in the siRNA‐NC group ,siRNA‐coa3 group and blank control group (P<0 .05) .The results of Western blot showed that in siRNA‐LT1 group the inhibitory rate of expression of LT protein in the three time points were 43 .1% ,18 .4% and 5 .0% ,re‐spectively ;in the siRNA‐LT2 group were 38 .2% ,15 .4% and 30 .1% ,respectively .Conclusion The specific siRNA could inhibit the expression of LT gene in vitro .

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. .

Presence of enterotoxigenic Staphylococcus aureus in artisan fruit salads in the city of San Luis, Argentina

An increase in the consumption of fruit juices and minimally processed fruits salads has been observed in recent years all over the world. In this work, the microbiological quality of artisan fruit salads was analysed. Faecal coliforms, Salmonella spp, Shigella spp, Yersinia enterocolitica and Escherichia coli O157:H7 were not detected; nevertheless, eleven strains of Staphylococcus aureus were isolated. By multiplex PCR, all isolates showed positive results for S. aureus 16S rRNA gene and 63.6% of them were positive for sea gene. Furthermore, PCR sea positive strains were able to produce the corresponding enterotoxin. Finally, the inactivation of these strains in fruit salads by nisin, lysozyme and EDTA, was studied. EDTA produced a total S. aureus growth inhibition after 60 h of incubation at a concentration of 250 mg/L. The presence of S. aureus might indicate inadequate hygiene conditions during salad elaboration; however, the enterotoxigenicity of the strains isolated in this study, highlights the risk of consumers' intoxication. EDTA could be used to inhibit the growth of S. aureus in artisan fruit salads and extend the shelf life of these products.

A surveillance of enteropathogens in piglets from birth to seven days of age in Brazil / Levantamento dos enteropatógenos de leitões do nascimento a sete dias de idade no Brasil

The purpose of the study was to evaluate the real importance of anaerobic enteropathogens and rotavirus in contrast to more common agents as cause of diarrhea in piglets within the first week of life. Sixty 1- to 7-day-old piglets, 30 diarrheic and 30 non-diarrheic (control), from 15 different herds were selected, euthanized and necropsied. Samples of the jejunum, ileum, colon, cecum and feces were collected from the piglets and analyzed to determine the presence of the following enteropathogens: enterotoxigenic Escherichia coli (ETEC), Clostridium perfringens types A and C, Clostridium difficile, rotavirus and Isospora suis. Among diarrheic piglets, 23.3% were positive for C. difficile, 70% for C. perfringens type A cpb2+, 14.3% for rotavirus and 10% for ETEC. Among non-diarrheic control piglets, 10% were positive for C. difficile, 76.7% for C. perfringens type A cpb2+, 0% for rotavirus, 3.3% for ETEC and 3.3% for I. suis. C. perfringens type C was not detected in any of the animals. Histological lesions characteristic of C. difficile, E. coli and rotavirus were observed. However, no C. perfringens type A suggestive lesions were detected. There was a positive correlation between mesocolon edema and the presence of C. difficile toxins. Although C. perfringens type A cpb2+ was the most frequently detected enteropathogen, there was no association between its presence and diarrhea or macro or microscopic changes. C. difficile and Rotavirus were the most relevant pathogens involved with neonatal diarrhea in this study, and histopathology associated with microbiological test proved to be the key to reach a final diagnosis.

O objetivo do presente estudo foi avaliar a real importância de enteropatógenos anaeróbios e rotavirus em comparação à outros agentes mais comuns como causa de diarreia em leitões até cinco dias de idade. Leitões com 0 a 7 dias de vida, 30 diarreicos e 30 não diarreicos (controles) de 15 granjas diferentes foram eutanasiados e necropsiados. Amostras de jejuno, íleo, colon e ceco foram coletadas e submetidas à detecção dos seguintes enteropatógenos: Escherichia coli enterotoxigênica (ETEC), Clostridium perfringens, Clostridium difficile, rotavirus e Isospora suis. Entre os animais diarréicos, 23.3% foram positivos para C. difficile, 70% para C. perfringens tipo A cpb2+, 14.3% para rotavirus e 10% para ETEC. Entre os leitões não-diarréicos, 10% foram positivos para C. difficile, 76.7% para C. perfringens tipo A cpb2+, 3.3% para ETEC e 3.3% for I. suis. C. perfringens tipo C não foi detectado em nenhum animal. Lesões histológicas características de C. difficile, E. coli e rotavirus foram observadas. Por outro lado, nenhuma lesão sugestiva de C. perfringens foi detectada. Foi possível observar uma correlação positiva entre edema de mesocolon e presença das toxinas A/B. Apesar de C. perfringens tipo A cpb2+ ter sido o patógeno mais encontrado, nenhuma associação com lesões foi encontrada. C. difficile e Rotavirus foram os agentes mais relevantes associados à diarreia neonatal, e ficou demonstrada a relevância de associação de histopatologia com testes de detecção microbiológica para se firmar um diagnóstico.

Prevalence of seg, seh and sei Genes among Clinical and Nasal Staphylococcus aureus Isolates in Palestine.

Aims: To investigate the presence of the staphylococcal enterotoxin genes seg, seh and sei among clinical and nasal isolates. Place and Duration of Study: Department of Biology and Biotechnology, An-Najah N. University, Palestine, in 2011. Methodology: A total 124 S. aureus isolates were collected, forty three were nasal and 81 were clinical isolates. PCR technique was used to detect enterotoxin genes seg, seh and sei, mecA gene and analysis of SCCmec types. Enterotoxigenic strains were also typed using coagulase typing kit. Results: Fifty two (41.9%) isolates were positive for one or more of these enterotoxin genes. The prevalence of toxin genes among S. aureus isolated from nasal swabs 25/43 (58.1%) was higher than those isolated from clinical samples 27/81 (33.3%). Combination of the toxin genes was noted only in MSSA isolate from both nasal swabs and clinical samples. Distribution of toxin genes in MSSA isolates was higher (49.5%) than those in MRSA isolates (21.2%). SCCmec typing showed that the MRSA enterotoxigenic strain were belonged to types II, III and IVa. MRSA strains were found to belong to coagulase serotypes II, III and VII, while MSSA strains were belonged to serotypes II-VII. In nasal samples, 16/25 (64.0%) of enterotoxigenic strains showed the genotype seg+/sei+, while in clinical samples 1/27 (3.7%), 1/27 (3.7%) and 3/27 (11.1%) of enterotoxigenic strains showed the genotypes seg+/seh+, seg+/sei+ and seg+/seh+/sei+, respectively. This study showed that the majority of the isolates 42/124 (33.9%) were seg+, while none of nasal strains harbored seh gene. Conclusion: The prevalence of seg, seh and sei genes in the S. aureus isolated from nasal swabs differed significantly from those obtained from clinical samples, as well as the prevalence of the same genes in MSSA differed significantly from those in MRSA. In addition, S. aureus isolates from clinical and nasal swabs could serve as a possible reservoir of newly described seg, seh and sei genes.

Biodiversity and Enterotoxigenic Potential of Staphylococci Isolated from Raw and Spontaneously Fermented Camel Milk.

Aims: To characterise the diversity, genotypic and phenotypic properties of coagulase negative and coagulase positive staphylococci from camel milk. Place and Duration of Study: Laboratory of Food Biotechnology, Department of Health Sciences and Technology (D-HEST), Swiss Federal Institute of Technology, Zurich, Switzerland, between July 2009 and June 2011. Methodology: Staphylococci isolated from 59 raw and spontaneously fermented camel milk (suusac) samples from Kenya and Somalia were identified, pheno- and genotypically characterized. Preliminary screening of colonies was done by catalase test, Gram staining reactions, clumping factor/protein A and microscopy. Further identification was done by 23S rDNA species PCR, thermostable nuclease gene (nuc) PCR and rep-PCR followed by staphylococcal genus ID32 Staph system and coagulase negative species specific PCR. PCR amplification of the genes encoding capsular polysaccharides cap5 and cap8, and staphylococcal enterotoxins SEA to SEE and SEG to SEJ was also carried out. Results: From a total of 235 BP medium isolates, staphylococci were 146 (62 %) of which, 66 (45 %) were Staphylococcus aureus. S. epidermidis accounted for 43 % of the coagulase negative staphylococci (CNS). The rest of the CNS were 25 % S. simulans, 16.3 % S. saprophyticus, 2.5 % S. haemolyticus, 2.5% S. hyicus, 2.5 % S. xylosus, 2.5 % S. lentus, 1.3 % S. carnosus and 1.3 % S. microti. Capsular polysaccharide gene cap5 was present in 15 % and cap8 in 23 % of the S. aureus isolates. Enterotoxin genes were detected in 47 % of the staphylococci with sej in 33 %, seb in 6 %, sed in 5 % and seg in 3 % of the isolates. Within the species enterotoxin genes were detected in 100 %, 64.7 %, 38.5 % and 22.7 % of the S. simulans, S. epidermidis, S. sapropyticus and S. aureus respectively. Conclusion: The diversity of CNS is remarkable and the prevalence of enterotoxin genes amongst CNS and CPS further informs generalizations for other milk and hygienic situations in similar production environment.

Epidemiological Relationship of Enterotoxigenic Escherichia coli and Enteroaggregative E. coli Isolated from Patients with Diarrhea in Seoul

Of total 1,438 specimens of patients with diarrhea in Seoul, 2011, 217 samples (15%) were found pathogenic Escherichia coli that included 192 strains (89%) of enterotoxigenic E. coli (ETEC) and enteroaggregative E. coli (EAEC). The highest isolation rate for ETEC and EAEC was found in August and September. Sixty two pathogenic E. coli strains (34 ETEC and 28 EAEC strains) were selected from 175 strains (94 ETEC and 81 EAEC strains) isolated in August and September. Of 94 strains characterized for ETEC phenotype, 76 (81%) expressed heat-stable toxin (ST) only. Antimicrobial susceptibility test was carried out by using sixteen types of antibiotics. A high level of antimicrobial resistance to tetracycline (57%), ampicillin and ticarcillin (54%) was observed among EAEC isolates while the highest resistance rate of ETEC was found for nalidixic acid (47%), followed by tetracycline (32%). As to the antimicrobial susceptibility test, EAEC showed the complicated multi-drug resistant patterns in which the resistance was higher than ETEC. Pulsed-field gel electrophoresis (PFGE) was carried out to examine the genetic relatedness among ETEC and EAEC isolates. Except for 11 strains, 51 strains were divided by eight pulsotypes. In PFGE analysis, isolates from foodborne disease outbreaks in August and September 2011 showed close relation.

Field evaluation of Enterotoxigenic Escherichia coli-specific bacteriophage (PhiCJ19) as a feed additive / 대한수의학회지

Field efficacy of enterotoxigenic Escherichia coli-specific phage (PhiCJ19) as a feed additive was evaluated in weaning piglets. Fifty-four piglets at 3~4 weeks old were allocated in three different groups and two of them were fed with bacteriophage at different concentrations (10(6) PFU/kg feed and 10(8) PFU/kg feed, respectively) for 30 days. Body weight and feed intake were measured at 10 days interval and body condition and fecal score were inspected every day. Based on the measurement, feed conversion rate (FCR) and average daily gain (ADG) of each group during 30 days were analyzed. The analysis suggests that the bacteriophage may help the improvement of FCR and ADG at 10(8) PFU/kg of bacteriophage feeding group in 30 days. A result from analysis of fecal score indicates that the bacteriophage also may help to relieve the intermittent diarrhea in post-weaning stage. Those results suggest that bacteriophage might help the growth of piglets in post-weaning stage.