Diarrhea outbreak in Pernambuco, Brazil, associated with a heat-stable cytotoxic enterotoxin produced by Aeromonas caviae / O surto de diarreia em Pernambuco, Brasil é associado com uma enterotoxina citotóxica termo-estável produzida por Aeromonas caviae

SUMMARYIn the present study enterotoxic and cytotoxic activities of twenty Aeromonas caviaestrains were examined. They originated from fecal specimens of patients with acute diarrhea during an outbreak in Brazil in 2004. Culture supernatants of fourteen strains (70%) caused fluid accumulation in rabbit ileal intestinal loops and in suckling mice assays, and also showed a cytotoxic activity in Vero and Caco-2 cells. The enterotoxic and cytotoxic factors were heat-stable after culture supernatants treatment at 100 ºC. The results revealed that A. caviaestrains produce a putative diarrheagenic virulence factor, a heat-stable cytotoxic enterotoxin that could be linked to the diarrhea outbreak that took place in Brazil.

RESUMOEm 2004 ocorreu um surto de diarreia aguda no Estado de Pernambuco, Brasil. Setenta por cento (14 dos 20) dos sobrenadantes de cultura de Aeromonas caviae,isoladas neste episódio induziram acúmulo de líquido em testes de alça ligada de intestino de coelhos, assim como em teste em camundongos recém-nascidos. Os mesmos sobrenadantes mostraram também atividade citotóxica em células de Vero e Caco-2, mas não em células HeLa e HEp2. As atividades enterotóxicas e citotóxicas mantiveram-se mesmo após o aquecimento a 100 ºC dos sobrenadantes de cultura. Este trabalho revela a expressão de um provável fator diarreiogênico: uma enterotoxina-citotóxica termo-estável, produzida por A. caviaeque pode ser associada ao surto de diarreia ocorrido no Brasil. Atualmente estamos purificando esta enterotoxina termo-estável, com o objetivo de elucidar seu papel como fator de virulência na diarreia causada por A. caviae.

[Effect of zataria multiflora boiss. essential oil on enterotoxin production by staphylococcus aureus ATCC 6538]

Staphylococcal food poisoning does not result from the ingestion of S. aureus per se, but rather from enterotoxins which are pre-formed within the food. There is no report on effect of Zataria multiflora Boiss. essential oil on enterotoxin production by this microorganism. The aim of this study was to evaluate the effect of different concentrations of Zataria multiflora Boiss. essential oil on enterotoxin production by S. aureus. This study was done as a factorial model using different concentrations of EO [0, 0.005, 0.015 and 0.03%].The bacterial growth was evaluated during 43 days of incubation and enterotoxin production was analyzed using a enterotoxin assay kit. The growth of the organism was completely inhibited by the EO=0.03%. Enterotoxin production was not affected by sub-inhibitory concentration of EO at level of 0.005%, while it was significantly [p < 0.05] inhibited by increasing subinhibitory concentration of EO to 0.015%. The adverse effect of sub-inhibitory concentration of Zataria multiflora Boiss. EO on enterotoxin production by S. aureus, demonstrated in this study, suggests the potential application of this EO as a natural antimicrobial in foods

Enterotoxigenic and nontoxigenic Bacteroides fragilis strains isolated in Brazil

The presence of enterotoxigenic Bacteroides fragilis and nontoxigenic B. fragilis (NTBF) among 109 strains isolated from 1980-2008 in Brazil were investigated by PCR. One strain, representing 0.9 percent of the total analyzed strains, harbored the bft gene which was identified as bft-1 isoform based on PCR-RFLP and sequencing. Forty-nine strains (44.9 percent) exhibited the NTBF pattern III which possesses the flanking region required for pathogenicity island acquisition in which the bftgene is codified. These data reinforce the potential of B. fragilis as an emerging enteropathogen in our country.

Production, characterization, and application of antibodies against heat-labile type-I toxin for detection of enterotoxigenic Escherichia coli

Strains of enterotoxigenic Escherichia coli (ETEC) are responsible for significant rates of morbidity and mortality among children, particularly in developing countries. The majority of clinical and public health laboratories are capable of isolating and identifying Salmonella, Shigella, Campylobacter, and Escherichia coli O157:H7 from stool samples, but ETEC cannot be identified by routine methods. The method most often used to identify ETEC is polymerase chain reaction for heat-stable and heat-labile enterotoxin genes, and subsequent serotyping, but most clinical and public health laboratories do not have the capacity or resources to perform these tests. In this study, polyclonal rabbit and monoclonal mouse IgG2b antibodies against ETEC heat-labile toxin-I (LT) were characterized and the potential applicability of a capture assay was analyzed. IgG-enriched fractions from rabbit polyclonal and the IgG2b monoclonal antibodies recognized LT in a conformational shape and they were excellent tools for detection of LT-producing strains. These findings indicate that the capture immunoassay could be used as a diagnostic assay of ETEC LT-producing strains in routine diagnosis and in epidemiological studies of diarrhea in developing countries as enzyme linked immunosorbent assay techniques remain as effective and economical choice for the detection of specific pathogen antigens in cultures.

Enteropathogenic bacteria and enterotoxin-producing Staphylococcus aureus isolated from ready-to-eat foods in Khon Kaen, Thailand.

The objective of this study was to investigate the microbiological quality of ready-to-eat food in the Municipality of Khon Kaen, Thailand. Four categories of 186 food samples were collected: (1) high heat food; (2) low heat food; (3) no heat food; and, 4) on-site prepared fruit juices and beverages. Of the food samples, 145 (78%) failed to meet acceptable microbiological standards, including fruit juice and beverages (100%), no heat food (91.7%), low heat food (81.7%) and high heat food (57.9%). The most frequent bacterial indexes indicating unacceptability were the most probable number (MPN) of coliforms (78%), the bacterial colony count (58%), and the MPN of E. coli (46%). Pathogenic bacteria were found in 6.5% of food samples. Salmonella, Vibrio cholerae non O1 and Aeromonas hydrophila were found in 4.3, 1.6 and 0.5% of the total food samples, respectively. The serovars of Salmonella found in food were S. Derby, S. Give, S. Krefield, S. Paratyphi B, S. Verchow, S. Lexington and S. Senftenberg. Staphylococcus aureus concentrations of >10(2) CFU/g and >10(5) CFU/g were found in 10.8% and 1.1% of the food samples. Enterotoxin types AB and A of S. aureus were found in 2.7% of the food samples. These results indicate that more than half of the ready-to-eat foods tested in Khon Kaen municipality did not meet microbiological national standards and many kinds of enteropathogenic bacteria were found, suggesting food stalls may be a source of foodborne disease.

Prevalence of enterotoxigenic Escherichia coli-associated diarrhoea and carrier state in the developing world.

This study assesses the importance of enterotoxigenic Escherichia coli (ETEC) as a diarrhoeal agent in developing countries. Odds ratios were calculated for incurring ETEC-associated diarrhoea based on data reported between 1970 and 1999. Carriage of ETEC was associated with diarrhoea in children aged less than five years, except for hospitalized infants aged 0-11 month(s) and children aged 1-4 year(s) at outpatient clinics. Two hundred and eighty million episodes of diarrhoea due to ETEC were seen yearly among those aged less than five years, and close to 50 million children of this age group were asymptomatic carriers of ETEC. Every 7th diarrhoeal episode in children aged less than one year and close to 25% of diarrhoeal cases in children aged 1-4 year(s) were due to ETEC. A child born in a developing country is likely to experience 0.5 diarrhoeal episodes per year caused by ETEC until the age of five years, after which the yearly incidence drops to 0.1. To conclude, ETEC remains an important diarrhoeal pathogen among children in the developing world.

Portación de Staphylococcus aureus enterotoxigénicos en manipuladores de alimentos / Carriage of enterotoxigenic Staphylococcus aureus in food handlers

BACKGROUND: One of the most common pathogens causing alimentary toxi-infections is Staphylococcus aureus (S aureus). The presence of S aureus in food, indicates flaws during food manipulations. For this reason most sanitary norms require the detection of S aureus carriers. AIM: To determine the carriage rate of enterotoxin producing S aureus strains in food handlers, and to evaluate the antibiotic susceptibility to six antimicrobial agents. MATERIALS AND METHODS: A total of 102 food handlers from 19 restaurants in Santiago, were analyzed. Samples for microbiological analysis were obtained with a swab from the retropharynx. RESULTS: S aureus grew in 35 out of the 102 samples obtained (34 per cent). Further analysis revealed that 19/35 (54 per cent) strains were able to produce enterotoxins. Therefore the corrected carriage rate was 19 per cent (19/102). The most frequently detected enterotoxin was the type A (12/19). All S aureus isolates were resistant to penicillin and susceptible to oxacillin, clindamycin, kanamycin, vancomycin and linezolid. CONCLUSIONS: The carriage rate of S aureus in food handlers in similar to the rate reported in the general population in our country. These results confirm the need for education and training programs in food safety, directed to food handlers.

Production of heat-labile enterotoxin by strains of Aeromonas veronii bv veronii.

Three isolates of Aeromonas veronii bv veronii (2 environmental, one blood infection) were examined to see if they produce any enterotoxin and, if so, to determine its characteristics. Two isolates caused fluid accumulation in the initial rabbit ileal loop tests. The other strain did so after a single passage through the rabbit ileal loop. All the isolates showed gradual enhancement of fluid secretion after each subsequent passage. Inocula of 1 x 10(4) viable cells and 0.25 mL of culture filtrate caused fluid accumulation comparable to those of toxigenic Vibrio cholerae 569B. The enterotoxic activity was inactivated at higher temperature, and showed biological activity over a wide range of pH. The only histopathological change observed was depletion of mucous from goblet cells. The findings of the study indicate that strains of A. veronii bv veronii produce a heat-labile, pH-stable diarrhoeagenic factor without causing any damage to the intestinal mucosa.

Effect of norepinephrine on growth of Salmonella and its enterotoxin production.

Salmonella typhimurium was cultured in presence or absence of norepinephrine in conditioned media. Two conditioned media containing bovine and pig serum were prepared. Supplementation of fresh cultures with norepinephrine (5 x 10(-5) M per mL of medium) resulted in ten-fold increase in growth as compared to controls. No significant difference in growth of organisms in media containing bovine and pig serum was observed. Growth was more in culture incubated under shaking condition than in non-shaking condition. Enterotoxin production increased by two to eight-folds in the medium supplemented with norepinephrine.

Methicillin-resistant Staphylococcus aureus in human milk

We collected and analyzed 500 samples of human milk, from five Brazilian cities (100 from each) to detect methicillin-resistant strains of Staphylococcus aureus (MRSA) producing enterotoxins. We found 57 strains of MRSA, and the mecA gene, responsible for resistance, was detected in all of them using a specific molecular probe. We examined 40 strains for the presence of four enterotoxins, after selecting a subset that included all strains from each region, except for the largest sample, from which 10 were randomly selected. Among these two presented enterotoxin B, and growth in human colostrum and trypicase soy broth. After 5 h of incubation at 37§C, population sizes were already higher than 9.4 x 105 UFC/ml and enterotoxin was released into culture medium and colostrum. Our results stress the importance of hygiene, sanitary measures, and appropriate preservation conditions to avoid the proliferation of S. aureus in human milk.

Enterotoxigenic Escherichia coli: An overview

Enterotoxigenic Escherichia coli is an important cause of traveler's diarrhea and diarrheal illnesses in children in the developing world. In this presentation we will focus on the main virulence attributes of this pathogenic category of E. coli, and discuss the evolution of studies conducted in our laboratory

Virulence factors of aeromonads--an emerging food borne pathogen problem.

Aeromonas spp. possess a number of virulence properties which are considered responsible for intestinal and extra-intestinal infections in human beings and also for a wide variety of infections in animals. The paper discusses current status of colonization and toxic factors of Aeromonas spp., especially in relation to food and biochemical markers. Future research needs are also identified.

Immunization against the colonization factor antigen I of enterotoxigenic Escherichia coli by administration of a bivalent Salmonella typhimurium aroA strain

An expression plasmid (pCFA-1) carrying the cfaB gene that codes for the enterotoxigenic Escherichia coli (ETEC) fimbrial adhesin colonization factor antigen I(CFA/I) subunit was constructed and used to transform a derivative of the attenuated Salmonella typhimurium aroA vaccine strain SL3261 carrying an F'lacl(q). Treatment of the transformed strain with isopropyl-beta-D-thiogalactopyranoside (IPTG) resulted in elevated in vitro expression of the CFA/I subunit. Although flagellar function and lipopolysaccharide (LPS) synthesis were similar in both the parental and the recombinant strains, spleen colonization was reduced in the recombinant strain. AII BALB/c mice parenteally inoculated with the recombinant strain developed significant anti-CFA/I and anti-LPS serum antibody titers (P<0.05). Moreover, 2 of 5 mice orally inoculated with the engineered Salmonella strain developed anti-CFA/I intestinal IgA (P>0.05) while 4/5 of the same mice developed anti-LPS (P<0.05). The results indicate that the vaccine strain elicited an antibody response against the bacterial host both after oral and intravenous immunization while the response against the CFA/I antigen was significant only after inoculation by the intravenous route.

Differential kinetic patterns for Shiga toxin production by Escherichia coli

El objetivo de este trabajo fue estudiar la cinetica de produccion de los distintos tipos de toxinas Shiga (Stx1; Stx2c) asociadas a Escherichia coli, en cepas de referencia y aisladas de pacientes con Sindrome Uremico Hemolitico (SUH). Las cepas fueron cultivadas en caldo Penassay e incubadas a 37§ con agitacion (200 rpm), tomandose muestras a distintos tiempos (1,5; 3; 9 y 24 horas) para determinar el crecimiento bacteriano y la citotoxicidad libre y asociada a celulas. Para Stx1, a las 3 horas de incubacion, la relacion entre la concentracion intracelular y la extracelular (ic/ec), estuvo comprendida entre 32 y 200 veces. A las 24 horas, ambas concentraciones se igualaron o (ec) resulto 2 veces mayor, dependiendo de la cepa estudiada. Sin embargo, tanto la actividad citotoxica libre como la asociada a celulas presentaron titulos muy bajos. Esto indico una perdida de actividad durante la fase estacionaria que podria deberse al cese de la sintesis de Stx1 o la accion de enzimas proteoliticas. Para Stx2, a las 3 horas (ic) fue igual o 2 veces superior a (ec), a las 34 horas (ec) fue 16 a 32 veces superior a (ic). Para Stx2c, (ec) aumento logaritmicamente con un rendimiento maximo a las 5 horas, permaneciendo luego constante hasta las 24 horas. En ese tiempo (ic) fue dos veces superior a (ec). El estudio de la cinetica de produccion de Stx por cepas de E. coli aisladas de pacientes con SUH demostro que correspondian al tipo Stx2. Estos resultados fueron confirmados por ensayos de citotoxicidad especifica en celulas Vero y por hibridacion con sondas geneticas. El tipo de Stx es considerado uno de los mayores factores de riesgo en la evolucion a SUH en pacientes infectados con E. coli enterohemorragicos. Por lo tanto, la disminucion de la citotoxicidad de Stx1, durante la fase estacionaria de crecimiento, podria explicar la mayor frecuencia de accion entre SUH y cepas de E. coli productoras de Stx2 en distintos paises, incluyendo Argentina

Rapid identification of colonization factor antigens I & II of enterotoxigenic Escherichia coli by coagglutination test.

Specific antisera for colonization factor antigens (CFA/I and CFA/II) were adsorbed to Staphylococcus aureus Cowan I strain, ATCC 12598 to make coagglutination (CoA) reagents for detection of CFAs in enterotoxigenic Esch. coli (ETEC) isolates. Among 1782 strains of Esch. coli isolated from patients with acute diarrhoea, 238 (13.4%) strains exhibited CFA expression. Most prevalent CFA/I positive serogroups were 015, 0148, 0153, 020, 0128, 0114 and 078. CFA/II was detected among isolates of serogroup 080, 085, 06 and 08. Ten ETEC isolates each of serogroups 04, 07, 061, 068, 0117 and 0158 did not show presence of CFA/I or CFA/II. CoA technique proved an appropriate, rapid diagnostic tool which can be used for screening large number of Esch. coli isolates in epidemiological studies.

Staphylococci in breast milk from women with and with and without mastitis

Verificou-se a presença de Staphylococci em leite materno procedente de 19 mulheres, nove consideradas sadias e dez apresentando sintomas clínicos de mastite. Staphylococci foram detectados em todas as amostras de leite, com contagens variando de 10² a 10 a quarta ufc/mL e revelando uma significativa variedade de colônias atípicas e típicas, estas últimas observadas em todas as placas de ágar Baird-Parker, exceto em uma amostra. Staphylococcus aureus, coagulase e termonuclease positivos e fermentadores de manitol em anaerobiose, estavam presentes em 13 amostras. Várias estirpes näo apresentaram uma ou duas,outras evidenciaram todas e uma näo apresentou nenhuma das características bioquímicas citadas. De um total de 19 cepas, 8 sintetizavam quantidade detectáveis de enenterotoxinas pelo método de imunodifusäo em gel e algumas mostraram-se produtoras de pequenas quantidades de enterotoxinas e de toxina da síndrome do choque tóxico, conforme detectado através do método de aglutinaçäo reversa passiva em latex

Enterotoxigenic enteric bacteria causing secretory diarrhoea.

Two hundred and fifty enteric bacteria isolated from cases of secretory diarrhoea of all age groups were studied for their enterotoxigenicity and prevalence of drug resistance. The principal pathogens were Escherichia coli 44.4%, Vibrio cholerae 28.8%, Salmonella typhimurium 19.2% and Campylobacter jejuni 2.4%. 104 (42.6%) strains were enterotoxigenic; V. cholerae (100%), Escherichia coli (25.2%) and Non-E. coli enterobacteria (6.5%). While 89.3% and 100% Enterotoxigenic Escherichia coli and Salmonella typhimurium strains were multi drug resistant, 40% and 100% respectively showed transfer of R-plasmids to standard receipt strains. In V. cholerae multi drug resistance was observed in only 5.5% strains.

Enterotoxin of Aeromonas sp: some culture conditions which affect its production

Cepas de Aeromonas sobria e Aeromonas hydrophila, isoladas de diferentes alimentos, foram submetidas ao teste de produçäo de enterotoxina (teste do camundongo recém-nascido) usando diferentes meios de cultura (BHI, TSI e CAYE) e condiçöes de cultivo (temperatura, aeraçäo e volume de meio de cultura em relaçäo à capacidade do frasco). Os resultados mostraram que näo houve produçäo de toxina no meio CAYE. A presença de enterotoxina foi detectada em baixa temperatura (16o.C) e em volumes mais elevados de meio (60 ml) e em culturas estacionárias, sugerindo que a aeraçäo e a temperatura de incubaçäo, embora näo críticos, säo fatores importantes na produçäo de enterotoxinas por Aeromonas

Correlation of phospholipase A & enterotoxin production by Salmonella typhimurium with reference to virulence parameters.

Seventy strains of S. typhimurium along with 10 strains each of S. typhi and S. paratyphi B, 3 strains of S. enteritidis and 2 strains of S. senftenberg were examined for phospholipase A (PLA) activity. S. typhimurium positive for PLA activity (17.14%) were subjected to study of other virulence parameters like enterotoxin production, haemolytic activity, surface hydrophobicity and antibiotic sensitivity. A high degree of correlation was observed between PLA activity and enterotoxin production, antibiotic sensitivity and cell surface hydrophobicity respectively.