Identification of Staphylococcus spp. isolated during the ripening process of a traditional minas cheese / Identificação de Staphylococcus spp. isolados durante o processo de maturação de um queijo-de-minas tradicional

The population dynamics of Staphylococcus spp. was studied during the ripening of Canastra Minas cheese at three farms located in the State of Minas Gerais, Brazil. The presence of coagulase (coa), thermonuclease (nuc), and enterotoxin (sea, seb, sec, and sed) genes was investigated in Staphylococcus strains isolated during the 60-day cheese-ripening period. The presence of the staphylococcal enterotoxins A, C, and D was also investigated in the cheese samples. Cheese samples that were matured for 0, 7, 15, 30, and 45 days presented staphylococci counts from 10³ to 10(8)cfu/g. All isolates considered coagulase-positive by physiological tests had the coa gene. However, no association was observed between the results obtained with biochemical tests and those obtained by PCR using gene-specific primers for coagulase-negative strains. Coagulase and thermonuclease genes occurred simultaneously in 41.3 percent of Staphylococcus spp. tested. None of the investigated Staphylococcus strains expressed enterotoxins SEA, SEB, SEC, and SED. Enterotoxins A, C, and D were not detected in any of the cheese samples.

Estudou-se a dinâmica das populações de Staphylococcus spp. durante a maturação do queijo Canastra, em três fazendas localizadas no estado de Minas Gerais. A presença dos genes que codificam para a produção das enzimas coagulase (coa), termonuclease (nuc) e produção de enterotoxinas (sea, seb, sec e sed), em linhagens de Staphylococcus isoladas durante os 60 dias de maturação do queijo foi analisada. Também foi investigada a presença de enterotoxina estafilocócica A, C e D nas amostras de queijo. As amostras de queijo com 0, 7, 15, 30 e 45 dias de maturação apresentaram contagens de Staphylococcus spp. entre 10³ e 10(8)ufc / g. Todos os isolados coagulase positivo nos testes fisiológicos apresentaram o gene coa. Não foi observada associação entre os resultados obtidos com os testes bioquímicos e aqueles obtidos com a PCR usando iniciadores gene-específicos para linhagens coagulase negativa. Os genes da coagulase e termonuclease ocorreram simultaneamente em 41,3 por cento dos Staphylococcus spp. testados. Nenhum dos isolados de Staphylococcus apresentou os genes que codificam para a produção das enterotoxinas SEA, SEB, SEC ou SED. As enterotoxinas A, C ou D não foram detectadas em nenhuma das amostras de queijo analisadas.

Staphylococcus aureus and food poisoning

Food-borne diseases are of major concern worldwide. To date, around 250 different food-borne diseases have been described, and bacteria are the causative agents of two thirds of food-borne disease outbreaks. Among the predominant bacteria involved in these diseases, Staphylococcus aureus is a leading cause of gastroenteritis resulting from the consumption of contaminated food. Staphylococcal food poisoning is due to the absorption of staphylococcal enterotoxins preformed in the food. Here, we briefly review the latest data on staphylococcal enterotoxins and some papers exemplifying the interactions between S. aureus and the food matrix; environmental factors affecting staphylococcal enterotoxin production are discussed

Development of two novel nontoxic mutants of Escherichia coli heat-labile enterotoxin

Escherichia coli heat-labile enterotoxin (LT) is composed of catalytic A and non-catalytic homo-pentameric B subunits and causes diarrheal disease in human and animals. In order to produce a nontoxic LT for vaccine and adjuvant development, two novel derivatives of LT were constructed by a site-directed mutagenesis of A subunit; Ser63 to Tyr63 in LTS63Y and Glu110, Glu112 were deleted in LT delta 110/112. The purified mutant LTs (mLTs) showed a similar molecular structural complex as AB5 to that of wild LT. In contrast to wild-type LT, mLTs failed to induce either elongation activity, ADP-ribosyltransferase activity, cAMP synthesis in CHO cells or fluid accumulation in mouse small intestine in vivo. Mice immunized with mLTs either intragastrically or intranasally elicited high titers of LT-specific serum and mucosal antibodies comparable to those induced by wild-type LT. These results indicate that substitution of Ser63 to Tyr63 or deletion of Glu110 and Glu112 eliminate the toxicity of LT without a change of AB5 conformation, and both mutants are immunogenic to LT itself. Therefore, both mLTs may be used to develop novel anti-diarrheal vaccines against enterotoxigenic E. coli.

Enterotoxigenicidad de campylobacter jejuni subsp. jejuni y su relación con aumento de AMPc y alteración electrolítica en el intestino de ratas / The relationship of campylobacter jejuni subsp. jejuni enterotoxigenicity and the increase of cAMP and electrolytic alterations in the rat intestine

Background: Small intestine alterations produced by the enterotoxigenic capacity of Campylobacter jejuni subsp. jejuni are similar to the hydric, electrolytic and pathological changes caused by choleraic and thermolabile Escherichia coli toxins. Aim: To study the enterotoxigenic capacity of 4 strains of Campylobacter jejuni subsp. jejuni using the intestinal loop model. Material and methods: Rat intestinal loops were inoculated with culture filtrates of the four strains. Enterotoxigenicity was assessed by fluid accumulation, the increase in Na+ and Cl- in the loop fluid, and cAMP increases in loop tissues. An enterotoxigenic Escherichia coil strain and sterile Brucella both were used as positive and negative controls, respectively. Results: The filtrates of two strains produced fluid accumulation in the loops, significantly increased Na+ and Cl - secretion to the intestinal lumen and increased tissue cAMP levels. Conclusions: Some strains of Campylobacter jejuni subsp. jejuni are able to show enterotoxigenicity in vivo, increasing cAMP levels in the intestinal cells and altering electrolyte exchange mechanisms

Toxigenicity & drug sensitivity of Vibrio mimicus isolated from patients with diarrhoea.

In a total of 720 faecal specimens from patients with secretory diarrhoea, vomiting, dehydration, gastroenteritis, cholera and cholera like illnesses, 18 strains of V. mimicus were isolated as pure culture. These were characterized for various toxin types and virulence factors using conventional in vitro and in vivo assays. Labile and stable toxins were elaborated by 15 and 2 strains respectively by ligated rabbit ileal loop (RIL) and suckling mouse assays. While 15 of the whole cell culture elaborated labile toxin, only 7 strains produced the same when culture filtrate was tested in RIL assay. Culture filtrates of 15 strains exhibited vascular permeability factor (PF) on adult rabbit skin, none of the strains were invasive as indicated by Sereny's test. Culture supernatants of all strains produced a cytotoxic factor to Vero and Chinese hamster ovary cells. Four of the 18 strains (22%) were resistant to multiple drugs (a combination of 3 or more drugs). The results emphasize the significance of continuous screening and identification of V. mimicus and to include in the differential diagnosis of patients with acute diarrhoea.

Mechanism of action of cholera toxin & other toxins.

Vibrio cholerae produce a variety of extracellular products that have deleterious effects on eukaryotic cells. The massive diarrhoea produced by V. cholerae is caused by cholera toxin (CT). CT is composed of 1A and 5B units. CT causes a significant amount of fluid secretion and haemorrhage in the ligated rabbit ileal loops. Its action involves the role of various biochemical pathways. CT acts by activation of adenylate cyclase-cAMP system located at the basolateral membrane of intestinal epithelial cells. The increase in cyclic AMP levels is mainly responsible for the altered transport of Na+ and Cl-. Besides activating cAMP, CT is also known to act through release of prostaglandins and involvement of intramural nerves. Besides CT, other bacterial toxins like Escherichia coli LT, Salmonella toxin, Shigella toxin and Campylobacter toxin also possess A-B structure. The structure and function of E. coli LT resembles closely that of CT. Most of the bacterial toxins exert their effect through involvement of ADP-ribosylating proteins whereas other toxins involve guanylate cyclase system, calcium and protein kinases for their ultimate action.

Salmonella in foods of animal origin: enterotoxigenicity and antibiogram.

A total of 82 strains of Salmonella belonging to 17 different serotypes recovered from foods of animal origin were in this study. Out of 39 cell free culture supernatants (CFCS), tested for enterotoxigenicity, 26 (66.67%) were positive in rabbit ileal loops. None were found positive for enterotoxigenicity in infant mouse model. According to the disc diffusion methods of testing, gentamycin, nalidixic acid and chloramphenicol were found to be most effective against Salmonella organism. Erythromycin and oxytetracycline were least effective.

Salmonella typhimurium enterotoxin mediated fluid secretion.

Unidirectional Na+ and Cl- fluxes were studied in rats treated with S. typhimurium enterotoxin (S-LT). There was net absorption of Na+ and Cl- in the control group, while in the toxin treated animals there was net secretion of Na+ and Cl- (P less than 0.001). There was no change in the transport of D-glucose in the toxin treated group as compared to the control animals. The Na+, K(+)-ATPase pump was unaltered in the S-LT treated animals (198.67 +/- 11.23 nmoles Pi/mg protein/min) as compared to the control group (189.93 +/- 10.09 nmoles Pi/mg protein/min). There was no change in the unidirectional fluxes of Ca+2 in the S-LT treated animals as compared to the control animals, suggesting no change in the permeability of the S-LT treated intestinal membrane to Ca+2.

Enterotoxin induced diarrhoea--an update.

The pathogenic personality or the criteria required to be a successful pathogen, of enteric bacteria includes, among others, the ability to produce potent proteins which by different intracellular mechanisms elicit what we overtly see as diarrhoea. Enteropathogens belonging to several genera like Vibrio, Escherichia, Shigella, Salmonella, Campylobacter, Aeromonas and Yersinia include species capable of elaborating strikingly similar exotoxins which seem to share common mechanisms of action involving specific receptor binding, internalization of the toxin followed by interaction with an intracellular target. It is now clear that there are several families of structurally, functionally and immunologically identical bacterial enterotoxins. In this communication, we have reviewed the recent developments on the various families of structurally homologous and antigenically cross reacting enteric toxins.

Sindrome de intoxicación por entero-toxinas / Syndrome of poisoning for enterotoxins

Se evalúan en este trabajo 1.000 casos de un síndrome clínico observados y estudiados en mi consultorio durante el año 1984. Este síndrome es el motivo más frecuente en la consulta médica práctica. El diagnóstico según la nomenclatura médica actual, sería "colon irritable", una frase poco feliz, ya que está confundiendo desde hace casi medio siglo. Los médicos catalogan generalmente el padecimiento según el cuadro clínico predominante en el momento de la consulta o emergencia. Ya que el término "colon irritable" es usado para simplificar en dos palabras un síndrome clínico sumamente polifacético y polisintomático, sería en mi opinión, mucho más correcto diagnósticarlo según sus causas desencadenantes como, por ejemplo, lo fue en estos casos, la intolerancia a algunos alimentos, que puede provocar en ciertos individuos, genética y ambientalemte predispuestos, en cuadro clínico tóxico a veces sumamente dramático causado por exo-o por endotoxinas o por ambas a la vez. El término "colon irritable" se dejaría solo para los casos cuya sintomatología sería relacionada exclusivamente con estas dos palabras para evitar de esta manera la confunsión reinante. Este síndrome no es tampoco tan inocente como pareciera a la primera vista, ya que es la causa de muchas calamidades del genéro humano provocando toda clase de violencias y de conflictos por la irritabilidad caracterológica y estados depresivos con los cuales cursa frecuentemente

Enterotoxinas y hemolisinas en Klebsiella pneumoniae / Enterotoxins and hemolysins in Klebsiella pneumoniae

Se estudió la capacidad de producir exterotoxinas en cepas de Klebsiellas pneumoniae aisladas de lactantes internados con septicemia y diarrea. La actividad enterotóxica se investigó mediante las pruebas del ratón lactante y del intestino ligado de conejo, determinando respectivamente el incremento del peso intestinal y la acumulación de fluido en los segmentos ligados. Los resultados indicaron que las bacterias provenientes de sangre, intestino y líquido cefalorraquídeo presentaron efecto semejante al observado con Escherichia coli enteropatógena, pero con mayor reacción inflamatoria. Las klebsiella se aislaron inicialmente de materia fecal y luego de sangre, originando finalmente meningitis, que resultó mortal en cuatro de los ocho casos estudiados. La entetoxina posee actividad hemolítica, pero difiere de la hemolisina activa en presencia de agentes reductores, porque tiene un rango de especies sensibles más amplio; no se inactiva por contacto con el oxígeno; no requiere de grupos sulfhidrilos libres para hemolizar eritrocitos; no pierde actividad a 60-C y sólo está presente en un número reducido de cepas con klebolisina, que es la hemolisina activada por agentes reductores como el 2 mercaptoetanol