DIARRHEA OUTBREAK IN PERNAMBUCO, BRAZIL, ASSOCIATED WITH A HEAT-STABLE CYTOTOXIC ENTEROTOXIN PRODUCED BY Aeromonas caviae.

In the present study enterotoxic and cytotoxic activities of twenty Aeromonas caviae strains 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. caviae strains produce a putative diarrheagenic virulence factor, a heat-stable cytotoxic enterotoxin that could be linked to the diarrhea outbreak that took place in Brazil.

INTRODUCTION

Aeromonas spp. are Gram-negative rods that belong to the Aeromonadaceae family, which are widely spread across nature and can be found in a great variety of places including salt and drinking water, soil, sewage systems and uncooked or refrigerated foods6. Aeromonas is considered to be a common pathogen of fishes and following the 1970s these microorganisms have also been considered to be human pathogens8 , 13. Acute diarrheal disease after the ingestion of contaminated water and food is common in many countries9. The virulence factors produced by these species, such as enterotoxins, hemolysins, cytotoxins, and adhesins, are the main determinants of human enteropathological processes6.

In 2004, there was an outbreak of acute diarrhea in Sao Bento do Una, Pernambuco State, Brazil5, in which V. cholerae O1 and Aeromonas ssp. in ISR 16S-23S and RAPD amplification were recovered; all V. cholerae revealed homogeneous profiles and presence of potential virulence genes, however, it was observed that Aeromonas spp. were highly heterogeneous. It was concluded, then, that V. cholerae O1 was probably the responsible agent for the diarrhea outbreak. Nevertheless, the participation of Aeromonas was not ruled out11.

Therefore, the present study aimed to verify the possible diarrheagenic virulence factors of A. caviae, because it is the most prevalent specie (ca. 41%) of all the isolated Aeromonas spp.11.

Twenty A. caviae were cultivated in Müller Hinton broth (Difco Lab. USA) with the aid of a shaker, at 37 ºC for 16 h. The culture supernatants were analyzed for enterotoxic activity by the suckling mice assay1 . Three neonatal Balb/C mice, two to four- days-old, were used for each sample. A 50 mL volume of each sample was administered to each mouse, intragastrically. After three hours, the mice were executed following recommended procedures and the whole intestines were removed. Both the intestines and remaining corpse were weighed to calculate the ratio between the intestines (I) and the remaining corpse (B): I/B. Assays were performed in sets of three. Results were considered positive when the ratio results were ≥ 0.091.

Additionally, a rabbit intestinal loop test2 was conducted using white adult male New Zealand rabbits weighing 1.5-2.0 kg. The intestinal lumen was rinsed three times with saline solution, then, series of intestines were exteriorized through a midline incision; ligated intestinal segments (loops) of about 5 cm of length, separated by a 1 to 2 cm interloop. Each loop was filled with 1 mL of the sample solution. A control loop injected with sterile culture medium was included. Rabbits were maintained for 18 h at room temperature following euthanasia. The fluid accumulation in the intestinal loops was measured as the ratio between the weight of the loop (in grams) and length (in centimeters). Ratio results > 0.2 were regarded as a positive response2. The assays were performed in sets of three. Sterile culture medium (Müller Hinton broth) was used as a negative control loop and the positive control was the cultured supernatant of A. veronii biovar sobria AS 3110.

The culture supernatants that were positive in the suckling mice assay, meaning 70% (14 of 20), were also positive in the rabbit intestinal loop test2, confirming the enterotoxic activity of A. caviae isolates.

The cytotoxic activity of A. caviae culture supernatants was detected as described7 in Vero (African green monkey kidney), Caco-2 (human intestinal carcinoma), HeLa (human uterus carcinoma) and HEp-2 (human larynx carcinoma) (ATCC, Rockville, MD, USA) cell lines cultivated in tissue culture flasks with Eagle's minimum essential medium (MEM, Nutricell, Campinas, SP, Brazil) supplemented with 10% (v/v) of fetal bovine serum (FBS). The filter-sterilized culture supernatants were inoculated in a confluent monolayer (serial two fold dilutions). Then, the plates were incubated at 37 ˚C in a 5% CO2 chamber. The culture filtrates of A. sobria AS-69, a non cytotoxin-producing strain10, and MEM medium were used as negative controls. Cell monolayer morphology was observed using an inverted microscope.

The same A. caviae culture supernatants, in which enterotoxic activity was observed, induced morphological alterations in Vero and Caco-2 cells after three hours of culture filtrates inoculation, inducing cellular elongation, rounding, loosening of intercellular junctions and detachment were observed, leading to cellular death (Fig.1). These observations resemble those previously described4, in which bacterial suspensions of A. hydrophila, A. veronii biovar sobria and A. caviae were assayed in cultured Vero and CHO cells. Despite the similarities of the cytotoxic effects, injuries and destruction induced on cells, we observed that the cytotoxin present in our samples was found free in the cultured supernatants.

Fig. 1

Aspects of the cytotoxic effects of A. caviaecultured supernatants submitted to heat-stability treatment in Caco-2 and Vero cells. A - Cytotoxic assay with heat-treated culture supernatants of Aeromonas caviae in Caco-2 human intestinal cells, indicating cellular vacuolation and monolayer destruction after 20 min. After 20 h, the monolayer was completely destroyed and detached. B - Caco-2 control cells. C - Vero cells treated with the heat stable enterotoxic cytotoxin exhibited rounding, cell-to-cell leakage of cellular membrane junctions and nuclear condensation. D - Vero control cells. Magnifications 430X.

The cytotoxic and enterotoxin activities of the A. caviae culture supernatants were not affected by heat treatment at 60 ºC and 100 ºC for 15 min. These assays demonstrated that the cytotoxic-enterototoxic activity produced by these A. caviae is heat-stable.

The description of a heat-stable cytotoxic activity produced by A. caviae in HEp-2 cells has already been described12, but our culture supernatants did not exhibit cytotoxic effects on HEp-2 or HeLa cells (data not shown). The most intense effects were observed in Caco-2 cells, a human intestinal cell lineage, reinforcing its specific activity in intestinal cells in these in vitro assays.

Therefore, this work reveals the expression of a heat-stable cytotoxic enterotoxin by A. caviae that could be associated with the diarrhea outbreak that took place in Pernambuco, Brazil. It could be a new diarrheagenic virulence factor not yet described among the Aeromonas species. The purification of this heat-stable cytotoxic enterotoxin is currently being conducted to identify its chemical nature, aiming to elucidate its role in the enteropathogenic activities induced by A. caviae.