Francisella tularensis is a highly infectious, facultative intracellular bacterium which causes epidemics of tularemia in both humans and mammals at regular intervals. The natural reservoir of the bacterium is largely unknown, although it has been speculated that protozoa may harbor it. To test this hypothesis, Acanthamoeba castellanii was cocultured with a strain of F. tularensis engineered to produce green fluorescent protein (GFP) in a nutrient-rich medium. GFP fluorescence within A. castellanii was then monitored by flow cytometry and fluorescence microscopy. In addition, extracellular bacteria were distinguished from intracellular bacteria by targeting with monoclonal antibodies. Electron microscopy was used to determine the intracellular location of F. tularensis in A. castellanii, and viable counts were obtained for both extracellular and intracellular bacteria. The results showed that many F. tularensis cells were located intracellularly in A. castellanii cells. The bacteria multiplied within intracellular vacuoles and eventually killed many of the host cells. F. tularensis was found in intact trophozoites, excreted vesicles, and cysts. Furthermore, F. tularensis grew faster in cocultures with A. castellanii than it did when grown alone in the same medium. This increase in growth was accompanied by a decrease in the number of A. castellanii cells. The interaction between F. tularensis and amoebae demonstrated in this study indicates that ubiquitous protozoa might be an important environmental reservoir for F. tularensis.

Diarrhoea is one of leading causes of morbidity and mortality worldwide. Recent estimations suggested the number of deaths is close to 2.5 million. This study examined the causative agents of diarrhoea in children under 5 years of age in suburban areas of Khartoum, Sudan. A total of 437 stool samples obtained from children with diarrhoea were examined by culture and PCR for bacteria, by microscopy and PCR for parasites and by immunoassay for detection of rotavirus A. Of the 437 samples analysed, 211 (48 %) tested positive for diarrhoeagenic Escherichia coli, 96 (22 %) for rotavirus A, 36 (8 %) for Shigella spp., 17 (4 %) for Salmonella spp., 8 (2 %) for Campylobacter spp., 47 (11 %) for Giardia intestinalis and 22 (5 %) for Entamoeba histolytica. All isolates of E. coli (211, 100 %) and Salmonella (17, 100 %), and 30 (83 %) isolates of Shigella were sensitive to chloramphenicol; 17 (100 %) isolates of Salmonella, 200 (94 %) isolates of E. coli and (78 %) 28 isolates of Shigella spp. were sensitive to gentamicin. In contrast, resistance to ampicillin was demonstrated in 100 (47 %) isolates of E. coli and 16 (44 %) isolates of Shigella spp. In conclusion, E. coli proved to be the main cause of diarrhoea in young children in this study, followed by rotavirus A and protozoa. Determination of diarrhoea aetiology and antibiotic susceptibility patterns of diarrhoeal pathogens and improved hygiene are important for clinical management and controlled strategic planning to reduce the burden of infection.

Vibrio cholerae species are extracellular, waterborne, gram-negative bacteria that are overwhelmed by predators in aquatic environments. The unencapsulated serogroup V. cholerae O1 and encapsulated V. cholerae O139 cause epidemic and pandemic outbreaks of cholera. It has recently been shown that the aquatic and free-living amoeba Acanthamoeba castellanii is not a predator to V. cholerae O139; rather, V. cholerae O139 has shown an intracellular compatibility with this host. The aim of this study was to examine the ability of V. cholerae O1 classical and El Tor strains to grow and survive in A. castellanii. The interaction between A. castellanii and V. cholerae O1 strains was studied by means of amoeba cell counts and viable counts of the bacteria in the absence or presence of amoebae. The viable count of intracellularly growing bacteria was estimated by utilizing gentamicin assay. Confocal microscopy and electron microscopy were used to determine the intracellular localization of V. cholerae in A. castellanii. The results showed that V. cholerae O1 classical and El Tor strains grew and survived intracellularly in the cytoplasm of trophozoites, and that the bacteria were also found in the cysts of A. castellanii. The interaction showed a facultative intracellular behaviour of V. cholerae O1 classical and El Tor strains and a possible role of A. castellanii as an environmental host of V. cholerae species.