BackgroundThis study was conducted to determine the etiology of diarrhoea in a hospital setting in Kolkata. Active surveillance was conducted for 2 years on two random days per week by enrolling every fifth diarrhoeal patient admitted to the Infectious Diseases and Beliaghata General Hospital in Kolkata.ResultsMost of the patients (76.1%) had acute watery diarrhoea in association with vomiting (77.7%) and some dehydration (92%). Vibrio cholerae O1, Rotavirus and Giardia lamblia were the important causes of diarrhoea. Among Shigella spp, S. flexneri 2a and 3a serotypes were most predominantly isolated. Enteric viruses, EPEC and EAEC were common in children <5 year age group. Atypical EPEC was comparatively higher than the typical EPEC. Multidrug resistance was common among V. cholerae O1 and Shigella spp including tetracycline and ciprofloxacin. Polymicrobial infections were common in all age groups and 27.9% of the diarrhoea patients had no potential pathogen.ConclusionsIncrease in V. cholerae O1 infection among <2 years age group, resistance of V. cholerae O1 to tetracycline, rise of untypable S. flexnerii, higher proportion of atypical EPEC and G. lamblia and polymicrobial etiology are some of the emerging trends observed in this diarrhoeal disease surveillance.

Summary Intracellular replication of Brucella requires the VirB complex, which is highly similar to conjugative DNA transfer systems. In this study, we show that Brucella internalizes into macrophages by swimming on the cell surface with generalized membrane ruffling for several minutes, after which the bacteria are enclosed by macropinosomes. Lipid raft‐associated molecules such as glycosylphosphatidylinositol (GPI)‐anchored proteins, GM1 gangliosides and cholesterol were selectively incorporated into macropinosomes containing Brucella. In contrast, lysosomal glycoprotein LAMP‐1 and host cell transmembrane protein CD44 were excluded from the macropinosomes. Removing GPI‐anchored proteins from the macrophage surface and cholesterol sequestration markedly inhibited the VirB‐dependent macropinocytosis and intracellular replication. Our results suggest that the entry route of Brucella into the macrophage determines the intracellular fate of the bacteria that is modulated by lipid raft microdomains.

1.Of the nineteen plants screened, six were found to contain large quantities of condensed tannins. Black locust (Robinia pseudo-dcucia), bush clover (Lespedezu bicolor), wistaria (Wistaria floribundu) and Japanese knotgrass (Reynoutria juponicu) were used for the present experiment. Tannins of the investigated plants were fractionated into three or four molecular forms, according to the degree of polymerization, by chromatography on a column of Sephadex LH-20.2. The protein-precipitating capacity of the fractionated tannins increased with the increase in degree of polymerization. The inhibitory effect of tannins on trypsin (EC 3 . 4 . 2 1 .4), a-amylase (EC 3 . 2 . 1 . 1) and lipase (EC 3 . 1 . 1 .3) activities in vitro also increased with the increase in degree of polymerization. The digestion of tannin-bovine serum albumin complex by trypsin was related to the degree of polymerization of tannins complexed.3. Inclusion of black locust tannins in the diet (10 g/kg) depressed the activities of trypsin and a-amylase in the upper, middle and lower parts of the intestine of the rats, but the lipase activity was increased in the middle part and remained unaffected in the upper and lower parts. It is presumed that the tannins have little affinity for lipase. Barry (1985) reported also that the liveweight gain and voluntary intake were low for sheep grazing high-tannin forage. Tannins are classified into hydrolysable and condensed types and the inhibitory effect on trypsin activity is more marked with condensed tannins than with hydrolysable tannins (Tamir & Alumot, 1969). It is also suggested that the principal tannins found in forage leaves are of the proanthocyanidin type formed by the polymerization of flavan-3,4-diols either alone or in combination with other flavonoids such as catechins (McLleod, 1974;Jones et al. 1976). 4.The present work was undertaken to isolate tannins from the leaves of black locust (Robinia pseudo-Acacia), bush clover (Lespedeza bicolor), wistaria ( WistariaJEoribunda) and Japanese knotgrass (Reynoutria juponica) which were found to have proanthocyanidin-type tannins in a survey of tannin-containing plants, and to investigate the effect of isolated tannins on trypsin (EC 3 . 4 . 2 1 .4), a-amylase (EC 3 . 2 . 1 . 1) and lipase (EC 3 . 1 . I .3) in vitro and in the intestine of rats. EXPERIMENTAL Survey of the plants containing condensed tanninsAnthocyanidin-formation and protein-precipitation tests were used for the survey of condensed tannin-containing plants.

The heat-stable enterotoxin (ST) produced by enterotoxigenic Escherichia coli is an extracellular peptide toxin that evokes watery diarrhea in the host. Two types of STs, STI and STII, have been found. Both STs are synthesized as precursor proteins and are then converted to the active forms with intramolecular disulfide bonds after being released into the periplasm. The active STs are finally translocated across the outer membrane through a tunnel made by TolC. However, it is unclear how the active STs formed in the periplasm are led to the TolC channel. Several transporters in the inner membrane and their periplasmic accessory proteins are known to combine with TolC and form a tripartite transport system. We therefore expect such transporters to also act as a partner with TolC to export STs from the periplasm to the exterior. In this study, we carried out pulse-chase experiments using E. coli BL21(DE3) mutants in which various transporter genes (acrAB, acrEF, emrAB, emrKY, mdtEF, macAB, and yojHI) had been knocked out and analyzed the secretion of STs in those strains. The results revealed that the extracellular secretion of STII was largely decreased in the macAB mutant and the toxin molecules were accumulated in the periplasm, although the secretion of STI was not affected in any mutant used in this study. The periplasmic stagnation of STII in the macAB mutant was restored by the introduction of pACYC184, containing the macAB gene, into the cell. These results indicate that MacAB, an ATP-binding cassette transporter of MacB and its accessory protein, MacA, participates in the translocation of STII from the periplasm to the exterior. Since it has been reported that MacAB cooperates with TolC, we propose that the MacAB-TolC system captures the periplasmic STII molecules and exports the toxin molecules to the exterior.

We purified the toxin of Aeromonas sobria capable of inducing a positive response in the mouse intestinal loop assay. The purified toxin showed a positive response not only in the loop assay but also in a hemolytic assay. Subsequently, we cloned the toxin gene and demonstrated that the product of this gene possessed both hemolytic and enterotoxic activities. These results showed that the enterotoxin of A. sobria possesses hemolytic activity. Nucleotide sequence determination of the toxin gene and amino acid sequence analysis of the purified toxin revealed that it is synthesized as a precursor composed of 488 amino acid residues, and that the 24 amino-terminal amino acid residues of the precursor is removed in the mature toxin. As antiserum against the purified toxin neutralized the fluid accumulation induced by living cells not only of A. sobria but also of A. hydrophila, this and antigenically related toxin(s) are thought to play an essential role in the induction of diarrhea by these organisms. The toxin-injured Chinese hamster ovary (CHO) cells induced the release of intracellular lactose dehydrogenase (LDH). The release of LDH from CHO cells and the lysis of erythrocytes by the toxin were repressed by the addition of dextran to the reaction solution, indicating that the toxin forms pores in the membranes and that the cells were injured by the osmotic gradient developed due to pore formation. However, the histopathological examination of intestinal cells exposed to the toxin showed that it caused fluid accumulation in the mouse intestinal loop without causing cellular damage.