WIPF2 promotes Shigella flexneri actin-based motility and cell-to-cell spread.

Shigella flexneri is an intracellular pathogen that disseminates in colonic epithelial cells through actin-based motility and formation of membrane protrusions at cell-cell contacts, that project into adjacent cells and resolve into vacuoles, from which the pathogen escapes, thereby achieving cell-to-cell spread. Actin nucleation at the bacterial pole relies on the recruitment of the nucleation-promoting factor N-WASP, which activates the actin nucleator ARP2/3. In cells, the vast majority of N-WASP exists as a complex with WIP. The involvement of WIP in N-WASP-dependent actin-based motility of various pathogens, including vaccinia virus and S. flexneri, has been highly controversial. Here, we show that WIPF2 was the only WIP family member expressed in the human colonic epithelial cell line HT-29, and its depletion impaired S. flexneri dissemination. WIPF2 depletion increased the number of cytosolic bacteria lacking actin tails (non-motile) and decreased the velocity of motile bacteria. This correlated with a decrease in the recruitment of N-WASP to the bacterial pole, and among N-WASP-positive bacteria, a decrease in actin tail-positive bacteria, suggesting that WIPF2 is required for N-WASP recruitment and activation at the bacterial pole. In addition, when motile bacteria formed protrusions, WIPF2 depletion decreased the number of membrane protrusions that successfully resolved into vacuoles.

Effect of duration of diarrhoea and predictive values of stool leucocytes and red blood cells in the isolation of different serogroups or serotypes of Shigella.

Diarrhoeal stools from 11,358 patients were microscopically examined for the presence of white and red blood cells (WBC and RBC) and cultured for enteric pathogens. Of the 3895 patients who had Shigella as sole pathogens, 2681 (72.3%) showed the presence of both WBC and RBC and the remainder had only WBC (P less than 0.001). The presence of both WBC and RBC was as good a predictor of shigellosis as the presence of greater than 25 WBC per high power field (hpf) of the microscope with or without RBC. However, the best predictor of shigellosis (positive predictive value 85%, negative predictive value 83%) was the presence of greater than 25 WBC/hpf and the presence of RBC regardless of their number. More patients infected with S. dysenteriae 1, S. flexneri and S. boydii shed both WBC and RBC than those infected with S. sonnei, most of whom shed WBC only (P less than 0.001). A greater number of patients infected with S. dysenteriae 1 shed more WBC and RBC than those infected with S. flexneri (P less than 0.001). The same trend was found when patients infected with S. flexneri were compared with those infected with S. boydii (though the difference was not statistically significant) and when patients infected with S. boydii were compared with patients infected with S. sonnei (P less than 0.001). There was a progressive decline in the isolation rate of Shigella as the duration of dysentery, before stool culture, increased.

First detection and sequence analysis of the bla-CTX-M-15 gene in Lebanese isolates of extended-spectrum-beta-lactamase-producing Shigella sonnei.

The emergence in Shigella species of extended-spectrum beta-lactamases (ESBL) that impart resistance to third-generation cephalosporins is a growing concern world-wide. So far, however, ESBL-producing Shigella have only been reported seven times, albeit from seven different countries. In Lebanon, three ESBL-producing clinical isolates of S. sonnei were recovered from 30 cases of shigellosis diagnosed between July 2004 and October 2005. All three were found to be resistant to amoxycillin, cefotaxime, ceftazidime, aztreonam, trimethoprim/sulphamethoxazole, gentamicin, and kanamycin. Each harboured the bla-CTX-M gene, and the results of sequence analysis indicated this to be of the bla-CTX-M-15 type and encoded on a 70-kb plasmid, flanked by an insertion element (ISEcp1). The bla-TEM-1 gene was also detected on the chromosomes of two of the ESBL-producing isolates. Class-2 integrons containing dhfr1, aadA1 and sat1 genes were detected on the chromosomes of all three isolates but not on the plasmids. Fluoroquinolone-modifying factors [QnrA, QnrB, QnrS or AAC(6')-Ib-cr] were not detected. The results of RAPD analysis, combined with data on antimicrobial susceptibility, indicated that each isolate was unique. In conclusion, the emergence of ESBL-producing isolates of S. sonnei has been demonstrated for the first time in Lebanon. The resistance of these isolates to third-generation cephalosporins was mediated by the CTX-M-15 enzyme, which was plasmid-encoded.