Antimicrob Agents Chemother, 2003 Mar, 47(3), 1137 - 9 In vitro susceptibilities of Shigella flexneri and Streptococcus pyogenes to inner gel of Aloe barbadensis Miller; Ferro VA et al.; Aloe barbadensis Miller (or Aloe vera) has widespread use in health products, and despite numerous reports on the whole plant, little work has been performed on the inner gel, which has been used extensively in these products . This report describes the in vitro susceptibilities of two bacteria to this component. Infect Immun, 2003 Mar, 71(3), 1551 - 6 Effects of ectopically expressed neuronal Wiskott-Aldrich syndrome protein domains on Rickettsia rickettsii actin-based motility; Harlander RS et al.; Neuronal Wiskott-Aldrich syndrome protein (N-WASP) and the actin-related protein 2/3 (Arp2/3) complex have emerged as critical host proteins that regulate pathogen actin-based motility . Actin tail formation and motility in Listeria monocytogenes require the Arp2/3 complex but bypasses N-WASP signaling . Motility of Shigella flexneri and vaccinia virus requires both N-WASP and the Arp2/3 complex . Functional roles for these cytoskeletal regulatory proteins in actin-based motility of Rickettsia rickettsii have not been established . In this study, functional domains of N-WASP tagged with green fluorescent protein that have characterized effects on Shigella and vaccinia virus actin-based motility were ectopically expressed in HeLa cells infected with R . rickettsii to assess their effects on rickettsial motility . S . flexneri-infected cells were used as a control . Expressed N-WASP domains did not localize to R . rickettsii or their actin tails . Expression of N-WASP missing the VCA domain (for "verprolin homology, cofilin homology, and acidic domains"), which acts as a dominant-negative form of N-WASP, completely inhibited actin-based motility of S . flexneri while only moderately inhibiting motility of R . rickettsii . Similarly, expression of the VCA domain, which acts as a dominant-negative with respect to Arp2/3 complex function, severely inhibited actin-based motility of S . flexneri (no motility observed in the majority of expressing cells) but only moderately inhibited R . rickettsii motility . These results, taken together with the differential effects on motility observed upon expression of other N-WASP domains, suggest that actin-based motility of R . rickettsii is independent of N-WASP and the Arp2/3 complex. Infect Immun, 2003 Mar, 71(3), 1352 - 60 Genetic characterization and immunogenicity of coli surface antigen 4 from enterotoxigenic Escherichia coli when it is expressed in a Shigella live-vector strain; Altboum Z et al.; The genes that encode the enterotoxigenic Escherichia coli (ETEC) CS4 fimbriae, csaA, -B, -C, -E, and -D', were isolated from strain E11881A . The csa operon encodes a 17-kDa major fimbrial subunit (CsaB), a 40-kDa tip-associated protein (CsaE), a 27-kDa chaperone-like protein (CsaA), a 97-kDa usher-like protein (CsaC), and a deleted regulatory protein (CsaD') . The predicted amino acid sequences of the CS4 proteins are highly homologous to structural and assembly proteins of other ETEC fimbriae, including CS1 and CS2, and to CFA/I in particular . The csaA, -B, -C, -E operon was cloned on a stabilized plasmid downstream from an osomotically regulated ompC promoter . pGA2-CS4 directs production of CS4 fimbriae in both E . coli DH5alpha and Shigella flexneri 2a vaccine strain CVD 1204, as detected by Western blot analysis and bacterial agglutination with anti-CS4 immune sera . Electron-microscopic examination of Shigella expressing CS4 confirmed the presence of fimbriae on the bacterial surface . Guinea pigs immunized with CVD 1204(pGA2-CS4) showed serum and mucosal antibody responses to both the Shigella vector and the ETEC fimbria CS4 . Among the seven most prevalent fimbrial antigens of human ETEC, CS4 is the last to be cloned and sequenced . These findings pave the way for CS4 to be included in multivalent ETEC vaccines, including an attenuated Shigella live-vector-based ETEC vaccine. Infect Immun, 2003 Mar, 71(3), 1255 - 64 Structural characterization of the N terminus of IpaC from Shigella flexneri; Harrington AT et al.; The primary effector for Shigella invasion of epithelial cells is IpaC, which is secreted via a type III secretion system . We recently reported that the IpaC N terminus is required for type III secretion and possibly other functions . In this study, mutagenesis was used to identify an N-terminal secretion signal and to determine the functional importance of the rest of the IpaC N terminus . The 15 N-terminal amino acids target IpaC for secretion by Shigella flexneri, and placing additional amino acids at the N terminus does not interfere with IpaC secretion . Furthermore, amino acid sequences with no relationship to the native IpaC secretion signal can also direct its secretion . Deletions introduced beyond amino acid 20 have no effect on secretion and do not adversely affect IpaC function in vivo until they extend beyond residue 50, at which point invasion function is completely eliminated . Deletions introduced at amino acid 100 and extending toward the N terminus reduce IpaC's invasion function but do not eliminate it until they extend to the N-terminal side of residue 80, indicating that a region from amino acid 50 to 80 is critical for IpaC invasion function . To explore this further, the ability of an IpaC N-terminal peptide to associate in vitro with its translocon partner IpaB and its chaperone IpgC was studied . The N-terminal peptide binds tightly to IpaB, but the IpaC central hydrophobic region also appears to participate in this binding . The N-terminal peptide also associates with the chaperone IpgC and IpaB is competitive for this interaction . Based on additional biophysical data, we propose that a region between amino acids 50 and 80 is required for chaperone binding, and that the IpaB binding domain is located downstream from, and possibly overlapping, this region . From these data, we propose that the secretion signal, chaperone binding region, and IpaB binding domain are located at the IpaC N terminus and are essential for presentation of IpaC to host cells during bacterial entry; however, IpaC effector activity may be located elsewhere. J Biol Chem, 2003 May 9, 278(19), 17103 - 7 Epub 2003 Feb 05. Helical structure of the needle of the type III secretion system of Shigella flexneri; Cordes FS et al.; Gram-negative bacteria commonly interact with animal and plant hosts using type III secretion systems (TTSSs) for translocation of proteins into eukaryotic cells during infection . 10 of the 25 TTSS-encoding genes are homologous to components of the bacterial flagellar basal body, which the TTSS needle complex morphologically resembles . This indicates a common ancestry, although no TTSS sequence homologues for the genes encoding the flagellum are found . We here present an approximately 16-A structure of the central component, the needle, of the TTSS . Although the needle subunit is significantly smaller and shares no sequence homology with the flagellar hook and filament, it shares a common helical architecture ( approximately 5.6 subunits/turn, 24-A helical pitch) . This common architecture implies that there will be further mechanistic analogies in the functioning of these two bacterial systems. Semin Nephrol, 2003 Jan, 23(1), 88 - 106 Pathology of renal diseases in the tropics; Boonpucknavig V et al.; Renal diseases unique to the tropics are those that occur in association with infectious diseases including dengue hemorrhagic fever, typhoid fever, shigellosis, leptospirosis, lepromatous leprosy, malaria, opisthorchiasis, and schistosomiasis . These renal complications can be classified on the basis of their clinical and pathologic characteristics into acute transient reversible glomerulonephritis, chronic progressive irreversible glomerulonephritis, amyloidosis, and acute renal failure (ARF) resulting from acute tubular necrosis, acute tubulointerstitial nephritis, and thrombotic microangiopathy . Certain primary glomerular diseases including immunoglobulin (Ig) M nephropathy and focal segmental and global glomerulosclerosis are prevalent in some tropical countries . Renal complications of venomous snakebites also are common in the tropics . This article discusses and summarizes important works in the literature in respect to the clinical syndromes, pathologic features, and pathogenesis of tropical renal diseases both in humans and experimental animal models . J Antimicrob Chemother, 2003 Feb, 51(2), 427 - 9 Growing antimicrobial resistance of Shigella isolates; Ashkenazi S et al.; Appropriate antibiotic treatment of shigellosis depends on identifying resistance patterns . By studying Shigella isolates in central Israel from 1998 to 2000, and comparing with the period 1991-1992, we identified a significantly increased resistance to tetracycline (from 23% to 87%, P < 0.00001), high resistance to trimethoprim-sulfamethoxazole (94%) and ampicillin (85%) and emerging resistance to quinolones (0.5-2%) . These data mandate local monitoring of resistance and its consideration in empirical therapy. Epidemiol Infect, 2002 Dec, 129(3), 445 - 50 Use of AFLP, plasmid typing and phenotyping in a comparative study to assess genetic diversity of Shigella flexneri strains; Herrera S et al.; Shigella flexneri infections are one of the main causes of acute diarrhoea in Cuba . Twenty strains isolated from sporadic cases in nine different Cuban provinces were characterized . Serotyping, antibiotic-resistance typing, plasmid-typing and AFLP-typing were used to determine their suitability for use in epidemiological studies of S . flexneri . The predominant serotypes were serotype 6 (35%) and serotype 2 (35%) . Eleven different plasmid profiles were detected (Diversity Index = 0.92) . AFLP-typing discriminated 12 different patterns (DI = 0.95), these patterns were not coincident with plasmid-typing patterns . Both techniques combined distinguished 14 patterns among the 20 studied strains (DI = 0.99) . There was no consistent relationship between plasmid-typing and AFLP-typing patterns or antibiotic-resistance typing patterns . Ninety-five percent of S . flexneri strains were multiresistant. Antimicrob Agents Chemother, 2003 Feb, 47(2), 727 - 34 Molecular epidemiology of the SRL pathogenicity island; Turner SA et al.; The Shigella resistance locus (SRL), which is carried on the SRL pathogenicity island (PAI) in Shigella flexneri 2a YSH6000, mediates resistance to the antibiotics streptomycin, ampicillin, chloramphenicol, and tetracycline . In the present study, we investigated the distribution and structural variation of the SRL and the SRL PAI in 71 Shigella isolates and 28 other enteric pathogens by PCR and Southern analysis . The SRL and SRL-related loci, although absent from the other enteric pathogens evaluated in this study, were found to be present in a number of Shigella isolates . SRL PAI markers were also present in the majority of strains carrying the SRL and SRL-related loci . PCR linkage studies with six of these strains demonstrated that the SRL is carried on elements similar in structure and organization to the YSH6000 SRL PAI, consistent with the hypothesis that the SRL PAI may be involved in the spread of multiple-antibiotic resistance in these strains. Bioorg Med Chem, 2003 Mar 6, 11(5), 781 - 8 Rational design and synthesis of peptide ligands for an anti-carbohydrate antibody and their immunochemical characterization; Johnson MA et al.; Molecular mimics of carbohydrates present an alternative source of compounds to target pathways involving protein-carbohydrate interactions . Certain peptides act as molecular mimics of carbohydrates in binding to anti-carbohydrate antibodies . A series of potential peptide ligands for the anti-carbohydrate antibody SYA/J6, directed against Shigella flexneri Y, was designed by molecular modeling based on a crystal structure of the antibody complex with a carbohydrate-mimetic peptide . These octapeptides were synthesized using solid-phase peptide synthesis, and their recognition by the antibody was investigated . The results shed light on the nature of peptide-carbohydrate mimicry. Mol Microbiol, 2003 Feb, 47(3), 825 - 38 An extended role for the nucleoid structuring protein H-NS in the virulence gene regulatory cascade of Shigella flexneri; Beloin C et al.; The H-NS nucleoid structuring protein has been shown previously to play a negative role in controlling virulence gene expression in Shigella flexneri by repressing transcription of the virF and virB regulatory genes and the VirF-dependent icsA structural gene under non-permissive growth conditions . Here, we show that H-NS also acts at the promoters of the VirB-dependent structural genes in the regulatory cascade . H-NS protein binds to the promoter regions in vivo and in vitro . The promoters were shown physically and by in silico analysis to contain regions of DNA curvature, a feature of H-NS binding sites . H-NS binding sites were determined by DNase I footprinting at the icsB and the virA promoters . The locations of these sites were consistent with a role for H-NS as a transcription repressor . The VirB-dependent structural gene promoters were found to respond directly to the H-NS repressor, revealing a level of control that is additional to that exerted by the H-NS-dependent virB activator gene . Moreover, the promoters were sensitive to the level of VirB protein in the cell, requiring a threshold level of VirB to be reached before becoming active . A model is discussed in which the levels of expression of the structural genes reflect the outcome of competition between the countervailing regulatory activities of the H-NS and VirB proteins. Vaccine, 2003 Jan 17, 21(5-6), 333 - 40 Immune responses elicited against multiple enterotoxigenic Escherichia coli fimbriae and mutant LT expressed in attenuated Shigella vaccine strains; Barry EM et al.; Shigella and enterotoxigenic Escherichia coli (ETEC) continue to be important causes of diarrheal disease in infants and young children in developing countries and are major etiologic agents of traveler's diarrhea . Since attenuated strains of Shigella have been developed as live oral vaccines against shigellosis, we have adapted these attenuated Shigella strains to serve as carriers of ETEC antigens, thereby constituting a hybrid vaccine . Since protective immunity against ETEC is largely directed against fimbrial antigens (of which there are multiple antigenic types), we have individually expressed four different ETEC fimbriae, including CFA/I, CS2, CS3, and CS4, using deltaguaBA attenuated Shigella vaccine strain CVD 1204 as a prototype live vector . Following mucosal (intranasal) immunization of guinea pigs, serum IgG and mucosal IgA responses were elicited against each fimbrial type . An additional strain was constructed expressing a detoxified version of the human ETEC variant of heat labile toxin (LThK63) . Following mucosal immunization of guinea pigs with a mixed inoculum containing five Shigella strains each expressing a different ETEC antigen, immune responses were observed against each ETEC antigen plus the Shigella vector. Vaccine, 2003 Jan 30, 21(7-8), 644 - 8 Immunogenicity of an HIV-1 gag DNA vaccine carried by attenuated Shigella; Xu F et al.; The use of live attenuated invasive bacteria as a carrier for DNA-based vaccines has been reported recently . In this study, we used a Shigella flexneri serotype 2a rfbF mutant for immunization of a DNA vaccine coding for HIV-1 SF2 Gag . The recombinant bacterial vector delivered gag DNA to mammalian cells in vitro resulting in Gag protein expression, and was found to have a low level of pathogenicity among a number of Shigella cell spread defective mutants tested . Intranasal immunization of mice with live recombinant bacterial cells induced a gag-specific cellular immune response similar to that seen with i.m . injection of naked DNA . Importantly, a strong boosting effect was observed in mice primed with DNA, suggesting utility of bacterial vectors in prime-boost vaccination regimens. Eur J Clin Microbiol Infect Dis, 2002 Dec, 21(12), 887 - 9 Epub 2002 Dec 10. Retrospective analysis of the first clonal outbreak of nalidixic acid-resistant Shigella sonnei shigellosis in Israel; Dagan D et al.; Reported here is a retrospective molecular analysis of the isolates recovered from the first outbreak of nalidixic acid (NA)-resistant Shigella sonnei shigellosis to occur in Israel . The outbreak affected 94 children . In the retrospective analysis, a total of 13 NA-resistant isolates and five NA-susceptible isolates recovered during the outbreak period were examined . Restriction fragment length polymorphism profiles obtained by digestion with BamHI, PvuI, HinfI or SmaI yielded identical profiles for all 18 isolates . All NA-resistant strains had an identical plasmid profile, but this profile differed from that displayed by the susceptible strains . In all of the NA-resistant strains a 304 bp fragment in the gyrA gene coding for a region associated with NA resistance was sequenced and showed a single point mutation, Ser83-->Phe . In this outbreak, the isolates of NA-resistant Shigella sonnei belonged to a single clone and NA resistance was associated with a point mutation in the gyrA gene. Clin Microbiol Infect, 2002 Dec, 8(12), 803 - 5 Pyosalpinx: not always a sexual transmitted disease? Pyosalpinx caused by Plesiomonas shigelloides in an immunocompetent host; Roth T et al.; Plesiomonas shigelloides are ubiquitous Gram-negative bacteria that are found in fresh or marine water, particularly in tropical or warm climates; they were recently implicated in diarrhoeal disease . Patients usually present with a history of recent travel to tropical regions or consumption of uncooked seafood . Extraintestinal disease has rarely been reported, occurring generally in neonates or immunocompromised patients, and is often fatal . We report a case of right pyosalpinx due to P . shigelloides acquired by swimming in contaminated water . Laparoscopic salpingectomy led to a good outcome. Mol Microbiol, 2003 Jan, 47(2), 519 - 27 Putrescine or a combination of methionine and arginine restores virulence gene expression in a tRNA modification-deficient mutant of Shigella flexneri: a possible role in adaptation of virulence; Durand JM et al.; The wild-type strain YSH6000 of Shigella flexneri growing in minimal medium contains the modified nucleoside epoxy-Q (oQ) in a subset of tRNAs . This nucleoside is lacking in tRNA from a tgt mutant of this bacterium . When these bacteria are growing in minimal medium, the expression of virulence genes is 10-fold lower in the tgt mutant than in the wild type, although only a twofold reduction in the expression of these virulence factors is observed in broth . Such a strong media-dependant expression of virulence genes was not observed in the wild type . Accordingly, the level of the positive regulator of virulence, VirF, is much lower in the mutant than in the wild type . However, the transcription of the virF gene in minimal medium is the same in the wild type and in the tgt mutant . As the undermodification of tRNA is not affected by the quality of the growth medium, we conclude that such an environmental change in growth conditions partly restores virulence gene expression by counteracting poor translation of the virF mRNA mediated by an oQ-deficient tRNA . Virulence gene expression is partly restored in the tgt mutant by the addition of a mixture of arginine and methionine . Addition of the polyamine putrescine, synthesis of which is metabolically related to that of arginine and methionine, has a comparable stimulatory effect on virulence gene expression . These results not only suggest a role for amino acids and polyamines in the environmental regulation of virulence gene expression in S . flexneri, but also demonstrate a strong and specific involvement of tRNA modifications, and especially oQ, in the adaptation of virulence gene expression to the nutritional quality of the growth medium. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Jan, 35(1), 49 - 54 {Simultaneous expression of CS3 colonization factor antigen and LT-B/ST fusion enterotoxin antigen of enterotoxigenic Escherichia coli by attenuated Shigella flexneri 2a}; Liu TT et al.; Enterotoxigenic Escherichia coli (ETEC) causes watery dehydrating diarrhea in infants in developing countries, and is the most common cause of travelers diarrhea . It has been known that the colonazition factor antigens (CFAs) and enterotoxins are important virulence factors of ETEC, and these two kinds of proteins should be included in any effective vaccine against ETEC . In this study, a host-plasmid lethal balancing system was constructed based on asd gene in an avirulent strain of S.flexneri to express CS3 antigens and the fusion LT-B/ST enterotoxins of Escherichia coli . Both of these antigens were expressed steadily in the S . flexneri vector without any antibiotic markers . Antibodies against CS3, LT, ST and LPS of Shigella were detected in sera of mice that were immunized with recombinant bacteria either oragastrically (o.g.) or intranasally (i.n.) . SIgA against CS3 and enterotoxins were detected simultaneously in feces of mice . This work is helpful for constructing multivalent recombinant vaccine for prevention of bacterial diarrhea. J Clin Microbiol, 2003 Jan, 41(1), 421 - 3 Changes in patterns of antimicrobial susceptibility and integron carriage among Shigella sonnei isolates from southwestern Korea during epidemic periods; Oh JY et al.; Shigella sonnei isolates from southwestern Korea during the epidemic periods of 1998 to 2000 were genetically related . The antimicrobial susceptibilities of the outbreak-related isolates changed annually . All isolates carried class 2 integrons, and the outbreak-related isolates from 1999 also carried class 1 integrons . The antimicrobial susceptibilities of S . sonnei isolates are readily changed by antibiotic selective pressures, and integrons are responsible for resistance to antimicrobial agents commonly used to treat shigellosis. J Clin Microbiol, 2003 Jan, 41(1), 110 - 7 Phenotypic and genotypic characterization of provisional serotype Shigella flexneri 1c and clonal relationships with 1a and 1b strains isolated in Bangladesh; Talukder KA et al.; The serotypes of 144 strains of Shigella flexneri serotype 1 (serotypes 1a, 1b, and 1c) isolated from patients attending the Dhaka treatment center of the International Centre for Diarrhoeal Disease Research, Bangladesh, between 1997 and 2001 were serologically confirmed by using commercially available antisera and a panel of monoclonal antibodies specific for S . flexneri group and type factor antigen (MASF) . Among serotype 1 isolates, the prevalence of provisional serotype S . flexneri 1c increased from 0 to 56% from 1978 to 2001 in Bangladesh . Detailed biochemical studies revealed that none of the strains of serotype 1 produced indole, while all the strains fermented mannose, mannitol, and trehalose . Twenty percent of the serotype 1c and all the serotype 1a strains fermented maltose and 53% of the serotype 1c strains and 60% of the serotype 1a strains fermented arabinose, whereas all serotype 1b strains were negative for fermentation of these sugars . Only 18% of serotype 1b strains were resistant to nalidixic acid, and most of the serotype 1c and 1b strains were resistant to ampicillin, tetracycline, and trimethoprim-sulfamethoxazole . All the strains of serotypes 1a and 1b and about 88% of the serotype 1c strains were found to be invasive by the Sereny test, had a 140-MDa plasmid, and had Congo red absorption ability . Plasmid profile analysis showed that 26% of the strains of serotype 1 contained identical patterns . Most of the serotype 1c strains (72%) had the 1.6-MDa plasmid, which was not found in either serotype 1a or 1b strains . A self-transmissible middle-range plasmid (35 to 80 MDa) was found in some strains carrying the multiple-antibiotic-resistance gene . Pulsed-field gel electrophoresis analysis yielded three types (types A, B, and C) with numerous subtypes among the serotype 1c strains, whereas serotypes 1b and 1a yielded only one type for each serotype, and those types were related to the types for serotype 1c strains . Ribotyping analysis yielded three patterns for serotype 1c strains and one pattern each for serotype 1a and 1b strains which were similar to the patterns for the serotype 1c strains . Overall analysis of the results concluded that subserotype 1c is closely related to serotypes 1a and 1b . Furthermore, the high rate of prevalence of serotype 1c necessitates the commercial production of antibody against this subserotype to allow the determination of the actual burden of shigellosis caused by provisional serotype 1c. J Infect Dis, 2003 Jan 1, 187(1), 87 - 95 Epub 2002 Dec 13. Human lactoferrin impairs virulence of Shigella flexneri; Gomez HF et al.; Lactoferrin is a glycoprotein present in most human mucosal secretions, including human milk . Lactoferrin is bacteriostatic in low iron media and, in some settings, bactericidal . Lactoferrin impairs ability of Shigella flexneri serotype 5 strain M90T to invade HeLa cells . To determine the mechanism by which lactoferrin decreases invasiveness of Shigella organisms, its effect on the major virulence proteins responsible for bacterial uptake by host cells was evaluated . Lactoferrin induced degradation of invasion plasmid antigens IpaB and, to a lesser extent, IpaC, the key proteins responsible for bacteria-directed phagocytosis by mammalian cells . The lipid A-binding N-terminal portion of lactoferrin (residues 1-33) induces release of invasion antigens but does not induce degradation of IpaBC . Lactoferrin does not directly degrade previously released invasion plasmid antigens but works by making IpaBC susceptible to breakdown by surface-expressed protease(s). Can J Microbiol, 2002 Oct, 48(10), 903 - 10 Phylogenetic study and identification of human pathogenic Vibrio species based on partial hsp60 gene sequences; Kwok AY et al.; The use of hsp60 gene sequences for phylogenetic study and identification of pathogenic marine vibrios was investigated . A 600-bp partial hsp60 gene was amplified by PCR and sequenced from 29 strains representing 15 Vibrio species within the family Vibrionaceae . Sequence comparison of the amplified partial hsp60 gene revealed 71-82% sequence identity among different Vibrio species and 96-100% sequence identity among epidemiologically distinct strains with the same species designation . This degree of discrimination allows unambiguous differentiation of all Vibrio species included in the current study from each other, as well as from Aeromonas hydrophila and Plesiomonas shigelloides, which are often misidentified as Vibrio species by conventional biochemical methods . Based on the hsp60 gene sequences, two previously unidentified shrimp isolates were found to be more closely related to Vibrio alginolyticus (93-94% sequence identity) than to Vibrio parahaemolyticus (89% sequence identity), whereas 16S rRNA gene analysis was unable to differentiate among these closely related species (95-97% sequence identity) . Our results indicate that the hsp60 gene may be a useful alternative target for phylogenetic analysis and species identification of marine Vibrios to complement more conventional identification systems. Pediatr Infect Dis J, 2002 Dec, 21(12), 1136 - 41 Multicenter, randomized, double blind clinical trial of short course versus standard course oral ciprofloxacin for Shigella dysenteriae type 1 dysentery in children; Zimbabwe et al.; BACKGROUND: Shigella dysenteriae type 1 (Sd1) dysentery is associated with serious morbidity and mortality . Treatment is either costly or complicated by the emergence of bacterial resistance . This study compared the efficacy and safety of short course (3 days) and standard course (5 days) oral ciprofloxacin in children with Sd1 dysentery . METHODS: A multicenter, randomized, double blind, controlled clinical trial . Children between 1 and 12 years of age with Sd1 dysentery were randomized to receive oral ciprofloxacin suspension 15 mg/kg every 12 h for 3 days followed by placebo for 2 days or ciprofloxacin suspension for 5 days . Treatment success was defined as either resolution of illness or marked improvement on Day 6 of study . Bacteriologic cure was defined as failure to grow Sd1 on a stool sample collected on Day 6 . Clinical assessments and joint examinations were performed at baseline, at discharge and 2 weeks later . RESULTS: Of 253 patients enrolled 128 were assigned to short course ciprofloxacin and 125 to standard course ciprofloxacin . The success rates were 65 and 69% for short and standard course ciprofloxacin, respectively . All patients had bacteriologic cure, and all Sd1 isolates were susceptible to ciprofloxacin . No bacteriologic relapses occurred during the study period . Eight patients reported arthralgia . All patients had normal joint function including at follow-up . CONCLUSION: In this study twice daily ciprofloxacin for 3 days achieved clinical cure rates similar to those for the same dosage given for 5 days in children with Sd1 dysentery. Biochemistry, 2002 Dec 24, 41(51), 15410 - 4 New UDP-GlcNAc C4 epimerase involved in the biosynthesis of 2-acetamino-2-deoxy-L-altruronic acid in the O-antigen repeating units of Plesiomonas shigelloides O17; Kowal P et al.; Plesiomonas shigelloides is a ubiquitous waterborne pathogen responsible for diseases such as diarrhea and bacillary dysentery, commonly afflicting infants and children . This bacterium is endowed with an O-antigen gene cluster consisting of 10 consecutive reading frames . One of these, designated wbgU (orf3), has been overexpressed and biochemically characterized to show that it encodes a uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) C4 epimerase, only the second microbial enzyme characterized to have this activity . Epimerization is an equilibrium reaction resulting in a 70:30 ratio of UDP-GlcNAc to uridine diphosphate-N-acetylgalactosamine (UDP-GalNAc), irrespective of the initial substrate . The K(m) values for UDP-GalNAc and UDP-GlcNAc are 131 microM and 137 microM, respectively . WbgU is also capable of converting nonacetylated derivatives but with much lower efficiency . It contains a tightly bound nicotinamide adenine dinucleotide {NAD(H)} molecule and requires no other cofactors for activity . We propose here that this enzyme catalyzes the first of the three transformations in the biosynthetic pathway of 2-acetamino-2-deoxy-L-altruronic acid, an unusual sugar present in the O-specific side chains of lipopolysaccharide of P . shigelloides O17 and its close relative Escherichia coli Sonnei. Symp Ser Soc Appl Microbiol, 2002, (31), 136S - 143S Quantitative assessment of risk reduction from hand washing with antibacterial soaps; Gibson LL et al.; The Centers for Disease Control and Prevention have estimated that there are 3,713,000 cases of infectious disease associated with day care facilities each year . The objective of this study was to examine the risk reduction achieved from using different soap formulations after diaper changing using a microbial quantitative risk assessment approach . To achieve this, a probability of infection model and an exposure assessment based on micro-organism transfer were used to evaluate the efficacy of different soap formulations in reducing the probability of disease following hand contact with an enteric pathogen . Based on this model, it was determined that the probability of infection ranged from 24/100 to 91/100 for those changing diapers of babies with symptomatic shigellosis who used a control product (soap without an antibacterial ingredient), 22/100 to 91/100 for those who used an antibacterial soap (chlorohexadine 4%), and 15/100 to 90/100 for those who used a triclosan (1.5%) antibacterial soap . Those with asymptomatic shigellosis who used a non-antibacterial control soap had a risk between 49/100,000 and 53/100, those who used the 4% chlorohexadine-containing soap had a risk between 43/100,000 and 51/100, and for those who used a 1.5% triclosan soap had a risk between 21/100,000 and 43/100 . The adequate washing of hands after diapering reduces risk and can be further reduced by a factor of 20% by the use of an antibacterial soap . Quantitative risk assessment is a valuable tool in the evaluation of household sanitizing agents and low risk outcomes. J Bacteriol, 2002 Dec, 184(24), 6751 - 9 Identification of the cis-acting site involved in activation of promoters regulated by activity of the type III secretion apparatus in Shigella flexneri; Mavris M et al.; Bacteria of Shigella spp . use a virulence plasmid-encoded type III secretion (TTS) system to invade the colonic epithelium in humans . The activity of the TTS apparatus is tightly regulated in the wild-type strain and is induced upon contact of bacteria with epithelial cells, whereas it is deregulated, i.e., constitutively active, in some mutants . Under conditions of deregulated secretion, approximately 20 proteins are secreted, including VirA, OspB to OspG, and at least three members of the IpaH family, all of which are encoded by the virulence plasmid . Conditions inducing or deregulating the activity of secretion also induce the transcription of virA and four ipaH genes . The transcription of virA and ipaH9.8 requires both MxiE, a transcriptional activator of the AraC family, and IpgC, the chaperone of IpaB and IpaC, acting as a coactivator . Using reporter plasmids containing lacZ transcriptional fusions, we showed that the ipaH7.8 . ipa4.5 . ospC1, and ospF promoters are activated under conditions of deregulated secretion and that both MxiE and IpgC are necessary and sufficient for their activation in both Shigella flexneri and Escherichia coli . Promoter mapping and deletion analysis of the ipaH9.8 . virA, and ospC1 promoters identified a 17-bp motif, the MxiE box, which overlaps the -35 region and is essential for the activation of these promoters . The presence of eight MxiE boxes on the virulence plasmid suggests that 11 genes encoding secreted proteins may be regulated by the activity of secretion . We also present evidence that at least one ipaH gene that is carried by the chromosome is controlled by MxiE and IpgC. Infect Immun, 2002 Dec, 70(12), 7050 - 3 Lactoferrin protects rabbits from Shigella flexneri-induced inflammatory enteritis; Gomez HF et al.; Shigella species cause bacillary dysentery in humans by invasion, intracellular multiplication, spread to adjacent cells, and induction of brisk inflammatory responses in the intestinal epithelium . In vitro data suggest that lactoferrin, a glycoprotein present in human mucosal secretions, has a role in protection from bacterial enteric infections . We sought to determine the activity of lactoferrin in vivo, using the concentration present in human colostrum, to investigate its effect on the development of clinical and pathological evidence of inflammation in a rabbit model of enteritis . Lactoferrin protected rabbits infected with Shigella flexneri from developing inflammatory intestinal disease . Typical histological changes in ill animals included villous blunting with sloughing of epithelial cells, submucosal edema, infiltration of leukocytes, venous congestion, and hemorrhage . Lactoferrin at a concentration normally found in human colostrum blocks development of S . flexneri-induced inflammatory enteritis. Rev Med Chil, 2002 Sep, 130(9), 1021 - 6 {Comparison of Shigella susceptibility to commonly used antimicrobials in the Temuco Regional Hospital, Chile 1990 - 2001}; Boehme C et al.; BACKGROUND: The permanent surveillance of antimicrobial susceptibility of Shigella sp in the Temuco Regional Hospital, allowed us to define the empirical use of antimicrobials in dysenteric syndrome . AIM: To study antimicrobial susceptibility of Shigella strains collected from 1997 to May 2001 and compare the results with those reported in 1990 . MATERIAL AND METHODS: Two hundred and seventeen Shigella strains, coming from stool cultures of pediatric patients, were studied . RESULTS: In the period 1989-1990 Shigella flexneri was the main species isolated (83%) whereas, in the period 1997-2001, Shigella sonnei (55.8%) predominated . In the second period, an increase of antimicrobial resistance, as compared with the period 1989-1990, was observed for ampicillin (74.5 and 42% respectively), for cotrimoxazol (57.5 and 45% respectively) and tetracycline (64 and 8% respectively) . Chloramphenicol resistance increased from 0 to 57.5% . In the second period no resistance to ciprofloxacin was detected . There was simultaneous resistance to four drugs in 30% of the strains, predominating multiresistance in S flexneri (52.1%) . CONCLUSIONS: In the two periods studied, a significant increase was detected in the resistance of Shigella strains to antimicrobials. J Biol Chem, 2003 Jan 31, 278(5), 2792 - 8 Epub 2002 Nov 08. Structure-function analysis of invasion plasmid antigen C (IpaC) from Shigella flexneri; Kueltzo LA et al.; Shigella flexneri causes a self-limiting gastroenteritis in humans, characterized by severe localized inflammation and ulceration of the colonic mucosa . Shigellosis most often targets young children in underdeveloped countries . Invasion plasmid antigen C (IpaC) has been identified as the primary effector protein for Shigella invasion of epithelial cells . Although an initial model of IpaC function has been developed, no detailed structural information is available that could assist in a better understanding of the molecular basis for its interactions with the host cytoskeleton and phospholipid membrane . We have therefore initiated structural studies of IpaC, IpaC I', (residues 101-363 deleted), and IpaC Delta H (residues 63-170 deleted) . The secondary and tertiary structure of the protein was examined as a function of temperature, employing circular dichroism and high resolution derivative absorbance techniques . ANS (8-anilino-1-napthalene sulfonic acid) was used to probe the exposure of the hydrophobic surfaces under different conditions . The interaction of IpaC and these mutants with a liposome model (liposomes with entrapped fluorescein) was also examined . Domain III (residues 261-363) was studied using linker-scanning mutagenesis . It was shown that domain III contains periodic, sequence-dependent activity, suggesting helical structure in this section of the protein . In addition to these structural studies, investigation into the actin nucleation properties of IpaC was conducted, and actin nucleation by IpaC and some of the mutants was exhibited . Structure-function relationships of IpaC are discussed. Biochemistry, 2002 Nov 19, 41(46), 13575 - 86 Molecular recognition of oligosaccharide epitopes by a monoclonal Fab specific for Shigella flexneri Y lipopolysaccharide: X-ray structures and thermodynamics; Vyas NK et al.; The antigenic recognition of Shigella flexneri O-polysaccharide, which consists of a repeating unit ABCD {-->2)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->3)-beta-D-GlcpNAc-(1-->}, by the monoclonal antibody SYA/J6 (IgG3, kappa) has been investigated by crystallographic analysis of the Fab domain and its two complexes with two antigen segments (a pentasaccharide Rha A-Rha B-Rha C-GlcNAc D-Rha A' and a modified trisaccharide Rha B-Rha C-GlcNAc D in which Rha C* is missing a C2-OH group) . These complex structures, the first for a Fab specific for a periodic linear heteropolysaccharide, reveal a binding site groove (between the V(H) and V(L) domains) that makes polar and nonpolar contacts with all the sugar residues of the pentasaccharide . Both main-chain and side-chain atoms of the Fab are used in ligand binding . The charged side chain of Glu H50 of CDR H2 forms crucial hydrogen bonds to GlcNAc of the oligosaccharides . The modified trisaccharide is more buried and fits more snugly than the pentasaccharide . It also makes as many contacts (approximately 75) with the Fab as the pentasaccharide, including the same number of hydrogen bonds (eight, with four being identical) . It is further engaged in more hydrophobic interactions than the pentasaccharide . These three features favorable to trisaccharide binding are consistent with the observation of a tighter complex with the trisaccharide than the pentasaccharide . Thermodynamic data demonstrate that the native tri- to pentasaccharides have free energies of binding in the range of 6.8-7.4 kcal mol(-1), and all but one of the hydrogen bonds to individual hydroxyl groups provide no more than approximately 0.7 kcal mol(-1) . They further indicate that hydrophobic interactions make significant contributions to binding and, as the native epitope becomes larger across the tri-, tetra-, pentasaccharide series, entropy contributions to the free energy become dominant. Di Yi Jun Yi Da Xue Xue Bao, 2001, 21(12), 935 - 937 gyrA gene mutations in quinolone-resistant Shigellae flexneri; Xiang Q et al.; OBJECTIVE: To study the resistance mechanism of quinolone against Shigellae flexneri . METHODS: The N-terminal coding region of gyrA gene of 38 quinolone-resistant Shigellae flexneri were amplified by PCR and mutations detected by single- strand conformation polymorphism (SSCP), followed by sequence analysis . RESULTS: Eight isolates were found to be positive for gyrA mutations, and DNA sequence analysis of gyrA gene revealed 2 mutations that resulted in changes of the amino acids (Ser-83--Leu, Asp-87--Gly) . CONCLUSION: gryA gene mutations is closely related to high resistance of Shigellae flexneri to quinolone. Indian J Med Res, 2002 Jan, 115, 11 - 3 Shigella isolation in Vellore, south India (1997-2001); Jesudason MV; Shigellosis has been a major cause of dysentery for many years at Vellore, south India . In the last two years the number of Shigella being isolated from samples of faeces from patients with diarrhoea has decreased (5% isolation rate in 1997 to 3.9% in 2001), although the microbiological methods and media used have not changed . Also, the nalidixic acid (NA) resistance has increased for S . sonnei (now 94%) . This is noteworthy, since NA has been recommended for the empirical treatment of patients suspected to have shigellosis and this concept needs to be reconsidered based on available data. Carbohydr Res, 2002 Oct 8, 337(18), 1633 - 40 Conformation of the branched O-specific polysaccharide of Shigella dysenteriae type 2: molecular mechanics calculations show a compact helical structure exposing an epitope which potentially mimics galabiose; Rosen J et al.; Conformational analyses of the branched repeating unit of the O-antigenic polysaccharide of Shigella dysenteriae type 2 have been performed with molecular mechanics MM3 . A filtered systematic search on the trisaccharide alpha-D-GalNAc-(1-->3)-{alpha-D-GlcNAc-(1-->4)}-alpha-D-GalNAc forming the branch, shows essentially a single favored conformation . Also, the downstream alpha-D-GalNAc-(1-->4)-alpha-D-Glc linkage is sterically constrained . The alpha-D-Glc-(1-->4)-beta-D-Gal moiety, however, forms a more flexible link region between the branch points, and shows a 90 degrees bend similar to what is known for the galabiose moiety occurring in globo-glycolipids . The calculations indicate that consecutive repeating units in their minimum energy conformation arrange in a helical structure with three repeating units per turn . This helix is very compact and appears to be stabilized by hydrophobic interactions involving the N-acetyl groups at the branch points . Random conformational search suggests the existence of another helical structure with four repeating units per turn . It appears possible that the alpha-D-Glc-(1-->4)-beta-D-Gal moiety, which is exposed on the surface of the helical structures, can evade recognition by the immune system of the host by the mimicry of globo structures. Obstet Gynecol, 2002 Nov, 100(5 Pt 2), 1063 - 5 Shigellosis complicating preterm premature rupture of membranes resulting in congenital infection and preterm delivery; Rebarber A et al.; BACKGROUND: The association of chorioamnionitis with preterm birth is well established . Intra-amniotic infection complicates 13-60% of preterm premature rupture of membranes (PROM) with enteric gram-negative pathogens accounting for 20-40% of recoverable organisms . However, the source of enteric pathogens leading to premature birth has been poorly characterized . CASE: A 36-year-old multiparous woman presented at 2567 weeks with preterm PROM . She reported a 5-day history of bloody, mucous diarrhea . A fourth cesarean delivery was performed secondary to the onset of labor . Fetal blood, placental membrane, and vaginal pool cultures revealed the presence of Shigella sonnei . With appropriate antibiotic therapy, the patient was discharged home on postoperative day 5 . Neonatal stool cultures revealed evidence of in utero fetal transmission . CONCLUSION: It is prudent to treat pregnant patients with clinical symptoms suggestive of shigellosis because this pathogen can result in preterm PROM and preterm delivery . Neonatal testing is indicated if maternal disease is suspected before delivery. FEMS Microbiol Lett, 2002 Oct 29, 216(1), 39 - 42 Identification of shiga toxin-producing bacteria by a new immuno-capture toxin gene PCR; Luo W et al.; Infections with bacteria producing shiga toxin are responsible for widespread disease and for the death of a large number of people . In the present study, we have developed a rapid method of high specificity for the detection of Shigella dysenteriae by combining immuno-capture of the bacteria and polymerase chain reaction (PCR) amplification of their toxin gene . We compared the sensitivity of our new method, referred to as immuno-capture toxin gene PCR (iTGPCR), with a conventional TGPCR (cTGPCR) method in detecting S . dysenteriae . Approximately 100 colony forming units (CFU) of bacteria in a volume of 400 microl were divided into 20 tubes with 5 CFU (20 microl) . One group of 10 tubes was analyzed by iTGPCR and the other by cTGPCR amplification . All were positive in the 10 tubes using iTGPCR but, in contrast, only half were positive in the 10 tubes with the cTGPCR method . This method was used to detect S . dysenteriae type I in sewage samples without the normal tedious preparation methods . These findings suggest that iTGPCR gives enhanced test sensitivity, and allows determination of pathogen serotype, and differentiation of toxin-producing strains from non toxin-producing strains. Res Microbiol, 2002 Sep, 153(7), 461 - 8 Histone-like proteins and the Shigella invasivity regulon; Prosseda G et al.; The contribution of histone-like proteins to the transcriptional regulation of virulence gene networks is a common feature among pathogenic bacteria . In this article we review current knowledge about the regulative role of major histone-like proteins in the silencing/activation of the invasivity phenotype of Shigella, the etiological agent of bacillary dissentery. Biomedica, 2002 Sep, 22(3), 272 - 9 {Acute diarrhea outbreak caused by Shigella flexneri at a school in Madrid, Cundinamarca: phenotypic and genotypic characterization of the isolates}; Hidalgo M et al.; Shigellosis is an acute diarrhoeal disease that is the main cause of morbidity and mortality in developing countries . In 1997, the Colombian Instituto Nacional de Salud Microbiology Group organized a network surveillance program with the country's Public Health Laboratories (PHLs) to monitor the principal etiological agents responsible for acute diarrhoeal disease . In May, 2001, the PHL of the state of Cundinamarca reported a food poisoning outbreak involving an elementary school community . The main goal of the Microbiology Group involvement was to establish the molecular relationships among the isolates from the outbreak by phenotypic and genotypic methods of characterization . Stool cultures were obtained from 22 of 195 affected individuals . The Microbiology Group confirmed the identification of the isolates by biochemical and serological probes . The antimicrobial susceptibilities were tested against the following battery of antibiotics: chloramphenicol, trimehoprim-sulfamethozazole, cefotaxime, gentamicin, ampicillin and ciprofloxacin . The isolates were subjected to pulsed field gel electrophoresis (PFGE) using the following CDC (U.S . Centers for Disease Control) protocols: Xbal restriction enzyme, Shigella sonnei CDC F2353 as the reference standard, and lambda phage as a molecular weight marker . In 15 of 22 (68%) stool cultures Shigella was recovered, all isolates were identified as Shigella flexneri serotype 6 biotype Newcastle with the same antimicrobial susceptibility profile . PFGE showed that 3 (20%) isolates were identical (100% genetic similarity) and the other 12 (80%) were very closely related (genetic similarity between 86-98%) . The network system permitted the INS ready access to the isolates and the implementation of the PFGE permitted a quantitative characterization of the clonal relationship among the isolates from the outbreak. Epidemiol Infect, 2002 Oct, 129(2), 235 - 43 Shifting serotypes, plasmid profile analysis and antimicrobial resistance pattern of shigellae strains isolated from Kolkata, India during 1995-2000; Dutta S et al.; One hundred and sixty-six shigellae strains, isolated from stool samples of paediatric patients (< 5 years old) at a Childrens' Hospital in Kolkata, India during the period of 1995-2000 were examined for serotyping, drug resistance pattern and plasmid profiles . Sh . flexneri (58 %) was found to be commonest isolate of total shigellae, followed by Sh . sonnei (28 %), Sh . boydii (9%) and Sh . dysenteriae (5%) . This profile of species was in sharp contrast to the picture obtained before 1995, when Sh . dysenteriae 1 predominated over Sh . flexneri . In Sh . flexneri strains, Sh . flexneri 2a (35%) was the most prevalent serotype, following Sh . flexneri 3a (31%), Sh . flexneri 6 (14%), Sh . flexneri 2b (11%) and Sh . flexneri 4 (9%) . Resistance patterns of the strains to 12 commonly used antimicrobial agents and minimum inhibitory concentrations (MICs) of the antibiotics were also tested . All strains were found uniformly susceptible to norfloxacin, but more than 90% strains were resistant to tetracycline, co-trimoxazole and 67% strains were resistant to ampicillin . Resistance to amoxicillin, chloramphenicol and nalidixic acid was found in 55% (range 45-74%), 46% (range 40-60%) and 29% (range 15-40%) strains respectively . Overall, shigellae strains showed statistically significant increase in resistance against tetracycline, nalidixic acid and furazolidone (P < 0.05) over the years of this study . This indicates decreased efficacy of furazolidone, cotrimoxazole and nalidixic acid for the empirical treatment of shigellosis in Kolkata . Although a few strains showed intermediate susceptibility to ciprofloxacin (4%) and cefotaxime (10%) by disk diffusion test, but the MICs of those antibiotics were within the normal limits . Almost 57% of the strains were resistant to four or more drugs with high MICs of the antibiotics . Plasmid profile analysis revealed presence of large plasmid of 220 kb in majority of the strains except in Sh . sonnei and a correlation between presence of smaller plasmids and shigellae serotypes . Hence this study reports epidemiological change of shigellae species in Kolkata, India with regard to serotypes and antibiotic resistance patterns. FEMS Microbiol Lett, 2002 Oct 8, 215(2), 237 - 42 Doc-mediated cell killing in Shigella flexneri using a C1/LacI controlled expression system; Schofield DA et al.; In this report we describe the development of a highly stringent and dually regulated promoter system for Shigella flexneri . Dual regulation was provided by utilizing a promoter susceptible to control by the bacteriophage P1 temperature-sensitive C1 repressor that in turn was under the transcriptional control of LacI . The level of induction/repression ratios observed was up to 3700-fold in S . flexneri . The general utility of this promoter system was evaluated by demonstrating that the bacteriophage P1 post-segregational killer protein Doc mediates a bactericidal effect in S . flexneri . This represents the first report of Doc (death on curing)-mediated killing in this Gram-negative species. Mol Med, 2002 Jul, 8(7), 405 - 16 ILG1 : a new integrase-like gene that is a marker of bacterial contamination by the laboratory Escherichia coli strain TOP10F'; Tian W et al.; BACKGROUND: Identification of differentially expressed genes between normal and diseased states is an area of intense current medical research that can lead to the discovery of new therapeutic targets . However, isolation of differentially expressed genes by subtraction often suffers from unreported contamination of the resulting subtraction library with clones containing DNA sequences not from the original RNA samples . MATERIALS AND METHODS: Subtraction using cDNA representational difference analysis (RDA) was performed on human B cells from normal or common variable immunodeficiency patients . The material remaining after the subtraction was cloned and individual clones were sequenced . The sequence of one clone with similarity to integrases (ILG1, integrase-like gene-1) was used to obtain the full length cDNA sequence and as a probe for the presence of this sequence in RNA or genomic DNA samples . RESULTS: After five rounds of cDNA RDA, 23.3% of the clones from the resulting subtraction library contained Escherichia coli DNA . In addition, three clones contained the sequence of a new integrase, ILG1 . The full length cDNA sequence of ILG1 exhibits prokaryotic, but not eukaryotic, features . At the DNA level, ILG1 is not similar to any known gene . At the protein level, ILG1 has 58% similarity to integrases from the cryptic P4 bacteriophage family (S clade) . The catalytic domain of ILG1 contains the conserved features found in site-specific recombinases . The critical residues that form the catalytic active site pocket are conserved, including the highly conserved R-H-R-Y hallmark of these recombinases . Interestingly, ILG1 was not present in the original B cell populations . By probing genomic DNA, ILG1 could only be detected in the E . coli TOP10F' strain used in our laboratory for molecular cloning, but not in any of its precursor strains, including TOP10 . Furthermore, bacteria cultured from the mouth of the laboratory worker who performed cDNA RDA were also positive for ILG1 . CONCLUSIONS: In the course of our studies using cDNA RDA, we have isolated and identified ILG1, a likely active site-specific recombinase and new member of the bacteriophage P4 family of integrases . This family of integrases is implicated in the horizontal DNA transfer of pathogenic genes between bacterial species, such as those found in pathogenic strains of E . coli, Shigella, Yersinia, and Vibrio cholera . Using ILG1 as a marker of our laboratory E . coli strain TOP10F', our evidence suggests that contaminating bacterial DNA in our subtraction experiment is due to this laboratory bacterial strain, which colonized exposed surfaces of the laboratory worker . Thus, identification of differentially expressed genes between normal and diseased states could be dramatically improved by using extra precaution to prevent bacterial contamination of samples. Cell Microbiol, 2002 Sep, 4(9), 613 - 26 Selection of Shigella flexneri candidate virulence genes specifically induced in bacteria resident in host cell cytoplasm; Bartoleschi C et al.; We describe an in vivo expression technology (IVET)-like approach, which uses antibiotic resistance for selection, to identify Shigella flexneri genes specifically activated in bacteria resident in host cell cytoplasm . This procedure required construction of a promoter-trap vector containing a synthetic operon between the promoterless chloramphenicol acetyl transferase (cat) and lacZ genes and construction of a library of plasmids carrying transcriptional fusions between S . flexneri genomic fragments and the cat-lacZ operon . Clones exhibiting low levels (<10 micro g ml-1) of chloramphenicol (Cm) resistance on laboratory media were analysed for their ability to induce a cytophatic effect--plaque--on a cell monolayer, in the presence of Cm . These clones were assumed to carry a plasmid in which the cloned fragment acted as a promoter/gene which is poorly expressed under laboratory conditions . Therefore, only strains harbouring fusion-plasmids in which the cloned promoter was specifically activated within host cytoplasm could survive within the cell monolayer in the presence of Cm and give a positive result in the plaque assay . Pai (plaque assay induced) clones, selected following this procedure, were analysed for intracellular (i) beta-galactosidase activity, (ii) proliferation in the presence of Cm, and (iii) Cm resistance . Sequence analysis of Pai plasmids revealed genes encoding proteins of three functional classes: external layer recycling, adaptation to microaerophilic environment and gene regulation . Sequences encoding unknown functions were also trapped and selected by this new IVET-based protocol. Infect Immun, 2002 Nov, 70(11), 6355 - 64 Shigella flexneri DegP facilitates IcsA surface expression and is required for efficient intercellular spread; Purdy GE et al.; A degP mutant of Shigella flexneri was identified in a screen for insertion mutants that invaded cultured cells but did not form wild-type plaques in monolayers . The degP mutant SM1100 invaded Henle cells at wild-type levels and induced apoptosis in macrophages but formed smaller plaques than those formed by wild-type S . flexneri in confluent monolayers of Henle and Caco-2 cells . The proportion of SM1100 bacteria with IcsA localized to the bacterial pole, a process required for actin polymerization into actin "tails," was reduced compared to results with wild-type bacteria . The reduction in proper IcsA localization may account for the reduced plaque size of the degP mutant . Although DegP is a protease, the protease activity of S . flexneri DegP was not required for IcsA localization or the formation of plaques in Henle cell monolayers . DegP was also required for efficient polar IcsA localization in E . coli expressing icsA . In addition, the growth or survival of SM1100 was compromised compared to that of the wild type at elevated temperatures and in acidic conditions. Scand J Infect Dis, 2002, 34(9), 692 - 3 Septic shock associated with Shigella flexneri dysentery; Beigelman A et al.; Septic shock is a very unusual presentation of Shigella infection . We describe a 3-y-old child who developed severe septic shock and severe encephalopathy during an episode of dysentery caused by Shigella flexneri. Scand J Infect Dis, 2002, 34(9), 687 - 9 Plesiomonas shigelloides sepsis in a thalassemia intermedia patient; Tzanetea R et al.; Bacteremia due to Plesiomonas shigelloides was associated with rapidly fulminant septicemia, disseminated intravascular coagulation and massive adrenal hemorrhage in a splenectomized patient suffering from thalassemia intermedia who was treated with hydroxyurea . P . shigelloides was isolated in blood cultures; despite a vigorous combination of antibiotics the patient died after 24 h in the ICU . Lethal sepsis due to P . shigelloides has not previously been reported in Greece. Curr Biol, 2002 Sep 17, 12(18), 1617 - 22 The WH1 and EVH1 domains of WASP and Ena/VASP family members bind distinct sequence motifs; Zettl M et al.; A complex of N-WASP and WASP-interacting protein (WIP) plays an important role in actin-based motility of vaccinia virus and the formation of filopodia . WIP is also required to maintain the integrity of the actin cytoskeleton in T and B lymphocytes and is essential for T cell activation . However, in contrast to many other N-WASP binding proteins, WIP does not stimulate the ability of N-WASP to activate the Arp2/3 complex . Although the WASP homology 1 (WH1) domain of N-WASP interacts directly with WIP, we still lack the exact nature of its binding site . We have now identified and characterized the N-WASP WH1 binding motif in WIP in vitro and in vivo using Shigella and vaccinia systems . The WH1 domain, which is predicted to have a similar structural fold to the Ena/VASP homology 1 (EVH1) domain, binds to a sequence motif in WIP (ESRFYFHPISD) that is very different from the EVH1 proline-rich DL/FPPPP ligand . Interaction of the WH1 domain of N-WASP with WIP is dependent on the two highly conserved phenylalanine residues in the motif . The WH1 binding motif we have identified is conserved in WIP, CR16, WICH, and yeast verprolin. Przegl Epidemiol, 2002, 56(2), 285 - 92 {Dysentery in Poland in 2000}; Stypulkowska-Misiurewicz H et al.; The notified number of dysentery cases is still falling down since the year 1995 when bacteriological examination of feces for diagnostic purposes lost free of charge status obtained in 1928 under the legislation act of infectious disease investigation and obligatory registration . Only 121 dysentery cases were notified in the year 2000 (incidence 0.31/100,000 population), while 292 cases were notified in 1999 (incidence 0.76/100,000) . No one death case was notified . Only 2 outbreaks (one due to S . flexneri 4a and one due to S . sonnei), both involving 26 patients, were notified in 2000, less than in 1999 when four outbreaks involving 146 patients were registered . In the year 2000, S . sonnei was the dominant etiological agent in 80% of notified dysentery cases and S . flexneri only in 20% of cases was the next . S . dysenteriae 3 was found only in one patient S . boydii in no one . All Shigella strains were susceptible to nitrofurans (ex.nifuroxaside), gentamicin, nalidixic acid, cefotaxime, and imipenem . Only two strains, including the one important from India, were susceptible to co-trimoxasole, but not to doxycycline. Microbes Infect, 2002 Sep, 4(11), 1125 - 32 Escherichia coli in disguise: molecular origins of Shigella; Lan R et al.; Shigella, which still stands as a genus with four species today, in reality belongs to the extremely diverse species Escherichia coli . There are several lineages of Shigella strains derived through independent acquisition of the pINV virulence plasmid . The chromosomally determined phenotypic properties of Shigella result from convergent evolution during niche adaptation, most due to loss of function, some from negative selection pressure. Microbes Infect, 2002 Sep, 4(11), 1121 - 4 Epithelial inflammation response induced by Shigella flexneri depends on mucin gene expression; Nutten S et al.; The protective effects of different mucin gene profiles on gut protection were assessed by the evaluation of TNFalpha production by intestinal epithelial cells infected by Shigella flexneri . Three HT-29 cell lines were used: HT29-G(-) (enterocyte-like cells, secreting no mucins), HT29-FU (highly expressing MUC2 and MUC4) and HT29-MTX (highly expressing MUC3 and MUC5AC) . These cells were infected either by an invasive (M90T) or the control isogenic (BS176) strains of S . flexneri, and TNFalpha mRNA production was quantified by competitive PCR . In the HT29-G(-) cells, M90T induced an increased production of TNFalpha mRNA compared to BS176, giving a TNFalpha ratio of 5.6 +/- 3.3 . In contrast, similar levels of TNFalpha mRNA were detected in HT29-FU and HT29-MTX cells stimulated with either M90T or BS176, giving ratios of 1.4 +/- 1.3 and 1.0 +/- 0.1, respectively . The results suggest that mucin genes have abilities to protect epithelial cells against S . flexneri . Furthermore, the difference in the TNFalpha ratio between the HT29-FU and HT29-MTX cells suggests distinct protective effects for these two mucin-secreting epithelial cells. EMBO J, 2002 Oct 1, 21(19), 5069 - 78 Conversion of PtdIns(4,5)P(2) into PtdIns(5)P by the S.flexneri effector IpgD reorganizes host cell morphology; Niebuhr K et al.; Phosphoinositides play a central role in the control of several cellular events including actin cytoskeleton organization . Here we show that, upon infection of epithelial cells with the Gram-negative pathogen Shigella flexneri, the virulence factor IpgD is translocated directly into eukaryotic cells and acts as a potent inositol 4-phosphatase that specifically dephosphorylates phosphatidylinositol 4,5-bisphosphate {PtdIns(4,5)P(2)} into phosphatidylinositol 5-monophosphate {PtdIns(5)P} that then accumulates . Transfection experiments indicate that the transformation of PtdIns(4,5)P(2) into PtdIns(5)P by IpgD is responsible for dramatic morphological changes of the host cell, leading to a decrease in membrane tether force associated with membrane blebbing and actin filament remodelling . These data provide the molecular basis for a new mechanism employed by a pathogenic bacterium to promote membrane ruffling at the entry site. Indian J Pediatr, 2002 Aug, 69(8), 687 - 95 Management of diarrhoea in practice; Singh UK et al.; Diarrhoea, a major cause of morbidity and mortality can be produced by a variety of etiological factors . Management protocol includes assessment of the child, physical examination, lab-evaluation, assessment of severity of dehydration and rehydration therapy using either of the following - WHO - ORS, Home available fluids (HAF), sugar salt solution (SSS), improve WHO-ORS, Amino acid fortified ORS, rice based ORS, low osmolarity ORS . Intravenous fluids are required if patients can't accept orally . Commonly observed electrolyte disturbances are hypernatremia, hyponatremia and hypokalemia . Concussion is a common problem and can result due to electrolyte imbalance, cavernous sinus thrombosis, associated meningitis, shigella encephalopathy and hypoglycemia in undernourished children . Treatment includes i.v . diazepam and i.v . glucose and correction of electrolyte imbalance . Additional treatment interventions include antimicrobial drugs including antibiotics, antimotility drugs, absorbents, nutritional and micro and macro nutrient supplementation. Glimpse, 1997 Jun, 19(2), 2 - 5 Scientific achievements of the Centre, 1991-1995, in research on child survival; High-protein diet facilitates growth of children after shigellosis; PIP: Health workers randomly assigned 69 2-5 year old children recovering from shigellosis at the hospital of the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), to receive either a high-protein diet (150 kcal/kg/day with protein making up 15% of total calories) or a standard control diet (150 kcal/kg/day with protein making up 7.5% of total calories) . They received this diet for 21 days . At the end of the study, children in the high-protein diet group were significantly taller and weighed considerably more than those in the standard diet group (p ,01) . Moreover, this height spurt was maintained at 3 and 6 months (p .03 and .001, respectively) . These children also gained significantly more weight (p .01) . Prealbumin and retinol-binding protein increased significantly more among the children in the high-protein diet group than among the controls (p .01), but the mean serum albumin increases were the same . Thus this biochemical parameter suggested that the body used the extra protein to make more of the essential body proteins at a faster rate to make up for the negative nitrogen balance caused by diarrhea during acute shigellosis . Significant increases in somatomedin-C occurred in the high-protein group in comparison to the control group (p .01), indicating that the high-protein diet stimulated growth and effective protein repletion . The children in the high-protein group experienced considerably more of an increase in the fat-free mass than the others (p .01), but this mass was the same for both groups . therefore, the high-protein diet increased more deposition of fat in the muscle tissue than fat tissue . In conclusion, ICDDR,B recommended that physicians use high-protein foods with adequate calories to manage children with shigellosis to help them recover their growth quickly . Glimpse . 1992 Nov-Dec;14(6):1, 4. Persistent diarrhoea still a serious and difficult problem; Diarrhoea management: drug treatment; PIP: The author proposes general guidelines for the use of drugs in the treatment of diarrhea . It is stressed that antidiarrheal drugs should never be used for the routine management of acute diarrhea, especially in children under 5 years of age; rather, fluid and electrolyte replacement should be the primary treatment . Antibiotics are not indicated in patients with watery diarrhea unless cholera is suspected, in which case tetracycline or another antimicrobial agent should be used . In patients with dysentery, especially children who are febrile or appear seriously ill, antimicrobials such as ampicillin or co-trimoxazole should be given . The choice of antibiotic should be based on known antimicrobial sensitivity patterns of Shigella strains in the geographic area . Antimotility drugs may give some symptomatic relief in adults, but are contraindicated in children under 5 years of age because of the risk of depressed respiration and altered consciousness . The antisecretory drugs chlorpromazine and berberine have not yet proven practical for routine use . Glimpse, 1982 Jan, 4(1), 2 - 4 Utilization of nutrients during and after diarrhoea; Training activities at the Centre; PIP: This article describes the international training workshops conducted by the International Center for Diarrheal Disease Research, Bangladesh (ICDDR,B) . During May 4-15, 1997, an international training course was conducted on the Laboratory Diagnosis of Common Diarrheal Disease Agents . The aim was to update the knowledge and skills of health professionals . 11 participants learned procedures for isolation and identification of pathogens, preparation of culture media, and laboratory safety . It is expected that those trained will spread their knowledge to others upon return to their home countries . A 4-week introductory course was conducted on Epidemiology and Biostatistics . The aim was to train persons from national institutions and nongovernmental organizations (NGOs) how to plan, design, analyze, and undertake epidemiological studies . A 2-week course was held on Emergency Response to Cholera and Shigella Epidemics . 12 participants from international NGOs that respond to disaster situations gained skills in managing epidemics and learning how to identify and apply measures to reduce morbidity and mortality from acute diarrhea in emergency and disaster situations . Participants learned how to recognize and manage patients with cholera and shigellosis and how to prepare an action plan for treatment and control purposes . This course consisted of theoretical lectures, hands-on training, and practical training . The Center's success in intervening in the Rwandan cholera epidemic justifies continued training for next year as well . 13 participants attended a 2-week course on Clinical Management of Diarrheal Diseases . Afr Health, 1997 Mar, 19(3), 9 - 10 Shigella dysenteriae type 1 in Zimbabwe; Nathoo KJ; PIP: Shigella dysenteriae type 1 (SD1) is an unusually virulent enteric pathogen which causes endemic or epidemic dysentery with high death rates . SD1 has been responsible for large-scale regional outbreaks of dysentery in Africa, Central America, and south Asia . Shigella dysentery epidemics have been reported in Africa since the 1800s and overall, more than 250 million people in the African region are at risk and subject to a case fatality rate of possibly 1-10% . In Zimbabwe, the 1994 national incidence of dysentery was 13.0/1000 and the national case fatality rate was 4.5% . The transmission of SD1, its clinical presentation, management, complications, the role of the laboratory, outbreak identification and adequate preparedness, and the prevention of infection are discussed . Child Health Dialogue . 1996;(3-4):5. Dealing with diarrhoea; Fontaine O; PIP: Children with severe dehydration, persistent diarrhea with dehydration, or bloody diarrhea with no signs of improvement must be hospitalized . In-patient care for a child with severe dehydration includes rapid intravenous (IV) fluid therapy . Children who can still drink should be given oral rehydration salts (ORS) solution while the health worker sets up the IV drip . Children with difficulty drinking should be given ORS as soon as the IV fluid therapy restores their ability to drink (within 3-4 hours for babies, or 1-2 hours for older children), since ORS amends mineral deficiencies more effectively than the IV fluids . The IV drip should be re-administered if the child still exhibits dehydration after 3 hours for older children or 6 hours for babies . If improvement is noted, health workers should encourage the mother to administer ORS and to breast feed frequently . Hospital personnel should observe the child for at least 6 hours before discharge . This allows them to be sure that mothers can maintain the child's fluid balance . Children with diarrhea for more than 14 days face malnutrition or death . Any child with persistent diarrhea who exhibits moderate or severe malnutrition and signs of dehydration and is less than 4 months old needs to be admitted to a hospital . Management of persistent diarrhea involves fluid replacement, appropriate diet, and treatment of associated infections, if needed . ORS is usually effective for persistent diarrhea, although in a few cases poor absorption of glucose may necessitate initial rehydration with IV therapy . Breast feeding is encouraged for infants . Older infants and young children should eat 6 times a day as soon as they are able to eat . Recommended diets for these children are a low lactose diet (milk, yogurt, or curds; cooked rice; oil; sugar/glucose) and a low starch and no lactose diet (eggs, chicken, or fish; cooked rice; oil; sugar/glucose) . Children with serious infections may require nasogastric feeding at first . Shigella bacteria tend to be responsible for dysentery . Children with this bloody diarrhea should be treated with an antibiotic . If their condition does not improve and they are malnourished, less than 1 year old, were initially dehydrated, or have recently had measles, they need to be hospitalized . Drugs to reduce frequency of stools should never be given in cases of bloody diarrhea . Older babies and children should be given an extra meal and supplementary vitamins and minerals each day for two weeks . Glimpse, 1994 Sep-Oct, 16(5), 3 - 4 Cholera epidemic among Rwandan refugees: experience of ICDDR,B in Goma, Zaire; Siddique AK; PIP: In July 1994, one of the worst cholera epidemics broke out among the nearly a million Rwandan refugees in Goma, eastern Zaire . The United Nations High Commission for Refugees estimated that nearly 12,000 people died during the epidemic . The International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B) sent an eight-member medical team to Goma headed by Dr AK Siddique, a senior scientist of the Center and head of the Epidemic Control Preparedness Program, Dacca, Bangladesh . During their two-week stay, the team, in collaboration with UNICEF and the Ministry of Health, Zaire, conducted epidemiological assessment, operated a temporary treatment center and provided technical advice on case management of cholera and shigellosis to other health workers . The team also set up a microbiology laboratory in Goma to identify the pathogens responsible for the epidemic and their drug sensitivity patterns . The team visited a number of temporary treatment facilities in two of the five camp sites and provided technical advice to the health-care providers . They also visited treatment facilities in Goma city, where an estimated 200,000 refugees were affected by the epidemic . Deaths from cholera even in the treatment centers were much higher than expected . The overall case-fatality rate in the treatment centers was nearly 15% . Laboratory investigations showed that the initial epidemic was indeed caused by Vibrio cholerae strains resistant to tetracycline and doxycycline . By the first week of August, the number of cholera cases was declining, but the number of dysentery cases was increasing rapidly . Predominantly Shigella dysenteriae type 1 was responsible, which was resistant to most drugs used for treating shigellosis, except mecillinam . Inappropriate rehydration therapy and inadequate experience of health workers failed to prevent deaths . The team took over the operation of temporary treatment center at Katindo in Goma city with one of the highest case-fatality rates (14.5%) and could reduce the fatality rate to less than 1% . Dialogue Diarrhoea, 1988 Dec, (35), 4 - 5 Mechanisms of diarrhoea and why they matter; Cutting WA; PIP: Diarrhea involves a significant increase in excreted fluid volume, due either to a failure of the bowel to reabsorb or absorb fluid or to a great increase in fluid secreted into the bowel . To cause diarrhea, pathogenic organisms must be swallowed, survive the acid in the stomach, colonize the small bowel, and stick to the enterocytes . In secretory diarrhea, Vibrio cholerae bacteria and enterotoxigenic Escherichia coli enter the enterocytes, stimulate adenylatecyclase, and cause a chain of reactions that results in the secretion of sodium and chloride ions (electrolytes)--accompanied by water--into the lumen of the bowel . In invasive diarrhea, Shigella bacteria not only colonize the surface of the small bowel but they also penetrate and invade the mucous membrane . Many enterocytes are destroyed, blood vessels may rupture, and white cells are excreted as pus along with blood and tissue fluid . Rotaviruses, a common cause of acute diarrhea in small children, penetrate the bowel in patches, killing many enterocytes and thus reducing the surface for absorption . Replacement of lost water and electrolytes is the 1st priority in the treatment of diarrhea . Examination of the diarrhea stools for blood can help to identify invasive diarrhea . Children with Shigella dysentery who seems seriously ill require specific antibiotics in addition to rehydration . J Food Prot, 2002 Sep, 65(9), 1417 - 21 Effect of organic acids and temperature on survival of Shigella flexneri in broth at pH 4; Zaika LL; The survival of bacterial pathogens in acidified foods depends not only on the hydrogen ion concentration, but also on the type of acid and the storage temperature . Shigella flexneri is a foodborne pathogen that is acid tolerant . The survival of S . flexneri 5348 in brain heart infusion broth supplemented with 0.04 M acetic, citric, lactic, malic, or tartaric acid and adjusted to pH 4 with HCI or NaOH was studied . The control medium was brain heart infusion broth adjusted to pH 4 with HCI . Stationary-phase cells were inoculated into media at initial populations of 6 to 7 log10 CFU/ml and incubated at 4, 19, 28, and 37 degrees C . A two-phase linear inactivation model was applied to plate count data to derive lag times (tL) and slopes of the curves, from which D-values and time required for a 4-log10 decrease in population (T4D) were calculated . In all cases, survival increased with decreasing temperature . For each acid, tL, the D-value, and T4D increased with decreasing temperature . All acids inhibited S . flexneri to some extent but to differing degrees as follows: lactic acid, acetic acid > citric acid, malic acid, tartaric acid > HCl . The T4D values for the control medium and for media containing acetic, citric, lactic, malic, and tartaric acids were 64, 47, 50, 34, 58, and 52 h, respectively, at 37 degrees C and 2,607, 1,498, 1,905, 1,346, 1,726, and 2,134 h, respectively, at 4 degrees C . The results of this study indicate that organic acids may aid in the inactivation of Shigella . However, these data also suggest that foods stored at or below room temperature containing low levels (< 1%) of acids could cause illness if contaminated with Shigella. J Bacteriol, 2002 Oct, 184(19), 5348 - 57 Three modifications in the D and T arms of tRNA influence translation in Escherichia coli and expression of virulence genes in Shigella flexneri; Urbonavicius J et al.; The modified nucleosides 2'-O-methylguanosine, present at position 18 (Gm18), 5-methyluridine, present at position 54 (m(5)U54), and pseudouridine, present at position 55 (Psi55), are located in the D and T arms of tRNAs and are close in space in the three-dimensional (3D) structure of this molecule in the bacterium Escherichia coli . The formation of these modified nucleosides is catalyzed by the products of genes trmH (Gm18), trmA (m(5)U54), and truB (Psi55) . The combination of trmH, trmA, and truB mutations resulting in lack of these three modifications reduced the growth rate, especially at high temperature . Moreover, the lack of three modified nucleotides in tRNA induced defects in the translation of certain codons, sensitivity to amino acid analog 3,4-dehydro-DL-proline, and an altered oxidation of some carbon compounds . The results are consistent with the suggestion that these modified nucleosides, two of which directly interact in the 3D structure of tRNA by forming a hydrogen bond between Psi55 and Gm18, stabilize the structure of the tRNA . Moreover, lack of Psi55 in tRNA of human pathogen Shigella flexneri leads to a reduced expression of several virulence-associated genes. Cell Motil Cytoskeleton, 2002 Oct, 53(2), 81 - 8 Actin-based phagosome motility; Zhang F et al.; Despite abundant evidence of actin's involvement at the particle internalization stage of phagocytosis, little is known about whether phagosomes undergo the same type of actin-based motility as observed with endocytic vesicles or such intracellular pathogens as Listeria and Shigella . By employing video microscopy to follow the fate of latex bead-containing phagosomes within the cytoplasm of bone marrow macrophages, we have made the novel observation of actin-based phagosome motility . Immunofluorescence microscopy confirmed that phagosomes containing IgG-opsonized, bovine serum albumin (or BSA) -coated or uncoated latex beads all formed actin-rich rocket tails that persisted only during a brief, 1-2 min period of actin-based motility . Average speeds of actin-based phagosome motility were 0.13 +/- 0.06 microm/s for IgG-coated beads, 0.14 +/- 0.04 microm/s for BSA-coated beads, and 0.11+/- 0.03 microm/s for uncoated beads . Moreover, the speeds and motile-phase duration of each type of phagosome were comparable to the behavior of pinosomes {Merrifield et al., 1999: Nat . Cell Biol . 1:72-74.} . Determination of optimal conditions for observing and analyzing actin-based phagosome motility should facilitate future investigations of phagocytosis and phagosome maturation . N Z Med J, 2002 Jun 21, 115(1156), 281 - 3 Geographically separate outbreaks of shigellosis in Auckland, New Zealand, linked by molecular subtyping to cases returning from Samoa; Hill PC et al.; AIM: To investigate simultaneous outbreaks of Shigella sonnei gastroenteritis occurring in February 2001 at a health camp for socially deprived children and an elderly care facility . METHODS: Those with symptoms were interviewed using a standardised questionnaire . Cases were defined as having at least three loose stools over a 24 hour period and stool samples requested . A case-control study investigating routes of transmission was performed at the health camp . Environmental investigations of food safety and hygiene were conducted at each facility . RESULTS: At the camp, 15 (37%) students and 15 (28%) staff met case criteria . Contact with human faeces (OR 4.0; 95% confidence interval 1.0-16.3; p = 0.05) and, for staff, eating camp food (OR 6.9; 1.0-5.0; p = 0.06) were shown to be independent risk factors for illness . At the elderly care facility, four (19%) residents and four (25%) staff met case criteria . Molecular subtyping confirmed that the outbreaks were related to each other and to other cases in travellers returning from Samoa to Auckland and other New Zealand cities over a four month period . CONCLUSION: Molecular subtyping is of considerable use in communicable disease investigation, providing strong evidence for links between outbreaks . With expanded technological capability, New Zealand could perform routine molecular subtyping of selected organisms to improve the detection and the investigation of regional and inter-regional outbreaks of infection. EMBO J, 2002 Sep 2, 21(17), 4449 - 57 Initial steps of Shigella infection depend on the cholesterol/sphingolipid raft-mediated CD44-IpaB interaction; Lafont F et al.; Shigellosis is an acute inflammatory bowel disease caused by the enteroinvasive bacterium SHIGELLA: Upon host cell-Shigella interaction, major host cell signalling responses are activated . Deciphering the initial molecular events is crucial to understanding the infectious process . We identified a molecular complex involving proteins of both the host, CD44 the hyaluronan receptor, and Shigella, the invasin IpaB, which partitions during infection within specialized membrane microdomains enriched in cholesterol and sphingolipids, called rafts . We also document accumulation of cholesterol and raft-associated proteins at Shigella entry foci . Moreover, we report that Shigella entry is impaired after cholesterol depletion using methyl-beta-cyclodextrin . Finally, we find that Shigella is less invasive in sphingosid-based lipid-deficient cell lines, demonstrating the involvement of sphingolipids . Our results show that rafts are implicated in Shigella binding and entry, suggesting that raft-associated molecular machineries are engaged in mediating the cell signalling response required for the invasion process. Rev Argent Microbiol, 2002 Apr-Jun, 34(2), 104 - 6 {Isolation of Plesiomonas shigelloides from a pediatric patient}; Turco M et al.; We report the isolation of P . shigelloides in the stools of a child with acute diarrhea living in the surroundings of Buenos Aires who usually drank untreated water . P . shigelloides has been implicated as an etiological agent in many sporadic cases of diarrhea, probably associated with drinking untreated water, eating uncooked seafood or foreign travel. Microbiology, 2002 Aug, 148(Pt 8), 2519 - 29 Enteroinvasive Escherichia coli virulence-plasmid-carried apyrase (apy) and ospB genes are organized as a bicistronic operon and are subject to differential expression; Santapaola D et al.; In Shigella flexneri and enteroinvasive Escherichia coli (EIEC) the expression of the virulence-plasmid(pINV)-carried potential pathogenesis-associated apy gene, which encodes apyrase (ATP diphosphohydrolase), is regulated by the same regulators that govern the expression of virulence genes . To understand the transcriptional organization of the apy gene, the authors sequenced an 8023 bp PstI fragment of the pINV of EIEC strain HN280, which encompasses apy as well as its adjacent genes . The PstI fragment displays 99% identity with the corresponding fragment of pWR100, the pINV of S . flexneri strain M90T, and contains four genes . One of these genes, ospB, encodes a secreted protein of unknown activity and is located immediately upstream of apy . Analyses of sequence, Northern hybridization, RT-PCR and primer extension data and transcriptional fusions indicated that ospB and apy are co-transcribed as a 2 kb bicistronic, temperature-regulated mRNA from an upstream promoter that precedes ospB . The 2 kb mRNA is post-transcriptionally processed in the intercistronic ospB-apy region, leading to the considerable accumulation of a more stable 1 kb apy-specific mRNA (half-life of 2.2+/-0.3 min, versus 27+/-4 s for the 2 kb transcript) . Upon temperature induction, peak expression of the ospB-apy operon occurs when bacteria enter into the late phases of bacterial growth, where the apy-specific transcript was found to be much more prevalent if compared to the ospB-apy transcript. J Appl Microbiol, 2002, 93(3), 479 - 86 Acid tolerance of Shigella sonnei and Shigella flexneri; Bagamboula CF et al.; AIMS: Determination of the behaviour of Shigella sonnei and Sh . flexneri under acid conditions . METHODS AND RESULTS: The growth and survival of Shigella spp . (9 isolates) in acidified Brain Heart Infusion (BHI) (pH 5.0-3.25 with pH intervals of 0.25) was determined after 6, 24 and 30 h incubation at 37 degrees C . Subsequently, survival of shigellae was studied in apple juice and tomato juice stored at 7 degrees C and 22 degrees C for up to 14 days and in strawberries and a fresh fruit salad, kept at 4 degrees C for 4 and 48 h . CONCLUSIONS: The minimum pH for growth in acidified BHI for Sh . flexneri and Sh . sonnei was, respectively, pH 4.75 and pH 4.50 . Survival in fruit juices and fresh fruits depended upon their pH, the type of strain and the incubation temperature . Shigella spp . Survived for up to 14 days in tomato juice and apple juice stored at 7 degrees C . The shortest survival time (2-8 d) was observed in apple juice at 22 degrees C . Sh . sonnei but not Sh . flexneri was recovered after 48 h from strawberries and fruit salad kept at 4 degrees C . SIGNIFICANCE AND IMPACT OF THE STUDY: Acid foods, especially if kept at refrigeration temperatures, support survival of Shigella spp . and may cause Shigella food poisoning. FEMS Microbiol Lett, 2002 Aug 6, 213(2), 257 - 64 Identification of a putative pathogenicity island in Shigella flexneri using subtractive hybridisation of the S . flexneri and Escherichia coli genomes; Walker JC et al.; The genetic differences between the human pathogen, Shigella flexneri, and the non-pathogenic Escherichia coli were investigated in an attempt to identify pathogenicity islands (PAIs) in the S . flexneri genome . Genomic subtraction identified a large unique region of DNA which was present in S . flexneri serotype 2a but absent from E . coli K-12 . This 42-kb DNA segment was localised to the S . flexneri chromosome and was found to contain a number of elements often associated with PAIs including: insertion sequence elements, bacteriophage genes, and a previously identified Shigella virulence gene (criR) . These findings indicate that this region may form a new PAI in the S . flexneri genome. Appl Environ Microbiol, 2002 Aug, 68(8), 3908 - 13 Isolation of Shigella dysenteriae type 1 and S . flexneri strains from surface waters in Bangladesh: comparative molecular analysis of environmental Shigella isolates versus clinical strains; Faruque SM et al.; Bacillary dysentery caused by Shigella species is a public health problem in developing countries including Bangladesh . Although, shigellae-contaminated food and drinks are often the source of the epidemic's spread, the possible presence of the pathogen and transmission of it through environmental waters have not been adequately examined . We analyzed surface waters collected in Dhaka, Bangladesh, for the presence of shigellae by a combination of PCR assays followed by concentration and culturing of PCR-positive samples . Analysis of 128 water samples by PCR assays for Shigella-specific virulence genes including ipaBCD, ipaH, and stx1 identified 14 (10.9%) samples which were positive for one or more of these virulence genes . Concentration of the PCR-positive samples by filtration followed by culturing identified live Shigella species in 11 of the 14 PCR-positive samples . Analysis of rRNA gene restriction patterns (ribotype) showed that the environmental isolates shared ribotypes with a collection of clinical isolates, but in contrast to the clinical isolates, 10 of the 11 environmental isolates were either negative or carried deletions in the plasmid-encoded invasion-associated genes ipaB, ipaC, and ipaD . However, all environmental Shigella isolates were positive for the chromosomal multicopy invasion-associated gene ipaH and all Shigella dysenteriae type 1 isolates were positive for the stx1 gene in addition to ipaH . This study demonstrated the presence of Shigella in the aquatic environment and dispersion of different virulence genes among these isolates which appear to constitute an environmental reservoir of Shigella-specific virulence genes . Since critical virulence genes in Shigella are carried by plasmids or mobile genetic elements, the environmental gene pool may contribute to an optimum combination of genes, causing the emergence of virulent Shigella strains which is facilitated in particular by close contact of the population with surface waters in Bangladesh. J Bacteriol, 2002 Aug, 184(16), 4409 - 19 MxiE regulates intracellular expression of factors secreted by the Shigella flexneri 2a type III secretion system; Kane CD et al.; The mxi-spa locus on the virulence plasmid of Shigella flexneri encodes components of the type III secretion system . mxiE, a gene within this locus, encodes a protein that is homologous to the AraC/XylS family of transcriptional regulators, but currently its role in pathogenesis remains undefined . We characterized the virulence phenotype of a nonpolar mxiE mutant and found that this mutant retained the ability to invade mammalian cells in tissue culture and secrete Ipas (type III effectors required for host cell invasion), although it was less efficient than wild-type Shigella at cell-to-cell spread . Despite its invasive properties in culture, the mxiE mutant was completely avirulent in an animal model . Potential targets for MxiE activation were identified by using promoter-green fluorescent protein fusions, and gene expression was examined under various growth conditions . Six MxiE-regulated genes were discovered: ospB, ospC1, ospE2, ospF, virA, and ipaH(9.8) . Notably, activation of these genes only occurred within the intracellular environment of the host and not during growth at 37 degrees C in liquid culture . Interestingly, all of the MxiE-regulated proteins previously have been shown to be secreted through the type III secretion system and are putative virulence factors . Our findings suggest that some of these Osp proteins may be involved in postinvasion events related to virulence . Since bacterial pathogens adapt to multiple environments during the course of infecting a host, we propose that Shigella evolved a mechanism to take advantage of a unique intracellular cue, which is mediated through MxiE, to express proteins when the organism reaches the eukaryotic cytosol. Emerg Infect Dis, 2002 Aug, 8(8), 862 - 4 Shigellosis linked to sex venues, Australia; O'Sullivan B et al.; From January 1 to July 31, 2000, 148 cases of Shigella infection were reported in New South Wales, Australia, compared with an annual average of 95 cases . Of reported cases, 83% were confirmed as Shigella sonnei biotype G infections; 80% were in homosexual men . Visiting a sex venue in the 2 weeks before onset of illness was the only factor significantly associated with shigellosis. Anal Biochem, 2002 Aug 1, 307(1), 131 - 7 Quadrupole ion-trap mass spectrometry to locate fatty acids on lipid A from Gram-negative bacteria; Kussak A et al.; The structure of lipid A released by mild acid hydrolysis from lipopolysaccharide from two strains of Shigella flexneri with different degrees of acylation was characterized using electrospray ionization (ESI) and ion-trap mass spectrometry . The lipid A was analyzed underivatized with ESI in negative-ion mode . With multiple stages of fragmentation (MS(n)), both the degree of acylation and the positions of the fatty acids on the disaccharide backbone could be determined . It was possible to determine the degree of acylation by the MS(n) technique, where in each MS stage the parent ion was an ion where one fatty acid had been eliminated . One way to determine the location of the fatty acids was by identifying cross-ring fragments of the reducing sugar from parent ions containing different numbers of fatty acids . Another was by identifying a possible charge-driven release of fatty acids situated close to a phosphate group . The fatty acids were otherwise eliminated by a charge-remote fragmentation mechanism . The combined data show the usefulness of ion-trap mass spectrometers for this type of analysis. Kansenshogaku Zasshi, 2002 Jun, 76(6), 466 - 9 {A fatal case of shigellosis in an elderly patient}; Imamura S et al.; A 71 year-old male, with no recent history of travelling abroad and a past history of lung cancer two years prior to presentation, which had been successfully treated, developed a sudden onset of watery diarrhea more than ten times a day on February 26, 2001, which gradually became bloody . The next day he visited the Department of Integrated Medicine of the Tokyo Metropolitan Komagome Hospital by ambulance because his consciousness was deteriorating and he was hospitalized . He was hypotensive on admission, and a dopamine preparation was used throughout . The peripheral WBC was 3,800/microliter and the lymphocyte count was 76/microliter which thus suggested the presence of cellular immune suppression . HIV was not tested . He died seven hours after admission . His stool culture yielded a growth of Shigella flexneri 2a, and a blood culture on admission was sterile . No verocyte toxin-producing Esherichia coli was not detected . The causes of death in cases with shigellosis have been reported in the literature to be an electrolyte imbalance, septicemia and disseminated intravasucular coagulation (DIC) in developed countries . Our present case was considered to be a debilitated patient complicated with hemolytic uremic syndrome due to an infection with Shigella bacteria which resulted in death despite performing intensive treatments. Tidsskr Nor Laegeforen, 2002 Jun 20, 122(16), 1568 - 72 {Health hazards for immigrants when travelling to their home countries}; Brunvatne R et al.; Vacations in the home country are important and positive events in the lives of immigrants, events that allow them to maintain contact with their culture, relatives and friends . However, vacations also carry certain health risks, though these risks can to some degree be prevented . Infectious disease is the greatest risk . Some children and adolescents also run the risk of female genital mutilation, forced marriage, and the risk og being left behind in the home country against their will . Among the notifiable diseases registered with the Norwegian Surveillance System for Communicable Diseases (MSIS), five stand out as having a higher incidence in people of foreign background than in people of Norwegian origin: malaria, hepatitis A, shigella infection, typhoid and paratyphoid fever . This higher incidence is partly the result of less use of pre-travel vaccines and malaria prophylaxis . Immigrants as a group are exposed to varied risks and should be given high priority in relation to vaccines and malaria prophylaxis for travel abroad . High priority should also be given to preventive health measures designed to reduce the risk of female genital mutilation and other violations against children and young people on visit to their country of origin. Infect Immun, 2002 Aug, 70(8), 4379 - 88 Identification of chromosomal Shigella flexneri genes induced by the eukaryotic intracellular environment; Runyen-Janecky LJ et al.; Upon entry into the eukaryotic cytosol, the facultative intracellular bacterium Shigella flexneri is exposed to an environment that may necessitate the expression of particular genes for it to survive and grow intracellularly . To identify genes that are induced in response to the intracellular environment, we screened a library containing fragments of the S . flexneri chromosome fused to a promoterless green fluorescent protein gene (gfp) . Bacteria containing promoter fusions that had a higher level of gfp expression when S . flexneri was intracellular (in Henle cells) than when S . flexneri was extracellular (in Luria-Bertani broth) were isolated by using fluorescence-activated cell sorting . Nine different genes with increased expression in Henle cells were identified . Several genes (uhpT, bioA, and lysA) were involved in metabolic processes . The uhpT gene, which encoded a sugar phosphate transporter, was the most frequently isolated gene and was induced by glucose-6-phosphate in vitro . Two of the intracellularly induced genes (pstS and phoA) encode proteins involved in phosphate acquisition and were induced by phosphate limitation in vitro . Additionally, three iron-regulated genes (sufA, sitA, and fhuA) were identified . The sufA promoter was derepressed in iron-limiting media and was also induced by oxidative stress . To determine whether intracellularly induced genes are required for survival or growth in the intracellular environment, we constructed mutations in the S . flexneri uhpT and pstS genes by allelic exchange . The uhpT mutant could not use glucose-6-phosphate as a sole carbon source in vitro but exhibited normal plaque formation on Henle cell monolayers . The pstS mutant had no apparent growth defect in low-phosphate media in vitro but formed smaller plaques on Henle cell monolayers than the parent strain . Both mutants were as effective as the parent strain in inducing apoptosis in a macrophage cell line. Epidemiol Infect, 2002 Jun, 128(3), 533 - 5 Prevalence of the genes for shigella enterotoxins 1 and 2 among clinical isolates of shigella in Israel; Yavzori M et al.; Two enterotoxins, shigella enterotoxin 1 (SHET1) and shigella enterotoxin 2 (SHET2) have been recently characterized and are believed to play a role in the clinical manifestation of shigellosis . One hundred and twenty-one isolates of Shigella spp . of 13 different serotypes and variants and 10 isolates of enteroinvasive Escherichia coli (EIEC) isolated in Israel, were examined by polymerase chain reaction for the presence of SHET1 and SHET2 genes . SHET1 was only prevalent among isolates of S . flexneri 2a while SHET2 was found in all the serotypes that were tested except for several isolates of S . flexneri 1b that lost their virulence plasmid during storage . In addition, we found that the S . flexneri 2a vaccine strain T-32 Istrati contains the gene encoding for SHET1 but not that encoding for SHET2, suggesting that the latter is located within a large deletion occurring in the 140 Mda plasmid of this S . flexneri 2a non-invasive vaccine strain. J Clin Microbiol, 2002 Jul, 40(7), 2490 - 7 Phenotypic and genotypic characterization of serologically atypical strains of Shigella flexneri type 4 isolated in Dhaka, Bangladesh; Talukder KA et al.; Twenty-one atypical Shigella flexneri type 4 strains isolated from patients attending the Dhaka treatment center of the International Centre for Diarrhoeal Disease Research, Bangladesh, were extensively characterized and compared with S . flexneri serotypes 4a and 4b . The atypical strains agglutinated only with the type antigen factor 4 and did not agglutinate with any group factors, thereby excluding their characterization into serotype 4a or 4b . Of the 21 strains, 85.7% did not ferment mannitol but were able to ferment most of the sugars, whereas the remaining 14.3% strains fermented mannitol but were unable to ferment most of the sugars . Most of the strains were resistant to ampicillin, tetracycline, and trimethoprim-sulfomethoxazole . All of the strains harbored the 140-MDa plasmid, had the ipaH gene, had the sen gene (encoding Shigella enterotoxin 2), had the ability to bind Congo red, and were positive for keratoconjunctivitis in the guinea pig eye, attesting their invasive properties . All of the strains contained a middle-range plasmid (35 to 62 MDa) as well as a number of stable small plasmids, yielding mainly two plasmid profiles which were different from those of 4a and 4b strains . Conjugation and curing experiments suggested that the middle-range plasmids harbored a self-transferable multiple antibiotic resistance marker . Pulsed-field gel electrophoresis analysis of all of the tested strains yielded two types with numerous subtypes, whereas ribotyping yielded only two types which were completely different from those of types 4a and 4b . This study concluded that two different clones of atypical S . flexneri type 4 exist and strongly suggests that these are new subserotypes of S . flexneri that await further serological classification. J Travel Med, 2002 May-Jun, 9(3), 137 - 40 Enterotoxigenic Escherichia coli as cause of diarrhea among Mexican adults and US travelers in Mexico; Bouckenooghe AR et al.; BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is the most common pathogen identified in travelers to Mexico with diarrhea . There have been few recent studies looking at the etiology of diarrhea in travelers compared with the local resident population . METHODS: We compared enteric pathogens isolated in two populations experiencing acute diarrhea acquired in Guadalajara, Mexico and also compared clinical illness caused by the principal pathogen, ETEC . RESULTS: A single and 2 enteropathogens were detected in 107 (23%) and 8 (2%), respectively, of 457 Mexicans in 1995 and 1997, and 37 (29%) and 2 (2%), respectively, of 127 US adults in 1997 . The most common pathogen was ETEC in both groups (11% of Mexican, 19% of US adults), although more common in the US travelers group (p =.0017) . Shigella spp and Cryptosporidium spp were less common in the Mexican (<1% and <1%, respectively) than in the travelers group (6% and 3%, respectively) (p <.001 and p =.002, respectively) . Entamoeba histolytica was more often found in the Mexican group (4% Mexican, 0% US adults; p =.027) . CONCLUSION: ETEC is the most common pathogen among travelers and Mexican residents in this study . The duration of untreated diarrhea due to ETEC was significantly shorter among Mexicans (49 hours in Mexican, 94 hours in US adults; p =.0004), as was the average number of unformed stools passed over 4 days (Mexicans 8.8 versus travelers 17.9 stools; p =.0009 Nippon Rinsho, 2002 Jun, 60(6), 1089 - 94 {Effect of cytokines on the expression of Shiga toxin toxicity}; Nakane A et al.; Shiga toxins(Stxs), which are produced by enterohemorrhagic Escherichia coli and Shigella dysenteriae serotype I, induce proinflammatory cytokines including tumor necrosis factor-alpha, interleukin(IL)-1 beta, IL-6, interferon-gamma, and chemokines such as IL-8 in intestinal epithelial cells, vascular endothelial cells, and monocytes/macrophages in vitro and in kidneys and spleen in vivo . Cytokines induced by Stxs and lipopolysaccharide enhance the toxicity of Stxs via up-regulation of the expression of Gb3, a Stx receptor, and infiltration of neutrophils . Stxs bind to neutrophils and transmigrate across intestinal mucosa and are transported to the target organs through bloodstreams . Stxs induce cytokines in vascular endothelial cells and peripheral blood monocytes and may injure organ tissues, finally resulting in hemolytic uremic syndrome and encephalopathia. Prep Biochem Biotechnol, 2002 May, 32(2), 143 - 55 Purification of lactoperoxidase from creek-water buffalo milk and investigation of kinetic and antibacterial properties; Ozdemir H et al.; Water buffalo lactoperoxidase (WBLP) was purified with Amberlite CG 50 H+ resin, CM Sephadex C-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography from skim milk . All purification steps of the WBLP were shown with SDS-PAGE and Rz (A412/A280) controlled the purification degree of the enzyme . Rz value for the purified WBLP was 0.8 . To determine purification steps and kinetic properties, the activity of enzyme was measured by using 2,2-azino-bis-(3-ethylbenzthiazoline-6 sulfonic acid) diammonium salt (ABTS) as a choromogenic substrate at pH=6 . Km, Vmax, optimum pH, and optimum temperature for the WBLP were found by means of graphics for ABTS as substrates . Optimum pH and optimum temperature of the WBLP were 6 and 60 degrees C, respectively . Km value at optimum pH and optimum temperature for the WBLP was 0.82 mM . Vmax value at optimum pH and optimum temperature was 13.7 micromol/mL x min . Km value at optimum pH and 25 degrees C for the WBLP was 0.77 mM . Vmax value at optimum pH and 25 degrees C was 4.83 micromol/mL x min . The purified WBLP was found to have high antibacterial activity in a thiocynate-H2O2 medium for some pathogenic bacteria, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginose, Shigella sonnei, Staphylococcus saphrophyticus, Staphylococcus epidermidis, and Shigella dysenteriae and compared with well known antibacterial substances such as tetracycline, penicillin, and netilmicine. Vet Microbiol, 2002 Jul 22, 87(4), 341 - 52 Shiga toxin genes in avian Escherichia coli; Parreira VR et al.; The purpose of this study was to determine the presence of stx genes in avian pathogenic Escherichia coli (APEC) . We examined 97 APEC isolates: 34 from lesions of avian cellulitis, 31 from avian septicemia, 13 from swollen head syndrome (SHS) in chickens, and 19 from diseased turkeys . We also examined five isolates from the feces of healthy chickens . All 102 E . coli isolates were tested for the presence of stx genes by PCR amplification and by colony blots using probes specific for stx1 and stx2 . Fifty-three percent (52) of the 97 APEC carried stx gene sequences: one isolate carried stx2 sequences, two carried both stx1 and stx2 sequences, and the remaining 49 isolates carried only stx1 sequences . Twenty-six isolates were positive by both hybridization and PCR amplification, 10 were positive by PCR only, and 16 were positive by hybridization only . All the stx-positive isolates were negative by PCR for the eae and E-hlyA genes . The five isolates from healthy chickens were all negative for stx . All 13 SHS isolates were positive for the stx1 gene and had low titres for cytotoxicity in the Vero cell assay (VCA) . Other stx-positive isolates were negative in the VCA . The stx1 gene from one SHS E . coli isolate was cloned and sequenced and shown to be identical to that of the stx gene of Shigella dysenteriae . The observations indicate that stx1 gene sequences are widespread among APEC but that cytotoxicity on Vero cells is uncommon. Cell Microbiol, 2002 Jun, 4(6), 367 - 81 Shigella flexneri regulates tight ju