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PubMed1995

Structural elucidation of the O-antigen lipopolysaccharide from two strains of Plesiomonas shigelloides that share a type-specific antigen with Shigella flexneri 6, and the common group 1 antigen with Shigella flexneri spp and Shigella dysenteriae 1.

Sugar and methylation analyses of native polysaccharides together with one-dimensional 1H- and 13C-NMR spectroscopy revealed that the two polysaccharides from strains 22074 and 12254 of Plesiomonas shigelloides are identical. The structure of the polysaccharide from strain 22074 was deduced from a uronic acid degradation and by NMR spectroscopy where heteronuclear multiple bond connectivity and two-dimensional nuclear Overhauser effect spectroscopy experiments established the pentasaccharide repeating unit as-->4)-alpha-D-GalpA-(1-->3)-alpha-D-GlcpNAc-(1-->3)-alpha-L- Rhap-(1-->2)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->.

Antigens, Bacterial↗
PubMed2002

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.

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.

Anti-Bacterial Agents↗
PubMed1975

Superiority of MacConkey's agar over salmonella-shigella agar for isolation of Shigella dysenteriae type 1.

The efficiency of MacConkey's agar in the isolation of various types of Shigella was compared with that of salmonella-shigella (SS) agar during an extensive 18-month outbreak of disease caused by Shigella. In all, 1,580 isolates of Shigella were obtained from 12,307 rectal swab and fecal samples of patients with diarrhea and their contacts by direct plating onto MacConkey's and SS agars. Shigella dysenteriae type 1 and Shigella flexneri constituted 55 percent and 33 percent of all isolates, respectively, with a smaller number of Shigella boydii and Shigella sonnei. MacConkey's agar was superior to SS agar in the detection of S. dysenteriae type 1; 83 percent of the isolates were detectable on MacConkey's agar, compared with 40 percent on SS agar. In contrast, 84 percent of S. flexneri isolates were detectable on SS agar, compared with 51 percent on MacConkey's agar alone. These findings confirm that, for the culture of fecal specimens thought to contain S. dysenteriae type 1, one of the media used should be noninhibitory. Shigella was isolated at a consistently and significantly higher rate from fecal samples than from rectal swabs.

Bacteriological Techniques↗
PubMed1993

Rapid detection of Shigella dysenteriae and Shigella flexneri in faeces by an immunomagnetic assay with monoclonal antibodies.

A rapid and sensitive method for the detection of Shigella dysenteriae type 1 and Shigella flexneri serotypes in faeces based on capture of the bacteria with immunomagnetic particles is described. The particles were coated with either of two different monoclonal antibodies specific for the O-antigens of Shigella dysenteriae type 1 and Shigella flexneri serotypes. Captured bacteria were detected by an enzyme immunoassay with O-antigen specific rabbit antiserum. The whole assay required 2 to 3 hours to perform and the sensitivity limit was 10(3) cfu/ml as determined by viable cell counting. One hundred and fifty enterobacteria strains, including 100 Shigella strains from a strain collection, and 302 fresh faecal samples were used for the study. All Shigella dysenteriae type 1 and Shigella flexneri culture-positive faecal samples were positive in the immunomagnetic assay. In addition 18 of 252 culture-negative faecal samples were positive. The immunomagnetic assay was compared with latex agglutination and indirect immunofluorescence using culture as the reference method. The immunomagnetic assay had a sensitivity of 100%, latex agglutination a sensitivity of 72% with 28% false-negative results, and indirect immunofluorescence a sensitivity of 95%. The immunomagnetic assay was superior in sensitivity since it also detected Shigella in faecal samples up to two days after antibiotic therapy had been started, which both latex agglutination and indirect immunofluorescence failed to do. The high sensitivity in detecting live and dead bacteria, and the ease of performance of the immunomagnetic assay render it an attractive method for detection of Shigella.

Animals↗
PubMed1999

Prevalence of Shigella enterotoxins 1 and 2 among Shigella strains isolated from patients with traveler's diarrhea.

Shigella spp. are known primarily as a cause of bacillary dysentery. However, in an initial phase, numerous patients exhibit watery diarrhea that may or may not be followed by dysentery. New virulence factors associated with the species of Shigella have recently been described. These are enterotoxins 1 and 2 of Shigella (ShET-1 and ShET-2, respectively). The aim of the present study was to determine the prevalence of ShET-1 and ShET-2 in species of Shigella isolated from patients with traveler's diarrhea. During the period from 1993 to 1998, stool samples from 500 travelers with diarrhea were cultured for the isolation of Shigella spp. and other enteropathogens. The detection of ShET-1 and ShET-2 was performed by a PCR technique with specific primers. Among a total of 51 strains of Shigella isolated during this period (22 S. flexneri, 26 S. sonnei, and 3 S. dysenteriae strains), at least one enterotoxin was detected in 31 (60.78%) strains; 2 (9.09%; both of which were S. flexneri strains) produced only ShET-1, while 21 (41.17%; 3 S. flexneri, 15 S. sonnei, and 3 S. dysenteriae strains) produced ShET-2. Furthermore, 8 (15.69%) of 22 S. flexneri strains presented both enterotoxins. Our results show that the prevalence of ShET-2 was high in all the Shigella species studied and confirm that ShET-1 is detected only in S. flexneri.

Bacterial Toxins↗
PubMed2006

Molecular characteristics of class 1 and class 2 integrons and their relationships to antibiotic resistance in clinical isolates of Shigella sonnei and Shigella flexneri.

OBJECTIVES: To analyse the gene cassettes and determine the roles of class 1 and class 2 integrons in antibiotic-resistant strains of Shigella sonnei (n=31) and Shigella flexneri (n=33). METHODS: Various molecular techniques, including PCR and Southern-blotting analysis, were used to analyse various markers of class 1 and class 2 integrons in these 64 S. sonnei and S. flexneri isolates collected in Hangzhou, China. The gene cassette arrays in integrons were identified by DNA sequencing and/or restriction fragment length polymorphism. Two genomic DNA fragments, one containing intI1 from a S. flexneri isolate that contains intI1 but lacks 3'-conserved region and another containing intI2 from a S. sonnei isolate, were cloned into pUC19 vectors and sequenced. The links between integron gene cassette arrays and antibiotic resistance were analysed. RESULTS: Class 2 integrons were present in 80.6% (25/31) of the S. sonnei isolates and 87.9% (29/33) of the S. flexneri isolates. All of these integron 2-positive isolates contained constant gene cassette arrays of dfrA1+sat1+aadA1 which confer resistance to trimethoprim and streptomycin. It was demonstrated that the class 2 integron was located in the Tn7 region inside the attTn7 locus downstream of glmS in Shigella. Class 1 integrons were found in 9.4% (6/64) of Shigella spp. isolates. An atypical class 1 integron without a 3'-conserved segment on the Shigella chromosome, termed Shigella atypical class 1 integron (SAI), was present in 84.9% (28/33) of S. flexneri isolates. The SAI contained two gene cassettes, bla(OXA30) and aadA1; however, the SAI conferred resistance to ampicillin, but not to streptomycin, in Escherichia coli host. The bla(OXA30) and aadA1 cassettes of the SAI seemed to be always coordinately excised or integrated. CONCLUSIONS: Multiple and complex mechanisms involving mobile genetic elements in class 1 and class 2 integrons and antibiotic resistance have been developed in the evolution of Shigella strains.

Ampicillin Resistance↗
PubMed2001

Sequence analysis of four Shigella boydii O-antigen loci: implication for Escherichia coli and Shigella relationships.

Shigella strains are in reality clones of Escherichia coli and are believed to have emerged relatively recently (G. M. Pupo, R. Lan, and P. R. Reeves, Proc. Natl. Acad. Sci. USA 97:10567-10572, 2000). There are 33 O-antigen forms in these Shigella clones, of which 12 are identical to O antigens of other E. coli strains. We sequenced O-antigen gene clusters from Shigella boydii serotypes 4, 5, 6, and 9 and also studied the O53- and O79-antigen gene clusters of E. coli, encoding O antigens identical to those of S. boydii serotype 4 and S. boydii serotype 5, respectively. In both cases the S. boydii and E. coli O-antigen gene clusters have the same genes and organization. The clusters of both S. boydii 6 and S. boydii 9 O antigens have atypical features, with a functional insertion sequence and a wzx gene located in the orientation opposite to that of all other genes in S. boydii serotype 9 and an rmlC gene located away from other rml genes in S. boydii serotype 6. Sequences of O-antigen gene clusters from another three Shigella clones have been published, and two of them also have abnormal structures, with either the entire cluster or one gene being located on a plasmid in Shigella sonnei or Shigella dysenteriae, respectively. It appears that a high proportion of clusters coding for O antigens specific to Shigella clones have atypical features, perhaps indicating recent formation of these gene clusters.

Bacterial Proteins↗
PubMed2005

Identification and characterization of Shigella boydii 20 serovar nov., a new and emerging Shigella serotype.

Analysis of 163 putative Shigella isolates from Canada and the USA showed biochemical reactions consistent with Shigella species, although none of the isolates reacted with antiserum raised against any of the well-established or provisional Shigella serotypes. All these isolates, provisionally designated serotype SH108, were positive for the ipaH gene and the invasion-associated locus. All fermented mannitol, were serologically indistinguishable from each other and showed no reaction in antisera prepared against Escherichia coli serotypes O1 to O181. PCR-RFLP analysis of the genes involved in O-antigen synthesis revealed a common pattern among these isolates that was distinct from recognized Shigella serotypes and E. coli. Between 1999 and 2003, isolates from across Canada were submitted to the National Laboratory for Enteric Pathogens for antibiotic susceptibility testing, phage typing and PFGE. These assays revealed heterogeneity among the members of this serotype. Antimicrobial susceptibility testing with seven antibiotics identified six profiles, with 90 % (45/50) of the isolates resistant to four or more antibiotics and 72 % (36/50) resistant to five or more. All isolates were typable using a panel of 16 phages, with 11 different phage types (PTs) represented. The most common PTs found were PT 3 (64 %), PT 6 (10 %) and PT 16 (6 %). Analysis of XbaI-restricted genomic DNA revealed 16 highly related patterns that were not readily distinguishable from those obtained for some other Shigella serotypes. The World Health Organization Collaborating Center for Shigella has added serotype SH108 to the Shigella scheme as S. boydii serotype 20 (serovar nov.). Strain SH108 (isolate 99-4528) is the reference strain for this serotype.

Bacterial Typing Techniques↗
PubMed2001

Attenuated Shigella flexneri 2a Delta guaBA strain CVD 1204 expressing enterotoxigenic Escherichia coli (ETEC) CS2 and CS3 fimbriae as a live mucosal vaccine against Shigella and ETEC infection.

To construct a prototype hybrid vaccine against Shigella and enterotoxigenic Escherichia coli (ETEC), the genes encoding the production of ETEC CS2 and CS3 fimbriae were isolated and expressed in attenuated Shigella flexneri 2a guaBA strain CVD 1204. The CS2 cotA to -D genes, isolated from ETEC strain C91F, and the CS3 cstA to -H genes, subcloned from plasmid pCS100, were cloned into ~15-copy-number-stabilized pGA1 behind the osmotically regulated ompC promoter, resulting in high expression of both fimbriae. Under nonselective in vitro growth conditions, pGA1-CS2 and pGA1-CS3 were stable in CVD 1204, exhibiting a plasmid loss of only approximately 1% per duplication. Expression of CS2 and CS3 reduced the invasiveness of Shigella for HeLa cells and slowed the intracellular growth rate. Guinea pigs immunized intranasally with CVD 1204(pGA1-CS2) or CVD 1204(pGA1-CS3), or with a mixture of these strains, developed secretory immunoglobulin A (IgA) in tears and serum IgG antibodies against Shigella lipopolysaccharide, CS2, and CS3 antigens. Moreover, the animals were protected against keratoconjunctivitis following conjunctival challenge with virulent S. flexneri 2a strain 2457T. Animals immunized with Shigella expressing CS2 or CS3 developed serum antibodies that agglutinated Shigella as well as an ETEC strain bearing the homologous fimbriae, whereas animals immunized with combined CVD 1204(pGA1-CS2) and CVD 1204(pGA1-CS3) developed antibodies that agglutinated all three test strains. These observations support the feasibility of a multivalent vaccine against shigellosis and ETEC diarrhea consisting of multiple Shigella live vectors expressing relevant ETEC antigens.

Animals↗
PubMed1992

Concentration of milk secretory immunoglobulin A against Shigella virulence plasmid-associated antigens as a predictor of symptom status in Shigella-infected breast-fed infants.

We conducted a prospective, community-based study of healthy breast-fed Mexican infants to determine the protective effects of anti-Shigella secretory IgA antibodies in milk. Milk samples were collected monthly, and stool culture specimens were obtained weekly and at the time of episodes of diarrhea. Nineteen breast-fed infants were found to have Shigella flexneri, Shigella boydii, or Shigella sonnei in stool samples. Ages of the 10 infants with symptomatic infection and the nine with asymptomatic infection did not differ significantly. Milk samples collected up to 12 weeks before infection were evaluated by enzyme-linked immunosorbent assay for secretory IgA antibodies against lipopolysaccharides of S. flexneri, S. boydii serotype 2, S. sonnei, and virulence plasmid-associated antigens. The geometric mean titers of anti-Shigella antibodies to virulence plasmid-associated antigens in milk received before infection were eightfold higher in infants who remained well than in those in whom diarrhea developed. The significance of milk secretory IgA directed against lipopolysaccharide was less clear. We conclude that human milk protects infants against symptomatic shigella infection when it contains high concentrations of secretory IgA against virulence plasmid-associated antigens.

Antigens, Bacterial↗
PubMed2002

Acid tolerance of Shigella sonnei and Shigella flexneri.

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.

Beverages↗
PubMed1992

Detection of Shigella dysenteriae type 1 and Shigella flexneri in feces by immunomagnetic isolation and polymerase chain reaction.

A combination of immunomagnetic separation (IMS) and a polymerase chain reaction (PCR) procedure was used for direct isolation and identification of Shigella dysenteriae type 1 and Shigella flexneri from feces. Immunomagnetic particles were coated with monoclonal antibody MASFB, which is specific for a common epitope of the O polysaccharides of S. dysenteriae type 1 and S. flexneri. Bacteria bound to the beads were boiled in water, and target DNA was amplified with a primer pair specific for a gene coded on the invasion-associated locus (ial) of the large virulence plasmid of all four Shigella spp. and enteroinvasive strains of Escherichia coli. A 320-bp DNA fragment was generated and detected by an alkaline phosphatase-conjugated probe. Nonviable cells were also captured and detected by this technique. The method is simple and fast (7 h) and has a detection limit of ca. 10 Shigella organisms per g in fecal samples. The combined IMS-PCR assay correctly identified all 57 samples carrying S. dysenteriae type 1 and 68 samples carrying S. flexneri from 238 fecal specimens and also permitted detection of 17 samples carrying Shigella spp. from 113 specimens from diarrheal patients in whom shigellae were not detected by conventional culture.

Antibodies, Monoclonal↗
PubMed1978

Immunochemical characteristics of Shigella sonnei and serotype 6 Shigella flexneri lipopolysaccharides and enterobacterial common antigen.

Immunochemical studies on Shigella sonnei and serotype 6 Shigella flexneri 0 antigens (lipopolysaccharides) and enterobacterial common antigen (ECA) isolated from Shigella sonnei were carried out. Oligosaccharide structure of 0-specific chain of serotype 6 Shigella fiexneri lipopolysaccharide was defined and beta-L-rhamnosyl-1,3-N-acetyl-D-galactosamin as immunodeterminant of type VI specificity was recognized. The structures of core regions of lipopolysaccharides isolated from R mutants of both Shigella subgroups were established. On the base of the serological and structural results it has been suggested that these core regions are identical and very close to RI core structure of E. coli C. The effective method of isolation and purification of enterobacterial common antigen from Shigella sonnei was elaborated and its immunological properties as well as chemical character defined.

Antigens, Bacterial↗
PubMed2006

Distribution of Shigella enterotoxin genes and secreted autotransporter toxin gene among diverse species and serotypes of shigella isolated from Andaman Islands, India.

We studied the prevalence and distribution of the newly described genes for Shigella enterotoxins (ShET1 and ShET2, encoded by set and sen genes) and secreted auto-transporter toxin (encoded by sat gene) in clinical isolates from the Andaman Islands, India. A total of 153 Shigella isolates obtained from hospitalized patients during 1994-2004 were analysed. These isolates included all the four species of Shigella (S. dyseteriae-29, S. flexneri-75, S. sonnei-38, S. boydii-5) that belonged to diverse serotypes (including serologically untypable-6) and each serotype included a wide variety of genotypes. Each isolate underwent polymerase chain reaction (PCR) for detection of set, sen and sat genes employing specific primers. We found the set gene in all S. flexneri 2a and 2b isolates (41 of 41, 100%) but not outside S. flexneri serotype 2. The sen gene was well distributed among all species and serotypes but its presence was apparently low at 49.1% (75 of 153), probably because of the loss of the large plasmid that harbours the gene in 76 of the 78 (97.4%) sen negative isolates. Also, all S. flexneri 2 isolates (including 2a and 2b serotypes) had the sat gene. It was present in 96% (72 of 75) of S. flexneri, in 6.9% (2 of 29) of S. dysenteriae, in 20% (1 of 5) of S. boydii, and in 33.3% (2 of 6) of untypable Shigella, but not in (0 of 38) S. sonnei. This study provides initial data on the prevalence and distribution of of the set, sen and sat genes in a wide variety of Shigella isolated over a 10-year period. Our results suggest a greater prevalence of the set and sat genes in S. flexneri 2 isolates than previously thought and might help in future pathochip designs.

Bacterial Toxins↗
PubMed2003

Safety and immunogenicity of Shigella sonnei-CRM9 and Shigella flexneri type 2a-rEPAsucc conjugate vaccines in one- to four-year-old children.

BACKGROUND AND OBJECTIVE: Shigella conjugate vaccines have been shown to be safe, immunogenic and efficacious in adult volunteers. We have now investigated the safety and immunogenicity of investigational Shigella sonnei and Shigella flexneri 2a conjugate vaccines in 1- to 4-year-old children, the age group at greatest risk for shigellosis. METHODS: The O-specific polysaccharides of S. sonnei and S. flexneri 2a, the two most common shigellae from patients in Israel, were bound to medically useful carrier proteins to form conjugates. Eighty healthy 1- to 4-year-olds were randomized to receive two 0.5-ml im injections 6 weeks apart of either S. sonnei-CRM(9) or S. flexneri 2a-rEPA(succ). Blood was taken before, 6 weeks after the first injection, 4 weeks after the second injections and 2 years after immunization for assay of IgG anti-lipopolysaccharide, diphtheria toxin and Pseudomonas aeruginosa exotoxin A antibodies by enzyme-linked immunosorbent assay. RESULTS: During an 8-day surveillance period after each immunization, low fever (37.8-39.0 degrees C) lasting only 24 to 48 h occurred in 2 of 40 recipients after the first injection and 4 of 40 recipients after the second injection of S. flexneri 2a-rEPA(succ) and in 2 of 38 of S. sonnei-CRM(9) after the second injection; no fever was detected after the first injection. Liver function tests were normal in all vaccinees. S. sonnei-CRM(9) elicited a >4-fold rise in IgG anti-LPS in 92.1% and S. flexneri 2a-rEPA(succ) in 85% (P < 0.0001) after the second injection; both conjugates elicited type-specific booster responses. At 2 years the geometric mean concentrations of both IgG anti-lipopolysaccharides were significantly higher than preimmunization levels. A >4-fold rise of IgG anti-diphtheria (65.8%) and IgG anti-ETA (77.5%) was observed. CONCLUSION: These experimental Shigella conjugate vaccines were safe and immunogenic in 1- to 4-year-old children.

Age Factors↗
PubMed1990

Therapeutic value of Gramurin (oxolinic acid) in Shigella infections and Shigella-negative dysentery syndrome.

A total of 10 Shigella-positive and 10 Shigella-negative dysentery syndrome patients were treated with daily 2-g Gramurin doses for 3 days. Favourable clinical results could be obtained in response to the therapy. The complaints of the patients ceased within 48 hours. Bacterial negativity was recorded on days 3, 4, and 5 after cessation of therapy in Shigella flexneri-positive cases (5 cases), while in case of Shigella sonnei positivity bacterium negativity could be obtained within the abovementioned period in only 3 out of the 5 cases. The presented results of Gramurin therapy in Shigella infections and Shigella-negative dysentery syndrome are virtually congruent with the earlier reported favourable observations of the authors with the drug in these conditions.

Adolescent↗