Shigella Diversity and Changing Landscape: Insights for the Twenty-First Century.

Shigella is a pathovar of Escherichia coli comprising four groups, Shigella flexneri, Shigella sonnei, Shigella dysenteriae, and Shigella boydii, each of them, with the exception of S.sonnei, comprising several serotypes. Shigella accounts for the majority of dysentery causing infections occurring world-wide each year. Recent advancements in the Shigella field have led to a better understanding of the molecular mechanisms underlying host epithelial cell invasion and immune cell function manipulation, mainly using S. flexneri as a model. Host-cell invasion is the final step of the infection process, as Shigella's virulence strategy relies also on its ability to survive hostile conditions during its journey through the gastro-intestinal tract, to compete with the host microbiota and to cross the intestinal mucus layer. Hence, the diversity of the virulence strategies among the different Shigella species has not yet been deeply investigated, which might be an important step to understand the epidemiological spreading of Shigella species worldwide and a key aspect for the validation of novel vaccine candidates. The recent development of high-throughput screening and sequencing methods will facilitate these complex comparison studies. In this review we discuss several of the major avenues that the Shigella research field has taken over the past few years and hopefully gain some insights into the questions that remain surrounding this important human pathogen.

Análisis del patrón epidemiológico de la shigelosis en Barcelona entre 1988 y 2012: ¿es una infección de transmisión sexual emergente? / Analysis of the epidemiological pattern of Shigellosis in Barcelona between 1988 and 2012: is it an emerging sexually transmitted infection?

INTRODUCCIÓN: El objetivo de este estudio fue describir la evolución y las características epidemiológicas de los pacientes con shigelosis durante 25 años en una gran ciudad. MÉTODOS: La shigelosis es una enfermedad de declaración obligatoria en España desde 1988. Se analizan los casos de residentes en Barcelona incluidos en el registro entre 1988-2012. Se presenta un análisis descriptivo según sexo, edad, vía de transmisión y especies de Shigella. Se realizó un análisis de tendencias y de series temporales. RESULTADOS: De los 559 casos analizados, el 60,15% correspondían a hombres. Se observó un incremento sostenido de la tendencia en hombres desde 2008 (p < 0,05), sobre todo a expensas de los de hombres que no tenían antecedentes de toXIInfección alimentaria ni de viajes a zonas endémicas. El incremento de la tendencia fue mayor en hombres de 21 a 60 años, tanto para S. flexneri (desde 2009) como para S. sonnei (desde 2003). En 2012 se observó que, en los hombres con S. flexneri, el 63% tenían sexo con hombres. CONCLUSIONES: Se detectó un incremento de la tendencia en los casos en hombres que no tenían antecedentes de toXIInfección alimentaria ni de viajes a zonas endémicas. Este incremento apunta a un cambio en el patrón de la shigelosis, pasando a ser predominantemente masculina, y cuyo mecanismo principal serían las relaciones sexuales

INTRODUCTION: The aim of the study was to analyze the incidence, management and cost associated to hematological and dermatological adverse effects (AE) in chronic hepatitis C patients on triple therapy (TT) with telaprevir (TVR) or boceprevir (BOC). METHODS: An analysis was made on the data recorded on patients who started treatment with TVR or BOC associated with peginterferon alfa and ribavirin in a 12-week follow-up period. RESULTS: Fifty-three patients were included (TVR n = 36; BOC n = 17). Thrombocytopenia (83% TVR vs. 88% BOC) followed by neutropenia (89% TVR vs. 82% BOC) were the most common AE. Dermatological AE were observed in 32% of patients. Eleven patients required treatment discontinuation (all of them received TVR), and toxicity was the main reason for discontinuation (64%). The percentage of patients who required supportive treatment for management of AE was 66%. The most used supportive treatment was erythropoietin. Eight patients required emergency health care, and 2 were hospitalized due to AE. Total cost of additional supportive resources was 32,522 Euros (625 [SD = 876] Euros/patient) (TVR 759 [SD = 1,022] Euros/patient vs. BOC 349 [SD = 327] Euros/patient; P > .05). Patients with grade iii-iv toxicity required greater supportive care with higher costs, compared to patients with grade i-ii toxicity (849 [SD = 1,143] Euros/patient vs. 387 [SD = 397] Euros/patient; P = .053). CONCLUSION: The addition of new protease inhibitors to conventional treatment leads to a higher incidence of hematological AE in our study, compared to data described in clinical trials. The elevated incidence of AE involves the use of supportive care, increasing total costs of therapy

Studies on Shigella boydii infection in Caenorhabditis elegans and bioinformatics analysis of immune regulatory protein interactions.

Shigella boydii causes bacillary dysentery or shigellosis and generates a significant burden in the developing nations. S. boydii-mediated infection assays were performed at both physiological and molecular levels using Caenorhabditis elegans as a host. Continuous exposure of worms to S. boydii showed a reduced life span indicating the pathogenicity of Shigella. Quantitative Real-Time PCR analysis was performed to analyze the expression and regulation of host specific candidate-antimicrobial genes (clec-60, clec-87, lys-7), which were expressed significantly during early infection, but weakened during the latter hours. Increased mortality of mutant RB1285 by S. boydii and Shigella flexneri indicated the role of lys-7 during Shigella infection. Protein-protein interactions (PPIs) database was used to analyze the interaction of immune proteins in both C. elegans and humans. In addition, the expression and regulation were revealed about immune genes (clec-61, clec-62, clec-63, F54D5.3 and ZK1320.2), which encode several intermediate immune protein partners (CLEC-61, CLEC-62, CLEC-63, F54D5.3, ZK1320.2, W03D2.6 and THN-2) that interact with LYS-7 and CLEC-60 and were found to play a role in C. elegans immune defense against S. boydii infections. Similarly, the immune genes that are specific to the human defense system, which encode IGHV4-39, A2M, LTF, and CD79A, were predicted to be expressed with LYZ and MBL2, thus indicating their regulation during Shigella infections. Our results using the lowest eukaryotic model system and human database indicated that the major players involved in immunity-related processes appear to be common in cases of Shigella sp. mediated immune responses. This article is part of a Special Issue entitled: Computational Methods for Protein Interaction and Structural Prediction.

The type III effectors NleE and NleB from enteropathogenic E. coli and OspZ from Shigella block nuclear translocation of NF-kappaB p65.

Many bacterial pathogens utilize a type III secretion system to deliver multiple effector proteins into host cells. Here we found that the type III effectors, NleE from enteropathogenic E. coli (EPEC) and OspZ from Shigella, blocked translocation of the p65 subunit of the transcription factor, NF-kappaB, to the host cell nucleus. NF-kappaB inhibition by NleE was associated with decreased IL-8 expression in EPEC-infected intestinal epithelial cells. Ectopically expressed NleE also blocked nuclear translocation of p65 and c-Rel, but not p50 or STAT1/2. NleE homologues from other attaching and effacing pathogens as well OspZ from Shigella flexneri 6 and Shigella boydii, also inhibited NF-kappaB activation and p65 nuclear import; however, a truncated form of OspZ from S. flexneri 2a that carries a 36 amino acid deletion at the C-terminus had no inhibitory activity. We determined that the C-termini of NleE and full length OspZ were functionally interchangeable and identified a six amino acid motif, IDSY(M/I)K, that was important for both NleE- and OspZ-mediated inhibition of NF-kappaB activity. We also established that NleB, encoded directly upstream from NleE, suppressed NF-kappaB activation. Whereas NleE inhibited both TNFalpha and IL-1beta stimulated p65 nuclear translocation and IkappaB degradation, NleB inhibited the TNFalpha pathway only. Neither NleE nor NleB inhibited AP-1 activation, suggesting that the modulatory activity of the effectors was specific for NF-kappaB signaling. Overall our data show that EPEC and Shigella have evolved similar T3SS-dependent means to manipulate host inflammatory pathways by interfering with the activation of selected host transcriptional regulators.

Identification of a group of shigella-like isolates as Shigella boydii 20.

Infections by Shigella species are an important cause of diarrhoeal disease worldwide. Of 4198 Shigella isolates received by the French National Reference Centre for Escherichia coli and Shigella, 180 from patients with diarrhoea and dysentery in 2000-2004 did not react with any available polyclonal rabbit antisera used to identify the established Shigella serogroups. This study describes the molecular and phenotypic characteristics of these isolates in seroagglutination tests, molecular serotyping (rfb-RFLP and fliC-RFLP), ribotyping, detection of invasivity and enterotoxins genes, and antibiotic sensitivity. All isolates gave biochemical reactions typical of Shigella boydii, were mannitol-positive and indole-negative. They all carried invasion-associated genes, enterotoxin 2 [ShET-2] and an IS630 sequence. They had a unique ribotype that was distinct from all other Shigella and E. coli patterns. Further characterization by rfb-RFLP clearly distinguished this serogroup from all other Shigella or E. coli O-groups. The fliC-RFLP pattern corresponded to P4, an F-pattern which is associated with 10 different serogroups of S. boydii. A new antiserum prepared against strain 00-977 agglutinated all 180 isolates and cross-agglutination and absorption studies with anti-00-977 serum and anti-CDC 99-4528 (reference for the newly described S. boydii serogroup 20) serum showed identical antigenic structure. Furthermore, strains 00-977 and CDC 99-4528 had the same molecular serotype, ribotype and virulence genes.

Structural and molecular characterization of Shigella boydii type 16 O antigen.

Shigella is a well-known human pathogen causing dysentery and their typing is solely based on the O antigens. We investigated the chemical structure and gene cluster of Shigella boydii type 16 O antigen. As judged by sugar and methylation analyses along with NMR spectroscopy data, the O antigen has an O-acetylated branched pentasaccharide repeating O unit, which consists of two D-mannose residues (D-Man), one residue each of d-glucuronic acid (D-GlcA), N-acetylglucosamine (D-GlcNAc) and D-galactose (D-Gal), and the structure of the O unit was established. The O antigen gene cluster of S. boydii type 16 was identified and shown to contain putative genes for the synthesis of GDP-D-Man, genes encoding sugar transferases, O unit flippase (Wzx) and O antigen polymerase (Wzy) as expected. The function of the wzy gene was characterized by mutation test. Genes specific to S. boydii type 16 O antigen gene cluster were identified by screening 186 Escherichia coli and Shigella type strains, and can be used to develop PCR assays for detection of type 16 strains.

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.

Isolation and characterization of provisional serovar Shigella boydii E16553 from diarrhoeal patients in Bangladesh.

In previous studies with strains of the Shigella dysenteriae provisional serovars E22383 and E23507 from diarrhoeal stools from patients in Bangladesh, two strains of Shigella species were identified as Shigella boydii provisional serovar E16553 by a reference laboratory. Further tests with an antiserum to an international type strain of the provisional serovar E16553 identified an additional 15 isolates. None of the isolates reacted with antisera to the established Shigella serovars or any other provisional serovars reported so far and all showed biochemical reactions typical of S. boydii. All of the isolates harboured the 140 MDa invasion plasmid, had the ipaH gene and produced keratoconjunctivitis in the guinea pig eye. All isolates were susceptible to ampicillin, sulfamethoxazole-trimethoprim, nalidixic acid, ciprofloxacin and mecillinam but eight strains were resistant to tetracycline. A single PFGE type (type A) was shown for all 17 clinical isolates, indicating a common source of origin. The pulsotype of the Bangladeshi isolates was closely related to that of a Japanese strain but was different from that of the type strain. On the basis of these biochemical, serological and virulence markers, and diverse geographical origin, it is recommended that the provisional status of serovar E16553 be changed and that it be included in the international serotyping classification scheme as S. boydii 19.

Correlation between Congo red binding and contact haemolysin production in Shigella species.

Haemolytic strains of Shigella dysenteriae type 1, Shigella flexneri, Shigella boydii and Shigella sonnei cultured on Congo red agar produced pigmented colonies (Pcr+) whereas nonhaemolytic strains produced white colonies and did not bind Congo red (Pcr-). S. flexneri-1 haemolysin negative mutant (lacking plasmid) of haemolysin positive prototroph also did not bind Congo red and produced nonpigmented colonies. Among the twelve strains of Shigella included in this study, the characteristics of Congo red binding, plasmid profile and haemolytic activity appeared to be correlated. Congo red binding occurred comparatively more by haemolysin-producing strains. Congo red binding can be used as a quick and reliable method for virulence traits of pathogens, including haemolysin activity.

Shigella em alimentos / Shigella in the foods

Comenta-se nesta breve revisao a participacao e o significado de Shigella em processos de infeccao alimentar. Sao tambem abordados as caracteristicas do microrganismo, seus fatores de virulencia e determinancia genetica.Aspectos epidemiologicos da infeccao, bem como, condicoes de crescimento e metodos de deteccao em alimentos, sao tambem abordados

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.

Characterization of Aeromonas caviae antigens which cross-react with Shigella boydii 5.

Live and boiled cells of 16 strains of Aeromonas caviae, isolated from patients with diarrhea, agglutinated with Shigella boydii 5 antiserum in a slide test. Further studies with seven selected strains showed agglutination with boiled cells in a tube test. Lipopolysaccharide antigen extracted from one of these strains cross-reacted with S. boydii 5 in enzyme-linked immunosorbent assay and immunoblot studies. Either all or the majority of the seven strains possessed properties deemed to be diarrheagenic.

[A new serotype of Shigella boydii].

Two strains which belong to the same serotype of Shigella were isolated from the bloody-pus stool of two patients (in 1986) and is reported in this paper. The results were identical both showing agglutination in low titer with serotype 8 of S. dysenteriae and serotype 4 of S. boydii when the two strains were checked well with all kinds of diagnostic antisera and vice versa, ie the antisera produced by the two strains were also checked well with sera prepared with the representative strains of all Shigella spp. No cross agglutination with O6, O7, and O150 of E. coli were found. Consequently, It appears to be a new serotype of Shigella. These two strains possess the ability of causing keratitis in guinea-pigs as well as invading epithelial cells, the DNA of both strains in agarose-electrophoresis showed a large plasmid, indicating that they are virulent strains possessing invasive ability. It was concluded that these two strains belonged to Shigella boydii as they fermented mannitol and non-related antigenically with Shigella flexneri. Since serotype 1-18 of S. boydii have been reported recently, we propose that this new serotype should be serotype 19 of Shigella boydii.

Congo red binding and salt aggregation as indicators of virulence in Shigella species.

Smooth strains of Shigella dysenteriae type 1, Shigella flexneri, Shigella boydii, and Shigella sonnei which form pigmented colonies (Pcr+) on Congo red agar were virulent in the Sereny test. Smooth variants unable to bind Congo red (Pcr-) were avirulent. Measurements of dye uptake from solution showed that S. dysenteriae type 1 bound the most dye, followed in order of uptake by S. flexneri, S. boydii, and S. sonnei. Using the salt aggregation test (SAT) to determine cell surface hydrophobicity, we found the same order of species. The SAT could not, however, detect differences in surface properties between Pcr+ and Pcr- pairs of isogenic smooth strains. Enteroinvasive Escherichia coli strains used in the study showed SAT and Congo red-binding properties which were similar to those of the S. flexneri strains. A direct correlation was found between pigment-binding ability and the presence of the large 140-megadalton plasmid in S. flexneri, enteroinvasive E. coli, and S. boydii but not in S. dysenteriae type 1 or S. sonnei strains. Congo red interacted with outer membranes and outer membrane proteins of S. dysenteriae type 1 but not with lipopolysaccharides. However, rough mutants of Shigella species deficient in lipopolysaccharides bound Congo red and formed pigmented colonies, showing that dye binding as a virulence assay may be misinterpreted in such cases. There was complete correlation of the Pcr+ phenotype with virulence in the smooth strains in this study, suggesting that Congo red binding can be utilized as a quick and reliable alternative to the Sereny test.

Large plasmid in Shigella boydii is responsible for epithelial cell penetration.

A large plasmid in a virulent Shigella boydii 5 strain was transferred to plasmid-cured avirulent strains of S. boydii 5, S. boydii 12, S. sonnei form II, and Escherichia coli K12. The transconjugants acquired the ability to invade tissue culture cells, which indicated that the large plasmid in S. boydii is responsible for epithelial cell invasiveness.

Comparison of DNA probes and the Sereny test for identification of invasive Shigella and Escherichia coli strains.

Forty-two Shigella and 29 Escherichia coli strains were screened for invasiveness in the Sereny test and for hybridization with two recently described DNA probes for the invasiveness plasmid. Both probes produced identical results. All Sereny-positive strains hybridized with both DNA probes. Three Sereny-negative strains also hybridized with the probes, suggesting that there are strains containing the invasiveness plasmid that are not pathogenic in animal models.

[The cathode bound group antigen of dysentery-provoking escherichieae (author's transl)]. / Das kathodisch wandernde Gruppenantigen ruhrerregender Escherichieen

Antigens from disrupted cells of dysentery-provoking and of non-enteropathogenic Escherichieae were submitted to immunoelectrophoresis on cellulose acetate stripes at pH 8.0. Among 6 immune sera produced for this purpose by immunizing rabbits against desintegrated dysentery bacteria, only one contained a precipitine reacting with an antigen similar to the "generic antigen" of BELAYA. This - at pH 8.0 - cathode-bound group antigen (KGA) could not only be found in virulent but also in 5 attenuated cultures and in 5 from 6 avirulent strains of several dysentery types. Only the - apathogenic - type culture 1111/55 of dysentery-provoking E. coli O 136 showed no KGA-reaction. Some sources of methodical errors responsible for false outcomes of immunopherogrammes have been discussed.