Reverse vaccinology approach for the identification and characterization of outer membrane proteins of Shigella flexneri as potential cellular- and antibody-dependent vaccine candidates

Bacteriemia por Shigella flexneri en dos lactantes / Shigella flexneri bacteremia in children: two case report and review of literature

Resumen Shigella spp. es el aislamiento microbiológico más frecuente en las diarreas bacterianas en Argentina. Clínicamente puede causar desde una diarrea acuosa hasta disentería. En forma infrecuente causa complicaciones extraintestinales, con una incidencia de bacteriemia desde 0,4 a 7,3%; asociado a factores de riesgo como niños menores de un año de edad e inmunodeficiencias, entre otros. Presentamos los casos clínicos de dos lactantes con bacteriemia por Shigella flexneri, que consultaron por fiebre y diarrea, uno de ellos con diagnóstico de inmunodeficiencia primaria.

Shigella spp. is the most frequent micro-biological isolation in bacterial diarrhea in Argentina. It causes a watery diarrhea or dysenteric disease. It rarely causes extraintestinal problems. It has an incidence of bacteremia of 0,4-7,3%, and its appearance compels us to look for associated risk factors, as children under one year of age and immunodeficiency, among others. We describe two children with Shigella flexneri bacteremia. They presented with fever and diarrhea. One of them had primary immune deficiency.

The influence of glycerol upon L. reuteri activity against enteropathogens / A influência do glicerol sobre a atividade de L. Reuteri contra enteropatógenos

BACKGROUND: Probiotics containing L. reuteri are popular for treating and preventing bacterial gastrointestinal infections. L. reuteri, produces reuterin, an antibiotic that inhibits gram-negative bacteria. Reuterin production is the result of glycerol fermentation by L reuteri. Although L. reuteri is normally present in the gastrointestinal system, only small amounts of glycerol are usually available; therefore, the production of reuterin may not occur and this could reduce the effectiveness of the probiotic supplement. Our objective is to identify the minimum concentrations of glycerol required for L. reuteri to exert an inhibitory effect on enteropathogenic enterobacteriaceae. METHOD: Samples containing 10(8) colony forming units (CFU) of L. reuteri DSM17938 (Colikids®, Ache, Brazil/BioGaia, Sweden) were grown with varying concentrations of glycerol (0.05-5%). 10(6) CFU of E.coli (CDC0126/INCQS/FIOCRUZ), Shigella flexneri (ATCC/120022), S. enterica (ATCC6539) and Y. enterocolitica (ATCC9610) were inoculated with L. reuteri in the different glycerol concentrations. Each enterobacteria and glycerol 5% without L. reuteri cultures were used as positive control groups. RESULTS: All bacteria were completely inhibited at higher ranges of glycerol concentrations (0.2-5%) and grew at lower concentrations (0.05-0.1%). CONCLUSION: L. reuteri requires at least 0.2% of glycerol to completely inhibit enterobacterial growth. These preliminary findings may influence the current method of use of probiotic supplements. The antibiotic activity of L. reuteri may have potential clinical use against important enteropathogens.

CONTEXTO: Os probióticos que contêm L. reuteri são populares para tratar e prevenir infecções bacterianas do trato gastrointestinal. L. reuteri produz reuterina, um antibiótico que inibe bactérias gram-negativas. A produção de reuterina depende da presença de quantidades adequadas de glicerol, cuja fermentação resulta na produção do antibiótico. Embora L. reuteri esteja normalmente presente no sistema GI, apenas pequenas quantidades de glicerol estão geralmente disponíveis; portanto, a produção de reuterina pode não ocorrer o que poderia reduzir a eficácia do suplemento probiótico. Nosso objetivo é identificar as concentrações mínimas de glicerol necessárias para que L. reuteri exerça um efeito inibitório nas enterobacteriaceas enteropatogênicas. MÉTODO: 108 CFU de L. reuteri DSM17938 (Colikids®, Ache, Brasil / BioGaia, Suécia) cresceu com concentrações variáveis de glicerol (0,05-5%). Foram inoculadas 106 UFC de E. coli (CDC0126 / INCQS / FIOCRUZ), Shigella flexneri (ATCC / 120022), S. enterica (ATCC6539) e Y. enterocolitica (ATCC9610) com L. reuteri nas diferentes concentrações de glicerol. Cada enterobacteria e glicerol 5% sem culturas de L. reuteri foram utilizadas como grupos de controle positivo. RESULTADOS: Todas as bactérias foram completamente inibidas em maiores concentrações de glicerol (0.2-5%) e cresceram em concentrações mais baixas (0.05-0.1%). CONCLUSÃO: L. reuteri requer pelo menos 0,2% de glicerol para inibir completamente o crescimento de enterobacteria. Essas descobertas preliminares podem influenciar o método atual de uso de suplementos de probióticos. A atividade antibiótica de L. reuteri pode ter potencial uso clínico contra importantes enteropatógenos.

Genome Sequencing Analysis of Atypical Shigella flexneri Isolated in Korea

OBJECTIVES: An atypical Shigella flexneri strain with a plural agglutination pattern [i.e., reacting not only with serum samples containing type antigen II but also with serum samples containing group antigens (3)4 and 7(8)] was selected for genome sequencing, with the aim of obtaining additional comparative information about such strains. METHODS: The genomic DNA of atypical S. flexneri strain NCCP 15744 was sequenced using an Ion Torrent PGM sequencing machine (Life Technologies, USA). The raw sequence data were preprocessed and reference-assembled in the CLC Assembly Cell software (version 4.0.6; CLC bio, USA). RESULTS: Ion Torrent sequencing produced 1,450,025 single reads with an average length of 144 bp, totaling ~209 Mbp. The NCCP 15744 genome is composed of one chromosome and four plasmids and contains a gtrX gene. Among the published genome sequences of S. flexneri strains, including 2457T, Sf301, and 2002017, strain NCCP 15744 showed high similarity with strain 2002017. The differences between NCCP 15744 and 2002017 are as follows: i) NCCP 15744 carries four plasmids whereas 2002017 carries five; ii) 19 genes (including CI, CII, and cro) were lost in the SHI-O genomic island of NCCP 15744 and six genes were gained as compared with strain 2002017. CONCLUSION: Strain NCCP 15744 is genetically similar to 2002017, but these two strains have different multilocus sequence types and serotypes. The exact reason is unclear, but the 19 lost genes may be responsible for the atypical seroconversion of strain NCCP 15744.

Characterization of Extended-Spectrum β-Lactamase Genes of Shigella flexneri Isolates With Fosfomycin Resistance From Patients in China

BACKGROUND: The emergence of fosfomycin resistance and extended-spectrum β-lactamase (ESBL) genes is a serious threat to public health and a new challenge in shigellosis treatment. The purpose of this study was to identify fosfomycin resistance and characterize β-lactamase genes in fos-carrying isolates of Shigella flexneri from patients in China. METHODS: A total of 263 S. flexneri isolates were collected from 34 hospitals in the Anhui Province of China during September 2012-September 2015 and screened for fosA3, fosA, and fosC2 by PCR amplification and sequencing. The fos-carrying isolates were then screened for β-lactamase genes. The clonal relationships between fosA3-carrying isolates, the transmissibility of fosfomycin resistance, replicon types of plasmids carrying fosfomycin resistance genes and other associated resistance genes were investigated. RESULTS: Twenty-five of the 263 isolates (9.5%) showed resistance to fosfomycin, and 18 (6.8%) were positive for fosA3. None of the isolates was positive for fosA or fosC2. Seventeen of the isolates carrying fosA3 (94%) were CTX-M producers (seven CTX-M-55, five CTX-M-14, and five CTX-M-123), while three (16.7%) were TEM producers (TEM-1).Sixteen (88.9%) fosA3-carrying isolates exhibited multi-drug resistance. The replicon types of the 13 fosA3-carrying plasmids were IncF (n=13), IncHI2 (n=3), IncIl-Ir (n=2), and IncN (n=1). CONCLUSIONS: Our results indicated that fosA3 could spread through plasmids in S. flexneri isolates, along with the bla(CTX-M) and bla(TEM), which facilitate its quick dispersal. To the best of our knowledge, this is the first report of CTX-M-123-type ESBLs in S. flexneri isolates from patients in China.

Processo autofágico no reconhecimento de Escherichia coli enteroinvasora: um possível mecanismo da degradação bacteriana por células epiteliais / Autophagy in recognition of Enteroinvasive Escherichia coli: a possible bacterial degradation mechanism by epithelial cells

O reconhecimento de bactérias invasoras pelas células hospedeiras através do processo autofágico é um fator chave na determinação da infecção bacteriana. Escherichia coli enteroinvasora (EIEC) possui uma proteína, denominada IcsB, que em estudos em Shigella, é responsável pela inativação deste processo de degradação bacteriana. Uma vez que EIEC expressa menos IcsB do que S. flexneri, nos propusemos a investigar o processo autofágico na infecção por EIEC, utilizando as técnicas de mutação gênica por inserção, western-blot, microscopia de fluorescência e eletrônica de transmissão e microarray. Verificamos que a proteína IcsB é um fator de virulência importante na camuflagem de EIEC, pois quando pouco ou nada expresso, há um maior reconhecimento da bactéria pelas células hospedeiras, favorecendo sua menor disseminação. Isto corrobora não somente com a transcrição gênica, mas com a importância da sequência de nucleotídeos deste gene, uma vez que a cepa de E. coli SM124/13 complementada com o icsB de Shigella se mostrou mais eficiente na disseminação dentro da célula hospedeira. De forma interessante, IcsB apresentou um papel inédito na regulação da resposta inflamatória das células HeLa, onde a ausência de IcsB em EIEC promoveu uma intensa perturbação na homeostase da célula hospedeira, com aumento da secreção de IL-6, IL-8 e morte celular. Adicionalmente, ficou evidente que a célula eucariótica responde de maneira distinta frente a infecção por EIEC e Shigella flexneri. EIEC provavelmente ativou o processo autofágico em células humanas de forma não canônica. Nossa hipótese seria de que EIEC é reconhecida pelo processo autofágico, podendo ser este um importante fenômeno de reconhecimento bacteriano que colabore para a menor disseminação intracelular de EIEC, e assim tornar sua doença mais branda, quando comparada com a infecção por Shigella

The invasive bacteria recognition by host cells through autophagy is a key factor for determining bacterial infection. Enteroinvasive Escherichia coli (EIEC) express a protein IcsB, which in Shigella, is known for inactivating the bacterial degradation process. Once EIEC showed less expression of icsB when compared to S. flexneri, we proposed to investigate the autophagy caused by EIEC infection, using techniques such as gene mutation by insertion, western blot, fluorescence microscopy, transmission electron microscopy and microarray. Our results showed that IcsB protein is an important virulence factor in EIEC because it causes a camouflage of the bacteria in the eukaryotic cell. When there is a low expression of the protein, the cell recognition of the invasive bacteria is high, decreasing the bacteria dissemination. This found confirms the importance of the gene transcription and the gene sequence, since the strain E. coli SM124/13, complemented with icsB from Shigella, showed higher dissemination efficiency inside of the host cell. Interestingly, IcsB showed a new role on regulating the inflammatory response in Hela cells. The absence of IcsB in EIEC generated an intense disturbance of the cell homeostasis, increased the secretion of IL-6 and IL-8, and caused cell death. Additionally, our results revealed that eukaryotic cell infected by EIEC or Shigella flexneri showed distinguish responses. In EIEC infection, the autophagy was activated in human cells, but not in a conventional mode. Our hypothesis is that EIEC is recognized by autophagy, being an important cell process for bacterial recognition. This process can cause a decrease in the intracellular spread of EIEC making the infection less severe when compared to the infection caused by Shigella

Infection of Extended-Spectrum β-Lactamase Producing Shigella flexneri in Children Attending a Childcare Center in Korea

Shigella is a common cause of bacterial enteritis worldwide. Shigella sonnei accounts for 90% of Shigella infections and Shigella flexneri is rarely reported in Korea. Although the incidence of Shigella infection has decreased, the incidence of organisms with antibiotic resistance has gradually increased in Korea. An outbreak of extended-spectrum β-lactamase (ESBL)-producing S. sonnei in children was reported in Korea; however, ESBL-producing S. flexneri has not yet been reported. We report the first two cases of multidrug-resistant CTX-M-14-producing S. flexneri infections in Korean children.

Manual de monitoramento da qualidade dos registros de enfermagem na assistência domiciliar / Manual de monitoreo de la calidad de los registros de enfermería en la atención domiciliaria / Manual for monitoring the quality of nursing home care records

RESUMO Objetivo: construir e validar um instrumento para monitorar a qualidade dos registros de enfermagem no Programa de Assistência Domiciliar (PAD) em um hospital universitário. Método: estudo metodológico envolvendo a elaboração de um manual e submetido à validação de conteúdo por seis juízes sob consenso ≥ 80%. A coleta ocorreu em 2012 por meio de questionário contendo: evolução de enfermagem, diagnóstico e prescrição de enfermagem e normas para os registros da equipe de enfermagem preconizadas pelo Conselho Regional de Enfermagem-SP e pela instituição. Os itens do manual foram julgados de acordo com as variáveis - relevância, pertinência, clareza e simplicidade. Resultados: das 39 proposições 100% atingiram consenso ≥ 80% em relevância, pertinência e clareza; 92,3% em simplicidade. Os itens sono/repouso, mobilidade e checagem nas atividades prescritas não atingiram consenso mínimo favorável, sendo aprimorados pelas sugestões dos juízes. Conclusão: acreditamos que o instrumento possibilitará a melhoria dos processos de trabalho no PAD. .

RESUMEN Objetivo: construir y validar un instrumento para monitorear la calidad del registros de enfermería en Programa de Atención Domiciliaria (PAD) de un Hospital Universitario. Metodo: estudio metodológico. Fue construido un manual y sometió a validación de contenido por seis jueces bajo el consenso ≥80%. La recogida currió en 2012, con un cuestionario que contiene: evolución de enfermería, diagnóstico y prescripción de enfermería y normas para los registros del personal de enfermaria estabelecidas por Consejo Regional de Enfermería-SP y por la institución. Los artículos del manual fueran juzgadso conforme las variables relevancia, pertinencia, claridad y sencillez. Resultados: de las 39 proposiciones 100% alcanzó consenso ≥ 80% en la relevancia, pertinencia y claridad; 92,3% en la simplicidad. Los itens sueño/resto, movilidad y verificar las actividades prescritas no alcanzó consenso favorable, siendo mejoradas por las sugerencias de los jueces. Conclusión: creemos que el instrumento permitirá la mejora de los procesos de trabajo en PAD. .

ABSTRACT Objective: to build and validate an instrument aimed at monitoring the quality of nursing records in the Home Care Program (HCP) of a university hospital. Method: methodological study involving the elaboration of a manual, whose content was later submitted to six experts for validation, reaching a ≥ 80% consensus. The data collection process was carried out in 2012 by means of a questionnaire comprised of the following issues: nursing evolution, nursing diagnosis, and nursing prescription, and standards for the nursing team recommended by the Regional Nursing Council of São Paulo and by the assessed institution. Manual items were judged according to the following variables: relevance, pertinence, clarity and simplicity. Results: of the 39 propositions, 100% achieved ≥ 80% agreement in the relevance, pertinence and clarity variables; 92.3% in the simplicity variable. Sleep/rest, Mobility and Check-out variables did not reach a favorable minimum consensus in the prescribed activities and were improved following suggestions from the experts. Conclusion: we believe that the instrument will enable the improvement of the HCP’s work process. .

Antisuero AA479: nuevo reactivo para la serotipificación de variantes atípicas se Shigella flexneri / AA479 antiserum: new reagent for the serotype characterization of atypical variants of Shigella flexneri

Shigella flexneri is divided into 13 serotypes based on the combination of antigenic determinants present in the O-antigen. A new O-antigen modification with phosphoethanolamine has been identified. The presence of this antigenic determinant (called E1037) is recognized by monoclonal antibody MASF IV-1. Given the increasing incidence of these new variants and the difficulty in supplying the monoclonal antibody to our country, we produced a polyclonal antiserum (AA479) through immunization with a S. flexneri Xv strain. The antiserum specificity was assessed by slide agglutination against isolates from clinical cases and a culture collection representing all Shigella serotypes. The results obtained demonstrated a 100% correlation between AA479 absorbed antiserum and monoclonal antibody MASF IV-1. The availability of AA479 antiserum in every public hospital in Argentina will allow us to identify atypical S. flexneri isolates in order to strengthen Shigella surveillance in our country and to compare with global epidemiological dat

Shigella flexneri se divide en al menos 13 serotipos sobre la base de la combinación de determinantes antigénicos presentes en el antígeno O. Se identificó una nueva modificación del antígeno O con fosfoetanolamina. La presencia de este determinante antigénico (denominado E1037) es reconocida por el anticuerpo monoclonal MASF IV-1. Teniendo en cuenta la incidencia creciente de estas nuevas variantes y la dificultad en la provisión del anticuerpo monoclonal para nuestro país, se elaboró un antisuero de tipo policlonal (AA479) mediante la inmunización con un cultivo de S. flexneri Xv. La especificidad del antisuero se evaluó por aglutinación en lámina con aislamientos clínicos y cultivos de colección, con lo que quedaron representados todos los serotipos de Shigella. Los resultados obtenidos demostraron una correlación del 100% entre el antisuero AA479 absorbido y el anticuerpo monoclonal MASF IV-1. La disponibilidad del antisuero AA479 en todos los hospitales públicos de Argentina permitirá identificar los aislamientos atípicos de S. flexneri; de esta forma se podrá fortalecer la vigilancia de Shigella en nuestro país y comparar con los datos epidemiológicos a nivel global

Analysis of the drug resistance and the integron resistance gene cassette's characteristics of Shigella flexneri / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate the correlation between Shigella flexneri multi-drug resistance and drug resistance gene cassette of integrons.</p><p><b>METHOD</b>All 79 strains of Shigella flexneri were isolated from the feces of children ranged in age from 6 months to 14 years in some hospitals of Jinan, between May 2009 and April 2012.The resistance was detected by Kirby Bauer agar diffusion method, 1, 2 and 3 integron gene was amplified by PCR, the variable region of positive strains treated with enzyme digestion and determined by Series Analysis.</p><p><b>RESULT</b>Among 79 Shigella flexneri strains, the resistance rate was 91% (72/79) to ampicillin, chloramphenicol, tetracycline, streptomycin, 70% (55/79) to sulfamethoxazole/trimethoprim, 30% (24/79), 23% (18/79), 33% (26/79) and 32% (25/79) to cefotaxime, ceftazidime, ciprofloxacin and levofloxacin.All 79 strains were susceptible to cefoxitin, imipenem, cefoperazone/sulbactam. The common drug resistance pattern is ampicillin tetracycline-chloramphenicol-streptomycin, accounted for 91% (72/79); 91% (72/79) strains carried integrons of class 1, 86% (68/79) strains carried integrons of class 2, No intI3 was detected. The resistance to ampicillin, streptomycin, tetracycline, chloramphenicol of atypical class 1 integron positive strains was significantly higher than the negative strains (χ² = 35.96, P<0.01). The sequencing results:dfrV was detected in class 1 integron variable regions of 9 strains, dfrA17-aadA5 in 2 strains, blaOXA-30-aadA1 in 70 strains, 2 strains were not detected resistance gene cassette, all resistance gene cassettes were dfrA1-sat1-aadA1 in class 2 integron variable regions.</p><p><b>CONCLUSION</b>The muti-drug resistance of Shigella flexneri in Jinan was closely associated with integrons.</p>

Shigella sonnei: ¨Est ocurriendo un cambio en nuestro medio? / Shigella sonnei: is a change occurring in our middle?

Objetivos: Determinar los serogrupos de Shigella como agentes etiol¢gicos de diarrea. Dise¤o: Estudio descriptivo, retrospectivo, anal¡tico. Instituci¢n: Instituto de Medicina Tropical, Universidad Nacional Mayor de San Marcos, Lima, Per£, y Hospital Nacional Daniel A. Carri¢n, Callao, Per£. Material biol¢gico : Aislamientos de Shigella sp. en coprocultivos. Intervenciones: Se revis¢ los aislamientos de Shigella sp. en coprocultivos, durante los a¤os 2010 y 2011, en la base de datos WHONET 5.6 del hospital, tanto de pacientes adultos como pedi tricos, ambulatorios, hospitalizados y atendidos en emergencia. Se distribuy¢ la Shigella por serogrupos. Principales medidas de resultados : Frecuencia absoluta y frecuencia relativa de Shigella sp. Resultados: El a¤o 2010 se aisl¢ 64 Shigellas sp.; 41 correspondieron al serogrupo flexneri (64,1 por ciento) y 20 al serogrupo sonnei (31,3 por ciento). En el a¤o 2011 se aisl¢ 90 Shigellas sp.; 36 correspondieron a serogrupo flexneri (40 por ciento) y 50 al serogrupo sonnei (55,6 por ciento). Las diferencias encontradas son estad¡sticamente significativas. Conclusiones: Shigella sonnei fue el serogrupo predominante en nuestro hospital.

Objectives: To determine Shigella serogroups as diarrhea etiological agents. Design: Descriptive, retrospective, analytical study. Setting: Instituto de Medicina Tropical, Universidad Nacional Mayor de San Marcos, Lima, Peru, and Hospital Nacional Daniel A. Carrion, Callao, Peru. Biologic material: Shigella sp. isolates in coprocultures. Interventions: Shigella sp. isolates in coprocultures of outpatient, hospitalized and emergency adult and pediatric patients attended during years 2010 and 2011were reviewed at the hospital WHONET 5.6 database. Shigella were distributed by serogroups. Main outcome measures : Absolute and relative frequency of Shigella sp. Results: Sisty-four Shigellas sp were isolated in 2010; 41 corresponded to flexneri serogroup (64.1 per cent) and 20 to sonnei serogroup (31.3 per cent). Ninety Shigellas sp. were isolated in 2011; 36 corresponded to flexneri serogroup (40 per cent) and 50 to sonnei serogroup (55,6 per cent). Differences were statistically significant. Conclusions: Shigella sonnei was the predominant serogroup at our hospital.

Shigella flexneri Inhibits Intestinal Inflammation by Modulation of Host Sphingosine-1-Phosphate in Mice

Infection with invasive Shigella species results in intestinal inflammation in humans but no symptoms in adult mice. To investigate why adult mice are resistant to invasive shigellae, 6~8-week-old mice were infected orally with S. flexneri 5a. Shigellae successfully colonized the small and large intestines. Mild cell death was seen but no inflammation. The infected bacteria were cleared 24 hours later. Microarray analysis of infected intestinal tissue showed that several genes that are involved with the sphingosine-1-phosphate (S1P) signaling pathway, a lipid mediator which mediates immune responses, were altered significantly. Shigella infection of a human intestinal cell line modulated host S1P-related genes to reduce S1P levels. In addition, co-administration of S1P with shigellae could induce inflammatory responses in the gut. Here we propose that Shigella species have evasion mechanisms that dampen host inflammatory responses by lowering host S1P levels in the gut of adult mice.

Identification of Bacterial Flora on Cellular Phones of Dentists

Dental professionals are repeatedly exposed to many microorganisms present in both blood and saliva. Thus, dental professionals are at a greater risk of acquiring and spreading infections, and the implementation of infections control guidelines is necessary. Cellular phones have become a necessary device for communicating in hospitals. Cellular phones contaminated with bacteria may serve as a fomite in the transmission of pathogens by the hands of medical personnel. Nevertheless, studies about rate and levels of bacterial contamination of cellular phones have been extremely limited with regards to dental personnel. The purpose of this study was to identify bacterial flora on the cellular phones of dentists by a molecular biological method using the 16S rRNA cloning and sequencing method. We acquired total 200 clones from dentists' cell phones and identified the bacterial species. Pseudomonas (34.6%), Lactobacillus (18.5%), Azomonas (11.5%), and Janthinobacterium (6%) were the dominant genera on dentists' cell phones. The oral bacteria identified were Anaerococcus lactolyticus, Gibbsiella dentisursi, Lactobacills leiae, Streptococcus mitis, Streptococcus oligofermentans, and Streptococcus sanguinis. Pathogenic bacteria and opportunistic pathogens such as Carnobacterium funditum, Raoultella planticola, Shigella flexneri, Lactobacillus iners, Staphylococcus aureus, and Staphylococcus epidermidis were also identified.

HYPOXIC STRESS, HEPATOCYTES AND CACO-2 VIABILITY AND SUSCEPTIBILITY TO Shigella flexneri INVASION / Stress hipóxico, viabilidade e susceptibilidade de hepatócitos e células Caco-2 à invasão por Shigella flexneri

SUMMARY Inflammation due to Shigella flexneri can cause damage to the colonic mucosa and cell death by necrosis and apoptosis. This bacteria can reach the bloodstream in this way, and the liver through portal veins. Hypoxia is a condition present in many human diseases, and it may induce bacterial translocation from intestinal lumen. We studied the ability of S. flexneri to invade rat hepatocytes and Caco-2 cells both in normoxic and hypoxic microenvironments, as well as morphological and physiological alterations in these cells after infection under hypoxia. We used the primary culture of rat hepatocytes as a model of study. We analyzed the following parameters in normoxic and hypoxic conditions: morphology, cell viability, bacterial recovery and lactate dehydrogenase (LDH) released. The results showed that there were fewer bacteria within the Caco-2 cells than in hepatocytes in normoxic and hypoxic conditions. We observed that the higher the multiplicity of infection (MOI) the greater the bacterial recovery in hepatocytes. The hypoxic condition decreased the bacterial recovery in hepatocytes. The cytotoxicity evaluated by LDH released by cells was significantly higher in cells submitted to hypoxia than normoxia. Caco-2 cells in normoxia released 63% more LDH than hepatocytes. LDH increased 164% when hepatocytes were submitted to hypoxia and just 21% when Caco-2 cells were in the same condition. The apoptosis evaluated by Tunel was significantly higher in cells submitted to hypoxia than normoxia. When comparing hypoxic cells, we obtained more apoptotic hepatocytes than apoptotic Caco-2 cells. Concluding our results contribute to a better knowledge of interactions between studied cells and Shigella flexneri. These data may be useful in the future to define strategies to combat this virulent pathogen. .

RESUMO A inflamação causada por Shigella flexneri pode causar danos à mucosa do cólon e morte celular por necrose e apoptose. Esta bactéria pode atingir a corrente sanguínea por esta via e o fígado através da veia porta. A hipóxia é uma condição presente em muitas doenças humanas, podendo induzir a translocação bacteriana a partir do lúmen intestinal. Nós estudamos a capacidade de S. flexneri invadir hepatócitos de rato e células Caco-2 nos microambientes de normóxia e hipóxia, bem como as alterações morfológicas e fisiológicas dessas células após a infecção sob hipóxia. Utilizamos a cultura primária de hepatócitos de ratos como modelo de estudo. Nós analisamos os seguintes parâmetros em condições de normóxia e hipóxia: morfologia, viabilidade celular, recuperação bacteriana e liberação de lactato desidrogenase (LDH). Os resultados mostraram menor quantidade de bactérias dentro das células Caco-2 do que em hepatócitos em condições de normóxia e hipóxia. Nós observamos que quanto maior foi a multiplicidade de infecção (MOI), maior também foi a recuperação bacteriana em hepatócitos. A condição hipóxica foi capaz de diminuir a recuperação de bactérias dos hepatócitos. A citotoxicidade avaliada pela liberação de LDH foi significativamente maior em células submetidas à hipóxia do que normóxia. As células Caco-2 em normóxia produziram 63% mais LDH do que os hepatócitos. O LDH aumentou 164% quando os hepatócitos foram submetidos à hipoxia e apenas 21% quando as células Caco-2 estavam na mesma condição. A apoptose avaliada por TUNEL foi significativamente ...

Bacteriemia por Shigellaflexneri serotipo 6 en pacientes con SIDA: Presentación de dos casos clínicos / Bacteraemia due to Shigella flexneri serotype 6 in two patients with AIDS

Infections due to Shigella usually remain localized to the digestive tract and are self-limited. Bacteremia is a potentially lethal complication that can occur in immunocompromised patients. We describe two episodes of bacteremia caused by Shigella in two adults with AIDS. In both patients, S. flexneri was recovered from stool and blood samples. The isolates belonged to serotype 6, were resistant only to trimethoprim-sulfamethoxazole and showed a similar band profile by pulsed-field gel electrophoresis. Patients received prolonged antimicrobial treatment with a favorable outcome. There were no cases of diarrhea in other individuals admitted to the emergency room. We hypothesized that patient No. 2 was infected at the hospital from patient No. 1. However, we could not establish the way of transmission. Although rare, it is important to take into account the possible occurrence of bacteremia due to Shigella or other bacterial enteropathogens in immunocompromised patients with diarrhea.

Las infecciones por Shigella spp., en general, permanecen localizadas en el tracto digestivo y tienen una evolución autolimitada. La bacteriemia es una complicación potencialmente letal que ocurre en pacientes con algún tipo de inmunocompromiso. Presentamos dos casos de bacteriemia causadas por Shigella en dos adultos con SIDA. En ambos pacientes, se recuperó Shigella flex-neri en muestras de deposiciones y sangre. Los aislados correspondieron al serotipo 6, fueron resistentes sólo a cotrimoxazol y mostraron un perfil de bandas similar por PFGE. Los pacientes recibieron tratamiento antimicrobiano prolongado y la evolución fue favorable. No se registraron otros casos de diarrea en individuos admitidos en el servicio de emergencia. La hipótesis fue que el paciente 2 adquirió la infección en el hospital a partir del paciente 1. Sin embargo, no pudimos establecer el modo de transmisión. Aunque poco frecuente, es importante tener presente la ocurrencia de bacteriemia por Shigella spp. o por otros enteropatógenos bacterianos en pacientes inmunocomprometidos con diarrea.

Expression of Shigella flexneri ipaB gene in tobacco

Shigellosis is a leading cause of diarrhea in many developing countries and although the disease can be controlled and managed with antibiotics, the constant emergence of resistant species requiring ever newer antibacterial drugs make development of an effective vaccine necessary. The bacteria are highly contagious and since immunity to Shigella is serotypespecific a multi-serotype vaccine is required for adequate protection. Proteins encoded by Shigella invasion plasmid, which are part of the Type Three Secretion System [TTSS] of this bacteria, are good candidate as vaccine targets since they are both immunogenic and conserved between different Shigella species. The advent of molecular farming, which is a low cost system, has opened up new venues for production of recombinant proteins. In view of the difficulties encountered in expressing IpaB in Escherichia coli [E. coli], the feasibility of the expression of this protein in tobacco has been investigated. The ipaB gene was cloned in place of the Hygromycin gene in pCambia1304 containing GFP as a reporter gene. The vector was then transferred into competent Agrobacterium tumefaciens [A. tumefaciens] strain LBA4404 which was used for agro-infiltration of Nicotiana tobaccum [N. tobaccum] leaves. Transformation was confirmed by expression of GFP. The gene was also cloned in pBAD/geneIII A and transformed E. coli host containing the construct was induced using different amounts of L-arabinose as inducer. Expression of IpaB gene by both hosts was determined by Western blotting using anti-IpaB monoclonal antibody. The data obtained showed that IpaB was expressed in plant leaves but expression in E. coli was not detectable. This study showed that N. tobaccum is capable of expressing this protein without its specific chaperon and in levels detectable by Western blotting

Surveillance of Antimicrobial Susceptibility Patterns among Shigella Species Isolated in China during the 7-Year Period of 2005-2011

BACKGROUND: Shigella is a frequent cause of bacterial dysentery in the developing world. Treatment with antibiotics is recommended for shigellosis, but the options are limited due to globally emerging resistance. This study was conducted to determine the frequency and pattern of antimicrobial susceptibility of Shigella in China. METHODS: We studied the antimicrobial resistance profiles of 308 Shigella spp. strains (260 S. flexneri, 40 S. sonnei, 5 S. boydii, and 3 S. dysenteriae) isolated from fecal samples of patients (age, from 3 months to 92 yr) presenting with diarrhea in different districts of Anhui, China. The antimicrobial resistance of strains was determined by the agar dilution method according to the CSLI guidelines. RESULTS: The most common serogroup in the Shigella isolates was S. flexneri (n=260, 84.4%), followed by S. sonnei (n=40, 13.0%). The highest resistance rate was found for nalidixic acid (96.4%), followed by ampicillin (93.2%), tetracycline (90.9%), and trimethoprim/sulfamethoxazole (80.8%). Among the isolates tested, 280 (91.0%) were multidrug resistant (resistant to > or =2 agents). The most common resistance pattern was the combination of ampicillin, tetracycline, and trimethoprim/sulfamethoxazole (70.8%). Resistance to ampicillin and tetracycline were more common among S. flexneri than among S. sonnei isolates. CONCLUSIONS: S. flexneri is predominant in Anhui, China, and its higher antimicrobial resistance rate compared with that of S. sonnei is a cause for concern. Continuous monitoring of resistance patterns is necessary to control the spread of resistance in Shigella. The recommendations for antimicrobial treatment must be updated regularly based on surveillance results.

Virulence genes and pathogenicity of Shigella flexneri Xv isolated in Beijing / 中华流行病学杂志

<p><b>OBJECTIVE</b>To understand the biochemical characteristics, virulence genes and pathogenicity of Shigella flexneri Xv isolated in Beijing.</p><p><b>METHODS</b>61 strains of S. flexneri Xv isolated from diarrhea patients in Beijing were systematically determined through biochemical reactions and serological tests. Application of PCR technique in detection of virulence genes on ipaH, sen, virF, ial and pulsed-field gel electrophoresis (PFGE) was used to identify the related characteristics and on rat lung slices to determine its pathogenicity.</p><p><b>RESULTS</b>All of the S. flexneri Xv could ferment glucose, mannitol, melibiose and arabinose. Using serum agglutination, we found that the antigen structure was (IV: 7, 8). IpaH, sen, virF and ial that carried rates of virulence genes appeared to be 100%, 81.97%, 75.41% and 80.30%, respectively. Among 61 strains of S. flexneri Xv, the PFGE typing of Shigella bacteria could be divided into 25 belt types while the results from rat lung slices showed inflammatory change of Xv.</p><p><b>CONCLUSION</b>S. flexneri Xv was found that it carried high rate of Shigella virulence genes, exhibiting genetic polymorphism and highly invasive.</p>