AIM To investigate the specific pathogenesis ofO-polysaccharide (O--PS) which is on the outermembrane of lipopolysaccharides (LPS) fromShigella fi^eri.METHODS The O--PS was isolated and purifiedfrom Shigella nexneri 5...AIM To investigate the specific pathogenesis ofO-polysaccharide (O--PS) which is on the outermembrane of lipopolysaccharides (LPS) fromShigella fi^eri.METHODS The O--PS was isolated and purifiedfrom Shigella nexneri 5 MgoT by enzymatichydrolysis and gel chromatography. Effects ofO--PS were observed by in vitro experiment,(HeLa cell Culture ), and in vivo experiment(rabbit lieal loop assay).RESULTS ID vitro and in vivo e-cP6riments withthe purified O--PS from Shigells flexnefi revealedthat the O--PS alone was toxic to Hela cells andcaused mucosal inflammation and hemorrhagicexudation in lieal loop of rabbit.DISCUSSION O--PS might b6 one of the factorscausing diarrhea and its mechanism wasdifferent from endotoxin reaction of LPS. Themolecular mechanism of O-PS need furtherstudies.展开更多
BACKGROUND Shigella flexneri(S.flexneri)is a major pathogen causing acute intestinal infection,but the systematic oxidative damage incurred during the course of infection has not been investigated.AIM To investigate t...BACKGROUND Shigella flexneri(S.flexneri)is a major pathogen causing acute intestinal infection,but the systematic oxidative damage incurred during the course of infection has not been investigated.AIM To investigate the incurred systemic RNA oxidative damage and the diagnostic value of RNA oxidative metabolites during S.flexneri-induced intestinal infection.METHODS In this study,a Sprague-Dawley rat model of acute intestinal infection was established by oral gavage with S.flexneri strains.The changes in white blood cells(WBCs)and cytokine levels in blood and the inflammatory response in the colon were investigated.We also detected the RNA and DNA oxidation in urine and tissues.RESULTS S.flexneri infection induced an increase in WBCs,C-reactive protein,interleukin(IL)-6,IL-10,IL-1β,IL-4,IL-17a,IL-10,and tumor necrosis factorα(TNF-α)in blood.Of note,a significant increase in urinary 8-oxo-7,8-dihydroguanosine(8-oxo-Gsn),an important marker of total RNA oxidation,was detected after intestinal infection(P=0.03).The urinary 8-oxo-Gsn level returned to the baseline level after recovery from infection.In addition,the results of a correlation analysis showed that urinary 8-oxo-Gsn was positively correlated with the WBC count and the cytokines IL-6,TNF-α,IL-10,IL-1β,and IL-17α.Further detection of the oxidation in different tissues showed that S.flexneri infection induced RNA oxidative damage in the colon,ileum,liver,spleen,and brain.CONCLUSION Acute infection induced by S.flexneri causes increased RNA oxidative damage in various tissues(liver,spleen,and brain)and an increase of 8-oxo-Gsn,a urinary metabolite.Urinary 8-oxo-Gsn may be useful as a biomarker for evaluating the severity and prognosis of infection.展开更多
AIM: To profile the immunogenic proteins of Shigella flexneri (S. flexneri) expressed during human infection using a proteomic approach. METHODS: Soluble and membrane protein extractions of S. flexneri 2457T were sepa...AIM: To profile the immunogenic proteins of Shigella flexneri (S. flexneri) expressed during human infection using a proteomic approach. METHODS: Soluble and membrane protein extractions of S. flexneri 2457T were separated by two-dimensional gel electrophoresis (2-DE). Proteins were transferred to PVDF membrane and immunoblotted with sera from shigellosis patients. Reactive protein spots were matched to Coomassie stained gels run in parallel, cut out and trypsin digested. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS) was used to determine the peptide mass fingerprints, which were searched in the MASCOT database to identify the protein. RESULTS: A total of 8 immunoreactive proteins were successfully identified from the Coomassie stained gels in three repeats. Six of these proteins have not previously been reported as immunogenic in S. flexneri. These proteins could be potential candidates for vaccine or attenuation studies. CONCLUSION: Soluble and membrane proteins of S. flexneri 2457T have been screened by 2-DE and immunoblotting with sera from shigellosis patients. Eight proteins are identified as immunogenic.展开更多
AIM: To screen the immunogenic membrane proteins ofShigella flexneri 2a 2457T.METHODS: The routine two-dimensional polyacrylamidegel electrophoresis (2-DE) and Western blotting werecombined to screen immunogenic prote...AIM: To screen the immunogenic membrane proteins ofShigella flexneri 2a 2457T.METHODS: The routine two-dimensional polyacrylamidegel electrophoresis (2-DE) and Western blotting werecombined to screen immunogenic proteins of S. flexneri2a 2457T. Serum was gained from rabbits immunizedwith the same bacteria. Immunogenic spots werecut out from the polyacrylamide gel and digested bytrypsin in-gel. Matrix-assisted laser desorption/ionizationtime of flight-mass spectrometry (MALDI-TOF-MS)was performed to determine the molecular weight ofpeptides. Electrospray ionization (ESI-MS/MS) wasperformed to determine the sequences of the interestingpeptides.RESULTS: A total of 20 spots were successfullyidentified from Coomassie brilliant blue stained gelsrepresenting 13 protein entries, 5 known antigens and8 novel antigens. A hypothetical protein (YaeT) wasdetected, which might be a candidate target of vaccine.CONCLUSION: Membrane proteins of S. flexneri 2a2457T were successfully observed by 2-DE. Severalknown and novel antigens were identified by massspectrum.展开更多
Shigella flexneri is an infectious pathogen that causes dysentery to human, which remains a serious threat to public health, particularly in developing countries. In this study, the global protein expression patterns ...Shigella flexneri is an infectious pathogen that causes dysentery to human, which remains a serious threat to public health, particularly in developing countries. In this study, the global protein expression patterns of S. flexneri during transition from exponential growth to stationary phase in vitro were analyzed by using 2-D PAGE combined with MALDI-TOF MS. In a time-course experiment with five time points, the relative abundance of 49 protein spots varied significantly. In- terestingly, a putative outer membrane protein YciD (OmpW) was almost not detected in the exponential growth phase but became one of the most abundant proteins in the whole stationary-phase proteome. Some proteins regulated by the global regulator FNR were also significantly induced (such as AnsB, AspA, FrdAB, and KatG) or repressed (such as AceEF, OmpX, SodA, and SucAB) during the growth phase transition. These proteins may be the key effectors of the bacterial cell cycle or play important roles in the cellular maintenance and stress responses. Our expression profile data provide valuable information for the study of bacterial physiology and form the basis for future proteomic analyses of this pathogen.展开更多
Aim: Shigella flexneri (S. flexneri) is a gram-negative enterobacterium responsible for severe intestinal end systemic infection in humans. The bacteria can reach the liver due to degeneration of the colonic epitheliu...Aim: Shigella flexneri (S. flexneri) is a gram-negative enterobacterium responsible for severe intestinal end systemic infection in humans. The bacteria can reach the liver due to degeneration of the colonic epithelium. Hypoxia is present in many human diseases and can induce the expression of the transcription factor HIF-1alpha that may have a cell protective role. The influence of hypoxia and HIF-1alpha on bacterial infection, studied in this work, is unclear. Hypoxia inducible factor-1alpha (HIF-1alpha) is a transcription factor that acts as a master regulator of gene expression induced by hypoxia. Methods: We compared the ability of S. flexneri to invade rat hepatocytes in primary culture both in normoxic and hypoxic conditions. We evaluated TNF-alpha released by hepatocytes, apoptosis rate and HIF-1alpha expression by confocal microscopy as well as real time PCR technique. Results: We showed that S. flexneri invaded less hepatocytes previously submitted to 24 h hypoxia (6.5% O2) than those cultivated in normoxia (21% O2). S. flexneri also induced HIF-1α expression in hepatocytes, TNF-α secretion and apoptosis. Conclusion: a) Hypoxia alone was not a stimulus to TNF-α secretion, but induced cell apoptosis and HIF-1α expression;b) S. flexneri was able to invade rat hepatocytes and hypoxia apparently influenced significantly bacterial cell invasiveness;c) HIF-1α was expressed in hypoxic conditions, and it was also stimulated by S. flexneri.展开更多
This study investigated antibacterial and antibiofilm activity of the combined use of phenyllactic acid(PLA)and bacteriocin XJS01 against Shigella flexneri_14.The minimum inhibitory concentration(MIC)of PLA and XJS01 ...This study investigated antibacterial and antibiofilm activity of the combined use of phenyllactic acid(PLA)and bacteriocin XJS01 against Shigella flexneri_14.The minimum inhibitory concentration(MIC)of PLA and XJS01 against S.flexneri_14 was 2.45 mg/mL and 18.75μg/mL,respectively.Growth and kill kinetics assays showed that the combined use of 1/2MIC PLA plus 1/2MIC XJS01 had a better activity against planktonic S.flexneri_14 compared to treatment with PLA and XJS01 used singly(1/2MIC and 2MIC).Cellular biochemical and morphological analysis revealed the remarkable ability of the combination in disrupting cell appearance and promoting deformation of planktonic S.flexneri_14 compared to single use.Moreover,S.flexneri_14 biofilm formation was inhibited and degraded by the combination,which showed a more remarkable antibiofilm activity than PLA and XJS01 when used singly.This study demonstrates the synergistic antibacterial activity of PLA and XJS01 against S.flexneri_14 in either planktonic or biofilm states in foods.展开更多
Comparative genome analysis is performed between Shigella flexneri 2a strain 301 and its close relatives, the nonpathogenic E. Coli K-12 strain MG1655. Result shows that there are 136 DNA segments whose size is larger...Comparative genome analysis is performed between Shigella flexneri 2a strain 301 and its close relatives, the nonpathogenic E. Coli K-12 strain MG1655. Result shows that there are 136 DNA segments whose size is larger than 1000 bp absent from Shigella flexneri 2a strain 301, which is up to 717253 bp in total length. These deleted segments altogether contain 670 open reading frames (ORFs). Prediction of these ORFs indicates that there are 40% genes of unknown function. The other genes of definite functions encode metabolic enzymes, structure proteins, transcription regulatory factors and some elements correlated with horizontal transfer. Here we compare the complete genomic sequences of the two closely related species, which differ in pathogenic phenotype. To our knowledge, this not only reveals the difference of genomic sequence between the two important enteric pathogens for the first time, but also provides valuable clues to further researches in its process of physiological activity, pathogenesis and the evolution of enteric bacteria.展开更多
In order to overcome the defects of difficult gene operations in low-copy suicide plasmid pCVD442, Gateway technology was applied in the construction process of recombinant plasmid for gene knockout in this study. Wit...In order to overcome the defects of difficult gene operations in low-copy suicide plasmid pCVD442, Gateway technology was applied in the construction process of recombinant plasmid for gene knockout in this study. With this improved knockout system, we inactivated sitC gene, which is associated with iron transport in Shigella flexneri 2a strain 301, to yield the mutant, MTS. The functional detection of the mutant was performed at the level of culture medium, cell and animal experiment, respectively. The gene expression profiles were compared with DNA microarray between the mutant and the wild type under iron-restricted conditions. The results showed that MTS grew obviously less well than the wild-type strains in L broth containing 150 μmol/L iron chelator DIP(2,2′-dipyridyl). Addition of iron or manganese to the cultures stimulated the growth of MTS to wild-type levels in rich culture medium. In either the experiment on the ability of intracellular multiplication and cell-to-cell spread in HeLa and U937 cell lines, or the experiment on keratoconjunctivitis in guinea pigs, MTS showed no obvious changes in virulence compared with the parental strain Sf301. When 65 μmol/L DIP was added to the cultured HeLa cells, the ability of intracellular multiplication of MTS reduced about 51.6% as compared with that of Sf301. The analysis of expression profiles under iron-limited condition showed that MTS was more sensitive for the change of iron deficiency than Sf301. There are 106 more up-regulated genes in MTS than in wild-type strains, which are involved in membrane transportation, amino acid metabolism and uncategorized function genes, while down-regulated genes are mainly in- volved in energy and carbohydrate metabolism. Under low iron conditions, the expression levels of known iron-transport associated genes generally increased. Additionally, the number of these genes and their increase amplitude in MTS are more than those in Sf301. Together, these results confirmed that Sit iron-transport system is important for the growth of Shigella.展开更多
An in vivo expression technology (IVET) was applied to screen s.flexneri 2a genes induced after invasion of epithelial cells, and virulence-related genes were further identified by mutational analysis. Thirteen intrac...An in vivo expression technology (IVET) was applied to screen s.flexneri 2a genes induced after invasion of epithelial cells, and virulence-related genes were further identified by mutational analysis. Thirteen intracellular induced genes were identified with a HeLa cell infection model. Of these, two were identified as alkylation-related genes; one was related to metabolism; one encoded a transcriptional regulator; three were identified as insertion elements; three ap- peared to be antisense to genes involved in the transmethylation,biosyntheseis, and phos- photransferase system;and three were predicted to encode polypeptides with unknown functions. Intracellular survival assavs showed that the mutants of alkA,citC and wcaJ genes had lower capability of intracellular replication or survival than the the wild-type strain.The results indicated that alkA, citC and wcaJ genes could take part in the intracellular survival or replication of S. flexneri 2a and the capability of intracellular survival or replication could be one of the major virulence elements. However, the yaiC mutant was able to survive in the murine infection assay but almost not in HeLa cell infection assay. Very possibly, yaiC gene was involved in the other mechanism of S. flexneri virulence. This study might lead to a better understanding of the intra- cellular survival or proliferation process of S. flexneri 2a and perhaps provide insights into the pathogenicity of this pathogen.展开更多
Introduction: Salmonella and Shigella are gram-negative bacilli that are resistant to most antibiotics and play an important role in an etiology of diarrhea disease. The aim of the study was to determine the prevalenc...Introduction: Salmonella and Shigella are gram-negative bacilli that are resistant to most antibiotics and play an important role in an etiology of diarrhea disease. The aim of the study was to determine the prevalence of Salmonella spp and Shigella spp isolated from stool and blood samples in the city of N’Djamena. Materials and Method: This was a prospective study conducted in the four district hospitals of N’Djamena from 14 July 2022 to 31 December 2022. A questionnaire form was drawn up to collect the information sent to the study patients. The samples were analyzed at the CHU de la Mère et de l’Enfant, Labo-Redes laboratory according to their protocols and the standard of the antibiogram committee of the French microbiology society. Results: Of the 803 biological samples analyzed, 39 were positive for Salmonella spp and Shigella spp, including 15 for Salmonella and 24 for Shigella, giving an overall prevalence rate of 4.85%. Borehole water, uncooked food and lack of access to a latrine constitute a risk of being infected by Salmonella spp and Shigella spp species. Of the 8 antibiotics tested, Salmonella spp and Shigella spp strains showed good sensitivity to nalidixic acid (100% for Salmonella and 90 for Shigella) and to ciprofloxacin (90.9% for Salmonella and 75% for Shigella). Resistance to ampicillin was found in 81.81% of Salmonella species and 78.57% of Shigella species, as was resistance to chloramphenicol (81.81% of Salmonella species and 67.85% of Shigella species). Similarly, cleanliness of the service and equipment is an essential factor in preventing Salmonella and Shigella infections.展开更多
Objective To investigate the characteristic of integrons and the relationship between integrons and antimicrobial resistance in Shigella spp. Methods Ninety Shigella strains (83 S. flexneri and 7 S. sonnei) were isola...Objective To investigate the characteristic of integrons and the relationship between integrons and antimicrobial resistance in Shigella spp. Methods Ninety Shigella strains (83 S. flexneri and 7 S. sonnei) were isolated from the stools of patients in China. Susceptibility to 8 antimicrobials was tested for all isolated strains. PCR, RFLP and sequencing analysis of integrons were applied to all of them. Results High prevalence of multi‐drug resistance (95.6%) was identified. Of the isolates 79 (87.8%) carried integrase genes of class 1 integron (3.3%), class 2 integron (10.0%) or both (74.4%). No intI3 was detected in the tested isolates. The prevalence of intI2 was significantly higher in isolates with multi‐drug resistance to at least 3 antibiotics than that in isolates with resistance to 2 and less antibiotics (P<0.05). Gene cassettes dfrA17‐aadA5, dfrA12‐orfF‐aadA2 of class 1 integron and dfrA1‐sat1‐aadA1 of class 2 integron were identified. Conclusion The class 2 integron may play a role in the emergence of multi‐drug resistance in Shigella spp.展开更多
Objective: We aimed to evaluate the combined antibacterial effects of allicin in combination with levofloxacin and ceftriaxone on Shigella isolated from the intestinal tract in vitro. Materials and Methods: Using a ch...Objective: We aimed to evaluate the combined antibacterial effects of allicin in combination with levofloxacin and ceftriaxone on Shigella isolated from the intestinal tract in vitro. Materials and Methods: Using a checkerboard design, broth microdilution assay was used to test the effects of the compounds on the organism. We also determined the MIC of the two groups of antibacterial drugs against 30 strains of Shigella and calculated the fractional inhibitory concentration(FIC) index, to judge the combination effect. Result: After the combined application of allicin and ceftriaxone the MIC decreased significantly. Distribution of the FIC index was as follows: FIC ≤0.5, accounting for 10%; 0.5< FIC ≤1.0, accounting for 60%; 1 < FIC ≤2, accounting for 30%; FIC >2, percentage is zero. After combined application of allicin and levofloxacin, distribution of FIC index was as follows: FIC≤0.5, ratio is zero; 0.5< FIC ≤1, accounting for 56.7%; 1 < FIC ≤2, accounting for 43.3%; FIC >2, ratio is zero. Conclusion: After the combined use of ceftriaxone, levofloxacin, and allicin, most of the tests showed synergistic effects and additive effects on Shigella, while some of them showed no correlation and no antagonistic effect.展开更多
Background: Shigella is one of the most serious pathogens associated with bloody diarrhea in children. The empiric antibiotic therapy of enteric illness with blood streaked stool leads to emergence of multi drug resis...Background: Shigella is one of the most serious pathogens associated with bloody diarrhea in children. The empiric antibiotic therapy of enteric illness with blood streaked stool leads to emergence of multi drug resistant (MDR) Shigella. The condition gets exacerbated by presence of integrons that facilitate the horizontal spread. Virulence genes associated with MDR Shigella modulate the patient outcome, particularly in children. Objectives: The present study was aiming at isolation of MDR Shigella from children with diarrheal sickness and characterization of those isolates as regarding presence of class 1 integrase and other virulence genes. Methods: Four hundred and ninety patients under the age of five suffering from diarrheal illness were examined for presence of Shigella in their stool specimens. MDR Shigella was determined using the antibiotic susceptibility testing by disc diffusion method;those isolates were tested for presence of class 1 integrase by PCR. Multiplex PCR assay was used to determine the presence of virulence genes, virA, ial, sen, set1A, set1B, sat, ipaBCD, ipaH and stx in the MDR Shigella isolates. Results: The isolation rate of Shigella from pediatric patients was 5.3%. Most of the isolated Shigella (57.7%) were from infants between 12 and 23 month. 73.1% of the identified Shigella were MDR. intI1 gene was present in 78.9% of MDR isolates. Muliplex PCR revealed that ipaH and ipaBCD, virA, sat, ial, set1A and set1B, sen were detected in 94.7%, 78.9%, 73.7%, 68.4%, 42.1%, 36.8% of the MDR Shigella isolates respectively. Conclusion: The MDR isolates represented a considerable percentage of Shigella detected in pediatric patients. Presence of intI1 gene in most of MDR Shigella reflects the higher possibility of resistant strains spread. Existence of a variety of virulence genes in those isolates is an important indicator of serious disease outcome.展开更多
Objective:To investigate the antimicrobial resistance patterns and prevalence of integrons in Shigella species isolated from children with diarrhea in southwest Iran.Methods:In this study,1530 stool samples were colle...Objective:To investigate the antimicrobial resistance patterns and prevalence of integrons in Shigella species isolated from children with diarrhea in southwest Iran.Methods:In this study,1530 stool samples were collected from children under 15 years with diarrhea referred to teaching hospitals in Ahvaz and Abadan,southwest Iran.Shigella spp.were identified by standard biochemical tests and PCR.The antibiotic resistance pattern of all Shigella isolates was determined by the disk diffusion method and minimum inhibitory concentration(MIC)by E-test.Results:Of 1530 stool samples,91(5.9%,91/1530)were positive for Shigella spp.the most common Shigella isolates were Shigella flexneri 47(51.6%,47/1530).Antibiotic susceptibility tests showed that the highest antibiotic resistance was related to trimethoprimsulfamethoxazole(87.9%,80/91)and ampicillin(86.8%,79/91).Multiplex PCR results revealed that 56%and 86.9%of Shigella isolates carried integron classⅠand integron classⅡgenes,respectively.None of the isolates included the integron classⅢgene.Conclusions:The high prevalence of multi-drug resistance in Shigella isolates in our area increases the concerns about dissemination of the antibiotic-resistant isolates in this bacterium.展开更多
基金Project supported by the National Natural Science Foundation of China,No.39370040.
文摘AIM To investigate the specific pathogenesis ofO-polysaccharide (O--PS) which is on the outermembrane of lipopolysaccharides (LPS) fromShigella fi^eri.METHODS The O--PS was isolated and purifiedfrom Shigella nexneri 5 MgoT by enzymatichydrolysis and gel chromatography. Effects ofO--PS were observed by in vitro experiment,(HeLa cell Culture ), and in vivo experiment(rabbit lieal loop assay).RESULTS ID vitro and in vivo e-cP6riments withthe purified O--PS from Shigells flexnefi revealedthat the O--PS alone was toxic to Hela cells andcaused mucosal inflammation and hemorrhagicexudation in lieal loop of rabbit.DISCUSSION O--PS might b6 one of the factorscausing diarrhea and its mechanism wasdifferent from endotoxin reaction of LPS. Themolecular mechanism of O-PS need furtherstudies.
基金Supported by the National Key R&D Program of China,No.2018YFC2000300and CAMS Innovation Fund for Medical Sciences,No.2018-I2M-1-002.
文摘BACKGROUND Shigella flexneri(S.flexneri)is a major pathogen causing acute intestinal infection,but the systematic oxidative damage incurred during the course of infection has not been investigated.AIM To investigate the incurred systemic RNA oxidative damage and the diagnostic value of RNA oxidative metabolites during S.flexneri-induced intestinal infection.METHODS In this study,a Sprague-Dawley rat model of acute intestinal infection was established by oral gavage with S.flexneri strains.The changes in white blood cells(WBCs)and cytokine levels in blood and the inflammatory response in the colon were investigated.We also detected the RNA and DNA oxidation in urine and tissues.RESULTS S.flexneri infection induced an increase in WBCs,C-reactive protein,interleukin(IL)-6,IL-10,IL-1β,IL-4,IL-17a,IL-10,and tumor necrosis factorα(TNF-α)in blood.Of note,a significant increase in urinary 8-oxo-7,8-dihydroguanosine(8-oxo-Gsn),an important marker of total RNA oxidation,was detected after intestinal infection(P=0.03).The urinary 8-oxo-Gsn level returned to the baseline level after recovery from infection.In addition,the results of a correlation analysis showed that urinary 8-oxo-Gsn was positively correlated with the WBC count and the cytokines IL-6,TNF-α,IL-10,IL-1β,and IL-17α.Further detection of the oxidation in different tissues showed that S.flexneri infection induced RNA oxidative damage in the colon,ileum,liver,spleen,and brain.CONCLUSION Acute infection induced by S.flexneri causes increased RNA oxidative damage in various tissues(liver,spleen,and brain)and an increase of 8-oxo-Gsn,a urinary metabolite.Urinary 8-oxo-Gsn may be useful as a biomarker for evaluating the severity and prognosis of infection.
文摘AIM: To profile the immunogenic proteins of Shigella flexneri (S. flexneri) expressed during human infection using a proteomic approach. METHODS: Soluble and membrane protein extractions of S. flexneri 2457T were separated by two-dimensional gel electrophoresis (2-DE). Proteins were transferred to PVDF membrane and immunoblotted with sera from shigellosis patients. Reactive protein spots were matched to Coomassie stained gels run in parallel, cut out and trypsin digested. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS) was used to determine the peptide mass fingerprints, which were searched in the MASCOT database to identify the protein. RESULTS: A total of 8 immunoreactive proteins were successfully identified from the Coomassie stained gels in three repeats. Six of these proteins have not previously been reported as immunogenic in S. flexneri. These proteins could be potential candidates for vaccine or attenuation studies. CONCLUSION: Soluble and membrane proteins of S. flexneri 2457T have been screened by 2-DE and immunoblotting with sera from shigellosis patients. Eight proteins are identified as immunogenic.
基金Supported by the Capital "248" Key Innovation Project, No. H010210360119, State Basic Research Development Program of China No. 973 Program, G1999054103 and 2005CB22904 and National Natural Science Foundation of China No. 30470101
文摘AIM: To screen the immunogenic membrane proteins ofShigella flexneri 2a 2457T.METHODS: The routine two-dimensional polyacrylamidegel electrophoresis (2-DE) and Western blotting werecombined to screen immunogenic proteins of S. flexneri2a 2457T. Serum was gained from rabbits immunizedwith the same bacteria. Immunogenic spots werecut out from the polyacrylamide gel and digested bytrypsin in-gel. Matrix-assisted laser desorption/ionizationtime of flight-mass spectrometry (MALDI-TOF-MS)was performed to determine the molecular weight ofpeptides. Electrospray ionization (ESI-MS/MS) wasperformed to determine the sequences of the interestingpeptides.RESULTS: A total of 20 spots were successfullyidentified from Coomassie brilliant blue stained gelsrepresenting 13 protein entries, 5 known antigens and8 novel antigens. A hypothetical protein (YaeT) wasdetected, which might be a candidate target of vaccine.CONCLUSION: Membrane proteins of S. flexneri 2a2457T were successfully observed by 2-DE. Severalknown and novel antigens were identified by massspectrum.
基金This work was supported by the National Key Basic Research Program of China （973 Program, No. 2005CB522904） the National Natural Science Foundation of China （No. 30470101）.
文摘Shigella flexneri is an infectious pathogen that causes dysentery to human, which remains a serious threat to public health, particularly in developing countries. In this study, the global protein expression patterns of S. flexneri during transition from exponential growth to stationary phase in vitro were analyzed by using 2-D PAGE combined with MALDI-TOF MS. In a time-course experiment with five time points, the relative abundance of 49 protein spots varied significantly. In- terestingly, a putative outer membrane protein YciD (OmpW) was almost not detected in the exponential growth phase but became one of the most abundant proteins in the whole stationary-phase proteome. Some proteins regulated by the global regulator FNR were also significantly induced (such as AnsB, AspA, FrdAB, and KatG) or repressed (such as AceEF, OmpX, SodA, and SucAB) during the growth phase transition. These proteins may be the key effectors of the bacterial cell cycle or play important roles in the cellular maintenance and stress responses. Our expression profile data provide valuable information for the study of bacterial physiology and form the basis for future proteomic analyses of this pathogen.
文摘Aim: Shigella flexneri (S. flexneri) is a gram-negative enterobacterium responsible for severe intestinal end systemic infection in humans. The bacteria can reach the liver due to degeneration of the colonic epithelium. Hypoxia is present in many human diseases and can induce the expression of the transcription factor HIF-1alpha that may have a cell protective role. The influence of hypoxia and HIF-1alpha on bacterial infection, studied in this work, is unclear. Hypoxia inducible factor-1alpha (HIF-1alpha) is a transcription factor that acts as a master regulator of gene expression induced by hypoxia. Methods: We compared the ability of S. flexneri to invade rat hepatocytes in primary culture both in normoxic and hypoxic conditions. We evaluated TNF-alpha released by hepatocytes, apoptosis rate and HIF-1alpha expression by confocal microscopy as well as real time PCR technique. Results: We showed that S. flexneri invaded less hepatocytes previously submitted to 24 h hypoxia (6.5% O2) than those cultivated in normoxia (21% O2). S. flexneri also induced HIF-1α expression in hepatocytes, TNF-α secretion and apoptosis. Conclusion: a) Hypoxia alone was not a stimulus to TNF-α secretion, but induced cell apoptosis and HIF-1α expression;b) S. flexneri was able to invade rat hepatocytes and hypoxia apparently influenced significantly bacterial cell invasiveness;c) HIF-1α was expressed in hypoxic conditions, and it was also stimulated by S. flexneri.
基金supported by Yunnan Fundamental Research Projects(Grant No.202101BE070001-046)the Natural Science Foundation of China(31960286).
文摘This study investigated antibacterial and antibiofilm activity of the combined use of phenyllactic acid(PLA)and bacteriocin XJS01 against Shigella flexneri_14.The minimum inhibitory concentration(MIC)of PLA and XJS01 against S.flexneri_14 was 2.45 mg/mL and 18.75μg/mL,respectively.Growth and kill kinetics assays showed that the combined use of 1/2MIC PLA plus 1/2MIC XJS01 had a better activity against planktonic S.flexneri_14 compared to treatment with PLA and XJS01 used singly(1/2MIC and 2MIC).Cellular biochemical and morphological analysis revealed the remarkable ability of the combination in disrupting cell appearance and promoting deformation of planktonic S.flexneri_14 compared to single use.Moreover,S.flexneri_14 biofilm formation was inhibited and degraded by the combination,which showed a more remarkable antibiofilm activity than PLA and XJS01 when used singly.This study demonstrates the synergistic antibacterial activity of PLA and XJS01 against S.flexneri_14 in either planktonic or biofilm states in foods.
基金supported by the State“973”Key Basic Research Program(Grant No.G1999054103)the State“863”High-Tech Project(Grant No.Z19-02-05-01)+1 种基金Beijing Innovation Engineering(Grant No.955020700)the North China Pharmaceutical Corporation(NCPC)
文摘Comparative genome analysis is performed between Shigella flexneri 2a strain 301 and its close relatives, the nonpathogenic E. Coli K-12 strain MG1655. Result shows that there are 136 DNA segments whose size is larger than 1000 bp absent from Shigella flexneri 2a strain 301, which is up to 717253 bp in total length. These deleted segments altogether contain 670 open reading frames (ORFs). Prediction of these ORFs indicates that there are 40% genes of unknown function. The other genes of definite functions encode metabolic enzymes, structure proteins, transcription regulatory factors and some elements correlated with horizontal transfer. Here we compare the complete genomic sequences of the two closely related species, which differ in pathogenic phenotype. To our knowledge, this not only reveals the difference of genomic sequence between the two important enteric pathogens for the first time, but also provides valuable clues to further researches in its process of physiological activity, pathogenesis and the evolution of enteric bacteria.
基金This work was supported by the High Technology Project(Grant No.2001AA223011)the State Key Basic Research Program(Grant No.G1999054105).
文摘In order to overcome the defects of difficult gene operations in low-copy suicide plasmid pCVD442, Gateway technology was applied in the construction process of recombinant plasmid for gene knockout in this study. With this improved knockout system, we inactivated sitC gene, which is associated with iron transport in Shigella flexneri 2a strain 301, to yield the mutant, MTS. The functional detection of the mutant was performed at the level of culture medium, cell and animal experiment, respectively. The gene expression profiles were compared with DNA microarray between the mutant and the wild type under iron-restricted conditions. The results showed that MTS grew obviously less well than the wild-type strains in L broth containing 150 μmol/L iron chelator DIP(2,2′-dipyridyl). Addition of iron or manganese to the cultures stimulated the growth of MTS to wild-type levels in rich culture medium. In either the experiment on the ability of intracellular multiplication and cell-to-cell spread in HeLa and U937 cell lines, or the experiment on keratoconjunctivitis in guinea pigs, MTS showed no obvious changes in virulence compared with the parental strain Sf301. When 65 μmol/L DIP was added to the cultured HeLa cells, the ability of intracellular multiplication of MTS reduced about 51.6% as compared with that of Sf301. The analysis of expression profiles under iron-limited condition showed that MTS was more sensitive for the change of iron deficiency than Sf301. There are 106 more up-regulated genes in MTS than in wild-type strains, which are involved in membrane transportation, amino acid metabolism and uncategorized function genes, while down-regulated genes are mainly in- volved in energy and carbohydrate metabolism. Under low iron conditions, the expression levels of known iron-transport associated genes generally increased. Additionally, the number of these genes and their increase amplitude in MTS are more than those in Sf301. Together, these results confirmed that Sit iron-transport system is important for the growth of Shigella.
文摘An in vivo expression technology (IVET) was applied to screen s.flexneri 2a genes induced after invasion of epithelial cells, and virulence-related genes were further identified by mutational analysis. Thirteen intracellular induced genes were identified with a HeLa cell infection model. Of these, two were identified as alkylation-related genes; one was related to metabolism; one encoded a transcriptional regulator; three were identified as insertion elements; three ap- peared to be antisense to genes involved in the transmethylation,biosyntheseis, and phos- photransferase system;and three were predicted to encode polypeptides with unknown functions. Intracellular survival assavs showed that the mutants of alkA,citC and wcaJ genes had lower capability of intracellular replication or survival than the the wild-type strain.The results indicated that alkA, citC and wcaJ genes could take part in the intracellular survival or replication of S. flexneri 2a and the capability of intracellular survival or replication could be one of the major virulence elements. However, the yaiC mutant was able to survive in the murine infection assay but almost not in HeLa cell infection assay. Very possibly, yaiC gene was involved in the other mechanism of S. flexneri virulence. This study might lead to a better understanding of the intra- cellular survival or proliferation process of S. flexneri 2a and perhaps provide insights into the pathogenicity of this pathogen.
文摘Introduction: Salmonella and Shigella are gram-negative bacilli that are resistant to most antibiotics and play an important role in an etiology of diarrhea disease. The aim of the study was to determine the prevalence of Salmonella spp and Shigella spp isolated from stool and blood samples in the city of N’Djamena. Materials and Method: This was a prospective study conducted in the four district hospitals of N’Djamena from 14 July 2022 to 31 December 2022. A questionnaire form was drawn up to collect the information sent to the study patients. The samples were analyzed at the CHU de la Mère et de l’Enfant, Labo-Redes laboratory according to their protocols and the standard of the antibiogram committee of the French microbiology society. Results: Of the 803 biological samples analyzed, 39 were positive for Salmonella spp and Shigella spp, including 15 for Salmonella and 24 for Shigella, giving an overall prevalence rate of 4.85%. Borehole water, uncooked food and lack of access to a latrine constitute a risk of being infected by Salmonella spp and Shigella spp species. Of the 8 antibiotics tested, Salmonella spp and Shigella spp strains showed good sensitivity to nalidixic acid (100% for Salmonella and 90 for Shigella) and to ciprofloxacin (90.9% for Salmonella and 75% for Shigella). Resistance to ampicillin was found in 81.81% of Salmonella species and 78.57% of Shigella species, as was resistance to chloramphenicol (81.81% of Salmonella species and 67.85% of Shigella species). Similarly, cleanliness of the service and equipment is an essential factor in preventing Salmonella and Shigella infections.
基金supported by the Research Fund from Ministry of Health of the People’s Republic of China, WKJ2007‐2‐024
文摘Objective To investigate the characteristic of integrons and the relationship between integrons and antimicrobial resistance in Shigella spp. Methods Ninety Shigella strains (83 S. flexneri and 7 S. sonnei) were isolated from the stools of patients in China. Susceptibility to 8 antimicrobials was tested for all isolated strains. PCR, RFLP and sequencing analysis of integrons were applied to all of them. Results High prevalence of multi‐drug resistance (95.6%) was identified. Of the isolates 79 (87.8%) carried integrase genes of class 1 integron (3.3%), class 2 integron (10.0%) or both (74.4%). No intI3 was detected in the tested isolates. The prevalence of intI2 was significantly higher in isolates with multi‐drug resistance to at least 3 antibiotics than that in isolates with resistance to 2 and less antibiotics (P<0.05). Gene cassettes dfrA17‐aadA5, dfrA12‐orfF‐aadA2 of class 1 integron and dfrA1‐sat1‐aadA1 of class 2 integron were identified. Conclusion The class 2 integron may play a role in the emergence of multi‐drug resistance in Shigella spp.
文摘Objective: We aimed to evaluate the combined antibacterial effects of allicin in combination with levofloxacin and ceftriaxone on Shigella isolated from the intestinal tract in vitro. Materials and Methods: Using a checkerboard design, broth microdilution assay was used to test the effects of the compounds on the organism. We also determined the MIC of the two groups of antibacterial drugs against 30 strains of Shigella and calculated the fractional inhibitory concentration(FIC) index, to judge the combination effect. Result: After the combined application of allicin and ceftriaxone the MIC decreased significantly. Distribution of the FIC index was as follows: FIC ≤0.5, accounting for 10%; 0.5< FIC ≤1.0, accounting for 60%; 1 < FIC ≤2, accounting for 30%; FIC >2, percentage is zero. After combined application of allicin and levofloxacin, distribution of FIC index was as follows: FIC≤0.5, ratio is zero; 0.5< FIC ≤1, accounting for 56.7%; 1 < FIC ≤2, accounting for 43.3%; FIC >2, ratio is zero. Conclusion: After the combined use of ceftriaxone, levofloxacin, and allicin, most of the tests showed synergistic effects and additive effects on Shigella, while some of them showed no correlation and no antagonistic effect.
文摘Background: Shigella is one of the most serious pathogens associated with bloody diarrhea in children. The empiric antibiotic therapy of enteric illness with blood streaked stool leads to emergence of multi drug resistant (MDR) Shigella. The condition gets exacerbated by presence of integrons that facilitate the horizontal spread. Virulence genes associated with MDR Shigella modulate the patient outcome, particularly in children. Objectives: The present study was aiming at isolation of MDR Shigella from children with diarrheal sickness and characterization of those isolates as regarding presence of class 1 integrase and other virulence genes. Methods: Four hundred and ninety patients under the age of five suffering from diarrheal illness were examined for presence of Shigella in their stool specimens. MDR Shigella was determined using the antibiotic susceptibility testing by disc diffusion method;those isolates were tested for presence of class 1 integrase by PCR. Multiplex PCR assay was used to determine the presence of virulence genes, virA, ial, sen, set1A, set1B, sat, ipaBCD, ipaH and stx in the MDR Shigella isolates. Results: The isolation rate of Shigella from pediatric patients was 5.3%. Most of the isolated Shigella (57.7%) were from infants between 12 and 23 month. 73.1% of the identified Shigella were MDR. intI1 gene was present in 78.9% of MDR isolates. Muliplex PCR revealed that ipaH and ipaBCD, virA, sat, ial, set1A and set1B, sen were detected in 94.7%, 78.9%, 73.7%, 68.4%, 42.1%, 36.8% of the MDR Shigella isolates respectively. Conclusion: The MDR isolates represented a considerable percentage of Shigella detected in pediatric patients. Presence of intI1 gene in most of MDR Shigella reflects the higher possibility of resistant strains spread. Existence of a variety of virulence genes in those isolates is an important indicator of serious disease outcome.
基金supported by the Vice-Chancellor for Research grant(Grant No.U98-564)of Abadan University of Medical Science
文摘Objective:To investigate the antimicrobial resistance patterns and prevalence of integrons in Shigella species isolated from children with diarrhea in southwest Iran.Methods:In this study,1530 stool samples were collected from children under 15 years with diarrhea referred to teaching hospitals in Ahvaz and Abadan,southwest Iran.Shigella spp.were identified by standard biochemical tests and PCR.The antibiotic resistance pattern of all Shigella isolates was determined by the disk diffusion method and minimum inhibitory concentration(MIC)by E-test.Results:Of 1530 stool samples,91(5.9%,91/1530)were positive for Shigella spp.the most common Shigella isolates were Shigella flexneri 47(51.6%,47/1530).Antibiotic susceptibility tests showed that the highest antibiotic resistance was related to trimethoprimsulfamethoxazole(87.9%,80/91)and ampicillin(86.8%,79/91).Multiplex PCR results revealed that 56%and 86.9%of Shigella isolates carried integron classⅠand integron classⅡgenes,respectively.None of the isolates included the integron classⅢgene.Conclusions:The high prevalence of multi-drug resistance in Shigella isolates in our area increases the concerns about dissemination of the antibiotic-resistant isolates in this bacterium.
