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The COVID-19 pandemic presents a serious public health challenge in all countries. However, repercussions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections on future global health are still being investigated, including the pandemic's potential effect on the emergence and spread of global antimicrobial resistance (AMR). Critically ill COVID-19 patients may develop severe complications, which may predispose patients to infection with nosocomial bacterial and/or fungal pathogens, requiring the extensive use of antibiotics. However, antibiotics may also be inappropriately used in milder cases of COVID-19 infection. Further, concerns such as increased biocide use, antimicrobial stewardship/infection control, AMR awareness, the need for diagnostics (including rapid and point-of-care diagnostics) and the usefulness of vaccination could all be components shaping the influence of the COVID-19 pandemic. In this publication, the authors present a brief overview of the COVID-19 pandemic and associated issues that could influence the pandemic's effect on global AMR.
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A set of 12 novel hydroxamate compounds (NHCs), structurally designed as inhibitors of histone deacetylase (HDAC) enzyme, were synthesized at our facility. These were adamantane derivatives with N-hydroxyacetamide as pharmacophore, and each of these compounds was tested for potentiating activity on fluconazole. The concentration of fluconazole which completely inhibited (concentration of complete inhibition [CCI]) the growth of Candida albicans ATCC 90028 and C. albicans ATCC 64550 was determined by micro-dilution method in the absence and presence of NHCs. The CCI of fluconazole without the NHC combination was 64 µg/ml and 1024 µg/ml against C. albicans ATCC 90028 and C. albicans ATCC 64550, respectively. The majority of the NHCs potentiated the fluconazole activity markedly as CCI of fluconazole against C. albicans ATCC 90028 reduced to 0.25 µg/ml. Similarly, CCI of fluconazole against C. albicans ATCC 64550 reduced to 4-8 µg/ml in combination with majority of NHCs while the best activity was displayed by the compound 1 with a reduction of CCI to 0.5 µg/ml. The study results revealed the potential usage of hydroxamate derivatives, structurally designed as HDAC inhibitors to enhance the activity of fluconazole against C. albicans.
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Emergence of extended-spectrum ß-lactamase (ESBL) and fluoroquinolone resistance among ocular Enterobacteriaceae is increasing in higher frequency. Therefore, studies are being carried out to understand their multidrug resistance pattern. A total of 101 Enterobacteriaceae isolates recovered from various ocular diseases in a tertiary eye care center at Chennai, India during the period of January 2011 to June 2014 were studied. Forty one randomly chosen isolates were subjected to antibiotic susceptibility by minimum inhibitory concentration (MIC) and genotypic analysis. Of them, 16 were ESBL producers, one was carbapenemase producer and four were resistant to ertapenem which could be due to porin loss associated with AmpC production, and 17 were resistant to fluoroquinolones. Sixteen isolates harbored ESBL genes in which 14 had more than one gene and none of them were positive for blaNDM-1 gene. QNR genes were detected in 18 isolates. ESBL producers were predominantly isolated from conjunctiva. A high degree of ESBL production and fluoroquinolone resistance is seen among the genus Klebsiella sp. Hence, monitoring the rate of ESBL prevalence plays a vital role in the administration of appropriate intravitreal antibiotics to save the vision and also to reduce the development of drug resistance in ocular pathogens.
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BACKGROUND: Microbial resource orchid is a collection of Gram-positive and Gram-negative clinical isolates sourced from different hospitals and diagnostic laboratories in India. We determined the antibiotic susceptibility of a set of Gram-negative Enterobacteriaceae and non-fermenter clinical isolates from microbial resource orchid, collected during the period of 2002-2012 against commonly used antibiotics. MATERIALS AND METHODS: A total of 247 Gram negative strains consisting of 142 Enterobacteriaceae and 105 non-fermenters from microbial resource orchid were selected for determining minimum inhibitory concentration against ß-lactams, aminoglycosides, quinolone, and tetracycline by agar dilution method as per clinical and laboratory standards institute guidelines. RESULTS: All the isolates had high resistance to ampicillin, piperacillin, ceftazidime, gentamicin, tetracycline, and ciprofloxacin. Pseudomonas aeruginosa showed moderate resistance to carbapenems. CONCLUSION: This study demonstrated the high level of antibiotic resistance among the strains collected under microbial resource orchid and further, such data and the strains can be used in new chemical entities profiling.
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Candida albicans is a mucosal commensal organism capable of causing superficial (oral and vaginal thrush) infections in immune normal hosts, but is a major pathogen causing systemic and mucosal infections in immunocompromised individuals. Azoles have been very effective anti-fungal agents and the mainstay in treating opportunistic mold and yeast infections. Azole resistant strains have emerged compromising the utility of this class of drugs. It has been shown that azole resistance can be reversed by the co-administration of a histone deacetylase (HDAC) inhibitor, suggesting that resistance is mediated by epigenetic mechanisms possibly involving Hos2, a fungal deacetylase. We report here the cloning and functional characterization of HOS2 (High Osmolarity Sensitive) , a gene coding for fungal histone deacetylase from C. albicans. Inhibition studies showed that Hos2 is susceptible to pan inhibitors such as trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), but is not inhibited by class I inhibitors such as MS-275. This in vitro enzymatic assay, which is amenable to high throughput could be used for screening potent fungal Hos2 inhibitors that could be a potential anti-fungal adjuvant. Purified Hos2 protein consistently deacetylated tubulins, rather than histones from TSA-treated cells. Hos2 has been reported to be a putative NAD+ dependent histone deacetylase, a feature of sirtuins. We assayed for sirtuin activation with resveratrol and purified Hos2 protein and did not find any sirtuin activity.
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Staphylococcus aureus (S. aureus) is important both as a nosocomial and community acquired pathogen causing various degrees of infections. Typing S. aureus has been a question that is still being addressed. Bacteriophage typing is still used as a golden standard for typing though molecular methods are investigated. In developing countries where neither molecular typing nor the bacteriophage typing methods can be routinely used, the recently developed capsular typing method can be considered as screening method. We compared capsular typing with bacteriophage typing of the strains isolated in Gulbarga, India. We observed that the typeability of capsular typing was significantly higher (96%) among the phage typed strains, and the predominant capsular type in the region was type-8. The data so generated can be used to group S. aureus based on capsules both as a screening prior to bacteriophage typing, and to identify capsular candidate for developing prophylactic and therapeutic alternatives.
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Escherichia coli K1 is the most common Gram-negative bacillary organism causing neonatal meningitis. E. coli K1 binding to and invasion of human brain microvascular endothelial cells (HBMECs) is a prerequisite for its traversal of the blood-brain barrier (BBB) and penetration into the brain. In the present study, we identified NlpI as a novel bacterial determinant contributing to E. coli K1 interaction with HBMECs. The deletion of nlpI did not affect the expression of the known bacterial determinants involved in E. coli K1-HBMEC interaction, such as type 1 fimbriae, flagella, and OmpA, and the contribution of NlpI to HBMECs binding and invasion was independent of those bacterial determinants. Previous reports have shown that the nlpI mutant of E. coli K-12 exhibits growth defect at 42 degrees C at low osmolarity, and its thermosensitive phenotype can be suppressed by a mutation on the spr gene. The nlpI mutant of strain RS218 exhibited similar thermosensitive phenotype, but additional spr mutation did not restore the ability of the nlpI mutant to interact with HBMECs. These findings suggest the decreased ability of the nlpI mutant to interact with HBMECs is not associated with the thermosensitive phenotype. NlpI was determined as an outer membrane-anchored protein in E. coli, and the nlpI mutant was defective in cytosolic phospholipase A(2)alpha (cPLA(2)alpha) phosphorylation compared to the parent strain. These findings illustrate the first demonstration of NlpI's contribution to E. coli K1 binding to and invasion of HBMECs, and its contribution is likely to involve cPLA(2)alpha.
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Adhesión Bacteriana/fisiología , Encéfalo/microbiología , Células Endoteliales/microbiología , Proteínas de Escherichia coli/fisiología , Escherichia coli/fisiología , Lipoproteínas/fisiología , Meningitis por Escherichia coli/microbiología , Proteínas de la Membrana Bacteriana Externa/fisiología , Western Blotting , Encéfalo/irrigación sanguínea , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/fisiología , Flagelos/genética , Flagelos/fisiología , Fosfolipasas A2 Grupo IV/fisiología , Humanos , Recién Nacido , Lipoproteínas/genética , MicrocirculaciónRESUMEN
OBJECTIVES: In the pursuit of developing a second-generation oxazolidinone, we have identified OCID0050 as a novel oxazolidinone with enhanced activity against bacterial strains resistant to methicillin, vancomycin and linezolid. METHODS: MIC and MBC determinations were performed according to CLSI guidelines. Linezolid-resistant bacterial strains were generated in-house; inoculum effect, pH effect and kill kinetics experiments were performed as per standard protocols. RESULTS AND CONCLUSIONS: OCID0050 demonstrated better inhibitory potency against many of the tested clinically significant strains by generally showing 2-4-fold lower MICs than linezolid. In addition, it has higher inhibitory activity against linezolid-resistant strains.
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Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Oxazolidinonas/farmacología , Animales , Farmacorresistencia Bacteriana , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Viabilidad Microbiana/efectos de los fármacosRESUMEN
Escherichia coli K1 is the most common Gram-negative organism causing neonatal meningitis. Binding to human brain microvascular endothelial cells (HBMEC) is an essential step for E. coli K1 traversal of the blood-brain barrier. In this study, we examined expression profiles of E. coli K1 strain RS218 during its binding to HBMEC. Comparison of HBMEC-bound E. coli K1 with collagen-bound E. coli revealed more than one hundred genes whose expression patterns were significantly changed in HBMEC-bound E. coli K1, but not in collagen-bound E. coli K1. These genes are involved mainly in cell surface decorations, cellular function, and nitrogen metabolism. The roles of several representative genes including frdA, clpB, carA, and ompT in HBMEC binding were verified with their isogenic mutants, which exhibited significantly less HBMEC binding capability compared to that of the parent strain. This transcriptome analysis provided us with the first genomic-level view of E. coli and HBMEC interactions.
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Encéfalo/metabolismo , Endotelio Vascular/metabolismo , Escherichia coli/genética , Perfilación de la Expresión Génica , Escherichia coli/metabolismo , HumanosRESUMEN
Polysaccharide (PS) and tetanus toxoid (TT) protein conjugate vaccines were prepared using O-acetylated (OAc+), O-acetyl negative (OAc(-)) and chemically de-O-acetylated (de-OAc) meningococcal W135 PS. The PSs were activated by periodate oxidation and coupled to hydrazine derivatized TT. High performance anion exchange chromatography of acid hydrolysates of periodate activated W135 PSs, showed that galactose residues in OAc+ PS were more sensitive to the periodate oxidation step than they were in the OAc(-) PS or de-OAc PS. Mouse antisera against OAc(-)-TT conjugate vaccines recognized both OAc(-) and OAc+ PS by ELISAs and had high bactericidal titers against both OAc+ and OAc(-) W135 strains. Purified high molecular weight (HMW) conjugates showed higher PS to protein ratios in OAc(-)-TT(HMW) and de-OAc-TT(HMW) indicating better conjugation efficiency than OAc+-TT(HMW) conjugate. Antisera against the HMW fractions gave higher bactericidal titers than antisera against unfractionated conjugates. Inhibition ELISAs indicated that OAc(-) and OAc+ HMW conjugates induced antibodies that bound both OAc+ and OAc(-) PS. Thus, for W135, PS O-acetylation does not contribute a dominant immunogenic epitope. The OAc(-) PS may be a good starting material for preparing W135 PS-TT conjugate vaccines using periodate oxidation.
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Vacunas Bacterianas/inmunología , Neisseria meningitidis Serogrupo W-135/inmunología , Polisacáridos Bacterianos/inmunología , Toxoide Tetánico/inmunología , Acetilación , Animales , Anticuerpos Antibacterianos/inmunología , Vacunas Bacterianas/química , Actividad Bactericida de la Sangre/inmunología , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Epítopos/inmunología , Femenino , Hidrólisis , Infecciones Meningocócicas/inmunología , Infecciones Meningocócicas/prevención & control , Ratones , Neisseria meningitidis Serogrupo W-135/química , Oxidación-Reducción , Ácido Peryódico , Polisacáridos Bacterianos/química , Toxoide Tetánico/química , Vacunas Conjugadas/química , Vacunas Conjugadas/inmunologíaRESUMEN
Tenacious adherence and autoaggregation of wild-type Actinobacillus actinomycetemcomitans strains jeopardize reliability of determined cell concentrations, e.g. for studies on bacteria-host interactions. We first compared the efficacy of two methods, an indirect and a direct method, for homogenizing cell suspensions of a wild-type, autoaggregating (SA269) strain and of a non-autoaggregating laboratory variant (ATCC 43718) used as a reference. Since the direct method left visible clumps in SA269 suspension, only the indirect method was further tested. In serial dilutions of the homogenized cell suspensions of strains SA269 and ATCC 43718, the OD600 values (R2=0.99, R2=0.99, respectively) and protein concentrations (R2=0.93, R2=0.95, respectively) correlated significantly (all P<0.002) with the dilution factor. There were no differences (P>0.05) in the bacterial viable counts between the two strains or between suspending solutions, i.e., PBS and water, the cell concentrations demonstrating 1x10(9) cells/ml at OD600=1. Repeated microscopic cell counts did not differ (P>0.05) from each other. Large aggregates occurred as 1% of cell units counted. Dispersing bacterial mass indirectly to solution leads to homogeneous cell suspensions with repeatable cell concentrations. Viability of A. actinomycetemcomitans was also maintained when cells were suspended in water.
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Aggregatibacter actinomycetemcomitans/fisiología , Aggregatibacter actinomycetemcomitans/crecimiento & desarrollo , Aggregatibacter actinomycetemcomitans/ultraestructura , Recuento de Colonia Microbiana , Microscopía Electrónica , Estadísticas no ParamétricasRESUMEN
We have previously shown that outer membrane protein A (OmpA) and type 1 fimbriae are the bacterial determinants involved in Escherichia coli K1 binding to human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. In investigating the role of OmpA in E. coli K1 binding to HBMEC, we showed for the first time that ompA deletion decreased the expression of type 1 fimbriae in E. coli K1. Decreased expression of type 1 fimbriae in the ompA deletion mutant was largely the result of driving the fim promoter toward the type 1 fimbrial phase-OFF orientation. mRNA levels of fimB and fimE were found to be decreased with the OmpA mutant compared to the parent strain. Of interest, the ompA deletion further decreased the abilities of E. coli K1 to bind to and invade HBMEC under the conditions of fixing type 1 fimbria expression in the phase-ON or phase-OFF status. These findings suggest that the decreased ability of the OmpA mutant to interact with HBMEC is not entirely due to its decreased type 1 fimbrial expression and that OmpA and type 1 fimbriae facilitate the interaction of E. coli K1 with HBMEC at least in an additive manner.
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Proteínas de la Membrana Bacteriana Externa/genética , Encéfalo/microbiología , Endotelio Vascular/microbiología , Escherichia coli/patogenicidad , Fimbrias Bacterianas/metabolismo , Encéfalo/irrigación sanguínea , Capilares/citología , Capilares/microbiología , Células Cultivadas , Escherichia coli/genética , Fimbrias Bacterianas/genética , Eliminación de Gen , Perfilación de la Expresión Génica , Humanos , Familia de Multigenes , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras GenéticasRESUMEN
Group B streptococcus (GBS), the leading cause of neonatal meningitis, has been shown to invade human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. GBS invasion of HBMEC has been shown to require the host cell actin cytoskeleton rearrangements. The present study examined the mechanisms underlying actin cytoskeleton rearrangements that are involved in type III GBS invasion of HBMEC. We showed that type III GBS invasion was inhibited by genistein, a general tyrosine kinase inhibitor (mean 54% invasion decrease at 100 microM), and LY294002, a phosphatidylinositol 3 (PI3) kinase inhibitor (mean 70% invasion decrease at 50 microM), but not by PP2, an inhibitor of the Src family tyrosine kinases. We subsequently showed that the focal adhesion kinase (FAK) was the one of the host proteins tyrosine phosphorylated by type III GBS. Over-expression of a dominant negative form of the FAK C-terminal domain significantly decreased type III GBS invasion of HBMEC (mean 51% invasion decrease). In addition, we showed that FAK phosphorylation correlated with its association of paxillin, an adapter protein of actin filament, and PI3-kinase subunit p85. This is the first demonstration that FAK phosphorylation and its association with paxillin and PI3 kinase play a key role in type III GBS invasion of HBMEC.
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Barrera Hematoencefálica/enzimología , Barrera Hematoencefálica/microbiología , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Infecciones Estreptocócicas/enzimología , Infecciones Estreptocócicas/microbiología , Streptococcus agalactiae/patogenicidad , Actinas/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Western Blotting , Cromonas/farmacología , Citoesqueleto/metabolismo , Citoesqueleto/microbiología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Células Endoteliales/microbiología , Activación Enzimática , Proteína-Tirosina Quinasas de Adhesión Focal/antagonistas & inhibidores , Genisteína/farmacología , Humanos , Morfolinas/farmacología , Paxillin/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacologíaRESUMEN
BACKGROUND: Escherichia coli K1 is the most common gram-negative bacterium causing neonatal meningitis, but the mechanisms by which E. coli K1 causes meningitis are not clear. METHODS: We identified 22 E. coli RS218-derived genomic islands (RDIs), using a comparative genome analysis of meningitis-causing E. coli K1 strain RS218 (O18:K1:H7) and laboratory K-12 strain MG1655. Series of RDI deletion mutants were constructed and examined for phenotypes relevant to E. coli K1 meningitis. RESULTS: We identified 9 RDI deletion mutants (RDI 1, 4, 7, 12, 13, 16, 20, 21, and 22) that exhibited defects in meningitis development. RDI 16 and 21 mutants had profound defects in the induction of a high level of bacteremia in neonatal rats, and RDI 4 mutants exhibited a moderate defect in the induction of bacteremia. RDI 1 and 22 mutants showed defects in the ability to invade human brain microvascular endothelial cells (HBMECs), and RDI 12 mutants were defective in the ability to bind to HBMECs. RDI 13 and 20 mutants were defective in the ability to both bind to and invade HBMECs. RDI 7 mutants were defective in the induction of bacteremia and in the ability to both bind to and invade HBMECs. CONCLUSIONS: These results provide a framework for the future discovery and analysis of bacteremia and meningitis caused by E. coli K1 strain RS218.
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Escherichia coli/genética , Islas Genómicas , Meningitis por Escherichia coli/microbiología , Animales , Bacteriemia/microbiología , Secuencia de Bases , Escherichia coli/aislamiento & purificación , Femenino , Eliminación de Gen , Humanos , Masculino , Datos de Secuencia Molecular , Embarazo , Complicaciones Infecciosas del Embarazo/microbiología , Ratas , Ratas Sprague-Dawley , Factores de Virulencia/genéticaRESUMEN
In a search for novel bioactive cell surface structures of periodontal pathogens, it was found that sera from two patients with Actinobacillus actinomycetemcomitans-associated infections reacted strongly at 17 kDa on immunoblots of A. actinomycetemcomitans outer-membrane protein (OMP) preparations. The 17 kDa antigen was also recognized by anti-CsgA (Escherichia coli curli major subunit) antibody. The 17 kDa A. actinomycetemcomitans protein was identified as peptidoglycan-associated lipoprotein (PAL; AaPAL) by two-dimensional immunoblotting and subsequent sequence analysis by mass spectrometry and bioinformatics tools. AaPAL was an OMP and a lipoprotein, and it had an OmpA-like domain. In a group of middle-aged subjects (n = 26), serum reactivity to AaPAL was associated with the presence of periodontitis but not with the oral detection of A. actinomycetemcomitans. Both human sera and rabbit antisera against three different types of antigens, the gel-purified AaPAL, A. actinomycetemcomitans whole-cell antigens, and CsgA, recognized putative PALs of oral haemophili in addition to AaPAL. The results demonstrated that the novel AaPAL is a conserved bacterial lipoprotein. It is expressed in vivo and is strongly immunoreactive. The antigenic cross-reactivity found between AaPAL and oral haemophili may enhance local and systemic immuno-inflammatory reactions in periodontitis.
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Infecciones por Actinobacillus/inmunología , Aggregatibacter actinomycetemcomitans/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Lipoproteínas/inmunología , Peptidoglicano/inmunología , Periodontitis/microbiología , Infecciones por Actinobacillus/microbiología , Adulto , Anciano , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Secuencia de Bases , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Periodontitis/inmunología , Proteómica/métodos , ConejosRESUMEN
BACKGROUND: Central nervous system (CNS) tuberculosis is a serious, often fatal disease that disproportionately affects young children. It is thought to develop when Mycobacterium tuberculosis breaches the blood-brain barrier (BBB), which is composed of tightly apposed brain microvascular endothelial cells. However, the mechanism(s) involved in this process are poorly understood. METHODS: To better understand these processes, we developed an in vitro model of M. tuberculosis BBB infection using primary human brain microvascular endothelial cells. RESULTS: M. tuberculosis was found to both invade and traverse the model BBB significantly more than did M. smegmatis (a nonpathogenic mycobacterium). Invasion by M. tuberculosis across the BBB required host-cell actin cytoskeletal rearrangements. By microarray expression profiling, we found 33 M. tuberculosis genes to be highly up-regulated during the early stages of invasion of the BBB by M. tuberculosis; 18 of them belong to a previously described in vivo-expressed genomic island (Rv0960-Rv1001). Defined M. tuberculosis isogenic transposon mutants for the up-regulated genes Rv0980c, Rv0987, Rv0989c, and Rv1801 were found to be deficient in their ability to invade the BBB model. CONCLUSIONS: We developed an in vitro model of M. tuberculosis BBB infection and identified M. tuberculosis genes that may be involved in CNS invasion.
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Barrera Hematoencefálica/microbiología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , Tuberculosis del Sistema Nervioso Central/genética , Tuberculosis del Sistema Nervioso Central/microbiología , Actinas/análisis , Encéfalo/irrigación sanguínea , Encéfalo/microbiología , Encéfalo/ultraestructura , Capilares/citología , Capilares/microbiología , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Endotelio Vascular/microbiología , Expresión Génica , Genes Bacterianos , Humanos , Modelos Biológicos , Mutación , Mycobacterium tuberculosis/ultraestructura , Tuberculosis del Sistema Nervioso Central/patología , Virulencia/genéticaRESUMEN
Bacterial interaction with specific host tissue may contribute to its propensity to cause an infection in a particular site. In this study, we examined whether meningitis-causing Escherichia coli K1 interaction with human brain microvascular endothelial cells, which constitute the blood-brain barrier, differed from its interaction with non-brain endothelial cells derived from skin and umbilical cord. We showed that E. coli K1 association was significantly greater with human brain microvascular endothelial cells than with non-brain endothelial cells. In addition, human brain microvascular endothelial cells maintained their morphology and intercellular junctional resistance in response to E. coli K1. In contrast, non-brain endothelial cells exhibited decreased transendothelial electrical resistance and detachment from the matrix upon exposure to E. coli K1. These different responses of brain and non-brain endothelial cells to E. coli K1 may form the basis of E. coli K1's propensity to cause meningitis.
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Encéfalo/citología , Células Endoteliales/metabolismo , Células Endoteliales/microbiología , Escherichia coli/fisiología , Piel/citología , Cordón Umbilical/citología , Adhesinas de Escherichia coli , Toxinas Bacterianas , Barrera Hematoencefálica/citología , Células Cultivadas , Impedancia Eléctrica , Escherichia coli/genética , Proteínas de Escherichia coli , Proteínas Fimbrias , Humanos , Mutación/genética , Factores de TiempoRESUMEN
Microbial penetration of the blood-brain barrier (BBB) into the central nervous system is essential for the development of meningitis. Considerable progress has been achieved in understanding the pathophysiology of meningitis, however, relatively little is known about the early inflammatory events occurring at the time of bacterial crossing of the BBB. We investigated, using real-time quantitative PCR, the expression of the neutrophil chemoattractants alpha-chemokines CXCL1 (Groalpha) and CXCL8 (IL-8), and of the monocyte chemoattractant beta-chemokine CCL2 (MCP-1) by human brain microvascular endothelial cells (HBMEC) in response to the meningitis-causing E. coli K1 strain RS218 or its isogenic mutants lacking the ability to bind to and invade HBMEC. A nonpathogenic, laboratory E. coli strain HB101 was used as a negative control. CXCL8 was shown to be significantly expressed in HBMEC 4 hours after infection with E. coli K1, while no significant alterations were noted for CXCL1 and CCL2 expression. This upregulation of CXCL8 was induced by E. coli K1 strain RS218 and its derivatives lacking the ability to bind and invade HBMEC, but was not induced by the laboratory strain HB101. In contrast, no upregulation of CXCL8 was observed in human umbilical vein endothelial cells (HUVEC) after stimulation with E. coli RS218. These findings indicate that the CXCL8 expression is the result of the specific response of HBMEC to meningitis-causing E. coli K1.
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Células Endoteliales/citología , Escherichia coli/crecimiento & desarrollo , Interleucina-8/genética , Encéfalo/irrigación sanguínea , Encéfalo/citología , Línea Celular , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CXCL1 , Quimiocinas CXC/genética , Células Endoteliales/metabolismo , Células Endoteliales/microbiología , Escherichia coli/clasificación , Escherichia coli/genética , Regulación de la Expresión Génica , Humanos , Meningitis por Escherichia coli/microbiología , Mutación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad de la Especie , Factores de TiempoRESUMEN
Dietary zinc deficiency is common in developing as well as developed countries. Endothelial cells (EC) lining the inner surface of peripheral blood vessels are sensitive to zinc deficiency and lose structural integrity when exposed to culture media low in zinc or to zinc chelators. In contrast, we demonstrate here that human brain microvascular EC (HBMEC), which constitute the blood-brain barrier (BBB), resist zinc depletion and respond by enhancing their barrier function. This response was specific for HBMEC and did not occur in non-brain EC, such as human umbilical vein endothelial cells, human aortic endothelial cells, and human iliac vein endothelial cells. Our results suggest the presence of specific mechanisms to counteract zinc deficiency at the BBB, likely involving HBMEC junctional complexes. Understanding the mechanisms involved in this unique response might provide means to modulate the BBB dysfunction associated with neurological disorders such as stroke, multiple sclerosis, and Alzheimer's disease.