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1.
Brucella spp. are Gram-negative, facultative intracellular coccobacilli that cause one of the most frequently encountered zoonosis worldwide. Humans naturally acquire infection through consumption of contaminated dairy and meat products and through direct exposure to aborted animal tissues and fluids. No vaccine against brucellosis is available for use in humans. In this study, we tested the ability of orally inoculated gamma-irradiated B. neotomae and B. abortus RB51 in a prime-boost immunization approach to induce antigen-specific humoral and cell mediated immunity and protection against challenge with virulent B. abortus 2308. Heterologous prime-boost vaccination with B. abortus RB51 and B. neotomae and homologous prime-boost vaccination of mice with B. neotomae led to the production of serum and mucosal antibodies specific to the smooth LPS. The elicited serum antibodies included the isotypes of IgM, IgG1, IgG2a, IgG2b and IgG3. All oral vaccination regimens induced antigen-specific CD4+ and CD8+ T cells capable of secreting IFN-γ and TNF-α. Upon intra-peritoneal challenge, mice vaccinated with B. neotomae showed the highest level of resistance against virulent B. abortus 2308 colonization in spleen and liver. Experiments with different doses of B. neotomae showed that all tested doses of 109, 1010 and 1011 CFU-equivalent conferred significant protection against the intra-peritoneal challenge. However, a dose of 1011 CFU-equivalent of B. neotomae was required for affording protection against intranasal challenge as shown by the reduced bacterial colonization in spleens and lungs. Taken together, these results demonstrate the feasibility of using gamma-irradiated B. neotomae as an effective and safe oral vaccine to induce protection against respiratory and systemic infections with virulent Brucella. 相似文献
2.
Soon RL Velkov T Chiu F Thompson PE Kancharla R Roberts K Larson I Nation RL Li J 《Analytical biochemistry》2011,(2):273-283
Fluorescence assays employing semisynthetic or commercial dansyl-polymyxin B have been widely employed to assess the affinity of polycations, including polymyxins, for bacterial cells and lipopolysaccharide (LPS). The five primary γ-amines on diaminobutyric acid residues of polymyxin B are potentially derivatized with dansyl-chloride. Mass spectrometric analysis of the commercial product revealed a complex mixture of di- or tetra-dansyl-substituted polymyxin B. We synthesized a mono-substituted fluorescent derivative, dansyl[Lys]1polymyxin B3. The affinity of polymyxin for purified gram-negative LPS and whole bacterial cells was investigated. The affinity of dansyl[Lys]1polymyxin B3 for LPS was comparable to polymyxin B and colistin, and considerably greater (Kd < 1 μM) than for whole cells (Kd ∼ 6–12 μM). Isothermal titration calorimetric studies demonstrated exothermic enthalpically driven binding between both polymyxin B and dansyl[Lys]1polymyxin B3 to LPS, attributed to electrostatic interactions. The hydrophobic dansyl moiety imparted a greater entropic contribution to the dansyl[Lys]1polymyxin B3–LPS reaction. Molecular modeling revealed a loss of electrostatic contact within the dansyl[Lys]1polymyxin B3–LPS complex due to steric hindrance from the dansyl[Lys]1 fluorophore; this corresponded with diminished antibacterial activity (MIC ?16 μg/mL). Dansyl[Lys]1polymyxin B3 may prove useful as a screening tool for drug development. 相似文献
3.
The last decade has witnessed increasing research on dissemination of bacterial pathogens in their hosts and on the processes that underlie bacterial spread and growth during organ colonization. Here, we discuss work on the mouse model of human typhoid fever caused by Salmonella enterica serovar Typhimurium. This has revealed the use of several routes of systemic dissemination that result in colonization and growth within the spleen and liver, the major sites of bacterial proliferation. We also highlight techniques that enable in vivo analysis of the infecting population at the spatiotemporal and single cell levels. These approaches have provided more detailed insights into the events underlying the dynamics of Salmonella replication, spread and clearance within host organs and tissues. 相似文献
4.
《Microbes and infection / Institut Pasteur》2009,11(4):476-483
Inhalation is a common route for Brucella infection. We investigated whether Brucella species can invade and replicate within alveolar (A549) and bronchial (Calu-6 and 16HBE14o-) human epithelial cells. The number of adherent and intracellular bacteria was higher for rough strains (Brucella canis and Brucella abortus RB51) than for smooth strains (B. abortus 2308 and Brucella suis 1330). Only smooth strains exhibited efficient intracellular replication (1.5–3.5 log increase at 24 h p.i.). A B. abortus mutant with defective expression of the type IV secretion system did not replicate. B. abortus internalization was inhibited by specific inhibitors of microfilaments, microtubules and PI3-kinase activity. As assessed with fluorescent probes, B. abortus infection did not affect the viability of A549 and 16HBE14o- cells, but increased the percentage of injured cells (both strains) and dead cells (RB51) in Calu-6 cultures. LDH levels were increased in supernatants of Calu-6 and 16HBE14o- cells infected with B. abortus RB51, and to a lower extent in Calu-6 infected with B. abortus 2308. No apoptosis was detected by TUNEL upon infection with smooth or rough B. abortus. This study shows that smooth brucellae can infect and replicate in human respiratory epithelial cells inducing minimal or null cytotoxicity. 相似文献
5.
Zhiqiang Li Shuli Wang Shujuan Wei Guangli Yang Chunmei Zhang Li Xi Jinliang Zhang Yanyan Cui Junfang Hao Huan Zhang Hui Zhang 《Microbial biotechnology》2022,15(6):1811-1823
Protective efficiency of a combination of four recombinant Brucella abortus (B. abortus) proteins, namely, ribosomal protein L7/L12, outer membrane protein (OMP) 22, OMP25 and OMP31, was evaluated as a combined subunit vaccine (CSV) against B. abortus infection in RAW 264.7 cell line and murine model. Four proteins were cloned, expressed and purified, and their immunocompetence was analysed. BALB/c mice were immunized subcutaneously with single subunit vaccines (SSVs) or CSV. Cellular and humoral immune responses were determined by ELISA. Results of immunoreactivity showed that these four recombinant proteins reacted with Brucella-positive serum individually but not with Brucella-negative serum. A massive production of IFN-γ and IL-2 but low degree of IL-10 was observed in mice immunized with SSVs or CSV. In addition, the titres of IgG2a were heightened compared with IgG1 in SSV- or CSV-immunized mice, which indicated that SSVs and CSV induced a typical T-helper-1-dominated immune response in vivo. Further investigation of the CSV showed a superior protective effect in mice against brucellosis. The protection level induced by CSV was significantly higher than that induced by SSVs, which was not significantly different compared with a group immunized with RB51. Collectively, these antigens of Brucella could be potential candidates to develop subunit vaccines, and the CSV used in this study could be a potential candidate therapy for the prevention of brucellosis. 相似文献
6.
Polymyxin B inhibits phorbol 12-myristate 13-acetate, but not chemotactic factor, induced effects in rabbit neutrophils 总被引:2,自引:0,他引:2
The addition of the amphipathic polycationic antibiotic polymyxin B to a suspension of rabbit neutrophils results in inhibiton of the agonist (secretion of secondary granules) and antagonist (inhibition of chemotactic factor induced degranulation) properties of phorbol 12-myristate 13-acetate. On the other hand, polymyxin B does not inhibit the degranulation of the neutrophils that is induced by chemotactic factors. These results imply that the role of protein kinase C in the initiation of neutrophil functions in response to the addition of chemotactic factors is less critical than previously thought. In addition, the reversal of the inhibitory properties of phorbol esters by polymyxin B indicates that the former are mediated by the ability of the tumor promoters to activate protein kinase C. These results thus strengthen the hypothesis that protein kinase C plays important roles in the regulation (as contrasted to initiation) of neutrophil functions. 相似文献
7.
Antimicrobial susceptibility testing with the last-resort antibiotics polymyxins (polymyxin B and colistin) is associated with several methodological issues. Currently, broth microdilution (BMD) is recommended for colistin and polymyxin B. BMD is laborious and the utility of alternative methods needs to be evaluated for polymyxin B susceptibility testing. In this study, using BMD as a reference method, the performance of agar dilution (AD) and MIC test strips (MTS) were evaluated in polymyxin B susceptibility testing. BMD, AD and MTS were used to determine MICs of 193 clinical isolates of Escherichia coli. Seventy-nine were positive for the polymyxin resistance gene mcr-1. Method performances were evaluated based on pair-wise agreements with the reference method (BMD) and statistical testing. AD and MTS showed an unacceptable number of very major errors (VMEs) compared with BMD, 9·3 and 10·7%, respectively. The essential agreement (EA) was low for AD (49·7%), but high for MTS (97·8%). However, statistical testing showed that MTS tended to yield a one-step lower MIC (P < 0·01) compared with BMD. The discordances observed with MTS and AD in comparison with BMD for polymyxin B susceptibility testing for E. coli suggest their inapplicability in routine testing. A large number of isolates clustered around the susceptibility breakpoint (2–4 mg l−1) and several mcr-1 positive isolates (17%) were determined as susceptible with BMD. A screening breakpoint for mcr-1 of 2 mg l−1 should also be considered. 相似文献
8.
Background
Brucellaspp. are highly similar, having identical 16S RNA. However, they have important phenotypic differences such as differential susceptibility to antibiotics binding the ribosome. Neither the differential susceptibility nor its basis has been rigorously studied. Differences found among other conserved ribosomal loci could further define the relationships among the classicalBrucellaspp. 相似文献9.
Yueli Wang Honghuan Li Zhenyu Xu Jihai Yi Wei Li Chuang Meng Huan Zhang Xiaoyu Deng Zhongchen Ma Yong Wang Chuangfu Chen 《Microbial biotechnology》2023,16(7):1524-1535
Exosomes, membrane vesicles released extracellularly from cells, contain nucleic acids, proteins, lipids and other components, allowing the transfer of material information between cells. Recent studies reported the role of exosomes in pathogenic microbial infection and host immune mechanisms. Brucella-invasive bodies can survive in host cells for a long time and cause chronic infection, which causes tissue damage. Whether exosomes are involved in host anti-Brucella congenital immune responses has not been reported. Here, we extracted and identified exosomes secreted by Brucella melitensis M5 (Exo-M5)-infected macrophages, and performed in vivo and in vitro studies to examine the effects of exosomes carrying antigen on the polarization of macrophages and immune activation. Exo-M5 promoted the polarization of M1 macrophages, which induced the significant secretion of M1 cytokines (tumour necrosis factor-α and interferon-γ) through NF-κB signalling pathways and inhibited the secretion of M2 cytokines (IL-10), thereby inhibiting the intracellular survival of Brucella. Exo-M5 activated innate immunity and promoted the release of IgG2a antibodies that protected mice from Brucella infection and reduced the parasitaemia of Brucella in the spleen. Furthermore, Exo-M5 contained Brucella antigen components, including Omp31 and OmpA. These results demonstrated that exosomes have an important role in immune responses against Brucella, which might help elucidate the mechanisms of host immunity against Brucella infection and aid the search for Brucella biomarkers and the development of new vaccine candidates. 相似文献
10.
Serologic evidence of Brucella infection in pinnipeds along the coast of Hokkaido,the northernmost main island of Japan
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Erika Abe Kazue Ohishi Tsuyoshi Ishinazaka Kei Fujii Tadashi Maruyama 《Microbiology and immunology》2017,61(3-4):114-122
Brucella infection in Hokkaido was serologically surveyed in four species of pinnipeds inhabiting Cape Erimo during 2008–2013 and the Shiretoko Peninsula in 1999 by ELISA using Brucella abortus and B. canis as antigens. Anti‐Brucella positive sera showed higher absorbance to B. abortus than B. canis in almost all samples. Anti‐B. abortus antibodies were detected in serum samples from 24% (n = 55) of Western Pacific harbor seals (Phoca vitulina stejnegeri) in Cape Erimo and from 66% (n = 41) of spotted seals (P. largha), 15% (n = 20) of ribbon seals (Histriophoca fasciata) and 18% (n = 17) of Western Steller's sea lions (Eumetopias jubatus jubatus) in the Shiretoko Peninsula. Anti‐Brucella antibodies were detected at higher absorbance in 1‐ to 4‐year‐old harbor seals than in the pups and mature animals, suggesting either that Brucella infection mainly occurs after weaning or that it is maternally transmitted to pups with premature or suppressed immunity. Anti‐Brucella antibodies were detected in both immature and mature spotted seals and ribbon seals, with higher absorbance in the former. The antibodies were detected only in mature Western Steller's sea lions. Western blot analysis of the serum samples showed some differences in band appearances, namely discrete versus smeary, and in the number of bands, indicating that multiple different Brucella may be prevalent in pinnipeds in Hokkaido. Alternatively, the Brucella of pinnipeds may have some intra‐species diversity. 相似文献
11.
Brucella spp noncanonical LPS: structure, biosynthesis, and interaction with host immune system 总被引:1,自引:0,他引:1
Patrícia Gomes Cardoso Gilson Costa Macedo Vasco Azevedo Sergio Costa Oliveira 《Microbial cell factories》2006,5(1):13-11
Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and non-professional
phagocytes, and cause abortion in domestic animals and undulant fever in humans. Several species are recognized within the
genus Brucella and this classification is mainly based on the difference in pathogenicity and in host preference. Brucella strains may occur as either smooth or rough, expressing smooth LPS (S-LPS) or rough LPS (R-LPS) as major surface antigen.
This bacterium possesses an unconventional non-endotoxic lipopolysaccharide that confers resistance to anti-microbial attacks
and modulates the host immune response. The strains that are pathogenic for humans (B. abortus, B. suis, B. melitensis) carry a smooth LPS involved in the virulence of these bacteria. The LPS O-chain protects the bacteria from cellular cationic
peptides, oxygen metabolites and complement-mediated lysis and it is a key molecule for Brucella survival and replication in the host. Here, we review i) Brucella LPS structure; ii) Brucella genome, iii) genes involved in LPS biosynthesis; iv) the interaction between LPS and innate immunity. 相似文献
12.
The Brucella cell envelope contains the zwitterionic phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Synthesis of PC occurs exclusively via the PC synthase pathway, implying that the pathogen depends on the choline synthesized by the host cell to form PC. Notably, PC is necessary to sustain a chronic infection process, which suggests that the membrane lipid content is relevant for Brucella virulence. In this study we investigated the first step of PE biosynthesis in B. abortus, which is catalyzed by phosphatidylserine synthase (PssA). Disruption of pssA abrogated the synthesis of PE without affecting the growth in rich complex medium. In minimal medium, however, the mutant required choline supplementation for growth, suggesting that at least PE or PC is necessary for Brucella viability. The absence of PE altered cell surface properties, but most importantly, it impaired several virulence traits of B. abortus, such as intracellular survival in both macrophages and HeLa cells, the maturation of the replicative Brucella-containing vacuole, and mouse colonization. These results suggest that membrane phospholipid composition is critical for the interaction of B. abortus with the host cell. 相似文献
13.
Temperature dependence of the binding of endotoxins to the polycationic peptides polymyxin B and its nonapeptide
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The interaction between endotoxins-free lipid A and various lipopolysaccharide (LPS) chemotypes with different sugar chain lengths-and the polycationic peptides polymyxin B and polymyxin nonapeptide has been investigated by isothermal titration calorimetry between 20 and 50 degrees C. The results show a strong dependence of the titration curves on the phase state of the endotoxins. In the gel phase (<30 degrees C for LPS and <45 degrees C for lipid A), an endothermic reaction is observed, for which the driving force is an entropically driven endotoxin-polymyxin interaction, due to disruption of the ordered water structure and cation assembly in the lipid A backbone and adjacent molecules. In the liquid crystalline phase (>35 degrees C for LPS and >47 degrees C for lipid A) an exothermic reaction takes place, which is mainly due to the strong electrostatic interaction of the polymyxins with the negative charges of the endotoxins, i.e., the entropic change DeltaS is much lower than in the gel phase. For endotoxins with short sugar chains (lipid A, LPS Re, LPS Rc) the stoichiometry of the polymyxin binding corresponds to pure charge neutralization; for the compounds with longer sugar chains (LPS Ra, LPS S-form) this is no longer valid. This can be related to the lower susceptibility of the corresponding bacterial strains to antibiotics. 相似文献
14.
Smooth virulent Brucella abortus strain 2308 (S2308) causes zoonotic brucellosis in cattle and humans. Rough B. abortus strain RB51, derived from S2308, is a live attenuated cattle vaccine strain licensed in the USA and many other countries. Our previous report indicated that RB51, but not S2308, induces a caspase-2-dependent apoptotic and necrotic macrophage cell death. Dendritic cells (DCs) are professional antigen presenting cells critical for bridging innate and adaptive immune responses. In contrast to Brucella-infected macrophages, here we report that S2308 induced higher levels of apoptotic and necrotic cell death in wild type bone marrow-derived DCs (WT BMDCs) than RB51. The RB51 and S2308-induced BMDC cell death was regulated by caspase-2, indicated by the minimal cell death in RB51 and S2308-infected BMDCs isolated from caspase-2 knockout mice (Casp2KO BMDCs). More S2308 bacteria were taken up by Casp2KO BMDCs than wild type BMDCs. Higher levels of S2308 and RB51 cells were found in infected Casp2KO BMDCs compared to infected WT BMDCs at different time points. RB51-infected wild type BMDCs were mature and activated as shown by significantly up-regulated expression of CD40, CD80, CD86, MHC-I, and MHC-II. RB51 induced the production of cytokines TNF-α, IL-6, IFN-γ and IL12/IL23p40 in infected BMDCs. RB51-infected WT BMDCs also stimulated the proliferation of CD4(+) and CD8(+) T cells compared to uninfected WT BMDCs. However, the maturation, activation, and cytokine secretion are significantly impaired in Casp2KO BMDCs infected with RB51 or Salmonella (control). S2308-infected WT and Casp2KO BMDCs were not activated and could not induce cytokine production. These results demonstrated that virulent smooth strain S2308 induced more apoptotic and necrotic dendritic cell death than live attenuated rough vaccine strain RB51; however, RB51, but not its parent strain S2308, induced caspase-2-mediated DC maturation, cytokine production, antigen presentation, and T cell priming. 相似文献
15.
16.
George Constantopoulos Robert M. Eiben Irwin A. Schafer 《Journal of neurochemistry》1978,31(5):1215-1222
Glycosaminoglycans, lipids and lysosomal enzymes were measured in brain, liver and spleen of a patient with mucopolysaccharidosis Type III B (α-N-Acetylglucosaminidase deficiency). The glycosaminoglycan content of the brain gray and white matter, leptomeninges, spleen and liver of the patient was 4, 3, 10, and 100 times greater than that of the respective tissues of normal controls. Partially degraded heparan sulfate, the concentration of which increased 17 times in the brain, accounted for the increased glycosaminoglycan content of all tissues. The concentration of the gangliosides GM2, GM3 and GD3 was markedly increased in the gray matter, and to a smaller degree in the white matter. Ceramide dihexoside was also increased in the gray matter of the patient with MPS III B. The activity of α-N-Acetylglucosaminidase was absent from the brain and the liver and greatly diminished in the spleen. β-Glucuronidase. β-glucosaminidase and α-l -iduronidase were more active than normally and the activity of α-galactosidase and β-galactosidase was markedly reduced. 相似文献
17.
Control of Salmonella enteritidis infections in poultry by polymyxin B and trimethoprim. 总被引:2,自引:0,他引:2
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Antimicrobial compounds were screened in vitro in Trypticase soy broth for antimicrobial activity against a virulent strain of Salmonella enteritidis. Of the several compounds tested, polymyxin B showed the strongest inhibition in vitro, preventing growth at a concentration of less than or equal to 10 micrograms/ml. Polymyxin B administered in the drinking water was effective in vivo for preventing infections in 1-day-old chickens but did not remove established infections in 1-week-old chickens. It was found that trimethoprim, which was not active in vitro, prevented colonization and removed existing infections in 1-day-old chickens when it was administered together with polymyxin B sulfate. Enrichment cultures in which selenite-cystine and tetrathionate broth media were used showed that chickens given a combination of 100 micrograms of polymyxin B sulfate per ml and 250 micrograms of trimethoprim per ml 24 h prior to oral inoculation with 10(8) to 10(9) CFU were negative for S. enteritidis after 7 days. Established infections (10(5) to 10(6) CFU/g of feces) in 1-week-old chickens were eliminated by treatment with the polymyxin-trimethoprim system. This antimicrobial agent treatment may be useful for preventing colonization in poultry and for eliminating S. enteritidis from infected flocks. 相似文献
18.
Antimicrobial compounds were screened in vitro in Trypticase soy broth for antimicrobial activity against a virulent strain of Salmonella enteritidis. Of the several compounds tested, polymyxin B showed the strongest inhibition in vitro, preventing growth at a concentration of less than or equal to 10 micrograms/ml. Polymyxin B administered in the drinking water was effective in vivo for preventing infections in 1-day-old chickens but did not remove established infections in 1-week-old chickens. It was found that trimethoprim, which was not active in vitro, prevented colonization and removed existing infections in 1-day-old chickens when it was administered together with polymyxin B sulfate. Enrichment cultures in which selenite-cystine and tetrathionate broth media were used showed that chickens given a combination of 100 micrograms of polymyxin B sulfate per ml and 250 micrograms of trimethoprim per ml 24 h prior to oral inoculation with 10(8) to 10(9) CFU were negative for S. enteritidis after 7 days. Established infections (10(5) to 10(6) CFU/g of feces) in 1-week-old chickens were eliminated by treatment with the polymyxin-trimethoprim system. This antimicrobial agent treatment may be useful for preventing colonization in poultry and for eliminating S. enteritidis from infected flocks. 相似文献
19.
The toxic activity ofBrucella melitensis andPseudomonas aeruginosa lipopolysaccharides as well as their behavior as immunogens, mitogens, and interferon inducers have been studied. Although
their toxicities were very similar, the former molecule was incapable of eliciting a primary immune response in mice. Rabbit
hyperimmunization gave titers half of those obtained withP. aeruginosa lipopolysaccharide. Optimal mitogenic responses of spleen cell cultures were obtained using 10–50 μg/ml and 50–100 μg/ml
ofPseudomonas andBrucella lipopolysaccharide, respectively, giving the latter a lower stimulation of3H-thymidine uptake. Interferon titers induced in chickens byBrucella lipopolysaccharide were three times lower than those obtained withPseudomonas lipopolysaccharide. 相似文献
20.
目的分析儿童活体肝移植术后常见致病菌的分布特点及其对常用抗菌药物的耐药性情况,为合理使用抗生素提供参考。方法回顾性分析重庆医科大学附属儿童医院自2006年6月至2009年12月术后监护的42例儿童活体肝移植患儿送检共152份标本进行菌株鉴定及药敏试验。结果经培养共分离出66株致病菌,阳性率为43.4%,以呼吸道来源为主,占71.2%。66株致病菌中革兰阴性菌(G^-)63.6%,革兰阳性菌(G^+)27.3%,真菌9.1%。常见G^-菌为铜绿假单胞菌、阴沟肠杆菌、大肠埃希菌和肺炎克雷伯菌肺炎亚种。常见G^+菌为金黄葡萄球菌和肺炎链球菌。真菌主要为白色假丝酵母菌。药敏结果显示细菌耐药性明显增强,G^-菌中阴沟肠杆菌、大肠埃希菌和肺炎克雷伯菌肺炎亚种对美洛培南、亚胺培南100%敏感,对环丙沙星、左旋氧氟沙星、阿米卡星和氯霉素敏感在85.7%以上;超广谱β-内酰胺酶(ESBLs)菌株检出率为66.7%100%。铜绿假单胞菌仅对环丙沙星、左旋氧氟沙星、庆大霉素、阿米卡星和多粘菌素敏高度感度,敏感度在91.6%以上。G^+菌中金黄色葡萄菌对万古霉素、替考拉宁、利奈唑胺、呋喃妥因、阿米卡星和环丙沙星100%敏感,β-内酰胺酶检出率为100%。肺炎链球菌对万古霉素、利奈唑胺、头孢西丁、美洛培南、左旋氧氟沙星和头孢吡肟均有83.3%以上的敏感性,对于阿莫西林仍有66.7%的敏感性,但对于大环内酯类100%耐药。白色假丝酵母菌对两性霉素B 100%敏感。结论儿童活体肝移植术后常见致病菌以G^-菌为主,多为多重耐药菌株,含有β-内酰胺酶抑制剂及碳青霉烯类抗生素是治疗该类细菌的有效抗生素。万古霉素是G^+菌感染的有效抗生素。两性霉素B是真菌感染的有效药物。为避免耐药率上升,临床应合理使用抗生素。 相似文献