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1.
J Clin Microbiol ; 54(11): 2774-2785, 2016 11.
Article in English | MEDLINE | ID: mdl-27605711

ABSTRACT

Population-based studies on Staphylococcus aureus nasal colonization are scarce. We examined the prevalence, resistance, and molecular diversity of S. aureus in the general population in Northeast Germany. Nasal swabs were obtained from 3,891 adults in the large-scale population-based Study of Health in Pomerania (SHIP-TREND). Isolates were characterized using spa genotyping, as well as antibiotic resistance and virulence gene profiling. We observed an S. aureus prevalence of 27.2%. Nasal S. aureus carriage was associated with male sex and inversely correlated with age. Methicillin-resistant S. aureus (MRSA) accounted for 0.95% of the colonizing S. aureus strains. MRSA carriage was associated with frequent visits to hospitals, nursing homes, or retirement homes within the previous 24 months. All MRSA strains were resistant to multiple antibiotics. Most MRSA isolates belonged to the pandemic European hospital-acquired MRSA sequence type 22 (HA-MRSA-ST22) lineage. We also detected one livestock-associated MRSA ST398 (LA-MRSA-ST398) isolate, as well as six livestock-associated methicillin-susceptible S. aureus (LA-MSSA) isolates (clonal complex 1 [CC1], CC97, and CC398). spa typing revealed a diverse but also highly clonal S. aureus population structure. We identified a total of 357 spa types, which were grouped into 30 CCs or sequence types. The major seven CCs (CC30, CC45, CC15, CC8, CC7, CC22, and CC25) included 75% of all isolates. Virulence gene patterns were strongly linked to the clonal background. In conclusion, MSSA and MRSA prevalences and the molecular diversity of S. aureus in Northeast Germany are consistent with those of other European countries. The detection of HA-MRSA and LA-MRSA within the general population indicates possible transmission from hospitals and livestock, respectively, and should be closely monitored.


Subject(s)
Carrier State/epidemiology , Nasal Cavity/microbiology , Staphylococcal Infections/epidemiology , Staphylococcus aureus/classification , Staphylococcus aureus/isolation & purification , Adult , Age Factors , Aged , Aged, 80 and over , Carrier State/microbiology , Cluster Analysis , Cohort Studies , Drug Resistance, Bacterial , Female , Genetic Variation , Genotype , Genotyping Techniques , Germany/epidemiology , Humans , Male , Middle Aged , Molecular Epidemiology , Molecular Typing , Prevalence , Sex Factors , Staphylococcal Infections/microbiology , Staphylococcal Protein A/genetics , Staphylococcus aureus/genetics , Virulence Factors/genetics , Young Adult
2.
Proteomics ; 16(20): 2667-2677, 2016 10.
Article in English | MEDLINE | ID: mdl-27324828

ABSTRACT

Staphylococcus aureus is a frequent commensal but also a dangerous pathogen, causing many forms of infection ranging from mild to life-threatening conditions. Among its virulence factors are lipoproteins, which are anchored in the bacterial cell membrane. Lipoproteins perform various functions in colonization, immune evasion, and immunomodulation. These proteins are potent activators of innate immune receptors termed Toll-like receptors 2 and 6. This study addressed the specific B-cell and T-cell responses directed to lipoproteins in human S. aureus carriers and non-carriers. 2D immune proteomics and ELISA approaches revealed that titers of antibodies (IgG) binding to S. aureus lipoproteins were very low. Proliferation assays and cytokine profiling data showed only subtle responses of T cells; some lipoproteins did not elicit proliferation. Hence, the robust activation of the innate immune system by S. aureus lipoproteins does not translate into a strong adaptive immune response. Reasons for this may include inaccessibility of lipoproteins for B cells as well as ineffective processing and presentation of the antigens to T cells.


Subject(s)
Adaptive Immunity , B-Lymphocytes/immunology , Bacterial Proteins/immunology , Lipoproteins/immunology , Staphylococcal Infections/immunology , Staphylococcus aureus/immunology , T-Lymphocytes/immunology , Adult , B-Lymphocytes/microbiology , Cells, Cultured , Cytokines/immunology , Female , Gene Expression Regulation , Healthy Volunteers , Humans , Immunoglobulin G/immunology , Male , Middle Aged , Proteome/immunology , Proteomics , Staphylococcal Infections/microbiology , T-Lymphocytes/microbiology , Virulence Factors/immunology , Young Adult
3.
J Infect Dis ; 212(5): 830-8, 2015 Sep 01.
Article in English | MEDLINE | ID: mdl-25737563

ABSTRACT

INTRODUCTION: Though Staphylococcus aureus is a major pathogen, vaccine trials have failed. In contrast, class-switched antibodies specific to S. aureus are common, implying immune memory formation and suggesting a large pool of S. aureus-reactive helper T-cells. OBJECTIVE: To elucidate the cellular arm of S. aureus-specific immune memory, the T-cell response in humans was characterized. METHODS: The proliferative response of human peripheral blood mononuclear cells (PBMCs) to S. aureus antigens and the frequency of S. aureus-specific T-cells were quantified by (3)H-thymidine incorporation; cytokine release was measured by flow cytometry. RESULTS: Staphylococcus aureus particles and extracellular proteins elicited pronounced proliferation in PBMCs of healthy adults. This reflected a memory response with high frequencies of T-cells being activated by single S. aureus antigens. The whole S. aureus-specific T-cell pool was estimated to comprise 3.6% of T-cells with 35-fold differences between individuals (range, 0.2%-5.7%). When exposed to S. aureus antigens, the T-cells released predominantly but not solely T helper (Th)1/Th17 cytokines. CONCLUSIONS: The large number of S. aureus antigen-reactive memory T-lymphocytes is likely to influence the course of S. aureus infection. To enable rational vaccine design, the naturally acquired human T-cell memory needs to be explored at high priority.


Subject(s)
Immunologic Memory , Staphylococcus aureus/immunology , T-Lymphocytes/immunology , Adult , Antigens, Bacterial/immunology , Cell Proliferation , Cytokines/metabolism , Flow Cytometry , Humans , Isotope Labeling , Leukocytes, Mononuclear/immunology
4.
Microb Cell Fact ; 11: 29, 2012 Mar 01.
Article in English | MEDLINE | ID: mdl-22380513

ABSTRACT

BACKGROUND: The lipase subfamilies I.1 and I.2 show more than 33% homology in the amino acid sequences and most members share another common property that their genes are clustered with the secondary genes whose protein products are required for folding the lipase into an active conformation and secretion into the culture medium. In previous studies, the lipase (LipA) and its chaperone (LipB) from Ralstonia sp. M1 were overexpressed in E. coli and the lipase was successfully refolded in vitro. The purpose of this study was to enhance the production of the active lipase LipA from Ralstonia sp. M1 in the heterologous host E. coli without in vitro refolding process, using two-plasmid co-expression systems and dual expression cassette plasmid systems. RESULTS: To produce more active lipase from Ralstonia sp. M1 in E. coli without in vitro refolding process but with the help of overexpression of the chaperone (LipB1 and LipB3 corresponding to 56-aa truncated and 26-aa truncated chaperone LipB), six different expression systems including 2 two-plasmid co-expression systems (E. coli BL21/pELipABa + pELipB1k and BL21/pELipABa + pELipB3k) and 4 dual expression cassette plasmid systems (BL21/pELipAB-LipB1a, BL21/pELipAB-LipB3a, BL21/pELipA-LipB1a, and BL21/pELipA-LipB3a) were constructed. The two-plasmid co-expression systems (E. coli BL21/pELipABa + pELipB1k and BL21/pELipABa + pELipB3k) produced the active lipase at a level of 4 times as high as the single expression cassette plasmid system E. coli BL21/pELipABa did. For the first time, the dual expression cassette plasmid systems BL21/pELipAB-LipB1a and BL21/pELipAB-LipB3a yielded 29- and 19-fold production of the active lipase in comparison with the single expression cassette plasmid system E. coli BL21/pELipABa, respectively. Although the lipase amount was equally expressed in all these expression systems (40% of total cellular protein) and only a small fraction of the overexpressed lipase was folded in vivo into the functional lipase in soluble form whereas the main fraction was still inactive in the form of inclusion bodies. Another controversial finding was that the dual expression cassette plasmid systems E. coli BL21/pELipAB-LipB1a and E. coli/pELipAB-LipB3a secreted the active lipase into the culture medium of 51 and 29 times as high as the single expression cassette plasmid system E. coli pELipABa did, respectively, which has never been reported before. Another interesting finding was that the lipase form LipA6xHis (mature lipase fused with 6× histidine tag) expressed in the dual expression cassette plasmid systems (BL21/pELipA-LipB1a and BL21/pELipA-LipB3a) showed no lipase activity although the expression level of the lipase and two chaperone forms LipB1 and LipB3 in these systems remained as high as that in E. coli BL21/pELipABa + pELipB1k, BL21/pELipABa + pELipB3k, BL21/pELipAB-LipB1a, and BL21/pELipAB-LipB3a. The addition of Neptune oil or detergents into the LB medium increased the lipase production and secretion by up to 94%. CONCLUSIONS: Our findings demonstrated that a dual expression cassette plasmid system E. coli could overproduce and secrete the active chaperone-dependent lipase (subfamilies I.1 and I.2) in vivo and an improved dual expression cassette plasmid system E. coli could be potentially applied for industrial-scale production of subfamily I.1 and I.2 lipases.


Subject(s)
Bacterial Proteins/biosynthesis , Biotechnology/methods , Escherichia coli/metabolism , Lipase/biosynthesis , Molecular Chaperones/metabolism , Plasmids/metabolism , Bacterial Proteins/genetics , Lipase/genetics , Molecular Chaperones/genetics , Plasmids/genetics , Ralstonia/enzymology
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