<meta charset="utf8"></head><body>Optimierte In-vivo-Modelle für intrazelluläre S. aureus-Infektionsstudien

<ol>
	<li>Sutton, J. A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+cell+wall+structure+and+dynamics+during+host-pathogen+interaction.">Staphylococcus aureus cell wall structure and dynamics during host-pathogen interaction.</a> PLoS Pathog. 17 (3), e1009468 (2021).</li><li>Yang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+flow+assay+of+methicillin-resistant+Staphylococcus+aureus+using+bacteriophage+cellular+wall-binding+domain+as+recognition+agent.">Lateral flow assay of methicillin-resistant Staphylococcus aureus using bacteriophage cellular wall-binding domain as recognition agent.</a> Biosens Bioelectron. 182, 113189 (2021).</li><li>Howden, B. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+host+interactions+and+adaptation.">Staphylococcus aureus host interactions and adaptation.</a> Nat Rev Microbiol. 21 (6), 380-395 (2023).</li><li>Guo, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+and+small+colony+variants-an+update+on+Staphylococcus+aureus+strategies+toward+drug+resistance.">Biofilm and small colony variants-an update on Staphylococcus aureus strategies toward drug resistance.</a> Int J Mol Sci. 23 (3), 1241 (2022).</li><li>Huitema, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+escape+strategies+of+Staphylococcus+aureus+in+persistent+cutaneous+infections.">Intracellular escape strategies of Staphylococcus aureus in persistent cutaneous infections.</a> Exp Dermatol. 30 (10), 1428-1439 (2021).</li><li>Pidwill, G. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+population+expansion+of+Staphylococcus+aureus+occurs+due+to+escape+from+a+finite+number+of+intraphagocyte+niches.">Clonal population expansion of Staphylococcus aureus occurs due to escape from a finite number of intraphagocyte niches.</a> Sci Rep. 13 (1), 1188 (2023).</li><li>Wang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+Staphylococcus+aureus+infection.">Autophagy in Staphylococcus aureus infection.</a> Front Cell Infect Microbiol. 11, 750222 (2021).</li><li>Arya, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+an+antivirulence+agent+targeting+the+master+regulator+of+virulence+genes+in+Staphylococcus+aureus.">Identification of an antivirulence agent targeting the master regulator of virulence genes in Staphylococcus aureus.</a> Front Cell Infect Microbiol. 13, 1268044 (2023).</li><li>M&#252;nzenmayer, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+sae-regulated+and+agr-regulated+factors+on+the+escape+of+Staphylococcus+aureus+from+human+macrophages.">Influence of sae-regulated and agr-regulated factors on the escape of Staphylococcus aureus from human macrophages.</a> Cell Microbiol. 18 (8), 1172-1183 (2016).</li><li>Chin, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+lugdunensis+uses+the+agr+regulatory+system+to+resist+killing+by+host+innate+immune+effectors.">Staphylococcus lugdunensis uses the agr regulatory system to resist killing by host innate immune effectors.</a> Infect Immun. 90 (10), e0009922 (2022).</li><li>Podkowik, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quorum-sensing+agr+system+of+Staphylococcus+aureus+primes+gene+expression+for+protection+from+lethal+oxidative+stress.">Quorum-sensing agr system of Staphylococcus aureus primes gene expression for protection from lethal oxidative stress.</a> Elife. 12, RP89098 (2024).</li><li>Purves, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Air+pollution+induces+Staphylococcus+aureus+usa300+respiratory+tract+colonization+mediated+by+specific+bacterial+genetic+responses+involving+the+global+virulence+gene+regulators+agr+and+sae.">Air pollution induces Staphylococcus aureus usa300 respiratory tract colonization mediated by specific bacterial genetic responses involving the global virulence gene regulators agr and sae.</a> Environ Microbiol. 24 (9), 4449-4465 (2022).</li><li>Wittekind, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+novel+protein+scra+acts+through+the+saers+two-component+system+to+regulate+virulence+gene+expression+in+Staphylococcus+aureus.">The novel protein scra acts through the saers two-component system to regulate virulence gene expression in Staphylococcus aureus.</a> Mol Microbiol. 117 (5), 1196-1212 (2022).</li><li>Pidwill, G. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+macrophages+in+Staphylococcus+aureus+infection.">The role of macrophages in Staphylococcus aureus infection.</a> Front Immunol. 11, 620339 (2020).</li><li>Lang, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photoconvertible+reporter+system+for+bacterial+metabolic+activity+reveals+that+Staphylococcus+aureus+enters+a+dormant-like+state+to+persist+within+macrophages.">A photoconvertible reporter system for bacterial metabolic activity reveals that Staphylococcus aureus enters a dormant-like state to persist within macrophages.</a> mBio. 13 (5), e0231622 (2022).</li><li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+macrophages+and+biofilm+during+Staphylococcus+aureus-associated+implant+infection:+Difficulties+and+solutions.">Interactions between macrophages and biofilm during Staphylococcus aureus-associated implant infection: Difficulties and solutions.</a> J Innate Immun. 15 (1), 499-515 (2023).</li><li>Sun, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcal+virulence+factor+hlgb+targets+the+endoplasmic-reticulum-resident+e3+ubiquitin+ligase+amfr+to+promote+pneumonia.">Staphylococcal virulence factor hlgb targets the endoplasmic-reticulum-resident e3 ubiquitin ligase amfr to promote pneumonia.</a> Nat Microbiol. 8 (1), 107-120 (2023).</li><li>Mulcahy, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+autophagy+and+apoptosis+facilitates+intracellular+survival+of+Staphylococcus+aureus+in+human+neutrophils.">Manipulation of autophagy and apoptosis facilitates intracellular survival of Staphylococcus aureus in human neutrophils.</a> Front Immunol. 11, 565545 (2020).</li><li>Cai, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+facilitates+its+survival+in+bovine+macrophages+by+blocking+autophagic+flux.">Staphylococcus aureus facilitates its survival in bovine macrophages by blocking autophagic flux.</a> J Cell Mol Med. 24 (6), 3460-3468 (2020).</li><li>Ahmad-Mansour, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+toxins:+An+update+on+their+pathogenic+properties+and+potential+treatments.">Staphylococcus aureus toxins: An update on their pathogenic properties and potential treatments.</a> Toxins. 13 (10), 677 (2021).</li><li>Davis, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+I+manage+a+patient+with+MRSA+bacteremia.">How I manage a patient with MRSA bacteremia.</a> Clin Microbiol Infect. 28 (2), 190-194 (2022).</li><li>Fait, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+response+and+adaptation+to+vancomycin.">Staphylococcus aureus response and adaptation to vancomycin.</a> Adv Microb Physiol. 85, 201-258 (2024).</li><li>Kelly, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+accumulation+of+antibiotics+to+Staphylococcus+aureus+in+phagosomes+of+live+macrophages.">Measurement of accumulation of antibiotics to Staphylococcus aureus in phagosomes of live macrophages.</a> Angew Chem Int Ed Engl. 63 (3), e202313870 (2024).</li><li>Rowe, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recalcitrant+Staphylococcus+aureus+infections:+Obstacles+and+solutions.">Recalcitrant Staphylococcus aureus infections: Obstacles and solutions.</a> Infect Immun. 89 (4), e00694-e00720 (2021).</li><li>Lehar, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+antibody-antibiotic+conjugate+eliminates+intracellular+S.+aureus.">Novel antibody-antibiotic conjugate eliminates intracellular S. aureus.</a> Nature. 527 (7578), 323-328 (2015).</li><li>Peyrusson, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Staphylococcus+aureus+persisters+upon+antibiotic+exposure.">Intracellular Staphylococcus aureus persisters upon antibiotic exposure.</a> Nat Commun. 11 (1), 2200 (2020).</li><li>Pollitt, E. J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+infection+dynamics.">Staphylococcus aureus infection dynamics.</a> PLoS Pathog. 14 (6), e1007112 (2018).</li><li>Sanders, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aseptic+laboratory+techniques:+plating+methods.">Aseptic laboratory techniques: plating methods.</a> J Vis Exp. 11 (63), e3064 (2012).</li><li>Qin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody-antibiotic+conjugate+targeted+therapy+for+orthopedic+implant-associated+intracellular+S.+aureus+infections.">Antibody-antibiotic conjugate targeted therapy for orthopedic implant-associated intracellular S. aureus infections.</a> J Adv Res. 65, 239-255 (2024).</li><li>Andie, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methicillin-resistant+Staphylococcus+aureus.">Methicillin-resistant Staphylococcus aureus.</a> Nat Rev Dis Primers. 31 (4), 18034 (2018).</li></ol>

<table><tbody><tr><td>24-well plate</td><td>Corning Incorporated, USA</td><td>3524</td><td></td></tr><tr><td>4 % paraformaldehyde solutione</td><td>BBI, UK</td><td>E672002-0500</td><td></td></tr><tr><td>6-well plate</td><td>Corning Incorporated, USA</td><td>3516</td><td></td></tr><tr><td>Beef extract powder</td><td>BBI, UK</td><td>A600114-0500</td><td></td></tr><tr><td>Biohazard safety equipment</td><td>Heal force, China</td><td>VS-1300L-u</td><td></td></tr><tr><td>Cell incubator</td><td>ESCO, Singapore</td><td>CCL-170B-8</td><td></td></tr><tr><td>Cell scraper</td><td>Nest</td><td>710001</td><td></td></tr><tr><td>Centrifuge M1416R</td><td>RWD, China</td><td>M1416R</td><td></td></tr><tr><td>Centrifuge tube</td><td>Guanghou Labselect, China</td><td>CT-002-50A</td><td></td></tr><tr><td>Confocal laser scanning microscope (CLSM)</td><td>Zeiss, Germany</td><td>880</td><td></td></tr><tr><td>Confocal petri dish</td><td>Biosharp, China</td><td>BS-20-GJM</td><td></td></tr><tr><td>DAPI dye</td><td>Shanghai Beyotime, China&amp;nbsp;</td><td>C1006</td><td></td></tr><tr><td>DIL working fluid</td><td>Shanghai Beyotime, China&amp;nbsp;</td><td>C1991S</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium</td><td>Thermo Gibco, USA</td><td>C11995500BT</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Hyclone</td><td>SV30208.02</td><td></td></tr><tr><td>Gentamycin</td><td>Shanghai Sangon, China</td><td>B540724-0010</td><td></td></tr><tr><td>Incubator</td><td>Shanghai Hengzi, China</td><td>HDPF-150</td><td></td></tr><tr><td>Lysozyme</td><td>Beijing Solarbio, China</td><td>L9070</td><td></td></tr><tr><td>MRSA252</td><td>Third Military Medical University, China</td><td>null</td><td></td></tr><tr><td>MRSA252(GPF)</td><td>Third Military Medical University, China</td><td>null</td><td></td></tr><tr><td>Penicillin and Streptomycin</td><td>Shanghai Beyotime, China&amp;nbsp;</td><td>C0222</td><td></td></tr><tr><td>Phosphate Buffer Solution</td><td>Shanghai Beyotime, China&amp;nbsp;</td><td>ST476</td><td></td></tr><tr><td>Saline</td><td>Sichuan Kelun, China</td><td>null</td><td></td></tr><tr><td>Sodium chloride</td><td>Shanghai Macklin, China</td><td>S805275</td><td></td></tr><tr><td>Starch soluble</td><td>Shanghai Sangon, China</td><td>A500904-0500</td><td></td></tr><tr><td>Triton X-100</td><td>Shanghai Beyotime, China&amp;nbsp;</td><td>P0096-100ml</td><td></td></tr><tr><td>Tryptic Soy Agar (TSA) plates</td><td>Beijing AOBOX Biotechnology Co., LTD,China</td><td>02-130</td><td></td></tr><tr><td>Tryptic Soy Broth (TSB) medium</td><td>Beijing AOBOX Biotechnology Co., LTD,China</td><td>02-102K</td><td></td></tr><tr><td>Tryptone</td><td>OXOID, UK</td><td>LP0042B</td><td></td></tr><tr><td>Vancomycin</td><td>Shanghai Beyotime, China&amp;nbsp;</td><td>ST2807-250mg</td><td></td></tr><tr><td>RAW264.7 cell</td><td>USA, ATCC</td><td>null</td><td></td></tr></tbody></table>

development and assessment of intracellular infection models for staphylococcus aureus

<meta charset="utf8"></head><body>Entwicklung und Bewertung von intrazellulären Infektionsmodellen für Staphylococcus aureus

Entwicklung und Bewertung von intrazellulären Infektionsmodellen für Staphylococcus aureus

S. aureus can invade and persist within host cells, including immune cells, which allows it to evade immune detection and clearance. This intracellular ...

development-assessment-intracellular-infection-models-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Development and Assessment of Intracellular Infection Models for Staphylococcus aureus

273 Aufrufe.  Southwest University. Staphylococcus aureus (S. aureus) hat die Fähigkeit, sich im ganzen Körper zu verbreiten und anhaltende und wiederkehrende Infektionen zu verursachen. Um diese Prozesse besser zu verstehen, wird in dieser Studie ein intrazelluläres Infektionsmodell für S. aureus etabliert. Dieses Modell wird eine entscheidende Grundlage für die Untersuchung der Mechanismen hinter intrazellulären Infektionen liefern.

Serie: Entwicklung und Bewertung von intrazellulären Infektionsmodellen für Staphylococcus aureus

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

Die Verwendung von Drip Flow und Rotating Disk-Reaktoren für Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

Forschung

Immunologie und Infektion

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the United States. Among soft-tissue infections, myositis, pyomyositis, and necrotizing fasciitis have been increasingly reported in association with MRSA arising from the community. To understand the interplay between MRSA and host immunity leading to more severe infection, the availability of animal models is critical, permitting the study of host and bacterial factors. Several infection models have been introduced to assess the pathogenesis of S. aureus during superficial skin infection. Here, we describe a subcutaneous infection model that examines the skin, subcutaneous, and muscle pathologies.

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

Murine skin and soft tissue infection model is utilized for assessing the virulence function of methicillin resistant Staphylococcus aureus (MRSA) and the host immunological responses. Here, we presented a subcutaneous infection model for skin and soft tissue infection.

Subkutane Infektion von Methicillin-resistenten Staphylococcus Aureus (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the ...

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

Die Infektion von Zebrafisch-Embryos mit Intrazelluläre bakterielle Krankheitserreger

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus (MRSA) in Rat

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments4-6. The increase in infections due to methicillin-resistant S. aureus (MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs7, 8. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

Experimental rat endocarditis model due to methicillin-resistant S. aureus.

Experimentelle Endokarditis Modell von Methicillin-resistenten<em&gt; Staphylococcus aureus</em&gt; (MRSA) in Ratten

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

Staphylococcus aureus Wachstum mit menschlichen Hämoglobins als Eisenquelle

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

Multiplex PCR Assay f

Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

Studieren Wechselwirkungen von<em&gt; Staphylococcus aureus</em&gt; Mit Neutrophilen durch Durchflusszytometrie und Mikroskopie Zeitraffer

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

Targeting Biofilm Assoziierte<em&gt; Staphylococcus aureus</em&gt; Verwendung von Resazurin Based Drug-Anfälligkeit Assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

Genotypisierung<em&gt; Staphylococcus aureus</em&gt; Durch ribosomalen Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

Methodik für die Studie des horizontalen Gentransfers in<em&gt; Staphylococcus aureus</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

Entwicklung und Bewertung von intrazellulären Infektionsmodellen für Staphylococcus aureus

Zusammenfassung

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Studieren Wechselwirkungen von

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