Typisierung und Expression von bakteriellen Exotoxinen am Beispiel von Staphylococcus aureus

Staphylococcus aureus (S. aureus) ist ein vielfältiges und potenziell lebensbedrohliches Bakterium. Bei Infektionen ist es wichtig die Virulenz- und Resistenzgene schnell zu erkennen, um eine angepasste Therapie zu initiieren. Mikroarrays bieten eine optimale Plattform zur geno- und phänotypischen Charakterisierung von Bakterien und somit für die Entwicklung, Validierung und Herstellung klinisch bedeutsamer Schnelltests. In dieser Arbeit wurden DNA-Mikroarrays zur Genotypisierung von S. aureus eingesetzt, die die Klassifizierung und Zuordnung einzelner Isolate zu Verwandtschaftsgruppen sowie den Nachweis vorhandener Virulenz- und Resistenzgene ermöglichen. In zwei Populationsstudien wurden Stämme in Trinidad & Tobago und in Norwegen untersucht. Diese zeigen epidemiologisch unterschiedliche Situationen, die scheinbar auf verschiedenen geografischen und sozioökonomischen Gegebenheiten beruhen. Es wurden ebenfalls Protein-Mikroarrays für den Nachweis von S. aureus Toxinen hergestellt. Zum qualitativen Nachweis vom Virulenzfaktor Panton-Valentine Leukocidin (PVL) wurde mit Hilfe spezifisch gefertigter Protein-Mikroarrays ein Lateral-Flow Test entwickelt und validiert. Weiterhin wurden zwei Protein-Mikroarrays entwickelt, mit denen die in vitro Expressionen der Exotoxine PVL und Hämolysin alpha quantitativ messbar sind. Mit deren Anwendung konnte ein Zusammenhang zwischen der in vitro Toxinexpression und dem genetischen Hintergrund der jeweiligen Stämme nachgewiesen werden. Beim Vergleich der in vitro gebildeten Konzentrationen und den klinischen Krankheitsbildern, die die Isolate zum Teil hervorgerufen haben, konnte keine Korrelation festgestellt werden. Für den parallelen phänotypischen Nachweis mehrerer S. aureus Toxine (PVL, Toxic Shock Syndrome Toxin, Hämolysin alpha & beta, Enterotoxine A & B, Staphylokinase) und Proteine (Penicillin-bindendes Protein 2a, Staphylococcal Protein A) in einem Experiment wurde ein Multiplex-Protein-Mikroarray entwickelt und validiert

PVL overexpression due to genomic rearrangements and mutations in the S. aureus reference strain ATCC25923

OBJECTIVE: ATCC25923 is a Staphylococcus aureus strain that is positive for the Panton Valentin leukocidin. It has been used for decades as reference strain. We observed that two separately maintained clones of ATCC25923 ("G477 and G478") differed grossly in the expression of this toxin. For that reason, both clones were sequenced using an Illumina MiSeq instrument. After assembling, the final sequences were analyzed and mapped to a previously published ATCC25923 sequence (GenBank CP009361) using bl2seq from the NCBI Blast2 package.RESULTS: The genomes of G477 and G478 size 2778,859 and 2792,213 nucleotides, respectively. Both genomes include a circular plasmid of 27,490 nucleotides. The sequence of the G477 chromosome maps nearly exactly to CP009361. G478 has a slightly larger size because of the presence of an additional transposable element tnp13k. The second copy of that tnp13k element is located in an intergenic region between the genes mazF and rsbU. The sequences of the ATCC25923 clones G477 and G478 differ mainly in the insertion of a second tnp13k element between the genes mazF and rsbU. That insertion may lead to a different transcription of that genome region resulting in upregulation of the expression of the Panton-Valentine leukocidin in the ATCC25923 clone G478.</p

Direct, Specific and Rapid Detection of Staphylococcal Proteins and Exotoxins Using a Multiplex Antibody Microarray.

BackgroundS. aureus is a pathogen in humans and animals that harbors a wide variety of virulence factors and resistance genes. This bacterium can cause a wide range of mild to life-threatening diseases. In the latter case, fast diagnostic procedures are important. In routine diagnostic laboratories, several genotypic and phenotypic methods are available to identify S. aureus strains and determine their resistances. However, there is a demand for multiplex routine diagnostic tests to directly detect staphylococcal toxins and proteins.MethodsIn this study, an antibody microarray based assay was established and validated for the rapid detection of staphylococcal markers and exotoxins. The following targets were included: staphylococcal protein A, penicillin binding protein 2a, alpha- and beta-hemolysins, Panton Valentine leukocidin, toxic shock syndrome toxin, enterotoxins A and B as well as staphylokinase. All were detected simultaneously within a single experiment, starting from a clonal culture on standard media. The detection of bound proteins was performed using a new fluorescence reading device for microarrays.Results110 reference strains and clinical isolates were analyzed using this assay, with a DNA microarray for genotypic characterization performed in parallel. The results showed a general high concordance of genotypic and phenotypic data. However, genotypic analysis found the hla gene present in all S. aureus isolates but its expression under given conditions depended on the clonal complex affiliation of the actual isolate.ConclusionsThe multiplex antibody assay described herein allowed a rapid and reliable detection of clinically relevant staphylococcal toxins as well as resistance- and species-specific markers

Population structure of Staphylococcus aureus from Trinidad & Tobago.

It has been shown previously that high rates of methicillin- and mupirocin-resistant Staphylococcus aureus exist in the Caribbean islands of Trinidad and Tobago, as well as a high prevalence of Panton-Valentine leukocidin-positive S. aureus. Beyond these studies, limited typing data have been published. In order to obtain insight into the population structure not only of MRSA but also of methicillin-susceptible S. aureus, 294 clinical isolates collected in 2012/2013 were typed by microarray hybridisation. A total of 15.31% of the tested isolates were MRSA and 50.00% were PVL-positive. The most common MSSA strains were PVL-positive CC8-MSSA (20.41% of all isolates tested), PVL-positive CC152-MSSA (9.52%) and PVL-positive CC30-MSSA (8.84%) while the most common MRSA were ST239-MRSA-III&SCCmer (9.18%) and ST8-MRSA-IV, "USA300" (5.78%). 2.38% of characterised isolates belonged to distinct strains likely to be related to "Staphylococcus argenteus" lineages. The population structure of S. aureus isolates suggests an importation of strains from Africa, endemicity of PVL-positive MSSA (mainly CC8) and of ST239-MRSA-III, and a recent emergence of the PVL-positive CC8-MRSA-IV strain "USA300"

Specific cut-off values for each target.

<p>*Anti-PVL_2 Antibody detects exclusive epitopes of lukF-P83. Positive signals for two of the anti-PVL antibodies, 1, 2 or 3, indicate for the target lukF-PV.</p><p>Specific cut-off values for each target.</p

Alpha Toxin yields by CC/ST affiliation.

<p>Alpha Toxin yields by CC/ST affiliation.</p

Alpha Toxin yields and clinical outcome (veterinary isolates excluded).

<p>Alpha Toxin yields and clinical outcome (veterinary isolates excluded).</p

<i>Staphylococcus aureus In Vitro</i> Secretion of Alpha Toxin (hla) Correlates with the Affiliation to Clonal Complexes

<div><p>The alpha toxin of <i>Staphylococcus aureus</i> is a pore forming toxin that penetrates host cell membranes causing osmotic swelling, rupture, lysis and subsequently cell death. Haemolysin alpha is toxic to a wide range of different mammalian cells; i.e., neurotoxic, dermonecrotic, haemolytic, and it can cause lethality in a wide variety of animals. In this study, the <i>in vitro</i> alpha toxin production of 648 previously genotyped isolates of <i>S. aureus</i> was measured quantitatively using antibody microarrays. Isolates originated from medical and veterinary settings and were selected in order to represent diverse clonal complexes and defined clinical conditions. Generally, the production of alpha toxin <i>in vitro</i> is related to the clonal complex affiliation. For clonal complexes CC22, CC30, CC45, CC479, CC705 and others, invariably no alpha toxin production was noted under the given <i>in vitro</i> conditions, while others, such as CC1, CC5, CC8, CC15 or CC96 secreted variable or high levels of alpha toxin. There was no correlation between alpha toxin yield and clinical course of the disease, or between alpha toxin yield and host species.</p></div

Layout of the protein microarray.

<p>(A) Positions of each substance on the chip. (B) Legend to the probes. (C) Picture of a processed fluorescent microarray. (D) Bar graph with signal intensities for the expressed proteins.</p

Alpha Toxin yields and <i>agr</i> group affiliation.

<p>Alpha Toxin yields and <i>agr</i> group affiliation.</p