Allergy—A New Role for T Cell Superantigens of Staphylococcus aureus?

Staphylococcus aureussuperantigens (SAgs) are among the most potent T cell mitogensknown.They stimulate large fractions of T cells by cross-linking their T cell receptor withmajor histocompatibility complex class-II molecules on antigen presenting cells, resulting in Tcell proliferation and massive cytokine release. To date, 26 different SAgs have been described in thespeciesS. aureus; they comprise the toxic shock syndrome toxin (TSST-1), as well as 25 staphylococcalenterotoxins (SEs) or enterotoxin-like proteins (SEls). SAgs can cause staphylococcal food poisoningand toxic shock syndrome and contribute to the clinical symptoms of staphylococcal infection. Inaddition, there is growing evidence that SAgs are involved in allergic diseases. This review providesan overview on recent epidemiological data on the involvement ofS. aureusSAgs and anti-SAg-IgEin allergy, demonstrating that being sensitized to SEs—in contrast to inhalant allergens—is associatedwith a severe disease course in patients with chronic airway inflammation. The mechanisms by whichSAgs trigger or amplify allergic immune responses, however, are not yet fully understood. Here, wediscuss known and hypothetical pathways by which SAgs can drive an atopic diseas

Intracellular Persisting Staphylococcus aureus Is the Major Pathogen in Recurrent Tonsillitis

BACKGROUND: The two major indications for tonsillectomy are recurrent tonsillitis (RT) and peritonsillar abscess (PTA). Unlike PTAs, which are primarily treated surgically, RT is often cured by tonsillectomy only after a series of failed drug therapy attempts. Although the bacteriological background of RT has been studied, the reason for the lack of success of conservative therapeutic approaches is not well understood. METHODS: In a prospective study, tonsil specimens from 130 RT patients and 124 PTA patients were examined for the presence of extra- and intracellular bacteria using antibiotic protection assays. Staphylococcus aureus isolates from RT patients were characterized by pulsed-field gel electrophoresis (PFGE), spa-typing and MSCRAMM-gene-PCR. Their ability for biofilm formation was tested and their cell invasiveness was confirmed by a flow cytometric invasion assay (FACS), fluorescent in situ hybridization (FISH) and immunohistochemistry. FINDINGS: S. aureus was the predominant species (57.7%) in RT patients, whereas Streptococcus pyogenes was most prevalent (20.2%) in PTA patients. Three different assays (FACS, FISH, antibiotic protection assay) showed that nearly all RT-associated S. aureus strains were located inside tonsillar cells. Correspondingly, the results of the MSCRAMM-gene-PCRs confirmed that 87% of these S. aureus isolates were invasive strains and not mere colonizers. Based upon PFGE analyses of genomic DNA and on spa-gene typing the vast majority of the S. aureus isolates belonged to different clonal lineages. CONCLUSIONS: Our results demonstrate that intracellular residing S. aureus is the most common cause of RT and indicate that S. aureus uses this location to survive the effects of antibiotics and the host immune response. A German translation of the Abstract is provided as supplementary material (Abstract S1)

Staphylococcus aureus nasal colonization among dental health care workers in Northern Germany (StaphDent study)

Methicillin-resistant Staphylococcus aureus (MRSA) can colonize dental patients and students, however, studies on the prevalence of MRSA and methicillin-susceptible S. aureus (MSSA) among dental health care workers (DHCW) including use of personal protective equipment (PPE) are scarce. We conducted an observational study (StaphDent study) to (I) determine the prevalence of MRSA and MSSA colonization in DHCW in the region of Mecklenburg Western-Pomerania, Germany, (II) resolve the MSSA population structure to gain hints on possible transmission events between co-workers, and (III) clarify use of PPE. Nasal swabs were obtained from dentists (n?=?149), dental assistants (n?=?297) and other dental practice staff (n?=?38). Clonal relatedness of MSSA isolates was investigated using spa typing and, in some cases, whole genome sequencing (WGS). PPE use was assessed by questionnaire. While 22.3% (108/485) of the participants were colonized with MSSA, MRSA was not detected. MSSA prevalence was not associated with size of dental practices, gender, age, or duration of employment. The identified 61 spa types grouped into 17 clonal complexes and four sequence types. Most spa types (n?=?51) were identified only once. In ten dental practices one spa type occurred twice. WGS data analysis confirmed a close clonal relationship for 4/10 isolate pairs. PPE was regularly used by most dentists and assistants. To conclude, the failure to recover MRSA from DHCW reflects the low MRSA prevalence in this region. Widespread PPE use suggests adherence to routine hygiene protocols. Compared to other regional HCW MRSA rates the consequent usage of PPE seems to be protective

Toxin exposure and HLA alleles determine serum antibody binding to toxic shock syndrome toxin 1 (TSST-1) of Staphylococcus aureus

Life-threatening toxic shock syndrome is often caused by the superantigen toxic shock syndrome toxin-1 (TSST-1) produced by Staphylococcus aureus. A well-known risk factor is the lack of neutralizing antibodies. To identify determinants of the anti-TSST-1 antibody response, we examined 976 participants of the German population-based epidemiological Study of Health in Pomerania (SHIP-TREND-0). We measured anti-TSST-1 antibody levels, analyzed the colonization with TSST-1-encoding S. aureus strains, and performed a genome-wide association analysis of genetic risk factors. TSST-1-specific serum IgG levels varied over a range of 4.2 logs and were elevated by a factor of 12.3 upon nasal colonization with TSST-1-encoding S. aureus. Moreover, the anti-TSST-1 antibody levels were strongly associated with HLA class II gene loci. HLA-DRB1*03:01 and HLA-DQB1*02:01 were positively, and HLA-DRB1*01:01 as well as HLA-DQB1*05:01 negatively associated with the anti-TSST-1 antibody levels. Thus, both toxin exposure and HLA alleles affect the human antibody response to TSST-1

Immunantwort gegen Superantigene bei Staphylococcus aureus Carriern

ZUSAMMENFASSUNG Staphylococcus aureus ist der häufigste Erreger nosokomialer Infektionen, und die zunehmende Antibiotikaresistenz dieser Spezies ist besorgniserregend. Zugleich besiedelt S. aureus die Nasenschleimhaut bei circa 35% der gesunden Bevölkerung, so genannten Carriern. Die nasale Besiedlung mit S. aureus ist ein wichtiger Risikofaktor für die Entstehung von S. aureus Infektionen, die zumeist durch den kolonisierenden Stamm verursacht werden. Dies unterstreicht die Bedeutung von Wirtsfaktoren für den Ausgang von S. aureus-Wirtsinteraktionen. Trotz der klinischen Bedeutung der nasalen Besiedlung ist über die humorale Immunantwort des Wirtes auf eine solche Besiedlung mit Staphylokokken wenig bekannt. Darum stand die anti-S. aureus Antikörperantwort von Carriern und Nichtcarriern im Mittelpunkt dieser Arbeit. Staphylokokken-Superantigene (SAg) dienten in unseren Studien als Indikatorantigene. SAg sind Virulenzfaktoren mit außergewöhnlicher Variabilität in der Spezies S. aureus und wirken als hoch potente T-Zellmitogene. Es sind inzwischen 19 verschiedene SAg-Genloci in der Spezies S. aureus bekannt, aber es gibt bisher nur wenige umfassende Untersuchungen zu ihrer Verteilung. Deshalb haben wir zunächst fünf Multiplex-PCRs zum Nachweis aller 19 SAg-Gene etabliert. Mit dieser robusten Hochdurchsatzmethode haben wir das SAg-Genmuster von mehr als 300 Isolaten untersucht, darunter 107 nasale Isolate von S. aureus Carriern und 88 Blutkulturisolate von Krankenhauspatienten aus Vorpommern. Die SAg-Genmuster waren sehr heterogen, was sich durch die Lokalisation der SAg-Gene auf mobilen genetischen Elementen (MGE), wie genomischen Inseln, Pathogenitätsinseln, Phagen und Plasmiden, erklären lässt. Die meisten Isolate (80%) besaßen SAg-Gene, im Durchschnitt fünf bis sechs, und die SAg des Enterotoxin-Genclusters (egc) waren bei weitem die häufigsten. Außerdem beobachteten wir eine strikte Korrelation zwischen dem Vorhandensein von SAg-Genen und der T-Zell-mitogenen Potenz von klinischen Isolaten. SAg-kodierende MGEs können durch zwei verschiedene Mechanismen verbreitet werden: horizontaler Transfer durch Bakteriophagen und vertikale Übertragung auf Tochterzellen. Um die Verteilung der SAg innerhalb unserer S. aureus-Population zu untersuchen, wurde die klonale Verwandtschaft der S. aureus-Isolate mittels spa-Genotypisierung bestimmt. Interessanterweise waren die SAg-kodierenden MGEs nicht zufällig verteilt, sondern eng an die klonalen Linien gekoppelt. Jede dieser klonalen Linien war durch eine bestimmte Kombination von SAg-Genen charakterisiert. Diese Befunde implizieren, dass die simultane Bestimmung von Virulenzgen-Profilen und dem genetischen Hintergrund die Trennschärfe von genetischen Untersuchungen von S. aureus Virulenzmechanismen erheblich erhöht. Durch den Vergleich der Virulenzgene von kommensalen und invasiven Isolaten mit dem gleichen genetischen Hintergrund ließ sich für das exfoliative Toxin D (bzw. die kodierende Pathogenitätsinsel) eine Bedeutung für die Invasivität von S. aureus aufzeigen, für SAg dagegen mit großer Wahrscheinlichkeit ausschließen. Es ist bekannt, dass neutralisierende Serumantikörper gegen die SAg SEA, SEB, SEC, SED und TSST-1 in gesunden Individuen häufig vorkommen. Die neutralisierenden Antikörper gegen die erst kürzlich beschriebenen SAg oder gegen komplexe SAg-Cocktails, wie sie von klinischen Isolaten sezerniert werden, wurden bisher jedoch noch nicht bestimmt. Darum haben wir über 100 Seren in Neutralisationsassays auf ihre SAg-neutralisierende Kapazität hin untersucht. Wir beobachteten eine erhebliche Heterogenität und erstaunlich große „Lücken“ in der anti-SAg-Antikörperantwort. Besonders egc-SAg wurden nur selten inhibiert (5-10%), während zwischen 32 und 86% der getesteten Seren „klassische“ SAg neutralisierten. Diese “egc-Lücke” in den SAg-neutralisierenden Antikörperprofilen gesunder Individuen war unerwartet, weil die egc-SAg mit Abstand am häufigsten vorkommen. Wir konnten zeigen, dass die „egc-Lücke“ wahrscheinlich nicht auf unterschiedliche T-Zellaktivierung durch egc-SAg im Vergleich zu klassischen SAg zurückzuführen ist, sondern auf eine unterschiedliche Regulation der SAg-Genexpression. S. aureus Carrier haben ein erhöhtes Risiko eine S. aureus Bakteriämie zu entwickeln, welche in den meisten Fällen durch den kolonisierenden Stamm verursacht wird. Erstaunlicherweise zeigte eine große prospektive klinische Studie, dass eine Bakteriämie bei Carriern eine deutlich geringere Letalität hat als bei Nichtcarriern. Um dieses Paradoxon zu erklären, haben wir die Hypothese aufgestellt, dass bei Carriern eine partielle Immunität gegen den kolonisierenden Stamm zu ihren verbesserten Überlebensraten beitragen könnte. Hierzu haben wir SAg als stamm-spezifische Indikatorantigene eingesetzt. Seren von persistierenden Carriern neutralisierten SAg ihres eigenen kolonisierenden Stammes mit signifikant höherer Effizienz als Seren von Nichtcarriern. Diese Antikörperantwort der Carrier war stammspezifisch, da sich die Antikörperantwort der Carrier gegen andere SAg nicht von der der Nichtcarrier unterschied. Somit löst die Besiedlung mit S. aureus eine starke, stammspezifische Antikörperantwort gegen Staphylokokken-SAg aus. Wir vermuten, dass bei Carriern neutralisierende Antikörper, die gegen SAg oder andere Staphylokokken-Virulenzfaktoren gerichtet sind, einen partiellen Schutz bei systemischen Infektionen gewähren. Dies könnte die bessere Prognose von Carriern mit einer S. aureus-Bakteriämie erklären. Außerdem implizieren unsere Ergebnisse, dass der Schlüssel zum Verständnis der Pathogenese von S. aureus Erkrankungen eher in der Identifizierung von Wirtsfaktoren als von bakteriellen Faktoren liegt. Solche Wirtsfaktoren können beispielsweise der Immunstatus oder Genpolymorphismen sein, die zu Kolonisierung, der Infektionsanfälligkeit oder dem Ausgang einer Erkrankung beitragen. Die Behandlung von S. aureus Bakteriämien mit gepoolten Immunglobulinen erbrachte keinen deutlichen Erfolg. Unsere Ergebnisse deuten darauf hin, dass „maßgeschneiderte“ Cocktails aus monoklonalen Antikörpern gegen verschiedene S. aureus Virulenzfaktoren therapeutisch wirksamer sein könnten.SUMMARY To date, Staphylococcus aureus is the most common cause of nosocomial infections and the species is becoming increasingly resistant to antibiotics. Beyond this, S. aureus colonises the nasal mucosa of circa 35% of the healthy population, so-called carriers. Importantly, S. aureus nasal carriage is a major risk factor for the development of S. aureus infections, which are commonly caused by the colonising strain. This underlines the importance of host factors for the outcome of S. aureus-host interactions. Despite the clinical importance of nasal carriage, little is known about humoral immune responses triggered by colonisation. Therefore, this thesis was focussed on the anti-staphylococcal antibody responses of S. aureus carriers and noncarriers. Staphylococcal superantigens (SAgs) served as indicator antigens for our studies. SAgs are virulence factors with extraordinary variability in the species S aureus and act as extremely potent T cell mitogens. To date, 19 different SAg gene loci are known in the species S. aureus, but molecular-epidemiological studies on the distribution of these genes are limited. Therefore, we established five multiplex PCRs for the detection of all known SAgs. With this robust and high-throughput technique we analysed the SAg gene patterns of more than 300 isolates, including 107 nasal isolates of S. aureus carriers and 88 blood culture isolates of hospital patients from Western Pomerania. The SAg gene patterns were highly heterogeneous, which can be explained by their localisation on mobile genetic elements (MGE), such as genomic islands, pathogenicity islands, phages and plasmids. Most isolates (~80%) harboured SAg genes, on average five to six, and SAgs of the enterotoxin gene cluster (egc) were by far the most prevalent. Additionally, we observed a strict correlation between the presence of SAg genes and the T cell mitogenic potency of clinical isolates. SAg-encoding MGEs can be distributed by two distinct mechanisms: horizontal transfer by bacteriophages and vertical transmission to daughter cells. To investigate the distribution of SAg genes within the S. aureus population, we determined the clonal relationship of our isolates by spa genotyping. Interestingly, SAg-gene encoding MGEs were not randomly distributed, but rather closely linked to clonal lineages. Each clonal lineage was characterised by defined combinations of SAg genes. These data suggest that the simultaneous assessment of virulence gene profiles and the genetic background strongly enhances the discriminatory power of genetic investigations into the mechanisms of S. aureus virulence. Indeed, the comparison of virulence genes within each clonal complex indicated a role in invasiveness for some MGEs, e.g. the exfoliative toxin D-encoding pathogenicity island, while rendering it unlikely for SAgs. It is known that neutralising serum antibodies against the SAgs SEA, SEB, SEC, SED and TSST-1 are frequently present in healthy individuals. However, the neutralising antibody profiles against more recently described SAgs or complex SAg cocktails as secreted by clinical isolates had not been determined so far. Therefore, we screened more than 100 sera for their SAg neutralising capacity with a neutralisation assay. We observed a marked heterogeneity and surprisingly large “gaps” in the neutralising capacity. Interestingly, the egc SAgs were inhibited only rarely (5-10%), whereas between 32 and 86% of the tested sera neutralised “classical” SAgs. This “egc gap” in the SAg-neutralising antibody profiles of healthy individuals was unexpected, since egc SAgs are by far the most prevalent SAgs. We could demonstrate that the “egc gap” is probably not due to different T cell activating properties of egc SAgs compared to classical SAgs, but rather to a differential regulation of SAg gene expression. S. aureus carriers have an increased risk of developing an S. aureus bacteraemia, which is in most cases caused by the colonising strain. Intriguingly, a large prospective clinical trial revealed a considerably higher mortality in noncarriers with invasive S. aureus strains compared to carriers with invasive disease. To explain these paradoxical findings, we hypothesised that in carriers partial immunity against the colonising strain may contribute to their improved outcome. We used SAgs as strain-specific indicator antigens. Importantly, sera from persistent carriers neutralised SAgs of their colonising strain with significantly higher efficiency than sera from noncarriers. This antibody response was strain-specific, since the antibody response of carriers against other SAgs did not differ from that of noncarriers. Thus, colonisation with S. aureus confers a strong and strain-specific antibody response against staphylococcal SAgs. We suggest that in carriers neutralising antibodies directed against SAgs and other staphylococcal virulence factors confer partial protection during systemic infections. This could explain the better prognosis of carriers with S. aureus bacteraemia compared to noncarriers. Moreover, our data imply that the key to understanding the pathogenesis of S. aureus disease may lie in the identification of host factors rather than bacterial factors. Such host factors could be the immune status and gene polymorphisms that contribute to colonisation, susceptibility to infection and outcome of infection. Finally, while the treatment of S. aureus bacteraemia with pooled immunoglobulins was performed in the past without significant success, our findings on strain-specific antibody profiles suggest that therapies with customised cocktails of monoclonal antibodies could have a higher efficacy

Development and evaluation of a rapid strategy to determine enterotoxin gene content in Staphylococcus aureus

Enterotoxins of S. aureus are important molecules displaying superantigenic properties. To date no less than 18 enterotoxins have been identified in S. aureus and their role has been documented in very diverse diseases. Using available nucleotide sequence information, we developed a rapid and automated PCR-based approach to evaluate enterotoxin content in S aureus. We studied a collection of S. aureus strains previously analyzed for enterotoxins gene content and report a perfect correlation between simplex and multiplex PCR assays for the presence of all enterotoxin genes described so far. The determination of enterotoxin content relies on 4 multiplex PCR tubes whose amplification products are resolved by a rapid microcapillary electrophoresis. Automated analysis of the PCR profiles evaluates for the presence of the 18 enterotoxin genes in less than 3 h and at moderate cost. Finally, the use of enterotoxin gene content for genotyping purpose was compared to multi-locus variable number of tandem repeat assay and spa genotyping. Analysis revealed an important homogeneity of the genetic backgrounds for strains harboring the egc cluster as well as a large diversity for strains harboring other enterotoxins but lacking the egc cluster. A combined genotyping method that includes rapid enterotoxin content determination appears informative for various epidemiological survey purposes

Omics Approaches for the Study of Adaptive Immunity to Staphylococcus aureus and the Selection of Vaccine Candidates

Staphylococcus aureus is a dangerous pathogen both in hospitals and in the community. Due to the crisis of antibiotic resistance, there is an urgent need for new strategies to combat S. aureus infections, such as vaccination. Increasing our knowledge about the mechanisms of protection will be key for the successful prevention or treatment of S. aureus invasion. Omics technologies generate a comprehensive picture of the physiological and pathophysiological processes within cells, tissues, organs, organisms and even populations. This review provides an overview of the contribution of genomics, transcriptomics, proteomics, metabolomics and immunoproteomics to the current understanding of S. aureus‑host interaction, with a focus on the adaptive immune response to the microorganism. While antibody responses during colonization and infection have been analyzed in detail using immunoproteomics, the full potential of omics technologies has not been tapped yet in terms of T-cells. Omics technologies promise to speed up vaccine development by enabling reverse vaccinology approaches. In consequence, omics technologies are powerful tools for deepening our understanding of the “superbug” S. aureus and for improving its control

Staphylococcus aureus Interferes with Streptococci Spatial Distribution and with Protein Expression of Species within a Polymicrobial Oral Biofilm

We asked whether transient Staphylococcus aureus in the oral environment synergistically interacts with orally associated bacterial species such as Actinomyces oris, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus mutans, and Veillonella dispar (six-species control biofilm 6S). For this purpose, four modified biofilms with seven species that contain either the wild type strain of the S. aureus genotype (USA300-MRSA WT), its isogenic mutant with MSCRAMM deficiency (USA300-MRSA ΔMSCRAMM), a methicillin-sensitive S. aureus (ST72-MSSA-) or a methicillin-resistant S. aureus (USA800-MRSA) grown on hydroxyapatite disks were examined. Culture analyses, confocal-laser-scanning microscopy and proteome analyses were performed. S. aureus strains affected the amount of supragingival biofilm-associated species differently. The deletion of MSCRAMM genes disrupted the growth of S. aureus and the distribution of S. mutans and S. oralis within the biofilms. In addition, S. aureus caused shifts in the number of detectable proteins of other species in the 6S biofilm. S. aureus (USA300-MRSA WT), aggregated together with early colonizers such as Actinomyces and streptococci, influenced the number of secondary colonizers such as Fusobacterium nucleatum and was involved in structuring the biofilm architecture that triggered the change from a homeostatic biofilm to a dysbiotic biofilm to the development of oral diseases