Temperate bacteriophages play an important role in the pathogenicity of Staphylococcus aureus, for instance, by mediating the horizontal gene transfer of virulence factors. Here we established a classification scheme for staphylococcal prophages of the major Siphoviridae family based on integrase gene polymorphism. Seventy-one published genome sequences of staphylococcal phages were clustered into distinct integrase groups which were related to the chromosomal integration site and to the encoded virulence gene content. Analysis of three marker modules (lysogeny, tail, and lysis) for phage functional units revealed that these phages exhibit different degrees of genome mosaicism. The prevalence of prophages in a representative S. aureus strain collection consisting of 386 isolates of diverse origin was determined. By linking the phage content to dominant S. aureus clonal complexes we could show that the distribution of bacteriophages varied remarkably between lineages, indicating restriction-based barriers. A comparison of colonizing and invasive S. aureus strain populations revealed that hlb-converting phages were significantly more frequent in colonizing strains.Staphylococcus aureus asymptomatically colonizes the anterior nares of humans but also causes a wide spectrum of acute and chronic diseases. Most of the dissimilarity between S. aureus strains is due to the presence of mobile genetic elements such as plasmids, bacteriophages, pathogenicity islands, transposons, and insertion sequences (2,14,19,23). Many virulence factors are encoded on such mobile elements (3,6,17,26,27,35). In particular, bacteriophages play an important role in the pathogenicity of S. aureus either by carrying accessory virulence factors such as Panton-Valentine leukocidin (PVL) (encoded by the luk-PV operon), staphylokinase (encoded by sak), enterotoxin A (encoded by sea), and exfoliative toxin A (encoded by eta) or by interrupting chromosomal virulence genes such as those for ␤-hemolysin (hlb) and lipase (geh) upon insertion. Additionally, phages are the primary vehicle of lateral gene transfer between S. aureus strains, providing the species with the potential for broad genetic variation. We could show that phages increase the genome plasticity of S. aureus during infection, facilitating the adaptation of the pathogen to various host conditions (11,12).Despite the obvious importance of phages for the biology of S. aureus, epidemiological data on the prevalence of phages in this species are limited (28, 33). More than 80 genome sequences of staphylococcal bacteriophages and prophages are available in the public genome databases. Most published S. aureus phages belong to the Siphoviridae family of temperate, tailed bacterial viruses. Traditionally, S. aureus phages were characterized according to their lytic activity, morphology, and serological properties (1, 28). Today, the temperate phages in clinical S. aureus isolates can by identified with a multiplex PCR strategy, which is based on sequence differences between viral genes codin...

Staphylococcus aureus is both a successful human commensal and a major pathogen. The elucidation of the molecular determinants of virulence, in particular assessment of the contributions of the genetic background versus those of mobile genetic elements (MGEs), has proved difficult in this variable species. To address this, we simultaneously determined the genetic backgrounds (spa typing) and the distributions of all 19 known superantigens and the exfoliative toxins A and D (multiplex PCR) as markers for MGEs. Methicillin-sensitive S. aureus strains from Pomerania, 107 nasal and 88 blood culture isolates, were investigated. All superantigenencoding MGEs were linked more or less tightly to the genetic background. Thus, each S. aureus clonal complex was characterized by a typical repertoire of superantigen and exfoliative toxin genes. However, within each S. aureus clonal complex and even within the same spa type, virulence gene profiles varied remarkably. Therefore, virulence genes of nasal and blood culture isolates were separately compared in each clonal complex. The results indicated a role in infection for the MGE harboring the exfoliative toxin D gene. In contrast, there was no association of superantigen genes with bloodstream invasion. In summary, we show here that the simultaneous assessment of virulence gene profiles and the genetic background increases the discriminatory power of genetic investigations into the mechanisms of S. aureus pathogenesis.

A clinically important adverse drug reaction, heparin-induced thrombocytopenia (HIT), is induced by antibodies specific for complexes of the chemokine platelet factor 4 (PF4) and the polyanion heparin. Even heparin-naive patients can generate anti-PF4/heparin IgG as early as day 4 of heparin treatment, suggesting preimmunization by antigens mimicking PF4/heparin complexes. These antibodies probably result from bacterial infections, as (1) PF4 bound charge-dependently to various bacteria, (2) human heparin-induced anti-PF4/heparin antibodies cross-reacted with PF4-coated Staphylococcus aureus and Escherichia coli, and (3) mice developed anti-PF4/heparin antibodies during polymicrobial sepsis without heparin application. Thus, after binding to bacteria, the endogenous protein PF4 induces antibodies with specificity for PF4/polyanion complexes. These can target a large variety of PF4-coated bacteria and enhance bacterial phagocytosis in vitro. The same antigenic epitopes are expressed when pharmacologic heparin binds to platelets augmenting formation of PF4 complexes. Boosting of preformed B cells by PF4/ heparin complexes could explain the early occurrence of IgG antibodies in HIT. We also found a continuous, rather than dichotomous, distribution of anti-PF4/heparin IgM and IgG serum concentrations in a cross-sectional population study (n ‫؍‬ 4029), indicating frequent preimmunization to modified PF4. PF4 may have a role in bacterial defense, and HIT is probably a misdirected antibacterial host defense mechanism. (Blood. 2011;117(4): 1370-1378) IntroductionThe chemokine platelet factor 4 (PF4, CXCL4) is stored within platelet ␣-granules 1 and released during platelet activation. Although its biologic role is poorly understood, PF4 commands attention in clinical medicine because it binds charge-dependently to the anticoagulant heparin, one of the most frequently used anticoagulants in clinical medicine, thereby neutralizing heparin's anticoagulant effect, 2,3 but also forming highly antigenic multimolecular complexes. [4][5][6] The resulting antibody response 7 induces the most frequent immune-mediated adverse drug reaction involving human blood cells, heparin-induced thrombocytopenia (HIT). 8 The pathogenic antibodies bind to PF4/heparin complexes at the platelet surface, and the resulting immune complexes induce Fcreceptor-mediated platelet activation and enhanced thrombin generation. 8 In a subset of patients, this causes thrombocytopenia and triggers paradoxical thrombosis, which is aggravated by continuation of heparin treatment.The immune response of HIT has several atypical features. Even in patients who receive heparin for the first time, there is rapid induction (as early as 4 days) of anti-PF4/heparin antibodies of IgG isotype. Moreover, IgG antibodies are the predominant class of immunoglobulins formed. 9,10 We and others discovered that up to 50% of patients after cardiopulmonary bypass surgery and 20% to 30% of orthopedic surgery patients (many of whom have not been previously exposed to heparin) devel...

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Background and Purpose-Recent studies have attributed the increased infection vulnerability of patients with stroke to stroke-induced immunosuppression. We have therefore explored the immunological changes in patients with ischemic stroke. Methods-Blood from 46 patients with stroke was analyzed by fluorescent-activated cell sorter to determine leukocyte subsets. To identify changes that represent clinically relevant immunosuppression, we compared patients who developed infection within 14 days after stroke with those who did not. Results-Stroke induced a dramatic and immediate loss of T-lymphocytes, most pronounced within 12 hours after stroke onset. Only patients with subsequent infection exhibited a delay in the recovery of CD4ϩ T-lymphocyte counts. Conclusions-Our data suggest that a loss of CD4ϩ T cell function contributes to the stroke-induced immunosuppression.The CD4ϩ T cell count on the day after stroke may emerge as a predictive marker for poststroke infection allowing, early identification of patients at risk. (Stroke. 2008;39:237-241.)

Mobile genetic elements (MGEs) encoding virulence and resistance genes are widespread in bacterial pathogens, but it has remained unclear how they occasionally jump to new host species. Staphylococcus aureus clones exchange MGEs such as S. aureus pathogenicity islands (SaPIs) with high frequency via helper phages. Here we report that the S. aureus ST395 lineage is refractory to horizontal gene transfer (HGT) with typical S. aureus but exchanges SaPIs with other species and genera including Staphylococcus epidermidis and Listeria monocytogenes. ST395 produces an unusual wall teichoic acid (WTA) resembling that of its HGT partner species. Notably, distantly related bacterial species and genera undergo efficient HGT with typical S. aureus upon ectopic expression of S. aureus WTA. Combined with genomic analyses, these results indicate that a ‘glycocode’ of WTA structures and WTA-binding helper phages permits HGT even across long phylogenetic distances thereby shaping the evolution of Gram-positive pathogens.

The two-component system SaeRS consisting of the histidin kinase SaeS and the response regulator SaeR is known to act on virulence gene expression in Staphylococcus aureus. In order to get a more comprehensive picture on SaeR-regulated genes, we studied the contribution of the two-component system on global gene expression by using both the proteomic and transcriptomic approach. Altogether, a loss of SaeRS resulted in a decreased amount of at least 17 extracellular proteins and two cell surface-associated proteins, among them several important virulence factors such as HlgA, HlgB, HlgC, LukF, and LukM. SaeRS activates the expression of these genes at the transcriptional level. The amount of the five proteins Aur, SspA, SsaA, Plc, and GlpQ was negatively influenced by SaeRS. However, the transcription of the corresponding genes was not affected by the two-component system. SaeRS had also no measurable influence on the transcription of the regulatory genes agr, sarA, arlRS, and sigB that contribute to the regulation of SaeRS-dependent virulence factors identified in this investigation. Our results clearly show that SaeRS is strongly involved in the tight temporal control of virulence factor expression in S. aureus. Its precise role within the regulatory network remains to be determined.Staphylococcus aureus is a gram-positive bacterium that colonizes the anterior nares of at least one-third of the human population but also causes a variety of infections ranging from superficial lesions, such as wound infections and abscesses, to severe systemic infections such as bacteremia, endocarditis, pneumonia, and osteomyelitis. The pathogenicity of this organism largely depends on the successful adaptation to the human host and the environmentally coordinated expression of virulence factors. The expression of virulence factors in S. aureus is regulated during the growth cycle by a network of interacting regulators (for a review, see reference 41). The best-characterized virulence-associated regulons thus far are the agr regulon (accessory gene regulator), the SarA regulon (staphylococcal accessory regulator), the B regulon (alternative sigma factor), the Rot regulon (regulator of toxins), and the ArlRS regulon (autolysis-regulated locus) (7,15,20,37,47,60,61).The sae locus was first described by Giraudo et al. (27) following the characterization of a Tn551 insertional mutant of S. aureus RC161. sae is a regulatory locus that consists of four open reading frames, two of them encode the response regulator and the sensor kinase, respectively (23). Two additional open reading frames coding for hypothetical proteins are probably important for the functionality of the sae operon (42, 56).

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