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Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is one of the worldwide concerns of antimicrobial chemotherapy. An accumulation of ten patients in five families (A-E) suffering from skin and soft tissue infection (SSTI) of CA-MRSA was experienced in 2012, in Fuchu-shi, Tokyo, Japan. Molecular epidemiological investigation was performed for the 10 MRSA strains obtained from 8 children and 2 of their parents to assess endemic patterns of CA-MRSA in the community. Results of molecular typing, presence of toxin genes and antimicrobial susceptibilities were analyzed combined with the patients' clinical information. Each family had its own unique MRSA strain: A, ST30-SCCmec IVd; B, ST8-SCCmec IVd; C, ST8-SCCmec IVa; D, ST8-SCCmec IVl; E, ST8-SCCmec IVl and ST858-SCCmec IVl. Seven strains from the families A-C carried Panton-Valentine leukocidin gene. Three strains from the families D and E carried toxic shock syndrome toxin gene. Strains belonged to the same family demonstrated genetically related banding patterns of pulsed-filed gel electrophoresis. The family C experienced intrafamilial transmission of USA300-0114. Our data showed the MRSA clones disseminating in this community were highly diverse. They contained USA300-0114 clone, the rapidly distributing clone in the world, as well as MRSA clones identified in Japan. Our results suggested intrafamilial transmission of MRSA could be initial phenomenon of wide transmission in a community, therefore CA-MRSA SSTI in children and their family members should be monitored closely in order to notice the spread of highly pathogenic and transmittable strains.

Gram-positive bacteria cause a broad spectrum of infection-related diseases in both immunocompetent and immunocompromised hosts, ranging from localized infections to severe systemic conditions such as septic and toxic shock syndromes. This situation has been aggravated by the recent emergence of multidrug-resistant strains, thus stressing the need for alternative therapeutic approaches. One such possibility would be modulating the host's immune response. Herein, the potential use of a soluble form of the scavenger-like human lymphocyte receptor CD6 (shCD6) belonging to an ancient family of innate immune receptors has been evaluated. shCD6 can bind to a broad spectrum of gram-positive bacteria thanks to the recognition of highly conserved cell wall components (lipoteichoic acid [LTA] and peptidoglycan [PGN]), which are essential for their viability and pathogenicity and are not amenable to antibiotic resistance. shCD6 has in vitro inhibitory effects on both bacterial growth and Toll-like receptor-mediated inflammatory response induced by LTA plus PGN. In vivo infusion of shCD6 improves survival on mouse models of septic shock by Staphylococcus aureus (either multidrug-resistant or -sensitive) or their endotoxins (LTA + PGN) or exotoxins (TSST-1). These results support the use of shCD6 and/or other scavenger-like immune receptors in the treatment of severe gram-positive-induced infectious conditions.

Every adaptive immune response requires costimulation through the B7/CD28 axis, with CD28 on T-cells functioning as principal costimulatory receptor. Staphylococcal and streptococcal superantigen toxins hyperstimulate the T-cell-mediated immune response by orders of magnitude, inducing a lethal cytokine storm. We show that to elicit an inflammatory cytokine storm and lethality, superantigens must bind directly to CD28. Blocking access of the superantigen to its CD28 receptor with peptides mimicking the contact domains in either toxin or CD28 suffices to protect mice effectively from lethal shock. Our finding that CD28 is a direct receptor of superantigen toxins broadens the scope of microbial pathogen recognition mechanisms.

Noninvasive vaginal infections by Staphylococcus aureus strains producing the superantigen TSST-1 can cause menstrual toxic shock syndrome (mTSS). With the objective of exploring the basis for differential susceptibility to mTSS, the relative responsiveness to TSST-1 of healthy women has been investigated. Peripheral blood mononuclear cells from healthy donors were incubated with purified TSST-1 or with the T-cell mitogen phytohemmaglutinin (PHA), and proliferation was measured. The concentrations of TSST-1 and PHA required to elicit a response equivalent to 15% of the maximal achievable response (EC15) were determined. Although with PHA, EC15 values were comparable between donors, subjects could be classified as being of high, medium, or low sensitivity based on responsiveness to TSST-1. Sensitivity to TSST-1-induced proliferation was associated with increased production of the cytokines interleukin-2 and interferon-γ. When the entire T lymphocyte population was considered, there were no differences between sensitivity groups with respect to the frequency of cells known to be responsive to TSST-1 (those bearing CD3(+) Vβ2(+)). However, there was an association between sensitivity to TSST-1 and certain HLA-class II haplotypes. Thus, the frequencies of DR7DQ2, DR14DQ5, DR4DQ8, and DR8DQ4 haplotypes were greater among those with high sensitivity, a finding confirmed by analysis of responses to immortalized homozygous B cell lines. Collectively, the results reveal that factors other than neutralizing antibody and the frequency of Vβ2(+) T lymphocytes determine immunological responsiveness to TSST-1. Differential responsiveness of lymphocytes to TSST-1 may form the basis of interindividual variations in susceptibility to mTSS.

OBJECTIVES It is common wisdom that persistent carriage of Staphylococcus aureus is more frequent in young children than in adults. The objectives of this study were to assess the S. aureus temporal carriage pattern among a healthy community of pre-school children, with concomitant description of genotype diversity, toxin-encoding genes and antibiotic resistance. METHODS Among 333 children 3-6 years of age, S. aureus nasopharyngeal carriage was assessed over one school year by culture of three sequential nasopharyngeal aspirates. Identification, methicillin resistance and toxin production profile were determined by PCR. Genotyping was performed by spa sequencing and multilocus sequence typing (MLST). RESULTS Out of 830 samples collected, 286 (34%) yielded S. aureus from 185 carriers (55%). Based on consecutive genotype analysis, only 40/268 (15%) children could be classified as persistent carriers, and the remaining 118 (44%) showed intermittent carriage. spa typing revealed 82 types clustered into 13 spa clonal complexes (CCs). Fourteen strains isolated from 11 (3%) children were methicillin-resistant S. aureus (MRSA), half of these strains belonged to the commonly hospital-associated spa t008-ST8-SCCmec IV. Methicillin-susceptible S. aureus (MSSA) were genotypically more diverse. Toxic shock syndrome toxin and egc1/2 complexes were highly prevalent (24%). Contrastingly, Panton-Valentine leucocidin (PVL) was carried only by three MSSA strains (0.6% of children). Exfoliative toxins were detected in 10 (3.5%) MSSA strains, of which 5 were related to the impetigo clone CC121. CONCLUSIONS Although S. aureus nasopharyngeal carriage was high among healthy pre-school children, persistent carriage seems to be less frequent than previously reported. The prevalence of MRSA carriage was 3%, but was not associated with PVL.

Objectives: This prospective cohort study evaluates colonization dynamics and molecular characteristics of methicillin-susceptible and - resistant Staphylococcus aureus (MSSA/MRSA) in a German general population.Methods: Nasal swabs of 1878 non-hospitalized adults were screened for S. aureus. Participants were screened thrice in intervals of 6-8 months. Isolates were characterized by spa and agr typing, mecA and mecC possession, respectively, and PCRs targeting virulence factors.Results: 40.9% of all participants carried S. aureus at least once while 0.7% of the participants carried MRSA (Mainly spa t011). MSSA isolates (n =1359) were associated with 331 different spa types; t084 (7.7%), t091 (6.1%) and t012 (71, 5.2%) were predominant. Of 206 participants carrying S. aureus at all three sampling time points, 14.1% carried the same spa type continuously; 5.3% carried different spa types with similar repeat patterns, but 80.6% carried S. aureus with unrelated spa types. MSSA isolates frequently harboured genes encoding enterotoxins (sec: 16.6%, seg: 63.1%, sei: 64.5%) and toxic shock syndrome toxin (tst: 17.5%), but rarely Panton-Valentine leukocidin (lukS-PV IlukF-PV: 0.2%).Conclusions: MSSA colonizing human nares in the community are clonally highly diverse. Among those constantly carrying S. aureus, clonal lineages changed over time. The proportion of persistent S. aureus carriers was lower than reported elsewhere. (C) 2016 Elsevier GmbH. All rights reserved.

Comparative genomics were used to assess genetic differences between Staphylococcus aureus strains derived from infected animals versus colonized or infected humans. A total of 77 veterinary isolates were genetically characterized by high-throughput amplified fragment length polymorphism (AFLP). Bacterial genotypes were introduced in a large AFLP database containing similar information for 1,056 human S. aureus strains. All S. aureus strains isolated from animals in close contact with humans (e.g., pet animals) were predominantly classified in one of the five main clusters of the AFLP database (cluster I). In essence, mastitis-associated strains from animals were categorized separately (cluster IVa) and cosegregated with bacteremia-associated strains from humans. Distribution of only 2 out of 10 different virulence genes differed across the clusters. The gene encoding the toxic shock syndrome protein ( tst ) was more often encountered among veterinary strains ( P < 0.0001) and even more in the mastitis-related strains ( P< 0.0001) compared to human isolate results. The gene encoding the collagen binding protein ( cna ) was rarely detected among invasive human strains. The virulence potential, as indicated by the number of virulence genes per strain, did not differ significantly between the human- and animal-related strains. Our data show that invasive infections in pets and humans are usually due to S. aureus strains with the same genetic background. Mastitis-associated S. aureus isolated in diverse farm animal species form a distinct genetic cluster, characterized by an overrepresentation of the toxic shock syndrome toxin superantigen-encoding gene.

ITALIC! Staphylococcus aureusbiofilms can be found on vaginal epithelia, secreting toxins and causing inflammation. The co-vaginal species ITALIC! Lactobacilluscan alter staphylococcal-induced epithelial secretion of inflammatory cytokines and quench staphylococcal toxic shock syndrome toxin-1 secretion. It is hypothesized that these effects of lactobacilli require direct physical contact between lactobacilli, staphylococci and the epithelium. Indeed, lactobacilli only reduced ITALIC! S. aureus-induced inflammatory cytokine expression when allowed physical contact with vaginal epithelial cells. Furthermore, a reduction in toxic shock syndrome toxin-1 secretion only occurred when a probiotic ITALIC! Lactobacillusstrain was allowed contact, but not when being physically separated from ITALIC! S. aureus Bacterial-probe atomic force microscopy demonstrated that lactobacilli and staphylococci strongly adhere to epithelial cells, while lactobacilli adhere stronger to staphylococci than staphylococci to each other, giving lactobacilli opportunity to penetrate and reside in staphylococcal biofilms, as visualized using confocal laser scanning microscopy with fluorescence ITALIC! in situhybridization probes. These results identify that physical contact and biochemical signaling by lactobacilli are intrinsically linked mechanisms that reduce virulence of ITALIC! S. aureusbiofilm.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important pathogens in the hospital environment. Monitoring of this pathogen by molecular characterization and phenotypic methods is important for the development of suitable infection control measures and proper therapy design. In this study, our aim was to investigate the molecular epidemiological characteristics of MRSA bloodstream isolates obtained from patients hospitalized at Ankara University Ibn-i Sina Hospital in a 10-year period (2002-2012) and monitor the possible changes. A total of 134 isolates were characterized according to their antimicrobial susceptibility profiles, biofilm formation capabilities, accessory gene regulator (agr) locus types, presence of genes encoding Panton-Valentine leukocidin (PVL), staphylococcal enterotoxins A-J (SEs A-J), toxic shock syndrome toxin, sasX, and genes associated with biofilm formation (icaD, icaA, IS256) by polymerase chain reaction. The staphylococcal cassette chromosome mec (SCCmec) types of isolates were also defined and their clonal relationships were investigated by pulsed-field gel electrophoresis (PFGE) analysis and multilocus sequence typing was performed for representative isolates obtained by PFGE.The majority of the isolates were resistant to rifampin (100%), ciprofloxacin (97%), tetracycline (97.7%), and gentamicin (94.7%); 100% carried type-III SCCmec and 89.5% were agr type-1. All the isolates were negative for PVL, and sasX genes while all of them carried the icaD, icaA, and IS256 genes. The most common SE was enterotoxin A (97%). Four major PFGE patterns with the dominance of one pattern and seven unique patterns were obtained. All the representative PFGE isolates (n = 11) belonged to sequence type 239.We have documented the characteristics of the dominant MRSA clone in our hospital, which was a PVL (-), sasX (-) ST239 clone carrying sea (+) with type-III SCCmec, and type-1 agr locus.

Staphylococcus aureus is the leading etiologic agent of orthopedic implant infections. Here a ribocluster of 27 S. aureus strains underwent further molecular characterization and subtyping by multilocus sequence typing (MLST) and spa-typing. This cluster had been detected by automated ribotyping (with EcoRI as restriction enzyme) of 200 S. aureus isolates from periprosthetic infections come for revision at the Rizzoli Orthopaedic Institute. The ribocluster, consisting of agr type III isolates, with a 74% co-presence of bone sialoprotein-binding (bbp) and collagen-binding (cna) genes, turned out devoid of mecA and IS256 and exhibited a high prevalence of toxic shock syndrome toxin gene (tst, 85%). Sequences achieved by spa typing and MLST were analyzed by BURP and goeBURST. Two predominant spa types, t012 (32%) and t021 (36%), and one predominant sequence type, ST30 (18/27, 67%), a Staphylococcus aureus lineage spread worldwide and regarded as the ancestor of MLST CC30, were identified. Two new sequence types (ST2954, ST2960) and one new spa type (t13129) were detected for the first time. BURP clustered the isolates into two spa clonal complexes, CC021/012 (22/27, 81%) and CC166 (4/27, 15%), plus one singleton, while goeBURST recognized solely MLST CC30. Interestingly, the 27-strains cluster detected by ribotyping corresponded exactly to CC30.

Introduction: Staphylococcus pseudintermedius is coagulase-positive species of the Staphylococcus intermedius group. It is an opportunistic pathogen that can cause infection in various parts of the body and has a zoonotic potential. Although studies on the pathogenicity and epidemiology of S. pseudintermedius are limited, it is known that this bacterium has several virulence factors, including toxins. These toxins can be classified into three main groups: pyrogenic toxins with superantigenic properties such as toxic shock syndrome toxin and staphylococcal enterotoxins, exfoliative toxins, and cytotoxins such as hemolysins and leukocidins. Methodology: In this study, the occurrence of eight toxin genes (sea, sec, tst, SIET, EXI, LuK F-I, Luk S-I, and hlg ƴ) was examined by PCR in 58 isolates of S. pseudintermedius from four domestic animal species. Results: All S. pseudintermedius isolates had at least one of the eight toxin genes. The predominant toxin genes were Luk S-I (95%), Luk F-I (91%), and EXI (91%), and the least prevalent gene was hlg ƴ (5%). Significant association (p = 0.0175) was found between the occurrence patterns of genes hlg ƴ and Luk F-I. Conclusions: The frequent occurrence of these genes in S. pseudintermedius obtained from diseased animals indicates that these toxins may play an important role in the pathogenesis of infection among domestic animals.

Introduction: Recent studies suggest that IgE to Staphylococcus aureus (SA) enterotoxins represent a risk factor for severe asthma even in asthmatic patients considered non atopic. SA enterotoxins can stimulate specific IgE responses but, acting as superantigens, they can promote a polyclonal IgE response, airway inflammation, and bronchial hyperresponsiveness. In comparison with the measurement of serum specific IgE, Basophil Activation Test (BAT) can give more relevant results because it measures only functional IgE capable of activating basophils. BAT for SA enterotoxins has never been performed until to now. Methods: We recruited 35 patients with severe asthma treated according to GINA guidelines. They were tested for skin prick test to common aeroallergens. Total and specific IgE to SA enterotoxins (ImmunoCAP) were measured. Nasal swabs and sputum cultures were obtained. Basophil activation tests (BAT) using CD203c expression was done after stimulation with different concentrations of enterotoxins A, B, and toxic shock syndrome toxin (Sigma). Results: BAT for at least one of SA enterotoxin was positive in 13 among 35 severe asthmatic patients (37%), higher in non atopic than in atopic asthmatic patients (41% vs 33%). Specific IgE to SA enterotoxins were detected in 19 patients ( 54%). No relationship was observed between SA nasal colonization and the presence of IgE or the positivity of basophil activation test for SA enterotoxins. Conclusions: In this study we demonstrate the involvement of specific IgE mechanisms in severe asthmatic patients sensitised to staphylococcus enterotoxins. The potential benefit of anti-IgE therapy in this subgroup of severe asthmatic patients has to be investigated.

We describe a patient presenting with fulminant monomicrobial necrotizing fasciitis (NF) and toxic shock syndrome (TSS) caused by Staphylococcus aureus producing toxic shock syndrome toxin-I (TSST-I). To our knowledge, TSST-I positivity combined with NF has been described only once in previous literature. We discuss the clinical and pathophysiological aspects of both necrotizing fasciitis and toxic shock syndrome and provide a comprehensive overview of causative micro-organisms and the different toxins they produce. We conclude that intravenous immunoglobulin (IVIG) treatment might be considered not only in streptococcal but also in staphylococcal NF.

Toxic shock syndrome toxin 1 (TSST-1) is a Staphylococcus aureus superantigen that has been implicated in both menstrual and nonmenstrual toxic shock syndrome (TSS). Despite the important role of TSST-1 in severe human disease, a comprehensive understanding of staphylococcal regulatory factors that control TSST-1 expression remains incomplete. The S. aureus exotoxin expression (Sae) operon contains a well-characterized two-component system that regulates a number of important exotoxins in S. aureus , although regulation of TSST-1 by the Sae system has not been investigated. We generated a defined deletion mutant of the Sae histidine kinase sensor ( saeS ) in the prototypic menstrual TSS strain S. aureus MN8. Mutation of saeS resulted in a complete loss of TSST-1 expression. Using both luciferase reporter experiments and quantitative real-time PCR, we demonstrate that the Sae system is an important transcriptional activator of TSST-1 expression. Recombinant SaeR was able to bind directly to the tst promoter to a region containing two SaeR consensus binding sites. Although the stand-alone SarA transcriptional regulator has been shown to be both a positive and a negative regulator of TSST-1, deletion of sarA in S. aureus MN8 resulted in a dramatic overexpression of TSST-1. As expected, mutation of agr also reduced TSST-1 expression, but this phenotype appeared to be independent of Sae. A double mutation of saeS and sarA resulted in the loss of TSST-1 expression. This work indicates that the Sae system is a dominant and direct transcriptional activator that is required for expression of TSST-1. IMPORTANCE The TSST-1 superantigen is an exotoxin, produced by some strains of S. aureus , that has a clear role in both menstrual and nonmenstrual TSS. Although the well-characterized agr quorum sensing system is a known positive regulator of TSST-1, the molecular mechanisms that directly control TSST-1 expression are only partially understood. Our studies demonstrate that the Sae two-component regulatory system is a positive transcriptional regulator that binds directly to the TSST-1 promoter, and furthermore, our data suggest that Sae is required for expression of TSST-1. This work highlights how major regulatory circuits can converge to fine-tune exotoxin expression and suggests that the Sae regulatory system may be an important target for antivirulence strategies.

Methicillin resistant Staphylococcus aureus (MRSA) is a major human pathogen responsible for several life threatening conditions. MRSA have the ability to acquire resistance to several antimicrobial agents and phage therapy is one potential option to treat this pathogen. The aim of the study was to isolate and characterize bacteriophages effective against a wide range of methicillin-resistant Staphylococcus aureus (MRSA). A mixture of ten MRSA isolates was used for the isolation of phage from wastewater treatment plants. Three phages were selected for further characterization. All three phages belong to the Siphoviridae family and have long non-contractile flexible tails. The three phages showed a wide host range against S. aureus. Phages E¸SA1 and E¸SA2 were resistant to a pH range from 4-10 while E¸SA3 has a pH range from 3-11. DNA from all three phages was resistant to digestion by endonuclease enzymes such as EcoRI and AccI. There was a high degree of mosaicism among the three virulent phages and with their ancestor phages of Siphoviridae due to their non-uniform access to the common genetic pool by horizontal gene transfer and recombination. Since some of the staphylococcal toxins are phage encoded, the presence of genes for such toxins was tested by performing polymerase chain reaction and all three phages lacked genes for any of the staphylococcal toxins, including staphylococcal enterotoxins (sea, seb, sec and see), exfoliating toxins (eta and etb) and the toxic shock syndrome toxin (tst), therefore these bacteriophage are suitable candidates for future use in phage therapy against MRSA.

We analyzed 353 Staphylococcus aureus bloodstream isolates from 2004 to 2009 to identify dominant genotypes, changes over time, and associations between genotype, phenotype, and clinical parameters. The isolates were genotyped with regard to spa type and presence of Panton-Valentine leukocidin and toxic shock syndrome toxin 1-encoding genes. A high level of genetic diversity was detected. All but three isolates were methicillin sensitive. Interestingly, spa clonal complex 021 showed a weak association with higher all-cause hospital mortality.

SUMMARYThis study determined the genetic background of virulence and resistance genes of MRSA ST398 in Austria. From 2004 up to 2008 a total of 41 human isolates of MRSA ST398 were investigated for virulence and resistance gene patterns using DNA microarray chip analysis. Highly similar virulence gene profiles were found in 29 (70·7%) of the isolates but genes encoding Panton–Valentine leukocidin, enterotoxins, or toxic shock syndrome toxin were not detected. Genes conferring resistance to tetracycline and erythromycin-lincosamide were common as all but one of the isolates exhibited tetM and/or tetK, which are involved in tetracycline resistance, and 12 (29·9%) were positive for ermC, conferring resistance to erythromycin/lincosamide. SplitsTree analysis showed that 40 isolates were closely related. Changes in virulence and resistance gene patterns were minimal over the observed time period.

Background: The main etiologic agent of toxic shock syndrome is the toxic shock syndrome toxin-1 (TSST-1) protein secreted by Staphylococcus aureus. Diagnosis of toxic shock syndrome is difficult and is significantly underdiagnosed in young children with burns due to the nonspecific presentation coupled with a rapid deterioration in patient condition. Methods: The lytic and cytolytic activity of a number of clinical and laboratory TSST-1-positive strains of methicillin-susceptible S. aureus (101, 253, 279 and RN4282, respectively) and Pseudomonas aeruginosa PAO1 strain were tested in vitro using an assay designed to assess the relative exotoxin activity of bacteria using phospholipid vesicles and a T cell toxicity assay. In addition, the activity of lytic exotoxins such as δ-toxin and the secretion of nonlytic TSST-1 toxin from S. aureus was measured using the vesicle assay and Western blotting over the 20-hour growth of TSST-1-positive S. aureus culture. Results: Both the vesicle and T cell assays suggest a lytic exotoxin-mediated mechanism of vesicle rupture and T cell death, with high levels of vesicle lysis and T cell toxicity. It is important to note that the clinical TSST-1-positive methicillin-susceptible S. aureus strains exhibited lytic exotoxin production as well as TSST-1 expression as confirmed by Western blot. Conclusion: We suggest that there is no correlation between the expression of TSST-1 and lack of exotoxin production. We also suggest that apurulence in an S. aureus-infected burn wound in a child should not be used to rule out toxic shock syndrome.

Bacterial resistance to multiple antibiotics is a health problem. Essential oils (EOs) possess antibacterial properties and have been screened as potential sources of novel antimicrobial compounds. Terpenes and terpenoids are components derived from EOs. Some of these EOs show inhibitory activity against Staphylococcus aureus. Carvacrol has specific effects on S. aureus and Staphylococcus epidermidis. Perilla oil suppresses expression of α-toxin, Staphylococcus enterotoxin A and B and toxic shock syndrome toxin. Geraniol shows good activity in modulating drug resistance in several gram-negative species. EOs could act as biopreservatives, reducing or eliminating pathogenic bacteria and increasing the overall quality of animal and vegetable food products. Although clinical studies are scarce, the uses of EOs for topical administration and as penetration enhancers for antiseptics are promising. Little information exists for oral administration.

–The worldwide spread of community acquired Staphylococcus aureus (CASA) skin infection is becoming an emerging problem. This study was conducted to delineate the virulence properties and characteristics of Staphylococcus aureus isolated from children belonging to low socio-economic classes in Midnapore town, West Bengal, India. Samples were collected from affected children by aseptic means from areas of infection with proper medical intervention. These samples were confirmed first as Staphylococcus aureus by PCR amplification of 16S rRNA. Further morphology, antibiotic susceptibility, capability of secreting several enzymes, biofilm forming abilities was studied. PCR was carried out for detection of the presence of enterotoxins (seA and seB), exfoliatin toxins (etA and etB) and toxic shock syndrome toxin (tssT) genes. Results reveal that 33% community acqured Staphylococcus aureus strains were methicillin resistant and 22% were vancomycin resistant and those strains were capable of biofilm formation. Besides, all of the pathogenic and non-pathogenic strains were harboring exfoliatin toxin gene (etA). So it can be concluded that inducible multidrug resistant community acquired Staphylococcus aureus with their diverse pathogenic toxic potentials giving emerging alarm in that geographical location in India. Keywords––Staphylococcus aureus, Staphylococcal Scalded Skin Syndrome, Staphylococcal toxins, vancomycin, biofilm.

Enhancement of susceptibility to lipopolysaccharide (LPS; endotoxin) is a defining characteristic of Staphylococcus aureus superantigens. At the time of this publication, there are 24 identified staphylococcal superantigens (SAgs), some of which have yet to be fully characterized. Testing the capacity of superantigens to potentiate LPS sensitivity is essential to characterize the role of these proteins in disease development. Here we describe how to perform studies of the enhancement of LPS-induced toxic shock syndrome in rabbits. This protocol also provides information on a second important activity of superantigens: the production of fever.

Background Methicillin-resistant Staphylococcus aureus (MRSA) causes serious, often life-threatening, infections. Exotoxins such as alpha-hemolysin (AH), Panton Valentine leukocidin (PVL), and Toxic shock syndrome toxin 1 (TSST-1) mediate pathogenesis and inhibition of toxin production is an important consideration in choosing appropriate treatments. Vancomycin is recommended for severe MRSA infections; however, increasing vancomycin resistance, poor clinical outcomes and nephrotoxicity are serious concerns. Thus newer agents are needed, including those that block bacterial toxin production. In the current study, we compared the effects of sub-inhibitory doses (sub-MIC) of two folic acid inhibitor antibiotics (iclaprim, trimethoprim) with cell wall-active agents (nafcillin, vancomycin) on transcription and translation of AH, PVL and TSST-1 in two clinical MRSA isolates. Methods Community-acquired MRSA strains 1560 (a USA400 strain; AH+, TSST-1+, PVL-) and 04014 (CDC strain 368–04; AH+, TSST-1-, PVL+) were studied. MICs were determined by standard microbroth dilution. Gene expression was studied by northern blotting and/or qRT-PCR; toxins were quantitated by ELISA (PVL and TSST-1) and rabbit erythrocyte lysate assay (AH). Results In agreement with our previous findings, nafcillin increased production of AH, TSST-1, and PVL compared with untreated control cultures. In both MRSA strains, iclaprim and trimethoprim delayed the onset of mRNA production and shifted its peak production to later time points. Both iclaprim and trimethoprim suppressed AH production in both strains of MRSA and delayed, but did not reduce, maximal TSST-1 production in MRSA1560. Trimethoprim significantly increased maximal PVL production over both untreated and iclaprim-treated cultures. Conclusion The folic acid antagonist antibiotics, iclaprim and trimethoprim, altered both mRNA synthesis dynamics and protein toxin production in MRSA at concentrations below those that inhibit bacterial growth. These results, plus the fact that iclaprim is 15-fold more active than trimethoprim (MICs = 0.13 and 2.0 ug/ml, respectively), provide additional rationale for the use of iclaprim to treat complicated MRSA infections. Disclosures A. Bryant, Motif Biosciences: Grant Investigator, Research grant; D. Huang, Motif Bio: Employee, Salary; D. Stevens, Motif Biosciences: Grant Investigator, Research grant

WOS: 000300079900027 PubMed: 22160510 Coagulase-negative staphylococci (CNS) are the most prevalent mastitis pathogens. However, virulence characteristics of CNS have not been well determined. The presence of genes for enterotoxins (sea-sej), toxic shock syndrome toxin-1 (tst), the exfoliative toxins (eta, etb), Panton-Valentine leukocidin (pvl) and mecA of CNS species isolated from cows and ewes with subclinical mastitis was investigated in this study. A total of 121 CNS (81 cows, 40 ewes) representing 18 different Staphylococci species were examined by PCR, and 38.1% (33 cows and 13 ewes) of CNS isolates had one or more se genes. The difference between percentages for SE toxin genes of CNS strains isolated from cows (40.7%) and ewes (32.5%) was not statistically significant (P>0.05; chi(2) = 0.380). It was found that S. simulans isolates had the highest prevalent se genes. Furthermore, the most common SE gene types was seh-sej. In this study, none of the isolates harbored the toxic shock syndrome toxin gene (tsst) and the exfoliative toxin genes (eta, etb). Five cow (6.17%) and three ewe CNS (7.5%) isolates had mecA gene. Three cow (3.7%) and two ewe CNS (5.0%) isolates had pvl gene. In conclusion, the present study showed that CNS species isolated from cows and ewes could serve as potential reservoir of se, mecA, and pvl genes.

Background Toxins contribute to the pathogenicity of Staphylococcus aureus infections by inducing a dysregulated inflammatory response. This study evaluated the impact of anti-staphylococcal antibiotic exposures over an increasing concentration range on cytokine production from peripheral blood mononuclear cells (PBMCs) after S. aureus toxin exposures. Methods Human PBMCs were suspended in complete Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal bovine serum at 10(6) cells/mL with 100 ng/mL S. aureus toxic shock syndrome toxin-1 (TSST-1), staphylococcal enterotoxin A (SEA), α-toxin or Panton-Valentine leucocidin (PVL). Vancomycin, trimethoprim/sulfamethoxazole, tigecycline, daptomycin, linezolid, clindamycin and azithromycin were added at a concentration range of 0.5-100 mg/L. Cytokine [interleukin-1β (IL-1β), IL-6, IL-8, interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α)] concentrations were measured in duplicate by ELISA following exposure and were compared with response with toxin alone. Results At concentrations approximating serum C(max), tigecycline decreased IL-6 by 52%-57% and IFN-γ production by 43%-53% compared with toxin alone (P ≤ 0.05) and linezolid inhibited TNF-α by 12%-35% and IL-8 by 25%-42% (P ≤ 0.02). However, trimethoprim/sulfamethoxazole increased TNF-α and IL-8 production (P = 0.002). Clindamycin, daptomycin, vancomycin and azithromycin had no consistent significant effect at approximate serum C(max) concentrations. All antibiotics had a concentration-dependent effect on cytokine production, with tigecycline, clindamycin and trimethoprim/sulfamethoxazole being the most potent inhibitors of cytokine production at concentrations exceeding 25 mg/L. Conclusions S. aureus toxins stimulate production of inflammatory cytokines in PBMCs. Antimicrobials with high tissue penetration, including tigecycline, clindamycin, trimethoprim/sulfamethoxazole and linezolid, reduced cytokine production, which, along with their antimicrobial effects, may have importance in the therapeutic outcome of severe infections.

To investigate a potential mechanism by which superantigens could induce glucocorticoid insensitivity in chronic rhinosinusitis (CRS) patients.Prospective cohort study.Tertiary medical center.Sinonasal polyps were obtained from CRS patients with nasal polyps (CRSwNP; 20 without recurrence, 18 with recurrent NP followed for 1.5-2.0 years) and nasal mucosa from 16 CRS patients without nasal polyps (CRSsNP). Specimens were tested by enzyme-linked immunosorbent assay for staphylococcal exotoxins (SEs) including SEA, SEB, SEC, SED, and toxic shock syndrome toxin type-1 (TSST-1) and assessed by immunohistochemistry for glucocorticoid receptor (GR) α and β, and the GRβ/GRα ratio was analyzed.In CRSwNP, 13 of 18 (72.22%) subjects with subsequently recurrent NP, 11 of 20 (55.00%) subjects without NP recurrence, and 1 of 16 (6.25%) CRSsNP subjects with positive reactions for SEs were obtained. There were no positive results in controls. The expressions of GRβ in 3 CRS groups and controls were significantly different (all P.05), and a similar increasing tendency of the GRβ/GRα ratio was found among groups besides the comparison of CRSwNP versus recurrent NP groups (P = .053). Furthermore, there was a clear trend of increased GRβ expression in the enzyme-linked immunosorbent assay (ELISA)-positive samples compared with ELISA-negative samples. Concerning GRα, the expression was enhanced significantly just in toxin-positive recurrent NP versus controls (P = .048), but the relative induction of GRβ was much higher, thereby leading to a higher GRβ/GRα ratio.Bacterial superantigens may contribute to glucocorticoid insensitivity through induction of GRβ, which appears to be a marker of steroid insensitivity in CRSwNP.

To investigate the distribution of staphylococcal enterotoxin-like (SEl) genes in Staphylococcus aureus from food, a total of 154 S. aureus isolates from ready-to-eat (RTE) foods in Korea were analyzed by mutiplex PCR for the detection of the following 9 staphylococcal enterotoxin-like genes; sek, sel, sem, sen, seo, sep, seq, ser, and seu. Seventy-nine isolates (51.3%) were found to have at least one of SEl genes. The major SEl genes were sek, sem, sen, and seq. Other SEl genes found in the isolates were seo (21 isolates, 13.6%), seu (12 isolates, 7.8%), sep (8 isolates, 5.2%), sel (7 isolates, 4.5%), and ser (2 isolates, 1.3%). Most (95%) of the isolates with staphylococcal enterotoxin (SE) genes were also carried SEl genes. The genes seg, sei, sem, and sen were all detected in the same isolates. Ninety-seven % of isolates with seg+sei+sem+sen also contained seo or seu. Ninety-four % of isolates with sea+seh were found to coexist with sek+seq. The toxic shock syndrome toxin gene, tst-1, was found in all isolates with egc-2, including seg, sei, sem, sen, and seu. The coexistence of SE and SEl genes in S. aureus isolates from RTE foods can be explained by the mobile genetic elements. Because of the mobile genetic element, SE and SEl genes of S. aureus in foods may be transferable to nontoxigenic S. aureus and other food pathogens. Additional studies must be conducted to prevent spread of pathogenic genes such as enterotoxin gene.

Aims: To elucidate the stability of superantigenic activity and pathogenesis of toxic shock syndrome toxin 1 (TSST-1) and staphylococcal enterotoxin A (SEA) against heating and digestive enzymes. Methods and Results: Purified TSST-1 and SEA were treated with heating, pepsin and trypsin that are related to food cooking, stomach and intestine conditions. The integrity, superantigenic activity and toxicity of treated TSST-1 and SEA were analysed by Western blotting, spleen cell culture, cytokine assay and toxic shock models. Both TSST-1 and SEA showed strong resistance to heating, pepsin and trypsin digestion. Furthermore, the treated TSST-1 showed significant higher induction of interferon-γ and toxic shock compared with that of SEA. Pepsin- or trypsin-digested TSST-1 fragments still showed significant superantigenic and lethal shock toxicities. Conclusions: The superantigenic activity of TSST-1 was stable to heating and digestive enzymes. Pepsin- and trypsin-digested TSST-1 fragments still showed superantigenic and lethal shock activities, indicating that digested TSST-1 could cross epithelial cells and induce systemic toxicity. Significance and Impact of the Study: This study found, for the first time, that pepsin- or trypsin-digested smaller TSST-1 retained significant superantigenic and lethal shock activities. The different resistance of TSST-1 and SEA participates in the different pathogenic activities during food poisoning and toxic shock syndrome.

Background The Centers for Disease Control and Prevention (CDC) and others reported that methicillin-resistant S. aureus (MRSA) are significant causes of serious human infections, including pulmonary illnesses. We investigated the role played by superantigens in lung-associated lethal illness in rabbits. Methods A rabbit model was established to investigate the potential role played by superantigens, staphylococcal enterotoxin B (SEB), staphylococcal enterotoxin C (SEC), and toxic shock syndrome toxin-1 (TSST-1). Rabbits received intrabronchial community-associated (CA) MRSA strains USA200 (TSST-1(+)), MW2 (SEC(+)), c99-529 (SEB(+)), or purified superantigens. Some rabbits were preimmunized against superantigens or treated with soluble high-affinity T cell receptors (Vβ-TCR) to neutralize SEB and then challenged intrabronchially with CA-MRSA or superantigens. Results Rabbits challenged with CA-MRSA or superantigens developed fatal, pulmonary illnesses. Animals preimmunized against purified superantigens, or treated passively with Vβ-TCRs and then challenged with CA-MRSA or superantigens, survived. Lung histological analysis indicated that nonimmune animals developed lesions consistent with necrotizing pneumonia after challenge with CA-MRSA or purified superantigens. Superantigen-immune animals or animals treated with soluble Vβ-TCRs did not develop pulmonary lesions. Conclusions Superantigens contribute to lethal pulmonary illnesses due to CA-MRSA; preexisting immunity to superantigens prevents lethality. Administration of high-affinity Vβ-TCR with specificity for SEB to nonimmune animals protects from lethal pulmonary illness resulting from SEB(+) CA-MRSA and SEB.

This study provides what we believe to be the first report of the presence of EMRSA-15 and its variants isolated from nasal swabs from 13 healthy and diseased individuals in India. The majority of the isolates belonged to staphylococcal cassette chromosome mec (SCCmec) type IV and spa type t852, whilst four isolates were non-typable and heterotypic for the presence of the mecA gene. All non-typable isolates were positive for the orfX gene by PCR and belonged to spa types t005 and t2986. They may have variant SCCmec cassettes indicating genetic changes occurring in the Indian EMRSA-15. All isolates were positive for Panton–Valentine leukocidin and toxic shock syndrome toxin, which is a cause for concern. In addition to soft-tissue infections, the EMRSA-15 isolates from patients were also responsible for meningitis and brain abscesses, which is quite rare.

Aims: The effect of subminimal inhibitory concentrations (sub-MICs) of cefalexin, ciprofloxacin and roxithromycin was investigated on some virulence factors [e.g. coagulase, Toxic Shock Syndrome Toxin 1 (TSST-1) and biofilm formation] expressed by Staphylococcus aureus biofilms. Methods and Results: Biofilms were grown with and without the presence of 1/16 MIC of antibiotics on Sorbarod filters. Eluate supernatants were collected, and coagulase and TSST-1 production were evaluated. Coagulase production was reduced in eluates exposed to roxithromycin when compared to control, while TSST-1 production was reduced in biofilms exposed to cefalexin and to a lesser extent, ciprofloxacin. In addition, the ability of Staph. aureus to produce biofilm in microtitre plates in the presence of sub-MIC antibiotics indicated that cefalexin induced biofilm formation at a wide range of sub-MICs. TSST-1 produced from the challenged and control biofilms was purified, and its proliferative activity was studied on single cell suspension of mouse splenocytes using MTS/PMS assay. No significant difference in the activity between the treated toxin and the control has been observed. Conclusions: Antibiotics at sub-MIC levels interfere with bacterial biofilm virulence expression depending on the type and concentration of antibiotic used. Significance and Impact of the Study: The establishment of sub-MICs of antibiotics in clinical situations may result in altered virulence states in pathogenic bacteria.

Summary Background Staphylococcus aureus Enterotoxin B (SEB) has immunomodulatory effects in allergic airway disease. The potential contribution of SEB to the sensitization process to allergens remains obscure. Objective In order to study the effects of staphylococcal-derived toxins on the sensitization to ovalbumin (OVA) and induction of allergic airway inflammation, we have combined the nasal application of OVA with different toxins. Methods Nasal applications of OVA and saline, SEA, SEB, toxic shock syndrome toxin (TSST)-1, protein A or lipopolysaccharide (LPS) were performed on alternate days from day 0 till 12. On day 14, mice were killed for the evaluation of OVA-specific IgE, cytokine production by mediastinal lymph node (MLN) cells and bronchial hyperreactivity (BHR) to inhaled metacholine. The effect of SEB on dendritic cell (DC) migration and maturation, and on T cell proliferation was evaluated. Results Concomitant endonasal application of OVA and SEB resulted in OVA-specific IgE production, whereas this was not found with SEA, TSST-1, protein A, LPS or OVA alone. Increased DC maturation and migration to the draining lymph nodes were observed in OVA/SEB mice, as well as an increased T cell proliferation. Bronchial inflammation with an influx of eosinophils and lymphocytes was demonstrated in OVA/SEB mice, together with hyperresponsiveness and the production of IL-4, IL-5, IL-10 and IL-13 by MLN stimulated with OVA. Conclusions Our data demonstrate that SEB facilitates sensitization to OVA and consecutive bronchial inflammation with features of allergic asthma. This is likely due to augmentation of DC migration and maturation, as well as the allergen-specific T cell proliferation upon concomitant OVA and SEB application. Cite this as: W. Huvenne, I. Callebaut, M. Plantinga, J. A. J. Vanoirbeek, O. Krysko, D. M. A. Bullens, P.Gevaert, P. Van Cauwenberge, B. N. Lambrecht, J. L. Ceuppens, C. Bachert and P. W. Hellings, Clinical & Experimental Allergy, 2010 (40) 1079–1080.

Background: Staphylococcus aureus is the major causative agent of hospital-acquired and community-acquired infections. These bacteria produce a wide variety of exotoxins, including Toxic Shock Syndrome Toxin (TSST) and virulence factors, which are thought to contribute to its pathogenic potential. Objectives: The aim of this study was to identify tsst gene in S. aureus isolated from patients and healthy carriers. Methods: In this cross-sectional study, a total of 60 humanS. aureus isolates were collected from individuals referred to Shahid Beheshti hospital (patients, n = 40) and healthy farm workers (n = 20) in Hamadan province of Iran. Thereafter, DNA samples were extracted using the phenol-chloroform method and the samples were investigated for tsst gene using a specific PCR assay. Results: The DNA fragment corresponding to the tsst gene (326 bp) was observed in 45% (9 out of 20) of S. aureus isolated from healthy farm workers; while, 22.5% (9 out of 40) of patients’ isolates were found to be positive for tsst gene, which indicated that in total 30% of the isolates possessed this gene. Conclusions: The results of the present study showed the high prevalence of the tsst gene among S. aureus isolated from healthy farm workers and patients. Therefore, appropriate precautions must be considered to decrease the risk of transmission of such isolates to other humans.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains have emerged as serious health threats in the last 15 years. They are associated with large numbers of atopic dermatitis skin and soft tissue infections, but when they originate from skin and mucous membranes, have the capacity to produce sepsis and highly fatal pulmonary infections characterized as necrotizing pneumonia, purpura fulminans, and postviral toxic shock syndrome. This review is a discussion of the emergence of 3 major CA-MRSA organisms, designated CA-MRSA USA400, followed by USA300, and most recently USA200. CA-MRSA USA300 and USA400 isolates and their methicillin-sensitive counterparts (community-associated methicillin-sensitive S aureus) typically produce highly inflammatory cytolysins alpha-toxin, gamma-toxin, delta-toxin (as representative of the phenol soluble modulin family of cytolysins), and Panton Valentine leukocidin. USA300 isolates produce the superantigens enterotoxin-like Q and a highly pyrogenic deletion variant of toxic shock syndrome toxin 1 (TSST-1), whereas USA400 isolates produce the superantigens staphylococcal enterotoxin B or staphylococcal enterotoxin C. USA200 CA-MRSA isolates produce small amounts of cytolysins but produce high levels of TSST-1. In contrast, their methicillin-sensitive S aureus counterparts produce various cytolysins, apparently in part dependent on the niche occupied in the host and levels of TSST-1 expressed. Significant differences seen in production of secreted virulence factors by CA-MRSA versus hospital-associated methicillin-resistant S aureus and community-associated methicillin-sensitive S aureus strains appear to be a result of the need to specialize as the result of energy drains from both virulence factor production and methicillin resistance.

The prevalence of antibodies to Toxic Shock Syndrome Toxin (TSST-1) in a Swedish healthy control population was investigated using an enzyme-linked immunosorbent assay (ELISA). 88% of the control group above the age of 10 showed positive antibody levels as compared to 31% of those who were under 10 years old. These results indicate a very common normal exposure to TSST-1 during early life and also identify the small risk-group of potential TSS-patients. Patients with S. aureus endocarditis and septicemia showed slightly higher antibody levels as compared to the controls (p less than 0.05). The difference was in part due to 3/4 septicemia patients, infected with TSST-1 producing strains, who showed very high antibody levels. None of these 4 patients developed any signs of TSS. 5/5 menstrual associated TSS-patients were negative in the ELISA in serial serum samples as were 3/5 non-menstrual associated TSS-patients. The TSST-1 ELISA is proposed for identifying chiefly young women at risk of acquiring menstrual related Toxic Shock Syndrome.

Objectives To investigate whether superantigens (SAgs) are involved in the development of Kawasaki disease (KD) by examining SAg genes in the stool of patients with KD. Study design Stool specimens were obtained from 60 patients with KD and 62 age-matched children (36 children with acute illness and 26 healthy children). Total DNA was extracted from these stool samples. Using polymerase chain reaction, we examined genes of 5 SAgs: streptococcal pyrogenic exotoxin-A (SPE-A), SPE-C, SPE-G, SPE-J, and toxic shock syndrome toxin-1. Results At least 1 of the 5 SAg genes was detected in 42 (70%) specimens from patients with KD, 14 (38.9%) from the febrile group, and 7 (26.9%) from the healthy group. The detection rate between subjects with and without KD was of at least 1 of the 5 SAg genes ( P P = .002). Conclusions SAg may be involved in the development of KD; data suggest that multiple SAgs may trigger KD.

Microarrays were used to extensively characterise 155 Staphylococcus aureus isolates obtained from asymptomatic carriers from Saxony, Germany, in order to determine clonal complex affiliation, as well as the carriage of clinically relevant genes. Isolates belonged to 20 different clonal complexes (CCs). The most common CC was CC8 (18.71%), followed by CCs 15, 30 and 45. Three isolates (1.94%) were methicillin-resistant S. aureus (MRSA). Beta-lactamase was common (70.97%), but other resistance genes were found only sporadically. Genes encoding superantigens were abundant. The enterotoxin cluster egc was found in 45.81% of isolates. The toxic shock syndrome toxin gene tst was detected in 14.84% of isolates and 17.42% harboured enterotoxin A alleles (sea, sea-N315). Contrarily, Panton-Valentine leukocidin (lukS/F-PV) was rare, being found in only one methicillin-susceptible CC30 isolate. Its low prevalence in asymptomatic carriers might emphasise a pathogenetic significance in patients with skin and soft tissue infections. Most microbial surface components recognising adhesive matrix molecules of the host (MSCRAMMs) genes were nearly ubiquitously present. However, two MSCRAMM genes, cna (collagen adhesion) and sasG (surface protein G), were detected in only some CCs. These data provide an insight into its pathogenesis, especially when compared to isolates from patients with defined clinical conditions. They might also be helpful for the design of a future vaccine.

Enterotoxigenic Staphylococcus aureus frequently contaminate milk and milk products causing food poisoning. Staphylococcus aureus were isolated from bovine, ovine and caprine milk and milk filters from 78 dairy production holdings supplying the farmhouse cheese sector in Ireland, using standard culture methods. Molecular methods were applied to study the distribution of genes encoding staphylococcal enterotoxins and toxic shock syndrome toxin in the collection. Multilocus variable number tandem repeat analysis was used to subtype the collection. One hundred and two Staphylococcus aureus (54 milk filters and 48 bulk milk) were recovered from apparently healthy animals; half of the isolates were toxigenic. Our findings are discussed in light of the risks posed to public health.

BACKGROUND. Persistent carriers have a higher risk of Staphylococcus aureus infections than noncarriers but a lower risk of bacteremia-related death. Here, the role played by anti-staphylococcal antibodies was studied. METHODS. Serum samples from 15 persistent carriers and 19 noncarriers were analyzed for immunoglobulin (Ig) G, IgA, and IgM binding to 19 S. aureus antigens, by means of Luminex technology. Nasal secretions and serum samples obtained after 6 months were also analyzed. RESULTS. Median serum IgG levels were significantly higher in persistent carriers than in noncarriers for toxic shock syndrome toxin (TSST)-1 (median fluorescence intensity [MFI] value, 11,554 vs. 4291; P < .001) and staphylococcal enterotoxin (SE) A (742 vs. 218; P < .05); median IgA levels were higher for TSST-1 (P < .01), SEA, and clumping factor (Clf) A and B (P < .05). The in vitro neutralizing capacity of anti-TSST-1 antibodies was correlated with the MFI value (R(2) = 0.93) and was higher in persistent carriers (90.6% vs. 70.6%; P < .05). Antibody levels were stable over time and correlated with levels in nasal secretions (for IgG, R(2) = 0.87; for IgA, R(2) = 0.77). CONCLUSIONS. Antibodies to TSST-1 have a neutralizing capacity, and median levels of antibodies to TSST-1, SEA, ClfA, and ClfB are higher in persistent carriers than in noncarriers. These antibodies might be associated with the differences in the risk and outcome of S. aureus infections between nasal carriers and noncarriers.

The aim of this study was to examine the genotype properties of Staphylococcus aureus of phage type 187 strains that constitute a separate group among the strains of S. aureus. Sixteen strains were collected from the hospital patients (n=12) and the healthy carriers (n=4) in 13 medical centres in Poland during 1991 and 2005. Biotyping, antibiotic susceptibility, phage typing, detection the genes of enterotoxins and toxic shock syndrome toxin, genotyping of chromosomal DNA by pulsed-field gel electrophoresis (PFGE), also amplification and restriction analysis of the coagulase (coa) and the protein A genes (spa) (PCR/restriction fragment length polymorphism (RFLP)) was tested. The results of this study showed that all staphylococcus of phage type 187 belonged to the human biotype (A) and appeared to be sensitive to all of the tested antibiotics, including methicillin (MSSA). Finding out the toxin genes showed that almost all of them (93.8%) had the enterotoxin C gene (sec) and TSST-1 gene (tst). The PFGE typing proved that the phage type 187 strains (except for one) constitute one PFGE type. These results and the identical restriction patterns in the PCR/RFLP method, also the same biotype, sensitivity to antibiotics and the presence genes of the same type of toxins confirmed that the phage type 187 strains constitute one clone within our country. Additionally, the fact that almost all of them have the enterotoxin genes and tst gene allows to consider them the strains of potentially high virulence.

Most clinical isolates of Staphylococcus aureus harbour genes encoding superantigenic toxins that bind the Vbeta domain of T-cells, but little information is available concerning superantigenic toxin production during staphylococcal toxic shock syndrome (TSS) and septic shock. This prospective study investigated 14 patients with staphylococcal TSS or septic shock; the toxin gene profile of each isolate was determined and flow-cytometry was used to identify the discriminant Vbeta signature (DVbetaS) of each superantigenic toxin in vitro. Attempts were also made to identify in-vivo production of superantigenic toxin DVbetaS in patients' blood. The DVbetaS identified in vitro were: toxic shock syndrome toxin (TSST)-1, Vbeta 2; staphylococcal enterotoxin (SE), Vbeta 9, Vbeta 22; SEB, Vbeta 3, Vbeta 14, Vbeta 17; SED, Vbeta 1, Vbeta 8; egc, Vbeta 5.3, Vbeta 7.1, Vbeta 9, Vbeta 23; and SElK, Vbeta 5.1. The DVbetaS of TSST-1 and SEB were detected in patients with menstrual and non-menstrual TSS, respectively, whereas no Vbeta signature was detected during septic shock. All patients with septic shock (but only one patient with TSS) had lymphopenia and/or impaired cellular immunity. Detection of a superantigenic toxin DVbetaS may help to show which toxin is produced during staphylococcal TSS, thus confirming the diagnosis and hastening the administration of anti-toxin therapy. In contrast, this approach failed to demonstrate superantigenic toxin involvement in cases of septic shock. In this latter condition, a superantigenic toxin may not be produced by S. aureus, or its production may occur without expansion of targeted T-cells because of T-cell apoptosis and/or anergy.

Staphylococcus aureus is a commensal organism that colonizes up to 50% of humans [1, 2]. The organism most often colonizes the anterior nares and, from there, may colonize other body surfaces, including other mucous membranes and damaged skin. S. aureus causes a wide variety of human illnesses, including scalded skin syndrome, toxic shock syndrome (TSS), and necrotizing pneumonia [3-9]. The ability of S. aureus to cause human disease depends on the production of cell-surface adhesins, antiphagocytic factors, and secreted exotoxins, whose functions appear to be both securing nutrients for the microbes and delaying function of the immune system [10-12]. Among the secreted factors is a large family of superantigen exotoxins [8]. Staphylococcal superantigens include staphylococcal enterotoxins, classically the common causes of food poisoning and nonmenstrual TSS, and TSS toxin 1 (TSST-1), the cause of both menstrual and nonmenstrual TSS [8]. Staphylococcal enterotoxin serotypes A–E (SEA–SEE) and SEG–SEQ have been well described in the literature. SEA–SEE and SEI are capable of causing vomiting and diarrhea when administered to monkeys and, thus, are correctly referred to as staphylococcal enterotoxins [13]. The remaining staphylococcal enterotoxins either lack emetic activity (SEG, SEK, SEL, and SEQ) or have not been tested for emetic activity. According to the suggestions of a recent nomenclature committee, these superantigens are more correctly designated as staphylococcal enterotoxin–like (SEl) (SEl-G, -H, -J, -K, -L, -M, -N, -O, -P, and -Q) [14]. Superantigens are defined by their ability to stimulate cytokine release from both T cells and macrophages [15]. The proteins bind to relatively invariant regions of major histocompatibility complex II molecules on antigen-presenting cells, and they cross-bridge with certain variable regions of the beta-chains of T cell receptors (Vb-TCR) [16]. Each superantigen has a relatively unique subset of Vb-TCR interactions. For example, TSST-1 stimulates only T cells bearing Vb2-TCRs, but these account for only ∼10% of the total repertoire of T cells in humans [16]. However, during acute TSS, T cells bearing Vb2-TCRs may proliferate in a skewed manner, such that the activated T cells account for 60% of the patients’ T cells [17]. The massive cytokine release by both T cells and macrophages accounts for the most-severe manifestations of superantigen-mediated illnesses [8, 15, 16]. Atopic dermatitis is a T cell–mediated skin disease that can significantly compromise quality of life, because patients experience sleep disturbances, social embarrassment, and emotional distress. S. aureus infection contributes to the worsening of skin inflammation in atopic dermatitis [18-20]. These organisms have been shown to produce superantigens, including SEA, SEB, SEC, and TSST-1. However, the full spectrum of superantigens produced by S. aureus isolates that infect patients with atopic dermatitis has not been examined previously. Furthermore, superantigens have been demonstrated to induce corticosteroid resistance of T cells in vitro [21]. This could contribute to difficulty in management of atopic dermatitis, because topical corticosteroids are the most common medication used for treatment of atopic dermatitis. The present study, therefore, was undertaken to characterize S. aureus isolates from patients with steroid-resistant atopic dermatitis with regard to their ability to produce superantigens.

Recent reports suggest that staphylococcal exotoxins, acting as superantigens, activate T cells with subsequent massive proliferation, thereby contributing to the etiology of chronic rhinosinusitis with nasal polyps (CRSwNP). The objectives of this study are (1) to demonstrate directly the presence of staphylococcal exotoxins in nasal mucosa and sinonasal polyp tissue, and (2) provide indirect evidence of the effect of superantigens on the T cells expressing the target of superantigen, i.e., the β variable chain receptor (TCRBV) in polyp tissue and peripheral blood of patients with CRSwNP. Sinonasal polyp tissue and peripheral blood specimens were obtained from 37 patients with chronic rhinosinusitis (22 patients with bilateral nasal polyps, 15 without nasal polyps) and 12 normal subjects for comparative negative controls. Tissue specimens were assayed by enzyme-linked immunosorbent assay (ELISA) for the most common staphylococcal exotoxins (A–D) and toxic shock syndrome toxin type 1. Fresh tissue and blood samples were analyzed by flow cytometry to determine the expression of TCRBV. In the CRSwNP subjects 12 of 22 samples (54.54%) demonstrated reactivity for at least 1 staphylococcal exotoxin, while 2 of the 12 were positive for 2 toxins. There were no positive results in the CRS without nasal polyps or control groups. There was a clear trend of increased TCRBV expression in the ELISA-positive group both for tissue and blood specimens. Staphylococcal superantigens were present in the nasal cavity of patients with CRSwNP with a high percentage of TCRBV, which suggests the possibility of superantigens as etiological agents of CRSwNP.

High mobility group box-1 (HMGB-1) is a DNA-binding protein secreted by activated monocytes and has been identified as a key late mediator of endotoxic shock. We investigated the regulation of HMGB-1 in human peripheral blood mononuclear cells (PBMCs) following stimulation with the staphylococcal superantigen, toxic shock syndrome toxin-1 (TSST-1), and found that TSST-1, like LPS, induced the secretion of HMGB-1 from human PBMC. However, unlike monocyte-driven sepsis caused by endotoxin, translocation and secretion of HMGB-1 mediated by TSST-1 was dependent on the presence of both activated T cells and monocytes. Furthermore, we show that nuclear HMGB-1 is released from TSST-1 stimulated T cells. This finding presents a basis for investigating the potential of targeting HMGB-1 for the treatment of toxic shock syndrome, and provides further insight on the role of HMGB-1 in the cross-talk between activated monocytes and T cells.

Staphylococcus aureus is a common pathogen which can colonise and infect not only man, but also domestic animals. Especially, infection of cattle is of high economic relevance as S. aureus is an important causal agent of bovine mastitis. In the present contribution, a DNA microarray was applied for the study of 144 different gene targets, including resistance genes and genes encoding exotoxins, in S. aureus isolated from cows. One hundred and twenty-eight isolates from Germany and Switzerland were tested. These isolates were assigned to 20 different strains and nine clonal complexes. The majority of isolates belonged either to apparently closely related clonal complexes 8, 25, and 97 (together 34.4%) or were related to the sequenced bovine strain RF122 (48.4%). Notable characteristics of S. aureus of bovine origin are the carriage of intact haemolysin beta (in 82% of isolates tested), the absence of staphylokinase (in 89.1%), the presence of allelic variants of several exotoxins such as toxic shock syndrome toxin and enterotoxin N, and the occurrence of the leukocidin lukF-P83/lukM (in 53.1%). Two isolates were methicillin-resistant S. aureus (MRSA). One of them was a clonal complex 8 MRSA related to the epidemic MRSA strain Irish 01. The other one belonged to ST398/spa-type 34 resembling a newly emerging MRSA strain which has been described to occur in humans as well as in domestic animals. The presence of these two strains highlights the possibility of transfers of S. aureus strains between different host species.

Extracellular protein toxins contribute to the pathogenesis of a wide variety of Staphylococcus aureus infections. The present study investigated the effects that cell-wall active antibiotics and protein-synthesis inhibitors have on transcription and translation of genes for Panton-Valentine leukocidin, alpha-hemolysin, and toxic-shock syndrome toxin 1, in both methicillin-sensitive and methicillin-resistant S. aureus. Subinhibitory concentrations of nafcillin induced and prolonged mRNA for Panton-Valentine leukocidin, alpha-toxin, and toxic-shock syndrome toxin 1 and increased toxin production. In contrast, clindamycin and linezolid markedly suppressed translation, but not transcription, of toxin genes. These results suggest (1) that protein-synthesis inhibition is an important consideration in the selection of antimicrobial agents to treat serious infections caused by toxin-producing gram-positive pathogens and (2) that, by inducing and enhancing toxin production, inadvertent use of beta-lactam antibiotics to treat methicillin-resistant S. aureus infections may contribute to worse outcomes.

Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) prevalence among companion animals and veterinary personnel (VP) was investigated. Strains' molecular characteristics were evaluated in order to assess S. aureus transmission. Specimens (224) from colonized and infected sites of 102 animals (92 dogs, 10 cats) and 18 VP were collected during 2012 and 2013. Antibiotic susceptibility was performed by the disk diffusion method and Etest. mecA, mecC, tst (toxic shock syndrome toxin) and lukF/lukS-PV (Panton-Valentine leukocidin, PVL) genes were investigated by PCR. Genotypes were identified by Multi Locus Sequence Typing (MLST), Staphylococcal Cassette Chromosome mec (SCCmec), accessory gene regulator group (agr), spa and Pulsed Field Gel Electrophoresis (PFGE). S. aureus prevalence among pets and VP was 36.3% (37/102) and 38.9% (7/18), respectively. Younger companion animals, those living in rural areas, having a disease upon admission or Coagulase-negative staphylococci co-carriage showed significantly higher prevalence of S. aureus isolation (p0.05). Twenty-six pets and five VP carried PVL-positive S. aureus. In total, 60 S. aureus strains were recovered (53 from pets, seven from VP) of which 16 were MRSA (26.7%), 12 mecA- and four mecC-positive. MRSA showed higher resistance rates against other antimicrobials as compared to methicillin-susceptible ones. Strains were classified by MLST in 13 STs, with the predominance of ST80 and ST15. In MRSA, SCCmec types II, IV and XI were identified. The most frequent spa types were t5559 and t7558. Fifty-six strains were classified into 15 PFGE types. Comparison of genetic markers shows that identical or very similar strains disseminate among animals and VP. Companion animals harbor PVL-positive clones constituting a possible source for transmission to humans.