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Background: Staphylocoagulase (SCG) is a virulence factor of Staphylococcus aureus, one of the most lethal pathogens of our times. The complex of SCG with prothrombin (SCG/ProT) can clot fibrinogen, and SCG/ProT-induced fibrin and plasma clots have been described to show decreased mechanical and lytic resistance, which may contribute to septic emboli from infected cardiac vegetations. At infection sites, neutrophils can release DNA and histones, as parts of neutrophil extracellular traps (NETs), which in turn favor thrombosis, inhibit fibrinolysis and strengthen clot structure. Objectives: To characterize the combined effects of major NET-components (DNA, histone H1 and H3) on SCG/ProT-induced clot structure, mechanical and lytic stability. Methods: Recombinant SCG was used to clot purified fibrinogen and plasma. The kinetics of formation and lysis of fibrin and plasma clots containing H1 or core histones+/-DNA were followed by turbidimetry. Fibrin structure and mechanical stability were characterized with scanning electron microscopy, pressure-driven permeation, and oscillation rheometry. Results: Histones and DNA favored the formation of thicker fibrin fibers and a more heterogeneous clot structure including high porosity with H1 histone, whereas low porosity with core histones and DNA. As opposed to previous observations with thrombin-induced clots, SCG/ProT-induced fibrin was not mechanically stabilized by histones. Similarly to thrombin-induced clots, the DNA-histone complexes prolonged fibrinolysis with tissue-type plasminogen activator (up to 2-fold). The anti-fibrinolytic effect of the DNA and DNA-H3 complex was observed in plasma clots too. Heparin (low molecular weight) accelerated the lysis of SCG/ProT-clots from plasma, even if DNA and histones were also present. Conclusions: In the interplay of NETs and fibrin formed by SCG, DNA and histones promote structural heterogeneity in the clots, and fail to stabilize them against mechanical stress. The DNA-histone complexes render the SCG-fibrin more resistant to lysis and thereby less prone to embolization. [ABSTRACT FROM AUTHOR]

BackgroundStaphylocoagulase (SCG) is a virulence factor of Staphylococcus aureus, one of the most lethal pathogens of our times. The complex of SCG with prothrombin (SCG/ProT) can clot fibrinogen, and SCG/ProT-induced fibrin and plasma clots have been described to show decreased mechanical and lytic resistance, which may contribute to septic emboli from infected cardiac vegetations. At infection sites, neutrophils can release DNA and histones, as parts of neutrophil extracellular traps (NETs), which in turn favor thrombosis, inhibit fibrinolysis and strengthen clot structure.ObjectivesTo characterize the combined effects of major NET-components (DNA, histone H1 and H3) on SCG/ProT-induced clot structure, mechanical and lytic stability.MethodsRecombinant SCG was used to clot purified fibrinogen and plasma. The kinetics of formation and lysis of fibrin and plasma clots containing H1 or core histones+/-DNA were followed by turbidimetry. Fibrin structure and mechanical stability were characterized with scanning electron microscopy, pressure-driven permeation, and oscillation rheometry.ResultsHistones and DNA favored the formation of thicker fibrin fibers and a more heterogeneous clot structure including high porosity with H1 histone, whereas low porosity with core histones and DNA. As opposed to previous observations with thrombin-induced clots, SCG/ProT-induced fibrin was not mechanically stabilized by histones. Similarly to thrombin-induced clots, the DNA-histone complexes prolonged fibrinolysis with tissue-type plasminogen activator (up to 2-fold). The anti-fibrinolytic effect of the DNA and DNA-H3 complex was observed in plasma clots too. Heparin (low molecular weight) accelerated the lysis of SCG/ProT-clots from plasma, even if DNA and histones were also present.ConclusionsIn the interplay of NETs and fibrin formed by SCG, DNA and histones promote structural heterogeneity in the clots, and fail to stabilize them against mechanical stress. The DNA-histone complexes render the SCG-fibrin more resistant to lysis and thereby less prone to embolization.

Staphylocoagulase (Coa) plays a critical role in the pathogenicity of Staphylococcus aureus (S. aureus). The present study was undertaken to investigate the underlying mechanism which helicid (HEL) suppressed the virulence factor Coa, as well as to assess the synergistic inhibitory effects of HEL in conjunction with antibiotics, thereby establishing the potential of HEL as an antibacterial adjuvant. We employed coagulation and biofilm assays to comprehensively assess the inhibitory impact of HEL on S. aureus pathogenicity. The thermal shift assay demonstrated that HEL exerted a direct impact on the protein stability of Coa, evidenced by a 6°C change in melting temperature (ΔTm) at a concentration of 100 μM. HEL binding to Coa proteins was further validated by molecular dynamics simulations and fluorescence quenching. Molecular docking and point mutation assays identified S23 and D112 as crucial binding sites for HEL and Coa. Furthermore, HEL has been observed to potentiate the bactericidal properties of ceftaroline fosamil (CEF-F), concurrently diminishing the resistance exhibited by S. aureus towards CEF-F, as demonstrated by antibiotic synergy tests and resistance induction assays. The combination of HEL and CEF-F effectively reduced the number of bacteria and improved the survival of both Galleria mellonella larvae and mice. Additionally, a significant decrease was observed in the levels of TNF-α, IL-6, and IFN-γ in mice broncho-alveolar lavage fluid (BALF). Ultimately, our findings confirmed that the direct binding of HEL to Coa could diminish the pathogenicity of S. aureus. Moreover, the combination with CEF-F substantially reduced the lethality associated with S. aureus-infected pneumonia and extended the efficacy of the antibiotic., Competing Interests: Declaration of Competing Interest The authors affirm that there are no conflicts of interest regarding this study., (Copyright © 2024. Published by Elsevier B.V.)

Introduction: Staphylococcus aureus is one of the critical pathological bacteria. This bacterium had developed a variety of genetic mutations that made it resistant to drugs and more harmful to humans. In addition, all attempts to design a specific vaccine against S. aureus have failed. Therefore, this experiment was designed as a trial for vaccine production, by using a photodynamic treatment (PDT) through partial biological inhibition. The PDT of bacteria mainly focused on reducing the activity of staphylocoagulase (SC), which has a protective feature for bacteria. This study aimed to examine the photodynamic effect of combining a specific wavelength of a laser and a certain dilution photosensitizer, methylene blue (MB) dye. The possible PDT effect on the inhibition of pathogenic enzymatic activity was predicted. This study also aimed to evaluate the inhibitory effect of PDT on the total bacterial account (viability) simultaneously with SC assay. Methods: A 650nm wavelength diode laser was used with 100 mW output power and 2 minutes of exposure time. Dye dilutions were 50, 100, 150 and 200 µg/mL. The viability of bacteria after and before laser treatment was calculated using single plate-serial dilution spotting methods. The activity of SC was detected by using human plasma for 4 hours incubation of crude-substrate interaction. Results: The results revealed a significant decrease in enzyme activity and colony-forming units (CFU) after irradiating bacterial suspension with 150 g/mL MB, as well as a decline in CFU. However, irradiation with a laser alone showed a significant increase in SC activity and CFU for the same exposure time. Conclusion: Besides reducing the production of SC activity, PDT significantly inhibited the viability of S. aureus. The application of MB photosensitizer at a concentration of 150 g/mL in combination with a laser wavelength of 650 nm resulted in a complete decrease in the SC activity value as well as the viability of bacteria. [ABSTRACT FROM AUTHOR]

The prevalence of Staphylococcus aureus as an aggressive pathogen resistant to multiple antibiotics causing nosocomial and community-acquired infections is increasing with limited therapeutic options. Macrolide-lincosamide streptogramin B (MLSB) family of antibiotics represents an important alternative therapy for staphylococcal infections. This study was conducted over a period of five years from August 2013 to July 2018 to investigate the prevalence and molecular epidemiology in Iran of inducible resistance in S. aureus. In the current study, 126 inducible methicillin-resistant S. aureus (MRSA) (n = 106) and methicillin-sensitive S. aureus (MSSA) (n = 20) isolates were characterized by in vitro susceptibility analysis, resistance and virulence encoding gene distribution, phenotypic and genotypic analysis of biofilm formation, prophage typing, S. aureus protein A locus (spa) typing, staphylocoagulase (SC) typing, staphylococcal cassette chromosome mec (SCCmec) typing, and multilocus sequence typing. Of the 126 isolates, 76 (60.3%) were classified as hospital onset, and 50 (39.7%) were classified as community onset (CO). Biofilm formation was observed in 97 strains (77%). A total of 14 sequence types (STs), 26 spa types, 7 coagulase types, 9 prophage types, 3 agr types (no agr IV), and 9 clonal complexes (CCs) were identified in this study. The prevalence of the inducible MLSB (iMLSB) S. aureus increased from 7.5% (25/335) to 21.7% (38/175) during the study period. The iMLSB MRSA isolates were distributed in nine CCs, whereas the MSSA isolates were less diverse, which mainly belonged to CC22 (7.95%) and CC30 (7.95%). High-level mupirocin-resistant strains belonged to ST85-SCCmec IV/t008 (n = 4), ST5-SCCmec IV/t002 (n = 4), ST239-SCCmec III/t631 (n = 2), and ST8-SCCmec IV/t064 (n = 2) clones, whereas low-level mupirocin-resistant strains belonged to ST15-SCCmec IV/t084 (n = 5), ST239-SCCmec III/t860 (n = 3), and ST22-SCCmec IV/t790 (n = 3) clones. All the fusidic acid–resistant iMLSB isolates were MRSA and belonged to ST15-SCCmec IV/t084 (n = 2), ST239-SCCmec III/t030 (n = 2), ST1-SCCmec V/t6811 (n = 1), ST80-SCCmec IV/t044 (n = 1), and ST59-SCCmec IV/t437 (n = 1). The CC22 that was predominant in 2013–2014 (36% of the isolates) had almost disappeared in 2017–2018, being replaced by the CC8, which represented 39.5% of the 2017–2018 isolates. This is the first description of temporal shifts of iMLSB S. aureus isolates in Iran that identifies predominant clones and treatment options for iMLSB S. aureus–related infections.

Staphylococcus aureus is the leading cause of infective endocarditis (IE). While the role of S. aureus cell-wall associated protein clumping factor A (ClfA) in promoting IE has been already demonstrated, that of the secreted plasma-clotting factors staphylocoagulase (Coa) and von Willebrand factor-binding protein (vWbp) has not yet been elucidated. We investigated the role of Coa and vWbp in IE initiation in rats with catheter-induced aortic vegetations, using Lactococcus lactis expressing coa, vWbp, clfA or vWbp/clfA, and S. aureus Newman Δcoa, ΔvWbp, ΔclfA or Δcoa/ΔvWbp/ΔclfA mutants. vWbp-expression increased L. lactis valve infection compared to parent and coa-expressing strains (incidence: 62%, versus 0% and 13%, respectively; P 0.05 versus ΔclfA mutant). Coa does not support the initial colonization of IE (in L. lactis) without other key virulence factors and vWbp contributes to initiation of IE (in L. lactis) but is marginal in the present of ClfA.

The prevalence of Staphylococcus aureus as an aggressive pathogen resistant to multiple antibiotics causing nosocomial and community-acquired infections is increasing with limited therapeutic options. Macrolide-lincosamide streptogramin B (MLSB) family of antibiotics represents an important alternative therapy for staphylococcal infections. This study was conducted over a period of five years from August 2013 to July 2018 to investigate the prevalence and molecular epidemiology in Iran of inducible resistance in S. aureus. In the current study, 126 inducible methicillin-resistant S. aureus (MRSA) (n = 106) and methicillin-sensitive S. aureus (MSSA) (n = 20) isolates were characterized by in vitro susceptibility analysis, resistance and virulence encoding gene distribution, phenotypic and genotypic analysis of biofilm formation, prophage typing, S. aureus protein A locus (spa) typing, staphylocoagulase (SC) typing, staphylococcal cassette chromosome mec (SCC mec) typing, and multilocus sequence typing. Of the 126 isolates, 76 (60.3%) were classified as hospital onset, and 50 (39.7%) were classified as community onset (CO). Biofilm formation was observed in 97 strains (77%). A total of 14 sequence types (STs), 26 spa types, 7 coagulase types, 9 prophage types, 3 agr types (no agr IV), and 9 clonal complexes (CCs) were identified in this study. The prevalence of the inducible MLSB (iMLSB) S. aureus increased from 7.5% (25/335) to 21.7% (38/175) during the study period. The iMLSB MRSA isolates were distributed in nine CCs, whereas the MSSA isolates were less diverse, which mainly belonged to CC22 (7.95%) and CC30 (7.95%). High-level mupirocin-resistant strains belonged to ST85-SCC mec IV/t008 (n = 4), ST5-SCC mec IV/t002 (n = 4), ST239-SCC mec III/t631 (n = 2), and ST8-SCC mec IV/t064 (n = 2) clones, whereas low-level mupirocin-resistant strains belonged to ST15-SCC mec IV/t084 (n = 5), ST239-SCC mec III/t860 (n = 3), and ST22-SCC me c IV/t790 (n = 3) clones. All the fusidic acid–resistant iMLSB isolates were MRSA and belonged to ST15-SCC mec IV/t084 (n = 2), ST239-SCC mec III/t030 (n = 2), ST1-SCC mec V/t6811 (n = 1), ST80-SCC mec IV/t044 (n = 1), and ST59-SCC mec IV/t437 (n = 1). The CC22 that was predominant in 2013–2014 (36% of the isolates) had almost disappeared in 2017–2018, being replaced by the CC8, which represented 39.5% of the 2017–2018 isolates. This is the first description of temporal shifts of iMLSB S. aureus isolates in Iran that identifies predominant clones and treatment options for iMLSB S. aureus –related infections. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is the leading cause of infective endocarditis (IE). While the role of S. aureus cell-wall associated protein clumping factor A (ClfA) in promoting IE has been already demonstrated, that of the secreted plasma-clotting factors staphylocoagulase (Coa) and von Willebrand factor-binding protein (vWbp) has not yet been elucidated. We investigated the role of Coa and vWbp in IE initiation in rats with catheter-induced aortic vegetations, using Lactococcus lactis expressing coa, vWbp, clfA or vWbp/clfA, and S. aureus Newman Δcoa, ΔvWbp, ΔclfA or Δcoa/ΔvWbp/ΔclfA mutants. vWbp-expression increased L. lactis valve infection compared to parent and coa-expressing strains (incidence: 62%, versus 0% and 13%, respectively; P < 0.01). Likewise, expression of clfA increased L. lactis infectivity (incidence: 80%), which was not further affected by co-expression of vWbp. In symmetry, deletion of the coa or vWbp genes in S. aureus did not decrease infectivity (incidence: 68 and 64%, respectively) whereas deletion of clfA did decrease valve infection (incidence: 45%; P = 0.03 versus parent), which was not further affected by the triple deletion Δcoa/ΔvWbp/ΔclfA (incidence: 36%; P > 0.05 versus ΔclfA mutant). Coa does not support the initial colonization of IE (in L. lactis) without other key virulence factors and vWbp contributes to initiation of IE (in L. lactis) but is marginal in the present of ClfA. [ABSTRACT FROM AUTHOR]

The C-terminal repeat domain of staphylocoagulase that is secreted by the S. aureus is believed to play an important role interacting with fibrinogen and promotes blood clotting. To study this interaction by NMR, full assignment of each amide residue in the HSQC spectrum was required. Despite of the short sequence of the repeat construct, the HSQC spectrum contained a substantial amount of overlapped and exchange broadened resonances, indicating little secondary or tertiary structure. This caused severe problems while using the conventional, amide based NMR method for the backbone assignment. With the growing interest in small apparently disordered proteins, these issues are being faced more frequently. An alternative strategy to improve the backbone assignment capability involved carbon direct detection methods. Circumventing the amide proton detection offers a larger signal dispersion and more uniform signal intensity. For peptides with higher concentrations and in combination with the cold carbon channels of new cryoprobes, higher fields, and sufficiently long relaxation times, the disadvantage of the lower sensitivity of the C nucleus can be overcome. Another advantage of this method is the assignment of the proline backbone residues. Complete assignment with the carbon-detected strategy was achieved with a set of only two 3D, one 2D, and a HNCO measurement, which was necessary to translate the information to the HSQC spectrum. [ABSTRACT FROM AUTHOR]

Staphylocoagulase, a protein produced by S. aureus , play major role in blood coagulation and investigations are in advance to discover more staphylocoagulase producing species. The present study demonstrates the identification of a coagulase producing bacteria and isolation, purification and characterization of the protein. The bacteria was identified using 16S rDNA sequencing and phylogenetic investigation, classified the bacteria as Staphylococcus sp. MBBJP S43 with Genbank accession number KX907247. Tube test and Chromozym TH assay were used to study enzyme activity and comparison was made with five standard coagulase positive strains. The SEM images of the fibrin threads provide evidence of coagulation. The optimum temperature for enzyme activity was 37 °C and pH of 6.5–7.5. Glucose and lactose as a carbon source and ammonium chloride as nitrogen source greatly influenced the bacterial growth. Staphylocoagulase has been purified to homogeneity (766 fold) by 80% (NH 4 ) 2 SO 4 precipitation, Sephadex G–75 gel filtration, DEAE anion exchange chromatography, and HPLC using C18 column. SDS PAGE revealed the molecular weight of the protein to be approximately 66 kD and FTIR spectra of the purified protein demonstrated the presence of α helical structure. Present study revealed that the Staphylococcus sp. MBBJP S43 strain is a potential staphylocoagulase producing bacteria. [ABSTRACT FROM AUTHOR]

Staphylocoagulase, an extracellular protein secreted by Staphylococcus aureus, has been used as an epidemiological marker. At least 12 serotypes and 24 genotypes subdivided on the basis of nucleotide sequence have been reported to date. In this study, we identified a novel staphylocoagulase nucleotide sequence, coa310, from staphylococcal food poisoning isolates that had the ability to coagulate plasma, but could not be typed using the conventional method. The protein encoded by coa310 contained the six fundamental conserved domains of staphylocoagulase. The full-length nucleotide sequence of coa310 shared the highest similarity (77.5%) with that of staphylocoagulasetype (SCT) XIa. The sequence of the D1 region, which would be responsible for the determination of SCT, shared the highest similarity (91.8%) with that of SCT XIa. These results suggest that coa310 is a novel variant of SCT XI. Moreover, we demonstrated that coa310 encodes a functioning coagulase, by confirming the coagulating activity of the recombinant protein expressed from coa310. This is the first study to directly demonstrate that Coa310, a putative SCT XI, has coagulating activity. These findings may be useful for the improvement of the staphylocoagulase-typing method, including serotyping and genotyping. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a leading cause of skin and soft tissue infections, foreign body infections, and infective endocarditis. In case of endovascular infection with S. aureus, higher rates of cardiac valve destruction, embolic complications, severe sepsis, and death occur. The unique capacity of S. aureus to induce clotting has been known for over a century; however, its role in virulence has long been controversial. S. aureus secretes two coagulases, staphylocoagulase and vonWillebrand factor binding protein that both activate prothrombin to generate fibrin. A better understanding of the molecular mechanisms as well as the new strategies to target the coagulases have highlighted their importance in S. aureus virulence. Coagulase activity is essential for the formation of S. aureus--fibrin--platelet microaggregates and for the homing of S. aureus to the vascular wall under flow. Absence or inhibition of S. aureus coagulase activity improved outcome in disease models of skin infection, sepsis, catheter infection, and endocarditis. Here, we review how the manipulation of the host's hemostatic system contributes to the disease-causing potential of S. aureus and discuss the S. aureus coagulases as promising targets for novel therapeutic strategies. [ABSTRACT FROM AUTHOR]

In the coagulation cascade during host infection by MRSA, staphylococci are blocked within a fibrin clot mesh, and very hard to clear these pathogens by host tissues and also enable the pathogen to cause lethal sepsis. To overcome this complication, this work aimed at synthesis of pyrazolylpyridine analogs as potential anti-superbug agents and using a simple synthetic protocol. The synthesized pyrazolylpyridine analogs were characterized using spectroscopic techniques like FTIR, NMR, and mass spectroscopy. All synthesized pyrazolylpyridine analogs were evaluated for the in-vitro antibacterial activity. The antibacterial activity was further confirmed with cellular content leakage and measurement of potassium efflux. The anti-biofilm activity was studied qualitatively and quantitatively with crystal violet dye technique. The compounds binding ability with 1NU7 and 1MWT proteins were tested using molecular docking and further analogs were exposed to dynamic simulations. The synthesized compounds were also inspected for their ADMET analysis. It was observed that 4(a g) series showed a potential antibacterial activity compared to 3(a g) analogs to standard drug vancomycin. The MIC value of 4e was found to 30±0.4 µg/mL and also showed an excellent anti-biofilm activity. The revealed data was strongly correlated with molecular docking studies on staphylocoagulase enzyme (PDB: 1NU7) and Penicillin-binding protein 2a (PBP2a) (PDB: 1MWT) with a good docking score, and glide score with high binding energy. By dynamic simulation it was obtained that 4d exhibit stable interaction with staph-coagulase enzyme up to 20 ns with PBP2a protein, and compound 4e exhibited stable interaction at both active and allosteric site. The potent pyrazolylpyridine was designed and synthesized to treat infectious agent MRSA. Among the analogs synthesized, compound 4e showed antibacterial activity validated by insilico studies. In this study, experimental results show significant anti-coagulase, anti-biofilm and antimicrobial activity against MRSA by synthesised compound 4e. This leads to the compound 4e can serve as a potent drug against MRSA in upcoming days. [Display omitted] [ABSTRACT FROM AUTHOR]

The treatment of Methicillin-resistant staphylococcus aureus (MRSA) infections has become challenging due to the growth of multidrug resistance in the bacteria. Here we report the synthesis of pyridine-coupled pyrazoles as an antimicrobial agent against MRSA. A series of pyridine coupled pyrazoles were synthesized and synthesized compounds were characterized using FT-IR, 1H NMR, and Mass spectroscopy. The ADMET results of all the 14 active compounds are interpreted. To identify the potent compound the synthesized compounds screened for minimum inhibitory concentrations against MRSA and compared with standard drug vancomycin. Among the synthesized compounds 6d exhibited good antibacterial activity with MIC value 21 μg/mL, bacterial cell membrane damage study was studied potassium efflux, and cellular content leakage assay. Anticoagulant study for the potent compound also studied and validated by molecular docking and molecular dynamics simulation studies. The docking study of the synthesized compound was carried out and the study depicted that the pyridine ring of all the analogues binds with the various amino acids in the binding pocket of the active site of the Staphylocoagulase and PBP2a protein of MRSA. [Display omitted] • Pyrazole dipyridine analogues were successfully synthesized and characterized. • The potent Pyrazole dipyridine was designed and synthesized to treat infectious agent MRSA. • Evaluated for antibacterial activity, anti-coagulase and anti-biofilm of potent analogue against MRSA was studied. • Molecular docking, molecular dynamics simulation were performed for PBP2a and Staphylocoagulase. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus (S. aureus) is a frequent cause of catheter-related infections. S. aureus secretes the coagulases staphylocoagulase and von Willebrand factor–binding protein, both of which form a staphylothrombin complex upon binding to prothrombin. Although fibrinogen and fibrin facilitate the adhesion of S. aureus to catheters, the contribution of staphylothrombin-mediated fibrin has not been examined. In this study, we use a S. aureus mutant lacking both coagulases (Δcoa/vwb) and dabigatran, a pharmacological inhibitor of both staphylothrombin and thrombin, to address this question. Genetic absence or chemical inhibition of pathogen-driven coagulation reduced both fibrin deposition and the retention of S. aureus on catheters in vitro. In a mouse model of jugular vein catheter infection, dabigatran reduced bacterial load on jugular vein catheters, as well as metastatic kidney infection. Importantly, inhibition of staphylothrombin improved the efficacy of vancomycin treatment both in vitro and in the mouse model. [ABSTRACT FROM AUTHOR]

Abstract: Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) sequence type 9 (ST9) is a potential source of zoonotic infection for humans. In this study, we investigated and compared the virulence profiles of MRSA ST9 isolates from healthy swine and human clinical origins. A total of 152 MRSA ST9 isolates, including 147 LA-MRSA isolates and 5 human clinical isolates, were studied for the accessory gene regulator (agr) and 20 enterotoxin genes (se), exfoliatoxin gene, and tst gene. The evolutionary history of staphylocoagulase (SC) in Taiwan MRSA ST9 was reconstructed based on phylogenetic and population genetics. The predominant type of LA-MRSA ST9 isolates (78.9%) was agr-II that differed from the predominant agr-I human clinical MRSA strains in Taiwan and the LA-MRSA ST398 lineage from Europe. Forty-nine percent of the LA-MRSA ST9 isolates carried a combination of enterotoxin gene cluster-2 (egc-2, seg, sei, sem, sen, seo, and seu) and tst. In addition, the Taiwan LA-MRSA ST9 and the human clinical ST9-MRSA contained a novel SC XIc subtype and had a unique history of evolution indicating a recent common ancestor. These findings suggest a cross-species transmission of this emerging ST9-SC XIc MRSA between swine and human. [Copyright &y& Elsevier]

Abstract: Staphylocoagulases (SCs) have been classified by the differences in antigenicity using a serological method. We have developed a system to classify them based on the nucleotide differences in SC genes (coa). The system was composed of three multiplex PCRs (M-PCRs): M-PCR:A, identifying types III, IV, VII, and VIII; M-PCR:B, identifying types I, II, V, and VI; M-PCR:C, identifying three subtypes of type VI. In this study, we found that coa genes of the serotype VI were not identical, but classified into three subtypes based on the nucleotide differences, especially in D2 and the central region: VIa, the coa gene carried by stp12 from human; and VIb and VIc, the coa genes carried by strains IFH556 and IFH514 isolated from bovine raw milk. The primer pair used in M-PCR:B was designed to identify all three subtypes of type VI coa. The results showed that coa types of 154 out of 155 Staphylococcus aureus strains from various origins assigned by M-PCR:A and B were identical to those obtained by serological methods, leaving a serotype IV strain unclassifiable. All 73 type VI strains were classified into one of three subtypes by M-PCR:C. Furthermore, we found that type VIa and VIb strains carried characteristic pyrogenic toxin superantigen genes, while no toxin genes were identified in type VIc strains, suggesting the correlation between the subtype of type VI coa gene and the carriage of genomic islands. Our results showed that these M-PCRs are convenient methods for SC typing that might be useful for epidemiological studies. [Copyright &y& Elsevier]

Staphylocoagulase (SC) is a major phenotypic determinant of Staphylococcus aureus. Serotype of SC (coagulase type) is used as an epidemiological marker and 10 types (I–X) have been discriminated so far. To clarify genetic diversity of SC within a single and among different serotype(s), we determined approximately 1500 bp-nucleotide sequences of SC gene encoding D1, D2, and central regions (N-terminal half and central regions of SC; SCNC) for a total of 33 S. aureus strains comprising two to three strains from individual coagulase types (I–VIII, X) and 10 strains which were not determined as previously known SC serotypes (ND-strains). Amino acid sequence identities of SCNC among strains with a single coagulase type of II, III, IV, V, VI and X were extremely high (more than 99%), whereas lower identity (56–87%) was observed among different types. In contrast, within a single coagulase type of I, VII, or VIII, sequence divergence was found (lowest identity; 82%). SCNC sequences from the ND-strains were discriminated into two genetic groups with an identity of 71% to each other (tentatively assigned to genotypes [XI] and [XII]), and exhibited less than 86% sequence identities to those of most known coagulase types. All the types [XI] and [XII] strains were methicillin susceptible and belonged to different sequence types from those of coagulase types I–X strains reported so far by multilocus sequence typing. These findings indicated genetic heterogeneity of SC in coagulase types I, VII, and VIII strains, and the presence of two novel SC genotypes related to antigenicity of SC serotypes. [ABSTRACT FROM AUTHOR]

Staphylocoagulase (SC) secreted byStaphylococcus aureusis a potent non-proteolytic activator of the blood coagulation zymogen prothrombin and the prototype of a newly establishedzymogenactivator andadhesionprotein (ZAAP) family. The conformationally activated SC·prothrombin complex specifically cleaves fibrinogen to fibrin, which propagates the growth of bacteria-fibrin-platelet vegetations in acute bacterial endocarditis. Our recent 2.2 Å X-ray crystal structures of an active SC fragment [SC(1-325)] bound to the prothrombin zymogen catalytic domain, prethrombin 2, demonstrated that SC(1-325) represents a new type of non-proteolytic activator with a unique fold. The observed insertion of the SC(1-325) N-terminus into the ‘Ile 16’ cleft of prethrombin 2, which triggers the activating conformational change, provided the first unambiguous structural evidence for the ‘molecular sexuality’ mechanism of non-proteolytic zymogen activation. Based on the SC(1-325) fold, a new family of bifunctional zymogen activator and adhesion proteins was identified that possess N-terminal domains homologous to SC(1-325) and C-terminal domains that mediate adhesion to plasma or extracellular matrix proteins. Further investigation of the ZAAP family may lead to new insights into the mechanisms of bacterial factors that hijack zymogens of the human blood coagulation and fibrinolytic systems to promote and disseminate endocarditis and other infectious diseases. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus encodes a secreted von Willebrand factor-binding protein (vWbp) of 482 amino acids. The N-terminal part of this protein is homologous to staphylocoagulase and therefore we investigated whether vWbp has coagulating activity. Recombinant vWbp was shown to coagulate human and porcine plasma efficiently, but was less active against plasma from other species. The coagulation efficiency was concentration dependent, and could be inhibited by specific antibodies against vWbp. Furthermore, the species-specific coagulation by vWbp depended on the interaction with prothrombin. This interaction also resulted in specific cleavage of vWbp, releasing the C-terminal part from the coagulating domain. [Copyright &y& Elsevier]

Fibrin (Fbn) deposits are a hallmark of staphylocoagulase (SC)-positive endocarditis. Binding of the N terminus of Staphylococcus aureus SC to host prothrombin triggers formation of an active SC·prothrombin∗ complex that cleaves host fibrinogen to Fbn. In addition, the C-terminal domain of the prototypical SC contains one pseudorepeat (PR) and seven repeats (R1 → R7) that bind fibrinogen/Fbn fragment D (frag D) by a mechanism that is unclear. Here, we define affinities and stoichiometries of frag D binding to C-terminal SC constructs, using fluorescence equilibrium binding, NMR titration, alanine scanning, and native PAGE. We found that constructs containing the PR and single repeats bound frag D with K D ∼50 to 130 nM and a 1:1 stoichiometry, indicating a conserved binding site bridging the PR and each repeat. NMR titration of PR-R7 with frag D revealed that residues 22 to 49, bridging PR and R7, constituted the minimal peptide (MP) for binding, corroborated by alanine scanning, and binding of labeled MP to frag D. MP alignment with the PR-R and inter-repeat junctions identified critical conserved residues. Full-length PR-(R1 → R7) bound frag D with K D ∼20 nM and a stoichiometry of 1:5, whereas constructs containing the PR and various three repeats competed with PR-(R1 → R7) for frag D binding, with a 1:3 stoichiometry. These findings are consistent with binding at PR-R and R-R junctions with modest inter-repeat sequence variability. CD of PR-R7 and PR-(R1 → R7) suggested a disordered flexible structure, allowing binding of multiple fibrin(ogen) molecules. Taken together, these results provide insights into pathogen localization on host fibrin networks., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

There have been few studies focused on the molecular characterization of invasive Staphylococcus aureus strains in patients with diabetes in Iran. In the present study, 20 invasive S. aureus strains recovered from the patients with diabetes characterized by the virulence and resistance analysis, biofilm formation, staphylocoagulase (SC) typing, S. aureus protein A locus (spa) typing staphylococcal cassette chromosome mec (SCC mec) typing, and multilocus sequence typing (MLST). Virulence gene detection indicated a high prevalence of strains encoding the pvl genes (50%), a low prevalence of the tst and seg gene (each of them was 5%) and a markedly high prevalence of fnbB (95%), fnbA (85%) , icaD (75%), icaA (65%). A total of 3 coagulase types (III, 85%; II, 10%; V, 5%), 2 agr types (I, 90%; II 10%) and 2 SCC mec types (IV, 65%; III, 35%) and four different clones namely ST8-MRSA-IV/t008 (50%) (USA300), ST239-MRSA-III/t030 (35%), ST5-MRSA-IV/t002 (10%), and ST45-MRSA-IV/t038 (5%) were detected in this study. Eighty-five percent of the isolates were biofilm producers. All the 4 high-level mupirocin resistance (HLMUPR) strains belonged to CC/ST8-MRSA-IV/t008 clone and carried mupA gene. Fusidic acid-resistant isolate belonged to ST239-SCC mec III/t030 clone. One vancomycin-intermediate resistance isolates was detected in our study, which belonged to ST5-MRSA-IVt002. Circulating clone in MRSA strains (USA300) isolated from the patients with diabetes highlighting the possibility of transmission of these microorganisms' clones between hospital, community, and environments. However, further studies require providing critical insights into the importance of continued controlling and treatment of S. aureus infections in patients with diabetes. • First genetic diversity study on S. aureus strains isolated from patients with diabetes in Iran. • Genetic diversity of MRSA strains with a majority of USA300 was observed • Dominant genetic lineages of MRSA isolates and strong biofilm formation ability was reported. [ABSTRACT FROM AUTHOR]