Neobavaisoflavone Inhibits Biofilm Formation and α-Toxin Activity of Staphylococcus aureus.

Neobavaisoflavone had antimicrobial activities against Gram-positive multidrug-resistant (MDR) bacteria, but the effect of neobavaisoflavone on the virulence and biofilm formation of S. aureus has not been explored. The present study aimed to investigate the possible inhibitory effect of neobavaisoflavone on the biofilm formation and α-toxin activity of S. aureus. Neobavaisoflavone presented strong inhibitory effect on the biofilm formation and α-toxin activity of both methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains at 25 µM, but did not affect the growth of S. aureus planktonic cells. Genetic mutations were identified in four coding genes, including cell wall metabolism sensor histidine kinase walK, RNA polymerase sigma factor rpoD, tetR family transcriptional regulator, and a hypothetical protein. The mutation of WalK (K570E) protein was identified and verified in all the neobavaisoflavone-induced mutant S. aureus isolates. The ASN501, LYS504, ILE544 and GLY565 of WalK protein act as hydrogen acceptors to form four hydrogen bonds with neobavaisoflavone by molecular docking analysis, and TRY505 of WalK protein contact with neobavaisoflavone to form a pi-H bond. In conclusion, neobavaisoflavone had excellent inhibitory effect on the biofilm formation and α-toxin activity of S. aureus. The WalK protein might be a potential target of neobavaisoflavone against S. aureus.

Enterotoxigenic methicillin resistant Staphylococcus aureus contamination in salted fish from Gwadar Balochistan / Contaminação de Staphylococcus aureus resistente à meticilina enterotoxigênica em peixes salgados de Gwadar Balochistan

Staphylococcus aureus is an important foodborne pathogen associated to food intoxication and other multiple infections in human being. Its presence in salted food is a serious issue due to its salt tolerance potential. A study was conducted to analyze the presence of enterotoxins producing drug resistance S. aureus in salted sea fish from Gwadar. Freshly persevered samples (n=50) of salted fish were subjected to analyze the presence of S. aureus using 16S rRNA and Nuc genes primers. The isolates were then evaluated for drug resistance and enterotoxins producing potential using specific primers for MecA (methicillin resistance gene), (SEA) staphylococcal enterotoxin A and (SEB) staphylococcal enterotoxin B genes. Total 13/50 (26%) of the samples were found positive for the presence of S. aureus, preliminary confirmed with biochemical profiling and finally with the help of target genes presence. The isolates were found showing 100% resistant to methicillin, which were molecularly confirmed by the presence of MecA gene present in genome. The isolates 5/13 (38%) were positive for SEA and 3/13 (23%) for SEB genes, whereas 2/13 (15%) were confirmed having both SEA and SEB genes in its genome. It was also confirmed that all the isolates were capable to form biofilm over the glass surfaces. It was concluded that the study confirmed the presence of enterotoxigenic methicillin resistance Staphylococcus aurous (MRSA) in salted fish product, that poses gross food safety concern. Preventive and control measures are necessary to handle this serious food safety concern.

Staphylococcus aureus é um importante patógeno de origem alimentar associado à intoxicação alimentar e outras infecções múltiplas em seres humanos. Sua presença em alimentos salgados é um problema sério devido ao seu potencial de tolerância ao sal. Um estudo foi realizado para analisar a presença de enterotoxinas produtoras de resistência a drogas S. aureus em peixes salgados do mar de Gwadar. Amostras recém-perseveradas (n = 50) de peixes salgados foram submetidas à análise da presença de S. aureus usando os primers dos genes 16S rRNA e Nuc. Os isolados foram então avaliados quanto à resistência a drogas e potencial de produção de enterotoxinas usando primers específicos para os genes MecA (gene de resistência à meticilina), (SEA) enterotoxina A estafilocócica e (SEB) enterotoxina B estafilocócica genes. Um total de 13/50 (26%) das amostras foi considerado positivas para a presença de S. aureus, confirmadas preliminarmente com perfis bioquímicos e finalmente com a ajuda da presença de genes-alvo. Os isolados foram encontrados com 100% de resistência à meticilina, os quais foram confirmados molecularmente pela presença do gene MecA no genoma. Os isolados 5/13 (38%) foram positivos para SEA e 3/13 (23%) para genes SEB, enquanto 2/13 (15%) foram confirmados tendo os genes SEA e SEB em seu genoma. Também foi verificado que todos os isolados foram capazes de formar biofilme sobre as superfícies de vidro. Concluiu-se que o estudo confirmou a presença de Staphylococcus aurous resistente à meticilina enterotoxigênica (MRSA) em produtos de peixe salgado, o que representa uma grande preocupação para a segurança alimentar. Medidas preventivas e de controle são necessárias para lidar com essa grave preocupação com a segurança alimentar.

Transparent Anti-SARS-CoV-2 and Antibacterial Silver Oxide Coatings.

Transparent antimicrobial coatings can maintain the aesthetic appeal of surfaces and the functionality of a touch-screen while adding the benefit of reducing disease transmission. We fabricated an antimicrobial coating of silver oxide particles in a silicate matrix on glass. The matrix was grown by a modified Stöber sol-gel process with vapor-phase water and ammonia. A coating on glass with 2.4 mg of Ag2O per mm2 caused a reduction of 99.3% of SARS-CoV-2 and >99.5% of Pseudomonas aeruginosa, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus compared to the uncoated glass after 1 h. We envisage that screen protectors with transparent antimicrobial coatings will find particular application to communal touch-screens, such as in supermarkets and other check-out or check-in facilities where a number of individuals utilize the same touch-screen in a short interval.

Comparative Assessment of Bacteriophage and Antibiotic Activity against Multidrug-Resistant Staphylococcus aureus Biofilms.

Problems connected with biofilm-related infections and antibiotic resistance necessitate the investigation and development of novel treatment strategies. Given their unique characteristics, one of the most promising alternatives to conventional antibiotics are bacteriophages. In the in vitro and in vivo larva model study, we demonstrate that phages vB_SauM-A, vB_SauM-C, and vB_SauM-D are effective antibiofilm agents. The exposure of biofilm to phages vB_SauM-A and vB_SauM-D led to 2-3 log reductions in the colony-forming unit number in most of the multidrug-resistant S. aureus strains. It was found that phage application reduced the formed biofilms independently of the used titer. Moreover, the study demonstrated that bacteriophages are more efficient in biofilm biomass removal and reduction in staphylococci count when compared to the antibiotics used. The scanning electron microscopy analysis results are in line with colony forming unit (CFU) counting but not entirely consistent with crystal violet (CV) staining. Additionally, phages vB_SauM-A, vB_SauM-C, and vB_SauM-D can significantly increase the survival rate and extend the survival time of Galleria mellonella larvae.

Secondary Amine Pendant ß-Peptide Polymers Displaying Potent Antibacterial Activity and Promising Therapeutic Potential in Treating MRSA-Induced Wound Infections and Keratitis.

Interest in developing antibacterial polymers as synthetic mimics of host defense peptides (HPDs) has accelerated in recent years to combat antibiotic-resistant bacterial infections. Positively charged moieties are critical in defining the antibacterial activity and eukaryotic toxicity of HDP mimics. Most examples have utilized primary amines or guanidines as the source of positively charged moieties, inspired by the lysine and arginine residues in HDPs. Here, we explore the impact of amine group variation (primary, secondary, or tertiary amine) on the antibacterial performance of HDP-mimicking ß-peptide polymers. Our studies show that a secondary ammonium is superior to either a primary ammonium or a tertiary ammonium as the cationic moiety in antibacterial ß-peptide polymers. The optimal polymer, a homopolymer bearing secondary amino groups, displays potent antibacterial activity and the highest selectivity (low hemolysis and cytotoxicity). The optimal polymer displays potent activity against antibiotic-resistant bacteria and high therapeutic efficacy in treating MRSA-induced wound infections and keratitis as well as low acute dermal toxicity and low corneal epithelial cytotoxicity. This work suggests that secondary amines may be broadly useful in the design of antibacterial polymers.

The Untargeted Phytochemical Profile of Three Meliaceae Species Related to In Vitro Cytotoxicity and Anti-Virulence Activity against MRSA Isolates.

BACKGROUND: A high mortality rate is associated with about 80% of all infections worldwide, mainly due to antimicrobial resistance. Various antimicrobial and cytotoxic activities have been proposed for Meliaceae species. This study aimed to evaluate the in vitro anti-virulence and cytotoxic effect of the leaf extracts of Aphanamixis polystachya, Toona ciliata and Melia azedarach against five MRSA strains and on three cancer cell lines, followed by biological correlation to their encompassed phytoconstituents. MATERIAL AND METHODS: We explored three plants of this family against a panel of Methicillin-resistant Staphylococcus aureus (MRSA) strains and several cancer cell lines to select the most promising candidates for further in vivo and preclinical studies. The phytochemical composition was evaluated by UHPLC-QTOF-MS untargeted profiling. Cell viability was assessed by SRB assay. Minimum Inhibitory Concentration was carried out by using the agar micro-dilution technique. Inhibition of biofilm formation and preformed biofilm disruption were assessed spectrophotomertically, according to the Sultan and Nabil method (2019). RESULTS: A total of 279 compounds were putatively annotated to include different phytochemical classes, such as flavonoids (108), limonoids/terpenoids (59), phenolic acids (49) and lower-molecular-weight phenolics (39). A. polystachya extract showed the most potent cytotoxic activity against Huh-7, DU-145 and MCF-7 cell lines (IC50 = 3, 3.5 and 13.4 µg mL-1, respectively), followed by M. azedarach, with no effect recorded for T. ciliata extract. Furthermore, both A. polystachya and M. azedarach extracts showed promising anti-virulence and antimicrobial activities, with A. polystachya being particularly active against MRSA. These two latter extracts could inhibit and disrupt the biofilm, formed by MRSA, at sub-lethal concentrations. Interestingly, the extracts inhibited hemolysin-α enzyme, thus protecting rabbit RBCs from lysis. A. polystachya extract reduced the pigmentation and catalase enzyme activity of tested pigmented strains better than M. azedarach at both tested sub-MICs. Consequently, susceptibility of the extract-treated cells to oxidant killing by 200 mM H2O2 increased, leading to faster killing of the cells within 120 min as compared to the extract-non-treated cells, likely due to the lower antioxidant-scavenging activity of cells exhibiting less staphyloxanthin production. CONCLUSION: These findings suggested that both A. polystachya and M. azedarach natural extracts are rich in bioactive compounds, mainly limonoids, phenolics and oxygenated triterpenoids, which can combat MRSA biofilm infections and could be considered as promising sources of therapeutic cytotoxic, antibiofilm and anti-virulence agents.

Prevalence of Panton Valentine Leucocidin gene containing Staphylococcus aureus in pus samples from Paediatric patients.

Staphylococcus (Staph) aureus containing Panton Valentine Leucocidin (PVL) gene are spreading in the whole world. This gene encodes PVL toxin that has lytic effect on WBCs contributing to the low immunity of the body. It also causes pus formation in various places of the body. This study was conducted to understand the effect of PVL positive Staph aureus in causing purulent infections in children between the age of one day to 15 years. Pus samples from various sites of the body from children between the age of one day to 15 years were taken. The number of pus samples containing Staph aureus was 45. These were collected over a period of one year, from October 2, 2017 to September 30, 2018, at the Shaikh Zayed Hospital, Lahore. A total of 27 (60%) PVL samples were positive Staph aureus. Prevalence of PVL gene was noted to be high in MSSA 9(64%), wound swabs 18(75%), in isolates from orthopaedic department 6(75%), indoor 21(63%), and in males 18(66%). Our study showed that most of the Staph aureus samples that were obtained from pus samples from children had PVL gene in their genome. This percentage is very high. To control its spread, we need to treat not only the patients but also their close contacts. The main objective to conduct this study was to assess the prevalence of PVL positive Staph aureus strain in our local setup. Paediatric age group was selected because it is the most vulnerable group and pus samples were chosen because this strain causes recurrent purulent infections.

Identification of structurally diverse menaquinone-binding antibiotics with in vivo activity against multidrug-resistant pathogens.

The emergence of multidrug-resistant bacteria poses a threat to global health and necessitates the development of additional in vivo active antibiotics with diverse modes of action. Directly targeting menaquinone (MK), which plays an important role in bacterial electron transport, is an appealing, yet underexplored, mode of action due to a dearth of MK-binding molecules. Here we combine sequence-based metagenomic mining with a motif search of bioinformatically predicted natural product structures to identify six biosynthetic gene clusters that we predicted encode MK-binding antibiotics (MBAs). Their predicted products (MBA1-6) were rapidly accessed using a synthetic bioinformatic natural product approach, which relies on bioinformatic structure prediction followed by chemical synthesis. Among these six structurally diverse MBAs, four make up two new MBA structural families. The most potent member of each new family (MBA3, MBA6) proved effective at treating methicillin-resistant Staphylococcus aureus infection in a murine peritonitis-sepsis model. The only conserved feature present in all MBAs is the sequence 'GXLXXXW', which we propose represents a minimum MK-binding motif. Notably, we found that a subset of MBAs were active against Mycobacterium tuberculosis both in vitro and in macrophages. Our findings suggest that naturally occurring MBAs are a structurally diverse and untapped class of mechanistically interesting, in vivo active antibiotics.

Differences Between Methicillin-susceptible Versus Methicillin-resistant Staphylococcus aureus Infections in Pediatrics: Multicenter Cohort Study Conducted in Bogotá, Colombia, 2014-2018.

BACKGROUND: The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) has changed in recent years. The present article is intended to establish differences between clinical, laboratory and imaging findings and outcomes of MSSA and MRSA infections, as well as among subgroups of infection such as skin and soft tissue infection, osteoarticular, bacteremia or pneumonia in a pediatric population from Bogota, Colombia. METHODS: Retrospective cohort study using clinical records of patients under 18 years of age treated at the participating centers in Bogota, Colombia, between 2014 and 2018. The first positive S. aureus culture was studied. MSSA and MRSA were compared. The χ2 test, Fisher exact test, and Kruskal-Wallis test were calculated, and the statistical significance was presented using the difference and its 95% CI. RESULTS: Five hundred fifty-one patients were included; 211 (38%) corresponded to MRSA and 340 (62%) to MSSA for a total of 703 cultures. A significantly higher probability of having an MSSA infection than MRSA was found in patients with previous heart disease (3.3% vs. 0.5%), neurologic disease (5.9% vs. 2.5%), recent major surgeries (11% vs. 5%) or who has an implanted device (11% vs. 4%). In contrast, in severe MRSA infections (bacteremia, osteoarticular infections and pneumonia), a higher rate of complications was seen (admission to the pediatric intensive care unit, mechanical ventilation and vasoactive support), and in osteoarticular MRSA, more than 1 surgery per case was seen (89% vs. 61%). Laboratory results and mortality were similar. CONCLUSIONS: MRSA was associated with a more severe course in bacteremia, osteoarticular infections and pneumonia. Some classical risk factors associated with MRSA infections were found to be related to MSSA. In general, with the exception of skin and soft tissue infection, there was an increased risk of pediatric intensive care unit admission and mechanical and inotropic support with MRSA in a pediatric population.

Anti-biofilm and anti-inflammatory effects of Lycosin-II isolated from spiders against multi-drug resistant bacteria.

Currently, multidrug-resistant bacteria are rapidly increasing worldwide because of the misuse or overuse of antibiotics. In particular, few options exist for treating infections caused by long-persisting oxacillin-resistant strains and recently proliferating carbapenem-resistant strains. Therefore, alternative treatments are urgently needed. The antimicrobial peptide (AMP) Lycosin-II is a peptide consisting of 21 amino acids isolated from the venom of the spider Lycosa singoriensis. Lycosin-II showed strong antibacterial activity and biofilm inhibition effects against gram-positive and gram-negative bacteria including oxacillin-resistant Staphylococcus aureus (S. aureus) and meropenem-resistant Pseudomonas aeruginosa (P. aeruginosa) isolated from patients. In addition, Lycosin-II was not cytotoxic against human foreskin fibroblast Hs27 or hemolytic against sheep red blood cells at the concentration of which exerted antibacterial activity. The mechanism of action of Lycosin-II involves binding to lipoteichoic acid and lipopolysaccharide of gram-positive and gram-negative bacterial membranes, respectively, to destroy the bacterial membrane. Moreover, Lycosin-II showed anti-inflammatory effects by inhibiting the expression of pro-inflammatory cytokines that are increased during bacterial infection in Hs27 cells. These results suggest that Lycosin-II can serve as a therapeutic agent against infections with multidrug-resistant strains.

Staphylococcal Phages Adapt to New Hosts by Extensive Attachment Site Variability.

Bacterial pathogens commonly carry prophages that express virulence factors, and human strains of Staphylococcus aureus carry Sa3int phages, which promote immune evasion. Recently, however, these phages have been found in livestock-associated, methicillin-resistant S. aureus (LA-MRSA). This is surprising, as LA-MRSA strains contain a mutated primary bacterial integration site, which likely explains why the rare integration events that do occur mostly happen at alternative locations. Using deep sequencing, we show that after initial integration at secondary sites, Sa3int phages adapt through nucleotide changes in their attachment sequences to increase homology with alternative bacterial attachment sites. Importantly, this homology significantly enhances integrations in new rounds of infections. We propose that promiscuity of the phage-encoded tyrosine recombinase is responsible for establishment of Sa3int phages in LA-MRSA. Our results demonstrate that phages can adopt extensive population heterogeneity, leading to establishment in strains lacking bona fide integration sites. Ultimately, their presence may increase virulence and zoonotic potential of pathogens with major implications for human health. IMPORTANCE A growing number of humans are being infected by antibiotic resistant Staphylococcus aureus originating from livestock. The preference of S. aureus for humans or animals is in part determined by factors encoded by viruses (phages) that reside in the bacterial genome. Here, we reveal a process by which phages adapt to and become integrated in new strains of S. aureus lacking the preferred phage integration site. We propose that this is due to the relaxed specificity of a phage-encoded enzyme called recombinase. As this recombinase is used by many other phages, our results might have implications for a broader range of phages. Importantly, the adaptation described here enables S. aureus to jump between host organisms and increases its zoonotic threat.

Bisdemethoxycurcumin Reduces Methicillin-Resistant Staphylococcus aureus Expression of Virulence-Related Exoproteins and Inhibits the Biofilm Formation.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen of nosocomial infection, which is resistant to most antibiotics. Presently, anti-virulence therapy and anti-biofilm therapy are considered to be promising alternatives. In the current work, we investigated the influence of bisdemethoxycurcumin (BDMC) on the virulence-related exoproteins and the biofilm formation using a reference strain and clinic isolated strains. Western blotting, quantitative RT-PCR, and tumor necrosis factor (TNF) release assay were performed to assess the efficacy of BDMC in reducing the expression of Staphylococcus enterotoxin-related exoproteins (enterotoxin A, enterotoxin B) and α-toxin in MRSA. The anti-biofilm activity of BDMC was evaluated through a biofilm inhibition assay. The study suggests that sub-inhibitory concentrations of BDMC significantly inhibited the expression of sea, seb, and hla at the mRNA level in MRSA. Moreover, the expression of virulence-related exoproteins was significantly decreased by down-regulating accessory gene regulator agr, and the inhibition of biofilms formation was demonstrated by BDMC at sub-inhibitory concentrations. Consequently, the study suggests that BDMC may be a potential natural antibacterial agent to release the pressure brought by antibiotic resistance.

Biochanin A as an α-hemolysin inhibitor for combating methicillin-resistant Staphylococcus aureus infection.

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant pathogen that poses a significant risk to global health today. In S. aureus, α-hemolysin is an important virulence factor as it contributes to the capacity of the bacteria to infect the host. Here, we showed that biochanin A (bioA), an isoflavone present in red clover, cabbage and alfalfa, effectively inhibited hemolytic activity at a dose as low as 32 µg/mL. Further, western blot and RT-qPCR data showed that bioA reduced the production and expression of MRSA hemolysin in a dose-dependent manner. In addition, when different concentrations of bioA were added to a coculture system of A549 cells and S. aureus, it could significantly decrease cell injury. Importantly, the in vivo study showed that bioA could protect mice from pneumonia caused by a lethal dose of MRSA, as evidenced by improving their survival and reducing the number of bacterial colonies in lung tissues, the secretion of hemolysin into alveolar lavage fluid and the degree of pulmonary edema. In conclusion, biochanin A protected the host from MRSA infection by inhibiting the expression of the hemolysin of MRSA, which may provide experimental evidence for its development to a potential anti-MRSA drug.

Optimized Silica-Binding Peptide-Mediated Delivery of Bactericidal Lysin Efficiently Prevents Staphylococcus aureus from Adhering to Device Surfaces.

Staphylococcal-associated device-related infections (DRIs) represent a significant clinical challenge causing major medical and economic sequelae. Bacterial colonization, proliferation, and biofilm formation after adherence to surfaces of the indwelling device are probably the primary cause of DRIs. To address this issue, we incorporated constructs of silica-binding peptide (SiBP) with ClyF, an anti-staphylococcal lysin, into functionalized coatings to impart bactericidal activity against planktonic and sessile Staphylococcus aureus. An optimized construct, SiBP1-ClyF, exhibited improved thermostability and staphylolytic activity compared to its parental lysin ClyF. SiBP1-ClyF-functionalized coatings were efficient in killing MRSA strain N315 (>99.999% within 1 h) and preventing the growth of static and dynamic S. aureus biofilms on various surfaces, including siliconized glass, silicone-coated latex catheter, and silicone catheter. Additionally, SiBP1-ClyF-immobilized surfaces supported normal attachment and growth of mammalian cells. Although the recycling potential and long-term stability of lysin-immobilized surfaces are still affected by the fragility of biological protein molecules, the present study provides a generic strategy for efficient delivery of bactericidal lysin to solid surfaces, which serves as a new approach to prevent the growth of antibiotic-resistant microorganisms on surfaces in hospital settings and could be adapted for other target pathogens as well.

Antifungal Treatment Aggravates Sepsis through the Elimination of Intestinal Fungi.

Prophylactic antifungal therapy is widely adopted clinically for critical patients and effective in reducing the morbidity of invasive fungal infection and improves outcomes of those diagnosed patients; however, it is not associated with higher overall survival. As intestinal commensal fungi play a fundamental role in the host immune response in health and disease, we propose that antifungal therapy may eliminate intestinal fungi and aggravate another critical syndrome, sepsis. Here, with murine sepsis model, we found that antifungal therapy with fluconazole dismissed intestinal fungal burden and aggravated endotoxin-induced but no gram-positive bacteria-induced sepsis. Nevertheless, antifungal therapy did not exert its detrimental effect on germ-free mice. Moreover, colonizing more commensal fungi in the mouse intestine or administration of fungal cell wall component mannan protected the mice from endotoxin-induced sepsis. On the molecular level, we demonstrated that antifungal therapy aggravated endotoxin sepsis through promoting Gasdermin D cleavage in the distal small intestine. Intestinal colonization with commensal fungi inhibited Gasdermin D cleavage in response to lipopolysaccharide challenge. These findings show that intestinal fungi inhibit Gasdermin D-mediated pyroptosis and protect the mice from endotoxin-induced sepsis. This study demonstrates the protective role of intestinal fungi in the pathogenesis of endotoxin-induced sepsis in the laboratory. It will undoubtedly prompt us to study the relationship between antifungal therapy and sepsis in critical patients who are susceptible to endotoxin-induced sepsis in the future.

Increased Risk of Thrombocytopenia and Death in Patients with Bacteremia Caused by High Alpha Toxin-Producing Methicillin-Resistant Staphylococcus aureus.

Alpha toxin (Hla) is a major virulence factor of Staphylococcus aureus that targets platelets but clinical data on Hla pathogenesis in bacteremia (SAB) is limited. We examined the link between in vitro Hla activity and outcome. Study isolates obtained from 100 patients with SAB (50 survivors; 50 non-survivors) were assessed for in vitro Hla production by Western immunoblotting in a subset of isolates and Hla activity by hemolysis assay in all isolates. Relevant demographics, laboratory and clinical data were extracted from patients' medical records to correlate Hla activity of the infecting isolates with outcome. Hla production strongly correlated with hemolytic activity (rs = 0.93) in vitro. A trend towards higher hemolytic activity was observed for MRSA compared to MSSA and with high-risk source infection. Significantly higher hemolytic activity was noted for MRSA strains isolated from patients who developed thrombocytopenia (median 52.48 vs. 16.55 HU/mL in normal platelet count, p = 0.012) and from non survivors (median 30.96 vs. 14.87 HU/mL in survivors, p = 0.014) but hemolytic activity of MSSA strains did not differ between patient groups. In vitro Hla activity of MRSA strains obtained from patients with bacteremia is significantly associated with increased risk for thrombocytopenia and death which supports future studies to evaluate feasibility of bedside phenotyping and therapeutic targeting.

Decursinol Angelate Mitigates Sepsis Induced by Methicillin-Resistant Staphylococcus aureus Infection by Modulating the Inflammatory Responses of Macrophages.

The herbal plant Angelica gigas (A. gigas) has been used in traditional medicine in East Asian countries, and its chemical components are reported to have many pharmacological effects. In this study, we showed that a bioactive ingredient of A. gigas modulates the functional activity of macrophages and investigated its effect on inflammation using a sepsis model. Among 12 different compounds derived from A. gigas, decursinol angelate (DA) was identified as the most effective in suppressing the induction of TNF-α and IL-6 in murine macrophages. When mice were infected with a lethal dose of methicillin-resistant Staphylococcus aureus (MRSA), DA treatment improved the mortality and bacteremia, and attenuated the cytokine storm, which was associated with decreased CD38+ macrophage populations in the blood and liver. In vitro studies revealed that DA inhibited the functional activation of macrophages in the expression of pro-inflammatory mediators in response to microbial infection, while promoting the bacterial killing ability with an increased production of reactive oxygen species. Mechanistically, DA treatment attenuated the NF-κB and Akt signaling pathways. Intriguingly, ectopic expression of an active mutant of IKK2 released the inhibition of TNF-α production by the DA treatment, whereas the inhibition of Akt resulted in enhanced ROS production. Taken together, our experimental evidence demonstrated that DA modulates the functional activities of pro-inflammatory macrophages and that DA could be a potential therapeutic agent in the management of sepsis.

Clonality, virulence genes, and antibiotic resistance of Staphylococcus aureus isolated from blood in Shandong, China.

BACKGROUND: Bloodstream infection (BSI) caused by Staphylococcus aureus (S. aureus) can be life-threatening and pose a great challenge to infection control and clinical treatment. However, little information exists regarding the characterization of S. aureus in BSI patients in Shandong, China. To identify the clonality, virulence genes, and antibiotic resistance of S. aureus in blood, a total of 101 nonrepetitive blood isolates were collected. The antibiotic resistance phenotypes were determined, and virulence genes were analyzed with polymerase chain reaction (PCR). Finally, the genetic relatedness was investigated with Staphylococcus chromosomal cassette mec (SCCmec) typing for methicillin-resistant S. aureus (MRSA) isolates, Staphylococcal protein A (spa), and multilocus sequence typing (MLST) for all of 101 isolates. RESULTS: Of the 101 S. aureus isolates, 24 MRSA isolates and 77 methicillin-susceptible S. aureus (MSSA) isolates were identified. Overall, MRSA isolates had higher resistance rates than MSSA isolates when exposed to any of the 15 antibiotics tested in this study except for trimethoprim/sulfamethoxazole. Among the 17 virulence genes tested in this study, hla, hld, and hlg could be detected in all isolates. MRSA isolates were more likely to carry seb and hlb genes, while MSSA isolates were more likely to carry seg and sei genes. Thirty-five sequence types (STs) and 49 spa types were identified, of which ST59-t437 and ST398-t571 were the most abundant. These two genotypes were also the most abundant ST-spa types in MRSA and MSSA isolates, but their abundances shifted over time, with ST398-t571 being the predominant genotype from 2016 to 2017, and ST59-t437 from 2018 to 2020. Besides, all the ST59-t437 isolates harbored hlgb gene, whereas most (88.9%) ST398-t571 did not. In addition, twenty-four MRSA isolates were subject to SCCmec typing. SCCmec IVa was the most prevalent SCCmec type, and all the ST59-t437 MRSA isolates were SCCmec IVa. We also observed 15 new STs, and some of them were MRSA. CONCLUSION: These findings provide additional observations and epidemiological data for blood S. aureus isolates, which can improve future infection-control measures and aid in potential clinical treatments in hospitals and other clinical settings.

Characterization of antibiotic resistance and virulence genes of ocular methicillin-resistant Staphylococcus aureus strains through complete genome analysis.

Virulence-factor encoding genes (VFGs) and antimicrobial resistance genes (ARGs) of ocular Methicillin-Resistant Staphylococcus aureus (MRSA), are the reason behind the common cause of severe and untreatable ocular infection and are largely unknown. The unavailability of the complete genome sequence of ocular MRSA strains hinders the unambiguous determination of ARGs and VRGs role in disease pathogenesis and their genomic location. To fulfill this critical need, we achieved the high-quality complete genome of four ocular MRSA strains (AMRF3 - AMRF6) by combining MinION nanopore sequencing technology, followed by polishing with Illumina sequence reads. We obtained a single chromosome and a plasmid in each strain. Sequence typing revealed that AMRF3 and AMRF5 strains harbored ST772, whereas AMRF4 and AMRF6 harbored ST 2066. All plasmids carried heavy metal cadmium resistance genes cadC and cadD, while cadA was detected only in the plasmid pSaa6159 of AMRF4 and AMRF6 strains. Further, pSaa6159 contains a complete Tn552 transposon with beta-lactamase genes, blaI, blaR1, and blaZ. Interestingly, pSaa6159 in AMRF6 carried five copies of Tn552 transposon. Several exotoxins and enterotoxins were identified across ocular MRSA strains and ST2066 strains found to be not carried any enterotoxins; this finding suggests that these two strains are exotoxigenic. Besides, ST2066 strains carried serine proteases (splA, splB, splD, splE and spIF) and exotoxin (seb and set 21) for their virulence, while ST772 carried antimicrobial resistance genes (blaZ, dfrG, msrA, mphC and fosB) and enterotoxin sec for virulence, suggesting sequence type-specific resistance and virulence. Also, we identified many VFGs and ARGs, that provided multi-drug resistance, enterotoxigenic, exotoxigenic, biofilm-forming, host tissue adhesion and immune response evasion in ocular MRSA strains. Thus, our study provides a better insight into the genomes of ocular MRSA strains that would provide more effective treatment strategies for ocular MRSA infection.