RESUMEN

Introduction. Staphylococcus aureus is a leading agent in community-acquired bacteraemia (CAB) and has been linked to elevated mortality rates and methicillin resistance in Costa Rica.Gap statement and aim. To update and enhance previous data obtained in this country, we analysed the clinical manifestations of 54 S. aureus CAB cases in a tertiary hospital and delineated the sequence types (STs), virulome, and resistome of the implicated isolates.Methodology. Clinical information was retrieved from patient files. Antibiotic susceptibility profiles were obtained with disc diffusion and automated phenotypic tests. Genomic data were exploited to type the isolates and for detection of resistance and virulence genes.Results. Primary infections predominantly manifested as bone and joint infections, followed by skin and soft tissue infections. Alarmingly, 70% of patients continued to exhibit positive haemocultures beyond 48 h of treatment modification, with nearly a quarter requiring mechanical ventilation or developing septic shock. The 30-day mortality rate reached an alarming 40%. More than 60% of the patients were found to have received suboptimal or inappropriate antibiotic treatment, and there was an alarming tendency towards the overuse of third-generation cephalosporins as empirical treatment. Laboratory tests indicated elevated creatinine levels, leukocytosis, and bandaemia within the first 24 h of hospitalization. However, most showed improvement after 48 h. The isolates were categorized into 13 STs, with a predominance of representatives from the clonal complexes CC72 (ST72), CC8 (ST8), CC5 (ST5, ST6), and CC1 (ST188). Twenty-four isolates tested positive for mecA, with ST72 strains accounting for 20. In addition, we detected genes conferring acquired resistance to aminoglycosides, MLSB antibiotics, trimethoprim/sulfamethoxazole, and mutations for fluoroquinolone resistance in the isolate collection. Genes associated with biofilm formation, capsule synthesis, and exotoxin production were prevalent, in contrast to the infrequent detection of enterotoxins or exfoliative toxin genes.Conclusions. Our findings broaden our understanding of S. aureus infections in a largely understudied region and can enhance patient management and treatment strategies.

Asunto(s)

Antibacterianos , Bacteriemia , Infecciones Comunitarias Adquiridas , Pruebas de Sensibilidad Microbiana , Infecciones Estafilocócicas , Staphylococcus aureus , Centros de Atención Terciaria , Humanos , Costa Rica/epidemiología , Centros de Atención Terciaria/estadística & datos numéricos , Infecciones Comunitarias Adquiridas/microbiología , Infecciones Comunitarias Adquiridas/epidemiología , Infecciones Comunitarias Adquiridas/mortalidad , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/epidemiología , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/mortalidad , Bacteriemia/microbiología , Bacteriemia/epidemiología , Bacteriemia/mortalidad , Bacteriemia/tratamiento farmacológico , Masculino , Staphylococcus aureus/genética , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/aislamiento & purificación , Persona de Mediana Edad , Femenino , Anciano , Adulto , Antibacterianos/uso terapéutico , Antibacterianos/farmacología , Anciano de 80 o más Años , Adulto Joven , Adolescente , Factores de Virulencia/genética , Niño

RESUMEN

Staphylococcus aureus can develop antibiotic resistance and evade immune responses, causing infections in different body sites. However, the metabolic changes underlying this process are poorly understood. A variant strain, C1V, was derived from the parental strain C1 by exposing it to increasing concentrations of vancomycin in vitro. C1V exhibited a vancomycin-intermediate phenotype and physiological changes compared to C1. It showed higher survival rates than C1 when phagocytosed by Raw264.7 cells. Metabolomics analysis identified significant metabolic differences pre- and post-induction (C1 + SC1 vs. C1V + SC1V: 201 metabolites) as well as pre- and post-phagocytosis (C1 vs. SC1: 50 metabolites; C1V vs. SC1V: 95 metabolites). The variant strain had distinct morphological characteristics, decreased adhesion ability, impaired virulence, and enhanced resistance to phagocytosis compared to the parental strain. Differential metabolites may contribute to S. aureus ' resistance to antibiotics and phagocytosis, offering insights into potential strategies for altering vancomycin nonsusceptibility and enhancing phagocyte killing by manipulating bacterial metabolism.

Asunto(s)

Antibacterianos , Metabolómica , Fagocitosis , Staphylococcus aureus , Vancomicina , Vancomicina/farmacología , Ratones , Animales , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Fagocitosis/efectos de los fármacos , Células RAW 264.7 , Antibacterianos/farmacología , Virulencia , Infecciones Estafilocócicas/microbiología , Pruebas de Sensibilidad Microbiana , Resistencia a la Vancomicina/genética , Metaboloma/efectos de los fármacos , Adhesión Bacteriana/efectos de los fármacos , Adaptación Fisiológica

RESUMEN

Aim: The epigenome influences gene regulation and phenotypes in response to exposures. Epigenome assessment can determine exposure history aiding in diagnosis.Materials & methods: Here we developed and implemented a machine learning algorithm, the exposure signature discovery algorithm (ESDA), to identify the most important features present in multiple epigenomic and transcriptomic datasets to produce an integrated exposure signature (ES).Results: Signatures were developed for seven exposures including Staphylococcus aureus, human immunodeficiency virus, SARS-CoV-2, influenza A (H3N2) virus and Bacillus anthracis vaccinations. ESs differed in the assays and features selected and predictive value.Conclusion: Integrated ESs can potentially be utilized for diagnosis or forensic attribution. The ESDA identifies the most distinguishing features enabling diagnostic panel development for future precision health deployment.

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This article introduces ESDA, a new analytic tool for integrating multiple data types to identify the most distinguishing features following an exposure. Using the ESDA, we were able to identify signatures of infectious diseases. The results of the study indicate that integration of multiple types of large datasets can be used to identify distinguishing features for infectious diseases. Understanding the changes from different exposures will enable development of diagnostic tests for infectious diseases that target responses from the patient. Using the ESDA, we will be able to build a database of human response signatures to different infections and simplify diagnostic testing in the future.

Asunto(s)

COVID-19 , Epigenómica , Aprendizaje Automático , Staphylococcus aureus , Humanos , Epigenómica/métodos , Staphylococcus aureus/genética , COVID-19/virología , COVID-19/genética , SARS-CoV-2/genética , Epigenoma , Subtipo H3N2 del Virus de la Influenza A/genética , Bacillus anthracis/genética , Algoritmos , Epigénesis Genética , Transcriptoma , Infecciones por VIH/genética , Gripe Humana/genética

RESUMEN

Staphylococcus aureus is the most common cause of skin and soft tissue infections (SSTIs) with Methicillin-Resistant S. aureus (MRSA) strains being a major contributor in both community and hospital settings. S. aureus relies on metabolic diversity and a large repertoire of virulence factors to cause disease. This includes α-hemolysin (Hla), an integral player in tissue damage found in various models, including SSTIs. Previously, we identified a role for the Spx adapter protein, YjbH, in the regulation of several virulence factors and as an inhibitor of pathogenesis in a sepsis model. In this study, we found that YjbH is critical for tissue damage during SSTI, and its absence leads to decreased proinflammatory chemokines and cytokines in the skin. We identified no contribution of YjbI, encoded on the same transcript as YjbH. Using a combination of reporters and quantitative hemolysis assays, we demonstrated that YjbH impacts Hla expression and activity both in vitro and in vivo. Additionally, expression of Hla from a non-native promoter reversed the tissue damage phenotype of the ΔyjbIH mutant. Lastly, we identified reduced Agr activity as the likely cause for reduced Hla production in the ΔyjbH mutant. This work continues to define the importance of YjbH in the pathogenesis of S. aureus infection as well as identify a new pathway important for Hla production.

Asunto(s)

Proteínas Bacterianas , Toxinas Bacterianas , Regulación Bacteriana de la Expresión Génica , Proteínas Hemolisinas , Staphylococcus aureus , Transactivadores , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/genética , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/inmunología , Toxinas Bacterianas/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Staphylococcus aureus/patogenicidad , Staphylococcus aureus/inmunología , Staphylococcus aureus/genética , Ratones , Animales , Transactivadores/genética , Transactivadores/metabolismo , Infecciones Cutáneas Estafilocócicas/microbiología , Infecciones Cutáneas Estafilocócicas/inmunología , Infecciones Cutáneas Estafilocócicas/patología , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/inmunología , Piel/microbiología , Piel/patología , Piel/inmunología , Factores de Virulencia/genética , Humanos , Infecciones de los Tejidos Blandos/microbiología , Infecciones de los Tejidos Blandos/inmunología , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Citocinas/metabolismo , Citocinas/inmunología , Citocinas/genética

RESUMEN

Bicarbonate and CO2 are essential substrates for carboxylation reactions in bacterial central metabolism. In Staphylococcus aureus, the bicarbonate transporter, MpsABC (membrane potential-generating system) is the only carbon concentrating system. An mpsABC deletion mutant can hardly grow in ambient air. In this study, we investigated the changes that occur in S. aureus when it suffers from CO2/bicarbonate deficiency. Electron microscopy revealed that ΔmpsABC has a twofold thicker cell wall thickness compared to the parent strain. The mutant was also substantially inert to cell lysis induced by lysostaphin and the non-ionic surfactant Triton X-100. Mass spectrometry analysis of muropeptides revealed the incorporation of alanine into the pentaglycine interpeptide bridge, which explains the mutant's lysostaphin resistance. Flow cytometry analysis of wall teichoic acid (WTA) glycosylation patterns revealed a significantly lower α-glycosylated and higher ß-glycosylated WTA, explaining the mutant's increased resistance towards Triton X-100. Comparative transcriptome analysis showed altered gene expression profiles. Autolysin-encoding genes such as sceD, a lytic transglycosylase encoding gene, were upregulated, like in vancomycin-intermediate S. aureus mutants (VISA). Genes related to cell wall-anchored proteins, secreted proteins, transporters, and toxins were downregulated. Overall, we demonstrate that bicarbonate deficiency is a stress response that causes changes in cell wall composition and global gene expression resulting in increased resilience to cell wall lytic enzymes and detergents.

Asunto(s)

Bicarbonatos , Pared Celular , Staphylococcus aureus , Staphylococcus aureus/metabolismo , Staphylococcus aureus/genética , Bicarbonatos/metabolismo , Pared Celular/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Estrés Fisiológico , Regulación Bacteriana de la Expresión Génica , Dióxido de Carbono/metabolismo

RESUMEN

The human body harbors a substantial population of bacteria, which may outnumber host cells. Thus, there are multiple interactions between both cell types. Given the common presence of Staphylococcus aureus in the human body and the role of Th17 cells in controlling this pathogen on mucous membranes, we sought to investigate the effect of α-hemolysin, which is produced by this bacterium, on differentiating Th17 cells. RNA sequencing analysis revealed that α-hemolysin influences the expression of signature genes for Th17 cells as well as genes involved in epigenetic regulation. We observed alterations in various histone marks and genome methylation levels via whole-genome bisulfite sequencing. Our findings underscore how bacterial proteins can significantly influence the transcriptome, epigenome, and phenotype of human Th17 cells, highlighting the intricate and complex nature of the interaction between immune cells and the microbiota.

Asunto(s)

Toxinas Bacterianas , Epigénesis Genética , Proteínas Hemolisinas , Staphylococcus aureus , Células Th17 , Staphylococcus aureus/inmunología , Staphylococcus aureus/genética , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/inmunología , Proteínas Hemolisinas/metabolismo , Humanos , Células Th17/inmunología , Células Th17/metabolismo , Toxinas Bacterianas/inmunología , Toxinas Bacterianas/genética , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Metilación de ADN , Diferenciación Celular , Transcriptoma

RESUMEN

Staphylococcus aureus is a major cause of neonatal infections in various anatomical sites, resulting in high morbidity and mortality in The Gambia. These clinical infections are often preceded by nasal carriage of S. aureus, a known risk factor. To determine whether potential sources of newborn S. aureus infections were from carriage, and to characterize S. aureus present in different anatomical sites (blood, ear, eye, umbilical cord, skin, pus, oropharynx, breast milk and vagina), we performed whole-genome sequencing of 172 isolates from clinical sites as well as from healthy and unhealthy carriage. A random selection of mothers (n = 90) and newborns (n = 42) participating in a clinical trial and testing positive for S. aureus were considered for this study. Sequence data were analyzed to determine S. aureus multilocus sequence types and selected antimicrobial and virulence gene profiles. Our findings revealed that in The Gambia, ST15 is the dominant sequence type associated with both carriage and clinical infection. In addition, S. aureus isolates causing clinical infection among neonates were genetically similar to those colonizing their oropharynx, and the different anatomical sites were not found to be uniquely colonized by S. aureus of a single genomic profile. Furthermore, while S. aureus associated with clinical infection had similar antimicrobial resistance gene profiles to carriage isolates, only hemolysin and adhesive factor virulence genes were significantly higher among clinical isolates. In conclusion, this study confirmed S. aureus oropharyngeal colonization among neonates as a potential source of clinical infection in The Gambia. Hence, interventions aiming to reduce neonatal clinical infections in The Gambia should consider decreasing oropharyngeal S. aureus carriage.Trial registration The trial was registered at ClinicalTrials.gov NCT03199547.

Asunto(s)

Portador Sano , Infecciones Estafilocócicas , Staphylococcus aureus , Humanos , Gambia/epidemiología , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/epidemiología , Recién Nacido , Portador Sano/microbiología , Portador Sano/epidemiología , Staphylococcus aureus/genética , Staphylococcus aureus/aislamiento & purificación , Staphylococcus aureus/clasificación , Femenino , Secuenciación Completa del Genoma , Tipificación de Secuencias Multilocus , Genómica , Factores de Virulencia/genética , Genoma Bacteriano , Masculino , Adulto , Antibacterianos/farmacología , Antibacterianos/uso terapéutico

RESUMEN

OBJECTIVE: The purpose of this study is a new update on the resistance profile, Macrolide-Lincosamide-Streptogramin B resistance mechanisms and biofilm formation in the Staphylococcus aureus isolated from health care workers (HCWs) nasal carriage at a children's teaching hospital in Babol (Northern Iran). RESULTS: A total of 143 non-repetitive nasal swab samples were collected from volunteers, where 53.8% (n; 77/143) were HCWs, 33.6% (n; 48/143) medical students, and 12.6% (n; 18/143) resident students. The prevalence of nasal carriers of S. aureus was 22.4% (n; 32/143), among them, 40.6% (n; 13/32) were identified as methicillin-resistant Staphylococcus aureus (MRSA( carriers. Antimicrobial susceptibility testing showed that erythromycin (68.8%, n; 22/32) and ciprofloxacin (15.6%, n; 5/32) had the highest and lowest resistance rate, respectively. The frequency of resistance genes in the strains was as follows; ermC (n; 17/32, 53.1%), ermA (n; 11/32, 34.4%), ermB (n; 6/32, 18.7%), ereA (n; 3/32, 9.4%). Moreover, 50.0% (n; 16/32), 28.1% (n; 9/32) and 21.8% (n; 7/32) of isolates were strongly, weakly and moderately biofilm producer, respectively. Macrolides-lincosamides-streptogramins B (MLSB) antibiotic resistance among S. aureus isolates from HCWs nasal carriage have found significant prevalence rates throughout the globe. It is crucial to remember that the development of biofilms and MLS B antibiotic resistance are both dynamic processes.

Asunto(s)

Antibacterianos , Biopelículas , Portador Sano , Clindamicina , Personal de Salud , Staphylococcus aureus Resistente a Meticilina , Pruebas de Sensibilidad Microbiana , Infecciones Estafilocócicas , Staphylococcus aureus , Humanos , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Clindamicina/farmacología , Antibacterianos/farmacología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/aislamiento & purificación , Staphylococcus aureus/fisiología , Staphylococcus aureus/genética , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/fisiología , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/epidemiología , Portador Sano/microbiología , Irán , Masculino , Adulto , Femenino , Eritromicina/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Farmacorresistencia Bacteriana/genética

RESUMEN

Staphylococcus aureus is a notorious pathogen predominantly involved in skin and soft tissue infections, exhibiting a distinct innate sex bias. This study explores the influence of testosterone on the virulence of S. aureus and elucidates its underlying mechanisms. Utilizing a skin abscess model in intact and castrated male mice, we assessed the effects of testosterone on S. aureus pathogenicity. Compared to controls, castrated mice showed significantly reduced abscess sizes and decreased bacterial loads, highlighting the role of testosterone in modulating the severity of S. aureus infections. In vitro experiments revealed that testosterone enhances the hemolytic activity, cytotoxicity, and oxidative stress resistance of S. aureus. Real-time quantitative PCR analysis showed a significant upregulation of the genes encoding α-hemolysin (hla) and phenol-soluble modulin (psmα). Importantly, testosterone treatment significantly enhanced the expression of the accessory gene regulator (Agr) quorum-sensing system components (agrC, agrA, agrB, agrD), while the SaeRS system (saeR, saeS, and sbi) exhibited only slight changes. Gene knockout experiments revealed that deletion of agrC, rather than saeRS and agrBD, abolishes the testosterone-induced enhancement of hemolysis and gene expression, underscoring the key role of AgrC. Molecular docking simulations indicated a direct interaction between testosterone and AgrC protein, with a strong binding affinity at the active site residue SER201. This study provides new insights into the mechanistic basis of how testosterone enhances the pathogenicity of S. aureus, potentially contributing to increased male susceptibility to S. aureus infections and offering a targeted approach for therapeutic interventions.

Asunto(s)

Proteínas Bacterianas , Infecciones Estafilocócicas , Staphylococcus aureus , Testosterona , Masculino , Testosterona/farmacología , Testosterona/metabolismo , Animales , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/metabolismo , Ratones , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Virulencia , Infecciones Estafilocócicas/microbiología , Transactivadores/genética , Transactivadores/metabolismo , Regulación Bacteriana de la Expresión Génica , Percepción de Quorum , Simulación del Acoplamiento Molecular , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Absceso/microbiología , Hemólisis , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/genética

RESUMEN

Staphylococcus aureus acts both as a colonizing commensal bacterium and invasive pathogen. Nasal colonization is associated with an increased risk of infection caused by the identical strain. In patients with atopic dermatitis (AD), the degree of S. aureus colonization is associated with the severity of the disease. Here, we comparatively analyzed the in vivo transcriptional profile of S. aureus colonizing the nose and non-diseased skin (non-lesional skin) as opposed to the diseased skin (lesional skin-defined here as infection) of 12 patients with AD. The transcriptional profile during the asymptomatic colonization of the nose closely resembled that of the lesional skin samples for many of the genes studied, with an elevated expression of the genes encoding adhesion-related proteins and proteases. In addition, the genes that modify and remodel the cell wall and encode proteins that facilitate immune evasion showed increased transcriptional activity. Notably, in a subgroup of patients, the global virulence regulator Agr (accessory gene regulator) and downstream target genes were inactive during nasal colonization but upregulated in the lesional and non-lesional skin samples. Taken together, our results demonstrate a colonization-like transcriptional profile on diseased skin and suggest a role for the peptide quorum sensing system Agr during the transition from asymptomatic nasal colonization to skin colonization/infection.

Asunto(s)

Dermatitis Atópica , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Piel , Infecciones Estafilocócicas , Staphylococcus aureus , Dermatitis Atópica/microbiología , Dermatitis Atópica/genética , Humanos , Staphylococcus aureus/genética , Piel/microbiología , Piel/metabolismo , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/genética , Femenino , Masculino , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Adulto , Transcriptoma , Infecciones Cutáneas Estafilocócicas/microbiología , Infecciones Cutáneas Estafilocócicas/genética , Mucosa Nasal/microbiología , Transactivadores

RESUMEN

The RNA-based study provides an excellent indication of an organism's gene expression profile. Obtaining high-yield and high-purity RNA from Gram-positive and acid-fast bacteria is difficult without high-end kits and facilities. We optimised effective and simple protocol for RNA isolation that is a combination of enzymatic, physical and chemical treatment to disrupt cells. We successfully isolated high quality intact total RNA with yields ranging from 23.13 ± 0.40 to 61.51 ± 0.27 µg and the 260/280 purity ratio of 1.95 ± 0.01 to 2.05 ± 0.01 from Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Mycobacterium smegmatis. These results represents a significantly enhanced yield and purity compared to other combination of techniques which we performed. Compared to previous studies the yield obtained by this method is high for the studied organisms. Furthermore the yielded RNA was successfully used for downstream applications such as quantitative real time PCR. The described method can be easily optimised and used for various bacteria.

Asunto(s)

ARN Bacteriano , ARN Bacteriano/genética , ARN Bacteriano/aislamiento & purificación , Staphylococcus aureus/genética , Staphylococcus aureus/aislamiento & purificación , Bacterias Grampositivas/genética , Bacterias Grampositivas/aislamiento & purificación , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/aislamiento & purificación , Enterococcus faecalis/genética , Enterococcus faecalis/aislamiento & purificación , Mycobacterium smegmatis/genética

RESUMEN

The ESKAPE family, comprising Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., poses a significant global threat due to their heightened virulence and extensive antibiotic resistance. These pathogens contribute largely to the prevalence of nosocomial or hospital-acquired infections, resulting in high morbidity and mortality rates. To tackle this healthcare problem urgent measures are needed, including development of innovative vaccines and therapeutic strategies. Designing vaccines involves a complex and resource-intensive process of identifying protective antigens and potential vaccine candidates (PVCs) from pathogens. Reverse vaccinology (RV), an approach based on genomics, made this process more efficient by leveraging bioinformatics tools to identify potential vaccine candidates. In recent years, artificial intelligence and machine learning (ML) techniques has shown promise in enhancing the accuracy and efficiency of reverse vaccinology. This study introduces a supervised ML classification framework, to predict potential vaccine candidates specifically against ESKAPE pathogens. The model's training utilized biological and physicochemical properties from a dataset containing protective antigens and non-protective proteins of ESKAPE pathogens. Conventional autoencoders based strategy was employed for feature encoding and selection. During the training process, seven machine learning algorithms were trained and subjected to Stratified 5-fold Cross Validation. Random Forest and Logistic Regression exhibited best performance in various metrics including accuracy, precision, recall, WF1 score, and Area under the curve. An ensemble model was developed, to take collective strengths of both the algorithms. To assess efficacy of our final ensemble model, a high-quality benchmark dataset was employed. VacSol-ML(ESKAPE) demonstrated outstanding discrimination between protective vaccine candidates (PVCs) and non-protective antigens. VacSol-ML(ESKAPE), proves to be an invaluable tool in expediting vaccine development for these pathogens. Accessible to the public through both a web server and standalone version, it encourages collaborative research. The web-based and standalone tools are available at http://vacsolml.mgbio.tech/.

Asunto(s)

Antígenos Bacterianos , Vacunas Bacterianas , Aprendizaje Automático , Antígenos Bacterianos/inmunología , Humanos , Vacunas Bacterianas/inmunología , Klebsiella pneumoniae/inmunología , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/patogenicidad , Enterococcus faecium/inmunología , Enterococcus faecium/genética , Staphylococcus aureus/inmunología , Staphylococcus aureus/genética , Acinetobacter baumannii/inmunología , Pseudomonas aeruginosa/inmunología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Biología Computacional/métodos , Enterobacter/inmunología , Enterobacter/genética , Vacunología/métodos

RESUMEN

Detection methods based on CRISPR/Cas12a have been widely developed in the application of pathogenic microorganisms to guarantee food safety and public health. For sensitive detection, the CRISPR-based strategies are often in tandem with amplification methods. However, that may increase the detection time and the process may introduce nucleic acid contamination resulting in non-specific amplification. Herein, we established a sensitive S. aureus detection strategy based on the CRISPR/Cas12a system combined with DNAzyme. The activity of Cas12a is blocked by extending the spacer of crRNA (bcrRNA) and can be reactivated by Mn2+. NH2-modified S. aureus-specific aptamer was loaded on the surface of Fe3O4 MNPs (apt-Fe3O4 MNPs) and MnO2 NPs (apt-MnO2 NPs) by EDC/NHS chemistry. The S. aureus was captured to form apt-Fe3O4 MNPs/S. aureus/apt-MnO2 NPs complex and then MnO2 NPs were etched to release Mn2+ to activate DNAzyme. The active DNAzyme can cleave the hairpin structure in bcrRNA to recover the activity of the CRISPR/Cas system. By initiating the whole detection process by generating Mn2+ through nanoparticle etching, we established a rapid detection assay without nucleic acid extraction and amplification process. The proposed strategy has been applied in the ultrasensitive quantitative detection of S. aureus and has shown good performance with an LOD of 5 CFU/mL in 29 min. Besides, the proposed method can potentially be applied to other targets by simply changing the recognition element and has the prospect of developing a universal detection strategy.

Asunto(s)

Técnicas Biosensibles , Sistemas CRISPR-Cas , ADN Catalítico , Staphylococcus aureus , Staphylococcus aureus/aislamiento & purificación , Staphylococcus aureus/genética , ADN Catalítico/química , Técnicas Biosensibles/métodos , Límite de Detección , Compuestos de Manganeso/química , Humanos , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/diagnóstico , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/genética , Aptámeros de Nucleótidos/química , Óxidos/química , Endodesoxirribonucleasas

RESUMEN

Membrane vesicles (MVs) are produced by species across all domains of life and have diverse physiological functions as well as promising applications. While the mechanisms for vesiculation in Gram-negative bacteria are well-established, the genetic determinants and regulatory factors responsible for MV biogenesis in Gram-positive bacteria remain largely unknown. Here, we demonstrate that a Q225P substitution in the alternative sigma factor B (SigB) triggers MV production in Staphylococcus aureus strain Newman by hindering the specific binding of SigB to the asp23 promoter, thereby repressing expression of alkaline shock protein 23 (Asp23). Isogenic deletion of asp23 also promotes MV formation in Newman, confirming the critical roles played by sigB and asp23 in modulating S. aureus vesiculation. While bacterial growth and cytoplasmic membrane fluidity are not impaired, mutation of asp23 weakens the cell wall and enhances autolysis, consistent with decreased expression of alpha-type psm and lrgAB that modulate murein hydrolase activity. TEM and proteomic analysis show that Newman and asp23 deletion mutant generate MVs with nearly identical morphology and composition, but virulence-associated factors are significantly enriched in MVs from the asp23 mutant. Overall, this study reveals novel genetic determinants underlying S. aureus vesiculation and advances the understanding of the physiology of MV biogenesis in S. aureus.

Asunto(s)

Proteínas Bacterianas , Pared Celular , Staphylococcus aureus , Staphylococcus aureus/metabolismo , Staphylococcus aureus/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Pared Celular/metabolismo , Factor sigma/metabolismo , Factor sigma/genética , Regulación Bacteriana de la Expresión Génica , Vesículas Extracelulares/metabolismo

RESUMEN

Staphylococcus aureus infection and colonization in patients may be transmitted to healthcare providers and the environment and subsequently cause healthcare-associated infections in other patients. Pathogenic S. aureus strains produce virulence factors, such as Panton-Valentine Leukocidin (PVL), that contribute to the severity of infections and aid in their spread. The emergence of antimicrobial resistance (AMR) is additional concern with respect to S. aureus infection. In this study, the virulence genes and antibiotic resistance profiles of S. aureus were characterized from patients' clinical isolates, healthcare workers' (HCWs') nasal colonization screenings, and the environment at a tertiary healthcare hospital in Addis Ababa, Ethiopia. A total of 365 samples were collected from September 2021 to September 2022: 73 patients' clinical specimens, 202 colonization screenings from HCWs, and 90 hospital environment's swabs. Fifty-one (25.2%) HCW and 10/90 (11.1%) environment S. aureus isolates were identified. Among the 134 isolates, 10 (7.5%) were methicillin-resistant S. aureus (MRSA). Three (4.1%), five (9.8%), and two (20.0%) of the MRSA isolates were identified from the patients, HCWs, and the environment, respectively. Overall, 118 (88.1%) were ampicillin and penicillin resistant; 70 (52.2%) were trimethoprim sulfamethoxazole resistant; and 28 (20.9%) were erythromycin resistant. S. aureus isolates from patients were more resistant to antibiotics than isolates from HCWs or the hospital environment (p<0.05). A total of 92/134 (68.6%) isolates possessed the lukfF-PV gene, which was identified in 62 (85.0%), 26 (51.0%), and 4 (40.0%) of the patient, HCWs, and the environment, respectively. The proportion of lukfF-PV gene containing S. aureus isolated from patient samples was statistically significant. Four (40.0%) of the MRSA isolates also had the lukfF-PV gene. The identification of highly AMR and virulence factors from patients, HCWs and the environment is concerning. Further studies are needed to identify potential transmission links and improve infection prevention and control.

Asunto(s)

Antibacterianos , Personal de Salud , Infecciones Estafilocócicas , Staphylococcus aureus , Centros de Atención Terciaria , Humanos , Etiopía/epidemiología , Femenino , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/epidemiología , Infecciones Estafilocócicas/tratamiento farmacológico , Adulto , Masculino , Staphylococcus aureus/genética , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/aislamiento & purificación , Antibacterianos/farmacología , Persona de Mediana Edad , Pruebas de Sensibilidad Microbiana , Adolescente , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Adulto Joven , Factores de Virulencia/genética , Leucocidinas/genética , Niño , Exotoxinas/genética , Preescolar , Infección Hospitalaria/microbiología , Infección Hospitalaria/epidemiología , Farmacorresistencia Bacteriana/genética , Lactante , Anciano , Toxinas Bacterianas

RESUMEN

Foodborne illnesses caused by Salmonella and Staphylococcus aureus are a significant public health concern, leading to societal and economic repercussions. It is important to develop a simple and straightforward bacteria detection and identification method. A triple-probe multiplex rolling circle amplification technique has been developed to simultaneously detect Salmonella Typhimurium and S. aureus. This method utilizes fluorophore-labeled long padlock probes targeting S. Typhimurium invA and S. aureus glnA specific genes, along with a pH-based detection approach for direct visual identification. The multiplex hyperbranched saltatory rolling circle amplification assay at 30 °C has showed promising results with synthetic targets within 30 min and real bacteria within 2 h after establishing the detection settings. The assay is specific for S. aureus and S. Typhimurium, with a limit of detection of 39 µM for fluorescence and 78 µM for colorimetric. In the simulative test of this method for the detection of S. Typhimurium and S. aureus in milk, the limit of detection for the fluorescence signal after 2 h of amplification was 10 CFU/mL and 5 CFU/mL, respectively. The detection method was evaluated to be stable enough to detect pathogen for 3.29 months. Consequently, this triple-probe-multiplex rolling circle amplification method displays notable specificity, sensitivity, as well as ease of interpretation when testing food samples for harmful pathogens.

Asunto(s)

Microbiología de Alimentos , Técnicas de Amplificación de Ácido Nucleico , Salmonella typhimurium , Staphylococcus aureus , Técnicas de Amplificación de Ácido Nucleico/métodos , Staphylococcus aureus/aislamiento & purificación , Staphylococcus aureus/genética , Salmonella typhimurium/aislamiento & purificación , Salmonella typhimurium/genética , Microbiología de Alimentos/métodos , Leche/microbiología , Animales , Límite de Detección

RESUMEN

BACKGROUND: Burns are a problem that affects millions of individuals around the world. OBJECTIVE: This research aimed to analyze the genetic characteristics and antibiotic resistance of Staphylococcus aureus strains isolated from patients with burns. Identifying the genetic variations of three local strains of Staphylococcus aureus isolated from burns. MATERIALS AND METHODS: Swab samples were collected from eighty sources (burns). Using sterile swabs containing media collected from patients treated at Baqubah Teaching Hospital between July 2022 and the end of September 2022, these samples were then cultured on blood agar and brain heart infusion agar. A total of twenty-four hours were spent incubating the cultured samples in an aerobic environment at 37 °C. During this time, isolated growing colonies showed characteristic growth, color, and hemolysis, while suspicious colonies were cultured for further identification. RESULTS: Our results indicated the presence of several polymorphisms that were distributed in the investigated samples. However, almost all observed variations were concentrated only in the S2 isolates. The construction of phylogenetic trees confirmed this notion by positioning these S2-based amplicons to distinct categories within Staph. aureus organisms. Furthermore, the phylogenetic tree offered additional tools for the guaranteed identity of the samples that were analyzed. Consequently, the utilization of the PCR-sequencing approach in three DNA samples belonging to these local bacterial isolates has resulted in the confirmation of the identity of this strain. However, particular emphasis should be placed on S2 isolate as it has special variants that differ from its mates, in terms of its metabolic as well as phylogenetic consequences. Therefore, S2 isolates may represent a new strain that requires a whole genome sequencing strategy to validate its identity within Staph. aureus organisms. S.aureus resistance was 100% (Augmentin and Tetracycline), and 90% (Azithromycin and Trimethoprim), while Cefotaxime and Chloramphenicol recorded (75%, and 85%) respectively.

Asunto(s)

Antibacterianos , Quemaduras , Farmacorresistencia Bacteriana , Pruebas de Sensibilidad Microbiana , Filogenia , Infecciones Estafilocócicas , Staphylococcus aureus , Staphylococcus aureus/genética , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/aislamiento & purificación , Quemaduras/microbiología , Humanos , Antibacterianos/farmacología , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/tratamiento farmacológico , Farmacorresistencia Bacteriana/genética

RESUMEN

Infections caused by Staphylococcus aureus pose a significant global public problem. Therefore, new antibiotics and therapeutic strategies are needed to combat this pathogen. This investigation delves into the effects of iclaprim, a newly discovered inhibitor of folic acid synthesis, on S. aureus virulence. The phenotypic and genotypic effects of iclaprim were thoroughly examined in relation to virulence factors, biofilm formation, and dispersal, as well as partial virulence-encoding genes associated with exoproteins, adherence, and regulation in S. aureus MW2, N315, and ATCC 25923. Then, the in vivo effectiveness of iclaprim on S. aureus pathogenicity was explored by a Galleria mellonella larvae infection model. The use of iclaprim at sub-inhibitory concentrations (sub-MICs) resulted in a reduction of α-hemolysin (Hla) production and a differential effect on the activity of coagulase in S. aureus strains. The results of biofilm formation and eradication assay showed that iclaprim was highly effective in depolymerizing the mature biofilm of S. aureus strains at concentrations of 1 MIC or greater, however, inhibited the biofilm-forming ability of only strains N315 and ATCC 25923 at sub-MICs. Interestingly, treatment of strains with sub-MICs of iclaprim resulted in significant stimulation or suppression of most virulence-encoding genes expression. Iclaprim did not affect the production of δ-hemolysin or staphylococcal protein A (SpA), nor did it impact the total activity of proteases, nucleases, and lipases. In vivo testing showed that sub-MICs of iclaprim significantly improves infected larvae survival. The present study offered valuable insights towards a better understating of the influence of iclaprim on different strains of S. aureus. The findings suggest that iclaprim may have potential as an anti-virulence and antibiofilm agent, thus potentially mitigating the pathogenicity of S. aureus and improving clinical outcomes associated with infections caused by this pathogen. KEY POINTS: • Iclaprim effectively inhibits α-hemolysin production and biofilm formation in a strain-dependent manner and was an excellent depolymerizing agent of mature biofilm • Iclaprim affected the mRNA expression of virulence-encoding genes associated with exoproteins, adherence, and regulation • In vivo study in G. mellonella larvae challenged with S. aureus exhibited that iclaprim improves larvae survival.

Asunto(s)

Antibacterianos , Biopelículas , Larva , Pruebas de Sensibilidad Microbiana , Infecciones Estafilocócicas , Staphylococcus aureus , Factores de Virulencia , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/patogenicidad , Staphylococcus aureus/genética , Biopelículas/efectos de los fármacos , Animales , Factores de Virulencia/genética , Antibacterianos/farmacología , Virulencia/efectos de los fármacos , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/tratamiento farmacológico , Larva/microbiología , Mariposas Nocturnas/microbiología , Proteínas Hemolisinas/genética , Ácido Fólico/farmacología , Ácido Fólico/biosíntesis , Antagonistas del Ácido Fólico/farmacología , Coagulasa/metabolismo , Modelos Animales de Enfermedad , Pirimidinas

RESUMEN

Cellular processes require precise and specific gene regulation, in which continuous mRNA degradation is a major element. The mRNA degradation mechanisms should be able to degrade a wide range of different RNA substrates with high efficiency, but should at the same time be limited, to avoid killing the cell by elimination of all cellular RNA. RNase Y is a major endoribonuclease found in most Firmicutes, including Bacillus subtilis and Staphylococcus aureus. However, the molecular interactions that direct RNase Y to cleave the correct RNA molecules at the correct position remain unknown. In this work we have identified transcripts that are homologs in S. aureus and B. subtilis, and are RNase Y targets in both bacteria. Two such transcript pairs were used as models to show a functional overlap between the S. aureus and the B. subtilis RNase Y, which highlighted the importance of the nucleotide sequence of the RNA molecule itself in the RNase Y targeting process. Cleavage efficiency is driven by the primary nucleotide sequence immediately downstream of the cleavage site and base-pairing in a secondary structure a few nucleotides downstream. Cleavage positioning is roughly localised by the downstream secondary structure and fine-tuned by the nucleotide immediately upstream of the cleavage. The identified elements were sufficient for RNase Y-dependent cleavage, since the sequence elements from one of the model transcripts were able to convert an exogenous non-target transcript into a target for RNase Y.

Asunto(s)

Bacillus subtilis , Regulación Bacteriana de la Expresión Génica , División del ARN , Estabilidad del ARN , ARN Bacteriano , Staphylococcus aureus , Staphylococcus aureus/genética , Staphylococcus aureus/enzimología , Bacillus subtilis/genética , Bacillus subtilis/enzimología , Bacillus subtilis/metabolismo , ARN Bacteriano/metabolismo , ARN Bacteriano/genética , Estabilidad del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Endorribonucleasas/metabolismo , Endorribonucleasas/genética , Conformación de Ácido Nucleico , Secuencia de Bases

RESUMEN

Staphylococcus aureus is responsible for a substantial number of invasive infections globally each year. These infections are problematic because they are frequently recalcitrant to antibiotic treatment. Antibiotic tolerance, the ability of bacteria to persist despite normally lethal doses of antibiotics, contributes to antibiotic treatment failure in S. aureus infections. To understand how antibiotic tolerance is induced, S. aureus biofilms exposed to multiple anti-staphylococcal antibiotics are examined using both quantitative proteomics and transposon sequencing. These screens indicate that arginine metabolism is involved in antibiotic tolerance within a biofilm and support the hypothesis that depletion of arginine within S. aureus communities can induce antibiotic tolerance. Consistent with this hypothesis, inactivation of argH, the final gene in the arginine synthesis pathway, induces antibiotic tolerance. Arginine restriction induces antibiotic tolerance via inhibition of protein synthesis. In murine skin and bone infection models, an argH mutant has enhanced ability to survive antibiotic treatment with vancomycin, highlighting the relationship between arginine metabolism and antibiotic tolerance during S. aureus infection. Uncovering this link between arginine metabolism and antibiotic tolerance has the potential to open new therapeutic avenues targeting previously recalcitrant S. aureus infections.

Asunto(s)

Antibacterianos , Arginina , Biopelículas , Infecciones Estafilocócicas , Staphylococcus aureus , Arginina/metabolismo , Antibacterianos/farmacología , Animales , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Ratones , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Vancomicina/farmacología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Femenino , Farmacorresistencia Bacteriana/genética , Farmacorresistencia Bacteriana/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Hidrolasas/metabolismo , Hidrolasas/genética , Proteómica