RESUMO
Central metabolic pathways control virulence and antibiotic resistance, and constitute potential targets for antibacterial drugs. In Staphylococcus aureus the role of the pentose phosphate pathway (PPP) remains largely unexplored. Mutation of the 6-phosphogluconolactonase gene pgl, which encodes the only non-essential enzyme in the oxidative phase of the PPP, significantly increased MRSA resistance to ß-lactam antibiotics, particularly in chemically defined media with physiologically-relevant concentrations of glucose, and reduced oxacillin (OX)-induced lysis. Expression of the methicillin-resistance penicillin binding protein 2a and peptidoglycan architecture were unaffected. Carbon tracing and metabolomics revealed extensive metabolic reprogramming in the pgl mutant including increased flux to glycolysis, the TCA cycle, and several cell envelope precursors, which was consistent with increased ß-lactam resistance. Morphologically, pgl mutant cells were smaller than wild-type with a thicker cell wall and ruffled surface when grown in OX. The pgl mutation reduced resistance to Congo Red, sulfamethoxazole and oxidative stress, and increased resistance to targocil, fosfomycin and vancomycin. Levels of lipoteichoic acids (LTAs) were significantly reduced in pgl, which may limit cell lysis, while the surface charge of pgl cells was significantly more positive. A vraG mutation in pgl reversed the increased OX resistance phenotype, and partially restored wild-type surface charge, but not LTA levels. Mutations in vraF or graRS from the VraFG/GraRS complex that regulates DltABCD-mediated d-alanylation of teichoic acids (which in turn controls ß-lactam resistance and surface charge), also restored wild-type OX susceptibility. Collectively these data show that reduced levels of LTAs and OX-induced lysis combined with a VraFG/GraRS-dependent increase in cell surface positive charge are accompanied by significantly increased OX resistance in an MRSA pgl mutant.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/metabolismo , Via de Pentose Fosfato/genética , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Oxacilina/farmacologia , Parede Celular/metabolismo , Monobactamas/metabolismo , Resistência beta-Lactâmica/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Testes de Sensibilidade MicrobianaRESUMO
Prolonging the clinical effectiveness of ß-lactams, which remain first-line antibiotics for many infections, is an important part of efforts to address antimicrobial resistance. We report here that inactivation of the predicted d-cycloserine (DCS) transporter gene cycA resensitized methicillin-resistant Staphylococcus aureus (MRSA) to ß-lactam antibiotics. The cycA mutation also resulted in hypersusceptibility to DCS, an alanine analogue antibiotic that inhibits alanine racemase and d-alanine ligase required for d-alanine incorporation into cell wall peptidoglycan. Alanine transport was impaired in the cycA mutant, and this correlated with increased susceptibility to oxacillin and DCS. The cycA mutation or exposure to DCS were both associated with the accumulation of muropeptides with tripeptide stems lacking the terminal d-ala-d-ala and reduced peptidoglycan cross-linking, prompting us to investigate synergism between ß-lactams and DCS. DCS resensitized MRSA to ß-lactams in vitro and significantly enhanced MRSA eradication by oxacillin in a mouse bacteremia model. These findings reveal alanine transport as a new therapeutic target to enhance the susceptibility of MRSA to ß-lactam antibiotics.
Assuntos
Alanina/metabolismo , Antibacterianos/farmacologia , Ciclosserina/farmacologia , Resistência a Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , beta-Lactamas/farmacologia , Animais , Antimetabólitos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas Bacteriológicas , Transporte Biológico , Feminino , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Staphylococcus aureus Resistente à Meticilina/genética , Camundongos , Mutação , Polissacarídeos/química , Polissacarídeos/metabolismo , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologiaRESUMO
BACKGROUND: Clinical Microbiology is a core subject in medical undergraduate curricula. However, students struggle to cover the content and clinically contextualise basic microbiology. Our aim was to evaluate student engagement with new e-learning material and to investigate the impact it had on examination performance in a Clinical Microbiology module. METHODS: An online resource was designed to support didactic teaching in a Fundamentals of Clinical Microbiology module. One cohort of students had access to the online material (2017/2018 class) and the other did not (2016/2017 class). Each cohort sat the same multiple-choice question (MCQ) and short-note question (SNQ) examination papers and the impact of engagement with the online resource and examination performance was analysed. RESULTS: Both groups were of the same academic standard prior to beginning the module. In the 2017/2018 cohort, 227/309 (73.5%) students had ≥80% engagement with the content. Students engaged most with the index of pathogens and pathogen focused clinical cases related to diverse genera and families of clinically important microorganisms. A statistically higher difference in the mean percentage grade in both the MCQ and SNQ examinations was seen for 2017/2018 compared to 2016/2017 cohort. For the MCQ examination, the 2017/2018 cohort were on average 5.57% (95% confidence interval (CI): 3.92 to 7.24%; P < 0.001) higher, and for the SNQ examination the 2017/2018 cohort were on average 2.08% (95% CI: 0.74 to 3.41%; P = 0.02) higher. When the results were adjusted for previous examination performance, for every percentage increase in online engagement the grade in the SNQ examination only increased by 0.05% (95% CI: 0.02 to 0.08) on average. CONCLUSIONS: These findings suggest students engage with e-learning when studying and that such activities may help students perform better in assessments.
Assuntos
Competência Clínica , Instrução por Computador , Educação de Graduação em Medicina , Avaliação Educacional , Microbiologia/educação , Estudantes de Medicina , Educação de Graduação em Medicina/métodos , Feminino , Humanos , Internet , MasculinoRESUMO
Here, we demonstrate that antimicrobial peptides (AMPs) are an effective antibiofilm treatment when applied as catheter lock solutions (CLSs) against S. aureus biofilm infections. The activity of synthetic AMPs (Bac8c, HB43, P18, Omiganan, WMR, Ranalexin, and Polyphemusin) was measured against early and mature biofilms produced by methicillin-resistant S. aureus and methicillin-susceptible S. aureus isolates from patients with device-related infections grown under in vivo-relevant biofilm conditions. The cytotoxic and hemolytic activities of the AMPs against human cells and their immunomodulatory potential in human blood were also characterized. The D-Bac8c2,5Leu variant emerged as the most effective AMP during in vitro studies and was also highly effective in eradicating S. aureus biofilm infection when used in a CLS rat central venous catheter infection model. These data support the potential use of D-Bac8c2,5Leu, alone or in combination with other AMPs, in the treatment of S. aureus intravenous catheter infections.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Infecções Relacionadas a Cateter/tratamento farmacológico , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Peptídeos/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Animais , Citocinas/sangue , Modelos Animais de Doenças , Humanos , Testes de Sensibilidade Microbiana , Peptídeos Cíclicos/farmacologia , Ratos , Ratos Sprague-Dawley , Vancomicina/administração & dosagemRESUMO
Innovative approaches to the use of existing antibiotics is an important strategy in efforts to address the escalating antimicrobial resistance crisis. We report a new approach to the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections by demonstrating that oxacillin can be used to significantly attenuate the virulence of MRSA despite the pathogen being resistant to this drug. Using mechanistic in vitro assays and in vivo models of invasive pneumonia and sepsis, we show that oxacillin-treated MRSA strains are significantly attenuated in virulence. This effect is based primarily on the oxacillin-dependent repression of the accessory gene regulator quorum-sensing system and altered cell wall architecture, which in turn lead to increased susceptibility to host killing of MRSA. Our data indicate that ß-lactam antibiotics should be included in the treatment regimen as an adjunct antivirulence therapy for patients with MRSA infections. This would represent an important change to current clinical practice for treatment of MRSA infection, with the potential to significantly improve patient outcomes in a safe, cost-effective manner.
Assuntos
Antibacterianos/uso terapêutico , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Oxacilina/uso terapêutico , Infecções Estafilocócicas/tratamento farmacológico , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Modelos Animais de Doenças , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Resistência a Meticilina , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Camundongos , Testes de Sensibilidade Microbiana , Oxacilina/farmacologia , Pneumonia Estafilocócica/tratamento farmacológico , Pneumonia Estafilocócica/microbiologia , Percepção de Quorum/genética , Sepse/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Virulência/efeitos dos fármacosRESUMO
Objectives: Biofilm infections of intravascular catheters caused by Staphylococcus aureus may be treated with catheter lock solutions (CLSs). Here we investigated the antibacterial activity, cytotoxicity and CLS potential of 5-hydroxyethyl-3-tetradecanoyltetramic acid (5HE-C14-TMA) compared with the related compounds 3-tetradecanoyltetronic (C14-TOA) and 3-tetradecanoylthiotetronic (C14-TTA), which are variants of quorum sensing signalling molecules produced by Pseudomonas aeruginosa . Methods: Antibacterial activity and mechanism of action of 5HE-C14-TMA, C14-TOA and C14-TTA were determined via MIC, bacterial killing, membrane potential and permeability assays. Susceptibility of S. aureus biofilms formed in the presence of plasma in vitro was investigated, MTT cytotoxicity testing was undertaken and cytokine release in human blood upon exposure to 5HE-C14-TMA and/or S. aureus biofilms was quantified. The effectiveness of 5HE-C14-TMA as CLS therapy in vivo was assessed using a rat intravascular catheter biofilm infection model. Results: MICs of 5HE-C14-TMA, C14-TOA and C14-TTA ranged from 2 to 4 mg/L. 5HE-C14-TMA and C14-TTA were bactericidal; all three compounds perturbed the staphylococcal membrane by increasing membrane permeability, depolarized the transmembrane potential and caused ATP leakage. Cytotoxicity and haemolytic activity were compound and target cell type-dependent. 5HE-C14-TMA reduced S. aureus biofilm viability in a dose-dependent manner in vitro and in vivo and did not trigger release of cytokines in human blood, but inhibited the high levels of IL-8 and TNF-α induced by S. aureus biofilms. Conclusions: 5HE-C14-TMA, C14-TOA and C14-TTA are membrane-active agents. 5HE-C14-TMA was the most potent, eradicating S. aureus biofilms at 512-1024 mg/L both in vitro and in vivo as a CLS.
Assuntos
Antibacterianos/uso terapêutico , Infecções Relacionadas a Cateter/tratamento farmacológico , Pirrolidinonas/uso terapêutico , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Dispositivos de Acesso Vascular/microbiologia , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Infecções Relacionadas a Cateter/microbiologia , Descoberta de Drogas , Humanos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana , Pseudomonas aeruginosa/metabolismo , Pirrolidinonas/farmacologia , Percepção de Quorum , Ratos , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/fisiologia , Vancomicina/farmacologiaRESUMO
High-level resistance to antimicrobial drugs is a major factor in the pathogenesis of chronic Staphylococcus aureus biofilm-associated, medical device-related infections. Antimicrobial susceptibility analysis revealed that biofilms grown for ≤ 24 hours on biomaterials conditioned with human plasma under venous shear in iron-free cell culture medium were significantly more susceptible to antistaphylococcal antibiotics. Biofilms formed under these physiologically relevant conditions were regulated by SaeRS and dependent on coagulase-catalyzed conversion of fibrinogen into fibrin. In contrast, SarA-regulated biofilms formed on uncoated polystyrene in nutrient-rich bacteriological medium were mediated by the previously characterized biofilm factors poly-N-acetyl glucosamine, fibronectin-binding proteins, or autolytic activity and were antibiotic resistant. Coagulase-mediated biofilms exhibited increased antimicrobial resistance over time (>48 hours) but were always susceptible to dispersal by the fibrinolytic enzymes plasmin or nattokinase. Biofilms recovered from infected central venous catheters in a rat model of device-related infection were dispersed by nattokinase, supporting the important role of the biofilm phenotype and identifying a potentially new therapeutic approach with antimicrobials and fibrinolytic drugs, particularly during the early stages of device-related infection.
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Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Infecções Relacionadas a Cateter/microbiologia , Coagulase/metabolismo , Fibrinolíticos/metabolismo , Staphylococcus aureus/enzimologia , Staphylococcus aureus/fisiologia , Adulto , Animais , Antibacterianos/metabolismo , Infecções Relacionadas a Cateter/prevenção & controle , Modelos Animais de Doenças , Humanos , Ratos Sprague-Dawley , Staphylococcus aureus/efeitos dos fármacosRESUMO
Central metabolic pathways controls virulence and antibiotic resistance, and constitute potential targets for antibacterial drugs. In Staphylococcus aureus the role of the pentose phosphate pathway (PPP) remains largely unexplored. Mutation of the 6-phosphogluconolactonase gene pgl, which encodes the only non-essential enzyme in the oxidative phase of the PPP, significantly increased MRSA resistance to ß-lactam antibiotics, particularly in chemically defined media with glucose, and reduced oxacillin (OX)-induced lysis. Expression of the methicillin-resistance penicillin binding protein 2a and peptidoglycan architecture were unaffected. Carbon tracing and metabolomics revealed extensive metabolic reprogramming in the pgl mutant including increased flux to glycolysis, the TCA cycle, and several cell envelope precursors, which was consistent with increased ß-lactam resistance. Morphologically, pgl mutant cells were smaller than wild-type with a thicker cell wall and ruffled surface when grown in OX. Further evidence of the pleiotropic effect of the pgl mutation was reduced resistance to Congo Red, sulfamethoxazole and oxidative stress, and increased resistance to targocil, fosfomycin and vancomycin. Reduced binding of wheat germ agglutinin (WGA) to pgl was indicative of lower wall teichoic acid/lipoteichoic acid levels or altered teichoic acid structures. Mutations in the vraFG or graRS loci reversed the increased OX resistance phenotype and restored WGA binding to wild-type levels. VraFG/GraRS was previously implicated in susceptibility to cationic antimicrobial peptides and vancomycin, and these data reveal a broader role for this multienzyme membrane complex in the export of cell envelope precursors or modifying subunits required for resistance to diverse antimicrobial agents. Altogether our study highlights important roles for the PPP and VraFG/GraRS in ß-lactam resistance, which will support efforts to identify new drug targets and reintroduce ß-lactams in combination with adjuvants or other antibiotics for infections caused by MRSA and other ß-lactam resistant pathogens. Author summary: High-level resistance to penicillin-type (ß-lactam) antibiotics significantly limits the therapeutic options for patients with MRSA infections necessitating the use of newer agents, for which reduced susceptibility has already been described. Here we report for the first time that the central metabolism pentose phosphate pathway controls MRSA resistance to penicillin-type antibiotics. We comprehensively demonstrated that mutation of the PPP gene pgl perturbed metabolism in MRSA leading to increased flux to cell envelope precursors to drive increased antibiotic resistance. Moreover, increased resistance was dependent on the VraRG/GraRS multienzyme membrane complex previously implicated in resistance to antimicrobial peptides and vancomycin. Our data thus provide new insights on MRSA mechanisms of ß-lactam resistance, which will support efforts to expand the treatment options for infections caused by this and other antimicrobial resistant pathogens.
RESUMO
SARS-CoV-2 vaccination promises to improve outcomes for patients with COVID-19 pneumonia (most notably those with advanced age and at high risk for severe disease). Here, we examine serum 25-Hydroxyvitamin D (25(OH)D) status and outcomes in both old (>70 years) and young vaccinated (n = 80) and unvaccinated (n = 91) subjects, who were hospitalized due to COVID-19 pneumonia in a single center (Connolly Hospital Dublin). Outcomes included ICU admission and mortality. Serum 25(OH)D levels were categorized as D30 (<30 nmol/L), D40 (30-49.99 nmol/L) and D50 (≥50 nmol/L). In multivariate analyses, D30 was independently associated with ICU admission (OR: 6.87 (95% CI: 1.13-41.85) (p = 0.036)) and mortality (OR: 24.81 (95% CI: 1.57-392.1) (p = 0.023)) in unvaccinated patients, even after adjustment for major confounders including age, sex, obesity and pre-existing diabetes mellitus. While mortality was consistently higher in all categories of patients over 70 years of age, the highest observed mortality rate of 50%, seen in patients over 70 years with a low vitamin D state (D30), appeared to be almost completely corrected by either vaccination, or having a higher vitamin D state, i.e., mortality was 14% for vaccinated patients over 70 years with D30 and 16% for unvaccinated patients over 70 years with a 25(OH)D level greater than 30 nmol/L. We observe that high mortality from COVID-19 pneumonia occurs in older patients, especially those who are unvaccinated or have a low vitamin D state. Recent vaccination or having a high vitamin D status are both associated with reduced mortality, although these effects do not fully mitigate the mortality risk associated with advanced age.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Humanos , Idoso , Idoso de 80 Anos ou mais , SARS-CoV-2 , COVID-19/prevenção & controle , Vitamina D , Vitaminas , Hospitais , VacinaçãoRESUMO
INTRODUCTION: Sepsis is a life-threatening organ dysfunction that is caused by a dysregulated host response to the infection. Urosepsis contributes up to 25% of all sepsis cases. An important part of the management of urosepsis is to rule out possible surgical causes such as urolithiasis, obstructive uropathy, or abscess formation along the urogenital tract. OBJECTIVE: The aim of this study is to look at whether urological conditions and recent urological surgery contribute significantly to all patients admitted with urosepsis. METHODS: A total of 2679 urine cultures and 654 blood cultures performed in Connolly Hospital Emergency Department were reviewed between 2016 and 2018. Patients were included if they had a matching urine culture and blood culture performed within 24âhours of admission. A retrospective chart review was performed for all patients included in the study. RESULTS: Our study included 85 patients admitted with urosepsis between 2016 and 2018. The average age was 70.3âyears (21-100âyears), in which 61% (n = 52) of patients were female, 18% (n = 16) had a long-term indwelling catheter, 11.8% (n = 10) were admitted as urosepsis with a urological condition. The most common urological condition predisposing patients to urosepsis in this study was bladder outlet obstruction secondary to benign prostatic hyperplasia. A total of 4.7% (n = 4) of patients died during their admission. The complications as a result of urosepsis included a prostatic abscess, a psoas abscess, an ileus, an infected cyst, and 1 case of emphysematous pyelonephritis. CONCLUSION: In this study, the majority of patients admitted with urosepsis did not have an underlying urological condition or recent urological instrumentation. Clinicians should be aware of potential complications as a result of a urosepsis.
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INTRODUCTION: Our hospital found itself at the epicentre of the Irish COVID-19 pandemic. We describe the organisational challenges faced in managing the surge and identified risk factors for mortality and ICU admission among hospitalised SARS-CoV-2-infected patients. METHODS: All hospitalised SARS-CoV-2 patients diagnosed between March 13 and May 1, 2020, were included. Demographic, referral, deprivation, ethnicity and clinical data were recorded. Multivariable regression, including age-adjusted hazard ratios (HR (95% CI), was used to explore risk factors associated with adverse outcomes. RESULTS: Of 257 inpatients, 174 were discharged (68%) and 39 died (15%) in hospital. Two hundred three (79%) patients presented from the community, 34 (13%) from care homes and 20 (8%) were existing inpatients. Forty-five percent of community patients were of a non-Irish White or Black, Asian or minority ethnic (BAME) population, including 34 Roma (13%) compared to 3% of care home and 5% of existing inpatients, (p < 0.001). Twenty-two patients were healthcare workers (9%). Of 31 patients (12%) requiring ICU admission, 18 were discharged (58%) and 7 died (23%). Being overweight/obese HR (95% CI) 3.09 (1.32, 7.23), p = 0.009; a care home resident 2.68 (1.24, 5.6), p = 0.012; socioeconomically deprived 1.05 (1.01, 1.09), p = 0.012; and older 1.04 (1.01, 1.06), p = 0.002 were significantly associated with death. Non-Irish White or BAME were not significantly associated with death 1.31 (0.28, 6.22), p = 0.63 but were significantly associated with ICU admission 4.38 (1.38, 14.2), p = 0.014 as was being overweight/obese 2.37 (1.37, 6.83), p = 0.01. CONCLUSION: The COVID-19 pandemic posed unprecedented organisational issues for our hospital resulting in the greatest surge in ICU capacity above baseline of any Irish hospital. Being overweight/obese, a care home resident, socioeconomically deprived and older were significantly associated with death, while ethnicity and being overweight/obese were significantly associated with ICU admission.
Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/mortalidade , Feminino , Mortalidade Hospitalar , Hospitalização , Hospitais , Humanos , Irlanda , Masculino , Pandemias , Fatores de RiscoRESUMO
BACKGROUND AND AIMS: Urosepsis accounts for up to 20-30% of all sepsis cases; however, increasing antimicrobial resistance is posing a significant threat to patient's outcomes. The aim of this study was to look at the prevalence of multi-drug resistant (MDR) organisms in patients admitted with urosepsis and their effect on the treatment and outcome of patients in our hospital. METHODS: A total of 2679 urine cultures and 654 blood cultures performed in Connolly Hospital Emergency Department were reviewed between 2016 and 2018. Patients were included if they had a matching urine culture and blood culture performed within 24 h of admission. We compared patient demographics and underlying co-morbidities between patients admitted with urosepsis secondary to MDR organisms and non-MDR organisms. RESULTS: Our study included 85 patients admitted with urosepsis. The most common causative pathogen was Escherichia coli, and 34.1% (n = 29) of pathogens were classified as an MDR organism. Patients admitted with urosepsis from long-term care facilities were 2.3 times more likely to have urosepsis due to a MDR organism compared with patients admitted from the community. Patients admitted with urosepsis secondary to a MDR organism were also more likely to have co-morbidities such as diabetes and dementia. CONCLUSION: The high rate of antimicrobial resistance in patients admitted with urosepsis poses a challenge in prescribing the most appropriate antibiotics. It is crucial that prescribers follow local antibiotic guidelines for the treatment of urosepsis and are cognisant of the risk of specific patient groups presenting with urosepsis due to MDR organisms.
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Antibacterianos/uso terapêutico , Sepse/tratamento farmacológico , Idoso , Antibacterianos/farmacologia , Feminino , Humanos , Masculino , Prevalência , Estudos RetrospectivosRESUMO
PURPOSE OF REVIEW: Healthcare-associated or nosocomial infection (HCAI) is of increasing importance to healthcare providers and the public. Surveillance is crucial but must be adjusted for risk, especially when used for interhospital comparisons or for public reporting. RECENT FINDINGS: Surveillance data are increasingly adjusted for risk factors for HCAI if meaningful comparisons are made between institutions or across national boundaries. Postdischarge surveillance is important in detecting those infections that may not present to the institution in which the original procedure occurred. Caution is urged when comparing data from two sources, for example, an active surveillance program and administrative datasets. The public reporting of HCAI rates can assist in improving the quality of healthcare, but to date there is little evidence that this is happening. In the United States, a number of states have introduced mandatory reporting of HCAIs, but there is considerable variation in what data are released, how these are reported and the rigor of the validation of the dataset. SUMMARY: The consumerization of healthcare requires a response from healthcare providers to engage with the public on how, when and what risk-adjusted surveillance data to release. Information campaigns are required to ensure the public understand any publicly released data and its limitations.
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Infecção Hospitalar/epidemiologia , Infecção Hospitalar/prevenção & controle , Instalações de Saúde , Vigilância de Evento Sentinela , Notificação de Doenças , Humanos , Risco Ajustado , Estados UnidosRESUMO
The Staphylococcus aureus FnBPA and FnBPB proteins promote acid-induced biofilm accumulation. Meticillin-resistant S. aureus (MRSA) isolates from device-related infections with both fnbA and fnbB produced significantly more biofilm than isolates with either gene alone. Under mildly acidic growth conditions, FnBP-mediated biofilm and fnbA and fnbB transcript levels were substantially higher during growth at 37 degrees C than at 30 degrees C. Thus, in addition to a lowered pH, carriage of both fnbA and fnbB and growth at 37 degrees C promote MRSA biofilm development, further supporting a role for the FnBPA and FnBPB surface proteins in the pathogenesis of MRSA device-related infections.
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Adesinas Bacterianas/genética , Biofilmes , Regulação Bacteriana da Expressão Gênica/fisiologia , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Infecções Relacionadas à Prótese/microbiologia , Estudos Retrospectivos , Infecções Estafilocócicas/microbiologia , TemperaturaRESUMO
Prevention of device related infections due to Staphylococcus aureus biofilms on devices represents a significant challenge. Such infections have recently been shown to be dependent on the coagulation pathway via activation of pro-thrombin and fibrin production. Three direct-thrombin inhibitors, argatroban, hirudin and dabigatran, were examined to determine their effect on preventing S. aureus biofilm on plastic biochip surfaces under shear stress using an in vivo relevant model of infection. Surface functionalization of polyurethane discs via dityrosine covalent crosslinking with hirudin was performed and changes in bacterial density and microscopic appearances determined. The three direct-thrombin inhibitors prevented S. aureus biofilm formation on plasma-coated surfaces treated with these agents. Coating of polyurethane with one of these agents, hirudin, significantly inhibited biofilm formation on the modified surface. These findings reveal the exciting potential for coating biomaterial surfaces with direct thrombin inhibitors to prevent staphylococcal binding and subsequent device-related infections.
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Antibacterianos/farmacologia , Antitrombinas/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Materiais Revestidos Biocompatíveis , Equipamentos e Provisões/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Humanos , Microscopia Eletrônica de Varredura , Poliuretanos/química , Poliuretanos/farmacologia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/ultraestrutura , Propriedades de Superfície , Trombina/metabolismoRESUMO
Device-associated infections involving biofilm remain a persistent clinical problem. We recently reported that four methicillin-resistant Staphylococcus aureus (MRSA) strains formed biofilm independently of the icaADBC-encoded exopolysaccharide. Here, we report that MRSA biofilm development was promoted under mildly acidic growth conditions triggered by the addition of glucose to the growth medium. Loss of sortase, which anchors LPXTG-containing proteins to peptidoglycan, reduced the MRSA biofilm phenotype. Furthermore introduction of mutations in fnbA and fnbB, which encode the LPXTG-anchored multifunctional fibrinogen and fibronectin-binding proteins, FnBPA and FnBPB, reduced biofilm formation by several MRSA strains. However, these mutations had no effect on biofilm formation by methicillin-sensitive S. aureus strains. FnBP-promoted biofilm occurred at the level of intercellular accumulation and not primary attachment. Mutation of fnbA or fnbB alone did not substantially affect biofilm, and expression of either gene alone from a complementing plasmid in fnbA fnbB mutants restored biofilm formation. FnBP-promoted biofilm was dependent on the integrity of SarA but not through effects on fnbA or fnbB transcription. Using plasmid constructs lacking regions of FnBPA to complement an fnbAB mutant revealed that the A domain alone and not the domain required for fibronectin binding could promote biofilm. Additionally, an A-domain N304A substitution that abolished fibrinogen binding did not affect biofilm. These data identify a novel S. aureus biofilm phenotype promoted by FnBPA and FnBPB which is apparently independent of the known ligand-binding activities of these multifunctional surface proteins.
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Adesinas Bacterianas/fisiologia , Biofilmes/crescimento & desenvolvimento , Staphylococcus aureus/fisiologia , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Antibacterianos/farmacologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Concentração de Íons de Hidrogênio , Resistência a Meticilina , Fenótipo , Estrutura Terciária de Proteína , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Fatores de TempoRESUMO
PURPOSE: The treatment of device-related infections is challenging and current anti-microbial compounds have poor anti-biofilm activity. We aimed to identify and characterize novel compounds effective in the eradication of Staphylococcus aureus biofilms. METHODOLOGY: Two novel compounds, MMV665953 {1-(3-chloro-4-fluorophenyl)-3-(3,4-dichlorophenyl)urea} and MMV665807{5-chloro-2-hydroxy-N-[3-(trifluoromethyl)phenyl]benzamide}, effective in killing S. aureus biofilms, were identified by screening of the open access 'malaria box' chemical library. The minimum bactericidal concentrations, half-maximal inhibition concentration (IC50) values and minimal biofilm killing concentrations effective in the killing of biofilm were determined against meticillin-resistant S. aureus and meticillin-sensitive S. aureus. Fibrin-embedded biofilms were grown under in vivo-relevant conditions, and viability was measured using a resazurin-conversion assay and confocal microscopy. The potential for the development of resistance and cytotoxicity was also assessed. RESULTS: MMV665953 and MMV665807 were bactericidal against S. aureus isolates. The IC50 against S. aureus biofilms was at 0.15-0.58 mg l-1 after 24 h treatment, whereas the concentration required to eradicate all tested biofilms was 4 mg l-1, making the compounds more bactericidal than conventional antibiotics. The cytotoxicity against human keratinocytes and primary endothelial cells was determined as IC50 7.47 and 0.18 mg l-1 for MMV665953, and as 1.895 and 0.076 mg l-1 for MMV665807. Neither compound was haemolytic nor caused platelet activation. MMV665953 and MMV665807 derivatives with reduced cytotoxicity exhibited a concomitant loss in anti-staphylococcal activity. CONCLUSION: MMV665953 and MMV665807 are more bactericidal against S. aureus biofilms than currently used anti-staphylococcal antibiotics and represent a valuable structural basis for further investigation in the treatment of staphylococcal biofilm-related infections.
Assuntos
Antimaláricos/farmacologia , Benzamidas/farmacologia , Biofilmes/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacosRESUMO
The major Staphylococcus aureus autolysin, Atl, has been implicated in attachment to surfaces and release of extracellular DNA during biofilm formation under laboratory conditions. Consistent with this, polyclonal antibodies to the amidase and glucosaminidase domains of Atl inhibited in vitro biofilm formation. However, in a murine model of device-related infection the community-associated S. aureus strain USA300 LAC JE2 established a successful infection in the absence of atl These data indicate that Atl activity is not required for biofilm production in this infection model and reveal the importance of characterizing the contribution of biofilm phenotypes to virulence under in vivo conditions.
Assuntos
Biofilmes , Infecções Relacionadas a Cateter/microbiologia , N-Acetil-Muramil-L-Alanina Amidase/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/patogenicidade , Animais , Modelos Animais de Doenças , Camundongos , Staphylococcus aureus/genética , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismo , VirulênciaRESUMO
Antibiotic resistance and biofilm-forming capacity contribute to the success of Staphylococcus aureus as a human pathogen in both healthcare and community settings. These virulence factors do not function independently of each other and the biofilm phenotype expressed by clinical isolates of S. aureus is influenced by acquisition of the methicillin resistance gene mecA. Methicillin-sensitive S. aureus (MSSA) strains commonly produce an icaADBC operon-encoded polysaccharide intercellular adhesin (PIA)-dependent biofilm. In contrast, the release of extracellular DNA (eDNA) and cell surface expression of a number of sortase-anchored proteins, and the major autolysin have been implicated in the biofilm phenotype of methicillin-resistant S. aureus (MRSA) isolates. Expression of high level methicillin resistance in a laboratory MSSA strain resulted in (i) repression of PIA-mediated biofilm production, (ii) down-regulation of the accessory gene regulator (Agr) system, and (iii) attenuation of virulence in murine sepsis and device infection models. Here we review the mechanisms of MSSA and MRSA biofilm production and the relationships between antibiotic resistance, biofilm and virulence gene regulation in S. aureus.