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Staphylococcus epidermidis has emerged as an important opportunistic pathogen causing orthopedic-device-related infections (ODRI). This study investigated the association of genome variation and phenotypic features of the infecting S. epidermidis isolate with the clinical outcome for the infected patient. S. epidermidis isolates were collected from 104 patients with ODRI. Their clinical outcomes were evaluated, after an average of 26 months, as either "cured" or "not cured." The isolates were tested for antibiotic susceptibility and biofilm formation. Whole-genome sequencing was performed on all isolates, and genomic variation was related to features associated with "cured" and "not cured." Strong biofilm formation and aminoglycoside resistance were associated with a "not-cured" outcome (P = 0.031 and P < 0.001, respectively). Based on gene-by-gene analysis, some accessory genes were more prevalent in isolates from the "not-cured" group. These included the biofilm-associated bhp gene, the antiseptic resistance qacA gene, the cassette chromosome recombinase-encoding genes ccrA and ccrB, and the IS256-like transposase gene. This study identifies biofilm formation and antibiotic resistance as associated with poor outcome in S. epidermidis ODRI. Whole-genome sequencing identified specific genes associated with a "not-cured" outcome that should be validated in future studies. (The study has been registered at ClinicalTrials.gov with identifier NCT02640937.).
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Antibacterianos/uso terapêutico , Biofilmes/crescimento & desenvolvimento , Genoma Bacteriano/genética , Equipamentos Ortopédicos/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus epidermidis/genética , Aminoglicosídeos/uso terapêutico , Articulação do Tornozelo/microbiologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Fêmur/microbiologia , Fíbula/microbiologia , Articulação do Quadril/microbiologia , Humanos , Articulação do Joelho/microbiologia , Meticilina/farmacologia , Resistência a Meticilina/genética , Testes de Sensibilidade Microbiana , Estudos Prospectivos , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/isolamento & purificação , Tíbia/microbiologia , Transativadores/genética , Resultado do TratamentoRESUMO
Staphylococcus epidermidis is a common cause of biomedical device-associated infections. Agr is the major quorum sensing system in staphylococci and regulates virulence factors. Four agr-specificity groups exist in S. epidermidis, and chronic S. epidermidis infections are hypothesised to select for agr-negative phenotypes. Therefore, we investigated S. epidermidis strains from prosthetic joint- and catheter-associated infections to establish i) whether an infection selects for an agr-negative phenotype; ii) the importance of PSMγ and iii) if the agr-specificity group is infection dependent. S. epidermidis nasal isolates from healthy volunteers were used as controls. The distribution of agr-specificity groups was significantly different between infection and control episodes, but did not distinguish between the infection types. PSMγ secretion was used to determine agr-activity and HPLC analysis showed that 44% of prosthetic and 32% of catheter-associated episodes produced no PSMγ in comparison to 8% of the control strains. However, PSMγ expression did not always correlate with RNAIII up-regulation, indicating that PSMγ synthesis is likely influenced by additional post-transcriptional control. The data suggests chronic S. epidermidis infections favour agr-specificity group 1 but the results suggest that they do not select for an agr-negative phenotype. Further studies are required to explore the mechanisms underlying the selection and survival of these S. epidermidis phenotypes isolated from biomedical device-associated infections.
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Biofilmes/crescimento & desenvolvimento , Infecções Relacionadas a Cateter/microbiologia , Infecções Relacionadas à Prótese/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/fisiologia , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Toxinas Bacterianas/análise , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Biomarcadores , RNA Bacteriano/análise , RNA Bacteriano/genética , RNA Bacteriano/fisiologia , Staphylococcus epidermidis/patogenicidadeRESUMO
BACKGROUND: A major complication of using medical devices is the development of biofilm-associated infection caused by Staphylococcus epidermidis where polysaccharide intercellular adhesin (PIA) is a major mechanism of biofilm accumulation. PIA affects innate and humoral immunity in isolated cells and animal models. Few studies have examined these effects in prosthetic joint infection (PJI). METHODS: This study used ex vivo whole blood modelling in controls together with matched-serum and staphylococcal isolates from patients with PJI. RESULTS: Whole blood killing of PIA positive S. epidermidis and its isogenic negative mutant was identical. Differences were unmasked in immunosuppressed whole blood pre-treated with dexamethasone where PIA positive bacteria showed a more resistant phenotype. PIA expression was identified in three unique patterns associated with bacteria and leukocytes, implicating a soluble form of PIA. Purified PIA reduced whole blood killing while increasing C5a levels. In clinically relevant staphylococcal isolates and serum samples from PJI patients; firstly complement C5a was increased 3-fold compared to controls; secondly, the C5a levels were significantly higher in serum from PJI patients whose isolates preferentially formed PIA-associated biofilms. CONCLUSIONS: These data demonstrate for the first time that the biological effects of PIA are mediated through C5a in patients with PJI.
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Artrite/microbiologia , Atividade Bactericida do Sangue , Complemento C5a/metabolismo , Interações Hospedeiro-Patógeno , Polissacarídeos Bacterianos/metabolismo , Infecções Relacionadas à Prótese/microbiologia , Staphylococcus epidermidis/fisiologia , Humanos , Staphylococcus epidermidis/crescimento & desenvolvimento , Staphylococcus epidermidis/isolamento & purificação , Staphylococcus epidermidis/metabolismoRESUMO
Whole blood models are rapid and versatile for determining immune responses to inflammatory and infectious stimuli, but they have not been used for bacterial discrimination. Staphylococcus aureus, S. epidermidis and Escherichia coli are the most common causes of invasive disease, and rapid testing strategies utilising host responses remain elusive. Currently, immune responses can only discriminate between bacterial 'domains' (fungi, bacteria and viruses), and very few studies can use immune responses to discriminate bacteria at the species and strain level. Here, whole blood was used to investigate the relationship between host responses and bacterial strains. Results confirmed unique temporal profiles for the 10 parameters studied: IL-6, MIP-1α, MIP-3α, IL-10, resistin, phagocytosis, S100A8, S100A8/A9, C5a and TF3. Pairwise analysis confirmed that IL-6, resistin, phagocytosis, C5a and S100A8/A9 could be used in a discrimination scheme to identify to the strain level. Linear discriminant analysis (LDA) confirmed that (i) IL-6, MIP-3α and TF3 could predict genera with 95% accuracy; (ii) IL-6, phagocytosis, resistin and TF3 could predict species at 90% accuracy and (iii) phagocytosis, S100A8 and IL-10 predicted strain at 40% accuracy. These data are important because they confirm the proof of concept that host biomarker panels could be used to identify bacterial pathogens.
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The exposure of human lung and skin to carbon black (CB) is continuous due to its widespread applications. Current toxicological testing uses 'healthy' cellular systems; however, questions remain whether this mimics the everyday stresses that human cells are exposed to, including infection. Staphylococcus aureus lung and skin infections remain prevalent in society, and include pneumonia and atopic dermatitis, respectively, but current in vitro toxicological testing does not consider infection stress. Therefore, investigating the effects of CB co-exposure in 'stressed' infected epithelial cells in vitro may better approximate true toxicity. This work aims to study the impact of CB exposure during Staphylococcus aureus infection stress in A549 (lung) and HaCaT (skin) epithelial cells. Physicochemical characterisation of CB confirmed its dramatic polydispersity and potential to aggregate. CB significantly inhibited S. aureus growth in cell culture media. CB did not induce cytokines or antimicrobial peptides from lung and skin epithelial cells, when given alone, but did reduce HaCaT and A549 cell viability to 55% and 77%, respectively. In contrast, S. aureus induced a robust interleukin (IL)-8 response in both lung and skin epithelial cells. IL-6 and human beta defensin (hßD)-2 could only be detected when cells were stimulated with S. aureus with no decreases in cell viability. However, co-exposure to CB (100 µg/mL) and S. aureus resulted in significant inhibition of IL-8 (compared to S. aureus alone) without further reduction in cell viability. Furthermore, the same co-exposure induced significantly more hßD-2 (compared to S. aureus alone). This work confirms that toxicological testing in healthy versus stressed cells gives significantly different responses. This has significant implications for toxicological testing and suggests that cell stresses (including infection) should be included in current models to better represent the diversity of cell viabilities found in lung and skin within a general population. This model will have significant application when estimating CB exposure in at-risk groups, such as factory workers, the elderly, and the immunocompromised.
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Staphylococcus aureus and Staphylococcus epidermidis biofilms cause chronic infections due to their ability to form biofilms. The excretions/secretions of Lucilia sericata larvae (maggots) have effective activity for debridement and disruption of bacterial biofilms. In this paper, we demonstrate how chymotrypsin derived from maggot excretions/secretions disrupts protein-dependent bacterial biofilm formation mechanisms.
Assuntos
Adesinas Bacterianas/metabolismo , Biofilmes/efeitos dos fármacos , Quimotripsina/metabolismo , Dípteros/enzimologia , Staphylococcus aureus/fisiologia , Staphylococcus epidermidis/fisiologia , Animais , Larva/enzimologiaRESUMO
Fifty-six α-hemolytic streptococcal isolates were identified using MALDI Biotyper MS (Bruker Daltonics), API 20 Strep (bioMérieux), and BD Phoenix (Becton, Dickinson). The gold standard for identification was 16S rRNA gene sequence analysis with 16S-23S rRNA intergenic spacer sequencing. The following percentages of isolates were correctly identified to the species level: MALDI Biotyper, 46%; BD Phoenix, 35%; and API 20 Strep, 26%.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Análise de Sequência de DNA/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Infecções Estreptocócicas/diagnóstico , Infecções Estreptocócicas/microbiologia , Streptococcus/classificação , Streptococcus/isolamento & purificação , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Intergênico/química , DNA Intergênico/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Humanos , RNA Ribossômico 16S/genética , Streptococcus/química , Streptococcus/genéticaRESUMO
BACKGROUND: The skin commensal and opportunistic pathogen Staphylococcus epidermidis is a leading cause of hospital-acquired and biomaterial-associated infections. The polysaccharide intercellular adhesin (PIA), a homoglycan composed of ß-1,6-linked N-acetylglucosamine residues, synthesized by enzymes encoded in icaADBC is a major functional factor in biofilm accumulation, promoting virulence in experimental biomaterial-associated S. epidermidis infection. Extracellular mucous layer extracts of S. epidermidis contain another major polysaccharide, referred to as 20-kDa polysaccharide (20-kDaPS), composed mainly out of glucose, N-acetylglucosamine, and being partially sulfated. 20-kDaPS antiserum prevents adhesion of S. epidermidis on endothelial cells and development of experimental keratitis in rabbits. Here we provide experimental evidence that 20-kDaPS and PIA represent distinct molecules and that 20-kDaPS is implicated in endocytosis of S. epidermidis bacterial cells by human monocyte-derived macrophages. RESULTS: Analysis of 75 clinical coagulase-negative staphylococci from blood-cultures and central venous catheter tips indicated that 20-kDaPS is expressed exclusively in S. epidermidis but not in other coagulase-negative staphylococcal species. Tn917-insertion in various locations in icaADBC in mutants M10, M22, M23, and M24 of S. epidermidis 1457 are abolished for PIA synthesis, while 20-kDaPS expression appears unaltered as compared to wild-type strains using specific anti-PIA and anti-20-kDaPS antisera. While periodate oxidation and dispersin B treatments abolish immuno-reactivity and intercellular adhesive properties of PIA, no abrogative activity is exerted towards 20-kDaPS immunochemical reactivity following these treatments. PIA polysaccharide I-containing fractions eluting from Q-Sepharose were devoid of detectable 20-kDaPS using specific ELISA. Preincubation of non-20-kDaPS-producing clinical strain with increasing amounts of 20-kDaPS inhibits endocytosis by human macrophages, whereas, preincubation of 20-kDaPS-producing strain ATCC35983 with 20-kDaPS antiserum enhances bacterial endocytosis by human macrophages. CONCLUSIONS: In conclusion, icaADBC is not involved in 20-kDaPS synthesis, while the chemical and chromatographic properties of PIA and 20-kDaPS are distinct. 20-kDaPS exhibits anti-phagocytic properties, whereas, 20-kDaPS antiserum may have a beneficial effect on combating infection by 20-kDaPS-producing S. epidermidis.
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Macrófagos/imunologia , Macrófagos/microbiologia , Fagocitose , Polissacarídeos Bacterianos/metabolismo , Staphylococcus epidermidis/imunologia , Staphylococcus epidermidis/metabolismo , Células Cultivadas , Elementos de DNA Transponíveis , Perfilação da Expressão Gênica , Humanos , Peso Molecular , Mutagênese Insercional , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/genética , Polissacarídeos Bacterianos/imunologiaRESUMO
Staphylococcus epidermidis is found naturally on the skin but is a common cause of persistent orthopaedic device-related infections (ODRIs). This study used a pan-genome and gene-by-gene approach to analyse the clonality of whole genome sequences (WGS) of 115 S. epidermidis isolates from 55 patients with persistent ODRIs. Analysis of the 522 gene core genome revealed that the isolates clustered into three clades, and MLST analysis showed that 83% of the isolates belonged to clonal complex 2 (CC2). Analysis also found 13 isolate pairs had different MLST types and less than 70% similarity within the genes; hence, these were defined as re-infection by a different S. epidermidis strain. Comparison of allelic diversity in the remaining 102 isolates (49 patients) revealed that 6 patients had microevolved infections (>7 allele differences), and only 37 patients (77 isolates) had a 'true' persistent infection. Analysis of the core genomes of isolate pairs from 37 patients found 110/841 genes had variations; mainly in metabolism associated genes. The accessory genome consisted of 2936 genes; with an average size of 1515 genes. To conclude, this study demonstrates the advantage of using WGS for identifying the accuracy of a persistent infection diagnosis. Hence, persistent infections can be defined as 'true' persistent infections if the core genome of paired isolates has ≤7 allele differences; microevolved persistent infection if the paired isolates have >7 allele differences but same MLST type; and polyclonal if they are the same species but a different MLST type.
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BACKGROUND: Recently, antimicrobial peptides (AMPs) have been investigated for their use in cancer therapy. They have been reported to selectively target and kill cancer cells whilst leaving normal healthy cells unaffected. Certain Anura AMPs have expressed selective cytotoxicity against tumour cells. AIM: To test the potential of Anura AMPs bombinin H2, bombinin H4, temporin A, and temporin L for use as therapeutic agents for non-small cell lung carcinoma (NSCLC). METHODS: Cytotoxic effects on NSCLC cell lines A549 and Calu-3 and normal epithelial cell line Beas-2B were tested using the CellTox Green Cytotoxicity Assay. Their haemolytic effects on human erythrocytes were also tested for their clinical relevance. Cell membrane profiling, using MALDI-TOF, was performed to ascertain if membrane characteristics of the NSCLC and Beas-2B cell lines may contribute to the AMPs mode of action. RESULTS: Bombinin H4 (100-1.5 µM, p < 0.05) and temporin A (100-50 µM, p < 0.05) showed selective cytotoxicity towards the NSCLC cell lines. Furthermore, they exhibited low levels of haemolytic activity (bombinin H4, 0.061%; temporin A, 0.874%) comparable to untreated cells. Cell membrane profiling showed the phospholipid composition of normal epithelial cell line Beas-2B to be divergent from the cancerous cell lines. However, there was an overlap in the phospholipid profiles of the NSCLC cell lines supporting the hypothesis that the AMPs may have a selective affinity via the membrane composition of cancerous cell lines. CONCLUSION: These results suggest that bombinin H4 and temporin A show potential for application in lung cancer therapies. Further in vitro and in vivo studies are required to develop a greater understanding of their use as anticancer agents.
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Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of ventilator-associated pneumonia (VAP). Patients with VAP have poorly functioning neutrophils, related to increased levels of the complement fragment C5a. The antibiotic linezolid has been useful in controlling MRSA-related VAP infections; however clinical benefit does not always correlate with antimicrobial effect, suggesting the possibility of immunomodulatory properties. Here the effects of linezolid on healthy and dysfunctional neutrophils (modelled by C5a-induced injury) was investigated. Functional assays (killing, phagocytosis, transmigration, and respiratory burst) were used to assess the effects of pre-, co- and post-incubating linezolid (0.4-40 mg/L) with healthy neutrophils relative to those with C5a-induced injury. C5a decreased neutrophil killing, and phagocytosis of MRSA. Furthermore, C5a significantly decreased neutrophil transmigration to IL-8, but did not affect respiratory burst. Co-incubation of linezolid significantly improved killing of MRSA by dysfunctional neutrophils, which was supported by concomitant increases in phagocytosis. Conversely linezolid impaired killing responses in healthy neutrophils. Pre- or post-incubation of linezolid prior or following C5a induced injury had no effect on neutrophil function. This study suggests that linezolid has immunomodulatory properties that protect human neutrophils from injury and provides insight into its mode of action beyond a basic antibiotic.
Assuntos
Complemento C5a/metabolismo , Linezolida/uso terapêutico , Neutrófilos/efeitos dos fármacos , Antibacterianos/uso terapêutico , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Pneumonia Associada à Ventilação Mecânica/tratamento farmacológico , Pneumonia Associada à Ventilação Mecânica/metabolismo , Pneumonia Associada à Ventilação Mecânica/microbiologia , Explosão Respiratória/efeitos dos fármacosRESUMO
Medical device-associated infections, most frequently caused by Staphylococcus epidermidis and Staphylococcus aureus, are of increasing importance in modern medicine. The formation of adherent, multilayered bacterial biofilms is crucial in the pathogenesis of these infections. Polysaccharide intercellular adhesin (PIA), a homoglycan of ß-1,6-linked 2-acetamido-2-deoxy-D: -glucopyranosyl residues, of which about 15% are non-N-acetylated, is central to biofilm accumulation in staphylococci. It transpires that polysaccharides - structurally very similar to PIA - are also key to biofilm formation in a number of other organisms including the important human pathogens Escherichia coli, Aggregatibacter (Actinobacillus) actinomycetemcomitans, Yersinia pestis, and Bordetella spp. Apparently, synthesis of PIA and related polysaccharides is a general feature important for biofilm formation in diverse bacterial genera. Current knowledge about the structure and biosynthesis of PIA and related polysaccharides is reviewed. Additionally, information on their role in pathogenesis of biomaterial-related and other type of infections and the potential use of PIA and related compounds for prevention of infection is evaluated.
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Chronic infections are commonly associated with biofilms formed by bacteria such as Staphylococcus epidermidis. With the increase in antibiotic resistant bacteria, maggot debridement therapy has been reintroduced for the treatment of chronic wounds. Studies have shown that the excretion/ secretions (ES) of Lucilia sericata larvae (maggots) contain many bioactive compounds which may contribute to the efficacy of maggot therapy. The present study evaluates the effect of L. sericata ES on the formation and disruption of S. epidermidis 1457 and 5179-R1 biofilms. These strains employ either polysaccharide intercellular adhesin (PIA) or accumulation associated protein (Aap) for intercellular adhesion. A semiquantitative biofilm assay was used to measure the formation/disruption of S. epidermidis 1457 and 5179-R1 biofilms by ES. ES activity was characterized according to concentration, incubation time and temperature, thermal stability, and size. Immunofluorescence microscopy was used to ascertain the effect of ES on PIA and Aap. In the presence of ES, S. epidermidis 1457 and 5179-R1 nascent biofilm formation was inhibited, and pre-formed biofilms disrupted. ES activity was temperature and time dependent, inactivated by heat treatment, and disruption depended on the mechanism of intercellular adhesion. The molecule(s) responsible was >10 kDa in size and appeared to have protease or glucosaminidase activity. ES interferes with S. epidermidis biofilm formation, specifically degrading factors employed in biofilm accumulation, which would increase bacterial susceptibility to antibiotics and the host's immune system. In purified form, ES-factors may have general applicability for the treatment or prevention of chronic biofilm infections caused by staphylococci.
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Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Fatores Biológicos/farmacologia , Dípteros/metabolismo , Staphylococcus epidermidis/efeitos dos fármacos , Adesinas Bacterianas/metabolismo , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Aderência Bacteriana/efeitos dos fármacos , Fatores Biológicos/química , Fatores Biológicos/metabolismo , Dípteros/embriologia , Estabilidade de Medicamentos , Imunofluorescência , Larva/metabolismo , Peso Molecular , Staphylococcus epidermidis/crescimento & desenvolvimento , Staphylococcus epidermidis/metabolismo , Temperatura , Fatores de TempoRESUMO
This study describes the analysis of DNA from heat-killed (boilate) isolates of Mycobacterium tuberculosis from two UK outbreaks where DNA was of sub-optimal quality for the standard methodologies routinely used in microbial genomics. An Illumina library construction method developed for sequencing ancient DNA was successfully used to obtain whole genome sequences, allowing analysis of the outbreak by gene-by-gene MLST, SNP mapping and phylogenetic analysis. All cases were spoligotyped to the same Haarlem H1 sub-lineage. This is the first described application of ancient DNA library construction protocols to allow whole genome sequencing of a clinical tuberculosis outbreak. Using this method it is possible to obtain epidemiologically meaningful data even when DNA is of insufficient quality for standard methods.
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DNA Bacteriano/genética , Genoma Bacteriano/genética , Mycobacterium tuberculosis/genética , Polimorfismo de Nucleotídeo Único , Tuberculose/microbiologia , Criança , Surtos de Doenças , Saúde Global , Humanos , Tipagem de Sequências Multilocus , Tuberculose/epidemiologia , Sequenciamento Completo do GenomaRESUMO
Orthopedic device-related infection (ODRI), including both fracture-related infection (FRI) and periprosthetic joint infection (PJI), remain among the most challenging complications in orthopedic and musculoskeletal trauma surgery. ODRI has been convincingly shown to delay healing, worsen functional outcome and incur significant socio-economic costs. To address this clinical problem, ever more sophisticated technologies targeting the prevention and/or treatment of ODRI are being developed and tested in vitro and in vivo. Among the most commonly described innovations are antimicrobial-coated orthopedic devices, antimicrobial-loaded bone cements and void fillers, and dual osteo-inductive/antimicrobial biomaterials. Unfortunately, translation of these technologies to the clinic has been limited, at least partially due to the challenging and still evolving regulatory environment for antimicrobial drug-device combination products, and a lack of clarity in the burden of proof required in preclinical studies. Preclinical in vivo testing (i.e. animal studies) represents a critical phase of the multidisciplinary effort to design, produce and reliably test both safety and efficacy of any new antimicrobial device. Nonetheless, current in vivo testing protocols, procedures, models, and assessments are highly disparate, irregularly conducted and reported, and without standardization and validation. The purpose of the present opinion piece is to discuss best practices in preclinical in vivo testing of antimicrobial interventions targeting ODRI. By sharing these experience-driven views, we aim to aid others in conducting such studies both for fundamental biomedical research, but also for regulatory and clinical evaluation. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:271-287, 2019.
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Experimentação Animal/normas , Fixação Interna de Fraturas/efeitos adversos , Fixadores Internos/efeitos adversos , Infecções Relacionadas à Prótese , Animais , Anti-Infecciosos/administração & dosagem , Modelos Animais , Infecções Relacionadas à Prótese/microbiologia , Projetos de PesquisaRESUMO
BACKGROUND: Synthesis of the Staphylococcus aureus peptidoglycan pentaglycine interpeptide bridge is catalyzed by the nonribosomal peptidyl transferases FemX, FemA and FemB. Inactivation of the femAB operon reduces the interpeptide to a monoglycine, leading to a poorly crosslinked peptidoglycan. femAB mutants show a reduced growth rate and are hypersusceptible to virtually all antibiotics, including methicillin, making FemAB a potential target to restore beta-lactam susceptibility in methicillin-resistant S. aureus (MRSA). Cis-complementation with wild type femAB only restores synthesis of the pentaglycine interpeptide and methicillin resistance, but the growth rate remains low. This study characterizes the adaptations that ensured survival of the cells after femAB inactivation. RESULTS: In addition to slow growth, the cis-complemented femAB mutant showed temperature sensitivity and a higher methicillin resistance than the wild type. Transcriptional profiling paired with reporter metabolite analysis revealed multiple changes in the global transcriptome. A number of transporters for sugars, glycerol, and glycine betaine, some of which could serve as osmoprotectants, were upregulated. Striking differences were found in the transcription of several genes involved in nitrogen metabolism and the arginine-deiminase pathway, an alternative for ATP production. In addition, microarray data indicated enhanced expression of virulence factors that correlated with premature expression of the global regulators sae, sarA, and agr. CONCLUSION: Survival under conditions preventing normal cell wall formation triggered complex adaptations that incurred a fitness cost, showing the remarkable flexibility of S. aureus to circumvent cell wall damage. Potential FemAB inhibitors would have to be used in combination with other antibiotics to prevent selection of resistant survivors.
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Proteínas de Bactérias/genética , Parede Celular/química , Inativação Gênica , Genes Bacterianos , Staphylococcus aureus/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Biologia Computacional , Teste de Complementação Genética , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Óperon , Polissacarídeos/química , Staphylococcus aureus/metabolismo , Staphylococcus aureus/ultraestrutura , Temperatura , Transcrição GênicaRESUMO
Nosocomial staphylococcal foreign-body infections related to biofilm formation are a serious threat, demanding new therapeutic and preventive strategies. As the use of biofilm-associated factors as vaccines is critically restricted by their prevalence in natural staphylococcal populations we studied the distribution of genes involved in biofilm formation, the biofilm phenotype and production of polysaccharide intercellular adhesin (PIA) in clonally independent Staphylococcus aureus and Staphylococcus epidermidis strains isolated from prosthetic joint infections after total hip or total knee arthroplasty. Biofilm formation was detected in all S. aureus and 69.2% of S. epidermidis strains. Importantly, 27% of biofilm-positive S. epidermidis produced PIA-independent biofilms, in part mediated by the accumulation associated protein (Aap). Protein-dependent biofilms were exclusively found in S. epidermidis strains from total hip arthroplasty (THA). In S. aureus PIA and proteins act cooperatively in biofilm formation regardless of the infection site. PIA and protein factors like Aap are of differential importance for the pathogenesis of S. epidermidis in prosthetic joint infections (PJI) after THA and total knee arthroplasty (TKA), implicating that icaADBC cannot serve as a general virulence marker in this species. In S. aureus biofilm formation proteins are of overall importance and future work should focus on the identification of functionally active molecules.
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Adesinas Bacterianas/metabolismo , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Infecções Relacionadas à Prótese/microbiologia , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/isolamento & purificação , Staphylococcus epidermidis/isolamento & purificação , Prótese de Quadril/efeitos adversos , Humanos , Prótese do Joelho/efeitos adversos , Polissacarídeos Bacterianos/metabolismo , Infecções Relacionadas à Prótese/etiologiaRESUMO
PURPOSE: Nasal colonization with methicillin-resistant Staphylococcus aureus (MRSA) is poorly described for surgeons, despite the increased exposure to nosocomial pathogens and at-risk patients. This study investigated the molecular epidemiology and antimicrobial resistance of 26 MRSA isolates cultured from the nares of an international cross-sectional study of 1166 human and 60 veterinary surgeons. METHODOLOGY: All isolates were subjected to agr, spa and multilocus sequence typing, and the presence of 22 virulence factors was screened for by PCR. Additionally, biofilm-forming ability, haemolytic activity, staphyloxanthin production and antibiotic resistance were determined. The genome of a rifampicin-resistant MRSA was sequenced. RESULTS: Approximately half of the isolates belonged to well-described clonal lineages, ST1, ST5, ST8, ST45 and ST59, that have previously been associated with severe infections and increased patient mortality. Two of the three veterinarian MRSA belonged to epidemic livestock-associated MRSA clonal lineages (ST398 and ST8) previously associated with high transmission potential between animals and humans. The isolates did not display any consistent virulence gene pattern, and 35â% of the isolates carried at least one of the Panton-Valentine leukocidin (lukFS-PV), exfoliative toxin (eta) or toxic shock syndrome (tst) genes. Resistance to rifampicin was detected in one veterinarian isolate and was found to be due to three mutations in the rpoB gene. CONCLUSION: Surgeons occupy a critical position in the healthcare profession due to their close contact with patients. In this study, surgeons were found to be colonized with MRSA at low rates, similar to those of the general population, and the colonizing strains were often common clonal lineages.
Assuntos
Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Nariz/microbiologia , Cirurgiões , Médicos Veterinários , Fatores de Virulência/genética , Toxinas Bacterianas/genética , Biofilmes , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Estudos Transversais , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla/genética , Feminino , Genoma Bacteriano , Genótipo , Humanos , Internacionalidade , Masculino , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Fenótipo , Reação em Cadeia da Polimerase , Rifampina/farmacologia , Análise de Sequência de DNA , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologiaRESUMO
Staphylococcus epidermidis is a permanent member of the normal human microbiota, commonly found on skin and mucous membranes. By adhering to tissue surface moieties of the host via specific adhesins, S. epidermidis is capable of establishing a lifelong commensal relationship with humans that begins early in life. In its role as a commensal organism, S. epidermidis is thought to provide benefits to human host, including out-competing more virulent pathogens. However, largely due to its capacity to form biofilm on implanted foreign bodies, S. epidermidis has emerged as an important opportunistic pathogen in patients receiving medical devices. S. epidermidis causes approximately 20% of all orthopedic device-related infections (ODRIs), increasing up to 50% in late-developing infections. Despite this prevalence, it remains underrepresented in the scientific literature, in particular lagging behind the study of the S. aureus. This review aims to provide an overview of the interactions of S. epidermidis with the human host, both as a commensal and as a pathogen. The mechanisms retained by S. epidermidis that enable colonization of human skin as well as invasive infection, will be described, with a particular focus upon biofilm formation. The host immune responses to these infections are also described, including how S. epidermidis seems to trigger low levels of pro-inflammatory cytokines and high levels of interleukin-10, which may contribute to the sub-acute and persistent nature often associated with these infections. The adaptive immune response to S. epidermidis remains poorly described, and represents an area which may provide significant new discoveries in the coming years.
RESUMO
Bacterial species comprise related genotypes that can display divergent phenotypes with important clinical implications. Staphylococcus epidermidis is a common cause of nosocomial infections and, critical to its pathogenesis, is its ability to adhere and form biofilms on surfaces, thereby moderating the effect of the host's immune response and antibiotics. Commensal S. epidermidis populations are thought to differ from those associated with disease in factors involved in adhesion and biofilm accumulation. We quantified the differences in biofilm formation in 98 S. epidermidis isolates from various sources, and investigated population structure based on ribosomal multilocus typing (rMLST) and the presence/absence of genes involved in adhesion and biofilm formation. All isolates were able to adhere and form biofilms in in vitro growth assays and confocal microscopy allowed classification into 5 biofilm morphotypes based on their thickness, biovolume and roughness. Phylogenetic reconstruction grouped isolates into three separate clades, with the isolates in the main disease associated clade displaying diversity in morphotype. Of the biofilm morphology characteristics, only biofilm thickness had a significant association with clade distribution. The distribution of some known adhesion-associated genes (aap and sesE) among isolates showed a significant association with the species clonal frame. These data challenge the assumption that biofilm-associated genes, such as those on the ica operon, are genetic markers for less invasive S. epidermidis isolates, and suggest that phenotypic characteristics, such as adhesion and biofilm formation, are not fixed by clonal descent but are influenced by the presence of various genes that are mobile among lineages.