RESUMEN
Topical antibiotic preparations, such as fusidic acid (FA) or mupirocin, are used in the prevention and treatment of superficial skin infections caused by staphylococci. Previous genomic epidemiology work has suggested an association between the widespread use of topical antibiotics and the emergence of methicillin resistant Staphylococcus aureus in some settings. In this study, we provide experimental proof of co-selection for multidrug resistance in S. aureus following exposure to FA or mupirocin. Through targeted mutagenesis and phenotypic analyses, we confirmed that fusC carriage confers resistance to FA, and mupA carriage confers high-level resistance to mupirocin in multiple S. aureus genetic backgrounds. In vitro experiments demonstrated that carriage of fusC and mupA confer a competitive advantage in the presence of sub-inhibitory concentrations of FA and mupirocin, respectively. Further, we used a porcine skin colonisation model to show that clinically relevant concentrations of topical antibiotics can co-select for presence of unrelated antimicrobial resistance determinants, such as mecA, blaZ, and qacA, in fusC or mupA harbouring S. aureus These findings provide valuable insights on the role of acquired FA or mupirocin resistance in co-selecting for broader antibiotic resistance in S. aureus, prompting greater need for judicious use of topical antibiotics.
RESUMEN
Staphylococcus aureus small-colony variants (SCVs) are associated with unusually chronic and persistent infections despite active antibiotic treatment. The molecular basis for this clinically important phenomenon is poorly understood, hampered by the instability of the SCV phenotype. Here we investigated the genetic basis for an unstable S. aureus SCV that arose spontaneously while studying rifampicin resistance. This SCV showed no nucleotide differences across its genome compared with a normal-colony variant (NCV) revertant, yet the SCV presented the hallmarks of S. aureus linked to persistent infection: down-regulation of virulence genes and reduced hemolysis and neutrophil chemotaxis, while exhibiting increased survival in blood and ability to invade host cells. Further genome analysis revealed chromosome structural variation uniquely associated with the SCV. These variations included an asymmetric inversion across half of the S. aureus chromosome via recombination between type I restriction modification system (T1RMS) genes, and the activation of a conserved prophage harboring the immune evasion cluster (IEC). Phenotypic reversion to the wild-type-like NCV state correlated with reversal of the chromosomal inversion (CI) and with prophage stabilization. Further analysis of 29 complete S. aureus genomes showed strong signatures of recombination between hsdMS genes, suggesting that analogous CI has repeatedly occurred during S. aureus evolution. Using qPCR and long-read amplicon deep sequencing, we detected subpopulations with T1RMS rearrangements causing CIs and prophage activation across major S. aureus lineages. Here, we have discovered a previously unrecognized and widespread mechanism of reversible genomic instability in S. aureus associated with SCV generation and persistent infections.
Asunto(s)
Inestabilidad Cromosómica , Cromosomas Bacterianos , Fenotipo , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Translocación Genética , Inversión Cromosómica , Orden Génico , Genoma Bacteriano , Hemólisis , Humanos , Fagos de Staphylococcus/fisiología , Staphylococcus aureus/virologíaRESUMEN
[This corrects the article DOI: 10.1371/journal.pgen.1006246.].
RESUMEN
Staphylococcus aureus is found in the nasal cavity of up to 30% of the human population. Persistent nasal carriage of S. aureus is a risk factor for influenza virus-induced secondary bacterial pneumonia. There is limited understanding of the factors that cause S. aureus to shift from the upper to the lower respiratory tract and convert from a commensal organism to an invasive pathogen. Here we show that neutrophils actively prevent S. aureus dissemination. Establishment of a mouse model of localized S. aureus nasal carriage revealed variations in the longevity of persistence of S. aureus isolates. Improved persistence within this site was associated with reduced nasal inflammation, less neutrophil egress into the airways and reduced neutrophil-bacteria association. Neutrophil depletion of mice with localized S. aureus nasal carriage triggered the development of an invasive S. aureus infection. Moreover, utilizing a model of influenza-induced staphylococcal pneumonia we showed that treatment with granulocyte-colony-stimulating factor, a potent enhancer of neutrophil number and function, significantly reduced bacterial loads in the lung and improved disease outcomes. These data reveal that neutrophils play an important and active role in confining S. aureus to the upper respiratory tract and highlight the use of approaches that improve neutrophil function as effective strategies to attenuate morbidity associated with staphylococcal pneumonia.
Asunto(s)
Pulmón/microbiología , Neutrófilos/inmunología , Neumonía Estafilocócica/inmunología , Animales , Ratones , Nariz/microbiología , Infecciones por Orthomyxoviridae , Staphylococcus aureusRESUMEN
Coagulase-negative staphylococci (CoNS) represent one of the major causes of health care- and medical device-associated infections. Emerging antimicrobial resistance has complicated the treatment of systemic infections caused by CoNS. Here, we describe the prevalence of antimicrobial resistance in clinical CoNS strains from a tertiary care hospital over a 4-year period, and we observed a significant increase in resistance to daptomycin. Notably, Staphylococcus capitis accounted for the majority of these daptomycin-resistant (DAP-R) CoNS. To further investigate the mechanisms of daptomycin resistance in CoNS, daptomycin-susceptible clinical strains of S. capitis and Staphylococcus epidermidis underwent in vitro daptomycin exposure to generate DAP-R CoNS mutants. Unlike that seen with Staphylococcus aureus, alteration of cell surface charge was not observed in the DAP-R CoNS strains, but biofilm formation was compromised. Whole-genome sequencing analysis of the DAP-R CoNS strains identified single nucleotide polymorphisms (SNPs) in walKR, the essential two-component regulatory system controlling cell wall biogenesis. PCR and sequencing of walK and walR from 17 DAP-R CoNS clinical isolates identified seven nonsynonymous mutations. The results were confirmed by the recreation of the walK SNP in S. epidermidis, which resulted in reduced susceptibility to daptomycin and vancomycin. This study highlights the significance of CoNS in evolving daptomycin resistance and showed that walKR is shared among the staphylococcal species and is involved in antibiotic resistance development. Notably, we did not observe mutations in genes responsible for phospholipid biosynthesis or an altered cell surface charge, suggesting that reduced daptomycin susceptibility in CoNS may emerge in a fashion distinct from that in S. aureus.
Asunto(s)
Antibacterianos/farmacología , Daptomicina/farmacología , Farmacorresistencia Bacteriana/genética , Staphylococcus capitis/genética , Staphylococcus epidermidis/genética , Sustitución de Aminoácidos/genética , Proteínas Bacterianas/genética , Biopelículas/crecimiento & desarrollo , Infección Hospitalaria/microbiología , Histidina Quinasa/genética , Humanos , Pruebas de Sensibilidad Microbiana , Polimorfismo de Nucleótido Simple/genética , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/efectos de los fármacos , Staphylococcus capitis/efectos de los fármacos , Staphylococcus capitis/aislamiento & purificación , Staphylococcus epidermidis/efectos de los fármacos , Staphylococcus epidermidis/aislamiento & purificación , Centros de Atención Terciaria , Vancomicina/farmacologíaRESUMEN
Staphylococcus lugdunensis is a coagulase negative bacterial pathogen that is particularly associated with severe cases of infectious endocarditis. Unique amongst the coagulase-negative staphylococci, S. lugdunensis harbors an iron regulated surface determinant locus (isd). This locus facilitates the acquisition of heme as a source of nutrient iron during infection and allows iron limitation caused by "nutritional immunity" to be overcome. The isd locus is duplicated in S. lugdunensis HKU09-01 and we show here that the duplication is intrinsically unstable and undergoes accordion-like amplification and segregation leading to extensive isd copy number variation. Amplification of the locus increased the level of expression of Isd proteins and improved binding of hemoglobin to the cell surface of S. lugdunensis. Furthermore, Isd overexpression provided an advantage when strains were competing for a limited amount of hemoglobin as the sole source of iron. Gene duplications and amplifications (GDA) are events of fundamental importance for bacterial evolution and are frequently associated with antibiotic resistance in many species. As such, GDAs are regarded as evolutionary adaptions to novel selective pressures in hostile environments pointing towards a special importance of isd for S. lugdunensis. For the first time we show an example of a GDA that involves a virulence factor of a Gram-positive pathogen and link the GDA directly to a competitive advantage when the bacteria were struggling with selective pressures mimicking "nutritional immunity".
Asunto(s)
Endocarditis Bacteriana/microbiología , Hierro/metabolismo , Infecciones Estafilocócicas/metabolismo , Staphylococcus lugdunensis/metabolismo , Variaciones en el Número de Copia de ADN/genética , Endocarditis Bacteriana/genética , Duplicación de Gen , Sitios Genéticos/genética , Hemo/genética , Hemo/metabolismo , Hemoglobinas/genética , Hemoglobinas/metabolismo , Humanos , Proteínas Reguladoras del Hierro/genética , Proteínas Reguladoras del Hierro/metabolismo , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/patología , Staphylococcus lugdunensis/patogenicidad , Propiedades de SuperficieRESUMEN
Topical antibiotics, such as mupirocin and fusidic acid, are commonly used in the prevention and treatment of skin infections, particularly those caused by staphylococci. However, the widespread use of these agents is associated with increased resistance to these agents, potentially limiting their efficacy. Of particular concern is the observation that resistance to topical antibiotics is often associated with multidrug resistance, suggesting that topical antibiotics may play a role in the emergence of multidrug-resistant (MDR) strains. New Zealand (NZ) has some of the highest globally recorded rates of topical antibiotic usage and resistance. Using a combination of Pacific Biosciences single-molecule real-time (SMRT) whole-genome sequencing, Illumina short-read sequencing, and Bayesian phylogenomic modeling on 118 new multilocus sequence type 1 (ST1) community Staphylococcus aureus isolates from New Zealand and 61 publically available international ST1 genome sequences, we demonstrate a strong correlation between the clinical introduction of topical antibiotics and the emergence of MDR ST1 S. aureus We also provide in vitro experimental evidence showing that exposure to topical antibiotics can lead to the rapid selection of MDR S. aureus isolates carrying plasmids that confer resistance to multiple unrelated antibiotics, from within a mixed population of competitor strains. These findings have important implications regarding the impact of the indiscriminate use of topical antibiotics.
Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/efectos de los fármacos , Administración Tópica , Teorema de Bayes , Farmacorresistencia Bacteriana Múltiple/genética , Ácido Fusídico/farmacología , Genoma Bacteriano/efectos de los fármacos , Genoma Bacteriano/genética , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Mupirocina/farmacología , Nueva Zelanda , Infecciones Cutáneas Estafilocócicas/tratamiento farmacológico , Infecciones Cutáneas Estafilocócicas/microbiología , Staphylococcus aureus/genéticaRESUMEN
Coagulase-negative staphylococci (CoNS), such as Staphylococcus capitis, are major causes of bloodstream infections in neonatal intensive care units (NICUs). Recently, a distinct clone of S. capitis (designated S. capitis NRCS-A) has emerged as an important pathogen in NICUs internationally. Here, 122 S. capitis isolates from New Zealand (NZ) underwent whole-genome sequencing (WGS), and these data were supplemented with publicly available S. capitis sequence reads. Phylogenetic and comparative genomic analyses were performed, as were phenotypic assessments of antimicrobial resistance, biofilm formation, and plasmid segregational stability on representative isolates. A distinct lineage of S. capitis was identified in NZ associated with neonates and the NICU environment. Isolates from this lineage produced increased levels of biofilm, displayed higher levels of tolerance to chlorhexidine, and were multidrug resistant. Although similar to globally circulating NICU-associated S. capitis strains at a core-genome level, NZ NICU S. capitis isolates carried a novel stably maintained multidrug-resistant plasmid that was not present in non-NICU isolates. Neonatal blood culture isolates were indistinguishable from environmental S. capitis isolates found on fomites, such as stethoscopes and neonatal incubators, but were generally distinct from those isolates carried by NICU staff. This work implicates the NICU environment as a potential reservoir for neonatal sepsis caused by S. capitis and highlights the capacity of genomics-based tracking and surveillance to inform future hospital infection control practices aimed at containing the spread of this important neonatal pathogen.
Asunto(s)
Farmacorresistencia Bacteriana Múltiple/genética , Sepsis Neonatal/microbiología , Staphylococcus capitis/genética , Antibacterianos/farmacología , Coagulasa/genética , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Genómica/métodos , Humanos , Recién Nacido , Unidades de Cuidado Intensivo Neonatal , Sepsis Neonatal/tratamiento farmacológico , Nueva Zelanda , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Staphylococcus capitis/efectos de los fármacosRESUMEN
Objectives: Daptomycin non-susceptibility in Staphylococcus aureus can emerge via the accumulation of single or multiple mutations, each resulting in a slight increase in the daptomycin MIC. The daptomycin-non-susceptible phenotype may include other features such as daptomycin tolerance. This study identifies S. aureus genomic regions that frequently develop mutations following prolonged daptomycin exposure but have not been previously associated with daptomycin non-susceptibility. Methods: Sequence variations in the same eight loci independently observed following 28 day parallel serial passages of S. aureus J01 in daptomycin were introduced in isolation into S. aureus J01. MICs were determined by microbroth dilution. Daptomycin killing and tolerance were determined by kill curve analysis. Results: Single mutations in snoF, hmp1, sspA, rimP, hepT, rsh, map1 and amaP had only a modest impact on the daptomycin MIC (≤2-fold). In contrast, individual mutation in several of these regions resulted in pronounced changes to daptomycin tolerance. Conclusions: This study demonstrates that less characterized mutations in S. aureus following daptomycin exposure do not result in significant daptomycin susceptibility changes, but rather allow for enhanced survival characteristics during treatment. This sheds new light on genetic adaptations that may play a role in persistent infection. Further studies are needed to elucidate the prevalence of these mutations in clinical isolates.
Asunto(s)
Antibacterianos/farmacología , Daptomicina/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Staphylococcus aureus Resistente a Meticilina/genética , Análisis Mutacional de ADN , ADN Bacteriano/genética , Genes Bacterianos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , FenotipoRESUMEN
UNLABELLED: De novo guanine biosynthesis is an evolutionarily conserved pathway that creates sufficient nucleotides to support DNA replication, transcription, and translation. Bacteria can also salvage nutrients from the environment to supplement the de novo pathway, but the relative importance of either pathway during Staphylococcus aureus infection is not known. In S. aureus, genes important for both de novo and salvage pathways are regulated by a guanine riboswitch. Bacterial riboswitches have attracted attention as a novel class of antibacterial drug targets because they have high affinity for small molecules, are absent in humans, and regulate the expression of multiple genes, including those essential for cell viability. Genetic and biophysical methods confirm the existence of a bona fide guanine riboswitch upstream of an operon encoding xanthine phosphoribosyltransferase (xpt), xanthine permease (pbuX), inosine-5'-monophosphate dehydrogenase (guaB), and GMP synthetase (guaA) that represses the expression of these genes in response to guanine. We found that S. aureus guaB and guaA are also transcribed independently of riboswitch control by alternative promoter elements. Deletion of xpt-pbuX-guaB-guaA genes resulted in guanine auxotrophy, failure to grow in human serum, profound abnormalities in cell morphology, and avirulence in mouse infection models, whereas deletion of the purine salvage genes xpt-pbuX had none of these effects. Disruption of guaB or guaA recapitulates the xpt-pbuX-guaB-guaA deletion in vivo In total, the data demonstrate that targeting the guanine riboswitch alone is insufficient to treat S. aureus infections but that inhibition of guaA or guaB could have therapeutic utility. IMPORTANCE: De novo guanine biosynthesis and purine salvage genes were reported to be regulated by a guanine riboswitch in Staphylococcus aureus We demonstrate here that this is not true, because alternative promoter elements that uncouple the de novo pathway from riboswitch regulation were identified. We found that in animal models of infection, the purine salvage pathway is insufficient for S. aureus survival in the absence of de novo guanine biosynthesis. These data suggest targeting the de novo guanine biosynthesis pathway may have therapeutic utility in the treatment of S. aureus infections.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Guanina/biosíntesis , Purinas/metabolismo , Riboswitch , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/metabolismo , Animales , Proteínas Bacterianas/genética , Femenino , Humanos , Ratones , Staphylococcus aureus/genéticaRESUMEN
The capacity for intracellular survival within phagocytes is likely a critical factor facilitating the dissemination of Staphylococcus aureus in the host. To date, the majority of work on S. aureus-phagocyte interactions has focused on neutrophils and, to a lesser extent, macrophages, yet we understand little about the role played by dendritic cells (DCs) in the direct killing of this bacterium. Using bone marrow-derived DCs (BMDCs), we demonstrate for the first time that DCs can effectively kill S. aureus but that certain strains of S. aureus have the capacity to evade DC (and macrophage) killing by manipulation of autophagic pathways. Strains with high levels of Agr activity were capable of causing autophagosome accumulation, were not killed by BMDCs, and subsequently escaped from the phagocyte, exerting significant cytotoxic effects. Conversely, strains that exhibited low levels of Agr activity failed to accumulate autophagosomes and were killed by BMDCs. Inhibition of the autophagic pathway by treatment with 3-methyladenine restored the bactericidal effects of BMDCs. Using an in vivo model of systemic infection, we demonstrated that the ability of S. aureus strains to evade phagocytic cell killing and to survive temporarily within phagocytes correlated with persistence in the periphery and that this effect is critically Agr dependent. Taken together, our data suggest that strains of S. aureus exhibiting high levels of Agr activity are capable of blocking autophagic flux, leading to the accumulation of autophagosomes. Within these autophagosomes, the bacteria are protected from phagocytic killing, thus providing an intracellular survival niche within professional phagocytes, which ultimately facilitates dissemination.
Asunto(s)
Autofagia/fisiología , Proteínas Bacterianas/metabolismo , Células Dendríticas/microbiología , Infecciones Estafilocócicas/inmunología , Transactivadores/metabolismo , Animales , Bacteriemia/metabolismo , Bacteriemia/microbiología , Western Blotting , Células de la Médula Ósea/microbiología , Células Cultivadas , Modelos Animales de Enfermedad , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/inmunologíaRESUMEN
In this study, we investigated the cell wall-anchored fibronectin-binding proteins SpsD and SpsL from the canine commensal and pathogen Staphylococcus pseudintermedius for their role in promoting bacterial invasion of canine progenitor epidermal keratinocytes (CPEK). Invasion was examined by the gentamicin protection assay and fluorescence microscopy. An ΔspsD ΔspsL mutant of strain ED99 had a dramatically reduced capacity to invade CPEK monolayers, while no difference in the invasion level was observed with single mutants. Lactococcus lactis transformed with plasmids expressing SpsD and SpsL promoted invasion, showing that both proteins are important. Soluble fibronectin was required for invasion, and an RGD-containing peptide or antibodies recognizing the integrin α5ß1 markedly reduced invasion, suggesting an important role for the integrin in this process. Src kinase inhibitors effectively blocked internalization, suggesting a functional role for the kinase in invasion. In order to identify the minimal fibronectin-binding region of SpsD and SpsL involved in the internalization process, recombinant fragments of both proteins were produced. The SpsD520-846 and SpsL538-823 regions harboring the major fibronectin-binding sites inhibited S. pseudintermedius internalization. Finally, the effects of staphylococcal invasion on the integrity of different cell lines were examined. Because SpsD and SpsL are critical factors for adhesion and invasion, blocking these processes could provide a strategy for future approaches to treating infections.
Asunto(s)
Proteínas Bacterianas/metabolismo , Enfermedades de los Perros/microbiología , Células Epiteliales/microbiología , Fibronectinas/metabolismo , Infecciones Estafilocócicas/veterinaria , Staphylococcus/metabolismo , Animales , Proteínas Bacterianas/genética , Línea Celular , Pared Celular/genética , Pared Celular/metabolismo , Enfermedades de los Perros/metabolismo , Perros , Unión Proteica , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiología , Staphylococcus/genética , Staphylococcus/patogenicidad , VirulenciaRESUMEN
Accurately estimating parasite transmission success and subsequent infection load has important ramifications for a wide range of disease-related questions and research disciplines. Recent interest in the role of parasites in amphibian population declines and deformities, for instance, has prompted increased interest in approaches to quantify infection and pathology. Here, we introduce a novel method of fluorescently labeling trematode cercariae and optimize its application to interactions between the pathogenic trematode Ribeiroia ondatrae and amphibian hosts (Lithobates sphenocephalus). We then compare the efficacy of this method with two other approaches commonly used to assess infection in second intermediate hosts necropsy and tissue clearing with a focus on accuracy, precision, and bias. Dye (Invitrogen, BODIPY® FL C12) concentrations of <200 nM and DMSO solvent concentrations <0.2% offered a highly visible, long-lasting marker with no detectable effects on cercariae survival or metacercariae establishment. Among methods, the necropsy approach yielded the highest proportion of detected parasites and the lowest standard error around the mean. However, the fluorescent labeling method offered highly similar results (r = 0.99), with an estimated 75% of administered parasites establishing successfully. At low to moderate parasite exposure dosages, tissue clearing was comparable to the other two methods, but tended to underpredict infection at the higher exposures (i.e., 'proportional bias'), likely because individual parasites became more difficult to distinguish. Conceptually, these findings illustrate that initial infection within amphibian hosts is a consistent, linear function of exposure dosage, suggesting that parasite density dependence does not regulate initial establishment. From an applied standpoint, our results offer a methodological foundation for subsequent research using fluorescently labeled parasites, which offer distinct advantages such as allowing in vivo parasite tracking within living hosts.
Asunto(s)
Ranidae/parasitología , Trematodos/fisiología , Infecciones por Trematodos/veterinaria , Animales , Cercarias/efectos de los fármacos , Cercarias/fisiología , Dimetilsulfóxido/farmacología , Colorantes Fluorescentes/farmacología , Larva/parasitología , Dosificación Letal Mediana , Distribución Aleatoria , Método Simple Ciego , Caracoles , Solventes/farmacología , Factores de Tiempo , Trematodos/efectos de los fármacos , Infecciones por Trematodos/diagnóstico , Infecciones por Trematodos/parasitologíaRESUMEN
Staphylococcus aureus asymptomatically colonises the anterior nares, but the host and bacterial factors that facilitate colonisation remain incompletely understood. The S. aureus surface protein ClfB has been shown to mediate adherence to squamous epithelial cells in vitro and to promote nasal colonisation in both mice and humans. Here, we demonstrate that the squamous epithelial cell envelope protein loricrin represents the major target ligand for ClfB during S. aureus nasal colonisation. In vitro adherence assays indicated that bacteria expressing ClfB bound loricrin most likely by the "dock, lock and latch" mechanism. Using surface plasmon resonance we showed that ClfB bound cytokeratin 10 (K10), a structural protein of squamous epithelial cells, and loricrin with similar affinities that were in the low µM range. Loricrin is composed of three separate regions comprising GS-rich omega loops. Each loop was expressed separately and found to bind ClfB, However region 2 bound with highest affinity. To investigate if the specific interaction between ClfB and loricrin was sufficient to facilitate S. aureus nasal colonisation, we compared the ability of ClfBâºS. aureus to colonise the nares of wild-type and loricrin-deficient (Lorâ»/â») mice. In the absence of loricrin, S. aureus nasal colonisation was significantly impaired. Furthermore a ClfBâ» mutant colonised wild-type mice less efficiently than the parental ClfB⺠strain whereas a similar lower level of colonisation was observed with both the parental strain and the ClfBâ» mutant in the Lorâ»/â» mice. The ability of ClfB to support nasal colonisation by binding loricrin in vivo was confirmed by the ability of Lactococcus lactis expressing ClfB to be retained in the nares of WT mice but not in the Lorâ»/â» mice. By combining in vitro biochemical analysis with animal model studies we have identified the squamous epithelial cell envelope protein loricrin as the target ligand for ClfB during nasal colonisation by S. aureus.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Coagulasa/metabolismo , Proteínas de la Membrana/metabolismo , Nariz/microbiología , Staphylococcus aureus/metabolismo , Adhesinas Bacterianas/genética , Animales , Adhesión Bacteriana/fisiología , Línea Celular , Células Epiteliales/microbiología , Femenino , Humanos , Queratina-10/metabolismo , Lactococcus lactis/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Estructura Terciaria de ProteínaRESUMEN
BACKGROUND: The community-associated methicillin-resistant S. aureus (CA-MRSA) ST93 clone is becoming dominant in Australia and is clinically highly virulent. In addition, sepsis and skin infection models demonstrate that ST93 CA-MRSA is the most virulent global clone of S. aureus tested to date. While the determinants of virulence have been studied in other clones of CA-MRSA, the basis for hypervirulence in ST93 CA-MRSA has not been defined. RESULTS: Here, using a geographically and temporally dispersed collection of ST93 isolates we demonstrate that the ST93 population hyperexpresses key CA-MRSA exotoxins, in particular α-hemolysin, in comparison to other global clones. Gene deletion and complementation studies, and virulence comparisons in a murine skin infection model, showed unequivocally that increased expression of α-hemolysin is the key staphylococcal virulence determinant for this clone. Genome sequencing and comparative genomics of strains with divergent exotoxin profiles demonstrated that, like other S. aureus clones, the quorum sensing agr system is the master regulator of toxin expression and virulence in ST93 CA-MRSA. However, we also identified a previously uncharacterized AraC/XylS family regulator (AryK) that potentiates toxin expression and virulence in S. aureus. CONCLUSIONS: These data demonstrate that hyperexpression of α-hemolysin mediates enhanced virulence in ST93 CA-MRSA, and additional control of exotoxin production, in particular α-hemolysin, mediated by regulatory systems other than agr have the potential to fine-tune virulence in CA-MRSA.
Asunto(s)
Infecciones Comunitarias Adquiridas/microbiología , Infecciones Comunitarias Adquiridas/patología , Expresión Génica , Proteínas Hemolisinas/biosíntesis , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Infecciones Cutáneas Estafilocócicas/microbiología , Infecciones Cutáneas Estafilocócicas/patología , Animales , Australia , Toxinas Bacterianas/biosíntesis , Toxinas Bacterianas/genética , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Prueba de Complementación Genética , Genoma Bacteriano , Proteínas Hemolisinas/genética , Humanos , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Ratones , Ratones Endogámicos BALB C , Análisis de Secuencia de ADNRESUMEN
Staphylococcus aureus bacteremia causes significant morbidity and mortality. Treatment of staphylococcal infections is hindered by widespread antibiotic resistance, and attempts to develop an S. aureus vaccine have failed. Improved S. aureus treatment and infection prevention options require a deeper understanding of the correlates of protective immunity. CD4+ T cells have been identified as key orchestrators in the defense against S. aureus, but uncertainties persist regarding the subset, polarity, and breadth of the memory CD4+ T-cell pool required for protection. Here, using a mouse model of systemic S. aureus infection, we discovered that the breadth of bacterium-specific memory CD4+ T-cell pool is a critical factor for protective immunity against invasive S. aureus infections. Seeding mice with a monoclonal bacterium-specific circulating memory CD4+ T-cell population failed to protect against systemic S. aureus infection; however, the introduction of a polyclonal and polyfunctional memory CD4+ T-cell pool significantly reduced the bacterial burden. Our findings support the development of a multi-epitope T-cell-based S. aureus vaccine, as a strategy to mitigate the severity of S. aureus bacteremia.
Asunto(s)
Bacteriemia , Linfocitos T CD4-Positivos , Infecciones Estafilocócicas , Staphylococcus aureus , Animales , Bacteriemia/inmunología , Bacteriemia/microbiología , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/inmunología , Ratones , Linfocitos T CD4-Positivos/inmunología , Células T de Memoria/inmunología , Memoria Inmunológica , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Femenino , Vacunas Estafilocócicas/inmunología , Índice de Severidad de la EnfermedadRESUMEN
The past decade has seen an increase in the prevalence of sequence type (ST) 45 methicillin-resistant Staphylococcus aureus (MRSA), yet the underlying drivers for its emergence and spread remain unclear. To better understand the worldwide dissemination of ST45 S. aureus, we performed phylogenetic analyses of Australian isolates, supplemented with a global population of ST45 S. aureus genomes. Our analyses revealed a distinct lineage of multidrug-resistant ST45 MRSA harbouring qacA, predominantly found in Australia and Singapore. Bayesian inference predicted that the acquisition of qacA occurred in the late 1990s. qacA was integrated into a structurally variable region of the chromosome containing Tn552 (carrying blaZ) and Tn4001 (carrying aac(6')-aph(2")) transposable elements. Using mutagenesis and in vitro assays, we provide phenotypic evidence that qacA confers tolerance to chlorhexidine. These findings collectively suggest both antimicrobial resistance and the carriage of qacA may play a role in the successful establishment of ST45 MRSA.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina , Infecciones Estafilocócicas , Humanos , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus/genética , Teorema de Bayes , Filogenia , Infecciones Estafilocócicas/epidemiología , Proteínas de Transporte de Membrana/genética , Proteínas Bacterianas/genética , AustraliaRESUMEN
Staphylococcus aureus is a pulmonary pathogen associated with substantial human morbidity and mortality. As vaccines targeting virulence determinants have failed to be protective in humans, other factors are likely involved in pathogenesis. Here we analysed transcriptomic responses of human clinical isolates of S. aureus from initial and chronic infections. We observed upregulated collagenase and proline transporter gene expression in chronic infection isolates. Metabolomics of bronchiolar lavage fluid and fibroblast infection, growth assays and analysis of bacterial mutant strains showed that airway fibroblasts produce collagen during S. aureus infection. Host-adapted bacteria upregulate collagenase, which degrades collagen and releases proline. S. aureus then imports proline, which fuels oxidative metabolism via the tricarboxylic acid cycle. Proline metabolism provides host-adapted S. aureus with a metabolic benefit enabling out-competition of non-adapted strains. These data suggest that clinical settings characterized by airway repair processes and fibrosis provide a milieu that promotes S. aureus adaptation and supports infection.
RESUMEN
Health care-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) forms biofilm in vitro that is dependent on the surface-located fibronectin binding proteins A and B (FnBPA, FnBPB). Here we provide new insights into the requirements for FnBP-dependent biofilm formation by MRSA. We show that expression of FnBPs is sustained at high levels throughout the growth cycle in the HA-MRSA strain BH1CC in contrast to laboratory strain SH1000, where expression could be detected only in exponential phase. We found that FnBP-mediated biofilm accumulation required Zn(2+), while the removal of Zn(2+) had no effect on the ability of FnBPA to mediate bacterial adherence to fibrinogen. We also investigated the role of FnBPA expressed on the surface of S. aureus in promoting biofilm formation and bacterial adhesion to fibrinogen. The minimum part of FnBPA required for ligand binding has so far been defined only with recombinant proteins. Here we found that the N1 subdomain was not required for biofilm formation or for FnBPA to promote bacterial adherence to fibrinogen. Residues at the C terminus of subdomain N3 required for FnBPA to bind to ligands using the "dock, lock, and latch" mechanism were necessary for FnBPA to promote bacterial adherence to fibrinogen. However, these residues were not necessary to form biofilm, allowing us to localize the region of FnBPA required for biofilm accumulation to residues 166 to 498. Thus, FnBPA mediates biofilm formation and bacterial adhesion to fibrinogen using two distinct mechanisms. Finally, we identified a hitherto-unrecognized thrombin cleavage site close to the boundary between subdomains N1 and N2 of FnBPA.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Fibrinógeno/metabolismo , Fibronectinas/metabolismo , Staphylococcus aureus Resistente a Meticilina/metabolismo , Infecciones Estafilocócicas/microbiología , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/aislamiento & purificación , Adhesión Bacteriana , Biopelículas/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Quelantes del Hierro/farmacología , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/fisiología , Ácido Pentético/farmacología , Estructura Terciaria de Proteína , Proteínas Recombinantes , Trombina/metabolismo , Zinc/metabolismoRESUMEN
Staphylococcus lugdunensis is a commensal of humans and an opportunistic pathogen. It can cause an aggressive form of infective endocarditis in healthy humans akin to Staphylococcus aureus. Here we compared the virulence of the genome-sequenced S. lugdunensis strain N920143 to S. aureus in an experimental rat endocarditis model. N920143 caused a milder course of disease with lower levels of bacteraemia and smaller endocardial vegetations than S. aureus strain Newman. However, vegetations were comparable to those produced by S. aureus MRSA strain COL. Little is known about virulence factors of S. lugdunensis as systems to manipulate the bacterium genetically are currently limited. Here, we report a method for electroporation of S. lugdunensis with plasmid DNA and demonstrate that the low efficiency of transformation is due to the activity of a conserved type I restriction-modification system. To streamline the transformation process, we constructed SL01B, an E. coli strain expressing the hsdM/hsdS genes of N920143. Modified plasmid DNA isolated from SL01B transformed S. lugdunensis strains from clonal complexes 1 and 2 efficiently. A deletion mutant of N920143 lacking sortase A was significantly less virulent than the wild-type in the endocarditis model. Mutants defective in single surface proteins Fbl or vWbl were not significantly different from the wild-type but showed trends towards reduced virulence.