RESUMO
Uncertainty tolerance (UT) is integral to healthcare. Providers' responses to medical uncertainty has ramifications on the healthcare system, the healthcare provider and the patient. Understanding healthcare providers' UT, is important for improving patient-care outcomes. Understanding whether and to what extent it is possible to modulate individuals' perceptions and responses to medical uncertainty, can provide insights into mechanisms for support for training and education. The objectives of this review were to further characterize moderators of healthcare UT and explore moderator influences on the perceptions and responses to uncertainty experienced by healthcare professionals. Framework analysis of qualitative primary literature was conducted on 17 articles, focusing on the impacts of UT on healthcare providers. Three domains of moderators were identified and characterized relating to the healthcare provider's personal attributes, patient-derived uncertainty and the healthcare system. These domains were further categorized into themes and subthemes. Results suggest these moderators influence perceptions and responses to healthcare uncertainty across a spectrum ranging from positive to negative to uncertain. In this way, UT could be a state-based construct within healthcare settings and is contextually determined. Our findings further characterize the integrative model of uncertainty tolerance (IMUT) (Hillen Social Science and Medicine 180, 62-75, 2017) and provide evidence for the relationship between moderators and their influences on cognitive, emotional and behavioral responses to uncertainty. These findings provide a foundation for understanding the complex nature of the UT construct, add to theory development, and provide groundwork for future research exploring appropriate support for training and education in healthcare fields.
Assuntos
Atenção à Saúde , Pessoal de Saúde , Humanos , Incerteza , Pessoal de Saúde/psicologia , EscolaridadeRESUMO
Disease control in animal production systems requires constant vigilance. Historically, the application of in-feed antibiotics to control bacteria and improve performance has been a much-used approach to maintain animal health and welfare. However, the widespread use of in-feed antibiotics is thought to increase the risk of antibiotic resistance developing. Alternative methods to control disease and maintain productivity need to be developed. Live vaccination is useful in preventing colonization of mucosa-dwelling pathogens by inducing a mucosal immune response. Native poultry isolate Ligilactobacillus agilis La3 (previously Lactobacillus agilis) has been identified as a candidate for use as a live vector to deliver therapeutic proteins such as bacteriocins, phage endolysins, or vaccine antigens to the gastrointestinal tract of chickens. In this study, the complete genome sequence of L. agilis La3 was determined and transcriptome analysis was undertaken to identify highly expressed genes. Predicted promoter regions and ribosomal binding sites from constitutively expressed genes were used to construct recombinant protein expression cassettes. A series of double-crossover shuttle plasmids were constructed to facilitate rapid selectable integration of expression cassettes into the Lagilis La3 chromosome via homologous recombination. Inserts showed 100% stable integration over 100 generations without selection. A positive relationship was found between protein expression levels and the predicted strength of the promoters. Using this system, stable chromosomal expression of a Clostridium perfringens antigen, rNetB, was demonstrated without selection. Finally, two recombinant strains, Lagilis La3::P eft -rnetB and Lagilis La3::P cwah -rnetB, were constructed and characterized, and they showed potential for future application as live vaccines in chickens.IMPORTANCE Therapeutic proteins such as antigens can be used to prevent infectious diseases in poultry. However, traditional vaccine delivery by intramuscular or subcutaneous injection generally has not proven effective for mucosa-dwelling microorganisms that live within the gastrointestinal tract. Utilizing live bacteria to deliver vaccine antigens directly to the gut immune system can overcome some of the limitations of conventional vaccination. In this work, Ligilactobacillus agilis La3, an especially effective gut colonizer, has been analyzed and engineered with modular and stable expression systems to produce recombinant proteins. To demonstrate the effectiveness of the system, expression of a vaccine antigen from poultry pathogen Clostridium perfringens was monitored over 100 generations without selection and found to be completely stable. This study demonstrates the development of genetic tools and novel constitutive expression systems and further development of L. agilis La3 as a live delivery vehicle for recombinant proteins.
Assuntos
Proteínas de Bactérias/genética , Vacinas Bacterianas/imunologia , Expressão Gênica/imunologia , Genoma Bacteriano , Lactobacillus/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Clostridium perfringens/fisiologia , Lactobacillus/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vacinas Atenuadas/imunologiaRESUMO
ß-Aminopeptidases have the unique capability to hydrolyze N-terminal ß-amino acids, with varied preferences for the nature of ß-amino acid side chains. This unique capability makes them useful as biocatalysts for synthesis of ß-peptides and to kinetically resolve ß-peptides and amides for the production of enantiopure ß-amino acids. To date, six ß-aminopeptidases have been discovered and functionally characterized, five from Gram-negative bacteria and one from a fungus, Aspergillus Here we report on the purification and characterization of an additional four ß-aminopeptidases, one from a Gram-positive bacterium, Mycolicibacterium smegmatis (BapAMs), one from a yeast, Yarrowia lipolytica (BapAYlip), and two from Gram-negative bacteria isolated from activated sludge identified as Burkholderia spp. (BapABcA5 and BapABcC1). The genes encoding ß-aminopeptidases were cloned, expressed in Escherichia coli, and purified. The ß-aminopeptidases were produced as inactive preproteins that underwent self-cleavage to form active enzymes comprised of two different subunits. The subunits, designated α and ß, appeared to be tightly associated, as the active enzyme was recovered after immobilized-metal affinity chromatography (IMAC) purification, even though only the α-subunit was 6-histidine tagged. The enzymes were shown to hydrolyze chromogenic substrates with the N-terminal l-configurations ß-homo-Gly (ßhGly) and ß3-homo-Leu (ß3hLeu) with high activities. These enzymes displayed higher activity with H-ßhGly-p-nitroanilide (H-ßhGly-pNA) than previously characterized enzymes from other microorganisms. These data indicate that the new ß-aminopeptidases are fully functional, adding to the toolbox of enzymes that could be used to produce ß-peptides. Overexpression studies in Pseudomonas aeruginosa also showed that the ß-aminopeptidases may play a role in some cellular functions.IMPORTANCE ß-Aminopeptidases are unique enzymes found in a diverse range of microorganisms that can utilize synthetic ß-peptides as a sole carbon source. Six ß-aminopeptidases have been previously characterized with preferences for different ß-amino acid substrates and have demonstrated the capability to catalyze not only the degradation of synthetic ß-peptides but also the synthesis of short ß-peptides. Identification of other ß-aminopeptidases adds to this toolbox of enzymes with differing ß-amino acid substrate preferences and kinetics. These enzymes have the potential to be utilized in the sustainable manufacture of ß-amino acid derivatives and ß-peptides for use in biomedical and biomaterial applications. This is important, because ß-amino acids and ß-peptides confer increased proteolytic resistance to bioactive compounds and form novel structures as well as structures similar to α-peptides. The discovery of new enzymes will also provide insight into the biological importance of these enzymes in nature.
Assuntos
Aminopeptidases/genética , Proteínas de Bactérias/genética , Burkholderia/genética , Proteínas Fúngicas/genética , Mycobacteriaceae/genética , Yarrowia/genética , Aminopeptidases/metabolismo , Proteínas de Bactérias/metabolismo , Burkholderia/metabolismo , Proteínas Fúngicas/metabolismo , Cinética , Mycobacteriaceae/metabolismo , Especificidade por Substrato , Yarrowia/metabolismoRESUMO
BACKGROUND: Clostridium perfringens causes a range of diseases in animals and humans including necrotic enteritis in chickens and food poisoning and gas gangrene in humans. Necrotic enteritis is of concern in commercial chicken production due to the cost of the implementation of infection control measures and to productivity losses. This study has focused on the genomic analysis of a range of chicken-derived C. perfringens isolates, from around the world and from different years. The genomes were sequenced and compared with 20 genomes available from public databases, which were from a diverse collection of isolates from chickens, other animals, and humans. We used a distance based phylogeny that was constructed based on gene content rather than sequence identity. Similarity between strains was defined as the number of genes that they have in common divided by their total number of genes. In this type of phylogenetic analysis, evolutionary distance can be interpreted in terms of evolutionary events such as acquisition and loss of genes, whereas the underlying properties (the gene content) can be interpreted in terms of function. We also compared these methods to the sequence-based phylogeny of the core genome. RESULTS: Distinct pathogenic clades of necrotic enteritis-causing C. perfringens were identified. They were characterised by variable regions encoded on the chromosome, with predicted roles in capsule production, adhesion, inhibition of related strains, phage integration, and metabolism. Some strains have almost identical genomes, even though they were isolated from different geographic regions at various times, while other highly distant genomes appear to result in similar outcomes with regard to virulence and pathogenesis. CONCLUSIONS: The high level of diversity in chicken isolates suggests there is no reliable factor that defines a chicken strain of C. perfringens, however, disease-causing strains can be defined by the presence of netB-encoding plasmids. This study reveals that horizontal gene transfer appears to play a significant role in genetic variation of the C. perfringens chromosome as well as the plasmid content within strains.
Assuntos
Clostridium perfringens/genética , Clostridium perfringens/fisiologia , Enterite/microbiologia , Evolução Molecular , Variação Genética , Animais , Galinhas/microbiologia , Cromossomos/genética , Enterite/complicações , Necrose/complicações , Plasmídeos/genéticaRESUMO
Clostridioides (Clostridium) difficile is a major bacterial pathogen of both humans and animals. Several species of pathogenic clostridia are known to harbour large plasmids with combinations of virulence, antibiotic resistance and metabolism determinants. Small cryptic plasmids have been previously identified in C. difficile, but there is a lack of recent work examining the prevalence and heterogeneity of plasmids in this diverse bacterial species. A survey of clinical and historical isolates of C. difficile showed that several strains carry large plasmids. Following whole-genome sequencing of these diverse strains, 42-47â¯kb plasmids with high nucleotide identity were found to be carried in 4.9% (nâ¯=â¯451) of isolates, with no firm connection to the strain backgrounds. These plasmids appear to have arisen as a result of recombination with a bacteriophage, but contain key plasmid features, such as a putative plasmid replication and partitioning locus. As no virulence factors or antibiotic resistance determinants were identified, further work is required to identify the selective advantage that must exist for the host isolates to maintain these large plasmids.
Assuntos
Bacteriófagos/genética , Clostridioides difficile/genética , Clostridioides difficile/patogenicidade , Fases de Leitura Aberta , Plasmídeos/química , Fatores de Virulência/genética , Técnicas de Tipagem Bacteriana , Clostridioides difficile/classificação , Clostridioides difficile/metabolismo , Replicação do DNA , Variação Genética , Filogenia , Plasmídeos/metabolismo , Recombinação Genética , Análise de Sequência de DNA , Virulência , Fatores de Virulência/metabolismoRESUMO
The cytochrome P450 monooxygenases (P450s) catalyze a vast array of oxygenation reactions that can be useful in biocatalytic applications. CYP101J2 from Sphingobium yanoikuyae is a P450 that catalyzes the hydroxylation of 1,8-cineole. Here we report the crystallization and X-ray structure elucidation of recombinant CYP101J2 to 1.8 Å resolution. The CYP101J2 structure shows the canonical P450-fold and has an open conformation in the absence of substrate. Analysis of the structure revealed that CYP101J2, in the absence of substrate, forms a well-ordered substrate-binding channel that suggests a unique form of substrate guidance in comparison to other bacterial 1,8-cineole-hydroxylating P450 enzymes. Proteins 2017; 85:945-950. © 2016 Wiley Periodicals, Inc.
Assuntos
Proteínas de Bactérias/química , Cicloexanóis/química , Sistema Enzimático do Citocromo P-450/química , Monoterpenos/química , Sphingomonadaceae/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Cicloexanóis/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Eucaliptol , Expressão Gênica , Hidroxilação , Modelos Moleculares , Monoterpenos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sphingomonadaceae/enzimologia , Especificidade por SubstratoRESUMO
Clostridium perfringens is a gastrointestinal pathogen capable of causing disease in a variety of hosts. Necrotic enteritis in chickens is caused by C. perfringens strains that produce the pore-forming toxin NetB, the major virulence factor for this disease. Like many other C. perfringens toxins and antibiotic resistance genes, NetB is encoded on a conjugative plasmid. Conjugative transfer of the netB-containing plasmid pJIR3535 has been demonstrated in vitro with a netB-null mutant. This study has investigated the effect of plasmid transfer on disease pathogenesis, with two genetically distinct transconjugants constructed under in vitro conditions, within the intestinal tract of chickens. This study also demonstrates that plasmid transfer can occur naturally in the host gut environment without the need for antibiotic selective pressure to be applied. The demonstration of plasmid transfer within the chicken host may have implications for the progression and pathogenesis of C. perfringens-mediated disease. Such horizontal gene transfer events are likely to be common in the clostridia and may be a key factor in strain evolution, both within animals and in the wider environment.IMPORTANCEClostridium perfringens is a major gastrointestinal pathogen of poultry. C. perfringens strains that express the NetB pore-forming toxin, which is encoded on a conjugative plasmid, cause necrotic enteritis. This study demonstrated that the conjugative transfer of the netB-containing plasmid to two different nonpathogenic strains converted them into disease-causing strains with disease-causing capability similar to that of the donor strain. Plasmid transfer of netB and antibiotic resistance was also demonstrated to occur within the gastrointestinal tract of chickens, with approximately 14% of the isolates recovered comprising three distinct, in vivo-derived, transconjugant types. The demonstration of in vivo plasmid transfer indicates the potential importance of strain plasticity and the contribution of plasmids to strain virulence.
Assuntos
Galinhas , Infecções por Clostridium/veterinária , Clostridium perfringens/genética , Conjugação Genética , Transferência Genética Horizontal , Doenças das Aves Domésticas/microbiologia , Animais , Infecções por Clostridium/microbiologia , Clostridium perfringens/patogenicidade , Trato Gastrointestinal/microbiologia , Plasmídeos/genética , VirulênciaRESUMO
Clostridium perfringens produces an extensive repertoire of toxins and extracellular enzymes, many of which are intimately involved in the progression of disease and are encoded by genes on conjugative plasmids. In addition, many C. perfringens strains can carry up to five of these conjugative toxin or antimicrobial resistance plasmids, each of which has a similar 35kb backbone. This conserved backbone includes the tcp conjugation locus and the central control region (CCR), which encodes genes involved in plasmid regulation, replication and partitioning, including a parMRC partitioning locus. Most conjugative plasmids in C. perfringens have a conserved replication protein, raising questions as to how multiple, closely related plasmids are maintained within a single strain. Bioinformatics analysis has highlighted the presence of at least 10 different parMRC partitioning system families (parMRCA-J) in these plasmids, with differences in amino acid sequence identity between each ParM family ranging from 15% to 54%. No two plasmids that encode genes belonging to the same partitioning family have been observed in a single strain, suggesting that these families represent the basis for plasmid incompatibility. In an attempt to validate the proposed parMRC incompatibility groups, genetically marked C. perfringens plasmids encoding identical parMRCC or parMRCD homologues or different combinations of parMRCA, parMRCC and parMRCD family homologues were introduced into a single strain via conjugation. The stability of each plasmid was determined using an incompatibility assay in which the plasmid profile of each strain was monitored over the course of two days in the absence of direct selection. The results showed that plasmids with identical parMRCC or parMRCD homologues were incompatible and could not coexist in the absence of external selection. By contrast, plasmids that encoded different parMRC homologues were compatible and could coexist in the same cell in the absence of selection, with the exception of strains housing parMRCC and parMRCD combinations, which showed a minor incompatibility phenotype. In conclusion, we have provided the first direct evidence of plasmid incompatibility in Clostridium spp. and have shown experimentally that the compatibility of conjugative C. perfringens plasmids correlates with the presence of parMRC-like partitioning systems of different phylogenetic subfamilies.
Assuntos
Actinas/genética , Proteínas de Bactérias/genética , Clostridium perfringens/genética , Conjugação Genética , DNA Topoisomerase IV/genética , Regulação Bacteriana da Expressão Gênica , Plasmídeos/química , Proteínas Repressoras/genética , Actinas/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Sequência de Bases , Clostridium perfringens/efeitos dos fármacos , Clostridium perfringens/metabolismo , Replicação do DNA , DNA Topoisomerase IV/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Resistência Microbiana a Medicamentos/genética , Loci Gênicos , Plasmídeos/metabolismo , Replicon , Proteínas Repressoras/metabolismoRESUMO
We report the isolation and characterization of three new cytochrome P450 monooxygenases: CYP101J2, CYP101J3, and CYP101J4. These P450s were derived from Sphingobium yanoikuyae B2, a strain that was isolated from activated sludge based on its ability to fully mineralize 1,8-cineole. Genome sequencing of this strain in combination with purification of native 1,8-cineole-binding proteins enabled identification of 1,8-cineole-binding P450s. The P450 enzymes were cloned, heterologously expressed (N-terminally His6 tagged) in Escherichia coli BL21(DE3), purified, and spectroscopically characterized. Recombinant whole-cell biotransformation in E. coli demonstrated that all three P450s hydroxylate 1,8-cineole using electron transport partners from E. coli to yield a product putatively identified as (1S)-2α-hydroxy-1,8-cineole or (1R)-6α-hydroxy-1,8-cineole. The new P450s belong to the CYP101 family and share 47% and 44% identity with other 1,8-cineole-hydroxylating members found in Novosphingobium aromaticivorans and Pseudomonas putida Compared to P450cin (CYP176A1), a 1,8-cineole-hydroxylating P450 from Citrobacter braakii, these enzymes share less than 30% amino acid sequence identity and hydroxylate 1,8-cineole in a different orientation. Expansion of the enzyme toolbox for modification of 1,8-cineole creates a starting point for use of hydroxylated derivatives in a range of industrial applications. IMPORTANCE: CYP101J2, CYP101J3, and CYP101J4 are cytochrome P450 monooxygenases from S. yanoikuyae B2 that hydroxylate the monoterpenoid 1,8-cineole. These enzymes not only play an important role in microbial degradation of this plant-based chemical but also provide an interesting route to synthesize oxygenated 1,8-cineole derivatives for applications as natural flavor and fragrance precursors or incorporation into polymers. The P450 cytochromes also provide an interesting basis from which to compare other enzymes with a similar function and expand the CYP101 family. This could eventually provide enough bacterial parental enzymes with similar amino acid sequences to enable in vitro evolution via DNA shuffling.
Assuntos
Cânfora 5-Mono-Oxigenase/isolamento & purificação , Cânfora 5-Mono-Oxigenase/metabolismo , Cicloexanóis/metabolismo , Monoterpenos/metabolismo , Esgotos/microbiologia , Sphingomonadaceae/enzimologia , Biotransformação , Cânfora 5-Mono-Oxigenase/classificação , Cânfora 5-Mono-Oxigenase/genética , Citrobacter/enzimologia , Citrobacter/genética , Transporte de Elétrons , Escherichia coli/genética , Eucaliptol , Genoma Bacteriano , Hidroxilação , Microbiologia Industrial , Ligação Proteica , Pseudomonas putida/enzimologia , Pseudomonas putida/genética , Proteínas Recombinantes/metabolismo , Sphingomonadaceae/genética , Sphingomonadaceae/isolamento & purificação , Sphingomonadaceae/metabolismoRESUMO
The investigation of genomic variation between Clostridium perfringens isolates from poultry has been an important tool to enhance our understanding of the genetic basis of strain pathogenicity and the epidemiology of virulent and avirulent strains within the context of necrotic enteritis (NE). The earliest studies used whole genome profiling techniques such as pulsed-field gel electrophoresis to differentiate isolates and determine their relative levels of relatedness. DNA sequencing has been used to investigate genetic variation in (a) individual genes, such as those encoding the alpha and NetB toxins; (b) panels of housekeeping genes for multi-locus sequence typing and (c) most recently whole genome sequencing to build a more complete picture of genomic differences between isolates. Conclusions drawn from these studies include: differential carriage of large conjugative plasmids accounts for a large proportion of inter-strain differences; plasmid-encoded genes are more highly conserved than chromosomal genes, perhaps indicating a relatively recent origin for the plasmids; isolates from NE-affected birds fall into three distinct sequence-based clades while non-pathogenic isolates from healthy birds tend to be more genomically diverse. Overall, the NE causing strains are closely related to C. perfringens isolates from other birds and other diseases whereas the non-pathogenic poultry strains are generally more remotely related to either the pathogenic strains or the strains from other birds. Genomic analysis has indicated that genes in addition to netB are associated with NE pathogenic isolates. Collectively, this work has resulted in a deeper understanding of the pathogenesis of this important poultry disease.
Assuntos
Infecções por Clostridium/veterinária , Clostridium perfringens/genética , Enterite/veterinária , Variação Genética , Doenças das Aves Domésticas/microbiologia , Aves Domésticas/microbiologia , Animais , Toxinas Bacterianas/genética , Técnicas de Tipagem Bacteriana/veterinária , Cromossomos Bacterianos/genética , Infecções por Clostridium/microbiologia , Clostridium perfringens/isolamento & purificação , Eletroforese em Gel de Campo Pulsado/veterinária , Enterite/microbiologia , Enterotoxinas/genética , Genômica , Tipagem de Sequências Multilocus/veterinária , Plasmídeos/genéticaRESUMO
Clostridium difficile is a major nosocomial pathogen, causing gastrointestinal disease in patients undergoing antibiotic therapy. This bacterium contains many extrachromosomal and integrated genetic elements, with recent genomic work giving new insights into their variability and distribution. This review summarises research conducted in this area over the last 30 years and includes a discussion on the functional contributions of these elements to host cell phenotypes, as well as encompassing recent genome sequencing studies that have contributed to our understanding of their evolution and dissemination. Importantly, we also include a review of antibiotic resistance determinants associated with mobile genetic elements since antibiotic use and the spread of antibiotic resistance are currently of significant global clinical importance.
Assuntos
Clostridioides difficile/genética , Farmacorresistência Bacteriana/genética , Animais , Sequência de Bases , Clostridioides difficile/efeitos dos fármacos , Conjugação Genética , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Genoma Bacteriano , Humanos , Plasmídeos/genética , Análise de Sequência de DNARESUMO
IMPORTANCE: There has been a decrease in healthcare-associated Clostridioides difficile infection in Australia, but an increase in the genetic diversity of infecting strains, and an increase in community-associated cases. Here, we studied the genetic relatedness of C. difficile isolated from patients at a major hospital in Melbourne, Australia. Diverse ribotypes were detected, including those associated with community and environmental sources. Some types of isolates were more likely to carry antimicrobial resistance determinants, and many of these were associated with mobile genetic elements. These results correlate with those of other recent investigations, supporting the observed increase in genetic diversity and prevalence of community-associated C. difficile, and consequently the importance of sources of transmission other than symptomatic patients. Thus, they reinforce the importance of surveillance for in both hospital and community settings, including asymptomatic carriage, food, animals, and other environmental sources to identify and circumvent important sources of C. difficile transmission.
Assuntos
Clostridioides difficile , Infecções por Clostridium , Infecção Hospitalar , Animais , Humanos , Clostridioides difficile/genética , Infecções por Clostridium/epidemiologia , Genômica , Infecção Hospitalar/epidemiologia , AustráliaRESUMO
Hospital-acquired diarrhoea (HAD) is common, and often associated with gut microbiota and metabolome dysbiosis following antibiotic administration. Clostridioides difficile is the most significant antibiotic-associated diarrhoeal (AAD) pathogen, but less is known about the microbiota and metabolome associated with AAD and C. difficile infection (CDI) with contrasting antibiotic treatment. We characterised faecal microbiota and metabolome for 169 HAD patients (33 with CDI and 133 non-CDI) to determine dysbiosis biomarkers and gain insights into metabolic strategies C. difficile might use for gut colonisation. The specimen microbial community was analysed using 16 S rRNA gene amplicon sequencing, coupled with untargeted metabolite profiling using gas chromatography-mass spectrometry (GC-MS), and short-chain fatty acid (SCFA) profiling using GC-MS. AAD and CDI patients were associated with a spectrum of dysbiosis reflecting non-antibiotic, short-term, and extended-antibiotic treatment. Notably, extended antibiotic treatment was associated with enterococcal proliferation (mostly vancomycin-resistant Enterococcus faecium) coupled with putative biomarkers of enterococcal tyrosine decarboxylation. We also uncovered unrecognised metabolome dynamics associated with concomitant enterococcal proliferation and CDI, including biomarkers of Stickland fermentation and amino acid competition that could distinguish CDI from non-CDI patients. Here we show, candidate metabolic biomarkers for diagnostic development with possible implications for CDI and vancomycin-resistant enterococci (VRE) treatment.
Assuntos
Clostridioides difficile , Infecções por Clostridium , Humanos , Clostridioides difficile/genética , Disbiose , Multiômica , Diarreia , Antibacterianos/efeitos adversos , Biomarcadores , Infecções por Clostridium/diagnóstico , Proliferação de Células , HospitaisRESUMO
Clostridium perfringens causes a wide range of diseases in a variety of hosts, due to the production of a diverse set of toxins and extracellular enzymes. The C. perfringens toxins play an important role in pathogenesis, such that the presence and absence of the toxins is used as a typing scheme for the species. In recent years, several new toxins have been discovered that have been shown to be essential or highly correlated to diseases; these include binary enterotoxin (BecAB), NetB and NetF. In the current study, genome sequence analysis of C. perfringens isolates from diverse sources revealed several putative novel toxin homologs, some of which appeared to be associated with potential mobile genetic elements, including transposons and plasmids. Four novel toxin homologs encoding proteins related to the pore-forming Leukocidin/Hemolysin family were found in type A and G isolates. Two novel toxin homologs encoding proteins related to the epsilon aerolysin-like toxin family were identified in Type A and F isolates from humans, contaminated food and turkeys. A novel set of proteins related to clostridial binary toxins was also identified. While phenotypic characterisation is required before any of these homologs can be established as functional toxins, the in silico identification of these novel homologs on mobile genetic elements suggests the potential toxin reservoir of C. perfringens may be much larger than previously thought.
RESUMO
The American Society for Microbiology's curricular guidelines for Introductory Microbiology highlighted key laboratory skills in the isolation, visualization and identification of microorganisms as core learning objectives in the discipline. Since the publication of these guidelines in 2012, there has been a paucity of diagnostic assessment tools in the literature that can be used to assess competencies in the microbiology laboratory. This project aimed to establish a laboratory competency examination for introductory microbiology, with tasks specifically aligned to laboratory skills and learning outcomes outlined in curricular guidelines for microbiology. A Laboratory Competency Examination assessing student skills in light microscopy, Gram-staining, pure culture, aseptic technique, serial dilution, dilution calculations and pipetting was developed at The University of Queensland, Australia. The Laboratory Competency Examination was field-tested in a large introductory microbiology subject (â¼400 students), and student performance and learning gains data were collected from 2016 to 2017 to evaluate the validity of the assessment. The resulting laboratory assessment is presented as an endpoint diagnostic tool for assessing laboratory competency that can be readily adapted towards different educational contexts.
Assuntos
Currículo/normas , Microbiologia/educação , Competência Profissional/normas , Queensland , Estudantes de MedicinaRESUMO
PROBLEM ADDRESSED: Clostridium perfringens is the etiological agent of necrotic enteritis in chickens. As necrotic enteritis is a gastrointestinal disease, the interactions of pathogenic C. perfringens strains with the complex microbiota of the gastrointestinal tract may influence disease development and severity of disease. OBJECTIVE: In this study the interactions of a pathogenic strain of C. perfringens, WER-NE36, with the microbiota of broilers was investigated to determine whether the pre-existing microbiota could influence disease outcomes in the necrotic enteritis challenge model. Methods and approach: Faecal microbiota compositions were measured before and after C. perfringens challenge and caecal microbiota was also characterised at necropsy. The microbiota profiles from individual birds were related back to the degree of necrotic enteritis that each bird developed. RESULTS: Under the experimental conditions used the pre-existing microbiota did not have an effect on disease outcomes. However, C. perfringens challenge was shown to have a significant effect on the microbiota of broilers, regardless of disease status, by displacement of commensal clostridia. CONCLUSIONS: The microbiota signature after challenge resembled that of lower productivity birds, supporting the finding that physically obvious disease (necrotic lesions), as well as dysbiosis, are associated with shifts in gut microbiota and affect broiler performance, increasing costs to the poultry industry.
Assuntos
Infecções por Clostridium/veterinária , Clostridium perfringens/patogenicidade , Disbiose , Enterite/etiologia , Enterite/microbiologia , Microbioma Gastrointestinal , Necrose/microbiologia , Animais , Galinhas , Infecções por Clostridium/etiologia , Infecções por Clostridium/microbiologia , Infecções por Clostridium/patologia , Clostridium perfringens/genética , Clostridium perfringens/isolamento & purificação , Enterite/patologia , Interações Microbianas , Doenças das Aves Domésticas/etiologia , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/patologia , RNA Ribossômico 16SRESUMO
ß-Aminopeptidases are a unique group of enzymes that have the unusual capability to hydrolyze N-terminal ß-amino acids from synthetic ß-peptides. ß-Peptides can form secondary structures mimicking α-peptide-like structures that are resistant to degradation by most known proteases and peptidases. These characteristics of ß-peptides give them great potential as peptidomimetics. Here, the X-ray crystal structure of BcA5-BapA, a ß-aminopeptidase from a Gram-negative Burkholderia sp. that was isolated from activated sludge from a wastewater-treatment plant in Australia, is reported. The crystal structure of BcA5-BapA was determined to a resolution of 2.0â Å and showed a tetrameric assembly typical of the ß-aminopeptidases. Each monomer consists of an α-subunit (residues 1-238) and a ß-subunit (residues 239-367). Comparison of the structure of BcA5-BapA with those of other known ß-aminopeptidases shows a highly conserved structure and suggests a similar proteolytic mechanism of action.
Assuntos
Aminopeptidases/química , Proteínas de Bactérias/química , Burkholderia/química , Peptídeos/química , Peptidomiméticos/química , Motivos de Aminoácidos , Aminopeptidases/genética , Aminopeptidases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Burkholderia/enzimologia , Burkholderia/isolamento & purificação , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Peptídeos/síntese química , Peptídeos/metabolismo , Peptidomiméticos/síntese química , Peptidomiméticos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteólise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Águas Residuárias/microbiologiaRESUMO
Horizontal gene transfer by mobile genetic elements plays an important role in the evolution of bacteria, allowing them to rapidly acquire new traits, including antibiotic resistance. Mobile genetic elements such as conjugative and mobilizable transposons make up a considerable part of the C. difficile genome. While sequence analysis has identified a large number of these elements, experimental analysis is required to demonstrate mobility and function. This chapter describes the experimental methods utilized for determining function and transfer of mobile genetic elements in C. difficile including detection of the circular transfer intermediate and the analysis and confirmation of mobile genetic element transfer to recipient cells.
Assuntos
Clostridioides difficile/genética , Conjugação Genética , Elementos de DNA Transponíveis , Transferência Genética Horizontal , Genoma Bacteriano , Reação em Cadeia da Polimerase/métodos , Antibacterianos/farmacologia , Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/metabolismo , Primers do DNA/química , Primers do DNA/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Circular/genética , DNA Circular/metabolismo , Eritromicina/farmacologia , Plasmídeos/química , Plasmídeos/metabolismo , Rifampina/farmacologia , Tetraciclina/farmacologia , Tianfenicol/farmacologiaRESUMO
Clostridium difficile is well recognized as the leading cause of antibiotic-associated diarrhea, having a significant impact in both health-care and community settings. Central to predisposition to C. difficile infection is disruption of the gut microbiome by antibiotics. Being a Gram-positive anaerobe, C. difficile is intrinsically resistant to a number of antibiotics. Mobile elements encoding antibiotic resistance determinants have also been characterized in this pathogen. While resistance to antibiotics currently used to treat C. difficile infection has not yet been detected, it may be only a matter of time before this occurs, as has been seen with other bacterial pathogens. This review will discuss C. difficile disease pathogenesis, the impact of antibiotic use on inducing disease susceptibility, and the role of antibiotic resistance and mobile elements in C. difficile epidemiology.
RESUMO
The worldwide emergence of epidemic strains of Clostridium difficile linked to increased disease severity and mortality has resulted in greater research efforts toward determining the virulence factors and pathogenesis mechanisms used by this organism to cause disease. C. difficile is an opportunist pathogen that employs many factors to infect and damage the host, often with devastating consequences. This review will focus on the role of the 2 major virulence factors, toxin A (TcdA) and toxin B (TcdB), as well as the role of other putative virulence factors, such as binary toxin, in C. difficile-mediated infection. Consideration is given to the importance of spores in both the initiation of disease and disease recurrence and also to the role that surface proteins play in host interactions.