RESUMO
Staphylococcus aureus is the most prevalent cystic fibrosis (CF) pathogen. Several phenotypes are associated with worsened CF clinical outcomes including methicillin-resistance and small-colony-variants. The inoculum effect (IE) is characterized by reduced ß-lactam susceptibility when assessed at high inoculum. The IE associates with worse outcomes in bacteremia and other high-density infections, and may therefore be relevant to CF. The prevalence of IE amongst a CF cohort (age ≥18 years), followed from 2013 to 2016, was investigated. Yearly methicillin-sensitive S. aureus (MSSA) isolates were screened at standard (5 × 105 CFU/mL) and high (5 × 107 CFU/mL) inoculum against narrow-spectrum anti-Staphylococcal ß-lactams and those with anti-pseudomonal activity common to CF. A ≥ 4-fold increase in minimum inhibitory concentration between standard and high inoculum defined IE. Isolates underwent blaZ sequencing and genotyping and were compared against published genomes. Fifty-six percent (99/177) of individuals had MSSA infection. MSSA was observed at ≥105 CFU/mL in 44.8% of entry sputum samples. The prevalence of the IE was 25.0%-cefazolin; 13.5%-cloxacillin; 0%-meropenem; 1.0%-cefepime; 5.2%-ceftazidime; and 34.4%-piperacillin-tazobactam amongst baseline MSSA isolates assessed. blaZ A associated with cefazolin IE (P = 0.0011), whereas blaZ C associated with piperacillin-tazobactam IE (P < 0.0001). Baseline demographics did not reveal specific risk factors for IE-associated infections, nor were long-term outcomes different. Herein, we observed the IE in CF-derived MSSA disproportionally for cefazolin and piperacillin-tazobactam and this phenotype strongly associated with underlying blaZ genotype. The confirmation of CF being a high density infection, and the identification of high prevalence of MSSA with IE in CF supports the need for prospective pulmonary exacerbation treatment studies to understand the impact of this phenotype.
Assuntos
Fibrose Cística , Infecções Estafilocócicas , Adulto , Humanos , Adolescente , Meticilina/farmacologia , Meticilina/uso terapêutico , Cefazolina/farmacologia , Staphylococcus aureus/genética , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Estudos Prospectivos , Fibrose Cística/tratamento farmacológico , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/epidemiologia , Monobactamas/farmacologia , Combinação Piperacilina e Tazobactam/uso terapêutico , Ceftazidima/farmacologia , Antibióticos beta Lactam , Testes de Sensibilidade MicrobianaRESUMO
BACKGROUND: CF is traditionally assessed in clinic. It is unclear if home monitoring of young people with CF is feasible or acceptable. The COVID-19 pandemic has made home monitoring more of a necessity. We report the results of CLIMB-CF, exploring home monitoring's feasibility and potential obstacles. METHODS: We designed a mobile app and enrolled participants with CF aged 2-17 years and their parents for six months. They were asked to complete a variety of measures either daily or twice a week. During the study, participants and their parents completed questionnaires exploring depression, anxiety and quality of life. At the end of the study parents and participants completed acceptability questionnaires. RESULTS: 148 participants were recruited, 4 withdrew prior to starting the study. 82 participants were female with median (IQR) age 7.9 (5.2-12 years). Median data completeness was 40.1% (13.6-69.9%) for the whole cohort; when assessed by age participants aged ≥ 12 years contributed significantly less (15.6% [9.8-30%]). Data completeness decreased over time. There was no significant difference between parental depression and anxiety scores at the start and the end of the study nor in CFQ-R respiratory domain scores for participants ≥ 14 years. The majority of participants did not feel the introduction of home monitoring impacted their daily lives. CONCLUSIONS: Most participants felt home monitoring did not negatively impact their lives and it did not increase depression, anxiety or decrease quality of life. However, uptake was variable, and not well sustained. The teenage years pose a particular challenge and further work is required.
Assuntos
Fibrose Cística/terapia , Aplicativos Móveis , Monitorização Fisiológica/métodos , Monitorização Fisiológica/psicologia , Qualidade de Vida , Adolescente , Ansiedade , COVID-19/epidemiologia , Criança , Pré-Escolar , Depressão , Estudos de Viabilidade , Feminino , Humanos , Masculino , Pandemias , SARS-CoV-2RESUMO
OBJECTIVE: To compare the gut microbiome profile (by way of taxon analysis and indices of ß- and α-diversity) and inflammatory markers (C-reactive protein [CRP], interleukin-6[IL-6] and tumour necrosis factor-α [TNF-α]) of obsessive-compulsive disorder (OCD) outpatients and non-psychiatric community controls. METHODS: We collected morning stool and blood samples from 21 non-depressed, medication-free OCD patients and 22 age- and sex-matched non-psychiatric community controls. Microbiota analysis was performed using Illumina sequencing of the V3 region of 16S rRNA; serum CRP samples were analysed using immunoturbidimetry and plasma IL-6/TNF-α were examined by high-sensitivity ELISA. Multiple comparisons were corrected for using the false discovery rate (α = 0.05). RESULTS: Compared to controls, the OCD group presented lower species richness/evenness (α-diversity, Inverse Simpson) and lower relative abundance of three butyrate producing genera (Oscillospira, Odoribacter and Anaerostipes). Compared to controls, mean CRP, but not IL-6 and TNF-α, was elevated OCD patients. CRP revealed moderate to strong associations with psychiatric symptomatology. CONCLUSION: To our knowledge, this is the first investigation of the gut microbiome in OCD. In addition, our findings lend further support for the potential association of inflammation and OCD. These results suggest the gut microbiome may be a potential pathway of interest for future OCD research.
Assuntos
Microbioma Gastrointestinal , Transtorno Obsessivo-Compulsivo , Humanos , Inflamação , Projetos Piloto , RNA Ribossômico 16S/genéticaRESUMO
BACKGROUND: Analysis of "emerging" pathogens in cystic fibrosis (CF) lung disease has focused on unique pathogens that are rare in other human diseases or are drug resistant. Escherichia coli is recovered in the sputum of up to 25% of patients with CF, yet little is known about the epidemiology or clinical impact of infection. METHODS: We studied patients attending a Canadian adult CF clinic who had positive sputum cultures for E coli from 1978 to 2016. Infection was categorized as transient or persistent (≥3 positive sputum cultures, spanning >6 months). Those with persistent infection were matched 2:1 with age, sex, and time-period controls without history of E coli infection, and mixed-effects models were used to assess pulmonary exacerbation (PEx) frequency, lung function decline, hospitalization, and intravenous antibiotic days. RESULTS: Forty-five patients (12.3%) had E coli recovered from sputum samples between 1978 and 2016, and 18 patients (40%) developed persistent infection. Nine patients (24%) had PEx at incident infection, and increased bioburden was predictive of exacerbation (P = .03). Risk factors for persistent infection included lower nutritional status (P < .001) and lower lung function (P = .009), but chronic infection with Pseudomonas aeruginosa was protective. There was no difference in annual lung function decline, need for hospitalization or intravenous antibiotics, or risk of PEx in patients with persistent infection. CONCLUSIONS: Persistent E coli infection was frequent and was more common in CF patients with low nutritional status and lung function. However, this does not predict clinical decline. Multicenter studies would allow better characterization of the epidemiology and clinical impact of E coli infection.
RESUMO
A diverse microbiota exists within the airways of individuals with non-cystic fibrosis bronchiectasis (nCFB). How the lung microbiome evolves over time, and whether changes within the microbiome correlate with future disease progression is not yet known. We assessed the microbial community structure of 133 serial sputa and subsequent disease course of 29 nCFB patients collected over a span of 4-16 years using 16S rRNA paired-end sequencing. Interestingly, no significant shifts in the microbial community of individuals were observed during extended follow-up suggesting the microbiome remains relatively stable over prolonged periods. Samples that were Pseudomonas aeruginosa culture positive displayed markedly different microbial community structures compared to those that were positive for Haemophilus influenzae. Importantly, patients with sputum of lower microbial community diversity were more likely to experience subsequent lung function decline as defined by annual change in ≥-1 FEV1% predicted. Shannon diversity values <1 were more prevalent in patients with FEV1 decline (P = 0.002). However, the relative abundance of particular core microbiota constituents did not associate with risk of decline. Here we present data confirming that the microbiome of nCFB individuals is generally stable, and that microbiome-based measurements may have a prognostic role as biomarkers for nCFB.
Assuntos
Brônquios/microbiologia , Bronquiectasia/microbiologia , Microbiota , Adulto , Idoso , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bronquiectasia/tratamento farmacológico , Feminino , Humanos , Estudos Longitudinais , Masculino , Microbiota/efeitos dos fármacos , Pessoa de Meia-IdadeRESUMO
5-lipoxygenase (5-LO) catalyzes the conversion of arachidonic acid (AA) into pro-inflammatory leukotrienes. N-3 PUFA like eicosapentaenoic acid are subject to a similar metabolism and are precursors of pro-resolving mediators. Stearidonic acid (18:4 n-3, SDA) is a plant source of n-3 PUFA that is elongated to 20:4 n-3, an analogue of AA. However, no 5-LO metabolites of 20:4 n-3 have been reported. In this study, control and 5-LO-expressing HEK293 cells were stimulated in the presence of 20:4 n-3. Metabolites were characterized by LC-MS/MS and their anti-inflammatory properties assessed using AA-induced autocrine neutrophil stimulation and leukotriene B4-mediated chemotaxis. 8hydroxy9,11,14,17-eicosatetraenoic acid (Δ17-8-HETE) and 8,15-dihydroxy-9,11,13,17-eicosatetraenoic acid (Δ17-8,15-diHETE) were identified as novel metabolites. Δ17-8,15-diHETE production was inhibited by the leukotriene A4 hydrolase inhibitor SC 57461A. Autocrine neutrophil leukotriene stimulation and neutrophil chemotaxis, both BLT1-dependent processes, were inhibited by Δ17-8,15-diHETE at low nM concentrations. These data support an anti-inflammatory role for Δ17-8,15-diHETE, a novel 5-LO product.
Assuntos
Anti-Inflamatórios/metabolismo , Araquidonato 5-Lipoxigenase/metabolismo , Ácidos Hidroxieicosatetraenoicos/biossíntese , Leucotrieno B4/biossíntese , Neutrófilos/enzimologia , Ácido Araquidônico/metabolismo , Ácido Eicosapentaenoico/análogos & derivados , Ácido Eicosapentaenoico/metabolismo , Feminino , Células HEK293 , Humanos , MasculinoRESUMO
The realisation that microbes regarded as beneficial to the host can impart effects at sites distant from their habitat, has raised many possibilities for treatment of diseases. The objective of a workshop hosted in Turku, Finland, by the International Scientific Association for Probiotics and Prebiotics, was to assess the evidence for these effects and the extent to which early life microbiome programming influences how the gut microbiota communicates with distant sites. In addition, we examined how probiotics and prebiotics might affect the skin, airways, heart, brain and metabolism. The growing levels of scientific and clinical evidence showing how microbes influence the physiology of many body sites, leads us to call for more funding to advance a potentially exciting avenue for novel therapies for many chronic diseases.
Assuntos
Doença Crônica/terapia , Prebióticos/administração & dosagem , Probióticos/administração & dosagem , Animais , Microbioma Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/microbiologia , Humanos , Prebióticos/análise , Probióticos/químicaRESUMO
Achromobacter species are increasingly being detected in cystic fibrosis (CF) patients, with an unclear epidemiology and impact. We studied a cohort of patients attending a Canadian adult CF clinic who had positive sputum cultures for Achromobacter species in the period from 1984 to 2013. Infection was categorized as transient or persistent (≥50% positive cultures for 1 year). Those with persistent infection were matched 2:1 with age-, sex-, and time-matched controls without a history of Achromobacter infection, and mixed-effects models were used to assess pulmonary exacerbation (PEx) frequency and lung function decline. Isolates from a biobank were retrospectively assessed, identified to the species level by nrdA sequencing, and genotyped using pulsed-field gel electrophoresis (PFGE). Thirty-four patients (11% of those in our clinic), with a median age of 24 years (interquartile range [IQR], 20.3 to 29.8 years), developed Achromobacter infection. Ten patients (29%) developed persistent infection. Persistence did not denote permanence, as most patients ultimately cleared infection, often after years. Patients were more likely to experience PEx at incident isolation than at prior or subsequent visits (odds ratio [OR], 2.7 [95% confidence interval {CI}, 1.2 to 6.7]; P = 0.03). Following persistent infection, there was no difference in annual lung function decline (-1.08% [95% CI, -2.73 to 0.57%] versus -2.74% [95% CI, -4.02 to 1.46%]; P = 0.12) or the odds of PEx (OR, 1.21 [95% CI, 0.45 to 3.28]; P = 0.70). Differential virulence among Achromobacter species was not observed, and no cases of transmission occurred. We demonstrated that incident Achromobacter infection was associated with a greater risk of PEx; however, neither transient nor chronic infection was associated with a worsened long-term prognosis. Large, multicenter studies are needed to clarify the clinical impact, natural history, and transmissibility of Achromobacter.
Assuntos
Achromobacter/isolamento & purificação , Fibrose Cística/complicações , Infecções por Bactérias Gram-Negativas/epidemiologia , Achromobacter/classificação , Achromobacter/genética , Adolescente , Adulto , Feminino , Seguimentos , Infecções por Bactérias Gram-Negativas/patologia , Humanos , Masculino , América do Norte/epidemiologia , Prevalência , Testes de Função Respiratória , Estudos Retrospectivos , Resultado do Tratamento , Adulto JovemRESUMO
The European and Developing Countries Clinical Trials Partnership (EDCTP) was established in 2003 to accelerate the development of medical interventions for tuberculosis (TB), human immunodeficiency virus (HIV) and malaria, with a particular focus on Phase II and III clinical trials. Between 2003 and 2015, the first EDCTP programme committed 65.6 million to research on TB and TB-HIV co-infection. The programme made a significant contribution to the first three elements of the DOTS TB control strategy, which mobilised European and African funding for TB-related research and generated important evidence on TB diagnostics and treatment regimens. As well as informing the development of international policy on TB diagnosis and treatment, the programme also significantly enhanced the capacity of countries in sub-Saharan Africa to undertake clinical trials and associated clinical research. The lessons learned from the first EDCTP programme have informed the development of a second, expanded EDCTP programme, EDCTP2, which was launched in 2014, and is due to run until 2024. One key lesson is the need for continued partnerships to fight the global threat of TB.
Assuntos
Antituberculosos/administração & dosagem , Política de Saúde , Cooperação Internacional , Tuberculose/terapia , África Subsaariana/epidemiologia , Coinfecção , Países em Desenvolvimento , Terapia Diretamente Observada , Infecções por HIV/epidemiologia , Infecções por HIV/terapia , Humanos , Internacionalidade , Malária/epidemiologia , Malária/terapia , Desenvolvimento de Programas , Avaliação de Programas e Projetos de Saúde , Tuberculose/diagnóstico , Tuberculose/epidemiologiaRESUMO
Microbial communities reside in healthy tissues but are often disrupted during disease. Bacterial genomes and proteins are detected in brains from humans, nonhuman primates, rodents and other species in the absence of neurological disease. We investigated the composition and abundance of microbiota in frozen and fixed autopsied brain samples from patients with multiple sclerosis (MS) and age- and sex-matched nonMS patients as controls, using neuropathological, molecular and bioinformatics tools. 16s rRNA sequencing revealed Proteobacteria to be the dominant phylum with restricted diversity in cerebral white matter (WM) from MS compared to nonMS patients. Both clinical groups displayed 1,200-1,400 bacterial genomes/cm3 and low bacterial rRNA:rDNA ratios in WM. RNAseq analyses showed a predominance of Proteobacteria in progressive MS patients' WM, associated with increased inflammatory gene expression, relative to a broader range of bacterial phyla in relapsing-remitting MS patients' WM. Although bacterial peptidoglycan (PGN) and RNA polymerase beta subunit immunoreactivities were observed in all patients, PGN immunodetection was correlated with demyelination and neuroinflammation in MS brains. Principal component analysis revealed that demyelination, PGN and inflammatory gene expression accounted for 86% of the observed variance. Thus, inflammatory demyelination is linked to an organ-specific dysbiosis in MS that could contribute to underlying disease mechanisms.
Assuntos
Encéfalo/microbiologia , Doenças Desmielinizantes/microbiologia , Disbiose/microbiologia , Esclerose Múltipla/microbiologia , Proteobactérias/isolamento & purificação , Substância Branca/microbiologia , Actinobacteria/classificação , Actinobacteria/genética , Actinobacteria/isolamento & purificação , Adulto , Idoso , Idoso de 80 Anos ou mais , Autopsia , Encéfalo/patologia , Estudos de Casos e Controles , Cianobactérias/classificação , Cianobactérias/genética , Cianobactérias/isolamento & purificação , DNA Bacteriano/genética , Doenças Desmielinizantes/patologia , Disbiose/patologia , Feminino , Humanos , Inflamação , Masculino , Microbiota/genética , Pessoa de Meia-Idade , Esclerose Múltipla/patologia , Análise de Componente Principal , Proteobactérias/classificação , Proteobactérias/genética , RNA Ribossômico 16S/genética , Substância Branca/patologiaRESUMO
BACKGROUND AND AIMS: Non-alcoholic fatty liver disease is now the leading liver disease in North America. The progression of non-alcoholic fatty liver disease to the inflammatory condition, non-alcoholic steatohepatitis is complex and currently not well understood. Intestinal microbial dysbiosis has been implicated in the development of non-alcoholic fatty liver disease and progression of non-alcoholic steatohepatitis. Volatile organic compounds are byproducts of microbial metabolism in the gut that may enter portal circulation and have hepatotoxic effects contributing to the pathogenesis of non-alcoholic steatohepatitis. To test this hypothesis, we measured volatile organic compounds in cecal luminal contents and portal venous blood in a mouse model of non-alcoholic steatohepatitis. METHODS: Gas chromatography-mass spectrometry analysis was conducted on cecal content and portal vein blood for volatile organic compound detection from mice fed a methionine and choline deficient diet, which induces non-alcoholic steatohepatitis. The colonic microbiome was studied by 16S rRNA gene amplification using the Illumina MiSeq platform. RESULTS: Sixty-eight volatile organic compounds were detected in cecal luminal content, a subset of which was also present in portal venous blood. Importantly, differences in portal venous volatile organic compounds were associated with diet-induced steatohepatitis establishing a biochemical link between gut microbiota-derived volatile organic compounds and increased susceptibility to non-alcoholic steatohepatitis. CONCLUSION: Our model creates a novel tool to further study the role of gut-derived volatile organic compounds in the pathogenesis of non-alcoholic steatohepatitis.
Assuntos
Inflamação/microbiologia , Fígado/irrigação sanguínea , Hepatopatia Gordurosa não Alcoólica/sangue , Hepatopatia Gordurosa não Alcoólica/microbiologia , Veia Porta/microbiologia , Compostos Orgânicos Voláteis/análise , Animais , Bactérias/isolamento & purificação , Células Cultivadas , Modelos Animais de Doenças , Inflamação/patologia , Mediadores da Inflamação/análise , Fígado/microbiologia , Fígado/patologia , Macrófagos/microbiologia , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Microbiota , Hepatopatia Gordurosa não Alcoólica/patologia , Veia Porta/patologiaRESUMO
The microbiome is the complex collection of microorganisms, their genes, and their metabolites, colonizing the human and animal mucosal surfaces, digestive tract, and skin. It is now well known that the microbiome interacts with its host, assisting in digestion and detoxification, supporting immunity, protecting against pathogens, and maintaining health. Studies published to date have demonstrated that healthy individuals are often colonized with different microbiomes than those with disease involving various organ systems. This review covers a brief history of the development of the microbiome field, the main objectives of the Human Microbiome Project, and the most common microbiomes inhabiting the human respiratory tract, companion animal digestive tract, and skin in humans and companion animals. The main changes in the microbiomes in patients with pulmonary, gastrointestinal, and cutaneous lesions are described.
Assuntos
Microbiota , Animais , Trato Gastrointestinal/microbiologia , Humanos , Pulmão/microbiologia , Animais de Estimação , Sistema Respiratório/microbiologia , Pele/microbiologiaRESUMO
The monitoring of epidemic Pseudomonas aeruginosa is important for cystic fibrosis (CF) infection control. The prairie epidemic strain (PES) is common in western Canadian CF clinics. Using whole-genome sequencing, we identified a novel genomic island and developed a PCR assay for PES. Against a collection of 186 P. aeruginosa isolates, the assay had 98% sensitivity and 100% specificity.
Assuntos
Fibrose Cística/complicações , Reação em Cadeia da Polimerase , Infecções por Pseudomonas/diagnóstico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/classificação , Pseudomonas aeruginosa/genética , Eletroforese em Gel de Campo Pulsado , Genoma Bacteriano , Humanos , Tipagem de Sequências Multilocus/métodos , Reação em Cadeia da Polimerase/métodos , Técnica de Amplificação ao Acaso de DNA Polimórfico , Sensibilidade e EspecificidadeRESUMO
An increase in P13 Kinase activity and an associated reduction in histone deacetylase activity may contribute to both relative steroid insensitivity in patients with severe eosinophilic asthma and impaired macrophage scavenger function and susceptibility to recurrent infective bronchitis that may, in turn, contribute to further steroid insensitivity.
Assuntos
Asma/complicações , Asma/metabolismo , Infecções Bacterianas/complicações , Infecções Bacterianas/microbiologia , Bronquite/complicações , Bronquite/microbiologia , Adolescente , Adulto , Idoso , Asma/diagnóstico , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Escarro/enzimologia , Escarro/imunologia , Adulto JovemRESUMO
BACKGROUND: Epidemic P. aeruginosa (ePA) infections are common in cystic fibrosis (CF) and have been associated with accelerated clinical decline. Factors associated with ePA are unclear, and evidence based infection control interventions are lacking. METHODS: We prospectively collect all bacterial pathogens from adult CF patients. We performed PA strain typing on retrospectively collected enrollment samples and recent isolates to identify patients infected with ePA. All patients attending our clinic were approached to complete a survey on infection control knowledge, beliefs and exposures. We analyzed responses of those with ePA relative to the entire cohort without ePA as well as those infected with unique strains of P. aeruginosa to assess for risk factors for ePA and differences in infection control knowledge, beliefs or behaviours. RESULTS: Of 144 participants, 30 patients had ePA (two Liverpool epidemic strain, 28 Prairie epidemic strain), 83 % of which had established infection prior to transition to the adult clinic. Risk of concomitant infecting pathogens was no different between groups although, Staphylococcus aureus and non-tuberculous mycobacteria were less common in those with ePA. Patients with ePA were more likely to have attended CF-camp and have a history of CF fundraising. Patients with ePA did not differ with respect to beliefs regarding pathogens or transmission risk, except they believed indirect contact posed little risk. Furthermore, patients with ePA were more likely to continue to associate with others with CF despite extensive counselling. Use of peer-peer online networking was minimal in both groups. CONCLUSION: Infections with ePA are closely linked to past exposures, now routinely discouraged. As socialization is the greatest risk factor for ePA, infection control strategies for ePA must focus on discouraging face-to-face interactions amongst CF patients. As peer support remains a desire amongst patients, investment in technologies and strategies that enable indirect communication and support are required.
Assuntos
Fibrose Cística/psicologia , Conhecimentos, Atitudes e Prática em Saúde , Controle de Infecções , Infecções por Pseudomonas/psicologia , Pseudomonas aeruginosa , Adulto , Coinfecção/epidemiologia , Fibrose Cística/epidemiologia , Epidemias , Feminino , Humanos , Masculino , Infecções por Mycobacterium não Tuberculosas/epidemiologia , Micobactérias não Tuberculosas , Grupo Associado , Infecções por Pseudomonas/epidemiologia , Infecções por Pseudomonas/microbiologia , Estudos Retrospectivos , Fatores de Risco , Apoio Social , Infecções Estafilocócicas/epidemiologia , Staphylococcus aureus , Adulto JovemRESUMO
BACKGROUND: The application of molecular based diagnostics in sepsis has had limited success to date. Molecular community profiling methods have indicated that polymicrobial infections are more common than suggested by standard clinical culture. A molecular profiling approach was developed to investigate the propensity for polymicrobial infections in patients predicted to have bacterial sepsis. RESULTS: Disruption of blood cells with saponin and hypotonic shock enabled the recovery of microbial cells with no significant changes in microbial growth when compared to CFU/ml values immediately prior to the addition of saponin. DNA extraction included a cell-wall digestion step with both lysozyme and mutanolysin, which increased the recovery of terminal restriction fragments by 2.4 fold from diverse organisms. Efficiencies of recovery and limits of detection using Illumina sequencing of the 16S rRNA V3 region were determined for both viable cells and DNA using mock bacterial communities inoculated into whole blood. Bacteria from pre-defined communities could be recovered following lysis and removal of host cells with >97% recovery of total DNA present. Applying the molecular profiling methodology to three septic patients in the intensive care unit revealed microbial DNA from blood had consistent alignment with cultured organisms from the primary infection site providing evidence for a bloodstream infection in the absence of a clinical lab positive blood culture result in two of the three cases. In addition, the molecular profiling indicated greater diversity was present in the primary infection sample when compared to clinical diagnostic culture. CONCLUSIONS: A method for analyzing bacterial DNA from whole blood was developed in order to characterize the bacterial DNA profile of sepsis infections. Preliminary results indicated that sepsis infections were polymicrobial in nature with the bacterial DNA recovered suggesting a more complex etiology when compared to blood culture data.
Assuntos
Bactérias/classificação , Bactérias/genética , Sangue/microbiologia , Coinfecção/diagnóstico , DNA Bacteriano/genética , Técnicas de Diagnóstico Molecular/métodos , Sepse/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Coinfecção/microbiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Ribossômico 16S/genética , Sepse/microbiologia , Adulto JovemRESUMO
Early-life stress is a determinant of vulnerability to a variety of disorders that include dysfunction of the brain and gut. Here we exploit a model of early-life stress, maternal separation (MS) in mice, to investigate the role of the intestinal microbiota in the development of impaired gut function and altered behaviour later in life. Using germ-free and specific pathogen-free mice, we demonstrate that MS alters the hypothalamic-pituitary-adrenal axis and colonic cholinergic neural regulation in a microbiota-independent fashion. However, microbiota is required for the induction of anxiety-like behaviour and behavioural despair. Colonization of adult germ-free MS and control mice with the same microbiota produces distinct microbial profiles, which are associated with altered behaviour in MS, but not in control mice. These results indicate that MS-induced changes in host physiology lead to intestinal dysbiosis, which is a critical determinant of the abnormal behaviour that characterizes this model of early-life stress.
Assuntos
Ansiedade/microbiologia , Comportamento Animal , Colo/microbiologia , Disbiose/microbiologia , Microbioma Gastrointestinal , Privação Materna , Estresse Psicológico/microbiologia , Tonsila do Cerebelo/metabolismo , Animais , Ansiedade/metabolismo , Ansiedade/psicologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteína C-Reativa/metabolismo , Colo/inervação , Corticosterona/metabolismo , Dopamina/metabolismo , Disbiose/metabolismo , Disbiose/psicologia , Microbioma Gastrointestinal/genética , Sequenciamento de Nucleotídeos em Larga Escala , Hipocampo/metabolismo , Sistema Hipotálamo-Hipofisário/metabolismo , Camundongos , Modelos Animais , Plexo Mientérico , Norepinefrina/metabolismo , Peroxidase/metabolismo , Sistema Hipófise-Suprarrenal/metabolismo , RNA Ribossômico 16S/genética , Serotonina/metabolismo , Estresse Psicológico/metabolismo , Estresse Psicológico/psicologiaRESUMO
The polymicrobial nature of invasive pyogenic infections may be underestimated by routine culture practices, due to the fastidious nature of many organisms and the loss of viability during transport or from prior antibacterials. Pyrosequencing was performed on brain and liver abscesses and pleural fluid and compared to routine culture data. Forty-seven invasive pyogenic infection samples from 44 patients [6 intracerebral abscess (ICA), 21 pyogenic liver abscess (PLA), and 18 pleural fluid (PF) samples] were assayed. Pyrosequencing identified an etiologic microorganism in 100 % of samples versus 45 % by culture, p <0.01. Pyrosequencing was also more likely than traditional cultures to classify infections as polymicrobial, 91 % versus 17 %, p <0.001. The median number of genera identified by pyrosequencing compared to culture was 1 [interquartile range (IQR) 1-3] versus 0 (IQR 0-1) for ICA, 7 (IQR 1-15) versus 1 (IQR 0-1) for PLA, and 15 (IQR 9-19) versus 0 (IQR 0-1) for PF. Where organisms were cultured, they typically represented the numerically dominant species identified by pyrosequencing. Complex microbial communities are involved in invasive pyogenic infection of the lung, liver, and brain. Defining the polymicrobial nature of invasive pyogenic infections is the first step towards appreciating the clinical and diagnostic implications of these complex communities.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Abscesso Encefálico/microbiologia , Empiema/microbiologia , Abscesso Hepático Piogênico/microbiologia , Streptococcus/genética , Adolescente , Adulto , Idoso , Carga Bacteriana , Criança , Pré-Escolar , Técnicas de Cultura , DNA Bacteriano/genética , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/isolamento & purificação , Humanos , Pessoa de Meia-Idade , Derrame Pleural/microbiologia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Infecções Estreptocócicas/microbiologia , Streptococcus/classificação , Streptococcus/isolamento & purificação , Adulto JovemRESUMO
Establishing the risk of human infection is one of the goals of public health. For bacterial pathogens, the virulence and zoonotic potential can often be related to their host source. Escherichia coli bacteria are common contaminants of water associated with human recreation and consumption, and many strains are pathogenic. In this study, we analyzed three promoter-containing intergenic regions from 284 diverse E. coli isolates in an attempt to identify molecular signatures associated with specific host types. Promoter sequences controlling production of curli fimbriae, flagella, and nutrient import yielded a phylogenetic tree with isolates clustered by established phylogenetic grouping (A, B1, B2, and D) but not by host source. Virulence genes were more prevalent in groups B2 and D isolates and in human isolates. Group B1 isolates, primarily from nonhuman sources, were the most genetically similar, indicating that they lacked molecular adaptations to specific host environments and were likely host generalists. Conversely, B2 isolates, primarily from human sources, displayed greater genetic distances and were more likely to be host adapted. In agreement with these hypotheses, prevalence of σ(S) activity and the rdar morphotype, phenotypes associated with environmental survival, were significantly higher in B1 isolates than in B2 isolates. Based on our findings, we speculate that E. coli host specificity is not defined by genome-wide sequence changes but, rather, by the presence or absence of specific genes and associated promoter elements. Furthermore, the requirements for colonization of the human gastrointestinal tract may lead to E. coli lifestyle changes along with selection for increased virulence.