RESUMO
Given the increase in resistance to antibacterial agents, there is an urgent need for the development of new agents with novel modes of action. As an interim solution, it is also prudent to reinvestigate old or abandoned antibacterial compounds to assess their efficacy in the context of widespread resistance to conventional agents. In the 1970s, much work was performed on the development of peptide mimetics, exemplified by the phosphonopeptide, alafosfalin. We investigated the activity of alafosfalin, di-alanyl fosfalin and ß-chloro-L-alanyl-ß-chloro-L-alanine against 297 bacterial isolates, including carbapenemase-producing Enterobacterales (CPE) (n = 128), methicillin-resistant Staphylococcus aureus (MRSA) (n = 37) and glycopeptide-resistant enterococci (GRE) (n = 43). The interaction of alafosfalin with meropenem was also examined against 20 isolates of CPE. The MIC50 and MIC90 of alafosfalin for CPE were 1 mg/L and 4 mg/L, respectively and alafosfalin acted synergistically when combined with meropenem against 16 of 20 isolates of CPE. Di-alanyl fosfalin showed potent activity against glycopeptide-resistant isolates of Enterococcus faecalis (MIC90; 0.5 mg/L) and Enterococcus faecium (MIC90; 2 mg/L). Alafosfalin was only moderately active against MRSA (MIC90; 8 mg/L), whereas ß-chloro-L-alanyl-ß-chloro-L-alanine was slightly more active (MIC90; 4 mg/L). This study shows that phosphonopeptides, including alafosfalin, may have a therapeutic role to play in an era of increasing antibacterial resistance.
Assuntos
Antibacterianos , Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecium/crescimento & desenvolvimento , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Peptídeos , Fosfoproteínas , Antibacterianos/química , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Peptídeos/química , Peptídeos/farmacologia , Fosfoproteínas/química , Fosfoproteínas/farmacologiaRESUMO
RATIONALE: Aspiration of infective subglottic secretions causes ventilator-associated pneumonia (VAP) in mechanically ventilated patients. Mechanisms underlying subglottic colonization in critical illness have not been defined, limiting strategies for targeted prevention of VAP. OBJECTIVES: To characterize subglottic host defense dysfunction in mechanically ventilated patients in the ICU; to determine whether subglottic mucin contributes to neutrophil phagocytic impairment and bacterial growth. METHODS: Prospective subglottic sampling in mechanically ventilated patients (intubated for four or more days), and newly intubated control patients (intubated for less than 30 min); isolation and culture of primary subglottic epithelial cells from control patients; laboratory analysis of host innate immune defenses. MEASUREMENTS AND MAIN RESULTS: Twenty-four patients in the ICU and 27 newly intubated control patients were studied. Subglottic ICU samples had significantly reduced microbiological diversity and contained potential respiratory pathogens. The subglottic microenvironment in the ICU was characterized by neutrophilic inflammation, significantly increased proinflammatory cytokines and neutrophil proteases, and altered physical properties of subglottic secretions, including accumulation of mucins. Subglottic mucin from ICU patients impaired the capacity of neutrophils to phagocytose and kill bacteria. Phagocytic impairment was reversible on treatment with a mucolytic agent. Subglottic mucus promoted growth and invasion of bacterial pathogens in a novel air-liquid interface model of primary human subglottic epithelium. CONCLUSIONS: Mechanical ventilation in the ICU is characterized by substantial mucin secretion and neutrophilic inflammation. Mucin impairs neutrophil function and promotes bacterial growth. Mucolytic agents reverse mucin-mediated neutrophil dysfunction. Enhanced mucus disruption and removal has potential to augment preventive benefits of subglottic drainage.
Assuntos
Inflamação/imunologia , Inflamação/fisiopatologia , Mucinas/imunologia , Neutrófilos/imunologia , Respiração Artificial/efeitos adversos , Adulto , Idoso , Estado Terminal , Feminino , Glote/imunologia , Glote/fisiopatologia , Humanos , Imunidade Inata/imunologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Adulto JovemRESUMO
Isolation of nontuberculous mycobacteria (NTM) from the sputum of patients with cystic fibrosis (CF) is challenging due to overgrowth by rapidly growing species that colonize the lungs of patients with CF. Extended incubation on Burkholderia cepacia selective agar (BCSA) has been recommended as an expedient culture method for the isolation of rapidly growing NTM in this setting. The aim of this study was to assess five selective media designed for the isolation of Burkholderia cepacia complex, along with two media designed for the isolation of mycobacteria (rapidly growing mycobacteria [RGM] medium and Middlebrook 7H11 agar), for their abilities to isolate NTM. All seven media were challenged with 147 isolates of rapidly growing mycobacteria and 185 isolates belonging to other species. RGM medium was then compared with the most selective brand of BCSA for the isolation of NTM from 224 sputum samples from patients with CF. Different agars designed for the isolation of B. cepacia complex varied considerably in their inhibition of other bacteria and fungi. RGM medium supported the growth of all isolates of mycobacteria and was more selective than any other medium. NTM were recovered from 17 of 224 sputum samples using RGM medium, compared with only 7 samples using the most selective brand of BCSA (P = 0.023). RGM medium offers a superior option, compared to other selective agars, for the isolation of rapidly growing mycobacteria from the sputum of patients with CF. Furthermore, the convenience of using RGM medium enables routine screening for rapidly growing NTM in all submitted sputum samples from patients with CF.
Assuntos
Técnicas Bacteriológicas/métodos , Meios de Cultura/química , Fibrose Cística/complicações , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Micobactérias não Tuberculosas/isolamento & purificação , Escarro/microbiologia , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Programas de Rastreamento/métodosRESUMO
BACKGROUND: Necrotising enterocolitis (NEC) and late-onset sepsis (LOS) are the leading causes of death among preterm infants in the developed world. This study aimed to explore the serum proteome and metabolome longitudinally in preterm infants with NEC or LOS, matched to controls. METHODS: Nineteen patients (10 cases, 9 controls) were included. A sample 14 d prior to and following, as well as at disease diagnosis, was included for cases. Controls had serum matched at diagnosis for corresponding case. All samples (n = 39) underwent shotgun proteomic analysis, and 37 samples also underwent metabolomics analysis using ultra performance liquid chromatography-tandem mass spectrometry. RESULTS: The proteomic and metabolomic profiles of serum were comparable between all infants. Eight proteins were associated with NEC and four proteins were associated with LOS. C-reactive protein was increased in all NEC patients at diagnosis. CONCLUSION: No single protein or metabolite was detected in all NEC or LOS cases which was absent from controls; however, several proteins were identified which were associated with disease status. The differing expression of these proteins between diseased infants potentially relates to differing pathophysiology of disease. Thus, it is unlikely a single biomarker exists for NEC and/or LOS.
Assuntos
Enterocolite Necrosante/sangue , Doenças do Prematuro/sangue , Metaboloma , Proteoma/metabolismo , Sepse/sangue , Biomarcadores/sangue , Proteínas Sanguíneas/química , Estudos de Casos e Controles , Cromatografia Líquida , Perfilação da Expressão Gênica , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Espectrometria de Massas em TandemRESUMO
PURPOSE OF REVIEW: In newborns, interactions between the host and the microbiome operate synergistically, modulating host immune function and shaping the microbiome. Next generation molecular sequencing methodologies in tandem with modeling complex communities allow insights into the role of the microbiome in health and disease states. Infection-related disease states in which dysbiosis is integral include late-onset sepsis (LOS) and necrotizing enterocolitis (NEC), which still cause deaths and morbidity. Understanding microbiomic interactions may lead to alternative prevention, monitoring or treatment strategies, and modulation of long-term health outcomes especially in the preterm population. Recent studies have advanced understanding of the microbiome in NEC and LOS. RECENT FINDINGS: Mechanisms of host-microbiome interaction have been demonstrated. Patterns of microbiomic change in association with NEC and LOS have been observed, with community changes dominated by Proteobacteria and Firmicutes appearing to precede NEC, and very early microbiomic signatures influencing LOS. Data on viral and fungal elements are emerging. SUMMARY: Greater understanding of the neonatal bowel microbiome may allow tailored clinical practice and therapeutic intervention. Data handling and interpretation is challenging. Mechanistic studies of clinical interventions that affect the gut microbiome are important next steps.
Assuntos
Bacteriemia/microbiologia , Enterocolite Necrosante/microbiologia , Fezes/microbiologia , Trato Gastrointestinal/microbiologia , Interações Hospedeiro-Patógeno/imunologia , Microbiota , Bacteriemia/imunologia , Bacteriemia/prevenção & controle , Enterocolite Necrosante/imunologia , Enterocolite Necrosante/prevenção & controle , Trato Gastrointestinal/imunologia , Humanos , Recém-Nascido , Probióticos/uso terapêutico , Sepse/microbiologiaRESUMO
BACKGROUND: Chronic airway infection contributes to the underlying pathogenesis of non-cystic fibrosis bronchiectasis (NCFBr). In contrast to other chronic airway infections, associated with COPD and CF bronchiectasis, where polymicrobial communities have been implicated in lung damage due to the vicious circle of recurrent bacterial infections and inflammation, there is sparse information on the composition of bacterial communities in NCFBr. Seventy consecutive patients were recruited from an outpatient adult NCFBr clinic. Bacterial communities in sputum samples were analysed by culture and pyrosequencing approaches. Bacterial sequences were analysed using partial least square discrimination analyses to investigate trends in community composition and identify those taxa that contribute most to community variation. RESULTS: The lower airway in NCFBr is dominated by three bacterial taxa Pasteurellaceae, Streptococcaceae and Pseudomonadaceae. Moreover, the bacterial community is much more diverse than indicated by culture and contains significant numbers of other genera including anaerobic Prevotellaceae, Veillonellaceae and Actinomycetaceae. We found particular taxa are correlated with different clinical states, 27 taxa were associated with acute exacerbations, whereas 11 taxa correlated with stable clinical states. We were unable to demonstrate a significant effect of antibiotic therapy, gender, or lung function on the diversity of the bacterial community. However, presence of clinically significant culturable taxa; particularly Pseudomonas aeruginosa and Haemophilus influenzae correlated with a significant change in the diversity of the bacterial community in the lung. CONCLUSIONS: We have demonstrated that acute exacerbations, the frequency of exacerbation and episodes of clinical stability are correlated, in some patients, with a significantly different bacterial community structure, that are associated with a presence of particular taxa in the NCFBr lung. Moreover, there appears to be an inverse relationship between the abundance of P. aeruginosa and that of of H. influenzae within the NCFBr lung bacterial community. This interaction requires further exploration.
Assuntos
Bactérias/classificação , Bactérias/isolamento & purificação , Biota , Bronquiectasia/microbiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Técnicas Bacteriológicas , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Análise de Sequência de DNA , Escarro/microbiologiaRESUMO
The aim of this study was to determine the ability of a disc susceptibility test using faropenem (10 µg) to predict carbapenemase activity in Enterobacteriaceae. A collection of 166 isolates of carbapenemase-producing Enterobacteriaceae (CPE) and 82 isolates of Enterobacteriaceae that produced other ß-lactamases was compiled from diverse sources. Disc susceptibility testing was performed using the CLSI/EUCAST methodology with discs of faropenem (10 µg), temocillin (30 µg), and four carbapenems (each 10 µg). A further prospective evaluation of the faropenem disc susceptibility test was performed using 205 consecutive isolates referred to a United Kingdom reference laboratory in parallel with molecular methods for carbapenemase detection. Of 166 isolates of CPE, 99% showed growth up to the edge of a 10-µg faropenem disc compared with only 6% of other ß-lactamase producers (sensitivity, 99%; specificity, 94%). A "double zone" around 10-µg faropenem discs was frequently associated with OXA-48 producers. Of the carbapenems, the most useful agent was imipenem, where a zone diameter of ≤ 23 mm as a predictor of carbapenemase activity had a sensitivity of 99% and a specificity of 85%. The presence of no zone of inhibition around a 30-µg temocillin disc was a consistent feature of strains producing OXA-48 carbapenemase. For 205 isolates of Enterobacteriaceae referred to a United Kingdom reference laboratory, growth up to a 10-µg faropenem disc correctly identified 84 of 86 carbapenemase producers (98% sensitivity), with a specificity of 87%. Disc susceptibility testing using faropenem (10 µg) is a simple, convenient, and highly predictive screening test for carbapenemase-producing Enterobacteriaceae.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/enzimologia , beta-Lactamases/metabolismo , beta-Lactamas/farmacologia , Enterobacteriaceae/isolamento & purificação , Enterobacteriaceae/metabolismo , Infecções por Enterobacteriaceae/microbiologia , Humanos , Testes de Sensibilidade Microbiana/métodos , Sensibilidade e Especificidade , Reino UnidoRESUMO
The aim of this study was to investigate the polymicrobial communities in an adult Cystic Fibrosis population stratified by gender and the most common CFTR mutation, F508del. In this pilot study, DNA was extracted from sputum samples of 29 adult patients (16 male: 13 female) with an F508del mutation in a stable clinical state. Universal primers were used to amplify DNA from bacterial and fungal communities and the resulting fragments were analysed by denaturing gradient gel electrophoresis. Bacterial profiles showed a significant effect of gender (P = 0.046) and P. aeruginosa carriage (P = 0.034) on community structure. Bacterial communities were found to be randomly assembled. Fungal community analysis found that F508del homozygous patients had a greater diversity than heterozygous patients (P = 0.007). This study indicates that the bacterial lung communities of adult CF patients are randomly assembled but have distinct gender based differences. Furthermore, the fungal communities colonising the CF lung are more diverse in F508 homozygotes. This is the first paper to identify a reduced bacterial diversity in female patients with CF and to implicate more severe CFTR genotypes with increased risk of infection with multiple fungal species.
Assuntos
Bactérias/isolamento & purificação , Infecções Bacterianas/microbiologia , Biota , Coinfecção/microbiologia , Fibrose Cística/complicações , Fungos/isolamento & purificação , Micoses/microbiologia , Bactérias/classificação , Bactérias/genética , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fungos/classificação , Fungos/genética , Homozigoto , Humanos , Dados de Sequência Molecular , Análise de Sequência de DNA , Fatores SexuaisRESUMO
Four chromogenic media for carbapenemase-producing Enterobacteriaceae (CPE) and two selective broths were challenged with a collection of Enterobacteriaceae with well-defined ß-lactamases and 100 stool samples. With low inocula of 130 isolates of CPE, the sensitivities of the four chromogenic media were as follows: Brilliance CRE, 78%; chromID Carba, 91%; chromID ESBL, 96%; and Colorex KPC, 56%. The corresponding sensitivities of Trypticase soy broth plus ertapenem or meropenem were 78% and 47%, respectively.
Assuntos
Proteínas de Bactérias/metabolismo , Técnicas Bacteriológicas/métodos , Compostos Cromogênicos/metabolismo , Meios de Cultura/química , Enterobacteriaceae/enzimologia , beta-Lactamases/metabolismo , Humanos , Sensibilidade e EspecificidadeAssuntos
Microbioma Gastrointestinal/imunologia , Transplante de Células-Tronco Hematopoéticas , Imunodeficiência Combinada Severa/microbiologia , Imunodeficiência Combinada Severa/terapia , Microbioma Gastrointestinal/genética , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/microbiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Lactente , Masculino , Metaboloma/imunologia , Imunodeficiência Combinada Severa/imunologia , Fatores de TempoRESUMO
Formalin-Fixed Paraffin-Embedded (FFPE) tissues are routinely collected, archived, and used for clinical diagnosis, including maternal and neonatal health. Applying FFPE samples to microbiota research would be beneficial to reduce preparation, storage and costs associated with limited available frozen samples. This research aims to understand if FFPE fetal membrane samples are comparable to frozen tissues, which are the current gold standard for DNA microbiota analysis. Extracted DNA from nine matched paired patients were sequenced by Illumina sequencing of the V4 16S rRNA gene region. This included duplicate frozen amnion and chorion fetal membrane rolls or FFPE combined amniochorionic samples. Negative controls of surrounding wax blocks and DNA extraction reagents were processed alongside samples using identical methods. DNA quality and quantity was assessed by NanoDrop, agarose gel electrophoresis and Bioanalyzer. Decontam and SourceTracker were integrated into microbiota analysis to identify the presence of contaminating sources. The bacterial profile and nine genera differed between FFPE and frozen fetal membranes. There were no differences in bacterial profiles between FFPE samples and corresponding wax negative controls, with 49% of bacteria in FFPE fetal membrane samples matched to the source origin of paraffin wax, and 40% originating from DNA extraction reagent sources. FFPE samples displayed high fragmentation and low quantity of extracted DNA compared to frozen samples. The microbiota of FFPE fetal membrane samples is influenced by processing methods, with the inability to differentiate between the microbiota of the tissue sample and the surrounding wax block. Illumina sequencing results of FFPE and frozen fetal membrane samples should not be compared using the methods employed here. Variation could be influenced by limitations including storage time, DNA extraction and purification methods. To utilise FFPE fetal membrane samples in microbiota research then contamination prevention and detection methods must be included into optimised and standardised protocols, with recommendations presented here.
Assuntos
Formaldeído , Microbiota , Bactérias , DNA , Membranas Extraembrionárias , Humanos , Recém-Nascido , Microbiota/genética , Inclusão em Parafina/métodos , RNA Ribossômico 16S/genética , Fixação de Tecidos/métodosRESUMO
Agricultural soils are heterogeneous environments in which conditions affecting microbial growth and diversity fluctuate widely in space and time. In this study, the molecular ecology of the total bacterial and free-living nitrogen-fixing communities in soils from the Nafferton Factorial Systems Comparison (NFSC) study in northeast England were examined. The field experiment was factorial in design, with organic versus conventional crop rotation, crop protection, and fertility management factors. Soils were sampled on three dates (March, June, and September) in 2007. Total RNA was extracted from all soil samples and reverse transcribed. Denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR) were used to analyze nifH and 16S rRNA genes in order to study free-living diazotrophs and the total bacterial community, respectively. Crop rotation was shown to have a significant effect on total bacterial diversity (and that of free-living N fixers) (P ≤ 0.001). On all three dates, nifH activity was higher in the conventional crop rotation. In contrast, qPCR analysis of free-living N fixers indicated significantly higher levels of activity in conventionally fertilized plots in June (P = 0.0324) and in plots with organic crop protection in September (P = 0.0143). To our knowledge, the effects of organic and conventional farming systems on free-living diazotrophs have never been studied. An increased understanding of the impacts of management practices on free-living N fixers could allow modifications in soil management practices to optimize the activity of these organisms.
Assuntos
Agricultura/métodos , Bactérias/classificação , Bactérias/metabolismo , Conservação dos Recursos Naturais , Fixação de Nitrogênio , Microbiologia do Solo , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Produtos Agrícolas/crescimento & desenvolvimento , Eletroforese em Gel de Gradiente Desnaturante , Inglaterra , Fabaceae/crescimento & desenvolvimento , Hordeum/crescimento & desenvolvimento , Fixação de Nitrogênio/genética , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico 16S/genética , Solo/análiseRESUMO
Sediment cores taken from Great Slave Lake, Canada, were analysed to investigate their metabolically active microbial populations and geochemistry. The amplification of cDNA detected metabolically active bacterial (50 separate bands) and archaeal (49 separate band) communities. The bacterial communities were further resolved indicating active actinobacterial and γ-proteobacterial communities (36 and 43 individual bands respectively). Redundancy discriminate analysis and Monte Carlo permutation testing demonstrated the significant impact of geochemical parameters on microbial community structures. Geochemical analyses suggest that the upper 0.4 m represents soil weathering and erosion in the lake catchment. An increase in organic carbon in the lower core suggests either more primary productivity, indicating warmer climate conditions, associated with Holocene Climatic Optimum conditions pre 5,000 years BP or change from a reducing environment in the lower core to an oxidizing environment during more recent deposition. Drivers for bacterial, archaeal and actinobacterial community structures were sediment particle size, and its mineral composition. Depth also significantly affected γ- proteobacterial community structure. In contrast the organic carbon content did not significantly shape the microbial community structures within the sediment. This study indicates that geochemical parameters significantly contribute to microbial community structure in these sediments.
Assuntos
Archaea/classificação , Bactérias/classificação , Biodiversidade , Sedimentos Geológicos/química , Sedimentos Geológicos/microbiologia , Minerais/análise , Archaea/genética , Archaea/isolamento & purificação , Bactérias/genética , Bactérias/isolamento & purificação , Canadá , Metagenoma , Estatística como AssuntoAssuntos
Disbiose/microbiologia , Trato Gastrointestinal/microbiologia , Doença Enxerto-Hospedeiro/microbiologia , Transplante de Células-Tronco Hematopoéticas , Microbiota/imunologia , Imunodeficiência Combinada Severa/microbiologia , Antibacterianos/uso terapêutico , Antifúngicos/uso terapêutico , Antivirais/uso terapêutico , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Disbiose/imunologia , Fezes/microbiologia , Feminino , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/virologia , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Doença Enxerto-Hospedeiro/virologia , Humanos , Lactente , Recém-Nascido , Estudos Longitudinais , Masculino , Micoses/tratamento farmacológico , Micoses/imunologia , Micoses/microbiologia , Agonistas Mieloablativos/uso terapêutico , Projetos Piloto , Imunodeficiência Combinada Severa/imunologia , Imunodeficiência Combinada Severa/terapia , Imunodeficiência Combinada Severa/virologia , Condicionamento Pré-Transplante , Viroses/tratamento farmacológico , Viroses/imunologia , Viroses/virologiaRESUMO
INTRODUCTION: It is widely debated whether fetal membranes possess a genuine microbiome, and if bacterial presence and load is linked to inflammation. Chorioamnionitis is an inflammation of the fetal membranes. This research focussed on inflammatory diagnosed histological chorioamnionitis (HCA) and aimed to determine whether the bacterial load in fetal membranes correlates to inflammatory response, including histological staging and inflammatory markers in HCA. METHODS: Fetal membrane samples were collected from patients with preterm spontaneous labour and histologically phenotyped chorioamnionitis (HCA; n = 12), or preterm (n = 6) and term labour without HCA (n = 6). The bacterial profile of fetal membranes was analysed by sequencing the V4 region of the 16S rRNA gene. Bacterial load was determined using qPCR copy number/mg of tissue. The association between bacterial load and bacterial profile composition was assessed using correlation analysis. RESULTS: Bacterial load was significantly greater within HCA amnion (p = 0.002) and chorion (p = 0.042), compared to preterm birth without HCA. Increased bacterial load was positively correlated with increased histological staging (p = 0.001) and the expression of five inflammatory markers; IL8, TLR1, TLR2, LY96 and IRAK2 (p=<0.050). Bacterial profiles were significantly different between membranes with and without HCA in amnion (p = 0.012) and chorion (p = 0.001), but no differences between specific genera were detected. DISCUSSION: Inflammatory HCA is associated with infection and increased bacterial load in a dose response relationship. Bacterial load is positively correlated with HCA severity and the TLR signalling pathway. Further research should investigate the bacterial load threshold required to generate an inflammatory response in HCA.
Assuntos
Corioamnionite/microbiologia , Membranas Extraembrionárias/microbiologia , Microbiota/fisiologia , Adulto , Carga Bacteriana , Feminino , Idade Gestacional , Humanos , Gravidez , Estudos RetrospectivosRESUMO
The cell envelopes of gram-positive bacteria contain structurally diverse membrane-anchored macroamphiphiles (lipoteichoic acids and lipoglycans) whose functions are poorly understood. Since regulation of membrane composition is an important feature of adaptation to life at higher temperatures, we have examined the nature of the macroamphiphiles present in the thermophilic actinomycetes Thermobifida fusca and Rubrobacter xylanophilus. Following hot-phenol-water extraction and purification by hydrophobic interaction chromatography, Western blotting with a monoclonal antibody against lipoteichoic acid strongly suggested the presence of a polyglycerophosphate lipoteichoic acid in T. fusca. This structure was confirmed by chemical and nuclear magnetic resonance analyses, which confirmed that the lipoteichoic acid is substituted with beta-glucosyl residues, in common with the teichoic acid of this organism. In contrast, several extraction methods failed to recover significant macroamphiphilic carbohydrate- or phosphate-containing material from R. xylanophilus, suggesting that this actinomycete most likely lacks a membrane-anchored macroamphiphile. The finding of a polyglycerophosphate lipoteichoic acid in T. fusca suggests that lipoteichoic acids may be more widely present in the cell envelopes of actinomycetes than was previously assumed. However, the apparent absence of macroamphiphiles in the cell envelope of R. xylanophilus is highly unusual and suggests that macroamphiphiles may not always be essential for cell envelope homeostasis in gram-positive bacteria.
Assuntos
Actinobacteria/metabolismo , Lipopolissacarídeos/metabolismo , Streptomycetaceae/metabolismo , Ácidos Teicoicos/metabolismo , Actinobacteria/classificação , Actinobacteria/genética , Parede Celular/metabolismo , Cromatografia Gasosa , Cromatografia por Troca Iônica , Ácidos Graxos/isolamento & purificação , Ácidos Graxos/metabolismo , Amplificação de Genes , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/metabolismo , Lipopolissacarídeos/isolamento & purificação , Filogenia , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/isolamento & purificação , Streptococcus agalactiae/genética , Streptococcus agalactiae/isolamento & purificação , Streptococcus agalactiae/metabolismo , Streptomycetaceae/classificação , Streptomycetaceae/genética , Ácidos Teicoicos/isolamento & purificaçãoRESUMO
Concatenated sequence analysis with 16S rRNA, rpoB and fusA genes identified a bacterial strain (IRBG74) isolated from root nodules of the aquatic legume Sesbania cannabina as a close relative of the plant pathogen Rhizobium radiobacter (syn. Agrobacterium tumefaciens). However, DNA:DNA hybridization with R. radiobacter, R. rubi, R. vitis and R. huautlense gave only 44%, 5%, 8% and 8% similarity respectively, suggesting that IRBG74 is potentially a new species. Additionally, it contained no vir genes and lacked tumour-forming ability, but harboured a sym-plasmid containing nifH and nodA genes similar to those in other Sesbania symbionts. Indeed, IRBG74 effectively nodulated S. cannabina and seven other Sesbania spp. that nodulate with Ensifer (Sinorhizobium)/Rhizobium strains with similar nodA genes to IRBG74, but not species that nodulate with Azorhizobium or Mesorhizobium. Light and electron microscopy revealed that IRBG74 infected Sesbania spp. via lateral root junctions under flooded conditions, but via root hairs under non-flooded conditions. Thus, IRBG74 is the first confirmed legume-nodulating symbiont from the Rhizobium (Agrobacterium) clade. Cross-inoculation studies with various Sesbania symbionts showed that S. cannabina could form fully effective symbioses with strains in the genera Rhizobium and Ensifer, only ineffective ones with Azorhizobium strains, and either partially effective (Mesorhizobium huakii) or ineffective (Mesorhizobium plurifarium) symbioses with Mesorhizobium. These data are discussed in terms of the molecular phylogeny of Sesbania and its symbionts.
Assuntos
Rhizobium/genética , Nódulos Radiculares de Plantas/microbiologia , Sesbania/microbiologia , Aciltransferases/análise , Aciltransferases/genética , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , DNA Bacteriano/análise , DNA Bacteriano/genética , Fixação de Nitrogênio , Oxirredutases/análise , Oxirredutases/genética , Fator G para Elongação de Peptídeos/análise , Fator G para Elongação de Peptídeos/genética , Filogenia , Plasmídeos/análise , Plasmídeos/genética , RNA Ribossômico 16S/análise , RNA Ribossômico 16S/genética , Rhizobium/ultraestrutura , Nódulos Radiculares de Plantas/ultraestrutura , Alinhamento de Sequência , Análise de Sequência de DNA , Sesbania/ultraestrutura , Especificidade da Espécie , SimbioseRESUMO
Necrotising enterocolitis (NEC) and sepsis are serious diseases of preterm infants that can result in feeding intolerance, the need for bowel resection, impaired physiological and neurological development, and high mortality rates. Neonatal healthcare improvements have allowed greater survival rates in preterm infants leading to increased numbers at risk of developing NEC and sepsis. Gut bacteria play a role in protection from or propensity to these conditions and have therefore, been studied extensively using targeted 16S rRNA gene sequencing methods. However, exact epidemiology of these conditions remain unknown and the role of the gut microbiota in NEC remains enigmatic. Many studies have confounding variables such as differing clinical intervention strategies or major methodological issues such as the inability of 16S rRNA gene sequencing methods to determine viable from non-viable taxa. Identification of viable community members is important to identify links between the microbiota and disease in the highly unstable preterm infant gut. This is especially important as remnant DNA is robust and persists in the sampling environment following cell death. Chelation of such DNA prevents downstream amplification and inclusion in microbiota characterisation. This study validates use of propidium monoazide (PMA), a DNA chelating agent that is excluded by an undamaged bacterial membrane, to reduce bias associated with 16S rRNA gene analysis of clinical stool samples. We aim to improve identification of the viable microbiota in order to increase the accuracy of clinical inferences made regarding the impact of the preterm gut microbiota on health and disease. Gut microbiota analysis was completed on stools from matched twins (n = 16) that received probiotics. Samples were treated with PMA, prior to bacterial DNA extraction. Meta-analysis highlighted a significant reduction in bacterial diversity in 68.8% of PMA treated samples as well as significantly reduced overall rare taxa abundance. Importantly, overall abundances of genera associated with protection from and propensity to NEC and sepsis such as: Bifidobacterium; Clostridium, and Staphylococcus sp. were significantly different following PMA-treatment. These results suggest non-viable cell exclusion by PMA-treatment reduces bias in gut microbiota analysis from which clinical inferences regarding patient susceptibility to NEC and sepsis are made.
Assuntos
Bactérias/classificação , Enterocolite Necrosante/microbiologia , Microbioma Gastrointestinal , Trato Gastrointestinal/microbiologia , Azidas , Bactérias/genética , Bactérias/isolamento & purificação , Viés , Biodiversidade , DNA Bacteriano/genética , Enterocolite Necrosante/epidemiologia , Enterocolite Necrosante/mortalidade , Fezes/microbiologia , Humanos , Lactente , Recém-Nascido Prematuro , Reação em Cadeia da Polimerase , Probióticos/uso terapêutico , Propídio/análogos & derivados , RNA Ribossômico 16S/genética , Taxa de SobrevidaRESUMO
The short and long-term impact of birth mode on the developing gut microbiome in neonates has potential implications for the health of infants. In term infants, the microbiome immediately following birth across multiple body sites corresponds to birth mode, with increased Bacteroides in vaginally delivered infants. We aimed to determine the impact of birth mode of the preterm gut microbiome over the first 100 days of life and following neonatal intensive care unit (NICU) discharge. In total, 867 stool samples from 46 preterm infants (21 cesarean and 25 vaginal), median gestational age 27 weeks, were sequenced (V4 region 16S rRNA gene, Illumina MiSeq). Of these, 776 samples passed quality filtering and were included in the analysis. The overall longitudinal alpha-diversity and within infant beta-diversity was comparable between cesarean and vaginally delivered infants. Vaginally delivered infants kept significantly more OTUs from 2 months of life and following NICU discharge, but OTUs lost, gained, and regained were not different based on birth mode. Furthermore, the temporal progression of dominant genera was comparable between birth modes and no significant difference was found for any genera following adjustment for covariates. Lastly, preterm gut community types (PGCTs) showed some moderate differences in very early life, but progressed toward a comparable pattern by week 5. No PGCT was significantly associated with cesarean or vaginal birth. Unlike term infants, birth mode was not significantly associated with changes in microbial diversity, composition, specific taxa, or overall microbial development in preterm infants. This may result from the dominating effects of NICU exposures including the universal use of antibiotics immediately following birth and/or the lack of Bacteroides colonizing preterm infants.
RESUMO
BACKGROUND: Late onset sepsis (LOS) in preterm infants is associated with considerable morbidity and mortality. While studies have implicated gut bacteria in the aetiology of the disease, functional analysis and mechanistic insights are generally lacking. We performed temporal bacterial (n = 613) and metabolomic (n = 63) profiling on extensively sampled stool from 7 infants with LOS and 28 matched healthy (no LOS or NEC) controls. RESULTS: The bacteria isolated in diagnostic blood culture usually corresponded to the dominant bacterial genera in the gut microbiome. Longitudinal changes were monitored based on preterm gut community types (PGCTs), where control infants had an increased number of PGCTs compared to LOS infants (P = 0.011). PGCT 6, characterised by Bifidobacteria dominance, was only present in control infants. Metabolite profiles differed between LOS and control infants at diagnosis and 7 days later, but not 7 days prior to diagnosis. Bifidobacteria was positively correlated with control metabolites, including raffinose, sucrose, and acetic acid. CONCLUSIONS: Using multi-omic analysis, we show that the gut microbiome is involved in the pathogenesis of LOS. While the causative agent of LOS varies, it is usually abundant in the gut. Bifidobacteria dominance was associated with control infants, and the presence of this organism may directly protect, or act as a marker for protection, against gut epithelial translocation. While the metabolomic data is preliminary, the findings support that gut development and protection in preterm infants is associated with increased in prebiotic oligosaccharides (e.g. raffinose) and the growth of beneficial bacteria (e.g. Bifidobacterium).