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Clostridiodes difficile (C. difficile) was ranked an "urgent threat" by the Centers for Disease Control and Prevention (CDC) in 2019. C. difficile infection (CDI) is the most common healthcare-associated infection (HAI) in the United States of America as well as the leading cause of antibiotic-associated gastrointestinal disease. C. difficile is a gram-positive, rod-shaped, spore-forming, anaerobic bacterium that causes infection of the epithelial lining of the gut. CDI occurs most commonly after disruption of the human gut microflora following the prolonged use of broad-spectrum antibiotics. However, the recurrent nature of this disease has led to the hypothesis that biofilm formation may play a role in its pathogenesis. Biofilms are sessile communities of bacteria protected from extracellular stresses by a matrix of self-produced proteins, polysaccharides, and extracellular DNA. Biofilm regulation in C. difficile is still incompletely understood, and its role in disease recurrence has yet to be fully elucidated. However, many factors have been found to influence biofilm formation in C. difficile, including motility, adhesion, and hydrophobicity of the bacterial cells. Small changes in one of these systems can greatly influence biofilm formation. Therefore, the biofilm regulatory system would need to coordinate all these systems to create optimal biofilm-forming physiology under appropriate environmental conditions. The coordination of these systems is complex and multifactorial, and any analysis must take into consideration the influences of the stress response, quorum sensing (QS), and gene regulation by second messenger molecule cyclic diguanosine monophosphate (c-di-GMP). However, the differences in biofilm-forming ability between C. difficile strains such as 630 and the "hypervirulent" strain, R20291, make it difficult to assign a "one size fits all" mechanism to biofilm regulation in C. difficile. This review seeks to consolidate published data regarding the regulation of C. difficile biofilms in order to identify gaps in knowledge and propose directions for future study.
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Biofilmes/crescimento & desenvolvimento , Clostridioides difficile/crescimento & desenvolvimento , Clostridioides difficile/patogenicidade , Infecções por Clostridium/patologia , Humanos , VirulênciaRESUMO
Enterococci are robust gram-positive bacteria that are found in a variety of surroundings and that cause a significant number of healthcare-associated infections. The genus possesses a high-efficiency pheromone-responsive plasmid (PRP) transfer system for genetic exchange that allows antimicrobial-resistance determinants to spread within bacterial populations. The pCF10 plasmid system is the best characterised, and although other PRP systems are structurally similar, they lack exact functional homologues of pCF10-encoded genes. In this review, we provide an overview of the enterococcal PRP systems, incorporating functional details for the less-well-defined systems. We catalogue the virulence-associated elements of the PRPs that have been identified to date, and we argue that this reinforces the requirement for elucidation of the less studied systems.
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Proteínas de Bactérias/genética , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Enterococcus faecalis/genética , Infecções por Bactérias Gram-Positivas/microbiologia , Feromônios/fisiologia , Plasmídeos/genética , Animais , Conjugação Genética , Humanos , VirulênciaRESUMO
Enterococci, which are on the WHO list of priority pathogens, are commonly encountered in hospital acquired infection and are becoming increasing significant due to the development of strains resistant to multiple antibiotics. Enterococci are also important microorganisms in the environment, and their presence is frequently used as an indicator of faecal pollution. Their success is related to their ability to survive within a broad range of habitats and the ease by which they acquire mobile genetic elements, including plasmids, from other bacteria. The enterococci are frequently present within a bacterial biofilm, which provides stability and protection to the bacterial population along with an opportunity for a variety of bacterial interactions. Enterococci can accept extrachromosomal DNA both from within its own species and from other bacterial species, and this is enhanced by the proximity of the donor and recipient strains. It is this exchange of genetic material that makes the role of biofilms such an important aspect of the success of enterococci. There remain many questions regarding the most suitable model systems to study enterococci in biofilms and regarding the transfer of genetic material including antibiotic resistance in these biofilms. This review focuses on some important aspects of biofilm in the context of horizontal gene transfer (HGT) in enterococci.
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Biofilmes , Enterococcus , Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Enterococcus/genética , Transferência Genética HorizontalRESUMO
At present, anti-virulence drugs are being considered as potential therapeutic alternatives and/or adjuvants to currently failing antibiotics. These drugs do not kill bacteria but inhibit virulence factors essential for establishing infection and pathogenesis through targeting non-essential metabolic pathways reducing the selective pressure to develop resistance. We investigated the effect of naturally isolated plant compounds on the repression of the quorum sensing (QS) system which is linked to virulence/pathogenicity in Pseudomonas aeruginosa. Our results show that trans-cinnamaldehyde (CA) and salicylic acid (SA) significantly inhibit expression of QS regulatory and virulence genes in P. aeruginosa PAO1 at sub-inhibitory levels without any bactericidal effect. CA effectively downregulated both the las and rhl QS systems with lasI and lasR levels inhibited by 13- and 7-fold respectively compared to 3- and 2-fold reductions with SA treatment, during the stationary growth phase. The QS inhibitors (QSI) also reduced the production of extracellular virulence factors with CA reducing protease, elastase and pyocyanin by 65%, 22% and 32%, respectively. The QSIs significantly reduced biofilm formation and concomitantly with repressed rhamnolipid gene expression, only trace amount of extracellular rhamnolipids were detected. The QSIs did not completely inhibit virulence factor expression and production but their administration significantly lowered the virulence phenotypes at both the transcriptional and extracellular levels. This study shows the significant inhibitory effect of natural plant-derived compounds on the repression of QS systems in P. aeruginosa.
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Acroleína/análogos & derivados , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Percepção de Quorum/efeitos dos fármacos , Ácido Salicílico/farmacologia , Fatores de Virulência/genética , Acroleína/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Espaço Extracelular/metabolismo , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/patogenicidade , Percepção de Quorum/genética , Virulência/efeitos dos fármacos , Fatores de Virulência/metabolismoRESUMO
BACKGROUND: The field of diagnostics continues to advance rapidly with a variety of novel approaches, mainly dependent upon high technology platforms. Nonetheless much diagnosis, particularly in developing countries, still relies upon traditional methods such as microscopy. Biological material, particularly nucleic acids, on archived glass slides is a potential source of useful information both for diagnostic and epidemiological purposes. There are significant challenges faced when examining archived samples in order that an adequate amount of amplifiable DNA can be obtained. Herein, we describe a model system to detect low numbers of bacterial cells isolated from glass slides using (laser capture microscopy) LCM coupled with PCR amplification of a suitable target. RESULTS: Mycobacterium smegmatis was used as a model organism to provide a proof of principle for a method to recover bacteria from a stained sample on a glass slide using a laser capture system. Ziehl-Neelsen (ZN) stained cells were excised and catapulted into tubes. Recovered cells were subjected to DNA extraction and pre-amplified with multiple displacement amplification (MDA). This system allowed a minimum of 30 catapulted cells to be detected following a nested real-time PCR assay, using rpoB specific primers. The combination of MDA and nested real-time PCR resulted in a 30-fold increase in sensitivity for the detection of low numbers of cells isolated using LCM. CONCLUSIONS: This study highlights the potential of LCM coupled with MDA as a tool to improve the recovery of amplifiable nucleic acids from archived glass slides. The inclusion of the MDA step was essential to enable downstream amplification. This platform should be broadly applicable to a variety of diagnostic applications and we have used it as a proof of principle with a Mycobacterium sp. model system.
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DNA Bacteriano/isolamento & purificação , Microscopia Confocal/métodos , Mycobacterium smegmatis/isolamento & purificação , DNA Bacteriano/genética , Vidro/análise , Humanos , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium smegmatis/classificação , Mycobacterium smegmatis/genética , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Coloração e Rotulagem/instrumentaçãoRESUMO
Global climate changes during the Cenozoic (65.5-0 Ma) caused major biological range shifts and extinctions. In northern Europe, for example, a pattern of few endemics and the dominance of wide-ranging species is thought to have been determined by the Pleistocene (2.59-0.01 Ma) glaciations. This study, in contrast, reveals an ancient subsurface fauna endemic to Britain and Ireland. Using a Bayesian phylogenetic approach, we found that two species of stygobitic invertebrates (genus Niphargus) have not only survived the entire Pleistocene in refugia but have persisted for at least 19.5 million years. Other Niphargus species form distinct cryptic taxa that diverged from their nearest continental relative between 5.6 and 1.0 Ma. The study also reveals an unusual biogeographical pattern in the Niphargus genus. It originated in north-west Europe approximately 87 Ma and underwent a gradual range expansion. Phylogenetic diversity and species age are highest in north-west Europe, suggesting resilience to extreme climate change and strongly contrasting the patterns seen in surface fauna. However, species diversity is highest in south-east Europe, indicating that once the genus spread to these areas (approximately 25 Ma), geomorphological and climatic conditions enabled much higher diversification. Our study highlights that groundwater ecosystems provide an important contribution to biodiversity and offers insight into the interactions between biological and climatic processes.
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Anfípodes/classificação , Evolução Biológica , Mudança Climática , Filogenia , Anfípodes/genética , Animais , Teorema de Bayes , Ecossistema , Europa (Continente) , Geografia , Água Subterrânea , Irlanda , Dados de Sequência Molecular , Reino UnidoRESUMO
Faecal pollution of water by bacteria has a negative effect on water quality and can pose a potential health hazard. Conventional surveillance of microbial water quality relies on the analysis of low-frequency spot samples and is thus likely to miss episodic or periodic pollution. This study aimed to investigate the potential of filter-feeding sponges for time-integrated biomonitoring of microbial water quality. Laboratory trials tested the effects of different ratios of bacterial abundance and the sequence of exposure on bacterial retention by the freshwater sponge Ephydatia fluviatilis (Linnaeus, 1759) to establish its potential to indicate bacterial exposure. Gemmule grown sponges were simultaneously exposed to Escherichia coli and Enterococcus faecalis but at different ratios (Trial 1) or individually exposed to each bacterial species but in different sequential order (Trial 2). The E. coli and E. faecalis retained in each sponge was quantified by culture on selective agars. Data analysis was conducted using the Kruskal-Wallis test and/or the Mann-Whitney U test to compare between the numbers of bacteria retained in each treatment. Additionally, the Wilcoxon matched-paired signed-rank test was used for comparison of the different bacterial abundances retained within each individual sponge. Sponges from all trials retained E. coli and E. faecalis in small numbers relative to the exposure (<0.05â% Trial 1 and <0.07â% Trial 2) but exhibited higher retention of E. coli. Higher abundance of either bacterial species resulted in significantly lower (P<0.005) retention of the same species within sponges (Trial 1). An initial exposure to E. coli resulted in significantly higher (P=0.040) retention of both bacterial species than when sponges were exposed to E. faecalis first (Trial 2).Bacterial retention by sponges was neither quantitatively representative of bacterial abundance in the ambient water nor the sequence of exposure. This implies either selective filtration or an attempt by sponges to prevent infection. However, freshwater sponges may still be useful in biomonitoring as qualitative time-integrated samplers of faecal indicator bacteria as they detect different bacteria present in the water even if their quantities cannot be estimated.
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The rapid emergence of multidrug-resistant pathogens worldwide has raised concerns regarding the effectiveness of conventional antibiotics. This can be observed in ESKAPE pathogens, among others, whose multiple resistance mechanisms have led to a reduction in effective treatment options. Innovative strategies aimed at mitigating the incidence of antibiotic-resistant pathogens encompass the potential use of biosurfactants. These surface-active agents comprise a group of unique amphiphilic molecules of microbial origin that are capable of interacting with the lipidic components of microorganisms. Biosurfactant interactions with different surfaces can affect their hydrophobic properties and as a result, their ability to alter microorganisms' adhesion abilities and consequent biofilm formation. Unlike synthetic surfactants, biosurfactants present low toxicity and high biodegradability and remain stable under temperature and pH extremes, making them potentially suitable for targeted use in medical and pharmaceutical applications. This review discusses the development of biosurfactants in biomedical and therapeutic uses as antimicrobial and antibiofilm agents, in addition to considering the potential synergistic effect of biosurfactants in combination with antibiotics. Furthermore, the anti-cancer and anti-viral potential of biosurfactants in relation to COVID-19 is also discussed.
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Aquatic habitats are particularly susceptible to chemical pollution, such as antimicrobials, from domestic, agricultural, and industrial sources. This has led to the rapid increase of antimicrobial resistance (AMR) gene prevalence. Alternate approaches to counteract pathogenic bacteria are in development including synthetic and biological surfactants such as sodium dodecyl sulfate (SDS) and rhamnolipids. In the aquatic environment, these surfactants may be present as pollutants with the potential to affect biofilm formation and AMR gene occurrence. We tested the effects of rhamnolipid and SDS on aquatic biofilms in a freshwater stream in Northern Ireland. We grew biofilms on contaminant exposure substrates deployed within the stream over 4 weeks. We then extracted DNA and carried out shotgun sequencing using a MinION portable sequencer to determine microbial community composition, with 16S rRNA analyses (64,678 classifiable reads identified), and AMR gene occurrence (81 instances of AMR genes over 9 AMR gene classes) through a metagenomic analysis. There were no significant changes in community composition within all systems; however, biofilm exposed to rhamnolipid had a greater number of unique taxa as compared to SDS treatments and controls. AMR gene prevalence was higher in surfactant-treated biofilms, although not significant, with biofilm exposed to rhamnolipids having the highest presence of AMR genes and classes compared to the control or SDS treatments. Our results suggest that the presence of rhamnolipid encourages an increase in the prevalence of AMR genes in biofilms produced in mixed-use water bodies.
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Anti-Infecciosos , Tensoativos , RNA Ribossômico 16S/genética , Tensoativos/farmacologia , Anti-Infecciosos/farmacologia , Água Doce , BiofilmesRESUMO
Skin tanning, either by exposure to natural sunlight or through use of UV sunbeds, has become a popular practice in the US, where it is estimated that approximately 1 million times per day someone in the US uses UV radiation for skin tanning, equating to 30 million Americans (circa 10% of the US population) who use a tanning bed. As well as exposing the host to periods of UV radiation, such practices also expose commensal skin bacteria, including Staphylococcus aureus, to such UV radiation. Previous work has indicated that environmental stresses on bacteria may lead to an upregulation of stress responses, in an attempt for the organism to combat the applied stress and remain viable. UV light may act as an environmental stress on bacteria, and so it was the aim of this study to examine the effect of UVc light on the antibiotic susceptibility of commensal skin bacteria, to determine if UV radiation would increase the antibiotic resistance of such skin flora and thus lead to a potential skin flora with increased antibiotic resistance. Previously, it has been shown that UVc light has a greater mutational effect on bacteria compared to lower-energy UV forms, including UVa and UVb light. Therefore, we decided to employ UVc light in our study to amplify the potential for mutational events occurring in skin staphylococci organisms (n=8) including methicillin-sensitive Staphylococcus aureus (n=2), methicillin-resistant Staphylococcus aureus (n=4), and coagulase-negative staphylococci (Staphylococcus haemolyticus) (n=2) were exposed to varying degrees of sublethal radiation via UVc light, and their minimum inhibitory concentration (MIC) susceptibility was determined by broth dilution assay against three classes of commonly used antibiotics, namely ß-lactams (penicillin), macrolides (erythromycin), and fluoroquinolones (ciprofloxacin). There was no significant difference between antibiotic susceptibility before UVc exposure and until maximum sublethal stress, prior to cell death due to fatal UVc exposure with the cells. These results indicate that UV environmental stress/exposure does not upregulate antibiotic resistance, and therefore these data indicate that UVc radiation does not lead to a more antibiotic-resistant population in the staphylococci organisms post-exposure.
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Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Staphylococcus/efeitos dos fármacos , Staphylococcus/efeitos da radiação , Raios Ultravioleta , Fluoroquinolonas/farmacologia , Macrolídeos/farmacologia , Staphylococcus/classificação , beta-Lactamas/farmacologiaRESUMO
OBJECTIVES: Ciprofloxacin is the most frequently used member of the fluoroquinolones during initial eradication therapy of Pseudomonas aeruginosa, as well as during acute pulmonary exacerbations. However, its long-term effect on the susceptibility of the commensal flora within the cystic fibrosis (CF) airways has not yet been examined. The aim of this study was therefore to examine the consequence of oral ciprofloxacin usage on the resistance of the commensal viridans group streptococci (VGS), in terms of MICs and mutational analysis of the quinolone resistance-determining regions (QRDRs). METHODS: The MICs of ciprofloxacin, efflux activities and amino acid substitutions in the QRDRs for 190 isolates of VGS, originating from the sputa of adult CF patients who had been exposed constantly to ciprofloxacin, were examined. VGS organisms included Streptococcus salivarius, Streptococcus mitis, Streptococcus sanguinis, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus infantis, Streptococcus gordonii, Streptococcus anginosus, Streptococcus cristatus, Streptococcus australis and Streptococcus mutans. Ciprofloxacin susceptibility was determined by broth microdilution and QRDRs within the gyrA, gyrB, parC and parE gene loci were explored using sequence analysis. RESULTS: Twenty-seven (14.2%) streptococcal isolates were resistant to ciprofloxacin (MICs ≥8 mg/L) and 21 (11.1%) had reduced susceptibility (MICs 4 mg/L). As a comparator, clinically non-significant and non-invasive VGS organisms were examined in 12 consecutive non-CF patients in the community, where no resistance to ciprofloxacin was observed. Five novel QRDR PCR assays were developed to elucidate mutations within the CF VGS population, where there were six positions, which corresponded to previously reported quinolone resistance responsible mutations, and eight novel potential QRDR resistance mutations. Double mutations in gyrA and parC/parE led to MICs of 16 to >64 mg/L, while single mutations in parC or parE resulted in MICs of 8-32 mg/L and 8 mg/L, respectively. The mean homologies of each species to Streptococcus pneumoniae R6 were: gyrA, 70.3%-95%; gyrB, 69.6%-96.2%; parC, 76.1%-94.8%; and parE, 70.7%-94.7%. The close relatives of S. pneumoniae, S. mitis and S. oralis, showed high similarity for all four genes (more than 86%). CONCLUSIONS: Treatment of P. aeruginosa with oral ciprofloxacin in patients with CF may concurrently reduce antibiotic susceptibility in the commensal VGS flora, where these organisms may potentially act as a reservoir of fluoroquinolone resistance gene determinants for newly acquired and antibiotic-susceptible pathogens, particularly the Streptococcus milleri group.
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Antibacterianos/farmacologia , DNA Girase/genética , DNA Topoisomerase IV/genética , Farmacorresistência Bacteriana , Quinolonas/farmacologia , Estreptococos Viridans/genética , Adulto , Antibacterianos/efeitos adversos , Antibacterianos/uso terapêutico , Ciprofloxacina/efeitos adversos , Ciprofloxacina/farmacologia , Ciprofloxacina/uso terapêutico , Fibrose Cística/complicações , DNA Bacteriano/química , DNA Bacteriano/genética , Uso de Medicamentos , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Quinolonas/efeitos adversos , Quinolonas/uso terapêutico , Análise de Sequência de DNA , Infecções Estreptocócicas/microbiologia , Estreptococos Viridans/isolamento & purificaçãoRESUMO
Enterococci and biofilm-associated infections are a growing problem worldwide, given the rise in antibiotic resistance in environmental and clinical settings. The increasing incidence of antibiotic resistance and its propagation potential within enterococcal biofilm is a concern. This requires a deeper understanding of how enterococcal biofilm develops, and how antibiotic resistance transfer takes place in these biofilms. Enterococcal biofilm assays, incorporating the study of antibiotic resistance transfer, require a system which can accommodate non-destructive, real-time experimentation. We adapted a Gene Frame® combined with fluorescence microscopy as a novel non-destructive platform to study the conjugal transfer of vancomycin resistance in an established enterococcal biofilm.A multi-purpose fluorescent in situ hybridisation (FISH) probe, in a novel application, allowed the identification of low copy number mobile elements in the biofilm. Furthermore, a Hoechst stain and ENU 1470 FISH probe identified Enterococcus faecium transconjugants by excluding Enterococcus faecalis MF06036 donors. Biofilm created with a rifampicin resistant E. faecalis (MW01105Rif) recipient had a transfer efficiency of 2.01 × 10-3; double that of the biofilm primarily created by the donor (E. faecalis MF06036). Conjugation in the mixed enterococcal biofilm was triple the efficiency of donor biofilm. Double antibiotic treatment plus lysozyme combined with live/dead imaging provided fluorescent micrographs identifying de novo enterococcal vancomycin resistant transconjugants inside the biofilm. This is a model system for the further study of antibiotic resistance transfer events in enterococci. Biofilms promote the survival of enterococci and reduce the effectiveness of drug treatment in clinical settings, hence giving enterococci an advantage. Enterococci growing in biofilms exchange traits by means of horizontal gene transfer, but currently available models make study difficult. This work goes some way to providing a non-destructive, molecular imaging-based model system for the detection of antibiotic resistance gene transfer in enterococci.
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Nasal pathogen detection sensitivities can be as low as 70% despite advances in molecular diagnostics. This may be linked to the choice of sampling method. A diagnostic test accuracy review for sensitivity was undertaken to compare sensitivity of swabbing to the nasopharynx and extracting nasal aspirates, using the PRISMA protocol, Cochrane rapid review methodology, and QUADAS-2 risk of bias tools, with meta-analysis of included studies. Sensitivities were calculated by a consensus standard of positivity by either method as the 'gold standard.' Insufficient sampling methodology, cross sectional study designs, and studies pooling samples across anatomical sites were excluded. Of 13 subsequently eligible studies, 8 had 'high' risk of bias, and 5 had 'high' applicability concerns. There were no statistical differences in overall sensitivities between collection methods for eight different viruses, and this did not differ with use of PCR, immunofluorescence, or culture. In one study alone, Influenza H1N1(2009) favored nasopharyngeal swabs, with aspirates having 93.3% of the sensitivity of swabs (p > 0.001). Similarly equivocal sensitivities were noted in reports detecting bacteria. The chain of sampling, from anatomical site to laboratory results, features different potential foci along which sensitivity may be lost. A fair body of evidence exists that use of a different sampling method will not yield more respiratory pathogens.
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The aim of this review was to provide an update on the complex relationship between manure application, altered pathogen levels and antibiotic resistance. This is necessary to protect health and improve the sustainability of this major farming practice in agricultural systems based on high levels of manure production. It is important to consider soil health in relation to environment and land management practices in the context of the soil microflora and the introduction of pathogens on the health of the soil microbiome. Viable pathogens in manure spread on agricultural land may be distributed by leaching, surface run-off, water source contamination and contaminated crop removal. Thus it is important to understand how multiple pathogens can persist in manures and on soil at farm-scale and how crops produced under these conditions could be a potential transfer route for zoonotic pathogens. The management of pathogen load within livestock manure is a potential mechanism for the reduction and prevention of outbreaks infection with Escherichia coli, Listeria Salmonella, and Campylobacter. The ability of Campylobacter, E. coli, Listeria and Salmonella to combat environmental stress coupled with their survival on food crops and vegetables post-harvest emphasizes the need for further study of these pathogens along with the emerging pathogen Providencia given its link to disease in the immunocompromised and its' high levels of antibiotic resistance. The management of pathogen load within livestock manure has been widely recognized as a potential mechanism for the reduction and prevention of outbreaks infection but any studies undertaken should be considered as region specific due to the variable nature of the factors influencing pathogen content and survival in manures and soil. Mediocre soils that require nutrients could be one template for research on manure inputs and their influence on soil health and on pathogen survival on grassland and in food crops.
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Pseudomonas aeruginosa uses quorum sensing (QS) to modulate the expression of several virulence factors that enable it to establish severe infections. The QS system in P. aeruginosa is complex, intricate and is dominated by two main N-acyl-homoserine lactone circuits, LasRI and RhlRI. These two QS systems work in a hierarchical fashion with LasRI at the top, directly regulating RhlRI. Together these QS circuits regulate several virulence associated genes, metabolites, and enzymes in P. aeruginosa. Paradoxically, LasR mutants are frequently isolated from chronic P. aeruginosa infections, typically among cystic fibrosis (CF) patients. This suggests P. aeruginosa can undergo significant evolutionary pathoadaptation to persist in long term chronic infections. In contrast, mutations in the RhlRI system are less common. Here, we have isolated a clinical strain of P. aeruginosa from a CF patient that has deleted the transcriptional regulator RhlR entirely. Whole genome sequencing shows the rhlR locus is deleted in PA80 alongside a few non-synonymous mutations in virulence factors including protease lasA and rhamnolipid rhlA, rhlB, rhlC. Importantly we did not observe any mutations in the LasRI QS system. PA80 does not appear to have an accumulation of mutations typically associated with several hallmark pathoadaptive genes (i.e., mexT, mucA, algR, rpoN, exsS, ampR). Whole genome comparisons show that P. aeruginosa strain PA80 is closely related to the hypervirulent Liverpool epidemic strain (LES) LESB58. PA80 also contains several genomic islands (GI's) encoding virulence and/or resistance determinants homologous to LESB58. To further understand the effect of these mutations in PA80 QS regulatory and virulence associated genes, we compared transcriptional expression of genes and phenotypic effects with isogenic mutants in the genetic reference strain PAO1. In PAO1, we show that deletion of rhlR has a much more significant impact on the expression of a wide range of virulence associated factors rather than deletion of lasR. In PA80, no QS regulatory genes were expressed, which we attribute to the inactivation of the RhlRI QS system by deletion of rhlR and mutation of rhlI. This study demonstrates that inactivation of the LasRI system does not impact RhlRI regulated virulence factors. PA80 has bypassed the common pathoadaptive mutations observed in LasR by targeting the RhlRI system. This suggests that RhlRI is a significant target for the long-term persistence of P. aeruginosa in chronic CF patients. This raises important questions in targeting QS systems for therapeutic interventions.
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Proteínas de Bactérias/metabolismo , Fibrose Cística/microbiologia , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/fisiologia , Percepção de Quorum , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Variação Genética , Genômica , Humanos , Mutação/genética , Filogenia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Seaweeds are potentially sustainable crops and are receiving significant interest because of their rich bioactive compound content; including fatty acids, polyphenols, carotenoids, and complex polysaccharides. However, there is little information on the in vivo effects on gut health of the polysaccharides and their low-molecular-weight derivatives. Herein, we describe the first investigation into the prebiotic potential of low-molecular-weight polysaccharides (LMWPs) derived from alginate and agar in order to validate their in vivo efficacy. We conducted a randomized; placebo-controlled trial testing the impact of alginate and agar LWMPs on faecal weight and other markers of gut health and on composition of gut microbiota. We show that these LMWPs led to significantly increased faecal bulk (20-30%). Analysis of gut microbiome composition by sequencing indicated no significant changes attributable to treatment at the phylum and family level, although FISH analysis showed an increase in Faecalibacterium prausnitzii in subjects consuming agar LMWP. Sequence analysis of gut bacteria corroborated with the FISH data, indicating that alginate and agar LWMPs do not alter human gut microbiome health markers. Crucially, our findings suggest an urgent need for robust and rigorous human in vivo testing-in particular, using refined seaweed extracts.
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BACKGROUND: Pseudomonas aeruginosa is considered to grow in a biofilm in cystic fibrosis (CF) chronic lung infections. Bacterial cell motility is one of the main factors that have been connected with P. aeruginosa adherence to both biotic and abiotic surfaces. In this investigation, we employed molecular and microscopic methods to determine the presence or absence of motility in P. aeruginosa CF isolates, and statistically correlated this with their biofilm forming ability in vitro. RESULTS: Our investigations revealed a wide diversity in the production, architecture and control of biofilm formation. Of 96 isolates, 49% possessed swimming motility, 27% twitching and 52% swarming motility, while 47% were non-motile. Microtitre plate assays for biofilm formation showed a range of biofilm formation ability from biofilm deficient phenotypes to those that formed very thick biofilms. A comparison of the motility and adherence properties of individual strains demonstrated that the presence of swimming and twitching motility positively affected biofilm biomass. Crucially, however, motility was not an absolute requirement for biofilm formation, as 30 non-motile isolates actually formed thick biofilms, and three motile isolates that had both flagella and type IV pili attached only weakly. In addition, CLSM analysis showed that biofilm-forming strains of P. aeruginosa were in fact capable of entrapping non-biofilm forming strains, such that these 'non-biofilm forming' cells could be observed as part of the mature biofilm architecture. CONCLUSIONS: Clinical isolates that do not produce biofilms in the laboratory must have the ability to survive in the patient lung. We propose that a synergy exists between isolates in vivo, which allows "non biofilm-forming" isolates to be incorporated into the biofilm. Therefore, there is the potential for strains that are apparently non-biofilm forming in vitro to participate in biofilm-mediated pathogenesis in the CF lung.
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Biofilmes , Fibrose Cística/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/fisiologia , Análise de Variância , Aderência Bacteriana , Criança , Genótipo , Humanos , Microscopia Eletrônica de Varredura , Fenótipo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/isolamento & purificação , Técnica de Amplificação ao Acaso de DNA Polimórfico , Infecções Respiratórias/microbiologiaRESUMO
Fresh human sewage was examined from a sewage treatment plant for the presence of members of the Burkholderia cepacia complex (BCC) of bacterial organisms and confirmed the presence of viable B. cenocepacia and B. vietnamiensis, by a combination of cultural, phenotypic and genotypic techniques. Both these organisms are important respiratory pathogens for patients with cystic fibrosis (CF). Presently, the survival dynamics of these organisms in sewage effluent and sludge is, as yet, unknown. Therefore, as this study represents the first report of these CF pathogens in sewage and until such survival data is available, careful risk assessment needs to be undertaken in relation to the end use application of potentially contaminated sewage and where such material comes into association with non-colonised patients with cystic fibrosis, so that any potential transmission of these pathogens from sewage to patient is assessed and minimised/eliminated.
Assuntos
Burkholderia/isolamento & purificação , Esgotos/microbiologia , Eliminação de Resíduos Líquidos , Burkholderia/classificação , Burkholderia/genética , Fibrose Cística/microbiologia , Exposição Ambiental , Genótipo , Humanos , Irlanda , Fenótipo , Medição de Risco , Análise de SobrevidaRESUMO
Antibiotic resistant bacteria from faecal pollution sources are pervasive in aquatic environments. A facilitating role for the emergence of waterborne, multi-drug resistant bacterial pathogens has been attributed to biofiltration but had not yet been substantiated. This study investigated the effect of filtration and gut passage in Daphnia spp. on conjugal transfer of resistance genes in Enterococcus faecalis. In vivo conjugation experiments involved a vancomycin-resistant donor strain bearing a plasmid-borne vanA resistance gene, and two vancomycin-susceptible and rifampicin-resistant recipient strains in the presence of Daphnia magna or Daphnia pulex. Results showed successful transfer of the vanA resistance gene from donor to recipient; gene identity was confirmed by PCR and DNA sequencing. There was no significant difference in the number of transconjugants recovered from D. magna and D. pulex. However, transconjugant numbers differed by one order of magnitude between recipient strains. Transconjugant numbers from D. magna were also significantly different between treatments with ingestion of individual phytoplankton species before filtration of bacteria. The highest transfer efficiency calculated from excreted transconjugants was 2.5â¯×â¯10-6. This proof of concept for facilitation of horizontal gene transfer by a filter feeding organism provides evidence that Daphnia can disseminate antibiotic resistant transconjugants in the environment.
Assuntos
Daphnia/fisiologia , Farmacorresistência Bacteriana/genética , Enterococcus faecalis/fisiologia , Animais , Monitoramento Ambiental , Transferência Genética Horizontal , Plasmídeos , Vancomicina , Resistência a Vancomicina , Zooplâncton/fisiologiaRESUMO
The use of coliforms and Escherichia coli as indicator species for assessing the quality of water is well established and a large variety of methods based on ß-galactosidase (B-GAL) activity, inherent to the microbes within this classification, have arisen to enable their detection and enumeration. Chlorophenol red (CPR) is widely used as a chromogenic label, but its capacity for translation to electroanalytical devices has yet to be fully explored. The CPR moiety is capable of undergoing oxidation at carbon substrates (+0.7â¯V) giving rise to a variety of phenolic intermediates. Electrochemical, XPS and enzymatic techniques were employed to characterise the underpinning chemistry and the intermediate identified as a 1,2-quinone derivative in which the chlorine substituent is retained. The latter was found to accumulate at the electrode and, in contrast to the parent CPR, was found to be detected at a significantly less positive potential (+0.3â¯V). Bacterial hydrolysis of a CPR labelled substrate was demonstrated with the 1,2-quinone oxidation product found to accumulate at the electrode and detected using square wave voltammetry. Proof of concept for the efficacy of the alternative electrode pathway was established through the detection of E.coli after an incubation time of 2.5â¯h with no interference from the labelled substrates.