RESUMO
Inflammatory bowel disease (IBD) is a chronic inflammatory condition that affects humans and several domestic animal species, including cats and dogs. In this study, we have analyzed duodenal organoids derived from canine IBD patients using quantitative proteomics. Our objective was to investigate whether these organoids show phenotypic traits of the disease compared with control organoids obtained from healthy donors. To this aim, IBD and control organoids were subjected to quantitative proteomics analysis via liquid chromatography-mass spectrometry. The obtained data revealed notable differences between the two groups. The IBD organoids exhibited several alterations at the levels of multiple proteins that are consistent with some known IBD alterations. The observed phenotype in the IBD organoids to some degree mirrors the corresponding intestinal condition, rendering them a compelling approach for investigating the disease and advancing drug exploration. Additionally, our study revealed similarities to some human IBD biomarkers, further emphasizing the translational and comparative value of dogs for future investigations related to the causes and treatment of IBD. Relevant proteins such as CALU, FLNA, MSN and HMGA2, which are related to intestinal diseases, were all upregulated in the IBD duodenal organoids. At the same time, other proteins such as intestinal keratins and the mucosal immunity PIGR were depleted in these IBD organoids. Based on these findings, we propose that these organoids could serve as a valuable tool for evaluating the efficacy of therapeutic interventions against canine IBD.
Assuntos
Doenças Inflamatórias Intestinais , Intestinos , Cães , Animais , Humanos , Gatos , Doenças Inflamatórias Intestinais/veterinária , Animais Domésticos , Duodeno , OrganoidesRESUMO
Antimicrobial peptides (AMPs) are key components of innate immune defenses. Because of the antibiotic crisis, AMPs have also come into focus as new drugs. Here, we explore whether prior exposure to sub-lethal doses of AMPs increases bacterial survival and abets the evolution of resistance. We show that Escherichia coli primed by sub-lethal doses of AMPs develop tolerance and increase persistence by producing curli or colanic acid, responses linked to biofilm formation. We develop a population dynamic model that predicts that priming delays the clearance of infections and fuels the evolution of resistance. The effects we describe should apply to many AMPs and other drugs that target the cell surface. The optimal strategy to tackle tolerant or persistent cells requires high concentrations of AMPs and fast and long-lasting expression. Our findings also offer a new understanding of non-inherited drug resistance as an adaptive response and could lead to measures that slow the evolution of resistance.
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Peptídeos Catiônicos Antimicrobianos/farmacologia , Resistência Microbiana a Medicamentos/fisiologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Bactérias/metabolismo , Meliteno/farmacologia , Polissacarídeos/metabolismoRESUMO
Unicellular organisms have the prevalent challenge to survive under oxidative stress of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2). ROS are present as by-products of photosynthesis and aerobic respiration. These reactive species are even employed by multicellular organisms as potent weapons against microbes. Although bacterial defences against lethal and sub-lethal oxidative stress have been studied in model bacteria, the role of fluctuating H2O2 concentrations remains unexplored. It is known that sub-lethal exposure of Escherichia coli to H2O2 results in enhanced survival upon subsequent exposure. Here we investigate the priming response to H2O2 at physiological concentrations. The basis and the duration of the response (memory) were also determined by time-lapse quantitative proteomics. We found that a low level of H2O2 induced several scavenging enzymes showing a long half-life, subsequently protecting cells from future exposure. We then asked if the phenotypic resistance against H2O2 alters the evolution of resistance against oxygen stress. Experimental evolution of H2O2 resistance revealed faster evolution and higher levels of resistance in primed cells. Several mutations were found to be associated with resistance in evolved populations affecting different loci but, counterintuitively, none of them was directly associated with scavenging systems. Our results have important implications for host colonisation and infections where microbes often encounter reactive oxygen species in gradients.
Assuntos
Escherichia coli/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Resistência a Medicamentos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Evolução Molecular , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Nephridiophagids are unicellular fungi (Chytridiomycota), which infect the Malpighian tubules of insects. While most life cycle features are known, the effects of these endobionts on their hosts remain poorly understood. Here, we present results on the influence of an infection of the cockroach Blattella germanica with Nephridiophaga blattellae (Ni = Nephridiophaga-infected) on physical, physiological, and reproductive fitness parameters. Since the gut nematode Blatticola blattae is a further common parasite of B. germanica, we included double infected cockroaches (N + Ni = nematode plus Ni) in selected experiments. Ni individuals had lower fat reserves and showed reduced mobility. The lifespan of adult hosts was only slightly affected in these individuals but significantly shortened when both Nephridiophaga and nematodes were present. Ni as well as N + Ni females produced considerably less offspring than parasite-free (P-free) females. Immune parameters such as the number of hemocytes and phenoloxidase activity were barely changed by Nephridiophaga and/or nematode infections, while the ability to detoxify pesticides decreased. Quantitative proteomics from hemolymph of P-free, Ni, and N + Ni populations revealed clear differences in the expression profiles. For Ni animals, for example, the down-regulation of fatty acid synthases corroborates our finding of reduced fat reserves. Our study clearly shows that an infection with Nephridiophaga (and nematodes) leads to an overall reduced host fitness.
Assuntos
Blattellidae , Quitridiomicetos , Animais , Feminino , Hemolinfa , Insetos , Estágios do Ciclo de VidaRESUMO
BACKGROUND: Fluoroquinolones such as ciprofloxacin induce the mutagenic SOS response and increase the levels of intracellular reactive oxygen species (ROS). Both the SOS response and ROS increase bacterial mutagenesis, fuelling the emergence of resistant mutants during antibiotic treatment. Recently, there has been growing interest in developing new drugs able to diminish the mutagenic effect of antibiotics by modulating ROS production and the SOS response. OBJECTIVES: To test whether physiological concentrations of N-acetylcysteine, a clinically safe antioxidant drug currently used in human therapy, is able to reduce ROS production, SOS induction and mutagenesis in ciprofloxacin-treated bacteria without affecting antibiotic activity. METHODS: The Escherichia coli strain IBDS1 and its isogenic mutant deprived of SOS mutagenesis (TLS-) were treated with different concentrations of ciprofloxacin, N-acetylcysteine or both drugs in combination. Relevant parameters such as MICs, growth rates, ROS production, SOS induction, filamentation and antibiotic-induced mutation rates were evaluated. RESULTS: Treatment with N-acetylcysteine reduced intracellular ROS levels (by â¼40%), as well as SOS induction (by up to 75%) and bacterial filamentation caused by subinhibitory concentrations of ciprofloxacin, without affecting ciprofloxacin antibacterial activity. Remarkably, N-acetylcysteine completely abolished SOS-mediated mutagenesis. CONCLUSIONS: Collectively, our data strongly support the notion that ROS are a key factor in antibiotic-induced SOS mutagenesis and open the possibility of using N-acetylcysteine in combination with antibiotic therapy to hinder the development of antibiotic resistance.
Assuntos
Acetilcisteína/farmacologia , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Fluoroquinolonas/farmacologia , Mutagênese/efeitos dos fármacos , Resposta SOS em Genética/efeitos dos fármacos , Ciprofloxacina/farmacologia , Farmacorresistência Bacteriana/genética , Escherichia coli/fisiologia , Proteínas de Escherichia coli/genética , Testes de Sensibilidade Microbiana , Taxa de Mutação , Espécies Reativas de Oxigênio/análiseRESUMO
BACKGROUND: In nature, microorganisms have to adapt to long-term stressful conditions often with growth limitations. However, little is known about the evolution of the adaptability of new bacteria to such environments. Pseudomonas aeruginosa, an opportunistic pathogen, after natural evaporation of seawater, was shown to be trapped in laboratory-grown halite crystals and to remain viable after entrapment for years. However, how this bacterium persists and survives in such hypersaline conditions is not understood. RESULTS: In this study, we aimed to understand the basis of survival, and to characterise the physiological changes required to develop salt tolerance using P. aeruginosa as a model. Several clones of P. aeruginosa were rescued after 14 years in naturally evaporated marine salt crystals. Incubation of samples in nutrient-rich broth allowed re-growth and subsequent plating yielded observable colonies. Whole genome sequencing of the P. aeruginosa isolates confirmed the recovery of the original strain. The re-grown strains, however, showed a new phenotype consisting of an enhanced growth in growing salt concentration compared to the ancestor strain. The intracellular accumulation of K+ was elicited by high concentration of Na+ in the external medium to maintain the homeostasis. Whole transcriptomic analysis by microarray indicated that 78 genes had differential expression between the parental strain and its derivative clones. Sixty-one transcripts were up-regulated, while 17 were down-regulated. Based on a collection of single-gene knockout mutants and gene ontology analysis, we suggest that the adaptive response in P. aeruginosa to hyper-salinity relies on multiple gene product interactions. CONCLUSIONS: The individual gene contributions build up the observed phenotype, but do not ease the identification of salinity-related metabolic pathways. The long-term inclusion of P. aeruginosa in salt crystals primes the bacteria, mediating a readjustment of the bacterial physiology to growth in higher salt concentrations. Our findings provide a starting point to understand how P. aeruginosa, a relevant environmental and pathogenic bacterium, survives to long-term salt stress.
Assuntos
Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/fisiologia , Tolerância ao Sal/fisiologia , Água do Mar/microbiologia , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Ontologia Genética , Genes Bacterianos/genética , Homeostase , Redes e Vias Metabólicas/genética , Redes e Vias Metabólicas/fisiologia , Fenótipo , Pseudomonas aeruginosa/genética , Salinidade , Tolerância ao Sal/genética , Sais , Estresse Fisiológico , Sequenciamento Completo do GenomaRESUMO
Cationic antimicrobial peptides are ubiquitous immune effectors of multicellular organisms. We previously reported, that in contrast to most of the classic antibiotics, cationic antimicrobial peptides (AMPs) do not increase mutation rates in E. coli Here, we provide new evidence showing that AMPs do not stimulate or enhance bacterial DNA recombination in the surviving fractions. Recombination accelerates evolution of antibiotic resistance. Our findings have implications for our understanding of host-microbe interactions, the evolution of innate immune defences, and shed new light on the dynamic of antimicrobial-resistance evolution.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Escherichia coli/genética , Recombinação Genética , Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Escherichia coli/efeitos dos fármacos , Recombinação Genética/efeitos dos fármacosRESUMO
Pseudomonas aeruginosa is the causative agent of chronic respiratory infections and is an important pathogen of cystic fibrosis patients. Adaptive mutations play an essential role for antimicrobial resistance and persistence. The factors that contribute to bacterial mutagenesis in this environment are not clear. Recently it has been proposed that cationic antimicrobial peptides such as LL-37 could act as mutagens in P. aeruginosa. Here we provide experimental evidence that mutagenesis is the product of a joint action of LL-37 and free iron. By estimating mutation rate, mutant frequencies and assessing mutational spectra in P. aeruginosa treated either with LL-37, iron or a combination of both we demonstrate that mutation rate and mutant frequency were increased only when free iron and LL-37 were present simultaneously. Colistin had the same effect. The addition of an iron chelator completely abolished this mutagenic effect, suggesting that LL-37 enables iron to enter the cells resulting in DNA damage by Fenton reactions. This was also supported by the observation that the mutational spectrum of the bacteria under LL-37-iron regime showed one of the characteristic Fenton reaction fingerprints: C to T transitions. Free iron concentration in nature and within hosts is kept at a very low level, but the situation in infected lungs of cystic fibrosis patients is different. Intermittent bleeding and damage to the epithelial cells in lungs may contribute to the release of free iron that in turn leads to generation of reactive oxygen species and deterioration of the respiratory tract, making it more susceptible to the infection.
Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Resistência Microbiana a Medicamentos/genética , Mutagênese/efeitos dos fármacos , Infecções por Pseudomonas/genética , Pseudomonas aeruginosa/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Dano ao DNA/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Ferro/farmacologia , Mutação , Taxa de Mutação , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/patogenicidade , Espécies Reativas de Oxigênio/metabolismo , CatelicidinasRESUMO
Cationic antimicrobial peptides are ancient and ubiquitous immune effectors that multicellular organisms use to kill and police microbes whereas antibiotics are mostly employed by microorganisms. As antimicrobial peptides (AMPs) mostly target the cell wall, a microbial 'Achilles heel', it has been proposed that bacterial resistance evolution is very unlikely and hence AMPs are ancient 'weapons' of multicellular organisms. Here we provide a new hypothesis to explain the widespread distribution of AMPs amongst multicellular organism. Studying five antimicrobial peptides from vertebrates and insects, we show, using a classic Luria-Delbrück fluctuation assay, that cationic antimicrobial peptides (AMPs) do not increase bacterial mutation rates. Moreover, using rtPCR and disc diffusion assays we find that AMPs do not elicit SOS or rpoS bacterial stress pathways. This is in contrast to the main classes of antibiotics that elevate mutagenesis via eliciting the SOS and rpoS pathways. The notion of the 'Achilles heel' has been challenged by experimental selection for AMP-resistance, but our findings offer a new perspective on the evolutionary success of AMPs. Employing AMPs seems advantageous for multicellular organisms, as it does not fuel the adaptation of bacteria to their immune defenses. This has important consequences for our understanding of host-microbe interactions, the evolution of innate immune defenses, and also sheds new light on antimicrobial resistance evolution and the use of AMPs as drugs.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Mutagênese/imunologia , Vertebrados/imunologia , Animais , Anti-Infecciosos/imunologia , Peptídeos Catiônicos Antimicrobianos/imunologia , Bactérias/imunologia , Imunidade Inata/imunologiaRESUMO
Genetic constraints can block many mutational pathways to optimal genotypes in real fitness landscapes, yet the extent to which this can limit evolution remains to be determined. Interestingly, mutator bacteria elevate only specific types of mutations, and therefore could be very sensitive to genetic constraints. Testing this possibility is not only clinically relevant, but can also inform about the general impact of genetic constraints in adaptation. Here, we evolved 576 populations of two mutator and one wild-type Escherichia coli to doubling concentrations of the antibiotic cefotaxime. All strains carried TEM-1, a ß-lactamase enzyme well known by its low availability of mutational pathways. Crucially, one of the mutators does not elevate any of the relevant first-step mutations known to improve cefatoximase activity. Despite this, both mutators displayed a similar ability to evolve more than 1000-fold resistance. Initial adaptation proceeded in parallel through general multi-drug resistance mechanisms. High-level resistance, in contrast, was achieved through divergent paths; with the a priori inferior mutator exploiting alternative mutational pathways in PBP3, the target of the antibiotic. These results have implications for mutator management in clinical infections and, more generally, illustrate that limits to natural selection in real organisms are alleviated by the existence of multiple loci contributing to fitness.
Assuntos
Antibacterianos/farmacologia , Cefotaxima/farmacologia , Resistência Microbiana a Medicamentos/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Evolução Molecular , Mutação , Alelos , Relação Dose-Resposta a Droga , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Genéticos , Pirofosfatases/genética , Pirofosfatases/metabolismoRESUMO
Non-heritable, phenotypic drug resistance toward antibiotics challenges antibiotic therapies. Characteristics of such phenotypic resistance have implications for the evolution of heritable resistance. Diverse forms of phenotypic resistance have been described, but phenotypic resistance characteristics remain less explored than genetic resistance. Here, we add novel combinations of single-cell characteristics of phenotypic resistant E. coli cells and compare those to characteristics of susceptible cells of the parental population by exposure to different levels of recurrent ampicillin antibiotic. Contrasting expectations, we did not find commonly described characteristics of phenotypic resistant cells that arrest growth or near growth. We find that under ampicillin exposure, phenotypic resistant cells reduced their growth rate by about 50% compared to growth rates prior to antibiotic exposure. The growth reduction is a delayed alteration to antibiotic exposure, suggesting an induced response and not a stochastic switch or caused by a predetermined state as frequently described. Phenotypic resistant cells exhibiting constant slowed growth survived best under ampicillin exposure and, contrary to expectations, not only fast-growing cells suffered high mortality triggered by ampicillin but also growth-arrested cells. Our findings support diverse modes of phenotypic resistance, and we revealed resistant cell characteristics that have been associated with enhanced genetically fixed resistance evolution, which supports claims of an underappreciated role of phenotypic resistant cells toward genetic resistance evolution. A better understanding of phenotypic resistance will benefit combatting genetic resistance by developing and engulfing effective anti-phenotypic resistance strategies. IMPORTANCE: Antibiotic resistance is a major challenge for modern medicine. Aside from genetic resistance to antibiotics, phenotypic resistance that is not heritable might play a crucial role for the evolution of antibiotic resistance. Using a highly controlled microfluidic system, we characterize single cells under recurrent exposure to antibiotics. Fluctuating antibiotic exposure is likely experienced under common antibiotic therapies. These phenotypic resistant cell characteristics differ from previously described phenotypic resistance, highlighting the diversity of modes of resistance. The phenotypic characteristics of resistant cells we identify also imply that such cells might provide a stepping stone toward genetic resistance, thereby causing treatment failure.
Assuntos
Ampicilina , Antibacterianos , Farmacorresistência Bacteriana , Escherichia coli , Fenótipo , Análise de Célula Única , Ampicilina/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Farmacorresistência Bacteriana/efeitos dos fármacos , Testes de Sensibilidade MicrobianaRESUMO
Introduction: Bloodwork is a widely used diagnostic tool in veterinary medicine, as diagnosis and therapeutic interventions often rely on blood biomarkers. However, biomarkers available in veterinary medicine often lack sensitivity or specificity. Mass spectrometry-based proteomics technology has been extensively used in the analysis of biological fluids. It offers excellent potential for a more comprehensive characterization of the plasma proteome in veterinary medicine. Methods: In this study, we aimed to identify and quantify plasma proteins in a cohort of healthy dogs and compare two techniques for depleting high-abundance plasma proteins to enable the detection of lower-abundance proteins via label-free quantification liquid chromatography-mass spectrometry. We utilized surplus lithium-heparin plasma from 30 healthy dogs, subdivided into five groups of pooled plasma from 6 randomly selected individuals each. Firstly, we used a commercial kit to deplete high-abundance plasma proteins. Secondly, we employed an in-house method to remove albumin using Blue-Sepharose. Results and discussion: Among all the samples, some of the most abundant proteins identified were apolipoprotein A and B, albumin, alpha-2-macroglobulin, fibrinogen beta chain, fibronectin, complement C3, serotransferrin, and coagulation factor V. However, neither of the depletion techniques achieved significant depletion of highly abundant proteins. Despite this limitation, we could detect and quantify many clinically relevant proteins. Determining the healthy canine proteome is a crucial first step in establishing a reference proteome for canine plasma. After enrichment, this reference proteome can later be utilized to identify protein markers associated with different diseases, thereby contributing to the diagnosis and prognosis of various pathologies.
RESUMO
Acute haemorrhagic diarrhoea is a common complaint in dogs. In addition to causes like intestinal parasites, dietary indiscretion, intestinal foreign bodies, canine parvovirus infection, or hypoadrenocorticism, acute haemorrhagic diarrhoea syndrome (AHDS) is an important and sometimes life-threatening differential diagnosis. There is some evidence supporting the link between Clostridium perfringens toxins and AHDS. These toxins may be partially responsible for the epithelial cell injury, but the pathogenesis of AHDS is still not fully understood. Recent studies have suggested that severe damage to the intestinal mucosa and associated barrier dysfunction can trigger chronic gastrointestinal illnesses. Besides bloodwork and classical markers for AHDS such as protein loss and intestinal bacterial dysbiosis, we focused mainly on the plasma-proteome to identify systemic pathological alterations during this disease and searched for potential biomarkers to improve the diagnosis. To accomplish the goals, we used liquid chromatography-mass spectrometry. We compared the proteomic profiles of 20 dogs with AHDS to 20 age-, breed-, and sex-matched control dogs. All dogs were examined, and several blood work parameters were determined and compared, including plasma biochemistry and cell counts. We identified and quantified (relative quantification) 207 plasmatic proteins, from which dozens showed significantly altered levels in AHDS. Serpina3, Lipopolysaccharide-binding protein, several Ig-like domain-containing proteins, Glyceraldehyde-3-phosphate dehydrogenase and Serum amyloid A were more abundant in plasma from AHDS affected dogs. In contrast, other proteins such as Paraoxonase, Selenoprotein, Amine oxidases, and Apolipoprotein C-IV were significantly less abundant. Many of the identified and quantified proteins are known to be associated with inflammation. Other proteins like Serpina3 and RPLP1 have a relevant role in oncogenesis. Some proteins and their roles have not yet been described in dogs with diarrhoea. Our study opens new avenues that could contribute to the understanding of the aetiology and pathophysiology of AHDS.
Assuntos
Doenças do Cão , Proteoma , Cães , Animais , Proteômica , Hemorragia Gastrointestinal/microbiologia , Síndrome , Diarreia/microbiologia , Doenças do Cão/patologiaRESUMO
Introduction/objective: Suppression of the SOS response in combination with drugs damaging DNA has been proposed as a potential target to tackle antimicrobial resistance. The SOS response is the pathway used to repair bacterial DNA damage induced by antimicrobials such as quinolones. The extent of lexA-regulated protein expression and other associated systems under pressure of agents that damage bacterial DNA in clinical isolates remains unclear. The aim of this study was to assess the impact of this strategy consisting on suppression of the SOS response in combination with quinolones on the proteome profile of Escherichia coli clinical strains. Materials and methods: Five clinical isolates of E. coli carrying different chromosomally- and/or plasmid-mediated quinolone resistance mechanisms with different phenotypes were selected, with E. coli ATCC 25922 as control strain. In addition, from each clinical isolate and control, a second strain was created, in which the SOS response was suppressed by deletion of the recA gene. Bacterial inocula from all 12 strains were then exposed to 1xMIC ciprofloxacin treatment (relative to the wild-type phenotype for each isogenic pair) for 1 h. Cell pellets were collected, and proteins were digested into peptides using trypsin. Protein identification and label-free quantification were done by liquid chromatography-mass spectrometry (LC-MS) in order to identify proteins that were differentially expressed upon deletion of recA in each strain. Data analysis and statistical analysis were performed using the MaxQuant and Perseus software. Results: The proteins with the lowest expression levels were: RecA (as control), AphA, CysP, DinG, DinI, GarL, PriS, PsuG, PsuK, RpsQ, UgpB and YebG; those with the highest expression levels were: Hpf, IbpB, TufB and RpmH. Most of these expression alterations were strain-dependent and involved DNA repair processes and nucleotide, protein and carbohydrate metabolism, and transport. In isolates with suppressed SOS response, the number of underexpressed proteins was higher than overexpressed proteins. Conclusion: High genomic and proteomic variability was observed among clinical isolates and was not associated with a specific resistant phenotype. This study provides an interesting approach to identify new potential targets to combat antimicrobial resistance.
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Animal fodder is routinely complemented with antibiotics together with other food supplements to improve growth. For instance, sepiolite is currently used as a dietary coadjuvant in animal feed, as it increases animal growth parameters and improves meat and derived final product quality. This type of food additive has so far been considered innocuous for the development and spread of antibiotic resistance. In this study, we demonstrate that sepiolite promotes the direct horizontal transfer of antibiotic resistance plasmids between bacterial species. The conditions needed for plasmid transfer (sepiolite and friction forces) occur in the digestive tracts of farm animals, which routinely receive sepiolite as a food additive. Furthermore, this effect may be aggravated by the use of antibiotics supplied as growth promoters.
Assuntos
Ração Animal , Bactérias/efeitos dos fármacos , Bactérias/genética , Resistência Microbiana a Medicamentos/genética , Aditivos Alimentares/farmacologia , Transferência Genética Horizontal/efeitos dos fármacos , Silicatos de Magnésio/farmacologia , Plasmídeos/genética , Transformação Bacteriana/efeitos dos fármacos , Animais , Animais Domésticos/genética , Animais Domésticos/microbiologia , Antibacterianos/farmacologia , Bactérias/classificação , Testes de Sensibilidade Microbiana , Transformação Bacteriana/genéticaRESUMO
One of the most terrible consequences of Darwinian evolution is arguably the emergence and spread of antibiotic resistance, which is becoming a serious menace to modern societies. While spontaneous mutation, recombination and horizontal gene transfer are recognized as the main causes of this notorious phenomenon; recent research has raised awareness that sub-lethal concentrations of antibiotics can also foster resistance as an undesirable side-effect. They can produce genetic changes by different ways, including a raise of free radicals within the cell, induction of error-prone DNA-polymerases mediated by SOS response, imbalanced nucleotide metabolism or affect directly DNA. In addition to certain environmental conditions, subinhibitory concentrations of antimicrobials may increase, even more, the mutagenic effect of antibiotics. Here, we review the state of knowledge on antibiotics as promoters of antibiotic resistance.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Farmacorresistência Bacteriana , Mutagênicos/farmacologia , Mutação , Seleção Genética , Animais , Transferência Genética Horizontal , Humanos , Recombinação GenéticaRESUMO
Hypermutation may accelerate bacterial evolution in the short-term. In the long-term, however, hypermutators (cells with an increased rate of mutation) can be expected to be at a disadvantage due to the accumulation of deleterious mutations. Therefore, in theory, hypermutators are doomed to extinction unless they compensate the elevated mutational burden (deleterious load). Different mechanisms capable of restoring a low mutation rate to hypermutators have been proposed. By choosing an 8-oxoguanine-repair-deficient (GO-deficient) Escherichia coli strain as a hypermutator model, we investigated the existence of genes able to rescue the hypermutable phenotype by multicopy suppression. Using an in vivo-generated mini-MudII4042 genomic library and a mutator screen, we obtained chromosomal fragments that decrease the rate of mutation in a mutT-deficient strain. Analysis of a selected clone showed that the expression of NorM is responsible for the decreased mutation rate in 8-oxoguanine-repair-deficient (mutT, mutY, and mutM mutY) strains. NorM is a member of the multidrug and toxin extrusion (MATE) family of efflux pumps whose role in E. coli cell physiology remains unknown. Our results indicate that NorM may act as a GO-system backup decreasing AT to CG and GC to TA transversions. In addition, the ability of NorM to reduce the level of intracellular reactive oxygen species (ROS) in a GO-deficient strain and protect the cell from oxidative stress, including protein carbonylation, suggests that it can extrude specific molecules-byproducts of bacterial metabolism-that oxidize the guanine present in both DNA and nucleotide pools. Altogether, our results indicate that NorM protects the cell from specific ROS when the GO system cannot cope with the damage.
Assuntos
Antiporters/metabolismo , DNA Glicosilases/deficiência , Reparo do DNA , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Guanina/análogos & derivados , Peróxido de Hidrogênio/farmacologia , Mutação , Antiporters/genética , Dano ao DNA , DNA Glicosilases/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Guanina/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Família Multigênica , FenótipoRESUMO
Pseudomonas aeruginosa is a versatile opportunistic pathogen causing a wide variety of hospital-acquired acute infections in immunocompromised patients as well as chronic respiratory infections in patients suffering from cystic fibrosis or other chronic respiratory diseases. Several traits contribute to its ability to colonize and persist in the lungs of chronically infected patients, including development of high resistance to antimicrobials and hypermutability, biofilm growth, and alginate hyperproduction, or a customized pathogenicity, which may include the loss of classical virulence factors and metabolic changes. Here we argue that a combination of both intrinsic and environmental mutagenesis leads to a high number of mutant variants in the population. The conducive environment then triggers a positive feedback loop leading to adaptation and persistence of P. aeruginosa, rendering these chronic infections almost impossible to eradicate.
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Fibrose Cística/complicações , Mutagênese , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/patogenicidade , Infecções Respiratórias/microbiologia , Adaptação Biológica , Biofilmes , Doença Crônica , Farmacorresistência Bacteriana , Interação Gene-Ambiente , Humanos , Taxa de Mutação , Fenótipo , Infecções por Pseudomonas/complicações , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiologia , Infecções Respiratórias/complicaçõesRESUMO
The role of the gut microbiome in developing Inflammatory Bowel Disease (IBD) in humans and dogs has received attention in recent years. Evidence suggests that IBD is associated with alterations in gut microbial composition, but further research is needed in veterinary medicine. The impact of IBD treatment on the gut microbiome needs to be better understood, especially in a breed-specific form of IBD in Yorkshire Terriers known as Yorkshire Terrier Enteropathy (YTE). This study aimed to investigate the difference in gut microbiome composition between YTE dogs during disease and remission and healthy Yorkshire Terriers. Our results showed a significant increase in specific taxa such as Clostridium sensu stricto 1, Escherichia-Shigella, and Streptococcus, and a decrease in Bacteroides, Prevotella, Alloprevotella, and Phascolarctobacterium in YTE dogs compared to healthy controls. No significant difference was found between the microbiome of dogs in remission and those with active disease, suggesting that the gut microbiome is affected beyond clinical recovery.
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Microbioma Gastrointestinal , Doenças Inflamatórias Intestinais , Microbiota , Humanos , Cães , Animais , Doenças Inflamatórias Intestinais/microbiologia , BacteroidetesRESUMO
Excessive discharge of quaternary ammonium disinfectants such as benzalkonium chloride (BAC) into aquatic systems can trigger several physiological responses in environmental microorganisms. In this study, we isolated a less-susceptible strain of Aeromonas hydrophila to BAC, designated as INISA09, from a wastewater treatment plant in Costa Rica. We characterized its phenotypic response upon exposure to three different concentrations of BAC and characterized mechanisms related to its resistance using genomic and proteomic approaches. The genome of the strain, mapped against 52 different sequenced A. hydrophila strains, consists of approximately 4.6 Mb with 4,273 genes. We found a massive genome rearrangement and thousands of missense mutations compared to the reference strain A. hydrophila ATCC 7966. We identified 15,762 missense mutations mainly associated with transport, antimicrobial resistance, and outer membrane proteins. In addition, a quantitative proteomic analysis revealed a significant upregulation of several efflux pumps and the downregulation of porins when the strain was exposed to three BAC concentrations. Other genes related to membrane fatty acid metabolism and redox metabolic reactions also showed an altered expression. Our findings indicate that the response of A. hydrophila INISA09 to BAC primarily occurs at the envelop level, which is the primary target of BAC. Our study elucidates the mechanisms of antimicrobial susceptibility in aquatic environments against a widely used disinfectant and will help better understand how bacteria can adapt to biocide pollution. To our knowledge, this is the first study addressing the resistance to BAC in an environmental A. hydrophila isolate. We propose that this bacterial species could also serve as a new model to study antimicrobial pollution in aquatic environments.