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1.
Int J Mol Sci ; 22(5)2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33668925

RESUMO

Lipid A of Gram-negative bacteria is known to represent a central role for the immunological activity of endotoxin. Chemical structure and biosynthetic pathways as well as specific receptors on phagocytic cells had been clarified by the beginning of the 21st century. Although the lipid A of enterobacteria including Escherichia coli share a common structure, other Gram-negative bacteria belonging to various classes of the phylum Proteobacteria and other taxonomical groups show wide variety of lipid A structure with relatively decreased endotoxic activity compared to that of E. coli. The structural diversity is produced from the difference of chain length of 3-hydroxy fatty acids and non-hydroxy fatty acids linked to their hydroxyl groups. In some bacteria, glucosamine in the backbone is substituted by another amino sugar, or phosphate groups bound to the backbone are modified. The variation of structure is also introduced by the enzymes that can modify electrostatic charges or acylation profiles of lipid A during or after its synthesis. Furthermore, lipid A structure can be artificially modified or engineered by the disruption and introduction of biosynthetic genes especially those of acyltransferases. These technologies may produce novel vaccine adjuvants or antagonistic drugs derived from endotoxin in the future.


Assuntos
Engenharia Genética , Bactérias Gram-Negativas/metabolismo , Lipídeo A/química , Acilação , Genes Bacterianos , Bactérias Gram-Negativas/genética
2.
J Infect Public Health ; 14(4): 478-483, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33743369

RESUMO

The emergence of multidrug-resistant Gram negative bacteria has given rise to significant therapeutic challenges. These pathogens may have developed resistance to tigecycline, which is an alternative antibiotic used empirically in the treatment of serious infections. The objectives of this study were to identify the in-vitro activity of tigecycline against multidrug-resistant Gram negative strains isolated from clinical specimens and their related genes, at a university hospital. For this, 150 clinical isolates of multidrug-resistant Gram negative cultures from various clinical specimens were collected. Bacterial isolates were cultured, identified and their antibiotic susceptibilities were determined. Polymerase chain reaction was performed to amplify AcrB, AmpC, RamR, MexR, AdeB, TetA genes. Results revealed that all isolates were multidrug-resistant. The resistance of isolates was 91.4% to aztreonam, 94.6% to piperacillin, 34% to imipenem, 38.7% to meropenem, 71.3% to levofloxacin, 97.3% to ceftriaxone, 94.7% to cefepime, 9.3% to colistin, 78% to tetracycline, 21.4% to tigecycline and 68% to trimethoprim. AcrB, AmpC, RamR, MexR, AdeB, TetA genes were present in multidrug-resistant Gram negative bacteria. AcrB, RamR, TetA genes were related to tigecycline resistance. It is concluded that infections caused by multidrug-resistant Gram negative bacteria occur at a high rate. Most isolates were multi drug resistant, with 21.4% being resistant to tigecycline.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Tigeciclina/farmacologia , DNA Bacteriano/genética , Bactérias Gram-Negativas/genética , Humanos , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase/métodos
3.
Nat Commun ; 12(1): 1171, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33608525

RESUMO

Direct cloning represents the most efficient strategy to access the vast number of uncharacterized natural product biosynthetic gene clusters (BGCs) for the discovery of novel bioactive compounds. However, due to their large size, repetitive nature, or high GC-content, large-scale cloning of these BGCs remains an overwhelming challenge. Here, we report a scalable direct cloning method named Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination (CAPTURE) which consists of Cas12a digestion, a DNA assembly approach termed T4 polymerase exo + fill-in DNA assembly, and Cre-lox in vivo DNA circularization. We apply this method to clone 47 BGCs ranging from 10 to 113 kb from both Actinomycetes and Bacilli with ~100% efficiency. Heterologous expression of cloned BGCs leads to the discovery of 15 previously uncharacterized natural products including six cyclic head-to-tail heterodimers with a unique 5/6/6/6/5 pentacyclic carbon skeleton, designated as bipentaromycins A-F. Four of the bipentaromycins show strong antimicrobial activity to both Gram-positive and Gram-negative bacteria such as methicillin-resistant Staphylococcus aureus, vancomycinresistant Enterococcus faecium, and bioweapon Bacillus anthracis. Due to its robustness and efficiency, our direct cloning method coupled with heterologous expression provides an effective strategy for large-scale discovery of novel natural products.


Assuntos
Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Clonagem Molecular/métodos , Endodesoxirribonucleases/genética , Integrases/genética , Recombinação Genética , Actinobacteria/genética , Actinobacteria/metabolismo , Produtos Biológicos/metabolismo , Vias Biossintéticas/genética , DNA Bacteriano , Enterococcus faecium/genética , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/metabolismo , Staphylococcus aureus Resistente à Meticilina/genética , Família Multigênica , Streptomyces/genética
4.
J Med Microbiol ; 70(3)2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33448924

RESUMO

Introduction. Gram-negative bacteria are a common source of infection both in hospitals and in the community, and antimicrobial resistance is frequent among them, making antibiotic therapy difficult, especially when these isolates carry carbapenem resistance determinants.Hypothesis/Gap Statement. A simple method to detect all the commonly found carbapenemases in Germany was not available.Aim. The aim of this study was to develop a multiplex PCR for the rapid and reliable identification of the most prevalent carbapenemase-encoding genes in Gram-negative bacteria in Germany.Methodology. Data from the German Gram-negative reference laboratory revealed the most prevalent carbapenemase groups in Germany were (in order of prevalence): bla VIM, bla OXA-48, bla OXA-23, bla KPC, bla NDM, bla OXA-40, bla OXA-58, bla IMP, bla GIM, bla GES, ISAba1-bla OXA-51, bla IMI, bla FIM and bla DIM. We developed and tested two multiplex PCRs against 83 carbapenem-resistant Gram-negative clinical isolates. Primers were designed for each carbapenemase group within conserved regions of the encoding genes obtained from publicly available databases. Multiplex-1 included the carbapenemase groups bla VIM, bla OXA-48, bla OXA-23, bla KPC, bla NDM and bla OXA-40, while multiplex-2 included bla OXA-58, bla IMP, bla GIM, bla GES, ISAba1-bla OXA-51 and bla IMI.Results. In the initial evaluation, all but one of the carbapenemases encoded by 75 carbapenemase-positive isolates were detected using the two multiplex PCRs, while no false-positive results were obtained from the remaining eight isolates. After evaluation, we tested 546 carbapenem-resistant isolates using the multiplex PCRs, and all carbapenemases were detected.Conclusion. A rapid and reliable method was developed for detection and differentiation of 12 of the most prevalent carbapenemase groups found in Germany. This method allows for the rapid testing of clinical isolates prior to species identification and does not require prior phenotypical characterization, constituting a rapid and valuable tool in the management of infections in hospitals.


Assuntos
Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Bactérias Gram-Negativas , Infecções por Bactérias Gram-Negativas/microbiologia , Reação em Cadeia da Polimerase/métodos , beta-Lactamases/genética , Genes Bacterianos , Alemanha , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/epidemiologia , Humanos , Prevalência
5.
Int J Food Microbiol ; 340: 109045, 2021 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-33465548

RESUMO

Extended use of antibiotics in dairy farming for therapeutic and prophylactic reasons, but also the higher prevalence of antibiotic resistant bacteria (ARB) in the farm environment raised the concern of consuming raw cow's milk and its derived products. The aim of this study was to predict by shotgun metagenomic analyses the presence of antibiotic resistance genes (ARGs) mainly correlated with Gram-negative bacteria in antibiotic residue free raw cow's milk derived exclusively from healthy animal from South Tyrol (Northern Italy), chosen as a model system. Assessment of shotgun metagenomic data of reconstructed scaffolds, revealed the existence of Pseudomonas spp. as the most abundant Gram-negative species in the raw cow's milk samples bearing ARGs. Besides, ARGs also linked to lactic acid bacteria such as Lactococcus sp. and Lactobacillus sp. ARGs correlated to microbiome found in milk samples conferred resistance towards aminoglycoside-streptothricin, beta-lactamase, macrolide, tetracycline, carbapenem, cephalosporin, penam, peptide, penem, fluoroquinolone, chloramphenicol and elfamycin antibiotics. Further bioinformatic processing included de-novo reassembly of all metagenomic sequences from all milk samples in one, to reconstruct metagenome assembled genomes (MAGs), which were further used to investigate mobile genetic elements (MGE). Analyses of the reconstructed MAGs showed that, MAG 9 (Pseudomonas sp1.) contained the oriT gene (origin of transfer gene) needed for transferring virulent factors. Although the presence of Pseudomonas is common in raw cow's milk, pasteurization treatment reduces their survivability. Nevertheless, attention should be paid on Pseudomonas spp. due to their intrinsic resistance to antibiotics and their capability of transferring virulent factors to other bacteria.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Bactérias Gram-Negativas/efeitos dos fármacos , Leite/microbiologia , Animais , Bovinos , Feminino , Genes Bacterianos , Bactérias Gram-Negativas/genética , Itália , Metagenoma , Microbiota , Pseudomonas/efeitos dos fármacos , Pseudomonas/genética
6.
Nat Commun ; 12(1): 369, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33446644

RESUMO

Lipopolysaccharides are important components of the bacterial cell envelope that among other things act as a protective barrier against the environment and toxic molecules such as antibiotics. One of the most widely disseminated pathways of polysaccharide biosynthesis is the inner membrane bound Wzy-dependent pathway. Here we present the 3.0 Å structure of the co-polymerase component of this pathway, WzzB from E. coli solved by single-particle cryo-electron microscopy. The overall architecture is octameric and resembles a box jellyfish containing a large bell-shaped periplasmic domain with the 2-helix transmembrane domain from each protomer, positioned 32 Å apart, encircling a large empty transmembrane chamber. This structure also reveals the architecture of the transmembrane domain, including the location of key residues for the Wzz-family of proteins and the Wzy-dependent pathway present in many Gram-negative bacteria, explaining several of the previous biochemical and mutational studies and lays the foundation for future investigations.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Polissacarídeos Bacterianos/química , Microscopia Crioeletrônica , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/metabolismo , Modelos Moleculares , Polissacarídeos Bacterianos/metabolismo , Regiões Promotoras Genéticas , Domínios Proteicos
7.
Anal Chem ; 93(2): 928-935, 2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33320524

RESUMO

It is predicted that the antibiotic resistance crisis will result in an annual death rate of 10 million people by the year 2050. To grapple with the challenges of the impending crisis, there is an urgent need for novel and rapid diagnostic tools. In this study, we developed a novel monoclonal antibody-named mAb-EspB-B7-that targets the EspB protein, a component within the bacterial type 3 secretion system (T3SS), which is mainly expressed in Gram-negative pathogens and is essential for bacterial infectivity. We found that mAb-EspB-B7 has high affinity and specificity toward recombinant and native EspB proteins; is stable over a range of pH levels, temperatures, and salt concentrations; and retains its functionality in human serum. We identified the epitope for mAb-EspB-B7 and validated it by competitive enzyme-linked immunosorbent assay (ELISA). Since this epitope is conserved across several T3SS-harboring pathogens, mAb-EspB-B7 holds great potential for development as an active component in precise and rapid diagnostic tools that can differentiate between commensal and pathogenic bacterial strains. To this end, we integrated the well-characterized monoclonal antibody into an electrochemical biosensor and demonstrated its high specificity and sensitivity capabilities in detecting pathogenic bacterial T3SS-associated antigens as well as intact bacteria. We foresee that in the near future it will be possible to design and develop a point-of-care biosensor with multiplexing capabilities for the detection of a panel of pathogenic bacteria.


Assuntos
Anticorpos Monoclonais/sangue , Técnicas Biossensoriais , Técnicas Eletroquímicas , Bactérias Gram-Negativas/genética , Testes Imediatos , Sistemas de Secreção Tipo III/sangue , Ensaio de Imunoadsorção Enzimática , Bactérias Gram-Negativas/patogenicidade , Humanos , Concentração de Íons de Hidrogênio , Temperatura , Sistemas de Secreção Tipo III/genética
8.
BMC Bioinformatics ; 21(Suppl 14): 367, 2020 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-32998698

RESUMO

BACKGROUND: Essential genes are those genes that are critical for the survival of an organism. The prediction of essential genes in bacteria can provide targets for the design of novel antibiotic compounds or antimicrobial strategies. RESULTS: We propose a deep neural network for predicting essential genes in microbes. Our architecture called DEEPLYESSENTIAL makes minimal assumptions about the input data (i.e., it only uses gene primary sequence and the corresponding protein sequence) to carry out the prediction thus maximizing its practical application compared to existing predictors that require structural or topological features which might not be readily available. We also expose and study a hidden performance bias that effected previous classifiers. Extensive results show that DEEPLYESSENTIAL outperform existing classifiers that either employ down-sampling to balance the training set or use clustering to exclude multiple copies of orthologous genes. CONCLUSION: Deep neural network architectures can efficiently predict whether a microbial gene is essential (or not) using only its sequence information.


Assuntos
Bactérias/genética , Genes Essenciais , Redes Neurais de Computação , Área Sob a Curva , Análise por Conglomerados , Códon , Bactérias Gram-Negativas/genética , Bactérias Gram-Positivas/genética , Curva ROC
9.
PLoS One ; 15(10): e0240085, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33075077

RESUMO

INTRODUCTION: Bacterial pathogens are often involved in dermatitis in reptiles. Exact identification of reptile-specific but otherwise uncommon bacterial species may be challenging. However, identification is crucial to evaluate the importance of the detected bacterial species. OBJECTIVE: The aim of this study was to assess the number of aerobic bacterial isolates cultured from skin-derived samples of reptiles which were not reliably identified by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), and to determine their identity. MATERIAL AND METHODS: Routine bacterial diagnostics were performed on 235 skin samples, and 417 bacterial isolates were analysed by MALDI-TOF MS. The isolates were grouped into categories based on their first score: category I (≥ 2.00), category II (≥ 1.70 and < 2.00), and category III (< 1.70). Isolates from category III were further investigated by 16S rRNA gene sequencing and the following criteria were applied: query cover 100%, e-value rounded to 0.0 and sequence identity (%) > 98.00% for genus identification, and > 99.00% for species identification. RESULTS: The majority of bacterial isolates were in category I (85.1%) or category II (8.4%). In category III (6.5%) results achieved at first by MALDI-TOF MS corresponded to the results of the molecular analysis in 8.0% of isolates at the species level and in 24.0% at the genus level. Bacterial isolates classified as category III were heterogenic in genus (e.g. Chryseobacterium, Devriesea, Pseudomonas, Staphylococcus, Uruburuella), and some have only been described in reptiles so far. CONCLUSIONS: Most of the aerobic bacterial isolates cultured from reptile skin achieved high scores by MALDI-TOF MS. However, in the majority of category III isolates MALDI-TOF MS results were different from those of the molecular analysis. This strengthens the need to carefully examine low-scored results for plausibility and to be familiar with the occurrence and morphology of relevant reptile-specific bacterial species (e.g. Devriesea agamarum) as well as with the limits of the database used.


Assuntos
Bactérias Aeróbias/isolamento & purificação , Répteis/microbiologia , Pele/microbiologia , Animais , Bactérias Aeróbias/química , Bactérias Aeróbias/genética , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/isolamento & purificação , Bactérias Gram-Positivas/metabolismo , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
10.
Ecotoxicol Environ Saf ; 203: 111037, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32888596

RESUMO

Glacier studies as of late have ruffled many eyeballs, exploring this frigid ecology to understand the impact of climate change. Mapquesting the glaciers led to the discovery of concealed world of "psychrophiles" harboring in it. In the present study, the antibiotic resistance genes (ARGs) and heavy metal resistance genes (MRGs) were evaluated through both the culture-dependent and culture-independent methods. Samples were collected from two different glaciers, i.e., debris-covered glacier (Changme Khangpu) and debris-free glacier (Changme Khang). Functional metagenomics of both the glacier samples, provided evidence of presence of resistant genes against various antibiotic groups. Bacitracin resistant gene (bacA) was the predominant ARG in both the glaciers. MRGs in both the glacier samples were diversified as the genes detected were resistant against various heavy metals such as arsenic, tungsten, mercury, zinc, chromium, copper, cobalt, and iron. Unique MRGs identified from Changme Khangpu glacier were resistant to copper (cutA, cutE, cutC, cutF, cueR, copC, and copB) and chromium (yelf, ruvB, nfsA, chrR, and chrA) whereas, from Changme Khang glacier they showed resistance against cobalt (mgtA, dmef, corD, corC, corB, and cnrA), and iron (yefD, yefC, yefB, and yefA) heavy metals. ARGs aligned maximum identity with Gram-negative psychrotolerant bacteria. The cultured bacterial isolates showed tolerance to high concentrations of tested heavy metal solutions. Interestingly, some of the antibiotic resistant bacterial isolates also showed tolerance towards the higher concentrations of heavy metals. Thus, an introspection of the hypothesis of co-occurrence and/co-selection of ARGs and MRGs in such environments has been highlighted here.


Assuntos
Adaptação Biológica/genética , Antibacterianos/toxicidade , Resistência Microbiana a Medicamentos/genética , Poluentes Ambientais/toxicidade , Genes Bacterianos/efeitos dos fármacos , Camada de Gelo/microbiologia , Metais Pesados/toxicidade , Adaptação Biológica/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/genética , Camada de Gelo/química , Índia , Metagenômica , Siquim
11.
J Med Microbiol ; 69(9): 1132-1144, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32812863

RESUMO

Introduction. While colonization by Staphylococcus aureus in haemodialysis patients has been assessed, knowledge about colonization by beta-lactam-resistant Gram-negative bacilli is still limited.Aim. To describe clinical and molecular characteristics in haemodialysis patients colonized by S. aureus (MSSA-MRSA) and beta-lactam-resistant Gram-negative bacilli in an ambulatory renal unit.Methodology. The study included patients with central venous catheters in an outpatient haemodialysis facility in Medellín, Colombia (October 2017-October 2018). Swab specimens were collected from the nostrils and skin around vascular access to assess colonization by S. aureus (MSSA-MRSA). Stool samples were collected from each patient to evaluate beta-lactam-resistant Gram-negative bacilli colonization. Molecular typing included PFGE, multilocus sequence typing (MLST), spa typing and enterobacterial repetitive intergenic consensus-PCR (ERIC). Clinical information was obtained from medical records and personal interview.Results. A total of 210 patients were included in the study. S. aureus colonization was observed in 33.8 % (n=71) of the patients, 4.8 % (n=10) of which were colonized by methicillin-resistant S. aureus. Stool samples were collected from 165 patients and of these 41.2 % (n=68) and 11.5 % (n=19) were colonized by extended-spectrum-beta-lactamase-producing (ESBL) and carbapenem-resistant bacilli, respectively. Typing methods revealed high genetic diversity among S. aureus and ESBL-producing Gram-negative bacilli (ESBL-GNB). Antibiotic use and hospitalization in the previous 6 months were observed in more than half of the studied population.Conclusion. The high colonization by ESBL-GNB in haemodialysis patients shows evidence for the need for stronger surveillance, not only for S. aureus but also for multidrug-resistant bacilli in order to avoid their spread. Additionally, the high genetic diversity suggests other sources of transmission outside the renal unit instead of horizontal transmission between patients.


Assuntos
Antibacterianos/farmacologia , Infecção Hospitalar/microbiologia , Bactérias Gram-Negativas/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/microbiologia , Lactamas/farmacologia , Resistência beta-Lactâmica , Idoso , Fezes/microbiologia , Feminino , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Diálise Renal , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/isolamento & purificação
12.
PLoS One ; 15(8): e0237260, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32857755

RESUMO

OBJECTIVE: Infections caused by multidrug-resistant Gram-negative bacilli (MDR-GNB) are a major issue in intensive care. The intestinal and oropharyngeal microbiota being the reservoir of MDR-GNB. Our main objective was to assess the link between the composition of the intestinal microbiota and the tracheal and intestinal colonization by MDR-GNB, and also by Enterococcus spp. and yeasts. METHODS: We performed a 2-month prospective, monocentric cohort study in the medical intensive care unit of our hospital. Patients ventilated >3 days and spontaneously passing feces were included. A fecal sample and an endotracheal aspiration (EA) were collected twice a week. MDR-GNB but also Enterococcus faecium and yeasts (as potential dysbiosis surrogate markers) were detected by culture methods. The composition of the intestinal microbiota was assessed by 16S profiling. RESULTS: We collected 62 couples of feces and EA from 31 patients, including 18 feces and 9 EA positive for MDR-GNB. Forty-eight fecal samples were considered for 16S profiling. We did not observe a link between the diversity and the richness of the intestinal microbiota and the MDR-GNB intestinal relative abundance (RA). Conversely, we observed a negative link between the intestinal diversity and richness and the RA of Enterococcus spp. (p<0.001). CONCLUSION: The fecal MDR-GNB RA was not associated to the diversity nor the richness of the intestinal microbiota, but that of Enterococcus spp. was.


Assuntos
Enterococcus faecium/isolamento & purificação , Bactérias Gram-Negativas/isolamento & purificação , Intestinos/microbiologia , Traqueia/microbiologia , Adulto , Idoso , Cuidados Críticos , Enterococcus faecium/genética , Face/microbiologia , Feminino , Microbioma Gastrointestinal , Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Leveduras/genética , Leveduras/isolamento & purificação , Adulto Jovem
13.
DNA Cell Biol ; 39(9): 1473-1477, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32721230

RESUMO

Global antibiotic resistance, driven by intensive antibiotic exposure/abuse, constitutes a serious challenge to all health care, particularly in an era when new antimicrobial development has slowed to a trickle. Recently, we published work demonstrating the discovery and partial mechanism of action of a novel bactericidal agent that is effective against both gram-positive and gram-negative multidrug-resistant bacteria. This drug, called AB569, consists of acidified nitrite (A-NO2-) and EDTA, of which there is no mechanism of resistance. Using both chemistry-, genetic-, and bioinformatics-based techniques, we first discovered that AB569 was able to generate bactericidal levels of nitric oxide (NO), while the EDTA component stabilized S-nitrosyl thiols, thereby furthering NO and downstream reactive nitrogen species production. This elegant chemistry triggered a paralytic downregulation of vital genes using RNA-seq involved in the synthesis of DNA, RNA, ATP, and protein in the representative ESKAPE pathogen, Pseudomonas aeruginosa.


Assuntos
Antibacterianos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Antibacterianos/química , Farmacorresistência Bacteriana , Ácido Edético/química , Bactérias Gram-Negativas/genética , Bactérias Gram-Positivas/genética , Nitritos/química
14.
Environ Monit Assess ; 192(6): 376, 2020 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-32417981

RESUMO

Multidrug resistance mediated by ß-lactamase in Gram-negative bacilli is a serious public health problem. Sewers are considered reservoirs of multiresistant bacteria due to presence of antibiotics that select them and favor their dissemination. The present study evaluated the antibiotic resistance profile and ß-lactamases production in Gram-negative bacilli isolates from hospital sewage and urban wastewater treatment plants (UWWTP) in Brazil. Bacteria were isolated and identified with biochemical tests. Antibiotic susceptibility testing was performed by the disk-diffusion method and detection of extended-spectrum ß-lactamase and carbapenemases by enzymatic inhibitor and conventional PCR. Differences in resistance to amoxicillin clavulanic, aztreonam, cefepime, and cefotaxime were observed in hospital sewage compared with urban sewage (p < 0.05). The multidrug-resistant phenotype was observed in 33.3% of hospital sewage isolates (p = 0.0025). ß-lactamases genes were found in 35.6% of isolates, with the most frequent being blaKPC and blaTEM (17.8%), and blaSHV and blaCTX-M (13.3% and 8.9%, respectively). The data obtained are relevant, since the bacteria detected are on the priority pathogens list from the World Health Organization and hospital sewage could be released untreated into the municipal collection system, which may favor the spread of resistance. Changes in hospital sewage discharge practices, as well as additional technologies regarding effluent disinfection in the UWWTP, can prevent the spread of these bacteria into the environment and negative impact on water resources.


Assuntos
Monitoramento Ambiental , Bactérias Gram-Negativas , Eliminação de Resíduos de Serviços de Saúde , Águas Residuárias , beta-Lactamases , Antibacterianos , Brasil , Cidades , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/genética , Hospitais , Testes de Sensibilidade Microbiana
15.
Nat Commun ; 11(1): 2607, 2020 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-32451375

RESUMO

Quantification of pathogen and host biomarkers is essential for the diagnosis, monitoring, and treatment of infectious diseases. Here, we demonstrate sensitive and rapid quantification of bacterial load and cytokines from human biological samples to generate actionable hypotheses. Our digital assay measures IL-6 and TNF-α proteins, gram-negative (GN) and gram-positive (GP) bacterial DNA, and the antibiotic-resistance gene blaTEM with femtomolar sensitivity. We use our method to characterize bronchoalveolar lavage fluid from patients with asthma, and find elevated GN bacteria and IL-6 levels compared to healthy subjects. We then analyze plasma from patients with septic shock and find that increasing levels of IL-6 and blaTEM are associated with mortality, while decreasing IL-6 levels are associated with recovery. Surprisingly, lower GN bacteria levels are associated with higher probability of death. Applying decision-tree analysis to our measurements, we are able to predict mortality and rate of recovery from septic shock with over 90% accuracy.


Assuntos
Citocinas/sangue , DNA Bacteriano/sangue , Choque Séptico/imunologia , Choque Séptico/microbiologia , Asma/imunologia , Asma/microbiologia , Carga Bacteriana , Biomarcadores/análise , Biomarcadores/sangue , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Líquido da Lavagem Broncoalveolar/microbiologia , Estudos de Casos e Controles , Citocinas/análise , DNA Bacteriano/genética , Árvores de Decisões , Genes Bacterianos , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/isolamento & purificação , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Interleucina-6/análise , Interleucina-6/sangue , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Multiplex/estatística & dados numéricos , Prognóstico , Sensibilidade e Especificidade , Choque Séptico/mortalidade , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/sangue , Resistência beta-Lactâmica/genética
16.
Arch Microbiol ; 202(7): 1839-1848, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32447433

RESUMO

As a part of studying the effect of deoxygenation, eutrophication and acidification on bacterial diversity, strain HWN-4T was isolated from tube well water and characterized. The draft genome sequencing of strain HWN-4T revealed a genome size of 5,774,764 bp and the annotation indicated 5102 coding sequences including 66 RNA genes. Strain HWN-4T is Gram negative, rod-shaped, motile in the log phase, catalase and oxidase positive, and the major fatty acids and respiratory quinone present are C10:0 3-OH, C14:0 3OH/C16:1 iso I, C16:1 ω7c/C16:1 ω6c, C16:0 and C17:0 cyclo and ubiquinone-8, respectively. The phylogenetic analyses, based on 16S rRNA gene sequence, indicated that strain HWN-4T is a member of the genus Mitsuaria. The average nucleotide identity (ANI) and genome-to-genome similarity between strain HWN-4T and all other species/strains of the genus Mitsuaria are less than (%) 95.0 and 70.0, respectively. This confirms the status of strain HWN-4T as a novel species. The species status is further confirmed by phenotypic differences exhibited by strain HWN-4T with other members of the same genus. Based on the collective differences exhibited by strain HWN-4T with other members of the genus Mitsuaria, the name Mitsuaria chitinivorans sp. nov. is proposed. Further, the diagnostic signature nucleotides were identified in the 16S rRNA gene sequences of members of the genera Mitsuaria, Pelomonas and Roseateles, that distinctly differentiate them and support an emendation of the genera. Besides, phylogenetic and structural characterization of chitinases from members of the genus Mitsuaria was performed. The type strain of Mitsuaria chitinivorans sp. nov. is HWN-4T = LMG 28685T = KTCC 42483T.


Assuntos
Biodegradação Ambiental , Bactérias Gram-Negativas/classificação , Filogenia , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/análise , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/isolamento & purificação , Hibridização de Ácido Nucleico , Fosfolipídeos/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Especificidade da Espécie
17.
Nat Commun ; 11(1): 2381, 2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32404906

RESUMO

Many bacteria employ a type III secretion system (T3SS) injectisome to translocate proteins into eukaryotic host cells. Although the T3SS can efficiently export heterologous cargo proteins, a lack of target cell specificity currently limits its application in biotechnology and healthcare. In this study, we exploit the dynamic nature of the T3SS to govern its activity. Using optogenetic interaction switches to control the availability of the dynamic cytosolic T3SS component SctQ, T3SS-dependent effector secretion can be regulated by light. The resulting system, LITESEC-T3SS (Light-induced translocation of effectors through sequestration of endogenous components of the T3SS), allows rapid, specific, and reversible activation or deactivation of the T3SS upon illumination. We demonstrate the light-regulated translocation of heterologous reporter proteins, and induction of apoptosis in cultured eukaryotic cells. LITESEC-T3SS constitutes a new method to control protein secretion and translocation into eukaryotic host cells with unparalleled spatial and temporal resolution.


Assuntos
Proteínas de Bactérias/metabolismo , Células Eucarióticas/metabolismo , Bactérias Gram-Negativas/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Proteínas de Bactérias/genética , Linhagem Celular Tumoral , Citosol/metabolismo , Citosol/microbiologia , Células Eucarióticas/microbiologia , Regulação Bacteriana da Expressão Gênica , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/fisiologia , Humanos , Luz , Microscopia de Fluorescência , Optogenética/métodos , Transporte Proteico/efeitos da radiação , Análise Espacial , Sistemas de Secreção Tipo III/genética , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismo , Yersinia enterocolitica/fisiologia
18.
BMC Infect Dis ; 20(1): 308, 2020 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-32334517

RESUMO

BACKGROUND: Diphtheria outbreaks occurred in endemic areas and imported and indigenous cases are reported in UE/EEA. Because of the high infectiveness and severity of the disease, early and accurate diagnosis of each suspected case is essential for the treatment and management of the case and close contacts. The aim of the study was to establish simple and rapid testing methods based on Loop-Mediated Isothermal Amplification (LAMP) assay for the detection of Corynebacterium diphtheriae and differentiation between toxigenic and non-toxigenic strains. METHODS: Corynebacterium diphtheriae and Corynebacterium ulcerans isolates from the National Institute of Public Health-National Institute of Hygiene collection were used for the development of LAMP assay for the diagnosis of diphtheria and nontoxigenic C. diphtheriae infections. Various colorimetric methods for visualization of results were investigated. Sensitivity and specificity of the assay were examined using a collection of DNA samples from various gram-positive and gram-negative bacteria. RESULTS: The LAMP assay for tox and dtxR genes was developed. The sensitivity and specificity of the assay were calculated as 100%. The detection limit was estimated as 1.42 pg/µl concentration of DNA template when the reaction was conducted for 60 min. However, the detection limit was lowered 10 times for every 10 min of reduction in the time of incubation during the reaction. Positive results were successfully detected colorimetrically using hydroxynaphthol blue, calcein, QuantiFluor, and lateral flow Milenia HybriDetect dipsticks. CONCLUSION: The assay developed in the study might be applied for point-of-care testing of diphtheria and other C. diphtheriae infections as well as for other infections caused by diphtheria-toxin producing Corynebacterium species. It is highly sensitive, specific, inexpensive, easy to use, and suitable for low-resource settings.


Assuntos
Colorimetria/métodos , Corynebacterium diphtheriae/genética , Difteria/microbiologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Proteínas de Bactérias/genética , Colorimetria/instrumentação , Corynebacterium diphtheriae/isolamento & purificação , Proteínas de Ligação a DNA/genética , Bactérias Gram-Negativas/genética , Bactérias Gram-Positivas/genética , Humanos , Limite de Detecção , Testes Imediatos
19.
Prostate ; 80(7): 577-587, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32162709

RESUMO

BACKGROUND: The pathogens responsible for chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS; NIH category III) are not currently known. the present study utilized high-throughput next-generation sequencing to screen for potential pathogens associated with NIH category III CP (CP III). METHODS: This study included 33 patients with CP III and 30 healthy men, from which one sample each of urethral secretions and expressed prostatic secretion (EPS) was collected. High-throughput next-generation sequencing was performed to detect the sequence variations and the relative abundance of the bacterial 16S ribosomal variable region and fungal internal transcribed spacer region in all samples. Bioinformatics software and databases were used for data analysis, and differences with P < .05 were considered statistically significant. RESULTS: Unweighted pair group method with arithmetic mean (UPGMA) cluster analysis, principal component analysis (PCA), and Spearman's rank correlation showed that the microbial compositions of the urethral secretions and EPS collected from the same subject were essentially the same. CONCLUSIONS: No potential pathogens were identified in diagnosed patients with CP III. The EPS may be free from bacteria before and after infection. Changes in the urinary tract microbiome may disrupt the microecological balance of the urinary system, thereby leading to CP III. Conversely, the true pathogens of CP III may not be prokaryotic or eukaryotic microorganisms, Future research may involve the evaluation of noncellular microbes.


Assuntos
Prostatite/microbiologia , Infecções Bacterianas/microbiologia , Estudos de Casos e Controles , Doença Crônica , Análise por Conglomerados , Fungos/genética , Fungos/isolamento & purificação , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Micoses/microbiologia , Dor Pélvica/microbiologia , Análise de Componente Principal , RNA Ribossômico 16S/genética
20.
Appl Environ Microbiol ; 86(10)2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32198174

RESUMO

Resistance to the "last-resort" antibiotics, such as carbapenems, has led to very few antibiotics being left to treat infections by multidrug-resistant bacteria. Spread of carbapenem resistance (CR) has been well characterized for the clinical environment. However, there is a lack of information about its environmental distribution. Our study reveals that CR is present in a wide range of Gram-negative bacteria in the coastal seawater environment, including four phyla, eight classes, and 30 genera. These bacteria were likely introduced into seawater via stormwater flows. Some CR isolates found here, such as Acinetobacter junii, Acinetobacter johnsonii, Brevundimonas vesicularis, Enterococcus durans, Pseudomonas monteilii, Pseudomonas fulva, and Stenotrophomonas maltophilia, are further relevant to human health. We also describe a novel metallo-ß-lactamase (MBL) for marine Rheinheimera isolates with CR, which has likely been horizontally transferred to Citrobacter freundii or Enterobacter cloacae In contrast, another MBL of the New Delhi type was likely acquired by environmental Variovorax isolates from Escherichia coli, Klebsiella pneumoniae, or Acinetobacter baumannii utilizing a plasmid. Our findings add to the growing body of evidence that the aquatic environment is both a reservoir and a vector for novel CR genes.IMPORTANCE Resistance against the "last-resort" antibiotics of the carbapenem family is often based on the production of carbapenemases, and this has been frequently observed in clinical samples. However, the dissemination of carbapenem resistance (CR) in the environment has been less well explored. Our study shows that CR is commonly found in a range of bacterial taxa in the coastal aquatic environment and can involve the exchange of novel metallo-ß-lactamases from typical environmental bacteria to potential human pathogens or vice versa. The outcomes of this study contribute to a better understanding of how aquatic and marine bacteria can act as reservoirs and vectors for CR outside the clinical setting.


Assuntos
Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Água Doce/microbiologia , Bactérias Gram-Negativas/efeitos dos fármacos , Organismos Aquáticos , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , New South Wales
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