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1.
Rev Soc Bras Med Trop ; 52: e20190237, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31508785

RESUMO

INTRODUCTION: The increased use of colistin against infections caused by Acinetobacter baumannii and Pseudomonas aeruginosa has resulted in colistin resistance. The purpose of this study was to detect plasmid-mediated mcr-1 gene in colistin-resistant A. baumannii and P. aeruginosa isolates. METHODS: A total of 146 clinical isolates of A. baumannii (n = 62) and P. aeruginosa (n = 84) were collected from the four largest tertiary care hospitals in Peshawar, Pakistan. All bacterial isolates were phenotypically screened for multidrug resistance using the Kirby-Baur disc diffusion method. The minimum inhibitory concentration (MIC) of colistin in all isolates was phenotypically performed using dilution methods. mcr-1 gene was detected through polymerase chain reaction and the nucleotide sequence of amplicon was determined using Sanger sequencing. RESULTS: Approximately 96.7% A. baumannii and 83.3% P. aeruginosa isolates were resistant to multiple antibiotics. Colistin resistance was found in 9.6% (6/62) of A. baumannii and 11.9% (10/84) of P. aeruginosa isolates. Among 16 colistin resistant isolates, the mcr-1 gene was detected in one A. baumannii (1.61% of total isolates; 16.6% of colistin resistant isolates) and one P. aeruginosa strain (1.19% of total isolates; 10% of colistin resistant isolates). Nucleotide BLAST showed 98-99% sequence similarity to sequences of the mcr-1 gene in GenBank. CONCLUSIONS: Our study reports, for the first time, the emergence of plasmid-mediated mcr-1-encoded colistin resistance in multidrug resistant strains of A. baumannii and P. aeruginosa. Further large scales studies are recommended to investigate the prevalence of this mode of resistance in these highly pathogenic bacteria.


Assuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/genética , Proteínas de Bactérias/genética , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Acinetobacter baumannii/efeitos dos fármacos , Farmacorresistência Bacteriana , Humanos , Testes de Sensibilidade Microbiana , Paquistão , Plasmídeos/genética , Pseudomonas aeruginosa/efeitos dos fármacos
2.
Klin Lab Diagn ; 64(8): 497-502, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31479607

RESUMO

The growing prevalence of metallo-ß-lactamase (MBL)-producing Pseudomonas aeruginosa in nosocomial pathogen populations has been attributed to their clonal spread, and/or horizontal transfer of MBL determinants in mobile genetic elements, including integrons. To characterize the genetic background of the beta-lactamase VIM-2 encoding gene in the population of carbapenemresistant (Carba-R) P. aeruginosa clinical isolates.The detection of class 1 integrons was performed by PCR. Typing of the class 1 integrons containing the blaVIM gene cassette was performed by the PCR-restriction fragment length polymorphism (RFLP) approach followed by sequencing of variable regions of class 1 integrons. Five types of the blaVIM-2-carrying integrons were identified: ST654-isolates accounting for more than 50% of the Carba-R population harbored In56; ST235-isolates contained In559 (26% Carba-R isolates); ST111-isolates (19% Carba-R isolates) were characterized by carrying In59-like integron; two ST235-isolates harbored In59 and In249 each. Except In56, carrying the only blaVIM-2-gene cassette, all other identified integron types harbored the genes of resistance to trimethoprim and/or aminoglycosides. No new types of integrons were identified in the P. aeruginosa clinical isolates. The observed correlation of the integron type with specific STs indicates a clonal dissemination of significant resistance determinant producers - ST111, ST654 and ST235 epidemic lines. The features of the integron variable regions can be used for the epidemiological characterization of clinical P. aeruginosa isolates.


Assuntos
Carbapenêmicos/farmacologia , Farmacorresistência Bacteriana , Integrons , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , beta-Lactamases/genética , Antibacterianos/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos
3.
Mem Inst Oswaldo Cruz ; 114: e190105, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31389522

RESUMO

BACKGROUND: Healthcare-associated infections caused by bacteria such as Pseudomonas aeruginosa are a major public health problem worldwide. Gene regulatory networks (GRN) computationally represent interactions among regulatory genes and their targets. They are an important approach to help understand bacterial behaviour and to provide novel ways of overcoming scientific challenges, including the identification of potential therapeutic targets and the development of new drugs. OBJECTIVES: The goal of this study was to reconstruct the multidrug-resistant (MDR) P. aeruginosa GRN and to analyse its topological properties. METHODS: The methodology used in this study was based on gene orthology inference using the reciprocal best hit method. We used the genome of P. aeruginosa CCBH4851 as the basis of the reconstruction process. This MDR strain is representative of the sequence type 277, which was involved in an endemic outbreak in Brazil. FINDINGS: We obtained a network with a larger number of regulatory genes, target genes and interactions as compared to the previously reported network. Topological analysis results are in accordance with the complex network representation of biological processes. MAIN CONCLUSIONS: The properties of the network were consistent with the biological features of P. aeruginosa. To the best of our knowledge, the P. aeruginosa GRN presented here is the most complete version available to date.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Redes Reguladoras de Genes , Pseudomonas aeruginosa/genética , Genoma Bacteriano , Família Multigênica , Infecções por Pseudomonas/genética , Valores de Referência
4.
J Agric Food Chem ; 67(31): 8581-8589, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31321975

RESUMO

Intermediates in aromatic amino acid biosynthesis can serve as substrates for the synthesis of bioactive compounds. In this study we used two intermediates in the shikimate pathway of Escherichia coli, chorismate and anthranilate, to synthesize three bioactive compounds: 4-hydroxycoumarin (4-HC), 2,4-dihydroxyquinoline (DHQ), and 4-hydroxy-1-methyl-2(1H)-quinolone (NMQ). We introduced genes for the synthesis of salicylic acid from chorismate to supply the substrate for 4-HC and the gene encoding N-methyltransferase for the synthesis of N-methylanthranilate from anthranilate. Polyketide synthases and coenzyme (Co)A ligases were tested to determine the optimal combination of genes for the synthesis of each compound. We also tested several constructs and identified the best one for increasing levels of endogenous substrates for chorismate, anthranilate, and malonyl-CoA. With the use of these strategies, 255.4 mg/L 4-HC, 753.7 mg/L DHQ, and 17.5 mg/L NMQ were synthesized. This work provides a basis for the synthesis of diverse coumarin and quinoline derivatives with potential medical applications.


Assuntos
4-Hidroxicumarinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica , Policetídeo Sintases/genética , Quinolinas/metabolismo , 4-Hidroxicumarinas/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ácido Corísmico/metabolismo , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Photorhabdus/enzimologia , Photorhabdus/genética , Policetídeo Sintases/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Quinolinas/química , ortoaminobenzoatos/metabolismo
5.
Nat Commun ; 10(1): 2931, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270321

RESUMO

The virulence of Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, is regulated by many transcriptional factors (TFs) that control the expression of quorum sensing and protein secretion systems. Here, we report a genome-wide, network-based approach to dissect the crosstalk between 20 key virulence-related TFs. Using chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq), as well as RNA-seq, we identify 1200 TF-bound genes and 4775 differentially expressed genes. We experimentally validate 347 of these genes as functional target genes, and describe the regulatory relationships of the 20 TFs with their targets in a network that we call 'Pseudomonas aeruginosa genomic regulatory network' (PAGnet). Analysis of the network led to the identification of novel functions for two TFs (ExsA and GacA) in quorum sensing and nitrogen metabolism. Furthermore, we present an online platform and R package based on PAGnet to facilitate updating and user-customised analyses.


Assuntos
Proteínas de Bactérias/genética , Pseudomonas aeruginosa/genética , Fatores de Transcrição/genética , Proteínas de Bactérias/metabolismo , Imunoprecipitação da Cromatina , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Fatores de Transcrição/metabolismo , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
6.
J Microbiol Biotechnol ; 29(6): 839-844, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31154751

RESUMO

Anthranilate derivatives have been used as flavoring and fragrant agents for a long time. Recently, these compounds are gaining attention due to new biological functions including antinociceptive and analgesic activities. Three anthranilate derivatives, N-methylanthranilate, methyl anthranilate, and methyl N-methylanthranilate were synthesized using metabolically engineered stains of Escherichia coli. NMT encoding N-methyltransferase from Ruta graveolens, AMAT encoding anthraniloyl-coenzyme A (CoA):methanol acyltransferase from Vitis labrusca, and pqsA encoding anthranilate coenzyme A ligase from Pseudomonas aeruginosa were cloned and E. coli strains harboring these genes were used to synthesize the three desired compounds. E. coli mutants (metJ, trpD, tyrR mutants), which provide more anthranilate and/or S-adenosyl methionine, were used to increase the production of the synthesized compounds. MS/MS analysis was used to determine the structure of the products. Approximately, 185.3 µM N-methylanthranilate and 95.2 µM methyl N-methylanthranilate were synthesized. This is the first report about the synthesis of anthranilate derivatives in E. coli.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , ortoaminobenzoatos/metabolismo , Vias Biossintéticas , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Coenzima A-Transferases/genética , Coenzima A-Transferases/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Engenharia Metabólica , Metiltransferases/genética , Metiltransferases/metabolismo , Mutação , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Proteínas Recombinantes/metabolismo , Ruta/enzimologia , Ruta/genética , Vitis/enzimologia , Vitis/genética , ortoaminobenzoatos/química
7.
J Environ Sci (China) ; 83: 123-132, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31221375

RESUMO

The long-term persistence of antibiotic resistance in the environment, especially in drinking water, is a public health concern. Expression of an efflux pump, an important mechanism of resistance to antibiotics, usually confers a fitness cost in bacteria. In this study, we aimed to determine why antibiotic resistance conferred by overexpression of an efflux pump persisted in low-nutrient environments (TOC < 10 mg/L) such as drinking and source water in which antibiotic selective pressure might be very low or even absent. Competition experiments between wild-type Pseudomonas aeruginosa and ciprofloxacin-resistant mutants revealed that the fitness cost of ciprofloxacin resistance significantly decreased (p < 0.05) under low-nutrient (0.5 mg/L total organic carbon (TOC)) relative to high-nutrient (500 mg/L TOC) conditions. Mechanisms underlying this fitness cost were analyzed. The mexD gene expression in resistant bacteria (cip_3 strain) was significantly lower (p < 0.05) in low-nutrient conditions, with 10 mg/L TOC ((8.01 ±â€¯0.82)-fold), than in high-nutrient conditions, with 500 mg/L TOC ((48.89 ±â€¯4.16)-fold). Moreover, rpoS gene expression in resistant bacteria ((1.36 ±â€¯0.13)-fold) was significantly lower (p < 0.05) than that in the wild-type strain ((2.78 ±â€¯0.29)-fold) under low-nutrient conditions (10 mg/L TOC), suggesting a growth advantage. Furthermore, the difference in metabolic activity between the two competing strains was significantly smaller (p < 0.05) in low-nutrient conditions (5 and 0.5 mg/L TOC). These results suggest that nutrient levels are a key factor in determining the persistence of antibiotic resistance conferred by efflux pumps in the natural environment with trace amounts or no antibiotics.


Assuntos
Água Potável/microbiologia , Resistência Microbiana a Medicamentos/genética , Pseudomonas aeruginosa/genética , Poluentes da Água/análise , Ciprofloxacino , Água Potável/química , Aptidão Genética
8.
Microbiol Res ; 223-225: 137-143, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178047

RESUMO

Pseudomonas aeruginosa is an opportunistic pathogen with high clinical relevance for hospital infections of patients. Accumulating DNA sequencing results of clinical P. aeruginosa isolates have revealed frequent mutations in lasR gene, which encodes the highest arches component of quorum-sensing system (QS). We analyzed the sequencing data of lasR gene from a large collection of cystic fibrosis (CF) P. aeruginosa isolates. Our systematical analyses revealed that single nucleotide polymorphisms (SNPs) selection in lasR gene were largely constrained by codon-usage frequency. As a whole, SNP-substituted codons encoding unconserved amino acid resulted in unfavored codons with relatively low codon-usage frequency, while those associating with conserved amino acid were not strictly regulated in such way. These SNPs substitutions gives rise to diverse functional LasR isoforms and contributes to the relative growth fitness of recombinant lasR variant strains. Our survey reveals a novel pattern of SNPs selections in lasR gene of CF isolates. Our findings could be served as a powerful resource for understanding adaptive mechanism of clinical isolates under environmental constrains and developing anti-bacteria drugs for CF patients.


Assuntos
Proteínas de Bactérias/genética , Códon/genética , Fibrose Cística/microbiologia , Polimorfismo de Nucleotídeo Único/genética , Pseudomonas aeruginosa/genética , Transativadores/genética , Sequência de Aminoácidos , Regulação Bacteriana da Expressão Gênica , Isoformas de Proteínas , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/patogenicidade , Percepção de Quorum/genética , Alinhamento de Sequência , Análise de Sequência de DNA
9.
Prep Biochem Biotechnol ; 49(8): 800-806, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31156029

RESUMO

In this study, azurin, a bacteriocin with anticancer property, was produced by food-grade Lactococcus lactis using the Nisin Controlled Gene Expression (NICE) System. In addition, the antibacterial and cytotoxic properties of recombinant azurin in the culture supernatant were also investigated. Azurin gene from Pseudomonas aeruginosa was cloned into the pNZ8149 vector and the resulting recombinant DNA was transformed into food grade L. lactis NZ3900. The expression of azurin protein was induced by the optimum concentration of nisin for 3 h. Inhibition zones for Escherichia coli and Bacillus cereus were observed at 5.0 and 10 mg/mL concentrations of lyophilized supernatants containing azurin, but no inhibition zone at azurin-free lyophilized supernatants. When HUVEC, HT29, HCT116, and MCF7 cell lines were treated with lyophilized culture supernatants with azurin or without azurin, cell viability decreased with increasing concentrations of the supernatant. Furthermore, the supernatants containing azurin showed more anti-proliferative effect than the azurin-free supernatants. This work provides a practicable method to produce recombinant azurin in the food grade L. lactis strain. As a result, the recombinant L. lactis strain, producing azurin, can be used in the investigation of food biopreservatives and in the development of a therapeutic probiotic.


Assuntos
Azurina/genética , Clonagem Molecular/métodos , Lactococcus lactis/genética , Pseudomonas aeruginosa/genética , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Azurina/farmacologia , Bacillus cereus/efeitos dos fármacos , Linhagem Celular , Escherichia coli/efeitos dos fármacos , Amplificação de Genes , Humanos , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Transformação Genética
10.
Genome Biol Evol ; 11(1): 1780-1796, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31173069

RESUMO

The diversification of microbial populations may be driven by many factors including adaptation to distinct ecological niches and barriers to recombination. We examined the population structure of the bacterial pathogen Pseudomonas aeruginosa by analyzing whole-genome sequences of 739 isolates from diverse sources. We confirmed that the population structure of P. aeruginosa consists of two major groups (referred to as Groups A and B) and at least two minor groups (Groups C1 and C2). Evidence for frequent intragroup but limited intergroup recombination in the core genome was observed, consistent with sexual isolation of the groups. Likewise, accessory genome analysis demonstrated more gene flow within Groups A and B than between these groups, and a few accessory genomic elements were nearly specific to one or the other group. In particular, the exoS gene was highly overrepresented in Group A compared with Group B isolates (99.4% vs. 1.1%) and the exoU gene was highly overrepresented in Group B compared with Group A isolates (95.2% vs. 1.8%). The exoS and exoU genes encode effector proteins secreted by the P. aeruginosa type III secretion system. Together these results suggest that the major P. aeruginosa groups defined in part by the exoS and exoU genes are divergent from each other, and that these groups are genetically isolated and may be ecologically distinct. Although both groups were globally distributed and caused human infections, certain groups predominated in some clinical contexts.


Assuntos
ADP Ribose Transferases/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Genética Populacional , Pseudomonas aeruginosa/genética , Fluxo Gênico , Genoma Bacteriano , Filogenia , Recombinação Genética
11.
J Med Microbiol ; 68(6): 952-956, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31107204

RESUMO

Exploiting the immunosuppressive, analgesic and highly addictive properties of morphine could increase the success of a bacterial pathogen. Therefore, we performed sequence similarity searches for two morphine biosynthesis demethylases in bacteria. For thebaine 6-O-demethylase and codeine O-demethylase, we found strong alignments to three (Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii) of the six ESKAPE pathogens (Enterococcus faecalis, Staphylococcus aureus, K. pneumoniae, A. baumannii, P. aeruginosa and Enterobacter species) that are commonly associated with drug resistance and nosocomial infections. Expression of the aligned sequence found in P. aeruginosa (NP_252880.1/PA4191) is upregulated in isolates obtained from cystic fibrosis patients. Our findings provide putative mechanistic targets for understanding the role of morphine in pathogenicity.


Assuntos
Acinetobacter baumannii/enzimologia , Infecção Hospitalar/microbiologia , Enterobacter/enzimologia , Klebsiella pneumoniae/enzimologia , Oxirredutases O-Desmetilantes/genética , Pseudomonas aeruginosa/enzimologia , Staphylococcus aureus/enzimologia , Acinetobacter baumannii/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Codeína/metabolismo , Enterobacter/genética , Humanos , Klebsiella pneumoniae/genética , Derivados da Morfina/metabolismo , Alcaloides Opiáceos/administração & dosagem , Pseudomonas aeruginosa/genética , Alinhamento de Sequência , Staphylococcus aureus/genética , Tebaína/metabolismo
12.
BMC Res Notes ; 12(1): 244, 2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036061

RESUMO

OBJECTIVES: Diabetic foot ulcers (DFUs) often lead to hospital admissions, amputations and deaths; however, there is no up-to-date information on microbial isolates from DFUs and no mention of utilization of molecular techniques in Sub-Saharan Africa. We conducted a cross-sectional study among 83 adult patients at a tertiary hospital in Kenya over 12 months. The study aimed to isolate, identify bacteria, their antibiotic susceptibility patterns in active DFUs, and to compare standard microbiological methods versus a real-time PCR commercial kit in the detection of Staphylococcus aureus DNA and methicillin-resistant S. aureus (MRSA) DNA. RESULTS: Eighty swabs (94%) were culture-positive; 29% were Gram-positive and 65% were Gram-negative. The main organisms isolated were S. aureus (16%), Escherichia coli (15%), Proteus mirabilis (11%), Klebsiella pneumoniae (7%) and Pseudomonas aeruginosa (7%). The bacterial isolates showed resistance to commonly used antibiotics such as ampicillin, amoxicillin, cefepime, ceftazidime, cefuroxime, clindamycin, erythromycin, piperacillin-tazobactam, tetracycline and trimethoprim-sulphamethoxazole (TMPSMX). Thirty-one percent of the S. aureus isolated and 40% of the Gram-negatives were multi-drug resistant organisms (MDROs). There was a high prevalence of nosocomial bacteria. MRSA were not identified using culture methods but were identified using PCR. PCR was more sensitive but less specific than culture-based methods to identify S. aureus.


Assuntos
Antibacterianos/uso terapêutico , Infecções Bacterianas/diagnóstico , Pé Diabético/diagnóstico , Farmacorresistência Bacteriana Múltipla , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/epidemiologia , Infecções Bacterianas/microbiologia , Técnicas de Tipagem Bacteriana , Cefalosporinas/uso terapêutico , Clindamicina/uso terapêutico , Estudos Transversais , Pé Diabético/tratamento farmacológico , Pé Diabético/epidemiologia , Pé Diabético/microbiologia , Escherichia coli/classificação , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Humanos , Quênia/epidemiologia , Klebsiella pneumoniae/classificação , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Macrolídeos/uso terapêutico , Staphylococcus aureus Resistente à Meticilina/classificação , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Testes de Sensibilidade Microbiana , Penicilinas/uso terapêutico , Proteus mirabilis/classificação , Proteus mirabilis/efeitos dos fármacos , Proteus mirabilis/genética , Proteus mirabilis/isolamento & purificação , Pseudomonas aeruginosa/classificação , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Staphylococcus aureus/classificação , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Sulfanilamidas/uso terapêutico
13.
Vet Microbiol ; 231: 169-176, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30955805

RESUMO

Purulent disease is the main factor that prevents the population increase of forest musk deer in artificial breeding, and especially the intracorporal suppurative lesions in late-stage with complex bacterial communities normally bring more difficulties for veterinary treatment. Although it is well-recognized that Pseudomonas aeruginosa and Escherichia coli are the two main bacterial pathogens which can be frequently co-isolated from the lung pus of forest musk deer, few studies have explored the interspecific relationship and coexistent mechanism of the two species. In this study, we identified a P. aeruginosa strain MYL-2, which harbored a loss-of-function mutation in the central regulator (LasR) of quorum-sensing (QS) system, from the lung pus of a dying forest musk deer with co-infecting E. coli strain MYL-58. Interestingly, P. aeruginosa MYL-2 could coexist with E. coli MYL-58 compared to the dominant role of lasR-intact P. aeruginosa strain MYL-1 in the competitive experiments. The results of in vitro coevolution assay further revealed that the QS-mediated competitive advantage of P. aeruginosa MYL-1 would be decreased along with the enrichment of lasR mutants in the communities, and P. aeruginosa could finally coexist with E. coli by forming a relatively stable equilibrium. Therefore, these findings provide an evolutionary explanation for the coexistence of P. aeruginosa and E. coli in the suppurative lesions of forest musk deer, and may also contribute to further understanding the pathology of animal purulent disease and the development of novel veterinary therapy.


Assuntos
Cervos/microbiologia , Infecções por Escherichia coli/veterinária , Pulmão/microbiologia , Infecções por Pseudomonas/veterinária , Infecções Respiratórias/veterinária , Animais , Evolução Molecular Direcionada , Escherichia coli/genética , Escherichia coli/patogenicidade , Pulmão/patologia , Microbiota , Mutação , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Percepção de Quorum , Infecções Respiratórias/microbiologia , Supuração/microbiologia , Virulência
14.
PLoS Comput Biol ; 15(4): e1006507, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30973869

RESUMO

The identification of genes essential for bacterial growth and survival represents a promising strategy for the discovery of antimicrobial targets. Essential genes can be identified on a genome-scale using transposon mutagenesis approaches; however, variability between screens and challenges with interpretation of essentiality data hinder the identification of both condition-independent and condition-dependent essential genes. To illustrate the scope of these challenges, we perform a large-scale comparison of multiple published Pseudomonas aeruginosa gene essentiality datasets, revealing substantial differences between the screens. We then contextualize essentiality using genome-scale metabolic network reconstructions and demonstrate the utility of this approach in providing functional explanations for essentiality and reconciling differences between screens. Genome-scale metabolic network reconstructions also enable a high-throughput, quantitative analysis to assess the impact of media conditions on the identification of condition-independent essential genes. Our computational model-driven analysis provides mechanistic insight into essentiality and contributes novel insights for design of future gene essentiality screens and the identification of core metabolic processes.


Assuntos
Genes Essenciais , Redes e Vias Metabólicas/genética , Biologia Computacional , Meios de Cultura , Elementos de DNA Transponíveis , Bases de Dados Genéticas/estatística & dados numéricos , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala/estatística & dados numéricos , Humanos , Modelos Genéticos , Mutagênese , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo
15.
Expert Rev Med Devices ; 16(5): 413-420, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30957585

RESUMO

OBJECTIVES: Endogenous and exogenous infection of the biliary tract could occur during endoscopic retrograde cholangiopancreatography. METHODS: Bile samples of patients with hepatobiliary diseases, and swab samples of elevator channel samples of duodenoscope and washing instruments were prepared simultaneously and cultured aerobically and anaerobically. Antimicrobial susceptibility of the most common characterized bacterial species was tested, and their genetic relatedness was analyzed by multiple locus variable number of tandem repeats method. RESULTS: Contamination with Pseudomonas aeruginosa was detected in 38.2% of the elevator channels' and 26.6% of the bile samples. Staphylococcus aureus, Enterococcus spp., Staphylococcus epidermidis, Escherichia coli, Enterobacter spp., and Clostridium perfringenes were among other bacterial isolates in the elevator channel swab samples. Highest antimicrobial resistance rate among P. aeruginosa isolates from the bile and swab samples were detected against gentamicin (100% and 73%, respectively), while the lowest one was measured to piperacillin-tazobactam (25% and 0%, respectively). Out of the 27 distinct MLVA profiles, relatedness of P. aeruginosa strains in the bile samples compared with those from the elevators was shown in three series of the samples. CONCLUSION: Identity of P. aeruginosa strains among the bile and elevator channel samples showed possibility of cross-contamination among patients even at distinct time intervals. Expert opinion: Bacterial infection is considered as main complications of ERCP. Entry of bacteria into the biliary tract via contaminated device and its related instruments and their proliferation in this tissue could cause serious infections. To prevent this side effect, reprocessing of medical equipment via standard cleaning and disinfection procedures are needed. Our results showed incompliance of methods used for endoscope cleaning and disinfection procedure. Although host risk factors, such as sphincterotomy, could increase rate of infection with different types of bacteria, their ability for formation of biofilm and spore, which could help them to resist disinfectants and washing procedures seems to be main cause of persistent colonization and transmission among different patients. New standards for disinfection compared with currently used methods and use of materials to eliminate formation of bacterial microcolonies seem to be necessary to prevent cross-contamination.


Assuntos
Bactérias/genética , Colangiopancreatografia Retrógrada Endoscópica , Duodenoscópios , Repetições Minissatélites/genética , Bactérias/classificação , Bactérias/isolamento & purificação , Bile/microbiologia , Desinfecção , Resistência Microbiana a Medicamentos , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Pseudomonas aeruginosa/genética
16.
MBio ; 10(2)2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30992347

RESUMO

Biogenesis of the outer membrane of Gram-negative bacteria depends on dedicated macromolecular transport systems. The LolABCDE proteins make up the machinery for lipoprotein trafficking from the inner membrane (IM) across the periplasm to the outer membrane (OM). The Lol apparatus is additionally responsible for differentiating OM lipoproteins from those for the IM. In Enterobacteriaceae, a default sorting mechanism has been proposed whereby an aspartic acid at position +2 of the mature lipoproteins prevents Lol recognition and leads to their IM retention. In other bacteria, the conservation of sequences immediately following the acylated cysteine is variable. Here we show that in Pseudomonas aeruginosa, the three essential Lol proteins (LolCDE) can be replaced with those from Escherichia coli The P. aeruginosa lipoproteins MexA, OprM, PscJ, and FlgH, with different sequences at their N termini, were correctly sorted by either the E. coli or P. aeruginosa LolCDE. We further demonstrate that an inhibitor of E. coli LolCDE is active against P. aeruginosa only when expressing the E. coli orthologues. Our work shows that Lol proteins recognize a wide range of signals, consisting of an acylated cysteine and a specific conformation of the adjacent domain, determining IM retention or transport to the OM.IMPORTANCE Gram-negative bacteria build their outer membranes (OM) from components that are initially located in the inner membrane (IM). A fraction of lipoproteins is transferred to the OM by the transport machinery consisting of LolABCDE proteins. Our work demonstrates that the LolCDE complexes of the transport pathways of Escherichia coli and Pseudomonas aeruginosa are interchangeable, with the E. coli orthologues correctly sorting the P. aeruginosa lipoproteins while retaining their sensitivity to a small-molecule inhibitor. These findings question the nature of IM retention signals, identified in E. coli as aspartate at position +2 of mature lipoproteins. We propose an alternative model for the sorting of IM and OM lipoproteins based on their relative affinities for the IM and the ability of the promiscuous sorting machinery to deliver lipoproteins to their functional sites in the OM.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Escherichia coli/genética , Lipoproteínas/metabolismo , Transporte Proteico , Pseudomonas aeruginosa/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Ácido Aspártico/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Lipoproteínas/genética , Pseudomonas aeruginosa/genética
17.
Genome Biol Evol ; 11(5): 1385-1397, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30980662

RESUMO

Bacterial pathogens evolve during the course of infection as they adapt to the selective pressures that confront them inside the host. Identification of adaptive mutations and their contributions to pathogen fitness remains a central challenge. Although mutations can either target intergenic or coding regions in the pathogen genome, studies of host adaptation have focused predominantly on molecular evolution within coding regions, whereas the role of intergenic mutations remains unclear. Here, we address this issue and investigate the extent to which intergenic mutations contribute to the evolutionary response of a clinically important bacterial pathogen, Pseudomonas aeruginosa, to the host environment, and whether intergenic mutations have distinct roles in host adaptation. We characterize intergenic evolution in 44 clonal lineages of P. aeruginosa and identify 77 intergenic regions in which parallel evolution occurs. At the genetic level, we find that mutations in regions under selection are located primarily within regulatory elements upstream of transcriptional start sites. At the functional level, we show that some of these mutations both increase or decrease transcription of genes and are directly responsible for evolution of important pathogenic phenotypes including antibiotic sensitivity. Importantly, we find that intergenic mutations facilitate essential genes to become targets of evolution. In summary, our results highlight the evolutionary significance of intergenic mutations in creating host-adapted strains, and that intergenic and coding regions have different qualitative contributions to this process.


Assuntos
Adaptação Biológica , DNA Intergênico , Evolução Molecular , Interações Hospedeiro-Patógeno/genética , Pseudomonas aeruginosa/genética , Mineração de Dados , Farmacorresistência Bacteriana/genética , Regulação Bacteriana da Expressão Gênica , Genes Essenciais , Mutação , Regiões Promotoras Genéticas
18.
Microb Pathog ; 131: 128-134, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30959097

RESUMO

Quorum sensing (QS) is the cell density dependent communication network which coordinates the production of pathogenic determinants in majority of pathogenic bacteria. Pseudomonas aeruginosa causes hospital-acquired infections by virtue of its well-defined QS network. As the QS regulatory network in P. aeruginosa regulates the virulence determinants and antibiotic resistance, attenuating the QS system seems to be influential in developing next-generation anti-infective agents. In the current study, the QS attenuation potential of a flavonoid, mosloflavone was investigated against P. aeruginosa virulence and biofilm formation. Mosloflavone inhibited the pyocyanin production, LasB elastase and chitinase by 59.52 ±â€¯2.74, 35.90 ±â€¯4.34 and 61.18 ±â€¯5.52% respectively. The QS regulated biofilm formation and development was also reduced when supplemented with sub-MIC of mosloflavone. The gene expression studies of mosloflavone using RT-PCR depicted its ability to down-regulate the expression levels of QS regulated virulence genes such as lasI (60.64%), lasR (91.70%), rhlI (57.30%), chiC (90.20%), rhlA (47.87%), rhlR (21.55%), lasB (37.80%), phzM (42.40%), toxA (61.00%), aprA (58.4%), exoS (78.01%), algD (46.60%) and pelA (50.45%). The down-regulation of QS virulence phenotypes by mosloflavone could be attributed to its binding affinity with the QS regulatory proteins, LasR and RhlR by competitively inhibiting the binding of natural autoinducers as evidenced from simulation studies. Mosloflavone also exhibited promising potential in controlling bacterial infection in Caenorhabditis elegans model system, in vivo. The anti-biofilm and anti-QS potential of mosloflavone in the current study illustrated the candidature of mosloflavone as a promising biocide.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Flavonoides/farmacologia , Fenótipo , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Alginatos , Animais , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Caenorhabditis elegans , Quitinases/metabolismo , Modelos Animais de Doenças , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Glicolipídeos/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Metaloendopeptidases/genética , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/genética , Piocianina/metabolismo , Transativadores/genética , Virulência/efeitos dos fármacos , Virulência/genética , Fatores de Virulência/genética
19.
Environ Pollut ; 250: 262-273, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30999203

RESUMO

Dibenzofuran (DBF) derivatives have caused serious environmental problems, especially those produced by paper pulp bleaching and incineration processes. Prominent for its resilient mutagenicity and toxicity, DBF poses a major challenge to human health. In the present study, a new strain of Pseudomonas aeruginosa, FA-HZ1, with high DBF-degrading activity was isolated and identified. The determined optimum conditions for cell growth of strain FA-HZ1 were a temperature of 30 °C, pH 5.0, rotation rate of 200 rpm and 0.1 mM DBF as a carbon source. The biochemical and physiological features as well as usage of different carbon sources by FA-HZ1 were studied. The new strain was positive for arginine double hydrolase, gelatinase and citric acid, while it was negative for urease and lysine decarboxylase. It could utilize citric acid as its sole carbon source, but was negative for indole and H2S production. Intermediates of DBF 1,2-dihydroxy-1,2-dihydrodibenzofuran, 1,2-dihydroxydibenzofuran, 2-hydroxy-4-(3'-oxo-3'H-benzofuran-2'-yliden)but-2-enoic acid, 2,3-dihydroxybenzofuran, 2-oxo-2-(2'-hydrophenyl)lactic acid, and 2-hydroxy-2-(2'-hydroxyphenyl)acetic acid were detected and identified through liquid chromatography-mass analyses. FA-HZ1 metabolizes DBF by both the angular and lateral dioxygenation pathways. The genomic study identified 158 genes that were involved in the catabolism of aromatic compounds. To identify the key genes responsible for DBF degradation, a proteomic study was performed. A total of 1459 proteins were identified in strain FA-HZ1, of which 100 were up-regulated and 104 were down-regulated. A novel enzyme "HZ6359 dioxygenase", was amplified and expressed in pET-28a in E. coli BL21(DE3). The recombinant plasmid was successfully constructed, and was used for further experiments to verify its function. In addition, the strain FA-HZ1 can also degrade halogenated analogues such as 2, 8-dibromo dibenzofuran and 4-(4-bromophenyl) dibenzofuran. Undoubtedly, the isolation and characterization of new strain and the designed pathways is significant, as it could lead to the development of cost-effective and alternative remediation strategies. The degradation pathway of DBF by P. aeruginosa FA-HZ1 is a promising tool of biotechnological and environmental significance.


Assuntos
Benzofuranos/metabolismo , Poluentes Ambientais/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Dioxigenases/genética , Dioxigenases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Resíduos Industriais , Redes e Vias Metabólicas/genética , Proteômica , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento
20.
Molecules ; 24(7)2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30979013

RESUMO

Rhamnolipids are a mixture of the homologs species due to variations in the rhamnose units and ß-hydroxy fatty acid moieties, mainly including Rha-C10-C10, Rha-Rha-C10-C10, and Rha-C10. In this study, strain P. aeruginosa YM4 was selected for its capacity to efficiently produce di-rhamnolipid (Rha-Rha-C10-C10) as the predominant component with soybean oil and glycerol as carbon source, accounting for 64.8% and 85.7% of total products, respectively. The critical micelle concentration (CMC) of rhamnolipid products varies with the content of di-rhamnolipid, whereby lower CMC values corresponding to higher di-rhamnolipid contents. The rhamnolipids containing 85.7% di-rhamnolipid had the lowest CMC value of 50 mg/L. Accordingly the viscosity-reducing efficiency and oil-washing efficiency of rhamnolipids increased with higher di-rhamnolipid component. At a concentration of 500 mg/L, the rhamnolipids containing 85.7% di-rhamnolipid worked best and showed 82.5% oil-washing efficiency, which offered great promise for applications in enhanced oil recovery. The results showed the variation of structure and composition of rhamnolipids had a significant effect on their application.


Assuntos
Glicolipídeos/biossíntese , Poluição por Petróleo/prevenção & controle , Pseudomonas aeruginosa/metabolismo , Ramnose/biossíntese , Carbono/química , Ácidos Graxos/química , Glicerol/química , Glicolipídeos/química , Humanos , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Ramnose/química , Óleo de Soja/química , Tensoativos/química
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