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1.
PLoS Pathog ; 16(9): e1008867, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32925969

RESUMO

Surface attachment, an early step in the colonization of multiple host environments, activates the virulence of the human pathogen P. aeruginosa. However, the downstream toxins that mediate surface-dependent P. aeruginosa virulence remain unclear, as do the signaling pathways that lead to their activation. Here, we demonstrate that alkyl-quinolone (AQ) secondary metabolites are rapidly induced upon surface association and act directly on host cells to cause cytotoxicity. Surface-induced AQ cytotoxicity is independent of other AQ functions like quorum sensing or PQS-specific activities like iron sequestration. We further show that packaging of AQs in outer-membrane vesicles (OMVs) increases their cytotoxicity to host cells but not their ability to stimulate downstream quorum sensing pathways in bacteria. OMVs lacking AQs are significantly less cytotoxic, suggesting these molecules play a role in OMV cytotoxicity, in addition to their previously characterized role in OMV biogenesis. AQ reporters also enabled us to dissect the signal transduction pathways downstream of the two known regulators of surface-dependent virulence, the quorum sensing receptor, LasR, and the putative mechanosensor, PilY1. Specifically, we show that PilY1 regulates surface-induced AQ production by repressing the AlgR-AlgZ two-component system. AlgR then induces RhlR, which can induce the AQ biosynthesis operon under specific conditions. These findings collectively suggest that the induction of AQs upon surface association is both necessary and sufficient to explain surface-induced P. aeruginosa virulence.


Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa , Quinolonas/farmacologia , Percepção de Quorum/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Virulência/metabolismo , Células A549 , Animais , Humanos , Camundongos , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade
2.
PLoS One ; 15(8): e0237752, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32817626

RESUMO

Pseudomonas aeruginosa remains a leading cause of nosocomial and serious life-threatening infections, and contributes to increased mortality in immunocompromised individuals. P. aeruginosa infection triggers host immune response and often provokes potent inflammatory mediators, which do not necessarily eradicate the causative pathogen. On the other hand, it causes severe airway damage and eventually decreased lung function. Such unfavorable outcomes of inflammatory injury have necessitated the development of novel effective agents that can combat with P. aeruginosa-mediated inflammation. Herein, we investigated the potential of quercetin in regulating P. aeruginosa-induced inflammation, with particular emphasized on the interleukin-1ß (IL-1ß). Our results showed that quercetin exerted the potent inhibitory activity against the production of IL-1ß in macrophages infected by live P. aeruginosa PAO1, without exhibiting cytotoxicity. According to our settings, such the potent inhibitory activity of quercetin was clearly demonstrated through its ability to efficiently inhibit IL-1ß during P. aeruginosa infection, pre- or even post-infection. In addition, quercetin strongly suppressed MAPK signaling pathway by inhibiting phosphorylation of the p38 MAPK and JNK2, and molecular docking study supported well with this observation. Moreover, quercetin reduced the NLRP3 expression and inhibited the P. aeruginosa-mediated cleavage of caspase-1 as well as mature IL-1ß. These results thus indicated that quercetin inhibition of P. aeruginosa-induced IL-1ß production is mediated by suppressing the initial priming step and by inhibiting the NLRP3 inflammasome activation. Taken together, our findings demonstrated the promising regulatory activity of quercetin against IL-1ß production in P. aeruginosa-infected macrophages, and indicated that quercetin has the potential to be effective as a novel therapeutic agent for treatment of P. aeruginosa-induced inflammation.


Assuntos
Interleucina-1beta/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Infecções por Pseudomonas/tratamento farmacológico , Quercetina/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamassomos/efeitos dos fármacos , Inflamassomos/genética , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/patogenicidade , Transdução de Sinais/efeitos dos fármacos
4.
PLoS One ; 15(7): e0235345, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32667930

RESUMO

This study was carried out to investigate the risks of simultaneous exposure to pesticide residues and bacteria contaminants in locally produced fresh vegetables and vegetables in Tanzania. A total of 613 samples were analyzed for pesticide residues, out of which 250 were also analyzed for bacterial contamination. Overall, 47.5% had pesticide residues, 74.2% exceeded Maximum Residue Levels (MRLs). Organophosphorus (95.2%), organochlorines (24.0%), pyrethroids (17.3%), and carbamates (9.2%) residues dominated. MRL values were mostly exceeded in tomatoes, onions, watermelons, cucumbers, Chinese cabbage, and sweet paper. Tetramethrin (0.0329-1.3733 mg/kg), pirimiphos-methyl (0.0003-1.4093 mg/kg), permethrin (0.0009-2.4537 mg/kg), endosulfan (beta) (0.0008-2.3416 mg/kg), carbaryl (0.0215-1.5068 mg/kg), profenofos (0.0176-2.1377 mg/kg), chlorpyrifos (0.0004-1.2549 mg/kg) and dieldrin (0.0011-0.5271 mg/kg) exceeded MRLs. The prevalence of bacteria contamination was high (63.2%). Enterobacter (55.6%) Pseudomonas aeruginosa (32.4%), E. coli (28.2%), Citrobacter (26.8%), Klebsiella oxytoca (14.8%), and Salmonella (7.7%) were isolated. Furthermore, 46.4% tested positive for both pesticide residues and bacterial contaminants. Vegetables from farms (60.7%) contained more dual contaminants than market-based vegetables (41.8%). This may have resulted from excessive pesticide use and unhygienic handling of fresh fruits and vegetables at production level. Binary logistic regression showed that fresh fruits and vegetables with pesticide residues were 2.231 times more likely to have bacteria contaminants (OR: 2.231; 95% CI: 0.501, 8.802). The contamination levels of pesticide residues and bacterial contaminants could be perceived as a serious problem as most fresh fruits and vegetables recorded values of pesticide residues far above the MRLs with pathogenic bacteria isolated in higher proportions. MRLs was higher in most vegetables consumed raw or semi-cooked such as watermelons, carrots, cucumber, tomatoes, onion and sweet paper. There is an urgent need to develop pesticide monitoring and surveillance systems at farmer level, educating farmers and promoting the use of greener pesticides to mitigate the health effects of pesticides and bacterial contaminants.


Assuntos
Contaminação de Alimentos/análise , Frutas/efeitos adversos , Praguicidas/efeitos adversos , Verduras/efeitos adversos , Citrobacter/isolamento & purificação , Citrobacter/patogenicidade , Enterobacter/isolamento & purificação , Enterobacter/patogenicidade , Frutas/química , Frutas/microbiologia , Humanos , Hidrocarbonetos Clorados/efeitos adversos , Hidrocarbonetos Clorados/química , Klebsiella oxytoca/isolamento & purificação , Klebsiella oxytoca/patogenicidade , Resíduos de Praguicidas/efeitos adversos , Resíduos de Praguicidas/química , Praguicidas/química , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/patogenicidade , Piretrinas/efeitos adversos , Piretrinas/química , Salmonella/isolamento & purificação , Salmonella/patogenicidade , Verduras/química , Verduras/microbiologia
5.
PLoS Genet ; 16(6): e1008848, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32530919

RESUMO

Pseudomonas aeruginosa colonizes the airways of cystic fibrosis (CF) patients, causing infections that can last for decades. During the course of these infections, P. aeruginosa undergoes a number of genetic adaptations. One such adaptation is the loss of swimming motility functions. Another involves the formation of the rugose small colony variant (RSCV) phenotype, which is characterized by overproduction of the exopolysaccharides Pel and Psl. Here, we provide evidence that the two adaptations are linked. Using random transposon mutagenesis, we discovered that flagellar mutations are linked to the RSCV phenotype. We found that flagellar mutants overexpressed Pel and Psl in a surface-contact dependent manner. Genetic analyses revealed that flagellar mutants were selected for at high frequencies in biofilms, and that Pel and Psl expression provided the primary fitness benefit in this environment. Suppressor mutagenesis of flagellar RSCVs indicated that Psl overexpression required the mot genes, suggesting that the flagellum stator proteins function in a surface-dependent regulatory pathway for exopolysaccharide biosynthesis. Finally, we identified flagellar mutant RSCVs among CF isolates. The CF environment has long been known to select for flagellar mutants, with the classic interpretation being that the fitness benefit gained relates to an impairment of the host immune system to target a bacterium lacking a flagellum. Our new findings lead us to propose that exopolysaccharide production is a key gain-of-function phenotype that offers a new way to interpret the fitness benefits of these mutations.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Vias Biossintéticas/genética , Fibrose Cística/complicações , Fibrose Cística/microbiologia , Flagelos/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Mutação , Polissacarídeos Bacterianos/biossíntese , Pseudomonas aeruginosa/patogenicidade , Seleção Genética
6.
BMC Bioinformatics ; 21(1): 159, 2020 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-32349677

RESUMO

BACKGROUND: Genomic islands are associated with microbial adaptations, carrying genomic signatures different from the host. Some methods perform an overall test to identify genomic islands based on their local features. However, regions of different scales will display different genomic features. RESULTS: We proposed here a novel method "2SigFinder ", the first combined use of small-scale and large-scale statistical testing for genomic island detection. The proposed method was tested by genomic island boundary detection and identification of genomic islands or functional features of real biological data. We also compared the proposed method with the comparative genomics and composition-based approaches. The results indicate that the proposed 2SigFinder is more efficient in identifying genomic islands. CONCLUSIONS: From real biological data, 2SigFinder identified genomic islands from a single genome and reported robust results across different experiments, without annotated information of genomes or prior knowledge from other datasets. 2SigHunter identified 25 Pathogenicity, 1 tRNA, 2 Virulence and 2 Repeats from 27 Pathogenicity, 1 tRNA, 2 Virulence and 2 Repeats, and detected 101 Phage and 28 HEG out of 130 Phage and 36 HEGs in S. enterica Typhi CT18, which shows that it is more efficient in detecting functional features associated with GIs.


Assuntos
Algoritmos , Genoma Bacteriano , Ilhas Genômicas/genética , Genômica/métodos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Salmonella enterica/genética , Salmonella enterica/patogenicidade , Virulência
7.
Nucleic Acids Res ; 48(11): 5967-5985, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32406921

RESUMO

During infection of a host, Pseudomonas aeruginosa orchestrates global gene expression to adapt to the host environment and counter the immune attacks. P. aeruginosa harbours hundreds of regulatory genes that play essential roles in controlling gene expression. However, their contributions to the bacterial pathogenesis remain largely unknown. In this study, we analysed the transcriptomic profile of P. aeruginosa cells isolated from lungs of infected mice and examined the roles of upregulated regulatory genes in bacterial virulence. Mutation of a novel regulatory gene pvrA (PA2957) attenuated the bacterial virulence in an acute pneumonia model. Chromatin immunoprecipitation (ChIP)-Seq and genetic analyses revealed that PvrA directly regulates genes involved in phosphatidylcholine utilization and fatty acid catabolism. Mutation of the pvrA resulted in defective bacterial growth when phosphatidylcholine or palmitic acid was used as the sole carbon source. We further demonstrated that palmitoyl coenzyme A is a ligand for the PvrA, enhancing the binding affinity of PvrA to its target promoters. An arginine residue at position 136 was found to be essential for PvrA to bind palmitoyl coenzyme A. Overall, our results revealed a novel regulatory pathway that controls genes involved in phosphatidylcholine and fatty acid utilization and contributes to the bacterial virulence.


Assuntos
Proteínas de Bactérias/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Genes Bacterianos/genética , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Animais , Arginina/metabolismo , Sequência de Bases , Imunoprecipitação da Cromatina , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Ligantes , Camundongos , Modelos Moleculares , Mutação , Ácido Palmítico/metabolismo , Palmitoil Coenzima A/metabolismo , Fosfatidilcolinas/metabolismo , Pneumonia Bacteriana/microbiologia , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/genética , Transcriptoma , Virulência/genética
8.
J Vis Exp ; (156)2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32150161

RESUMO

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of chronic wound infections in diabetic and hospitalized patients. There is an urgent need for chronic infection models to aid in the investigation of wound pathogenesis and the development of new therapies against this pathogen. Here, we describe a protocol that uses delayed inoculation 24 hours after full-thickness excisional wounding. The infection of the provisional wound matrix present at this time forestalls either rapid clearance or dissemination of infection and instead establishes chronic infection lasting 7-10 days without the need for implantation of foreign materials or immune suppression. This protocol mimics a typical temporal course of post-operative infection in humans. The use of a luminescent P. aeruginosa strain (PAO1:lux) allows for quantitative daily assessment of bacterial burden for P. aeruginosa wound infections. This novel model may be a useful tool in the investigation of bacterial pathogenesis and the development of new therapies for chronic P. aeruginosa wound infections.


Assuntos
Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/patogenicidade , Infecção dos Ferimentos/microbiologia , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Pseudomonas/patologia , Infecção dos Ferimentos/patologia
9.
PLoS One ; 15(3): e0230199, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32155223

RESUMO

OBJECTIVES: Infective endocarditis (IE) with non-HACEK Gram-negative (GN) organisms is rare, but associated with poor outcomes. The purpose of this study was to quantify the microbiology, treatment strategies, and frequency of poor outcomes in patients with non-HACEK GN IE. MATERIALS: Retrospective cohort of adults with definite non-HACEK GN IE from 1/11-1/19. The primary endpoint was poor patient outcome, defined as a composite of all-cause death or infection-related readmission within 90-days of index infection. RESULTS: 43 patients were included: 51% patients were men, and the median (IQR) age was 40 (31-50) years. Forty patients reported injection drug use. The most common organisms were Pseudomonas aeruginosa (68%) and Serratia marcescens (9%). Seventy-six percent of patients received definitive combination therapy; the most common antibiotics used in combination with a ß-lactam were aminoglycosides (50%) and fluoroquinolones (34%). Three patients discontinued combination therapy due to toxicity. Twelve-month, all-cause mortality and readmission was 30% and 54%, respectively. In multivariable logistic regression, variables independently associated with composite poor outcome were receipt of fluoroquinolone-based IE combination therapy and septic shock. CONCLUSIONS: Long-term mortality and readmission rates were high. Patients who received fluoroquinolone-based IE combination therapy more frequently developed poor outcomes than those who did not.


Assuntos
Endocardite/epidemiologia , Endocardite/mortalidade , Endocardite/terapia , Adulto , Antibacterianos/uso terapêutico , Estudos de Coortes , Endocardite Bacteriana/epidemiologia , Endocardite Bacteriana/mortalidade , Endocardite Bacteriana/terapia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pseudomonas aeruginosa/patogenicidade , Estudos Retrospectivos , Serratia marcescens/patogenicidade , Resultado do Tratamento , Estados Unidos/epidemiologia
10.
BMC Infect Dis ; 20(1): 225, 2020 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-32183752

RESUMO

BACKGROUND: Otitis media is inflammation of the middle ear, comprising a spectrum of diseases. It is the commonest episode of infection in children, which often occurs after an acute upper respiratory tract infection. Otitis media is ranked as the second most important cause of hearing loss and the fifth global burden of disease with a higher incidence in developing worlds like Sub-Saharan Africa and South Asia. Therefore, this systematic review is aimed to quantitatively estimate the current status of bacterial otitis media, bacterial etiology and their susceptibility profile in sub-Saharan Africa. METHODS: A literature search was conducted from major databases and indexing services including EMBASE (Ovid interface), PubMed/MEDLINE, Google Scholar, ScienceDirect, Cochrane Library, WHO African Index-Medicus and others. All studies (published and unpublished) addressing the prevalence of otitis media and clinical isolates conducted in sub-Saharan Africa were included. Format prepared in Microsoft Excel was used to extract the data and data was exported to Stata version 15 software for the analyses. Der-Simonian-Laird random-effects model at a 95% confidence level was used for pooled estimation of outcomes. The degree of heterogeneity was presented with I2 statistics. Publication bias was presented with funnel plots of standard error supplemented by Begg's and Egger's tests. The study protocol is registered on PROSPERO with reference number ID: CRD42018102485 and the published methodology is available from http://www.crd.york.ac.uk/CRD42018102485. RESULTS: A total of 33 studies with 6034 patients were included in this study. All studies have collected ear swab/discharge samples for bacterial isolation. The pooled isolation rate of bacterial agents from the CSOM subgroup was 98%, patients with otitis media subgroup 87% and pediatric otitis media 86%. A univariate meta-regression analysis indicated the type of otitis media was a possible source of heterogeneity (p-value = 0.001). The commonest isolates were P. aeruginosa (23-25%), S. aureus (18-27%), Proteus species (11-19%) and Klebsiella species. High level of resistance was observed against Ampicillin, Amoxicillin-clavulanate, Cotrimoxazole, Amoxicillin, and Cefuroxime. CONCLUSION: The analysis revealed that bacterial pathogens like P. aeruginosa and S. aureus are majorly responsible for otitis media in sub-Saharan Africa. The isolates have a high level of resistance to commonly used drugs for the management of otitis media.


Assuntos
Infecções Bacterianas/microbiologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Otite Média/microbiologia , África ao Sul do Saara/epidemiologia , Infecções Bacterianas/tratamento farmacológico , Criança , Humanos , Otite Média/tratamento farmacológico , Prevalência , Infecções por Pseudomonas/epidemiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/patogenicidade , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade
11.
PLoS Pathog ; 16(3): e1008444, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32208458

RESUMO

Cystic fibrosis (CF) is a genetic disease that affects mucin-producing body organs such as the lungs. Characteristic of CF is the production of thick, viscous mucus, containing the glycoprotein mucin, that can lead to progressive airway obstruction. Recently, we demonstrated that the presence of mucin induced a rapid surface adaptation in motile bacteria termed surfing motility, which data presented here indicates is very different from swarming motility. Pseudomonas aeruginosa, the main colonizing pathogen in CF, employs several stress coping mechanisms to survive the highly viscous environment of the CF lung. We used motility-based assays and RNA-Seq to study the stringent stress response in the hypervirulent CF isolate LESB58 (Liverpool Epidemic Strain). Motility experiments revealed that an LESB58 stringent response mutant (ΔrelAΔspoT) was unable to surf. Transcriptional profiling of ΔrelAΔspoT mutant cells from surfing agar plates, when compared to wild-type cells from the surfing edge, revealed 2,584 dysregulated genes. Gene Ontology and KEGG enrichment analysis revealed effects of the stringent response on amino acid, nucleic acid and fatty acid metabolism, TCA cycle and glycolysis, type VI secretion, as well as chemotaxis, cell communication, iron transport, nitrogen metabolic processes and cyclic-di-GMP signalling. Screening of the ordered PA14 transposon library revealed 224 mutants unable to surf and very limited overlap with genes required for swarming. Mutants affecting surfing included two downstream effector genes of the stringent stress response, the copper regulator cueR and the quinolone synthase pqsH. Both the cueR and pqsH cloned genes complemented the surfing deficiency of ΔrelAΔspoT. Our study revealed insights into stringent stress dependency in LESB58 and showed that surfing motility is stringently-controlled via the expression of cueR and pqsH. Downstream factors of the stringent stress response are important to investigate in order to fully understand its ability to colonize and persist in the CF lung.


Assuntos
Proteínas de Bactérias , Proteínas de Ligação a DNA , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa , Sistemas do Segundo Mensageiro , Estresse Fisiológico , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade
12.
Pathog Dis ; 78(1)2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-32167551

RESUMO

BACKGROUND: Pseudomonas aeruginosa possesses an array of virulence genes ensuring successful infection development. A two-partner secretion system Exolysin BA (ExlBA) is expressed in the PA7-like genetic outliers consisting of ExlA, a pore-forming toxin and ExlB transporter protein. Presence of exlBA in multidrug-resistant (MDR) strains has not been investigated, particularly in the strains isolated from wounded soldiers. METHODS: We screened whole genome sequences of 2439 MDR- P. aeruginosa strains for the presence of exlBA. We compiled all exlBA positive strains and compared them with a diversity set for demographics, antimicrobial profiles and phenotypic characteristics: surface motility, biofilm formation, pyocyanin production and hemolysis. We compared the virulence of strains with comparable phenotypic characteristics in Galleria mellonella. RESULTS: We identified 33 exlBA-positive strains (1.5%). These strains have increased antibiotic resistance, they are more motile, produce more robust biofilms and have comparable pyocianin production with the diversity set despite the phenotypic differences within the group. In in vivo infection models, these strains were less virulent than Type III Secretion System (T3SS) positive counterparts. CONCLUSIONS: exlBA-positive strains are wide spread among the PA7-like outliers. While not as virulent as strains possessing T3SS, these strains exhibit phenotypic features associated with virulence and are still lethal in vivo.


Assuntos
Exotoxinas/genética , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/fisiologia , Antibacterianos/farmacologia , Biofilmes , Farmacorresistência Bacteriana Múltipla , Exotoxinas/metabolismo , Genoma Bacteriano , Genômica/métodos , Humanos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/patogenicidade , Virulência/genética , Fatores de Virulência/genética
14.
Lett Appl Microbiol ; 70(5): 372-379, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32048742

RESUMO

Pseudomonas aeruginosa is a prominent member of emerging waterborne pathogens. The environmental reservoirs of multi-resistant phenotypes and other virulence factors in this bacterium are poorly understood. Our study aimed to determine the virulence properties of P. aeruginosa isolated from Roraima Sur Cave (RSC) waters at Guayana Highlands. Based on the best identification at species level by biochemical tests, 16S rRNA sequencing and phylogenetic inferences, one RSC isolate named LG11 was characterized for virulence properties in comparison with P. aeruginosa reference strains. PCR amplification of alginate, elastase, exoenzyme S, exotoxin A, neuraminidase and Quorum-Sensing genes showed a high virulence potential in LG11. This isolate demonstrated multi-resistance to ceftriaxone, tigecycline and imipenem. Pyocyanin production was greater in LG11 (0·478 µg ml-1 ) than the strain ATCC 10145 (0·316 µg ml-1 ), but the highest pigment concentration (2·140 µg ml-1 ) was displayed by the clinical strain CVCM 937 (P = 0·000175). Pronounced biomass production on granite and glass (P < 0·05) and well-developed biofilms indicated the ability of P. aeruginosa from RSC to colonize surfaces found in human and healthcare environments. These data suggest that waters from pristine ecosystems such as RSC could be reservoirs of this opportunistic bacterium carrying important virulence properties with potential epidemiological implications. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows for the first time the occurrence of virulence genes and multi-resistance to antimicrobials in Pseudomonas aeruginosa isolated from cave waters at Guayana Highlands. These findings, together with the biofilm formation on surfaces found in human and healthcare settings, suggest public health risks and the potential of these virulence properties to be transferred from or to native populations in waters. Our results provide important insights to the current knowledge of P. aeruginosa in the environment, setting the basis for future studies driven to assess reservoirs of multi-resistant bacteria and virulence features unknown in pristine ecosystems.


Assuntos
Cavernas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/patogenicidade , Fatores de Virulência/genética , Microbiologia da Água , Antibacterianos/farmacologia , Biofilmes/crescimento & desenvolvimento , Farmacorresistência Bacteriana/efeitos dos fármacos , Ecossistema , Testes de Sensibilidade Microbiana , Filogenia , Pseudomonas aeruginosa/isolamento & purificação , Piocianina/biossíntese , Percepção de Quorum , RNA Ribossômico 16S/genética , Venezuela , Virulência
15.
PLoS Pathog ; 16(2): e1008311, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32040500

RESUMO

Pseudomonas aeruginosa is one of the leading causes of nosocomial pneumonia and its associated mortality. Moreover, extensively drug-resistant high-risk clones are globally widespread, presenting a major challenge to the healthcare systems. Despite this, no vaccine is available against this high-concerning pathogen. Here we tested immunogenicity and protective efficacy of an experimental live vaccine against P. aeruginosa pneumonia, consisting of an auxotrophic strain which lacks the key enzyme involved in D-glutamate biosynthesis, a structural component of the bacterial cell wall. As the amounts of free D-glutamate in vivo are trace substances in most cases, blockage of the cell wall synthesis occurs, compromising the growth of this strain, but not its immunogenic properties. Indeed, when delivered intranasally, this vaccine stimulated production of systemic and mucosal antibodies, induced effector memory, central memory and IL-17A-producing CD4+ T cells, and recruited neutrophils and mononuclear phagocytes into the airway mucosa. A significant improvement in mice survival after lung infection caused by ExoU-producing PAO1 and PA14 strains was observed. Nearly one third of the mice infected with the XDR high-risk clone ST235 were also protected. These findings highlight the potential of this vaccine for the control of acute pneumonia caused by this bacterial pathogen.


Assuntos
Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Vacinas Atenuadas/imunologia , Administração Intranasal , Animais , Anticorpos Antibacterianos/imunologia , Feminino , Imunidade nas Mucosas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pneumonia Bacteriana/imunologia , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Sistema Respiratório/imunologia , Infecções Respiratórias/imunologia , Vacinas Atenuadas/farmacologia
17.
Infect Immun ; 88(5)2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32041787

RESUMO

Phagocytosis is the key mechanism for host control of Pseudomonas aeruginosa, a motile Gram-negative, opportunistic bacterial pathogen which frequently undergoes adaptation and selection for traits that are advantageous for survival. One such clinically relevant adaptation is the loss of bacterial motility, observed within chronic infections, that is associated with increased antibiotic tolerance and phagocytic resistance. Previous studies using phagocytes from a leukocyte adhesion deficiency type 1 (LAD-I) patient identified CD18 as a putative cell surface receptor for uptake of live P. aeruginosa However, how bacterial motility alters direct engagement with CD18-containing integrins remains unknown. Here we demonstrate, with the use of motile and isogenic nonmotile deletion mutants of two independent strains of P. aeruginosa and with CRISPR-generated CD18-deficient cell lines in human monocytes and murine neutrophils, that CD18 expression facilitates the uptake of both motile and nonmotile P. aeruginosa However, unexpectedly, mechanistic studies revealed that CD18 expression was dispensable for the initial attachment of the bacteria to the host cells, which was validated with ectopic expression of complement receptor 3 (CR3) by CHO cells. Our data support that surface N-linked glycan chains (N-glycans) likely facilitate the initial interaction of bacteria with monocytes and cooperate with CD18 integrins in trans to promote internalization of bacteria. Moreover, talin-1 and kindlin-3 proteins promote uptake, but not binding, of P. aeruginosa by murine neutrophils, which supports a role for CD18 integrin signaling in this process. These findings provide novel insights into the cellular determinants for phagocytic recognition and uptake of P. aeruginosa.


Assuntos
Antígenos CD18/metabolismo , Integrinas/metabolismo , Fagócitos/metabolismo , Fagócitos/microbiologia , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Animais , Células CHO , Linhagem Celular , Cricetulus , Camundongos , Monócitos/metabolismo , Monócitos/microbiologia , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Ligação Proteica/fisiologia , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/microbiologia , Talina/metabolismo , Células Th1
18.
Appl Microbiol Biotechnol ; 104(5): 2179-2191, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31900562

RESUMO

O-acetylation of alginate produced by the opportunistic human pathogen Pseudomonas aeruginosa significantly contributes to its pathogenesis. Three proteins, AlgI, AlgJ and AlgF have been implicated to form a complex and act together with AlgX for O-acetylation of alginate. AlgI was proposed to transfer the acetyl group across the cytoplasmic membrane, while periplasmic AlgJ was hypothesised to transfer the acetyl group to AlgX that acetylates alginate. To elucidate the proposed O-acetylation multiprotein complex, isogenic knockout mutants of algI, algJ and algF genes were generated in the constitutively alginate overproducing P. aeruginosa PDO300 to enable mutual stability studies. All knockout mutants were O-acetylation negative and complementation with the respective genes in cis or trans restored O-acetylation of alginate. Interestingly, only the AlgF deletion impaired alginate production suggesting a link to the alginate polymerisation/secretion multiprotein complex. Mutual stability experiments indicated that AlgI and AlgF interact independent of AlgJ as well as impact on stability of the alginate polymerisation/secretion multiprotein complex. Deletion of AlgJ did not destabilise AlgX and vice versa. When the alginate polymerase, Alg8, was absent, then AlgI and AlgF stability was strongly impaired supporting a link of the O-acetylation machinery with alginate polymerisation. Pull-down experiments suggested that AlgI interacts with AlgJ, while AlgF interacts with AlgJ and AlgI. Overall, these results suggested that AlgI-AlgJ-AlgF form a multiprotein complex linked via Alg8 to the envelope-spanning alginate polymerisation/secretion multiprotein complex to mediate O-acetylation of nascent alginate. Here, we provide the first insight on how the O-acetylation machinery is associated with alginate production.


Assuntos
Alginatos/metabolismo , Proteínas de Bactérias/metabolismo , Complexos Multiproteicos/metabolismo , Pseudomonas aeruginosa/metabolismo , Acetilação , Alginatos/química , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Técnicas de Inativação de Genes , Teste de Complementação Genética , Complexos Multiproteicos/genética , Periplasma/metabolismo , Polimerização , Ligação Proteica , Estabilidade Proteica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade
19.
Nat Commun ; 11(1): 543, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992714

RESUMO

Health care-associated infections such as Pseudomonas aeruginosa bacteremia pose a major clinical risk for hospitalized patients. However, these systemic infections are presumed to be a "dead-end" for P. aeruginosa and to have no impact on transmission. Here, we use a mouse infection model to show that P. aeruginosa can spread from the bloodstream to the gallbladder, where it replicates to extremely high numbers. Bacteria in the gallbladder can then seed the intestines and feces, leading to transmission to uninfected cage-mate mice. Our work shows that the gallbladder is crucial for spread of P. aeruginosa from the bloodstream to the feces during bacteremia, a process that promotes transmission in this experimental system. Further research is needed to test to what extent these findings are relevant to infections in patients.


Assuntos
Bacteriemia/microbiologia , Bacteriemia/transmissão , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/transmissão , Pseudomonas aeruginosa/patogenicidade , Animais , Bacteriemia/patologia , Modelos Animais de Doenças , Epitélio/microbiologia , Fezes/microbiologia , Feminino , Vesícula Biliar/microbiologia , Vesícula Biliar/patologia , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/patologia , Humanos , Intestinos/microbiologia , Intestinos/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pneumonia/microbiologia , Infecções por Pseudomonas/patologia , Sistemas de Secreção Tipo III
20.
J Vis Exp ; (155)2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31984966

RESUMO

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of these products are naturally produced by the organisms, other products require genetic engineering of the organism to increase the yields of production. Avirulent strains of Escherichia coli have traditionally been the preferred bacterial species for producing biopharmaceuticals; however, some products are difficult for E. coli to produce. Thus, avirulent strains of other bacterial species could provide useful alternatives for production of some commercial products. Pseudomonas aeruginosa is a common and well-studied Gram-negative bacterium that could provide a suitable alternative to E. coli. However, P. aeruginosa is an opportunistic human pathogen. Here, we detail a procedure that can be used to generate nonpathogenic strains of P. aeruginosa through sequential genomic deletions using the pEX100T-NotI plasmid. The main advantage of this method is to produce a marker-free strain. This method may be used to generate highly attenuated P. aeruginosa strains for the production of commercial products, or to design strains for other specific uses. We also describe a simple and reproducible mouse model of bacterial systemic infection via intraperitoneal injection of validated test strains to test the attenuation of the genetically engineered strain in comparison to the FDA-approved BL21 strain of E. coli.


Assuntos
Deleção de Genes , Engenharia Genética/métodos , Infecções por Pseudomonas/genética , Pseudomonas aeruginosa/patogenicidade , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Virulência
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