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1.
Mol Genet Genomics ; 296(2): 299-312, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33386986

RESUMO

Pseudomonas syringae pv. tabaci 6605 (Pta6605) is a causal agent of wildfire disease in host tobacco plants and is highly motile. Pta6605 has multiple clusters of chemotaxis genes including cheA, a gene encoding a histidine kinase, cheY, a gene encoding a response regulator, mcp, a gene for a methyl-accepting chemotaxis protein, as well as flagellar and pili biogenesis genes. However, only two major chemotaxis gene clusters, cluster I and cluster II, possess cheA and cheY. Deletion mutants of cheA or cheY were constructed to evaluate their possible role in Pta6605 chemotaxis and virulence. Motility tests and a chemotaxis assay to known attractant demonstrated that cheA2 and cheY2 mutants were unable to swarm and to perform chemotaxis, whereas cheA1 and cheY1 mutants retained chemotaxis ability almost equal to that of the wild-type (WT) strain. Although WT and cheY1 mutants of Pta6605 caused severe disease symptoms on host tobacco leaves, the cheA2 and cheY2 mutants did not, and symptom development with cheA1 depended on the inoculation method. These results indicate that chemotaxis genes located in cluster II are required for optimal chemotaxis and host plant infection by Pta6605 and that cluster I may partially contribute to these phenotypes.


Assuntos
Histidina Quinase/genética , Proteínas Quimiotáticas Aceptoras de Metil/genética , Pseudomonas aeruginosa/fisiologia , Pseudomonas syringae/fisiologia , Tabaco/microbiologia , Quimiotaxia , Resistência à Doença , Deleção de Genes , Histidina Quinase/metabolismo , Proteínas Quimiotáticas Aceptoras de Metil/metabolismo , Família Multigênica , Filogenia , Doenças das Plantas/microbiologia , Pseudomonas aeruginosa/patogenicidade , Pseudomonas syringae/patogenicidade , Virulência
2.
J Vis Exp ; (166)2020 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-33369598

RESUMO

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural integrity. The nosocomial pathogen, Pseudomonas aeruginosa, is known to adopt a biofilm mode of growth, causing chronic pulmonary infection in patients with cystic fibrosis (CF). The computer program, COMSTAT, is a useful tool for quantifying antimicrobial-induced changes in P. aeruginosa biofilm architecture by extracting data from three-dimensional confocal images. However, standardized operation of the software is less commonly addressed, which is important for optimal reporting of biofilm behavior and cross-center comparison. Thus, the aim of this protocol is to provide a simple and reproducible framework for quantifying in vitro biofilm structures under varying antimicrobial conditions via COMSTAT. The technique is modeled using a CF P. aeruginosa isolate, grown in the form of biofilm replicates, and exposed to tobramycin and the anti-Psl monoclonal antibody, Psl0096. The step-by-step approach aims to reduce user ambiguity and minimize the chance of overlooking crucial image-processing steps. Specifically, the protocol emphasizes the elimination of subjective variations associated with the manual operation of COMSTAT, including image segmentation and the selection of appropriate quantitative analysis functions. Although this method requires users to spend additional time processing confocal images prior to running COMSTAT, it helps minimize misrepresented biofilm heterogenicity in automated outputs.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Software , Biofilmes/crescimento & desenvolvimento , Fluorescência , Humanos , Processamento de Imagem Assistida por Computador , Microscopia Confocal , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/fisiologia
3.
PLoS Pathog ; 16(12): e1009126, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33351859

RESUMO

Embedded in an extracellular matrix, biofilm-residing bacteria are protected from diverse physicochemical insults. In accordance, in the human host the general recalcitrance of biofilm-grown bacteria hinders successful eradication of chronic, biofilm-associated infections. In this study, we demonstrate that upon addition of promethazine, an FDA approved drug, antibiotic tolerance of in vitro biofilm-grown bacteria can be abolished. We show that following the addition of promethazine, diverse antibiotics are capable of efficiently killing biofilm-residing cells at minimal inhibitory concentrations. Synergistic effects could also be observed in a murine in vivo model system. PMZ was shown to increase membrane potential and interfere with bacterial respiration. Of note, antibiotic killing activity was elevated when PMZ was added to cells grown under environmental conditions that induce low intracellular proton levels. Our results imply that biofilm-grown bacteria avoid antibiotic killing and become tolerant by counteracting intracellular alkalization through the adaptation of metabolic and transport functions. Abrogation of antibiotic tolerance by interfering with the cell's bioenergetics promises to pave the way for successful eradication of biofilm-associated infections. Repurposing promethazine as a biofilm-sensitizing drug has the potential to accelerate the introduction of new treatments for recalcitrant, biofilm-associated infections into the clinic.


Assuntos
Biofilmes/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Prometazina/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Animais , Tolerância a Medicamentos/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Pseudomonas
4.
J S Afr Vet Assoc ; 91(0): e1-e6, 2020 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-33054249

RESUMO

Although Pseudomonas aeruginosa (P. aeruginosa) can infect both animals and humans, there is a paucity of veterinary studies on antimicrobial resistance of P. aeruginosa in South Africa. Secondary data of canine clinical cases presented at the hospital from January 2007 to December 2013 was used. The following information was recorded: type of sample, the date of sampling and the antimicrobial susceptibility results. Frequencies, proportions and their 95% confidence intervals were calculated for all the categorical variables. In total, 155 P. aeruginosa isolates were identified and included in this study. All the isolates were resistant to at least one antimicrobial (AMR), while 92% were multi-drug resistant (MDR). Most isolates were resistant to lincomycin (98%), penicillin-G (96%), orbifloxacin (90%), trimethoprim-sulfamethoxazole (90%) and doxycycline (87%). A low proportion of isolates was resistant to imipenem (6%), tobramycin (12%), amikacin (16%) and gentamicin (18%). A high proportion of MDR-P. aeruginosa isolates was resistant to amoxycillin-clavulanic acid (99%), tylosin (99%), chloramphenicol (97%) and doxycycline (96%). Few (6%) of MDR-P. aeruginosa isolates were resistant to imipenem. Pseudomonas aeruginosa was associated with infections of various organ systems in this study. All P. aeruginosa isolates of P. aeruginosa exhibited resistance to ß-lactams, fluoroquinolones and lincosamides. Clinicians at the hospital in question should consider these findings when treating infections associated with P. aeruginosa.


Assuntos
Doenças do Cão/microbiologia , Farmacorresistência Bacteriana Múltipla , Infecções por Pseudomonas/veterinária , Pseudomonas aeruginosa/efeitos dos fármacos , Centros Médicos Acadêmicos , Animais , Antibacterianos/classificação , Antibacterianos/farmacologia , Cães , Hospitais Veterinários , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/fisiologia , África do Sul
5.
Nat Commun ; 11(1): 5395, 2020 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-33106492

RESUMO

Tit-for-tat is a familiar principle from animal behavior: individuals respond in kind to being helped or harmed by others. Remarkably some bacteria appear to display tit-for-tat behavior, but how this evolved is not understood. Here we combine evolutionary game theory with agent-based modelling of bacterial tit-for-tat, whereby cells stab rivals with poisoned needles (the type VI secretion system) after being stabbed themselves. Our modelling shows tit-for-tat retaliation is a surprisingly poor evolutionary strategy, because tit-for-tat cells lack the first-strike advantage of preemptive attackers. However, if cells retaliate strongly and fire back multiple times, we find that reciprocation is highly effective. We test our predictions by competing Pseudomonas aeruginosa (a tit-for-tat species) with Vibrio cholerae (random-firing), revealing that P. aeruginosa does indeed fire multiple times per incoming attack. Our work suggests bacterial competition has led to a particular form of reciprocation, where the principle is that of strong retaliation, or 'tits-for-tat'.


Assuntos
Proteínas de Bactérias/metabolismo , Evolução Biológica , Pseudomonas aeruginosa/fisiologia , Sistemas de Secreção Tipo VI/metabolismo , Vibrio cholerae/fisiologia , Proteínas de Bactérias/genética , Pseudomonas aeruginosa/genética , Sistemas de Secreção Tipo VI/genética , Vibrio cholerae/genética
6.
Proc Natl Acad Sci U S A ; 117(37): 22967-22973, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32868444

RESUMO

Hospital-acquired infections are a global health problem that threatens patients' treatment in intensive care units, causing thousands of deaths and a considerable increase in hospitalization costs. The endotracheal tube (ETT) is a medical device placed in the patient's trachea to assist breathing and delivering oxygen into the lungs. However, bacterial biofilms forming at the surface of the ETT and the development of multidrug-resistant bacteria are considered the primary causes of ventilator-associated pneumonia (VAP), a severe hospital-acquired infection for significant mortality. Under these circumstances, there has been a need to administrate antibiotics together. Although necessary, it has led to a rapid increase in bacterial resistance to antibiotics. Therefore, it becomes necessary to develop alternatives to prevent and combat these bacterial infections. One possibility is to turn the ETT itself into a bactericide. Some examples reported in the literature present drawbacks. To overcome those issues, we have designed a photosensitizer-containing ETT to be used in photodynamic inactivation (PDI) to avoid bacteria biofilm formation and prevent VAP occurrence during tracheal intubation. This work describes ETT's functionalization with curcumin photosensitizer, as well as its evaluation in PDI against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli A significant photoinactivation (up to 95%) against Gram-negative and Gram-positive bacteria was observed when curcumin-functionalized endotracheal (ETT-curc) was used. These remarkable results demonstrate this strategy's potential to combat hospital-acquired infections and contribute to fighting antimicrobial resistance.


Assuntos
Antibacterianos/farmacologia , Curcumina/farmacologia , Intubação Intratraqueal/instrumentação , Pneumonia Associada à Ventilação Mecânica/prevenção & controle , Antibacterianos/química , Biofilmes/efeitos dos fármacos , Curcumina/química , Humanos , Intubação Intratraqueal/efeitos adversos , Pneumonia Associada à Ventilação Mecânica/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologia
7.
PLoS One ; 15(9): e0237851, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32877414

RESUMO

This study examined the antibacterial effect of protoporphyrin IX-ethylenediamine derivative (PPIX-ED)-mediated photodynamic antimicrobial chemotherapy (PPIX-ED-PACT) against Pseudomonas aeruginosa in vitro and in vivo. PPIX-ED potently inhibited the growth of Pseudomonas aeruginosa by inducing reactive oxygen species production via photoactivation. Atomic force microscopy revealed that PPIX-ED-PACT induced the leakage of bacterial content by degrading the bacterial membrane and wall. As revealed using acridine orange/ethidium bromide staining, PPIX-ED-PACT altered the permeability of the bacterial membrane. In addition, the antibacterial effect of PPIX-ED-PACT was demonstrated in an in vivo model of P. aeruginosa-infected wounds. PPIX-ED (100 µM) decreased the number of P. aeruginosa colony-forming units by 4.2 log10. Moreover, histological analysis illustrated that the wound healing rate was 98% on day 14 after treatment, which was 10% higher than that in the control group. According to the present findings, PPIX-ED-PACT can effectively inhibit the growth of P. aeruginosa in vitro and in vivo.


Assuntos
Antibacterianos/uso terapêutico , Fotoquimioterapia , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/fisiologia , Infecção dos Ferimentos/tratamento farmacológico , Infecção dos Ferimentos/microbiologia , Animais , Antibacterianos/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/efeitos da radiação , Etilenodiaminas/química , Etilenodiaminas/farmacologia , Etilenodiaminas/uso terapêutico , Feminino , Luz , Camundongos , Camundongos Endogâmicos BALB C , Viabilidade Microbiana/efeitos dos fármacos , Modelos Biológicos , Células NIH 3T3 , Fotodegradação , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Protoporfirinas/química , Protoporfirinas/farmacologia , Protoporfirinas/uso terapêutico , Pseudomonas aeruginosa/efeitos da radiação , Cicatrização/efeitos dos fármacos
8.
Med Microbiol Immunol ; 209(6): 669-680, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32880037

RESUMO

The ability of bacteria to aggregate and form biofilms impairs phagocytosis by polymorphonuclear leukocytes (PMNs). The aim of this study was to examine if the size of aggregates is critical for successful phagocytosis and how bacterial biofilms evade phagocytosis. We investigated the live interaction between PMNs and Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Staphylococcus epidermidis using confocal scanning laser microscopy. Aggregate size significantly affected phagocytosis outcome and larger aggregates were less likely to be phagocytized. Aggregates of S. epidermidis were also less likely to be phagocytized than equally-sized aggregates of the other three species. We found that only aggregates of approx. 5 µm diameter or smaller were consistently phagocytosed. We demonstrate that planktonic and aggregated cells of all four species significantly reduced the viability of PMNs after 4 h of incubation. Our results indicate that larger bacterial aggregates are less likely to be phagocytosed by PMNs and we propose that, if the aggregates become too large, circulating PMNs may not be able to phagocytose them quickly enough, which may lead to chronic infection.


Assuntos
Biofilmes , Escherichia coli/fisiologia , Neutrófilos/fisiologia , Fagocitose , Pseudomonas aeruginosa/fisiologia , Staphylococcus aureus/fisiologia , Staphylococcus epidermidis/fisiologia , Escherichia coli/ultraestrutura , Humanos , Pseudomonas aeruginosa/ultraestrutura , Pele/microbiologia , Staphylococcus aureus/ultraestrutura , Staphylococcus epidermidis/ultraestrutura
9.
APMIS ; 128(12): 647-653, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32794206

RESUMO

IL-2 is a pro-inflammatory and a Th1 inducing cytokine, which is important for activation of the cell-mediated immunity. IL-2 in serum and sputum has been observed to be reduced in cystic fibrosis (CF) patients. The present IL-2 treatment study of Pseudomonas aeruginosa (PA) lung infected mice was performed in order to evaluate the effect of IL-2 supplement. One hundred-and-twenty female BALB/c mice were divided into three groups: 1) IL-2 treatment/infection (TIG), 2) non-treatment/infection (NTIG), and 3) IL-2 treatment/non-infection (TNIG). The mice were challenged intra-tracheally with PA (PAO579) embedded in seaweed alginate to resemble the biofilm mode of growth. At day 0 and 1, the treatment groups received IL-2 s.c. Mice were killed on day 1 or 2, and cytokine production, lung pathology, and quantitative lung bacteriology were estimated. IL-2 treatment of infected mice reduced the number of mice with signs of macroscopic lung pathology at day 2 (p < 0.05). The reduced macroscopic pathology was paralleled by a reduced IL-1ß and TNF-α. In contrast, an increased PMN response at day 2 was observed in the IL-2 treated mice (p < 0.01). This was preceded by a significantly higher degree of microscopic inflammation at day 1 (p < 0.02). The IL-12 levels decreased in both groups of infected mice at day 2 (p < 0.01), however, significantly more in the IL-2 treated mice (p < 0.02). In accordance, but surprisingly, IFN-γ was significantly reduced in the IL-2 treated PA infected group at day 2 (p < 0.05). The present results indicate that early IL-2 treatment prolongs the PMN response but also reduces pro-inflammatory IL-1ß and TNF-α and macroscopic signs of pathology.


Assuntos
Interleucina-2/administração & dosagem , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/fisiologia , Animais , Fibrose Cística/tratamento farmacológico , Fibrose Cística/imunologia , Fibrose Cística/microbiologia , Fibrose Cística/patologia , Citocinas/genética , Citocinas/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/genética
10.
PLoS Genet ; 16(8): e1008783, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32813693

RESUMO

Pseudomonas aeruginosa and Candida albicans are opportunistic pathogens whose interactions involve the secreted products ethanol and phenazines. Here, we describe the role of ethanol in mixed-species co-cultures by dual-seq analyses. P. aeruginosa and C. albicans transcriptomes were assessed after growth in mono-culture or co-culture with either ethanol-producing C. albicans or a C. albicans mutant lacking the primary ethanol dehydrogenase, Adh1. Analysis of the RNA-Seq data using KEGG pathway enrichment and eADAGE methods revealed several P. aeruginosa responses to C. albicans-produced ethanol including the induction of a non-canonical low-phosphate response regulated by PhoB. C. albicans wild type, but not C. albicans adh1Δ/Δ, induces P. aeruginosa production of 5-methyl-phenazine-1-carboxylic acid (5-MPCA), which forms a red derivative within fungal cells and exhibits antifungal activity. Here, we show that C. albicans adh1Δ/Δ no longer activates P. aeruginosa PhoB and PhoB-regulated phosphatase activity, that exogenous ethanol complements this defect, and that ethanol is sufficient to activate PhoB in single-species P. aeruginosa cultures at permissive phosphate levels. The intersection of ethanol and phosphate in co-culture is inversely reflected in C. albicans; C. albicans adh1Δ/Δ had increased expression of genes regulated by Pho4, the C. albicans transcription factor that responds to low phosphate, and Pho4-dependent phosphatase activity. Together, these results show that C. albicans-produced ethanol stimulates P. aeruginosa PhoB activity and 5-MPCA-mediated antagonism, and that both responses are dependent on local phosphate concentrations. Further, our data suggest that phosphate scavenging by one species improves phosphate access for the other, thus highlighting the complex dynamics at play in microbial communities.


Assuntos
Antibiose , Candida albicans/fisiologia , Etanol/metabolismo , Fosfatos/metabolismo , Pseudomonas aeruginosa/fisiologia , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Pseudomonas aeruginosa/metabolismo , Transdução de Sinais , Transcriptoma
11.
PLoS One ; 15(7): e0236599, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32722685

RESUMO

The increasing prevalence of carbon nanotubes (CNTs) as components of new functional materials has the unintended consequence of causing increases in CNT concentrations in aqueous environments. Aqueous systems are reservoirs for bacteria, including human and animal pathogens, that can form biofilms. At high concentrations, CNTs have been shown to display biocidal effects; however, at low concentrations, the interaction between CNTs and bacteria is more complicated, and antimicrobial action is highly dependent upon the properties of the CNTs in suspension. Here, impact of low concentrations of multiwalled CNTs (MWCNTs) on the biofilm-forming opportunistic human pathogen Pseudomonas aeruginosa is studied. Using phase contrast and confocal microscopy, flow cytometry, and antibiotic tolerance assays, it is found that sub-lethal concentrations (2 mg/L) of MWCNTs promote aggregation of P. aeruginosa into multicellular clusters. However, the antibiotic tolerance of these "young" bacterial-CNT aggregates is similar to that of CNT-free cultures. Overall, our results indicate that the co-occurrence of MWCNTs and P. aeruginosa in aqueous systems, which promotes the increased number and size of bacterial aggregates, could increase the dose to which humans or animals are exposed.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Nanotubos de Carbono/química , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Suspensões
12.
Huan Jing Ke Xue ; 41(2): 831-838, 2020 Feb 08.
Artigo em Chinês | MEDLINE | ID: mdl-32608744

RESUMO

Because of the massive discharge of nitrogenous wastewater, the eutrophication of a water body is becoming increasingly serious, and how to effectively remove nitrogen from this wastewater remains an urgent problem to be solved. In this study, due to disadvantages in the traditional biological nitrogen removal process, such as complex and long procedures, high energy consumption, weak impact resistance, and N2O release, the nitrogen removal theory by heterotrophic nitrification was further analyzed by discussing the physiological-biochemical, heterotrophic nitrification-aerobic denitrification, and N2O production characteristics of a high-efficiency heterotrophic nitrifying bacteria Pseudomonas aeruginosa YL. Results show that the strain YL had an eminent heterotrophic nitrification and aerobic denitrification ability, and NH4+-N, NO2--N, and NO3--N with concentration of 100 mg·L-1 could be completely removed during the 24-hour incubation period. There was almost no intermediate product in the process of heterotrophic nitrification, however when NO3--N was used as nitrogen source, the accumulation of NO2--N reached 25.55 mg·L-1. Meanwhile, the successful expression of denitrification genes napA, nirK, and nosZ further confirmed the aerobic denitrification ability of strain YL. Gaseous nitrogen products accounted for about 30%-40% of removed TN in the heterotrophic nitrification-aerobic denitrification process by strain YL, and N2 was the main denitrification product. When NH4+-N, NO2--N, and NO3--N were used as the sole nitrogen source, N2 production amounted to 3.46, 3.49, and 3.36 mg, respectively. In contrast, only small amounts of N2O were produced in the denitrification process by strain YL, and the total amount was 6.63 µg when NH4+-N was the nitrogen source, which was much lower than in the cases of NO2--N and NO3--N as the sole nitrogen source. In addition, high C/N, low pH, high temperature, high NH4+-N, and high NO2--N conditions could result in more N2O generation. Nevertheless, most environmental factors had little effect on N2O production of strain YL, and the maximum N2O production was significantly lower than that of short-cut nitrification system and autotrophic nitrification system. These results demonstrated that strain YL exhibited excellent abilities of nitrogen removal, N2O emission control, and tolerance to environmental conditions, and could be an effective candidate for treating wastewater without secondary air pollution.


Assuntos
Desnitrificação , Processos Heterotróficos , Nitritos/metabolismo , Pseudomonas aeruginosa/fisiologia , Aerobiose , Genes Bacterianos , Nitrificação , Nitrogênio/metabolismo
13.
14.
J Vis Exp ; (159)2020 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-32510504

RESUMO

Swarming is a form of surface motility observed in many bacterial species including Pseudomonas aeruginosa and Escherichia coli. Here, dense populations of bacteria move over large distances in characteristic tendril-shaped communities over the course of hours. Swarming is sensitive to several factors including medium moisture, humidity, and nutrient content. In addition, the collective stress response, which is observed in P. aeruginosa that are stressed by antibiotics or bacteriophage (phage), repels swarms from approaching the area containing the stress. The methods described here address how to control the critical factors that affect swarming. We introduce a simple method to monitor swarming dynamics and the collective stress response with high temporal resolution using a flatbed document scanner, and describe how to compile and perform a quantitative analysis of swarms. This simple and cost-effective method provides precise and well-controlled quantification of swarming and may be extended to other types of plate-based growth assays and bacterial species.


Assuntos
Pseudomonas aeruginosa/fisiologia , Estresse Fisiológico , Imagem com Lapso de Tempo , Antibacterianos/farmacologia , Bacteriófagos/fisiologia , Análise Custo-Benefício , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/virologia , Imagem com Lapso de Tempo/economia
15.
Arch Microbiol ; 202(8): 2181-2188, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32519021

RESUMO

Bacterial quorum sensing (QS) system regulates the production of most costly but sharable extracellular products (public goods) in a growth-phase-dependent manner, and the development of this energy-intensive process is susceptible to environmental changes. However, the role of nutrient factors in dominating the QS-mediated cooperative interaction and intracellular metabolism still remains less understood. Here we studied the performance of QS system by growing Pseudomonas aeruginosa under different nutrient and culture conditions. The results of comparative-transcriptomic analyses revealed that carbon source-limitation was the main factor suppressing the activation of QS system, and a substantial number of public-good-encoding genes were induced when phosphorus is limiting in short-term culture. By contrast, although the QS regulation of P. aeruginosa in all the cultures was generally decreased along with the enrichment of QS-deficient individuals during evolution, limitation of different nutrient factors had discrepant effects in directing the formation of population structure by coordinating the production of public goods and primary metabolism, especially the starch and sucrose metabolism. These findings demonstrate the pleiotropy of QS regulation in balancing the development of cooperative behavior and metabolism, and provide a reference for further understanding the role of QS system in causing persistent infections.


Assuntos
Pseudomonas aeruginosa/fisiologia , Percepção de Quorum/fisiologia , Meios de Cultura/farmacologia , Nutrientes/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos
16.
J Vet Sci ; 21(3): e46, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32476320

RESUMO

BACKGROUND: High concentrations of particulate matter less than 2.5 µm in diameter (PM2.5) in poultry houses is an important cause of respiratory disease in animals and humans. Pseudomonas aeruginosa is an opportunistic pathogen that can induce severe respiratory disease in animals under stress or with abnormal immune functions. When excessively high concentrations of PM2.5 in poultry houses damage the respiratory system and impair host immunity, secondary infections with P. aeruginosa can occur and produce a more intense inflammatory response, resulting in more severe lung injury. OBJECTIVES: In this study, we focused on the synergistic induction of inflammatory injury in the respiratory system and the related molecular mechanisms induced by PM2.5 and P. aeruginosa in poultry houses. METHODS: High-throughput 16S rDNA sequence analysis was used for characterizing the bacterial diversity and relative abundance of the PM2.5 samples, and the effects of PM2.5 and P. aeruginosa stimulation on inflammation were detected by in vitro and in vivo. RESULTS: Sequencing results indicated that the PM2.5 in poultry houses contained a high abundance of potentially pathogenic genera, such as Pseudomonas (2.94%). The lung tissues of mice had more significant pathological damage when co-stimulated by PM2.5 and P. aeruginosa, and it can increase the expression levels of interleukin (IL)-6, IL-8, and tumor necrosis factor-α through nuclear factor (NF)-κB pathway in vivo and in vitro. CONCLUSIONS: The results confirmed that poultry house PM2.5 in combination with P. aeruginosa could aggravate the inflammatory response and cause more severe respiratory system injuries through a process closely related to the activation of the NF-κB pathway.


Assuntos
Material Particulado/efeitos adversos , Pneumonia/etiologia , Infecções por Pseudomonas/fisiopatologia , Pseudomonas aeruginosa/fisiologia , Animais , Peso Corporal , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Subunidade p50 de NF-kappa B/metabolismo , Material Particulado/classificação , Pneumonia/induzido quimicamente , Pneumonia/microbiologia , Organismos Livres de Patógenos Específicos , Fator de Necrose Tumoral alfa/metabolismo
17.
J Vis Exp ; (159)2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32478753

RESUMO

Antimicrobial resistance, a major consequence of diagnostic uncertainty and antimicrobial overprescription, is an increasingly recognized cause of severe infections, complications, and mortality worldwide with a huge impact on our society and on the health system. In particular, patients with compromised immune systems or pre-existing and chronic pathologies, such as cystic fibrosis (CF), are subjected to frequent antibiotic treatments to control the infections with the appearance and diffusion of multidrug resistant isolates. Therefore, there is an urgent need to address alternative therapies to counteract bacterial infections. Use of bacteriophages, the natural enemies of bacteria, can be a possible solution. The protocol detailed in this work describes the application of phage therapy against Pseudomonas aeruginosa infection in CF zebrafish embryos. Zebrafish embryos were infected with P. aeruginosa to demonstrate that phage therapy is effective against P. aeruginosa infections as it reduces lethality, bacterial burden and pro-inflammatory immune response in CF embryos.


Assuntos
Fibrose Cística/microbiologia , Fibrose Cística/terapia , Embrião não Mamífero/microbiologia , Terapia por Fagos , Infecções por Pseudomonas/terapia , Pseudomonas aeruginosa/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/microbiologia , Animais , Antibacterianos/farmacologia , Bacteriófagos/fisiologia , Citocinas/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Mediadores da Inflamação/metabolismo , Microinjeções , Morfolinos/farmacologia , Terapia por Fagos/efeitos adversos , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Reprodutibilidade dos Testes
18.
J Vis Exp ; (160)2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32597860

RESUMO

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.


Assuntos
Ar , Anti-Infecciosos/farmacologia , Brônquios/patologia , Técnicas de Cocultura , Células Epiteliais/microbiologia , Macrófagos/microbiologia , Pseudomonas aeruginosa/fisiologia , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Contagem de Colônia Microbiana , Citocinas/metabolismo , Impedância Elétrica , Células Epiteliais/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Cinética , L-Lactato Desidrogenase/metabolismo , Macrófagos/efeitos dos fármacos , Permeabilidade , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Células THP-1 , Tobramicina/farmacologia
19.
PLoS One ; 15(6): e0235059, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32574199

RESUMO

BACKGROUND: To support effective antibiotic selection in empirical treatments, infection control interventions, and antimicrobial resistance containment strategies, many medical institutions collect antimicrobial susceptibility test data conducted at their facilities to prepare cumulative antibiograms. AIM: To evaluate how the setpoints of duplicate isolate removal period and data collection period affect the calculated susceptibility rates in antibiograms. METHODS: The Sakai City Medical Center is a regional core hospital for tertiary emergency medical care with 480 beds for general clinical care. In this study, all the Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae isolates collected at the Sakai City Medical Center Clinical Laboratory between July 2013 and December 2018 were subjected to antimicrobial susceptibility tests and the resulting data was analyzed. FINDINGS: The longer the duplicate isolate removal period, the fewer the isolates are available for every bacterial species. Differences in the length of the duplicate isolate removal period affected P. aeruginosa susceptibility rates to ß-lactam antibiotics by up to 10.8%. The setpoint of the data collection period affected the antimicrobial susceptibility rates by up to 7.3%. We found that a significant change in susceptibility could be missed depending on the setting of the data collection period, in preparing antibiogram of ß-lactam antibiotics for P. aeruginosa. CONCLUSIONS: When referring to antibiograms, medical professionals involved in infectious disease treatment should be aware that the parameter values, such as the duplicate isolate removal period and the data collection period, affect P. aeruginosa susceptibility rates especially to ß-lactam antibiotics. And antibiogram should be updated within the shortest time period that is practically possible, taking into account restrictions such as numbers of specimen.


Assuntos
Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Testes de Sensibilidade Microbiana/normas , Pseudomonas aeruginosa/efeitos dos fármacos , Algoritmos , Serviço Hospitalar de Emergência , Escherichia coli/isolamento & purificação , Escherichia coli/fisiologia , Hospitalização/estatística & dados numéricos , Humanos , Klebsiella pneumoniae/isolamento & purificação , Klebsiella pneumoniae/fisiologia , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/fisiologia , Centros de Atenção Terciária , Fatores de Tempo
20.
PLoS One ; 15(6): e0235093, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32584878

RESUMO

Bacterial biofilms are one of the major issues in the treatment of chronic infections such as chronic wounds, where biofilms are typically polymicrobial. The synergy between species can occur during most polymicrobial infections, where antimicrobial resistance enhances as a result. Furthermore, self-produced extracellular polymeric substance (EPS) in biofilms results in a high tolerance to antibiotics that complicates wound healing. Since most antibiotics fail to remove biofilms in chronic infections, new therapeutic modalities may be required. Disruption of EPS is one of the effective approaches for biofilm eradication. Therefore, degradation of EPS using enzymes may result in improved chronic wounds healing. In the current study, we investigated the efficacy of trypsin, ß-glucosidase, and DNase I enzymes on the degradation of dual-species biofilms of Pseudomonas aeruginosa and Staphylococcus aureus in a wound-like medium. These species are the two most common bacteria associated with biofilm formation in chronic wounds. Moreover, the reduction of minimum biofilm eradication concentration (MBEC) of meropenem and amikacin was evaluated when combined with enzymes. The minimum effective concentrations of trypsin, ß-glucosidase, and DNase I enzymes to degrade biofilms were 1 µg/ml, 8 U/ml, and 150 U/ml, respectively. Combination of 0.15 µg/ml trypsin and 50 U/ml DNase I had a significant effect on S. aureus-P. aeruginosa biofilms which resulted in the dispersal and dissolution of all biofilms. In the presence of the enzymatic mixture, MBECs of antibiotics showed a significant decrease (p < 0.05), at least 2.5 fold. We found that trypsin/DNase I mixture can be used as an anti-biofilm agent against dual-species biofilms of S. aureus-P. aeruginosa.


Assuntos
Antibacterianos/farmacologia , Biofilmes , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/fisiologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/fisiologia , Infecção dos Ferimentos/tratamento farmacológico , Ferimentos e Lesões/microbiologia , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Quimioterapia Combinada , Humanos , Infecção dos Ferimentos/microbiologia
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