RESUMO
Background: The presence of resistant and potentially virulent bacterial strains in a veterinary hospital environment is a neglected problem. Pseudomonas aeruginosa is an opportunistic microorganism present and circulating in the veterinary hospital environment, of clinical importance and zooanthroponotic transmission of P. aeruginosa has also been reported. The aim of this study was to characterize the population of P. aeruginosa present in a veterinary hospital environment by evaluating their resistance profile and biofilm production. Materials, Methods & Results: A total of 306 samples were collected from the veterinary hospital environment (swabs from consultation tables, surgical tables, door handles, hospitalization cages, stethoscopes, thermometers, and muzzles). The isolates were biochemically identified as belonging to the species Pseudomonas aeruginosa through nitrate to nitrite reduction, motility and oxidase test, growth at 42°C, pigment production, and alkalinization of acetamide. Antimicrobial resistance was tested using the minimum inhibitory concentration (MIC) test. Twenty seven isolates of P. aeruginosa were obtained, with a frequency of 8.8%. The detection of beta-lactamase production and biofilm formation genes by polymerase chain reaction (PCR). Two multidrug resistant (MDR) and 3 single-drug resistant (SDR) strains of P. aeruginosa were identified. Furthermore, it was observed that the strains carried genes related to beta-lactamase production (TEM and CTX-M group 25) and biofilm production (pelA, pslA, ppyR). Discussion: Pseudomonas aeruginosa is considered a major cause of opportunistic hospital infections, as it causes significant morbidity and mortality in immunosuppressed individuals, both in...(AU)
Assuntos
Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/isolamento & purificação , Farmacorresistência Bacteriana , beta-Lactamas , Biofilmes , Hospitais Veterinários , BrasilRESUMO
Background: The presence of resistant and potentially virulent bacterial strains in a veterinary hospital environment is a neglected problem. Pseudomonas aeruginosa is an opportunistic microorganism present and circulating in the veterinary hospital environment, of clinical importance and zooanthroponotic transmission of P. aeruginosa has also been reported. The aim of this study was to characterize the population of P. aeruginosa present in a veterinary hospital environment by evaluating their resistance profile and biofilm production. Materials, Methods & Results: A total of 306 samples were collected from the veterinary hospital environment (swabs from consultation tables, surgical tables, door handles, hospitalization cages, stethoscopes, thermometers, and muzzles). The isolates were biochemically identified as belonging to the species Pseudomonas aeruginosa through nitrate to nitrite reduction, motility and oxidase test, growth at 42°C, pigment production, and alkalinization of acetamide. Antimicrobial resistance was tested using the minimum inhibitory concentration (MIC) test. Twenty seven isolates of P. aeruginosa were obtained, with a frequency of 8.8%. The detection of beta-lactamase production and biofilm formation genes by polymerase chain reaction (PCR). Two multidrug resistant (MDR) and 3 single-drug resistant (SDR) strains of P. aeruginosa were identified. Furthermore, it was observed that the strains carried genes related to beta-lactamase production (TEM and CTX-M group 25) and biofilm production (pelA, pslA, ppyR). Discussion: Pseudomonas aeruginosa is considered a major cause of opportunistic hospital infections, as it causes significant morbidity and mortality in immunosuppressed individuals, both in...
Assuntos
Farmacorresistência Bacteriana , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/isolamento & purificação , Biofilmes , Brasil , Hospitais Veterinários , beta-LactamasRESUMO
Neutrophil extracellular traps (NETs) are networks of decondensed chromatin loaded with antimicrobial peptides and enzymes produced against microorganisms or biochemical stimuli. Since their discovery, numerous studies made separately have revealed multiple triggers that induce similar NET morphologies allowing to classify them as lytic or non-lytic. However, the variability in NET composition depending on the inducer agent and the local milieu under similar conditions has been scarcely studied. In this work, a comparative study was conducted to evaluate structural and enzymatic divergences in NET composition induced by biochemical (phorbol myristate acetate [PMA] and hypochlorous acid [HOCl]) and microbiologic (Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa) stimuli, along with the presence of plasma from healthy donors or patients with systemic lupus erythematosus (SLE). The results showed a differential composition of DNA and the antimicrobial peptide cathelicidin (LL37) and a variable enzymatic activity (neutrophil elastase, cathepsin G, myeloperoxidase) induced by the different stimuli despite showing morphologically similar NETs. Additionally, SLE plasma´s presence increased DNA and LL37 release during NET induction independently of the trigger stimulus but with no enzymatic activity differences. This work provides new evidence about NET composition variability depending on the inducer stimulus and the local milieu.
Assuntos
Armadilhas Extracelulares/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Neutrófilos/metabolismo , Biomarcadores/análise , Biomarcadores/metabolismo , Candida albicans/imunologia , Estudos de Casos e Controles , Catelicidinas/análise , Catelicidinas/metabolismo , Catepsina G/análise , Catepsina G/metabolismo , Células Cultivadas , Armadilhas Extracelulares/imunologia , Voluntários Saudáveis , Humanos , Ácido Hipocloroso/imunologia , Elastase de Leucócito/análise , Elastase de Leucócito/metabolismo , Lúpus Eritematoso Sistêmico/sangue , Neutrófilos/imunologia , Peroxidase/análise , Peroxidase/metabolismo , Cultura Primária de Células , Pseudomonas aeruginosa/imunologia , Staphylococcus aureus/imunologia , Acetato de Tetradecanoilforbol/imunologiaRESUMO
Pseudomonas aeruginosa is considered an opportunistic pathogen of great clinical importance. The clearance of this bacterium occurs through recognition of the pathogen by innate immune system receptors, leading to a lung inflammatory response. However, this response must be controlled via immunoregulatory pathways. In this study, we evaluate the role of endogenous murine IL-10 after acute infection with the virulent strain P. aeruginosa PA14. To assess the role of IL-10, intratracheal infection with the PA14 strain was performed in C57BL/6 or IL-10 KO mice. The PA14 strain was recovered in both types of animals, although IL-10 KO mice presented a higher number of viable bacteria in the lung when compared to the C57BL/6 group. Histopathological and stereological analyses showed that IL-10 KO mice had higher tissue damage and inflammatory infiltrate when compared to control animals. The activity of MMP-9 but not MMP-2, as well as IL-6 and TNF-α expression, were augmented in the lungs of infected animals and was much more evident in IL-10 KO animals when compared to the other analyzed groups. This work indicates that endogenous IL-10 control P. aeruginosa infection, the expression of pro-inflammatory genes, MMP-9 activity and histopathological processes of the infectious process in question.
Assuntos
Interleucina-10/imunologia , Pulmão , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Animais , Imunidade , Pulmão/imunologia , Pulmão/patologia , Metaloproteinase 9 da Matriz/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Pseudomonas aeruginosa, bactéria ubíqua e versátil, pode se comportar como um patógeno oportunista, com ampla capacidade adaptativa, por múltiplos fatores de virulência e resistência. Como agente patogênico nas infecções pulmonares em pacientes com fibrose cística (FC), é motivo de prognóstico ruim, aumento de hospitalizações e altas taxas de morbimortalidade, sendo quase impossível a sua erradicação, ao evoluírem para a cronicidade. Globalmente, é notável o aumento nos índices de amostras não sensíveis aos carbapenêmicos e a múltiplos antimicrobianos, essenciais à terapêutica. Assim, avaliamos temporalmente a susceptibilidade aos antimicrobianos e a presença de amostras hipermutáveis (HPM) em P. aeruginosa de diferentes morfotipos, não sensíveis aos carbapenêmicos (PANSC), obtidas de pacientes FC com infecção pulmonar crônica, acompanhados em dois centros de referência no Rio de Janeiro. De 2007 a 2016, a análise retrospectiva, através dos resultados obtidos no teste de disco-difusão (TDD), permitiu selecionar amostras de PANSC incluídas neste trabalho. Usando os resultados obtidos no TDD, foi definida a susceptibilidade a outros antimicrobianos, bem como os fenótipos de resistência, multi-(MDR), extensivo-(XDR) e pandroga resistentes (PDR). Adicionalmente, determinou-se a concentração inibitória mínima (CIM) para imipenem (IPM), meropenem (MEM), doripenem (DOR) e polimixina (POL). Através de teste fenotípico, foi calculada a frequência de mutação espontânea e as amostras hipermutáveis foram caracterizadas. O sequenciamento de genoma total (SGT) foi realizado em seis amostras de diferentes morfotipos, incluindo uma variante fenotípica rara, a small colony variant (SCV). Essas amostras foram recuperadas em dois episódios de exacerbação do paciente. Foram investigadas a clonalidade, resistência a antimicrobianos e virulência. Das 143 amostras, de 18 pacientes (9 pediátricos e 9 adultos), os resultados do TDD apontaram taxas de não susceptibilidade superiores a 44% para gentamicina, amicacina, tobramicina e ciprofloxacina, e maiores de 30 % para POL. Pela determinação da CIM, quase a totalidade (96%) das amostras foram não sensíveis a IMP, seguidos de 56% para MEM e 44% para DOR. Analisando-se a distribuição dos valores da CIM50 e CIM90 nos dois grupos de pacientes, os valores para IMP foram maiores entre as amostras dos pacientes pediátricos, equivalendo a 32 µg/mL e 64 µg/mL, respectivamente. Cerca de 25%, 37% e 6% eram MDR, XDR e PDR, respectivamente. Aproximadamente 12% eram HPM, e mais da metade destas foram XDR. Após o SGT, as seis amostras, recuperadas do caso clínico foram classificadas em um novo sequence type (ST2744), com a presença de genes de resistência adquiridos blaPAO, blaOXA-50, aph(3')-Iib, fosA, catB7 e crpP, apresentando mutações em genes codificadores de porinas e bombas de efluxo. Entretanto, não foram observados marcadores genéticos clássicos exclusivos para os fenótipos SCV e HPM. Este é o primeiro relato de P. aeruginosa SCV na FC, no Brasil. A vigilância epidemiológica de P. aeruginosa é crucial para a conduta terapêutica, bem como para o sucesso da resposta do paciente e erradicação da infecção pulmonar, justificando o uso de técnicas fenotípicas e moleculares na detecção dos mecanismos de resistência e virulência desse microrganismo na FC.
Pseudomonas aeruginosa, a ubiquitous and versatile bacterium, can behave as an opportunistic pathogen, with strong adaptive capacity, due to multiple virulence and resistance factors. As a pulmonary infection pathogen in patients with cystic fibrosis (CF), it is related with poor prognosis, increased hospitalizations and high rates of morbidity and mortality, and the eradication is almost impossible, especially after chronicity. The increase rates of isolates non-susceptible to carbapenem and multiple antimicrobials, essentials to therapy, have been observed worldwide. Therefore, we assessed the antimicrobial susceptibility and the presence of hypermutability (HPM) in non-susceptible to carbapenem P. aeruginosa (PANSC) isolates from different morphotypes, obtained from CF patients with chronic pulmonary infection, followed at two reference centers in Rio de Janeiro. Using the results obtained by disk-diffusion test (DDT) between 2007 to 2016, we select 143 PANSC and susceptibility to other antimicrobials was defined, as well as the resistance phenotypes, multi- (MDR), extensive- (XDR) and pandrug resistant (PDR). Additionally, the minimum inhibitory concentration (MIC) for imipenem (IPM), meropenem (MEM), doripenem (DOR) and polymyxin (POL) was determined. Hypermutable isolates were characterized by determination of mutation frequency. Whole genome sequencing (WGS) was performed in six morphotypes isolates, including the small colony variant (SCV), a rare variant phenotype. These isolates were recovered in two exacerbation episodes. Clonality, antimicrobial resistance and virulence were investigated. Of the total (143 isolates) isolated from 18 patients (9 pediatric and 9 adults), non-susceptibility rates above than 44% for gentamicin, amikacin, tobramycin and ciprofloxacin, and more than 30% for POL were observed. Almost all (96%) of the isolates were non-susceptible to IPM by MIC determination, followed by 56% for MEM and 44% for DOR. MIC50 (32 µg/mL) and MIC90 (64 µg/mL) rates for IPM were higher among pediatric patient isolates and 25%, 37% and 6% were MDR, XDR and PDR, respectively. 12% of all isolates were classified as HPM and more than half were categorized as XDR. Using WGS, the six isolates recovered from the clinical case, were identified as a new sequence type (ST2744). Acquired resistance genes blaPAO, blaOXA-50, aph (3')-Iib, fosA, catB7 and crpP and mutations in encoding genes for porins and efflux pumps, was annotated. None exclusive classic genetic markers related to SCV and HPM phenotypes were not observed. This is the first Brazilian report of P. aeruginosa SCV in CF. Our results highlight the importance of epidemiological surveillance in P. aeruginosa. The application of phenotypic and molecular techniques to investigate resistance and virulence mechanisms, can contribute to therapeutic success in CF.
Assuntos
Humanos , Masculino , Feminino , Criança , Adolescente , Adulto , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/imunologia , Carbapenêmicos/uso terapêutico , Farmacorresistência Bacteriana/efeitos dos fármacos , Infecções por Pseudomonas/fisiopatologia , Tobramicina/farmacologia , Amicacina/farmacologia , Gentamicinas/farmacologia , Ciprofloxacina/farmacologia , Imipenem/farmacologia , Polimixinas/farmacologia , Fibrose Cística , Doripenem/farmacologia , Meropeném/farmacologia , Pulmão/fisiopatologiaRESUMO
Pseudomonas aeruginosa (PA) is an opportunistic human pathogen responsible for causing serious infections in patients with cystic fibrosis. Infections caused by PA are difficult to treat and eradicate due to intrinsic and added resistance to antibiotic therapy. Therefore, it is necessary to establish effective prevention strategies against this infectious agent. In this study, a combination of immunoinformatic tools was applied to predict immunogenic and immunodominant regions in the structure of exotoxins commonly secreted as virulence factors in PA infection (ExoA, ExoS, ExoT, ExoU and ExoY). The peptides derived from exotoxins were evaluated for the potential affinity for human leukocyte antigen (HLA) I and HLA-II molecules, antigenicity score and toxicity profile. From an initial screening of 941 peptides, 13 (1.38%) were successful in all analyzes. The peptides with relevant immunogenic properties were mainly those derived from Exo A (10 / 76.9%). All peptides selected in the last analysis present a high population coverage rate based on the interaction of HLA alleles (95.36 ± 7.83%). Therefore, the peptides characterized in this study are recommended for in vitro and in vivo studies and can provide the basis for the rational design of a vaccine against PA.
Assuntos
Exotoxinas/química , Exotoxinas/imunologia , Antígenos HLA/química , Peptídeos/química , Peptídeos/imunologia , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/imunologia , Sequência de Aminoácidos , Toxinas Bacterianas/imunologia , Simulação por Computador , Epitopos/química , Epitopos/imunologia , Antígenos HLA/imunologia , Humanos , Imunogenicidade da Vacina , Simulação de Acoplamento Molecular , Conformação Proteica , Infecções por Pseudomonas/imunologia , Vacinas/química , Vacinas/imunologia , Fatores de Virulência/química , Fatores de Virulência/imunologiaRESUMO
Pseudomonas aeruginosa is one of the most common opportunistic pathogens causing respiratory infections in hospitals. Vancomycin, the antimicrobial agent usually used to treat bacterial nosocomial infections, is associated with gut dysbiosis. As a lung-gut immunologic axis has been described, this study aimed to evaluate both the immunologic and histopathologic effects on the lungs and the large intestine resulting from vancomycin-induced gut dysbiosis in the P. aeruginosa pneumonia murine model. Metagenomic analysis demonstrated that vancomycin-induced gut dysbiosis resulted in higher Proteobacteria and lower Bacteroidetes populations in feces. Given that gut dysbiosis could augment the proinflammatory status of the intestines leading to a variety of acute inflammatory diseases, bone marrow-derived macrophages were stimulated with cecal content from dysbiotic mice showing a higher expression of proinflammatory cytokines and lower expression of IL-10. Dysbiotic mice showed higher levels of viable bacteria in the lungs and spleen when acutely infected with P. aeruginosa, with more lung and cecal damage and increased IL-10 expression in bronchoalveolar lavage. The susceptible and tissue damage phenotype was reversed when dysbiotic mice received fecal microbiota transplantation. In spite of higher recruitment of CD11b+ cells in the lungs, there was no higher CD80+ expression, DC+ cell amounts or proinflammatory cytokine expression. Taken together, our results indicate that the bacterial community found in vancomycin-induced dysbiosis dysregulates the gut inflammatory status, influencing the lung-gut immunologic axis to favor increased opportunistic infections, for example, by P. aeruginosa.
Assuntos
Disbiose/etiologia , Microbioma Gastrointestinal/imunologia , Intestinos/microbiologia , Pulmão/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/imunologia , Vancomicina/toxicidade , Animais , Antibacterianos/toxicidade , Modelos Animais de Doenças , Disbiose/patologia , Fezes/microbiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Intestinos/imunologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/efeitos dos fármacosRESUMO
Currently, the world health sector faces a big problem due to the increase of bacterial strains resistant to antibiotics. In 2017, the World Health Organization reported a list of resistant bacteria, among which Pseudomonas aeruginosa was present. This opportunistic pathogen is associated to nosocomial infections, and no effective vaccines against this bacterium have been found. Larrea divaricata Cav. (jarilla) is a shrub highly distributed in America and widely used in folk medicine. In our laboratory, cross-reactivity of antibodies obtained from the recognition of jarilla proteins against proteins from gram-negative bacteria has been demonstrated. The objective of this study was to study the cross-reactivity of anti-L. divaricata antibodies with P. aeruginosa extracellular proteins in order to find an innocuous prophylactic therapy against this nosocomial pathogen. We observed that antibodies generated by proteins from jarilla crude extract recognized antigenic determinants present in extracellular proteins of P. aeruginosa. However, further studies are needed to investigate the neutralizing capacity of these antibodies on the specific enzymatic proteins involved in the pathogenicity of this bacterium.
Assuntos
Reações Cruzadas/imunologia , Larrea/química , Larrea/imunologia , Mimetismo Molecular , Extratos Vegetais/imunologia , Proteínas de Plantas/imunologia , Pseudomonas aeruginosa/imunologia , Animais , Anticorpos/imunologia , Ensaio de Imunoadsorção Enzimática , Imunidade Humoral , Imunoglobulina G/imunologia , Larrea/metabolismo , Camundongos , Extratos Vegetais/química , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificaçãoRESUMO
Fever is a hallmark of infections and inflammatory diseases, represented by an increase of 1-4°C in core body temperature. Fever-range hyperthermia (FRH) has been shown to increase neutrophil recruitment to local sites of infection. Here, we evaluated the impact of a short period (1 h) of FRH (STFRH) on pro-inflammatory and bactericidal human neutrophil functions. STFRH did not affect neutrophil spontaneous apoptosis but reverted the lipopolysaccharide (LPS)-induced anti-apoptotic effect compared with that under normothermic conditions. Furthermore, STFRH accelerated phorbol myristate acetate (PMA)-induced NETosis evaluated either by the nuclear DNA decondensation at 2 h post-stimulation or by the increase in extracellular DNA that colocalized with myeloperoxidase (MPO) at 4 h post-stimulation. Increased NETosis upon STFRH was associated with an increase in reactive oxygen species (ROS) production but not in autophagy levels. STFRH also increased NETosis in response to Pseudomonas aeruginosa challenge but moderately reduced its phagocytosis. However, these STFRH-induced effects did not influence the ability of neutrophils to kill bacteria after 4 h of co-culture. STFRH also significantly reduced neutrophil capacity to release the pro-inflammatory cytokines chemokine (C-X-C motif) ligand 8/interleukin 8 (CXCL8/IL-8) and IL-1ß in response to LPS and P. aeruginosa challenge. Altogether, these results indicate that a short and mild hyperthermal period is enough to modulate neutrophil responses to bacterial encounter. They also suggest that fever spikes during bacterial infections might lead neutrophils to trigger an emergency response promoting neutrophil extracellular trap (NET) formation to ensnare bacteria in order to wall off the infection and to reduce their release of pro-inflammatory cytokines in order to limit the inflammatory response.
Assuntos
Armadilhas Extracelulares/imunologia , Febre/imunologia , Interleucina-1beta/imunologia , Interleucina-8/imunologia , Neutrófilos/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Armadilhas Extracelulares/microbiologia , Feminino , Febre/microbiologia , Febre/patologia , Humanos , Masculino , Neutrófilos/microbiologia , Neutrófilos/patologia , Infecções por Pseudomonas/patologiaRESUMO
Objective: To determine the efficacy of levofloxacin loaded niosomes in treating Sprague Dawley rats infected with Pseudomonas aeruginosa (ATCC 27853). Design and Methodology: Three groups of six (6) animals were infected with a known dose of the pathogen i.e. Pseudomonas aeruginosa via the intraperitoneal (ip) route. At six (6) hours post infection the infected animals were treated with drug free niosomes (control), free levofloxacin (conventional) and levofloxacin trapped in niosomes (ip). Blood was collected via tail snips at days 0,1,3,5,7 and 10 for complete blood counts and viable bacterial counts by colony forming units (CFU/µl). At day 10 the animals were sacrificed and samples from the kidney, liver and spleen were examined for bacterial counts. Results: All animals in the control group succumbed to the infection; one animal from the conventional group died. All niosome treated animals survived. The mean lymphocyte count (X109) was lower for the niosome (7.258±1.773) versus conventional (17.684±10.008) (p<0.03) treated groups at day ten (10). Neutrophil counts (X109) were lower for the niosome (2.563±1.609) versus conventional (6.2±6.548) p<0.02) treated groups. The CFUs in the bloodstream were similar for both treatment groups; the niosome treated group showed greater reduction in liver, kidney and spleen CFUs versus the conventional group (1.33±2.074) vs (5.8± 3.74) (p< 0.043), (1.5±2.35) vs (9.6±8.65) (p< 0.038) and (3.8 4.71) vs (25.6 14.66) (p<0.007) respectively. Conclusions: Further work is recommended on niosomes as a drug delivery system to treat intracellular infections.