Resistência aos antimicrobianos e diversidade genética de variantes fenotípicas de Pseudomonas aeruginosa não sensíveis aos carbapenêmicos recuperadas de pacientes com fibrose cística com infecção pulmonar crônica: uma análise temporal / Antimicrobial resistance and genetic diversity of phenotypic variants of carbapenem non susceptible Pseudomonas aeruginosa recovered from patients with cystic fibrosis with chronic lung infection: a temporal analysis

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.

Tecidos de poliéster e suas implicações para confecção de jalecos / Polyester cloths and their implications for making white coats

A literatura científica reporta a contaminação microbiana dos jalecos utilizados por profissionais da saúde, no entanto não há consenso relacionando seu uso com a redução da exposição microbiana por risco ocupacional. O objetivo desta pesquisa foi avaliar tecidos de poliéster (oxford e microfibra) utilizados na confecção de jalecos, quanto à função de barreira física contra fluido e bactérias, nas perspectivas e desafios do controle de infecção na área da saúde. Trata-se de um estudo do tipo experimental / laboratorial in vitro realizado em três etapas. Na primeira etapa, os tempos de passagem do fluido através dos tecidos foram cronometrados e registrados em segundos desde o início do escoamento do fluido até as formações e quedas das últimas gotas. Na segunda etapa (microbiológica), inóculos padronizados das bactérias padrão de Staphylococcus aureus (ATCC 25923) e Pseudomonas aeruginosa (ATCC 27853) foram adicionadas ao fluido. Decorrida a passagem do fluido através dos tecidos, alíquotas de 50µL in natura e diluídas (10-1 a 10-5) foram semeadas na superfície de placas de Petri (60x15mm) com meios de cultura seletivos, incubadas a 37°C por 24h e o número de unidades formadoras de colônia das bactérias expresso por mililitro do fluido (UFC/mL). Na terceira etapa, as características estruturais dos tecidos e a retenção bacteriana foram analisadas por meio de microscopia eletrônica de varredura (MEV). Os dados obtidos foram submetidos aos testes de normalidade (Kolmogorov-Smirnov e Shapiro-Wilk) e, posteriormente, ao teste de U de Mann-Whitney por meio do software IBM SPSS Statistics (versão 25) e nível de significância ?=5%. A comparação entre as medianas dos tempos de passagem do fluido através dos tecidos de oxford e microfibra demonstrou diferença estatisticamente significante (p<0,001) independente das variáveis envolvidas (tecidos limpo ou limpo e passado, e tecidos autoclavado ou não autoclavado). Na etapa microbiológica, não foi observada diferença entre as medianas das cargas bacterianas dos tecidos de oxford e microfibra após a passagem do fluido com S. aureus (p=0,056) e P. aeruginosa (p=0,320). As análises por MEV permitiram evidenciar estruturas com formas irregulares e de cristal, bem como espaços (macroporos) entre os fios dos tecidos de oxford, que permitiram um menor tempo de passagem do fluido através do tecido. No entanto, não foi constatada a presença bacteriana na superfície dos tecidos. Em conclusão, diante dos dois tipos de tecidos utilizados na confecção de jalecos, o de microfibra apresentou maior tempo de passagem do fluido comparado ao de oxford, em decorrência das diferenças estruturais desses tecidos. Entretanto, a função de barreira física bacteriana após a passagem do fluido através dos tecidos não foi observada, o que reforça a necessidade de substituição do jaleco quando esse entra em contato com fluidos biológicos, visando à biossegurança: controle de contaminação/infecção na área da saúde

Scientific literature reports contamination of white coats used by health professionals, but there is not a consensus relating its usage to reduction of microbial exposure by occupational risk. The objective of this study was to evaluate Oxford and microfiber cloths used for making white coats, regarding its function as physical barrier to fluid and bacteria, in perspectives and challenges of infection control in health field. It is an in vitro experimental / laboratory study carried out in three stages. In the first stage, fluid passage times through the pieces of cloths were measured and registered in seconds since the beginning of fluid flow until formations and falls of the last drops. In the second stage (microbiological), standardized inocula of standard bacteria of Staphylococcus aureus (ATCC 25923) and Pseudomonas aeruginosa (ATCC 27853) were added to the fluid. After the passage of fluid through the pieces of cloths, in natura and diluted 50µL aliquots (10-1 to 10-5) were seeded on the surface of Petri dishes (60x15mm) with selective culture mediums, incubated at 37°C for 24h and the number of colony forming units of bacteria expressed by milliliter of fluid (CFU/mL). In the third stage, structural characteristics of cloths and bacterial retention were analyzed through scanning electron microscopy (SEM). The obtained data were submitted to normality tests (Kolmogorov-Smirnov and Shapiro-Wilk) and, later, to Mann-Whitney U test through IBM SPSS Statistics (version 25) software and ?=5% significance level. Comparison between medians of the fluid passage time through oxford and microfiber cloths showed statistically significant difference (p<0.001) independent of the involved variables (clean or clean and ironed cloths, and autoclaved or non-autoclaved cloths). In the microbiological stage, difference was not observed between medians of bacterial loads of Oxford and microfiber cloths after the passage of the fluid with S. aureus (p=0.056) and P. aeruginosa (p=0.320). The analyses by SEM allowed evidence structures with irregular and crystal shapes as well as gaps (macropores) between the threads of pieces of Oxford cloth, that allowed a shorter fluid passage time through the cloth. However, bacterial presence on the surface of cloths were not noticed. In conclusion, before the two types of cloths used for making white coats, the microfiber one presented longer fluid passage time compared to the Oxford one, due to the structural differences of these cloths. However, the functionality as bacterial physical barrier after fluid passage through the pieces of cloths were not observed, which reinforces the need to replace the white coat when it comes in contact with biological fluids, aiming at biosafety: contamination/infection control in health field

Human monocytes tolerant to LPS retain the ability to phagocytose bacteria and generate reactive oxygen species

Tolerance to lipopolysaccharide (LPS) occurs when animals or cells exposed to LPS become hyporesponsive to a subsequent challenge with LPS. This mechanism is believed to be involved in the down-regulation of cellular responses observed in septic patients. The aim of this investigation was to evaluate LPS-induced monocyte tolerance of healthy volunteers using whole blood. The detection of intracellular IL-6, bacterial phagocytosis and reactive oxygen species (ROS) was determined by flow cytometry, using anti-IL-6-PE, heat-killed Staphylococcus aureus stained with propidium iodide and 2',7'-dichlorofluorescein diacetate, respectively. Monocytes were gated in whole blood by combining FSC and SSC parameters and CD14-positive staining. The exposure to increasing LPS concentrations resulted in lower intracellular concentration of IL-6 in monocytes after challenge. A similar effect was observed with challenge with MALP-2 (a Toll-like receptor (TLR)2/6 agonist) and killed Pseudomonas aeruginosa and S. aureus, but not with flagellin (a TLR5 agonist). LPS conditioning with 15 ng/mL resulted in a 40 percent reduction of IL-6 in monocytes. In contrast, phagocytosis of P. aeruginosa and S. aureus and induced ROS generation were preserved or increased in tolerant cells. The phenomenon of tolerance involves a complex regulation in which the production of IL-6 was diminished, whereas the bacterial phagocytosis and production of ROS was preserved. Decreased production of proinflammatory cytokines and preserved or increased production of ROS may be an adaptation to control the deleterious effects of inflammation while preserving antimicrobial activity.

Immunosuppressive effect of purified pyocyanine pigment on T-lymphocytes viability against experimental infection with hydatid cyst protoscolices

The effect of pyocyanine pigment, which was isolated and purified from Pseudomonas aeruginosa, on specific lymphocytes viability inside the body of white male Balb/c mice against the experimental secondary hydatidosis and the infectivity of protoscolices was studied in comparison with negative control mice groups, phosphate buffered saline [PBS] and positive control group [immunoferon]. Four groups of male Balb/c mice were intraperitoneally [IP] inoculated with four purified concentrations of pyocyanine [25, 50, 75, 100 micro m/ml]. Seven days later, they were given the same concentrations as a booster dose of the pigment, then 7 days later they were intraperitoneally infected with 2000 protoscoleces/ mL [PBS] as a challenge dose. The fifth group was intraperitoneally inoculated with 1ml of sterile PBS and used as a negative control group, while the sixth group was intraperitoneally inoculated with 100 micro mg/ml immunoferon and received the challenge dose of 2000 protoscoleces/ ml PBS and served as the positive control group. The concentrations of 50, 75 and 100 micro m/ml of this pigment had suppressive effect on these specific immune response cells. This effect was statistically significant [p<0.01] after six weeks from the challenge dose with intraperitoneal protoscolices infection. This effect revealed that the protoscolices infectivity increased due to suppression viability of T lymphocytes, while the immunoferon showed a significant stimulation of these specific cellular cells, which decrease the protoscolices infectivity in comparison with higher pigment concentrations. Pyocyanine is a toxic pigment causing suppression of T-cells activity, especially at higher concentrations which allow protoscolices development and growth

effect of combination of pseudomonas aeruginosa alginate and an immunomodulator protein of garlic on opsonophagocytosis in murine model

Chronic pulmonary infection in patients with cystic fibrosis is predominantly due to infection by mucoid strains of Pseudomonas aeruginosa. Mucoid P. aeriginosa is due to the production of exopolysaccharide called also alginate. Alginate in addition to interference with the clearance of lung has antiphagocytic property. Optimal killing activity of P. aeruginosa requires opsonic antibodies. Since immunomodulatory effects of garlic on enhancing phagocytic activity has been proved, in this study the effect of combination of alginate and an immunomodulator protein of garlic on production of opsonic antibodies against P. aeruginosa mucoid exopolysaccharide has been investigated. Alginate was extracted from a 72-hour culture of P. aeruginosa strain 8821M and then DNasel, RNaseA and Proteinase k were added. Subsequently, alginate was purified with gel filtration chromatography by sephacryl S-400. Female BALB/c mice aged 6-8 weeks were divided into five groups and injected subcutaneously on days 0, 7, 14 with either alginate, garlic, alginate- garlic, R10 or alginate-R10 and opsonophagocytic killing activity was calculated in each group. The purified alginate contained 34.6 micro g/ml uronic acid, 0.5 micro g/ml nucleic acid, 1.45 micro g/ml protein and 0.08 micro g/ml LPS. Opsonophagocytic killing activity after immunization with R10, alginate and their combination showed significant increases of respectively 69%, 67% and 82% comparing to the control group. Combination of P.aeruginosa alginate and immunomodulator fraction of garlic enhances immunogenicity to P.aeroginosa by the elicitation of opsonic antibodies in BALB/c mice

immunology of pseudomonal human persistent pyuria

Pseudomonas aeruginosa was diagnosed in association with human persistent pyuria. It was noted among immunoreactive and immunocompromized patients. The test patient showed high; total serum protein, total circulating globulin and high mucosal globulin concentrations than those of normal subjects. The immunoglobulin concentration for the classes IgG IgM and IgA were higher than those normal subjects. The albumin - globulin ratio was lower than those of normal subjects. Circulating anti. Pseudomans aeruginosa specific agglutinins were of titer median value of 400 mucosal specific agglutinins, however, they were with median titer values of 40. in immunreactive patients. While they were of titer median values of 240, and 40 in the immunocomproruized patients. Likewise significant migration inhibition index LIF were noted in peripheral blood leukocytes and Mucosul leucocytes in immunreactive and nonsignificant in the immunocompromized patient. This can be due to the presence of an immunodominant T dependent and/or T independent epitopes

Imunidade humoral em pacientes com fibrose cística / Humoral immunity of patients with cystic fibrosis

Pacientes com FC frequentemente såo acometidos por infecçöes pulmonares, principalmente por P. aeruginosa tanto da variedade nåo mucóide como mucóide. Esta última, quando se instala, é acompanhada geralmente de deterioraçåo progressiva da funçåo pulmonar. Detectam-se nestes pacientes, anticorpos para o microorganismo e diversos produtos por ele produzidos. A imnunoeletroferese cruzada tem sido tradicionalmente utilizada para pesquisa de precipitinas para P. aeruginosa. Esta surgem pouco após o início do episódio infeccioso e guardam correlaçåo com a cronicidade do processo, quando se mostram mais numerosoas. Em pacientes infectados por P. auruginosa mucóide, o número de precipitinas costuma ser maior que nos infectados pela variedade nåo-mucóide. As precipitinas habitualmente nåo surgem nos pacientes apenas colonizados, servindo assim para distinguir casos de colonizaçåo dos de infecçåo, e indicando a oportunidade do emprego de antibioticoterapia nestes pacientes. O número de precipitinas se reduz após controle do processo infeccioso. Outras técnicas vêm sendo desenvolvidas com sucesso, para a pesquisa de anticorpos para P. aureginosa, visando a obtençåo de maior sensibilidade e especificidade, abrindo caminho para novas possibilidades de investigaçåo

Proteasas de Pseudomonas aeruginosa: caracterización parcial y relaciones inmunológicas con proteasas de diferentes taxas / Proteases of pseudomonas aeruginosa: partial characterization and immunological relations with proteases of different rates

El presente trabajo tuvo el propósito de aislar, caracterizar y estudiar inmunológicamente, dos enzimas extracelulares proteolíticas de una cepa de Pseudomonas aeruginosa (Pa) tomada como prototipo, para luego compararlas con las producidas por cepas de la misma o diferentes especies bacterianas. La cepa prototipo mostró por cromatografía de intercambio iónico las dos enzimas proteolíticas identificadas como A y B, sobre la base de su mayor o menor electronegatividad. La enzima A fue activa sobre caseína y no sobre elastina y necesitó fuerza iónica adicional para eluir del intercambiador. la enzima B fue activa sobre caseína y elastina y no necesitó fuerza iónica adicional para su elución. Fue la responsable del 80% o más de la actividad total. Con las dos enzimas obtenidas por poliacrilamida, se inmunizaron conejos, obteniéndose antisueros (As A y As B), con los que se efectuaron reacciones inmunológicas. Los As A y As B inmunoprecipaitaron las enzimas respectivas, tanto de la solución enzimática concentrada como de las obtenidas por cromatografía. Estos dos componentes enzimáticos no mostraron identidad inmunológica. Los antisueros homólogos fueron capaces de inhibir la actividad enzimática in vitro. Seis cepas adicionales de Pa mostraron un comportamiento inmunoenzimático similar a la prototipo. Dos de Serratia marcescens, aisladas de pacientes, mostraron por zimograma una especie proteolítica coincidente con la A de Pa, activa sólo sobre caseína y que reaccionó solamente con el As A. Una cepa de Pseudomonas fluorescens conservada en laboratorio no mostró actividad en el zimograma y tampoco reactividad con los As A y As B

Anticuerpos hemaglutinantes contra las enzimas elastasa y proteasa de Pseudomonas aeruginosa en población normal de Maracaibo / Hemagglutinating antibodies for elastase and protease enzymes of Pseudomona aeruginosa in normal population of Maracaibo

Se estudiaron 311 sujetos de ambos sexos, aparentemente sanos, a quienes se les determinó el título de anticuerpos dirigidos contra las enzimas elastasa y proteasa de Pseudomonas aeruginosa, siguiendo la técnica de hemaglutinación pasiva en microtitulación, encontrándose que todos ellos (100%) mostraron títulos por debajo de 1:80

Estudo da atividade antimicrobiana de extrato de folha de Aristolochia gigantea Mart e Zucc / Study of the antimycrobial activity of extracts from the leaves of Aristolochia gigantea Mart and Zucc

Foram investigadas as atividades antibacteriana e antifúngica dos extratos éter de petróleo e alcaloídico de folhas de Aristolochia gigantea Mart e Zucc, pelo método de difusäo em agar. O extrato éter de petróleo mostrou atividade contra uma bactéria Gram-positiva. O extrato alcaloídico apresentou atividade contra duas bactérias Gram-positivas. Uma Gram-negativa, näo apresentando qualquer atividade antifúngica.

Estudo da atividade antibacteriana de extratos de espécies de aristolochiaceas brasileiras / Study of the antibacterial activity of extracts from Brazilian Aristolochiaceae especies

Foram testadas as atividades antibacterianas de extratos de folhas, flores, caule e raizes de Aristolochia gigantea Mart e Zucc e de Aristolochia glandulosa Ferrari, frente a bactérias Gram-positivas e Gram-negativas, pelo método de difusäo em placa. Cinco extratos mostraram-se ativos.