Pseudomonas aeruginosa isolated from the environment of a Veterinary Academic Hospital in Brazil - resistance profile

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)

Pseudomonas aeruginosa isolated from the environment of a Veterinary Academic Hospital in Brazil - resistance profile

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...

Structural differences of neutrophil extracellular traps induced by biochemical and microbiologic stimuli under healthy and autoimmune milieus.

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.

The role of IL-10 in immune responses against Pseudomonas aeruginosa during acute lung infection.

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.

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.

In silico integrative analysis predicts relevant properties of exotoxin-derived peptides for the design of vaccines against Pseudomonas aeruginosa.

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.

Vancomycin-induced gut dysbiosis during Pseudomonas aeruginosa pulmonary infection in a mice model.

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.

Molecular mimicry between Larrea divaricata Cav. plant and a reference strain of Pseudomonas aeruginosa.

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.

Short-Term Fever-Range Hyperthermia Accelerates NETosis and Reduces Pro-inflammatory Cytokine Secretion by Human Neutrophils.

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.

Efficacy of levofloxacin loaded non-ionic surfactant vesicles (niosomes) in a Pseudomonas aeruginosa infection model in Sprague Dawley rats

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.

Secretory IgA-mediated immune response in saliva and early detection of Pseudomonas aeruginosa in the lower airways of pediatric cystic fibrosis patients.

Pseudomonas aeruginosa (Pa) detection in the paranasal sinuses may help to prevent or postpone bacterial aspiration to the lower airways (LAW) and chronic lung infection in cystic fibrosis (CF). We assessed the ability of an ELISA test for measurement of specific Pa secretory IgA (sIgA) in saliva (a potential marker of sinus colonization) to early detect changes in the Pa LAW status (indicated by microbiological sputum or cough swab culture and specific serum IgG levels) of 65 patients for three years, in different investigation scenarios. Increased sIgA levels were detected in saliva up to 22 months before changes in culture/serology. Patients who remained Pa-positive had significantly increased sIgA levels than patients who remained Pa-negative, both at the baseline (39.6 U/mL vs. 19.2 U/mL; p = 0.02) and at the end of the follow-up (119.4 U/mL vs. 25.2 U/mL; p < 0.001). No association was found between sIgA levels in saliva and emergence or recurrence of Pa in the LAW. A positive median sIgA result in the first year of follow-up implied up to 12.5-fold increased risk of subsequent Pa exposure in the LAW. Our test detected early changes in the P. aeruginosa LAW status and risk of exposure to P. aeruginosa in the LAW with two years in advance. Comparison with sinus culture is needed to assess the test's ability to identify CF patients in need of a sinus approach for Pa investigation, which could provide opportunities of Pa eradication before its aspiration to the lungs.

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

Ação dos extratos de hamamélis e abacateiro sobre cepas clínicas resistentes de Klebsiella pneumoniae e Pseudomonas aeruginosa / Action of Hamamelis and Avocado extracts on clinical strains of resistant Klebsiella pneumoniae and Pseudomonas aeruginosa

A resistência adquirida pelas bactérias aos antibióticos de amplo espectro tornou-se uma ameaça à saúde global. A necessidade de encontrar fármacos que consigam combater micro-organismos multirresistentes tem se tornado um grande desafio. Neste cenário, as plantas medicinais são promissoras por terem propriedades antimicrobianas eficazes, devido à presença de compostos fitoquímicos com atividades biológicas diversas. O objetivo desse estudo foi avaliar a ação antimicrobiana dos extratos glicólicos de Hamamelis virginiana (hamamélis) e de Persea americana (abacateiro) sobre cepas multirresistentes de Klebsiella pneumoniae e Pseudomonas aeruginosa. A avaliação da atividade antimicrobiana dos extratos vegetais foi realizada em sete cepas clínicas de K. pneumoniae e de P. aeruginosa, em comparação com uma cepa de referência de K. pneumoniae (ATCC 4352) e de P. aeruginosa (ATCC 15442). Para a determinação das concentrações inibitórias mínimas (CIM) e bactericida mínima (CBM) dos extratos de abacateiro e de hamamélis foi utilizado o método de microdiluição em caldo, segundo NCCLS. Após obtenção destes resultados, foi verificada a ação dos extratos sobre biofilmes monomicrobianos de oito cepas de K. pneumoniae e de P. aeruginosa (1 cepa ATCC e 7 cepas clínicas resistentes). Após o período de 48 horas para a formação do biofilme, os extratos foram adicionados separadamente, pelo período de cinco minutos, na concentração efetiva pré-determinada (CBM) e concentrações superiores. Posteriormente, os biofilmes foram lavados e mensurados por dois diferentes testes, em que foram avaliadas a biomassa do biofilme pelo cristal violeta e a viabilidade dos micro-organismos pelo teste de MTT. Os experimentos foram realizados com n=10, utilizando duas repetições para cada cepa/extrato. Os dados foram analisados estatisticamente pelo método ANOVA, complementado pelo Teste de Tukey, com nível de significância de 5% (p≤0.05). Os resultados demonstraram uma significativa atividade antibacteriana de ambos os extratos contra a cepapadrão e as cepas clínicas multirresistentes de K. pneumoniae e P. aeruginosa. Os extratos de abacateiro e de hamamélis, em todas as concentrações, demonstraram uma redução estatisticamente significativa na biomassa formada para pelo menos alguma das cepas clínicas de K. pneumoniae e P. aeruginosa testada. Para avaliação da viabilidade celular, ambos os extratos, na concentração de 200 mg/mL, demonstraram redução estatisticamente significativa nas cepas multirresistentes avaliadas de K. pneumoniae e P. aeruginosa. Como conclusão, os extratos glicólicos de H. virginiana e P. americana apresentaram ação antimicrobiana, resultando em concentrações com capacidade bactericida e significativa redução antibiofilme formados pelas cepas multirresistentes e ATCC de K. pneumoniae e P. aeruginosa(AU)

The resistance created by bacteria to broad-spectrum antibiotics has become a threat to global health. The need to find drugs that can combat multidrug-resistant microorganisms has become a major challenge. In this scenario, medicinal plants are promising because they have effective antimicrobial properties due to the presence of phytochemical compounds with diverse biological activities. The objective of this study was to evaluate the antimicrobial action of the glycolic extracts of Hamamelis virginiana (hamamelis) and Persea americana (avocado) on multiresistant strains of Klebsiella pneumoniae and Pseudomonas aeruginosa. The evaluation of the antimicrobial activity of the plant extracts was carried out in seven clinical strains of K. pneumoniae and P. aeruginosa, compared to an strain of K. pneumoniae (ATCC 4352) and P. aeruginosa (ATCC 15442). The broth microdilution method, according to NCCLS, was used to determine the minimum inhibitory (MIC) and bactericidal (MBM) concentrations of the extracts of avocado and witch hazel. After obtaining these results, the extracts on monomicrobial biofilms of eight strains of K. pneumoniae and P. aeruginosa (1 strain ATCC and 7 resistant clinical strains) were verified. After the 48 hour period for biofilm formation, the extracts were removed, with a five-minute interval, in the effective exercise session (MBC) and in the upper sets. Afterwards, the biofilms were washed and measured by two different testicles, in which the biofilm biomass was evaluated by the violet glass and the viability of the microorganisms by the MTT test. The experiments were performed with n = 10, using two replicates for each strain / extract. The data were analyzed statistically by the ANOVA method, complemented by the Tukey test, with a significance level of 5% (p≤0.05). The results demonstrated an antibacterial activity of both extracts against the standard strain and as the clinical strains of K. pneumoniae and P. aeruginosa. The extracts of avocado and witch hazel at all concentrations demonstrated a statistically significant reduction in the biomass formed for at least some of the clinical strains of K. pneumoniae and P. aeruginosa tested. For evaluation of cell viability, both extracts, at a concentration of 200 mg / mL, demonstrated a statistically significant reduction in the multiresistant strains evaluated for K. pneumoniae and P. aeruginosa. In conclusion, the glycolic extracts of H. virginiana and P. americana showed antimicrobial action, resulting in concentrations with bactericidal capacity and significant antibiofilm reduction formed by the multiresistant strains and ATCC of K. pneumoniae and P. aeruginosa(AU)

Prevalence, Antimicrobial Susceptibility, and Clonal Diversity of Pseudomonas aeruginosa in Chronic Wounds.

PURPOSE: Our purposes in this study were to (1) identify Pseudomonas aeruginosa strains collected from swabs of chronic wounds, (2) evaluate the susceptibility of P. aeruginosa strains to various antimicrobials, (3) detect the presence of virulence factors exoenzyme S (exoS) and exoenzyme U (exoU) in P. aeruginosa strains, and (4) evaluate wound colonization by P. aeruginosa via pulsed-field gel electrophoresis (PFGE). DESIGN: Descriptive research using a quantitative approach. SAMPLE AND SETTING: Swabs from 43 adults with chronic wounds treated in an outpatient setting in Niterói City, Brazil, were included using convenience sampling. METHODS: Swabs were collected at 2 points during treatment, 30 to 45 days apart. P. aeruginosa isolates were identified using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Antimicrobial susceptibility testing was performed using the disk diffusion method. The presence of exoS and exoU genes was evaluated using polymerase chain reaction. Genotyping diversity was determined through PFGE. RESULTS: Forty-eight P. aeruginosa isolates were detected in chronic wounds, and 3 were multidrug resistant (6%). Resistance to aztreonam and ciprofloxacin was observed in 48% and 27% of isolates, respectively. The presence of the exoS gene was verified in 54% of isolates, and 27% were positive for the exoU gene. In most wounds, P. aeruginosa strains had the same genetic characteristics at the 2 time points analyzed, indicating that the wound beds remained colonized. CONCLUSIONS: P. aeruginosa was present in 75% of tested chronic wound samples, and the same clones persisted for more than 1 month. In addition, most bacteria contained virulence genes that were associated with high potential to establish infection. The use of silver in chronic wounds may be associated with multidrug resistance in P. aeruginosa; therefore, it is important to avoid colonization by these bacteria.

Selenoprotein T is required for pathogenic bacteria avoidance in Caenorhabditis elegans.

Selenoprotein T (SELENOT) is an endoplasmatic reticulum (ER)-associated redoxin that contains the amino acid selenocysteine (Sec, U) within a CXXU motif within a thioredoxin-like fold. Its precise function in multicellular organisms is not completely understood although it has been shown in mammals to be involved in Ca2+ homeostasis, antioxidant and neuroendocrine functions. Here, we use the model organism C. elegans to address SELENOT function in a whole organism throughout its life cycle. C. elegans possess two genes encoding SELENOT protein orthologues (SELT-1.1 and SELT-1.2), which lack Sec and contain the CXXC redox motif instead. Our results show that a Sec→Cys replacement and a gene duplication were two major evolutionary events that occurred in the nematode lineage. We find that worm SELT-1.1 localizes to the ER and is expressed in different cell types, including the nervous system. In contrast, SELT-1.2 exclusively localizes in the cytoplasm of the AWB neurons. We find that selt-1.1 and selt-1.2 single mutants as well as the double mutant are viable, but the selt-1.1 mutant is compromised under rotenone-induced oxidative stress. We demonstrate that selt-1.1, but not selt-1.2, is required for avoidance to the bacterial pathogens Serratia marcescens and Pseudomonas aeruginosa. Aversion to the noxious signal 2-nonanone is also significantly impaired in selt-1.1, but not in selt-1.2 mutant animals. Our results suggest that selt-1.1 would be a redox transducer required for nociception and optimal organismal fitness. The results highlight C. elegans as a valuable model organism to study SELENOT-dependent processes.

Efeito do extrato de Curcuma longa L. sobre infecções in vitro por Staphylococcus aureus, Pseudomonas aeruginosa e Candida albicans em macrófagos murinos (RAW 264.7) / Effect of Curcuma longa L. extract on Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans in vitro infections in murine macrophages

Os seres humanos vivem em constante relação com micro-organismos, comensais e patogênicos, que podem ameaçar a homeostase do hospedeiro. Espécies patogênicas apresentam mecanismos capazes de contornar as barreiras de defesa do hospedeiro, facilitando sua disseminação e proliferação nos tecidos invadidos. Cepas resistentes aos antimicrobianos disponíveis surgem diariamente, por isso, é relevante encontrar métodos alternativos para controla-las. Produtos de plantas medicinais vem sendo investigados para essa finalidade. No presente estudo, foi avaliada a capacidade do extrato de C. longa em controlar infecções in vitro por S. aureus, P. aeruginosa e C. albicans em macrófagos murinos (RAW 264.7). Para isto, foram aplicadas as concentrações inibitórias mínimas (CIM) do extrato vegetal nas infecções e por meio de análise da fagocitose foi analisado a contribuição delas para a eliminação destes micro-organismos. A viabilidade dos macrófagos foi analisada por meio de teste colorimétrico com corante vermelho neutro. Foram checadas a produção de citocinas inflamatórias (IL-1ß, IL-6, TNF-α e IL-10) e óxido nítrico (NO), por ELISA e com reagente de Griess, respectivamente. Os resultados foram analisados por ANOVA e Tukey's Test (P ≤ 0.05). Foi verificado que o extrato de C. Longa auxiliou de maneira efetiva os macrófagos na fagocitose dos micro-organismos avaliados, apresentando reduções significativas de unidades formadoras de colônia por mililitro (UFC/mL), e atuou mediação da síntese de citocinas e NO. Os macrófagos apresentaram-se viáveis durante a infecção. Desta forma, foi constatado que o extrato de C. longa foi capaz de auxiliar in vitro os macrófagos na eliminação de S. aureus, P. aeruginosa e C. albicans, além de proporcionar efeito imunomodulador na síntese de IL-1ß, TNF-α, IL-10 e NO, no intuito de controlar as infecções microbianas in vitro(AU)

Humans live in constant contact with microorganisms, commensals and pathogens, which may threaten host homeostasis. Pathogenic species present mechanisms able circumventing the host defense barriers, facilitating their dissemination and proliferation in invaded tissues. Antimicrobial-resistant strains emerge daily, then it is important finding an alternative method to control them. Medicinal plant products have been investigated for this purpose. In this study, the ability of C. longa extract to control in vitro infections by S. aureus, P. aeruginosa and C. albicans in murine macrophages (RAW 264.7) was evaluated. For this, the minimum inhibitory concentrations (MIC) of the plant extract were used in the infections. Phagocytosis was analyzed to check if they would contribute to eliminate these microorganisms. The viability of the macrophages was analyzed by colorimetric test using neutral red dye. Productions of inflammatory cytokines (IL-1ß, IL-6, TNF-α and IL-10) and nitric oxide (NO) were verified during infection by ELISA and Griess's reagent, respectively. The results were analyzed by ANOVA and Tukey's Test (P ≤ 0.05). C. Longa L. extract effectively assisted the macrophages in the phagocytosis of the evaluated microorganisms, presenting significant reductions of colony-forming units per milliliter (CFU/mL), and mediated the synthesis of cytokines and NO. In addition, macrophages were viable during infection. C. Longa L. extract assisted in vitro the macrophages in the elimination of S. aureus, P. aeruginosa, and C. albicans, promoting immunomodulatory effect in the synthesis of IL-1ß, TNF-α, IL-10 and NO, in order to control microbial infections in vitro(AU)

Elimination of Pseudomonas aeruginosa through Efferocytosis upon Binding to Apoptotic Cells.

For opportunistic pathogens such as Pseudomonas aeruginosa, the mucosal barrier represents a formidable challenge. Infections develop only in patients with altered epithelial barriers. Here, we showed that P. aeruginosa interacts with a polarized epithelium, adhering almost exclusively at sites of multi-cellular junctions. In these sites, numerous bacteria attach to an extruded apoptotic cell or apoptotic body. This dead cell tropism is independent of the type of cell death, as P. aeruginosa also binds to necrotic cells. We further showed that P. aeruginosa is internalized through efferocytosis, a process in which surrounding epithelial cells engulf and dispose of extruded apoptotic cells. Intracellularly, along with apoptotic cell debris, P. aeruginosa inhabits an efferocytic phagosome that acquires lysosomal features, and is finally killed. We propose that elimination of P. aeruginosa through efferocytosis is part of a host defense mechanism. Our findings could be relevant for the study of cystic fibrosis, which is characterized by an exacerbated number of apoptotic cells and ineffective efferocytosis.

MIF Promotes Classical Activation and Conversion of Inflammatory Ly6C(high) Monocytes into TipDCs during Murine Toxoplasmosis.

Macrophage migration inhibitory factor (MIF) mediates immunity against Toxoplasma gondii infection by inducing inflammatory cytokines required to control the parasite replication. However, the role of this inflammatory mediator in the cell-mediated immune response against this infection is still poorly understood. Here, we used T. gondii-infected WT and Mif (-/-) mice to analyze the role of MIF in the maturation of CD11b(+) and CD8α (+) dendritic cells (DCs). We found that MIF promotes maturation of CD11b(+) but not CD8α (+) DCs, by inducing IL-12p70 production and CD86 expression. Infected Mif (-/-) mice showed significantly lower numbers of TNF and inducible nitric oxide synthase- (iNOS-) producing DCs (TipDCs) compared to infected WT mice. The adoptive transfer of Ly6C(high) monocytes into infected WT or Mif (-/-) mice demonstrated that MIF participates in the differentiation of Ly6C(high) monocytes into TipDCs. In addition, infected Mif (-/-) mice display a lower percentage of IFN-γ-producing natural killer (NK) cells compared to WT mice, which is associated with reducing numbers of TipDCs in Mif (-/-) mice. Furthermore, administration of recombinant MIF (rMIF) into T. gondii-infected Mif (-/-) mice restored the numbers of TipDCs and reversed the susceptible phenotype of Mif (-/-) mice. Collectively, these results demonstrate an important role for MIF inducing cell-mediated immunity to T. gondii infection.

Influence of inflammatory response, infection, and pulmonary function in cystic fibrosis.

AIMS: Recurrent infections and activation of the inflammatory response affect the prognosis of cystic fibrosis (CF). We investigated the relationship between inflammatory response, infection, and pulmonary function in CF. MAIN METHODS: A clinical-cross-sectional study was conducted with 86 subjects: control group (CG, n=31, the same age and sex of the CF group), and CF group (CFG, n=55, age: 1-16 years), further distributed into CFG negative or positive bacteriology (CFGB(-)/CFGB(+)), and CFG negative or positive Pseudomonas aeruginosa (CFGPa(-)/CFGPa(+)). Using the Wald test, multiple linear regression (95% confidence interval) was performed between CG and CFG, and between CG and each of the CF subgroups (CFGB(-)/CFGB(+) and CFGPa(-)/CFGPa(+)). The inflammatory markers evaluated were myeloperoxidase (MPO), adenosine deaminase (ADA) activities, interleukin-1beta (IL-1ß), tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP), nitric oxide metabolites (NOx) levels, and total and differential leukocyte counts. KEY FINDINGS: After adjusting for sex and age, CFG compared to CG revealed an increase of MPO, IL-1ß (P<0.001 in all subgroups), and CRP: CFG (P=0.002), CFGB(-) (P=0.007), CFGB(+) (P=0.009), CFGPa(-) (P=0.004) and CFGPa(+) (P=0.020). NOx (P=0.001, P<0.001), leukocytes (P=0.002, P=0.001), and neutrophils (P=0.003, P<0.001) were increased in CFGB(+) and CFGPa(+), respectively. A negative correlation between FEV1 and leukocytes (P=0.008) and FEV1 and neutrophils (P=0.031) resulted in CFG. SIGNIFICANCE: The inflammatory response characterized by the increase of MPO, IL-1ß, and CRP is determinant for CF. Also leukocytosis due to neutrophilia determines the pulmonary function deficiency in this disease.

Serum antibodies to Pseudomonas aeruginosa in cystic fibrosis as a diagnostic tool: a systematic review.

BACKGROUND: A systematic literature review of the last 40 years on the research of serum antibodies to Pseudomonas aeruginosa in cystic fibrosis and its utility as a diagnostic tool. METHODS: Research papers in English, Portuguese, and Spanish were accessed through electronic databases (PubMed, Medline, LILACS, and SciELO). RESULTS: 26 studies were assessed. ELISA technique was the most commonly used technique to detect serum P. aeruginosa antibodies. The most consistent results were those in which the response against the antigen St-Ag:1-17 was evaluated. The accuracy levels of the ELISA technique remain controversial, but most studies showed a good correlation between antibody titers and microbiological culture. CONCLUSIONS: The detection of serum antibodies to P. aeruginosa shows capacity for early detection of this pathogen and potential utility and viability of incorporation in the diagnostic routine of patients with cystic fibrosis.