Analysis of biofilm production by isolated bacteria in a neonatal unit

Neonatal infections caused by biofilm-producing microorganisms are more persistent and difficult to treat. Klebsiella spp. are important nosocomial pathogens often involved in outbreaks in Neonatal Intensive Care Units (NICUs). These pathogens are capable of producing biofilm on biotic and abiotic surfaces, increasing their permanence on the surfaces. The aim of this study was to detect biofilm production in samples of Klebsiella pneumoniae and Klebsiella oxytoca from the environment, hands of health professionals and bloodstream from a NICU. The detection of biofilm production was performed by microplate technique, using crystal violet for biomass quantification, extracellular matrix safranin and (2,3-Bis - (2- Methoxy-4-Nitro-5-Sulfophenyl) - 2H - Tetrazolium – 5 - Carboxanilide) for evaluation of metabolic activity. In addition, Colony Forming Units (CFU) were counted to quantify the number of viable cells present in the biofilm. In total, 33 samples of Klebsiella spp. were used in the study, being 28 samples of Klebsiella pneumoniae and five samples of Klebsiella oxytoca. All samples were strong biofilm producers, but 14 samples produced more biofilm than control, in at least one of the three colorimetric methods used and 15 were less efficient in this production. The biofilm production of the other samples were not statistically significant. Comparing the biofilm production produced by the two species, Klebsiella pneumoniae was more efficient in biomass and extracellular matrix production than Klebsiella oxytoca. As for the metabolic activity evaluated in the biofilm of the two species, there were no statistically significant differences, as well as the number of viable cells present in the biofilm produced by the two species. Biofilm production by Klebsiella spp. guarantees a longer permanence of these pathogens in the unit, due to the greater protection guaranteed by the extracellular matrix production. The longer the stay of these microorganisms in the unit, the greater the chance of the newborn acquiring an infection. This study shows the importance of biofilm-producing microorganisms in a NICU and the use of three colorimetric and quantitative methods for the evaluation of different biofilm parameters, which are cheap and effective tools for detecting bacterial biofilm production.CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorFAPEMIG - Fundação de Amparo a Pesquisa do Estado de Minas GeraisDissertação (Mestrado)Infecções neonatais causadas por microrganismos produtores de biofilme são mais persistentes e de difícil tratamento. Klebsiella spp. são importantes patógenos nosocomiais frequentemente envolvidos em surtos nas Unidades de Terapia Intensiva Neonatais (UTINs). Esses patógenos são capazes de produzir biofilme em superfícies bióticas e abióticas, aumentando a sua permanência nas superfícies. O objetivo desse estudo foi detectar a produção de biofilme em amostras de Klebsiella pneumoniae e Klebsiella oxytoca provenientes do ambiente, mãos dos profissionais de saúde e corrente sanguínea de neonatos internados uma UTIN. A detecção da produção de biofilme foi feita pela técnica de microplaca, utilizando cristal violeta para quantificação da biomassa, safranina para matriz extracelular e (2,3-Bis-(2- Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) para avaliação da atividade metabólica. Além disso, as Unidades Formadoras de Colônia (UFC) foram contadas para a quantificação do número de células viáveis presentes no biofilme. No total, foram utilizadas no estudo 33 amostras de Klebsiella spp., sendo 28 amostras de Klebsiella pneumoniae e cinco de Klebsiella oxytoca. Todas as amostras foram forte produtoras de biofilme, porém 14 amostras produziram mais biofilme do que o controle, em pelo menos um dos três métodos colorimétricos utilizados e 15 foram menos eficientes nessa produção. A produção de biofilme das demais amostras não foram estatisticamente significantes. Comparando a produção de biofilme produzida pelas duas espécies, Klebsiella pneumoniae foi mais eficiente na produção de biomassa e matriz extracelular do que Klebsiella oxytoca. Já a atividade metabólica avaliada no biofilme das duas espécies, não houve diferenças estatisticamente significantes, assim como o número de células viáveis presentes no biofilme produzidas pelas duas espécies. A produção de biofilme por Klebsiella spp. garante uma maior permanência desses patógenos na unidade, devido a uma maior proteção garantida pela produção da matriz extracelular. Quanto maior o tempo de permanência desses microrganismos na unidade, maiores são as chances do neonato adquirir uma infecção. Esse estudo mostra a importância de microrganismos produtores de biofilme em uma UTIN e a utilização de três métodos colorimétricos e um quantitativo para a avaliação de diferentes parâmetros do biofilme, que constituem-se como ferramentas baratas e eficazes para a detecção da produção de biofilme bacteriano

In vitro and in vivo studies of brazilian red propolis: antibacterial, antiparasital and antiviral potential against pathogenic microorganisms

Infectious diseases continue to be an important cause of thousands of deaths annually around the world. Therefore, the objective of this study was to evaluate the antimicrobial, antiviral and antiparasitic activity of Brazilian Red Propolis (BRP) against microorganisms that cause endodontic infections, Chikungunya virus and Toxoplasma gondii, using in vitro, in vivo and in silico approaches, in addition to carrying out evaluations toxicology of these products. Antimicrobial activity was determined by the broth dilution method and synergistic activity by the checkerboard assay. Antibiofilm activity was evaluated by staining with 2% crystal violet and counting microorganisms. In vivo infection was performed on Caenorhabditis elegans AU37 larvae and in silico analysis was performed using molecular docking. The effect on modulating the growth of T. gondii was evaluated using a β-galactosidase colorimetric assay. In the first study, BRP demonstrated antiperiodontopathogenic activity against all microorganisms evaluated, as well as antibiofilm activity against monospecies and multispecies biofilms. Furthermore, it was able to inhibit Chikungunya virus infection by up to 97%. In C. elegans, propolis did not demonstrate toxicity at the therapeutic concentrations determined in the study. In the second study, BRP demonstrated antibacterial and antifungal activity against all microorganisms included. The crude extract demonstrated additive activity in combination with amphotericin B against C. albicans (ATCC 28366). Furthermore, the BRP samples significantly inhibited biomass production and reduced the number of viable cells in the biofilm, in addition to reducing cell aggregation and causing damage to the cell wall of the microorganisms present in the biofilm. The highest concentrations of the crude extract and Gutiferone E of BRP increased the survival of infected and treated larvae and reduced the risk of death. The isolated substances Gutiferone E and Oblongifolin B inhibited the intracellular proliferation of T. gondii and demonstrated several targets of action against bacteria and T. gondii in silico analysis. The results presented in the two articles are promising and constitute a basic study so that in the future BRP can be used as a medicine against clinically relevant microorganisms.FAPEMIG - Fundação de Amparo a Pesquisa do Estado de Minas GeraisTese (Doutorado)As doenças infecciosas continuam a ser uma causa importante de milhares de mortes anualmente em todo o mundo. Portanto, o objetivo desse estudo foi avaliar a atividade antimicrobiana, antiviral e antiparasitária da Própolis Vermelha brasileira (PVB) frente a microrganismos causadores de infecções endodônticas, vírus Chikungunya e Toxoplasma gondii, utilizando abordagens in vitro, in vivo e in silico além de realizar avaliações toxicológicas desses produtos. A atividade antimicrobiana foi determinada pelo método de diluição em caldo e a atividade sinérgica pelo ensaio checkerboard. A atividade antibiofilme foi avaliada pela coloração com cristal violeta a 2% e contagem de microrganismos. A infecção in vivo foi realizada em larvas de Caenorhabditis elegans AU37 e a análise in silico foi realizada pelo docking molecular. O efeito na modulação do crescimento de T. gondii foi avaliado através de ensaio colorimétrico de β-galactosidase. No primeiro estudo, a PVB demonstrou atividade antiperiodontopatogênica frente a todos os microrganismos avaliados, bem como atividade antibiofilme frente a biofilmes monoespécies e multiespécies. Além disso, foi capaz de inibir até 96% a infecção pelo vírus Chikungunya. Em C. elegans, a própolis não demonstrou toxicidade nas concentrações terapêuticas determinadas no estudo. No segundo estudo, a PVB demonstrou atividade antibacteriana e antifúngica frente a todos os microrganismos avaliados. Além disso, as amostras de PVB inibiram de forma significativa a produção de biomassa e reduziu o número de células viáveis do biofilme, além de diminuir a agregação celular. As maiores concentrações do extrato bruto e Gutiferona E aumentaram a sobrevivência das larvas infectadas e tratadas e reduziram o risco de morte. As substâncias isoladas Gutiferona E e Oblongifolina B inibiram a proliferação intracelular de T. gondii e demonstraram diversos alvos de ação frente as bactérias e T. gondii na análise in silico. Os resultados apresentados nos dois artigos são promissores e constituem-se como um estudo base, para que futuramente a PVB possa ser utilizada como medicamento frente a microrganismos de relevância clínica.2026-07-1

Combining Essential Oils with Each Other and with Clotrimazole Prevents the Formation of <i>Candida</i> Biofilms and Eradicates Mature Biofilms

Fungal infections by Candida spp. are opportunistic and most often occur in individuals with some predisposing factor. Essential oils (EO) have anti-Candida potential, being a therapeutic alternative to be explored, especially for superficial and mucosal candidiasis. The objective was to analyze the synergistic potential between the EO of Citrus limon, Cupressus sempervirens, Litsea cubeba and Melaleuca alternifolia, and each of them with clotrimazole, to inhibit in vitro the formation and eradication of Candida spp. biofilms. Added to this, the survival of Caenorhabditis elegans was evaluated after exposure to EO, clotrimazole and their synergistic combinations. Anti-Candida activity was determined by microdilution for the substances alone and in EO–EO and EO–clotrimazole combinations. The combinations were performed by the checkerboard method, and the reduction in the metabolic activity of biofilms was determined by the viability of MTT/menadione. C. elegans larvae survival was evaluated after 24 h of exposure to EO, clotrimazole and synergistic combinations. The minimum inhibitory concentration (MIC) of EO ranged from 500 to >4000 µg/mL. The lowest MIC (500 µg/mL) was for C. sempervirens and L. cubeba on a C. krusei isolate; for clotrimazole, the MIC ranged from 0.015 to 0.5 µg/mL. Biofilm inhibition and eradication both ranged from 1000 to >4000 µg/mL. The lethal concentration (LC50) of C. limon, L. cubeba and M. alternifolia was 2000 µg/mL for C. elegans, while for C. sempervirens and clotrimazole, it was not determined within the concentration limits tested. In combination, more than 85% of the larvae survived M. alternifolia–clotrimazole, M. alternifolia–L. cubeba, C. sempervirens–clotrimazole and C. sempervirens–C. limon combinations. This study is the first, to our knowledge, to present a synergistic relationship of EO–EO and EO–clotrimazole combinations on Candida spp. biofilms

Exploring the antifungal, antibiofilm and antienzymatic potential of Rottlerin in an in vitro and in vivo approach

Abstract Candida species have been responsible for a high number of invasive infections worldwide. In this sense, Rottlerin has demonstrated a wide range of pharmacological activities. Therefore, this study aimed to evaluate the antifungal, antibiofilm and antivirulence activity of Rottlerin in vitro against Candida spp. and its toxicity and antifungal activity in vivo. Rottlerin showed antifungal activity against all yeasts evaluated, presenting Minimum Inhibitory and Fungicidal Concentration (MIC and MFC) values of 7.81 to > 1000 µg/mL. Futhermore, it was able to significantly inhibit biofilm production, presenting Biofilm Inhibitory Concentration (MICB50) values that ranged from 15.62 to 250 µg/mL and inhibition of the cell viability of the biofilm by 50% (IC50) from 2.24 to 12.76 µg/mL. There was a considerable reduction in all hydrolytic enzymes evaluated, with emphasis on hemolysin where Rottlerin showed a reduction of up to 20%. In the scanning electron microscopy (SEM) analysis, Rottlerin was able to completely inhibit filamentation by C. albicans. Regarding in vivo tests, Rottlerin did not demonstrate toxicity at the therapeutic concentrations demonstrated here and was able to increase the survival of C. elegans larvae infected. The results herein presented are innovative and pioneering in terms of Rottlerin’s multipotentiality against these fungal infections

Inanimate Surfaces and Air Contamination with Multidrug Resistant Species of Staphylococcus in the Neonatal Intensive Care Unit Environment

Background: Contamination of the hospital environment with multi-resistant (MDR) Staphylococcus increases the risk of infection. The aim of this study is to identify the MDR species of Staphylococcus on inanimate surfaces, in air, and in clinical samples, and analyze the risk factors that correlate with the occurrence of infections in a Neonatal Intensive Care Unit. Methods: Samples of inanimate surfaces and air were taken using a premoistened swab (0.9% sodium chloride) and spontaneous air sedimentation, respectively. The clinical isolates were recovered from infected neonates. The isolates (environmental and clinical) were identified by matrix-assisted laser desorption ionization-time of flight and the resistance profile was calculated using the disk diffusion agar technique. Results: In total, 181 isolates were obtained, 93 from (surfaces), 18 from the air, and 70 clinical samples. S. epidermidis was the most frequent species (66.8%), and the failure rate in air cleaning was 100%. More than 60% of the isolates were MDR, and the majority of clinical isolates (60.4%) had a resistance profile identical to that of the environmental isolates. Conclusion: Staphylococcus spp. were found in most of the analyzed samples, with a high frequency of MDR isolates, demonstrating the importance of the hospital environment as a reservoir, and the need for infection control measures, and rational use of antimicrobials