Sensibilidad antimicrobiana y detección de genes de virulencia ipaH, ial, set1A, set1B y Stx en aislados clínicos de Shigella

El género Shigella comprende bacilos de las especies S. flexneri, S. sonnei, S. boydii y S. dysenteriae, productores de disentería bacilar o shigelosis. La virulencia y patogenicidad del género se caracteriza por la presencia de una alta multirresistencia a antibióticos y variedad de factores de virulencia que permiten la infección al hospedero. El objetivo de este estudio fue establecer la sensibilidad antimicrobiana y detectar los genes de virulencia “Invasion plasmid antigen H“, ipaH; “Invasion-associated locus”, ial; “Shigella toxin” Stx; “Shigella enterotoxin 1A”, set1A; y “Shigella enterotoxin 1B”, set1B en aislados clínicos de Shigella spp.; para el efecto se analizaron 79 aislados obtenidos de Zurita & Zurita laboratorios y del Hospital Vozandes Quito. Mediante serotipaje, se obtuvieron 3 especies: S. flexneri (63,29%), S. sonnei (29,11%), y S. boydii (7,59%). La sensibilidad antimicrobiana se analizó siguiendo el método de Kirby- Bauer y las recomendaciones del “Clinical and Laboratory Standars Institute”, CLSI. La resistencia obtenida fue a tetraciclina (96,20%), ampicilina (91,14%), trimetoprim/sulfametoxazol (86,08%) y cloranfenicol (84,81%). El análisis de presencia de genes de virulencia se realizó mediante la reacción en cadena de la polimerasa (PCR) utilizando iniciadores previamente descritos. Se determinó una alta prevalencia de los genes ipaH (91,14%) e ial (82,28%), los genes codificantes de enterotoxina 1 (set1A y set1B) se encontraron en un 34,18%. Los resultados obtenidos demuestran la existencia de una multirresistencia a antibióticos y cepas altamente virulentas..

Sensibilidad antimicrobiana entre los serogrupos de Shigellae aislados en la ciudad de Quito-Ecuador

The pathotype Shigellae includes bacillus from the species S. flexneri, S. sonnei, S. boydii and S. dysenteriae which are causatives of the disease known as bacillary dysentery or shigellosis. The pathogenicity of the genus is characterized by the presence of high antibiotic resistance. The aim of this study was to identify the species and antimicrobial susceptibility in clinical isolates of Shigellae. We analyzed 79 isolates obtained from Zurita & Zurita Laboratorios and Vozandes Hospital in the city of Quito. Three species were obtained by serotyping, using polyclonal antisera: S. flexneri (n=50; 63,29%), S. sonnei (n=23; 29,11%), and S. boydii (n=6; 7,59%). The antimicrobial susceptibility was analyzed using the Kirby Bauer method and recommendations from the “Clinical and Laboratory Standards Institute” (CLSI). Most of the isolates showed resistance to tetracycline (n=76; 96,20%), ampicillin (n=75; 94,94%), trimethoprim/sulfamethoxazole (n=68; 86,08%) and chloramphenicol (n=67; 84,81%). The results showed the incidence of multidrug resistance to antibiotics commonly used for treating shigellosis, and the presence of three species of Shigellae.El patotipo Shigellae comprende bacilos de las especies S. flexneri, S. sonnei, S. boydii y S. dysenteriae, causantes de la enfermedad conocida como disentería bacilar o shigelosis. La patogenicidad del género se caracteriza por la presencia de una alta multirresistencia a antibióticos. El objetivo de este estudio fue identificar las especies y la sensibilidad a antimicrobianos en aislados clínicos de Shigellae. Se analizaron 79 aislados obtenidos de Zurita & Zurita Laboratorios y del Hospital Vozandes en la ciudad de Quito. Mediante serotipaje, con el uso de antisueros policlonales, se obtuvieron 3 especies: S. flexneri (n=50; 63,29%), S. sonnei (n=23; 29,11%), y S. boydii (n=6; 7,59%). La sensibilidad antimicrobiana se analizó siguiendo el método de Kirby-Bauer y las recomendaciones del “Clinical and Laboratory Standards Institute” (CLSI). La mayoría de las cepas mostraron resistencia a tetraciclina (n=76; 96,20%), ampicilina (n=75; 94,94%), trimetoprim/sulfametoxazol (n=68; 86,08%) y cloranfenicol (n=67; 84,81%). Los resultados obtenidos demostraron la incidencia de multirresistencia a antibióticos comúnmente utilizados para el tratamiento de shigelosis, y la presencia de 3 especies de Shigellae

Caracterización físico-química y análisis de la capacidad antimicrobiana de mieles de abeja sin aguijón (Tribu Meliponini) en Ecuador

La miel, como producto natural, ha sido utilizada y estudiada debido a sus propiedades nutricionales, antioxidantes y antimicrobianas. La miel de melipónidos o abejas sin aguijón es considerada exótica, y ha sido estudiada por tener un gran potencial nutricional y terapéutico especialmente por sus propiedades antioxidantes y antimicrobianas superiores a la miel de abeja común Apis mellifera. En este estudio se identificaron las diferentes especies de abejas sin aguijón y se caracterizaron las mieles que producen La calidad de las mieles se analizó por medio de parámetros físico-químicos y microbiológicos. La composición química de la miel fue determinada cuantificando azúcares, vitamina C, ácidos orgánicos, polifenoles, flavonoides, proteínas, aminoácidos y minerales. La actividad antioxidante fue analizada por medio de los métodos DPPH y FRAP. La actividad antimicrobiana se determinó sobre bacterias Gram positivas y Gram negativas en estado planctonico y en biopeliculas preformadas. Se identificaron 7 géneros y 12 especies de abejas sin aguijón en un total de 39 mieles recolectadas en tres regiones geográficas del Ecuador. El análisis físico-químico determinó que las mieles de abejas sin aguijón poseen un contenido de cenizas de 0,51 gr 100 gr-1, conductividad eléctrica de 0,17 mS cm-1, humedad de 27,35 gr 100 gr-1, color ámbar extra claro, pH 3,49, acidez libre de 105,12 meq Kg-1, acidez total de 420,20 meq Kg-1, actividad diastasa de 15,26 °Gothe, HMF de 31,17 mg Kg-1 y azúcares reductores de 72,72 gr 100 gr-1. La calidad microbiológica demostró la presencia de microorganismos aerobios mesófilos, hongos y levaduras en valores tolerables para el consumo. No se presentaron microrganismos de la familia Enterobacteriaceae y Salmonella spp., y los microorganismos Clostridia sulfito reductores se presentaron en 6 mieles. La composición química de las mieles fue de azúcares totales (75,30 gr 100 gr-1), con mayor presencia de fructosa (40,79 gr 100 gr-1), seguido por glucosa (24,94 gr 100 gr-1) y sacarosa (3,90 gr 100 gr-1). Vitamina C en un rango de 2,29 μg gr-1 a 63728,53 μg gr-1. Fenoles de 92,92 mg GAE 100 gr-1 y flavonoides de 60,37 mg CE 100 gr-1. Ácidos orgánicos totales de 1,72 mg 100 gr-1 con mayor cuantificación de ac. oxálico (0,52 mg 100 gr-1), seguido por ac. cítrico (0,48 mg 100 gr-1) y en menores concentraciones los ácidos: acético (0,44 mg 100 gr-1) y láctico (0,34 mg 100 gr-1). De los 23 minerales examinados se cuantificaron K (0,42 mg gr-1), Ca (0,22 mg gr-1), Na (0,16 mg gr-1), Mg (0,07 mg gr-1), Mn (0,05 mg gr-1), y Fe (0,01 mg gr-1). La concentración total de proteínas totales fue de 3,22 mg BSA 100 gr-1, mientras que la cuantificación de los aminoácidos totales fue de 13,54 mg 100 gr-1. Los aminoácidos que se cuantificaron en mayor cantidad fueron leucina (4,24 mg 100 gr-1), prolina (2,57 mg 100 gr-1), histidina (1,79 mg 100 gr-1) y valina (1,07 mg 100 gr-1). La capacidad antioxidante se determinó con valores de DPPH de 14,96 μmol TE 100 gr-1 y FRAP de 72,11 μmol TE 100 gr-1. La cuantificación de compuestos con actividad antimicrobiana se determinó para el H2O2 de 0,67 nmol y de GOx de 1,34 nmol/min mL-1. La actividad antimicrobiana sobre bacterias planctónicas se determinó en un MIC 10,92 gr mL-1 y con el tratamiento de las mieles con catalasa y proteinasa K se determinó que todas las mieles perdieron su actividad antimicrobiana con un MIC ˃20,00 gr mL-1. La actividad antimicrobiana sobre biopelículas preformadas se determinó en una reducción de crecimiento de las biopelículas del 20% Se determinó que el uso combinado de miel y antibióticos reduce las biopelículas preformadas en un 100%. Los análisis de microscopía demostraron que las mieles de abejas sin aguijón reducen la viabilidad de las biopelículas mediante la destrucción de la matriz extracelular, la reducción del tamaño celular y la modificación de la forma de la pared celular de las bacterias que conforman la biopelícula. La biodiversidad de las especies de abejas sin aguijón influye en las características físico-químicas de las mieles sin embargo en general se conservan sus propiedades manteniendo su composición característica de un pH ácido y alta humedad. La composición química de las mieles de melipónido le atribuye propiedades nutricionales, antioxidantes y antimicrobianas que la convierten en un alimento polifuncional con un alto potencial para su uso en la industria alimenticia y farmacéutica.Honey, as a natural product, has been used and studied due to its nutritional, antioxidant and antimicrobial properties. Honey from meliponids or stingless bees is considered exotic, and has been studied for having great nutritional and therapeutic potential, especially for its antioxidant and antimicrobial properties which are superior to honey from the common bee Apis mellifera. In this study, the different species of stingless bees were identified and the honey they produce was characterized. The quality of the honey was analyzed by means of physical-chemical and microbiological parameters. The chemical composition of honey was determined by quantifying sugars, vitamin C, organic acids, polyphenols, flavonoids, proteins, amino acids and minerals. The antioxidant activity was analyzed by means of the DPPH and FRAP methods. The antimicrobial activity was determined on Gram positive and Gram negative bacteria in the planktonic state and on preformed biofilms. Seven genera and 12 species of stingless bees were identified in a total of 39 honeys collected in three geographical regions of Ecuador. The physical-chemical analysis determined that honey from stingless bees has an ash content of 0.51 g 100 g-1, electrical conductivity of 0.17 mS cm-1, humidity of 27.35 g 100 g-1, extra light amber color, pH 3.49, free acidity of 105.12 meq Kg-1, total acidity of 420.20 meq Kg-1, diastase activity of 15.26 °Gothe, HMF of 31.17 mg Kg-1 and reducing sugars of 72.72 g 100 g-1. The microbiological quality showed the presence of mesophilic aerobic microorganisms, fungi and yeasts in tolerable values for consumption. There were no microorganisms of the Enterobacteriaceae family and Salmonella spp., and the sulfite-reducing Clostridia microorganisms were present in 6 honeys. The chemical composition of the honeys was total sugars (75.30 g 100 g-1), with a greater presence of fructose (40.79 g 100 g-1), followed by glucose (24.94 g 100 g-1) and sucrose (3.90 g 100 g-1). Vitamin C in a range of 2.29 μg g-1 to 63728.53 μg g-1. Phenols of 92.92 mg GAE 100 g-1 and flavonoids of 60.37 mg CE 100 g-1. Total organic acids of 1.72 mg 100 g-1 with greater quantification of oxalic acid (0.52 mg 100 g-1), followed by citric acid (0.48 mg 100 g-1) and in lower concentrations the acids: acetic (0.44 mg 100 g-1) and lactic (0.34 mg 100 g-1). Of the 23 minerals examined, K (0.42 mg gr-1), Ca (0.22 mg gr-1), Na (0.16 mg gr-1), Mg (0.07 mg gr-1), Mn (0.05 mg gr-1), and Fe (0.01 mg gr-1) were quantified. The total concentration of total proteins was 3.22 mg BSA 100 g-1, while the quantification of total aminoacids was 13.54 mg 100 g-1. The aminoacids that were quantified in greater quantity were leucine (4.24 mg 100 g-1), proline (2.57 mg 100 g-1), histidine (1.79 mg 100 g-1) and valine (1.07 mg 100 g-1). The antioxidant capacity was determined with DPPH values of 14.96 μmol TE 100 g-1 and FRAP values of 72.11 μmol TE 100 g-1. The quantification of compounds with antimicrobial activity was determined for H2O2 of 0.67 nmol and GOx of 1.34 nmol/min mL-1. The antimicrobial activity on planktonic bacteria was determined at a MIC of 10.92 g mL-1 and with the treatment of the honeys with catalase and proteinase K it was determined that all the honeys lost their antimicrobial activity with a MIC ˃20.00 g mL-1. The antimicrobial activity on preformed biofilms was determined to reduce biofilm growth by 20%. It was determined that the combined use of honey and antibiotics reduced preformed biofilms by 100%. Microscopy analyzes showed that stingless bee honeys reduce the viability of biofilms by destroying the extracellular matrix, reducing cell size, and changing the shape of the cell wall of the bacteria that make up the biofilm. The biodiversity of the species of stingless bees influences the physical-chemical characteristics of the honeys, however, in general, their properties are preserved, maintaining their characteristic composition of an acidic pH and high humidity. The chemical composition of meliponid honeys gives the nutritional, antioxidant and antimicrobial properties that make them a multifunctional food with a high potential for use in the food and pharmaceutical industries

Honey quality parameters, chemical composition and antimicrobial activity in twelve Ecuadorian stingless bees (Apidae: Apinae: Meliponini) tested against multiresistant human pathogens

[EN] Ecuadorian honey samples of eight genera and 12 stingless bee species from three geographic regions (Andes, Amazon and Pacific) were studied for their physicochemical parameters, chemical composition, mineral elements and antimicrobial activity. Meliponine honey is acidic and has a high water content, but our study revealed substantial chemical variation. Oxytrigona mellaria was highest in proteins, while Melipona sp. had the most free amino acid content. Five species of honey contained vitamin C, which was highest in O. mellaria. The most abundant minerals were the macronutrients, potassium and calcium. All honey inhibited microbial growth in gram-negative and gram-positive multiresistant human pathogens, with O. mellaria and Trigona silvestriana being most effective against both bacteria

Molecular Identification of the Human Pathogen Amphimerus sp. in the Freshwater Snail Aroapyrgus sp. in Ecuador

International audienceHere, we report for the first time the snail intermediate host for the Amphimerus liver fluke, a foodborne trematodiasis. In Ecuador, Amphimerus of the Opisthorchiidae family, infects humans, cats, and dogs, in the tropical Pacific-coast region. Opisthorchiidae comprising also Clonorchis sinensis , Opisthorchis sp., and Metorchis sp., have complex life cycles involving a definitive and two intermediate hosts. We identified morphologically and investigated the presence and prevalence of Amphimerus cercaria and DNA in freshwater snails collected in a human-amphimeriasis endemic region in Ecuador, extracted DNA from snail tissue and emerged cercariae, performed real-time polymerase chain reaction (PCR) with the newly developed primers and probe amplifying the Amphimerus ribosomal internal transcribed spacer 2 (ITS2) region, and sequenced the amplified DNA fragment. We collected 2,800 snails, characterized four species Aroapyrgus sp., Melanoides tuberculata , Biomphalaria cousini , and Aplexa marmorata , isolated three cercariae morphotypes. Of the 640 snails analyzed by qPCR, only Aroapyrgus and one of the three cercariae resulted positive, at a 15% infection prevalence. Polymerase chain reaction revealed that the Aroapyrgus snail and cercaria-morphotype-3 corresponded to Amphimerus , but not to C. sinensis , Fasciola hepatica , or Paragonimus mexicanus . The sequence of amplified DNA product matched that of human-isolated Amphimerus . This finding constitutes the first documentation that Aroapyrgus sp. is the first intermediate host for the Amphimerus sp. that infect humans in Ecuador. The ITS2–gene PCR and sequencing analysis demonstrated a high prevalence of snail infection and proved useful for detecting the infection in snails, which findings can help the establishment of suitable control programs against transmission in any endemic region of interest

Effect of thermal liquefaction on quality, chemical composition and antibiofilm activity against multiresistant human pathogens of crystallized eucalyptus honey

Thermal liquefaction is a conventional method used by beekeepers to liquefy crystallized honey. However, an abusive use of heat may affect its quality, chemical composition and bioactivity. The purpose of this study was to investigate the effect of thermal liquefaction on the quality, chemical composition and antibiofilm properties of eucalyptus honey. Thermal liquefaction (at 45 and 60 ◦C) did not affect the honey’s quality; however, a significant reduction in the reducing capacity, total phenolic content and hydrogen peroxide content was observed. At 60 ◦C, a significant reduction in the honey’s ability to inhibit biofilm formation was observed in Pseudomonas aeruginosa, as well as a reduction in its ability to remove preformed biofilms in both Staphylococcus aureus and Pseudomonas aeruginosa. Structural changes in biofilm architecture caused by honey were not affected by thermal treatment. Therefore, we recommend liquefaction at 45 ◦C as the most convenient for honey liquefaction without affecting its characteristics

Influence of Botanical Origin and Chemical Composition on the Protective Effect against Oxidative Damage and the Capacity to Reduce In Vitro Bacterial Biofilms of Monofloral Honeys from the Andean Region of Ecuador

Three types of monofloral honey from the Andean regions of Ecuador (Avocado, Eucalyptus, and Rapeseed honey) were analyzed to determine their floral origin, physicochemical parameters, chemical composition, antioxidant capacity, and their capacity to reduce in vitro bacterial biofilms. The chemical composition varied considerably depending on floral origin. The highest values of bioactive compounds were found in Avocado honey, classified as dark amber in color, while the lowest values were found in Eucalyptus honey followed by Rapeseed honey, both classified as extra light amber. When compared to Eucalyptus and Rapeseed honey, Avocado honey showed a more effective superoxide scavenging activity, chelating metal ions capacity, and a higher ability to protect human erythrocyte membranes against lipid peroxidation. For antimicrobial activity, the hydrogen peroxide content and the capacity to inhibit the biofilm formation, and to remove preformed biofilm from Staphylococcus aureus and Klebsiella pneumoniae was determined. Avocado honey showed the highest values of hydrogen peroxide content, as well as the highest capacity to reduce in vitro bacterial biofilms. A correlation between color vs. phenolics content vs. superoxide scavenging activity vs. chelating metal ions capacity, and the capacity to protect human erythrocyte membranes against lipid peroxidation was found

Evaluation of the polyphenolic profile of native Ecuadorian stingless bee honeys (Tribe: Meliponini) and their antibiofilm activity on susceptible and multidrug-resistant pathogens: An exploratory analysis

Biofilms are associated with infections that are resistant to conventional therapies, contributing to the antimicrobial resistance crisis. The need for alternative approaches against biofilms is well-known. Although natural products like stingless bee honeys (tribe: Meliponini) constitute an alternative treatment, much is still unknown. Our main goal was to evaluate the antibiofilm activity of stingless bee honey samples against multidrug-resistant (MDR) pathogens through biomass assays, fluorescence (cell count and viability), and scanning electron (structural composition) microscopy. We analyzed thirty-five honey samples at 15% (v/v) produced by ten different stingless bee species (Cephalotrigona sp., Melipona sp., M. cramptoni, M. fuscopilosa, M. grandis, M. indecisa, M. mimetica, M. nigrifacies, Scaptotrigona problanca, and Tetragonisca angustula) from five provinces of Ecuador (Tungurahua, Pastaza, El Oro, Los Ríos, and Loja) against 24-h biofilms of Staphylococcus aureus, Klebsiella pneumoniae, Candida albicans, and Candida tropicalis. The present honey set belonged to our previous study, where the samples were collected in 2018–2019 and their physicochemical parameters, chemical composition, mineral elements, and minimal inhibitory concentration (MIC) were screened. However, the polyphenolic profile and their antibiofilm activity on susceptible and multidrug-resistant pathogens were still unknown. According to polyphenolic profile of the honey samples, significant differences were observed according to their geographical origin in terms of the qualitative profiles. The five best honey samples (OR24.1, LR34, LO40, LO48, and LO53) belonging to S. problanca, Melipona sp., and M. indecisa were selected for further analysis due to their high biomass reduction values, identification of the stingless bee specimens, and previously reported physicochemical parameters. This subset of honey samples showed a range of 63–80% biofilm inhibition through biomass assays. Fluorescence microscopy (FM) analysis evidenced statistical log reduction in the cell count of honey-treated samples in all pathogens (P <0.05), except for S. aureus ATCC 25923. Concerning cell viability, C. tropicalis, K. pneumoniae ATCC 33495, and K. pneumoniae KPC significantly decreased (P <0.01) by 21.67, 25.69, and 45.62%, respectively. Finally, scanning electron microscopy (SEM) analysis demonstrated structural biofilm disruption through cell morphological parameters (such as area, size, and form). In relation to their polyphenolic profile, medioresinol was only found in the honey of Loja, while scopoletin, kaempferol, and quercetin were only identified in honey of Los Rios, and dihydrocaffeic and dihydroxyphenylacetic acids were only detected in honey of El Oro. All the five honey samples showed dihydrocoumaroylhexose, luteolin, and kaempferol rutinoside. To the authors’ best knowledge, this is the first study to analyze stingless bees honey-treated biofilms of susceptible and/or MDR strains of S. aureus, K. pneumoniae, and Candida species