Chromogenic, Biochemical and Proteomic Identification of Yeast and Yeast-like Microorganisms Isolated from Clinical Samples from Animals of Costa Rica.

Yeast infections are challenging human and animal medicine due to low rates of detection and the emergence of unknown ecology isolates. The aim of this study was to verify the biochemical identification of yeasts and yeast-like microorganisms obtained from animals comparing the results with chromogenic media and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF MS). Between January and August 2023, yeast and yeast-like isolates from samples of animals with suspicion of mycosis were identified using Vitek® 2 Compact, Brilliance® Candida Agar and MALDI Biotyper® MSP. A total of 39 cases were included, and 45 isolations were obtained. Cryptococcus neoformans (15.5%, 7/45), Meyerozyma guilliermondii (13.3%, 6/45), Candida parapsilosis (11.1%, 5/45), Candida albicans and Candida tropicalis (8.9%, each one 4/45) were the most identified organisms. There was full agreement with the three identification methods in 71.1% (32/45) of the isolates, disagreement on species in 17.8% (8/45), disagreement on genus and species in 6.7% (3/45) and, in 4.4% (2/45), there was no matched pattern in MALDI-TOF to compare the results. Biochemical methods are a good option in laboratories where proteomics are not available, and chromogenic media enhances diagnostics by detecting mixed infections. Surveillance must be implemented to improve the detection of agents shared between humans and animals.

Anovaginal Colonization by Group B Streptococcus and Streptococcus anginosus among Pregnant Women in Brazil and Its Association with Clinical Features.

Streptococcus agalactiae (Group B Streptococcus; GBS) is a leading cause of neonatal invasive disease worldwide. GBS can colonize the human gastrointestinal and genitourinary tracts, and the anovaginal colonization of pregnant women is the main source for neonatal infection. Streptococcus anginosus, in turn, can colonize the human upper respiratory, gastrointestinal, and genitourinary tracts but has rarely been observed causing disease. However, in the last years, S. anginosus has been increasingly associated with human infections, mainly in the bloodstream and gastrointestinal and genitourinary tracts. Although anovaginal screening for GBS is common during pregnancy, data regarding the anovaginal colonization of pregnant women by S. anginosus are still scarce. Here, we show that during the assessment of anovaginal GBS colonization rates among pregnant women living in Rio de Janeiro, Brazil, S. anginosus was also commonly detected, and S. anginosus isolates presented a similar colony morphology and color pattern to GBS in chromogenic media. GBS was detected in 48 (12%) while S. anginosus was detected in 17 (4.3%) of the 399 anovaginal samples analyzed. The use of antibiotics during pregnancy and history of urinary tract infections and sexually transmitted infections were associated with the presence of S. anginosus. In turn, previous preterm birth was associated with the presence of GBS (p < 0.05). The correlation of GBS and S. anginosus with relevant clinical features of pregnant women in Rio de Janeiro, Brazil, highlights the need for the further investigation of these important bacteria in relation to this special population.

Development of a rapid assay for ß-etherase activity using a novel chromogenic substrate.

Biocatalytic processes play a crucial role in the valorization of lignin; therefore, methods enabling the monitoring of enzymes such as ß-etherases, capable of breaking ß-O-4 aryl-ether bonds, are of significant biotechnological interest. A novel method for quantifying ß-etherase activity was developed based on the ß-ester bond formation between a chromophore and acetovainillone. The chromogenic substrate ß-(ρ-nitrophenoxy)-α-acetovanillone (PNPAV), was chemically synthesized. Kintetic monitoring of ρ-nitrophenolate release at 410 nm over 10 min, using recombinant LigF from Sphingobium sp SYK-6, LigF-AB and LigE-AB from Althererytrobacter sp B11, yielded enzimatic activities of 404. 3 mU/mg, 72 mU/mg, and 50 mU/mg, respectively. This method is applicable in a pH range of 7.0-9.0, with a sensitivity of up to 50 ng of enzyme, exhibiting no interference with lipolytic, glycolytic, proteolytic, and oxidoreductase enzymes.

Chromogenic Escherichia coli reporter strain for screening DNA damaging agents.

The presence of pollutants in soil and water has given rise to diverse analytical and biological approaches to detect and measure contaminants in the environment. Using bacterial cells as reporter strains represents an advantage for detecting pollutants present in soil or water samples. Here, an Escherichia coli reporter strain expressing a chromoprotein capable of interacting with soil or water samples and responding to DNA damaging compounds is validated. The reporter strain generates a qualitative signal and is based on the expression of the coral chromoprotein AmilCP under the control of the recA promoter. This strain can be used simply by applying soil or water samples directly and rendering activation upon DNA damage. This reporter strain responds to agents that damage DNA (with an apparent detection limit of 1 µg of mitomycin C) without observable response to membrane integrity damage, protein folding or oxidative stress generating agents, in the latter case, DNA damage was observed. The developed reporter strain reported here is effective for the detection of DNA damaging agents present in soils samples. In a proof-of-concept analysis using soil containing chromium, showing activation at 15.56 mg/L of Cr(VI) present in soil and leached samples and is consistent with Cr(III) toxicity at high concentrations (130 µg). Our findings suggest that chromogenic reporter strains can be applied for simple screening, thus reducing the number of samples requiring analytical techniques.

Evaluation of Chromogenic Culture Media for Rapid Identification of Gram-Positive Bacteria Causing Mastitis.

The present study aimed to evaluate the diagnostic performance specificity (Sp), sensitivity (Se), positive predictive value (PPV), negative predictive value (NPV), and accuracy (Acc) of two chromogenic culture media for rapid identification of Gram-positive bacteria causing subclinical mastitis (SCM) in dairy cows. For this, the performance of chromogenic culture media Gram-positive (GP) and Staphylococcus (Staph) (CHROMagar ™, Paris-France) was evaluated in milk samples collected from: (1) lactating cows with SCM (n = 504), and (2) cows in the post-partum period (PP) (7 ± 3 days post-partum; n = 536). Rapid identification of Gram-positive bacteria in chromogenic media was performed by visual inspection of colony colors after 24 h of incubation at 37°C. Bacterial identification by MALDI-TOF mass spectrometry was considered the reference methodology for calculating: Acc, Se, Sp, PPV, NPV, and Cohen's Kappa coefficient of agreement (k). The chromogenic media GP showed high Acc for Strep. agalactiae/dysgalactiae identification in both samples of SCM (Se: 89.1%; Sp: 96.3% and Acc: 95.6%) and of cows in PP (Se: 100%; Sp: 99.0% and Acc: 99.1%). Similar results were observed for Strep. uberis/Enterococcus spp. identification (Se: 90.5%; Sp: 92.5% and Acc: 92.3%) in SCM samples and Se: 100%; Sp: 99.6% and Acc: 99.6% in samples of PP cows using the GP media. However, the GP chromogenic media showed low Se (25.0% in SCM samples and 50.0% in samples of cows in PP) for Staph. aureus identification, despite Sp and Acc were high (Sp: 98.3% and Acc: 95.4% in SCM and Sp samples: 99.4% and Acc: 98.9% in PP cow samples). Staph culture media showed high Acc for Staph. aureus identification (Se: 80.0%; Sp: 98.8% and Acc: 98.0% in SCM samples and Se: 66.7%; Sp: 100% and Acc: 99.6% in PP cow samples), although the low prevalence of Staph. epidermidis and Staph. saprophyticus limit inferences about the performance of identifying these pathogens in Staph media. In conclusion, despite the limitation of the GP media for identification of Staph. aureus, GP, and Staph chromogenic media obtained satisfactory diagnostic performance results for the rapid identification of the main Gram-positive pathogens associated with SCM.

Evaluation of chromogenic culture media for rapid identification of microorganisms isolated from cows with clinical and subclinical mastitis.

This study aimed to evaluate the diagnostic performance (specificity, Sp; sensitivity, Se; accuracy; positive predictive value; negative predictive value; and Cohen's kappa coefficient, κ, of agreement) of chromogenic culture media for rapid identification of microorganisms isolated from cows with clinical (CM) and subclinical mastitis (SCM). For this, 2 experiments were carried out: evaluation of (1) biplate, and (2) triplate of chromogenic culture media for rapid identification of mastitis-causing microorganisms. For the evaluation of diagnostic performance, identification of microorganisms by MALDI-TOF mass spectrometry was considered the standard methodology. In experiment 1, 476 milk samples collected from cows with CM and 660 from cows with SCM were evaluated by inoculation in 2 selective chromogenic culture media (CHROMagar) for gram-positive bacteria and another for gram-negative bacteria. In experiment 2, 476 milk samples from cows with CM and 500 from cows with SCM were evaluated by inoculation in triplate chromogenic culture media (Smartcolor2, Onfarm), selective for Streptococcus and Strep-like organisms, Staphylococcus, and gram-negative bacteria. In experiment 1 for the CM samples, the use of biplates with gram-positive and gram-negative culture media showed Se that ranged from 0.56 (0.32-0.81; Staphylococcus aureus) to 0.90 (0.80-0.99 Streptococcus uberis), Sp varied from 0.94 (0.92-0.96; Strep. uberis) to 1.00 (Prototheca spp. or yeast), and κ ranged from 0.47 (0.26-0.67; Staph. aureus) to 0.84 (0.78-0.9; Escherichia coli). The Se of biplates for SCM samples ranged from 0.50 (0.15-0.85; E. coli) to 0.94 (0.87-1.00; Staph. aureus), Sp varied from 0.95 (0.93-0.97; Strep. uberis) to 0.99 (0.98-1.00; Staph. aureus and Strep. Agalactiae or dysgalactiae), and κ ranged from 0.18 (0.00-0.40; Escherichia coli) to 0.88 (0.80-0.95; Staph. aureus). In experiment 2, the Se of the triplate chromogenic media in CM samples ranged from 0.09 (0.00-0.26; Serratia spp.) to 0.94 (0.85-1.00; Klebsiella spp. and Enterobacter spp.), Sp varied from 0.94 (0.92-0.96; Strep. agalactiae and Strep. dysgalactiae) to 1.00 (Serratia spp.) and κ ranged from 0.07 (0.00-0.24; Serratia spp.) to 0.85 (0.75-0.94; Klebsiella spp. and Enterobacter spp.). For SCM samples, the use of the triplate with the chromogenic culture media showed Se that varied from 0.25 (0.10-0.40; Lactococcus spp.) to 1.00 (Strep. Agalactiae or dysgalactiae), Sp ranged from 0.92 (0.90-0.94; Strep. Agalactiae and Strep. dysgalactiae) to 0.99 (0.98-1.00; Klebsiella spp. and Enterobacter spp.), and κ varied from 0.28 (0.00-0.72; E. coli) to 0.72 (0.60-0.82; Staph. aureus). Our results suggest that the diagnostic accuracy of the biplate and triplate of chromogenic culture media varies according to pathogen, and the results of chromogenic culture media may be useful for rapid decision-making on mastitis treatment protocols of the main mastitis-causing microorganisms, but their use for implementation of mastitis control measures will depend on each farm specific needs.

Bacteriophage-Based Biosensing of Pseudomonas aeruginosa: An Integrated Approach for the Putative Real-Time Detection of Multi-Drug-Resistant Strains.

During the last decennium, it has become widely accepted that ubiquitous bacterial viruses, or bacteriophages, exert enormous influences on our planet's biosphere, killing between 4-50% of the daily produced bacteria and constituting the largest genetic diversity pool on our planet. Currently, bacterial infections linked to healthcare services are widespread, which, when associated with the increasing surge of antibiotic-resistant microorganisms, play a major role in patient morbidity and mortality. In this scenario, Pseudomonas aeruginosa alone is responsible for ca. 13-15% of all hospital-acquired infections. The pathogen P. aeruginosa is an opportunistic one, being endowed with metabolic versatility and high (both intrinsic and acquired) resistance to antibiotics. Bacteriophages (or phages) have been recognized as a tool with high potential for the detection of bacterial infections since these metabolically inert entities specifically attach to, and lyse, bacterial host cells, thus, allowing confirmation of the presence of viable cells. In the research effort described herein, three different phages with broad lytic spectrum capable of infecting P. aeruginosa were isolated from environmental sources. The isolated phages were elected on the basis of their ability to form clear and distinctive plaques, which is a hallmark characteristic of virulent phages. Next, their structural and functional stabilization was achieved via entrapment within the matrix of porous alginate, biopolymeric, and bio-reactive, chromogenic hydrogels aiming at their use as sensitive matrices producing both color changes and/or light emissions evolving from a reaction with (released) cytoplasmic moieties, as a bio-detection kit for P. aeruginosa cells. Full physicochemical and biological characterization of the isolated bacteriophages was the subject of a previous research paper.

Bactéria cromogênica associada a manchas negras / Black-spotted chromogenic bactéria

Objetivos: Este trabalho tem como objetivo avaliar a associação das bactérias Prevotella Melaninogênica e Actinomyces ao biofilme escuro em dentes permanentes. Relato de Caso: Um paciente apresentou manchas enegrecidas ao longo do contorno cervical dos dentes foi convidado a participar do presente estudo. Foi coletado com o auxílio de uma cureta estéril, uma parte deste biofilme que transferido para um eppendorff contendo soro fisiológico. O material coletado foi agitado em um Vórtex por 15 segundos e em seguida, inoculadas em 100 microlitros (µl) da solução em um meio de ágar sangue suplementado com Canamicina 5%, seguido de cultivo por 72 horas. A partir do crescimento bacteriano, as mesmas foram isoladas em ágar macconkey, chocolate e sangue em anaerobiose. Em seguida foram realizados testes de bioquimismo, utilizando-se os meios MIO (motilidade, indol e ornitina), citrato, TSI (triplo açúcar com ferro) e LIA (ágar lisina com ferro). Conclusão: Através das características clínicas associadas ao bioquimismo empregado nos testes, podemos concluir que a bactéria que está associada as manchas negras presentes no paciente é do gênero prevotella spp(AU)

Objectives: This study aims to evaluate the association of Prevotella Melaninogenic and Actinomyces bacteria with dark biofilm in permanent teeth. Case Report: A patient with black spots along the cervical contour of the teeth was invited to participate in the present study. After its clarification and acceptance, completing the IC, was collected with the aid of a sterile curette, a part of this biofilm that was inserted into an eppendorff containing saline. The collected material was vortexed for 15 seconds and then inoculated into 100 microliters (µl) of the solution in a 5% kanamycin supplemented blood agar medium, followed by cultivation for 72 hours. From bacterial growth, they were isolated on macconkey agar, chocolate and anaerobic blood. Biochemical tests were then performed using MIO (motility, indole and ornithine), citrate, TSI (triple sugar with iron) and LIA (iron lysine agar) media. Conclusion: Through the clinical characteristics associated with the biochemism employed in the tests, we can conclude that the bacteria that is associated with black spots present in the patient is of the genus prevotella spp(AU)

Validación del ensayo FVIII: C cromogénico en una plataforma automatizada

El objetivo de este trabajo fue realizar la validación analítica del método cromogénico (FVIII:Ccro) en la plataforma ACL TOP y correlacionarlo con el método coagulable en una etapa (FVIII:Ccoag). El estudio de validación (EP5-A2, EP6-A2 y comparación de métodos por EP-9) se realizó para la curva de rango normal-bajo (CRNB): aproximadamente entre 10-150 UI/dL de FVIII y de rango muy bajo (CRMB): aproximadamente entre 0-10 UI/dL. Los resultados de repetitividad (CVr) y precisión intermedia (CVi) fueron menores del 6% y comparables a los informados por el fabricante para otras plataformas. El rango de medición analítica fue de 11-129 UI/dL con CRNB, y se extrapoló a 0,3 UI/dL al utilizar la CRMB. Para la CRNB FVIII:Ccro mostró buena correlación con FVIII:Ccoag: r: 0,98, pendiente: 0,982 (0,961-1,003), ordenada al origen: -0,3 (-1,1-0,5), sesgo: -2,0%. Para CRMB se obtuvo un r de 0,96, pendiente: 0,921 (0,855-0,988), ordenada al origen: -0,07 (-0,35-0,20), sesgo: -10,2%. Sólo 4 pacientes presentaron niveles discrepantes entre ambos métodos. La determinación de FVIII:C por el método cromogénico automatizado en la familia ACL TOP fue comparable con FVIII coagulable en una etapa en el rango analítico evaluado. El FVIII:Ccro automatizado puede utilizarse para el diagnóstico y seguimiento del tratamiento de los pacientes hemofílicos.

The objective of this work was to perform the analytical validation of the chromogenic method (FVIII:Ccro) on the ACL TOP platform correlating with one stage assay (FVIII:Ccoag). The validation study (EP5-A2, EP6-A2 and comparison of methods by EP-9) was performed for the low-normal range curve (CRNB): approximately between 10-150 IU/dL of FVIII and very low range (CRMB): approximately between 0-10 IU/dL. The results of CVr (repeatability) and CVi (intermediate precision) were lower than 6% and comparable to those reported by the manufacturer for other platforms. The analytical measurement range was 11-129 IU/dL, extrapolated to 0.3 IU/dL using the CRMB. For CRNB FVIII:Ccro showed good correlation with FVIII:Ccoag: r: 0.98, slope: 0.982 (0.961-1.003), intercept: -0.3 (-1.1-0.5), bias: -2.0%. For CRMB: r: 0.96 was obtained, pending: 0.921 (0.855-0.988), intercept: -0.07 (-0.35-0.20), bias: -10.2%. Only 4 patients presented discrepant levels between both methods. The automated chromogenic FVIII assay in the ACL TOP family is comparable with one stage coagulable FVIII in the analytical range studied. The FVIII:Ccro automated can be used for the diagnosis and monitoring of the treatment of hemophilic patients.

O objetivo deste trabalho foi a validação analítica do método cromogênico (FVIII:Ccro) na plataforma ACL TOP correlacionando-se com o método coagulável numa etapa (FVIII:Ccoag). O estudo de validação (EP5-A2, EP6-A2 e comparação de métodos por EP-9) foi realizado para a curva de faixa normal-baixa (CRNB) aproximadamente entre 10 e 150 Ul/dL de FVIII e de faixa muito baixa (CRMB): aproximadamente entre 0 e 10 UI/dL. Os resultados de Repetitividade (CVr) e precisão intermediária (CVi) foram inferiores a 6% e comparáveis aos descritos pelo fabricante para outras plataformas. A faixa de medição analítica foi de 11-129 UI/dL com CRNB extrapolando-se para 0,3 UI/dL utilizando a CRMB. Para a CRNB FVIII:Ccro houve boa correlação com o FVIII: Ccoag: r: 0,98, inclinação: 0,982 (0,961-1,003), ordenada na origem: -0,3 (-1,1-0,5), Viés: -2,0%. Para CRMB: foi obtido um r: 0,96, pendente: 0,921 (0,855-0,988), ordenado na origem: -0,07 (-0,35-0,20), Viés: -10,2%. Apenas quatro pacientes apresentaram níveis discrepantes entre os dois métodos. A determinação de FVIII:C pelo método cromogênico automatizado na família ACL TOP foi comparável ao FVIII coagulável em um estágio na faixa analítica avaliada. FVIII: O FVIII:Ccro automatizado pode ser utilizado para o diagnóstico e seguimento do tratamento dos pacientes hemofílicos.

Incidencia de Escherichia coli O157: H7 en heces de rumiantes lactantes con síndrome diarreico / Incidence of Escherichia coli 0157: H7 in feces of lactating ruminants with diarrheal syndrome

RESUMEN Objetivo. Identificar Escherichia coli O157:H7 presente en heces diarreicas de rumiantes lactantes con síndrome diarreico y seguridad de ingesta de calostro. Materiales y métodos. Se realizó un muestreo de 316 rumiantes durante el período de agosto 2015 a marzo 2016 en los municipios de Río Grande, General Enrique Estrada, Morelos y Calera de Víctor Rosales del estado de Zacatecas, 67 de bovinos, 183 de ovinos y 66 de caprinos. Resultados. Se identificaron en medio cromogénico CHROMagarTM: 260 coliformes, 78 Escherichia coli O157:H7, 16 Proteus spp., y 25 colonias de bacterias sin identificar con este medio, encontrándose una incidencia de Escherichia coli O157:H7 de 22.03% en los cuatro municipios. Conclusiones. Escherichia coli O157:H7 es la segunda bacteria encontrada en heces de rumiantes con un 22% de incidencia, la cual es un factor de riesgo de muerte en rumiantes lactantes (menos de 21 días de nacidos) causando pérdidas económicas y riesgo para la salud de la población del estado de Zacatecas.

ABSTRACT Objective. To identify Escherichia coli 0157:H7 present in diarrheal feces of lactating ruminants with diarrheal syndrome and safety of colostrum intake. Materials and methods. A feces sampling of 316 ruminants was carried out during the period of August 2015 to March 2016 in the municipalities of Río Grande, General Enrique Estrada, Morelos and Calera de Victor Rosales of the state of Zacatecas, obtained from 67 cattle, 183 sheep and 66 goats. Results. The following were identified in CHROMagarTM chromogenic medium: 260 coliforms, 78 Escherichia coli 0157:H7, 16 Proteus spp. and 25 colonies of unidentified bacteria, finding an incidence of Escherichia coli 0157:H7 of 22.03% in the four municipalities. Conclusions. Escherichia coli 0157:H7 is the second bacteria found in ruminant feces with an incidence of 22%, which is a mortality risk factor in lactating ruminants (less than 21 days old), causing economic loss and health risk for the population of the state of Zacatecas.

Data associated with the characterization and presumptive identification of Bacillus and related species isolated from honey samples by using HiCrome Bacillus agar.

The dataset described in this paper provides information on the morphological features of 24 different species of the genera Bacillus, Paenibacillus, Brevibacillus, Lysinibacillus, and Rummeliibacilluswhen growing in HiCrome Bacillus agar. The species studied are common contaminants of honey. In support to the recent publication entitled "HiCrome Bacillus agar for presumptive identification of Bacillus and related species isolated from honey samples" (2), a collection of 197 bacterial isolates belonging to 24 different species of aerobic spore-forming bacteria have been screened for their colony appearance and color and any substrate color change of HiCrome Bacillus agar at 24 and 48 h of incubation. Two simple flowcharts utilizing a combination of colony and media characteristics in the chromogenic medium and a set of simple biochemical and morphological tests were developed for quick presumptive identification.

Utility of CHROMagar mSuperCARBA for surveillance cultures of carbapenemase-producing Enterobacteriaceae.

Culture of carbapenemase-producing Enterobacteriaceae (CPE) as part of active surveillance is one of the most useful strategies for successful infection control programmes. Our objective was to compare the recently introduced CHROMagar mSuperCARBA agar for CPE detection in surveillance cultures from perineal swabs with the US Centers for Disease Control and Prevention method. Our results showed that this agar is a useful and affordable alternative (sensitivity 93.05%, specificity 96.21%, diagnostic accuracy 95.2%) to detect CPE in hospital settings.

Efecto de una combinación de nutrientes e inhibidores del crecimiento bacteriano sobre el desarrollo de Salmonella / Effect of a combination of nutrients and inhibitors of bacterial growth on the development of Salmonella

La selección de bases nutritivas para los medios de cultivo está relacionada con el microorganismo objeto de estudio y el propósito del medio. Los inhibidores del crecimiento bacteriano en los medios selectivos y diferenciales pueden interferir en el desarrollo del microorganismo de interés. Por ello se requiere un balance entre sustancias promotoras e inhibidoras del crecimiento bacteriano, sobre todo en bacterias que pueden encontrarse a muy bajas concentraciones o sometidas a diferentes condiciones de estrés durante el almacenamiento de las muestras que las contiene, como Salmonella. El objetivo consistió en evaluar el efecto de una combinación de nutrientes de diferentes orígenes y de inhibidores del crecimiento de bacterias grampositivas sobre el desarrollo de Salmonella. Se seleccionaron 52 cepas: incluyendo Salmonella, otras bacterias y levaduras. Se determinó la capacidad nutricional de mezclas de bases nutritivas registrando el incremento de la biomasa mediante técnica espectrofotométrica. Se comprobó la capacidad de recuperación e inhibición de dos variantes experimentales con diferentes inhibidores mediante la determinación de parámetros cuantitativos, y se comparó la productividad de la formulación final con un medio cromogénico. Salmonella mostró un crecimiento abundante en las variantes con diferentes combinaciones nutricionales, se logró la inhibición de un grupo de microorganismos y la productividad de la composición final fue superior a 0.80. La mezcla de bases nutritivas de diferentes orígenes y las sales biliares como inhibidor de crecimiento de bacterias grampositivas resulta una combinación eficaz para promover el crecimiento de Salmonella cuando estas bacterias se encuentran en baja concentración.

The selection of nutrient bases depends on the microorganism and the purpose of the medium. Inhibitors of bacterial growth are of great importance in selective and differential media and may interfere in the growth of the microorganism of interest. An adequate balance between promoter substances and inhibitors of bacterial growth is thus required, especially for bacteria that could be found either at very low concentrations or those subject to different stress conditions during storage of the samples containing them, such as Salmonella. The aim was to evaluate the effect of a combination of nutrients of different origins and inhibitors of grampositive bacteria on the development of Salmonella serotypes. 52 Salmonella strains and a representation of other bacteria and yeasts were selected. The nutritional capacity of the composition was determined by spectrophotometric technique formulating variants with mixtures of nutritive bases, and recording the increase in biomass. Recovery capacity and inhibition of two experimental variants with different inhibitors were quantitatively tested. The productivity of the final formulation was compared with a chromogenic medium (Oxoid, England). Salmonella showed abundant growth in the variants made with different nutrient combinations. Both experimental formulations showed their ability to recover microorganisms of interest. The final composition showed productivity values higher than 0.80 in both variants. The mixture of nutrient bases and bile salts as an inhibitor of growth of grampositive bacteria was an effective combination, capable of stimulating the growth of Salmonella genus when these bacteria are found at low concentration.

CXXC5 expression in prostate cancer: implications for cancer progression.

Identification of genes specifically deregulated in prostate adenocarcinoma may lead to discovery of new oncogenes/tumour suppressors with clinical relevance for diagnosis, prognosis and/or therapy. CXXC5 is a gene encoding a retinoid-inducible nuclear factor, whose overexpression in breast tumours, metastatic malignant melanomas and papillary thyroid carcinoma has been recently reported. We previously found differential expression of CXXC5 transcripts in metastatic prostate cancer cell lines of both rat and human origin. However, knowledge on the expression of this gene in benign or malignant human prostate tissue is lacking. The aim of this study was to determine the mRNA and protein expression pattern of CXXC5 in human benign prostate tissue, proliferative inflammatory atrophy, high-grade prostatic intra-epithelial neoplasia and prostate cancer, using qPCR, chromogenic in situ hybridization and immunohistochemistry. Our results showed that protein levels determined by immunohistochemistry were in agreement with transcript levels observed by chromogenic in situ hybridization. CXXC5 mRNA and protein expressions were significantly higher in prostate cancer, high-grade prostatic intra-epithelial neoplasia, and proliferative inflammatory atrophy, compared to benign prostate tissue. Significantly, within the same tissue specimens, CXXC5 staining was stronger in malignant acini than in matched adjacent, benign acini; immunostaining for this protein was mainly localized to the nucleus of benign epithelial cells and both the nucleus and cytoplasm of malignant epithelial cells. Our findings suggest that CXXC5 may play a role in the process of prostate carcinogenesis. Additional studies are required to determine the biological and clinical significance of CXXC5 in prostate cancer development and/or progression.

Determinación de hierro sérico: Evolución del desempeño de los laboratorios participantes del Sub-Programa PEEC-Hematología / Iron serum determination: evolution of performance of participating laboratories of the PEEC-Hematology Sub-Program / Determinação de ferro sérico: Evolução do desempenho dos laboratórios participantes do Sub-Programa PEEC-hematologia

El objetivo de este trabajo fue determinar la evolución del desempeño analítico en la determinación de hierro sérico, de los laboratorios participantes del Sub- Programa PEEC-Hematología (PEEC-H) del Programa de Evaluación Externa de Calidad Prof. Dr. Daniel Mazziotta de la Fundación Bioquímica Argentina, mediante el análisis de los resultados de ferremia en 6 encuestas (E) realizadas en los meses de julio entre los años 2010 y 2015 (E 77, 81, 85, 89, 93 y 97). Hasta el 2011 se utilizaban métodos con y sin desproteinización, siendo estos últimos los más utilizados (94%). En 2015 en la red de laboratorios se emplearon solamente métodos directos sin desproteinización, siendo los colorimétricos los más utilizados (aproximadamente 95%). El Desvío Relativo Porcentual aceptable (DRPa) fue de ±10% en todas las encuestas analizadas. El 56% de los laboratorios tuvieron un desempeño promedio aceptable en las E 77, 81 y 85, evolucionando 3 años después, a 70% en las E 89, 93 y 97. Según estas consideraciones, al presente no es necesario ajustar el DRPa para el analito hierro, ya que con este valor los laboratorios aún deben trabajar para lograr una mejoría en su desempeño.

The aim of this work was to evaluate the evolution of the analytical performance of serum iron determination by the laboratories participating in the Sub- Program PEEC-Hematology (PEEC-H) EQAS Program Prof. Dr. Daniel Mazziotta of the Argentine Biochemical Foundation. To this end, results of serum iron determinations from July 2010 to July 2015 (surveys #77, 81, 85, 89, 93 and 97) were used. Up to 2011, there were methods both with and without deproteinization, the latter being the most used (94%). In 2015, only one commercial method without deproteinization was used, with colorimetric methods employed in 95% of the cases. In all the surveys analyzed, the acceptable DRP was ±10%. In surveys 77, 81 and 85, 56% of the laboratories had an acceptable performance percentage, and it evolved to a 70% in the surveys 89, 93 and 97, three years later. According to these considerations, there is no need to adjust the acceptable DRP for the iron analyte. In this way, laboratories will continue to work in order to improve their performance.

O objetivo deste estudo foi determinar a evolução do desempenho analítico na determinação de ferro sérico, dos laboratórios participantes no Sub-Programa PEEC-Hematologia (PEEC-H) do Programa de Avaliação Externa de Qualidade Prof. Dr. Daniel Mazziotta da Fundación Bioquímica Argentina, através da análise dos resultados de ferremia em 6 pesquisas de opinião (E) realizadas nos meses de julho entre os anos 2010 a 2015 (Pesquisa No. 77, 81, 85, 89, 93 e 97). Até 2011 eram empregados métodos com e sem desproteinização, sendo os colorimétricos os mais utilizados (aproximadamente 95%). O Desvio Relativo Percentual aceitável (DRPa) foi de ±10% em todas as pesquisas analisadas. 56% dos laboratórios tiveram desempenho médio aceitável nas pesquisas 77, 81 e 85, progredindo para 70% nas pesquisas de 89,93 e 97, 3 anos mais tarde. De acordo com estas considerações, hoje não é necessário ajustar o DRPa para o analito ferro, visto que com esse valor os laboratórios ainda devem trabalhar para alcançar uma melhoria no seu desempenho.

Optical devices for the detection of cyanide in water based on ethyl(hydroxyethyl)cellulose functionalized with perichromic dyes.

Films of three polymers, based on ethyl(hydroxyethyl)cellulose functionalized with protonated perichromic dyes, were used for anion sensing. The polymer functionalized with protonated Brooker's merocyanine acts as a chromogenic/fluorogenic system for the selective detection of cyanide in water. An increase of >28 times was verified for the fluorescence lifetime of the sensing units in the polymer in comparison with protonated Brooker's merocyanine in water. Moreover, an increase in the pKa values was verified for the sensing units in the polymers. Data suggest that the hydrocarbonic polymeric chains provide an adequate microenvironment to protect the sensing unit from bulk water. The other polymer, functionalized with an iminophenol, also showed high selectivity for cyanide (detection limit=9.36×10-6molL-1 and quantification limit=3.12×10-5molL-1). The polymer functionalized with azophenol units is unable for the detection of cyanide, due to the low pKa value verified for its chromogenic units.

Analysis of streptokinase by validated liquid chromatography methods and correlation with an in vitro bioassay.

Reversed-phase and size-exclusion liquid chromatography methods were validated for the assessment of streptokinase. The reversed-phase method was carried out on a Jupiter C4 column (250 mm × 4.6 mm id) maintained at 25°C. The mobile phase consisted of 50 mM sodium sulfate solution pH 7.0 and methanol (90:10, v/v), run isocratically at a flow rate of 0.8 mL/min. The size-exclusion method was carried out on a Protein KW 802.5 column (300 mm × 8.0 mm id), at 25°C. The mobile phase consisted of 40 mM sodium acetate solution pH 7.0, run isocratically at a flow rate of 1.0 mL/min. Retention times were 19.3 min, and 14.1 min, and calibration curves were linear over the concentration range of 0.25-250 µg/mL (25.75-25 750 IU/mL) (r2 = 0.9997) and 5-80 µg/mL (515-8240 IU/mL) (r2 = 0.9996), respectively, for reversed-phase and size exclusion, with detection at 220 and 204 nm. Chromatographic methods were employed in conjunction with the in vitro bioassay for the content/potency assessment of Streptokinase, contributing to improve the quality control and ensure the efficacy of the biotherapeutic.

Development of fast and simple chromogenic methods for glucan phosphatases in-gel activity assays.

Glucan phosphatases are essential for normal starch degradation in plants and glycogen metabolism in mammals. Here we develop two chromogenic methods for the detection of glucan phosphatase activity in situ after non denaturing poliacrylamide gel electrophoresis; one method uses pNPP and the second one applies BCIP/NBT. The assays are sensitive, fast, simple, reliable and cost-effective preventing the use of radioactive or fluorogenic compounds. Taking advantage of an efficient separation method combined with the reported assays it is possible to obtain information about oligomeric state of the active enzymes as well as to simultaneously detect glucan substrate binding and phosphatase activity.

Recuperación y diferenciación de aeromonas con CromoCen AE y CromoCen AGN / Recovery and differentiation of Aeromonas with CromoCen AE and CromoCen AGN

INTRODUCCIÓN: en la actualidad las especies del género Aeromonas han emergido como un problema de salud pública, son ellas los agentes etiológicos de las enfermedades diarreicas con el aumento de la atención médica por años. Los procedimientos convencionales para su diagnóstico son muy engorrosos, laboriosos y duraderos. Una nueva metodología que emplea medios de cultivo cromogénicos ha permitido la simplificación y aceleración de su diagnóstico, que ofrece resultados altamente específicos. OBJETIVO: estudiar el efecto de la combinación de diferentes agentes selectivos de los microorganismos grampositivos sobre el aumento de la capacidad de recuperación, cuantificación y diferenciación de las especies de Aeromonas. MÉTODOS: se estudió el efecto inhibidor de la combinación de agentes selectivos (desoxicolato de sodio (0,05-0,2 g·L-1), sales biliares (0,65 g·L-1), verde brillante (0,025-0,03 g·L-1), cristal violeta (0,001-0,01 g·L-1) y sulfito de sodio (0,8 g·L-1) sobre los microorganismos grampositivos, así como la capacidad de recuperación, cuantificación y diferenciación de las especies de Aeromonas. Como base se utilizó la formulación de CromoCen AGN, sin el desoxicolato de sodio. RESULTADOS: los valores de las productividades de los medios CromoCen AE y CromoCen AGN a partir del inóculo 1,5 × 102 UFC·mL-1 resultaron, para: A. hydrophila 116,8 % y 23,9 %, A. caviae 100,8 % y 3,95 %, A. bestiarium 93,6 % y 28,8 %, A. culicicola 85,1 % y 66,12 %, A. veronii 116,7 % y 59,2 %, A. popoffi 86,56 % y 13,2 %, A. trota 94,8 % y 11,25 % y para A. eucrinophila 103,9 % y 2,80 %. La nueva composición cromogénica logró la diferenciación de los microorganismos por sus características culturales: color, forma, superficie, bordes en las colonias y proteólisis del medio circundante. CONCLUSIONES: la combinación de diferentes agentes selectivos para la inhibición de los microorganismos grampositivos coadyuvo el aumento de la capacidad de recuperación, cuantificación y diferenciación de las especies de Aeromonas.

INTRODUCTION: Species of the genus Aeromonas are a current public health problem, for they are the etiological agents responsible for the growing incidence of diarrheal diseases requiring medical care. Conventional procedures for their diagnosis are very complicated, laborious and time-consuming. A new methodology based on the use of chromogenic culture media allows diagnostic simplification and acceleration, yielding highly specific results. OBJECTIVE: Study the effect of combining several selective agents for gram-positive microorganisms upon an increased capacity for recovery, quantification and differentiation of Aeromonas species. METHODS: Assessment was conducted of the inhibiting effect of combined selective agents (sodium deoxycholate (0.05-0.2 g·L-1), bile salts (0.65 g·L-1), brilliant green (0.025-0.03 g·L-1), crystal violet (0.001-0.01 g·L-1) and sodium sulfite (0.8 g·L-1)) on gram-positive microorganisms, as well as their capacity for recovery, quantification and differentiation of Aeromonas species. The base used was the CromoGen AGN formulation without sodium deoxycholate. RESULTS: Productivity values for the media CromoCen AE and CromoCen AGN based on inoculation of 1.5 × 102 CFU·mL-1 were 116.8 % and 23.9 % for A. hydrophila, 100.8 % and 3.95 % for A. caviae, 93.6 % and 28.8 % for A. bestiarium, 85.1 % and 66.12 % for A. culicicola, 116.7 % and 59.2 % for A. veronii, 86.56 % and 13.2 % for A. popoffi, 94.8 % and 11.25 % for A. trota, and 103.9 % and 2.8 0% for A. eucrinophila. The new chromogenic composition enabled differentiation of microorganisms based on their cultural characteristics: color, shape, surface, colony borders and environmental proteolysis. CONCLUSIONS: Combination of various selective agents for the inhibition of grampositive microorganisms led to an increased capacity for recovery, quantification and differentiation of Aeromonas species.

Efecto de una combinación nutritiva y selectiva con sustratos cromogénicos para diagnóstico de cocos grampositivos / Effect of a selective nutrient combination of chromogenic substrates for diagnosis of Grampositive cocci

INTRODUCCIÓN: la reemergencia de infecciones por bacterias grampositivas y el aumento de su patogenicidad, requiere de un diagnóstico microbiológico rápido y certero. En BioCen se desarrolló una composición cromogénica para el aislamiento, cultivo y diferenciación rápida y presuntiva de microorganismos grampositivos por medio de reacciones cromogénicas específicas, donde las bacterias gramnegativas se encuentran inhibidas de manera parcial o total. OBJETIVO: evaluar el efecto de la combinación de bases nutritivas, inhibidores selectivos y sustratos cromogénicos para aumentar la selectivad y capacidad diferencial para especies de los géneros Enterococcus, Streptococcus y Staphylococcus de importancia clínica. MÉTODOS: se evaluaron 21 cepas microbianas de la American Type Culture Collection y 24 aislamientos clínicos de Streptococcus, Enterococcus y Staphylococcus y otros microorganismos gramnegativos. Se evaluaron diferentes combinaciones de bases nutritivas, acetato de talio, ácido nalidíxico y sustratos cromogénicos para la promoción del crecimiento y diferenciación de las bacterias grampositivas. Se evaluó la funcionalidad microbiológica y se le determinaron los parámetros de calidad diagnóstica. RESULTADOS: la combinación de bases nutritivas permitió el desarrollo de los microorganismos grampositivos, en 24 h y su diferenciación por reacciones cromogénicas específicas. El crecimiento de los microorganismos gramnegativos fue inhibido por la acción del acetato de talio (0,014 g·L-1) y ácido nalidíxico (0,008 g·L-1), excepto Proteus mirabilis y Pseudomonas aeruginosa, cuyas características morfológicas no interfieren en la diferenciación de los microorganismos diana. La sensibilidad, especificidad y exactitud diagnósticas fueron del 100 %. CONCLUSIÓN: la combinación de las bases nutritivas, los inhibidores selectivos y los sustratos cromogénicos permitió el desarrollo y diferenciación de especies de los microorganismos evaluados. La inoculación en el medio cromogénico de microorganismos diana y no diana y la diferenciación de aquellas cepas donde se detectó color similar de las colonias por medio de pruebas complementarias rápidas, le confirió al medio elevadas sensibilidad, especificidad y exactitud diagnóstica.

INTRODUCTION: reemergence of Grampositive bacteria infections and the rise of their pathogenicity require a quick and accurate microbiological diagnosis. BioCen has developed a chromogenic composition for isolation, culturing and rapid and presumptive differentiation of gram-positive microorganisms through specific chromogenic reactions in which the inhibition of gramnegative bacteria is partial or total. OBJECTIVE: to evaluate the effect of a combination of nutrient bases, selective inhibitors and chromogenic substrates to increase the selectivity and differential capacity to detect Enterococcus, Streptococcus and Staphylococcus species of clinical importance. METHODS: twenty one microbial strains from the American Type Culture Collection and 24 clinical isolates of Enterococcus, Streptococcus and Staphylococcus and of other gramnegative microorganisms were evaluated. Various combinations of nutrient bases, thallium acetate, nalidixic acid and chromogenic substrates were also assessed for the promotion, growth and differentiation of grampositive bacteria. The microbiological functionality was evaluated whereas the diagnostic quality parameters were determined. RESULTS: the combination of nutrient bases allowed the development of grampositive microorganisms in 24 hours and their differentiation through specific chromogenic reactions. The growth of gramnegative microorganisms was inhibited by the thallium acetate (0.014 g·L-1) and nalidixic acid (0,008 g·L-1) except for Proteus mirabilis and Pseudomonas aeruginosa whose morphological characteristics do not interfere with differentiation of target microorganisms. Sensitivity, specificity and accuracy for diagnosis were 100 %. CONCLUSIONS: the combination of nutrient bases, selective inhibitors and chromogenic substrates allowed the development and differentiation of the evaluated microorganism species. The inoculation of target and non-target microorganisms in the chromogenic medium and the differentiation of those strains where a similar color of the colonies was detected by means of supplementary rapid tests provided the medium with high diagnostic sensitivity, specificity and accuracy.