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1.
Gac. méd. Méx ; 157(1): 113-115, ene.-feb. 2021. tab
Article in Spanish | LILACS | ID: biblio-1279084

ABSTRACT

Resumen Introducción: Clostridioides difficile causa diarrea y colitis pseudomembranosa. Su diagnóstico se realiza con la detección de glutamato-deshidrogenasa (GDH) o las toxinas A y B y se confirma con pruebas de amplificación de ácidos nucleicos. Objetivo: Definir si la determinación de GDH es redundante a la de las toxinas. Métodos: Estudio observacional retrospectivo de muestras fecales de pacientes con sospecha de infección por Clostridioides difficile. Las toxinas y GDH se determinaron mediante inmunocromatografía. Se realizó una simulación bayesiana con los cocientes de probabilidad; se consideró significativo un valor de p < 0.05. Resultados: Se analizaron 329 resultados de GDH y toxinas A y B. Se encontró una prevalencia de infección de Clostridioides difficile de 18.2 %. La sensibilidad y especificidad de la prueba de GDH fue de 0.90 y 0.89, respectivamente. El cociente de probabilidad positivo fue de 8.9 y el negativo, de 0.11. Conclusiones: Un resultado negativo de GDH disminuye considerablemente la probabilidad de infección, pero no la descarta. La detección de toxinas de Clostridioides difficile puede ser necesaria en instituciones donde la amplificación de ácidos nucleicos no es económica o accesible.


Abstract Introduction: Clostridioides difficile causes diarrhea and pseudomembranous colitis. Its diagnosis is made with glutamate dehydrogenase (GDH) or toxins A and B detection and is confirmed with nucleic acid amplification tests. Objective: To define if GDH determination is redundant to that of toxins. Methods: Retrospective, observational study in diarrheal stools of patients with suspected Clostridioides difficile infection. Toxins and GDH were determined by immunochromatography. Bayesian simulation was performed with likelihood ratios; a p-value < 0.05 was regarded as significant. Results: 329 GDH and toxin A and B results were analyzed. Clostridioides difficile infection prevalence was 18.2 %. Sensitivity and specificity of the GDH test were 0.90 and 0.89, respectively. Positive likelihood ratio was 8.9, and negative was 0.11. Conclusions: A negative GDH result considerably reduces the probability of infection but does not rule it out. Clostridioides difficile toxins detection may be necessary in institutions where nucleic acid amplification is not affordable or accessible.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Bacterial Proteins/analysis , Bacterial Toxins/analysis , Clostridioides difficile , Clostridium Infections/diagnosis , Enterotoxins/analysis , Feces/chemistry , Biomarkers/analysis , Likelihood Functions , Prevalence , Retrospective Studies , Bayes Theorem , Sensitivity and Specificity , Clostridium Infections/epidemiology , Diarrhea/microbiology , Feces/enzymology , Glutamate Dehydrogenase/analysis
2.
Braz. j. microbiol ; 49(4): 914-918, Oct.-Dec. 2018. tab
Article in English | LILACS | ID: biblio-974286

ABSTRACT

ABSTRACT The global emergence of carbapenemases led to the need of developing new methods for their rapid detection. The aim of this study was to evaluate the performance of the rapid tests for carbapenemase-producing and non-producing Enterobacteriaceae. Carbapenem non-susceptible Enterobacteriaceae from a surveillance study submitted to a multiplex real time PCR for carbapenemase detection were included in this study. The isolates were subjected to the rapid phenotypic tests Carba NP, Blue-Carba and Carbapenem Inactivation Method (CIM). A total of 83 carbapenemase-producing (43) and non-producing (40) isolates were included in the study. The sensitivity/specificity were 62.7%/97.5%, 95.3%/100%, and 74.4%/97.5% for Carba NP, Blue-Carba and CIM, respectively. Both Carba NP and Blue-Carba presented their final results after 75 min of incubation; the final results for CIM were obtained only after 8 h. Failure to detect OXA-370 carbapenemase was the main problem for Carba NP and CIM assays. As the Blue-Carba presented the highest sensitivity, it can be considered the best screening test. Conversely, CIM might be the easiest to perform, as it does not require special reagents. The early detection of carbapenemases aids to establish infection control measures and prevent carbapenemases to spread reducing the risk of healthcare associated infections and therapeutic failure.


Subject(s)
Humans , Bacterial Proteins/analysis , beta-Lactamases/analysis , Enterobacteriaceae/enzymology , Enterobacteriaceae Infections/microbiology , Enzyme Assays/methods , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , beta-Lactamases/genetics , beta-Lactamases/metabolism , Brazil , Carbapenems/pharmacology , Polymerase Chain Reaction , Sensitivity and Specificity , Enterobacteriaceae/isolation & purification , Enterobacteriaceae/drug effects , Enterobacteriaceae/genetics , Enterobacteriaceae Infections/diagnosis , Anti-Bacterial Agents/pharmacology
3.
Braz. j. microbiol ; 49(4): 885-890, Oct.-Dec. 2018. tab, graf
Article in English | LILACS | ID: biblio-974312

ABSTRACT

ABSTRACT In this study, the performance of the "RESIST-3 O.K.N. K-SeT" (Coris BioConcept, Gembloux, Belgium) immunochromatographic assay was evaluated in 132 Klebsiella pneumoniae comprising 102 carbapenem resistant and 30 carbapenem susceptible isolates. Genotypically known isolates of Gram negative bacteria (n = 22) including various species were also tested by the assay as controls. The isolates tested by the immunochromatographic assay and also were run PCR for bla KPC, bla IMP, bla VIM, bla NDM, and bla OXA-48. The rates of bla NDM, bla OXA-48, and bla KPC in carbapenem resistant isolates were found at 52.9%, 39.2%, and 2.0%, respectively. Both bla NDM and bla OXA-48 were found in six (5.9%) isolates. The results of the assay showed 100% concordance with those obtained by PCR in 132 K. pneumoniae. The agreement between the two methods was found to be identical at the isolate level. The assay also correctly detected all genotypically known isolates of Escherichia coli, Serratia marcescens, Citrobacter freundii, Enterobacter cloacae, K. pneumoniae carrying bla KPC, bla NDM, and/or bla OXA-48. On the other hand, the assay did not exhibit any cross-reaction in control isolates harboring bla IMP and bla VIM. We conclude that the RESIST-3 O.K.N. K-SeT is a reliable, rapid, and user friendly test and we recommend it for routine diagnostic laboratories.


Subject(s)
Humans , Bacterial Proteins/analysis , beta-Lactamases/analysis , Klebsiella Infections/microbiology , Immunoassay/methods , Klebsiella pneumoniae/enzymology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Turkey , beta-Lactamases/metabolism , Carbapenems/pharmacology , Polymerase Chain Reaction , Klebsiella pneumoniae/isolation & purification , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/chemistry , Anti-Bacterial Agents/pharmacology
5.
Braz. j. microbiol ; 48(4): 774-781, Oct.-Dec. 2017. tab, graf
Article in English | LILACS | ID: biblio-889161

ABSTRACT

ABSTRACT Food-borne diseases, caused by the pathogenic bacteria, are highly prevalent in the world. Salmonella is one of the most important bacterial genera responsible for this. Salmonella Enteritidis (SE) is one of the non-typhoid Salmonellae that can be transmitted to human from poultry products, water, and contaminated food. In recent years, new and rapid detection methods such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) have been developed. In this study, recombinant FliC (rFliC) was produced to be used as an antigen. The immunization was conducted in mice with the purified recombinant FliC (rFliC). The mice were subcutaneously immunized with rFliC and elicited significant rFliC specific serum IgG antibodies. An indirect ELISA system was established for the detection of Salmonella Enteritidis. Our results confirmed that the recombinant flagellin can be one of the excellent indicators for the detection of Salmonella Enteritidis.


Subject(s)
Humans , Animals , Mice , Enzyme-Linked Immunosorbent Assay/methods , Flagellin/analysis , Salmonella enteritidis/isolation & purification , Antibodies, Bacterial/analysis , Antibodies, Bacterial/immunology , Antigens, Bacterial/analysis , Antigens, Bacterial/immunology , Bacterial Proteins/analysis , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Flagellin/genetics , Flagellin/immunology , Mice, Inbred BALB C , Salmonella enteritidis/genetics , Salmonella enteritidis/immunology
6.
Braz. j. microbiol ; 48(2): 242-245, April.-June 2017. tab, graf
Article in English | LILACS | ID: biblio-839378

ABSTRACT

Abstract The modified Carba NP test presented here may be a valuable tool for laboratories interested in investigating a large number of carbapenemase-producing bacteria in a less-costly way. The test was evaluated against 48 carbapenemase-producing and carbapenemase-non-producing gram-negative bacteria. No false–positive results were obtained, but false-negative results were observed with OXA-23- and GES-carbapenemase-producing isolates. Aeromonas sp. are not testable by Modified Carba NP.


Subject(s)
Bacterial Proteins/analysis , beta-Lactamases/analysis , Bacteriological Techniques/methods , Gram-Negative Bacteria/enzymology , False Negative Reactions
7.
Braz. j. microbiol ; 48(2): 225-231, April.-June 2017. graf
Article in English | LILACS | ID: biblio-839393

ABSTRACT

Abstract Streptococcus pneumoniae is one of the most frequent opportunistic pathogens worldwide. DNA processing protein A (DprA) is an important factor involved in bacterial uptake and DNA integration into bacterial genome, but its role in S. pneumoniae virulence remains unclear. The aim of this study was to characterize the effects of the pneumococcal dprA gene on the pathogenesis of S. pneumoniae. To construct a dprA-deficient pneumococcal strain, the dprA gene of the S. pneumoniae strain D39 was inactivated. The virulence of this dprA-deficient strain, designated ΔD39, was compared with that of the wild-type strain by evaluating their respective capabilities to adhere to human pulmonary epithelial cells (PEC-A549) and by analyzing their choline-binding protein expression levels. In addition, the expression profiles of genes associated with virulence and host survival assays were also conducted with the mutant and the wild-type strain. Our results indicate that the capability of ΔD39 to adhere to the PEC-A549 airway cells was significantly lower (p < 0.01) compared with D39. Additionally, the 100-KD choline-binding protein was not detected in ΔD39. The addition of competence-stimulating peptide (CSP) lead to a significantly reduction of psaA mRNA expression in the dprA-deficient mutant and an increased level of psaA transcripts in the wild-type strain (p < 0.01). The median survival time of mice intraperitoneally infected with ΔD39 was significantly higher (p < 0.01) than that of mice infected with D39. The results of this study suggest that DprA has a significant effect on virulence characteristics of S. pneumoniae by influencing the expression of choline-binding protein and PsaA.


Subject(s)
Humans , Animals , Pneumococcal Infections/pathology , Streptococcus pneumoniae/pathogenicity , Bacterial Proteins/metabolism , Bacterial Adhesion , Virulence Factors/analysis , Membrane Proteins/metabolism , Pneumococcal Infections/microbiology , Streptococcus pneumoniae/genetics , Bacterial Proteins/analysis , Bacterial Proteins/genetics , Survival Analysis , Cell Line , Virulence Factors/genetics , Disease Models, Animal , Epithelial Cells/microbiology , Gene Knockout Techniques , Membrane Proteins/genetics , Mice
8.
Braz. j. microbiol ; 48(2): 211-217, April.-June 2017. tab, graf
Article in English | LILACS | ID: biblio-839365

ABSTRACT

Abstract Pseudomonas aeruginosa is an opportunistic pathogen that causes frequently nosocomial infections, currently becoming more difficult to treat due to the various resistance mechanisms and different virulence factors. The purpose of this study was to determine the risk factors independently associated with the development of bacteremia by carbapenem-resistant P. aeruginosa, the frequency of virulence genes in metallo-β-lactamases producers and to evaluate their ability to produce biofilm. We conducted a case–control study in the Uberlândia Federal University – Hospital Clinic, Brazil. Polymerase Chain Reaction was performed for metallo-β-lactamases and virulence genes. Adhesion and biofilm assays were done by quantitative tests. Among the 157 strains analyzed, 73.9% were multidrug-resistant, 43.9% were resistant to carbapenems, 16.1% were phenotypically positive for metallo-β-lactamases, and of these, 10.7% were positive for blaSPM gene and 5.3% positive for blaVIM. The multivariable analysis showed that mechanical ventilation, enteral/nasogastric tubes, primary bacteremia with unknown focus, and inappropriate therapy were independent risk factors associated with bacteremia. All tested strains were characterized as strongly biofilm producers. A higher mortality was found among patients with bacteremia by carbapenem-resistant P. aeruginosa strains, associated independently with extrinsic risk factors, however it was not evident the association with the presence of virulence and metallo-β-lactamases genes.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Pseudomonas aeruginosa/genetics , Pseudomonas Infections/epidemiology , Bacterial Proteins/genetics , beta-Lactamases/genetics , Bacteremia/epidemiology , Biofilms/growth & development , beta-Lactam Resistance , Virulence Factors/genetics , Pseudomonas aeruginosa/isolation & purification , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/enzymology , Pseudomonas Infections/microbiology , Bacterial Proteins/analysis , beta-Lactamases/analysis , Brazil/epidemiology , Case-Control Studies , Survival Analysis , Polymerase Chain Reaction , Risk Factors , Bacteremia/microbiology
9.
Braz. oral res. (Online) ; 31: e39, 2017. tab, graf
Article in English | LILACS | ID: biblio-839507

ABSTRACT

Abstract The present study compared IgA specificity against oral streptococci in colostrum and saliva samples. Sixty-two mother-and-child pairs were included; samples of colostrum (C) and saliva (MS) were collected from the mothers and saliva samples were collected from babies (BS). The specificity of IgA against Streptococcus mutans and S. mitis were analyzed by western blot. Only 30% of babies’ samples presented IgA reactivity to S. mutans, while 74 and 80% of MS and C, respectively, presented this response. IgA reactivity to S. mutans virulence antigens (Ag I/II, Gtf and GbpB) in positive samples showed differences between samples for Gtf and especially for GbpB (p < 0.05), but responses to Ag I/II were similar (p > 0.05). The positive response of Gtf-reactive IgA was different between C (90%) and MS (58%) samples (p < 0.05), but did not differ from BS (p > 0.05). GbpB was the least detected, with 48 and 26% of C and MS, and only 5% of BS samples presenting reactivity (p > 0.05). Eight percent of MS and C samples presented identical bands to SM in the same time-point. In conclusion, the differences of IgA response found between C and MS can be due to the different ways of stimulation, proliferation and transportation of IgA in those secretions. The colostrum has high levels of IgA against S. mutans virulence antigens, which could affect the installation and accumulation process of S. mutans, mainly by supplying anti-GbpB IgA to the neonate.


Subject(s)
Humans , Female , Infant, Newborn , Saliva/immunology , Streptococcus mutans/immunology , Immunoglobulin A, Secretory/analysis , Immunoglobulin A, Secretory/immunology , Colostrum/immunology , Streptococcus mitis/immunology , Saliva/microbiology , Bacterial Proteins/analysis , Bacterial Proteins/immunology , Virulence , Enzyme-Linked Immunosorbent Assay , Glycoproteins/analysis , Glycoproteins/immunology , Blotting, Western , Analysis of Variance , Colostrum/microbiology , Glucosyltransferases/analysis , Glucosyltransferases/immunology , Mothers , Antibody Formation/immunology , Antigens, Bacterial/analysis , Antigens, Bacterial/immunology
10.
Braz. j. microbiol ; 47(2): 394-402, Apr.-June 2016. tab, graf
Article in English | LILACS | ID: lil-780824

ABSTRACT

Abstract Clostridium difficile has emerged as an increasingly important nosocomial pathogen and the prime causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis in humans. In addition to toxins A and B, immunological studies using antisera from patients infected with C. difficile have shown that a number of other bacterial factors contribute to the pathogenesis, including surface proteins, which are responsible for adhesion, motility and other interactions with the human host. In this study, various clostridial targets, including FliC, FliD and cell wall protein 66, were expressed and purified. Phage antibody display yielded a large panel of specific recombinant antibodies, which were expressed, purified and characterised. Reactions of the recombinant antibodies with their targets were detected by enzyme-linked immunosorbent assay; and Western blotting suggested that linear rather than conformational epitopes were recognised. Binding of the recombinant antibodies to surface-layer proteins and their components showed strain specificity, with good recognition of proteins from C. difficile 630. However, no reaction was observed for strain R20291—a representative of the 027 ribotype. Binding of the recombinant antibodies to C. difficile M120 extracts indicated that a component of a surface-layer protein of this strain might possess immunoglobulin-binding activities. The recombinant antibodies against FliC and FliD proteins were able to inhibit bacterial motility.


Subject(s)
Humans , Bacterial Proteins/analysis , Clostridioides difficile/genetics , Clostridium Infections/microbiology , Antibodies, Bacterial/analysis , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Recombinant Proteins/analysis , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Gene Expression , Blotting, Western , Clostridioides difficile/isolation & purification , Clostridioides difficile/immunology , Clostridium Infections/diagnosis , Ribotyping , Antibodies, Bacterial/genetics , Antibodies, Bacterial/immunology
11.
Article in English | WPRIM | ID: wpr-149376

ABSTRACT

BACKGROUND/AIMS: To evaluate a new monoclonal antibody for Helicobacter pylori urease in gastric tissue. METHODS: A total of 107 volunteers were enrolled. All subjects underwent a 13C-urea breath test and esophagogastroduodenoscopy. Gastric aspirates were analyzed for pH and ammonia. Six biopsy specimens in the gastric antrum and body were obtained for a rapid urease test and histology. The new monoclonal antibody-based H. pylori urease test (HPU) was performed to rapidly and qualitatively detect urease in two biopsy specimens. RESULTS: H. pylori infection was diagnosed in 73 subjects. The sensitivity and specificity of the HPU was 89% and 74%, respectively. The subjects were divided into two groups: one with true-positive and true-negative HPU results (n = 90) and the other with false-positive and false-negative HPU results (n = 17). Across all subjects, ammonia levels were 900.5 +/- 646.7 and 604.3 +/- 594.3 mumol/L (p > 0.05), and pH was 3.37 +/- 1.64 and 2.82 +/- 1.51 (p > 0.05). Sensitivity was higher in the presence of atrophic gastritis or intestinal metaplasia. CONCLUSIONS: HPU detected H. pylori in approximately 10 min. Gastric aspirate ammonia and pH levels did not affect the test results. Sensitivity was good in the presence of atrophic gastritis or intestinal metaplasia.


Subject(s)
Adult , Antibodies, Monoclonal/immunology , Bacterial Proteins/analysis , Biomarkers/analysis , Biopsy , False Negative Reactions , False Positive Reactions , Female , Gastritis, Atrophic/diagnosis , Helicobacter Infections/diagnosis , Helicobacter pylori/enzymology , Humans , Immunologic Tests , Male , Metaplasia , Middle Aged , Predictive Value of Tests , Pyloric Antrum/microbiology , Reproducibility of Results , Time Factors , Urease/analysis , Workflow
12.
Article in English | WPRIM | ID: wpr-85719

ABSTRACT

We tested correlations between anti-Helicobacter pylori IgG and IgA levels and the urease test, anti-CagA protein antibody, degree of gastritis, and age. In total, 509 children (0-15 years) were enrolled. Subjects were stratified as 0-4 years (n = 132), 5-9 years (n = 274), and 10-15 years (n = 103) and subjected to the urease test, histopathology, ELISA, and western blot using whole-cell lysates of H. pylori strain 51. The positivity rate in the urease test (P = 0.003), the degree of chronic gastritis (P = 0.021), and H. pylori infiltration (P < 0.001) increased with age. The median titer for anti-H. pylori IgG was 732.5 IU/mL at 0-4 years, 689.0 IU/mL at 5-9 years, and 966.0 IU/mL at 10-15 years (P < 0.001); the median titer for anti-H. pylori IgA was 61.0 IU/mL at 0-4 years, 63.5 IU/mL at 5-9 years, and 75.0 IU/mL at 10-15 years (P < 0.001). The CagA-positivity rate was 26.5% at 0-4 years, 36.5% at 5-9 years, and 46.6% at 10-15 years for IgG (P = 0.036), and 11.3% at 0-4 years, 18.6% at 5-9 years, and 23.3% at 10-15 years for IgA (P < 0.001). Anti-H. pylori IgG and IgA titers increased with the urease test grade, chronic gastritis degree, active gastritis, and H. pylori infiltration. Presence of CagA-positivity is well correlated with a high urease test grade and high anti-H. pylori IgG/IgA levels.


Subject(s)
Adolescent , Antibodies, Bacterial/blood , Antigens, Bacterial/analysis , Bacterial Proteins/analysis , Blotting, Western , Child , Child, Preschool , Chronic Disease , Enzyme-Linked Immunosorbent Assay , Female , Gastritis/pathology , Helicobacter Infections/blood , Helicobacter pylori/isolation & purification , Humans , Immunoglobulin A/blood , Immunoglobulin G/blood , Infant , Infant, Newborn , Male , Severity of Illness Index , Urease/metabolism
13.
Salud pública Méx ; 57(4): 352-357, jul.-ago. 2015. ilus, tab
Article in Spanish | LILACS | ID: lil-760500

ABSTRACT

Objetivo. Comparar la concordancia entre cultivo, histología y prueba rápida de la ureasa para el diagnóstico de infección por Helicobacter pylori, así como la relación de hallazgos histopatológicos y frecuencia de positividad entre dichos procedimientos diagnósticos. Material y métodos. Estudio de pruebas diagnósticas. Población de sujetos con endoscopía digestiva y toma de muestras gástricas antrales en un hospital de especialidades en México. Se realizó prueba rápida de la ureasa (una muestra), histología (dos muestras) y cultivo (dos muestras). Análisis estadístico con coeficiente de Kappa. Resultados. Se estudiaron 108 sujetos: 28 (25.9%) hombres y 80 (74.1%) mujeres; la edad promedio fue 49.1 (DE 15.1) años. El coeficiente de Kappa fue 0.729 y 0.377 entre cultivo con histología y prueba rápida de la ureasa respectivamente; asimismo, el coeficiente de Kappa fue 0.565 entre histología y prueba rápida de la ureasa. Conclusiones. La fuerza de concordancia fue mayor entre histología con cultivo y la prueba rápida de la ureasa, por lo cual la histología es lo más recomendable en la práctica clínica para la detección de la infección por Helicobacter pylori.


Objective. Compare the strength of concordance between culture, histology, rapid urease test for diagnosis of Helicobacter pylori infection and histopathological findings relationship and frequency of positivity among such diagnostic procedures. Materials and methods. Diagnostic test study. The study population were subjects with endoscopy and take samples of gastric antral. Rapid urease test (one sample), histology (two samples) and culture (two samples), and histopathological findings of gastric mucosa were performed. Statistical design with Student's t, Fisher exact test, Kappa coefficient. Results. We reviewed 108 subjects, 28 (25.9%) men, 80 (74.1%) women, mean age was 49.1 years (SD 15.1). The Kappa coefficient was 0.729 and 0.377 between culture with histology and rapid urease test, respectively; likewise the Kappa coefficient was 0.565 between histology and rapid urease test. Conclusions. The strength of concordance was higher between histology with culture and rapid urease test; the most recommended being histology in clinical practice for the detection of Helicobacter pylori infection.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Helicobacter pylori/isolation & purification , Helicobacter Infections/diagnosis , Gastritis/diagnosis , Pyloric Antrum/microbiology , Bacterial Proteins/analysis , Urease/analysis , Cross-Sectional Studies , Prospective Studies , Reproducibility of Results , Helicobacter pylori/growth & development , Bacteriological Techniques , Gastroscopy , Gastric Mucosa/microbiology , Gastritis/microbiology
15.
Rev. Soc. Bras. Med. Trop ; 48(2): 162-169, mar-apr/2015. tab, graf
Article in English | LILACS | ID: lil-746224

ABSTRACT

INTRODUCTION: Epidemiological data on the prevalence of extended-spectrum β-lactamases (ESBLs) are scarce in Brazil despite the fact that these data are essential for empirical treatment and control measures. The objective of this study was to evaluate the prevalence of different ESBLs by type and distribution in a tertiary hospital in southern Brazil. METHODS: We evaluated 1,827 enterobacterial isolates between August 2003 and March 2008 isolated from patients at a tertiary hospital. Samples were identified using a Vitek automated system and were confirmed by biochemical testing. The identified ESBL strains were characterized by phenotypic methods, polymerase chain reaction (PCR), and sequencing. Genetic similarities were evaluated by pulsed-field gel electrophoresis. RESULTS: It was 390 (21.3%) ESBL-producing strains, which expressed the ESBLs CTX-M (292), SHV (84), CTX and SHV (10), TEM (2), and PER (2). CONCLUSIONS: The prevalence of ESBL-expressing strains was high, especially in Klebsiella pneumoniae and Enterobacter spp. CTX-M was the predominant type of ESBL observed, and its genetic variability indicates a polyclonal distribution. .


Subject(s)
Humans , Enterobacteriaceae/enzymology , beta-Lactamases/biosynthesis , Brazil , Bacterial Proteins/analysis , Bacterial Proteins/genetics , Electrophoresis, Gel, Pulsed-Field , Enterobacteriaceae/drug effects , Enterobacteriaceae/genetics , Enterobacteriaceae/isolation & purification , Genotype , Microbial Sensitivity Tests , Polymerase Chain Reaction , Tertiary Care Centers
16.
Biol. Res ; 48: 1-11, 2015. ilus, tab
Article in English | LILACS | ID: biblio-950778

ABSTRACT

BACKGROUND: Insects have developed resistance against Bt-transgenic plants. A multi-barrier defense system to weaken their resistance development is now necessary. One such approach is to use fusion protein genes to increase resistance in plants by introducing more Bt genes in combination. The locating the target protein at the point of insect attack will be more effective. It will not mean that the non-green parts of the plants are free of toxic proteins, but it will inflict more damage on the insects because they are at maximum activity in the green parts of plants. RESULTS: Successful cloning was achieved by the amplification of Cry2A, Cry1Ac, and a transit peptide. The appropriate polymerase chain reaction amplification and digested products confirmed that Cry1Ac and Cry2A were successfully cloned in the correct orientation. The appearance of a blue color in sections of infiltrated leaves after 72 hours confirmed the successful expression of the construct in the plant expression system. The overall transformation efficiency was calculated to be 0.7%. The amplification of Cry1Ac-Cry2A and Tp2 showed the successful integration of target genes into the genome of cotton plants. A maximum of 0.673 µg/g tissue of Cry1Ac and 0.568 µg/g tissue of Cry2A was observed in transgenic plants. We obtained 100% mortality in the target insect after 72 hours of feeding the 2nd instar larvae with transgenic plants. The appearance of a yellow color in transgenic cross sections, while absent in the control, through phase contrast microscopy indicated chloroplast localization of the target protein. CONCLUSION: Locating the target protein at the point of insect attack increases insect mortality when compared with that of other transgenic plants. The results of this study will also be of great value from a biosafety point of view.


Subject(s)
Animals , Bacterial Proteins/genetics , Recombinant Fusion Proteins , Chloroplasts/genetics , Insect Control/methods , Gossypium/genetics , Endotoxins/genetics , Hemolysin Proteins/genetics , Lepidoptera , Bacillus thuringiensis , Bacterial Proteins/analysis , Insecticide Resistance/genetics , Immunohistochemistry , Gene Expression/genetics , Chloroplasts/metabolism , Polymerase Chain Reaction , Microscopy, Phase-Contrast , Plants, Genetically Modified , Cloning, Molecular , DNA Primers , Plant Leaves/genetics , Transgenes/physiology , Endotoxins/analysis , Gene Fusion , Hemolysin Proteins/analysis , Insecticides , Larva
17.
Braz. j. phys. ther. (Impr.) ; 18(6): 538-543, 09/01/2015. tab, graf
Article in English | LILACS | ID: lil-732351

ABSTRACT

BACKGROUND: The adapted arcometer has been validated for use in adults. However, its suitability for use in children can be questioned given the structural differences present in these populations. OBJECTIVE: To verify the concurrent validity, repeatability, and intra- and inter-reproducibility of the adapted arcometer for the measurement of the angles of thoracic kyphosis and lumbar lordosis in children. METHOD: Forty children were evaluated using both sagittal radiography of the spine and the adapted arcometer. The evaluations using the arcometer were carried out by two trained evaluators on two different days. In the statistical treatment, the intraclass correlation coefficient (ICC), Pearson's product moment correlation, Spearman's rho, the paired t test, and Wilcoxon's test were used (α=.05). RESULTS: A moderate and significant correlation was found between the x-ray and the adapted arcometer regarding thoracic kyphosis, but no correlation was found regarding lumbar lordosis. Repeatability and intra-evaluator reproducibility of the thoracic kyphosis and lumbar lordosis were confirmed, which was not the case of inter-evaluator reproducibility. CONCLUSION: The adapted arcometer can be used to accompany postural alterations in children made by the same evaluator, while its use for diagnostic purposes and continued evaluation by different evaluators cannot be recommended. Further studies with the aim of adapting this instrument for use in children are recommended. .


Subject(s)
Bacterial Proteins/analysis , Membrane Glycoproteins/analysis , Membrane Glycoproteins/genetics , Prodigiosin/biosynthesis , Serratia marcescens/metabolism , Centrifugation, Density Gradient , Electrophoresis, Polyacrylamide Gel , Molecular Weight , Membrane Glycoproteins/biosynthesis , Solubility , Sarcosine/analogs & derivatives , Serratia marcescens/analysis
18.
Braz. j. microbiol ; 45(3): 933-936, July-Sept. 2014. ilus, tab
Article in English | LILACS | ID: lil-727023

ABSTRACT

This paper presents a method to estimate the biomass of Spirulina cultivated on solid medium with sugarcane bagasse as a support, in view of the difficulty in determining biomass concentrations in bioprocesses, particularly those conducted in semi-solid or solid media. The genus Spirulina of the family Oscillatoriaceae comprises the group of multicellular filamentous cyanobacteria (blue-green microalgae). Spirulina is used as fish feed in aquaculture, as a food supplement, a source of vitamins, pigments, antioxidants and fatty acids. Therefore, its growth parameters are extremely important in studies of the development and optimization of bioprocesses. For studies of biomass growth, Spirulina platensis was cultured on solid medium using sugarcane bagasse as a support. The biomass thus produced was estimated by determining the protein content of the material grown during the process, based on the ratio of dry weight to protein content obtained in the surface growth experiments. The protein content of the biomass grown in Erlenmeyer flasks on surface medium was examined daily to check the influence of culture time on the protein content of the biomass. The biomass showed an average protein content of 42.2%. This methodology enabled the concentration of biomass adhering to the sugarcane bagasse to be estimated from the indirect measurement of the protein content associated with cell growth.


Subject(s)
Biomass , Bacterial Proteins/analysis , Culture Media/chemistry , Microbiological Techniques/methods , Spirulina/chemistry , Spirulina/growth & development , Cellulose , Saccharum
19.
Article in English | WPRIM | ID: wpr-163728

ABSTRACT

We evaluated the new C. DIFF QUIK CHEK COMPLETE (CD COMPLETE; TechLab, USA), which is a rapid membrane enzyme immunoassay that uses a combination of glutamate dehydrogenase (GDH) antigen and toxin A and B detection. A total of 608 consecutive loose stool specimens collected from the patients with suspected Clostridium difficile infection (CDI) from August to December 2012 were subjected to the CD COMPLETE and VIDAS Clostridium difficile A & B (VIDAS CDAB; bioMerieux, France). Their performances were compared with a toxigenic culture as a reference. Stool specimens that were culture-negative and CD COMPLETE- or VIDAS CDAB-positive were analyzed by using an enrichment procedure. In comparison to the toxigenic cultures, sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) were 63.6%, 98.0%, 76.1%, and 96.4%, respectively, for the CD COMPLETE-toxin and 75.5%, 97.4%, 72.5%, and 97.8%, respectively, for the VIDAS CDAB. In comparison to the enriched C. difficile cultures, the sensitivity, specificity, PPV, and NPV for the CD COMPLETE-GDH were 91.0%, 92.4%, 70.5%, and 98.1%, respectively. The CD COMPLETE is a reliable method for the diagnosis of CDI and provides greater sensitivity than toxin enzyme immunoassay alone. Furthermore, the CD COMPLETE-GDH has advantages over direct culture in detecting C. difficile.


Subject(s)
Bacterial Proteins/analysis , Bacterial Toxins/analysis , Clostridium Infections/diagnosis , Clostridioides difficile/enzymology , Enterotoxins/analysis , Feces/microbiology , Glutamate Dehydrogenase/analysis , Humans , Immunoenzyme Techniques , Reagent Kits, Diagnostic , Sensitivity and Specificity
20.
Clin. biomed. res ; 34(1): 47-52, 2014. tab
Article in Portuguese | LILACS | ID: biblio-834451

ABSTRACT

INTRODUÇÃO: A disseminação de Klebsiella pneumoniae carbapenemase (KPC) no Brasil e a recente detecção de bactérias produtoras de New Delhi metalo-β-lactamase (NDM-1) em hospital terciário do sul do Brasil indicam a necessidade da avaliação da presença destas enzimas em enterobactérias resistentes a carbapenêmicos (ERC).OBJETIVO: Avaliar prevalência de carbapenemases nas ERC em quatro hospitais terciários de Porto Alegre, por meio de PCR multiplex em tempo real. MÉTODOS: Estudo descritivo, período de abril a dezembro de 2013. Isolados bacterianos de pacientes internados foram identificados pelo sistema automatizado VITEK 2, com realização do teste de suscetibilidade aos antimicrobianos. Amostras com isolados de ERC foram encaminhadas ao laboratório de referência para análise por PCR em tempo real para identificação de carbapenemases. RESULTADOS: Total de 701 isolados. As ERC predominantes foram K. pneumoniae (47% das amostras positivas) e Enterobacter cloacae (18%). As carbapenemases mais frequentes foram KPC (48%), OXA-48-like (3%) e NDM (2%). Em 47% das amostras não foi identificado o mecanismo de resistência. Isolados originados de culturas de vigilância foram associados com maior positividade para carbapenemases do que isolados de amostras clínicas (p<0,0001). Isolados de ERC pertencentes ao grupo Proteae (Proteus spp., Morganella spp., Providencia spp.) foram associados a menor positividade para carbapenemase do que isolados de outras ERC (p<0,0001). CONCLUSÃO: KPC foi a carbapenemase mais frequentemente detectada. A circulação de uma enzima OXA-48-like foi demonstrada, um achado novo e preocupante. O achado da carbapenemase NDM também é preocupante devido ao seu potencial de disseminação. Esses dados e outros estudos poderão contribuir para um entendimento maior da epidemiologia das ERC.


BACKGROUND: The spread of Klebsiella pneumoniae carbapenemase (KPC) in Brazil and the recent detection of bacteria producing New Delhi metallo-β-lactamase (NDM-1) in a tertiary care hospital in Porto Alegre indicate the need to evaluate the presence of these enzymes in carbapenem-resistant Enterobacteriaceae (CRE). AIM: To evaluate the prevalence of carbapenemases in CRE in four tertiary care hospitals in Porto Alegre using multiplex real-time PCR.METHODS: Descriptive study from April to December 2013. Bacterial isolates from hospitalized patients were identified by VITEK 2 automated system, with antimicrobial susceptibility testing. Samples with CRE isolates were sent to the reference laboratory for analysis using real-time PCR for identification of carbapenemases. RESULTS: Total of 701 isolates. The predominant CRE were K. pneumoniae (46% of positive samples) and Enterobacter cloacae (18%). The most frequent carbapenemases were KPC (48%), OXA-48-like (3%), and NDM-1 (2%). In 47% of the samples no carbapenemase was identified. Isolates originated from surveillance cultures were associated with higher positivity for carbapenemases than isolates from clinical samples (p<0.0001). CRE isolates belonging to the Proteae group (Proteus spp., Morganella spp., Providencia spp.) were associated with less positivity for carbapanemase than isolates of other CRE (p<0.0001). CONCLUSION: KPC was the most frequently detected carbapenemase. The movement of an OXA-48-like enzyme was demonstrated, a novel and worrisome finding. The finding of carbapenemase NDM is also worrisome due to its dissemination potential. These data and further studies may contribute to a better understanding of the epidemiology of CRE.


Subject(s)
Carbapenems/antagonists & inhibitors , Drug Resistance, Bacterial , Enterobacteriaceae/isolation & purification , Enterobacteriaceae Infections/microbiology , Bacterial Proteins/isolation & purification , Cross-Sectional Studies , Clinical Enzyme Tests/methods , Bacterial Proteins/analysis , Multiplex Polymerase Chain Reaction , Real-Time Polymerase Chain Reaction
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