RESUMO
Laccase is an exothermic enzyme with copper in its structure and has an important role in biodegradation by providing oxidation of phenolic compounds and aromatic amines and decomposing lignin. The aim of this study is to reach maximum laccase enzyme activity with minimum cost and energy through optimization studies of Proteusmirabilis isolated from treatment sludge of a textile factory. In order to increase the laccase enzyme activities of the isolates, medium and culture conditions were optimized with the study of carbon (Glucose, Fructose, Sodium Acetate, Carboxymethylcellulose, Xylose) and nitrogen sources (Potassium nitrate, Yeast Extract, Peptone From Soybean, Bacteriological Peptone), incubation time, pH, temperature and Copper(II) sulfate concentration then according to the results obtained. Response Surface Method (RSM) was performed on six different variables with three level. According to the data obtained from the RSM, the maximum laccase enzyme activity is reached at pH 7.77, temperature 30.03oC, 0.5 g/L CuSO4, 0.5 g/L fructose and 0.082 g/L yeast extract conditions. After all, the laccase activity increased 2.7 times. As a result, laccase activity of P. mirabilis can be increased by optimization studies. The information obtained as a result of the literature studies is that the laccase enzymes produced in laboratory and industrial scale are costly and their amounts are low. This study is important in terms of obtaining more laccase activity from P.mirabilis with less cost and energy.
Assuntos
Meios de Cultura , Lacase , Proteus mirabilis , Esgotos , Temperatura , Indústria Têxtil , Lacase/metabolismo , Proteus mirabilis/enzimologia , Proteus mirabilis/isolamento & purificação , Proteus mirabilis/metabolismo , Proteus mirabilis/genética , Esgotos/microbiologia , Concentração de Íons de Hidrogênio , Meios de Cultura/química , Resíduos Industriais , Nitrogênio/metabolismo , Carbono/metabolismo , Biodegradação AmbientalRESUMO
Infection by Proteus mirabilis causes urinary stones and catheter incrustation due to ammonia formed by urease (PMU), one of its virulence factors. Non-enzymatic properties, such as pro-inflammatory and neurotoxic activities, were previously reported for distinct ureases, including that of the gastric pathogen Helicobacter pylori. Here, PMU was assayed on isolated cells to evaluate its non-enzymatic properties. Purified PMU (nanomolar range) was tested in human (platelets, HEK293 and SH-SY5Y) cells, and in murine microglia (BV-2). PMU promoted platelet aggregation. It did not affect cellular viability and no ammonia was detected in the cultures' supernatants. PMU-treated HEK293 cells acquired a pro-inflammatory phenotype, producing reactive oxygen species (ROS) and cytokines IL-1ß and TNF-α. SH-SY5Y cells stimulated with PMU showed high levels of intracellular Ca2+ and ROS production, but unlike BV-2 cells, SH-SY5Y did not synthesize TNF-α and IL-1ß. Texas Red-labeled PMU was found in the cytoplasm and in the nucleus of all cell types. Bioinformatic analysis revealed two bipartite nuclear localization sequences in PMU. We have shown that PMU, besides urinary stone formation, can potentially contribute in other ways to pathogenesis. Our data suggest that PMU triggers pro-inflammatory effects and may affect cells beyond the renal system, indicating a possible role in extra-urinary diseases.
Assuntos
Proteus mirabilis/enzimologia , Proteus mirabilis/patogenicidade , Urease/metabolismo , Urease/toxicidade , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Técnicas In Vitro , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/microbiologia , Modelos Moleculares , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/microbiologia , Neurotoxinas/química , Neurotoxinas/metabolismo , Neurotoxinas/toxicidade , Sinais de Localização Nuclear , Agregação Plaquetária/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Urease/química , Virulência/fisiologiaRESUMO
This study aimed to characterize multidrug-resistant Proteus mirabilis clones carrying a novel class 1 integron-borne blaIMP-1 In1359 was inserted into a large conjugative plasmid that also carried blaCTX-M-2 The production of carbapenemases in Enterobacteriaceae that are intrinsically resistant to polymyxins and tigecycline is very worrisome, representing a serious challenge to clinicians and infection control teams.
Assuntos
Regulação Bacteriana da Expressão Gênica , Integrons , Plasmídeos/química , Proteus mirabilis/genética , beta-Lactamases/genética , Antibacterianos/farmacologia , Brasil/epidemiologia , Carbapenêmicos/farmacologia , Células Clonais , Farmacorresistência Bacteriana Múltipla/genética , Humanos , Testes de Sensibilidade Microbiana , Plasmídeos/metabolismo , Polimixinas/farmacologia , Infecções por Proteus/tratamento farmacológico , Infecções por Proteus/epidemiologia , Infecções por Proteus/microbiologia , Infecções por Proteus/transmissão , Proteus mirabilis/efeitos dos fármacos , Proteus mirabilis/enzimologia , Proteus mirabilis/isolamento & purificação , Centros de Atenção Terciária , Tigeciclina/farmacologia , beta-Lactamases/metabolismoRESUMO
INTRODUCTION: Infections caused by Klebsiella pneumoniae carbapenemase (KPC)-producing isolates pose a major worldwide public health problem today. METHODS: A carbapenem-resistant Proteus mirabilis clinical isolate was investigated for plasmid profiles and the occurrence of ß-lactamase genes. RESULTS: The isolate exhibited resistance to ertapenem and imipenem and was susceptible to meropenem, polymyxin, and tigecycline. Five plasmids were identified in this isolate. DNA sequencing analysis revealed the presence of bla KPC-2 and bla TEM-1 genes. An additional PCR using plasmid DNA confirmed that bla KPC-2 was present in one of these plasmids. CONCLUSIONS: We report the detection of bla KPC-2 in P. mirabilis in Brazil for the first time. This finding highlights the continuous transfer of bla KPC between bacterial genera, which presents a serious challenge to the prevention of infection by multidrug-resistant bacteria.
Assuntos
Antibacterianos/farmacologia , Proteus mirabilis/enzimologia , beta-Lactamases/genética , Brasil , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Humanos , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase , Proteus mirabilis/efeitos dos fármacosRESUMO
The aims of this study were to evaluate the in vitro activity of extended-spectrum cephalosporins (ESC) in non-inducible AmpC enterobacteria throµgh phenotypic and genotypic characterization of the mechanisms of resistance (ESBL, plasmid-mediated AmpC and KPC) and to evaluate the interpretation criteria proposed by the existing recommendations and the new breakpoints established by the CLSI and the EUCAST. Susceptibility tests and PCR multiplex for b/aSHV and b/aCTX-M and amplification using specific primers was performed. One hundred sixty nine resistant isolates: K/ebsie//a pneumoniae (95), Escherichia co/i (55), and Proteus mirabi/is (19) were recovered. ESC resistance was 56.2 %, 32.6%, and 11.2 %, respectively. ESBL was detected in 152 (90 %) isolates, plasmid-mediated AmpC in 12 (7 %) and KPC in 5 (3 %). The CLSI 2009 recommendations and the breakpoints sµggested by the CLSI 2010 and the EUCAST for ceftriaxone were efficacious to detect ESBL, whereas the different breakpoints for ceftazidime presented discrepancies. The CLSI 2010 breakpoints only detected 55 % of the ESBL-producing isolates due to the endemic presence of CTX-M ESBLs in our country. Regarding the plasmid-mediated AmpC producers, the recommendations of the CLSI 2010 and the EUCAST 2010 proved to be more efficient than the old ones.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Cefalosporinas/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Testes de Sensibilidade Microbiana/normas , Proteus mirabilis/efeitos dos fármacos , beta-Lactamases/genética , Cefepima , Ceftazidima/farmacologia , Ceftriaxona/farmacologia , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Humanos , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Estudos Prospectivos , Infecções por Proteus/microbiologia , Proteus mirabilis/enzimologia , Proteus mirabilis/genética , Proteus mirabilis/isolamento & purificação , Sociedades Científicas/normasRESUMO
Los objetivos de este estudio fueron determinar la actividad in vitro de las cefalosporinas de espectro extendido frente a aislamientos clínicos de enterobacterias sin AmpC inducible y evaluar la utilidad de las normativas propuestas por el CLSI 2009 y de los puntos de corte recomendados por el CLSI 2010 y el EUCAST 2010. El análisis incluye la caracterización feno y genotípica de los mecanismos de resistencia. En todos los aislamientos se realizó un antibiograma semicuantitativo y se determinó la CIM por dilución en agar. Asimismo, se realizó la detección fenotípica de p-lactamasas de espectro extendido (BLEE), de AmpC plasmídica (AmpCp) y de carbapenemasas de tipo KPC. En los aislamientos que fueron resistentes a las cefalosporinas de espectro extendido (CEE) se evaluó, mediante PCR múltiple para b/aSHV y b/aCTX-M y PCR con cebadores específicos, el tipo de p-lactamasa pre-valente y la presencia de KPC. Se recuperaron de pacientes 169 aislamientos resistentes a CEE: 95 de K/ebsie//a pneumoniae, 55 de Escherichia co/i y 19 de Proteus mirabi/is. La resistencia a CEE se verificó en el 56,2 %; 32,6 % y 11,2 % de estos conjuntos de aislamientos, respectivamente. Se detectó el fenotipo BLEE en 152 aislamientos (90 %), el fenotipo AmpCp en 12 (7 %) y el KPC en 5 (3 %). Las recomendaciones del CLSI 2009 y los puntos de corte del CLSI 2010 y del EUCAST 2010 para la ceftriaxona permitieron detectar eficientemente las BLEE, mientras que para la ceftacidima, con los puntos de corte del CLSI 2010 solo se detectó el 55 % de las BLEE. Esta discrepancia en los porcentajes de resistencia a ceftriaxona y a ceftacidima se relaciona con la presencia de CTX-M en nuestro medio. Los nuevos puntos de corte detectaron con mayor eficiencia las enzimas de tipo AmpCp.(AU)
The aims of this study were to evaluate the in vitro activity of extended-spectrum cephalosporins (ESC) in non-inducible AmpC enterobacteria throµgh phenotypic and genotypic characterization of the mechanisms of resistance (ESBL, plasmid-mediated AmpC and KPC) and to evaluate the interpretation criteria proposed by the existing recommendations and the new breakpoints established by the CLSI and the EUCAST. Susceptibility tests and PCR multiplex for b/aSHV and b/aCTX-M and amplification using specific primers was performed. One hundred sixty nine resistant isolates: K/ebsie//a pneumoniae (95), Escherichia co/i (55), and Proteus mirabi/is (19) were recovered. ESC resistance was 56.2 %, 32.6%, and 11.2 %, respectively. ESBL was detected in 152 (90 %) isolates, plasmid-mediated AmpC in 12 (7 %) and KPC in 5 (3 %). The CLSI 2009 recommendations and the breakpoints sµggested by the CLSI 2010 and the EUCAST for ceftriaxone were efficacious to detect ESBL, whereas the different breakpoints for ceftazidime presented discrepancies. The CLSI 2010 breakpoints only detected 55 % of the ESBL-producing isolates due to the endemic presence of CTX-M ESBLs in our country. Regarding the plasmid-mediated AmpC producers, the recommendations of the CLSI 2010 and the EUCAST 2010 proved to be more efficient than the old ones.(AU)
Assuntos
Humanos , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Cefalosporinas/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli , Klebsiella pneumoniae , Testes de Sensibilidade Microbiana/normas , Proteus mirabilis , beta-Lactamases/genética , Ceftazidima/farmacologia , Ceftriaxona/farmacologia , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Estudos Prospectivos , Infecções por Proteus/microbiologia , Proteus mirabilis/enzimologia , Proteus mirabilis/genética , Proteus mirabilis/isolamento & purificação , Sociedades Científicas/normasRESUMO
Los objetivos de este estudio fueron determinar la actividad in vitro de las cefalosporinas de espectro extendido frente a aislamientos clínicos de enterobacterias sin AmpC inducible y evaluar la utilidad de las normativas propuestas por el CLSI 2009 y de los puntos de corte recomendados por el CLSI 2010 y el EUCAST 2010. El análisis incluye la caracterización feno y genotípica de los mecanismos de resistencia. En todos los aislamientos se realizó un antibiograma semicuantitativo y se determinó la CIM por dilución en agar. Asimismo, se realizó la detección fenotípica de p-lactamasas de espectro extendido (BLEE), de AmpC plasmídica (AmpCp) y de carbapenemasas de tipo KPC. En los aislamientos que fueron resistentes a las cefalosporinas de espectro extendido (CEE) se evaluó, mediante PCR múltiple para b/aSHV y b/aCTX-M y PCR con cebadores específicos, el tipo de p-lactamasa pre-valente y la presencia de KPC. Se recuperaron de pacientes 169 aislamientos resistentes a CEE: 95 de K/ebsie//a pneumoniae, 55 de Escherichia co/i y 19 de Proteus mirabi/is. La resistencia a CEE se verificó en el 56,2 %; 32,6 % y 11,2 % de estos conjuntos de aislamientos, respectivamente. Se detectó el fenotipo BLEE en 152 aislamientos (90 %), el fenotipo AmpCp en 12 (7 %) y el KPC en 5 (3 %). Las recomendaciones del CLSI 2009 y los puntos de corte del CLSI 2010 y del EUCAST 2010 para la ceftriaxona permitieron detectar eficientemente las BLEE, mientras que para la ceftacidima, con los puntos de corte del CLSI 2010 solo se detectó el 55 % de las BLEE. Esta discrepancia en los porcentajes de resistencia a ceftriaxona y a ceftacidima se relaciona con la presencia de CTX-M en nuestro medio. Los nuevos puntos de corte detectaron con mayor eficiencia las enzimas de tipo AmpCp.(AU)
The aims of this study were to evaluate the in vitro activity of extended-spectrum cephalosporins (ESC) in non-inducible AmpC enterobacteria throAgh phenotypic and genotypic characterization of the mechanisms of resistance (ESBL, plasmid-mediated AmpC and KPC) and to evaluate the interpretation criteria proposed by the existing recommendations and the new breakpoints established by the CLSI and the EUCAST. Susceptibility tests and PCR multiplex for b/aSHV and b/aCTX-M and amplification using specific primers was performed. One hundred sixty nine resistant isolates: K/ebsie//a pneumoniae (95), Escherichia co/i (55), and Proteus mirabi/is (19) were recovered. ESC resistance was 56.2 %, 32.6%, and 11.2 %, respectively. ESBL was detected in 152 (90 %) isolates, plasmid-mediated AmpC in 12 (7 %) and KPC in 5 (3 %). The CLSI 2009 recommendations and the breakpoints sAggested by the CLSI 2010 and the EUCAST for ceftriaxone were efficacious to detect ESBL, whereas the different breakpoints for ceftazidime presented discrepancies. The CLSI 2010 breakpoints only detected 55 % of the ESBL-producing isolates due to the endemic presence of CTX-M ESBLs in our country. Regarding the plasmid-mediated AmpC producers, the recommendations of the CLSI 2010 and the EUCAST 2010 proved to be more efficient than the old ones.(AU)
Assuntos
Humanos , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Cefalosporinas/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Testes de Sensibilidade Microbiana/normas , Proteus mirabilis/efeitos dos fármacos , beta-Lactamases/genética , Ceftazidima/farmacologia , Ceftriaxona/farmacologia , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Estudos Prospectivos , Infecções por Proteus/microbiologia , Proteus mirabilis/enzimologia , Proteus mirabilis/genética , Proteus mirabilis/isolamento & purificação , Sociedades Científicas/normasRESUMO
Los objetivos de este estudio fueron determinar la actividad in vitro de las cefalosporinas de espectro extendido frente a aislamientos clínicos de enterobacterias sin AmpC inducible y evaluar la utilidad de las normativas propuestas por el CLSI 2009 y de los puntos de corte recomendados por el CLSI 2010 y el EUCAST 2010. El análisis incluye la caracterización feno y genotípica de los mecanismos de resistencia. En todos los aislamientos se realizó un antibiograma semicuantitativo y se determinó la CIM por dilución en agar. Asimismo, se realizó la detección fenotípica de p-lactamasas de espectro extendido (BLEE), de AmpC plasmídica (AmpCp) y de carbapenemasas de tipo KPC. En los aislamientos que fueron resistentes a las cefalosporinas de espectro extendido (CEE) se evaluó, mediante PCR múltiple para b/aSHV y b/aCTX-M y PCR con cebadores específicos, el tipo de p-lactamasa pre-valente y la presencia de KPC. Se recuperaron de pacientes 169 aislamientos resistentes a CEE: 95 de K/ebsie//a pneumoniae, 55 de Escherichia co/i y 19 de Proteus mirabi/is. La resistencia a CEE se verificó en el 56,2 %; 32,6 % y 11,2 % de estos conjuntos de aislamientos, respectivamente. Se detectó el fenotipo BLEE en 152 aislamientos (90 %), el fenotipo AmpCp en 12 (7 %) y el KPC en 5 (3 %). Las recomendaciones del CLSI 2009 y los puntos de corte del CLSI 2010 y del EUCAST 2010 para la ceftriaxona permitieron detectar eficientemente las BLEE, mientras que para la ceftacidima, con los puntos de corte del CLSI 2010 solo se detectó el 55 % de las BLEE. Esta discrepancia en los porcentajes de resistencia a ceftriaxona y a ceftacidima se relaciona con la presencia de CTX-M en nuestro medio. Los nuevos puntos de corte detectaron con mayor eficiencia las enzimas de tipo AmpCp.
The aims of this study were to evaluate the in vitro activity of extended-spectrum cephalosporins (ESC) in non-inducible AmpC enterobacteria throµgh phenotypic and genotypic characterization of the mechanisms of resistance (ESBL, plasmid-mediated AmpC and KPC) and to evaluate the interpretation criteria proposed by the existing recommendations and the new breakpoints established by the CLSI and the EUCAST. Susceptibility tests and PCR multiplex for b/aSHV and b/aCTX-M and amplification using specific primers was performed. One hundred sixty nine resistant isolates: K/ebsie//a pneumoniae (95), Escherichia co/i (55), and Proteus mirabi/is (19) were recovered. ESC resistance was 56.2 %, 32.6%, and 11.2 %, respectively. ESBL was detected in 152 (90 %) isolates, plasmid-mediated AmpC in 12 (7 %) and KPC in 5 (3 %). The CLSI 2009 recommendations and the breakpoints sµggested by the CLSI 2010 and the EUCAST for ceftriaxone were efficacious to detect ESBL, whereas the different breakpoints for ceftazidime presented discrepancies. The CLSI 2010 breakpoints only detected 55 % of the ESBL-producing isolates due to the endemic presence of CTX-M ESBLs in our country. Regarding the plasmid-mediated AmpC producers, the recommendations of the CLSI 2010 and the EUCAST 2010 proved to be more efficient than the old ones.
Assuntos
Humanos , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Cefalosporinas/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Testes de Sensibilidade Microbiana/normas , Proteus mirabilis/efeitos dos fármacos , beta-Lactamases/genética , Ceftazidima/farmacologia , Ceftriaxona/farmacologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Estudos Prospectivos , Infecções por Proteus/microbiologia , Proteus mirabilis/enzimologia , Proteus mirabilis/genética , Proteus mirabilis/isolamento & purificação , Sociedades Científicas/normasRESUMO
In the last years, Enterobacteriaceae such as Klebsiella pneumoniae, Proteus mirabilis and Escherichia coli, have acquired resistance to third-generation cephalosporins (C3G) because of the presence of plasmid-mediated AmpC ß-lactamases. The aim of this work was to detect plasmid AmpC enzymes and to investigate the predominant types in our region. Between March and July 2009, 733 consecutive isolates of Enterobacteriaceae derived from hospitals and outpatient centers were studied. Susceptibility testing was performed by disk diffusion; one P. mirabilis and three E. coli strains showed resistance to cephamycins (cefoxitin) and C3G. An E-test to determine MIC and a synergy test by aminophenylboronic disks were performed. Enzymatic activity against cefoxitin was confirmed by a microbiological assay. A polymerase chain reaction (PCR) for the detection of plasmid-mediated ampC genes of different groups was performed and a 462-bp amplicon was obtained when using primers directed against the CIT group; the obtained sequences were compared to blaCMY-2 sequences, showing 100% identity. The emergence of CMY-2-type plasmid-mediated AmpC ß-lactamases indicated the importance of implementing systematic monitoring of these resistances to avoid potential clinical and epidemiological consequences.
Assuntos
Proteínas de Bactérias/análise , Escherichia coli/enzimologia , Proteus mirabilis/enzimologia , Fatores R/genética , beta-Lactamases/análise , Sequência de Aminoácidos , Argentina , Proteínas de Bactérias/genética , Cefoxitina/farmacologia , Resistência às Cefalosporinas/genética , Cefalosporinas/farmacologia , DNA Bacteriano/genética , Enterobacteriaceae/enzimologia , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Infecções por Enterobacteriaceae/microbiologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase , Infecções por Proteus/microbiologia , Proteus mirabilis/efeitos dos fármacos , Proteus mirabilis/genética , Proteus mirabilis/crescimento & desenvolvimento , Proteus mirabilis/isolamento & purificação , Homologia de Sequência de Aminoácidos , Infecções Urinárias/microbiologia , beta-Lactamases/química , beta-Lactamases/genéticaRESUMO
OBJECTIVE: To compare the efficacy of four phenotypic methods for the identification of strains producing extended-spectrum ß-lactamases isolated from urine cultures. MATERIALS AND METHODS: Comparative cross-sectional study. 147 strains isolated from positive urine cultures for Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis between January and February 2009 in the National Institute of Health of Children underwent a screening test, those which resulted positive were processed for confirmatory testing through the four phenotypic methods evaluated. RESULTS: Out of the 147 strains, 43 (29.3%) were suspicious in the screening tests. Using the method described by the Clinical and Laboratory Standards Institute (CLSI) as a reference standard for this study, 27 strains (62.8%) were positive, with similar results using Jarlier's method. On the other hand, using Hodge's and tridimensional methods 23 (53.5%) of samples were positive. The evaluation of the confirmation methods in comparison to the one described by CLSI, showed a sensitivity and specificity of 100% for Jarlier's method, on the other hand, for Hodge's and tridimensional methods we found a sensitivity of 85.2% and 100%, respectively. CONCLUSIONS: All the evaluated methods showed a high efficacy, without significant differences, so they could all be used according to the available facilities of each clinical laboratory. Nevertheless, due to its advantages besides the technical aspect, like costs, easiness and feasibility of its application, we recommend the use of Jarlier's method.