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1.
Rev. Soc. Bras. Med. Trop ; 51(1): 88-93, Jan.-Feb. 2018. tab
Article in English | LILACS | ID: biblio-1041448

ABSTRACT

Abstract INTRODUCTION: Here, we determined the genes encoding antibiotic resistance enzymes and virulence factors and evaluated the genetic relationship between Enterobacter spp. isolated from different clinical samples. METHODS: A total of 57 clinical isolates of Enterobacter spp. were tested for the production of extended-spectrum β-lactamases (ESBLs), carbapenemase, and AmpC using phenotypic and genotypic methods. RESULTS: The most common ESBLs and AmpC β-lactamases were bla TEM (63.3%) and bla EBC (57.7%), respectively. The most prevalent virulence gene was rpos (87.7%). The random amplified polymorphic DNA (RAPD) patterns of strains were genetically unrelated. CONCLUSIONS: RAPD polymerase chain reaction analysis revealed high genetic diversity among isolates.


Subject(s)
Humans , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , beta-Lactamases/genetics , Escherichia coli/drug effects , Feces/microbiology , Anti-Bacterial Agents/pharmacology , Phenotype , Bacterial Proteins/drug effects , beta-Lactamases/biosynthesis , Polymerase Chain Reaction , Clone Cells , Drug Resistance, Multiple, Bacterial , beta-Lactams/adverse effects , Escherichia coli/enzymology , Escherichia coli/genetics , Disk Diffusion Antimicrobial Tests , Genotype , Iran
2.
Rev. chil. infectol ; 34(5): 476-484, oct. 2017. tab, graf
Article in Spanish | LILACS | ID: biblio-899745

ABSTRACT

Resumen En la actualidad, la diseminación de enterobacterias productoras de carbapenemasas se considera un grave problema en clínica debido al fracaso en el tratamiento de las infecciones que ellas producen. Entre las carbapenemasas, la enzima KPC se ha diseminado mundialmente y ha sido identificada en las principales especies de enterobacterias relacionadas con infecciones asociadas a la atención en salud, con claro predominio de Klebsiella pneumoniae a nivel mundial. El gen blaKPC es transportado, principalmente, por el transposón Tn4401, detectado en diversas especies de enterobacterias con distintos secuencio-tipo (ST) y diferente origen geográfico. Adicionalmente, se han descrito nuevas plataformas genéticas que se distinguen del Tn4401 original debido a inserciones y deleciones de otros genes. Los plásmidos que albergan el gen blaKPC pueden ser del tipo conjugativo y no conjugativo movilizable, y además contener otros determinantes genéticos de resistencia. Las cepas productoras de KPC pueden presentar diversos niveles de resistencia a los carbapenémicos, debido a la participación de mecanismos adicionales como diferente grado de expresión de porinas y bombas de expulsión asociados con la producción de β-lactamasas de espectro extendido y/o AmpC. Sin embargo, las carbapenemasas, con KPC como la enzima más frecuente, otorgan grados de resistencia más elevados.


The dissemination of carbapenemase-producing Enterobacteriaceae is currently considered a serious clinical problem due to the failure in the treatment of infections produced by them. Among the carbapenemases, the enzyme KPC has spread worldwide and has been identified in the main enterobacterial species related with healthcareassociated infections, although Klebsiella pneumoniae is the predominant specie. The blaKPC gene is transported, mainly by the transposon Tn4401, detected in various enterobacterial species of different sequence types (ST) and geographical origin. In addition, new genetic platforms that are distinguished, from Tn4401 because of insertions or deletions of other genes have been described. Plasmids containing the blaKPC gene can be conjugative and mobilizable non-conjugative plasmids, and can carry other genetic determinants of resistance. The KPC-producing strains may have different levels of resistance to carbapenems, due to the involvement of additional mechanisms such as different expression levels of porins and efflux pumps associated with the production of extended spectrum β-lactamases and/or AmpC. However, the carbapenemases, with KPC as the most common enzyme, provide higher levels of resistance.


Subject(s)
Bacterial Proteins/biosynthesis , beta-Lactamases/biosynthesis , Klebsiella pneumoniae/enzymology , Bacterial Proteins/genetics , beta-Lactamases/genetics , Microbial Sensitivity Tests , Drug Resistance, Bacterial/genetics , Carbapenem-Resistant Enterobacteriaceae , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/genetics , Anti-Bacterial Agents/pharmacology
3.
Rev. chil. infectol ; 34(4): 326-332, ago. 2017. tab, graf
Article in Spanish | LILACS | ID: biblio-899719

ABSTRACT

Resumen Introducción: La detección de bacilos gramnegativos productores de carbapenemasas es compleja, existiendo actualmente varios test disponibles. La confirmación mediante la caracterización molecular de la enzima no está disponible en todos los laboratorios del país. Objetivo: Plantear una estrategia rápida, eficiente y sencilla para la detección y confirmación de carbapenemasas en cepas de bacilos gramnegativos. Material y Métodos: Se utilizaron 39 aislados productores y ocho no productores de carbapenemasas para evaluar los test fenotípicos Carba NP, CarbAcineto NP, Blue-Carba y validar el test molecular Xpert® Carba-R directo de la colonia en comparación con RPC convencional. Resultados: La sensibilidad para Carba NP, CarbAcineto NP y Blue-Carba fue de 79,5; 87,2 y 84,6%, respectivamente; mientras que la especificidad fue de 100; 100 y 87,5%, respectivamente. La concordancia entre RPC convencional y Xpert® Carba-R fue de 100%. El límite de detección para Xpert® Carba-R fue diferente según el tipo de carbapenemasa: 40,8 ufc/reacción par KPC y NDM y 30,6 ufc/reacción para VIM. Discusión: En aislados con susceptibilidad disminuida a carbapenémicos se propone realizar un tamizaje con CarbAcineto NP, para luego caracterizar la carbapenemasa con Xpert® Carba-R y adoptar las medidas de contención específica: para cada caso.


Introduction: The detection of carbapenemase-producing gram negative bacilli is complicated, because there are available multiple options of test. The confirmation of the enzyme by molecular characterization is not available in all laboratories in our country. Objective: To propose a fast, efficient and simple strategy to detect and confirm CPB. Materials and Methods: 39 CPB isolates and 8 non-producing were used to evaluate the phenotypic test Carba NP, CarbAcineto NP and Blue-Carba, validating the test Xpert® Carba-R, to be used directly with bacterial colonies with conventional PCR. Results: The sensitivity of Carba NP, CarbAcineto NP and Blue-Carba was 79,5; 87,2 y 84,6%, respectively; and specificity was 79.5; 87.2 and 84.6%, respectively. The limit of detection of Xpert® Carba-R was different for each carbapenemasa: 40.8 ufc/reaction to KPC and NDM and 30.6 ufc/reaction to VIM. Discussion: On isolates with decreased susceptibility to carbapenems we propose to use as screening the test CarbAcineto NP, follow by Xpert®Carba-R to characterize the carbapenemase and adopt specific infection control measures.


Subject(s)
Humans , Bacterial Proteins/biosynthesis , beta-Lactamases/biosynthesis , Gram-Negative Aerobic Bacteria/isolation & purification , Gram-Negative Aerobic Bacteria/enzymology , Anti-Bacterial Agents/pharmacology , Phenotype , Microbial Sensitivity Tests , Polymerase Chain Reaction , Bacteriological Techniques , Sensitivity and Specificity , Gram-Negative Aerobic Bacteria/drug effects
4.
Braz. j. microbiol ; 47(3): 647-657, July-Sept. 2016. tab, graf
Article in English | LILACS | ID: lil-788974

ABSTRACT

ABSTRACT The purpose of this study was to isolate, purify and optimize the production conditions of an organic solvent tolerant and thermostable lipase from Acinetobacter sp. AU07 isolated from distillery waste. The lipase production was optimized by response surface methodology, and a maximum production of 14.5 U/mL was observed at 30 ºC and pH 7, using a 0.5% (v/v) inoculum, 2% (v/v) castor oil (inducer), and agitation 150 rpm. The optimized conditions from the shake flask experiments were validated in a 3 L lab scale bioreactor, and the lipase production increased to 48 U/mL. The enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography and the overall yield was 36%. SDS-PAGE indicated a molecular weight of 45 kDa for the purified protein, and Matrix assisted laser desorption/ionization time of flight analysis of the purified lipase showed sequence similarity with GDSL family of lipases. The optimum temperature and pH for activity of the enzyme was found to be 50 ºC and 8.0, respectively. The lipase was completely inhibited by phenylmethylsulfonyl fluoride but minimal inhibition was observed when incubated with ethylenediaminetetraacetic acid and dithiothreitol. The enzyme was stable in the presence of non-polar hydrophobic solvents. Detergents like SDS inhibited enzyme activity; however, there was minimal loss of enzyme activity when incubated with hydrogen peroxide, Tween 80 and Triton X-100. The kinetic constants (Km and Vmax) revealed that the hydrolytic activity of the lipase was specific to moderate chain fatty acid esters. The Vmax, Km and Vmax/Km ratio of the enzyme were 16.98 U/mg, 0.51 mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate.


Subject(s)
Organic Chemicals , Solvents , Bacterial Proteins/isolation & purification , Bacterial Proteins/biosynthesis , Acinetobacter/enzymology , Lipase/isolation & purification , Lipase/biosynthesis , Organic Chemicals/chemistry , Solvents/chemistry , Substrate Specificity , Temperature , Bacterial Proteins/chemistry , Enzyme Stability , Kinetics , Chromatography, Ion Exchange , Enzyme Activation , Extracellular Space/enzymology , Hydrogen-Ion Concentration , Ions , Lipase/chemistry , Lipolysis , Metals , Molecular Weight
5.
Braz. j. med. biol. res ; 49(4): e5178, 2016. graf
Article in English | LILACS | ID: biblio-951664

ABSTRACT

A bacterial strain (PAP04) isolated from cattle farm soil was shown to produce an extracellular, solvent-stable protease. Sequence analysis using 16S rRNA showed that this strain was highly homologous (99%) to Brevibacillus laterosporus. Growth conditions that optimize protease production in this strain were determined as maltose (carbon source), skim milk (nitrogen source), pH 7.0, 40°C temperature, and 48 h incubation. Overall, conditions were optimized to yield a 5.91-fold higher production of protease compared to standard conditions. Furthermore, the stability of the enzyme in organic solvents was assessed by incubation for 2 weeks in solutions containing 50% concentration of various organic solvents. The enzyme retained activity in all tested solvents except ethanol; however, the protease activity was stimulated in benzene (74%) followed by acetone (63%) and chloroform (54.8%). In addition, the plate assay and zymography results also confirmed the stability of the PAP04 protease in various organic solvents. The organic solvent stability of this protease at high (50%) concentrations of solvents makes it an alternative catalyst for peptide synthesis in non-aqueous media.


Subject(s)
Animals , Organic Chemicals/metabolism , Peptide Hydrolases/biosynthesis , Bacterial Proteins/biosynthesis , Brevibacillus/metabolism , Peptide Hydrolases/chemistry , Soil Microbiology , Solvents , Temperature , Time Factors , Bacterial Proteins/chemistry , Enzyme Stability , Cattle , Culture Media , Electrophoresis, Polyacrylamide Gel , Brevibacillus/isolation & purification , Hydrogen-Ion Concentration
7.
Braz. j. infect. dis ; 18(6): 678-680, Nov-Dec/2014.
Article in English | LILACS | ID: lil-730427

ABSTRACT

Nosocomial infections caused by carbapenem-resistant Acinetobacter baumannii isolates have reached epidemic levels in past decades. Currently this microorganism is responsible for outbreaks of difficult eradication and with high mortality rates worldwide. We herein report a rare case of an OXA-72-producing A. baumannii isolate colonizing a 47-year-old male patient with peritonitis due to abdominal stab wound, four years earlier than the first report of this carbapenemase in Acinetobacter pittii in Colombia. Although OXA-72 presents a low prevalence compared with OXA-23, our study demonstrated that A. baumannii isolates carrying the blaOXA-72 gene were present in the hospital environment in Colombia and could act as a reservoir for further spread to other Acinetobacter species, like A. pittii, causing carbapenem-resistance.


Subject(s)
Humans , Male , Middle Aged , Acinetobacter Infections/microbiology , Acinetobacter baumannii/isolation & purification , Bacterial Proteins/biosynthesis , beta-Lactamases/biosynthesis , Acinetobacter baumannii/drug effects , Acinetobacter baumannii/enzymology , Colombia , Microbial Sensitivity Tests , Molecular Typing
9.
Einstein (Säo Paulo) ; 12(3): 282-286, Jul-Sep/2014. tab, graf
Article in English | LILACS | ID: lil-723932

ABSTRACT

Objective To analyze the profile of patients with microorganisms resistant to carbapenems, and the prevalence of the enzyme Klebsiella pneumoniae carbapenemase in interobacteriaceae. Methods Retrospective descriptive study. From the isolation in bacteriological tests ordered by clinicians, we described the clinical and epidemiological characteristics of patients with enterobacteria resistants to carbapenems at a university hospital, between March and October 2013. Results We included 47 isolated patients in this study, all exhibiting resistance to carbapenems, including 9 patients who were confirmed as infected/colonized with K. pneumoniae carbapenemase. Isolation in tracheal aspirates (12; 25.5%) predominated. The resistance to ertapenem, meropenem, and imipenem was 91.5%, 83.0% and 80.0%, respectively. Aminoglycosides was the class of antimicrobials that showed the highest sensitivity, 91.5% being sensitive to amikacin and 57.4% to gentamicin. Conclusion The K. pneumoniae carbapenemase was an important agent in graun isotaling in hospital intection. The limited therapeutic options emphasize the need for rapid laboratory detection, as well as the implementation of measures to prevent and control the spread of these pathogens. .


Objetivo Analisar o perfil dos pacientes que apresentaram microrganismos com resistência aos carbapenêmicos, e a prevalência da enzima Klebsiella pneumoniae carbapenemase em enterobactérias. Métodos Estudo retrospectivo descritivo. A partir do isolamento em exames bacteriológicos solicitados pelos clínicos, descrevemos as características clínicas e epidemiológicas dos pacientes que apresentaram enterobactérias resistentes aos carbapenêmicos entre março e outubro de 2013 em um hospital universitário. Resultados Foram incluídos 47 pacientes isolados, todos apresentando resistência aos carbapenêmicos, dos quais 9 tiveram confirmação de infecção/colonização por K. pneumoniae carbapenemase. Ocorreu predomínio de isolamento em aspirados traqueais (12; 25,5%). A resistência ao ertapenem, meropenem e imipenem foi de 91,5%, 83,0% e 80,0%, respectivamente. Os aminoglicosídeos foram a classe de antimicrobianos que apresentou maior sensibilidade, sendo 91,5% sensível a amicacina e 57,4% a gentamicina. Conclusão A K. pneumoniae carbapenemase constituiu um importante patógeno hospitalar em isolamento crescente nesse nosocômio. As limitadas opções terapêuticas reforçam a necessidade de uma rápida detecção laboratorial, assim como a implementação de medidas de prevenção e controle da disseminação desses patógenos. .


Subject(s)
Adolescent , Adult , Child , Female , Humans , Male , Young Adult , Bacterial Proteins/biosynthesis , Cross Infection/microbiology , Drug Resistance, Bacterial , Enterobacteriaceae/enzymology , Klebsiella Infections/enzymology , Klebsiella pneumoniae/enzymology , beta-Lactamases/biosynthesis , Carbapenems/therapeutic use , Enterobacteriaceae Infections/enzymology , Enterobacteriaceae Infections/microbiology , Enterobacteriaceae/drug effects , Enterobacteriaceae/isolation & purification , Hospitals, Teaching/statistics & numerical data , Klebsiella Infections/microbiology , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/isolation & purification , Microbial Sensitivity Tests , Polymerase Chain Reaction , Retrospective Studies
10.
Indian J Exp Biol ; 2014 Aug; 52(8): 773-780
Article in English | IMSEAR | ID: sea-153758

ABSTRACT

A major impediment in chemotherapy of Tuberculosis (TB) is the persistence of M. tuberculosis in a latent or dormant state, possibly perpetuated by paucity of oxygen within the lung granuloma. Proteome analysis of the anaerobically persisting microbe could therefore provide novel targets for drugs against latent TB infection (LTBI). An Indian clinical isolate of M. tuberculosis was cultured under aerobic and anaerobic conditions following Wayne’s hypoxia model and its cytosolic proteins were resolved by two-dimensional gel electrophoresis (2DE). Peptide mass fingerprinting of 32 differentially expressed spots using MALDI TOF-TOF MS-MS resulted in identification of 23 proteins. Under the anaerobic culture conditions, expression of 12 of these proteins was highly suppressed (>2 fold reduction in spot volumes), with 4 of them (GrpE, CanB, MoxR1 and Eis) appearing as completely suppressed since corresponding spots were not detectable in the anaerobic sample. On the other hand, 4 proteins were highly expressed, with two of them (Wag31 and GroES) being uniquely expressed under anaerobic conditions. Suppression of Eis could make the anaerobically persisting bacilli susceptible to the aminoglycoside antibiotics which are known to be acetylated and inactivated by Eis. Although all 4 over-expressed proteins can be considered as putative drug targets for LTBI, Wag31 appears particularly interesting in view of its role in the cell wall biogenesis.


Subject(s)
Anaerobiosis , Antigens, Bacterial/biosynthesis , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/biosynthesis , Cell Culture Techniques , Cytosol/metabolism , Gene Expression Regulation, Bacterial , Heat-Shock Proteins/antagonists & inhibitors , Heat-Shock Proteins/biosynthesis , Humans , Latent Tuberculosis/drug therapy , Latent Tuberculosis/microbiology , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/pathogenicity , Proteome , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
12.
Biomédica (Bogotá) ; 33(4): 546-553, Dec. 2013. ilus
Article in English | LILACS | ID: lil-700473

ABSTRACT

Introduction: Helicobacter pylori strains expressing cytotoxic CagA protein are more commonly associated with peptic ulceration, atrophic gastritis and gastric adenocarcinoma than those lacking CagA. Determination of anti-CagA antibodies, therefore, acquires a relevant clinical significance in the serological detection of H. pylori infection and disease risk prediction. However, the CagA-serology has been questioned due to the differences found in their performance evaluations in different populations. Objective: To obtain a recombinant CagA fragment useful for serodiagnosis of H. pylori infection Methods: A fragment of the cagA gene was cloned into a prokaryotic T7 RNA polymerase expression vector. A recombinant C-terminal His 6 -tagged CagA was expressed, subsequently solubilized with urea and purified by immobilized metal affinity chromatography. The performance of the recombinant protein was evaluated using 180 human serum samples with an in-house Western blot assay compared to the Helicoblot 2.1 reference test. Results: The expressed His 6 -tagged CagA showed an immunoreactive 80kDa band as was revealed by SDS-PAGE and Western blot analysis using two different specific anti-CagA polyclonal antibodies. The recombinant protein was successfully purified obtaining a 93% of purity. The performance analysis of the purified recombinant antigen showed good immunoreactivity and exhibited values of sensitivity, specificity and accuracy of 88.1%, 100% and 92.7%, respectively. Conclusion: The CagA fragment of the study may constitute a useful tool for serological diagnosis of CagA-positive H. pylori infection.


Introducción. Las cepas de Helicobacter pylori que expresan la citotoxina CagA, se asocian más frecuentemente con úlcera péptica, gastritis atrófica y adenocarcinoma gástrico que las que carecen de esta citotoxina. Por lo anterior, el determinar la presencia de anticuerpos anti-CagA adquiere gran importancia clínica en la detección serológica de la infección por H. pylori y la predicción del riesgo de enfermedades. Sin embargo, los métodos serológicos que emplean CagA han sido cuestionados debido a las diferencias encontradas en las evaluaciones de su desempeño en diversas poblaciones. Objetivo. Obtener un fragmento recombinante de la proteína CagA para el serodiagnóstico de la infección por H. pylori . Materiales y métodos. Un fragmento del gen cagA fue clonado en un vector de expresión procariota que contenía el promotor de la T7 ARN polimerasa. El fragmento de la proteína CagA con seis histidinas en la región C-terminal, se expresó, se solubilizó con urea y se purificó por cromatografía de afinidad con iones metálicos inmovilizados. El desempeño de la proteína recombinante se evaluó empleando un método in house de Western Blot y 180 sueros humanos. Los resultados se compararon con la prueba de referencia Helicoblot 2.1. Resultados. La proteína CagA expresada mostró una banda inmunorreactiva de 80 kDa en el Western Blot al emplear dos anticuerpos policlonales anti-CagA específicos. La proteína recombinante fue purificada hasta un 93 % de pureza y el análisis de desempeño del antígeno recombinante purificado mostró buena inmunorreacción y exhibió valores de sensibilidad, especificidad y exactitud de 88,1 %, 100 % y 92,7 %, respectivamente. Conclusiones. El fragmento de la proteína CagA del estudio puede constituir una herramienta útil para el diagnóstico serológico de la infección por cepas de H. pylori positivas para CagA.


Subject(s)
Adolescent , Adult , Humans , Middle Aged , Young Adult , Antigens, Bacterial/blood , Bacterial Proteins/blood , Helicobacter pylori , Helicobacter Infections/blood , Helicobacter Infections/diagnosis , Antigens, Bacterial/biosynthesis , Antigens, Bacterial/genetics , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Cloning, Molecular , Gene Expression , Helicobacter pylori/genetics , Helicobacter pylori/metabolism , Recombinant Proteins , Serologic Tests
13.
Rev. argent. microbiol ; 45(3): 185-90, set. 2013.
Article in Spanish | LILACS, BINACIS | ID: biblio-1171786

ABSTRACT

Sixty-four colistin-resistant Klebsiella pneumoniae isolates recovered from clinical specimens from 57 patients admitted to Hospital de Clinicas Jose de San Martin during the period 2010-2012 were studied to describe the microbiological and epidemiological characteristics and factors associated with the emergence of colistin-resistance. Fifty-four colistin-susceptible K. pneumoniae isolates from the same period were also included in the study. The genetic relatedness among the isolates was studied by a PCR assay. Fifty percent of the resistant isolates were KPC-2 producers, 45.3


were ESBL producers and 4.7


only showed resistance to aminopenicilins. All KPC-producers (resistant and susceptible to colistin) were genotipically indistinguishable except for one, whereas the presence of 7 clonal types, which were different from the ones identified in the colistin-susceptible isolates, were detected among ESBL producers. The previous use of colistin was the main factor associated with the acquisition of resistance, and in the case of non-KPC producers the stay in ICU was another significant factor observed. Colistin resistance emerged in our hospital in the year 2010, reaching 3


in nosocomial isolates and maintaining this rate in successive years, due to the selection of resistant subpopulations in the epidemic clonal type in KPC-producers and due to the dispersion of colistin-resistant clonal types in non-KPC producing-isolates.


Subject(s)
Colistin/pharmacology , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/enzymology , Bacterial Proteins/biosynthesis , beta-Lactamases/biosynthesis , Drug Resistance, Bacterial , Female , Humans , Klebsiella Infections/epidemiology , Male , Bacterial Proteins/classification , beta-Lactamases/classification
14.
Indian J Exp Biol ; 2013 Apr; 51(4): 336-342
Article in English | IMSEAR | ID: sea-147599

ABSTRACT

Proteases are shown to have greener mode of application in leather processing for dehairing of goat skins and cow hides. Production of protease by submerged fermentation with potent activity is reported using a new isolate P. aeruginosa MTCC 10501. The production parameters were optimized by statistical methods such as Plackett-Burman and response surface methodology. The optimized production medium contained (g/L); tryptone, 2.5; yeast extract, 3.0; skim milk 30.0; dextrose 1.0; inoculum concentration 4%: initial pH 6.0; incubation temperature 30 °C and optimum production at 48 h with protease activity of 7.6 U/mL. The protease had the following characteristics: pH optima, 9.0; temperature optima 50 °C; pH stability between 5.0-10.0 and temperature stability between 10-40 °C. The protease was observed to have high potential for dehairing of goat skins in the pre- tanning process comparable to that of the chemical process as evidenced by histology. The method offers cleaner processing using enzyme only instead of toxic chemicals in the pre-tanning process of leather manufacture.


Subject(s)
Animals , Bacterial Proteins/biosynthesis , Culture Media , Endopeptidases/biosynthesis , Goats , Hydrogen-Ion Concentration , Industrial Microbiology/methods , Industry , Models, Statistical , Peptones/chemistry , Pressure , Pseudomonas aeruginosa/metabolism , Tanning , Temperature , Yeasts/chemistry
15.
Einstein (Säo Paulo) ; 10(4): 439-441, Oct.-Dec. 2012.
Article in English | LILACS | ID: lil-662468

ABSTRACT

OBJECTIVE: To evaluate ertapenem disk performance to predict Klebsiella pneumonie carbapenemase production by Gram-negative bacilli. METHODS: All Gram-negative bacilli isolated between January 2010 and June 2011 were tested by disk diffusion (OxoidTM) for sensitivity to ertapenem, meropenem and imipenem. Resistant or intermediate sensitivity strains (diameter <22 mm for ertapenem) were also tested for the blaKPC gene by polymerase chain reaction. Disk predictive positive value for Klebsiella pneumoniae carbapenemase and specificity were calculated. RESULTS: Out of the 21839 cultures performed, 3010 (13.78%) were positive, and Gram-negative bacilli were isolated in 708 (23.52%) of them. Zone of inhibition diameter for ertapenem disk was <22 mm for 111 isolates, representing 15.7% of all Gram-negative isolates. The PCR assay for blaKPC detected 40 Klebsiella pneumoniae carbapenemase-producing strains. No strains intermediate or resistant to meropenem and imipenem were sensitive to ertapenem. The ertapenem disk presented a positive predictive value of 36% to predict blaKPC and 89% specificity. CONCLUSION: The resistance of Gram-negative bacilli detected by disk diffusion against ertapenem does not predict Klebsiella pneumoniae carbapenemase production. Other mechanisms, such as production of other betalactamases and porin loss, may be implicated. The need to confirm the presence of the blaKPC is suggested. Therefore, ertapenem was a weak predictor for discriminating strains that produce Klebsiella pneumoniae carbapenemase.


OBJETIVO: Avaliar o desempenho do disco de ertapenem para predizer micro-organismos produtores de Klebsiella pneumoniae carbapenemase. MÉTODOS: Bacilos Gram-negativos isolados em cultura entre janeiro de 2010 e junho de 2011 foram testados por disco-difusão (OxoidTM) para ertapenem, meropenem e imipenem. As cepas consideradas intermediárias ou resistentes (halo<22mm) para ertapenem foram encaminhadas para a pesquisa do blaKPC por reação em cadeia da polimerase. Calcularam-se o valor preditivo positivo e a especificidade do disco. RESULTADOS: Foram realizadas 21.839 culturas nesse período, sendo 3.010 (13,78%) positivas. Bacilos Gram-negativos foram isolados em 708 (23,52%) destas. A zona de inibição do disco de ertapenem foi <22mm para 111 (15,67%) dos isolados. A pesquisa do blaKPC caracterizou 40 cepas produtoras de Klebsiella peneumoniae carbapenemase. Não houve nenhum caso de disco intermediário ou resistente para meropenem ou imipenem com ertapenem sensível. O valor preditivo positivo foi de 36% e a especificidade calculada do disco de ertapenem para produção de Klebsiella pneumoniae carbapenemase foi de 89% em nosso serviço. CONCLUSÃO: A resistência ao disco de ertapenem não define bacilo produtor de Klebsiella pneumoniae carbapenemase. Mecanismos, como produção de outras betalactamases e perda de porinas, podem estar implicados. Sugerese a necessidade da confirmação da presença do gene blaKPC. O ertapenem, portanto, mostrou-se fraco preditor para discriminar cepas produtoras de Klebsiella pneumoniae carbapenemase.


Subject(s)
Humans , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/biosynthesis , Disk Diffusion Antimicrobial Tests/methods , Klebsiella pneumoniae/enzymology , beta-Lactamases/biosynthesis , beta-Lactams/pharmacology , beta-Lactam Resistance , Brazil , Bacterial Proteins/analysis , Gram-Negative Bacteria/isolation & purification , Klebsiella pneumoniae/drug effects , Microbial Sensitivity Tests , Polymerase Chain Reaction , Predictive Value of Tests , beta-Lactamases/analysis
16.
Indian J Med Microbiol ; 2012 Jul-Sept; 30(3): 323-331
Article in English | IMSEAR | ID: sea-143978

ABSTRACT

Background: Culture filtrate proteins (CFPs) of Mycobacterium tuberculosis are potential vaccine candidates. Objective: The aim was to study the influence of iron levels on CFPs and assess the immuno-protective potential of defined antigenic fractions from high (8 μg Fe/mL) and low iron (0.02 μg Fe / mL) cultures of M. tuberculosis. Materials and Methods: The CFPs of M. tuberculosis from high (CFP-high) and low (CFP-low) iron conditions were first compared to identify iron-regulated proteins and then fractionated to obtain ten antigen pools (CF-Ags H1- H5 and L1-L5) that were used to assess the immune response of TB patients and normal healthy controls. Results: Iron limitation resulted in the up-regulation of two novel iron-regulated low-molecular-weight proteins Irp-1 (in CF-Ag L4) and Irp-2 (in CF-Ag L5) and repression of two ESAT proteins (identified with monoclonal antibody HYB 76.8). The median stimulation indices (SIs) against most of the CF-Ags were high in pulmonary TB patients. The CF-Ags L1 and L2 showed statistically significant SI (P values of 0.0027 and 0.0029 respectively); the % case recognition was high with these antigens as well as with L4 ( P = 0.0275). IFN-γ in response to these CF-Ags was significantly high in the endemic normals; maximal expression was seen with CF-Ag L5 (median value of 233 pg mL -1 ) that was higher than the corresponding H5 (140 pg mL -1 ) and H3 and L3 (205 and 206 pg mL -1 respectively). Conclusions: CF-Ags L5, H3 and L3 showed immuno-protective potential in this geographical location.


Subject(s)
Adult , Antigens, Bacterial/biosynthesis , Antigens, Bacterial/immunology , Bacterial Proteins/biosynthesis , Bacterial Proteins/immunology , Female , Humans , Iron/metabolism , Male , Mycobacterium tuberculosis/immunology , Mycobacterium tuberculosis/metabolism , T-Lymphocytes/immunology , Tuberculosis/immunology , Tuberculosis/microbiology
18.
Rev. chil. infectol ; 29(2): 224-228, abr. 2012. ilus, tab
Article in Spanish | LILACS | ID: lil-627237

ABSTRACT

Carbapenem resistance in Enterobacteriaceae is an emerging problem worldwide. Among the mechanisms involved are the production of ESBLs or AmpC associated with porins loss or the presence of carbapenemases. Among these, the KPC betalactamase has become especially relevant given its rapid spread.In this article we present the first case of isolation of a strain of KPC producer Klebsiella pneumoniae at a hospital in Santiago, in a patient coming from Italy, with a history of multiple hospitalizations for treatment of non-Hodgkin lymphoma and subjected to several cycles of chemotherapy and hemodialysis. The strain was isolated from a urine culture on the seventh day of the patient's arrival to Chile. The isolate was resistant to quinolones, aminoglycosides, cephalosporins and carbapenems, retaining only susceptibility to tigecycline and colistin. In phenotypic test it was found to have positive Hodge test and positive synergy with carbapenems/boronic acid. Polymerase chain reaction demonstrated the presence of beta-lactamases TEM, SHV and KPC-2. None other Class A serine-carbapenemase or metallo-bectalactamases were present.


La resistencia a carbapenémicos en enterobacterias es un fenómeno emergente en el mundo. Entre los mecanismos implicados se encuentra la producción de BLEE o AmpC asociados a alteraciones de las porinas o la presencia de carbapenemasas. Entre éstas, las de tipo KPC han cobrado especial relevancia dada su rápida diseminación. Se presenta el primer caso de aislamiento de una cepa de Klebsiella pneumoniae productora de KPC en un hospital de Santiago- Región Metropolitana, en un paciente trasladado desde Italia, con el antecedente de múltiples hospitalizaciones para tratamiento de un linfoma no Hodgkin y sometido a varios ciclos de quimioterapia y hemodiálisis. La cepa fue aislada desde un urocultivo al séptimo día de la llegada del paciente a Chile. Fue resistente a quinolonas, aminoglucósidos, cefalosporinas y carbapenémicos, sólo manteniendo sensibilidad a tige-ciclina y colistin. Por test fenotípicos resultó tener test de Hodge positivo y sinergia positiva con carbapenémicos más ácido borónico. Por reacción de polimerasa en cadena se demostró la presencia de β-lactamasas tipo TEM, SHV y KPC-2/KPC-3, no detectándose la presencia de otras serino-carbapenemasas de clase A o de metalo-β-lactamasas.


Subject(s)
Adult , Humans , Male , Bacterial Proteins/biosynthesis , Klebsiella Infections/microbiology , Klebsiella pneumoniae/enzymology , beta-Lactam Resistance/genetics , beta-Lactamases/biosynthesis , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Chile , Carbapenems/pharmacology , Italy , Klebsiella pneumoniae/drug effects , Lymphoma, Non-Hodgkin/drug therapy , Microbial Sensitivity Tests , beta-Lactamases/genetics
19.
Article in English | IMSEAR | ID: sea-136347

ABSTRACT

Background & objectives: A cyclic lipopeptide, surfactin produced by a strain of Bacillus subtilis subsp. subtilis (VCRC B471) was found to exhibit activity against both the larval and pupal stages of mosquitoes. The present study was aimed at increasing the production of the mosquitocidal metabolite by modifying the conventional medium. Methods: Enhancement of mosquitocidal metabolite production was attempted by replacing the existing micronutrients of the conventional NYSM and supplementing the medium with additional amounts of glucose. The LC50 value of culture supernatant (CS) against the larval and pupal stages of Anopheles stephensi was determined. Crude mosquitocidal metabolite (CMM) was separated from the CS, identified by MALDI-TOF analysis and its LC50 dosage requirement for the pupal stage of the above mosquito species determined. Results: The medium containing a new composition of micronutrients and glucose up to 1 per cent resulted in increased metabolite production. The LC50 value of the CS obtained in the improved medium against larvae and pupae of An. stephensi was 5.57 and 0.71 μl/ml, respectively. The yield of CMM was doubled in the improved medium. MALDI-TOF analysis revealed that the CMM was surfactin. Interpretation & conclusions: The new improved medium enhanced the production of mosquitocidal metabolite as the dosage required for inciting 50 per cent mortality among the pupal stages of mosquitoes was only half of that required when the metabolite was produced in the conventional medium. The mosquitocidal metabolite was identified as surfactin, a cyclic lipopeptide and biosurfactant.


Subject(s)
Animals , Bacillus subtilis/growth & development , Bacillus subtilis/metabolism , Bacterial Proteins/biosynthesis , Bacterial Proteins/chemistry , Bacterial Proteins/pharmacology , Culicidae/drug effects , Culture Media/chemistry , Humans , Insecticides , Lipopeptides/biosynthesis , Lipopeptides/chemistry , Lipopeptides/pharmacology , Peptides, Cyclic/biosynthesis , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology
20.
Braz. j. infect. dis ; 15(1): 69-73, Jan.-Feb. 2011. tab
Article in English | LILACS | ID: lil-576789

ABSTRACT

In 2008 isolates of KPC-producing Klebsiella pneumoniae (KPC-KPN) were detected for the first time at Hospital Heliópolis, São Paulo, Brazil. The aim of this study was to characterize the clinical and microbiological outcomes of infections caused by KPC-KPN. A historical cohort of patients from whom KPC-KPN strains were isolated was performed. Isolates were identified as resistant to ertapenem by automated broth microdilution system and screened as carbapenemase producers by the modified Hodge test. The beta-lactamase resistance gene blaKPC was detected by PCR. The genetic relatedness of isolates was determined by PFGE. The study provides early clinical experience in treating KPC-KPN infections in a Brazilian tertiary center.


Subject(s)
Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/biosynthesis , Klebsiella Infections/microbiology , Klebsiella pneumoniae/enzymology , beta-Lactamases/biosynthesis , Brazil , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/genetics , Microbial Sensitivity Tests
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