Optimization studies on laccase activity of Proteus mirabilis isolated from treatment sludge of textile industry factories : Optimization of laccase activity of Proteus mirabilis.

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.

Proteus mirabilis Urease: Unsuspected Non-Enzymatic Properties Relevant to Pathogenicity.

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.

Dissemination of Multidrug-Resistant Proteus mirabilis Clones Carrying a Novel Integron-Borne blaIMP-1 in a Tertiary Hospital.

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.

Detection of bla KPC-2 in Proteus mirabilis in Brazil.

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.

[Resistance to extended-spectrum cephalosporins in non-inducible AmpC enterobacteria: evaluation of the new MIC breakpoints]. / Resistencia a cefalosporinas de espectro extendido en enterobacterias sin AmpC inducible: Evaluación de los nuevos puntos de corte.

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.

Resistencia a cefalosporinas de espectro extendido en enterobacterias sin AmpC inducible: Evaluación de los nuevos puntos de corte / Resistance to extended-spectrum cephalosporins in non-inducible AmpC enterobacteria: Evaluation of the new MIC breakpoints

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)

Resistencia a cefalosporinas de espectro extendido en enterobacterias sin AmpC inducible: Evaluación de los nuevos puntos de corte / Resistance to extended-spectrum cephalosporins in non-inducible AmpC enterobacteria: Evaluation of the new MIC breakpoints

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)

Resistencia a cefalosporinas de espectro extendido en enterobacterias sin AmpC inducible: Evaluación de los nuevos puntos de corte / Resistance to extended-spectrum cephalosporins in non-inducible AmpC enterobacteria: Evaluation of the new MIC breakpoints

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.

[CMY-2-type plasmid-mediated AmpC ß-lactamases emerging in Tucumán, Argentina]. / ß-lactamasas AmpC plasmídicas tipo CMY-2 emergentes en Tucumán, Argentina.

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.

[Comparison of four phenotypic methods to detect extended-spectrum betalactamases]. / Comparación de cuatro métodos fenotípicos para la detección de beta-lactamasas de espectro extendido.

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.

Proteus mirabilis isolates of different origins do not show correlation with virulence attributes and can colonize the urinary tract of mice.

Proteus mirabilis has been described as an aetiological agent in a wide range of infections, playing an important role in urinary tract infections (UTIs). In this study, a collection of P. mirabilis isolates obtained from clinical and non-clinical sources was analysed in order to determine a possible correlation between origin, virulence factors and in vivo infectivity. Isolates were characterized in vitro, assessing several virulence properties that had been previously associated with P. mirabilis uropathogenicity. Swarming motility, urease production, growth in urine, outer-membrane protein patterns, ability to grow in the presence of different iron sources, haemolysin and haemagglutinin production, and the presence and expression of diverse fimbrial genes, were analysed. In order to evaluate the infectivity of the different isolates, the experimental ascending UTI model in mice was used. Additionally, the Dienes test and the enterobacterial repetitive intergenic consensus (ERIC)-PCR assay were performed to assess the genetic diversity of the isolates. The results of the present study did not show any correlation between distribution of the diverse potential urovirulence factors and isolate source. No significant correlation was observed between infectivity and the origin of the isolates, since they all similarly colonized the urinary tract of the challenged mice. Finally, all isolates showed unique ERIC-PCR patterns, indicating that the isolates were genetically diverse. The results obtained in this study suggest that the source of P. mirabilis strains cannot be correlated with pathogenic attributes, and that the distribution of virulence factors between isolates of different origins may correspond to the opportunistic nature of the organism.

[Enzymatic resistance to beta lactam antibiotics within the genus Proteus and evaluation of Proteus mirabilis phenotypes and genotypes for resistance to third- and fourth-generation cephalosporins]. / Resistencia enzimática a betalactámicos en el género Proteus y evaluación de los fenotipos y genotipos de resistencia a cefalosporinas de tercera y cuarta generación en Proteus mirabilis.

INTRODUCTION: The aim of this study was to evaluate betalactam resistance within the genus Proteus and characterize the betalactamases responsible for this resistance. METHODS: We analyzed 99 strains (87, P. mirabilis; 10 P. vulgaris, and 2, P. penneri) isolated from patients at one University Hospital. Antibiotic susceptibility tests were performed according to NCCLS recommendations. Presence of extended spectrum betalactamases (ESBL) was inferred by both double disk diffusion tests and minimum inhibitory concentration (MIC) of third and fourth generation cephalosporins alone and in the presence of clavulanic acid. Isoelectric points (pI) of the enzymes were estimated by isoelectrofocusing and the presence of the encoding genes was confirmed by polymerase chain reaction (PCR). RESULTS: A broad spectrum betalactamase could be detected in those isolates (28%) resistant to penicillin and first generation cephalosporins while CTX-M-2 enzyme could be detected in P. mirabilis isolates resistant to third and fourth generation cephalosporins (18%). One of the P. vulgaris displayed reduced susceptibility to cefotaxime due to an enzyme of pI 7.4, while resistance to cefotaxime in one P. penneri was related to an enzyme of pI 6.8. Both enzymes were active on cefotaxime (1,000 mg/l) in the iodometric assay. CONCLUSION: The broad extended spectrum betalactamase within genus Proteus was TEM-1, while CTX-M-2 was the ESBL responsible for the third and fourth generation cephalosporins in P. mirabilis. In P. vulgaris and P. penneri this resistance was associated with the hyperproduction of the chromosomal encoded betalactamase.

ZapA, a possible virulence factor from Proteus mirabilis exhibits broad protease substrate specificity.

The opportunistic bacterium Proteus mirabilis secretes a metalloprotease, ZapA, considered to be one of its virulence factors due to its IgA-degrading activity. However, the substrate specificity of this enzyme has not yet been fully characterized. In the present study we used fluorescent peptides derived from bioactive peptides and the oxidized beta-chain of insulin to determine the enzyme specificity. The bradykinin- and dynorphin-derived peptides were cleaved at the single bonds Phe-Ser and Phe-Leu, with catalytic efficiencies of 291 and 13 mM/s, respectively. Besides confirming already published cleavage sites, a novel cleavage site was determined for the beta-chain of insulin (Val-Asn). Both the natural and the recombinant enzyme displayed the same broad specificity, demonstrated by the presence of hydrophobic, hydrophilic, charged and uncharged amino acid residues at the scissile bonds. Native IgA, however, was resistant to hydrolysis by ZapA.

Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis.

The protease ZapA, secreted by Proteus mirabilis, has been considered to be a virulence factor of this opportunistic bacterium. The control of its expression requires the use of an appropriate methodology, which until now has not been developed. The present study focused on the replacement of azocasein with fluorogenic substrates, and on the definition of enzyme specificity. Eight fluorogenic substrates were tested, and the peptide Abz-Ala-Phe-Arg-Ser-Ala-Ala-Gln-EDDnp was found to be the most convenient for use as an operational substrate for ZapA. A single peptide bond (Arg-Ser) was cleaved with a Km of 4.6 microM, a k cat of 1.73 s-1, and a catalytic efficiency of 376 (mM s)-1. Another good substrate for ZapA was peptide 6 (Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Gln-EDDnp) which was cleaved at a single bond (Phe-Ser) with a Km of 13.6 microM, a k cat of 3.96 s-1 and a catalytic efficiency of 291 (mM s)-1. The properties of the amino acids flanking the scissile bonds were also evaluated, and no clear requirement for the amino acid residue at P1 was found, although the enzyme seems to have a preference for a hydrophobic residue at P2.

Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis

The protease ZapA, secreted by Proteus mirabilis, has been considered to be a virulence factor of this opportunistic bacterium. The control of its expression requires the use of an appropriate methodology, which until now has not been developed. The present study focused on the replacement of azocasein with fluorogenic substrates, and on the definition of enzyme specificity. Eight fluorogenic substrates were tested, and the peptide Abz-Ala-Phe-Arg-Ser-Ala-Ala-Gln-EDDnp was found to be the most convenient for use as an operational substrate for ZapA. A single peptide bond (Arg-Ser) was cleaved with a Km of 4.6 µM, a k cat of 1.73 s-1, and a catalytic efficiency of 376 (mM s)-1. Another good substrate for ZapA was peptide 6 (Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Gln-EDDnp) which was cleaved at a single bond (Phe-Ser) with a Km of 13.6 µM, a k cat of 3.96 s-1 and a catalytic efficiency of 291 (mM s)-1. The properties of the amino acids flanking the scissile bonds were also evaluated, and no clear requirement for the amino acid residue at P1 was found, although the enzyme seems to have a preference for a hydrophobic residue at P2.

Chromogenic detection of aminoglycoside phosphotransferases.

A coupled chromogenic reaction (based on an agar overlay combining NADH, pyruvate kinase, lactate dehydrogenase, phosphoenolpyruvate, ATP, and kanamycin sulfate with thiazolyl blue-phenazine methosulfate for detection of NADH consumption) was optimized for the detection of aminoglycoside phosphotransferases (APHs). When used after analytical isoelectrofocusing of bacterial extracts from APH-producing strains, this method revealed one band in each of two strains with a genetically confirmed APH (3') I and two bands in another strain with both APH (3') I and APH (3') VI, whereas no bands were detected in susceptible control strains or in aminoglycoside-resistant microorganisms without APH genes.

A review of protease secretion instability in Proteus mirabilis

Proteus mirabilis secreta uma única protease que é uma metalo-enzima alcalina. Descrevemos aqui alguns fatores fisiológicos que afetam a secreçäo de protease em clones isolados. Mais de cem diferentes isolados clínicos foram examinados quanto a capacidade de secretar protease. Verificou-se que algumas linhagens herdam o determinante de protease como um caráter estável (säo denominadas linhagens estáveis); outras linhagens exigem um fenótipo muito instável (denominadas linhagens instáveis) com 10% das colônias-filhas perdendo o caráter de produçäo de protease. Nunca foi observada a reversäo do fenótipo protease negativa. Por outro lado, a introduçäo de fatores R em células produtoras de protease reduziu a atividade proteásica em cerca de 50%, o que sugere uma interferência na expressäo e/ou secreçäo de protease. Embora haja evidências de que um locus da protease tenha localizaçäo extracromossômica, näo foi demosntrada a presença de plasmídios nas linhagens instáveis. Um modo que pode explicar os vários dados é apresentado

Influence of RP4 plasmid on extracellular protease secretion by Proteus mirabilis.

The transference by conjugation of protease genetic information between Proteus mirabilis strains only occurs upon mobilization by a conjugative plasmid such as RP4 (Inc P group). Upon receiving the RP4 plasmid, the level of proteolytic activity of the protease-excreting P. mirabilis is reduced to about 50%. A similar phenomenon occurs when the protease character is mobilized by the RP4 plasmid from the above transconjugant to a non-protease-excreting recipient strain. The molecular mechanism underlying the interference of R plasmids with proteolytic activity remains to be elucidated but there is evidence suggesting that some alteration in the bacterial envelope might be involved.