Effect of calcium on Pseudomonas aeruginasa and Bacillus cereus metabolites / Efeito do cálcio nos metabólitos de Pseudomonas aeruginasa e Bacillus cereus

The effects of Calcium (Ca+²) on virulence and some parameters should be analyzed in this study. Pseudomonas aeruginosa Gram (-) and Bacillus cereus Gram (+) were used. Both bacteria are soil bacteria. In this study; the effect of Ca+² on protease, amylase, LasB elastolytic assay, H2O2, pyorubin and biofilm on metabolites of these bacteria were investigated during 24 hour time. In this study, the effect of Ca+² on the production of some secondary metabolites on P. aeruginosa and B. cereus was investigated and presented for the first time by us.

Os efeitos do cálcio (Ca+²) na virulência e alguns parâmetros devem ser analisados neste estudo. Pseudomonas aeruginosa Gram (-) e Bacillus cereus Gram (+) foram usados. Ambas as bactérias são bactérias do solo. Neste estudo, o efeito do Ca+² sobre a protease, amilase, ensaio elastolítico LasB, H2O2, piorubina e biofilme nos metabólitos dessas bactérias foram investigados durante 24 horas. Neste estudo, o efeito do Ca+² na produção de alguns metabólitos secundários em P. aeruginosa e B. cereus foi investigado e apresentado pela primeira vez por nós.

Biofilm production by clinical isolates of Pseudomonas aeruginosa and structural changes in LasR protein of isolates non biofilm-producing

ABSTRACT Introduction: Biofilm production is an important mechanism for the survival of Pseudomonas aeruginosa and its relationship with antimicrobial resistance represents a challenge for patient therapeutics. P. aeruginosa is an opportunistic pathogen frequently associated to nosocomial infections, especially in imunocompromised hosts. Objectives: Analyze the phenotypic biofilm production in P. aeruginosa isolates, describe clonal profiles, and analyze quorum sensing (QS) genes and the occurrence of mutations in the LasR protein of non-biofilm producing isolates. Methods: Isolates were tested for biofilm production by measuring cells adherence to the microtiter plates. Clonal profile analysis was carried out through ERIC-PCR, QS genes were by specific PCR. Results: The results showed that 77.5% of the isolates were considered biofilm producers. The results of genotyping showed 38 distinct genetic profiles. As for the occurrence of the genes, 100% of the isolates presented the lasR, rhlI and rhlR genes, and 97.5%, presented the lasI gene. In this study nine isolates were not biofilm producers. However, all presented the QS genes. Amplicons related to genes were sequenced in three of the nine non-biofilm-producing isolates (all presenting different genetic similarity profile) and aligned to the sequences of those genes in P. aeruginosa strain PAO1 (standard biofilm-producing strain). Alignment analysis showed an insertion of three nucleotides (T, C and G) causing the addition of an amino acid valine in the sequence of the LasR protein, in position 53. Conclusion: The modeling of the resulting LasR protein showed a conformational change in its structure, suggesting that this might be the reason why these isolates are unable to produce biofilm.

High genetic diversity among Pseudomonas aeruginosa and Acinetobacter spp. isolated in a public hospital in Brazil

In Brazil and other regions of the world, Pseudomonas aeruginosa and Acinetobacter spp. have emerged as important agents of nosocomial infection and are commonly involved in outbreaks. The main objective of the present study was to evaluate the genetic relationship among P. aeruginosa and Acinetobacter spp. isolated from patients in a public university hospital in northwestern Paraná, Brazil, and report their antimicrobial resistance profile. A total of 75 P. aeruginosa and 94 Acinetobacter spp. isolates were phenotypically identified and tested for antibiotic susceptibility using automated methodology. Polymyxin B was tested by disk diffusion for P. aeruginosa. Metallo-β-lactamase (MBL) was detected using a disk approximation test. Genotyping was performed using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR). Approximately 55% of the P. aeruginosa isolates and 92% of the Acinetobacter spp. isolates were multiresistant, but none were MBL-producers. ERIC-PCR revealed the presence of small clusters of carbapenem-resistant Acinetobacter spp., most likely OXA-type carbapenemase producers. Furthermore, high genetic diversity in P. aeruginosa and Acinetobacter spp. clinical isolates was observed, suggesting that cross-transmission is not very frequent in the studied hospital.

No Brasil, bem como em outras regiões do mundo, Pseudomonas aeruginosa e Acinetobacter spp. surgiram como importantes agentes de infecção nosocomial e são comumente envolvidos em surtos. O objetivo principal deste estudo foi descrever a relação genética de P. aeruginosa e Acinetobacter spp. isoladas de pacientes internados em hospital universitário público do noroeste do Paraná - Brasil e reportar o perfil de resistência dessas bactérias. Um total de 75 P. aeruginosa e 94 Acinetobacter spp. isolados foi fenotipicamente identificado e testado para a suscetibilidade aos antibióticos por metodologia automatizada. A polimixina B foi testada por difusão em disco para P. aeruginosa. Metalo-β-lactamase (MBL) foi detectada por disco-aproximação. Análise genotípica foi realizada por enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR). Aproximadamente 55% dos isolados de P. aeruginosa e 92% de Acinetobacter spp. isolados foram multirresistentes, mas nenhum foi produtor de MBL. Os resultados de ERIC-PCR revelaram pequenos grupamentos de Acinetobacter spp. resistentes aos carbapenêmicos, provavelmente pela produção de carbapenemases do tipo OXA. Além disso, alta diversidade genética entre os isolados de P. aeruginosa e Acinetobacter spp. foi observada, sugerindo que a transmissão cruzada destas espécies bacterianas não é muito frequente em nosso hospital.

Study of Pseudomonas aeruginosa causing ventilator associated pneumonia.

Ps. aeruginosa is a frequent and prominent cause of nosocomial pneumonia especially in persons on assisted ventilation in the intensive care units. In a year long surveillance of ventilator associated pneumonia (VAP) we isolated 42 strains from broncho alveolar lavage samples collected and processed from 102 patients. By pyocin typing 40 of the 42 strains could be typed into 39 types but this designation changed each time the test was repeated. SDS-PAGE analysis of the whole cell proteins grouped the 42 strains of Ps. aeruginosa into 20 groups. After ribotyping, using an 18 mer DIG labelled oligonucleotide to the conserved region of 16S rRNA gene, the strains were designated into 18 types. The major type contained 8 isolates, but there was no clustering of isolates, indicating that each infecting strain was acquired separately and not from a common source. It would, therefore, appear that cross infection with a single clone was not the predominant mode of Ps. aeruginosa infection causing VAP in our ICU.

Producción y sensibilidad a microcinas en cepas de Pseudomonas aeruginosa / Pseudomonas aeruginosa microcins: production and sensibility

Se aislaron cepas de Pseudomonas aeruginosa de distintos procesos infecciosos con el objeto de estudiar la capacidad de producir microcinas y la sensibilidad a las elaboradas por otras cepas. Se investigaron por el método de estrías cruzadas en condiciones mínimas de crecimiento y en agar nutritivo, siendo más adecuado el primero. Se analizó el efecto de la presencia de metionina en medio mínimo. El tamaño molecular de las bacteriocinas se estimó con membranas capaces de retener moléculas mayores de 8000 daltons. Los resultados obtenidos indicaron una alta incidencia de cepas productoras de microcinas (85,7 por ciento). La sensibilidad a las mismas fue variada desde 0 a 10 cepas susceptible. Al mantener P. aeruginosa en el laboratorio se redujo el espectro de actividad, mientras que las recientemente aisladas inhibieron mayor número de cepas. Se observó también que disminuyó la sensibilidad a microcinas al conservarse las cepas por más de seis meses. Es conveniente realizar tipificación mediante microcinas con bacterias mantenidas por poco tiempo en cultivo

Ausencia de actividad antimicrobiana de un extracto acuoso liofilizado de Aloe vera (Sabila) / Absence of antimicrobial activity of lyophilized aqueous extract of Aloe vera (sabila)

Se estudio la actividad antimicrobiana de dos concentraciones (10 y 50 mg/mL) de un extracto acuoso liofilizado de hojas de Aloe vera (sabila), mediante el sistema de ensayo de difusion en agar, con una bateri aminima de cepas de microorganismos compuesta por cuatro bacterias (Staphylococcus aureus, Bacillus subtilils, Escherichia coli y Pseudomonas aeruginosa) y una levadura (Candida albicans). Los resultados indican que solo frente al Staphylococcus aureus se obteine una ligera actividad inhibitoria, al compararla con la que produce el control positivo (estreptomicina). Para el resto de los microorganismos estudiados la respuesta es negativa. Estos resultados permiten desestimar el uso del extracto acuoso liofilizado de Aloe vera como antimicrobiano, en tanto que sugiere explorar este efecto con otro tipo de extracto con el objetivo de avalar o no la utilizacion de esta planta como antimicrobiano

Actividad antimicrobiana del Schinus terebentifolius Raddi (copal) / Antimicrobial activity of Schinus terebentifolius Raddi (copal)

Se estudio la actividad antimicrobiana de diferentes concentraciones de un extracto fluido (etanol al 30 porciento) de hojas de Schinus terebenthifolius Raddi (copal) con una bateria minima de cepas de microorganismos que incluye Staphylococcus aureus y Bacillus subtilis, como grampositivo, escherichia coli y Pseudomonas aeruginosa, como gramnegativo y la levadura, Candida albicans, mediante el metdo de difusion en agar. Los resultados obtenidos indican que en la menor concentracion utilizada, del 10 porciento del extracto fluido, no se aprecia inhibicion de ninguno de los microorganismos evaluados, mientras que en las concentraciones del 50 y el 100 porciento del extracto fluido hay respuesta de inhibicion frente a las bacterias grampositiva y gramnegativa, pero no asi con la levadura. Estos resultados contribuyen a ratificar experimentalmente el uso tradicional que se hace de esta planta como antimicrobiano, ademas sugiere la elaboracion de formas farmaceuticas que permitan ampliar con mayor eficiencia su utilizacion para este objetivo

Cellular distribution of hexa and trivalent chromium in Pseudomonas aeruginosa.

Binding of Cr to P. aeruginosa whole cells increased with an increase in positive charge of the chromium complexes used. Distribution of Cr bound from aqueous solutions of potassium dichromate--a Cr(VI) salt and basic chromium sulphate (BCS)--a Cr(III) salt [used for tanning which has 25% of Cr2O3 on weight basis and is of 33% basicity (one hydroxyl/Cr atom)] was more in soluble fraction (protein and nucleic acids) than in insoluble fraction (lipids, polyphosphates, polysaccharides, lipopolysaccharides and mucopeptides). Electron spin resonance spectrum of the cells exposed to hexavalent chromium revealed the presence of Cr(III) ion demonstrating a reduction of the same by the Pseudomoas. The infrared spectra of chromium exposed cell envelopes support that protein carboxylic sites and proteins are involved in binding Cr(III) and predominantly lipids in Cr(VI). Bound chromium could be removed chemically and the cells could then be reused as a biosorbent.