Efficacy of combinations of colistin with other antimicrobials involves membrane fluidity and efflux machinery.

OBJECTIVE: Despite its use was abandoned several decades ago, the polycationic peptide colistin has become the last hope to treat severe infections caused by multidrug-resistant Gram-negative bacteria. Thus, the development of colistin resistance may seriously compromise the efficacy of treatment. Moreover, colistin has high toxicity being dose dependent. A potentially effective strategy to avoid resistance may be to combine colistin with other antimicrobials. This may help in the rescue of old antimicrobials and in reducing toxic undesired effects. METHODS: Antimicrobial susceptibility determination, efflux machinery function measurements in different conditions and measurement of inhibition of the extrusion by colistin were performed. Moreover, modifications of anisotropy of the membranes by using fluorescent dyes was accomplished. RESULTS: Sub-inhibitory concentrations of colistin have a synergistic effect with several antimicrobials that act intracellularly (targeting protein synthesis and DNA replication). This effect was demonstrated through the uptake increases of acridine orange. in Pseudomonas aeruginosa, Escherichia coli and Acinetobacter baumanii but also in an intrinsically colistin-resistant species as Serratia marcescens. Measurements of the anisotropy of bacterial membranes, as a measure of membrane fluidity, showed significant changes indicative of colistin activity. CONCLUSION: The alterations in the cellular efflux machinery that resulted in higher intracellular concentrations of acridine orange, and likely of other antimicrobials combined with data of membrane fluidity and measured synergism in vitro allow us to envisage the use of these combinations to fight infections caused by multidrug-resistant bacteria.

Killing effect of nanoencapsulated colistin sulfate on Pseudomonas aeruginosa from cystic fibrosis patients.

Pseudomonas aeruginosa frequently infects the respiratory tract of cystic fibrosis (CF) patients. Multidrug-resistant phenotypes and high capacity to form stable biofilms are common. Recent studies have described the emergence of colistin-resistant isolates in CF patients treated with long-term inhaled colistin. The use of nanoparticles containing antimicrobials can contribute to overcome drug resistance mechanisms. The aim of this study was to explore antimicrobial activity of nanoencapsulated colistin (SLN-NLC) versus free colistin against P. aeruginosa clinical isolates from CF patients and to investigate their efficacy in biofilm eradication. Susceptibility of planktonic bacteria to antimicrobials was examined by using the broth microdilution method and growth curve assay. Minimal biofilm eradication concentration (MBEC) and biofilm prevention concentration (BPC) were determined to assess antimicrobial susceptibility of sessile bacteria. We used atomic force microscopy (AFM) to visualize treated and untreated biofilms and to determine surface roughness and other relevant parameters. Colistin nanoparticles had the same antimicrobial activity as free drug against planktonic bacteria. However, nanoencapsulated colistin was much more efficient in the eradication of biofilms than free colistin. Thus, these formulations have to be considered as a good alternative therapeutic option to treat P. aeruginosa infections.

Effects of photodynamic therapy on Enterococcus faecalis biofilms.

Microbial biofilms are involved in almost all infectious pathologies of the oral cavity. This has led to the search for novel therapies specifically aimed at biofilm elimination. In this study, we used atomic force microscopy (AFM) to visualize injuries and to determine surface roughness, as well as confocal laser scanning microscopy (CLSM) to enumerate live and dead bacterial cells, to determine the effects of photodynamic therapy (PDT) on Enterococcus faecalis biofilms. The AFM images showed that PDT consisting of methylene blue and a 670-nm diode laser (output power 280 mW during 30 s) or toluidine blue and a 628-nm LED light (output power 1000 mW during 30 s) induced severe damage, including cell lysis, to E. faecalis biofilms, with the former also causing an important increase in surface roughness. These observations were confirmed by the increase in dead cells determined using CLSM. Our results highlight the potential of PDT as a promising method to achieve successful oral disinfection.

Class 1 integrons in environmental and clinical isolates of Pseudomonas aeruginosa.

The aims of this study were to ascertain the presence and spread of class 1 integrons amongst environmental and clinical isolates of Pseudomonas aeruginosa and to characterise their variable regions. A total of 76 isolates (56 clinical and 20 environmental) were studied. The presence of plasmids was explored, and polymerase chain reaction (PCR) was used for integron detection. All amplicons were sequenced. PCR detected class 1 integrons in 26 of the 56 clinical isolates; environmental isolates were integron-free. No plasmids were found, thus all the integrons found are possibly on the chromosome. Most isolates presented one amplicon, except PA110514 and PA116136, which showed two PCR products each. Variable regions revealed that 18 strains carried only one gene involved in aminoglycoside resistance, whereas in 3 strains gene cassettes were not found. The most prevalent cassettes amongst isolates were those encoding aminoglycoside adenyltransferase B (aadB). Several of the strains had acquired the same or a highly similar cassette array as those detected in geographically distant P. aeruginosa. This finding suggests that contact with bacterial reservoirs contributes to the evolution of this pathogen towards multiresistance. Empty structures found may represent a reservoir increasing the capacity to adapt to the environment. However, these integrons are not retained when the selective pressure disappears. It is hypothesised that integrons containing gene cassettes are crucial vehicles for the rapid horizontal transfer of resistance. If this is so, reduced use of antibiotics may lead to a significant decrease in the carriage of integrons amongst P. aeruginosa strains.

Synergism between outer membrane proteins and antimicrobials.

Antibiotic-resistant bacteria are becoming one of the most important problems in health care because of the number of resistant strains and the paucity of new effective antimicrobials. Since antibiotic-resistant bacteria will continue to increase, it is necessary to look for new alternative strategies to fight against them. It is generally accepted that Gram-negative bacteria are intrinsically less susceptible than Gram-positive bacteria to antimicrobials. The main reason is that Gram-negative bacteria are surrounded by a permeability barrier known as the outer membrane (OM). Hydrophilic solutes most often cross the OM through water-filled channels formed by a particular family of proteins known as porins. This work explores the possibility of using exogenous porins to lower the required amounts of antibiotics (ampicillin, ciprofloxacin, cefotaxime, clindamycin, erythromycin, and tetracycline). Porins had a bactericidal effect on Escherichia coli cultures, mainly in the logarithmic phase of growth, when combined with low antibiotic concentrations. The use of different antibiotic-porin mixtures showed a bactericidal effect greater than those of antibiotics and porins when used separately. It was possible to observe different behaviors according to the antibiotic type used.

Quality of dietary control in phenylketonuric patients and its relationship with general intelligence.

OBJECTIVES: Assessment of the quality of dietary treatment of phenylketonuria (PKU) patients and investigation of its relationship with the general intelligence of the patients. METHODS: Cross-sectional and longitudinal study of 105 PKU treated patients. The index of dietary control (IDC) was calculated as the phenylalanine (Phe) data reduction in half-year medians and the mean of all medians throughout the patient's life. We calculated four different IDCs related to age: IDC-A (< 6 years), IDC-B (6-12 years), IDC-C (13-18 years) and IDC-D (> 18 years). To evaluate the fluctuation of Phe values we calculated the standard error of the estimate of the regression of Phe concentration over age. Development quotient was calculated with the Brunet-Lezine test (< 4 years). Intelligence quotient was evaluated with the Kaufman Bit Intelligence Test (K-Bit), Wechsler Intelligence Scale for Children-Revised (WISC-R) and Wechsler Adult Intelligence Scale Third Edition (WAIS III). RESULTS: Cross-sectional study: The IDC in age groups were significantly different and so were the number of patients with good, acceptable and poor IDC related to age (p < 0.001). Sampling frequency was good in 72, acceptable in 23 and poor in 10 patients. The general intelligence (101 +/- 10) correlated negatively with the IDC (p < 0.0001) and Phe fluctuations (p < 0.004). Longitudinal study: Significant differences were observed between the IDC through the patients' lifetime except in the adolescent/adult period. CONCLUSIONS: 85% of PKU patients showed good/acceptable quality of dietary control. General intelligence correlates with the IDC at all ages, which highlights the importance of good control to achieve good prognosis.

Quality of dietary control in phenylketonuric patients and its relationship with general intelligence / Calidad del control de la dieta en pacientes fenilcetonúricos y su relación con la inteligencia general

Objectives: Assessment of the quality of dietary treatment of phenylketonuria (PKU) patients and investigation of its relationship with the general intelligence of the patients. Methods: Cross-sectional and longitudinal study of 105 PKU treated patients. The index of dietary control (IDC) was calculated as the phenylalanine (Phe) data reduction in half-year medians and the mean of all medians throughout the patient's life. We calculated four different IDCs related to age: IDC-A (< 6 years), IDC-B (6-12 years), IDC-C (13-18 years) and IDC-D (> 18 years). To evaluate the fluctuation of Phe values we calculated the standard error of the estimate of the regression of Phe concentration over age. Development quotient was calculated with the Brunet-Lezine test (< 4 years). Intelligence quotient was evaluated with the Kaufman Bit Intelligence Test (K-Bit), Wechsler Intelligence Scale for Children-Revised (WISC-R) and Wechsler Adult Intelligence Scale Third Edition (WAIS III). Results: Cross-sectional study: The IDC in age groups were significantly different and so were the number of patients with good, acceptable and poor IDC related to age (p < 0.001). Sampling frequency was good in 72, acceptable in 23 and poor in 10 patients. The general intelligence (101 ± 10) correlated negatively with the IDC (p < 0.0001) and Phe fluctuations (p < 0.004). Longitudinal study: Significant differences were observed between the IDC through the patients' lifetime except in the adolescent/adult period. Conclusions: 85% of PKU patients showed good/acceptable quality of dietary control. General intelligence correlates with the IDC at all ages, which highlights the importance of good control to achieve good prognosis (AU)

Objetivos: Evaluación de la calidad del control dietético en pacientes con fenilcetonuria (PKU) e investigación de su relacióncon el nivel de inteligencia. Métodos: Estudio transversal y longitudinal de 105 pacientes PKU en tratamiento dietético. El índice de control de la dieta (IDC) se ha calculado como la reducción de los valores de fenilalanina (Phe) a las medianas de cada 6 meses y la media de todas las medianas a lo largo de la vida del paciente. Se han calculado cuatro diferentes IDC según la edad: IDC-A (< 6 años), IDC-B (6-12 años), IDC-C (13-18 años) and IDC-D (> 18 años). Para evaluar las fluctuaciones de los valores de Phe hemos calculado el error estándar de la regresión estimada de la concentración de Phe según la edad. El índice de inteligencia se ha evaluado mediante el test de Brunet-Lezine (< 4 años) y el coeficiente de inteligencia mediante Kaufman Bit Intelligence Test (K-Bit), Wechsler Intelligence Scale for Children-Revised (WISC-R) y Wechsler Adult Intelligence Scale Third Edition (WAIS III). Resultados: Estudio transversal: El IDC en los diferentes grupos de edad es significativamente diferente y también lo son el número de pacientes con un IDC bueno, aceptable y malo en relación con la edad (p < 0,001). La frecuencia de controles de Phe fue buena en 72 pacientes, aceptable en 23 y mala en 10. La inteligencia general (101 ± 10) se correlaciona negativamente con el IDC (p < 0,0001) y con las fluctuaciones de Phe (p < 0,004). Estudio longitudinal: Se han observado diferencias significativas entre ICD a lo largo de la vida de los pacientes a excepción del período adolescencia/edad adulta. Conclusiones: El 85% de pacientes PKU mostraron una calidad del control de la dieta buena/aceptable. Los niveles de inteligencia general se correlacionan con el IDC en todas las edades, lo que muestra la importancia del buen control de la dieta para lograr un buen pronóstico (AU)

Aplicación del protocolo de actuación para embarazadas con fenilcetonuria clásica: tres gestaciones con éxito / Application of a protocol for pregnant women with classic phenylketonuria: three successful pregnancies

El síndrome de fenilcetonuria materna (SFM) aparece en hijos de madres afectadas por hiperfenilalaninemia grave o moderada con concentraciones plasmáticas elevadas de fenilalanina(Phe) durante los meses previos y/o la gestación. Cursa con malformaciones similares al síndrome alcoholico fetal y es especialmente frecuente la aparición de microcefalia y retardomental y del desarrollo. La planificación y los controles estrictos de Phe durante el embarazo son fundamentales para evitar la aparición de SFM. Presentamos un protocolo de seguimiento y nuestra experiencia en tres embarazos que han finalizado con éxito

Maternal phenylketonuria syndrome(MPS)occurs in children whose mothers have severe or moderate hyperphenylalaninemia and present high plasma phenylalanine levels during the months prior to and/or throughout pregnancy. MPS presents with malformations similar to those associated with fetal alcohol syndrome, the most common of which are microcephaly, congenital heart defects and mental and developmental retardation. Planning and strict control of phenylalanine levels during pregnancy are essential for the prevention of the development of MPS. We present a follow-up protocol and our experience with three successful pregnancies in women with phenylketonuria

Do adult patients with phenylketonuria improve their quality of life after introduction/resumption of a phenylalanine-restricted diet?

AIM: To evaluate the possible influence of dietary treatment on the quality of life of adult patients with PKU (phenylketonuria) following late introduction or resumption of a Phe-restricted diet. METHODS: Fifteen adult patients with classical PKU (10F, 5M; mean age: 27.5 y, range: 16.4-37.5 y) were selected for the study. These patients had either resumed a restricted diet after a period of discontinuation, or were placed on a restricted diet after late diagnosis. All of them were interviewed about their quality of life using a 24-item questionnaire. RESULTS: The index of dietary control was poor (median Phe: 954 micromol/L) in 8/15 patients, regular (Phe: 514 micromol/L) in 4/15 and good (Phe: 354 micromol/L) in 3/15 patients. Fifty-three percent of patients reported that their state of health was very good, 47% described it as good, and 40% felt that their present health on-diet was better than it had been off-diet; 53% believed that they were calmer, quieter and less easily upset and 40% were more alert and were more able to maintain attention while on-diet. Thirty-three percent of the patients felt happier, and 27% felt more vital; 20% thought that they were less impulsive and aggressive, and that they were now less argumentative than before. Sixty percent of the patients felt that their quality of life had improved on-diet compared with the situation off-diet. CONCLUSION: More than half of our patients believed that their quality of life improved with a Phe-restricted diet; they reported feeling calmer, quieter, and less easily upset. Only 47% attained regular to good dietary control.