RESUMEN
A series of gramicidin S derivatives 4-15 are presented that have four ornithine residues as polar protonated side chains and two central hydrophobic amino acids with unaltered turn regions. These peptides were screened against human erthrocytes and our standard panel of Gram negative- and Gram positive bacteria, including four MRSA strains. Based on the antibacterial- and hemolytic data, peptides 13 and 14 have an improved biological profile compared to the clinically applied topical antibiotic gramicidin S.
Asunto(s)
Antibacterianos/química , Gramicidina/análogos & derivados , Gramicidina/química , Antibacterianos/síntesis química , Antibacterianos/farmacología , Eritrocitos/efectos de los fármacos , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Gramicidina/síntesis química , Gramicidina/farmacología , Hemólisis , Humanos , Pruebas de Sensibilidad Microbiana , Péptidos/síntesis química , Péptidos/química , Péptidos/farmacologíaRESUMEN
In this paper, we describe the crystal structure of previously reported ring-extended gramicidin S (GS) derivative 2 (GS14K4), containing a d-amino acid residue in one of the ß-strand regions. This structure is in agreement with a previously reported modeling study of the same molecule. The polar side chain of the additional d-amino acid residue is positioned at the same face of the molecule as the hydrophobic side chains, and we believe that because of this compound 2 is considerably less hydrophobic than extended GS derivatives in which the strand regions are exclusively composed of l-amino acids. Using this backbone structure as our benchmark we prepared a small series of ring-extended GS analogues featuring sugar amino acid dipeptide isosteres of varied hydrophobicity at the turn region. We show that via this approach hydrophobicity of extended GS analogues can be tuned without affecting the secondary structure (as observed from NMR and CD spectra). Biological evaluation reveals that hydrophobicity correlates to cell toxicity, but still bacteriolysis is induced with GS analogues that are too hydrophilic to efficiently lyse human red blood cells.
Asunto(s)
Antibacterianos/síntesis química , Gramicidina/análogos & derivados , Antibacterianos/química , Antibacterianos/farmacología , Dicroismo Circular , Cristalografía por Rayos X , Eritrocitos/efectos de los fármacos , Gramicidina/química , Gramicidina/farmacología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Pruebas de Sensibilidad Microbiana , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaAsunto(s)
Resistencia a la Meticilina , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Casas de Salud/estadística & datos numéricos , Infecciones Estafilocócicas/epidemiología , Anciano de 80 o más Años , Humanos , Países Bajos/epidemiología , Prevalencia , Infecciones Estafilocócicas/microbiologíaRESUMEN
Monobenzylated sugar amino acids (SAAs) that differ in ether ring size (containing an oxetane, furanoid, and pyranoid ring) were synthesized and incorporated in one of the ß-turn regions of the cyclo-decapeptide gramicidin S (GS). CD, NMR spectroscopy, modeling, and X-ray diffraction reveal that the ring size of the incorporated SAA moieties determines the spatial positioning of their cis-oriented carboxyl and aminomethyl substituents, thereby subtly influencing the amide linkages with the adjacent amino acids in the sequence. Unlike GS itself, the conformational behavior of the SAA-containing peptides is solvent dependent. The derivative containing the pyranoid SAA is slightly less hydrophobic and displays a diminished haemolytic activity, but has similar antimicrobial properties as GS.
Asunto(s)
Aminoácidos/química , Antiinfecciosos/química , Antiinfecciosos/síntesis química , Gramicidina/química , Oligopéptidos/química , Oligopéptidos/síntesis química , Secuencia de Aminoácidos , Amino Azúcares , Dicroismo Circular , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Difracción de Rayos XRESUMEN
Ring extended Gramicidin S analogues containing adamantane amino acids and six cationic residues were designed and evaluated. Systematic replacement of the hydrophobic residues with adamantane amino acids resulted in a small set of compounds with varying amphipathic character. It was found that the amphipathicity of these compounds is correlated to their biological activity. Several bacterial strains including MRSA strains were shown to be killed by the novel peptides. The most potent antibacterial peptides are tetradecameric GS analogues containing six positives charges and two adamantane moieties.
Asunto(s)
Adamantano/química , Aminoácidos/química , Antiinfecciosos/química , Gramicidina/análogos & derivados , Secuencia de Aminoácidos , Antiinfecciosos/farmacología , Cationes/química , Dicroismo Circular , Gramicidina/farmacología , Interacciones Hidrofóbicas e Hidrofílicas , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacosRESUMEN
The cyclic cationic antimicrobial peptide gramicidin S (GS) is an effective topical antibacterial agent that is toxic for human red blood cells (hemolysis). Herein, we present a series of amphiphilic derivatives of GS with either two or four positive charges and characteristics ranging between very polar and very hydrophobic. Screening of this series of peptide derivatives identified a compound that combines effective antibacterial activity with virtually no toxicity within the same concentration range. This peptide acts against both Gram-negative and Gram-positive bacteria, including several MRSA strains, and represents an interesting lead for the development of a broadly applicable antibiotic.
Asunto(s)
Adamantano/química , Adamantano/farmacología , Antibacterianos/química , Antibacterianos/farmacología , Eritrocitos/efectos de los fármacos , Gramicidina/química , Gramicidina/farmacología , Hemólisis/efectos de los fármacos , Péptidos/química , Péptidos/farmacología , Secuencia de Aminoácidos , Aminoácidos , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Humanos , Modelos Moleculares , Permeabilidad , Relación Estructura-ActividadRESUMEN
The cyclic decapeptide gramicidin S (GS) was used as a model for the evaluation of four turn mimetics. For this purpose, one of the D-Phe-Pro two-residue turn motifs in the rigid cyclic beta-hairpin structure of GS was replaced with morpholine amino acids (MAA 2-5), differing in stereochemistry and length of the side-chain. The conformational properties of the thus obtained GS analogues (6-9) was assessed by using NMR spectroscopy and X-ray crystallography, and correlated with their biological properties (antimicrobial and hemolytic activity). We show that compound 8, containing the dipeptide isostere trans-MAA 4, has an apparent high structural resemblance with GS and that its antibacterial activity against a panel of Gram positive and -negative bacterial strains is better than the derivatives 6, 7 and 9.
Asunto(s)
Aminoácidos/química , Antibacterianos/química , Antibacterianos/farmacología , Dipéptidos/química , Gramicidina/química , Gramicidina/farmacología , Morfolinas/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Espectroscopía de Resonancia Magnética , Estructura Molecular , Conformación Proteica , Relación Estructura-ActividadAsunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Gramicidina/análogos & derivados , Gramicidina/farmacología , Aminoácidos/química , Bacterias/efectos de los fármacos , Carbohidratos/química , Eritrocitos/efectos de los fármacos , Hemólisis/efectos de los fármacos , Humanos , Estructura Molecular , Estructura Secundaria de ProteínaRESUMEN
Loloatin C is a cyclic cationic antimicrobial peptide which is active against gram positive as well as certain gram negative bacteria. Unfortunately, it is equally potent against human erythrocytes. To probe the structure-activity relationship of this promising antibiotic peptide, amino acid substitution and/or incorporation of a constraint sugar amino acid dipeptide isoster has been applied. Six new derivatives have been synthesized using SPPS and their solution structure investigated using NMR studies. Finally, the antimicrobial and the hemolytic activities have been determined.
Asunto(s)
Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/química , Péptidos Cíclicos/química , Secuencia de Aminoácidos , Antibacterianos/síntesis química , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/síntesis química , Péptidos Catiónicos Antimicrobianos/farmacología , Eritrocitos/efectos de los fármacos , Hemólisis , Humanos , Pruebas de Sensibilidad Microbiana , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/farmacología , Relación Estructura-ActividadRESUMEN
BACKGROUND: Molecular typing of methicillin-resistant Staphylococcus aureus (MRSA) is required to study the routes and rates of transmission of this pathogen. Currently available typing techniques are either resource-intensive or have limited discriminatory ability. Multiple-locus variable number tandem repeat analysis (MLVA) may provide an alternative high throughput molecular typing tool with high epidemiological resolution. METHODOLOGY/PRINCIPAL FINDINGS: A new MLVA scheme for S. aureus was validated using 1681 S. aureus isolates collected from Dutch patients and 100 isolates from pigs. MLVA using 8 tandem repeat loci was performed in 2 multiplex PCRs and the fluorescently labeled PCR products were accurately sized on an automated DNA sequencer. The assessed number of repeats was used to create MLVA profiles consisting of strings of 8 integers that were used for categorical clustering. MLVA yielded 511 types that clustered into 11 distinct MLVA complexes which appeared to coincide with MLST clonal complexes. MLVA was at least as discriminatory as PFGE and twice as discriminatory as spa-sequence typing. There was considerable congruence between MLVA, spa-sequence typing and PFGE, at the MLVA complex level with group separation values of 95.1% and 89.2%. MLVA could not discriminate between pig-related MRSA strains isolated from humans and pigs, corroborating the high degree of relationship. MLVA was also superior in the grouping of MRSA isolates previously assigned to temporal-spatial clusters with indistinguishable SpaTypes, demonstrating its enhanced epidemiological usefulness. CONCLUSIONS: The MLVA described in this study is a high throughput, relatively low cost genotyping method for S. aureus that yields discrete and unambiguous data that can be used to assign biological meaningful genotypes and complexes and can be used for interlaboratory comparisons in network accessible databases. Results suggest that MLVA offsets the disadvantages of other high discriminatory typing approaches and represents a promising tool for hospital, national and international molecular epidemiology.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina/genética , Secuencias Repetidas en Tándem , Electroforesis en Gel de Campo Pulsado , Colorantes Fluorescentes , Reacción en Cadena de la PolimerasaRESUMEN
Methicillin-resistant Staphylococcus aureus sequence type 398 (ST398 MRSA) was identified in Dutch pigs and pig farmers. ST398 methicillin-susceptible S. aureus circulates among humans at low frequency (0.2%) but was isolated in 3 human cases of bacteremia (2.1%; p = 0.026). Although its natural host is probably porcine, ST398 MRSA likely causes infections in humans.
Asunto(s)
Resistencia a la Meticilina , Meticilina/farmacología , Staphylococcus aureus/clasificación , Staphylococcus aureus/efectos de los fármacos , Porcinos/microbiología , Animales , Antibacterianos/farmacología , Portador Sano , Humanos , Países Bajos , Nariz/microbiología , Exposición Profesional , Infecciones Estafilocócicas/epidemiología , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/veterinaria , Staphylococcus aureus/genéticaRESUMEN
An outbreak of community-acquired methicillin-resistant Staphylococcus aureus occurred among members and close contacts of a soccer team. Typing of the isolates showed the outbreak was caused by the well-known European ST80-IV strain. To our knowledge, this is the first report of an outbreak of this strain among members of a sports team.
Asunto(s)
Brotes de Enfermedades , Resistencia a la Meticilina , Fútbol , Infecciones Estafilocócicas/epidemiología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/efectos de los fármacos , Adolescente , Adulto , Electroforesis en Gel de Campo Pulsado , Femenino , Humanos , Pruebas de Sensibilidad Microbiana , Países Bajos/epidemiología , Infecciones Estafilocócicas/transmisión , Staphylococcus aureus/clasificación , Staphylococcus aureus/aislamiento & purificaciónRESUMEN
BACKGROUND: Sporadic cases of CA-MRSA in persons without risk-factors for MRSA carriage are increasing. CASE PRESENTATION: We report a MRSA cluster among family members of a pig-farmer, his co-workers and his pigs. Initially a young mother was seen with mastitis due to MRSA. Six months later her baby daughter was admitted to the hospital with pneumococcal otitis. After staying five days in hospital, the baby was found to be MRSA positive. At that point it was decided to look for a possible source, such as other family members and house-hold animals, including pigs on the farm, since those were reported as a possible source of MRSA earlier. Swabs were taken from the throat and nares of family members and co-workers. A veterinarian obtained swabs from the nares, throat and perineum of 10 pigs. Swabs were cultured following a national protocol to detect MRSA that included the use of an enrichment broth. Animal and human strains were characterized by PFGE, spa-typing, MLST analysis, SSCmec, AGR typing, and the detection for PVL, LukM, and TSST toxin genes. Three family members, three co-workers, and 8 of the 10 pigs were MRSA positive. With the exception of the initial case (the mother) all persons were solely colonized, with no signs of clinical infections. After digestion with SmaI, none of the strains showed any bands using PFGE. All isolates belonged to spa type t108 and ST398. CONCLUSION: 1. This report clearly shows clonal spread and transmission between humans and pigs in the Netherlands. 2. MLST sequence type 398 might be of international importance as pig-MRSA, since this type was shown earlier to be present in epidemiologically unrelated French pigs and pig-farmers. 3. Research is needed to evaluate whether this is a local problem or a new source of MRSA, that puts the until now successful Search and Destroy policy of the Netherlands at risk.
Asunto(s)
Crianza de Animales Domésticos , Infecciones Comunitarias Adquiridas/transmisión , Resistencia a la Meticilina , Infecciones Estafilocócicas/transmisión , Infecciones Estafilocócicas/veterinaria , Staphylococcus aureus/efectos de los fármacos , Porcinos/microbiología , Adulto , Animales , Animales Domésticos , Proteínas Bacterianas/genética , Técnicas de Tipificación Bacteriana , Portador Sano/microbiología , Infecciones Comunitarias Adquiridas/microbiología , Electroforesis en Gel de Campo Pulsado , Femenino , Humanos , Recién Nacido , Masculino , Mastitis/microbiología , Resistencia a la Meticilina/genética , Cavidad Nasal/microbiología , Análisis de Secuencia de ADN , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/clasificación , Staphylococcus aureus/genética , Staphylococcus aureus/aislamiento & purificación , Enfermedades de los Porcinos/microbiología , Enfermedades de los Porcinos/transmisión , ZoonosisRESUMEN
This paper describes the design and synthesis of gramicidin S (GS) analogues 10a-c containing arylated sugar amino acids (SAAs) as a replacement of one of the two (D)Phe-Pro beta-turn regions. The cyclic, amphiphilic peptides adopt a beta-sheet conformation featuring an unusual reverse turn induced by the SAAs. The altered turn region induces a slight distortion of the antiparallel beta-sheet, as compared to GS; the overall geometry however closely resembles that of the nonarylated GS analogue 1. GS analogues 10a-c proved to be as active as the parent GS itself as antibacterial agents and are equally efficient in lysing red blood cells. These properties are in sharp contrast to the diminished biological activity displayed by 1. We conclude that the presence of aromaticity in the turn regions of GS derivatives is required for biological activity, whereas the native conformation of the beta-hairpin is not. Our findings may guide future research toward efficient and nonhemolytic GS analogues for combating bacterial infections.
Asunto(s)
Aminoácidos/química , Amino Azúcares/química , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Productos Biológicos/farmacología , Eritrocitos/efectos de los fármacos , Gramicidina/farmacología , Hemólisis/efectos de los fármacos , Acrilatos/química , Secuencia de Aminoácidos , Antibacterianos/síntesis química , Productos Biológicos/síntesis química , Gramicidina/síntesis química , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Fenilalanina/química , Prolina/química , Estructura Secundaria de ProteínaRESUMEN
Multienzyme multiplex PCR-amplified fragment length polymorphism (ME-AFLP) typing is a reliable and simple method for typing of bacterial species. In this study we analyzed two well-documented strain collections of Staphylococcus aureus and compared ME-AFLP typing results with results of various other typing methods. The discriminatory power of ME-AFLP was found comparable to pulsed-field gel electrophoresis, and typing results were highly concordant. ME-AFLP typing presents a suitable method for prescreening of large strain collections. Furthermore, the obtained typing patterns were found to cluster according to the staphylococcal cassette chromosome mec types of the strains.
Asunto(s)
Electroforesis en Gel de Campo Pulsado/métodos , Resistencia a la Meticilina , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Longitud del Fragmento de Restricción , Staphylococcus aureus/clasificación , Staphylococcus aureus/efectos de los fármacos , Proteínas Bacterianas/genética , Técnicas de Tipificación Bacteriana , Cromosomas Bacterianos , Brotes de Enfermedades , Humanos , Infecciones Estafilocócicas/epidemiología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genéticaRESUMEN
The design and synthesis of analogues of the cyclic beta-sheet gramicidin S (GS), having additional functionalities in their turn regions, is reported. The monomeric GS analogues were transformed into dimers and their activities towards biological membranes, through antimicriobial and hemolytic assays, were evaluated. Finally, conductivity measurements have been performed to elucidate ion channel forming properties.
Asunto(s)
Antibacterianos/síntesis química , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Gramicidina/síntesis química , Gramicidina/farmacología , Antibacterianos/química , Dimerización , Gramicidina/química , Hemólisis , Canales Iónicos/química , Pruebas de Sensibilidad Microbiana , Conformación Molecular , Sensibilidad y Especificidad , Relación Estructura-ActividadRESUMEN
A practical gram-scale and high-yielding synthesis of the antimicrobial peptide gramicidin S is presented. An Fmoc-based solid-phase peptide synthesis protocol is employed for the generation of the linear decapeptide precursor, which is cyclized in solution to afford the target compound. The versatility of our method is demonstrated by the construction of eight gramicidin S analogues (15a-h) having nonproteinogenic sugar amino acid residues (4-7) incorporated in the turn regions.
Asunto(s)
Aminoácidos/síntesis química , Antibacterianos/síntesis química , Carbohidratos/síntesis química , Gramicidina/síntesis química , Aminoácidos/farmacología , Antibacterianos/farmacología , Bacillus cereus/efectos de los fármacos , Carbohidratos/farmacología , Enterococcus faecalis/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Gramicidina/farmacología , Humanos , Pruebas de Sensibilidad Microbiana , Staphylococcus/efectos de los fármacosRESUMEN
The epidemiology of vancomycin-resistant Entero- coccus faecium (VREF) in Europe is characterized by a large community reservoir. In contrast, nosocomial outbreaks and infections (without a community reservoir) characterize VREF in the United States. Previous studies demonstrated host-specific genogroups and a distinct genetic lineage of VREF associated with hospital outbreaks, characterized by the variant esp-gene and a specific allele-type of the purK housekeeping gene (purK1). We investigated the genetic relatedness of vanA VREF (n=108) and vancomycin-susceptible E. faecium (VSEF) (n=92) from different epidemiologic sources by genotyping, susceptibility testing for ampicillin, sequencing of purK1, and testing for presence of esp. Clusters of VSEF fit well into previously described VREF genogroups, and strong associations were found between VSEF and VREF isolates with resistance to ampicillin, presence of esp, and purK1. Genotypes characterized by presence of esp, purK1, and ampicillin resistance were most frequent among outbreak-associated isolates and almost absent among community surveillance isolates. Vancomycin-resistance was not specifically linked to genogroups. VREF and VSEF from different epidemiologic sources are genetically related; evidence exists for nosocomial selection of a subtype of E. faecium, which has acquired vancomycin-resistance through horizontal transfer.
Asunto(s)
Resistencia a la Ampicilina/genética , Enterococcus faecium/efectos de los fármacos , Genes MDR/genética , Resistencia a la Vancomicina/genética , Brotes de Enfermedades , Enterococcus faecium/genética , Genotipo , Humanos , Reacción en Cadena de la PolimerasaRESUMEN
The synthesis of novel gramicidin S analogues having additional functionalities in the turn region by employing a biomimetic approach is described. The preservation of beta-sheet character in all analogues was established by NMR and the biological activity was evaluated.
Asunto(s)
Gramicidina/análogos & derivados , Gramicidina/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Gramicidina/síntesis química , Pruebas de Sensibilidad Microbiana , Imitación Molecular , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de ProteínaRESUMEN
A multilocus sequence typing (MLST) scheme has been developed for Enterococcus faecium. Internal fragments from seven housekeeping genes of 123 epidemiologically unlinked isolates from humans and livestock and 16 human-derived isolates from several outbreaks in the United States, the United Kingdom, Australia, and The Netherlands were analyzed. A total of 62 sequence types were detected in vancomycin-sensitive E. faecium (VSEF) and vancomycin-resistant E. faecium (VREF) isolates. VSEF isolates were genetically more diverse than VREF isolates. Both VSEF and VREF isolates clustered in host-specific lineages that were similar to the host-specific clustering obtained by amplified fragment length polymorphism analysis. Outbreak isolates from hospitalized humans clustered in a subgroup that was defined by the presence of a unique allele from the housekeeping gene purK and the surface protein gene esp. The MLST results suggest that epidemic lineages of E. faecium emerged recently worldwide, while genetic variation in both VREF and VSEF was created by longer-term recombination. The results show that MLST of E. faecium provides an excellent tool for isolate characterization and long-term epidemiologic analysis.