Structure and antimicrobial activity of phloroglucinol derivatives from Achyrocline satureioides.

The new prenylated phloroglucinol α-pyrones 1-3 and the new dibenzofuran 4, together with the known 23-methyl-6-O-demethylauricepyrone (5), achyrofuran (6), and 5,7-dihydroxy-3,8-dimethoxyflavone (gnaphaliin A), were isolated from the aerial parts of Achyrocline satureioides. Their structures were determined by 1D and 2D NMR spectroscopic studies, while the absolute configuration of the sole stereogenic center of 1 was established by vibrational circular dichroism measurements in comparison to density functional theory calculated data. The same (S) absolute configuration of the α-methylbutyryl chain attached to the phloroglucinol nucleus was assumed for compounds 2-6 based on biogenetic considerations. Derivatives 7-16 were prepared from 1 and 5, and the antimicrobial activities of the isolated metabolites and some of the semisynthetic derivatives against a selected panel of Gram-positive and Gram-negative bacteria, as well as a set of yeast molds, were determined.

Multicomponent synthesis of antibacterial dihydropyridin and dihydropyran embelin derivatives.

A series of dihydropyran and dihydropyridin embelin derivatives were synthesized through a novel and straightforward one-pot protocol based on a three-component reaction with embelin, aldehydes, and cyclic enaminones as synthetic imputs. The type of substituent on the nitrogen atom of the ß-enaminone is key to obtain nitrogenated or oxygenated rings. The obtained compounds were active against Gram-positive bacteria, including multiresistant Staphylococcus aureus clinical isolates.

Molecular epidemiology of plasmid-mediated high-level mupirocin resistance in methicillin-resistant Staphylococcus aureus in four Spanish health care settings.

Mupirocin is used for the decolonization of methicillin-resistant Staphylococcus aureus (MRSA). High-level mupirocin resistance (Hi-Mup(R)) is of concern, having been associated with therapeutic failure. Our main objective was to assess the emergence and mode/s of spread of Hi-Mup(R) in the MRSA population recovered between 2002 and 2009 in four health care settings in the Pontevedra province, northwest Spain. Five hundred and fifty consecutive clinical MRSA isolates were obtained and screened for antimicrobial susceptibility. Isolates were stratified into multidrug-resistant (MDR) and non-MDR. High-level mupirocin resistant MRSA were characterized by genotyping and plasmid analysis. Thirty-one MRSA (5.6%) exhibited Hi-Mup(R). No association was detected between Hi-Mup(R) and MDR but isolates displaying Hi-Mup(R) were more likely to be resistant to gentamicin and tobramycin. Four main MRSA clones were identified: ST125/t067/PFGE A, ST36/t018/PFGE D, ST8/t008/PFGE B, and ST72/t148 or t3092/PFGE B. Each isolate carried the Hi-Mup(R)ileS2-encoding gene on plasmids and ten plasmid types were distinguished based on unique IS257-ileS2 configurations. Some plasmid types were successfully disseminated among the MRSA clones. Remarkably, six plasmid types were acquired by the predominant genotype ST125/t067/PFGE A. In conclusion, molecular characterization of MRSA isolates combined with the rapid typing of ileS2-encoding plasmids through determination of IS257-ileS2 configurations have proved to be a powerful strategy to address the molecular epidemiology of Hi-Mup(R). The transmission of a diverse set of ileS2-carrying plasmids promoted the emergence of the resistance, with a limited role of clonal expansion in its dispersion.

Molecular epidemiology and characterization of virulence genes of community-acquired and hospital-acquired methicillin-resistant Staphylococcus aureus isolates in Colombia.

OBJECTIVE: To determine the molecular epidemiology and presence of virulence genes in community-acquired (CA) and hospital-acquired (HA) methicillin-resistant Staphylococcus aureus (MRSA) isolates and their relationship to clinical outcomes. METHODS: An observational and prospective study of infections caused by MRSA was conducted between June 2006 and December 2007 across seven hospitals in three Colombian cities. MRSA isolates were analyzed for SCCmec. Also, pulsed-field gel electrophoresis and multilocus sequence typing were performed and 25 virulence genes were identified. RESULTS: Two hundred and seventy isolates were collected from 262 adult hospital patients with MRSA infections. Overall, 68% of the isolates were classified as HA-MRSA and 32% as CA-MRSA. We identified differences in the patterns of virulence genes: 85% of HA-MRSA isolates possessed the enterotoxin gene cluster (egc), whereas 92% of CA-MRSA isolates possessed the lukF-PV/lukS-PV genes. Multivariate analysis showed an increased risk of mortality for seg (p=0.001, odds ratio 4.73) and a protective effect for eta (p=0.018, odds ratio 0.33). CONCLUSIONS: Our study confirms that three clones of MRSA predominantly circulate in Colombia: a Chilean clone, a pediatric clone that causes HA-MRSA infections, and a USA300-related clone (SCCmec IVc) in CA-MRSA infections, which differ in the content of clinically important virulence genes. This study confirms that PVL is not a determinant of severity or mortality in CA-MRSA infections.

Achyrofuran is an antibacterial agent capable of killing methicillin-resistant vancomycin-intermediate Staphylococcus aureus in the nanomolar range.

Currently, there is a pressing need for novel antibacterial agents against drug-resistant bacteria, especially those which have been common in our communities and hospitals, such as methicillin-resistant Staphylococcus aureus (MRSA). The South American plant Achyrocline satureioides ("Marcela") has been widely used in traditional medicine for a number of diseases, including infections. Several crude extracts from this plant have shown good antimicrobial activities in vitro. In the search for the active principle(s) that confers these antimicrobial activities, we have processed the dichloromethane extract from the aerial parts of the plant. One of the isolated compounds showed extraordinary antibacterial activities against a set of clinically relevant Gram-positive strains that widely differ in their antibiogram profiles. This compound was identified as achyrofuran on the basis of its spectroscopic and physical data. We determined the MIC to be around 0.1 µM (0.07 µg/ml) for the reference methicillin-resistant and vancomycin-intermediate S. aureus strain NRS402. Moreover, nanomolar concentrations of achyrofuran killed 10(6) bacteria within 12 h. Based on the presence of the 2,2'-biphenol core, we further studied whether achyrofuran killed bacteria through a mechanism of action similar to that reported for the naturally occurring antibiotic MC21-A. Indeed, we found that achyrofuran was not bacteriolytic by itself although it greatly compromised membrane impermeability as determined by increased SYTOX Green uptake.

Genetic diversity of community-associated methicillin-resistant Staphylococcus aureus isolated from Tenerife Island, Spain.

With the recent detection of MRSA (methicillin-resistant Staphylococcus aureus) infections in patients lacking health care-related risk factors, the term community-acquired MRSA (CA-MRSA) has been become widely recognised. Many cases of CA-MRSA spreading to the community have been described worldwide. The aim of this study was to determine the features of CA-MRSA isolates from Tenerife Island. Toward this end, one hundred MRSA isolates were collected from eight different health regions, and their molecular features were investigated. This study revealed a wide variety of MRSA clones, including an emergent ST: ST1434 (CC8) and two new spa types, t7575 (ST125) and t7678 (ST22). The PVL genes were found in only five isolates belonging to unrelated lineages, ST8, ST30 and ST22, which could indicate at least three independent introductions of PVL(+) strains to Tenerife. Moreover, we detected that hospital MRSA clones, like EMRSA-15 and EMRSA-16, had spread to the community and are now circulating in both environments. Therefore, in our study, the CDC's rules were not specific enough to distinguish CA-MRSA from HA-MRSA. Thus, we think that the current epidemiological information is not enough to discriminate between both MRSAs, and it is necessary for prevention guidelines to include the routine determination of at least the genetic background, the antimicrobial susceptibility profile, and the PVL genes of each MRSA isolate.

New Staphylococcus aureus genetic cluster associated with infectious osteomyelitis.

Diverse genotyping methods, including multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA), pulsed field gel electrophoresis (PFGE), and multilocus sequence typing (MLST), were used for genotyping Staphylococcus aureus in samples recovered from a clinical case of osteomyelitis. An unexpected genetic diversity of strains was determined, including four new sequence types (ST 1521, 1522, 1628 and 1629) belonging to the same genetic lineage, implying the appearance of a new subgroup derived from clonal complex CC121 isolated from that hospital. A close phylogenetic relationship among the STs was demonstrated, reflecting a possible diversifying evolution process. To our knowledge, there have no been previous reports of staphylococcal genetic variability observed within a single individual with such a high degree of variation. These findings emphasize the need for infection control measures to monitor the high genetic variability continuously occurring in this often dangerous infectious agent.

New Staphylococcus aureus genetic cluster associated with infectious osteomyelitis

Diverse genotyping methods, including multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA), pulsed field gel electrophoresis (PFGE), and multilocus sequence typing (MLST), were used for genotyping Staphylococcus aureus in samples recovered from a clinical case of osteomyelitis. An unexpected genetic diversity of strains was determined, including four new sequence types (ST 1521, 1522, 1628 and 1629) belonging to the same genetic lineage, implying the appearance of a new subgroup derived from clonal complex CC121 isolated from that hospital. A close phylogenetic relationship among the STs was demonstrated, reflecting a possible diversifying evolution process. To our knowledge, there have no been previous reports of staphylococcal genetic variability observed within a single individual with such a high degree of variation. These findings emphasize the need for infection control measures to monitor the high genetic variability continuously occurring in this often dangerous infectious agent (AU)

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PCR-based amplification of heterogeneous IS257-ileS2 junctions for molecular monitoring of high-level mupirocin resistance in staphylococci.

OBJECTIVES: Plasmids encoding the ileS2 gene are responsible for the wide spread of high-level mupirocin resistance in staphylococci, and consequent clinical and epidemiological problems. We investigated the location of insertion sequence IS257 flanking ileS2 in different plasmids and developed a method for molecular typing of the IS257-ileS2 spacer regions. METHODS: Nine ileS2-encoding plasmids (i.e. pPR1-pPR9) classified into distinct structural groups (i.e. S1-S4) were analysed. Complete DNA sequences between IS257s flanking ileS2 were determined. A PCR-based amplification scheme was designed for the simultaneous amplification of up- and down-stream IS257-ileS2 regions. The method was applied to a total of 90 high-level mupirocin-resistant clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA). RESULTS: pPR1-pPR9 possessed IS257s flanking ileS2. Plasmids of each structural group showed a unique configuration of IS257-ileS2 spacer regions. The PCR-based method permitted accurate typing of the heterogeneous IS257-ileS2 up- and down-stream junctions, and the differentiation of plasmids of each group. The results obtained corresponded with previous plasmid typing based on restriction enzyme analyses and ileS2 locus hybridization polymorphs. The application of the PCR-based method to a diverse collection of MRSA isolates carrying ileS2-encoding plasmids demonstrated its versatility and revealed extraordinary heterogeneity in the IS257-ileS2 spacers. CONCLUSIONS: The devised PCR-based scheme offers a rapid, simple and effective approach for typing IS257-ileS2 configurations present on ileS2-encoding plasmids. Hopefully, it could serve as a useful molecular epidemiological tool to control high-level mupirocin resistance.

Evaluation of multiple-locus variable-number tandem-repeat analysis for typing a polyclonal hospital-acquired methicillin-resistant Staphylococcus aureus population in an area where such infections are endemic.

Multiple-locus variable-number tandem-repeat analysis (MLVA) was performed with 292 methicillin-resistant Staphylococcus aureus (MRSA) isolates previously characterized by pulsed-field gel electrophoresis, multilocus sequence typing, and staphylococcal cassette chromosome mec typing. Quantitative correspondence analyses showed the best correlation between data when an >or=80% cutoff was applied to MLVA. We confirmed the validity of MLVA for identification of related strains in a polyclonal MRSA population.

Complete nucleotide sequence and comparative analysis of pPR9, a 41.7-kilobase conjugative staphylococcal multiresistance plasmid conferring high-level mupirocin resistance.

We have sequenced the conjugative plasmid pPR9, which carries the ileS2 gene, which had contributed to the dissemination of high-level mupirocin resistance at our institution. The plasmid backbone shows extensive genetic conservation with plasmids belonging to the pSK41/pGO1 family, but comparative analyses have revealed key differences that provide important insights into the evolution of these medically important plasmids and high-level mupirocin resistance in staphylococci and highlight the role of insertion sequence IS257 in these processes.

Nosocomial infections caused by community-associated methicillin-resistant Staphylococcus aureus in Colombia.

BACKGROUND: Community-associated methicillin-resistant Staphylococcus aureus strains (CA-MRSA) have emerged as the causative agent of health care-associated infections. METHODS: An observational and prospective study was carried out in 5 hospitals in Bogotá, Colombia; severe MRSA infections were identified, and their origin led to classification as health care-associated (HA-MRSA), community-associated, or nosocomial infections. MRSA isolates were analyzed by SCCmec, pulsed-field gel electrophoresis, multilocus sequence typing, and virulence factors. RESULTS: Twenty-six (10.4%) CA-MRSA nosocomial infection-causing strains (eg, USA300) were detected in 250 MRSA infection isolates in mainly primary bacteremia and surgical site infections. The mortality related to nosocomial infection by CA-MRSA was 27%. CONCLUSION: The presence of nosocomial infection by CA-MRSA in Colombia was confirmed.

Molecular characterization of Alternaria alternata causing ocular infection: detection of IGS-RFLP intraspecific polymorphism.

A case of fungal keratitis is presented in which corneal scrapings were obtained for microbiological studies, including morphological identification and molecular characterization of the etiologic agent. Comparative sequence analyses of the Internal Transcribed Spacer domain of 5.8S and 26S regions of nuclear rDNA showed 100% identity with different species of Alternaria and PCR-RFLP analysis of Intergenic Spacer regions revealed intraspecific variation. The combination of morphological and molecular characters resulted in the unambiguous identification of the causal agent as Alternaria alternata. Treatment with antifungals contributed to the improvement in the patient's lesions.

High-level mupirocin resistance within methicillin-resistant Staphylococcus aureus pandemic lineages.

The methicillin-resistant Staphylococcus aureus (MRSA) population in the Hospital Universitario Nuestra Señora de Candelaria over a 5-year period (1998 to 2002) was marked by shifts in the circulation of pandemic clones. Here, we investigated the emergence of high-level mupirocin resistance (Hi-Mup(r)). In addition to clonal spread, transfer of ileS2-carrying plasmids played a significant role in the dissemination of Hi-Mup(r) among pandemic MRSA lineages.

PCR protocol for specific identification of Candida nivariensis, a recently described pathogenic yeast.

Candida nivariensis is a recently described pathogenic yeast closely related to Candida glabrata. We developed a specific set of oligonucleotide primers based on the internal transcribed spacer regions of the rRNA gene for the rapid identification of C. nivariensis. PCR with these primers amplified a 206-bp amplicon from C. nivariensis.

Seasonal microbial ribotype shifts in the sulfurous karstic lakes Cisó and Vilar, in northeastern Spain / Cambios estacionales en los ribotipos microbianos en los lagos cársticos y sulfurosos Cisó y Vilar, en el nordeste de España

Spatio-temporal changes in two sulfurous lakes from the karstic area of Banyoles (Girona, Spain), holomictic lake Cisó and meromictic lake Vilar, were studied over one year. Samples were collected at different depths from the two lakes on the same days, during each of the four seasons, and several physico-chemical variables (temperature, light, pH, conductivity, sulfide, oxygen concentration, pigment concentrations, etc.) were measured. To fingerprint bacterial populations from each sample, DNA was extracted, bacterial 16S rRNA genes were amplified by PCR, and restriction fragment length polymorphism (RFLP) analyses of the total bacterial 16S rDNAs were performed. Each 16S rDNA pool was independently digested with three restriction endonucleases (AluI, HinfI, and RsaI) and separated electrophoretically. More restriction fragments were obtained from the Lake Vilar samples than from the Lake Cisó samples. Moreover, intrasample and intersample differences were observed in each lake. RFLP patterns were compared by scoring similarities using the Jaccard coefficient and then building a multidimensional scaling (MDS) map from the resulting similarities matrix. In both lakes, results indicated that seasonality was mostly responsible for the observed fluctuations in the RFLP patterns, while the effect of stratification was less pronounced (AU)

Se estudió a lo largo de un año el cambio espacio-temporal que se produjo en dos lagos sulfurosos de la zona cárstica de Banyoles (Girona, España), el lago Cisó, holomíctico, y el Lago Vilar, meromíctico. Se tomaron muestras a diferentes profundidades en los dos lagos los mismos días durante las cuatro estaciones y se midieron algunas variables fisicoquímicas (temperatura, luz, pH, conductividad, sulfuro, concentraciones de oxígeno y de pigmentos, etc.). Para obtener la impronta genética de las poblaciones bacterianas de cada muestra, se extrajo el DNA, se amplificaron los genesdel 16S rRNA mediante PCR y se analizó el polimorfismo en la longitud de los fragmentos de restricción (RFLP) del total de 16S rDNA bacteriano. Los diferentes conjuntos de 16S rDNA bacteriano fueron digeridos de manera independiente con tres endonucleasas de restricción (AluI, HinfI, y RsaI) y separados por electroforesis. Se obtuvieron más fragmentos de restricción de las muestras del lago Vilar que del Cisó. Además, en cada lago se observaron también diferencias dentro de cada muestra y entre las diferentes muestras. Luego se compararon los patrones de RFLP puntuando las similitudes mediante el coeficiente Jaccard y la creación un mapa de escalamiento multidimensional (MDS) a partir de la matriz de similitudes resultante. Los resultados indicaron que la estacionalidad era la principal causa de las fluctuaciones observadas en los patrones de RFLP en ambos lagos, mientras que el efecto de la estratificación era menos pronunciado (AU)

Phenotypic and molecular characterization of Candida nivariensis sp. nov., a possible new opportunistic fungus.

The new species Candida nivariensis, isolated from the clinical samples of three patients in Spain over a 3-year period, is presented here. This species can be easily differentiated from Candida glabrata, the closest genetic species, by different colony color on CHROMagar and by its ability to ferment trehalose. The analyses of the internal transcribed spacer region and the D1-D2 region of the 26S rRNA gene sequences support a new species designation.

Outbreak of Shigella sonnei in a rural hotel in La Gomera, Canary Islands, Spain.

Shigella sonnei is a significant cause of gastroenteritis in both developing and industrialized countries. Knowledge of the diversity and antimicrobial susceptibility of the bacterium may be helpful in the management of both individual cases and outbreaks. This study was undertaken to evaluate the molecular epidemiology of an outbreak of diarrhea due to S. sonnei. The outbreak involved 14 of 28 (50%) tourists in a small rural hotel in La Gomera, Canary Islands, Spain. All of the S. sonnei isolates recovered had the same antimicrobial susceptibility and pulsed-field gel electrophoresis patterns, suggesting that the outbreak was produced by a single strain.

Outbreak of Shigella sonnei in a rural hotel in La Gomera, Canary Islands, Spain / Brote de Shigella sonnei en un hotel rural de La Gomera (Islas Canarias, España)

Shigella sonnei is a significant cause of gastroenteritis in both developing and industrialized countries. Knowledge of the diversity and antimicrobial susceptibility of the bacterium may be helpful in the management of both individual cases and outbreaks. This study was undertaken to evaluate the molecular epidemiology of an outbreak of diarrhea due to S. sonnei. The outbreak involved 14 of 28 (50%) tourists in a small rural hotel in La Gomera, Canary Islands, Spain. All of the S. sonnei isolates recovered had the same antimicrobial susceptibility and pulsed-field gel electrophoresis patterns, suggesting that the outbreak was produced by a single strain (AU)

Shigella sonnei es una causa significativa de gastroenteritis, tanto en países en desarrollo como industrializados. El conocimiento de la diversidad de esa bacteria y de su sensibilidad a los antimicrobianos puede ser una ayuda en el tratamiento de casos individuales y de brotes infecciosos. Este estudio se realizó para evaluar la epidemiología molecular de un brote de diarrea debido a S. sonnei. El brote afectó a 14 de los 28 (50%) turistas en un pequeño hotel rural en La Gomera, Islas Canarias, en España. Todos los aislados de S. sonnei recuperados presentaron el mismo patrón de sensibilidad a los antimicrobianos y el mismo patrón de electroforesis en gel de campo pulsado, lo cual indica que el brote fue causado por una sola cepa (AU)