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Recently the CLSI recommended a disk diffusion method and breakpoints for meningococci which include breakpoints derived for nalidixic acid which serve as surrogate markers for gyrase A mutations associated with diminished fluoroquinolone susceptibility. This study presents the application of this methodology to a panel of 57 meningococcal strains isolated in Spain that include all levels of susceptibility to ciprofloxacin. In conclusion, the most useful method to predict isolates with gyrA mutations that decrease the activity of fluoroquinolones is the use of 30-microg nalidixic acid disks.

Objectives: To assess the ciprofloxacin resistance of Neisseria meningitidis isolated from 1999 through 2006 in Spain, susceptibility testing was conducted on 5300 isolates., Methods: Ten isolates showed MICs of ciprofloxacin ranging between 0.06 and 0.25 mg/L, and they were characterized by analysis for mutations within the quinolone resistance determining regions (QRDRs) in the gyrA, gyrB, parC and parE genes. Mutations in the mtrR gene were also analysed., Results and Conclusions: Single mutations in gyrA appeared to be the main mechanism involved, Thr-91-->Ile being the most frequent substitution seen. Two meningococci had four different gyrA substitutions. No mutations in the QRDRs of the parC and gyrB genes were detected, and three strains showed a His-495-->Asn substitution in the parE gene. In addition, two different alterations in the mtrR gene affecting the expression of the MtrCDE efflux system were identified which may also contribute to the reduced susceptibility to quinolones seen in three strains.

PNC-27, a synthetic peptide, is derived from the TP53-HDM2 binding domain that include TP53 amino acids 12-26 linked with 17 amino acids from the antennapedia protein transference domain. This peptide induces membrane rupture in tumor cells through toroidal pores formation and has motivated several experimental studies; nonetheless, its mechanism of biological action remains unknown to date. Herein, we present a theoretical study at the Hartree-Fock and density functional theory (B3LYP) levels of theory of TP53 protein residues 12-26 (PPLSQETFSDLWKLL) in order to characterize its electronic structure and physicochemical properties. Our results for atomic and group charges, fitted to the electrostatic potential (ESP) show important reactive sites (L14, S15, T18, S20, L25, and L26), suggesting that these amino acids are exposed to nucleophilic and electrophilic attacks. Analysis of bond orders, intramolecular interactions and of several global reactivity descriptors, such as ionization potentials, hardness, electrophilicity index, dipole moments, total energies, frontier molecular orbitals (HOMO-LUMO), and electrostatic potential, led us to characterize active sites and the electronic structure and physiochemical features that taken together may be important in understanding the specific selectivity for this peptides type's cancer-cell membrane lysis properties.

Pharmacophore-based structural identification, synthesis, and structure-activity relationships of a new class of muscarinic M3 receptor antagonists, the diaryl imidazolidin-2-one derivatives, are described. The versatility of the discovered scaffold allowed for several structural modifications that resulted in the discovery of two distinct classes of compounds, specifically a class of tertiary amine derivatives (potentially useful for the treatment of overactive bladder by oral administration) and a class of quaternary ammonium salt derivatives (potentially useful for the treatment of respiratory diseases by the inhalation route of administration). In this paper, we describe the synthesis and biological activity of tertiary amine derivatives. For these compounds, selectivity for the M3 receptor toward the M2 receptor was crucial, because the M2 receptor subtype is mainly responsible for adverse systemic side effects of currently marketed muscarinic antagonists. Compound 50 showed the highest selectivity versus M2 receptor, with binding affinity for M3 receptor Ki = 4.8 nM and for M2 receptor Ki = 1141 nM. Functional in vitro studies on selected compounds confirmed the antagonist activity toward the M3 receptor and functional selectivity toward the M2 receptor.

Meningococcal disease is a serious and rapidly progressing illness. It is therefore very important to monitor changes in the level of antibiotic susceptibility among clinical isolates. Different aspects such as interpretation of laboratory results, determination of breakpoints predicting treatment failure as well as definition of susceptibility levels in clinical samples using molecular methods are critical points for the surveillance of antibiotic resistance in Neisseria meningitidis. Within the strategic framework outlined by the EU.MenNet project, several objectives were identified to analyze 'The spread of antibiotic resistant meningococci in Europe', including the extent of antimicrobial resistance, its association with particular meningococcal lineages and geographical areas, as well as molecular characterization of the mechanisms involved, particularly in penicillin resistance. A heterogeneous figure for the frequency of intermediate resistance to penicillin appears across Europe. This heterogeneity may reflect different clonal lineages and/or uneven access to antibiotics in each country. In addition, the use of different criteria for the methodology used for definition cannot be avoided. The description of five specific changes associated with intermediate resistance to penicillin also allows the design of PCR-based tools to objectify results and for application in clinical samples.

Introduction: The aim of this study was to assess the evolution of the population MICs for various antimicrobial drugs against Neisseria meningitidis isolates obtained from asymptomatic carriers during a chemoprophylaxis campaign carried out for an epidemic outbreak of meningococcal disease in Nerva, a small town in Huelva province (Spain)., Material and Methods: A nasopharyngeal carrier study including 427 people was carried out to determine the incidence rate of the epidemic strain among the general population. On the basis of the results, chemoprophylaxis with rifampicin was administered to the population aged 15 to 29 years (age group showing the highest incidence of the epidemic strain among carriers) living in Nerva. Three months later a new carrier study was performed (507 people) to evaluate the effects of chemoprophylaxis. Given the evolution of the outbreak, seven months later a new intervention was required with ciprofloxacin chemoprophylaxis; a second carrier study (399 people) was performed two months later to evaluate its effect., Results: The number of strains isolated during the three carrier studies was 59 (13.8%), 33 (6.5%), and 22 (5.5%), respectively. Analysis of the changes in the MIC50 and MIC90 for the various antibiotics from the first to the second carrier study (rifampicin chemoprophylaxis) showed statistical differences only in the distribution of rifampicin MICs. Similarly, when changes from the second to the third study were analyzed (ciprofloxacin chemoprophylaxis), significant variations were detected for the cefotaxime MICs. Nevertheless, although there were changes in the MICs, the percentages of susceptibility from the beginning to the end of the study did not vary., Conclusions: Massive chemoprophylaxis in the age group with the highest incidence of the epidemic strain among carriers did not clearly modify the antibiotic susceptibility of the isolates. However, a slight increase in the MIC50 and MIC90 was observed for rifampicin after the first chemoprophylactic intervention and for cefotaxime at the end of the study. Consecutive chemoprophylactic interventions with rifampicin and ciprofloxacin had an impact on the percentage of meningococcal carriers in the overall population, with a clearly decreasing trend.

We report the case of an infant affected with congenital systemic juvenile xanthogranuloma scheduled for central venous access system implantation (Port-a-Cath) and a liver and bone marrow biopsy. The patient had impaired liver function, thrombocytopenia, and coagulopathy which was refractory to daily fresh-frozen plasma and platelet infusions: 80 microg x kg(-1) dose(-1) of recombinant factor VIIa (rFVIIa) was administered i.v. every 2 h starting 30 min before the procedure and ending 6 h afterwards. Very minor bleeding was observed during the procedure. In conclusion, rFVIIa therapy was effective as prophylaxis for both invasive procedures in this patient with a coagulopathy which was refractory to other different therapies.

Speciation is generally regarded to result from the splitting of a single lineage. An alternative is hybrid speciation, considered to be extremely rare, in which two distinct lineages contribute genes to a daughter species. Here we show that a hybrid trait in an animal species can directly cause reproductive isolation. The butterfly species Heliconius heurippa is known to have an intermediate morphology and a hybrid genome, and we have recreated its intermediate wing colour and pattern through laboratory crosses between H. melpomene, H. cydno and their F1 hybrids. We then used mate preference experiments to show that the phenotype of H. heurippa reproductively isolates it from both parental species. There is strong assortative mating between all three species, and in H. heurippa the wing pattern and colour elements derived from H. melpomene and H. cydno are both critical for mate recognition by males.

Pulsed field gel electrophoresis (PFGE), multiplex PCR and multilocus sequence typing (MLST) methods were used for genotyping study of seventy-three L. monocytogenes isolates collected in Poland between 2000 and 2002 from human, food, environment and a diseased goat. The multiplex PCR, which is an alternative method to classical serotyping, divided the isolates into four PCR groups, IIa (42.5%), IIb (27.4%), IIc (4.1%) and IVb (26%). The isolates displayed 33 distinct PFGE profiles. Twenty eight strains were further characterised by MLST based on sequence analyses of seven housekeeping genes. The combined sequence analyses revealed a total of 10 different allelic profiles from which 3 were not described earlier. It is intended that results obtained in this study will be the first data of a national database of L. monocytogenes genotypes in Poland.

Synthetic methodologies aiming at the creation of new phosphorus-centered functional groups are reported, as well as applications to the field of nucleotide chemistry. Thus, difluorophosphonothioate-based, ionic reagents 3b and 3d are shown to allow the stereocontrolled and efficient synthesis of phosphonodifluoromethyl analogues of nucleoside-3'-phosphates. An alternate, radical approach describes the use of hypophosphorous acid to stereoselectively link two furanosyl units in positions 3 and 5, and to provide an access to alpha,alpha-difluoro-H-phosphinates. These intermediates are shown to be precursors to the corresponding fluorinated phosphonic acids, phosphonothioic acids and variously substituted phosphinates.

The performance of a multiplex PCR assay that separates the four major serovars of the pathogenic Listeria monocytogenes into four distinct PCR groups was evaluated through a multicenter typing study. Identical panels of 90 Listeria isolates were distributed to five participating laboratories that were blind to the nature of the isolates. Isolates were characterized using the previously standardized protocol. Overall concordance was 96.6 to 100%, sufficient for the assay to be used as an alternative to serotyping and confidently applied in laboratories involved in L. monocytogenes typing.

The PorA protein is a potential candidate as a vaccine component against meningococcal disease. However, this protein experiences antigenic variation and is subject to phase variations to evade immune selective pressure. In this study, the mechanisms responsible for altered expression of the PorA protein were analysed in 50 non-subtypable strains isolated from patients with meningococcal disease in Spain. The porA gene was amplified from 47 of the 50 strains. The majority of isolates were not recognized by the subtyping panel, as a result of non-synonymous base changes in the variable regions of the porA gene. Two of these strains revealed a premature stop codon before the variable region VR1 of PorA due to a single base-pair substitution at position 109 of the porA coding region. Another two presented a homopolymeric tract of eight adenine residues in the coding region, producing a DNA strand-slippage mechanism and PorA phase variation.

Testing of susceptibility to penicillin G by E-test and sequencing of an internal fragment of the penA gene were done for 43 meningococcal strains. Those strains for which the MIC was >/=0.094 micro g/ml showed mosaic alleles, so 0.094 micro g/ml is suggested as the penicillin G intermediate breakpoint when E-test is used.

The transpeptidase encoding region of the penA gene was sequenced in 44 meningococcal strains (41 serogroup C [23 characterized as serotype 2b and 18 as serotype 2a] and 3 serogroup B [B:2b:P1.2,5]). All strains were characterized by multilocus sequence typing and were determined to be susceptible or intermediate resistant to penicillin (Pen(s) or Pen(i), respectively). A high degree of homology was found among the penA alleles identified in the Pen(s) strains. All the Pen(i) C:2b strains, which belonged to 2 different clonal complexes, showed the same penA gene allele. This fact suggests that 1 of the clonal complexes acquired that allele, spreading it to the other by horizontal transfer. The same allele also was found in the B:2b strains studied, indicating that 1 of the Pen(i) C:2b strains underwent a capsular switching event. A different mosaic penA allele was identified in the Pen(i) C:2a strains, which belonged to the ET37 cluster.

A mass immunization campaign for 18-month to 19-year-olds was undertaken in Spain in 1996-1997 because of an epidemic of serogroup C meningococcal disease associated with a C:2b:P1.2,5 strain belonging to the A4 lineage. Surveillance for the "capsule-switching" phenomenon producing B:2b:P1.2,5 isolates was undertaken. Of 2,975 meningococci characterized, B:2b:P1.2,5 and B:2b:P1.2 antigenic combinations were found in 18 isolates; 15 meningococci were defined as serogroup B belonging to the A4 lineage.

Objective: To analyze the etiology, clinical picture, diagnostic methods, surgical treatment and follow up of a series of six patients with large abdominal pseudocyst as a complication of ventriculo-peritoneal shunts for cerebrospinal fluid relief., Design: Study of a series of cases., Patients and Methods: We studied six patients from 1992 to 1995 with large abdominal pseudocyst; there were four women and two men, with range of age from nine to 19 years, and average of 15.9 years. Diagnosis was supported by clinical findings, ultrasound and computed tomographic scan., Results: Meningeal tuberculosis was the cause of hydrocephalus, in five cases and neurocysticercosis in one. The most common symptoms were diffuse abdominal pain, nausea and vomiting. All of them presented with abdominal distention and a soft mass filled with liquid that occupied most of the abdominal cavity without evidence of shunt malfunction. All patients underwent surgical treatment for excision of the pseudocyst and relocation of the catheter of the shunt, without morbidity on mortality. In the follow up we have not experienced recurrence of the pseudocyst in any case., Conclusion: According to our experience, we propose that in a patient with a history of meningeal tuberculosis and ventriculo-peritoneal shunt that presents with an abdominal mass should be investigated for the occurrence of a pseudocyst. The ultrasound and tomographic scan are of great value for diagnosis. Surgical treatment through an open laparotomy is the best option for these patients. Morbidity and mortality are considered low, without evidence of recurrence.

The effectiveness of Agrobacterium radiobacter K84, 0341, and a K84 non-agrocin-producing mutant (K84 Agr) in biological control of crown gall on rootstocks of stone fruit trees was determined in three experiments. In experiment 1, K84 and 0341 controlled crown gall on plum plants in soil inoculated with two strains of Agrobacterium tumefaciens resistant to agrocin 84. In experiment 2, K84 controlled crown gall on peach plants in soils inoculated with strains of A. tumefaciens sensitive or resistant to agrocin 84 or with a mixture of both. However, the effectiveness of K84 was higher against the sensitive strain than against the resistant strain. There was a residual effect of K84 from one year to another in soil inoculated with the sensitive strains. In experiment 3, K84 and K84 Agr controlled crown gall on plum and peach plants in soils inoculated with strains of A. tumefaciens sensitive or resistant to agrocin 84. The control afforded by K84 was higher than that provided by K84 Agr against the sensitive strain but was similar against the resistant strain.