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A novel class of (P^N^C) pincer ligands capable of stabilizing elusive gold(III) species is reported here. Straightforward access to (P^N^C)gold(III) hydroxo, formate, and hydride complexes has been streamlined by first incorporating a cycloauration step devoid of toxic metals or harsh conditions. The resulting gold complexes exhibit remarkable stability in solution as well as in the solid state under ambient conditions, which enabled their characterization by X-ray diffraction analyses. Interestingly, the influence of the ligand allowed the preparation of gold(III)-hydrides using mild hydride donors such as H-Bpin, which contrasts with sensitive super hydrides or strong acids and cryogenic conditions employed in previous protocols. A detailed bonding characterization of these species is complemented by reactivity studies.

One of the main hallmarks of Alzheimer’s Disease is the formation of ß-amyloid plaques, whose formation may be enhanced by metal binding or the appearance of familial mutations. In the present study, the simultaneous effect of familial mutations (E22Q, E22G, E22K, and D23N) and binding to metal ions (Cu(II) or Al(III)) is studied at the Aß42monomeric and fibrillar levels. With the application of GaMD and MD simulations, it is observed that the effects of metal binding and mutations differ in the monomeric and fibrillar forms. In the monomeric structures, without metal binding, all mutations reduce the amount of a-helix and increase, in some cases, the ß-sheet content. In the presence of Cu(II) and Al(III) metal ions, the peptide becomes less flexible, and the ß-sheet content decreases in favor of forming a-helix motifs that stabilize the system through interhelical contacts. Regarding the fibrillar structures, mutations decrease the opening of the fiber in the vertical axis, thereby stabilizing the S-shaped structure of the fiber. This effect is, in general, enhanced upon metal binding. These results may explain the different Aß42aggregation patterns observed in familial mutations.

CO2reduction (CO2RR) can convert CO2into feedstock for the chemical industry. In aqueous CO2electrolysis a key challenge is how to combine the CO2educt with a neutral or alkaline electrolyte and achieve a stable cell operation. We propose a novel cell design and operation mode based on a catalyst coated cation exchange membrane: a cationic acid (NH4+), with a volatile conjugate base (NH3), replaces the protons usually present for ion transport. The approach avoids a high proton concentration at the cathode catalyst while still removing all products within the gas phase. In this paper different cell concepts are investigated to identify a pathway to a stable, efficient and scalable operation mode. In a completely novel cell design a FECO> 50% was already maintained for over 35 h at 50 mA cm−2, and at 200 mA cm−2a cell voltage of 3.6 V (FECO> 60%) was achieved. Surprisingly, ammonium oxidation at the anode was fully supressed under the reaction conditions.

ABSTRACTThe aim of this study was to analyze the factors associated with conventional contact tracing (CCT) and molecular epidemiology (ME) methods in assessing tuberculosis (TB) transmission, comparing the populations studied and the epidemiological links established by both methods. Data were obtained from TB case and CCT registries, and ME was performed using IS6110-based restriction fragment length polymorphism (RFLP) analysis and mycobacterial interspersed repetitive unit 12 (MIRU12) typing as a secondary typing method. During two years (2003 and 2004), 892 cases of TB were reported, of which 687 (77%) were confirmed by culture. RFLP analysis was performed with 463 (67.4%) of the 687 isolated strains, and MIRU12 types in 75 strains were evaluated; 280 strains (60.5%) had a unique RFLP pattern, and 183 (39.5%) shared patterns, grouping into 65 clusters. CCT of 613 (68.7%) of 892 cases detected 44 clusters involving 101 patients. The results of both CCT and ME methods yielded 96 clusters involving 255 patients. The household link was the one most frequently identified by CCT (corresponding to 80.7% of the cases clustered by this method), whereas nonhousehold and unknown links were associated with 94.1% of the strains clustered by ME. When both methods were used in 351 cases (39.3%), they showed the same results in 214 cases (61%). Of the remainder, 106 (30.2%) were clustered only by ME, 19 (5.5%) were clustered only by CCT, and 12 (3.4%) were clustered by both methods but into different clusters. Patients with factors potentially associated with social problems were less frequently studied by CCT (P= 0.002), whereas patients of <15 years of age, most with negative cultures, were less frequently studied by ME (P= 0.005). Significant differences in the populations studied by ME versus CCT were observed, possibly explaining the scarce correlation found between the results of these methods. Moreover, ME allowed the detection of nonhousehold contact relationships, whereas CCT was more useful for tracing transmission chains involving patients of <15 years of age. In conclusion, the two methods are complementary, suggesting the need to improve the methodology of contact study protocols.

The Calabria-Peloritani Arc southern terrane is a stack of crystalline basement nappes, some of them provided with a widely outcropping Alpine sedimentary cover, sealed by clastics of the Stilo-Capo d'Orlando Formation (SCOF). New field observations in the Stilo area lead to define a Pignolo Formation as a sedimentary cycle predating the emplacement of the uppermost nappe (Stilo Unit) of the tectonic pile. It includes the well-known Lithothamnium and larger foraminifers bearing calcarenites, previously interpreted as a basal member of the SCOF. The biostratigraphic revision of both formations, together with recently published data about other preorogenic deposits, point to a stacking of the whole terrane between the Aquitanian and the middle-late Burdigalian. A comparison between the sedimentary cycles characterising the Calabria-Peloritani southern terrane during the Oligocene-Early Miocene and those almost coeval of the Betic-Rifian internal units highlights their quite similar evolution. Thus it is reliable that both the orogenic belts originated from contiguous paleogeographic realms. These considerations confirm that the present western Mediterranean Chains were originally segments of a continuous orogenic belt disrupted by the opening of the Balearic and Tyrrhenian basins.

Stratified coatings are used to provide properties at a surface, such as hardness or refractive index, which are different from underlying layers. Although time-savings are offered by self-assembly approaches, there have been no methods yet reported to offer stratification on demand. Here, we demonstrate a strategy to create self-assembled stratified coatings, which can be switched to homogeneous structures when required. We use blends of large and small colloidal polymer particle dispersions in water that self-assemble during drying because of an osmotic pressure gradient that leads to a downward velocity of larger particles. Our confocal fluorescent microscopy images reveal a distinct surface layer created by the small particles. When the pH of the initial dispersion is raised, the hydrophilic shells of the small particles swell substantially, and the stratification is switched off. Brownian dynamics simulations explain the suppression of stratification when the small particles are swollen as a result of reduced particle mobility, a drop in the pressure gradient, and less time available before particle jamming. Our strategy paves the way for applications in antireflection films and protective coatings in which the required surface composition can be achieved on demand, simply by adjusting the pH prior to deposition.

To understand the molecular basis of exocytosis in human neutrophils, the role of syntaxin 6 and SNAP-23 in neutrophil degranulation was examined. Human syntaxin 6 was cloned and identified as a 255-amino acid protein with a carboxy-terminal transmembrane region and two coiled-coil domains. Syntaxin 6 was localized mainly in the plasma membrane of human resting neutrophils, whereas SNAP-23 was located primarily in the mobilizable tertiary and specific granules. SNAP-23 was translocated to the cell surface, colocalizing with syntaxin 6, on neutrophil activation. In vitro binding studies established that SNAP-23 binds to syntaxin 6. Coimmunoprecipitation assays indicated that SNAP-23 interacts with syntaxin 6 in vivo, and this interaction was dramatically increased on neutrophil activation. Antibodies against SNAP-23 inhibited Ca++ and GTP-?-S–induced exocytosis of CD67-enriched specific granules, but they hardly affected exocytosis of the CD63-enriched azurophilic granules, when introduced into electropermeabilized neutrophils. Anti–syntaxin 6 antibodies prevented exocytosis of both CD67- and CD63-enriched granules in electropermeabilized neutrophils. These data show that syntaxin 6 and SNAP-23 are involved in human neutrophil exocytosis, demonstrating that vesicle SNAP receptor-target SNAP receptor (v-SNARE– t-SNARE) interactions modulate neutrophil secretion. Syntaxin 6 acts as a target for secretion of specific and azurophilic granules, whereas SNAP-23 mediates specific granule secretion.

ABSTRACTHaemophilus parasuisis the agent responsible for causing Gla¨sser's disease, which is characterized by fibrinous polyserositis, polyarthritis, and meningitis in pigs. In this study, we have characterized native outer membrane proteins with affinity to porcine transferrin (NPAPT) from H. parasuisserovar 5, Nagasaki strain. This pool of proteins was used as antigen to developed two vaccine formulations: one was adjuvanted with a mineral oil (Montanide IMS 2215 VG PR), while the other was potentiated with a bacterial neuraminidase from Clostridium perfringens. The potential protective effect conferred by these two vaccines was compared to that afforded by two other vaccines, consisting of recombinant transferrin-binding protein (rTbp) A or B fragments from H. parasuis, Nagasaki strain, and by a commercially available inactivated vaccine. Five groups of colostrum-deprived piglets immunized with the vaccines described above, one group per each vaccine, and a group of nonvaccinated control animals were challenged intratracheally with a lethal dose (3 × 108CFU) of H. parasuis, Nagasaki strain. The two vaccines containing rTbps yielded similar results with minimal protection against death, clinical signs, gross and microscopic lesions, and H. parasuisinvasion. In contrast, the two vaccines composed of NPAPT antigen and commercial bacterin resulted in a strong protection against challenge (without deaths and clinical signs), mild histopathological changes, and no recovery of H. parasuis, thus suggesting their effectiveness in preventing Gla¨sser's disease outbreaks caused by serovar 5.

ABSTRACTPunctual mutations in the TEM-1 or TEM-2 gene may lead to inhibitor-resistant-TEM (IRT) ß-lactamases with resistance to ß-lactam-ß-lactamase inhibitor combinations and susceptibility to cephalosporins. The aim of this work was to analyze the current epidemiology of IRT ß-lactamases in contemporary clinical Escherichia coli. Isolates were prospectively collected in our hospital (2007 and 2008) from both outpatients (59.8%) and hospitalized patients (40.2%). The genetic relationships of the isolates were determined by XbaI pulsed-field gel electrophoresis, multilocus sequence typing, and phylogenetic group analysis. IRT genes were sequenced and located by hybridization, and the incompatibility group of the plasmids was determined. From a total of 3,556 E. coliisolates recovered during the study period, 152 (4.3%) showed reduced susceptibility to amoxicillin-clavulanate, with 18 of them producing IRT enzymes (0.5%). These were mostly recovered from urine (77.8%). A high degree of IRT diversity was detected (TEM-30, -32, -33, -34, -36, -37, -40, and -54), and the isolates were clonally unrelated but were mostly associated with phylogenetic group B2 (55.5%). In 12 out of 16 (75%) isolates, the blaIRTgene was plasmid located and transferred by conjugation in 9 of them, whereas chromosomal localization was demonstrated in 4 isolates (25%). The sizes of the plasmids ranged from 40 kb (IncN) to 100 kb (IncFII, IncFI/FIIA), and they showed different restriction patterns by restriction fragment length polymorphism analysis. Unlike extended-spectrum ß-lactamase producers, the frequency of IRT producers remains low in both community and hospital settings, with most of them causing urinary tract infections. Although blaIRTgenes are mainly associated with plasmids, they can be also located in the chromosome. Despite this situation, clonal expansion and/or gene dispersion was not observed, denoting the independent emergence of these enzymes.

ABSTRACTThe aim of this study was to analyze trends in adult invasive pneumococcal disease (IPD) due to macrolide-resistant strains and to study the evolution of serotypes, genotypes, and macrolide-resistant determinants of strains collected in a prospective study between 1999 and 2007 in Barcelona, Spain. IPD due to macrolide-resistant strains of serotypes included in the 7-valent conjugate vaccine (PCV7) decreased from 2.16/100,000 (pre-PCV7 period, 1999 to 2001) to 0.80/100,000 (late-PCV7 period, 2005 to 2007) (P= 0.001), whereas IPD due to macrolide-resistant strains of non-PCV7 serotypes increased from 1.08/100,000 to 2.83/100,000 (P< 0.001). These changes were related to a fall of clones of PCV7 serotypes (ST81 [P< 0.05], ST90, ST315, and ST17) and an increase in new clones of serotypes 19A and 24F (ST230) and 33F (ST717) in the late-PCV7 period. The most common phenotype was MLSB(90.9%), related to the erm(B) gene. The frequent association between MLSBphenotype and tetracycline resistance [tet(M) gene], was related to transposons of the Tn916-family such as Tn6002or Tn3872. In conclusion, overall adult IPD rates due to macrolide-resistant pneumococci stabilized between 1999 and 2007 in Barcelona. The decrease in macrolide-resistant PCV7 pneumococci was balanced by the increase in macrolide-resistant non-PCV7 pneumococci.

ABSTRACTIn response to epidemic levels of serogroup B meningococcal disease in Cuba during the 1980s, the VA-MENGOC-BC vaccine was developed and introduced into the National Infant Immunization Program in 1991. Since then the incidence of meningococcal disease in Cuba has returned to the low levels recorded before the epidemic. A total of 420 Neisseria meningitidisstrains collected between 1983 and 2005 in Cuba were analyzed by multilocus sequence typing (MLST). The set of strains comprised 167 isolated from disease cases and 253 obtained from healthy carriers. By MLST analysis, 63 sequence types (STs) were identified, and 32 of these were reported to be a new ST. The Cuban isolates were associated with 12 clonal complexes; and the most common were ST-32 (246 isolates), ST-53 (86 isolates), and ST-41/44 (36 isolates). This study also showed that the application of VA-MENGOC-BC, the Cuban serogroup B and C vaccine, reduced the frequency and diversity of hypervirulent clonal complexes ST-32 (vaccine serogroup B type-strain) and ST-41/44 and also affected other lineages. Lineages ST-8 and ST-11 were no longer found during the postvaccination period. The vaccine also affected the genetic composition of the carrier-associated meningococcal isolates. The number of carrier isolates belonging to hypervirulent lineages decreased significantly after vaccination, and ST-53, a sequence type common in carriers, became the predominant ST.

ABSTRACTThe mitogen-activated protein kinase (MAPK) Sty1 is essential for the regulation of transcriptional responses that promote cell survival in response to different types of environmental stimuli in Schizosaccharomyces pombe. In fission yeast, three distinct eukaryotic initiation factor 2a (eIF2a) kinases, two mammalian HRI-related protein kinases (Hri1 and Hri2) and the Gcn2 ortholog, regulate protein synthesis in response to cellular stress conditions. In this study, we demonstrate that both Hri1 and Hri2 exhibited an autokinase activity, specifically phosphorylated eIF2a, and functionally replaced the endogenous Saccharomyces cerevisiaeGcn2. We further show that Gcn2, but not Hri1 or Hri2, is activated early after exposure to hydrogen peroxide and methyl methanesulfonate (MMS). Cells lacking Gcn2 exhibit a later activation of Hri2. The activated MAPK Sty1 negatively regulates Gcn2 and Hri2 activities under oxidative stress but not in response to MMS. In contrast, Hri2 is the primary activated eIF2a kinase in response to heat shock. In this case, the activation of Sty1 appears to be transitory and does not contribute to the modulation of the eIF2a kinase stress pathway. In strains lacking Hri2, a type 2A protein phosphatase is activated soon after heat shock to reduce eIF2a phosphorylation. Finally, the MAPK Sty1, but not the eIF2a kinases, is essential for survival upon oxidative stress or heat shock, but not upon MMS treatment. These findings point to a regulatory coordination between the Sty1 MAPK and eIF2a kinase pathways for a particular range of stress responses.

ABSTRACTMolecular epidemiology studies have allowed the identification of the methicillin (meticillin)-resistant (MRSA) and methicillin-susceptible (MSSA) clonal complexes (CCs) and clones of Staphylococcus aureuscirculating in a Spanish hospital recently. Of 81 isolates tested, 32.1% were MRSA. Most of them carried staphylococcal cassette chromosome mec(SCCmec) IVc (88.5%) and belonged to CC5 (88.5%; multilocus sequence typing types ST125 [mainly associated with spatype t067], ST5, and ST228). A higher diversity was found among MSSA isolates (67.9%). Eighty percent shared the genetic background of major MRSA lineages (CC5 [38.2%; ST125 and ST5], CC30 [25.5%; ST30], CC45 [14.5%; ST45 and ST47], and CC8 [1.8%; ST8]), but CC12, CC15, CC51, and CC59 were also detected. Many exotoxin genes were present in each of the 81 isolates, independent of whether they were involved in sepsis (11 to 22) or other types of infections (13 to 21), and they appeared in 73 combinations. The relevant data are that (i) all isolates were positive for hemolysin and leukotoxin genes (98.8% for lukEDand 25.9% for lukPV); (ii) all contained an enterotoxin gene cluster (egcwith or without seu), frequently with one or more genes encoding classical enterotoxins; (iii) about half were positive for tstand 95% were positive for exfoliatin-encoding genes (eta, etb, and/or etd); and (iv) the four agrgroups were detected, with agrII(55.6%) and agrIII(23.5%) being the most frequent. Taken together, results of the present study suggest a frequent acquisition and/or loss of exotoxin genes, which may be mediated by efficient intralineage transfer of mobile genetic elements and exotoxin genes therein and by eventual breakage of interlineage barriers.

ABSTRACTPotassium and Na+effluxes across the plasma membrane are crucial processes for the ionic homeostasis of cells. In fungal cells, these effluxes are mediated by cation/H+antiporters and ENA ATPases. We have cloned and studied the functions of the two ENA ATPases of Ustilago maydis, U. maydisEna1 (UmEna1) and UmEna2. UmEna1 is a typical K+or Na+efflux ATPase whose function is indispensable for growth at pH 9.0 and for even modest Na+or K+tolerances above pH 8.0. UmEna1 locates to the plasma membrane and has the characteristics of the low-Na+/K+-discrimination ENA ATPases. However, it still protects U. maydiscells in high-Na+media because Na+showed a low cytoplasmic toxicity. The UmEna2 ATPase is phylogenetically distant from UmEna1 and is located mainly at the endoplasmic reticulum. The function of UmEna2 is not clear, but we found that it shares several similarities with Neurospora crassaENA2, which suggests that endomembrane ENA ATPases may exist in many fungi. The expression of ena1and ena2transcripts in U. maydiswas enhanced at high pH and at high K+and Na+concentrations. We discuss that there are two modes of Na+tolerance in fungi: the high-Na+-content mode, involving ENA ATPases with low Na+/K+discrimination, as described here for U. maydis, and the low-Na+-content mode, involving Na+-specific ENA ATPases, as in Neurospora crassa.

A simple method that allows selective positioning of nanoparticles into mesoporous monolayer or multilayer thin films is presented. This technique applies UV lithography in order to bring about in situ light-induced reduction of silver in templated cavities of TiO2. The nanoparticle lithography presented here provides a novel approach to hierarchical lithography patterning for multifunctional devices.

ABSTRACTAt our institution, the prevalence of clinical isolates of Clostridium difficilewith resistance to metronidazole is 6.3%. We observed that initial metronidazole MICs of 16 to 64 mg/liter against toxigenic, primary fresh C. difficileisolates, as determined by agar dilution, decreased to 0.125 mg/liter after the isolates were thawed. In this study, we examined the possibility of heterogeneous or inducible resistance. Totals of 14 metronidazole-resistant and 10 metronidazole-susceptible clinical isolates of toxigenic C. difficilewere studied. The isolates were investigated for the presence of nimgenes by PCR. After the isolates were thawed, susceptibility testing was done by agar dilution, by disc diffusion using a 5-µg metronidazole disc, and by the Etest method. An experiment for determining the effect of prolonged exposure to metronidazole was applied to all resistant isolates and to susceptible control strains. None of the isolates presented the nimgenes. All initially metronidazole-resistant C. difficileisolates became susceptible after thawing; however, they presented slow-growing subpopulations within the inhibition zones of both the disk and the Etest strip. All metronidazole-susceptible isolates remained homogeneously susceptible by both methods. After prolonged exposure in vitro to metronidazole, no zone of inhibition was found around the 5-µg disk in any of the metronidazole-resistant isolates, and the MICs as determined by the Etest method ranged from 0.125 to >256 mg/liter, with colonies growing inside the inhibition zone. Our results indicate that (i) resistance to metronidazole was not due to the presence of nimgenes, (ii) resistance to metronidazole in toxigenic C. difficileisolates is heterogeneous, and (iii) prolonged exposure to metronidazole can select for in vitro resistance. We recommend routine performance of the disk diffusion method (5-µg metronidazole disk) with primary fresh C. difficileisolates in order to ensure that metronidazole-heteroresistant populations do not go undetected.

ABSTRACTTo improve the microbiological diagnosis of device-related osteoarticular infections, we have developed a protocol based on the sonication of device samples, followed by concentration and inoculation of the sonicate in a broad variety of media in a quantitative manner. Sixty-six samples from 31 patients were included in the study (17 of them with clinical diagnosis of infection). The sonication procedure had a sensitivity of 94.1%, which is better than that of conventional cultures (88.2%). One case of contamination and six cases of unexpected positive cultures were detected (specificity of 42.8%): two of these were considered to represent true infection, while the other four were considered to be nonsignificant (corrected specificity of 50%), although the clinical importance of these isolates is questionable. When we analyzed the number of CFU, no breakpoint between significant and nonsignificant isolates could be established. Based on our results, the procedure of sonication of retrieved implants is better than conventional cultures for the diagnosis of device-related infections. The significance of some isolates in patients without clinical infection remains uncertain. However, they may become pathogens and cannot be routinely considered to be contamination.

ABSTRACTA new myosin motor-like chitin synthase gene, chsVb, has been identified in the vascular wilt fungus Fusarium oxysporumf. sp. lycopersici. Phylogenetic analysis of the deduced amino acid sequence of the chsVbchitin synthase 2 domain (CS2) revealed that ChsVb belongs to class VII chitin synthases. The ChsVb myosin motor-like domain (MMD) is shorter than the MMD of class V chitin synthases and does not contain typical ATP-binding motifs. Targeted disrupted single (?chsVb) and double (?chsV?chsVb) mutants were unable to infect and colonize tomato plants or grow invasively on tomato fruit tissue. These strains were hypersensitive to compounds that interfere with fungal cell wall assembly, produced lemon-like shaped conidia, and showed swollen balloon-like structures in hyphal subapical regions, thickened walls, aberrant septa, and intrahyphal hyphae. Our results suggest that the chsVbgene is likely to function in polarized growth and confirm the critical importance of cell wall integrity in the complex infection process of this fungus.

SUMMARYIn recent years, the increasing number of donors from different regions of the world is providing a new challenge for the management and selection of suitable donors. This is a worldwide problem in most countries with transplantation programs, especially due to the increase in immigration and international travel. This paper elaborates recommendations regarding the selection criteria for donors from foreign countries who could potentially transmit tropical or geographically restricted infections to solid-organ transplant recipients. For this purpose, an extensive review of the medical literature focusing on viral, fungal, and parasitic infections that could be transmitted during transplantation from donors who have lived or traveled in countries where these infections are endemic has been performed, with special emphasis on tropical and imported infections. The review also includes cases described in the literature as well as risks of transmission during transplantation, microbiological tests available, and recommendations for each infection. A table listing different infectious agents with their geographic distributions and specific recommendations is included.

ABSTRACTWe present here the biochemical and genetic characterization and antimicrobial susceptibility of 58 isolates of Aerococcus viridansisolated in pure culture from different clinical specimens of normally sterile body sites of pigs. A. viridansisolates were commonly susceptible to ß-lactam antimicrobials and exhibited a great genetic heterogeneity as determined by pulsed-field gel electrophoresis typing. The results indicate that A. viridansmight be included in the list of possible etiological agents causing disease in pigs.

ABSTRACTIn this multicenter study, the reliability of two nonradiometric, fully automated systems, the MB/BacT and BACTEC MGIT 960 systems, for testing the susceptibilities of 82 Mycobacterium tuberculosisstrains to isoniazid, rifampin, ethambutol, and streptomycin was evaluated in comparison with the radiometric BACTEC 460TB system. The arbitration of discrepant results was done by the reanalysis of the strain, the determination of the MIC, and the molecular characterization of some resistance determinants. The overall level of agreement with BACTEC 460TB results was 96% with the MB/BacT test and 97.2% with the BACTEC MGIT 960 system. With both methods, the level of agreement with BACTEC 460TB results was 96.3% for isoniazid, 98.8% for rifampin, and 98.8% for ethambutol. The level of agreement for streptomycin was 90.2% with MB/BacT and 97.5% with BACTEC MGIT 960. Overall, there were 11 very major errors and 2 major errors with the MB/BacT method and 5 very major errors and 2 major errors with the BACTEC MGIT 960 system. In general, the MB/BacT and BACTEC MGIT 960 systems showed good performance for susceptibility testing with first-line antituberculosis drugs.

ABSTRACTVisceral leishmaniasis (VL) caused by Leishmania infantumis a common disease in human immunodeficiency virus (HIV)-infected people in the Mediterranean basin. However, most such cases are asymptomatic, and little information about the prevalence of these infections in HIV-infected individuals is available. The aim of this study was to assess the prevalence of subclinical infection and the relationship between several Leishmaniainfection markers by noninvasive methods in asymptomatic HIV-infected patients from Southern Spain. Ninety-two HIV-infected patients, who were consecutively attended at the participant hospitals in 2004, were invited to participate in this study. These patients were asymptomatic and without any history of cutaneous or visceral leishmaniasis. Leishmaniakinetoplast DNA (kDNA) was amplified from peripheral blood samples from 28 (30.4%) of these HIV-infected subjects. Sera from three (3.5%) patients tested positive for Leishmaniaby an enzyme-linked immunoassay. Two patients (2.4%) showed a specific 16-kDa band by Western blotting. In contrast, none of the patients showed a positive agglutination of urine. The leishmanin skin test was positive for four (4.3%) patients. None of the patients with a PCR-positive result showed a positive reaction by enzyme-linked immunoassay or by specific bands in Western blotting or had a positive leishmanin skin test. In conclusion, L. infantumkDNA was detected in a large proportion of asymptomatic HIV-infected patients, although a demonstrable cellular or humoral immune response to this parasite was not shown. Conversely, Leishmaniaantigen in urine was not detected in these patients.

ABSTRACTEight optochin-susceptible (Opts) alpha-hemolytic (viridans) streptococcus isolates were characterized at the molecular level. These isolates showed phenotypic characteristics typical of both viridans streptococci and Streptococcus pneumoniae. Comparison of the sequence of housekeeping genes from these isolates with those of S. pneumoniae, Streptococcus mitis, Streptococcus oralis, and Streptococcus pseudopneumoniaesuggested that the Optsisolates corresponded to streptococci of the mitis group. Besides, the Optsstreptococci were negative by a Gen-Probe AccuProbe pneumococcus test and hybridized with specific pneumococcal probes (lytAand ply) but also with ant, a gene not present in most S. pneumoniaestrains. Moreover, the isolates were insoluble in 1% sodium deoxycholate but completely dissolved in 0.1% deoxycholate. Sequence analysis of the lytAgene revealed that the Optsstreptococci carried lytAalleles characteristic of those present in nonpneumococcal streptococci of the mitis group. The determination of the partial nucleotide sequence embracing the atpoperon encoding the FoF1H+-ATPase indicated that the optochin susceptibility of the isolates was due to the acquisition of atpC, atpA, and part of atpBfrom S. pneumoniaeby horizontal gene transfer.

ABSTRACTWe sought here to review the present definition of catheter-related bloodstream infections (CR-BSI) due to coagulase-negative staphylococci (CNS) by comparing the routine phenotypic methods with a genotypic procedure that considers different morphotypes. Our phenotypic characterization of CNS isolates included routine identification with biotype and antibiotype. The genotypic diagnosis was based on longer incubation periods with the consideration of all morphotypes and molecular typing by pulsed-field gel electrophoresis techniques. We prospectively selected 61 episodes of suspected CR-BSI by CNS occurring during 1 year, based on the presence of a compatible clinical setting and the isolation of one or more CNS from blood and catheter tip. Of these episodes, 47 (77%) were identified as true episodes of CR-BSI based on the presence of microorganisms of the same genotype in the blood and on the catheter tip. The sensitivity, specificity, positive predictive, negative predictive, accuracy, positive likelihood ratio, and negative likelihood ratio values obtained by different phenotypic microbiological approaches to establish the diagnosis of CR-BSI were as follows: identity at species level (78.7%, 85.7%, 94.9%, 54.5%, 80.3%, 5.51, and 0.25, respectively); identity of species and biotype (59.6%, 92.9%, 96.6%, 40.6%, 67.2%, 8.34, and 0.44, respectively); identity of species and antibiotype (61.7%, 92.9%, 96.7%, 41.9%, 68.8%, 8.64, and 0.41, respectively); and identity of species, biotype, and antibiotype (48.9%, 92.9%, 95.8%, 35.1%, 59%, 6.85, and 0.55, respectively). Our study demonstrates the inaccuracy of the diagnosis of CNS CR-BSI when the current definition based on conventional routine microbiological practice is followed. A new definition of CNS CR-BSI is necessary, at least as an epidemiological and research tool.

ABSTRACTRho GTPases are regulators of signaling pathways that control actin organization and cell polarity processes in all eukaryotic cells. In Schizosaccharomyces pombe, Rho4p is involved in the regulation of septum degradation during cytokinesis. Here we show that Rho4p participates in the secretion of the glucanases Eng1p and Agn1p, which are responsible for the septum degradation. First, eng1+or agn1+overexpression suppressed the rho4?multiseptation phenotype, and simultaneous overproduction of Rho4p and Eng1p or of Rho4p and Agn1p caused a dramatic lysis. Second, Rho4p was not necessary for Eng1p-mediated glucanase activity as measured in cell extracts; however, rho4?cells have a lower level of (1,3)-ß-d-glucanase activity in the culture medium. Additionally, Eng1- or Agn1-green fluorescent protein did not properly localize to the septum in rho4?cells grown at 37°C. There was a decreased amount of these enzymes in the cell wall and in the culture medium of rho4?cells at 37°C. These results provide evidence that Rho4p is involved in the regulation of Eng1p and Agn1p secretion during cytokinesis.

ABSTRACTA set of 84 Staphylococcus aureusisolates collected from the milk of cows with subclinical mastitis in Asturias (a cattle region of Spain) and six control strains were tested for sequences of genes encoding hemolysins (hla, hlb, hld, hlg, and hlg-2), leukotoxins (lukPV, lukM, and lukED), toxic shock syndrome toxin (tst), and enterotoxins (seato see, segto ser, and seu) by conventional and multiplex PCR. It was found that 84, 83, 11, and 39 isolates carried some type of hl, luk, tst, or segene, respectively, which were arranged in 14 exotoxin genotypes. All of the isolates were negative for lukPV, hlg, sea, sed, see, sej, sek, sep, seq, and ser. Two gene groupings could be related with pathogenicity islands—[lukED, seg, sei, sem, sen, seo ± seu] with Saß-1 and [tst, sec, sel] with SaPIbov, present in 45 and 13.1% of the isolates, respectively—while 11.9% of them carried both islands. Only one contained seb(together with ?Saß-1), and another contained seh(together with lukED). The isolates were also analyzed by pulsed-field gel electrophoresis performed with SmaI. Thirty-nine SmaI profiles (similarity coefficient [S] = 0.94 to 0.21) were differentiated; 12, 1, and 10 of these, respectively, were generated by isolates presumptively carrying Saß-1, SaPIbov, or both. Five SmaI profiles (S= 0.8) formed a cluster, which contained 20 and 10 isolates carrying one (?Saß-1) or both islands. These data show the high frequency of genes encoding cytotoxins and pyrogenic toxin superantigens, their relationship with pathogenicity islands, and their distribution among a diversity of genetic types of S. aureusrelated to subclinical mastitis.

ABSTRACTWe used spoligotyping and restriction fragment length polymorphism (RFLP) of the IS6110-insertion sequence to study the molecular epidemiology of multidrug-resistant (MDR) tuberculosis in Spain. We analyzed 180 Mycobacterium tuberculosiscomplex isolates collected between January 1998 and December 2000. Consecutive isolates from the same patients (n= 23) always had identical genotypes, meaning that no cases of reinfection occurred. A total of 105 isolates (58.3%) had unique RFLP patterns, whereas 75 isolates (41.7%) were in 20 different RFLP clusters. Characterization of the katGand rpoBgenes showed that 14 strains included in the RFLP clusters did not actually cluster. Only 33.8% of the strains isolated were suggestive of MDR transmission, a frequency lower than that for susceptible strains in Spain (46.6%). We found that the Beijing/W genotype, which is prevalent worldwide, was significantly associated with immigrants. The 22 isolates in the largest cluster corresponded to the Mycobacterium bovisstrain responsible for two nosocomial MDR outbreaks in Spain.

ABSTRACTA Streptomyces albusforearm actinomycetoma that could not be identified by culture was properly identified at the species level by study of an internal fragment of the heat shock protein gene, comparative sequence analysis of 16S ribosomal DNA (rDNA), and the hypervariable ?-region of the 16S rDNA.

ABSTRACTThe levels of genetic relatedness of 139 Stenotrophomonas maltophiliastrains recovered from 105 hospitalized non-cystic fibrosis patients (51% from medical wards, 35% from intensive care units, and 14% from surgical wards) and 7 environmental sources in the same hospital setting during a 4-year period were typed by the pulsed-field gel electrophoresis (PFGE) technique. A total of 99 well-defined distinct XbaI PFGE patterns were identified (Simpson's discrimination index, 0.996). The dendrogram showed a Dice similarity coefficient ranging from 28 to 80%. Two major clusters (I and II), three minor clusters (III, IV, and V), and two independent branches were observed when using a 36% Dice coefficient, indicating a high diversity of genetic relatedness. It is of note that 84% of strains were grouped within two major clonal lineages. No special cluster gathering was found among strains belonging to the same sample type specimen, patients' infection or colonization status, and ward of precedence. Despite this fact, three different clones (A, B, and C) recovered from respiratory samples from six, three, and two patients, respectively, and two clones, D and E, in two bacteremic patients each, were identified. Isolation of an S. maltophiliastrain belonging to the clone A profile in a bronchoscope demonstrated a common source from this clone. This study revealed a high genetic diversity of S. maltophiliaisolates despite their origin from a single hospital, which may be related to the wide environmental distribution of this pathogen. However, few clones could be transmitted among different patients, yielding outbreak situations.

ABSTRACTDrug resistance in Mycobacterium tuberculosiscomplex strains is solely due to chromosomal mutations that could affect bacterial virulence. Molecular epidemiology studies have shown that resistant strains are less likely to be clustered than susceptible strains. However, a few multidrug-resistant (MDR) M. tuberculosiscomplex strains have been described as causing outbreaks, suggesting that they have restored virulence or increased transmission. One of the biggest MDR tuberculosis outbreaks documented to date was caused by the B strain of M. bovis. Restriction fragment length polymorphism fingerprinting revealed that the B strain contains two copies of IS6110. Here, we mapped and sequenced the regions flanking the two copies of IS6110in the B strain. Ligation-mediated PCR showed that one of these IS6110copies is located within the promoter region of phoP, a transcriptional regulator that is essential for M. tuberculosisvirulence. We used PCR to screen 219 MDR M. tuberculosiscomplex strains (90.4% of all MDR isolates) isolated in Spain between 1998 and 2002 and found that the B strain was the only strain that contained a copy of IS6110in the phoPpromoter. To determine whether IS6110affects phoPpromoter activity in the B strain, we individually cloned the phoPgene and its promoter region (including IS6110from the B strain and the equivalent region from M. tuberculosiswithout IS6110as a control) into a mycobacterial replicative plasmid and transformed M. smegmatiswith the resulting plasmid. Primer extension analysis showed that phoPtranscription was strongly upregulated when the promoter region contained IS6110, as in the case of the B strain.

ABSTRACTThe echinocandin caspofungin is a new antifungal drug that blocks cell wall synthesis through inhibition of ß-(1-3)-glucan synthesis. Saccharomyces cerevisiaecells are able to tolerate rather high caspofungin concentrations, displaying high viability at low caspofungin doses. To identify yeast genes implicated in caspofungin tolerance, we performed a genome-wide microarray analysis. Strikingly, caspofungin treatment rapidly induces a set of genes from the protein kinase C (PKC) cell integrity signaling pathway, as well as those required for cell wall maintenance and architecture. The mitogen-activated protein kinase Slt2p is rapidly activated by phosphorylation, triggering signaling through the PKC pathway. Cells lacking genes such as SLT2, BCK1, and PKC1, as well as the caspofungin target gene, FKS1, display pronounced hypersensitivity, demonstrating that the PKC pathway is required for caspofungin tolerance. Notably, the cell surface integrity sensor Wsc1p, but not the sensors Wsc2-4p and Mid2p, is required for sensing caspofungin perturbations. The expression modulation of PKC target genes requires the transcription factor Rlm1p, which controls expression of several cell wall synthesis and maintenance genes. Thus, caspofungin-induced cell wall damage requires Wsc1p as a dedicated sensor to launch a protective response through the activated salvage pathway for de novo cell wall synthesis. Our results establish caspofungin as a specific activator of Slt2p stress signaling in baker's yeast.

The KDEL receptor is a Golgi/intermediate compartment-located integral membrane protein that carries out the retrieval of escaped ER proteins bearing a C-terminal KDEL sequence. This occurs throughout retrograde traffic mediated by COPI-coated transport carriers. The role of the C-terminal cytoplasmic domain of the KDEL receptor in this process has been investigated. Deletion of this domain did not affect receptor subcellular localization although cells expressing this truncated form of the receptor failed to retain KDEL ligands intracellularly. Permeabilized cells incubated with ATP and GTP exhibited tubular processes-mediated redistribution from the Golgi area to the ER of the wild-type receptor, whereas the truncated form lacking the C-terminal domain remained concentrated in the Golgi. As revealed with a peptide-binding assay, this domain did not interact with both coatomer and ARF-GAP unless serine 209 was mutated to aspartic acid. In contrast, alanine replacement of serine 209 inhibited coatomer/ARF-GAP recruitment, receptor redistribution into the ER, and intracellular retention of KDEL ligands. Serine 209 was phosphorylated by both cytosolic and recombinant protein kinase A (PKA) catalytic subunit. Inhibition of endogenous PKA activity with H89 blocked Golgi-ER transport of the native receptor but did not affect redistribution to the ER of a mutated form bearing aspartic acid at position 209. We conclude that PKA phosphorylation of serine 209 is required for the retrograde transport of the KDEL receptor from the Golgi complex to the ER from which the retrieval of proteins bearing the KDEL signal depends.

ABSTRACTThe usefulness of single-enzyme amplified-fragment length polymorphism (AFLP) analysis for the subtyping of Mycobacterium kansasiitype I isolates was evaluated. This simplified technique classified 253 type I strains into 12 distinct clusters. The discriminating power of this technique was high, and the technique easily distinguished between the epidemiologically unrelated control strains and our clinical isolates. Overall, the technique was relatively rapid and technically simple, yet it gave reproducible and discriminatory results. This technique provides a powerful typing tool which may be helpful in solving many questions concerning the reservoirs, pathogenicities, and modes of transmission of these isolates.

ABSTRACTA new mycobacteriophage-based technique (PhageTek MB) was compared with standard culture and staining techniques for diagnosis of pulmonary tuberculosis. A total of 2,048 respiratory specimens from 1,466 patients collected from February 2000 to March 2001 were studied by both (i) conventional methods (direct microscopic examination [auramine-rhodamine fluorochrome], and culture in BacT/ALERT 3D and solid media) and (ii) the PhageTek MB assay. This phenotypic test utilizes specific mycobacteriophages to detect the presence of live Mycobacterium tuberculosiscomplex organisms within a decontaminated clinical sample. Overall, 205 (10%) specimens were positive for mycobacteria (134 patients): 144 (70.2%) M. tuberculosisisolates and 61 (29.8%) nontuberculous mycobacterium isolates (30 Mycobacterium kansasii, 12 Mycobacterium xenopi, 9 Mycobacterium gordonae, 7 Mycobacterium aviumcomplex, 2 Mycobacterium chelonae, and 1 Mycobacterium fortuitumisolate). PhageTek MB was more likely to give a positive result with specimens in which high numbers of acid-fast bacilli were observed on the smear. The sensitivity, specificity, and positive and negative predictive values of this mycobacteriophage-based technique versus culture for M. tuberculosiswere 58.3, 99.1, 83.2, and 96.9%, respectively. PhageTek MB is a rapid (48-h), specific, safe, and easy-to-perform test. According to the prevalence of the disease in the population studied, the test would require improved sensitivity in order to be used as a screening test for routine diagnosis of respiratory tuberculosis in our setting.

ABSTRACTIn the corn smut fungus Ustilago maydis, pathogenic development is initiated when two compatible haploid cells fuse and form the infectious dikaryon. Mating is dependent on pheromone recognition by compatible cells. In this report, we set out to evaluate the relationship between the cell cycle and the pheromone response in U. maydis. To achieve this, we designed a haploid pheromone-responsive strain that is able to faithfully reproduce the native mating response in nutrient-rich medium. Addition of synthetic pheromone to the responsive strain induces the formation of mating structures, and this response is abolished by mutations in genes encoding components of the pheromone signal transduction cascade. After recognition of pheromone, U. maydiscells arrest the cell cycle in a postreplicative stage. Visualization of the nucleus and microtubule organization indicates that the arrest takes place at the G2phase. Chemical-induced cell cycle arrest and release in the presence of pheromone further support this conclusion.

ABSTRACTß-Glucans are the main components of the fungal cell wall. Fission yeast possesses a family of ß-glucan synthase-related genes. We describe here the cloning and characterization of bgs3+, a new member of this family. bgs3+was cloned as a suppressor of a mutant hypersensitive to Echinocandin and Calcofluor White, drugs that interfere with cell wall biosynthesis. Disruption of the gene is lethal, and a decrease in Bgs3p levels leads to rounded cells with thicker walls, slightly reduces the amount of the ß-glucan, and raises the amount of a-glucan polymer. These cells finally died. bgs3+is expressed in vegetative cells grown in different conditions and during mating and germination and is not enhanced by stress situations. Consistent with the observed expression pattern, Bgs3-green fluorescence protein (GFP-Bgs3p) was found at the growing tips during interphase and at the septum prior to cytokinesis, always localized to growth areas. We also found GFP-Bgs3p in mating projections, during the early stages of zygote formation, and at the growing pole during ascospore germination. We conclude that Bgs3p localization is restricted to growth areas and that Bgs3p is a glucan synthase homologue required for cell wall biosynthesis and cell elongation in the fission yeast life cycle.

ABSTRACTThe genetic diversity of Vibrio choleraeO1 strains from Argentina was estimated by random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE). Twenty-nine isolates carrying the virulence genes ctxA, zot, ace, and tcpAappeared to represent a single clone by both typing methods; while 11 strains lacking these virulence genes exhibited several heterogeneous RAPD and PFGE patterns. Among the last group, a set of isolates from the province Tucuma´n showed a single RAPD pattern and four closely related PFGE profiles. These strains, isolated from patients with diarrhea, did not produce the major V. choleraeO1 virulence determinants, yet cell supernatants of these isolates caused a heat-labile cytotoxic effect on Vero and Y-1 cells and elicited significant variations on the water flux and short-circuit current in human small intestine mounted in an Ussing chamber. All these effects were completely abolished by incubation with a specific antiserum against El Tor hemolysin, suggesting that this virulence factor was responsible for the toxic activity on both the epithelial cells and the small intestine specimens and may hence be involved in the development of diarrhea. We propose “Tucuma´n variant” as the designation for this new cluster of cholera toxin-negative V. choleraeO1 strains.

ABSTRACTEarly diagnosis of tuberculosis and screening of other mycobacteria is required for the appropriate management of patients. We have therefore developed a 5'-exonuclease fluorogenic PCR assay in a single-tube balanced heminested format that simultaneously detects Mycobacterium tuberculosiscomplex (MTC) and members of the Mycobacteriumgenus (MYC) using the 16S ribosomal DNA target directly on clinical samples. One hundred twenty-seven clinical samples (65 smear negative and 62 smear positive) with a positive culture result from 127 patients were tested, including 40 negative control specimens. The finding of both a positive MTC and probe value and a positive MYC probe value confirmed the presence of MTC or mycobacteria with a 100% positive predictive value. However, a negative value for MTC or MYC did not discount the presence of mycobacteria in the specimen. Interestingly, the addition of the MYC probe allowed the diagnosis of an additional 7% of patients with tuberculosis and rapid screening of nontuberculous mycobacteria (NTM). Thus, over 75% of the patients were diagnosed with mycobacterial disease by PCR. The sensitivity was much higher on smear-positive samples (90.3%) than smear-negative samples (49.2%) and was slightly higher for MTC than NTM samples. With regard to the origin of the sample, MTC pulmonary samples gave better results than others. In conclusion, we believe this test may be useful for the rapid detection of mycobacteria in clinical samples and may be a valuable tool when used together with conventional methods and the clinical data available.

ABSTRACTToxoplasma gondiiis a common life-threatening opportunistic infection. We used experimental murine T. gondiiinfection to optimize the PCR for diagnostic use, define its sensitivity, and characterize the time course and tissue distribution of experimental toxoplasmosis. PCR conditions were adjusted until the assay reliably detected quantities of DNA derived from less than a single parasite. Forty-two mice were inoculated intraperitoneally with T. gondiitachyzoites and sacrificed from 6 to 72 h later. Examination of tissues with PCR and histology revealed progression of infection from blood to lung, heart, liver, and brain, with PCR consistently detecting parasites earlier than microscopy and with no false-positive results. We then evaluated the diagnostic value of this PCR assay in human patients. We studied cerebrospinal fluid and serum samples from 12 patients with AIDS and confirmed toxoplasmic encephalitis (defined as positive mouse inoculation and/or all of the Centers for Disease Control clinical diagnostic criteria), 12 human immunodeficiency virus-infected patients with suspected cerebral toxoplasmosis who had neither CDC diagnostic criteria nor positive mouse inoculation, 26 human immunodeficiency virus-infected patients with other opportunistic infections and no signs of cerebral toxoplasmosis, and 18 immunocompetent patients with neurocysticercosis. Eleven of the 12 patients with confirmed toxoplasmosis had positive PCR results in either blood or cerebrospinal fluid samples (6 of 9 blood samples and 8 of 12 cerebrospinal fluid samples). All samples from control patients were negative. This study demonstrates the high sensitivity, specificity, and clinical utility of PCR in the diagnosis of toxoplasmic encephalitis in a resource-poor setting.

ABSTRACTUsing a repetitive extragenic palindromic PCR (REP-PCR), we genotypically characterized strains causing nosocomial Acinetobacter baumanniiinfections and analyzed the source of bacteremia in 67 patients from an institution in which infections by this bacterium were endemic. Six different genotypes were found, including 21, 27, 3, 9, 3, and 4 strains. The probable source of bacteremia, according to clinical and/or microbiological criteria, was known in 42 patients (63%): respiratory tract (n= 19), surgical sites (n= 12), intravascular catheters (n= 5), burns (n= 3), and urinary tract (n= 3). The definite source of bacteremia, according to REP-PCR, could be established in 30 (71%) out of the 42 patients with strains from blood and other sites; in these cases clinical and microbiological criteria for the source of bacteremia were thus confirmed. In the remaining 12 patients (29%) the probable source was refuted by the REP-PCR method. The definite sources of bacteremia according to genotype were as follows: respiratory tract in 13 patients (31%), surgical sites in 8 (19%), intravascular catheters in 4 (9%), burns in 3 (7%), and urinary tract in 2 (5%). A comparison of strains from blood cultures and other sites with regard to their REP-PCR and antimicrobial resistance profiles was also made. Taking the REP-PCR as the “gold standard,” the positive predictive value of antibiotype was 77% and the negative predictive value was 42%. In summary, the utility of the diagnosis of the source of nosocomial A. baumanniibacteremia using clinical and/or microbiological criteria, including antibiotyping, is limited, as demonstrated by REP-PCR.

ABSTRACTCandida albicansis able to respond to environmental changes by inducing a distinct morphological program, which is related to the ability to infect mammalian hosts. Although some of the signal transduction pathways involved in this response are known, it is not clear how the environmental signals are sensed and transmitted to these transduction cascades. In this work, we have studied the function of GPA2, a new gene from C. albicans, which encodes a G-protein a-subunit homologue. We demonstrate that Gpa2 plays an important role in the yeast-hypha dimorphic transition in the response of C. albicansto some environmental inducers. Deletion of both alleles of the GPA2gene causes in vitro defects in morphological transitions in Spider medium and SLAD medium and in embedded conditions but not in medium containing serum. These defects cannot be reversed by exogenous addition of cyclic AMP. However, overexpression of HST7, which encodes a component of the filament-inducing mitogen-activated protein kinase (MAPK) cascade, bypasses the Gpa2 requirement. We have obtained different gain-of-function and loss-of-function mutant alleles of the GPA2gene, which we have introduced in several C. albicansgenetic backgrounds. Our results indicate that, in response to environmental cues, Gpa2 is required for the regulation of a MAPK signaling pathway.

ABSTRACTThe isolation of a capsid intertypic poliovirus recombinant from a child with vaccine-associated paralytic poliomyelitis is described. Virus 31043 had a Sabin-derived type 3-type 2-type 1 recombinant genome with a 5'-end crossover point within the capsid coding region. The result was a poliovirus chimera containing the entire coding sequence for antigenic site 3a derived from the Sabin type 2 strain. The recombinant virus showed altered antigenic properties but did not acquire type 2 antigenic characteristics. The significance of the presence in nature of such poliovirus chimeras and the consequences for the current efforts to detect potentially dangerous vaccine-derived poliovirus strains are discussed in the context of the global polio eradication initiative.

ABSTRACTENG1(YNR067c), a gene encoding a new endo-1,3-ß-glucanase, was cloned by screening a genomic library with a DNA probe obtained by PCR with synthetic oligonucleotides designed according to conserved regions found between yeast exo-1,3-ß-glucanases (Exg1p, Exg2p, and Ssg1p). Eng1p shows strong sequence similarity to the product of the Saccharomyces cerevisiae ACF2gene, involved in actin assembly “in vitro,” and to proteins present in other yeast and fungal species. It is also related to plant glucan-binding elicitor proteins, which trigger the onset of a defense response upon fungal infection. Eng1p and Acf2p/Eng2p are glucan-hydrolyzing proteins that specifically act on 1,3-ß linkages, with an endolytic mode of action. Eng1p is an extracellular, heavily glycosylated protein, while Acf2p/Eng2p is an intracellular protein with no carbohydrate linked by N-glycosidic bonds. ENG1transcription fluctuates periodically during the cell cycle; maximal accumulation occurs during the M/G1transition and is dependent on the transcription factor Ace2p. Interestingly, eng1deletion mutants show defects in cell separation, and Eng1p localizes asymmetrically to the daughter side of the septum, suggesting that this protein is involved, together with chitinase, in the dissolution of the mother-daughter septum.

ABSTRACTThe M phenotype is by far the most common mechanism of erythromycin resistance among Streptococcus pyogenesisolates in Spain. A geographic analysis of the relationship between within-country differences in the prevalence of M-type resistance to erythromycin in S. pyogenesand the level of consumption of 14- and 15-membered macrolides within different provinces was carried out. From 1998 to 1999, a nationwide multicenter surveillance study yielded 2,039 consecutive pharyngeal isolates of S. pyogenes. Data on antibiotic consumption for the same period were gathered from IMS Health, and the corresponding daily defined doses per 1,000 inhabitants per day were calculated according to the Anatomic Therapeutic Classification index. Macrolide use was subdivided into dosages given three times a day (TID), twice a day (BID), or once a day (OD). Spearman nonparametric correlation coefficients (R) were calculated, and variables proving to be significantly associated (P< 0.1) were introduced into a linear regression model. The total consumption of macrolides presented a significant correlation with the prevalence of resistance (R= 0.527; P= 0.032). Neither TID nor BID macrolide consumption showed significant correlations. Only OD consumption had a significance below 0.1. These data are consistent with the hypothesis that only the total consumption of macrolides influences the local rates of M-type erythromycin resistance in S. pyogenes,and subgroups of macrolides seem to have an additive rather than a selective effect by contributing to increasing the final amount of macrolides used. Local variations in total consumption were associated only with BID consumption (R= 0.849; P= 0.004). The simple linear regression with total macrolide consumption showed a considerable determination coefficient (R2= 0.678; P= 0.006). The model explains up to 68% of the measured variation and is clearly better as a predictor of the prevalence of resistance than the mere mean is. By solving the regression equation, the resultant value of 2.2 defined doses per 1,000 inhabitants per day fits with the existence of a critical threshold of selective pressure.

ABSTRACTA diagnostic protocol was started to study the etiology of pulmonary infiltrates in immunosuppressed patients. The diagnostic yields of the different techniques were analyzed, with special emphasis on the importance of the sample quality and the role of rapid techniques in the diagnostic strategy. In total, 241 patients with newly developed pulmonary infiltrates within a period of 19 months were included. Noninvasive or invasive evaluation was performed according to the characteristics of the infiltrates. Diagnosis was achieved in 202 patients (84%); 173 patients (72%) had pneumonia, and specific etiologic agents were found in 114 (66%). Bronchoaspirate and bronchoalveolar lavage showed the highest yields, either on global analysis (23 of 35 specimens [66%] and 70 of 134 specimens [52%], respectively) or on analysis of each type of pneumonia. A tendency toward better results with optimal-quality samples was observed, and a statistically significant difference was found in sputum bacterial culture. Rapid diagnostic tests yielded results in 71 of 114 (62.2%) diagnoses of etiological pneumonia.

ABSTRACTCHAT and Cox type 1 live-attenuated poliovirus strains were developed in the 1950s to be used as vaccines for humans. This paper describes their characterization with respect to virulence, sensitivity for growth at high temperatures, and complete nucleotide and amino acid sequences. The results are compared to those for their common parental wild virus, the Mahoney strain, and to those for two other poliovirus strains derived from Mahoney, the Sabin 1 vaccine strain and the mouse-adapted LS-a virus. Analysis of four isolates from cases of vaccine-associated paralytic poliomyelitis related to the CHAT vaccine revealed genetic and phenotypic properties of the CHAT strain following replication in the human gut. CHAT-VAPP strain 134 contained a genome highly evolved from that of CHAT (1.1% nucleotide differences), suggesting long-term circulation of a vaccine-derived strain in the human population. The molecular mechanisms of attenuation and evolution of poliovirus in humans are discussed in the context of the global polio eradication initiative.

Nitric oxide (NO) may act as a neuroprotector or neurotoxic agent in dopamine neurons, depending on cell redox status. We have investigated the effect of several thiolic antioxidants, glutathione (GSH), its cell permeable analog GSH ethyl ester (GSHEE), and the GSH synthesis precursor l - N -acetyl cysteine (L-NAC), as well as non-thiolic antioxidants like ascorbic acid (AA) and uric acid, on NO-induced toxicity in fetal midbrain cultures. The cultures were treated for 8-24 h with neurotoxic doses of the NO donor diethylamine/nitric oxide complex sodium DEA/NO (200-400 μM) and/or antioxidants. Thiolic antioxidants, at equimolar concentrations, added at the same time or previous to DEA/NO, protected from cell death, from tyrosine hydroxylase (TH) positive cell number decrease and from intracellular GSH depletion, induced by DEA/NO, without increasing intracellular GSH content. In these conditions, S -nitrosothiol compound formation was detected in the culture media. Protection disappeared when antioxidants were supplied 30 min after NO treatment. Nevertheless, non-thiolic antioxidants, AA and uric acid, with similar peroxynitrite scavenging activity to thiolic antioxidants, and free radical-scavenging enzymes as catalase and Cu/Zn-superoxide dismutase, which prevent extracellular peroxynitrite ion formation, and 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron), which prevents intracellular peroxynitrite ion formation, did not rescue cell cultures from neurotoxicity induced by NO. In addition, AA exacerbated DEA/NO-induced toxicity in a dose-dependent manner from 200 μM AA. The present results suggest that only antioxidants with thiol group exert neuroprotection from NO-induced toxicity in fetal midbrain cultures, probably by direct interaction of NO and thiol groups, resulting in NO blocking. On the other hand, some classical antioxidants, like AA, exacerbate neurotoxicity due to NO.

ABSTRACTAkv1-99, a variant of Akv murine leukemia virus, induces B-cell lymphomas with nearly 100% incidence and a mean latency period of 12 months after injection into newborn NMRI mice. PCR amplification and sequence analyses of DNA flanking integrated proviruses revealed proviral insertion into the N-ras/unr(upstream of N-ras) locus in 2 out of 13 B-cell lymphomas, both of which appeared clonal by Southern blotting analysis. These two tumors showed increased expression levels of N-rasby Northern blotting, as did a third tumor shown by reverse transcriptase PCR to have a nonclonal provirus integration located in the same area. However, no significant changes in expression were observed when using a specific probe for the unrgene. All proviruses were integrated in the same transcriptional orientation as unrand N-rasgenes. By promoter insertion, the two Akv1-99 proviruses integrated between exon -1 and exon 1 of N-rasgave rise to two different spliced products, whereas the provirus integrated into unrused only an exon skipping pattern. The absence of mutations of the N-rascodons 12, 13, 18, and 61 suggests that activation of the proto-oncogene is exclusively due to overexpression by retroviral promoter insertion, and furthermore, Northern blot analyses indicate that the expression of unris unaffected by N-rasoverexpression even in the case where the unrgene itself is the target of proviral insertion. Thus, altogether our findings indicate that overexpression of N-rasplays a role in development of murine leukemia virus-induced B-cell lymphomas, leaving the expression of the tightly linked unrgene unaltered.